Metabolic Biochemistry, Volume - T. P. Mommsen

Preface
The idea of editing a series of volumes on The Biochemistry and Molecular Bi-
ology of Fishes was born out of the present-day lack of a forum for state-of-the-art
review articles in this rapidly expanding field of research. On the one hand, researchers
and students in this area always find themselves combing the literature on general
(rat-dominated) biochemistry before discovering short and usually incomplete and
disappointing coverage of the situation in the piscine setting. On the other hand, the
rapidly expanding volume and quality of the primary literature in fish biochemistry
and molecular biology supply convincing evidence for a maturing field. This discipline
is no longer the younger sibling of rat or human biochemistry but has recently led to
a number of major conceptual breakthroughs; for this reason, and because its activ-
ity domain is sometimes nonoverlapping with 'mainstream' biochemistry, the field is
certainly ripe and ready for a review series of its own.
Comparative biochemistry and molecular biology and comparative physiology as
disciplines by definition use organisms as a special kind of experimental parameter
for probing general mechanisms and principles of function. In theory this approach is
relatively blind to phylogenetic boundaries, but in practise the realities of funding and
availability of experimental material greatly narrow the field of play. As a result, two
phylogenetic groups - - the insects and the fishes - - have over the last several decades
provided the bulk of the experimental data base in these disciplines. Interestingly,
although comparative biochemistry in many ways grew out of comparative physiol-
ogy, the growth and development of these two activities in the insect field have to
major extent proceeded along independent paths. By contrast, the comparative phys-
iology and biochemistry of fishes have not been so independent of one another and
the tendency has been for the former to envelope the latter. We believe that the cur-
rent conceptual developments in the fields as well as the simple logistics of dealing
with massive data bases make this the right time for the reality of independence to
match the perception of independence, which we feel is another important rationale
for this review series.
Our goal is to provide researchers and students with a pertinent information source
from theoretical and experimental angles. To be useful to students, theoreticians,
and experimentalists alike, contributing authors are urged to emphasize concepts
as well as to relate experimental results to the biology of the animals, to point out
controversial issues, and todelineate as much as is possible directions for future
research.
Peter W. Hochachka
Thomas P. Mommsen
Vancouver and Victoria, B.C.
Contributors
Hiroki Abe, Department of Food Science and Nutrition, Kyoritsu Women's University,
1- 710 Motohachioji, Hachioji, Tokyo 193, Japan (Chapter 14)
James S. Ballantyne, Department of Zoology, University of Guelph, Guelph, Ontario,
Canada NI G 21/11 (Chapter 10)
Andrew H. Bass, Section of Neurobiology and Behavior, Cornell University, Ithaca,
New York 14853, USA (Chapter 12)
Ralf Bastrop, Universiti~tRostock, Fachbereich Biologie, Zoologisches Institut, Univer-
sitiitsplatz 2, D-02500 Rostock 1, Germany (Chapter 7)
Richard W. BriU, Southwest Fisheries Science Center, Honolulu Laboratory, National
Marine Fisheries Service, National Oceanic and Atmospheric Administration, Hon-
olulu, Hawaii 96822-2396, USA (Chapter 1)
Stephen EJ. Brooks, Nutrition Research Division, Health Canada, Tunney's Pasture,
Ottawa, Ontario, Canada K1A OL2 (Chapter 13)
C.G. Carter, Department of Aquaculture, University of Tasmania, PO Box 1214,
Lauceston, Tasmania 7250, Australia (Chapter 8)
Nathan L. Collie, Department of Biological Sciences, Texas Tech University, Lubbock,
Texas 79409-3131, USA (Chapter 9)
Ronaldo P. Ferraris, Department of Physiology, University of Medicine and Dentistry
of New Jersey,, New Jersey Medical School, Newark, New Jersey 07103-2714, USA
(Chapter 9)
Glen D. Foster, Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa,
Ontario, Canada KIN 6N5 (Chapter 4)
Edward M. Goolish, National Oceanic and Atmospheric Administration, Southwest
Fisheries Science Center, La Jolla, California 92038, USA and Scripps Institution
of Oceanography, Center for Marine Biotechnology and Biomedicine, University of
California, San Diego, La Jolla, California 92093, USA (Chapter 15)
Joaquim Guti6rrez, Departament de Bioquimica i Fisiologia, Universitat de Barcelona,
Unitat de Fisiologia Animal F, Av. Diagonal, 645, E-08071 Barcelona, Spain (Chap-
ter 17)
Peter W. Hochachka, Department of Zoology, University of British Columbia, Vancou-
ver, British Columbia, Canada V6T 2A9 (Chapter 1)
viii Contributors
D.E Houlihan, Department of Zoology, University of Aberdeen, Aberdeen, TiUydrone

Avenue, Aberdeen AB9 2TN, Scotland, UK (Chapter 8)
Karl Jiirss, Universiti~t Rostock, Fachbereich Biologie, Zoologisches Institut, Univer-
sitiitsplatz 2, 1)-02500 Rostock 1, Germany (Chapter 7)
Odile Mathieu-Costello, Department of Medicine, Universityof California, San Diego,
La Jolla, California 92093-0623, USA (Chapter 1)
I.D. McCarthy, Department of Zoology, University of Aberdeen, Aberdeen, Tillydrone
Avenue, Aberdeen AB9 2TN, Scotland, UK (Chapter 8)
Thomas P. Mommsen, Department of Biochemistry and Microbiology, University of
Victoria, P.O. Box 3055, Victoria, British Columbia, Canada VSW3P6 (Chapter 12)
9Thomas W. Moon, Department of Biology, University of Ottawa, 30 Marie Curie,
Ottawa, Ontario, Canada KIN 6N5 (Chapter 4)
Christopher D. Moyes, Department of Biology, Queen's University, Kingston, Ontario,
Canada K7L 3N6 (Chapter 16)
Isabel Navarro, Departament de Bioquimica i Fisiologia, Universitat de Barcelona,
Unitat de Fisiologia Animal F,, Av. Diagonal, 645, E-08071 Barcelona, Spain (Chap-
ter 17)
Bernd Pelster, lnstitut ffir Physiologie, Ruhr-Universitdt Bochum, D.44780 Bochum,
Germany (Chapter 5)
Jean-Francois Rees, Laboratory of Animal Physiology, Catholic University of Louvain,
Croix du Sud 5, B-1348 Louvain-la-Neuve, Belgium (Chapter 18)
Kenneth B. Storey, Departments of Biology and Chemistry, Carleton University,
Ottawa, Ontario, Canada KIS 5B6 (Chapter 13)
Eric M. Thompson, Laboratoire de Biologie Cellulaire, Unit~ de Biologie du D~-
veloppement, Institut National de la Recherche Agronomique, F-78352 Jouy-en-Josas,
France (Chapter 18)
Guido van den Thillart, Institute of Evolutionary and Ecological Sciences, Animal
Physiology, Gorlaeus Laboratories, University of Leiden, PO Box 9502, 2300 RA
Leiden, The Netherlands (Chapter 3)
Douglas R. Tother, NERC Unit of Aquatic Biochemistry, School of Natural Sciences,
University of Stirlinb StirlingFK9 4LA, Scotland, UK (Chapter 6)
Marcel van Raaij, Institute of Evolutionary and Ecological Sciences, Animal Physiol-
ogy, Gorlaeus Laboratories, University of Leiden, PO Box 9502, 2300 RA Leiden, The
Netherlands (Chapter 3)
Patrick J. Walsh, Marine Biology and Fisheries Division, Rosenstiel School of Marine
and Atmospheric Sciences, Universityof Miami, 4600 Rickenbacker Causeway, Miami,
Florida 33149-1098, USA (Chapter 12)
Contributors ix
Jean-Michel Weber, Biology Department, University of Ottawa, 30 Marie Curie,

Ottawa, Ontario, Canada KIN 6N5 (Chapter 2)
Timothy G. West, Department of Zoology, Cambridge University, Downing Street,
Cambridge, CB2 EJ3, UK (Chapter 16)
Harold H. Zakon, Department of Zoology, Patterson Laboratory, The University of
Texas, Austin, Texas 78712, USA (Chapter 11)
Georges Zwingelstein, Laboratoire Maritime de Physiologie, Institut Michel Pacha,
Universit~ de Lyon, 1337 Corniche Michel Pacha, Tamaris, F-83500 La Seyne sur Mer,
France (Chapter 2)
Abbreviations
AA Amino acid(s) HK Hexokinase

AChR Acetylcholine receptor HPLC High performance liquid
ACI'H Adrenocorticotropic hormone chromatography
AIaAT Alanine aminotransferase HSP Heat-shock protein
ALD Aldolase IDL Intermediate density lipoproteins
AQ Ammonia quotient LCAT Lecithin:cholesterol acyl transferase
AS Atlantic salmon cell line LDH Lactate dehydrogenase
AspAT Aspartate aminotransferase LDL Low density lipoproteins
BBMV Brushborder membrane vesicles LT Leukotrienes
BCAAT Branched-chain amino acid LX Lipoxins
aminotransferase ME Malic enzyme
BCKAD Branched-chain a-ketoacid MT 17oe-Methyltestosterone
dehydrogenase NEAA Non-essential amino acids
BF-2 Bluegill fry cell line NMJ Neuromuscular junction
BiP Immunoglobulin binding protein NMR Nuclear magnetic resonance
BLMV Basolateral membrane vesicles ODC Ornithine decarboxylase
cAMP Y,5'-cyclic adenosine-monophosphate PAF Platelet activating factor
CCO Cytochrome C oxidase PC Pyruvate carboxylase
CHSE-214 Chinook salmon epithelium cell line PCA Perchloric acid
CPK Creatine phosphokinase PCr Phosphocreatine
CPT Carnitine palmitoyl transferase PDG Phosphate-dependent glutaminase
CS Citrate synthase 6PGDH 6-Phosphogluconate dehydrogenase
DAG Diacylglycerol PEPCK Phosphenolpyruvate carboxykinase
DHT 5a-Dihydrotestosterone PFK-I Phosphofructokinase- 1
DMF Dimethylformamide PG Prostaglandins
DMSO Dimethylsulfoxide PG I Phosphoglucose isomerase
E2 17fl-Estradiol PGK Phosphoglycerate kinase
EAA Essential amino acid(s) PK Pyruvate kinase
EDTA Ethylenediaminetetraacetic acid PKA Protein kinase A
EO Electric organ PKC Protein kinase C
EOD Electric organ discharge PMN Pacemaker nucleus
EPO Erythropoietin PtdA Phosphatidic acid
FAA Free amino acid(s) PtdCho Phosphatidylcholine
FABP Fatty acid binding protein PtdEtn Phosphatidylethanolamine
FBPase Fructose 1,6-bisphosphatase Ptdlns Phosphatidylinositol
FFA Free fatty acid(s) PtdSer Phosphatidylserine
FG Fast glycolytic (muscle fiber) PUFA Polyunsaturated fatty acids
FHM Fathead minnow cell line RQ Respiratory quotient
FOG Fast oxidative glycolytic (muscle fiber) RT-2 Rainbow trout germ cell line
G6Pase Glucose 6-phosphatase RTG Rainbow trout gonad cell line
G6PDH Glucose 6-phosphate dehydrogenase SDA Specific dynamic action
GABA Gamma-aminobutyrate SO Slow oxidative (muscle fiber)
GAPDH Glyceraldehyde 3-phosphate T3 3,5,3? -Triiodo-L-thyronine
dehydrogenase TAG Triacylglycerol
GDH Glutamate dehydrogenase TF Turbot fin cell line
GLP Glucagon-like peptide TPI Triosephosphate isomerase
GPase Glycogen phosphorylase TRH thyrotropin releasing hormone
oeGPDH ~-Glycerophosphate dehydrogenase TX Thromboxanes
GSase Glycogen synthase VHDL Very high density lipoproteins
HDL High density lipoproteins VLDL Very low-density lipoprotein
HEPE 12-Hydroxyeicosapentaenoate XDH Xanthine dehydrogenase
HETE 12-Hydroxyeicosatetraenoate XO Xanthine oxidase
Hochachka and Mommsen (eds.), Biochemistry and molecular biology of fishes, vol. 4
9 1995 Elsevier Science B.V. All rights reserved.
CHAPTER 1
Design for a high speed path for oxygen:

tuna red muscle ultrastructure and
vascularization
ODILE MATHIEU-COSTELLO, RICHARD W. ]]RILL * AND

PETER W. HOCHACHKA **
Department of Medicine, University of California, San Diego, La JoUa, CA 92093-0623, U.S.A.,
* Southwest Fisheries Science Center, Honolulu Laboratory, National Marine Fisheries Service,
National Oceanic and Atmospheric Administration, Honolulu, HI 96822-2396, U.S.A. and
**Department of Zoology, University of British Columbia, Vancouver, B.C., Canada V6T 2,49
I. Introduction
II. Materials and methods
1. Animals
2. Tissue preparation
3. Morphometry
III. Results and discussion
Acknowledgements
IV. References
I. Introduction
Because it is one of the most aerobic muscles in fish, the red muscle of tuna
is of particular interest to study strategies and constraints in structural designs
for high 02 flux from capillary to muscle fiber mitochondria. Tuna can maintain
extremely high aerobic metabolic rates and reach high swimming speeds 4. The tuna
red muscle is well known to operate at higher than ambient water temperature by
conserving heat via the central counter-current heat exchange (for review, see ref.
36), and white muscle lactate turnover rates after exercise are known to be closer
to those found in mammals than in other fish 1'39. In this chapter, we summarize
our morphometric findings on the three-dimensional arrangement of the capillary
network and its relationships with fiber ultrastructure in red muscle of skipjack
tuna, Katsuwonus pelamis, in comparison to highly aerobic skeletal muscles of birds
and mammals. Muscles designed for high sustainable activity (hummingbird and
bat flight muscles as well as the red muscle of tuna) are all composed of only
one population of very highly aerobic fibers, instead of the mosaic of fiber types
with different metabolic pattern found in the vast majority of skeletal muscles.
This homogeneity allows one to specifically examine capillary-fiber geometrical
relationships across species, in particular vascular supply in relation to muscle
2 O. Mathieu-CosteUo, R.W. Brilland RW. Hochachka
fiber aerobic capacity in cases of very high demand for 02 flux. As summarized
further in this chapter, previous studies showed striking similarities in structural
design for high 02 flux in hummingbird and bat flight muscles despite several
differences in capillary-fiber geometry2s,29. In fish as in birds, red blood cells are
nucleated and less deformable than mammalian red cells, but they can be larger
than bird red cells, and fishes operate at different body temperature than both
birds and mammals. Thus, it is of particular interest: (1) to examine capillary-fiber
structural arrangement in the red muscle of one of the most athletic fishes known;
and (2) to compare it with that in highly aerobic skeletal muscles of birds and
mammals.
II. Materials and methods
While the details of methods used here have been described elsewhere 22, it is
important to briefly highlight aspects that are relevant to properly explain the
results.
1. Animals
Five Skipjack tuna (Katsuwonus pelamis); body mass 1.5-2 kg; fork length 43-44
cm) were purchased from local commercial fishermen and held in outdoor 10 m
diameter holding tanks supplied with continuously flowing seawater (25 4- 1~
at the Kewalo Research Facility (National Marine Fisheries Service, Honolulu,
Hawaii).
2. Tissuepreparation
After the tunas had been netted and anesthetized, muscle peffusion fixation with
glutaraldehyde fixative (four animals) or infusion with Batson's casting material
(one animal) were performed following procedures and subsequent tissue process-
ing described elsewhere in detail22. Transverse and longitudinal sections (1 /zm
thick) of perfusion-fixed tissue were used for light microscopy morphometry of
capillarity and fiber size. Ultrathin transverse sections (50-70 nm) were examined
with a Zeiss 10 transmission electron microscope and sampled for morphometry of
fiber ultrastructure. Samples injected with casting material were examined with a
Stereoscan 360 scanning electron microscope (Cambridge Instrument).
3. Morphometry
Sarcomere length was measured on longitudinal sections, after careful control

of the angle of each section 19. Fiber cross-sectional area, capillary diameter and
capillary number around a fiber were measured on transverse sections with an image
analyzer. Capillary numbers per fiber sectional area in transverse and longitudinal
sections were collected by point-counting, and the data were used to estimate
Design for a high speed path for oxygen: tuna red muscle ultrastructure and vascularization 3
the degree of orientation of capillaries and capillary length per fiber volume 18.
Capillary-to-fiber ratio (i.e. capillary number per fiber number) was computed as
the product of capillary density (i.e. number per fiber cross-sectional area) and
mean fiber cross sectional area. Capillary surface per fiber volume was obtained
by intersection-counting on vertical (i.e. longitudinal) sections using a cycloid
grid 2. Capillary-to-fiber perimeter ratio in transverse section, which is an index of
the size of the capillary-fiber interface 25 was measured by intersection-counting
in transverse sections 21, and capillary surface per fiber surface estimated as the
product of capillary-to-fiber perimeter ratio and an orientation coefficient c'(K',O)
as described elsewhere 25.
The volume of mitochondria per volume of muscle fiber was estimated by stan-
dard point-counting 22, and mitochondrial volume per/zm fiber length calculated as
the product of mitochondrial volume density and fiber cross-sectional area. Where
appropriate, data on fiber size and capillary density were normalized to sarcomere
length, in order to compare morphological data between muscles, independent of
the particular length at which each sample was fixed and therefore examined. A
normalizing sarcomere length of 2.1/zm was chosen because it is in the mid-range
of the sarcomere lengths where maximal tension is developed in skeletal muscles,
and it is within the range of operating sarcomere lengths in hindlimb muscles of
mammal during terrestrial locomotion (range, 1.7-2.7 ~m) 6, wing muscles of bird
during wing beat cycle (1.7-2.3/zm) 5 and red muscle in fish during swimming at
slow speed (1.9-2.2/zm) 35.
III. Results and discussion
Figure la-c illustrates the high capillary density, small fiber size and high mi-
tochondrial volume density previously reported in red muscle of tuna 3,1~ In
longitudinal sections (Fig. lb), we found a large number of capillaries cut in trans-
verse or oblique section, as well as branches running perpendicular to the muscle
fiber axis. This suggested the presence of capillary manifolds in tuna red muscle,
as previously found in the highly aerobic pectoralis muscle of pigeon 2~ Figure 2a,b
illustrate the remarkable similarity between the appearance of capillary manifolds
in tuna red muscle (Fig. 2a) and pigeon pectoralis muscle (Fig. 2b). In that study,
Potter and coworkers 34 showed that these capillary branches oriented perpendicular
to the muscle fiber axis are venular capillaries which form dense manifolds around
groups of muscle fibers. The examination of microcorrosion casts of tuna red muscle
also showed that capillaries form a dense envelope of blood around muscle fibers
(Fig. 2c).
The functional implications of the particular arrangement of venular capillaries
in those muscles are not fully understood. Capillary manifolds could facilitate an
increased vascular supply to and from the muscle fibers at the venular end of
the network where substrates and 02 content are lowest and metabolite concen-
tration highest. They could also be related to other functional aspects such as
heat dissipation and/or the blood pumping action of the muscle during flight in
4 O. Mathieu-Costello, R.W. BriU and RW. Hochachka
Fig. 1. Fine structure of tuna red muscle, a and b: light micrographs of portions of muscle bundles
in transverse and longitudinal sections, respectively, c: electron micrograph of transverse section of
muscle fibers and adjacent capillaries (c). Capillaries are empty after the fixation by vascular perfusion.
Note large capillary density and small fiber size (a-c), large number of capillary branches running
perpendicular to the muscle fiber axis (b) and high density of mitochondria, M (c). From ref. 22.
birds. Interestingly, however, capillary manifolds were found in flight muscle of

hummingbird ~, but not in bat 24,29. The fact that they were found in tuna red muscle
also suggest possible rheological implications since in fish, as in bird, red blood cells
are nucleated and less deformable than mammalian red cells. Another possibility
in tuna is transfer of heat from the muscle at the venular end of the network, as it
possibly favors heat removal in bird flight muscle 2~
Table 1 summarizes morphometric data on capillarity and fiber ultrastrueture
in red muscle of tuna compared with tuna white muscle, and aerobic muscles of
birds and mammals with large differences in aerobic capacities. In tuna red muscle,
fiber cross-sectional area was small (~500/~m 2) but not as small as in ultimate
cases of high aerobic capacity in bird and mammal. In hummingbird and bat flight
muscles, average fiber cross-sectional area was ~200 and 300/~m 2, respectively, in
tissues similarly prepared. Note that the number of capillaries per number of fibers
was similar in tuna red muscle and hummingbird flight muscle (~1.6). However,
Fig. 2. Examples of capillary manifolds, a: light micrograph in a longitudinal section of tuna red muscle.
b and c: scanning electron micrographs of vascular corrosion casts examined perpendicular to the
surface of the manifold in pigeon flight muscle (b) and in cross-section in tuna red muscle (c). Note
the remarkable similarity between the appearance in tuna (a) and pigeon (b) muscles, and the dense
envelope formed by capillaries around muscle fibers (c). Based on fiber dimensions, two muscle fibers
(A and B) could be contained in the empty space in c. From refs. 22 (a,c) and 34 (b).
because of the difference in fiber size, there was a huge difference in capillary
numerical density between the muscles. The number of capillaries per mm 2 fiber
cross-sectional area at 2.1/zm sarcomere length was 3400 in tuna red muscle and
8000 in flight muscle of hummingbird.
Capillary length density is an important estimate of capiUarization which ac-
counts for capillary geometry, and determines capillary volume and surface area
available for exchange per unit volume of fiber and mitochondria. Figure 3 shows
estimates of the degree of capillary orientation, expressed as the percentage added
6 O. Mathieu-CosteUo, R. 11/..
BriU and R W. Hochachka
r~ 0
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Design for a high speed path for oxygen: tuna red muscle ultrastructure and vascularization
100
80 Rat
60 ~ ; u n o red mu.clo
~0 \e e Bat
4.0 Oqlt% p,,~omll.
20 Pigeon
O . , . 9 . , . | . i . 9 - , .
1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8

Sarcomere length,/~m
Fig. 3. Plot of the degree of orientation of capillaries (expressed as the percentage added to capillary
length by tortuosity and branching, compared with straight, unbranched capillaries oriented parallel
to the muscle fiber axis) against sarcomere length in red muscle of tuna (solid circle) compared with
group mean values (:I:SE) in highly aerobic muscles of birds and mammals. From refs. 22 (tuna), 28
(hummingbird) and 29 (bat). Relationships in rat hindlimb (solid line) and pigeon pectoralis muscles
(broken line) are from refs. 25 and 20, respectively.
to capillary length by tortuosity and branching, against sarcomere length in the

muscles listed in Table 1. It is now well established that both fiber size 7,11,26 and
the degree of orientation of capillaries 19'27,33 are functions of sarcomere length. As
a consequence, capillary density in transverse sections can underestimate capillary
length per fiber volume by a different percentage (e.g. 10-70% in rat muscles;
see solid line in Fig. 3) depending on the sarcomere length at which samples are
fixed and therefore examined. Figure 3 also shows that the degree of orientation of
capillaries can vary between muscles and/or animals, for example bat flight muscle
compared with rat soleus, or bird compared with mammal. Therefore, it may not be
appropriate to compare muscle capillarity based on capillary densities in transverse
sections alone even if all samples are fixed at the same sarcomere length 2~
It is interesting to note that in tuna red muscle, the contribution of tortuosity
and branching to capillary length was similar to that in rat muscles (Fig. 3) in
spite of the different geometry. In contrast to tuna and bird muscles 22,28 skeletal
muscles of mammals showed no evidence of capillary manifolds 29. The different
contribution of capillary tortuosity and branching to capillary length in tuna red
muscle compared with bird muscles (Fig. 3) may be related to differences in fiber
size, yielding differences in length and/or number of branches in manifolds between
the muscles. In addition, capillary length density in tuna red muscle and pigeon
pectoralis muscle were remarkably similar (4100 mm/mm 3) in spite of the different
degree of capillary orientation, capillary-to-fiber ratio and fiber size. It was less than
half the capillary length density seen in ultimate cases such as the flight muscles of
hummingbird and small bats (Table 1).
8 O. Mathieu-CosteUo, R.W. Brilland R W. Hochachka
E
E || ,,1 ,,, ,,,,,, , , ,,,,
E lOOOO
9 . b,t,,d r,t pectoroli8
E - - - heart HbirdT].. 9
...'T" Bat
E 8ooo
:3
0
>
~. 6000
t)
J~
t= Pigeon T..." "" Tuna ~k --
4O00
.. ~l~lllp~ ~
J=
r 2000 Rat..'" ~
l)
.,m
9O "Tuna white muscle

o 0 i , ,, I ,li,, I - * - 9
Q,
e a , 10 20 30 40
0 Mitochondriol volume / fiber volume,
Fig. 4. Plot of capillary length per fiber volume against mitochondrial volume density in red muscle of
tuna (solid circle) compared with tuna white muscle (open circle), and group mean values (4-SE) in
highly aerobic muscles of birds (solid triangle) and mammals. From refs. 22 (tuna), 28 (hummingbird),
29 (bat and rat), 20 and 23 (pigeon). Linear relationship in bat pectoralis, bat hindlimb and rat M.
soleus (r = 0.993) is from ref. 29. Linear relationship in mammalian heart (r = 0.85) is from ref. 14.
Our measurements of fiber size in red and white muscle of skipjack tuna are
within the range of values reported by others, although direct comparison is often
difficult because of differences in tissue preparation or because sarcomere length is
not reported. To our knowledge, capillary density (e.g. capillary-to-fiber ratio and
number per fiber cross-sectional area) or geometry in tuna red muscle had never
been reported prior to our studies. Comparison with data in red muscle of other
fishes 8,17,3~ revealed that neither was fiber size the smallest, nor capillary number
around a fiber or capillary density the highest in red muscle of tuna. Similarly,
mitochondrial volume density in tuna red muscle (28.5-35%; this study and ref. 16)
was high, but not the highest, for fish muscle. The highest mitochondrial volume
density for fish (45.5%) has been reported in red muscle of anchovy 17.
The comparison of capillary length per fiber volume at a given mitochondrial vol-
ume density showed that values in tuna red muscle were as great as in mammalian
heart and about half those in highly aerobic muscles of bird and mammals (Fig.
4). For example, capillary length per fiber volume at 30% mitochondrial volume
density was 4300 mm -2 in tuna red muscle 22 compared with 7600 mm -2 in bat and
rat muscles 29. It is interesting to note that in flight muscle of bird (hummingbird
and pigeon), capillary length per unit volume of mitochondria was similar to that
in bat and rat hindlimb (Fig. 4). There were about 25 km capillaries per ml of
mitochondria in those muscles compared to 14 km in tuna red muscle. The different
capillary geometry does not account for the different relationship between capillary
length per fiber volume and mitochondrial volume density in tuna compared with
highly aerobic muscles of birds and mammals (capillary manifolds were found both
in tuna red muscle and bird flight muscle).
On average, about one third of fiber mitochondrial volume was subsarcolemmal
in tuna red muscle. This fraction was less than in flight muscles and more than
in rat soleus, where subsarcolemmal mitochondria represented about one half and
less than one fifth of the fractional volume of mitochondria, respectively (Table 1).
In other words, comparison of highly aerobic muscles in fish, bird and mammal
shows that the proportion of subsarcolemmal mitochondria is not greater in muscle
with greater fiber size. Rather the opposite is observed, bat and hummingbird flight
muscles (with the smallest fiber size) showing the greatest relative proportion of
subsarcolemmal mitochondria. Interestingly the red muscle of anchovy, with the
greatest reported volume density of mitochondria for fish skeletal muscle (45.5%),
also showed a much greater fiber cross-sectional area (1115 /tm2; ref. 17) than
tuna and other highly aerobic muscles (Table 1). This also indicated that intrafiber
diffusion distances to mitochondria are not necessarily reduced in highly aerobic
muscles of fish. A relatively large proportion of subsarcolemmal mitochondria (25%
of total mitochondrial fractional volume) was found in tuna white muscle (Table 1).
It was similar to that in bat hindlimb and almost as large as in pigeon pectoralis
and tuna red muscle, i.e. muscles with much smaller fiber size and much greater
proportion of interfibrillar mitochondria than in white muscle of tuna. Thus, a great
ratio of subsarcolemmal relative to interfibrillar mitochondria is not necessarily a
characteristic of highly aerobic muscles.
Another important parameter to consider when assessing the three-dimensional
arrangement of capillaries relative to the muscle fibers and its impact on the
geometry of blood-tissue exchange, is capillary-fiber surface. Traditionally, muscle
potential for 02 flux had been viewed in terms of intercapillary and diffusion
distances. In contrast, recent experimental and theoretical evidence (see ref. 13 for
review) suggested an important role of the capillary-fiber interface in determining
02 flux rates in working red muscles. Cryomicrospectroscopy measurements of
myoglobin saturation in quick-frozen red muscles have shown that the major pO2
drop from capillary into a cross-section through the muscle fiber occurs within a
few microns subjacent to the capillary and further decline towards the center of
the fiber is very shallow because of myoglobin facilitated diffusion 9. In this context,
capillary-to-fiber surface, i.e. the size of the capillary-fiber interface, is an aspect of
capillary-fiber structure which needs to be also considered when assessing muscle
capacity for 02 flux from capillary to fiber mitochondria. As pointed out by Sullivan
and Pittman 3s, matching 02 supply and demand in muscles can be achieved by
nature via different strategies. It can change fiber size (which affects capillary
surface per fiber volume) or capillary-fiber contact area (i.e. capillary-fiber surface)
or both.
Figures 5 and 6 show the relationships between capillary surface per fiber
volume and mitochondrial volume density (Fig. 5) and capillary-fiber surface and
mitochondrial volume per unit length of fiber (Fig. 6) in red muscle of tuna
compared with highly aerobic muscles of bird and mammal. Capillary surface
density at a given volume density of mitochondria was smaller in tuna red muscle
10 O. Mathieu-CosteUo, R.W. BriU and R14/..Hochachka
E
E
(~ 160 9. . bat and rat
E
E 140 Pectoralis
E 120 Hbird. ~:[-~~(~t
:3
o
> 100
L
Hindlimb
9
.Q
(P
u
80
60
40
Bot .."
.,~.-~ ,.',. Tuna
0 Rat .."
1:: 20 el4 red muscle
M . . tuno ..hit. muscl.,
~, 0 9 I 9 l ,
0 0 10 20 30 40
Q.
0
13 Mitochondriol volume / fiber volume,
Fig. 5. Plot of capillary surface per fiber volume against mitochondrial volume density in red muscle
of tuna (solid circle) compared with tuna white muscle (open circle) and group mean values (:I:SE) in
highly aerobic muscles of bird and mammal. From refs. 22 (tuna), 28 (hummingbird) and 29 (bat and
rat). Linear relationship (r = 0.99) in bat M. pectoralis, bat hindlimb and rat M. so/eus (dotted line) is y
= 3.4 x +2.4.
Q)
o 0.6 ....
0
e9
&
.r
0.4 A A't*
e===
o 6
e 9149 0
<>
0 0.2
0
s o s 0 ~
r 0 0
o 0.0 ,,.
.--_ 0 50 1 O0 150 200 250
Q.
C) Mitochondrial volume //~m fiber length
o
Fig. 6. Plot of capillary surface per fiber surface against mitochondrial volume per unit length of
muscle fiber (i.e. mitochondrial volume density multiplied by fiber cross-sectional area) in tuna red
muscle (solid circle) compared with rat soleus (open diamond), flight muscle of hummingbird (solid
triangle) and bat (solid diamond) and group mean value (:I:SE) in bat hindlimb. From refs. 22 (tuna),
28 (hummingbird) and 29 (bat and rat).
Design ]'or a high speed path ]:or oxygen: tuna red muscle ultrastructure and vascularization 11
(Fig. 5). This was due to the smaller capillary length density in tuna (Fig. 4) while
capillary diameter was similar ("4/xm) among muscle groups. It is also interesting
to note the similar capillary surface per unit volume of mitochondria in highly
aerobic flight muscles (bat and hummingbird) and in bat hindlimb and rat soleus
muscles ('-'3400 cm 2 per ml of mitochondria). In comparison, the value in tuna red
muscle was only "~1800 cm 2 (Fig. 5). In contrast, capillary surface per fiber surface
at a given mitochondrial volume per unit length of fiber was similar in tuna red
muscle and rat M. soleus and it was about half that in the flight muscles of bat and
hummingbird (Fig. 6). Interestingly, the ratio between capillary-to-fiber surface and
mitochondrial volume per unit length of fiber in the most highly aerobic muscle in
fish, i.e. the red muscle of anchovy (calculated from ref. 17; see ref. 22) was also
close to that in tuna red muscle and it was more than half those in flight muscles
of bat and hummingbird. This suggests consistent differences in the size of the
capillary-to-fiber interface relative to the mitochondrial volume to be supplied per
unit length of fiber in extremely highly aerobic muscle of fish compared with bird
and mammal.
The greater capillary-fiber surface ratio in flight muscles at a given mitochondrial
volume per unit length of fiber suggests an increased capacity for 02 flux. It is con-
sistent with the greater respiratory rates of mitochondria in flying hummingbirds 37
(7-10 ml 02 per ml mitochondria per min) compared with locomotory muscles of
mammals running at VO2max (ref. 15) (5 ml O2/ml mitochondria/min). It supports
the idea of an important role of the capillary-fiber interface in determining 02 flux
rates in working red muscles 9. In tuna red muscle, capillary-fiber surface at a given
volume of mitochondria per unit length of fiber was similar to that in rat soleus
(Fig. 6), in spite of the lower capillary surface per unit volume of mitochondria in
tuna (Fig. 5). Measurements of maximal respiratory rates of tuna red muscle mito-
chondria in vitro 31, yielded estimates of maximal in vivo mitochondrial respiratory
rates at least 3-5 times lower in tuna red muscle than in mammals 22. The reason for
this difference is not fully understood. The differences in operating temperatures
between the muscles could play a role, since accounting for plausible Q l0 values
yield maximal respiratory rates in tuna close to those in mammal 22. However, other
explanations are also possible including the up-regulation of protein and amino
acid metabolism in fish muscle compared with other vertebrates 12 which may re-
quire greater mitochondrial volume densities for the enzymes of amino acid and
protein turnover. Both substrate and heat transfer may also require an increased
capillary-fiber surface in tuna independently of 02 transfer per se 22.
In summary, examination of capillary-to-fiber geometry in tuna red muscle
displays both similarities and differences with features found in the most highly
aerobic muscles of birds and mammal. Three features seem prominent in the
design for high flux paths for oxygen: (1) small fiber size; (2) high capillary density;
and (3) high mitochondrial density, but in tuna these are not as pronounced as
in hummingbird and bat flight muscles. Additionally, a particular arrangement of
capillary manifolds seem required in birds and tuna but not in mammals. Perhaps
because of constraints of function at different temperatures, capillary length per
unit volume of mitochondria is substantially shorter in red muscle of tuna than in
12 O. Mathieu-Costello, R. W. Brill and R W. Hochachka
skeletal muscles of both bird and mammal over a wide range of aerobic capacities.
Similarly, capillary-to-fiber surface appears to be systematically smaller in highly
aerobic muscles of fish than in flight muscle of birds and mammals for the volume
of mitochondria to be supplied per unit length of fiber. Whether those differences
are related to differences in mitochondrial properties or capillary function or both,
remains to be determined.
Acknowledgements. Supported by Grant 5PO1 HL-17731 from the National

Institutes of Health, U.S.A.
II4. References
1. Arthur, P.G., T.G. West, R.W. Brill, P.M. Schulte and P.W. Hochachka. Recovery metabolism
of skipjack tuna (Katsuwonus pelamis) white muscle: rapid and parallel changes in lactate and
phosphocreatine after exercise. Can. I. Zool. 70: 1230-1239, 1992.
2. Baddeley, A.J., H.J.G. Gundersen and L.M. Cruz-Orive. Estimation of surface area from vertical
sections./. Microsc. 142: 259-276, 1986.
3. Bone, Q. Myotomal muscle fiber types in Scomber and Katsuwonus. In: The Physiological Ecolo~ of
Tunas, edited by G.D. Sharp and A.E. Dizon, New York, Academic Press, pp. 183-205, 1978.
4. Bushnell, P.G. and R.W. Brill. Responses of swimming skipjack (Katsuwonus pelamis) and yellowfin
(Thunnus albacares) tunas to acute hypoxia, and a model of their cardiorespiratory function. PhysioL
Zool. 64: 787-811, 1991.
5. Cutts, A. Sarcomere length changes in the wing muscles during the wing beat cycle of two bird
species, l. Zool. London (.4) 209: 183-185, 1986.
6. Dimery, N.J. Muscle and sarcomere lengths in the hind limb of the rabbit (Otyctolagus cuniculus)
during a galloping stride, l. Zool. London (tl) 205: 373-383, 1985.
7. Dulhunty, A.E and C. Franzini-Armstrong. The relative contributions of the folds and caveolae
to the surface membrane of frog skeletal muscle fibres at different sarcomere lengths. I. Physiol.
(London) 250: 513-539, 1975.
8. Dunn, J.E, W. Davison, G.M.O. Maloiy, P.W. Hochachka and M. Guppy. An ultrastructural and
histochemical study of the axial musculature in the African lungfish. Cell Tissue Res. 220: 599-609,
1981.
9. Gayeski, T.E.J. and C.R. Honig. O2 gradients from sarcolemma to cell interior in red muscle at
maximal VO2. Am. I. Physiol. 251: H789-H799, 1986.
10. George, J.C. and E.D. Stevens. Fine structure and metabolic adaptation of red and white muscles
in tuna. Env. Biol. Fish. 3: 185-191, 1978.
11. Gray, S.D. and E.M. Renkin. Microvascular supply in relation to fiber metabolic type in mixed
skeletal muscles of rabbits. Microvasc. Res. 16: 406-425, 1978.
12. Hochachka, P.W. and G.N. Somero. Biochemical Adaptation, New Jersey, Princeton University
Press, 1984.
13. Honig, C.R., Gayeski, T.E.J. and Groebe, IC Myoglobin and oxygen gradients. In: The Lung, edited
by R.G. Crystal, J.B. West, P.J. Barnes, N.S. Cherniack and E.R. Weibel. New York: Raven Press, p.
1489-1496, 1991.
14. Hoppeler, H. and S.R. Kayar. Capillarity and oxidative capacity of muscles. News Physiol. Sci. 3:
113-116, 1988.
15. Hoppeler, H. and S.L. Lindstedt. Malleability of skeletal muscle in overcoming limitations: struc-
tural elements. J. Exp. BioL 115: 355-364, 1985.
16. Hulbert, W.C., M. Guppy, B. Murphy and P.W. Hochachka. Metabolic sources of heat and power in
tuna muscles. I. Muscle fine structure, l. Exp. Biol. 82: 289-301, 1979.
17. Johnston, I.A. Quantitative analyses of ultrastructure and vascularization of the slow muscle fibres
of the anchovy. Tissue Cell 14: 319-328, 1982.
18. Mathieu, O., L.M. Cruz-Orive, H. Hoppeler and E.R. Weibel. Estimating length density and
quantifying anisotropy in skeletal muscle capillaries. I. Microsc. 131: 131-146, 1983.
19. Mathieu-Costello, O. Capillary tortuosity and degree of contraction or extension of skeletal muscles.
Microvasc. Res. 33: 98-117, 1987.
20. Mathieu-Costello, O. Morphometric analysis of capillary geometry in pigeon pectoralis muscle. Am.
J. Anat. 191: 74-84, 1991.
21. Mathieu-Costello, O. Morphometry of the size of the capillary-to-fiber interface in muscles. Adv.
Exp. Med. Biol, 345: 661-668, 1994.
22. Mathieu-Costello, O., P.J. Agey, R.B. Logemann, R.W. Brill and P.W. Hochachka. Capillary-fiber
geometrical relationships in tuna red muscle. Can. J. Zool. 70: 1218-1229, 1992.
23. Mathieu-CosteUo, O., P.J. Agey, R.B. Logemann, M. FIorez-Duquet and M.H. Bernstein. Effect of
flying activity on capillary-fiber geometry in pigeon flight muscle. Tissue Cell, 26: 57-73, 1994.
24. Mathieu-Costello, O., P.J. Agey and J.M. Szewczak. Capillary-fiber geometry in pectoralis muscles
of one of the smallest bats. Respir. Physiol., 95: 155-169, 1994.
25. Mathieu-Costello, O., C.G. Ellis, R.E Potter, I.C. MacDonald and A.C. Groom. Muscle capillary-
to-fiber perimeter ratio: morphometry. Am. J. Physiol. 261: H 1617-H 1625, 1991.
26. Mathieu-Costello, O., D.C. Poole and R.B. Logemann. Muscle fiber size and chronic exposure to
hypoxJa.Adv. Exp. Med. Bio1248: 305-311, 1989.
27. Mathieu-Costello, O., R.E Potter, C.G. Ellis and A.C. Groom. Capillary configuration and fiber
shortening in muscles of the rat hindlimb: correlation between corrosion casts and stereological
measurements. Microvasc. Res. 36: 40-55, 1988.
28. Mathieu-Costello, O., R.K. Suarez and P.W. Hochachka. Capillary-to-fiber geometry and mitochon-
drial density in hummingbird flight muscle. Respir. Physiol. 89:113-132, 1992.
29. Mathieu-Costello, O., J.M. Szewczak, R.B. Logemann and P.J. Agey. Geometry of blood-tissue
exchange in bat flight muscle compared with bat hindlimb and rat soleus muscle. Am. J. Physiol.
262: R955-R965, 1992.
30. Mosse, P.R.L. The distribution of capillaries in the somatic musculature of two vertebrate types
with particular reference to teleost fish. Cell Tissue Res. 187: 281-303, 1978.
31. Moyes, C.D., O. Mathieu-Costello, R.W. Brill and P.W. Hochachka. Mitochondrial metabolism of
cardiac and skeletal muscles from a fast (Katsuwonus pelamis) and a slow (Cyprinus carpio) fish.
Can. J. Zool. 70: 1246-1253, 1992.
32. Poole, D.C. and O. Mathieu-Costello. Analysis of capillary geometry in rat sub-epicardium and
sub-endocardium. Am. J. Physiol. 259: H204-H210, 1990.
33. Potter, R.E and A.C. Groom. Capillary diameter and geometry in cardiac and skeletal muscle
studied by means of corrosion casts. Microvasc. Res. 25: 68-84, 1983.
34. Potter, R.E, O. Mathieu-Costello, H.H. Dietrich and A.C. Groom. Unusual capillary network
geometry in a skeletal muscle, as seen in microcorrosion casts of M. pectoralis of pigeon. Microvasc.
Res. 41: 126-132, 1991.
35. Rome, L.C. and A.A. Sosnicki. The influence of temperature on mechanics of red muscle in carp. J.
Physiol. (London) 427: 151-169, 1990.
36. Stevens, E.D. and Neill, W.H. Body temperature relations of tunas, especially skipjack. In: Fish
Physiology. Vol. VII, Locomotion, edited by W.S. Hoar and D.J. Randall, New York, Academic
Press, p. 315-359, 1978.
37. Suarez, R.K., J.R.B. Lighton, G.S. Brown and O. Mathieu-Costello. Mitochondrial respiration in
hummingbird flight muscles. Proc. Natl. Acad. Sci. USA 88: 4870-4873, 1991.
38. Sullivan, S.M. and R.N. Pittman. Relationship between mitochondrial volume density and capillarity
in hamster muscles. Am. J. Physiol. 252: H149-H155, 1987.
39. Weber, J.-M., R.W. Brill and P.W. Hochachka. Mammalian metabolite flux rates in a teleost: lactate
and glucose turnover in tuna. Am. J. Physiol. 250: R452-R458, 1986.
Hochachka and Mommsen (eds.), Biochemistryand molecularbiology of fishes, vol. 4
CHAPTER 2
Circulatory substrate fluxes and their

regulation
JEAN-MICHEL WEBER AND GEORGES ZWINGELSTEIN *

Biology Department, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada K l N 6N5 and
* Laboratoire Maritime de Physiologic, Institut Michel Pacha, Universit~ de Lyon, 1337 Comiche
Michel Pacha, Tamaris, F-83500 La Seyne sur Mer, France
I. Introduction
II. Why and how to measure metabolite fluxes in vivo?
III. Basic regulatory mechanisms
IV. Lactate fluxes
V. Glucose fluxes
VI. Amino acid fluxes
VII. Lipid fluxes
VIII. References
I. Introduction
Multicellular life can only be sustained if selected metabolic fuels, end-products,

and anabolic precursors are transported between cells at the appropriate rates
and times. In vertebrates, most inter-tissue metabolite exchange depends on the
cardiovascular system and, consequently, the regulation of circulatory substrate fluxes
plays a crucial role in achieving homeostasis.
Fishes are no exception. They must constantly adjust rates of blood metabolite
turnover to coordinate biochemical processes involved in maintenance, growth,
reproduction, locomotion and various responses to environmental stresses. As a
group of vertebrates, however, they must use distinct metabolic strategies mainly
imposed by their aquatic environment and high protein intake 48,83. Both, proteins
and lipids dominate fish energy metabolism because low amounts of carbohydrates
are ingested and their absorption is rather limited 42. Surprisingly, most of the
detailed information concerning metabolite fluxes of fish deals with carbohydrates
even though they often represent a very small fraction of these organism's total
energy budget. This bias can be explained by: (1) an imitation of mammalian
studies where carbohydrates can play a major role; and (2) the relative simplicity of
carbohydrate biochemistry compared with lipids and proteins.
In this chapter, we examine the main metabolic substrates found in the systemic
circulatory system of fish (Fig. 1) and review what is presently known about the
modulation of their fluxes. Plasma concentrations of these major substrates are
16 J.-M. Weber and G. Zwingelstein
TRANSPORT
STORAGEAND
TRANSFORMATION
EXCHANGE WITH
ENVIRONMENT
INTESTINE
RFtEE 90LUW3~" [~GLYCEROL
ACIOiS
~T~A~
ADIPOSE
GILLS
SKi~ETAL I~SCI, F.S
SKIN
~iOTEIV.BOUNQ FATTYACIDS 1
TRIACYLGLYCEROLS HEART
PHOSFtlOUPIOS
~DNEY STEROLESTERS
BRAIN
" I I
!
GONADS ..... i
Fig. 1. Major soluble and protein-bound circulatory fuels in fish: sources and destinations.
summarized in Table 1. The secondary circulation47.119 is not discussed separately

because no metabolite measurements from this compartment are yet available.
Also, the fluxes of several systemic substrates have never been measured directly.
In such cases, we suggest important avenues for future work and provide indirect
estimates whenever possible.
II. Why and how to measure metabolite fluxes in vivo ?
All body constituents are constantly produced and utilized 44, and circulatory
metabolites are therefore kept in a dynamic state, undergoing constant turnover.
For decades, however, changes in plasma concentration have been used to draw
quantitative conclusions about rates of substrate release into the circulation and up-
take therefrom. Such conclusions are often not valid because concentration changes
only indicate an imbalance between release and uptake, and major variations in
flux can potentially occur while concentration stays constant 12s. Fortunately, flux
and concentration of individual metabolites usually vary in parallel and some qual.
itative information about flux can be gained from the direction of concentration
changes.
Modem access to various metabolic tracers has opened the door for the direct
measurement of fluxes in vivo on a routine basis and mammalian biology has greatly
benefited from this approach. In contrast, relatively few whole organism turnover
studies have been attempted in fish with the two major techniques presently
available: bolus injection and continuous infusion. The terminology, experimental
procedures and calculations necessary to carry out reliable flux measurements have
Circulatory substrate fluxes and their regulation 17
TABLE 1
Resting concentrations of major plasma substrates in fish
Metabolite g 1-1 /zmol m1-1

Directly available (no hydrolysis required)
Glucose 123,131 0.2- 2.5 1 -14
Lactate 9,26,71,123 0.01- 0.2 0.1- 2
Amino acids 54,77,92,95 0.3 - 2.3 2 -14
Fatty acids 59,64,131 0.1 - 1.5 0.1- 5.4
Only available after hydrolysis
Triacylglycerols64'94,131 1 -11 1 -12
Phospholipids 19,94 4 -10 7 -12
Total proteins 1~ 28 -35 m
Molar concentrations were calculated using average molecular weights of 160 (amino acids), 280 (fatty
acids), 880 (triacylglycerols), and 780 (phospholipids).
been described in detail by Hetenyi 44, Katz 51-53, Okajima 81 and their coworkers
and by Wolfe 128, amongst others.
The bolus injection technique has almost been used exclusively in fish studies
because it only requires a single catheter for both, tracer injection and blood sam-
piing. In contrast, continuous infusion takes two catheters to allow simultaneous
infusion and sampling, and the added difficulties associated with surgical placing
and maintenance of two lines have encouraged fish biologists to opt for the simpler
experimental design of bolus injection 123. This is unfortunate because much more
information could be obtained from continuous infusion where consecutive mea-
surements of flux are possible in a single experiment under steady or non-steady state
conditions (i.e. even when metabolite concentration varies during the experiment).
A more complete understanding of flux regulation in fish will require common
use of continuous infusion and the development of easier double catheterization
techniques should make this possible.
III. Basic regulatory mechanisms
How does the organism alter metabolite turnover rate in response to different
stresses? In a study on the regulation of plasma metabolite fluxes in exercising
Thoroughbred horses, blood flow and plasma metabolite concentration were pro-
posed as the coarse and fine control, respectively 125,126. There is no reason to
believe that flux regulation follows different principles in teleosts. Blood flow is the
coarse control because its changes will affect all plasma fuels to the same extent.
Metabolite concentration represents the fine control because modifying it for in-
dividual substrates will allow the modulation of flux for each fuel independently.
This way, the respective contribution of each substrate to total metabolism can be
affected by its relative concentration in the circulation, and a positive correlation
between circulating concentration and turnover rate has been demonstrated for a
LACTATE
[•'• SWIMMING
X
=)
_J
c~,.s. I
It. TROUT I ~ST
IrLouNoER~
~,.o,, I
CONCENTRATION
Fig. 2. Relationships between plasma lactate concentration and flux in several species of teleosts. Note
the positive correlation between the slope of this relationship and cardiac output.
variety of metabolites in all species studied to date. In addition, the slope of the
relationship between concentration and flux increases as cardiac output rises 126.
The regulating roles of cardiac output and circulating metabolite concentration
can be demonstrated for teleosts in the case of lactate. Figure 2 shows how the
slope varies between species and experimental conditions. Slopes range between 0.7
and 3.6 in resting teleosts where changes in lactate concentration were elicited by
hypoxia or previous heavy exercise21,29,71. In contrast, during exercise, rainbow trout
(Oncorhynchus mykiss) show a slope of 5.2 (ref. 123): almost twice the value found
during hypoxia29. This large difference can be explained by the fact that cardiac
output is much higher in swimming animals than in resting hypoxic fish. Finally,
it is not surprising to find the highest slope of 15.1 in skipjack tuna (Katsuwonus
pelamis), a species with a cardiac output more than 7 times higher than rainbow
trout 32.
There is no doubt that the above analysis of flux regulation is still extremely
primitive. Potentially important biochemical signals and direct neural effects have
not even been mentioned here because their influence has not been investigated in
fish. Presumably, some of these factors will affect fluxes indirectly by changing blood
flow or circulating concentration of the metabolite of interest. Several hormones
are bound to play important regulating roles and their investigation should be a
priority in future research.
IV. Lactate fluxes

Lactate has occupied a prominent position in studies of hypoxia and muscle
metabolism for a very long time. In the last 20 years, tracer experiments have al-
lowed to establish that its fluxes were much higher than for other plasma substrates,
even in resting organisms, and that it could become an important metabolic fuel
TABLE 2
Lactate turnover rate in post-absorptive teleosts
Species Mass Rt Predicted Rt for Predicted mammal Rt/

(kg) (/zmol kg -1 mammal of same size Measured fish Rt
min -1)
Anguilla rostrata 26 O.180 0.50 145 290
Platichthys stellatus 71 0.335 0.76 112 147
Oncorhynchus kisutch 71 0.275 1.33 122 92
Ictalurus punctatus 21 0.800 2.25 78 35
Oncorhynchus mykiss 29 0.350 2.80 110 39
Oncorhynchus mykiss 123 0.322 4.41 114 26
Katsuwonus pelamis TM 1.420 112 61 0.54
Predicted values for mammals of equivalent body mass were calculated as follows: Rt -- 70.78 Mb 0'42,
where Rt = lactate turnover rate in ttmol kg -I min -1, and Mb ffi body mass in kg (modified from
reference 123).
for oxidative tissues in mammals 76,122. This new picture of lactate metabolism has
attracted the attention of fish biologists, and resting lactate turnover rates have been
measured in several teleost species (Table 2). Except for tuna, the lactate fluxes
of fish range from 0.5 /~mol kg -1 rain -1 in eels (Anguilla sp.) to 4.4/tmol kg -1
min -1 in rainbow trout (Oncorhynchus mykiss). Enough information is available
from mammals to derive an allometric equation expressing the relationship between
resting lactate turnover rate and body mass in this vertebrate group 123. We have
used this equation to compare lactate fluxes in teleosts and mammals of equivalent
size (Table 2). This comparison shows that turnover rate is 26 to 290 times lower
in fish than in mammals, but skipjack tuna (Katsuwonuspelamis), the only scombrid
measured to date, stands out as a clear exception with fluxes exceeding those of
mammals TM.
Ratios between lactate turnover and oxygen consumption rates of teleosts and
mammals of the same size are similar, suggesting that the metabolic role of lactate
is equivalent in all resting vertebrates when the effect of body mass is taken into
account 123. This conclusion may not hold during exercise because patterns of lactate
exchange between skeletal muscle and the circulation are so strikingly different
in fish and mammals. After strenuous swimming for example, lactate is released
extremely slowly from fish white muscle 113 and this typical pattern of retention
found in all teleosts is exaggerated in bottom-dwelling, sedentary s p e c i e s 114'121.
Therefore, during exercise, lactate oxidation may account for different proportions
of total VO2 in mammals, pelagic and benthic fish.
Stresses of different kinds are known to stimulate lactate fluxes in mammals, but
little information is available for fish. Nonetheless, fasting, hypoxia and exercise
have all been shown to increase lactate turnover rate in some teleosts. In American
eels, long-term fasting causes a 2.5-fold rise in turnover rate 26, but the effect of food
restriction has never been quantified in other fish species. Similarly, the effect of
hypoxia has only been measured in rainbow trout where turnover rate increased by
20 J.-M. Weberand G. Zwingelstein
seven-fold at an environmental p O 2 of 4 kPa (ref. 29). Exercise studies have been

limited by the steady state assumption of the bolus injection technique. Two steady
state situations have been investigated to date: prolonged aerobic swimming and
recovery from strenuous, anaerobic exercise. During sustained exercise, circulatory
lactate transport between tissues could be a convenient way to shuttle carbohydrate
energy between body compartments 76, and white to red muscle lactate exchange was
hypothesized as a potential mechanism to support energy metabolism in active red
muscle of fish. However, lactate flux only increases by two-fold during sustainable
swimming in trout, showing that such a mechanism does not play a significant role
in this species 123. In recovery from exhaustive exercise, fluxes are also elevated
to accelerate the disposal of large lactate loads accumulated during anaerobic
work. After strenuous swimming, a 3- and 9-fold increase in turnover rate was
measured in flounder and salmon, respectively71. In neither species were recovery
fluxes sufficiently high to explain the time course of decrease in muscle lactate
concentration, suggesting that a fraction of the total lactate load never leaves white
muscle and is metabolized in situ.
V Glucosefluxes
Rates of glucose turnover have been measured in several species at rest (Table 3).
They were determined under steady state conditions and therefore represent both,
rates of glucose production (Ra) and disappearance from the circulation (Rd) at
the whole organism level. The liver accounts for most of the glucose produced,
but fish kidneys can probably also make a significant contribution unlike their
mammalian counterpart 55.7s,1~ The relative importance of liver and kidney has not
been quantified in vivo, and is likely to depend upon species, diet, and level of
activity.
Measured rates of glucose t u r n o v e r (Rt) are upper estimates of glucose oxidation
TABLE 3
Glucose turnover rate (Rt) in resting, post-absorptive teleosts
Species Rt
Dicentrarchus labrax3s 0.6
Oncorhynchus mykiss 27'3s 1.0
Oncorhynchus mykiss 2 1.1
Paralabrax sp. 14,15 2.1
Oncorhynchus kisutch 65 2.2
Hemitriptems ameticanus 118 3.6
Hoplias malabaricus67 3.9
Pleuronectes platessa 12 5.7
Katsuwonus pe/.am/s124 15.3
Anguilla rostrata26 56
Turnover rates are given in/~mol glucose kg -1 min -1.
(Rox) because not all glucose leaving the circulation is usually oxidized. Rox glucose
has not been quantified directly in vivo, but comparative recovery of expired 14CO2
after bolus injection of different 14C-substrates shows that glucose is oxidized
at much lower rates than fatty acids and amino acids except for glycine 37,115.
Unfortunately, the experimental approach used in these studies does not provide
absolute rates of oxidation. Continuous infusion of 14C-substrates after priming
the CO2/bicarbonate pool, and monitoring 14CO2 production will be needed to
measure such rates. Then, comparing R~,, t and Rox values will allow to determine
what percentage of total glucose turnover is oxidized.
Except for eel and tuna, glucose turnover rates of fish range between 0.6 and
5.7 /zmol kg -1 min -1 (Table 3). These values are 20-100 times lower than for
resting mammals of equivalent size ~24. The lower body temperature and lower
metabolic rate of fish may account for this difference. The high glucose fluxes of
tuna can be explained by their 'mammalian' metabolic rates and greater reliance on
carbohydrates for energy metabolism, but it remains unclear why eels should have
the ability to support even higher turnover rates than tuna or mammals. Species
showing high turnover rates appear to have a better ability to maintain steady blood
glucose concentrations 1~ However, the main factors involved in the regulation of
glucose fluxes have not been investigated thoroughly. The evidence available to
date suggests that the regulatory mechanisms of fish operate very slowly (hours)
compared with mammals (minutes). Hepatic glucose production only shuts down
1-2 h after glucose loading in Paralabrax 14. Also, indirect evidence from changes
in circulating glucose concentration suggests that insulin45,1~~and glucagon ~11 take
at least 30 min to start modifying fluxes and that their effect lasts for several
hours. Elevated plasma cortisol has no effect on the glucose turnover of rainbow
trout (cortisol injection) 2 and sea raven (high cortisol induced by chronic stress) 118.
Similarly, subjecting trout to 3 h of low water pO2 (4 kPa) had no effect on their
glucose flux29, mainly because elevated plasma catecholamines tend to abolish the
inhibitory effect of hypoxia 129. In future work, quantifying the respective effects
of circulating glucose, insulin, glucagon and other hormones will require the use
of continuous infusion and, eventually, the 'glucose clamp' technique 44 should be
adapted for fish experiments.
Because several tissues rely exclusively on glucose for energy metabolism, some
attention has been devoted to potentially limiting glucose fluxes during fasting. The
most dramatic effect has been shown in American eels where a 10-fold decrease
in glucose turnover was measured after 15 months of food deprivation 26. Shorter
studies in other species provide conflicting results. A 30% reduction in glucose flux
was observed in Paralabrax 14 and Hoplias67,but Hemitripterus 118 and Dicentrarchus 38
showed an 80 and 320% increase, respectively. These species differences are quite
puzzling and a closer look at the combined effects of several factors including size,
age, diet, locomotory habits, and temperature may provide an explanation.
The effect of exercise on glucose turnover rate has not been investigated in fish.
However, West et al. 127 have recently used deoxyglucose to quantify glucose uptake
of individual tissues from the circulation. This exciting approach will allow to deter-
mine the relative contribution of different organs to whole-animal glucose turnover
and it opens the door for a detailed investigation of fish glucose metabolism in vivo.
In a first series of experiments on trout, these authors have shown that exercise
causes a 28-fold increase in red muscle glucose utilization but has no effect on
cardiac muscle. Interestingly, glucose utilization only accounts for less than 10% of
the oxidative metabolism of these two tissues during swimming 127.
VI. A m i n o acid fluxes
Proteins represent a very important source of energy in teleosts 16, and rates of
nitrogen excretion have been used to quantify protein catabolism 117. Different stud-
ies have concluded that amino acid oxidation accounts for 14-85% of total 1(/IO2
depending on species, feeding status, and level of activity 18'56'57'116. Despite this
well-known dependence on protein for energy metabolism, very few researchers
have tried to measure rates of circulatory amino acid turnover and oxidation.
Furthermore, reports to date are qualitative only, providing relative rates between
substrates or experimental conditions. Borer and colleagues estimated that ala-
nine, glutamate, and aspartate fluxes of Paralabrax were equivalent to mammalian
values 15, confirming the much higher relative importance of amino acid catabolism
to total MO2 in fish than in mammals, because of the large metabolic rate difference
between these two groups of animals. The turnover rate of the three amino acids
measured was not affected by 72 days of fasting 15.
Measurements of 14CO2production after injection of t4C-substrates show that
circulating glutamate, alanine, leucine, and phenylalanine are oxidized much
more rapidly than glucose in resting fish37. This is also true in swimming trout
where leucine becomes the preferred amino acid substrate for oxidation in ac-
tive muscles nS. As expected, non-essential amino acids are generally favored over
essential amino acids 120.
A significant fraction of total flux is channeled through gluconeogenesis. Teleosts
have evolved a relatively high capacity for converting amino acids to glucose and
this has been interpreted as a strategy to synthesize enough mucopolysaccharides
for mucus production in organisms with little dietary carbohydrates 15.
Two very interesting situations where amino acid fluxes should be particularly
high have not been investigated so far: elasmobranchs and migrating salmon.
Elasmobranchs have no significant ability to oxidize lipids outside the livers.1~
Therefore, during sustained locomotion, they should derive most of their energy
from amino acid oxidation as indicated by their high capacity to metabolize glu-
tarnine in muscle mitochondria 22. Similarly, salmon is known to depend almost
exclusively on protein catabolism in the last stages of long migrations, after carbo-
hydrate and lipid reserves have been depleted 3.16. In sockeye salmon (Oncorhynchus
nerka), several amino acids appear to be converted to alanine before inter-organ
transport 77, suggesting that alanine fluxes are much higher in migrating than in
non-migrating teleosts. In addition, amino acid fluxes should increase throughout
migration as white muscle proteins are progressively catabolized via the indirect
action of androgens 4,s, and proteolytic agents such as cathepsins 130.
Circulatory substratefluxes and their regulation 23
Finally, no amino acid flux measurement should be attempted in fish without

considering the large concentration gradient between red cell and plasma. Amino
acids are three 11 to over 200 times 34 more concentrated inside fish erythrocytes than
in plasma, and, therefore, the specific activity measured in plasma or whole blood
will be different, leading to the calculation of distinct flux rates. Each experimental
situation should be considered individually before selecting plasma or whole blood
because concentration gradients and red cell membrane transport kinetics are so
variable between species and amino acids.
I~I. Lipid fluxes

To our knowledge, plasma lipid fluxes have never been measured in fish at the
whole organism level even though fat represents a critical source of ATP in these
animals. The following analysis will focus on indirect and qualitative information
to point out promising directions and potential difficulties for future research. The
major circulatory lipids of f i s h - free fatty acids (FFA) and triacylglycerols (TAG)
- and their sites of appearance and disappearance are summarized in Fig. 3. Also,
many important aspects of circulatory lipid transport in fish have been reviewed by
Sheridan 98.
Lipid substrates are shuttled between tissues either as FFA (rapid delivery) or
as TAG and phospholipids (slow delivery). Most of our discussion will deal with
INTESTINE
CATABOLIC
DIETARY UPIDS TISSUES
STORAGE i
TISSUES
TG+PL _
..........
~' F ~
FFA ~ TG + PL
UVER
Fig. 3. Source, destination and composition of plasma lipids. FABP ffi fatty acid binding protein; FFA
= free fatty acids; HSL = hormone sensitive lipase; LPL = lipoprotein lipase; PL = phospholipids; TG
= triacylglycerols; VLDL = very low density lipoproteins.
24 J..M. Weber and G. Zwingelstein
the relatively simple situation of FFA rather than with other lipids (TAG, glyceryl
ethers, phospholipids, free and esterified cholesterol) whose complex circulatory
transport involves ehylomierons, VLDL, HDL, and LDL9s. Even in mammals, the
present understanding of these compounds' kinetics is still very limited.
In fish, 20--30% of circulating FFA are unsaturated with chain lengths of 20 and
22 carbons 1~176 The major function of these polyunsaturated FFA is to act as
precursors of membrane phospholipids 13,1n and eicosanoid compounds 6,66,88,while
shorter chain fatty acids (C18 and less) are used primarily for energy metabolism.
Therefore, C20 and C22 acids should have much lower turnover rates than the
FFA involved in oxidative pathways. Also, one would expect that swimming will
have a much more pronounced effect on the flux of 'short' acids than on C20 and
longer FFA. The choice of an appropriate marker fatty acid for measuring FFA
fluxes for different purposes and under different conditions should take the above
considerations into account.
Plasma FFA concentration is approximately one order of magnitude higher
in teleosts than in elasmobranchs and holocephalans (see Table 4). In addition,
and contrary to eyclostomes and teleosts28,4~ elasmobranchs lack albumin-like
plasma proteins 33, and they are incapable of oxidizing fatty acids in other tissues
than in liver7,22,1~ The FFA fluxes of sharks, skates and rays should therefore
be significantly reduced in view of their remarkably limited capacity to transport
and metabolize lipids. The high cardiac output of elasmobranehs (53 v e r s u s 17
ml kg-lmin -1 in resting dogfish and trout, respectively5~ can only partially
compensate for their low plasma FFA (approximately 0.15 v e r s u s 1.2 mM). With the
same relative extraction from plasma, dogfish would only be able to support less
than half the FFA delivery rate of trout.
TABLE 4
Total plasma free fatty acid concentration in teleosts, elasmobranchs, and a holocephalan
Species FFA
(~mol m1-1)
Teleosts
Oncorhynchus mykiss 43 1.52
Salvelinus alpinus 39 2.11
Dicentrarchus labrax 131 1.10
Mullus surmuletus 131 1.42
Scomber scombrus 131 1.22
Gadus aeglefinus 59 1.54
Gadus morhua 59 1.28
Elasmobranchs
Scyliorhinus canicula 131 0.15
Squalus acanthias 131 0.15
Raja rad/ata 59 0.09
Etmoptems spinax59 0.29
Holoeephalan
Chimaera monstrosa 59 0.17
T h e effect of exercise on p l a s m a FFA fluxes should be very different b e t w e e n

species b e c a u s e teleosts show very diverse swimming abilities and lipid s t o r a g e
s t r a t e g i e s 1~ S o m e species store m o s t of their T A G in l o c o m o t o r y muscles (e.g.
herring, Clupea harengus), o t h e r s c o n c e n t r a t e T A G in liver (e.g. cod, G a d u s m o r h u a )
or in a d i p o s e tissue 98. Table 5 lists a few species to illustrate muscle/liver s t o r a g e
TABLE 5
Total lipid content of liver and muscle (% lipid per g tissue wet weight)
Species Liver Muscle

Oncorhynchus mykiss 1 5 4
Oncorhynchus nerka 17 7 15
Clupea harengus 17 2 11
Scomber scombrus 17 8 13
AnguiUa anguiUa70 12 18
Dicentrarchus labrax a 18 3
Gadus morhua 36 63 0.3
Gadus aeglefinus 82 63 0.4
a G6rard Brichon, unpublished results.
TABLE 6
Effects of hormones on plasma FFA concentration indicating similar changes in FFA fluxes
Hormone Effect on plasma FFA

Insulin 63,74 Decrease
Glucagon46 Increase
Glucagon23,60,91,109 No effect
Catecholamines3~ Increase
Catecholamines31 Decrease
Catecholamines86 No effect
ACTH 75 Increase
ACTH 3~176 No effect
Somatostatin99,101 Increase
Urotensin I199,1~ Increase
Arginine vasotocin49,68 Increase
Arginine vasotocin 68 Decrease
Thyroid hormone8~176 Increase
Thyroid hormone96 No effect
Cortisol 2~ Increase
Cortiso196 No effect
Sex steroid analog 1~ Increase
Growth hormone69,75 Increase
Prolactin61,75 Increase
26 J..M. Weber and G. Zwingelstein
options. During swimming, teleosts favoring hepatic and adipose storage will have
to supply most FFA to their working muscles v/a the circulation. Such species
should therefore increase plasma FFA fluxes to a much larger extent than fish with
considerable TAG reserves in their muscles.
A variety of hormones are potentially involved in the regulation of plasma FFA
fluxes. In mammals, flux and concentration are positively correlated 41,s4 and fish
should be no different. The direction of hormonal effects on FFA concentration
and flux are probably also identical in this group of vertebrates. Table 6 summarizes
the potential effects of several hormones on the turnover rate of circulating FFA.
It is interesting to note that some hormones will not regulate FFA fluxes in all
species because their effects are tissue specific 9s. For example, catecholamines
stimulate lipolysis in fish hepatocytes 97, but have no effect on their adipocytes 79.
Consequently, catecholamines should increase FFA turnover in species storing TAG
in the liver, but they should play no regulating role in species using mostly adipose
tissue for lipid storage.
Finally, the study of circulating triacylglycerol, phospholipids, and cholesterol
promises to be extremely complex, but experimental difficulties should be overcome
TABLE 7A
Total lipoprotein concentration and respective percent contribution of VLDL, LDL and HDL in
plasma. All values were measured by ultracentrifugation methods
Species Lipoproteins VLDL LDL HDL

(gl -~) (~) (~)
Oncorhynchus mykiss
juvenile 1~ 13-17 18 64 18
adult 24,25.35 23-26 7 36 57
Oncorhynchus nerka93 7 26 38 37
Sardinops caerulae 62 8 13 15 72
Myxine glutinosa 72 29 57 24 19
Latimeria chalumnae 72 14 77 14 9
Scyliorhinus canicula 72 2 14 75 11
Centrophorus squamosus 73 7 61 34 6
Conger vulgaris72 7 67 33 -
TABLE 7B
Average lipid and protein composition of fish lipoproteins (% lipoprotein wet weight)
Proteins Phospho- Free ..... Cholesteryl Triacyl-

lipids a cholesterol esters ,,
glycerols
Chylomicron 2 8 1 2 84
VLDL 13 15 7 18 43
LDL 26 23 9 14 25
HDL 49 26 6 12 10
a Phospholipids (mostly phosphatidylcholine and sphingomyelin). In Latimeria, Scyliorhinus, and Conger,
13 tO 70% of the triacylglycerol fraction is made of alkyldiacylglycerols72,73. Values calculated from
references 24, 25, 35, 62, 72, 73, 93, 100, and 105.
because, in certain species, these compounds can represent 10 to 40 times the energy
stored in circulating FFA (see Table 1). They are first transported as chylomicrons
before being stored in liver or adipose tissue where they can be converted to
lipoproteins and released back in the circulation 98. Measuring the fluxes of these
compounds will be a real challenge because the relative contribution of VLDL,
LDL, and HDL to total plasma lipoproteins varies greatly between species (Table
7A), and each class of lipoproteins has a different composition (Table 7B). For
these two major reasons, great care will have to be taken in choosing adequate
lipid tracers and modes of administration to decipher specific aspects of plasma
lipoprotein kinetics.
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Hochachka and Mommsen (eds.), Biochemistryand molecular biologyof fishes, vol. 4
CHAPTER 3
Endogenous fuels; non-invasive v e r s u s

invasive approaches
GUIDO VAN DEN THILLART AND MARCEL VAN RAAIJ

Institute of Evolutionary and Ecological Sciences, Animal Physiology, Gorlaeus Laboratories,
University of Leiden, P.O. Box 9502, 2300 RA Leiden, The Netherlands
I. Introduction
II. Quantifying endogenous fuels: destructive methods
1. Handling stress
2. Tissue damage
3. Tissue extraction
4. Storage
5. Measurement
III. Non-destructive approaches
1. Cannulation
2. Calorimetry
3. Nuclear magnetic resonance spectroscopy
IV. Storage of endogenous fuels
1. High energy phosphates
2. Carbohydrates
3. Lipids
4. Proteins and amino acids
V. Mobilization of endogenous fuels
1. Hypoxia and anoxia
2. Exercise
3. Starvation and migration
VI. Summary
VII. References
I. Introduction
Life is a condition that requires non-equilibrium conditions, since all processes pro-
ceed only when free energy is converted into entropy. A state of non-equilibrium is
kept at the expense of free energy: energy consumption for maintenance and activity
ultimately result in heat production, since the organism itself hardly changes. The
conversion of energy forms such as from chemical energy to kinetic energy are al-
ways coupled with increase of entropy, normally resulting in heat production. In the
case of exercise on a hometrainer the conversion-efficiency from chemical to kinetic
energy is about 25%, so 75% is lost as heat. Of course, kinetic energy dissipates in the
end as heat, so all free energy is then lost as heat. For metabolic pathways the conser-
vation of the free energy in the form of ATP has not always the same efficiency37. We
can distinguish high and low efficiency pathways depending on the amount of free
34 G. ,,an den ThiUartand M. van Raaij
energy loss22. We may ask ourselves why nature did not develop only high efficiency
pathways in order to minimize energy losses. The answer to this question is: reac-
tion rate. The higher the energy loss, the faster the reaction can proceed. So, high
efficiency processes are necessarily slow because they proceed near the equilibrium
condition, and low efficiency processes are fast and are hardly influenced by changes
in substrate and product levels because they operate far from equilibrium 37.
In order to keep an organism in a state of non-equilibrium and to enable a
large number of physiological processes, a constant energy input is needed. The
sources for this energy input are the substrates for fermentation and oxidative pro-
cesses. From a thermodynamic point of view, cells possess three types of reactions:
endergonic, equilibrium and exergonic reactions. Most anabolic and homeosta-
sis reactions are endergonic and require reactions coupled with ATP hydrolysis.
While instead most exergonic reactions are coupled with ATP synthesis of which
the most important are: the creatine kinase reaction; the pyruvate kinase and the
3-phosphoglycerate kinase reactions of the glycolysis; and the oxidative phosphory-
lation. The substrates for these three processes are respectively: creatine phosphate,
glycogen (glucose) and NADH. Although the first two can be considered as fuels,
the last substrate is in fact an intermediate occurring at fairly low concentrations
normally below 10 ~M 31,n9 and can therefore hardly be viewed as a fuel. Instead,
the substrates for the NADH generating processes should be considered as fuels for
the oxidative phosphorylation. NADH is generated mainly by three processes: (1)
the/~-oxidation of fatty acids; (2) the Krebs cycle; and (3) the glycolytic pathway.
The substrates for these processes are lipids, proteins and sugars, and can therefore
be considered as the fuels for the oxidative phosphorylation. When we define a fuel
as a compound that acts as a substrate for an ATP producing pathway, and that
can be stored to some extent, we should include anaerobic processes as well, and
consider both ATP and PCr as fuels.
Biochemically speaking we know only two types of ATP synthesis: (1) chemically
driven reactions like the pyruvate kinase reaction (substrate phosphorylation); and
(2) electrochemically driven reactions like the H+-driven ATP synthesis in the inner
mitochondrial membrane. The latter is more important from a quantitative point of
view, i.e. 18 times as much ATP is produced by the mitochondria than by glycolysis
during complete degradation of glucose. However, the energy generating processes
under anaerobic condition are for most animals crucial for survival, since oxygen
shortage is a regularly occurring phenomenon either on the tissue level (due to
ischemia or high consumption level) or particularly with fish on the organismal level
(due to low environmental 02) 22,43,1~176 Therefore, although the total ATP pro-
duction capacity of anaerobic processes is limited in comparison with that of oxida-
tive processes, we feel compelled to discuss the fuels for both pathways separately.
II. Q u a n t i f y i n g e n d o g e n o u s fuels: destructive m e t h o d s
The quantification of endogenous fuels and metabolites especially for the purpose of
describing physiological processes, is difficult because measurement always implies
Endogenous [uels; non-invasive versus invasive approaches 35
interference. It is therefore crucial for the interpretation of the data to know

to what level the process under study is disturbed by the determination of a
certain parameter. Thus far most measurements are based on destructive methods
(chemical and enzymatic reactions), for which an extract is required. Few people
realize the problems associated with tissue sampling, extraction, and metabolite
measurement. Depending upon the metabolite in question, the applied method of
sampling, and the method of extraction, the concentration may vary by more than
an order of magnitude, which should make us at least very cautious with respect
to the interpretation of the results. The number of different procedures indicate
already how difficult it is to obtain reliable metabolite concentrations. Since most
metabolite measurements are based on chemical reactions with and/or purification
of the compound in question, a solution of the metabolite must be obtained from a
previous tissue extraction. We can distinguish 3 phases in an extraction procedure,
and within each phase a number of steps (Fig. 1).
Phase I refers to the way the animal is handled: how it is taken out of its
box and manipulated in order to obtain tissue samples. Animals may be killed by
electrocution 76, by a blow on the head, by decapitation, by anesthesia, a combination
of these or even by immersion in liquid nitrogen a9,136 (see also Table 1). The major
problem in this phase is to prevent struggling of the animal, particularly when one is
interested in resting values. Certainly electrocution is the poorest and anesthesia the
best way to reach resting values. On the other hand, one should take into account
that during the period necessary to reach anesthesia, animals (after exposure to
anoxia or exercise) have time for recovery (5-10 min).
Phase II is the sampling phase. In this phase samples are taken by a biopsy
needle, or by dissection. This takes time, depending on the skill of the operator
I Precauti~ I
I sampling I
I freezing I
I extracti~ 1 l~r+ ~AI [~wder-~AI I~I
/
1 , high speed mixer/centrifugation I
Fig. 1. Steps in tissue extraction. At every step artifacts may develop, disturbing the final metabolic
picture. Only very critical consideration of the procedures may result in an acceptable estimation.
36 G. van den Thillart and M. van Raaij
TABLE 1
Muscle lactate under 'resting' conditions
Species Lactate lmmobilisation Sampling

(/zmol g-l)
Trout49 15 Liquid N2 Excision at-20~
Trout/carp 95 14 A + blow a Tissue in liquid N2
Trout98 13 Blow Tissue in liquid N2
Trout1~ 1 A Freeze clamp
Trout25 6 Decapitation Freeze clamp
Tuna 5 7 A + blow Freeze clamp
Cod16 5 Blow Freeze clamp
Perch76 3.5 Electrocution Tissue in liquid N2
Human 1~ 3 A + biopsy Liquid N2, freeze dry
Eel 124 2.4 A + curare Freeze clamp
Goldfish112 1.5 A Freeze clamp
Dog 17 1.2 A + isol. prep. Freeze clamp
a A = anaesthetic.
and the number and kind of tissues that have to be sampled. To reduce the loss of
precious time in this phase, dissection is sometimes carried out on frozen tissue.
Normally the sampling phase is terminated by freeze-clamping the tissue at liquid
nitrogen temperature (-195.8~ 1~ Sometimes this step is left out, and the
tissue is extracted immediately in cold perchloric acid (PCA). Freezing is however
the best way to 'freeze' metabolism and bring it within a few milliseconds to a
complete stop 138. Obviously this is important when one is dealing with processes
that have high reaction rates.
Phase III includes extraction and denaturation of the sample. In order to extract
and denaturate the sample properly, it is necessary to pulverize the frozen sample
together with the extraction medium to a fine powder 1~2,~5. This way the time for
denaturation is minimized, and total surface area for extraction is maximized. This
step can be left out only when slow metabolic processes are studied. Although acid
will eventually hydrolyze compounds like ATP and PCr, the rate of hydrolysis is
only a few percent per day, and therefore in most cases negligible. Extraction and
denaturation occur during thawing, therefore it is obligatory to mix the powdered
tissue with the PCA during thawing thoroughly.
At each step artifacts may be introduced, sometimes leading to spurious results.
We can distinguish five conditions where artifacts are likely to develop: handling
stress, tissue excision, tissue extraction, tissue and extract storage, and metabolite
measurement.
1. Handling stress
Except for blood sampling from cannulated fish, tissue sampling leads to handling
stress, since few animals will voluntarily give up a part of their body for scientific
Endogenous fuels; non-invasive versus invasive approaches 37
inspection. So the animals have to be anesthetized and/or killed quickly in order

to prevent extreme struggling which will otherwise certainly lead to significant
changes of the metabolite profile. Large animals offer the possibility to use biopsy
needles, although this type of sampling has its restrictions, too, because of local
tissue damage (see below). Handling stress is a behavioral type of stress, a stress
reaction initiated by the central nervous system either via direct stimulation or
indirectly via hormones (adrenaline and cortisol). A large number of physiological
reactions are activated under these conditions, all aimed at preparing the animal
for an outburst of activity by redirecting bloodflow to the muscles, stimulating
heartbeat and ventilation, increasing muscle tone, etc. All these activities have their
effects on tissue metabolism, the more so if the animal is already engaged in strug-
gling.
Handling stress can be overcome only if the animal is not able to respond
to the sampling; this can be reached either by surprise, by anesthesia, or by a
probing technique that is not sensed by the animal. Animals are not easily surprised,
certainly not on the level of tissue sampling. The fastest method is needle biopsy,
this technique is often used with experiments on humans and larger mammals 1~
but also on fish this technique has been applied 5. It can be carried out fast enough
to reduce handling stress, certainly if a local anesthetic is applied, although due to
local tissue damage sampling-artifacts cannot be completely prevented (see below).
The major problem with anesthesia is the delay; it takes time for the anesthetic to
take effect. During this delay recovery processes may take place, especially when
they are fast, the original metabolic picture may change completely during a delay
of 5-10 minutes. Besides this delay, anesthesia has also side-effects on metabolism
such as erythrocyte swelling 96. Repeated anesthesia also changes markedly the
levels of blood borne metabolites 12. In the case of resting metabolism anesthesia
is the best approach, since the disturbance will be minimal, and no recovery
processes are to be expected. In the case of exercise, a fast blow, followed by
decapitation, should be preferred in order to prevent recovery during the delay of
the anesthetic. The delay problem may be overcome by infusion of an anesthetic via
a cannula inserted in the aorta. Cannulation of the dorsal aorta is widely used for
acid-base and blood-gas studies, its application for metabolic studies is restricted
since only blood can be sampled. As far as we know cannulation has never been
used in order to reach a very fast anesthesia after exercise. In some papers on
fish metabolism, the experimental fish were inactivated/killed with electrical shock,
obviously this method has its limitations, since it will likely stimulate the whole
animal and particularly the muscles, thus leading to significant changes in the
metabolite profiles. It has been described several times that premortem stress not
only reduces the levels of glycogen, PCr and ATP, but also dramatically accelerates
the postmortem degradation rate in comparison with anesthetized fish 29'103.
The best way to overcome handling stress is to use non-invasive and non-
destructive (physical) probing techniques which are not sensed by the animal. The
technique currently available is in vivo NMR (nuclear magnetic resonance), which
will be discussed separately.
2. Tissue damage
To acquire a piece of tissue, it must be excised, which obviously causes damage to

the cells. In addition to local damage, nerves are cut and/or damaged, leading to
stimulation of the adjacent tissue via spinal reflexes, thus resulting in general activa-
tion of the excised tissue. The metabolic rate of certain tissues can thus be increased
enormously, therefore one should employ two different strategies: (1) suppress
activation as much as possible; and (2) inactivate the sample as fast as possible.
Activation particularly of muscle and neural tissue can be reduced significantly by
anesthesia, and muscle relaxants. For example, muscle from anesthetized eel (An-
guilla anguilla) responds immediately to incision, only by intracardial injection of
curare the spinal reflexes can be suppressed 124. Recently, Arthur and collaborators 5
applied lidocaine at the spinal cord of skipjack tuna (Katsuwonus pelamis) - after
previous MS 222 anesthesia - to suppress spinal reflexes during biopsy sampling.
To inactivate the sample, freeze-clamping is the ultimate procedure. The aim of
sampling is to have a momentary view of the metabolite levels. Using aluminium
blocks cooled by liquid nitrogen (-195.8~ Wollenberger and colleagues 138
demonstrated that a piece of tissue can be metabolically put to a standstill within a
few msec. This seems fast enough, especially since muscle contractions are slower.
The dimensions of the tongs determine the sample size, normally 0.1-2.0 g. With
enlarged tongs even whole animals can be freeze-clamped to a weight of about 6 g
(refs. 2, 60, 87, 136). Freeze-clamping is not always used, some authors immerse the
samples 9s or even whole animals in liquid nitrogen 36.49. This technique is inferior to
the clamping method, because the time needed to completely deep-freeze a sample
may take several minutes or longer depending on the size. Temperature equili-
bration depends on distance, temperature-difference and heat-transfer capacity.
The equilibration time is exponentially related to both distance and heat transfer
capacity, so the distance should be minimal (<2 mm), and the heat transfer should
be maximal (copper or aluminium clamps). Particularly the conductance of heat
through ice or water appears very slow, which makes the freezing rate extremely
size dependent ~3s.
An important problem that remains, is the time period needed for cutting out
the tissue, a procedure that takes 10-200 s. A problem still hard to overcome since
most tissues are difficult to excise. The application of needle-biopsy is mainly suited
for muscle and possibly liver of large animals. Tissue preparation after freezing is
not advisable, because freezing of a whole animal cannot be carried out fast enough.
3. Tissue extraction
In order to extract metabolites from the tissue properly, one should make a very
fine homogenate to reduce the diffusion barriers (maximal surface area), to break
the protein-substrate bonds, and most importantly to block all enzyme activities.
The technique often used is the one described by Williamson and Corkey137;
this technique employs ethanol as an antifreeze and perehlorie acid (PCA) as a
denaturation agent. The (frozen and powdered) tissue is then homogenized at
-15~ A low temperature is necessary to minimize enzyme activities during the

interval between the onset of thawing and complete denaturation. Too often this
technique is not consistently applied, probably because its rationale is not well
understood. Between -0.8 and -5~ glycolysis and hydrolysis of high energy bonds
proceed at higher rates than at room temperature 9,42,77,8~ The reason for this is that
between -0.8 and -5~ the intracellular water is only partially frozen. Ice crystals
rupture the membranes, resulting in a release of Ca 2+ from the sarcoplasmatic
reticulum and the extracellular space. The presence of Ca 2+ causes a striking
activation of glycolysis and of myosin ATPase. The formation of ice (crystals) also
increases the concentrations of enzymes, ions, substrates and modulators to very
high values in the remaining water phase, which of course greatly stimulates the
enzymatic reactions. The rate of ATP hydrolysis of fish muscle in the 'critical
freezing zone' can be 200 times as high as the rate at room temperature 9,77.
Denaturation at low temperature can be achieved by the use of an ethanol/
perchloric acid mixture, which should be pulverized in liquid nitrogen together
with the tissue. When the concentration of ethanol is sufficiently high (30% v/v)
the medium melts between -15 and -20~ without the formation of ice crystals.
Denaturation thus starts at a low temperature below the critical freezing zone. Still,
we should realize that at the moment a protein can be denaturated, it can at same
time catalyze its own reaction. Since the denaturation process takes time, it will
never be possible to completely exclude enzymatic reactions during denaturation.
Denaturation is a stochastic process, so it will take time to denaturate all proteins.
Therefore we should try to reduce the reaction rates as much as possible, this holds
especially for ATPases and creatine kinase since the activities of these enzymes are
rather high. Enzymes that use ATP as a substrate, need Mg 2+ as a cofactor. It is
possible to chelate both Ca 2+ and Mg 2+ to F- and EDTA. The addition of these
substances to the extraction medium results in almost 100% recovery of ATP and
PCr which was checked by adding standards to the frozen tissue/PCA powder 115.
This procedure is however not applicable to whole animal extractions. The reason
appears to be the presence of Ca 2+ and Mg 2+ salts from the skeleton and the
high concentrations of digestive enzymes. The problem can be solved by extracting
separately the carcass and the intestines, adding the appropriate amount of EDTA
and F - to the extraction medium (van den Thillart and Nieveen, unpublished).
Another solution is to freeze dry the sample at -40~ followed by PCA extraction.
This way the interfering enzymes are dehydrated and therefore not active before
denaturation. Low lactate levels and high PCr levels are found with this technique
in muscle 1~ as well as in whole animals 87.
4. Storage
Metabolites in frozen tissues are not stable at -20~ nucleotide levels and free
fatty acid contents 28 change at this temperature, apparently due to enzyme activities.
Therefore storage in liquid nitrogen or at liquid nitrogen temperature is advisable,
since even at temperatures lower than -20~ conversions are reported.
In most extracts, we have found trace amounts of enzyme activities, particularly
activities of ereatine kinase were always present. This means that after some time
equilibrium conditions will be reached in the extract. For example, in the presence
of ATPases and ereatine kinase, ATP will be buffered by creatine phosphate: [PCr]
decreases, while [ATP] remains constant. Metabolite levels in the extract are not
stable often due to the presence of enzymes, therefore the best way to store extracts
is in liquid nitrogen. Of course some metabolites are more stable than others, and
extracts are not always the same with respect to traces of enzyme activity. In the
tissue extracts we have analyzed for nueleotides by HPLC, we found <2% change
in nueleotide concentration over a 12 hour period at room temperature, indicating
a rather stable preparation. However this is not always the ease. The stability of the
extracts, and the efficiency of the extraction procedure has to be checked frequently,
particularly since many artifacts may occur.
5. Measurement
There are many different ways of measuring metabolites. The techniques are mostly
well described and tested, but under restricted conditions, and with pure solutions.
Extracts, however, contain a large number of mostly unknown compounds which
were never tested before. Also, the presence of trace amounts of enzymes may
disturb the measurements. Therefore the use of internal standards at least in a few
samples of a series is highly recommended in order to test the recovery. Recovery
should be tested in two ways: (1) addition of internal standards to the tissue powder
to test the overall recovery; and (2) addition of internal standards to the extract, to
test the efficiency of the analysis.
In the discussion about (destructive) measurements and the impact of the differ-
ent artifacts, it is important to take into account the relative level of disturbance.
For example, when glyeolysis was active during the sampling and extraction proce-
dure, then it is obvious that the relative change of glycogen is much smaller than the
change in the level of lactate. Glycogen may have decreased from 10 to 9/zmol 1-1
(-10%), while at the same time lactate increased from 0.5 to 2.5/zmol 1-1 (+400%).
Similarly, a significant change in the concentration of free fatty acids can develop in
frozen tissues, because the concentrations are very low, while in contrast the fluxes
and also the concentrations of triglyeerides are normally high 12~ Thus the impact of
the sampling and extraction procedure on the final data is not always the same.
111. Non-destructive approaches
As we have seen above, destructive techniques have quite a few drawbacks; apart
from the handling stress and the extraction artifacts, an additional problem is the
fact that for each single measurement one animal is needed. Thus, usually a large
number of experimental animals is required for statistical reasons. There are a few
techniques that allow us to overcome these problems: eannulation, calorimetry, and
in vivo NMR.
1. Cannulation
For cannulation, an indwelling catheter is usually inserted in the dorsal aorta

under anesthesia according to the method of Soivio et al. 97. After a two day
recovery in a narrow box, experiments can be carried out on the fish, and blood
samples can be drawn without disturbing the animal. The low levels of cortisol and
catecholamines found under these conditions indicate that there is indeed a low
stress level 121. Cannulation particularly of the dorsal aorta is often applied in acid/
base studies, other applications are rare. For metabolic studies cannulation can be
used for turnover studies, i.e. lactate and glucose 13, but also to follow certain blood
constituents as metabolic parameters. For example, changes in lactate, glucose,
amino acids, fatty acids, and hormones have been used in this respect to follow
the metabolic response of fish upon exercise, hypoxia, and acid exposure. More
complicated cannulation applications, such as the determination of A - V differences
over certain organs, are almost exclusively found in the mammalian literature.
This is likely due to the difficulty of reaching small blood vessels in fish. Still, for
evaluating the metabolic function of organs, such studies are obligatory. Results
from studies on isolated organs, and certainly on isolated cells, have always some
bias in comparison to whole animal studies; perfusion is never the same, neural
and hormonal control is cut off, and cell surface is changed, etc. Cannulation can
of course not be applied for the measurement of metabolites within the different
tissues. However, the technique is important for the determination of the turnover
of both endogenous and exogenous substrates.
2. Calorimetry
According to the laws of thermodynamics, all the available energy liberated by

conversion of substrates is converted to heat, other products, and activity. Under
steady state conditions, activity dissipates as heat, and we have to consider only
the substrates and products. By means of calorimetry, it is possible to calculate
the amount and the kind of substrates used by the animal for its heat production,
i.e. the use of sugars, proteins, and lipids can be calculated. This is based on the
equations that can be elaborated from the different fermentation and oxidation re-
actions taking place within the animal 36,126. Calorimetry refers to the measurement
of heatflow, which can be carried out directly and indirectly; for direct calorimetry
one has to use a calorimeter, while for indirect calorimetry one calculates the
heatflux from oxygen consumption, and CO2 and NH3 excretion. Calorimetry is a
non-invasive and non-destructive method that can be carried out without excessive
stress for the experimental animals. Recently, it has been shown that non-invasive
glycogen determination is possible from accurate respiration studies 72. Normally
the animal may stay alive, although for some calculations additional end products
should be measured like urea, lactate, and ethanol. Under conditions when the
animal is not in an aerobic steady state, a combination of direct and indirect
calorimetry reveals the fraction of each pathway for the total energy production 128.
Actually, this is the only way to quantify with a direct method the total contribution
of anaerobiosis to the energy production.
Calorimetry has some restrictions, which makes this technique not easily accessi-
ble. In the first place, the animals must be in a steady state, because it takes a while
before a thermal, gas, and metabolite equilibrium is reached. In the second place,
we must assume that no other reactions take place other than those used for the
calculations. And finally the method is slow, and expensive.
Unlike mammalian respirometry, RQ (respiratory quotient) measurements of
fish do not give sufficient information on substrate utilization. This is because
protein oxidation by NH3 excreting animals results in RQ = 0.96, which is not
distinguishable from carbohydrate oxidation with RQ = 1.00. Therefore also NH3
excretion should be measured. Measurement of CO2 and NH3 production by fish
is difficult. CO2 measurements are disturbed by the HCO 3 buffer in the water.
The NH3 production is about 10% of the 02 consumption. Furthermore, the
accumulation of CO2 and NH3 is directly related to the O2 concentration change
in the water, which is small due to the low solubility of oxygen. Thus, some skill is
essential to measure CO2 and NH3 accurately enough in order to obtain reliable RQ
and AQ values (RQ, respiratory quotient = ~'CO2/'v'O2; AQ, ammonia quotient =
VNH3/VO2).
In contrast, oxygen consumption measurements are relatively easy to obtain,
because of the high sensitivity of the currently available oxygen electrodes 38. The
oxygen consumption data are mostly used as a measure of the energy flow of the
animal. This is based on the assumption that the animal is completely aerobic,
and that the animal uses the same mixed substrate for its oxidation reactions. Of
course both conditions will never be completely fulfilled; animals do use different
substrates, anaerobic pathways, and incomplete oxidation reactions from time to
time. However, for an estimation of energy flow oxygen consumption can be used,
as long as its limitations are not forgotten.
Both direct and indirect calorimetry on fish is seldom applied. Based on the few
available data, an overview has recently been published 127.
3. Nuclear magnetic resonance spectroscopy
With in vivo NMR we are able to look inside tissues without incurring damage or
changing the metabolite pattern. The technique is based on radiowave absorption/
emission in a high magnetic field (see Volume 3 of this series, Chapter 50). As with
all spectroscopic techniques, NMR is non-destructive: the chemical composition of
the sample does not change during the measurement. Because the radiowaves and
magnetic fields penetrate through living tissue, the technique is suited for in vivo
applications. For metabolic studies three different types of nuclei are available: 1H,
13C and 31p. These nuclei occur in numerous metabolites, which then in principle
can be measured with NMR spectroscopy. There is, however, a great difference
between high resolution NMR (analysis of a solution in a test tube) and in vivo
NMR (whole animal or tissue within the sensitive area) with respect to resolution
and sensitivity. Mainly because of peak broadening, the detection threshold for
the different compounds that can be detected by in vivo NMR is much lower
than with high resolution NMR. The biggest problems occur with 1H- and 13C-
NMR. With 1H-NMR there is an enormous interference of water and lipids on
the metabolite signals: a 104-fold difference in signal strength. In addition, the
spectral band available for all the different signals is small, resulting in overlap of
most signals. New pulse editing techniques are being developed which will make
1H-NMR available for many applications in the near future. The drawback of 13C-
NMR is the low sensitivity of the nucleus, and the fact that the natural abundance
of 13C is only 1.1%. The sensitivity can be enlarged by 13C-coupled 1H-NMR, and
the concentration of 13C can be increased by the use of 13C enriched compounds.
Still, the techniques is difficult and expensive. There are a few applications for very
small animals and in vitro systems (not for fish).
The above mentioned problems do not occur with 31P-NMR, which makes this
the most widely used technique for in vivo NMR at the moment. While in vivo 1H-
and 13C-NMR have a high potential for future metabolic studies, the applicability
of in vivo 31p-NMR for fish studies has been demonstrated already by several
publications 15,11~ In vivo 31p-NMR spectroscopy allows non-invasive and
non-destructive measurement of phosphorus-containing compounds such as sugar
phosphates, inorganic phosphate, IMP, phosphodiesters, creatine phosphate, and
ATP. Furthermore, from the spectral position of the inorganic phosphate peak, the
intracellular pH can be calculated 11~ and from pH, ATP, Pi, and PCr, the free
concentration of ADP can be calculated 11~ The most striking observations
with in vivo NMR are the low Pi (1-0.5 mM) and the high PCr/total creatine ratio
(>90%) in muscle tissue in comparison with standard measurements. The low Pi
value, sometimes even below the detection limit of 0.5 mmol 1-1 (Fig. 1), indicates
a high phosphorylation potential, which is to be expected under aerobic and resting
conditions 31,11~ A high phosphorylation potential should go together with a high
PCr/total creatine ratio. The low PCr/total creatine ratios observed in the literature,
led to the suggestion that creatine should play a role in osmoregulation 7, however,
it has been found that with very careful sampling and extraction of muscle tissue a
much higher degree of creatine phosphorylation can be obtained 5,87,1~
Figure 2 shows the inorganic phosphate section of the in vivo 31P-NMR spectrum
of eel (A. anguiUa) muscle, after 24 h in a flow-cell under resting and normoxic
conditions. It demonstrates the effect of exposure to respectively 100, 10, 6 and
3% air-saturated water. Evidently, under normoxia, the Pi is almost invisible, which
means a concentration lower than 0.5 mM. Under hypoxic conditions, the Pi peak
increases, shifts and broadens, indicating respectively increasing concentration,
decreasing pH, and compartments with different pH. In addition to the low Pi level,
the high degree of phosphorylation of creatine (90%) is typical for in vivo 31P-NMR
spectra. In comparison with 'destructive' methods, the NMR technique shows that
the phosphorylation potential of the cytosol of resting aerobic tissues is very high.
The effect of an anesthetic on the energy balance of a fish is clearly demonstrated
by Chiba and coworkers is. In a simple in vivo NMR experiment, these authors
showed that the phosphorylation potential of loach (Cobitis biwae) muscle increases
markedly during anesthesia.
, I,, j, I,,,, L , , , , i , ,
5.5 5.0 4.5 4.0
PPN
Fig. 2. Part of a series of in vivo 31p-NMR spectra from the rump muscle of eel. The eel was exposed
to 100, 10, 6, and 3% air-saturated water, respectively. Under normoxic conditions the Pi signal was
absent; during hypoxia the Pi peak increases markedly. The shift to the right indicates a decrease of the
intracellular pH from 7.605 to 7.100.
When a piece of muscle tissue is excised, we find with in vivo NMR a significant
lower PCr level together with a higher Pi concentration than in the muscle of an
intact animal 115. This indicates that the sampling procedure must be responsible
for this difference. Even the use of an anesthetic appears insufficient to prevent
activation of muscle catabolism during and following excision. This follows also from
the skipjack tuna (IC pelamis) experiments of Arthur and colleagues 5" Although the
application of the biopsy sampling coupled with a spinal block is impressive, and
creatinc phosphorylation reaches 75% (the highest so far), the levels of PCr are not
high enough and the lactate level is not low enough. The shortfall from the expected
values, based on NMR data, can be caused by tissue damage, freezing time, and
extraction technique. Since we observed metabolic activation in excised tissue with
NMR, tissue damage appears to be the most likely reason. Similar biopsy studies
on humans 1~ show lactate levels around 3 raM, and crcatine phosphorylation
of 60-70%. Freeze clamping a whole animal 2~176 and muscle pieces, result in
lactate levels below 1 mM. Further, blood lactate levels in fish are low under
resting conditions (<0.5 mM, refs. 13, 16, 121). In conclusion, it is likely that
the effects of tissue damage cannot be completely overcome by the application
of anesthesia. Since PCr/total creatine ratios are seldom presented, the lactate
concentration appears to be a sensitive indicator for the level metabolic rest of the
muscle tissue. In Table 1, a list is presented of lactate levels in muscle from several
species, obtained under different conditions. The lowest levels are obtained when
an anesthetic is used in combination with very fast sampling and freeze clamping.
IV. Storage of endogenousfuels

1. High energy phosphates
When endogenous fuels are considered as those substances that contribute to cellu-
lar energy metabolism, the ultimate forms of energy are phosphorylated adenylates
(especially ATP) and creatine-phosphate. ATP levels may vary considerably between
various tissues and species. Values of about 1/zmol g-I are normally found in brain
and liver25,98,131 while heart and red muscles contain ATP levels in the range of
2-5/zmol g-1 (refs. 25, 98, 111). However, the highest ATP levels (up to 10/zmol
g-l) are mostly observed in white muscle 5,25,76. In addition, white muscles also
contain considerable amounts of PCr (20-30/zmol g-1)5,25,76, whereas in heart and
red muscle moderate levels are observed (10-20/zmol g-l). PCr is also present in
significant amounts in the brain; almost no PCr is found in the liver25,~.
In vertebrates, PCr is a directly available high-energy phosphate reservoir stored
generally in the cytosol although in muscle it may be locally concentrated near
myosin ATPase 8,86. As discussed earlier, PCr stabilizes [ATP] via the creatine kinase
reaction 11~ Although this pathway can generate ATP at high velocity, which is
favourable in fast twitch white muscle specialized for burst work, the amounts of
ATP and PCr provide only a minor reserve in terms of useful energy equivalents.
Even high levels of ATP and PCr can provide only enough energy to support a few
seconds of burst activity. Standard metabolic rate, however, can be supported for a
few hours.
2. Carbohydrates
Especially in carnivorous fish species, carbohydrates are generally not a major fuel
for cellular energy metabolism. However, during hypoxia or burst activity carbohy-
drate may become the preferential substrate. Carbohydrates are essentially stored in
the form of glycogen, a polymer of glycosyl units with linear t~(1-4) and branched
u(1-6) linkages and is generally found in the cytosol in the form of small granules
called fl-particles. Glycogen can also be stored in larger a-particles and specialized
glycogen bodies which are observed only in tissues which store huge amounts of
glycogen. As in mammals, the primary storage site in fish is the liver or hepatopan-
creas. Hepatic glycogen contents are extremely variable between fish species and
even between individuals; values from about 2/zmol glycosyl units g-1 wet weight
46 G. van den Thillan and M. van Raaij
TABLE 2
Glycogen contents of fish liver and muscles
Species Liver White muscle Red muscle

(ttmoles glucosylunits per gram of tissue wet weight)
Atlantic c o d 54 2 - -
Atlantic cod 54 39 - -
European eel 124 15 2 -
American eel69 49 12 14
Flounder79 50 6 -
S u n f i s h 111 111 3 -
Rainbow trout79 120 6 -
Rainbow trout25 134 18 16
Common carp1~176 556 - 14
Cutthroat trout 1~1 667 4 -
Mullet2 750 - -
Mackerels2 816 9 41
Goldfish111 (20~ 750 13 32
Goldfish132 (20~ 967 26 -
Goldfish132 (4~ 1366 27 -
Crucian carp45 (summer) 139 22 -
Crucian carp45 (winter) 1806 194 -
to extraordinary levels of 1800/~mol g-1 are observed (Table 2). Liver glycogen
may serve to provide exogenous blood glucose as is demonstrated in the anaerobic
goldfish 93 but not in hypoxic rainbow trout 25. White and red muscles contain signifi-
cantly less glycogen than the liver, sometimes more than a magnitude in difference.
Although the white muscle is specialized for burst type exercise which can be re-
garded as typically anaerobic, this tissue does not necessarily store more glycogen
than the red fibers 25'69'111. However, in very active pelagic fish (e.g. tuna) white mus-
cle glycogen levels may reach values above 100/~mol g-1 (ref. 5). In heart muscle,
glycogen levels are mostly 2-4 times higher to those in the red muscle 25.52,Ts.s3,n4. It
has been observed that, in contrast to the situation in mammals, the fish heart may
use carbohydrates as a major energy fuel 93,94. Other tissues may all contain variable
amounts of glycogen and the concentration in the brain in particular may have a high
impact on the hypoxia tolerance of fish species. In a comparative study on rainbow
trout (Oncorhynchus mykiss) and brown bullhead catfish ( I c t a l ~ nebulosus), the
brain of the anoxia-tolerant catfish contained about five times as much glycogen as
the anoxia-intolerant rainbow trout 23. This implicates the importance of glycogen
as an endogenous fuel in brain, heart and muscles during emergency situations like
environmental hypoxia, anoxia or exhaustive exercise. On the basis of glycogen con-
tents and the total mass of the tissue, both liver and white muscle glycogen stores
respectively are important as energy reserves.
Glycogen contents of fish tissues are highly influenced by environmental factors
including O2-availability, temperature, season and dietary composition. Hypoxia
acclimated goldfish store about twice as much glycogen in their muscle tissues
Endogenousfuels; non-invasiveversusinvasiveapproaches 47
compared to normoxic individuals although liver glycogen is not affected, possi-

bly because maximal glycogen deposition was already established 111. In flounder
(Platichthys flesus), the deposition of heart glycogen was markedly increased af-
ter three weeks of moderate hypoxia63. Glycogen in liver and white muscle of
cold-adapted goldfish (Carassius auratus, 4"C) was significantly higher than that in
20"C acclimated animals 132. This is probably of ecological relevance since Crucian
carp (Carassius carassius), a highly anoxia tolerant species from the same genus,
which can survive for months in anoxic ponds during winter, store huge amounts
of glycogen in their muscles and liver at the beginning of the winter but deplete
these stores during summer 45 (Table 1). Beside these abiotic factors, glycogen levels
are affected also by the composition of dietary intake. When trout (O. mykiss) were
forced fed orally with high doses of glucose, liver and muscle glycogen levels were
rapidly increased 18 while increased levels of dietary protein also stimulated glycogen
storage probably by increased gluconeogenesis 13~
3. Lipids
Lipids are a major energy fuel for fish tissues and most tissues posses the necessary
machinery for catabolism of fatty acids at high rates 51. As in other vertebrates,
lipids used for energy metabolism are stored in fish mainly as triglycerides, although
some species use wax-esters or alkoxydiacylglycerols exclusively or in combination
with triglycerides 91. Lipids appear to be stored in several depot organs which
contrasts to the situation in mammals in which lipids are stored almost exclusively
in adipose tissues. The most important storage sites in fish are mesenteric fat, liver
and muscles. The deposition, mobilization and composition of lipids in fish tissues
have been reviewed by Sheridan 91 and Henderson and Tother 41 and the reader is
directed to these publications for detailed information (el. chapter 6, this volume).
Adipose tissue is found in the viscera of a number of species including rainbow
trout (Oncorhynchus mykiss), coho salmon (Oncorhynchus kisutch), perch (Perca
fluviatilis), carp (Cyprinus carpio) and goldfish (Carassius auratus) and high lipid
contents which amount to more than 80% on a wet weight basis may be observed
occasionally 41. Visceral lipid contents of 15-45% wet weight are frequently ob-
served, although it must be mentioned that most of the literature deals with fish fed
commercial diets 41. In contrast to these fatty viscera, relatively lipid-poor viscera are
found in snakeheads 9~ Mesenteric fat deposits are markedly increased by excessive
dietary intake 134. Considering the high lipid concentrations and the relative contri-
bution to total body weight (up to 10%), visceral lipid depots are quantitatively a
major fuel reserve which releases free fatty acids (FFA) to the blood thus supplying
exogenous fuels for most other tissues.
Dietary lipids and other lipogenic substrates are processed in the liver which is
the primary 'lipogenic factory' of the fish body 91. Part of this lipid is transported to
other body compartments, but a significant portion is stored directly in the liver. Fat
contents of fish livers generally range from 3% to about 25% wet weight. Although
lipid contents reaching as high as 50% may occasionally be observed in fresh water
fish, really 'fatty' livers are found in benthic marine species like cod (60-70% wet
48 G. van den ThiUan and M. van Raaij
weight) 41. Liver lipids can be mobilized readily, thereby providing lipoproteins and
FFA for utilization in peripheral tissues.
Lipid levels in muscle tissues are generally lower than those in liver, although
some species exhibit extreme triglycefide stores in their muscles 85. It is known for
some time now that red muscle stores about 5 times more lipid than white muscle
and triglycedde deposits in the form of lipid droplets are found in the cytosol as well
as the interstitial space 33'34'91. Although generally overlooked, in several species a
relative large amount of lipid is also present superposed on the anterior red muscle
below the skin. While liver and mesenteric fat may be regarded as storage sites for
the total body, the lipid which is stored in and around the red muscle probably
has a direct endogenous function. Since this tissue is well peffused, contains a
high density of mitochondria, displays substantial tdglyceride lipase activity and
may posses a [FFA] higher than the blood, the lipid in this muscle is used for the
energy metabolism in situ, especially during low speed sustained swimming (see
below).
Other tissues like kidney, heart and brain are all highly capable of burning fatty
acids St, but their lipid contents are mostly moderate supporting local metabolism
only.
4. Proteins and amino acids
Proteins contribute an important part to the general fish diet, especially in carniv-
orous species. After feeding a protein rich diet, the concentration of amino acids
(AA) is increased in all tissues with the rate of protein synthesis, gluconeogenesis
and AA catabolism increasing accordingly Is. The deposition of proteins is difficult
to measure although some techniques using radioactive labeled AA have been
employed 11,27,46. The study of protein catabolism is hampered by the absence of
effective techniques but some insights can be obtained from the differences between
the rates of synthesis and growth6,1s. Since the fish body contains a large amount
of protein, changes in concentrations are mostly small and may not reach statistical
significance or are simply the result of other changes in the tissue (e.g. water or lipid
content). Instead, most studies are directed to measurement of AA metabolism or
NH~ production. However, it must be noted that the latter can also be influenced
by adenylate breakdown during hypoxia or high intensity exercise 122.
Amino acids are an important energy-source for fish tissues and the literature
on this subject is vast and numerous. This conclusion is based on amino acid
decarboxylation 74,1~ deamination 122,123, enzyme patterns and activities 2,1s,~,7~ and
nutrition studies 47,13~ However, in spite of all these data, the quantitative impact
of AA on total oxidative energy metabolism is incompletely understood, especially
since large species differences are observed.
The protein content of the fish body amounts generally to 10 to 20% of fresh
weight and is therefore the main body constituent since 60-80% of body weight is
water. The liver protein content is generally lower than 10% wet weight although
slightly higher amounts have been observed in eels21,69. The rate of protein synthesis
in the liver of rainbow trout was found to be the highest of all tissues studied
(liver > intestine > gills > muscle) 133, however, a considerable part of these
proteins are designated to the plasma. The levels of free AA are highest in the
liver and kidney and amount to approximately 20-60/zmol g-I (taurine excluded),
although levels below 10/zmol g-1 have also been reported 68. The major amino
acids in the liver are glutamate, glycine, proline and alanine, as well as taurine. The
amount of AA is very sensitive to the nutritional status of the animal and varies
with species, dietary history and the time of sampling after feeding 18. Nevertheless,
within an individual, the [AA] is mostly higher in the liver than in other tissues
which suggests a major role for the liver in AA metabolism. It was observed that
50-70% of the total NH3 production originates from the liver 81 and, moreover,
hepatectomized eels showed a reduced capacity to deaminate an excess of AA
which may occur after feeding 56. In accordance with the role of the liver with regard
to carbohydrate and lipid stores, amino acid deamination will provide the necessary
substrates for either gluconeogenesis or lipogenesis within the liver. Although liver
protein turnover is relatively high, the proteins of fish liver are not often mobilized
and since the liver mass does not exceed 3% of total body weight, liver proteins
cannot be regarded as a major energy store (see mobilization section).
Protein levels in muscles are mostly higher than those observed in other tissues.
Values up to 20% wet weight are normally observed and generally white muscle
contains more protein than the red muscle (Table 3). Muscle amino acids are
mostly present in slightly lower concentrations compared to liver ranging from 6
to approximately 50/zmol g-I (Table 3). Glutamate, alanine and glycine are the
major contributors to the total AA pool similar to liver. Also asparagine, glutamine
and in particular histidine may be present in high amounts in muscle 117,119. Beside
high levels of histidine, muscles may also contain high levels of other histidine-
derived imidazole compounds, trimethylamine, sarcosine and sulphur-containing
polypeptides. However, these compounds serve primarily as biological buffers or
osmoregulators and are probably not important as fuels for energy metabolism 122.
Although liver and kidney contain the highest concentrations of AA, the (white)
muscles provide by far the largest reservoir of amino acids on the basis of total
body weight. In addition, substantial proteinase activity can be found in muscle
tissues 4'64'68. Therefore this huge amount of energy storage may be important in
times of starvation or migration (see below).
I4. Mobilization of endogenous fuels

Energy metabolism is fuelled by a mixture of substrates which can be provided
by two sources: (1) dietary intake and absorption from the intestine; and (2)
endogenous, stored fuels. When dietary intake is absent (starting about 48 h after
the last meal) the animal must rely on its fuel stores. In the following section
we will discuss the mobilization of endogenous fuels as occurs during a number
of conditions (hypoxia-anoxia, exercise, starvation, migration) which may all be
encountered by fish in their natural environment.
50 G. van den Thillan and M. van Raaij
TABLE 3
Protein and amino acid contents of red and white muscle in teleostean fishes
Species Muscle type Protein T ot al amino acids

(% wet weight) (~tmol g - l )
Carp W h i t e 19 4 6.3
W h i t e 11 14 -
W h i t e 99 20 -
R e d 99 17 -
Goldfish W h i t e 119 - 30
R e d 119 - 25
W h i t e 117 - 36
R e d 117 - 30
Sockeye salmon W h i t e 6s - 10
R e d 6s - 8.8
Chum salmon White 4 15-20 -
Rainbow trout White ~ 19 -

D a r k 99 16 -
W h i t e 98 - 40
D a r k 9s - 36
European eel W h i t e 21 27 -
American eel W h i t e 69 15 -
R e d 69 19 -
Cod W h i t e 13~ 10 -
Char W h i t e 99 21 -
D a r k 99 18 -
Tilapia W h i t e 99 19 -
R e d 99 17 -
See text for species names.
1. Hypoxia and anoxia
Since fish live in a medium which essentially contains a low amount of dissolved
oxygen, they can easily encounter hypoxic or even anoxic environments. Environ-
mental hypoxia or anoxia can be caused by high temperature in tropical stagnant
waters, by excessive algal growth/decay or by ice covering water ponds during winter
time. The primary physiological reactions associated with hypoxia are hyperventi-
lation, bradycardia, increased stroke volume of the heart, increased gill peffusion,
redirection of blood flows and improved oxygen affinity of hemoglobin 43 ,44 , 105 . The
metabolic changes during moderate hypoxia are often transient since most fish are
able to adapt to environmental hypoxia 1~ Therefore, we will focus our attention
on the effects on metabolic energy stores during acute severe hypoxia and anoxia.
When the oxygen delivery to any tissue is hampered or inadequate, due to
ischemia, hypoxia or anoxia, the rate of oxidative ATP generation cannot keep pace
with the rate by which it is consumed. To provide the cell of a supplementary amount
of ATP two possibilities are present: (1) Stabilization of the ATP concentration by
PCr stores and (2) anaerobic glycolysis.
Although ATP levels may vary between different tissues or species, its con-
centration is quite stable during normal oxidative metabolism. The rates of ATP
consumption and regeneration are tightly regulated at the cellular level by redox
and energy state and at the level of tissues by neural and hormonal input. There-
fore, the concentration of ATP will not be affected to any great extent. During the
absence of oxygen, PCr will transfer its high-energy phosphate to ADP which can
proceed at very high velocity and is regulated primarily by the concentrations of
H + and ADP 110,125. Using in vivo 31P-NMR, Van den Thillart and colleagues 115
demonstrated that ATP levels in the white muscle of anoxic carp (Cyprinus carpio)
were maintained at control levels until PCr dropped to <20% of the control value.
Interestingly, PCr in the same tissue of the anoxic goldfish was only reduced to 55%
of the normoxic value and ATP levels were unaffected. Similarly, ATP levels in the
white muscle of trout (O. mykiss) exercised to exhaustion are significantly decreased
which is accompanied by a depletion of PCr to <20% of the control value 89.
However, in the white muscle of submaximally exercised tuna, PCr decreased to
values of about 15% of those in resting fish, while ATP was almost unaffected 5.
The recovery of PCr is a rapid process and may be complete even when ATP levels
are still depressed 89,115. The rationale for this observation is the low activity of the
purine nucleotide cycle, which converts the accumulated IMP into ATP 115 (see also
Chapter 16 of this volume). Thus, PCr is a high-phosphate energy-store which is
rapidly depleted when cellular energy requirements cannot be fully met by oxidative
and anaerobic metabolism which occurs during acute hypoxia, anoxia or intensive
exercise 110.
During oxygen shortage, the oxidative phosphorylation and electron transport
chain is inhibited resulting in a change of the redox state of the tissue and an
impaired mitochondrial ATP synthesis. One possibility to generate ATP apart from
the electron transport chain is anaerobic glycolysis. Compared to normal oxidative
metabolism this pathway is very inefficient since only 2 mol ATP are synthesized
per mole glucose fermented whereas 38 mol ATP are obtained when glucose would
be fully oxidated. Therefore, the flux through the glycolytic chain either has to
be increased (Pasteur effect) or the metabolic activity of the tissue has to be
depressed in order to adjust ATP consuming and producing processes. Since the
efficiency of the glycolysis is rather low with respect to ATP to glucose ratio, the
pathway proceeds at relatively high velocities. A prerequisite for proper cellular
function is the maintenance of redox balance. Thus, pathways have evolved in
which dehydrogenating reactions are coupled to hydrogenating reactions. The most
common end product of anaerobic glycolysis is lactate.
As discussed above, carbohydrates become the major if not only substrate for en-
ergy metabolism during anaerobiosis, whereas catabolism of fatty acids and amino
acids becomes inhibited due to the absence of an appropriate coupling between de-
hydrogenating and hydrogenating reactions. The importance of anaerobic glycolysis
is indicated by the occurrence of high levels of lactate dehydrogenase in most fish
52 G. ,an de,, Thillan and M. van Raaij
tissues 32'39'107. Glycogen mobilization during severe hypoxia or anoxia is observed

in rainbow trout ~, flounder (Platichthys flesus) 52, cutthroat trout (Oncorhynchus
clarki) and sunfish (Lepomis sp.) 4~ eel (,4. anguilla) n4, carp (Cyprinus carpio) 95,
goldfish (Carassius auratus) n1.132 and Crucian carp (Carassius carassius) 45. The
white muscle is the first tissue to become hypoxic because of its relatively low per-
fusion and utilizes mainly endogenous glycogen; the uptake of exogenous glucose
is of minor importance since fish tissues contain only low activities of inducible
hexokinase 57. The lactate diffuses into the circulation where it becomes a suitable
substrate for other tissues (e.g. heart and red muscle). At more severe hypoxia,
glycogen depletion also occurs in heart, brain and red muscle 2.52.124. Generally,
glycogen in white and red muscle, heart and brain is mobilized during hypoxia
or anoxia for metabolism in situ. In contrast, glycogenolysis in the liver is initi-
ated mostly to release glucose to the blood which results in hyperglycemia. This
phenomenon is observed in anoxia tolerant cyprinids 65,93. Depletion of liver glyco-
gen is the major energy source during long term anoxia in goldfish and crucian
carp which is associated with an increased glucose concentration in liver and
blood 45,93,111. However, in hypoxic trout such a mobilization of liver glycogen
and hyperglycemia is not always observed ~. At present, it is not known whether
this can be attributed to the moderate hypoxic conditions which may be insuf-
ficient to induce the necessary stimuli for glycogenolysis or to a difference in
metabolic organization. Beside the production of lactate as an anaerobic end prod-
uct, alanine and succinate may accumulate during oxygen deprivation in several
tissues43.105.
Since anaerobic metabolism generally results in the accumulation of acidic end
products, this way of energy production is only suitable for a limited period. A very
special strategy, however, is employed by cyprinids of the genus Carassius and to
some extent by Rutilus rutilus 1~ These fish are able to couple anaerobic glycolysis
to the production of the neutral end product ethanol which is easily excreted to the
environment 5~ and, in addition, they are capable of a considerable depression
of their metabolic rates 128. Although in terms of energy equivalents this pathway
may be 'wasteful' since the carbon skeletons of ethanol are lost, this strategy
enables the animals to survive long periods of environmental anoxia especially at
low temperatures 45'113.
The catabolism of fatty acids is severely impaired during anoxia as was demon-
strated in several mammalian tissues 55'71. As a result, FFA levels and their interme-
diates accumulate due to inhibition of the/~-oxidation ~1. In addition, the release of
stress-related hormones such as catecholamines and cortisol stimulate lipolytic ac-
tivity which augments the FFA increase 55. However, in fish this effect is completely
absent and a significant decrease of plasma FFA by 50% may be observed, which can
be attributed to a decrease of lipolytic activity26,65. Although the exact mechanism
of this process is not resolved, experiments of Farkas 26 and recent studies in our
laboratory suggest a special regulatory role for catecholamines and in particular for
norepinephrine during anoxia (Van Raaij, thesis 1994). Although the catabolism of
lipids is blocked during anoxia, lipogenesis may proceed. During the last decade, it
has been frequently proposed that fatty acids may serve as anaerobic end products
in anoxia tolerant cyprinids 43'1~ At present, the quantitative importance of such

a process is unknown. However, recently we succeeded to demonstrate the presence
of 'low-flux' fatty acid synthesis in anoxic goldfish using radiolabeled substrates (Van
Raaij, thesis 1994).
Amino acid metabolism in general is depressed during oxygen restriction because
the flux through the citric acid cycle is diminished. Nevertheless, some metabolic
turnover of AA is observed during anoxia (e.g. conversion of aspartate to succinate)
which is mostly related to coupling of redox reactions. It has been suggested that AA
catabolism may be of some significance in anoxic goldfish 1~ Interestingly, Van
der Boon and coworkers 1~8 demonstrated that some constituents of the myofibriUar
proteins in the white muscle of goldfish are depleted during anoxia which indicates
protein breakdown. Hence, the role of protein and amino acid metabolism in
hypoxic and anoxic fish has to be evaluated in future research.
2. Exercise
During exercise, the metabolic turnover in the muscles increases, sometimes by

more than two orders of magnitude. Metabolic fuels have to be mobilized rapidly
in order to provide the necessary substrates for energy production. The inten-
sity of the exercise and the oxygen availability in the tissue determine to a
great extent the preference of substrate utilization whereas white and red mus-
cles show different metabolic strategies with respect to exercise. During sustained
swimming and submaximal exercise, lipids and proteins are the preferred fuels.
The bulk of energy production is thought to be derived from AA catabolism
although surprisingly few quantitative studies have been performed. Already in
1968, Krueger et al. 58 concluded that coho salmon swimming at 6 BL s -1 de-
rived 55% of their energy from protein catabolism whereas this contribution was
estimated to amount 80% at higher speeds. Van den Thillart 1~ demonstrated
that protein oxidation contributed 80% to energy metabolism in resting rainbow
trout and even increased to 90% during sustained swimming. Ultimately, Kutty 59
noted that the level of NH + excretion by exercised Tilapia mossambica (now:
Oreochromis mossambicus) in relation to oxygen consumption was high enough to
propose that the energy production of these fish was totally supported by protein
catabolism. In exercised rainbow trout, the total pool of free AA was significantly
increased in liver, brain, and white and red muscle 98. These data indicate the
quantitative importance of protein oxidation during exercise and call for further
investigation.
Lipid catabolism is thought to be an important contributor to aerobic metabolism
at rest and during sustained exercise but again only few quantitative data are
available. The importance of lipid oxidation especially in the red muscle (see
above), was proposed already in the studies of Alexander 1. Histochemical studies
later demonstrated the excellent capacity of red muscle for lipid catabolism 33.34. It
was demonstrated that muscle lipid content of coho salmon was decreased after
several hours of intensive exercise 58. The greatest decreases were found for the
fatty acids 18 : 1, 16:0 and 16:1 which indicates the mobilization of triglycerides. At
higher speeds (>7 BL s-1) the fish were rapidly exhausted and specific losses were
observed for the fatty acids 18:2, 20:4 and 22:6. It is now well known that these
fatty acids are associated with phospholipid hydrolysis and since these lipid classes
arc normally hardly mobilized 41, this observation suggests that some tissue damage
did occur. Nevertheless, it was estimated that during submaximal exercise, coho
salmon would derive 45% of its energy from lipid catabolism whereas this value was
decreased to about 15% during exhaustive exercise. The latter value however, may
be of less significance because the authors did not account for glycogen utilization
which was certainly present during exhaustive exercise. In mackerel (Trachuna
symme~cus), it was observed that red muscle used endogenous triglycerides for
sustained swimmings2 while this was not noted in white muscle. In resting rainbow
trout, lipid oxidation may contribute about 20% to the energy metabolism whereas
this value was about 10% during sustained swimming ~~ From the kinetics of
arterially infused radiolabeled substrates, Van den Thillart 1~ concluded that a
preferential oxidation of endogenous substrates occurred during the first hours of
sustained aerobic exercise. Recently, S~ingerss demonstrated that the lipid content
and the fine structure of red muscle showed considerable species-specific variation.
The red muscle of Danube bleak, a cyprinid which is known to be a sustained
swimmer, contained more then 10% lipid on a wet weight basis whereas in the Asp,
a piscivorous predator which performs mainly burst type exercise, lipid contents
of only 2% were observed. From these observations, S~ingerss proposed that these
variations in lipid content are associated with a difference in swimming behavior.
Interestingly, a positive statistical correlation between the amount of red muscle
and mobility was reported ~.
During intensive exhausting exercise or during burst type exercise, as can occur
frequently in the white muscle of active pelagic or piscivorous species, the pattern
of substrate utilization is different. Since oxygen supply is inadequate during these
conditions, the changes in fuel mobilization resemble those during hypoxia with the
result that metabolic flux is increased by several-fold. Thus, endogenous PCr and
glycogen are the preferred substratcs during intensive exercise (see Hypoxia sec-
tion). However, also during submaximal exercise, the white muscle may use endoge-
nous glycogen as a substrate for anaerobic glycolysis resulting in the production of
lactate. This area of fish physiology is well-documented 5'2~176176176
and we will only give an outline describing the general findings. The degree of
glycogen depletion and the accumulation of lactate arc positively correlated with
the intensity and the duration of the exercise24. Mobilization of glycogen may
be extremely rapid and values of about 40 ~mol g-1 s-1 have been reported 24.
Glycogenolysis in the muscle tissues is probably initiated by increased levels of Ca 2+
which is released from the sarcoplasmatic reticulum. These ions act upon protein
kinase which subsequently activates glycogen phosphorylasc 44. The lactate formed
in the white muscle diffuses to some extent to the blood, although the degree
of this process seems to be species specific. Active pelagic fish, are sometimes
called 'lactate-releasers' since their blood lactate levels increase significantly during
exercise while in benthic inactive species blood lactate is elevated only modestly.
The latter species have therefore been called , lactate-non-releasers ,79 ' 104 . As stated
above, lactate released into the circulation is a preferred substrate for aerobic
metabolism in other tissues or for gluconeogenesis in the liver ~~
3. Starvation and migration

A number of fish species will encounter prolonged periods of starvation during their
life, often seasonally dependent and associated with migration and reproduction.
The utilization of endogenous fuels is dependent on the length of the starvation
period and the species under investigation. A considerable number of papers have
been published on this area during the last 30 years. In this section we will describe
the generalities that have arisen from this research emphasizing the importance of
mobilizing endogenous fuels for energy metabolism.
With respect to the mobilization of glycogen stores, the fish species studied
so far may be divided in two categories: species which do or do not mobilize
glycogen during the initial phase of starvation. Glycogen utilization during the
first stage of starvation was found in common carp (C. carpio), roach (R. rutilus),
killifish (Fundulus heteroclitus), rainbow trout (O. mykiss) and brown trout (Salmo
trutta). During wintering of common carp, Takeuchi and Ishii 1~176 found that liver
and (white) muscle glycogen levels were reduced by 65 and 80% respectively.
Glycogen contents of the carcass of juvenile roach were decreased from 5.1 to
about 1.6 /zmol g-1 after about three weeks and remained at this level during
the remainder of the starvation period (50 days) 67. Similarly, whole body glycogen
was depleted by 50% after 5 days of starvation in juvenile rainbow trout ss while
glucose availability was significantly enhanced. The liver glycogen pool of killifish
was depleted by over 90% after fasting for five weeks 62. Recently, Navarro et al. 75
demonstrated glycogen depletion in the liver and muscle of brown trout of about 80
and 20% respectively already after eight days of starvation, although after 4 weeks
the glycogen contents were partly restored (from gluconeogenesis). In common
carp, starved for about one month, no significant mobilization of liver glycogen is
observed whereas after three months, these stores were depleted to 20% of the
initial value 73. Thus carp may display an intermediate response. Representatives
of the second category are migrating sockeye salmon (Oncorhynchus nerka) and
fasting European (Anguilla anguiUa) and American (A. rostrata) eels. During their
migration to the spawning grounds, the liver and muscle glycogen levels of salmon
are not significantly changed and are in fact increased just prior to spawning 3~ The
glycogen content of liver and red muscle of American eel was not affected after
six months of starvation while white muscle glycogen was decreased by 40% (ref.
69). Similarly, Larsson and Lewander 61 showed that liver glycogen of European eels
was not decreased during the first three months of starvation but was decreased by
40% after about five months. Muscle glycogen was not changed at all. However,
these observations do not rule out the possibility of carbohydrate catabolism since
liver glycogen may be continuously replaced by gluconeogenesis as was proposed
by Larsson and Lewander 61. Increased gluconeogenetic activity from lactate and
alanine was observed in migrating salmon 3~ Liver glycogen contents of fasting cod
were extremely low 54 (Table 2) and although these levels were reduced during
56 G. van den ThiUan and M. van Raaij
starvation, liver glycogen is probably of minor quantitative importance in this

species.
In the second category, glycogen is only used as energy source at prolonged
starvation when the availability of other fuels (e.g. lipids) is reduced. In the first
category, glycogen utilization may be of transient importance during the first stage
of starvation until the mechanisms for utilization of other substrates are activated.
Lipids and protein are then the major fuels for energy metabolism.
Lipid mobilization is a common strategy in fasting fish although there appears
to be some variation with respect to target tissues. Visceral lipid depots are easily
mobilized and decline almost immediately after cessation of feeding 4s. Especially
in salmonids, the mesenteric fat deposits are the first to be depleted 4s,75. The
visceral index of brown trout decreased from 8% of total body weight to about
5% after starvation for one week 7s. A similar mechanism may be operative in
fasting American eel (tt. rostrata) as well since a significantly increased [FFA]
was observed in the plasma while liver and muscle lipids were not affected 69.
Similarly, increased plasma [FFA] was also found in fasting plaice (Pleuronectus
platessa) 13s. Lipid mobilization from liver and red muscle was observed in European
eel (,4. anguilla) 21'61, rainbow trout 48,84, and carp 19,73. The lipid depletion of these
tissues is much more gradual then of visceral depots and will contribute to the
energy metabolism during the whole starvation period. In general, there appears
to a preferential mobilization of saturated fatty acids from visceral depots while
saturates and monounsaturates are derived from liver and muscle lipids. These fatty
acids indicate the mobilization of triglycerides, the polyunsaturated fatty acids of
the phospholipid fraction are usually retained 41'134.
It is generally believed that during prolonged starvation, proteins are the major
source of energy. The nitrogen loss of several tissues of common carp was found to
decrease in the following order: muscle > spleen > kidney > liver > intestine 19.
In particular the large mass of white muscle is believed to be a huge reservoir of
energy equivalents. Protein utilization was observed in the muscles of starving eel
(A. rostrata) 69 and in migrating sockeye salmon (O. nerka) 68. In both species, the
'insoluble' myofibrillar proteins were mostly affected, whereas the 'soluble' fraction
was relatively unaffected. Based on changes in enzyme activities (especially alanine
aminotransferase) M6ndez and Wieser 67 concluded that protein catabolism was
enhanced also in fasting juvenile roach (Rutilus rutilus). Thus, amino acids may be
mobilized from tissues proteins, especially in white muscle, and serve as substrates
for energy metabolism either directly via catabolism in situ or via gluconeogenesis.
The latter process requires inter-organ transport, since the highest gluconeogenetic
activities are found in the liver. It has indeed be observed that the white muscle of
migrating salmon releases relatively large amounts of alanine 68 and, moreover, glu-
coneogenesis from alanine was increased in hepatocytes from migrating salmon 3~ as
well as from fasted rainbow trout 14. Increased protein catabolism during starvation
is enabled by increased activities of proteolytic enzymes like cathepsin and other
acid and neutral proteinases during migration of salmonids 3'4'68. In addition, it was
found that the rate of protein synthesis in the white muscles of fasting rainbow trout
and carp was decreasedl 1,53.
In conclusion, the energy metabolism in fasting and migrating fish is fuelled

primarily by amino acids and fatty acids. However, glycogen may be an additional
substrate either during the first stage of starvation or only during prolonged periods
of starvation.
VI. Summary
Fuels are compounds that act as substrates for ATP producing pathways and can be
stored to a certain extent. Fuels for anaerobic processes are ATP, PCr and glycogen.
These anaerobic processes have a limited capacity, however, their survival value
is very high, since they allow either a very high energy flux (exercise) for a short
period, or a low energy flux during hypoxia/anoxia for a long period. Fuels for
aerobic processes are sugars, lipids and proteins. During catabolism they produce
NADH and FADH2 which are the ultimate substrates for the electron transport
chain and mitochondrial ATP synthesis. The quantitation of fuels is fundamental
to the understanding of energy metabolism. Most methods are of a destructive
nature and cause as such a series of possible artifacts: stress due to handling and
sampling, tissue damage, and incomplete metabolite extraction and denaturation
of proteins. Most problems occur with those pathways where a high energy flux
can be generated such as in muscle, i.e. it is very difficult to obtain low levels
for lactate, and a high PCr/total creatine ratio. Another source of interference is
where low metabolite levels occur together with high fluxes; this is found with FFA:
low levels are easily disturbed due to lipolysis. Non-destructive techniques preclude
most but not all problems. The most promising technique is in vivo NMR, which
allows metabolite measurements without invasive or destructive actions. Particularly
important is the finding that under resting conditions the phosphorylation potential
of muscle is very high, resulting in >90% phosphorylation of creatine. Furthermore
a review is given of the range of different fuels occurring in different tissues and
different species. Obviously glycogen is quantitatively of minor importance in fish.
The major energy source is protein, although lipids are in some species of equal
importance. Fuel mobilization under hypoxia, exercise and starvation is discussed.
Under conditions where anaerobic metabolism is activated, PCr and glycogen are
the major fuels, while for long term exercise and starvation, both lipids and proteins
are the predominant source of energy.
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Hochachka and Mommsen (eds.), Biochemistryand molecularbiology of fishes, vol. 4
CHAPTER 4
Tissue carbohydrate metabolism,

gluconeogenesis and hormonal and
environmental influences
THOMAS W. MOON AND GLEN D. FOSTER

Department of Biology, Ottawa-Carleton Institute of Biology, University of Ottawa, Ottawa, Ontario
KIN 6N5, Canada
I. Introduction
II. Liver
1. The general organization of hepatic metabolism
2. Environmental adaptations
2.1. Fasting
2.2. Hypoxia
2.3. Temperature
2.4. Stress
2.5. Seasonality
3. Hormone signal transduction pathways
III. Kidney
1. The organization of kidney metabolism
IV. Skeletal muscle and heart
1. Myotomal muscle
2. Cardiac muscle
V. Brain
VI. Red blood cells
VII. Conclusions
VIII. References
I. Introduction
Carbohydrates are key to the metabolism of all vertebrates, including fish species.
The diversity of lifestyles and habitats selected by fish has resulted in significant
differences in the way species handle and partition dietary carbohydrates within
their bodies. Few generalities can be presented, and the reader is directed to
Love 9~ for a review of some of the issues which may be involved. Early studies by
Leibson 87, Plisetskaya and Kuz'mina 134, and Palmer and Ryman 123 introduced the
ideas of motor activities affecting carbohydrate disposition and glucose intolerance,
but papers since have generally lost the comparative perspective which is critical
to our understanding these species differences. The idea that at least carnivorous
fishes (e.g. salmonids) have a poor tolerance to carbohydrate has been revisited
66 T.W. Moon and G.D. Foster
recently 133,177. It is clear that this is an issue in the metabolic biochemistry of

fishes, but more information is needed. In particular, the sensitivities of hormonal
release to circulating carbohydrate and the specific tissue hormone receptors and
species with non-carnivorous life styles need to be examined. Although this review
will mention a variety of species, many have similar nutrient requirements and a
strong comparative approach is needed before an understanding of this issue will be
possible.
It remains unclear whether any fish tissues require or use preferentially carbohy-
drate as a source of energy. The red blood cell and kidney cortex of mammals have
a strict glucose requirement based upon their anaerobic metabolic profile. Fish red
cells are nucleated and may contain a few mitochondria, and it is reported that
90% of the resting nucleoside triphosphate is produced aerobically 33. Red cells of
the sea raven (Hemitripterus americanus) also have an aerobic metabolism fueled by
exogenous glucose x46. Lamprey (Petromyzon marinus) brain tissue minces do utilize
glucose 4s and the trout brain is thought to use primarily exogenous glucose to sup-
plement its low endogenous glycogen reserves 27. Glucose/glycogen is critical for the
maintenance of cardiac performance in the Atlantic hagfish (Myxfne glutinosa) 151.
Yet in no case is the quantitative significance of carbohydrates in overall tissue
metabolism clearly defined.
The ability of tissues to utilize a particular nutrient is dependent on the perme-
ability of the cell to it and its cellular metabolism to ensure adequate membrane gra-
dients. Carbohydrates such as glucose and lactate poorly penetrate the hydrophobic
cell membrane, and transport is generally carrier-mediated. Fish tissues such as
red cells and liver, with few exceptions, show non-saturable uptake kinetics with
respect to these metabolites (red cells 16~ liver171). Hexokinase (HK) activities, the
enzyme responsible for glucose phosphorylation and the maintenance of the mem-
brane gradient for glucose, are generally low in fish tissues (see Tables 2-8), and
although there is some evidence for its modulation by diet 37, there is no evidence
for a mammalian-type glucokinase in fish. Unfortunately, our knowledge of glucose
transporters in fish tissues is poor, with the possible exception of red cells 3s,lss,
an area which could help identify the mechanism of 'glucose intolerance' in
fish.
Fish tissues do contain glycogen in varying quantities (Table 1) indicating that
the inability of tissues to take-up glucose or maintain plasma glucose content is
compensated for by other metabolic pathways. Gluconeogenesis is the pathway
responsible for de novo glucose and glycogen synthesis (glyconeogenesis) from pre-
cursors including lactate, amino acids, glycerol, and fructose 1~ The importance
of this pathway to tissue carbohydrate homeostasis in fish has been the subject
of several recent reviews97,1~ The high dietary protein requirements of fish9~
should provide adequate substrate for this pathway and there is extensive evidence
for the modulation of gluconeogenic rates by intrinsic and extrinsic factors. The
prevalence of proteins in the carnivorous diet and their use as an energy source
together with the generally lowered energy demands of fish n4, may have alleviated
the strict need for carbohydrate as a key energy source to many fish species. The
Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences 67
TABLE 1
Glycogen contents of various fish tissues
Species/Condition Liver Kidney White muscle Red muscle Heart Brain

Hagfish
fed 15.039 3039 - 2257
fasted; 1 month 1.8 0.72 -
fasted; 4 months 4.7 36 -
anoxia - 0.9557
Skate 6311~ 9.011~
Crucian carp
winter/normoxic 150066,117 167117
summer 5566 22
Catfish 600118 _ 19118 140118 211118 1627

winter/fed 8012~ 3.7 4.2120 12.912o 4212o _
fasted; 60 days 67 - 1.7 7.3 30
spring 31 2.4 3.3 7.9 23
anoxic n D
_ _ 627
Perch
fed 62145 15.345
fasted; 7 weeks 182 10.8
Flounder
normoxic 6476 16TM 45 TM
hypoxic 18 4 36
Tuna 4178 93178 24178
American eel
fed 49 l~ 121o6 141o6 _ 10136
fasted, 6 months 53 7 11 -
Rainbow trout
normoxic 3526, 2000 u 5.126 _ _ 3.027
anoxic _ _ _ 1.527
Values are given in/zmol glucosyl units g-I tissue. These are representative values and not intended
to be comprehensive. Superscript numbers represent reference numbers. If values are not followed by
superscript numbers, reference is identical to that located above in the same column.
role of carbohydrates may, therefore, be for short-term responses to acute stress

situations 19 and/or as a last resort.
The purpose of this review, therefore, is to provide an overview of recent studies
on carbohydrate metabolism in fish using a tissue approach. An overriding thesis in
the review is that carbohydrate metabolism has a significant role in fish, and that this
function becomes more understandable when considered in light of perturbations of
the system. The question of whole fish carbohydrate homeostasis will be examined
only in terms of its importance to the individual tissue. An attempt will be made
to update those previous reviews on fish metabolism (e.g. refs. 19, 61, 176), and by
doing so, to identify those areas which need further research.
68 T.W.Moon and G.D. Foster
II. Liver
The liver is a central organ of metabolism and is key to the regulation of carbohy-
drate metabolism in all vertebrate classes. A huge literature is available and it is not
our intent to be all-inclusive, but to select those areas of recent interest.
1. The general organization of hepatic metabolism

The ability of fish to metabolize carbohydrate and the enzymes required for this
purpose are clearly understood. A general listing of recently published enzyme
activities in fish livers is provided on Table 2. A complete enzyme profile is not
available for each fish, but as methods become more available, such holes will dis-
appear. The overall importance of carbohydrate metabolism in the fish liver as well
as the relative importance of glycolysis and gluconeogenesis, however, are less well
defined. For instance, hepatectomy in the fiver lamprey (Lampetra fluviatilis) s6 and
the Pacific hagfish (Eptatretus stouti) 69 affected neither survival nor basal blood glu-
cose concentrations, leading to the suggestion that liver carbohydrate metabolism
may not be critical to whole body carbohydrate homeostasis. These studies, how-
ever, did not consider the ability of these species to tolerate environmental stress,
such as hypoxia, exercise, fasting, or temperature changes, nor do they explain the
ubiquity of carbohydrate-metabolizing pathways in fish livers. We suggest that while
the importance of carbohydrate metabolism in the whole animal energy budget may
be less than in other vertebrate classes, carbohydrate metabolism is critical and
becomes so during adaptive responses to the environment.
Liver glycogen contents are extremely variable in fish (Table 1). Agnathans
generally have levels below 20/zmol g-l, while elasmobraneh and teleost values
range from 20 to 2000/zmol g-1. Measured glycogen values for fed rainbow trout
(Oncorhynchus mykiss) range from 35 (ref. 26) to 2000 (ref. 84)/zmol g-1. This
variability may reflect sampling procedures, strain and/or life history differences,
or even the method of analysis. The Crucian carp (Carassius carassius) has liver
glycogen concentrations above 1500/zmol g-1 liver, and during periods of high
glycogen content, the liver may reach 15% of body weight66. This gives a total liver
carbohydrate store of over 20,000/zmol 100 g-1 body weight, or 4% of body weight!
A similar analysis using other teleosts gives values no higher than 1500/zmol g-1
body weight (0.3%). While other teleosts do not demonstrate these high amounts of
liver glycogen, the liver still contains as much as 50% of the whole animal glycogen
stores, assuming skeletal muscle contains the rest.
The hormones glucagon, glucagon-like peptide (GLP), and catecholamines all
increase gluconeogenesis and glycogenolysis in a variety of species by inhibition
of pyruvate kinase (PK) and phosphofructokinase-1 (PFK-1) activities and in-
creasing phosphoenolpyruvate earboxykinase (PEPCK) and glycogen phosphorylase
(GPase) activities 23'42'44'47'56'98'100'102'103'118'119'130'167.These enzymes have all been
reported in most species studied (Table 2), and most are controlled by reversible
phosphorylation-dephosphorylation mechanisms. The vasoactive peptides vaso-
tocin and isotocin also increase glycogenolysis and glueoneogenesis in three species
where they have been tested, with the effects very pronounced in the American
eel (Anguilla rostrata) 111. Elevated plasma cortisol concentrations stimulate gluco-
neogenesis in some teleosts, possibly by increasing the availability of gluconeogenic
substrates (amino acids) and activation of specific liver enzymes 1~8. It is not clear
to this point whether this is a direct affect of cortisol or it is mediated by other
glucoregulatory hormones 169.
Insulin, the only hypoglycemic hormone in mammals, acts to counteract glucagon-
stimulated gluconeogenesis and glucose production 65, with little or no effect on
basal (no hormone) rates. This situation with insulin-counteracting the effects of
glucagon on gluconeogenesis and glycogenolysis has been reported for American
eel 42 and sea raven 41'46 hepatocytes. Petersen et al. 130, however, reported an inhibi-
tion of basal (no hormone) gluconeogenic flux by insulin in trout hepatocytes, with
a concurrent inhibition of PK activity. This inhibitory effect of insulin in the absence
of glucagon has been found in no other teleost species nor in mammals where PK
is unaffected 65. In fact, insulin actually increased gluconeogenic flux in hepatocytes
isolated from sea raven 41,46, American eel 42, and hagfish 49. These actions of insulin
occurred through an enhanced inhibitory effect of ATP on PFK-1 in all three
species. No effects of insulin on PK and PEPCK were found in sea raven hep-
atocytes. Insulin also decreased glucagon-stimulated glycogenolysis and decreased
glycogenolysis below basal (no hormone) levels in both sea raven and American eel
hepatocytes 41,42. It is apparent that the actions of insulin differ between species and
with respect to the carbohydrate pathways in the liver.
Mammalian hepatocytes show metabolic heterogeneity such that periportal cells
catalyze glucose release and gluconeogenesis while perivenous cells preferentially
take up glucose for glycogenesis and lipogenesis 77. No evidence for this mammalian
form of heterogeneity has been found in the catfish 121 or the trout 99 liver. However,
density gradient separation of cell types demonstrated more oxidative and gluco-
neogenic scope existed in the less dense cells of trout liver 99, and greater overall
enzyme activities in the less dense cells of the Gulf toadfish (Opsanus beta) 1~
An interesting finding in the toadfish study was that although enzyme activities
were higher in the less dense cells, the heavier cells exhibited 2.5-4 times more
metabolic activity than the less dense population. Similarly, the distinctively large
lobe of the hagfish liver was found to be more metabolically active than the small
lobe 49. Metabolic heterogeneity may exist in fish livers, although rather than a
segregation of metabolic pathways existing, as in the mammal, the heterogeneity
may be more subtle or simply reflect differential overall metabolic activities of the
cells.
A review of substrate utilization has been published previously 1~ and the
general principles will not be covered here. This section will concentrate on recent
findings that relate to an understanding of the fate of certain substrates, as well take
a comparative look at some interesting differences found in some species.
Lactate is generally the most readily utilized substrate in fish liver 1~
leading to the suggestion that the liver is important in the Cori cycle 157 (Cori
cycle is: muscle lactate ~ liver glucose ~ muscle glycogen). However, in starry
flounder (Platichthys stellatus) 96 , American eel 21 and skipjack tuna (Katsuwonus
~ ~ IL~
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pelamis) 178, in vivo lactate turnover studies showed no more than 5% of the lactate
label was incorporated into blood glucose, indicating low Cori cycle activity. The
fate and function of lactate and other C-3 molecules in the blood remains unclear,
other than their use as oxidative substrates for immediate energy production
in peripheral tissues. Recent studies of the hormonal regulation of hepatocyte
metabolism may shed some light on this question. As discussed above, increased
flux of C-3 precursors such as lactate and alanine to C-6 endproducts resulted
when isolated hepatocytes of hagfish, sea raven, and American eel hepatocytes were
exposed to insulin, and these observations led to the hypothesis that a major role
of liver carbohydrate metabolism is to convert C-3 substrates available from the
diet either to liver energy stores or to glucose for export to peripheral tissues for
storage (see refs. 41, 46). Thus, rather than the liver functioning as an intermediate
organ in maintaining carbon cycling, it may function more to provide a reservoir
of energy stores, namely glycogen. As such, the use of exogenous substrates and
the regulation of their use will be directed more towards energy storage than
towards blood euglycemia. This issue is called the 'glucose paradox' or the indirect
pathway for glycogen production and has recently been reviewed 14a. Unfortunately,
this issue is complicated by isotope methodology, and it is not clear what the
contribution of this pathway compared to the direct pathway (glucose to glycogen)
is to glycogen production. The fish system may be a good model to test these
ideas.
Lactate use by hepatocytes is generally greater than other substrates, although
there are some notable exceptions. In sea raven hepatocytes, lactate flux to glucose
is lower, if present at all, than the flux of serine and alanine 41,174, and in skate
(Raja erinacea) 1~ and skipjack tuna 16 hepatocytes, lactate conversion rates are
lower than for alanine. Low lactate utilization in sea raven hepatocytes may be due
to the cytoplasmic/mitochondrial localization of PEPCK ls7 or to low LDH titers
in the liver 174 (Table 2). PEPCK activities in sea raven liver, however, are 100%
mitochondria1188; thus, there are no significant redox problems and lactate oxidation
is as high as alanine and serine oxidation 41, indicating active flux through LDH.
Furthermore, the LDH isozyme pattern in sea raven liver is not different from other
teleosts 188. The low use of lactate in skipjack tuna 16 and skate 1~ suggests: (1) an
inactive Cori cycle, with the liver function directed towards the conversion of amino
acids (dietary source or muscle catabolism) to endproducts in the liver rather than
the liver functioning as an intermediate cycling organ; and (2) elasmobranchs may
have a minimal reliance on carbohydrate metabolism, using fats and ketone bodies
to a greater extent. As such, the use of lactate (carbohydrate) may be reduced. At
this time, there is no clear understanding of why species have particular preferences
for substrates, but experiments on transport characteristics may assist in a better
understanding of these differences.
Liver metabolism not only demonstrates species differences and substrate prefer-
ences, but also responds to a variety of environmental perturbations. Again, this
review is not intended to cover all aspects of this topic, but to select those aspects
which focus on carbohydrate changes and update previous reviews 11e.157.
2.1. Fasting
Enzyme changes and flux studies with fasting generally reflect a stimulation of
gluconeogenesis 39,45,46,54,1~ Increased glucose production in isolated hepato-
cytes from fasted fish is sometimes found 46, although not always 45. The decreased
glucose production is probably due to the confounding fact of the negative glyco-
gen balance found in isolated hepatocytes 97,112 and the general decrease in liver
glycogen levels found in fish (e.g. refs. 45, 46, 54, 115, 147, 169) which will decrease
glycogenolysis43,97. GPase activities are generally unaltered during a fast in eel,
trout, or yellow perch 42,45,1~ although GSase activities decreased in fasted trout 169.
The use of glycogen during a fast appears to be species-specific. There are different
strategies for dealing with a fast, some of which include maintaining glycogen levels
at the expense of protein and/or lipid9~ As noted on Table 1, some species
demonstrated significant changes in liver glycogen with fasting (perch, hagfish),
while others do not (American eel, catfish). Hansen and Abraham 5s showed that
fasting shifted the pattern of metabolite incorporation in vivo, favoring gluconeo-
genesis from alanine and serine. These experiments indicate the importance of
both in vivo and in vitro approaches to the study of fish liver metabolism during
fasting.
While enzyme activities and flux demonstrate stimulated gluconeogenic activity
as a function of tissue weight, analysis of the data in terms of total liver potential
shows a decreased metabolic activity in the livers of yellow perch and trout, thus
indicating a hypometabolic response to fasting45. A study of sea raven metabolism,
however, showed that following a 6 week 46 or 8 week 169 fast, hepatocyte flux was not
lowered after taking into account changes in the hepatosomatic index. Therefore,
the hypometabolic response to fasting is not a general feature of all teleosts, but
has been shown in only a few species. Dormancy or metabolic depression have been
shown in a few fish species as a function of low temperatures ee,173, but its precise
mechanistic basis has yet to be identified.
Titers of insulin, glucagon and glucagon-like peptide (GLP) generally decrease
during a fast in a variety of fish species 54,55,6~176 although in some of
these species the glucagon or GLP to insulin ratio(s) increased 54,1~ indicating
a hormonal profile suitable for stimulated gluconeogenesis. Most studies have em-
ployed endpoint quantification of hormones, but it is clear that hormone titers vary
considerably during the fasting period and depending upon the time of year the
experiments are conducted 1~ Guti6rrez and Plisetskaya 55 showed that follow-
ing a 40 day fast, insulin binding capacity to the salmon liver was increased, but
the affinity to the membranes was actually decreased. They concluded, therefore,
that specific binding of insulin to the liver cell is unchanged following a fast of
this duration even though insulin titers decreased. The additional complication of
annual cycles 53 and of diet and feeding times 127 on plasma insulin and glucose must
also be considered when designing these experiments, but seldom are. Glucagon
receptors have recently been identified in bullhead (Ictalurus nebulosus) and eel
hepatocytes 189, but how these are affected by nutritional status is not known. GLP
binding has yet to be demonstrated in any fish system.
Foster and M o o n 46 showed that the sensitivity of sea raven hepatocyte carbo-
hydrate metabolism to insulin was unchanged following a 6 week fast, although
glucagon sensitivity was increased. They concluded that this increased sensitivity al-
lowed the hepatocyte to respond to the lower levels of glucagon presumably present
in the circulation, while the unchanged sensitivity to insulin reflected a reduced
metabolic impact of insulin. This study is in contrast to that reported by Klee et a1.82
which showed that glycogenolysis could be induced by epinephrine but not glucagon
in trout fasted for 3 weeks; both hormones stimulated glycogenolysis in 1 week
fasted trout. These studies suggested that at least glucagon may be critical only at
certain times in the fasting process, and this could be related to the binding of this
hormone to sites on the hepatocyte. Further studies to identify receptor binding
and the physiological consequences of fasting on these parameters is needed before
the known physiological changes in hormone titers can be interpreted at the tissue
level.
The duration of the fast has an affect on the liver changes that are measured,
but few studies have followed these changes. Rainbow trout exhibited an initial
increase in PEPCK and FBPase activities in the early stages of a fast, while in the
later stages activities of these enzymes declined ~3. Navarro and her colleagues ~5
showed an initial (3-5 day) attempt to maintain plasma glucose levels in brown
trout (Salmo trutta), followed by hypoglycemia. These changes were accompanied
by an initial increase in the glucagon" insulin ratio, which disappeared at the later
stage of the fast. Other studies by the group of Guti6rrez 9,1~ have followed
plasma hormone and metabolite concentrations during the fasting period. These
carp and trout studies suggested at least 2 fasting stages; an initial stage with few
changes followed by a later stage with major changes. Their studies showed that
the timing of these stages are species dependent, but the experiments are key as
they suggest that the length of the fast is critical in determining the physiological
response. Canals et al. 17 showed an increased alanine oxidation in isolated trout
hepatocytes following a 2 week fast, but at 3 weeks this parameter was decreased
below initial values. It is probable that there is an initial hypermetabolism in the
early stages of a fast followed by a hypometabolic response to longer term fasts
(see above). The fact that glycogen levels decrease in a fast, but do not completely
disappear suggest that glycogen may be liberated in the very first phase of the fast,
when glucagon concentrations are elevated 115. The levels will then stabilize when
the fast becomes extended and glucagon titers decrease. It may be expected that in
the last phases of a long term fast carbohydrate stores will finally be liberated, after
lipid and protein pools are exhausted.
Re-feeding results in a number of changes in the liver, most of which reflect a
repletion of glycogen reserves. For example, re-fed cod (Gadus morhua) showed an
overshoot in glycogen stores 8, and catfish (Rhamdia hilarii) liver slices showed an
increased flux of 14C-glucose to glycogen91. These anabolic effects are consistent
with an increased specific binding of insulin to liver membranes in salmon 55. Tilapia
(Oreochromis [Sarotherodon] mossambicus) showed a less pronounced recovery of
liver glycogen levels, with the reserves still below fed values even after 30 days of
re-feeding 13s. The mechanisms involved in glycogen recovery are not clear. In tilapia
liver, GPase a activities were reduced during the fast, and remained low at 15 days
of re-feeding. However, activities of this enzyme had recovered to control values
even though the glycogen stores had not fully recovered 13a. Re-feeding mudskipper
showed no change in the GPase %a activities, although total activities increased 88.
It is probable that more is involved than simple changes in enzyme activities in
the recovery of glycogen reserves. Blasco and collaborators 9 have reported that the
changes noted in plasma hormones during fasting are returned to normal by 12
days of re-feeding in carp, concomitant with a complete recovery of liver reserves.
Certainly the re-establishment of hormone titers are involved in these progresses,
and the study by Sheridan and Mommsen 147 demonstrated that at least insulin,
glucagon and GLP returned to prefasted levels within 2 weeks of re-feeding. Given
that plasma insulin and glucagon levels peak within 2 h post-feeding in brown
trout ~4, it is clear that fish can respond rapidly to changes in their nutritional state.
2.2. Hypoxia
Numerous fish species experience prolonged periods of low oxygen availability and/
or complete anoxia. Furthermore, specific tissues may experience hypoxia during
periods of strenuous exercise. The response to low oxygen availability (hypoxia) is
either metabolic depression (e.g. the mudskipper) is and/or the anaerobic use of car-
bohydrates, and the response can be tissue-specific ls4. Hypoxic plaice demonstrated
reduced serum glucose 18~ but blood glucose was maintained in trout through cate-
cholamine inhibition of PK and stimulation of GPase, effects that were blocked in
the presence of propranolo1184. Claireaux and Dutil 2~ also implicated catecholamine
release with hypoxia in cod. However, Dunn and Hochachka 29 found no increase in
glucose turnover in 3 h hypoxie trout, although lactate turnover was increased.
Anoxia did not change glucose production in liver pieces of trout or bullheads s9,
suggesting the absence of a Pasteur effect and the existence of metabolic depres-
sion. Consistent with these effects, liver from anoxic goldfish showed decreased
OPase activities and decreased fructose 2,6-bisphosphate levels 154, again suggesting
metabolic depression and the absence of a Pasteur effect. As this metabolic de-
pression was not apparent in all tissues 59,1s4, the whole animal does not necessarily
go hypometabolic. Instead, the metabolic depression was specific to the fiver, im-
plicating an active protection of liver glycogen stores during anoxia/hypoxia. The
protection of liver glycogen stores has been reported in air-exposed American eels
(Moon et al., in preparation), hypoxic cod 2~ the anoxic Crucian carp 117, and 1
h hypoxic mudskippers (Channa punctata) 1st, although liver glycogen decreased
in the anoxic flounder 76 (see Table 1). Activities of liver GPase are key to these
changes. GPase activities declined and glycogen was stable with air-exposure, but
by 3 days of air exposure, glycogen levels in American eel fiver drop dramatically,
GPase a activities rose, liver lactate levels increased and eels begin to die (Moon
et al., in preparation). Death in the anoxia-tolerant Crucian carp (after 18 days
of anoxia) was correlated with near zero content of liver glycogen 117. Again, there
was a reciprocal relationship between liver glycogen content and GPase activities
in carp 66. Long term hypoxia (3 days) in the mudskipper also resulted in decreased
liver glycogen reserves, although GPase activities were not affected ls6. Furthermore,
the survival time under anoxia is dependent on the initial liver glycogen reserves TM,
and these levels showed seasonal fluctuations 66. At least in the carp and goldfish,
extended anoxia is associated with altered glycolytic endproducts (ethanol, acetic
acid) 63. The protection of glycogen may be necessary to maintain a readily utilizable
energy source during prolonged hypoxia/anoxia (as was suggested for fasting, see
above) in order to respond to the superimposition of another (secondary) stress.
Re-submergence experiments (Moon et al., in preparation) showed a rapid rise in
liver glycogen content in eels, further supporting the importance of liver glycogen
protection and maintenance; similar studies have not apparently been done with the
carp. van den Thillart and Verbeek 166 correlated an oxygen debt during recovery
from anoxia to a replenishment of glycogen pools in trout. It may be postulated that
liver glycogen is maintained as a last-resort fuel, and depletion of this reserve results
in death. Work is required to understand why depletion of this reserve may result in
death and why liver glycogen is protected during early stages of anoxia/hypoxia.
2.3. Temperature
Some fish species thermally compensate metabolic activity at decreased temper-
atures by increasing enzyme activities, and this is thought to be a major aspect
of temperature acclimation 62,78-a~ Furthermore temperature-dependent effects on
specific enzymes, such as PK and PFK-1, reflect stimulated pathway flux1,62. Recent
studies have examined effects of temperature on in vitro liver metabolism and
how changes in in vivo hormone titers may affect liver metabolism. Total glucose
production in trout liver pieces showed some thermal compensation, but not in
bullheads 59. Seibert 143 found glucose production in the absence of glycogen deple-
tion in trout hepatocytes at low temperatures, while as the incubation temperature
increased glycogen depletion rates met and eventually exceeded glucose production
rates. Thus, thermal compensation of trout hepatocyte glucose production may
be through gluconeogenesis, as low incubation temperatures inhibit the in vitro
depletion of glycogen. A similar study must be performed on non-compensating
species to see if such changes are really adaptive. Sea raven hepatocytes showed
an exceptionally large Q10 value (Q10=29) for alanine gluconeogenesis between 10
and 2"C, indicating an inhibited gluconeogenic potential at lower temperatures 174,
which is inconsistent with the observations on trout hepatocytes 143, but this species
may not compensate.
A number of studies have examined the relationship between temperature and
nutritional and seasonal effects. Walsh and colleagues 173 compared 15*C-fed eel
liver enzymes to the same enzymes from eels adapted to 5, 10 and 15"C but fasted
for 6 months. They reported that enzymes and metabolite reserves were similar
between the 5*C-fasted and 15~ eels, but significantly lower in the 10 and
15*C-fasted fish. These studies concluded that torpor (metabolic depression) can
have a significant metabolic impact on an animal's energetics and that thermal
compensation need not occur, nor may it be relevant in all species. Studies with the
golden ide (Leuciscus idus melanotus) found that liver glycogen stores increased at
76 T.;E.Moon and G.D. Foster
low temperatures compared to lipids if ide were fed a natural diet 137. The question
raised by this study is that of natural versus artificial diets and this aspect has not
been adequately addressed in fish.
Blasco et al.ll reported in carp that circulating glucagon was elevated at high
acclimation temperatures (28~ compared to 15~ controls. In association to this,
liver (and brain) glycogen levels were reduced; no changes, however, were found
with low acclimation temperatures. They concluded that glucagon is important in
the mobilization of carbohydrates during a high temperature stress. Different re-
suits were found with the sea bream (Chrysophrys major), a warm-water species,
where both liver glycogen and hepatosomatic index increased at high acclimation
temperatures lsl. Prosser and coworkers 136attempted to identify the hormonal mech-
anisms involved in temperature adaptations in catfish hepatocytes. They found greater
rates of protein synthesis at high temperatures were inhibited by thyroxin, but were
unable to identify any other factor which might be responsible. More studies are
required to elucidate the hormonal adjustments involved in temperature effects on
carbohydrate metabolism, but such studies must consider the appropriate physiolog-
ical hormone content, so more whole fish hormone studies will also be required.
2. 4. Stress
Stress may increase the importance of carbohydrate metabolism in the whole
animal energy budget, and blood glucose and lactate generally increase during
stress 5. The precise mechanism(s) involved are speculative and complicated by
secondary factors, some of which are difficult if not impossible to control. Cortisol
is thought to be an important stress hormone, but its effects may be both direct and
indirect through the actions of other glucoregulatory hormones. In general, cortisol
does have a glucoregulatory action in some fish speciess but its actions on the
isolated hepatocyte are minimal4~ Factors including nutritional state will alter the
metabolic response of trout, presumably due to changes in substrate availability (see
above) 17~ Studies on salmonids do suggest that cortisol alters liver enzyme profiles
to enhance gluconeogenic potentials 168, but similar results are not seen in all cases.
The actions of this hormone on liver cell metabolism need to be re-examined, and
new experimental designs developed. Of all the glucoregulatory hormones found in
fish, this is the most inconsistent.
2.5. Seasonality
A full discussion of seasonality and carbohydrate metabolism is beyond the scope
of this review, but a few comments are important for completeness. As discussed
above, seasonal differences in glycogen contents in the Crucian carp are found,
and these differences correlate with the ability of the carp to withstand anoxia~s,117.
Glycogen content generally decrease through the winter, when food consumption
is low~ or during the spawning season, as is seen in the catfish 12~ and increase
when fish are actively feeding. But in many of these cases, glycogen content can be
very high as the species enters the winter season (see Table 1). Seasonal changes
in hormone responses are also common. Hormone responsiveness is found to
decrease in cold seasons4~176 which may reflect an impaired receptor or
transduction system. Mommsen and M o o n 103 found a decreased cAMP response

to glucagon in March as compared with November in eel hepatocytes. Consistent
with this observation, glycogenolysis was less responsive to glucagon in March.
A consideration of seasonality is critical in both designing experiments and in
interpreting their results.
Environmental parameters seldom function in isolation, and it is critical that
their interactive effects on liver carbohydrate metabolism be studied. As many of
these parameters change with season, such as temperature, feeding, and possibly
predatory stress, controlled seasonal studies would be very helpful in understand-
ing these biochemical adaptations to the environment. Kent and colleagues 8~ have
attempted to place temperature effects on liver characteristics into an ecological
framework by studying naturally reared catfish. They found their study was con-
founded by other seasonal factors such as nutrition, as well as reproduction and
photoperiod. More studies of this kind are required before the complexity of fish
liver metabolism can be fully understood.
3. Hormone signal transduction pathways
Recent work with fish liver has been focused on understanding the signal transduc-
tion pathways of hormones. To date the most interesting finding is the significant
species differences in the quantitative and qualitative responses to hormones.
Glucagon stimulates cAMP concentrations in fish livers, although there is not al-
ways a good correlation between the physiological response (e.g. glycogenolysis or
gluconeogenesis) and the rise in cAMP 1~ The vasoactive peptides, vasotocin and
isotocin, have also been found to stimulate glycogenolysis through the activation of
cAMP 111.
Catecholamines in mammals act through either al- or fl-adrenoceptors 32. The
absence of classic a-antagonist binding and studies showing that ~l-antagonists were
many-times more effective than a-antagonists in competitive displacement studies,
suggested fl-adrenoceptors predominated in the liver of fish species 7~ Other stud-
ies, however, found that the phenylephrine (a classic a-adrenergic agonist)-induced
glycogenolysis in isolated hepatocytes of eel and catfish could not be totally ex-
plained by this agonist acting in the fish liver as a ~l-agonist 15'111. More recent
studies have found epinephrine induced an increase in intracellular free calcium
concentrations ([Ca2+]i) (refs. 108, 187). Such changes in [Ca2+]i in mammalian
hepatocytes result from the activation of a-adrenoceptors 32, and these are the first
studies to support the existence of an t~-adrenoceptor system on fish hepatocytes.
Moon et al. 108, however, were unable to link changes in [Ca2+]i with the activation
of glycogenolysis, the classic mammalian metabolic effector of changes in [Ca2+]i
(ref. 32). In addition, this change in [Ca2+]i is very species dependent 187, and more
studies are needed to more completely identify the signalling processes occurring in
these hepatocyte systems.
The liver is critical to the carbohydrate status of fish as demonstrated by
the patterns of response to environmental perturbations and hormones. There
are significant species differences in these patterns, but whether these reflect
evolutionary differences or simply the prior history of the fish used is unclear.
Future experiments must consider the plethora of factors which may affect the
collected data, and carefully design experiments to eliminate as many factors as
possible. Even so, papers must clearly identify the history of the fish being used
and how the fish were held prior to and during the experiment. Without such
information, it is difficult to interpret this huge literature. Certainly patterns are
emerging, but as new species are explored, it is critical to provide such information.
III. Kidney
1. The organization of kidney metabolism
Fish kidney carbohydrate metabolism has been poorly studied, as has its response to
environmental conditions. Few generalizations are possible, although in all species
examined, glycolytic enzymes are active (Table 3). For example, cod, trout, and
flatfish HK and PK activities are greater than in the fiver, and PFK-1 activities are
equivalent s3. A similar situation exists for the kidney of the salmon 1~ flounder 76,
and the more primitive skate ~1~ Thus, the fish kidney appears to have an active
glycolytic pathway. Kidney does have glycogen, but the content is much less than
that found in liver in species where it has been analyzed (Table 1).
The gluconeogenic pathway is present in this tissue, although its activity and
contribution to whole animal glucose metabolism has often been questioned.
TABLE 3
Kidney enzymes from selected fish species
Enzyme Skate 11~ Sturgeon 152 Rainbow Atlantic Flounder s3 Cod s3 Marlin 156
trout 83 salmon 105
IO~ 20~ 15~ IO~ 15~ 15~ 25~
HK 0.39 0.79 0.38 ND 0.12 0.07 -

PFK-1 0.37 0.10 0.59 - 0.73 1.09 -
PK 15.0 - 23.0 16.1 9.6 7.8 -
LDH 6.9 39.0 - 110 - - 1.64
PC 0.085 . . . . . .
PEPCK ND - 0.33 0.044 0.32 0.37 0.53
FBPase 0.96 0.13 0.57 0.92 0.82 0.13 1.36
GPase 0.17 - - 2.14 - - -
G6PDH 1.06 - - 4.21 - - -
ME 0.55 - - 0.28 - - -
HOAD 0.86 0.26 - 0.33 - 5.54 -
MDH 35.1 - - 113 - - -
CS 2.05 2.9 - 0.35 - 12.1 -
GDH - 2.19 - 2.14 - - -
GOT - 16.8 - 5.84 - - -
GPT - 1.1 - 23.5 - - -
Values are/tmol min -1 g-I wet tissue weight at the temperature noted.
N D - none detected; - - - not assayed. Temperature refers to assay temperature.
FBPase and PEPCK activities are found in salmon 1~ marlin (Makaira nigricans)156,
cod, flatfish, and trout 83, and flounder 76. FBPase, but not PEPCK, was found in the
skate 11~ In comparison to the liver, the gluconeogenic enzymes tend to be lower
in activity (Table 3), which, together with the greater activities of the glycolytic
enzymes, suggests a smaller gluconeogenic potential for this tissue. Glucose 6-
phosphatase (G6Pase) in agnathans is an exception; the activity of this enzyme is
3.3- and 1.1-times greater than its counterpart in the liver in the hagfish and the
lamprey, respectively 132. This compares with values in the salmon of 0.08-times 132
and in the flounder of 0.54-times 76 that of the liver.
Gluconeogenic flux in kidney slices has been estimated in flounder 76, salmon 1~
and skate 1~ Gluconeogenesis occurred in salmon kidney with rates from alanine
being similar to those in liver (0.04/zmol g-1 h-i; 10oC); lactate gluconeogenesis
was 0.46/zmol g-1 h-1 which is 50% of that in the liver, but a full 10-times above
that for alanine. This tissue has significant gluconeogenic potential, even though,
as the authors point out, PEPCK activities are only 3% of that in the liver. This
discrepancy between rate and enzyme activity may reflect an assay problem rather
than a real enzyme activity difference. Jorgensen and Mustafa 76 measured flounder
PEPCK in the forward direction and found activities equivalent to those in the
liver, and rates of lactate gluconeogenesis identical to the liver (0.36 /zmol g-1
h-i; at 10~ The kinetics of kidney PEPCK are unknown, but it is possible that
measurement in the reverse direction will lead to an underestimation of activities.
The skate kidney appears different from that of the teleosts with respect to
gluconeogenesis. Mommsen and Moon 1~ report very low rates of gluconeogenesis
(0.01 /zmol g-1 h-i; at 100C), with the majority of the alanine and lactate being
oxidized (2.5 gmol g-1 h-i); no PEPCK activities could be detected in this tissue.
They concluded that the kidney in this animal has no significant gluconeogenic
function.
Hexose monophosphate shunt enzymes were reported in the skate kidney 11~
but only G6PDH, and not 6PGDH, was found in the salmon kidney l~ (see Table
3). The glycogen metabolizing system in fish kidney has been poorly studied. In
the skate, GPase activities are present, as are substantial quantities of glycogen 1~
Oxidation of lactate and alanine is no greater in the kidney of the salmon than the
liver, and this is consistent with the relatively low activities of citrate synthase l~
Given the high glycolytic enzyme activities found in the teleost kidney, it may be
postulated that anaerobic glycolysis is a significant source of energy for kidney
function. The low gluconeogenic as compared with oxidative potential of the skate
kidney, and the fact that citrate synthase (CS) activities are 7-times more active
than the liver enzyme, all suggest that kidney metabolism is oriented towards
oxidationl05, ll0.
Various oxidative and gluconeogenic substrates have been examined in the
flounder 76, salmon 1~ and skate 1~ kidney. Lactate, alanine, and serine were all
metabolized to both CO2 and glucose (alanine and serine were not examined in
flounder). The distribution of carbon between CO2 and glucose varies between
substrates and species. In salmon, gluconeogenesis from lactate is approximately
10-times greater than from alanine (see above), whereas in the skate gluconeogenic
flux is the same for the two substrates. In terms of carbon distribution, 75% of
the lactate carbon is converted to glucose in the salmon as compared to 2% in the
skate. With respect to alanine, 28% of the carbon is converted to glucose in the
salmon as compared to 3% in the skate. The substrate that showed the greatest
relative conversion to glucose in the skate kidney slice was serine, with 12% of
the carbons reaching glucose. Mammalian kidney utilizes lactate and glutamate but
not alanine or serine for glucose production sl, indicating subtle differences exist
in these pathways between these two groups. Few kidney carbohydrate metabolism
studies have been performed on fish, and questions that remain to be answered
include the use of various substrates, the glueoneogenie potential of the tissue and
its importance in whole animal glucose production, the predominant pathways for
energy supply in the tissue, and glycogen metabolism.
Z Environmental adaptations
The impact of environmental factors on kidney metabolism has been poorly studied.
Morata et al. 113 reported increased activities of PEPCK and G6Pase in trout kidney
after 60 days of fasting, while FBPase activities were unaffected. A long term
fast results in increased gluconeogenesis in the mammalian kidney, with glucose
production from this tissue accounting for up to 50% of whole animal glucose
production sl. The results of Morata et aL 113 are consistent with the mammalian
situation, but no metabolic studies have been performed to corroborate the enzyme
changes. JCrgensen and Mustafa 76 reported that the flounder kidney maintained a
stable energy charge during extended hypoxia, and suggested that this tissue may
replace the liver as the principal gluconeogenic organ during hypoxia.
It is clear that many metabolic questions remain unanswered with regards to the
fish kidney. Few species have been studied and in even fewer eases are changes in
function related to a change in the condition of the fish. It does appear, however,
that this tissue has a reduced metabolic potential compared to the liver, but that it
can be gluconeogenic in at least a few species of teleosts.
II(. Skeletal muscle and heart
1. Myotomal muscle
Johnston and Altringham 71 have reviewed the organization of fish myotomal mus-
cle, the energetics of muscle contraction, and adaptations of fish muscle in Volume
1 of this series. The use of carbohydrate as an energy source for muscle contraction
has not been directly reviewed and is complicated by the diversity of fish species
and their lifestyles. In general, sustained swimming is powered by red muscle and
burst swimming by white muscle and the oxidation of fatty acids and the anaerobic
breakdown of glycogen, respectively. Glycogen is found in red muscle in quanti-
ties slightly less than white muscle (Table 1), and studies have reported glycogen
breakdown and lactate accumulation during red muscle activities 12s'lss'164, but fatty
TABLE 4
W h i t e m u s c l e e n z y m e s f r o m s e l e c t e d fish s p e c i e s
Enzyme S k a t e 11~ H a g f i s h 39 R a i n b o w t r o u t 83,155 P e r c h 45 G o l d f i s h 165 M a r l i n 156

10oC ' 10oc 15~ 20~ 10~ iSoC 25~
HK 0.058 - 0.06 - 0.056 -

PFK-1 17.4 5.8 9.8 - - -
PK 49.8 98.0 110 - 39.0 1105
LDH 110 305 - 2723 260 2697
PC 0.032 - - - 0.10 ND
PEPCK ND 0.043 ND - - 0.90
FBPase 0.076 0.40 0.13 - 0.25 2.39
GPase 1.9 - - 51.4 23.0 -
G6PDH 0.044 ND - - 0.06 -
ME 0.096 0.08 - - 0.60 0.60
IDH - 1.74 . . . .
HOAD 0.037 2.0 - - - 0.70
MDH 12.0 31.4 - - 115 562
CS 0.63 0.63 - - - 2.71
V a l u e s a r e / t m o l m i n -1 g - 1 w e t t i s s u e w e i g h t a t t h e t e m p e r a t u r e n o t e d .
ND = none detected; - = not assayed. Temperature refers to assay temperature.
acids and proteins (amino acids) are thought to be the principal metabolizable
fuel for red m u s c l e 71'164'179'182. Increased swimming speed elevates blood flow to
muscle la2 and endurance 25 and sprint 126 exercise training does increase the aerobic
capacity of fish myotomal muscle, but the contribution of endogenous substrates to
energy production are probably greater than blood-borne substrates during muscle
con traction 164,179.
Enzyme activities reported in red and white myotomal fish muscle are noted
on Tables 4 and 5. Parkhouse and colleagues 12s reported that GPase and PFK-1
are the primary glycolytic flux regulating enzymes in both red and white muscle
of rainbow trout under all levels of exercise when glycogen contents were high.
GPase total and %a values increased with exhaustive exercise in trout white
but not red muscles 155. At low glycogen contents, HK and glyceraldehyde 3-P
dehydrogenase-phosphoglycerate kinase (GAPDH-PGK)/LDH became the primary
control points 125, suggesting that recovery metabolism is quite distinct from that
during exercise and requires exogenous glucose. This latter idea is contrary to the
general belief that teleost glucose turnover rates are insufficient to account for
glycogen synthesis 2 .6. .7.29
. 89 177,179.
Anaerobic glycogen breakdown to lactate is the hallmark of exhaustive exercise
in fishes. Glycogen content per g tissue in white muscle is generally below that
found in the liver with some exceptions, the most notable being the tuna (Table
1). The principal question plaguing fish physiologists/biochemists over the years has
been the fate of this lactate following exercise. Wood and Perry la2 discussed this
issue and have shown two patterns of handling the 'metabolic acid load' (AH+m)
and the lactate load (ALa-) generated during exhaustive exercise. The more active
species (e.g. tuna, trout, dogfish) increase blood AH+m and ALa- immediately
TABLE 5
Red muscle enzymes from selected fish species

. . . . . . .
Enzyme Skate n ~ Sturgeon 152 Rainbow trout s3,155 Goldfish 165 Marlin 156
10*C 20"C 15"C; 20"C 15"C " 25"c
. . . . .
HK 1.27 0.008 0.14 4.40 --

PFK-1 16.8 0.49 3.76 ... . .
PK 52.0 - 110 61.0 316

LDH 59.3 40.0 0.09 400 617
PC 0.10 - - 0.38 ND
PEPCK ND - - - ND
FBPase 0.054 0.094 ND 0.20 0.68
GPase - - 15.3 24.0
G6PDH 0.079 - 0.18 17.6
ME - - - 1.30 4.40
HOAD 0.095 0.34 - - 17.4
MDH 94.7 3.15 - 330 336
CS 15.9 - - - 18.3
. . . . . . . . .
Values a r e / x m o l min -1 g - I wet tissue weight at the temperature noted.

ND --- none detected; - ffi not assayed. Temperature refers to assay temperature.
following exercise, but ALa- exceeds AH+m and persists in the blood for a greater
time period. Sluggish species (e.g. flounder, sole, skate, sea raven) demonstrate only
a minor ALa-, always below AH+m. These latter species are thought to retain
metabolically generated lactate within the muscle. In all cases except the skipjack
tuna 17s, fish species require 12-24 h to clear a metabolic lactate load 6,96,122,142.
The principal question addressed in each study is what processes are involved
in glycogen repletion following exhaustive exercise. The classic mammalian pattern
known as the Cori cycle which can account for 30-55% of the post-exercise glycogen
repletion 92,178, apparently does not operate in f i s h 6'21'96. Lactate turnover is directly
related to plasma lactate concentrations, but even in the skipjack tuna where
values of turnover are similar to those reported in mammals, Cori cycle activity
is estimated to account for <5% of glycogen repletion 178. Lactate utilization by
isolated fish hepatoeytes is well below that required to explain muscle glycogen
repletion 172, and even if this were not the ease, glucose uptake into trout white
muscle 179 represents a small fraction of trout glucose turnover 29.
These inconsistencies and the fact that a stoichiometry between lactate disap-
pearance and glycogen repletion does exist in fish muscle, has led most investiga-
tors to propose that muscle glyeogenesis from lactate must account for glycogen
repletion 2,6,96,142. The precise mechanism for muscle glyeogenesis from lactate is
not dear, although the enzymes PEPCK and malie enzyme (ME) and/or pyruvate
carboxylase (PC) are thought to be important 92. As Table 4 indicates, these three
enzymes occur in few species and are present in low activities where measured/
present. Only in goldfish 165 and possibly marlin 156 have all enzymes required for
glycogenesis been reported. Activities of PK are high relative to other glyeolytie
enzymes (Table 4), and a recent study by Sehulte et at 142 has proposed that a
high [ATP]/[ADP] ratio resulting from a substantial decrease in [ADP] during
early recovery in trout could drive PK in the reverse direction thus eliminating
the need for these other enzymes. This solution to glycogen repletion has been
previously suggested by others for higher vertebrate muscle glycogenesis. In their
recent review, however, McDermott and Bonen 92 concluded that the pathway for
glyco(neo)genesis in vertebrate muscle is 'far from being delineated' (p. 174) and
certainly need not be identical to gluconeogenesis in the liver. This is still an
incomplete story and new approaches are required, but the separation of myotomal
muscle into distinct red and white fibre groups makes the fish an ideal model for
such studies.
Endurance exercise training has been shown to modify myotomal muscle struc-
ture, enzyme activities and aerobic capacities (see refs. 25, 71). Pearson and
collaborators 126 reported that sprint trained trout performed better, accumulated
more lactate and depleted less ATP without differences in glycogen and phospho-
creatine depletion compared to untrained trout. In addition, trained trout repleted
glycogen more quickly. They concluded that trained trout increased exogenous glu-
cose use to conserve endogenous energy reserves, implying changes both in muscle
capillary density25 and enzyme activities71. Such studies do not suggest changes in
metabolites used for exercise, but imply an increased efficiency of their use.
Environmental conditions have been shown to modify swimming performance
by remodelling the myotomal muscles of a number of species71. Low temperature
acclimation increases the volume of the aerobic fiber type, recruitment patterns and
improves swimming performance in goldfish, green sunfish, chain pickerel, carp,
stripped base and flounder, but not in salmonids (reviewed in refs. 52, 73, 74).
it has been argued by Guderley and Blier52 that these species differences may
reflect the species thermal tolerance and temperatures for optimum locomotion.
Changes in performance at low temperatures are also related to changes in enzyme
activities and concentrations of energy stores in these muscles (glycogen, lipid),
and an apparent switch of metabolism away from carbohydrate to fats 75. The
response of goldfish muscle to hypoxia is modified by thermal acclimation, with
weaker responses noted at low temperatures 165. Hypoxia in tenth (Tinca tinca) was
associated with a decline in aerobic capacities of myotomal muscle and a decrease
in the threshold for activating anaerobic glycolysis, but a reduction in the use of
exogenous compared to endogenous carbohydrate sources 72.
The metabolic system of fish myotomal muscle is an example of matching supply
with demand 71, and as environmental conditions change, demands will change.
Studies need to precisely identify the metabolic consequences of the adaptability of
fish muscles, to better understand these supply-demand issues.
2. Cardiac muscle
The fish heart has different metabolic demands, and its metabolism would be
expected to differ from that of myotomal muscle. Enzymes of glycogen metabolism,
glycolysis, and the pentose phosphate shunt are present in all fish hearts examined
(Table 6). PEPCK activities have not been detected in any fish heart examined.
Activities of PFK-1 and PK resemble those found in skeletal muscle, while activities
TABLE 6
C a r d i a c m u s c l e e n z y m e s f r o m selected fish species
Enzyme H a g f i s h s7 S k a t e 11~ Dogfish is~ S e a r a v e n 15~ E e l p o u t 150 Cod s7 M a r l i n 156

15"(2 10"C 15"C 15"C 15"C 15"C 25"C
ilK 1.70 1'13 " 4.35 3.53 . . . . . 2.45 6.23
PFK-1 18.8 3.74 10.4 1.83 1.17 9.63 m
PK 36.0 46.7 66.7 51.9 36.3 58.9 104

LDH 114 67.8 251 217 128 33O 708
PC - 0.10 - - - ND
PEPCK - ND - - - ND
FBPase - 0.12 - - - ND
GPase a 3.48 . . . .
6PDH - 0.42 - - -
ME - 1.35 - - - m
2.5
HOAD 8.28 0.13 1.78 4.09 1.35 5.54 8.63
MDH - 67.3 - - 226 332
CS 6.92 4.10 21.3 16.9 12.8 12.1 14.2
Values a r e / z m o l m i n -1 g - l w e t tissue w e i g h t at t h e t e m p e r a t u r e n o t e d .
N D - n o n e d e t e c t e d ; - - n o t assayed. T e m p e r a t u r e refers to assay t e m p e r a t u r e .
a R e p r e s e n t s glycogen p h o s p h o r y l a s e a, r a t h e r t h a n total activity.
of FBPase arc approximately an order of magnitude lower in the heart than the
myotomal muscle (Tables 4-6). These observations and the absence of PEPCK and
PC suggest that the fish heart is one of the most glycolytically directed of the fish
tissues. Glycogen concentrations in fish heart are generally 30-90 ~tmol g-1 (Table
1), which are relatively high compared to higher vertebrates. This may reflect a less
advanced system to maintain oxygen and substrate supply to the heart necessitating
the use of endogenous substrates under stress (hypoxic) conditions, and a coronary
circulation is not found in all fish species 24. Driedzic and Hart 2s showed that sea
raven heart function can be maintained in the absence of substrates, but that
iodoacetate damaged performance. They concluded, therefore, that glycogen is
consumed through glycolysis in the absence of substrates; GPase is present in the
fish heart (Table 6).
The mammalian heart metabolizes glucose, lactate, free fatty acids and ketone
bodies, but fatty acid is the principal fuel (see ref. 150). This reliance upon fatty
acids is related to the high aerobic efficiency of ATP production from this substrate.
In contrast, glucose may be as important or more so as a fuel for the fish heart.
Driedzic and Hart 28 demonstrated that the addition of glucose was equally effective
as palmitate in preventing contractile failure in the perfuscd sea raven heart. In the
more primitive hagfish heart, glucose outcompctes palmitate oxidation, and the ad-
dition of palmitate alone to isolated hearts resulted in decreased performance and
decreased tissue glycogen content sT,re. These authors concluded that carbohydrates
were an obligatory aerobic substrate for hagfish heart metabolism, and presented
two explanations. Either the dependence upon carbohydrate is an adaptation to
hypoxia in this species, or it may reflect a primitive feature of the chordate heart;
as animals evolved a more active lifestyle, the importance of glycolysis diminished.
Palmitate decreased cardiac performance in the skate, glucose was without effect
and only ketone bodies had a significant positive impact on heart performance 28.
While glucose did not affect skate heart performance, the addition of iodoacetate
dramatically decreased heart performance, suggesting glycogen utilization is impor-
tant in maintaining heart function in the absence of exogenous substrates 28. The
presence of HK in the elasmobranch heart (Table 6) does provide the potential for
glycogen synthesis. Sidell et al. 150 examined a variety of temperate teleost species
and have found a positive correlation between maximal myofibriUar ATPase, an
index of ATP demand, and both HK and carnitine palmitoyl transferase activities.
Their analysis indicated that unlike mammals where fatty acid use increased as
work capacities expand, teleosts have developed no preference for fatty acid or
carbohydrate use depending on power requirements.
Lactate, in addition to fatty acids and glucose, is also an important substrate for
teleost heart function. Lanctin et al. 85 showed greater oxidation rates of lactate than
glucose in the isolated trout heart, a fact confirmed by Milligan 94. Lactate supple-
mentation was sufficient to maintain sea raven cardiac performance in the presence
of oxaloacetate 28. Furthermore, Milligan and Farrel195 showed that under aerobic
conditions lactate was capable of providing sufficient energy at high workloads,
and its transport was carrier-mediated. In addition, both glucose and lactate oxi-
dation, and glycogenolysis, in the trout heart are adrenergically regulated through
fl-receptors 94. A negative correlation was reported in sea ravens between body size
and both heart oxygen consumption and PK activities, while LDH activities scale
positively 31 This has lead to the hypothesis that large individuals may utilize more
lactate than smaller ones, but this has yet to be critically tested.
Fish hearts and in particular contractile performance have been assessed under
numerous environmental conditions. For example, hearts from different species
have differing abilities to withstand hypoxic stress. Hagfish cardiac performance is
not affected by a 3 h hypoxia stress 149, and sea raven hearts withstand anoxia better
than ocean pout even though performance in both decrease by 1 h 163. The decline
in performance in these two teleosts was found to be related to acidosis rather than
ATP depletion, with the sea raven having a better buffering capacity possibly due to
the low myoglobin content of pout hearts 15~ These studies demonstrated adequate
endogenous fuel reserves to maintain ATP concentrations during hypoxia, and the
importance of carbohydrate in hagfish cardiac metabolism.
Driedzic and colleagues studied the effects of temperature transitions on heart
energy metabolism in a variety of fish species (sea raven, pike, eel, white and
yellow perch, and bass). They reported that HK activities are greatly decreased in
acute high to low temperature transitions, whereas fatty acid metabolizing enzymes
decreased to a lesser extent, or were completely protected 4,144,145. Furthermore,
acclimation to low temperatures resulted in increased fatty acid metabolizing
enzyme activities and oxygen consumption was increased with palmitoleate but
compromised with glucose additions 144. These data support an increased fatty
acid catabolism in cold temperatures. They did find in sea raven hearts, however, a
decreased palmitate oxidation at 5~ as compared to 15~ in the absence of changes
in glucose oxidation. Thus, the relative importance of these metabolic fuels at low
temperatures is not dear, and these inconsistencies may relate to the use of these
substrates for other muscle functions including the maintenance of Ca 2+ levels
rather than energy production per se. Milligan94 has reported that the response
of trout myoeytes to #-adrenergie stimulation was enhanced at warm tempera-
tures.
Species specific responses of cardiac metabolism to temperature changes have
been reported. Eel and bass heart function is compromised at low temperatures,
while that of pike, yellow and white perch is not 4,145. Consistent with these results,
cardiac ATPase and fatty acid metabolizing enzyme activities decreased to a greater
extent with a temperature change from 15 to 5"C in the eel and bass heart as
compared with the others. Thus, in the compensating species, carbohydrate as an
energy source decreased in favor of fatty acids. It is interesting to note that while
ATPase, HK and fatty acid metabolizing enzyme changes differed between species,
CS activities were protected in all species. Even though carbohydrate and fatty acid
pathways may be differentially affected, the Krebs cycle is in all cases protected
from temperature effects.
There are few common features with regard to teleost cardiac metabolism
except that hearts can utilize either glucose or fatty acids. The primitive hagfish
use only carbohydrates, while elasmobranchs are unique in their use of ketones,
consistent with this ability in other tissues and their presence in the blood. All
hearts examined contain glycogen, which probably relates to the relatively poor
oxygenation/circulation conditions of the cardiac tissues of many fish hearts. The
most important question is what factors are key to substrate selection. The recent
preparation of isolated myocytes by Milligan94 may be a useful metabolic model to
address these issues. In addition, work by Stewart et al. 153 with fatty acid binding
proteins in heart may lead to a better understanding of fuel use in this tissue of
fishes.
I4. B r a i n
Extrapolation from other vertebrates has assumed that the fish brain is a glucose-
consuming tissue. While there is some evidence for this, little attention has been
given to other possible energy substrates and pathways. Indirect evidence for this
glucose-consuming capability comes from in vivo studies. When active fish that
contain relatively low levels of brain glycogen are made hypoglyeemie by insulin
injection, convulsions and death resulted, similar to the mammalian situation sT.
Also, Washburn et aL 177 have reported that rainbow trout brain has the highest
glucose utilization rate per unit weight of all tissues examined and as high as
reported for rat; these values were found to be higher in female than male trout.
Direct in vitro studies have shown glucose uptake in brain tissue; incubation of trout
and bullhead brain pieces in the presence of glucose resulted in a net uptake of
glucose59, but in no ease was glucose released, except in the sea lamprey~.
Glycogen levels in the brain of fish (Table 1) are generally higher than their
mammalian counterparts, and it has been shown that under hypoglycemic, anoxic,
and ischemic stress that this energy store is or may be mobilized. Glycogen values
greater than 100 ~mol g-I have been reported for the e e l 36 and the lamprey4a,132,
and values for most fish species are generally above mammalian levels93. A notable
exception is the rainbow trout where levels are in the range reported for mammals 27.
While the high glycogen levels suggest a greater importance of this compound in
energy metabolism in most teleosts as compared with mammals, little attention has
been focused on the regulation of its synthesis and degradation. Glycogen synthase
(GSase) and GPase, the two enzymes of glycogen metabolism, have been reported
in the brain of the adult lamprey4a. Total activities of GSase are at least 10-times
higher than the total GPase activities (Table 7), in contrast to the rat brain where
GPase activities are 20- to 50-times more active than GSase 93. The regulation of
these enzymes in fish is unknown, although the a and b forms appear to exist in the
lamprey brain 48.
The catabolism of endogenous glycogen is confused by the complexity of the
brain itself and the location of the glycogen in the brain. Rovainen 14~ reported
that brain glycogen in the larval lamprey (P. marinus) is localized primarily to
the meninges rather than the brain itself. In fact, the 'cleaned brain' (brain less
meninges) contained 33% of the glycogen content of the whole brain, while
the meninges contained 170% more. Rovainen ~4~ postulated that the meninges
catabolize glycogen to glucose which is used by the neural tissues, as evidenced by
activities of glucose 6-phosphatase (Table 7) and high levels of glucose production
in the meninges compared with the whole brain. Similar studies have yet to be done
in teleosts, although incubation of trout and catfish brain pieces did not exhibit any
net glucose production 59. Studies with adult lampreys also demonstrate that whole
TABLE 7
B r a i n e n z y m e s f r o m selected fish species
Enzyme L a m p r e y 48 S t u r g e o n 39 R a i n b o w t r o u t 83 F l o u n d e r s3 C o d s3 Goldfish154
11~ 20~ 15~ 15~ 15~ 15~
HK - 0.66 0.54 0.01 0.12 -
PFK-1 - 0.08 2.42 2.96 2.68 7.0
PK 16.5 - 35.2 27.9 54.0 -
LDH 326 60.0 . . . .
PEPCK 1.7 - ND ND ND -
FBPase ND 0.068 ND ND ND -
G6Pase 0.25 a . . . . .
GSase 0.05 . . . . .
GPase 0.55 . . . . 45.0
G6PDH ND 0.04 . . . .
HOAD - 0.05 . . . .
CS - 3.3 . . . .
Values a r e t t m o l m i n - l g-1 wet tissue w e i g h t at t h e t e m p e r a t u r e n o t e d .

N D = n o n e d e t e c t e d ; - -- n o t assayed. T e m p e r a t u r e refers to assay t e m p e r a t u r e .
a Larval l a m p r e y ( r e f e r e n c e 140).
brains liberate glucose when incubated without substrate r and this release is
blocked by insulin 48.
Activities of hexose monophosphate shunt enzymes are reported in the mam-
malian brain 93, possibly to provide an alternative oxidative route for glucose or to
provide reducing equivalents for brain lipid metabolism. G6PDH was not detected
in the lamprey brain ~ but was in the sturgeon (Acipenserfulvescens)152;it has been
examined in very few species, so the existence of this pathway is uncertain.
Adult lamprey brain pieces convert both glucose and lactate to glycogen~. The
flux from glucose to glycogen is enhanced by insulin under certain conditions. The
existence of a gluconeogenic pathway in mammalian brain tissue is unlikely given
the absence of PEPCK activities64 although FBPase is expressed in these tissues93.
PEPCK, but not FBPase, activities are found in adult lamprey brainsr no fish
species has been reported to have both PEPCK and FBPase activities in the brain
(see Table 7). It seems unlikely that the teleost brain is capable of gluconeogenesis,
although more species and experiments need to be undertaken.
While lactate is converted to glycogen in the lamprey brain, the majority of this
substrate is converted to CO2 (ref. 48). The oxidation of lactate is 2- to 4-times
greater than that of glucose in this system. Thus, lactate is a better oxidative
substrate than glucose in this preparation, but its quantitative importance is not
known. The use of other substrates including amino acids and ketone bodies have
not been examined in fish, although ketone bodies have been shown to increase
during anoxia in trout brain 2:.
Few studies have examined the effects of environmental factors on brain carbohy-
drate metabolism. DiAngelo and Heath 27 reported a correlation between glycogen
content and anoxia-tolerance in trout and bullhead, similar to studies on other
vertebrates ~39. Using a minced brain preparation from these two species, Heath 59
further suggested that bullhead brain relies more on endogenous reserves (glyco-
gen) and possibly a depressed metabolism to tolerate anoxia, while the trout brain
uses exogenous glucose to supplement its limited glycogen reserves. Given the re-
cent interest in brain and depressed metabolism in mammals, the fish brain appears
to be an ideal organ to examine the mechanisms of anoxic-tolerance.
Brain glycogen content in goldfish and sardines was maximal during the winter
months and increased with cold acclimation 14. The explanation for this cold-
induced increase in brain glycogen is far from clear. These authors suggest it may
be related to a general decrease in brain metabolism during the cold, but this is
not consistent with the relative thermal independence of brain metabolism reported
by Heath 59. Certainly coordination is required regardless of temperature, so again
further studies are needed to address this interesting paradox. In this same vein,
the importance of pesticides and other toxicants on brain metabolism need to be
investigated. One study using lindane and eels found significant changes in brain
metabolites ~. This should be a fruitful future area for toxicology studies.
Thus, the fish brain remains a poorly studied tissue although it offers many
interesting comparative and environmental challenges. Certainly carbohydrates are
important metabolic fuels, but little is known of their compartmentalization, pattern
of use, or qualitative significance, all areas for further study.
111. Red blood cells
The oxygen consumption of the nucleated, mature red blood cells of fish is
considered high relative to that of the mature mammalian cell. Values range from
2 to 10 /zmol 02 l-lcells h -1 in fish, or 3- to 10-times that of mammalian red
cells 13'116. In fact, Nikinmaa 116 has suggested that the fish red cell is metabolically
more comparable to the reticulocyte, the stage in mammalian erythropoiesis just
prior to the loss of regular cellular organelles. Although these rates are low
compared to other tissues of fish, they account for 2-5% of the resting whole-trout
oxygen consumption 3~ 175.
The question of what fuels this oxygen consumption is far from clear. The mature
red cell of most mammals has a high glucose transport capacity which does not
limit glycolysis, and anaerobic glycolysis is considered to meet all of the energetic
needs of the cell 116,185. Fish red cells aregenerally poorly permeable to glucose.
Red cells from rainbow trout 12,161, paddyfield eel (Monopterus albus) 161 and carp
(Cyprinus carpio) 160 are considered to be deficient of o-glucose transport. A study
of Amazonian fish species by Kim and Isaacks 81 showed significant glucose uptake
in only 2 of 5 species. Species which do show high glucose permeability (and in
some cases cytochalasin-B-sensitivity) are the river lamprey (L. fluviatilis) 16~ the
Pacific hagfish (E. stouti) 68, the sea perch (Embiotaca lateralis) 67, and the Japanese
eel TM. Equilibration times for glucose across red cells in most studies are from
a few minutes (e.g. lungfish) al to hours (e.g. trout) 129, with the exception being
hagfish where they are a few seconds 68. Ferguson and Storey 34 showed that the
transmembrane glucose gradient was 9, favoring extracellular concentrations. The
Pacific hagfish glucose transporter has now been isolated and found to have close
structural similarities with the human erythrocyte glucose transporter GLUT-1 (ref.
185). Studies such as this one should help to better understand differences between
red cell transport functions.
Nikinmaa 116 stated that with the possible exception of the hagfish, glucose
transport limits glucose metabolism in fish nucleated red cells. Enzymes from fish
red cells have been reported from five studies, and in most eases, all the glycolytic
enzymes are present (Table 8). Two studies suggested that HK limits flux, but most
studies support PFK as the rate limiting enzyme 34,5~ Unfortunately, only the study
of Pesquero et al. 129 has examined glucose transport, oxygen consumption, glucose
oxidation and enzymes simultaneously. This study reported 3-O-methylglucose (3-
O-MG) transport rates of 430 nmol g-1 hb h -1, oxygen consumption rates of 70
nmol g-1 hb h-1 and HK activities of 760 nmol g-lhb h-l; transport in brown
trout red cells does appear to limit glucose oxidation. This species is considered to
have poor glucose permeabilities, and unfortunately no studies have examined these
parameters in cells considered to have greater permeabilities (e.g. Japanese eel red
cells have a transport rate as much as 200-times that of trout) 161.
The data with respect to oxidation is confusing and again experiments have
used primarily trout red cells rather than a red cell system which is metabolically
more active (e.g. eel) 61. Walsh et al. 175 found CO2 production rates from 14C-
labeled substrates in the order glucose > lactate > > alanine > oleate. If the
TABLE 8
Red blood cell enzymes from selected fish species
Enzyme Rainbow trout34.175 Brown trout129 Perch3 Sea r a v e n 146

15oc34 15oc175 15oc 25oc 15"C
-HK 2.7 - 0.76 0.08 0.25
PFK-1 1.6 ND 0.2 1.4 0.69
Aldolase 4.3 - - 5.2 0.15
G3PDH 22.4 - - 27.3
PK 32.1 29.6 9.7 13.2 1.00
LDH 50.5 27.2 30.0 233 1.03
PEPCK ND ND - - . . .
FBPase ND ND - -
GPase ND ND - -
G6PDH 14.1 24.4 8.1 20.6
IDH 2.9 4.4 ND -
MDH 64.6 32.0 - - 14.9
CS 0.58 ND ND - 0.38
Values are/~mol min-1 g-I hemoglobin except for sea raven which is/zmol min-1 m1-1 RBC at the
temperature noted.
ND = none detected;- = not assayed.
rates of all 4 substrates were summed, they represented less than 15% of the
total oxygen consumption rate. They suggested other substrates were involved,
and as their experiments used whole trout blood, endogenous compounds could
not be excluded. There was significant hexose monophosphate shunt activity in
this preparation (Table 8), but this makes the oxygen consumption rates even
more difficult to interpret. They did report a concentration-dependent increase
in CO2 production with glucose. Pesquero et ai.129 reported that total oxygen
consumption was independent of glucose or pyruvate addition, and at I mM glucose,
CO2 production represented less than I% of total oxygen consumption. Significant
hexose monophosphate shunt activities were found as a-cyano-3-hydroxycinnamate,
a compound which blocks pyruvate transfer into the mitochondria, decreased CO2
production only by 33%. In neither study 129,17s could CS activities be detected
(Table 8), although only one other study has reported CS activities. Activities of
this enzyme are low relative to that of other measurable enzymes. Also, Pesquero
et at. 129 reported mitochondria 'were practically absent' (p. 450) as shown by EM
images. Only in the study by Sephton et al. 146 was there a direct correlation between
oxygen consumption and CO2 production rates from glucose and this was in the
sea raven. This strongly supports the aerobic metabolism of glucose as the principal
source of energy in these cells. Washburn and colleagues 177 have also shown that
glucose utilization rates of trout red cells are greater than muscle but well below
those of brain and gonad. Certainly the oxidative capacities of fish red cells are far
from clear and more studies must be done in this area.
The reliance upon anaerobic glycolysis for energy production of the mature
mammalian red cell means that lactate must represent a major metabolic endprod-
uct (see ref. 116). Even though it has been estimated by Ferguson et ai.35 that over
90% of the energy for the trout red cell is generated aerobically, lactate does appear
as a major metabolic endproduct in most studies. Walsh et al. 175 demonstrated that
lactate production represented 28% of the total glucose metabolism, even with high
blood oxygenation. Values reported by Pesquero et al. 129 exceeded 90% in brown
trout. They showed convincingly that, as in mammalian red cells, older trout red
cells are less metabolically active and that their preparation consisted primarily
of 'old' cells; this could account for the lowered oxygen consumption and glucose
oxidation rates compared to those of Walsh et al. 175. Sephton et al. 146 also reported
that rinsed, resuspended red cells of sea raven had a lower oxygen consumption
than whole blood, but under no aerobic condition did these cells produce lactate.
Whether other sources of lactate such as glycogen 34, or possibly carbon recycling 146
are important are not clear.
It seems, therefore, that the basic metabolic organization of the fish red cell
remains to be clearly elucidated. A number of conflicting studies exist, but we must
await studies on red cells which are more permeable to glucose than trout cells.
Certainly gluconeogenesis does not occur, but even the presence of an active Krebs
cycle is uncertain (Table 8), even though oxygen consumption rates are considered
'high '13,116. The sea raven red cell is the only clear story, but this is an unusual
species in its own right 116.
An extensive literature indicates that catecholamines modify the red cells of many
fish species both in vivo and in vitro 128,159. The red cell response to epinephrine in-
volves the activation of the Na+/H + exchanger with the ultimate effect of maintain-
ing blood oxygen transport by increasing the affinity and/or capacity of hemoglobin
to bind oxygen. This complex process involves an elevation of intraceUular pH
(pHi), cell swelling and decreased intracellular nucleoside triphosphates resulting
from increased ATP utilization by the Na+-pump. As plasma catecholamine levels
rise under a variety of environmental disturbances including hypoxia and exercise 159,
such changes in nucleoside triphosphate levels would be expected to change red cell
metabolism.
Under oxygenated conditions, Ferguson and Boutilier 33 reported a tight coupling
between nucleoside triphosphate utilizing and producing processes in adrenergic-
stimulated trout red cells. Tufts and Boutilier 162 reported that at rest, 20% of
cellular energy was used by the Na+-pump, while this value increased to 43% in
the presence of isoproterenol. Given that adrenergic stimulation increased oxygen
consumption in trout red cells and ATP levels did not fall, Boutilier and Ferguson 13
argued that oxidative phosphorylation is adequate to supply the needed energy.
Only when oxygen was limiting did ATP levels fall, but the small decrease in ATP
and the small increase in lactate suggested metabolism was depressed in these cells.
Unfortunately, the substrate utilized by these cells under either condition was not
identified.
Pesquero et al. 129 found that isoproterenol increased 3-O-MG uptake in brown
trout red cells, but glucose metabolism to CO2 and lactate increased by 2- and
4-fold, respectively. Thus, aerobic glucose metabolism does not appear to fuel
increased energy requirements. Sephton et al. 146 reported that substrates other than
glucose were izwolved in isoproterenol-stimulated sea raven red cells. In both of
92 Zig. Moon and (7.1). Foster
these studies, the in vitro incubation of red cells under hypoxie conditions resulted
in increased lactate and decreased nueleoside triphosphate levels. During exercise,
Wood et al. 183 showed that trout red cells increased the utilization of lactate as a
result of higher plasma lactate concentrations and the effects of epinephrine on red
cell metabolism.
The consistency and physiological importance of the red cell response to adren-
ergic stimulation makes this system ideal to study the importance of substrate type,
availability and metabolism in a cell system. Such studies are critical to unravel the
complexities of what was thought to be a relatively simple metabolic system. Also, it
is critical that systems other than trout red cells are used as experimental models, as
these cells may have quite distinct membrane permeabilities relative to red cells of
other species.
VII. C o n c l u s i o n s
This review was not meant to be all-inclusive, but to look at specific fish tissues,
the use of carbohydrates by these tissues and how utilization rates and patterns
may be altered by environmental and hormonal conditions. Much information is
available, but there are significant deficiencies in many areas, especially the kidney
and brain. Liver, and in particular hepatoeytes, are well studied, but virtually
nothing is known of hepatic function in non-carnivorous species. Muscle and red
cells represent good model systems, but the evidence for glyeogenesis and oxidative
metabolism, respectively, are circumstantial at best. To address these deficiencies,
the comparative approach is key and the use of species of different life histories
and life-styles essential. When it comes to hormone-binding, trout hepatoeytes seem
to have a dearth of specific binding sites, yet in many eases these cells respond
metabolically; why? What accounts for the significant species differences raised in
this paper, or should we expect even more differences than we actually see?
Fish represent a diverse vertebrate class with a very long evolutionary history.
As fish biochemists/physiologists we need to begin to use these differences to better
understand this group of animals and vertebrates evolution as a whole.
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CHAPTER 5
Metabolism of the swimbladder tissue
BERND PELSTER
Institut far Physiologic, Ruhr-Universiti~t Bochum, D-44780 aochum, Germany
I. Introduction
II. Histology of the swimbladder
III. Energy metabolism
1. Aerobic metabolism
2. Anaerobic glycolysis
3. Pentose phosphate shunt
4. Synopsis
IV. Metabolism and gas deposition: the single concentrating effect
V. Control of swimbladder metabolism
VI. Role of carbonic anhydrase
VII. Metabolism of rete capillaries
VIII. Antioxidants
IX. References
I. Introduction
Many fish posses a gas-filled swimbladder as a hydrostatic organ in order to

achieve neutral buoyancy. As a compliant bladder it obeys Boyle's law and changes
pressure and volume with changes in hydrostatic pressure, i.e. with water depth.
The hydrostatic pressure increases by about 1 atmosphere for every 10 m of water
depth. To keep the bladder volume constant and thus to remain neutrally buoyant
during vertical migrations a descending fish has to deposit gas into the bladder to
compensate for the compression of the bladder, an ascending fish has to resorb gas
to compensate for the expansion of the bladder caused by the changes in hydrostatic
pressure. Neutral buoyancy usually is achieved with the bladder making up some
5% or 8% of the body volume in marine and freshwater fish, respectively.
The swimbladder is filled only by diffusion of gases from the blood or from the
swimbladder epithelium into the lumen 19,49. Therefore, at great depths very high
gas partial pressures must be generated in the blood in order to maintain a positive
pressure gradient towards the lumen, although the pressure of gases dissolved in
water remains close to 1 atmosphere, irrespective of the water depth is.
The initial step in the generation of very high partial pressures is the secretion
of metabolites into the blood which decrease the physical solubility of gases in
the blood or liberate gases from a chemical binding site (the single concentrating
effect) 34. According to Henry's law the decrease in solubility results in an increase
102 B. Pelster
in gas partial pressures. In a counter-current system, the rete mirabile of the

swimbladder, the venous blood is then brought into close contact to its own arterial
supply, in which gas partial pressures are not yet elevated. Along the partial
pressure gradients gas molecules diffuse back to the arterial side and the initial
increase in gas partial pressure (single concentrating effect) is further enhanced
by back-diffusion and counter-current concentration. Depending on the magnitude
of the single concentrating effect and on the properties of the counter-current
exchanger finally gas pressures of several hundred atmospheres can be achieved,
sufficient to explain the occurrence of fishes with a gas-filled swimbladder at a depth
of several thousand meters 31034,62.
A crucial step in the functioning of the swimbladder thus is the production and
release of metabolites that modify the gas carrying properties of blood passing
the tissue. The morphological arrangement of the various layers of swimbladder
tissue provides close contact of the cells producing these metabolites, the gas
gland cells, to the vascular system, and also ensures accumulation of gas molecules
in the bladder, and not in the surrounding tissues. A brief discourse on the
morphology and histology of the bladder, therefore, appears to be worthwhile
before concentrating on the metabolism of the swimbladder tissue and especially of
the gas gland cells.
II. Histology of the swimbladder
The swimbladder originates as an unpaired dorsal outgrowth of the foregut. During

development the connection to the gut may persist (physostome fishes), but in many
species it is completely lost and the swimbladder is a closed gas cavity (physoclist
fishes). In adult teleosts the structural diversity in general swimbladder morphology
is remarkable 61. Talking about swimbladders, usually, the secretory part of the
bladder is envisaged, although it should be kept in mind that in most species either
a separate resorbing bladder is present, or a special section of the secretory bladder
can be closed off by muscular activity and is designed to allow for the resorption
of gas. As the resorption of gases occurs by diffusion along the diffusion gradients
and does not include any special metabolic features, I will focus my attention on the
secretory part of the swimbladder.
The wall of the secretory bladder consists of a number of thin tissue layers (Fig.
1), usually including layers of smooth muscle cells. The terminology of Fange 17
distinguishes between the inner epithelium, muscularis mucosae, submucosa and
tunica externa. The tunica externa represents a dense connective tissue capsule. The
submucosa may be impregnated with guanine crystals29,36 or include layered lipid
membranes 6, providing a low gas permeability of the swimbladder wall and thus
reducing diffusional loss of gases. In physostome fishes the muscularis mucosae
mainly consists of smooth muscle cells, but muscle cells may also be present in the
swimbladder wall of physoclist fishes.
In the innermost layer, the swimbladder epithelium, the so-called gas gland cells
are found. While in the eel (Anguilla) gas gland cells are spread over the whole
Metabolism of the swimbladder tissue 103
Fig. 1. Tissue layers of the swimbladder wall (secretory part) of the European eel, AnguiUa anguilla, s =
serosa; s m = submucosa; m = museularis mucosae; ep = epithelium; c = capillary. (from Dornl3; with
permission).
internal epithelium of the secretory bladder, in many species (Perca, Gadus) gas
gland cells are clustered together, forming a massive complex of several cell layers.
In some species the compact gas gland is the result of an extensive secondary folding
of the single-layered epithelium (Gobius, Syngnathus) 19,65. Gas gland cells usually
are in intimate contact to the vascular system, either by an extensive capillary
network underlying the epithelium, as in the eel, or by close proximity of a massive
gas gland and the rete mirabile.
Gas gland cells are cylindrical or cubical with a size ranging from 10-25 /zm
to giant cells of 50-100/zm or even more, but the size of the cells appears not
to be correlated to the water depth at which fish normally live 17,4~ The cells
are polarized with some small microviUi on the luminal side, while the basal side
often is more densely vacuolated and shows a number of infoldings, known from
other secretory or resorbing tissues, which, however, always lack mitochondria. The
meaning of these foldings is not yet understood 9,13,42. The variable density of the
granulated plasma of gas gland cells may represent variable functional states, and
not necessarily indicates presence of different cell types (Fig. 2) 13,42.
Gas gland cells are characterized by the presence of only few filamentous or
elongated mitochondria with few tubular cristae 9,13,26,42. The expression of further
organelles such as Golgi apparatus, endoplasmatic reticulum and ribosomes is not
consistent among different species, but they often are only poorly developed and
may even be missing completely in some species, although Jasinski and Kilarski 26
and Morris and Albright 42 found a well developed Golgi complex in Perca fluviatilis
104 R Pelster
Fig. 2. Schematic drawing of gas gland cells of the swimbladder epithelium of the European eel; (from
Dorn13; with permission).
and in Fundulus heteroclitus gas gland cells, respectively. The density of the Golgi
complex and of related vesicular structures may increase during periods of gas
deposition, as observed in Perca fluviatilis and in Acerina cemua 26. A general
feature again is the presence of lipid droplets or even lipid-containing vacuoles. A
comprehensive review on the significance of lipids for the achievement of neutral
buoyancy has recently been published in this series s4.
III. Energy metabolism
Studies on the metabolism of swimbladder tissue almost exclusively concentrate

on the swimbladder epithelium and the gas gland complex. In gas gland cells the
metabolites are produced that in blood provoke the reduction of gas solubility
or release gases from their chemical binding sites and thus are crucial for the
functioning of the swimbladder. Very little is known about the metabolism of
muscular cells or the connective tissues. According to our present knowledge,
however, they are not involved in the process of gas deposition and it can be
assumed, that their metabolism is comparable to the metabolism known from
similar cells in other tissues. Due to the morphology of the swimbladder, it is almost
impossible to obtain a clean preparation of gas gland cells without contamination
of smooth muscle cells, connective tissue or endothelial cells from the capillary
network, which should be kept in mind when discussing the metabolism of gas gland
cells.
TABLE 1
Activities of selected enzymes of aerobic metabolism in gas gland tissue
Enzyme Anguilla anguilla 4a Gadus morhua Opsanus beta 63

(25~ (10"C)16 (23oc)5 (24oc)
Citrate synthase 1.1 4- 0.2 2.27 4. 0.21 ND 0.14 4- 0.05
Malate dehydrogenase 51.7 4. 3.9 159.55 4. 27.56 163 4- 5 9.69 4. 1.09
Fumarase ND ND 1.47 + 0.1 ND
C~ochrome oxidase ND 1.06 4. 0.09 21.5 4. 3.1 ND
C~ochrome oxidase 0.074 a 0.041 a
Activities are given in ttmol min -1 g-1 fresh mass (4- S.E.). ND = not determined.
a From reference 22.
1. Aerobic metabolism
Mitochondria, representing the structural basis for aerobic metabolism, are not
abundant in the swimbladder epithelium. Accordingly, activities of enzymes of the
tricarboxylic acid cycle or the respiratory chain are very low (Table 1), even when
compared with a purely non-oxidative tissue, the white muscle. In the cod, Gadus
morhua, for example, cytochrome oxidase activity in gas gland cells represents only
20% of the activity found in white muscle tissue s .
Somewhat surprising is, therefore, the result of Ball et al.2, who measured an
oxygen uptake of about 0.3 ml g-1 h-1 at a temperature of 30~ for gas gland
tissue, but also for slices of heart muscle, by employing the manometric Warburg
procedure. On the other hand, oxygen uptake could not be stimulated by the
addition of glucose and was several times lower than the lactate production in
the same preparations. Similar results were obtained for cod gas gland, where
catecholamines or acetylcholine stimulated lactate formation, but did not increase
oxygen uptake of the tissue 16. In vitro oxygen uptake was sensitive to changes in the
sodium concentration and a decrease in sodium or a replacement of NaCI by KCI in
the saline solution significantly decreased oxygen consumption.
Oxygen uptake has also been measured in saline-perfused swimbladder prepara-
tions 46 and in situ in anesthetized, immobilized European eels (Anguilla anguilla) 5~
In situ oxygen consumption of the swimbladder tissue of the European eel was
about 3.8-times higher than in saline-perfused swimbladder preparations, indicating
an increase in metabolic activity during periods of gas deposition. In saline-perfused
swimbladders gas deposition was not measurable, while in the anesthetized eels
gas was deposited at a rate of 0.48 ml h -1. Mass specific oxygen uptake of the
active swimbladder tissue is in the same range as oxygen consumption of the whole
organism under resting conditions. Oxygen uptake of resting fish at a temperature
of 15-20~ varies between 15 and 65 nmol g-1 rain-1 (refs. 27, 53), and the weight-
specific oxygen uptake of swimbladder tissue amounts to approximately 22-45 nmol
g-~ min -1, assuming a mass of the swimbladder tissue of about 1-2 g for a 400-600
g eel. If the difference in oxygen consumption of saline-peffused and gas depositing
swimbladders in situ represents the transition from resting to active state, oxygen
106 B. Pelster
uptake of the swimbladder tissue is lower than in other tissues, as suggested by the
presence of only few mitochondria. The unsuccessful attempts to stimulate oxygen
uptake of gas gland tissue in vitro 2.16 thus may indicate that gas gland cells are
already stimulated maximally during preparation.
Like the oxygen consumption, the rate of CO2 formation in the active gas
depositing swimbladder in situ was about 3.9-times higher than the CO2 formation
of saline-perfused swimbladders, and in both preparations the rate of oxygen
consumption of the swimbladder tissue was much lower than the rate of CO2
formation 46.5~ Therefore, beside aerobic metabolism another metabolic pathway
must be available for the formation of CO2 in the swimbladder tissue (see below).
Furthermore, in vitro 2 and in situ 5~ lactate formation clearly surmounts the rate
of oxygen consumption, indicating a predominant role of anaerobic glyeolysis in the
swimbladder tissue.
2. Anaerobic glycolysis
Energy production via glycolysis may be fueled by glycogen stores or by extracellular

glucose. Activity of phosphorylase, key enzyme for the breakdown of intracellular
stores of glycogen, has not been measured, but several histological s.~3,17,26,42,Ss and
biochemical studies 11'17 demonstrated the presence of glycogen in gas gland cells.
Copeland s and F~inge17 reported a decrease in glycogen content of gas gland cells
during swimbladder inflation, and it was originally believed that glycogen was the
main source for the metabolism of these cells. Comparison of the measured rates
of lactate production with the amount of glycogen stored in the cells, however,
revealed that glycogen could fuel the anaerobic glycolysis only for a few minutes 11,
and it is now generally accepted that gas gland metabolism is fueled mainly by blood
glucose 19.
In general, the enzyme activities of the anaerobic glycolytic pathway are quite
high and in the range of activities measured in white muscle (Table 2), indicating
a dominate role of this pathway. The production of acid by gas gland cells has
been known for many years 23,37, and analyzing the kinetic properties of lactate
dehydrogenase in nine species of marine fishes Gesser and Fange 22 concluded
that the gas gland enzyme predominantly is the muscle type enzyme, suited for
the formation of lactic acid. Gas gland tissue of various species incubated in vitro
has indeed been shown to produce large amounts of lactate. In experiments using
gas gland of scup, Stenotomus chrysops 2, 70-90% of the glucose taken up from
the incubation medium was balanced by lactate formation, and in the study of
D'Aoust 11 using gas gland of Sebastodes miniatus, glucose was almost completely
converted to lactate. As already observed for the aerobic metabolism, a decrease in
sodium concentration of the incubation medium also caused a decrease in lactate
formation in Stenotomus chrysops gas gland2.
It is also interesting to note that, although enzyme activities of the anaerobic
glycolysis in gas gland are comparable to white muscle, the maximal rate of lactate
formation observed in vitro is several times lower than in white muscle 16. In most
tissues the rate of anaerobic glycolysis decreases very much with increasing oxygen
TABLE 2
Activities of selected enzymes of anaerobic glycolysis in gas gland tissue
Enzyme Anguilla anguilla48 Gadusmorhua Opsanus beta 63

(25~ (10~ (23oc)5 (24oc)
Hexokinase 1.2 4. 0.2 16.13 4- 1.33 1.45 4. 0.11 ND
Phosphofructokinase 10.1 4- 1.3 26.51 4- 1.44 0.71 4- 0 . 0 5 23.964- 2.78
Phosphoglucose isomerase ND ND ND 29.15 4- 1.82
Glyceraldehydephosphate 79.8 4- 1.7 ND ND 29.97 4- 3.52
dehydrogenase
Glycerolphosphate ND ND 4.80+ 0.48 ND
dehydrogenase
Pyruvate kinase 123 4- 22 209.4 4- 23.9 345 4- 25 ND
Lactate dehydrogenase 190 4- 51 1203 4. 99 1154 4- 46 173.8 4. 18.4
Lactate dehydrogenase 46 a 134 a
Activities are given in/zmol min -1 g-I fresh mass (4- S.E.). ND = not determined.
a From reference 22.
t e n s i o n ( P a s t e u r effect). I n c u b a t i o n of gas gland cells with 9 5 % oxygen a n d 5 % CO2

( t e l 2) o r e v e n with h y p e r b a r i c oxygen p r e s s u r e (51 a t m o s p h e r e s ) 11, however, did
n o t result in a r e d u c e d rate of lactate f o r m a t i o n , clearly indicating a b s e n c e of the
P a s t e u r effect.
P r o d u c t i o n o f lactate has b e e n shown in a R i n g e r - p e r f u s e d p r e p a r a t i o n of the
E u r o p e a n eel, but, as o b s e r v e d for Stenotomus chrysops gas gland in vitro 2, it did n o t
c o m p l e t e l y b a l a n c e the c o n c o m i t a n t u p t a k e of glucose, indicating that p a r t of the
glucose was shifted to s o m e o t h e r m e t a b o l i c pathway 46.
In vivo, lactate f o r m a t i o n has only b e e n d e m o n s t r a t e d in two species, n a m e l y
the b a r r a c u d a a n d the E u r o p e a n eel, w h e r e b l o o d samples w e r e o b t a i n e d by b l o o d
vessel p u n c t u r e , b u t b l o o d flow could not be m e a s u r e d . As s h o w n in Table 3
the v e n o - a r t e r i a l c o n c e n t r a t i o n difference varies b e t w e e n 1 a n d 5 m m o l 1-1, a n d
t h e highest lactate c o n c e n t r a t i o n m e a s u r e d in a s w i m b l a d d e r b l o o d s a m p l e was
TABLE 3
Lactate release from gas gland tissue into the blood measured as concentration difference between
venous efltux from (re) and arterial influx into (ai) the swimbladder tissue, and the highest lactate
concentration value measured in blood samples obtained by blood vessel puncture
Species A Lactate (re - ai) Lactatemax

(/zmol m1-1) (/tmol m1-1)
European eel 33 1.3 4- 1.4 15.1
European eel6~ 2.6 4- 1.8 20.9 a
European eel 3~ 2.0 4- 1.5 11.4 a
Barracuda 14 4.4 4- 1.0 12.6
Values are presented as means 4- SD.
a Indicates blood samples obtained from the swimbladder pole of the rete after passage of the gas gland
(vi), where the highest lactate concentration is expected to occur. European eel = Anguilla anguilla L.;
barracuda = Sphyraena barracuda.
108 R Pdster
20.9 mmol 1-1. In a recent study, lactate and glucose balance of the active, gas de-
positing swimbladder of the eel could be measured in situ revealing that about 75-
80% of glucose taken up from the blood were converted to lactate 51. As aerobic me-
tabolism cannot account for the difference in glucose uptake and lactate secretion,
this study again indicates that part of the glucose is shifted to additional metabolic
pathways. A likely candidate appears to be the pentose phosphate shunt 46.
3. Pentose phosphate shunt
Freshly deposited gas may contain remarkable fractions of CO2, sometimes even up
to 30% (ref. 64). Therefore, the assumption that CO2 originates almost exclusively
from the HCO 3 pool of the blood has been questioned, and as the respiratory
exchange ratio of gas gland tissue usually exceeds 1 (see above) aerobic metabolism
cannot be held responsible. Fringe 18 included the pentose phosphate shunt as a
possible origin of CO2 in his schema of gas gland metabolism, but did not present
experimental evidence except for activities of two enzymes of the shunt, measured
in gas gland of the cod 5. In vitro traces of uniformly labeled, but not of C3,4 labeled,
glucose were converted to CO2 by gas gland of Sebastodes miniatus, probably in the
pentose phosphate shunt 11. Glucose uptake of the tissue, however, was completely
balanced by the formation of lactate and the author concluded that the pentose
phosphate shunt was of no significance for the tissue.
Gas gland tissue of various fishes indeed is characterized by the presence of
key enzymes of the pentose phosphate shunt (Table 4), and in European eel
their activity is even comparable to the activity measured in liver tissue, which is
known for the presence of this shunt 48. More direct evidence for the existence of
metabolic pathways generating CO2 without concomitant oxygen consumption was
provided by analyzing the gas exchange and glucose metabolism of saline-perfused
eel swimbladder preparations 46 and of the active, gas depositing swimbladder in
situ 5~ In both preparations, the production of CO2 by the tissue by far exceeded
the rate of oxygen uptake and the glucose uptake could not be balanced by
lactate formation, indicating that part of the glucose was metabolized in additional
metabolic pathways, presumably the pentose phosphate shunt.
In a recent study, the involvement of this pathway in gas gland glucose metabo-
lism could be demonstrated for Opsanus beta 63. The ratio of CO2 formed from C1
TABLE 4
Activities of selected enzymes of the pentose phosphate shunt of gas gland tissue
. . . . . . . .
Enzyme Anguilla anguilla 4s Gadus morhua 5 Opsanus beta 63

. . . . . .
Glucose-6-phosphate dehydrogenase 2.7 4- 0.5 1.99 4- 0.11 2.22 + 0.29

6-Phosphogluconate dehydrogenase 0.7 4- 0.2 0.92 4- 0.07 1.89 4- 0.12
Transaldolase ND ND 0.29 4- 0.04
Transketolase ND ND 0.10 4- 0.02
Activities are given in/tmol min -1 fresh mass (4- S.E.), measured at 23-25"C. ND = not determined.
and C6 labeled glucose reached a value of 3.4 under normoxia and of 6.4 under
hyperoxia, clearly indicating a significant contribution of the pentose phosphate
shunt to glucose metabolism.
4. Synopsis
A summary of the metabolic pathways involved in gas gland metabolism is given

in Fig. 3. Glucose, representing the main fuel, and oxygen are removed from the
blood. The main fraction of glucose is converted to lactate, which in turn is released
into the blood. Part of the glucose is shifted t o the pentose phosphate shunt
and decarboxylated in the 6-phosphogluconate dehydrogenase reaction. Along the
diffusion gradient, the CO2 diffuses into the swimbladder lumen, but also into the
blood 46. Only a very small fraction of glucose is oxidized in the aerobic metabolism,
also forming CO2.
Fig. 3. Present concept for pathways of glucose metabolism and CO2 formation in gas gland cells. AGI
= anaerobic glycolysis; CA = carbonic anhydrase; GA-3-P = glyceraldehyde-3-phosphate; G-6-P =
glucose-6-phosphate; PPS = pentose phosphate shunt; TCA = tricarboxylicacid cycle.
110 B. Pelster
TABLE 5
Contribution of various metabolic pathways to total glucose metabolism and total CO2 production of
gas gland tissue of the European eel, Anguilla anguilla, at 22-24~
Total Lactate Aerobic Pentose phosphate

formation metabolism shunt
Glucose (/zmol min -1) 0.72 0.58 0.007 0.14
CO2 (#mol min -1) 0.20 - 0.041 0.16
Glucose uptake and CO2 formation not accounted for by aerobic or anaerobic glycolysis are listed
under pentose phosphate shunt, although it cannot be excluded that some glucose was converted to
glycogen (see text for further explanations). Data from references 50 and 51.
A quantitative assessment of the contribution of these pathways to glucose me-

tabolism in the eel swimbladder is given in Table 5. According to the oxygen con-
sumption of swimbladder tissue only 1% of the glucose removed from the blood is
completely oxidized in the aerobic metabolism, leaving 99% for anaerobic glycolysis
and the pentose phosphate shunt. Aerobic metabolism can explain only 20% of the
CO2 formation. To account for the additional CO2 about 22% of the glucose has
to be decarboxylated in the pentose phosphate shunt, leaving the main fraction of
glucose metabolism for anaerobic glycolysis. Depending on the reaction sequence,
glucose shifted to the pentose phosphate shunt may, after decarboxylation in the 6-
phosphogluconate dehydrogenase reaction, finally also be converted to lactate if the
pentose is metabolized to glyceraldehyde-3-phosphate and thus enter the glycolytic
pathway (Fig. 3). Nevertheless, these results clearly demonstrate that the main frac-
tion of glucose is fermented directly in the anaerobic glycolysis because glucose
uptake from the blood by far exceeds the formation of CO2 in the tissue.
IV. Metabolism and gas deposition: the single concentrating effect
The importance of gas gland metabolism for swimbladder function can be demon-
strated by plotting the rate of lactate formation or the acidification of the blood
during passage of the gas gland tissue versus the rate of gas deposition: gas depo-
sition increases with increasing acidification of the blood as well as with increasing
lactate formation (Fig. 4). Lactic acid and CO2 are released from gas gland cells and
in blood initiate the single concentrating effect, the first step in the generation of
high gas partial pressures and in gas deposition, for inert gases and also for oxygen
and CO2.
The increase in blood lactate concentration reduces the physical solubility of
gases in blood via the salting-out effect, which is of special importance for inert
gases. The increase in lactate concentrations measured in vivo ranges between 1
and 5 mmol 1-1 with maximum lactate levels of about 21 mmol 1-1 (Table 3),
resulting in a solubility reduction of only 1% or even less45,47, but nevertheless may
be sufficient to allow for a 10- to 15-fold increase in inert gas partial pressure in the
counter-current system of the rete mirabile31.
Fig. 4. Correlation between blood acidification, represented by the ven0-arterial pH differenceS~

or lactate release by the gas gland cellssl (bottom) and the rate of gas deposition in the swimbladder of
the European eel (Anguilla anguilla).
Oxygen in blood is bound to hemoglobin, and protons reduce the oxygen affinity
of the hemoglobin (Bohr effect). Hemoglobin of fish with a swimbladder usually
is characterized by the Root effect, that is by a reduction in oxygen-carrying
capacity with increasing proton concentration 57. At low pH, Root hemoglobins
are fixed in the deoxygenated state and do not bind oxygen even at high oxygen
tensions 52 56 The release of lactic acid and of CO2 from gas gland cells acidifies
. .
the blood and thus provokes a release of oxygen from the hemoglobin via the
Root effect. According to the pH values measured in swimbladder blood about
40% of the hemoglobin may be deoxygenated during passage of the swimbladder
112 B. Pelster
tissue, resulting in a tremendous increase in gas partial pressure s2. In the rete
mirabile this large single concentrating effect may be multiplied v/a back-diffusion
and counter-current concentration, allowing for the generation of a gas pressure of
several hundred atmospheres 31,34,62.
V. Control of swimbladder metabolism

Gas gland metabolism is of pivotal importance for swimbladder function and with
increasing acid production and acid release of the cells, the rate of gas deposition
increased 5~ while inhibition of lactate production by injection of oxamie acid, an
inhibitor of lactate dehydrogenase, into the bladder drastically decreased the rate
of gas deposition 11. The description of 'active' and 'inactive' gas glands in the
literature 17,1s,26 and the significantly reduced rate of acid secretion under hypoxic
conditions in the eel swimbladders~ clearly indicate the presence of an effective
control system.
Cutting the vagus abolishes the deposition of gas, and the swimbladder metab-
olism thus appears to be under vagal control 4.zl,3s. The presence of aeetylcholine
esterase in gas gland tissue and the inhibitory effect of atropine on gas deposition
may indicate that cholinergic fibers are involved, adrenergic fibers could not be
detected 1,2~ Attempts to stimulate gas deposition by parasympathicomimetic
drugs or by electrical stimulation of the vagus, however, have been unsuccessful 17.
Very little is known about control mechanisms at the cellular level. In gas gland
of the bluegill sunfish (Lepomis macrochirus) presence of glucagon in the incubation
medium stimulated the formation of lactate from internal glycogen stores 12. The
concentration of glucagon applied, however, appears to be quite high (30 mg 1-1),
and the main fuel for metabolism is blood borne glucose and not the internal
glycogen stores (see above). Epinephrine has been shown to have n o effect on
lactate production in bluegill sunfish 12, while a significant stimulation has been
observed in cod gas gland in vitro 16. The possible importance of epinephrine for
regulation of the metabolism is not obvious because adrenergic fibers have not
been detected in gas gland tissue, and a stimulatory effect on metabolism would
counteract the well known inhibitory effect of catecholamines on swimbladder
perfusion 19.
The important feature of the metabolism is the production of acid (lactic acid
and CO2) and its release into the blood stream. An interesting question is the
mechanism of acid release and its implications on the metabolism. Analysis of
the acid base changes in blood during passage of the gas gland tissue revealed
that proton extrusion mainly occurs via CO2 after dehydration of HCO~, probably
with recycling of the HCO 3 mediated by an anion exchanger (Fig. 3). Only about
25% of the total acidification of the blood can be ascribed to proton release 32. If
proton release occurs along a proton gradient, the intracellular pH must be low
because pH values of 6.5-7.0 have been measured in the blood 32,6~ A high buffer
capacity of gas gland cells63 will provide stability of the internal milieu, but will
not change possible gradients for proton extrusion. At low pH, glycolytic activity is
Metabolism of the swimbladdertissue 113
depressed in most tissues, but Ewart and Driedzic ~6 reported a 3-times higher rate
of lactate production at pH 6.5 compared to pH 7.8 at 10*C for cod gas gland, which
may indicate special adaptations of glycolytic enzymes to Operate at low pH. A
comparison of white muscle and gas gland phosphofructokinase, however, revealed
no difference in the pH optimum 48.
Beside the acid, the gas gland metabolism also has to cope with a high tension
of oxygen. Presence of oxygen usually diminishes or even abolishes the production
of lactate in the anaerobic glycolysis, which would impair gas deposition into the
swimbladder. Accordingly, gas gland tissue does not show the Pasteur effect 11.
Control site usually is the enzyme phosphofructokinase and one may speculate
that this step is by-passed in glucose metabolism by shifting glucose towards the
pentose phosphate shunt and entering the glycolytic pathway at the glyceraldehyde-
3-phosphate level (Fig. 3). The low activities of the enzymes transaldolase and
transketolase are not in favor of this hypothesis63 (Table 4), and the dominating role
of anaerobic glycolysis has been demonstrated by the comparison of glucose uptake
and CO2 formation (see above).
With the pentose phosphate shunt contributing no more than 20-25% to glucose
metabolism, we need to look for control mechanisms in the anaerobic glycolytic
pathway and one possibility appears to be that the decrease in phosphofructokinase
activity typically observed on oxygenation is prevented by the action of some
regulatory cofactor. The inhibitory effect of increasing ATP concentrations on the
gas gland enzyme is not different from the effect on the white muscle enzyme48,
but cellular ATP levels in vivo are unknown and there are other cofactors or
metabolites, like citrate or ADP, which could be of importance.
VI. Role o f carbonic anhydrase
Based on biochemical measurements, the presence of carbonic anhydrase in swim-

bladder epithelium has been known for a long time 11,17,39,59. Histological analysis
also demonstrated the presence of carbonic anhydrase in the swimbladder epithe-
lium of the eel, and the staining was especially intense in the basal region of the
cells 13. More detailed studies on the localization are not yet available and it is un-
known whether a membrane bound enzyme, perhaps facing the extracellular space,
is present.
Results demonstrating the presence of carbonic anhydrase in the rete mirabile
are equivocal. Biochemical studies indicate presence of the enzyme in rete
membranes 17,39,s9. The very high activity of carbonic anhydrase in red cells, how-
ever, renders the measurement of enzyme activities in tissue homogenates very
sensitive to contaminations with erythrocytes, and histological studies failed to
demonstrate the enzyme in rete membranes 13.
The significance of carbonic anhydrase for swimbladder function has been
verified in a number of studies: inhibition of the enzyme by injection of sulfonamide
into the bladder decreases gas deposition 17'39.59. Kutchai 35 observed a decrease
in lactate production after inhibition of carbonic anhydrase, but an interpretation
114 B. Pelster
Fig. 5. Possible pathways for proton transfer from gas gland cells to the erythrocytos and the role of
carbonic anhydrase. CA = carbonic anhydrase; Hb = hemoglobin.
of his results is somewhat difficult because at a concentration of 10-4 mol 1-1,

employed in this study, acetazolamide is known to inhibit anion exchange.
Figure 5 presents a possible sequence of reactions elucidating the role of
carbonic anhydrase for swimbladder function. To initiate the single concentrating
effect, protons, generated in the anaerobic metabolism, must be excreted into the
blood. Lactic acid formation clearly dominates the formation of CO2 in the pentose
phosphate shunt, and an increase in proton concentration in the gas gland cells will
shift the equilibrium of the reaction
H + + HCO~ ~x - CO2 + H20 (1)
towards formation of CO2. Uncatalyzed this reaction is very slow, but presence
of carbonic anhydrase in the cell results in an immediate equilibration, providing
the highly diffusive gas CO2. CO2 easily crosses cell membranes and enters the
erythrocytes. In the red cells, the reverse reaction occurs, again catalyzed by
carbonic anhydrase, liberating the H +, which in turn will liberate oxygen from the
hemoglobin via the Root effect. HCO~ returns to the plasma in exchange for 121-
by the Band III anion exchanger. To avoid a HCO]" depletion of the gas gland cells,
the molecule must be recovered from the plasma, closing the circle. Entry of the
negatively charged HCO]" into the cell may at least partly compensate for the loss of
negative charges due to the extrusion of lactate into the blood. Another possibility
for lactate release would be non-ionic diffusion as lactic acid, which appears to be
not very likely24.
VII. Metabolism ofrete capillaries

The common eel has a bipolar rete mirabile, allowing access to blood vessels
proximal and distal to the rete as well as complete isolation of the rete mirabile.
Rasio 55 took advantage of this morphology and analyzed the glucose metabolism
of isolated rete mirabile preparations. Anaerobic glycolysis appears to be the main
root for glucose metabolism and lactate formation accounted for more than 95% of
the glucose uptake of about 0.4 mmol g-1 rain-l, 1.9% was converted to CO2, and
less than 1% incorporated into glycogen or lipids. The ratio of 1.8 for CO2 formed
from C1 and C6 labeled glucose indicates the presence of the pentose phosphate
shunt in rete cells, although not as pronounced as in gas gland tissue. The main
fraction of CO2 appears to originate in the aerobic metabolism as indicated by
the decrease in CO2 formation under cyanide inhibition of cytochrome oxidase.
Elevated glucose concentrations in the incubation medium, stimulated all pathways
of glucose metabolism, and especially the incorporation into glycogen and lipids5s.
The metabolism with predominance of anaerobic glycolysis and presence of the
pentose phosphate shunt thus shows some similarities to gas gland metabolism, and
enzyme activities measured in the rete mirabile indeed are similar to those reported
for gas gland tissue 4a.
Fill. Antioxidants
Oxygen being the main gas in the swimbladder of most fishes, tissues of this
organ are exposed to almost constant hyperoxia and probably face the highest
oxygen tensions ever encountered in nature. Most tissues are highly sensitive to
hyperbaric oxygen and for lung tissue, for example, development of edema and
proliferation of certain cells have been described during exposure to only one or
two atmospheres of oxygen, finally resulting in a loss of the respiratory function 1~
The swimbladder epithelium obviously is not severely affected by hyperoxia and
it would be interesting to know whether special metabolic adaptations avert the
damage usually caused by oxygen radicals.
Activities of enzymes involved in the breakdown of oxygen radicals like catalase,
superoxide dismutase and glutathione peroxidase have been measured in the gas
gland tissue of a number of fishes. Catalase and glutathione peroxidase activity were
low and not elevated compared to other tissues, only superoxide dismutase activ-
ity was significantly higher than in other tissues and the activity increased during
exposure to hyperoxic conditions 43 '44 . In eel swimbladder epithelium, glutathione
reductase activity has also been detected, while it was not measurable in muscle tis-
sue (B. Pelster and P. Scheid, unpublished result). The swimbladder of trout (Salmo
116 B. Pelster
trutta) also appears to contain antioxidant mechanisms which inhibit peroxidative

oxidation of polyunsaturated fatty acids 7.
The importance of the pentose phosphate shunt for the formation of CO2 has
already been discussed, it may also be of great significance for the degradation
of oxygen radicals. In some reactions of this pathway NADPH is formed, which
has to be reoxidized so as not to stop the shunt. Radical-oxidizing enzymes like
glutathione reductase, however, use NADPH as a coenzyme, and the presence of
the pentose phosphate shunt thus may also be linked to the defense from oxygen
toxicity. In lung tissue the activity of the pentose phosphate shunt increases under
hyperoxia 3,2s. In gas gland of the toadfish Opsanus beta, the ratio of CO2 formed
from C1 and C6 labeled glucose was about twice as high under hyperoxia than under
normoxia, clearly indicating an increased contribution of the pentose phosphate
shunt to glucose metabolism under hyperoxic conditions 63 and proving its dual role
for swimbladder function.
IX. References
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of fishes. Acta Physiol. $cand. 22: 224-230, 1951.
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Biol. Bull, 108: 1-17, 1955.
3. Bassett, D.J.P. and A.B. Fisher. Glucose metabolism in rat lung during exposure to hyperbaric 02.
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5. Bostr6m, S.L., R. Fiinge and R.G. Johansson. Enzyme activity patterns in gas gland tissue of the
swimbladder of the cod (Gadus morthua). Comp. Biochem. Physiol. 43B: 473-478, 1972.
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8. Copeland, D.E. The stimulus of the swimbladder reflex in physoclistous teleosts. J. Exp. Zool. 120:
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mikroskopische Untersuchungen. Z. f. Zellforschung 55: 849-912, 1961.
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gas and oxygen.Adv. Biol. Med. Phys. 11: 231-244, 1967.
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Hochachka and Mommsen (eds.), Biochemistryand molecularbiology offishes, vol. 4
CHAPTER 6
Glycerophospholipid metabolism
DOUGLAS R. TOCHER
NERC Unit of Aquatic Biochemistry, School of Natural Sciences, University of Stirling, Stirling FK9
4LA, Scotland, U.K.
I. Introduction
II. Biosynthesis, turnover and catabolism
III. Digestion, absorption and transport
1. Digestion and absorption
2. Transport
2.1. Transport from intestine to liver
2.2. Transport between liver and extra-hepatic tissues
2.3. Vitellogenin
IV. Composition
1. Content
2. Head group composition
3. Fatty acyl composition
3.1. Total glycerophospholipids
3.2. Glycerophospholipid classes
4. Molecular species
5. Dietary effects
6. Adaptation to environmental factors
6.1. Temperature
6.2. Salinity and hydrostatic pressure
V. Roles
1. Structural roles
2. Metabolic roles
2.1. Eicosanoid metabolism
2.1.1. Species and tissue distribution of eicosanoids in fish
2.1.2. Range of eicosanoids in fish
2.1.3. Stimuli for production of eicosanoids
2.1.4. Fatty acid precursors of eicosanoids
2.1.5. Glycerophospholipid sources of precursor fatty acids
2.1.6. Functions of eicosanoids
2.2. The phosphoinositide cycle
2.3. Other metabolism
2.3.1. Protein kinase C
2.3.2. Platelet-activating factor
3. Nutritional roles
3.1. Embryonic development
3.2. Larval diet
VI. Conclusions and perspectives
VII. References
120 D.R. Tocher
I. Introduction
Glycerophospholipids are the major class of complex lipids characterized by a

glycerol backbone with one of the primary hydroxyl groups (sn-3) esterified to
phosphoric acid (Fig. 1). The secondary hydroxyl group in glycerophospholipids (sn-
2) is always esterified to a long-chain fatty acid, which in the majority of instances
is monounsaturated or polyunsaturated. The sn-1 hydroxyl group is most commonly
esterified to another fatty acid, generally saturated or monounsaturated, forming
the diacyl glycerophospholipids (or phosphoglycerides). However, the sn-1 position
can also contain a long aliphatic chain in c/s adS-unsaturated ether linkage in the
case of the plasmalogens (alk-l-enyl acyl derivatives), or a saturated aliphatic chain
in simple ether linkage (alkyl acyl derivatives) (Fig. 1).
Phosphatidic acid (PtdA), a quantitatively minor glycerophospholipid, is nonethe-
less an important intermediate in the biosynthesis of glycerophospholipids and can
be regarded as the simplest. In PtdA the glycerol backbone is esterified to two fatty
acids and phosphoric acid (Fig. 1). The quantitatively important glyeerophospho-
lipids contain nitrogenous bases, esterified to the phosphoric acid, such as choline
(phosphatidylcholine, PtdCho), ethanolamine (phosphatidylethanolamine, PtdEtn)
and serine (phosphatidylserine, PtdSer) or polyalcohols, such as inositol (phos-
phatidylinositol, Ptdlns) or glycerol (phosphatidylglycerol) (Fig. 1). Cardiolipin
CH2-O-H CH2-O-CO-RI
I I
CH-O-H CH-O-CO-R2
I I
CH2-O-H CH2-O-H
Glycerol Diacylglycerol(DAG)
CH2-O-CO-RI CH2-O-CO-RI
I I
CH-O-CO-R2 CH-O-CO-R2
I I
CH2-O-P-O-H CH2-O-P-O-X
Phosphatidic acid (PtdA) Diacyl glycerophospholipid
CH2-O-R1 CH2-O-C-C-R1
I I
CH-O-CO-R2 CH-O-CO-R2
I I
CH2-O-P--O-X CH2-O-P-O-X
1-O-alkyl-2-acyl-glycerophospholipid 1-O-alk-1'-enyl-2-acyl-glycerophospholipid
R1 Long-chainaliphatic group, usuallysaturated or monounsaturated
- -
R2 - Long-chain aliphatic group, usuallypolyunsaturated or monounsaturated

P -- PO2H
X - choline (-CH2CH2N+(CH3)3), ethanolamine (-CH2CH2NH~),
serine (-CH2CTI(NH+)COO-), myo-inositol(-C6H11O5),
glycerol (-CH2CH(OH)CH2OH) or phosphatidylglycerol.
Fig. 6.1. Basic structures of the glycerophospholipids,their precursors and head groups.
Glycerophospholipidmetabolism 121
(diphosphatidylglycerol) is formed when the phosphate groups of two phospha-

tidic acid molecules are bridged by a third glycerol moiety esterified at the 1 and
3 positions. All the glycerophospholipids are important components of biological
membranes and are not found in high concentrations elsewhere in the cell.
Glycerophospholipids, phosphoglycerides and phospholipids are notsynonymous
terms although the terminologies are still used in a confused manner today. Not all
glycerophospholipids are phosphoglycerides, as the term phosphoglyceride should,
strictly speaking, be reserved for the diacyl derivatives alone and should not include
the ether-linked derivatives. Similarly, not all phospholipids are glycerophospho-
lipids. For instance, sphingomyelin contains phosphorus and so is a phospholipid
but the phosphoric acid is esterified to a sphingosine backbone and not glycerol
and so is correctly termed a sphingolipid. This chapter deals exclusively with the
metabolism of the major classes of glycerophospholipids (PtdCho, PtdEtn, PtdSer
and Ptdlns), including the ether-linked derivatives, although, due to the dearth of
information on the metabolism of the ether-linked derivatives in fish, the focus
will be on the diacyl derivatives. I have endeavored to maintain this nomen-
clature throughout this chapter, but there are many instances where the use of
phospholipid rather than glycerophospholipid has been more appropriate in the
discussion of previous work and therefore for simplicity the term 'phospholipid' is
often used.
II. Biosynthesis, turnover a n d catabolism
The pathways of glycerophospholipid biosynthesis have not been extensively studied

or elucidated in fish83. However, the existing evidence strongly suggests that the
same pathways operate in fish as in mammals. Holub et al. 1~176 demonstrated the
existence of glycerol-3-phosphate acyltransferase in the liver of rainbow trout (On-
corhynchus mykiss). When liver microsomes were incubated with sn-[U-14C]glycerol-
3-phosphate in the presence of activated fatty acid, palmitoyl-CoA, 77% of the
radioactivity was recovered in total glycerophospholipids with the remainder re-
covered in neutral lipids. PtdA and lysoPtdA were also labeled, supporting the
conclusion that glycerophospholipid and lipid biosynthesis in general proceeded via
a PtdA intermediate in fish.
The presence of cytidine diphosphate (CDP)-choline-l,2-diacylglycerol choline
phosphotransferase has been demonstrated in the microsomes of trout liver 1~
and brain and liver from goldfish (Carassius auratus) 129. The synthesis of Ptd-
Cho from 14C-CDP-choline and 1,2-diacylglycerol (diolein) in the presence of
Mg2+established that the CDP-choline pathway for the biosynthesis of PtdCho, as
studied in detail in mammals, also operated in fish 101'129. There have been few
studies in fish to fully characterize the biosynthetic pathways for PtdCho, PtdEtn,
PtdSer, PtdIns and cardiolipin or the pathways, known in mammals, for intercon-
version between the glycerophospholipids. However, in a recent study, the de novo
pathways of glycerophospholipid biosynthesis were investigated in trout hepatocytes
and the activities of CDP-choline and CDP-ethanolamine phosphotransferases,
122 D.R. Tocher
PtdEtn-methyltransferase (PtdEtn ~ PtdCho) and PtdSer-decarboxylase (PtdSer

PtdEtn) were demonstrated 9~
In mammals, ether-linked glycerophospholipids are formed solely via the dihy-
droxyacetonr phosphate pathway (see ref. 194). Briefly, fatty alcohol is formed
by the NADPH reduction of fatty acyl-CoA. Fatty alcohol then reacts with fatty
acyldihydroxyacetone phosphate to form alkyldihydroxyaeetone phosphate which
is then reduced, specifically with NADPH, to form alkylglycerophosphate. The
enzymes responsible for the synthesis and reduction of alkyldihydroxyacetone phos-
phate are located in the peroxisomes. Reaction with fatty acyl-CoA, removal of
phosphate and reaction with CDP-base results in the formation of alkyl glyc-
erophospholipid. Alk-l-enyl acylglycerophospholipids (plasmalogens) are formed
by oxidation of the corresponding alkyacylglycerophospholipid by a microsomal
enzyme requiring NADPH and molecular oxygen. Little of the above pathway
has been characterized in fish, but the available evidence from studies with spiny
dogfish (Squalus acanthias) appears to suggest that the biosynthesis of ether-linked
glycerophospholipids in fish is via a pathway similar to that outlined above (see
ref. 194).
A review of muscle lipase activities in various fish species indicated that the cat-
alytic hydrolysis of phospholipids was primarily under the control of phospholipases
A1 and A2 205. Intracellular phospholipase A activities have been demonstrated in
muscle tissue from rainbow trout 112, pollock (Gadus pollachius) 11, winter flounder
(Pseudopleuronectes americanus) z~ and Atlantic cod (Gadus morhua) 48. Neas and
Hazel 154-156 studied the activity of phospholipase A2 towards PtdCho in the micro-
somes of trout liver. Activity of phospholipase C has been demonstrated directly
in isolated olfactory cilia from the channel catfish (Ictalurus punctatus) 39 and has
been implicated indirectly in other tissues by the demonstration of a phosphoinosi-
tide cycle (see section V.2.2)1~176 but it appears that phospholipasr D activity has
not been investigated in fish. Holub et al. ~02 showed that trout liver microsomes
also contained acylCoA: 1-acyl-sn-glycero-3-phosphorylcholine acyltransferase ac-
tivity. Therefore, enzymes required for partial catabolism of glycerophospholipids
and for the reacylation of lyso-glycerophospholipids, and thus for the turnover of
glycerophospholipids, have been demonstrated in fish.
Catabolism of ether-linked glycerophospholipids hinges on the cleavage of the
ether bond. Enzymic cleavage of the O-alkyl bond has been demonstrated in fish 194.
The cleavage is considered to occur in two steps, whereby the alkyl bond is first
oxidized to alk-l-enyl via a reaction involving NADPH, molecular oxygen and a
pteridine cofactor, before cleavage to generate fatty aldehyde 194. The above enzyme
system has yet to be directly studied in fish, and so it is not known if it will also
cleave plasmalogens. Plasmalogenases, as described in mammalian brain, do not
appear to have been studied in fish 194.
The specificities of the enzymes involved in both de novo synthesis of the glyc-
erophospholipids and in the turnover processes of deacylation/reacylation with
respect to both head group and fatty acyl chains have important consequences in
maintaining the normal glycerophospholipid class composition, the fatty acyl dis-
tribution among the glyeerophospholipids, and in the adaptation to environmental
Glycerophospholipid metabolism 123
changes. Some of the more direct enzyme studies, discussed above, addressed this
problem in relation to environmental temperature 1~176 However, there has
been a considerable amount of data obtained from more indirect studies of the
effects of environment on glycerophospholipid metabolism and this is summarized
later (see section IV.6).
III. Digestion, absorption and transport
I. Digestionand absorption
Depending upon the precise nature of the diet, a significant and potentially large
and consistent portion of the lipid component in the natural food of fishes will
be biomembrane lipids, primarily glycerophospholipids. Unfortunately, the lack
of a discrete pancreas in most teleost species has hampered studies on intesti-
nal lipolysis in fish. In consequence, even less is known about the digestion
and absorption of dietary glycerophospholipids than is known about the biosyn-
thetic pathways. There are virtually no studies on the intestinal digestion of
glycerophospholipids in fish, but it could be presumed that the mechanisms are
similar to those in mammals. Therefore dietary glycerophospholipids are presum-
ably digested by pancreatic or intestinal phospholipases resulting in the forma-
tion of 1-acyl lyso-glycerophospholipids and free fatty acids that are absorbed by
the intestinal mucosal cells 96'198. Mankura et al. studied the hydrolysis of L-1-
palmitoyl-2-[1-14C]arachidonyl-3-sn-glycerophosphatidylcholine by carp hepatopan-
creas preparations TM. They found phospholipase A2 activity distributed in all the
subcellular fractions, although the highest activity was located in the 10,000 g su-
pernatant. The activity was dependent upon Ca 2+ and bile salt, consistent with a
pancreatic enzyme, but had a conflicting acidic pH optimum of 5.0 TM.Whether this
phospholipase activity reflects an intestinal activity or an intracellular phospholipase
is, therefore, unclear. Recent work has suggested that cod pyloric caeca/pancreas
contains a single, bile salt-activated, lipase activity with a wide substrate speci-
ficity including triacylglycerols, steryl esters, fatty acid methyl esters and carboxyl
esters 79,8~ Whether this enzyme is also active towards phospholipid and whether
cod intestine actually lacks phospholipase A2 activity is unclear.
The concentration of lyso-glycerophospholipids is very low in fish plasma and
so it has also been assumed that the majority of lysophospholipid is re-esterified
within the intestinal mucosa before export into the circulatory system. However,
studies on the incorporation of [1- 14C]palmitate and [U-14C]L-glycerol-3-phosphate
into lipids in carp (Cyprinus carpio) intestinal homogenates in the presence of CTP,
CDP-choline and CDP-ethanolamine showed that glycerophospholipid biosynthe-
sis proceeded via PtdA and diacylglycerol (DAG) intermediates 11~ Therefore,
mechanisms may exist in fish intestinal mucosa for the synthesis of glycerophos-
pholipids from moieties more degraded than lyso-glycerophospholipids. Iijima and
coworkers l~ have also studied the absorption of radioactivity from [l:4C]dioleoyl
PtdCho force-fed to carp. At 20-28 h after dosing, radioactivity in the lipids of
124 D.R. Tocher
plasma lipoproteins was primarily associated with triacylglycerols followed by Ptd-

Cho and free fatty acids l~ The radioactivity associated with the sn-1 position of
PtdCho was more than twice that associated with the sn-2 position 1~ These data
support the hypothesis that the majority of glycerophospholipids are digested and
absorbed v/a 1-acyl lyso-glycerophospholipid intermediates with re-estedfication
before export from the intestinal cells.
Z Transport
There are several reviews that between them cover the area of lipid transport in
fish very thoroughly 16,66,n6,2~ This section will give an overview of the subject
particularly focusing on the role of glycerophospholipids in lipoprotein structure
and function without major consideration of the apoprotein constituents.
2.1. Transport from intestine to liver

As in mammals, glycerophospholipids are transported from the intestine in the form
of lipoproteins. The re-esterification reactions occur primarily in the endoplasmic
reticulum leading to the production of chylomicron-like and very low density
lipoprotein (VLDL)-like particles in the lumen as observed in carp 162, tench (Tinca
tinca) 163 and trout 1s,2~176 Studies in trout showed that lipid load and degree of
unsaturation affected the relative production of the lipoproteins, with high dietary
lipid and polyunsaturated fatty acids (PUFA) leading to the production of larger
chylomicrons, whereas high dietary saturated fatty acids resulted in the production
of smaller VLDL particles 2~ The effects of glycerophospholipid content and
composition on the production of intestinal lipoproteins has not been studied.
However, as trout chylomicrons contain about 8% phospholipid and trout VLDL
contains approximately 21% phospholipid ~,67,69,211, it is possible that variable
proportions of dietary phospholipid could be accommodated by varying the relative
proportions of the intestinal lipoproteins produced.
In mammals, the intestinal lipoproteins are transported from the intestine almost
exclusively v/a the lymphatic system. It appears that in fish such as trout and
tench the majority of the intestinal lipoproteins are similarly transported via the
lymphatic system31,32,163,21~before appearing in the circulatory system47'204,212 and
delivery to the liver. In carp, however, it was shown that intestinal lipoproteins may
be transported exclusively and directly to the liver v/a the portal system 162. This
pathway may also operate for a portion of intestinal lipoproteins in other fish such
as trout and tench.
2. 2. Transport between liver and extra-hepatic tissues

Due to their amphipathic nature, glycerophosholipids are integral components of
the plasma lipoproteins responsible for the transport of the neutral lipid classes such
as triacylglycerol and steryl esters that are insoluble in aqueous solvents. As well
as chylomicrons and VLDL, mammalian plasma also contains low density lipopro-
teins (LDL), intermediate density lipoproteins (IDL) and high density lipoproteins
(HDL) 13~ The lipoproteins vary in size and structure, as well as in protein'lipid
ratios and in the relative proportions of the different lipid classes leading to the
density differences which have been used traditionally to separate and classify the
different types 13~ Fish plasma contains a similar range of lipoproteins 16,66,2~ Al-
though there are differences in detail, the general size, structure and composition
of the plasma lipoproteins are comparable throughout the vertebrates, including
fish 46. In most fish virtually all glycerophospholipid is transported in the plasma in
the form of lipoprotein but analysis of an albumin-like protein from carp plasma
showed that it contained 22% lipid of which 15% was phospholipid, predominantly
PtdCho 151. This accounted for approximately 50% of the total plasma lipid in carp,
with the remainder being transported via the lipoproteins ~5~
Total phospholipids account for 8, 21, 25 and 29% of the total weight of chylomi-
crons, VLDL, LDL and HDL, respectively47,67,69,211. However, as a percentage of
the total lipids, the proportion of total phospholipids in trout lipoproteins ranges
from under 9% in chylomicrons to 23% in VLDL, 35% in LDL and 53% in
H D L 47'67'69'211. Similar levels of phospholipids were found in serum VLDL, LDL
and HDL from Pacific sardine (Sardinops caerulea) 125, HDL from pink salmon (On-
corhynchus gorbusha) 158 and HDL from chum salmon (Oncorhynchus keta) 8. Most
of the above studies were performed with fed fish. Iijima et al. 1~ compared the
lipid composition of carp plasma lipoproteins under starved and fed conditions. The
carp lipoproteins contained proportionally more phospholipid than trout, salmon
or sardine lipoproteins with the total lipid from VLDL, LDL and HDL containing
30, 58 and 82% phospholipid, respectively, in fed fish 1~ The proportion of phos-
pholipid in the total lipid was not significantly altered by starvation in the carp 1~
In a later study, lijima et al. l~ studied the absorption and transport of radioactivity
from [1-14C]dioleoyl PtdCho fed to carp. At 20-28 h after dosing, radioactivity was
primarily associated with HDL followed by LDL and VLDL 1~
Chylomicrons are produced exclusively in the intestine, but although some VLDL
can also be synthesized in the gut as described above, the majority of VLDL in
the plasma is synthesized in the liver, at least in rainbow trout ~aa,2~ The major
enzymes of lipoprotein metabolism and remodelling, including lipoprotein lipase
(LPL) and hepatic lipase (HL), have been shown in trout and cod tissues 34,36.
Lecithin:cholesterol acyl transferase (LCAT), a plasma enzyme which catalyzes the
esterification of cholesterol using fatty acid from PtdCho, has been demonstrated
in the plasma of trout 35, carp 11a and char (Salvelinus alpinus) 57. Lecithin:alcohol
acyltransferase, which catalyzes the transfer of an acyl group from PtdCho to long-
chain alcohols has been shown in carp plasma 139 and may be, at least partially,
responsible for the surprisingly high level of circulating wax ester reported in that
species 139,14~ In addition, intermediate density lipoprotein (IDL), a fraction with
density between that of VLDL and LDL has been fractionated from trout serum 14.
The presence of the whole spectrum of lipoproteins, including IDL, and the
enzymes described above strongly suggests that lipoprotein remodelling processes,
as characterized in mammals, also occur in fish. Therefore, triacylglycerols in
chylomicrons and VLDL are hydrolyzed by LPL and HL at tissue sites with
the hydrolysis products being absorbed. Excess surface constituents, including
phospholipids, 'bud off' as nascent HDL particles (similar to HDL3), which can
126 D.R. Tocher
also be secreted by the liver. HDL3 can take up free cholesterol from peripheral
tissues which is then esterified by the action of LCAT resulting in the production
of mature HDL (HD~). Remnants of ehylomierons and VLDL hydrolysis can be
taken up by the liver, but further action by LPL and HL leads to the formation
of LDL via IDL.
As in mammals, the relative proportions of the plasma lipoproteins in fish can
vary from species to species, but is a constant characteristic of each species depend-
ing upon dietary status. In trout, HDL is the predominant class ranging from 0.5-2.3
g/dl, followed by LDL (0.2-1.1 g/dl) and then VLDL (0.1-0.7 g/dl) 47,67,69,211. HDL
was also the main lipoprotein class in carp 6, sea bass (Dicentrarchus labrax) 193, pink
salmon (O. gorbusha) 158, chum salmon ls2 and channel catfish 136. HDL appeared to
be absent from the plasma of carp 15~ The relative amounts of HDL, LDL and
VLDL vary with age, nutrition and sexual cycle66.
In common with most vertebrates, PtdCho appears to be the predominant
glycerophospholipid class in fish lipoproteins 46,158. However, the precise phos-
pholipid class composition in fish plasma lipoproteins has been rarely reported
in the above studies. Similarly, the precise fatty acid compositions of individ-
ual glycerophospholipid classes have not been extensively studied. The fatty acid
compositions of total lipids and total phospholipids have been reported. Fish
lipoproteins generally contain higher levels of PUFA, particularly (n-3)PUFA, than
the corresponding mammalian lipoproteins ~i,nT.158,211,2n. In trout the phospholipid
fractions from all lipoproteins were particularly rich in 16:0 and 22:6n-3, and total
(n-3)PUFA was higher and total (n-6)PUFA lower, in phospholipids in comparison
with triacylglycerols47'67'69'127. However, the levels of (n-3)PUFA in the cholesteryl
ester fractions of LDL and HDL exceeded those of the phospholipids 47,67,69,127. The
exact fatty acid composition of the plasma lipoproteins were affected by diet, both
acutely, particularly with chylomierons and VLDL after a meal, and chronically as
seen with essential fatty acid-deficiency in trout 7~
The apoprotein compositions of fish lipoproteins are similar to mammalian
lipoproteins with the major apoproteins being apoprotein A (I and II) in HDL,
apoprotein B in LDL and mixtures of apoproteins B, C, and E in VLDL and A, B
and C in chylomicrons6,13,15,16,'ui'47,136,ls8,lsS'211.There are few direct studies on the
functions of the different apoproteins in fish, but it is likely that they have the same
metabolic functions such as receptor binding (apoproteins B and E) and enzyme
activation (AI and LCAT; CII and LPL) as in mammalian systems~37. Consistent
with this, it was shown that trout adipose tissue LPL was activated by the apoprotein
fraction of trout HDL (mainly AI and C)ss.
There are virtually no reports of studies on the uptake of phospholipid from
the plasma lipoproteins in fish. However, by analogy with the system characterized
in mammals, phospholipids can probably be taken up into the tissues by two or
three main mechanisms 137. Probably the most important pathway, quantitatively,
is receptor-mediated endocytosis via B/E and E receptors. These are important
pathways for LDL (apo B), VLDL- and ehylomieron-remnants (apo B and E) and
HDL (apo E, especially in HDL1, an apo E-rich variant). These receptors are
found on various tissues including liver. In mammals the precise tissue distribution
varies between different species. Probably all the lipoproteins, but particularly LDL
and HDL, can be taken up by tissues via non-specific pinocytosis. For instance, in
liver approximately 30% of LDL uptake is via a non-receptor-mediated pathway.
Finally, it may be that surface components of VLDL and chylomicrons, including
phospholipids, may be taken up or exchanged via direct interaction with the
endothelial cell membranes in the tissues.
2.3. Vitellogenin (very high density lipoprotein)

Another lipoprotein class in fish important in the transport of phospholipids
is vitellogenin which is only found in mature oviparous females or estrogen-
injected fish 43,55,166,217,248. Vitellogenin has a density higher than HDL and has
been designated very high density lipoprotein I (VHDL 1)149 and contains about
80% protein and 20% lipid in rainbow trout 43,7~ sea trout (Salmo trutta) 166
and goldfish 61,1~ The lipid is predominantly phospholipid (about 65-70% of total
lipid) 7~176 and rich in (n-3)PUFA 7~ particularly 22:6n-3, which accounts
for 20% of total fatty acids in trout vitellogenin 127. There are few data on the
detailed class compositions of fish vitellogenins. Vitellogenin is synthesized in
the liver and is transported to the ovary during the first stage of oogenesis
termed vitellogenesis 247,248. Vitellogenin is taken up intact by receptor-mediated
micropinocytosis 2~ into the developing oocytes where it is cleaved into a phosphate-
rich protein, phosvitin and a lipid-rich protein, lipovitellin 111,2~
Lipovitellin in trout eggs was composed of 77% protein and 23% lipid with a lipid
class composition similar to HDL 212. Cod roe lipovitellin had higher lipid content
than the trout at over 40% with 70% of the lipid being glycerophospholipids 226. Of
the total glycerophospholipids in cod lipovitellin, 67% was PtdCho and 22% was
PtdEtn with 4% each of PtdSer and PtdIns 226.
During the early stages of vitellogenesis, VLDL in the plasma may also be
increased in response to estrogen 187, and may also be taken up into the developing
oocytes by receptor-mediated endocytosis 248, at least in eggs with high triacylglyc-
erol content and lipid droplets. Recently it has been confirmed that winter flounder
contains a further high density lipoprotein (VHDL II), originally called Pk A as its
density and relationship to vitellogenin was unknown 216, that is taken up in vivo by
the ovary of viteUogenic females 149.
114 Composition
The net result of the many metabolic pathways discussed in Sections II and III
is the composition of glycerophospholipids in the tissues. The metabolism is a
very dynamic situation, of course, but the glycerophospholipid composition is more
stable provided the environmental conditions and diet are reasonably constant.
There are many papers reporting the glycerophospholipid composition of fish
tissues, and the effects of diet and environmental factors. A comprehensive review
of these areas is beyond the scope of this article. The reader is directed to some
other reviews that cover various aspects of glycerophospholipid composition in
128 D.R. Tocher
fish 1-5'96'194'198. This section contains some generalizations that can be made about
fish glycerophospholipid composition focusing on some features that have been of
particular interest in our own laboratory.
1. Content
It is difficult to generalize about the glycerophospholipid content of fish tissues
for several reasons. One of the major reasons is that studies have tended to report
contents in a variety of ways, such as mg/g tissue or as percentage of weight of tissue,
in both cases either wet or dry weights, or as percentage of total lipids. Irrespective
of the method used, nutritional status of the fish will have an effect as variation
in neutral lipid content will influence the results obtained. This is complicated by
tissue differences. Other than adipose tissue, some fish, such as cod and halibut
(Hippoglossus hippoglossus) store significant amounts of lipid in the liver, whereas
in others, such as mackerel (Scomber scombrus) and capelin (Mallotus villosus),
deposits of lipid between skin and muscle can account for a large proportion of the
fish's total reserves 96.
In snakehead (Channa sp.) fillet, eviscerated body of the guppy (Poecilia retie.
ulata) and carp muscle, total phospholipids accounted for 21-26% of the total
lipid s6,96. Phospholipids were present in goldfish muscle mitochondria at almost
2 mg/g muscle e42. In several studies on trout, phospholipids ranged from approx-
imately 45-75% of the total lipid in liver 9~. In chum and coho (Oncorhynchus
kisutch) salmon livers, phospholipids were consistently less than 40 and 30%, re-
spectively, whereas in parr and smolt cherry salmon (Oncorhynchus masou) livers,
phospholipids predominated 96. About 58% of the total lipid in goldfish intestinal
tissue was phospholipid 144.
It was reported in two studies on goldfish brain that phospholipids accounted
for 3.9% of the tissue weight 129, and 47% of the total lipid 189. In contrast, total
lipid from trout and cod brains was approximately 75% total polar lipids, predom-
inantly glycerophospholipids 2~. In sea bass on a variety of diets, the proportion
of phospholipids in the brain varied between 65 and 71% 17s. Phospholipids in
coelacanth (Latimeria chalumnae) brain amounted to over 41 mmol/g wet weight of
tissue 223. In a study of many marine fish including elasmobranchs and teleosts, total
phosphorus in the brains varied between 460 and 2650 mg/g fresh weight 123. Total
polar lipids, predominantly glycerophospholipids, made up 60 and 82% of the total
lipid in retinal tissue from trout and cod 228. However in whole eyes from guppy,
phospholipids were only 28% of the total lipid s6.
Relatively high levels of polar lipids, predominantly glyeerophospholipids, are
generally associated with fish eggs containing relatively low lipid contents 96. There-
fore, the roe of some species of marine fish including cod, herring (Clupea haren-
gus), haddock (Melanogrammus aeglefinus), whiting (Merlangus merlangus) and
saithe (Pollachius virens) were relatively rich in phospholipids, which accounted for
61-72% of the total lipid 23~ However, in sand eel (Ammodytes lancea) roe which
was richer in lipid and contained distinct oil globules, total lipid contained only 23%
phospholipid 23o.
2. Head group composition

The class composition of glycerophospholipids in fish tissues can be far more easily
generalized than content. PtdCho is almost invariably the major glycerophospho-
lipid class with PtdEtn almost invariably being the second most abundant. PtdCho
accounted for between 47 and 84% and PtdEtn accounted for between 16 and 34%,
of the total phospholipids in a range of tissues including muscle, liver, gill and
intestines from various fish species 96. This is a similar situation to that in mammals,
and also like mammals, the main exception to this is brain tissue where the per-
centage of PtdEtn can exceed that of PtdCho, as reported for brains from cod and
trout 228, coelacanth 223, rohu (Labio rohita)2~ and a range of elasmobranchs and
teleosts 13s. However, other studies have found PtdCho exceeding PtdEtn in brain
tissues from pike (Esox lucius) and carp 153, hake (Merluccius hubbsi) and sea bass
(Acanthustius brasilianus)12 and a range of fishes from the Caribbean 123.
PtdSer ranged from 2 to 9%, and Ptdlns ranged from 3 to 8% in several
tissues from various species of fish 86,88,96,128,129. The levels of PtdSer exceed those
of Ptdlns in fish neural tissues 96,123'129,153 and trout brush border membranes 12a,
whereas the opposite was true in trout gills 88 and liver86, goldfish liver 129 and the
roes from several marine fish 23~ Polyphosphoinositides, Ptdlns4P and Ptdlns4,5P2,
constituted 1.0 and 0.9 mol% of the total phospholipids in the lesser spotted
dogfish (Scyliorhinus canicula) rectal gland 29,2~ and were also found in cod gills3~
Cardiolipin ranged from 3 to 4% in trout liver and gills to over 8% in trout intestinal
brush border membranes 86,88,128. As in mammals, relatively high levels (6-11%) of
cardiolipin were associated with mitochondria in goldfish gill5a and muscle 242.
Many aspects of ether-linked glycerides including the glycerophospholipid deriva-
tives in marine animals, including fish, were the subject of recent reviews 45,194. Plas-
malogens (alkenylacyl derivatives) were detected in small amounts in total hepatic
lipid of the spiny dogfish 138. In the non-myelinated olfactory nerve of the garfish
(Lepisosteus osseus), ethanolamine, choline and serine phospholipids contained 58,
1.4 and 2.5% of the totals as plasmalogens 44. In goldfish brains, ethanolamine and
choline phospholipids contained 43 and 9% as plasmalogens 63. Bonito (Euthynnus
pelamis) white muscle contained 3-6% and 4-8% of total choline and ethanolamine
glycerophospholipids as plasmalogens 17~ Ethanolamine plasmalogen accounted for
12-13% of the total ethanolamine glycerophospholipids in trout spleen and gill,
about 6% of the total in kidney and <1% of the total in liver241. Less than 1%
choline plasmalogens were found in trout tissues 24~. Plasmalogens have also been
reported in fish gills38'16~ carp muscle mitochondria 251, goldfish optic nerve 142 and
smooth dogfish (Mustelus canis) erythrocyte membranes 25~
About 3% alkylacylphospholipids were detected in the total lipids of anchovy
(Engraulis mordax) TM. Alkylacylphospholipids accounted for 8 and 12%, respec-
tively, of total ethanolamine and choline glycerophospholipids from garfish olfac-
tory nerve 44. Bonito white muscle contained 1.4 and 0.6% of total choline and
ethanolamine glycerophospholipids as the alkyacyl derivatives 17~ 1-O-alkyl-2-acyl-
sn-glycero-3-phosphocholine accounted for 10% of total choline glycerophospho-
lipids in trout gill, kidney and spleen 241. In the trout, 1-O-alkyl-2-acyl-sn-glycero-3-
130 D.R. Tocher
phosphoethanolamine was only found in spleen where it accounted for 6% of total

ethanolamine glycerophospholipids241.
3. Fatty acyl composition
The fatty acyl composition of glycerophospholipids and the effects of diet, en-
vironment, pollutants, toxins and drugs etc. is one of the most studied areas in
lipid metabolism in fish. Therefore, there is a considerable amount of data in the
literature. However, as diet so markedly affects fatty acid compositions, only the
compositions of fish caught in their natural habitats, rather than captive fish, are
reviewed briefly in this section. Readers are directed to several review articles that
more comprehensively cover fatty acid compositions in fish tissues2,3,5,96,198.
3.1. Totalglycerophospholipids
Total phospholipids from tissues of wild fish are characteristically rich in PUFA
which can account for almost 60% of the fatty acids present s4,96. Phospholipids con-
tain higher proportions of PUFA, lower saturated fatty acids and similar amounts of
monoenoic fatty acids compared with triacylglycerol. The PUFA of phospholipids
are of longer chain length than those in neutral lipids with the ratio of C20 +
C22 PUFA to C1a PUFA 4-10 times greater than in neutral lipida4,96. In most
cases 22:6n-3 is the major PUFA of total phospholipid, followed by 20:5n-396. The
proportion of 20:4n-6 in total phospholipid is variable and can be occasionally
higher than 20:5n-364. Other common C20 and (222 PUFA include 22:5n-3, 20:4n-3,
20:3n-3, 20:2n-6, 20:3n-6, 22:4n-6 and 22:5n-6, present in variable amounts in phos-
pholipids, but rarely exceeding 2-3% of the total fatty acids. The C1s PUFA are
dominated by 18:3n-3 and 18:2n-6, with small percentages of 18:4n-3 and 18:3n-6.
The level of 18:2n-6 can frequently exceed that of 18:3n-3s4,96. Furan fatty acids, a
relatively common component of fish neutral lipids, are almost totally absent from
phospholipids s4,174. The ether linked chains in alkenylacyl and alkylacyl derivatives
are almost exclusively 16:0, 18:0 and 18:144,17~ .
The phospholipids of freshwater species tend to contain higher proportions of
saturated fatty acids and Cls PUFA and lower levels of C20 and (222 PUFA, than
the equivalent lipids from marine fish96. Ackman pointed out that, for fish from
northerly latitudes, it is the high proportions of C1s PUFA, rather than the low
C20 and C22 PUFA, that are typical of freshwater species 1. The lower (n-3) to
(n-6)PUFA ratio in freshwater fish (1.6 to 2.0) in comparison to marine fish (7.8 to
18.5) was also noted 1,2.96.
3.2. Glycerophospholipid classes

Studies on various tissues from several species have indicated that there is a
pattern for the distribution of fatty acids among the glycerophospholipids which
generally holds true despite tissue differences and extreme dietary effects. PtdCho
is characterized by having high 16:0 (higher than any other class) and lower PUFA
(i.e. lower than PtdEtn and PtdSer). PtdEtn is characterized by having intermediate
levels of saturated and monounsaturated fatty acids and high levels of PUFA which
are relatively evenly split between C20 and C22 PUFA. PtdSer is characterized by
high 18:0 and high PUFA, predominantly C22 PUFA. PtdIns also has high 18:0, but
relatively lower PUFA which is predominantly C20, particularly 20:4n-6, and a low
(n-3) to (n-6) ratio.
The above pattern has been most frequently observed in various neural tissues
including trout and cod brains and retinas 22a, turbot (Scophthalmus maximus)
brain 148, herring brain 146, carp and pike brain and spinal cord 153, hake and sea
bass brain and spinal cord 12, and brains from Caribbean fish 123. However, the
same pattern of distribution was observed in whole turbot 132, cod gills and dogfish
rectal glands 29, marine fish eggs23~ trout spleen 241 and the electric organ of
some elasmobranchs 19~ It is noteworthy that this pattern of distribution was
clearly demonstrated by various long established cultured fish cell lines including
rainbow trout gonad (RTG-2), turbot fin (TF), Atlantic salmon (Salmo salar) (AS),
chinook salmon (Oncorhynchustshawytscha) embryo (CHSE-214), bluegill (Lepomis
macrochirus) fry (BF-2) and fathead minnow (Pimephales promelas) (FHM) 239.
Some of these cells lines have been in culture for over 25 years 239.
As indicated earlier, this pattern of distribution holds despite some differences
between species and tissues, such as lower PUFA, especially 22:6n-3, in spinal
cords 12,153 and dogfish rectal gland 29 or higher PUFA in retinas, especially in-
creased 22:6n-3 in PtdCho 228, or lower (n-3) to (n-6) ratios as in some Caribbean
fish brains 123 and the cell lines which have been cultured in mammalian sera 239.
Similarly, several studies on Atlantic salmon showed that dietary effects did not
abolish these patterns 2~ nor did direct supplementation of PUFA to cultured fish
ce11s225,227,229.
Fish glycerophospholipids have some features worthy of additional comment with
respect to fatty acid composition. The composition of Ptdlns in fish, so similar to
that in mammals, showing selective retention of 20:4n-6, has suggested a potential
role for this class in eicosanoid metabolism (see section V.2.1) 29,230. The high
levels of 22:6n-3 in fish neural tissues (as indicated by high 22:6n-3 to 20:5n-3 ratios)
parallels the situation in mammals 199. In particular, the high level of PtdEtn coupled
with the very high levels of 22:6n-3 in PtdEtn in fish neural tissues may suggest
a special role for this glycerophospholipid class in neural functions. Fish neural
tissues are an ideal system in which to study this possibility. PtdCho in fish neural
tissues is unusual in that it contains relatively large amounts of the very-long-chain
monoene 24:112,25,29,153,228. This may be to compensate for the lower amounts of
sphingomyelin, which is rich in long chain monoenes, found in fish neural tissues in
comparison to mammalian neural tissues 199'228.
4. Molecularspecies
It has been increasingly appreciated recently that it is not simply the fatty acyl
composition of glycerophospholipids that is important, but also the particular
combinations of fatty acids, i.e. the molecular species, that are present within
the individual glycerophospholipid classes. In an early study the possible fatty
acid molecular species of cod PtdCho and PtdEtn were estimated based on fatty
132 D.R. Tocher
acid compositions alone 169. However, the first analysis of the molecular species
composition of fish PtdCho appeared in 1985221. In this study the experimenters
used HPLC to analyze diacylglycerol acetate derivatives which had been prepared
from diacylglycerol produced by the action of bacterial phospholipase C on PtdCho
purified from the dorsal muscle of 12 species of fish. The principal molecular
species in muscle PtdCho from most species, including marine and freshwater
species, was 16:0-22:6 (i.e. 16:0 in the 1-position and 22:6 in the 2-position) 221.
However, the two flatfish studied were different in that the main molecular species
of their muscle PtdCho was 16:0-20:5. The di-PUFA molecular species 20:5-22:6
and 22:6-22:6 were significant components of muscle PtdCho, particularly in the
marine fish, although they were not quantified in that study221. Some two dozen
different molecular species of muscle PtdCho were identified and quantified in
chum salmon 22~ In addition to the major di-PUFA species mentioned above, minor
amounts of several other di-PUFA species including 20:5-20:5, 20:5-22:5, 22:5-
22:6 and 20:4-22:5 were reported 22~ These workers also reported surprisingly high
percentages of molecular species with PUFA in the 1-position and a saturated or
monoenoic fatty acid in the 2-position, such as 22:6-16:0 and 22:6-18:122~
The molecular species of total glycerophospholipids was determined in bonito
white muscle by selected-ion monitoring gas chromatography/mass spectrometry
using electron impact ionization 171. The predominant molecular species were 16:0-
22:6n-3 (--,36%), 18:1-22:6n-3 (~,20%) and 16:0-18:1 ('-,13%) with the di-PUFA
species 22:6n-3-22:6n-3 and 20:5n-3-22:6n-3 accounting for almost 9 and 6%,
respectively 171. About 17 different species were identified, but species with PUFA
in the 1-position and a saturated or monoenoic fatty acid in the 2-position were not
found. Ohshima et al. used the same method to analyze the molecular species com-
position of 1-O-alk-l'-enyl-2-acylglycerophospholipids (plasmalogens) from bonito
white muscle 172. Less than a dozen species were found, with the predominant
species being 16:0"-22:6n-3 (alkenyl"-acyl), which accounted for over 57% of the
total, followed by 16:0"-18:1 (,~15%) and 18:0"-22:6n-3 (,-.13%) 172.
Hazel and Zerba investigated the molecular species compositions of PtdCho and
PtdEtn from trout mitochondrial and microsomal membranes using gas chromatog-
raphy of trimethylsilyl ethers of the diacylglycerols92. The predominant species
of PtdCho were 16:0-22:6, 16:0-18:1, 16:0-20:3 and 16:0-22:5, whereas the pre-
dominant species of PtdEtn were 18:1-20:4, 14:0-16:0, 18:0-22:6 and 18:1-22:692.
Mitochondria contained higher proportions of long-chain, polyunsaturated molec-
ular species of PtdEtn, but less of the corresponding species of PtdCho, than
microsomes 92.
Using 3,5-dinitrobenzoyl derivatives of glycerophospholipids and a combination
of three different isocratic solvent systems, Bell and coworkers have identified ap-
proximately 70 different molecular species in a range of different fish tissues23-25.2s.
In cod roe, four species 16:0-20:5n-3, 18:1-20:5n-3, 16:0-22:6n-3 and 18:1-22:6n-3
contributed 67 and 62% of the total species in PtdCho and PtdEtn, respectively23.
The 16:0-containing species predominated in PtdCho, whereas the 18:l-containing
species predominated in PtdEtn. These four species accounted for only 23% in cod
roe PtdIns, where 18:0-20:4n-6 was the predominant species at 37% of the total
molecular species23. Di-PUFA species totalled less than 3% in cod roe PtdCho and
PtdEtn, and were not found in Ptdlns.
In contrast, retina from rainbow trout and cod contained high levels of di-
PUFA molecular species25,28. Trout retina contained 14.6, 43.5 and 29.0% di-
PUFA species, almost all di-22:6n-3, in PtdCho, PtdEtn and PtdSer, respectively28.
In cod retina, di-22:6n-3 accounted for 29, 72 and 60% of the total molecular
species in PtdCho, PtdEtn and PtdSer, respectively25. Trout and cod brain tissue
also contained between 16 and 26% di-PUFA species in PtdEtn and PtdSer 25,28.
However, in brain, PtdCho contained only ~1% di-PUFA species, and the di-22:6n-
3 species accounted for less of the total di-PUFA species, compared to retina 25,28.
Fish brain PtdCho was also characterized by containing high percentages (9 and
13% in trout and cod, respectively) of 18:1-24:1/24:1-18:1 species25,28.
In cod muscle and liver, saturated fatty acid-PUFA and monounsaturated fatty
acid-PUFA species predominated, particularly 16:0-20:5 and 16:0-22:6 in PtdCho,
16:0-22:6 and 18:1-22:6 in PtdEtn and 18:0-22:6 and 18:1-22:6 in PtdSer 25. Muscle
had the widest range of di-PUFA species, at least 6, totalling 21 and 38% in PtdCho
and PtdEtn, respectively. Liver contained the lowest amount of di-PUFA species of
the four tissues studied in cod 25. Ptdlns in cod muscle and liver had very low levels
of di-PUFA species24. In cod liver Ptdlns, 49% was the 18:0-20:4n-6 species and
in retina the major species of Ptdlns were 16:0-20:4n-6 (26%) and 18:0-20:4n-6
(24%) 24. However, in cod brain Ptdlns, 18:0-20:5n-3 was the most abundant species
(41%) and in muscle 40% was 18:0-22:6n-324. The finding that 18:0-20:4n-6 was
not the predominant species in Ptdlns in all fish tissues was particularly noteworthy.
In all the above studies, di-saturated fatty acid species were notably uncommon.
5. Dietary effects
The main effects of diet on glycerophospholipids are related to fatty acid composi-
tion and these are briefly summarized here.
Algae commonly feature in the diet of the early life stages of some freshwater
fish. Freshwater algae contain higher levels of C18 PUFA than C20 or Czz PUFA,
although 20:5n-3 can be present in significant amounts in some diatoms 96,198.
However, 22:6n-3 is rarely found. Aquatic insects are a major food source for
freshwater fish, particularly salmonids, and although C18 PUFA predominate in
these insects, 20:4n-6 and 20:5n-3 can account for up to 7 and 25%, respectively,
of the total fatty acids96. Therefore, the lipids at the different trophic levels of the
freshwater food chain are characterized by 18:2n-6, 18:3n-3, 20:5n-3 and 20:4n-6.
In contrast, the marine food chain contains lipids in which 18:3n-3, 20:5n-3 and
22:6n-3 predominate and which have low levels of (n-6)PUFA 195. Therefore, these
differences in the dietary input between the freshwater and marine environments
are primarily responsible for the differences in glycerophospholipid fatty acid
composition observed in freshwater and marine fish described above 195,198.
As eluded to above, the PUFA composition of glycerophospholipids in fish tissues
is highly dependent upon dietary PUFA composition 254.255 . The absence of PUFA
in the diet, leads to the deposition of 20:3n-9, derived from the desaturation and
134 D.R. Tocher
elongation of 18:1n-9, in tissue phospholipids in salmonids 255,256. Feeding increased

levels of 18:3n-3 to trout resulted in deposition of 18:3n-3 in phospholipids and
particularly neutral lipids, whereas the level of its conversion products, 20:5n-3 and
22:6n-3, increased in phospholipids 254. The level of 22:6n-3 in phospholipids is also
directly dependent on its level in the diet 257. Similarly, when increased levels of
18:2n-6 are included in the diet of salmonids, it is incorporated into neutral lipids
and to a lesser extent phospholipids, whereas its conversion products 20:4n-6 and
22:5n-6 are particularly incorporated into phospholipids 255-257. This may not be the
case in fish that lack the full spectrum of desaturases (A6, A5 and A4, see ref.
95), such as the turbot which appears to lack specifically the A5 desaturase 132,197.
Low or absent A5 desaturase activity may be a common characteristic of marine
fish or fish that are extreme carnivores, although there are few fish species in which
the desaturase complement has been sufficiently characterized. In addition, dietary
studies are lacking in turbot and other species that may lack A5 desaturase activity.
However, in cultured turbot cells, 22:6n-3 and 22:5n-6 could not be produced by
supplementing 18:3n-3 and 18:2n-6, respectively, to the cultures 229.
6. Adaptation to environmental factors
Due to the implications for homeoviscous adaptation of biological membranes of

poikilothermic animals, this is a subject which has been the focus of considerable
attention in fish over the years. This section is a brief summary of the main
findings on lipid adaptations to environment in fish. For a detailed account of the
experimental data and a very comprehensive review of the literature, the reader is
directed to recent articles by Hazel and Williams91, Hazel 89 and Volume 5 of this
series 98. There are several other reviews specifically on the effects of temperature
on membrane lipids in fish51,53,87.
6.1. Temperature
Changes in membrane composition and phospholipid structure in response to
cold acclimation include changes in head group composition, acyl chain composi-
tion, phospholipid structure and molecular species composition 87. Cold acclimation
has been associated with increased proportions of PtdEtn and decreased propor-
tions of PtdCho 42,63'144'242. The proportion of plasmalogens, primarily alkenylacyl-
glycerophosphoethanolamine, also decreased, particularly in neural membranes, in
response to cold ac Climation 63 . Cold adaptation resulted in reduced proportions
of saturated fatty acids and increased proportions of PUFA and to a lesser extent
monounsaturated fatty acids 42,5~ Increased proportions of (n-3) and/or (n-6)
PUFA (often at the expense of (n-9) acids) and increased average acyl chain
length have also been reported 242. Changes in glycerophospholipid and fatty acid
metabolism directly at the cellular level in response to temperature have also been
demonstrated in fish cells in culture 233.234.
Structural changes induced by cold acclimation include increased proportions
of PUFA at the 2-position, increased unsaturation at the 1-position and generally
increased proportions of highly unsaturated molecular species 144. These changes
are perhaps linked to gross compositional changes. However, a further adaptation

to environmental changes is the redistribution of fatty acyl chains into different
glycerophospholipid molecular species, which can have significant effects on the
properties of membranes without gross compositional changes. For instance, the
phase behavior of 18:0-18:1 PtdCho (Tin = +3"C) is totally different to that of a
binary (1:1) mixture of 18:0-18:0 PtdCho and 18:1-18:1 PtdCho (two transitions
at -22"C and 30-53"C) despite having an identical fatty acyl chain composition la4.
Hazel and Z e r b a 92 showed that PtdCho in mitochondrial membranes from trout
acclimated to 5"C contained proportions of 16:0-22:6n-3 and 16:0-20:5n-3 that
were 2- and 3-fold greater, respectively, than those from 20*C-acclimated fish. The
effects of increasing the proportion of 16:0-22:6n-3 PtdCho was investigated in
model membrane systems 94. Increased 16:0-22:6n-3 PtdCho tended to fluidize the
membranes and reduced the temperature sensitivity of electrolyte permeation 94.
Whereas the compositional changes, briefly summarized above, are well doc-
umented, the enzymic mechanisms behind these changes are yet to be fully
elucidated. Fatty acid desaturase activities, including A9, A6 and A5, are gen-
erally increased in response to lowered environmental temperature 59,85,161.2~176
and this alone could account for the increased degree of unsaturation in membrane
glycerophospholipids. The deacylation/reacylation reactions of glycerophospholipid
turnover have been studied to determine the effects of temperature 93,133. Studies
on the microsomal acyl-CoA:lysoPtdCho acyltransferase from liver of thermally
acclimated rainbow trout have suggested that, although the activity may not be
affected by temperature, the substrate specificities may vary and so contribute to
restructuring 133. A recent study on the pathways of de novo biosynthesis of glyc-
erophospholipids showed that the activities of CDP-choline and CDP-ethanolamine
phosphotransferase were reduced at 5"C, whereas PtdSer decarboxylase activity was
increased 9~ The overall synthesis of PtdCho depended more on temperature than
that of PtdEtn and so the ratio PtdCho: PtdEtn synthesis positively correlated with
temperature 9~ This may be a mechanism underpinning the increased proportions
of PtdEtn and decreased proportions of PtdCho in cold-acclimated fish.
6. 2. Salinity and hydrostatic pressure

The major difference between freshwater and sea water is the high concentration
of inorganic salts in the latter and so changes in the lipids of fish transferred from
freshwater to sea water in the laboratory can be considered as adaptive responses
to environmental salinity. In the wild, anadromous fish make the change from
freshwater to sea water and vice versa but these changes are usually accompanied
by great changes to the diet, including starvation. Therefore, changes in the lipids of
wild anadromous fish will not be included here.
The proportion of phospholipids to neutral lipids increased in all tissues of the
guppy during sea water adaptation 56. Tissue specific changes occurred in the relative
proportions of the phospholipid classes, with the proportion of PtdEtn increasing in
gill, intestine, liver and eye and PtdCho decreasing in the kidney of the fish in sea
water 56. However, there were no changes in.the relative proportions of phospholipid
classes in brush-border membranes from the intestine of rainbow trout transferred
136 D.R. Tocher
from freshwater to sea water ns. Similarly, phospholipid class composition of the
gills of Atlantic salmon was unaffected by keeping the fish in salinities from 0 to 2%
over 48 h 222.
In the guppy, the levels of 22:6n-3 and 20:4n-6 increased and decreased, respec-
tively, in PtdCho and, especially, PtdEtn of the digestive tract during sea water
adaptation 56. Similarly, the proportion of total (n-3)PUFA, almost entirely 22:6n-3,
in PtdCho from trout intestinal membranes increased from 17.7 to 33.4% within
one day of transfer from freshwater to sea water 128. There were concomitant de-
creases in the percentages of (n-6)PUFA and saturated fatty acids ns. In contrast,
the level of (n-3)PUFA, particularly 22:6n-3, decreased in gill PtdCho in response
to increased salinity in Atlantic salmon 222.
Studies have suggested that there are similarities between the adaptive responses
to low temperature and high pressures. Deep water and Antarctic fish both pos-
sessed elevated proportions of PUFA compared to tropical species 176. In a study
comparing the compositions of liver mitochondrial PtdEtn from 13 species of teleost
fish ranging in depth from 200 to 4000 m, the proportions of 16:0 and 18:0 decreased
in abundance with depth, whereas PUFA levels remained relatively constant and
monounsaturated fatty acid levels increased 52. Similar increases in the unsaturation
index with depth/pressure were observed in PtdCho and PtdSer 52. Changes in the
membrane lipid environment have been implicated in the pressure adaptation of
Na +/K+-ATPase in fish gills77.
V Roles
As the major lipid components in all biological membranes, glyeerophospholipids
have a crucial role to play in both the structure and function of all animal cells.
This is as true for fish as it is for mammals. Indeed, as fish are poikilothermie, it
could be argued that the lipid components of the biological membranes have an
additional important role in homeoviseous adaptation in relation to temperature.
The following is a brief review of some specific roles of glyeerophospholipids that
have, or may have, relevance to fish.
1. Structural roles
Glyeerophospholipids have the same central role in the structure of cell mem-
branes in fish as they do in mammals. The dynamic changes in the composition
and metabolism of the glycerophospholipids in biomembranes in response to en-
vironmental factors91, discussed above, is testament to their importance. Fish
membranes, however, are characterized by high levels of (n-3)PUFA, including
22:6n-3. This is particularly the case in fish neural membranes where, like virtually
all vertebrate brains and retinas, 22:6n-3 is the predominant fatty acid 1~. It is
now established that 22:6n-3 is essential for the proper development and function
of mammalian neural membranes 19'159. This is probably equally true in fish as we
have recently established that 22:6n-3 is rapidly taken up by turbot brain when the
post-larvae were weaned from a 22:6-poor diet to a 22:6-rich diet 147,148. Biochemical
studies showed that the incorporation of 22:6 was particularly avid in PtdEtn 238.
Recent biophysical studies have begun to elucidate some of the physical and struc-
tural properties that this acid, when esterified in glycerophospholipids, may impart
to membranes.
Increasing the number of double bonds in long chain fatty acids of a given chain
length constrains the structures of the molecules such that they form increasingly
compact conformations 9. This effect is maximal in 22:6n-3 whose minimum energy
conformation is a relatively compact helix or 'angle iron' structure with an overall
length shorter even than a saturated fatty acid, such as 18:0 9. In fish neural tissues,
the 22:6n-3 is mainly esterified to PtdEtn and PtdSer, and a considerable portion of
this is in the form of di-22:6n-3 species (see above). Given the marked helicity of
22:6n-3, it can be deduced that cell membranes, such as retinal rod outer segments,
that are rich in di-22:6n-3PtdEtn, have a very specialized phospholipid bilayer,
probably with novel liquid crystalline properties. Precisely how this specialization
relates to the function of rhodopsin, and the retina in general, is not known in detail.
It has been considered, on the basis of physicochemical studies of mammalian rod
outer segment membranes, that the abundance of di-22:6n-3PtdEtn maintains the
membrane bilayer with the required balance between fluidity and rigidity necessary
to accommodate very rapid protein conformational changes initiated by the c/s-
trans conversion undergone by the retinal chromophore of rhodopsin 62. In addition,
it has recently been deduced on theoretical grounds that the minimum energy
form of 22:6n-3 is conformationally stable over a wide range of temperature,
so that membrane bilayers rich in 22:6n-3 may be essentially 'buffered' against
environmental change, with obvious advantages to their associated physiological
processes 185. The foregoing scenario can be extended in principle to encompass
very fast conformational changes undergone by other signal transducers in cell
membranes, e.g. those involved in ion current generation, thus accounting for the
abundance of 22:6n-3 in neural tissue glycerophospholipids generally.
It is well established in mammals, that phospholipids are asymmetrically dis-
tributed in cell membranes, with choline-containing phospholipids, PtdCho and
sphingomyelin, being concentrated in the outer leaflet and PtdEtn, PtdSer and, to
a lesser extent, PtdIns being concentrated in the inner leaflet of the membrane 6~
This aspect of membrane structure has not been well studied in fish. However,
the localizations of the amine-containing glycerophospholipids, PtdEtn and PtdSer,
were studied in the photoreceptor membranes of the retina from walleye pollock
(Theragra chalcogramma) and an asymmetric distribution was apparent 113.
2. Metabolic roles
2.1. Eicosanoid metabolism

Eicosanoids are a range of highly bioactive molecules so named because they are
derived, via the action of dioxygenase enzymes, primarily from the C20 PUFA
20:3n-6, 20:4n-6 and 20:5n-3. Two main enzymes are involved: (1) cyclooxygenase,
which produces cyclic oxygenated derivatives, collectively called prostanoids, in-
138 D.R. Tocher
eluding prostaglandins (PG), prostacyclins (PG I) and thromboxanes (TX); and (2)
lipoxygenases, which produce linear oxygenated derivatives including hydroperoxy
and hydroxy fatty acids, leukotrienes (LT) and lipoxins (LX). Eieosanoids are pro-
duced by a wide range of tissues in response to a variety of extracellular stimuli.
Glyeerophospholipids are important as the source of the substrate fatty acids for
the cyclooxygenase and lipoxygenase enzymes.
The pathway from extraeellular stimulus to the production of eieosanoids forms
a cascade termed the 'arachidonic acid cascade' as araehidonie acid (20:4n-6) is the
primary precursor in mammals. Briefly, activation of cell surface receptors results
in the production of free precursor acid either via phospholipase A2 activity or
via the sequential action of phospholipase C and DAG lipase. The activation of
phospholipase A2 may occur through elevation of intraceUular Ca 2+ (it is activated
in vitro by high Ca2+levels) or it may be regulated by a G protein, as phospholipase
C appears to be in the phosphoinositide cycle4~ It appears that the increased
concentration of the free precursor acid is itself the key stimulus for the activity
of the eyclooxygenase or lipoxygenase enzymes214. The pathways for the synthesis
of individual eieosanoids are complex with many different steps2t4 and will not be
discussed in detail here, although work is progressing in characterization of these
pathways, particularly lipoxygenase pathways, in fish.
In fish there is little mechanistic data on most of the steps involved in this
cascade, particularly up to the production of free fatty acid precursor. However, due
to the very obvious differences in the C20 PUFA composition of the glyeerophos-
pholipids between mammals and fish, there has been considerable interest in the
general production of eicosanoids in fish. Although the roles in the cascade of the
glycerophospholipids themselves are not well understood, eieosanoid metabolism is
nonetheless an important aspect of glycerophospholipid metabolism. The following
sections offer a brief review, therefore, of the existing knowledge of eicosanoids in
fish with particular emphasis on glycerophospholipids. Henderson and Tother ~ give
a more complete review on eieosanoid metabolism in fish in relation to essential
fatty acid metabolism.
2.1.1. Species and tbsue distribution of eicosanoids in fish Prostanoids have

been found in a large range of freshwater fish, including c a r p 17'49'120'164~ , rain-
bow trout rig, brown trout 49, brook trout (Salvelinus fontinalb) 8t'82, tench 49, tilapia
(Tilapia mossambica [Oreochromis mossambicus]) tT, Asian catfish (Heteropneustes
fossili~ and Clarias batrachus) t7, pond loach (Mbgumus anguillacaudatua) 1~, eel
(Anguilla anguilla) 2ta, sheat-fish (Parasilurus asotus) 164 and goldfishsl, and marine
fish, such as plaice (Pleuronectes platessa) 7, turbot 97, dogfish 192, flounder (Par.
alichthys olivaceus) 143, black (Acanthopagrus schlegeli) and red (Pagrus major) sea
breams 143, black rockfish (Sebastes schlegeli) t43 and Atlantic salmon 2~ Virtually
every fish tissue so far studied has shown eyclooxygenase activity, including gills,
kidney, spleen, intestine, stomach, liver, heart and isolated eardiomyoeytes, skeletal
muscle, brain, fin, skin, air sac, ovaries and ovarian fluid, testes and milt, blood
thrombocytes and leukoeytes20,22,49,u9,120,164.16s,167,16s.In some of the above studies
exogenous fatty acid precursors were added, but synthesis of prostanoids from
endogenous fatty acids has been shown in ovarian tissues 168, gills49, leukocytes 22,
isolated cardiac myocytes2~ and testes, liver, heart, intestine, brain, air sac and
kidney 164. Gills were found to have the greatest capacity for the synthesis of
prostanoids from exogenously added fatty acid precursors 49,167, whereas intestine,
heart, air sac and skin synthesized more prostanoids from endogenous substrates
than gill164.
Lipoxygenase products have been identified in tissues from American eel
(AnguiUa rostrata)183, rainbow trout 73'75'76'177'179-181'235, dogfish 178'192, plaice 232,
salmon 21'22'191, and catfish, carp, rudd (Scardinius erythrophthalmus) and tilapia
(Oreochromis niloticus) 191. Tissues studied include gills 21,22,73,75,183, skin 76, whole
blood 21'22'178'179, blood total leukocytes 177,191,192, peripheral blood neutrophils 232,
tissue macrophages 18~ and brain cells 23s.
A third group of eicosanoids, epoxides formed by the action of cytochrome P-450
enzymes, have been demonstrated in mammalian systems214. To date, there has
been no report of these derivatives being found in fish tissues. However, this is
probably simply because they have not been looked for. By analogy with how the
characterization of lipoxygenase products in the fish field lagged slightly behind the
mammalian field and how similar the fields appear today, it is likely that epoxide
derivatives will be discovered and characterized in fish tissues in the future.
2.1.2. Range of eicosanoids in fish PGE 17'49'120'143'164'167'192,PGD 17'120'143'192,and

PGF 17'49'120'143'167'169'192have most commonly been reported for many fish tissues96.
TXB, the stable metabolite of TXA, was the major prostanoid producedby rainbow
trout thrombocytes 119'12~and Atlantic salmon blood 22 and was also produced by
salmon cardiomyocytes2~ and isolated gill ceUs 22, and dogfish leukocytes 192. No
metabolites of PGI were produced by dogfish leukocytes, but 6-keto-PGFl~, the
stable metabolite of PGI2, was detected in Atlantic salmon blood, gill cells and
cardiomyocytes2~
The 12-1ipoxygenase product, 12-hydroxy fatty acid, was the major lipoxygenase
product from trout gill75, skin 76, head kidney macrophages 179,1sl and brain cells23s.
It was also reported in studies with plaice neutrophils 232. The 12-1ipoxygenase
activity has now been found in the gills and skin of 14 species of fish and well
characterized in rainbow trout gill 1~ The presence of 15-1ipoxygenase in fish tissue
has also been indicated by the presence of 15-hydroxy products in trout brain
cells235 and dogfish blood cells 178. The 15-1ipoxygenase activity has been identified
and more thoroughly characterized in various fish gills72. The 5-hydroxy products
of the 5-1ipoxygenase were found in studies with plaice neutrophils 232 and rainbow
trout head kidney macrophages lsl and brain cells235. LTB has been the most
widely reported lipoxygenase product, being produced by various tissues including
plaice neutrophils 232, dogfish leukocytes 192, trout macrophages ls~ leukocytes 177,
whole blood 179 and brain cells23s. The peptido-leukotrienes, LTC, D and E, were
produced by American eel gill tissue 183. Various other di- and tri-hydroxy-products
have also been noted in fish tissues71,73,74,17a. Lipoxins, LXA and LXB, were major
lipoxygenase products in rainbow trout head kidney macrophages 180.181. Head
kidney cells, mainly macrophages, from Atlantic salmon and carp also produced
140 D.R. Tocher
lipoxins, but the same preparations from catfish, rudd and tilapia did not produce
lipoxins 191.
Overall, it appears that fish produce the same wide range of eicosanoids as in
mammals. Also, as in mammals, the precise eieosanoid profile varies with tissue
and, to some extent, species. Although relatively few species have been investigated,
there appears to be no major differences between elasmobranehs and teleosts,
or between freshwater and marine fish. However, as more species are studied,
differences or patterns may become apparent.
2.1.3. Stimuli for production of eicosanoids In early studies, production of

eicosanoids in experimental systems in fish was most often stimulated simply
by the addition of exogenous fatty acid substrate %. More recently, eieosanoid
production has been stimulated by incubation with the calcium ionophore
A2318720''22,177-180'183'191'192'232'235.Therefore, a rise in intracellular Ca2+ion con-
eentration was effective in stimulating the production of a variety of eicosanoids in
a wide range of fish tissues. However, there have been no specific eicosanoid studies
using more physiological stimuli in fish.
2.1.4. Fatty acid precursors of eicosanoids Production of PGE2 and PGF2a from
endogenous 20:4n-6 in carp, tench and trout tissues 49,167 and PGE3 from en-
dogenous 20:5n-3 in tissues from carp and sheat-fish 164 have been reported. The
amounts of PGE3 produced were generally lower than those for endogenous
PGE2 production 49'1u. Production of 1-series prostaglandins from endogenous
20:3n-6 was not detected 49. Approximately 5-fold more 20:4n-6 was converted to
eicosanoids by plaice skin microsomes compared to 20:5n-37. Only prostanoids of
the 2-series (20:4n-6 products) and LTB4 were produced by dogfish leukocytes
exposed to calcium ionophore A23187192. Bell and colleagues 21,22 measured the
production of TXB2, 6-ketoPGFla, 12-hydroxyeicosatetraenoate (HETE) and LTB4
from endogenous 20:4n-6 and 12-hydroxyeicosapentaenoate (HEPE) and LTB5
from endogenous 20:5n-3 in Atlantic salmon tissues. In salmon fed a normal
diet the production of 12-HETE exceeded that of 12-HEPE in blood leuko-
cytes, whereas the levels of 12-HEPE in gills and LTB5 in blood leukocytes
exceeded the levels of the respective 20:4n-6 products 21'22. TXB2 and 6-keto-
PGFta were produced in isolated eardiomyocytes from Atlantic salmon 2~ Rainbow
trout blood cells stimulated with A23187 produced both LTB4 and LTB5 from
endogenous precursors, with LTB4 production exceeding that of LTB5179'180.Rain-
bow trout macrophages stimulated with A23187 produced LTB4 and LXA4, and
also LTB5 and LXA5 from endogenous 20:4n-6 and 20:5n-3, respectively 18~ with
the 20:4n-6 products predominating 181. Similarly, the levels of lipoxygenase prod-
ucts from 20:4n-6 exceeded those from 20:5n-3 in Atlantic salmon, carp and
tilapia 191.
More exogenously added [1-14C]20:4n-6 compared to [1-t4C]20:5n-3 was incor-
porated into prostaglandins by turbot tissues 97 . Incubation of thromboeytes from
several fish species with A23187 in the presence of [1-14C]20:4n-6 resulted in the
formation of labeled PGF2~, PGE2 and PGD2 in all cases 143. In contrast, when
the thrombocytes were stimulated with A23187 in the presence of [1-14C]20:5n-3,

production of PGE3 and PGD3 were only produced by one species 143. In plaice
neutrophils labeled with [1-14C]20:4 n-6 or [1-14C]20:5n-3231, the production of
labeled LTB4 and 12/15-HETE exceeded that of LTB5 and 12/15-HEPE, respec-
tively 232.
Overall, the results of these studies indicate that both 20:4n-6 and 20:5n-3
serve as eicosanoid precursors in fish tissues. However, in almost all cases the
evidence suggests that 20:4n-6 is the preferred substrate despite the preponderance
of 20:5n-3 in the tissue glycerophospholipids.
Dihomo-y-linolenic acid (20:3n-6) can serve as a substrate for fish cyclooxyge-
nase enzymes when added exogenously 49,167. However, there was no production of
labeled prostanoids when thrombocytes from several fish species were incubated
with [1-14C]22:6n-3 in the presence of A23187147. Lipoxygenase-derived deriva-
tives of endogenous 22:6n-3 have been reported in fish tissue 74. There have been
several studies characterizing different lipoxygenase-derived metabolites of exoge-
nously added 22:6n-3 in various fish tissues 73'75'76. It remains to be seen how
many of these are produced in vivo. The 12-1ipoxygenase derivative of 22:6n-3,
14-hydroxydocosahexaenoate (14-HDHE) was produced from endogenous 22:6n-3
in Atlantic salmon gill cells stimulated with A23187 22.
2.1.5. Glycerophospholipid sources of precursor fatty acids The precise glyc-

erophospholipid class that serves as a source of precursor fatty acids for eicosanoid
synthesis is still a controversial area in mammalian metabolism. The wide range
of tissues studied using a variety of stimuli has probably confused this area. Over-
all, it appears that one glycerophospholipid class may not be the sole supplier
of eicosanoid precursor in all systems. In contrast, this area is virtually unstud-
ied in fish. The evidence, summarized above, indicating, in all systems studied so
far, that 20:4n-6 is the predominant eicosanoid precursor in fish invites a very
obvious question. How do fish selectively utilize 20:4n-6 for eicosanoid synthesis
when the tissues are rich in 20:5n-3? One possible answer to this would be to
selectively concentrate the eicosanoid precursor in one glycerophospholipid species.
The characteristic fatty acid composition of Ptdlns in fish tissues, with high 20:4n-6,
led Sargent and coworkers to speculate that Ptdlns may be an ideal source of
eicosanoid precursor in fish 29'230. However, in a time course study on the loss of
radioactivity from labeled glycerophospholipids in plaice neutrophils in response to
A23187, the labeling appeared to decrease firstly in PtdCho and to a lesser extent
PtdEtn prior to loss of label from Ptdlns 226. A recent study examining 3H/14C ratios
in trout brain cells dual-labeled with [3H]20:4n-6 and [14C]20:5n-3, suggested that
the glycerophospholipid source of eicosanoid precursors was unlikely to be Ptdlns
and that PtdCho was the most likely source 235. If PtdCho is the preferred source
of eicosanoid precursor in fish, then the specificity for 20:4n-6 may result from
a 20:4n-6-specific phospholipase A2 or from the specificity of the cyclooxygenase
and lipoxygenase enzymes. These aspects are virtually unstudied in fish. However,
the 3H/14C ratio of released fatty acids obtained in the study above suggested that
there was no specificity for 20:4n-6 at the level of the phospholipase 235. There is
142 D.R. Tocher
conflicting evidence in fish regarding the possibility that the specificity may reside
mainly in the dioxygenase enzymes. In one study on sardine skin lipoxygenase, the
enzyme activity towards 20:4n-6 was twice that towards 20:5n-3145. Interestingly,
the enzyme was as active towards 22:6n-3 as it was towards 20:4n-6145. However,
the 12-1ipoxygenase activity from rainbow trout gill was equally active with 20:4n-6,
20:5n-3 and 22:6n-31~
2.1.6. Functions ofeicosanoids Considering the relatively large number of studies

characterizing eicosanoid production in fish, there are surprisingly few studies on
the functions of eicosanoids in fish. An exception to this is fish reproduction, where
the roles of prostanoids are well known 96. Prostaglandins are involved in inducing
ovulation, and may be involved in eliciting behavioral changes, such as spawning
activity96. This subject has been comprehensively reviewed previouslya1,219.
The production of thromboxane by rainbow trout thrombocytes 119,n~ Atlantic
salmon blood 21,22 and dogfish leukocytes 192, and the presence of prostacyclin (PGI)
in Atlantic salmon plasma 22 suggests that the control of thrombocyte aggregation
and blood clotting in fish may involve a "IXA/PGI balance as in mammals. Series-2
prostanoids have been shown to have various cardiovascular effects when injected
into fish 182'184'246. Similarly, series-1 prostanoids were also shown to have vasoactive
properties in snakehead 253. It is not known if these effects have any physiological
significance in fish.
The peptido-leukotrienes from eel gills had the same biological effects on
mammalian lung tissue as mammalian LTC4, and so they may play a role in
modulating gill function at a local level 183. An LTB extract from plaice neutrophils
stimulated with A23187 had chemotactic activity for plaice leukocytes 96. Similarly, it
was found that LTB4 enhanced the migration of fish leukocytes in vitro 1~ Therefore
LTB may play similar roles in inflammatory and immune processes as it does in
mammals.
2.2. Thephosphoinositidecycle
The phosphoinositide cycle, in which phosphorylated derivatives of PtdIns, such
as PtdIns4,5P2, are converted by the action of phospholipase C into two intracel-
lular second messengers, DAG and inositol phosphates (e.g. InsP3), in response
to various hormones and effectors is well characterized in mammals 33. Consid-
erably less is known about the phosphoinositide cycle in fish. However, studies
in various fish tissues have suggested that the basic components of the cycle do
operate in fish 10'39'78'196'207. In an early study in seawater eel gills, it was shown that
depression of salt secretion by 10-5 M adrenalin was accompanied by enhanced
turnover of inositol lipids78. Dogfish rectal gland is rich in inositol phospholipids
(reflecting the extensive plasma membranes in chloride cells) with PtdIns, Pt-
dIns4P and PtdIns4,SP2 comprising 9.1, 1.0 and 0.9% of total cellular phospholipid,
respectively 2~ Studies with 32p-labeled orthophosphate established that inositol
phospholipids are metabolically active in non-stimulated rectal glands, i.e. an active
inositol lipid cycle exists in the resting gland, and that elevation of intracellu-
lar cAMP depresses inositol lipid turnover 207. Interestingly, the situation in fish
is opposite to that in the avian salt gland where activation of salt secretion by
aeetyleholine is accompanied by enhanced turnover of inositol lipids99.
Cilia isolated from the olfactory epithelium of channel catfish were shown to con-
tain an active phospholipase C 39. Thg enzyme was shown to hydrolyze exogenously
added radioactive Ptdlns4,5P2, with IP3 the major inositol phosphate product 39.
The optimum pH was 6.7 compared to the more acidic optima of about pH 5
for mammalian Ptdlns-speeifie phospholipase C 39'186. Similar to the mammalian
phosholipase C activity, the activity in catfish cilia had multiple molecular forms
with Mr >100,000, 82,000 and 60,000 which compare reasonably well with the
molecular weights found in a range of mammalian tissues 39,186.
The metabolism of inositol phospholipids was studied in metabolically active
electrocytes from the electric ray (Discopyge tschudii) after labeling with myo-
[3H]Insl~ Ptdlns displayed the highest level of labeling which was inhibited by
lithium l~ Incubation of the labeled electrocytes over 24 h showed that labeled
inositol phosphates were produced in the rank order InsP > InsP3 > InsP2, and
that the presence of lithium ions enhanced the accumulation 1~ These results were
interpreted as indicating the participation of phospholipase C and of Li-sensitive
phosphatases in the modulation of phosphoinositide metabolism in the eleetrocytes
of the electric ray.
2.3. Other metabolism

2.3.1. Protein kinase C One consequence of an active phosphoinositide cycle is
the production of increased intracellular Ca 2+ and DAG, both activators of protein
kinase C 124. PtdSer also has an important metabolic function in mammals as a
specific activator of protein kinase C, along with DAG and Ca2+ions 124. Protein
kinase C has been found in trout spleen and dogfish rectal gland, and the activities
were stimulated by the addition of PtdSer 26. There was no difference between
PtdSer from bovine brain and PtdSer from trout liver (7.5-times greater 22:6n-3 and
very low (n-6)PUFA) in their ability to stimulate fish protein kinase C26. In contrast,
trout liver PtdSer was not as effective as bovine brain PtdSer in the activation of rat
spleen protein kinase C27. Studies using the calcium ionophore A23187 and phorbol
esters (analogues of DAG) in goldfish ovarian follicles and testes have implicated a
role for protein kinase C in the stimulation of steroidogenesis in these tissues 243'245.
2.3.2. Platelet-activatingfactor Platelet-activating factor (PAF), 1-O-alkyl-2-acetyl-

sn-glycero-3-phosphocholine, is a biologically active phospholipid synthesized by
inflammatory cells 215. In mammals, PAF is implicated in the activation and/or
aggregation of platelets and leukocytes and may be a mediator of hypotensive
activities as well as causing increased vascular permeability, vasoconstriction and
contraction of smooth muscle 21s. PAF synthesis has been demonstrated in gill,
kidney, liver and spleen of rainbow trout and its production was stimulated by
calcium ionophore A2318724~ The enzyme responsible for deactivation of PAF,
PAF acetylhydrolase, has been shown in fish serum 41. In mammals, the reacylation
of lyso-PAF is highly specific for arachidonic acid, and it is the 1-alky-2-arachidonyl-
glycerophosphocholine species that is the substrate for the synthesis of PAF via
144 D.R. Tocher
an arachidonate-specific phospholipase A2 that also produces arachidonate for

eicosanoid synthesis 2~s. The lipid substrates, most of the enzymes involved and their
specificities, and the roles of PAF, are all unknown in fish.
3. Nutritional roles
3.1. Embryonic development

Upon hatching, the larvae of many fish species are unable to feed as the intestinal
tract and mouthparts are not fully developed 37. Therefore, throughout embryogen-
esis in the egg and then early larval development up to first feed, the larvae gain
nutrition from the endogenous energy reserves of the yolk. In some fish, particularly
marine fish, phospholipids account for the great majority of lipid stored in the
egg23~ Phospholipids are not normally regarded as an energy source, and in most
situations this generalization is true. However, there is evidence that, during em-
bryonic and early larval development of some fish, phospholipids are preferentially
utilized not only for cell division and organogenesis but also for energy.
During development of rainbow trout, phospholipid was continuously metabo-
lized, whereas triacylglycerol was not utilized until after hatching 213. In the Atlantic
salmon, PtdCho, which originally accounted for over 94% of the total phospholipids
of the egg, was continuously metabolized so that in the fry a PtdCho:PtdEtn ratio
approaching that of salmon muscle was obtained s4. In the whitefish (Coregonus
sp.), total phospholipid decreased slightly and PtdCho decreased from 76 to 61%
of the total phospholipids by the time of hatching ~s7. During development of the
grass carp (Ctenopharyngodon sp.) phospholipids were utilized continuously during
development ~2e. The data suggested that a significant portion of the phospholipid
was utilized for energy ~22. PtdCho declined in absolute terms during embryonic
development of the Atlantic herring and cod, whereas catabolism of neutral lipid,
primarily triacylglycerol was initiated after hatching 6s,237.
Actual oxidation of the phospholipid fatty acids was not measured in any of
these studies. However, the data clearly show a decrease in the absolute amount
of phospholipid, usually PtdCho, during embryogenesis in the egg, particularly in
marine fish species. Therefore, more than simple redistribution of PtdCho from
lipid stores into larval tissue is occurring. One consequence of complete catabolism
of phospholipid for energy would be the loss of important PUFA. In herring,
however, there was selective retention of PUFA during embryonic development,
as PUFA produced by the hydrolysis of PtdCho were selectively retained in the
neutral lipid pool at the expense of monoenes 236. A similar process occurred in cod
eggs during embryonic development, where it was calculated that approximately
one third of the 22:6n-3 produced by PtdCho catabolism was incorporated into
triacylglycerol and sterol ester 65.
3.2. Larval diet

From the data obtained on lipid metabolism during embryonic development in cod
eggs, it was postulated that PtdCho, replete in (n-3)PUFA, should represent a major
portion of the lipid in artificial diets for fish larvae 6s. This was the same conclusion
as obtained from earlier work 114, although the experimental route to the con-
clusions was rather different. Several studies suggested that glycerophospholipids
were superior to triacylglycerols in promoting growth in larval ayu (Plecoglossus
altivelus) 115-117, red sea bream (Chrysophrys major) 114, white sturgeon (Acipenser
transmontanus) 1~ and cod 173. The addition of glycerophospholipids to the diets of
larval ayu also reduced the incidence of malformations 115. Kanazawa concluded
that glycerophospholipids containing a PUFA at position-2 and with an inositol or
choline head group were indispensable for normal growth and survival of larval
ayu 114. This requirement was in addition to the essential fatty acid requirement of
the fish.
The mechanism behind the growth-promoting ability of Ptdlns and PtdCho in
larval fish has never been conclusively established. Although glycerophospholipids
will present a higher percentage of PUFA in their total fatty acids compared to tria-
cylglyeerols, this itself cannot account for the effects as one of the growth-promoting
glycerophospholipids, soybean lecithin 117, is not a rich source of the (n-3)PUFA re-
quired in large amounts by larval fish 224. Furthermore, the inherent differences in
fatty acid compositions between PtdCho and Ptdlns suggest that supply of particular
fatty acids is not a factor. It may be that the glyeerophospholipids are superior to
triacylglycerols because, being more polar, they are more easily emulsified and,
therefore, more rapidly hydrolyzed and assimilated. Triacylglycerol assimilation may
be more susceptible to bile salt limitation than glycerophospholipid assimilation,
especially in larval fish fed diets rich in triacylglycerols whose digestive capacity may
not be fully developed. Definition of the development of larval hepatic, intestinal
and pancreatic functions in larval fish is required to elucidate this area.
Glycerophospholipids have a number of other effects - when added to formu-
lated pelleted diets - related to their polarity, and physical properties in general.
Glycerophospholipids may improve the mixing, binding and texture of pelleted di-
ets. The improved binding effects may help to reduce the leaching of water-soluble
vitamins or components of the diet, such as choline and inositol. With specific
reference to choline and inositol, supplying these essential components of the diet
in the form of glycerophospholipid may, therefore, be more efficacious. Finally, as
well as being more easily emulsified themselves, glycerophospholipids may improve
the emulsification of the neutral lipid in the diet and so aid the digestion and
absorption of all dietary lipid.
VI. Conclusions and perspectives
The class and fatty acid compositions of glycerophospholipids in fish tissues are now
well characterized. However, the enzymic systems underpinning the compositions,
including those responsible for the adaptation of the compositions in response
to dietary and environmental influences, are poorly characterized. More work is
required to elucidate the general pathways of glycerophospholipid biosynthesis,
catabolism (tissue and subcellular sites) and interconversion in fish. Fundamental
questions related to the digestion and absorption of glycerophospolipids remain
146 D.R. Tocher
unanswered. For instance, what are the intestinal enzymes that act on glycerophos-
pholipids, what are the absorbed products and how exactly are they metabolized
in the mucosal ceils? There is little information on the appearance of intestinal
phospholipases in larval fish, especially marine fish, which is a crucial question with
important consequences for aquaculture. Ontogenetic studies of the development
of hepatic, intestinal and pancreatic functions in larval fish are required in this area.
The specific uptake of glycerophospholipids from serum lipoproteins, including
enzyme involvements, mechanisms and tissue specificities are all virtually unknown
in fish. The significance and roles of individual molecular species of glycerophos-
pholipids needs to be studied, including the effects of diet and environmental factors
on specific molecular species. The precise mechanisms that allow 20:4n-6 to be the
main eicosanoid precursor in (n-3)PUFA-rich fish are still unclear. What are the
specificities of phospholipase A, cyclooxygenase, lipoxygenases and other enzymes
in the eicosanoid pathways? The operation of the Ptdlns cycle, including the tissues
and stimuli linked to the cycle, and the glycerophospholipid class specificities of
phospholipase C are unstudied in fish.
The above list of areas requiring study has a common link. The enzymes involved
in glycerophospholipid metabolism, such as phospholipases and acyltransferases
require isolation and characterization in fish tissues. Only then will it be possible to
answer most of the questions raised above.
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CHAPTER 7
Amino acid metabolism in fish
KARL JORSS AND RALF BASTROP

Universitdt Rostock, Fachbereich Biologic, Zoologisches lnstitut, Universiti~tsplatz 2, D-02500
Rostock 1, Germany
I. Introduction
II. Pools of free amino acids
III. Blood
IV. Intestine (gut)
V. Liver
VI. Kidney
VII. Muscle
VIII. Interorgan amino acid fluxes
Acknowledgement
IX. References
I. Introduction
From the nutritionist's standpoint, the animal organism has one common pool of
free amino acids (FAA or AA) into which all amino acids freed by the decomposi-
tion of food and body protein and all non-essential amino acids (NEAA) produced
by biosynthesis flow. In turn, this pool also feeds all reactions in which AA are con-
sumed (Fig. 1). However, this standpoint is too simplistic for a mechanistic analysis
of amino acid metabolism. The following facts must be taken into account: (1) the
total FAA pool is broken down by the cell membrane into an intracellular and an
extracellular pool; (2) the intraceUular pool is organ specific and probably also cell
specific. The intracellular pools of different organs are linked by the extraceUular
pool (blood circulation). The various organs can therefore interact with each other
as the nutritional status of the organism changes; (3) transport mechanisms are
needed for movement of amino acids from the extracellular to the intracellular
pool. They are also needed for the flow of amino acids from the 'milieu ext~rieur';
in this case both the intestinal epithelium (apical and basolateral cell membrane)
must be crossed instead of a single cell membrane; and (4) the composition of
the intracellular AA pool is strongly influenced by the enzymes present in the
cell and their transport across the cell membrane. The same factors also govern
the amino acid distribution among cellular compartments such as the cytosol and
mitochondda.
In the following we shall discuss mainly the roles and interactions of the organs in
the fish AA metabolism on the basis of our knowledge of the mammalian organism,
160 K. Jfrrss and R. Bastrop
,.,,,,,.
DIETARY PROTEINS BODY PROTEINS
[ 1
AMMONIA
<x-KETO ACIDS
GLUCOSE CO2, H20 LIPID

Fig. 1. Pool of free amino adds.
and draw attention to corresponding control points, since exhaustive descriptions

of the sequences of biochemical reactions in which AA are involved in fish have
already been published 27,136.145,149.To simplify the discussion, we shall consider the
amino acids as being metabolized mainly in just four organs: the intestine, liver,
kidney and skeletal muscle.
II. Pools of free amino acids

The only comparative analysis published so far about the AA pools in different
organs deals with the channel catfish (Ictaluruspunctatus), a freshwater species 155.
Presenting FAA concentrations in the serum, liver, muscle, gill, gut tract, kidney,
heart, brain and spleen after 48 h of fasting, this analysis permits the following
conclusions to be drawn.
1. Each organ has a specific FAA concentration.
2. In general, the liver and kidney contained the highest concentration, expressed
as #mol/g tissue, of most of the amino acids.
3. The total serum level of proteinogenic FAA of about 2.0/tmol/g is strikingly low
compared to that, say, of the liver (130/~mol/g) and muscle (16.5 #mol/g). This
indicates the importance of ATP dependent transport mechanisms for the uptake
of the various amino acids into the fish cell at least after the postabsorptive phase.
4. If the total amount of each AA is considered as split up among the different
organs, it can be seen that in the channel catfish the muscle contains the largest
fraction of them all except aspartic acid.
Amino acid metabolism in fish 161
Less comprehensive analyses dealing with other teleosts (e.g. rainbow trout 27)
lead to the same conclusions.
IlL B l o o d
The intracellular AA pools of the different organs are linked by the blood circu-
lation system. It is striking that the blood of the channel catfish contains less than
1% of the total of each proteinogenic amino acid after 48 h of starvation ~ss. This
fraction naturally increases more or less temporarily after feeding 1~ The blood
itself has an intracellular AA pool (mainly in the red blood cells, RBC) and an
extracellular pool (plasma). An interaction between erythrocytes and the plasma
may play a role in convectional AA transport. The AA concentrations in the plasma
relative to those in the cells of the various organs and the variations in these ratios
after feeding by the fish are also of interest.
In carp (Cyprinus carpio), coho salmon (Oncorhynchus kisutch), rainbow trout
(Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus), plasma contains
47.5, 58.2, 52.6 and 9.0% of the sum of the essential amino acids (EAA), respectively
and 32.8, 38.3, 33.4 and 22.3%, respectively, of the sum of the NEAA. The
remainder resides in the intracellular pool. Ogata and Arai 111 calculated the FAA
content of erythrocytes from the levels found in plasma and whole blood. Their
studies also revealed that intraceUular concentrations of taurine (2-aminoethane
sulfonic acid), aspartic acid and glutamic acid were the highest (> 10-fold) relative
to those found in the plasma. The AA must be actively transported across the
membrane to achieve the concentrations found in the RBC. An Na+-dependent
mechanism which transports L-serine has already been identified in the erythrocytes
of brown trout, Salmo trutta 44.
The species-specific nature of the intracellular and extracellular AA distribution
is shown by the fact that the lowest intracellular FAA concentrations were found as a
rule in the coho salmon. Moreover, the concentrations of all EAA in erythrocytes of
the channel catfish is distinctly higher (>3-fold) than in the plasma. It is conceivable
that erythrocytes perform a transport function particularly for amino acids whose
concentrations in the RBC change rapidly after feeding. The faster an amino acid
can enter the erythrocytes, the more probable it seems that the RBC are involved in
its transport, thereby increasing the transport capacity of the blood for that AA.
The blood amino acid pattern reflects the net results of digestion, absorption and
subsequent utilization. The free amino acid pattern in the plasma is influenced par-
ticularly by the composition of the protein in the food 48. This applies especially to
the EAA. Plasma FAA levels generally undergo a marked increase after feeding to
reach a peak level or a plateau, then return to prefeeding values. The time at which
the peak post-feeding concentrations are reached may vary considerably among fish
species and experimental conditions. A corresponding correlation has been found
between dietary and systemic free essential amino acids at least in the rainbow
trout 1~ the carp 115 and the channel catfish 153. Wilson and coworkers 153 interpret
this as a form of metabolic conservation of EAA to ensure adequate circulation
162 g. Jarss and R. Bastrop
levels for protein synthesis, whereas the NEAA in the diet are metabolized and
altered to a great extent.
Postprandial changes in plasma and erythrocyte FAA levels have been studied in
parallel only for the carp. According to Ogata 1~ the RBC appear to be implicated
in convectional transport only for tyrosine, phenylalanine, tryptophan, histidine,
isoleucine and leucine. These EAA may be particularly easily transferred from the
plasma to erythrocytes and back. They probably enter the cell mainly by diffusion
because, except for tyrosine, their concentrations in the plasma and in erythrocytes
scarcely differ 111. More species should be studied to define more precisely the
function of erythrocytes in the transport of FAA. Knowledge of these mechanisms
involved in the exchange of FAA between the plasma and erythrocytes will probably
be of decisive importance in this question, although attention should focus on
its capacity and speed. Attention should also be given to AA consumption by
erythrocytes for metabolism in addition to the question of their transport function.
Moderate activities of glutamate dehydrogenase (GDH, EC 1.4.1.2), aspartate
aminotransferase (AspAT, EC 2.6.1.1) and alanine aminotransferase (AIaAT, EC
2.6.1.2), the most important enzymes implicated in AA catabolism, have been found
in the erythrocytes of the rainbow trout 36. However, alanine oxidation rates are
quite low in trout erythrocytes 144. In carp erythrocytes, glutamine is more important
than glucose as an oxidative substrate. Its importance in this respect is exceeded
only by lactate and pyruvate 13~ Glutamate, aspartate and isoleucine are oxidized
to a much smaller extent. The oxidation of glutamate was only one tenth that of
glutamine in the presence of other substrates. Tiihonen and Nikinmaa ~3~ attribute
this to the properties of the carp erythrocyte membrane. The report that catfish
hepatocytes produce five times more ammonia when using glutamine as a substrate
than when using glutamate substantiates these findings 16.
IV. Intestine (guO

The intestine plays a special role in maintaining AA homeostasis. The passage of
the AA from the intestinal milieu across the intestinal wall into the circulation
involves transceUular transport. When considering the absorption process, it is nec-
essary to distinguish between uptake of AA by the brush-border membrane and
its basolateral release into the circulation. In addition, depending on the physio-
logical status of the fish, basolateral transport mechanisms selectively remove FAA
from the circulation for organ-specific AA metabolism. When considering the AA
requirements of the intestine, the rapid turnover of enterocytes and their proteins
must be taken into account besides the AA needed for intermediary metabolism.
The general anatomical structure of the fish intestinal wall is similar to that of the
higher vertebrate intestine. An absorptive epithelium faces the luminal contents
and is responsible for the transport of nutrients (e.g. amino acids), ions and water.
However, in contrast to mammals, the intestinal epithelium of fish is not divided
into villus and crypt regions 37. According to Vernier 141, a regional differentiation of
the intestine in teleost fish gives rise to an anterior intestine, in which amino acids
and peptides are absorbed, and a posterior intestine where proteins are absorbed in
macromolecular form. Epithelial cells of the posterior intestine are characterized by
a heterophagous process for dietary proteins. Vernier ~4~ also attributes nutritional
as well as immunological functions to the posterior intestine. Basically, amino acids
can cross the apical and basolateral cell membrane with the aid of Na+-dependent
carriers, Na+-independent carriers or simple diffusion. Studies of amino acid up-
take into intact epithelia and brush-border membrane vesicles (BBMV) have shown
that the transapical step consists of 'active' accumulation. Much less is known
about the mechanisms of AA transport across the basolateral membrane ~2~ In
the eel (Anguilla anguilla), the intestinal brush-border transport of all AA tested
(a-aminoisobutyric acid, a-(methylamino)isobutyric acid, N-methylglycine, alanine,
glycine, glutamic acid, lysine, phenylalanirie and proline) was performed by Na +-
dependent secondary transport systems. Na+-independent systems were detected
for alanine, glycine and lysine. An additional significant diffusional transfer across
the brush border was also observed for all of the above amino acids. At least
four distinct Na+-dependent carrier mechanisms exist in the brush-border of the
ee118,127,128,142:
1. A cationic system transporting exclusively lysine and arginine.

2. A second specific system for the anionic amino acids glutamic acid and aspartic
acid.
3. A third mechanism responsible for the transport of proline and N-methylated
amino acids; this system is inhibited completely by alanine and partly by pheny-
lalanine.
4. A neutral amino acid system accepting most neutral AA such as alanine, glycine,
serine and cysteine. Comparable systems also exist in the brush border of the
mammalian small intestine s8.
Like the mammals, fish also have multiple transporters with overlapping speci-
ficities. The Na+-dependent nutrient transport mechanisms are energized in the
final analysis by Na+/K+-ATPase. The intracellular Na + level is maintained at a
low level by this enzyme, which pumps Na+across the basolateral membrane. The
energy produced by ATP hydrolysis and invested in the Na + pump is thus stored
in the inwardly directed Na + gradient. A group of carrier proteins in the mucosal
membrane provide the means by which the Na + influx is coupled to the 'uphill'
movement of nutrients (AA) into the cell. This mechanism can only function if
K +channels are present for recycling the K + ions, and these were recently found by
Loretz and Fourtner as in the basolateral membrane of intestinal epithelial cells of
a goby (Gillichthys mirabilis). Thus, the intestinal intake of amino acids is possibly
controlled at least at three points (Fig. 2): (1) carrier protein; (2) Na+/K+-ATPase;
and (3) K + channel.
In this context it is interesting to note that Reshkin and coworkers 119 induced par-
allel changes in the apical glucose transporters and the basolateral Na+/K+-ATPase
of the upper intestine (anterior intestine) of tilapias (Oreochromis mossambicus) by
means of hormone treatment. The apical enterocyte membrane of fish transports
dipeptides besides FAA 12'118. A comparative study into the uptake of phenylalanine
164 K. Jarss and R. Bastrop
LUMEN
ii i iiiiii i
9~| a .
i I
BLOOD
Fig. 2. Intestinal intake of amino acids.
and glycyl-L-phenylalanine into BBMV of the anterior intestinal epithelium of Ore-

ochromis mossambicus acclimated to sea water showed that glycyl-L-phenylalanine
is transported intact by a cationic-independent facilitated diffusion mechanism dur-
ing which the dipeptide is rapidly hydrolyzed in the vesicle. The dipeptide transport
mechanism is independent of the systems carrying L-phenylalanine ns. Such systems
might perform an important function during the early stages of digestion when
peptide concentrations in the intestinal lumen are high. The transepithelial amino
acid absorption system requires exit pathways across the basolateral membrane.
It must be assumed that basolateral uptake mechanisms also exist. Their function
would be to nourish the epithelial cells from the blood when amino acid intake
from the intestinal lumen is limited. Less is known about the mechanisms serving
the transport across the basolateral membrane of the vertebrate intestine than
about the transport mechanisms of the apical membrane because the former are
less accessible for experimentation ~9'Ss'n~ The fact that FAA are transported into
the blood on the one hand while, as we have seen, transport mechanisms must
simultaneously exist for the uptake of amino acids out of the circulation on the
other is an additional complication.
Experiments with basolateral membrane vesicles (BLMV) from the intestinal
epithelium of seawater-adapted European eels (Anguilla anguilla) have shown that
the mechanisms vary considerably. Among the six free L-AA that were tested,
only proline and glutamic acid appeared to be transported through the basolateral
membrane by Na+-dependent carriers. The simultaneous presence of an inward
Na +gradient and an outward K + gradient was necessary for glycine, while alanine,
lysine and phenylalanine were transported by Na+-independent facilitated diffusion
and, apparently, simple diffusion n~ Owing to the ionic gradients existing in vivo,
Amino acid metabolism in ftsh 165
ion-dependent transport mechanisms will not serve the translocation of AA into the
circulation. They must in some way be linked to the specific metabolic requirements
of the enterocytes and serve the active uptake of glutamic acid and glycine from
the circulation into the intestinal epithelial cell. The decisive processes taking
place in the basolateral membrane and serving as the last step in the transcellular
translocation of amino acids from the intestinal lumen into the circulation are
probably facilitated diffusion and free diffusion. Facilitated diffusion by means of
carriers and free diffusion are possible due to the accumulation of FAA in the
intestinal epithelial cell and may, in the final analysis, therefore also be dependent
upon the step energized by Na+/K+-ATPase, i.e. the Na+-dependent uptake of free
amino acids across the apical membrane. This raises the question of the degree to
which the various A.A, transport mechanisms of the intestine are involved in the
control of AA homeostasis, because the luminal milieu varies according to diet
composition and time after feeding. In addition, the regulation mechanism must
affect both the brush border and the basolateral transport mechanisms.
Experimental results have been published only for brush-border transport in
fish. Buddington and coworkers 14 compared proline uptake across the brush border
membrane in nine fish species of differing natural diets which all received the
same diet. The proline transport rates varied much less among species than
glucose uptake, since species with different natural diets still have similar protein
requirements. In adaptation experiments with Oreochromis mossambicus, a protein-
rich and a protein-deficient diet revealed no differences in the maximum rate
of proline transport 131. In other words, regulation apparently serves to match
transporter activity to a running average of diet composition over many days.
However, fish might also possess a basic set of AA transporter proteins, and their
activity would be capable of instantaneous modulation. This kind of regulation
mechanism would match the different AA transport systems to the current gut
content. Allosteric actions of amino acids on AA carriers are conceivable as
triggers.
The most important functions of the intestinal epithelial cells in quantitative
terms are probably proliferation, protein synthesis and the transport of solutes and
ions. All three of these are linked in two ways with amino acid metabolism or
transport: protein synthesis, the basic condition for the synthesis of enzymes and
transporter proteins, form the basis for cell proliferation. It requires a balanced
spectrum of FAA. Protein synthesis and transport functions, like regulation pro-
cesses involving phosphorylation, require ATP, which is probably obtained to a great
extent by the oxidation of amino acids. Of the total protein synthesis taking place in
a rainbow trout weighing 80 g one hour after the last feeding, the proportion found
in the whole digestive tract is 31% at 10~ and 39% at 18~ However, only 10%
of this is deposited 33,34. Thus, in absolute terms, the digestive tract has a higher
protein turnover than any other fish organ. The presence of key enzymes in the gut
implies that the oxidation of FAA accounts for a substantial fraction of the ATP
production. Wilson 152 studied the tissue distribution of the three most important
enzymes of amino acid catabolism in the channel catfish. The activities of alanine
aminotransferase and aspartate aminotransferase per gram organ fresh weight and
166 K. J~irss and R. Bastrop
per mg protein were distinctly lower in the intestine than in the kidney and liver.
However, consideration of the total organ activities per 100 g body weight revealed
that gut glutamate dehydrogenase was quantitatively the most important enzyme
(see below).
Liver Gut tract
(U/IO0 g fish weight) (U/100 g fish weight)
AspAT 40.7 18.2
AIaAT 28.0 10.3
GDH 3.4 4.9
Since this applies only to GDH, it does not necessarily imply that transdeamination
plays an important role. As stated earlier, active basolateral uptake of glutamic acid
from the blood has been observed in the eel (Anguilla anguilla). It is also interesting
that, of 22 AA tested in the sea water adapted Japanese eel (Anguilla japonica),
only L-glutamine, L-glutamic acid, L-alanine and v-alanine stimulated ion and
water transport in the intestine 2. GDH serves to introduce glutamine and glutamate
carbons into the citrate cycle in the form of a-ketoglutarate. Neither of these AA
appears to undergo transamination in this case because, unlike in the case of ~.-
alanine, the transport effects were not inhibited by aminooxyacetate. Interestingly,
the rate of glutamine and glutamate uptake into the intestinal epithelial cell
probably limits the transport effect of these AA in the Japanese eel and not
their intraceUular enzymatic turnover 3. The effect of v-alanine is particularly
interesting and requires further study. D-Alanine is probably deaminated oxidatively
to pyruvate by a D-amino-oxidase. Such an enzyme has been found in the pyloric
caeca of the rainbow trout 3s. Another important fact was reported by Mural et
al.l~ a considerable temporary (3-6 h) increase in the ammonia concentration was
observed in the hepatic portal vein of force-fed rainbow trout receiving a casein diet
relative to that in starved fish. This transient effect had almost vanished within 12 h,
whereas the aspartate and glutamate levels did not increase.
In summary, the intestine (gut tract) of fish has a high AA requirement relative
to that of the whole organism due to its high protein turnover. Moreover, FAA must
be oxidized on a considerable scale in the gut, so that during the postabsorptive
phase certain amino acids must be extracted from the circulation. This probably
applies to glutamine and glutamic acid. This implies that the gut is also involved in
the ammoniogenesis of the fish to an extent that has yet to be defined. Glutamine is
an important energy source for the mammalian intestine, which extracts it from the
blood. However, in mammals the intestine releases alanine into the circulation22,35.
Gluconeogenesis from FAA and the channeling of the carbon skeletons of FAA into
fat synthesis probably fail to play a role in the actual mucosal epithelium.
V. Liver
The function of the liver is very closely intertwined with that of the intestine
in that it filters the products of digestion and releases them into the circulation
for use by other organs. The amino acids transported through the basolateral
membranes of the enterocytes pass along the hepatic portal system to the liver of
the fish. The postprandial changes in the FAA have been studied in the plasma and
hepatopancreas of the carp (Cyprinus carpio11~ and in the plasma and liver of the
rainbow trout is~ In the case of the carp, a close correlation was found particularly
between EAA in the diet, in the blood plasma and in the hepatopancreas four
hours after the last feeding. In the rainbow trout, however, the FAA in the liver
remained fairly stable throughout the postprandial period. Taking the postprandial
changes in the AA concentrations into account, it was found that in the trout
the liver concentrations of taurine, aspartie acid, glutamic acid, glutamine and
alanine were several times higher (5- to 33-fold) than the plasma concentrations.
Van der Boon and colleagues 134 reported similar differences between blood and
liver concentrations of taurine, aspartic acid, glutamic acid, glutamine, asparagine
and glycine in the goldfish (Carassius auratus). It can safely be assumed that
ATP-dependent mechanisms are involved in AA uptake from the circulation. Such
mechanisms can possibly regulate the metabolism of various amino acids in the
fish liver in the same way, for instance, as alanine metabolism is regulated in the
mammalian liver 24,32. However, Haschemeyer 5~ showed that the uptake of a mixture
of 15 radioactively marked amino acids supplied through the hepatic portal vein is
very rapid in the oyster toadfish, Opsanus tau. AA uptake rates into the intracellular
space peak within one minute at a body temperature of 24"C and within about
two minutes at ll*C. It is therefore improbable that AA transport limits protein
synthesis in the liver. This need not apply to other reactions, however. The amino
acyl tRNA synthetases are known to have much higher substrate affinities than
amino acid catabolizing enzymes 145.
According to Campbell and coworkers 16, the higher rates of ammoniogenesis
from glutamate, glutamine and aspartate by isolated mitochondria than by the
whole cell suggest that the transport of these three amino acids may be limiting
for their metabolism by intact catfish hepatocytes. Since the liver is the central site
of intermediary metabolism, it is not surprising that it is also the most important
organ governing AA homeostasis in the fish. Hepatic parenchymal cells are the
main site of transdeamination 91,146 and gluconeogenesis 82 ,93 ,96 ,98,100 Moreover, the
liver is an organ with a high protein turnover 33,34. Blood AA levels rise considerably
in hepatectomized eels (Anguilla anguilla and Anguilla japonica) 67,77, while sham
operated and hepatectomized eels do not differ in their levels of ammonia excretion.
If L-alanine is injected into starved fish, ammonia excretion increases perceptibly
only in the sham-operated specimens 77. In fed fish, ammonia is produced mainly in
the liver, whereas in the postabsorptive state, kidney, musculature and intestine also
contribute significantly to total amino acid deamination. As a rule, activities of AA
catabolizing enzymes are high in the liver 136,145,152. Apart from direct deamination
of a few AA by specific enzymes 27, the main pathway for ammoniogenesis in
the liver is transdeamination (Fig. 3). The purine nucleotide cycle88, which is an
important route for release of ammonia in fish muscle, is of little quantitative
significance for ammoniogenesis in the fish liver 17,135.
The transfer of the ammonia to c~-ketoglutarate (2-oxoglutarate) is a basic
168 K. 1arss and R. Bastrop
AMINO ACID ot-KETOGLUTARATE NADH
AMINOTRANSFERASES GLUTAMATE DEHYDROGENASE

m,,. , , .,
+
~-KETO ACID GLUTAMATE NAD + H_O
z
Fig. 3. "l~ansdeamination.
prerequisite for transdeamination. In the liver of the goldfish (Carassius auratus)

glutamate is formed rapidly from alanine, tyrosine, aspartate and branched-chain
AA. Ammonia from histidine is also transferred quickly to a-ketoglutarate 135
in either of two ways. Ammonia may be liberated by histidase and transferred
to a-ketoglutarate by glutamate dehydrogenase as suspected by van Waarde 135.
Alternatively, the ammonia may be transferred to pyruvate by a histidine amino-
transferase. However, reports on the presence of the latter enzyme in the fish
liver are contradictory 27. Glutamate dehydrogenase (GDH), the key enzyme in
transdeamination, is located in the mitochondrial matrix. Ammoniogenesis by trans-
deamination is therefore a mitochondrial process. Being an allosteric enzyme,
GDH is the decisive point of control for catabolism in the AA metabolism. It
is inhibited by ATP and GTP and stimulated by ADP, AMP and leucine 39,53.1~.
In hepatocytes, AlaAT and AspAT are present both inside and outside of the
mitochondria 1~,147. If ammonia production from a particular AA is stimulated by
ADP, then transdeamination is probably involved. Similarly, inhibition of ammonia
production from a particular AA by the transamination inhibitor aminooxyacetate
can be regarded as a sign of the deamination of the AA through transdeamina-
tion. Experiments of this kind showed that aspartate and alanine are deaminated
by transdeamination in the mitochondria of catfish hepatocytes, whereas serine is
deaminated only to a small extent by this pathway. In the catfish hepatocyte, serine
is probably deaminated mainly by a cytosolic serine dehydratase (EC 4.2.1.13) 16. In
the rainbow trout hepatocyte, serine is reported to be mainly transaminated in the
gluconeogenesis process 147. However, the main route of serine catabolism in fish
liver is probably through the action of serine hydroxymethyltransferase (EC 2.1.2.1).
In this reaction, the serine reacts with tetrahydrofolate to form glycine and N ~-
N 10 -methylenetetrahydrofolate 27 ' 107 . In vitro, ammonia production from various AA
by hepatocytes of Ictalun~ punctatus decreases in the order asparagine, glutamine
> alanine, serine > aspartate, glutamate. The order for isolated mitoehondria is
Amino acid metabolismin fish 169
glutaminc > glutamate > alaninc, serine, aspartatc and asparaginc 16. The authors
drew two conclusions from this result: (1) the higher rates of ammoniogencsis
from glutamate, glutamine and aspartate by isolated mitochondria suggest that the
metabolism of these AA in the intact channel catfish hepatocyte is limited by their
transport through the hepatocyte membrane; and (2) the higher or equal rates of
ammoniogenesis from glutaminc, glutamate, aspartate and alanine by mitochondria
suggest that the mitochondrial compartment is the main site of their catabolism.
Conversely, the markedly higher rate of ammoniogenesis from asparagine by
intact hepatocytes than by isolated mitochondria implies that asparagine catabolism
takes place mainly in the cytosol, although only 24-34% of total asparaginase (EC
3.5.1.1) activity is found in the cytosolic fraction of channel catfish liver 17. The
teleost liver can produce ammonia from glutamine (Gin) and asparaginc (Asn)
at high rates 17,126,139. Both glutaminase I (EC 3.5.1.2) and glutaminase II (EC
2.6.1.5) are present in the teleost liver 17,2~ The phosphate dependent glutaminase
I predominates quantitatively and is located in the hepatocyte mitochondria. The
intracellular localization of asparaginase is apparently still not known exactly 16. At
any rate, both enzymes catalyze reactions yielding substrates that can flow directly
into transdeamination:
Asparaginase A s n + H 2 0 ~ Asp + NH3 (1)
Glutaminase I Gin + H 2 0 ~ Glu + NH3 (2)
Finally, all or some carbon atoms of many AA end up in pyruvate or certain
intermediates of the citric acid cycle. In carnivorous fish, where the natural diet is
high in protein and low in carbohydrate, amino acids constitute a major source of
energy 136. Liver cells utilize AA as an energy source either directly by oxidation of
the carbon skeleton or indirectly by conversion of the carbon skeleton to glucose.
AA can also be used in fat synthesis 149. Glutamic acid, both from the body protein
of the fish 45'154 and from food protein 1~ is probably the AA present in the largest
quantities. In whole body tissue of fish, this is followed by aspartic acid, lysine,
leucine and alanine (g/100 g amino acid). It should be remembered that glutamate
and aspartate can be produced from glutamine and asparaginc respectively in a
single, hydrolytic step. In view of the quantitative predominance of glutamic acid
in protein and its close link to the citric acid cycle, one might think that these
AA would be used mainly for oxidation. This also applies to a lesser degree to
aspartic acid and alanine. On the other hand, all three of these AA are glucogenic.
Moreover, oxidation is the only possibility for the essential ketogenic AA lysine
and leucine. All these AA or their carbon skeletons can naturally be used in the
synthesis of protein and fat respectively.
The turbot (Scophthalmus maximus) oxidizes about twice as much glutamic
acid and alaninc as it does leucine and phenylalanine, regardless of the protein
content of its diet 26. High glutamic acid and alanine oxidation rates have also
been reported in the hepatopancreas of the carp (Cyprinus carpio L.; refs. 105
and 106). In an experiment with alanine, histidine, proline, lysine, leucine, serine,
asparagine, glycine and valine it was found that hepatocytes of rainbow trout
oxidized mainly asparagine, serine and alanine 42. The main AA oxidized by little
skate (Raja erinacea) hepatocytes was glutamate followed at some distance by
glutamine, aspartate, alanine, proline and serine 1~ Summing up, it can be stated
that, despite all species-specific differences, glutamic acid, aspartic acid, alanine,
serine, asparagine and glutamine are the most important AA in fish liver with
respect to oxidation.
Gluconeogenesis in the liver or in isolated hepatocytes of the fish is among the
main topics of current research 93.9s,1~176
The most important AA for gluconeogenesis
in the fish liver appear to be alanine, glutamic acid, serine and aspartic acid 56,1~176
whereby alanine and glutamic acid appear to be channeled preferably into this
pathway. In the fish liver, gluconeogenesis from serine 147 and aspartic acid 117 is
less important in quantitative terms than that from alanine. Alanine obtained
by proteolysis in the musculature forms the starting point for de novo glucose
synthesis in the liver of migrating salmon (Oncorhynchus sp.; refs. 41 and 95). In the
teleost Paralabrax clathratus, gluconeogenesis from alanine and glutamate increases
3.9- and 2.2-fold respectively in the course of starvation for 14 days, whereas
gluconeogenesis from aspartate decreases 16.7-fold 7.
The question remains how the AA are distributed between direct oxidation via
the citric acid cycle and gluconeogenesis in the liver? In terms of the stoichiometry
of the carbon, glucose synthesis from alanine dominates over oxidation to CO2 in
hepatocytes from sockeye salmon (Oncorhynchus nerka) 41. The same also applies
to alanine in hepatocytes of the American eel (Anguilla rostrata); but in these cells
aspartate is converted to CO2 rather than used for glucose synthesis4~ After a major
analytic effort, Jungas and coworkers 69 came to the following conclusion regarding
the liver of omnivorous mammals: the amount of ATP produced by AA oxidation is
roughly the same as the amount needed to convert amino acid carbon to glucose.
According to these authors, the daily supply of amino acids in the diet cannot be
totally oxidized to CO2 in the liver, mainly because ATP production would then
exceed demand in the liver. At present, no such budget can be formulated for fish
owing to the lack of experimental data.
In fish, the liver is also the main site of fat synthesis m. Although protein
would seem to the major natural source of carbon for fatty acid synthesis in
fish, little is known concerning the flow of amino acid carbon into fat 55.m. After
intraperitoneal injection of U-14C-labeled glutamate into juvenile carp (Cyprinus
carpio), much more radioactivity was found in hepatopancreatic lipid than in
glycogen. [U-14C]Glucose was hardly incorporated into hepatopancreatic lipid l~
Correspondingly, more [14C]alanine than [14C]glucose was converted into fatty acids
by slices of rainbow trout liver in vitro54. However, radioactivity from [14C]aspartate
was not incorporated into fatty acids in hepatocytes from the eelAnguilla rostrata ~7.
The distribution of AA in the fish liver between protein synthesis on the one
hand and oxidation, gluconeogenesis and lipogenesis on the other is probably
influenced strongly by nutritional status and the consequent hormone balance. The
most important exogenous factors influencing AA homeostasis in the liver are
food quantity and composition. Intracellular AA concentrations are also strongly
influenced by salinity in euryhaline teleosts64.
It has often been shown that the liver enzymes serving AA metabolism in omnivo-
rous mammals possess a certain degree of adaptability to food composition 47. After
analyzing the corresponding experimental findings for fish, Cowey and Walton 27
came to the conclusion that 'there is little effect on the activities of amino acid-
catabolizing enzymes'. At this point, we consider this conclusion a little premature
for the following reasons: (1) too few species have been studied so far; (2) in the
studies published to date, the units chosen to express enzyme activities were often
unsuitable for such generalized conclusions; and (3) the experimental conditions
(e.g. diets in which protein was replaced by poorly metabolizable carbohydrate) led
in some cases to inappropriate conclusions.
To assess the importance of changes in liver enzyme activity for the whole fish
organism it is necessary to consider the total liver activities relative to body weight
(U/g liver fresh weight x liver-somatic index (%) -- U/100 g body weight). Conclu-
sions relating to the liver cell itself can be obtained by expressing enzyme activities
relative to DNA levels. The decisive enzymes for transdeamination, i.e. glutamate
dehydrogenase, aspartate aminotransferase and alanine aminotransferase, should
be considered on the basis of our own results in this respect. It should be noted
that aminotransferase activities were all expressed as total activities (cytosolic +
mitochondrial isoenzymes). It is therefore possible that any adaptive effect would
affect only one isoenzyme, and the activity of this isoenzyme would in turn change
more relative to the total activity. GDH activity is usually measured as glutamate
formation in the presence of the allosteric effector ADE An adaptive change in
GDH activity, should it reflects a change in the amount of enzyme, must be con-
sidered specially because the activity of the existing molecules of this aUosteric
enzyme can already be greatly modulated by ligands 53. In a two factor (feeding
x salinity) experiment 73 with tilapia, Oreochromis mossambicus, starvation reduced
the activities (U/100 g body weight) of AspAT and AIaAT in the liver to less than
half of the original levels. GDH activity was reduced almost to half the original
level. Salinity, which strongly affects the free amino acid concentration in the liver
of O. mossambicus 4, had no effect on the activities of these enzymes 73. A series
of experiments with rainbow t r o u t 72,74-76 led to quantitatively similar results as far
as salinity effects are concerned. Food deprivation also significantly reduced GDH
activity (U/mg DNA) per liver cell in carp (Cyprinus carpio) 5. The activity of GDH
in the rainbow trout liver cell appears to play a different role of the enzyme is
regulated by a route differing from that observed in other teleostean fishes. After
starvation or a reduced ration, the activity of the trout liver enzyme stabilizes at the
level of a well nourished fish, whereas AspAT activity is slightly, but significantly,
reduced71,75, 76.
Generally reduced activities of important AA catabolism enzymes were found
in the livers of tilapia and carp in accordance with the lower NH3 excretion by
starved fish reported by Fromm 43 and Infante 66. This suggests that the activity of
this metabolic pathway is reduced as a result of starvation. The fact that there
is no loss of GDH activity (U/100 g body weight) in the liver of starved rainbow
trout can only mean that the liver plays a greater role in gluconeogenesis in this
species than in others. After intraperitoneal injection of [U- 14C]glutamate into
172 K. Jiirss and R. Bastrop
rainbow trout after a 30 day starvation, De la Higuera and Cardenas 56 found

more radioactivity in blood glucose and liver glycogen than in fish receiving a
high protein diet. Gluconeogenesis from [14C]alanine increases by similar amounts
in rainbow trout fed a high protein diet or if the animals are starved 2s. In
ammoniotelic fish, gluconeogenesis is linked to ammoniogenesis by a route that
appears to be functionally similar to the way gluconeogenesis is linked to urea cycle
in ureotelic mammals 69. The function of gluconeogenesis is supported by the fact
that gluconeogenesis from pyruvate and alanine is activated by leucine in the same
way as GDH in Anguilla japonica 52,53.
Although the role of transdeamination must be sought primarily in connec-
tion with gluconeogenesis during starvation, in fish receiving a high protein diet
transdeamination is probably mainly responsible for channelling AA to the citric
acid cycle and fatty acid synthesis despite the increased level of gluconeogenesis
under these conditions. The oxidation of AA increases considerably when fish are
fed a high protein diet 26,146. In rainbow trout, this is accompanied by increased
conversion of food protein into body fat 74'83 if the fish are given an isoenergetic
protein rich, but low fat diet, the liver activities (expressed in units per 100 g
body weight) of AspAT and AIaAT increase substantially7~ This also applies to
GDH and the lipogenic enzymes which, under these conditions, are also adjusted
to a higher activity level (Table 1). This effect is most obvious when the activities
obtained for the diets A and C are expressed as ratios. Similar adaptive response
of enzymes involved in transdeamination will be scarcely discernible, if at all, when
the protein in the diet is replaced by carbohydrate (see ref. 27 for a review of
various findings). In other words, the enzymes implicated in transdeamination in
the teleost liver respond adaptively in several fish species, although not to the
same degree as has been recorded for mammals 47. Some enzymes metabolizing
amino acids are also involved in biosynthetic pathways. As the case of ornithine
decarboxylase (ODC; EC 4.1.1.17) demonstrates, such enzymes may also be subject
to marked adaptive changes in fish. ODC is the first and rate-limiting enzyme in
polyamine biosynthesis. After a 12 clay starvation period, hepatic ODC activity in
TABLE 1
Effect of diet on glutamate dehydrogenase and NADPH-generating enzymes in rainbow trout

(Oncorhynchus mykiss) liver
Diet A Diet B Diet C

Raw protein (% of dry weight) 24.0 46.2 57.8
Raw fat (% of dry weight) 25.0 12.1 6.7
Enzyme Enzyme activity in units/mg DNA Ratio C/A
Glutamate dehydrogenase 11.7 4- 1.0 12.2 4- 1.1 18.0 4- 0.8 1.5
Glucose 6-phosphate dehydrogenase 3.1 4- 0.1 3.9 4- 0.4 9.8 4- 1.2 3.2
6-Phosphogluconate dehydrogenase 1.7 4- 0.1 2.2 4- 0.3 5.6 4- 0.7 3.3
Glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) and 6-phosphogluconate dehydrogenase (E.C.
1.1.1.43) are considered lipogenic enzymes, since they produce cytosolic NADPH equivalents required
for fatty acid synthesis 46. Data from Bastrop and Jiirss (unpublished results).
A m i n o acid metabolism in fish 173
brook charr (trout), Salvelinus fontinalis, increased from almost zero to about 60
pmol CO2/h/mg protein within 24 h after refeeding 6.
VI. Kidney
Formally, amino acid homeostasis in the kidney (excretory part) is similar to that
in the intestine. Amino acids not only enter the tubular cells from the lumen,
but also from the peritubular blood. This flux opposes the cellular exit of AA of
luminal origin. Amino acids taken from both sides of the tubular cell enter the same
pool. Owing to the specific enzymes in the renal cell, certain AA may be removed
from the pool or the concentrations of certain NEAA may increase is a result of
synthesis. In the channel catfish (I. punctatus), the concentrations of most FAA are
quantitatively about the same in both the kidney and the liver. Only the methionine
concentration is about 2.6 times as high in the kidney as in the liver in this species ls5.
In the rainbow trout, in contrast, the methionine concentration in the liver is higher
by a factor of 5 than in the kidney 149. The differences between FAA concentrations
liver and kidney are altogether more pronounced in the rainbow trout than in the
channel catfish.
Renal taurine and alanine concentrations are very high in the latter species (> 20
/zmol/g) 155. Relatively high glutamate concentrations are feature common to the
renal FAA pools of the catfish (14/zmol/g), rainbow trout (6.9/tmol/g) and goldfish
(2.3 /.tmol/g) 79'149'155. The concentrations of most FAA are higher in the kidney
than in the plasma of all three of these teleost species. ATP-dependent transport
mechanisms are probably mainly responsible for maintaining the concentration
difference. This applies equally to FAA extracted from the ultrafiltrate and to any
taken up from the peritubular blood vessels.
The process of AA absorption from the ultrafiltrate appears to be more efficient
in fish than in mammals because the carp and channel catfish excrete fewer AA
in their urine than humans 112. In vertebrates, the amino acids filtered out at the
glomeruli are re-absorbed in the early proximal tubule 12s. The FAA concentration
differences between the ultrafiltrate, tubulus cell and blood plasma suggest that
an uphill gradient has to be overcome during amino acid resorption across the
brush-border membrane, whereas the peritubular exit step is a downhill process.
Eveloff and colleagues 31 published a comparative study of the BBMV in the kidney
and intestine of winter flounder, Pseudopleuronectes americanus. The uptake of
L-alanine was stimulated by sodium and inhibited by the addition of 10 mmol
L-phenylalanine in BBMV from both organs. Much of what we know about FAA
uptake into the intestinal epithelial cells probably also applies in principle to the
less studied cells of the proximal tubulus of the fish kidney. The peritubular exit of
amino acids is not well understood even in mammals 125. Active transport has been
shown to be involved in the peritubular uptake of glutamine, glycine, taurine and
acidic AA for protein synthesis, gluconeogenesis and ammoniogenesis at least in
the case of mammals 125.
In the case of P. americanus it was found that taurine is actively secreted in
174 K. larss and R. Bastrop
the tubulus. This also applies to species of fishes with aglomerular kidneyss. The
movement of taurine across the peritubular membrane was identified as the Na +
and Cl- dependent concentrating step in taudne secretion 124. Unlike fish, mammals
do not actively secrete taurine in the kidney 125. In most species, cellular, organ
and whole body taurine concentrations are regulated by transport; biosynthesis and
metabolism are of only minor importance in this respect 6s. This invites one to
conclude that, since taudne is of great importance to fish as an osmotic effector,
the ability to secrete it actively through the kidney represents a specially developed
control point for regulating the amount of taurine present in fish. Taurine enters
the fish organism with food and is probably also synthesized in the fish liver by a
pathway resembling that found in mammals 157.
If expressed in units per g fresh weight, the activities of the three most important
transdeamination enzymes (glutamate dehydrogenase, aspartate aminotransferase
and alanine aminotransferasr in the kidney of Ictalur~ punctatus 152, Cyprinus
carpio and Oncorhynchus mykiss 122 are close to those in the liver. In the teleost
kidney, AIaAT and AspAT are distributed extra and intramitochondrially in the
same ways as in the liver79. In an experiment in which the organ-somatic index was
used to express activities relative to 100 g fish weight, Jfirss and colleagues7s showed
that GDH capacities were slightly higher in the kidney of rainbow trout than in
the liver, while renal AspAT activity was twice as high as the liver AspAT level. In
Salmo salar, however, renal GDH and AspAT activities expressed in U/g were only
about one tenth as high as in the liver~.
At any rate, as in the liver, transdeamination appears to be quantitatively the
main process by which ammonia is formed in the fish kidney. The purine nucleotide
cycle does not appear to contribute to ammonia production in the kidney of the
fish79'146. GDH is regulated by an aUosteric process and, therefore, is also important
controlling AA metabolism in the kidney. Bittorf 9 compared the hepatic and renal
GDH of the rainbow trout after nearly 150-fold purification. Both renal and
hepatic forms of the enzyme were inhibited by ATP (1 mmol/l), GTP (1 retool/l),
malate (5 retool/l) and isocitrate (5 mmol/l). Among 15 AA (administered at a
final concentration of 5 mmol/1) tested, only leueine activated hepatic and renal
GDH. The renal activity was activated to similar degrees in both directions: in the
presence of leucine, the rate of oxidative deamination was enhanced to 197% of
control. Conversely, reductive amination was increased to 212%, respectively. Liver
GDH was activated much more strongly towards reduetive amination: oxidative
deamination increased to 139% of control, while reduetive amination reached
313%.
Hughes et al. 62 found that, among five Salvelinus namaycush tissues tested (an-
terior kidney, posterior kidney, gill, liver and muscle), specific activity was highest
for branched-chain amino acid aminotransferase (EC 2.6.1.42; BCAAT) in the pos-
terior kidney. Similar results relating to this extra and intramitoehondrial enzyme
have been obtained for other teleost specieszT. BCAAT catalyzes the first step in the
catabolism of leueine, isoleucine and valine, in which the amino group is transferred
either to a-ketoglutarate or to the corresponding branched-chain a-ketoaeid. By
synthesizing glutamate, this enzyme leads to the oxidative deamination of branched-
chain AA (BCAA) by GDH. The ketoacids remaining after transamination serve

as substrates for irreversible oxidative decarboxylation, which is the second step
in the BCAA degradation pathway and is catalyzed by branched-chain a-ketoacid
dehydrogenase (EC 1.2.4.4; BCKAD) a multienzyme complex located on the in-
ner surface of the inner mitochondrial membrane 49. Unlike BCAAT, BCKAD is a
highly regulated enzyme at least in mammals. Among the AA-degrading enzymes
only BCKAD and phenylalanine hydroxylase are regulated by phosphorylation-
dephosphorylation mechanisms 49. BCKAD has so far been studied only in the liver
and red muscle of the A. rostrata 97 and in the liver of the rainbow trout 3~ The
reaction catalyzed by BCKAD yields the corresponding CoA compounds:
BCAAT BCKAD
leucine - , a-ketosiocaproate --, isovaleryl-CoA (3)
isoleucine - , a-keto-~l-methylvalerate - , r (4)
valine - , a-ketoisovalerate - , isobutyryl-CoA (5)
The subsequent reactions are comparable to those involved in fatty acid oxidation 49.
Naturally, these reaction sequences do not necessarily all take place completely in
the kidney of fish. Part of the ketoacids from transamination of the branched-chain
AA can leave the renal cells and be translocated to other tissues by the circulation.
The activities of serine catabolizing enzymes in the kidney and liver respectively
of the rainbow trout reported by Walton and Cowey 148 yield the following activity
quotients between these organs (Table 2). The direct deamination of serine is
catalyzed by serine dehydratase in the cytosol:
Serine dehydratase serine --, pyruvate + NH + + H 2 0 (6)
In other words, the available tissue distribution of the serine catabolizing enzymes
indicate that the kidney is an important site of serine catabolism in fish. The
mammalian kidney releases serine into the circulation 1. A major comparative study
of ammoniotelic teleost species 28 showed that specific arginase (EC 3.5.3.1) activity
in the kidney was exceeded only by that in the liver. However, if the arginase
activities in the various organs are compared with the total arginase activity in the
whole fish, it is found that renal arginase activity in fed rainbow trout is only about
one third lower than in the liver 21,76. Van Waarde and Kesbeke ~39 report that in the
TABLE 2
Serine catabolizing enzymes in rainbow trout liver and kidney
Kidney/liver activity ratio

Serine dehydratase (E.C 4.2.1.13) 2.1
Serine pyruvate transaminase (E.C. 2.6.1.51) 0.5
Serine-hydroxy methyltransferase (E.C. 2.1.2.1) 0.3
Data from Walton and Cowey14s.
176 K. larss and R. Bastrop
goldfish (Carassius aumtus) renal asparaginc synthetase and glutamine synthetase

activities are 0.6- and 2.1-fold as high as in the liver. They could not detect
asparaginase (EC 3.5.1.1) in the kidney of this species. Mitochondrial glutaminase
I (EC 3.5.1.1) is found with relatively high activities in the kidneys of various
fish species 79'146. Altogether, it is evident that the kidney possesses the enzymes
needed for the degradation of substantial amounts of AA. The catabolized FAA
are probably only oxidized there and used for gluconeogenesis, whereby changes in
the proportions between the AA will naturally also occur due to the synthesis of
N A A and the release of degradation products into the circulation. The first step
towards channeling the AA to oxidation or gluconeogenesis is direct deamination
(e.g. of histidine) and/or indirect transdeamination. As in other vertebrates 8~
ammoniogenesis in the fish kidney could serve a special function in acid/base
regulation. Glutamine serves as a source of ammonia for regulation of the acid/base
balance in mammalian kidneys~ Pequin 113 and Pequin and Serfaty 114 showed that
the blood ammonia level increases from 0.68 to 4.60/zg/ml during passage through
the kidney. In view of the previously mentioned results achieved by Kenyon 77 with
the hepatectomized eel it can be assumed that the kidney contributes appreciably to
total ammoniogenesis in the postabsorptive fish.
In obligate air-breathers such as Arapaima gigas, the renal contribution is proba-
bly highest of all regardless of nutritional status. In this species, the enzymes found
in the unusually large kidney which is not divided into head and trunk regions 57 sug-
gest that transdeamination is the main form of ammoniogenesis. Kidney and liver
homogenates from Stizostedion vitreum deaminated more glutamine and asparagine
than any of the other 18 AA tested by Stieber and Cvancara 126. However, according
to King and Goldstein 79, intact renal cells (teased renal tubules) from the goldfish
(C. auratus) produced most ammonia from aspartate. These authors report that AA
were utilized in the following order for ammoniogenesis: aspartate > > alanine >
glutamine > glycine > glutamate. After incubating kidney slices from the elasmo-
branch Squalus acanthias with AA, King and Goldstein 7s found that the quantitative
sequence for ammonia formation was glutamine > > aspartate, glutamate, alanine,
glycine. However, when analyzing the results of experiments with intact renal cells,
it is necessary to take possible variations in the transport capacity of the renal cell
into account.
According to King and Goldstein 78'79 acidosis leads to an ammoniogenic re-
sponse in the kidney of the goldfish and dogfish. The authors suspect that ammonia
production in the kidney of Squalus acanthias is regulated by the relative activities
of glutaminase and glutamine synthetase working in a substrate cycle between glu-
tamine and glutamate + NH +. Such a cycle is scarcely conceivable for the teleost
kidney owing to the low glutamine synthetase activity79. Ammonia is produced
in the teleost kidney and is released mainly into the circulation (for subsequent
excretion via the gills), but is also excreted to a lesser degree with the urine 116.
The remaining carbon skeletons are oxidized directly in the citric acid cycle or
used for gluconeogenesis. In view of the enzymes it possesses, the kidney must be
considered second only to the liver in importance as a site of gluconeogenesis s2'96.
Slices of posterior kidney from Salmo salar, like liver slices from the same species,
channel much more alanine derived carbon into oxidation (to CO2) than into glu-
coneogenesis. The gluconeogenic flux in the kidney, on a gram wet weight basis, is
somewhat lower than in the liver 96. However, since it has been shown in the case of
the liver that the distribution of AA between oxidation and gluconeogenesis varies
according to the specific AA and is also species specific, the above findings can not
be generalized. The experiments performed by Jiirss and colleagues 75,76 with the
rainbow trout show that renal amino acid catabolism decreases relative to that of
the liver after several weeks of starvation. Total renal activities of GDH, AspAT
and arginase were markedly suppressed at the end of the starvation period, while
the corresponding hepatic activities remained almost unchanged. After experiments
with plaice, Pleuronectes platessa, Moon and Johnston 99 came to the conclusion that
the role of the liver in amino acid metabolism is maintained and slightly enhanced
by food deprivation, whereas that of the kidney is reduced. In the rainbow trout, re-
nal GDH and AspAT activities scarcely respond to diets with different protein:lipid
ratios, whereas activities of these enzymes in the liver increase significantly as the
protein content of the diet increases 74. Lupianez et al. 89 obtained corresponding
results for the different responses of GDH and AIaAT activities in the rainbow trout
kidney and liver to increasing diet protein content also when protein was replaced
by carbohydrate. However, the kidney seems to be important for the metabolism of
branched-chain AA. Hughes et al. 63 showed that the addition of leucine or valine
to the diet induced a significant increase in the activity of branched-chain amino
acid transferase only in the posterior kidney of Salvelinus namaycush, and not in the
muscle or liver. Leucine therefore appears also to be an important substrate in the
teleost kidney.
VII. M u s c l e
The somatic muscle system accounts for over 50% of the body weight of most fish 151
and thus possesses the largest FAA pool in absolute terms lss. Many fish species
possess both red and white musculature, but in most species red muscle accounts
only for about 6% of the total muscle weight 87. Therefore, only the white muscle
represents the largest FAA pool in fish. It is evident from the enzyme profile that
red muscle is aerobic, while the white muscle is anaerobic 13,29. The morphological
and biochemical differences between the two muscle types form the basis for their
different functions. Electrophysiological studies show that during slow swimming
the propulsive force is brought about entirely by red muscle and that, as the
swimming velocity increases, white muscle is progressively recruited 61. With respect
to protein turnover, the muscle differs fundamentally from other organs. According
to in vivo experiments undertaken by Fauconneau and Arna133,34 only about 33%
of total protein synthesis in fed rainbow trout takes place in the muscle. However,
52-76% of the synthesized protein is deposited in the muscle. This is considerably
more than in such metabolically active organs as the liver or intestinal tract, which
deposit only about 5 and 11% of the synthesized protein, respectively 33,34. The
red and white musculature differ in this respect, too. The red muscle of rainbow
trout retains 30.4% of the synthesized muscle, whereas white muscle retains no less
than 79.7% ~~ It can therefore be concluded that fewer amino acids flow into the
FAA pool from protein degradation in the muscle of non-starved fish than in other
organs. Since neither lipogenesis nl nor gluconeogenesis from amino acidss2,1~ play
a quantitatively important role in fish muscle, the main physiological purposes of
AA catabolism in the muscle must be two-fold. First, to support ATP production
for locomotion, and second, to supply other organs with substrates during periods
of depletion.
The different functions of the two muscle types are also reflected in quali-
tative and quantitative distinctions between their AA pools. Van der Boon and
coworkers z33 report the following quantitative AA sequences in the goldfish after
6 days of starvation: in white muscle histidine is most prevalent, followed in de-
creasing abundance by glutamine, glutamate, asparagine, glycine and alanine. In red
muscle, the sequence is: His > Gin > A s n > Gly > Ala > Glu. Individual ratios for
white over red muscle are: His = 1.7, Asn = 1.4, Gln = 1.1, Gly = 2.8, Glu = 0.28,
Ala = 2.8. The concentrations of these and a few other AA in the circulation system
of the goldfish are low, and the high intracellular concentrations in the muscle must
therefore be achieved by means of active transport mechanisms or, in a few cases,
synthesis. Since, in addition, taurine reaches its highest concentration (>20/~mol/g
wet weight) in the muscle of the goldfish - as it does in many other fish species
- the FAA pool of the goldfish muscle can be considered typical of cyprinids 134. It
is interesting to note that the taurine concentration is high in the liver of rainbow
trout, whereas its concentration in the muscle is relatively low~Ss.
AIaAT, AspAT, GDH and branched-chain amino acid transferase (BCAAT) ac-
tivities are many times higher in the red, oxidative muscle of teleosts than in the
white, glycolytic muscle99'135. The highest AspAT activities are found in the red
muscle and heart of fishs3'135. The intra and extramitochondrial AspAT in this
case, forming a component of the malate-aspartate shuttle, very probably serves
the transport of electrons. Of the three enzymes catabolizing serine, low activities
of serine-hydroxy methyltransferase have been detected in the red muscle of the
rainbow trout only1~. Van Waarde 135 reports that aspartate is the only actively
transaminated amino acid in the epaxial white muscle of goldfish, whereas signifi-
cant transamination of aspartate and the branched chain AA leucine, isoleucine and
valine takes place in the lateral red muscle. Asparagine and glutamine synthetase
activities are higher in the red muscle of the goldfish than in the white muscle, while
asparaginase and glutaminase activities were too low to be measured (activity <0.05
units) 139. Phosphate-dependent glutaminase (EC 3.5.1.2) has been detected in mi-
tochondria isolated from the mudskipper (Periophthalmus chrysospilos68). While red
muscle shows a much greater ability to catabolize AA than white muscle 135, the
anaerobic white muscle needs glycogen to perform work. One of the functions
of the AA pool in the white muscle of fish is probably to provide a reserve AA
supply for other organs and tissues. In salmonids, the bulk of the energy used for
sustained swimming (with the red muscle) is obtained by oxidizing amino acids.
Van den Thillart 132 injected 14 C-labeled glucose, palmitate or glutamate, alanine
and leucine individually into rainbow trout (O. mykiss) and measured the rates of
Amino acid metabolism in/ish 179
oxygen consumption and CO2 production at rest and while swimming at 80% of the
maximum speed. Glucose oxidation was extremely low in both resting and actively
swimming fish. Protein and lipid oxidation were estimated to provide 80 and 20%
respectively of the total substrates oxidized in the whole fish at rest and 90 and 10%
respectively during sustained swimming. The rate of oxidation of leucine increased
much more during the transition from the resting to the swimming state than that
of alanine and glutamate.
Amino acids must be deaminated before their carbon skeletons can enter the
oxidative metabolism. Two major pathways have been described for the liberation
of ammonia from amino acids: the mitochondrial glutamate dehydrogenase route
using glutamate and the cytosolic purine nueleotide cycle using aspartate as the
ultimate substrate. Intact aerobic red muscle mitochondria from goldfish (Carassius
auratus) produce large amounts of aspartate and small quantifies of ammonia
when incubated with glutamate, ADP and orthophosphate. Aspartate is the only
nitrogenous end product during anaerobiosis 136. Malate, IMP, AMP and NH3 levels
increased in the white muscle after direct electrical stimulation of the goldfish
myotome 14~ whereas aspartate decreased. The concentration of lactate in the red
muscle increased 2.6-fold, while the corresponding increase in the white muscle
was 13.2-fold. However, blood ammonia levels did not change. The results cited
here suggest that the enzymes of the purine nucleotide cycle play some role in
NH3 production, but do not prove that the purine nucleotide cycle supplies the
NH3 from the muscle for excretion through the gills. Mommsen and Hoehachka 94
obtained data sets which gave additional insight into the role of white muscle purine
nucleotide cycle in the rainbow trout by measuring white muscle metabolites after
exhaustion and during the time of their recovery. The changes in the metabolite
concentrations in the exhausted muscle were similar to those reported by van
Waarde and Kesbeke 14~ for electrically stimulated goldfish muscle. The specific
dynamics of ATP, IMP, NH3, malate and aspartate during the 25 h recovery period
led Mommsen and Hochachka to the conclusion that the purine nucleotide cycle
as such cannot operate as a functional unit. It seems more probable that two
temporally and functionally separate phases exist.
Phase 1. Exhaustive exercise: A M P 2- + H 2 0 ---, IMP 2- + NH3. T h e NH3 can
accept protons released during the cleavage of ATP and thus serve as an H+buffer
with the concomitant formation of NH +" NH3 + H + ---, NH +.
Phase 2. The bulk of the NH + is retained in the white muscle and transferred to
a-ketoglutarate by GDH during recovery. Transamination yields aspartate, and the
proton picked up from the medium by NH3 is released backinto the medium when
aspartate is added to IMP.
Only further experiments will show whether this reinterpretation for the func-
tion of the components of purine nucleotide cycle is correct. However, from the
results presented by Mommsen and Hochachka 94 it seems certain that the purine
nucleotide cycle of the muscle does not contribute appreciably to ammoniogenesis
for excretion. Experiments performed by Ip and coworkers 20.68 with mitochondria
prepared from the lateral muscle of different species of mudskippers (Perioph.
thalmus chrysospilos, Boleophthalmus boddaerti, Periophthalmodon schlosseri) also
180 K. :l~rss and R. Bastrop
suggest that the purine nucleotide cycle does not play a leading role in the net am-
moniogenesis. Muscle mitochondria preparations from all three species produced
most ammonia after the addition of glutamine, but only very small amounts after
the addition of alanine or aspartate. No ammonia at all was produced when serine
was added. The quotients of the ammonia produced with the two most important
substrates (glutamine/glutamate) were 34.0 for P. chrysospilos 35.0 for B. boddaerti
and 36.0 for P. schlosseri, respectively. Since glutamine can supply only twice as
much ammonia as glutamate, it must be concluded that glutamine enters the mito-
chondria more quickly and/or to some extent produces ammonia more quickly and
without transdeamination. Aminooxyacetate and bromofuorate (an inhibitor of glu-
tamate dehydrogenase) completely inhibit ammonia production from glutamate and
aspartate in all three species. In 1?.chrysospilos, ammonia production from glutamine
was curtailed to 21% of control by bromofuorate and reduced to 32% of control
by aminooxyacetate 6s. Apart from illustrating the importance of transdeamination
in ammoniogenesis from glutamine, these findings suggest that the permeability
of the internal mitochondrial membrane may not be the same for glutamine and
glutamate (transport mechanisms). Moreover, in view of the inhibitory effect of
aminooxyacetate, an as yet undetected aminotransferase in the fish muscle must
be involved in ammoniogenesis from glutamate. Finally, we must remark, without
commenting on their quantitative significance, that the musculature may contribute
to ammonia excretion by fish in one of the two following ways: (1) direct release into
the circulation system of ammonia from transdeamination (in view of the enzyme
profile, predominantly in the red musculature) and breakdown of the glutamine
(white muscle; ref. 133); or (2) transport of the amino group to the liver in the
form of alanine or glutamine (cf. section VIII). Clearly, the enzymatic machinery
responsible for the production of ammonia from glutamine requires more detailed
investigation.
VII. Interorgan flux of amino acids

The physiological necessity of an amino acid flux between organs stems from
the biochemical specialization of the various tissues and organs and the need to
guarantee supplies of their substrates during the postabsorptive status and long
periods of food deprivation. Hormones are doubtless important for regulating the
parts played by the organs in AA metabolism, but their role will not be discussed
here. Any discussion on interorgan amino acid nutrition presupposes knowledge
of the blood circulation, transport mechanisms of the cell membrane, and AA
metabolism in the various tissues and organs. In the case of fish, our knowledge in
these areas is far from complete and based on only a few species. Therefore, this
section will discuss problems rather than present known facts. Unlike that of fish,
the flow of amino acids between organs in mammals has been a topic of metabolic
research for many years 5'22'23'35'69'92. It therefore seems appropriate to take the
mammalian interorgan AA flux as a starting point for considering its counterpart in
fish. Theoretically, of course, the fate of every single AA should be analyzed, but this
Amino acid metabolism in .fish 181
has not yet been done even for mammals. However, the quantitatively important
fluxes of alanine, glutamine and branched-chain amino acids (BCAA) have been
analyzed quite extensively. In the mammalian system, AA interconversion takes
place after the amino acids from a protein rich diet have been taken up into the
mucosal cells. In other words, the liver receives a mixture of AA processed by
the intestine, whereas the peripheral organs receive a different AA mixture from
the liver. In the mammalian mucosal cells, glutamine, glutamate and aspartate are
converted into alanine, citrulline, ornithine, proline and ammonia 92. Except for the
synthesis of citrulline and ornithine, a similar transformation appears to take place
in fish. Citrulline and ornithine are synthesized in the mammalian intestine and
likely fuel the hepatic urea cycle. This route of carbon/nitrogen flux could therefore
be useful only for ureotelic fish such as a species of tilapia living in an alkaline lake
(Oreochromis alcalicus grahami156)and the Gulf toadfish, Opsanus beta 143. However,
nothing is known in these two species about the role of the intestine in this respect.
Murai et al. 1~ report that levels of alanine, proline and ammonia in the hepatic
portal vein increase rapidly regardless of diet composition in force-fed rainbow
trout receiving a defined casein or amino acid diet. Surprisingly, considerably less
glutamate and aspartate appear in the plasma than might be expected from the AA
content of the diet. This finding suggests that glutamate and aspartate are oxidized
(intestinal ammoniogenesis) or converted into proline and alanine. Glutamate is
the precursor for the biosynthesis of proline 129. At present, however, no enzymes
are known to be involved in proline biosynthesis in fish 27.
Alanine might be produced from glutamate and pyruvate by transamination, and
ammonia is probably liberated from glutamate by oxidative deamination and from
aspartate by transdeamination. As noted in section IV, intestinal GDH, AIaAT and
AspAT activities are sufficiently high for these reactions. It is therefore evident that
the AA mixture in the hepatic portal vein of fish has been modified by the intestinal
metabolism. Murai and colleagues 1~ also measured the differences between the
AA concentrations in the hepatic portal vein and the hepatic vein. AA uptake by
the liver was very intense for up to 12 h after feeding. The intensity of hepatic
uptake accounted for almost half of the amounts absorbed in the portal blood
for almost all EAA, including the BCAA. In mammals, however, the liver extracts
only small amounts of BCAA from the portal blood 49. Perhaps the fish liver can
utilize BCAA better than the mammalian liver. This is suggested not only by the
findings of Murai and coworkers 1~ but also by the enzymes present in the fish liver.
Upon being taken up into the intracellular hepatic AA pool, BCAA are used for
protein synthesis or undergo transamination to yield BCKA and glutamate. In the
rat, BCAAT activity in the liver amounts to only 11 or 12% of the activity found in
skeletal muscle49. Hepatic BCAAT activity in Salvelinus namaycush is about 57% of
the activity found in the muscle 62, and Moon 97 reports that BCAAT activity in the
liver of Anguilla rostrata is even higher than in red muscle. However, activities of the
second enzyme in BCAA catabolism, BCKAD, are almost equal in the liver and red
muscle of A. rostrata, whereas in mammals its activity in the muscle is only 5-8% of
that found in the liver97.
Of the NEAA, the liver extracts quantitatively appreciable amounts of alanine
and proline from the portal blood in the rainbow trout ~~ Although a considerable
amount of information is available concerning the catabolism of alanine in the fish
liver (section V), proline has not been studied in this respect zT. Its carbon skeleton
could be used for glutamate synthesis 129 and thus ultimately flow into the large liver
pools of glutamate and glutaminr The interaction between liver and musculature
in AA homeostasis is seen most distinctly in the postabsorptivr fish and during food
deprivation. It is important to note that reversals in direction while fasting are not
characteristic of the major AA fluxes2z. Proteolysis in the muscle is the main source
of AA for other organs after starvation. Houlihan and colleagues s9 reported that
white muscle contributes 66% to the whole body protein loss during starvation.
This means that white muscle is mainly responsible for the maintenance of plasma
AA concentrations during food deprivation. In view of the concentration gradient,
the transport of the AA from the muscle into the circulation probably does not
consume ATE Blasco r al. 11 published a detailed study into the variation in time
of plasma AA levels in starved carp (C. carpw) without, unfortunately, analytically
distinguishing glutamine from glutamate. According to Ogata and Arai 111, the
glutamine concentration in the plasma of the carp is 28 times higher than the
glutamate concentration. We shall therefore assume that the changes recorded
by Blasco et al. 11 for glutamine plus glutamate refer only to glutamine. If this
assumption is correct, it would mean that the glutamine concentration in the carp
plasma was elevated from the 8th to the 50th day of starvation. The alaninr
concentration was abnormally high only from the 5th to the 19th day. BCAA levels
in the plasma of the starved carp were significantly depressed during the first week
of starvation. The plasma leucine and isoleucine concentrations in these fish were
significantly higher than normal from the 19th to the 50th day of starvation.
Muscle is the most important tissue for the oxidation of BCAA in mammals 2z,49
and fish 13z. This applies particularly to the red muscle owing to its special role
in sustained swimming, its blood flow and its enzyme profile. From the results
published by Blasco et al. 11, we can conclude that the demand for BCAA exceeds
proteolytic BCAA liberation in the carp during the first week of starvation. Accord-
ing to Loughna and Goldspink s6, protein degradation in the white muscle of starved
rainbow trout does not increase until after about one week. The increased amounts
of glutamine and alanine very probably entering the blood stream from the muscle
during starvation might sometimes be destined for organs. In mammals, for in-
stance, glutamine may be supplied to the intestine and kidneys 1'9~ Under starvation
conditions, alanine must be the main substrate for de novo glucose synthesis in the
liver. Hayashi and Ooshiro 51 observed that 14C-glucose synthesis from 14C-alanine
increased by 100% in A. japonica during starvation for periods ranging from 1 to
2 months. Moreover, alanine is known to be the single important source for de
novo synthesis of glucose in the liver of migrating sockeye salmon (Oncorhynchus
nerka) 41,95. There are signs that alanine is synthesized at the expense of other AA
in the fish muscle. Knapp and Wieser sl showed that the alanine concentration in
the muscle of starved rudd (Scardinus erythrophthalmus) increased 2.5-fold, whereas
the concentrations of other AA decreased. Leech and colleagues 84 reported that
the tail muscle of the spiny dogfish (Squalus acanthias) releases alanine after fasting
for one day. Moreover, in vitro experiments with isolated pelvic fin muscles revealed
that alanine biosynthesis was dependent on a source of added isoleucine or leucine,
but not glucose 84.
The following sequences of reactions seem conceivable for the synthesis of ala-
nine and glutamine in the light of what we know about mammalian metabolism 49.
Because a-ketoglutarate is an efficient amino-acceptor for BCAAT, BCAA amino
groups could be used to form glutamate which in the presence of pyruvate and
alanine aminotransferase can readily pass BCAA amino groups on to form alanine.
The amino group of the BCAA would then be carded to the liver by alanine for
transdeamination. In addition, in the presence of oxaloacetate and aspartate amino-
transferase, glutamate can, via aspartate, liberate the amino groups of BCAA as
ammonia via the purine nucleotide cycle, and ammonia can be transformed into glu-
tamine by glutamine synthetase. Carbon skeletons (pyruvate and a-ketoglutarate)
are needed for the de novo synthesis of alanine and glutamine. Pyruvate could be
produced by glycolysis and ketoglutarate could be taken from the citric acid cycle,
into which the carbon skeletons of the BCAA might also flow. The enzyme profile
(BCKAD) of the fish liver suggests that some of the branched-chain a-ketoacids
derived from the BCAA in the muscle are used in the fish liver in the same way
as Harper et al. 49 describe their use in mammals. The kidney has scarcely been
mentioned in this brief discussion of the interorgan amino acid flux owing to lack of
data. Its position in the AA homeostasis of the fish urgently needs studying.
Acknowledgement. The authors wish to express their thanks to Brian Patchett,

Intertext Rostock, for his help in translating the manuscript.
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Hochachka and Mommsen (eds.), Biochemistryand molecularbiologyoffishes, vol. 4
CHAPTER 8
Protein synthesis in fish
D.E HOULIHAN,C.G. CARTER*AND I.D. MCCARTHY

Department of Zoology, University of Aberdeen, TUlydroneAvenue, Aberdeen AB9 2TN, Scotland,
U.K. and * Department of Aquaculture, University of Tasmania, P.O. Box 1214, Launceston,
Tasmania 7250, Australia
I. Introduction
II. Measurement of rates of protein turnover
III. General model of protein turnover in fish
IV. Individual variation in protein turnover
V. Protein synthesis and free pool amino acid concentrations
VI. Energy cost of protein synthesis
VII. Species comparisons
VIII. Life history protein turnover
IX. Protein synthesis in tissues and cells
X. Conclusions
Acknowledgements
XI. References
I. Introduction
This chapter will discuss rates of protein synthesis in fish. It will principally explore
nutrition and protein synthesis in the whole animal and discusses the impact that
protein synthesis has upon rates of oxygen consumption. We believe that the
emphasis for future studies of protein metabolism should be on the individual
animal as it is becoming increasingly clear that the rate of food consumption
of the individual is a major determinant of individual rates of protein synthesis.
Before discussing protein consumption, growth and synthesis of individual fish it is
necessary to critically examine current methods for their measurement.
Possible relationships between rates of protein synthesis and oxygen consumption
have been the source of much discussion in mammals and are also an area of some
uncertainty in fish. However, an understanding of protein synthesis rates can act
as a mechanistic explanation for the linkage between energy consumption and
growth rate most commonly expressed in the energy budget formulation. It provides
a resolution of the incorrect assumption contained in models of fish (and other
heterotrophs) growth, such as the Putter-von Bertalanffy model, that growth and
metabolism are independent processes competing for the same energy source 1~
Higher metabolism does not necessarily imply that less energy is available for the
synthesis of proteins. Estimation of the energy cost of protein synthesis therefore
forms a major part of this chapter.
192 D.F. Houlihan, C.G. Carterand LD. McCarthy
Protein synthesis can be viewed at a number of levels. Whole-animal values can

be integrated into descriptions of assimilation/growth or assimilation/metabolism
patterns in different fish species and will be the focus of this chapter. The measure-
ment of protein synthesis rates in body organs and tissues can provide information
on the extent to which differences exist between various tissues and offer a challenge
in understanding the integration of organ metabolism into whole animal physiology.
For example, we still do not understand the significance of the ditferenees in rate of
mitochondrial protein synthesis between different tissues in fish53.
However, it is probably extremely hazardous to move between these levels to
draw conclusions on the effects of protein synthesis on oxygen consumption. For
example, it is unlikely that the stimulation in synthesis of individual proteins, except
perhaps actin and myosin, will impinge upon whole animal energetics. It has been
estimated that metallothionein synthesis in metal-challenged daphnids represents a
very small proportion of oxygen consumption ~2.
II. Measurement o f rates o f protein turnover
In all animals there is a continual cycle of synthesis and degradation of protein, in

conditions where the rate of protein synthesis exceeds the rate of degradation there
will be a net gain of protein and a net loss occurs when degradation rates exceed
synthesis rates 114.
The majority of methods for estimating protein synthesis measure the flux of an
amino acid or nitrogen 1~ This involves the use of tracer substances, i.e. amino
acids labelled with an isotope, that are given in a single dose or by continuous
infusion (reviewed in refs. 5, 104, 122, 129). The measurements, parameters and
formulae that are commonly employed in studies of protein growth, synthesis
and degradation and which will be used in this chapter are described in Table
1. Most often protein synthesis rates are expressed in fractional terms, i.e. the
proportion of the protein mass synthesised per day (ks, Table 1). The term protein
turnover was used by Waterlow and coworkers 122 to describe both protein synthesis
and degradation depending on conditions. The definition of protein turnover is
dependent on nutritional status; under maintenance conditions where there is no
net loss or gain of body protein, fractional rates of protein synthesis (ks) and
degradation (kd) will be equal and equivalent to the rate of turnover (kt), i.e. ks -
kd -- kt. Under conditions of protein loss the rate of degradation exceeds synthesis
and the rate of turnover will be equal to the synthesis rate, i.e. ks - kt; during
weight gain, de novo synthesis and resynthesis of degraded proteins contribute to
synthesis so turnover will be equal to degradation, kd -- kt (refs. 56, 127).
Almost all estimates of protein synthesis in fish have been made by measuring the
in vivo incorporation of a radiolabelled essential amino acid into body protein using
either constant infusion (e.g. refs. 31, 49, 112) or a single flooding dose (e.g. refs.
37, 40, 54, 59, 103) to administer the radiolabel. The available data for in vivo whole
animal estimates of protein synthesis, measured by a variety of methods, in rainbow
trout, Oncorhynchus mykiss (Walbaum), are collected in Table 2. Despite the range
Protein synthesis in fish 193
TABLE 1
Measurements, parameters and formulae used to describe protein turnover
Measurement Parameter Formula

Protein consumed Protein consumption rate [g prot. consumed/g fish prot./day] x 100
(g) kr: % day -1
Protein-nitrogen Protein-nitrogen kr x A EpN
absorption efficiency absorption rate
(AEpN: %) ka: % day -1
Protein content Protein growth rate [(In Pf - In Po)/time] x 100
(g) kg: % day -1
Specific radioactivity Protein synthesis rate [(Sb/Sa) x (1440/incorporation time)] x 100
of bound (Sb) and free ks: day -1
pool (Sa) phenylalanine
DPM nmo1-1
Protein synthesis Protein degradation rate kd = k , - kg
(k~) kd: day -1
RNA content (rag) Capacity for prot. synthesis (mg RNA/g protein)
Protein content (g) Cs: mg RNA g protein -1
Capacity for protein RNA Activity (ks x 10)/Cs
synthesis (Cs) kRNA: g protein synthesised
Protein synthesis g-1 RNA day -l
Protein consumption Protein retention efficiency (kg/kr ) x 1O0
Protein growth kg/kr : %
Protein synthesis Anabolic stimulation efficiency (kslkr)x 100
Protein consumption ks/kr : %
Protein growth Synthesis retention efficiency (kg/ks)x 100
Protein synthesis ks~k,:
Abbreviation used: Prot. = protein.
in methods and experimental variables, the maximum values for whole animal
fractional rates of protein synthesis are around 4-5% for rainbow trout of less than
200 g. In each of these studies, there was a range of measured synthesis rates which
is likely to be explained by differences in the nutritional status of individual fish
(see below). Although in vivo incorporation is the most commonly used method to
measure rates of protein synthesis, several studies have used isolated polyribosomes
from fish epaxial muscle to make in vitro measurements of rates of protein synthesis
(e.g. refs. 65, 71, 72).
In order to avoid the problems of variable free pool specific radioactivity and
amino acid recycling, the majority of studies in fish have used the flooding dose
method of Garlick and coworkers 43 to introduce the radiolabel in a single high dose
injection. The most commonly used radiolabel is 3H-phenylalanine and although
previous work has shown that a flooding dose of this amino acid does not affect
rates of synthesis 73,85, more work is needed in order to confirm this assump-
tion.
194 D.E Houlihan, C.G. Caner and LD. McCarthy
a)
1200
lOOO
|
4ram
.o
OD
q; 6oo
J=
Q.
_o 4oo
0
E
2OO
0 - I ' 9 ,, , L I , _ I
0 60 120 180 240 300 360

Time (minutes)
b)
~--~ 12oo
r
,1~ lOOO
t:,.
.?
o 8oo
E
I=
600
0 .... I , . . , .. 9 i 9
0 6o 120 180 24o 3o0 36o
Time (minutes)
9
2.5
sT. 2.0
1~ 1.5
~ 1.0
~ 0.5
0.00 60 120 180 240 300 360
Time (minutes)
TABLE 2
Comparison of whole animal fractional rates of protein synthesis in fed rainbow trout, Oncorhynchus
rnykiss, measured by different methods
Method Synthesis Weight Temperature Meals Reference

(% day -1) (g) (*C) (day -1 )
Caudal vein infusion 2.3-5.1 80 10 2 32
Flooding dose injection a 2.1 (4- 0.2) 2000 8 1 37
Flooding dose injection b 4.4 (4- 0.5) 50 11 1 40
Flooding dose injection b 1.2-4.5 80 10 1 82
Flooding dose injection b 0.6-1.5 200 14 1 c
Fed 15N-protein 1.5-4.5 117 14 1 19
a Injection of L-[U-14C]-arginine.
b Injection of L-[2,6-3H]-phenylalanine.
c McCarthy et al., in preparation.
The measurements that are needed for the successful calculation of protein
synthesis rates using the flooding dose technique are shown in Fig. 1. The data are
taken from the white muscle of 80 g rainbow trout at 10*C (McCarthy et aL, in
preparation). The concentration of phenylalanine in the tissues after the flooding
injection can (and should) be measured in order to demonstrate flooding of the
tissue free pool. Assuming a free phenylalanine concentration in the white muscle
of rainbow trout of 74 nmol g-1 wet weight 2~ a 12-fold increase above normal levels
is achieved following injection (Fig. la). Measurement of the protein-bound and free
pool phenylalanine-specific radioactivities are required to calculate the fractional
rate of protein synthesis (Table 1). It is important to construct time courses for the
specific radioactivity of the free and the protein-bound phenylalanine. Following
injection, the specific radioactivity of the free pool must be elevated and either
remain stable or show a slow linear decline over the incorporation period (Fig.
lb) in order to calculate synthesis rates 43,~. The labelling of the body protein
must be linear over the incorporation time (Fig. l c) and from the intercept of the
regression line it is possible to estimate how soon after injection the radiolabel
began to be incorporated into body protein. Several studies have validated the use
of the flooding dose method in fish at a range of water temperatures and using an
incorporation time of between 40 min and 6 h 16'41'54'59'73'123.
The flooding dose technique has been used to measure rates of protein synthesis
in fish from 1 g up to 500 g in weight by either intraperitoneal or intravenous
injection 16,54,59,75,123. Recently this technique has been used to measure synthesis
rates in fish of less than 1 g either by microinjection into file peritoneal cavity78,86
Fig. 1. (a) The mean white muscle free pool phenylalanine concentration (nmol Phe g wet wt-1), (b)
the mean white muscle free pool phenylalanine-specific radioactivity (Sa, DPM nmol Phe -1) and (c)
the mean white muscle protein-bound phenylalanine-specific radioactivity (S b, DPM nmol Phe -1) for
rainbow trout (80 g, 10oc)_ at various time intervals following a single caudal vein injection of 135 mM
phenylalanine and L-[2,6- 3H]phenylalanine (McCarthy et al. in preparation). The data are presented as
mean4-SEM for each time interval.
196 D.E Houlihan, C.G. Carter and I.D. McCarthy
or by adapting the method of Fauconneau 3~ and bathing juvenile fish in a flooding

dose of 3H-phenylalanine53'ss. The flooding dose method has also been applied
to measure in vivo rates of protein synthesis in isolated organs and in vitro
in cells54,59,s3,s4,1~176 As a result of its ease of application and adaptability,
coupled with a sensitive and selective method of extraction and measurement of
phenylalanine nS, the flooding dose technique has become a valuable tool in the
study of protein synthesis in fish.
Recently our attention has turned to measurements of protein synthesis using
stable isotopes, principally 15N, which have been used extensively in the study of
protein synthesis in mammals (e.g. refs. 109, 122, 129) as well as for one ectotherm,
the mussel, Mytilus edulis L.5~ Initial results for rainbow trout indicate that protein
synthesis rates obtained from feeding 15N enriched protein and collecting ammonia
are similar to those obtained with radiolabcUed amino acids (Table 2). Feeding
15N enriched protein is advantageous because it allows non-invasive and non-
destructive measurements of protein synthesis which can be repeated on the same
animal. The use of stable isotopes should be used increasingly in the study of
protein synthesis in fish. This would enable us to answer questions such as, how
do changes in environmental conditions affect the protein turnover of the same
fish? Furthermore, being able to 'track' the same fish provides the opportunity to
measure ontogenetic changes in protein synthesis and construct synthesis/weight
relationships for the same fish.
In contrast to our detailed understanding of the mechanisms of protein synthesis
(e.g. ref. 99), relatively little is known about the mechanisms of protein degradation.
Several of the cellular pathways concerned with protein degradation have been
identified 93,117, however, our understanding of the processes involved is far from
complete and this has made quantification of the process difficult. In endotherms
two main isotopic methods have been used to estimate in vivo protein degradation
rates. The first involves measuring the loss of radiolabel from prelabelled proteins
with time. However, this method can be complicated by amino acid reutilisation 122.
An alternative isotopic method has involved constant infusion of radiolabel (e.g.
3H-leucinc) for a known period followed by the measurement of the amino acid flux
and the increase during the infusion period of the 3H labelling of body water 42.
Due to the complications of direct measurement, most commonly protein degra-
dation has been estimated from the difference between synthesis and growth, i.e.
kd = ks - k s (refs. 53, 91, 93). Since in fish protein synthesis is measured over
a period of minutes or hours and protein growth over a period of days or weeks,
this calculation can only be an estimate of the true value. However, recent work
has shown little diurnal variation in the synthesis rates in the skeletal muscle of
rats 1~ or sea bass, Dicentrarchus labrax L.7s measured over a 24 h period. Fuller and
colleagues 42 found a highly significant correlation between protein degradation esti-
mated from the difference between synthesis and growth and from the whole-animal
flux of 3H-leucine. These results have given confidence in the use of this technique
to estimate in vivo protein degradation rates in fish. The range of in vitro meth-
ods that are available to measure rates of protein degradation has recently been
reviewed .
Reliable determinations of the whole-body rates of protein growth, loss or

accretion, are not particularly easy to calculate either since initial values for
an individual fish cannot be measured directly (but see Ellis29). However, this
measurement is a necessary prerequisite in understanding rates of protein turnover.
In long term studies the best method seems to be to use frequent weighings of
individual fish (fresh weights) combined with the regular analysis of the protein
content of whole animals sacrificed during the course of the growth estimate.
III. General model of protein turnover in fish

As a starting point for the more complete analysis of protein and amino acid
metabolism, we have adapted the model of Millward and Rivers 9~ to describe the
Consumed protein nitrogen

6.75 mmol N
J Faecal loss
I , I 1.01mmolN
Absorbed protein nitrogen
5.74 mmol N
Free amino acid pool I Total nitrogen

loss
3.59 mmol
,,7,
l T I
I
I
I
Protein synthesis
4.97 mmol N
I Protein degradation
2.82 mmol N
I
T I
J Protein pool I I I I I I I I I I | | ~
I
I
1'13.0mmo'N!
Protein growth
2.15 mmol N
(a)
Fig. 2. Daily protein-nitrogen flux for immature 80 g rainbow trout (Oncorhynchusmykiss) at 10~ based
on the model of Millward and Rivers 9~ The data presented are for: (a) a growing trout fed a ration of
1.4% body weight per day of a commercial diet; or (see Fig. 2b).
198 D.F. Houlihan, C.G. Carter and I.D. McCarthy
j i
Free amino acid pool Total nitrogen

loss
I 2.52 mmo;"N I 1 -0.68 mmol N
!
!
!
i,,,, i , i |
I
Protein synthesis Protein degradation :

I
I
1.22 mmol N 1.90 mmol N ,
I
i i , II
T '
!
] I [ ..............
T ,,
Protein growth
-0.68 mmol N
(b)
i i| ii
Fig. 2. (continued from Fig. 2a) (b) a starving trout (McCarthy et at. unpublished data). Protein-nitrogen
= protein/5.85 (ref. 45). Apparent absorption etficiency is calculated assuming 85% absorption 6~ and
the white muscle total free amino acid pool2o, in preparation) is used as an indication of the whole body
free amino acid pool.
daily protein nitrogen flux in a growing immature rainbow trout following feeding
(Fig. 2). The loss of nitrogen is shown as a single figure, although further subdivision
of loss via ammonia from amino acid oxidation, urea and other nitrogenous
molecules ultimately derived from the catabolism of non-protein substrates or via
proteins lost in, for example, mucus, scales and digestive enzymes9~ would give
a more complete picture. At the heart of the model is the relationship between
dietary amino acid intake, the free amino acid pool and the protein pool linked via
protein synthesis, protein degradation and amino acid metabolism (transamination/
deamination followed by oxidation and or synthesis of non-protein molecules). The
model allows ratios between its components to be calculated. The nitrogen balance
for a feeding trout indicates that 15% of the consumed nitrogen is lost v/a the
faeces, 53% lost (the majority excreted as ammonia) and the protein nitrogen
retention efficiency (kg/kr, Table 1) is 32%. (Fig. 2a). The anabolic stimulation
efficiency (ks/kr) indicates that 74% of the consumed or 87% of the absorbed amino
acids are used in protein synthesis. In an earlier report Houlihan and colleaguess5
estimated that for each gram of protein eaten by cod, 1 g of protein was synthesised.
Thus the cod values are close to the model presented here for rainbow trout. This
high proportion of the absorbed amino acids which are synthesised into proteins
may be the first phase of the animal's response to the dietary amino acid influx,
followed later by protein breakdown.
Not all the synthesised protein is retained as growth, the synthesis retention
efficiency (ks~ks, Table 1) is 43% and indicates an amount equivalent to 57% of the
Protein synthesisin fish 199
newly synthesised protein nitrogen is degraded and returns to the free amino acid
pool, to be oxidised and excreted, or recycled in the synthesis of protein. It is likely
that the processes of protein degradation follow at some time after the synthesis
although experimental evidence for the time course of synthesis versus degradation
remains to be elucidated. The frequency and size of meals clearly play a pivotal role
in determining the daily protein synthesis and degradation rates.
It may come as a surprise to realise that the dietary amino acids supplied by a
normal sized meal are approximately double the size of the whole animal free pool.
The evidence is that tissue free pools remain relatively stable after a meal and there-
fore the relative importance of protein synthesis in maintaining this homeostasis is
evident. What we need to know now is what determines the relative rates of amino
acid oxidation and protein synthesis and this is discussed further below.
Compared with a growing animal, the rates of synthesis and degradation are
substantially lower in a starving trout (Fig. 2b). The majority of amino acids from
protein breakdown are used as energy substrates and the ratio between fractional
rates of protein degradation and synthesis indicates that only 36% are recycled into
protein. Reduced fractional rates of protein synthesis rates during starvation have
been found in almost all animals, including fish (e.g. refs. 16, 34, 55) and means that
turnover rates are lower in starved than in fed animals.
In summary, this kind of model provides a useful framework for describing
whole animal nitrogen flux and emphasises the importance of measuring dietary
intake. However, it represents a simplification by the assumption that there is one
free amino acid pool and one protein pool. Different tissues respond to a meal at
different rates; protein synthesis may remain elevated for more than 24 h after a
meal in the white muscle 83.
I V I n d i v i d u a l variation in protein turnover
We can move on from this static model of nitrogen flux to test relationships
between rates of food consumption and protein turnovers6. The data set we have
chosen comes from experiments where the rate of food consumption of individual
rainbow trout (measured by radiography) a1,116 have been combined with whole
animal protein synthesis measurements. Rates of protein growth increase linearly
with increasing protein consumption rates (Fig. 3a) and in general about 70-80%
of the observed variation in growth rate between individual fish can be explained
by differences in food consumption 15,16,23,s~ There is also a linear relationship
between protein consumption and protein synthesis which can be termed the
anabolic stimulation of protein synthesis (Fig. 3b) and which is thought to be the
result of the combination of dietary amino acid and hormonal stimulation of protein
synthesis84,s9'114, mediated through an increase in the number of ribosomes and/or
an alteration in ribosomal activitys3. The individual response to a given protein
intake, in terms of the magnitude of synthesis is clearly variable (Fig. 3b).
Whole-animal rates of protein degradation are independent of ration level
(Fig. 3c) in contrast to previous results. Some studies have suggested that protein
200 D.E Houlihan, C.G. Cannerand LD. McCarthy
e)
2.0
1.5
o
o
'~ 1.0 o
e~ 0 0 0 0
0.5
9
a_ v
-1.0
-1.5
1 2 3 4 5 6
Protein Consumption (kr, % day-1)
b)
0
4 0
m
~ A
o o o
:~
c~
- g
2~
n_ 1
0 I I I I i ' *
o 1 2 3 4 $ 6 7
Protein Consumption (kr, % day .1)
c)
4
o
"0 , o
o
o o
o ooO~ o o
o
o o o
o
v 1
. s i i i , i
1 2 3 4 S 6 7
Protein Consumption (kr, % day-1)

degradation is stimulated by increased consumption 55,89, whilst others have found

rates of protein degradation to be independent of ration 16'52'82,95. It has been
suggested that degradation rates are genetically determined, whilst synthesis rates
are more responsive to nutritional status and environmental influences ~8.
Not all the variation in growth is explained by variation in consumption (Fig. 3a)
and individual fish can be eating similar amounts of food and yet show markedly
different growth rates. Recently we have examined how individual differences
in protein turnover can help to explain differences in protein-nitrogen retention
efficiency 16,78,82. Seven pairs of rainbow trout consuming similar amounts of food,
but growing at different rates, were selected from the group in Fig. 3a. The
relationship between consumption and growth for these fish is shown in Fig. 4a.
The mean protein-nitrogen retention efficiencies (Table 1) for the two groups of
fish were 37.7 ~ 3.4% for the fish growing at a faster rate for a given food intake
and 16.7 4- 2.2% for the fish growing at a slower rate for a given rate of food
intake. There were no consistent differences in mean rates of protein synthesis but
individuals with a higher protein-nitrogen retention efficiency were found to have
reduced rates of protein degradation (Fig. 4b). Consequently, the trout with lower
retention efiiciencies will have a higher cost of growth per gram of protein retained.
This supports the finding that individual rates of protein degradation efficiency
are negatively correlated with protein-nitrogen retention efficiency in grass carp,
Ctenopharyngodon idella (Val.) 16 and with growth efficiency in chickens 118. It has
been suggested by Hawkins 51 that individual differences in protein degradation are
an indication of genotype-dependent differences in maintenance requirements and
that this is a major influence on individual differences in performance between
individuals.
Superimposed on the influence of genotype on individual differences in protein
turnover and growth performance is the modification of an individual's response
as a result of social interaction. The social structure of juvenile salmonid fish is
characterized by aggressive behaviour between individuals and the formation of
dominance hierarchies 88. In Fig. 5 we attempt to model the relationship between
social rank, protein turnover and growth performance of individual fish within a
dominance hierarchy. This model is based upon experimental data obtained from
groups of laboratory-reared rainbow trout and Atlantic salmon, Salmo salar L.,
hand-fed a variety of different ration levels.
Disproportional food acquisition between individuals within the group is a
common feature of social hierarchies with dominant individuals gaining preferential
access to food 88, consuming a larger share of the group meal (Fig. 5a) 79 and growing
at a faster rate compared to subordinates (Fig. 5b) 1. In Fig. 5a, the distribution of
food between the individuals within the group would appear to indicate a linear
Fig. 3. The relationship between the fractional rate of protein consumption (kr, % day-1) and: (a)
the fractional rate of protein growth (ks, % day-l); (b) the fractional rate of protein synthesis (ks, %
day- 1 ); and (c) the fractional rate of protein degradation (kd, % day-Z) for individual rainbow trout
(80 g, 10~ s2. The regression equations are: (a) y - 0.366x - 0.347 (Rz ffi 0.736, n ffi 37, p <0.001);
and (b) y = 0.445x + 1.686 (Re -- 0.736, n ffi 37, p <0.001).
202 D.E Houlihan, C.G. Carterand LD. McCarthy
a)
2
a . v 0
.,f 0
I
1
I
2
, &
3
, |
4
, JI
S
Protein Consumption (kr, %. day "1)
b)
(o
"0
i , eO
Ix. i 9 9 i . t i
o
0 10 20 30 40 SO eO
Protein Retention Efficiency (kg/kr, %)
Fig. 4. (a) The consumption-growth relationship (expressed on a fractional basis as a percentage of the
final protein mass per day) for seven pairs of fish consuming similar amounts of food but growing at
different rates 7s. The efficient fish, i.e. individuals growing at a faster rate for a given food intake, are
indicated by the closed circles and the inefficient fish indicated by the open circles respectively. (b) The
relationship between protein retention efficiency (protein growth/protein consumption, ks/kr) and the
fractional rate of protein degradation for the seven pairs of fish in (a).
dominance hierarchy which have been reported for small groups of social fish of
between 4 and 10 individuals 8s.97. However, the hierarchical structure may be more
complex in larger groups of fish and non-linear hierarchies in large social groups of
fish have been reported9'~. The low ranking of subordinate fish may impose a long
term chronic stress on individuals (Fig. 5c) (reviewed by Jobling) 6a and influence
both growth performance (Fig. 5d) and immunocompetence (Fig. 5f). Dominance
has been associated with reduced metabolic costs and increased growth efficiency
(Fig. 5d) 1,s7. It has been suggested that increased stress, mediated v/a cortisol,
inhibits protein synthesis and stimulates protein degradation in fish (reviewed in
Van der Boon et al.7). Decreased rates of degradation in dominant fish exhibiting
A
Im
A
16 "O
i 14
i QO,
Q,
o
e
,0,
" nnO CC
0
41
0 9 9 9
IIIIIIIIII
"'_., _.,nnOOOO!9 9 r 9
Fish
, ,. : ,. , : ,
C
.1.
w
4;
O
b.
O.
Share of Group Meal (%)
d)
o
.=
.....
._u
Ic : :=
ILl
g
fn
Share of Group Meal (%) Share of Group Meal (%)
o) 0
S~
Shaire of Group Meal (%) Share of Group Meal (%)
Fig. 5. A model showing the predicted influence of social rank on growth performance and immuno-
competence of individual fish within a group hierarchy. In this model social rank is assigned on the
basis of an individual's share of the group meal (a). The predicted relationship between share of group
meal and protein growth (% day-l), stress response, protein retention efficiency (ka/kr, %), synthesis
retention efficiency (kg/ks, %) and immunocompetence are shown in parts b-f respectively.
204 D.E Houlihan, C.G. Carter and LD. McCarthy
higher growth rates will therefore result in an increase in the percentage of the
synthesized protein that is retained as growth (Fig 5e).
The effect of social rank on the immunoeompetenee of individual fish within
a group environment is still unclear. Much of the available data compares the
responses of the dominant and subordinate individual within pairs of fish. These
results have suggested that exposure to long term stress results in reduced im-
munocompetence in subordinate fish24,~~ However, in these studies the single
subordinate fish is the sole focus of the aggressive behaviour of the dominant and
this would serve to maximise the physiological stress imposed on that individual. In
a large group the dominant fish may be more stressed by having to maintain their
position. The immunological performance of individuals within a group environ-
ment is still little studied, especially in relation to individual feeding rates. Recent
work has suggested that within a group the immunocompetence of dominant fish
is reduced (Fig. 5f) (Thompson et al., in preparation). This may be due to the
immunosuppressive effect of increased consumption by dominant fish or it may
represent a trade-off whereby growth is maximised in the more active dominant fish
at the expense of the immune system. The combined measurement of consumption,
protein turnover and immunocompetence in individual fish of known social rank
within a group will enable us to further examine the interplay between social rank
and growth performance and to investigate whether these metabolic trade-offs do
occur.
Thus in summary we find that the measurement of rates of protein synthesis
in fish needs to be approached in an holisticfashion, considering the individual's
ability to obtain food together with metabolism and growth efficiency. Using
this approach, particularly with repeated, long-term measurements using stable
isotopes, protein synthesis rates will give us a firm basis for understanding the role
of protein turnover and in designing selection programmes. For example, we can
already suggest that maximisation of protein synthesis rates is not the mechanism
guaranteeing fast growth rate, rather it is reduced turnover which correlates with
increased growth rate. We are only at the threshold of investigating the implications
of these studies on for example, hormonal control, longevity and disease resis-
tance.
V. Proteinsynthesisand freepool amino acid concentrations

Despite the small size of the free amino acid pool in large fish, equivalent to
approximately 2% of the protein pool and the large influx of dietary amino acids,
approximately double the free pool size, there is comparatively little change in free
amino acid concentrations in fish tissues following a mealz~176 This is true for
both the total and the essential free amino acid pools (Fig. 6). It is interesting to
note that although white muscle and liver have very different fractional rates of
protein synthesis56, the free pool concentrations are very similar. Only the plasma
free pool shows large changes in amino acid concentration lzs ; in rainbow trout
the maximum concentration is twice the prefeeding leveP z~ The stability of the
Fig. 6. Free amino acid concentrations (t~mol/g tissue wet weight) in the white muscle and liver of
rainbow trout (200 g, 12"C) over a 24 h period after feeding2~ The data presented are the total
essential amino acid (white bars) and total free amino acid (cross-hatched bars) concentrations in
the liver and the total essential amino acid (hatched bars) and total free amino acid (black bars)
concentrations in the white muscle respectively.
tissue free pools is strongly indicative of a homeostatic control mechanism. Indeed,

Millward and Rivers 9~ proposed that the accumulation of essential amino acids
may be harmful and are therefore maintained a low concentrations. It has been
suggested that enzymes involved in amino acid metabolism have a relatively high
Km (ref. 22) whereas aminoacyl-tRNA synthetases have relatively low Km values
a n d t h e r e f o r e p r o t e i n synthesis will be ' p r e f e r r e d '26. H o w e v e r , q u a n t i t a t i v e e v i d e n c e
for this hypothesis is still lacking.
In fish the major pathways of amino acid utilization are through protein synthesis
or deamination and oxidation to ammonia. The model of protein-nitrogen flux
indicated 87% of absorbed amino acids were partitioned into protein synthesis.
In catfish, Ictalurus punctatus, approximately 21% of the total nitrogen in an
essential amino acid infused was excreted as ammonia over the 24 h following
infusion 1~ In unfed catfish the application of cycloheximide, to inhibit translation,
led to an immediate increase in ammonia excretion which had doubled after four
hours and suggested the importance of synthesis in regulating free amino acid
concentrations 1~ It is very important to qualify these conclusions by investigating
the flux of an individual amino acid. In rainbow trout 20-40% of the leucine
metabolised in a day was oxidised 33,34 and it was demonstrated that the majority of
this essential amino acid was utilised in protein synthesis.
Oxygen consumption, ammonia excretion and protein synthesis all show post-
prandial increases 1~ The sequential pattern of all three variables has not
been determined simultaneously in any fish but all the evidence suggests that the
206 D.E Houlilm~ C.G. Carter and I.D. McCarthy
postprandial stimulation of protein synthesis is the major contributor to the post-

prandial increase in oxygen consumption 1~ The few data available suggest
that the oxygen consumption and ammonia excretion peaks are not synchronous.
In catfish the ammonia peak was at 4 h and the oxygen consumption peak at 8
h after an infused meal 1~ In grass carp (Ctenopharyngodon idella) AQ values (mg
ammonia/mg oxygen) peaked 5 h after feeding and indicated a relative increase in
ammonia excretion which preceded the peak in oxygen consumption x3. Brown and
Cameron ~~ also demonstrated that after cycloheximide administration, ammonia
was excreted following a meal without an increase in oxygen consumption. Thus,
overlapping peaks with maximum values of ammonia excretion preceding those of
oxygen consumption and protein synthesis would be predicted.
Reeds and Davis ~~ concluded that in endotherms it is rare to encounter cir-
cumstances in vivo in which the amino acid concentrations fall to levels that might
inhibit translation. It has been suggested that amino acid imbalances may lead to
increased amino acid oxidation and nitrogen excretion in fish3s,66,68 and examples of
amino acid imbalances on protein synthesis rates are provided by Fauconneau 31 and
Fauconneau et al.3s. Grass carp which were fed only lettuce and which had positive
protein growth rates had a lower fractional rate of protein synthesis, lower synthesis
retention efficiency, lower nitrogen retention efficiency and consequently a higher
loss of nitrogen than grass carp fed a nutritionally balanced diet. Although rates of
protein consumption were similar on the two diets it was not known whether these
results were due to lettuce being deficient in methionine, the low energy content of
lettuce or the low digestibility of lettuce protein 16.17. Such studies emphasise that
much more work is needed to establish relationships between diet composition,
protein turnover and amino acid metabolism. This question is of importance in the
theoretical understanding for the use of lipids in commercial diets as a method of
protein sparing6.
In conclusion, analyses of amino acid fluxes in fish provide valuable support for
separate estimates of rates of protein synthesis. The apparent relative constancy of
the tissue free pools points to control mechanisms of protein synthesis and amino
acid oxidation which are little understood. Perhaps the most intriguing feature is the
similarity of the tissue free pools in different tissues with widely differing fractional
rates of protein synthesis. This implies that the fluxes of amino acids, be they
diffusional or active, into different tissues must be closely linked with the amino
acid sinks of synthesis and oxidation.
VI. Energy cost of protein synthesis

An alternative approach to the bioenergetic analysis of inputs and outputs in fish is
to quantify the energy cost of the various life processes. There have been a number
of attempts at quantifying the energy cost of protein synthesis but even in mammals
it is an area of some uncertainty. One approach (termed the 'direct' by Waterlow
and Millward nl) is to measure the rates of protein synthesis and multiply these by
a protein synthesis cost factor, a theoretical cost of protein synthesis has frequently
been employed. The minimum theoretical cost assumes that 4 ATP equivalents
are required for each peptide bond synthesised which, with a molecular weight of
peptides of 110, results in a cost of 36 mmol ATP g-I protein synthesised 1~ An
additional ATP has been included for transport processe s giving a value of 8.3
mmol 02 g-~ protein synthesised (assuming 6 mmol ATP synthesised per mmol
oxygen) 1~ It is important to note that the theoretical cost of peptide bond synthesis
appears to be independent of temperature, body size, etc. Using theoretical values,
protein synthesis has been estimated to account for a minimum of between 23 and
42% of the total oxygen consumption in cod, Gadus m o r h u a 59'75 and between 11
and 22% in the more active grass carp 16. In a variety of mammals, protein synthesis
was calculated to account for 20-25% of the resting heat production 124.
An alternative approach (termed the 'indirect' approach by Waterlow and
Millward m ) is to compare protein synthesis rates and oxygen consumption in
a variety of conditions and to calculate the oxygen consumption as a ratio of
protein synthesis. A close relationship between oxygen consumption, growth and
protein synthesis have been found for a variety of animals including fish 48'52'62'121.
Comparisons between energy cost of protein synthesis in mammals estimated stoi-
chiometrically and indirectly indicate that the latter give values approximately 5-fold
higher than the former m, and generally aerobic costs of protein synthesis appear
to vary widely (Table 3). Some of the data on energy costs of protein synthesis
have been determined by measuring oxygen consumption and protein synthesis
before and after the application of a protein synthesis inhibitor such as cyclohex-
imide and have given values close to theoretical, e.g. juvenile tilapia, Oreochromis
mossambicus 57. Cycloheximide-sensitive protein synthesis and oxygen consumption
suggest that from 80 to 87% of oxygen consumption is used for protein synthesis in
trout hepatocytes and juvenile tilapia.
A possible explanation for these disconcertingly large variations in protein
synthesis costs and the suggestion that different tissues have different energetic
costs for protein synthesis 2,47,48,77 may lie in the observation that energy costs of
protein synthesis appear to be related to the rate of protein synthesis, the higher
the rate of synthesis the lower the aerobic cost (Table 3). An example of decreasing
aerobic costs of protein synthesis with increasing rates of synthesis in isolated fish
cells is given in Fig. 7a. These variable costs would arise if there were fixed costs
at low rates of protein synthesis together with the peptide bond formation cost.
In isolated fish hepatocytes 100 and other fish cells 113 plots of oxygen consumption
and protein synthesis do reveal a large intercept (oxygen consumption) at zero
protein synthesis. The slope of the line relating oxygen consumption may approach
the theoretical peptide bond formation cost. Thus as the rate of protein synthesis
increases the fixed costs make a decreasing contribution to the aerobic costs which
at high protein synthesis rates will approach theoretical values (Fig. 7a).
Variable protein synthesis costs would explain the apparent plateau of oxygen
consumption with increasing protein synthesis rates found in a comparison of differ-
ent fish cell types (Fig. 7b). Recent data from juvenile fish have also demonstrated
that there is no further increase in oxygen consumption at growth rates above
8-10% per day 125,~26 and it has been suggested that variable protein synthesis
208 D.E Houlihan, C G. Carter and I.D. McCarthy
50"
L. 40"
Macrophages BF-2
eoe
C ~
30" RTG-2
m Q.
C
20"
Scale Cells
10"
c
O i i I iii I |i,i ii ,,i "I I I I II I 9
(a) 0 1 2 3 4 5 6 7 8 9 10
Protein Synthesis (ks) (% day"1)

Fig. 7. a: mean (-I-SEM) fractional rates of protein synthesis (% day-1) and oxygen consumption (nmoi
02 mg-I protein h -1) of fish cells. (From Smith and Houlihan 1 1 3 .)
250
le cells
2oo
@,j r ~
9mm I ~
~o lso
2
" ~ BF-2
, I I ,, t i
0
(t~ O 1 2 3 4
Absolute protein synthesis rate

(mg protein synthesized per g protein per hour)
Fig. 7. b: mean (+SEM) rates of protein synthesis and cycloheximide sensitive costs of protein synthesis
for fish cells. (From Smith and Houlihan113.)
TABLE 3
Energetic costs for protein synthesis from different sources
Species ks Oxygen consumption Method Reference

(% day-1) (mmol02 g-1 protein)
- 8 Theoretical 62
Calf-muscle 0.7- 1.5 450 Correlation 47
Pig-muscle 1 - 6 158-575 Correlation 2
Trout hepatocytes 1 - 8.5 37-138 Correlation 100
Whole sheep 3 - 5 49 Correlation 48
Blue mussel 3 - 8 25 Correlation 52
Juvenile nase 14 -25 25 Correlation 58
Sheep hepatocytes 30 -50 7 Correlation 77
Trout hepatocytes 1 - 9 111-583 Inhibition with cycloheximide 100
Trout scale cells 0.5 217 Inhibition with cycloheximide 113
RTG-2 1.1 133 Inhibition with cycloheximide 113
Larval herring 3 98 Inhibition with cycloheximide a
BF-2 9.1 11 Inhibition with cycloheximide 113
Trout macrophages 2.9 46 Inhibition with cycloheximide 113
Chickens 29 12 Inhibition with cycloheximide 4
Juvenile tilapia 30 6 Inhibition with eycloheximide 57
a Houlihan et al., in preparation.
costs could contribute to this apparent cost-free growth 56. What these fixed cost
components are remains speculative, but constant activation of t R N A as well as
the production of r R N A which remains remarkably constant regardless of the rate
of protein synthesis 98 most probably contribute to this fixed energy expenditure.
It has been suggested by Cooper and Gibson 25 that accumulation of r R N A in the
cytoplasm occurs only at half the rate at which it is synthesised in the nucleus. This
would mean that the cost of r R N A synthesis would be underestimated by at least
one half.
The high proportion of the energy budget for protein synthesis in hepatocytes
indicates that there is not much energy left for other purposes. Estimates of the cost
of the N a + / K + ATPase pump vary from 26% of the total energy expenditure in rat
diaphragm muscle to 40% and in calf muscle46,47 to 6% in perfused rat liver 39 and
adipose tissue 21. In isolated trout hepatocytes, it was estimated that the Na+/K +
ATPase pump contributes 2.8% of the oxygen consumption 1~176
In summary, the aerobic costs of protein synthesis, even using minimum theo-
retical values appears to make a large contribution to the oxygen consumption of
inactive fish. Allocation of the costs to the individual tissues is complicated, as it
will be the product of the mass of protein in each tissues, the tissue-specific rate
of protein synthesis and the tissue-specific cost 56. However, in terms of the whole
animal, it is interesting to relate the cost of protein synthesis to the cost of growth.
Thus, in the example of the rainbow trout given above (Figs. 3 and 4) animals with
similar rates of protein synthesis and hence similar protein synthesis costs (unless
there are large differences in body composition) will have widely different costs of
growth when the retention efficiency of synthesised proteins vary.
210 D.E Houlihan, C. G. Carter and I.D. McCarthy
VII. Species comparisons

Comparisons of protein growth/synthesis/degradation between fish species are
complicated because a single species-specific value for protein synthesis is not
justifiable. Also differences in water temperature influence at least protein growth
and synthesis rates. In addition, fish show indeterminate growth which means there
is no maximum or 'adult' size as would be found in endotherms. What we can do
is to compare the available data from different species in terms of the relationships
between protein consumption, growth and synthesis and recognise that conclusions
may be confused because of differences in body size, temperature or diet (Table 4).
An interesting observation is that from the limited data there seems to be a
positive correlation between the rate of protein synthesis at zero growth (mainte-
nance synthesis, k, Cm)) and the maximum synthesis rate (k, Cmax)) calculated at the
maximum ration or growth rate (Table 4: n = 9; r = 0.99; p <0.0001). This implies
that fish need a high maintenance synthesis rate to allow a high protein synthetic
scope.
It is also noteworthy that although the anabolic stimulation efficiency and
the synthesis retention efficiency vary widely between species, they are negatively
correlated (n = 7; r = -0.93; p < 0.001). When consumption stimulates a high
TABLE 4
Comparison of indices of protein turnover between different species of fish
Species Temp. Weight Diet PE/TE kr(max) ks(m) ks(max) ks 'ks/kr ks~ks 'ks/kr
(~ (g) (%) (% per day) (%)
Seawater
Atlantic 10 300 C 69.8 7.3 1.4 3.8 2.0 52.0 53.0 22.4
cod55,59
Atlantic 13 180 SP 50.4 5.3 1.4 3.8 1.7 71.7 44.7 32.1
salmon i s
Sea bass 78 18 8 FM 59.9 5.2 0.2 1.7 1.3 32.7 76.5 25.0
18 8 GM 59.9 6.0 0.2 1.6 1.3 26.7 81.3 21.7
Freshwater
Rainbow 10 80 C 58.8 6.0 2.0 4.4 1.9 73.3 43.2 31.7
trout 82,9 12 250 C 56.1 4.0 0.7 1.3 0.8 32.5 61.5 20.0
Grass carp 16 22 23 SP 42.1 6.0 1.3 2.9 1.7 48.3 58.6 28.3
22 27 L 53.0 - 1.3 1.9 0.3 - 15.8 -
Nase 58 20 0.01 Artemia - - 13.7 31.7 17.0 - 54.0 -
, , , ,
Diets: C = commercial diet; SP = semi-purified experimental diet; FM = fishmeal based semi-purified

diet; GM = Greaves meal based semi-purified diet; L = lettuce.
PE/TE = protein energy to total energy of diets; krCmax) -- the maximum measured protein consump-
tion rate; ks(m) = predicted fractional rate of protein synthesis at maintenance; ks(max) = predicted
fractional rate of protein synthesis at maximum ration or growth; kg - protein growth rate; ks/kr --
anabolic stimulation efficiency; kg/ks = synthesis retention efficiency; ks/kr = protein-nitrogen retention
efficiency.
Species: Atlantic cod (Gadus morhua); Atlantic salmon (Salmo salar); sea bass (Dicentrarchus/abrax);
rainbow trout (Oncorhynchus myk/ss); grass carp (Ctenopharyngodon idella); nase (Chondrostoma nasus).
a McCarthy et al., in preparation.
rate of protein synthesis, less of the protein is retained as growth. Conversely, when
there is a low anabolic stimulation a larger proportion of the synthesised protein
is retained. This results in broadly similar nitrogen retention efticiencies between
species. The data for the rainbow trout suggest the differences between high or low
anabolic stimulation efficiency are not species-specific but due to the experimental
conditions. The diet fed to cod has a higher protein to energy ratio than salmon diet
and this may partly explain the lower anabolic stimulation of cod, proportionally
more of the dietary amino acid is oxidised and less partitioned into synthesis. It
is noteworthy that omnivorous and herbivorous fish are able to utilise a wider
range of diets than carnivores 119. Compared with trout, higher synthesis, growth
and nitrogen retention efficiency are expected for herbivorous grass carp feeding on
a high carbohydrate diet.
These species comparisons point the way for further studies. We need to measure
synthesis at defined points in the ration-growth curve constructed under optimum
conditions for each species. These points might be at maintenance (zero growth),
maximum growth (nitrogen retention) efficiency (optimum growth) and at maximum
growth. The problem is that the optimum conditions under which maximum growth
can be achieved are not known for the majority of fish species. Indeed, we cannot
yet answer the question as to whether maximum nitrogen retention efficiency varies
between speciesa.
VIII. L i f e history p r o t e i n t u r n o v e r
Recently more information has become available on the protein synthesis rates of
larval and juvenile fish and gives a more complete picture of protein turnover over
the life history of fish and the influence of environmental variables.
The rate of many physiological variables are proportional to body weight ac-
cording to the relationship Y = aX b, where a is a constant and b is the weight
exponent 54. For feeding fish the decline in weight-specific rates with increasing
body size are similar for growth, whole animal fractional synthesis and growth
rates and white muscle fractional rates of synthesis and degradation (Table 5).
In its usual form the yon Bertalanffy growth function results from the integra-
tion: d W / d t = H W d - K W m, where W is body weight and H W d and K W m
are synthesis (or anabolism) and the degradation (or catabolism) of body sub-
stances respectively; in both components body weight effects are recognised by
the use of exponents. The exponents d and rn have been assumed to be 2/3
and 1 respectively94,1~ Reiss 1~ equates anabolism with assimilated energy and
catabolism with average metabolic rate. As we have argued above, metabolic rate
cannot be taken as an index of catabolism. The exponents in Table 5 for protein
synthesis and degradation are more appropriate and suggest that both anabolism
and catabolism scale in proportion to between, approximately, 0.7 and 0.6 of body
weight.
Very high fractional rates of protein synthesis have been reported for some
juvenile fish with efliciencies of retention of synthesised proteins of between 6
212 D.E Houlihan, C.G. Carterand LD. McCarthy
TABLE 5
Effects of body weight W (g) on the ingestion rate, specific growth rate, endogenous nitrogen
excretion, protein synthesis rates and protein degradation rate using the formula:
log Y - log a + b log X for whole animals and white muscle. All parameters are in weight-specific units
Species Parameter Slope

(r) (b)
Various species t~ Ingestion rate -0.25
Various salmonids ~ Growth rate -0.3 to -0.45
Tilapia 57 Protein synthesis -0.50
Grass carp 14, a Endogenous nitrogen excretion -0.37
Rainbow trout 61 Protein synthesis -0.26
Rainbow trout 61 Protein growth -0.25
Trout white muscle 54 Protein synthesis -0.49
Trout white muscle 54 Protein degradation -0.42
a Calculated for unfed grass carp.
and 9% (refs. 35, 36). On the other hand using the techniques of either bathing
the juvenile fish in tritiated phenylalanine or microinjection of the radiolabelled
amino acids, much lower fractional rates of protein synthesis have been found and
efficiencies of retention are around 40-50% (refs. 57, 58, 86). We need more work
on fish larvae to resolve these differences.
Environmental fluctuations effect rates of protein turnover in fish and the effects
of temperature have recently been discussed 41,56. Koehn and Bayne~~ argued that
the cost of increased protein turnover due to environmental stress would be
reflected in less energy being available for growth. The effect of environmental
stress on protein turnover in fish has been little studied. However, in a recent study
with dab, Limanda limanda (L.), exposed to sewage sludge for three months, it
was found that the sludge treated animals exhibited lower protein growth rates and
increased protein turnover compared to control fish~. This is the first study in fish
which demonstrates that an environmental stress may have a physiological cost in
the form of increased protein turnover.
IX. Protein synthesis in tissues and cells
This review has concentrated on protein synthesis rates in whole bodies of fish
because we believe that it unlikely that we will make much sense out of protein
synthesis rates of individual tissues or proteins by just pulling fish out of a tank
with no regard to the details of their previous nutritional history. We also suspect
that the individual tissues and proteins will follow, more or less, the pattern evident
for the whole animal. There is good evidence for this in the white muscle (see
below). It is also important to recognise that relatively few proteins make up a large
part of the individual's total protein pool. Thus from estimates of the amounts of
contractile and ribosomal proteins in the body of a rainbow trout it is found that
they make up a third of the total bodyprotein (Table 6).
TABLE 6
Contribution of specific proteins to total whole animal protein in an 80-g rainbow trout (Oncorhynchus
mykiss) with a protein content of 9.2 g
g % of total protein
Total muscle proteins 5.31 57.72
Contractile 2.53 27.47
Ribosomal 0.94 10.22
Plasma 0.001 0.01
Collagen 0.36 3.91
Elastin 0.10 1.09
Muscle protein calculated from Houlihan et al.54. MyofibriUarprotein/muscleprotein ffi 0.476 (reference
28). Ribosomal protein/muscle protein ffi 0.177 (reference 27). Plasma proteins ffi 25.4 mg 100 m1-1
(reference 3). Blood volume -- 5.4 ml 100 g-I wet weight111.Connective tissue protein/total protein =
0.05; connective tissue = 78.5% collagen, 21.5% elastin74.
Recent information of protein synthesis rates in fish tissues has been reviewed
by Houlihan 53 and Houlihan and coworkers 56. Fauconneau and colleagues 37 found
a highly significant positive relationship between whole animal specific growth rates
and white muscle synthesis for rainbow trout collected from a number of studies.
With the addition of more data the white muscle fractional synthesis rates ( W M . ks,
% day -l) can be predicted for rainbow trout from specific growth rate (SGR, %
day-t), temperature (T, *C) and wet weight (W, g): W M . ks = 1.53 + 0.48 SGR
- 0.10 T - 1.86 10 -4 W (n = 14; R2= 0.865; p <0.0001). With an increasing
data base it should be possible to construct a similar relationship to describe whole
animal rates of synthesis from white muscle protein synthesis rates.
X. Conclusions
This review has emphasised 'supply side' economics in determining protein syn-
thesis in fish. We believe that there is a good reason for doing this because if
food consumption is not carefully monitored then the interpretation of any changes
in protein turnover under different conditions may be severely compromised. In
addition we have also emphasised the importance of the individual differences in
rates of protein turnover and attempted to show that there are already indications
of different strategies within species and possibly between species. It is justifi-
able to speculate that in rainbow trout some individuals are following a 'growth
maximisation' strategy. This is achieved not by maximising protein synthesis rates
but by reducing protein degradation rates. Other individuals may be following a
'turnover maximisation' strategy, not necessarily by reducing protein synthesis but
by increasing rates of protein degradation.
We are still not clear if there are 'energy minimisation' strategies, i.e. low
maintenance costs and possibly low protein synthesis costs in individuals. The
evidence points to the need for a high maintenance if growth rates are to be high
and therefore high maintenance may be a feature of both growth and turnover
214 D.E Houlihan, CO. Carterand I.D. McCarthy
maximisation. There is evidence for some trade-offs in these strategies in that

there may be reduced immunoeompetence in the 'growth maximisation' strategy.
The evidence is that protein synthesis rates are highest after the arrival of dietary
amino acids and that it is the timing and extent of the subsequent release of
amino acids from protein degradation before the next meal which is a decisive
factor controlling growth rates. It is still not dear whether the source of increased
ammonia production after a meal is the amino acids from the meal or pre-existing
proteins and amino acids.
The likelihood of variable protein synthesis costs introduces some uncertainty in
calculations of the contribution that protein synthesis makes to oxygen eonsurnption
in fish. However, it is likely that the costs of protein synthesis are an important factor
controlling the 'demand side' of energeties. For two fish synthesising proteins at
similar rates, costs of protein synthesis will be the same but the fish which retains the
highest proportion of the synthesised proteins will have the lowest costs of growth.
In the context of estimating protein growth costs, two strands from this review are
worth picking out. Flatfish living in a stressful environment synthesise proteins at a
similar rates as control animals but because of increased protein breakdown exhibit
reduced protein growth rates; their impaired immunocompetenee may or may not
be related to this increased turnover (section VII) 6~176 In contrast, dominant
salmonids in a group may exhibit reduced protein turnover, either as a consequence
or a result of their dominant social position (section IV). These dominant fish
may have reduced immunocompetenee. The costs of growth are higher in the
environmentally stressed animals and lower in the dominant fish but in the face of a
disease challenge increased protein turnover may prove vitally important despite the
energy burden it imposes. If this review does nothing more than direct the focus of
future research into integrating energy costs of protein turnover with the functional
significance of the proteins that are being turned over it will have achieved the aims
of the authors.
Acknowledgements. We are grateful for financial support from the Agriculture

and Food Research Council, the British Council, the Ministry of Agriculture
Fisheries and Food, the Natural Environmental Research Council and the Science
and Engineering Research Council.
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CHAPTER 9
Nutrient fluxes and regulation in fish

intestine
NATHAN L. COLLIE AND RONALDO P. FERRARIS *

Department of Biological Sciences and Institute for Biotechnology, Texas Tech University, Lubbock,
TX 79409-3131, U.S.A. and *Department of Physiology, University of Medicine and Dentistry of
New Jersey, New Jersey Medical School, Newark, NJ 07103-2714, U.S.A.
I. Introduction
II. A primer on nutrient transport pathway characteristics
III. Mechanisms of sugar absorption
1. Brush-border transport
2. Molecular biology of the Na+/D-glueose transporter
3. Site-density of Na+/D-glucose transporters in fish and other vertebrates
4. Basolateral transport
4.1. Basolateral transporter isoforms
IV. Mechanisms of amino acid (AA) transport
1. Amino acid entry across the brush-border membrane
1.1. Na+-dependent uptake pathways
1.2. Na+-independent uptake pathways
1.3. Na + : AA coupling ratios in AA uptake pathways
2. Transport across the enterocyte basolateral membrane
V. Regulation of nutrient transport
1. Adaptations to diet
1.1. Genetic adaptations
1.2. Phenotypic adaptations
2. Hormonal regulation
2.1. Steroids
2.1.1. Anabolic steroids
2.1.2. Interrenal steroids
2.2. Growth hormone (GI-I)
2.3. Thyroid hormones
VI. Summary and perspectives
Acknowledgements
VII. References
I. Introduction
Studies on intestinal nutrient absorption have increasingly turned to the question

of how transport processes are regulated (for reviews, see refs. 8, 17-20, 26, 53
and 54). This shift in emphasis was possible, in part, because extensive work over
the past three decades helped characterize nutrient transport mechanisms 11,16,45,79.
Much of the early work on fish intestine that led to a basic outline of how nutrients
move from the gut lumen to blood was summarized almost 10 years ago 24. This
222 N.L. Collie and R.R Fetraris
review focuses on research since then on mechanisms of glucose and amino acid
(AA) transport and its regulation in fish intestine.
We begin by examining the cellular mechanisms responsible for nutrient ab-
sorption, as a logical background for discussing adaptive changes in transport
mechanisms. Recent advances in the molecular biology of glucose transport and its
relevance to fish intestine are explored. However, work in this area for AA trans-
port has only recently emerged 5~176 and no information is yet available in fish
species. Despite these advances in mammalian species, we ask whether our knowl-
edge of transport mechanisms is sufficient in fish to pinpoint which mechanisms
underlie adaptive changes in nutrient absorption.
In the final section we discuss the adaptive regulation of nutrient absorption,
emphasizing two major aspects: adaptations to diet and hormonal regulation.
We conclude by pointing out problems that remain unsolved and that future
studies might profitably address. Related topics in intestinal nutrient absorption
not covered explicitly here have been reviewed recently, including organismal
aspects of digestion in fish 76, comparisons of techniques and nutrient transport
models 67, intestinal development 8.2~ ecology and nutrient absorption 19,54, intesti-
nal metabolism 11.32,67,and signal transduction in enterocytes 1~
II. A primer on nutrient transport pathway characteristics
For readers whose primary interests lie outside epithelial transport, we first present
a general, concise introduction to nutrient transport pathways, before turning to
specific topics on glucose and AA absorptive mechanisms. As described below,
these absorptive mechanisms can be distinguished by a minimum of four transport
characteristics.
First, nutrients may enter or exit absorptive cells by diffusion (non-mediated) or
by a membrane transporter, which mediates solute movement. A plot of transport
rate versus nutrient concentration is linear for diffusion and curvilinear (saturable)
for mediated routes. Since some finite diffusion occurs for all solutes, the latter
curvilinear relationship will always have a linear component superimposed on the
nonlinear, mediated component.
Second, carrier-mediated transport can be further resolved into facilitated (pas-
sive) or active mechanisms requiring energy input to drive nutrient entry against
an electrochemical gradient. For sugars and AAs entering absorptive cells, the
energy source for active uptake is provided secondarily by the Na + gradient, and
maintained by Na + pumps (Na+/K+-ATPases) spanning the basolateral membrane.
Hence, facilitated and active transport mechanisms are typically differentiated by
the absence and presence of Na + dependency, respectively. However, other ionic
requirements for active uptake have been reported, such as C1- in nutrient uptake
by the marine teleost, Boops salpa, though the anion's mechanistic role is unclear 4's.
Third, the specificity of carrier-mediated transport for related but discrete
chemical classes of nutrients (e.g. acidic versus basic AAs) refines transport into
additional uptake pathways. Measuring the uptake of a radiolabeled test solute
Nutrient fluxes and regulation in fish intestine 223
in the presence of one or more potential competitors is an effective method of

identifying which chemically related solutes share a given pathway.
Fourth, particularly with the advent of membrane vesicle preparations, it has
been possible to identify the coupling ratio (stoichiometry) between the number
of Na + ions that enter the enterocyte per nutrient molecule transported. This is
important for discriminating between different transporters as well as for estimating
the gradients of nutrients that can be established across membrane barriers.
Below we use these various characteristics to describe the absorptive pathways of
nutrients entering the enterocyte from the gut lumen and exiting the cell across the
basolateral membrane.
III. Mechanisms o f sugar absorption
Fish possess the hydrolytic enzymes (amylase, maltase, sucrase and lactase) nee-
essary to digest dietary carbohydrates into their component monosaccharides D-
glucose, D-galactose and D-fructose 3,63. These monosaccharides are then trans-
ported transcellularly by brush-border carriers from the intestinal lumen into the
cell, and then by basolateral carriers from the cytoplasm into the blood. There is no
significant absorption of monosaccharides through the paracellular pathway 19,3~
1. Brush-border transport
In fish small intestine, early studies have shown that o-glucose and o-galactose ab-
sorption is mutually competitive, Na+-dependent, energy-dependent, concentrative
and electrogenic 24. More recent studies 5,68,81 using membrane vesicles from fish
intestine not only have confirmed results from those early studies, but also have
shown that the D-glucose transport system in fish intestine is phlorizin-inhibitable
and exhibits steric requirements similar to those exhibited by mammalian small
intestine. The o-glucose transport system can be inhibited by D-galactose and
o-glucose analogs like t~- and ~l-methyl-glucoside, but not by 2-deoxy-D-glucose,
a o-glucose analog transported only by the basolateral, Na+-independent glucose
transport system, nor by inositol, a cyclohexanol sugar which is also a vitamin. Thus,
the functional characteristics of the D-glucose transport system in fishes closely
parallels those found in mammals. The molecular basis for these properties is the
Na+/o-glucose transporter, whose ion dependency, substrate specificity and phlo-
rizin inhibition characterize those of the brush-border transport of D-glucose in fish
intestines.
2. Molecular biology of the Na +~o-glucose transporter
An intestinal Na+ /D -glucose transporter has been recently cloned from rabbit
ileum 38. The properties of the protein deduced from the eDNA are identical
to those of the native brush-border membrane protein identified in biochemical
experiments 44,47. Coady and coworkers 12 then used the eDNA encoding the Na+/
224 N.L Collie and R.P. Ferraris
D-glucose transporter to examine the distribution of homologous mRNA in vari-

ous classes of animals. The mRNA extracted from pyloric ceca of rainbow trout
hybridized with radiolabeled cDNA from the cloned rabbit Na+/D-glucose trans-
porter. Trout mRNA showed a distinct but relatively faint single band at 2.9 kb;
the faint band may reflect a relatively low abundance of mRNA coding for the
transporter. This is not surprising because v-glucose absorption rates 7.23 and site
density of Na +/D-glucose transporters (see below) are much lower in fish compared
to mammalian intestine. Thus, the mRNA encoding the Na+/D-glucose transporter
is widely distributed among vertebrates, has been conserved in evolution, and may
even be structurally related to the Na +/proline transporter from Escherichia r 39.
Based on its predicted amino acid sequence, the Na+/D-glucose transporter
is thought to traverse the plasma membrane 11 times 3s. The amino terminu~ is
located intracellularly, the carboxyl terminus, extracellularly. The protein consists
of 662 amino acid residues, and has a molecular weight of about 75 kDa. A long,
hydrophilic, intracellular region linking membrane spans 10 and 11, and another
long, highly polar, extracellular region linking spans 7 and 8 are believed involved in
D-glucose binding. The activity of the Na+/D-glucose transporter in situ in the brush-
border membrane requires the simultaneous presence of four intact, independent,
identical subunits arranged as a homotetramer 7s. The gene encoding the human
intestinal Na+ /D -glucose transporter is found on chromosome 22 (ref. 37).
3. Site-density o f Na +~D-glucose transporters in fish and other vertebrates
Because the D-glucose carrier of fish intestine is phlorizin-sensitive, we have at-

tempted to measure the site density of these transporters by D-glucose-inhibitable,
specific phlorizin binding (Ferraris and Vinnakota, unpublished observations).
Ligand-binding analysis of specific phlorizin binding by isolated evened sleeves
of catfish intestine reveals that the apparent atfmity (apparent because of unstirred
layer effects) of specific phlorizin binding to the transporter is similar to that found
in desert iguanas and laboratory mice (Table 1). Site density of the fish transporter
is 3-6 times less than in mammals, but 3 times greater than in the iguana. The Ql0
TABLE 1
Site density of Na+/D-glucose cotransporters in the small intestine of fish, reptiles and mammals
Catfish Iguanaa Mouseb Woodrat*

Site density (pmol/mg) 0.190 0.056 "1.08 0.52
Total site number (pmol) 131 62 2100 1450
Total glucose uptake (mmol/min) 0.24c 2 17 10
Turnover number/site (min-i) 1800c 32,300 8100 6900
Apparent Kd (m) 0.33 0.19 0.51 3.03
"From Ferraris et al.29
b Ferraris et al.28
c 24oc; iguana, mouse and woodrat glucose uptake were determined at 37~ Turnover number was
calculated by dividingtotal glucoseuptake by total site number.
values for carrier-mediated glucose uptake is about 2.3, and is similar in the three
species (turtle, iguana and woodrat) 56 studied so far. If this Q10 value were applied
to the catfish results in Table 1, then catfish glucose uptake would be 0.72/zmol/min,
and turnover number would be 5400 min -1. Thus, low glucose absorption rates in
fish intestine may be due to a combination of lower site density of Na+ /D -glucose
transporters as well as lesser amounts of absorptive tissue, even when body size
differences are taken into account.
There are a number of questions about the Na+/o-glucose transporter that
remain unresolved. First, the amino acid sequence, molecular weight, and glyco-
sylation sites of the transporter remain unknown for any fish species. Second, the
sequence homology among classes of fish (cyclostomes, elasmobranchs and bony
fishes) and among vertebrates may yield interesting information about the evolution
of nutrient transport mechanisms. Third, the relative contribution of biochemical
factors (i.e. site density of transporters, turnover number) as well as anatomical
factors (mucosal folds, villi, microvilli) to species differences in glucose absorption
should be investigated to explain the observed variation among vertebrates.
4. Basolateral transport
The basolateral D-glucose transport system in fish is also functionally similar to that
described for mammals. Transport of D-glucose and o-galactose in tilapia intestine
(Oreochromis mossambicus) occurs by stereospecific, facilitated diffusion 69, and
transport rate is quantitatively less than that in mammals and birds. It is not known
whether differences in absorption rate is due to differences in site number or in
turnover number per site. Basolateral glucose transport is also osmotically reactive,
Na+-independent, and can be inhibited by phloretin and cytochalasin B, but not
by phlorizin 69. Preferred substrates are D-galactose and 2-deoxy-o-glucose. Thus,
these characteristics typify both the fish and mammalian glucose transporter.
Unlike the Na+/D-glucose transporter found only in specialized epithelial cells
(usually the small intestine and kidney of vertebrates, the nutrient absorbing gills6s
and integument 92 of certain invertebrates), facilitative glucose transporters are
present on the surface of almost all cells in organisms as diverse as cyanobacteria,
algae, yeasts, protozoa, mouse, rat and man 4~ Its main function is to accelerate
the translocation of glucose across the lipid bilayer along its concentration gradient.
Facilitative D-glucose transporters are found in almost all cells because of the
central importance of D-glucose delivery to cell metabolism. In certain specialized
epithelial tissues like the small intestine, transporters can also function to transport
D-glucose from the cell to extraceUular fluids.
4.1. Basolateral transporter isoforms

There are five isoforms of the facilitated glucose transporter which differ in their
tissue distribution, hormonal control, kinetic properties and substrate regulation 2.
Two isoforms which predominate in the small intestine are thought to contain 501
or 524 amino acid residues. The facilitated glucose transporter traverses the plasma
membrane 12 times, and both the amino and carboxyl termini are intracellular 6~
226 N.L. Collie and R.P. Ferraris
The conserved polar amino acids found in membrane span number 7 are thought to
be involved in glucose transloeation.
For fish basolateral glucose transporters, the number of isoforms and their DNA
and protein sequence have yet to be determined. Transporter site density, measured
by eytoehalasin B binding, and turnover number of each site are important indices
that will help explain species differences in basolateral glucose transport rates in
fish and in other vertebrates.
IE'. Mechanisms of amino acid (AA) transport
Following a meal intraluminal protein digestion releases small peptides and free
AAs, which are then absorbed by several active and passive mechanisms. Multiple
pathways handle the absorption of the more than 20 different AAs found in the fish
gut lumen, taken up first across the brush-border membrane and then exiting across
the basolateral membrane in series.
1. Amino acid entry across the brush-bordermembrane

Three general mechanisms of AA uptake exist in most cells, including fish ente-
roeytes: (1) passive diffusion; (2) passive, Na+-independent transporters; and (3)
active, Na+-dependent transporters. The latter two mechanisms involve recognition
of specific transported solutes by membrane proteins and, hence, can be further
subdivided into uptake pathways based on the classes of AA they transport. In
mammals where the pathways have been systematically studied (e.g. mouse, rabbit,
and rat; compared in ref. 55), at least four separate Na+-dependent transporters
handle neutral, basic (cationic), acidic (anionic), and imino acids, plus 2 additional
Na+-independent carriers (one for neutral and basic and one for acidic AAs). In
contrast, the data in fish intestine are less dear, because the uptake pathways stud-
ied are divided among diverse species, fed different diets, in various developmental
states, under assorted environmental conditions.
1.1. Na +-dependent uptakepathways

Studies on AA uptake specificity reveal several possible pathways in different
fish species (Table 2). In killifish (Fundulus heteroclitus) anterior intestine, Miller
and Kinter 59 described one diffusive and two Na+-dependent components of
eyeloleueine (a nonmetabolizable leueine analog) transport. Of the latter two,
the primary pathway (60% of the total uptake) was common to all neutral
AAs tested, whereas the second (20%) recognized both neutral o- and L-a-
aminocarboxylic acids, but not fl-alanine or taurine. Lack of stereospeeifieity is
not uncommon among AA transporters. Christensen ~ points out that the 'weak'
specificity may simply represent the best compromise for a limited number of
transporters that must handle all AAs, 'our largest group of mutually analogous
nutrients'. In contrast, rainbow trout (Oncorhynchus mykiss) intestine exhibited
stereospeeific leueine uptake 49. Only L-form neutral AAs inhibited [14C]-leueine
TABLE 2
Brush-border membrane amino acid uptake pathways in fish intestine
Species Pathway Accepted substrates Excluded substrates Comments

Na +.dependent
Killifish 59 Neutral AAs Leu, Ala, Pro, AIB, - Only neutral AAs
#-Ala,taurine tested; D- and
L-isomers accepted
Killifish 59 Neutral Leu, Ala, Pro fl-Ala, taurine
a-amino-carboxylic
acids
Rainbow trout 49 Neutral Leu, Met, Val Acidic and basic Possibly 2 neutral
AAs, D-Leu pathways
European eel sl,89 Neutral Ala, Gly MeAIB
European eel s7,89 Imino Pro, MeAIB Basic, acidic Ala acts as non-com-
petitive inhibitor
European eel aa Basic Lys, Arg Gly, BCH, MeAIB, Accepts Leu, Met,
Ala, ~l-Ala Cys, Pro
Tilapia 7~ Phenylalanine Phe, Ala, Met MeAIB
Na +-independent
European eel a~ Basic/Neutral Lys, Arg, Gly, Cys, pro, MeAIB, Similar to mammalian
Ser, Ala, Leu, Met, BCH, fl-Ala "L-system" a
Phe
Tilapia70 Dipeptide Glycyl-L-Phenylala- Phe, Gly, Ala,
nine Met, Pro
All substrate AAs are listed using standard 3-letter abbreviations and refer to the L-isomer unless oth-
erwise noted, fl-Ala = ~-Alanine; AIB = a-aminoisobutyric acid; MeAIB = a-(methylamino)isobutyric
acid; BCH = 2-amino-2-norbornane carboxylic acid.
a The pathway in eels differs from the epithelial L-system (as defined in Reference 79) in excluding the
model substrate, BCH. Species: European eel (AnguiUa anguUla); rainbow trout (Oncorhynchus myk/ss);
tilapia (Oreochromis mossambicus); killifish (Fundulus heteroclitus).
transport. Hence, stereospecificity of analogous transporters may vary in different

species.
More recent competition studies have begun to refine multiple uptake pathways
in additional species. As shown in Table 2, the most extensive work has been
on European eel (Anguilla anguilla) 81,87-89 using brush-border membrane vesicles
(BBMV).
Na+-dependent uptake comprised 3 pathways in eel intestine. The first trans-
ported neutral AAs and excluded the archetypical imino acid, a-(methylamino)iso-
butyric acid (MeAIB) 89. A second transported imino acids (proline and MeAIB) but
excluded basic and acidic AAs 87. Unlike the intestinal imino transporter in rabbit
and guinea pig 6a,79, the eel pathway also accepted alanine. However, when proline
and alanine interactions were re-examined in a second paper, Vilella et al. 89 found
that alanine was a noncompetitive inhibitor of proline uptake. This may indicate an
allosteric site on the Na+/proline transporter that binds alanine and modulates pro-
228 N.L. Collie and R.R Ferraris
line uptake. The third pathway was largely specific for basic AAs ~. However, since
lysine uptake was inhibited strongly by long-chain and sulfur-containing AAs (e.g.
leucine, methionine, and eysteine), one or more additional neutral AA transporters
may exist that also accept lysine.
Phenylalanine uptake has been characterized recently in tilapia (O. mossambicus)
intestine 7~ The Na+-dependent pathway showed a preference for methionine and
alanine, a moderate acceptance for proline and glycine, and a complete exclusion of
MeAIB. This specificity pattern closely resembles the PHE carrier described for the
rabbit ileum brush border 79.
1.2. Na +-independent uptake pathways

Vilella and colleagues8s described a facilitated transport component for lysine
uptake in eel BBMV. Basic AAs were the strongest competitors (75% inhibition),
followed by long-chain and aromatic neutral AAs (60%), and then by short-chain
neutral AAs (50%). Imino acids exhibited essentially no inhibition. This pattern was
distinct from the Na+-dependent pathway, which showed only weak inhibition by
aromatics, no inhibition for short-chain neutrals, but moderate inhibition by imino
acids.
An entirely different type of facilitated transporter, one for peptides in tilapia
BBMV, has been reported by Reshkin and Ahearn 7~ Intestinal peptide uptake
across the brush border has been known for some time in mammals 1,27, but was pre-
viously unstudied in fish intestine. Uptake of the dipeptide glycyl-L-phenylalanine
proved to be Na+-independent, unlike the Na+-energized phenylalanine transport
measured in the same tissue. None of the AAs accepted by the phenylalanine trans-
porter caused inhibition of dipeptide uptake. Additional evidence that the peptide
was carried by a transporter distinct from the phenylalanine uptake pathway was the
markedly different kinetic constants exhibited for the two solutes. The Km and Vmax
for glycyl-L-phenylalanine uptake were, respectively, 10- and 5-fold higher than the
corresponding constants for phenylalanine transport. Despite the lower affinity of
the dipeptide transporter, the authors suggest that dipeptide uptake may make
a significant contribution to AA delivery to the blood particularly when luminal
peptide levels are high after a meal. Unfortunately, little is known about peptide
concentrations in the gut lumen during the feeding cycle.
1.3. Na + :AA coupling ratios in AA uptake pathways

Few studies have examined the stoichiometry of Na+-coupled nutrient cotransport
in fish intestine. When AA uptake is measured as a function of external Na +
concentration, Scatchard or Hill plots of the data permit estimates of the number of
Na+binding sites per AA translocated 86. Using this approach in eel BBMV, Vilella
et ai.88 found a coupling ratio of two or more Na + ions per AA. In the same species,
Na+-dependent proline cotransport exhibited a 1-1 stoichiometry for Na+/proline
uptake, further supporting separate uptake pathways for lysine and proline ss. By
comparison proline uptake in rabbit jejunum BBMV occurs by an imino carrier
that binds two Na + for each proline translocated 8~ Taken together with the
above-described carrier specificities, the data suggest that fish and mammals may
possess functionally different transporters even for closely related AA uptake

pathways.
2. Transport across the enterocyte basolateral membrane
Once AAs cross the brush-border membrane, they are metabolized, used in protein
synthesis, or diffuse unchanged to the basolateral membrane. It is clear that this
membrane must have transport properties distinct from the brush border to support
the net absorption of nutrients from lumen to blood. Only recently have cell
membrane fractionation techniques been used to isolate basolateral membrane
vesicles (BLMV) in fish intestine. Reshkin and coworkers 72 prepared BLMV from
European eel intestine and identified three carrier-mediated AA transport pathways
for six L-AAs surveyed: (1) Na+-dependent carriers for proline and glutamate; (2)
Na+-independent transporters for alanine, lysine, and phenylalanine; and (3) a
glycine transporter that required both an inwardly directed (blood-to-cell) Na +
gradient and an outwardly directed K + gradient.
All three basolateral pathways could serve as influx routes supplying the entero-
cyte with AAs for metabolism and protein synthesis, such as in the post-absorptive
state or during fasting. The Na+-independent pathway presumably serves a dual
role by also facilitating the exit of absorbed AAs into the blood. Since the specificity
of these pathways was not tested, each component might be resolved further into
multiple pathways serving specific AA classes.
In summary, intestinal AA transporters are functionally different from those of
non-polarized animal cells; and, intestinal transporters from divergent vertebrate
groups, such as fish and mammals, are also dissimilar in their specificities for some
related classes of AAs. This dissimilarity points out the need to characterize AA
transport pathways more extensively in fish species. Currently, we have a detailed,
though incomplete, description of AA entry and exit pathways for one fish species,
the carnivorous eel. Information about nutrient transporter characteristics in other
species would facilitate our understanding of the regulation of nutrient absorption
and metabolism. We now turn to a consideration of known patterns and possible
signals for transport regulation.
14. Regulation o f nutrient transport
The adaptive regulation of nutrient uptake in vertebrates has been the subject of
several recent reviews8,14,18-2~ so we summarize here the patterns for fish
before focusing on hormones as regulatory signals.
1. Adaptations to diet
Adaptations to diet can be categorized broadly as genetic (fixed, hard-wired) or

phenotypic (reversible), and the mechanisms operating within these categories, as
either specific or nonspecific.
1.1. Genetic adaptations

Nonspecific. Nonspecific mechanisms are those which, if altered, will affect the ab-
sorption of all nutrients. The tendency for herbivores to have long, thin intestines,
and for carnivores to have thick mucosa or pyloric ceca are examples of geneti-
cally fixed adaptations of nonspecific mechanisms in fishes. Herbivorous fish like
the common carp (Cyprinus carpio), grass carp (Ctenopharyngodon idella), milkfish
(Chanos chanos) and tilapia (O. mossambicus) have intestinal length/fork length
ratios ranging from 1.9 to 5.8; carnivorous fish like trout (Oncorhynchus mykiss) and
striped bass (Morone saxatilis) have lesser ratios of 0.46-0.49; and omnivorous fish
like sturgeon (Acipenser sp.) and catfish (Ictahm~ sp.) have typically intermediate
ratios of 0.87-1.60 (refs. 7, 25). Another nonspecific mechanism, the passive perme-
ability of the intestine to various nutrients does not seem to vary significantly among
fish species and diet sl.
Specific adaptations. Because sugars and AAs are transported by discrete mem-
brane proteins, their uptake rates can be modulated specifically and independently.
Among species with diverse feeding habits, there are genetically fixed differences in
the expression of specific nutrient transporters, and in the ability of those species to
regulate that expression. When each group is fed its natural diet, herbivorous fishes
have faster rates of glucose transport per unit weight of intestine than carnivorous
ones 23,51. Buddington and coworkers 7 affirmed the existence of these genetically
fixed adaptations of specific mechanisms to diet by feeding eight species of fish the
same diet (thereby eliminating possible phenotypic adaptations). They determined
intestinal transport rates of D-glucose and L-proline and found that the intestinal
uptake capacity for D-glucose was much higher in herbivores, intermediate for
omnivores, and lowest for carnivores. Differences in proline uptake were less pro-
nounced, perhaps because even species on diverse diets have roughly similar protein
requirements 7. However, the latter conclusion does not suggest that AA uptake is
not genetically regulated, for two reasons. First, smaller fish had higher proline
uptake rates than did larger fish of the same species fed the same diet. Presumably,
fish may be genetically hard-wired to manage the variable protein requirements of
different developmental states (e.g. rapid growth or transformations in juveniles) 13.
Second, the pattern for one AA (proline) may not be representative of changes
occurring in other AA uptake pathways.
In contrast to omnivores, carnivorous rainbow trout have a limited ability to in-
crease glucose uptake in response to changes in dietary carbohydrate 9,43. Apparently,
differences in regulatory ability among fish species on diverse natural diets may be
fixed genetically. Nevertheless, a recent study using hormones to stimulate trout
growth indicates that endocrine signals may override the genetic limits on glucose
uptake even in carnivorous fish is (discussed below under Hormonal regulation).
1.2. Phenotypic adaptations

Phenotypic, reversible adaptations to diet primarily involve specific mechanisms.
Omnivorous fish like carp 6,7 and tilapia ss can adapt to changes in dietary sugar by
alterations in Vmax of glucose uptake. Glucose uptake rate per mg of gut tissue
were 1.73-fold higher in carp proximal intestine fed a 24% glucose diet compared
with those on the glucose-free diet. Nonspecific increases in gut mass and length
contributed a smaller fraction to the overall higher glucose uptake capacities in carp
fed the high-glucose diet. Similarly, tilapia fed a 60% carbohydrate diet showed a
two-fold increase in the Vmax for BBMV glucose uptake over values in fish fed a
17% carbohydrate diet s5. Unlike the experiments with carp, there was no effect of
dietary carbohydrate on gut mass. Thus, reversible changes in glucose uptake may
occur mainly at the level of transporter density in the brush border, since alterations
in Vmax correlate well with glucose transporter site density (see Mechanisms of sugar
absorption, section III).
Phenotypic adaptations of AA uptake to diet are well-known from studies in
mammals, but the patterns are more complex, befitting the greater diversity of
AA transporters versus that for sugar uptake. In essence the gut has evolved an
absorptive strategy that is a compromise between obtaining available AAs as a
source of both calories and essential nitrogen, and avoiding the toxicity of essential
AA at high dietary levels 18,26. For the nonessential and nontoxic AA proline, one
would expect the induction of its transporter with increased dietary proline levels, as
long as the benefits (either calories or essential nitrogen) exceeded the biosynthetic
costs of maintaining the transport (e.g. transporter synthesis, cation driving forces).
Indeed, this pattern proved to be the case for mouse intestine 57. Other AAs showed
a complex relationship between dietary protein levels and uptake rate. Is AA uptake
inducible by diet in fish, with a similarly complex pattern?
With minor exceptions, the answer is unknown. Few studies have directly ex-
amined nutrient transport adaptations by varying diet in the same species. In the
omnivore tilapia (O. mossambicus), proline uptake by BBMV was unaffected by
feeding diets differing in protein (4% fish meal vs 65%) 85. The only report affirming
proline transport induction by diet is paradoxical. When adult rainbow trout were
fed diets differing not in protein but in carbohydrate (0 and 24%), only proline
uptake capacity was stimulated 6. Glucose uptake was unchanged. Buddington 6 sug-
gested that trout, perhaps unable to distinguish between energy supplied by AA or
glucose, may respond with enhanced AA uptake to any dietary increase in digestible
energy. This explanation and additional questions regarding dietary induction of
other AA transport pathways await further testing.
2. Hormonal regulation
One of the most intriguing questions in regulatory biology concerns the signals that
switch on or off adaptational responses. An obvious, logical choice for signals reg-
ulating nutrient transport would be luminal or blood concentrations of nutrients
themselves. However, this has not been explored in fish directly, though the gen-
eral topic has been reviewed for mammalian intestine (see refs. 18, 26 and 53). We
focus here instead on endocrine signals, a second plausible choice as mediators of
transport regulation. Hormones integrate diverse physiological processes that con-
trol growth, development, reproduction, and environmental adaptation. We limit our
discussion to three types of hormones for which data support a regulatory role in nu-
trient transport by the gut: steroids, growth hormone (GH), and thyroid hormones.
232 N.L Collieand R.P. Ferraris
2.1. Steroids
The sex steroids 17a-methyltestosterone (MT) and 17/3-estradiol (E2) as well as
the interrenal steroid cortisol have been implicated in the control of nutrient
absorption.
2.1.1. Anabolic steroids. MT, a potent growth-promoter in fish, improves food

conversion efficiency, protein digestibility, and protein assimilation 21,46. In a series
of publications on rainbow trout intestine, Habibi and coworkers found that MT
and, to a lesser extent, E2 stimulated leucine transport in vitro 34,3s and in vivo 33.
MT and E2 were both effective stimulators if given by injection over a 10-day
period. However, MT alone enhanced transport when added directly to everted gut
sac incubations for periods as short as 20 rain 3s. Several methodological problems
confounded a precise understanding of where and how theses steroids might
influence leucine absorption. For example, the Tween 80 solvent (the vehicle for
the steroids added directly to incubations) by itself caused significant changes in
leucine transport. The mucosal uptake of leucine was measured under steady state,
not initial rate, conditions and in the presence of unstirred water layers, both of
which cause significant errors in transport rate calculations (see refs. 52 and 53). It
is also unknown what effect restraining unanesthetized fish for in vivo perfusions
might have on nutrient uptake, but is certain to elevate plasma cortisol levels7s,s4.
More recently, observations of short-term MT actions have been extended
to glucose absorption in tilapia intestine. Hazzard and Ahearn 36 added various
doses of MT to the serosal side of upper intestine for 30 rain, before measuring
transepithelial glucose fluxes under open-circuit conditions. MT caused a two-
fold increase in net glucose absorption, owing to stimulation of the mucosal-to-
serosal flux. The response was dose-dependent, being maximal at 15 ng MT/ml of
Ringer solution. The short time-course of MT effects adds to mounting evidence
that steroids exert nongenomic actions on a variety of target tissues, including
intestine64.66, 73.
Long-term treatments (5-11 months) of seawater-adapted tilapia with MT-
supplemented diets were also shown to stimulate glucose uptake in BBMV. At a
growth-promoting dietary dose (10 mg/kg food), MT elicited four distinct transport
effects compared with controls: the maximal glucose transport rate (Vmax) increased
2 fold; glucose passive permeability increased 53%; Na+/K+-ATPase activity rose
2.5 fold; and, the coupling ratio of Na+-glucose cotransport increased from 1:1
to 2:1. These changes suggest that the hormonally mediated adaptations occur
at several levels in glucose transport mechanisms. Transporter induction (implied
by the elevated Vmax), higher permeability, and higher coupling ratio would all
accelerate glucose entry into the enterocyte, especially at luminal glucose concen-
trations higher than those in the blood. Since Na+influx through the transporter
would also increase, the elevated Na+-pump activity might prevent dissipation of
the Na+-gradient. Thus, the driving force for nutrient uptake would be maintained
even at high transport rates.
Collectively, the trout and tilapia data suggest that stimulation of nutrient
absorption represents one mechanism contributing to MT's anabolic actions in fish.
Nutrientfluxes and regulationin fish intestine 233
2.1.2. Interrenal steroids. Glucocorticoids, which mobilize fuels during stress

in vertebrates, also play a developmental role in modulating intestinal enzyme
levels in suckling mammals (see ref. 41). In migratory salmonids, by comparison,
cortisol serves a developmental function in preparing fresh water salmon parr
for seawater adaptation during the parr-smolt metamorphosis 31,77. Collie and
Stevens 16 found that cortisol stimulates L-proline uptake in pyloric ceca of coho
salmon (Oncorhynchus kisutch) prior to seawater migration. When salmon were
implanted with cortisol pellets for two weeks, the Vmax and Km of proline uptake
were increased two-fold, concomitant with a significant rise in plasma cortisol,
compared with controls receiving cholesterol implants. Hence in fish as well as in
mammals, glucoeorticoids appear to regulate digestive adaptations during ontogeny.
2.2. Growthhormone (GH)

Somatic growth in juvenile vertebrates is accelerated by GH, requiring a boost in di-
etary requirements for calories and protein. In salmon undergoing parr-smolt trans-
formation, the energetic costs of rapid growth are compounded by caloric needs to
fuel migration and seawater adaptation. Like anabolic steroids, GH stimulates food
consumption and more efficient conversion of ingested nutrients into body tissue 21.
Two studies offer evidence that a component of this enhanced digestive efficiency
is stimulation of nutrient transport. Bovine GH pellets implanted into coho salmon
(0. kisutch) increased the Vmax of intestinal proline uptake and stimulated mucosal
mass per cm of gut length 16. Both specific and nonspecific mechanisms, therefore,
combined to yield greater AA uptake in growth-stimulated salmon. A second study
confirmed this effect, but in juvenile striped bass (M. saxatilis) a2. GH injections
for 3-4 weeks elevated Na+-dependent and Na+-independent AA in tissue slices
and BBMV. The earliest effect, observed two days after a single injection, was an
increased Vmax of Na+-dependent uptake. An slower increase in mucosal mass
per serosal surface area later augmented AA uptake further. Thus, enhanced AA
uptake may be an early adaptation to growth stimulation, ensuring an adequate fuel
supply for anabolic metabolism.
2.3. Thyroid hormones

Nutrient transport is regulated under many divergent circumstances, so we might
not expect to find one signal common to all of them, even when transport changes
in the same direction. But for some related subset of conditions, such as accelerated
growth, whether occurring naturally or artificially induced, a 'unifying' signal is
plausible. Some evidence supports the thyroid hormone 3,5,3'-triiodo-L-thyronine
(T3) as one candidate signal regulating energy balance in a variety of anabolic
states 22.
Above we discussed two examples of hormonal growth enhancement (MT and
GH), which were accompanied by increased nutrient absorption. If T3 signals
intestinal adaptation under these anabolic conditions, we could make two testable
a priori predictions. First, T3 levels should be elevated in these conditions. Second,
T3 treatment itself should reproduce some or all of the effects on nutrient transport
elicited by MT and GH.
234 N.L. Collie and R.P. Ferraris
The first prediction appears to hold in some species, because fish receiving
androgens or GH show increased T3 levels (reviewed in ref. 22). Unfortunately, "1"3
levels were not reported in any of the above-mentioned papers examining nutrient
transport in response to MT or GH. However, in a companion study to the Reshkin
et al.7~ paper, tilapia from the same MT-fed treatment groups did in fact exhibit
elevated T3 levels~. It appears likely, then, that both MT and GH increase plasma
T3 concentrations and stimulate nutrient uptake.
The second prediction has been tested in tilapia by Reshkin et a/. 71 and, more
recently, in rainbow trout by Collie et al. is. In the study by Reshkin et a/. 71, which
examined MT's effects on glucose transport, tilapia were also fed T3-supplemented
diets in graded doses. All parameters stimulated by MT were similarly affected by
the optimal T3 dose (1 mg/kg feed), including body mass, brush-border glucose
Vmax and Km, passive glucose permeability, Na+/K+-ATPase activity, and the
Na + "glucose coupling ratio. The striking match between MT and "['3 actions
supports the prediction that "1"3can mimic MT's effects on glucose transport.
Similarly, in rainbow trout intestine, dietary T3 was able to reproduce most of
GH's effects on glucose and proline uptake across the brush border is. Two weeks
of either ovine GH injections (0.2-2.0/zg/g bwt, every 4th day) or T3-supplemented
diets (10-20 mg/kg feed) stimulated Na+-dependent glucose and proline uptake
per mg of wet gut mass. After 2 weeks of treatment, there were as yet no effects on
body size or on gut mueosal mass. Hence, the early effects of both treatments are
consistent with a change in nutrient transporter activity, rather than a nonspeeific
mechanism such as mueosal growth. By 6 weeks of treatment, however, both
hormones caused significant increases in body mass and mueosal mass per cm of
intestine.
As in tilapia, our second prediction holds for trout intestine, that T3 can
reproduce the intestinal nutrient changes elicited by a second growth-promoter,
GH. It should be noted that neither study excludes (3H- and MT-speeitie effects
that are independent of T3's actions. However, the results support the hypothesis
of T3 as a common signal underlying enhanced nutrient absorption in growth-
stimulated fish.
Fl. Summary andperspectives

We began by considering the nutrient transport mechanisms for glucose and
AAs, asking if the transport pathways are sufficiently known to permit detailed
studies about their regulation. Fish intestine shares many fundamental nutrient
transport properties with mammalian intestine, but we cannot tacitly assume that
this is uniformly the case. Particularly for AA transport, there are many unsolved
questions about the number and specificity of the pathways involved. For both
glucose and AA absorption, the basolateral membrane remains the 'dark side of
the epithelium '91. Its transporters are beginning to yield to membrane isolation
techniques that provide vesicles for transport characterization. Too infrequently
have such techniques been used to investigate fish intestine.
With the cloning of glucose transporters on both 'sides' of mammalian en-

terocytes, there is now much excitement over the prospect that a transporter's
protein domains (i.e. AA sequences) can be linked with the transport functions they
perform. The recent reports of cloned AA transporters offer the possibility of asso-
ciating unambiguously a specific protein with the class of AAs it transports 5~176
Significant sequence homology appears to exist among transporters in different
vertebrate groups 12,39,40. Hence, sequence information in mammals may be used to
create eDNA probes and identify related transporters in fish intestine.
As our understanding of transport mechanisms improves, so should our ability
to answer questions of the regulation of intestinal transport. We conclude by
emphasizing below several areas where future research might be directed.
(1) Dietary carbohydrate and species. Herbivorous and omnivorous fish adapt
to dietary carbohydrate levels with altered glucose uptake rates, but carnivores
(e.g. trout) appear less able to up-regulate glucose transport 2~ However, certain
hormone treatments (GH, T3) seem capable of increasing glucose absorption even
in carnivores. Higgs and coworkers 42 report that dietary T3 treatment offsets the
reduced growth rates of rainbow trout on high-carbohydrate diets. Is intestinal
glucose transport induced by differing carbohydrate levels in T3-supplemented diets
or is the gut responding directly to T3 itself as the signal? Which steps in glucose
absorption are regulated by T3 ?
(2) Induction of AA transport. Intestinal proline uptake varies less than does
glucose uptake to changes in dietary composition 2~ However, there have been no
direct tests of altering specific AA levels in the diet to look for induction of specific
AA transport pathways. The results could provide important, practical input into
the design of fish diets that may boost AA absorption and promote rapid growth.
(3) Steroids and nutrient transport. Androgens and interrenal steroids promote
glucose and AA transport, but the mechanism remains unclear. The rapid ef-
fects of androgens offers a fruitful model to explore nongenomic mechanisms of
action on nutrient transport. In addition, the interactions of steroids with other
endocrine signals, such as T3, needs clarification before the proximate signals for
transport regulation can be identified. How are these signals transduced within the
enterocyte?
(4) Intestinal absorption and fish growth. Rapid growth and enhanced nutrient
transport are correlated, but it is unclear whether intestinal adaptation leads or
simply responds to the growth of other tissues. The early response of the gut to
hormonal growth stimulation may suggest the former, but time-course studies of
changes in absorptive function relative to the function and growth of other tissues
seem especially warranted.
Acknowledgements. We wish to thank our mutual colleagues for generously

sharing ideas and manuscripts during the preparation of this review. Portions of
our work were supported by the California Sea Grant College Program, Project
R/A-71, and NIH Grant DK 42973. The U.S. Government is authorized to produce
and distribute reprints for governmental purposes notwithstanding any copyright
notation that may appear hereon.
236 N.L Collie and R.R Ferraris
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O 1995 Elsevier Science B.V. All rights reserved.
C H A P T E R 10
Metabolic organization of thermogenic

tissues of fishes
JAMES S. BALLANTYNE
Department of Zoology, University of Guelph, Guelph, Ontario, N I G 2W1 Canada
I. Introduction
II. Metabolism of endothermic muscle of sharks
III.Metabolism of endothermic muscle of tuna
IV. Metabolism of billfish brain heater
V. Summary and prospectus
Acknowledgements
VI. References
I. Introduction
Living systems catalyze many of the exothermic chemical reactions found in nature.
Although the amount of heat produced by a chemical reaction cannot be changed,
by coupling one reaction with another, the amount of heat released can be reduced
by conserving the energy in chemical bonds. Uncontrolled combustion yields few
useful intermediate molecules and much energy is lost as heat, consequently, much
of the early evolution of life on this planet must have been involved with optimizing
systems to minimize heat loss. Only much later in the evolution of life did the
controlled production of heat become a metabolic option and confer survival
advantages on organisms. With metabolism optimized to trap as much energy as
possible as ATP, two mechanisms of heat production are possible. One involves
bypassing ATP synthesis and the other involves 'wasting' ATP through the action of
ATPases.
Bypassing ATP synthesis occurs in mammalian brown adipose tissue through
the action of a proton channel, thermogenin. This protein allows protons to enter
the mitochondria without participating in ATP synthesis. This leak partially col-
lapses the membrane potential and proton gradient across the inner mitochondrial
membrane. The leaks themselves are merely signals used to turn on the metabolic
processes generating heat. Heat is produced when substrates are oxidized in Krebs
cycle to pump protons out of the mitochondrial matrix to reestablish the membrane
potential. This allows the mitochondria to respire at high rates without the need
to phosphorylate ADP. Proton leaks probably occur in all mitochondrial mem-
branes and may be a design feature of some endothermic mitochondria. Brand and
242 ,/.,5. Ballantyne
colleagues 12 have shown the membranes of mitochondria from ectothermie reptiles

to be less leaky than those of similar sized mammals at the same temperature. In
these cases the leak is nompecific and a property of the membrane. Such 'basal'
leaks may be important thermogenic mechanisms but, since they are properties of
entire membranes not specific channels, regulation of the rates would be problem-
atic requiting long-term changes in membrane properties. Such changes do occur
and are mediated by thyroid hormones in mammals 13. Leaks mediated by specific
channels, however, offer the advantage of being more rapidly modulated.
In addition to bypassing mitochondrial ATP synthesis, another mechanism for
generating heat is to release the energy trapped in ATP itself. A variety of ATPases
exist that could be marshalled for this function. Two of the major ones are transport
ATPases: Na+/K+-ATPase and Ca2+-ATPase. A third ATPase, myosin ATPase, is
involved in energy transduetion during muscle contraction. In understanding the
design of thermogenic mechanisms it is important to account for all of these
processes.
The use of heat production to elevate and control body temperature is most
widespread among terrestrial organisms. Insects, birds and mammals all have
representatives capable of sustaining higher than ambient body temperatures.
Since the low thermal conductivity of air provides a favorable environment for
the development of endothermy, some of these endotherms can be quite small
(less than one gram for some insects). Endothermy in the aquatic environment is
confined to larger organisms due to the high thermal conductivity of water. The
smallest endothermie fish weigh about 1-2 kg or 1000 times more than the smallest
terrestrial endothermic vertebrate (2 g).
Endothermic tissues have been documented in the large species of three groups
of fishes: the lamnid sharks, the billfishes, and the tunas. Large size alone is not
a sufficient condition for endothermy in fishes. Specific anatomical features have
been developed to enable warm-bodied fish to retain heat as well as shunt it to
other tissues (see ref. 8 for review). Bone ~1 has suggested that the constancy of
temperature, not simply elevated temperature, was the critical factor in the design of
warm tissues. Constancy requires regulation to control the rate of heat production,
the capacity for sustained heat production and the ability to control heat loss. The
metabolic production of heat must occur at faster rates than it is dissipated if the
tissue temperature is to rise above ambient. If the capacity to produce heat exceeds
the rate at which it is dissipated, regulation of the reactions producing heat must be
strict to prevent tissue temperature from rising to dangerous levels.
The tissues of fish may be heated by intracellular as well as extracellular
chemical reactions. The viscera of lamnid sharks 17 and tunas 16 have been shown
to have higher than ambient temperatures. Heat production in these tissues is
an extracellular phenomenon based on the exothermic hydrolysis of protein and
triglyeerides 16. As an extraeellular process, the normal mechanisms of metabolic
regulation do not operate to control the rates of heat production. Since digestion
of food is the source of heat, regulation of heat production in these tissues may
simply involve regulation of heat dissipation through control of the vascular heat
exchangers. Nevertheless, the involvement of the cells of the visceral tissues in heat
Metabolic organization of thermogenic tissues o.ffishes 243
production cannot be ruled out and further studies of the metabolism of these
tissues are required.
Intracellular processes of heat production are better understood and will be the
primary focus of this review. In fish, every tissue producing significant amounts of
heat by intracellular processes is a muscle. These include a modified extraocular
muscle in the head of billfish and the lateral red muscles of tunas and lamnid
sharks. Insights into the organization of metabolism of thermogenie tissues of fishes
is, therefore, dependent on an understanding of the metabolism of fish red muscle.
The goal of this review is to summarize the current state or our knowledge relating
to the organization of metabolism and its regulation in thermogenic tissues of fishes.
This review will rely heavily on studies of the metabolism of nonthermogenic fish
muscle to elucidate the function of the metabolism of thermogenic tissues.
II. Metabolism of warm muscle of sharks

Among the elasmobranchs, endothermy occurs in one family, the Lamnidae, with
only 5 species. These sharks are large, fast swimming predators that successfully
compete with, and feed upon, marine mammals. While the metabolic rates of most
elasmobranchs are lower than those of comparable teleost fishes 14 studies of one
endothermie lamnid, the shortfin mako shark (Isurus oxyrhinchus), indicate it has
a metabolic rate comparable to that of some Warm-bodied tunas 2s. The primary
source of heat in lamnid sharks is the lateral red muscle 51. The heat produced by
this tissue is used to warm the blood which passes through the brain and eyes 9 and
viscera 17 as well as the muscle itself. The sources of heat in elasmobranch muscle
are the processes associated with muscle contraction. The two major transport
ATPases (Na+/K+-ATPase and Ca2+-ATPase) must be involved as well as the
myosin ATPase. The oxidation of substrates to supply ATP for these enzymes
produces most of the heat.
The elasmobranchs differ from virtually all other groups of fish in that fatty
acids are not important oxidative substrates in muscle. The levels of enzymes of
oxidation of fatty acids, carnitine acyl transferases, are undetectable in the muscle
of nonendothermic sharks 4~ and isolated red muscle mitochondria do not oxidize
fatty acids or acyl carnitines 2,19,4~ The explanation for the absence of lipid oxidation
in elasmobranch red muscle has been attributed to the absence of the fatty acid
carrier protein in the blood 22. The levels of plasma nonesterified fatty acids in
elasmobranchs are low by comparison with those of some species of fishes4,22,52
but are far higher in concentration than is possible based on their solubility. In the
absence of albumin, it has been suggested 4 that fatty acids must be carried by other
lipid fractions of the plasma and consequently may not be delivered to the tissues
with sufficient rapidity to sustain muscle contraction.
The large lipid reserves in the liver of elasmobranchs, could be an accessi-
ble energy source for red muscle provided the carbon is transported in another
form. Ketone bodies have been suggested to be major fuels for elasmobranch red
muscle 2.19,4~ Ketone body levels in elasmobranch plasma are high and increase
244 J.S. BaUantyne
TABLE 1
Substrate oxidation by mitochondria isolated from 'cold' and 'warm' elasmobranch red muscle
Substrate State 3' rate of respir'ation

Raja erinacea 19 Squalus acanthias 19 lsurus oxyrinchus 2
Glutamine "" 19.18 ~- 1.77 (7) 45.65 4- 4.97 (5) ' 49.84 4- 2.21 (3)
Glutamate 9.32 4- 1.49 (7) 26.31 4- 7.61 (6) 45.94 4- 13.81 (3)
Proline 8.16 + 0.99 (7) 28.47 4- 2.46 (4) 38.43 4- 41.16 (2)
/~-Hydroxybutyrate 8.85 4- 1.20 (6) 54.04 4- 5.15 (6) 46.53 -4- 3.92 (3)
Pyruvate 9.70 4- 1.65 (6) 27.51 4. 7.64 (4) 34.34 4- 12.53 (3)
a-Glycerophosphate ND ND ND
Palmitoyl-L-carnitine ND ND ND
Endogenous 3.61 4- 0.43 (7) 3.71 4- 0.88 (5) 3.72 4- 2.24 (3)
Values are means 4- SEM with the number of mitochondrial preparations given in parentheses.
Respiration is expressed in nmol O2/min/mg protein. Rates for little skate (Raja erinacea) and spiny
dogfish (Squalus acanthias) were determined at 10~ and the rates for mako shark (Isutus oxyrinchus)
were determined at 20~ ND = Not detected.
during starvation 52. The source of plasma ketone bodies is likely hepatic lipid since
liver is capable of the oxidation of fatty acids in elasmobranchs 3,43,44 and Anderson 1
has shown ketone body formation from palmitoyl-CoA by liver mitochondria. Ke-
togenesis from amino acids has been demonstrated in elasmobranch hepatocytes38
and may play an important role in supplying substrates for muscle contraction.
In addition to ketone bodies, amino acids may be important direct energy sources
in thermogenic red muscle of elasmobranchs tg. Three of the major substrates
oxidized by elasmobranch red muscle mitochondria are amino acids (glutamine,
glutamate and proline; Table 1). The high protein diet of elasmobranchs would
make reliance on amino acids as an energy source for muscle contraction a viable
alternative to lipid.
Of the amino acids oxidized, glutamine in particular, appears to be a major
metabolic substrate in elasmobranch red muscle2,19. Plasma glutamine levels in the
dogfish shark (Squalus acanthias) are about 12/~M and in the muscle itself levels
are about 0.6 mM. This 50 fold gradient indicates active uptake of this amino
acid 19 although nothing is known of the mechanism of transport of this amino acid
across the sarcolemma in elasmobranchs. One possibility is a Na + coupled symport
mechanism similar to that found in mammalian muscle 32. Once inside the cell,
glutamine is converted to glutamate via phosphate dependent glutaminase (PDG).
PDG levels in elasmobranch red muscle are higher than those found in muscle
of any species (Table 2) and rival levels in the mammalian kidney 19. The pathway
for oxidation of glutamine is thought to be autocatalytic, in that mitochondrial
malic enzyme converts some of the amino acid carbon to pyruvate providing acetyl
CoA to condense with the remaining carbon in oxaloacetate to form citrate. Since
this reaction involves the production of NADH, there is no loss of ATP synthetic
capacity when some of the malate is converted to pyruvate instead of oxaloacetate.
Since glutamate is also oxidized by elasmobranch red muscle mitochondria, and
glutamate is an intermediate in the oxidation of glutamine, some coordination of
Metabolic organization of thermogenic tissues of fishes 245
TABLE 2
Activities of glutarninase and glutamine synthetase in red muscle of fishes
Species Glutaminase Glutamine synthetase

Hagfish 19 0.39 NA
Skate 19 1.96 0.27
Dogfish 19 3.26 NA
Bowfin 18 0.22 0.09
Lake char 18 0.23 0.01
The units are/xmoles of substrate converted per minute per gram wet weight of tissue. All measurements
were made at 10*C. Hagfish -- Petromyzon marinus, Skate = Raja erinacea, Dogfish ffi Squalus acanthias,
Bowfin ffi Amia calva, Lake char ffi Salvelinus namaycush. NA ffi Not analyzed.
the catabolism of these two substrates is required especially if their oxidation serves
two separate purposes. The oxidation of glutamine by elasmobranch red muscle
mitochondria is regulated, in part, by a compartmentation of glutamate. Several
lines of evidence support this conclusion. Chamberlin and Ballantyne 19 have shown
that oxidation of exogenous glutamate is inhibited by aminooxyacetate, an inhibitor
of pyridoxal phosphate-dependent transaminases while glutamine oxidation is not.
This indicates that glutamate entering from outside the mitochondrion has different
access to certain matrix enzymes than glutamate generated inside the mitochon-
drion from glutamine via PDG. Glutamate derived from glutamine is catabolized via
glutamate dehydrogenase (GDH) not aspartate aminotransferase (AspAT). Studies
of similar systems in teleost fishes indicate aspartate production from glutamate is
much greater than that from glutamine 18 and CO2 production from glutamine is
much higher than that from glutamate even though respiration rates with the two
substrates are similar (M. Gerrits and J.S. Ballantyne, unpublished data).
The physical nature of the compartmentation can only be speculated upon.
In one possible configuration, the membrane carrier for glutamine is coupled to
PDG which is, in turn, coupled to GDH (Fig. 1). Transfer of glutamate formed
by PDG directly to GDH occurs without release of glutamate. This glutamate,
therefore, does not have access to AspAT, the other mitochondrial enzyme using
glutamate as a substrate. GDH converts glutamate to a-ketoglutarate which may
enter Krebs cycle for oxidation. In the other glutamate pool, glutamate entering via
the glutamate/aspartate carrier has immediate access to AspAT and a-ketoglutarate
is again produced. This a-ketoglutarate pool may not be the same as that produced
by GDH and, therefore, may not enter the Krebs cycle (region 7 in Fig. 1).
Glutamate carbon must exit as ot-ketoglutarate to permit continuous operation of
the malate-aspartate shuttle. Compartmentation of glutamate may occur simply to
ensure the exit of its carbon as a-ketoglutarate. Thus by compartmentalizing these
two functions of glutamate, cytoplasmic redox can be balanced while oxidation of
amino acids such as glutamine and proline may proceed unaffected, in this scheme,
cytosolic glutamate enters the mitochondria only for redox balance. Glutamine is
oxidized to supply energy for ATP synthesis.
Oxidation of glutamine in elasmobranch red muscle could potentially compete
246 J.$. Ballantyne
Fig. 1. Compartmentation of glutamate metabolism in thermogenic tissues of fishes. Some intermediates

are omitted for clarity. 1 = glutamine carrier; 2 = phosphate-dependent glutaminase; 3 = glutamate
dehydrogenase; 4 = malate/a-ketoglutarate carrier; 5 = glutamate aspartate carrier; 6 = aspartate
amino transferase; 7 = segment of Krebs cycle. AAT = aspartate aminotransferase; MDH = malate
dehydrogenase; ME = malic enzyme.
with oxidation of other substrates such as fl-hydroxybutyrate. As indicated above,

oxidation of glutamine occurs via GDH in the mitochondrial matrix. Oxidation
of/5-hydroxybutyrate also occurs in the mitochondrial matrix via the action of
/3-hydroxybutyrate dehydrogenase. Both enzymes require NAD to convert their
substrate into the compound entering Krebs cycle. Competition for mitochondrial
matrix NAD may, therefore, occur (Fig. 2). A similar situation has been studied
in the mammalian kidney, where it has been found that elevated levels of ketone
bodies inhibit glutamine catabolism 3s. Inhibition is due to increased formation of
citrate due to higher rates of acetyl CoA condensation with oxaloacetate during
ketone body metabolism. The elevated levels of a-ketoglutarate that result inhibit
GDH resulting in the accumulation of glutamate and the inhibition of PDG (Fig.
2). It has also been suggested that c~-ketoglutarate may inhibit the glutamine
carder 24 in mammalian kidney. Similar studies are required to establish if the
regulatory mechanism operating in the mammalian kidney applies to elasmobranch
red muscle.
fl-Hydroxybutyrate may compete with pyruvate for entry into the mitochondria
since both substrates are transported on the monocarboxylate carrier. Since amino
Metabolic organization of thermogenic tissues of iishes 247
Fig. 2. Competition between ketone bodies and glutamine as oxidative substrates in thermogenic tissues
of fishes. Some intermediates are omitted for clarity. 1 = mitochondrial monocarboxylate carrier; 2 = fl-
hydroxybutyrate dehydrogenase; 3 = 3-oxoacid CoA transferase; 4 = acetoacetyl CoA acetyltransferase;
5 = pyruvate dehydrogenase; 6 = citrate synthase; 7 = aconitase; 8 = isocitrate dehydrogenase; 9 =
glutamine transporter; 10 = phosphate-dependent glutaminase; 11 = glutamate dehydrogenase; 12 =
ot-ketoglutarate dehydrogenase; 13 = succinyl CoA synthetase; 14 = succinate dehydrogenase; 15 =
fumarase; 16 = malate dehydrogenase 17 = malic enzyme.
acids and ketone bodies are important energy sources for elasmobranch muscle,
carbohydrate may thus be spared from oxidation. Little is known of the interaction
between ketone body and carbohydrate metabolism in elasmobranchs. Plasma
glucose levels are lower in elasmobranchs compared to teleost fishes s2 and muscle
mitochondria oxidize pyruvate at lower rates than those of glutamine and fl-
hydroxybutyrate (Table 1). Hexokinase levels in red muscle of elasmobranchs are
relatively high 39 perhaps implying glucose can be taken up by the tissue to replenish
carbohydrate depletion during anaerobic glycolysis. Cytoplasmic redox balance
when carbohydrate is catabolized aerobically is achieved through the malate-
aspartate shuttle rather than the ct-glycerophosphate shuttle based on the absence
of mitochondrial oxidation of t~-glycerophosphate (Table 1).
The rates of substrate oxidation per mg mitochondrial protein of the red muscle
mitochondria of the highly active shortfin mako shark are not higher than those
of less active elasmobranchs such as the little skate or the dogfish shark (Table 1).
The substrate preference for oxidation by mitochondria is similar (Table 1) and the
degree of coupling of oxidation to phosphorylation as indicated by the respiratory
248 J.S. Ballantyne
control ratio is similar2 suggesting heat production is due to rapid oxidation of

substrates and high myosin ATPase activity not through a high proton leak as occurs
in the brown adipose tissue of mammals. The basal proton leak in warm-bodied
elasmobranch muscle has, however, not been determined.
The contributions of Na+/K+-ATPase and Ca2+-ATPase likewise have not been
determined in this tissue but may be expected to resemble those of other muscles
and thus contribute relatively little to heat production 2~
III. Metabolism of warm muscle of tuna
The red muscle of tuna is involved in 'basal' swimmingr with heat production a
by-product of locomotion. Tunas by contrast with elasmobranchs are representatives
of a recently evolved group of fishes, the teleosts. One important difference in the
metabolism of the endothermic red muscle of the tunas compared to that of the
elasmobranchs is the use of lipid as an energy source. The presence of a fatty acid
carrier protein, albumin, in the blood allows efficient transport of fatty acids to red
muscle for oxidation.
Red muscles of skipjack (Katsuwonus pelamis) 3~ and kawakawa tuna (Euthynnus
affin/s)23 have been shown to contain significant amounts of fat and glycogen.
Reliance on exogenous glucose as indicated by hexokinase levels is moderate in
red muscle compared to white27. The studies of Weber and colleaguess~ indicate
plasma glucose turnover rates for tuna are higher than those reported for any other
fish species. The extent of utilizationof glucose by red muscle for thermogenesis
remains to be determined. The studies of tuna red muscle by Moyes et al.42 indicate
similar substrate preferences to other teleost red muscle mitochondria. Pyruvate
and palmitoyl carnitine are oxidized at the highest rates indicating lipids and
carbohydrates are preferred energy sources. Hulbert et al.31 report abundant lipid
droplets in skipjack tuna (K. pelamis) but Mathieu-Costello and coworkers 36 find
few lipid droplets in red muscle of the same species. Such differences likelyreflect
seasonal changes in food abundance (cf.Chapter I, thisvolume).
The role of amino acids as energy sources in tuna muscle, has not been examined.
Since glutamine has been found to be a major oxidative substrate in aU other fish
red muscle examined to date2,1s,19 the importance of glutamine metabolism in
one of the most metabolically active fish tissueswould be of considerable interest.
The regulation of amino acid catabolism is likelyto resemble that found in other
teleosts~s as well as elasmobranchs 19.
Although it is assumed that tunas lack the ability to use ketone bodies, this
has never been demonstrated. Tissues of other teleostshave been found to oxidize
ketone bodies including /3-hydroxybutyrate as energy sources2 and the enzyme
/~-hydroxybutyrate dehydrogenase has been demonstrated in a variety of teleost
tissues (ref.34a).
The special regulatory requirements to control one of the most metabolically
active fish tissues have been the subject of investigation for many years. Redox
balance is required for aerobic glycolysis and although this may be supplied
in part by the malate-aspartate shuttle, the u-glycerophosphate shuttle is also

likely to be important since the levels of ct-glycerophosphate dehydrogenase are
high in tuna red muscle 27 compared to other fish species such as carp 41. The
rationale for a reliance on this hydrogen shuttle compared to the malate-aspartate
shuttle is unknown but may be speculated upon. It may simply be faster to
balance cytoplasmic redox without transporting substrates into the mitochondria.
The malate-aspartate shuttle involves the transport of two metabolites into the
mitochondrial matrix in exchange for two other metabolites (Fig. 1). The u-
glycerophosphate shuttle does not require the transport of u-glycerophosphate into
the matrix the mitochondrial form of the enzyme is accessible to the cytoplasm.
Transport mechanisms are frequently flux controlling and elimination of substrate
transport may help enhance the rates of redox balance. Alternatively, the use of
the a-glycerophosphate shuttle may be related to the mechanism of coupling the
respiration rate of the mitochondria to the rate of muscle contraction and ADP
supply. Mitochondrial respiration in vitro is controlled in part by availability of ADP.
ADP levels in tuna red muscle are lower in steady-state swimming than in 'resting'
fish 27. While regulation of mitochondrial respiration rates by ADP availability may
suffice in low performance tissues such as liver, other tissues may require a more
rapid initiation of respiration than can be provided by ADP. Calcium may provide
such a signal. Calcium stimulates the mitochondrial oxidation of u-glycerophosphate
in mammals (see ref. 28 for review), insects 29 and other fish 2. This has not been
demonstrated in tuna red muscle mitochondria. Calcium release in muscle cells
precedes the accumulation of ADP and may provide a more rapid signal for the
initiation of mitochondrial respiration. Such a rapid response time may only be
critical in warm-bodied fish pushing the limits of metabolism, t~-Glycerophosphate
levels in red muscle do not change from rest to steady-state 27 perhaps implying
stimulation by elevated calcium levels occurs allosterically with a decrease in the
apparent Km for u-glycerophosphate as has been described for brown adipose
tissue mitochondria 15. Although the kinetics of tuna red muscle t~-GPDH have
been studied 3~ the effects of calcium remain to be examined. Competition between
o~-glycerophosphate dehydrogenase and lactate dehydrogenase (LDH) for NADH
is a function of pH and other factors 26. At low pH, LDH balances redox with lactate
accumulation, while at high pH, u-GPDH balances redox 26.
Another function of a-glycerophosphate dehydrogenase may be the in situ re-
cycling of glycerol released during fatty acid oxidation. Glycerol may be used to
re-esterify fatty acids or as a source of carbon for gluconeogenesis. Brown adipose
tissue, another tissue using lipid as an energy source, has high levels of mitochon-
drial u-glycerophosphate dehydrogenase 15 and glycerol kinase 6. Gluconeogenesis
from glycerol may be an important function of these enzymes. Re-esterification
of fatty acids also requires glycerol-3-phosphate and may provide a mechanism to
prevent excessive accumulation of free fatty acids. The levels of glycerol kinase in
tuna red muscle have not been determined but should be high.
Lactate may serve as an energy source for red muscle especially following periods
of burst activity in white muscle. Lactate accumulation in white muscle of tuna may
be as high as 90 mM 27. The levels of LDH in red muscle of tuna are low (500 units
250 J.$. Ballantyne
per gram tissue fresh weight; 1 unit defined as the amount of enzyme producing 1
/~mol of product per min at the given assay conditions) measured in the forward
(pyruvate to lactate) direction 27. Since the reverse direction (lactate to pyruvate)
direction is generally ten- to fifteen-fold lower in activity in tuna (J.S. Ballantyne,
unpublished data) and functioning well below maximal rates, lactate to pyruvate
flux would likely be low and not sufficient to maintain high rates of Krebs cycle
activity.
Several studies have attempted to explain the apparent high metabolic rate of this
tissue. The red muscle fibres have a mitochondrial volume density of 28.5% 36. This
is not substantially different from values obtained for a variety of nonendothermic
fish muscles (e.g. 31% for trout red muscle34). The activities of citrate synthase
per mg mitochondrial protein of tuna red muscle are similar to those of carp red
muscle 42.
The extreme aerobic capacity of tuna red muscle is apparently due to a com-
bination of factors including the larger red muscle mass, the high mitochondrial
density and an increased membrane surface area 42. Perhaps most importantly, the
elevated temperatures of tuna red muscle plays a role in the enhancement of power
production 33 and heat production.
The studies of Moyes and colleagues 42 indicate the red muscle mitochondria of
tuna are well coupled although as in the case of the elasmobranch endothermic red
muscle the basal proton leak was not quantified.
IV. Metabolism of billfish brain heater

The red muscle of tunas and lamnid sharks, generates excess heat as part of its
normal function in locomotion. In these cases, the lateral red muscle is a very active
tissue in very active fish. Heat is produced by ATP hydrolysis and mitochondrial
oxidation of substrates. In contrast, the swordfish (Xiphiasgladius) and other billfish
are relatively inactive and do not generate significant amounts of heat in their
lateral red muscle. They do have warm brains and eyes due to the presence of a
modified extraocular muscle s. Much of the anatomy and biology of this tissue has
been extensively reviewed by Blocks. Of all thermogenic tissues of fishes, the brain
heater is by far the best characterized, but the regulation of metabolism in this
tissue has not been examined in detail.
The brain heater organ has lost much of its contractile apparatus. That com-
ponent of heat production derived from myosin ATPase activity is, therefore, lost.
Accompanying the loss of contractile elements, is an enormous proliferation of
mitochondfia with up to 70% of the cell volume being occupied by mitochondria 7.
The lack of contractile function has lead to the speculation that heat production in
this tissue is by a mechanism similar to that of mammalian brown adipose tissue.
In brown adipose tissue, a proton leak v/a a specific proton channel 'thermogenin'
effectively uncouples the mitochondria resulting in high rates of respiration with
no requirement for the phosphorylation of ADE Substrate oxidation consequently
proceeds at high rates to pump protons out of the mitochondria. The high rates of
Metabolic organization of thetmogenic tissues of lishes 251
TABLE 3
Substrate oxidation by mitochondria isolated from swordfish (Xiphias gladius) brain heater organ
Substrate State 3 rate of respiration

Endogenous 18.41 4- 8.97 (3)
Glutamine 148.81 4. 50.39 (4)
Glutamate 167.16 4. 48.10 (4)
Proline 37.48 -4- 16.56 (4)
/~-Hydroxybutyrate 60.32 4- 27.34 (4)
Pyruvate 151.28 4. 61.80 (4)
Palmitoyl-L-carnitine 143.70 4- 49.80 (4)
a-Glycerophosphate (5 mM 4- 1 mM CaCI2) 167.03 4- 91.16 (4)
Values are means 4- SEM with the number of mitochondrial preparations given in parentheses.
Respiration is expressed in nmol O2/min/mg protein. Rates were determined at 20°C. Data are taken
from reference 2.
substrate oxidation are the source of heat. Block7 reports being unable to detect the
proton channel 'thermogenin' in this tissue. Freshly isolated mitochondria of the
heater tissue of the swordfish are tightly couplede indicating the capability for ATP
synthesis. As in the two other thermogenic fish tissues examined above, the basal
proton leak has not been quantified in billfish brain heater mitochondria.
In spite of the absence of a specific proton channel, the mitochondria of the
biUfish brain heater resemble those of mammalian brown adipose tissue in several
respects. The substrates oxidized are similar. Swordfish brain heater has high
levels of citrate synthase, carnitine palmitoyl transferase and 3-hydroxyacyl CoA
dehydrogenase49 and palmitate is readily oxidized by isolated mitochondria (Table
3) indicating substantial reliance on lipid as an energy source for thermogenesis.
Brown adipose tissue mitochondria also rely on lipid as an energy source (see
ref. 45 for review). Ketone bodies are also oxidized by swordfish heater (Table
3) and brown adipose tissue 21. Teleost fishes have been thought to be unable to
oxidize ketone bodies 52 although more primitive fishes such as elasmobranchs 19,52,
holosteans 4s and chondrosteans 47 do use ketone bodies as an energy source. A
recent investigation has revealed the presence of fl-hydroxybutyrate dehydrogenase
in several species of teleost fishes (ref. 34a). Obviously the role of ketone bodies in
teleost fishes needs to be reexamined. Amino acids also serve as an energy source
in binfish brain heater with glutamine playing a prominent role (Table 3) as occurs
in other fish muscle. Glutamine is also an important oxidative substrate in brown
adipose tissue where PDG levels are higher than in liver21. The role of glutamine
oxidation in brown adipose tissue remains to be explained. Competition between
ketone bodies and amino acids such as glutamine may occur as indicated above for
elasmobranch red muscle. Carbohydrate oxidation may also play an important role
in the energetics of the biUfish heater organ. Both Tullis et al. 49 and Ballantyne et
al. 2 report high levels of hexokinase in this tissue and glycogen levels are highs. High
rates of a-glycerophosphate oxidation in both bill heater organ and brown adipose
tissue is another unusual parallel between these two tissues and may indicate similar
mechanisms for controlling heat production.
252 I.s. BaUantyne
The loss of the contractile apparatus removes another ATPase and its signal
(ADP) that could be involved in heat production. In the absence of a specific
mitochondrial proton leak channel, some other means must be provided to al-
low regulation of heat production. The brain heater has substantial amounts of
sarcoplasmie reticulum (SR) and Block7 has suggested that the calcium ATPase
associated with this organeUe provides the ADP required to initiate mitoehondrial
respiration. Electrical or hormonal stimulation of the muscle cell initiates calcium
release from the SR. The calcium pump in the membrane of the SR pumps calcium
back into the SR at the cost of ATP hydrolysis. Continued release of calcium main-
tains a constant supply of ADP to maintain mitochondrial respiration at high rates.
The signal for enhanced mitoehondrial respiration rates could be calcium itself
rather than ADP. A more rapid response could be elicited from the mitoehondria
if calcium were the primary signal. ATP is only hydrolyzed to ADP after calcium
is released from the sareoplasmie retieulum. Calcium entry into the mitoehondria
would, therefore, precede that of ADP. At least three key mitochondrial enzymes
are activated by intramitoehondrial calcium (pyruvate dehydrogenase, isoeitrate
dehydrogenase, a-ketoglutarate dehydrogenase) (Fig. 3). Calcium activation of , -
ketoglutarate dehydrogenase and isoeitrate dehydrogenase has been demonstrated
in trout heart mitochondria 37. Although Ca 2+ activation of pyruvate dehydrogenase
in fish muscle has not been demonstrated, this enzyme may also be Ca 2+ activated
in brain heater organ mitoehondria since several mammalian forms of the enzyme
are activated by mieromolar calcium~. Ballantyne et al. 2 also found substantial
calcium-stimulated oxidation of a-glyeerophosphate in swordfish brain heater mito-
ehondria. High levels of t~-glycerophosphate dehydrogenase have been associated
with redox balance during aerobic glycolysis. Calcium stimulation of respiration
may be an important mechanism for stimulating mitoehondrial metabolism before
ADP levels drop as discussed above. Such a coupling of intraeellular calcium levels
to mitochondrial respiration provides a link between the proposed mechanism 7 for
stimulation of heat production in the tissue. Brown adipose tissue mitoehondria
also have substantial levels of calcium activated t~-glyeerophosphate 15. The site of
action of calcium stimulation is a-glyeerophosphate dehydrogenase 15. Calcium acts
allosterically to reduce the apparent Km for a-glycerophosphate 15. Regulation at
the site of mitoehondrial a-glyeerophosphate dehydrogenase may be important in
sparing glycogen or glucose as has been suggested for mammalian brown adipose
tissue. Inhibition of this enzyme by aeyl CoA in mammalian brown adipose tissue
reduces flux through glyeolysis15. This inhibition would also result in the accumu-
lation of the glycerol 3-phosphate required for esterifieation of fatty acids to form
triglyeerides. The levels of glycerol kinase in swordfish brain heater organ 2 indicate
in situ re-esterifieation of fatty acids may occur in this tissue.
A final similarity between mammalian brown adipose tissue and swordfish brain
heater is the rate of heat production. Similar rates of heat production of swordfish
heater organ have been calculated using either mitoehondrial respiration rates 2 or
enzyme activities49. The rates of heat production of swordfish heater organ rivals
that of mammalian brown adipose tissue (0.5 W/g) 2. The amount of heat produced
varies with the substrate oxidized (0.08 W/g for palmitate versus 0.37 W/g for
Fig. 3. Summary of the metabolic organization of swordfish brain heater organ. Dashed lines indicate
potential sites of regulation due to calcium and other effectors. Some intermediates are omitted for
clarity. 1 = plasma membrane sodium dependent glutamine carrier; 2 = plasma membrane sodium de-
pendent monocarboxylate carrier; 3 = fatty acid transport; 4 = sodium dependent glucose transporter; 5
= Na +/K+-ATPase; 6 = acyl CoA synthetase; 7 = carnitine acyl transferase complex; 8 = mitochondrial
monocarboxylate carrier; 9 = mitochondrial glutamine carrier; 10 = phosphate-dependent glutaminase;
11 = glutamate dehydrogenase; 12 ffi fl-hydroxybutyrate dehydrogenase; 13 = mitochondrial calcium
efltux pathway; 14 = mitochondrial Na+/H+-exchanger; 15 ffi acetyl-CoA acetyltransferase; 16 = 3-
oxoacid CoA-transferase; 17 = malic enzyme; 18 = pyruvate dehydrogenase; 19 = citrate synthase;
20 = malate dehydrogenase; 21 = fumarase; 22 = succinate dehydrogenase; 23 = succinyl CoA syn-
thetase; 24 = mitochondrial Ca2+/Na+-exchanger; 25 = electron transport chain with proton pumps; 26
= t~-ketoglutarate dehydrogenase; 27 = isocitrate dehydrogenase; 28 = aconitase; 29 = mitochondrial
proton leak; 30 = ATP synthetase; 31 = adenylate exchanger; 32 = mitochondrial calcium influx; 33 =
sarcoplasmic reticulum calcium ATPase; 34 = sarcoplasmic reticulum calcium channel; 35 = glycogen
phosphorylase; 36 = phosphofructokinase-1; 37 = cytoplasmic glycerol 3-phosphate dehydrogenase;
38 = triacylglycerol lipase; 39 = glycerol kinase; 40 = mitochondrial glycerol 3-phosphate dehydro-
genase; 41 = aldolase; 42 = glyceraldehyde phosphate dehydrogenase; 43 = phosphoglycerate kinase,
phosphoglyceromutase, enolase, pyruvate kinase.
pyruvate) 2. Such considerations may explain the choice of substrates oxidized in this
tissue but the similarities of the mechanisms regulating substrate oxidation remain
to be determined.
There are several sites of heat production in cells. The two major ATPases in
the swordfish brain heater are the transport ATPases, Na+/K+-ATPase and the
254 ZS. BaUantyne
Ca2+-ATPase. Using ouabain binding studies Block and Franzini-Atmstrong 1~ have

demonstrated high densities of Na+/K+-ATPase in billfish. Without quantitative
estimates of the activity of these two ATPases, it is difficult to assess their respective
contributions to thermogenesis.
In mammalian muscle the ratio of CaZ+-ATPase to Na+/K+-ATPase is between
25 and 125 (see ref. 20 for review). At rest these two pumps are probably responsible
for similar fractions (about 5%) of the total energy exchange of the muscle2~
During activation of mammalian muscle, calcium release from SR is enhanced
30 fold compared to Na + influx and K + efflux2~ increasing the contribution of
calcium cycling to the total energy turnover. Under these conditions calcium cycling
may stimulate 20-50% of total heat produced 2~ Of this the Ca2+-ATPase would
contribute 1/3 of the heat with the remaining 2/3 due to heat release during
substrate catabolism to produce ATP 2.
In summary, heat production in billfish brain heater involves a variety of ther-
mogenic processes (Fig. 3). Substrate (glutamine, ketone bodies and glucose; sites
1, 2, and 4 in Fig. 3 respectively) entry into the cell at the expense of the Na +
gradient will increase Na+/K+-ATPase (site 5 in Fig. 3) activity as would elec-
trical stimulation of the muscle. This would stimulate mitochondrial respiration
by generating ADE The entry of ketone bodies and pyruvate (site 8 in Fig. 3)
into the mitoehondrial matrix at the expense of the membrane potential and
proton gradient would stimulate mitochondrial respiration to pump protons out
and reestablish these gradients. Hormonal and/or electrical stimulation of the
cell resulting in calcium release from the SR would activate glycogen phospho-
rylase (site 35 in Fig. 3) enhancing glycolysis. Redox balance for this pathway
(the t~-glycerophosphate shuttle) would be simultaneously activated by cytoplasmic
calcium stimulation of mitochondrial a-glycerophosphate oxidation (site 40 in Fig.
3). Ca2+-ATPase in the SR (site 33 in Fig. 3) would pump calcium back into the
SR and produce ADP stimulating mitochondrial respiration. Calcium entry into
the mitochondrial matrix would stimulate respiration in several ways. Activation
of three mitochondrial dehydrogenases (pyruvate dehydrogenase, a-ketoglutarate
dehydrogenase and isocitrate dehydrogenase; sites 18, 27, 26 in Fig. 3 respectively)
may occur. Calcium efflux from mitochondria in exchange for Na + (site 24 in
Fig. 3) would stimulate mitochondrial respiration when Na + efflux is coupled to
proton influx (site 14 in Fig. 3). Proton influx collapses the proton gradient and
must be redressed by pumping protons out of the mitochondrial matrix (site 25
in Fig. 3.)
It is unlikely that all of these mechanisms operate simultaneously but more infor-
mation is needed to establish which processes operate under specific conditions.
The Similarities between mammalian brown adipose tissue and billfish heater
organ in both metabolism and metabolic regulation are a remarkable parallel
development. The marshalling of the existing metabolic potential of fish muscleto
produce a similar thermogenir tissue with a design similar to that developed from
white fat cells in the mammals indicates the constraints placed on the 'design' of a
new tissue using 'hardwired' metabolic pathways.
Metabolic organizationof thermogenictissueso]'1ishes 255
V. S u m m a r y a n d prospectus
The thermogenic tissues of fishes are similar in many respects. All are highly
aerobic muscles with high mitochondrial densities. In the tuna and elasmobranchs,
myosin ATPase combines with the two major transport ATPases (Ca2+-ATPase and
Na+/K+-ATPase) to provide ADP to stimulate mitochondrial respiration while in
the swordfish brain heater organ the Na+/K+-ATPase and the Ca2+-ATPase assume
most of this function. Measurements of all three ATPases in fish thermogenic
tissues would help quantify the relative importance of each of these. In the
brain heater tissue calcium may also provide a signal to initiate high rates of
mitochondrial respiration. Although ATPases figure prominently in the mechanisms
of heat production, ATP hydrolysis itself is only responsible for about one third
of the heat produced, the majority being released during mitochondrial substrate
oxidation 2.
Redox is likely balanced by the malate-aspartate shuttle in all systems with
the potential for redox balance via the t~-glycerophosphate shuttle operating in
swordfish brain heater and tuna red muscle but not in the elasmobranch.
Ketone bodies and amino acids are the major oxidative fuels of the endothermic
red muscle of the lamnid sharks. Lipid, carbohydrate and amino acids are possible
energy sources in the endothermic tissues of the teleosts. Some regulation presum-
ably occurs when more than one substrate is available but these processes have not
been investigated in any fish thermogenic tissues.
Even though the current evidence suggests that mitochondria isolated from
endothermic tissues of warm-bodied fishes are well coupled 2. the existence of a
basal proton leak higher than found in nonendothermic tissues cannot be ruled out.
Temperature may also influence proton permeability. In spite of the importance
of temperature to the metabolism of these fishes there are very few studies of the
effects of temperature on the metabolism of the relevant tissues. Future studies
directed at the thermal sensitivity of enzymes and isolated mitochondria should be
undertaken.
Very little is known of the hormonal regulation of heat production. Block8 has
suggested that catecholamines are involved in billfish heater organ and the role of
calcium as a signal supports this view. It is also possible that thyroid hormone in in-
volved in mediating heat production in these fish as occurs in mammals. Ballantyne
and colleagues 5 have shown rapid stimulation of mitochondrial respiration in red
muscle of fish after administration of triiodothyronine.
The regulation of fish red muscle metabolism is poorly understood. Differences
in apparent metabolic complexity exist between the more primitive elasmobranchs
and the more advanced teleosts but the mechanisms of regulating even the simplest
of these metabolic units have not been adequately addressed. The current evidence
suggests that the mechanisms of metabolic regulation of the endothermic fishes do
not differ from the nonendothermic counterparts.
In spite of the difficulty in obtaining these largely pelagic fishes as experimen-
tal animals they will continue to be important models for the understanding of
metabolism in fishes, because they push the limits of metabolism.
256 J.S. BaUantyne
Acknowledgements. I would like to acknowledge useful discussions with Mary

Chamberlin, Tom Singer, Martin Gerrits and Chris Moyes. Studies from the author's
laboratory are supported by the Natural Sciences and Engineering Research Council
of Canada through research grants and by a University Research Fellowship.
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31. Hulbert, W.C., M. Guppy, B. Murphy and P.W. Hochachka. Metabolic sources of heat and power in
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42. Moyes, C.D., O.A. Mathieu-Costello, R.W. Brill and P.W. Hochachka. Mitochondrial metabolism
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258 J.S. Ballontyne
47. Singer, T.D. and V.G. Mahadevappa and J.S. Ballantyne. Aspects of the energy metabolism in the
lake sturgeon, Acipenser fluvescens: with special emphasis on lipid and ketone body metabolism.
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52. Zammit, V.A. and E.A. Newsholme. Activities of enzymes of fat and ketone body metabolism and
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fish. Biochem. J. 184: 313-322, 1979.
CHAPTER 11
Electric organs: structure, physiology,

hormone-sensitivity, and biochemistry
HAROLD H. ZAZON
Department of Zoology, Patterson Laboratory, The University of Texas, Austin, TX 78712, U.S.A.
I. Introduction
II. Distribution
III. Structureand neural control
IV. EOD variation and species communication
V. Functionalorganization of the EO in weaklyelectric pulse-type fish
VI. Functionalorganization of the EO in weaklyelectric wave-typefish
VII. Sex differences in and hormonal modulation of the EOD
VIII. Biochemistryof electric organs: acetylcholine receptor and related molecules
IX. Biochemistryof electric organs: muscle-specific proteins
X. Biochemistryof electric organs: intermediate filaments
Xl. Proteinsorting and targeting
XII. Futuredirections
XIII. References
I. Introduction
Electric organs (EOs) are a uniquely piscine adaptation. Strongly electric fish,
whose discharges are used to stun prey or attackers, were familiar to the ancient
Mediterranean civilizations and native tribes of South America and Africa. The
EOs of these fish have played key roles in the neurosciences from demonstrating
to eighteenth century scientists that animals could generate electricity to being the
tissue of choice for recent biochemical identification and molecular cloning of the
acetylcholine receptor, the sodium channel, and other important molecules 59 .66 - 68 .
Beside the more familiar strongly electric eel, Electrophorus, or the Torpedo ray,
a far greater number of fish possess weak EOs the discharges of which cannot be
sensed by us without the aid of electronic amplification. These fish, notably the
gymnotiform and rnormyriform teleosts, are nocturnally active and frequently live
in murky habitats. They use their EOs, often in preference to visual cues 18, to
locate objects around them and to communicate with each other (for reviews on
electroreception see refs. 17, 36 and 42). In this chapter, I review the electric organs
of both strongly and weakly electric fish in terms of their distribution, functional
organization, hormone-sensitivity, and biochemistry. The reader is encouraged to
consult previous reviews on the electric organ 5,9,1~
260 H.H. Zakon
Fig. 1. Summary diagram of known groups of electric fish. A line drawing of a representative fish from
each group, the structure of its electrocytes, and a sample EOD are given. The dots on the electrocytes
indicate sites of electromotoneuron termination. The s or w near each fish indicates whether the electric
organ generates a strong or weak discharge. The EODs are shown with positive polarity up. From ref.
5.
II. Distribution
EOs are found in seven unrelated groups of extant fishes suggesting that they have
probably evolved at least that many times. (Fig. 1). In every group but one the
EO is embryonically derived from a muscle precursor 2325444647
.... . EOs develop from
different muscles in the different taxa, however, strengthening the argument for
their multiple origins. The fact that EOs are all myogenic argues less for a common
ancestor than for the idea that muscle tissue is easily converted into EO.
Electric organs: structure, physiology, hormone-sensitivity, and biochemistry 261
Among the elasmobranchs strong EOs are found in two families of rays (Torpedi-
noidae, Rajidae) which are not closely related. In addition, the muscles that give rise
to the EO and the source of the motor innervation differ between these two families
further suggesting an independent evolution of the EO within elasmobranchs 2,t~
The strong EO of the Torpenoidae, best studied in the genus Torpedo, is apparently
defensive. The weaker EO of the Rajidae is too weak to be defensive but may
function in social communication 14.
A strong EO is found in the catfish family Malapteruridae. The EO of this
species derives from muscles of the body wall and is a thin jacket of cells that
entirely surrounds the body from head to tail fin44. Weak EOs have evolved
independently in the members of the genus Synodontis (the upside-down catfish).
The EO of Synodontis is the most muscle-like of all EOs so far described. As
well as possessing sarcomeres, the electrocytes have a distinct but poorly organized
T-tubule system, and retain origination and insertion points on bone and swim
bladder 31. While not well-studied, the EOD of this group is thought to function
in social communication and electrolocation 31. The independent origin of the EOs
in these 2 groups of catfish is further suggested by their innervation: the EO of
Malapterurus is innervated by 2 giant motoneurons one on each side of the rostral
spinal cord, while a pool of smaller medullary motoneurons innervate the EO of the
synodontids 15,31.
Well-known to neuroethologists, weak EOs are found in the two independently
derived lineages of electric fish (Gymnotiformes, Mormyriformes). Many elegant
behavioral studies have elucidated the role of the EO in social communication and
electrolocation in these fish 17,35,36. One member of the gymnotiforms, the electric
eel (Electrophorus electricus), possess a strong electric organ as well. The EO of one
exceptional family of gymnotiforms, the Apteronotidae, is comprised of the axons
of the electromotoneurons in adult fish 1~ (see section VI). However, even these
fish are the exception that proves the rule as they possess a muscle-derived EO as
larvae but lose it as they mature 47. In fact, all weakly electric gymnotiforms and
mormyriforms may possess a myogenic larval EO for the first 2 months of life after
which time it degenerates as the adult EO develops46,47,8~
Last, the perciform teleosts, the Uranoscopidae, have evolved an EO from the
oculomotor muscles. The EO of these fish is innervated by a large cluster of
electrotonically coupled motoneurons, formerly of the oculomotor nucleus, which
have been co-opted for a new function 54. The uranoscopid EO is not used for
communication or electrolocation since they have no electrosensory system. They
are believed to discharge the EO into their mouth to aid in immobilizing captured
but struggling prey.
IlL Structure and neural control
Generally EOs are composed of columns of large cells oriented along the same
axis and ensheathed in connective tissue. This arrangement channels the flow of
current along the axis of the organ and out into the water. Myogenically derived
262 H.H. Zakon
eleetrocytes are flattened and wafer-like, long and tubular, or barrel-shaped (Fig.
1). The electroeytes in a column are usually innervated on the same face, so that
when that face is depolarized by the release of neurotransmitter, current flows into
the innervated face and out of the uninnervated face of each eleetroeyte along the
column 8.
The EO in all species is innervated by a distinct pool of motoneurons (eleetro-
motoneurons) which, like somatomotoneurons, are nicotinic eholinergie neurons.
These may be spinal or cranial motoneurons depending on the group. A well-
defined medullary pacemaker nucleus controls the electromotoneurons in both
gymnotiform and mormyriform electric fish 13. The pool of pacemaker neurons are
electrotonically coupled to each other and to the output neurons of the pacemaker
nucleus which typically make extensive diverging contacts with a large number of
motoneurons. This tight coupling and high degree of divergence optimizes syn-
chronous firing of the electrocytes. Pacemaker cells have been identified in some
other groups as well and they may well be a general feature of eleetromotor systems
to insure coordinate activation of the eleetromotoneurons and electrocytes.
IV. EOD variation and species communication

The EODs of gymnotiform and mormyriform teleosts are species-specific 41'42'49. In
addition, there may be sex differences, age differences (larva v e r s u s adult), status
differences (dominant v e r s u s submissive individuals), and individual differences in
the waveform of the d i s c h a r g e 22'32'37'39'58'80'81 (Fig. 2). While only a few studies
have investigated the potential behavioral relevance of this variation, the ones that
have suggest that they are discriminable to conspecifics and may convey behaviorally
relevant information 39'49'5~
EODs can be classified as either pulse-type, in which the pulses are generated
irregularly and pulse duration is short compared to the interval between pulses, or
wave-type, in which the EO is driven with extreme regularity and where the pulse
duration is about equal to the interval between pulses (Fig. 2). Pulse- and wave-type
EODs are found in both orders of weakly electric fish.
A 13
w
Fig. 2. The EODs of a male and female (A) pulse fish (Hypopomus brevirostris) and (B) wave fish
(Sternopygusmacrurus). From ref. 84.
I4. Functional organization of the EO in weakly electric pulse-type fish

Pulse-type EODs differ between species in the number and polarity of their phases
and by differences in the amplitude and duration of each phase. Sex differences
within a species depend on variations in the duration and amplitudes of each phase
(Fig. 2). As will be described below, the number of phases and their polarities are
determined by fixed parameters as the site of innervation, the number of excitable
faces and, in the mormyrids, the geometry of the electrocyte. The duration and
amplitude of each phase, however, depend on more subtle properties like the
duration and amplitude of the action potentials generated by each face.
While the generation of a mono- or di-phasic EOD is essentially identical
in gymnotid and mormyrid fish, the mechanisms by which EODs with three or
four phases are produced is achieved differently. Gymnotiform electrocytes are
structurally simple and generate only mono- or biphasic pulses. The multiphasic
gymnotiform EOD is built from the discharges of a number of functionally distinct
electric organs that fire with phase delays. Diversity in the multiphasic mormyrid
EOD, on the other hand, comes from the synchronous activation of a single
population of electrocytes each of which possesses complex morphological and
physiological membrane specializations.
The simplest EOD is a monophasic pulse as given by Electrophorus. The Elec-
trophorus electrocyte is a simple rectangular cell with a posterior face that is
innervated and generates action potentials, and a highly invaginated anterior face
that is electrically inexcitable 45,64. Upon being depolarized by synpatic input the
posterior face fires an action potential during which positive current (Na + ions) en-
ters. The head-positive monophasic EOD pulse is generated when this current flows
headward along each column of electrocytes during the posterior membrane's spike.
This occurs in both the main electric organ, which generates the eel's high-voltage
pulse, as well as in Sach's organ, which generates the low voltage pulse. Intracellular
recordings of electrocytes from other gymnotiforms and mormyrids which generate
monophasic EOD pulses have shown this to be generally true of them all.
Both faces of the electrocytes are excitable in those species that produce a
diphasic discharge. First, the posterior face fires an action potential during which
current flows toward the head. The flow of current through the anterior membrane
depolarizes it causing it to spike and current then flows tailward. The alternate flow
of current headward and tailward gives theEOD its diphasic shape.
For an electrocyte with two excitable faces to generate a diphasic discharge
it is imperative that the two faces do not spike simultaneously or the currents
flowing in opposite directions will cancel out. It seems to be a general property
of such electrocytes that the innervated membrane has a lower threshold than the
uninnervated side (such that the uninnervated side is not brought to threshold by
the synaptic input) allowing the innervated face to fire first.
The two faces may differ in other excitable properties as well. In some instances
the two faces produce spikes of different duration; if there is an asymmetry in spike
duration it is the face which fires second and which does not have to worry about
colliding with a spike, that usually produces the longer duration spike.
264 H.H. Zakon
Some gymnotiforms possess so-called accessory organs which are physically

distinct from the main organ. The EOD of Steatogenys elegans, for example, is
diphasir over most of its body but triphasir with an early negative wave, over its
head. Bennett 9,1~ has shown that the small head-negative component is generated
by a small EO running along the lower jaw (the submental organ). The electrocytes
of this organ are innervated on their anterior faces which are the only spiking
face and thus produce monophasir head negative potentials. The discharge of the
submental organ is in phase with the rest of the EOD but is activated slightly earlier
making the EOD triphasir Similar accessory organs have been described in other
gymnotiform species 9,1~
The most complex gymnotiform EOD thus far studied is that of Uymnoms carapo
which has four distinct components ~1,19,~,s7,78. In this species, the EO runs from
the pectoral girdle to the tip of the tail; it appears as a continuous organ but it
includes three functionally distinct regions. The most rostral portion of the organ
contains electrocytes which are innervated on both anterior and posterior faces.
The posterior face generates an action potential but the anterior face gives only a
synaptir potential. The electrocytes in the midbody region are also innervated on
both faces. Here, both faces are capable of spiking, but the anterior face has a lower
threshold and fires first. The largest part of the EO, that found in the tail, follows
the typical gymnotiform pattern of innervation only on the posterior face. Both
faces generate action potentials and the posterior face has the lower threshold.
When the EO is normally activated, the anterior face of the most rostral set of
electrocytes produces an EPSP first, which generates a small slow head negative
component. Then, the anterior faces of the midbody electrocytes spike causing a
larger head negative wave. The posterior faces of the midbody electrocytes fire at
about the same time as the posterior faces of the electrocytes in the tail and these
generate a large head positive component, the most pronounced component of the
EOD. Finally, the anterior faces of the electrocytes in the tail fire generating the
last head negative component.
The electrocytes of mormyrids, on the other hand, are flattened, thin and bear
prominent stalks (Fig. 1). The organization of the stalks is species-dependent and
is critical to the generation of an EOD with the proper number of phases 6'12.
The simplest electrocyte bears a single stalk that fuses with one face (usually the
posterior) of the electrocyte. The action potential is generated in the stalk and
propagated into the posterior membrane, which causes headward current flow,
followed by a depolarization of the anterior membrane and tailward current flow.
This results in a diphasir spike according to the principles discussed above for the
gymnotiform electrocyte.
In other species, a complex network of stalks may penetrate the main body of
the electrocyte and fuse with it on the other side. For example, the stalk may
appear on the anterior side of the electrocyte where it is innervated, it may then
penetrate the main body of the electrocyte where it fuses with the posterior face.
In these electrocytes a spike is generated in the stalk and proceeds along the
stalk in a tailward direction resulting in a head-negative EOD phase. The spike
then propagates into the posterior membrane of the electrocyte depolarizing it and
causing it to generate an action potential followed by the generation of an action

potential from the anterior face. These events result in a triphasic action potential
with an initial head-negative wave.
Perhaps the most impressive electrocytes are those of the mormyrid Stoma-
torhinus corneti. In this species the stalk penetrates the body of the electrocyte
from the posterior side, emerges from the anterior face and repenetrates it, finally
emerging again from and fusing with the posterior face. The movement of currents
alternately in a headward, then tailward direction in the stalks followed by the usual
headward and tailward movement when the posterior and anterior membranes are
sequentially depolarized results in an EOD with 4 phases.
VI. Functional organization of the EO in weakly electric wave-type fish

Wave-type EODs differ between species and individuals within a species by their
frequency. Stemopygus, the species with the lowest EOD frequency discharges at
50-200 Hz while some of the apteronotids discharge in excess of 1 kHz. The sole
wave-type mormyriform fish, Gymnarchus niloticus, discharges around 300 Hz. The
resting EOD frequency of wave-type fish is extremely regular varying only by a few
tenths of a percent 16. Yet, EOD frequency may be modulated by up to 25% during
social interactions.
The production of the EOD in wave-type fish is relatively simple and is similar
in both gymnotids and mormyrids. It is easiest to consider a wave-type EOD as a
pulse-type EOD that is extremely regular (Figs. 1 and 2). If the durations of the
EO pulses are about the same as those of the inter-pulse interval, the EOD will be
periodic and nearly sinusoidal.
In the wave-type fish with myogenic EOs (Eigenmannia, Stemopygus, Gymnar-
chus) the EO generates a monophasic pulse as occurs in Electrophorus. The EOD
frequency of a wave-type EOD is determined by the firing frequency of the pace-
maker nucleus. However, in order for the waveform to approximate a sinewave,
the EO pulse duration must also vary. So, for example, the EOD frequency of
Stemopygus sp. varies from 50 to 200 Hz and EO pulse duration varies from 4 to
14 ms (Fig. 2). These two parameters covary so that the fish with the lowest EOD
frequency has the longest pulse duration 62,63. These two independent parameters
may be manipulated by hormonal modulation (see below).
The Apteronotidae, the sole group of electric fish without a myogenic EO, also
generate a wave-type discharge. Some members of the Apteronotidae generate
EOD frequencies in excess of 1 kHz. The electromotor system is electrotonically
coupled from the PMN to the electromotoneurons 69. It is thought that their
myogenic EO is lost in order to eliminate the one obligatory chemical synapse in
the electromotor circuitry that might fail at these high frequencies.
The unique EO of the Apteronotidae is composed of bundles of electromo-
toneuron axons which project forward within the EO and then make a hairpin turn
posteriorly; they end blindly within the bundle since their muscle-derived target
cells have degenerated (Fig. 1). Each axon possess three to half a dozen specialized
266 H.H.Zakon
nodes of Ranvier just before the hairpin turn and at the blind end. The specialized
node is 5-20/zm in length (which is one or two orders of magnitude longer than
a normal node of Ranvier) and has no Na+channels 69,79. The absence of excitable
channels at this node near the hairpin turn prevents current flowing into the axon
from the previous node from depolarizing this patch of membrane. Instead, current
flows outward, eapaeitatively coupled to the extraeellular space through the exten-
sive membrane area. The summation of the outward currents from large numbers of
simultaneously active nodes generates the first phase of the EOD. The movement
of the action potential into the blind end of the axon and of current out of the
specialized nodes into extraeellular space generates the second phase of the EOD 79.
It would be interesting to determine the mechanisms by which Na+ehannels are
selectively targeted to most nodes of Ranvier, but selectively eliminated from the
large specialized nodes.
VII. Sex differences in and hormonal modulation of the EOD
Sex differences in the EOD waveform have been reported for a large enough sample
of weakly electric fish, that it may turn out to be the rule. These sex differences
are under the control of gonadal steroids: they disappear after gonadeetomy or
in captivity under conditions when sex steroid titers fall, and they are induced by
treatment with sex steroids or gonadotropin 4,7'51'52'62'63'sS-sT.
In both gymnotiform and mormyriform pulse fish the duration of some or all
components of the pulse are longer in males than in females and juveniles captured
in the field during breeding season 3s,51. The mormyrids Brienomyrus brachyistius and
Gnathonemuspetersii have been particularly well studied. While the EOD of Brieno-
myrus is triphasie and that of Gnathonemus has four phases 3,7,51. In both species the
major components of the EOD pulses of females and juveniles are broadened after
implantation with a variety of androgens, including the non-aromatizable androgen
5-a-dihydrotestosterone (DHT). On the other hand implantation with an estrogen,
17/5-estradiol, has little effect.
Similar results have been obtained with two species of the pulse gymnotiform Hy-
popomus. In both eases the EOD pulse is diphasie and the sex difference is primarily
in the second phase 29'4~ (Fig. 2). As in mormyrids, the EOD pulse of Hypopomus
occidentalis can be influenced with DHT treatment but not by estrogen 29.
Along with the sex difference in the EOD pulse, the EO itself usually shows
morphological sex differences in pulse fishs,6. In both groups the eleetroeytes of
males are larger than those of females and they may have additional membrane
infoldings. In the hypopomids tail size is also sexually dimorphie with males
having longer or thicker tails 29,4~ In both groups of pulse fish androgen treatment
of females and juveniles increases the size of the electroeytes. Presumably as
a consequence of increases in eleetroeyte size, tail size also increases in the
Hypopomids. It is not obvious whether this is strictly a byproduct of mechanisms
underlying the production of the EOD or whether sex differences in tail morphology
may provide additional visual cues.
... *2 A
Ip
E
4.
0
-1 I , !
20 .'o
§ 20
B
N
z 0
r
0
~ -20
~-40
<i
-60 ~ _ I ,
,o 20 4o io
Ooy
Fig. 3. Changes in EOD pulse duration (A) and frequency (B) in Stemopygus implanted with dihy-
drotestosterone (solid lines) or empty control (dashed lines) capsules. Note the concurrent lowering of
EOD frequency and broadening of EOD pulse. From ref. 63.
Sex differences in EOD frequency occur in three species of gymnotiform wave

fish commonly studied. In Stemopygus and Eigenmannia sexually mature males have
lower EOD frequencies than females 3~176 There is a negative correlation between
androgen level and EOD frequency in gonadally recrudescing Stemopygus in the
field and gonadotropin hormone-treated fish in the laboratory 85,s7. Additionally, a
variety of androgens lower EOD frequency and broaden the EO pulse in female
and juvenile Stemopygus (Fig. 3) and 17fl-estradiol is reported to raise EOD
frequency 6~
The apteronotids show interesting species differences in sexual dimorphism of
EOD frequency. The EOD frequencies of sexually mature female Apteronotus
leptorhynchus are lower than those of males 61. When A. leptorhynchus of either
sex are treated with testosterone, their EOD frequency is lowered and this effect
is antagonized by inhibitors of the aromatase enzyme (M. Zucker and H. Zakon,
unpublished results). This result suggests that testosterone acts via its conversion
to an estrogen. In support of this hypothesis, EOD frequency is lowered in this
species by treatment with 17~l-estradiol 61 (M. Zucker and H. Zakon, unpublished
results). On the other hand, the congeneric species A. albifrons shows a sexual
dimorphism of EOD frequency that is opposite and more similar to that seen in
the other gymnotid wave fish: male EOD frequency is lower than that of females
(M. Zucker and H. Zakon, unpublished results). The detailed endocrinology is still
being studied in this species.
268 H.H. Zakon
[ 200 nA
1,0o~
300 ~ t
Fig. 4. Variation is sodium current kinetics of electrocytes from unsexed juvenile Sternopygus (EOD
frequency of each fish is given next to the voltage-clamp traces). Each family of curves was generated
by voltage-clamping the electrocytes from -50 to 0 mV (unpublished data courtesy of M.B. Ferrari, and
from ref. 84).
A knowledge of the action of steroids on the ion currents of the EO may prove
useful in understanding how steroids act on ion currents in central neurons or
other excitable cellss4. While intracellular recordings before and after androgen
treatment have been made from the EO of fish from 3 genera (Brienomyrus,
Sternopygus, Hypopomus), the identification of the ion currents underlying the action
potential has only been done in Sternopygus. In this species, an inward rectifying
K +current sets the resting potential, the rising phase of the action potential depends
entirely on a Na+current, and the falling phase of the spike depends primarily on
the inactivation rate of the Na+current and, in some cells, a delayed rectifying
K + current 24. Interestingly, the inactivation kinetics of the Na +current varies among
electrocytes of fish with high versus low EOD frequencies. In other words, in fish
with a low frequency EOD and broad action potentials, the Na+current inactivates
slowly whereas in those fish that generate higher frequency EODs with short
action potentials (Fig. 4). Long-term (3 week) androgen treatment slows down the
inactivation rate of the Na+current, which apparently is the mechanism by which
the EOD pulse becomes broader after androgen treatment (Ferrari and Zakon, in
preparation).
VIII. Biochemistry of electric organs: acetylcholine receptor and related

molecules
The rich innervation and large size of the EOs of Torpedo and Electrophorus have
made them critical in providing large amounts of purified synaptic membranes
for efforts to elucidate the structure of molecules involved in cholinergic synaptic
transmission55, 59,67, 68, 72.
Based on molecular weight and antigenic profile, the acetylcholine receptor
(AChR) of the Torpedo EO has been shown to comprise 4 subunits in the same
stoichiometry as that of mammalian muscle (approximately 40 (c~), 50 (/I), 57 (y),
65 (8) kDa in a 2" 1" 1" 1 ratio) 67,68,72 (Fig. 5). The AChR of Electrophorus, while
less well studied, has also been isolated 55. When extracted under conditions that
normally isolate all subunits in Torpedoor mammalian muscle, only 3 subunits (c~, fl,
y) appear. Under altered conditions, however, a 8 subunit is recovered. While this
suggests subtle differences in the organization of the AChR between these species,
the similarity of subunit composition, and antigenicity across Torpedo,Electrophorus
and mammals highlights the highly conserved nature of the AChR.
A distinct AChR subunit type (E) is expressed in mature mammalian muscle (the
8 subunit is only expressed early in muscle development and in the extrajunctional
region of mature mammalian muscle after denervation) 34. Such a subunit does not
seem to occur in Torpedo EO nor is there any indication of denervation-induced
Fig. 5. Autoradiograms of antibodies against various proteins of Torpedo EO. The name of the antibodies
are given below each lane. The leftmost lane contains antibodies against subunits of the ACh receptor,
followed by lanes with antibodies against the 43 and 53 kDa proteins, which are associated with the
ACh receptor, and a 90 kDa protein, which is likely to be the Na +/K+-ATPase. From ref. 26.
270 H.H.Zakon
changes in the TorpedoEO AChR 82'83. However, since the developmental dynamics
of AChR subunits in Torpedomuscle are not known it is not yet clear whether these
are tissue or species differences.
In addition to the AChR, a number of other proteins are enriched in the
postsynaptie membrane fraction of Torpedo EO and have also been found at the
mammalian neuromuscular junction (Fig. 5). The most prominent of these is a 43
kDa molecule which is found in approximately equimolar concentrations as the
AChR and is co-localized with it in the membrane 26,27,s2,a3. Attention was initially
drawn to this protein when it was shown that after it, and other, proteins were
extracted from Torpedo membranes, the AChRs no longer remained clustered but
diffused throughout the membrane. It has since been shown that this molecule
induces clustering of the AChR when messenger RNA for it and the AChR are
injected into Xenopus oocytes 28. Interestingly, the 43 kDa protein is regulated
differently during development than the AChR: both molecules are markedly
increased in abundance following innervation of immature electroc~es, but while
AChR mRNA is up-regulated by 30-fold following denervation of adult organs, the
43 kDa protein is increased by only 2 to 3 fold 27.
Torpedo EO contains a number of other proteins most likely important to regu-
lation of the synaptic membrane as, for example, dystrophin and dystrophin-related
protein 2~ Both of these molecules, which are localized to the synaptic membrane
are also found in mammalian muscle (Fig. 6). While the distribution of dystrophin
at the mammalian neuromuscular junction and within Torpedo EO is quite similar,
the highly similar protein, dystrophin-related protein is found in high levels in both
mammalian and Torpedo neuromuscular junctions, but only at low density in the
synaptic membrane of the electric organ 2~ The lack of dystrophin has been impli-
cated as causal in Duchenne muscular dystrophy although it is unclear what role it
plays in normal muscle physiology, or how muscle pathology results from its absence.
At the present the function of dystrophin-related protein is also unclear.
IX. Biochemistry of electric organs: muscle-specific proteins

Sarcomeres often form in the developing EO. While well-formed sarcomeres are
maintained in the mature EOs of some species, they disappear in others. It
would be informative to study the molecular processes underlying differentiation
of the electrocytes in a range of species to determine where along the pathway of
differentiation muscle development is halted or compromised. An understanding
of the molecular events controlling the suppression of the muscle phenotype in
electrocytes could be helpful in discerning the causes of muscle wasting in various
human dystrophies. It is known, for example, that two of the muscle regulatory
genes (MyoD and my5) are expressed in Torpedoelectrocytes 65.
Mature electrocytes of the gymnotiform Sternopygushave no sarcomeres and do
not express proteins like myosin and tropomyosin 7~ On the other hand, during
regeneration of the electric organ the developing electrocytes express these protein
for about a week and then these proteins gradually becomes undetectable (J.M.
Fig. 6. Localization of dystrophin to the Torpedo electrocyte. A cryosectioned electrocyte is double-

labeled with a-bungarotoxin to localize the ACh receptor (A) and an anti-dystrophin antibody (A').
Using immunoelectron microscopy, dystrophin can be localized just below the plasma membrane on the
innervated surface of the electrocyte. From ref. 74.
Patterson and H.H. Zakon, unpublished results). In addition, after the terminal
portions of the EO are amputated to induce regeneration, the intact electrocytes
near the wound transiently express myosin. This last observation indicates that
at least portions of the suppressed muscle program may be reactivated in a
differentiated electrocyte.
F-actin is observed in the EO of the gymnotiforms Stemopygus (J. Patterson and
H. Zakon, unpublished results) and Electrophorus77 where it is in highest density at
the innervated end of the electrocyte. Two isoforms of actin are expressed in mam-
malian muscle and these come from separate genes. One form is sarcomeric actin
and the other form, cytoplasmic actin, participates in the subsynaptic cytoskeletal
framework 33. Both forms are believed to exist in the Electrophorus electrocyte based
upon 2 dimensional gel analysis 73. It would be interesting to know whether the
sarcomeric actin is functional or merely a vestige of the electrocyte's myogenic
lineage.
F-actin is also abundant in electrocytes from early embryonic Torpedo but falls
to low levels thereafter. This shift in abundance is paralleled by a disruption and
disappearance of organized myofibrils 25. In fact, with the exception of some F-actin
along the non-innervated face, most of the F-actin detected after homogenization of
the adult EO may actually be from nerve terminals rather than electrocytes 4s,53,83. It
is interesting that no F-actin is localized to the postsynaptic cytoplasmic surface of
272 H.H. Zakon
the neuro-electroeyte junction as has been reported for mammalian neuromuscular

junctions 33. Since the distribution of F-action in Torpedo muscle is not known, it is
not clear whether this reflects a difference between the postsynaptie membranes of
muscles and eleetroeytes in this species or whether this is a species difference.
X. Biochemistry of electric organs: intermediate filaments

A number of intermediate filament proteins have been observed in the EO. The
cytoplasm of the Torpedo electrocyte contains a meshwork of intermediate filaments
which have been identified as desmin 48,75. These filaments extend throughout the
cytoplasm and beneath both innervated and uninnervated membranes. In this it
differs from desmin in rat muscle which is concentrated at the synaptic membrane
and, less so, at Z bands 75. The desmin from the EO and muscle of Torpedo are
believed to be identical 7s.
Desmin has been biochemically or immunocytochemically identified in the EO
and muscle of Electrophorus, Stemopygus and Hypopomus pinnicaudatus 21 (J. Pat-
terson and H. Zakon, in preparation). It is possible that EO and muscle express
different isoforms of the protein as muscle and EO are differentially stained by a
range of monoclonal antibodies against mammalian desmin (J. M. Patterson and
H.H. Zakon, in preparation). Five isoforms of desmin have been identified in the
Electrophorus EO, but since Electrophorus muscle has not yet been studied, it is
not known whether any of them are unique to the electric organ 21. At this time
it is not certain whether these electrophoretie variants represent the products of
different genes, different splice products of a single gene, or the same protein with
differing degrees of phosphorylation 21. Certainly, the fact that there is a single
desmin gene in mammalian muscle 71 and that the isoforms from the Electrophorus
EO have identical molecular weights and protease digestion patterns argues for the
last alternative.
Electrocytes are usually typified by the proteins they lack when compared to
muscle (i.e. myosin). However, one recently identified keratin-like protein is found
in EO, but not muscle, of Stemopygus7~ Both the EO and skin label intensely
with an antibody to acidic keratins (AE1). Western blots indicate that this antibody
recognizes a molecule of the same molecular weight in skin and EO (J.M. Patterson,
personal communication). While the definitive identification of this molecule awaits
its sequencing, these results suggest that it is a keratin. This result is intriguing since
it is the first example of extensive production of a keratin in a cell from a myogenic
lineage. It is not known how widely this keratin-like molecule is found among the
gymnotiforms.
XI. Protein sorting and targeting

One of the most striking aspects of electrocytes is the organization of their
plasmalemmas into distinct faces. For example, all electrocytes possess innervated
faces that are rich in molecules involved with cholinergic transmission (see above)
and uninnervated faces laden with Na+/K+-ATPase 1,26. This cellular feature and
the electrocyte's tractable size make them potentially fruitful cells in which to study
how proteins are differentially sorted and inserted into membranes.
Recent studies on the EO of Torpedo have discovered an asymmetry in the or-
ganization of microtubules within the electrocytes resulting in selective associations
with the subsynaptic space 43. Furthermore, a particular guanosine triphosphate-
binding protein (Rab6p) which is believed to be involved in exocytosis has been
localized within the Golgi apparatus and, most abundantly, within post-Golgi vesi-
cles that are localized beneath the synaptic membrane. These vesicles are no longer
localized there if the microtubular network underlying the synaptic membrane is
disrupted 43. These, and other, results suggest that there is a specific intracellular
network composed of microtubules which associate with the intermediate filament
proteins of the subsynaptic membrane and convey proteins specifically bound to this
membrane.
The question of subcellular localization of membrane constituents is just be-
ginning to receive attention using the electrocytes as a model system. Beside
the elegant work already done future questions include detailing how the Na+/
K+-ATPase molecules are selectively ferried to the non-innervated membrane. In
addition, those electrocytes that generate action potentials from only a single face
must have a mechanism whereby ion channel proteins are delivered to and inserted
into only the active face. The process by which ion channels are sorted and targeted
cannot be pursued in the EO of Torpedo since these cells do not generate action
potentials. Instead, these questions can be addressed using the electrocytes of other
spedes such as Electrophorus or Stemopygus.
XII. Future directions
EOs have played important roles in understanding the mechanisms of synaptic

activation and electrical excitability. I would like to emphasize three areas in
which EOs can continue to make key contributions to the cellular and molecular
organization of excitable cells in the future.
First, the extensive variation in the excitability properties of electrocytes across,
or even within, species allows one to study how membrane excitability is regulated
and how proteins are targeted to a particular membrane. For example, how is
the AChR with its entourage of other proteins selectively targeted to a particular
membrane in those species with a single active face in the electrocyte? The dorsal
row of electrocytes of Gymnotus is innervated on both faces but only the posterior
face generates action potentials. What causes the AChR to be present at both faces,
but Na +channels only to one?
Second, how do steroid hormones modulate the ion currents of the EO? The
kinetics of the Na+current is modulated after androgen treatment in Sternopygus.
This is the first example of androgen action on any ion current and the first example
of steroidal modulation of Na+current kinetics. Is the Na+current also modulated
274 H.H. Zakon
by androgens in the mormyrids which have independently evolved EOs? If so, are
the cellular and molecular processes underlying these modifications similar in the 2
taxa?
Last, the independent evolution of a myogenic EO in seven taxa offers a series
of interesting natural experiments in the molecular processes by which muscle can
selectively suppress parts of the muscle program while retaining others.
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Hochachka and Mommsen (eds.), Biochemistry and molecular biology offishes, vol. 4
CHAPTER 12
Biochemical and molecular aspects of

singing in batrachoidid fishes
PATRICK J. WALSH, THOMAS P. MOMMSEN * AND ANDREW H . BASS **
Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science,
4600 Rickenbacker Causeway, Miami, FL 33149, U.S.A., *Department of Biochemistry and
Microbiology, University of Victoria, P.O Box 3055, Victoria, British Columbia V8W 3P6, Canada
and ** Section of Neurobiology and Behavior, Cornell University, Ithaca, IVY 14853, U.S.A.
I. Introduction
II. Contraction mechanics
III. Cell ultrastructure and contractile proteins
IV. Metabolism
V. Hormonal regulation
VI. Prospects for future research
Acknowledgement
VII. References
I. Introduction
Fishes of the family Batrachoididae (toadfish and midshipman) have a rather

simple, but none the less remarkable, neuromuscular system for generating sounds.
Central to the production of sound in these species is a heart-shaped swimbladder
which has two large strips of muscle, one on each side. Sound is produced when
neural signals, originating in the brain, trigger simultaneous contraction of the
muscles against the taut surface of the swimbladder. The finely tuned (pun fully
intended) neuromuscular system of this family presents researchers with several
interesting avenues of inquiry. First, the muscle is among the fastest in the animal
kingdom. Thus comparative study of this tissue will yield interesting insights into
muscle structure/function relationships in general.
The sonic muscle is used to generate defensive sounds (grunts) by both sexes, as
well as a mate call (boatwhistle of toadfish, hum of midshipman) used exclusively by
males to attract females to their nest for mating. This differential use of the muscle
in the sexes has led to a clear sexual dichotomy (even a trichotomy in one species)
in several facets of the neuromuscular set up, presenting a perfect opportunity for
a second line of research into the neuroendocrine basis of sex-related vocal traits.
In both of these areas, scant attention has been paid to the metabolic adaptations
underlying muscle function. It is the aim of this chapter to summarize what is
known about the metabolic biochemistry of this unique muscle system, set upon a
280 P.J. Walsh, T.P. Mommsen and A.H. Bass
foundation of the only slightly larger body of information on ultrastructure of the

muscle and biochemistry of the contractile apparatus.
In an effort to come to a unifying picture of sonic muscle function we may meld
information from different species and genera within this family. However, because
this organ has such direct and obvious consequences to reproduction (and hence
future composition of the gene pool), we caution the reader to be mindful that
the species differences may ultimately yield more clues to the importance of sonic
muscle function than species similarities. In essence, we view comparative metabolic
study of the neuromuscular system within this family (and perhaps comparison
with singing muscles in other phyla), in direct conjunction with study of behavioral
ecology, as a third fruitful avenue for investigation. Indeed, populations within the
same species may turn out to be the most appropriate comparative unit for study of
this system.
II. Contraction mechanics
The extremely rapid contraction mechanics of the 'superfast' Batrachoidid sonic

muscles were first documented in the oyster toadfish (Opsanus tau) by Skoglund30,31.
Upon electrical stimulation of the motor nerve, he detected contractions within 0.5
ms of the action potential, which reached a peak within 5-8 ms, and muscle
relaxation was complete within an additional 5-7 ms. We now know that this cycle
of contraction/relaxation is among the fastest in the animal kingdom. By way of
comparison, peak contraction is reached in insect flight muscle in 15 ms, and in
vertebrate fast muscle in 20 ms25,zs. The paired muscles contract in synchrony
and there is less than a 0.5 ms delay in the onset of depolarization between
the cranial and caudal ends of the muscle. The number of vibrations per second
determines the fundamental frequency of vocalizations and is established by: (1) the
sonic motor pathway in the brain: and (2) a temperature dependent phenomenon
with frequency increasing along with temperature 4,s. Thus, at about 15~ the
fundamental frequency for vocalizations is in the range of 100 s-1 (Hz). Duration
does not seem to be temperature dependent 16 and for grunts ranges from 70 to
200 ms and for boatwhistles from 500 to 700 ms ~~ The hum vocalization was first
described by Ibara and coworkers 27 who estimated the duration of single hums to be
up to 60 rain. Remarkably, during mating season, toadfish and midshipman males
can produce these sounds for hours at a time.
Both intra- (male-male) and inter- (male-female) sex differences exist for
sound duration (grunts versus hums) as well as for the fundamental frequency
of vocalizations in the plainfin midshipman, Porichthys notatus. Midshipman have
three classes of sexually mature adults: females and two male reproductive morphs
referred to as Type I and II males (for review, see ref. 2). "I~e I males are about
40% larger in body size, have a 9-fold smaller gonad weight to body weight ratio
(gonadosomatic index), and build and defend nest sites in the intertidal zone.
Type I males also have the most extensive vocal repertoire; they generate the long
duration hums to attract females to their nest and trains of grunts during agonistic
Biochemical and molecular aspects of singing in batrachoidid fishes 281
encounters with conspecifics 1~ Type II males 'sneak' or 'satellite' spawn when

females are in a nest with Type I males 7,1~ Type II males, like females, have never
been observed to build nests; nor do they hum or appear to generate any sounds
during prespawning behavior. Type II males and females do however infrequently
generate low amplitude, isolated grunts when they are housed together in crowded
conditions. The vocalizations of Type II males and females are also about 20%
lower in fundamental frequency than those of Type I males (hums and grunts) 1~
which is determined by the rhythmic firing properties of the central sonic motor
pathway 4,5. Although only male toadfish generate boatwhistles during reproductive
encounters, comparable sex differences do not appear to exist in the fundamental
frequency of vocalizations between males and females (see ref. 5 for discussion).
III. Cell ultrastructure and contractile proteins
The innervation of sonic muscle fibers appears to be adapted for rapid and syn-
chronous contraction. Gainer and Klancher 22 first noted polyaxonal and multiple in-
nervation along the entire length of the muscle in O. tau, allowing simultaneous and
distributed action potentials. Sonic motoneurons located in the caudal brainstem
and rostral spinal cord form a pair of sonic motor nuclei that are contiguous along
the midline. Sonic motoneurons are cholinergic, i.e. they contain acetylcholine, as
most other vertebrate motoneurons 8. The axons of sonic motoneurons exit the brain
via ventral occipital nerve roots to ipsilateraUy innervate each sonic muscle where
they form terminal boutons along the surface of muscle fibers 3,4. The ultrastructure
of sonic nerve terminals (or boutons) and neuromuscular junctions (NMJs) has
been described in midshipman 2~ Sonic NMJs resemble those of other vertebrates
in having distinct pre- and postsynaptic membranes separated by a basal lamina,
and active zones with clusters of clear, round synaptic vesicles (--~0.04-0.06 /zm
diameter). Mitochondria and glycogen deposits are scattered throughout terminal
boutons and, more infrequently, dense core vesicles (--,0.08/zm diameter). Unlike
tetrapods, the NMJs lack postjunctional folds as is the case for other fish striated
muscle, excepting perhaps the sonic muscle of weakfish, Cynoscion regalis29. There
are intra- (Type I versus Type II males) and inter- (females versus Type I males) sex-
ual dimorphisms in the sonic nerve terminals. The terminal boutons of Type I males
are larger and lie in a trough along a muscle fiber's surface. The larger terminal size
of Type I males parallels the increased size of their muscle fibers. There are also
sex dimorphisms in synaptic vesicle density, glycogen content, myelination and the
number of terminal boutons per innervation site. Interestingly, the terminals of Type
I males had the fewest number of synaptic vesicles which correlates with the higher
level of activity of the sonic motor system in the mate calling Type I males. Prelimi-
nary findings also indicated that for all three adult morphs, specimens maintained in
captivity beyond the breeding season had elevated vesicle densities which would cor-
relate with what we assume to be the lessened activity of the motor system outside
the breeding season (a decrease in vocalizations has never been directly assessed
since midshipman move far offshore to deep water during the nonbreeding season).
282 RJ. Walsh, T.R Mommsen and A.H. Bass
Sonic muscle fibers have a number of ultrastructural features that distinguish

them from other striated muscle fibers 1,6,14,21. For example, the sarcoplasmic
reticulum is greatly exaggerated, e.g. constituting nearly 30% of the muscle fiber
volume in O. tau 21, and very rich in calcium content 32. Other distinguishing features
include the small diameter of their myofibrils (300-400/~m), reduced area of the
T-tubule system, the confinement of mitochondria to a central core and a thin
peripheral rim of sarcoplasm, and the almost perfect alignment across adjacent
myofibrils of the Z, A, H, and I bands and of triads (T-tubule plus terminal
cisternae of sarcoplasmic reticulum). Fawcett and Revel 14 considered these features
to be related to the physiological properties of the sonic muscle, namely their high
speed of contraction (also see ref. 1).
Sex dimorphisms have also been reported for the ultrastructural features of sonic
muscle fibers. In R notatus, the sonic muscle fibers and myofibrils of Type I males are
distinct from those of females and Type II males, which are similar to each other.
First, the sonic muscle mass of l~pe I males, when corrected for body size, is almost
600% greater than either females or Type II males. Second, this is paralleled by a
300-400% increase in the number, and a 500% increase in the diameter, of muscle
fibers. Third, there is a dramatic dimorphism in the phenotype of individual fibers.
Thus, the sonic fibers of Type I males have a central donut-shaped ring of myofibrils
bordered centrally and peripherally by a zone of sarcoplasm that is densely filled
with mitochondria 6. The cross sectional area of the myofibril-containing zone is 60-
100% greater in 7 ~ e I males; the sarcoplasm-containing zone is 900% greater n.
The sarcoplasmic reticulum which separates adjacent myofibrils also appears to be
more highly branched in Type I males, while their Z-lines are nearly 20-fold wider6!
The special features of the Type I male fibers and myofibrils are assumed to be
adaptations related to their humming abilities 6. The muscle fibers of females and
Type II males resemble those of male and female toadfish, as well as other species
of sonic fish 6.
More modest sex dimorphisms have been reported for muscle mass in toad-
fish 15.34. In O. tau, the larger muscle mass in males (~40%) is paralleled by a 47%
increased fiber number 18. One report states that sonic muscle fibers are 15% larger
in females18; another that fiber diameter is the same for females and males 1. Other
sex dimorphisms include the number of mitochondria (although still relatively low
compared to Type I male midshipman fibers), and the surface area of components
of the T-tubule system (see ref. 1 for details).
The specialized contractile and related proteins have been studied to some extent
in the sonic muscle of O. tau. In accordance with the exaggerated sarcoplasmic
reticulum, Appclt et al. 1 have recently demonstrated a high content of Ca 2+ ATPase
(responsible for calcium sequestration in the SR) and of foot protein (part of
the functional calcium-release channel at the SR-T-tubule junction) in the sonic
muscle as compared with other muscles. ATPase values were not different between
males and females, and density of feet was slightly higher in females than in males
(although this was a calculated parameter with no measured error).
Notable differences in other sonic muscle proteins also are apparent. Hamoir
et al. 25 found that parvalbumin content in O. tau is about twice as high as that
found in typical teleost skeletal muscle. The high parvalbumin content appears to
correlate with the speed of the contraction cycle, and it is believed that parvalbumin
serves as a cytoplasm/SR calcium shuttle and is involved in the rapid relaxation
times. Interestingly, parvalbumin subtype Illf predominates in the sonic muscle
instead of the typical pattern of three subtypes (including lllf) in fish trunk
muscle. This parvalbumin has been purified from sonic muscle, its crystallography
examined 24,35, and its amino acid sequence determined 13. It does not appear to be
unusual compared to other fish IIIf parvmodulins 13, so we may conclude that the
differential expression of parvmodulin types, rather than a fundamental change in
amino acid sequence, is sufficient to adapt this molecule to the requirements of
superfast contraction.
The myosin light chains (LC2) (i.e. the 'head' of the thick filaments which
contains the ATPase activity and which mechanically interacts with the thin actin
filaments) of the sonic muscle migrate much faster in 8 M urea PAGE than
do corresponding proteins from the trunk fast muscle 23,2s, and have a heavier
molecular weight 26. However, the significance of these differences is still unknown.
Clearly, the molecular architecture of this muscle is unique. Further molecular
study will undoubtedly yield interesting insights. Unfortunately in many of these
other protein studies, no mention of sex of fish was made, If these authors have
recorded these observations it would be useful for them to reexamine their data for
sex differences. Given the other manifold sex-related differences in this muscle, it
would be interesting to see if these proteins also exhibited sex differences.
114. Metabolism
Details on the metabolic machinery which powers the remarkable contractile

abilities of the sonic muscle are far from complete. Fine and Pennypaeker 17
examined the histoehemical properties of the sonic muscle in O. tau in order to type
the muscle. Based on their observations, a positive stain for ATPase after alkaline
preincubation, and positive NAD diaphorase, they characterized the muscle as
Type lla, or biochemically speaking as fast oxidative glycolytic (FOG). This is
distinguished from two other typical muscle types: in fish Type I or slow oxidative
(SO) (e.g. lateral line red muscle), and q'~,pe lib or fast glycolytic (FG) (e.g. caudal
white muscle). Fine and Pennypacker 17 also noted several features of the muscle
structure which appear to favor short diffusion distances for oxygen (e.g. small
fiber size, large number of sarcoplasmic pockets where mitochondria are clustered).
Glycogen content in the sonic muscle is about twice that in the body muscle, and
fat content of over six times greater, with no apparent differences between sexes 19.
Although one would expect high glycogen in either FOG or FG fibers, the presence
of the high lipid content (which can only be metabolized aerobically) strongly
supports the typing as FOG. These authors further suggest that the sonic muscle
is a fatigue resistant fiber (FR) and this clearly fits with the abilities of males to
produce sounds for hours at a time.
In the Gulf toadfish (O. beta), sonic muscle mass increases with body mass, but
284 RJ. Walsh, T.R Mommsen and A.H. Bass
Fig. 1. Correlation between sonic muscle mass and body mass in males (open squares) and females
(closed diamonds) of the Gulf toadfish (Opsanus beta). Double logarithmic plot. Toadfish were collected
by shrimp trawl around Miami, Florida. Linear regressions are: males, y -- -1.732+0.912x, r = 0.99;
females, y - -1.882+0.913x, r - 0.97. The mean weight for males was 143.24-14.2 g (n - 50) and for
females 164.64-17.2 (n = 47), t - 0.96. The y-intercepts are significantly different at p <0.001. From
ref. 34, with permission.
as mentioned above, the mass of sonic muscle is higher in males than in females.
Interestingly, the slope of the two log-log regression plots is identical for the
two sexes (Fig. 1), but the two lines have significantly different y-intercepts. This
indicates that the relative sex difference in sonic muscle mass is determined at a
point outside of the fish range analyzed in our data; and this difference was already
apparent in the smallest specimen sampled (7.3 g total weight). From this point on,
sonic muscle mass increases to a similar degree in both sexes.
Remarkably, mitochondrial densities in O. tau are rather lower than typical white
muscle 1, and activities of the mitochondrial matrix enzyme, citrate synthase, are
lower than skeletal muscle for O. beta 33'34. However for both parameters, males of
O. beta have significantly higher values than females. In addition, when we measured
the mass-specific aerobic capacity of the sonic muscle, as indicated by the activity
of citrate synthase (EC 4.1.3.7), we noticed that the activity scales differently in
males and in females. The activity of citrate synthase per gram of tissue wet weight,
which can be taken as an approximate reflection of the abundance of mitochondria
in the tissue, increases in males, but clearly decreases in females, while in the
smaller (largely immature) fish, the y-intercepts are similar (Fig. 2). It can be
concluded that, in contrast to sonic muscle mass, the differentiation of CS activities
occurs during later stages in development and maturation. Therefore it seems that
it is the degree of aerobic potential that enables males to vocalize for extended
periods 34.
While the picture for the toadfish can be summarized as 'fast, more oxidative
fibers in males than in females with immediate mass differentiation and later
developmental differentiation in oxidative potential', the situation is quite different
in the plainfm midshipman (Porichthys notatus). In this species, the male sonic
muscles are much more oxidatively oriented than the female counterparts. Table 1
Fig. 2. Correlation between mass specific activity of citrate synthase (units per gram sonic muscle mass)
and body mass in males (open squares) and females (closed diamonds) of the Gulf toadfish (Opsanus
beta). Toadfish were collected by shrimp trawl around Miami, Florida. Linear regressions are: males, y
= 1.159+0.006x, r ffi 0.51; females, y = 1.477+0.001x, r = 0.26. The slopes are significantly different
at p <0.001. From ref. 34, with permission.
TABLE 1
Enzyme activities in muscles of plainfin midshipman (Porichthys notatus)
Enzyme Sonic Sonic White Heart

(females) (males) (males) (males)
Citrate synthase 7.5 4- 1.1 44 4. 5.6a 1.8 4. 0.5 21 4. 2.2
Malate dehydrogenase 254 4- 53 984 4- 115 a 123 4. 15 995 4- 64
Lactate dehydrogenase 56 4. 3.2 94 4- 5.9 115 4- 4.0 491 4"66
Aspartate aminotransferase 48 4. 9.6 253 4. 34 a 14 4. 3.2 130 4- 14
Glucose 6-phosphate 0.224- 0.1 0.164- 0.03 0.03 4. 0.01 0.45 + 0.12
dehydrogenase
Malic enzyme 0.72 4- 0.1 1.23 4- 0.18 0.15 4. 0.03 1.394. 0.07
Enzymes were measured under Vmax conditions at room temperature, as outlined in reference 33.
Animals were collected subtidally at the Bamfield Marine Station, British Columbia. Activities are given
as units (mean 4- SEM) per gram tissue fresh weight for 4 or 5 independent observations.
a Significantly different (p < 0.05) than the corresponding value in the female sonic muscle (Student's
t-test). 1 Unit is defined as the amount of enzyme producing 1 /xmol of product per min. Enzymes
are: citrate synthase (EC 4.1.3.7); Malate dehydrogenase (EC 1.1.1.37); Lactate dehydrogenase (EC
1.1.1.27); Aspartate aminotransferase (EC 2.6.1.1); Glucose 6-phosphate dehydrogenase (NADP-linked,
EC 1.1.1.49) and Malic enzyme (malate dehydrogenase-decarboxylating (EC 1.1.1.40). Data from TP.
Mommsen, K. Nickolichuck and A.H. Bass (unpublished).
gives a short overview over enzymatic capacities of some of the muscles in question,
while Table 2 presents the corresponding values for some of these muscles in
toadfish (O. beta). At this point, we have no indication about size and maturation-
dependent differentiation in P. notatus sonic muscle, but it is clear from the data
in Table 1 that much larger sex-dependent differences exist between males and
females. Judging from the enzymatic machinery, the female sonic muscle could be
classified together with the white skeletal muscle, while the male sonic muscle is
clearly more oxidative that the generally 'most-oxidative' muscular tissue in fishes,
the heart. Obviously, toadfish and midshipmen have taken different metabolic
286 P.J. Walsh, T.P. Mommsen and A.H. Bass
TABLE 2
Enzyme activities in muscles of Gulf toadfish (Opsanus beta)
Enzyme Sonic Sonic White White

(females) (males) (females) (males)
Citrate synthase 1.51 4- 0.14 2.68 4- 0.31 a 0.44 4- 0.06 0.42 4- 0.05
Malate dehydrogenase 202 4- 11 399 4. 24a 35.3 4- 3.3 41.5 4- 5.4
Lactate dehydrogenase 482 4- 47 333 4- 36 a 292 4- 23 356 4- 23.4
Aspartate aminotransferase 22.3 4- 1.6 26.2 4- 2.9 ND 24.1
Enzymes were measured under Vmax conditions at room temperature, as outlined in references 33 and
34. Animals were collected by shrimp-trawl around Miami, Florida. Activities are given as units (mean
4- SEM) per gram tissue fresh weight for 6 or 7 independent observations (sonic muscles) and 13 to
19 observations for white muscles. 1 Unit is defined as the amount of enzyme producing 1/~mol of
product per min. For enzyme classification, see legend to Table 1. Data from reference 33. ND - not
determined.
a Indicates significantly different from female sonic muscle (p < 0.05). Enzymes in white muscles were
not significantly different from each other. Data recalculated from references 33 and 34.
routes to achieve similar physiological goals, the production of sound. While in

both cases, clearly a superfast, generally oxidative, fiber was committed to the job,
a large latitude is apparent in the degree of oxidative potential. Again, an in depth
analysis of calling behavior of toadfish versus midshipman may help to shed light on
these different biochemical strategies. In an initial study on muscle size in mature;
male midshipmen, we also noticed that size, expressed as sonic muscle somatic
index (mass of sonic muscle expressed as a percentage of body weight), undergoes
systematic changes in the course of the annual cycle, with a clear peak at about
1.4% of body weight during spawning/guarding time in June (T.R Mommsen and
K. Nickolichuck, unpublished observations). Since this peak coincides further with
a peak in oxidative capacity of the tissue, it is clear that the fish control not only
muscle size but also metabolic machinery.
Given that white muscle in toadfish (or other teleosts for that matter) is
not an aerobic tissue, and that mitochondrial densities and enzyme activities
are even lower in toadfish sonic muscle, how is prolonged contraction possible?
One potential mechanism is that the muscle fibers, although capable of repeated
superfast contraction, may have a much lower work load than a typical skeletal
muscle used in an escape response to power a fish against the drag of water.
A reduced work load probably is only a partial answer to this paradox. As
described earlier, the sarcoplasmic zone of the sonic muscle fibers of R notatus is
densely filled with mitochondria and is 900% larger in Type I males compared to
either Type II males or females. Mitochondrial content in Type I males is also clearly
far greater than in either male or female O. tau (compare Figs. 2 and 3 from ref.
6 to Figs. 1 and 2 from ref. 14). Appelt and colleagues I comment on the relatively
small volume of mitochondria in toadfish sonic fibers (1% in females and 4% in
males), which seems low given the continuous activity levels during the breeding
season. Thus, it appears that in ~ e I male midshipman, there may be a better
correlation between mitochondrial content and extended periods of vocal activity
than in the toadfish, and also, given the interesting trimorphism in midshipman, this
species is perhaps a more interesting candidate for biochemical and metabolic study
than the toadfishes.
More than likely the sonic muscle will exhibit unique mechanisms and further
species differences for metabolic regulation in the areas of substrate preference
during different types of vocalizations, rapidity of substrate mobilization, transport
of metabolites across mitochondrial membranes, etc. A wide range of experimental
approaches are possible. We recommend that initial experiments focus on classical
metabolic analysis in which enzyme activities, equilibrium constants, and mass ac-
tion ratios of metabolic intermediates are measured under different physiological
circumstances. Two physiological systems might show special promise. Neurophysi-
ologists have worked with induced sound production in these species for many years
by stimulating regions of the brain. One could perform such stimuli for varying
lengths, on different sexes, in different seasons, etc., and freeze clamp muscles
for biochemical measurements and analyses (e.g. cross-over plots). Laboratory or
field fish for which degree of sound production can be documented would also
make excellent subjects. Finally, in an evolutionary sense, successfully mated males
(which conveniently stay with the young in their nests for several weeks) could be
biochemically compared to their less successful counterparts.
V. Hormonal regulation
It is obvious from the above discussions that there are differences between
males, females, and sneaker males in a variety of morphological and biochemi-
cal aspects. In midshipman, recent light and electron microscopic studies demon-
strate the hormonal sensitivity of the sonic muscle. Androgens (testosterone, l l-
ketotestosterone), but not estrogens (17fl-estradiol) induce nearly a 100% increase
in sonic muscle weight in juvenile males and juvenile females, but only a 50%
increase in Type II males (adult females have not been studied). The increase
in muscle mass is most closely paralleled by an increase in the cross sectional
area of the mitochondria-filled sarcoplasm9,11. The results suggest that sarcoplasm/
mitochondrial volume may vary seasonally in Type I males depending on circulating
plasma levels of steroid hormones. This suggestion is supported by the above men-
tioned observation of a clear circannual change in sonic muscle mass in a Northern
population of midshipmen (T.P. Mommsen and K. Nickolichuck, unpublished ob-
servation). The hormone-sensitivity of muscle mass was confirmed for adult females
and extended to include the oxidative capacity of the sonic muscle. In a northern
population of P. notatus, intraperitoneal injection of 17-methyltestosterone deposits
leads to significant increases in sonic muscle mass, coupled with an increase in
mass-specific aerobic capacity (T.P. Mommsen and A. Bass, unpublished results).
Conversely, treatment of adult Type I males with estradiol for a few weeks resulted
in significant decreases in the overall mass of the sonic muscle. Obviously, a number
of different muscle parameters are under hormonal control in midshipmen, includ-
ing muscle mass, oxidative machinery, sarcoplasm/mitochondrial volume, and every
single one of these would make an interesting object of study.
288 RI. Walsh, T.R Mommsen and A.H. Bass
VI. Prospectsfor future research

The sonic muscles of batraehoidid fishes represent excellent systems with which to
study metabolic regulation and adaptation over both short and long time courses.
The study of the mechanisms of rapid mobilization of enzymes and substrates which
fuel contraction will certainly yield insights into basic metabolic control theory.
The unique 'female-type' sonic muscle in mature 'sneaker' males certainly present
researchers with an ideal to study hormone receptors (expression and function) and
control of muscle differentiation by sex hormones. Additionally, study of the role of
sex hormones in control of metabolism on seasonal and life-history time scales will
yield insights into mechanisms of endocrine action and the evolutionary biology of
sexual dimorphisms.
Acknowledgement. The authors' research is supported by NSF, NIH (U.S.A.)

and NSERC (Canada).
VII. References
1. Appelt, D., V. Shen and C. Franzini-Armstrong. Quantitation of Ca ATPase, feet and mitochondria
in superfast muscle fibres from the toadfish, Opsanus tau. J. Muscle Res. Cell Motil. 12: 543-552,
1991.
2. Bass, A.H. Dimorphic male brains and alternative reproductive tactics in a vocalizing fish. Trends
Neurosci. 15: 139-145, 1992.
3. Bass, A~ and K. Andersen. Inter- and intrasexual dimorphisms in the vocal control system of a
teleost fish: motor axon number and size. Brain Behav. Evol. 37: 204-214, 1991.
4. Bass, A.H. and R. Baker. Sexual dimorphisms in the vocal control system of a teleost fish:
morphology of physiologically identified neurons. J. Neurobiol. 21: 1155-1168, 1990.
5. Bass, A.H. and R. Baker. Evolution of homologous vocal control traits. Brain Behau Ecol. 38:
240-254, 1991.
6. Bass, A.H. and M.A~ Marchaterre. Sound-generating (sonic) motor system in a teleost fish
(Porichthys notatus): sexual polymorphism in the ultrastructure of myofibrils. J. Comp. Neurol.
286: 141-153, 1989.
7. Brantley, R.K. Ontogeny of Inter- and Intra-Specific Sexual Dimorphism in a Vocalizing Fish: Be-
havioral, Morphological and Endocrine Correlates. Ph.D. dissertation, Cornell University, 161 pp.,
1992.
8. Brantley, R.IL and A.H. Bass. lntrasexual dimorphisms in a sound producing fish: alternative
reproductive morphs? Soc. Neurosci. Abstr. 14: 691, 1988.
9. Brantley, R. and A. Bass. Sexual polymorphisms and androgen sensitivity of sound-generating
muscle in a vocalizing fish. $oc. NeuroscL Abstr. 16: 323, 1990.
10. Brantley, R.IL and A.H. Bass. Alternative male spawning tactics and acoustic signals in the plainfin
midshipman fish Porichtys notatus Girard Teleostei Batrachoididae. Ethology 96: 213-232, 1994.
11. Brantley, R.K., M.A. Marchaterre and A.H. Bass. Androgen effects on vocal muscle structure in a
teleost fish with inter- and intra-sexual dimorphism. J. MorphoL 216: 305-318, 1993.
12. Brantley, R., J. Tseng and A. Bass. The ontogeny of inter- and intrasexual vocal muscle dimorphism
in a sound-producing fish. Brain Behav. Evol. 42: 336-349, 1993.
13. Collin, C.G.S and N. Gerardin-Otthiers. The amino acid sequence of the parvalbumin from the very
fast swimbladder muscle of the toadfish (Opsanus tau). Comp. Biochem. Physiol. 93B: 49-55, 1989.
14. Fawcett, D.W. and J.P. Revel. The sarcoplasmic reticulum of a fast-acting fish muscle. J. Biophys.
Biochem. Cytol. 10: 89-102, 1961.
15. Fine, M.L. Sexual dimorphism of the growth rate of the swimbladder of the toadfish, Opsanus tau.
Copeia 3: 483-490, 1975.
16. Fine, M.L. Seasonal and geographic variation of the mating call of the oyster toadfish. Oecologia
36: 45-57, 1978.
17. Fine, M.L. and K.R. Pennypacker. Histochemical typing of sonic muscle from the oyster toadfish.
Copeia 1: 130-134, 1988.
18. Fine, M.L., N.M. Burns and TM. Harris. Ontogeny and sexual dimorphism of sonic muscle in the
oyster toadfish. Can. J. Zool. 68: 1374-1381, 1990.
19. Fine, M.L., K.R. Pennypacker, K.A. Drummond and C.R. Blem. Concentration and location of
metabolic substrates in fast toadfish sonic muscle. Copeia 4: 910-915, 1986.
20. Fluet, A. and A. Bass. Sexual dimorphisms in the vocal control system of a teleost fish: ultrastructure
and neuromuscular junctions. Brain Res. 531: 312-317, 1990.
21. Franzini-Armstrong, C. and G. Nunzi. Junctional feet and particles in the triads of a fast-twitch
muscle fibre. J. Muscle Res. Cell Motil. 4: 233-252, 1983.
22. Gainer, H. and Klancher, J.E. Neuromuscular junctions in a fast-contracting fish muscle. Comp.
Biochem. Physiol. 15: 159-165, 1965.
23. Hamoir, G. and B. Focant. Proteinic differences between the sarcoplasmic reticulums of the
superfast swimbladder and the fast skeletal muscles of the toadfish Opsanus tau. Mol. Physiol. 1:
353-359, 1981.
24. Hamoir, G., O. Dideberg and P. Charlier. Crystallization and preliminary X-ray data for parvalbumin
IIIf of Opsanus tau.J. Mol. Biol. 153: 487-489, 1981.
25. Hamoir, G., N. Gerardin-Otthiers and B. Focant. Protein differentiation of the superfast swimblad-
tier muscle of the toadfish, Opsanus tau. J. Mol. Biol. 143: 155-160, 1980.
26. Huriaux, E, E Lefebvre and B. Focant. Myosin polymorphism in muscles of the toadfish, Opsanus
tau. J. Muscle Res. Cell Motil. 4: 223-232, 1983.
27. Ibara, R.M., L.T Penny, A.W. Ebeling, G. Van Dykhuizen and G. CaiUiet. The mating call of the
plainfin midshipman fish, Porichthys notatus. In: Predators and Prey in Fishes, edited by D.L.G.
Noakes et al., The Hague, Dr. W. Junk Publishers, pp. 205-212, 1983.
28. Kuffler, S.W. and E.M. Vaughan Williams. Properties of the 'slow' skeletal muscle of frog. J. Physiol.
121: 318-340, 1953.
29. Ono, R.D. and S.G. Poss. Structure and innervation of the swim bladder musculature in the
weakfish, Cynoscion regalis (Teleostei: Scianenidae). Can. J. Zool. 60: 1955-1967, 1982.
30. Skoglund, C.R. Neuromuscular mechanisms of sound production in Opsanus tau. BioL Bull. 117:
438, 1959.
31. Skoglund, C.R. Functional analysis of swim bladder muscles engaged in sound production of the
toadfish. J. Biophys. Biochem. Cytol. 10: 187-200, 1961.
32. Somlyo, A.V., H. Shuman and A.R Somlyo. Composition of sarcoplasmic reticulum in situ by
electron probe X-ray microanalysis. Nature 268: 556-558, 1977.
33. Walsh, RJ., C. Bedolla and TP. Mommsen. Reexamination of metabolic potential in the toadfish
sonic muscle. J. Exp. Zool. 241: 133-136, 1987.
34. Walsh, P.J., C. Bedolla and TP. Mommsen. Scaling and sex-related differences in toadfish (Opsanus
beta) sonic muscle enzyme activities. Bull. Mar. Sci. 45: 68-75, 1989.
35. Wery, J.P., O. Dideberg, R Charlier and C. Gerday. Crystallization and structure at 3.2 A resolution
of a terbium parvalbumin. FEBS Lett. 182: 103-106, 1985.
Hochachka and Mommsen (eds.), Biochemistry and molecular biology of fishes, voi. 4
CHAPTER 13
Is glycolytic rate controlled by the

reversible binding of enzymes to subcellular
structures?
STEPHEN P.J. BROOKS AND KENNETH B. STOREY *

Nutrition Research Division, Health Canada, Tunney's Pasture, Ottawa, Ontario, Canada K1A OL2,
9Departments of Biology and Chemistry, Carleton University, Ottawa, Ontario, K1S 5B6 Canada
I. Introduction
II. In vivo enzyme-subcellular binding experiments
III. The search for a metabolic trigger
IV. In vitro measurement of enzyme binding
V. In vitro kinetic studies
VI. Acknowledgements
VII. References
I. Introduction
In order to maintain homeostasis under widely differing metabolic conditions

cells must tightly regulate individual metabolic pathways. In the classical view of
cellular biochemistry, the control of metabolic pathways is achieved through the
reversible regulation of key enzymes of the pathway. These key enzymes are found
at the beginning or the ends of pathways so that overall flux may be governed
through control of only a few enzymes. For example, in the classical view, glycolytic
flux is controlled primarily through allosteric regulation of four enzymes: glycogen
phosphorylase (GP), hexokinase (HK), phosphofructokinase (PFK) and pyruvate
kinase (PK). Glycogen phosphorylase, HK and PFK are found at the start of the
glycolytic sequence whereas PK is one of the terminal glycolytic enzymes. In fish,
all four enzymes show several features common to many regulatory enzymes: they
catalyze high energy steps that are (essentially) irreversible, they catalyze reactions
which involve a high energy intermediate and they are multi-subunit enzymes. They
are also allosterically regulated by several different compounds which serve to link
enzyme activity to the energy state of the cell 34.
The complexity of controls governing glycolytic enzyme activity (in this review
we will confine ourselves to the enzymes of glycolysis) becomes apparent when one
considers the widely different metabolic states that fish can endure. For example,
muscle ATP production during the initiation of burst swimming in salmonid fish
increases 10-15 fold when compared to slow swimming c o n t r o l s 22,49,56,63,65. On the
292 S.RJ. Brooks and K.B. Storey
other hand, metabolic rates in severely hypoxic and anoxic goldfish and carp drop
to levels only 30% of their respective control values 23,ss-61. Both metabolic states
demand a drastic reorganization of metabolism to balance energy-producing and
energy-consuming reactions. It is this balance that is mediated through control of
enzyme activities. Several different levels of enzyme control have been identified
which can regulate glycolytic rates including: (1) changes in the concentration of the
allosteric activators of GP, HI(, PFK and pK21'22'28'3~ (2) changes in cellular
pH towards more acidic values during prolonged periods of exercise22,49; and (3)
changes in enzyme phosphorylation (see Chapter 51, Volume 3 of this series).
More recently a fourth method of glycolytic control has been proposed: the
reversible binding of glycolytic enzymes to subcellular structures to form multi-
enzyme complexes 7-1~ In situations where a rapid change in glycolytic
rate occurs (during burst swimming in trout, for example) the formation of a
multi-enzyme complex would represent a quick and easily reversible mechanism
to control glycolytic rates. In mammals, several multi-enzyme complexes have
been shown to occur. For example, the enzymes involved in DNA and RNA
replication, protein biosynthesis, glycogen metabolism, amino acid metabolism,
fatty acid metabolism, and the citric acid cycle have all been shown to exist in
an aggregated state sS. In fish, a correlation between changes in glycolytic enzyme
binding to cellular particulate matter (a crude preparation containing mitochondria,
lysosomes, nuclei, F-actin and tubulin polymers) and changes in metabolic demand
have also been observed (Fig. 1; refs. 7, 8, 23, 43). The potential kinetic advantages
of enzyme-enzyme and enzyme-subcellular structure complexes are numerous and
100
["' i rested
l exhausted
8 0 I-
.i0
:3 6 0 I-
0
rn
9 I l *
#0 -
20
0
PFK ALD GAPDH PGK PK LDH CK
Fig. 1. Changes in glycolytic enzyme binding in trout (Oncorhynchus myk/ss) skeletal muscle. Enzyme
binding in rested animals (open bars) and animals exercised to exhaustion (closed bars) was measured
using the Dilution method. *Significantly different from rested at the P <0.05 level. Adapted from ref. 7.
Is glycolytic rate controlled by the reversible binding of enzymes to subceUular structures? 293
include increased enzyme activity through localized regions of higher substrate

concentrations and localized regions of higher allosteric activator concentrations:
multi-enzyme complexes should catalyze reactions faster than soluble enzymes.
However, a direct cause and effect relationship between increased glycolytic enzyme
binding and increased flux through glycolysis has not been demonstrated.
This chapter examines the evidence for and against the formation of a func-
tional glycolytic complex. We will define the glycolytic complex as a multi-enzyme
structure containing all the enzymes necessary to metabolize glucose to pyruvate
(the complete glycolytic sequence). In order for the complex to be considered as
functional, three criteria must be satisfied: (1) the enzymes must bind together to
form a complex; (2) the enzymes must be kinetically active when bound in the
multi-enzyme complex; and (3) the magnitude of the change in enzyme binding
must account for the magnitude of the change in glycolytic flux.
Over the past several years it has become clear that some glycolytic enzymes are
bound to subcellular structures or to other enzymes in vivo. These associations may
indeed regulate the rates of individual glycolytic enzymes. Why then is there so much
resistance to the concept of a glycolytic 'metabolon'? As we shall see below, part
of the answer comes from kinetic studies of bound glycolytic enzymes. Specifically,
bound enzymes are often inhibited when compared with their soluble counterparts
even though measurements of enzyme binding before and after metabolic stress
indicate that the bound enzymes should be more active 12. Results such as these
illustrate the sharp discrepancy between kinetic and binding data and suggest that a
kinetically active enzyme complex does not exist (see criteria 1 and 2, above). In the
present review, we will critically examine all the available data on glycolytic enzyme
complex formation and present an overall view of the complex as it is presently
understood. It is hoped that a balanced treatment has been achieved that will allow
the reader to form his or her own conclusions regarding the existence of the glycolytic
complex and the in vivo kinetic benefits that such a complex would exhibit.
II. In vivo enzyme-subcellular binding experiments
We have chosen to call experiments that measured enzyme binding before and after
a physiological stress 'in vivo' experiments although the actual bound/free measure-
ments are usually performed after tissue homogenization and centrifugation. The
following is a summary of procedures used to assess the in vivo bound/free status
of glycolytic enzymes. The most convincing evidence in favor of enzyme-subcellular
binding interactions in vivo has come from experiments using the Dilution method.
In this technique 17, muscles are homogenized in an iso-osmotic sucrose solution
(250 mM sucrose, zero ionic strength) and centrifuged to separate bound and free
enzyme fractions 189 bound enzymes are found in the pellet, free enzymes in the
supernatant (refer to Volume 3 of this series for a detailed protocol and analysis of
the Dilution method). Because the technique is rapid, it provides a good method for
assaying the distribution between free and bound enzymes under defined metabolic
conditions (Fig. 1). Variations of this technique have incorporated changes in cen-
294 s.P.J. Brooksand K.B. Storey
trifugation time and speed24,3a,S2as well as changes in the ionic strength of the
homogenization medium 16. Using this method, changes in glycolytic enzyme bind-
ing have been measured in exercised 7,a, starved43, and anoxic23 fish muscle as well
as in other animals under a variety of metabolic states 9.1~ These studies
demonstrated a positive correlation between increased enzyme association with
particulate matter and increased energy demand in skeletal muscle7.8,17,23 as well
as between decreased binding and decreased energy demand 9't~ By showing
that: (1) enzyme binding changes in response to changes in metabolic demand; and
(2) the direction of change correlates with the metabolic flux in situations of both
increasing and decreasing metabolic rate, these data argue for both the existence
and the functional significance of a glycolytic enzyme complex. The correlation
between increased (or decreased) glycolytic demand and increased (or decreased)
enzyme binding suggests that increased enzyme binding promotes an increase in
glycolytic flux. This result predicts that glycolytic enzymes would be kinetically
activated in the complex when compared with their soluble counterparts and limits
us to considering only this possibility. This latter point is emphasized because it is
essential that we remember it when examining the in vitro experiments on enzyme
binding (see below).
Although a majority of in vivo experiments have been performed with the
Dilution methodology, questions about its validity rule out its use as an absolute
identifier of enzyme complexes. Specifically, the Dilution method dilutes the cellular
milieu 4-5 fold (depending on the procedure) prior to measurement of enzyme
binding. This means that the cellular ionic strength and protein concentration have
been diluted to a value that is 20-25% of the in vivo value before the identification
of bound and free enzymes. This dilution may artificially increase (or promote)
enzyme binding because enzymes bind largely through ionic interactions 8. The
effects of diluting the cellular ionic strength on enzyme binding have been measured
for rainbow trout (Oncorhynchus mykiss) white muscle PFK and glyceraldehyde
3-phosphate dehydrogenase (GAPDH) by adding various salts and observing the
outcome s. Fig. 2 shows this effect for PFK. Note that the binding is extremely
sensitive to increasing salt concentrations so that at physiological concentrations of
ATP, ADP, fructose 6-phosphate, lactate, Ca 2+, Mg2+, and KCI virtually 90% of the
PFK and GAPDH activities are free s. Studies such as these have called the Dilution
method into question and suggest that the absolute amount of enzyme bound may
not reflect the true value in vivo (see Table 1).
In order to better assess the enzyme bound/free distribution in vivo, several other
methods have also been developed. These differ widely in their ability to reproduce
cytoplasmic conditions so that results obtained with them must be interpreted
accordingly. The other methods include:
(1) the Press Juice method1'2'4;

(2) the Minced and Spun procedure4;
(3) Glutaraldehyde Fixation39;
(4) Detergent Solubilization43'54;
(5) diffusion measurements with fluorescently labeled proteins *~.
Is glycolytic rate controlled by the reversible binding of enzymes to subcellular structures? 295
lO0
13
80
LLI
tu 60
n-
il
0 MIIATP
= F-6P
o~ 40
,., C~CI 2
= KCI
20
0 20 40 60 80 I00 120
[EFFECTOR] (rn_M)
Fig. 2. Release of rainbow trout PFK from particulate matter by salts and physiological metabolites.
Increasing concentrations of a 1:1 mixture of MgCl2 and ATP (MgATP, open circles), fructose 6-
phosphate (F-6P, closed squares), CaCI2 (open squares) and KCI (closed circles) were added to trout
muscle particulate matter suspended in 250 mM sucrose. Supernatant (Free) PFK activity was measured
after centrifugation at 12,000 g for 5 rain. Adapted from ref. 8.
TABLE 1
Comparison of glycolytic enzyme binding in rainbow trout (Oncorhynchus mykiss) white skeletal muscle
estimated using three different methodologies
Enzyme Dilution method Quickly pressed method Rapid minced and spun
(% bound) (% bound) (% bound)
PFK 95.3 70.5 81.2
ALD 32.2 28.5 13.8
GAPDH 73.1 10.0 20.8
LDH 17.3 1.0 16.1
Percentage of bound enzyme was measured in exhausted trout white muscle. Description of method-
ologies is found in the text. Data modified from Brooks and Storey 8.
Abbreviations: ALD = aldolase; PFK = phosphofructokinase-1; GAPDH = glyceraldehyde 3-
phosphate dehydrogenase; LDH = L-lactate dehydrogenase.
A short discussion on the merits of these techniques is provided below.

The Minced and Spun method involves mincing tissue in a Waring blender
and centrifuging the resulting homogenate for 20-24 h at 100,000 g to separate
particulate matter and cellular juice fractions 1. This technique has the advantage
that it does not dilute cell contents during the homogenization step; the ionic
strength and the protein concentration of the cellular juice are maintained at values
found in vivo. However, the lengthy centrifugation step allows changes in cellular
metabolite levels to occur. Thus, enzyme binding is ultimately measured under
conditions where ATP, creatine phosphate and cellular pH are lower than that found
in vivo and the lactate concentration is higher because of anaerobic fermentation.
Changes in ATP and phosphate concentrations and pH are known to alter the
binding of aldolase (ALD) 4, GAPDH 2~ and PFK s (Fig. 2). A recent modification
296 s.PJ. Brooks and K.B. Storey
of this technique permits a more rapid measurement of enzyme binding. The tissue
is minced finely with a razor blade and centrifuged for 2 h at 100,000 g (Rapid
Minced and Spun method). The distribution of the enzyme activities between
particulate matter and supernatant is calculated from the observed activities in each
phase, and the known water content of the tissue (Table 1)a. Although this latter
technique drastically reduces the length of time required to prepare the samples,
it still involves a lengthy period of centrifugation which allows changes in cellular
conditions to occur.
The Pressed Juice technique is similar to that of the Minced and Spun method
with the omission of the mincing step; muscle strips are centrifuged at 100,000 g
for 24-48 h and enzyme activities in the supernatant (pressed juice) and pellet
(dehydrated tissue) are measured. This method suffers the same drawbacks as the
Minced and Spun method and also from a filter effect due to the presence of the
plasma membrane. As a consequence of this, the free enzyme concentrations from
pressed juice experiments are always significantly lower than those obtained by the
Minced and Spun method 2,a. For example, more than 40% of the total ALD and
lactate dehydrogenase (LDH) activity was not recovered after repeated washing of
'pressed muscle strips' in high ionic strength buffer2. A modified version of this
method involves pressing muscle strips at 264 tons/in. 2 in a French Pressure Cell for
15 s (Quickly Pressed methodS). The free enzyme concentration is obtained from
the pressed juice (taking into account the extracellular volume) and the percentage
bound is calculated from the total activity measured in non-pressed strips. The large
pressures employed in this technique should negate any filter effect caused by the
presence of the plasma membrane (see Table 1).
The measurement of enzyme binding by Glutaraldehyde Fixation of proteins has
been used to determine the degree of enzyme association in synaptosomes 39. The
procedure involves incubating an intact synaptosome preparation with increasing
glutaraldehyde concentrations for 10 rain, washing the syaaptosomes, and measur-
ing the activity that precipitates after centrifugation. Problems with this technique
are: (1) whole tissues cannot be used as all cells must be equally exposed to the
glutaraldehyde solution; (2) the time required to isolate cells often changes their
metabolic profiles; and (3) since the cytoplasm is approximately 30% protein by
weight (which corresponds to a protein concentration of 330 mg/ml) 27 the crowding
of the cytoplasmic proteins in vivo may lead to glutaraldehyde-induced covalent
protein-protein linkages between pairs of enzymes that may be physically close but
may not be actually bound together.
The Detergent Solubilization technique involves washing cells in Triton X-100
followed by centrifugation to identify bound enzymes43. Specifically, excised tissue
is finely minced with a knife and stirred in 3 vol. of a high ionic strength buffer
containing 0.05% Triton X-100. This suspension is centrifuged at 1000 g for 10 min
and the pellet re-extracted twice. The bound fraction represents the enzyme activity
remaining in the pellet after the third wash and the free enzyme activity is the
total of the activities in the three supernatants. The rationale underlying the use of
this procedure comes from a desire to minimize the mechanical disruption of the
cytoplasmic matrix during the extraction stage 19. A plot of the percentage of activity
TABLE 2
Comparison of the Dilution method and the Detergent solubilization method for measuring glycolytic
enzyme binding in turtle (Pseudemys scripta) brain
Enzyme Dilution method Detergent solubilization

(% bound) (% bound)
PFK 18 53
ALD 21 46
GAPDH 21 42
PGK 31 40
PK 19 46
LDH 27 49
Percentage of free enzyme was measured in brain from resting turtles. Data from Duncan and Storey 24.
phosphate dehydrogenase; PGK = phosphoglycerate kinase; PK -- pyruvate kinase; LDH -- L-lactate
dehydrogenase.
solubilized at each wash showed that the third wash contributes only 10% to the
total free activity suggesting that most of the free enzyme activity has been removed
by this step. This procedure is rapid allowing the measurement of bound and free
enzyme profiles in tissue samples from animals under a variety of metabolic con-
ditions. However, the results obtained with this procedure are difficult to interpret
because of two major problems: (1) the presence of the plasma membrane may in-
hibit free movement of enzyme from the cytosol to the buffer; and (2) cells situated
at the center of the minced tissue are not likely to be exposed to Triton X-100.
These problems may lead to an overestimation of the proportion of bound enzyme
activity as illustrated in studies with anoxic turtle brain (Table 2). The Detergent
method gives approximately twice the bound activity when compared with the
Dilution method (which itself overestimates the degree of enzyme binding, Table 1).
In general, the procedures used to measure enzyme binding vary widely in their
ability to reproduce cytoplasmic conditions and, consequently, results obtained
using them must be interpreted accordingly. Note that the limitations of these
techniques all favor overestimation of the amount of bound activity. Note also
that these techniques do not offer any information on the subcellular location of
the bound enzymes: enzymes may be randomly distributed along one or several
different organelles or bound together in a functional complex in vivo (particulate
matter is composed of fractions of several different organelles). In the absence of
any further experimentation, one may conclude that a glycolytic complex had been
obtained which was bound to particulate matter.
A recent study from our laboratory of enzyme complex formation in fish white
muscle illustrates the problems inherent in many of these techniques. We showed
that PFK, ALD, GAPDH and phosphoglycerate kinase (PGK) binding increased
within 30 s of the initiation of burst swimming in trout when measured with the
Dilution method 7. In a later report 8, we measured the binding of PFK and GAPDH
using three other techniques: polyethylene glycol precipitation; a modification of the
Minced and Spun method (Rapid Minced and Spun method); and a modification
298 s.P..j. Brooksand K.B. Storey
of the Pressed Juice method (Quickly Pressed method) to reduce the procedural
limitations discussed above (Table 1). These newer techniques showed that the
correlation between increased binding of GAPDH and increased length of exercise,
as measured by the Dilution method, was an artifact; GAPDH was greater than
75% free when measured using the newer techniques as opposed to 25% free
using the Dilution method. PFK binding was also reduced from 95% bound by the
Dilution method to 70% bound by the Quickly Pressed method (Table 1). These
results suggest that experimentally observed changes in enzyme binding may reflect
limitations in the methodology and not actual in vivo changes in enzyme binding s.
I l L The search for a m e t a b o l i c trigger

The identification of a metabolic trigger that mediates changes in enzyme binding
in vivo is an important part of the identification of a functional glycolytic complex.
The discovery of such a signal will immediately validate the complex by linking
physiological changes in muscle metabolism to changes in enzyme binding through
a known mediator. The absence of a signal compound does not, in and of itself,
mean that an enzyme complex does not exist in muscle tissue. When combined with
other data that suggest that enzyme binding is an artifact, however, the absence of
a defined signal compound would argue against the existence of a complex. The
experiments of this section were performed either with cell free homogenates or
by the Dilution method. Despite the limitations of this method, it is effective for
monitoring relative changes in the binding of glycolytic enzymes. This is because
changes in the putative signal compound should reproduce changes in enzyme
binding already measured using the Dilution method. Unfortunately, to date, a
putative signal that mediates changes in glycolytic enzyme binding in vivo has
not been identified. Cell-free studies with trout white muscle 7.s demonstrated that
neither decreased pH, nor increased or decreased calcium concentrations could
reproduce the changes in PFK, ALD and GAPDH binding observed after exercise.
One can also show that changes in cellular pH occur much later than changes in
enzyme binding in exercised trout muscle in vivo 7.
Many of the in vivo studies on the putative metabolic trigger were performed
with isolated tissues of the whelk (Bus)con caniculatum), a marine gastropod,
because: (1) isolated tissues of this species respond to environmental anoxia in
a fashion identical to that of the whole animal; and (2) it is relatively easy to
alter cellular conditions in these tissues through manipulation of the incubation
medium 1~ Results with isolated whelk tissues confirmed and extended the trout
studies. In particular, these experiments showed that enzyme binding decreased
when metabolic rates were depressed (during anoxia) but at the same time tissue
pH had also decreased. This effect was exactly opposite to that observed when the
cellular pH of the isolated tissues was artificially altered by incubating tissues in
buffers of lower or higher pH (ref. 10). It is also opposite to the effect observed in
exercised trout muscle 7 and to in vitro studies on pH and enzyme binding 8,20 ,36,53 ; in
both these cases enzyme binding increased with decreasing cellular pH.
Studies with isolated whelk tissues also demonstrated that changes in enzyme
binding did not respond to changes in the concentration of protein kinase second
messengers (cAMP, cGMP, Ca2+; see ref. 10) or to changes in the concentration of
enzyme substrates and/or products. Enzyme substrates were altered by incubating
isolated whelk tissues in the presence of iodoacetic acid which inhibited GAPDH
activity. Addition of iodoacetic acid caused a 10-100 fold increase in PFK and ALD
substrates and a two fold lowering of PK substrates but did not alter the binding of
any of these enzymes when measured with the Dilution method 13.
Ultimately, the fact that a cellular signal that regulates enzyme binding has
not been identified could argue either for and against the existence of a functional
glycolytic complex. On the one hand, the difficulty of characterizing a signal suggests
that enzyme binding is specifically controlled by an intricate mechanism as yet to be
identified. On the other hand, the lack of an identified cellular signal may indicate
that enzyme binding is created by diluting a fragile cellular condition which cannot
be reproduced in vitro.
IV. In vitro measurement o f enzyme binding
In vitro experiments on enzyme binding can only provide a limited amount of

information on the glycolytic complex. In general, such studies make use of purified
systems often containing only two or three components. Therefore, although they
provide details of the physical interaction between an enzyme and a single type
of subcellular structure, they cannot, by themselves, show that a glycolytic com-
plex exists. Problems with in vitro experiments also arise. Experiments measuring
enzyme-particulate matter binding are often difficult to interpret because enzymes
may be bound to one of several different subceUular structures including: the
plasma membrane 5~ the microsomal fraction 14, F-actin polymers 6 and/or the outer
mitochondrial membrane 64. Furthermore, enzymes may bind to different locations
on the same subcellular structure. For example, PFK can bind either F-actin 62 or
troponin C (a muscle fibril protein 4~) suggesting that a unique myofibril binding site
does not exist for PFK. The localization of individual glycolytic enzymes to all of
these structures makes it difficult to unequivocally conclude that all enzymes are
binding to a single subcellular location; a unique subcellular binding site may not
exist for each enzyme. The results obtained from reconstituted systems are more
straightforward since defined species are present. They are, however, artificial by
nature and do not give an idea of the true enzyme distribution in vivo.
Most experimenters study enzyme binding to filamentous actin (F-actin) as the
base structure for formation of a glycolytic enzyme complex. This is because: (1)
F-actin binds the largest number of enzymes with the highest affinity; and (2)
histochemical studies have localized most of the bound enzymes to the I-band of
muscle tissue (see ref. 26 and references therein).
Other problems with the in vitro analysis of enzyme binding include the fact that
studies are conducted at low ionic strengths and neutral pH because physiological
ionic strengths completely dissociate the complexes 4,8,2~ This shows that the
300 S. t?.I. Brooks and K.B. Storey
interactions are largely ionic in nature and suggests that they do not occur in vivo
(see also section II and Fig. 2). In vitro studies are also, by necessity, performed
at protein concentrations well below those found in vivo. The effect of decreased
protein concentration is unclear but mathematical calculations 11 suggest that the
percentage of enzymes associated with F-actin will increase with increasing protein
concentration.
Both whole tissue extracts and isolated proteins have been used to quantify en-
zyme binding. These studies have shown that almost all glyeolytie enzymes bind to
F-actin or particulate matter under low ionic strength conditions. Specificity of the
enzyme-F-actin interactions is suggested by four different lines of evidence. Firstly,
studies of the ALD-F-aetin interaction 62 and the PFK-F-aetin interaction 42,44
demonstrated that the binding phenomenon was saturable and apparently involved
specific protein-protein interactions 47. Secondly, some enzymes are released from
F-actin by relatively low concentrations of their substrates. This suggests the in-
volvement of enzyme active sites (see section V) and may indicate the existence of a
signal compound for enzyme release from F-actin. For example, the binding of PFK
to F-actin was inhibited by low concentrations of ATP and ADP in rabbit muscle 42
and bass white muscle 53 but was not inhibited by fructose 6-phosphate 42,53. Binding
of ALD to bovine skeletal muscle filaments and F-actin was competitively inhibited
by its substrate fructose 1,6-bisphosphate 4,31. GAPDH binding was inhibited by in-
creasing concentrations of fructose 1,6-bisphosphate 39. PK binding to mierotubules
was decreased specifically by phosphoenolpyruvate 29 and LDH binding to partic-
ulate matter was reduced in the presence of NADH 36. Ca 2+ ions also inhibit the
binding of PFK 4~ ALD 32 and GAPDH 8. The demonstration that specific inhibitors
of the enzyme-F-aetin interaction exist (i.e. enzyme substrates and produets) argues
for a physiologically relevant process. Thirdly, enzyme binding (specifically PFK) is
dependent on the degree of enzyme phosphorylation suggesting that binding may
be sensitive to cellular signals that change enzyme phosphorylation patterns 44. And
fourthly, the binding of PFK 8,~, ALD 4, GAPDH 2~ and PK 1~ to particulate matter
and F-actin is affected by changes in cellular pH with increased binding at lower pH
values.
Although several of the in vitro binding experiments suggest that glyeolytie
enzymes bind specifically to F-actin, remember that it is extremely difficult to
extrapolate these results to the situation in vivo. For example, although enzyme
binding may be sensitive to low concentrations of substrates at low ionic strength,
this sensitivity may disappear at the higher ionic strengths in vivo. Please refer
to sections II and III for a complete discussion of the limitations of the binding
experiments since many of the same caveats apply.
7. I n vitro kinetic studies
The kinetic studies of enzyme-F-actin complexes are perhaps the most important
because they determine how a bound enzyme or a glyeolytie complex would
function in vivo. Specifically, kinetic studies show whether an enzyme is activated
or inhibited when bound to F-actin. If a glycolytic complex is bound to F-actin

and if this complex is responsible for higher glycolytic flux, the enzymes in the
complex should be at least as active as their soluble counterparts. Note that kinetic
studies of individual enzyme-F-actin complexes cannot, by themselves, indicate
exactly how a multi-enzyme complex would behave in vivo. This is because the
juxtaposition of enzyme active sites in a multi-enzyme structure should create
a localized compartment of higher substrate and product concentrations. The
creation of a localized compartment will mean higher enzyme activities for bound
enzymes because these enzymes 'see' higher substrate concentrations than the
soluble enzyme. Such substrate effects are different, however, from the effects of
enzyme binding on kinetic constants. In the latter case, changes in enzyme kinetic
parameters are due solely to enzyme-F-actin binding. These changes may result
either from a direct interaction between an active site (or an allosteric site) and
F-actin, or from a conformational change in the enzyme induced by binding. In
the former case, competitive inhibition with respect to a substrate (or activator) is
observed whereas in the latter case, either the maximal activity and/or the affinity
for substrate may be affected.
Although the in vitro binding studies were criticized because the experiments
were carried out at low ionic strength, this does not apply to the majority of
kinetic experiments; as a general rule, the kinetic experiments were performed
under conditions that more closely reflected the in vivo ionic strength. However,
as with the binding experiments, the total protein concentration in the kinetic
experiments was much lower than that found in vivo because of the difficulty in
measuring enzyme activities at high concentrations. The effect of lower protein
concentrations on the enzyme kinetic patterns is different for different enzymes.
For example, PFK is more polymerized at higher protein concentrations 4s and the
polymerized enzyme has a higher affinity for fructose 6-phosphate and a lower
susceptibility to ATP inhibition 35. Allosteric activators which stimulate activity
at low PFK concentrations do not work at high protein concentrations 5 or in
permeabilized cells 3 because activity is already maximal. Studies on liver PK kinetics
in the presence of polyethylene glycol indicate that PK would also be activated at
higher protein concentrations 46. These studies illustrate the importance of protein
concentration in determining overall kinetic effects and may indicate that activation
of PFK by F-actin at low protein concentration in vitro may not be operative at the
higher PFK concentrations in vivo.
Table 3 presents the kinetic consequences of enzyme-F-actin binding for the
enzymes of glycolysis. In only one case, that of the PFK-F-actin interaction, was the
enzyme activated by enzyme binding. All other enzymes were inhibited when bound
to F-actin. In the case of PFK, activation resulted from a lower Km for fructose 6-
phosphate and slightly higher inhibition constant for MgATP 42,44. The combination
of a higher affinity for fructose 6-phosphate and a reduced ATP inhibition resulted
in an overall PFK activation at low concentrations of fructose 6-phosphate in the
presence of F-actin. Thus, the kinetic studies of PFK-F-actin complexes agree with
the in vivo and in vitro binding data which suggest that PFK binding increases during
periods of high glycolytic activity to increase glycolytic flux. In the case of ALD,
302 S.P..J.Brooksand K.R Storey
TABLE 3
Kinetic effect of F-actin binding on selected glycolytic enzymes
Enzyme Overall effect Specific changes

PFK Activation ATP and citrate less effective inhibitors
AMP and Pi better activators
Higher affinity for substrate fructose 6-phosphate 42,44
ALD Inhibition Direct competition of F-actin for substrate TM
TPI Not known
GAPDH Inhibition Lower affinity for substrate glyceraldehyde 3-phosphate 2~
PGK Not known
PGM Not known
Enolase Not known
PK Inhibition Lower affinityfor substrate phosphoenolpyruvate15
LDH Inhibition Lzwer affinityfor substrate NADH 36
phosphate dehydrogenase; TPI = triosephosphate isomerase; PGK = phosphoglycerate kinase; PK
= pyruvate kinase; LDH = L-lactate dehydrogenase; PGM = phosphoglucomutase.
F-actin competed directly with fructose 1,6-bisphosphate for ALD 4,31 so that the
F-actin-bound ALD was completely inactive. The binding of GAPDH 2~ PK is and
LDH 36 to F-actin also inhibited enzyme activity by increasing the Km value for the
enzyme substrates.
The anomalous kinetic responses of bound enzymes argue strongly against the
concept of a functional glycolytic complex. If glycolytic flux increases as a result
of an increase in enzyme binding (as expected from the in vivo studies) one
would expect that all enzymes in the complex were, at least, as active as their
soluble counterparts. The fact that the majority of bound enzymes are inhibited
suggests that enzyme binding is not a mechanism of enzyme activation but rather a
negative regulatory mechanism: binding reduces glycolytic flux. These conclusions
are supported by a recent report which found that binding of glycolytic enzymes
to subcellular structures in red blood cells was a negative control mechanism to
s l o w glycolysis 33. Other studies showed that sonicated cells had a much higher rate
of glycolysis than did intact or permeabilized cells indicating that the existence of
an ordered cellular structure inhibited glycolysis37. The kinetic studies of F-actin-
bound enzymes suggests that a similar, inhibitory role for enzyme binding may exist
in muscle tissue.
VI. Conclusion
Do the enzyme-F-actin complexes identified in the present review meet the criteria
initially set out for identifying a functional glycolytic complex?
(1) The enzymes must readily associate under conditions of physiological ionic
strength and protein concentrations. An abundance of physical evidence suggests that
a proportion of many of the cytosolic glycolytic enzymes are bound to F-actin in
Is glycolytic rate controlled by the reversible binding of enzymes to subcellular structures ? 303
TABLE 4
Calculated estimates of the percentages of enzymes associated with F-actin in vivo
Enzyme % Bound
PFK 27
ALD 3
TPI 7
GAPDH 7
PGK 0.04
PK 60
LDH 2
Calculations based on in vitro dissociation constants and in vivo ionic strength and protein
concentrations 11.
vivo. This is supported by in vitro studies demonstrating enzyme binding at low

ionic strength as well as by in vivo studies using a wide variety of methodologies.
Although it is extremely difficult to measure protein associations in vivo because
most techniques dilute protein and/or ionic strength, the most reliable methods
indicate that a defined percentage of some enzymes is associated with particulate
matter (Table 1). Mathematical extrapolations using in vitro binding constants also
suggest that a large percentage of PFK and PK would be bound to F-actin (Table 4).
However, the fact that enzymes may associate with F-actin in vitro (or in vivo)
does not necessarily indicate that a functional kinetic complex exists. F-actin is
abundant in muscle and it is possible that enzymes are randomly distributed
along the filament. This argument illustrates the major problem with the in vivo
studies: they fail to localize bound enzymes to a single type of subceUular structure.
Demonstrating that enzymes associate with particulate matter is not proof of a
functional complex. If other (non-glycolytic) enzymes bind to F-actin a random
distribution of several enzymes throughout the subcellular matrix will result. The
percentage of enzyme binding may increase/decrease in response to increased/
decreased glycolytic flux but this does not necessarily indicate the formation of a
functional glycolytic complex. Glycolytic enzymes must be localized to one specific
structure before a functional complex formation is considered a possibility.
(2) The enzymes must be kinetically active when bound and formation of the
complex must confer a kinetic advantage to the bound enzymes. The kinetic studies
of the enzyme-F-actin complexes show that neither condition was fulfilled for
ALD, GAPDH, PK and LDH. In fact, ALD is completely inhibited when bound to
F-actin.
(3) The magnitude of the change in enzyme binding must account for the magnitude
of the change in glycolytic flux. A consideration of the situation in trout skeletal
muscle shows that this condition is not fulfilled. During the early stages of exercise,
glycolytic flux in trout white muscle increased by 10-15 f o l d 21'49'56'63'65. Changes
in glycolytic enzyme binding during exercise (Table 1) showed that the largest
increases were on the order of 50% (PFK and GAPDH). Unless the activities
of the bound enzymes are much higher than those of the soluble enzymes the
relatively small increase in enzyme binding cannot support the large increase in
304 S.RJ. Brooks and K.B. Storey
glycolytic flux. Bound PFK may have a much higher activity than the soluble enzyme
because binding changes PFK sensitivity to inhibitors and activators (the effects are
synergistic). This is not true for ALD, GAPDH, PK and LDH: all these enzymes
are inhibited when bound. These results suggest that changes in ALD, GAPDH, PK
and LDH enzyme binding are not related to changes in glyeolytie flux in vivo.
Does enzyme binding play a role in modulating glyeolytie rates? PFK binds
tightly to F-aetin under in vivo conditions of high ionic strength, high protein
concentrations, and in vivo pH. Binding of PFK to F-actin increases in response to
increased glyeolytie flux. PFK is also activated by binding to F-aetin and this effect
is modulated by reversible phosphorylation. All these factors suggest that binding of
PFK is intimately linked to the energy state of the cell. In light of the fact that the
other glycolytic enzymes are inhibited when bound to F-actin, what function could
this binding have? It may serve to localize PFK in the vicinity of the myosin ATPase
where it would be more susceptible to changes in the adenosine nueleotide tri-/di-
phosphate pool. A greater interaction between PFK and the myosin ATPase would
exist because a shorter distance between these enzymes would lead to a localized
higher nucleotide pool concentration. Thus, during periods of high glyeolytie flux,
localized low ATP concentrations and high ADP and phosphate concentrations
(brought about through muscle myosin ATPase activity) would activate PFK by:
(1) reducing the concentration of the PFK inhibitor ATP; and (2) increasing the
concentration of the PFK activators ADP and phosphate. Because PFK is a key
regulatory enzyme of the cell, changes in its activity would result in large changes
in the overall glyeolytie flux. A closer correspondence between PFK activity and the
myosin ATPase activity would, therefore, provide a tighter link between glyeolytie
flux and ATP demand resulting in quicker response in glycolytie rates to energy
needs in the working muscle.
Acknowledgements. Work on glycolytic complexes continues in the laboratory of

K.B.S. thanks to research grants from NSERC (Canada) and NIH (U.S.A., GM
43796). The authors also wish to thank J.M. Storey for editorial criticisms.
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myofibrils in the red and white muscle of the Barred Sand Bass Paralabrax nebulifer (Serranidae). jr.
Exp. Biol. 137: 13-27, 1988.
54. Shearwin, K., C. Nanhua and C. Masters. Interaction between glycol)tic enzymes and cytoskeletal
structure - the influence of ionic strength and molecular crowding. Biochem. Int. 21: 53-60, 1990.
55. Srere, P.A. Complexes of sequential metabolic enzymes. Ann. Rev. Biochem. 56: 89-124, 1987.
56. Storey, K.B. Metabolic consequences of exercise in organs of rainbow trout. J. EXp. Zool. 260:
157-164, 1991.
57. Storey, ICB. and J.M. Storey. Facultative metabolic rate depression: molecular regulation and
biochemical adaptation in anaerobiosis, hibernation, and estivation. 0.. Rev. Biol. 65: 145-174, 1990.
58. Van den Thillart, G. Adaptations of fish energy metabolism to hypoxia and anoxia. Mol. Physiol. 2:
49-61, 1982.
59. Van den Thillart, G., E Kesbeke and A. van Waarde. The influence of anoxia on the energy
metabolism of goldfish Carassias auratus (L.) Comp. Biochem. Physiol. 59A: 329-336, 1976.
60. Van Waversveld, J., A.D.E Addink and G. van den Thillart. Simultaneous direct and indirect
calorimetry on normoxic and anoxic goldfish. J. E ~ . Biol. 142: 325-335, 1989.
61. Van Waversveld, J., A.D.E Addink and G. van den Thillart. The anaerobic energy metabolism of
goldfish determined by simultaneous direct and indirect calorimetry during anoxia and hypoxia. J.
Comp. Physiol. 159B: 263-268, 1989.
62. Walsh, T.P., D.J. Winzor, EM. Clarke, C.J. Masters and D.J. Morton. Binding of aldolase to
aetin-containing filaments. Biochem. J. 186: 89-98, 1980.
rainbow trout (Salmo gairdneri) during and after bursts of activity. J. Comp. Physiol. 155B: 485-492,
1985.
64. Wilson, J.E. Brain hexokinase, the prototype ambiquitous enzyme. Cute. Top. Cell. Regul. 16: 1-44,
1980.
65. Wokoma, A. and I.A. Johnston. Anaerobic metabolism during activity in the rainbow trout ($almo
gairdneri). Experientia 39: 1366-1367, 1983.
CHAPTER 14
Histidine-related dipeptides: distribution,

metabolism, and physiological function
HIROKI ABE
Department of Food Science and Nutrition, Kyoritsu Women's University, 1-710Motohachioji,
Hachioji, Tokyo 193, Japan
I. Introduction
II. Distribution of free L-histidine and related dipeptides in fish tissues
Ill. Effects of physiological conditions on the dipeptide concentrations in fish muscle
IV. Metabolicturnover of L-histidine in fish
V. Metabolismand interorgan transport of the dipeptides in fish
VI. Physiologicalfunctions of histidine-related dipeptides as proton buffers
VII. References
I. Introduction
Animal tissue has long been known to contain quite a few different types
of L-histidine-related dipeptides (imidazole dipeptides). The dipeptides refer
to carnosine (fl-alanyl-L-histidine), anserine (fl-alanyl-Tr-methyl-L-histidine), bale-
nine (/5-alanyl-r-methyl-L-histidine, also called ophidine), and homocarnosine (y-
aminobutyryl-L-histidine). Of these dipeptides, carnosine, anserine, and balenine
may occur in vertebrate skeletal muscle in large amounts depending on the species.
Fig. 1 shows the structures of these muscle dipeptides.
+ CO0-
1 i Carnoslne
0 H,,N.~N~.H
+ CO0-
t I 3 N ' ~ N H ' ~
I i Anserine
0 C H3"N'~N'~H
+ CO0-
1 i BalenIne
0 H,,N,~N~cH9
Fig. 1. Chemical structures of histidine-related dipeptides.
310 H. Abe
Camosine was first isolated from meat (came) extract by Gulewitsch and
Amiradzibi 27 in 1900 and the structure was established by Baumann and Ingvaldsen 14.
Anserine was isolated from goose (Anseriformes) muscle in 1929 by Ackermann and
eoworkers n and identified as ~-alanyl-l-methyl-L-histidine. In the ease of balenine,
however, confusion has arisen over its structure. The compound was first isolated in
1962 by Pocchiari and colleagues 67 from the muscle of a baleen whale, Balaenoptera
sp., hence the name. Long before this discovery, Imamura 3s, in 1939, isolated a
dipeptide named ophidine (derived from Ophiophagus sp.) from snake muscle and
it was wrongly identified as/5-alanyl-2-methyl-L-histidine43. The correct structure of
ophidine was finally established by Nakai and Tsujigado 6~ in 1965 and found to be
identical to that of balenine. Thus, the name of the dipeptide was finally unified into
balenine sg. However, the confusion about the name goes on unabated today, since
some people still use the term ophidine and, not to be outdone, ophidine is identified
as ~-alanyl-2-methyl-L-histidine even in Chemical Abstracts.
The nomenclature of methyl derivatives of L-histidine has also been somewhat
confusing because the numbering system of the imidazole ring of L-histidine was
reversed in the chemical and the biochemical literature. IUPAC-IUB 39 finally, in
1974, recommended that the imidazole ring nitrogen close to the a-carbon of L-
histidine should be named as pros (Tr) and that far from the a-carbon as tele (r).
Thus, formerly 1-methyl-L-histidine should be designated as ~r-methyl-L-histidine
and the 3-methyl derivative as r-methyl-.
Since the discovery of these dipeptides, various methods have been developed to
determine their concentrations in animal tissues. These preparative and analytical
methods were reviewed by Crush 23. Supplanting early methods using paper chro-
matography and cation exchange chromatography, modified techniques of amino
acid analysis for physiological fluid were used in the 1970s2~ Finally, fast
and accurate determination methods using ion-exchange and reversed phase high-
performance liquid chromatography (HPLC) were adopted in the early 1980s1'9.1s.
Fig. 2 shows a typical separation of histidine-related compounds by ion-exchange
HPLC.
Extensive studies have been undertaken to date on the distribution, metabolism,
and physiological functions of these dipeptides in animal tissues. Table 1 represents
a brief summary of the distribution of histidine and the dipeptides in the skeletal
muscle of various animals. Although the distribution of the dipeptides is restricted
in the muscle of invertebrate and most white-fleshed fishes such as percid and
pleuronectid fishes, large amounts of these dipeptides are found in the wide variety
of vertebrate muscles. Their distribution, however, lacks any phylogenetic basis.
Balenine, for instance, which is found only in trace amount in most vertebrate
muscles, is abundant in the muscle of snakes and whales. Camosine, on the
other hand, is rather common in vertebrate muscles, but some fishes and birds
contain much higher amounts of anserine than camosine. In spite of such high
concentrations of these dipeptides in animal muscle, their physiological functions
have not been satisfactorily elucidated.
Free L-histidine which is one of the constituents of camosine is abundant in
every tissues of animals because it is a proteinaceous amino acid, although it is
Histidine-related dipeptides: distribution, metabolism, and physiological function 311
,.?,
r--'-
I I III I I I IN I II I am
0 10 20 30
RETENTION TIME (min)
Fig. 2. Separation of authentic histidine-related compounds (2 nmol each) by high-performance liq-
uid chromatography. Conditions: chromatograph, Shimadzu LC-6A; column, Nucleosil 5-SB cation-
exchanger (diameters: 4.6 x 250 ram, Nagel); mobile phase, 90 mM KI-12PO4 (pH 5.0) containing 10%
methanol; temperature, 55"C; flow rate, 1 ml/min; detection, UV 210 nm. Crn = creatinine; His ffi
L-histidine; Car - carnosine; ~r-meHis = rr-methyl-L-histidine; ~-meHis = r-methyl-L-histidine; Ans
ffi anserine; Bal ffi balenine.
normally below 1 mM in animal muscles. As seen in Table 1, however, several

fishes such as tunas and mackerels, salmon, and carp contain large amount of free
L-histidine in their white muscle. Physiological function of this large amount of
L-histidine has also been obscure. In contrast to these compounds, levels of rr- and
r-methyl-histidine and homocarnosine are very low in animal muscles. Therefore, in
the following, we focus our attention mainly on free L-histidine, carnosine, anserine,
and balenine.
II. Distribution of free L-histidine and related dipeptides in fish tissues

Since the early work of Lukton and Olcotd s, there have been many reports
on the distribution of these dipeptides in fish muscle. Table 2 shows a typical
distribution of these dipeptides in fish and whale muscles containing large amounts
of the dipeptides mainly determined during this decade. Marine pelagic high-speed
swimmers with myoglobin-rich dark flesh, such as tuna, sardine, mackerel, contain
312 H. Abe
TABLE 1
Distribution of histidine-related dipeptides in animal skeletal muscles
Animals Histidine Carnosine Anserine Balenine

Invertebrates 4- 4- 4- 4-
Fish
Elasmobranchii + + 1-,-33 +
Teleostei
Scombrinae 20,,- 120 ~ 10 ~ 120 4-
Salmoninae ~10 + 7~42 4-
Anguillinae + 7-,,25 + +
Cyprininae 4~ 25 + + 4-
Percinae + 4- 4- 4-
Amphibians
Frog + 2~17 + 4-
Reptiles
Snakes + l,-d0 + 2,-,13
Crocodiles, turtles + + 1-,- 7 4-
Birds
Pigeon, chicken + ,-,13 5,,~43 4-
Mammals
Kangaroo + 2"-, 4 8,-,16 4-
Baleen whales + 6-,-13 + 50-~65
Toothed whales + 10~20 1~ 6 16-,,25
Seals + 9,-,26 3,-, 5 4-
Cat, lion + 4~ 16 6~ 18 4-
Ox, bison + 10,-,20 1-~ 4 +
Sheep, goat + 2-,- 8 5~ 9 +
Horse + -~34 + +
Pig + 12~21 -,,1 ~,2
Rabbit + ~2 14-, 19 4-
Deer + ~-4 -,,14 ~4
Rat + 4,-, 6 3,~ 9 4-
Apes + 17---23 + 4-
Human + 2~ 8 ~2 4-
Values are given in/zmol per g muscle weight. + = < 1/zmol g-1 muscle; 4- = trace or not determined.
Data compiled from references 1-3, 6, 7, 20, 21, 23, 72, 75-79, and 82.
copious amounts of free L-histidine in their white muscle. This is an extraordinary

feature absent in other animals thus far examined 44,ts,75. The L-histidine content
in the white muscle ranged from 15 to 94 ?tmol/g in dark-fleshed fishes, from 4
to 20/~mol/g in 'intermediate' fishes, and from 0.07 to 1/~mol/g in white-fleshed
species 3. Carp muscle is an exception in that it contains rather high concentration
of L-histidine 2, though muscle color is white. The level in red muscle of these fishes,
however, is much lower than that of white muscle as seen in Table 2.
Together with L-histidine, carnosine, anserine, and balenine are also observed in
the muscle of various fishes as well as other vertebrate. In contrast to L-histidine,
contents of these histidine-related compounds do not depend on the muscle color 3
but are highly species specific3'23. These dipeptides may be widely distributed in fish
TABLE 2
Free L-histidine and related dipeptides in fish white and red muscles and whale muscle
Classification Species n Muscle His Car Ans Bal

Fishes
Clupeioidei Clupeidae, sardine 3 6 White 39.6 + + +
Red 9.64 + + +
Salmonoidei Salmonidae, salmon 59,69 (4 species) - White 4.59 0.56 24.5 -
Rainbow trout (j)2,4 6 White 0.41 + 16.8 +
Rainbow trout (C) 4 5 White 2.57 + 17.2 +
Red 0.59 + 3.02 +
Osmeridae, smelt (wild) 75 7 White 1.80 + 5.30 +
Osmeridae, smelt (cultured) 6 White 2.38 + 1.59 +
Cyprinoidei C~rinidae, carp 2 3 White 10.1 + + +
Others2(6 species) - White 5.10 + + +
Cobitidae, pond loach 2 3 White 4.52 + <0.16 +
Anguilloidei Anguillidae, Japanese eel 2 10 White 0.37 18.3 <0.3 <0.15
Congridae, conger eel 2 2 White 4.35 5.16 <0.85 <0.3
Exocoetoidei Exocoetidae, flyingfish (A) 3 5 White 31.1 + 8.43 +
Red 1.75 + 1.59 +
Scombroidei Scombridae, tunas 79,s0 (4 species) - White 62.9 + 27.3 -
Red il.0 + 7.92 -
Skipjack tuna 0 ) 3 3 White 89.5 4.52 15.4 +
Red 17.3 0.69 3.99 +
Skipjack tuna (H) 6 5 White 93.5 2.90 51.1 +
Red 13.3 0.56 7.31 +
Kawakawa (H) 6 3 White 75.4 1.09 27.7 +
Red 6.51 0.35 4.48 +
Common mackerel 3 6 White 24.9 <0.24 <0.05 +
Red 9.88 <0.06 + +
Histiophoridae, blue marlin (H) 7 5 White 15.9 2.64 105 +
Red 4.79 + 21.1 +
Carangidae, horse mackerel 3 6 White 11.2 + + +
Red 2.35 + + +
Mugiloidei Sphyraenidae, Japanese barracuda 3 5 White 4.86 + <0.95 +
Whales 7s
Mystacoceti Fin whale 5 Dorsal 0.11 5.74 0.20 61.0
(Baleen whale) Sei whale 5 Dorsal 0.13 9.35 0.47 72.9
Little piked whale 3 Dorsal 0.24 5.92 1.44 78.0
Odontoceti Sperm whale 2 Dorsal 0.18 8.89 4.37 0.10
(Toothed whale) Pilot whale 2 Dorsal 0.17 1.11 1.59 23.0
Values are presented as averages in /zmol g-1 muscle. + = trace; - = not determined. His = L-
histidine; Car = carnosine; Ans = anserine; Bal = balenine. Numbers in parentheses are species
numbers averaged. Place of capture: (A) = Atlantic Ocean; (J) = Japan; (H) = Hawaii. Fishes without
a designation were all caught around Japan.
muscles, a l t h o u g h the distribution of b a l e n i n e may be limited. T h e c o n c e n t r a t i o n s

o f b a l e n i n e a r e v e r y l o w in m o s t fish m u s c l e s , b u t q u i t e h i g h in t h e s k e l e t a l m u s c l e
of several whales, and no systematic differences are apparent between baleen and
toothed whales.
314 H. Abe
In fish red muscle concentrations of histidine-related peptides are generally low,

corresponding to one tenth to one quarter of their concentration in white. As far
as the dipeptide distribution is concerned, the concentrations in white muscle are
closely related to the swimming ability of the fish species.
These compounds in fish muscle showed no significant difference among muscle
portions of the dorsal or ventral muscle, though dorsal muscle showed slightly
higher levels3. Individual difference of the concentration of these compounds is
small in a given species, and, as results for histidine in rainbow trout (Oncorhynchus
mykiss) and for anserine in skipjack tuna (Katsuwonuspelamis) show, rather sub-
stantial differences between regional populations may exist (Table 2). Generally,
fishes belonging to the same suborder group, however, reveal quite similar distribu-
tion patterns of these compounds irrespective of the family as seen in Salmonidae,
Cyprinidae, and Anguillidae2. Other fishes with less pronounced swimming ability
contain much smaller amounts of these compounds in their white muscle.
In contrast to the large amounts of these compounds in fish skeletal muscle,
the levels are extremely low in cardiac muscle as well as smooth muscles, such as
stomach and intestine. In the skipjack tuna, only small amounts of the dipeptides
are found in non-muscular tissue, such as liver, brain, gill, kidney, spleen, pylorie
caecum, and ovary3. Blood levels of these compounds are also very limited and
well below concentrations determined in skeletal muscle tissue. Therefore, the
distributions of these compounds in fish tissues clearly suggest that these compounds
in fish muscle play a role in swimming function and performance.
Among all fishes examined to date, skipjack tuna Katsuwonuspelamis and Pacific
blue marlin Makaira nigricansexemplify one end of the extremes. Live skipjack tuna
(1-2 kg body weight) captured off Hawaii on hook and line showed large amounts
of L-histidine and anserine and lesser amount of carnosinr in their white muscle.
The level of L-histidine was on average 93.5/~mol/g reaching a maximum of 105
/zmol/g, which is highest value measured for all species thus far examined. Ansefine
was similarly abundant, reaching a maximum of about 69.1 ~mol/g, with an average
concentration of 51.1/~mol/g (ref. 6). The total content of these compounds thus
approaches 150/~mol/g in the white muscle.
In contrast to skipjack tuna, Pacific blue marlin (50-100 kg body weight), caught
off Hawaii during the Annual Billfish Tournament, showed the highest anserine
content of all animals thus far examined, with an average value of 105 ~mol/g, and
a maximum of 120/~mol/g (ref. 7). The combined total of histidine and related
compounds in marlin white muscle reached above 120/~mol/g.
The levels in the red muscle of both species, however, amount to 15-30%
of those attained in the white muscle. Fish white muscle is known to be a
highly anaerobic muscle and thought to be employed only during the burst swim-
ming. In contrast, the red muscle is used during, generally slower-speed, sustain-
able swimming33's4. In this connection, tunas arc considered to be the 'ultimate
teleosts '33 because of their ability to reach high burst swimming speeds 25,s7 and
maintain muscle temperatures as high as 10~ above ambient temperatures 25.71 .
The ability to engage in burst swimming of tunas and marlin is sustained by the el-
evated capacities for anaerobic glycolysis in the white muscle, typically represented
Histidine-related dipeptides: distribution, metabolism, and physiologicalfunction 315
by the activity of lactate dehydrogenase, which in tuna is the highest so far found in
any animal 28. During burst swimming, large amounts of lactate, nearly 100 #mol/g
muscle, are accumulated as an end product in tuna white muscle 29.
Therefore, the highest amounts of L-histidine and related dipeptides in the
anaerobic white muscle of tunas and billfishes may indicate that these dipeptides
contribute to the extreme ability for anaerobic burst swimming. A critical analysis
the data collated in Tables 1 and 2 clearly leads to the conclusion of a positive
correlation between the concentration of these dipeptides and the ability of a given
species for the anaerobic exercise such as burst swimming, sprint running, flying,
and diving.
III. Effects of physiological conditions on the dipeptide concentrations in

fish muscle
It may be quite difficult to understand the unknown physiological function of a com-
pound in spite of the unequivocal distribution of the compound in tissue. Fishes,
however, should be most suitable experimental animals to unveil the physiological
role(s) of these dipeptides, because of the wide spectrum of their distribution pat-
terns is found in fish species, fish comprise two functionally and spatially different
muscle types (white and red), and fish can adapt and acclimate to various physio-
logical and ecological environments. Thus, in this section I would like to examine
the effects of physiological conditions on the concentrations of these dipeptides for
obtaining some clues for their physiological roles. A number of independent obser-
vations have been reported in the literature and these may lead to a unified inter-
pretation.
As seen in Table 2, the content of anserine in the white muscle of cultured smelt-
fish Plecoglossusaltivelis is lower than that determined for their feral counterparts.
Hawaiian skipjack tuna contains much higher concentrations of anserine than the
Japanese counterpart. Existing large concentration differences of these dipeptides
in different subpopulation of one species, suggest that environmental factors/life
style factors may play an important role. It has been reported that carnosine in chi-
nook salmon (Oncorhynchus tshawytscha) decreased when the fish were maintained
on a histidine-deficient diet, while anserine did not 47. Similarly, the anserine level
was also shown to be kept fairly constant during spawning migration of sockeye
salmon (Oncorhynchus nerka), at a time when the white muscle concentration of
L-histidine decreased sharply 22,59,92,93.
Because of these substantial concentrations and their obvious contribution to
intramuscular osmotic pressure, the potential role of these compounds in os-
moregulation in fish muscle needs to be examined. As shown in Fig. 3, carnosine
concentrations in the muscle of Japanese eels (Anguillajaponica) were largely in-
dependent of the external salinity 9,36, though L-histidine was slightly increased in
fish adapted to half-strength and full-strength seawater. Along with the increase
of external salinity total amino acids in the white muscle of rainbow trout (On-
corhynchus mykiss) increased about two-fold, mainly accounted for by increases in
316 H. Abe
2.0r w ", = = 125 i i i ...... = ='|

'/ / I I' Eel-Car 'i
/ ~ 15 ir~T Trout,_~Ar~
3
o
9 r/
//Trout-His\
l, okX..Imx
/ / a -H"
,.
/9 s -
(--) i (~.-9Eel=His
9. n ~ 0l i ' i , ,
FW SW FW FW SW FW
Fig. 3. Changes of L-histidine and dipeptides in the muscle of eel, rainbow trout, and Japanese dace
during the acclimation to sea water. Mean and the standard deviation of three fish are shown. FW =
freshwater; SW = seawater. See the legend of Fig. 2 for additional abbreviations. Redrawn from ref. 9.
non-imidazole amino acids such as glyeine and alanine 9. Histidine as well as lysine
and arginine also increased about 5-fold of the fresh water level in half-strength
seawater and seawater (Fig. 3). These increases in histidine, however, correspond
to only 4% of the total augmentation of the tissue amino acid pool. Anserine in
trout white muscle was maintained at an almost constant level during the seawater
acclimation, although it was slightly increased in seawater-adapted specimens. This
is also the case for histidine in a cyprinid, Japanese dace Leuciscus haRonennsis.
Because eel and trout contain fairly large amounts of carnosine and anserine,
respectively, relative to the total amino acid pool, it might be predicted that these
dipeptides contribute to the basal tissue osmotic pressure. The contribution of
the dipeptides to osmoregulation of the white muscle is, however, likely to be
negligible.
Fish usually show appreciably high tolerance to prolonged starvation, and an
analysis on changes in histidine and dipeptide behavior during starvation may
deliver important clues as to the general function of these compounds in teleostean
fishes. During the starvation of eel and rainbow trout for 200 and 62 days,
respectively, white muscle L-histidine decreased significantly in both species9 as
depicted in Fig. 4. This is also the case for Japanese dace (Leuciscus hakonennsis), a
species containing large amounts of free L-histidine9. Camosine in eel and anserine
in trout, on the Other hand, showed significantly small changes during starvation,
with eamosine in eel gradually decreasing after 100 days starvation. The decrease of
free L-histidine may be related to this compound serving as a direct energy source
for skeletal muscle itself during starvation. As described above, Mommsen et al. 59
showed that free L-histidine in white muscle of sockeye salmon plummeted to one
2.5 0.5
2.0 0.4 ~
9 I:" == I - I-I ir
lo .
1.0 - -* -0.2 "~
....... I ~E
4-,
"0"5
C: r '\. 4r~ "~:q 0'1~"0E
o $" -e u
u 0 , n 0
25 l I 'l I I I
L -
E15 ., /o
m . Trout-
":I0 /
0
4"OC5"
U ~-*"~"*Dace-His -
0 I I I I I I
0 40 80 120 160 200
Days
Fig. 4. Changes of the level of L-histidine and dipeptides in the white muscle of eel, rainbow trout, and
Japanese dace during starvation. Mean and the standard deviation for three fish are shown. Redrawn
from ref. 9.
tenth of control values during the spawning migration upstream, while the anserine
content did not change at all. This may be due to the forced starvation of this
species during the prolonged upstream migration.
Much more dramatic changes with starvation are seen in skipjack tuna 6, a species
living in the fast lane compared with the trout, eel or dace. Table 3 shows L-histidine
and related dipeptides in white and red muscles of starved tuna. The well-fed
controls just after catch are the same individuals as shown in Table 2. Individual
difference in the concentration of each compound was rather small for control tuna,
and the combined total pool size in the muscle was quite similar among individuals.
Although during starvation rather large individual difference in each compound is
found, histidine levels in white muscle decreased significantly on average compared
to those in controls, suggesting this amino acid is utilized as an energy source or a
glucose source during starvation.
318 H. Abe
TABLE 3
Effect of starvation on the levels of L-histidine and related dipeptides in white and red muscle of
skipjack tuna (Katsuwonuspelamis)
Days after White muscle Red muscle

capture His Car Arts Total His Car Ans Total
Control 0 93.5 2.90 51.1 147.4 13.3 0.556 7.31 21.3
(n = 5) 4-9.2 4-1.66 4-10.2 4-3.5 4-1.1 4-0.299 4-1.28 4-2.1
Tuna no.
1 1 79.9 14.7 19.1 113.7 1.05 0.439 0.580 2.07
2 1 87.7 5.89 40.6 134.3 6.59 0.994 5.51 13.1
3 2 74.1 4.94 47.8 126.8 8.67 0.713 9.12 18.6
4 2 69.1 12.1 34.3 111.5 10.9 2.66 7.91 21.5
5 2 40.3 7.70 38.3 86.3 7.21 1.57 8.90 18.0
6 3 65.3 13.9 36.5 115.7 9.71 3.04 9.00 21.8
7 5 31.5 10.6 64.0 106.1 3.92 1.96 9.78 16.2
8 12 3.46 24.9 58.8 87.2 3.59 4.86 13.3 21.9
Mean 56.4 11.8 42.4 110.7 6.46 2.03 8.01 16.7
4- SD 4-26.9" 4-5.9' 4-13.4 4-16.0"* 4-3.16"* 4-1.37" 4-3.46 4-6.2
Average values given in/zmol g-l muscle; * = p < 0.01; ** ffi p < 0.001. From ref. 6.
Carnosine, on the other hand, showed a marked increase, whereas anserine

showed no large changes. Tunas 7 and 8, which were starved for 5 and 12 days
after capture, respectively, showed an extremely large decrease in histidine and an
increase in carnosine, suggesting at least a part of histidine appears to be converted
into carnosine and anserine. Anserine concentrations were practically identical to
those in the controls, though it decreased at the initial stage of starvation. This
tendency was also observed for skipjack tuna red muscle. Kawakawa (Euthynnus
affinis), a species closely related to skipjack tuna, also showed similar results, though
the degree of change with starvation was somewhat attenuated 6.
The total concentration of these compounds was rather similar among individuals
even during starvation, decreasing by 30-40% of the control fish. Thus, a metabolic
response to starvation appears to be the defence of the total pool size of histidine-
related compounds. This implies that the physiological roles of free L-histidine
accumulated specifically in tuna white muscle - as well as other scombroid fishes
- is the same as those of the related dipeptides. The drawback of free L-histidine is
that, as described above, it is more metabolically reactive than the dipeptides. That
is presumably why the conversion of histidine into more metabolically inert forms,
such as carnosine and anserine, is necessary.
Histidine and its related dipeptides show only minor changes during exhaustion,
an observation that holds for tuna muscle 6 as well as for the rat 2~
With increasing body mass, large accumulation of histidine and anserine occur/ed
in the early stages of growth of carp and trout, respectively 4. Irrespective of the
actual compound, these substances increase with growth of the fish and reach a
plateau well before their adult size for both species. Similar data have also been
obtained from mammals and birds 26'3~
TABLE 4
Half-lives of NC-histidine in rainbow trout (Oncorhynchus mykiss) and skipjack tuna (Katsuwonus
pelamis)
Tissue Species Half-life (h)

at 4"C a at 14"C b at 24"C a
Blood Rainbow trout 2.0 1.0
Skipjack tuna 1.4 0.72
White muscle Rainbow trout 181 90
Skipjack tuna 132 66
Red muscle Skipjack tuna 105 53
a Measured at the given temperature.
b Calculated for 14"C, using an estimated value for Q l0 of 2.0. Compiled from references 6 and 8.
ll:. Metabolic turnover of L-histidine in fish

From the above considerations, it is expected that the turnover rate of L-histidine in
fish muscle is fast and that of dipeptides is slow. Therefore, the metabolic turnover
of 14C-histidine was examined on the blood and muscle of skipjack tuna and
rainbow trout 6,8. In Table 4, metabolic half-lives of 14C-histidine are summarized.
As the experimental temperatures were 4 and 24"C for trout and tuna, respectively,
calculated half-lives at 14"C are also shown in Table 4 after making a simple Q10
correction of 2.
After bolus intraarterial injection of 14C-labeled histidine into the aorta of trout
and tuna, the label was rapidly removed from blood in both species. The metabolic
half-lives were 2.0 4- 0.17 and 0.72 4- 0.124 h for trout and tuna, respectively. This
difference between the two species disappears if one makes a simple Q10 correction,
in which case the half-life of histidine in tuna is longer than that in trout. These
results suggest that the uptake of the amino acid by the tissues is fairly rapid. At the
same time, histidine washout from trout white muscle after intramuscular injection
and absorption from the peritoneal cavity after intraperitoneal injection is also
found to be fairly fast in trout.
In free swimming tuna, if the label was administered into white muscle, the
decay of the radioactivity from the muscle was slow, whereas the label was taken
up by red muscle promptly after injection (Fig. 5). The metabolic half-lives of
L-histidine in white and red muscles were 66 and 53 h, respectively, which implies
much faster turnover in tuna than in trout muscle (181 h). Again, this difference
is temperature dependent and disappears after the above temperature correction
to 14"C, resulting in a slower rate for the tuna. Thus, the half-life of L-histidine
in muscle may be almost the same irrespective of the level of the amino acid in
the muscle. Since skipjack tuna maintains muscle temperatures significantly above
ambient temperatures, this fast turnover in tuna muscle suggests that histidine
metabolism is probably quite rapid under the normal conditions of this species at
sea. These muscle turnovers in fish, on the other hand, are much slower than those
in rat gastroenemius muscle, showing a half-life of 3.6 h (ref. 82).
320 H. Abe
I - " I ............. I - I
m
A m
B
IO
'o
~4
X
D
o,
, I ....... I i, i
0 20 40 60 80 ! O0 0 20 40 60 80 1 O0
TIME (11)
Fig. 5. Total and specific radioactivities in white and red muscles of skipjack tuna after intramuscular
injection of L-[U-14C]histidine (30 #Ci/tuna). Each point represents one skipjack tuna. A -- white
muscle; B -- red muscle. O - Total activity; O--" specific radioactivity of histidine. From ref. 6.
Metabolic turnovers of the dipeptides have not been examined for fish muscle,
but the half-lives of the dipeptides were reported to be of the order of 3 weeks in rat
muscle 31,s2 and 4 weeks in chick31, respectively. Judging from the rate of synthesis de-
scribed later, the turnover of the dipeptides in fish muscle is also expected to be slow.
E~Metabolism and interorgan transport of the dipeptides in fish

Metabolism of histidine-related dipeptides has been examined mainly on terrestrial
animals and little information has been available on fish. After intramuscular
injection of 14C-histidine into trout, the label was gradually incorporated into
muscle anserine s as shown in Fig. 6. Fourteen days after injection, the specific
radioactivity of anserinc reached the same level of that of histidine. In this case,
the incorporation of the label into r was small, but increased after 14C-
/5-alanine injection intramuscularly. The radioactivity of r was highest
one day after 14C-fl-alaninc injection and decreased gradually within eight days,
accompanied by the increase in anserine radioactivity.
As the conclusion of these experiments, therefore, the biosynthesis of anser-
ine should occur through carnosine as an intermediate step (Fig. 7). Carnosine
biosynthesis from histidine and fl-alanine was also confirmed in eel muscle ~. These
1.6 e ~. . . . i' "l e i I
1.4
A
~1.2
.,,,.
-1.-
~)
Zl.0
i-,
0.8 J
I,-
U
9< 0.6
0.4
" I,
0
T--
~o.2
, I I I i I
0 2 4 6 8 10 12 14 16 18
TIME (days)
Fig. 6. Ratio of specific radioactivity of anserine to that of L-histidine in the white muscle of rainbow
trout after intramuscular injection of L-[UJ4C]histidine (2/xCi/trout). Mean and the standard deviation
of three trout are shown on each day. From ref. 8.
experiments also revealed that the rate-limiting factor for the biosynthesis of the
dipeptides is the #-alanine level in muscle as is the case in mouse 55 and rat 82.
Anserine biosynthesis has been known to occur via two or three different pathways
in chick and rat. These pathways are: (1) direct condensation of rr-methyl-L-histidine
with fl-alanine by a carnosine synthetase-like enzymea1,41,s2,ss; (2) N-methylation of
carnosine by carnosine N-methyltransferaseS6; or (3) fl-alanyl transfer from carno-
sine to Jr-methyl-L-histidine. The biosynthetic pathway of anserine, however, is as
yet controversial even in terrestrial animals. At least in trout, the main pathway of
anserine biosynthesis may follow pathway (2) as described above.
The major organ responsible for the biosynthesis of the dipeptides is confirmed
to be skeletal muscle in chick and rat. As mentioned above, carnosine biosynthesis
also occurred in the white muscle of eel 86. Similarly, 14C-histidine was incorporated
into earnosine and anserine only in white and red muscle of trout and tuna 6,8. At
least in skipjack tuna, the rate of incorporation of the label into anserine is higher
in white muscle than red. Thus it can be concluded that the biosynthesis of these
dipeptides in the skeletal muscle is a generalized feature in vertebrate animals.
These dipeptides are known to be catabolized by carnosinase and anserinase (see
Fig. 7) and degraded to the corresponding constituents, histidine and methyl-L-
histidine. Anserinase is known to occur in the skeletal muscle of a codling (Gadus
322 H. Abe
Glucose
Glutamate
CO 2 + H 2 0
Histidlne ~:-Methylhistidine
Carnosine .... ~ Anserine
Fig. 7. Metabolic pathways of L-histidine-related compounds in fish. 1 = carnosine synthetase (EC

6.3.2.11); 2 = carnosinase (EC 3.4.13.3); 3 = carnosine N-methyltransferase (EC 2.1.1.22); 4 = anseri-
nase (EC 3.4.13.5).
callarias) 4~ Lenney and coworkers 46 purified N-acetyl-L-histidine deacetylase, which

is now considered to be identical to anserinase, from the brain of skipjack tuna and
showed the enzyme also acts on carnosine and anserine. In addition, the activity
of this enzyme was found in tuna ocular muscle, but not in the skeletal muscle. In
codling, however, weak activity of the enzyme was also located in skeletal muscle.
The properties of this enzyme are similar to those of hog kidney carnosinase 45.
Trout anserinase activity is highest in brain followed by eye and kidney94. The
activity in kidney is reported to be high in cod, pollack, smeltfish, and trout, but
undetectable in tuna, flounder, carp, and sardine sl. Also in mammals and birds,
the activities of carnosinase and anserinase are fairly high in kidney and olfactory
mucosa and low in liver and muscle 53,54,9~
As shown below for the rainbow trout, when large amounts of carnosine or anser-
ine are injected into white muscle, these dipeptides are degraded into L-histidine
and ~r-methyl-L-histidine, respectively. In contrast to the synthetic pathway of
anserine which involves carnosine as an intermediate, anserine is not catabolized v/a
carnosine to L-histidine 5. Major organs responsible for the dipeptide degradation
may be the kidney and the liver, even in fishs,6,s. Although methyl-L-histidine pro-
duced from anserine and balenine decomposition must be excreted without further
reutilization, L-histidine is known to be degraded to glutamic acid v/a urocanic
acid 42 and reutilized for energy or glucose sources or for protein synthesis (see Fig.
7). The activity of histidine degrading enzymes is also high in kidney and liver and
low or undetectable in muscle 42.
In fish, the organs responsible for the biosynthesis and degradation of these
dipeptides are different as described above. Thus, these facts give rise to interesting
problems concerning the interorgan transports of these dipeptides. As already
shown in Fig. 5, labeled histidine administered into tuna white muscle is promptly
transported into red muscle. The label was also, as shown above, washed out quickly
from trout muscle and intraperitoneal cavity into blood and from blood into tissues.
If the levels of these dipeptides which accumulated in large amounts in fish muscle
are closely regulated in the muscle, it would be anticipated that after administration
of large amounts of these dipeptides into muscle they may be decomposed in situ
or washed out into blood, transported to several other organs, and finally decom-
posed or excreted. To test this hypothesis, large amounts of carnosine and anserine
were injected intramuscularly into white muscle of small trout and their respective
metabolic fates were traced 5. Fig. 8 gives the results from anserine administration.
Almost the same data were obtained from carnosine injection.
60 c ~ r ' ~ r - - ' l ~ ~ / / - r ' e ' n - H - 1 - 1 , , , i ,~,', ,',', ,',',
RM
~ 20
z O~ ' ' ' ',;," "b" //"~

_o
~ 1 4 , k " ~ i ' ~ r ~ r ~ r -t,"r-r
An l
Z' ll I
2 4 6 8 24 96 240 2 4 6 8 24 96 240
TIME(h)
Fig. 8. Interorgan transport of anserine in rainbow trout and the degradation into 7r-methyl-L-histidine
after intramuscular injection of 100 rag/trout of anserine. WMA = anterior white muscle (injection
port); WMP - posterior white muscle; RM -- red muscle. Ans -- anserine; 7r-meHis -- 7r-methyl-L-
histidine. Each point represents one trout. Modified from ref. 5.
324 H. Abe
Anserine in the anterior white muscle (injection port) showed a sharp decline
2 min after injection and returned to the control level after 24 h. In the posterior
white muscle, however, the anserine level did not change and remained at a slightly
higher level than in the control. Red muscle anserine was about twice as high
20 min after injection, but decreased to the control level after 7 h. This fact
is consistent with that for tuna mentioned above. These were also the ease for
camosine injection.
After anserine injection, the levels of eamosine and L-histidine did not change
throughout the experimental period in all tissues and blood, suggesting anserine
is not degraded via carnosine as mentioned above. However, lr-methyl-L-histidine
increased about 10-fold in each muscle. The rate of this increase was rather rapid
in red muscle. Just after injection as shown in Fig. 8, a large amount of anserine
was washed out into blood and the level sharply declined to the control level after 5
h. The anserine leached out was incorporated into kidney, reaching a maximum 20
rain after injection. The incorporation was less pronounced in liver.
The anserine incorporated into kidney was immediately degraded to ~r-methyl-
L-histidine which reached a maximum 5 h after injection and kept this level until at
least 24 h after injection. Thus, kidney rather than liver seems to be responsible for
the catabolism of the dipeptides. The pattern of increase and decrease of 7r-methyl-
L-histidine was almost identical among liver, red muscle and blood, suggesting the
incorporation of the compound produced in kidney via blood.
In contrast to breakdown of anserine, carnosine may even be catabolized in
situ in muscle because the L-histidine level increased in every muscle prior to the
increase in kidney. These data clearly indicate that carnosine and anserine levels
must be closely regulated metabolically in fish white and red muscle and there may
be a close relationship between white and red muscle of fish. Therefore, large part
of L-histidine derived from fish diets may be absorbed from intestine into blood
and transported to several tissues and used for protein or energy sources. A small
part of the amino acid, however, may be incorporated into dipeptides in muscle and
accumulated in situ.
VI. Physiological functions of histidine-related dipeptides as proton

buffers
Many efforts have been carried out to date in order to clarify the physiological
functions of these dipeptides mainly for terrestrial animals. Earlier work has been
reviewed by Crush 23 and Christman 21. Five lines of evidence have been obtained
from these efforts:
(1) direct activators of some enzymes in the glycolytic pathway;

(2) enhancers for Ca2+-sensitive systems such as myofibriUar Mg2+-ATPase;
(3) oxygen or copper transporters by chelating action of these dipeptides;
(4) free radical scavengers;
(5) buffering components for protons.
TABLE 5
Apparent pK values of the histidine imidazole group in various naturally occurring compounds
Compound Temperature pK
(*c)
lmidazole 20 7.23
L-Histidine 20 6.21
~r-MethyI-L-histidine 20 6.62
r-MethyI-L-histidine 20 5.98
Carnosine 20 7.01
Anserine 20 7.15
Balenine 20 6.93
Homoearnosine 20 7.10
"'Bjpical" histidyl-imidazole in proteins 25 6.5
Adjacent to acidic ( - ) group 25 7-8
Adjacent to basic (+) group 25 5-6
Compiled from references 10 and 35.
Of these functions, we have focussed on the buffering capacity of these com-

pounds accumulated in large amounts in vertebrate skeletal muscle.
Maintaining the intracellular pH of skeletal muscle is critically important during
the anaerobic burst locomotion of the animals, because otherwise the fall of pH due
to proton production as a result of ATP consumption 34 will cause glycolytic enzymes
to cease their actions 35. Since the early work of Bate-Smith ~3, the importance
of non-bicarbonate buffering of vertebrate muscle has been emphasized by many
researchers 7,17.19,24,35,70,74.
The intracellular buffering capacity in vertebrate muscle is dominated by the
imidazole group of L-histidine, which occurs as histidine residues on protein, as
free L-histidine, and as histidine-related dipeptides. One of the two nitrogens of
imidazole ring on histidine molecule can be protonated at physiological pH ranges
and act as a buffering component for protons produced during the anaerobic muscle
exercise. Table 5 represents the apparent pK values determined on histidine-related
compounds at 20~ All the p K values fell into the range of pH 6-7.3, indicating
the appropriate p K characteristics for buffering constituents in the physiological pH
range which is pH 6.8-7.2 at 20~ in the muscle cytosol 7~
Other than the free imidazole compounds, histidine residues on proteins and
inorganic phosphate are considered to be good buffers (Table 5). Other candidates
are organic phosphate compounds such as nucleotides and sugar phosphates and
several organic acids such as citrate and succinate only when they exist in large
amounts in muscle cytosol.
In aerobic exercise, protons produced along with the ATP consumption are
turned over through oxidative phosphorylation and not accumulated in the intra-
cellular fluid 34. In anaerobic metabolism during 02 limitation typically occurred in
the fast-twitch white muscle of fishes, however, large quantities of proton would
accumulate and lower the muscle pH 34,35. Consequently, all the animals performing
anaerobic exercise must evolve the means maintaining the muscle pH as far as
326 H. Abe
possible to enhance the anaerobic capacity. This is the reason why fish white muscle
have a higher buffering capacity compared to other tissues such as slow-twitched
red muscle.
Proton buffering capacity (termed fl value) due to non-bicarbonate buffering
compounds was defined as the /~moles of sodium hydroxide required per gram
tissue to change the p H by one unit 7,13,19. This unit is termed a 'slyke' based on
the original technique of Van Slyke 85. Buffering capacities of several vertebrate
muscles are listed in Table 6. Individual variation of the buffering capacity in a
species was small compared to the large variations in interspecific comparisons.
T h e buffering capacity was typically high in the muscle of marine diving m a m m a l s
TABLE 6
Buffering capacity (fl) of skeletal muscles of marine and terrestrial mamals and birds and various fishes
Species n Buffering capacity

(/~mol NaOH/pH. g muscle; pH 6-7)
Marine mammals and birds
Little piked whale 1 111
Spotter porpoise 5 84.1
Northern fur seal 1 79.2
Harbor seal 4 76.2
Weddell seal 4 72.1
Sea otter adult 3 70.6
Adelie penguin 1 70.0
California sea lion 2 61.2
California gray whale calf 1 47.5
Terrestrial mammals and birds
Chicken, pectoralis minor 1 82.8
Pig, psoas muscle 1 78.6
Pig, biceps femoris 1 63.2
Ox, biceps femoris 1 69.0
Rabbit 3 66.9
Dog 50.2
Warm-bodied fishes and related species
Skipjack tuna, white muscle 5 136
Skipjack tuna, red muscle 5 68.8
Frigate mackerel, white muscle 109
Albacore, white muscle 107
Yellowfin, white muscle 105
Black skipjack, white muscle 102
Blue marlin, white muscle 5 96.4
Blue marlin, red muscle 5 56.8
Pelagic ~ h e s
Common mackerel, white muscle 5 81.3
Common mackerel, red muscle 5 51.2
Rainbow trout, white muscle 5 63.3
Rainbow trout, red muscle 5 30.1
Mean of 11 species, white muscle 63.3
Deep sea fishes
Mean of 9 species, white muscle 46.0
Data compiled from references 7, 19, and 64.
and warm-bodied tuna fishes. Marine mammals showed a higher average fl value
than terrestrial mammals. This may relate to the anaerobic capacity for prolonged
breathhold diving. Castellini and Somero 19 clearly revealed that the buffering
capacity of mammalian muscle correlated strongly with muscle myoglobin content,
which is more abundant in the muscle of marine mammals. The capacity also
correlated with muscle lactate dehydrogenase activity 19.
Among the fish species listed in Table 6, the white skeletal muscle of warm-
bodied tuna fishes and related species, which are noted for their extremely high
burst swimming abilities, showed the highest buffering capacity. These fishes possess
the highest muscle lactate dehydrogenase activities as well as the highest muscle
buffering capacities in any animals ever been known 19. Deep sea fish species on
average, on the other hand, possess markedly lower buffering capacities and the
lactate dehydrogenase activities in their muscle. Pelagic fishes, actively swimming
predators and foragers such as mackerel and trout, showed the intermediate value
in buffering capacity and lactate dehydrogenase activity between the other two fish
groups. Intraspecific difference of buffering capacity is again very small compared
to the large interspecific variations.
Aerobic slow-twitch red muscle is found to have lower buffering capacities than
white muscle from the same species as shown in Table 6 for skipjack tuna, blue
marlin, common Japanese mackerel (Scomberjaponicus), and rainbow trout. The
capacity in red muscle is almost half of that in white muscle. These data clearly
indicated that the non-bicarbonate buffering capacity in animal skeletal muscle is
strongly correlated with the anaerobic locomotory ability of the animal species and
the anaerobic capacity of the muscle.
The buffering capacities and carnosine content in human skeletal muscle were
also reported to be significantly higher for anaerobically trained elite athletes
(sprinters and rowers) than more aerobically trained marathoners or untrained
men 66. Thus, the high buffering capacity in the vertebrate skeletal muscle is
considered to be a universal strategy for enhancing the anaerobic exercise capability
beyond the species.
In view of the correlation between buffeting capacity and anaerobic capability, it
is appropriate to know what compounds in the vertebrate skeletal muscle actually
participate in the elevation of the muscle buffering capacity. Fig. 9 represents
the brief summary of muscle homogenate buffering and the major compounds
contributing to the buffering in the pH range of 6.5-7.5. Almost the same tendency
was also the case for the pH range 6.0-7.0. Contribution of contractile proteins is as
low as several percent to the homogenate buffering. Histidine residues in the muscle
soluble proteins, however, contribute from 9 to 38% relative to the homogenate
buffering. This contribution is rather high for dark-fleshed fish muscle containing
much myoglobin, such as tuna and mackerel. Not all of the histidine residues in the
native proteins, however, can contribute to the total muscle buffering 64.
Buffering capacities of histidine-related compounds of skipjack tuna and marlin
white muscle are as high as 40 and 60%, respectively, relative to their muscle
buffering. This is also the case for whale skeletal muscle in which case the con-
tribution was about 25%. The ratio is also high for bovine, porcine, and chicken
328 H. Abe
Fig. 9. Contributions of proteins, inorganic phosphate, and histidine-related compounds to the buffering
capacity of muscle homogenate from several vertebrates. Buffering capacity is represented as #mol
NaOH required per g of muscle to change the pH by one unit over 6.5-7.5. WM = white muscle; RM =
red muscle; SM = skeletal muscle; BF - biceps femoris; PSM = psoas muscle; PM = pectoralis minor.
From ref. 64.
muscle, ranging from 12 to 23%, but only 1 to 6% for carp and flounder white
muscle containing below 10/zmol/g muscle of histidine-related compounds. For red
muscle, however, the ratio is much lower than that for corresponding white muscle,
as seen in tuna and martin red muscle.
The ratio of inorganic phosphate buffering to total muscle buffeting capacity in
tuna and marlin white muscle and whale skeletal muscle is relatively low, while in
the white muscle of trout, carp, and flounder which contain only small amounts of
histidine-related compounds the ratio is significantly high, ranging from 50 to 80%.
This may stem from rather species independent content of inorganic phosphate in
muscle. The phosphate buffering, however, would be overestimated because inor-
ganic phosphate is liberated from organic phosphate, such as creatine phosphate
or ATP during the postmortem changes of animal muscle. The contribution of
total buffering capacities of histidine-related compounds and inorganic phosphate
to muscle homogenate buffeting is from 50 to 83%, except for mackerel red muscle
and the muscle of whale, ox, and pig in which mammalian case rather high con-
tribution is attributed to unknown compounds. These unknown compounds may
comprise some nucleotides, organic acids, and/or taurine 64.
As no large difference was observed in the phosphate concentration and buffer-
ing capacity among animal muscles thus far examined, inorganic phosphate may
contribute to the basic muscle buffering together with the muscle proteins. Thus,
the large variation of muscle buffering capacity would mainly be attributable to the
levels of histidine-related compounds in animal muscle. These data and the previous
d a t a 7,19,24,37 clearly indicate that the accumulation of histidine-related compounds
in animal muscle enlarge the muscle buffering capacity and therefore the muscle
anaerobic capability. This is realized in some animals such as tunas, billfishes, and
marine mammals.
Judging from these results, it is apparent that the large amounts of histidine-
related dipeptides in fish muscle enhance the muscle buffering capacity. However,
every fish as well as invertebrate contains small amounts of all of these dipeptides
in their muscles (unpublished data). Moreover, we cannot find any clear difference
among the properties as buffer constituent of these three dipeptides 1~ Thus, we
can conclude that these dipeptides have some other physiological roles related to
the muscle functions and several animals adapted to the anaerobic muscle exercise
have evolved to select and accumulate one or some of them for the muscle buffer
components.
Several other possible physiological roles mentioned above are worthy of atten-
tion from these considerations. These dipeptides may play a role in the regulation
of glycolytic enzymes 37 and myofibrillar ATPase 12,65, and in the stimulation of con-
traction of muscle fibers 15,6a. Other workers also suggested that as a chelator these
dipeptides are involved in the intracellular transport of copper for activation of
cytochrome oxidase and in the regulation of anaerobic glycolysis 16.
More recently, N-acetylated forms of histidine related compounds were found to
be distributed together with carnosine and anserine in cardiac and skeletal muscles,
brain, and eye lens of several vertebrates 61-63. In vertebrate brain, carnosine has
been known to be a neurotransmitter in the olfactory system 5~ In the animal
skeletal and cardiac muscle, these dipeptides and their N-acetylated forms are
attracting much attention due to their ability to enhance the calcium sensitivity
of contractile proteins 32,58, their t~-adrenergic antagonist properties 57, and their
scavenging activity for oxygen free-radicals 49. Careful considerations should be
given to these attractive hypotheses in the future.
VII. References
1. Abe, H. Determination of L-histidine-related compounds in fish muscles using high-performance
liquid chromatography. Bull. Jan. Soc. Sci. Fish. 47: 139, 1981.
2. Abe, H. Distribution of free L-histidine and related dipeptides in the muscle of fresh-water fishes.
3. Abe, H. Distribution of free L-histidine and its related compounds in marine fishes. Bull. Jpn. Soc.
Sci. Fish. 49: 1683-1687, 1983.
4. Abe, H. Effect of growth on the concentration of L-histidine and anserine in the white muscle of
carp and rainbow trout. Bull. Jpn. Soc. Sci. Fish. 53: 1657-1661, 1987.
5. Abe, H. Interorgan transport and catabolism of carnosine and anserine in rainbow trout. Comp.
6. Abe, H., R.W. Brill and P.W. Hochachka. Metabolism of L-histidine, carnosine, and anserine in
skipjack tuna. Physiol. Zool. 59: 439-450, 1986.
7. Abe, H., G.P. Dobson, U. Hoeger and W.S. Parkhouse. Role of histidine-related compounds to
intraceUular buffering in fish skeletal muscle. Am. J. Physiol. 249: R449-R454, 1985.
8. Abe, H. and P.W. Hochachka. Turnover of 14C-labelled L-histidine and its incorporation into
carnosine and anserine in rainbow trout. Bull. Jpn. Soc. Sci. Fish. 53: 1089-1094, 1987.
330 H. Abe
9. Abe, H. and S. Ohmama. Effect of starvation and seawater acclimation on the concentration of free
L-histidine and related dipeptides in the muscle of eel, rainbow trout and Japanese dace. Comp.
10. Abe, H. and E. Okuma. Effect of temperature on the buffering capacities of histidine-related
compounds and fish skeletal muscle. Bull. 7p.n. Soc. Sci. Fish. 57: 2101-2107, 1991.
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muskulatur. Z. Physiol. Chem. 183: 1-10, 1929.
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336-343, 1938.
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Biol. Chem. 35: 263-276, 1918.
15. Boldyrev, A.A. and V.B. Petukhov. Localization of carnosine effect on the fatigued muscle prepara-
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16. Brown, C.E. Interactions among carnosine, anserine, ophidine and copper in biochemical adapta-
tion. Z. Theor. Biol. 88: 245-256, 1981.
17. Burton, R.E Intracellular buffering. Respir. Physiol. 33: 51-58, 1978.
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extractives.Anal. Biochem. 39: 181-187, 1971.
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24. Davey, C.L. The significance of carnosine and anserine in striated skeletal muscle. Arch. Biochem.
Biophys. 89: 303-308, 1960.
25. Dizon, A.E., R.W. BriU and H.S.H. Yuen. Correlations between environment, physiology, and
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edited by G.D. Sharp and A.W. Dizon, New York, Academic Press, pp. 233-259, 1978.
26. Fisher, D.E., J.E Amend and D.H. Strumeyer. Anserine and carnosine in chicks, rat pups, and
duckling: comparative ontogenetic observations. Comp. Biochem. Physiol. 56B: 367-370, 1977.
27. Gulewitsch, W. and S. Amiradzibi. Ober das Carnosine, eine neue organische Base des Fleischex-
traktes. Bet. Dtsch. Chem. Ges. 33: 1902-1908, 1900.
28. Guppy, M. and P.W. Hochachka. Controlling the highest lactate dehydrogenase activity known in
nature. Am. J. Physiol. 234: R136-R140, 1978.
29. Guppy, M., W.C. Hulbert and P.W. Hochachka. Metabolic sources of heat and power in tuna
muscles. II. Enzyme and metabolite profiles. Y. E~. Biol. 82: 303-320, 1979.
30. Hama, T., N. Tamaki, H. lizumi and M. Kita. Observation on the changes of ~-alanine, anserine and
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9 1995ElsevierScienceB.V.All rightsreserved.
CHAPTER 15
The metabolic consequences of body size
EDWARD M. GOOLISH
National Oceanic and Atmospheric Administration, Southwest Fisheries Science Center, La Jolla,
CA 92038, U.S.A. and Scripps Institution of Oceanography, Center for Marine Biotechnology and
Biomedicine, University of California, San Diego, La JoUa, CA 92093, U.S.A.
I. Introduction
II. Theoretical explanations for the negative allometry of aerobic metabolism
1. Muscle contraction time
2. The effect of body size on the rate of oxygen delivery
III. Symmorphosisand the scaling of individual respiratory traits
1. Blood oxygen carrying capacity
2. Gill surface area
3. Cardiac output
4. Tissue respiration and enzyme activity
IV. Tissue-levelexceptions to negative allometry in aerobic metabolism
V. The scaling of maximum whole-body aerobic capacity
VI. Aerobicallometry: a comparison with hypoxia
VII. Effects of body size on anaerobic metabolism
1. Enzymatic evidence
2. Power requirements during burst swimming
3. Constraints on anaerobic scaling
VIII. Behavioral and ecological implications of metabolic scaling
Acknowledgements
IX. References
I. I n t r o d u c t i o n
How should the body size of an organism affect the metabolic biochemistry oc-
curring within individual cells? There are no obvious direct effects of body size
on cell metabolism, since the size of individual cells remains relatively unchanged.
What does change with increased body size, however, is the interaction between the
cells and the environment. With larger body size cellular metabolism is increasingly
removed from the environment by distance and, therefore, also by time. In his
analysis of the interaction between animal metabolism and the environment, EE.J.
Fry 37 defined as limiting factors those identities which govern metabolic rate by
restricting the supply or removal of materials in the metabolic chain. In the case of
aerobic metabolism this would include such things as oxygen, CO2, and substrate
(e.g. glucose). The distancing between the cell and environment which occurs with
increased body size will result in a decreased rate of delivery of these limiting
factors. The purpose of this review is to discuss how this limitation in the delivery
336 E.M. Goolish
of materials can be expected to influence the physiology and cellular metabolism of

fish.
II. Theoretical explanations for the negative allometry of aerobic

metabolism
The scaling of aerobic metabolism has been the subject of extensive theoretical
and empirical study 14,11~ It is not my intent here to review this material in
detail, however some theoretical overview is necessary to understand the patterns
of metabolic allometry in fish and how they are different from other groups of
animals. Many issues remain controversial, even including what the actual value
is for the scaling exponent b (where 17o2 ffi a Weightb). Peters 11~ analyzed a data
set of 146 mammalian species and obtained a mean intraspeeific value for b of
0.74, confirming the 3/4 law or Kleiber's rules3. Heusner 62 examined a similar set of
data for mammals and concluded that the actual interspecific value for b is 0.68,
in accordance with theories based on surface area. The available information for
fish is summarized in Fig. 1 as a frequency distribution of the intraspecific scaring
exponent b for standard, i.e. basal, metabolism. The data are taken from early
reviews ~,7s,~~176176 together with new values reported over the last two decades
as referenced in the legend. In studies where estimates of b were obtained under
varied conditions (such as at different temperatures, salinities or rations), each
value of b was included individually. The mean of 104 reported values for b is 0.790
with a standard error of 0.011. This mean value for b is significantly higher than
Fig. 1. Frequency distribution of the intraspecific scaling exponent b for the standard metabolic rate
(SMR) of the fish, where SMR - a Weight o, b - 0.79, n -- 104. Values taken from refs. 4, 10, 15, 20,
21, 24, 25, 30, 44, 60, 66, 69, 78, 80, 81, 98, 100, 104, 106, 107, 111, 122, 140, 156, 160 and 161.
The metabolic consequences of body size 337
both 0.67 (p <0.001) and 0.75 (p <0.01). For now let us just recognize that aerobic
metabolism displays negative allometry in fish and consider the mechanisms which
have been proposed to explain this relationship.
Theoretical explanations for the decrease in metabolism with size can be divided
into two categories; those based on the rate of energy use by animals, and those
based on the rate of energy production (i.e. the delivery of oxygen and other
metabolites). Included in the first group are mechanisms such as changes in
tissue composition with size, the energetic costs of homeothermy, and the energy
demand by muscles during locomotion 26,s3,Ss,11~ The arguments made by this group
of explanations are for the most part scientifically valid, however, their lack of
generality suggests that they are not fundamental mechanisms for the negative
allometry of metabolism. In this regard fish provide a useful comparison and test
of these theories. For example, the locomotion model proposed by McMahon 94
is based on terrestrial animals, laws of elastic criteria, and the observation that
larger animals are progressively stockier. Most fish are neutrally buoyant and do
not experience the types of structural stresses during locomotion on which this
model is based; and yet their metabolism also shows negative allometry. Similarly,
most fish are not endothermic, and therefore are not subjected to the decrease
in relative homeothermic costs with increased size. The universality of aerobic
allometry suggests that it is not the result of changes in energy demand, which will
be different for different groups of organisms, but rather the result of decreased
energy production (i.e. a 'supply-side' limitation).
1. Muscle contraction time
One area of theory which has been developed to account for the negative allometry
in metabolic rate is based on the maximum rate of energy use by the skeletal muscles
during locomotion 94,128. A particular line of reasoning among these 'demand-side'
theories notes that the rate of muscle contraction decreases with increased body
size, which suggests a concomitant decrease in muscle metabolism. And, since
muscle mass is such a large proportion of whole-body mass, overall metabolic rate
will also presumably decline. Theoretical-estimates of the maximum velocity of
shortening by mammalian muscle fibers do indicate negative allometry 63,86,95 with
a recent empirical study reporting a scaling exponent of -0.18 for slow fibers 123.
In the cod (Gadus morhua) 3, muscle mass, myotome cross-sectional area, and fiber
length scale geometrically, whereas twitch contraction time is proportional to L ~
L ~ Furthermore, as individuals of this species increase in size, the cycle frequency
for maximum power output decreases from 12.5 to 5 Hz, or as L -~ (~, W -0.17,
ref. 2). Wardle 15~ also observed that the contraction time of white lateral muscle
more than doubled with increased body size for a variety of teleosts ranging in size
from 10 to 75 cm. Assuming that contraction time was reflected in the frequency
of tail-beats during swimming, Wardle used this relationship to account for the
scaling of maximum swimming speed among fish. Although all of these observations
appear to be valid, they are weakened as explanations for metabolic allometry for
two reasons. Firstly, the universality of metabolic allometry, even among sessile
338 E.M. Goolish
species with little or no muscle tissue, suggests that the scaling is not solely the
result of locomotor costs. Secondly, even with muscle respiration rate decreasing
with increased size, one would expect that this aerobic capacity would be exploited
by the fish for other fitness-related processes such as growth or reproduction.
2. The effectof body sizeon the rate of oxygen delivery
The limitation to material delivery imposed by increased size is illustrated here

using oxygen. The same principles hold true, however, for all other materials whose
rate of delivery or removal will affect metabolism; e.g. CO2, NH3, and organic
substrates. It is tempting to consider oxygen as the most limiting factor on the basis
that some other components can be stored within in the body. For example, glucose
can be stored either in the liver or locally as glycogen. However, similar adaptations
also exist for oxygen in the form of cellular myoglobin and the splenic storage
of erythrocytes. Ultimately, all of the materials involved in metabolism are under
similar limitations of delivery rate for sustained aerobic energy production.
The movement of oxygen from the environment to mitochondria in fish can
be divided into three general steps; uptake by diffusion at the gills, convective
transport in the blood, and finally diffusion again into the cell (Fig. 2). We should
ask, to begin with, how an increase in body size would affect each of these
individual steps, and secondly how much potential capacity exists at each step
to compensate for the effects of body size. The uptake of oxygen at the gills
by diffusion is a function of the surface area available, the diffusion distance,
the diffusion coefficient, and the concentration difference across the respiratory
surface. Assuming structural similarity, among these factors only surface area would
be expected to change systematically and appreciably with increased size; scaling
as Weight2/3. This decrease, however, could be compensated for by increasing
the proportion of tissue allocated for respiration or by increasing the structural
Fig. 2. Summary of respiratory steps involved in the delivery of oxygen from the environment to the
cells of fish. The proportion of total energy demand supplied by either aerobic or anaerobic energy
production will vary with tissue type and body size.
complexity of the respiratory surface. Respiratory surface area can and does display
isometric scaling for mammals 154, amphibians 143, and insects s2, as well as for some
fish 67'71'125. The area available for oxygen uptake, therefore, does not appear to be
the major limitation for aerobic metabolic scaling.
The convective transport of oxygen in the circulatory system is determined in
general by the oxygen carrying capacity of the blood, the proportion of oxygen
unloaded at the tissues, and the volume of blood per gram of tissue which can be
delivered in a given unit of time. There is no underlying reason to expect either
oxygen carrying capacity or the percent of unloading to be greatly affected by body
size. However, the volume of blood which can be delivered to a gram of tissue over
a given time interval will be dramatically affected by body size. Total blood volume
displays isometric scaling for animals in general 121 and for fish in particular 67, which
means that a gram of tissue in a large fish is supplied by the same volume of blood
(approximately 50/zl) as in a very small fish. Therefore, as has been eloquently
noted by Coulson and colleagues 18,19, the rate of oxygen delivery to a unit of tissue
will be inversely related to blood circulation time, which can vary by several orders
of magnitude. For mammals 136, circulation time increases as body weight raised to
a power of 0.79.
Consider, for example, the situation in a 100-cm fish and a 2-cm fish. If blood
velocity is 1-cm/s and independent of size 58, during a 100-s interval an average
gram of tissue in the large fish will be supplied with a single unloading of blood
oxygen (i.e. 2 x 50 cm). During the same time interval in the small fish, an average
gram of tissue will be supplied with the oxygen from 50 unloadings. Since orders
of magnitude increases in blood velocity are improbable, this physical distancing
and its effect on oxygen and metabolite delivery appears to be the most direct and
inescapable consequence of increased body size.
The final step in the movement of oxygen from the environment to the mitochon-
dria involves diffusion from the capillaries into the cell. Changes in body size would
not be expected to directly affect processes at this level since cell size and capillary
structure are relatively unchanged.
Of the three general steps in oxygen transfer (uptake by diffusion, convective
transport and diffusion into the cell), it is the convective transport by the circulatory
system that appears most clearly limiting. Increased capacity for non-limiting steps,
e.g. diffusion into the cell, would not increase the overall rate of metabolism;
nor would increased capacity for the use of oxygen such as mitoehondrial density.
However, compensation for decreased oxygen delivery capacity with increased
size might be expected to occur for components of the limiting step, convective
transport.
III. Symmorphosis and the scaling of individual respiratory traits

If the overall rate of oxygen delivery and use decreases with increased body size,
how would this be expected to affect the scaling of the individual steps involved
in respiration? The concept of symmorphosis provides a framework for addressing
340 E.M. Goolish
this question. As originally proposed by Taylor and Weibe1141 the hypothesis of

symmorphosis states that 'the formation of structural elements is regulated to
satisfy but not exceed the requirements of the functional system'. When applied to
allometrie variation among mammals ls4, the hypothesis of symmorphosis appears to
be acceptable for all internal compartments of the respiratory system (blood, heart,
muscle capillaries, and mitoehondria). However, syrnmorphosis does not seem to be
supported by data on the lung, which forms the interface to the environment.
It makes intuitive sense that selection would operate towards a system where
individual steps in a multi-step process such as respiration would be matched.
It is not appropriate, however, to test the concept of symmorphosis on single
structural parameters4~ the entire structural system must be considered in context.
Consider for example the Antarctic ieefish (Chaeniehthyidae) whose blood lacks
hemoglobin 67. The decrease in oxygen carrying capacity which results should not
necessarily be expected to be accompanied by a decrease in the capacity of
other components of oxygen transfer, such as cardiac output. In fact, relative
heart size and cardiac output in these fish is extraordinarily high61, as a probable
compensation for the absence of hemoglobin. The proper question to ask when
applying symmorphosis is whether capacity at one functional level, e.g. circulatory
transport, is matched to the capacity of other functional levels, such as the aerobic
capacity of the tissues.
1. Blood oxygen carrying capacity
Whichever process limits oxygen delivery and use, symmorphosis predicts that all
other steps will decrease in capacity with increased body size to match this rate.
However, to the extent that it is physically possible, positive eompenstaion may
be expected for components of that process which is limiting. If, as discussed
above, convective transport and increased blood circulation time is the limiting
factor in aerobic respiration, then one would not expect to see a decrease but
rather a compensatory increase in its various components. This appears to be the
situation for fish. Fig. 3 summarizes data on the sealing of hematoerit values for
fish, and the pattern that emerges is that larger fish have higher concentrations of
circulating erythrocytes. The associated increase in hemoglobin concentration, and
higher blood oxygen carrying capacity which would result from this, may mitigate
some of the effects of increased blood circulation time.
The hematoerit values presented in Fig. 3 were not obtained from eannulated fish
but from fish which would have experienced some degree of physical activity during
sampling. Stress in general and physical activity in particular are known to cause
an increase in the hematoerit values of fish 132. It is not dear then, if the reported
positive allometry in hematoerit is a general pattern existing in resting fish or if it is
the result of activity during sampling and the release of erythroeytes from the spleen
or other organs. The scaling of spleen weight in fish has, in fact, been reported to
display positive allometry 67, with a mean sealing exponent for five species of 1.20.
A study of splenic contraction in tilapia (Oreochromis niloticus) 16z further suggests
that the allometry in hematocrit occurs as the result of handling and the energy
70
60
40-~ oj "S
.g
0 .... ~ 1 7 6 " S
0
30
E
Q
20-
10
' ' ' ; I .... I .... I .... I ' ; ' ' | ' ' " ' ! 1 ' ' ' " ' ' 1 ....
1~ 5 10 15 20 25 30 35 40
Length (cm)
Fig. 3. Effects of body size on the hematocrit values reported for fish. Intraspecifically, larger fish
appear to have higher concentrations of circulating erythroeytes and also, presumably, increased oxygen-
carrying capacity. Data are for tilapia 124 (Sarotherodon mossambica; O, now Oreochromis mossambicus),
largemouth bass 17 (Microptems salmoides; ~ ) , American plaice 130 (Hippoglossoides platessoides; - - -),
North Sea plaice 120 (Pleuronectes platessa; 0), cutthroat trout 85 (Salmo (Oncorhynchus) clarki; 0),
rainbow trout 38 (Oncorhynchus mykiss; ..... ), European sea bass 38 (Dicentrarchus labrax; ...), tilapia 119
(+), and Ophiocephalus sp.119(O). Length-weight transformations for rainbow trout and European sea
bass were made using the data from refs. 153 and 138, respectively.
demands of physical activity. Relative spleen weight and total spleen hemoglobin
content were determined for a wide size-range of fish before and after 3 minutes of
forced exercise (Fig. 4). The amount of hemoglobin (i.e. erythrocytes) released into
the blood increased dramatically with increased body size, presumably resulting in
increased oxygen carrying capacity. It appears, in fact, that small fish (less than 6
g) were able to accommodate the increased energy demands of swimming activity
without any release of splenic erythrocytes at all. This pattern, where the energetic
costs of swimming seem relatively minor to small fish, is one that occurs at many
levels as we will see later in the context of anaerobic scaling.
A further way that fish could compensate for changes in oxygen delivery capacity
with increased size is by altering blood oxygen affinity. Half-saturation pressures
(P s0) decrease with increased body size for birds 88, lizards 113 and mammals 88. The
decrease in blood oxygen affinity with decreased body size that this represents
is thought to favor unloading of oxygen in small individuals with a high aerobic
demand. The relationship for snakes appears to be the opposite, with an increase
in oxygen affinity with decreasing body size 112. This unique allometry has been
342 E.M. Goolish
~ 0.1
_a Before
>,
r 0.01
m
.0
o
mm
E
After
c
!
o. 0.001 9 , , , , , ,
1 110 ....... I
100 ........ 1000
Weight (g)
Fig. 4. Quantity of spleen hemoglobin in tilapia, Oreochmmis niloticus, before and after activity-induced
splenic contraction. Oxygen carrying capacity is augmented by an increase in circulating erythrocytes
for large fish but not for small ones. From ref. 162.
attributed to differences in pulmonary gas concentrations which are produced by

the specialized lung morphology and breathing movements of snakes. No such
multi-species data set on the scaling of blood oxygen affinity exists for fish. A recent
study of Dover sole (Microstomuspacificus) captured off the Coast of California
did report a pattern of increased blood oxygen affinity with increased size, with
P50 values decreasing from 4 torr at 100 g to approximately 2 tort at 1500 g (ref.
148). However, this species makes an ontogenetic vertical migration from shallow
water into the Oxygen Minimum Zone where oxygen concentrations fall to as low
as 0.30 mg/l (ref. 73). It is not yet clear, therefore, if this allometric relationship
can be generalized to all fish. Multiple hemoglobins are common among fish11s,
and changes in the frequency of particular forms with increased size and age have
been reported for many species including herring (Clupea harengus )159, salmonlSS,
Seorpaeniehthyes9~ and skate 91. Whether these shifts in hemoglobin have any
functional significance with regard specifically to body size is not known.
Compensation by larger fish for decreased oxygen delivery capacity could also
occur through an increase in tissue myoglobin concentration. This, like the apparent
increase in splenic storage, would provide the tissue of larger fish with a greater
reservoir of oxygen to use during periods of high energy demand. Increased
concentrations of myoglobin in larger species have been reported for the heart
and skeletal muscle of mammals22, with a sealing exponent of 1.31 calculated for
whole-body content t. A similar pattern is seen for the aerobic tissue of fish, with
cardiac myoglobin content displaying positive allometry for both salmonids 32 and
tuna ~16. No information exists for the skeletal muscle of fish, however it is known
that for some species the proportion of myoglobin-rieh red muscle to total muscle
increases as fish become larger46'54.
2. Gill surface area

Respiratory surface area can and does scale isometrically, or nearly so, for many
animals. Interspecific data are most complete for mammals where scaling exponents
of 0.95 and 1.19 have been reported for mammals in general and wild species,
respectively 41. A recent analysis of total pulmonary diffusing capacity for animals
ranging in size from a mouse to cow indicates a scaling exponent of 1.084 (ref.
154). Studies of fish have also found that gill surface area can scale isometrically, at
least within a species. Such results have been reported for the mackerel (Trachurus
trachurus71; b = 1.17), the carp (Cyprinus carpio99; b ~ 1.0), the dragonet (Cal-
lionymus/yra71; b = 1.52), an erythrinid (Hoplias malabaricus35; b = 1.18), and the
icefish (Chaenocephalus aceratus67; b = 1.09). Taken together these results suggest
that relative respiratory surface area need not necessarily decline with increased
body size, and that it may not be a limiting factor in oxygen delivery.
In general, however, intraspecific scaling exponents for the gill surface area of
fish are mostly between 0.8 and 0.9 (refs. 99, 107), with actual values of b ranging
widely from as low as 0.60 to the examples cited above which display positive
allometry. The fact that most species display negative allometry seems unusual
since information for other groups of animals, and some fish, suggests that there is
no physical constraint to respiratory surface area increasing in proportion to body
size. A decrease in relative gill surface area could be explained, however, if the
concept of symmorphosis is correct in its assertion that the various steps involved in
respiration are matched. The decreased capacity for oxygen delivery and use with
increased body size may require less respiratory surface area for oxygen uptake.
Pauley 1~ has proposed that low values of b for gill surface area, near 0.6, are
observed for small species of fish but that values of b approach unity for species
with increased maximum body size. His arguments are based in general on the
yon Bertalantiy growth equation, and on the allometry of oxygen requirements for
growth. However, no clear explanation is given for why the value of b should differ
between small and large species. The evidence supporting a relationship between
the value of b for gill surface area and maximum body size is equivocal 1~ and the
relationship might be dismissed were it not for a similar pattern which appears to
exist between the value of b for metabolic rate and maximum body size. A definitive
analysis of this latter relationship is precluded by the indeterminate nature of fish
growth, and the difficulty in objectively defining the maximum size of a species.
Also, most studies only include individuals over a portion of the species' size
range, and relatively few small or large species have been studied. Furthermore,
temperature appears to influence the value of b (ref. 44), and the studies cited in
Fig. I have been conducted at a variety of ambient temperatures.
If we focus, however, on those studies which have examined only small species
of fish (less than about 5 g), we find that all report very low scaling exponents
for metabolic rate compared to the overall mean value for fish of 0.79 (Fig. 1).
The earliest of these, b = 0.71, was derived by combining data for three species
of Cyprinodontiformes all less than 1.4 g (ref. 160). Even lower values have been
reported for the pupfish (Cyprinodon macularius81; b = 0.63), the mosquitofish
344 E.M. Goolish
(Gambusia affin/$97; b = 0.64), a Cyprinid minnow (Phoxinus phoxintt$2~ b =

0.55), and the sand goby (Gobius minutust~ b = 0.62). While the value of b for
metabolism varies widely for the majority of fish studied (i.e. species of intermediate
size), the consistently low values for small species does suggest that a single value of
b may not be representative of all fish and that the actual value of b may increase
with the maximum size of the species. Supporting such a relationship are values
of b for gill surface area, such as that for one of the word's smallest species, the
Philippine goby (Mistichthys luzonensisl~ b = 0.60).
Recent studies which have re-analyzed the mammalian literature have inde-
pendently come to the same conclusion, that the actual value of b for a species
may depend on its maximum size. Jurgens 7s re-examined the data of Bartels 5 and
found that the value of b was significantly lower in small mammals (0.60) than in
large mammals (0.77). A re-analysis 7s of the data of Hayssen and Lacy59 revealed
values of b for small and large mammals of 0.65 and 0.86, respectively. Finally, the
metabolic data of Elgar and Harvey29 indicated a scaling exponent of 0.65 for small
mammals and one of 0.86 for large species.
Why metabolic rate should display greater allometry over the life-span of a small
species compared to a larger one is not dear, however with regard to fish two possible
explanations are most apparent. The first concerns the relatively large influence of
growth rate on the metabolism of fish and other ectotherms. For although the basal
metabolism of fish is approximately an order of magnitude lower than mammals,
organismal-level rates of growth in these groups can be similar. Hence, among fish
and other ectotherms ingestion and growth rates will largely determine an individ-
ual's overall rate of metabolism 4s's2'l~176 If we assume that, for species of all size,
initial larval rates of growth are at some similar maximum, and that at that their re-
spective adult size growth rates are also the same (i.e. zero), then the same decrease
in growth rate (and therefore metabolism) must occur over a much more narrow size
range for smaller species. This would result in more severe negative allometry for
small species and a lower value for the metabolic scaling exponent b.
A second interpretation of higher scaling exponents in larger species involves the
energetic cost of swimming and how it is affected by body size. The hydrodynamics
of locomotion in water are such that the power required to overcome drag increases
dramatically with larger size lsl. Scaling exponents of approximately 1.17 and 1.34
have been calculated for sustained and sprint swimming, respectively 152. In larger
species, such as salmon, higher swimming costs in larger individuals are indicated
by scaling exponents for oxygen consumption during aerobic activity which are 1.0
or higher ~~ This increase in aerobic scope, and the resulting size-independence
of active metabolism, may also be reflected in relative isometry for standard
metabolism in large species. Small species would not require this increased aerobic
scope, and would therefore display greater negative allometry.
3. Cardiac output
The cardiac output of fish is determined by contraction frequency and by stroke

volume, the latter of which is presumably limited by heart size. As discussed earlier
for skeletal muscle, increases in body size would be expected to result in increased
muscle contraction times and, therefore, decreased contraction frequencies. While
such changes in the skeletal muscle may not limit overall metabolism, decreases
in cardiac frequency with increased size would directly influence the whole-body
rate of oxygen delivery by its effect on cardiac output. Data for mammals, birds,
and lizards do indicate negative allometry for cardiac frequency 11~ with most
relationships showing the rate decreasing as approximately W -~ Limited data
for the heart of the skate, Raja erinacea, also shows a decrease in frequency
with increased body size 1~ The relative size of the heart is not under any such
constraint, and scales according to an exponent near 1.0 for most groups of animals
including fish 13'33'53'72. The scaling of total cardiac output has not been examined
systematically for fish, but it almost certainly displays negative allometry as is
the case among mammals 57 (b ~ 0.75). Compensation for decreased frequency
by increasing relative heart size is limited, of course, since the heart itself will
have increasingly higher oxygen requirements. This is largely the same conclusion
reached by Jones 74, who analyzed the metabolic costs of the cardiac and branchial
pumps in fish and their possible role in limiting maximum oxygen consumption.
The effect of negative allometry in cardiac output on oxygen delivery (let us
assume it is near W~ would be in addition to the decrease in oxygen transport
in large fish due to increased circulation time. By simple dimensional analysis, the
effect of increased circulation time on the mean rate of oxygen delivery is equivalent
to W 0"67. The scaling exponents for cardiac output and increased circulation time,
when combined, would suggest that the overall rate of oxygen delivery should
increase according to W~176 It is perhaps surprising then, that the decrease in
metabolic rate with increased body size is as small as it is (scaling as W~ and
even more remarkable that the maximum metabolic rate during activity can remain
unaffected by size 11, scaling with a b-value near 1.0.
At least two explanations could account for this discrepancy. Firstly, larger fish
may compensate for the effects of size on cardiac output and circulation time
by increasing capacity at other steps in the delivery of oxygen. Some evidence
for this possibility has already been discussed, in that larger fish appear to have
greater hematocrit values and higher myoglobin concentrations. Gill surface area
may also scale with higher b-values in larger species. However, the potential for
such compensation seems limited, and it is not likely that increasing capacity at
a non-limiting step (such as oxygen uptake at the gills) would have any effect
on the overall rate of delivery. A second explanation is that small fish have the
capacity to deliver oxygen at a rate in excess of what is needed by the tissues. While
this is in contradiction to the concept of symmorphosis, it is not unreasonable to
expect that, ontogenetically, small fish are under the developmental constraint to
possess the respiratory structure which will be required as large adults when physical
limitations will decrease the potential for oxygen delivery. Small species would be
less affected by these considerations and, therefore, might be expected to display
lower scaling exponents for metabolic rate. As discussed above, this appears to be
the case. The resolution of these issues would be benefited by applying the concept
of symmorphosis to the study of fish, by testing whether individual respiratory
346 E.M. Goolish
processes are matched, and by identifying limiting steps in oxygen delivery. The
plasticity afforded by fish would be an advantage in such studies. For example,
drastic growth retardation (i.e. stunting) is possible for fish, where by maintaining
fish at cold temperatures and with little food they can be raised to adult age but
maintained at juvenile size. Scaling studies on such populations may be able to
partly separate ontogenetic and actual size effects.
4. Tissue respiration and enzyme activity
There are two ways that an individual fish could accommodate a decrease in whole-
body metabolic rate with increased size. One way is to shift tissue proportions
away from those with high aerobic capacities to those with low aerobic capacities,
and this is a pattern which is observed for intraspecific (or ontogenetic) allometry.
Essentially all of the tissues of the common carp (C. carpio) display negative
aUometry in weight except for the tissue with the lowest weight-specific metabolic
capacity, the muscle tissue 53,1~176 From 0.10 g to adult size muscle mass increases
from approximately 30% to over 60% of total body mass. This shift in tissue
proportions is in accord with energy requirements during ontogeny, i.e. high food
processing and growth capacity when small, and high swimming costs at larger sizes.
Although these changes do not provide a mechanistic explanation of metabolic
aUomctry, they can partly account for the observed whole-body decrease in aerobic
metabolism within a species.
In the case of interspecific aUometry only adult fish are being considered, all
having a growth rate near zero. Any shift of energy use away from the viscera,
therefore, would be expected to be less severe or to not occur at all. In the absence
of such shifts in tissue proportions, increased body size must be accompanied by
decreases in weight-specific aerobic capacity for individual tissues. Unfortunately,
almost no information exists for fish on the scaling of tissue aerobic capacity among
species of different adult size. One study of succinate dehydrogenase in the brain
tissue of 13 species of marine fish96 reported activity decreasing as W -~ However,
it was not clear that the individual fish examined represented the maximum size for
each species.
The remaining data which exist on the scaling of aerobic enzyme activity,
represents intraspecific studies, and most of this information is for muscle tissue.
This literature, and data on in vitro rates of oxygen consumption by tissues, indicates
a general pattern of negative allometry with most scaling exponents for weight-
specific activity ( = b - 1) ranging from -0.15 to -0.30. A summary of data for
the aerobic enzyme citrate synthase (CS) in white epaxial muscle indicated a mean
scaling exponent of -0.23 for 18 species 16. Variability in the value of b was large,
however, ranging from near isometry (-0.06) to allometry as severe as -0.68. In the
skeletal muscle of the toadfish (Opsanus beta) 149 CS activity scales with exponents of
-0.24 and -0.18 for males and females, respectively. A large data set has recently
been collected for the white muscle CS activity of a flatfish, the California halibut
(Paralichthys californicus) 1~ CS activity declines with a scaling exponent of -0.15
for individuals ranging in size from 0.2 to 1073 g (Fig. 5A).
10
i
.1
' ' 9'~
1000
4p 9
t
100
E 10
=L
3:
Q
--I
B
I
I I0 I00 I000 10000
Body weight (g)
Fig. 5. The scaling of citrate synthase (CS) and lactate dehydrogenase (LDH) activity in the white
muscle tissue of a flatfish, the California halibut (Pamlichthys califomicus). Scaling exponents are -0.15
and 0.46 for CS and LDH, respectively. From ref. 147.
An analysis of total muscle, i.e. red and white, cytochrome c oxidase (CCO)
activity in the common carp showed no decrease in weight-specific activity with
increased size 53. This result, and a similar one for in vitro oxygen consumption 146,
could be explained by an increase in the proportion of red muscle tissue with
increased size which has been demonstrated for fish46. This increase in red muscle
tissue, if found to be general among fish, may be an important compensation for
decreased weight-specific metabolism with increased size.
Negative allometry is also the most common scaling pattern for the aerobic
metabolism of other tissues, but again with some exceptions and with widely ranging
scaling exponents. The CCO activity of the carp brain 53 and the pooled CS activity
of brain tissue for two Paralabrax species 133 both decrease in larger fish, scaling
as -0.27 and -0.09, respectively. However, no effect of body size was reported
for the brain CS activity of the Dover sole 134. Weight-specific CCO activity in the
intestine of carp 53 decreases as W -~ and in vitro rates of oxygen consumption
were also reported to decrease for the gills (b = -0.14 ) and kidney (b = -0.15) of
the cutthroat trout (Salmo clarki, now Oncorhynchus clarki) 68. Similarly, liver CCO
activity in the American plaice (Hippoglossoides platessoides) TM was found to scale
as W -0"14. One factor which may be influencing these slopes is the relative cost
of growth in each tissue and how that is affected by body size. Fractional rates of
348 E.M. Goolish
protein synthesis in tissues of the rainbow trout (Oncorhynchus mykiss, previously

Salmo gairdneri) display negative aHometry7~ with scaling exponents ranging from
-0.14 in the ventricle to -0.49 in white muscle tissue.
Both the oxygen consumption rate and CCO activity of the heart of the sea
raven (Hemitripterus americanus) decrease with increased size33, displaying scaling
exponents of-0.51 and -0.13, respectively. However, five other cardiac enzymes,
both aerobic and anaerobic, were found to be size independent. More species
need to be examined before the general scaling pattern for heart tissue is clear,
but there may be reasons to expect its response to be unique from other tissues.
Highly aerobic tissue such as this under continuous energy demand, and with
luminal peffusion, may be maintained at a more constant level of aerobic capacity.
Compensation for changes in cardiac output demand in fish appears to be made
more by increasing the relative size of the heart, and therefore presumably stroke
volume. The activity of cardiac enzymes in the almost mammalian-like tuna are
no higher than in inactive species12; compensation occurs by increasing heart size.
Similarly, compensation by fish for cold temperature occurs by increasing heart
size45, and the increased energy demands of feeding and growth are accompanied
by increases in heart size but not by changes in weight-specific enzyme activity52.
Hochachka et al.65 have proposed that the oxidative capacity of muscle tissue may
be limited by mitochondria volume densities, and this principle may be operating in
fish cardiac muscle.
Finally, enzyme activities were recently obtained for whole-body homogenates of
larval Atlantic menhaden (Brevoortia tyrannus) ranging in size from approximately
1 to 10 mg 117. Weight-specific CS and malate dehydrogenase (MDH) activities
displayed sharp negative allometry, scaring with exponents of -0.785 and -0.805,
respectively. However, this is a period of substantial ontogenetic development, and
these extreme exponents may reflect changes in tissue proportion as well as actual
scaling effects.
II4. Tissue-level exceptions to negative allometry in aerobic metabolism

The only requirement concerning metabolism, given the organismal limitations on
oxygen delivery, is that the sum of aerobic capacity for all tissues must decline with
increased body size. However, there is no physical reason why the metabolism of
all tissues must decline, or that the metabolism of each tissue must decline at the
same rate. Specialized patterns of tissue-specific scaling will occur as is beneficial
to the species depending on its unique physiological, behavioral and ecological
requirements. Hence, there are numerous exceptions to the general pattern of
allometry (i.e. W~ and nearly all possible values for the intraspecific scaring
exponent b have been reported for various tissues.
In larval fish, even positive allometry has been observed for muscle CS activity,
with scaling exponents of 0.21 and 0.24 reported for northern anchovy (Engraulis
mordax) and Pacific sardine (Sardinops sago.x), respectively76. These increases in
aerobic capacity were attributed to the increased energetic costs of swimming
during that period, and presumably prior to the level when delivery constraints
would limit oxidative metabolism. Subsequent studies are suggestive of an inflection
point at approximately 100 mg for the northern anchovy77. Much variability would
be expected for the scaling of liver metabolism because of its role in energy storage,
and this appears to be the case. During the period of smoltification in Atlantic
salmon (Salmo salar), for example, liver CCO activity actually increases with larger
body size 8. Patterns such as this most likely reflect periods of energy mobilization
rather than scaling effects.
Perhaps one of the most interesting exceptions to the general relationship of
negative allometry in aerobic metabolism is the sex-related differences in scaling for
enzymes in the sonic muscle of the toadfish, O. beta 149. This muscle, in association
with the swimbladder, is used by the males to generate a mating call during certain
seasons of the year. In females sonic muscle CS activity declines with increased
body size as expected, however over the same size range in males sonic muscle
CS activity increases several-fold (Fig. 6A). The relationships are linear and do
not fit the usual logarithmic transformation well, but when expressed this way for
comparative purposes indicate scaling exponents of -0.13 and 0.43 for females
and males, respectively. Here is a clear demonstration that fish can overcome the
constraints of increased body size by altering enzyme levels, and presumably also
perfusion rates, to maintain and even increase aerobic energy production when
there is a strong selective advantage to do so. An expected consequence of this
increase, of course, is that some other tissue and physiological process must show
greater than normal negative allometry for whole-body allometry to be maintained.
An additional notable exception to the general scaling paradigm concerns the
tissue with the highest known aerobic capacity among vertebrates, which occurs not
in a small mammal but among large species of fish. This tissue is in the heater
organ of the endothermic scombroid fishes, and in some species it displays higher
CS activity than even that reported for hummingbird flight muscle 9. High aerobic
enzyme activity in this tissue is associated with the highest known mitochondrial
volume (63-70%), made possible in part by the absence of contractile filaments.
This observation further supports the proposal that mitochondrial volume may be
the limiting factor in cellular oxidative capacity. Scaling information is limited,
and confounded by other factors, but it appears that among species which possess
this heater organ tissue aerobic capacity for adults displays the usual negative
allometry. The effects of body size within a species are more complex. Based on
the scaling of heat loss and thermal inertia one would expect that the requirements
for heat production would be greater in smaller individuals. However, because it is
not practical to maintain elevated temperatures when small, young individuals of
endothermic species do not even possess well-developed heater organs. This is also
reflected in the fact that the heat-generating red muscle of endothermic scombroids
displays positive allometry 54. Therefore, it is probable that if the entire size range
of individuals within a species is considered, heater tissue aerobic capacity would
display positive allometry at least over some initial size range. This would be a
pattern similar to that which seems to occur during the ontogeny of locomotor
muscle tissue in larval fish 77. The heater organ system of scombroids further
350 E.M. Goolish
Fig. 6. Sex-related differences in metabolic scaling for the sonic muscle tissue of the toadfish, Opsanus
beta. A: effect of body size on muscle citrate synthase activity for female (e, b ffi -0.13) and male (o,
b ffi 0.43) toadfish. B: effect of body size on muscle lactate dehydrogenase activity for female (O, b ffi
-0.09) and male (m, b ffi - 0.24) toadfish. From ref. 149 with permission.
demonstrates how it is possible for fish to overcome, if only locally, the restrictions
imposed on tissue metabolism by body size.
V. The scaling of maximum whole-body aerobic capacity

We began this discussion by considering the allometry of standard metabolism
in fish, which appears to scale as W~ followed by an analysis of what factors
may limit aerobic capacity in larger individuals. If the mechanisms presented are
correct, however, then the limitations they impose should be reflected even more
directly in the scaling of maximum whole-body aerobic metabolism. This appears
to be the case for mammals where both basal and maximum aerobic metabolism
scale approximately as W~ (ref. 142). Maximum aerobic metabolism in fish,
as with mammals, has been determined during maximum sustainable locomotor
activity (= active metabolism). Among fish, however, maximum metabolic rate
has been reported to scale isometrically, or even more remarkably with positive
allometry 11,14~ It seems inconsistent, therefore, to develop scaling theory to
explain negative metabolic allometry when maximum aerobic capacity, i.e. oxygen
consumption, is unaffected by size or actually increases per gram of tissue in larger
individuals.
Considering the universality of negative aerobic allometry, the most likely res-
olution to this paradox is that the active metabolism of fish does not represent
the maximum metabolic rate for all sizes of fish. That this should be the case is
suggested, firstly, by aerobic locomotor muscle proportions. Unlike mammals where
locomotor muscle comprises nearly half oftotal body mass 1~ fish typically possess
only about 3% of body mass as aerobic locomotor muscle - and many species have
none. The relative amount of red muscle in fish also appears to decrease in smaller
individuals 46,s4. A second indication is that theoretical estimates of the energetic
costs of swimming in fish increase dramatically with body size. Even accounting
for a length-specific decrease in performance, the power requirements to overcome
drag at maximum sustainable speeds have been predicted to increase as W 1"17 (ref.
152). Furthermore, since we are discussing mostly intraspecific comparisons here,
we must also consider the scaling of feeding and growth which would be expected
to be a larger component of energy allocation among small (i.e. young) fish.
One approach to this issue would be a complete partitioning of aerobic capacity
to individual tissues and, by inference, to physiological function. This has been done
for the common carp using CCO activity as a measure of tissue-specific aerobic
metabolism 4a,53. The proportion of whole-body CCO activity contributed by each of
the white muscle, the red muscle, and the viscera, are summarized in ~ig. 7 for male
fish ranging in size from approximately 2 to 2200 g. The first thing to note is that
most of the aerobic capacity in these fish (40-65%) occurs in the characteristically
anaerobic white muscle tissue. This is aerobic capacity which is not even called
upon during the sustained swimming activity used to measure maximum metabolic
rate. It is employed, however, following exhaustive anaerobic activity when energy
is required for recovery processes. This large aerobic capacity in the white muscle
can account for the observation that oxygen consumption rates following exhaustive
exercise can exceed that during aerobic swimming in adult fish 135.
The next largest component of whole-body CCO activity in male carp is the
summed activity for the viscera, i.e. those tissues involved in food processing and
growth. In the smallest fish the viscera contributes almost 40% to whole-body CCO
activity, with this proportion declining with size to equal the contribution of red
muscle in 55 cm fish at approximately 15%. This pattern suggests that the rate of
oxygen consumption following a meal (Specific Dynamic Action, SDA) may exceed
that for active metabolism, a function of red muscle, in small individuals but not for
352 E.M. Goolish
70
60 White muscle
A
>,
lo
0
,-, 50
0
.c3: 40
Viscera
~. 3O
~
~ 20
0
U
10
0 " 110 " 2'0 " 3'0 " ~0 " 5~) " 60
Length (cm)
Fig. 7. Tissue-specific partitioning of aerobic capacity in male carp, Cyprinus carpio, as measured by
cytochrome c oxidase (CCO) activity. In small fish the summed aerobic capacity of the viscera, used in
food processing, is far greater than that which occurs in the red aerobic locomotor muscle. From refs.
48 and 53.
larger ones. The only data available for the scaling of SDA supports this prediction.
The maximum postprandial rate of metabolism in the largemouth bass (Micropterus
salmoides) is higher than that during sustained aerobic swimming for individuals up
to 100 g, after which active metabolism far exceeds maximum S D A 139.
It appears then that fish differ from mammals in that a variety of physiological
activities can be responsible for maximum aerobic metabolism among individuals of
different size. Unique characteristics which contribute to this include an extremely
small minimum size (mg), a buoyant medium which permits large anaerobic muscle
mass, no maintenance of elevated body temperature in small individuals, and,
having a lower metabolism, their energy allocation is influenced to a larger degree
by the costs of growth. Active metabolism as it has been determined is an accurate
measure of the energetic costs of swimming, but it does not seem to represent
the scaling of maximum aerobic metabolism. When the aerobic contribution of
other physiological activities are included, such as the elevated metabolism of
postprandial young fish, it is likely that maximum aerobic capacity will conform to
the expected relationship of negative aUometry.
I/7. Aerobic allometry: a comparison with hypoxia

Nearly all of the mechanisms which have been proposed to account for the negative
allometry in aerobic metabolism, such as respiratory surface area or circulation time,
are based on the decreased rate of oxygen delivery to the tissue. From the cells
perspective, then, the effects of increased body size are little different than those
imposed by environmental hypoxia. For this reason, it is interesting to compare a few
of the metabolic adjustments made by fish to these two respiratory challenges.
Beginning at the gills, compensation for hypoxic conditions occurs largely by
increasing the amplitude of ventilation, and ventilation volume 43. This indicates
that under hypoxia oxygen delivery is more diffusion-limited than perfusion-limited,
as is the case under normoxie conditions 89,126. That no systematic pattern similar to
this is observed with increased body size suggests that oxygen uptake at the gill, e.g.
surface area, is not the limiting factor for scaling. During hypoxic exposure blood
oxygen carrying capacity is increased by higher hematocrit levels and higher oxygen
affinity (a lower P50). Hematocrit levels also seem to increase with body size (Fig.
3), however, in contrast to hypoxia this appears to be more of an episodic increase
in larger fish as energy demands require. Whether there are general patterns for
the scaling of Pso among fish is still not clear, but any effects of size if they exist
are likely to be small and not of the kind which could overcome the influence of
increased circulation time.
Predicting changes in cardiac function is more complicated, because although
higher cardiac output may increase the rate of oxygen delivery, it will also in-
crease metabolic costs. Hypoxic bradycardia during acute exposure is often fully
compensated for by an increase in stroke volume to maintain cardiac output 34,
however long-term studies at clearly limiting low oxygen concentrations are rare.
A recent study of Dover sole maintained at 0.50 mg/1 for 8 weeks suggests that
when metabolic rate is permanently depressed, relative heart size decreases 51. The
decrease in cardiac output which would result may facilitate oxygen uptake at the
gills or, alternatively, the reduction in cardiac tissue may represent a meaningful
energy savings. No such changes in relative heart size occur as a result of scaling,
however an almost certain decrease in heart rate would similarly lower total cardiac
output. Increased cardiac myoglobin concentrations appear to be associated both
with hypoxia tolerance among species 23, and increased body size 32'116.
A study of long-term acclimation to hypoxia by the killifish (Fundulus heteroclitus)
reported only selected and transient changes in liver enzyme activity56. However,
other respiratory adjustments such as hematocrit and oxygen affinity were observed
which may have provided nearly complete compensation. A more severe and longer
exposure to hypoxia is encountered by the Dover sole during its ontogenetic migra-
tion into the Oxygen Minimum Zone. At a size of approximately 300 g it encounters
the lowest oxygen concentrations (0.3-0.5 mg/l), and it is in the muscle of these fish
that sharp decreases are observed in both aerobic and anaerobic enzyme activity
(Fig. 8) 147. This response is very different than the general scaling pattern of enzyme
activity, where with increased size decreases occur in aerobic enzymes but increases
are seen for anaerobic metabolism. The limiting influence of oxygen delivery during
hypoxia does not appear to allow anaerobic energy production by the muscle during
locomotor activity. In contrast, the positive allometry for anaerobic metabolism
suggests that, although oxygen delivery may be limiting metabolism in larger indi-
viduals, these fish retain the necessary unused aerobic capacity to repay the oxygen
debt following an anaerobic episode.
354 E.M. Goolish
Fig. 8. White muscle citrate synthase (CS) and lactate dehydrogenase (LDH) activities from Dover
sole, Microstomus paciIfcus, ranging in size from 2 to 2000 g. Shallow-water fish (<200 m), in water of
higher oxygen content, are indicated with open circles. CS and LDH activities for these fish scale with
exponents of-0.36 and 0.14, respectively. From reL 147.
VII. Effects of body size on anaerobic metabolism

1. Enzymaticevidence
Anaerobic metabolism is not, at least initially, constrained by the delivery rate of
oxygen and other materials as is true for aerobic metabolism. We would, therefore,
expect it to be influenced by body size differently that aerobic metabolism and this is
indeed true for certain tissues. Anaerobic energy production in fish generally occurs
through one of two pathways: (1) the high-power, low efficiency lactate pathway
used primarily by muscle tissue during extreme activity; and (2) the low-power,
higher efficiency pathway leading to ethanol which is employed by some cyprinids
during environmental anoxia. The scaling of this second pathway has not been
investigated, however it would not be surprising to find that it scales with the
negative allometry of aerobic metabolism. This is expected because it is usually
activated during episodic exposure to low oxygen, and would therefore have to
supply the higher whole-body maintenance requirements of smaller fish.
The lactate pathway is called upon to produce energy at rates greater than that
possible aerobically during periods when high swimming performance is critical 49.
The first evidence that this form of anaerobic metabolism does not decrease but
in fact usually increases with body size came from the scaling of weight-specific
white muscle glycolytic enzyme activities. Mean intraspecific scaling exponents of
0.35 and 0.21 were reported for lactate dehydrogenase (LDH) and pyruvate kinase
(PK), respectively, among 13 species of marine fish 133. A later study of LDH in four
Antartic mesopelagic fish 144 found their activity to scale with exponents of 0.23,
0.76, 0.95 and 1.05; while the macrourid, Coryphaenoides armatus 129, displayed a
value of 0.66. Similar positive allometry has been observed interspecifically for the
locomotor muscle LDH activity of mammals 31. In contrast to muscle tissue, brain
LDH activity in fish is not higher in larger fish 133,134.This suggests that the changes
seen in muscle are the result of muscle function and the costs of locomotion, and
not simply reflecting whole-body changes in metabolism. How the scaling of these
anaerobic enzyme activities are regulated is largely unknown, and this issue remains
as a challenge for molecular biologists in the next decade.
Positive allometry in muscle anaerobic potential is believed to be the result of
an increase in the relative power required by larger fish to swim at burst or sprint
speeds. This has led to a comparison of the scaling exponents for LDH among two
active pelagic species, the kelp bass (Paralabrax clathratus) and rainbow trout (O.
mykiss), and a benthic flatfish, the Dover sole 134. The scaling exponents for bass and
trout were both positive (b = 0.41 and 0.40, respectively), while the weak-swimming
Dover sole displayed a negative value of -0.44. However, the decrease in muscle
LDH seen for larger Dover sole appears to be the result of factors other than
just body size. As discussed earlier, the Dover sole is one of a group of species
which make an ontogenetic vertical migration into the Oxygen Minimum Zone
off the coast of California. A more complete data set has recently been collected
for this species which indicates that once large fish enter the Oxygen Minimum
Zone there is a dramatic environmentally-induced depression in metabolic rate
as demonstrated by sharp decreases in muscle CS and LDH activities (Fig. 8) 147.
If only shallow-water fish are considered (<200 m), occurring in water of higher
oxygen, a significant increase in LDH activity is observed in larger individuals (Fig.
8A, b = 0.14), as is typical for most other species. Furthermore, recent data for
another flatfish, the halibut (P. califomicus), indicates clear positive allometry in
white muscle LDH activity with a scaling exponent of 0.46 (Fig. 5B) 147. The halibut
is a extremely strong swimmer, which suggests that the scaling of anaerobic potential
is determined more specifically by muscle function and swimming performance than
by whether a species is pelagic or benthic.
The increase in activity of anaerobic enzymes in the white muscle of larger fish
would be expected to result in faster production of and/or higher concentrations
356 E.M. Goolish
1600
50 ee 9
/" - 1400
9 /
(n
40
- 1200 (o
A
9
E
o
. ." / . // ... - 1000
E
~L 3o
v
Q
9/ " /// -800 --
>,,
f,:) m.
20 / -600 .>_.
e,d
9 / t.)
/ ca
/ - 400 3:
r~
10 / .J
/
-200
j
0 i i ~ i i 0
0 5 1~) 15 20 25 310 35 40 45
Length (cm)
Fig. 9. Comparison of scaling relationships for white muscle lactate dehydrogenase (LDH) activity (- - -)
and the concentration of lactate (O) in the white muscle tissue of rainbow trout, Oncorhynchus myk/ss,
following 6 min of maximal burst activity. LDH activity is from ref. 134, and muscle lactate from ref. 46.
of lactate following maximal burst swimming. A review of the literature 4s indicates

such a pattern of higher white muscle lactate concentrations after exhaustive activity
in larger species and, for salmonids, in those studies examining larger individuals.
This question has also been addressed directly in a study of rainbow trout~s, a
species in which positive allometry in white muscle LDH activity has also been
demonstrated 134 (b = 0.40, Fig. 9). Larger rainbow trout have higher muscle lactate
concentrations when anesthetized (approximately 30 s of stress), and they also
produce lactate at a faster rate when chased to maximal activity~. White muscle
lactate concentrations following 6 min of maximal activity are significantly higher
in larger rainbow trout, scaling as W~ (Fig. 9). From this one might expect that
larger fish would display positive allometry for white muscle buffeting capacity, and
this has also been observed for kelp bass 134 (b = 0.064). Evidence of increased
anaerobic potential during activity has also been reported among a variety of other
ectotherms 7,39,114, us.
2. Power requirements during burst swimming
The increase in anaerobic potential of the muscle of larger fish is believed to

be the result of the relative increase with size in the power which is required
to overcome drag at burst swimming speeds. Estimates of the scaling exponent
for this power requirement range from 1.22 to 1.53, depending on which model
is used and whether flow is considered laminar or turbulent 133. In these models
length-specific burst performance is assumed to vary little with size. These estimates
of the scaling exponent for burst power requirements are near those exponents
representing the scaling of total muscle LDH activity 133 (~ = 1.55), supporting the
original hypothesis. Further analysis has also considered the aerobic contribution of
the red muscle to burst power requirements, and compared these two in absolute
terms 48. In this analysis weight-specific power output by red muscle is taken to be
4.25 W/kg, and the proportion of red muscle tissue to whole-body mass is 3%. The
scaling function which results for total red-muscle power output is shown in Fig.
10, together with the power required to swim at burst speeds as estimated from
a simple Newtonian equation for drag. The difference between these two curves,
which represents the anaerobic scope, appears to be near zero for small fish (1-3
cm) after which it increases with larger body size.
That there may be a minimum size threshold for the use of anaerobic metabolism
during swimming is also suggested by the length-specific scaling of sustained
(aerobic) and burst swimming performance 48. Burst swimming appears to be little
affected by size, with a velocity of 10 body lengths/s being representative of most
sizes. Aerobic swimming performance, limited by oxygen delivery, decreases with
size from over 12 body lengths/s in small fish to less than 3 body lengths/s in fish
greater than 40 cm. These functions intersect for small fish, again at approximately
1-3 cm, which provides further evidence that very small fish can swim at the
maximum speeds possible from muscle mechanics without the need for anaerobic
energy production. Large and preferential increases in the anaerobic enzyme
activity of muscle are also observed during this period 27,28,36,64,77.
Limited data are also available for the scaling of anaerobic enzyme activity in
other fish muscle tissues. LDH activity in the sea raven heart (H. americanus)
increases as W ~ while the aerobic enzyme CCO declines 33 (b = -0.13). The
sea raven heart differed from the muscle of this species, however, by displaying
negative allometry in pyruvate kinase (PK) activity. From these results the authors
propose that the hearts of larger fish may be more effective at utilizing exogenously
produced lactate. New data for the heart tissue of Dover sole indicates a similar
pattern 148, with scaling exponents of 0.22 and -0.17 for LDH and CS activities,
respectively.
In contrast to this, LDH activity in the sonic muscle of the toadfish displays
negative allometry 149 for both males (b = -0.24) and females (b = -0.09).
The scaling patterns for LDH activity in heart tissue and for sonic muscle tissue
provide an important comparison to white epaxial muscle. They suggest that
the hydrodynamic explanation proposed for locomotor muscle is only one of
many mechanisms involved in anaerobic scaling, and that like aerobic metabolism
anaerobic metabolism is very tissue-specific. Information on the scaling of LDH in
other tissues would be useful, particularly for those like the swimbladder where its
anaerobic role is central to tissue function.
3. Constraints on anaerobic scaling
The anaerobic potential of fish muscle appears to increase with body size for most
active species. Is there an upper limit to this source of energy production, and
358 E.M. Goolish
Fig. 10. Theoretical scaling functions for the power requirements during burst swimming (Pb) and
the maximum power output from the red muscle tissue of a typical fish (Pro). In small fish power
requirements at burst speeds appear to be met by the aerobic power output of red muscle, without any
anaerobic contribution. From ref. 48.
will that limit influence the swimming performance of large fish? This issue has
been addressed for mammals65, where the upper limit was considered to be set by
a compromise between myofilament volume densities, and the combined volume
densities of glycogen, intracellular buffering components and glycolytic enzymes.
In fish, muscle glycogen concentrations are relatively constant with regard to size
at about 1% of muscle weight sT. Total anaerobic capacity among species will, as
a result, scale approximately as muscle mass, i.e. as W1"~ We have already seen,
however, that the estimated power requirements to swim at burst speeds increase
as W 1"5 ( ~ L 4"4, Fig. 10). Therefore, once a fish reaches the size when all of its
anaerobic reserves are required, it will not be able to increase its rate of anaerobic
metabolism (scope) without suffering a decline in swimming velocity or stamina.
This analysis has been applied to the rainbow trout 46, a species for Which the scaring
of total anaerobic capacity has been determined, and it suggests a marked decrease
in length-specific swimming performance even over the size range of 5-50 cm.
Glycogen concentrations much higher than 1% are seen in liver tissue, which has
led to the proposal that muscle glycogen is maintained low as a 'brake' to glycolytic
activity, and the subsequent acidification and osmotic perturbation 65.
A second apparent limitation to the positive allometry of anaerobic metabolism is
the decrease in aerobic capacity of larger fish which is required to repay the 'oxygen
debt'. Following exhaustive activity energy is required for, among other things, the
metabolizing of lactate and the resynthesis of glycogen. The rates of these processes
are dependent on aerobic capacity and therefore also o n size 47,84'145. No data exist
for very large fish, however attempts have been made to estimate what these rates
would be for large animals based on a proportionality with metabolic rate. Assuming
an aerobic scaling exponent of 0.75, for example, the time required for complete
restoration of muscle glycogen in an 800 cm whale shark has been estimated at over
10 days 4s. This suggests that, from an ecological view, reliance on anaerobically
powered burst swimming is not a practical option for very large fish. This is in
agreement with the observed swimming behavior of these animals which is slow
and periodic, i.e steady. Because of the limitation imposed by the scaling of muscle
glycogen, and the further limitation of aerobic recovery, the scaling of anaerobic
metabolism in very large fish might not show continued positive allometry. There
appears to be an intermediate body size which is optimal for the useful exploitation
of anaerobic energy production during activity.
VIII. Behavioral and ecological implications of metabolic scaling

The scaling of aerobic and anaerobic metabolism may be a consequence of lower-
level processes, but they will in themselves also have an effect on organismal-
level characteristics such as behavior and ecology. Specifically regarding muscle
metabolism, these scaling relationships suggest that very small fish (i.e. larvae) are
able to swim at their maximum speeds with little or no anaerobic contribution.
Such fish would have unlimited stamina. However, because the relative costs of
swimming decrease with smaller body size, the aerobic scope of these small fish
due to activity is very narrow. This is reflected in the fact that standard metabolism
displays negative allometry (W~ while active metabolism is unaffected by size or
for some species may actually increase with size. As a result of these relationships,
and the fact that anaerobic metabolism is negligible, the entire range of power
exerted by small fish during activity has been measured at only 4- to 6-fold in
salmonids 156, and just 2- to 3-fold for larval cyprinids 157. Aerobic scope during
activity increases with body size, but more significant is the increased contribution
of anaerobic metabolism. When converted to units of oxygen consumption 6, the
anaerobic energy production during the first 30 s of sprint activity can be greater
than 7000 mg O2/kg/h in large salmonids 137. Therefore, considering also the decline
in standard metabolism, the range in activity power in large fish is not 2- to 3-fold
as seen for small fish but over 150-fold.
This dramatic scaling of the range and potential variance in the rate of energy
use during activity would be expected to influence behavior, particularly foraging
behavior. If we consider the sit-and-wait through active continuum, the continual
aerobic swimming of small fish over a narrow power range characterizes them as
active foragers. With larger size, however, behavior consists more and more of
periods of low metabolism broken by intermittent episodes of very high power
output (anaerobic metabolism). This activity pattern suggests that the mode of
foraging by fish will become increasingly sit-and-wait with increased body size. Such
a change in foraging will, in turn, affect other ecological characteristics including
habitat choice and prey choiceS~ and as such be a factor determining ontogenetic
niche shifts 155. This predicted shift in foraging behavior does appear to occur
360 E.M. Goolish
among large generalist species. With increased body size foraging switches from
zooplankton, to littoral invertebrates, and finally to active prey of large relative size,
fish 42'79'92'93. As discussed earlier, however, beyond some optimal size anaerobic
metabolism during activity may become less important, and the animals would again
display sustained aerobic foraging behavior with low variance in power output. This
does characterize the behavior of some of the largest fish, and also whales, which
filter prey from the water at a nearly uniform speed.
Acknowledgments. The studies discussed here were made possible by a grant

from the National Science Foundation (BSR-8700153), and support for writing was
provided by the National Research Council of the National Academy of Sciences.
I would also like to thank Dr. Russ Vetter and Eric Lynn (N.O.A.A., Southwest
Fisheries Science Center) for contributing unpublished data on the enzymatic
sealing of Dover sole and halibut.
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CHAPTER 16
Exercise metabolism of fish
CHRISTOPHER D. MOYES AND TIMOTHY G . WEST *

Department of Biology, Queen's University, Kingston, Ontario, K7L 3N6 Canada and *Department
of Zoology, Cambridge University, Downing Street, Cambridge CB2 3EJ, U.K.
I. Introduction
1. The typical fish!
2. Fuel selection
II. Steady-state swimming
1. Exercise metabolism
2. Protein and amino acid utilization
3. Carbohydrate utilization
3.1. Muscle glycogen
3.2. Circulatory glucose
3.3. Lactate
4. Utilization of fat fuels
5. Summary of fuel oxidation
III. Burst exercise
2. Carbohydrate metabolism
3. Phosphagens
4. Exhaustion: the influence of basal glycogen
5. Recovery metabolism
6. Carbohydrate metabolism in recovery from burst exercise
6.1. Fate of lactate
6.2. Route of glycogen synthesis
7. Fuel for recovery metabolism
8. Energy metabolism
8.1. Phosphocreatine
8.2. Adenylates
9. Why is recovery so slow?
10. Summary of burst exercise and recovery
IV. References
I. Introduction
The arrangement of fish muscle into separate, homogeneous fiber types has long
been recognized as advantageous for studying exercise metabolism (see refs. 9, 24,
46). Red muscle is recruited at steady-state swim speeds whereas white muscle is
used in short-term burst exercise. As muscle is a high proportion of whole body mass,
its recruitment imposes extraordinary demands on whole body metabolism. This fact
alone allows confident estimates of the potential contribution of specific fuels, de-
pots and pathways in exercise. Our first goal is to integrate empirical studies with
368 C.D. Moyes and T.G. West
a tissue-mass based model of fish exercise metabolism which sets theoretical limits
on which fuels and pathways can be physiologically relevant in exercise. When com-
paring the many studies of fish exercise metabolism, markedly different responses to
exercise are observed under conditions which would, at least superficially, appear to
be very similar. These variations are usually attributed to unquantifiable differences
in species, stocks, acclimation conditions or exercise regimes. Our second goal is to
extract information from these variable responses to exercise in order to generate
a model which takes into account inter-individual and inter-species differences in
metabolic status. Finally, this review should clearly point to critical deficiencies in the
current understanding of exercise and recovery metabolism.
1. The typical fish!
Fish demonstrate body forms and locomotory patterns which defy simple general-
izations. We consider a salmoniform to be the 'typical fish' solely as a framework
for discussion about relationships between anatomy and metabolic performance.
Most studies of fish exercise metabolism deal with salmonids, because of their wide
availability to researchers and commercial importance in locations where research is
supported 23,49,55,59,60,63,64,75,78,81-84,93,94,101,114. Physiologically, they perform better
than most species in both steady state exercise and burst exercise.
When fish other than salmonids are used, differences from the salmonid response
can be highly informative about the fundamental mechanisms which underlie
metabolic control of exercise. Across species, a 20-fold range in aerobic capacity of
skeletal muscle is observed~6. The differences in rate of recovery from burst exercise
spans two orders of magnitude in those few species which have been examined74.
Also, the fundamental differences in the fuels utilized by muscle in osteichthians
and chondrichthians 73 have not been explored in relation to exercise.
2. Fuel selection
Fuel preference studies are an attempt to quantify the contribution of the various
carbon substrates to changes in tissue energy demand. There have been few direct
in vivo studies of fuel selection by fish muscle. However, we can gain initial insight
into the possible metabolic partitioning of different substrates during sustained
exercise, burst exercise and exercise recovery from whole-body sites and levels of
the main storage substrates. Representative salmonid tissue masses and metabolite
levels, per g organ mass and per kg body mass, are summarized in Table I. These
metabolite levels represent the maximum mobilizable fuels available to a non-
feeding salmonid. Our first attempt to assess the relative importance of metabolic
fuels and storage depots in the normal post-absorptive animal involves a comparison
of the demands imposed by the exercise (Table 2) to the quantities of the putative
fuels available (Table 1). Where physiologically significant quantities of fuels exist,
rates of substrate supply, competition between pathways and the influence of
regulatory factors will determine the pattern of fuel utilization. Unfortunately, few
studies address these situations.
Exercise metabolism offish 369
TABLE 1
Tissue masses and glycogen stores of a resting salmonid
ECF Liver Adipose White Red

tissue muscle muscle
g tissue/kg body mass 250 15 20 600 70
Glycogen (as glucosyl)
(/zmol/g tissue) 0 100 -200 - 40 40
(~mol/kg body mass) 0 1.5 - 3.0 - 24 2.8
Glucose
(/zmol/g tissue) 2 -5 2 - 5 - 2 2
(/zmol/kg body mass) 0.5 -2.0 0.03- 0.075 - 1.2 0.14
Lactate
(~mol/g tissue) 7 3 3 5
(/zmol/kg body mass) 1.75 0.045 1.8 0.35
Triglyceride
(/~mol/g tissue) 22 10 - 30 800 15 40
0tmol/kg body mass) 5.5 0.15- 0.45 16 9 3
Fatty acids
(/~mol/g tissue) 3 3 2 2
(/zmol/kg body mass) 0.75 0.045 1.2 0.14
Protein (as amino acyl)
(/zmol/g tissue) 2000 1000 1000 500
(/zmol/kg body mass) 500 15 600 35
Amino acids
(~mol/g tissue) 40 - 50 9
(~mol/kg body mass) 10 30 0.63
ECF = extracellular fluid. Data were compiled from various sources (references 30, 42, 54, 60, 66,
81, 86, 96, 108, and 117). These values are estimates for total fuels and are not necessarily available
metabolically.
These analyses focus on maximal capacities for flux which are different both
qualitatively and quantitatively for red and white muscle recruitment. As exercise
becomes less intense or prolonged, the less abundant fuels and depots may become
increasingly important. Where no direct studies have been done, indirect studies
using enzyme analysis and isolated mitochondria can give insights into the capacity
and preference for fuels in exercise and recovery.
II. Steady-state swimming
The differences between red and white muscle morphology, orientation, ultrastruc-
ture and innervation that allow recruitment at different swim speeds have been
reviewed elsewhere,94690
, . Fish are able to swim in a continuous csteady-state , fash-
ion over a broad range of speeds. A e r o b i c red muscle is recruited almost exclusively
TABLE 2
A comparison of metabolite changes expected to occur if skeletal muscle is recruited at maximal work
levels in fish red and white muscle
. . . . Red muscle .... Wllite muscle ......

. . . . . . . . . . . . .
icon, o tput
Maximal work* (roW/g) 5 - 8 25 - 35
ATP utilization b (/~mol/min/g) 18 -29 90 -126
Aerobically generated A TP c
Oxygen (O2) consumed:
(/~mol/min/g muscle mass) 3.0 - 4.8 15 - 21
(/~mol/min/kg body mass) 0.21 - 0.34 9 - 12.6
Glucose utilized:
(ttmol/min/g muscle mass) 0.50-0.81 4.2- 5.8
(ttmole/min/kg body mass) 0.035- 0.057 2.5- 3.5
Anaerobically generated A TP d
Lactate produced:
(~tmol/min/g muscle mass) 12.0 -19.4 60 - 84
(/~mol/min/kg body mass) 0 . 8 4 - 1.36 36.0- 50.4
9 Determined in vitro for bullrout (Myoxocephalus scorpius L.).
b Assuming -500 kJ/mole 02, 20% mechanical efficiency and 6 ATPIO2.
c Assumes red muscle is 7% body mass and white muscle 60% body mass. A yield of 36 tool ATP/mol
~lucose is assumed.
1.5 ATP/lactate formed implies endogenous glycogen is utilized. If exogenous glucose were the fuel,
the values would be 50% greater (1 ATP/lactate formed).
in fish swimming up to 80% of fatigue or critical speed (Uc~,), beyond which the
glyeolytie white rnusele begins to be recruited as peak levels of whole-body aerobic
demand are approached 46's7'9~176 Studies of swimming physiology often have the
fish perform at speeds close to 80% of Ucr,, both in recognition of the muscle
recruitment pattern and because of the utility of Ucrit a s a standard measure of
aerobic performance. We can evaluate the potential role of circulatory fuels during
swimming using estimates of red muscle oxidative capacity and circulatory turnover
of metabolites. In the absence of such measurements, we can analyze potential rates
of depletion of circulatory and intramuscular substrate stores in support of high
intensity red muscle recruitment using the values in Tables 1 and 2.
2. Protein and amino acid utilization
In fish, dietary protein levels of 30-50% (w/w) seem optimal for growth2~ With
protein providing a potentially considerable portion of the total caloric intake
in omnivores, it is not surprising that daffy routine metabolism may be covered
in large part by amino acid catabolism. On average, it is estimated that amino
acid oxidation accounts for 10-20% of maintenance energy costs~.s3. This value
can possibly double after long-term starvation and may be elevated considerably,
on a shorter-term basis, immediately after feeding53. With starvation, increased
degradation of muscle-protein is most evident after body reserves of fat have been
depleted 8. Similarly, amino acid utilization seems important in migratory salmonids
late in the run when other fuel stores have been nearly exhausted 66. Taken together,
these observations suggest that in normal (fed, non-migratory) fish the availability
of amino acids for routine oxidative metabolism is influenced by post-feeding
absorptive mechanisms moreso than by the actual turnover of body-protein.
The effects of activity metabolism on protein or amino acid dynamics in fish have
not been examined extensively. Continuous low level swimming increases protein
turnover and degradation in feeding rainbow trout (Oncorhynchus mykiss) 41, but the
proportion of amino acids utilized for swimming energetics has not been determined
directly. In sockeye salmon (Oncorhynchus nerka) starved for 22 days, an indirect
assessment of whole-body amino acid utilization (ammonia excretion) indicated
that, despite a probable increase in protein degradation, daily increases in routine
metabolism were seemingly not supported by amino acids as long as reserves of
fat and carbohydrate were available 11,53. Ammonia excretion largely reflects amino
acid transdeamination in the liver 194 and will therefore over-estimate rates of
amino acid oxidation by the extent that the resulting carbon skeletons are used in
gluconeogenesis, which increases during activity and starvation (see ref. 99). In any
case, it seems likely that the higher aerobic demands of muscle resulting from a rest
to exercise transition, in both normal and food-deprived fish, would rely even more
on non-protein related energy reserves since the tissue pools of free amino acids are
small (Table 1) and the oxygen cost of ATP production from fat and carbohydrate
oxidation is about 30% lower than that of amino acids 2a. In support of this, it
has been noted that oxidation of neutral amino acids (serine, glycine, alanine)
does not occur at detectable rates in mitochondria isolated from carp (Cyprinus
carpio) red and white muscle 72. In addition, one study of in vivo substrate oxidation
rates indicated that glutamate, alanine and leucine, the latter classed as a branch-
chained amino acid which is possibly oxidized at elevated rates in exercising skeletal
muscle 51,52, seemed to contribute very little to whole-body oxidative metabolism of
rainbow trout (O. mykiss) swimming at 80% of U~rit (ref. 102).
Exercising fish may be able to draw on dietary amino acids after feeding as ox-
idative substrates for swimming. However, confirmation of such use of amino acids
awaits more detailed examination of post-feeding fluxes and oxidation rates. In most
studies of swimming metabolism the researcher avoids post-feeding energetics by
design in order to minimize inter-individual variability in basal oxygen consumption.
Direct circulatory infusions of amino acid mixtures (see ref. 100) could be a more
useful experimental approach for looking at the effects of amino acid availability
on fuel selection in fish. In this way it may be possible to mimic post-feeding levels
of plasma amino acids while at the same time eliminate potential influences of gut
absorptive rate on whole-body metabolism.
3. Carbohydrate utilization
There are potentially three ways to supply carbohydrate to exercising fish red
muscle (Fig. 1): (1) mobilization of intramuscular glycogen reserves; (2) uptake
of circulatory glucose derived from hepatic glueoneogenesis/glycogenolysis; and (3)
oxidation of lactate produced from white muscle glycogen.
372 CD. Moyes and T.G. West
WHITEMUSCLE PLASMA LIVER

i azJ,ooGi ', I "
v
G-6-P
|
< Glucose<
=
~ruvate
I
,-~ Laetm , > Lactate
10Pmi~/min
J'aoa A, i I
I
I
I
Glucose<
10umol/m
GlA se
..
iI ~
iI II
G-6-P< Glucosc< Amh;o (;~c.ro,

l
8 35-56l~nd/min Acids
i
pyn!,,,vate< Lact=e< , ~ Lactate

70-112~ n
i
tI
!
RED MUSCLE i
!
Fig. 1. Major routes for circulatory carbohydrate delivery (bold arrows) to red muscle in vivo. Glucose
is released from the liver, while at high work rates (>80% of Ucrit) circulatory lactate is derived
from white muscle glycogen. In a 1 kg fish (70 g of red muscle/kg), maximal rates of circulatory
glucose or lactate delivery (from turnover measurements z6,1~ cannot match substrate demand of 56
and 112/zmol/min/kg, respectively (calculated from O2 consumption, Table 2). Combined use of all
carbohydrate stores is also of little relevance since in vivo estimates of lactate and glucose contributions
to total oxidation are minimal l~
3.1. Muscle glycogen

Intramuscular glycogen is not expected to be the primary fuel for steady state
exercise since total red muscle glycogen is a fairly small depot (Table 1). if used
exclusively, red muscle glycogen would support sustained maximal aerobic swim-
ming for less than 1 h (see Tables 1 and 2). It is likely however, that many fish
species deviate somewhat from our 'typical-fish' scenario. In carp (Carassius caras.
sius), red muscle glycogen content appears to be 2-8 fold higher than that of white
muscle 44,47,5~ providing some evidence for a possibly greater reliance on intra-
muscular carbohydrate for swimming metabolism than is expected in salmonids.
Increased mobilization of endogenous red muscle glycogen has been shown in
carp during graded levels of swimming intensity48. Glycogen appears to be a mi-
nor oxidative fuel for long-term aerobic exercise, although it may augment fuel
supply, particularly at higher intensities and in the early phases of a rest to work
transition 97.
3.2. Circulatory glucose

The capacity for red muscle to utilize glucose is indicated indirectly from activities
of hexokinase that generally range from 0.5 to 1.5 ~mol glucose/g tissue/min or
total activity of 35-100/zmol/min in a 1 kg fish21,45. From the maximum oxidative
demand of the total red muscle mass (Table 1) it can be calculated that similar rates
of glucose oxidation are required for maximal red muscle activity (35-60/zmol/
min/kg body mass). As an estimate of fuel use, hexokinase measurements suggest
that glucose flux alone could account for the maximal rate of oxygen consumption
of fish red muscle. Crabtree and Newsholme 21 have similarly estimated that the
glucose utilizing capacity of trout (O. mykiss) red muscle could meet the aerobic
fuel demands. Can the delivery of circulatory glucose keep up with the fuel demand
during exercise?
Glucose release from the liver of fish results from net gluconeogenic flux and
glycogenolysis. Both processes are regulated by the action of various hormones
and functional peptides 67,68,85,88,99,117, but it is not known if one pathway domi-
nates hepatic glucose production in vivo. The responsiveness of glycogen stores to
catecholamines 6,88,117 coupled with the observation that plasma levels of these hor-
mones do not change during submaximal exercise 15,89, suggests that glucose derived
from hepatic glycogen may not be different from resting conditions. In any case,
liver glycogen levels alone (Table 1) could not support extended periods of maximal
aerobic swimming. Regardless of the pathway of hepatic glucose production, the
rate of production and release to plasma in vivo is probably within the range of
1-10/zmol/min/kg, estimating from plasma turnover in teleosts under different ex-
perimental conditions (reviewed in ref. 31). Unfortunately, reliable determinations
of salmonid glucose utilization during exercise are not available. In Fig. 1, we have
used the upper end of the range of available data (measured in hypoxic trout 26) as a
possible maximum turnover rate for exercising fish to illustrate the likely imbalance
between plasma glucose supply and red muscle demand. It is of course expected
that glucose in transit though the plasma compartment is distributed to more tissues
than just red muscle. However, in using turnover as an initial maximal estimate
of red muscle uptake, it is apparent that glucose delivery would have to greatly
exceed 10/xmol/min/kg to match the oxidative demand for substrate during high
intensity aerobic exercise. Glucose is oxidized at low whole-body rates in swimming
trout 1~ but rates of 2-deoxyglucose uptake in vivo verify that glucose utilization is
a minimal part of total substrate oxidation in specifically the working red muscle 112.
Red muscle glucose utilization at 80% of Ucrit averaged 21 nmol/min/g tissue mass,
a rate which accounted for <10% of expected red muscle oxygen consumption.
The capacity of red muscle to use glucose (estimated from hexokinase activity) may
mean that red muscle uptake can be expanded beyond the low level observed in
exercising trout, perhaps in periods of hypoxia or low lipid availability. However,
dependence of red muscle on plasma glucose during maximal aerobic exercise
would seem to be always limited by plasma availability since turnover apparently
cannot match aerobic demand for substrate.
It would be of interest to examine turnover and red muscle utilization of glu-
cose simultaneously during exercise. Since plasma glucose is poorly regulated in
fish 31'38'65's~ in vivo uptake of glucose in fish tissues might be expected to correlate
with parameters that affect glucose availability (e.g. plasma concentration, turnover
or changes in regional blood flow). Increased glucose utilization in trout red muscle
during exercise 112 is perhaps related simply to blood flow redistribution to mainly
the red fiber masssT. Direct comparisons of turnover and tissue utilization during
exercise would further identify major sites of glucose disposal during exercise and
could confirm that glucose utilization in red muscle is low, perhaps inhibited, in
vivo so that it may be spared for use in other tissues, as suggested from total red
muscle uptake in a 1 kg trout (about 1.5/zmol/min/70 g red muscle 112) compared to
our best estimate of turnover (10/zmol/min/kg body mass). More investigation of
plasma glucose turnover and uptake during exercise, starvation and hypoxia may re-
veal tissues and conditions in which glucose is quantitatively more important in vivo.
3.3. Lactate
Another route for the delivery of exogenous carbohydrate fuel to working oxida-
tive muscle may be via the steady-state release of lactate from white glycolytie
muscle 1~ In mammals, the fate of such lactate is predominantly oxidation resulting
presumably from a shuttling of lactate from glycolytie to oxidative tissues v/a the
circulation 12. It is intriguing to consider that white muscle glycogen stores in fish
could supply lactate to red muscle in the same manner. The total white muscle
mass represents a substantial glycogen store, about 20-fold greater than liver (Table
1), that could support hours of intense steady-state swimming (again, assuming
exclusive delivery of the lactate released to red muscle and a red muscle demand of
70-112 ttmol/min/kg body mass; Fig. 1). If such a pathway operated, lactate must
be produced by white muscle at adequate rates, which would be reflected in plasma
lactate turnover, assuming a conventional circulatory transfer.
Several species are known to recruit white muscle at high steady state swimming
speeds but there is no evidence that the white muscle metabolism under these
conditions is primarily aerobic. If for example the aerobic demands of high intensity
red muscle activity (29/~mol ATP/min/g, Table 2) were imposed on the recruited
white muscle its aerobic metabolism could provide <15% of the ATP (e.g. rainbow
trout white muscle mitochondria can produce 3.5/zmol ATP/min/g75). (In some
species, such as skipjack tuna (Katsuwonus pelamis), the mitoehondrial content
and presumably aerobic capacity of white muscle is exceptionally high, 2-fold and
5-fold that of trout 75 and carp (Cyprinus carpio) 76, respectively, even without the
higher body temperatures considered.) The possibility that lactate produced by
white muscle could serve as fuel for working red muscle might be evident even
in the initial stages of white muscle recruitment. Hence, studies in which lactate
dynamics have been examined in swimming rainbow trout 1~176 have imposed a
high level of sustained exercise (85% of Ucrit) with the expectation that any white
muscle recruitment would be fueled primarily by anaerobic glycolysis.
The problem with relying on circulatory lactate as a red muscle fuel seems to
be the same as that stated for exogenous glucose. As reiterated in Fig. 1, turnover
simply cannot match the oxidative demand of red muscle. Lactate turnover would
have to increase by about 10-fold to cover maximal red muscle metabolic rates
calculated for a 1 kg fish (Table 2). Empirical studies indicate that, on average, the
lactate turnover rate during sustained exercise at 80% Ucrit doubles over resting fish
(to about 10/zmol/min/kg body mass), similar to that in a salmonid recovering from
burst exercise63, but this change does not occur consistently in individual fish 1~
Thus, lactate remains of minor importance as an oxidative fuel during an endurance
swim 1~ although it may be more important at higher levels of Ucr,. Under these
conditions, however, the expected increases in both white muscle recruitment and
whole-body oxygen consumption make predictions difficult.
Although elevated rates of lactate oxidation are predicted in swimming trout 1~
this does not necessarily reflect red muscle metabolic events. These turnover exper-
iments merely point out the fate of the lactate which appears in, and disappears
from, the plasma compartment and are not inconsistent with the limited whole-body
dependence on lactate as a fuel. As in mammals, oxidation may in fact be its
major fate in vivo since hepatic gluconeogenesis from circulatory lactate is minimal
in most instances and in a variety of species63,1~176 Although other fuels seem
more important for overall metabolism, tissues that contribute relatively little to
the absolute change in metabolic rate with the onset of exercise could still utilize
lactate as an oxidizable fuel. Trout myocardium for example, increases its oxygen
consumption several-fold at high work intensities, but could rely heavily on circula-
tory lactate turnover because of its small relative mass and fractional contribution
to whole-body oxygen consumption during swimming 58,112. This possibility requires
further investigation.
4. Utilization of fat fuels
Currently, the major gap in our understanding of fuel utilization during steady-state
exercise concerns the relative importance of lipid fuels (Fig. 2). In total, the body
stores of triacylglycerol (TAG) represent the largest energy reserve in fish which
could, if delivered exclusively to the red muscle, supply enough free fatty acid
(FFA) to fuel about 100 h of maximal aerobic red muscle contraction in a 1 kg
animal (assumes about 23/,mol O2 utilized per/~mol fatty acid oxidized). However,
there are few empirical measurements that help to identify which of major depots
are mobilized in vivo during exercise. Such studies are complicated by the fact
that TAG is deposited in, and mobilized from, muscle, liver and the viscera96.
Quantitative approaches are needed to increase our awareness of the importance
of circulating FFA, extramuscular depot fats and of the dynamics of skeletal muscle
intracellular and pericellular TAG stores. Presently, any conclusions regarding the
376 C D Moyes and T.G. West
VISCERAL FAT LIVER

i, i i .v,.. ,, ,, ,
Iazyooa H 1
I
..-- Glucose< Glucose~"

i/
A
%
- - - .~P,Ivcero~-
FFA
I 9
L I
Lactate
Lactate
I
~G-6-P
/
/
/,./
Glycerol-3-P- > ,, rate
@@ / '@~
Glycerol
RED MUSCLE
Fig. 2. Interactions between the major triacylglyceride (TAG) depots in the provision of fat fuel to
working red muscle. FFA delivered to red muscle from visceral fat, liver and possibly white muscle enter
FFA and TAG pools prior to oxidation. Glycerol, released from intramuscular lipolysis, is used in TAG
synthesis (liver and visceral fat) and in hepatic gluconeogenesis. Total body fat stores could possibly
support 4--5days of continuous maximalred muscleactivity(see text).
relative importance of the various lipid stores, and lipid fuels in general, are limited
to studies which focus on either changes in lipid content (e.g. observations of
whole-body and organ fat depletion during starvation/migration,842 - 44) or on in
vitro enzyme measurements and mitochondrial capacities for fatty acid utilization.
Such studies support a major role for lipids in steady-state exercise in fish.
Although hepatic and skeletal muscle TAG contents are similar (per g tissue),
the smaller liver can be viewed as a minor storage organ with respect to steady-state
exercise, regardless of the kinetics of hepatic mobilization. On a quantitative basis,
Exercise m e t a b o l i s m o f f i s h 377
visceral fat deposits are the richest store of lipid per g tissue, but skeletal muscle,
by virtue of its mass possesses similar TAG stores per kg body mass (Table 2).
Each of these stores might be drawn upon during aerobic exercise. The degree to
which the white muscle and visceral fat may supply FFA to red muscle should be
reflected in estimates of circulatory delivery. However, one study of in vivo palmitate
oxidation suggests that circulatory fatty acids contribute minimally to the total
oxidative fuel required by trout swimming at 85% of tl 1~ Possible delays in the
"-"crit"
equilibration of labeled circulatory precursor (14C-palmitate) with the intracellular
TAG pool at the site of utilization may result in underestimates of circulatory
FFA oxidation 36, but it is not known if this is a problem in fish at different levels
of activity. Among endothermic species, the likelihood that intramuscular lipid
supports aerobic energy demands, at least to some extent, is supported by the
observations of increased stores in aerobically trained muscle 4~ and by the proximity
and presumed functional association between mitochondria and intracellular lipid
droplets in oxidative fibers 33,4~ The possibility that intramuscular TAG serves
simultaneously as a sink for circulatory fuels and a source of mitochondrial substrate
needs to be evaluated more closely in fish.
As with most vertebrate oxidative muscles, red muscle from teleost demonstrates
a high capacity to utilize FFA, as indicated by mitochondrial oxidation rates 72'76'77.
While this in itself reveals little about the relative importance of fatty acids,
comparisons of mitochondrial oxidation rates and carnitine palmitoyl transferase
(CPT) activities between species and tissues are more illuminating. As in aerobic
training of mammalian muscles, where an increase in the capacity to utilize fatty
acids is apparent, a comparison of fish species and muscle types reveals that CPT
activity increases with aerobic capacity of the tissue 76. This change is due to both
an increase in quantity of mitochondria and CPT activity per unit mitoehondria.
Thus, fatty acids may become relatively more important in tissues (white muscle
versus red) and species (carp versus tuna) with greater aerobic capacities 76. This
agrees fundamentally with conclusions from various comparative studies which
have examined the fuel demands in endotherms and concluded that adaptation for
increased aerobic capacity is related to an increased dependence on fat fuels 25,11~
One point of interest is that chondrichthian red muscle, unlike that of teleosts,
lacks demonstrable capacity to utilize FFA as indicated by low activities of CPT and
an inability of isolated mitochondria to oxidize FFA in a variety of forms (short,
medium and long chain fatty acids and fatty acyl carnitinesT3). The fuel used in
place of fatty acids in these species has not been established, but is thought to be
primarily ketone bodies.
In mammals, ketone body utilization by skeletal muscle increases with circulating
concentrations which rise primarily during starvation (e.g. ref. 91). Teleost skele-
tal muscle apparently has no capacity to oxidize fl-hydroxybutryate and minimal
capacity to utilize acetoacetate 72 ,76,77 . Conversely, chondrichthian red muscle lacks
the capacity for FFA oxidation, but does possess an impressive capacity to oxidize
ketone bodies. If ketones functionally replace FFA in muscle metabolism, many,
as yet undemonstrated, differences in whole-body lipid metabolism are expected in
these species 77.
The expectation that changes in red muscle aerobic demand are dominated by
FFA oxidation in fish may mean that FFA inhibits carbohydrate utilization, as seen
in heart preparations 92 and in vivo in mammals 29. In this ease FFA availability
may spare glucose for use in other tissues. This is supported only indirectly by the
possibility that red muscle glucose uptake in a 1 kg trout may be a small proportion
of the expected total glucose turnover during exercise (discussed earlier). Ketone
body use in ehondriehthians has been suggested to have a similar sparing effect on
glucose 69. Investigations which emphasize FFA kinetics in concert with effects on
glucose uptake and oxidation (and reciprocal studies of glucose effects on lipolysis
and FFA oxidation) in exercise and starvation, or FFA limitation, will help describe
the nature of fuel interactions in fishes.
5. Summary of fuel oxidation
The utilization of fat fuels in fish during sustained exercise is implied indirectly
from enzyme measurements, mitoehondrial flux capacities and an indication that
the expansion of aerobic capacity across tissue-types and species seems associated
with a greater capacity for mitoehondrial transport of long-chain fatty acids. Fat
stores are the richest energy reserve in the whole-animal, but direct quantitative
determinations of in vivo lipolysis, fat oxidation and effects of fat availability on fuel
use at high intensity steady-state exercise are needed to verify preference for fat in
the whole-animal. Low in vivo rates of circulatory glucose and lactate utilization are
consistent with potential reliance on fat in oxidative muscle, although a potentially
important role for lactate in smaller oxidative tissue masses (e.g. heart) cannot
be dismissed. Muscular oxidation of amino acids may be important after feeding,
but amino acid availability for continuous exercise seems otherwise limited, except
late in migration. Chondriehthians are one group that deviate from the typical
fish model presented in that they are possibly reliant on ketone bodies for activity
metabolism moreso than fatty acids.
IlL Burst exercise
White muscle is recruited when the power output needed to achieve relatively high
swim speeds exceeds the capacity of red muscle. In the natural world, burst exercise
is important iv escape from predators, capture of prey and migration against
swift water currents. Experimentally, burst exercise is achieved in a swim tunnel
or by manually chasing the fish. Neither of these techniques can be expected to
realistically duplicate fully the physiological events occurring in nature 11~. Swimming
to exhaustion is typically the endpoint of a burst exercise regime. The need to reach
a standard state to reduce experimental variation associated with interindividual
differences in motivation is usually thought more important than the fact that this
type of exercise would rarely be expected to occur in nature. Although most studies
report to swim fish to near-exhaustion, comparison of parameters such as tissue

lactate suggests that even this end-point may be somewhat subjective.
2. Carbohydrate metabolism
Simply stated, the high power outputs of white muscle exceed the capacity of
mitochondrial metabolism to meet the demand. However, it should not be inter-
preted that burst exercise is fuelled anaerobically because white muscle has too few
mitochondria (<2% volume density, ref. 46). The metabolic cost of achieving the
maximal power outputs of white muscle determined in vitro, 126 ~mol ATP/min/g
(Table 2), is well above white muscle aerobic capacity (3.5 /zmol ATP/min/g for
trout, ref. 75). As the capacity for a very aerobic muscle such as skipjack tuna heart
(42/zmol ATP/min/g, ref. 76) is also well below this ATP demand, it can be argued
that even if a comparable mitochondrial volume density (30%) could be achieved in
white muscle, it would still be inadequate to meet the demands of burst exercise. In
fact, a large proportion of white muscle intracellular space is necessarily devoted to
myofibrils precluding the packing of mitochondria to the volume densities observed
in more aerobic muscles39,111. Since there is not enough white muscle mitochondrial
capacity to meet the demands of burst exercise, questions concerning the adequacy
of oxygen delivery are largely irrelevant.
Most of the ATP used in burst exercise is obtained from muscle glycogen and
glycolysis. It has been suggested that exogenous glucose may be an important fuel
during burst exercise 84. This is highly unlikely except, perhaps, at the very lowest
intensities for reasons related to the high rate of demand and the large mass
recruited. Extramuscular sources of glucose and glycogen are minor compared to
the white muscle stores of approximately 24 /~mol glucosyl units/kg body mass
(Table 1). The plasma glucose pool size is small, (Table 1) and circulating levels
change little in exercise or recovery59. Liver typically has significant glycogen
content/g tissue but as total hepatic glycogen represents less than 10% of the
intra-muscular depot (Table 1) and it does not change much with exercise59,62, it is
unlikely to be an important source of glucose for burst exercise. The minimal role
of circulatory glucose in burst exercise has been directly demonstrated in vivo using
the glucose analogue 2-deoxyglucose. At high swim speeds glucose uptake rates of
0.3-0.4 nmol/min/g occur 112 compared to lactate production rates of approximately
2 ~mol/min/g 74. It should be noted that this lactate production rate is averaged over
an extended exercise regime, consisting of alternating periods of burst and gliding.
if the in vitro predictions of ATP demand in burst exercise are valid, instantaneous
glucose uptake rates of 63/zmol/min/g would be required (Table 2).
3. Phosphagens
An integrated picture of the regulatory control of burst exercise is far from complete.
As the rate of utilization of ATP increases, pathways for synthesis of ATP must be ac-
tivated. Catabolism of immediately available ATP leads to an increase in ADE This
shifts the mass action ratio of the creatine phosphokinase (CPK) reaction toward
ATP production, with concomitant decrease in phosphoereatine and a net release

of inorganic phosphate (Pi). When the ATP-buffering role of phosphoereatine is ex-
ceeded, breakdown of ATP to IMP (inosine 5'-monophosphate) by AMP deaminase
occurs. The net result is a decrease in ATP with a increase in IMP (1 mol/mol ATP),
ammonia (1 tool/tool) and Pi (1 tool/tool) 24,64. It has not been shown that Pi re-
leased from phosphoereatine breakdown and ATP catabolism is retained within the
white muscle. Considering the importance of Pi in metabolic regulation, improved
resolution of the changes in Pi occurring in recovery would be most useful.
While the changes in adenylates and Pi are important signals for stimulation
of glycogenolysis and glyeolysis, they would also be expected to stimulate white
muscle respiration during exercise. As mentioned previously, it is unlikely that the
ATP produced by mitochondria would form a significant proportion of the total
ATP utilized. However, the stimulation of mitochondria during exercise may cause
longer lasting effects and have implications for recovery, when mitoehondrial ATP
production is critical. It is known, for instance that exercise induces dephosphory-
lation (activation) of pyruvate dehydrogenase75. If this activation persists, it could
have important consequences to regulation of fuel selection in recovery.
4. Exhaustion: the influence of basal glycogen
Experimentally, a burst exercise regime usually continues until the fish is exhausted.
At this point, the fish is still capable of performing slow, steady state exercise but is
unable to burst. Although most studies involve this type of protocol, dramatic differ-
ences in the metabolite status at exhaustion are obvious. Several studies employ fish
possessing low resting glycogen levels, which may be related to seasonal variations,
dietary status, size or age23,64,93. Exercising these fish may actually deplete white
muscle glycogen completely in at least some fibers (<1 /zmol/g) and exhaustion
apparently occurs when the muscle is starved for the carbon fuel. In studies using
fish with higher initial glycogen levels (>20/zmol glueosyl/g) similar exercise pro-
tocols result in greater changes in adenylates and lactate 84,94. The actual cause of
exhaustion when adequate glycogen occurs may be more related to disturbances
of excitation-contraction coupling 113. Not surprisingly, the patterns of recovery in
these two groups are different as well. In 'carbon starved' exhaustion, recovery of
metabolite status tends to be slower, most obvious with phosphocreatine. As yet,
no comprehensive study has directly addressed the influence of basal glycogen on
exercise and recovery.
5. Recovery metabolism
The exercise-induced imbalances in metabolite levels return to resting levels in

minutes to several hours, depending on the metabolite. When a fish is deemed
recovered is dependent on which metabolite is examined. Perhaps the most useful
parameter on which to base the rate of recovery of carbohydrate status is lactate.
The development of techniques to best sample fish is based upon minimization
of lactate accumulation in 'resting' fish94,1~ Lactate generally returns to resting
levels at the slowest rate. It is easily measured with little error arising from
tissue extraction (little binding, highly acid-soluble). As resting levels are very low,
exercise-induced changes with respect to rest are 10-fold or more. This contrasts
to the situation with glycogen, where resting levels are highly variable between
individuals, and exercise-induced changes are often less than 30% of the total
glycogen level.
Changes in lactate, however, offer little insight into the recovery of energy
status. The most meaningful energy parameter incorporates some expression of
free adenylate concentrations, but these determinations require much more analysis
(total ATP, ADP, AMP, pHi, creatine and phosphocreatine) than phosphocreatine,
the more commonly determined parameter. Changes in phosphocreatine reflect the
adenylate status (free MgATP/MgADP) through CPK, which is assumed to be near
equilibrium in vivo 18. It should be emphasized that phosphocreatine determination
using freeze-clamped tissue typically underestimates the in vivo level. Using NMR,
van Waarde and colleagues 1~176 estimate creatine is 80% phosphorylated in vivo,
whereas the best estimates from freeze clamp studies are 62% in salmonids 94, 75%
in tuna 2 and 70% with in vitro muscle fibers 7~ While absolute levels are difficult to
determine with confidence, the relative changes within a study are probably realistic.
Those studies which measure phosphocreatine yet neglect to determine creatine are
less useful for assessing changes in energy status and very difficult to compare with
other studies. Also, pH/must be considered in the analysis as acidosis will depress
phosphocreatine through mass action effects on CPK, even when ATP/ADP is
constant. Basing assessment of adenylate status on phosphocreatine changes alone
would lead to erroneous conclusions 94.
6. Carbohydrate metabolism in recovery from burst exercise
6.1. Fate of lactate

White muscle post-exercise must reduce lactate levels as well as replenish glycogen
stores in preparation for the next exercise bout. In mammals, lactate may be
oxidized or used as a substrate for hepatic gluconeogenesis or skeletal muscle
glycogen synthesis directly. Its fate may be highly dependent on muscle type,
species, pH and hormonal conditions 6,7,1~176 In reptiles, lactate produced in
white muscle is largely transferred to red muscle for glycogen resynthesis 32. We
believe that in fish, with few exceptions, lactate oxidation is minimal. The majority
of lactate post-exercise is converted to glycogen and this occurs directly within the
white muscle 74.
It is clear that lactate disappearance is not due primarily to oxidation in white
muscle or any tissue as whole animal respiration would greatly exceed that observed
in vivo 93. Lactate may be utilized by tissues such as heart sa, but its oxidation would
have little impact on whole body lactate levels. We believe that in recovery lactate
disappearance accounts for glycogen reappearance 94, most obvious when single fish
are repeatedly sampled 2. This is a critical, and somewhat controversial, conclusion
as several studies conclude otherwise based on either of two lines of evidence:
(1) lactate disappearance apparently does not parallel glycogen re-appearance with
382 CD. Moyes and T.(7. West
the required stoichiometry; and (2) glycogen does not recover to pre-exercise
levels. However, the relationship between white muscle lactate and glycogen is
very difficult to address where terminal samples constitute the time-course, due
primarily to variability in resting glycogen levels. We feel that if the following
two criteria are not met in a study it is very diftieult to establish the relationship
between lactate and glycogen in recovery. Firstly, as lactate is most certainly derived
primarily from muscle glycogen, moles of lactate produced cannot exceed twice the
moles of glycogen utilized. Variability in glycogen levels can be so great that, at
least in one study84, there was an apparent increase in lactate during exercise that
is more than double that possibly arising from glycogen. When the stoiehiometry
between glycogen disappearance and lactate appearance is violated during exercise,
the relationship in recovery is more difficult to address with confidence. Secondly,
glycogen and lactate in unexercised controls at the end of the study must be
similar to fish prior to exercise. In studies which employ small fish with relatively
low glycogen levels (e.g. ref. 93), there is an apparent depletion of glycogen in
both exercised and control fish, suggesting significant utilization of white muscle
carbohydrate for basal metabolism.
As a consequence of the high and variable glycogen levels typically observed, lim-
ited oxidation of lactate (<20%) would be very difficult to detect. Indeed some fuel
must be oxidized by white muscle mitochondria to support net glycogen resynthesis
in recovery. Given the relatively low capacity for both hepatic glueoneogenesis and
white muscle glucose transport (see next section), any metabolic strategy that leads
to substantial depletion of white muscle carbohydrate seems maladaptive. As will
be discussed in section 7, it is likely that lipid oxidation fuels recovery metabolism
despite high levels of lactate 75.
Several indirect and direct studies suggest that lactate conversion to glycogen
does not involve extramuscular tissues but occurs directly within the white muscle.
In mammals, the fiver may be an important site of glueoneogenesis 19 (but see also
ref. 12), but the importance of the Coil cycle in fish is dubious due to low hepatic
glueoneogenie capacity and limited capacity for glucose uptake by white muscle.
The in vitro hepatic glueoneogenie rate of toadfish ~~ and tuna 14 is a small fraction
of the observed rate of glycogen replenishment. In vivo, [14C]-laetate injected into
the blood remains primarily as lactate in skipjack tuna (/E pelamis) 1~ and coho
salmon (Oncorhynchus kisutch) 63, arguing against a significant influence of fiver, or
any other gluconeogenic tissue. A recent study concludes that, in channel catfish
(Ictalurus punctatus), lactate is primarily released from white muscle 16. Estimation
of intraceUular lactate levels, based on eompartmentation analysis, suggests a peak
of no greater than 1 /zmol/g (1563 /zmol/kg body mass). As this is far below
the lactate level induced in most fish exercise studies, either the white muscle
lactate pool was underestimated, and consequently the degree of release into the
blood, or this species does not exercise as intensely as others (J.N. Cameron,
personal communication). In other species lactate turnover is a small fraction
of the rate of lactate disappearance from white muscle (tuna 2'1~ trout63). Even
if adequate gluconeogenic potential existed, glucose uptake, as indicated by 2-
deoxyglucose transport, by white muscle is too low (rainbow trout, 2.5 nmol/min/g,
T.G. West, unpublished) to account for the observed rate of glycogen repletion
(25-50 nmol/min/g) 6~ Thus, both extra-muscular glucose production and white
muscle glucose uptake are low compared to the observed rate of glycogen repletion.
Since glycogen recovers to control levels, it follows that this pathway occurs within
white muscle where lactate is retained. Furthermore, the smaller fraction that is
released following exercise may be taken up by white muscle in the latter stages of
recovery, relegating the plasma compartment to a short term lactate storage site 2.
6. 2. Route of glycogen resynthesis

While it is likely that white muscle glycogen resynthesis is the major fate of
lactate, the pathway by which this occurs is not established. At the beginning of
the gluconeogenic pathway, key enzymes responsible for net reversal of glycolysis
in liver are absent from white muscle. All vertebrate white muscles examined
lack pyruvate carboxylase 22. Alternate pathways in mammals and amphibia have
been suggested to involve malic enzyme plus phosphoenolpyruvate carboxykinase 7
(PEPCK) and reversal of pyruvate kinase 27 (PK). The situation appears more
straightforward in fish because PEPCK does not occur at detectable levels in white
muscle 74, except in marlin (Makaira nigricans) 98. Reversal of PK is thought unlikely
in liver because mass action ratios suggest it is too far from equilibrium to catalyze
a net reversal under physiological conditions. Pyruvate kinase in resting skeletal
muscle is much closer to equilibrium than in liver, due to higher free ATP/ADP
ratios (liver ~20, ref. 71 versus 200 in rainbow trout white muscle, ref. 94). In
recovering trout white muscle, the ATP/ADP ratio increases to approximately 2000
(ref. 94). Increases in pyruvate (6-fold in trout 98, 20-fold in tuna 2) would also favor
PK reversal. Changes in phospoenolpyruvate, the product for the reverse direction
have not been measured in recovery, but realistic estimates suggest that PK could be
close to equilibrium post-exercise 94. No direct evidence for PK reversal as the route
of glycogen resynthesis in muscle has been presented in any muscle. Examination
of PK activity in those species for which lactate disappearance rates are available
reveals an interesting relationship consistent with such a role for PK 74. Fig. 3
compares the rate of disappearance of white muscle lactate following exercise of
several fish species against the maximal velocity of PK (forward). A mean of 0.05%
of PK maximal velocity would be required for lactate conversion to glycogen at
the observed rates. Dyson and coworkers 27 suggest that PK could be driven in its
reverse direction at a maximal rate of about 2% of the forward maximal rate, well
above that which would be required in fish white muscle. Whatever the route of
glycogen production from lactate, mitochondrially produced ATP is required to fuel
net glycogen resynthesis.
Z Fuelforrecovery metabolism
We have argued that the early stages of recovery may be fuelled by glycolytic ATP
production, but it is expected that glycogen resynthesis and most of the recovery
costs will be met by mitochondrial ATP production. This stimulation of aerobic
metabolism is indicated by the excess post-exercise oxygen consumption 93, although
r
C
~A
o5
eo~U.1
.~_ 0
-o E
4~ v
y o 4
r
r
r
0.ol .2 I. . . . . . . . . . . . I
1 O0 1000
pyruvate kinase activity
(/~mol/min/g)
Fig. 3. Relationship between the rate of recovery from burst exercise (lactate disappearance) and
pyruvate kinase activity in fish white muscle. Enzyme activities were determined at the temperature
at which the recovery studies were performed. 1 = Starry flounder (Platichthys stellatus) recovery
rate 61 and p _y~vate kinase activity 74 ; 2 -- Sea raven (Hemitripterus americanus)57 ,74 ; 3 -- Northern pike
(Esox lucius)74,95; 4,5 - Rainbow trout (Oncorhynchus myk/ss)6~ 6 -- Skipjack tuna (Katsuwonus
pe/am/~) 2,34.
this typically exceeds the identifiable costs of recovery in fish93 and mammals 5.
The question of fuel oxidized under these conditions has only recently been
addressed 5,6,75. It is paradoxical that, under conditions where lactate is present at
very high concentrations, fat appears to be the metabolic fuel utilized in recovery
metabolism. This has been shown in mammals using arteriovenous metabolite dif-
ferences and respiratory quotients across exercised muscle groups 3,6. Difficulty in
obtaining a viable isolated perfused preparation has prevented a direct assessment
of respiratory quotients or metabolite changes across fish white muscle. An early
report examining whole animal respiratory exchange ratios, suggested that carbohy-
drate may be the fuel utilized by fish under similar conditions, but it is unlikely that
respiratory gases are in equilibrium at this time 63. Indirect studies using isolated
trout white muscle mitochondria suggest that fatty acid oxidation may be preferred
fuel in recovery. Fish white muscle mitochondria, as in mammals, oxidize pyru-
vate based fuels (e.g. lactate) in preference to fatty acids when each is presented
singly4,72 but when presented together, fatty acid oxidation inhibits utilization of
pyruvate 75. Control of fuel selection at the mitochondrial level in skeletal mus-
cle is primarily through regulation of pyruvate dehydrogenase. Although pyruvate
dehydrogenase is subject to both allosteric (stimulation, inhibition) and covalent
(activation, inactivation) regulation 35, it is likely that inhibition of pyruvate dehy-
drogenase is achieved allostericaUy in isolated white muscle mitochondria in vitro
and in recovering fish in vivo 75. As white muscle respiration rates in recovering
fish are a low proportion of the oxidative capacity, even a modest fatty acid supply
would be capable of meeting the needs of recovery metabolism 75.
8. Energy metabolism
During exercise, dramatic changes in the phosphagens occur which allow ATP to be
produced at very high rates to meet the demands of burst exercise. Phosphocreatine
is depleted as the free ATP/ADP ratio decreases and the tissue becomes acidotic 94.
A fraction of the ATP pool is catabolized to IMP via AMP deaminase 64,94. In
general the changes in phosphagens are greater than in mammals. In recovery, there
is a modest energy cost associated with re-establishing the phosphagen levels 93. Any
changes in free adenylates must be integrated with carbohydrate metabolism as they
are important regulators of both carbohydrate metabolism and the mitochondrial
pathways which must be used to provide ATP for net glycogen resynthesis.
8.1. Phosphocreatine
Few studies of fish burst exercise and recovery provide detailed time courses of
changes in phosphocreatine and these are limited to even fewer species (skipjack
tuna 2, salmonids23, 64,59,84,93,94) . As phosphocreatine resynthesis occurs as a conse-
quence of re-establishment of appropriate free ATP/ADP ratio, with consideration
of the intracellular pH, it provides some insight into the changes in adenylate status.
Most rainbow trout studies find phosphocreatine levels to recover quickly (<2 h),
however, two studies find phosphocreatine recovery to be much slower 23,64. Phos-
phocreatine resynthesis cannot be regulated separately, so delayed resynthesis does
not represent a shift in the intracellular metabolic priorities, but is indicative of a
persistent disturbance in either pH or free ATP/ADP. In both of the studies which
demonstrate this slow recovery of phosphocreatine, glycogen is severely depleted by
the end of exercise (< 1/zmol/g). Also, in studies where time-courses are sufficiently
fine and glycogen is not depleted, lactate production is observed to continue after
cessation of exercise (e.g. ref. 93). Taken together, these two observations suggest
that the earliest phase of recovery metabolism, which includes phosphocreatine
resynthesis, is dependent, paradoxically, on anaerobic glycolysis 23. Those individuals
which deplete glycogen in exercise, may possess inadequate glycolytic potential to
allow a fast recovery of phosphocreatine, indicative of depressed free ATP/ADP.
Immediately following exercise the free ATP/ADP ratio is low 94 and stimulatory
to both glycolytic and mitochondrial pathways. If post-exercise conditions are such
that mitochondrial metabolism is differentially suppressed, glycolytic ATP would be
required to meet the non-gluconeogenic demands of recovery. One possible such
effector is Pi, which is expected to be high post-exercise and inhibits mitochondrial
respiration at expected post-exercise levels 75. Coincident with recovery of phospho-
creatine concentration is net utilization of Pi. Thus, recovery of phosphocreatine
may be a prerequisite of utilization of mitochondrial pathways for ATP synthesis
and in situ glycogen resynthesis.
8.2. Adenylates
Investigation of recovery of adenylate status as an indicator of energy metabolism
must take into account the metabolite species present. Much of the acid-extractable
ADP pool in white muscle is bound to myofibrils. Thus, changes in total ADP
tell little about energy metabolism. Calculations of unbound adenylate species can
be performed assuming CPK and adenylate kinase are near equilibrium and their
metabolites are homogeneously distributed within the intraceUular space. When
only acid-extractable adenylates are considered, the minor changes observed mask
much greater changes occurring in the metabolically-relevant free pool. At present
only one study estimates the changes in free adenylates following exercise in fish
white muscle 94. Although total ATP is low for many hours following exercise, the
energy status (e.g. free ATP/ADP) recovers quickly and is even elevated above
rest 94. Both the quick recovery and apparent overshoot have important implications
in the integration of the post-exercise recovery pathways. Mitochondrial ATP
synthesis is stimulated by a decrease in ATP/ADP ratio, yet in recovery, when
an increase in whole animal respiration is known to occur, the ATP/ADP ratio
increases 94. If this ratio was the sole parameter to consider, a decrease in white
muscle respiration would be expected from the increase in ATP/ADP observed post-
exercise. It is likely that the increase in Pi stimulates respiration to provide ATP to
fuel recovery metabolism 75. Acidosis stimulates mitochondrial respiration when P~
is limiting, possibly by increasing the proportion of diprotonated Pi present. Thus,
in less extreme, more physiological exercise, when less Pi is expected to accumulate,
cytosolic acidosis would be expected to stimulate mitochondrial ATP production 75.
Again, it must be stressed that the changes in P~ expected from net phosphocreatine
breakdown and AMP deaminase activity have not been assessed directly.
Although energy status recovers quickly, total adenylate concentrations take
much longer ~'94. Resynthesis of ATP from IMP requires the re-aminating arm of
the purine nucleotide cycle64. During high intensity exercise, ATP is stoichiometri-
cally converted to IMP. As this IMP remains within the cell, there is no requirement
for adenosine synthesis for ATP production following exercise. The reaminating
arm of the purine nucleotide cycle involves two enzymes and requires input of
aspartate as the nitrogen source and GTP as phosphate and energy source 64.
9. Why is recovery so slow?
The earliest studies on recovery metabolism suggested that the rate of recovery
from burst exercise in fish was very slow by mammalian standards (e.g. refs. 37
and 56). Recent work using skipjack tuna suggests that this generalization was
more related to the choice of species 2. Lactate disappearance from skipjack tuna
white muscle following burst exercise occurs at rates within the range observed
in mammalian studies but in mammals, much of the lactate is released from the
working tissue and oxidized elsewhere 6,13. Thus, when one considers the difference
in the fate of lactate, tuna white muscle 2 can convert lactate to glycogen as
fast or faster than mammalian skeletal muscle even though the rate of lactate
disappearance is greater in mammals 6. When the full range of fish species studied is
compared, the rates of lactate disappearance span two orders of magnitude 74. The
biochemical basis of the interspecies differences has not been established. The rate
of lactate disappearance correlates with the aerobic capacity of the white muscle 74.
Resynthesis of glycogen post-exercise demands mitochondrial ATP production
and white muscle is a mitochondria-poor tissue but the simplest explanation for
this relationship, that maximal mitochondrial ATP producing capacity limits the
rate of glycogen resynthesis, is unlikely75,94. The high free ATP/ADP observed
throughout recovery would actually suppress mitochondrial ATP production to
approximately 10-30% of the maximal capacity7s. The high free ATP/ADP ratios,
while suppressing mitochondrial respiration, may be required to drive PK in the
reverse direction for net glycogen resynthesis94. As was mentioned previously, the
activity of PK also correlates with the rate of recovery across these same species
(Fig. 3). Direct demonstration that the capacity for PK reversal limits the rate of
recovery across species has proven elusive.
10. Summary of burst exercise and recovery
Burst exercise is fuelled by glycogen breakdown to lactate. Reliance on circulatory

glucose is minimal. Adenylates and phosphocreatine undergo much greater changes
than are observed in mammals. Although total ATP remains low for several
hours, post-exercise energy status, as indicated by phosphocreatine/creatine or free
adenylate ratios, recovers more quickly. In species or stocks with severe glycogen
depletion, the time required for recovery of energy status is extended. Reduction
of Pi through post-exercise glycolysis and net phosphocreatine synthesis may be a
prerequisite for rapid recovery. Resynthesis of glycogen occurs primarily in white
muscle and requires much longer to recover than does energy status. Recovery is
faster in species possessing: (1) more aerobic white muscle; and (2) more pyruvate
kinase. Unlike mammals, oxidation is thought not to be a quantitatively important
fate of white muscle lactate post-exercise. Glycogen resynthesis occurs in white
muscle, and probably through pyruvate kinase, reversed in vivo by high pyruvate and
free ATP/ADP ratios. Direct evidence for this or any route of glycogen resynthesis
is presently lacking. Glycogen resynthesis is probably fuelled by mitochondrial
oxidation of fatty acids, sparing lactate for glycogen resynthesis.
Note added in proof
Recent studies (A. Pagnotta, L. Brooks and L. Milligan. Potential regulatory

roles of cortisol in recovery from exhaustive exercise in rainbow trout. Can. J. Zool.,
in press) have shown that the rate of recovery from burst exercise is profoundly
influenced by circulating cortisol levels. If exercised fish are allowed to swim during
recovery, cortisol levels remain low and recovery rate increases 2--4 fold.
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92. Saddik, M. and G.D. Lopaschuk. Myocardial triglyceride turnover and contribution to energy
substrate utilization in isolated working rat hearts. I. Biol. Chem. 226: 8162-8170, 1991.
93. Scarabello, M., G.J.E Heigenhauser and C.M. Wood. The oxygen debt hypothesis in juvenile
rainbow trout after exhaustive exercise. Resp. Physiol. 84: 245-259, 1991.
recovery of white muscle. J. Exp. Biol. 166: 181-195, 1992.
95. Schwalme, K. and W.C. McKay. The influence of angling-induced exercise on the carbohydrate
metabolism of northern pike (Esox lucius L.). J. Comp. Physiol. 156B: 67-75, 1985.
97. Spriet, L.L., L. Berardinucci, D.R. Marsh, C.B. Campbell and TE. Graham. Glycogen content has
no effect on skeletal muscle glycogenolysis during short-term tetanic stimulation. J. Appl. Physiol.
68: 1883-1888, 1990.
98. Suarez, R.K., M.D. Mallet, C. Daxboek and P.W~Hochachka. Enzymes of energy metabolism and
gluconeogenesis in the Pacific blue marlin, Makaira nigricans. Can. J. Zool. 64: 694-697, 1986.
99. Suarez, R.K. and T.P. Mommsen. Gluconeogenesis in teleost fishes. Can. I. Zool. 65: 1869-1882,
1987.
100. Tappy, L., IC Acheson, S. Normand, D. Schneeberger, A. Thelin, C. Pachiaudi, J.-P. Riou and E.
Jequier. Effects of infused amino acids on glucose production and utilization in healthy human
subjects. Am. J. Physiol. 262: E826-E833, 1992.
I01. Tang, Y. and R.(3. Boutilier. White muscle intracellular acid-base and lactate status following
exhaustive exercise: a comparison between freshwater- and seawater-adapted rainbow trout. J.
Exp. Biol. 156: 153-171, 1991.
102. Van den Thillart, G. Energy metabolism of swimming trout (Salmo gairdneri). J. Comp. Physiol.
156B: 511-520, 1986.
103. Van den ThiUart, (3., A. Van Waarde, H.J. Muller, C. Erkelens, A. Addink and J. Lugtenburg.
Fish muscle energy metabolism measured by in vivo 31p-NMR during anoxia and recovery. Am. J.
Physiol. 256: R922-R929, 1989.
104. Van Waarde, A. Aerobic and anaerobic ammonia production by fish. Comp. Biochem. Physiol. 74B:
675-684, 1983.
105. Van Waarde, A., G. van den Thillart, H.J. Muller, C. Erklelens, A. Addink and J. Lugtenburg.
Functional coupling of glycolysis and phosphocreatine utilization in anoxic fish muscle -an in vivo
alp NMR study. J. Biol. Chem. 265: 914-923, 1990.
106. Walsh, P.J. An in vitro model of post-exercise hepatic gluconeogenesis in the gulf toadfish Opsanus
beta. J. Exp. Biol. 147: 393-406, 1989.
107. Webb, P.W. The swimming energetics of trout I. Oxygen consumption and swimming efficiency. J.
Exp. Biol. 55: 521-540, 1971.
108. Weber, J.-M. Effect of endurance swimming on the lactate kinetics of rainbow trout. J. Exp. Biol.
158: 463-476, 1991.
109. Weber, J.-M., R.W. Brill and P.W. Hochachka. Mammalian metabolite flux rates in teleost: lactate
392 CD. Moyes and 7:.(;. West
1 I0. Weber, J.-M. Pathways for oxidative fuel provisions to working muscles: ecological implications of
maximal supply fimitations. Hrperientia 48: 557-564, 1992.
III. Weibel, E.R. Design and performance of muscular systems: an overview. J. Exp. Biol. 115: 405--412,
1985.
112. West, T.G., P.O. Arthur, R.IC Suarez, C.J. Doll and P.W. Hochachka. In vivo utilization of glucose
by heart and Iocomotory muscles of exercising rainbow trout (Oncorhynchus mykiss). J. Exp. Biol.,
177: 63-79, 1993.
113. Westserblad, H., J.A. Lee, J. Lannergen and D.G. Allen. Cellular mechanisms of fatigue in skeletal
muscle. AtrL J. Physiol. 261: C195-C209, 1991.
rainbow trout (Salmo gairdneri) during and after bursts of activity. J. Cornp. Physiol. 155B: 485--492,
1985.
115. Wokoma, A. and I.A. Johnston. Lactate production at high sustainable cruising speeds in rainbow
trout (Salmo gairdneri Richardson). J. Exp. Biol. 90: 361-364, 1981.
116. Wood, C.M. Acid base and ion balance, metabolism, and their interactions after exhaustive
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CHAPTER 17
Fasting and starvation
ISABEL NAVARRO AND JOAOUIM GUTIl~RREZ

Depanament de Bioqufmica i Fisiologia, Universitat de Barcelona, Unitat de Fisiologia, Avda.
Diagonal, 645, E-08071 Barcelona, Spain
I. Introduction
II. Carbohydrate mobilization
1. Liver glycogen
2. Blood glucose
3. Muscle glycogen
III. Lipid mobilization
1. Liver
2. Intestinal fat
3. Muscle lipids
4. Lipid classes
4.1. Free fatty acids
5. Influence of starvation on lipolysis and lipogenesis
IV. Protein mobilization
1. Liver proteins
2. Muscle proteins
3. Plasma protein and amino acids
V. Endocrine regulation
1. Insulin
2. Glucagon and glucagon-like peptide
3. Glucocorticoids
4. Growth hormone
5. Thyroid hormones
VI. General conclusions
Acknowledgements
VII. References
I. Introduction
Fish can survive for a long time without food and for many species a fasting period
forms part of the natural life cycle. Winter months, spawning migration and/or pre-
spawning stage can all be naturally non-feeding periods. Thus, numerous species
can starve for many months and then recover fully after refeeding. Therefore,
these species are well adapted to mobilize their metabolic reserves and even body
constituents to survive periods of food deprivation.
Specific effects of starvation on metabolism are dependent on multiple variables;
including the species under consideration, the preferential tissues for metabolic
stores, the quantity stored and their availability as well as distinct routes of mobi-
lization. In this sense, the pre-fasting diet may exert substantial influence on the
394 I. Navarro and I. Guti~rrez
metabolic events initiated by fasting75. It is also important to differentiate between

experimental and natural fasting, because natural fasting can be accompanied by
other, compounding factors, such as gonadal growth, low temperatures, etc. Exper-
imental fasting effects are likely dependent on endogenous and exogenous factors.
For instance, the season chosen, temperature and photoperiod, fish age and whether
or not the fasting is imposed during a reproductive period will all have substantial
bearing on the experimental results. Therefore, all these factors have to be taken
into account when comparing fasting responses between species or even within
species.
Another important aspect when comparing the experimental results of fast-
ing periods in fishes is the way in which the data are expressed. Machado and
eoworkers 114, for instance, pointed out that many discrepancies found in the liter-
ature can be explained because conclusions are obtained on the basis of changes
in the concentrations of tissue metabolites, being more informative than changes in
total amounts stored in tissue.
Similarly, Black and Love 15 found a great deal of clear information when data
were considered in relation to one another. In the same vein, Foster and Moon 55
indicated the advantage of using body weight and tissue DNA content as reference
points for metabolic and enzymatic parameters.
The aim of this chapter is to give a brief overview of gross metabolic and
enzymatic changes observed in fasting and starving fish. We have chosen the storage
substrate as the organizing principle, before focusing on various parameters of
endocrine regulation during fasting and starvation in fishes.
II. Carbohydrate mobilization
1. Liver glycogen
Carbohydrates are stored as glycogen in liver normally amounting to 1-6% of liver
weight, although some species, such as carp (Cyprinus carpio), can accumulate values
exceeding 10%. When required, hepatic glycogen is enzymatieaUy broken down and
transported to the extrahepatie tissues as glucose. On arrival at its target cells,
glucose is either metabolized or reconverted into glycogen. Unfortunately, liver
glycogen determination has a number of inherent problems which may complicate
the interpretation of observed changes in this particular parameter under some
experimental conditions. First, variation in the amount of liver glycogen is large
between individuals; and second, as the example of the carp shows, concentrations
may be exceptionally high. Because liver glycogen is normally determined in
terminal samples, it is inherently diftieult to determine (or detect) small changes in
liver glycogen and often even large decreases may be missed due to large individual
variability. Usually, glycogen depletion is a continuous process from the beginning
of fasting 1~ In most species, liver glycogen is mobilized early in experimental
fasting in fish; in fact, partially because of the ease of mobilization, liver glycogen
is generally the first substrate to be used during fasting. At 5, 8, 15, 20 days
Fasting and starvation 395
TABLE 1
Variation of body parameters and liver and muscle components in fed and fasted trout (Salmo tmtta
fario)
Fed (days) Fasted (days)

1 15 30 50 8 30 50
Body parameters
Weight (g) 163 175 174 201 135 134 121
Length (cm) 22.7 22.0 24.4 25.3 21.6 23.8 23.7
HSI (%) 1.24 1.09 1.03 1.08 0.99 0.83 a,b 0.77 a,b
MLI (gcm -1) 3.6 4.2 3.9 4.41 3.2 a 3.1 a 2.9 a,b
VI (%) 8.4 9.1 6.1 9.9 5.0 a'b 4.5 a'b 3.6 a,b
Liver composition
Glycogen (%) 4.6 3.9 3.9 2.5 0.9 a,b 0.9 a,b 0.6 a,b
Protein (%) 14.3 15.1 15.3 15.4 16.7 a'b 16.7 a,b 16.1 a,b
Lipids (%) 3.7 3.3 4,0 4.3 3.7 3.8 3.6 a
Water (%) 75.1 75.1 74.6 75.3 76.0 a,b 75.5 a 76.2 a,b
P-DNA (mg 100 g-l) 27.8 35.4 b 36.2 b 39.1 b 43.0 a,b 51.6 a,b 57.3 a,b
Muscle composition
Glycogen (%) 0.24 0.27 0.27 0.30 0.20 0.10 a,b 0.09 a,b
Protein (%) 19.7 19.2 19.6 19.3 18.8 18.8 18.2 a,b
Lipid (%) 1.5 1.6 1.4 1.7 1.5 1.1 1.0 a
Water (%) 77,8 77.7 77.5 76.9 78.1 77.8 78.5 a
P-DNA (mg 100 g-l) 2.5 2.7 2.2 1.9 3.3 2.4 2.1
Results are means from 10 fish in each group.
a Indicates significantly different (p < 0.05) from fed fish at the same point (Duncan's test).
b Indicates significantly different (p < 0.05) from the fed group at day 1 of the experiment (Duncan's
test). Abbreviations: HSI ffi Hepatosomatic index (gram liver weight 100 g-1 body weight); MLI =
Index of muscle weight (g) over length (era); VI ffi Index of viscera weight (g) without liver per 100
g body weight; P-DNA ffi DNA phosphorous (mg) per 100 g tissue fresh weight. Maximum standard
error of individual obervations were below 15%, in most cases below 10% of the mean. Adapted from
Navarro et al. TM.
fasting, significant decreases in glycogen have been described in several species of

teleostean fishes 65,75,114,115,135,169 o r elasmobranchs 47. Table 1 shows the effects of
15, 30 and 50 days of starvation on liver and muscle glycogen in brown trout (Salmo
trutta fario ).
However, several species undergo prolonged periods of fasting in their natural
environment with little depletion of liver glycogen. Pacific sockeye salmon (On-
corhynchus nerka) migrate some 1000 km without decrease in this reserve 58. Fasted
European eels (Anguilla anguilla) did not show a change in liver glycogen after
95 days fasting 96. As mentioned above, carp (C. carpio) accumulate exceptionally
high levels of liver glycogen and after 12 months of starvation still maintain 6% of
glycogen in their livers 192. Nagai and Ikeda 133 indicated that 22 days fasting in carp
(C. carpio) did not provoke any change in liver glycogen (10.65%) and it was only
after 100 days that a clear decrease was observed (1.55%). Such an obvious defence
of hepatic glycogen, even after long periods of starvation, may be explained by a
compensatory increase in the rate of gluconeogenesis from non-carbohydrate pre-
cursors, which has two effects, namely: (1) that hepatocyte glycogen can be spared;
396 L Navarro and J. Guti~rrez
TABLE 2
Variation of body parameters and liver and muscle components in fed and fasted yellow perch (Perca
flavescens)
Fed Fasted for 7 weeks

Body parameters
Condition factor 1.26 1.13"
Hepatosomatic index (%) 1.99 0.85 a
Plasma
Glucose (mM/l) 4.27 3.03 a
Liver composition
Glycogen (/zMol//~g DNA) 253 40.8'
Protein (mg//zg DNA) 43.9 28.0 a
Glucose (/zMol//zg DNA) 73.0 98.0
DNA (~g/g tissue weight) 2.4 4.5 a
Muscle composition
Glycogen (/xMol/ttg DNA) 69.4 43.3 a
Protein (mg//~g DNA) 883 700 a
Glucose (/zMol//zg DNA) 17.4 7.6 a
DNA (/xg/g tissue weight) 0.22 0.25
Values are means of 3 to 12 observations.
a Significantly different from fed control fish (~p < 0.05; Student's t-test).
Condition factor -- gram body weight per cm ~ body length. Adapted from Foster and Moon 5s.
and (2) that the hepatic production of glucose 6-phosphate can lead to an increased
carbon flux into glycogen. Increase in glyconeogenesis was suggested in A. an-
guilla after 95 days fasting from the increase of the hepatic glutamate-oxaloacetate
transaminase (aspartate aminotransferase, AspAT, EC 2.6.1.1), together with the
maintenance of glycogen. Liver gluconeogenesis was also enhanced in 6- or 7-week
fasted rainbow trout 126 (O. mykiss) or yellow perch 55 (Perca flavescens) (Table 2),
respectively. The percentage per wet tissue weight of key gluconeogenie enzymes
AspAT, phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) and lactate de-
hydrogenase (LDH, EC 1.1.1.27) significantly increased in yellow perch 55. These
data are in agreement with the observations of Morata and eoworkers 129 who
found increases in liver and kidney gluconeogenesis during the second month of
starvation in rainbow trout, and with those of French and coworkers 59,5a who
determined relative increases in PEPCK and LDH in starving and exercising rain-
bow trout and increases in PEPCK and glutamate-pyruvate transaminase (alanine
aminotransferase, AlaAT, EC 2.6.1.2) in migrating (starving) sockeye salmon.
It is clear that in the face of rapid proteolytie degradation, the activities of key
enzymes are somehow defended. However, the mechanism of this defence is not
clear until more detailed research, preferably at the molecular level, has been done.
For instance, it is not obvious from the descriptive studies mentioned above: (1)
whether these enzymes are protected against a generalized proteolytic attack; (2)
whether the proteolytic attack, which involves selective activation of eathepsin-like
proteases, is general in nature; or (3) whether compensatory increases in enzyme
synthesis are responsible for the apparent maintenance of such key enzymes. Thus,
this topic would make an ideal area to apply molecular biological techniques to
probe mRNA longevity, transcription rates and possible induction of key enzymes.
Of the enzymes mentioned, it is known from other vertebrates that transcription
of PEPCK is under multifaceted endocrine control, involving crosstalk between
different regulatory pathways. The applicability to piscine systems, however, re-
mains to be elucidated. At any rate, the nature of the key enzymes purportedly
preserved, more than hints at key metabolic pathways which retain importance and
metabolic flux throughout starvation. Applying a teleological view to intermediary
metabolism, all key enzymes support the idea of a drastically changed emphasis
on amino acid metabolism, possibly with concurrent increases in the importance of
gluconeogenesis. The transaminases can be considered feeder enzymes for carbon
into oxidative and gluconeogenic pathways, while PEPCK plays a pivotal role in
the flux of carbons from C3, C4 and C5 precursors (which can be derived from
most amino acids) into glucose. Nevertheless the often times noticed maintenance
of LDH poses a bit of a problem to the picture outlined above.
At any rate, however, given the decrease in liver glycogen, the overall contribu-
tion of glycogen to the total energy expenditure is comparatively small considering
the limited weight of the liver (hepatosomatic index ranging normally from 1 to 3%;
of. Tables 1 and 2).
2. Blood glucose
Although blood glucose in fishes is known to fluctuate widely between species and
also within species, within a given, uniform batch of individuals, blood glucose con-
centrations are maintained at a steady level during long periods of food deprivation.
It is thought that this apparent defence of blood glucose against fluctuations or
depletion occurs largely at the expense of liver glycogen, at least during the initial
stages of fasting. Hochachka and Sinclair77, for instance, found a decrease in liver
glycogen without modification of blood glucose in 14-day fasted rainbow trout.
Not surprisingly, glycemia profiles during fasting vary depending on the species
considered, and this is one area where other physiological factors mentioned above
are likely to exert substantial influence. In juvenile sea bass (Dicentrarchus labrax),
glycemia is decreased after a 5-day fast, and in juvenile brown trout (Salmo trutta
ratio) after a 10-day fast 65'135. Zammit and Newsholme 198, working on adults of the
same species, failed to observe changes in glycemia after imposing a 40-day fast.
It was only after 100 days of food deprivation that blood glucose levels decreased
significantly. The hagfish (Myxine glutinosa) seems to follow a similar pattern as the
teleosts, since this species maintained blood glucose throughout a three-week fast 43.
An entirely different picture emerges for an elasmobranch fish, exemplified by the
response of the common dogfish. In these animals (Scyliorhinus canicula), whose
metabolism is even less 'glucosocentric' than in teleostean fishes, plasma glucose
decreases rapidly during food deprivation and reaches a low of 0.52 mg/100 ml (0.03
mM) within eight days of food deprivation; however, later during starvation, blood
glucose levels recover and reach pre-fasting levels (12.05 mg/100 ml; 0.67 mM) after
67 days of fasting47.
In carp and eel, no changes in glycemia were observed after several weeks of
fasting 34,131. Starved Clarias lazera (a warm-water catfish) experienced no change
in blood glucose after four months, although this parameter decreased to 60%
after an additional three months of experimentation 68. Sluggish fish seem to be
able to maintain glucose levels for longer periods than more active species, likely
reflecting the comparatively smaller demand and hence turnover of glucose in these
species. As shown by Weber and coworkers 1~,~89, a strong positive correlation exists
between glucose turnover and assumed physical activity of teleostean fishes. In
the case of the extremely active skipjack tuna (Katsuwonus pelamis), for instance,
glucose turnover rates approach or even surpass those of many mammals (cf.
chapter 2, this volume).
In carp (C. carpio) an increase in glycemia was observed in early starvation 133.
It is possible to distinguish two phases in the maintenance of blood glucose during
fasting and starvation in fishes; one is that glucose sequestered from the plasma
pool is replenished by liver glyeogenolysis; the other is replenished through the
compensatory activation of glueoneogenesis, especially from amino acids mobilized
though preferential proteolysis of muscle 114. However, as the studies mentioned
above already reveal, a clear temporal or functional relationship between these
phases, as exists in many mammals, cannot be delineated. At any rate, a combination
of both phases is clearly involved in the stabilization of blood glucose during
starvation.
3. Muscle glycogen
The amount of glycogen stored in fish muscle falls into the range between 0.2 and
1.3% per wet weight and is thus generally assumed to make only a small potential
contribution to total energy expenditure ~14. However, it should be kept in mind,
that because of its immediate involvement in muscular activity, muscle glycogen is
a most volatile entity and its mobilization is more likely related to an increase in
muscular activity than to the fasting process. Not surprisingly, the rate of muscle
glycogen mobilization is positively correlated with ambient temperatures. In brown
trout (S. trutta fario) significant decreases in muscle glycogen were found at 8 days
fasting in summer, but only at 30 days fasting in winter (Table 1) 135. Similarly,
in rainbow trout (O. mfldss) muscle glycogen is lowest in summer 61 when high
temperature and likely increased activity of the fish combine to drain the muscle
glycogen stores.
In Atlantic cod (Gadus morhua), at early stages of starvation, both liver and
muscle glycogen were mobilized. But red muscle glycogen remained unchanged
during most of the depletion 15.
However, in several fish species glycogen in muscle is not mobilized and it
seems likely that this store is maintained at the expense of blood glucose which is
supplied by hepatic processes (gluconeogenesis, glycogenolysis). In juvenile sea bass
(Dicentrarchus labrax) fasted 22 clays in summer, muscle glycogen decreased very
little 65, while carp fasted 19 or 67 days did not show a depletion in muscle glycogen
levels is. Similarly, European eels fasted for 164 days maintained unchanged muscle
Fastingand starvation 399
glycogen concentrations 34. A comparable pattern seems to hold at least for one
species of elasmobranch: 82 days of fasting did not provoke a decrease in muscle
glycogen in S. canicula 47.
In conclusion, although important differences may exist in carbohydrate metab-
olism among teleostean species, a tendency to maintain stable glucose values dur-
ing starvation is observed. Concentration of liver glycogen varies between species,
but this is the main tissue for glycogen storage, and liver glycogenolysis accounts
for maintenance of glycemia during early starvation (weeks). Liver gluconeogenesis
from amino acids (AA) and lactate will supply glucose for later (months) stages of
starvation. A different picture emerges for the representative (S. canicula) elasmo-
branch, with lower dependence on glucose and more exaggerated lipid metabolism.
III. Lipid mobilization
Apart from the at times obvious changes in carbohydrates, many authors are
convinced that lipids are mobilized first. Lipid depletion during starvation has
been demonstrated in many species of fish, marine as well as freshwater, and this
literature has been comprehensively reviewed by Sargent and coworkers TM.
An inverse relationship exists between lipid and water contents and catabolized
lipids are replaced by an equal volume of water 1~ Thus, under conditions of
moderate starvation and lipid loss, body weight is maintained through the uptake of
water. This phenomenon is best exemplified (and exaggerated) in migrating sockeye
salmon (Oncorhynchus nerka): these animals use up their entire - substantial -
storage lipids in the first part of their migration, yet do not change body weight
appreciably during this phase 82. Values exceeding a water content of 81% are
reached in fish muscle, when only very low levels of liver lipids can be detected 1~
The strategy of unloading body storage lipids is similar in species classified as
fatty and those considered non-fatty, in that the bulk of lipid is used first, before
proteins are drawn upon. Lipids can be stored in liver, intestinal fat and muscle,
with the quantitative importance of each tissue varying between species. Salmonids
generally rely on the deposition of visceral fat, while many gadid species accumulate
hepatic storage lipid, and clupeiformes contain high contents of lipids in muscle.
Independent of the actual site of deposition, lipids will be mobilized from these
sites during starvation.
1. Liver
As mentioned above, the actual decrease of tissue lipid during fasting varies
between species depending on the tissue for lipids storage and also depending on
the strategy followed in mobilizing other reserves including carbohydrates. In the
Atlantic cod (Gadus morhua), for instance, liver lipids decreased drastically with
fasting; in fact this storage depot was exhausted even in the face of an appreciable
quantity of the initial glycogen reserve remaining 15. Carp could also be included in
this group of species that spare glycogen reserves to a certain degree. In carp (C.
400 L Navarro and Z Guti~rrez
carpio) after 19 days of fasting, an appreciable depletion on liver lipids occurred,

while the fish seemed to protect their entire store of hepatic glycogen throughout
this period ~7. In longer term experiments, where carp were fasted for 100 days, liver
lipid was exhausted, while glycogen level was retained at about 1.6% (ref. 133).
On the other hand, the rest of body fat reserves tends to be depleted before
liver lipid is mobilized in salmonids. Nevertheless, substantial decreases in hepatic
content of storage lipids can be detected in rainbow trout (O. mykiss) 115 or brown
trout (Salmo trutta fario) with prolonged fasting (Table 1) 135.
In sea bass (Dicentrarchus/abrax), a species which stores lipid predominantly
in mesenteric fat, fasting for 22 days 65 or 130 days 27 did not provoke significant
decreases in hepatic lipids. In contrast, Stirling 169 did notice decreases in liver lipids
from sea bass that were fasted for 26 and 60 days.
2. Intestinal fat
The major lipid storage site of many teleostean fishes is mesenteric fat. For
instance, lipid accumulated by rainbow trout (O. mykiss) is deposited preferentially
in perivisceral adipose tissue 16~ and it is this mesenterie fat that is first mobilized in
fasted rainbow trout. In this species, during 48 days fasting, more lipid was mobilized
from perivisceral depots than either liver or muscle 9~ Species with sustained
accumulation of mesenteric fat usually follow the same strategy of mobilizing this
lipid reserve preferentially. In Esox lucius intestinal lipids are mobilized before any
change in liver lipid is apparent a6. Similarly, in the sea bass (Dicentrarchus/abrax),
mesenteric lipid depots decrease by 41% after 15 days of fasting compared with
control fish65, an observation supporting earlier data in the same species 198.
3. Muscle lipids
In species storing lipids in muscle, mobilization of intramuscular lipid is initiated as
soon as food intake ceases. An excellent case showing this point is the freshwater
teleost Rhamdia hilarii. This catfish has no organized or visible adipose tissue in
the abdominal cavity and during fasting intramuscular lipid (average of 5% wet
weight) plays an important role for the overall energy requirement 114. In this
species, after 30 days of fasting, muscle fat was reduced to one-fifth of fed values,
which, considering that muscle constitutes 60-70% of fish body weight, represents
an important source of energy. In species devoid of intracellular storage lipid,
structural muscle lipid is usually retained until later phases of starvation when
protein structures begin to be broken down. In Gadus morhua, for instance, a
species that has little lipid stored in the skeletal muscle and considerable amounts
stored in the liver, this hepatic reserve is used first. Muscle protein is degraded and
partial destruction of structural lipids may occur at the final stages of starvation,
only when other sources of energy have been nearly consumed.
Muscle lipids in sea bass, which preferentially deposits lipids for storage in
mesenteric fat, failed to show significant changes during fasting 6s'169, while in
starving trout, muscle lipids underwent decreases after a 50-day fast 9~ but
experienced no changes during a 27-day fast. Muscle lipids in carp (Cyprinus carpio)
are not easily mobilized in fasting, possibly reflecting the sluggish activity pattern
of this species. Working on carp, Takeuchi and colleagues 17s failed to observe
variations in muscle lipids as a consequence of fasting. Identical results have been
reported for fasting plaice (Pleuronectes platessa) 91. However, it should not be
forgotten that the metabolic requirements of fasting species may be adjusted to the
amount and availability of storage substances accumulated around the body. Fasting
yellow perch (Perca flavescens), for instance, were found to be able to enter into
a stage of hypometabolism, thus decreasing metabolic output and sparing storage
substances 5s. Unfortunately, the potential for such metabolic 'down-regulation' has
not been widely appreciated in fishes and is likely overlooked unless stringent
controls are incorporated into the experimental protocols. Of course the possibility
of behavioral hypometabolic regulation in natural settings should not be overlooked,
and likely deserves more attention than it has garnered at this point. For instance,
lower rates of oxygen consumption and activities of energy metabolism enzymes
in deep-living fishes may reflect a combination of factors: reduced abundance of
food; low temperature and water oxygen concentration; and deprived light intensity,
which may account for reduced locomotory activity19s. At any rate, the potential
for hypometabolic regulation is well described for other vertebrates (hibernators,
estivators) and this is certainly an area of research where fish could add another
useful model to the analytical arsenal.
In fishes, red muscle contains more lipids than white muscle; for example, in
Atlantic salmon, white muscle has a lipid content of 2%, while red muscle has about
15% (ref. 108). However, dark muscle lipid is not easily mobilized, which may be
related to the special properties of these types of tissues. Red muscle is used for
sustained swimming and for activities such as maintaining body position against
currents, whereas white muscle is used intermittently, for sudden movements in
case of pursuit or escape. Some diminution in locomotory capacity during food
deprivation may be tolerable 195.
Although generally the muscle of elasmobranchs tends to contain more lipid than
that of teleostean fishes, the specific composition may indicate that the bulk of this
lipid represents structural rather than storage material and therefore is not easily
mobilized. In agreement with this hypothesis, 82 days of starvation did not provoke
significant changes on muscle lipids of S. canicula 47.
4. Lipid classes
During starvation-induced breakdown of lipids, different phases can be distin-

guished. The most accessible lipid store appears to be triacylglycerols (TAG,
triglycerides), well exemplified in the European eel (Anguilla anguilla). In this
species, triglycerides were the main energetic substrate during 95 days fasting96.
Other (structural) lipids, in contrast, are generally entirely spared or used only
during the later phases of food deprivation. As a result of focused breakdown of
triglycerides, the specific fatty acid composition changes in the course of starvation.
Since myristate (14:0), palmitoeate (16: 1), and oleate (18: 1) are mobilized prefer-
402 L Navarro and 1. Guti~rrez
entially, their abundance in the remaining triglyceride declines almost continuously

in the liver of A. anguilla in the course of starvation 35.
As a rule, triglycerides are mobilized before phospholipids during starvation 161,
reflecting the duality of storage versus structural lipid. As delineated for the
European eel liver above, there also is an apparent selectivity in the fatty acids
mobilized. In rainbow trout, saturated fatty acids were mobilized in preference to
other types of fatty acids from perivisceral fat deposits. Similarly, the bulk of fatty
acids depleted from liver and muscle lipids were 16:1, 18:1 and 20:1 (ref. 90). A
marked decrease in the 18: 1 content of body and liver of starved rainbow trout was
reported 161.
In salmon, spawning depletion resulted in utilization of long-chain mono-unsatu-
rated acids such as 20:1 and 22:1, while those of shorter chains and polyunsaturated
acids where consumed later s9. Levels of 20" 5 and 22' 6 were maintained or propor-
tionally increased in Fugu vermicularis porphyreus during early fasting72. In stock fish
(Merluccius capensis) a relative increase in the degree of unsaturation is noted during
starvation 19~ It should be kept in mind that the proportion of unsaturated fatty acids
as well as the degree of unsaturation exert pronounced effects on membrane fluidity.
Therefore, it is likely that the retention of certain fatty acids at the expense of others
is guided not only by the necessity to requisition carbons for oxidation but also by the
need to maintain structural integrity and fluidity of the membrane. As a result it can
be expected that the mobilization of unsaturated fatty acids from lipids will differ in
warm-water- and cold-water-adapted species.
Obviously, there is a natural limit to the amount of structural lipids that must be
retained. Below this threshold, essential membrane functions will be compromised
and the survival of the fish is in question 1~ In Perca flavescens, this limit seems
to be reached when only 2.2% of overall lipid is left. Below this critical value the
animals die 137.
4.1. Freefatty acids

The fasting-induced pattern of changes in plasma free fatty acids (FFAs) varies
between species, but clearly does not lead to the rapid and marked increase of
FFA familiar in starving mammals 13. Researchers noted decreases of FFA in 30-
and 90-day starved oyster toadfish (Opsanus tau) 176while rainbow trout experienced
increases 166 or no change under the same conditions. In the European eel (A.
anguiUa) no changes in plasma FFA were noticed during the initial 95 days of fast,
followed by a marked rise thereafter 96. A noticeable increase with 3 or 5 days fasting
is found in Limanda limanda 49. In sea bass (D. labrax) 40 days of starvation caused
an increase in plasma FFA concentration of about 65% along with a 3- to 7-fold
increase in plasma glycerol concentration 198 . In fasting catfish (R. hilarii) plasma
FFA increased two-fold during the first 30 days 114. However, initially FFA levels do
not increase, and authors suggested that all FFA produced was first utilized locally,
and it is not until later stages of starvation that a spillover of fatty acids into the
plasma compartment is noticed.
It is well known that in fasted teleosts, in stark contrast to mammals, the pro-
duction and utilization of ketone bodies does not play an important role 15'147'198.
In fact, it is under debate whether ketone body metabolism forms an integral

part of fish intermediary metabolism at any time. First, some studies showed that
3-hydroxybutyrate dehydrogenase (EC 1.1.1.28), a key enzyme in ketone body pro-
duction, was lacking in the livers of teleost fish 9'197'198. Nevertheless, LeBlaneh
and Ballantyne ~~ have demonstrated the presence of that enzyme in tissues
of some freshwater species such as goldfish (Carassius auratus), brown bullhead
(Ictalurus nebulosus), pike (Esox lucius) and crappie (Pomoxis nigromaculatus).
3-Hydroxybutyrate dehydrogenase activity has also been found in marine teleost
species: alewife (Alosa pseudoharengus), smelt (Osmerus mordax), and mummichog
(Fundulus heteroclitus). The levels of the enzyme in freshwater fishes are highest
in the liver and kidney, tissues known to be ketogenic in other vertebrates 9, which
could suggest a similar role of ketone bodies in fishes. But the levels of this key
enzyme in ketone body formation in the marine species studied did not display
the same tissue pattern, since the highest levels in the alewife were found in the
brain, while the heart of the smelt had the highest activity. Second, under normal
physiological conditions the concentrations of ketone bodies in tissues or plasma
and the rate of utilization are very low48 suggesting the idea of a minor role in
teleost metabolism. No data exist showing that ketone bodies make a substantial
contribution to the energetic requirements of teleost fishes during starvation.
On the other hand, the presence of the enzyme 3-hydroxybutyrate dehydrogenase
in elasmobranchs was observed in earlier studies 12s,198. The levels of this enzyme
and plasma levels of ketone bodies are both higher than in teleosts 47,198 and isolated
hepatocytes will readily use and convert added ketone bodies 119. These results in-
directly support an earlier contention that plasma transport of fatty acids may be
limited in elasmobranchs and other organisms lacking carder proteins such albumin.
Thus, ketone bodies may provide a more soluble lipid transport form 4,5,1~ Never-
theless, the role of ketone bodies in energy production and thus their contribution
to energy supply during fasting is doubtful. Contrasting with increments of plasma
ketone bodies observed in fasted mammals, plasma levels of these metabolites were
not significantly altered in starved skate (Raja clavata) 197, but a large increase was
observed at 36 days fasting in dogfish (Scyliorhinus canicula)47.
5. Influence of starvation on lipolysis and lipogenesis

Generally, information on lipid mobilization in lower vertebrates is scant. Lipolytic
activity has been found in salmonid adipose tissue, liver and red (dark) muscle 164.
In trout adipose tissue, the triglyceride lipase is subject to covalent modification
through phosphorylation, with activity increasing as the enzyme is phosphorylated
(K. Michelsen, J. Harmon and M. Sheridan, unpublished results). Activity of an acid
lipase of lysosomal origin was found in rainbow trout dark muscle 14 and adipose
tissue 16s. Bilinski and coworkers 14 suggested that lysosomal lipase from rainbow
trout lateral line muscle serves mainly in the mobilization of intracellular lipids for
internal use. Neutral lipase from rainbow trout adipose tissue was characterized 16s
and it is this enzyme which seems to play an important role in lipid mobilization in
adipose tissue and thus to supplying fatty acids to peripheral tissues 164.
404 L Navarro and J. Gutir
At the same time, as the lipolytic machinery is activated during starvation, the
activity of the lipogenic pathway seems to be curtailed, at least during the later
stages of food deprivation. In coho salmon (O. kisutch), for instance, the activities
of several lipogenic enzymes in liver remain unchanged for the first two days of
starvation, while significant decreases were noticeable after 23 days 1~ Usually,
cytosolic enzymes supplying reducing power in the form of NADPH are considered
'lipogenic'. These include two enzymes of the pentose phosphate shunt, namely glu-
cose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, as well
as malic enzyme (malate dehydrogenase-decarboxylating) and isocitrate dehydroge-
nase. The total NADPH available for FA synthesis in liver is lower in American eels
(Anguilla rostrata) fasted 4-6 months than in fed or freshly caught eels 2. In rainbow
trout liver the activities of the NADP-linked dehydrogenases were substantially
depressed after four weeks of food deprivation s . As other experiments show, the
activities of these NADP-generating enzymes, albeit not always all of them, are
governed by the dietary status of fishes 161. Abundance of fatty material, as in the
case of elevated concentrations of dietary lipid, decreases the activity of G6PDH
and ME in rainbow trout liver 1~ Conversely, feeding a diet high in carbohydrates
to channel catfish (lctalurus punctatus) results in increased hepatic titers of all four
NADPH-generating enzymes.
A more direct measure of lipogenic potential would be the analysis of ATP-
citrate lyase and particularly of acetyl-coenzyme A carboxylase, the committed step
in the synthesis of fatty acids. In the case of ATP-citrate lyase, activity of the
enzyme decreases significantly in carp (Cyprinus carpio) starved for two months
(H. Segner and R. B6hm, unpublished results). Unfortunately, the carboxylase
is not well described in piscine systems, and none of the existing studies 6~
analyzed carboxylase activity and phosphorylation status during different physio-
logical stages of the experimental animals. In the rat liver, the enzyme is under
stringent control though three different mechanism, all of which are known to be
influenced by diet and starvation: (1) allosteric effectors such as citrate; (2) covalent
modification through phosphorylation and dephosphorylation; and (3) enzyme con-
centration.
Liver slices from European eels fasted for 7-39 weeks incorporated 14C-acetate
into FA at lower rates (6- to 20-fold) than those found in fed eels 1. After 95 weeks
starvation, eel liver was still able of incorporate ~4C-acetate into FA, albeit at a
lower rate 1. Esox lucius starved for 2 months, converted ~4C-acetate preferentially
into unesterified fatty acids and phospholipids 94. It appears that during starvation,
emphasis is on the synthesis of structural lipids, while carbon will be preferentially
funnelled into storage lipids (triglycerides) after re-feeding.
In conclusion, lipids are stored as triglycerides in different tissues. But indepen-
dently of the tissue location, lipid mobilization occurs during fasting, simultaneously
or after carbohydrate mobilization, but always before protein degradation. In
teleosts, FFA are the main products of lipid mobilization, while in elasmobranchs
ketone bodies take their position. In comparison with carbohydrates, lipids repre-
sent an important source of energy due to the high depots accumulated and their
high caloric value (Table 3).
TABLE 3
Initial energy content in total liver and muscle, relative change and rate of energy loss between
different periods of fasting in carp (Cyprinus carpio)
Initial values Relative change (%)

(kcal) Day 1-8 Day 8-19
Liver
Glycogen 1.12 -57.0 - 18.1
Protein 0.84 - 18.6 - 13.8
Lipids 0.47 -12.4 -29.8
Total 2.42 - 35.2 - 19.2
Muscle
Glycogen 0.73 + 1.7 -6.5
Protein 21.7 -19.9 -22.4
Lipids 2.0 + 23.4 - 17.2
Total 24.4 - 15.8 - 21.2
Rate of energy loss
Liver (cal/h) 5.07 1.1
Muscle (cal/h) 22.9 16.5
Data are given as means for 8 fish in each group. From Blasco et al. 17.
IV. Protein mobilization

The sequence of mobilization of the different sources of energy during the course of
starvation seems to be very similar, in general terms, in the different species of fish.
Usually, protein reserves are spared at the beginning of fast, thus proteolysis occurs
only when more readily available energy reserves have been widely consumed such
as liver glycogen and lipid stores as it does in higher vertebrates. Then, as protein
is utilized, water moves in to take its place. Fish present special adaptations for
protein mobilization: high level of proteolytic enzymes in muscle, coupled with the
generalized ability to excrete excess nitrogen as ammonia or ammonium ion. Only
very few species go through the metabolic expense of synthesizing - at appreciable
metabolic cost - and excreting urea 123. The actual contribution protein makes to
meeting the overall energy requirements during fasting depends largely on the
species 187. It is trivial to note that the impact of starvation is felt sooner in active
than in sluggish fish. The activity of catheptic enzymes is greater and more rapid
autolysis is noticed in the muscle of such species as mackerel (Scomberscombrus) as
compared with carp (Cyprinuscarpio) or cod (Gadus morhua).
Different organs deplete endogenous protein to differing extents during starva-
tion. As detailed below, fish tend to mobilize more protein from white muscle than
from dark muscle. While increases in proteolytic activity with starvation have been
found in liver, kidney, spleen, and red muscle, not surprisingly, it is the white muscle
that experiences the largest increases in proteolytic activity.
In the initial phases of starvation in carp (Cyprinus carpio), proteolytic capacity
is lowest in muscle, and increasingly larger in spleen, liver, kidney and highest in
the intestine. This order in the series is most likely a reflection of normal tissue
406 L Navarro and Z Gutilrrez
turnover and synthetic demand of the tissue. As the period of food deprivation
lengthens, this order is almost reversed. Now intestine has the lowest proteolytic
activity while activity increases in the order: liver, kidney, spleen and white muscle.
Rates of protein turnover must be matched to the energy demands; allowing for the
dietary constraints that mark starvation, protein synthesis rates are adjusted to the
new requirements. Again, the white muscle appears to be the fish tissue that is most
sensitive to fasting and this tissue responds with a reduction in the rate of protein
synthesis after food deprivation 7~'1~ Protein synthesis rates of other tissues such as
the liver and gills have been found to be little affected by starvation 81,1s4,1~.
1. Liverproteins
In general, fasting exerts only limited effects on hepatic protein suggesting that
the bulk of this protein has vital functions 31,126. Observed increases in percentage
of liver protein is usually correlated with decreases in liver weight due to the
mobilization of other reserves in the course of starvation. These increases are
observed in carp 17, eel (,4. rostrata) 124 or brown trout (Salmo trutta fario) 13s after
different periods of fasting. Absolute values of the total quantity of the store in
the entire organ is a more meaningful physiological measurement, and analysis of
data in percentage can lead to erroneous interpretations. Thus, actually, the total
quantity of liver protein declines with fasting 17,13s.
Fasting exerts little influence on protein synthesis rate in liver, but degradation
increases under fasting conditions. Nevertheless, selective destruction of proteins
may occur. Effects of starvation on tissue enzyme activities are variable 3~ The
activity of gluconeogenic enzymes increases in numerous fasted teleost species 171.
Increases in the liver activities of alanine and aspartate aminotransferases has
been reported in many teleost species suggesting a stimulation of gluconeogenic
flux from amino acids 59.171. These changes are accompanied by increased activi-
ties of liver gluconeogenic enzymes including phosphoenolpyruvate carboxykinase
(PEPCK) 124,127,171.
Another analytical aspect that has to be taken into account is, again, the manner
in which the data are expressed and interpreted. Enzyme activity changes expressed
in terms of tissue weight are not always adequate, because the liver mass relative
to body size declines with fasting in many fish species, the total potential of the
liver for some metabolic pathways would be reduced. This is the case in the perch
(Perca flavescens) which shows a decrease in overall metabolism including the liver
gluconeogenic pathway. It is clear that, particularly with respect to the liver, the
use of tissue DNA content as a denominator for various parameters can result in a
different interpretation of the fasting response ss.
2. Muscle proteins
Fish are in general well adapted to withstand long periods of depletion and this is
probably because the muscle is rich in proteolytic enzymes which can mobilize the
body tissues for fuel when required 1~
The main effects of prolonged starvation are increases in muscle proteolysis

activity and mobilization of amino acids derived from muscle proteins for utilization
by more 'vital tissues'. During periods of extended starvation, catabolism of muscle
protein is initiated and the resultant amino acids are the major sources of energy for
the fish 1~ Muscle protein mobilization is produced after glycogen mobilization
when glycemia and lactate are in decline.
In some species, including Gadus morhua, muscle protein breakdown increases in
the early stages of starvation and maximum values were found 3-4 weeks into a fast.
Protein content starts to decline after liver lipid has fallen below a critical value.
Muscle protein is the most important body energy store, being used mostly during
prolonged starvation 31,187. During spawning migration of sockeye and chum salmon,
catabolism of muscle protein is preceded by 3- to 7-fold increases in the muscular
activities of the proteolytic enzymes (cathepsin D and carboxypeptidase A) 118.
In common with mammals, when fish muscle protein is degraded, contractile
and soluble proteins are removed preferentially, while connective tissue proteins
are used to a lesser extent. The consequence is that the connective fibers rich in
collagen are almost entirely spared from degradation. Thus, the proportions of
glycine, proline and hydroxyproline (the key amino acids in collagen) in muscle
protein increases during fasting.
The changes in protein concentration and composition during depletion may
be distinctly different in red and white locomotory muscles 1~ For example, in
the flounder (Pleuronectes platessa) white muscle loses a significant portion of
myofibrillar protein during starvation, but this part of muscle protein was conserved
in red muscle. Only mitochondrial and sarcoplasmic proteins were mobilized from
red muscle in this species.
The overall balance between synthesis and degradation is altered with fasting.
Some regulatory adjustments in protein metabolism are needed to bring about
these changes in muscle protein concentration and composition. First, rate of
protein turnover needs to be adapted to the new energy demands. Studies on
protein synthesis in rainbow trout 1~ and barred sand bass ~~ have shown that
white muscle protein synthesis rate declines to a plateau after 10-14 days with-
out food. Second, and very important, the turnover of specific proteins in each
muscle type must be regulated closely to ensure conservation of physiological ca-
pacity as we have also mentioned for liver protein. In this context, studies with
barred sand bass (Paralabrax nebulifer) by Lowery and Somero 1~ are illustrative.
Also, white muscle myofibrillar protein synthesis rate was more affected by starva-
tion than was the sarcoplasmic protein synthesis rate. Actin, a major myofibrillar
component (approximately 10% of total white muscle protein in fish) was very
sensitive to fasting (Table 4). That leaves the question of how the synthesis of
vital enzymes or other proteins is differentially regulated? For instance, the rel-
ative amounts of the different glycolytic enzymes are relatively well conserved
during periods of starvation, despite very large decreases in the total activities
of the enzymes 11s. The conservation of relative activities could be achieved by
different mechanisms. It seems that, at least in barred sand bass, differential
effects on synthetic rates may exist. Muscle enzymes present at high concentra-
408 I. Navarro and :I. Guti~rrez
TABLE 4
Changes in protein and actin concentrations during fasting in Pamlabrax nebulifer
Days fasting White muscle . . . . . Red muscle

protein (mg/g) % of fed protein (my/g) % of fed
rotein
0 192 4- 4 100 247 4- 12 100
5 174 4- 9 91 264 4- 12 107
10 177 4- 24 92 263 4. 12 107
16 135 4. 5" 71 222 4- 17 90
23 130 4. 3" 68 209 4. 11" 85
Actin
0 18.5 4. 1.0 100 13.3 4. 0.3 100
5 15.24. 0.5" 82 12.9 4. 0.4 97
10 15.6 4. 0.3" 84 13.5 4. 0.7 102
16 12.84. 0.2' 69 12.9 4. 0.5 97
23 13.0 4. 0.4" 70 12.7 4. 0.4 95
Values are means 4. SEM of four observations.
"Significantly different from fed values (p < 0.025). Adapted from Lowery and Somero 1~
tions, low specific activities and possessing long half-fives, show a diminution in
synthesis rate with fasting much more pronounced than that found in enzymes
present in lower concentrations and higher specific activities. In this way, the
protein turnover may be regulated such that the relative proportions of specific
proteins or enzymes are conserved. Unfortunately, methods of determining spe-
cific protein degradation rates present many problems, the most serious of which
is the re-incorporation of radiolabeled amino acids 196. The half-life of a protein,
consequence of both degradation and synthesis rates, is likely changed during
starvation.
3. Plasma protein and amino acids
There is a clear tendency for plasma proteins to decrease in fasting fish. In both
brown trout (S. trutta fario) and carp (C. carpio) the electrophoretic pattern of
plasma proteins is substantially altered compared with control fish after 30 days or
6 months fasting, respectively 1~ Plasma protein decreased in fasted mature carp
after five days of fasting tT. In addition, the overall concentration of plasma protein
is curtailed. In carp, for instance, a 6-month fast decreases plasma concentration
from 3.9 to 2.8% (ref. 106). Apparently, albumins are the first plasma proteins
to undergo reduction in concentration, followed by the alpha and beta globulins.
Gamma globulins, in contrast, were not utilized; in fact, since the overall protein
concentration decreases, the percentage of plasma proteins contributed by gamma
globulins is increased. A similar picture was observed in Gadus morhua during
starvation, while an extreme situation is noticed in sockeye salmon during the
spawning migration. The anorexic females of this species build up impressive levels
of vitellogenin in their plasma in the course of migration, but at the same time
deplete their plasma store of albumin in its entirety. Some authors have suggested
using albumin: globulin ratio as a nutritional indicator because of this sharp decline
in albumin during starvation 1~ But in any case, the plasma concentration of
different free amino acids is of greater interest than plasma protein levels since it
reflects results of whole body protein turnover.
The composition of the free circulating amino acid (AA) pool changes markedly
during fasting in all the species studied. In rainbow trout (O. mykiss) even a
relatively short (24 or 48 h) fasting period lead to a decrease in plasma essential
amino acids (EAA) but not in the non-essential AA ls6. The significant positive
correlation between the levels of plasma EAA and their respective concentrations
in the diet, as well as the lack of such a relationship for NEAA appears to be a
general phenomenon 16,32,148. The decline in EAA observed in plasma of carp or
trout at the onset of fasting may respond to the uptake of these AA by tissues
without a new contribution from diet, while NEAA levels are metabolized and
altered to a greater extent and can undergo extensive interconversions.
Of all the presumed essential AA, histidine dropped most precipitously in fasted
migrating salmon 118. It appears that histidine is utilized by the fish at high rates in
the early phases of migration, but the exact metabolic role of this amino acid is
still not known. In carp (Cyprinus carpio), branched-chain AAs (leucine, isoleucine
and valine) were affected most, decreasing by 44% after five days without food
(Fig. 1) 16. This decline suggests an increase of oxidation of these amino acids
during fasting 16,199. Nevertheless, after 19 days fasting a compensatory increase in
plasma AA was noticed in this species. This increase is largely due to increases in
branched-chain AAs. Of the non-essential AAs, alanine, glutamate and glutamine
levels are augmented with the two five-carbon amino acids experiencing the largest
2500
2000
~L
"" 1500
4-
o
4-
r
I000
o
u
500
O I I 1 9 9 I I
2 5 8 19 50 R
Day
Fig. 1. Variations in plasma essential amino acids (EAA) and non-essential amino acids (NEAA) levels
at different periods of starvation and refeeding (]2 days) in Cyprinus carpio. Control (filled circles),
starved (open circles), R = refed (for 12 days). Differences were determined by Duncan's multiple
r a n g e test. * Different from control (p <0.05); ** different from control (p <0.01); *** different from
preceding point (p <0.05); **** different from preceding point (p <0.01). From ref. 16, with permission.
increases (two-fold over control). The increase in plasma amino acids observed
in fasting carp after 19 days is a consequence of muscle proteolysis and thus,
it may be expected that generalized proteolysis of muscle proteins will provide
a balanced cross-section of all the AA. However, this is clearly not the case.
In starving dogfish (Squalus acanthias) alanine was selectively released into the
plasma draining the white muscle t~ Similarly, Mommsen and coworkers tls noticed
that alanine was the major AA released from white muscle of migrating sockeye
salmon for transport of amino acid derived carbon to other tissues. It therefore
appears that the main fate of AAs is transdeamination in situ to supply carbon
precursors for gluconeogenesis mainly as alanine and possibly also as glutamate
(which represent 46% of NEAA) 16. Both alanine and glutamate are excellent
gluconeogenic substrates in most teleostean fishes 129,157,171. While this has been
shown directly in isolated fish hepatocytes numerous times, this notion is further
supported by the marked depletion of the glutamate/glutamine pool at 50 days
fasting in carp, coinciding with tight maintenance of glycemia at this time t6.
Furthermore, other in vitro studies have demonstrated that fasting induced an
increase in uptake of L-alanine by trout (Salmo trutta) hepatocytes that showed
an inverse relationship with L-alanine plasma levels2~ Thus, all the above studies
support the metabolic model that glutamate and mainly alanine form fundamental
precursors for gluconeogenic pathway in fish tissues under fasting conditions.
In summary, during starvation, proteins are mobilized at the final stage. Different
tissues can contribute to the available pool of AAs, however, skeletal muscle
represents the largest store of protein and, as consequence of the percent of muscle
weight (with respect to body weight) it constitutes the main energy source during
prolonged starvation (Table 3). Blood AA from tissue proteolysis will be transported
to gluconeogenic tissues to maintain the necessary glycemic level.
V. Endocrine regulation
1. Insulin
Although the role of insulin in mammals under fasting conditions has been widely
analyzed, comparative studies on the regulation of fish metabolism by insulin during
food deprivation are relatively sparse. Nevertheless, of all the pancreatic hormones,
insulin has received the most attention in fish studies t+9. Initial studies on the role
of insulin in fish have concentrated on the direct effects of exogenous insulin on
blood metabolites and tissue energy reserves. The regulatory function of insulin in
fish during different nutritional conditions has been more clearly elucidated when
measurements of circulating levels became available. Initial data on plasma insulin
levels were successfully obtained by some authors in different teleost species 141.176.
These difficulties in measuring fish insulin levels can be explained by the fact that
the mammalian radioimmunoassay (RIA) components proved to be useless tools
in measuring fish insulins except in a few isolated cases. Cross-reactivities between
many fish insulins and antibodies to mammalian insulins are very weak, in spite of
the fact that piscine insulins are structurally remarkably close to other vertebrate
insulins 42. Thus, the development of RIAs with fish components or homologous
RIAs has been an essential step towards understanding the unique role of insulin
during fasting in fish. Antibodies raised against insulins isolated from species such as
scorpion fish, bonito, cod and anglerfish, together with their respective radiolabeled
equivalents, can be successfully used in RIAs to accurately determine insulin
levels of other fish species 63,141,178. Nevertheless, the development of a homologous
RIA for coho salmon (Oncorhynchus Idsutch) has been described lsl. Clearly such
homologous systems are to be favored, since they are less prone to erroneous data
and require less effort to verify concentration estimates.
The general actions of insulin in fish seem to be similar to other verte-
brates, in that insulin is a hypoglycemie hormone, lipogenie and promotes protein
synthesis 122,132. Just as in mammals, the circulating levels of insulin diminish dur-
ing fasting in fish. Table 5 shows the changes in plasma insulin levels in starved
elasmobranch and different teleost species.
An interesting phenomenon has been described for the eyclostomata. Working
on the Atlantic hagfish (Myxine glutinosa), Emdin 41 found that plasma insulin
levels declined within 1 month of starvation from 12 to 6 ng/ml. Concomitantly, a
decline of blood glucose and an almost complete exhaustion of glycogen reserves
in liver and skeletal muscle were observed. Surprisingly, in this species, insulin
lacked metabolic effects on the liver whereas the hormone had the expected~ but
rather small, effect in muscle, where it stimulates the synthesis of glycogen, protein
and lipids. It appears that the physiological role of insulin regulation of skeletal
muscle metabolism is similar to that in mammals while its effects in liver are
weak or absent. In fact, Emdin 41 considered liver of little significance in fasting or
other catabolic situations in this cyclostome. In contrast, more recently, studies on
hepatocytes isolated from this species of hagfish s7 reveal unusual effects of insulin
in liver carbohydrate metabolism, such as increases in gluconeogenesis and total
glucose production. Since it seems that glucagon is absent in the hagfish islet 42 an
ancient catabolic function of this hormone may still be present in this primitive
group of Agnathans. Similar contradictory insulin effects have also been described
in two species of teleosts 53,s4. Thus, it may be necessary to design appropriate
fasting studies since agnathan tissue metabolism and its regulation have been poorly
studied, and important evolutionary aspects could be elucidated from these studies.
Little work has been done on selachian physiology. The studies on the effects
of insulin in the dogfish shark (Squalus acanthias) suggest that the dogfish uses
ketone bodies as a primary fuel at rest. Glucose would apparently be synthesized by
gluconeogenesis to maintain muscle glycogen reserves. These carbohydrate stores
would be used for anaerobic glycolysis in situations where vigorous swimming is
needed. This hypothesis agrees with the opportunistic food habits of this carnivorous
fish altering between consumption of large amounts of food and relatively long
periods (about two weeks) of fasting 159.
Artificial food deprivation in an elasmobranch (Scyliorhinus canicula) leads to
plasma insulin decreases similarly to what is described in the more evolved teleosts.
Plasma insulin decrease progressively after 15 and 36 days of food deprivation
412 L Navarroand I. Guti~rrez
with a maintenance of low levels with prolonged fasting62. Plasma glucose levels
decreased initially, and recovered during the period of declining insulin titers.
It is interesting that the glucose decrease was paralleled by changes in plasma
amino acid levels at 36 days of fasting. A similar decrease in total plasma amino
acid levels has been also seen in other species of chondrichthyes such as Squalus
acanthias 1~ suggesting an increase of gluconeogenic processes would be favored by
the diminished plasma insulin levels. More studies are needed to identify the tissue
destination and metabolic pathway of the supposed de novo synthesized glucose and
possible regulation by insulin and other hormones.
Comparatively more studies have been performed in teleosts (Table 5). The
general picture emerging from these studies is a clear diminution of insulin plasma
levels in fish after just a few days of fasting. Plasma insulin decreased during fasting
in goldfish (Carassius auratus) 141. Serum insulin levels of fed animals were nearly
twice those found in 5-day fasted goldfish using codfish insulin as standard and
guinea pig anti-cod insulin serum and 12sI-labeled cod insulin as other components.
The same authors measured insulin secretion in islet incubations of oyster toadfish
(Opsanus tau) obtaining values very similar to those determined earlier in the same
species by Tashima and Cahil1176 using scombroid (bonito) insulin standards and
guinea pig anti-bovine insulin antibody. In fact, these were the first studies describ-
ing a decline of insulin levels caused by fasting in teleosts. Subsequently, Thorpe
and Ince 177 observed a very strong decline in plasma insulin in the rainbow trout
fasted for 7 days, a diminution of 76% in relation to fed animals, and in only 4
days fasted cod a decline of 61% was observed. Furthermore, upon refeeding, the
animals showed a clear recovery of the insulin levels after one week of food admin-
istration. Plisetskaya and coworkers m developed a homologous radioimmunoassay
to determine plasma insulin in salmonids. These authors measured significant de-
creases (in excess of 50%) in insulin levels in blood of coho salmon (O. kisutch)
after one or two weeks of fasting. The observed decreases are entirely reversed after
refeeding.
Long-term experiments of food deprivation in salmonids always induce a decline
in insulin plasma levels. In rainbow trout, a 6-week fast produced a decline in insulin
plasma levels. This fact, together with the relative changes in other pancreatic
hormones such glucagon and GLP, enhanced the gluconeogenic pathways in the
liver, that are activated after this period of fasting 126. This fact is also in concordance
with the observations that, after injection of insulin in starved rainbow trout, glucose
synthesis from alanine was drastically depressed, when compared with control fish28.
The role of insulin in the regulation of gluconeogenic processes is corroborated in
salmonids by in vitro experiments. In isolated rainbow trout hepatocytes, insulin
curtailed the rate of gluconeogenesis from lactate 146. This gluconeogenic inhibition
by insulin is accompanied by activation of pyruvate ldnase activity. However,
this pattern does not seem to be universal since in isolated hepatocytes of sea
raven (Hemitripterus americanus) porcine and fish insulins, contrary to expectations,
increased the flux rate of amino acids into glucose s2. More recent studies show
that this insulin-stimulated increase in gluconeogenesis in sea raven may be at least
partially related to an inhibition of the fructose-6-phosphatase activity ratio of 6-
TABLE 5
Plasma insulin levels of fed or fasted elasmobranch and teleostean fishes
State Insulin (ng/mi 4- SE)

Scyliorhinus canicula 62 Fasted 1d 0.77 4- 0.04
Fasted 15 d 0.64 4- 0.04
Fasted 36 d 0.25 4- 0.03
Fasted 82 d 0.30 4- 0.04
Carassius auratus 140 Fed 3.50 4- 0.30
Fasted 5d 1.90 4- 0.50
Cyprinus carpio 17 Fasted 1d 5.21 4- 0.68
Fasted 5d 2.27 4- 0.27
Fasted 50 d 2.23 + 0.29
Refed 12 d 5.93 4- 0.26
Fasted 16 d 8.28 4- 0.96
Gadus morhua 176 Fed 8.43 4- 0.90
Fasted 4d 3.28 4- 0.20
Fasted 7d 1.60 4- 0.44
Fasted 21 d 0.20 4- 0.13
Gadus morhua -/3 Fed 4.90 4- 0.82
Fasted 7d 1.90 4- 0.82
Fasted 21 d 0.50 4- 0.12
Dicentrarchus labrax Fasted I d 10.91 + 0.25
Fasted 22 d 8.68 4- 0.22
Oncorhynchus mykiss 176 Fed 5.65 4- 0.30
(Salmo gairdneri) Fasted 7d 2.20 4- 0.30
Oncorhynchus mykiss 1-/6 Fed 6.80 4- 0.60
(Salmo gairdneri) Fasted 7d 1.60 4- 0.30
Oncorhynchus myk/ss 12s Fed 12.10 4- 1.10
Fasted 42 d 2.00 4- 0.10
Fed 13.20 4- 1.50
Fasted 42 d 3.00 4- 0.10
Oncorhynchus kisutch 150 Fed 4.50 4- 0.80
Fasted 7d 1.40 4- 0.20
Oncorhynchus kisutch 150 Fed 4.30 4- 0.90
Fasted 21 d 0.90 4- 0.10
Salmo trutta (summer) TM Fasted 1d 4.85 4- 0.27
Fasted 3d 3.85 4- 0.21
Fasted 30 d 3.12 4- 0.11
Fasted 50 d 1.62 4- 0.14
Refed 8d 3.69 4- 0.32
Salmo trutta (winter) 134 Fasted 1d 5.48 4- 0.20
Fasted 8d 2.53 4- 0.18
Fasted 30 d 1.29 4- 0.23
Fasted 50 d 1.17 4- 0.23
Refed 8d 4.94 4- 0.23
414 L Navarro and J. Gutic~rrez
phosphofructo-l-kinase (PFK-1 EC 2.7.1.11). Phosphoenol-pyruvate carboxykinase

and pyruvate kinase (PK, EC 2.7.1.40) were not affected by insulin. PK is the most
probable point for critical hormonal regulation in the gluconeogenic process in
mammals. But caution must be used in interpreting these results as the relative
importance of the various enzymes in the regulation of these pathways in fish is
not yet clearly understood. A variable pattern is revealed in American eel (Anguilla
rostrata) hepatocytes, where insulin induced either increases or decreases in alanine
gluconeogenesis depending on the time of the year s3.
Other fasting experiments, with more dynamic measurements of insulin levels,
have been performed in teleosts. In juveniles of Pyrenean brown trout, plasma
insulin levels showed a progressive decrease (between 20 and 30%) after short-term
fasting (3, 8 or 15 days) or after a more prolonged starvation (a decrease of 67%
after 50 days). The decline of insulin titers was more rapid in summer than in
winter 135. This diminution of insulin levels was correlated with the mobilization
of tissue energy reserves. Thus low levels of insulin could favor a degradation
of liver glycogen during short-term fasting, together with the action of other
hormones. Knowing the anabolic function of insulin in protein and lipid metabolism
in fish 116.1~, a decline in plasma insulin may contribute to the decrease in fat and
protein reserves under fasting conditions in this species. A progressive decline of
insulin plasma levels was also associated with the mobilization of energy reserves in
sea bass, although, in that case, the role of glucagon seems to be more relevant 65.
Reduced plasma insulin levels following fasting are common in other species
of teleosts, including cod 173'177 and carp 17. In carp, insulin plasma levels always
decrease with fasting experiments, although the response of glucagon to fasting is
not so uniform in this species of teleost. Blasco and coworkers 17 found a two-fold
decrease in insulin with respect to control values, after 8 days of fasting. This rapid
decrease correlated nicely with the early mobilization of protein observed in that
study. In carp, as well as in trout fasting experiments, the levels of insulin returned
to control values after short periods (about 10 days) of refeeding. This fact suggests
a rapid and high adaptability of fish to the recovery from fasting periods 76,135.
Unfortunately, the above-mentioned changes in insulin concentration and their
correlation with metabolic output of fishes, are even less clear-cut under natural,
rather than experimentally imposed, periods of food deprivation. For example, dur-
ing the (anorexic) spawning migration of the pink salmon (Oncorhynchus gorbusha),
plasma levels of insulin remained stable or, if any changes were noted, plasma
levels were elevated. The elevation of plasma insulin, opposed to the expected
decline, in this unique situation may preserve the energy stores instead of an early
mobilization, thus enabling the fish to preserve sufficient metabolic reserves for
final maturation of gonadal products and the exhausting spawning upon arrival
at the spawning grounds ~2e. Another explanation for the elevated insulin titers
in pre-spawning non-feeding salmon or lamprey is a possible role of insulin as a
gonadotropic hormone similar to the one described for mammals 155.
To ultimately make the connection between changing insulin titers in plasma
during starvation and the altered metabolic status of fishes, it has become important
to examine the interactions of insulin with its receptor in fish tissues. While the
first studies on insulin receptor in fishes elegantly, if not conclusively, dealt with
evolutionary aspects of both the peptide and its receptor 1~176 more recent studies
have focused on functional receptor characterization and the varied influence of
different physiological states on insulin receptors in fish tissues. Specific insulin
binding to fish liver or muscle 66,67 appears to be lower than that reported in
mammals and birds 92,13~ In rainbow trout, a fast of 40 days caused a decline in
plasma insulin levels and an increase in the binding capacity (specific number sites)
in liver membranes, a situation reminiscent of increasing binding capacity for insulin
in mammals and birds after a short fast 3'167. However, at the same time, a decrease
in the binding affinity in relation to control fish was noted, and as a result, specific
insulin binding remained unchanged. This could be explained as a way to reduce
the anabolic fluxes in the liver. Refeeding of fasted fish for 15 days restored plasma
insulin levels and increased binding affinity with the presumed receptors attaining
a higher specific activity than in control fish. Nevertheless during short-term fasting
experiments (a few days) the number of binding sites seems to be a major regulating
factor: after 3-6 days of fasting no changes in insulin titers were observed, but
specific binding of insulin to the liver plasma membranes increased from 5.0 to
9.3% (ref. 66). We conclude that at the onset of fasting, insulin binding increases in
fish liver, possibly as a compensatory mechanism, until all the metabolic fluxes are
shifted towards catabolic and gluconeogenic processes, while a prolonged fast leads
to a stabilization of the binding of insulin to its receptors. Since hepatic metabolism
is strictly linked to the ratio of insulin to glucagon, the analysis of binding of other
hormones such glucagon to the liver will be a prerequisite to understand metabolic
regulation in the liver.
Insulin binding to muscle in fish and its regulation in response to fasting seem to
vary between species 14~ It is interesting that omnivorous species such as carp (C.
carpio) appear to have more numerous insulin receptors, with higher tyrosine kinase
activity than carnivorous species (trout (Salmo trutta), sea bream (Sparus aurata)).
The most striking difference was observed between carp and trout. In fed fish,
the number of insulin receptors was significantly higher in carp than in trout (Fig.
2). Fifteen days of fasting resulted in a decrease to 50% of the respective control
values in insulin binding to semipurified receptors of carp and trout muscle. This
observation is supported by the concomitant decreased rate of anabolic processes
and utilization of glucose by this tissue during fasting. After 30 days of fasting this
tendency is a special feature in carp 17,18.
In summary, a number of strategies are available to fish tissues to respond to
changes in insulin secretion rate and changes in plasma insulin titers. At least in
liver and in white skeletal muscle, the number of hormone binding sites or their
affinity can be adjusted, at times concurrently, with different nutritional states, thus
resulting in the observed metabolic alterations.
2. Glucagon and glucagon-like peptides
It was not until quite recently that attempts have been made to elucidate the role
of glucagon in fish under fasting conditions. Unlike insulin, glucagon levels in fish
416 I. Navarro and I. Guti~rrez
Fig. 2. Specific binding of insulin (inset) and binding capacity of muscle insulin receptors (main panel)
of starved fed trout (Salmo trutta fario) and carp (Cyprinus carpio). Bars with identical letters a r e n o t
significantly different at the 0.05 level. From ref. 140, with permission.
plasma can be measured successfully with a mammalian RIA ~, in spite of the

fact that an appreciable variability exists in fish glucagons42. What helps matters is
that key regions of the peptide are highly conserved26 and show many structural
similarities to the mammalian hormone. Nevertheless, some authors have used
homologous RIA systems to assay glucagon titers in teleostean fishes ~52.
In mammals, pancreatic insulin and glucagon act together in response to fasting.
After a short-term fasting, insulin titers decrease while glucagon levels increase
substantially resulting in a high molar ratio of glucagon/insulin. After a more
prolonged fast, glucagon levels remain high and plasma insulin concentrations
continue to decrease. Thus, the glucagon/insulin (G/I) molar ratio appears to be a
key regulatory parameter providing a link to the nutritional state of the organism 179.
In most mammals, a normal G/I molar ratio after an overnight fast would be
approximately 0.30. Undemutrition or a low carbohydrate meal will drive this molar
ratio up towards unity and in total starvation the G/I value will likely approach
1.5-2.0. When the animal is in a catabolic situation, glucagon is instrumental in
mediating the retrieval of stored nutrients at a time of need. On the other hand, in
an anabolir situation, such as after a meal, the excess of nutrients must be stored,
and in mammals G/I ratios may decrease to 0.1-0.05 or even lower if the diet
carbohydrate content is high.
In fasting experiments in fish, plasma hormone levels follow a somewhat dif-
ferent pattern. In fasted juvenile sea bass (D. /abr~) a steep increase in plasma
glucagon was found after 4 days of fasting (Table 6) (similar to that occurring in
higher vertebrates). This was accompanied by a stabilization of glucose levels. In
contrast to mammals, this increased hormone level is not maintained and glucagon
TABLE 6
Plasma glucagon levels in fed or fasted teleosts
State Glucagon (ng/ml 4- SE)

Dicentrarchus labrax 65 Fasted 1d 0.72 4- 0.14
Fasted 3d 1.31 4- 0.30
Fasted 8d 0.54 4- 0.11
Fasted 15 d 0.37 4- 0.05
Fasted 22 d 0.32 4- 0.05
Salmo trutta (summer) 135 Fasted 1d 0.65 4. 0.09
Fasted 3d 0.91 4- 0.03
Fasted 8d 0.51 4- 0.03
Fasted 15 d 0.46 4- 0.04
Fasted 30 d 0.25 4- 0.03
Fasted 50 d 0.25 4- 0.03
Refed 8d 0.52 4- 0.06
Salmo trutta (winter) 135 Fasted 1d 0.44 4-0.04
Fasted 3d 0.46 4- 0.05
Fasted 5d 0.55 4- 0.09
Fasted 8d 0.45 4- 0.03
Fasted 30 d 0.32 4- 0.03
Fasted 50 d 0.23 4- 0.03
Refed 8d 0.43 4- 0.04
Fasted 5d 0.28 4- 0.04
Fasted 50 d 0.14 4- 0.01
Refed 12 d 0.54 4- 0.05
Fasted 50 d 0.64 4- 0.04
Fasted 65 d 0.75 4- 0.12
Gadus morhua 7a Fed 1.10 4- 0.32
Fasted 7d 0.30 4- 0.09
Fasted 21 d 0.30 4- 0.03
Fasted 7d 0.30 4- 0.06
Fasted 21 d 0.40 4- 0.13
concentrations declined again after 8 days. An increase of glucagon levels has also
been observed in brown trout after 3 days of fasting 135, although this response was
dampened compared to sea bass. It is possible that species differences can account
for this difference in response, but surprisingly, no such increase was detected
following a two- to four-day fast in the rainbow trout, another salmonid with a very
similar lifestyle and diet as the brown t r o u t 152,173.
The changes in glucagon and insulin in this short-term fasting lead to a rise
in G/I molar ratio. Contrary to mammals, this ratio remains usually below 0.5.
Increases of G/I values from 0.1 to 0.2 after a four-day fast in sea bass or from
0.2 to 0.4 in three-day fasted brown trout have been observed 65,135. Immediately
following a meal in fish, G/I molar ratios do not decrease as clearly as in mammals.
High levels of insulin a few hours after ingestion in salmonids 76,134 are compensated
by subsequent elevation of glueagon levels which may be related to the high protein
content of the diet. Sea bass (Dicentrarchus labrax) fed a natural diet shows a similar
pattern of post-feeding hormonal changes 133.
In fasted sea bass and trout a correlation exists between high glueagon levels and
decreased fiver glycogen reserves. The glycogenolytie action of glueagon has been
demonstrated in vivo and in vitro in numerous species of teleosts, although the de-
gree of glyeogenolytie action can vary depending on the season. Glueagon injections
induced a decrease in liver glycogen in eel (,4. japonica) 22, coho salmon (O. kisutch)
or chinook salmon (O. tshawytscha) 152. Similar potent glyeogenolytie actions of
glueagon have been seen in isolated hepatoeytes from sea raven (Hemitripterus
americanus) 52, eoho salmon (O. kisutch) 156 or catfish (Ictalurus melas) 139. Thus it
can be envisaged that increasing plasma levels of glueagon at the beginning of
fasting may induce the mobilization of glycogen reserves.
In contrast to mammals, long-term fasting in fish is accompanied by a drop
in plasma glueagon (Table 6) and in glueagon-like peptide levels (Table 7) 126. The
relative changes in insulin and glueagon family peptides increased the G/I ratio from
0.11 in fed fish to 0.16 in fasted fish and GLP/I molar ratio from 0.08 to 0.25 after 6
weeks of fasting in eoho salmon. Moon and coworkers 126 concluded that the relative
hormonal changes are responsible for the observed activation of the glueoneogenic
pathway during the later stages of food deprivation. Fasting-dependent increases
in liver glueoneogenie capacity - as assessed by maximum enzyme activities of key
enzymes - have been described in different species of fish (reviewed in ref. 125).
In vitro experiments demonstrated that glucagon and especially GLP appear to be
key peptides for regulating hepatic gluconeogenesis in fish 17,12~ It is interesting
that this potent gluconeogenic effect of GLP has been found only in fish and not in
mammals, where only insulinotropie and intestinal-related functions of GLP have
been described 126. In fishes, a high GLP/I ratio appears to enhance gluconeogenie
processes and mobilization of carbohydrate reserves.
TABLE 7
Plasma glucagon-like peptide (GLP) levels in fed or fasted teleosts
State "GLP (ng/mi 4-SE)

Oncorhynchus mykiss 126 Fed 0.6 4- 0.10
Fasted 42 d 0.3 • 0.04
Oncorhynchus mykiss 15~ Fed 1.9 4- 0.40
Fasted 42 d 0.4 4- 0.02
Fasted 7d 0.2 4- 0.03
Fasted 21 d 0.2 4- 0.03
Fasted 7d 0.2 4- 0.06
Fasted 21 d 0.2 4- 0.06
A high G/I ratio associated with long-term fasting has also been described in
50-day starved brown trout (increase from 0.20 to 0.48) 135 but only in the summer
season and not in the winter. Immature carp food-deprived for 50 or 65 days
demonstrated an unusual increase of plasma glueagon levels 18. Nevertheless, these
increases in G/I ratios are not always clearly seen in fish during long-term fasting
experiments 17,65. It is quite apparent that generalizations are difficult at this point
and that many other factors, such as the physiological state or previous diet,
can influence hormone responses during fasting in fish. It is interesting that in
anorexic individuals of brown trout (during the period of reproduction) maintained
in captivity, a G/I ratio higher than unity was found in peripheral blood and portal
vein 21. Such a high molar ratio was never described before in fish. It is possible
that during natural processes of anorexia, the hormonal response is enhanced in
comparison to experimental fish.
Although it is clear that lipid reserves are mobilized during fasting in fish, the
role of glucagon (a strongly lipolytie hormone in mammals) and other members
of the glucagon family of hormones in this process needs to be elucidated. An
indication of lipolytic action of glucagon has been found in Esox lucius 85 or D.
labrax 142 where injections of glucagon induced an increase in plasma free fatty
acids. However, administration of glucagon remained without effect in Opsanus
tau 176 or eel (A. anguilla) 95. It appears that hepatic lipolysis in fish is mediated by
triacylglycerol lipase which hydrolyses stored triacylglycerol to glycerol and fatty
acids. Glucagon family peptides have been found to influence lipolysis in salmonids.
In vivo administration of glucagon or glucagon-like peptide induces an elevation of
plasma fatty acids accompanied by enhanced hepatic triaeylglycerol lipase activity,
although the effectiveness of GLP in lipid metabolism in salmon varies depending
on the time of the year 152. In vitro experiments support the role of glucagon in lipid
metabolism during fasting. It has been also demonstrated that glucagon acts directly
on the liver since glucagon stimulates triacylglycerol lipase activity in liver slices
as well as fatty acid and glycerol release into the culture medium. More recently,
Harmon and coworkers 7~ concluded that the increased lipolytic activity by glucagon
in trout hepatocytes is mediated by phosphorylation of the enzyme.
The previous nutritional state of fish can modulate hormonal mediated lipolysis:
trout basal hepatic lipolysis is enhanced in liver slices from four weeks fasted fish,
and glucagon-stimulated lipolysis was more pronounced in liver slices from these
food-deprived animals than in liver from fed fish. Surprisingly, glucagon failed to
affect hepatic lipolysis in the liver of six-week fasted animals 69, maybe as a result
of already advanced lipid store depletion, or alternatively, as a consequence of
alterations at the receptor level. Thus, all the above studies support the idea of
glucagon and GLP being key hormones in regulating lipid mobilization during
fasting in fish.
Although plasma glucagon titers give us important information about the role of
this hormone during fasting, receptor studies help in understanding the significance
of the hormonal regulation. Navarro and Moon 136 have recently characterized, for
the first time in fish, specific binding of glucagon in hepatocytes isolated from
two teleostean species, the American eel (A. rostrata) and the brown bullhead
420 L Navarro and Z Guti~rrez
(I. nebulosus). Two classes of binding sites have been described with apparent
dissociation constants (Kd) of 1.97 nM (high affinity) and 17.3 nM (low affinity)
for bullhead and 2.68 and 22.9 nM, respectively, for eel cells. These values are
approximately ten times higher than those generally reported for mammalian
hepatocytes 11,8~ This reflects the relatively slow association of glucagon binding in
these fish which may be attributed to intrinsic characteristics of ectothermic animals.
It is interesting that, a higher number of binding sites was found in the hepatocytes
from the eel (10,413/ce11) in comparison to the bullhead (3811/ce11). Mommsen and
M o o n 121 reported a higher increase in intracellular cAMP in response to the same
glucagon concentrations in eel compared to bullhead hepatocytes. This suggests
the existence of a correlation between the responsiveness of liver for glucagon
binding and adenylyl cyclase in these fish species. However, it should also be noted
that the eel is a species which fasts in captivity, while bullheads adapt to captivity
quickly and feed actively. Nevertheless, more studies are needed to elucidate if such
differences reflect species characteristics, or nutritional state. Liver cells of both
eel and bullhead were able to regulate the abundance of their own receptors, since
receptor number decreased by about 55% in both species with incubation of 100 nM
glucagon. This 'down regulation' has been described in mammalian species 79,16~
These findings raise the question as to whether plasma glucagon levels could
regulate hepatic glucagon receptors in vivo and especially in catabolic situations
such as fasting.
Variations in the number of receptors could help explain discrepancies de-
scribed in glucagon sensitivity for fasted chinook salmon (O. tshawytscha) 93, and for
sea raven (Hemitripterus americanus) 56. Glucagon-stimulated glycogenolysis in liver
pieces isolated from fed and from short-term (one-week) fasted salmon, but failed
to stimulate glycogenolysis in liver slices from long-term (three-week) fasted fish.
In contrast, epinephrine maintains its glycogenolytic action regardless of nutritional
state. In this fish species, a short fast induces an initial liver glycogen degrada-
tion while in longer term fasted salmon, mobilization ceases in the face of high
glucagon/insulin molar rates. Thus it appears likely that glucagon is modulating
fasting-associated adjustments in the metabolism of salmon by decreasing hormone
sensitivity. Thus these salmon are able to maintain some minimum pool of glycogen.
Distinctive hormonal features of the sea raven (Hemitripterus americanus) fasting
strategies distinguish it from those species in which glycogen is partially conserved
as in salmon. In six-week fasted sea raven, endogenous carbohydrate stores are used
preferentially for the production of glucose instead of gluconeogenic precursors.
Hepatocytes isolated from six-week fasted sea raven had an increased apparent
sensitivity of rates of total glucose production (presumably by glycogenolysis) to
glucagon. This phenomenon would retain the effectiveness of glucagon in the liver
and allow for increased glucose production in this species under fasting conditions.
All the findings mentioned above show that nutritional state modifies hormone
effects and may explain some of the seasonal differences in hormone action
previously reported for this and other fish species 52'53.
In conclusion, fasted fish have proven to be a good model to study glucagon-like
hormone actions. It seems that glucagon family peptides in fish are key hormones
in regulating energy reserves in fish under fasting conditions. Recent and detailed
studies on the glucagon-related hormones reveal fine adjustments to the nutritional
state of fish involving tissue receptors and responsiveness of target cells. Clearly
such studies are to be favored since they may help to understand the metabollic
mechanisms of fish adapting to adverse conditions.
3. Glucocorticoids
Administration of adrenocortical hormones, such as cortisol, to higher vertebrates

normally stimulates glueoneogenesis, provokes a rise in liver glycogen and leads to
an inhibition of glucose uptake in several peripheral tissues. Concomitantly, protein
catabolism is stimulated. However, the immediate importance of such hormones
under fasting conditions is under debate since generally, a decrease in cortisol
secretion in early fasting is observed 46. Nevertheless, these hormones may have a
supportive function in the control of normoglycemia, even in fasting conditions.
It appears that corticosteroid hormones could have similar actions in teleost
fish, but the importance of these hormones during fasting remains controversial.
With respect to carbohydrate metabolism, the studies performed in whole animals
concluded that cortisol administration resulted in enhanced liver gluconeogenesis
based mainly on tissue carbohydrate changes 19,97,132. Since activation of gluco-
neogenic processes during fasting has been demonstrated also in fish as well as in
higher vertebrates, cortisol may contribute to its regulation. In mammals, it has
been reported that cortisol may exert, in addition to the direct metabolic effects,
an indirect action on other hormones such as thyroid hormones, glucagon or cate-
cholamines. Also, some authors have suggested that the insulin/cortisol ratio could
regulate the direction of metabolic fluxes during fasting in fish 132. However, to date
none of the in vivo studies analyzing the role of corticosteroids in fish systems have
attempted to separate direct from permissive effects. Arguably the best example
of such permissive effects in mammals is the potentiating effect of corticosteroids
on glucagon's multiple actions, an area that remains to be analyzed with piscine
systems. Vijayan and colleagues 182- 184 have looked at the interactive effects of cor-
tisol and other glucoregulatory hormones. These authors demonstrated that cortisol
implantation for 7 days in fed sea raven (H. americanus) enhances the responsive-
ness of hepatocytes to the actions of epinephrine and insulin, but not glucagon, on
carbohydrate metabolism. Although hepatocytes from food-deprived (eight weeks)
animals showed enhanced responsiveness to pancreatic hormones, these effects
were not modified by cortisol implantation. These slow-release implants of steroid
hormones have been used successfully to evaluate the chronic effects of cortisol on
the physiology of teleosts 181,182,194.
The effects on carbohydrate metabolism in isolated fish hepatocytes are not
definitive. Recent studies show that cortisol increases hepatic activities of glycerol
kinase (GK, EC 2.7.1.30) and fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11)
in brook charr (Salvelinus fontinalis) indicating enhanced gluconeogenic potential
from glycerol 181. Metabolic flux in eel hepatocytes was altered by cortisol adminis-
tration, shifting the preferred gluconeogenic substrate from lactate to amino acids51
422 I. Navarro and J. Guti#rrez
as previously suggested by other authors 29,s7. Following administration of corti-

sol, activities of phosphoenolpyruvate carboxykinase and glutamate-oxaloacetate
transaminase (AspAT, EC 2.6.1.1) increased, suggesting an enhanced amino acid
gluconeogenesis. Surprisingly, the absolute rate of gluconeogenic substrates in-
corporation into glucose actually declined in hormone-injected fish51. However,
repeated injection of cortisol (which is considered a stress hormone itself), re-
quires repeated handling of specimens and may lead to superimposing metabolic
stress reactions mediated by catecholamines or permissive effects of cortisol plus
catecholamines (or others) skewing the experimental results. In addition, repeated
injection of cortisol may actually lead to increased turnover, i.e. speed-up removal
of the injected hormone and thus the separation of cortisol-induced effects from
those induced by fluctuating hormone titers, or drastically altered endogenous
turnover, is difficult.
Cortisol is known to stimulate glycogen synthesis in mammals, but in fish the data
are not clear-cut. At this point, it is not clear whether species differences hormone
application or other parameters are responsible for the observed inconsistent
results. In fed goldfish (C. auratus), for instance, cortisol failed to increase liver
glycogen concentration, but the hormone seemed to be effective in maintaining liver
glycogen content in fasting fish 17~ Administration of cortisol in the Japanese eel,
however, resulted in increased liver glycogen levels23.
Cortisol has been implicated in the regulation of protein metabolism. Adminis-
tration of cortisol in fed goldfish resulted in a loss of body weight and the hormone
accelerated the rate of loss of body weight in fasted animals 17~ Associated with this
observation, a rise in ammonia excretion and a two-fold increase in the concentra-
tion of alanine aminotransferase (AIaAT) in the liver were noted for treated fish.
This suggests that cortisol is important in controlling the utilization of tissue protein
during fasting at least in the goldfish. Similarly, Chart and Woo 23 demonstrated that
injections of cortisol induce a rise in liver transaminase activities in the eel, together
with an elevation of ammonia excretion, and hyperaminoacidemia; the latter is
likely a reflection of increased peripheral proteolysis. These observations coalesce
into the suggestion that in teleostean fishes, cortisol shifts the preference towards
amino acids as gluconeogenic substrates 29,51, conceivably together with increases in
proteolysis in extrahepatic tissues.
Studies on the lipid metabolism of the eel indicate that cortisol induces lipolysis,
increasing production and utilization of free fatty acids from triglycerides for energy
production 36,~~ Implantation of juvenile coho salmon with cortisol resulted in
lipid depletion, mainly in triacylglyerols accompanied by elevated lipase activity
in the liver, red muscle and mesenteric fat 163. Similarly, one step removed from
the immediate effects of cortisol, administration of mammalian adrenocorticotropic
hormone (AUrH) increased plasma FFA in goldfish within 6-24 h 116. However,
the same treatment failed to affect the concentration of plasma FFA in carp 45 and
rainbow trout 174. Unfortunately, no information is available on the interplay of
A u r H with lipid metabolism during fasting.
In natural fasting situations, such as during the non-feeding migration periods
in some fish, glucocorticoids are believed to play an important role, especially in
enhancing the gluconeogenic processes 132. Plasma glucocorticoid levels increase in

sturgeons (Acipenser galdenstadti) during the upstream migration period, particu-
larly in males and this increase in hormone titer is accompanied by a depletion of
the interrenal tissue.
During the anorexia period of migration of the sockeye salmon (O. nerka), a
six-fold increase in the concentration of corticosteroids has been observed. This fact
was accompanied by a massive (up to 60%) breakdown of body, primarily muscle,
protein 82'84. Other authors have also found high corticoid levels associated with
migration in salmon. McBride et al. 111 found that plasma cortisol levels increase
in pink salmon (Oncorhynchus gorbusha) during migration, reaching levels similar
to those normally associated with stressed fish7. It appears that this hyperadreno-
corticism is not directly associated with food deprivation, although it is clear that
the catabolic effect of the increased levels of corticosteroids would facilitate the
mobilization of energy from body reserves. On the other hand, it has been de-
scribed that teleost interrenals are able to metabolize androgenic steroid precursors
to testosterone and to synthesize progesterone 25,83. The enhanced activity of the
adrenal may contribute to the process of sexual maturation.
Experimental fasting studies made mainly in salmonids have shown an opposite
trend. In O. mykiss (Salmo gairdneri) plasma cortisol levels were not affected by
prolonged fasting (65 days), suggesting that this hormone is not involved in the
processes of energy mobilization in this species 115. In an attempt to simulate natural
migration conditions, continuous swimming of experimentally food-deprived coho
salmon (O. kisutch) changed neither plasma cortisol levels nor cortisol binding sites
in liver 184. However, plasma cortisol titers decrease with fasting in sea raven 183.
Although cortisol levels seem to be representative indicators of the fish responses
to stress, these are not greatly affected by fasting. Barton et al. 7, found that
plasma cortisol elevations in response to handling stress in juvenile chinook salmon
were not appreciably modified in 20-day fasted fish in comparison to controls.
Nevertheless, the hyperglycemia response to stress was lower in fasted than in fed
fish.
In summary, experimental fasting situations have not been conclusive in de-
termining the precise role of cortisol in fish, although some general trends such
as the activation of gluconeogenic pathway from amino acids, are apparent. Data
on glucocorticoid actions under fasting conditions in fish are scarce and plasma
variations are not always clearly correlated with the physiological state of the fish.
The interpretation of the data is complicated by the possible permissive effects of
cortisol on other hormone actions.
4. Growth hormone
Few studies have been performed on the role of growth hormone (GH) during
fasting in fish. In mammals, the plasma GH concentration increases with a short-
term fast. Similarly, very large increases in plasma GH levels have been reported
for trout after fasting. Periods of fasting of 20-30 days resulted in a 7-fold increase
in plasma GH, compared to controls in steelhead trout (Fig. 3) 6. The same species
424 I. Navarro and Z Guti~rrez
fasted for six weeks also maintain higher levels of growth hormone than comparable,
fed animals 44. It has been postulated that the increase in lipid mobilization is
needed to sustain energy production during periods of continuous activity and food
deprivation such during migration time 112. In concordance with this hypothesis,
Sheridan 163 has reported that GH stimulates an increase in lipolytic enzyme activity
in coho salmon parr.
Barret and McKeown 6 also observed an exaggerated GH response when starving
fish were exercised for 24 h in comparison with exercised fish maintained on a
normal feeding regime (Fig. 3).
Obviously fasting leads to a paradoxical situation in which starved animals
although having high levels of GH do not grow. GH acts indirectly through
insulin-like growth factors (IGFs) in most tissues. Thus, Sumpter and coworkers 172
working on rainbow trout suggest that the most probable mechanism to explain this
Fig. 3. Mean levels of plasma growth hormone (GH) of juvenile steelhead trout (Salmo gairdneti; now:
Oncorhynchus mykiss). Some treatment groups were starved for 30 days, others were forced to swim
at 1.5 body length per s for 24 h. Growth hormone levels were measured with a radioimmunoassay.
Reprinted from ref. 6, with permission.
paradoxical situation would be a suppression of insulin-like growth factors similar

to the situation described for mammals 24.
In vitro studies with tilapia (Oreochromis mossambicus), have shown that fasting
results in a substantial increase in GH release from pituitary cell incubations 158.
An interesting outcome from these studies on the regulation of fish GH secretion
during fasting is that the decline in glucose levels would be the signal to induce
hormone secretion as it does in mammals.
In conclusion, there is a clear response of growth hormone to fasting in fish,
although the specific functions of this hormone in the metabolic adjustments
demand further investigations.
5. Thyroid hormones
It is generally agreed that thyroid hormones, tri-iodothyronine (T3) and thyroxine

(T4), play an important role in vertebrates affecting a variety of processes such
as metabolism, growth, differentiation and reproduction. Studies on the action of
thyroid hormones are complicated because even in higher vertebrates thyroid hor-
mones mostly affect metabolism indirectly being required for the permissive actions
of other hormones. It seems that, as in mammals, T4 in fish acts mainly after conver-
sion to T3 which represents the active thyroid hormone at the level of target cells 33.
The production of T3 is increased during short-term overfeeding which is correlated
with an increased diet-induced thermogenesis in mammals. On the contrary, plasma
levels of T3 decrease during fasting. Some of the known metabolic effects of thyroid
hormones on mammals are the potentiation of the mobilization of fat reserves,
induction of hyperglycemia and activation of protein synthesis. The general picture
that emerges from studies on administration of thyroid hormones in fish is that
these hormones appear to regulate fish intermediary metabolism in a similar way as
in other vertebrates. However contradictory findings have been described depend-
ing on the hormonal dose, fish species or season (reviewed in refs. 98 and 153).
The thyroid hormone-induced increase in the metabolic rate, consistently reported
in the mammalian literature 11, has not been unequivocally proved in fish 145'193
Fasting effects on fish thyroid have been studied primarily in salmonids. Short-
term fasting for several days or more prolonged food deprivation for several
weeks induced a decrease of plasma levels of both T3 and T4 in Oncorhynchus
mykiss 50'99'115. A similar lowering of plasma thyroid hormone levels was found in
starved Platichthys sp. 138. In contrast, no significant changes or even increases in
plasma T4 have been described in S. fontinalis 37,74. Decreased thyroid activity in
response to fasting in teleost is indicated by histological changes observed in the
thyroid of starved Oncorhynchus nerka 11~and Anguilla anguilla 78.
Kinetic studies on thyroid hormones are not clearly correlated with changes
in plasma hormones although they demonstrate a reduction in thyroid activity in
fasted fish as well as in mammals. Since thyroid activity is generally related to a
well fed and optimal metabolic state of the animal, this reduction may represent
a homeostatic protective mechanism to prolong survival of the organism under
conditions of food deprivation. Surprisingly, fasting induces a reduction in T4 and
426 /. Navarro and J. Guti~rrez
T3 metabolic clearance similarly in fish or mammals39,74. Thus it appears that

fast-induced low levels of plasma T3 could be due to a reduction in peripheral
conversion of I"4 to T3 and its release to the plasma. The changes in blood hormone
levels may not depend solely on the balance between production and degradation.
A major complication is that, at least in mammals, food intake also influences the
plasma protein binding of thyroid hormones with attendant changes in total and
free hormone levels4~ Some in vivo studies support the hypothesis of decreased
mono-deiodination from 1"4 to T3. Injection of 125I-T4 in trout demonstrated that
the transformation of T4 to T3 is already reduced after 3 days of fasting3s,s~
The number of putative T3 receptors in the liver nuclei is also reduced by 3 days
of fasting is~ At least in salmonids, fasting may produce a peripheral hypothyroidism
by decreasing both T3 production and the density of hepatic T3 binding sites.
This general decrease in thyroid activity associated with fasting observed in
fish and also in higher vertebrates, may reflect the need to limit an exaggerated
metabolite mobilization of energy reserves ~5.
VI. General conclusions
In conclusion, there is no doubt that fish tissue metabolism is finely regulated under
fasting conditions by the actions of many hormones. Interpretation of experimental
data have to be done carefully, taking into consideration the previous nutritional
and physiological state of the fish. Especially in the case of starvation, laboratory
experiments, generally done on heavily inbred fish previously fed on a diet max-
imizing meat production, give quite different results to those done on naturally
starving fish. Thus, understanding how fish respond to fasting requires research
at multiple levels from molecular to ecological. Studies involving plasma hormone
levels and energy tissue reserves gives us general information on which metabolic
processes are favored during a catabolic fasting situation. Bearing in mind that fish
metabolism is the result of a complicated integration of multiple processes in the
whole animal, and numerous environmental factors, in vitro studies are preferred
in order to identify which metabolic pathways are activated during fasting and how
they are regulated by the specific actions of the hormones. Piscine cell systems are
a relatively new approach for studies at the cellular and metabolic level and could
be particularly valuable for integrating molecular and physiological studies. Recent
research at the molecular level, the principal ones being, for instance, the charac-
terization of fish tissue hormone receptors and the identification of new regulating
peptides, open new questions on the particular strategies of metabolic regulation
to survive food deprivation situations, an interesting field of research that demands
further investigation in fish models.
Acknowledgements This work was supported by grants from CAICYT (PB90-

0471), CICYT (PTR93-0026) and NATO collaborative research programme (CGR.-
921175). We thank the Departament of Medi Natural (Generalitat de Catalunya)
and Zoological Park of Barcelona for their help in supplying fish.
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9 1995Elsevier Science B.V. All rights reserved.
CHAPTER 18
Origins of luciferins: ecology of

bioluminescence in marine fishes
ERIC M. THOMPSON AND JEAN-FRANCOIS REES *

Laboratoire de Biologie CeUulaire, Unit~ de Biologic du Dl,veloppement, Institut National de la
Recherche Agronomique, ]ouy-en-]osas, France and * Laboratory of Animal Physiology, Catholic
University of Louvain, Croix du Sud 5, 1348 Louvain-la-Neuve, Belgium
I. Introduction
II. The functions of bioluminescence
III. Organization of the light organs
IV. The biochemistry of bioluminescence
1. The components of bioluminescent systems
1.1. Luciferins
1.2. Luciferases
2. Reaction mechanism
3. Energy transfer
V. The trophic transfer of marine luciferins
1. Ingestion
2. Intestinal absorption
3. Circulatory transport
4. Storage of luciferin
5. Recycling and de novo synthesis of luciferin
6. Retention
7. Targeting
VI. Origins of imidazolopyrazine luciferins in fish bioluminescence
VII. Conclusions
Acknowledgements
VIII. References
L Introduction
Enzyme-catalyzed light production, bioluminescence, is a very common property of

fishes inhabiting the mesopelagic realm of the oceans. In waters off Bermuda,
over 93% of the individuals and 66% of the species were reported to be
bioluminescent 4,11. On the other hand, relatively few epipelagic species (1-2%)
harbor luminescent organs 82 and no freshwater bioluminescent fish species have
been reported. The total absence of luminescent fishes in freshwater is some-
what enigmatic since environmental conditions similar to those of the dysphotic
zone occur in some lakes. Interestingly, one report suggested that bioluminescent
phenomena might occur in Lake Baikal 13, possibly involving fishes.
436 E.M. Thompson and I.-E Rees
As most luminescent species have evolved sophisticated light organs with differ-
entiated luminous cells (photoeytes) associated with complex lens-like bodies, filters
and reflectors 44, it seems evident that the production of light should provide some
major selective advantages to biolumineseent fish species. It is of particular interest
that research in the last twenty years has revealed that the luminescent system used
by a number of species are not strictly endogenous and that either the entire lumi-
nescent system (symbiotic luminescent bacteria) or the substrate of the photogenic
oxidation could be of exogenous origin. In the latter case, the lueiferins appear to be
imidazol0pyrazines derived from either eoelenterazine, a luciferin found in a wide
range of biolumineseent organisms or from the luciferin of the ostracod Vargula71.
It has been suggested, and demonstrated in the instance of the epipelagie species
Potichthys notatus, that fish are capable of utilizing active luciferin obtained 'from
the diet 119.
This article will review progress in the study of fish bioluminescence and discuss
the properties of these systems relative to the suggested roles of light production
in fish. The biochemical and evolutionary aspects of bacterial light organs have
been recently reviewed 42,76,s7, and these will not be discussed in the present review.
We will also describe the problems which fish need to overcome in order to
obtain these highly oxidizable substrates from their diet as well as to process
and store them. Finally, we will briefly consider the origins of biolumineseence
in marine fishes and the possible ultimate sources of the luciferins which they
utilize.
II. The functions of bioluminescence

The preponderance of luminescent fishes in the deep sea has rendered difficult the
study of the roles of bioluminescence in all but a very few species. As a consequence,
most hypotheses have been based on morphological and physical parameters such
as the anatomical disposition of the light organs and the spectral characteristics
of the fight emission 3~ The migration to surface waters of the midshipman fish
Porichthys, has allowed some observation on the use of the photophores in this
species s2. However, the large discrepancies in light organ morphology, location,
and control mechanisms, in different species, mean that generalizations remain
speculative.
If one considers that the functions of bioluminescence are strictly related to
the light emitted and not to any metabolic advantage that might be conferred by
luminescent activity, such as the regeneration of metabolic substrates in luminescent
bacteria 41, bioluminescent phenomena could be classified into two broad categories.
Each of these would place certain constraints on photophore distribution and the
physical properties of light production, such as the intensity, emission spectrum,
angular distribution, and kinetics.
In the first group, photophores are devoted to communication between individ-
uals, that is intra- and interspecific signaling related to feeding, defense, courtship
and schooling. One can assume that selection pressure is likely to have favored
Origins of luciferins: ecology of bioluminescence in marine fishes 437
mechanisms enabling fish to emit short-duration flashes of high intensity. A further

criterion would be an emission spectrum compatible with the visual acuity of the
receiver. The emission spectrum need not exactly match that of the ambient light
as contrast would favor detection. However, as not all wavelengths are similarly
absorbed, an emission spectrum peaking near the wavelength of optimal transmis-
sion in seawater would be beneficial, unless the luminescence is involved in very
short-range communication. This would allow the emission to propagate over long
distances, a critical parameter in a deep-sea environment characterized by a very
low density of organisms. By situating the peak of emission near the wavelength of
optimal transmission, modification of the spectral composition with distance is also
reduced. In other words, the spectral quality of the signal that could be perceived
would remain the same over most of its journey towards potential receivers, with
overall intensity decreasing with distance. Thus it does not seem surprising that in
most bioluminescent organisms living in the marine pelagic environment, variations
in the color of light do not seem to be widely used as an intra- and interspecific
recognition parameter.
While light emission oriented towards communication relies on detection by
other individuals, it has been suggested that light emission could also play a role
in making fish less visible to predators 21. CounteriUumination would allow the
fish to compensate for the contrast resulting from the absorption of downwelling
sunlight by its dorsal surface. In many fish, and also among squid and shrimp,
the photophores are located on the lateral and ventral sides of the animal, and
light emission is directed ventrally. The hatchetfish Argyropelecus with its large
photophores obliterating its ventral surface has been shown to fulfill many condi-
tions that are considered essential for an effective luminescent camouflage, that is,
the spectral composition and the angular distribution of the luminescence closely
matches that of the downwelling ambient light 29,3~ Further, the fish is equipped
with a preorbital photophore directing its emitted light into the eye ~ possibly
acting as a standard for comparison with background light. The effectiveness of
counterillumination would likely be restricted to regions of the mesopelagic zone
where the intensity of light penetration does not demand an excessive energetic
cost in operating such a system of camouflage. Optimally, the fish should be able
to produce a range of intensities similar to that of the ambient light at the depth
range the fish commonly inhabits. Thus the production of bright luminescence
would not be of interest for those species which are likely to favor the emission of
light with the same spectral composition as the ambient light at a steady matched
intensity.
A different interpretation for the predominant ventral location of light organs
among fishes and oceanic invertebrates has been proposed 7~ According to this
hypothesis, communication oriented ventrally may have been selected because of
reduced predation pressure as a result of a population decline in the deep sea.
Thus, fewer potential predators would be located below the fish to intercept and
home in on a luminescent signal. However, this would also imply fewer potential
intended receivers for the signal. This hypothesis has not been supported by any
solid experimental evidence.
438 E.M. Thompson and L-E Rees
III. Organization of the light organs

Whereas light organs colonized by luminescent bacterial symbionts are open glands
connected to the exterior, non-bacterial organs are mostly internal. Only in sear-
siid fish is the postcleithral photophore a secretory organ expelling luminescent
epithelial cells into seawater 4s. Although photophores vary considerably in shape,
size, and location among species, their general organization can be schematized as
consisting of photogenic tissue surrounded by accessory optical structures. The lu-
minescence originates from highly specialized cells known as photocytes. In some
species (e.g. Porichthys, Maurolicus, Gonostoma) photocytes show clear signs of se-
cretory activities 9,~~ but precisely how these relate to luminescence remains to be
clearly defined. The photogenic tissue is richly supplied by a capillary network and
is innervated. In Porichthys, the nerves are catecholaminergic fibers from the sym-
pathetic system 7,s6. Photocytes are generally surrounded by an interference reflector
composed of multiple layers of alternating high and low refractive index. The high
refractive index material is usually composed of guanine crystals 44,46 (Fig. 1). In ale-
pocephalid fish, however, the reflectors lack guanine ~2. The reflector directs light
emission by the photocytes towards a translucid lens-like body that concentrates
the light through the photophore's aperture. In myctophid fishes, this structure oc-
curs as a thickened portion of the overlying scale 66. In some species, photophores
completely lack any light-collimating structures and the orientation of the light is a
direct function of the shape and position of the reflector. Other accessories include
light-channeling structures and filters. Spectrally selective filters are particularly im-
portant in adjusting the composition of the fight emission. Since spectral adjustments
Fig. 1. Guanine crystals from the reflector of Porichthysnotatus photophores as seen by scanning
electron microscopy.The intracellular crystalswere expelled from damaged reflector cells. Scale bar: 1
/zm (photograph courtesy of T. Smith).
Fig. 2. Disposition of the light organs of Parapriacanthus ransonneti, tlo - thoracic light organ; st =
stomach; pc - pyloric caecae; m = transluscent keel muscle; alo = anal light organ; an = anus. F r o m
ref. 36.
are made at the expense of the overall light intensity, filtering is probably not the
optimal method for light emission implicated in signaling. For this purpose, energy
transfer seems the most cost-effective strategy for altering the emission spectrum
with no reduction in the peak intensity (see section IV, part 3). In contrast, selective
filtering would be particularly relevant to fish emitting light with a spectral compo-
sition closely matching that of low-intensity downwelling light. For example, in the
ventrally oriented photophores of the hatchetfish Atg~opelecus, the halfband width
is narrowed from 53 to 29 nm through pigmented filters overlying the photogenic tis-
sue with no loss of intensity at the emission maximum 127. Future experiments should
provide information on the relative occurrence of these two mechanisms among dif-
ferent species.
Apogonid and pempherid fish appear to have evolved a unique type of light
organ. The light organs are connected with the exterior via the digestive tract,
resembling to some extent, the organization of bacterial light organs. In Parapria-
canthus, the luminous organ system consists of a pair of ducts that are connected to
the pyloric caeca (Fig. 2) and a single duct adjacent to the anus and communicating
with the exterior through a small pore 36. The thoracic organ of Apogon ellioti is an
oval-shaped structure directly connected to the intestine. In all cases light passes
through the translucent keel muscle 36.
IV. The biochemistry of bioluminescence

1. The components of bioluminescent systems
In all fish species studied thus far, light appears to be produced through the oxida-
tion of an imidazolopyrazine substrate. Imidazolopyrazines are lipophilic molecules
A. Vargulalucifedn
B. Coelenterate-type luciferin (coelenterazine)
II
,N OH
Fig. 3. Chemical structure of imidazolopyrazine luciferins found in Vargula- and coelenterate-like

photogenic systems.
characterized by a central fused imidazolone-pyrazine nucleus covalently linked to

three side group substituents. Two types of imidazolopyrazine-based systems have
been identified among biolumineseent marine organisms (Fig. 3).
In the first system, the components cross-react with the luciferins and the lu-
ciferases found in luminous coelenterates. In the second, cross-reaction occurs with
the luciferin and luciferase of the marine ostracod l~rgu/a (formerly Cypridina).
No other system has yet been identified in fish with non-bacterial photophores.
The distribution of the two types of imidazolopyrazine systems among fish gen-
era is listed in Table 1. The r system is widely distributed,
whereas only a few species utilize a system similar to that of Vargula. It can
also be noted that species oxidizing coelenterazine inhabit the mesopelagie realm
TABLE 1
Distribution of coelenterate-type and Vargula-type luminescent systems among fishes
Coelenterate-type Vargula-type
Salmoniformes Batrachoidiformes
Platytroctidae Batrachoididae
Searsia sp. (E) 16 Porichthys sp. (E,S) 27
Stomiiformes Perciformes
Gonostomatidae Apogonidae
Cyclothone braueri (S)a Apogon eUioti (E,S) 1~
Sternoptychidae Pempheridae
Argyropelecus hemigymnus (E,S) 96.1~176 Parapriacanthus bercyformes (E,S) 39,59,60
Photichthyidae
Vinciguerria attenuata (E,S) 95
Yarella iUustris (E,S,D) 1~176
Melanostomiidae
Echiostoma barbetum (S)16
Malaeosteidae
Photostomias sp. (E) 16
Myctophiformes
Neoscopelidae
Neoscopelus microchir (E,S) 57.100
Myetophidae
Diaphus elucens (E,S,D) 57,1~176
Diaphus coeruleus (E,S,D) 1~176
Diaphus suborbitalis (D) s6
Lampadena sp. (S) 16
Myctophurn sp. (S) 56
Myctophum asperum (D)56
Benthosema fibulata (D) 56
The various components of the luminescent system (E ffi luciferase-like activity; S = luciferin and D --
stabilized derivatives of the luciferin) detected in each species are indicated. Superscript numbers refer
to the list of references.
a J.-E Rees, unpublished.
of the oceans whereas species utilizing Vargula luciferin are restricted to coastal
areas.
1.1. Luciferins
Both Vargula luciferin and coelenterate luciferin (coelenterazine) emit a bright
greenish-yellow fluorescence in organic solvents, such as methanol (~m~ = 535
nm) 1~ In aprotic solvents (DMSO, DMF), both substances are chemiluminescent
with very similar emission spectra peaking at 480 nm. However, the quantum yields
(~), the number of photons emitted per mole of substrate consumed, are very low
for chemiluminescent reactions. For example, the ~ of coelenterazine in DMSO
and DMF were shown to be 0.0021 and 0.0001, respectively52,1~
In the coelenterate-type system, the luciferin may be present in a stabilized form.
Stabilized derivatives of coelenterazine have been found in the tissues, mostly the
In Diaphus, Myctophum and
liver and pyloric caeca of a few species (Table 1)56,1~176
442 E.M. Thompson and J.-E Rees
Benthosema, the stabilized derivative is a glucuronide form in which a ketone group

is conjugated to D-glucopyranosyluronic acid s6. Coelenterazine has also been found
as a luciferyl-sulfate in the biolurninescent anthozoan Renillasl.s4. In this species,
the active luciferin can be regenerated by removal of the sulfate group in the
presence of phosphoadenosine and luciferyl-sulfokinase 26.
Apart from reversibly oxidized luciferinol Hs there are no known stabilized
derivatives of Vargula luciferin and it is assumed that the luciferin is stored in
its free form in the light organs. Since the luminescence of dried Vargula can
be rapidly triggered upon immersion in water it is probable that most of the
luminescent substrate is present in a free form that can readily react with the
luciferase. However, one cannot exclude the possibility that stabilized forms of
Vargula luciferin might exist in Vargula, and in fish, but have not thus far been
detected because of the hydrolysis of stabilizing groups during extraction under
acidic conditions.
1.2. Luciferases
Our knowledge of fish luciferases is extremely limited, when data are not completely
lacking. Some coelenterazine-based luciferase activity has been found in a few
species (Table 1). In Vinciguerria attenuata, coelenterate-type luciferase activity
was found in the body (including the light organs) but not in the digestive tract
and glands 95. In this species, weak luciferase activity was associated with cellular
membranes and was not affected by EDTA or various potential cofactors such as
ATP, or pyridine and flavin nucleotides. In Arg~opelecus, the luciferase activity
seems rather specific for coelenterazine as no fight was emitted upon mixing Vargula
luciferin and the fish luciferase 96. The highest levels of luciferase activity were
recorded in the digestive tract and the light organs of the fish, but luciferase
activity was also detected in other tissues. Since the luminescence of isolated
luminous organs from Arg~opelecus is completely inhibited by cyanide, a well-
known inhibitor of peroxidases, it is possible that the highly active peroxidase
detected in Arg~opelecus tissues 16 could play a role in the luminescence of this
species.
In Porichthys, Vargula-like luciferase appears to be restricted to the light organs.
Very little data is available on this luciferase 1~8. In Diaphus, some luciferase activity
against Vargula luciferin and coelenterazine was detected in extracts of the two large
nasal photophores 55'1~176 The luminescence reaction induced upon mixing Vargula
luciferin and Diaphus photophore extracts is first order and is independent of
luciferin concentration, just as in the Vargula reaction n~ Since Diaphus luciferase
was inhibited by rabbit antibodies raised against Vargula luciferase, portions of the
two enzymes may present similar epitopes n~
Cross-reaction with Vargula luciferin has also been observed in Apogon eUioti,
Arachmia fucata, A. lineolata, Rhabdamia cypselura, Parapriacanthus beryciformes
and R ransonneti photophores 34,37- 39,60,104. The luminescence produced by mixing
hot- and cold-water extracts of R. cypseluralight organs, and to a lesser extent those
of Parapriacanthusransonneti,was found to be potentiated by the addition of N A D
and NADP to the reaction mixture 34'37-39. The luciferases of Porichthys, Vargula and
Parapriacanthus appear to have similar molecular weights of approximately 65,000

Da, but differences were observed among the chromatographic, immunological and
kinetic properties of the enzymes 121.
2. Reaction mechanism
Because of the structural similarities between luciferins of the two imidazolopy-
razine systems, some weak cross-reactivity between their components may occur.
However, the efficiency of the luciferase-catalyzed luminous reaction is much higher
with the natural substrate of the enzyme. Weak cross-reactivity may explain con-
flicting reports on the nature of the luminescent system in the nasal photophores
of Diaphus which was first said to be of the Vargula-type12~ and later classified
as coelenterate-type 55'1~176Similarly, the coelenterate-type luciferase of the deed-
pod shrimp Oplophorus also cross-reacts with Vargula luciferin 12~ Very weak light
emission can also be observed when mixing coelenterazine and Vargula lucif-
erase 109.
No direct data are available on bioluminescent reaction mechanisms in fish.
However, it is likely that they are similar or identical to mechanisms described
in other organisms utilizing similar substrates (e.g. Va~ula and Renilla), and for
which data are available. All luminescent reactions described thus far involve the
oxidation of a substrate by molecular oxygen or, in a few cases, by a derivative
such as hydrogen peroxide 41. In imidazolopyrazine-based luminescence, molecular
oxygen is required in the reaction. The chemical mechanisms leading to the
photogenic oxidation of the luciferin appear very similar in Renilla and Vargula.
In both cases, it seems well established that the reaction of the luciferin with
oxygen leads to formation of a cyclic peroxide intermediate (dioxetanone) which
decomposes to generate sufficient energy for light emission (Fig. 4). The breakdown
of the dioxetanone intermediate produces CO2 and the corresponding oxyluciferin.
Since one oxygen atom is incorporated into the carbonyl product and another
into the oxyluciferin, both Vargula and Renilla luciferase can be considered as
monooxygenases 72.
Not all coelenterates possess a luminous system similar to that of Renilla. In some
species (Aequorea, Clythia, Obelia, etc.), the luminous system consists of a stable
complex composed of coelenterazine, oxygen and an apoprotein. The luminescence
of this precharged system (photoprotein) is triggered by the binding of calcium ions
to specific sites on the proteic moiety of the complex 1~ Although photoproteins are
structurally very different from the system found in Renilla, the reaction mechanism
is similar. In fish, oxygen appears necessary for all luminescent reactions described
thus far, suggesting that precharged systems similar to photoproteins do not occur
in this group 34'12~
The wavelength of light emission is not strictly dependent on the nature of
the luminescent substrate. It is the structure of the light-emitting complex, the
luciferase-bound oxyluciferin, that determines the spectral composition of the
luminescence. Physiological conditions (e.g. ions, pH) present during the transition
from the excited to the ground state also influence the final emission spectrum.
444 E.M. Thompson and Z-E Rees
HO
O - O
od I! R2
N N--
R1 N Rs
C02
N
"~ N
it
Fig. 4. Proposed mechanism for the oxidation of coelenterazine resulting in the production of light.
From ref. 72.
Thus organisms that share the same luciferin will not necessarily produce light with
identical emission spectra.
This was demonstrated in an elegant way in click-beetles by Wood and
collaborators 12s. In this group of r the luciferin is structurally identi-
cal to the benzothiazole molecule described in other insects 74, but the color of
the luminescence ranges from green to orange, depending on the species. It was
possible to shift the spectral composition of the light towards the lower or the upper
end of the spectrum by changing as little as three amino acids in the luciferase
amino acid sequence ns. Information on in vitro bioluminescence spectra in fish are
limited. An extract of Diaphus elucens light organs was found to catalyze the oxida-
tion of Vargula luciferin with an emission peak at 456 nm n~ whereas similar tests
carried out in Apogon revealed an emission maximum at 460 nm 1~ These values
are very similar to that of the luciferin-luciferase reaction of Vargula. The biolumi-
nescence spectra of in vitro reactions are very similar in both the coelenterate and
Vargula systems. In the Vargula system, the in vitro spectrum peaks at 456-465 nm
whereas corresponding values range from 465 to 480 nm for the coelenterate-type
system ~s. These values are very similar to that of the emission maxima for the
chemiluminescence of both compounds in aprotic solvents 94.
In contrast with the paucity of data on in vitro emission spectra, in vivo emission
spectra are available for many species 47,n7. Most in vivo bioluminescence spectra
are unimodal with the peak emission ranging from 460 to 490 nm. In some cases,
the spectrum shows two peaks as observed in Porichthys and Searsia. The bimodal
spectrum of Porichthys has been tentatively ascribed to energy transfer or to the
protonation of the oxyluciferin anion during the reaction ~7. In most cases, the
emission spectrum of the in vitro luciferin-luciferase reaction cannot be deduced

from in vivo data as the presence of filters and reflectors can alter the spectral
composition of the light emitted. At the molecular level, energy transfer can also
occur, considerably altering the luminescence emission spectrum.
3. Energy transfer
As mentioned above, in some species, the spectrum of the light emitted in vitro
when mixing luciferin and luciferase in the presence of oxygen does not match
that produced in vivo. Although filters and selective reflection can play a role in
such spectral shifts these can also result from energy transfer mechanisms. In these
systems, the energy liberated by the excited complex returning to the ground state
is not emitted directly but is passed on to other molecules. These energy-acceptors
are the actual emitters and the spectrum of the light will depend on their structure
and environment.
Energy transfer can proceed through either radiative or non-radiative mecha-
nisms, that is, the energy produced in the luciferin-luciferase complex is trans-
formed into light prior to its transfer to a fluorescent emitter or, the electronic
excitation is directly passed on to the emitter. Non-radiative energy transfer mech-
anisms can be very efficient with virtually no energy loss during transfer. If the
chemiluminescence yield of the emitter is higher than that of the excited oxy-
luciferin, the overall quantum yield of the reaction can be increased by energy
transfer, as observed in the coelenterate Renilla 124. Since these mechanisms render
possible the adaptation of a biolumineseent system to the emission of other wave-
lengths at very low cost, it is not surprising that energy transfer processes are found
in many bioluminescent organisms 125.
Energy transfer is known to occur in coelenterates using either a photoprotein
(e.g. Aequorea) or a conventional luciferin-luciferase system (e.g. Renilla). In
fish, energy transfer has been proposed to occur in the deep sea stomiatoid and
searsiid fish. In addition to the many small photophores scattered over their body,
stomiatoid fishes of the genera Pachystomias, Malacosteus and Aristostomias have
two pairs of larger light organs around the eyes. One pair located behind the
eyes emit blue light, the other pair underneath the eyes emit red light. The latter
pair of photophores contain a red fluorescent protein similar to a phycobiliprotein
which is absent from the first pair. Therefore, it has been suggested that the
red emission could result from energy transfer to this chromophore 17. As the
fluorescence spectrum of this phycobiliprotein does not match that of the in
vivo bioluminescence, it remains to be clearly demonstrated whether this pigment
functions as the in vivo emitter in these species. In the deep sea fish Searsia, the
bioluminescence of excreted luminous cells undergoes spectral changes during the
time course of the emission. Just after the onset of the luminescence, the spectrum
appears monophasic peaking at 478 nm. As light emission continues, a shoulder in
the spectrum appears at 408 nm and its relative intensity gradually increases with
time until it predominates 45. Although it has been suggested that these temporal
variations could be related to energy transfer mechanisms, it is also possible that
446 E.M. Thompson and Z-E Rees
the changes could be ascribed to progressive alterations in the environment of

the emitting species in expelled cells subjected to an hyperosmotic shock in sea-
water.
V. The trophic transfer of marine luciferins

Early biochemical studies revealing cross-reactivity and identity between compo-
nents of fish luminescent systems and those of invertebrates were quite surprising.
Organisms that were not phyllogenetically related were linked by common com-
ponents of their luminescent systems. The accumulation of examples of various
species sharing the Vargula- or the coelenterate-type system among cephalopods,
crustaceans, and fish, tended to rule out the possibility of evolutionary conver-
gence. Although the transfer of both the luciferin and luciferase from l~rgu/a to
apogonids was proposed in early work34, it now seems that most of these apparent
convergences are the result of the transfer of luciferin through trophic chains.
Experimental evidence of the occurrence of such a dietary transfer of lu-
ciferin was later provided in the fish Porichthys119 and also the mysid shrimp
Gnathophausia 31. While possibly explaining the widespread occurrence of imida-
zolopyrazines among marine bioluminescent organisms, this hypothesis has raised
other questions. For example, is it an advantageous strategy for an organism to
rely on exogenous sources, sometimes very scarce, for the supply of a highly la-
bile substance that is readily rendered inactive by oxygen? If only the luciferin is
transferred, did the various luciferases evolve independently towards the systems
found in present species? If so, what were the ancestors of the imidazolopyrazine
luciferases? As non-luminescent Porichthys in Puget Sound represent a successful
breeding population, what essential functions does bioluminescence serve in this
species?
These are intriguing questions for the biologist trying to understand the basis
for the evolution of bioluminescence in fish and invertebrates. At present, no clear
answer to these questions can be formulated, but recent data provide us with clues
to pathways which might have been involved. What are the adaptive responses
that would be required for such a dietary transfer mechanism to be efficient?
These include the ingestion of prey containing luciferin, transfer of luciferin to the
light organs, the possible recycling of the oxidized luciferin, the avoidance of loss
through body surfaces or the excretory system, and the development of luciferases
and bioluminescent control mechanisms. Fig. 5 summarizes the major problems
facing a fish using vicarious luciferin. We will now consider some of these possible
adaptations in more detail.
1. Ingestion
The first problem encountered by a luminescent fish species dependent on an exoge-
nous source of luciferin is ingestion of the source prey. In the case of fishes requiting
coelenterate-type luciferin, the dietary supply appears not to be problematic as this
Fig. 5. The metabolism of exogenous luciferin in fishes. This figure summarizes some of the problems
facing a hypothetically luminescent fish. 1 ffi ingesting prey containing the desired luciferin; 2 --
absorbing it from the intestines; 3 ffi protecting this very labile luminescent substrate during its transfer
via the circulatory system; 4 ffi storing luciferin (e.g. in the liver); 5 ffi recycling the oxidized luciferin
or resynthesizing de novo the active luciferin; 6 = avoiding losses through renal excretion and diffusion
through the gills; 7 ffi targeting the active luciferin to the light organs.
molecule seems to be abundant in the marine ecosystem. Coelenterazine is a rather

widespread molecule that is present in both luminescent and non-luminescent or-
ganisms from many phyla 16,1~176176 It has a worldwide geographical distribution and
is present in both pelagic and coastal areas of the oceans. Since the highest luciferin
concentrations occur in luminescent species ~~176176 it would be more efficient for the
fish to feed predominantly on other luminescent organisms using the same luciferin,
providing that all other nutritional requirements can be satisfied equally by either
luminescent or non-luminescent food items. Do luminescent fish feed selectively
on luminous prey organisms? This hypothesis might appear a little paradoxical
since the bright luminescent flashes produced by many planktonic organisms were
previously assigned roles in predator evasion 1,14,15,2s.
Feeding ecology studies analyzing the diet of mesopelagie fishes are few in
number and none has thus far investigated the role of bioluminescence in the
predator-prey relationship. In an attempt to analyze whether bioluminescent fish
prefer luminous to non-luminous prey, we have analyzed the proportion of bio-
luminescent organisms in the food of mesopelagic fish as reported in ecological
studies. Our analysis was restricted to studies reporting the composition of the fish
diet down to the genus level (Table 2). It is important to consider this data with
caution, as not all species within a genus are luminescent, and not all food items
could always be identified. Also, due to the low number of organisms which have
been studied for the presence of coelenterazine, the proportion of those actually
containing coelenterazine may be underestimated.
The data reveal that luminescent prey can make up a large part of the food items
TABLE 2.
Luminescent organisms in the diet of mesopelagic bioluminescent fish genera
Genus % luminescent % prey items with Reference

prey items a coelenterazine b
Valenciennellus 69-86 84--100 81
59-71 52- 78 22
47-70* 33- 44 50
Argyropelecus 53-89 58- 85 81
64" 31 50
12-24 92 63
Lampanyctus 22-42 65 81
31 * 100 50
5- 8 100 62
Gonostoma 52 * 65 50
60-81 36-- 70 22
Sternoptyx 10-50 17- 59 49
63
~ncig~erria 15-80 5- 43 22
From data reported in the literature.
a In some cases, large proportions of the food items could not be identified. The bioluminescent nature
of prey items was based on systematicdistribution data for bioluminescence4s. Genera were considered
luminescent only where the occurrence of bioluminescence has been clearly documented.
b From data in reference 16. Since only a few copepod genera were investigated for the presence of
coelenterazine in their tissues, and coelenterazine is likely to be the luminescent substrate in many
planktonic crustaceans, these values are probably underestimates.
*These values correspond to the percentage of luminescent organisms in the three most abundant food
items. Dashes indicate the absence of luminescent genera.
taken by these fishes. In some species this can represent 80% of ingested organisms.
Most of the luminescent prey consist of copepods, mainly Pleuromamma sp. and
euphausiids. While small individuals feed almost exclusively on small zooplankton
(mostly copepods), larger individuals tend to feed on euphausiids and even small
fishes. Coelenterazine is found to be present in the diet of all fish species for
which data are presented. It is remarkable that in some stomach content analyses
(Valenciennellus sp.), luminescent prey accounted for more than 70% of the diet and
all of these prey items are known to possess a coelenterazine-based luminescent
system 16.
While these data demonstrate that luminescent fishes ingest luminescent prey,
they do not signify that these fishes feed selectively on luminescent organisms.
Previous studies of the feeding ecology of fishes reveal large discrepancies in the
feeding behavior and regimes of mesopelagic fishes. While some are random feed-
ers, taking the nearest prey available, others show evidence of feeding selectivity.
Clarke suggested that sternoptychids and small gonostomatids feed by active, visual
searching and preferred prey were probably more visible in terms of body trans-
parency, pigmentation and size 22. He did not take bioluminescence into account in
this analysis. In Benthoseraa glaciale, there seems to be a clear preference for the
luminescent copepod Metridia lucens whereas the predominant copepod species,

Clausocalanus, living at the same depth as Gonostoma, appears to be avoided 97.
Thus, in summary, a large part of the food taken by mesopelagic fish consists
of luminescent organisms, mainly copepods and euphausiids. Although some fish
appear to feed randomly, other species selectively feed on particular luminescent
organisms from which they can obtain the luciferin for their own luminescence.
Since coelenterazine is also present in non-luminescent organisms, though in lower
quantities 1~ these might constitute accessory sources of the luminescent substrate,
meaning that exclusive feeding on luminescent prey is not essential.
The situation of fishes using Vargula luciferin is in sharp contrast with that of those
requiting coelenterazine. Although the precise sources of Vm~,ula luciferin remain
to be clarified, it seems likely that few organisms biosynthesize Vargula luciferin,
and the source may be limited to the ostracods themselves. The known geographic
distribution of Vargula luciferin is restricted to areas inhabited by Vargula. The fact
that ostracods are scavengers would probably reduce their ability to obtain large
amounts of active luciferin from the diet. Finally, Vargula produces bright flashes of
luminescence, resulting from the discharge of luciferin and luciferase into seawater,
a strategy which does not seem wholly compatible with dependence on an exogenous
source of a relatively rare molecule. This is in contrast to the secreted biolumines-
cence of the mysid shrimp Gnathophausia, a species which is known to acquire its
luciferin (coelenterazine) from the diet 31. In this case, the abundance of coelen-
terazine in the shrimp diet compensates for the loss of luciferin accompanying the
activity of the luminous glands. The possibility that no source of luciferin other than
Vargula exists is further supported by the sympatry of Vargula sp. and the distribution
of fishes using Vargula-like luciferin in their own bioluminescent reactions.
In the group perciformes, both luminescent apogonids and pempherids are sym-
patric with VargUla. However, specimens of Vargula are found only rarely in the
stomach contents of these fishes. Only a dozen out of more than 2000 Parapria-
canthus beryciformes specimens were found to contain some Vargula specimens 59,114
and Vargula have yet to be found in Apogon stomach contents 36. Quantitative data
on the yield of luciferin extracted from the pyloric caeca of 2300 Parapriacanthus
specimens revealed that on average, one specimen contained 10/zg of luciferin s9.
Considering that the luciferin content of Vargula hilgendorfii is about 1 p,g33, this
suggests that the ingestion of a few dozen ostracods should be sufficient to replenish
the luciferin stores.
While the linkage of luminescent Vargula with apogonid and pempherid biolu-
minescence seems compelling, the use of an exogenous source of luciferin, most
probably luminescent Vargula, has been most extensively studied in the batrachoidid
genus Porichthys.
Paleontological evidence indicates an Atlantic origin for the genus with a subse-
quent migration to the Pacific coasts of North and South America. In the Atlantic,
all species are functionally bioluminescent. Along the coast of Peru and Chile, one
species, Aphos porossus, is given separate generic status because it lacks the pho-
tophores characteristic of all other members of the group. All other morphological
characters are consistent with inclusion in the genus Porichthys. In the Northern
hemisphere, Porichthys notatus has the greatest range, extending from Vancouver
Island to the Southern tip of Baja California. However, this species is divided by
a distributional break off the coast of Oregon into two populations. The Northern
population, located mainly around Vancouver Island and in Puget Sound, is unable
to luminesce due to a lack of luciferin, despite possessing photophores which are
ultrastructurally indistinguishable from the Southern population 1~ It is of interest
to note that both the Northern population of R notatus and the SouthernA. porossus
are separated from their luminescent relatives by regions of cold seasonal upwelling
corresponding roughly to the spawning periods of these fishes and which may act as
boundaries for the habitat range of the appropriate luminescent prey.
In fact, the sparse data available concerning the range of luminescent Vargula
tsujii suggest a limit coincident with the Northern extreme of the California pop-
ulation of R notatus 64. Near this distributional boundary both luminescent and
non-luminescent individuals of R notatus can be found 126. Furthermore, it is pos-
sible to induce sustained luminescence capability in non-luminescent P. notatus
by feeding either purified Vargula luciferin or whole luminescent Vargula to the
fish 111'119. Feeding of a single luminescent ostracod is sufficient to establish a weak
luminescence capability in a previously non-luminescent individual. Despite this
strong circumstantial evidence concerning the source of luciferin in the Porichthys
diet, considerable stomach content analysis of P. notatus has yet to reveal the
presence of luminescent Vargula in the natural luminescent population 2'1~
At the Northern end of the range of the California population of P. notatus,
around the San Francisco Bay area, a more detailed analysis of the population struc-
ture relative to luminescent capability has suggested some avenues of research 11~
Maternal luciferin is known to be transferred to Porichthys eggs 109'119 and provides
an initial luciferin source for developing larvae and juveniles. In juveniles ranging
up to 80 mm in standard length (corresponding to the first year class) the relative
uniformity in luminescence capability in the San Francisco Bay region may reflect
this maternal transfer 11~ However, in the second year class, a distinct gradient
of decreasing luminescence capability is found from the South to the North. One
explanation may be that Southern juveniles are able to directly feed on lumines-
cent Vargula which are absent further North, thus maintaining sufficient luciferin
stores. In this regard, it has also been noted that juvenile P. notatus are attracted
by luminescent flashes78. In the third year class, a high proportion of luminescent
individuals is found throughout this region. This may result from the attrition of
non-luminescent individuals or perhaps an indirect dietary incorporation of Vargula
luciferin available to larger but not smaller fish. One possible source would be
cannibalism 11~ Young R notatus are an important dietary component of R myriaster,
a shallow water relative found further to the South, but there are as yet no data to
support the idea that P. notatus cannibalizes its own juveniles.
2. Intestinal absorption
Have luminescent fishes evolved specific absorption mechanisms for luciferins and
their metabolites? After prey have been ingested, they undergo mechanical and
Originsof luciferins:ecologyof bioluminescencein marinefishes 451
chemical digestion in the stomach. The acidic conditions present in the stomach
should protect luciferins from autoxidation. This seems consistent with previous
studies 1~176176
showing that relatively high amounts of active luciferin can be isolated
from fish stomachs, irrespective of whether or not these organisms are themselves
luminescent. Since enol-sulfate bonds are hydrolyzed at low pH, a proportion of
the known stabilized luciferin derivatives may be converted into active luciferin
during transit through the stomach. These active molecules would then be unstable
at the alkaline pH found in the intestines, and rapid absorption of luciferin would
be advantageous. Whether mesopelagic fishes have developed specific absorption
mechanisms for coelenterazine is at present very difficult to investigate because of
the problem of maintaining these fishes in captivity.
On the other hand, experiments carded out on shallow-water fishes suggest that
these possess selective absorption mechanisms for Vargula lueiferin. Perhaps the
most striking examples of adaptation are found in the pempherid and apogonid
fishes of the Indo-pacifie. In these species, the thoracic light organs communicate
directly with the digestive tract through small ducts connected either to the intestine
(Apogon sp.) or the pylorie caeca (Parapriacanthus sp.). The anal luminous organs of
these species, while not connected to the digestive tract, are closely associated with
the rectum, and this disposition probably allows fairly rapid transfer of luciferin to
these organs 36.
The close association of the light organs with the digestive tract probably allows
rapid and efficient transfer of luciferin to the light organs, but places constraints
on the number, and the localization of the photophores. Thus, such a strategy
becomes deficient in the case of a bioluminescent fish such as Porichthys, with more
than 700 photophores distributed over the head and trunk. In this genus, luciferin
must be absorbed through the intestinal wall and carded to the photophores via
the circulatory system. Recent studies suggest that Porichthys may have developed
specific intestinal absorption mechanisms for the uptake of Vargula luciferin 112,113.
Uptake of orally administered luciferin was compared in luciferin deficient, Puget
Sound, P. notatus, a non-luminescent relative, Opsanus beta, and a non-luminescent
unrelated fish, Paralabrax clathratus. When fed a single 7.6/.tg dose of luciferin, the
blood concentrations of luciferin 12 h later were 2.12, 0.56 and 0 ng/ml respectively.
When challenged with 6 such doses of luciferin at 4 day intervals, the blood luciferin
levels observed 16 h after the last feeding were 24, 1 and 0 ng/ml respectively.
Since the in vitro stability of luciferin in the blood of all three species was the same,
the differences in blood luciferin in vivo concentrations seem to reflect differential
intestinal absorption. It should be noted, however, that the overall efficiency of
luciferin uptake remained low in Puget Sound P. notatus. This was on the order of
0.5%, a figure confirmed when isolating 14C-labeled luciferin from photophores 112.
However, it would seem likely that potential natural sources of Vargula luciferin
in the diet of P. notatus are present in very low quantities at any given time.
Therefore, the evolution of a high affinity-low capacity system of luciferin uptake
may have been favored. There are as yet no data on whether naturally luminescent
California P. notatus are capable of the uptake and eventual utilization of the
oxidized products of Vargula luciferin. Should such a capability exist, the selective
pressure for the rapid intestinal uptake of active luciferin would be considerably
reduced.
3. Circulatory transport
Vargula luciferin and coelenterazine are very sensitive to molecular oxygen. Thus,
the fish is faced with the problem of protecting luciferin from autoxidation during
its circulatory transport to the photocytes. In Porichthys, luciferin is continuously
present in the blood 2~'n3. One could propose the involvement of a specific luciferin
binding protein, as found in the anthozoan Renilla 25, in the transport of the lumines-
cent substrate to the light organs. Experiments demonstrate that blood components
in Porichthys can effectively protect luciferin from autoxidation 113. However, no spe-
cific carrier for luciferin was identified in Porichthys blood, independent of whether
or not the fish were from a naturally luminescent or non-luminescent population,
or originally non-luminescent but subsequently induced with luciferin. Instead, it
seems that binding and protection of Vargula luciferin is a non-specific property of
fish blood, as this property is also found in the non-luminescent fishes Opsanus and
Paralabrax113.
Results suggest that Vargula luciferin can bind to either plasma components
or red blood cells. It has been calculated that the number of lueiferin molecules
binding to the erythroeyte surface was 2.5 x 107per cell. Since the binding capacity
of erythrocytes was not affected by a trypsin treatment luciferin appears to bind
directly to the membrane surface and not to groups protruding beyond the perme-
ability barrier. The nature of the heat-sensitive plasma components ensuring some
protection of the luciferin is not known. No data are available on the stability of
coelenterazine in the blood of mesopelagie fishes.
4. Storage of lucifetin
It is highly probable that the discontinuous luciferin supply should be compensated
for by some ability to store luciferin in a form and location from where it can be
rapidly mobilized when needed. The liver appears to contain large quantities of
coelenterazine in many mesopelagic fishes and, together with the photophores, are
the likely main storage sites for luciferin 1~176
Deep sea fish have a comparatively low
resting metabolism 24, and storage involving low energetic cost is likely to have been
favored. Among the most cost-effective storage mechanisms for such labile com-
pounds would be to transform the luciferin into a stable, inactive derivative, from
which the active form can be easily regenerated, rather than synthesizing binding
proteins or maintaining a low pH in intracellular compartments as demonstrated
in some other luminescent organisms 43. This seems to be the method selected
by organisms using coelenterazine, as natural, stabilized derivatives of this com-
pound have been shown to exist. Two different derivatives have been described,
an enol-sulfate form and one in which a keto-group is linked to a gluco-pyranosyl
moiety 52,s6. The enol-sulfate form has yet to be clearly identified in fish but its pres-
ence is well documented in the anthozoan, Renilla 52'54. It is particularly noteworthy
that transfer of gluco-pyranosyl acid is involved in the detoxification of exogenous

substances. Indeed, enzymes involved in glucuronoconjugation play a very impor-
tant role in vertebrates in neutralizing toxic compounds and other poorly soluble
substances prior to excretion through the renal system 1~ Glucoronoeonjugation
is known to take place in the liver of vertebrates. Thus it might be that these
very systems could be used for preserving labile compounds such as luciferins from
autoxidation and allow low-cost storage of these highly valuable substances. The
synthesis of enol-sulfate derivatives is also the mechanism apparently involved in
the protection of ascorbic acid against autoxidation in fish and crustaceans 77,s3,122.
Vertebrates appear to share this property with some invertebrates. Besides Re-
nilla, stabilized forms of coelenterazine, apparently the enol-sulfate derivative,
are also present in shrimp, both luminescent and non-luminescent 1~176176 These
luciferin derivatives can be synthesized by shrimp, as the non-luminous Crangon
septemspinosa and Pandalus danae, when injected with eoelenterazine, accumulated
coelenterazine enol-sulfate in the hepatopancreas 1~
Fish which use a luminescent system based on the incorporation of Vargula lu-
ciferin, do not have large stores of the luminescent substrate in their hepatic tissues,
nor do they seem to possess any stabilized derivative of the luciferin. In Porichthys,
the many photophores seem to be the main luciferin storage site, with luciferin
content in the liver attaining only 2.5-4% of that of the pooled light organs 27.
Although the possible roles of the translucid subocular gels remain enigmatic, these
structures were shown to contain rather large amounts of luciferin 27, and might thus
constitute important luciferin stores. In other epipelagic species (Apogon, Parapria-
canthus), the pyloric caeca and the light organs seem to be the main storage sites
for luciferin 36.
The nature of the mechanisms utilized in preserving the very labile Vargula
luciferin from autoxidation remain obscure. In Porichthys, the luciferin from pho-
tophore extracts elutes together with a high molecular weight compound exerting an
inhibitory action on the light reaction 118. Although the nature of this compound was
not elucidated, it might possibly play some role in the protection of luciferin against
autoxidation and also constitute part of the triggering mechanism for luminescence
as does the luciferin binding protein in Renilla 2~ Another possibility is that particu-
lar ionic conditions (e.g. low pH) might exist in the many large intracellular vesicles
suggested to contain the luminescent substrate in the photogenic cells 1~ Porichthys
photophores can be dissected out and retain their luminescent capacities for several
hours 7,93. When irradiated with near UV-light, they emit a bright greenish fluores-
cence, the intensity of which seems proportional to the amount of active luciferin
present in the photogenic cells 6. Recently, it has been observed that both the
fluorescent and luminescent properties of isolated photophores can be irreversibly
abolished by treating the light organ with glyceraldehyde 3-phosphate 91,92. This
glycolytic intermediate seems to control the luminescent activity of photocytes 92.
Since glyceraldehyde 3-phosphate seems to have no inhibitory action on the Vargula
luciferin-luciferase reaction in vitro (J.-E Rees, unpublished observation), under-
standing the action of this compound on the photophores could provide some clues
to the storage conditions of the luciferin within the photocytes.
5. Recycling and de novo synthesis of luciferin
We have seen that many bioluminescent fishes are exposed to luciferin in their diet,
and are capable of incorporating and possibly modifying this bioactive molecule
into stabilized derivatives. To what extent are bioluminescent fish capable of de novo
synthesis or recycling of these valuable molecules?
Conversion of oxyluciferin to luciferin has been demonstrated in vivo in the
firefly ss and it appears that in the bioluminescent squid, Watasenia scintillans,
oxyluciferin or etioluciferin may be returned to the liver for resynthesis of luciferin
or preluciferin ss. Among bioluminescent fishes the question has been most closely
investigated in Porichthys notatus.
Although recently, it has been found that luminescent capability can be extin-
guished in naturally luminescent juvenile California R notatus by massive repetitive
stimulations in an epinephrine bath 8~ suggesting, under these conditions, the re-
quirement for a continuous source of luciferin in this population, the finding that
feeding of a single luminescent ostracod was capable of establishing luminescence
capability in previously non-luminescent R notatus from Puget Sound s, raised the
question as to whether this could be explained by a simple incorporation of the
luciferin with no subsequent stimulation of de novo synthesis or recycling. A further
study was carried out to quantify the light yield from a number of Puget Sound P.
notatus, each of which was induced by feeding of a single 6.4/tg dose of purified
Vargula luciferin 111. Surprisingly, this single dose was capable of stimulating a lumi-
nescence capability which persisted for more than two years and a calculation of the
light produced during this period suggested that it surpassed the yield which could
be anticipated from the single initial feeding. In order to try and determine whether
this resulted from de novo synthesis or recycling of luciferin, ~4C-labeled Vargula
luciferin was synthesized and administered to non-luminescent R notatus in. When
luciferin was recovered from the photophores 7 weeks later it was found that the
labeled luciferin had indeed been directly incorporated and that its specific activity
remained unchanged, indicating that no de novo synthesis of luciferin had taken
place. To test whether recycling might explain the sustained luminescence capacity,
both oxyluciferin and etioluciferin were also administered to non-luminescent fish
but failed to stimulate a bioluminescent response ~n.
Thus several questions remain to be answered. Are Vargula 0xyluciferin and
etioluciferin unable to penetrate to a potential site of luciferin resynthesis in
Potichthys? Does Porichthys modify Vargula luciferin or its oxidation products so
that Vargula oxyluciferin or etioluciferin are not recognized? Is it first necessary
to induce luminescence capability with active Vargula luciferin before the oxidation
products can be utilized? Also, it cannot be completely ruled out that de novo
synthesis is tied to the presence of active luciferin in the fish but is restricted to
certain periods, such as during reproductive activity.
There are no indications as to whether mesopelagic fish possess any recycling
mechanism for coelenterazine. Nevertheless, one could attempt to compare their
theoretical needs with the amounts of coelenterazine that have been detected in
their tissues. Let us first consider the hatchetfishArgyropelecus hemigymnus. Accord-
Origins of luciferins:ecologyof bioluminescencein marinefishes 455
ing to the angular distribution and the spectral composition of the light produced by
their large, ventrally oriented photophores, this species is considered a good exam-
ple of a counterilluminating fish29'3~ Population studies have shown that this species
lives at depths ranging from 100 to 600 rn in the Northeastern Atlantic and 110 to
530 rn in the Mediterranean 3'4'97. The fish migrates to the upper layers (110-270 m)
at night 4,32,89,116. In the Strait of Messina, the depth range of A. hemigymnus was
reported to be 180-500 m (ref. 5). Light measurements 5,98 showed thatA, hemigym-
nus are present in an ambient irradiance extending from 1.5 x 10-5 to 8 x 10-3/zW
cm -2. Since the optimal use of the photophores for counterillumination requires that
the light output equal that of the ambient light level, a one cm 2 photophore should
be capable of producing from 3.6 x 107 to 1.9 x 101~photons s -1 (),max = 480 nm). If
we assume the quantum yield of the luminescence reaction to be equivalent to that of
the coelenterazine-based system of Renilla (~ -- 0.05), the amount of eoelenterazine
required would be between 7.2 x 108 and 3.9 x 1011 molecules per second. Thus,
a fish with a total emitting area of one cm 2 would consume 0.07 pmol coelenter-
azine per min when living at the lowest ambient light levels, and 38 pmol min -1 at
the brighter end of the inhabited irradiance range. The total coelenterazine content
of A. hemigymnus has been reported as ranging from undetectable levels up to 47
pmo116,96,1~ Thus, during the day, the amount of coelenterazine present in the fish
would allow the emission of a continuous glow with an intensity matching ambient
light for up to about 10 h at the deeper end of its depth range, whereas this could
persist for only 70 s at the upper irradiance levels. These values have been confirmed
by experiments carried out on isolated photophores. Pharmacological experiments 68
revealed that A. hemigymnus large ventral photophores stimulated by epinephrine
can produce a one-hour glow with a total yield of 1012 photons or the equivalent
of 0.4 ~W cm 2. No further light response could be elicited following this emission,
suggesting that this corresponds roughly to the total luminescence potential of the
isolated organ. Thus, the ventral photophores of a fish spending the day at the lower
end of the irradiance range could emit a sustained glow for about 8 hours with no
need to recycle the oxidized luciferin; on the other hand, camouflage in the upper ir-
radiance zone would only allow for the emission of a few short-duration flashes, and
would depend on the existence of recycling mechanisms for the oxidized luciferin.
However, at night, light intensities are reduced to 10-6-10 -9 of daytime levels 85.
Therefore, if light emission by Argyropelecus was to be used only at night, a single
pmol of coelenterazine could allow continuous luminescence for a period of years!
Furthermore, if we consider that most fish cannot distinguish a luminescent source
from background light when the ratio of the two intensities is as different as 1:10
(ref. 85), this would allow camouflage to be effective at a reduced luciferin consump-
tion rate (up to 90% economy). This strategy is supported by recent observations on
the ventrally oriented bacterial photophores of leiognathids. Measurements of the
absolute luminescence produced by Gazza minuta in response to increasing levels of
downwelling light revealed that, while the luminescence output also increased with
that of the background, it never exceeded 20% (range 2-19%) of the ambient light
level 7s. If this strategy is effective in dissimulating mesopelagic fishes from predators,
this would be a means of conserving luminescent substrates.
456 E.M. Thompson and L.F. Rees
Feeding studies on Arg~opelecus showed that the stomach contains an average of

4 prey items per specimen, and that ingestion rates are likely to be the order of a
few prey (mostly copepods) per day 22,50,81,97. Considering a coelenterazine content
of luminous copepods of 2-35 pmol per organism 16, the fish is likely to dispose
of a daily dietary supply of around 10-100 pmol of coelenterazine. This is clearly
not sufficient for the production of sustained luminescence at high ambient light
intensities unless some recycling of oxidized coelenterazine occurs. However, if the
fish were to restrict the use of their photophores to depths corresponding to diurnal
periods of low light irradiance, then the dietary supply of coelenterazine would be
sufficient and no recycling of the oxyluciferin would be required.
Among bioluminescent fishes, Arg~opelecus appears to contain particularly small
amounts of coelenterazine 16,96,1~176 The coelenterazine content of myctophids seems
to be up to three orders of magnitude greater 1~176 In these fishes, the emission
of a sustained glow of the same intensity as ambient light would pose tittle
problem, and the photophores would be effective over a wide intensity range. In
fact, the quantities of coelenterazine present would allow the fish to emit bright
flashes that could be used for communication. The application of electric stimuli
to mesopelagic fishes revealed great variations in the intensity and kinetics of the
triggered luminescent flashes79. The flash intensities extended from 5 x 10 9 to
8.7 • 1011 q s -1. However, the kinetics of evoked luminescence were very rapid,
lasting for no more than 4 seconds. As suggested by the rapid decrease in the
intensity of successive flashes, such high intensity luminescence could probably not
be produced over long periods, presumably due to the rapid depletion of luciferin
stores. Nonetheless, it remains possible that the quantities of luciferin in these
fishes would permit use in both counteriUumination and signaling. In this context,
it is of interest that among two species of Sternoptyx, S. diaphana, living at depths
ranging from 800 to 1500 m, was reported to produce flash intensities ten times that
of S. pseudobscura, even though the latter is known to occur at a shallower depth
range of 300-1000 m 79. However, one should keep in mind that the light emissions
observed were not spontaneous phenomena but resulted from artificial stimulation
of perturbed to moribund fishes brought relatively rapidly to atmospheric pressure.
Therefore, extrapolation of this data to in situ uses of luminescence remains
hazardous.
Although the above calculations suggest that numerous mesopelagic species
could rely completely on a dietary supply of coelenterazine, the possibility exists
that some species may recycle the oxidized luciferin. A possible recycling mechanism
for coelenterazine has been proposed involving the etioluciferin (coelenteramine)
and p-hydroxyphenylpyruvic acid (Fig. 6) 72. This reaction is the final step in a
chemical synthesis of coelenterazine 53. Whether or not this reaction occurs in vivo
remains to be demonstrated.
6. Retention
Experiments have demonstrated that non-luminescent specimens of Porichthys nora.

tus can be made bioluminescent by the ingestion of a few #g of Vargula luciferin, and
Origins of luciferins: ecology of bioluminescence in marine.fishes 457
IN ~ O H H jN~ jNH: ~CO2H

20 ~- R.~N~Rs + HO"
RI~N~Rs
~ Llght
~
- CO2 [Luciferase HO~
riC02H
0
H
HO:tC~
1% " -,,11o.
R~N~~Rs
.o c t "
Fig. 6. Hypothetical recycling mechanism for oxidized coelenterazine. From ref. 72.
that once induced, this capability persisted for more than 2 years ~ . Since luciferin
is found in significant concentrations in P. notatus blood 113, losses are very likely to
occur through the gills and kidneys. However, since de novo luciferin synthesis does
not seem to occur in this species 111, losses of luciferin would be very disadvantageous
and should be prevented to the greatest extent possible. The association of luciferin
with blood components described above may in part reduce losses due to diffusion.
On the other hand, increases in free luciferin in the circulation may not necessarily
result in increased excretion by the kidneys. The kidney structure in Porichthys is
likely to resemble that of its close relative Opsanus tau. In this species, the kidneys
consist exclusively of tubules with no glomerula 69 and excretory activity relies on
active secretion of solutes rather than filtration. The structure of Vargula luciferin,
in which a guanido group is attached to the imidazolopyrazine nucleus, makes the
molecule reasonably hydrophilic and may also reduce losses by limiting membrane
penetration (E.M. Thompson, unpublished observation).
Unfortunately, no data concerning coelenterazine retention in mesopelagic fishes
are available. In coelenterazine, the guanido group of Vargula luciferin is replaced
by a phenyl residue rendering the molecule more hydrophobic. Recent reports have
demonstrated that coelenterazine can easily diffuse across membranes 61. Thus, this
molecule would theoretically be free to diffuse across surfaces such as the gills
and be irreversibly lost in the surrounding water. However, since the abundance of
coelenterazine in the fish diet appears much higher than for Vargula luciferin, one
could expect that the mechanisms implicated in the retention of the luciferin and/or
its oxidation products should not necessarily be as performant as in the case of
Porichthys. Also, the transformation of coelenterazine into a more stable, and more
hydrophilic form, by sulfotransfer or glucuronoconjugation, could, by decreasing
coelenterazine's hydrophobicity, reduce membrane transit, and aid in retention of
the luminescent substrate.
Z Targeting
Once present in fish blood, luciferin must be transferred into the photogenic
cells where it can be used for light production. As coelenterazine is hydrophobic
it is able to diffuse across the cell membrane. However, passive diffusion of
the lucfferin would be slow and non-specific, and is unlikely to be the only
mechanism of luciferin uptake in the photocytes. Studies in Porichthys notatus,
suggest that highly specific lucifcrin uptake systems are present on the photocyte cell
membrane. In non-luminescent Puget Sound specimens, there is a delay of several
days after feeding with Var~/a luciferin, before lucifcrin begins to accumulate in
the photocytes, as judged both by bioluminescence capability and the onset of
photophore fluorescence in near ultraviolet lights,111,119. Since Vargu/a luciferin and
Podchthys luciferin appear to be very similar, if not identical 27,1~ it is unlikely that
this delay reflects a conversion of the luciferin, and it is more probable that the
presence of lucifcrin in the blood may be necessary to stimulate the synthesis of
lucifcrin receptors on the photocytes. The uptake mechanism appears to be highly
selective, as two lucifcrin analogues, one of them differing from Vargula luciferin
only by a guanido group, were unable to induce luminescence or photophore
fluorescence in Puget Sound E notatus in. Similar results were obtained by either
oral or intraperitoneal administration of these compounds, ruling out the possibility
that this only reflects specificity at the level of intestinal absorption. Since the
amounts of lucifcrin in P. notatus blood are in the ng/ml range, the photocytes are
likely to have a very high affinity uptake mechanism for luciferin.
Photophorcs are not the only targets for luciferin uptake. Female Porichthys no-
tatus lay over 100 eggs on the underside of rocks. Early observations demonstrated
that Vargula luciferin can be found in newly laid eggs and was detectable throughout
all developmental stages of luminous R notatus 119. At that time, the presence of Var-
gula lucifcrin in eggs and prefeeding larval stages was interpreted as a reflection of
the capacity of the fish for de novo synthesis of luciferin. However, subsequent work
suggests that the presence of lucifcrin in P. notatus eggs more likely involves the
transfer of maternal lucifcrin stocks into the eggs Ill. When female E notatus were
induced with Vargula luciferin, luminescence intensity showed a cyclic pattern with
low points corresponding to the spawning season. During this period the females
had swollen abdomens, characteristic of egg formation and the few eggs which
were laid were found to contain lucifcrin. Following this period, the luminescence
intensity of these females was found to recover, coincident with the reabsorption of
unlaid eggs. No similar cyclical pattern of luminescence intensity was observed in
induced males 111. The presence of maternal lucifcrin in the eggs, and the detection
of lucifcrase on day 28, just prior to release of the juveniles from the substrate 119,
means that the young juveniles possess a fully functional luminescent system when
they become free-swimming. Maternal transfer of luciferin does not appear to be
restricted to P. notatus, as in the hatchetfishArgyropelecushemigymnus, not only was
coelenterazine present in the egg bags, but its concentration was the highest among
all fish tissues, about twelve times that found in the light organs 96.
VI. Origins of imidazolopyrazine luciferins in fish bioluminescence

Earlier, we divided the functions of bioluminescence in fish into two broad cate-
gories. However, counterillumination and signaling need not necessarily be mutually
exclusive functions, both could be used in a given species, simply by varying the
intensity and kinetics of light emission. As previously discussed, the properties of
light propagation in seawater may have led to similar selective pressures on the
wavelength of emission for either function. Thus, it is important to note that the
chemiluminescent properties of both Vargula luciferin and coelenterazine corre-
spond well with the constraints imposed by transmission in the seawater medium.
The emission spectrum of imidazolopyrazine chemiluminescence is affected by
the electronic charges of the imidazolopyrazine moiety and of the substituent
side groups. At physiological pH, the emitter is the monoanion form of the
oxyluciferin 72 and the luminescence maximum is from 470 to 480 nm. The various
luciferases which have evolved do not appear to have significantly modified this
spectral distribution as many bioluminescent organisms, using either eoelenterazine
or Vargulaluciferin, have very similar emission spectra. In organisms which do emit
considerably different wavelengths of light, this seems to be achieved by transferring
the energy of the excited imidazolopyrazine luciferin to a secondary emitter such as
a fluorescent protein 124. Thus imidazolopyrazines are well suited for use in marine
bioluminescence, but do they have other properties that might account for their
widespread occurrence in the marine environment?
Besides the production of light, bioluminescent reactions using imidazolopy-
razines also consume oxygen. It has been suggested that primitive luciferases may
have evolved in order to utilize molecular oxygen directly as an electron acceptor
at the low oxygen tensions present in the primitive atmosphere 73,99. Initially, oxygen
would have been toxic to organisms which had evolved in an anaerobic environment.
Therefore, the presence of molecules with a high affinity for the neutralization of
oxygen would have become important. Further data have shown that imidazolopy-
razines also react with oxygen derivatives such as the superoxide anion (O2), and
singlet oxygen (102)67'84. These molecules are highly reactive and are deleterious
for many cellular functions. In seawater, significant concentrations of superoxide
anion and hydrogen peroxide are generated by photochemical processes 9~176
The highest levels of these molecules are found in the surface layers of the ocean
and decrease with depth 13~ In coastal waters at midday, the rate of superoxide
production 9~ can reach 5 x 10-7 mol 1-1 h -1 and its steady-state concentration is 2
x 10 -8 mol 1-1. Therefore, an effective defense against oxidative processes is essen-
tial for marine organisms. A byproduct of the proposed use of imidazolopyrazines
in these reactions would have been the emission of a weak chemiluminescence.
When Vargulaluciferin is placed in physiological media containing bovine serum,

there is a very weak catalysis of luminescence. This produces a very broad spectral
distribution with no distinct peak shape (Thompson, unpublished results). Thus, to
make effective adaptive use of the low light level produced in antioxidative pro-
cesses, would have involved the evolution of enzymes capable of: (1) improving the
quantum yield of the chemiluminescent reaction; and (2) reducing the interaction
of the excited state oxyluciferin with other molecules in counterproductive energy
transfer resulting in a degradation of spectral quality. The present day luciferases
are the fruit of this process and it will be of considerable interest to unravel the
various evolutionary origins of these enzymes. It has been suggested that biolu-
minescence may have as many as 30 independent origins during the course of
evolution 41.
We propose that antioxidative processes are the evolutionary roots of fish biolu-
minescence and that counterillumination was likely to be the first bioluminescent
function to arise from this origin. Selection based on predator avoidance would have
acted to increase the quantum yield of the chemiluminescent reactions and to orient
light emission in a ventral direction. Once established, further gains in luminescence
intensity, surpassing ambient light levels, and the development of finer controls on
the kinetics of bioluminescent emission, would have made communication possible.
The imidazolopyrazine luciferins are at the heart of this process, but are they syn-
thesized by the majority of marine bioluminescent fishes, or is the dietary transfer
we have discussed the predominant route accounting for the widespread use of
these molecules? If the latter explanation is correct, what are the ultimate sources
of these molecules in the marine environment?
As considered in section V, luminescent Vargula are most probably the source
of substrate for fishes exploiting Vargula lueiferin in their luminescent reactions.
However, the number of fish species using this system is small in comparison to
the number of marine fishes which are likely to make use of eoelenterazine. The
ultimate source(s) of coelenterazine are more difficult to pinpoint. However, the
organism(s) are likely to have a wide oceanic distribution, both coastal and open
water, and occupy a relatively low trophie level. It is not essential that the or-
ganism(s) be bioluminescent themselves. Copepods fit well with the above criteria
and bioluminescenee depletion studies seem to support this possibility. The abil-
ity of copepods (Pleuromamma, Gaussia) to recover biolumineseence ability after
their luminescence potential had been completely exhausted by overstimulation has
recently been investigated 65. Since both species excrete all components of their
luminous system into seawater 23 ,28 ,65 , it is noteworthy that unfed individuals of both
species fully recovered their luminescent potential within 24 hours. Similar experi-
ments carded out on the deeapod Gnathophausia revealed that this crustacean was
unable to synthesize coelenterazine in order to reestablish bioluminescence in the
absence of a dietary source of this molecule 31. In these experiments, one cannot be
certain that the depletion of copepod luminescence ability is due to the depletion of
luciferin, but these observations do suggest that luminescent eopepods synthesize 1u-
eiferin. The luciferin in these eopepods has been characterized as coelenterazine 16.
As luminescent copepods are consumed by a wide range of organisms, they could
Origins of luciferins:ecologyof bioluminescencein marinefishes 461
constitute the bulk of the luciferin sources for many luminescent fishes and inver-
tebrates. In opposition to this proposal is a report that the luminescence of the
copepod Metridia lucens was affected by its nutritional status 28. The intensity of
flashes decreased in starved specimens while remaining rather stable in individuals
which continued to feed. This observation does not rule out the possibility that M.
lucens is able to synthesize luciferin as the diminishing luminescence capability of
unfed specimens could also have resulted from overall physiological deterioration
of the animal. It is of interest that in this study, the control group was fed exclusively
on diatoms (Thalassiorira sp.). Thus, the possibility exists that diatoms could be
the source of luciferin, either directly or through the supply of some precursor, as
previously suggested 16.
VII. C o n c l u s i o n s
The relative scarcity of bioluminescence among terrestrial organisms has probably

contributed to a general view of such displays as aesthetically pleasing oddities.
However, relative to the terrestrial environment, the marine world presents a much
greater diversity of light regimes. For those fortunate enough to have studied
bioluminescence in the oceans, the pervasive importance of these phenomena in the
interaction of marine organisms with their environment is clear. Probably arising
initially as a response to environmental oxidative stress, the weak chemiluminescent
byproduct has been harnessed and developed into a variety of functions essential
for the survival and reproduction of a large number of marine fish species.
The more or less random phylogenetic distribution of bioluminescent organisms,
led to an initial view that bioluminescent systems had likely evolved completely in-
dependently on a number of occasions. The subsequent discovery that many of these
organisms were linked by the use of common luminescent substrates has stimulated
new and interesting avenues of research. Understanding of the trophic relationships
between bioluminescent fishes and source organisms containing luciferin, and the
characterization of the biochemical and physiological adaptations required for the
effective use of these bioactive exogenous molecules, have become important areas
of investigation. In this regard it is interesting to compare the imidazolopyrazine
luciferins with vitamins. As is the case with vitamins, at least some fishes must
acquire luciferins nutritionally 41. There are further similarities between luciferins
and vitamin C. Both imidazolopyrazines and ascorbic acid are susceptible to autox-
idation, stabilized forms of both types of molecule include enol-sulfate derivatives,
and both react with the superoxide anion 19,84. Although these similarities may be
coincidental, they might also indicate a more general strategy among organisms for
the use of certain exogenous compounds against undesired oxidative processes.
Thus, the study of bioluminescence in marine fishes presents challenging prob-
lems in a process which evolved as a response to environmental conditions, and
has, over evolutionary time, been exploited by fishes for the manipulation of their
surroundings. The knowledge we gain of these events will extend well beyond the
comprehension of the production of light.
Acknowledgements. This work was supported by the Fonds National de la

Recherche Scientifique (FNRS), Belgium, and the Institut National de la Recherche
Agronomique (INRA), France. Jean-Francois Rees is a Research Associate of the
FNRS.
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Species Index
Acanthopagms schlegeli, 138 Chimaera monstrosa, 24

Acanthustius brasilianus, 129 Chondrostoma nasus, 210
Acerina cernua, 104 Chrysophrys major, 76, 145
Acipenser galdenst~dti, 423 Clarias batrachus, 138
Acipenser sp., 230 Clarias lazera, 398
Acipenser transmontanus, 145 Clausocalanus sp., 449
Aequorea sp., 443, 445 Clupea harengus, 25, 128, 342
Alosa pseudoharengus, 403 Clythia sp., 443
Amia calva, 245 Cobitis biwae, 43
Ammodytes lancea, 128 Columbia livia, 6
Anguilla anguilla, 25, 36, 43ff., 50, 52, 55f., 103, Conger sp., 26
105, 107f., l lOf, 138, 163ff., 227, 395f., 401f, Conger vulgaris, 26
419, 425 Coregonus sp., 144
Anguilla japonica, 166f., 172, 315, 418 Coryphaenoides armatus, 355
Anguilla rostrata, 19f., 46, 50, 55f., 69, 139, 170, Crangon septemspinosa, 453
175, 181,404, 406, 414, 419 Ctenopharyngodon ideUa, 201, 206, 210
AnguUla sp., 19, 36, 102 Ctenophatyngodon sp., 144
Aphos porossus, 449f. Cyclothone braueri, 441
Apogon eUioti, 439, 441f. Cynoscion regalis, 281
Apogon sp., 444, 451, 453 Cypridina sp., 440
Apteronotus albifrons, 267 Cyprinodon macularius, 343
Apteronotus leptorhynchus, 267 Cyprinus carpio, 46f., 5Of., 55, 89, 123, 161, 169ff.,
Arachmia fucata, 442 174, 182, 230, 292, 343, 346, 352, 371, 374,
Arachmia lineolata, 442 394f., 398f., 401, 404f., 408f., 413, 415f.
Arapaima gigas, 76
Argyropelecus hemigymnus, 441, 454f, 459
Argyropelecus sp., 437, 439, 442, 448, 455f. Diaphus coeruleus, 441
Aristostomias, 445 Diaphus elucens, 441, 444
Diaphus suborbitalis, 441
Dicentrarchus labrax, 20, 24f., 126, 196, 210, 341,
Balaenoptera sp., 310 396, 398, 400, 402, 413, 416ff.
Benthosema fibulata, 441 Dicentrarchus sp., 21
Benthosoma glaciale, 448 Discopyge tschudii, 143
Benthosoma sp., 442
Boleophthalmus boddaerti, 179f.
Boops salpa, 222 Echiostoma barbetum, 441
Brevoortia tyrannus, 348 Eigenmannia, 265, 267
Brienomyrus brachyistius, 266 Electrophorus electricus, 261
Brienomyrus sp., 266, 268 Electrophorus sp., 259, 263, 265, 269, 271ff.
Busycon caniculatum, 299 Embiotaca lateralis, 89
Engraulis mordax, 129, 348
Eptatretus stouti, 68, 89
CaUionymus lyre,343 Eptesicus ~scus, 6
Camssius auratus,36, 46f.,50, 52, 121, 167f.,176, Escherichia coli, 224
179, 292, 403, 412f.,417, 422
Esox lucius, 129, 384, 400, 403f., 419
Carassius camssius, 46f.,52, 68, 372
Etmopterus spinax, 24
Carassius sp.,52
Centrophorus squamosus, 26 Euthynnus affinis, 248, 318
Chaenocephalus aceratus,343 Euthynnus pelamis, 129
Channa punctata, 74
Channa sp., 128 Fugu vermicularisporphyreus, 402
Chanos chanos, 230 Fundulus heteroclitus, 55, 104, 226f., 353, 403
468 Species Index
Gadus aegle]inus, 24f. Makaira nigricans, 314, 383

Gadus callarias, 321 Malacosteus sp., 445
Gadus morhua, 24f., 36, 46, 50, 73, 105, 107f., Malaptemrus sp., 261
122, 207, 210, 337, 398ff., 405, 407f., 413, Mallotus villosus, 128
417f. Maurolicus sp., 438
Gadus poUachius, 122 Melanogrammus aeglefinus, 128
Gadus sp., 103 Merlangus merlangus, 128
Gambusia affinis, 344 Meduccius capensis, 402
Gaussia sp., 460 Merluccius hubbsi, 129
Gazza minuta, 455 Metridia lucens, 449, 461
GiUichthys mirabilis, 163 Micropterus salmoides, 341, 352
Gnathonemus petersii, 266 Microstomuspaciftcus, 342, 354
Gnathonemus sp., 266 Misgurnus anguiUacaudatus, 138
Gnathophausia sp., 446, 449, 460 Miatichthys luzonensis, 343
Gobius minutus, 344 Monopterus albus, 89
Gobius sp., 103 Morone saxatilis, 230, 233
Gonostoma sp., 438, 448f. Mullus surmuletus, 24
Gymnarchus niloticus, 265 Mustelus canis, 129
Gymnarchus sp., 265 Myctophum asperum, 441
Gymnotus carapo, 264 Myctophum sp., 441f.
Gymnotus sp., 273 Mytilus edulis, 196
Myoxocephalus scorpius, 370
Myxine glutinosa, 26, 66, 396, 411
Hemitripterus americanus, 20, 66, 348, 357, 384,
412, 418, 420f. Neoscopelus microchir, 441
Hemitripterus sp., 20
Heteropneustesfossilis, 138
Hippoglossoidesplatessoides, 341, 347 Obelia sp., 443
Hippoglossus hippoglossus, 128 Oncorhynchus clarki, 46, 52, 341, 347
Hoplias malabaricus, 20, 343 Oncorhynchusgorbusha, 125f., 414, 423
Hoplias sp., 21 Oncorhynchus keta, 50, 125
Hypopomus brevirostris, 262 Oncorhynchus kisutch, 19f., 47, 128, 161,233, 382,
404, 41 lff., 418, 423
Hypopomus occidentalis, 266 Oncorhynchus masou, 128
Hypopomus pinnicaudatus, 272 Oncorhynchus mykiss, 18ft., 24ff., 36, 46f., 5Of.,
Hypopomus sp., 266, 268 55, 68, 121, 161, 172, 174, 178, 192, 195,
197, 210, 213, 226f., 230, 293, 293, 295f.,
Ictalurus melas, 418 314f., 319, 321, 323, 341, 348, 355f., 371,
lctalurus nebulosus, 46, 72, 403, 420 384, 396, 398, 400, 409, 413, 418, 423ff.
lctaluruspunctatus, 19, 122, 16Of., 168, 173f., 205, Oncorhynchus nerka, 22, 25f., 50, 55f., 170, 182,
382, 404 315, 371, 395, 399, 423, 425
Ictalurus sp., 230 Oncorhynchus sp., 170
Isurus oxyrhinchus, 243f. Oncorhynchus tshawytscha, 131, 315, 418, 420
Ophiocephalus sp., 341
Ophiophagus sp., 310
Katsuwonus pelamis, lf., 6, 18ft., 36, 38, 44, 69, Oplophorus sp., 443
248, 314, 318f., 374, 382, 284, 398 Opsanus beta, 69, 105, 107f., 116, 181, 283ff., 345,
349ff.
Labio rohita, 1, 129 Opsanus sp., 452
Lampadena sp., 441 Opsanus tau, 167, 280ff., 286, 402, 412, 419, 457
Lampanyctus sp., 448 Oreochromis alcalicus grahami, 181
Lampetra fluviatilis, 68, 89 Oreochromis [Sarotherodon] mossambicus, 53, 73,
Latimeria chalumnae, 26, 128 138, 163ff., 171, 207, 225, 227f., 230f., 341,
Latimeria sp., 26 425
Lepisosteus osseus, 129 Oreochromis niloticus, 139, 340, 342
Lepomis macrochin~s, 112, 131 Oreochromis sp., 50, 212
Lepomis sp., 46, 52 Osmerus mordax, 403
Leuciscus hakonennsis, 316
Leuciscus idus melanotus, 75 Pachystomias sp., 445
Limanda limanda, 212, 402 Pagrus major, 138
Species Index 469
Pandalus danae, 453 Salvelinus sp., 50

Paralabrax clathratus, 170, 355, 451 Sardinops caerulea, 26, 125
Paralabrax nebulifer, 407f. Sardinops sagax, 348
Paralabrax sp., 20ft., 347, 452 Sarotherodon, see Oreochromis
Paralichthys califomicus, 346f., 355 Scardinius erythrophthalmus, 139, 182
Paralichthys olivaceus, 138 Scomber japonicus, 327
Parapriacanthus beryciformes, 441f., 449 Scomber scombms, 24f., 128, 405
Pampriacanthus ransonneti, 439, 442f. Scophthalmus maximus, 131, 169
Parapriacanthus sp., 439, 443, 449, 451,453 Scyliorhinus canicula, 24, 26, 129, 396, 401, 403,
Parasilurus asotus, 138 411, 413
Perca flavescens, 396, 401f., 406 Scyliorhinus sp., 26
Perca fluviatilis, 47, 103f. Searsia sp., 441, 444f.
Perca sp., 36, 103 Sebastes schlegeli, 138
Periophthalmodon schlosseri, 179f. Sebastodes miniatus, 106, 108
Periophthalmus chrysospilos, 178ff. Selaphorus rufus, 6
Petromyzon marinus, 66, 87, 245 Sparus aurata, 415
Photostomias sp., 441 Sphymena barracuda, 107
Phoxinus phoxinus, 344 Squalus acanthias, 24, 122, 176, 182, 244f., 410ft.
Pimephales promelas, 131 Steatogenys elegans, 264
Platichthys flesus, 47, 52 Stenotomus chrysops, 106f.
Platichthys sp., 425 Sternoptyx diaphana, 456
Platichthys stellatus, 19, 69, 384 Stemoptyx pseudobscura, 456
Plecoglossus altivelis, 145, 315 Stemoptyx sp., 448, 456
Pleuromamma sp., 448, 460 Stemopygus macrums, 262
Pleuronectes platessa, 20, 56, 138, 177, 341, 401, Stemopygus sp., 265ff., 270ff.
407 Stizostedion vitreum, 176
Poecilia reticulata, 128 Stomatorhinus cometi, 256
PoUachius virens, 128 Syngnathus sp., 103
Pomoxis nigromaculatus, 403 Synodontis sp., 261
Porichthys myriaster, 450
Porichthys notatus, 280, 282, 284ff., 436, 438, 450ff., Thalassiorira sp., 461
454, 456ff. Themgra chalcogramma, 137
Porichthys sp., 436, 438, 441ff., 446, 449ff., 457 Tilapia, see Oreochromis
Pseudemys scripta, 298 Tinca tinca, 83, 124
Pseudopleuronectes americanus, 122, 173 Torpedo sp., 259, 262, 269ff.
Trachums sp., 46
Raja clavata, 403 Trachurus symmetricus, 54
Raja erinacea, 71, 170, 244f., 345 Trachurus trachurus, 343
Raja radiata, 24
Renilla sp., 442ff., 452ff. Valenciennellus sp., 448
Rhabdamia cypselum, 442 Vargula hilgendorJii, 449
Rhamdia hilarii, 73, 400, 402 Vargula sp., 436, 440ff., 449ff., 454, 456ff.
Rutilus rutilus, 52, 55f. Vargula tsujii, 450
Vinciguerria attenuata, 441ff.
Salmo clarki, see Oncorhynchus clarki Vinciguerria sp., 448
Salmo gairdneri, see Oncorhynchus mykiss
Salmo salar, 131, 176, 201, 210, 349 Watasenia scintiUans, 454
Salmo tmtta, 55, 73, 115, 127, 161, 410, 413, 415,
417
Salmo trutta fario, 395, 397f., 400, 406, 408, 416 Xenopus sp., 270
Salvelinus alpinus, 24, 125 Xiphias gladius, 250f.
Salvelinus fontinalis, 138, 173, 421, 425
Salvelinus namaycush, 174, 177, 181, 245 Yarella illustris, 441
Subject Index
A23187 cannulation, 37
eicosanoid production, 140 gas gland, 112
leukotrienes, 142 Acidification
platelet-activating factor synthesis, 143 gas gland, 111
protein kinase C, 143 vs. lactate release, 111
steroidogenesis, 143 Acidosis
Absorption ammoniogenesis, 176
glucose, 225 ATP/ADP ratio, 385
Absorptive cells mitochondrial respiration, 386
intestinal transport, 222 Aconitase
Accessory organs thermogenic tissue, 247
electric organ, 264 ACITI
Acetate effects on plasma fatty acids, 25
anoxia, 75 plasma fatty acids, 422
flux into fatty acids, 404 starvation, 422
flux into lipids, 404 F-Actin
Acetazolamide effects of enzyme binding on Kin, 301
gas gland metabolism, 114 enzyme association, 303
Acetoacetate enzyme binding, 292
utilization in muscle, 377 in electric organ~ 271f.
Acetoacetyl CoA acetyltransferase kinetic effects on glycolytic enzymes, 302
thermogenic tissue, 247 muscle, 408
N-Acetyl-L-histidine deacetylase phosphofructokinase binding, 299
anserinase, 322 protein synthesis, 192
N-Acetylation starvation, 408
histidine, 329 Actinopterygii
Acetyl-Coenzyme A carboxylase electric discharge, 260
covalent modification, 404 families of electric fish, 260
starvation, 404 Acyl-CoA: 1-acyl-sn-glycero-phosphorylcholine
Acetylcholine acyltransferase, 122
gas gland lactate formation, 105 Adenylate kinase
salt secretion, 143 calculation of free ADP
sonic motoneuron, 281 equilibrium, 386
Acetylcholine esterase Adenylate status
gas gland tissue, 112 muscle recovery, 380ff.
Acetylcholine receptor (AChR), 259ff. Adipose tissue, 47
antibodies, 269 acid lipase, 403
asymmetry in electric organ, 273 amino acids, 369
clustering, 270 catfish, 400
developmental dynamics, 270 fatty acids, 369
effects of electric organ denervation, 269 gadids, 399
localization in electrocyte, 271 glucose, 369
mRNA injection, 270 glycerophospholipid storage, 128
mRNA, 270 glycogen, 369
subunit composition, 269 lactate, 369
Acid lipase, 403 neutral lipase, 403
Acid protease protein, 369
starvation, 56 salmonids, 399
Acid secretion triacylglycerols, 369
hypoxia, 112 ADP
Acid-base calculation of free ADP from NMR, 43
472 Subject Index
ADP (continued) Alanine aminotransferase (GP'P, AAT), see also

muscle pool, 385 Aminotransferases
muscle recovery, 381 brain, 87
Adrenalin, see epinephrine effects of cortisol, 422
Adrenergic fibers gut, 166
gas gland, 112 heart, 84
Adrenoceptors kidney, 78
signal transduction, 77 liver, 70, 166, 396
Aerobic capacity, 346, 358 red blood cells, 162
effects of 17a-methyltestosterone, 287 red muscle, 82
endurance training, 83 starvation, 406
mass-specific, 284 white muscle, 81
red muscle, 250 D-Alanine
scaling, 346 stimulation of intestinal ion transport, 166
sonic muscle, 284 Albumin
tissue-specific partitioning, 352 elasmobranchs, 243, 403
Aerobic exercise, 377 starvation, 408
glucose utilization, 373 Albumin-like protein
Aerobic fibers phospholipid transport, 125
red muscle, 1 Aldolase
Aerobic metabolism, 335ff. association with F-actin, 303
capacity, 338 binding in turtle brain, 297
gas gland, 109 binding to subcellular muscle structures, 292
red blood cells, 90 effects of metabolites on enzyme binding,
respiration, 340 295, 300
scaling, 336ff. ~ kinetic effects of F-actin, 302
Aerobic scaling exponent, 359 loss during muscle preparation, 296
Aerobic scope red blood cells, 90
body size, 359 Algae
Aerobic swiming, 20 effects on glycerophospholipid composition,
Aglomerular kidney 133
taurine secretion, 174 1-O-alkyl- l'-enyl-2- acylglycerophospholipid
role in luciferin loss, 457 structure, 120
Air sac 1-O-alkyl-2-acylglycerophospholipid
cyclooxygenase, 138 structure, 120
prostanoid synthesis, 139 Alkylacylphosphofipids
Air-breathing fish olfactory nerve, 129
ammoniogenesis, 176 Alkyiglycerophosphate, 122
Alanine Allometry, 340
brush border uptake, 227 lactate turnover, 19
flux, 22 metabolic, 336ff.
flux to C6-products, 71 spleen, 340
gluconeogenesis, 410, 414 Allosteric activators
migration, 55 glycolytic flux, 292
QIO, 75 a-Amino-carboxylic acids
hepatic uptake, 410 brush border uptake, 227
hypoxia, 52 t~-Aminoisobutyrate
inhibitor of amino acid uptake, 227 intestinal transport, 163
muscle proteolysis, 170 a-Bungarotoxin
oxidation, 22, 371 localization of AChR, 271
fasting, 73 a-Globulins
kidney, 79 starvation, 408
QIO, 75 a-Glycerophosphate
red blood cells, 162 elasmobranch muscle mitochondrial substrate,
release from muscle, 182, 410 244
spawning migration, 22 oxidation in thermogenic tissue mitochon-
starvation, 409 dria, 251
stimulation of intestinal ion transport, 166 a-Glycerophosphate dehydrogenase
turnover, 22 vs. lactate dehydrogenase, 249
Subject Index 473
o~-Glycerophosphate dehydrogenase (continued) vs. protein synthesis, 165

brown adipose tissue, 249 Amino acid uptake
ot-Glycerophosphate shuttle, 247, 249 protein synthesis, 198
thermogenic tissue, 255 stimulation by growth hormone, 233
a-Ketoglutarate Amino acid utilization
mitochondrial transport, 245 deamination, 205
a-Ketoglutarate dehydrogenase oxidation, 205
control by calcium, 252 protein synthesis, 205
ot-(Methylamino)isobutyrate Amino acids
brush border uptake, 227 adipose tissue, 369
intestinal transport, 163 as endogenous fuel, 48
ct-Methyl-glucosides brush border uptake, 226f.
glucose transport, 223 carbon dioxide prodcution, 22
ot-Methyltestosterone concentration in plasma, 23
glucose permeability, 232 concentration in red blood cells, 23
leucine transport, 232 content of muscles, 50
Na+-glucose cotransport, 232 cortisol, 69
Na +/K+-ATPase, 232 extracellular fluid, 369
nutrient absorption, 232 gluconeogenesis, 22, 422
transporter induction, 232 gradient from liver to plasma, 167
Amino acid flux hepatectomy, 167
androgen action, 22 homeostasis, 199
measurement, 23 imbalance, 206
protein degradation, 196 infusion, 371
protein synthesis, 192 interorgan flux, 180f.
spawning migration, 22 intestinal transport, 222
Amino acid homeostasis ketogenesis, 244
role of intestine, 162f. kidney, 49, 173
Amino acid metabolism, 159ff. liver, 49, 166, 369
anoxia, 53 maintenance cost, 370
Km of enzymes, 205 mucus production, 22
muscle, 11, 177ff. muscle, 49
postprandial, 48 Na-amino acid coupling ratios, 228
regulation, 248 Na+-dependent uptake, 226
starvation, 177 Na+-independent transport, 228
D-Amino acid oxidase oxidation in elasmobranchs, 22
intestine, 166 oxidation, 21, 370
Amino acid oxidation, 22 plasma concentration at rest, 17
elasmobranch muscle mitochondria, 245 postprandial, 161, 199
flux, 22 red muscle, 369
intestine, 165 renal absorption, 173
nitrogen loss, 198 renal levels, 173
starvation, 409 starvation, 407
thermogenic tissue, 251 stereospecific uptake, 226
vs. oxygen consumption, 22 stimulation of protein synthesis, 199
Amino acid pool tissue pools in fasting, 160
homeostasis, 199, 205 transport, 226
postprandial, 204 utilization, 371
protein synthesis, 198 white muscle, 369
Amino acid recycling 2-Amino-2-norbonane carboxylic acid
prelabelled proteins, 196 brush border uptake, 227
protein synthesis, 193 Aminoacyl-tRNA synthetases
Amino acid transport Kms, 167, 205
basolateral membrane, 164 Aminooxyacetate
between red blood cells and plasma, 161 alanine transport, 166
brush border membrane vesicles, 163 aminotransferases, 168
induction, 235 ammonia production, 180
limits to hepatic protein synthesis, 167 glutamate oxidation, 245
Na+-dependence, 163 Aminotransferases, 171
474 Subject Index
Aminotransferases (continued) red muscle, 370

aminooxyacetate, 168 white muscle, 370
compartmentation, 168 Anaerobic glycolysis, 106
diet, 172 burst swimming, 314
kidney, 174 gas gland tissue, 106
starvation, 171 rete mirabile, 115
Ammonia Anaerobic metabolism, 359
amino acid oxidation, 205 burst swimming, 80
during kidney passage, 176 limitations, 358
excretion, 371 Anaerobic potential
hepatic portal vein, 166 muscle, 355
muscle, 380 Anaerobic scope, 357
nitrogen loss, 198 Anaesthesia, 35
protein synthesis, 196 erythrocyte swelling, 37
removal, 338 Androgens, 232
Ammonia excretion, 405 amino acid flux, 22
cydoheximide, 106, 205f. effects on nutrient transport, 233f.
effects of cortisol, 422 influence on electric organ discharge, 266ff.
exercise, 53 sonic muscle, 287
indirect calorimetry, 41 spawning migration, 22
postprandial increase, 205f. white muscle proteins, 22
postprandial, 214 Anoxia, 87
starvation, 167, 171 acetate, 75
Ammonia production amino acid metabolism, 53
aminooxyacetate, 180 brain ketone bodies, 88
bromofuorate, 180 enzyme binding, 298
from glutamine, 180 ethanol production, 75, 355
hepatocytes, 162 fatty add levels, 52
vs. glutamate oxidation, 162 fructose 2,6-bisphosphate, 74
vs. glutamine oxidation, 162 glucose production, 74
Ammonia quotient, 42 glycogen phosphorylase activity, 74
Ammoniogenesis lipogenesis, 52
acidosis, 176 liver glycogen, 75
air-breathing fish, 176 metabolic rate, 291
intestine, 166 oxygen debt, 75
liver mitochondria, 167 Pasteur effect, 74
muscle, 179 protein degradation, 53
AMP seasonality, 77
deaminase, 380, 386 tolerance in carp, 74
muscle recovery, 381, 385 Anoxic stress
muscle, 380 brain, 87
AMP deaminase, 386 Anserinase, 321f.
purine nucleotide cycle, 386 anserine degradation, 322
Amphibians brain, 322
distribution of histidine-related dipeptides, 312 muscle, 322
Amplitude Anserine
electric organ discharge, 263 biosynthesis, 321
Amylase degradation, 322f.
intestine, 223 from carnosine, 322
Anabolic steroids from injected histidine, 320
regulation of nutrient transport, 232, 235 growth, 318
Anabolic stimulation efficiency heart, 329
definition, 193 imidazole dipeptides, 309
protein synthesis, 198 imidazole pK value, 325
Anabolism incorporation into kidney, 324
scaling exponents, 212 incorporation into liver, 324
Anaerobic capacity interorgan transport, 323
correlation with muscle buffering, 327 red muscle, 313
Anaerobic energy production structure, 309
Subject Index 475
Anserine (continued) in glutamate metabolism, 246

white muscle, 313 kidney, 78
Anterior intestine liver, 70, 166, 396
amino acid absorption, 163 red blood cell, 162
peptide absorption, 163 red muscle, 82, 285
Anti-dystrophin antibody sonic muscle, 285f.
immunocytochemistry of electrocyte, 271 starvation, 406
Antioxidants toadfish heart, 285
gas gland, l15f. toadfish white muscle, 284ff.
origin of luminescence, 459 white muscle, 81, 284ff.
Aperture Aspartate-glutamate carrier
photophore, 438 in glutamate metabolism, 246
Apoproteins ATP
enzyme activation, 126 effects on glycolytic enzyme binding, 295
in luminous system, 443 hypoxic muscle, 51
lipid transport, 124 muscle recovery, 381
lipoproteins, 126 ATP buffering
receptor binding, 126 role of phosphocreatine, 380
Arachidonate ATP hydrolysis, 34
eicosanoid metabolism, 138f. heat production, 255
Arachidonic acid cascade, 138 during tissue extraction, 39
Arginase ATP production
kidney, 175 burst swimming, 291
liver, 175 ATP synthesis, 34
Arginine ATP utilization
brush border uptake, 227 red muscle, 370
intestinal transport, 163 white muscle, 370
osmoregulation, 316 ATP-citrate lyase
Arginine vasotocin starvation, 404
effects on plasma fatty acids, 25 ATP/ADP ratio
L-U-14C-Arginine glycogenesis, 82
protein synthesis, 195 in acidotic tissue, 385
Aromatase ATP synthesis, 386
electric organ discharge frequency, 267 ATPase
Ascorbate thermogenesis, 242
autoxidation, 453, 461 Atropine
Asparaginase gas deposition, 112
kidney, 176 Autoxidation
liver, 169 ascorbate, 453, 461
muscle, 178 imidazolopyrazines, 461
Asparagine synthetase luciferins, 461
kidney, 176
muscle, 178 Back diffusion
liver, 176 rete mirabile, 112
Aspartate swimbladder, 102
conversion to succinate, 53 Balenine
liver mitochondrial substrate, 167 degradation, 322
muscle, 160 HPLC, 311
purine nucleotide cycle, 386 imidazole dipeptides, 309
red blood cells, 161 imidazole pK value, 325
turnover, 22 red muscle, 313
Aspartate aminotransferase (GOT, DAT), see also structure, 309
Aminotransferases vertebrate muscle, 309
brain, 87 white muscle, 313
effects of cortisol, 422 Band Ill anion exchanger
elasmobranch muscle mitochondria, 245 red blood cell, 114
glutamate oxidation, 245 Basolateral glucose transport, 225, see glucose
gut, 166 transport
heart, 84, 285 Basolateral membrane
476 Subject Index
Basolateral membrane (continued) Birds

amino acid release, 162ff. distribution of histidine-related dipeptides, 312
transport processes, 222 muscle buffering capacity, 326
Na +/K+-ATpase, 222 Blood
Basolateral membrane vesicles acidification by gas gland, 111
amino acid transport, 164 circulation time, 339
Basolateral Na +/K+-ATpase, 163 glucose
Bat after hepatectomy, 68
flight muscle characteristics, 2 histidine-related dipeptides, 314
muscle morphometric data, 6 6-keto prostaglandin Fla, 139
Benzothiazole lipoxygenase products, 139
in click beetleluciferin, 444 nutrient transport from gut, 221
fl-Alanine velocity, 339
carnosine synthesis, 320 volume
incorporation into carnosine, 320 scaling, 339
intestinal transport, 226 Boatwhistle, 279
17fl-Estradiol Body mass
effect on electric organ discharge, 266f. lactate turnover, 19
,0-Globulins, 408 Body weight
starvation, 408 scaling with protein metabolism, 212
fl-Hydroxybutyrate Bohr effect
elasmobranch muscle mitochondrial substrate, gas gland, 111
244 Bolus (flooding dose) injection
elasmobranch muscle, 246 fractional rate of protein synthesis, 195
monocarboxylate carrier, 246 isolated cells, 196
oxidation in teleosts, 248 isolated organs, 196
oxidation in thermogenic tissue mitochon- metabolite turnover, 16f.
dria, 251 protein synthesis, 192
utilization in muscle, 377 Bouton
fl-Hydroxybutyrate dehydrogenase glycogen, 281
mitochondria, 246 mitochondria, 281
teleosts, 248 sonic muscle, 281
fl-Methyl-glucosides Boyle's law, 101
glucose transport, 223 Bradycardia
fl-Oxidation hypoxia, 50
fatty acids, 34 Brain
fl-Receptors amino acids, 53, 160
heart, 85 exercise, 53
BF-2 cell line amino acids in fasting, 160
cost of protein synthesis, 208f. anoxia, 88
fractional protein synthesis rate vs. oxygen anoxic stress, 87
consumption, 208 anserinase, 322
glycerophospholipid fatty acids, 131 carbon dioxide production, 89
Bicarbonate cyclooxygenase, 138
gas gland entry, 114 glucose uptake, 86
proton transfer, 114 glucose utilization, 66
source of carbon dioxide in gas gland, 108 glycerophospholipid composition, 128f.
Bile salts glycogen content, 46, 66f., 86
lipase, 123 glycolytic enzyme binding, 297
phospholipase A2 histidine-related dipeptides, 314
Billfishes 3-hydroxybutyrate dehydrogenase, 403
thermogenic tissue, 242 hypoglycemic stress, 87
Binding protein insulin hypoglycemia, 86
fatty acids, 86 ischemia, 87
luciferin, 452 ketone bodies, 88
Bioluminescence, 435ff. lactate dehydrogenase scaling, 355
energy transfer, 445 lipoxygenase products, 139
origins, 459 oxygen consumption, 89
spectral shifts, 445 prostanoid synthesis, 139
Subject Index 477
Brain (continued) glycolysis, 314

succinate dehydrogenase, 346 scaling, 358
Brain heater, see also thermogenic tissue, 243
ATPases, 253 Ca 2+ -ATPase
heat production, 252 elasmobranch muscle, 243
metabolism, 250ff sonic muscle, 282
Na +/K+-ATl'ase, 254 thermogenesis, 242, 252
Branched-chain or-ketoacid dehydrogenase Calcium
localization, 175 control of mitochondrial enzymes, 252
phosphorylation/dephosphorylation, 175 during tissue extraction, 39
Branched-chain amino acid aminotransferase eicosanoid production, 140
influence of diet, 177 enzyme binding, 299
tissue distribution, 174 in luminous systems, 443
Branched-chain amino acids inhibition of enzyme binding, 300
liver metabolism, 181 phospholipase A2, 138
muscle metabolism, 181 protein kinase C, 143
Branchial pump sonic muscle, 282
metabolic cost, 345 Calcium ionophore, see A23187
Bromofuorate Calorimetry, 41f.
ammonia production, 180 Camouflage
Brown adipose tissue, 249 bioluminescence, 455
heat production, 252 counterillumination, 437
ketone body oxidation, 251 photocytes, 437
proton channel, 241 cAMP
Brush border enzyme binding, 299
amino acid uptake, 226f. glucagon, 77
Na/glucose transporter, 223 isotocin, 77
transport phosphoinositide cycle, 142
amino acids, 165 seasonal response, 77
glucose, 165 vasotocin, 77
Brush border membrane Cannibalism
amino acid uptake, 162 source of luciferin, 450
glycerophospholipid headgroup composition, Cannulation
129 acid-base, 37
phospholipid changes with seawater adapta- blood gas analysis, 37
tion, 135 dorsal aorta, 37
vesicles, 227, 231 turnover studies, 41
amino acid transport, 163 Capacity for protein synthesis
dipeptide transport, 164 definition, 193
proline transport, 231 Capillaries
renal amino acid uptake, 173 degree of orientation, 7
intestinal amino acid uptake, 173 density, 3
Buffering gas gland rete mirabile, 115
histidine-related dipeptides, 324 geometry, 8
non-bicarbonate, 325 length density, 5
Buffering capacity length per fiber volume, 8
correlation with anaerobic capacity, 327 manifolds, 3f., 11
correlation with lactate dehydrogenase, 327 orientation, 3, 5
correlation with myoglobin content, 327 surface per fiber volume, 10
gas gland, 112 tortuisity & branching, 7
heart, 85 Capillarity
muscle homogenates, 328 vertebrate muscles, 6
white muscle, 356 Capillary density, 7
Burst exercise, 367, 375, 378 length density, 7f.
anaerobic metabolism, 80, 359 Capillary manifolds, 3ft.
ATP demand, 378 hummingbird flight muscle, 4
ATP production increase, 291 muscle, 11
creatine phosphate, 45 red muscle, 3f., 11
glycogen, 80 Capillary network
478 Subject Index
Capillary network (continued) as neurotransmitter, 329

gas gland, 103 biosynthesis, 320
photogenic tissue, 438 from histidine, 320, 322
Capillary to fiber ratio, 3 heart, 329
Capillary-fiber surface ratio, 11 imidazole dipeptides, 309
Carbohydrate homeostasis, 66 imidazole pK value, 325
Carbohydrate metabolism, 65ff. N-methylation, 321
red blood cells, 89f. osmoregulation, 315f.
thermogenic tissue, 251 red muscle, 313
Carbohydrates starvation, 317f.
as endogenous fuel, 45ff. structure, 309
Carbon dioxide synthetase-like enzyme, 321
from bicarbonate, 108 white muscle, 313
gas gland metabolism, 109 Carnosine N-methyltransferase
production carnosine biosynthesis, 320f.
indirect calorimetry, 41 Carrier-mediated transport, 222f.
swimbladder, 106 Carriers
amino acids, 22 basolateral membrane, 223
brain, 89 brush-border, 223
removal, 338 Catabolism
Carbonic anhydrase scaling exponents, 212
gas gland, 109 Catalase
gas gland metabolism, 113ff. gas gland, 115
red blood cell, 114 Catecholaminergic fibers
rete mirabile, 113 photogenic tissue innervation, 438
Carboxypeptidase A Catecholamines, 68
starvation, 407 anoxia, 52
Cardiac myoglobin effects on plasma fatty acids, 25
scaling, 342 gas gland lactate formation, 105
Cardiac output, 340 gluconeogenesis, 68
contraction frequency, 344 glucose turnover, 21
stroke volume, 344 glycogenolysis, 68
vs. lactate flux, 18 lipolysis, 52
Cardiac pump red blood cells, 91
metabolic cost, 345 role in thermogenic tissues, 255
Cardiolipin, 120f. signal transduction, 77
brush border membranes, 129 swimbladder perfusion, 112
gill, 129 Catfish
gill mitochondria, 129 Na+/glucose transporter, 224
liver, 129 Cathepsin
muscle mitochondria, 129 spawning migration, 22
Cardiomyocytes starvation, 56, 396, 405, 407
cyclooxygenase, 138 Catheterization,
6-keto prostaglandin Fla, 139 flux measurements, 17
prostaglandin synthesis, 140 cDNA
prostanoid synthesis, 139 Na+/glucose transporter, 223
thromboxane synthesis, 140 CDP-choline-l,2diacylglycerol choline phospho-
Cardiovascular system transferase
prostanoids, 142 brain microsomes, 121
Carnitine acyl transferase liver microsomes, 121
elasmobranchs, 243 temperature acclimation, 135
Carnitine palmitoyl transferase, 377 CDP-ethanolamine phosphotransferase
thermogenic tissue, 251 hepatocytes, 121
Carnivores, 211 temperature effects, 135
glucose uptake, 230 Cell lines
intestinal nutrient transport, 230 glycerophospholipid fatty acids, 131
Carnosinase, 322 Cell proliferation
muscle, 321 hyperoxia, 115
Carnosine, 318 Cephalopods
Subject Index 479
Cephalopods (continued) content of copepods, 456

luminescent system, 446 crustaceans, 446
cOMP decarboxylation, 444
enzyme binding, 299 diffusibility, 457
Chemiluminescence distribution in fishes, 441
bioluminescent systems, 444 enol-sulfate, 452
Chicken glucuronidation, 442
cost of protein synthesis, 209 liver, 442
Chloride cells luciferin regeneration, 442
phosphoinositide cycle, 142 luciferyl-sulfate, 442
Chloride dependence mechanism of oxidation, 444
taurine secretion, 174 peroxide formation, 444
transport, 222 pyloric caeca, 442
Cholesterol implants recycling mechanism, 454, 456f.
control for experiments with steroids, 233 stable derivatives, 452
Choline storage, 452
diet, 145 structure, 440
in glycerophospholipids, 120 Collagen
Cholinergic fibers percent of whole animal protein, 213
gas gland, 112 Communication
CHSE-214 bioluminescence, 436
glycerophospholipid fatty acids, 131 photophores, 436
Chylomicron-like particles short-range, 437
lipid transport, 124 Compartmentation
Chylomicrons aminotransferases, 168
composition, 25, 124 glutamate metabolism - schematic, 245
hydrolysis, 126 Consumption-growth relationship
intestinal production, 125 growth rates, 202
Circulation Continuous infusion
convective circulation, 338 metabolite turnover, 16f.
lactate exchange, 19 protein synthesis, 192
metabolic substrates, 15 Contractile protein
oxygen delivery, 338 percent of whole animal protein, 213
Citrate, 113 Contraction cycle
Citrate synthase parvalbumin content, 283
brain, 87 Contraction frequency, 344f.
gas gland tissue, 105 negative allometry, 345
heart, 84, 285 Convective transport, 340
liver, 70 amino acids, 161f.
mass specific activity, 285 oxygen, 339
muscle, 81f., 250, 285ff., 35411". Copepods
red blood cells, 90 bioluminescence, 448
red muscle, 82, 250, 355 coelenterazine content, 456
scaling in white muscle, 354 fish diet, 448
sonic muscle, 284ff. luciferin synthesis, 460
thermogenic tissue, 247, 251 Copper transport
toadfish heart, 285 histidine-related dipeptides, 324
toadfish white muscle, 284ff. Cori cycle, 69, 71
white muscle, 81, 284ff., 354f. Cortisol
Click beetle activation of liver enzymes, 69
luciferin, 444 amino acid availability, 69
recycling of luciferin, 454 carbohydrate metabolism, 76
Coelacanth chronic effects, 421
brain glycerophospholipid composition, 128 effect on liver enzymes, 69, 421
plasma lipoproteins, 25 effects on other hormones, 421
Coelenteramine effects on plasma fatty acids, 25
recycling of coelenterazine, 456 gluconeogenesis, 69, 421
Coelenterazine, 436, 460 glucose turnover, 21
cephalopods, 446 glucose uptake, 421
480 Subject Index
Cortisol (continued) brush border uptake, 227

handling stress, 37 Cytochalasin B
lipolysis, 52 glucose transport, 225
liver binding, 423 C~ochrome c oxidase
liver enzymes, 69, 76, 421 aerobic capacity, 352
parr-smolt transformation, 233 carp muscle, 352
proline transport, 233 carp viscera, 352
protein catabolism, 202, 421 copper, 329
protein synthesis, 202 gas gland tissue, 105, 115
seawater adaptation, 233 raven heart, 357
spawning migration, 423 Cytoplasmic actin, 271
starvation, 421
stress, 202, 232 D5 desaturase, 134
during perfusion experiments, 232 Deiodinase
Cost of protein synthesis, 208ff. diet, 426
Counter-current heat exchange 2-Deoxyglucose, 379
red muscle, 1 glucose transport, 223
Counter-current system glucose uptake, 21
gas gland, 110, 112 transport by white muscle, 382
single concentrating effect, 110 uptake in red muscle, 373
swimbladder, 102 Dephosphorylation
Counterillumination, 455 activation of pyruvate dehydrogenase, 380
photophores, 437 Depolarization
Creatine electric organ, 263
muscle recovery, 381 Desaturase, 134f.
phosphorylation, 43, 381 temperature acclimation, 135
Creatine phosphate Desaturation
exhausting exercise, 54 fatty acids, 134
hypoxic muscle, 51 Desmin
muscle, 45 electrocyte, 271
Creatine phosphokinase, 34 Detergent solubilization
binding to subcellular muscle structures, 292 bound vs. free enzymes, 294
calculation of free ADP, 386 Development
mass action ratio, 379 AChR, 270
Creatinine Diacylglycerol
HPLC, 311 phosphoinositide cycle, 142
Critical speed, see Ucri, protein kinase C, 143
Crocodiles structure, 120
distribution of histidine-related dipeptides, 312 Diacylglycerol lipase
Cross-over plot, 287 eicosanoid metabolism, 138
Crustaceans Diacylglycerophospholipid
luminescent system, 446 structure, 120
Current Diet
electric organ discharge, 263 choline, 145
Cyanide effects on glycerophospholipid composition,
gas gland cytochrome oxidase, 115 133
inhibition of luminescence, 442 glycerophospholipids, 145
a-Cyano-3-hydroxycinnamate inositol, 145
pyruvate transport, 90 intestinal transport, 230
Cycloheximide luciferin, 436
ammonia excretion, 205f. natural vs. artificial, 76
protein synthesis, 205 renal aminotransferase, 177
Cycloleucine renal glutamate dehydrogenase, 177
intestinal transport, 226 Dietary lipid
Cyclooxygenase protein sparing, 206
eicosanoid metabolism, 137f. Diffusion
dihomo-y-linolenic acid, 141 amino acid uptake, 164
tissue distribution, 138 coefficient, 338
Cysteine luciferin, 458
Subject Index 481
Diffusion distance, 338 phosphatidylinositol fatty acids, 131

intrafiber, 9 Eicosanoids, 24; see also individual group
oxygen flux, 9 distribution, 138
Digestion fatty acid precursors, 139f.
heat production, 242 function, 142
Digestive enzymes metabolism 137ff.
nitrogen loss, 198 production, 139
Digestive tract sources, 141
luciferase, 442 thrombocyte aggregation, 142
Dihomo-F-linolenic acid tissue distribution, 138
cyclooxygenase, 141 Elasmobranchs
Dihydrotestosterone albumin, 243, 403
effect on electric organ discharge, 266f. amino acid oxidation, 22
Dihydroxyacetone phosphate pathway carnitine acyl transferase, 243
ether-linked glycerophospholipids, 122 distribution of histidine-related dipeptides, 312
Dilution method electric discharge, 260
enzyme binding to subcellular muscle struc- erythrocyte plasmalogens, 129
tures, 293f. ether-linked glycerophospholipids, 122
Dioxygenase fasting, 397, 411
eicosanoid metabolism, 137f. fatty acid carrier protein, 243
Dipeptide transport fatty acid oxidation, 243, 377
brush-border membrane vesicles, 164 glutaminase, 244
intestine, 163f. glutamine oxidation, 22
Dipeptides, see also histidine-related dipeptides glycemia, 397, 412
brush border uptake, 227f. ketone bodies, 243, 377, 402f., 411
Diurnal variation kidney, 79
protein synthesis, 196 glutaminase, 176
DNA glutamine synthetase, 176
liver content with starvation, 395 leukocyte prostacyclin, 139
muscle content with starvation, 395 lipid oxidation, 22
starvation, 406 location of electric organs, 260
Dominance hierarchy mitochondrial substrates, 244
growth, 201 muscle
protein synthesis, 201 ATPases, 243
Dormancy carnitine acyl transferase, 243
low temperature, 72 lipid content, 401
Dorsal aorta cannulation, 37 mitochondria substrates, 244
Down-regulation phosphoinositide cycle, 142
metabolic, 401 plasma fatty acids, 24, 243
glucagon receptors, 420 plasma glucose, 247
Drag, 344, 356 plasma glutamine, 244
Duchenne muscular dystrophy red muscle mitochondria
dystrophin, 270 hexokinase, 247
Duration respiratory control ratio, 247
electric organ discharge, 263 role of insulin, 411
Dystrophin Elastin
in electric organ, 270 percent of whole animal protein, 213
localization in electrocyte, 271 Electric eel
Dystrophin-related protein electric organ, 261
in electric organ, 270 Electric fish
distribution, 260
Edema location of electrocytes, 260
hyperoxia, 115 Electric organ (EO), 259ff.
Eggs discharge, 260
glycerophospholipid composition, 128 evolution, 261
luciferin, 458 F-actin, 271
reabsorption, 458 from oculomotor muscle, 261
transfer of luciferin, 450 GTP binding protein, 273
Eicosanoid metabolism, 137ff. innervation, 269
482 Subject Index
Electric organ (EO) (continued) proteins, 48

keratin, 272 Endoplasmic reticulum
microtubules, 273 gas gland cells, 103
myogenic, 260, 265 Endothermic tissue
nicotinic cholinergic neurons, 262 fishes, 242
pacemaker nucleus, 262 heat production, 242
Electric organ discharge (EOD) Endothermy, 242
amplitude, 263 Endurance swim, 375
diphasic, 264 Endurance training
duration, 263 effects on enzymes, 83
effects of steroid hormones, 266f. aerobic capacity, 83
frequency, 265 Energy minimization
in fish families, 260 protein synthesis, 214
in pulse-type fish, 262. Energy transfer
in wave-type fish, 262 photoproteins, 445
monophasic, 263 Energy use, 337
pulse-type, 262f. Enterocytes
sex differences, 262f. basolateral membrane, 229
triphasic, 264 transport, 229
waveform, 262 turnover, 162
Electric ray Enzyme association
inositol lipid metabolism, 143 synaptosomes, 296
phospholipase C, 142 Enzyme binding
Electrocytes, 263 artifacts, 298
anatomy, 264 metabolic triggers, 298f.
desmin, 271 Epinephrine, see also catecholamines
EPSP, 264 effects of glucocorticoids, 421
expression of my5, 270 effects on luminescence, 454
expression of MyoD, 270 gas gland lactate formation, 112
expression of myosin, 270 gill inositol lipid turnover, 142
expression of tropomyosin, 270 glycogenolysis, 73, 420
F-actin, 271f. handling stress, 37
inositol phospholipid metabolism, 143 Epithelial cells
keratin-like protein, 272 photophore secretions, 438
Li-sensitive phosphatase, 143 Epoxides
location in fish, 260 eicosanoids, 139
myofibrils, 271 EPSP
myosin, 270, 272 electrocytes, 264
Na + current, 268 Erythrocytes, see also Red blood cells
phospholipase C, 143 concentration, 340
plasmalemma, 272 release from spleen, 341
sarcomere, 261 storage, 338
T-tubule system, 261 Escherichia coli
voltage-clamp, 268 Na +/proline transporter, 224
Z bands, 272 Essential amino acids
Electrolocation, 261 fasting, 409
Electromotor neuron, 265 oxidation, 22
location in electric fish, 260 plasma, 161
innervation of electric organ, 262 postprandial, 161, 204
Electroreception, 259 Estradiol
Elongation leucine transport, 232
fatty acids, 134 regulation of nutrient transport, 232
Emission spectrum sonic muscle, 287
photophores, 437 Estrogen, 232, 287
Endogenous fuels effect on electric organ discharge, 266tf.
amino acids, 48 sonic muscle, 287
carbohydrates, 45ff. vitellogenin, 127
high-energy phosphates, 45 VLDL, 127
lipids, 47 Ethanol
Subject Index 483
Ethanol (continued) Exocytosis

anoxia, 75, 354 electric organ, 273
cyprinids, 354 Golgi apparatus, 273
hypoxia, 52, 354 Extracellular fluid
Ethanolamine amino acids, 369
in glycerophospholipids, 120 fatty acids, 369
Ether-linked glycerophospholipids, 122 glucose, 369
dihydroxyacetone phosphate pathway, 122 glycogen, 369
elasmobranchs, 122 lactate, 369
Etioluciferin, 454 protein, 369
recycling of coelenterazine, 456 triacylglycerols, 369
Euphausids Extraocular muscle
fish diet, 448 thermogenic tissue, 250
Everted gut sac Eye
nutrient transport, 232 anserinase, 322
Evolution phospholipid changes with seawater adapta-
electric organs, 261 tion, 135
luminescence, 459ff. heater, 243
Na +/glucose transporter, 224
Excretion F-Actin, see actin
ammonia, 41, 53, 106, 167, 171, 205ff., 214, Facilitated glucose transport, 225
371, 405, 422 Faecal nitrogen loss
Exercise, 374 daily balance, 197
aerobic, 377 Fasting, see also starvation, 72ff., 393ff.
ammonia excretion, 53 alanine oxidation, 73
carbohydrate utilization, 371 amino acid entry, 229
catecholamines, 91 amino acid turnover, 22
dephosphorylation of pyruvate dehydrogenase, DNA, 406
380 fatty acid composition, 401
enzyme binding, 298 fatty acid depletion, 402
fatty acids, 53 fatty acid unsaturation, 402
free fatty acids, 376 free amino acids, 160
fuel selection, 368, 371 fructose 1,6-bisphosphatase, 73
glucose turnover, 21 glucagon-insulin ratio, 72
glycogen repletion, 82 glucose turnover, 21
glycolytic enzyme binding, 292, 303 glycogen phosphorylase, 72, 74
glycolytic flux, 303 growth hormone levels, 423
lactate load, 81f. hormone titers, 72
lactate removal, 82 hypermetabolism, 73
lactate turnover, 19 hypoglycemia, 73
lipid oxidation, 53 hypometabolism, 72f, 401.
metabolic acid load, 81f. XCF,424
metabolic control, 368 ketone bodies, 402f.
metabolism, 369 lactate turnover, 19
metabolite levels, 368 lipid mobilization, 56
phosphagens, 385 lipogenesis, 404
plasma lactate, 18 lipolysis, 403
protein fuels, 53 liver composition, 395
purine nucleotide cycle, 179 liver glycogen, 72, 394
Exhausting exercise, 378 muscle composition, 395
creatine phosphate, 54 muscle lipid, 401
dipeptides, 318 muscle protein synthesis, 56
glycogen, 54, 380 PEPCK, 73
glycogenolysis, 54 plasma fatty acids, 402
histidine, 318 triacylglycerols, 401
lactate, 54 Fat
lactate release, 55 tuna red muscle, 248
phosphocreatine, 380 sonic muscle, 283
purine nucleotide cycle, 179 Fat fuels, 377
484 Subject Index
Fat metabolism luminescent species, 436

recovery, 384 Fin
Fat oxidation, 378 alanine biosynthesis, 183
Fat synthesis cyclooxygenase, 138
from amino acids, 169 Firefly
from glutamate, 170 recycling of luciferin, 454
Fatigue, 370 Flight muscle
Fatigue resistance bat, 2
sonic fibers, 283 hummingbird, 2
Fatty acid desaturase, 134f. pigeon, 3
Fatty acid binding protein Flooding dose technique, see Bolus injection
transport, 23 Flux regulation, 18
elasmobranchs, 243 Food consumption
Fatty acid metabolism effects of growth hormone, 233
temperature effects, 134 Food protein
Fatty acid oxidation sonic muscle, 282
anoxia, 52 Foraging behaviour
elasmobranch muscle, 243, 377 metabolism, 359
heart, 84 Foregut
pyruvate utilization swimbladder, 102
recovery, 384 Fork length
red muscle, 80 vs. intestine, 230
teleost muscle, 378 Fractional rate of protein synthesis
white muscle, 384 methodology, 195
Fatty acid synthesis Free amino acid pool
starvation, 404 daily balance - feeding, 197
Fatty acids, see also PUFA daily balance - starving, 198
adipose tissue, 369 Free amino acids, 159ff.
as anaerobic end products, 52f. a-keto acids, 160
/I-oxidation, 34 intracellular pool, 159
binding proteins, 86 metabolic connections, 160
composition in starvation, 401 extracellular pool, 159
composition of glycerophospholipids, 132ff. Free fatty acids, see also Fatty acids
depletion in starvation, 402 exercise, 376
desaturation, 134 from viscera, 377
elongation, 134 muscle, 376
extracellular fluid, 369 red muscle, 48
flux, 23 utilization, 376
from acetate, 404 white muscle, 369
from amino acids, 170 Free radical scavenger
from glucose, 170 histidine-related dipeptides, 324
hormone effects on plama fatty acids, 25 Freeze clamping
liver, 369 tissue sampling, 38
membrane fluidity, 402 Frog
oxidation, 21 distribution of histidine-related dipeptides, 312
oxidative substrates, 243 Fructose
plasma concentration, 17, 24 intestinal transport, 223
red muscle, 369 Fructose 1,6-bisphosphatase
synthesis from acetate, 404 binding of aldolase, 300
unsaturation in starvation, 402 binding of glyceraldehyde 3-phosphate dehy-
white muscle, 369 drogenase, 300
Feeding brain, 87
amino acid pool size, 204 competition with F-actin, 301
essential amino acids, 204 effects of cortisol, 421
FHM cell line fasting, 73
glycerophospholipid fatty acids, 131 heart, 84
Fiber length, 337 iodoacetate, 299
Fiber size, 6 kidney, 78
Filters liver, 70
Subject Index 485
Fructose 1,6-bisphosphatase (continued) mitochondria, 103

red blood cells, 90 morphology schematic, 104
red muscle, 82 perfusion, 105
white muscle, 81 proton transfer schematic, 114
Fructose 2,6-bisphosphate fete mirabile, 110
anoxia, 74 ribosomes, 103
Fructose 6-phosphatase vagai control, 112
starvation, 414 Gas gland tissue
Fructose 6-phosphate enzyme activities, 105
iodoacetate, 299 glycogen, 106
Fuels glycolytic enzymes, 107
exercise, 368 mass-specific oxygen uptake, 105
mobilization, 49f. oxygen uptake, 105
selection, 371 respiratory chain, 105
Fumarase Gas partial pressure
gas gland tissue, 105 increase in gas gland, 110
thermogenic tissue, 247 Gastrocnemius
Furan fatty acids histidine turnover, 319
neutral lipids, 130 Genetic adaptations
nutrient transport, 230
G protein, 273 Gill
phospholipase A2, 138 amino acids in fasting, 160
Galactose branched-chain amino acid aminotransferase,
absorption, 223 174
intestinal transport, 223 cardiolipin, 129
y-Globulins cyclooxygenase, 138
starvation, 408 glycerophospholipid headgroup composition,
Gas 129
diffusional loss from gasbladder, 102 histidine-related dipeptides, 314
partial pressure, 102 inositol lipid turnover, 142
resorption in swimbladder, 102 6-keto prostaglandin Fla, 139
Gas deposition lipoxins, 139
acidification, 111 lipoxygenase products, 139
effects of sulfonamide, 113 loss of luciferin, 457
gas gland, ll0f. Na +/K +-ATpase
Gas gland pressure adapation, 136
acid production, 106, 112 membrane lipids, 136
anaerobic glycolysis, 106 phospholipid changes with seawater adapta-
antioxidants, 115f. tion, 135
bicarbonate entry, 114 platelet-activating factor synthesis, 143
buffering capacity, 112 prostanoid synthesis, 139
capillary network, 103 scaling, 343f.
carbon dioxide production, 106 surface area, 343ff.
cells, 102 symmorphosis, 343
contamination with other cells, 104 Gill mitochondria
counter-current system, 110 cardiolipin, 129
endoplasmic reticulum, 103 Gill perfusion
glucose utilization, 106 hypoxia, 50
glycolytic activity, 112 Glomerulus
Golgi apparatus, 103 role in luciferin loss, 457
hormonal control, 112 GLP, see Glucagon-like peptide, 68, 415ff.
hypoxia, 112 Glucagon, 415ff.
lactate dehydrogenase, 106 absence from hagfish gut, 411
lactate formation, 106, 115 binding sites, 420
lactate levels, 110 effects of glucocorticoids, 421
lipids, 104 effects on plasma fatty acids, 25
metabolic control, 112 gas gland lactate formation, 112
metabolism schematic, 109 gluconeogenesis, 68
microvilli, 103 glucose turnover, 21
486 Subject Index
Glucagon (continued) muscle uptake rates, 379

glycogenolysis, 68, 73, 418 oxidation, 20f., 162
lipolysis, 419 heart, 84
liver glycogen, 418 limits in brain, 89
plasma fatty acids, 419 red blood cells, 162
re-feeding, 74 permeability
receptors, 72f., 419f. effects of thyroid hormones, 234
down-regulation, 420 methyltestosterone effects, 232
lipid reserves, 419 red blood cells, 89
signal transduction, 77 plasma concentration at rest, 17
starvation, 412 plasma pool, 379
values for fish plasma, 417 plasma turnover, 373f.
Glucagon-like peptide (GLP) production, 20
gluconeogenesis, 68 anoxia, 74
glycogenolysis, 68 hypoxia, 74
levels in fish plasma, 418 liver, 21
plasma fatty acids, 419 red blood cell substrate, 162
re-feeding, 74 release
seasonal response, 77 brain, 88
starvation, 412, 418 insulin, 88
Glucagon/insulin ratio liver, 373
starvation, 416 stimulation of gas gland oxygen uptake, 105
Glucocorticoids, see also Cortisol storage, 338
effects on transport, 233 substrate for heart, 84
gluconeogenesis, 421 transport, 223
glucose uptake, 421 a-methyl-glucosides, 223
protein catabolism, 421 ~-methyl-glucosides, 223
starvation, 421 2-deoxyglucose, 223
Glucokinase, 66 liver, 66
Gluconeogenesis, 65ff., 397ff. methyltestosterone effects, 232
amino acids, 22, 170, 178, 406, 410 Na +/glucose transporter, 223
catecholamines, 68 red blood cells, 66, 89
cortisol, 69, 76, 422 transporter isoforms, 225
GLP, 68, 418 Vmax effects, 232
glucagon, 68, 418 uptake
glucocorticoids, 421 brain, 86
glycerol, 249 carnivores, 230
hagfish, 411 glucocorticoids, 421
insulin, 69 herbivores, 230
kidney, 176, 396 kinetics, 66
liver, 371, 373, 375ff., 396 stimulation, 231
non-carbohydrate precursors, 396 Vmax, 230
postprandial, 48 utilization
rate in fish liver, 382 brain, 66
spawning migration, 55 hagfish heart, 66
starvation, 396ff., 406, 410 red muscle, 370
thermal compensation, 75 white muscle, 370
vasoactive peptides, 68 vs. fatty acid oxidation, 21
vertebrate liver, 381 Glucose 6-phosphate
Gluconeogenic flux fasting, 396
liver, 373 flux into glycogen, 396
Glucose gas gland metabolism, 109
absorption, 223, 225 Glucose 6-phosphate dehydrogenase
methyltestosterone effects, 232 effects of diet, 172
effects of thyroid hormones, 234 gas gland tissue, 108
aerobic exercise, 373 kidney, 78
intestinal uptake, 225 sonic muscle, 285
intestine, 223 starvation, 404
loading, 21 toadfish heart, 285
Subject Index 487
Glucose 6-phosphate dehydrogenase (continued) Glutaminase

toadfish white muscle, 285 elasmobranch kidney, 176
Glucose disappearance, 20 elasmobranch red muscle, 244f.
Glucose intolerance, 65 glutamate metabolism, 246
Glucose metabolism hagfish red muscle, 245
gas gland tissue, 108 liver, 169
Glucose paradox, 71 muscle, 178
Glucose sparing red muscle mitochondria, 245
elasmobranchs, 378 teleost red muscle, 245
Glucose transporter thermogenic tissue, 247
hagfish brain, 89 Glutamine
intestine, 163 elasmobranch muscle mitochondrial substrate,
Glucose turnover, 372, 398 244
cannulation, 41 elasmobranch plasma, 244
glycogen synthesis, 81 intestinal substrate, 166
in post-absorptive teleosts, 20 liver mitochondrial substrate, 167
tuna, 248 oxidation
Glucose-clamp technique, 21 elasmobranch mitochondria, 22
Glucuronidation muscle, 248
coelenterazine, 442 red blood cells, 162
liver, 453 thermogenic tissue mitochondria, 251
GLUT-l, 89 red blood cell substrate, 162
Glutamate starvation, 409
elasmobranch muscle mitochondrial substrate, stimulation of intestinal ion transport, 166
244 subcellular compartmentation, 245
flux into lipid, 170 Olutamine carrier
hepatic oxidation, 170 glutamate metabolism, 246
liver mitochondrial substrate, 167 thermogenic tissue, 247
liver, 49 Glutamine synthetase
mammalian kidney, 80 elasmobranch kidney, 176
mitochondrial transport, 245 elasmobranch red muscle, 245
oxidation, 22, 251, 371 hagfish red muscle, 245
thermogenic tissue mitochondria, 251 kidney, 176
red blood cells, 161 liver, 176
starvation, 409 muscle, 176, 178, 245
stimulation of intestinal ion transport, 166 teleost red muscle, 245
subcellular compartmentation, 245 Olutathione peroxidase
turnover, 22 gas gland, 115
Glutamate dehydrogenase, 171 Glutathione reductase
adaptive changes, 171 gas gland, 115
allosteric regulation, 168 Glycemia, 398
brain, 87 elasmobranch starvation, 412
effects of diet, 172 starvation, 396, 407
glutamate metabolism, 246 Glyceraldehyde 3-phosphate
glutamate oxidation, 245 effects on luminescence, 453
gut, 166 gas gland metabolism, 109f.
heart, 84 Glyceraldehyde 3-phosphate dehydrogenase
kidney, 78, 174 association with F-actin, 303
liver, 70, 166 binding in turtle brain, 297
red blood cell, 162 binding to subcellular muscle structures, 292
red muscle, 82 effects of metabolites on enzyme binding, 300
starvation, 171 effects of methodology on enzyme binding,
white muscle, 81 295
Glutamate oxidation gas gland tissue, 107
aminooxyacetate inhibition, 245 glycolytic control, 81
hepatocytes, 162 kinetic effects of F-actin, 302
vs. ammonia production, 162 release from muscle particulate matter, 294
Glutamate-aspartate carrier Glycerol
in glutamate metabolism, 246 gluconeogenesis, 249, 421
488 Subject Index
Glycerol (continued) mobilization, 373

in glycerophospholipids, 120 hypoxia, 51
re-esterification, 249 muscle, 46, 248, 251, 283, 369, 395f.
structure, 120 reappearance following exercise, 381
Glycerol kinase recovery, 74, 369
brown adipose tissue, 249 muscle, 359
effects of cortisol, 421 red muscle, 248, 283, 369
thermogenic tissue, 252 repletion, 383
Glycerol-3-phosphate acyltransferase sonic muscle, 283
liver, 121 synthesis from lactate, 382
Glycerolphosphate dehydrogenase thermogenic tissue, 251
gas gland tissue, 107 tuna red muscle, 248
Glycerophospholipid metabolism white muscle, 369
temperature effects, 134 Glycogen metabolism
Glycerophospholipids, 120ft. heart, 83
absorption, 123 Glycogen phosphorylase
basic structures, 120 allosteric regulation, 291
biosynthesis, 121, 123 anoxia, 74
deacylation/reacylation, 122 exhausting exercise, 54
dietary effects, 133 fasting, 72, 74
digestion, 123 gas gland, 106
3,5-dinitrobenzoyl derivatives, 132 glycolytic control, 81
embryonic development, 144 hypoxia, 74
fatty acyl composition, 130 kidney, 78
functions, 136ff. propranolol, 74
growth, 145 role in glycogenolysis, 68
head group composition, 129f. Glycogen production
larval diet, 144 indirect pathway, 71
lipovitellin, 127 Glycogen synthase
metabolic roles, 137f. brain, 87
terminology, 121 heart, 84
transport, 123 liver, 70
vitellogenin, 127 Glycogen synthesis
Glycine brain, 88
brush border uptake, 227 effects of cortisol, 422
fasting, 407 exogenous glucose, 81
from serine, 168 heart, 85
muscle, 407 insulin, 88
oxidation, 21, 371 lactate, 88
Glycogen recovery, 385
adipose tissue, 369 Glycogenesis
body mass, 369 from lactate, 82
brain, 46, 66, 86 role of enzymes, 82
burst swimming, 80 with fasting, 396
catecholamine, 373 Glycogenolysis, 371
content of tissues, 67 Ca z+, 77
depletion in liver, 394 catecholamines, 68
extracellular fluid, 369 epinephrine, 73, 420
GLP, 418 exhausting exercise, 54
glucagon, 418 GLP, 68, 420
hagfish heart, 66 glucagon, 68, 73, 420
hagfish liver, 411 hormones, 68f, 73, 77, 420
hypoxia, 46 hypoxic liver, 52
in boutons, 281 insulin, 69
indirect calorimetry, 41 liver, 52, 373
insulin, 411 seasonality, 77
liver, 46, 67, 369, 394ff. starvation, 55, 420
liver, starvation, 400 vasoactive peptides, 68
migration, 400 Glycolysis, 34
Subject Index 489
Glycolysis (continued) purine nucleotide cycle, 386

activation by histidine-related dipeptides, 324 regulation of glutamate dehydrogenase, 174
activation by calcium, 39 GTP binding protein
ATP/ADP ratio, 385 electric organ, 273
during tissue extraction, 39 Guanine crystals
Glycolytic complex, 299, 301 reflectors, 438
definition, 293 swimbladder submucosa, 102
Glycolytic control Gut
muscle, 81 amino acids in fasting, 160
recovery, 81 nutrient transport, 221
Glycolytic enzyme binding Gymnotiform
effect of methodology, 295 electric fish, 26 If.
Glycolytic enzymes
binding to particulates, 292 Hagfish
multienzyme complex, 292 glucagon, 411
regulation. 291 glycemia, 397
starvation, 407 liver glycogen, 411
Glycolytic flux role of insulin, 411
regulation through binding, 302 Hagfish heart
F-actin binding, 301 glucose utilization, 66
Glycolytic metabolon, 293 Handling stress, 36f.
Glyconeogenesis, 66 Hatching
Glycyl-phenylalanine phospholipids, 144
brush border uptake, 227 lipid catabolism, 144
Golgi apparatus HDL (high density lipoproteins)
exocytosis, 273 average composition, 25
gas gland cells, 103f. plasma, 124
Gonadal steroids Head kidney
influence on electric organ discharge, 266 lipoxins, 139
Gonadectomy macrophages, 139
sex differences in electric organ discharge, Heart
266 N-acetylated histidine, 329
Growth amino acids in fasting, 160
capacity, 346 anserine, 329
glycerophospholipids, 145 aspartate aminotransferase, 285
histidine, 318 ATP turnover, 379
intestinal nutrient absorption, 235 t-receptors, 85
3H-leucine, 196 buffering capacity, 85
protein synthesis, 191 carbohydrate metabolism, 83ff.
stunting, 346 carnosine, 329
vs. metabolism, 191 citrate synthase, 285
Growth hormone cyclooxygenase, 138
amino acid uptake, 233 fatty acid oxidation, 84f.
dynamics, 424 glucose 6-phosphate dehydrogenase, 285
effects on food consumption, 233 glucose oxidation, 84
effects on plasma fatty acids, 25 glycogen content, 67, 84
lipid mobilization, 424 glycogen synthesis, 85
proline uptake, 233 3-hydroxybutyrate dehydrogenase, 403
regulation of nutrient transport, 23 I, 233 hypoxic stress, 85
starvation, 423 lactate dehydrogenase, 285
vs. thyroid hormones, 234 lactate oxidation, 85
Growth performance lactate transport, 85
chronic stress, 202 lactate utilization, 381
genotype, 201 malate dehydrogenase, 285
immunocompetence, 202f. malic enzyme, 285
social rank, 203f. myoglobin, 85
Growth rate, 213f., 344, 346 prostanoid synthesis, 139
scaling with body weight, 212 Heart size, 340
GTP scaling, 344f.
490 Subject Index
Heat exchangers, 1, 11, 242 vs. oxygen consumption, 207

heat dissipation, 242 seasonal response, "/7
Heat flux thermal compensation, 75
indirect calorimetry, 41 thyroid hormone, 76
Heat production Hepatopancreas
ATPase, 248 phospholipase A2, 123
hormonal regulation, 255 Herbivores, 211
intracellular processes, 243 glucose uptake, 230
myosin ATPase, 248 intestinal nutrient transport, 230
protein synthesis, 207 Heterogeneity
proton leak, 248 liver cells, 69
rate in thermogenic tissue, 252 Hexokinase, 52
resting, 207 brain, 87
thermogenic tissue, 242 gas gland tissue, 107
vs. substrate, 252 glycolytic control, 81
Heat transfer heart, 84
muscle, 11 in tissues, 66
Hematocrit kidney, 78
body size, 341 (figure) liver, 70
physical activity, 340 red blood cells, 90
scaling, 340, 345 red muscle, 82, 248
stress, 340 regulation, 291
Hemoglobin, 340 thermogenic tissue, 251
acidification, 111 tuna red muscle, 248
concentration, 340 white muscle, 81
hypoxia, 50 Hexosephosphate shunt
multiple forms in fish, 342 elasmobranch kidney, 79
oxygen affinity, 341 red blood cells, 90
Root effects, 111, 114 High-energy phosphates
spleen, 342 (figure) as endogenous fuel, 45
Henry's law, 101 Histidase
Hepatectomy liver, 168
blood glucose, 68 Histidine
glycogen balance, 72 absorption, 319
glycogen levels, 72 N-acetylation, 329
plasma amino acids, 167 degrading enzymes, 322
Hepatic portal vein growth, 318
amino acids, 181 heart, 329
postprandial ammonia, 166 hepatic metabolism, 168
Hepatocytes imidazole dipeptides, 309
alanine uptake, 410 in carnosine synthesis, 320
amino acid utilization, 167, 169f. into muscle anserine, 320
ammoniogenesis, 162, 167, 169 muscle, 49, 313
CDP-choline phosphotransferase, 121 osmoregulation, 315f.
CDP-ethanolamine phosphotransferase, 121 pK value, 325
cost of protein synthesis, 209 red blood cell transport, 162
glucagon binding, 420 red muscle, 313
gluconeogenesis, 167 starvation, 316ff., 409
glutamine oxidation, 162 turnover, 319
glycerophospholipid biosynthesis, 121 uptake by tissues, 319
glycogenolysis, 418 washout, 319
ketogenesis from amino acids, 244 white muscle, 313
Na +/K+-ATPase vs. oxygen consumption, 209 Histidine aminotransferase
oxygen consumption, 209 liver, 168
vs. protein synthesis, 209 Histidine-related dipeptides
phosphatidyl ethanolamine methyltransferase, as a-adrenergic antagonists, 329
122 buffering, 324
phosphatidyl serine decarboxylase, 122 calcium sensitivity, 329
protein synthesis, 76, 207 contractile proteins, 329
Subject Index 491
Histidine-related dipeptides (continued) fatty acid precursors, 140

copper transport, 324 12-Hydroxyeicosatetraenoate (HETE)
degradation, 322 fatty acid precursors, 140
enzyme activation, 324 3-Hydroxybutyrate dehydrogenase
free radical scavenger, 324 elasmobranchs, 403
glycolysis, 324 teleosts, 403
interorgan transport, 322 p-Hydroxyphenyipyruvate
Mg2+-ATPase, 324 recycling of coelenterazine, 456
non-muscular tissues, 314 Hydroxyproline
osmoregulation, 315 fasting, 407
oxygen transport, 324 muscle, 407
physiological roles, 324ff. Hyperamino acidemia
proton buffering, 324 cortisol, 422
red muscle, 313 Hyperglycemia
structures, 309 hypoxia, 52
white muscle, 313 thyroid hormones, 425
Holocephalan Hypermetabolism
plasma fatty acid concentration, 24 fasting, 73
Homeostasis Hyperoxia
amino acids, 162, 199 cell proliferation, 115
Homeothermy, 337 contribution of pentose shunt in gas gland, 109
cost, 337 edema, 115
Homeoviscous adaptation gas gland, 115
membranes, 134 Hyperventilation
Homocarnosine hypoxia, 50
imidazole dipeptides, 309 Hypoglycemia
imidazole pK value, 325 fasting, 73
vertebrate muscle, 309 role of insulin, 411
Hormonal control Hypoglycemic stress
fasting, 74, 410ft., 418, 424 brain, 87
sonic muscle parameters, 287 Hypometabolism
Hormone-sensitive lipase fasting, 72f.
in intertissue lipid exchange, 23 starvation, 401
HSI Hypothyroidism, 426
starvation, 397 Hypoxia, 374
Hummingbird acid secretion, 112
muscle characteristics, 2 aerobic allometry, 352
muscle morphometric data, 6 carbohydrate metabolism, 74f.
Hydrodynamics, 344 catecholamines, 91
Hydrogen shuttles enzyme activities, 74
a-glycerophosphate shuttle, 247, 249, 255 glucose flux, 21
malate-aspartate shuttle, 178, 247, 249, 255 glycogen mobilization, 51
thermogenic tissue, 255 heart glycogen, 47
tuna red muscle, 249 heart, 85
Hydroperoxy fatty acids hyperglycemia, 52
eicosanoid metabolism, 138f. hypoglycemia, 74
Hydroxy fatty acids lactate flux, 18
eicosanoid metabolism, 138f. lactate turnover, 19, 74
12-Hydroxy fatty acids liver glycogen, 47
distribution, 139 metabolic depression, 52
Hydroxyacyl-Coenzyme A dehydrogenase metabolic rate, 291
brain, 87 muscle glycogen, 46
heart, 84 normoglycemia, 74
kidney, 78 physiological effects, 50
liver, 70 thermal acclimation, 83
red muscle, 82
thermogenic tissue, 251 Icefish
white muscle, 81 hemoglobin, 340
12-Hydroxyeicosapentaenoate (HEPE) oxygen carrying capacity, 340
492 Subject Index
IDL, 124f. starvation, 410ff.

Imidazole values for fish plasma, 413
pK value, 325 Insulin binding
Imidazolopyrazine re-feeding, 73
autoxidation, 461 Insulin receptor
light production, 439 liver, 415
origin of luminescence, 459 muscle, 415f.
Imino acid transporter Insulin-like growth factors (IGFs)
brush border, 227 starvation, 424
Immunocompetence Interference reflector
feeding, 204 guanine crystals, 438
social rank, 203f. photogenic tissue, 438
IMP, see Inosine 5'-monophosphate Interorgan transport
Induction anserine, 323
amino acid transport, 235 Interrenal steroids
Infusion effects on intestinal transport, 233
metabolite turnover, 16f. metabolism, 423
Ingestion Intestinal absorption
scaling, 344 luciferin, 450ff.
luciferin, 446 Intestinal mucosa
Ingestion rate re-esterification of lysophospholipid, 123
scaling with body weight, 212 Intestine
Inosine 5'-monophosphate amino acid oxidation, 165
purine nucleotide cycle, 386 amino acid requirement, 166
muscle, 380, 385 amino acid transport, 222
myo-lnositol ammoniogenesis, 166
in glycerophospholipids, 120 chylomicron-like particles, 124
Inositol connection to light organ, 439, 451
diet, 145 cyclooxygenase, 138
Inositol phosphates dipeptide transport, 163f.
phosphoinositide cycle, 142 glucose transport, 222
Inositol phospholipid metabolism glycerophospholipid headgroup composition,
electrocytes, 143 129
Insects glycerophospholipid storage, 128
effects on glycerophospholipid composition, length, 230
133 lipoproteins, 124
endothermy, 242 Na+-pumps, 222
luminescence, 444 Na +/K+-ATPase, 222
Insulin nutrient absorption, 221
brain glucose release, 88 passive nutrient permeability, 230
brain glycogen synthesis, 88 phospholipases, 123
carbohydrate metabolism, 73 phospholipid changes with seawater adapta-
crossreactivity, 410 tion, 135f.
effects of glucocorticoids, 421 prostanoid synthesis, 139
effects on liver enzymes, 412, 414 protein turnover, 165f.
effects on plasma fatty acids, 25 proteolysis in starvation, 406
gluconeogenesis, 69 storage for triacylglycerols, 400f.
glucose turnover, 21 transport processes, 221
glycogenolysis, 69 VLDL, 124
gonadotropic action, 414 Intracellular pH
hypoglycemia, 86, 411 calculation from NMR, 43
lipogenesis, 411 glycolytic flux, 292
liver glycogen, 411 muscle recovery, 381
membrane at~nity, 72 Invertebrates
plasma dynamics, 41 lff. distribution of histidine-related dipeptides, 312
protein synthesis, 411 Iodoacetate
re-feeding, 74, 412 fructose 6-phosphate, 299
RIA, 410 glycolytic enzyme binding, 299
spawning migration, 414 heart performance, 84f.
Subject Index 493
Ionic strength elasmobranch hepatocytes, 244

dilution method for enzyme binding, 294 Ketone bodies, 71, 378
IP3 anoxia, 88
phosphoinositide cycle, 142 competition with glutamine in thermogenic
Ischemia tissue, 247
brain, 87 elasmobranch fuel, 243, 411
Islet elasmobranch red muscle, 377
hagfish, 411 oxidation
Isocitrate heater organ, 251
regulation of glutamate dehydrogenase, 174 brown adipose tissue, 251
lsocitrate dehydrogenase (NAD) oxidation in teleosts, 248
control by calcium, 252 starvation, 244, 402f.
thermogenic tissue, 247 substrate for heart, 85
Isocitrate dehydrogenase (NADP) teleost brain, 88
brain, 87 thermogenic tissue, 247
heart, 84 Kidney
liver, 70 aglomerular, 457
red blood cells, 90 alanine oxidation, 79
red muscle, 82 amino acid
starvation, 404 absorption, 173
white muscle, 81 catabolism, 177
Isoleucine fasting, 160
metabolic fate, 175 levels, 173
red blood cell transport, 162 ammoniogenesis, 174, 176
role in alanine biosynthesis, 183 anserinase, 322
starvation, 409 arginase, 175
Isometric scaling asparagine metabolism, 176
gill surface area, 343 blood ammonia, 176
respiration, 339 branched-chain amino acid metabolism, 175
lsoproterenol brush-border membranse vesicles
red cell 3-O-methyiglucose uptake, 91 amino acid uptake, 173
red cell metabolism, 91 carbohydrate metabolism, 78ff.
red cell Na+-pump, 91 catabolism of dipeptides, 324
Isotocin cyclooxygenase, 138
gluconeogenesis, 68 enzyme activities, 78
glycogenolysis, 68 ethanolamine plasmalogens, 129
signal transduction, 77 fasting, 80
free amino acids, 49
Juvenile fish gluconeogenesis, 79f., 176
cost of protein synthesis, 209 glucose 6-phosphate dehydrogenase
protein synthesis, 211 fasting, 80
vs. oxygen consumption, 207 glucose production, 20
glutamate dehdydrogenase, 174
K+ current glutamate utilization, 80
electrocytes, 268 glutaminase, 176, 244
K+-channels glutamine metabolism, 176
intestinal amino acid transport, 163 glycogen content, 67, 78
K+-gradient hexosephosphate shunt, 79
amino acid transport, 164 histidine degrading enzymes, 322
basolateral membrane, 229 histidine-related dipeptides, 314
Keel muscle 3-hydroxybutyrate dehydrogenase, 403
light organ, 439 hypoxia, 80
Keratin-like protein lactate oxidation, 79
electrocytes, 272 loss of luciferin, 457
6-Ketoprostaglandin Fla PEPCK
distribution, 139 fasting, 80
Keto-testosterone phosphatidylcholine/seawater adaptation, 135
sonic muscle, 287 prostanoid synthesis, 139
Ketogenesis proteolysis in starvation, 405
494 Subject Index
Kidney (continued) red muscle, 370

purine nucleotide cycle, 174 white muscle, 370
seawater adaptation, 135 red muscle, 249, 369
serine metabolism, 79, 175 release
taurine secretion, 173 gas gland, 107
Killifish white muscle, 374
amino acid transporter, 226 rest, 36, 380
intestinal transport, 226 retention, 19, 82
Kleiber's rule, 336 storage sites, 383
Krebs cycle, 34 sustained swimming, 80
gas gland metabolism, 109 swimbladder blood, 107
in glutamate metabolism, 246 transport, 20
proton pumps, 242 heart, 85
red blood cells, 91 liver, 66
red blood cells, 66
Lactase tuna white muscle, 249, 315
intestine, 223 turnover, 82, 372, 382
Lactate, 369 body mass, 19
accumulation in white muscle, 249 cannulation, 41
adipose tissue, 369 exercise, 19
brain, 88 fasting, 19
burst swimming, 80 fish species, 69
disappearance from white muscle, 384 hypoxia, 19, 74
disappearance, 82 in postabsortive teleosts, 19
disappearence from muscle, 381 red muscle, 1
dynamics, 375 stress, 19
effect of sampling methodology, 36 vs. oxygen consumption, 19
exchange between muscle & circulation, 19 uptake kinetics, 66
extracellular fluid, 369 utilization, 357
fate, 381 vs. flux, 18
flux to C6-products, 71 vs. lactate dehydrogenase, 356
formation white muscle, 249, 369, 384
gas gland, 105, 109f. Lactate dehydrogenase
acetylcholine-stimulated, 105 association with F-actin, 303
catecholamine-stimulated, 105 binding in turtle brain, 297
fuel for red muscle, 375 binding to subceUular muscle structures, 292
gas gland metabolism, 109 brain, 87, 355
gluconeogenesis correlation with buffering capacity, 327
effects of cortisol, 422 enzyme binding, 295, 300
gluconeogenesis, 412 gas gland, 106f.
hypoxia. 52 heart, 84
liver substrate, 69 isozymes, 71
liver, 369 kidney, 78
load kinetic effects of F-actin, 302
exercise, 81f. liver, 70f.
metabolism, 19, 82, 109, 371, 379 loss during muscle preparation, 296
body ma~s, 19 muscle, 81, 106, 285, 314, 354ff.
muscle, 380 muscle type in gas gland, 106
transport, 382 raven heart, 357
non-ionic diffusion, 115 red blood cells
oxidation, 371, 375 red muscle, 81
heart, 85 scaling in white muscle, 354f.
kidney, 79 scaling, 355
red blood cells, 162 sonic muscle, 285f., 357
substrate, 71 starvation, 396
plasma concentration at rest, 17 toadfish heart, 285
production toadfish white muscle, 285f.
exercise, 379 tuna white muscle, 314
red blood cells, 91 vs. a-glycerophosphate dehydrogenase, 249
Subject Index 495
Lactate dehydrogenase (continued) protein turnover, 211f.

vs. lactate concentration, 356 Light emission, 438
white muscle, 81, 285f. counterillumination, 437
Lactate pathway, 354f. photophores, 436
Lake Baikal spectral shift, 445f.
luminescent species, 435 temporal variation, 446
Laminar flow Light filtering, 439
power requirements for swimming, 356 Light organ
Lamnid sharks anatomy, 439
red muscle, 243 coelenterazine, 442
thermogenic tissue, 242 glucuronidation, 453
Larval fish guanine crystals, 438
cost of protein synthesis, 209 keel muscle, 439
glycerophospholipid diet, 144f. luciferase, 442
protein synthesis, 211 luciferin recycling, 454
PUFA, 144 luciferin storage, 453
LDL (low density lipoproteins) luminescent species, 436
average composition, 25 peroxidase, 442
plasma, 124 relation to intestine, 439
Lecithin: alcohol acyltransferase relation to pyloric caeca, 439
plasma, 125 storage of luciferin, 452
Lecithin: cholesterol acyltransferase Lindane
plasma, 125 brain metabolism, 88
Lens Lipase
aperture, 438 adipose tissue, 403
light organs, 438 bile-salt activated, 123
Leucine red muscle, 403
activation of gluconeogenesis, 172 Lipid
brush border uptake, 227 as endogenous fuel, 47
effect on branched-chain amino acid amino- availability, 374
transferase, 177 depletion
glutamate dehydrogenase, 168 cortisol, 422
growth, 196 starvation, 399f., 405
metabolic fate, 175 droplets
oxidation, 22, 205, 371 tuna red muscle, 248
protein synthesis, 196, 205 from acetate, 404
red blood cell transport, 162 gas gland, 104f.
regulation of glutamate dehydrogenase, 174 lipoproteins, 124
role in alanine biosynthesis, 183 liver composition, 395f.
starvation, 409 liver, 25
stereospecific intestinal uptake, 226 metabolism
transport hatching, 144
regulation by estradiol, 232 embryonic development, 144
regulation by methyl-testosterone, 232 mobilization, 400
3H-Leucine growth hormone, 424
growth, 196 muscle content, 25, 395f., 401
protein synthesis, 196 oxidation
Leukocytes elasmobranch liver, 244
chemotactic activity of leukotriene, 142 elasmobranch tissues, 243
cyclooxygenase, 138 embryonic development, 144
lipoxygenase products, 139 transport, 124
prostacyclin synthesis, 139 albumin-like protein, 125
prostanoid synthesis, 139 lipoproteins, 124f.
Leukotrienes Lipid reserves
chemotactic activity, 142 effects of thyroid hormones, 425
eicosanoid metabolism, 138f. glucagon, 419
fatty acid precursors, 140 starvation, 419
Lidocaine, 38 Lipogenesis
Life history anoxia, 52
496 Subject Index
Lipogenesis (continued) CDP-choline-l,2diacylglycerol choline phos-

from amino acids in muscle, 178 photransferase, 121
insulin,411 composition change in starvation, 395
perivenous cells,69 cortisol, 76
starvation,404 binding, 423
Lipogenic enzymes cyclooxygenase, 138
effectsof diet, 172 depletion of lipids, 400
Lipolysis, 123, 378 dipeptide degradation, 322
cortisol,422 DNA content in starvation, 406
effectsof growth hormone, 424 enzymes, 70
starvation,419 activation by cortisol, 69
Lipoprotein lipase,125 ethanolamine plasmalogens, 129
activationby apoproteins, 126 fasting, 72
in intertissuelipidexchange, 23 fat synthesis, 170
Lipoproteins fatty acid synthesis, 404
apoproteins, 126 fatty acids, 369
average composition, 25 fractional rate of protein synthesis, 204f.
contribution to plasma fraction,25 free amino acids, 49
lipidtransport, 124 fructose 6-phosphatase, 412
phosphatidylcholine, 126 gluconeogenesis, 170, 371, 375, 406, 412
plasma, 25 from amino acids, 170
PUFA, 126 gluconeogenic flux, 373
remodelling, 125 glucose, 369
uptake production, 20, 372
receptor-mediated, 126f. release, 373
pinocytosis, 127 transport, 66
vitellogenin, 127 glutamate dehydrogenase, 172
Lipovitellin glutamate production from amino acids, 168
lipid composition, 127 glycerol-3-phosphate acyltransferase, 121
glycerophospholipid headgroup composition,
Lipoxins
129
eicosanoid metabolism, 138f. glycerophospholipid storage, 128
gill, 139 glycogen, 46, 75, 338, 358, 369, 394, 405
macrophages, 139f. anoxia survival, 75
Lipoxygenase anoxia tolerance, 74
eicosanoid metabolism, 138f. content, 46, 67
fatty acid specificity, 142 fasting, 72
tissue products, 139 mobilization, 394
12-Lipoxygenase, 139 re-feeding, 73
15-Lipoxygenase, 139 seasonality, 76
Lithium-sensitive phosphatase starvation, 55
electrocytes, 143 glycogenolysis, 52, 373
Liver, 68ff. heterogeneity, 69
amino acids, 369 histidine degrading enzymes, 322
effects of salinity, 171 histidine metabolism, 168
exercise, 53 histidine-related dipeptides, 314
fasting, 160 3-hydroxybutyrate dehydrogenase, 403
ammoniogenesis, 49 insulin binding, 72
anserinase, 322 insulin receptor, 415
apoprotein receptors, 126 lactate, 369
arginase, 175 dehydrogenase, 71
as energy store, 71 transport, 66
branched-chain a -ketoacid dehydrogenase, iipase, 125, 422
175 lipids, 405
branched-chain amino acid aminotransferase, content, 25, 47
174 oxidation, 244
carbon cycling, 71 lipogenic enzymes, 172
cardiolipin, 129 lobes
carnosinase, 322 metabolic activity, 69
Subject Index 497
Liver (continued) peroxide formation, 443

mass in starvation, 406 plasma binding, 452
microsomal lipid synthesis, 121 quantum yields, 441
mitochondrial ammoniogenesis, 167 receptors, 458
mitochondrial phospholipids/temperature, 136 recycling, 447, 454
ornithine decarboxylase, 172 red cell binding, 452
periportal cells, 69 retention, 456
perivenous cells, 69 signalling, 456
phosphofructokinase- 1, 414 spectra, 441
phospholipid changes with seawater adapta- stomach, 451
tion, 135 storage, 447, 452
platelet-activating factor synthesis, 143 structures, 440
postprandial amino acids, 167 transport, 452
prostanoid synthesis, 139 trophic transfer, 446
protein, 369, 405f. uptake, 458
synthesis, 76, 167 Luciferin-binding protein, 452
synthesis vs. amino acid pool, 204f. Luciferinol, 442
proteolysis in starvation, 405 Luciferyl-sulfate
purine nucleotide cycle, 167 coelenterazine, 442
pyruvate kinase, 412 Luciferyl-sulfokinase
serine metabolism, 168 luciferin regeneration, 442
signal transduction, 77 Luminescence, see also bioluminescence
starvation, 405 effects of cyanide
storage for triacylglycerols, 399 effects of epinephrine, 454
thyroid hormone binding, 426 inhibition by cyanide, 442
transdeamination, 371 phylogenetic distribution, 461
triacylglycerols, 369, 399 quantum yield, 455
lipase, 419 starvation, 461
mobilization, 375 Luminescent organisms
VLDL synthesis, 125 fish diet, 448
Locomotion, 337 Luminescent species
Luciferase, 440ff. light organs, 436
amino acid sequence, 444 Luminescent system
chemiluminescence, 444 distribution in fishes, 441
coelenterazine recycling, 457 Lymphatic system
digestive tract, 442 transport of lipoproteins, 124
effects of glyceraldehyde 3-phosphate, 453 Lysine
light organs, 442 brush border uptake, 227f.
molecular weight, 443 intestinal transport, 163
monooxygenase, 443 osmoregulation, 316
Luciferin, 436, 440ff. Lysosomes
autoxidation, 451, 461 lipase, 403
binding protein, 452
blood, 457 Macrophages
counterillumination, 456 cost of protein synthesis, 208f.
de novo synthesis, 454 fractional protein synthesis rate vs. oxygen
diet, 446 consumption, 208
diffusion, 458 12-hydroxy fatty acids, 139
effects of glyceraldehyde 3-phosphate, 453 leukotrienes, 139
eggs, 458 lipoxins, 139f.
exogenous sources, 447ff. lipoxygenase products, 139
from oxyluciferin, 454 Maintenance cost
ingestion, 446 protein turnover, 214
intestinal absorption, 450 Maintenance synthesis, 210
losses from body, 457 Malate
maternal transfer, 450 regulation of glutamate dehydrogenase, 174
metabolism, 447 Malate aspartate shuttle, 178, 247, 249, 255
origins, 459 thermogenic tissue, 255
oxidation, 443 Malate dehydrogenase
498 Subject Index
Malate dehydrogenase (continued) Mesentery

brain, 87 lipase, 422
gas gland tissue, 105 Metabolic acid load
heart, 84, 285 exercise, 81f.
in glutamate metabolism, 246 Metabolic allometry, 336L
kidney, 78 Metabolic cost
liver, 70 branchial pump, 345
red blood cells, 90 cardiac pump, 345
red muscle, 82, 285f. Metabolic depression, 74E
sonic muscle, 285f. hypoxia, 52
thermogenic tissue, 247 low temperature, 72
toadfish heart, 285 Metabolic intermediates
toadfish white muscle, 285f. extraction, 39
white muscle, 81, 285L flux, 15
Malate/a-ketoglutarate carrier measurements, 40
in glutamate metabolism, 246 sampling, 38
Malformation storage, 39f.
role of glycerophospholipids, 145 turnover studies, 41
Malic enzyme (NADP) Metabolic pathway
brain, 87 efficiency, 33f.
glutamine oxidation, 244 regulation, 291
glycogenesis, 82 Metabolic rate, 359
heart, 84, 285 aerobic scaling exponent, 359
in glutamate metabolism, 246 anoxia, 291
in muscular glycogen synthesis, 383 burst swimming, 291
kidney, 78 effects of thyroid hormones, 425
liver, 70 hypoxia, 291
red muscle, 82, 285 large animals, 359
sonic muscle, 285 scaling, 345
starvation, 404 slow swimming, 291
thermogenic tissue, 247 Metabolic substrates
toadfish heart, 285 systemic circulation, 15ft.
toadfish white muscle, 285. Metabolism
white muscle, 81, 285 aerobic, 335ff.
Maltase allometry, 344
intestine, 223 basal, 336
Mammals down-regulation, 401
distribution of histidine-related dipeptides, 312 exercise, 368
endothermy, 242 fatty acid contribution, 24
muscle buffering capacity, 326 muscle, 337, 359
Manometry respiration rate, 338
gas gland metabolism, 105 routine, 371
Marine mammals scaling, 336ff.
distribution of histidine-related dipeptides, 312 standard metabolic rate, 336
muscle buffering capacity, 326 Methionine
Mate call brush border uptake, 227
toadfish, 281 protein consumption, 206
Maternal transfer protein synthesis, 206
luciferin, 450, 458ff. 3-O-Methylglucose
Membranes brain glucose transport, 89
composition, 134 uptake
electric organs, 269f. red cell, 91
temperature, 134 isoproterenol, 91
transporter, 222 Methyl-histidine
vesicles, see also basolateral membrane & excretion, 322
brush border membranes histidine-related dipeptides, 309ff.
preparations, 223 imidazole pK value, 325
Mesenteric fat, 47 N-Methylglycine
starvation, 56 intestinal transport, 163
Subject lndex 499
Mg2+-ATPase, 324 muscle fiber, 10

role of histidine-related dipeptides, 324 sonic fibers, 286
Microsome membranes Mitochondrial volume density
composition of phosphatidylcholine, 132 tuna red muscle, 250
composition of phosphatidylethanolamine, 132 vertebrate muscles, 7
Microsomes Monocarboxylate carrier
CDP-choline-l,2diacylglycerol choline phos- /~-hydroxybutyrate, 246
photransferase, 121 pyruvate transport, 246
lipid synthesis, 121 Monooxygenase
phospholipase A2 luciferase, 443
Microtubules Mormyriform
electric organ, 273 electric fish, 26 If.
Microvilli Morphometry
gas gland cells, 103 red muscle, 1
Midshipman Mosquitofish
sonic muscle, 279ff. metabolic rate, 344
sound production, 279ff. Motoneuron
Migration, see also Spawning migration cholinergic, 281
body weight, 399 innervation of electric organ, 262
lipid loss, 399 Motor nerve
lipid utilization, 376 sonic muscle, 280
vertical, 101, 342 MS222, 38
vitellogenin, 408 Mucopolysaccharides
Milt amino acid gluconeogenesis, 22
cyclooxygenase, 138 Mucus
Mitochondria nitrogen loss,198
amino acid oxidation, 371 Mucus production
/5-hydroxybutyrate dehydrogenase, 246 amino acid gluconeogenesis, 22
carnitine palmitoyl-Coenzyme A transferase, Muscle, see also Red muscle, White muscle, Sonic
377 muscle & Heart
flux capacities, 378 31P-spectra, 44
gas gland cells, 103 A C h R subunits,269
glutaminase, 169, 176, 178 adenylates, 380
glutamine oxidation, 22 A D P pool, 385
in boutons, 281 alanine biosynthesis,183
interfibrillar, 9 allometry, 346
membranes, 242 amino acid metabolism, 11, 177ff.
composition of phosphatidylcholine, 132 amino acid pool in starvation,160, 178
composition of phosphatidylethanolamine, aminotransfcrases, 178
132 ammoniogcnesis, 179
temperature effects on lipid composition, A M P deaminase, 380, 385
135 AMP, 380
mobilization in starvation, 407 anaerobic capacity,327
muscle glutaminase, 178 anserinase, 322
muscle, 371 ATP/ADP ratio,385
oxidative substrates, 244 branched-chain amino acid aminotransferase,
phosphorylation, 242 174, 178
proton leak, 241 buffering capacity,325ff.
quantity in red muscle, 377 starvation,316ff.
renal glutaminase, 176 capillarymanifolds, II
respiration, 242 catheptic enzymes, 405
acidosis, 386 composition change in starvation,395
thermogenic tissue, 252 contraction, 337
sonic muscle, 284 conversion into electricorgan, 260
subsarcolemmal, 9 creatine phosphate, 45
Mitochondrial density cyclooxygenase, 138
sonic muscle, 284 cytosol pH, 325
Mitochondrial volume, 3 differentiation,270
density, 3 fate of lactate,381
500 Subject Index
Muscle (continued) utilization in starvation, 400

fibers, 337 proteolysis, 170, 398, 410
cross-sectional area, 4 purine nucleotide cycle, 179
diffusion distance, 9 pyruvate, 383
ultrastructure, 4 respiration, 380
volume of mitochondria, 3 resting lactate, 36
fiber size, 3 starvation, 55, 160, 178, 398, 405f.
vs. subsarcolemmal mitochondria, 9 steady-state swimming, 367
free fatty acids, 376 storage for triacylglycerols, 399
gluconeogenesis from amino acids, 178 substrate transfer, 11
glucose uptake, 383 triacylglycerol mobilization, 375
glutamate dehydrogenase, 178 trimethylamine, 49
glutathione reductase, 115 twitch contraction time, 337
glycerophospholipid headgroup composition, utilization of ketone bodies, 377
129 weight
glycerophospholipid storage, 128 vs. glycogen, 358
glycogen, 46, 338, 358, 371, 380, 405 Muscular dystrophy
mobilization in starvation, 398 dystrophin, 270
starvation, 55 Mussel
elasmobranchs, 399 protein synthesis, 196
glycogen synthesis, 381, 383 stable isotopes, 196
glycogenolysis, 398 my5
glycolytic enzyme binding, 292 expression in electrocytes, 270
heat transfer, 11 myo-lnositol
histidine, 178 in glycerophospholipids, 120f., 129
histidine degrading enzymes, 322 metabolism in electrocytes, 143
IMP, 179, 380 Myoeardium
insulin receptor, 415f. oxygen consumption, 375
lactate, 380 work, 375
dehydrogenase, 327 Myocytes
exchange, 19 ~-adrenergic stimulation, 86
release/retention, 19, 82, 382 MyoD
lipases, 122 expression in electrocytes, 270
lipid, 25, 377, 405 Myofibrillar protein
content, 25 anoxia, 53
starvation, 56, 401 percent of whole animal protein, 213
iipogenesis from amino acids, 178 Myofibrils
malate-aspartate shuttle, 178 ADP pool, 385
metabolism, 337, 359 electrocytes, 271
mitochondria fasting, 407
cardiolipin, 129 free ADP, 386
ethanolamine plasmalogens, 129 Mg2+-ATPase, 324
modifed as heater organ, 250 role of histidine-related dipeptides, 324
morphometric data, 6 sonic muscle, 282
myoglobin, 327 space in white muscle, 379
NMR, 381 synthesis, 407
non-bicarbonate buffering, 325 Myofilament
oxidative fibers, 377 volume density, 358
phosphagens, 379 Myogenic electric organ, 265
phosphate, 380 Myoglobin, 342
polyribosomes, 193 correlation with buffering capacity, 327
protein, 405 heart, 85, 342
degradation, 178 oxygen storage, 338
metabolism, 11 red muscle, 311, 342
percent of whole animal protein, 213 scaling, 345
proportion of amino acids, 407 Myosin
retention, 178 in electrocytes, 270, 272
starvation, 406ff. protein synthesis, 192
synthesis, 178, 193 Myosin ATPase
Subject Index 501
Myosin ATPase (continued) intestine, 163, 222

activation during tissue extraction, 39 methyltestosterone effects, 232
creatine phosphate, 45 thermogenesis, 242, 248
elasmobranch muscle, 243 Na +/proline transporter
heater organ heat production, 248 E. coli, 224
phosphofructokinase, 304 intestine, 227
thermogenesis, 242, 248, 250 NAD(P)
Myosin light chains role in luminescence, 443
sonic muscle, 283 Nasal photophores, 443
Negative allometry, 337, 357
Na + ions standard metabolism, 359
electric organ discharge, 263 Neuromuscular junction
Na+-amino acid coupling ratios presence of dystrophin, 270
intestinal transport, 228 sonic muscle, 281
Na+-channel, 259, 265 Neutral amino acids
Na+-current intestinal transport, 163
electrocytes, 268 Neutral lipase, 403
Na+-dependent transport Neutral lipids
amino acids, 164, 226 furan fatty acids, 130
coupling ratio, 223 seawater adaptation, 135
intestine, 222 Neutral protease
stimulation by growth hormone, 233 starvation, 56
Na+-efflux Neutrophils
heater mitochondria, 254 eicosanoid biosynthesis, 141
Na+-gradient leukotrienes, 142
amino acid transport, 164 lipoxygenase products, 139
basolateral membrane, 229 Niche shift, 359
Na+-influx Nicotinic cholinergic neurons, 262
intestinal amino acid transport, 163 Nitrogen excretion, see also Ammonia
Na+-pump amino acid imbalance, 206
intestinal amino acid transport, 163 protein metabolism, 22
intestine, 163, 222 scaling with body weight, 212
isoproterenol, 91 Nitrogen flux
red blood cells, 91 protein synthesis, 192
Na +/glucose coupling Nitrogen loss
effects of thyroid hormones, 234 amino acid oxidation, 198
methyltestosterone effects, 232 ammonia production, 198
Na+/glucose transporter daily balance - feeding, 197
amino acid sequence, 224 daily balance - starving, 198
brush border, 223 digestive enzymes, 198
cDNA, 223 mucus, 198
evolution, 224 scales, 198
gene, 224 urea production, 198
pyloric caeca, 224 Nitrogen retention efficiency, 211
site density, 224 Nodes of Ranvier
subunits, 224 electric organ axon, 266
Na +/H +-exchanger Non-bicarbonate buffering
red blood cells, 91 muscle, 325, 327
Na +/K+-ATpase Non-essential amino acids, see also amino acids
amino acid transport, 164 fasting, 409
contribution to hepatocyte oxygen consump- oxidation, 22
tion, 209 plasma, 161
effects of thyroid hormones, 234 post-prandial, 161
elasmobranch muscle, 243 uptake from portal blood, 181
gill membrane lipids, 136 Non-mediated transport, 222
heater organ heat production, 248 Non-steady state conditions
in autoradiogram, 269 flux measurements, 17
in electric organs, 269, 273 Norepinephrine
intestinal amino acid transport, 163 anoxia, 52
502 SubjectIndex
Normoxia Na+/K+-ATPase in brain heater, 254

contribution of pentose shunt in gas gland, Ovarian fluid
109 cyclooxygenase, 138
Nuclear magnetic resonance, 37, 42ff. Ovarian follicles
muscle metabolism, 42 protein kinase C, 143
Nuclei steroidogenesis, 143
thyroid hormone binding, 426 Ovary
Nutrient absorption cyclooxygenase, 138
adaptive changes, 222 prostanoid synthesis, 139
growth, 235 histidine-related dipeptides, 314
intestine, 221 Ovulation
Nutrient transport eicosanoids, 142
adaptations to diet, 229f. Oxaloacetate
genetic adaptations, 230 heart performance, 85
intestine permeability, 230 Oxamate
regulation, 229 gas deposition, 112
stimulation by growth hormone, 233 gas gland lactate production, 112
Nutrient transporter Oxidative capacity
sequence homology, 235 white muscle, 384
Nutrition 3-Oxoacid CoA transferase
effects on hormones, 420 thermogenic tissue, 247
protein synthesis, 191 Oxygen
protein turnover, 192 affinity, 341f.
carrying capacity, 339ff.
Ocular muscle convective transport, 339
anserinase, 322 delivery, 338
electric organ, 261 in luminescence, 443
Olfactory cilia reaction with imidazolopyrazines, 461
phospholipase C, 122 storage, 338
Olfactory epithelium tension in gas gland, 113
phospholipase C, 122, 143 Oxygen consumption
Olfactory nerve amino acid oxidation, 22
plasmalogens, 129 basal, 371
alkylacylphospholipids, 129 brain, 89
Omnivores, 211 burst swimming, 359
glucose uptake, 230 gas gland tissue, 106
Oocytes indirect calorimetry, 41
injection of AChR mRNA, 270 post-exercise, 383
Oogenesis, 127 postprandial increase, 205f.
Ophidine protein synthesis, 191
HPLC, 311 red blood cells, 91
imidazole dipeptides, 309 red muscle, 370, 373
vertebrate muscle, 309 vs. lactate turnover, 19
Optical structures white muscle, 370
photogenic tissue, 438 Oxygen debt
Organ recovery, 75
protein synthesis, 192 Oxygen delivery, 339, 341, 343, 345
Orientation Oxygen flux
muscle capillaries, 7 capillary to muscle, If.
Omithine decarboxylase muscle, 9
starvation, 172 Oxygen minimum zone, 355
Osmoregulation Oxygen radicals
carnosine, 315 gas gland, 115
histidine-related dipeptides, 315 removal, 115
muscle amino acids, 315f. Oxygen transfer, 340
Ostracods Oxygen transport, 91
coelenterazine, 436 histidine-related dipeptides, 324
luciferin, 436 Oxygen uptake
Ouabain gas gland tissue, 105
Subject Index 503
Oxygen uptake (continued) Peroxidase

symmorphosis, 343 light organs, 442
Oxyluciferin, 443, 445, 454 Peroxisomes
conversion to luciferin, 454 metabolism of alkylglycerophosphate, 122
Pesticides
P450 enzymes brain metabolism, 88
epoxide formation, 139 pH
Pacemaker nucleus ATP/ADP ratio, 385
electric organ, 262 effects on glycolytic enzyme binding, 295
nicotinic cholinergic neurons, 262 Phase polarity
Palmitate electric organ discharge, 263
cardiac performance, 85 Phenotypie adaptations
oxidation in heart, 84f. intestinal transport, 230
substrate for heart, 84f. Phenylalanine
Palmitoyl-L-carnitine brush border uptake, 227f.
elasmobranch muscle mitochondrial substrate, free pool, 195
244 intraperitoneal injection, 195
oxidation intravenous injection, 195
thermogenic tissue mitochondria, 251 microinjection, 195
tuna red muscle, 248 oxidation, 22
Pancreas, 123 protein-bound pool, 195
Paracellular pathway red blood cell transport, 162
intestinal transport, 223 white muscle, 195
Parasympathicomimetic drugs 3H-Phenylalanine
gas deposition, 112 protein synthesis, 193
Parr-smolt transformation specific activity, 193
cortisol, 233 L-2,6-3H-Phenylalanine
Parvalbumin protein synthesis, 195
sonic muscle, 282f. specific activity, 195
Pasteur effect, 51 time course, 195
anoxia, 74 Phenylephrine
gas gland, 107, 113 glycogenolysis, 77
Pentose phosphate shunt, see also hexose phos- Phloretin
phate shunt glucose transport, 225
gas gland enzymes, 108 Phlorizin
gas gland metabolism, 109 glucose transport, 223f.
gas gland tissue, 108ff. Phorbol ester
heart, 83 protein kinase C, 143
liver, 108 Phosphagens
fete mirabile, 115 exercise, 385
starvation, 404 Phosphatase
PEPCK lithium-sensitive, 143
effects of cortisol, 422 Phosphate
fasting, 73 buffering, 325, 328
glycogenesis, 82 effects on glycolytic enzyme binding, 295
heart, 83 limitations, 386
in muscular glycogen synthesis, 383 muscle, 380
insulin, 69 homogenate buffering, 328
kidney, 78f. signal in 31P-NMR, 44
starvation, 396, 414 stimulation of respiration, 386
subcellular distribution, 71 utilization in recovery, 385
transcription, 397 Phosphatidic acid
Peptido-leukotrienes structure, 120
gill function, 142 Phosphatidyl ethanolamine methyltransferase
Periportal cells hepatocytes, 122
glucose release, 69 Phosphatidyl serine decarboxylase
Perivenous cells hepatocytes, 122
glycogenesis, 69 Phosphatidylcholine, 120f.
lipogenesis, 69 fatty acid composition of membranes, 132
504 Subject Index
Phosphatidylcholine (continued) release from muscle particulate matter, 295

fish tissues, 129 role in gluconeogenesis, 68
growth promotion, 145 starvation, 412
in lipovitellin, 127 temperature effects, 75
lipoproteins, 126 white muscle, 81, 113
temperature acclimation, 134f. 6-Phosphogluconate dehydrogenase
Phosphatidylethanolamine, 120f. carbon dioxide formation, 109
changes with seawater adaptation, 135 effects of diet, 172
fatty acid composition of membranes, 132 gas gland tissue, 108, 110
in fish tissues, 129 kidney, 79
in lipovitellin, 127 starvation, 404
liver mitochondria, 136 Phosphoglucose isomerase
temperature acclimation, 134f. gas gland tissue, 107
Phosphatidylglycerol, 120f. 3-Phosphoglycerate kinase, 34
Phosphatidylinositol, 120f. binding to subcellular muscle structures, 292
fatty acid composition, 131 binding in turtle brain, 297
growth promotion, 145 glycolytic control, 81
in fish tissues, 129 Phosphoglycerides, see glycerophospholipids
in lipovitellin, 127 teminology, 121
Phosphatidylserine, 120f. Phosphoinositide cycle, 122, 142f., 146
in fish tissues, 129 phospholipase C, 138
in lipovitellin, 127 Phospholipase A2
Phosphatidylserine decarboxylase bile salts, 123
temperature effects, 135 calcium, 138
Phosphoadenosine eicosanoid metabolism, 138
luciferin regeneration, 442 hepatopancreas, 123
Phosphocreatine microsomes, 122
anaerobic glycolysis, 385 Phospholipase C, 146
exercise, 385 eicosanoid metabolism, 138
muscle recovery, 381 molecular forms, 143
resynthesis, 385 olfactory cilia, 122
Phosphocreatine/creatine ratio olfactory epithelium, 143
NMR, 43 phosphoinositide cycle, 142
Phosphoenolpyruvate Phospholipase D, 122
binding of lactate dehydrogenase, 300 Phospholipases
binding of pyruvate kinase, 300 intestine, 123
in muscle recovery, 383 microsomes, 122
Phosphoenolpyruvate carboxykinase, see PEPCK muscle, 122
Phosphofructokinase-1 olfactory cilia, 122
association with F-actin, 303 Phospholipids
binding in turtle brain, 297 in intertissue lipid exchange, 23
binding to F-actin, 299 mobilization in starvation, 402
binding to subcellular muscle structures, 292 plasma concentration at rest, 17
binding to troponin C, 299 seawater adaptation, 135
brain, 87 starvation, 56
enzyme binding, 295, 300 terminology, 121
gas gland tissue, 107, 113 Phosphorylation/dephosphorylation
glycolytic control, 81 acetyl-coenzyme A carboxylase, 404
heart, 83f. branched-chain alpha-ketoacid dehydrogenase,
insulin, 69 175
kidney, 78 glycogen phosphorylase, 68
kinetic effects of F-actin, 302 glycolytic flux, 292
liver, 70 phosphofructokinase-1, 68, 300
myosin ATPase, 304 pyruvate kinase, 68
phosphorylation, 68, 300 triacylglycerol lipase, 403, 419
polymerization, 300 Phosvitin, 127
red blood cells, 90 Photocytes
red muscle, 82 luminescence, 436, 438
regulation, 291 Photogenic cells, 453
Subject Index 505
Photogenic oxidation, 436 white muscle, 129

Photogenic tissue Plasmologenase, 122
capillary network, 438 Platelet-activating factor
filter, 438 synthesis in tissues, 143
innervation, 438 Platelet-activating factor acetylhydrolase
lens, 438 plasma, 143
reflectors, 438 Polyethylene glycol
Photophores, 436, 442 pyruvate kinase kinetics, 301
light emission, 436 Polyribosomes
luciferin storage, 453 muscle protein synthesis, 193
nasal, 443 Polyunsaturated fatty acids, 24
secretions, 438 Portal system
spectral characteristics, 436 transport of intestinal lipoproteins, 124
Photoprotein Positive allometry
binding of calcium, 443 spleen weight, 340
Photoreceptor Posterior intestine
membrane glycerophospholipids, 137 immunological role, 163
Phycobiliprotein protein absorption, 163
photoprotein, 445 Power output
Physical activity weight specific, 357
hematocrit, 340 Preluciferin
Physoclists, 102 in luciferin recylcing, 454
Physostomes, 102 Pressure adaptation
Pigeon gill Na+/K+-ATPase, 136
pectoralis muscle, 3 Prey
muscle morphometric data, 6 luminescence, 447
Pinocytosis Prey immobilization
lipoprotein uptake, 127 electric organ, 261
Pituitary Progesterone
growth hormone, 424 metabolism, 423
Plasma Prolactin
amino acids, 23 effects on plasma fatty acids, 25
elasmobranch starvation, 412 Proline
fasting, 409 brush border uptake, 227
essential amino acids, 161 elasmobranch muscle mitochondrial substrate,
fatty acids 244
effects of ACTH, 422 fasting, 407
effects of hormones, 419 muscle, 407
starvation, 419 oxidation in thermogenic tissue mitochon-
glucose dria, 251
elasmobranch, 247 transporter
pool, 379 correlation with diet, 165
lecithin: alcohol acyltransferase, 125 uptake, 231
lecithin: cholesterol acyltransferase, 125 brush border membrane vesicles, 231
lyso-glycerophospholipids, 123 dependence on diet, 230
non-essential amino acids, 161 effects of growth hormone, 233
platelet-activating factor acetylhydrolase, 143 effects of steroids, 233
postprandial amino acid pool, 167, 204 effects of thyroid hormones, 234
protein stimulation, 231
percent of whole animal protein, 213 Propranolol
starvation, 408 glycogen phosphorylase, 74
substrates pyruvate kinase activity, 74
list of resting concentrations, 17 Prostacyclin (PGI)
wax esters, 125 blood clotting, 142
Plasmalemma eicosanoid metabolism, 138f.
in electrocytes, 272 elasmobranch leukocytes, 139
Plasmalogens, 120 fatty acid precursors, 140
brain, 129 thrombocyte aggregation, 142
olfactory nerve, 129 Prostaglandins
506 Subject Index
Prostaglandins (continued) plasma concentration at rest, 17

D series pool
distribution, 139 red muscle, 369
fatty acid precursors, 140 retention
E-series muscle, 178
distribution, 139 retention efficiency
fatty acid precursors, 140 definition, 193
eicosanoid metabolism, 138f. sparing
F-series dietary lipid, 206
fatty acid precursors, 140 white muscle, 369
distribution, 139 Protein kinase
ovulation, 142 enzyme binding, 299
spawning activity, 142 exhausting exercise, 54
vasoactive properties, 142 Protein kinase C
Prostanoids, 137 phosphoinositide cycle, 143
vasoactive properties, 142 Protein loss, 192
synthesis, 139 Protein synthesis, 191ft., 197ff.
tissue distribution, 139 cortisol, 202
Protein cost, 191,206ff., 214
adipose tissue, 369 direct approach, 206f.
as endogenous fuel, 48 indirect approach, 207
catabolism daily balance - feeding, 197
nitrogen excretion, 22 daily balance - starving, 198
consumption rate dietary amino acids, 199
definition, 193 diurnal variation, 196
content of muscles, 50 effects of thyroid hormones, 425
degradation rate essential amino acids, 205
definition, 193 fasting, 407
resynthesis, 192 muscle, 56
degradation, 192f., 371 heat production, 207
anoxia, 53 hormonal stimulation, 199
cortisol, 202 insulin, 411
muscle, 178 3H.leucine, 196
pathways, 196 limits through amino acid transport, 167
protein recycling, 199 liver, 48
role of genetics, 201 mass synthesized per day, 192
scaling with body weight, 212 methionine-deficient diet, 206
starvation, 199 muscle, 178
extracellular fluid, 369 nutrition, 191
growth oxygen consumption, 191, 207
daily balance- feeding, 197 postprandial, 48, 205f.
daily balance - starving, 198 rate
growth rate definition, 193
definition, 193 retention efficiency, 192
imidazole pK value, 325 definition, 193
liver, 369 growth, 198
composition, 395f. protein synthesis, 198
metabolism ribosomal activity, 199
effects of cortisol, 422 role of environment, 201
muscle, 11 role of nutrition, 201
mobilization scaling with body weight, 212
glucocorticoids, 421 sewage sludge diet, 212
starvation, 405 stable isotopes, 196
muscle content, 395L starvation, 199, 407
nitrogen thyroid hormones, 76, 425
daily flux - schematic, 197 tritiated amino acids, 195
daily absorption, 197 vs. amino acid transport, 165
oxidation Protein synthetic scope, 210
exercise, 53 Protein turnover
Subject Index 507
Protein turnover (continued) D-amino acid oxidase, 166

definition, 192f. coelenterazine, 442
environmental influence, 196 histidine-related dipeptides, 314
fasting, 407 luciferin storage, 453
formulae, 193 Na+/glucose transporter, 224
general model, 197 relation to light organ, 439
genotype, 201 Pyruvate
measurements, 193 elasmobranch muscle mitochondrial substrate,
nutritional status, 192 244
parameters, 193 fatty acid oxidation, 384
rates in fish species, 210 in muscle, 383
sewage sludge diet, 212 oxidation in red blood cells, 162
starvation, 199 oxidation in thermogenic tissue mitochon-
Protein-nitrogen absorption rate dria, 251
definition, 193 Pyruvate carboxylase
Protein-nitrogen retention efficiency brain, 87
growth, 201 glycogenesis, 82
Proteolysis heart, 84
muscle, 410 in muscular glycogen synthesis, 383
starvation, 396, 405, 410 kidney, 78
Proton liver, 70
gas gland metabolism, 109 muscle, 81f.
hemoglobin oxygen affinity, 111 Pyruvate dehydrogenase
Proton buffering activation, 380
histidine-related dipeptides, 324 control by calcium, 252
Proton extrusion dephosphorylation during exercise, 380
gas gland, 112 regulation in muscle, 384
Proton gradient, 34 thermogenic tissue, 247
brown adipose tissue, 241 Pyruvate kinase, 34
gas gland, 112 association with F-actin, 303
heater mitochondria, 254 binding in turtle brain, 297
proton release, 112 binding to subcellular muscle structures, 292
Proton influx brain, 87
heater mitochondria, 254 correlation with lactate disappearance, 384
Proton leak effects of metabolites on enzyme binding, 300
elasmobranch thermal tissue, 250 equilibrium conditions, 383
heater organ heat production, 248 gas gland tissue, 107
Proton transfer glycogenesis, 82
gas gland schematic, 114 heart, 83f.
PUFA (poly-unsaturated fatty acids) hypoxia, 74
dioxygenase, 137 in muscular glycogen synthesis, 383
eicosanoid metabolism, 137f. insulin, 69
embryonic development, 144 kidney, 78
in diet, 133 kinetic effects of F-actin, 302
lipoproteins, 124, 126 liver, 70
phospholipids, 130f. negative allometry, 357
starvation, 56 propranolol, 74
temperature acclimation, 134 raven heart, 357
Puget Sound, 450 red blood cells, 90
Pulse-type fish red muscle, 82
electric organ discharge, 262 regulation, 68, 291
Pupfish reversal, 83, 383f.
metabolic rate, 343 scaling, 355
Purine nucleotide cycle, 386 starvation, 412
kidney, 174 temperature effects, 75
liver, 167 white muscle, 81, 384
muscle, 179 Pyruvate oxidation
Putter-yon Bertalanffy model, 191 elasmobranch muscle mitochondria, 247
Pyloric caeca tuna red muscle, 248
508 Subject Index
Pyruvate transport metabolism, 91

monocarboxylate carrier, 246 oxidation, 91
red blood cells, 90 permeability, 89
transport, 66, 89
Quantum yield hexosemonophosphate shunt, 90
luminescence, 455 intracellular pH, 91
luciferins, 441 catecholamines, 91
lactate production, 91
lactate transport, 66
Rab6p luciferin binding, 452
in electric organ, 273 mitochondria, 66, 90
Rat soleus muscle
oxidative capacity, 90
morphometric data, 6
oxygen consumption, 91
Rate of energy loss proton transfer schematic, 114
starvation, 405
swelling, 37, 91
Rays Red muscle
electric organs, 261
Re-feeding, 73f., 173, 393, 412f. acid lipase, 403
anabolic effects, 73 aerobic capacity, 250
insulin binding, 73 amino acids, 369
liver glycogen, 73 content, 50
ornithine decarboxylase, 173 exercise, 53
plasma hormones, 74 oxidation, 178, 371
plasma insulin, 412 anaerobic energy production, 370
Receptor-mediated endocytosis anserine in starvation, 318
apoproteins, 126 anserine transport, 324
Receptors asparagine synthetase, 178
apoproteins, 126 ATP turnover, 370, 374
glucagon, 72f., 419f. branched-chain a-ketoacid dehydrogenase, 175
luciferin, 458 buffering capacity, 326f.
Recovery, 20 burst power requirements, 357
burst exercise, 375 capillary manifolds, 5
fat metabolism, 384 capillary surface per fiber volume, 10
fatty acid oxidation, 384 carnosine in starvation, 318
glycogen dynamics, 381L citrate synthase, 250
glycogen repletion, 82 cytochrome c oxidase, 352
glycogen synthesis in muscle, 385 depletion, 407
glycolytic control, 81 elasmobranch, 377
lactate dynamics, 380f. fatty acids, 369
lipid oxidation, 381 oxidation, 80
metabolites, 380 fine structure, 4
oxygen debt, 75 free fatty acids, 48
rate, 386 glucose, 369
Rectal gland oxidation, 373
glycerophospholipid headgroup composition, utilization, 370
129 glutaminase, 244f.
phosphoinositide cycle, 142 glutamine oxidation, 248
Recycling glutamine synthetase, 178, 245
luciferin, 454 glycogen, 46, 67, 80, 369, 372
Red blood cell heat production, 243
alanine oxidation, 162 hexokinase, 247, 373f.
amino acids, 161 histidine in starvation, 318
amino acid concentration, 23 histidine turnover, 320
amino acid metabolism enzymes, 162 histidine uptake, 320
amino acid utilization, 162 ketone bodies, 377
band Ill anion exchanger, 114 lactate, 369
carbohydrate metabolism, 89f. accumulation, 80
carbonic anhydrase, 114 as substrate, 249
ethanolamine plasmalogens, 129 exchange, 20
glucose production, 370
Subject Index 509
Red muscle (continued) exercised muscle, 384

lipase, 422 Rest
lipid content, 48, 401 glycolytic enzyme binding, 292
mass, 373 Rete mirabile
mitochondria, 371 capillaries, 115
morphometry, 1 carbonic anhydrase, 113
oxidation rates, 377 gas gland, 1I0
oxidative capacity, 370 swimbladder, 102
oxygen consumption, 179, 370, 373 Retina
power output, 357 di-PUFA, 133
protein, 369 rod membrane lipids, 137
content, 50 Ribosomal activity
proteolysis, 405 protein synthesis, 199
provision of fat fuel, 376f. Ribosomal protein
scaling, 342 percent of whole animal protein, 213
sealing of power output, 358 Ribosomes
serine hydroxymethyl transferase, 178 gas gland cells, 103
structural design, lff. protein synthesis, 199
sustained swimming, 80 RNA Activity
triacylglycerol lipase, 48 definition, 193
triacylglycerols, 369 Roe
uptake of 2-deoxyglucose, 373 glycerophospholipid headgroup composition,
work output, 370 129
Red muscle (tuna) Root effect
alpha-glycerophosphate dehydrogenase, 249 gas gland, 111
fat content, 248 red blood cell, 114
glycogen, 248 rRNA production
hexokinase, 248 protein synthesis, 209
hydrogen shuttles, 249 RTG-2 cell line
lipid droplets, 248 cost of protein synthesis, 208
morphometrie data, 6 fractional protein synthesis rate vs. oxygen
palmitoylcarnitine oxidation, 248 consumption, 208
pyruvate oxidation, 248 glycerophospholipid fatty acids, 131
Redox balance
elasmobranch muscle mitochondria, 245 Sach's organ, 263
Reflectors Salinity
guanine, 438 lipid composition, 135
lens, 438 Salt gland (birds)
luminescent species, 436 phosphoinositide cycle, 143
Regulation Sampling
glycolytic enzymes, 291 artifacts, 36
Reptiles freezing rate, 38
distribution of histidine-related dipeptides, 312 freeze clamping, 38
lactate oxidation, 381 techniques, 35f.
muscle glycogen synthesis, 381 handling stress, 36f.
Resorbing bladder, 102 tissue extraction, 35
Respiration Sand goby
isometric scaling, 339 metabolic rate, 344
role of phosphate, 386 Sarcomere
surface area, 339 in electric organ, 270
white muscle, 384 in electrocytes, 261
Respiratory area length, 3, 7
scaling, 343 Sarcomeric actin, 271
scaling exponents, 343 Sarcoplasmic protein
Respiratory chain synthesis, 407
gas gland tissue, 105 starvation, 407
Respiratory control ratio Sarcoplasmic reticulum
elasmobranch muscle mitochondria, 247 calcium release during tissue extraction, 39
Respiratory quotient, 42 mobilization in starvation, 407
510 Subject Index
Sarcoplasmic reticulum (continued) red muscle, 178

sonic muscle, 282 Serine-pyruvate transaminase
thermogenic tissue, 252 kidney, 175
Sarcosine, 49 liver, 175
Scale cells Serum
cost of protein synthesis, 208f. amino acids in fasting, 160
fractional protein synthesis rate vs. oxygen Sewage sludge diet
consumption, 208 protein growth, 212
Scales protein turnover, 212
nitrogen loss, 198 Sex differences
Scaling electric organ discharge, 262, 266f.
aerobic metabolism, 336ff. sonic muscle, 279ff.
anaerobic metabolism, 354ff. sound duration, 280
blood volume, 339 sound frequency, 280
circulation time, 339 Sex steroids
contraction frequency, 344 influence on electric organ discharge, 266
growth rate, 344 Sexual dimorphism
heart size, 344f. electric organ discharge frequency, 267
hematocrit, 340 sonic nerve terminals, 281
repiration, 339 Signal transduction pathways, 77
spleen, 340 Single concentrating effect, 101, 110f.
stroke volume, 344 gas gland, 112
Scaling exponent, 336, 344 Skin
frequency distribution for standard metabolic cyclooxygenase, 138
rate, 336 glycerophospholipid headgroup composition,
sustained swimming, 344 129
weight-specific activity, 346 glycerophospholipid storage, 128
sprint swimming, 344 keratin, 272
Seasonality lipoxygenase products, 139
anoxia, 76 lipoxygenase specificity, 142
carbohydrate metabolism, 76 Slow muscle fibers, 337
hormone responses, 76 Slyke
hypoxia tolerance, 74 muscle buffering, 326L
liver glycogen, 76 Smooth muscle cells
Seawater secretory bladder, 102
adaptation Snakes
cortisol, 233 distribution of histidine-related dipeptides, 312
hydrogen peroxide, 459 Sneaker males
superoxide anion, 459 toadfish, 281
Secretory activity Social communication
photocytes, 438 electric organs, 261
Secretory bladder, 102 Social rank
Serine growth performance, 203
brush border uptake, 227 immunocompetence, 203
in glycerophospholipids, 120 stress response, 203
metabolism, 175 Solubility
liver, 168 swimbladder gas, 101
oxidation, 371 Somatostatin
kidney, 79 effects on plasma fatty acids, 25
transport Sonic motoneuron, 281
Na +-dependent, 161 Sonic motor pathway, 281
red blood cells, 161 Sonic muscle, 279ff.
Serine dehydratase aspartate aminotransferase, 285
kidney, 175 ATPase, 283
liver, 168, 175 bouton, 281
Serine-hydroxymethyl transferase Ca 2+ ATPase, 282
kidney, 175 calcium content, 282
liver, 168 citrate synthase, 284f.
liver, 175 fatigue resistance, 283
Subject Index 511
Sonic muscle (continued) storage of erythrocytes, 338

fiber typing, 283 Sprint swimming
glucose 6-phosphate dehydrogenase, 285 scaling exponent, 344
glycogen content, 283 Standard metabolic rate, 336
lactate dehydrogenase, 285 scaling, 344, 359
malate dehydrogenase, 285 negative allometry, 359
malic enzyme, 285 Starvation, 393ff.
mitochondrial density, 284 actin levels, 408
mitochondrial volume, 286 alanine aminotransferaase, 406
motor nerve, 280 alanine release, 410
myofibrils, 282 amino acid gluconeogenesis, 170
myosin light chains, 283 amino acids, 370, 409
NAD diaphorase, 283 aminotransferases, 171
neuromuscular junction, 281 ammonia excretion, 167, 171
relaxation, 280 aspartate aminotransferase, 406
sarcoplasmic reticulum, 282 body protein loss, 182
T-tubule system, 282 elasmobranch fish, 255, 411
vs. muscle mass, 284 enzyme activities, 406
Z-line, 282 epinephrine, 420
Sonic muscle weight glucagon/insulin ratio, 416f.
androgens, 287 glucocorticoids, 421
estradiol, 287 glucose synthesis from alanine, 182
estrogen, 287 glucose turnover, 374
hormonal control, 287 glutamate dehydrogenase, 171
keto-testosterone, 287 glycogen utilization, 55
testosterone, 287 glycolytic enzymes, 407
Sonic nerve terminal (bouton) growth hormone levels, 423
midshipman, 281 histidine-related dipeptides, 317f.
sexual dimorphism, 281 ketone bodies, 377
Sound production, 279ff. lipid depletion, 376, 400
Spawning activity lipoprotein composition, 125
eicosanoids, 142 luminescence, 461
Spawning migration mobilization of mitochondria, 407
alanine flux, 22 mobilization of sarcoplasmic reticulum, 407
alanine gluconeogenesis, 22, 55, 170 muscle amino acids, 178
anserine, 315 muscle carnosine, 316ft.
carboxypeptidase A, 407 muscle histidine, 178, 316ft.
cathepsin D, 407 myofibrillar protein, 407
corticoids, 423 nitrogen loss, 56
fatty acid composition, 402 ornithine decarboxylase, 172
histidine, 315 plasma branched-chain amino acids, 182
insulin, 414 plasma proteins,. 408
lactate gluconeogenesis, 55 protein degradation, 56, 199, 407
lipid changes, 402 protein mobilization, 405
muscle proteolysis, 170 protein synthesis, 199, 406f.
Species difference protein turnover, 199
hormonal response, 77 renal amino acid metabolism, 177
Spectral shifts triacylglycerol lipase, 419
photoproteins, 445 Steady-state conditions,
Sphingomyelin, 121 flux measurements, 17
Spleen lactate turnover, 20
amino acids in fasting, 160 Steady-state swimming, 374
contraction, 340 red muscle, 367
cyclooxygenase, 138 Steroidogenesis
ethanolamine plasmalogens, 129 A23187, 143
hemoglobin, 341 protein kinase C, 143
histidine-related dipeptides, 314 Steroids
platelet-activating factor synthesis, 143 non-genomic actions, 232
scaling, 340 regulation of nutrient transport, 231s 235
512 SubjectIndex
Stoichiometry secretory bladder, 102

intestinal transport, 223 sonic muscle, 279ff.
Stomach sound production, 279ff.
cyclooxygenase, 138 tissue layers, 103
luciferin, 451 volume, 101
Storage Swimming metabolism, 371
luciferin, 452f. Symbionts
Stress bioluminescence, 438
carbohydrate metabolism, 76 Symmorphosis, 339ff., 345
hematocrit, 340 Sympathetic system
social rank, 203 photogenic tissue innervation, 438
subordinate fish, 202 Synaptic membranes
Stroke volume, 344 electric organ, 269f.
hypoxia, 50 Synaptosomes
Structural lipids enzyme association, 296
lipids, 402
starvation, 400 T-tubule system
Stunting, 346 in electrocytes, 261
Subordinate fish sonic muscle, 282
feeding, 201 Tail beat frequency, 337
growth, 201 Taurine
Subsarcolemmal mitochondria intestinal transport, 226
vs. muscle fiber size, 9 muscle buffering, 328
Substrate flux, 15 red blood cells, 161
Substrate transfer renal secretion, 173
muscle, 11 Teleosts
Subunits distribution of histidine-related dipeptides, 312
AChR, 269 muscle buffering capacity, 326
Succinate Temperature
from aspartate, 53 acclimation
hypoxia, 52 gluconeogenesis, 75
Succinate dehydrogenase protein synthesis, 76
scaling, 346 Q10, 75
thermogenic tissue, 247 enzyme activities, 75
Succinyl-CoA synthetase fiber volume, 83
thermogenic tissue, 247 heart performance, 85
Sucrase hypoxia, 83
intestine, 223 Krebs cycle, 86
Sulfonamide lipid metabolism, 134f.
carbonic anhydrase, 113 membrane composition, 134
Superoxide anion muscle recruitment, 83
in luminescence, 459 myocyte response, 86
in seawater, 459 phospholipid structure, 134
Superoxide dismutase swimming performance, 83
gas gland, 115 Testes
Sustained swimming, 80, 375 cyclooxygenase, 138
red muscle, 80 prostanoid synthesis, 139
scaling exponents, 3 ~ protein kinase C, 143
Swim tunnel, 378 steroidogenesis, 143
Swimbladder, see also gas gland Testosterone
acidification, 111 effect on electric organ discharge, 267
blood pH, 111 metabolism, 423
counter-current exchange, 102 sonic muscle, 287
gas partial pressure, 101 TF cell line
metabolism, 101ft. glycerophospholipid fatty acids, 131
origin, 102 Thermal compensation
perfusion gluconeogenesis, 75
catecholamines, 112 hepatocytes, 75
resorbing bladder, 102 Thermal tolerance
Subject Index 513
Thermal tolerance (continued) sonic muscle, 279ff., 357

locomotion, 83 sound production, 279ff.
Thermogenesis Torpor, 75
thyroid hormone, 425 Transaldolase
Thermogenic mechanisms, 242 gas gland tissue, 108, 113
Thermogenic tissue, 250ff. Transdeamination
ot-glycerophosphate oxidation, 251 liver, 371
amino acid oxidation, 251 Transketolase
carbohydrate oxidation, 251 gas gland tissue, 108, 113
contractile apparatus, 250 Transport
enzyme activities, 251 amino acids, 162
glycerol kinase, 252 carrier-mediated, 222
glycogen, 251 chloride-dependent, 222
hexokinase, 251 diffusion, 222
ketone bodies vs. glutamine, 247 epithelia, 222
ketone body oxidation, 251 glucose, 223
metabolic organization - schematic, 254 intestine, 221
mitochondrial respiration, 252 ionic requirements, 222
myosin ATPase, 250 luciferin, 452
proton leak, 251 monosaccharides, 223
sarcoplasmic reticulum, 252 Na+-dependent, 222
substrate oxidation, 251 non-mediated, 222
swordfish brain, 251 paracellular, 223
thermogenin, 251 phlorizin-inhibitable, 223
Thermogenin Transporter density, 231
brown adipose tissue, 241 Triacylglycerol lipase
swordfish thermogenic tissue, 251 phosphorylation, 403, 419
Threshold red muscle, 48
electrocytes, 263 starvation, 419
Thrombocytes Triacylglycerols, 369
A23187, 140f. adipose tissue, 369
aggregation embryonic development, 144
eicosanoids, 142 extracellular fluid, 369
cyclooxygenase, 138 in intertissue lipid exchange, 23
lipoxygenase, 141 liver, 369
prostaglandin synthesis, 140 mobilization during exercise, 376
Thromboxane plasma concentration at rest, 17
distribution, 139 red muscle, 369
eicosanoid metabolism, 138f. starvation, 401
fatty acid precursors, 140 stores, 375, 377
thrombocyte aggregation, 142 white muscle, 369
Thyroid hormones Tricarboxylic acid cycle, see also Krebs cycle
conversion of 3"4 to T3, 426 gas gland metabolism, 109
effects on plasma fatty acids, 25 3,5,Y-Triiodo-L-thyronine see thyroid hormones
metabolic effects, 425 Trimethylamine
mitochondrial respiration, 255 muscle, 49
protein synthesis, 76 Triosephosphate isomerase
receptors, 426 association with F-actin, 303
regulation of nutrient transport, 231, 233 Triton X-100
role in thermogenic tissues, 255 detergent solubilization technique, 296f.
starvation, 425f. tRNA
stimulation of nutrient uptake, 233 protein synthesis, 209
thermogenesis, 242, 425 Trophic transfer
Tissue protein synthesis, 192 luciferin, 446
Tissue extraction Tropomyosin
metabolites, 38f. in electrocytes, 270
temperature effects, 39 Troponin C
Tissue respiration, 346 phosphofructokinase binding, 299
Toadfish Tryptophan
514 Subject Index
Tryptophan (continued) density, 127

red blood cell transport, 162 lipid composition, 127
Tubulin migration, 408
enzyme binding, 292 receptor-mediated micropinocytosis, 127
Tunas starvation, 408
thermogenic tissue, 242 transport, 127
Turbulent flow uptake, 127
power requirements for swimming, 356 VLDL (very low density lipoproteins)
Turtle average composition, 25
glycolytic enzyme binding in brain, 297 composition, 124
Tween 80 estrogen, 127
leucine transport, 232 hepatic synthesis, 125
"l~vitch contraction time, 337 hydrolysis, 126
"l~rosine in intertissue lipid exchange, 23
red blood cell transport, 162 lipid transport, 124
"I~osine kinase receptor-mediated endocytosis, 127
insulin receptor, 415 uptake by oocytes, 127
Vocalization
Ucrit, 179, 370ff. frequency, 280
lactate turnover, 375 toadfish, 280
red muscle glucose utilization, 373 Voltage-clamp
Ultrastructure electrocytes, 268
vertebrate muscles, 6 Volume of mitochondria
Urea muscle fiber, 3
excretion, 405 yon Bertalanffy growth equation, 211, 343
nitrogen loss, 198
Urotensin II Warburg
effects on plasma fatty acids, 25 gas gland metabolism, 105
Water
Vagus liver content, 395f.
gas deposition, 112 muscle content, 395f.
Valine Wave-type fish
brush border uptake, 227 electric organ discharge, 262
effect on branched-chain amino acid amino- Waveform
transferase, 177 electric organ discharge, 262
metabolic fate, 175 Wax ester
starvation, 409 plasma, 125
Van Slyke, 326 Weakfish
Vargula luciferin, see also luciferin sonic muscle, 281
derivatives, 442 Weight gain
distribution in fishes, 441 protein turnover, 192
structure, 440 Weight, see scaling
Vascular heat exchangers, 242 Weight-specific activity
Vasoactive peptides scaling exponents, 346
gluconeogenesis, 68 Whales
glycogenolysis, 68 histidine-related dipeptides, 313
Vasotocin White muscle
gluconeogenesis, 68 aerobic capacity, 379
glycogenolysis, 68 amino acids, 315ff., 369
signai transduction, 77 content, 50
Vertical migration, 101 exercise, 53
Viscera oxidation, 371
cytochrome c oxidase, 352 anaerobic energy production, 370
triacylglycerol mobilization, 375 anserine in starvation, 318
Visceral fat, 47 anserine transport, 324
storage for triacylglycerols, 399f. aspartate aminotransferase, 285
supply of free fatty acids, 377 ATP utilization, 370
Vitellogenesis, 127 buffering capacity, 356
ViteUogenin burst swimming, 80
Subject Index 515
White muscle (continued) malic enzyme, 285

capillary surface per fiber volume, 10 mitochondria, 371
carnosine in starvation, 318 density, 284
citrate synthase, 284f., 354 volume density, 379
cytochrome c oxidase, 352 myofibril space, 379
depletion, 407 oxygen consumption, 370
fatty acids, 369 phenylalanine specific activity, 195
fraction rate of protein synthesis, 204f. plasmalogens, 129
fractional protein synthesis rate, 213 protein, 369
free phenylalanine, 195 content, 50
gas gland, 106 synthesis vs. amino acid pool, 204f.
glucose 6-phosphate dehydrogenase, 285 proteolysis, 405
glucose, 369 anoxia, 53
utilization, 370 release of lactate, 374
glycogen content, 46, 67, 80, 369, 371f., 374 scaling of protein metabolism, 212
histidine, 315ff. transport of deoxyglucose, 382
content, 312f. triacylglycerols, 369
starvation, 318 work output, 370
turnover, 320 White muscle- tuna
uptake, 320 morphometric data, 6
washout, 319 Work output
histidine-related dipeptides, 312f. red muscle, 370
lactate, 369, 371 white muscle, 370
accumulation, 80, 249
dehydrogenase, 285, 354 Xenopus oocytes
exchange, 19, 20 injection of AChR mRNA, 270
production, 370
retention, 19 Z bands
lipid content, 401 electrocytes, 272
malate dehydrogenase, 285 sonic muscle, 282

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Preface

D.E Houlihan, Department of Zoology, University of Aberdeen, Aberdeen, TiUydrone

Jean-Michel Weber, Biology Department, University of Ottawa, 30 Marie Curie,

AA Amino acid(s) HK Hexokinase

Design for a high speed path for oxygen:

ODILE MATHIEU-COSTELLO, RICHARD W. ]]RILL * AND

II. Materials and methods

Sarcomere length was measured on longitudinal sections, after careful control

III. Results and discussion

birds. Interestingly, however, capillary manifolds were found in flight muscle of

1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8

to capillary length by tortuosity and branching, against sarcomere length in the

9O "Tuna white muscle

Acknowledgements. Supported by Grant 5PO1 HL-17731 from the National

Circulatory substrate fluxes and their

JEAN-MICHEL WEBER AND GEORGES ZWINGELSTEIN *

Multicellular life can only be sustained if selected metabolic fuels, end-products,

summarized in Table 1. The secondary circulation47.119 is not discussed separately

II. Why and how to measure metabolite fluxes in vivo ?

Resting concentrations of major plasma substrates in fish

Metabolite g 1-1 /zmol m1-1

III. Basic regulatory mechanisms

IV. Lactate fluxes

Lactate turnover rate in post-absorptive teleosts

Species Mass Rt Predicted Rt for Predicted mammal Rt/

seven-fold at an environmental p O 2 of 4 kPa (ref. 29). Exercise studies have been

Glucose turnover rate (Rt) in resting, post-absorptive teleosts

VI. A m i n o acid fluxes

Finally, no amino acid flux measurement should be attempted in fish without

I~I. Lipid fluxes

T h e effect of exercise on p l a s m a FFA fluxes should be very different b e t w e e n

Species Liver Muscle

Hormone Effect on plasma FFA

Species Lipoproteins VLDL LDL HDL

Proteins Phospho- Free ..... Cholesteryl Triacyl-

Fisheries Ministry Report No. 42 (Oslo), 1959.

metabolites. Physiol. Rev. 63: 606-667, 1983.

Biochem. PhysioL 50B: 425-430, 1975.

Endogenous fuels; non-invasive v e r s u s

GUIDO VAN DEN THILLART AND MARCEL VAN RAAIJ

II. Q u a n t i f y i n g e n d o g e n o u s fuels: destructive m e t h o d s

interference. It is therefore crucial for the interpretation of the data to know

Muscle lactate under 'resting' conditions

Species Lactate lmmobilisation Sampling

inspection. So the animals have to be anesthetized and/or killed quickly in order

To acquire a piece of tissue, it must be excised, which obviously causes damage to

-15~ A low temperature is necessary to minimize enzyme activities during the

111. Non-destructive approaches

For cannulation, an indwelling catheter is usually inserted in the dorsal aorta

According to the laws of thermodynamics, all the available energy liberated by

3. Nuclear magnetic resonance spectroscopy

IV. Storage of endogenousfuels

Species Liver White muscle Red muscle

compared to normoxic individuals although liver glycogen is not affected, possi-

4. Proteins and amino acids

I4. Mobilization of endogenous fuels

Species Muscle type Protein T ot al amino acids

Chum salmon White 4 15-20 -

Rainbow trout White ~ 19 -

See text for species names.