205

Insulin restores GH responsiveness during lactation-induced

negative energy balance in dairy cattle: effects on expression of
IGF-I and GH receptor 1A
S T Butler, A L Marr, S H Pelton, R P Radcliff1, M C Lucy1
and W R Butler
Department of Animal Science, Cornell University, Ithaca, New York 14853, USA
1
Department of Animal Science, University of Missouri, Columbia 65211, Missouri, USA
(Requests for offprints should be addressed to W R Butler; Email: [email protected])

Abstract
Early lactation in dairy cattle is a period of severe negative in INS compared with CTL cows (2·330·14 vs
energy balance (NEB) characterized by reduced blood 0·270·14 ng/ml (S.E.M.); P<0·001) while blood glucose
glucose and insulin concentrations and elevated blood GH concentrations were not different between treatments
concentrations. The liver is refractory to GH during NEB (45·32·2 vs 42·52·2 mg/dl; P>0·1). Plasma IGF-I
and this uncoupling of the GH–IGF axis results in increased continuously during the insulin infusion, and
diminished plasma concentrations of IGF-I. Our objec- reached the highest concentrations at the end of the clamp,
tives were to examine the effects of insulin administration being almost 4-fold higher in INS compared with CTL
during the immediate postpartum period on plasma IGF-I cows (1174 vs 304 ng/ml; P<0·001). Hepatic
and GH concentrations and to examine the hepatic expression of GHR 1A and IGF-I mRNA was low in
expression of total GH receptors (all GH receptor tran- CTL cows, but was increased 3·6-fold (P<0·05) and
scripts), GH receptor 1A (GHR 1A) and IGF-I. In 6·3-fold (P<0·001) respectively in INS cows. By contrast,
addition, we examined adipose tissue for total GH receptor in adipose tissue the changes in gene expression in
and IGF-I mRNA levels to establish the effects of chronic response to insulin were reversed with decreases in both
hyperinsulinemia on an insulin-responsive peripheral total GHR and IGF-I mRNA. The expressions of GHR
tissue. Holstein cows (n=14) were subjected to either 1A and IGF-I mRNA in liver tissue were correlated in
a hyperinsulinemic–euglycemic clamp (insulin; INS) or INS (r=0·86; P<0·05), but not CTL cows (r=0·43;
saline infusion (control; CTL) for 96 h starting on day P>0·1). Insulin appears to be a key metabolic signal in
10 postpartum. Insulin was infused i.v. (1 µg/kg body coupling the GH–IGF axis, thus orchestrating a marked
weight per h), blood samples were collected hourly, and elevation in circulating IGF-I concentrations.
euglycemia was maintained by infusion of glucose. Insulin Journal of Endocrinology (2003) 176, 205–217
concentrations during the infusions were increased 8-fold

Introduction intake is increasing, but energy balance (EB) remains

negative due to the energetic costs of rising milk produc-
Circulating insulin-like growth factor-I (IGF-I) is pro- tion. During this time the liver becomes refractory to GH
duced primarily by the liver in response to growth (Vicini et al. 1991) and circulating IGF-I concentrations
hormone (GH) ( Jones & Clemmons 1995) and this rela- are dramatically reduced. The decline in circulating IGF-I
tionship forms the basis of the GH–IGF axis. GH receptors begins 2 weeks prior to parturition and is paralleled by a
(GHRs) are found in many tissues and the liver is the site decline in plasma insulin. Changes in plasma GH over the
of greatest abundance (Bornfeldt et al. 1989, Brameld et al. same period are opposite to that of IGF-I and insulin (Bell
1996, Edens & Talamentes 1998, Lucy et al. 1998). et al. 2000). The inability of GH to stimulate hepatic
Expression of the GHR and IGF-I genes in liver is acutely IGF-I production during periods of NEB is termed ‘GH
responsive to nutritional status (Bornfeldt et al. 1989, Pell resistance’ (Donaghy & Baxter 1996) and has been docu-
et al. 1993) and physiological state (Kobayashi et al. 1999a). mented in many species. It has been well established that
The early lactation period in dairy cattle is characterized hepatic GHR concentration is positively correlated with
by prolonged negative energy balance (NEB) where feed plasma IGF-I and the level of nutrition. Thus GHR

Journal of Endocrinology (2003) 176, 205–217 Online version via http://www.endocrinology.org

0022–0795/03/0176–205  2003 Society for Endocrinology Printed in Great Britain
206 S T BUTLER and others · Lactation-induced negative energy balance in cattle: insulin restores GH responsiveness

concentration may be a major regulatory mechanism for for a 96 h period starting at 1500 h on day 10 and finishing
determining activity within the GH–IGF axis (Breier at 1500 h on day 14. As a prophylactic measure, animals
1999). were treated with penicillin G procaine during the treat-
The hypoinsulinemia of early lactation is part of a series ment period (9106 units/day; The Butler Company,
of coordinated changes that occur around the time of Columbus, OH, USA). For the INS group, the target
parturition in support of lactation. Low plasma insulin glycemia for each cow (10%) was the average baseline
levels reduce glucose uptake by insulin-responsive periph- blood glucose concentration for that individual cow. The
eral tissues (adipose and muscle) and facilitate greater insulin infusate was prepared for each cow by dissolving
uptake of glucose by the mammary gland (Bauman & purified bovine pancreatic insulin (I-5500, lot 109H0967,
Elliot 1983), a tissue that is not insulin-responsive. Thus, it 28·3 USP units/mg; Sigma, St Louis, MO, USA) in
is not surprising that genetic advances for milk production 0·01 M HCl, followed by dilution with sterile saline
have resulted in lower levels of circulating insulin in containing plasma (1·25%) from that cow. The insulin
Holstein cows (Bonczek et al. 1988). As insulin is a solution prepared for each cow was calculated to provide
key signal of metabolic status, we hypothesized that an infusion rate of 1 µg/kg BW per h, and was infused via
the hypoinsulinemia associated with early lactation was a syringe pump (model SE 400; Vial Medical, Grenoble,
responsible for the specific down-regulation of GHR 1A France). During the insulin infusion, euglycemia was
in liver and that this in turn was responsible for maintained by infusion of glucose (50% w/v dextrose
the uncoupling of the GH–IGF axis. Here we describe solution; The Butler Company) from sterile bottles at
the effects of a hyperinsulinemic–euglycemic clamp in variable rates using a peristaltic pump (Micro/Macro Plum
lactating cows during the NEB of early lactation. An XL; Abbott Laboratories, Morgan Hills, CA, USA). Blood
elevation of plasma insulin within the physiological range samples were collected hourly during the 96 h infusion
increased hepatic expression of GHR 1A and IGF-I period. Blood glucose concentrations were deter-
mRNA resulting in a marked increase in plasma IGF-I mined immediately (SureStep Blood Glucose Monitoring
levels. System; Lifescan Inc., Milpitas, CA, USA) and the
infusion rate of glucose was adjusted if necessary. For CTL
animals, sterile saline was infused at a rate of 100 ml/h for
Materials and Methods 96 h, and blood glucose was measured every 4 h.
Animals and treatment
EB determination
All experimental procedures were approved by the
Cornell University Institutional Animal Care and Use EB was calculated daily from measurements of milk yield
Committee. Beginning on day 8 postpartum, 14 mature and dry matter intake (DMI), BW (weekly measurement),
lactating Holstein cows (61015 kg ( S.E.) body weight milk-fat percentage (twice-weekly measurement) and the
(BW)) were provided with free access to a total mixed calculated NEl value of the diet (Beam & Butler 1997).
ration formulated to have a net energy for lactation (NEl) Daily net energy consumed (NEconsumed) was increased for
typical for animals in this stage of lactation (1·63 Mcal the animals on the INS treatment by a variable amount
NEl/kg). Feed was offered every 2 h to minimize post- depending on the infusion rate of glucose required to
prandial variations in nutrient supply and water was freely maintain euglycemia. The estimated NEl value of glucose
available at all times. Daily samples of the feed offered was 3·66 Mcal/kg glucose (Léonard & Block 1997). The
were composited on a monthly basis for nutrient analysis estimated glucose NEl was multiplied by the kilograms of
(Dairy One Cooperative, Ithaca, NY, USA). Feed refusals glucose infused per day and added to the NEconsumed.
were weighed and discarded at 1200 h each day. Cows
were milked at 0600 and 1800 h daily, milk yield deter-
Plasma measurements
mined, and milk samples were collected for compositional
analysis twice weekly (Dairy One Cooperative). Plasma was collected and stored at
20 C four times daily
On day 8 postpartum, cows were randomly assigned to during days 8 and 9 (baseline period) and every 2 h during
either a hyperinsulinemic–euglycemic clamp (insulin; the 96 h infusion period. The samples were subsequently
INS) or saline infusion (control; CTL) treatment (n=7/ assayed for insulin, GH, IGF-I, free IGF-I and non-
treatment). For both treatments, three indwelling jugular esterified fatty acids (NEFA). Insulin concentrations were
catheters were inserted (Tygon Microbore Tubing; quantified daily on days 8 and 9 and every 2 h during
Norton Performance Plastic, Akron, OH, USA); two the 96 h infusion period by a double-antibody RIA (Linco
catheters on one side were used for infusion of solutions Research Inc., St Louis, MO, USA) as described
(insulin and glucose or saline) and the catheter on the (McGuire et al. 1995a). Plasma GH and IGF-I were
contralateral side was used to collect blood samples. measured daily on days 8 and 9, and every 6 h during the
Baseline measurements (four blood samples/day) were infusion period. Plasma GH was measured by RIA as
taken on days 8 and 9, and the treatments were imposed described previously (Rosemberg et al. 1989) with the
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 Lactation-induced negative energy balance in cattle: insulin restores GH responsiveness · S T BUTLER and others 207

exception that the bovine GH for iodination and standards Butler Company). An incision of approximately 1 cm was
was obtained from Pharmacia Animal Health (Kalamazoo, made through the skin and the biopsy instrument was used
MI, USA; lot 12, code 77-001). Plasma IGF-I concen- to pierce the intercostal muscles and peritoneum. The
trations were quantified by RIA following acid–ethanol liver was located and a 1–1·5 g sample was removed. The
extraction as previously described (Rosemberg et al. 1989) sample was immediately snap-frozen in liquid nitrogen
with the exception that IGF-I for iodination and standards and stored at –80 C. The incision site was sutured
was obtained from Monsanto Co. (St Louis, MO, USA; lot (Ethicon Inc., Somerville, NJ, USA), and treated topically
GTS-2) and the rabbit anti-human IGF-I (AFP4892898) with Scarlet Oil (Vedco Inc., St Joseph, MO, USA).
was obtained through the US National Hormone and Adipose tissue was collected essentially as described by
Peptide Program (Dr A F Parlow, Scientific Director). Houseknecht et al. (1995). Briefly, s.c. adipose tissue was
Free IGF-I (i.e. readily dissociable) was determined for collected by surgical biopsy from the tailhead region. The
plasma samples taken at 0, 48 and 96 h relative to the start area was shaved, and sanitized with Betadine and 70%
of infusions in a single assay using a commercially available ethanol. Lidocaine was administered around the biopsy
sandwich-type IRMA (DSL Inc., Webster, TX, USA) site. A 4–6 cm incision was made and s.c. adipose tissue
developed for measuring free IGF-I in human serum but removed (2–4 g), snap frozen in liquid nitrogen and stored
also validated for use in bovine fluids (Beg et al. 2001). at
80 C. The incision site was sutured, and treated
Plasma NEFA concentrations were quantified daily on topically with Scarlet Oil.
days 8 and 9, and every 12 h during the infusion period Total cellular RNA was isolated using the guanidinium
using an enzymatic assay (Wako Pure Chemical Industries thiocyanate–phenol–chloroform extraction procedure
Ltd, Osaka, Japan). (Chomczynski & Sacchi 1987) with modifications for
adipose tissue (Harris et al. 1993). The RNA samples were
dissolved in sterile water and concentrations determined
Western ligand blot of IGF-binding proteins (IGFBPs) by spectrophotometry at 260 nm (1 optical density
Plasma samples collected at 0, 48 and 96 h relative to the unit=40 µg RNA/ml). Integrity of the RNA was deter-
start of infusions were subjected to Western ligand blotting mined by staining a sample of denatured total RNA
as described by McGuire et al. (1995b) to evaluate the (15 µg) with ethidium bromide, followed by electro-
temporal changes in IGFBPs during the infusion period. phoresis on a 1·2% agarose-formaldehyde gel. Total RNA
Plasma proteins were denatured in loading buffer (13·3% extracts with intact 28S and 18S ribosomal RNA bands
SDS, 0·42 M Tris, 0·013% bromophenol blue, pH 6·5) were used in subsequent real-time RT-PCR assays.
at 100 C for 3 min and separated using discontinuous
SDS-PAGE at 175 V (double gel unit; Life Technologies
Real-time RT-PCR
Inc., Gaithersburg, MD, USA). Proteins were transferred
to a nitrocellulose membrane overnight at 10 V using The amounts of total GHR (GHRtot), GHR 1A, IGF-I
a plate electrode apparatus (BioRad Laboratories Inc., and cyclophilin mRNA in liver tissue were analyzed using
Hercules, CA, USA). Membranes were incubated for 16 h real-time RT-PCR. In adipose tissue, GHRtot, IGF-I and
with 125I-IGF-I, washed, and placed on a phosphoimager cyclophilin mRNA were quantified, but GHR 1A
screen for 48 h. Band intensities were quantified using mRNA was not measured because GHR 1A is a liver-
a Bio-Imaging Analyzer BAS 1000 (Fuji Photo Film specific transcript (Lucy et al. 1998). Total RNA (2·5 µg)
Co. Ltd, Tokyo, Japan). Abundance of the IGFBPs is was reverse transcribed using SuperScript First
expressed in arbitrary units. IGFBP-3 and IGFBP-2 were Strand Synthesis System for RT-PCR (Gibco BRL,
identified on the basis of molecular mass using the findings Gaithersburg, MD, USA). Probe and primer sets for
from other similar studies on dairy cows that identified bovine GHRtot, GHR 1A, IGF-I and cyclophilin
these proteins by immunoblot and found them to have a were designed using Primer Express Software (Applied
molecular mass and responsiveness to elevated insulin Biosystems, Foster City, CA, USA). The primer
similar to that which we observed (McGuire et al. 1995b, sequences were as follows: GHRtot forward, 5 -GGTA
Mashek et al. 2001). TGGATCTCTGGCAGCTG-3 ; GHRtot reverse, 5 -
CTCTGACAAGGAAAGCTGGTGTG-3 ; GHR 1A
forward, 5 -CCAGCCTCTGTTTCAGGAGTGT-3 ;
Tissue collection and RNA isolation GHR 1A reverse, 5 -TGCCACTGCCAAGGTCAAC-
Liver and adipose tissues were collected prior to termin- 3 ; IGF-I forward, 5 -TTGGTGGATGCTCTCCAG
ation of treatments after 96 h of infusion. The liver biopsy TTC-3 ; IGF-I reverse, 5 -GCACTCATCCACGATT
was carried out as described by Smith et al. (1997). Briefly, CCTGT-3 ; cyclophilin forward, 5 -CACCGTGTT
a biopsy site between the 11th and 12th ribs was shaved, CTTCGACATCG-3 ; and cyclophilin reverse, 5 -
sanitized with 7·5% povidone-iodine (Betadine; Purdue ACAGCTCAAAAGAGACGCGG-3 .
Frederick Co., Norwalk, CT, USA) and 70% ethanol, and The probes for each gene were purchased pre-labeled
anesthetized with lidocaine (lidocaine HCl, 2%; The with a 5 reporter dye (FAM (6-carboxy-fluorescein) or
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208 S T BUTLER and others · Lactation-induced negative energy balance in cattle: insulin restores GH responsiveness

VIC) and 3 quencher dye (TAMRA (6-carboxy- in CTL cows during the experimental period (2·330·14
tetramethyl-rhodamine)). Their sequences were as fol- vs 0·270·14 ng/ml; treatment P<0·001; treatment by
lows: GHRtot, 5 -6 FAM-TGGCAGGCTCCAGTGAT time, P<0·001; Fig. 1). Blood glucose concentrations,
GCTTTTTCT-TAMRA-3 ; GHR 1A, 5 -6 FAM-TC however, were not different between INS and CTL cows
CATACCTGTAGGACCAAGAGTCCAGCA-TAMR over the 96 h period (45·32·2 vs 42·52·2 mg/dl;
A-3 ; IGF-I, 5 -6 FAM-CTCGAGCAGTCGGAGGG P>0·1), and were maintained within10% of baseline
CGC-TAMRA-3 ; and cyclophilin, 5 -VIC-TGTCGAC values. The glucose infusion began 10 min after starting
GGCGAGCCCTTGG-TAMRA-3 . The probe for the insulin infusion at an initial infusion rate of 70 g/h.
GHR 1A spanned an intron splice site. Twenty-five The infusion rate necessary to maintain euglycemia gradu-
microliter reactions were prepared as recommended by the ally increased over the first 24 h, and remained relatively
manufacturer, using 100 nM probe and 500 nM primers constant thereafter at an average rate of 1641·4 g/h for
and the Taqman Universal PCR Master Mix (Applied the remaining 72 h.
Biosystems). Standards (high and low), water (no template Plasma IGF-I concentrations were increased by insulin
control) or samples were added to the reaction mixture. treatment (treatment, P<0·001; treatment by time,
The PCR amplifications and fluorescent detection P<0·001; Fig. 2). Concentrations were similar during the
were performed in triplicate using the ABI Prism baseline period (P>0·05), but started to increase in INS
7700 Sequence Detection System (Applied Biosystems). cows after infusions commenced. The IGF-I concen-
Real-time fluorescence measurements were taken and a trations were greater by 24 h and continued to rise until
threshold cycle (CT) value for each sample was calculated the end of the infusion, at which point IGF-I was 4-fold
by determining the point at which the fluorescence higher in INS compared with CTL cows (1174 vs
exceeded a threshold limit (10 times the standard deviation 304 ng/ml). Plasma free IGF-I concentrations were not
of the baseline). The CT value for each sample was different (0·590·017 vs 0·580·017 ng/ml; P>0·05)
proportional to the starting copy number in the PCR between treatment groups during the baseline period.
reaction (Heid et al. 1996). Sequence detection software During the infusion, free IGF-I was elevated in INS cows
(Applied Biosystems) was used to analyze the amplification compared with CTL cows (treatment by time, P<0·001),
plots. The CT for cyclophilin was used to standardize the with peak values at 48 h (0·770·017 vs 0·58
amount of sample RNA in the reaction, allowing the 0·017 ng/ml; P<0·05), and remained higher in INS
relative amounts of GHRtot, GHR 1A and IGF-I mRNA compared with CTL cows at 96 h (0·650·017 vs
to be calculated for each sample. 0·580·017 ng/ml; P<0·05).
Plasma GH concentrations were not different between
the treatments during the baseline period, but declined
Statistical analysis in both treatments over time (P<0·001). A trend was
Statistical analyses were performed using the statistical observed for GH to be lower in INS cows than in CTL
software package NCSS 2000 (NCSS, Kaysville, UT, cows (4·70·5 vs 5·90·5 ng/ml; treatment, P=0·11).
USA). The average CT for the GHRtot, GHR 1A and In INS cows, GH declined to its lowest levels by 36 h after
IGF-I genes were normalized to the average CT for the start of infusion and remained relatively constant
cyclophilin for each individual animal. The gene expres- thereafter. The CTL cows showed a more gradual decline
sion data was log transformed to generate equal variances in GH over the infusion period, eventually reaching a final
between treatments and subjected to one-way ANOVA. concentration similar to INS cows (Fig. 3).
All other parameters were analyzed using repeated- A representative Western ligand blot of the temporal
measures ANOVA with treatment and time as fixed profile of IGFBP in plasma is depicted in Fig. 4. The
factors nested within animal. Time included values during abundance of the binding proteins is expressed in arbitrary
the baseline and infusion periods (
48 to 96 h). Values units. The amount of IGFBP-3 in plasma, observed as a
for GH and free IGF-I were log transformed prior to 40–44 kDa doublet, was not different between INS and
analysis to generate a normal distribution. When the CTL cows at the start of treatment (P>0·05), but was
interaction between treatment and time was significant significantly increased during insulin treatment (P<0·005).
(P<0·05), pairwise comparisons of individual means were Plasma levels of IGFBP-3 were increased in INS com-
carried out using the Tukey–Kramer test. pared with CTL cows at 48 h (2581180 vs 1815180;
P<0·07) and 96 h (2726180 vs 1939180; P<0·06).
The amount of IGFBP-2 (34 kDa band) was similar for
INS and CTL cows at 0 h (P>0·05), but was decreased
Results during insulin infusion by 76% (23687 vs 96487;
P<0·001) and 83% (13287 vs 79587; P<0·001) at
Plasma measurements 48 and 96 h respectively (treatment, P<0·001; treatment
Plasma insulin concentrations (means S.E.M.) were by time, P<0·005). Two additional minor bands were
elevated approximately 8-fold in INS cows over the values observed to migrate with apparent molecular masses of
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 Lactation-induced negative energy balance in cattle: insulin restores GH responsiveness · S T BUTLER and others 209

Figure 1 Mean plasma insulin and blood glucose concentration during the 48 h baseline
and 96 h (solid bar) infusion periods. Upper panel: in INS cows, insulin infusion
commenced at time 0 and continued for 96 h, whereas CTL cows were infused with
saline. Plasma insulin levels were measured at
48 and
24 h, and every 2 h during the
infusion period in both treatment groups. Significant treatment (P<0·001) and treatment
by time (P<0·001) effects were observed (pooled S.E.M. =0·14 ng/ml). Lower panel: blood
glucose was measured every 6 h during the 48 h baseline period, and hourly during the
infusion period. Glycemia was maintained within10% of baseline values (dotted lines).
Blood glucose levels were not different between treatments (pooled S.E.M. =2·2 mg/dl; P>0·1).

29 and 24 kDa. Although the identity of these bands infusion period, plasma NEFA concentrations in INS cows
cannot be stated with any certainty, their molecular masses averaged 37% of the values found in CTL cows (20912
suggest that they might contain IGFBP-1 and IGFBP-4 vs 56332 nmol/ml).
respectively. Plasma concentration of the 29 kDa binding
protein was not different between treatments at 0 h
Gene expression
(P>0·05), but was lower in INS cows at 96 h (567·7 vs
997·7; treatment by time, P<0·001). Likewise, plasma The relative abundances of GHR 1A, IGF-I and GHRtot
concentration of the 24 kDa binding protein was similar mRNA in liver tissue are illustrated in Fig. 5. Insulin
between INS and CTL cows at 0 h (P>0·05), but was treatment caused a 3·6-fold increase in the level of GHR
reduced in INS cows at 96 h (324·9 vs 594·9; 1A mRNA (P<0·05). The levels of GHRtot mRNA were
treatment by time, P<0·005). not different between treatments (P>0·1). Relative to the
Plasma NEFA concentrations were similar between CTL cows, insulin treatment increased IGF-I mRNA
treatments during the baseline period (P>0·05), but levels in liver 6·3-fold (P<0·001). Across both treatments,
declined in INS cows during the infusion (treatment, levels of GHR 1A and IGF-I mRNA were highly
P<0·005; treatment by time, P<0·001). Over the 96 h correlated (r=0·80; P<0·001). Within treatments, the
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210 S T BUTLER and others · Lactation-induced negative energy balance in cattle: insulin restores GH responsiveness

Figure 2 Mean plasma IGF-I in INS and CTL cows during the 48 h baseline and 96 h
infusion periods. Plasma IGF-I levels were measured at
48 and
24 h, and every 6 h
during the infusion period for both treatments (pooled S.E.M. =4·9 ng/ml). Significant
treatment (P<0·001) and treatment by time (P<0·001) effects were observed. The solid
black bar represents the infusion period.

correlation was significant in INS cows (r=0·86; P<0·05), hepatic and adipose GHRtot mRNA levels in either
but not in CTL cows (r=0·43; P>0·1). The amount of treatment. Adipose GHRtot and adipose IGF-I mRNA
IGF-I mRNA in liver tissue was not correlated with levels were not correlated in either treatment.
GHRtot mRNA in either treatment.
The amount of GHRtot and IGF-I mRNA were lower
in adipose tissue compared with liver (P<0·001). Insulin DMI, milk production and EB
treatment decreased the abundance of GHRtot and IGF-I DMI, milk production and EB were similar between
mRNA in adipose tissue by 1·8-fold (P<0·05) and 3·4- treatments during the baseline period, but there was a
fold (P<0·05) respectively (Fig. 6). Adipose and liver reduction in DMI (treatment, P<0·01; treatment by time,
IGF-I mRNA levels were correlated in CTL cows P<0·001) and milk production (treatment, P<0·01; treat-
(r=0·83; P<0·05), but were not correlated in INS cows ment by time, P<0·001) in INS cows during the infusion
(r=
0·37; P>0·1). There was no correlation between period (Fig. 7). The net effect of these changes combined
with the NEl of glucose infused led to greater EB in the
INS compared with CTL cows (treatment P<0·005,
treatment by time, P<0·001). EB was 12·9 Mcal/day
greater for INS compared with CTL cows for the final 3
days of the infusion. DMI at 96 h (i.e. intake consumed
over the final 24 h of treatment) was correlated with IGF-I
mRNA in INS cows (r=0·82; P<0·05), but not in CTL
cows (r=0·22; P>0·1) and was correlated with GHR 1A
mRNA in both INS (r=0·69; P<0·1) and CTL cows
(r=0·91; P<0·01). There was a significant negative
relationship between plasma GH and DMI in INS cows
(r=
0·86; P<0·05), but not in CTL cows (r=
0·37;
P>0·1).

Discussion
Figure 3 Plasma GH concentrations during the baseline and
infusion periods in INS and CTL cows. Plasma GH was measured
at
48 and
24 h, and every 6 h during the 96 h infusion period
The most striking observation of this study was the 4-fold
(solid bar). The effects of treatment (P>0·1) and treatment by time increase in circulating IGF-I concentration in response to
(P>0·1) were not significant (pooled S.E.M. =0·54 ng/ml). a chronic elevation of plasma insulin during a period of
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 Lactation-induced negative energy balance in cattle: insulin restores GH responsiveness · S T BUTLER and others 211

Figure 4 Effect of treatments on plasma IGFBP at 0, 48 and 96 h relative to the start of

infusion on three representative cows per treatment. Plasma samples were analyzed by
Western ligand blot with 125I-IGF-I. Molecular masses (kDa) are indicated. See text for
details.

NEB-induced GH resistance. In accordance with the different modes of action (Boni-Schnetzler et al. 1991,
observed increase in plasma IGF-I, hepatic IGF-I mRNA Krishna et al. 1996). Regions of the rat IGF-I gene
levels were elevated 6·3-fold in INS cows. The increase in downstream of exon 1 are sensitive to nutritional status and
IGF-I during insulin infusion was associated with an in vitro transcription was reduced using nuclear extracts
increase in hepatic GHR 1A, a major GHR transcript in from diabetic compared with normal rats (Pao et al. 1995).
bovine liver tissue. We found GHR 1A was increased The molecular mechanisms by which insulin acts to
3·6-fold in INS cows, and that GHR 1A levels were increase hepatic IGF-I gene transcription have recently
correlated with IGF-I mRNA in INS but not CTL cows been elucidated. The ubiquitous transcription factor Sp1
(Fig. 5). It has been demonstrated previously that hepatic can bind to regions of the IGF-I gene, potentially making
GHR 1A and IGF-I mRNA decline in periparturient a substantial contribution to IGF-I gene expression in vivo
cows (Kobayashi et al. 1999a), leading to the suggestion (Zhu et al. 2000). Insulin has profound effects on concen-
that the reduction in GH action, and consequent trations of Sp1, both in vitro in hepatocytes and in vivo in
uncoupling of the GH–IGF axis, is due to reduced hepatic liver tissue of diabetic rats (Pan et al. 2001). In addition, a
GHR 1A (Kobayashi et al. 1999b). The results of this study recently identified insulin-responsive binding protein
indicate that insulin is an important metabolic signal for (IRBP) binds to a region of the rat IGF-I gene in close
the GH–IGF axis, coordinating the parallel increase in proximity to the Sp1-binding site (Kaytor et al. 2001b).
liver GHR 1A and IGF-I mRNA resulting in the marked Sp1 is known to be involved in heterotypic interactions
elevation in plasma IGF-I levels. Conversely, both GHRtot with many nuclear proteins and is thought to interact
and IGF-I mRNA levels in adipose tissue were reduced as directly with IRBP to promote insulin-induced hepatic
a result of the insulin treatment, indicating that insulin IGF-I expression (Kaytor et al. 2001a). Thus insulin
induces pleiotropic effects on the GH–IGF axis in differ- has a direct effect on hepatic IGF-I gene expression by
ent tissues. Two potential mechanistic scenarios exist to regulating IRBP and Sp1.
explain the observed increase in hepatic IGF-I mRNA: Insulin also appears to regulate GHR synthesis in
(i) the elevation in insulin had a direct effect on IGF-I several species. In humans, diabetes is associated with
gene expression; and (ii) the elevation in insulin stimulated GH resistance because hepatic GHR concentrations are
an increase in hepatic GHR expression, allowing GH to reduced and this is thought to be due to portal vein
act through its cognate receptor to mediate the increased insulinopenia (Hanaire-Broutin et al. 1996b). Continuous
IGF-I output. Information exists to indicate that insulin i.p. insulin infusion (allowing insulin absorption into the
has stimulatory effects on both of these mechanisms. portal vein) increased plasma GH-binding protein (an
Transcription of the IGF-I gene is regulated by multiple indicator of hepatic GHR concentrations) and returned
factors in various tissues (Adamo 1995). However, the GH responsiveness to normal levels (Hanaire-Broutin et al.
liver is the main source of circulating IGF-I (Sjögren et al. 1996a). Diabetic human patients with residual beta-cell
1999, Yakar et al. 1999) and hepatic IGF-I synthesis is secretion are capable of producing IGF-I in response to
regulated primarily by GH, nutritional status and insulin exogenous GH, whereas patients without residual beta-
(Thissen et al. 1994, Jones & Clemmons 1995, Phillips cell secretion are unresponsive to GH (Wurzburger et al.
et al. 1998). In cultures of primary rat hepatocytes, IGF-I 1995). In rats, diabetes leads to reduced liver GH binding
expression was increased by insulin alone (Boni-Schnetzler and insulin therapy restores GH binding to normal levels
et al. 1991, Pao et al. 1993, Krishna et al. 1996) and in an (Baxter et al. 1980). A recent study has demonstrated that
additive manner in combination with GH, indicating expression of the liver-specific bovine GHR 1A gene is
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212 S T BUTLER and others · Lactation-induced negative energy balance in cattle: insulin restores GH responsiveness

Figure 5 Relative levels of liver GHR 1A, IGF-I and GHRtot mRNA and the correlation
within each treatment for GHR 1A and IGF-I mRNA. Upper panel: RNA was extracted
from liver, and subjected to real-time RT-PCR using probes for GHRtot, GHR 1A, IGF-I
and cyclophilin. The GHRtot, GHR 1A and IGF-I mRNA data were corrected with
cyclophilin, and the value for CTL cows set at 1 for each gene. Expression of the
cyclophilin gene was not different between treatments (P>0·1). Values are means S.E.M.
Significance of difference from CTL is indicated by asterisks: *P<0·05; ***P<0·001. Lower
panel: a positive correlation was observed between GHR 1A and IGF-I mRNA in INS
cows (r=0·86; P<0·05), but not in CTL cows (r=0·43; P>0·1). The values used in the
correlation analysis were unadjusted data points.

regulated by the liver-enriched transcription factor likely in the light of results from other studies. Infusion of
HNF-4 ( Jiang & Lucy 2001a). Insulin is recognized as insulin and glucose to cows approaching positive EB
a positive modulator of the HNF transcription factor caused an increase in plasma IGF-I compared with cows
network, including HNF-4 (Duncan et al. 1998). infused with either saline or glucose (Léonard & Block
A chronic elevation in plasma insulin concentrations 1997). GH binding to hepatic microsomal membranes was
increased plasma IGF-I in dairy cows that were in positive enhanced in insulin-treated cows (Léonard et al. 1992),
EB (i.e. cows that were not GH-resistant) (McGuire et al. and GH binding was correlated with plasma insulin
1995b). Although the mechanism for the increased IGF-I (Léonard et al. 2001). These combined results imply that
was not addressed, a combination of direct insulin action insulin treatment increases circulating IGF-I, at least in
and greater sensitivity of hepatic tissue to GH seems most part, by increasing the number of GHRs in liver. These
Journal of Endocrinology (2003) 176, 205–217 www.endocrinology.org
 Lactation-induced negative energy balance in cattle: insulin restores GH responsiveness · S T BUTLER and others 213

Adipose tissue contains high levels of IGF-I mRNA and

is GH responsive (Peter et al. 1993). GH promotes lipolysis
by antagonizing the anti-lipolytic activities of adenosine
and enhancing the lipolytic response to catecholamines
(Houseknecht & Bauman 1997, Lanna & Bauman 1999).
High levels of circulating GH and low levels of insulin are
typical of early lactation, and their respective concen-
trations facilitate adipose tissue mobilization in support of
milk production. We found the expression of both GHRtot
and IGF-I mRNA in adipose tissue was reduced following
insulin treatment. These results indicate that high insulin
levels are inhibitory to adipose GHR expression and thus
antagonize the ability of adipose tissue to respond to GH.
This is consistent with the observation that genetic selec-
tion for increasing milk production is associated with
reduced plasma insulin levels and increased circulating GH
(Bonczek et al. 1988) and that higher producing animals
mobilize greater amounts of body reserves in early
lactation.
Despite the marked increase in liver GHR 1A mRNA
in response to insulin, hepatic GHRtot mRNA (mainly
GHR 1A, 1B and 1C) levels were not different between
the treatments indicating that transcript(s) other than
GHR 1A were down-regulated. It is important to note
Figure 6 Relative level of GHRtot and IGF-I mRNA in s.c. adipose
that transcription of the GHR gene is controlled by
tissue taken prior to terminating the infusions and the relationship multiple different promoters that could respond differently
between hepatic and adipose IGF-I mRNA levels. Upper panel: to the same signal. Although GHR 1B appears to be
RNA was extracted from adipose tissue and subjected to real-time unregulated, it is worthy of note that the promoter region
RT-PCR using probes for GHRtot, IGF-I and cyclophilin. The GHRtot of GHR 1B contains both negative and positive regulatory
and IGF-I mRNA data were corrected with cyclophilin, and the
value for GHRtot mRNA and IGF-I mRNA in CTL cows set at 1. elements influencing transcriptional activity ( Jiang et al.
Expression of the cyclophilin gene was not different between 2000). In addition, the various transcripts are controlled by
treatments (P>0·1). Values are means S.E.M. Significance of translational mechanisms, with GHR 1B being poorly
difference from CTL is indicated by an asterisk: *P<0·05. Lower translated and the other transcripts generally having high
panel: a positive correlation was observed between adipose and
liver IGF-I mRNA in CTL cows (r=0·83; P<0·05), but not in INS
translational efficiency in vitro ( Jiang & Lucy 2001b). The
cows (r=0·37; P>0·1). The values used in the correlation analysis physiological importance of changes in GHR transcripts
were unadjusted data points. other than GHR 1A in liver tissue are not well defined,
whereas the importance of hepatic GHR 1A expression in
relation to the functionality of the GH–IGF axis has been
clearly demonstrated (Kobayashi et al. 1999a, Smith et al.
results are in agreement with our observations of a parallel 2002). Thus it is possible that insulin increased GHR 1A
increase in IGF-I mRNA and GHR 1A following a transcription in liver via stimulation of liver-enriched
chronic elevation in plasma insulin. transcription factors (including HNF-4), and down-
The miniature Bos indicus represents a unique bovine regulated other transcripts in both tissues by as yet
model of Laron dwarfism. The dwarf phenotype is due undetermined mechanisms.
to low hepatic expression of GHR 1A, and conse- Pituitary GH release is primarily determined by
quent reduction in circulating IGF-I (Liu et al. 1999). hypothalamic GH-releasing hormone and somatostatin,
Circulating insulin levels are very low in these animals and but is also stimulated by ghrelin, and inhibited by plasma
they are unresponsive to exogenous GH in terms of IGF-I IGF-I via a negative feedback loop (Berelowitz et al. 1981,
release, but GH responsiveness is restored following insulin Kojima et al. 1999). GH release should be suppressed in
therapy, i.e. the hypoinsulinemia appears to be responsible the INS cows due to elevated plasma IGF-I. We observed
for the inability of GH to elicit an IGF-I response (Elsasser a decrease in GH levels in the INS cows after approxi-
et al. 1990). Thus, we propose that the increase in IGF-I mately 30 h that was maintained for the remainder of the
observed in the present study was not due to the effects of infusion but values were not different from saline-infused
insulin acting alone, but through a combination of insulin cows at the end of the infusion period (Fig. 3). This may
effects and insulin-induced restoration of the GH–IGF be ascribed to the reduced intake in the INS cows, and its
axis. attendant effects on clearance of GH from plasma and
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214 S T BUTLER and others · Lactation-induced negative energy balance in cattle: insulin restores GH responsiveness

Figure 7 Milk yield, DMI and EB over the 48 h baseline and 96 h infusion periods. The
solid bar represents the infusion period. Milk yield and DMI decreased during the infusion
period for INS cows (treatment, P<0·01; treatment by time, P<0·001). EB was improved in
the INS compared with CTL cows during the infusion period (treatment, P<0·005;
treatment by time, P<0·001).

ghrelin release. The metabolic clearance rate of GH in complex is much longer (12 h) than the half-lives of the
ruminants is slower during periods of chronic feed restric- lower molecular mass complexes (30–90 min), a shift from
tion (Trenkle 1976, Lapierre et al. 1992). Conflicting low molecular mass binding proteins to IGFBP-3 reduces
reports exist regarding the role of plasma insulin in the clearance rate of IGF-I (Thissen et al. 1994, Jones &
regulating circulating ghrelin (Caixas et al. 2002, Saad et al. Clemmons 1995). Thus, the shift in the relative propor-
2002), but ghrelin is clearly elevated during starvation in tions of IGFBP-2 and IGFBP-3 observed in this study
rats (Tschop et al. 2000) and in cows prior to feeding represents another mechanism by which insulin regulates
(Hayashida et al. 2001). It is clear that IGF-I acts as a plasma IGF-I concentrations. Although free IGF-I is
negative feedback regulator of GH release, but this effect rapidly cleared from the circulation ( Jones & Clemmons
was confounded in the present study by the ability of 1995), we observed an increase in free IGF-I dur-
insulin administration to depress DMI. ing hyperinsulinemia, as previously reported in humans
Insulin infusion altered IGFBP and increased free IGF-I (Frystyk et al. 1997). This reflects the effect of insulin to
concentrations in plasma. The marked reduction in reduce the presence of the low molecular mass complexes
IGFBP-2 is in agreement with numerous reports showing coincident with increased transcription and secretion of
that IGFBP-2 is inhibited by insulin (Brismar et al. 1988, IGF-I. The physiological significance of increasing the
Boni-Schnetzler et al. 1990, McGuire et al. 1995b, Scharf concentration of free IGF-I merits further study.
et al. 1996, Mashek et al. 2001). In contrast to previous In summary, we found that an elevation in plasma
reports (McGuire et al. 1995b, Mashek et al. 2001), we insulin within the physiological range during NEB
observed IGFBP-3 to be significantly higher in the increased plasma IGF-I by approximately 4-fold. The
insulin-treated cows. This is consistent with the identifi- increase in plasma IGF-I was associated with an increase in
cation of an insulin response element in the rat IGFBP-3 hepatic GHR 1A and IGF-I mRNA. To the best of our
gene (Villafuerte et al. 1997). Although the plasma con- knowledge, this is the first report to demonstrate that
centrations of the 29 kDa (potentially containing GHR 1A is responsive to circulating insulin levels. As
IGFBP-1) and 24 kDa (potentially containing IGFBP-4) GHR 1A is a major GHR transcript in liver ( Jiang &
binding proteins were low in all cows, significant reduc- Lucy 2001b), it is apparent that insulin plays a key role in
tions in both were observed with insulin treatment. The regulating hepatic GHR concentration. It is likely that
net effect of the insulin treatment was to reduce the insulin itself also contributed to the observed increase in
abundance of the lower molecular mass binding protein IGF-I by direct actions on the IGF-I gene. Based on our
complexes and increase the abundance of IGFBP-3, thus current observations of the effects of hyperinsulinemia
increasing the proportion of IGF-I circulating in the during NEB, we propose the model outlined in Fig. 8 to
150 kDa ternary complex. As the half-life of the ternary illustrate the interaction between plasma insulin and GH
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 Lactation-induced negative energy balance in cattle: insulin restores GH responsiveness · S T BUTLER and others 215

Figure 8 Model of the interaction between plasma insulin and the GH–IGF axis in liver and adipose tissue.
Insulin stimulates hepatic expression of both GHR and IGF-I, resulting in increased plasma IGF-I. High IGF-I
acts to decrease plasma GH. In adipose tissue, high plasma insulin is inhibitory to both GHR and IGF-I
expression. The reduced adipocyte GHR concentration coincident with low plasma GH results in reduced
GH-stimulated adipose tissue mobilization. During situations of low plasma insulin, including periods of NEB,
the opposite occurs. Hepatic expression of GHR and IGF-I is reduced, resulting in reduced plasma IGF-I and
increased plasma GH. In adipose tissue, GHR expression is up-regulated during hypoinsulinemia. The
increased adipocyte GHR concentration and high plasma GH levels result in increased adipose tissue
mobilization.

in influencing circulating IGF-I concentration and adipose and purified bovine GH were obtained through NHPP,
tissue mobilization. Further work is necessary to confirm NIDDK and Dr A F Parlow. S T B was a recipient of a
that experimentally induced hypoinsulinemia would result Walsh fellowship from Teagasc (the Irish Agriculture and
in the proposed outcome in liver and adipose tissue. Food Development Authority) in support of his PhD
However, it has already been demonstrated that a rapid program.
drop in insulin in periparturient cows is associated with a
decrease in hepatic GHR 1A expression (Kobayashi et al.
1999a). We propose that the hypoinsulinemia observed in References
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