The Journal of Nutrition

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Acute Dietary Protein Intake Restriction Is

Associated with Changes in Myostatin
Expression after a Single Bout of Resistance
Exercise in Healthy Young Men1,2
Tim Snijders,3,6 Lex B. Verdijk,3,6 Bryon R. McKay,4 Joey S.J. Smeets,3,6 Janneau van Kranenburg,3,6
Bart B.B. Groen,3,6 Gianni Parise,4 Paul Greenhaff,5 and Luc J.C. van Loon3,6*

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3
NUTRIM School for Nutrition, Toxicology, and Metabolism, Maastricht University, Maastricht, The Netherlands; 4Department of
Kinesiology, McMaster University, Hamilton, Ontario, Canada; and 5School of Biomedical Sciences, University of Nottingham, Queen’s
Medical Centre, Nottingham, UK

Abstract
Skeletal muscle satellite cells (SCs) play an important role in the myogenic adaptive response to exercise. It remains to be
established whether nutrition plays a role in SC activation in response to exercise. In the present study, we assessed
whether dietary protein alters the SC response to a single bout of resistance exercise. Twenty healthy young (aged 21 6
2 y) males were randomly assigned to consume a 4-d controlled diet that provided either 1.2 g protein
kg body
weight21
d21 [normal protein diet (NPD)] or 0.1 g protein
kg body weight21
d21 [low protein diet (LPD)]. On the second
day of the controlled diet, participants performed a single bout of resistance exercise. Muscle biopsies from the vastus
lateralis were collected before and after 12, 24, 48, and 72 h of post-exercise recovery. SC content and activation status
were determined using immunohistochemistry. Protein and mRNA expression were determined using Western blotting
and reverse transcription polymerase chain reaction. The number of myostatin + SCs decreased significantly at 12, 24, and
48 h (range, 214 to 249%; P < 0.05) after exercise cessation, with no differences between groups. Although the number
of myostatin + SCs returned to baseline in the type II fibers on the NPD after 72 h of recovery, the number remained low on
the LPD. At the 48 and 72 h time points, myostatin protein expression was elevated (86 6 26% and 88 6 29%,
respectively) on the NPD (P < 0.05), whereas it was reduced at 72 h (236 6 12% compared with baseline) in the LPD
group (P < 0.05). This study demonstrates that dietary protein intake does not modulate the post-exercise increase in SC
content but modifies myostatin expression in skeletal muscle tissue. This trial was registered at clinicaltrials.gov as
NCT01220037. J. Nutr. 144: 137–145, 2014.

Introduction
Normally, SCs remain in a quiescent state, but, in response to
Skeletal muscle stem cells, also known as satellite cells (SCs)7, are exercise and/or muscle damage, SCs can become activated and
thought to play an important role in the myogenic adaptive subsequently proliferate and differentiate. SCs differentiate to
response to exercise. SCs reside in a niche between the basal
form new myofibers, fuse with existing fibers, or return to a
lamina and the sarcolemma of their associated muscle fiber.
quiescent state, replenishing the resident pool of SCs through self-
renewal (1). The progression of SCs through the myogenic
1
Author disclosures: T. Snijders, L. B. Verdijk, B. R. McKay, J. S. J. Smeets, program is mediated by the expression of different myogenic
J. van Kranenburg, B. B. B. Groen, G. Parise, P. Greenhaff, and L. J. C. van Loon, regulatory factors (MRFs) [e.g., myogenic differentiation
no conflicts of interest.
2
Supplemental Figures 1–3 and Supplemental Table 1 are available from the
(MyoD), myogenic factor-5, myogenin, and Mrf4]. The activa-
‘‘Online Supporting Material’’ link in the online posting of the article and from the tion, proliferation, and differentiation processes of the SCs
same link in the online table of contents at http://jn.nutrition.org. represent important regulatory steps that ultimately determine
6
Present address: Department of Human Movement Sciences, Faculty of
their fate in supporting skeletal muscle fiber adaptation. In
Health, Medicine, and Life Sciences, Maastricht University, Maastricht, The
Netherlands. addition to the MRFs, myostatin has been identified as a key
7
Abbreviations used: BW, body weight; Ct, threshold cycle; DAPI, regulator in the SC response to exercise and/or muscle damage. In
4#,6-diamidino-2-fenylindool; En%, energy percent; LPD, low protein diet; animal muscle, reduced myostatin expression has been shown to
MRF, myogenic regulatory factor; MyoD, myogenic differentiation; NPD, normal
result in substantial muscle hypertrophy (2,3). More recent work
protein diet; SC, satellite cell; 1RM, one-repetition maximum.
* To whom correspondence should be addressed. E-mail: L.vanLoon@ by McKay et al. (4) suggests that, in humans, myostatin is a strong
maastrichtuniversity.nl. negative regulator of the SC myogenic response to an acute bout
ã 2014 American Society for Nutrition.
Manuscript received August 19, 2013. Initial review completed September 25, 2013. Revision accepted November 17, 2013. 137
First published online December 4, 2013; doi:10.3945/jn.113.183996.
of resistance exercise. Accordingly, because myostatin has been TABLE 1 Participant characteristics of healthy young men who
reported to negatively regulate SC activation and self-renewal (5), performed a single bout of resistance-type exercise and con-
inhibition of myostatin expression may represent an important sumed a normal or low protein diet for 4 d1
myogenic stimulus to allow skeletal muscle hypertrophy in
NPD group LPD group
response to prolonged resistance exercise training (6).
Prolonged resistance exercise training has been well estab- Age, y 22 6 1 21 6 1
lished as an effective interventional strategy to augment skeletal Anthropometrics
muscle mass and strength (7–9). A single bout of resistance Height, m 1.82 6 0.02 1.83 6 0.03
exercise increases muscle protein synthesis as well as breakdown Weight, kg 75.9 6 1.8 79.2 6 3.0
rates, allowing skeletal muscle to recondition (10–12). Ingestion BMI, kg/m2 23.0 6 0.6 23.7 6 0.8
of dietary protein after a single session of resistance exercise Body composition
further stimulates muscle protein synthesis rate and reduces Whole-body lean mass, kg 58.4 6 17.2 59.7 6 18.8
muscle protein breakdown, allowing net muscle protein accre- Leg lean mass, kg 21.1 6 0.4 22.3 6 0.9
tion (10,12). In accordance, it has been demonstrated that Leg fat, % 15.1 6 1.7 16.9 6 1.8
protein supplementation can augment the increase in skeletal 1RM muscle strength, kg

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muscle mass in response to prolonged resistance exercise Leg press 214 6 7 235 6 9
training (13). To turn short-term changes in net muscle protein Leg extension 137 6 4 138 6 5
balance into more long-term muscle fiber hypertrophy, addi-
1
Values are means 6 SEMs, n = 10 per group. LPD, low protein diet group; NPD,
tional myonuclei are prerequisite (14,15). Because myonuclei
normal protein diet group; 1RM, one-repetition maximum.
are postmitotic, skeletal muscle SCs are required to provide new
myonuclei during post-exercise recovery. It has been well
characterized that protein availability can significantly influence rest, a muscle biopsy was taken from the musculus vastus lateralis.
muscle protein synthesis rates after exercise (16–18), which may Thereafter, participants were provided with a standardized breakfast.
be translated into long-term benefits (13). It could be speculated Next, participants performed a single bout of resistance exercise,
that dietary protein can also modulate the SC response during consisting of 2 different exercises. Participants performed six sets of 10
post-exercise recovery in a feedforward manner as to prepare the repetitions at 75% 1RM on the horizontal leg press machine
(Technogym) and six sets of 10 repetitions at 75% 1RM on the leg
intrinsic potential of the muscle for long-term adaptation.
extension machine (Technogym). A resting period of 2 min between sets
However, no data exist on the influence of protein intake on the
was allowed. The entire protocol required ;40 min to complete. All
acute SC response after exercise. Therefore, in the present study, participants were verbally encouraged during the test to complete the
we assessed SC content and activation status combined with entire protocol. Before and after the exercise session, a 5–10 min warm
associated myocellular signaling during 3 d of recovery from a up/cooling down was performed on a cycle ergometer. At the end of the
single bout of resistance exercise while ingesting a normal exercise protocol the participants rested for 3 h in a supine position. At
protein [13 energy percent (En%)] diet (NPD) or low protein (<2 1230 h, participants received a standardized lunch, after which
En%) diet (LPD). We hypothesized that the acute SC response to participants were provided with a standardized dinner to be consumed
a single bout of exercise would be attenuated by an acute at 1800 h that evening. On the same day, participants reported back to
restriction of dietary protein intake. the laboratory at 2030 h for a second muscle biopsy sample collection
(t = 12 h). Next, muscle biopsies were taken in an overnight fasted state
exactly 24, 48, and 72 h after the start of the single exercise bout. During
the 72 h post-exercise recovery, all participants adhered to either a
Participants and Methods standardized NPD (13 En% protein) or LPD (<2 En% protein).
Participants. Twenty healthy male volunteers (aged 21 6 2 y; height,
1.82 6 0.01 m; bodyweight, 78 6 2 kg) participated in this study (Table 1). Diet control and physical activity. During the entire 4 d intervention
All participants were recreationally active, which was defined as period, all participants consumed a controlled diet. Participants were
participating in noncompetitive sporting activities being performed less randomly assigned to consume either an NPD (13 En% protein) or an
than three times a week. Participants were informed about the nature LPD (<2 En% protein) (Table 2). For each participant, the meals were
and risks of the experimental procedures before their written consent customized and were provided as prepackaged meals, drinks, and
was obtained. The study was approved by the Medical Ethical snacks. Energy intake was based on the equations by Harris and Benedict
Committee of the Maastricht University Medical Centre and complied (20), with a physical activity index of 1.4 (21). All volunteers refrained
with the guidelines set out in the Declaration of Helsinki. Exclusion from any sort of heavy physical exercise 3 d before the first test day until
criteria were defined that would preclude successful participation in the completion of the experiment.
exercise session, which included (silent) cardiac or peripheral vascular
disease and orthopedic limitations. After inclusion, all participants were
randomly assigned to consume either a controlled NPD or LPD. Body
composition (fat and fat-free mass) was determined by dual energy x-ray
TABLE 2 Specification of controlled dietary intake of healthy
absorptiometry scan (Hologic). Lean mass and percentage body fat were
young men who performed a single bout of resistance-type
determined on a whole-body level and for specific regions (e.g., trunk
exercise and consumed a normal or low protein diet for 4 d1
and legs). In addition, all participants performed a familiarization trial to
become experienced with the research protocol and the equipment.
NPD group LPD group
Proper lifting technique was demonstrated and then practiced by the
participants for each of the 2 lower-limb exercises (leg press and leg Energy, MJ/d 11 6 0.3 11 6 0.6
extension). Subsequently, maximal strength (one-repetition maximum Protein, g/d 88 6 2.7 11 6 0.4*
[1RM]) was estimated by using the multiple repetitions testing procedure
Carbohydrate, g/d 474 6 11 502 6 22
during 2 separate visits (19). All strength tests were completed at least 2
Fat, g/d 60 64 56 6 6
wk before the start of the experimental protocol.
1
Values are means 6 SEMs; n = 10 per group. *P , 0.0001, significantly
Protocol. At 0800 h, after 24 h of the controlled diet, participants different compared with NPD. LPD, low protein diet group; NPD, normal protein
reported to the laboratory after an overnight fast. After 30 min of supine diet group.

138 Snijders et al.

Muscle biopsy. Muscle biopsies were obtained from the middle region PCR system (Applied Biosystems), with 2 mL of cDNA (diluted five
of the vastus lateralis muscle 15 cm above the patella and ;2 cm away times) and Taqman Gene Expression Mastermix (Applied Biosytems) at
from the fascia by means of the percutaneous needle biopsy technique a total volume of 25 mL. Taqman primer/probe sets were obtained from
described by Bergstrom et al. (22). Muscle biopsies were carefully freed Applied Biosystems (Taqman Gene Expression Assays): myogenin,
from any visible fat and blood, with one part embedded in Tissue-Tek MyoD, and myostatin. The primers are listed in Supplemental Table 1.
(Sakura Finetek Europe) and rapidly frozen in liquid nitrogen cooled Each sample was run in duplicate. The housekeeping gene hydroxyme-
isopentane and another part frozen directly in liquid nitrogen, before thylbilane synthase was used as an internal control. The thermal cycling
being stored at 280°C for subsequent biochemical and histochemical conditions used included the following: 2 min at 50°C, 10 min at 95°C,
analysis. followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. The threshold
cycle (Ct) values of the target gene were normalized to Ct values of the
Immunohistochemical analyses. From all muscle biopsy samples, internal control hydroxymethylbilane synthase, and final results were
5-mm-thick cross-sections were cut at 220°C using a cryostat. Muscle calculated according to the 22ΔΔCt. The muscle biopsy attained before
samples collected before and after 12, 24, 48, and 72 h of post-exercise the single bout of exercise was used as a reference and given a value of 1,
recovery from each individual were mounted together on uncoated glass and relative fold changes at 12, 24, 48, and 72 h of post-exercise
slides and air dried for 3 h at room temperature before being stored at recovery were calculated.
220°C for subsequent analyses. All muscle cross-sections were stained
with antibodies against Pax7 (neat; Developmental Studies Hybridoma Western blotting. Each muscle sample frozen for biochemical analyses

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Bank), myostatin (near C terminus; 1:100; AB3239; Millipore) validated was homogenized in Tris buffer (50 mmol/L; 1 mmol/L EDTA, 1 mmol/L
previously to detect the C-terminal (active) myostatin (23,24), A4.951 EGTA, 0.1% Nonidet P-40, and 0.1% 2-mercaptoethanol, pH 7.4)
(myosin heavy chain type I; slow isoform; neat; Developmental Studies supplemented with the following protease and phosphatase inhibitors: 2 g/L
Hybridoma Bank], and laminin (1:1000; L8271; Sigma-Aldrich; and aprotinin, 2 g/L leupeptin, 300 mmol/L benzamidine, and 100 mmol/L
ab11575; Abcam). Appropriate secondary antibodies were applied: PMSF. After homogenization, each muscle extract was centrifuged for
Dylight 488 (1:500; Thermo Fisher Scientific), Alexa Fluor 594 (1:500; 5 min at 10,000 3 g (4°C). The pellet was homogenized with nuclear
Invitrogen), biotinylated secondary antibody (1:200; Vector Laborato- protein isolation buffer (20 mmol/L HEPES, 25% glycerol, 500 mmol/L
ries), streptavidin-594 fluorochrome (1:500; Invitrogen), Alexa Fluor NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 2 g/L aprotinin, 2 g/L
488 (1:500), Alexa Fluor 594 (1:500), Alexa Fluor 647 (1:500), and leupeptin, 300 mmol/L benzamidine, and 100 mmol/L PMSF, pH 7.9)
Alexa Fluor 350 (1:20). Nuclei were stained with 4,6-diamidino-2- and centrifuged for 5 min at 3000 3 g (4°C). After homogenization,
phenylindole [(DAPI) 0.238 mmol/L; Invitrogen]. The immunohisto- each muscle extract was centrifuged for 5 min at 10,000 3 g (4°C), and
chemical staining procedures were adapted from previously published sample buffer (final concentration, 60 mmol/L Tris, 5% glycerol, 20 g/L
methods (4). In short, for coimmunofluorescence staining (myosin heavy SDS, 0.1 mmol/L DTT, and 20 mg/L bromophenolblue) was added to
chain type I, laminin, Pax7, and myostatin), sections were fixed with 4% the supernatant. The supernatant was boiled for 5 min at 100° and
paraformaldehyde (Sigma-Aldrich) for 10 min, followed by several cooled on ice. The supernatant was used as the nuclear fraction.
washes in PBS. Sections were then covered for 60 min in a blocking Immediately before analyses, the muscle extraction sample was
solution containing 2% BSA, 5% FBS, 0.2% Triton X-100, and 0.1% warmed to 50°C and centrifuged for 1 min at 3000 3 g (room
sodium azide. After blocking, sections were incubated in the primary temperature). Total amount of sample loaded on the gel was based on
antibody at 4°C overnight. After several washes, sections were then weight (3.125 mg muscle per lane). Protein samples were run on a
incubated in the appropriate secondary antibodies. Sections were then 4–15% Criterion Tris-HCl gel (catalog No. 3450027; Bio-Rad) for 10 min
refixed in 4% paraformaldehyde to prevent migration of the secondary at 50 V (constant voltage) and 1 h 40 min at 120 V (constant voltage)
antibodies and reblocked in 10% goat serum in 0.01% Triton X-100 and transferred onto a polyvinylidene difluoride membrane in 1 h 30 min
(Sigma-Aldrich). The sections were then incubated in the second primary at constant 500 mA. Specific proteins were detected by incubation with
antibody, followed by incubation in the appropriate secondary antibody. specific antibodies in 1% BSA in 0.1% PBS–Tween 20 after blocking in
Sections were then washed with PBS, and DAPI was applied for nuclear 1% BSA in 0.1% PBS–Tween 20 (catalog No. A6003-25G; Sigma). The
staining. All stained muscle samples were viewed with the Nikon Eclipse antibodies used in this study were anti-MyoD (37 kDa; rabbit polyclonal
90i microscope outfitted with a high-resolution QImaging camera for IgG; sc-760; Santa Cruz Biotechnology), anti-myostatin (50 kDa; rabbit
fluorescence detection (Nikon Instruments), and images were captured polyclonal IgG; sc-6885-R; Santa Cruz Biotechnology), and anti-a-actin
and analyzed using the Nikon NIS Elements AR 3.0 software (Nikon (42 kDa; mouse monoclonal IgM; catalog No. A2172; Sigma). After
Instruments). SC quantification was conducted on $125 muscle fibers incubation, membranes were washed (three times for 5 min) in 0.1%
per participant per time point, and images were obtained with the 403 PBS–Tween 20 and incubated (1 h at room temperature) with the
objective. SCs were determined by identifying DAPI-positive (+) Pax7+ appropriate secondary antibody, polyclonal rabbit anti-mouse IgG–
cells within the myofiber (laminin). The number of SCs per muscle fiber, horseradish peroxidase (P0161; Dako), and polyclonal swine anti-rabbit
the proportion of SCs [number of SCs/(number of SC + number of IgG–horseradish peroxidase (P0399; Dako) dissolved in 1% BSA 0.1%
myonuclei) 3 100], and the number of SCs per fiber area (in square PBS–Tween 20. After a final wash step (three times for 5 min) in 0.1%
millimeters) were calculated for the type I and type II muscle fibers Tween 20–PBS, the membranes were incubated with Supersignal West
separately. Within each image, the number of myonuclei (number of Dura Extended Duration Substrate (catalog No. 340760; Thermo Fisher
myonuclei 2 number of SC) per muscle fiber were assessed for the type I Scientific) for 1 min. Visualization and quantification of the protein
and type II muscle fibers. In addition, SC activation was assessed by the bands was enabled by Bio-Rad universal hood II and Chemidoc XRS
presence of myostatin staining inside the SC. A DAPI+Pax7+myostatin+ camera and Quantity One 4.6.5 software, respectively (for additional
cell within the myofiber was considered a quiescent SC, and a representative images of the Western blot analyses, see Supplemental
DAPI+Pax7+myostatin-negative cell within in the myofiber was consid- Fig. 2).
ered to be an activated SC. Slides were masked for both group and time
point (for representative images of the staining, see Supplemental Fig. 1). Statistics. All data are expressed as means 6 SEMs. An independent
samples StudentÕs t test was used to compare differences between groups
Quantitative RT-PCR. Total RNA was isolated from 10–20 mg of at baseline. Furthermore, data were analyzed using a 2-factor repeated-
frozen muscle tissue using Trizol Reagent (Invitrogen), according to the measures ANOVA with group (NPD or LPD) as the between-participant
protocol of the manufacturer. Total RNA quantification was performed factor and time (before exercise and after 12, 24, 48, or 72 h of post-
spectrophotometrically at 260 nm (NanoDrop ND-1000 Spectropho- exercise recovery) and fiber type (type I vs. II) as within-participant
tometer; Thermo Fisher Scientific), and RNA purity was determined as factors. In the 2-factor repeated-measures ANOVA design, post-exercise
the ratio of readings at 260/280 nm. The first-strand cDNA was time points were compared with baseline values only, and BonferroniÕs
synthesized from 1 mg of RNA sample using the iScript cDNA synthesis corrections were applied to account for multiple comparisons to
kit (Bio-Rad). Taqman PCR was performed using the 7300 Real Time baseline. In case of significant time 3 group or fiber type 3 group

Dietary protein and the satellite cell response 139

interaction, additional analyses were performed for NPD and LPD
separately by the use of 1-factor repeated-measures ANOVA. In case of a
significant time 3 fiber type interaction, type I and type II muscle fibers
were analyzed separately, and BonferroniÕs correction was applied to
correct for multiple testing.
LeveneÕs test for equality of variance was used; in the case of unequal
variance, independent samples StudentÕs t test was performed for
unequal variances. Statistical significance was set at P < 0.05. All
calculations were performed using SPSS 19.0 (IBM).

Results
ParticipantsÕ characteristics. ParticipantsÕ characteristics are
presented in Table 1. No differences in age, weight, height, BMI,
whole-body lean mass, leg lean mass, leg fat percentage, 1RM
leg press, and 1RM leg extension were observed between groups.

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Dietary intake. No differences were observed in total energy
intake between groups (Table 2). Dietary protein intake was
significantly lower in the LPD compared with the NPD group
(P < 0.0001; Table 2). Participants in the NPD group con-
sumed 1.16 6 0.03 g protein
kg body weight (BW)21
d21, FIGURE 1 Number of satellite cells in type I (A) and type II (B) muscle
whereas participants in the LPD group consumed 0.14 6 0.01 g fibers before (pre) and after 12, 24, 48, and 72 h after a single bout of
protein
kg BW21
d21 (P < 0.0001; Table 2). resistance exercise in healthy young men who consumed a normal or
low protein diet for 4 d. Values represent means 6 SEMs, n = 10 per
group. *P , 0.05, significantly different compared with pre-exercise
Muscle fiber–type distribution and fiber area. No group values. Bars indicate that the effect of time is similar for both groups.
differences in the percentage of type I and type II muscle fibers LPD, low protein diet group; NPD, normal protein diet group.
and/or percentage of muscle area occupied by type I and type II
muscle fibers were observed between groups (Table 3). In both
groups, muscle fiber cross-sectional area and myonuclear domain post-exercise recovery when compared with baseline values (Fig.
size was significantly greater in type II compared with type I fibers 1; P < 0.0001). When SC content was expressed in proportion to
(Table 3; P < 0.05). No changes were observed in fiber-type the number of myonuclei or per square millimeter of fiber area,
distribution, myonuclear domain size, and/or muscle fiber size within SC content had increased significantly at 24, 48, and 72 h after
72 h of post-exercise recovery in either group (data not shown). exercise in both the NPD and LPD groups (Table 4; P < 0.01)

Myonuclear and SC content. No changes were observed in the Myostatin+ SCs. Before exercise, the proportion of myostatin+
number of myonuclei per type I and type II muscle fiber in both SCs was 76 6 3% and 76 6 4% in the type I and type II muscle
groups during post-exercise recovery (Supplemental Fig. 3). In fibers. No differences were observed between groups. The
contrast, the number of SCs per type I and type II muscle fiber number of myostatin+ SCs declined significantly in response to
was significantly greater at 24, 48, and 72 h compared with the single bout of resistance exercise in both groups (Fig. 2).
pre-exercise values in both the LPD and NPD groups (Fig. 1; After 12, 24, and 48 h of post-exercise recovery, both groups
P < 0.05). In addition, in both groups, we observed that type I showed a similar decline in the number of myostatin+ SCs per
muscle fiber SC content was increased within the first 12 h of muscle fiber (Fig. 2; P < 0.001). The decline was significantly
greater in the type II (249 6 5% in the NPD group and 240 6
TABLE 3 Muscle fiber–type composition and muscle fiber size 10% in the LPD group) compared with type I (226 6 6% in the
of healthy young men who performed a single bout of resistance- NPD group and 217 6 11% in the LPD group) muscle fibers at
type exercise and consumed a normal or low protein diet for 4 d1 12 and 24 h, respectively (P < 0.05). In the NPD group, the
number of myostatin+ SCs returned to baseline 72 h after the
Fiber type NPD group LPD group
single bout of exercise in both type I and II muscle fibers. In
Fiber–type distribution, % fiber no. 2
I 52 6 4 58 63 contrast, in the LPD group, the number of myostatin+ SCs
II 48 6 4 42 63 remained significantly depressed in the type II muscle fibers at
Fiber–type distribution, % CSA I 47 6 4 56 63 72 h of post-exercise recovery (see Fig. 2; P < 0.05).
II 53 6 4 44 63
Muscle fiber CSA, x1000 mm 2 I 6.3 6 0.5 6.2 6 0.3 mRNA expression. MyoD mRNA expression increased signif-
II 7.6 6 0.4* 6.9 6 0.3* icantly at 12 and 24 h after the single bout of resistance exercise,
Myonuclear content, n/fiber I 3.6 6 0.3 3.6 6 0.3 with no differences between groups (Fig. 3A; P < 0.05). In
II 3.5 6 0.2 3.4 6 0.3 addition, myogenin mRNA expression increased substantially
Myonuclear domain, x1000 mm2 I 1.8 6 0.1 1.8 6 0.1 during the first 12 h of post-exercise recovery (P < 0.05).
II 2.2 6 0.1* 2.1 6 0.2* Furthermore, in both groups, myogenin mRNA expression
remained elevated at 24 and 48 h after exercise cessation (Fig.
1
Values are means 6 SEMs, n = 10 per group. *P , 0.05, significantly different 3B; P < 0.05). In the NPD group, myostatin mRNA expression
compared with type I fibers. CSA, fiber cross-sectional area; LPD, low protein diet;
NPD, normal protein diet.
was significantly lower at 24 h (276%), 48 h (264%), and 72 h
2
Fiber type distribution calculated as a percentage by the number of muscle fibers (265%) when compared with pre-exercise expression levels
measured in the muscle biopsy sample. (Fig. 3C; P < 0.05). In contrast, the LPD group showed a smaller
140 Snijders et al.
 TABLE 4 Type I and II muscle fiber satellite cell content at baseline and 12, 24, 48, and 72 h after a
single bout of resistance-type exercise in healthy young men who consumed a normal or low protein diet
for 4 d1

Group Pre 12 h 24 h 48 h 72 h

Type I SCs, n/myonucleus NPD 2.9 6 0.5 3.4 6 0.4 4.5 6 0.5* 4.4 6 0.5* 4.5 6 0.6*
LPD 2.7 6 0.6 2.7 6 0.4 3.3 6 0.6* 3.9 6 0.4* 4.9 6 0.7*
Type II SCs, n/myonucleus NPD 3.2 6 0.6 3.2 6 0.4 4.2 6 1.0* 5.0 6 0.6* 5.1 6 0.8*
LPD 3.4 6 0.3 3.7 6 0.4 5.2 6 0.8* 5.0 6 0.6* 4.9 6 0.5*
Type I SCs, n/mm2 NPD 17.1 6 1.9 19.2 6 3.3 21.5 6 2.6* 22.9 6 2.0* 25.0 6 3.0*
LPD 14.7 6 1.9 15.6 6 2.0 17.4 6 2.2* 21.5 6 2.0* 22.8 6 2.5*
Type II SCs, n/mm2 NPD 13.3 6 0.8 14.2 6 1.0 18.7 6 1.1* 18.8 6 1.5* 21.3 6 2.1*
LPD 15.5 6 1.8 14.9 6 1.7 16.4 6 1.8* 21.5 6 1.6* 21.4 6 1.5*
1
Values are means 6 SEMs, n = 10 per group. *P , 0.05, significantly different compared with pre-exercise values. LPD, low protein diet;
NPD, normal protein diet; Pre, baseline; SC, satellite cell.

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reduction in myostatin mRNA expression, which was only During post-exercise recovery, dietary protein intake is
different from baseline after 48 h (240%) of post-exercise recovery essential to support the increase in myofibrillar muscle protein
(Fig. 3C; P < 0.05). synthesis, thereby allowing net muscle protein accretion
(10,12,18). However, for more long-term muscle adaptation,
Protein expression. MyoD protein expression was significantly SCs are required to provide additional myonuclei to allow
increased at 12 h after exercise with no differences between muscle fiber hypertrophy. Whether dietary protein intake is
groups (Fig. 4A; P < 0.05). In the NPD group, myostatin protein prerequisite to allow a proper SC response during recovery
expression was significantly elevated after 48 h (86 6 26%) and from a single bout of resistance exercise has not yet been
72 h (88 6 29%) of post-exercise recovery (Fig. 4B; P < 0.05). In investigated. We hypothesized that dietary protein intake and
contrast, myostatin protein expression was significantly reduced subsequent precursor availability drives the SC response to
(236 6 12%) in the LPD group at 72 h after exercise compared exercise in a feedforward signaling manner. Therefore, we
with baseline (Fig. 4B; P < 0.05). compared the post-exercise change in SC content between
participants consuming 1.2 g protein
kg BW21
d21 (13 En%
protein) and participants who restricted their dietary protein
Discussion intake to <0.1 g protein
kg BW21
d21 (<2 En% protein). In
healthy young men, dietary protein intake typically ranges
In the present study, we report that acute dietary protein intake
restriction does not affect the increase in SC content in type I and
type II muscle fibers after a single bout of resistance exercise. We
observed that the number of myostatin+ SCs declined substan-
tially during the first 48 h of post-exercise recovery, after which
they returned to baseline values. However, when dietary protein
intake was severely reduced, the number of myostatin+ SCs
remained suppressed in the type II muscle fibers. Correspond-
ingly, skeletal muscle myostatin protein expression was signif-
icantly lower during recovery from exercise when dietary
protein intake was restricted.
During post-exercise recovery, SCs are thought to be the sole
source for providing new myonuclei, thereby supporting skeletal
muscle reconditioning. In accordance, in the present study, we
show that SC content increases substantially within 72 h of post-
exercise recovery (Fig. 1). Expansion of the SC pool is consistent
with previous work showing similar changes in SC content
anywhere from 12 to 192 h after a single bout of exercise (4,25–
29). A rapid increase in SC content was observed in both type I
and type II muscle fibers during post-exercise recovery (Fig. 1).
Similar changes over time were observed when the SC pool size
in both type I and type II muscle fibers were expressed relative to
the number of myonuclei and/or per square millimeter (Table 4).
Interestingly, type I muscle fiber SC expansion occurred as early
FIGURE 2 Number of Mstn+SCs in type I (A) and type II (B) muscle
as 12 h into post-exercise recovery, whereas it took at least 24 h
fibers before (pre) and after 12, 24, 48, and 72 h after a single bout of
to observe a detectable expansion in type II muscle fiber SC
resistance exercise in healthy young men who consumed a normal or
content. This study confirms that changes in SC content in low protein diet for 4 d. Values represent means 6 SEMs, n = 10 per
response to a single bout of exercise are fiber-type specific group. *P , 0.05, significantly different compared with pre-exercise
(4,28,30,31). This emphasizes the importance of fiber type– values. Bars indicate that the effect of time is similar for both groups.
specific assessment of SC content and/or activation status in LPD, low protein diet group; Mstn+SC, myostatin-positive satellite
human skeletal muscle. cell; NPD, normal protein diet group.

Dietary protein and the satellite cell response 141

 The process of SC activation, proliferation, and differentia-
tion after exercise requires the orchestration of a group of
transcription factors collectively referred to as the MRFs (1). It
has been well established that MyoD acts to promote SC
proliferation and transition of cells into differentiation (36),
whereas myogenin is known to drive terminal differentiation
(37). In human skeletal muscle, MyoD and myogenin expression
have been reported to increase considerably during the first few
hours/days after a single bout of exercise. Consistent with
previous work (38–41), we report that MyoD mRNA and
protein expression increased after a single bout of resistance
exercise (Figs. 3A and 4A). In addition, we observed a sub-
stantial increase in myogenin mRNA expression (Fig. 3B), with
the peak value observed at 12 h after exercise cessation.
However, it is important to note that the 12 h post-exercise

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muscle biopsy sample was taken in the evening (2030 h) in a
post-prandial state, whereas all other muscle biopsy samples
were taken in the morning after an overnight fast. As such, we
cannot exclude a potential effect of the post-prandial vs. fasting
state on the changes observed at the 12-h time point. Nonethe-
less, no significant differences were observed between the NPD
and LPD groups in the increase in MyoD and myogenin
expression during post-exercise recovery. The exercise-induced
upregulation of MyoD and myogenin allows the SCs to become
activated and to progress through the cell cycle, thereby
proliferating to increase the SC pool size, supporting muscle
reconditioning. However, we observed no significant changes in
type I and type II muscle fiber myonuclear content, suggesting

FIGURE 3 Changes in MyoD (A), myogenin (B), and myostatin (C)

mRNA expression 12, 24, 48, and 72 h after a single bout of resistance
exercise in healthy young men who consumed a normal or low protein
diet for 4 d. Values represent means 6 SEMs, n = 10 per group. *P ,
0.05, significantly different compared with pre-exercise values. Bars
indicate that the effect of time is similar for both groups. LPD, low
protein diet group; MyoD, myogenic differentiation; NPD, normal
protein diet group; pre, baseline.

from 1.1 to 1.3 g protein
kg BW21
d21 (7,32–34). This is

considerably more than the 0.656 g protein
kg BW21
d21
that is considered to be the minimum amount of dietary protein
required to sustain skeletal muscle mass (35). Although the
protein intake restriction in the LPD intervention group is
severe and should not be translated to a normal physiologic
situation, it does provide a first insight in the potential
importance of protein intake on the post-exercise SC response.
To assess the influence of such an acute reduction in protein
intake per se on the post-exercise SC response, all participants
consumed the diet during the 4-d intervention period, starting
24 h before exercise. The current study shows that an acute FIGURE 4 MyoD (A) and myostatin (B) protein expression before (pre)
and after 12, 24, 48, and 72 h after a single bout of resistance exercise in
reduction in dietary protein intake does not affect the increase
healthy young men who consumed a normal or low protein diet for 4 d.
in muscle fiber SC content with an overall 64 6 19% and 52 6
Values represent means 6 SEMs, n = 10 per group. *P , 0.05,
23% increase in SC content during 72 h of recovery in the type I significant increase compared with pre-exercise value(s). #P , 0.05,
and II fibers, respectively, with no differences between groups significant decrease compared with pre-exercise value. Bar indicates that
(Fig. 1). These data are the first to show that the SC response to the effect of time is similar for both groups. A.U., arbitrary units; LPD,
exercise is essentially dissociated from dietary protein intake low protein diet group; Mstn, myostatin; MyoD, myogenic differentia-
and is likely evolutionary conserved. tion; NPD, normal protein diet group; pre, baseline.

142 Snijders et al.

that SCs have not yet differentiated to a detectable level within It has been hypothesized previously that myostatin expres-
the 72 h of post-exercise recovery in both groups (Supplemental sion is also regulated by nutritional intake. However, results
Fig. 3). from various animal studies on the influence of different
Besides the important role of MRFs in determining SC fate, nutritional protocols on myostatin expression yield conflicting
myostatin has been reported to be a strong negative regulator of results. Whereas some do observe changes in myostatin expres-
skeletal muscle growth in both animal (3,42,43) and human (44) sion in response to changes in nutrient intake (57–59), others do
models. Subsequently, myostatin has been hypothesized to act as not (60,61). This discrepancy can most likely be attributed to the
a negative regulator of SC activation (5). In support, myostatin different study designs, type of nutritional protocols used, and/
has been shown to be highly expressed in quiescent SCs and is or studied species. In human skeletal muscle, it has been shown
significantly downregulated during SC activation (45). Further- that muscle myostatin mRNA expression is affected by dietary
more, myostatin has been shown to completely block myoblast protein provision immediately before and after a single bout of
proliferation in C2C12 cells (46,47). However, previous in vivo resistance exercise (33,55). The present study is the first to assess
animal work on the proposed role of myostatin in SC function whether dietary protein intake alters myostatin expression on
has yielded many discrepant findings. Whereas some studies multiple levels (in SCs, mRNA, and protein expression) in
indicate that myostatin acts as a negative regulator of SC muscle biopsy samples taken at regular time points after a single

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activation (5,48,49), others have not been unable to confirm bout of resistance exercise. At baseline, no significant differences
these findings (50–52). The potential role of myostatin in SC were observed in the number of myostatin+ SC and/or protein
activation during a more complex physiologic setting, such as expression between groups. In contrast to the NPD group, we
post-exercise recovery, remains essentially unknown. Only one show that the number of myostatin+ SCs remained substantially
previous study colocalized, via immunohistochemistry, myosta- reduced in the type II muscle fibers in the LPD group throughout
tin within the SCs before and after performing a single bout of the entire recovery period (Fig. 2B). In addition, whereas in the
resistance exercise (4). This study by McKay et al. (4) demon- NPD group myostatin protein expression increased significantly
strated that the percentage of myostatin+ SCs in human skeletal at 48 and 72 h of post-exercise recovery, muscle myostatin
muscle decreases within the first 48 h after exercise. In addition, protein expression remained low in the LPD group (Fig. 4B).
they showed that the post-exercise decline in the number of These differences between groups are likely attributable to
myostatin+ SCs was accompanied by a concomitant increase in differences in protein intake per se but may also reflect a
the percentage of MyoD+ SCs and number of SCs in the S-phase response to the acute change in dietary protein intake in the LPD
of the cell cycle (4), indicative of an increase in SC activation. We group. Regardless, the results seem to indicate that, under
extend on these data (4) by assessing the number of myostatin+ conditions of insufficient protein intake, SC activation is
SCs and skeletal muscle myostatin expression for up to 72 h into maintained over a prolonged period of time during post-
post-exercise recovery. Our data show that, after the initial exercise recovery. Beside SC activation, myostatin has also
decline in the number of myostatin+ SCs within the first 48 h of been suggested to modulate post-exercise muscle synthesis, e.g.,
post-exercise recovery, the number of myostatin+ SCs increases through inhibiting mammalian target of rapamycin-induced
and returns to baseline levels after 72 h (Fig. 2A, B). The return protein synthesis (62,63). The muscle protein synthetic response
of the number of myostatin+ SCs toward baseline levels suggests has been reported to be transient and can be increased up to 48 h
a return of the SC to quiescence by 72 h (4,5). In accordance, after a single bout of exercise (64). In agreement, we show an
myostatin protein expression was shown to be increased at 48 increase in myostatin protein expression at 48 and 72 h of post-
and 72 h (86 6 26% and 88 6 29%, respectively) compared with exercise recovery, which may indicate that stimulation of muscle
pre-exercise values. In contrast, in the present study, we also protein synthesis after exercise has worn off. In contrast, when
observed that myostatin mRNA expression was downregulated at insufficient protein is ingested, we observed a significant decline
24, 48, and 72 h after performing a single bout of resistance in myostatin protein expression after 72 h of exercise cessation.
exercise. The apparent disconnect between the changes in We speculate that the altered myostatin response in the LPD
myostatin expression and the number of myostatin+ SCs is group may represent a compensatory attempt to allow muscle
explained by the fact that we use Western blotting and real-time reconditioning to occur when ample dietary protein-derived
PCR analyses to assess myostatin protein and mRNA expression amino acids become available.
in mixed muscle tissue, respectively. In contrast, using immuno- Although it remains to be established through which molec-
histochemistry, we assess the colocalization of myostatin within ular pathways the proposed feedback coupling between the
the SCs. As such, it is not surprising that the timing and/or post-exercise SC signaling response and food availability is
direction of changes in myostatin expression and the number of regulated, the present findings provide additional evidence that
myostatin+ SCs do not necessarily follow the same direction. post-exercise myostatin expression can be modulated by dietary
Moreover, even myostatin mRNA and protein expression levels protein intake. This opens up a wide range of new research
seem discongruent. It has been suggested previously that a SMAD questions in which we need to address how physical activity and
family member 7 (SMAD7)-dependent negative feedback loop nutrition interact to allow proper muscle reconditioning. Insight
exists through which increased myostatin protein expression is in this intricate interaction will be of key importance to support
associated with decreased myostatin mRNA expression (53,54). skeletal muscle mass maintenance and muscle adaptation in
However, such a relation cannot fully explain the observed both health and disease.
discrepancy between myostatin mRNA and protein in the present We conclude that SC content and activation status change in
study, especially at the 24-h time point. Clearly, although the a time-dependent and muscle fiber type-dependent manner
exercise-induced decrease in myostatin mRNA expression is in during 72 h of post-exercise recovery from a single bout of
line with previous reports (4,38,41,54–56), more research is resistance exercise. In addition, the post-exercise skeletal muscle
warranted to elucidate the intricate myostatin signaling during adaptive response is changed by the availability of dietary
post-exercise recovery in human skeletal muscle and more protein, as evidenced by an extended disinhibition of myostatin
specifically the expression or translocation of myostatin out of expression during post-exercise recovery when dietary protein
the SC. intake is restricted.
Dietary protein and the satellite cell response 143
Acknowledgments 17. Koopman R, Verdijk LB, Manders RJF, Gijsen AP, Gorselink M, Pijpers
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leucine stimulates muscle protein synthesis rates to the same extent in
and B.B.B.G. conducted the research; T.S., J.S.J.S., B.R.M., and young and elderly lean men. Am J Clin Nutr. 2006;84:623–32.
J.v.K. performed the immunohistochemical and biochemical 18. Tipton KD, Ferrando AA, Phillips SM, Doyle D Jr, Wolfe RR.
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Dietary protein and the satellite cell response 145