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doi: 10.1152/japplphysiol.00395.2012
113:206-214, 2012. First published 24 May 2012;J Appl Physiol
Garnham, John A. Hawley and Vernon G. Coffey
Donny M. Camera, Daniel W. D. West, Nicholas A. Burd, Stuart M. Phillips, Andrew P.
anabolic response to resistance exercise
Low muscle glycogen concentration does not suppress the
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Low muscle glycogen concentration does not suppress the anabolic response
to resistance exercise
Donny M. Camera,
1
Daniel W. D. West,
2
Nicholas A. Burd,
2
Stuart M. Phillips,
2
Andrew P. Garnham,
3
John A. Hawley,
1
and Vernon G. Coffey
1
1
Health Innovations Research Institute, School of Medical Sciences, RMIT University, Melbourne, Australia;
2
Exercise
Metabolism Research Group, Department of Kinesiology, McMaster University, Hamilton, Ontario, Canada; and
3
Exercise,
Muscle, and Metabolism Unit, School of Exercise and Nutrition Sciences, Deakin University, Melbourne, Australia
Submitted 26 March 2012; accepted in final form 22 May 2012
Camera DM, West DW, Burd NA, Phillips SM, Garnham AP,
Hawley JA, Coffey VG. Low muscle glycogen concentration does
not suppress the anabolic response to resistance exercise. J Appl
Physiol 113: 206 –214, 2012. First published May 24, 2012;
doi:10.1152/japplphysiol.00395.2012.—We determined the effect of
muscle glycogen concentration and postexercise nutrition on anabolic
signaling and rates of myofibrillar protein synthesis after resistance
exercise (REX). Sixteen young, healthy men matched for age, body
mass, peak oxygen uptake (V
˙O
2peak
) and strength (one repetition
maximum; 1RM) were randomly assigned to either a nutrient or
placebo group. After 48 h diet and exercise control, subjects under-
took a glycogen-depletion protocol consisting of one-leg cycling to
fatigue (LOW), whereas the other leg rested (NORM). The next
morning following an overnight fast, a primed, constant infusion of
L-[ring-
13
C
6
] phenylalanine was commenced and subjects completed
8 sets of 5 unilateral leg press repetitions at 80% 1RM. Immediately
after REX and 2 h later, subjects consumed a 500 ml bolus of a
protein/CHO (20 g whey ⫹40 g maltodextrin) or placebo beverage.
Muscle biopsies from the vastus lateralis of both legs were taken at
rest and 1 and 4 h after REX. Muscle glycogen concentration was
higher in the NORM than LOW at all time points in both nutrient and
placebo groups (P⬍0.05). Postexercise Akt-p70S6K-rpS6 phosphor-
ylation increased in both groups with no differences between legs
(P⬍0.05). mTOR
Ser2448
phosphorylation in placebo increased 1 h
after exercise in NORM (P⬍0.05), whereas mTOR increased
⬃4-fold in LOW (P⬍0.01) and ⬃11 fold in NORM with nutrient
(P⬍0.01; different between legs P⬍0.05). Post-exercise rates of
MPS were not different between NORM and LOW in nutrient
(0.070 ⫾0.022 vs. 0.068 ⫾0.018 %/h) or placebo (0.045 ⫾0.021 vs.
0.049 ⫾0.017 %/h). We conclude that commencing high-intensity
REX with low muscle glycogen availability does not compromise the
anabolic signal and subsequent rates of MPS, at least during the early
(4 h) postexercise recovery period.
skeletal muscle; muscle protein synthesis
SKELETAL MUSCLE GLYCOGEN CONCENTRATION exerts numerous
regulatory effects on cell metabolism in response to contraction
(23). Indeed, commencing endurance-based exercise with low
muscle glycogen availability has been shown to increase the
maximal activities of several oxidative enzymes in skeletal
muscle that promote endurance adaptation (21, 46). Although
the anabolic effects of resistance-based exercise on skeletal
muscle are well established (9), little is known regarding the
effects of altered muscle glycogen concentration availability on
the acute protein synthetic response to resistance exercise and
whether the summation of these responses may enhance or
attenuate training-induced adaptation.
The complex regulatory process of protein synthesis after
muscle contraction and/or protein ingestion includes activation
of the Akt-mTOR-S6K signaling pathway to initiate translation
(16, 38). Numerous studies have addressed the signaling re-
sponses to resistance exercise under a variety of nutritional
states (i.e., fasted/fed) (10, 43). However, the effects of muscle
glycogen availability have yet to be clearly elucidated. Work
by Creer and colleagues (11) showed an attenuation in Akt
phosphorylation during recovery when subjects commenced a
bout of moderate-intensity resistance exercise with low (⬃175
mmol/kg dry wt) vs. high (⬃600 mmol/kg dry wt) muscle
glycogen. Furthermore, contraction-induced translational sig-
naling may be suppressed when energy-sensing AMPK activity
is increased (1, 42). Wojtaszeski and coworkers (44) have
observed elevated resting and exercise-induced AMPK activity
when muscle glycogen levels were low (⬃160 mmol/kg dry
wt) compared with high (⬃910 mmol/kg dry wt). Moreover,
work from our laboratory also previously demonstrated low
muscle glycogen concentration has the capacity to alter basal
transcription levels of select metabolic and myogenic genes
(8). Thus the increased metabolic perturbation when exercising
in a low glycogen state might be expected to inhibit the
anabolic response to resistance exercise.
It is well accepted that protein intake following resistance
exercise is critical for optimizing many of the training-induced
adaptations in skeletal muscle (25). Ingestion of high-quality
protein has been shown to enhance translation initiation sig-
naling and maximally stimulate muscle protein synthesis rates
after resistance exercise (30, 35). Carbohydrate (CHO) inges-
tion provides substrate for muscle glycogen resynthesis, but
has no additive effect on rates of muscle protein synthesis after
resistance exercise (29). The capacity for protein-carbohydrate
coingestion in the early postexercise period to rescue any
putative attenuation of muscle protein synthesis when resis-
tance exercise is performed with low glycogen availability has
not been investigated. Accordingly, the primary aims of this
study were to determine the effect of 1) decreased muscle
glycogen concentration on the acute anabolic response after
resistance exercise performed in the fasted state; and 2) the
effect of protein/CHO supplementation on muscle cell signal-
ing and myofibrillar protein synthesis rates (19) following
exercise commenced with low muscle glycogen. We hypothe-
sized that low muscle glycogen concentration would suppress
the muscle anabolic response to resistance exercise but that
nutrient provision in the early recovery period after exercise
Address for reprint requests and other correspondence: V. Coffey, Exercise
Metabolism Group, School of Medical Sciences, RMIT Univ., PO Box 71,
Bundoora, Victoria 3083, Australia (e-mail: vernon.coffey@rmit.edu.au).
J Appl Physiol 113: 206–214, 2012.
First published May 24, 2012; doi:10.1152/japplphysiol.00395.2012.
8750-7587/12 Copyright ©2012 the American Physiological Society http://www.jappl.org206
at RMIT Library - Bundoora on August 12, 2012http://jap.physiology.org/Downloaded from
would restore muscle anabolism to a state that may promote
hypertrophy.
METHODS
Subjects
Sixteen healthy physically fit male subjects who had been partici-
pating in regular concurrent resistance and endurance training (⬃3⫻/
wk; ⬎1 yr) volunteered for this study. Subjects were randomly
assigned to either a nutrient [n⫽8, age 22.9 ⫾2.6 yr, body mass
80.6 ⫾8.8 kg, peak oxygen uptake (V
˙O
2peak
) 49.8 ⫾5.4
ml·kg
⫺1
·min
⫺1
, unilateral leg press one repetition maximum (1RM)
⬃141.7 ⫾4.6 kg] or placebo group [age 22.5 ⫾4.4 yr, body mass
78.2 ⫾4.7 kg, V
˙O
2peak
47.2 ⫾6.9 ml·kg
⫺1
·min
⫺1
, unilateral leg press
1RM ⬃141.8 ⫾0.8 kg; values are mean ⫾SD]. The experimental
procedures and possible risks associated with the study were ex-
plained to each subject, who all gave written informed consent before
participation. The study was approved by the Human Research Ethics
Committee of RMIT University.
Preliminary Testing
V
˙O
2peak
.Peak oxygen uptake was determined during an incremental
test to volitional fatigue on a Lode cycle ergometer (Groningen, The
Netherlands). The protocol has been described in detail previously
(24). In brief, subjects commenced cycling at a workload equivalent to
2 W/kg for 150 s. Thereafter, the workload was increased by 25 W
every 150 s until volitional fatigue (defined as the inability to maintain
a cadence ⬎70 revolutions/min). Throughout the test, which typically
lasted 12–14 min, subjects breathed through a mouthpiece attached to
a metabolic cart (Parvomedics, Sandy, UT) to determine oxygen
consumption.
Maximal strength. One repetition of maximal dynamic strength
(1RM) for each leg was determined on a plate loaded 45° leg press
machine (CalGym, Caloundra, Australia). Subjects completed the test
with feet placed at the bottom edge of the foot plate and range of
motion was 90° knee flexion/extension.
Familiarization to exercise training sessions. To familiarize sub-
jects to one-legged cycling (described subsequently), each subject
completed three familiarization sessions before the experimental
trial. These sessions consisted of 2 ⫻10 min bouts of one-legged
cycling, with a 2 min recovery period between repetitions. The
power output was gradually increased so that by the final session
subjects were performing one-legged cycling at ⬃75% of their
two-legged V
˙O
2peak
(37).
Diet/exercise control. Before the exercise depletion session (de-
scribed subsequently), subjects were instructed to refrain from exer-
cise training and vigorous physical activity and alcohol and caffeine
consumption for a minimum of 48 h. A CHO-based diet (⬃9 g/kg
body mass) was consumed 36 h before the one-legged exercise
depletion session. All food and drinks were supplied to subjects
prepackaged with a food checklist to record their daily intake.
One-legged glycogen depletion protocol. Subjects began a one-
legged cycling depletion session at a power output that elicited ⬃75%
of two-legged V
˙O
2peak
. The duration of each work bout was 10 min,
with 2 min rest between work bouts. Subjects maintained this work-
to-rest ratio until volitional fatigue. At this time, power output was
decreased by 10 W and subjects cycled at this (lower) work rate with
the same work-to-rest ratio until fatigue. After a 10-min rest, subjects
then completed 90-s one-leg maximal sprints on a Repco RE7100
Ergo (Altona North, Australia), with 60 s of recovery between work
bouts. This protocol was continued until volitional fatigue, defined as
the inability to maintain 70 revolutions/min. To further lower whole
body glycogen stores and minimize glycogen resynthesis in the LOW
leg, subjects completed 30 min of arm cranking on a Monark Rehab
Trainer 881E (Vansbro, Sweden). Following the exercise depletion
session, subjects were fed a low CHO (⬃1 g/kg body mass) evening
meal.
Experimental Testing Session
On the morning of an experimental trial, subjects reported to the
laboratory after a ⬃10-h overnight fast. After resting in the supine
position for ⬃15 min, catheters were inserted into the antecubital vein
of each arm and a baseline blood sample (⬃3 ml) was taken (Fig. 1).
A primed constant intravenous infusion (prime: 2 mol/kg; infusion:
0.05 mol·kg
⫺1
·min
⫺1
)ofL-[ring-
13
C
6
] phenylalanine (Cambridge
Isotopes Laboratories) was then administered. Under local anesthesia
(2–3 ml of 1% Xylocaine) a resting biopsy from the vastus lateralis of
both legs was obtained 1.5 h after commencement of the tracer
infusion using a 5-mm Bergstrom needle modified with suction. At
this time, two separate sites on each leg (⬃5 cm distal from each
other) were prepared for subsequent biopsies. Subjects then completed
a standardized unilateral warm-up (1 ⫻5 repetitions at 50% and 60%
1RM) on a leg-press machine before the resistance exercise testing
protocol was commenced. Resistance exercise consisted of eight sets
of five repetitions at ⬃80% of 1RM for each leg. The glycogen-
depleted leg (LOW) began the protocol, with ⬃60 s rest before the
rested normal leg (NORM) completed the same set. Each set was
separated by a 3-min recovery period during which the subject
remained seated on the machine. The training volume and intensity
and recovery interval were selected to provide sufficient anabolic/
hypertrophy stimulus and minimize metabolic perturbation and has
been used previously (7, 10). If the LOW leg could not complete the
Fig. 1. Schematic representation of the experimental trial. Subjects reported to the laboratory the evening before an experimental trial and performed a 1-legged
glycogen-depletion protocol to fatigue before consuming a low carbohydrate (CHO) meal. After an overnight fast, a constant infusion of L-[ring-
13
C
6
]
phenylalanine was commenced, and subjects completed 8 sets of 5 unilateral leg press repetitions at 80% one repetition maximum (1RM). Immediately after
resistance exercise (REX) and 2 h later, subjects consumed a 500-ml bolus of a protein/CHO beverage (20 g whey ⫹40 g maltodextrin) or placebo. Muscle
biopsies from both legs (vastus lateralis) were taken at rest and at 1 and 4 h after REX.
207Low Glycogen and Resistance Exercise •Camera DM et al.
J Appl Physiol •doi:10.1152/japplphysiol.00395.2012 •www.jappl.org
at RMIT Library - Bundoora on August 12, 2012http://jap.physiology.org/Downloaded from
repetitions, the NORM leg replicated the number of repetitions to
ensure the exercise was work matched and the weight was decreased
5% for subsequent sets. Immediately after the cessation of exercise
and 2 h postexercise, subjects ingested a 500 ml placebo (water,
artificial sweetener) or protein-CHO beverage (20 g whey protein, 40
g maltodextrin). The nutrient beverage was enriched with a small
amount of tracer (to 6.5% of L-[ring-
13
C
6
] phenylalanine) according
to the measured phenylalanine content of the beverage. Subjects
rested throughout a 240-min recovery period, and additional muscle
biopsies were taken 60 and 240 min postexercise and the samples
were stored at ⫺80°C until analysis. Blood samples were collected in
EDTA tubes at regular intervals during the postexercise recovery
period.
Analytical Procedures
Blood glucose and plasma insulin concentration. Whole blood
samples were immediately analyzed for glucose concentration using
an automated glucose analyzer (YSI 2300, Yellow Springs, OH).
Blood samples were then centrifuged at 1,000 gat 4°C for 15 min,
with aliquots of plasma frozen in liquid N
2
and stored at ⫺80°C.
Plasma insulin concentration was measured using a radioimmunoas-
say kit according to the manufacturer’s protocol (Linco Research).
Plasma amino acids and enrichment. Plasma amino acid concen-
trations were determined by HPLC from a modified protocol (34).
Briefly, 100 l of plasma was mixed with 500 l of ice cold 0.6 M
PCA and centrifuged at 15,000 rpm for 2 min at 4°C. The PCA was
neutralized with 250 l of 1.25 M potassium bicarbonate (KHCO
3
),
and the reaction was allowed to proceed on ice for 10 min. Samples
were then centrifuged at 15,000 rpm for 2 min at 4°C, and the
supernatant was separated from the salt pellet and subsequently
derivatized for HPLC analysis. Plasma [ring-
13
C
6
] phenylalanine
enrichments were determined as previously described (17).
Muscle glycogen. A small piece of frozen muscle (⬃20 mg) was
freeze-dried and powdered to determine muscle glycogen concentra-
tion (33). Freeze-dried muscle was extracted with 500 lof2M
hydrochloric acid (HCl), incubated at 100°C for 2 h, and then
neutralized with 1.5 ml of 0.67 M sodium hydroxide for subsequent
determination of glycogen concentration via enzymatic analysis with
fluorometric detection (Jasco FP-750 spectrofluorometer, Easton,
MD) at excitation 365 nm/emission 455 nm.
Western blots. Muscle samples were homogenized in buffer con-
taining 50 mM Tris·HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 10%
glycerol, 1% Triton X-100, 50 mM NaF, 5 mM sodium pyrophos-
phate, 1 mM DTT, 10 g/ml trypsin inhibitor, 2 g/ml aprotinin, 1
mM benzamidine, and 1 mM PMSF. Samples were spun at 18,000 g
for 30 min at 4°C, and the supernatant was collected for Western blot
analysis while the pellet was processed to extract the myofibrillar
enriched proteins (described below). After determination of protein
concentration using a BCA protein assay (Pierce, Rockford, IL),
lysate was resuspended in Laemmli sample buffer, separated by
SDS-PAGE, and transferred to polyvinylidine fluoride membranes
blocked with 5% nonfat milk, washed with 10 mM Tris·HCl, 100 mM
NaCl, and 0.02% Tween 20, and incubated with primary antibody
(1:1,000) overnight at 4°C on a shaker. Membranes were incubated
with secondary antibody (1:2,000), and proteins were detected via
enhanced chemiluminescence (Amersham Biosciences, Buckingham-
shire, UK; Pierce Biotechnology) and quantified by densitometry
(Chemidoc, BioRad, Gladesville, Australia). All sample (50 g) time
points for each subject were run on the same gel. Polyclonal anti-
phospho-Akt
Ser473
(no.9271), mTOR
Ser2448
(no. 2971), glycogen syn-
thase (GS)
Ser641
(no.3891), monoclonal anti-phospho-S6 ribosomal
protein
Ser235/6
(no.4856), AMPK␣
Thr172
(no. 2535), and AS160
(no.2670) were from Cell Signaling Technology (Danvers, MA).
Polyclonal anti-phospho-p70S6K
Thr389
(no. 04 –392) was from Milli-
pore (Temecula, CA). When commercially available, positive controls
(Cell Signaling Technology) were included confirming the band of
interest. Data are expressed relative to ␣-tubublin (no. 3873, Cell
Signaling Technology) in arbitrary units.
RNA Extraction and Quantification
Skeletal muscle tissue RNA extraction was performed using a
TRIzol-based kit according to the manufacturer’s directions (Invitro-
gen, Melbourne, Australia, Cat. No. 12183– 018A). Briefly, ⬃15 mg
of skeletal muscle tissue was removed from RNAlater-ICE solution
and homogenized in TRIzol. After elution through a spin cartridge,
extracted RNA was quantified using a QUANT-iT analyzer kit (In-
vitrogen, Cat. No. Q32852) according to the manufacturer’s direc-
tions.
Reverse Transcription and Real-Time PCR
First-strand complementary DNA (cDNA) synthesis was per-
formed using commercially available TaqMan Reverse Transcription
Reagents (Invitrogen) in a final reaction volume of 20 l. All RNA
samples and control samples were reverse transcribed to cDNA in a
single run from the same reverse transcription master mix. Serial
dilutions of a template RNA (AMBION; Cat. No. AM7982) were
included to ensure efficiency of reverse transcription and for calcula-
tion of a standard curve for real-time quantitative polymerase chain
reaction (RT-PCR). Quantification of mRNA (in duplicate) was per-
formed on a BioRad iCycler (BioRad). Taqman-FAM-labeled primer/
probes for atrogin (Cat. No. Hs01041408) and myostatin (Cat. No.
Hs00976237) were used in a final reaction volume of 20 l. PCR
conditions were 2 min at 50 °C for UNG activation, 10 min at 95°C,
then 40 cycles of 95°C for 15 s and 60°C for 60 s. Glyceraldehyde-
3-phosphate dehydrogenase (GAPDH) (Cat. No. Hs Hs99999905)
was used as a housekeeping gene to normalize threshold cycle (CT)
values. The relative amounts of mRNAs were calculated using the
relative quantification (⌬⌬CT) method (31).
Myofibrillar Protein Synthesis
Myofibrillar enriched proteins were isolated according to a
modified protocol (35). Briefly, the myofibrillar pellet was solubi-
lized in 0.3 M NaOH, precipitated in 1 M PCA, washed in ethanol,
and hydrolyzed overnight with 6 M HCl while being heated to
120°C. Liberated myofibrillar and plasma amino acids (for deter-
mination of L-[ring-
13
C
6
] phenylalanine enrichment) were purified
using cation-exchange chromatography (Dowex 50WX8 –200 res-
in; Sigma-Aldrich) and converted to their N-acetyl-n-propyl ester
derivatives for analysis by gas chromatography combustion-
isotope ratio mass spectrometry (GC-C-IRMS: Hewlett Packard
6890; IRMS model Delta Plus XP, Thermo Finnagan, Waltham,
MA). Intracellular free amino acids (IC) were extracted from a
separate piece of wet muscle (⬃20 mg) with ice-cold 0.6 M PCA.
Muscle was homogenized, and the free amino acids in the super-
natant were purified by cation-exchange chromatography and con-
verted to their heptafluorobutyric (HFB) derivatives before analy-
sis by GC-MS (models 6890 GC and 5973 MS; Hewlett-Packard,
Palo Alto, CA) as previously described (35).
Calculations
The rate of myofibrillar protein synthesis was calculated using the
standard precursor-product method: FSR (%/h) ⫽[(E
2b
⫺E
1b
)/
(E
IC
⫻t)]⫻100, where E
2b
⫺E
1b
represents the change bound protein
enrichment between two biopsy samples, E
IC
is the average enrich-
ment of intracellular phenylalanine between the two biopsy samples,
and tis the time between two sequential biopsies.
Statistical Analysis
All data were analyzed by two-way ANOVA (two factor: time ⫻
glycogen concentration) with Student-Newman-Keuls post hoc anal-
ysis. Statistical significance was established when P⬍0.05 (SigmaS-
208 Low Glycogen and Resistance Exercise •Camera DM et al.
J Appl Physiol •doi:10.1152/japplphysiol.00395.2012 •www.jappl.org
at RMIT Library - Bundoora on August 12, 2012http://jap.physiology.org/Downloaded from
tat for windows Version 3.11). Based on our a priori hypothesis that
anabolic responses to nutrient administration are significantly elevated
compared with placebo as shown previously (6, 15), we chose not to
make direct comparisons between nutrient and placebo interventions.
Data for Western blotting and mRNA abundance were log-trans-
formed prior to analysis. Log-transformed delta values between data
time points were also directly compared and converted to Cohen
effect sizes (ES). The default confidence interval was 90% to calculate
ES making the same assumptions about sampling distributions that
statistical packages use to derive Pvalues (26). We interpreted the
magnitude of the ES by using conventional threshold values of 0.2 as
the smallest effect, 0.5 as a moderate effect, and 0.8 as a large ES (26).
All data are expressed as arbitrary unit ⫾SD.
RESULTS
One-Legged Depletion Session and Muscle Glycogen
The time spent completing the one-legged depletion session
at an intensity of ⬃75% of two-legged V
˙O
2peak
was 100 ⫾3
min. Subjects also completed an average of 6 ⫾2 one-legged
maximal effort sprint repetitions.
As intended, the combination of the exercise depletion
protocol and dietary manipulation generated divergent muscle
glycogen levels that were higher in NORM than LOW at rest
for the placebo (382 vs. 176 mmol/kg dry wt; P⬍0.001) and
nutrient groups (383 vs. 184 mmol/kg dry wt; P⬍0.05;
Fig. 2). Glycogen concentration was decreased from rest in the
NORM leg in both groups at 1 and 4 h postexercise (P⬍0.05).
However, no significant change from rest was evident in the
LOW leg for either group. Muscle glycogen increased between
1 and 4 h postexercise in the LOW leg in the nutrient group
(⬃84 mmol/kg dry wt; P⬍0.01).
Plasma Insulin, Glucose, and Essential Amino Acids
There were main effects for plasma insulin and glucose
concentration in the nutrient but not the placebo group (P⬍
0.001; Fig. 3, Aand B). Peak blood insulin and glucose
concentrations occurred at 30 and 150 min postexercise (P⬍
0.001). Plasma essential amino acids (EAA) were elevated
about rest 150 min and 180 min (P⬍0.05) postexercise in the
nutrient group only (Fig. 3C).
Plasma Tracer Enrichments
Plasma L-[ring
13
C
6
] phenylalanine enrichment at rest and
60, 120, 180, and 240 min postexercise for nutrient and
placebo treatments were 0.042, 0.045, 0.055, and 0.049,
and 0.058, 0.054, 0.062, 0.057, 0.065, and 0.054 tracer-to-
tracee ratio: t/T, respectively. Linear regression analysis indi-
cated that the slopes of the plasma enrichments were not
significantly different from zero, demonstrating that isotopic
plateau was achieved.
Cell Signaling
Akt-mTOR-p70S6K-rpS6. There were main effects for
Akt
Ser473
phosphorylation for time and glycogen status (P⬍
0.05, Fig. 4A). Resting Akt phosphorylation was higher in
LOW than NORM in the placebo group and increased ⬃2-fold
1 h postexercise in NORM only (P⬍0.05, ES 0.75) before
Fig. 2. Muscle glycogen concentration at rest and during 4 h recovery after
resistance exercise (8 ⫻5 leg unilateral leg press at ⬃80% 1RM) and ingestion
of either 500 ml placebo or nutrient beverage immediately post and 2 h
postexercise in NORM and LOW glycogen legs. Values are mean ⫾SD. dw,
dry weight. Significantly different (P⬍0.05) vs. (a) rest, (b) 1 h and (*)
between treatments (NORM vs. LOW) at equivalent time point.
Fig. 3. Plasma insulin (A), blood glucose (B), and plasma essential amino acid
concentration (C) at rest and during 240 min recovery following resistance
exercise (8 ⫻5 leg unilateral leg press at ⬃80% 1RM) and ingestion of either
500 ml placebo or nutrient beverage immediately post and 2 h postexercise.
Values are mean ⫾SD. Significantly different (P⬍0.05) vs. (a) rest.
209Low Glycogen and Resistance Exercise •Camera DM et al.
J Appl Physiol •doi:10.1152/japplphysiol.00395.2012 •www.jappl.org
at RMIT Library - Bundoora on August 12, 2012http://jap.physiology.org/Downloaded from
returning to baseline at 4 h. Phosphorylation at rest was also
higher in the LOW compared with the NORM leg (P⫽0.058)
and increased ⬃10-fold in LOW and ⬃21-fold in NORM 1 h
after resistance exercise in the nutrient group (P⬍0.001, ES ⬎
1). Akt phosphorylation remained above resting levels follow-
ing 4 h recovery in the NORM leg only of the nutrient group
(P⬍0.05, ES ⬎1).
There were main effects for time and glycogen concentration
for mTOR
Ser2448
phosphorylation (P⬍0.05, Fig. 4B). mTOR
phosphorylation increased ⬃1-fold above rest at 1 and 4 h
postexercise in the NORM but not the LOW leg in the placebo
group (P⬍0.05, ES ⬃0.5). There was also disparity between
legs in the nutrient group that increased in the NORM com-
pared with LOW leg at 1 and 4 h recovery (P⬍0.05, ES ⬎1).
Phosphorylation in the nutrient group did increase above rest-
ing levels ⬃4-fold and ⬃1-fold in the LOW leg (P⬍0.01), an
effect that was more pronounced in the NORM leg (⬃11-fold
and ⬃4-fold, respectively; P⬍0.01).
p70S6K
Thr389
phosphorylation was higher at rest in the
LOW leg compared with NORM in placebo (P⬍0.05, Fig.
4C). The post– exercise phosphorylation response increased
above rest at 1 and4h(⬃5-fold) in NORM but not the LOW
leg (P⬍0.01; ES ⬃1). The comparison of p70S6K phos-
phorylation between legs at rest in the nutrient group ap-
proached significance and was increased above resting levels in
both legs at 1 h (LOW: ⬃45 fold, NORM: ⬃82 fold, ES ⬎1;
P⬍0.001) and 4 h (LOW: ⬃14 fold, NORM: ⬃16 fold; P⬍
0.001) during recovery from resistance exercise.
There were main effects for rpS6
Ser235/6
phosphorylation in
the nutrient but not placebo group (P⬍0.05, Fig. 4D). There
were ⬃4- and ⬃6-fold increases in rpS6
Ser235/6
phosphoryla-
tion in the LOW leg with placebo at 1 and 4 h, respectively
(P⬍0.05), and this effect was mirrored in NORM with
⬃15-fold increases at1h(P⬍0.001, ES 0.9) and4h(P⬍
0.01, ES 0.5). Resting rpS6
Ser235/6
phosphorylation was signif-
icantly elevated in the LOW compared with NORM leg (P⬍
0.05) in the nutrient group and increased in both legs 1 h after
resistance exercise and remained elevated following 4 h recov-
ery (P⬍0.001, Fig. 4D).
GS-AS160-AMPK. There were significant main effects for
GS
Ser641
phosphorylation for time and glycogen status in
placebo and nutrient groups (Fig. 5A). GS phosphorylation
was markedly higher in NORM than LOW at all time points
in the placebo condition (P⬍0.001). Following resistance
exercise phosphorylation decreased ⬃3- to 4-fold 1 h
postexercise in both legs (P⬍0.01) before increasing at 4
h in the LOW but not NORM leg (P⬍0.01, ES 0.4).
Similarly, phosphorylation was higher at all points in
NORM compared with LOW in the nutrient group (P⬍
0.05). There was a decrease from resting levels in the LOW
and NORM legs at 1 and 4 h postexercise (P⬍0.05) but GS
phosphorylation only increased between 1 and4hinthe
LOW leg (⬃5-fold, P⬍0.01, ES 0.6).
There were main effects for time in placebo and nutrient
groups for phospho-AS160 (P⬍0.05, Fig. 5B). AS160 in-
creased in the placebo condition at1h(⬃2-fold P⬍0.01, ES
0.9) and4h(⬃1-fold P⬍0.05, ES 0.9) after resistance
exercise in the LOW leg only. AS160 phosphorylation in the
nutrient group was increased at 1 h recovery in the LOW leg
(⬃4-fold, P⬍0.001) and both 1 and 4 h postexercise in the
NORM leg (⬃8-fold, P⬍0.001, ES 0.8; ⬃1 fold, P⬍0.05,
ES 0.5, respectively).
Fig. 4. Akt
Ser473
(A), mammalian target of
rapamycin (mTOR)
Ser2448
(B), p70S6K
Thr389
(C), and ribosomal protein S6 (rpS6)
Ser235/6
(D) phosphorylation in skeletal muscle at rest
and during 4 h postexercise recovery follow-
ing resistance exercise (8 ⫻5 leg unilateral
leg press at ⬃80% one 1RM). Images are
representative blots and values are expressed
relative to ␣-tubulin and presented in arbi-
trary units (mean ⫾SD, n⫽8). Significantly
different (P⬍0.05) vs. (a) rest, (b) 1 h, and
(*) between treatments (NORM vs. LOW) at
equivalent time point.
210 Low Glycogen and Resistance Exercise •Camera DM et al.
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AMPK
Thr172
phosphorylation was not different at any time
in placebo or nutrient groups (Fig. 5C).
Myofibrillar Protein Synthesis
There were no differences in the rates of myofibrillar protein
synthesis rates during the 1- to 4-h recovery period between
LOW and NORM in placebo (0.049 ⫾0.017 vs. 0.045 ⫾0.021
%/h) or nutrient (LOW vs. NORM: 0.068 ⫾0.018 vs. 0.070 ⫾
0.022 %/h) conditions (Fig. 6).
mRNA expression
Atrogin-myostatin. Atrogin mRNA abundance decreased in
the placebo group between rest and 4 h postexercise in the
LOW (⬃1.2-fold; P⬍0.05) and NORM leg (⬃1.8-fold; P⬍
0.01) and was also different between 1 and4hinNORM
(⬃1.4-fold; ES 0.5, P⬍0.01) (Fig. 7A). Likewise, atrogin
mRNA decreased from rest following 4 h recovery in the LOW
(⬃0.5-fold; P⬍0.001) and NORM leg (⬃2.6-fold, ES 0.6;
P⬍0.001) in the nutrient group. Atrogin was also different
between 1 and 4 h postexercise in the LOW (⬃0.5-fold, P⬍
0.001) and NORM leg (⬃2.2-fold, ES 0.25; P⬍0.001). The
mRNA abundance of atrogin was higher in the LOW leg
compared with NORM leg at the 4 h postexercise time point in
Fig. 5. Glycogen synthase (GS)
Ser641
(A), Akt substrate 160 kDa (AS160) (B),
and 5=-adenosine monophosphate-activated protein kinase (AMPK)
Thr172
(C)
phosphorylation in skeletal muscle at rest and during 4 h postexercise recovery
following resistance exercise (8 ⫻5 leg unilateral leg press at ⬃80% 1RM).
Images are representative blots and values are expressed relative to ␣-tubulin
and presented in arbitrary units (mean ⫾SD, n⫽8). Significantly different
(P⬍0.05) vs. (a) rest, (b) 1 h and (*) between treatments (NORM vs. LOW)
at equivalent time point.
Fig. 6. Myofibrillar protein fractional synthetic rates (FSR) during4hof
recovery after resistance exercise (8 ⫻5 leg unilateral leg press at ⬃80%
1RM) and ingestion of either 500 ml placebo or nutrient beverage immediately
post and 2 h postexercise in NORM and LOW glycogen legs. Values are
means ⫾SD.
Fig. 7. Atrogin (A) and myostatin (B) mRNA abundance at rest and during 4
h postexercise recovery following resistance exercise (8 ⫻5 leg unilateral leg
press at ⬃80% 1RM). Values are expressed relative to GAPDH and presented
in arbitrary units (mean ⫾SD, n⫽8). Significantly different (P⬍0.05) vs.
(a) rest, (b) 1 h and (*) between treatments (NORM vs. LOW) at equivalent
time point.
211Low Glycogen and Resistance Exercise •Camera DM et al.
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at RMIT Library - Bundoora on August 12, 2012http://jap.physiology.org/Downloaded from
the nutrient condition (P⬍0.01). Myostatin mRNA decreased
in the placebo group from rest to4hinLOW(⬃1.8-fold; P⬍
0.01) and NORM (⬃1.4-fold; P⬍0.001) and between 1 and
4 h recovery in NORM only (⬃0.8 fold; ES 0.33, P⬍0.01)
(Fig. 7B). In the nutrient condition, decreases in myostatin
mRNA expression were only observed in the NORM leg that
was reduced ⬃2.4-fold between rest and1h(ES⬃1, P⬍
0.01) and ⬃1-fold 1– 4 h during recovery from resistance
exercise (ES 0.8, P⬍0.05). Myostatin mRNA was higher in
the LOW leg compared with NORM leg at 4 h postexercise in
the nutrient group (P⬍0.01).
DISCUSSION
It is generally accepted that skeletal muscle adaptation to
repeated bouts of contractile activity are specific to the mode,
intensity, and duration of the exercise stimulus (9), but it is unclear
how changes in skeletal muscle glycogen availability may mod-
ulate nutrient-training interactions to promote or inhibit the adap-
tive response to resistance exercise. Here we report for the first
time that commencing a bout of strenuous resistance exercise with
low muscle glycogen concentration has negligible effects on
anabolic cell signaling and rates of muscle protein synthesis
during the early (4 h) postexercise recovery period. As expected,
ingestion of a protein/CHO beverage enhanced the anabolic re-
sponse to resistance exercise but failed to augment differences
between the normal and low glycogen legs.
Exercising in a low glycogen state presents a unique metabolic
challenge to skeletal muscle with few studies having investigated
the interaction of glycogen content and nutrient provision or their
effect on the adaptation response to resistance exercise. Hence the
primary novel finding of the present study was that rates of
myofibrillar protein synthesis between the NORM and LOW
glycogen legs during 1– 4 h recovery after resistance exercise
were not different (Fig. 6). This finding was unexpected given that
acute energy deficit has previously been reported to attenuate rates
of mixed muscle protein synthesis by ⬃19% (36), although the
metabolic perturbation with low glycogen in the current study
may have had less impact on cell energy status and thus failed to
modulate the myofibrillar protein synthetic response to low-
volume high-intensity resistance exercise. Nonetheless, our one
leg depletion protocol in combination with a low carbohydrate
meal was successful in creating divergence in resting muscle
glycogen concentration. Muscle glycogen content can be reduced
by ⬃25–40% following a single bout of resistance exercise (7,
39) compared with reductions of ⬃50% or greater after high-
intensity endurance exercise (7, 44). The distinct metabolic de-
mands with endurance exercise may make the adaptation response
in mitochondrial and CHO/fat metabolism more sensitive when
training with low glycogen, although any benefit to endurance
performance has yet to be established (28, 45, 46). In the present
study, glycogen availability in the LOW leg may have been
sufficient to complete the short periods of contractile activity with
long (3 min) recovery between sets without compromising myo-
fibrillar protein synthesis rates during recovery. Moreover, it is
possible that greater difference in glycogen availability is neces-
sary to generate differences in metabolic processes that might alter
muscle protein synthesis. However, even an endurance exercise
bout commenced with low glycogen has only modest effects on
muscle protein metabolism (3, 27).
The ingestion of carbohydrate postexercise does not increase
muscle protein synthesis in humans per se but we hypothesized
carbohydrate coingested with protein may have promoted the
anabolic response when muscle glycogen was compromised. In
the present study, the nutrient ingestion protocol resulted in
divergent plasma glucose, insulin, and amino acid profiles during
the 4-h recovery period (Fig. 3). However, we failed to observe an
effect of carbohydrate coingestion on anabolic signaling and rates
of myofibrillar protein synthesis despite moderate muscle glyco-
gen repletion during the early phase of recovery. Although insulin
has been suggested as a potential anabolic hormone that contrib-
utes to skeletal muscle accretion (2), recent evidence shows
insulin to play only a permissive role in muscle anabolism, at least
in young men (29). Despite the availability of carbohydrate for
restoring muscle glycogen and the associated increase in plasma
insulin levels during recovery in the low glycogen leg, there was
no difference in myofibrillar protein synthesis compared with the
normal leg. Nonetheless, our results provide further evidence of
the well-established capacity for amino acids to augment the
muscle protein synthesis response after resistance exercise follow-
ing an overnight fast.
Another novel finding of our study was that divergent glycogen
concentrations following the depletion protocol were associated
with differences in pre-exercise phosphorylation status of key
muscle cell signaling proteins that were generally ameliorated
after the resistance exercise bout. Acute changes in translation
initiation and glucose metabolism are stimulated by nutrient and
contractile overload and mediated, at least in part, through the
activation of the Akt-mTOR-S6K kinases (16, 38). We observed
elevated resting Akt
Ser473
phosphorylation in the LOW glycogen
leg (Fig. 4A) but this disparity did not extend to the postexercise
recovery period with similar responses between legs. In contrast,
Creer and colleagues (11) reported similar Akt phosphorylation at
rest and an attenuated postexercise response with low muscle
glycogen. The discrepancies between studies may reflect differ-
ences in protocols employed for generating divergent glycogen
concentration and the training status of the subjects, but is most
likely related to the timing of postexercise biopsies. Nonetheless,
it seems plausible that Akt-mediated signaling would be enhanced
to promote glucose transport and glycogen resynthesis at rest due
to low muscle glycogen, but strong contractile stimuli upregulates
the metabolic response uniformly regardless of glycogen status.
As might be expected, differences in markers of glucose uptake
glycogen synthesis and were observed at rest and postexercise
(Fig. 5). Glycogen synthase
Ser641
dephosphorylation (activation)
was significantly greater in LOW compared with NORM at every
time point in the nutrient and placebo groups (Fig. 5A). Moreover,
GS was significantly dephosphorylated in the LOW glycogen legs
of both groups 1 h after the resistance exercise bout. Considering
we previously showed no change in GS phosphorylation after
resistance exercise (7), this may indicate that low glycogen con-
centration is a critical factor for the capacity of low-volume,
high-intensity resistance exercise to exert any significant effect on
glycogen synthase activity and (re)synthesis. Postexercise in-
creases in AS160 phosphorylation were apparent with protein/
CHO ingestion but were not different between NORM and LOW
glycogen legs (Fig. 5B). Conversely, AS 160 phosphorylation
increased postexercise only in LOW from the placebo group. This
suggests any sensitivity AS 160 may exhibit to low glycogen
availability following resistance exercise is eliminated upon ade-
quate nutrient ingestion.
212 Low Glycogen and Resistance Exercise •Camera DM et al.
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There was no effect of glycogen status on mTOR
Ser2448
phos-
phorylation after resistance exercise in the placebo group, whereas
nutrient ingestion elevated mTOR above rest 1 h after exercise to
a greater extent in the NORM compared with the LOW leg (Fig.
4B). The increased phosphorylation of mTOR with protein/CHO
ingestion likely represents a synergistic effect mediated through
the insulin signaling cascade and capacity for amino acids to
directly activate mTOR through a putative interaction between the
Rag- and Rheb-GTPases (40). Although the disparity in the
magnitude of mTOR phosphorylation may indicate a modest
suppression due to low glycogen, there was still an ⬃4-fold
increase in mTOR phosphorylation in the LOW leg that was
sufficient to initiate activation of downstream proteins more prox-
imal to translation initiation. Moreover, the sustained elevation in
mTOR phosphorylation 4 h after resistance exercise in the nutrient
group was similar between NORM and LOW legs.
The AMPK complex has a glycogen binding domain that may
influence AMPK’s role as a cell energy sensor while also having
the capacity to negatively regulate mTOR activation (5, 20).
Previous work demonstrated increased mTOR phosphorylation
and muscle protein synthesis rates concomitant with elevated
AMPK activity following exercise, indicating that any putative
effect of AMPK on muscle protein synthesis in humans after
resistance exercise may only be modest (14). Regardless, we
failed to observe any changes in AMPK phosphorylation between
legs in the placebo or nutrient groups that might explain the
moderate difference in mTOR phosphorylation 1 h postexercise.
Moreover, we demonstrate similar phosphorylation status of reg-
ulatory targets of mTOR proximal to translation initiation indic-
ative of comparable activation despite disparity in glycogen con-
centration. This is in agreement with numerous previous studies
investigating translational signaling that show increases in p70
S6K and rpS6 phosphorylation during the early recovery period
following exercise and the augmented response with nutrient
provision after an overnight fast (12, 13, 30).
Consistent with the changes in cell signaling, muscle mRNA
responses of select genes associated with muscle proteolysis and
catabolism were relatively unchanged by muscle glycogen con-
centration. Muscle atrophy F-Box (MAFbx; also known as
atrogin-1) belongs to the ubiquitin proteasome pathway involved
in tagging contractile protein for degradation by cellular proteo-
somes (4, 18), whereas myostatin is a putative negative regulator
of muscle growth (41). The decrease in atrogin-1 mRNA abun-
dance at 4 h postexercise in NORM and LOW legs in the placebo
and nutrient groups (Fig. 7A) is similar to previous work from our
laboratory showing a decrease in atrogin mRNA 3 h after resis-
tance exercise (8). Likewise, the postexercise decrease in myo-
statin mRNA expression (Fig. 7B) is in accordance with previous
studies that have examined mRNA changes following resistance
exercise (22, 32). Interestingly, there was higher atrogin-1 and
myostatin expression in the LOW glycogen leg compared with the
normal glycogen leg after 4 h recovery in the nutrient group. To
our knowledge, this is the first study to investigate the interaction
of glycogen concentration and nutrients on catabolic genes after
resistance exercise. The possibility exists that the abundance of
exogenous CHO/amino acids and low muscle glycogen generated
a signal to “switch on” processes regulating muscle remodeling/
adaptation after exercise rather than preserving muscle protein by
suppressing breakdown without postexercise nutrient provision in
the placebo/fasted condition. However, it should be noted that the
mRNA abundance of atrogin and myostatin were not elevated
above resting levels and probably represents only a modest effect
on catabolic processes.
In conclusion, and in contrast to our original hypothesis, com-
mencing a bout of strenuous resistance exercise with low muscle
glycogen availability failed to attenuate anabolic signaling and
rates of myofibrillar protein synthesis compared with when the
same exercise bout was undertaken with normal glycogen avail-
ability. Protein-CHO supplementation also failed to mediate any
divergence in muscle protein synthesis between NORM and
LOW during recovery. Moreover, whereas we observed some
disparity between legs, undertaking exercise with low glycogen
did not induce an increase in select mRNA markers of catabolic
activity. Although we cannot rule out the possibility that alterna-
tive resistance training bouts employing different contraction
volumes and intensities might generate a more pronounced effect
of glycogen concentration on postexercise muscle cell signaling
and muscle protein synthesis rates, our findings indicate that
commencing resistance exercise with low muscle glycogen does
not impair this anabolic response in the early recovery period.
This is imperative when considering the potential for suboptimal
muscle glycogen situations when undertaking multiple high-
intensity exercise bouts in a day. Nonetheless, whereas low
glycogen availability may promote the aerobic training pheno-
type, we provide new information to show that modulating gly-
cogen concentration neither promotes nor inhibits the acute adap-
tation response after resistance exercise.
ACKNOWLEDGMENTS
We thank Fonterra Co-Operative Group Limited, Australia, for the gift of
whey protein for this study. We are grateful to Tracy Rerecich and Todd Prior
for their technical expertise.
GRANTS
This study was funded by the Australian Sports Commission through a
general collaborative grant awarded to V. G. Coffey and J. A. Hawley. D. M.
Camera is supported by a National Health and Medical Research Council
Postgraduate scholarship.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: D.M.C., D.W.W., N.A.B., A.P.G., and V.G.C. per-
formed experiments; D.M.C., D.W.W., N.A.B., S.M.P., and V.G.C. analyzed
data; D.M.C. and V.G.C. interpreted results of experiments; D.M.C. prepared
figures; D.M.C. and V.G.C. drafted manuscript; D.M.C., D.W.W., N.A.B.,
S.M.P., J.A.H., and V.G.C. edited and revised manuscript; D.M.C., D.W.W.,
N.A.B., S.M.P., J.A.H., and V.G.C. approved final version of manuscript;
S.M.P., J.A.H., and V.G.C. conception and design of research.
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