Journals of Gerontology: BIOLOGICAL SCIENCES
Cite journal as: J Gerontol A Biol Sci Med Sci. 2012 November;67(11):1170–1177
doi:10.1093/gerona/gls141
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Advance Access published on August 2, 2012
Muscle Protein Synthetic Responses to Exercise: Effects
of Age, Volume, and Intensity
Vinod Kumar,1,* Philip J. Atherton,1,* Anna Selby,1 Debbie Rankin,1 John Williams,1,3 Kenneth Smith,1
Natalie Hiscock,2 and Michael J. Rennie1
University of Nottingham, Graduate Entry Medicine and Health, Derby, UK.
2
Unilever Discover R & D, Colworth Science Park, Sharnbrook, UK.
3
Anaesthetic Department, Derby Hospitals NHS Foundation Trust, Derby, UK.
1
*These authors contributed equally to this work.
Address correspondence to Philip J. Atherton, PhD, Division of Metabolic and Molecular Physiology, School of Graduate Entry Medicine and Health,
Royal Derby Hospital, Uttoxeter Road, Derby, DE22 3DT, UK. Email: [email protected]
We explored the relationships between resistance exercise volume/intensity and muscle myofibrillar protein synthetic
(MPS) responses in young and older men. In a crossover design, four groups of six young (24 ± 6 years) and older (70 ± 5
years) men performed two volumes of resistance exercise: either 40% one repetition maximum (1RM) (3 × 14, then 6 ×
14 repetitions) or 75% 1RM (3 × 8, then 6 × 8 repetitions), such that at the same volume, work was identical between
intensities. Muscle biopsies were taken 0, 1, 2, and 4 hours after exercise to measure MPS via myofibrillar bound [1,213
C2]leucine and indices of mammalian target of rapamycin signaling by immunoblotting. In younger men, doubling
exercise volume produced limited added effects, whereas in older men, it resulted in greater MPS and p70S6 kinase
(p70S6KThr389) phosphorylation at both intensities, that is, MPS area under the curve: 75% (1× volume: 0.07 ± 0.01 vs 2×
volume: 0.14% ± 0.02% protein synthesized/4 hours (p < .001). Doubling exercise volume is a valid strategy to maximize
postexercise MPS in ageing.
Key Words: Protein synthesis—Ageing—Skeletal muscle.
Received November 1, 2011; Accepted April 18, 2012
Decision Editor: Rafael de Cabo, PhD
M
ost previous work on the response of myofibrillar protein synthetic (MPS) responses to resistance exercise shows that it is stimulated at exercise of
high intensity with a large number of sets/repetitions, for
example, 6–20 sets with 8–12 repetitions at 70%–80%
one repetition maximum (1RM), in fasted or fed conditions
(1–7). However, the relationship between intensity and volume of exercise for the stimulation of MPS is still not clear
because in previous studies, the effect of volume was not
explicitly examined, and many results are the aggregate
effects of feeding and exercise together. Furthermore, earlier work has mostly examined the responses of the synthesis
of mixed muscle proteins (1–3), whereas it is now becoming clear that myofibrillar protein responses to exercise
are usually relatively greater and sustained for longer than
those of sarcoplasmic fractions (8–10).
The control of increases in MPS after exercise has been the
subject of intense research. Although the proximal pathways
involved are complex and diffuse, there is little doubt that
activation of the mammalian target of rapamycin (mTOR)
and its downstream effectors, p70S6 kinase (p70S6K) and
eukaryotic initiation factor 4E binding protein 1 (4EBP1)
(11–14), ultimately co-ordinate the synthesis of skeletal
muscle protein in response to exercise and nutrition. This
involvement of mTOR was elegantly confirmed in a study
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demonstrating that administration of rapamycin suppressed
postexercise increases in MPS in humans (14). Accordingly,
the degree of increase in the phosphorylation of the mTOR
substrate p70S6K after a bout of resistance exercise has
been closely associated with that of exercise intensity, MPS,
and hypertrophy in both rat and human muscle (7,11,15).
However, it remains unclear whether downstream signaling
proteins such as p70S6K and 4EBP1 are differentially activated in response to resistance exercise of different volumes.
We recently showed that an acute bout of resistance
exercise at higher intensities (60%–90% 1RM) increases
MPS in the postabsorptive state over 1–2 hours postexercise
to a greater extent than that at lower intensities (20%–40%
1RM) but with a larger response in young than older men
(7). In that study, we kept the volume of exercise and total
external work output at different exercise intensities (ie,
percentage of 1RM × number of repetitions × number of
sets) constant such that we remain uninformed of how the
volume of exercise influences MPS in the postabsorptive
state in either young or old muscle. Thus, expanding on this
previous work (7), we sought to investigate the effect of
two different volumes of exercise, three versus six sets of
unilateral leg extension, at two different exercise intensities,
a lower intensity (40% 1RM) and a higher intensity
(75% 1RM), on MPS in healthy young and old men. We
AGEING, EXERCISE VOLUME, EXERCISE INTENSITY, AND MUSCLE PROTEIN SYNTHESIS
purposely decided to study subjects in the postabsorptive
state to distinguish the exercise-mediated responses from
those coupled to increased nutrient availability (7). We
hypothesized that there would exist a greater anabolic
sensitivity in young than older muscle such that in muscle of
young men, at a given intensity, there are modest thresholds
for responses of MPS and anabolic signaling beyond which
any additional volume of work would not induce further
increases in them and increasing the volume of exercise
would overcome “exercise desensitization” in muscles of
older men.
Methods
Recruitment and Screening
The study was approved by the University of Nottingham
Ethics Committee and complied with the Declaration of
Helsinki. Written informed consent was obtained from the
subjects after explaining the study procedure and associated
risks. Twenty-four men, both young (n = 12; mean ± standard error of mean [SEM], 24 ± 6 years, and older (n = 12;
mean ± SEM, 70 ± 5 years) men, were recruited through
newspaper and web advertisements. They were recreationally active, physically independent, and healthy overall, but
not currently or previously engaged in any formal resistance
training. Screening of the subjects, performed by an experienced clinician, included a clinical history, a physical examination, and an electrocardiogram. Blood was tested for full
blood count, coagulation profile, fasting blood glucose, and
markers of liver, kidney, and thyroid function. Subjects were
excluded if they had a history of cardiac, pulmonary, liver,
kidney, vascular or autoimmune disease, clotting disorders,
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uncontrolled hypertension, diabetes, thyroid disorders, obesity, anemia, cancer, alcohol abuse, obvious muscle wasting,
corticosteroid use, or inability to discontinue aspirin therapy.
Older subjects who had mild hypertension (<140/90 mm Hg)
controlled by medication were included in the study, but did
not take medication on the day of the study. The maximal
strength of the subjects’ dominant leg was measured approximately 1 week before the study on a free-weight leg extension machine (ISO leg extension, Leisure Lines [GB] Ltd),
and they were familiarized with the study exercise protocols.
Whole-body composition was measured by dual-energy
X-ray absorptiometry (GE Lunar Prodigy II, GE Healthcare).
Study Design
Subjects were randomly assigned to perform, at 3-month
intervals, first three and then six sets of an isotonic, unilateral leg extension and flexion exercise at one of two
different exercise intensities (either 40% or 75% of 1RM
(six per group and per intensity) in a randomized and balanced fashion. Although the work was matched between
intensities at the same volume, the protocols differed only
in volume of work. All subjects were studied after an
overnight fast. They were asked to refrain from any heavy
exercise for 72 hours before the study day. On the morning of the study (approximately 9 am), subjects had 18-g
polyethylene catheters inserted in the antecubital veins of
both arms, one for tracer infusion and the other for venous
blood sampling. Blood samples were taken according to
the protocol (Figure 1). Muscle biopsies were taken from
the m. vastus lateralis under sterile conditions using the
conchotome biopsy technique (16) with 1% lignocaine as
local anesthetic. The muscle tissue was washed in ice-cold
Figure 1. Study protocol for the measurement of myofibrillar protein synthesis and p70S6 kinase (p70S6K) phosphorylation to unilateral leg extension exercise
(six vs three sets) at 40% versus 75% 1RM in postabsorptive young and older men.
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KUMAR ET AL.
saline before blood, visible fat, and connective tissue were
removed, and then, it was immediately frozen in liquid
nitrogen and then stored at −80°C until further analysis.
A primed, continuous infusion (0.7 mg.kg–1, 1 mg.kg.h–1)
of [1,2-13C2]leucine tracer (99 atoms %; Cambridge
Isotopes Ltd, Cambridge, MA) was started (at 0 hours)
after the first biopsy and maintained until the end of the
study. After taking biopsies at rest, t = 0 and 2.5 hours in
the postabsorptive pre-exercise state, the subjects performed unilateral leg extensions at a moderate contraction
velocity (1–2 seconds concentric, 1–2 seconds eccentric),
with 3-minute rest between sets. Subjects who performed
unilateral leg extension at 40% 1RM subjects completed 3
sets × 14 repetitions or 6 sets × 14 repetitions, and those
who performed at 75% 1RM completed 3 sets × 8 repetitions or 6 sets × 8 repetitions. Four further muscle biopsies
were obtained from the exercised leg immediately after
and at 1, 2, and 4 hours following exercise. After the study,
cannulae were removed, and the subjects were fed (a
sandwich, chocolate bar, and drink of their choice) before
being allowed home.
Measures of MPS
Muscle tissue (approximately ~25 mg) was minced with
scissors in ice-cold homogenization buffer containing 50
mm Tris–HCl (pH 7.4), 1 mm EGTA, 1 mm EDTA, 10
mm β-glycerophosphate, 50 mm NaF, 0.5 mm activated
sodium orthovanadate (all Sigma-Aldrich, Poole, UK), and
a complete protease inhibitor cocktail tablet (Roche, West
Sussex, UK). The resulting homogenate was centrifuged at
3000g at 4°C for 20 minutes to precipitate the myofibrillar
fraction. The myofibrillar pellet was then solubilized with
0.3 M NaOH and centrifuged at 3000g for 20 minutes to
separate it from the insoluble collagen fraction. The soluble
myofibrillar protein was precipitated using ice-cold 1M
perchloric acid, the resulting pellet was washed twice with
70% ethanol and collected by centrifugation. Protein-bound
amino acids were released by acid hydrolysis in a Dowex
H+ resin slurry (0.05M HCl) at 110°C overnight. The amino
acids were then purified by ion exchange chromatography
on Dowex H+ resin. The amino acids were derivatized
as their n-acetyl-N-propyl esters as previously described
(8,17). Incorporation of [1,2-13C2]leucine into protein was
determined by gas chromatography–combustion–isotope
ratio mass spectrometry (Delta plus XP, Thermofisher
Scientific, Hemel Hempstead, UK) using our standard
techniques (18). The fractional synthesis rate (FSR) of
myofibrillar protein was determined from the incorporation
of [1,2-13C2]leucine, using the precursor labeling of
venous α-KIC between subsequent muscle biopsies as
previously described (19). Using venous KIC labeling to
reflect precursor labeling, that is, the leucyl-t-RNA is the
standard approach for this method (13,20), and it has been
demonstrated to closely reflect the leucyl-t-RNA labeling in
the postabsorptive state. In other experiments (unpublished
observations), we have established that the KIC enrichment
from the femoral vein on the exercised leg is in steady
state; thus, KIC labeling accurately reflects the intracellular
leucine labeling also in the exercising leg. The rate of
myofibrillar FSR (MPS) was calculated using the standard
precursor-product method: fractional protein synthesis
-1
( ks ,% h ) = L m /L p 1/t 100 , where L m is the
change in protein labeling between two biopsy samples, Lp
is the mean value over time of venous α-KIC labeling, and
t is the time between biopsies in hours.
Measures of Protein Phosphorylation
Immunoblotting was performed using our standard methods as previously described (20,21). Briefly, after finely
mincing the muscle tissue in ice-cold homogenization buffer
containing the appropriate phosphatase and protease inhibitors, sarcoplasmic proteins were separated from myofibrillar proteins by centrifugation at 3000g at 4°C. Proteins were
separated by electrophoresis at 200 V. h–1 for approximately
1 hour and transferred to 100% methanol permeabilized
0.2-mm polyvinylidene difluoride membranes for 45 minutes at 100 V. Membranes sections were then blocked for
1 hour with 5% BSA before overnight exposure at 4°C,
with gentle agitation, to a primary antibodies for p70S6K1
(Thr389) and (UK New England Biolabs) at 1:2000. The
next morning, membranes were exposed to antirabbit IgG
secondary at 1:2000 (UK New England Biolabs) for 1 hour
before exposure and densitometric quantification using
the Chemidoc XRS system (Bio-Rad Laboratories, Inc.
Hercules, CA).
Statistical Analysis
All data are reported as means ± SEM. All analyses were
made using GraphPad Prism version 5.0 or SPSS. Area under
the curve (AUC) data is presented as Rate of myofibrillar
protein synthesis × Time or percentage of total myofibrillar
protein synthesized in the period. One-way analysis of variance (ANOVA) with repeated measures was used to determine temporal differences in MPS at specific time points.
Area under the MPS and signaling time course curves (AUC)
were calculated and used to perform a three-way ANOVA
comparing cumulative AUC across age, intensity, and workload. Significance was accepted as p < .05.
Results
Subject Characteristics
The participants in this study were all healthy, recreationally active individuals (but engaged in no formal training
program) with no differences presenting between groups
in body weight (young, 72 ± 11 kg; older, 72 ± 16 kg), body
mass index (young, 22 ± 3 kg/m2; older, 23 ± 4 kg/m2), height
(young, 1.80 ± 0.07 m; older, 1.74 ± 0.06 m), DXA-derived
lean mass (young, 60 ± 11 kg; older, 52 ± 7 kg), or
DXA-derived fat-mass (young, 15 ± 7 kg; older, 19 ± 9 kg).
AGEING, EXERCISE VOLUME, EXERCISE INTENSITY, AND MUSCLE PROTEIN SYNTHESIS
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Figure 2. (A) Time course of the responses of myofibrillar protein synthesis (fractional synthetic rate, %. h−1) to three or six sets of unilateral leg resistance
exercise at 40% (left) or 75% (right) 1RM. *p < .05 increase above basal at both volumes. n = 6 young per group. (B) areas under the curves (AUC), that is, percentage of protein synthesized in 4 hours after three or six sets of unilateral leg extension exercise at 40% and 75% 1RM. n = 6 young per group. NS = not significant.
Thus, the only characteristics by which our groups could
be distinguished were age (young, 24 ± 6 years; older,
70 ± 5 years; p < .05) and 1RM leg extension force, which
was significantly diminished in our older subjects (young,
750 ± 225N; older, 392 ± 196 N; p < .05).
MPS and Anabolic Signaling in Younger Men
As previously reported (7), although the muscle of the
young subjects elicited little response to exercise at 40%
1RM (no significant increases in MPS at any time-point),
increasing the intensity at both workloads did significantly
elevate MPS above basal, 1–2 hours postexercise (ie, 75%:
0.12% ± 0.02% hours–1; p < .05; Figure 2B). Taking the
entire postexercise period together, the AUC for 2× volumes
of exercise was greater at 75% than 40% 1RM (Figure 2B;
p < .05) while increasing the volume of exercise at each
intensity did not supply any additional effect: 40% (AUC
1× volume: 0.07 ± 0.02 versus 2× volume 0.06% ± 0.02%
protein synthesized in 4 hours) and 75% (AUC 1× volume: 0.09 ± 0.03 versus 2× volume 0.14% ± 0.03% protein
synthesized in 4 hours; Figure 2B). In terms of signaling, although there was no effects on the phosphorylation
responses of p70S6K to resistance exercise at different volumes at 40% 1RM, increasing exercise volume from three
sets to six sets at 75% 1RM did result in a greater AUC
(p < .05) and sustained elevation of phosphorylated p70S6K
in young men, up to the last time point (4 hours postexercise; p < .05; Figures 3A and B).
MPS and Anabolic Signaling in Older Men
Despite there being no changes in MPS throughout
the time course at 1× volume during 40% or 75% 1RM
exercise, increasing to 2× volumes elicited significant
increases between 0–2 hours after exercise at both intensities
(Figure 4A). This also translated into significant differences
in the AUC (Figure 4A and 4B), whereby increasing the
volume of exercise at both 40% and 75% 1RM from three
to six sets significantly enhanced the MPS responses 40%
(AUC 1× volume: 0.01 ± 0.01 versus 2× volume 0.13% ±
0.03% protein synthesized in 4 hours; p < .01) and 75%
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KUMAR ET AL.
Figure 3. (A) Time course of the responses of phosphorylation of p70S6 kinase (p70S6K) (arbitrary units [AU]) as percentage basal values for each subject) to
three or six sets of unilateral leg resistance exercise at 40% (left) or 75% (right) 1RM, n = 6 young per group. *p < .05 from nonexercised condition; †p < .05 increase
above basal at both volumes. n = 6 young per group. (B) areas under the curves (AUC; ie, arbitrary units × hour) after three or six sets of unilateral leg extension
exercise at 40% and 75% 1RM in the younger subjects. n = 6 young per group.
(AUC 1× volume: 0.07 ± 0.01 versus 2× volume 0.14% ±
0.02% protein synthesized in 4 hours; p < .001). In terms
of signaling time course, although phosphorylated p70S6K
was unaffected by 1× volume of exercise, it was elevated
at 1 and 4 hours (p <.05) and 1 and 2 hours after exercise
(p < .05) in response to 2× volumes of 40% and 75%
1RM, respectively. In addition, the extent of increases in
postexercise p70S6K phosphorylation was significantly
higher after six sets of exercise in comparison to that after
three sets of exercise at both intensities in older men (AUC
both p = .05; Figure 5B). Finally, there were no differences
between AUC in response to 2× volumes of exercise at 40%
and 75% 1RM.
Differences in AUC of MPS Between Young and
Older Men
The AUC for MPS was greater in young versus older men
at 1× volume of 40% 1RM exercise, (p < .05), although
not at 75% 1RM. At 2× volume exercise, the older men
displayed a trend for greater MPS versus the young men
at 40% 1RM (p < .1). There were no differences in AUC
between young and older men at 75% 1RM.
Discussion
We report novel information concerning the responses of
MPS in young and older men to different volume of resistance exercise at two different intensities in the postabsorptive state. Increasing the volume of exercise from three to
six sets at a given intensity robustly enhances postexercise
MPS responses in older men, whereas it has relatively minimal (ie, nonsignificant approximately 50% increase in
AUC versus 110% in older men at 75% 1RM) additional
effect in young men. It has been previously demonstrated
that “anabolic blunting” is a pervasive feature of ageing muscle, which is revealed in a reduced sensitivity to
the anabolic effects of both feeding (22–25) and exercise
(7,25). These lower synthetic responses of older muscle
have been associated with inability to fully activate mTOR
AGEING, EXERCISE VOLUME, EXERCISE INTENSITY, AND MUSCLE PROTEIN SYNTHESIS
1175
Figure 4. (A) Time course of the responses of myofibrillar protein synthesis (fractional synthetic rate, %. h−1) to three or six sets of unilateral leg resistance exercise at 40% (left) or 75% (right) 1RM. *p < .05 from nonexercised condition. n = 6 old per group. (B) areas under the curves (AUC), that is, percentage of protein
synthesized in 4 hours after three or six sets of unilateral leg extension exercise at 40% and 75% 1RM . AUC greater for six sets at 40% (p < .01) and at 75% (p <
.001). n = 6 old per group.
signaling when compared with young muscle (7,23). We,
therefore, expected that the less sensitive muscle of older
men would require a greater anabolic stimulus (ie, to perform more work) to activate the protein synthetic machinery
sufficiently to achieve MPS rates comparable to those seen
in younger men. In concordance with this, we observed
that doubling the exercise volume at both 40% and 75% of
1RM, enhanced MPS rates such that they equaled the young
at 75% and even surpassed them at 40%.
The older men were weaker in terms of strength-related
performance (1RM young, 750 ± 225 N vs older, 392 ± 196
N) such that they were lifting considerably less weight
per unit cross-sectional area (given equal muscle mass by
DXA). Although this may explain the lack of sensitivity
at 1× volume of 75% 1RM exercise, why would MPS be
potentiated in older but not younger muscle after resistance
exercise at a lower intensity, such as 40%? According to
Henneman's size principle, during low-intensity muscular
contraction, slow twitch fibers with small motor units are
primarily recruited, whereas increasing muscular force
gradually recruits increasing numbers of type II fibers
(26). However, several studies have shown that in hypoxic
or fatiguing conditions, early recruitment of type II fibers
can occur with the effect of maintaining the muscular
force during low-intensity resistance exercise (27–29). In
comparison to young muscle, older muscle has reduced
fatigue resistance (30,31) such that a higher number of
repetitive muscular contractions even at low intensity
(40% 1RM) cause heightened metabolic stress (28) and
additional recruitment of type II fibers. Thus, increased
recruitment of larger type II fiber containing motor units
can perhaps explain increases in MPS in older, but not
younger men at 2× volumes of 40% 1RM exercise (32,33).
In terms of intramuscular “anabolic” signaling, the dissociation between p70S6K phosphorylation and MPS at
75% in the younger men further underlines the potential for
discordance between amplitude of signaling protein phosphorylation and biological end-points such as MPS (17).
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KUMAR ET AL.
Figure 5. (A) Time course of the responses of phosphorylation of p70S6 kinase (p70S6K) (arbitrary units [AU] as percentage of basal values for each subject)
to three or six sets of unilateral leg resistance exercise at 40% or 75% 1RM. *p < .05 from nonexercised condition. n = 6 old per group. (B) areas under the curves
(AUC; ie, arbitrary units × hour) after three or six sets of unilateral leg extension exercise at 40% and 75% 1RM. AUC greater for six sets at 40% (p < .05) and at
75% (p < .05). n = 6 old per group.
That said, we provide evidence for the existence of a link
between exercise volume and phosphorylation of p70S6K,
which is increased after resistance exercise to an extent
depending on the exercise volume at higher intensity in the
young group, and at both lower and higher intensities in the
older group.
In conclusion, we have found that in young men doubling
the volume of exercise at 40% had no additional effects and
at 75% 1RM only small additional effects, on the response
of MPS in the postabsorptive state; however, in older
men, it resulted greater responses at both intensities. The
results suggest that there is increased latency of response to
exercise in muscle of older men and increasing the volume
of exercise enhances the stimulatory effect of resistance
exercise on MPS, even at relatively low intensities, that
is, 40% of 1RM. Although we accept our sample sizes
are small and give limited resolution of the existence of
small cross-sectional differences in responses between
age-groups afforded by other studies, that is (25), our
present one-way findings (particularly, intensity crossover
within the older-groups) are clear and may have important
implications for the exercise recommendations for elderly
people, and the frail elderly in particular, for whom muscle
maintenance is of crucial importance and who may not
be capable of undertaking high-intensity exercise (>70%
1RM). Therefore, although further work is needed to define
the application of such an exercise strategy in overtly
sarcopenic groups, we conclude that older people would
perhaps benefit from doing moderate-intensity exercise of
a higher volume in order to help maintain/increase their
muscle mass.
Funding
This work was funded by Unilever PLC.
Acknowledgments
We thank Margaret Baker and Amanda Gates for their help with the
clinical studies.
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