3 séries plus anaboliques qu’une seule

Resistance exercise volume affects myofibrillar protein synthesis and anabolic signalling molecule phosphorylation in young men
Nicholas A. Burd
August 15, 2010 The Journal of Physiology, 588, 3119-3130.

We aimed to determine if any mechanistic differences exist between a single set (1SET) and multiple sets (i.e. 3 sets; 3SET) of resistance exercise by utilizing a primed constant infusion of [ring-13C6]phenylalanine to determine myofibrillar protein synthesis (MPS) and Western blot analysis to examine anabolic signalling molecule phosphorylation following an acute bout of resistance exercise. Eight resistance-trained men (24 ± 5 years, BMI = 25 ± 4 kg m−2) were randomly assigned to perform unilateral leg extension exercise at 70% concentric one repetition maximum (1RM) until volitional fatigue for 1SET or 3SET. Biopsies from the vastus lateralis were taken in the fasted state (Fast) and fed state (Fed; 20 g of whey protein isolate) at rest, 5 h Fed, 24 h Fast and 29 h

Fed post-exercise. Fed-state MPS was transiently elevated above rest at 5 h for 1SET (2.3-fold) and returned to resting levels by 29 h post-exercise.

However, the exercise induced increase in MPS following 3SET was superior in amplitude and duration as compared to 1SET at both 5 h (3.1-fold above rest) and 29 h post-exercise (2.3-fold above rest).

Phosphorylation of 70 kDa S6 protein kinase (p70S6K) demonstrated a coordinated increase with MPS at 5 h and 29 h post-exercise such that the extent of p70S6K phosphorylation was related to the MPS response (r = 0.338, P = 0.033). Phosphorylation of 90 kDa ribosomal S6 protein kinase (p90RSK) and ribosomal protein S6 (rps6) was similar for 1SET and 3SET at 24 h Fast and 29 h Fed, respectively. However, 3SET induced a greater activation of eukaryotic translation initiation factor 2Bε (eIF2Bε) and rpS6 at 5 h Fed. These data suggest that 3SET of resistance exercise is more anabolic than 1SET and may lead to greater increases in myofibrillar protein accretion over time.

J Physiol 588.16 (2010) pp 3119–3130 3119
Resistance exercise volume affects myofibrillar protein
synthesis and anabolic signalling molecule
phosphorylation in young men
Nicholas A. Burd1, Andrew M. Holwerda1, Keegan C. Selby1, DanielW. D.West1, AaronW. Staples1,
Nathan E. Cain1, Joshua G. A. Cashaback2, James R. Potvin2, Steven K. Baker3 and Stuart M. Phillips1
1ExerciseMetabolism Research Group and 2Occupational Biomechanics Laboratory, Department of Kinesiology,McMaster University, and 3Michael G.
DeGroote School of Medicine, Department of Neurology, McMaster University, Hamilton, Ontario, Canada
We aimed to determine if any mechanistic differences exist between a single set (1SET) and
multiple sets (i.e. 3 sets; 3SET) of resistance exercise by utilizing a primed constant infusion
of [ring-13C6]phenylalanine to determine myofibrillar protein synthesis (MPS) and Western
blot analysis to examine anabolic signalling molecule phosphorylation following an acute bout
of resistance exercise. Eight resistance-trained men (24±5 years, BMI=25±4 kgm−2) were
randomly assigned to performunilateral leg extension exercise at 70% concentric one repetition
maximum (1RM) until volitional fatigue for 1SET or 3SET. Biopsies from the vastus lateralis
were taken in the fasted state (Fast) and fed state (Fed; 20 g of whey protein isolate) at rest,
5 h Fed, 24 h Fast and 29 h Fed post-exercise. Fed-state MPS was transiently elevated above rest
at 5 h for 1SET (2.3-fold) and returned to resting levels by 29 h post-exercise. However, the
exercise induced increase in MPS following 3SET was superior in amplitude and duration as
compared to 1SET at both 5 h (3.1-fold above rest) and 29 h post-exercise (2.3-fold above rest).
Phosphorylation of 70 kDa S6 protein kinase (p70S6K) demonstrated a coordinated increase
with MPS at 5 h and 29 h post-exercise such that the extent of p70S6K phosphorylation was
related to the MPS response (r =0.338, P =0.033). Phosphorylation of 90 kDa ribosomal S6
protein kinase (p90RSK) and ribosomal protein S6 (rps6) was similar for 1SET and 3SET at
24 h Fast and 29 h Fed, respectively. However, 3SET induced a greater activation of eukaryotic
translation initiation factor 2Bε (eIF2Bε) and rpS6 at 5 h Fed. These data suggest that 3SET of
resistance exercise is more anabolic than 1SET and may lead to greater increases in myofibrillar
protein accretion over time.
(Received 10 May 2010; accepted after revision 24 June 2010; first published online 25 June 2010)
Corresponding author S. M. Phillips: Exercise Metabolism Research Group, Department of Kinesiology, McMaster
University, 1280 Main StreetWest, Hamilton, ON L8S 4K1, Canada. Email: .(JavaScript must be enabled to view this email address)
Abbrevations 1RM, one repetition maximum; 1SET, one set; 3SET, three sets; MPF, mean power frequency; MPS,
myofibrillar protein synthesis.
Introduction
The majority of studies examining muscle protein
synthesis following acute resistance exercise have utilized
high volume (i.e. ≥3 sets) bouts of exercise (Phillips et al.
1997; Kumar et al. 2008;Moore et al. 2009b). Presumably,
there is an exercise volume (i.e. load × repetitions)
dose–response that ultimately reaches a ceiling where the
stimulatory effects of more contractions would diminish.
A lack of difference between the acute rise in myofibrillar
protein synthesis (MPS) seen in young men who
had performed three versus six sets of resistance exercise
(Kumar et al. 2008) provides support for this concept.
We have proposed (Burd et al. 2009; Phillips et al. 2009)
that acute changes in muscle protein synthesis are predictive
of phenotypic adaptations; thus, it was of interest
to us to characterize changes inmuscle protein synthesis in
response to lower exercise volumes than previously tested.
We are unaware of any knowledge of the acute adaptations
of myofibrillar proteins to resistance exercise after lower
volumes of exercise, such as one or three sets. The efficacy
of single versus multiple sets of resistance training in
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2010 The Authors. Journal compilation C  2010 The Physiological Society DOI: 10.1113/jphysiol.2010.192856
3120 N. A. Burd and others J Physiol 588.16
inducing gains in muscle size and strength are equivocal
(Krieger, 2009, 2010; Ratamess et al. 2009; Winett et al.
2009). The conflicting results among studies may relate to
the large heterogeneity of response between participants in
response to resistance training (Hubal et al. 2005), coupled
with poor study design, and/or a heterogeneous training
status of the research subjects (Marx et al. 2001;Carpinelli,
2002).
Themechanisms that facilitatemuscle protein synthesis
following acute resistance exercise require the activation of
signalling moleculeswithin themTOR(mammalian target
of rapamycin) pathway (Kumar et al. 2008; Drummond
et al. 2009) or mitogen-activated protein kinase (MAPK)
signalling cascades (Williamson et al. 2003; Karlsson
et al. 2004; Tannerstedt et al. 2009). Whether MPS
is mediated by the convergence of Akt–mTOR and
MAPK signalling pathways on downstream targets such
as 70 kDa S6 protein kinase (p70S6K) and ribosomal
protein S6 (rpS6) to achieve maximal stimulation of
MPS still requires further examination. Furthermore, we
have recently demonstrated, following resistance exercise,
that eukaryotic translation initiation factor 2Bε (eIF2Bε)
phosphorylation (involved in ribosomal recycling) is
reduced in recreationally resistance-trained men (Glover
et al. 2008a); however, the extent of the impact of resistance
exercise volume on the activation of these anabolic factors
and the subsequent stimulation of MPS remains to be
investigated in humans.
Therefore, the purpose of this study was to examine
the extent to which resistance exercise performed for
one set (1SET) or three sets (3SET), with the
same relative workload, affected the amplitude and
early duration of MPS and phosphorylation of
anabolic signalling molecules. We utilized trained
subjects to overcome issues of training-induced
heterogeneity in motor unit firing rate and firing
synchrony (Sale, 1988). We also know that resistance
training can chronically elevate resting muscle protein
synthesis (Phillips et al. 2002; Kim et al. 2005) and
attenuate muscle protein breakdown that occurs in
response to an isolated bout of exercise (Phillips et al.
1999). Furthermore, resistance training can shorten the
duration and amplitude of muscle protein synthesis
following acute resistance exercise (Tang et al. 2008).
Thus, trained individuals need to maintain a relatively
‘unique’ exercise stimulus to promote continuing muscle
adaptation and as such increased volume of exercise may
be an important factor. Based on observations that high
volume resistance exercise can have long lasting effects
on muscle protein synthesis (Phillips et al. 1997), and
that anabolic signalling molecule activation is related to
(Kumar et al. 2008) and/or required for (Drummond et al.
2009)muscle protein synthesis,we hypothesized that 3SET
would induce a greater increase in MPS in both amplitude
and duration versus 1SET. We also hypothesized that the
increase inMPSwould be reflected in the extent of anabolic
signalling protein phosphorylation, in particular p70S6K
(Kumar et al. 2008; Terzis et al. 2008).
Methods
Subjects
Eight recreationally resistance-trained males (24.3±
1.6 years; 84.3±3.3 kg; body mass index
(BMI)=25.1±0.7 kgm−2) participated in this study.
Subjects were all habitually active and reported engaging
in lower body resistance exercise at least 1 time per
week for ≥1 year at the time of the study. Subjects were
informed about the experimental procedure to be used
as well as the purpose of the study and all potential risks
prior to obtaining written consent. All participants were
deemed healthy based on their response to a routine
medical screening questionnaire. The study was approved
by the local Research Ethics Board ofMcMasterUniversity
and Hamilton Health Sciences and conformed to all
standards for the use of human subjects in research as
outlined in the Declaration of Helsinki.
Experimental protocol
One week before any infusion trials, all subjects reported
to the laboratory for a familiarization session with
the exercise equipment and to establish their unilateral
1 repetition maximum (1RM) on each leg for knee
extension exercise (Hartman et al. 2007). Subjects’
unilateral 1RM for the right and left legs was 94.5±5.4 kg
and 92.3±5.0 kg, respectively (P =0.29). Each subject
recorded his dietary intake for 3 days prior to the
resting and exercise experimental infusion trial (trial 1).
A unilateral model, whereby each individual served as
his own rested control, was utilized to ensure that acute
changes in MPS following exercise and feeding were due
to these stimuli rather than inter-subject variability (i.e.
genetics and motivation).
On themorning of trial 1 (Fig. 1), participants reported
to the laboratory at 07.00 h after an overnight fast and
having refrained from any strenuous physical activity for
the previous 3 days. An 18-gauge catheter was inserted
in the antecubital vein of one arm for blood sampling.
After a baseline blood sample was drawn, a second
catheter was inserted in the contralateral arm for the
primed constant infusion (PHD2000;HarvardApparatus,
Natick, MA, USA) of L-[ring-13C6]phenylalanine (prime:
2 μmol kg−1; 0.05 μmol kg−1 min−1;Cambridge Isotopes,
Andover,MA, USA; Fig. 1) passed through a 0.2 μmfilter.
The subjects rested comfortably on a bed throughout
the infusions. At 3 h after the start of the infusion, a
single muscle biopsy was taken from the dominant leg to
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J Physiol 588.16 Resistance exercise volume and myofibrillar protein synthesis 3121
measure fasted rates of protein synthesis (Rest). Following
the biopsy, subjects’ legs were shaved with a hand razor
and cleaned with isopropyl alcohol prior to electrode
placement. Bipolar self-adhesive Ag–AgCl monitoring
electrodes (Kendall Meditrace 133, Chicopee, MA, USA)
were placed on the medial portion of the muscle bellies of
the vastus lateralis, vastus medialis, and rectus femoris
in line with the direction of muscle fibre orientation.
The reference electrode was placed on the head of the
fibula, for evaluation of electromyography (EMG) during
exercise. Subjects then performed a fatiguing bout of
unilateral leg extension exercise at 70% of their previously
established concentric 1RM. Subjects legs were
randomized to perform exercise volumes of 1SET or
3SET, balanced for leg dominance (based on strength)
in each condition, until volitional failure. Failure was
defined as the point at which the exercise could not be
completed or the subjects’ technique failed. For 3SET,
subjects performed three sets of exercise with 2min rest
between setswith one leg,whereas the other leg completed
a single set (1SET). The subjectswere instructed on proper
lifting cadence using verbal cues and a metronome set
to 50 beats min−1, which corresponded to 1 s concentric
muscle action, 0 s pause, and a 1 s eccentric muscle
action. A goniometer was positioned on the leg extension
machine to record knee joint angle. The derivative of the
flexion angle and angular velocity was used to identify
the concentric, isometric and eccentric phases of each
repetition.
After completion of the exercise subjects returned to the
resting position and lay supine while a blood sample was
collected. Then participants consumed a drink containing
20 g of whey protein isolate (Table 1), which was a
generous gift from Inbalance Nutrition Inc. (Burlington,
Table 1. Essential amino acid content of protein drinks (Fonterra
Alacen-895-I)
Essential amino acid g (100 g protein)−1
Isoleucine 6.2
Leucine 14.0
Lysine 11.1
Methionine 2.9
Phenylalanine 3.8
Threonine 5.0
Tryptophan 2.4
Valine 5.7
Histidine 2.1
ON, Canada). This protein dose has previously been
shown to maximally stimulate muscle protein synthesis
following resistance exercise (Moore et al. 2009a). To
minimize disturbances in isotopic equilibrium, the drinks
were enriched to 6% with tracer according to a measured
phenylalanine content of 3.5% in the whey protein.
Previouswork in our lab has shown that the tracer added to
protein in this manner is not absorbed at a rate appreciably
faster than the amino acids from digestion of the protein
itself. This is evidenced by a stable isotopic plateau in
both the blood and muscle pools (see Results), which
would not be the case if the isotope added to the protein
appeared more rapidly in the blood. Thus, this model and
approach satisfies all criteria for the precursor-product
approach and steady state equations. Trial 1was concluded
by obtaining bilateral biopsies at 5 h after completion of
unilateral resistance exercise. Subjectswere then instructed
to eat a meal that was representative of the meals they
previously recorded on the three-day dietary log, and this
meal was to be consumed no later than 22.00 h to ensure
Time (h)
Blood
Biopsy
Feed
Trial 1:
0 1 2 3 4 5 6
* * * * * *
7 8
* * * * *
Primed-continuous L-[ring-13C6]Phenylalanine Infusion
EX
* 0 1 2 3 4 5 6
* * * *
6.5
* * * * *
Trial 2: Primed-continuous L-[ring-13C6]Phenylalanine
Time (h)
Blood
Biopsy
Feed
Figure 1. Schematic diagram of the experimental
infusion protocols
Double arrows indicate bilateral biopsies were obtained
at corresponding time points.
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3122 N. A. Burd and others J Physiol 588.16
a 10 h fast prior to the beginning of the 24 h post-exercise
protein synthesis measurement (trial 2). Subjects also
refrained from any physical activity for the evening. In
the morning subjects returned to the laboratory for trial
2 and underwent the previously described infusion trial
procedures. Bilateral biopsies were obtained at 1.5 h after
the start of the infusion, followed by the consumption
of 20 g of tracer-enriched whey protein isolate drink.
Infusion trial 2was concluded by bilateral biopsies at 6.5 h.
Muscle biopsies were performed with a Bergstr¨om needle
that was custom-modified for manual suction under local
anaesthesia (2% xylocaine). Biopsy samples were blotted
dry and freed of any visible fat and connective tissue,
immediately frozen in liquid nitrogen and stored at−80◦C
until further analysis. Each biopsy was obtained through a
separate incision at least 3 cm from the previous incision.
Blood samples were drawn every 0.5–1 h of trial 1 and 2
and were processed as previously described (Moore et al.
2009a).
Blood analyses
Plasma L-[ring-13C6]phenylalanine enrichments were
determined as previously described (Glover et al. 2008b).
Blood amino acid concentrations were analysed by HPLC
as previously described (Moore et al. 2005). Blood glucose
concentrations were analysed using a blood glucosemeter
(OneTouch Ultra 2, Lifescan Inc., Milpitas, CA, USA)
within 5min of blood collection. Plasma insulin was
measured using a commercially available immunoassay
kit (ALPCO Diagnostics, Salem, NH, USA).
Electromyography analyses
The raw EMG signals were sampled at 1024 Hz,
using a custom-made bioamplifier, and were collected
with acquisition software (LabVIEW v 8.2, National
Instruments, Austin, TX, USA). All raw EMG signals
were digitized and stored on an external hard drive for
subsequent analysis. Repetitions were normalized to the
total time taken to perform the exercise, because of
the differing number of repetitions performed by each
of the subjects, such that the first and last repetitions
were represented as 0% and 100%, respectively. All
signal processing was performed off-line using custom
written software (LabVIEW). Signal amplitudes, reflective
of motor unit recruitment and their rate of discharge,
were assessed as the signal envelope. Specifically, the
raw signal was bandpass filtered between 20 and 500 Hz,
full-wave rectified and dual pass filtered using a 1.7 Hz low
pass cut off Butterworth filter. A moving average with a
250 ms sampling window was then used with an overlap
of 249 ms.
Signal amplitudes were normalized to the first
concentric phase of the first set, for both 1SET and 3SET
protocols. This was assumed to be at 70% activation, given
the 70% of 1RM load and previous literature indicating
a linear relationship between EMG amplitude and joint
moment (Babault et al. 2001). The average for each phase
of each repetition was modelled with a second order
polynomial regression equation. These data for each
10% interval of time were then plotted according to
the regression analysis. A fast Fourier transformation
was performed on each 250 ms window. The mean
power frequency (MPF), which approximates changes
in muscle fibre conduction velocity (Brody et al. 1991),
was then determined for each overlapping window, and
subsequently averaged for each repetition.
Muscle analyses
A piece of wet muscle (∼20 mg) was homogenized by
hand on ice using a Teflon-coated pestle in a standard
Western blotting homogenization buffer (10 μl mg−1): a
25mM Tris (pH 7.2) buffer containing 1mM Na3VO4,
50mM NaF, 40mM β-glycerolphosphate, 20mM sodium
pyrophosphate, 0.5% v/v Triton X-100, and Complete
Protease Inhibitor Mini-Tabs (Roche, Indianapolis, IN,
USA). The samples were centrifuged at 1500 g at 4◦C
for 10 min. The resultant supernatants were removed
and protein content was determined by the Bradford
assay. The myofibrillar and collagen pellet was stored at
−80◦C for future processing. Samples (25 μg of protein)
were loaded on 7.5 or 10% SDS-polyacrylamide gels
and then transferred to a PVDF membrane. Membranes
were blocked with 5% BSA (w/v) in Tris-buffered
saline with 0.1% Tween (v/v) (TBST), except p70S6K1
on Thr389 (2.5% milk for all conditions) and total
p70S6K1 (1.5% milk). Membranes were then incubated
overnight in primary antibody at 4◦C: p70S6K1 on
Thr389 (Santa Cruz Biotechnology, Inc., Santa Cruz,
CA, USA; no. 11759, 1:500); total p70S6K1 (Santa Cruz
Biotechnology, no. 9027, 1:500); rpS6 on Ser240/244
(Cell Signaling Technology, Inc., Danvers, MA, USA; no.
22155; 1:2000); total rpS6 (Cell Signaling Technology,
no. 2217; 1:2000); eIF2Bε on Ser539 (Genetex, San
Antonio, TX, USA; no. GTX24775, 1:6000); total eIF2Bε
(Cell Signaling Technology, no. 3595; 1:750); GSK3β
on Ser9 (Cell Signaling Technology, no. 9336S; 1:1000);
total GSK3β (Cell Signaling Technology, no. 9315;
1:6000); p38 MAPK on Thr180/Tyr182 (Cell Signaling
Technology, no. 9215S; 1:1000); total p38 MAPK (Cell
Signaling Technology, no. 9212, 1:1000); p90RSK1 on
Thr573 (Epitomics Inc., Burlingame, CA, USA; no.
2185-1; 1:500); total p90RSK1 (Cell Signaling Technology,
no. 9355, 1:1000); mTOR on Ser2448 (Cell Signaling
Technology, no. 2971; 1:1000): totalmTOR(Cell Signaling
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2010 The Authors. Journal compilation C  2010 The Physiological Society
J Physiol 588.16 Resistance exercise volume and myofibrillar protein synthesis 3123
Technology, no 2972; 1:1000). After washing in TBST,
membranes were incubated in horseradish peroxidase
(HRP)-linked anti-rabbit IgG secondary antibody (GE
Healthcare (Amersham Biosciences), Piscataway, NJ,USA;
no. NA934VS, 1:15,000), washed with TBST, and detected
by chemiluminescence (SuperSignalWest Dura Extended
Duration Substrate, ThermoScientific, no. 34075). Images
were developed using FluorChem SP Imaging system and
quantified by spot densitometry using ImageJ software.All
signalling protein responses were determined with n =8,
with the exception of mTOR (n =7; due to a smallmuscle
sample weight).
Myofibrillar proteins were isolated as previously
described (Moore et al. 2009b) and the hydrolysed
amino acids were purified using cation-exchange
chromatography (Dowex 50WX8-200 resin;
Sigma-Aldrich Ltd) and converted to their N-acetyl-npropyl
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,
USA) (Moore et al. 2009b). Intracellular amino acids
were extracted from a separate piece of wet muscle
(∼20 mg) with ice-cold 0.6 M perchloric acid. Muscle was
homogenized on ice with a Teflon-coated pestle and then
centrifuged at 12,000 g for 10 min at 4◦C. The supernatant
was then collected and this process was repeated two
more times. All three supernatants were combined and
taken as the intracellular amino acids and purified by
cation-exchange chromatography and converted to their
heptafluorobutyrate derivatives before analysis by GC-MS
(models 6890 GC and 5973 MS; Hewlett-Packard, Palo
Alto, CA, USA) as previously described (Moore et al.
2009b).
Calculations
The fractional synthetic rate (FSR) ofmyofibrillar proteins
was calculated using the standard precursor–product
method:
FSR (%h−1) = [E p2 − E p1]/E ic × 1/t × 100
Where Ep2 −Ep1 represents the change in bound protein
enrichment between two biopsy samples, Eic is the average
enrichment of intracellular phenylalanine between the two
biopsy samples and t is the time between biopsies. It
should be noted that the resting biopsy (Fast) obtained
during trial 1 represented Ep1 for both 1SET and 3SET
exercise legs and as such the exercise bout was included
in the calculation; this approach has been discussed
previously (Moore et al. 2009b). Furthermore, the
utilization of ‘tracer-naive’ subjects allowed us to use
the pre-infusion blood sample (i.e. mixed plasma protein
fraction) as the baseline enrichment (Ep1) for the
calculation of restingMPS(Fast). This approachmakes the
assumption that the ‘natural’ 13C enrichment (δ13CPDB)
in the blood reflects that of muscle protein; this is an
assumption that has been confirmed in our laboratory
and others’ (Heys et al. 1990; Nakshabendi et al. 1995;
West et al. 2009).
Statistics
A within-subject repeated measures design was utilized
for the current study. Differences in MPS and anabolic
signalling were tested by two-factor (condition × time)
analysis of variance (ANOVA) with repeated measures
on time factor. Acute exercise variables (repetitions, time
under tension, and volume per set within 3SET) were
analysed using a one-factor ANOVA. Differences in total
volume performed between conditions were determined
by Student’s paired t test. Blood glucose, plasma insulin,
and blood amino acid concentrations were analysed using
one-factor (time) repeated measures ANOVA. Linear
regression analyses were performed to assess the existence
of a linear fit between variables. Pearson’s r product
moment correlation was used to examine the relationship
between different variables (MPS and anabolic signalling
molecules). Tukey’s post hoc test was performed to
determine differences between means for all significant
main effects and interactions. All statistical analyses were
performed using SigmaStat 3.10.0 (Systat Software Inc.,
Point Richmond, CA, USA). For all analyses, differences
were considered significant at P <0.05. All results are presented
as means±standard error of the mean (S.E.M.).
Results
Resistance exercise
Acute resistance exercise variables are shown in Table 2.
There was no difference in the load utilized for 1SET
or all three sets of 3SET. There was also no difference
in the number of contractions, load, or volume load
for 1SET and set 1 of 3SET. The repetitions performed
during set 1 of 3SET were significantly greater than sets 2
(P <0.001) and 3 (P <0.001), and set 2 was significantly
greater than set 3 (P =0.009). Similar results were found
for volume load. The total volume performed for 3SET
(2183±154 kg) was significantly greater (P <0.001) than
for 1SET (942±97 kg).
Blood glucose, plasma insulin, and amino acid
concentrations
Blood glucose remained stable during infusion trials 1
and 2 (Table 3). Plasma insulin concentration peaked
(P <0.001) at 1 h post-drink ingestion for trial 1
(∼4.2-fold increase) and trial 2 (∼4.0-fold increase) but
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3124 N. A. Burd and others J Physiol 588.16
Table 2. Acute unilateral resistance exercise variables
1SET 3SET
Set 1 Set 2 Set 3
Load (kg) 68 ±4 64±4 64±4 64 ± 4
Repetitions 14 ±2 14±1 11± 1∗† 9 ± 1∗†§
Volume Load (kg) 942 ± 97 903 ± 59 698 ± 53∗ 582 ± 53∗†
Time under tension (s) 34 ±3 33±2 27± 2∗† 24 ± 2∗†
Values are means ± S.E.M. (n = 8). ∗Significantly different from 1SET, P < 0.05.
†Significantly different from Set 1 of 3SET, P < 0.05. §Significantly different from
Set 2 of 3SET, P < 0.009.
Table 3. Blood amino acid concentrations, blood glucose, and plasma insulin concentrations in the fasted-state and following
ingestion of 20 g of whey protein isolate during trial 1 and trial 2
After drink
Fast 0 h 0.5 h 1.0 h 1.5 h 2.0 h 3.0 h
Trial 1:
 EAA (μM) 563 ± 48 624 ± 60 757 ± 72 1096 ± 98∗ 900 ± 92∗ 858 ± 73 618 ± 53
Leucine (μM) 80 ±7 89± 7 125 ± 20 244 ± 29∗ 187 ± 16∗ 153 ± 14∗ 106 ± 10
Insulin (μUml−1) 4.4 ± 0.4 3.5 ± 0.6 13.0 ± 0.2∗ 18.5 ± 3.4∗ 6.9 ± 1.0∗ 5.1 ± 0.6 4.03 ± 0.6
Glucose (mM) 5.6 ± 0.2 – 5.6 ± 0.3 5.5 ± 0.1 5.6 ± 0.1 5.6 ± 0.4 5.4 ± 0.1
Trial 2:
 EAA (μM) 611 ± 47 600 ± 38 964 ± 63∗ 1156 ± 105∗ 889 ± 101∗ 756 ± 62 617 ± 60
Leucine (μM) 104 ± 10 99 ± 12 201 ± 19∗ 239 ± 30∗ 214 ± 35∗ 154 ± 20 120 ± 16
Insulin (μUml−1) 4.1 ± 0.5 4.2 ± 1.0 8.2 ± 1.3∗ 16.2 ± 2.5∗ 8.5 ± 2.2∗ 4.6 ± 0.6 –
Glucose (mM) 5.5 ± 0.2 – 5.2 ± 0.2 4.9 ± 0.1 5.0 ± 0.2 5.0 ± 0.2 4.7 ± 0.2
Values are means ± S.E.M. (n = 8). Drink comprised 20 g of whey protein isolate.  EAA are sum of His, Ile, Leu, Lys, Met, Phe,
Thr, Val (note: Cys not measured). ∗Significantly different from Fast, P < 0.05.
returnedtobasal levels by 2 h inboth trials.Bloodessential
amino acid (EAA) concentrations peaked (P <0.001) at
1 h post-drink ingestion for trial 1 (∼2-fold increase) and
trial 2 (∼1.9-fold increase) and returned to basal by 2 h in
both trials. Similarly, blood leucine concentration peaked
(P <0.001) at 1 h for trial 1 (∼3.0-fold increase) and
trial 2 (∼2.2-fold increase) and returned to basal by 3 h
and 2 h for trial 1 and trial 2, respectively.
Electromyography
The quadriceps muscles (i.e. vastus lateralis, vastus
medialis and rectus femoris) showed similar EMG results
and therefore only the vastus lateralis results are reported.
EMG amplitude for the concentric phase of exercise
(Fig. 2A) peaked at 50% of set completion time for 1SET
and the first and second sets of 3SET (P <0.001). The
amplitude of the third set of 3SET was not different from
0% set completion at any time point (P >0.05). EMG
amplitude was not different from the start of the set (0%
set completion) at 100%, 90%, or 70% set completion
for 1SET or the first and second set of 3SET, respectively
(P >0.05). The peak amplitude of the third set of 3SET
was significantly different from peak amplitude of 1SET
between 30 and 60% set completion (P <0.05). There was
a significant main effect of time (P <0.001) indicating a
decrease in isometricMPF from 0% (i.e. 1st repetition) to
100% (i.e. last repetition) set completion (Fig. 2B).
Plasma and Intracellular precursor enrichments
Intracellular precursor enrichment in the rested
fasted biopsy was 3.6±0.3 tracer/tracee. Intracellular
precursor enrichments were stable across time during
the infusion trial 1 for 1SET (3.9±0.3 tracer/tracee)
and 3SET (3.7±0.3 tracer/tracee) and during trial 2 for
1SET (3.8±0.2, 4.2±0.3 tracer/tracee at 1.5 and 6.5 h,
respectively; P >0.05) and 3SET (3.8±0.2 and 4.2±0.2
tracer/tracee at 1.5 and 6.5h respectively; P >0.05).
Furthermore, linear regression analysis indicated that the
slopes of the plasma enrichments were not significantly
different from zero during trial 1 or trial 2 (P >0.05),
suggesting that isotopic plateau was achieved and that
the use of the steady-state precursor product equation
was appropriate. These data also indicate that the tracer
added to the protein during ingestion did not appear in
the circulation more rapidly than the amino acids from
the protein (whey) itself and in fact enrichment was stable
during the period of incorporation.
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2010 The Authors. Journal compilation C  2010 The Physiological Society
J Physiol 588.16 Resistance exercise volume and myofibrillar protein synthesis 3125
Myofibrillar protein synthesis
Fed-stateMPS was elevated above Fast by 2.3- and 3.1-fold
at 5 h post-exercise for 1SET and 3SET, respectively
(both P <0.001; Fig. 3). However, the response at 5 h
post-exercise was significantly greater (P =0.008) for
3SET as compared to 1SET. MPS returned to a mean
value not different fromFast in 1SET at 29 h post-exercise
(P =0.36). However, MPS remained elevated by 2.3-fold
above Fast in 3SET (P =0.033) and was significantly
greater than 1SET at this same time point (P =0.001).
Signalling proteins
Phosphorylation of eIF2Bε (Fig. 4A) was significantly
decreased from fast by 37% at 5 h Fed for
3SET (P =0.031), whereas, phosphorylation of 90 kDa
ribosomal S6 protein kinase (p90RSK; Fig. 4B) was
significantly (P <0.05) greater at 24 h Fast than at Fast
by 120% and 116% for 1SET and 3SET, respectively.
Exercise increased phosphorylation of 70 kDa S6 protein
kinase (p70S6K; Fig. 4C) to a similar extent at 5h Fed
(P <0.05) by 61% and 64% above Fast for 1SET and
3SET, respectively. Furthermore, a latent feeding-induced
increase in p70S6K phosphorylation was observed at 29 h
Fed in 3SET such that this response was greater than Fast
(P <0.05) and 1SET at that same time point (P =0.031).
It was found that a significant relationship (r =0.338,
P =0.033) between the extent of p70S6K phosphorylation
and MPS existed (Fig. 5). Phosphorylation of ribosomal
protein S6 (rpS6) was greater than Fast at 5 h Fed for 3SET
(P =0.022). Also, rpS6 phosphorylation showed a trend
to be greater than Fast at 24 h Fast in 3SET (P =0.068).
Similar to p70S6K, a latent increase in rpS6 was also
demonstrated such that the response was greater than Fast
at 29 h Fed for 1SET (P =0.012) and 3SET (P =0.003).
Finally, there was no change in phosphorylation at any
time points for Akt, p38, mTOR, or Glycogen synthase
kinase (GSK) above Fast (data not shown).
Discussion
Our study is the first to describe the myofibrillar
protein synthetic response and the extent of anabolic
signalling molecule phosphorylation following resistance
exercise performed for only one set or for three sets of
contractions and to show a dose–response relationship
between exercise volume and the response of MPS. We
found that 1SET transiently enhanced fed-state MPS at 5 h
post-exercise and to a lesser extent than 3SET; however,
this minimal volume of exercise loses its stimulatory
effect the next day (i.e. 24–29 h). By contrast, 3SET
elevated fed-state myofibrillar protein synthesis at 5 h,
more than 1SET, and sustained the myofibrillar protein
synthetic response for at least 24 h post-exercise. The
mechanisms facilitating this response may be related,
in part, to phosphorylation (activation) of p70S6K and
its downstream target, rpS6, and the eventual recycling
of the ribosome by decreased eIF2Bε phosphorylation
(activation). Furthermore, p90RSK1 showed a latent
increase in phosphorylation at 24 hpost-exercise;however,
feeding had no stimulatory effect as p90RSK1 activation
returned to basal levels by 29 h post-exercise.
A novel finding of the current study is that the sustained
response of MPS after performing 3SET of resistance
exercise at 24–29 h suggests that a relatively low ‘dose’
of exercise conferred a ‘nutrient-sensitizing’ effect on
skeletal muscle late into the post-exercise recovery period
(i.e. at least 24 h later). This notion is supported by
our data demonstrating that the feeding-induced rise in
MPS is elevated 5 h following resistance exercise, whereas,
the response in the absence of exercise is transiently
elevated at 3 h but returns to basal levels in a non-exercise
control leg (Moore et al. 2009b). Therefore, it is now
0 20 40 60 80 100
0 70
80
90
100
110
1SET
3SET(1)
3SET(2)
3SET(3)
ab
ab
ab abab ab
ab
a c* c*c*c*
A Completion of Set (%)
Vastus Lateralis
Activation (%)
1SET 3SET(1) 3SET(2) 3SET(3)
0 20
40
60
First Repetition
Last Repetition
a B
bc d
MPF (Hz)
Figure 2. Increase in vastus lateralis EMG during the concentric
phase of resistance exercise and change in mean power
frequency during the isometric phase of resistance exercise
A, percentage increase in vastus lateralis EMG (activation) during the
concentric phase of resistance exercise. Numbers in parentheses of key
following 3SET indicate set number. B, the change in mean power
frequency (MPF) during the isometric phase of resistance exercise.
Times with different lower-case letters indicate significantly differences
from first repetition (0% completion): a for 1SET, b for 3SET(1), c for
3SET(2), d for 3SET(3), P < 0.05. ∗Significantly different from 1SET at
that time point, P < 0.05.
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2010 The Authors. Journal compilation C  2010 The Physiological Society
3126 N. A. Burd and others J Physiol 588.16
apparent that the lack of a stimulatory effect of feeding
on MPS following 1SET of resistance exercise at 24–29 h
post-exercise suggests that a certain threshold volume of
contractile activity is necessary to sensitize the muscle to
subsequent feeding.
Recent data (Kumar et al. 2008) have suggested that
the fall of fasted-state MPS at 2–4 h during post-exercise
recovery may have been due to the low dose of resistance
exercise volume utilized during their experimental trial.
However, the authors speculated that it was possible
that amino acid substrate simply became limiting and
ultimately resulted in the sharp decline in myofibrillar
protein synthesis. The current data shed some light
on this paradoxical finding by demonstrating that
resistance exercise volume is important, to some extent,
in maximizing the anabolic response to an exercise
stimulus. However, the ‘dose’ of exercise needed to confer
a lasting and meaningful effect on MPS is less than
commonly utilized in many investigations (i.e. 6–10 sets
× 10–12 repetitions), which have been shown to have
latent stimulatory effects for up to 48 h post-exercise
(Phillips et al. 1997). Furthermore, these data illustrate
that post-exercise feeding is important in sustaining the
myofibrillar protein synthetic response.
We have previously demonstrated that unilateral
resistance training alters the fed-state response of mixed
muscle protein synthesis such that a high load and a
high volume bout of exercise, 6 sets × 8–10 repetitions
at 80% 1RM, is incapable of sustaining the response at
28 h post-exercise following resistance training (Tang et al.
2008). The utilization of resistance-trained subjects in the
current study and the fact that a relatively low dose of
Fast 5h Fed 29h Fed
0.00
0.04
0.08
0.12
1SET
3SET
* ‡
†
* * ‡
Myofibrillar FSR (% •h-1)
1SET 3SET
0 500
1000
1500
2000
2500 ‡
Total Volume
Load (kg)
Figure 3. Myofibrillar protein fractional synthesis rate (FSR) at
rest and 5 h after protein ingestion after one set (1SET) or 3 sets
(3SET) of resistance exercise and 24–29 h later
Inset, volume load (kg times repetitions) performed during 1SET and
3SET of resistance exercise. ∗Significantly different from rest
(P < 0.05). †Significantly different from 29 h (P < 0.05). ‡Significantly
different from 1SET (P < 0.05).
exercise volume can sustain the fed-state response for
at least 24 h post-exercise would seem at odds with our
previous findings (Tang et al. 2008). This discrepancy may
be related to the notion that, in response to an isolated bout
of resistance exercise, quantitatively important changes
occurring in the myofibrillar protein fraction can become
non-detectable if exclusively examining themixed protein
synthetic response in trained subjects (Kim et al. 2005)
and this thesis is further supported by data demonstrating
that training preferentially stimulates the synthesis of
proteins specific to the exercise stimulus (Wilkinson
et al. 2008).
Evidence suggests that members of theMAPKsignalling
cascades (i.e. p38, Erk1/2, p90RSK1) are phosphorylated
in close temporal proximity to the resistance exercise bout
(Williamson et al. 2003; Creer et al. 2005; Drummond
et al. 2008; Tannerstedt et al. 2009). The latent increase in
phosphorylation of p90RSK1 at 24 h and the fact that
one of its targets, rpS6, was also phosphorylated to a
significant extent at 29 h post-exercise, suggests that the
MAPK signalling cascade may be involved in facilitating
translation initiation at further time points (i.e. ≥24 h)
following resistance exercise. Furthermore, the current
data suggest that there is redundancy in the signalling
pathways to activate rpS6 because p70S6K was also
phosphorylated the following day after acute resistance
exercise (Fig. 4C). Therefore, it could be that MAPK
and mTOR–p70S6K signalling cascades may converge to
promote the prolonged phosphoryalated (i.e. activated)
state of rpS6; however, more studies are warranted to
confirm this thesis. It is worth noting that the extent of
p70S6K phosphorylation has been shown to be related to
MPS (Kumar et al. 2008) and resistance exercise induced
increases in muscle mass (Terzis et al. 2008; Mayhew
et al. 2009). Our data further illustrate that the extent of
p70S6K phosphorylation can serve as a reasonable proxy
marker for the anabolic response to resistance exercise
(Fig. 5).
Translation initiation appears to be the primary locus of
control for muscle protein synthesis (Kubica et al. 2005).
During translation initiation, eIF2 recruits the initiator
methionyl-tRNA to the 40S ribosome in aGTP-dependent
manner. eIF2B catalyses the exchange of GDP for GTP on
eIF2 and thus renews the tRNAi binding capacity (Proud,
2005). We have recently demonstrated that reduced
phosphorylation of eIF2B’s catalytic ε subunit occurs at
6 h following resistance exercise in the fasted and fed states
(Glover et al. 2008a). Indeed, others have demonstrated
that resistance exercise had little effect on eIF2Bε
dephosphorylation within close temporal proximity (i.e.
≤1 h) to the resistance exercise bout (Camera et al. 2010).
However, the current data demonstrate that resistance
exercise induced a significant 37% dephoshorylation of
eIF2Bε at 5 h following 3SET, but not 1SET, of resistance
exercise (Fig. 4A). Therefore, it appears that resistance
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2010 The Authors. Journal compilation C  2010 The Physiological Society
J Physiol 588.16 Resistance exercise volume and myofibrillar protein synthesis 3127
-1.0 5h Fed 24h Fast 29h Fed
-0.5
0.0
0.5
1.0
1SET 3SET
* †
A eIF2B Ser539
(Fold change from rest)
5h Fed 24h Fast 29h Fed
0.0
0.5
1.0
1.5
2.0
* *
B p90RSK1 Thr573
(Fold change from rest)
5h Fed 24h Fast 29h Fed
0.0
0.5
1.0
1.5
* †
* *
C p70S6K Thr389
(Fold change from rest)
5h Fed 24h Fast 29h Fed
0.0
0.2
0.4
0.6
0.8
* *
* D
rps6 Ser240/244
(Fold change from rest)
Figure 4. Phosphorylation of eIF2BεSer539 (A), p90RSKThr573 (B), p70S6KThr389 (C) and rps6Ser240/244 (D)
following one set (1SET) or 3 sets (3SET) of resistance exercise in the fasted or fed states
Data are expressed as fold-change from rest. ∗Significantly different from Fast (P < 0.05). †Significantly different
from 1 set (1SET) within that time point (P < 0.05).
exercise increases eIF2Bε activity at later time points in
post-exercise recovery and that volume may be important
in achieving full activation. Indeed, it is important to
recognize that we have limited knowledge on the necessity
of activating particular anabolic signalling molecules (i.e.
eIF2Bε or p90RSK) to stimulate muscle protein synthesis.
It has been established that the phosphorylated states
of anabolic signalling molecules are not related to the
anabolic response (Greenhaff et al. 2008; Moore et al.
2009a) and thus certain anabolic signallingmolecules may
only be permissive but not stimulatory for muscle protein
synthesis.
Examination of vastus lateralis surfaceEMGamplitudes
during 1SET and 3SET reveals that the large
higher-threshold motor units were activated and their
associated type II fibres were recruited in both conditions.
Moreover, motor unit drop-out of these highly fatigable
fibres appeared to occur as the exercise set was nearing
100% completion. The recruitment of type II fibres is
important in eliciting maximal adaptations to resistance
exercise, as these fibres are highly responsive, and more
so than type I fibres, to resistance training insofar as
muscle hypertrophy is concerned (McCall et al. 1996;West
et al. 2010). It has been reported that phosphorylation
of p70S6K on Thr421/Ser424 (Koopman et al. 2006;
Tannerstedt et al. 2009) and on Ser389 (Tannerstedt et al.
2009) is greater in type II fibres. This notion, combined
with our data illustrating similar activation of p70S6K
at 5 h post-exercise, provides further support that 1SET
is capable of achieving maximal muscle activation as
compared to 3SET (Fig. 2A). However, the third set of
0 1 2 3 4
0.00
0.05
0.10
0.15
r = 0.34, P=0.033
p70S6K phosphorylation
(fold-change)
Myofibrillar FSR (% •h-1)
Figure 5. Relationship between myofibrillar protein synthesis
and the extent of phosphorylation of p70S6KThr389
There was a significant (P = 0.033) correlation between the degree of
phosphorylation (fold-change from basal) and myofibrillar protein
synthesis (FSR, % h−1).
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2010 The Authors. Journal compilation C  2010 The Physiological Society
3128 N. A. Burd and others J Physiol 588.16
3SET was significantly different from1SET between 30 and
60% completion of the exercise, which suggests significant
muscle fatigue was occurring, most likely in type II fibres
(Sale, 1987).
Our current methods do not allow us to discriminate
between fibre type-specificMPS; however, itwould appear
that the duration of muscle activation may be important
in sustaining the myofibrillar protein synthetic response.
Whether 1SET or 3SET induces a similar stimulation
of MPS in type II fibres and the subsequent increase
in type II fibre cross-sectional area following resistance
training remains to be seen. Lastly, mean power frequency
(MPF) showed a shift in the frequency, regardless of
condition, following the exercise sets (Fig. 2B). It has been
demonstrated that this frequency shift is associated with
fatigue and can be attributed to changes in muscle fibre
conduction velocity which result from decreases in pH
and metabolite accumulation with muscle fatigue (Brody
et al. 1991).
From a practical perspective it is important to
recognize that scientific studies are performed under
highly controlled conditions and help to decipher specific
mechanistic responses to different exercise perturbations.
In the current study we utilized loading parameters that
were of equal relative intensities, whereas repetitions and
load would be adjusted (i.e. progression) during repeated
bouts of exercise (i.e. training) and ultimately result in
increased volume load being applied during subsequent
bouts. Clearly a training study is required to delineate the
superiority of 1SET or 3SET for inducing hypertrophy.
In summary, our data demonstrate that fundamental
mechanistic differences exist between 1SET and 3SET
that may support the greater accretion of myofibrillar
proteins following 3SET of resistance exercise. The dose
of resistance exercise that results in a lasting stimulation
of MPS is less than is commonly utilized and that has
been reported (Phillips et al. 1997; Phillips et al. 1999).
Even 1SET of resistance exercise (∼14 contractions) at
a moderate intensity elicited a significant rise in MPS;
however, sustaining the exercise-induced rise in MPS
required a greater contraction volume.
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