Am J Physiol Endocrinol Metab 290: E731–E738, 2006.
First published November 1, 2005; doi:10.1152/ajpendo.00415.2005.
Anabolic signaling and protein synthesis in human skeletal muscle
after dynamic shortening or lengthening exercise
Daniel J. Cuthbertson,1,2 John Babraj,2 Kenneth Smith,3 Emilie Wilkes,3
Mark J. Fedele,4 Karyn Esser,4,5 and Michael Rennie2,3
1
Department of Medicine, Ninewells Hospital and Medical School; 2Division of Molecular Physiology, School of Life
Sciences, University of Dundee, Dundee, Scotland; 3School of Biomedical Sciences, Graduate Entry Medical School,
University of Nottingham, Derby City Hospital, Derby, United Kingdom; 4School of Kinesiology, University of Illinois,
Chicago Illinois; and 5Department of Physiology College of Medicine, University of Kentucky, Lexington, Kentucky.
Submitted 1 September 2005; accepted in final form 26 October 2005
Cuthbertson, Daniel J., John Babraj, Kenneth Smith, Emilie
Wilkes, Mark J. Fedele, Karyn Esser, and Michael Rennie. Ana-
bolic signaling and protein synthesis in human skeletal muscle after
dynamic shortening or lengthening exercise. Am J Physiol Endocrinol
Metab 290: E731–E738, 2006. First published November 1, 2005;
doi:10.1152/ajpendo.00415.2005.—We hypothesized a differential
activation of the anabolic signaling proteins protein kinase B (PKB)
and p70 S6 kinase (p70S6K) and subsequent differential stimulation of
human muscle protein synthesis (MPS) after dynamic shortening or
lengthening exercise. Eight healthy men [25 ⫾ 5 yr, BMI 26 ⫾ 3
kg/m⫺2 (means ⫾ SD)] were studied before and after 12 min of
repeated stepping up to knee height, and down again, while carrying
25% of their body weight, i.e., shortening exercise with the “up” leg
and lengthening exercise with contralateral “down” leg. Quadriceps
biopsies were taken before and 3, 6, and 24 h after exercise. After
exercise, over 2 h before the biopsies, the subjects ingested 500 ml of
water containing 45 g of essential amino acids and 135 g of sucrose.
Rates of muscle protein synthesis were determined via incorporation
over time of [1-13C]leucine (ⱕ6 h after exercise) or [1-13C]valine
(21–24 h after exercise) and phosphorylation of signaling proteins by
Western analysis. PKB and p70S6K phosphorylation increased ⬃3-
fold after 3 h and remained elevated at 6 and 24 h. After exercise, rates
of myofibrillar and sarcoplasmic protein synthesis were unchanged
over the period including exercise and 3 h of recovery but had
increased significantly at 6 (⬃3.0- and 2.4-fold, respectively) and 24 h
(⬃3.2- and 2.0-fold, respectively), independently of the mode of
exercise. Short-term dynamic exercise in either shortening or length-
ening mode increases MPS at least as much as resistance exercise and
is associated with long-term activation of PKB and p70S6K.
exercise-induced anabolism; myofibrillar, sarcoplasmic, and collagen
synthesis
A SINGLE BOUT OF RESISTANCE EXERCISE stimulates human mixed
muscle protein synthesis (MPS) within 4 h, and this effect
persists for up to 48 h (12, 27, 32). It has also been shown,
somewhat surprisingly given the lack of hypertrophy associ-
ated with dynamic exercise, that running and bicycling exer-
cise may also cause a marked increase in MPS (11, 25, 28, 38,
39). Compared with the rate in the fasted state, MPS is
stimulated to a greater extent after exercise by increasing the
availability of amino acids given either intravenously (4) or
orally (42). Recently, we showed that skeletal muscle collagen
synthesis is also acutely stimulated after both isokinetic resis-
Address for reprint requests and other correspondence: M. J. Rennie, School
of Biomedical Sciences, Graduate Entry Medical School, Univ. of Nottingham,
Derby City Hospital, Derby DE22 3DT, UK (michael.rennie@nottingham.ac.uk).
http://www.ajpendo.org 0193-1849/06 $8.00 Copyright © 2006 the American Physiological Society
tance exercise (29) and dynamic kicking exercise (28), and this
effect persists from 4 to 72 h after exercise (28).
The protein kinase B (PKB)-mammalian target of rapamycin
(mTOR) signaling pathway is known to regulate muscle
growth (5, 31) and is activated in response to a variety of
stimuli including increased mechanical load and resistance
exercise (2, 5, 8, 14, 30), amino acids (13), or a combination of
both (23). One of the downstream targets of this pathway is
p70 S6 kinase (p70S6K), which is reportedly involved in the
regulation of a subset of mRNAs that encode the translational
machinery (41). Activation of this signaling pathway in rat
muscle occurs in response to high-frequency electrical stimulation
mimicking resistance exercise, or resistance exercise itself (8, 30),
and in human muscle after resistance exercise (23, 34).
In rat muscle, PKB and p70S6K activation are reportedly
greater in response to lengthening rather than shortening con-
tractions (30), but the differential activation of signaling pro-
teins in response to different types of contractions has never
been examined in human muscle.
Maximal lengthening contractions are capable of generating
greater force than shortening contractions, possibly providing a
greater intensity of stimulus if force is a major determinant of
hypertrophy (rather than total work or ATP turnover). Thus it
might be expected that training with muscle lengthening exer-
cise should lead to greater increases in muscle mass and fiber
hypertrophy than training with shortening exercise, and indeed
this has been reported in some cases (19 –21, 24), but not
all (22).
The first study to address the question of possible differential
responses of muscle protein turnover according to mode of
contraction showed no difference in the extent of acute stim-
ulation of mixed MPS or muscle protein breakdown between
contraction types at 3, 24, and 48 h after resistance exercise
(32). In a subsequent study by Moore et al. (29), in which the
amount of external work done was equivalent, it was found that
rates of myofibrillar protein synthesis rose faster after exercise
in the muscle that had previously made lengthening contrac-
tions, the conclusion being that the extra work done by the
lengthening muscle had increased the response of muscle
protein synthesis, thus revealing a phenomenon not seen in the
original study by Phillips et al. (32).
We therefore now present a study conducted to evaluate the
effects, in the same individuals, of intense stepping exercise in
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E731
E732 MUSCLE ANABOLIC SIGNALING AND PROTEIN SYNTHESIS AFTER EXERCISE
which the muscles of one leg performed shortening exercise
while those of the contralateral leg performed lengthening
exercise. We chose this model to determine whether there were
possible differences between the responses to dynamic length-
ening and shortening exercise. Such differences might be
detectable with a more dynamic form of exercise carried out
continuously, rather than intermittently as during conventional
resistance exercise. We chose to measure the synthesis rates of
the myofibrillar proteins, the sarcoplasmic proteins, and muscle
extracellular matrix collagen to provide more specific informa-
tion on the responses than what was obtainable by analysis of
the overall synthesis rate of mixed muscle.
The hypotheses we tested here were that, in the quadriceps
of young, healthy, but untrained men, the mode of exercise,
(either muscle shortening or lengthening) would differentially
influence 1) activation of translational signaling proteins, 2)
stimulation of muscle myocellular protein synthesis, and 3)
muscle collagen synthesis, with the effects being more pro-
nounced after muscle lengthening vs. shortening. To do this,
we measured the phosphorylation states of two components of
the mTOR signaling pathway (PKB and p70S6K) and rates of
myofibrillar, sarcoplasmic, and collagen synthesis. We studied
the subjects in the fed, rather than fasted, state to elicit a
maximal anabolic response.
METHODS
Power Analysis
The coefficient of variation (CV) for repeated measures of muscle
protein labeling in the same sample is ⬃3.8%, using [1-13C]leucine or
[1-13C]valine to determine the rate of muscle protein synthesis. The
population CVs are ⬃10% for young men. For Western analysis we
commonly find CVs of ⬍20% in healthy young men. In fact, we have
had no difficulty in detecting significant differences in protein amount
or phosphorylation of 25% between groups of eight young men after
physiological interventions such as feeding. A power calculation,
Fig. 1. Experimental protocol.
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assuming a population variance of 15%, studied using laboratory
techniques with CVs of 15%, suggested that we need eight subjects to
detect (with 80% confidence at the 5% significance level) differences
of 20% between treatments (on a paired basis).
Subject Characteristics
Eight healthy men, 25 ⫾ 5 yr, 1.78 ⫾ 0.09 m, 81 ⫾ 10 kg, and
body mass index (BMI) 26 ⫾ 3 kg/m2, participated in the study. The
subjects were habitually active at a recreational level. They were
instructed to adhere to their usual diet and to refrain from strenuous
physical activity for 2 days before the study. The subjects were
informed of the experimental protocol both verbally and in writing
before giving their informed consent. The study protocol was ap-
proved by the Tayside Ethics Committee and was carried out accord-
ing to the Helsinki Declaration.
Experimental Protocol
The subjects attended the laboratory having fasted beginning at
2000 on the previous evening. A forearm vein of each arm was
cannulated at the antecubital fossae for infusion of stable isotope
tracer amino acids and for blood sampling. Four hours before exer-
cise, an infusion of [1-13C]leucine (prime 1.0 mg/kg, 0.8
mg 䡠 kg⫺1 䡠 h⫺1) was started and continued until 6 h postexercise (Fig.
1). Between 21 and 24 h after exercise, a separate infusion of
[1-13C]valine (prime 1.2 mg/kg, 1.0 mg 䡠 kg⫺1 䡠 h⫺1) was given. Two
separate amino acid tracers were used to exclude the need for an
additional muscle biopsy at 21 h and to determine baseline tissue
enrichment, the latter being determined from the zero time biopsy for
valine. Quadriceps muscle biopsies were taken, using 1% lignocaine
anesthetic and the conchotome technique (15) at the start and end of
the 4-h period preceding exercise from either leg and 3, 6, and 24 h
after exercise from both legs. All biopsies were taken using separate
incisions and made from distal to proximal areas of the quadriceps.
After 4 h at rest, the subjects stepped up with one leg onto a
knee-high box (of 0.55 m) and stepped down with the other leg at
⬃0.5 Hz for 6 min, carrying 25% body weight. The “up” and “down”
legs were randomly assigned by the investigators. A metronome and
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 MUSCLE ANABOLIC SIGNALING AND PROTEIN SYNTHESIS AFTER EXERCISE
verbal prompting guided the subjects to perform the exercise at the
correct cadence. After 2 min of rest, they performed two additional
3-min bouts. Thus the protocol lasted for a total of 16 min, 12 min of
which were during exercise. Work by the muscles raising the body
was calculated as the product of the total weight (body wt plus 25%
in backpack), the gravitational constant, the height of the step, and the
number of steps climbed within the 12 min. The total work done was
197 ⫾ 23 kJ with a power output of 274 ⫾ 32 W. Although a similar
amount of energy must have been absorbed by the leg supporting the
body during lowering, it is not possible to calculate the internal work
done by the muscles of this leg, although it is likely to have been
substantially less. On completion of the exercise bout, all subjects
reported being fatigued, were unable to continue further, and had to be
helped to a seat.
After exercise, in the 2 h preceding each biopsy, subjects were
given a 500-ml drink containing 45 g of essential amino acids (EAA)
and 135 g of sucrose. The amino acid-sucrose solution was given as
an initial aliquot of ⬃140 ml followed by six subsequent aliquots of
70 ml every 20 min. The amino acid composition reflected the amino
acid composition of muscle protein (42). Five percent of the total
leucine or valine dissolved in the drink was given as [1-13C]leucine or
[1-13C]valine, as appropriate, to maintain the isotopic labeling of the
plasma leucine and valine. This pattern of feeding was chosen to
maintain a sufficient supply of amino acids to the muscle. The
composition of the drinks, given over the three time periods, was
designed to meet the subjects’ 24-h energy (⬃9 MJ) and protein
requirements. No other feeding was allowed until the end of the study.
Analytical Methods
Plasma. Plasma was separated from whole blood by centrifugation
(300 g) immediately after collection. Labeling of leucine and valine
were measured in their tert-butyldimethylsilyl (t-BDMS) derivatives
(36) and that of ␣-ketoisocaproic acid (KIC) enrichment in its qui-
noxalinol-t-BDMS derivative (35). The plasma labeling of the keto-
acids of leucine and valine, KIC, and ketoisovaleric acid (KIV),
respectively, were taken as representing the value of the true precur-
sors (aminoacyl tRNA) for calculation of the fractional synthesis
rates.
Muscle
Myofibrillar and sarcoplasmic protein extraction. All procedures
were performed as previously described (6). Frozen muscle biopsy
samples (60 – 80 mg) were ground in liquid nitrogen, and the frozen
powder was transferred to 1.6 ml of homogenization buffer containing
protease inhibitors and phosphatase inhibitors [0.15 M NaCl, 0.1%
Triton, 0.02 M Tris, 50 ␮M DTT, 0.1 M EDTA, and 1 mM phenyl-
methylsulfonyl fluoride (PMSF)] to prevent dephosphorylation of the
signaling proteins (6). All procedures were performed on ice.
The homogenate was centrifuged at 1,600 g for 20 min to produce
a myofibrillar pellet and a supernatant containing the sarcoplasmic
fraction, which was removed. The myofibrillar pellet was washed and
centrifuged twice in a low-salt buffer and then washed twice with 70%
ethanol. The myofibrillar pellet was then solubilized in 0.3 N NaOH,
and an aliquot was removed for the determination of protein content
using the Bradford assay (Bio-Rad Laboratories, Hercules, CA). The
supernatant was subjected to high-speed centrifugation (7,000 g, 15
min), and the sarcoplasmic proteins were precipitated with ethanol
and then washed twice with buffer. The sarcoplasmic pellet was then
solubilized in 0.3 N NaOH, and an aliquot was removed for the
determination of protein content using the Bradford assay.
HCl (6 N) was added to the myofibrillar and sarcoplasmic pellets,
and the protein-bound amino acids were released by heating at 110°C
overnight. The HCl was evaporated under nitrogen, the amino acids
were purified by ion exchange chromatography on Dowex H⫹ resin,
and an aliquot was used to determine the rate of protein synthesis as
described below.
AJP-Endocrinol Metab • VOL
E733
Collagen extraction. Frozen muscle biopsy samples, weighing
between 20 and 30 mg, were powdered under liquid nitrogen and
extracted with 0.15 M NaCl buffer and centrifuged, with the
supernatant removed. Next, 0.7 M KCl was added to the pellet
containing myofibrillar proteins and collagen, which was also
centrifuged. The pellet containing collagen was then washed with
acetic acid and acetic acid-pepsin (0.1% wt/vol), dissolving imma-
ture collagen and procollagen and leaving an insoluble collagen
pellet, which was almost pure type I collagen, according to PAGE
analysis with immunoblotting. The myofibrillar, soluble, and in-
soluble collagen proteins were hydrolyzed in 0.1 M HCl-Dowex
H⫹ slurry at 110°C overnight, the liberated amino and imino acids
were separated using Dowex 50 W-X8 H⫹ ion exchange resin, and
an aliquot was used to determine the rate of protein synthesis as
described below.
Determination of the rate of muscle protein synthesis. The liberated
amino acids were dried and derivatized as N-acetyl-n-propyl esters for
measurement of tracer incorporation by gas chromatography-combus-
tion-isotope ratio mass spectrometry (Finnigan DeltaPlus XL; Finni-
gan, Bremen, Germany). Fractional synthetic rates (%/h) were calcu-
lated by comparing the incorporation of [13C]leucine or [13C]valine
over time into isolated protein fractions with the plasma KIC or KIV
enrichment, as previously described (40).
Western Blotting
Muscle (20 – 40 mg) was homogenized using a polytron in a buffer
containing 50 mM Tris 䡠 HCl, 100 mM sodium fluoride, 10 mM
EDTA, 2 mM EDTA, 1 mM bezamidine, 0.2 mM PMSF, 10 mg/ml
leupeptin hemisulfate, 10 mM Na3VO4, and 10 nM okadaic acid.
After homogenization, the protein concentration was determined us-
ing the DC protein assay (Bio-Rad Laboratories). PKB phosphoryla-
tion was quantified by using an antibody for phospho-PKB (Thr308
and Ser473) and total PKB (Cell Signaling Technologies, Beverly,
MA); p70S6K phosphorylation was quantified by using an antibody for
phospho-p70S6K (Thr389) and total p70S6K (Santa Cruz Biotechnol-
ogy, Santa Cruz, CA).
From the sarcoplasmic fraction, 40 ␮g of total protein in Laemmli
buffer were loaded per lane and subjected to SDS-PAGE (10% gel).
After electrophoretic separation, proteins were transferred to a poly-
vinylidene difluoride membrane, and the membranes were blocked in
5% powdered milk in Tris-buffered saline-1% Tween 20 for 1 h,
followed by overnight incubation at 4°C with the following primary
antibodies: phospho-PKB, phospho-p70S6K, p70S6K, and PKB. After
overnight incubation, the blot was washed and then probed with
anti-rabbit antibody (Vector Laboratories, Burlingame, CA) for 45
min at room temperature. The membranes were then washed, and
antibody binding was detected autoradiographically, using the Amer-
sham Life Sciences enhanced chemiluminesence detection kit. Den-
sitometric measurements were carried out using the FluorSmax Im-
ager with QuantityOne Software (Bio-Rad Laboratories). Once the
appropriate image was captured, membranes were stained with Pon-
ceau S to verify equal loading in all lanes.
All results were determined using the ratio of the intensity of the
phosphorylated form to the total intensity (both phosphorylated and
unphosphorylated forms). Data was normalized to the control preex-
ercise values and expressed in arbitrary units.
Data and Statistical Analysis
Data are expressed as means ⫾ SD (n ⫽ 8). The mean values of
subject characteristics were compared with an unpaired t-test, but
results from the plasma and muscle analyses were compared using
one-way ANOVA with the Bonferroni correction. The null hypothesis
was rejected at the 5% level (P ⬍ 0.05).
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E734 MUSCLE ANABOLIC SIGNALING AND PROTEIN SYNTHESIS AFTER EXERCISE
RESULTS
Metabolites and Tracer Labeling
Amino acid, glucose, and insulin concentrations. During the
periods of feeding, plasma glucose, insulin, and total and EAA
concentrations increased; initially, plasma concentrations of
non-EAA decreased slightly but remained stable thereafter
(Fig. 2).
KIC and KIV labeling. Plasma labeling of [13C]KIC and
13
[ C]KIV achieved plateaus (within 30 min of infusion) of
5.2 ⫾ 0.2 and 6.3 ⫾ 0.3% atom percent excess, respectively,
and remained steady throughout, even during oral ingestion of
amino acids.
Signaling Proteins
PKB phosphorylation. Compared with values before exer-
cise in the postabsorptive state, phosphorylation of Ser473 and
Thr308 increased markedly, peaking (3.2 ⫾ 1.6-fold, P ⬍
0.001) at 3 h and falling thereafter but remaining above
baseline at 6 h (2.3 ⫾ 1-fold, P ⬍ 0.05) and 24 h (2.5 ⫾
0.9-fold, P ⬍ 0.01; Fig. 3). There were no significant differ-
ences at any time points between the values of muscle PKB
phosphorylation in the two legs, which, therefore, have been
averaged.
Fig. 2. Plasma essential and nonessential amino acids (AA; A) and glucose and
insulin (B) concentrations for 8 young men. EAA, essential amino acids; CHO,
carbohydrate; NS, not significant. Values are means ⫾ SE; n ⫽ 8.
AJP-Endocrinol Metab • VOL
Fig. 3. p70 S6 kinase (p70S6K) and PKB (Ser473) phosphorylation. Values are
means ⫾ SD; n ⫽ 8. *P ⬍ 0.01; †P ⬍0.001; ‡P ⬍ 0.05. Representative
Western blots of p70S6K and phospho-PKB (Ser473; top) show increased
phosphorylation in a single subject. Pre-ex, preexercise.
p70S6K phosphorylation. Phosphorylation of p70S6K ac-
counted for 20% of the total amount of p70S6K (Fig. 3). At 3 h
after exercise, p70S6K phosphorylation had increased above
resting values by 3.5 ⫾ 2.1-fold in both legs, and this was
sustained up to 24 h (6 h, 3.4 ⫾ 1.8; 24 h, 3.4 ⫾ 1.5; all P ⬍
0.01). As for PKB phosphorylation, there were no differences
between the two legs, and so the values have been averaged.
Muscle Protein Synthesis
Myofibrillar protein synthesis. The fractional synthetic rate
of myofibrillar protein before exercise was 0.042 ⫾ 0.012%/h
(Fig. 4). There was no significant increase in protein synthesis
over the period between the preexercise biopsy and that at 3 h
after exercise in the muscle of either leg (lengthening, 0.051 ⫾
0.015%/h vs. shortening, 0.048 ⫾ 0.014%/h). However, the
rates of protein synthesis were significantly increased above
basal at 6 h (lengthening, 3.1 ⫾ 1-fold to 0.133 ⫾ 0.016%/h vs.
shortening, 2.8 ⫾ 0.7-fold to 0.118 ⫾ 0.023%/h; P ⬍ 0.001)
and 24 h (lengthening, 3.1 ⫾ 0.8-fold to 0.132 ⫾ 0.011%/h vs.
3.3 ⫾ 0.9-fold to 0.139 ⫾ 0.011%/h; P ⬍ 0.001). Because the
difference in the rates of myofibrillar protein synthesis between
the muscles of the two legs were not significant at any time
point, the values were averaged.
Sarcoplasmic protein synthesis. The fractional synthetic rate
of sarcoplasmic protein at rest was 0.061 ⫾ 0.007%/h (Fig. 4),
which was, as reported earlier (6), higher than the rate of
myofibrillar protein synthesis. As for myofibrillar protein syn-
thesis, no significant increase in sarcoplasmic protein synthesis
above basal was observed during exercise and at 3 h after
exercise (lengthening, 0.060 ⫾ 0.013%/h vs. shortening,
0.066 ⫾ 0.018%/h). However, as observed for the myofibrillar
fraction, sarcoplasmic protein synthesis was significantly stim-
ulated at 6 and 24 h, with no discernible difference in the
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 MUSCLE ANABOLIC SIGNALING AND PROTEIN SYNTHESIS AFTER EXERCISE
Fig. 4. Rates of myofibrillar and sarcoplasmic protein synthesis {FSR [frac-
tional synthetic rate (%/h)]} before and after exercise. Values are means ⫾ SD;
n ⫽ 8. *P ⬍ 0.001 vs. basal; †P ⬍ 0.001, different between 3 and 6 h; ‡P ⬍
0.001, different between 3 and 24 h (1-way ANOVA).
responses between the two types of contractile activity. Mean
rates of sarcoplasmic protein synthesis for lengthening and
shortening contractions were significantly elevated from base-
line at 6 h [2.4 ⫾ 0.7-fold to 0.146 ⫾ 0.028%/h vs. 2.3 ⫾
0.7-fold to 0.140 ⫾ 0.027%/h (P ⬍ 0.001)] and at 24 h [2.1 ⫾
0.3-fold to 0.125 ⫾ 0.019%/h vs. 1.9 ⫾ 0.9-fold to 0.117 ⫾
0.014%/h (P ⬍ 0.001)], respectively (Fig. 3). The stimulation
of rates of sarcoplasmic protein synthesis was ⬃20% less than
that of myofibrillar protein synthesis.
Collagen protein synthesis. As expected from recent work
(3), the fractional synthetic rate of collagen at rest was lower
than that for myofibrillar or sarcoplasmic proteins (0.016 ⫾
0.002 vs. 0.042 ⫾ 0.012 and 0.061 ⫾ 0.007%/h, respectively;
Fig. 5). At 3 h after exercise in both lengthening and shortening
legs, the rate of collagen synthesis increased significantly
above resting values (0.048 ⫾ 0.006 and 0.032 ⫾ 0.004%/h,
respectively; lengthening vs. rest P ⬍ 0.001, shortening vs. rest
P ⬍ 0.05, lengthening vs. shortening P ⬍ 0.05). At 6 h after
exercise, no further increase in the rate of collagen synthesis
from 3 h was observed in the muscle of the leg that had
undergone lengthening contractions (0.051 ⫾ 0.010 vs.
0.048 ⫾ 0.006%/h), but there was a rise in collagen synthesis
in the shortening muscle from 3 h (6 vs. 3 h, 0.058 ⫾ 0.012 vs.
0.032 ⫾ 0.004%/h; P ⬍ 0.001) so that any differences between
exercise type had disappeared by 6 h.
DISCUSSION
The major findings of this study are that, after intense
stepping exercise, 1) there are marked increases in the phos-
phorylation state of PKB and p70S6K within 3 h of the end of
exercise, which are largely sustained for up to 24 h; 2) there are
marked increases in the synthetic rates of myofibrillar and
sarcoplasmic protein synthesis, which are only apparent after
6 h but are largely sustained for up to 24 h; 3) the magnitude
of the increase in the rates for contractile and soluble protein
synthesis are larger than previously reported (see, e.g., Refs. 4,
12, 25–27) for any type of exercise; 4) the rates of muscle
collagen synthesis also rapidly increase, with increased rates
AJP-Endocrinol Metab • VOL
E735
being apparent within 3 h of exercise; and 5) there appears to
be no effect of the mode of contraction on the magnitude or
time course of the activation of the signaling proteins or on
rates of myofibrillar and sarcoplasmic protein synthesis.
In the present study, the increased phosphorylation of PKB
and p70S6K helps explain some, but not all, of the increases in
the rates of protein synthesis. After 3 h, phosphorylation of
PKB and p70S6K was increased when the rate of protein
synthesis was unchanged. However, at 6 and 24 h, the persist-
ing activation of the signaling proteins was in line with the
stimulation of muscle protein synthesis. The finding of a
prolonged activation of the signaling proteins in response to
exercise in the present study is in agreement with those of
several studies in rats (2, 18, 30), although Bolster et al. (8)
observed a much more transient response in the activation of
the signaling proteins in jumping rats in vivo. Previously,
studies examining activation of the signaling proteins after
exercise in human skeletal muscle demonstrated increases of
eIF4-BP1 and p70S6K phosphorylation for up to 4 h after an
acute bout of resistance exercise (23, 34). Measurements of
MPS were not made in the study by Karlsson et al. (23), but our
earlier work (34) showed that isometric exercise of the quad-
riceps muscle enhanced the effects on both anabolic signaling
and MPS.
In the present study, we observed a similar activation of the
signaling proteins with muscle lengthening or shortening,
whereas other workers studying rat muscle in vivo found a
greater activation of p70S6K or PKB with muscle lengthening
(2, 30). However, in those studies using rats, muscle contrac-
tions were induced by electrical stimulation (rather than vol-
untary exercise), which might have added an unknown factor.
The basal rates of myofibrillar and sarcoplasmic protein
synthesis and of collagen synthesis are similar to those previ-
ously reported (3, 13). The response of sarcoplasmic protein
synthesis to exercise in the fed state is the same as previously
reported after an acute bout of resistance exercise (26), but the
response of myofibrillar protein is substantially greater, possi-
bly because of the exhausting nature of the exercise, stepping
while carrying 25% of body weight. Whatever the reason for
Fig. 5. Rates of collagen synthesis [FSR (%/h)] before and after exercise.
Values are means ⫾ SD; n ⫽ 8. *P ⬍ 0.001 to basal; ⫹P ⬍ 0.05 to basal;
†P ⬍ 0.001, different between 3 and 6 h; ‡P ⬍ 0.05, different between
lengthening vs. shortening at 3 h (1-way ANOVA).
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E736 MUSCLE ANABOLIC SIGNALING AND PROTEIN SYNTHESIS AFTER EXERCISE
the increased response, the results support previous findings
(11, 25, 38, 39) that dynamic exercise is capable of inducing
significant increases in MPS.
The lack of an early rise (3 h after exercise) in the rates of
MPS requires explanation. In the rested state, a single oral
bolus of 10 g of EAA significantly stimulates myofibrillar and
sarcoplasmic protein synthesis after 3 h (13). Yet in the present
study, no stimulation of myofibrillar or sarcoplasmic protein
synthesis was observed during the 3-h period after the start of
exercise despite subjects being fed 45 g of EAA for 2 out of the
⬃3 h of the measurement. One possibility is that the normally
observed stimulation of MPS at 3 h after feeding was inhibited
after exercise due to a fall in muscle energy status (e.g.,
ATP/ADP ratio), as suggested previously (10).
Consistent with this, we recently showed that a pattern of
muscle stimulation likely to mimic dynamic long-term exercise
was associated with an inhibition of the PKB phosphorylation
of TSC2, an upstream regulator of mTOR, probably as a result
of activation of AMPK (1). However, if this were the case, the
effect is surprisingly long lasting, being maintained beyond the
16 min of the exercise protocol into a further 3 h of postexer-
cise recovery. Furthermore, at 3 h we observed no inhibitory
effect on anabolic signaling as reported to occur in rats after
exercise (7), but rather an activation of PKB and p70S6K.
Whatever the explanation, any inhibition on MPS during and
immediately after exercise did not affect synthesis of collagen,
possibly because this is not synthesized in myofibers, but in the
fibroblasts within the extracellular matrix of muscle.
The persistence of stimulation of MPS at 6 and 24 h with this
protocol is also a novel finding. An elevated rate of MPS after
resistance exercise normally persists for up to 48 h, but the
response slowly diminishes over this time (12, 32). However,
we observed a virtually undiminished elevation over 24 h with
a greater magnitude of stimulation of MPS observed (a three-
and twofold rise in myofibrillar and sarcoplasmic protein
synthesis, respectively), an effect not commonly observed in
previous studies. This may have been because of the intense
nature of the exercise protocol we used or the high rates of
amino acid ingestion. It has previously been shown that the
stimulation of MPS by an amino acid infusion declines rapidly
after 2.5 h despite continued amino acid availability (6). The
strategy we adopted to avoid this, of only providing nutrients
for 2 h in each measurement period, may have overcome any
tachyphylaxis of MPS to amino acid stimulation.
Nevertheless, the results of this study, like those of Phillips
et al. (32), showed there was no significant difference in
stimulation of MPS between muscles undergoing different
modes of contraction. However, in these studies comparisons
were made by using a model of resistance exercise that con-
sisted of eight sets of eight repetitions of raising or lowering
weights at 80% of up to one repetition, so that the amount of
external work done was substantially less in the muscles
lowering the weight, i.e., with lengthening contractions. Fur-
thermore, the studies were carried out in two groups of mixed
male and female subjects, with only four per group randomized
to one of the two modes of exercise, and all were studied in the
postabsorptive state. The rate of synthesis of mixed muscle,
rather than that of the specific muscle subfractions, was mea-
sured. It does seem that the lack of a greater effect is not due
to a lack of amino acid availability; instead, it may be due to a
difference in the intensity of work done by the muscles of the
AJP-Endocrinol Metab • VOL
two legs. When work is matched between legs operating while
shortening or lengthening, it is possible to see a rapid increase
in myofibrillar MPS after 4.5 h, and a greater area under the
myofibrillar time curve over 8.5 h after lengthening compared
with shortening contractions (29). In that study by Moore et al.
(29), the shortening and lengthening exercise were carried out
sequentially on the same day, with comparisons of rest and
exercise on different days. Also, all of the measurements of
rates of myofibrillar and collagen (but not sarcoplasmic) pro-
tein synthesis were made in the fed state. An alternative
interpretation of the Moore study is that it may have been the
provision of food, rather than the increased work done per se,
that stimulated the greater response, because both factors were
changed simultaneously. Also, the lengthening contractions
were carried out first, and although the timing of the biopsies
between legs was adjusted to account for the exercise time,
some unknown systemic factor may have influenced the result,
e.g., cytokines released differentially according to differential
stress in the two modes.
The greater overall stimulation of MPS observed in our
study relative to that seen in the study by Moore et al. (29),
irrespective of contraction type, requires explanation. The total
exercise time in the present study was much greater than in
many previous resistance exercise studies (12 min total exer-
cise vs. repetitions; see, e.g., Refs. 4, 12, 27, 32), and thus there
would have been a greater turnover of ATP. The hypothesis of
ATP turnover being an important determinant of the stimula-
tion of MPS is supported by the preliminary results of another
study by our group (9) in which exercise consisted of different
intensities and duration. Despite these differences, the total
work done was constant, and the exercise protocol stimulated
MPS markedly. However, if a disturbance of muscle energy
status is a major factor in stimulating the adaptive postexercise
response, it might be expected that lengthening contractions
would be less efficient at stimulating synthesis because of a
greater energetic efficiency and a supposedly smaller pertur-
bation in the ATP/ADP ratio. Recent studies have implicated
AMP protein kinase in regulating mTOR activity, linking
mTOR regulation with the energy status of the cell (16, 37).
The results of the present study provide evidence against this
idea because the extent of stimulation of MPS was identical,
even though the likely internal energy cost, borne presumably
by ATP hydrolysis, was less in the muscle of the lengthening
leg. In the present study, we may simply have achieved some
threshold value for total ATP turnover that resulted in the same
total stimulation of MPS, irrespective of mode.
Skeletal muscle collagen synthesis increased rapidly after
lengthening and shortening exercise. This response was greater
in the lengthened leg 3 h after exercise, which is in contrast to
the findings observed by Moore et al. (29) 4.5 h after exercise.
It has been demonstrated that as sarcomeres lengthen, the
collagen fibers are stretched into a linear confirmation (17), and
during repeated lengthening contraction some sarcomeres are
extended beyond the myofilament overlap (43). These sarco-
meres then remain extended, which leaves the collagen fibers
under increased tension. Therefore, even though less work is
done in lengthening contractions, there may be greater tension
in the extracellular matrix.
We have previously shown (33) that amino acids and resis-
tance exercise have a synergistic effect on the stimulation of
skeletal muscle protein synthesis. The synergism between
290 • APRIL 2006 • www.ajpendo.org
 MUSCLE ANABOLIC SIGNALING AND PROTEIN SYNTHESIS AFTER EXERCISE
amino acids and exercise on eIF4-BP1 and p70S6K activity that
we (33, 34) and others (23) observe provides a plausible
molecular mechanism for the enhanced effects of amino acids
on MPS postexercise (4, 42). The results from the present study
extend knowledge of the effects of feeding on postexercise
increases in signaling protein phosphorylation and protein
synthesis up to 24 h.
In addition, however, the results raise fascinating questions
about the effects of different types of exercise, dose responses of
anabolic signaling and MPS in relation to tension and duration of
exercise, and the possible inhibitory effect of very intense exercise
on MPS in the immediate postexercise period. Nevertheless, it
seems clear that, even in the fed state, lengthening and short-
ening exercise carried out in conditions in which the amount of
work done is not equalized, there is no difference in the
responses of anabolic signaling activation and MPS.
ACKNOWLEDGMENTS
We thank the subjects for their participation and the staff of the Clinical
Measurement Suite of Ninewells Hospital for their help.
GRANTS
This work was supported by grants to M. J. Rennie from the UK Medical
Research Council, UK Biotechnology and Biological Sciences Research Coun-
cil, US National Institute of Health (AR-49869), and The Wellcome Trust and
European Community (EXEGENESIS Integrated Project) and to K. Esser from
US National Institutes of Health (AR-45617).
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