Decrease in Akt/PKB signalling in human skeletal muscle by resistance exercise

Abstract

We analysed the effects of resistance exercise upon the phosphorylation state of proteins associated with adaptive processes from the Akt/PKB (protein kinase B) and the mitogen-activated protein kinase (MAPK) pathways. Nine healthy young men (21.7 +/- 0.55 year) performed 10 sets of 10 leg extensions at 80% of their 1-RM (repetition maximum). Muscle biopsies were taken from the vastus lateralis at rest, within the first 30 s after exercise and at 24 h post-exercise. Immediately post exercise, the phosphorylation states of Akt/PKB on Thr308 and Ser473 and 4E-BP1 on Thr37/46 (eukaryotic initiation factor 4E-binding protein 1) were decreased (-60 to -90%, P < 0.05). Conversely, the phosphorylation of p70(s6k) (p70 ribosomal S6 kinase) on Thr421/Ser424 was increased more than 20-fold (P < 0.05), and this was associated with a 10- to 50-fold increase in the phosphorylation of p38 and ERK1/2 (extracellular signal-regulated kinase) (P < 0.05). Twenty-four hours post-exercise the phosphorylation state of Akt/PKB on Thr308 was depressed, whereas the phosphorylation of p70(s6k) on Thr421/Ser424 and sarcoplasmic ERK1/2 were elevated. The present results indicate that high-intensity resistance exercise in the fasted state inhibits Akt/PKB and 4E-BP1 whilst concomitantly augmenting MAPK signalling and p70(s6k) on Thr421/Ser424.

Full text

Eur J Appl Physiol (2008) 104:57–65
DOI 10.1007/s00421-008-0786-7
123
ORIGINAL ARTICLE
Decrease in Akt/PKB signalling in human skeletal muscle
by resistance exercise
Louise Deldicque · Philip Atherton · Rekha Patel ·
Daniel Theisen · Henri Nielens · Michael J. Rennie ·
Marc Francaux
Accepted: 26 May 2008 / Published online: 6 June 2008
© Springer-Verlag 2008
Abstract We analysed the eVects of resistance exercise
upon the phosphorylation state of proteins associated with
adaptive processes from the Akt/PKB (protein kinase B)
and the mitogen-activated protein kinase (MAPK) path-
ways. Nine healthy young men (21.7 § 0.55 year) per-
formed 10 sets of 10 leg extensions at 80% of their 1-RM
(repetition maximum). Muscle biopsies were taken from
the vastus lateralis at rest, within the Wrst 30 s after exercise
and at 24 h post-exercise. Immediately post exercise, the
phosphorylation states of Akt/PKB on Thr308 and Ser473
and 4E-BP1 on Thr37/46 (eukaryotic initiation factor 4E-
binding protein 1) were decreased (¡60 to ¡90%,
P < 0.05). Conversely, the phosphorylation of p70
s6k
(p70
ribosomal S6 kinase) on Thr421/Ser424 was increased
more than 20-fold (P < 0.05), and this was associated with
a 10- to 50-fold increase in the phosphorylation of p38 and
ERK1/2 (extracellular signal-regulated kinase) (P < 0.05).
Twenty-four hours post-exercise the phosphorylation state
of Akt/PKB on Thr308 was depressed, whereas the phos-
phorylation of p70
s6k
on Thr421/Ser424 and sarcoplasmic
ERK1/2 were elevated. The present results indicate that
high-intensity resistance exercise in the fasted state inhibits
Akt/PKB and 4E-BP1 whilst concomitantly augmenting
MAPK signalling and p70
s6k
on Thr421/Ser424.
Keywords Cell Signaling · Protein synthesis ·
Resistance exercise · MAPK · p70s6k
Introduction
Adaptive remodelling of skeletal muscles is a feature of
chronic exercise training. Recent studies in humans have
identiWed the Akt/PKB (protein kinase B) and the MAPK
(mitogen-activated protein kinase) pathways as key cas-
cades in the regulation of skeletal muscle plasticity by exer-
cise (Cuthbertson et al. 2006; Dreyer et al. 2006; Eliasson
et al. 2006; Karlsson et al. 2004; Williamson et al. 2003).
Whereas activation of the Akt/PKB pathway is associated
with the modulation of translational eYciency, MAPK sig-
nalling apparently regulates sarcoplasmic and/or nuclear
transcription factors (Deldicque et al. 2005; Wang et al.
1998; Widegren et al. 2001).
Research into the regulation of Akt/PKB signalling by
exercise has produced contrasting results. A series of stud-
ies have demonstrated that contractile activity either posi-
tively or negatively regulates Akt/PKB activity
(Blomstrand et al. 2006; Creer et al. 2005; Dreyer et al.
2006; Terzis et al. 2008), others failed to Wnd any change
(CoVey et al. 2006; Creer et al. 2005; Deshmukh et al.
2006; Eliasson et al. 2006). Such inconsistencies likely reX-
ect exercise speciWcity and nutritional status, and even per-
haps phosphorylation site.
Whereas the eVect of exercise on Akt/PKB is not clear,
food consumption is a potent, indirect regulator of its activity
through associated insulin secretion (Brozinick and Birn-
baum 1998; Wojtaszewski et al. 2001). The activation of
Akt/PKB leads to the stimulation of mammalian target of
rapamycin (mTOR) and its downstream substrates such as
eukaryotic initiation factor 4E-binding protein 1 (4E-BP1)
L. Deldicque · D. Theisen · H. Nielens · M. Francaux (&)
Department of Physical Education and Rehabilitation,
Université catholique de Louvain, Place Pierre de Coubertin 1,
1348 Louvain-la-Neuve, Belgium
e-mail: marc.francaux@uclouvain.be
P. Atherton · R. Patel · M. J. Rennie
School of Graduate Entry Medicine and Health,
City Hospital, University of Nottingham, Derby, UK

58 Eur J Appl Physiol (2008) 104:57–65
123
and p70 ribosomal S6 kinase (p70
s6k
), two key regulators of
protein synthesis. 4E-BP1 is an inhibitor of the initiation
translation factor eukaryotic initiation factor 4E (eIF4E) and
when phosphorylated, eIF4E is released and can form the
multi-protein eIF4F complex (Gingras et al. 1999). The
assembly of this complex is necessary for cap-dependent ini-
tiation to continue. Activation of p70
s6k
requires a sequential
series of phosphorylation events and the eIF3 translation pre-
initiation complex serves an important function in organizing
and coordinating these events (Holz et al. 2005). Phosphory-
lation of Ser/Thr residues in the autoinhibitory domain, such
as at Thr 421 and Ser 424, is required for altering the confor-
mation of p70
s6k
and making Thr 389 and Thr 229 available
for phosphorylation, thereby fully activating p70
s6k
(Weng
et al. 1998). In vitro, Thr 389 is phosphorylated by mTOR
(Burnett et al. 1998) whereas phosphoinositide-dependent
kinase 1 (PDK1) is the kinase for Thr 229.
The MAPK family is composed of four members: extra-
cellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-ter-
minal kinase (p38, JNK) and extracellular signal-regulated
kinase 5 (ERK5). Once activated, they can phosphorylate
sarcoplasmic proteins or alternatively translocate to the
nucleus, where they phosphorylate muscle speciWc tran-
scription factors involved in muscle remodelling (Farooq
and Zhou 2004). ERK1/2 and p38 have been shown to be
involved in exercise-induced signalling in human skeletal
muscle (Long et al. 2004; Widegren et al. 1998; Yu et al.
2001). Cycling markedly increased ERK1/2 phosphoryla-
tion, albeit only transiently, whereas similar exercise has
been demonstrated to lead to a smaller but more persistent
increase in p38 activation (Widegren et al. 1998). ERK1/2
and p38 are also inXuenced by resistance exercise (Creer
et al. 2005; Karlsson et al. 2004), but a clear picture of the
regulatory pattern has not yet emerged. Indeed, MAPK sig-
nalling seems to be regulated diVerentially according to the
mode of contraction (concentric vs. eccentric), the intensity
of the exercise and the training status of the subjects (Wide-
gren et al. 2001).
Crosstalk between the MAPK pathway and p70
s6k
has
been reported in vitro. In H4 hepatoma cells (Mukhopad-
hyay et al. 1992) and in cardiomyocytes (Iijima et al. 2002;
Wang et al. 2001), MAPK proteins act to remove the inhi-
bition in the autoinhibitory domain of p70
s6k
by phosphory-
lating Thr 421/Ser 424. Such events are likely to allow
maximal kinase activity when in tandem with mTOR and
PDK1 phosphorylation (Weng et al. 1998). Whether this is
also the case in skeletal muscle has not yet been tested.
The aim of the present study was to test the hypothesis
that resistance exercise performed in a fasted state increases
the phosphorylation state of MAPK and p70
s6k
on Thr 421/
Ser 424, but does not modify the phosphorylation state of
Akt/PKB because feeding is required. The majority of prior
studies have focused upon the signalling of exercise during
the early phase of the recovery period. However, exercise is
known to alter protein metabolism for >2 days (Phillips
et al. 1997). Taking a biopsy at 24 h post-exercise contrib-
uted to the understanding of exercise signalling in the late
recovery period.
Materials and methods
Subjects
Nine healthy young men (age 21.7 § 0.55 year) partaking
in no formal resistance exercise regime, were recruited. All
subjects were given an oral and written account of the study
before signing a consent form. The protocol was approved
by the Ethic Committee of the Université catholique de
Louvain, and the investigation was performed according to
the principles outlined in the Declaration of Helsinki.
Experimental protocol
In the present study, we used remaining muscle samples
from the placebo group of a larger work the purpose of
which was to investigate the eVects of creatine supplemen-
tation on muscle signalling pathways and gene expression
(Deldicque et al. 2008). To investigate the eVects of resis-
tance exercise on the variables of interest, all study partici-
pants underwent three muscle biopsies, one at rest, one
immediately after exercise and one 24 h post-exercise. All
biopsies were taken after a 10 h overnight fast. The proce-
dure involved the administration of local anesthesia (1%
lidocaine) and a sample extraction from the mid portion of
the vastus lateralis muscle with a 4-mm Bergström biopsy
needle. Blood, macroscopically visible fat and connective
tissue were quickly removed, and the sample was immedi-
ately frozen in liquid nitrogen and stored at ¡80°C.
Before the experiment, subjects participated in a pre-test
to determine one repetition maximum (1-RM) for each leg
on a leg extension apparatus. The exercise consisted of a
one-leg knee extension movement from an angle of 90°–
160°. After a warm-up comprising three sets of ten repeti-
tions at 5 kg, the load was progressively increased until the
subject could not perform more than one single repetition.
Subjects were allowed 2 min rest between each set and
reached 1-RM within 5–6 trials.
Subjects were instructed to refrain from vigorous physi-
cal activity 2 days prior to and during the experimental
phase. Food intake on the evening preceding each muscle
biopsy was controlled by administering a standardised din-
ner (22% protein, 48% carbohydrate and 30% fat). On the
Wrst morning of the study, participants reported to the labo-
ratory, and a Wrst biopsy was taken at rest in a randomly
chosen control leg. The exercise was then performed with

Eur J Appl Physiol (2008) 104:57–65 59
123
the other leg after a warm-up of three sets of ten repetitions
at 5 kg. The main exercise session consisted of 10 sets of 10
repetitions at 80% of the 1-RM of the exercising leg which
corresponds to a mean value of positive work of
19,471 §1,403.5 J. The positive work of each repetition
was calculated by multiplying the moved mass by “g”
(9.81 m s
¡2
) and by the distance (height to which the mass
was raised). In this calculation, we neglected the friction
due to the pulleys. The subjects were instructed to lift the
weight (concentric phase) with both the control and the
exercising leg to a knee angle of 160° in 1 s and to lower
the weight only with the exercising leg (eccentric phase)
during the next 2 s. Each set of 10 repetitions lasted 30 s
and the rest in-between sets was 2 min 30 s. A second
biopsy was taken from the exercising leg within 30 s fol-
lowing the completion of the last repetition. A standardised
breakfast was given after the exercise session (7% protein,
74% carbohydrate and 19% fat). Each participant received
a standardised dinner in the evening (22% protein, 48%
carbohydrate and 30% fat). A third biopsy was taken 24 h
later from the exercising leg after a 10 h overnight fast at
2 cm-interval from the second one.
Protein extraction and cell fractionation
About 20–30 mg of frozen muscle were ground in a mortar
and homogenized in ice-cold hypotonic buVer [20 mM
Hepes, 5 mM sodium Xuoride, 1 mM sodium molybdate,
0.1 mM EDTA, 0.5% NP-40, protease inhibitor cocktail
(Roche Applied Science)] for 5 min on ice. The homoge-
nates were then centrifuged for 30 s at 10,000g. The super-
natant, containing the sarcoplasmic proteins, was stored at
¡80°C. The pellet was re-suspended in a buVer containing
20 mM Hepes, 5 mM sodium Xuoride, 1 mM sodium
molybdate, 0.1 mM EDTA, 20% glycerol, a protease inhib-
itor cocktail and the same volume of a saline buVer contain-
ing 20 mM Hepes, 5 mM sodium Xuoride, 1 mM sodium
molybdate, 0.1 mM EDTA, 20% glycerol, 0.8 M NaCl and
a protease inhibitor cocktail. The solution was then homog-
enized on a rotary mixer for 30 min at 4°C and centrifuged
for 10 min at 10,000g. The supernatant, containing the
nuclear proteins, was stored at ¡80°C. Sarcoplasmic and
nuclear protein concentrations were determined using a
protein assay kit (Bio-Rad Laboratories) with BSA as a
standard. Fraction purity was veriWed by immunoblotting
for nuclear histone 1 (anti-histone 1, 1:1,000, Santa Cruz).
The nuclear fraction was positive for histone 1 whereas the
sarcoplasmic was negative.
SDS/PAGE and immunoblotting
Cell lysates (70 g for sarcoplasmic proteins and 30 g for
nuclear proteins) were combined with Laemmli sample
buVer and separated by SDS/PAGE. After electrophoretic
separation at 40 mA, the proteins were transferred to a
PVDF membrane at 80 V for 4 h for a western blot analysis.
Membranes were then incubated in a 5% Blotto solution.
Subsequently, membranes were incubated with the follow-
ing antibodies (1:500) overnight at 4°C: phospho-Akt/PKB
Ser 473 (Cell Signaling), phospho-Akt/PKB Thr 308 (Cell
Signaling), total Akt/PKB (Cell Signaling), phospho-p70
s6k
Thr 389 (Santa Cruz), phospho-p70
s6k
Thr 421/Ser 424
(Santa Cruz), total p70
s6k
(Santa Cruz), phospho-p38 Thr
180/Tyr 182 (Cell Signaling), total p38 (Cell Signaling),
phospho-ERK1/2 Thr 202/Tyr 204 (Cell Signaling), total
ERK (Cell Signaling), phospho-eEF2 Thr 56 (Cell Signal-
ing), phospho-4E-BP1 Thr 37/46 (Cell Signaling), total 4E-
BP1 (Cell Signaling). Antibodies from Cell Signaling were
diluted in TBST containing 1% BSA and antibodies from
Santa Cruz were diluted in a 5% Blotto solution.
Membranes were washed in TBST and incubated for 1 h
at room temperature in a secondary antibody conjugated to
horseradish peroxidase (1:10,000, Cell Signaling). After an
additional three washes, chemiluminescence detection was
carried out using an Enhanced Chemiluminescent Western
blotting kit (ECL Plus, Amersham Biosciences). Then, the
membranes were stripped and re-probed with a total anti-
body to verify the relative amount of the analyzed proteins
through the whole experiment. The Wlms were then scanned
on an ImageScanner using the Labscan software and quan-
tiWed with the Image Master 1D Image Analysis Software
(Amersham Biosciences). The results represent the phos-
phorylated form of the protein. A value of 1 was arbitrarily
assigned to the pre-exercise conditions which were used as
a reference for the post-exercise values.
Cell culture experiments
C2C12 murine skeletal muscle myoblasts (ATCC, USA) were
seeded in 60 mm-diameter culture dishes and were grown in
Dulbeccos’s ModiWed Eagle Medium (DMEM, Gibco) sup-
plemented with 10% fetal bovine serum (Gibco), 1% penicil-
lin/streptomycin (5,000 U/5,000 g/ml; Gibco) and 100 M
non-essential amino acids (Gibco). When cells were 70% con-
Xuent, the proliferation medium was replaced by a diVerentia-
tion medium containing 1% horse serum (Gibco), 1%
penicillin/streptomycin (5,000 U/5,000 g/ml) and 100 M
non-essential amino acids. The diVerentiation medium was
replaced each day for 3 days. At this time, cells were incubated
for 30 min with either or both SB202190 (10 M, Sigma) and
PD098059 (50 M, Sigma) speciWc inhibitors of p38 and
ERK1/2, respectively. At the end of the incubation period,
cells were rinsed once with PBS and harvested in a lysis buVer
(pH 7.0) containing 20 mM Tris, 270 mM sucrose, 5 mM
EGTA, 1 mM EDTA, 0.1% Triton X-100, 1 mM sodium
orthovanadate, 50 mM sodium -glycerophosphate, 5 mM

60 Eur J Appl Physiol (2008) 104:57–65
123
sodium pyrophosphate, 50 mM sodium Xuoride, 1 mM 1,4-
dithiothreitol (DTT) and a protease inhibitor cocktail (Roche
Applied Science). The homogenate was immediately centri-
fuged at 10,000g for 10 min at 4°C. Protein concentration of
the supernatant was determined using the DC protein assay kit
(Bio-Rad Laboratories) with bovine serum albumin (BSA) as
a standard, before immunoblotting for p70
s6k
(Thr 421/Ser
424) as described.
Statistical analysis
The diVerence between pre-exercise and post-exercise con-
ditions was tested for signiWcance using an analysis of vari-
ance (ANOVA) on ranks for repeated measures (Friedman
test). When signiWcant, Student–Newman–Keuls post hoc
tests were applied. For the cell culture experiments, rank
sum tests were used to assess the diVerence between control
and inhibitory treatment. The signiWcance threshold was set
to P < 0.05. The results are presented as the means § SEM.
Results
InXuence of exercise on the phosphorylation state
of the Akt/PKB pathway intermediates
Immediately after exercise, the phosphorylation state of
Akt/PKB was decreased on both Ser 473 (¡60%, P <0.05,
Fig. 1a) and Thr 308 (¡70%, P < 0.05, Fig. 1b). This exer-
cise-induced inhibition was also observed for 4E-BP1 on
Thr 37/46 (¡90%, P <0.05, Fig.1e), with a similar trend
for p70
s6k
on Thr 389, although the signiWcance threshold
was not reached (Fig. 1c). Conversely, the phosphorylation
state of p70
s6k
on Thr 421/Ser 424 was increased by more
than 20-fold (P < 0.05, Fig. 1d).
Twenty-four hour post-exercise, the phosphorylation
state of Akt/PKB on Ser 473 and 4E-BP1 returned to basal
values, whereas the phosphorylation state of Akt/PKB on
Thr 308 remained depressed (P < 0.05, Fig. 1b) and the
phosphorylation state of p70
s6k
on Thr 421/Ser 424
remained elevated (P <0.05, Fig.1d). At this time, the
phosphorylation state of p70
s6k
on Thr 389 was markedly
elevated in two of the nine subjects, while it remained simi-
lar to basal values in the others.
Exercise did not modify the phosphorylation state of eEF2
on Thr 56 (Fig. 1f) and had no eVect on the expression of the
total form of Akt/PKB, 4E-BP1 and p70
s6k
(data not shown).
Exercise activates the p38 and the ERK1/2 MAPK
pathways
Exercise increased more than tenfold the phosphorylation
state of p38 at Thr 180/Tyr 182 in the sarcoplasm (P <0.05,
Fig. 2a) and more than 20-fold in the nucleus (P < 0.05,
Fig. 2b). After 1 day, these values returned to pre-exercise
levels. The phosphorylation state of ERK1/2 on Thr 202/
Tyr 204 was increased immediately after exercise about 50-
fold in the sarcoplasm (P < 0.05, Fig. 2c) and about 10-fold
in the nucleus (P < 0.05, Fig. 2d). Twenty-four hour after
exercise the phosphorylation state of ERK1/2 in the nucleus
had returned to basal values, whilst remaining elevated in
the sarcoplasm (P < 0.05). The total form of p38 and
ERK1/2, both in the sarcoplasm and in the nucleus, were
not aVected by exercise (data not shown).
Inhibition of p38 and ERK1/2 by using pharmacological
agents
A potential crosstalk between MAPK pathway and p70
s6k
was tested in myogenic C2C12 cells by using SB202190
(10 M), an inhibitor of p38, and PD098059 (50 M), an
inhibitor of ERK1/2 (Fig. 3). The phosphorylation state of
p70
s6k
on Thr 421/Ser 424 was decreased by 75%
(P < 0.05) when myotubes were incubated with SB202190
and by 20% with PD098059 (P < 0.05). Incubating the cells
with both inhibitors did not further decrease the phosphory-
lation state of p70
s6k
as compared with SB202190 alone
(¡75%, P <0.05).
Discussion
The present results have partially been presented in a previ-
ous study (Deldicque et al. 2008), the purpose of which was
to investigate the eVect of creatine coupled to an exercise
session on gene expression and Cell Signaling. The current
analyses emphasize the eVect of contractile activity alone
and show that resistance exercise of high-intensity and
comprising a large component of eccentric contraction
decreases the phosphorylation state of Akt/PKB when sub-
jects are in the fasted state. It is unlikely that the nutritional
status of the subjects is responsible for the drop we
observed in Akt/PKB phosphorylation. Blomstrand et al.
(2006) found that immediately after resistance exercise, the
phosphorylation state of Akt/PKB was decreased to the
same extent in the placebo group and in the group receiving
branched chain amino acids. The decrease in the Akt/PKB
phosphorylation state Wts well with the Wndings that exer-
cise in the fasted state decreases protein synthesis and
increases protein breakdown (Rennie and Tipton 2000;
Wolfe 2000). However, the phosphorylation state of Akt/
PKB has been found to increase after low intensity resis-
tance exercise (Creer et al. 2005). One could postulate that
high intensity (>80% 1-RM) and low intensity resistance
exercise induce opposite responses on Akt/PKB. One
potential candidate mediating the decrease in Akt/PKB

Eur J Appl Physiol (2008) 104:57–65 61
123
phosphorylation is the AMP-activated protein kinase
(AMPK). Indeed, AMPK activity has been shown to be
increased during high intensity resistance exercise (Dreyer
et al. 2006; Koopman et al. 2006) and to lead to a decrease
in the phosphorylation state of Akt/PKB on Ser 473 (Bol-
ster et al. 2002).
The type of contraction, concentric versus eccentric,
could also be an explanation for the discrepancies observed
after exercise on Akt/PKB. Eccentric exercise tends to
decrease Akt/PKB phosphorylation compared to concentric
exercise (Eliasson et al. 2006) and in our protocol, the
eccentric component was high. Eccentric contractions are
known to induce greater proteolysis and muscle damage
when compared to concentric contractions (Sorichter et al.
1995). Thus the type and the intensity of muscle contrac-
tion most likely trigger diVerent responses at the cellular
level.
Immediately after exercise, the phosphorylation state of
p70
s6k
on Thr 389 was slightly but not signiWcantly
depressed (Fig. 1c). This trend could have been the conse-
quence of decreased Akt/PKB phosphorylation. On the other
hand, a large increase was found in the phosphorylation at
Fig. 1 EVect of exercise on the
phosphorylation state of Akt/
PKB, p70
s6k
, 4E-BP1 and eEF2.
Phosphorylation states of Akt/
PKB on Ser 473 (a) and on Thr
308 (b), p70
s6k
on Thr 389 (c)
and on Ser 421/Thr 424 (d), 4E-
BP1 on Thr 37/46 (e) and eEF2
on Thr 56 (f) at rest, within 30 s
following exercise and 24 h after
exercise in the fasted state. A
representative immunoblot of
the phosphorylated form and the
total form is shown at the top of
each graph. Values of the phos-
phorylated form are expressed as
the means § SEM (n =9).
*P < 0.05 post-exercise versus
pre-exercise

62 Eur J Appl Physiol (2008) 104:57–65
123
Thr 421/Ser 424 (Fig. 1d). p70
s6k
possesses many sites of
phosphorylation, and to be fully activated, each site has to
be phosphorylated by speciWc kinases in a sequential man-
ner (Weng et al. 1998). The Wrst step in p70
s6k
activation
involves the phosphorylation of a cluster of (Ser/Thr) Pro
sites. The latter are situated in the autoinhibitory domain in
the carboxyl-terminal tail, including, amongst others, Thr
421 and Ser 424. The identity of the kinases phosphorylat-
ing these sites in vivo is currently not established. MAPK,
SAPK (stress-activated protein kinase) and cdc2 (or Cdk1,
cyclin dependent kinase 1) are capable of phosphorylating
them in vitro (Iijima et al. 2002; Mukhopadhyay et al.
1992; Wang et al. 2001, 1998). In the present study, both
p38 and ERK1/2 were increased immediately after exercise
and could therefore be responsible for the enhanced phos-
phorylation state of p70
s6k
observed on Thr 421/Ser 424.
In search of support for this hypothesis, we tested if
MAPK also contributed to the phosphorylation state of Thr
421/Ser 424 in myogenic cells, as already observed in H4
hepatoma (Mukhopadhyay et al. 1992) and cardiomyocytes
(Iijima et al. 2002; Wang et al. 2001). We measured the
phosphorylation state of p70
s6k
on Thr 421/Ser 424 after
having incubated C2C12 cells with SB202190 and
PD098059, speciWc inhibitors of p38 and ERK1/2,
Fi
g.
2
E
V
ect o
f
exerc
i
se on t
h
e
phosphorylation state of p38 and
ERK1/2. Phosphorylation state
of p38 on Thr 180/Tyr 182 and
ERK1/2 on Thr 202/Tyr 204 in
the cytoplasm (a, c) and in the
nucleus (b, d) at rest, within the
30 s following exercise and 24 h
after exercise in the fasted state.
A representative immunoblot of
the phosphorylated form and the
total form is shown at the top of
each graph. Values of the phos-
phorylated form are expressed as
the means § SEM (n =9).
*P < 0.05 post-exercise versus
pre-exercise
Fig. 3 EVect of the inhibition of p38 and ERK1/2 on the phosphory-
lation state of p70
s6k
in C2C12 cells. Phosphorylation state of p70
s6k
on
Ser 421/Thr 424 after addition of the inhibitor of p38 (SB202190,
10 M), the inhibitor of ERK1/2 (PD098059, 50 M) and both inhibi-
tors for 30 min in C2C12 cells. The experiments were carried out after
72 h of diVerentiation, when myotubes are formed. Values are
expressed as the means § SEM (n =4). *P <0.05 treatment versus
control

Eur J Appl Physiol (2008) 104:57–65 63
123
respectively (Fig. 3). The addition of each inhibitor or both
together decreased the phosphorylation state of Thr 421/Ser
424, indicating that in myogenic cells p38 and ERK1/2
contribute to the phosphorylation of these sites. Although
in vitro results are not directly translatable to in vivo Wnd-
ings and should thus be taken with caution, the current
results suggest that during exercise, p38 and ERK1/2 could
remove the autoinhibition of p70
s6k
by phosphorylating the
Wrst sites in the hierarchical activation of this kinase. As
suggested in Fig. 4, this priming of p70
s6k
possibly partici-
pates in the potentiation of protein synthesis in exercised
muscles when nutrients are provided during recovery
(Louis et al. 2003). Indeed, nutrients are required to achieve
a positive protein balance following exercise at least in part
by initiating signalling leading to the phosphorylation of
Thr 389 (Cuthbertson et al. 2006) and therefore conferring
full activation of p70
s6k
.
As illustrated in Fig. 1e, 4E-BP1 was dephosphorylated
immediately after exercise. Recently, two studies have
demonstrated similar Wndings (Dreyer et al. 2006; Koop-
man et al. 2006). This is in line with our results on the
phosphorylation of Akt/PKB and the fact that protein syn-
thesis is decreased immediately after exercise.
The majority of prior studies have focused upon the sig-
nalling of exercise during the early phase of the recovery
period. However, exercise is known to alter protein metab-
olism for up to 2 days (Phillips et al. 1997), which moti-
vated us to take a biopsy from our subjects at 24 h post-
exercise. To the best of our knowledge, there is only one
paper that has analysed the phosphorylation state of the
Akt/PKB pathway 24 h after exercise (Cuthbertson et al.
2006). In that study, phosphorylation of Akt/PKB on Ser
473 was increased two- to threefold, and, at the same time,
muscle protein synthesis was elevated. However, since the
subjects were not in a fasted state when the biopsies were
taken, these observations might not be directly attributable
to exercise. Our protocol made it possible to highlight the
speciWc eVect of exercise after 24 h since the biopsies were
taken after an overnight fast. The phosphorylation states of
intermediates of the MAPK and Akt/PKB pathways were
largely returned to pre-exercise values, except for Akt/PKB
on Thr 308, p70
s6k
on Thr 421/Ser 424 and the sarcoplas-
mic form of ERK1/2. These results are compatible with the
hypothesis that ERK1/2 is upstream, and a potential kinase
of p70
s6k
on Thr 421/Ser 424, since both ERK1/2 and
p70
s6k
on Thr 421/Ser 424 followed the same phosphoryla-
tion time-course.
By phosphorylating eukaryotic elongation factor 2
kinase (eEF2k), p70
s6k
renders it less active and raises the
inhibition exerted by eEF2k on eEF2 (Fig. 4). Therefore, a
decrease in eEF2 phosphorylation leads to its activation and
an enhanced elongation. In agreement with our observation
on the phosphorylation state of p70
s6k
on Thr 389, which
best reXects its activity, eEF2 phosphorylation was not
modulated by exercise (Fig. 1f). eEF2 has already been
found to be unaltered immediately after resistance exercise
but eEF2 was dephosphorylated at 1 and 2 h after (Dreyer
et al. 2006). It seems that exercise only transiently aVects
eEF2 during the recovery period since after 24 h, the phos-
phorylation state was similar to pre-exercise levels
(Fig. 1f). On the other hand, eEF2 phosphorylation was
increased during continuous endurance exercise and the
authors suggested a role of calcium in the control of eEF2
(Rose et al. 2005). In addition to p70
s6k
and calcium, AMP-
activated protein kinase (AMPK) has been shown to regu-
late eEF2 phosphorylation via eEF2 kinase in vitro (Hor-
man et al. 2002), although, it has not been conWrmed during
exercise in vivo (Rose et al. 2005). Further investigation is
needed to clarify the role of this elongation factor in the
control of protein synthesis in human skeletal muscle after
exercise.
Because MAPK can phosphorylate diVerent sarcoplasmic
targets or translocate to the nucleus, we measured the phos-
phorylation states of p38 and ERK1/2 in both compartments
(Fig. 2). Only two studies have analysed the sarcoplasmic
and the nuclear forms of p38 in human biopsies after endur-
ance exercise (Chan et al. 2004; McGee and Hargreaves
2004) and have demonstrated only moderate (two- to Wve-
fold) increases in the phosphorylation state of nuclear (Chan
et al. 2004) or both total and nuclear p38 (McGee and Harg-
reaves 2004). In comparison with those studies, we mea-
sured much larger increases in the phosphorylation state of
p38 and ERK1/2 (10- to 50-fold) both in the sarcoplasm and
in the nucleus. The greater response in our subjects is likely
due to the diVerent type of exercise. The regulation of
Fig. 4 Proposed model for exercise signalling in human skeletal mus-
cle. ERK1/2 extracellular signal-regulated kinases 1 and 2, Akt/PKB
protein kinase B, mTOR mammalian target of rapamycin, p70
s6k
p70
ribosomal S6 kinase, 4E-BP1 eukaryotic initiation factor 4E-binding
protein 1, eEF2k eukaryotic elongation factor 2 kinase, eEF2 eukary-
otic elongation factor 2

64 Eur J Appl Physiol (2008) 104:57–65
123
MAPK is indeed dependent on the mode and the intensity of
contractions (Widegren et al. 2001). It seems that for co-
activation of p38 and ERK1/2, high-intensity eccentric con-
tractions are required (Wretman et al. 2001). A session of
knee extensor resistance exercise consisting of 29 contrac-
tions at approximately 70% of the 1-RM was associated
with an increase in ERK1/2 phosphorylation with no eVect
on p38 (Williamson et al. 2003). On the other hand, 40 repe-
titions of leg press exercise at 80% of the 1-RM induced an
8-fold increase in the phosphorylation state of ERK1/2 and a
5-fold increase in the phosphorylation state of p38 (Karlsson
et al. 2004). Our exercise bout consisted in 100 repetitions at
80% of the 1-RM for the eccentric component, which could
explain the large amplitude of the response to exercise of
ERK1/2 and p38.
In summary, we report an inhibition of the Akt/PKB
pathway by resistance exercise performed in the fasted
state. Moreover our results show that p38 and ERK1/2 are
activated and suggest that there is a crosstalk between p38
and ERK1/2 and p70
s6k
on Thr 421/Ser 424. This priming
of p70
s6k
via MAPK could be an important step in potenti-
ating protein synthesis with feeding during the recovery
period from exercise. However, the present data do not give
a mechanism for Akt/PKB inhibition and p38 and ERK1/2
stimulation immediately after exercise. Further study is
warranted to describe these molecular phenomena.
Acknowledgments This work was supported by grants to MJ Rennie
from UK Biotechnology and Biological Sciences Research Council
(BB/X510697/1 and BB/C516779/1), US National Institute of Health
AR 49869, and the EC EXEGENESIS program and to M Francaux
from the Fonds de la Recherche ScientiWque Medicale (3.4574.03).
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