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MTOR signaling response to resistance exercise is altered by chronic resistance training and detraining in skeletal muscle


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Resistance training-induced muscle anabolism and subsequent hypertrophy occur most rapidly during the early phase of training and become progressively slower over time. Currently, little is known about the intracellular signaling mechanisms underlying changes in the sensitivity of muscles to training stimuli. We investigated the changes in the exercise-induced phosphorylation of hypertrophic signaling proteins during chronic resistance training and subsequent detraining. Male rats were divided into 4 groups: 1 bout (1B), 12 bouts (12B), 18 bouts (18B), and detraining (DT). In the DT group, rats were subjected to 12 exercise sessions, detrained for 12 days, and then were subjected to 1 exercise session before being sacrificed. Isometric training consisted of maximum isometric contraction was produced by percutaneous electrical stimulation of the gastrocnemius muscle every other day. Muscles were removed 24 h after the final exercise session. Levels of total and phosphorylated p70S6K, 4E-BP1, rpS6, and p90RSK levels were measured, and phosphorylation of p70S6K, rpS6, and p90RSK was elevated in the 1B group compared to control muscle (CON) after acute resistance exercise, while repeated bouts of exercise suppressed those phosphorylation in both 12B and 18B groups. Interestingly, these phosphorylation levels were restored following 12 days of detraining in the DT group. On the contrary, phosphorylation of 4E-BP1 was not altered with chronic training and detraining, indicating that with chronic resistance training, anabolic signaling becomes less sensitive to resistance exercise stimuli, but is restored after a short detraining period.
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doi: 10.1152/japplphysiol.01161.2012
114:934-940, 2013. First published 31 January 2013;J Appl Physiol
Fujita, Koichi Nakazato and Naokata Ishii
Riki Ogasawara, Koji Kobayashi, Arata Tsutaki, Kihyuk Lee, Takashi Abe, Satoshi
by chronic resistance training and detraining in skeletal
mTOR signaling response to resistance exercise is altered
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mTOR signaling response to resistance exercise is altered by chronic
resistance training and detraining in skeletal muscle
Riki Ogasawara,
Koji Kobayashi,
Arata Tsutaki,
Kihyuk Lee,
Takashi Abe,
Satoshi Fujita,
Koichi Nakazato,
and Naokata Ishii
The Research Organization of Science and Technology, Ritsumeikan University, Kusatsu, Shiga, Japan;
Faculty of Sport
and Health Science, Ritsumeikan University, Kusatsu, Shiga, Japan;
Department of Human and Engineered Environmental
Studies, University of Tokyo, Tokyo, Japan;
Graduate School of Health and Sport Science, Nippon Sport Science University,
Tokyo, Japan; and
Department of Health, Exercise Science and Recreation Management, University of Mississippi, Oxford,
Submitted 24 September 2012; accepted in final form 25 January 2013
Ogasawara R, Kobayashi K, Tsutaki A, Lee K, Abe T, Fujita S,
Nakazato K, Ishii N. mTOR signaling response to resistance exercise
is altered by chronic resistance training and detraining in skeletal
muscle. J Appl Physiol 114: 934 –940, 2013. First published January
31, 2013; doi:10.1152/japplphysiol.01161.2012.—Resistance train-
ing-induced muscle anabolism and subsequent hypertrophy occur
most rapidly during the early phase of training and become progres-
sively slower over time. Currently, little is known about the intracel-
lular signaling mechanisms underlying changes in the sensitivity of
muscles to training stimuli. We investigated the changes in the
exercise-induced phosphorylation of hypertrophic signaling proteins
during chronic resistance training and subsequent detraining. Male
rats were divided into four groups: 1 bout (1B), 12 bouts (12B), 18
bouts (18B), and detraining (DT). In the DT group, rats were sub-
jected to 12 exercise sessions, detrained for 12 days, and then were
subjected to 1 exercise session before being killed. Isometric training
consisted of maximum isometric contraction, which was produced by
percutaneous electrical stimulation of the gastrocnemius muscle every
other day. Muscles were removed 24 h after the final exercise session.
Levels of total and phosphorylated p70S6K, 4E-BP1, rpS6, and
p90RSK levels were measured, and phosphorylation of p70S6K, rpS6,
and p90RSK was elevated in the 1B group compared with control
muscle (CON) after acute resistance exercise, whereas repeated bouts
of exercise suppressed those phosphorylation in both 12B and 18B
groups. Interestingly, these phosphorylation levels were restored after
12 days of detraining in the DT group. On the contrary, phosphory-
lation of 4E-BP1 was not altered with chronic training and detraining,
indicating that, with chronic resistance training, anabolic signaling
becomes less sensitive to resistance exercise stimuli but is restored
after a short detraining period.
muscle hypertrophy; retraining; intracellular signaling; exercise
THE DYNAMIC AND PLASTIC NATURE of skeletal muscle allows it to
respond adaptively to changes in activity. Resistance exercise
accelerates muscle anabolism, and muscle proteins gradually
accumulate with repetition of this type of stimulus. Skeletal
muscle also has the ability to maintain cell homeostasis and
thus to respond adaptively to muscle contraction stimuli to
minimize cellular disturbances during subsequent stimulation.
This can be associated with the reduced anabolic response and
rate of growth of skeletal muscle observed with repetition of
the muscle contraction stimuli.
Resistance training is known to be a strong stimulus for
inducing muscle hypertrophy, but only a limited literature
exists that investigates the changes in phosphorylation status of
signaling proteins in response to resistance exercise in animal
models. In humans, resistance training-induced muscle anabo-
lism and subsequent hypertrophy occur most rapidly during the
early phases of training, becoming progressively slower with
time (33, 40). On the other hand, following a detraining period,
muscle adaptation responses may return to their initial levels,
and the effects of retraining on muscle growth are similar to
those observed during the initial phase of resistance training
(33, 37). However, the mechanisms underlying such changes in
the sensitivity of muscles to training stimuli are unclear.
The mammalian target of rapamycin (mTOR) signaling
pathway is recognized as a key regulator of translation initia-
tion and has been shown to be important in muscle protein
synthesis and muscle hypertrophy (2, 3, 13, 31, 36). Resistance
exercise is known to be one of the stimuli able to activate
mTOR signaling activity (6, 25). A recent study has clearly
demonstrated that mechanical load-induced muscle hypertro-
phy is fully dependent on mTOR signaling within the skeletal
muscle (17). However, resistance exercise also activates extra-
cellular signal-regulated kinase (ERK) 1/2 and its downstream
substrate p90 ribosomal S6 kinase (p90RSK), which are both
involved in a signaling pathway independent of mTOR activity
(6, 22, 29, 42). The ERK signaling pathway is also considered
to be a regulator of translation initiation and protein synthesis
(12, 13, 39). Therefore, studies investigating mTOR and ERK
signaling pathway activities should give insight into the mech-
anisms underlying resistance exercise-induced anabolic re-
The purpose of the present study was to examine whether the
activities (based on phosphorylation of signaling molecules) of
mTOR and ERK signaling pathways are altered with training
and detraining. To elucidate this adaptive response, a training
protocol was designed to induce maximal muscle contraction.
We hypothesized that the activation of the mTOR signaling
pathway in response to muscle contraction would be attenuated
after chronic training, even if muscle is contracted maximally,
but would recover after short-term detraining.
Animals. Twenty male Sprague-Dawley rats, 10 wk of age (356.1
4.4 g), were obtained from CLEA Japan (Tokyo, Japan). All animals
were housed individually in an environment maintained at 22–24°C
with a 12-h light-dark cycle and were allowed food and water ad
Address for reprint requests and other correspondence: R. Ogasawara, The Research
Organization of Science and Technology, Ritsumeikan Univ., 1-1-1 Nojihigashi,
Kusatsu, Shiga 525-8577, Japan (e-mail:
J Appl Physiol 114: 934–940, 2013.
First published January 31, 2013; doi:10.1152/japplphysiol.01161.2012.
8750-7587/13 Copyright
2013 the American Physiological Society http://www.jappl.org934
by Riki Ogasawara on April 2, 2013 from
libitum. Rats were randomly assigned to one of four groups: 1
exercise bout (1B), 12 exercise bouts (12B), 18 exercise bouts (18B),
and detraining (DT). In the DT group, rats were detrained 12 days
after completion of 12 exercise sessions and then completed 1 exer-
cise session. This study was approved by the Ethics Committee for
Animal Experiments at Nippon Sport Science University.
Muscle activation. Under isoflurane anesthesia, the hair was shaved
off the right lower leg of each rat, and the shaved legs were cleaned
with alcohol wipes. Rats were then positioned with their right foot on
the footplate (the ankle joint angle was positioned at 90°) in the prone
posture. The triceps surae muscle was stimulated percutaneously with
electrodes (Vitrode V, Ag/AgCl; Nihon Kohden, Tokyo, Japan),
which were cut to 10 mm 5 mm and connected to an electric
stimulator and an isolator (SS-104J; Nihon Kohden, Tokyo, Japan).
Resistance training protocol. Rats were acclimatized for 1 wk, and
the right gastrocnemius muscle was then isometrically trained every
other day (i.e., Monday, Wednesday, Friday, Sunday, Tuesday, Thurs-
day, etc.). The left gastrocnemius muscle served as an internal control.
For all exercise sessions, the gastrocnemius muscle was trained by
stimulating five contractions, with a 5-s interval between contractions,
per set for five sets, with 5-min rest intervals. The voltage (30 V)
and stimulation frequency (60 Hz) were adjusted to produce maximal
isometric tension. Before every exercise session, peak twitch torque
was measured. Torque signals were collected continuously at a sam-
pling rate of 1,024 Hz using a 16-bit analog-to-digital converter
(PowerLab/16SP; AD Instruments) and analyzed using Power Lab
Chart 5 software (AD Instruments). Twenty-four hours after the last
exercise session, rats were anesthetized and exsanguinated. Target
tissues were removed immediately after death. After the mass of each
tissue was measured, tissues were rapidly frozen in liquid N
stored at 80°C until use.
Western blotting analysis. Muscle samples were homogenized with
a polytron homogenizer in a homogenization buffer containing 100
mM Tris·HCl, pH 7.8, 1% NP40, 0.1% SDS, 0.1% sodium deoxy-
cholate, 1 mM EDTA, 150 mM NaCl, and protease and phosphatase
inhibitor cocktail (Thermo Fisher Scientific). Homogenates were cen-
trifuged at 15,000 g for 15 min at 4°C. The supernatant was removed,
and the protein concentration for each sample was determined using a
protein concentration determination kit (Protein Assay II; Bio-Rad,
Hercules, CA). The samples were diluted in 3 sample buffer (1.0%
vol/vol -mercaptoethanol [-ME], 4.0% wt/vol SDS, 0.16 M
Tris·HCl, pH 6.8, 43% vol/vol glycerol, and 0.2% wt/vol bromophe-
nol blue) and boiled at 85°C for 5 min. Using 10 –15% SDS-
polyacrylamide gels, 30 g of protein were separated by electropho-
resis and subsequently transferred to polyvinylidene difluoride
(PVDF) membranes. After transfer, the membranes were washed in
Tris-buffered saline containing 0.1% Tween-20 (TBST), and mem-
branes were then blocked with 3% BSA in TBST for 1 h at room
temperature. After blocking, membranes were washed and incubated
overnight at 4°C with primary antibodies, including phospho-p90RSK
(Thr573, catalog no. 9345), total p90RSK (catalog no. 9355), phos-
pho-p70S6 kinase (Thr389, catalog no. 9205), total p70S6 kinase
(catalog no. 9202), phospho-S6 ribosomal protein (Ser235/236, cata-
log no. 2211), phospho-S6 ribosomal protein (Ser240/244, catalog no.
2215), total S6 ribosomal protein (catalog no. 2217), phospho-4E-BP1
(Thr37/46, catalog no. 9459), and total 4E-BP1 (catalog no. 9452)
(Cell Signaling Technology, Danvers, MA). Membranes were then
washed again in TBST and incubated overnight with appropriate
secondary antibodies at 4°C. Chemiluminescent reagents (SuperSignal
West Dura; Pierce, Rockford, IL) were used to facilitate the detection of
protein bands. Images were scanned using a chemiluminescence de-
tector (AE6961; ATTO, Tokyo, Japan). After the scan, the mem-
branes were stained with Coomassie Blue to verify equal loading in all
lanes. Band intensities were quantified using a PC application (CS
Analyzer; ATTO). Samples from all eight experimental conditions
were run on the same gel, which allowed for the direct comparison
between conditions.
Statistical analysis. Changes in protein expression and muscle wet
weight were compared by two-way ANOVA [training status (group)
stimulation]. Changes in mechanical parameters were examined by
one-way ANOVA. Post hoc analyses were performed using t-tests
with the Benjamini and Hochberg false discovery rate correction for
multiple comparisons. All values were expressed as means SE.
Significance was accepted at P 0.05.
Animal characteristics. Rat characteristics are presented in
Table 1. Relative to the control muscle, neither muscle wet
weight nor its value relative to body weight in the exercised
muscle changed after a single bout of training. Muscle wet
weight and its value relative to body weight were increased
above control muscle after 12 and 18 training sessions by 8.6%
(P 0.01) and 10.7% (P 0.01), respectively. After 12
training sessions followed by 12 days of detraining, both
muscle wet weight and its value relative to body weight
remained equivalent to the posttraining level (P 0.03 vs.
control muscle).
Mechanical parameters. Table 2 shows changes in twitch
and tetanic parameters. The peak tetanic torque (Po) and peak
tetanic rate of force development (RFD) were increased with
training and were maintained at a similar level during the
subsequent detraining period. However, the Po relative to the
gastrocnemius muscle wet weight and the tetanic RFD relative
to Po were not changed during training and detraining periods.
Similarly, the peak twitch torque (Pt) was increased with
training, but the Pt relative to the gastrocnemius muscle wet
weight was not changed with training. On the other hand,
Table 1. Animal characteristics
1 Bout 12 Bouts 18 Bouts DT
BW, g 366.0 9.5 455.0 7.6† 473.1 15.3† 508.2 11.7†‡
GST WW, mg
RT 1,968.2 40.8 2,554.8 50.4*† 2,577.0 60.2*† 2,761.1 63.4*†
CON 1,956.7 53.8 2,353.2 21.9† 2,329.5 24.3† 2,538.2 58.6†‡§
GST WW/BW, mg/g
RT 5.28 0.09 5.62 0.06*† 5.45 0.05* 5.43 0.03*
CON 5.24 0.08 5.18 0.11 4.94 0.16 5.00 0.05
% Difference
RT vs. CON 0.69 1.29 8.62 2.53† 10.65 2.63† 8.80 0.93†
Values are means SE. DT, detraining; BW, body weight; GST, gastrocnemius muscle; WW, wet weight; RT, resistance-trained muscle; CON, control
muscle. *Significant difference vs. CON (P 0.05). †Significant difference vs. 1 bout (P 0.05). ‡Significant difference vs. 12 bouts (P 0.05). §Significant
difference vs. 18 bouts (P 0.05).
935Training, Detraining, and Muscle Anabolic Signaling Ogasawara R et al.
J Appl Physiol doi:10.1152/japplphysiol.01161.2012
by Riki Ogasawara on April 2, 2013 from
compared with the initial session, the twitch RFD was greater
at both the 18th training session and the 1st training session
after the 12-day detraining period, and the twitch RFD relative
to the Pt was greater at the 1st training session after the 12-day
detraining period than at the 18th training session.
p70S6 kinase. Phosphorylation at Thr389 of p70S6K was
elevated above control muscle (P 0.05) 24 h after the first
training session in the 1B group (Figs. 1A and 5). However,
repeated bouts of exercise blunted the level of phosphorylation
of p70S6K in the 12B or 18B groups, although this level was
still higher than in the control muscle (P 0.05). The p70
phosphorylation in response to a bout of exercise was restored
after 12 days of detraining in the DT group. Chronic training
increased total protein of p70S6K in the 12B or 18B groups,
whereas a short detraining period tended to decrease the total
protein of p70S6K (Figs. 1B and 5).
4E-BP1. No significant change in phosphorylation of 4E-BP1
at Thr37/46 was observed throughout the training and detraining
periods (P 0.10; Figs. 2A and 5). On the other hand, training
increased total 4E-BP1 protein in the 18B group (Figs. 2B and 5).
p90RSK. Acute muscle contraction increased phosphoryla-
tion of p90RSK at Thr573 in the 1B group, and a similar
Table 2. Mechanical parameters
1 Bout 12 Bouts 18 Bouts DT
Po, mN m 209.2 9.5 265.2 6.6† 279.0 6.5† 293.0 4.0†
Po/GST WW, mN m/g 106.3 4.2 103.8 1.6 108.4 2.5 106.3 2.7
Pt, mN m 90.0 6.3 100.9 4.2 108.0 3.5† 107.0 3.5
Pt/GST WW, mN m/g 45.8 3.2 39.5 1.3 42.0 1.8 38.8 1.3
Tetanic RFD, N m/s 3.79 0.32 5.01 0.20† 4.96 0.20† 4.99 0.23†
Tetanic RFD/Po, %Po/s 1,879.9 169.8 1,894.80 84.0 1,778.8 54.5 1,700.1 67.3
Twitch RFD, N m/s 3.62 0.28 4.03 0.17 4.36 0.15† 4.40 0.17†
Twitch RFD/Pt, %Pt/s 4,096.6 18.7 4,062.0 30.0 4,040.6 17.9 4,139.4 19.6*
Values are means SE. Po, peak tetanic torque; Pt, peak twitch torque; RFD, peak rate of force development. *Significant difference vs. 18 bouts (P 0.05).
†Significant difference vs. 1 bouts (P 0.05).
Fig. 1. Total and phosphorylated p70S6K 24 h after the respective training
protocols. Values are means SE. *Significant difference vs. control muscle
(P 0.05).
Significant difference vs. 1B (P 0.05).
Fig. 2. Total and phosphorylated 4E-BP1 24 h after the respective training.
Values are means SE. *Significant difference vs. control muscle (P 0.05).
Significant difference vs. 1B (P 0.05).
936 Training, Detraining, and Muscle Anabolic Signaling Ogasawara R et al.
J Appl Physiol doi:10.1152/japplphysiol.01161.2012
by Riki Ogasawara on April 2, 2013 from
increase was observed throughout the training and detraining
period in the 12B, 18B, and DT groups (all P 0.05; Figs. 3A
and 5). Chronic training increased total protein of p90RSK in
the 12B or 18B groups (P 0.05; Figs. 3B and 5). Total
protein of p90RSK was still elevated above control muscle but
tended to decrease after 12 days of detraining period (Figs. 3B
and 5).
S6 ribosomal protein. Acute exercise increased (P 0.05)
rpS6 phosphorylation status at both Ser235/236 (Figs. 4A and 5) and
Ser240/244 (Figs. 4B and 5) 24 h after the initial training session in
the 1B group. However, repeated bouts of exercise blunted the
level of phosphorylation of rpS6 at Ser235/236 in the 12B or
18B groups, although this level was still higher than the control
muscle (P 0.05), whereas no significant elevation in phos-
phorylation of rpS6 at Ser240/244 was observed after chronic
training in both the 12B and 18B groups. As in p70S6K, the
phosphorylation response of rpS6 was restored following de-
training in the DT group (P 0.02 vs. control muscle).
Chronic training increased total protein of p90RSK in the 12B or
18B groups, whereas a short detraining period tended to decrease
the total protein of p90RSK (Figs. 4C and 5).
Chronic muscle contraction induces a variety of metabolic
and morphological adaptations in skeletal muscle to maintain
homeostasis and minimize cellular disturbances during subse-
quent training sessions. Chronic adaptations are the result of
the cumulative effects of repeated bouts of exercise, and
certain molecular and cellular responses lead to specific adap-
tations. Therefore, acute exercise-induced molecular responses
are likely to be effective predictors of training outcomes.
However, it has been relatively unknown whether these re-
sponses were altered with chronic muscle contraction. In the
present study, we investigated the effects of chronic muscle
contraction (resistance training) and subsequent cessation of
training (i.e., detraining) on muscle anabolic signaling activi-
ties. Our main finding was that, although repeated bouts of
Fig. 3. Total and phosphorylated p90RSK 24 h after the respective training.
Values are means SE. *Significant difference vs. control muscle (P 0.05).
Significant difference vs. 1B (P 0.05).
Fig. 4. Total and phosphorylated rpS6 24 h after the respective training. Values are means SE. *Significant difference vs. control muscle (P 0.05).
Significant difference vs. 1B (P 0.05).
Fig. 5. Representative blots for the proteins. C, control (untrained) muscle; T,
trained muscle.
937Training, Detraining, and Muscle Anabolic Signaling Ogasawara R et al.
J Appl Physiol doi:10.1152/japplphysiol.01161.2012
by Riki Ogasawara on April 2, 2013 from
exercise blunted the phosphorylation of the mTOR down-
stream target p70S6K and rpS6, short-term detraining could
lead to a recovery of these. However, in our experiments, the
phosphorylation level of the signaling protein 4E-BP1, down-
stream of mTOR, was not significantly altered with training
and detraining.
Our rat isometric training model increased muscle mass (wet
weight) by 8.6% after 12 training sessions and by 10.7% after
18 training sessions. The extent of muscle hypertrophy in this
study was comparable to those in previous studies using direct
nerve stimulation (1, 20). Some signaling proteins (i.e.,
p70S6K and p90RSK) are known to be phosphorylated imme-
diately, and their peak activation state is often observed 3h
after resistance exercise. For example, previous studies have
reported that the peak activation or phosphorylation state of
p70S6K is observed 3 h after exercise (27, 32, 34). We
investigated the phosphorylation state of multiple signaling
proteins at 24 h after the initial training session. Therefore, we
may have missed the peak activation of some or all proteins.
However, endurance-type muscle contraction, which does not
result in muscle hypertrophy, is also known to increase phos-
phorylation of p70S6K during the early phase of the recovery
period but returns to basal level by 6 h after contraction (30).
Similarly, the rat squat training model has also been used as an
animal resistance exercise model, which results in increased
anabolic signaling activity (4, 11, 25). However, a previous
study using this model reported that p70S6K phosphorylation
was elevated for up to 12 h after exercise but returned to
preexercise levels by 24 h after exercise (7, 11, 21), and it has
not been reported to alter muscle weight as a result of repeated
bouts of exercise, probably because insufficient exercise load
or volume was added to muscle (14, 24). In contrast, certain
signaling proteins show relatively prolonged changes in their
activated or elevated phosphorylation state during a recovery
period (27, 32, 34), and in agreement with these data, we
observed elevated phosphorylation of p70S6K, p90RSK, and
rpS6 24 h after the initial training session. Similarly, studies
using direct electrical nerve stimulation have reported elevated
phosphorylation of these proteins for more than 24 h after
exercise (2, 18, 19). Therefore, acute resistance exercise-
induced activation of anabolic signaling pathways, especially
later events (i.e., 24 h after exercise) in the phosphorylation
cascade, may be important for determining the net anabolic
response and may be responsible for muscle hypertrophy
caused by repeated bouts of resistance exercise.
Acute contraction-induced kinase phosphorylation in skele-
tal muscle has been shown to be attenuated after chronic,
low-frequency, electrical stimulation-induced contractile activ-
ity, which leads to endurance training-like skeletal muscle
adaptation (26). However, relatively little is known about
changes in the activation of the mTOR signaling pathway with
chronic, high-frequency, electrical stimulation-induced maxi-
mal contractile activity. In the present study, we showed
attenuated phosphorylation of p70S6K and rpS6 with chronic
resistance exercise-like electrical stimulation inducing a max-
imal isometric contraction. Although few studies examined
change in phosphorylation status with training in animal study,
a previous study in humans examined the anabolic signaling
response to a bout of resistance exercise before and after 10 wk
of resistance training (41). This study demonstrated that the
duration of elevated Akt, p70S6K, and GSK3- phosphoryla-
tion was reduced in trained participants compared with un-
trained participants, and rpS6 phosphorylation, which was
elevated in the untrained state, was not increased after the
10-wk training period (41). Furthermore, another human study
reported that the phosphorylation of p70S6K and rpS6 did not
increase after a session of resistance exercise in highly resis-
tance-trained subjects (power lifters), whereas increases in
these phosphoproteins were observed in untrained subjects (8).
These results suggest that the responses of mTOR signaling
molecules to resistance exercise may be altered with chronic
resistance training, which agree with our current results in
animal model. On the other hand, in the present study, atten-
uated phosphorylation responses of p70S6K and rpS6 were
restored to the initial postexercise levels after 12 days of
detraining, and this recovery occurred without significant mus-
cle atrophy, suggesting that attenuated specific anabolic re-
sponses can be recovered after a short-term detraining period
without morphological changes to the muscle.
4E-BP1 as well as p70S6K are known to be key downstream
effectors of mTOR. Multisite phosphorylation of the transla-
tional repressor 4E-BP1 results in its dissociation from eIF4E,
thereby allowing eIF4E to assemble with eIF4G, facilitating
the recruitment of other translation initiation factors to form the
eIF4F complex and initiate cap-dependent translation (16).
Previous studies showed elevated 4E-BP1 phosphorylation
after resistance exercise (4, 38). However, changes in phos-
phorylation of 4E-BP1 and p70S6K in response to a bout of
exercise do not necessarily correspond, and some previous
studies have failed to find elevated phosphorylation of 4E-BP1
(9, 10, 23, 29). We also did not detect an elevated rate of
4E-BP1 phosphorylation 24 h after exercise throughout the
training and detraining periods, suggesting differential regula-
tion of 4E-BP1 and p70S6K. Future work is needed to clarify
the difference in regulation of the phosphorylation status of
4E-BP1 and p70S6K.
The ERK1/2 signaling pathway has the ability to regulate
proteins involved in the initiation and elongation stages of
mRNA translation in an mTOR-dependent and -independent
manner (28, 35, 43, 44). Recent studies indicated that ERK
signaling is involved in later phase anabolic responses after
resistance exercise (5, 6, 15). In the present study, we also
observed that phosphorylation of p90RSK, a downstream tar-
get of ERK1/2, was increased during the late-phase recovery
period after initial resistance exercise, as with p70S6K. How-
ever, different from p70S6K, increase in p90RSK phosphory-
lation in response to muscle contraction was not altered
throughout the training and detraining period, suggesting that
factors other than ERK signaling are mainly responsible for
changes in mTOR activation by chronic muscle contraction
and detraining.
The present study showed that the specific attenuation of
protein kinase phosphorylation in the skeletal muscle with
chronic resistance training was recovered after a short detrain-
ing period without muscle atrophy. These results suggest that
specific signaling may become less sensitive to exercise stim-
ulus even in muscle that is contracted maximally and that some
signaling proteins in the muscle can become resensitized after
a short detraining or nontraining period without a loss of
muscle mass. Therefore, short-term detraining may be an
effective and alternative intervention to maintain muscle re-
938 Training, Detraining, and Muscle Anabolic Signaling Ogasawara R et al.
J Appl Physiol doi:10.1152/japplphysiol.01161.2012
by Riki Ogasawara on April 2, 2013 from
sponsiveness or adaptation during the late phases of resistance
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: R.O., K.K., A.T., K.L., T.A., K.N., and N.I. concep-
tion and design of research; R.O., K.K., A.T., and K.L. performed experiments;
R.O. analyzed data; R.O., S.F., K.N., and N.I. interpreted results of experi-
ments; R.O. prepared figures; R.O. drafted manuscript; R.O., T.A., S.F., K.N.,
and N.I. edited and revised manuscript; R.O., K.K., A.T., K.L., T.A., S.F.,
K.N., and N.I. approved final version of manuscript.
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... While many of these studies described the anabolic signaling acutely after RE, only a few determined the acute signaling response after a period of training. In rat and human skeletal muscle, the acute phosphorylation is blunted after weeks of training, which indicates that skeletal muscle is somehow desensitized as a result of repeated resistance training [15,24]. Because long-term training programs may benefit from maintaining the anabolic signaling response on a high level, it would be of great interest to determine the time frame in which signaling will start to be reduced when RE is frequently carried out. ...
... After 10 days without RE, there was a significant re-increase in HSPB5 S59 phosphorylation and a reduction in desmin, indicating that skeletal muscle sarcomeres also rapidly respond to phases of unloading [26]. Because this rapid adaptation towards repeated RE may also affect the phosphorylation of anabolic signaling proteins [24], in muscle samples of 14 male subjects who were subjected to either progressive (PR) or constant (CO) loading by repeated RE, we analyzed whether-and in which time frame-reduced p mTOR S2448 , p p70S6k T421/S424 , and p S6 S235/236 in skeletal muscle may occur. We also analyzed whether cessation of RE for 10 days may reestablish this response, as reflected by a re-increased phosphorylation of mTOR, p70S6k, and rpS6. ...
... However, only a few studies have analyzed the impact of repeated RE on acute phosphorylation of components of the mTOR cascade without repeated biopsies in human skeletal muscle within this time frame [15,30,31]. Our study design in humans is very closely related to an excellent animal study, and our results broadly corroborate their findings [24]. The authors determined that, as early as after 12 repeated sessions of electrical stimulation of rat gastrocnemius muscle, the phosphorylation of rpS6, p70S6k, and p90S6k was significantly reduced, but recovered after 12 days of unloading [24]. ...
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The acute resistance exercise (RE)-induced phosphorylation of mTOR-related signaling proteins in skeletal muscle can be blunted after repeated RE. The time frame in which the phosphor-ylation (p) of mTOR S2448 , p70S6k T421/S424 , and rpS6 S235/236 will be reduced during an RE training period in humans and whether progressive (PR) loading can counteract such a decline has not been described. Aim: (1) To enclose the time frame in which pmTOR S2448 , prpS6 S235/236 , and pp70S6k T421/S424 are acutely reduced after RE occurs during repeated RE. (2) To test whether PR will prevent that reduction compared to constant loading (CO) and (3) whether 10 days without RE may re-increase blunted signaling. Methods: Fourteen healthy males (24 ± 2.8 yrs.; 1.83 ± 0.1 cm; 79.3 ± 8.5 kg) were subjected to RE with either PR (n = 8) or CO (n = 6) loading. Subjects performed RE thrice per week, conducting three sets with 10-12 repetitions on a leg press and leg extension machine. Muscle biopsies were collected at rest (T0), 45 min after the first (T1), seventh (T7), 13th (T13), and 14th (X-T14) RE session. Results: No differences were found between PR and CO for any parameter. Thus, the groups were combined, and the results show the merged values. prpS6 S235/236 and pp70s6k T421/S424 were increased at T1, but were already reduced at T7 and up to T13 compared to T1. Ten days without RE re-increased prpS6 S235/236 and pp70S6k T421/S424 at X-T14 to a level comparable to that of T1. pmTOR S2448 was increased from T1 to X-T14 and did not decline over the training period. Single-fiber immunohistochemistry revealed a reduction in prpS6 S235/236 in type I fibers from T1 to T13 and a re-increase at X-T14, which was more augmented in type II fibers at T13 (p < 0.05). The entity of myofibers revealed a high heterogeneity in the level of prpS6 S235/236 , possibly reflecting individual contraction-induced stress during RE. The type I and II myofiber diameter increased from T0 and T1 to T13 and X-T14 (p < 0.05) Conclusion: prpS6 S235/236 and pp70s6k T421/S424 reflect RE-induced states of desensitization and re-sensitization in dependency on frequent loading by RE, but also by its cessation.
... Finally, a resistance-like exercise protocol based on electrical stimulation was first developed in rats by Wong and Booth (1988) [34,35]; since then, their protocol has undergone several improvements [36], resulting in two major types of electrical stimulation in animal models: one in which the sciatic nerve is electrically stimulated via invasive procedures [2,37] and another in which the muscle is directly stimulated [11,38]. In the first model, electrical stimulation causes eccentric contractions of the tibialis anterior (causing a 7-14% hypertrophic response in 6 weeks); nevertheless, the majority of the lower leg muscles are stimulated simultaneously. ...
... The major difference in this study is the high intensity of training, in which the volume (10 sets with 12 repetitions), contraction/rest rate between contractions (3 s on and 2 s off), and resting time between sets (1 min) are, to our knowledge, the most intense in the literature [11][12][13][14]38,39]. This protocol design caused a rapid and strong hypertrophy (~30% hypertrophy) after only two weeks of training. ...
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Efficient and suitable animal models directed to skeletal muscle hypertrophy are highly needed; nevertheless, the currently available models have limitations, such as restricted hypertrophy outcome and prolonged protocols; thus, additional research is required. In this study, we developed an improved muscle training protocol for mice by directly stimulating the tibialis anterior (TA) muscle motor point using electrical stimulation. C57BL/6 adult male mice were separated into four groups: CTR (control groups for one and two weeks), ES1 (electrical stimulation for one week), and ES2 (electrical stimulation for two weeks). Following muscle training, TA was taken for further examination. The results demonstrated a steady increase in the fiber cross-sectional area as a result of muscle training (ES1, 14.6% and ES2, 28.9%, p < 0.0001). Two weeks of muscle training enhanced muscle mass and maximal tetanic force by 18 (p = 0.0205) and 30%, respectively (p = 0.0260). To assess the tissue remodeling response in this model, we evaluated satellite cell activity and observed an increase in the number of Pax-7-positive nuclei after one and two weeks of muscle training (both >2-fold, p < 0.0001). In addition, we observed an increase in the number of positive nuclei for MyoD after two weeks (2.6-fold, p = 0.0057) without fiber damage. Accordingly, phosphorylation of mTOR and p70 increased following two weeks of muscle training (17%, p = 0.0215 and 66%, p = 0.0364, respectively). The results indicate that this muscle training strategy is appropriate for promoting quick and intense hypertrophy.
... However, how these changes in signaling and muscle protein synthesis govern long term adaptions to exercise and orchestrate increases in muscle size is less clear. Indeed, chronic exposure to resistance type exercise in rats causes a decrease in anabolic signaling through mTORC1 which can be recovered through detraining 5 . Conversely, data in the context of sarcopenia suggests that age-related muscle loss is associated with an overactivation of mTORC1 that, in rodents, can be ameliorated through rapamycin treatment [6][7][8][9] . ...
... Our finding that chronic exposure to resistance type exercise results in a decrease in anabolic signaling is supported by a clinical trial from Phillips and colleagues, in which the authors observed a genetic signature consistent with mTORC1 inhibition that was correlated with gains in muscle mass over 20 weeks of resistance type exercise 10 . This is in line with data on the protein level from Ogasawara et al. in rats, who found that 12 bouts of resistance type exercise led to a decrease in S6K1 activity which could be reestablished through a 12-day break before the 13th bout 5,38 . Similar to our second experiment, Glynn et al. found that physically active rats had reduced baseline S6K1 (Thr389) levels compared to sedentary controls 39 . ...
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The molecular responses to acute resistance exercise are well characterized. However, how cellular signals change over time to modulate chronic adaptations to more prolonged exercise training is less well understood. We investigated anabolic signaling and muscle protein synthesis rates at several time points after acute and chronic eccentric loading. Adult rat tibialis anterior muscle was stimulated for six sets of ten repetitions, and the muscle was collected at 0 h, 6 h, 18 h and 48 h. In the last group of animals, 48 h after the first exercise bout a second bout was conducted, and the muscle was collected 6 h later (54 h total). In a second experiment, rats were exposed to four exercise sessions over the course of 2 weeks. Anabolic signaling increased robustly 6 h after the first bout returning to baseline between 18 and 48 h. Interestingly, 6 h after the second bout mTORC1 activity was significantly lower than following the first bout. In the chronically exercised rats, we found baseline anabolic signaling was decreased, whereas myofibrillar protein synthesis (MPS) was substantially increased, 48 h after the last bout of exercise. The increase in MPS occurred in the absence of changes to muscle fiber size or mass. In conclusion, we find that anabolic signaling is already diminished after the second bout of acute resistance type exercise. Further, chronic exposure to resistance type exercise training results in decreased basal anabolic signaling but increased overall MPS rates.
... Strength and endurance training cause significantly different or even opposite adaptations. Strength training causes skeletal muscle hypertrophy, by activating the mammalian/mechanistic target of the rapamycin (mTOR) signaling pathway, and neuromuscular responses [36,37], while aerobic training causes skeletal muscle oxidative and metabolic capacity to increase [38] by activating adenosine monophosphate (AMP)-activated protein kinase (AMPK). There is also evidence that AMPK interferes with mTOR signaling via tuberous sclerosis complex 2 (TSC2), suppressing protein synthesis [39]. ...
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The purpose of this study was to examine and compare the training and detraining effects of outdoor serial and integrated combined exercise programs on health, functional capacity, and physical fitness indices. Fifty-one untrained overweight/obese males (47 ± 4 y) were divided into a serial combined (SCG), an integrated combined (ICG), or a control (CG) group. The SCG and ICG implemented a 3-month training (3 sessions/week) consisting of walking and body weight exercises. The only difference between SCG and ICG was the sequence of aerobic and strength training. In SCG, the strength training was performed before aerobic training, while in ICG the aerobic and the strength training were alternated repeatedly in a predetermined order. Health, functional capacity, and physical fitness indices were measured before the training, following the termination of programs , and 1-month after training cessation. Following the training, both the SCG and ICG groups showed reduced blood pressure, heart rate, body fat, and waist-to-hip ratio (3-11%; p < 0.001), with improved respiratory function, muscle strength, aerobic capacity, flexibility, and balance (14-61%; p < 0.001). After 1-month of training cessation, significant reductions (p < 0.05) were observed in health indices and physical fitness without returning to baseline levels. However, there were no differences between SCG and ICG after training and training cessation (p > 0.05). In CG, all the above variables did not change. Furthermore, a great percentage of participants in both exercise groups (90%) reported high levels of enjoyment. In conclusion, both serial and integrated outdoor combined walking and body weight strength training programs are enjoyable and equally effective for improving health, functional capacity, and physical fitness indices in overweight/obese middle-aged males.
... In other words, fasttwitch muscles respond better to high-intensity exercise due to their higher rate of contraction and higher energy production [28]. While slow-twitch muscles are more efficient than fast-twitch muscles due to higher myoglobin concentrations, more capillaries, and higher mitochondrial enzyme activity [28][29][30]. Therefore, the researchers of the present study investigated the effect of mTORC1 pathway proteins on fast-twitch and slow-twitch skeletal muscle because HIIT training can have a dual nature (resistanceendurance). ...
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Abstract Purpose: In people with diabetes, one of the problems for patients is muscle wasting and inhibition of the protein synthesis pathway. This study aimed to evaluate the effects of HIIT on protein expression in two skeletal muscles, flexor hallucis longus (FHL) and soleus (SOL) in rats with type 2 diabetes mellitus (T2DM). Materials and Methods: Diabetes initially was induced by streptozotocin (STZ) and nicotinamide. Rats with type 2 diabetes were randomly and equally divided into control (n=6) and HIIT groups (n=6). After 8 weeks of training, the content of total and phosphorylated proteins of serine/threonine-protein kinases (AKT1), mammalian target of rapamycin (mTOR), P70 ribosomal protein S6 kinase 1 (P70S6K1), and 4E (eIF4E)-binding protein 1 (4E-BP1) in FHL and SOL muscles were measured by Western blotting. While body weight and blood glucose were also controlled. Results: In the HIIT training group, compared to the control group, a significant increase in the content of AKT1 (0.003) and mTOR (0.001) proteins was observed in the FHL muscle. Also, after 8 weeks of HIIT training, protein 4E-BP1 (0.001) was increased in SOL muscle. However, there was no significant change in other proteins in FHL and SOL muscle. Conclusion: In rats with type 2 diabetes appear to HIIT leading to more protein expression of fast-twitch muscles than slow-twitch muscles. thus likely HIIT exercises can be an important approach to increase protein synthesis and prevent muscle atrophy in people with type 2 diabetes.
... Resistance exercises improve skeletal muscle mass by muscle protein synthesis via the mTORC1 signaling pathway [12]. A previous study has demonstrated that LLE enhances mTORC1 signaling (Akt-mTOR-p70S6K) [10]. ...
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Loquat (Eriobotrya japonica (Thunb.) Lindl.) leaves are traditionally used to improve muscle weakness, but their effects on muscle protein synthesis require further research. Therefore, we aimed to investigate whether loquat leaf extract (LLE) enhances muscle contraction-induced activation of muscle protein synthesis signaling. Male Wistar rats (12 weeks old, n = 6/group) were categorized into water treatment (CON) and LLE treatment (LLE) groups. The rats were administered distilled water or LLE (1.5 g/kg/day) once a day by oral gavage for 7 days. On day 7, at 3 h post-LLE administration, the gastrocnemius muscle in the right leg of each rat was stimulated by electrical muscle stimulation (EMS) (100 Hz, 30 V) through five sets of 10 isometric contractions (7 s contraction, 3 s rest) with 3 min interset intervals. The rats were then sacrificed, and the gastrocnemius muscles of both legs were excised at 3 h post-EMS. The phosphorylation levels of mammalian target of rapamycin complex 1 (mTORC1) signaling pathway molecules (Akt, mTOR, and p70S6K) were determined by Western blotting. Regarding the muscle contraction-induced protein synthesis signaling pathway, Akt phosphorylation at Ser473 was not significantly different between the CON and LLE groups. mTOR phosphorylation at Ser2448 was increased by EMS but did not show a significant difference between the CON and LLE groups. p70S6K phosphorylation at Thr389 was significantly increased in response to EMS, whereas the LLE group showed significantly higher p70S6K phosphorylation at Thr389 than that in the CON group. This suggests that LLE enhances muscle contraction-induced activation of p70S6K phosphorylation in rat skeletal muscles.
... In support, it has recently been demonstrated that short-term accustomization (1-3 bouts) to high-frequency isometric in situ contractions, matched by absolute force-time integral, could reduce p70 S6K and 4E-BP1 phosphorylation (Kotani et al., 2021). Albeit assessed at a later time point, work by Ogasawara et al., also observed attenuated phosphorylation of p70 S6K, but not 4E-BP1, in rat gastrocnemius muscle post-stimulation (24 h) after 12 and 18 bouts of high-frequency in situ electrostimulation (Ogasawara et al., 2013). However, in contrast to our mode of stimulation, the in situ electrostimulation protocol applied in these studies were designed to resemble the motor recruitment pattern of resistance exercise, so outcomes are likely to differ. ...
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New findings: What is the central question of this study? The present study evaluated whether myofiber protein signaling responses to ex vivo dynamic contractions are altered by accustomization to voluntary endurance training in rats. What is the main finding and its importance? In response to ex vivo dynamic muscle contractions, canonical myofiber protein signaling pertaining to metabolic transcriptional regulation, as well as translation initiation and elongation, was not influenced by prior accustomization to voluntary endurance training in rats. Accordingly, intrinsic myofiber protein signaling responses to standardized contractile activity may be independent of prior exercise training in rat skeletal muscle. Abstract: Skeletal muscle training status may influence myofiber regulatory protein signaling in response to contractile activity. The current study employed a purpose-designed ex vivo dynamic contractile protocol to evaluate the effect of exercise-accustomization on canonical myofiber protein signaling for metabolic gene expression and for translation initiation and elongation. To this end, rats completed 8 weeks of in vivo voluntary running training versus no running control intervention, whereupon an ex vivo endurance-type dynamic contraction stimulus was conducted in isolated soleus muscle preparations from both intervention groups. Protein signaling responses by phosphorylation was evaluated by immunoblotting at 0 h and 3 h following ex vivo stimulation. Phosphorylation of AMPKα and its downstream target ACC, as well as phosphorylation of eEF2 was increased immediately following the dynamic contraction protocol (at 0 h). Signaling for translation initiation and elongation was evident at 3 h after dynamic contractile activity, as evidenced by increased phosphorylation of p70S6K and 4E-BP1, as well as a decrease in phosphorylation of eEF2 back to resting control-levels. However, prior exercise training did not alter phosphorylation of the investigated signaling proteins. Accordingly, protein signaling responses to standardized endurance-type contractions may be independent of training status in rat muscle during ex vivo conditions. The present findings add to our current understanding of molecular regulatory events responsible for skeletal muscle plasticity. This article is protected by copyright. All rights reserved.
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Resistance training has been known to have a positive effect on muscle performance in exercisers. Whole-body electromyostimulation (WB-EMS) is advertised as a smooth, time-efficient, and highly individualized resistance training technology. The purpose of this study is to evaluate the effects of WB-EMS training on maximum isometric elbow muscle strength and body composition in moderately trained males in comparison to traditional resistance training. The study was a randomized controlled single-blind trial. Twenty, moderately trained, male participants (25.15 ± 3.84, years) were randomly assigned to the following groups: a WB-EMS training group ( n = 11) and a traditional resistance training group (the control group [CG]: n = 9). Both training intervention programs consisted of 18 training sessions for six consecutive weeks. All subjects performed dynamic movements with the WB-EMS or external weights (CG). The primary outcome variables included maximum isometric elbow flexor strength (MIEFS), maximum isometric elbow extensor strength (MIEES) and surface electromyography amplitude (sEMG RMS ). Secondary outcomes involved lean body mass, body fat content, arm fat mass, and arm lean mass. ANOVAs, Friedman test and post hoc t -tests were used ( P = 0.05) to analyze the variables development after the 6-week intervention between the groups. Significant time × group interactions for MIEFS (η ² = 0.296, P Bonferroni = 0.013) were observed, the increase in the WB-EMS group were significantly superior to the CG [23.49 ± 6.48% vs. 17.01 ± 4.36%; MD (95% CI) = 6.48 (1.16, 11.80); d = 1.173, P = 0.020]. There were no significant differences were observed between interventions regarding MIEES, sEMG RMS and body composition. These findings indicate that in moderately trained males the effects of WB-EMS were similar to a traditional resistance training, with the only exception of a significantly greater increase in elbow flexor strength. WB-EMS can be considered as an effective exercise addition for moderately trained males.
We examined the effects of branched-chain amino acids (BCAA) and electrical pulse stimulation (EPS) on the mTORC1 pathway in muscle satellite cells (MSCs) isolated from branched-chain α-keto acid dehydrogenase kinase (BDK) knockout (KO) mice in vitro. MSCs were isolated from BDK KO and wild-type (WT) mice, proliferated, and differentiated into myotubes. BCAA stimulation increased the phosphorylation of p70 S6 kinase (p70S6K), a marker of protein translation initiation, in MSCs from WT and BDK KO mice, but the rate of the increase was higher in MSCs isolated from BDK KO mice. Contrarily, there was no difference in the increase in p70S6K phosphorylation by EPS. Acute BDK knockdown in MSCs from WT mice using shRNA decreased p70S6K phosphorylation in response to BCAA stimulation. Collectively, the susceptibility of mTORC1 to BCAA stimulation was elevated by chronic, but not acute, enhancement of BCAA catabolism.
Background Leucine has unique anabolic properties, serving as a nutrient signal that stimulates muscle protein synthesis. Objective We tested whether the leucine concentration is the only factor determining protein quality for muscle development. Methods We selected 3 dietary proteins: casein (CAS), egg white protein (EWP), and albumin (ALB), representing the leucine concentrations of ∼8.3%, 7.7%, and 6.7% of the total protein (wt:wt), respectively. In the chronic feeding experiment, these proteins were pair-fed to growing male Wistar rats [110–135 g body weight (BW)] for 14 d as a protein source, providing 10% of total energy intake, after which soleus and extensor digitorum longus (EDL) muscles were used to estimate muscle growth. In the acute administration experiment, we injected CAS, ALB, and EWP to rats by oral gavage (0.3 g protein/100 g BW), and after 1 or 3 h EDL muscle was excised for capillary electrophoresis-MS–based metabolomics. In another chronic feeding experiment, rats were pair-fed either CAS or a CAS diet supplemented with arginine to the same level as in the EWP diet for 14 d. Results At the end of the 14-d feeding, soleus and EDL muscle weight was 20% and 17% higher, respectively, when rats were fed EWP as compared with CAS (P < 0.05). In addition, the 14-d EWP diet increased the expression of p70S6K by 117% compared with CAS (P < 0.05). These results suggest the possibility that some amino acids (excluding leucine), derived from EWP, promote muscle growth. Metabolomics analysis showed that muscle arginine concentration, following acute protein administration, appeared to match muscle growth over the 14-d feeding period. In addition, 14-d arginine supplementation to a CAS diet increased EDL muscle weight by 15% when compared with the plain CAS diet (P < 0.05). Conclusions EWP promotes rat developmental muscle growth compared with CAS, which can be partly explained by the arginine-rich EWP.
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Elongation factor 2 kinase (eEF2k) phosphorylates and inactivates eEF2. Insulin induces dephosphorylation of eEF2 and inactivation of eEF2 kinase, and these effects are blocked by rapamycin, which inhibits the mammalian target of rapamycin, mTOR. However, the signalling mechanisms underlying these effects are unknown. Regulation of eEF2 phosphorylation and eEF2k activity is lost in cells in which phosphoinositide-dependent kinase 1 (PDK1) has been genetically knocked out. This is not due to loss of mTOR function since phosphorylation of another target of mTOR, initiation factor 4E-binding protein 1, is not defective. PDK1 is required for activation of members of the AGC kinase family; we show that two such kinases, p70 S6 kinase (regulated via mTOR) and p90RSK1 (activated by Erk), phosphorylate eEF2k at a conserved serine and inhibit its activity. In response to insulin-like growth factor 1, which activates p70 S6 kinase but not Erk, regulation of eEF2 is blocked by rapamycin. In contrast, regulation of eEF2 by stimuli that activate Erk is insensitive to rapamycin, but blocked by inhibitors of MEK/Erk signalling, consistent with the involvement of p90RSK1.
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To compare the effects of a periodic resistance training (PTR) program with those of a continuous resistance training (CTR) program on muscle size and function, 14 young men were randomly divided into a CTR group and a PTR group. Both groups performed high-intensity bench press exercise training [75 % of one repetition maximum (1-RM); 3 sets of 10 reps] for 3 days per week. The CTR group trained continuously over a 24-week period, whereas the PTR group performed three cycles of 6-week training (or retraining), with 3-week detraining periods between training cycles. After an initial 6 weeks of training, increases in cross-sectional area (CSA) of the triceps brachii and pectoralis major muscles and maximum isometric voluntary contraction of the elbow extensors and 1-RM were similar between the two groups. In the CTR group, muscle CSA and strength gradually increased during the initial 6 weeks of training. However, the rate of increase in muscle CSA and 1-RM decreased gradually after that. In the PTR group, increase in muscle CSA and strength during the first 3-week detraining/6-week retraining cycle were similar to that in the CTR group during the corresponding period. However, increase in muscle CSA and strength during the second 3-week detraining/6-week retraining cycle were significantly higher in the PTR group than in the CTR group. Thus, overall improvements in muscle CSA and strength were similar between the groups. The results indicate that 3-week detraining/6-week retraining cycles result in muscle hypertrophy similar to that occurring with continuous resistance training after 24 weeks.
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We aimed to determine if the time that muscle is under loaded tension during low intensity resistance exercise affects the synthesis of specific muscle protein fractions or phosphorylation of anabolic signalling proteins. Eight men (24 ± 1 years (sem), BMI = 26.5 ± 1.0 kg m(-2)) performed three sets of unilateral knee extension exercise at 30% of one-repetition maximum strength involving concentric and eccentric actions that were 6 s in duration to failure (SLOW) or a work-matched bout that consisted of concentric and eccentric actions that were 1 s in duration (CTL). Participants ingested 20 g of whey protein immediately after exercise and again at 24 h recovery. Needle biopsies (vastus lateralis) were obtained while fasted at rest and after 6, 24 and 30 h post-exercise in the fed-state following a primed, constant infusion of l-[ring-(13)C(6)]phenylalanine. Myofibrillar protein synthetic rate was higher in the SLOW condition versus CTL after 24-30 h recovery (P < 0.001) and correlated to p70S6K phosphorylation (r = 0.42, P = 0.02). Exercise-induced rates of mitochondrial and sarcoplasmic protein synthesis were elevated by 114% and 77%, respectively, above rest at 0-6 h post-exercise only in the SLOW condition (both P < 0.05). Mitochondrial protein synthesis rates were elevated above rest during 24-30 h recovery in the SLOW (175%) and CTL (126%) conditions (both P < 0.05). Lastly, muscle PGC-1α expression was increased at 6 h post-exercise compared to rest with no difference between conditions (main effect for time, P < 0.001). These data show that greater muscle time under tension increased the acute amplitude of mitochondrial and sarcoplasmic protein synthesis and also resulted in a robust, but delayed stimulation of myofibrillar protein synthesis 24-30 h after resistance exercise.
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Chronic mechanical loading (CML) of skeletal muscle induces compensatory growth and the drug rapamycin has been reported to block this effect. Since rapamycin is considered to be a highly specific inhibitor of the mammalian target of rapamycin (mTOR), many have concluded that mTOR plays a key role in CML-induced growth regulatory events. However, rapamycin can exert mTOR-independent actions and systemic administration of rapamycin will inhibit mTOR signalling in all cells throughout the body. Thus, it is not clear if the growth inhibitory effects of rapamycin are actually due to the inhibition of mTOR signalling, and more specifically, the inhibition of mTOR signalling in skeletal muscle cells. To address this issue, transgenic mice with muscle specific expression of various rapamycin-resistant mutants of mTOR were employed. These mice enabled us to demonstrate that mTOR, within skeletal muscle cells, is the rapamycin-sensitive element that confers CML-induced hypertrophy, and mTOR kinase activity is necessary for this event. Surprisingly, CML also induced hyperplasia, but this occurred through a rapamycin-insensitive mechanism. Furthermore, CML was found to induce an increase in FoxO1 expression and PKB phosphorylation through a mechanism that was at least partially regulated by an mTOR kinase-dependent mechanism. Finally, CML stimulated ribosomal RNA accumulation and rapamycin partially inhibited this effect; however, the effect of rapamycin was exerted through a mechanism that was independent of mTOR in skeletal muscle cells. Overall, these results demonstrate that CML activates several growth regulatory events, but only a few (e.g. hypertrophy) are fully dependent on mTOR signalling within the skeletal muscle cells.
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Sarcopenia, the loss of skeletal muscle mass during aging, increases the risk for falls and dependency. Resistance exercise (RE) training is an effective treatment to improve muscle mass and strength in older adults, but aging is associated with a smaller amount of training-induced hypertrophy. This may be due in part to an inability to stimulate muscle-protein synthesis (MPS) after an acute bout of RE. We hypothesized that older adults would have impaired mammalian target of rapamycin complex (mTORC)1 signaling and MPS response compared with young adults after acute RE. We measured intracellular signaling and MPS in 16 older (mean 70 ± 2 years) and 16 younger (27 ± 2 years) subjects. Muscle biopsies were sampled at baseline and at 3, 6 and 24 hr after exercise. Phosphorylation of regulatory signaling proteins and MPS were determined on successive muscle biopsies by immunoblotting and stable isotopic tracer techniques, respectively. Increased phosphorylation was seen only in the younger group (P< 0.05) for several key signaling proteins after exercise, including mammalian target of rapamycin (mTOR), ribosomal S6 kinase (S6K)1, eukaryotic initiation factor 4E-binding protein (4E-BP)1 and extracellular signal-regulated kinase (ERK)1/2, with no changes seen in the older group (P >0.05). After exercise, MPS increased from baseline only in the younger group (P< 0.05), with MPS being significantly greater than that in the older group (P <0.05). We conclude that aging impairs contraction-induced human skeletal muscle mTORC1 signaling and protein synthesis. These age-related differences may contribute to the blunted hypertrophic response seen after resistance-exercise training in older adults, and highlight the mTORC1 pathway as a key therapeutic target to prevent sarcopenia.
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The mammalian target of rapamycin (mTOR) is a protein kinase that, when present in a complex referred to as mTOR complex 1 (mTORC1), acts as an important regulator of growth and metabolism. The activity of the complex is regulated through multiple upstream signaling pathways, including those involving Akt and the extracellular-regulated kinase (ERK). Previous studies have shown that, in part, Akt and ERK promote mTORC1 signaling through phosphorylation of a GTPase activator protein (GAP), referred to as tuberous sclerosis complex 2 (TSC2), that acts as an upstream inhibitor of mTORC1. In the present study we extend the earlier studies to show that activation of the Akt and ERK pathways acts in a synergistic manner to promote mTORC1 signaling. Moreover, we provide evidence that the Akt and ERK signaling pathways converge on TSC2, and that Akt phosphorylates residues on TSC2 distinct from those phosphorylated by ERK. The results also suggest that leucine-induced stimulation of mTORC1 signaling occurs through a mechanism distinct from TSC2 and the Akt and ERK signaling pathways. Overall, the results are consistent with a model in which Akt and ERK phosphorylate distinct sites on TSC2, leading to greater repression of its GAP activity, and consequently a magnified stimulation of mTORC1 signaling, when compared with either input alone. The results further suggest that leucine acts through a mechanism distinct from TSC2 to stimulate mTORC1 signaling.
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For over 10 years, we have known that the activation of the mammalian target of rapamycin complex 1 (mTORC1) has correlated with the increase in skeletal muscle size and strength that occurs following resistance exercise. Initial cell culture and rodent models of muscle growth demonstrated that the activation of mTORC1 is common to hypertrophy induced by growth factors and increased loading. The further observation that high loads increased the local production of growth factors led to the paradigm that resistance exercise stimulates the autocrine production of factors that act on membrane receptors to activate mTORC1, and this results in skeletal muscle hypertrophy. Over the last few years, there has been a paradigm shift. From both human and rodent studies, it has become clear that the phenotypic and molecular responses to resistance exercise occur in a growth factor-independent manner. Although the mechanism of load-induced mTORC1 activation remains to be determined, it is clear that it does not require classical growth factor signaling.
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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 m2) 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 2B (eIF2B) 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.
The Ras-extracellular signal-regulated kinase (Ras-ERK) and phosphatidylinositol 3-kinase-mammalian target of rapamycin (PI3K-mTOR) signaling pathways are the chief mechanisms for controlling cell survival, differentiation, proliferation, metabolism, and motility in response to extracellular cues. Components of these pathways were among the first to be discovered when scientists began cloning proto-oncogenes and purifying cellular kinase activities in the 1980s. Ras-ERK and PI3K-mTOR were originally modeled as linear signaling conduits activated by different stimuli, yet even early experiments hinted that they might intersect to regulate each other and co-regulate downstream functions. The extent of this cross-talk and its significance in cancer therapeutics are now becoming clear.
Feeding protein after resistance exercise enhances the magnitude and duration of myofibrillar protein synthesis (MPS) over that induced by feeding alone. We hypothesized that the underlying mechanism for this would be a greater and prolonged phosphorylation of signalling involved in protein translation. Seven healthy young males performed unilateral resistance exercise followed immediately by the ingestion of 25 g of whey protein to maximally stimulate MPS in a rested and exercised leg. Phosphorylation of p70 ribosomal protein S6 kinase (p70S6K) was elevated (P<0.05) above fasted at 1 h at rest whereas it was elevated at 1, 3 and 5 h after exercise with protein ingestion and displayed a similar post-exercise time course to that shown by MPS. Extracellular regulated kinase1/2 (ERK1/2) and p90 ribosomal S6 kinase (p90RSK) phosphorylation were unaltered after protein ingestion at rest but were elevated (P < 0.05) above fasted early in recovery (1 h) and were greater for the exercised-fed leg than feeding alone (main effect; P < 0.01). Eukaryotic elongation factor 2 (eEF2) phosphorylation was also less (main effect; P<0.05) in the exercised-fed leg than in the rested leg suggesting greater activity after exercise. Eukaryotic initiation 4E binding protein-1 (4EBP-1) phosphorylation was increased (P<0.05) above fasted to the same extent in both conditions. Our data suggest that resistance exercise followed by protein feeding stimulates MPS over that induced by feeding alone in part by enhancing the phosphorylation of select proteins within the mammalian target of rapamycin (p70S6K, eEF2) and by activating proteins within the mitogen-activated protein kinase (ERK1/2, p90RSK) signalling.