J Appl Physiol 117: 1170–1179, 2014.
First published September 25, 2014; doi:10.1152/japplphysiol.00180.2014.
Alcohol impairs skeletal muscle protein synthesis and mTOR signaling in a
time-dependent manner following electrically stimulated muscle contraction
Jennifer L. Steiner and Charles H. Lang
Department of Cellular and Molecular Physiology, Pennsylvania State College of Medicine, Hershey, Pennsylvania
Submitted 24 February 2014; accepted in final form 21 September 2014
anabolic resistance; skeletal muscle metabolism; resistance exercise;
p70S6K1; 4E-binding protein
ACUTE AND CHRONIC ALCOHOL intoxication decreases rates of
skeletal muscle protein synthesis predominately in type II
muscle fibers at least in part through impairment of mammalian
target of rapamycin (mTOR)-dependent translation initiation
(27, 29 –31, 49). mTOR is a serine/threonine (Ser/Thr) kinase
that exists in two distinct protein complexes, mTORC1 and
mTORC2. Together these protein complexes represent a central metabolic integration point that regulates numerous cellular processes, including translation initiation and subsequently
protein synthesis (20). Upon activation, mTORC1 phosphorylates its primary downstream substrates, eukaryotic initiation
factor (eIF)-4E binding protein (4E-BP1) and p70S6K1
(S6K1). Multisite phosphorylation of 4E-BP1 is required for
the release of eIF4E (from 4E-BP1), resulting in initiation of
cap-dependent translation (17, 18, 39, 47). On the other hand,
phosphorylation of S6K1 by mTORC1 is specific to Thr389
Address for reprint requests and other correspondence: J. L. Steiner, Cellular
and Molecular Physiology (H166), Penn State College of Medicine, 500 Univ.
Drive, Hershey, PA 17033 (e-mail: [email protected]).
1170
which, in the conventional model, is facilitated by the conformation change induced by phosphorylation of the COOHterminal autoinhibitory domain containing Thr421 and Ser424 as
well as Ser411 and Ser418 (43, 50). S6K1 activates several
downstream substrates, including ribosomal protein S6 (rpS6),
eukaryotic elongation factor-2 (eEF2), programmed cell death
protein 4 (PDCD4), and eIF4B, which contribute to initiation
of translation, elongation, and presumably protein synthesis
(43, 52). Acute alcohol intoxication suppresses basal phosphorylation of rpS6 and 4E-BP1 and increases binding of the
translational repressor protein, 4E-BP1, with eIF4E (32, 34,
35). Additionally, the mTORC1-mediated anabolic response of
muscle to hormones [insulin and insulin-like growth factor-I
(IGF-I)] and nutrients (leucine) is suppressed by prior administration of an acute bolus of alcohol in a dose- and timedependent fashion (28, 32, 34, 53).
Muscle contraction stimulates protein synthesis and, when
repetitive in nature, muscle hypertrophy develops in an mTORdependent manner; however, additional signaling pathways
have also been implicated (7, 13, 19, 26, 38, 58). The precise
mechanism(s) of mTORC1 activation following a mechanical
stimulus like muscle contraction or stretch remains to be
determined, since it is independent of exogenous nutrients,
phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)
signaling, IGF-I responsive pathways, MEK/extracellular signal-regulated kinase (ERK) signaling, or changes in intracellular calcium levels (22–24, 38, 46, 48, 57–59). Most recently,
it was hypothesized that contraction-induced mTORC1 activation is mediated, at least in part, by diacylglycerol kinase
␨-regulated synthesis of phosphatidic acid (14, 59). Muscle
contraction induces phosphorylation of several substrates
within the mTORC1 signaling pathway, including S6K1, 4EBP1, mTOR, rpS6, eIF4B, and eEF2, in a time-dependent and
stimulus-specific manner (11, 13, 38, 48, 58). Several different
experimental paradigms result in increased mTOR signaling
and/or protein synthesis, including weighted or resistance exercise, muscle overload induced by synergistic muscle ablation, and electrically stimulated muscle contraction (6, 12, 26,
46, 48, 58). This last model, which uses electrical stimulation
to induce maximal muscle contractions, mimics the signaling
and hypertrophic responses observed following resistance exercise and has a potential clinical advantage, since it can be
performed on patients with physical limitations caused by
illness or disease (6, 12).
The effects of alcohol on electrically stimulated muscle
contraction-induced increases of skeletal muscle protein synthesis have not been investigated despite the direct clinical
relevance; stimulated muscle contraction is a potential therapeutic modality for those with chronic alcohol-induced myopathy that is characterized by similar molecular changes as those
occurring with acute intoxication (25, 32, 35). Therefore, the
8750-7587/14 Copyright © 2014 the American Physiological Society
http://www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 16, 2017
Steiner JL, Lang CH. Alcohol impairs skeletal muscle protein
synthesis and mTOR signaling in a time-dependent manner following electrically stimulated muscle contraction. J Appl Physiol 117: 1170 –1179, 2014. First published September 25, 2014;
doi:10.1152/japplphysiol.00180.2014.—Alcohol (EtOH) decreases
protein synthesis and mammalian target of rapamycin (mTOR)-mediated signaling and blunts the anabolic response to growth factors in
skeletal muscle. The purpose of the current investigation was to
determine whether acute EtOH intoxication antagonizes the contraction-induced increase in protein synthesis and mTOR signaling in
skeletal muscle. Fasted male mice were injected intraperitoneally with
3 g/kg EtOH or saline (control), and the right hindlimb was electrically stimulated (10 sets of 6 contractions). The gastrocnemius muscle
complex was collected 30 min, 4 h, or 12 h after stimulation. EtOH
decreased in vivo basal protein synthesis (PS) in the nonstimulated
muscle compared with time-matched Controls at 30 min, 4 h, and 12
h. In Control, but not EtOH, PS was decreased 15% after 30 min. In
contrast, PS was increased in Control 4 h poststimulation but remained unchanged in EtOH. Last, stimulation increased PS 10% in
Control and EtOH at 12 h, even though the absolute rate remained
reduced by EtOH. The stimulation-induced increase in the phosphorylation of S6K1 Thr421/Ser424 (20 –52%), S6K1 Thr389 (45–57%), and
its substrate rpS6 Ser240/244 (37–72%) was blunted by EtOH at 30
min, 4 h, and 12 h. Phosphorylation of 4E-BP1 Ser65 was also
attenuated by EtOH (61%) at 4 h. Conversely, phosphorylation of
extracellular signal-regulated kinase Thr202/Tyr204 was increased by
stimulation in Control and EtOH mice at 30 min but only in Control
at 4 h. Our data indicate that acute EtOH intoxication suppresses
muscle protein synthesis for at least 12 h and greatly impairs contraction-induced changes in synthesis and mTOR signaling.
Alcohol Decreases Muscle Contraction-Induced mTOR Signaling
METHODS
0.5
0.0
Con
EtOH
Con
Non-Stim
Protein synthesis
(nmol Phe/h/mg protein)
C
EtOH
D
1.0
0.5
EtOH
Non-Stim
1.0
0.5
0.0
Con
EtOH
Stim
Con
EtOH
Con
Non-Stim
1.5
Con
4 h Post-Contraction
1.5
Stim
12 h Post-Contraction
0.0
Protein synthesis
(nmol Phe/h/mg protein)
1.0
B
Δ from non-stimulated
gastrocnemius
(nmol Phe/h/mg protein)
Protein synthesis
(nmol Phe/h/mg protein)
30 min Post-Contraction
1171
EtOH
Stim
0.2
0.1
*
0.0
-0.1
Fig. 1. Protein synthesis following alcohol
and/or electrically stimulated muscle contraction. Rates of synthesis were assessed in
the muscle at 30 min (A), 4 h (B), and 12 h
(C) post-muscle contraction. The absolute
delta change was calculated by subtracting
the nonstimulated value from that of the
stimulated muscle from each animal and are
presented for both Control and Alcohol treatment groups at each time point (D). Shaded
bars, alcohol (EtOH)-treated mice (n ⫽
7–10); open bars, Control (Con) mice (n ⫽
7–10). Non-Stim, control condition; Stim,
muscle underwent electrically stimulated
muscle contraction. Horizontal bars, statistical differences between groups (P ⬍ 0.05)
(A, B, and C). *Statistical differences from
time-matched Control group (P ⬍ 0.05) (D).
Values are expressed as means ⫾ SE.
*
-0.2
Con EtOH
30 min
Con EtOH
4h
Con EtOH
12 h
J Appl Physiol • doi:10.1152/japplphysiol.00180.2014 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 16, 2017
Animals. Viral antibody-free male C57BL/6 mice aged 11–12 wk
old were purchased from Charles River Laboratories (Wilmington,
MA) and acclimated to the animal facility at the College of Medicine
at Penn State Hershey for at least 1 wk before experimental use. Mice
were housed in shoe-box cages with corn cob bedding under controlled environmental conditions (12:12-h light-dark) and were provided Teklad Global 2019 (Harlan Teklad, Boston, MA) and water ad
libitum until the start of the experiment. All experimental procedures
were performed in accordance with the National Institutes of Health
guidelines for the use of experimental animals and were approved by
the Institutional Animal Care and Use Committee of The Pennsylvania State University College of Medicine.
Experimental design and muscle contraction protocol. Before each
experiment, mice were fasted overnight with the muscle stimulation
protocol initiated between 8:00 A.M. and 1:00 P.M. the next day. To
minimize differences due to the length of fast, mice were randomly
assigned to either the Alcohol (n ⫽ 7–10) or Control (n ⫽ 7–10)
treatment and tested in an alternating fashion (i.e., one mouse from the
Control group followed by one from the Alcohol group). Mice were
administered alcohol at a dose of 3 g/kg via intraperitoneal injection
or were given an equal volume of 0.9% sterile saline (Control).
Administration of alcohol via intraperitoneal injection or oral gavage
results in similar absorption and clearance rates as evidenced by
analogous blood alcohol concentrations (BAC) (9). Alcohol was
administered before muscle contraction based on prior work showing
1.5
Steiner JL et al.
that pretreatment with alcohol antagonizes mTOR activation following other anabolic stimuli, including leucine, IGF-I, and insulin (27,
28, 53). Additionally, patients with chronic alcohol-induced myopathy
might have detectable circulating levels of alcohol when stimulated
muscle contraction would be performed as a therapeutic intervention,
making this a relevant preclinical model.
Immediately after alcohol treatment, mice were anesthetized via
isoflurane (2–3% in O2 with 1.5% maintenance). Hair was shaved
from the right leg/hip, and a small incision through the skin and
muscle was made for isolation of the sciatic nerve. Needle electrodes
were placed over the nerve, and maximal muscular contractions were
evoked using a current stimulator (model A365; World Precision
Instruments, Sarasota, FL) interfaced with Powerlab 4/35 and LabChart software (ADI Instruments, Colorado Springs CO). The stimulation protocol occurred as previously described by Baar and Esser
(6). Briefly, the protocol included 10 sets of 6 contractions (each
lasting 3 s) with 10 s rest between contractions and 60 s rest after each
set of 6 contractions (6). Pulse height was set to 6 volts and current to
1 mA to ensure maximal contractions were evoked. Each contraction
produced a lengthening action of the tibialis anterior and extensor
digitorum longus muscles, with a concomitant shortening of the
triceps surae complex (i.e., gastrocnemius, plantaris, and soleus). The
contralateral leg served as the nonstimulated control. At the cessation
of the protocol, mice were given 1 ml of warm sterile saline subcutaneously and allowed to recover for the specified amount of time (30
min, 4 h, or 12 h). Mice remained in the fasted state throughout the
recovery period but had free access to water. At the appropriate time
point, the gastrocnemius and plantaris muscle complex (referred to as
muscle from here on) was excised from the electrically stimulated and
nonstimulated leg and immediately placed between aluminum blocks
precooled to the temperature of liquid nitrogen. The gastrocnemius
and plantaris muscles were used, since they are predominately composed of type II muscle fibers and respond to stimulated muscle
contraction as well as experience the greatest decrement following
treatment with alcohol (6, 36, 44). Blood was collected from the vena
cava in heparinized syringes and centrifuged (10,000 g for 10 min) for
purpose of the present study was to determine whether acute
alcohol intoxication impairs mTOR-mediated protein synthesis
stimulated by in vivo muscle contraction. Because changes in
mTOR signaling and protein synthesis are time-dependent
following muscle contraction, and the effects of alcohol may
subside as it is metabolized over time, analysis was performed
at an early (30 min), intermediate (4 h), and late (12 h) time
point following muscle contraction.
A
•
1172
Alcohol Decreases Muscle Contraction-Induced mTOR Signaling
A
200
150
100
50
Con
EtOH
Stim
150
100
50
Con
EtOH
Non-Stim
Con
Stim
100
50
Con
EtOH
Non-Stim
Con
EtOH
Stim
250
F
30 min Post-Contraction
200
150
100
G
50
0
Con
EtOH
Con
Non-Stim
EtOH
Stim
EtOH
Contraction
- + - +
- - + +
S6K1 Thr389
S6K1 Thr421/Ser424
150
S6K1 Total
100
4E-BP1 Ser6 5
4E-BP1 Total
50
γ
β
α
4E-BP1 Total
0
EtOH
150
0
D
4E-BP1 Ser65/Total
(% Non-Stim Con)
4E-BP1
γ band/ α, β, γ bands
(% Non-Stim Con)
EtOH
Non-Stim
0
mTOR Ser2448/Total
(% Non-Stim Con)
Con
S6K1 Thr421/Ser424/Total
(% Non-Stim Con)
250
mTOR Ser2481/Total
(% Non-Stim Con)
S6K1 Thr389/Total
(% Non-Stim Con)
300
0
E
PDCD4 (Ser67) (Abcam, Cambridge, MA), eIF4B, eIF4B (Ser422),
eEF2, eEF2 (Thr56), ERK1/2 (Thr202/Tyr204), p42/44 MAPK, mTOR,
and mTOR (Ser2481 and Ser2448) were used. The FluorChem M
Multifluor System (ProteinSimple, San Jose, CA) was used for visualization following exposure to ECL reagent (Thermo Scientific,
Waltham, MA). Images were analyzed using AlphaView (ProteinSimple) and Image J software (National Institutes of Health).
Protein synthesis. A separate group of age-matched male C57BL/6
mice was used to determine the in vivo rate of muscle protein
synthesis in the gastrocnemius complex 30 min, 4 h, or 12 h postelectrically stimulated muscle contraction. All experimental protocols
were performed as described above except that at 30 min, 4 h, and 12
h after completion of electrically stimulated muscle contraction mice
were injected with L-[2,3,4,5,6-3H]phenylalanine (Phe; 150 mM, 30
␮Ci/ml; 0.5 ml) for an additional 15 min before tissue collection.
Mice were then anesthetized with isoflurane, and blood was collected
in heparinized syringes from the vena cava for measurement of
plasma Phe concentration and radioactivity. Muscles were excised and
immediately clamped between aluminum blocks precooled in liquid
nitrogen. High-performance liquid chromatography was used for the
measurement of specific radioactivity of plasma Phe levels in the supernatant from trichloroacetic acid-treated plasma extracts. The global rate
of [3H]Phe incorporation into protein within the muscle was assessed as
previously described by our laboratory (29, 56).
Blood alcohol concentration. The plasma alcohol concentration
was determined in all samples using a rapid analyzer (Analox Instru-
B
350
C
Steiner JL et al.
Con
EtOH
Non-Stim
Con
EtOH
Stim
mTOR Ser2481
mTOR Ser2448
150
mTOR Total
100
50
0
Con
EtOH
Non-Stim
Con
EtOH
Stim
Fig. 2. Effect of alcohol and electrically stimulated muscle contraction on mammalian target of rapamycin (mTOR) complex 1 signaling. p70S6K1 (S6K1, A and B),
4E-binding protein (4E-BP1, C and D), and mTOR (E and F) were assessed 30 min post-muscle contraction in the muscle. Bar graphs represent quantification of Western
blot images (G) normalized to the total amount of the respective protein with the control nonstimulated value set to 100%. Shaded bars, alcohol-treated mice (n ⫽ 9);
open bars, control mice (n ⫽ 9). Horizontal bars indicate statistical differences between groups (P ⬍ 0.05). Values are expressed as means ⫾ SE.
J Appl Physiol • doi:10.1152/japplphysiol.00180.2014 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 16, 2017
isolation of plasma. Both frozen tissue and plasma were stored at
⫺80°C until analyzed.
Experimental time points were specifically chosen based on: 1)
preliminary investigations showing 30 min was sufficient time to
allow for the induction of S6K1 Thr389 phosphorylation following
contraction; 2) previous literature confirming a significant anabolic
response by 30 min that is maximal between 3 and 12 h and
maintained for at least 16 h (6, 26, 38); and 3) literature supporting a
progressive time-dependent decrease in BAC following a peak at 60
min after intraperitoneal injection in mice, which corresponds to our
30-min time point (40).
Western blotting. One-half of the frozen gastrocnemius complex
(50 –90 mg) was homogenized using a glass mortar and pestle in 10
volumes of ice-cold buffer consisting of (in mmol/l): 50 HEPES, 0.1%
Triton X, 4 EGTA, 10 EDTA, 15 sodium pyrophosphate, 100 ␤-glycerophosphate, 25 sodium fluoride, 5 sodium orthovanadate, and 1
cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Tablet
(Roche Applied Science, Madison, WI) per 10 ml of buffer. Protein
concentration was quantified using Bio-Rad Protein Assay Dye reagent concentrate (Hercules, CA), and SDS-PAGE was carried out
using equal amounts of total protein per sample. The membranes were
incubated overnight at 4°C with primary antibody (Cell Signaling,
Beverly, MA, unless otherwise noted). Antibodies against Akt, Akt
(Ser473 and Thr308), PRAS40, PRAS40 (Thr246), S6K1, S6K1 (Thr389
and Thr421/Ser424), rpS6, rpS6 (Ser240/244 and Ser235/236), 4E-BP1
(Bethyl Laboratories, Montgomery, TX), 4E-BP1 (Ser65), PDCD4,
•
Alcohol Decreases Muscle Contraction-Induced mTOR Signaling
ments, Lunenburg, MA). Duplicate measurements were performed,
and the average is reported.
Statistical analysis. All data were analyzed using commercial
statistic software (SigmaPlot; Systat, San Jose, CA). Within each time
point (30 min, 4 h, 12 h) data were analyzed using a repeatedmeasures two-way ANOVA (contraction ⫻ alcohol) with StudentNeuman-Keuls post hoc tests when appropriate. Comparisons across
time points were not performed. Data are presented as means ⫾ SE
and considered significant when P ⬍ 0.05.
•
Steiner JL et al.
1173
RESULTS
Blood alcohol concentration. The BAC was measured in
plasma from all mice at each time point. Thirty minutes after
muscle contraction (⬃1 h after injection of alcohol), the BAC
was 66 ⫾ 3 mmol/l, which is equivalent to ⬃300 mg/dl (0.3%).
At the 4-h time point, the average BAC had dropped to 38 ⫾
3 mmol/l, which is equivalent to 175 mg/dl (0.17%). While
these BACs are high, similar levels have been reported in
trauma patients admitted to the emergency department (15, 21).
By 12 h, the BAC was below detectable levels in alcoholtreated mice. Lastly, no alcohol was detected in the plasma
from the saline-treated Control animals at any time point.
Muscle protein synthesis. Alcohol suppressed protein synthesis in both nonstimulated and stimulated muscle at 30 min
(⬃60%), 4 h (⬃75%), and 12 h (⬃40%) compared with
time-matched control values (Fig. 1, A, B, and C). Electrically
stimulated muscle contraction decreased protein synthesis 17%
at 30 min in Control mice, but this effect was not present in
Alcohol mice (Fig. 1A). At 4 h, muscle contraction significantly increased protein synthesis by 11% in Control mice, but
30 min Post-Contraction
A
B
Akt Thr308
Total Akt
150
AKT Ser473/Total
(% Non-Stim Con)
Akt Thr308/Total
(% Non-Stim Con)
150
100
50
0
Con
EtOH
Con
Non-Stim
C
Akt Ser473
Total Akt
100
50
0
EtOH
D
PRAS40 Thr246
50
0
Con
EtOH
Non-Stim
Con
EtOH
Stim
ERK1/2 Thr202/Tyr204/Total
(% Non-Stim Con)
PRAS40 Thr246/Total
(% Non-Stim Con)
100
EtOH
Con
EtOH
Stim
ERK1/2 Thr202/Tyr204
Total ERK
Total PRAS40
150
Con
Non-Stim
Stim
200
150
Fig. 3. Effect of alcohol and electrically stimulated muscle contraction on proteins upstream of mTORC1. After cessation of muscle contraction (30 min), protein kinase B
(Akt) Thr308 (A), Akt Ser473 (B), PRAS40
Thr246 (C), and extracellular signal-regulated
kinases (ERK)1/2 Thr202/Tyr204 (D) were assessed in the muscle. Bars graphs represent
quantification of Western blot images normalized to the total amount of respective
protein with the control nonstimulated value
set to 100%. Shaded bars, alcohol-treated
mice (n ⫽ 9); open bars, control mice (n ⫽
9). Horizontal bars indicate statistical differences between groups (P ⬍ 0.05). Values are
expressed as means ⫾ SE.
100
50
0
Con
EtOH
Non-Stim
Con
EtOH
Stim
J Appl Physiol • doi:10.1152/japplphysiol.00180.2014 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 16, 2017
this enhancement was not observed in Alcohol mice (Fig. 1B).
At the 12-h time point, muscle contraction increased protein
synthesis 12% in the stimulated muscle of both the Control and
alcohol-treated mice. However, the rate of synthesis in the
stimulated muscle from alcohol-treated mice remained lower
than the stimulated Control mice (Fig. 1C). To more clearly
present the magnitude of change induced by electrically stimulated muscle contraction in Control and Alcohol mice, Fig.
1D shows the change in absolute synthetic rates (stimulated
muscle minus nonstimulated muscle within an animal) for each
time point.
mTOR signaling 30 min post-muscle contraction. We next
assessed the phosphorylation of the primary mTORC1 substrates, S6K1 and 4E-BP1. Muscle contraction increased the
phosphorylation of Thr389 (158%) and Thr421/Ser424 (93%) on
S6K1 after 30 min, whereas alcohol prevented the stimulationinduced increase and reduced the magnitude of change compared with that observed in the stimulated leg of Controls (Fig.
2, A and B). Electrically stimulated muscle contraction also
increased the presence of the slower-migrating ␥-isoform of
4E-BP1 (20%) in Controls, whereas alcohol reduced migration
induced by stimulation compared with Controls (Fig. 2C). In
contrast, phosphorylation of Ser65 on 4E-BP1 in muscle from
Control was unchanged by stimulation, whereas the combination of alcohol and stimulated muscle contraction reduced its
phosphorylation at this early 30-min time point (Fig. 2D).
Phosphorylation of mTOR at the autocatalytic Ser2481 site and
PI3K/AKT-mediated residue, Ser2448, was not changed by
alcohol and/or muscle stimulation (Fig. 2, E and F).
1174
Alcohol Decreases Muscle Contraction-Induced mTOR Signaling
Steiner JL et al.
targets, PDCD4 and eEF2, remained unchanged by muscle
stimulation and/or acute alcohol intoxication (Fig. 4, C and D).
mTOR signaling 4 h post-muscle contraction. Four hours
after cessation of muscle contraction, the phosphorylation of
S6K1Thr389 (92%) and Thr421/Ser424 (115%), rpS6 Ser240/244
(430%), phosphorylation of the ␥-isoform of 4E-BP1 (111%),
4E-BP1 Ser65 (⬃80%), and mTOR Ser2448 (114%) was increased by stimulated muscle contraction in Control mice (Fig.
5, A–E). At this time point, alcohol prevented the stimulationinduced increase in the phosphorylation of each of these
proteins except for mTOR Ser2448 where no differences between the Control and Alcohol groups were detected (Fig. 5,
A–E). Furthermore, Akt Ser473 and Thr308, as well as mTOR
Ser2481, were unchanged by muscle contraction or alcohol at
this time point (Fig. 5H and Table 1). PRAS40 phosphorylation
on Thr246 was suppressed by alcohol but unchanged by muscle
contraction in either group (Fig. 5F), while conversely, the
phosphorylation of ERK Thr202/Tyr204 in the contracted muscle of alcohol-treated mice was decreased compared with
control mice (Fig. 5G). Proteins important in translation initiation and downstream of S6K1 phosphorylation, including
eEF2 Thr56, PDCD4 Ser67, and eIF4B Ser422, were unchanged
by both alcohol and muscle contraction at this time point (Fig.
5I and Table 1).
Signaling of primary mTOR substrates 12 h post-muscle
contraction. Measurement of selected mTORC1 substrates was
also performed 12 h after cessation of muscle contraction, a
time point at which no alcohol was present in the circulation.
The contraction-induced increase in S6K1 Thr389 (⬃600%)
30 min Post-Contraction
A
B
240/244
rpS6 Ser
eIF4B Ser422
Total eIF4B
Total rpS6
250
eIF4B Ser422/Total
(% Non-Stim Con)
200
100
0
Con
EtOH
Con
Non-Stim
C
200
150
100
50
0
EtOH
D
PDCD4 Ser6 7
EtOH
Con
EtOH
Stim
eEF2 Thr5 6
Total eEF2
150
eEF2 Thr56/Total
(% Non-Stim Con)
150
100
50
0
Con
Non-Stim
Stim
Total PDCD4
PDCD4 Ser67/Total
(% Non-Stim Con)
Fig. 4. Effect of alcohol and electrically stimulated muscle contraction on proteins downstream of S6K1. Ribosomal protein S6 (rpS6)
Ser240/244 (A), eukaryotic initiation factor
(eIF) 4B Ser422 (B), programmed cell death
protein 4 (PDCD4) Ser67 (C), and eukaryotic
elongation factor-2 (eEF2) Thr56 (D) were
assessed 30 min post-muscle contraction in the
muscle. Bars graphs represent quantification
of Western blot images normalized to the
total amount of respective protein with the
control nonstimulated value set to 100%.
Shaded bars, alcohol-treated mice (n ⫽ 9);
open bars, control mice (n ⫽ 9). Horizontal
bars indicate statistical differences between
groups (P ⬍ 0.05). Values are expressed as
means ⫾ SE.
rpS6 Ser240/244/Total
(% Non-Stim Con)
300
Con
EtOH
Non-Stim
Con
100
50
EtOH
Stim
J Appl Physiol • doi:10.1152/japplphysiol.00180.2014 • www.jappl.org
0
Con
EtOH
Non-Stim
Con
EtOH
Stim
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 16, 2017
The precise signaling cascade connecting mechanical stimulation to changes in mTORC1 signaling and then eventually
stimulating protein synthesis is still under investigation; however, signaling upstream and/or independent of mTOR activation may contribute (1, 38, 46). At 30 min post-electrical
muscle stimulation, phosphorylation of Akt Thr308 (12%) and
its downstream substrate in mTORC1, PRAS40 Thr246 (40%),
was decreased by stimulation, whereas phosphorylation of Akt
Ser473 was not altered (Fig. 3, A–C). Conversely, ERK Thr202/
Tyr204 phosphorylation was increased 53% in the stimulated
muscle of Control mice (Fig. 3D). Alcohol abrogated many of
these effects, since there was no change in the phosphorylation
of Akt Thr308 and Ser473 or PRAS40 Thr246 following stimulated muscle contraction in the presence of alcohol (Fig. 3,
A–C). In contrast, ERK Thr202/Tyr204 was increased by muscle
stimulation in alcohol-treated mice; however, the stimulationinduced increase was still blunted in Alcohol compared with
Control mice (Fig. 3D).
In response to phosphorylation by mTOR, S6K1 is activated
and signals to numerous substrates downstream that contribute
to different aspects of translation initiation and protein elongation (52). Electrically stimulated muscle contraction increased rpS6 Ser240/244 phosphorylation in gastrocnemius at 30
min in both control (110%) and alcohol-treated (100%) mice
(Fig. 4A). However, the magnitude of response to contraction
was reduced by alcohol (⬃40%). The contraction-induced
increase in the phosphorylation of eIF4B on Ser422 observed in
control mice (⬃80%) was not present in alcohol-treated mice
(Fig. 4B). The phosphorylation of two additional downstream
•
Alcohol Decreases Muscle Contraction-Induced mTOR Signaling
•
1175
Steiner JL et al.
4 h Post-Contraction
B
S6K1 Thr389
100
Con
EtOH
Non-Stim
Con
600
200
200
100
Con
EtOH
Non-Stim
Stim
E
Con
F
mTOR Total
mTOR Ser2448/Total
(% Non-Stim Con)
100
Con
EtOH
Non-Stim
Con
EtOH
100
Con
EtOH
Non-Stim
Stim
G
Con
S6K1 Thr421/Ser424
p=0.08
250
mTOR Ser2481
200
PRAS40 Total
50
0
Con
- - + +
- + - +
EtOH
Non-Stim
Stim
I
Stimulation
EtOH
ERK Total
EtOH
Stim
PRAS40 Thr246
EtOH
H
ERK Thr202/Tyr204
Con
100
200
0
0
EtOH
150
300
200
Con
Non-Stim
Stim
mTOR Ser2448
300
0
EtOH
Stimulation
EtOH
Con
EtOH
Stim
- - + +
- + - +
eEF2 Thr5 6
eEF2 Total
PDCD4 Ser6 7
308
150
Akt Thr
100
473
Akt Ser
50
Akt Total
PDCD4 Total
eIF4B Ser422
eIF4B Total
0
Con
EtOH
Non-Stim
Con
EtOH
Stim
Fig. 5. mTORC1 signaling in response to alcohol and electrically stimulated muscle contraction. After cessation of muscle contraction (4 h), S6K1Thr389 (A),
rpS6 Ser240/244 (B), 4E-BP1Ser65 (C), hyperphosphorylation of total 4E-BP1 (D), mTOR Ser2448 (E), PRAS40 Thr246 (F), and ERK1/2 Thr202/Tyr204 (G) were
assessed in the muscle. Bars graphs represent quantification of Western blots normalized to the total amount of respective protein with the control nonstimulated
value set to 100%. Representative images from Western blots for proteins listed in Table 1 and described in the text are shown in H and I. Shaded bars,
alcohol-treated mice (n ⫽ 8 –9); open bars, control mice (n ⫽ 9). Horizontal bars indicate statistical differences between groups (P ⬍ 0.05). Values are expressed
as means ⫾ SE.
and Thr421/Ser424 (220%), as well as rpS6 Ser240/244 (265%)
and 4E-BP1 Ser65 (⬃70%), persisted in Control animals at this
late time point (Fig. 6, A–D). Alcohol only suppressed the
phosphorylation of S6K1 Thr389 and rpS6 Ser240/244 in contracted muscle compared with Controls, whereas phosphorylation of S6K1 Thr421/Ser424 and 4E-BP1 Ser65 exhibited restored responsiveness to muscle contraction (Fig. 6, A–D).
Phosphorylation of ERK1/2 Thr202/Tyr204 was not altered by
contraction and/or alcohol intoxication at this time point (data
not shown).
Magnitude of change across time in S6K1 and rpS6
phosphorylation. While not a primary aim of this study, we
selected four representative samples within each treatment
group and submitted them to SDS-PAGE on the same gel for
visualization of differences in the magnitude of response to
muscle contraction and alcohol treatment over time (Fig. 7, A
J Appl Physiol • doi:10.1152/japplphysiol.00180.2014 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 16, 2017
γ
β
α
4E-BP1 Total
300
400
0
EtOH
400
4E-BP1 Ser65/Total
(% Non-Stim Con)
rpS6 Ser240/244/Total
(% Non-Stim Con)
S6K1 Thr389/Total
(% Non-Stim Con)
200
0
4E-BP1 γ band/α,β,γ bands
(% Non-Stim Con)
4E-BP1 Total
800
300
ERK1/2 Tyr202/Tyr204/Total ERK
(% Non-Stim Con)
4E-BP1 Ser65
rpS6 Total
S6K1 Total
D
C
rpS6 Ser240/244
PRAS40 Thr246/Total
(% Non-Stim Con)
A
1176
Alcohol Decreases Muscle Contraction-Induced mTOR Signaling
Table 1. Alcohol and/or electrically stimulated muscle
contraction-induced changes in phosphorylation of selected
proteins in muscle
Non-Stim, %
Stim, %,
Protein
Control
EtOH
Control
EtOH
S6K1 Thr421/Ser424/total
mTOR Ser2481/total
Akt Thr308/total
Akt Ser473/total
eEF2 Thr56/total
PDCD4 Ser67/total
eIF4B Ser422/total
100 ⫾ 4
100 ⫾ 10
100 ⫾ 13
100 ⫾ 12
100 ⫾ 8
100 ⫾ 15
100 ⫾ 13
103 ⫾ 4
83 ⫾ 5
74 ⫾ 7
70 ⫾ 14
100 ⫾ 14
94 ⫾ 7
147 ⫾ 32
215 ⫾ 20*
105 ⫾ 6
94 ⫾ 9
95 ⫾ 9
95 ⫾ 9
101 ⫾ 7
137 ⫾ 10
128 ⫾ 7ˆ
95 ⫾ 9
68 ⫾ 5
60 ⫾ 15
92 ⫾ 10
104 ⫾ 10
146 ⫾ 25
Values are means ⫾ SE. EtOH, alcohol; S6K1, p70S6K1; mTOR, mammalian target of rapamycin; Akt, protein kinasse B; eEF2, eukaryotic elongation factor 2; PDCD4, programmed cell death protein 4; eIF4B, eukaryotic
initiation factor 4B; Non-Stim, control condition; Stim, muscle underwent
electrically stimulated muscle contraction. *P ⬍ 0.05 compared with Control
Non-Stim value. ˆP ⬍ 0.05 compared with value from the Control Stim group.
Steiner JL et al.
DISCUSSION
The present study investigated the time-dependent effects of
acute alcohol intoxication on rates of protein synthesis and
mTORC1 signaling in skeletal muscle following electrically
stimulated muscle contraction. Alcohol decreased protein synthesis in the nonstimulated muscle at each time point (30 min,
4 h, and 12 h) compared with time-matched controls. Furthermore, alcohol prevented the muscle contraction-induced change
in synthesis observed in Control mice at 30 min and 4 h. Although
synthesis in the stimulated muscle was increased by a similar
percent in Control and Alcohol mice at the 12-h time point, the
absolute rate of protein synthesis remained suppressed in the
alcohol-treated muscle because of the alcohol-induced reduction of basal protein synthesis. Alcohol also suppressed the
phosphorylation of several proteins within the canonical
mTORC1 signaling cascade that were increased by stimulated
muscle contraction. However, activation of this pathway was
often discordant with changes in rates of synthesis in response
to muscle contraction and/or alcohol, highlighting the importance of measuring both responses.
Stimulated muscle contraction in control mice. Although
seemingly contradictory, the decrease in muscle protein synthesis observed at 30 min in Control mice is a well-recognized
phenomenon (5, 8, 38, 45, 51). Definitive mechanistic explanations are still lacking, but contributing factors likely include
12 h Post-Contraction
A
B
S6K1 Thr389
S6K1 Thr421/Ser424
Total S6K1
Fig. 6. Effect of alcohol and electrically stimulated muscle contraction on mTORC1 signaling. After muscle contraction (12 h),
S6K1Thr389 (A), S6K1 Thr421/Ser424 (B), rpS6
Ser240/244 (C), and 4E-BP1Ser65 (D) were assessed in muscle. Bars graphs represent quantification of Western blot images normalized
to the total amount of respective protein with
the control nonstimulated value of 100%.
Shaded bars, alcohol-treated mice (n ⫽ 8);
open bars, control mice (n ⫽ 8). Horizontal
bars indicate statistical differences between
groups (P ⬍ 0.05). Values are expressed as
means ⫾ SE.
S6K1 Thr389/Total
(% Non-Stim Con)
1000
800
600
400
200
0
Con
EtOH
Non-Stim
C
Con
EtOH
S6K1 Thr421/Ser424/Total
(% Non-Stim Con)
Total S6K1
300
200
100
0
Con
EtOH
Con
Non-Stim
D
EtOH
Stim
4E-BP1 Ser6 5
Total 4E-BP1
Total rpS6
500
250
4E-BP1 Ser65/Total
(% Non-Stim Con)
rpS6 Ser240/244/Total
(% Non-Stim Con)
400
Stim
rpS6 Ser240/ 244
400
300
200
100
0
500
Con
EtOH
Non-Stim
Con
EtOH
Stim
J Appl Physiol • doi:10.1152/japplphysiol.00180.2014 • www.jappl.org
200
150
100
50
0
Con
EtOH
Non-Stim
Con
EtOH
Stim
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 16, 2017
and B). Statistical analysis was not performed because of the
small sample size (n ⫽ 4); however, muscle contraction
evoked a similar level of activation of S6K1 Thr389 and rpS6
Ser240/244 at each time point in Control animals. As described
above, alcohol impaired this contraction-mediated increase at
each time point.
•
Alcohol Decreases Muscle Contraction-Induced mTOR Signaling
A
30 min
12 h
4h
S6K1 Thr389
S6K1 Thr389/Total
(% Non-Stim Con-30 min)
S6K1 Total
600
Con-Non
EtOH-Non
Con-Stim
EtOH-Stim
400
200
0
4h
30 min
12 h
12 h
4h
240/244
rpS6 Ser
rpS6 Ser240/244/Total
(% Non-Stim Con-30 min)
rpS6 Total
400
300
Control-Non
EtOH-Non
Control-Stim
EtOH-Stim
200
100
0
30 min
4h
12 h
Fig. 7. Comparison of the relative expression at each time point after alcohol
and electrically stimulated muscle contraction. S6K1 Thr389 (A) and rpS6
Ser240/244 (B) were measured at 30 min, 4 h, and 12 h postmuscle contraction.
Samples were resolved on the same gel, and Western blotting was performed.
Bars graphs represent quantification of Western blot images normalized to the
total amount of respective protein with all values set relative to the 30-min
control nonstimulated value set to 100%. Shaded bars, alcohol-treated mice
(n ⫽ 4); open bars, control mice (n ⫽ 4). Statistical analysis was not
performed, since the graph is only representative of 4 mice/group. Values are
expressed as means ⫾ SE.
the contraction-mediated decrease in Ca2⫹ release, enhanced
ATP turnover, and increased eEF2 phosphorylation (4, 5, 8, 38,
45, 51). The magnitude of the early contraction-induced decrease in synthesis is directly related to the stimulus intensity,
and as such we speculate that the maximal contractions produced by the current protocol resulted in a large decrease in
synthesis that was still recovering at the early (30-min) time
point (51). Similar to our findings, stretch of L6 myotubes
decreased protein synthesis at early time points before rates
returned to basal levels later in recovery (5).
Moreover, our data at 30 min postcontraction in control
muscle show a discordant response between rates of synthesis
and mTORC1 signaling, since protein synthesis was decreased
concomitant with increased phosphorylation of S6K1 and its
substrates eIF4B and rpS6. Despite similar findings being
reported in humans and ex vivo rodent muscle preparations (2,
3, 5, 45), mechanistic explanations and the physiological rel-
Steiner JL et al.
1177
evance of this seemingly dissonant response between signaling
and synthesis remains to be determined. Because the synthesis
rate had recovered and was increased in accordance with
mTORC1 activity at the 4- and 12-h time points (in Control
mice), it is possible that the early induction of these factors
is imperative for the later, long-lasting augmentation of
protein synthesis. In support of this speculation, treatment
with the mTOR inhibitor rapamycin before resistance exercise prevents the increase in protein synthesis in the postexercise period (13, 26).
Further highlighting the need for measuring rates of synthesis directly were our findings at both 30 min and 4 h showing
discordance in the phosphorylation of proteins within the
mTORC1 signaling pathway. This included increased phosphorylation of S6K1, rpS6, eIF4B, and 4E-BP1 at 30 min in
response to muscle stimulation while mTOR Ser2448 and
Ser2481, previously thought to be markers of mTOR activity,
remained unchanged at this time point. A similar relationship
has been reported following stretch of L6 skeletal myoblasts,
since mTOR Ser2448 was unchanged despite increased phosphorylation of S6K1 Thr389 and 4E-BP1 Thr37/46 (5). Currently, the importance of Ser2448 phosphorylation in the activation of mTOR is uncertain, since mutation of this residue to
alanine does not alter the kinase activity of mTORC1 (10).
However, there is also the possibility that phosphorylation of
S6K1 and/or its substrates occurred by an mTOR-independent
mechanism similar to that reported in rat epitrochlearis muscle
undergoing ex vivo electrical stimulation in the presence of the
mTOR inhibitor Torin1 (38). In summary, our findings from
Control animals in response to electrically stimulated muscle
contraction highlight the importance of assessing both signaling and protein synthesis to achieve a true representation of the
physiological impact of the stimulus.
Alcohol-related changes independent of muscle contraction.
The exact mechanism of the alcohol-induced decrease in basal
muscle protein synthesis has not been fully elucidated, although the concurrent impairment of mTORC1 signaling has
previously been reported, including decreased phosphorylation
of rpS6 Ser240/244 and 4E-BP1 Thr37/46 (28, 32, 34, 35). In
contrast, our current findings did not reveal significant differences in several mTORC1 substrates in the nonstimulated leg
following alcohol treatment, suggesting that mTOR-independent effects may be contributing to the observed reduction in
rates of muscle protein synthesis. However, not all investigations link mTORC1 activity to the regulation of basal protein
synthesis, since treatment with rapamycin decreases S6K1
activity but does not alter the basal rate of muscle protein
synthesis or translational activity (16, 26). Furthermore, alcohol may affect other aspects of translation initiation, including
reducing the formation of eIF4F complex (i.e., decrease eIF4EeIF4G binding), which would limit binding of the mRNA to
the 43S preinitiation complex (33). In chronic alcohol-fed
rats, the activity of eIF2B has also been significantly suppressed, effectively limiting translation initiation and protein synthesis, and could have contributed to the present
findings (36).
Additionally, differences in experimental design may have
contributed to the observed discrepancies from our past data.
Currently, mice were fasted overnight and remained fasted
until death (⬃16 –24 h later), whereas previous studies used
freely fed mice or overnight-fasted rats (28, 32, 34, 35). Such
J Appl Physiol • doi:10.1152/japplphysiol.00180.2014 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 16, 2017
B
30 min
•
1178
Alcohol Decreases Muscle Contraction-Induced mTOR Signaling
ACKNOWLEDGMENTS
We thank Maithili Navaratnarajah, Anne Pruznak, and Gina Deiter for
technical assistance. We also thank Dr. Sean Stocker for assistance with the
electrical stimulation protocol and Dr. Brad Gordon for helpful discussions.
GRANTS
This work was supported in part by National Institutes of Health Grants
R37-AA-011290 to C. H. Lang and F32-AA-23422 to J. L. Steiner.
Steiner JL et al.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: J.L.S. and C.H.L. conception and design of research;
J.L.S. performed experiments; J.L.S. and C.H.L. analyzed data; J.L.S. and
C.H.L. interpreted results of experiments; J.L.S. prepared figures; J.L.S.
drafted manuscript; J.L.S. and C.H.L. edited and revised manuscript; J.L.S. and
C.H.L. approved final version of manuscript.
REFERENCES
1. Anjum R, Blenis J. The RSK family of kinases: emerging roles in cellular
signalling. Nat Rev Mol Cell Biol 9: 747–758, 2008.
2. Areta JL, Burke LM, Ross ML, Camera DM, West DW, Broad EM,
Jeacocke NA, Moore DR, Stellingwerff T, Phillips SM, Hawley JA,
Coffey VG. Timing and distribution of protein ingestion during prolonged
recovery from resistance exercise alters myofibrillar protein synthesis. J
Physiol 591: 2319 –2331, 2013.
3. Atherton PJ, Etheridge T, Watt PW, Wilkinson D, Selby A, Rankin D,
Smith K, Rennie MJ. Muscle full effect after oral protein: time-dependent concordance and discordance between human muscle protein synthesis and mTORC1 signaling. Am J Clin Nutr 92: 1080 –1088, 2010.
4. Atherton PJ, Rennie MJ. It’s no go for protein when it’s all go. J Physiol
587: 1373–1374, 2009.
5. Atherton PJ, Szewczyk NJ, Selby A, Rankin D, Hillier K, Smith K,
Rennie MJ, Loughna PT. Cyclic stretch reduces myofibrillar protein
synthesis despite increases in FAK and anabolic signalling in L6 cells. J
Physiol 587: 3719 –3727, 2009.
6. Baar K, Esser K. Phosphorylation of p70(S6k) correlates with increased
skeletal muscle mass following resistance exercise. Am J Physiol Cell
Physiol 276: C120 –C127, 1999.
7. Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R,
Zlotchenko E, Scrimgeour A, Lawrence JC, Glass DJ, Yancopoulos
GD. Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol 3: 1014 –
1019, 2001.
8. Bylund-Fellenius AC, Ojamaa KM, Flaim KE, Li JB, Wassner SJ,
Jefferson LS. Protein synthesis versus energy state in contracting muscles
of perfused rat hindlimb. Am J Physiol Endocrinol Metab 246: E297–
E305, 1984.
9. Chen MM, Palmer JL, Ippolito JA, Curtis BJ, Choudhry MA, Kovacs
EJ. Intoxication by intraperitoneal injection or oral gavage equally potentiates postburn organ damage and inflammation (Abstract). Mediators
Inflamm 2013: 971481, 2013.
10. Copp J, Manning G, Hunter T. TORC-specific phosphorylation of
mammalian target of rapamycin (mTOR): phospho-Ser2481 is a marker
for intact mTOR signaling complex 2. Cancer Res 69: 1821–1827, 2009.
11. Deldicque L, Atherton P, Patel R, Theisen D, Nielens H, Rennie MJ,
Francaux M. Decrease in Akt/PKB signalling in human skeletal muscle
by resistance exercise. Eur J Appl Physiol 104: 57–65, 2008.
12. Drummond MJ, Dreyer HC, Fry CS, Glynn EL, Rasmussen BB.
Nutritional and contractile regulation of human skeletal muscle protein
synthesis and mTORC1 signaling. J Appl Physiol 106: 1374 –1384, 2009.
13. Drummond MJ, Fry CS, Glynn EL, Dreyer HC, Dhanani S, Timmerman KL, Volpi E, Rasmussen BB. Rapamycin administration in humans
blocks the contraction-induced increase in skeletal muscle protein synthesis. J Physiol 587: 1535–1546, 2009.
14. Foster DA. Regulation of mTOR by phosphatidic acid? Cancer Res 67:
1–4, 2007.
15. Friedman LS. Complications associated with blood alcohol concentration
following injury. Alcohol 48: 391–400, 2014.
16. Garelick MG, Mackay VL, Yanagida A, Academia EC, Schreiber KH,
Ladiges WC, Kennedy BK. Chronic rapamycin treatment or lack of
S6K1 does not reduce ribosome activity in vivo. Cell Cycle 12: 2493–
2504, 2013.
17. Gingras AC, Gygi SP, Raught B, Polakiewicz RD, Abraham RT,
Hoekstra MF, Aebersold R, Sonenberg N. Regulation of 4E-BP1
phosphorylation: a novel two-step mechanism. Genes Dev 13: 1422–1437,
1999.
18. Gingras AC, Raught B, Gygi SP, Niedzwiecka A, Miron M, Burley
SK, Polakiewicz RD, Wyslouch-Cieszynska A, Aebersold R, Sonenberg N. Hierarchical phosphorylation of the translation inhibitor 4E-BP1.
Genes Dev 15: 2852–2864, 2001.
J Appl Physiol • doi:10.1152/japplphysiol.00180.2014 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 16, 2017
a prolonged fast in mice causes weight loss, depletes glycogen,
and results in nearly undetectable levels of phosphorylated
proteins (e.g., S6K1 Thr389) within the mTORC1 pathway as
protein synthesis is suppressed to conserve energy (54). Thus,
we may have failed to detect potential alcohol-induced changes
in mTOR signaling in the basal condition, since protein detection was optimized to highlight changes induced by muscle
stimulation within the same animal.
Effects of alcohol and muscle contraction. The current
findings extend previous work showing alcohol administered
before other anabolic stimuli (refeeding, insulin, IGF-I, and
leucine) suppresses the induction of protein synthesis and
mTORC1 signaling (e.g., 4E-BP1 and S6K1 phosphorylation)
(28, 32, 34, 53). We hypothesized that the contraction-induced
increase in muscle protein synthesis would be maintained in
the presence of alcohol, since muscle contraction activates
mTORC1 independent of pathways previously identified to be
inhibited by alcohol, including PI3K/Akt and/or hormonal
stimulation (24, 48, 55). Contrary to expectations, our data
show that alcohol impairs the mechanical activation of
mTORC1 signaling similar to other previously reported anabolic stimuli (28, 32, 34, 53). Furthermore, protein synthesis
and signaling through mTORC1 (assessed by measurement of
the phosphorylation of S6K1 on Thr389 and rpS6 on Ser240/244)
remained suppressed by alcohol even after the BAC had
returned to undetectable levels at 12 h. Although not required,
the MEK/ERK pathway may also contribute to contractioninduced changes in muscle protein synthesis and mTOR signaling (41, 42, 46). Presently though, alcohol decreased
ERK1/2 phosphorylation in the nonstimulated and stimulated
muscle compared with Controls, and the increase in phosphorylation caused by stimulated muscle contraction was not sufficient to overcome the alcohol-induced suppression of protein
synthesis, arguing against a role for this pathway in the control
of protein synthesis.
Results from the current investigation suggest that alcohol
has a long-lasting effect on protein synthesis as well as mTOR
signaling induced by electrically stimulated muscle contraction, albeit the effects are not always concordant, especially at
earlier time points. Furthermore, we show that, even 12 h after
acute alcohol treatment, the rate of protein synthesis is still
suppressed compared with control animals, implying that an
episode of heavy drinking the night before could limit muscular adaptations from exercise performed the following morning. These findings also suggest that if muscle contraction is to
be administered in a therapeutic setting that chronic alcoholic
individuals should be encouraged to abstain before the exercise
session. Last, the impact of either a single incidence of acute
alcohol intoxication (i.e., binge drinking) or chronic alcohol
ingestion on muscle contraction-induced increases in muscle
mass over a more prolonged time frame remains to be determined.
•
Alcohol Decreases Muscle Contraction-Induced mTOR Signaling
Steiner JL et al.
1179
40. Livy DJ, Parnell SE, West JR. Blood ethanol concentration profiles: a
comparison between rats and mice. Alcohol 29: 165–171, 2003.
41. Ma L, Chen Z, Erdjument-Bromage H, Tempst P, Pandolfi PP.
Phosphorylation and functional inactivation of TSC2 by Erk implications
for tuberous sclerosis and cancer pathogenesis. Cell 121: 179 –193, 2005.
42. Ma L, Teruya-Feldstein J, Bonner P, Bernardi R, Franz DN, Witte D,
Cordon-Cardo C, Pandolfi PP. Identification of S664 TSC2 phosphorylation as a marker for extracellular signal-regulated kinase mediated
mTOR activation in tuberous sclerosis and human cancer. Cancer Res 67:
7106 –7112, 2007.
43. Magnuson B, Ekim B, Fingar DC. Regulation and function of ribosomal
protein S6 kinase (S6K) within mTOR signalling networks. Biochem J
441: 1–21, 2012.
44. Martin F, Ward K, Slavin G, Levi J, Peters TJ. Alcoholic skeletal
myopathy, a clinical and pathological study. Q J Med 55: 233–251, 1985.
45. Miranda L, Horman S, De Potter I, Hue L, Jensen J, Rider MH.
Effects of contraction and insulin on protein synthesis, AMP-activated
protein kinase and phosphorylation state of translation factors in rat
skeletal muscle. Pflugers Arch 455: 1129 –1140, 2008.
46. Miyazaki M, McCarthy JJ, Fedele MJ, Esser KA. Early activation of
mTORC1 signalling in response to mechanical overload is independent of
phosphoinositide 3-kinase/Akt signalling. J Physiol 589: 1831–1846,
2011.
47. Mothe-Satney I, Yang D, Fadden P, Haystead TA, Lawrence JC, Jr.
Multiple mechanisms control phosphorylation of PHAS-I in five (S/T)P
sites that govern translational repression. Mol Cell Biol 20: 3558 –3567,
2000.
48. O’Neil TK, Duffy LR, Frey JW, Hornberger TA. The role of phosphoinositide 3-kinase and phosphatidic acid in the regulation of mammalian
target of rapamycin following eccentric contractions. J Physiol 587:
3691–3701, 2009.
49. Preedy VR, Keating JW, Peters TJ. The acute effects of ethanol and
acetaldehyde on rates of protein synthesis in type I and type II fibre-rich
skeletal muscles of the rat. Alcohol Alcohol 27: 241–251, 1992.
50. Price DJ, Mukhopadhyay NK, Avruch J. Insulin-activated protein
kinases phosphorylate a pseudosubstrate synthetic peptide inhibitor of the
p70 S6 kinase. J Biol Chem 266: 16281–16284, 1991.
51. Rose AJ, Alsted TJ, Jensen TE, Kobberø JB, Maarbjerg SJ, Jensen J,
Richter EA. A Ca(2⫹)-calmodulin-eEF2K-eEF2 signalling cascade, but
not AMPK, contributes to the suppression of skeletal muscle protein
synthesis during contractions. J Physiol 587: 1547–1563, 2009.
52. Ruvinsky I, Meyuhas O. Ribosomal protein S6 phosphorylation: from
protein synthesis to cell size. Trends Biochem Sci 31: 342–348, 2006.
53. Sneddon AA, Koll M, Wallace MC, Jones J, Miell JP, Garlick PJ,
Preedy VR. Acute alcohol administration inhibits the refeeding response
after starvation in rat skeletal muscle. Am J Physiol Endocrinol Metab
284: E874 –E882, 2003.
54. Sokolović M, Sokolović A, Wehkamp D, Ver Loren van Themaat E, de
Waart DR, Gilhuijs-Pederson LA, Nikolsky Y, van Kampen AH,
Hakvoort TB, Lamers WH. The transcriptomic signature of fasting
murine liver (Abstract). BMC Genomics 9: 528, 2008.
55. Spangenburg EE, Le Roith D, Ward CW, Bodine SC. A functional
insulin-like growth factor receptor is not necessary for load-induced
skeletal muscle hypertrophy. J Physiol 586: 283–291, 2008.
56. Vary TC, Jefferson LS, Kimball SR. Amino acid-induced stimulation of
translation initiation in rat skeletal muscle. Am J Physiol Endocrinol
Metab 277: E1077–E1086, 1999.
57. Witkowski S, Lovering RM, Spangenburg EE. High-frequency electrically stimulated skeletal muscle contractions increase p70s6k phosphorylation independent of known IGF-I sensitive signaling pathways. FEBS
Lett 584: 2891–2895, 2010.
58. You JS, Frey JW, Hornberger TA. Mechanical stimulation induces
mTOR signaling via an ERK-independent mechanism: implications for a
direct activation of mTOR by phosphatidic acid. PLoS One 7: e47258,
2012.
59. You JS, Lincoln HC, Kim CR, Frey JW, Goodman CA, Zhong XP,
Hornberger TA. The role of diacylglycerol kinase zeta and phosphatidic
acid in the mechanical activation of mammalian target of rapamycin
(mTOR) signaling and skeletal muscle hypertrophy. J Biol Chem 289:
1551–1563, 2013.
J Appl Physiol • doi:10.1152/japplphysiol.00180.2014 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 16, 2017
19. Goodman CA. The role of mTORC1 in regulating protein synthesis and
skeletal muscle mass in response to various mechanical stimuli. Rev
Physiol Biochem Pharmacol 166: 43–95, 2014.
20. Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev
18: 1926 –1945, 2004.
21. Holmes JF, Adams C, Rogers P, Vu P. Successful conviction of
intoxicated drivers at a level I trauma center. West J Emerg Med 15:
480 –485, 2014.
22. Hornberger TA, Chien S. Mechanical stimuli and nutrients regulate
rapamycin-sensitive signaling through distinct mechanisms in skeletal
muscle. J Cell Biochem 97: 1207–1216, 2006.
23. Hornberger TA, Chu WK, Mak YW, Hsiung JW, Huang SA, Chien S.
The role of phospholipase D and phosphatidic acid in the mechanical
activation of mTOR signaling in skeletal muscle. Proc Natl Acad Sci USA
103: 4741–4746, 2006.
24. Hornberger TA, Stuppard R, Conley KE, Fedele MJ, Fiorotto ML,
Chin ER, Esser KA. Mechanical stimuli regulate rapamycin-sensitive
signalling by a phosphoinositide 3-kinase-, protein kinase B- and growth
factor-independent mechanism. Biochem J 380: 795–804, 2004.
25. Korzick DH, Sharda DR, Pruznak AM, Lang CH. Aging accentuates
alcohol-induced decrease in protein synthesis in gastrocnemius. Am J
Physiol Regul Integr Comp Physiol 304: R887–R898, 2013.
26. Kubica N, Bolster DR, Farrell PA, Kimball SR, Jefferson LS. Resistance exercise increases muscle protein synthesis and translation of eukaryotic initiation factor 2Bepsilon mRNA in a mammalian target of
rapamycin-dependent manner. J Biol Chem 280: 7570 –7580, 2005.
27. Kumar V, Frost RA, Lang CH. Alcohol impairs insulin and IGF-I
stimulation of S6K1 but not 4E-BP1 in skeletal muscle. Am J Physiol
Endocrinol Metab 283: E917–E928, 2002.
28. Lang CH, Frost RA, Deshpande N, Kumar V, Vary TC, Jefferson LS,
Kimball SR. Alcohol impairs leucine-mediated phosphorylation of 4EBP1, S6K1, eIF4G, and mTOR in skeletal muscle. Am J Physiol Endocrinol Metab 285: E1205–E1215, 2003.
29. Lang CH, Frost RA, Kumar V, Wu D, Vary TC. Impaired protein
synthesis induced by acute alcohol intoxication is associated with changes
in eIF4E in muscle and eIF2B in liver. Alcohol Clin Exp Res 24: 322–331,
2000.
30. Lang CH, Kimball SR, Frost RA, Vary TC. Alcohol myopathy: impairment of protein synthesis and translation initiation. Int J Biochem Cell
Biol 33: 457–473, 2001.
31. Lang CH, Kumar V, Liu X, Frost RA, Vary TC. IGF-I induced
phosphorylation of S6K1 and 4E-BP1 in heart is impaired by acute alcohol
intoxication. Alcohol Clin Exp Res 27: 485–494, 2003.
32. Lang CH, Lynch CJ, Vary TC. Alcohol-induced IGF-I resistance is
ameliorated in mice deficient for mitochondrial branched-chain aminotransferase. J Nutr 140: 932–938, 2010.
33. Lang CH, Lynch CJ, Vary TC. BCATm deficiency ameliorates endotoxin-induced decrease in muscle protein synthesis and improves survival
in septic mice. Am J Physiol Regul Integr Comp Physiol 299: R935–R944,
2010.
34. Lang CH, Pruznak AM, Deshpande N, Palopoli MM, Frost RA, Vary
TC. Alcohol intoxication impairs phosphorylation of S6K1 and S6 in
skeletal muscle independently of ethanol metabolism. Alcohol Clin Exp
Res 28: 1758 –1767, 2004.
35. Lang CH, Pruznak AM, Nystrom GJ, Vary TC. Alcohol-induced
decrease in muscle protein synthesis associated with increased binding of
mTOR and raptor: Comparable effects in young and mature rats (Abstract). Nutr Metab 6: 4, 2009.
36. Lang CH, Wu D, Frost RA, Jefferson LS, Kimball SR, Vary TC.
Inhibition of muscle protein synthesis by alcohol is associated with
modulation of eIF2B and eIF4E. Am J Physiol Endocrinol Metab 277:
E268 –E276, 1999.
37. Liu Q, Wang J, Kang SA, Thoreen CC, Hur W, Ahmed T, Sabatini
DM, Gray NS. Discovery of 9-(6-aminopyridin-3-yl)-1-(3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(1H)-one (Torin2) as a potent,
selective, and orally available mammalian target of rapamycin (mTOR)
inhibitor for treatment of cancer. J Med Chem 54: 1473–1480, 2011.
38. Liu Y, Vertommen D, Rider MH, Lai YC. Mammalian target of
rapamycin-independent S6K1 and 4E-BP1 phosphorylation during contraction in rat skeletal muscle. Cell Signal 25: 1877–1886, 2013.
39. Livingstone MB, M;. Rapamycin-insensitive mTORC1 activity controls eIF4E:4E-BP1 binding. F1000 Research 1, 2012 http://f1000r.es/
NM6hpo.
•