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Alcohol Ingestion Impairs Maximal Post-Exercise Rates of
Myofibrillar Protein Synthesis following a Single Bout of
Concurrent Training
Evelyn B. Parr
1
, Donny M. Camera
1
, Jose
´L. Areta
1
, Louise M. Burke
2
, Stuart M. Phillips
3
,
John A. Hawley
4,5
*, Vernon G. Coffey
6
1Exercise and Nutrition Research Group, School of Medical Sciences, RMIT University, Bundoora, Victoria, Australia, 2Department of Sports Nutrition, Australian Institute
of Sport, Canberra, ACT, Australia, 3Exercise Metabolism Research Group, Department of Kinesiology, McMaster University, Hamilton, Ontario, Canada, 4Exercise and
Nutrition Research Group, School of Exercise Science, Australian Catholic University, Fitzroy, Victoria, Australia, 5Research Institute for Sport and Exercise Sciences,
Liverpool John Moores University, Liverpool, United Kingdom, 6School of Exercise and Nutrition Sciences and Institute of Health and Biomedical Innovation, Queensland
University of Technology, Kelvin Grove, Queensland, Australia
Abstract
Introduction:
The culture in many team sports involves consumption of large amounts of alcohol after training/
competition. The effect of such a practice on recovery processes underlying protein turnover in human skeletal muscle are
unknown. We determined the effect of alcohol intake on rates of myofibrillar protein synthesis (MPS) following strenuous
exercise with carbohydrate (CHO) or protein ingestion.
Methods:
In a randomized cross-over design, 8 physically active males completed three experimental trials comprising
resistance exercise (865 reps leg extension, 80% 1 repetition maximum) followed by continuous (30 min, 63% peak power
output (PPO)) and high intensity interval (10630 s, 110% PPO) cycling. Immediately, and 4 h post-exercise, subjects
consumed either 500 mL of whey protein (25 g; PRO), alcohol (1.5 g?kg body mass
21
,1262 standard drinks) co-ingested
with protein (ALC-PRO), or an energy-matched quantity of carbohydrate also with alcohol (25 g maltodextrin; ALC-CHO).
Subjects also consumed a CHO meal (1.5 g CHO?kg body mass
21
) 2 h post-exercise. Muscle biopsies were taken at rest, 2
and 8 h post-exercise.
Results:
Blood alcohol concentration was elevated above baseline with ALC-CHO and ALC-PRO throughout recovery (P,
0.05). Phosphorylation of mTOR
Ser2448
2 h after exercise was higher with PRO compared to ALC-PRO and ALC-CHO (P,0.05),
while p70S6K phosphorylation was higher 2 h post-exercise with ALC-PRO and PRO compared to ALC-CHO (P,0.05). Rates
of MPS increased above rest for all conditions (,29–109%, P,0.05). However, compared to PRO, there was a hierarchical
reduction in MPS with ALC-PRO (24%, P,0.05) and with ALC-CHO (37%, P,0.05).
Conclusion:
We provide novel data demonstrating that alcohol consumption reduces rates of MPS following a bout of
concurrent exercise, even when co-ingested with protein. We conclude that alcohol ingestion suppresses the anabolic
response in skeletal muscle and may therefore impair recovery and adaptation to training and/or subsequent performance.
Citation: Parr EB, Camera DM, Areta JL, Burke LM, Phillips SM, et al. (2014) Alcohol Ingestion Impairs Maximal Post-Exercise Rates of Myofibrillar Protein Synthesis
following a Single Bout of Concurrent Training. PLoS ONE 9(2): e88384. doi:10.1371/journal.pone.0088384
Editor: Stephen E. Alway, West Virginia University School of Medicine, United States of America
Received October 13, 2013; Accepted January 6, 2014; Published February 12, 2014
Copyright: ß2014 Parr et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was, in part, funded by a grant from the Australian Sports Commission to LMB. The funders had no role in study design, data collection and
analysis, decision to publish or preparation of the manuscript. No additional external funding was received for this study.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: john.hawley@acu.edu.au
Introduction
The focus of the early post-exercise period (i.e., 1–8 h) is to
enhance physiological processes that are critical for reversing the
exercise-induced disturbances to homeostasis and physiological
function and for promoting adaptations to training [1]. Recom-
mended nutritional strategies to maximize recovery in skeletal
muscle include protein for enhancing rates of protein synthesis and
carbohydrate for replenishing glycogen stores [2,3]. Muscle
contraction and the intake of leucine-rich protein sources activate
independent but complimentary signaling responses that converge
at the mechanistic target of rapamycin (mTOR) to stimulate
protein translation enhancing rates of muscle protein synthesis [4–
6]. The ingestion of ,20–25 g of high quality protein soon after
exercise [7], repeated every 4 h [8] has been shown to maximise
the anabolic response in skeletal muscle.
The cultural environment surrounding some sports often
involves the intake of large amounts of alcohol after training and
competition, with athletes in several team sports being particularly
at risk of ‘‘binge drinking’’ practices [9–11]. Indeed, a number of
studies have reported that athletes are more likely than the general
population to drink alcohol to excess, with a large proportion
(,50–65%) consuming intakes above the threshold classified as
PLOS ONE | www.plosone.org 1 February 2014 | Volume 9 | Issue 2 | e88384
hazardous drinking [12,13]. The outcomes of binge drinking after
exercise are likely to include the direct effect of alcohol on
physiological processes as well as the indirect effect on the athlete’s
recovery due to not eating or resting adequately as a result of
intoxication. Although the concurrent consumption of carbohy-
drate can partially offset the deleterious effects of alcohol intake on
post-exercise glycogen resynthesis [14], the effect of alcohol
consumption on muscle protein synthesis is unknown.
Studies by Barnes and colleagues (2010, 2011) have investigated
the effects of post-exercise alcohol consumption on human muscle
function and performance [15,16]. However, data on the effects of
alcohol intake on skeletal muscle protein synthesis is limited to
work in rodents. These studies show that both acute and chronic
alcohol ingestion can have a detrimental effect on cell signaling
and protein synthesis in skeletal muscle [17–21]. The aim of the
current study was to determine the effect of alcohol intake on
anabolic cell signaling and rates of myofibrillar protein synthesis
(MPS) in humans during recovery from a bout of strenuous
exercise approximating stresses an athlete may experience in
training and performance for various team sports such as various
football and rugby codes, and court sports. We hypothesized that
compared to post-exercise protein intake, co-ingestion of alcohol
would down-regulate translation initiation signaling and decrease
rates of MPS.
Methods
Subjects
Eight healthy physically active male subjects (age 21.464.8 yr,
body mass (BM) 79.3611.9 kg, peak oxygen uptake (VO
2peak
)
48.164.8 mL?kg
21
?min
21
, leg extension one repetition maximum
(1RM) 104620 kg; values are mean 6SD) who had been
participating in regular exercise (3 times wk
21
for .6 months)
volunteered for this study. The experimental procedures and
possible risks associated with the study were explained to each
subject, who each gave written informed consent before partici-
pation.
Ethics statement
All subjects were informed of the purpose of the study, the
experimental procedures involved and all the potential risks
involved before giving written consent. No minors were involved
in this study as subjects were required to be 18 years of age at the
time of participation due to the legal age for alcohol consumption
in Australia. All subjects were deemed healthy based on their
response to a routine medical screening questionnaire. The study
was approved by the Human Research Ethics Committee of
RMIT University (43/11) and was carried out according to the
NHMRC National Statement on Ethical Conduct in Human
Research (2007) and the Australian Code for the Responsible
Conduct of Research (2007).
Study Design
The study employed a randomized counter-balanced, cross-
over design in which each subject completed bouts of consecutive
resistance, continuous and intermittent high-intensity exercise with
either post-exercise ingestion of alcohol-carbohydrate (ALC-
CHO), alcohol-protein (ALC-PRO) or protein only (PRO)
beverages on three separate occasions. Each experimental trial
was separated by a two week recovery period, during which time
subjects maintained their habitual physical activity pattern. Given
the data showing little/no effect of carbohydrate ingestion on
myofibrillar protein synthesis, the ALC-CHO treatment was used
as an iso-energetic control. The decision not to use parallel groups
was based on a within subject crossover design adding strength to
the interpretation and conclusions of the study but limited the total
number of treatments such that an exercise only trial was not
undertaken. Finally, we based our exercise protocol incorporating
the different metabolic stresses approximating those experienced
in team sports due to published reports of the increased incidence
of excessive alcohol consumption following performance in team/
group sport [13].
Preliminary Testing
VO
2
peak. VO
2peak
and peak power output (PPO) were
determined during an incremental test to volitional fatigue on a
Lode cycle ergometer (Groningen, The Netherlands). The
protocol has been described in detail previously [22].
Maximal strength. Quadriceps strength was determined on
a plate-loaded leg extension machine until the 1RM load was
established. Repetitions were separated by a 3-min recovery and
were used to establish the maximum load/weight that could be
moved through the full range of motion once, but not a second
time.
Diet/exercise control
For the 48 h prior to an experimental trial subjects were
instructed to refrain from strenuous exercise/training. Subjects
were provided pre-packaged food and drinks (,6000 kJ; 3.1 g
CHO?kg
21
BM, 0.5 g fat?kg
21
BM, 0.4 g protein?kg
21
BM) to be
consumed for the last meal prior to an experiment. A food diary to
record dietary intake was used to ensure adherence to the final
meal and overall daily intake for the 24 h prior to an experiment
day.
Experimental Procedure
The study employed a randomized cross-over design in which
each subject completed three experimental trials. Each trial was
separated by 14 d, during which subjects maintained their
habitual level of physical activity and their normal diet. The three
trials compared post-exercise protein synthesis with three different
treatments: a post-exercise feeding regimen providing protein
intake for optimal muscle protein synthesis [8] (2 feedings of 25 g
high quality protein at 0 and 4 h of recovery: PRO), a trial in
which the subjects consumed 1.5 g?kg
21
BM ethanol plus an
energy match for recommended protein feedings in the form of
carbohydrate (ALC-CHO), and ALC-PRO in which the same
amount of alcohol was consumed in addition to protein intake in
PRO also ingested at 0 and 4 h post-exercise (see Figure 1). All
trials involved a further standardised carbohydrate-rich meal
(1.5 g CHO?kg
21
BM) at 2 h post-exercise as post-event fuelling/
eating.
On the morning of an experimental trial, subjects reported to
the laboratory after a ,10-h overnight fast. After resting in a
supine position for ,15 min, catheters were inserted into the
antecubital vein of each arm and a baseline blood sample (,4 mL)
was taken from one arm. A primed constant intravenous infusion
(prime: 2 mmol?kg
21
; infusion 0.05 mmol?kg
21
min
21
)ofL-
[ring-
13
C
6
] phenylalanine (Cambridge Isotopes Laboratories,
Woburn, MA, USA) was then administered to the contralateral
arm for the duration of the experiment. Under local anaesthesia
(1% Xylocaine) a resting biopsy from the vastus lateralis of one leg
was obtained 3 h after commencement of the tracer infusion using
a 5-mm Bergstrom needle modified with suction, during the first
trial only. This procedure was undertaken once during subjects
first experimental trial to obtain resting fractional synthetic rates
using the previously validated single biopsy method [23]. During
subsequent trials tracer infusion commenced 1 h prior to the
Alcohol Impairs Muscle Recovery from Exercise
PLOS ONE | www.plosone.org 2 February 2014 | Volume 9 | Issue 2 | e88384
exercise protocol. The exercise bout incorporated the concurrent
stimuli of resistance, continuous and intermittent high-intensity
exercise to represent the key features of team sport activities. The
specific protocol involved a standardized warm-up (5 repetitions at
50% and 5 repetitions at 60% 1RM) on a leg extension machine
before the resistance exercise protocol was commenced. Resistance
exercise consisted of eight sets of five repetitions at ,80% of 1RM.
Each set was separated by a 3-min recovery period during which
the subject remained seated. After completion of the final set,
subjects rested for 5 min before commencing 30 min of contin-
uous cycling at ,63% PPO (,70% VO
2peak
). Upon completion,
subjects rested on the bike for 2 min before undertaking 10630 s
high intensity intervals at ,110% of PPO, with 30 s active
recovery (,50% PPO) between each work bout.
Immediately following exercise and after 4 h recovery, subjects
ingested a 500 mL solution of either protein (PRO, 25 g whey
protein powder; ISO8, Musashi, Melbourne, VIC Australia) or an
energy-match in the form of CHO (25 g maltodextrin, Interna-
tional Health Investments, Helensvale, QLD Australia). Further-
more, a CHO-based meal (1.5 g?kg
21
BM) was consumed ,2h
post-exercise, immediately after the muscle biopsy, according to
recommendations for post-exercise glycogen recovery [24].
Protein beverages included L-(ring-
13
C
6
] phenylalanine at 4% to
prevent marked disturbance in isotopic enrichment and to
maintain steady state enrichment. Blood (,4 mL) was collected
immediately post-exercise and at regular intervals (30–60 min)
throughout an 8 h recovery period, with additional muscle
biopsies from separate incisions taken at 2 and 8 h post-exercise.
Samples were stored at 280uC until analysis. The 8 h time frame
represents an important phase of post-exercise recovery [1] as well
as the period during which blood alcohol concentrations are likely
to be elevated by a post-event drinking binge [14]. The alcohol
dose in the present study represented the mean intake of alcohol
reported by team athletes during a drinking binge [9,10] and an
amount previously investigated in relation to post-exercise
refuelling [14]. The alcohol ingestion protocol (1.5 g?kg
21
BM;
1262 standard drinks) began 1 h post-exercise and was consumed
in 6 equal volumes of 1 part vodka (,60 mL) to four parts orange
juice (,240 mL, 1.8 g CHO?kg
21
BM) during a 3 h period. For
the PRO condition, orange juice was consumed with a matched
volume of water in place of the alcohol. Subjects ingested the
beverages within 5 min every 30 min.
Analytical Procedures
Blood glucose and plasma ethanol
concentrations. Whole blood samples (,25 mL) were immedi-
ately analysed for glucose concentrations using an automated
analyser (YSI 2300, Yellow Springs, OH, USA). Blood samples
were then centrifuged at 3,000 gat 4uC for 10 min, with aliquots
of plasma frozen and stored at 280uC. On a separate occasion,
plasma samples (,25 mL) were thawed and analysed for ethanol
concentration using an automated analyser (YSI 2900, Yellow
Springs, OH, USA).
Plasma amino acids and enrichment. Plasma amino acid
concentrations and enrichments were analyzed by gas chroma-
tography-isotope ratio mass spectrometry (MAT252; Finnigan,
Breman, Germany) using EZ:faast kit (Phenomenex, CA, USA).
Rates of Myofibrillar Protein Synthesis. A single pre-
infusion plasma sample, extracted by acetonitrile, was utilized as
the baseline enrichment in tracer naı
¨ve subjects [23]. For the one
non-tracer naı
¨ve subject a pre-infusion muscle biopsy was used for
baseline enrichment. Muscle tissue was processed as previously
described [7].
Calculations. The fractional synthetic rate (FSR) of myofi-
brillar protein synthesis was calculated using the standard
precursor–product method:
FSR(%:h{1)~½(E2b{E1b)=(EIC|t)|100
Where E2
b
-E1
b
represents the change in the bound protein
enrichment between two biopsy samples; E
IC
is the average
enrichment of intracellular phenylalanine between the two biopsy
samples; and tis the time between two sequential biopsies. The
inclusion of ‘tracer-naive’ subjects permitted use of the pre-
infusion blood sample (i.e. single biopsy method) as the baseline
enrichment (E1
b
) for the calculation of resting MPS.
Western Blots. Intracellular signaling proteins were extract-
ed, isolated and quantified as previously described [25]. The
amount of protein loaded in each well was 50 mg. Polyclonal anti-
phospho mechanistic target of rapamycin (mTOR) Ser2448
(no. 2971), elongation factor 2 (eEF2) Thr56 (no. 2331), 4E-BP1
Thr37/46 (no. 2855), monoclonal anti- 59adenosine monophos-
phate-activated protein kinase (AMPK) aThr172 (no. 2535) and
p70S6K Thr389 (no. 9234) were from Cell Signalling Technology
(Danvers, USA). Data represent the volume and intensity
quantified via densitometry and phosphorylation data and are
expressed relative to a-tubulin reference protein expression at the
equivalent time point on the same membrane (no. 3873, Cell
Signalling Technology, Danvers, USA) in arbitrary units. All
samples for each subject were run on the same gel.
Real Time PCR. Skeletal muscle (,20 mg) tissue RNA
extraction, reverse transcription and real-time polymerase chain
reaction (RT-PCR) was performed as previously described [25].
TaqMan-FAM labeled primer/probes (Applied Biosystems, Carls-
bad, CA, USA) for muscle ring finger 1 (MuRF-1) (Cat
Figure 1. Schematic representation of the experimental trial. Subjects reported to the laboratory after an overnight fast where a constant
infusion of L-[ring-
13
C
6
] phenylalanine was commenced (3 h in first trial; 1 h in trial 2/3), and subjects completed the concurrent exercise (865
repetitions at 80% one repetition maximum (1RM), 5 min rest, 30 min cycling at ,63% peak power output (PPO), 2 min rest, 10630 s high intensity
intervals at ,110% PPO). Immediately after exercise completion, and 4 h later, subjects consumed 500-mL of protein (25 g whey) or carbohydrate
(25 g maltodextrin).
doi:10.1371/journal.pone.0088384.g001
Alcohol Impairs Muscle Recovery from Exercise
PLOS ONE | www.plosone.org 3 February 2014 | Volume 9 | Issue 2 | e88384
No. Hs00261590) and Atrogin (Cat No. Hs01041408) were used
in a final reaction volume of 20 mL. Glyceraldehyde- 3-phosphate
dehydrogenase (GAPDH, HS9999- 9905_m1) was used as the
housekeeping gene. The relative amounts of mRNAs were
calculated using the relative quantification (DDCT) method [26].
Statistical Analysis
Blood, cell signaling and mRNA data were analyzed by two-
way ANOVA (two factor: time 6treatment) with repeated
measures and myofibrillar protein synthesis was analyzed by one-
way ANOVA with repeated measures. All data underwent
Student-Newman-Keuls post hoc analysis when P,0.05 (Sigma-
Plot for Windows; Version 12.5). All data are expressed as mean 6
SD and the level of statistical significance was set at P,0.05.
Results
Blood Alcohol and Glucose Concentration
There were main effects for time and treatment for blood
alcohol concentration (P,0.05; Figure 2). Blood alcohol concen-
tration peaked 4 h post-exercise (ALC-CHO 0.0596
0.017 g?100 mL
21
; ALC-PRO 0.05660.019 g?100 mL
21
) and
remained elevated above rest throughout the 8 h recovery period
(ALC-CHO: 0.023–0.059 g?100 mL
21
; ALC-PRO: 0.029–
0.056 g?100 mL
21
;P,0.05). Blood alcohol concentration was
higher (P,0.05) with ALC-CHO compared with ALC-PRO at
6 h (ALC-CHO: 0.055 g?100 mL
21
; ALC-PRO: 0.047 g?
100 mL
21
) and 8 h (ALC-CHO: 0.043 g?100 mL
21
; ALC-
PRO: 0.033 g?100 mL
21
) post-exercise. Blood glucose concentra-
tion increased above all time-points at 0.5 h (,17–41%) and 4.5 h
(,16–40%) in the ALC-CHO treatment (P,0.05; Figure 3) but
was not different from resting in ALC-PRO and PRO treatments.
The blood glucose concentration measured in the ALC-CHO
treatment was also different from ALC-PRO (,27–41%) and
PRO (,26–42%) at 0.5, 1, 4.5 and 5 h post-exercise (P,0.05).
Plasma Amino Acids Concentration
There were main effects for time and treatment for plasma
EAA, BCAA and leucine concentrations (P,0.05; Figure 4).
Protein intake increased AA concentration at 1 h post-exercise:
AA concentrations for ALC-PRO (EAA ,109%, BCAA ,118%,
leucine ,203%) and PRO (EAA ,151%; BCAA ,170%; leucine
,274%) treatments were different to all other time-points within
treatments (P,0.05). Post-exercise concentrations of EAA and
leucine with PRO were elevated above resting values at 1 h
(,39%), 2 h (,98%) and 6 h (,61%) time-points, respectively
(P,0.05). Leucine concentration remained above resting values
2h(,90%) and 6 h (,102%) post-exercise, and EAA and BCAA
were also higher than rest after 6 h recovery (EAA ,77%; BCAA
,38%) in the ALC-PRO treatment (P,0.05). Compared to ALC-
CHO treatments, AA concentration were higher for ALC-PRO
and PRO at 1 h (ALC-PRO: ,115–305%, PRO: ,163–394%),
2h(,56–168%, ,83–179%) and 6 h (,81–253%, ,75–181%)
post-exercise time-points (P,0.05). There were no changes in AA
concentration in the ALC-CHO treatment.
Intracellular and Plasma Tracer Enrichments
Phenylalanine enrichments showed a stable precursor pool
throughout the infusion period in all groups (Figure S1). Linear
regression analysis indicated that the intracellular (mean r
2
= 0.08)
and plasma (mean r
2
= 0.03) enrichments in ALC-CHO, ALC-
PRO and PRO treatments demonstrated isotopic plateau.
Cell Signaling
mTOR-p70S6K. There were main effects for time and
treatment for mTOR
Ser2448
phosphorylation (P,0.05,
Figure 5A). mTOR phosphorylation increased above rest at 2 h
(P,0.05) for all treatments (ALC-CHO: ,125%, ALC-PRO:
,157%, PRO: ,297%) and at 8 h (P,0.05) for ALC-CHO
(,111%) and ALC-PRO (,127%). mTOR phosphorylation with
PRO was higher (P,0.05) than ALC-CHO (,76%) and ALC-
PRO (,54%) at 2 and 8 h post-exercise, and PRO at 8 h post-
exercise (,168%).
There were main effects for time and treatment for
p70S6K
Thr389
phosphorylation (P,0.05, Figure 5B). p70S6K
phosphorylation was greater at 2 h (P,0.05) compared to rest and
8 h post–exercise in ALC-PRO (,418–585%) and PRO only
(,438–468%). p70S6K phosphorylation was also higher at 2 h
Figure 2. Blood alcohol levels after alcohol intake during
recovery following a single bout of concurrent training. Data
were analysed using a 2-way ANOVA with repeated measures and
Student-Newman-Keuls post hoc analysis. Values are mean 6SD.
Significant effect of treatme nt (P = 0.02), time (P,0.01) with no
interaction (P = 0.20). Significantly different (P,0.05) vs. (a) rest, and
(*) between treatments (ALC-CHO vs. ALC-PRO).
doi:10.1371/journal.pone.0088384.g002
Figure 3. Blood glucose concentrations before and duringre-
covery following a single bout of concurrent training. Drink =
25 g of whey protein (PRO and ALC-PRO) or 25 g maltodextrin (ALC-
CHO); Meal = 1.5 g?kg
21
BM. Data were analysed using a 2-way ANOVA
with repeated measures and Student-Newman-Keuls post hoc analysis.
Values are mean 6SD. Significant effect of treatment, time and
interaction (all P,0.01). Significantly different (P,0.05) (d) from 1 h
within treatment, (j) from 5 h within treatment, ($) between treatments
(ALC-CHO vs. ALC-PRO, PRO). ({) between treatments (ALC-CHO vs.
PRO), (`) between treatments (ALC-CHO vs. ALC-PRO).
doi:10.1371/journal.pone.0088384.g003
Alcohol Impairs Muscle Recovery from Exercise
PLOS ONE | www.plosone.org 4 February 2014 | Volume 9 | Issue 2 | e88384
post-exercise in PRO (,276%) and ALC-PRO (,242%) treat-
ments compared to ALC-CHO treatment (P,0.05).
eEF2-4E-BP1-AMPK. There were decreases in eEF2 phos-
phorylation (Figure 5C) below rest at 2 h and 8 h post-exercise in
ALC-CHO (,66–74%; P,0.05) and ALC-PRO (,61–67%; P,
0.05). No changes in 4E-BP1
Thr37/46
(Figure 5D) or AMPK
Thr172
phosphorylation [data not shown] were observed across treat-
ments or times.
Atrogene mRNA expression
There were increases above rest in MuRF-1 mRNA (Figure 6A,
P,0.05) at 2 h for all treatments (ALC-CHO: ,404%; ALC-
PRO: ,399%; PRO: ,474%). However, there were no
differences between treatments, and MuRF1 mRNA returned to
resting levels under all conditions after 8 h (P,0.05). There was a
main effect for time for atrogin-1 abundance (P,0.05, Figure 6B).
Atrogin-1 mRNA expression at 8 h decreased below rest (,37–
52%; P,0.05) and 2 h (,46–61%; P,0.05) with all treatments.
Rates of muscle protein synthesis
Rates of myofibrillar FSR were increased above rest
(0.02560.002%?h
21
) with ALC-CHO (0.03260.005%?h
21
,
,29%), ALC-PRO (0.03960.008% h
21
,,57%) and PRO
(0.05260.008%?h
21
,,109%) treatments throughout 2–8 h of
recovery (P,0.05; Figure 7). However, compared to PRO alone,
there was a hierarchical reduction in myofibrillar FSR with ALC-
PRO (24%, P,0.05) and ALC-CHO (37%, P,0.05). ALC-CHO
resulted in a lower FSR compared to ALC-PRO (,18%, P,0.05).
Discussion
The first novel finding of this study was that mTOR signaling
and rates of myofibrillar protein synthesis (MPS) following
concurrent resistance, continuous and intermittent high-intensity
exercise, designed to mimic the metabolic profile of many team
sports, were impaired during the early (8 h) recovery phase by the
ingestion of large amounts (1.5 gNkg
21
BM) of alcohol. These
outcomes were most evident (37% reduction in rates of MPS)
when alcohol was consumed in the absence of post-exercise
protein intake, as is likely to occur when intoxication reduces the
athlete’s compliance to sound recovery practices. However, a
second finding was that even when protein was consumed in
amounts shown to be optimally effective to stimulate MPS [8]
during post-exercise recovery, the intake of alcohol reduced MPS
by ,24%, representing only a partial ‘rescue’ of the anabolic
response compared with protein alone.
The alcohol consumption protocol used in the current study,
representing the mean intake of alcohol that has been self-reported
in several studies of binge drinking practices of team athletes
[9,10], elicited blood alcohol concentrations that exceeded the
0.05 g?100 mL
21
legal limit for driving in Australia (Figure 1).
Although peak post-exercise blood alcohol values were lower than
we have previously reported [14], such differences can, in part, be
explained by different alcohol ingestion protocols and feeding
regimens. The subtle differences in blood alcohol concentration
were likely a result of the different macronutrient composition
consumed and the aminoacidemia in PRO and ALC-PRO was
similar and significantly different to that seen with the carbohy-
drate treatment (Figure 4).
Despite alcohol having little effect on blood amino acid profiles,
myofibrillar FSR was significantly different between treatments
(Figure 7). The maximal FSR was measured when protein was the
only nutrient ingested, and is similar to other studies incorporating
resistance-type exercise with protein feeding [25,27]. However,
this study is the first to have measured FSR after consecutive bouts
of resistance, continuous and high-intensity exercise when alcohol
was consumed during recovery. While several studies examining
the effects of alcohol intake have been undertaken in rodents, the
relative quantity of alcohol administered in these investigations is
several fold higher than in the current human study [18,21,28–
30]. Furthermore, there are differences in techniques used to
measure rates of protein synthesis in animals versus humans.
Notwithstanding these differences, Lang et al. [28] reported a 25%
decrease in rates of muscle protein synthesis with alcohol
administration in rodents, a value in close agreement with the
current study. Our results show alcohol ingestion in humans
suppresses the elevated rates of protein synthesis in skeletal muscle
induced by exercise and protein ingestion.
Figure 4. Plasma EAA (A), BCAA (B), leucine (C) concentration
following a single bout of concurrent training. EAA – essential
amino acids; BCAA – branched-chain amino acids. Data were analysed
using a 2-way ANOVA with repeated measures and Student-Newman-
Keuls post hoc analysis. Values are mean 6SD. Significant effect of
treatment, time and interaction (all P,0.01) for (A), (B), and (C).
Significantly different (P,0.05) vs. (#) all timepoints for ALC-CHO and
ALC-PRO treatments, (*) vs. rest within treatments, and ($) compared to
ALC-CHO.
doi:10.1371/journal.pone.0088384.g004
Alcohol Impairs Muscle Recovery from Exercise
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The mechanistic target of rapamycin complex 1 (mTORC1) is a
central node for integrating nutrient (i.e. amino acid) and exercise/
contraction signal transduction [31,32]. Post-exercise phosphory-
lation of mTOR
Ser2448
was attenuated when alcohol was co-
ingested with either carbohydrate or protein compared to protein
ingestion alone. Interestingly, there was discordance in phosphor-
ylation responses between mTOR and its downstream signaling
targets (p70S6K and 4E-BP1). The mechanism through which
alcohol may attenuate mTOR complex 1 activity is still poorly
defined. Recent evidence has implicated several upstream
regulatory mechanisms of mTOR signaling including the Rag
family of GTPases [33,34], phosphatidic acid [35] and the DNA
damage response 2 (REDD2) protein [36]. The inhibitory effects
of alcohol on mTOR phosphorylation in skeletal muscle have
been attributed to increases in the mRNA/protein content of the
negative mTOR regulator REDD1 with acute intoxication and
that alcohol may also generate greater association of mTOR with
raptor to down regulate mRNA translation [20,37]. Thus it is
plausible that several mechanisms may act synergistically upstream
of mTOR in response to alcohol ingestion to modulate mTOR
activity. Nevertheless, our findings indicate that the observed
alcohol-induced attenuation of MPS was likely mediated, at least
in part, by effects on mTORC1-mediated signaling.
p70S6K enhances translation of mRNAs encoding ribosomal
proteins and elongation factors [38] and has been proposed as a
‘surrogate’ marker associated with rates of muscle protein synthesis
[39–42]. Lang and co-workers have previously shown reduced
p70S6K signaling following alcohol ingestion in rat skeletal muscle
[18,29]. We present new information in human skeletal muscle to
demonstrate the exercise and nutrient-induced increase in
Figure 5. mTOR
Ser2448
(A), p70S6K
Thr389
(B), eEF2
Thr56
(C), 4E-BP1
Thr37/46
(D) phosphorylation at rest and following a single bout of
concurrent training. Images are representative blots for each protein from the same subject and values are expressed relative to a-tubulin and
presented in arbitary units. Data were analysed using a 2-way ANOVA with repeated measures with Student-Newman-Keuls post hoc analysis. Values
are mean 6SD. Significant effect of time (P,0.01) and interaction (P = 0.02) but not treatment (P = 0.22) for (A); time (P,0.01) and interaction
(P = 0.02) but not treatment (P= 0.46) for (B); time (P,0.01) but not treatment (P = 0.14) or interaction (P = 0.56) for (C); no treatment (P = 0.86), time
(P = 0.24), or interaction (P = 0.77) effects for (D). Significantly different (P,0.05) vs. (a) rest, (e) ACL-PRO 8 h, (f) PRO 2 h, (g) PRO 8 h, and (*) 2 h
between treatments.
doi:10.1371/journal.pone.0088384.g005
Alcohol Impairs Muscle Recovery from Exercise
PLOS ONE | www.plosone.org 6 February 2014 | Volume 9 | Issue 2 | e88384
p70S6K phosphorylation is significantly reduced with alcohol
ingestion in the absence of the co-ingestion of protein. The
discordant mTOR-p70S6K phosphorylation with protein only
and protein feedings with alcohol is not unprecedented given we
[8] and others [43] have shown that mTOR-S6K phosphorylation
often parallels changes in MPS but does not always reflect either
the magnitude or duration of the increased MPS signal in humans.
An alternate mechanism through which alcohol may limit rates of
protein synthesis is endoplasmic reticulum stress and the resultant
unfolded protein response. Alcohol consumption generates oxida-
tive stress and inflammation and the potential to disrupt
endoplasmic reticulum homeostasis; a consequence of this
response is to limit the rate of protein synthesis [44,45]. The lack
of change in 4E-BP1
Thr37/46
phosphorylation following exercise
and between treatments contrasts previous findings in rodents
[18,20,28]. However, these differences may, in part, be explained
by the 2.3 fold greater relative alcohol administration in rodents
versus humans. Finally, it must be acknowledged that our data are
potentially limited by providing only a single ‘snapshot’ during
recovery and the possibility exists that our muscle biopsy time-
points failed to coincide with peak phosphorylation responses of
signal transduction. To the best of our knowledge, this is the first
Figure 6. MuRF-1 (A), Atrogin-1 (B) mRNA abundance at rest and following a single bout of concurrent training. Values are expressed
relative to GAPDH and presented in arbitrary units (mean 6SD, n = 7). Data were analysed using a 2-way repeated measures ANOVA with Student-
Newman-Keuls post hoc analysis. Significantly different (P,0.05) vs. (a) rest, (c,e,g) 8 h within treatments, and (b,d,f) 2 h within treatments.
doi:10.1371/journal.pone.0088384.g006
Alcohol Impairs Muscle Recovery from Exercise
PLOS ONE | www.plosone.org 7 February 2014 | Volume 9 | Issue 2 | e88384
study to investigate the effect of alcohol ingestion following
concurrent resistance, continuous and intermittent high-intensity
exercise in human skeletal and further studies are needed to better
understand the precise mechanisms through which alcohol
attenuates human skeletal muscle protein synthesis.
In contrast with the changes in cell signaling, muscle mRNA
responses of selected genes associated with muscle proteolysis and
catabolism were largely unchanged between treatments. MuRF-1
mRNA expression was elevated 2 h following exercise but had
returned to basal levels, by 8 h in all treatments. Whereas, atrogin-
1 mRNA expression did not change 2 h following exercise and was
significantly lower than rest and 2 h post-exercise at 8 h post-
exercise in all treatments. These results contrast findings by Vary
and colleagues [30] who found alcohol ingestion to increase
MuRF-1 and Atrogin-1 mRNA abundance in rat skeletal muscle.
Our data shows protein co-ingested with alcohol following exercise
induces comparable increases in atrogene mRNA expression
compared to protein ingestion alone in human skeletal muscle.
These increases are in agreement with previous findings demon-
strating increased atrogene mRNA expression following resistance
exercise [46,47]. Although we did not determine rates of muscle
protein breakdown, this process is up-regulated in mixed muscle
for up to 24 h after resistance exercise in the fasted state [48]. As
muscle damaging exercise has previously been reported to
decrease GLUT4 translocation and subsequent rates of muscle
glycogen resynthesis [49], the possibility that it also may impart a
negative effect on protein transporters and rates of protein
synthesis cannot be discounted. However, the atrogene results of
the current study indicate alcohol ingestion does not exert any
additional effects on ubiquitin ligase expression after exercise in
human skeletal muscle. Future studies investigating the time course
of atrogene expression and direct measures of skeletal muscle
proteasome activity and/or protein breakdown following alcohol
ingestion in humans are warranted.
In conclusion, the current data provide the novel observation
that alcohol impairs the response of MPS in exercise recovery in
human skeletal muscle despite optimal nutrient provision. The
quantity of alcohol consumed in the current study was based on
amounts reported during binge drinking by athletes. However,
published reports suggest intakes of some individuals can be
significantly greater [9,50], which is of concern for many reasons
related to health and safety [13]. Regrettably, there has been
difficulty in finding an educational message with alcohol
consumption related to sports performance that has resonance
with athletes. Given the need to promote protein synthesis that
underpins adaptation, repair and regeneration of skeletal muscle
the results of the current study provide clear evidence of impaired
recovery when alcohol is consumed after concurrent (resistance,
continuous and intermittent high-intensity) exercise even in the
presence of optimal nutritional conditions. We propose our data is
of paramount interest to athletes and coaches. Our findings
provide an evidence-base for a message of moderation in alcohol
intake to promote recovery after exercise with the potential to alter
current sports culture and athlete practices.
Supporting Information
Figure S1 Tracer enrichment of the muscle intra-
cellular protein pool (A) and blood plasma (B) following
a single bout of concurrent training.
(TIF)
Acknowledgments
The authors wish to thank Mr. Stephen Lane, Mr. Joshua Whittaker, Mr.
Jong-Sam Lee, Mr. William Manly, and Ms. Eliza Leverett for assistance
with experimental trials; Ms. Tracey Rerecich and Mr. Todd Prior for
their technical expertise; and the subjects for their time and effort.
Author Contributions
Conceived and designed the experiments: EBP LMB SMP JAH VGC.
Performed the experiments: EBP DMC JLA VGC. Analyzed the data:
EBP DMC JLA SMP JAH VGC. Contributed reagents/materials/analysis
tools: LMB SMP JAH VGC. Wrote the paper: EBP DMC JLA LMB SMP
JAH VGC.
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Alcohol Impairs Muscle Recovery from Exercise
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