Excessive alcohol (ethanol) ingestion is a major health risk and can lead to detrimental effects on most physiological systems in the body. Individuals who engage in regular physical activity have higher rates of alcohol use than sedentary individuals (25); this association also exists between engagement in vigorous physical activity and alcohol use (14). Furthermore, college athletes are at a greater risk for binge drinking than nonathletes (13) and over 54 percent report drinking alcohol during both their competitive and off seasons with approximately 49 percent drinking 5 or more drinks in 1 sitting (i.e., binging) (26). Although it is common for athletes to consume alcohol directly after training or events, almost 60% believe that their use of alcohol does not affect performance or overall health (26). However, postexercise alcohol ingestion negatively influences recovery from muscle damage and accentuates loss of dynamic and static strength (5,6). The underlying mechanism for this negative effect of alcohol on recovery has not been elucidated but research on alcohol ingestion in the absence of exercise suggests that reduced protein synthesis might be involved.
Protein synthesis provides the basis for adaptations (e.g., muscle growth and recovery) to resistance exercise (RE) training. Regulation of protein synthesis and subsequent muscle growth is complex, multifaceted, and involves several dependent and independent factors. Among these factors is the mammalian (or mechanistic) target of rapamycin (mTOR), a protein Ser-Thr kinase that resides within a multiprotein complex called mTOR complex 1 (mTORC1). The importance of mTORC1 signaling was demonstrated by Bodine et al. (7) who found that selectively blocking mTOR using rapamycin prevented contraction-induced muscle growth in rats. Thus, it seems that mTORC1 mediates an essential, although not the only, pathway for regulating skeletal muscle protein synthesis in response to RE (7). When activated, mTOR mediates phosphorylation of its downstream targets S6K1 and 4E-BP1, enhancing the translational efficiency of ribosomes, thus increasing protein synthesis (15). S6K1 is responsible for phosphorylation of the S6 subunit of the 40S ribosomal protein (29) resulting in stimulation of mRNA translation initiation. In this signaling pathway, mTORC1 also influences protein synthesis by removing the inhibitory effect of the translational regulator 4E-BP1. When 4E-BP1 is phosphorylated, it unbinds from eukaryotic translation initiation factor 4E (eIF4E), resulting in the formation of the translation initiation complex eIF4F (18), allowing for greater translation initiation.
The mechanical deformation of muscle fibers from RE is a potent stimulator for activation (phosphorylation) of the mTORC1 pathway (28). A direct relationship between increased mTORC1 signaling and RE has been shown in human and animal studies. Barr and Esser (3) conducted one of the first studies to find evidence of the role of muscle contraction in activating mTORC1 signaling. In that study, rats were attached with stimulating electrodes to produce high-resistance contractions, and it was found that S6K1 phosphorylation was increased at 3 and 6 hours postcontractions. Similarly, Atherton et al. (1) found that mTOR and S6K1 phosphorylation was increased in rats 3 hours after high-frequency stimulations that were designed to mimic RE. In men and women, it has been found that mTOR phosphorylation is increased at 1 and 2 hours post RE, and S6K1 is elevated at 2 hours post RE (11,12). The effect of RE on 4E-BP1 phosphorylation is less clear, as decreases, no changes, and increases in phosphorylation have been found during the first hour after RE (9,11). In summary, there is good evidence that muscle contractions increase mTOR, S6K1, and 4E-BP1 phosphorylation, which stimulates a central pathway for increased protein synthesis that is important for adaptations to RE training.
As with RE, alcohol affects the signal transduction of the mTORC1 pathway. In a series of studies, Lang et al. (21–24) investigated the effects of acute alcohol ingestion on mTORC1 signaling in rodents. Collectively, these studies showed that, at rest, alcohol ingestion induced a decrease in protein synthesis and a reduction in mTORC1 pathway activation, including reduced mTOR, S6K1, and 4E-BP1 phosphorylation. A reduction in mTORC1 pathway activation reduces ribosomal translation initiation which could, at least in part, explain the reduced protein synthesis found after alcohol ingestion. Only a single study (27) seems to have investigated the effects of acute alcohol ingestion on mTORC1 signaling in the context of exercise in humans. In that study, Parr et al. (27) found that ingestion of alcohol-attenuated protein synthesis and mTOR phosphorylation, but not S6K1 phosphorylation, from the collective stimulatory effects of combined endurance and RE and protein supplementation in men. The effect of acute alcohol ingestion on mTORC1 signaling has not been investigated with heavy RE or with women.
The activity of the mTORC1 signaling pathway is important for RE-induced protein synthesis and subsequent hypertrophy. Independently, RE and alcohol have opposite effects on mTORC1 signaling. Thus, knowledge of how alcohol modulates the RE-induced activation state of key components of the mTORC1 signaling cascade is important to better understand the potential negative implications of acute alcohol ingestion after heavy RE. To date, no study has examined the mTORC1 signaling in this context; therefore, the purpose of this study was to investigate the effect of alcohol ingestion on RE-induced mTORC1 signaling in men and women.
Experimental Approach to the Problem
To examine the acute effect of alcohol ingestion after RE on mTOR, S6K1, and 4E-BP1 activation (phosphorylation), a within subjects repeated-measures design was used. Ten resistance-trained males and 9 resistance-trained females completed 2 identical acute heavy RE trials (6 sets of Smith machine squats) (AHRET) as previously described (30). The AHRET sessions were separated by approximately 28 days (to allow for standardization of menstrual phase for the women). From 10 to 20 minutes, postexercise participants consumed either alcohol (alcohol condition) or no alcohol (placebo condition) diluted in an artificially sweetened and calorie-free beverage. All participants completed both conditions; conditions were assigned using randomization and were counter-balanced within each sex. Before exercise (PRE) and 3 hours (+3 hours) and 5 hours (+5 hours) hours postexercise, muscle tissue samples were obtained from the vastus lateralis by biopsies. Muscle samples were analyzed for phosphorylated mTOR, S6K1, and 4E-BP1.
Ten males (mean ± SD: 21–29 years, 83.8 ± 15.7 kg, 177 ± 7 cm, 14.8 ± 8.5% body fat) and 9 females (21–26 years, 60.1 ± 6.0 kg, 161 ± 4 cm, 26.8 ± 2.9% body fat) who were recreationally resistance trained participated in this study. To be considered recreationally resistance trained, the participants had to have completed at least 2 RE sessions per week that included the back squat for the previous 6 months. Participants were screened for relevant exclusion criteria, including: medical concerns, musculoskeletal problems, use of hormonal substances or medications such as androgenic-anabolic steroids, growth hormone, or glucocorticoids, or adherence to atypical diets that could confound the results of this study. In addition, female participants had to be eumenorrheic and not trying to become pregnant. To be included in the study, participants had to be 21–34 years of age and low-moderate consumers of alcohol. Participants completed the “Young Adult Alcohol Problems Screening Test” (17) and the “Alcohol Use Disorders Identification Test” (4)—written questionnaires on current and past alcohol use to screen for signs of alcohol abuse. Finally, to be included, participants could not have ingestion-induced metabolic intolerance nor be naive to alcohol ingestion. The study was approved by the University Institutional Review Board and the participants provided written informed consent to participate; all procedures were conducted in accordance with the Declaration of Helsinki.
Session 1: Anthropometric Measurements, Familiarization, and 1 Repetition Maximum
Approximately 1 week before the first exercise and alcohol/placebo session, participants reported to the laboratory for anthropometric measurements, familiarization, and 1 repetition maximum (1RM) determination. Body composition was measured using dual-energy X-ray absorptiometry (Lunar Prodigy; GE Healthcare, Fairfield, CT, USA). Participants wore only light athletic clothes and no shoes or metals for measurements of height, body weight, and body composition. Participants then performed a standardized warm-up consisting of light dynamic stretches (heel kicks, lunges, high knees, high kicks), and unweighted body squats. After the warm-up, participants were familiarized with the proper technique for performing the squat exercise using a Smith machine. The Smith machine allows only vertical translation of the bar. Linear bearings attached to either side of the bar allow it to slide up and down 2 steel shafts with minimal friction. Once participants had demonstrated proper technique in the Smith machine squat exercise, their 1RM strength was measured (19). Briefly, participants performed squats for 8–10 repetitions at ∼50% of their estimated 1RM followed by another set of 2–5 repetitions at ∼85% of estimated 1RM. Subsequently, 4–5 one repetition trials were used to determine the 1RM.
Sessions 2 and 3: Experimental Treatments
Each participant completed both experimental treatment conditions (alcohol and placebo) and thereby served as his/her own control. To account for hormonal variations during the menstrual cycle, women were “phased” so that they completed the treatment sessions during their early follicular phase (days 2–7 after the start of menses). Therefore, the treatments were performed approximately 28 days apart and administered in a balanced, randomized, crossover design. To prevent participants from anticipating treatment conditions, participants were blinded to the drink conditions and informed that they could potentially receive the same treatment on both treatment sessions. Participants were asked to record their dietary intake for the morning of the first treatment session (alcohol or placebo session) and to replicate that diet before the second treatment session.
On arrival to treatment sessions, participants confirmed that they had complied with the following presession instructions; refrain from: (a) eating or drinking anything (except for water) during the 2 hours leading up to the treatment session, (b) ingesting any alcohol for 84 hours before each treatment session, (c) consuming large amounts of caffeine (no more than 1 cup of coffee or equivalent allowed on the morning of the treatment session), (d) performing any RE or intense aerobic exercise for 96 hours before the session, and (e) donating blood within 8 weeks or donating plasma within 96 hours of the session. All sessions began at the same time of day for each participant (1,000–1,200 hours arrival time for all participants) to avoid confounding circadian influences.
Figure 1 contains an overview of the experimental treatment session timeline. On arrival at the laboratory on each treatment day, participants were screened for the presence of blood alcohol with a breathalyzer and were queried regarding compliance with study guidelines regarding diet, caffeine, alcohol, drugs, and exercise. Participants were asked to report to the laboratory in a euhydrated state. On arrival at the laboratory, hydration status was measured using urine refractometry; if participants presented with urine specific gravity ≥1.020 they were provided water to drink. After the hydration status was addressed, body weight was measured. Sixty-five minutes before performing the AHRET, participants consumed a standardized meal replacement drink (Ensure Plus; Abbott Laboratories, Abbott Park, IL, USA) containing 8 kcal per kg body mass.
Fifteen minutes before performing the AHRET (50 minutes after the meal), participants completed the standardized warm-up described above. Participants then completed 10 warm-up squats at 50% of 1RM followed by the AHRET. Briefly, the AHRET consisted of 6 sets of 10 repetitions of Smith machine squats starting at 80% of 1RM with 2 minutes of rest between sets. If participants were not able to complete 10 repetitions on their own, they were assisted by the researchers to do so and then the load was reduced for subsequent sets. The same load for each set was used for both treatment sessions to standardize total work performed in each session. After completion of the AHRET, participants rested in a comfortable reclining chair for the remainder of the session (next 5 hours) and were not allowed to sleep. Participants were permitted to read, write, or use a laptop/tablet during the rest period.
From 10 to 20 minutes post-AHRET, participants consumed either alcohol (alcohol condition) or no alcohol (placebo condition) in an artificially sweetened and calorie-free beverage, similar to procedures previously described (30). For the alcohol condition, participants ingested vodka (40% vol/vol alcohol; Smirnoff Co., Norwalk, CT, USA) diluted in water to a concentration of 15% vol/vol absolute alcohol to achieve a dose 1.09 g of alcohol per kg of fat-free body mass. The placebo condition substituted alcohol for an equal volume of water. The participants consumed one-tenth of the drink each minute during the 10-minute ingestion period. To reduce the ability of the participants to determine whether they were receiving the alcohol or placebo drink, the rim of the glass was smeared with a small amount of alcohol for both conditions. No adverse effects of the alcohol ingestion protocol were observed.
Muscle samples were obtained at PRE and 3 and 5 hours postexercise by biopsies. Three separate sites 3 cm apart on the same thigh (vastus lateralis) were used to obtain the 3 biopsies during a treatment session; the opposite thigh was used for the other session. Three separate sites were used to avoid confounding influences of local immune/inflammatory responses. Order of thigh biopsy was assigned in a randomized, crossover design. Each muscle sample was obtained from the superficial portion of the vastus lateralis using microbiopsy procedures. Briefly, the skin over the muscle was cleaned using betadine, and a local anesthetic (1% lidocaine w/o epinephrine) was injected under the skin and into the muscle. Then, a spring-driven Pro-Mag 14 gauge microbiopsy needle (Angiotech, Gainesville, FL, USA) was introduced past the fascia and the trigger mechanism was engaged. This advanced an inner cannula 22 mm to cut the tissue. After the needle was withdrawn, the needle puncture site was covered with sterile gauze and compression was applied to prevent bleeding. The site was then closed with a small adhesive bandage (i.e., Band-Aid); no suture was used. Muscle samples were immediately flash frozen in liquid nitrogen and stored at −80° C until later analysis. The biopsy procedure was well tolerated by the participants. One participant complained of local swelling and pain after the first biopsy procedure session. The participant was examined by our physician who did not find any medical concerns; the participant made a full recovery but was withdrawn from the study.
Muscle samples were homogenized after procedures similar to previously described (31). Briefly, muscle samples were homogenized on ice in a buffer (Tissue Extraction Reagent I; Invitrogen, Carlsbad, CA, USA) containing a protease and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) (10 μl buffer per mg muscle) using a mini glass tissue grinder (Kontes, Vineland, NJ, USA). The homogenate was gently mixed for 15 minutes at 4° C and subsequently centrifuged at 10,000g for 15 minutes at 4° C. The resultant supernatant was divided into several aliquots, flash frozen in liquid nitrogen, and stored at −80° C until Western blot analysis. Total protein concentration in the supernatant was determined immediately after muscle homogenization using the Direct Detect automated protein analyzer (Millipore, Billerica, MA, USA) according to manufacturer's instructions.
Supernatant containing 80 μg of protein was heated (95° C) for 5 minutes with an equal volume of loading buffer (BioRad, Hercules, CA, USA) and subsequently loaded into a precast 4–20% tris-glycine gel (BioRad). A molecular size standard (ladder) (BioRad) was loaded on the outside lane on either side of the gel. The protein was then separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the Mini-PROTEAN system (BioRad) at 200 V for 45 minutes at room temperature. All samples for each participant's biopsies were loaded onto the same gel and duplicate gels were used. After electrophoresis, samples were electrophoretically transferred onto a polyvinylidene difluoride membrane (BioRad) in a Mini Trans-Blot cell (BioRad) using 70 V for 2 hours at 4° C. Using the molecular weight standards, the membrane was cut into 3 pieces, with each piece containing one of the 3 targets of interest (mTOR, S6K1, and 4E-BP1) and 1 piece also containing the normalizer α-tubulin. The membrane pieces was blocked in milk (1 hour, 5% dry milk) and then incubated (overnight at 4° C, 5% BSA) with the appropriate rabbit antibodies (Cell Signaling Technology, Danvers, MA, USA) for phosphorylated mTOR at Ser2448 (#5536), S6K1 at Thr389 (#9234), and 4E-BP1 at Thr37/Thr46 (#2855) and subsequently incubated with an antirabbit horseradish peroxidase–linked secondary antibody (#7074; Cell Signaling Technology) (1 hour, 5% BSA). The membrane was then visualized using enhanced chemiluminescence and a C-Digit Blot Scanner (Li-Cor, Lincoln, NE, USA). The membrane piece containing α-tubulin was stripped of antibodies using Western ReProbe (G-Bioscience, St. Louis, MO, USA) and then probed for α-tubulin (#3873; Cell Signaling) using the immunoblotting procedure described above. Phosphorylated protein content was expressed relative to α-tubulin content (arbitrary units) for each particular sample and averaged across the duplicate membranes. The mean %CV between duplicates was 9.0%.
Although participants included both sexes, this study was not designed specifically to compare responses of men and women, and it was not powered, a priori, to do so. Therefore, data for each variable were analyzed separately for each sex using a 2-way analysis of variance (treatment × time point) with repeated measures on both factors (SPSS Statistics version 20; IBM, Chicago, IL, USA). The data meet assumptions for parametric statistics. Fisher's least significant difference post hoc test was used to determine pairwise differences. The alpha-level of significance was set at p ≤ 0.05 and data are displayed as mean ± SE unless otherwise indicated.
For men, there was a significant interaction (treatment × time point) effect for mTOR phosphorylation (F(2,18) = 6.713, p = 0.007). At +3 hours, mTOR phosphorylation was higher for placebo than for alcohol. For placebo, mTORC1 phosphorylation was significantly higher at +3 hours than at PRE. For women, there was a significant main effect for time (F(2,16) = 5.685, p = 0.014). mTOR phosphorylation was higher at +3 hours than at PRE and at +5 hours.
For men, there was a significant interaction (treatment × time point) effect for S6K1 phosphorylation (F(2,18) = 3.756, p = 0.043). For placebo, S6K1 phosphorylation was significantly higher at +3 hours compared with PRE and +5 hours. For women, a trend (p = 0.052) for time was found.
There were no significant effects found for 4E-BP1 phosphorylation in men or women. For men, a trend (p = 0.064) for an interaction (treatment × time point) effect was found (Figure 2).
The major findings of this study were that alcohol ingestion affected RE-mediated phosphorylation of the mTORC1 pathway in men but not in women. Considering the high rate of alcohol usage among athletes and other individuals who engage in regular recreational physical activity, the findings from this study provide important physiological insight regarding alcohol's effect on processes involved in protein synthesis during recovery from RE. The importance of mTORC1 signaling for muscle contraction–induced muscle growth (i.e., training-induced hypertrophy) was shown in an early study conducted by Bodine et al. (7). After rapamycin treatment to selectively block mTOR signaling, rat muscles were overloaded and compared with overloaded non–rapamycin-treated muscle. In the rapamycin-treated rats, mTORC1 signaling was decreased and muscle hypertrophy was blocked, with no atrophy in rapamycin-treated controls (7). These findings provide evidence that the increase of mTORC1 signaling is crucial to RE-induced skeletal muscle growth. The mTORC1 signaling pathway mediates protein synthesis in skeletal muscle (7) and RE increases phosphorylation of mTOR and its downstream targets (8). In contrast, alcohol ingestion (in the absence of exercise) reduces mTORC1 signaling and protein synthesis (21–23) but the collective effects of RE and alcohol has not been elucidated. It has previously been found that ingestion of alcohol attenuated protein synthesis and mTOR phosphorylation, but not S6K1 phosphorylation, from the collective stimulatory effects of combined endurance and RE and protein supplementation in men. To our knowledge, this is the first study to investigate the effects of postheavy RE alcohol ingestion on mTORC1 signaling in men and women.
mTOR is a protein Ser-Thr kinase that plays key roles in signaling pathways for cell growth and proliferation through its effects on ribosomal translation (10,16). A novel finding of this study was that alcohol ingestion prevented a RE-induced increase in mTOR phosphorylation at +3 hours postexercise in men. As expected, based on previous findings for men (11,12), for the placebo condition, mTOR phosphorylation was increased during the recovery phase (i.e., +3 hours). An interference of alcohol with mTOR phosphorylation from RE was also expected because, in the absence of exercise, acute alcohol ingestion reduces mTOR phosphorylation in male rats (21,24). The alcohol-induced reduction of protein synthesis signal transduction seems to be independent of the kinase directly upstream of mTOR. Phosphorylation of AKT, which leads to activation of mTOR (7), is not affected by alcohol treatment in male rats, whereas at the same time, mTOR phosphorylation is suppressed by the alcohol treatment (20). Therefore, it is important to note, that while alcohol ingestion in this study prevented the RE-induced increase of mTOR phosphorylation, the RE bout might have prevented an alcohol-induced decrease in mTOR phosphorylation. The findings of this study are similar to those of Parr et al. (27), who recently found that alcohol ingestion attenuated mTOR phosphorylation at 2 hours after combined endurance and RE and protein ingestion in men (27); however, in that study mTOR phosphorylation at 2 hours was increased compared with baseline in the alcohol condition. Thus, it seems that the ingestion of alcohol prevents or lessens the RE-induced increase in mTOR phosphorylation in men. This effect could have important implications for the signal transduction of the mTORC1 pathway and thus initiation of ribosomal translation and could, at least in part, help explain the reduced protein synthesis previously found with acute alcohol ingestion.
This study presents, for the first time, data on the effect of alcohol on exercise-induced mTOR phosphorylation in women. In women, mTOR phosphorylation was increased at +3 hours, but in contrast to the findings for men, mTOR phosphorylation was not affected by alcohol ingestion. Although the effect of alcohol ingestion on mTOR has not been previously investigated in women, the lack of an effect of alcohol ingestion on RE-induced mTOR phosphorylation in this study was a surprising finding. Acute alcohol ingestion has been found to reduce signaling downstream of mTOR (e.g., phosphorylation of ribosomal protein S6 and 4E-BP1) in female rats (22). Although only observed with chronic alcohol feeding, a sexual dimorphism in signaling downstream of mTOR seems to exist; female rats did not exhibit the decrease in signaling found in their male counterparts (22). Thus, alcohol ingestion immediately after heavy RE seems to have a greater influence on mTOR phosphorylation in men than in women.
S6K1, a downstream target of mTOR, mediates protein synthesis as a regulator of the translation of mRNAs encoding ribosomal proteins (2). The kinase activity of S6K1 is an important contributor to translation initiation, an essential rate-limiting step in protein synthesis (3). In this study, alcohol prevented a RE-induced increase in S6K1 phosphorylation at +3 hours in men. Resistance exercise is a potent stimulus for S6K1 phosphorylation in men (7,11,12). Thus, the increase found in the placebo condition is in agreement with previous findings. As hypothesized, alcohol ingestion impeded RE-induced S6K1 phosphorylation. Alcohol treatment, in the absence of exercise, has previously been found to reduce S6K1 phosphorylation in male rats (21,24). As in this study, these previous findings of impaired S6K1 phosphorylation with alcohol ingestion were associated with reduced mTOR phosphorylation. Because mTOR is an upstream regulator of S6K1 phosphorylation, the observed effect of alcohol on S6K1 phosphorylation in men might simply be due to reduced activation by mTOR. As with mTOR, it is important to note, that while alcohol ingestion in this study prevented the RE-induced increase of S6K1 phosphorylation, the RE bout might have prevented an alcohol-induced decrease in S6K1 phosphorylation.
The present findings for S6K1, in men, are in contrast to those of Parr et al. (27) who reported no inhibitory effect of alcohol on S6K1 phosphorylation from combined endurance and RE and protein ingestion in men. The amino acid leucine is a potent stimulator of mTOR. During recovery from exercise, Parr et al. (27) fed participants 2 large boluses of whey protein, which is rich in leucine; thus the stimulatory effect of leucine on S6K1 phosphorylation might have overwhelmed any negative effects of alcohol. This notion is supported by the lack of an increase in S6K1 phosphorylation from combined endurance and RE when carbohydrate and alcohol was ingested instead of protein and alcohol (27). In this study, for women, a trend (p = 0.052) for a main effect for time was found for increased S6K1 phosphorylation at +3 hours, but no effect of alcohol was evident. Previously, Dreyer et al. (11,12) have found that RE induced an increase in S6K1 phosphorylation in women at 2 hours postexercise. This increase was accompanied by an increase in mTOR phosphorylation at 1 and 2 hours postexercise. Thus, as for men, changes in S6K1 phosphorylation in women could simply due to an increase in phosphorylation of its upstream regulator mTOR. Regardless, alcohol ingestion in women did not seem to affect S6K1 phosphorylation.
When phosphorylated by mTOR, 4E-BP1 releases from eIF4E allowing for greater translation initiation. Despite the increases observed in 4E-BP1's upstream activator mTOR, there were no change from baseline found for 4E-BP1 phosphorylation at +3 hours or +5 hours for men or women, regardless of drink condition. Consistent with these findings, previous studies have observed no RE-induced changes in 4E-BP1 phosphorylation at 1 and 2 hours after exercise in men and women (11,12). In contrast to the lack of an effect observed with RE, acute alcohol ingestion independently reduces 4E-BP1 phosphorylation (21,22) in the hours after ingestion in males and females, an effect that could be attributed to the reduced mTOR activation caused by alcohol. Similar to this study, Parr et al. (27) found no change in 4E-BP1 phosphorylation after combined endurance and RE with and without alcohol ingestion in men. Although RE does not have a stimulatory effect on 4E-BP1 activation after exercise, RE might be able to prevent an alcohol-induced reduction in 4E-BP1 activation.
This study seems to be the first to investigate mTORC1 signaling at both +3 hours and +5 hours after RE. In the absence of alcohol ingestion (i.e., placebo condition), for men in this study, RE increased mTOR and S6K1 phosphorylation at +3 hours postexercise compared with PRE and that by +5 hours phosphorylation had returned to baseline levels; there was no change in 4E-BP1 phosphorylation across time points. In women, the same pattern was found for mTOR and 4E-BP1 but not S6K1 phosphorylation; as only a statistical trend (main effect of time: p = 0.052) for an increase in S6K1 phosphorylation was observed. These findings follow those of previous studies showing that mTORC1 signaling at 1 and 2 hours after exercise (in the absence of alcohol) is independent of sex (11,12). Combined, these findings provide important and novel information on the timecourse of changes in mTORC1 signaling after RE. Although mTORC1 signaling is important for protein synthesis and muscle growth, it is imperative to recognize that the regulation of protein synthesis and subsequent muscle growth is complex and multifaceted, and involves many factors beyond the mTORC1 signaling pathway.
In conclusion, the major findings of this study were that, although RE elicited similar mTORC1 signaling both in men and in women, alcohol ingestion seemed to attenuate RE-induced phosphorylation of the mTORC1 signaling pathway only in men. These findings in men are important because they suggest a potential mechanism underlying the attenuated RE-induced protein synthesis from alcohol ingestion previously observed for men. Future studies should further investigate the effect of alcohol ingestion on RE-induced signaling involved in regulating protein synthesis and exercise training adaptations, especially in women, because previous research has found that alcohol reduces protein synthesis in women despite the lack of changes in mTORC1 signaling found in this study.
Postexercise alcohol ingestion, including binge drinking, is common among athletes and many athletes believe that such consumption does not affect their performance. This study provides evidence that alcohol should not be ingested after RE because this ingestion could potentially hamper the desired muscular adaptations to RE by reducing anabolic signaling, at least in men. Although this study investigated the effects of alcohol ingestion immediately after RE, the ingestion of alcohol within the later time periods of post-RE muscle recovery (during rest hours after RE) could also potentially impair protein synthesis as evidence by several studies linking alcohol ingestion with reductions in mTORC1 signaling and decreased protein synthesis at rest (21–24). These findings, therefore, have direct practical implications for strength and conditioning professionals because they can use this information when advising their athletes and clients regarding alcohol use. The knowledge of these potential negative effects on the adaptation to resistance training could provide a substantive incentive to reduce the overall level of alcohol consumption or at least binge drinking for this segment of the population. Furthermore, this knowledge could potentially be used by athletes and others engaged in RE as a means to deflect peer-pressure to drink by showing the potential effect alcohol could have on their training outcomes.
This study was supported in part by a grant from the National Strength and Conditioning Association Foundation.
1. Atherton PJ, Babraj J, Smith K, Singh J, Rennie MJ, Wackerhage H. Selective activation of AMPK-PGC-1alpha or PKB-TSC2-mTOR
signaling can explain specific adaptive responses to endurance or resistance training-like electrical muscle stimulation. FASEB J 19: 786–788, 2005.
2. Avruch J, Belham C, Weng Q, Hara K, Yonezawa K. The p70 S6 kinase integrates nutrient and growth signals to control translational capacity. Prog Mol Subcell Biol 26: 115–154, 2001.
3. Baar K, Esser K. Phosphorylation of p70S6kcorrelates with increased skeletal muscle mass following resistance exercise. Am J Physiol 276: C120–C127, 1999.
4. Babor T, Higgins-Biddle J, Saunders J, Monteiro M. AUDIT: The alcohol use disorders identification test: Guidelines for use in primary care. In: Anonymous Geneva. Switzerland: World Health Organization, 2001.
5. Barnes MJ, Mundel T, Stannard SR. Post-exercise alcohol ingestion exacerbates eccentric-exercise induced losses in performance. Eur J Appl Physiol 108: 1009–1014, 2010.
6. Barnes MJ, Mundel T, Stannard SR. Acute alcohol consumption aggravates the decline in muscle performance following strenuous eccentric exercise. J Sci Med Sport 13: 189–193, 2010.
7. Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R, Zlotchenko E, Scrimgeour A, Lawrence JC, Glass DJ. 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. Bodine SC. mTOR
signaling and the molecular adaptation to resistance exercise. Med Sci Sports Exerc 38: 1950–1957, 2006.
9. Bolster DR, Kubica N, Crozier SJ, Williamson DL, Farrell PA, Kimball SR, Jefferson LS. Immediate response of mammalian target of rapamycin (mTOR
)-mediated signalling following acute resistance exercise in rat skeletal muscle. J Physiol 553: 213–220, 2003.
10. Caron E, Ghosh S, Matsuoka Y, Ashton-Beaucage D, Therrien M, Lemieux S, Perreault C, Roux PP, Kitano H. A comprehensive map of the mTOR
signaling network. Mol Syst Biol 6: 453, 2010.
11. Dreyer HC, Fujita S, Cadenas JG, Chinkes DL, Volpi E, Rasmussen BB. Resistance exercise increases AMPK activity and reduces 4E-BP1 phosphorylation and protein synthesis in human skeletal muscle. J Physiol 576: 613–624, 2006.
12. Dreyer HC, Fujita S, Glynn EL, Drummond MJ, Volpi E, Rasmussen BB. Resistance exercise increases leg muscle protein synthesis and mTOR
signalling independent of sex. Acta Physiol (oxf) 199: 71–81, 2010.
13. Ford JA. Alcohol use among college students: A comparison of athletes and nonathletes. Subst Use Misuse 42: 1367–1377, 2007.
14. French MT, Popovici I, Maclean JC. Do alcohol consumers exercise more? Findings from a national survey. Am J Health Promot 24: 2–10, 2009.
15. Hara K, Yonezawa K, Weng QP, Kozlowski MT, Belham C, Avruch J. Amino acid sufficiency and mTOR
regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. J Biol Chem 273: 14484–14494, 1998.
16. Harris TE, Lawrence JC Jr. TOR signaling. Sci STKE 2003: re15, 2003.
17. Hurlbut SC, Sher KJ. Assessing alcohol problems in college students. J Am Coll Health 41: 49–58, 1992.
18. Kimball SR, Farrell PA, Jefferson LS. Invited Review: Role of insulin in translational control of protein synthesis in skeletal muscle by amino acids or exercise. J Appl Physiol 93: 1168–1180, 2002.
19. Kraemer WJ, Fry AC. Strength testing: Development and evaluation of methodology. In: Physiological Assessment of Human Fitness. Maud P.J., Foster C., eds. Champaign, IL: Human Kinetics, 1995. pp. 115–138.
20. 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.
21. Lang CH, Frost RA, Deshpande N, Kumar V, Vary TC, Jefferson LS, Kimball SR. Alcohol impairs leucine-mediated phosphorylation of 4E-BP1, S6K1, eIF4G, and mTOR
in skeletal muscle. Am J Physiol Endocrinol Metab 285: E1205–E1215, 2003.
22. Lang CH, Frost RA, Vary TC. Skeletal muscle protein synthesis and degradation exhibit sexual dimorphism after chronic alcohol consumption but not acute intoxication. Am J Physiol Endocrinol Metab 292: E1497–E1506, 2007.
23. 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.
24. 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. Nutr Metab (Lond) 6: 4, 2009.
25. Moore MJ, Werch C. Relationship between vigorous exercise frequency and substance use among first-year drinking
college students. J Am Coll Health 56: 686–690, 2008.
26. NCAA Research Staff. Study of Substance Use Habits of College Student-athletes. Indianapolis, IN, 2012. pp: 7–8. Available at: http://www.ncaapublications.com/productdownloads/SAHS09.pdf
. Accessed August 2015.
27. Parr EB, Camera DM, Areta JL, Burke LM, Phillips SM, Hawley JA, Coffey VG. Alcohol ingestion impairs maximal post-exercise rates of myofibrillar protein synthesis following a single bout of concurrent training. PLoS One 9: e88384, 2014.
28. Spiering BA, Kraemer WJ, Anderson JM, Armstrong LE, Nindl BC, Volek JS, Maresh CM. Resistance exercise biology: Manipulation of resistance exercise programme variables determines the responses of cellular and molecular signalling pathways. Sports Med 38: 527–540, 2008.
29. Terada N, Patel HR, Takase K, Kohno K, Nairn AC, Gelfand EW. Rapamycin selectively inhibits translation of mRNAs encoding elongation factors and ribosomal proteins. Proc Natl Acad Sci U S A 91: 11477–11481, 1994.
30. Vingren JL, Hill DW, Buddhadev H, Duplanty AA. Post-resistance exercise ethanol
ingestion and acute tetosterone bioavailability. Med Sci Sports Exerc 45: 1825–1832, 2013.
31. Vingren JL, Kraemer WJ, Hatfield DL, Anderson JM, Volek JS, Ratamess NA, Thomas GA, Ho J, Fragala MS, Maresh CM. Effect of resistance exercise on muscle steroidogenesis. J Appl Physiol (1985) 105: 1754–1760, 2008.