The primary and novel finding from our study was that leucine-enriched EAA+CHO ingestion following resistance exercise simultaneously enhanced both mTOR signaling and mixed muscle protein synthesis in human subjects. Specifically, we observed a significant increase in S6K1 and 4E-BP1 phosphorylation when the leucine-enriched EAA+CHO solution was ingested following exercise relative to exercise alone. In addition, the leucine-enriched EAA+CHO-induced mTOR activation was associated with a 145% increase in mixed muscle protein synthesis compared with only a 41% increase observed in those subjects performing exercise alone.
Resistance exercise has a potent and acute effect on mTOR signaling (36
) and muscle protein synthesis (18
). However, our data suggest that signaling through mTOR is substantially increased during the postexercise recovery period when a leucine-enriched EAA+CHO solution is ingested. mTOR phosphorylation increases twofold with exercise alone, whereas phosphorylation increases fivefold when leucine-enriched EAA+CHO are ingested during recovery. However, we acknowledge that our findings are associative and do not establish cause and effect, since alternate signaling pathways such as MAPK or eukaryotic initiation factor-2 (eIF2) may be influencing translation initiation and elongation as well. In addition, glycogen status, which was not measured in this study, prior to or as a consequence of exercise may also be contributing or influencing muscle protein synthesis via GSK3. Further research is needed to accurately assess the potential of each signaling pathway to positively or negatively influence overall rates of muscle protein synthesis.
Downstream targets of mTOR signaling such as 4E-BP1 and S6K1 were also positively affected by the ingestion of the leucine-enriched EAA+CHO solution. In particular, 4E-BP1 phosphorylation following exercise alone had returned to base-line, whereas phosphorylation, when a leucine-enriched EAA+CHO solution was ingested, was significantly elevated above baseline, indicating enhanced translation initiation. In addition, and most remarkably, S6K1 phosphorylation was significantly enhanced above baseline and was measurably greater than the change observed in the exercise alone group. The substantial increase in S6K1 phosphorylation with the ingestion of the leucine-enriched EAA+CHO solution following exercise was even greater than that previously observed by our laboratory (21
) when subjects ingested an identical leucine-enriched EAA+CHO solution without any additional exercise stimulus. Evidence for amino acids (leucine in particular) working through a novel class 3 PI3K receptor (hVps34) have been previously reported (14
), potentially suggesting two separate points of convergence on mTOR activation (insulin receptor-PI3K-Akt-mTOR pathway and amino acid-hVps34-mTOR pathway).
Previous studies have demonstrated that performing resistance exercise (6
) or ingesting amino acids (26
) alone stimulates muscle protein synthesis; however, the combined effects of ingesting EAA following exercise appear to be more anabolic than either amino acids or exercise independently (12
). Other studies have shown that amino acid ingestion in combination with exercise stimulates components of the mTOR signaling pathway (7
). Using a less intense resistance exercise protocol compared with the current study, Karlsson et al. (25
) have shown that resistance exercise increases Ser424
phosphorylation of S6K1 and that ingestion of branched-chain amino acids further enhances that phosphorylation. The same group (25
) also measured S6K1 Thr389
phosphorylation and showed a significant and robust increase postexercise when branched-chain amino acids were ingested but no change with exercise alone. This is in contrast to our data, which show an increase in Thr389
phosphorylation in subjects performing exercise alone. The differences between studies in S6K1 phosphorylation at Thr389
may be partially explained by the difference in the exercise stimulus. For example, in the study by Karlsson et al. (25
), the number of sets (4 sets at 80% of a 1RM) was much less than our exercise protocol, which required subjects to perform 10 × 10 sets at 70% of their 1RM. Our data are in agreement with recent data showing an increase in mTOR (Ser2448
) and S6K1 (Thr389
) phosphorylation and muscle protein synthesis in subjects following ingestion of 10 g of EAA (16
). However, when we compare the phosphorylation data from the current study to our previous work (21
), it is clear that phosphorylation of mTOR and S6K1 was greater when the nutrients were ingested postexercise compared with ingestion of leucine-enriched EAA+CHO alone. Other groups also have shown a positive response on downstream components of mTOR signaling (4E-BP1 and S6K1 phosphorylation) following amino acid administration (22
Our data indicate that muscle protein synthesis is upregulated soon after resistance exercise and is further stimulated when a leucine-enriched EAA+CHO solution is ingested. These data are a progression of previous studies from our laboratory showing that FSR (a direct measure of amino acid incorporation into muscle protein) is acutely upregulated (within hours) and in association with components of the mTOR signaling pathway following exercise alone (18
) and ingestion of a leucine-enriched EAA+CHO solution (21
). These data are in opposition to the data reported by Cuthbertson et al. (17
), who showed muscle protein synthesis to be delayed by greater than 3 h following exercise. Although we are unable to explain the blunted response to exercise in that study (17
), several differences exist between studies. For example, their exercise protocol had subjects lift 25% of their body weight during repeated stepping exercise, whereas subjects in our study performed heavy resistance exercise (at or near 70% of 1RM). In addition, Cuthbertson et al. (17
) provided large doses of essential amino acids (45 g) and carbohydrate (135 g) 2 h before each biopsy. In our study, we provided ~20 g of “leucine”-enriched essential amino acids and 30 g of carbohydrate. The seemingly contradictory findings in addition to the differences in study design make comparisons difficult. It appears that further research is necessary to elucidate the contribution of nutrients and the timing of ingestion on signaling pathways influencing muscle protein synthesis in relation to exercise.
Although the ingestion of a leucine-enriched EAA+CHO solution following a single bout of resistance exercise had a profound effect on translation initiation (enhanced 4E-BP1 and S6K1 phosphorylation), it appeared to have no additive effect on translation elongation as measured by eEF2 phosphorylation. eEF2 phosphorylation was reduced during the 2 h of postexercise recovery (indicative of increased translation elongation) but was not further affected following the ingestion of the leucine-enriched EAA+CHO solution. This was interesting, because we previously showed that eEF2 phosphorylation was significantly decreased 1 h following ingestion of an identical leucine-enriched EAA+CHO solution without an exercise stimulus (21
). This may be due to our study design, given that others have shown that eEF2 phosphorylation changes rapidly (within minutes) and that the activation pattern is biphasic (23
We have previously shown that mixed muscle protein synthesis increased by 94% following leucine-enriched EAA+CHO ingestion in resting human subjects (21
). In the current study, we provided the identical leucine-enriched EAA+CHO solution to subjects 1 h following a single bout of resistance exercise and found that mixed muscle protein synthesis rates increased by 145% above baseline, whereas an increase of only 41% was measured in those subjects that performed the exercise without nutrition. Moreover, the positive change in FSR (a direct measure of muscle protein synthesis) was associated with positive changes in our mTOR signaling data, reflecting increases in translation initiation in those subjects who ingested the leucine-enriched EAA+CHO solution. Indeed, both mTOR and S6K1 phosphorylation was much higher in the subjects ingesting nutrients during postexercise recovery compared with those subjects ingesting nutrients without exercise (21
). Although the potential for a greater proportion of the anabolic response may have been due to ingestion of the EAA+CHO alone, our data support the concept that ingesting a leucine-enriched EAA+CHO solution during postexercise recovery potentially has an additive or synergistic effect on the exercise-induced anabolic response during recovery.
Although a great deal of research effort has implicated mTOR signaling to its downstream effectors, 4E-BP1 and S6K1, as a primary mechanism whereby translation initiation and elongation are activated, other signaling pathways exist (30
). While the research focus of our laboratory has centered on the mTOR pathway (18
), we acknowledge that many alternate pathways both dependent and independent of mTOR may exist and that further research is necessary to fully understand and characterize those pathways that also may be involved with postexercise anabolism associated with leucine and essential amino acids in general.
In summary, our data suggest that a leucine-enriched EAA+CHO solution ingested 1 h following a single bout of resistance exercise enhances muscle protein synthesis beyond exercise alone in association with enhanced mTOR, S6K1, and 4E-BP1 phosphorylation. Whereas others have measured signaling events following exercise and nutrient ingestion, to our knowledge this is the first study to also include direct measures of muscle protein synthesis during the acute phase of postexercise recovery. In addition, as the complexity of the signaling pathways controlling translation initiation and elongation are becoming more clear, we cannot rule out the possibility that other signaling pathways (30
) not measured in this study may also play a significant, if not more important, role than that of the mTOR pathway. In conclusion, our data suggest that ingestion of a leucine-enriched EAA+CHO solution 1 h following a single bout of resistance exercise augments human muscle protein synthesis, which may be partially explained by an increase in mTOR signaling.