In the present study, we chose to assess changes in the activity of Akt/mTOR pathway intermediates as markers of MPS in response to resistance exercise after ingesting 10 g of whey protein. As a result, we observed resistance exercise to effectively activate signaling intermediates of the Akt/mTOR pathway. Specifically, we demonstrated increased phosphorylation of IRS-1, AKT, and mTOR. Relative to their downstream targets, p70S6K was hyper-phosphorylated at 15 min post-exercise, whereas 4E-BP1 was hypo-phosphorylated at 15 min post-exercise. Conversely, we also observed that ingesting 10 g of whey protein was unable to induce a greater response in such kinase phosphorylation when compared to ingesting carbohydrate. Therefore, our results suggest that ingestion of 10 g of whey protein (5.25 g EAAs) is no different than an equal amount of carbohydrate at enhancing the activity of systemic and cellular signaling markers indicative of MPS following resistance exercise.
Resistance exercise and amino acids effectively stimulate MPS . Based on previous studies, the role that nutrient ingestion plays in activating the Akt/mTOR pathway [15, 18–20] is not completely understood, and may likely be related to the amount of amino acids available or whether co-ingested with carbohydrate. Previous studies have demonstrated that 20 g of whey protein (8.6 g EAAs)  and 10 g EAAs  maximally stimulated MPS, but that MPS was also increased even at whey protein doses of 5 g (2.2 g EAAs) and 10 g (4.3 g EAAs)  and an EAA dose of 5 g . When smaller amounts of EAAs (3-6 g) were ingested, with  and without  carbohydrate, the post-exercise increase in MPS was similar, but greater than basal or post-exercise fasted levels. In the present study, rather than assessing MPS, our interest was primarily focused on the extent with which 10 g of whey protein comprised of 5.25 EAAs would affect the activity of the Akt/mTOR pathway after resistance exercise when compared to carbohydrate alone and if this activity might also be systemically affected by either insulin or IGF-1. The reason for our interest was an attempt to discern if the 5.25 g of EAAs contained within 10 g of whey protein, without carbohydrate, was adequate to activate the Akt/mTOR compared to carbohydrate in response to a single bout of resistance exercise. Our interest was heightened by a previous study in which albumin protein intake at 10 g (4.3 g EAAs) significantly increased MPS, and maximally when 20 g (8.6 g EAAs) and 40 g (16.4 g EAAs) were ingested, yet none of the three concentrations had any affect on the activities of the Akt/mTOR pathway intermediates S6K1 (Thr389), rps6 (Ser240/244), or eIF2Bε (Ser539) at 60 and 240 min post-exercise . Despite previous evidence indicating otherwise , we were curious to determine if 10 g of whey protein would produce increases in other key Akt/mTOR signalling intermediates following resistance exercise.
It is evident that acute resistance exercise results in a significant increase in the rate of initiation of protein synthesis compared with resting muscle . It is suggested that signal transduction pathways control the rate of initiation of MPS, and appear to be the key factors in the hypertrophic process [34, 35]. Of particular importance is the complex myriad of signaling proteins, with Akt suggested to be a key regulator. Maximal activation of Akt occurs through phosphorylation of Ser473 and it appears that Akt may have a relatively short period of activation after an acute bout of resistance exercise . Research into the regulation of Akt signalling by exercise has produced conflicting results. A series of studies have demonstrated that contractile activity either positively or negatively regulates Akt activity [15, 37–39], while others failed to find any change [40–42]. In the current study, we found that resistance exercise and nutrient ingestion failed to induce a significant change in the phosphorylation of Akt.
Stimuli of the Akt pathway includes hormones and muscle contraction. Insulin  and IGF-I  bind to their respective membrane-bound receptors and subsequently activate phosphatidylinositol-3 kinase (PI-3K), an upstream activator for Akt phosphorylation. Quantification of circulating IGF-I levels has yielded inconsistent results, with levels being reported to decline , increase , or remain unchanged  after the onset of exercise. Furthermore, circulating IGF-1 has been shown to have no direct effect on muscle hypertrophy . In the current study, we observed no changes in serum IGF-1 following the exercise bout or due to nutrient ingestion, thereby suggesting hepatically-derived IGF-1 to have no appreciable effect on Akt pathway activation.
Serum insulin was increased in both groups. It is evident as to why insulin increased in the CHO group as 10 g of carbohydrate were ingested. In addition, the WP group also underwent a similar increase in insulin in the absence of ingested carbohydrate, which is in agreement with the insulin response previously demonstrated with 20 g of whey protein (10 g EAAs) . The Akt/mTOR signalling pathway is activated by insulin. Insulin binds with its receptor and leads to an increase in tyrosine phosphorylation of IRS-1 and eventually mTOR activation. In the present study, insulin significantly increased in both groups 30 min post-supplement ingestion and 15 min post-exercise, which was mirrored by significant increases in IRS-1 activation at 15 min post-exercise. Even though Akt phosphorylation was not significantly increased, activation of IRS-1 likely contributed to the observed increases in mTOR activation; however, this activity was not preferentially contingent on 10 g of whey protein ingestion.
mTOR is a 289 kDa serine/threonine kinase downstream of Akt and stimulates protein synthesis through downstream activation of p70S6K and 4E-BP1, providing a key point of convergence for both resistance exercise and amino acids . Amino acid ingestion has been shown to significantly enhance mTOR signalling [25, 50]. In the present study, the acute bouts of resistance exercise significantly increased mTOR and p70S6K activation at 15 min post-exercise, while a marked decrease in 4E-BP1 activation was also observed at 15 min post-exercise. While we observed mTOR activation to be enhanced by resistance exercise, the Akt/mTOR pathway signalling intermediates we assessed were unaffected by the provision of 10 g of whey protein comprised of 5.25 g EAAs.
Previous work has suggested that a minimal amount of 20 g is needed to stimulate MPS ; however, others have demonstrated positive effects utilizing a dosage as low as 6 g EAAs . Increases in MPS following resistance exercise have been observed when utilizing 10 g of whey protein; however, the protein supplement was co-ingested with 21 g of carbohydrate . However, it has recently been shown that approximately 5 g (2.2 g EAAs) and 10 g (4.2 g EAAs) of whey protein without carbohydrate significantly increased MPS 37% and 56%, respectively, over baseline. In this study, it was also shown that 20 g (8.6 g EAAs) maximally stimulated MPS following resistance exercise . Although, our results are supported by previous data which demonstrated that 20 g of albumin protein (8.6 g EAAs) enhanced MPS after resistance exercise, yet had no effects on activation of the mTOR pathway intermediates, S6K1, rps6, and eIF2Bε post-exercise , the dosage used in the current study (10 g whey protein, 5.25 g EAA) was apparently insufficient to reach an amino acid dosage capable of stimulating Akt/mTOR pathway activity.
A number of limitations exist in the current study. Firstly, we only assessed the relative changes in the phosphorlated levels of various Akt/mTOR pathway intermediates. Thus, these can only be used as markers indicative of MPS. We did not measure protein synthesis directly and thus caution needs to be taken when interpreting changes in phosphorylation status of signaling pathway intermediates to imply changes in human MPS, as this does not always determine functional changes. Secondly, no control was used and thus no direct comparison between isoenergetic carbohydrate and whey protein and resistance exercise could be made. However, previous research has clearly indicated that resistance exercise robustly activates Akt/mTOR signalling. Thirdly, only one dosage was used (10 g) and thus any comparison between other dosages cannot be made directly. Finally, our study focused on the early post-exercise recovery response in signalling and, therefore, we acknowledge the possibility that long-term activation of Akt/mTOR signalling and its downstream targets such as at 6, 24, or 48 hr post-exercise may be better indicators of muscle MPS over the course of a resistance training program.
In conclusion, the present study shows that ingestion of 10 g whey protein (5.25 g EAAs) prior to a single bout of lower body resistance exercise had no significant effect on activating systemic and cellular signaling intermediates of the Akt/mTOR pathway, otherwise indicative of MPS, in untrained men. Future research should examine the effects of dose response and timing of protein ingestion and compare the effects of various forms/fractions of proteins on post-exercise cell signalling responses to resistance exercise.