Participant characteristics are shown in Table 1. All subjects performed three sets of 12 repetitions of maximal knee extension on the Cybex machine. The groups were matched for age, BMI and leg strength.

As BCAA have been identified as potent promoters of protein synthesis above other amino acids, the BCAA composition of the WPI is reported in Table 2. BCAA comprised 25% of the total amino acids measured and specifically leucine contributed 14% of the total amino acids consumed by the WPI group. No amino acids were detected in the placebo supplement.

A single bout of maximal knee extension exercise, immediately proceeded by either WPI or placebo ingestion had no impact on the phosphorylation of Akt at Ser⁴⁷³ (Figure 1). In contrast, 2 h following WPI ingestion, mTOR demonstrated enhanced phosphorylation at the Ser²⁴⁴⁸ site (2.1-fold change).

There was a rise in mTOR Ser²⁴⁴⁸ 4 h following the ingestion of the placebo, though this failed to achieve significance (Figure 2). Of the possible downstream targets of mTOR, the phosphorylation of 4E-BP1 at Thr^37/46 was significantly increased 3.0-fold above resting values by WPI ingestion (Figure 3).

An alternative substrate for mTOR, p70^S6K was highly phosphorylated in response to WPI ingestion at 2 h recovery, increasing 6.2-fold (Figure 4). In turn rpS6, the downstream substrate of p70^S6K, displayed high within-subject variability and failed to achieve significant changes at any time point (Figure 5).

This study demonstrates the stimulatory effect of WPI supplementation on the activation of regulators of translational initiation, the key step in the commencement of cellular protein synthesis, following a single bout of resistance exercise. Ingestion of WPI (26.6 g) immediately at the completion of a single bout of strenuous leg resistance exercise, induced a greater response in the phosphorylation of mTOR, p70^S6K and 4E-BP1, compared to exercise plus placebo ingestion. The heightened responsiveness of these kinases was rapid, predominately occurring within 2 h of exercise completion. Furthermore, there was no evidence for a persistence of this beneficial action for WPI, at both 4 and 24 h following the exercise.

Resistance exercise provokes muscle hypertrophy through a number of purported signal transduction pathways, including activation of the PI(3)K/Akt pathway [13]. This is evident in both rat and in vitro studies [3,14,15]. Akt is a serine/threonine kinase involved in the regulation of cellular metabolism and has been shown to induce rapid skeletal muscle hypertrophy in vivo [16]. Phosphorylation of Ser⁴⁷³ is required for maximal activation of Akt and it appears that Akt may have a relatively short activation period after an acute bout of resistance exercise [17,18]. Human studies investigating the activation of Akt after resistance exercise are scarce and have shown an enhanced phosphorylation of Akt 1 h post-exercise, but not immediately after exercise [19,20]. Indeed, the supplementary ingestion of 100 g BCAA and exercise combined has been reported to induce a small, albeit significant, increase in Akt activation at 1 hr post-exercise [7]. In the current study, Ser⁴⁷³ phosphorylation of Akt was unaltered following either exercise or exercise combined with 26.6 g WPI ingestion at any of the measured time points.

mTOR is a 289 kDa serine/threonine kinase partially downstream of Akt and responsible for the complex integration of anabolic stimuli mediating cell growth. Chiefly, mTOR stimulates protein synthesis through downstream activation of p70^S6K and 4E-BP1, providing a key point of convergence for both resistance exercise and amino acids [5,8]. WPI ingestion significantly increased Ser²⁴⁴⁸ phosphorylation of mTOR 2 h post-exercise. Interestingly, the ingestion of 100 g BCAA as previously reported, prompted an increase in mTOR phosphorylation at 1 h post-exercise, but failed to persist to 2 h recovery [7]. Both leucine and Akt activate mTOR through phosphorylation of the Ser²⁴⁴⁸ site [8,21]. Amino acids have been described as “priming” molecules, whose phosphorylation of mTOR at Ser²⁴⁴⁸ is a prerequisite for further phosphorylation by Akt [22]. Furthermore, activation of mTOR can occur through two separate signalling pathways by either insulin induced activation of the PI(3)K/Akt pathway or an undefined Akt-independent pathway stimulated by amino acids [23,24,25]. One possible candidate is Vps34 (vacuolar protein sorting mutant 34), a class III PI3K (phosphoinositide 3-kinase), regulated by both resistance exercise and nutrient availability [26]. However the interaction between supplementary protein ingestion and resistance exercise on the regulation of Vps34 is not yet described.

Downstream of mTOR, activation of p70^S6K is strongly linked to muscle hypertrophy [27]. p70^S6K undergoes phosphorylation at multiple serine and threonine residues, with the Thr³⁸⁹ residue closely associated with complete activation of the kinase [28]. The acute resistance exercise bout performed by the placebo group was not sufficient to induce phosphorylation of p70^S6K during the time points measured. The provision of whey proteins in this study stimulated an early and robust activation of p70^S6K. These findings are consistent with those reported previously, in which BCAA ingestion increases phosphorylation of p70^S6K at Thr³⁸⁹ 2 h post-exercise [6]. Further scrutiny of these results revealed that the magnitude of response was far greater (approximately double) in this earlier trial. This may be due to the large 100 g dose of BCAA ingested, compared to 26.6 g of whey proteins administered in the current study. A dose-response of increasing leucine ingestion to increasing p70^S6K (Thr³⁸⁹) phosphorylation was observed in rats [29]. Moreover, it is suggested that full activation of p70^S6K is dependent on the provision of amino acids to elicit a downstream activation of rpS6 [6,30]. Thus, a modest dose of 26.6 g WPI taken in conjunction with a single bout of resistance exercise clearly changed the signalling response of p70^S6K, although larger doses of WPI may be required to have a downstream impact on rpS6 during exercise recovery. Exercise plus placebo also resulted in an increased, but not significant, phosphorylation of rpS6, however this occurred later in recovery at 4 h. It is unknown whether this time differential in rpS6 activation is of physiological relevance.

In addition to p70^S6K, the phosphorylation of 4E-BP1 is sensitive to amino acid provision [31]. The site-specific phosphorylation of Thr^37/46 is reported to be crucial for optimal activation of 4E-BP1 [32]. In this study a marked increase in 4E-BP1 phosphorylation at 2 h post-exercise was measured when subjects were fed WPI. No change in Thr^37/46 phosphorylation of 4E-BP1 was shown after exercise alone. This is consistent with a previous study that not only demonstrated no increase in 4E-BP1 phosphorylation in human skeletal muscle after an acute resistance bout, but a significant decrease in Thr^37/46 phosphorylation was observed immediately and 2 h post-exercise [30].

Whey proteins are a rich source of leucine (14%) and BCAA (26%) [33]. Previously our group has demonstrates that this modest dose (26.6g) WPI elicits a rapid rise in plasma amino acids, peaking around 1 hour following ingestion [34]. Consistent with this data and the demonstration that kinases central to translation initiation are enhance, data has been reported that the ingestion of supplementary WPI, either before or immediately following resistance exercise training enhances both muscularity and strength gains in young males undertaking resistance training programs [35,36,37]. In supporting these studies, our data provides cellular evidence of the efficacy of combined WPI ingestion and resistance exercise to activate translation initiation. Indeed, when consumed immediately at exercise cessation, a modest 26.6 g WPI induced a more rapid and heightened activation of three kinases, mTOR, 4E-BP1 and p70^S6K at 2 h recovery. This has important implications for athletes, as this dose can be readily incorporated into a variety of either beverage (as used in the current study) or food formulations. Future research must then be directed towards examining the chronic actions on either muscular mass or strength with equivalent doses of whey protein.

Fourteen healthy young men (aged 18–25 yr) who had not participated in regular resistance exercise within a year prior to commencing the study and were not currently using nutritional or other purported muscle building supplements were recruited. A medical history questionnaire was used to exclude subjects with a diagnosed condition or illness that would endanger subjects during strenuous exercise. All subjects were informed of the nature and possible risks of the experimental procedures, before their written informed consent was obtained. This study and all associated procedures was formally approved by the Deakin University Human Research Ethics Committee.

Subjects were randomly assigned to receive either a whey protein isolate (WPI) or placebo drink. The WPI powder contained 97% protein powder (of which 90% protein, 0.5% fat, 0.5% lactose, 5% moisture, 3.7% ash and 1.15% milk minerals), 2.9% artificial flavour and 0.1% aspartame sweetener, with a dose of 28.65 g powder prepared in 200 mL water (this dose delivered 26.6g protein per 200 mL, see Table 2). The protein powder component was a β-lactoglobulin-enriched whey protein isolate (WPI: NatraBoost^® BLG, Murray Goulburn Nutritionals, Brunswick, Victoria, Australia). The placebo powder was prepared in the same volume and contained the same quantity of artificial flavour and aspartame sweetener and both drinks looked identical.

The WPI and placebo supplements were analysed for amino acid composition as described previously [38]. Briefly, samples were hydrolysed in 6 M HCl to determine total amino acids (excluding tryptophan, cysteine, asparagine and glutamine). The hydrolysates were rotary evaporated to dryness to remove the HCl and reconstituted in sodium citrate buffer (pH 2.2), including internal standard norleucine. Samples were then run on HPLC (Water Inc.; Empower Pro, Version 2, Software, Waters Corporation) and amino acids were separated by ion-exchange column (Sodium, Part# WAT08002, Waters, USA) and detected by UV after a post-column ninhydrin reaction.

At least four days prior to the testing day, each subject completed a familiarization session on the Cybex NORM dynamometer (Cybex International Inc. UK). For the 24 h preceding, and the days of the trial, subjects consumed a standard controlled diet of 20% fat, 14% protein and 66% carbohydrate and abstained from alcohol, caffeine, tobacco and recreational exercise. Following an overnight fast, subjects reported to the exercise physiology laboratory for a resting muscle biopsy. Thirty minutes after the biopsy subjects completed three sets of 12 repetitions of maximal singled-legged knee extension exercise on the Cybex machine with a 2 minute rest between each set. Each repetition involved concentric and eccentric contraction of the knee extensors at a set speed of 60°/sec on the biopsied leg. Subjects were instructed to contract as hard as possible and were verbally encouraged throughout each set. On completion of the exercise, subjects immediately ingested the supplement and were required to remain resting in the laboratory for biopsies to be taken 2 h and 4 h post-exercise. Subjects were supplied with lunch and an evening meal to eliminate bias from their post-exercise meal and given instructions to avoid caffeine, dairy products and high protein foods (including eggs, chicken, fish and red meat). Subjects then returned to the laboratory the following morning following an overnight fast for a biopsy 24 h post-exercise. Needle biopsies were performed in the vastus lateralis muscle of the non-dominant leg, under aseptic conditions following local anesthesia (1% xylocaine). Excised muscle tissue from each biopsy was immediately frozen and stored in liquid nitrogen for subsequent analysis. To minimize the potential or interference of repeated biopsies, samples were collected at least 2 cm from previous biopsy sites.

Muscle samples (~30 mg) were homogenized in ice cold lysis buffer containing 20 mM Tris-HCL, 5 mM EDTA, 10 mM Na-pyrophosphate, 100 mM NaF, 2 mM Na₃VO₄, 1% igepal, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 3 mM benzamidine and 1 mM PMSF. Homogenates were rotated on ice for 1 h and then centrifuged at 13,000 x g for 10 min. The resulting supernatant was analyzed for total protein content using the BCA protein assay (Pierce Biotechnology, Inc. Rockford, IL).

Denatured total proteins from each sample in loading buffer were separated by electrophoresis by 5% (phospho-mTOR), 8% (phospho-Akt, phospho-p70^S6K) or 12% (phospho-4E-BP1, phospho-S6) SDS-PAGE and transferred to nitrocellulose membranes, or PVDF for phospho-4E-BP1 and phospho-S6. PVDF membranes were blocked by air drying and nitrocellulose membranes were blocked with 5% bovine-serum albumin in Tris-buffered saline for 1 h (phospho-mTOR) or 2 h (phospho-Akt, phospho-p70^S6K). All membranes were incubated overnight at 4°C with polyclonal rabbit anti-phospho-mTOR (Ser²⁴⁴⁸), phospho-p70^S6K (Thr³⁸⁹), phospho-S6 ribosomal protein (Ser^235/236), phospho-4E-PB1 (Thr^37/46) and phospho-Akt (Ser⁴⁷³) antibodies at 1:1,000 dilution (all from Cell Signaling Technology, Danvers, MA). Membranes were washed in TBST followed by 60 min incubation with anti-rabbit IgG conjugated to horse-radish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) then washed again. Immunoreactive bands were detected using enhanced chemiluminescence (Western Lightning Chemiluminescent Reagent, Perkin-Elmer, Boston, MA) and visualized on a Kodak Image Station 440CF, using Kodak ID 3.5 image analysis software (Perkin Elmer Life Sciences, Boston, MA).

Statistical analysis was performed using GraphPad Prism 4.1 (GraphPad Software, San Diego, CA). Means were compared using two-way ANOVA (repeated measures) and any significant differences analyzed using a Bonferroni post hoc test. Data is presented as mean ± standard error of the mean (SEM). A probability level of <0.05 was adopted throughout to determine statistical significance unless otherwise stated.