Muscle protein synthesis and mTORC1 signalling are concurrently stimulated following muscle contraction in humans. In an effort to determine whether mTORC1 signalling is essential for regulating muscle protein synthesis in humans, we treated subjects with a potent mTORC1 inhibitor (rapamycin) prior to performing a series of high-intensity muscle contractions. Here we show that rapamycin treatment blocks the early (1-2 h) acute contraction-induced increase (∼40%) in human muscle protein synthesis. In addition, several downstream components of the mTORC1 signalling pathway were also blunted or blocked by rapamycin. For instance, S6K1 phosphorylation (Thr421/Ser424) was increased post-exercise 6-fold in the control group while being unchanged with rapamycin treatment. Furthermore, eEF2 phosphorylation (Thr56) was reduced by ∼25% post-exercise in the control group but phosphorylation following rapamycin treatment was unaltered, indicating that translation elongation was inhibited. Rapamycin administration prior to exercise also reduced the ability of raptor to associate with mTORC1 during post-exercise recovery. Surprisingly, rapamycin treatment prior to resistance exercise completely blocked the contraction-induced increase in the phosphorylation of ERK1/2 (Thr202/Tyr204) and blunted the increase in MNK1 (Thr197/202) phosphorylation. However, the phosphorylation of a known target of MNK1, eIF4E (Ser208), was similar in both groups (P > 0.05) which is consistent with the notion that rapamycin does not directly inhibit MAPK signalling. We conclude that mTORC1 signalling is, in part, playing a key role in regulating the contraction-induced stimulation of muscle protein synthesis in humans, while dual activation of mTORC1 and ERK1/2 stimulation may be required for full stimulation of human skeletal muscle protein synthesis.

The loss of skeletal muscle mass during aging, sarcopenia, increases the risk for falls and dependence. Resistance exercise (RE) is an effective rehabilitation technique that can improve muscle mass and strength; however, older individuals are resistant to the stimulation of muscle protein synthesis (MPS) with traditional high-intensity RE. Recently, a novel rehabilitation exercise method, low-intensity RE, combined with blood flow restriction (BFR), has been shown to stimulate mammalian target of rapamycin complex 1 (mTORC1) signaling and MPS in young men. We hypothesized that low-intensity RE with BFR would be able to activate mTORC1 signaling and stimulate MPS in older men. We measured MPS and mTORC1-associated signaling proteins in seven older men (age 70+/-2 yr) before and after exercise. Subjects were studied identically on two occasions: during BFR exercise [bilateral leg extension exercise at 20% of 1-repetition maximum (1-RM) with pressure cuff placed proximally on both thighs and inflated at 200 mmHg] and during exercise without the pressure cuff (Ctrl). MPS and phosphorylation of signaling proteins were determined on successive muscle biopsies by stable isotopic techniques and immunoblotting, respectively. MPS increased 56% from baseline after BFR exercise (P<0.05), while no change was observed in the Ctrl group (P>0.05). Downstream of mTORC1, ribosomal S6 kinase 1 (S6K1) phosphorylation and ribosomal protein S6 (rpS6) phosphorylation increased only in the BFR group after exercise (P<0.05). We conclude that low-intensity RE in combination with BFR enhances mTORC1 signaling and MPS in older men. BFR exercise is a novel intervention that may enhance muscle rehabilitation to counteract sarcopenia.

We recently showed that resistance exercise and ingestion of essential amino acids with carbohydrate (EAA+CHO) can independently stimulate mammalian target of rapamycin (mTOR) signaling and muscle protein synthesis in humans. Providing an EAA+CHO solution postexercise can further increase muscle protein synthesis. Therefore, we hypothesized that enhanced mTOR signaling might be responsible for the greater muscle protein synthesis when leucine-enriched EAA+CHOs are ingested during postexercise recovery. Sixteen male subjects were randomized to one of two groups (control or EAA+CHO). The EAA+CHO group ingested the nutrient solution 1 h after resistance exercise. mTOR signaling was assessed by immunoblotting from repeated muscle biopsy samples. Mixed muscle fractional synthetic rate (FSR) was measured using stable isotope techniques. Muscle protein synthesis and 4E-BP1 phosphorylation during exercise were significantly reduced (P < 0.05). Postexercise FSR was elevated above baseline in both groups at 1 h but was even further elevated in the EAA+CHO group at 2 h postexercise (P < 0.05). Increased FSR was associated with enhanced phosphorylation of mTOR and S6K1 (P < 0.05). Akt phosphorylation was elevated at 1 h and returned to baseline by 2 h in the control group, but it remained elevated in the EAA+CHO group (P < 0.05). 4E-BP1 phosphorylation returned to baseline during recovery in control but became elevated when EAA+CHO was ingested (P < 0.05). eEF2 phosphorylation decreased at 1 and 2 h postexercise to a similar extent in both groups (P < 0.05). Our data suggest that enhanced activation of the mTOR signaling pathway is playing a role in the greater synthesis of muscle proteins when resistance exercise is followed by EAA+CHO ingestion.

BackgroundSarcopenia, the loss of skeletal muscle mass during aging, increases the risk for falls and dependency. Resistance exercise (RE) training is an effective treatment to improve muscle mass and strength in older adults, but aging is associated with a smaller amount of training-induced hypertrophy. This may be due in part to an inability to stimulate muscle-protein synthesis (MPS) after an acute bout of RE. We hypothesized that older adults would have impaired mammalian target of rapamycin complex (mTORC)1 signaling and MPS response compared with young adults after acute RE.MethodsWe measured intracellular signaling and MPS in 16 older (mean 70 ± 2 years) and 16 younger (27 ± 2 years) subjects. Muscle biopsies were sampled at baseline and at 3, 6 and 24 hr after exercise. Phosphorylation of regulatory signaling proteins and MPS were determined on successive muscle biopsies by immunoblotting and stable isotopic tracer techniques, respectively.ResultsIncreased phosphorylation was seen only in the younger group (P< 0.05) for several key signaling proteins after exercise, including mammalian target of rapamycin (mTOR), ribosomal S6 kinase (S6K)1, eukaryotic initiation factor 4E-binding protein (4E-BP)1 and extracellular signal-regulated kinase (ERK)1/2, with no changes seen in the older group (P >0.05). After exercise, MPS increased from baseline only in the younger group (P< 0.05), with MPS being significantly greater than that in the older group (P <0.05).ConclusionsWe conclude that aging impairs contraction-induced human skeletal muscle mTORC1 signaling and protein synthesis. These age-related differences may contribute to the blunted hypertrophic response seen after resistance-exercise training in older adults, and highlight the mTORC1 pathway as a key therapeutic target to prevent sarcopenia.

In this review we discuss current findings in the human skeletal muscle literature describing the acute influence of nutrients (leucine-enriched essential amino acids in particular) and resistance exercise on muscle protein synthesis and mammalian target of rapamycin complex 1 (mTORC1) signaling. We show that essential amino acids and an acute bout of resistance exercise independently stimulate human skeletal muscle protein synthesis. It also appears that ingestion of essential amino acids following resistance exercise leads to an even larger increase in the rate of muscle protein synthesis compared with the independent effects of nutrients or muscle contraction. Until recently the cellular mechanisms responsible for controlling the rate of muscle protein synthesis in humans were unknown. In this review, we highlight new studies in humans that have clearly shown the mTORC1 signaling pathway is playing an important regulatory role in controlling muscle protein synthesis in response to nutrients and/or muscle contraction. We propose that essential amino acid ingestion shortly following a bout of resistance exercise is beneficial in promoting skeletal muscle growth and may be useful in counteracting muscle wasting in a variety of conditions such as aging, cancer cachexia, physical inactivity, and perhaps during rehabilitation following trauma or surgery.

The mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) are important nutrient-and energy-sensing and signalling proteins in skeletal muscle. AMPK activation decreases muscle protein synthesis by inhibiting mTOR signalling to regulatory proteins associated with translation initiation and elongation. On the other hand, essential amino acids (leucine in particular) and insulin stimulate mTOR signalling and protein synthesis. We hypothesized that anabolic nutrients would be sensed by both AMPK and mTOR, resulting in an acute and potent stimulation of human skeletal muscle protein synthesis via enhanced translation initiation and elongation.We measured muscle protein synthesis and mTOR-associated upstream and downstream signalling proteins in young male subjects (n = 14) using stable isotopic and immunoblotting techniques. Following a first muscle biopsy, subjects in the 'Nutrition' group ingested a leucine-enriched essential amino acid-carbohydrate mixture (EAC). Subjects in the Control group did not consume nutrients. A second biopsy was obtained 1 h later. Ingestion of EAC significantly increased muscle protein synthesis, modestly reduced AMPK phosphorylation, and increased Akt/PKB (protein kinase B) and mTOR phosphorylation (P < 0.05). mTOR signalling to its downstream effectors (S6 kinase 1 (S6K1) and 4E-binding protein 1 (4E-BP1) phosphorylation status) was also increased (P < 0.05). In addition, eukaryotic elongation factor 2 (eEF2) phosphorylation was significantly reduced (P < 0.05). Protein synthesis and cell signalling (phosphorylation status) was unchanged in the control group (P > 0.05).We conclude that anabolic nutrients alter the phosphorylation status of both AMPK-and mTOR-associated signalling proteins in human muscle, in association with an increase in protein synthesis not only via enhanced translation initiation but also through signalling promoting translation elongation.

The relationship between mammalian target of rapamycin complex 1 (mTORC1) signaling and muscle protein synthesis during instances of amino acid surplus in humans is based solely on correlational data. Therefore, the goal of this study was to use a mechanistic approach specifically designed to determine whether increased mTORC1 activation is requisite for the stimulation of muscle protein synthesis following L-essential amino acid (EAA) ingestion in humans. Examination of muscle protein synthesis and signaling were performed on vastus lateralis muscle biopsies obtained from 8 young (25 ± 2 y) individuals who were studied prior to and following ingestion of 10 g of EAA during 2 separate trials in a randomized, counterbalanced design. The trials were identical except during 1 trial, participants were administered a single oral dose of a potent mTORC1 inhibitor (rapamycin) prior to EAA ingestion. In response to EAA ingestion, an ~60% increase in muscle protein synthesis was observed during the control trial, concomitant with increased phosphorylation of mTOR (Ser(2448)), ribosomal S6 kinase 1 (Thr(389)), and eukaryotic initiation factor 4E binding protein 1 (Thr(37/46)). In contrast, prior administration of rapamycin completely blocked the increase in muscle protein synthesis and blocked or attenuated activation of mTORC1-signaling proteins. The inhibition of muscle protein synthesis and signaling was not due to differences in either extracellular or intracellular amino acid availability, because these variables were similar between trials. These data support a fundamental role for mTORC1 activation as a key regulator of human muscle protein synthesis in response to increased EAA availability. This information will be useful in the development of evidence-based nutritional therapies targeting mTORC1 to counteract muscle wasting associated with numerous clinical conditions.

Drummond MJ, Glynn EL, Fry CS, Timmerman KL, Volpi E, Rasmussen BB. An increase in essential amino acid availability upregulates amino acid transporter expression in human skeletal muscle.