Christopher S. Fry

Skeletal muscle protein anabolic response to resistance exercise and essential amino acids is delayed with aging

Skeletal muscle loss during aging leads to an increased risk of falls, fractures, and eventually loss of independence. Resistance exercise is a useful intervention to prevent sarcopenia; however, the muscle protein synthesis (MPS) response to resistance exercise is less in elderly compared with young subjects. On the other hand, essential amino acids (EAA) increase MPS equally in both young and old subjects when sufficient EAA is ingested. We hypothesized that EAA ingestion following a bout of resistance exercise would stimulate anabolic signaling and MPS similarly between young and old men. Each subject ingested 20 g of EAA 1 h following leg resistance exercise. Muscle biopsies were obtained before and 1, 3, and 6 h after exercise to measure the rate of MPS and signaling pathways that regulate translation initiation. MPS increased early in young (1-3 h postexercise) and later in old (3-6 h postexercise). At 1 h postexercise, ERK1/2 MNK1 phosphorylation increased and eIF2alpha phosphorylation decreased only in the young. mTOR signaling (mTOR, S6K1, 4E-BP1, eEF2) was similar between groups at all time points, but MNK1 phosphorylation was lower at 3 h and AMP-activated protein kinase-alpha (AMPKalpha) phosphorylation was higher in old 1-3 h postexercise. We conclude that the acute MPS response after resistance exercise and EAA ingestion is similar between young and old men; however, the response is delayed with aging. Unresponsive ERK1/2 signaling and AMPK activation in old muscle may be playing a role in the delayed activation of MPS. Notwithstanding, the combination of resistance exercise and EAA ingestion should be a useful strategy to combat sarcopenia.

Rapamycin administration in humans blocks the contraction-induced increase in skeletal muscle protein synthesis

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.

Aging impairs contraction-induced human skeletal muscle mTORC1 signaling and protein synthesis

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.

Nutritional and contractile regulation of human skeletal muscle protein synthesis and mTORC1 signaling

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.

Blood flow restriction exercise stimulates mTORC1 signaling and muscle protein synthesis in older men

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.

Mammalian Target of Rapamycin Complex 1 Activation Is Required for the Stimulation of Human Skeletal Muscle Protein Synthesis by Essential Amino Acids

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.

An increase in essential amino acid availability upregulates amino acid transporter expression in human skeletal muscle

Essential amino acids (EAA) stimulate skeletal muscle mammalian target of rapamycin complex 1 (mTORC1) signaling and protein synthesis. It has recently been reported that an increase in amino acid (AA) transporter expression during anabolic conditions is rapamycin-sensitive. The purpose of this study was to determine whether an increase in EAA availability increases AA transporter expression in human skeletal muscle. Muscle biopsies were obtained from the vastus lateralis of seven young adult subjects (3 male, 4 female) before and 1-3 h after EAA ingestion (10 g). Blood and muscle samples were analyzed for leucine kinetics using stable isotopic techniques. Quantitative RT-PCR, and immunoblotting were used to determine the mRNA and protein expression, respectively, of AA transporters and members of the general AA control pathway [general control nonrepressed (GCN2), activating transcription factor (ATF4), and eukaryotic initiation factor (eIF2) alpha-subunit (Ser(52))]. EAA ingestion increased blood leucine concentration, delivery of leucine to muscle, transport of leucine from blood into muscle, intracellular muscle leucine concentration, ribosomal protein S6 (Ser(240/244)) phosphorylation, and muscle protein synthesis. This was followed with increased L-type AA transporter (LAT1), CD98, sodium-coupled neutral AA transporter (SNAT2), and proton-coupled amino acid transporter (PAT1) mRNA expression at 1 h (P < 0.05) and modest increases in LAT1 protein expression (3 h post-EAA) and SNAT2 protein expression (2 and 3 h post-EAA, P < 0.05). Although there were no changes in GCN2 expression and eIF2 alpha phosphorylation, ATF4 protein expression reached significance by 2 h post-EAA (P < 0.05). We conclude that an increase in EAA availability upregulates human skeletal muscle AA transporter expression, perhaps in an mTORC1-dependent manner, which may be an adaptive response necessary for improved AA intracellular delivery.

Excess Leucine Intake Enhances Muscle Anabolic Signaling but Not Net Protein Anabolism in Young Men and Women

Essential amino acids (EAA) stimulate skeletal muscle protein synthesis (MPS) in humans. Leucine may have a greater stimulatory effect on MPS than other EAA and/or decrease muscle protein breakdown (MPB). To determine the effect of 2 different leucine concentrations on muscle protein turnover and associated signaling, young men (n = 6) and women (n = 8) ingested 10 g EAA in 1 of 2 groups: composition typical of high quality proteins (CTRL; 1.8 g leucine) or increased leucine concentration (LEU; 3.5 g leucine). Participants were studied for 180 min postingestion. Fractional synthetic rate and leg phenylalanine and leucine kinetics were assessed on muscle biopsies using stable isotopic techniques. Signaling was determined by immunoblotting. Arterial leucine concentration and delivery to the leg increased in both groups and was significantly higher in LEU than in CTRL; however, transport into the muscle and intracellular availability did not differ between groups. MPS increased similarly in both groups 60 min postingestion. MPB decreased at 60 min only in LEU, but net muscle protein balance improved similarly. Components of mammalian target of rapamycin (mTOR) signaling were improved in LEU, but no changes were observed in ubiquitin-proteasome system signaling. Changes in light chain 3 and mTOR association with Unc-51-like kinase 1 indicate autophagy decreased more in LEU. We conclude that in 10 g of EAA, the leucine content typical of high quality proteins (~1.8 g) is sufficient to induce a maximal skeletal muscle protein anabolic response in young adults, but leucine may play a role in autophagy regulation.

Aging differentially affects human skeletal muscle microRNA expression at rest and after an anabolic stimulus of resistance exercise and essential amino acids

Sarcopenia, skeletal muscle loss during aging, is associated with increased falls, fractures, morbidity, and loss of independence. MicroRNAs (miRNAs) are novel posttranscriptional regulators. The role of miRNAs in cell size regulation after an anabolic stimulus in human skeletal muscle is unknown. We hypothesized that aging would be associated with a differential expression of skeletal muscle primary miRNA (pri-miRNA) and mature miRNA (miR). To test this hypothesis, we used real-time PCR and immunoblotting before and after an anabolic stimulus (resistance exercise + ingestion of a 20-g leucine-enriched essential amino acid solution) to measure the expression of muscle-specific miRNAs (miR-1, miR-133a, and miR-206), upstream regulators (MyoD and myogenin), and downstream targets [insulin-like growth factor-I, histone deacetylase-4, myocyte enhancing factor-2, and Ras homolog enriched in brain (Rheb)] in skeletal muscle of young and older men. Muscle biopsies were obtained at baseline and 3 and 6 h after exercise. At baseline, we found pri-miRNA-1-1, -1-2, -133a-1, and -133a-2 expression elevated in older compared with young men (P < 0.05). Pri-miRNA-1-2, -133a-1, and -133a-2 were reduced at 6 h after exercise only in the young men compared with baseline, whereas pri-miRNA-206 was elevated at different postexercise time points in older and young men (P < 0.05). Compared with baseline, miR-1 was reduced only in the young men, whereas Rheb protein was increased in both age groups after the anabolic stimulus (P < 0.05). We conclude that skeletal muscle primary and mature miRNA expression in young men is readily altered by an anabolic stimulus of resistance exercise + essential amino acid ingestion. However, aging is associated with higher basal skeletal muscle primary miRNA expression and a dysregulated miRNA response after the anabolic stimulus.

Bed rest impairs skeletal muscle amino acid transporter expression, mTORC1 signaling, and protein synthesis in response to essential amino acids in older adults

Skeletal muscle atrophy during bed rest is attributed, at least in part, to slower basal muscle protein synthesis (MPS). Essential amino acids (EAA) stimulate mammalian target of rapamycin (mTORC1) signaling, amino acid transporter expression, and MPS and are necessary for muscle mass maintenance, but there are no data on the effect of inactivity on this anabolic mechanism. We hypothesized that bed rest decreases muscle mass in older adults by blunting the EAA stimulation of MPS through reduced mTORC1 signaling and amino acid transporter expression in older adults. Six healthy older adults (67 ± 2 yr) participated in a 7-day bed rest study. We used stable isotope tracers, Western blotting, and real-time qPCR to determine the effect of bed rest on MPS, muscle mTORC1 signaling, and amino acid transporter expression and content in the postabsorptive state and after acute EAA ingestion. Bed rest decreased leg lean mass by ∼4% (P < 0.05) and increased postabsorptive mTOR protein (P < 0.05) levels while postabsorptive MPS was unchanged (P > 0.05). Before bed rest acute EAA ingestion increased MPS, mTOR (Ser(2448)), S6 kinase 1 (Thr(389), Thr(421)/Ser(424)), and ribosomal protein S6 (Ser(240/244)) phosphorylation, activating transcription factor 4, L-type amino acid transporter 1 and sodium-coupled amino acid transporter 2 protein content (P < 0.05). However, bed rest blunted the EAA-induced increase in MPS, mTORC1 signaling, and amino acid transporter protein content. We conclude that bed rest in older adults significantly attenuated the EAA-induced increase in MPS with a mechanism involving reduced mTORC1 signaling and amino acid transporter protein content. Together, our data suggest that a blunted EAA stimulation of MPS may contribute to muscle loss with inactivity in older persons.