Resistance exercise is a potent stimulator of muscle protein synthesis and muscle cell growth, with the increase in protein synthesis being detected within 2-3 h post-exercise and remaining elevated for up to 48 h. However, during exercise, muscle protein synthesis is inhibited. An increase in AMP-activated protein kinase (AMPK) activity has recently been shown to decrease mammalian target of rapamycin (mTOR) signalling to key regulators of translation initiation. We hypothesized that the cellular mechanism for the inhibition of muscle protein synthesis during an acute bout of resistance exercise in humans would be associated with an activation of AMPK and an inhibition of downstream components of the mTOR pathway (4E-BP1 and S6K1). We studied 11 subjects (seven men, four women) before, during, and for 2 h following a bout of resistance exercise. Muscle biopsy specimens were collected at each time point from the vastus lateralis. We utilized immunoprecipitation and immunoblotting methods to measure muscle AMPKα2 activity, and mTOR-associated upstream and downstream signalling proteins, and stable isotope techniques to measure muscle fractional protein synthetic rate (FSR). AMPKα2 activity (pmol min −1 (mg protein) −1 ) at baseline was 1.7 ± 0.3, increased immediately post-exercise (3.0 ± 0.6), and remained elevated at 1 h post-exercise (P < 0.05). Muscle FSR decreased during exercise and was significantly increased at 1 and 2 h post-exercise (P < 0.05). Phosphorylation of 4E-BP1 at Thr37/46 was significantly reduced immediately post-exercise (P < 0.05). We conclude that AMPK activation and a reduced phosphorylation of 4E-BP1 may contribute to the inhibition of muscle protein synthesis during resistance exercise. However, by 1-2 h post-exercise, muscle protein synthesis increased in association with an activation of protein kinase B, mTOR, S6K1 and eEF2.

Low-intensity resistance exercise training combined with blood flow restriction (REFR) increases muscle size and strength as much as conventional resistance exercise with high loads. However, the cellular mechanism(s) underlying the hypertrophy and strength gains induced by REFR are unknown. We have recently shown that both the mammalian target of rapamycin (mTOR) signaling pathway and muscle protein synthesis (MPS) were stimulated after an acute bout of high-intensity resistance exercise in humans. Therefore, we hypothesized that an acute bout of REFR would enhance mTOR signaling and stimulate MPS. We measured MPS and phosphorylation status of mTOR-associated signaling proteins in six young male subjects. Subjects were studied once during blood flow restriction (REFR, bilateral leg extension exercise at 20% of 1 repetition maximum while a pressure cuff was placed on the proximal end of both thighs and inflated at 200 mmHg) and a second time using the same exercise protocol but without the pressure cuff [control (Ctrl)]. MPS in the vastus lateralis muscle was measured by using stable isotope techniques, and the phosphorylation status of signaling proteins was determined by immunoblotting. Blood lactate, cortisol, and growth hormone were higher following REFR compared with Ctrl (P < 0.05). Ribosomal S6 kinase 1 (S6K1) phosphorylation, a downstream target of mTOR, increased concurrently with a decreased eukaryotic translation elongation factor 2 (eEF2) phosphorylation and a 46% increase in MPS following REFR (P < 0.05). MPS and S6K1 phosphorylation were unchanged in the Ctrl group postexercise. We conclude that the activation of the mTOR signaling pathway appears to be an important cellular mechanism that may help explain the enhanced muscle protein synthesis during REFR.

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.

. Effect of insulin on human skeletal muscle protein synthesis is modulated by insulin-induced changes in muscle blood flow and amino acid availability.

Muscle protein metabolism is resistant to insulin's anabolic effect in healthy older subjects. This is associated with reduced insulin vasodilation. We hypothesized that aerobic exercise restores muscle protein anabolism in response to insulin by improving vasodilation in older subjects. We measured blood flow, endothelin-1, Akt/mammalian target of rapamycin (mTOR) signaling, and muscle protein kinetics in response to physiological local hyperinsulinemia in two groups of older subjects following a bout of aerobic exercise (EX group: aged 70 ؎ 2 years; 45-min treadmill walk, 70% heart rate max) or rest (CTRL group: aged 68 ؎ 1 years). Baseline endothelin-1 was lower and blood flow tended to be higher in the EX group, but protein kinetics was not different between groups. Insulin decreased endothelin-1 (P < 0.05) in both groups, but endothelin-1 remained higher in the CTRL group (P < 0.05) and blood flow increased only in the EX group (EX group: 3.8 ؎ 0.7 to 5.3 ؎ 0.8; CTRL group: 2.5 ؎ 0.2 to 2.6 ؎ 0.2 ml ⅐ min ؊1 ⅐ 100 ml leg ؊1 ). Insulin improved Akt phosphorylation in the EX group and increased mTOR/S6 kinase 1 phosphorylation and muscle protein synthesis (EX group: 49 ؎ 11 to 89 ؎ 23; CTRL group: 58 ؎ 8 to 57 ؎ 12 nmol ⅐ min ؊1 ⅐ 100 ml leg ؊1 ) in the EX group only (P < 0.05). Because breakdown did not change, net muscle protein balance became positive only in the EX group (P < 0.05). In conclusion, a bout of aerobic exercise restores the anabolic response of muscle proteins to insulin by improving endothelial function and Akt/mTOR signaling in older subjects. Diabetes 56:1615-1622, 2007 S arcopenia is an age-dependent loss of skeletal muscle mass, strength, and quality, which may lead to weakness of the lower extremities, slowing of gait speed, and increased risk of falls (1-3). Sarcopenia is a multifactorial disorder, and evidence is accumulating that a reduced response of skeletal muscle to anabolic stimuli is an important contributing factor. For example, we and others (4,5) have previously shown that healthy aging is associated with a blunted muscle protein anabolic response to hyperaminoacidemia with hyperinsulinemia. Nonetheless, muscle protein synthesis and breakdown in the fasting state may not necessarily change with age (6) and may respond normally to a simple amino acid stimulus (7-10).Insulin is a potent anabolic stimulus for skeletal muscle. We have recently shown that physiological hyperinsulinemia increases skeletal muscle protein synthesis and anabolism in young healthy subjects, as long as blood flow and amino acid delivery to the muscle are stimulated by insulin (11). Thus, it appears that the insulin-induced modulation of muscle perfusion and nutrient availability is necessary for the anabolic response of muscle protein synthesis to insulin. Furthermore, insulin and amino acids both can stimulate the mammalian target of rapamycin (mTOR) signaling pathway (12). Specifically, insulin promotes the phosphorylation of Akt, an upstream regulator of mTOR, and enhances mTOR signaling to its down...

Fujita S, Rasmussen BB, Bell JA, Cadenas JG, Volpi E. Basal muscle intracellular amino acid kinetics in women and men. Am J Physiol Endocrinol Metab 292: E77-E83, 2007. First published August 8, 2006; doi:10.1152/ajpendo.00173.2006.-Sexual dimorphism in skeletal muscle mass is apparent, with men having more muscle mass and larger individual muscle cells. However, no sex-based differences have been detected in blood forearm phenylalanine turnover, although whole body leucine oxidation has been reported to be greater in men than in women. We hypothesized that sex differences in intracellular amino acid turnover may account for these discrepancies, with men having a higher intracellular turnover than women. We studied young, healthy women (women, n ϭ 8) and men (men, n ϭ 10) following an overnight fast. Phenylalanine, leucine, and alanine muscle intracellular kinetics were assessed using stable isotope methodologies, femoral arteriovenous blood sampling, and muscle biopsies. Muscle intracellular amino acid kinetics were reported relative to both leg volume and lean leg mass because of sex differences in leg volume and in muscle and fat distribution. When expressed per leg volume (nmol ⅐ min Ϫ1 ⅐ 100 ml leg volume Ϫ1 ), phenylalanine net balance (women: Ϫ16 Ϯ 4, men: Ϫ31 Ϯ 5), release from proteolysis in the blood (women: 46 Ϯ 9, men: 75 Ϯ 10) and intracellular availability (women: 149 Ϯ 23, men: 241 Ϯ 35), and alanine production, utilization, and intracellular availability were higher in men (P Ͻ 0.05). However, when the kinetic parameters were normalized per unit of lean leg mass, all differences disappeared. Muscle fractional synthetic rate was also not different between women and men. We conclude that there are no sex-based differences in basal muscle intracellular amino acid turnover when the data are normalized by lean mass. It remains to be determined if there are sex differences in intracellular amino acid metabolism following anabolic or catabolic stimuli. protein metabolism; muscle; sex; stable isotopes; gender SEXUAL DIFFERENCES IN HUMAN skeletal muscle mass are apparent. Muscle fiber type distribution is not different between women and men; however, cross-sectional area is larger for all fiber types in men (27,35). Interestingly, the largest muscle fibers in men appear to be fast-twitch type IIa fibers, whereas in women the slow-twitch type I fibers have the largest crosssectional area (27). Furthermore, during short-term limb immobilization, men and women have a similar reduction in muscle cell size, although women have larger reductions in strength (35).The sexual dimorphism in muscle mass is mainly attributed to endocrine differences between women and men because testosterone concentrations are usually ϳ10-fold higher in men (35). It has been shown that castration reduces muscle mass while testosterone administration at replacement or supraphysiological doses increases muscle mass in hypogonadal (3,5) and normal men (2), respectively. These effects are likely due to a stimulation of muscle protein syn...

Short- and long-term MBF responses to CPT, as an index for endothelium-related coronary vasomotion, can be measured reproducibly with (13)N-ammonia PET. In addition, the high interobserver reproducibility for repeat analysis of MBF values suggests the measurements to be largely operator independent.

Objective: To determine the effect of plasma glucose lowering on coronary circulatory function in type 2 diabetes mellitus. Methods: Twenty patients with type 2 diabetes and 18 weight-matched controls were studied. At baseline, myocardial blood flow (MBF) was measured with [13 N]ammonia and positron emission tomography at rest, during cold pressor testing (CPT), and during adenosine hyperaemia. In diabetic patients, MBF and blood chemistry were analysed again after 3 months of glucose-lowering treatment with glyburide and metformin. Results: Although hyperaemic MBF did not differ significantly between the patients and controls (1.81 (0.38) v 1.97 (0.43) ml/min/g; mean (SD)), the CPT-induced MBF increase (DMBF) was significantly less in diabetic patients than in controls (0.07 (0.07) v 0.25 (0.12) ml/min/g; p,0.001). Treatment with glyburide and metformin significantly decreased plasma glucose concentrations from 207 (76) to 134 (52) mg/dl (p,0.001). This decrease in plasma glucose was paralleled by a significant increase in DMBF in response to CPT (0.20 (0.16) from 0.07 (0.07) ml/min/g; p,0.001), which tended to be lower than in controls at baseline (0.20 (0.16) v 0.25 (0.12) ml/min/g; p = NS). The decrease in plasma glucose concentrations correlated significantly with the improvement in DMBF in response to CPT (r = 0.67, p,0.01). Conclusions: Type 2 diabetes mellitus is associated with abnormal MBF response to CPT, which can be significantly improved by euglycaemic control with glyburide and metformin. The close association between the decrease in plasma glucose concentration and the improvement in coronary vasomotor function in response to CPT suggests a direct adverse effect of raised plasma glucose concentration on diabetes-related coronary vascular disease.