Hans C. Dreyer

Resistance exercise increases AMPK activity and reduces 4E‐BP1 phosphorylation and protein synthesis in human skeletal muscle

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.

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.

Nutrient signalling in the regulation of human muscle protein synthesis

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.

Satellite cell numbers in young and older men 24 hours after eccentric exercise

We tested the hypothesis that the expansion of satellite cell numbers, 24 h after maximal eccentric knee extensor exercise, is blunted in older men. Muscle biopsies were obtained from the vastus lateralis of 10 young (23–35 years) and 9 older (60–75 years) men. Satellite cells were identified immunohistochemically using an antibody to neural cell adhesion molecule. After 92 maximal eccentric contractions, the mean number of satellite cells per muscle fiber increased to a greater extent among the young men (141%; P < 0.001) than older men (51%; P = 0.002) from preexercise levels. Similar results were obtained when satellite cells were expressed as a proportion of all sublaminar nuclei. We conclude that a single bout of maximal eccentric exercise increases satellite cell numbers in both age groups, with a significantly greater response among the young men. These data suggest that age‐related changes in satellite cell recruitment may contribute to muscle regeneration deficits among the elderly. Muscle Nerve 2006

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.

Human Muscle Gene Expression Following Resistance Exercise and Blood Flow Restriction

INTRODUCTION Blood flow restriction in combination with low-intensity resistance exercise (REFR) increases skeletal muscle size to a similar extent as compared with traditional high-intensity resistance exercise training. However, there are limited data describing the molecular adaptations that occur after REFR. PURPOSE To determine whether hypoxia inducible factor-1 alpha (HIF-1alpha) and REDD1 mRNA are expressed differently in REFR compared with low-intensity resistance exercise with no blood flow restriction (CONTROL). Secondly, to determine whether low-intensity resistance exercise is able to induce changes in mRNA expression of several anabolic and catabolic genes as typically seen with high-intensity resistance exercise. METHODS Six subjects were studied at baseline and 3 h after a bout of leg resistance exercise (20% 1RM) in REFR and CONTROL subjects. Each subject participated in both groups, with 3 wk separating each visit. Muscle biopsy samples were analyzed for mRNA expression, using qRT-PCR. RESULT Our primary finding was that there were no differences between CONTROL and REFR for any of the selected genes at 3 h after exercise (P > 0.05). However, low-intensity resistance exercise increased HIF-1alpha, p21, MyoD, and muscle RING finger 1 (MuRF1) mRNA expression and decreased REDD1 and myostatin mRNA expression in both groups (P < 0.05). CONCLUSION Low-intensity resistance exercise can alter skeletal muscle mRNA expression of several genes associated with muscle growth and remodeling, such as REDD1, HIF-1alpha, MyoD, MuRF1, and myostatin. Further, the results from REFR and CONTROL were similar, indicating that the changes in early postexercise gene expression were attributable to the low-intensity resistance exercise bout, and not blood flow restriction.

Essential amino acid and carbohydrate ingestion before resistance exercise does not enhance postexercise muscle protein synthesis

Ingestion of an essential amino acid-carbohydrate (EAA + CHO) solution following resistance exercise enhances muscle protein synthesis during postexercise recovery. It is unclear whether EAA + CHO ingestion before resistance exercise can improve direct measures of postexercise muscle protein synthesis (fractional synthetic rate; FSR). We hypothesized that EAA + CHO ingestion before a bout of resistance exercise would prevent the exercise-induced decrease in muscle FSR and would result in an enhanced rate of muscle FSR during postexercise recovery. We studied 22 young healthy subjects before, during, and for 2 h following a bout of high-intensity leg resistance exercise. The fasting control group (n = 11) did not ingest nutrients, and the EAA + CHO group (n = 11) ingested a solution of EAA + CHO 1 h before beginning the exercise bout. Stable isotopic methods were used in combination with muscle biopsies to determine FSR. Immunoblotting procedures were utilized to assess cell signaling proteins associated with the regulation of FSR. We found that muscle FSR increased in the EAA + CHO group immediately following EAA + CHO ingestion (P < 0.05), returned to basal values during exercise, and remained unchanged at 1 h postexercise. Muscle FSR decreased in the fasting group during exercise and increased at 1 h postexercise (P < 0.05). However, the 2 h postexercise FSR increased by approximately 50% in both groups with no differences between groups (P > 0.05). Eukaryotic elongation factor 2 phosphorylation was reduced in both groups at 2 h postexercise (EAA + CHO: 39 +/- 7%; fasting: 47 +/- 9%; P < 0.05). We conclude that EAA + CHO ingestion before resistance exercise does not enhance postexercise FSR compared with exercise without nutrients.

Pharmacological Vasodilation Improves Insulin-Stimulated Muscle Protein Anabolism but Not Glucose Utilization in Older Adults

OBJECTIVE Skeletal muscle protein metabolism is resistant to the anabolic action of insulin in healthy, nondiabetic older adults. This defect is associated with impaired insulin-induced vasodilation and mTORC1 signaling. We hypothesized that, in older subjects, pharmacological restoration of insulin-induced capillary recruitment would improve the response of muscle protein synthesis and anabolism to insulin. RESEARCH DESIGN AND METHODS Twelve healthy, nondiabetic older subjects (71 ± 2 years) were randomized to two groups. Subjects were studied at baseline and during local infusion in one leg of insulin alone (Control) or insulin plus sodium nitroprusside (SNP) at variable rate to double leg blood flow. We measured leg blood flow by dye dilution; muscle microvascular perfusion with contrast enhanced ultrasound; Akt/mTORC1 signaling by Western blotting; and muscle protein synthesis, amino acid, and glucose kinetics using stable isotope methodologies. RESULTS There were no baseline differences between groups. Blood flow, muscle perfusion, phenylalanine delivery to the leg, and intracellular availability of phenylalanine increased significantly (P < 0.05) in SNP only. Akt phosphorylation increased in both groups but increased more in SNP (P < 0.05). Muscle protein synthesis and net balance (nmol · min−1 · 100 ml · leg−1) increased significantly (P < 0.05) in SNP (synthesis, 43 ± 6 to 129 ± 25; net balance, −16 ± 3 to 26 ± 12) but not in Control (synthesis, 41 ± 10 to 53 ± 8; net balance, −17 ± 3 to −2 ± 3). CONCLUSIONS Pharmacological enhancement of muscle perfusion and amino acid availability during hyperinsulinemia improves the muscle protein anabolic effect of insulin in older adults.

Expression of growth-related genes in young and older human skeletal muscle following an acute stimulation of protein synthesis

Muscle growth is associated with an activation of the mTOR signaling pathway and satellite cell regulators. The purpose of this study was to determine whether 17 selected genes associated with mTOR/muscle protein synthesis and the satellite cells/myogenic program are differentially expressed in young and older human skeletal muscle at rest and in response to a potent anabolic stimulus [resistance exercise + essential amino acid ingestion (RE+EAA)]. Twelve male subjects (6 young, 6 old) completed a bout of heavy resistance exercise. Muscle biopsies were obtained before and at 3 and 6 h post RE+EAA. Subjects ingested leucine-enriched essential amino acids at 1 h postexercise. mRNA expression was determined using qRT-PCR. At rest, hVps34 mRNA was elevated in the older subjects (P < 0.05) while there was a tendency for levels of myoD, myogenin, and TSC2 mRNA to be higher than young. The anabolic stimulus (RE+EAA) altered mRNAs associated with mTOR regulation. Notably, REDD2 decreased in both age groups (P < 0.05) but the expression of Rheb mRNA increased only in the young. Finally, cMyc mRNA was elevated (P < 0.05) in both young and old at 6 h post RE+EAA. Furthermore, RE+EAA also increased expression of several mRNAs associated with satellite function in the young (P < 0.05), while expression of these mRNAs did not change in the old. We conclude that several anabolic genes in muscle are more responsive in young men post RE+EAA. Our data provide new insights into the regulation of genes important for transcription and translation in young and old human skeletal muscle post RE+EAA.