Statin treatment reduces leucine turnover, but does not affect endogenous production of beta-hydroxy-beta-methylbutyrate (HMB).

BACKGROUND: Statins, or hydroxy-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors, are one of the most commonly prescribed medications for lowering cholesterol. Myopathic side-effects ranging from pain and soreness to critical rhabdomyolysis are commonly reported and often lead to discontinuation. The pathophysiological mechanism is, in general, ascribed to a downstream reduction of Coenzyme Q10 synthesis. HMG-CoA is a metabolite of leucine and its corresponding keto acid α-ketoisocaproic acid (KIC) and ß-hydroxy-ß-methylbutyrate (HMB), however, little is known about the changes in the metabolism of leucine and its metabolites in response to statins. OBJECTIVE: We aimed to investigate if statin treatment has implications on the upstream metabolism of leucine to KIC and HMB, as well as on other branched chain amino acids (BCAA). DESIGN: 12 hyperlipidemic older adults under statin treatment were recruited. The study was conducted as a paired prospective study. Included participants discontinued their statin treatment for 4 weeks before they returned for baseline measurements (before). Statin treatment was then reintroduced, and the participants returned for a second study day 7 days after reintroduction (after statin). On study days, participants were injected with stable isotope pulses for measurement of the whole-body production (WBP) of all BCAA (leucine, isoleucine and valine), along with their respective keto acids and HMB. RESULTS: We found a reduced leucine WBP (22 %, p = 0.0033), along with a reduction in valine WBP (13 %, p = 0.0224). All other WBP of BCAA and keto acids were unchanged. There were no changes in the WBP of HMB. CONCLUSIONS: Our study shows that statin inhibition of HMG-CoA reductase has an upstream impact on the turnover of leucine and valine. Whether this impairment in WBP of leucine may contribute to the known pathophysiological side effects of statins on muscle remains to be further investigated.

Metabolic phenotyping using kinetic measurements in young and older healthy adults.

BACKGROUND: The aging process is often associated with the presence of sarcopenia. Although changes in the plasma concentration of several amino acids have been observed in older adults, it remains unclear whether these changes are related to disturbances in whole body production and/or interconversions. METHODS: We studied 10 healthy young (~22.7y) and 17 older adults (~64.8y) by administering a mixture of stable amino acid tracers in a pulse and in a primed constant infusion. We calculated whole body production (WBP) and metabolite to metabolite interconversions. In addition, we measured body composition, muscle function, and provided questionnaires to assess daily dietary intake, physical activity, mood (anxiety, depression) and markers of cognitive function. Plasma enrichments and metabolite concentrations were measured by GC- and LC-MS/MS and statistics were performed by student t-test. RESULTS: Older adults had a 11% higher body mass index (p=0.04) and 27% reduced peak leg extension force (p=0.02) than the younger group, but comparable values for muscle mass, mood and cognitive function. Although small differences in several plasma amino acid concentrations were observed, we found older adults had about 40% higher values of WBP for glutamine (221±27 vs. 305±21µmol/kgffm/h, p=0.03) and tau-methylhistidine (0.15±0.01 vs. 0.21±0.02µmol/kgffm/h, p=0.04), 26% lower WBP value for arginine (59±4 vs. 44±4µmol/kgffm/h, p=0.02) and a reduction in WBP (50%; 1.23±0.15 vs. 0.69±0.06µmol/kgffm/h, p=0.001) and concentration (25%; 3.5±0.3µmol/l vs. 2.6±0.2µmol/l, p=0.01) for ß-Hydroxy ß-Methylbutyrate. No differences were observed in protein catabolism. Clearance of arginine was decreased (27%, p=0.03) and clearance of glutamine (58%, p=0.01), leucine (67%, p=0.001) and KIC (76%, p=0.004) were increased in older adults. CONCLUSIONS: Specific differences exist between young and older adults in amino acid metabolism.

Determination of ß-hydroxy-ß-methylbutyrate concentration and enrichment in human plasma using chemical ionization gas chromatography tandem mass spectrometry.

Our objective was to develop a quick and simplified method for the determination of ß-Hydroxy-ß-methylbutyrate (HMB) and ɑ-ketoisocaproic acid (KIC) concentrations and enrichments by GC/MS/MS to determine the turnover rate of HMB in humans. In experiment 1, we provided a pulse of L-[5,5,5-2H3]leucine to younger adults in the postabsorptive state then collected blood samples over a 4h time period. In experiment 2, we provided a pulse of [3,4,methyl-13C3]HMB to older adults in the postabsorptive state then collected blood samples over a 3h time period. Plasma concentrations of KIC and HMB and MPE of KIC and HMB were determined by GC/MS/MS. Plasma enrichment of leucine was determined by LC/MS/MS. To determine plasma enrichment of [5,5,5-2H3]HMB and [3,4,methyl-13C3]HMB, samples were derivatized using pentafluorobenzyl bromide and analyzed using chemical ionization mode. The final methods used included multiple reaction monitoring of transitions 117.3>59.3 for M+0 and 120.3>59.3 for M+3. In experiment 1, peak MPE of Leu peaked at 9.76% generating a peak MPE of KIC at 2.67% and a peak HMB MPE of 0.3%. In experiment 2, the rate of appearance for HMB was 0.66µmol/kg ffm/h. We calculated that production of HMB in humans accounts for 0.66% of total leucine turnover.

The impact of postexercise essential amino acid ingestion on the ubiquitin proteasome and autophagosomal-lysosomal systems in skeletal muscle of older men.

Essential amino acid (EAA) ingestion enhances postexercise muscle protein synthesis, and, in particular, the anabolic response of older adults appears sensitive to the quantity of ingested leucine. The effect of leucine ingestion on muscle breakdown following resistance exercise (RE) is less understood. The purpose of this study was to identify the impact of postexercise leucine ingestion on the ubiquitin proteasome and autophagosomal-lysosomal systems following acute RE in older men. Subjects (72 ± 2 yr) performed RE and 1 h postexercise ingested 10 g of EAA containing a leucine quantity similar to quality protein (control, 1.8 g leucine, n = 7) or enriched in leucine (leucine, 3.5 g leucine, n = 8). Stable isotope infusion and muscle biopsies (vastus lateralis) obtained at rest and 2, 5, and 24 h postexercise were used to examine protein content (Western blot), mRNA expression (RT-quantitative PCR), and muscle protein fractional breakdown rate (FBR). Muscle-specific RING finger 1 mRNA increased in both groups at 2 and 5 h (P < 0.05). LC3 mRNA increased, and the LC3BII-to-LC3BI ratio decreased at all postexercise time points in control (P < 0.05). Conversely, LC3 mRNA only increased at 2 h, and the LC3BII-to-LC3BI ratio only decreased at 2 and 5 h in leucine (P < 0.05). Tumor necrosis factor receptor-associated factor-6 mRNA increased (P < 0.05) in control at 5 h. FBR was not statistically different between groups or from basal 24 h postexercise (P > 0.05). These data indicate that ingesting a larger quantity of leucine following RE may further reduce postexercise skeletal muscle autophagy in older men; however, it does not appear to influence the acute postexercise elevation in markers of the ubiquitin proteasome system or the breakdown of intact proteins.NEW & NOTEWORTHY The impact of postexercise leucine ingestion on processes of skeletal muscle breakdown in older adults is not well understood. Additional postexercise leucine ingestion appears to further reduce autophagy, but it does not interfere with the increase in ubiquitin proteasome system markers or the breakdown of intact proteins in skeletal muscle of older men. Postexercise leucine ingestion may promote a healthier protein pool and favorable muscle adaptations in older adults through greater accretion of myofibrillar proteins.

Protein Supplementation Has Minimal Effects on Muscle Adaptations during Resistance Exercise Training in Young Men: A Double-Blind Randomized Clinical Trial.

BACKGROUND: To our knowledge the efficacy of soy-dairy protein blend (PB) supplementation with resistance exercise training (RET) has not been evaluated in a longitudinal study. OBJECTIVE: Our aim was to determine the effect of PB supplementation during RET on muscle adaptation. METHODS: In this double-blind randomized clinical trial, healthy young men [18-30 y; BMI (in kg/m(2)): 25 ± 0.5] participated in supervised whole-body RET at 60-80% 1-repetition maximum (1-RM) for 3 d/wk for 12 wk with random assignment to daily receive 22 g PB (n = 23), whey protein (WP) isolate (n = 22), or an isocaloric maltodextrin (carbohydrate) placebo [(MDP) n = 23]. Serum testosterone, muscle strength, thigh muscle thickness (MT), myofiber cross-sectional area (mCSA), and lean body mass (LBM) were assessed before and after 6 and 12 wk of RET. RESULTS: All treatments increased LBM (P < 0.001). ANCOVA did not identify an overall treatment effect at 12 wk (P = 0.11). There tended to be a greater change in LBM from baseline to 12 wk in the PB group than in the MDP group (0.92 kg; 95% CI: -0.12, 1.95 kg; P = 0.09); however, changes in the WP and MDP groups did not differ. Pooling data from combined PB and WP treatments showed a trend for greater change in LBM from baseline to 12 wk compared with MDP treatment (0.69 kg; 95% CI: -0.08, 1.46 kg; P = 0.08). Muscle strength, mCSA, and MT increased (P < 0.05) similarly for all treatments and were not different (P > 0.10) between treatments. Testosterone was not altered. CONCLUSIONS: PB supplementation during 3 mo of RET tended to slightly enhance gains in whole-body and arm LBM, but not leg muscle mass, compared with RET without protein supplementation. Although protein supplementation minimally enhanced gains in LBM of healthy young men, there was no enhancement of gains in strength. This trial was registered at clinicaltrials.gov as NCT01749189.

Acute and chronic safety and efficacy of dose dependent creatine nitrate supplementation and exercise performance.

BACKGROUND: Creatine monohydrate (CrM) and nitrate are popular supplements for improving exercise performance; yet have not been investigated in combination. We performed two studies to determine the safety and exercise performance-characteristics of creatine nitrate (CrN) supplementation. METHODS: Study 1 participants (N = 13) ingested 1.5 g CrN (CrN-Low), 3 g CrN (CrN-High), 5 g CrM or a placebo in a randomized, crossover study (7d washout) to determine supplement safety (hepatorenal and muscle enzymes, heart rate, blood pressure and side effects) measured at time-0 (unsupplemented), 30-min, and then hourly for 5-h post-ingestion. Study 2 participants (N = 48) received the same CrN treatments vs. 3 g CrM in a randomized, double-blind, 28d trial inclusive of a 7-d interim testing period and loading sequence (4 servings/d). Day-7 and d-28 measured Tendo™ bench press performance, Wingate testing and a 6x6-s bicycle ergometer sprint. Data were analyzed using a GLM and results are reported as mean ± SD or mean change ± 95 % CI. RESULTS: In both studies we observed several significant, yet stochastic changes in blood markers that were not indicative of potential harm or consistent for any treatment group. Equally, all treatment groups reported a similar number of minimal side effects. In Study 2, there was a significant increase in plasma nitrates for both CrN groups by d-7, subsequently abating by d-28. Muscle creatine increased significantly by d-7 in the CrM and CrN-High groups, but then decreased by d-28 for CrN-High. By d-28, there were significant increases in bench press lifting volume (kg) for all groups (PLA, 126.6, 95 % CI 26.3, 226.8; CrM, 194.1, 95 % CI 89.0, 299.2; CrN-Low, 118.3, 95 % CI 26.1, 210.5; CrN-High, 267.2, 95 % CI 175.0, 359.4, kg). Only the CrN-High group was significantly greater than PLA (p < 0.05). Similar findings were observed for bench press peak power (PLA, 59.0, 95 % CI 4.5, 113.4; CrM, 68.6, 95 % CI 11.4, 125.8; CrN-Low, 40.9, 95 % CI -9.2, 91.0; CrN-High, 60.9, 95 % CI 10.8, 111.1, W) and average power. CONCLUSIONS: Creatine nitrate delivered at 3 g was well-tolerated, demonstrated similar performance benefits to 3 g CrM, in addition, within the confines of this study, there were no safety concerns.

Application of gas chromatography-tandem mass spectrometry (GC/MS/MS) for the analysis of deuterium enrichment of water.

Incorporation of deuterium from deuterium oxide ((2) H2 O) into biological components is a commonly used approach in metabolic studies. Determining the dilution of deuterium in the body water (BW) pool can be used to estimate body composition. We describe three sensitive GC/MS/MS methods to measure water enrichment in BW. Samples were reacted with NaOH and U-(13) C3 -acetone in an autosampler vial to promote deuterium exchange with U-(13) C3 -acetone hydrogens. Headspace injections were made of U-(13) C3 -acetone-saturated air onto a 30-m DB-1MS column in electron impact-mode. Subjects ingested 30 ml (2) H2 O, and plasma samples were collected. BW was determined by standard equation. Dual-energy X-ray absorptiometry scans were performed to calculate body mass, body volume and bone mineral content. A four-compartmental model was used to estimate body composition (fat and fat free mass). Full-scan experiments generated an m/z 45 peak and to a lesser extent an m/z 61 peak. Product fragment ions further monitored included 45 and 46 using selected ion monitoring (Method1), the 61 > 45 and 62 > 46 transition using multiple reaction monitoring (MRM; Method2) and the neutral loss, 62 > 45, transition (Method3). MRM methods were optimized for collision energy (CE) and collision-induced dissociation (CID) argon gas pressure with 6 eV CE and 1.5 mTorr CID gas being optimal. Method2 was used for final determination of (2) H2 O enrichment of subjects because of lower natural background. We have developed a sensitive method to determine (2) H2 O enrichment in BW to enable measurement of FM and FFM.

Leucine-enriched amino acid ingestion after resistance exercise prolongs myofibrillar protein synthesis and amino acid transporter expression in older men.

BACKGROUND: Postexercise protein or amino acid ingestion restores muscle protein synthesis in older adults and represents an important therapeutic strategy for aging muscle. However, the precise nutritional factors involved are unknown. OBJECTIVE: The purpose of this study was to determine the role of increased postexercise Leu ingestion on skeletal muscle myofibrillar protein synthesis (MyoPS), mammalian/mechanistic target of rapamycin complex 1 signaling, and amino acid transporter (AAT) mRNA expression in older men over a 24-h post-resistance exercise (RE) time course. METHODS: During a stable isotope infusion trial (l-[ring-(13)C6]Phe; l-[1-(13)C]Leu), older men performed RE and, at 1 h after exercise, ingested 10 g of essential amino acids (EAAs) containing either a Leu content similar to quality protein (control, 1.85 g of Leu, n = 7) or enriched Leu (LEU; 3.5 g of Leu, n = 8). Muscle biopsies (vastus lateralis) were obtained at rest and 2, 5, and 24 h after exercise. RESULTS: p70 S6 kinase 1 phosphorylation was increased in each group at 2 h (P < 0.05), whereas 4E binding protein 1 phosphorylation increased only in the LEU group (P < 0.05). MyoPS was similarly increased (∼90%) above basal in each group at 5 h (P < 0.05) and remained elevated (∼90%) at 24 h only in the LEU group (P < 0.05). The mRNA expression of select AATs was increased at 2 and 5 h in each group (P < 0.05), but AAT expression was increased at 24 h only in the LEU group (P < 0.05). CONCLUSIONS: Leu-enriched EAA ingestion after RE may prolong the anabolic response and sensitivity of skeletal muscle to amino acids in older adults. These data emphasize the potential importance of adequate postexercise Leu ingestion to enhance the response of aging muscle to preventive or therapeutic exercise-based rehabilitation programs. This trial was registered at clinicaltrials.gov as NCT00891696.

Activation of mTORC1 signaling and protein synthesis in human muscle following blood flow restriction exercise is inhibited by rapamycin.

Restriction of blood flow to a contracting muscle during low-intensity resistance exercise (BFR exercise) stimulates mTORC1 signaling and protein synthesis in human muscle within 3 h postexercise. However, there is a lack of mechanistic data to provide a direct link between mTORC1 activation and protein synthesis in human skeletal muscle following BFR exercise. Therefore, the primary purpose of this study was to determine whether mTORC1 signaling is necessary for stimulating muscle protein synthesis after BFR exercise. A secondary aim was to describe the 24-h time course response in muscle protein synthesis and breakdown following BFR exercise. Sixteen healthy young men were randomized to one of two groups. Both the control (CON) and rapamycin (RAP) groups completed BFR exercise; however, RAP was administered 16 mg of the mTOR inhibitor rapamycin 1 h prior to BFR exercise. BFR exercise consisted of four sets of leg extension exercise at 20% of 1 RM. Muscle biopsies were collected from the vastus lateralis before exercise and at 3, 6, and 24 h after BFR exercise. Mixed-muscle protein fractional synthetic rate increased by 42% at 3 h postexercise and 69% at 24 h postexercise in CON, whereas this increase was inhibited in the RAP group. Phosphorylation of mTOR (Ser(2448)) and S6K1 (Thr(389)) was also increased in CON but inhibited in RAP. Mixed-muscle protein breakdown was not significantly different across time or groups. We conclude that activation of mTORC1 signaling and protein synthesis in human muscle following BFR exercise is inhibited in the presence of rapamycin.

Insulin increases mRNA abundance of the amino acid transporter SLC7A5/LAT1 via an mTORC1-dependent mechanism in skeletal muscle cells.

Abstract Amino acid transporters (AATs) provide a link between amino acid availability and mammalian/mechanistic target of rapamycin complex 1 (mTORC1) activation although the direct relationship remains unclear. Previous studies in various cell types have used high insulin concentrations to determine the role of insulin on mTORC1 signaling and AAT mRNA abundance. However, this approach may limit applicability to human physiology. Therefore, we sought to determine the effect of insulin on mTORC1 signaling and whether lower insulin concentrations stimulate AAT mRNA abundance in muscle cells. We hypothesized that lower insulin concentrations would increase mRNA abundance of select AAT via an mTORC1-dependent mechanism in C2C12 myotubes. Insulin (0.5 nmol/L) significantly increased phosphorylation of the mTORC1 downstream effectors p70 ribosomal protein S6 kinase 1 (S6K1) and ribosomal protein S6 (S6). A low rapamycin dose (2.5 nmol/L) significantly reduced the insulin-(0.5 nmol/L) stimulated S6K1 and S6 phosphorylation. A high rapamycin dose (50 nmol/L) further reduced the insulin-(0.5 nmol/L) stimulated phosphorylation of S6K1 and S6. Insulin (0.5 nmol/L) increased mRNA abundance of SLC38A2/SNAT2 (P ≤ 0.043) and SLC7A5/LAT1 (P ≤ 0.021) at 240 min and SLC36A1/PAT1 (P = 0.039) at 30 min. High rapamycin prevented an increase in SLC38A2/SNAT2 (P = 0.075) and SLC36A1/PAT1 (P ≥ 0.06) mRNA abundance whereas both rapamycin doses prevented an increase in SLC7A5/LAT1 (P ≥ 0.902) mRNA abundance. We conclude that a low insulin concentration increases SLC7A5/LAT1 mRNA abundance in an mTORC1-dependent manner in skeletal muscle cells.

Deficiency in repair of the mitochondrial genome sensitizes proliferating myoblasts to oxidative damage.

Reactive oxygen species (ROS), generated as a by-product of mitochondrial oxidative phosphorylation, are particularly damaging to the genome of skeletal muscle because of their high oxygen consumption. Proliferating myoblasts play a key role during muscle regeneration by undergoing myogenic differentiation to fuse and restore damaged muscle. This process is severely impaired during aging and in muscular dystrophies. In this study, we investigated the role of oxidatively damaged DNA and its repair in the mitochondrial genome of proliferating skeletal muscle progenitor myoblasts cells and their terminally differentiated product, myotubes. Using the C2C12 cell line as a well-established model for skeletal muscle differentiation, we show that myoblasts are highly sensitive to ROS-mediated DNA damage, particularly in the mitochondrial genome, due to deficiency in 5' end processing at the DNA strand breaks. Ectopic expression of the mitochondrial-specific 5' exonuclease, EXOG, a key DNA base excision/single strand break repair (BER/SSBR) enzyme, in myoblasts but not in myotubes, improves the cell's resistance to oxidative challenge. We linked loss of myoblast viability by activation of apoptosis with deficiency in the repair of the mitochondrial genome. Moreover, the process of myoblast differentiation increases mitochondrial biogenesis and the level of total glutathione. We speculate that our data may provide a mechanistic explanation for depletion of proliferating muscle precursor cells during the development of sarcopenia, and skeletal muscle dystrophies.

Short-term bed rest increases TLR4 and IL-6 expression in skeletal muscle of older adults.

Bed rest induces significant loss of leg lean mass in older adults. Systemic and tissue inflammation also accelerates skeletal muscle loss, but it is unknown whether inflammation is associated to inactivity-induced muscle atrophy in healthy older adults. We determined if short-term bed rest increases toll-like receptor 4 (TLR4) signaling and pro-inflammatory markers in older adult skeletal muscle biopsy samples. Six healthy, older adults underwent seven consecutive days of bed rest. Muscle biopsies (vastus lateralis) were taken after an overnight fast before and at the end of bed rest. Serum cytokine expression was measured before and during bed rest. TLR4 signaling and cytokine mRNAs associated with pro- and anti-inflammation and anabolism were measured in muscle biopsy samples using Western blot analysis and qPCR. Participants lost ∼4% leg lean mass with bed rest. We found that after bed rest, muscle levels of TLR4 protein expression and interleukin-6 (IL-6), nuclear factor-κB1, interleukin-10, and 15 mRNA expression were increased after bed rest (P < 0.05). Additionally, the cytokines interferon-γ, and macrophage inflammatory protein-1ß, were elevated in serum samples following bed rest (P < 0.05). We conclude that short-term bed rest in older adults modestly increased some pro- and anti-inflammatory cytokines in muscle samples while systemic changes in pro-inflammatory cytokines were mostly absent. Upregulation of TLR4 protein content suggests that bed rest in older adults increases the capacity to mount an exaggerated, and perhaps unnecessary, inflammatory response in the presence of specific TLR4 ligands, e.g., during acute illness.

Protein blend ingestion following resistance exercise promotes human muscle protein synthesis.

High-quality proteins such as soy, whey, and casein are all capable of promoting muscle protein synthesis postexercise by activating the mammalian target of rapamycin (mTORC1) signaling pathway. We hypothesized that a protein blend of soy and dairy proteins would capitalize on the unique properties of each individual protein and allow for optimal delivery of amino acids to prolong the fractional synthetic rate (FSR) following resistance exercise (RE). In this double-blind, randomized, clinical trial, 19 young adults were studied before and after ingestion of ∼19 g of protein blend (PB) or ∼18 g whey protein (WP) consumed 1 h after high-intensity leg RE. We examined mixed-muscle protein FSR by stable isotopic methods and mTORC1 signaling with western blotting. Muscle biopsies from the vastus lateralis were collected at rest (before RE) and at 3 postexercise time points during an early (0-2 h) and late (2-4 h) postingestion period. WP ingestion resulted in higher and earlier amplitude of blood branched-chain amino acid (BCAA) concentrations. PB ingestion created a lower initial rise in blood BCAA but sustained elevated levels of blood amino acids later into recovery (P < 0.05). Postexercise FSR increased equivalently in both groups during the early period (WP, 0.078 ± 0.009%; PB, 0.088 ± 0.007%); however, FSR remained elevated only in the PB group during the late period (WP, 0.074 ± 0.010%; PB, 0.087 ± 0.003%) (P < 0.05). mTORC1 signaling similarly increased between groups, except for no increase in S6K1 phosphorylation in the WP group at 5 h postexercise (P < 0.05). We conclude that a soy-dairy PB ingested following exercise is capable of prolonging blood aminoacidemia, mTORC1 signaling, and protein synthesis in human skeletal muscle and is an effective postexercise nutritional supplement.

Rapamycin does not affect post-absorptive protein metabolism in human skeletal muscle.

UNLABELLED: Administration of the mTORC1 inhibitor, rapamycin, to humans blocks the increase in skeletal muscle protein synthesis in response to resistance exercise or amino acid ingestion. OBJECTIVE: To determine whether rapamycin administration influences basal post-absorptive protein synthesis or breakdown in human skeletal muscle. MATERIALS/METHODS: Six young (26±2 years) subjects were studied during two separate trials, in which each trial was divided into two consecutive 2 h basal periods. The trials were identical except during one trial a single oral dose (16 mg) of rapamycin was administered immediately prior to the second basal period. Muscle biopsies were obtained from the vastus lateralis at 0, 2, and 4 h to examine protein synthesis, mTORC1 signaling, and markers of autophagy (LC3B-I and LC3B-II protein) associated with each 2 h basal period. RESULTS: During the Control trial, muscle protein synthesis, whole body protein breakdown (phenylalanine Ra), mTORC1 signaling, and markers of autophagy were similar between both basal periods (p>0.05). During the Rapamycin trial, these variables were similar to the Control trial (p>0.05) and were unaltered by rapamycin administration (p>0.05). Thus, post-absorptive muscle protein metabolism and mTORC1 signaling were not affected by rapamycin administration. CONCLUSIONS: Short-term rapamycin administration may only impair protein synthesis in human skeletal muscle when combined with a stimulus such as resistance exercise or increased amino acid availability.

Skeletal muscle autophagy and protein breakdown following resistance exercise are similar in younger and older adults.

BACKGROUND: The loss of skeletal muscle mass and strength during aging, sarcopenia, increases the risk for falls and dependency. Resistance exercise (RE) training is effective at improving muscle mass and strength in older adults; however, aging is associated with reduced training-induced hypertrophy. Recent research has illustrated an impaired muscle protein synthetic response following an acute bout of RE in older adults but much less is known regarding the effect of acute RE on muscle protein breakdown (MPB). We hypothesize that the ubiquitin proteasome system and the autophagosomal-lysosomal system may regulate the overall rate of MPB during postexercise recovery. METHODS: Muscle biopsies of the vastus lateralis were sampled from 16 older (age = 70±2 years) and 16 younger (age = 27±2 years) participants at baseline and at 3, 6, and 24 hours following an acute bout of RE. In conjunction with stable isotopic techniques to measure MPB, we utilized immunoblotting and RT-PCR to examine protein and mRNA expression for key signaling molecules in both the ubiquitin proteasome system and the autophagosomal-lysosomal system. RESULTS: MuRF1 mRNA expression increased, whereas GABARAP mRNA decreased after RE in both younger and older adults (p < .05). The LC3B-II/LC3B-I protein ratio decreased in both groups after RE (p < .05), but MPB was not different 24 hour post-RE in either group (p > .05). CONCLUSIONS: Aging does not influence skeletal MPB, autophagy, or the ubiquitin proteasome system following an acute bout of RE. Therefore, targeting the muscle protein synthesis response to exercise may hold more promise in the prevention of sarcopenia.

PAX7+ satellite cells in young and older adults following resistance exercise.

INTRODUCTION: Resistance exercise (RE) stimulates a muscle protein anabolic response partially through enhanced satellite cell (SC) activity, however, age- and gender-related changes in SC content over a 24-h time course are not known. METHODS: Ten young (27 ± 2 years) men and women and 11 older (70 ± 2 years) men and women performed an acute bout of RE. Myofiber and SC characteristics were determined from muscle biopsies of the vastus lateralis using immunohistochemistry. Immunoblotting was used to determine phosphorylation of cyclin-dependent kinase-2 and protein expression of p27(Kip1) and cyclin D1. RESULTS: Pax7+ SC were significantly increased in young men 24 h following RE. Percent SC were significantly increased in older women at 6 and 24 h following RE. Aging decreased myonuclear domain and increased protein expression of p27(Kip1) . CONCLUSIONS: An acute bout of RE increases SC content in young men at 24 h and older women at 6 and 24 h.

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.

Reactive hyperemia is not responsible for stimulating muscle protein synthesis following blood flow restriction exercise.

Blood flow restriction (BFR) to contracting skeletal muscle during low-intensity resistance exercise training increases muscle strength and size in humans. However, the mechanism(s) underlying these effects are largely unknown. We have previously shown that mammalian target of rapamycin complex 1 (mTORC1) signaling and muscle protein synthesis (MPS) are stimulated following an acute bout of BFR exercise. The purpose of this study was to test the hypothesis that reactive hyperemia is the mechanism responsible for stimulating mTORC1 signaling and MPS following BFR exercise. Six young men (24 ± 2 yr) were used in a randomized crossover study consisting of two exercise trials: low-intensity resistance exercise with BFR (BFR trial) and low-intensity resistance exercise with sodium nitroprusside (SNP), a pharmacological vasodilator infusion into the femoral artery immediately after exercise to simulate the reactive hyperemia response after BFR exercise (SNP trial). Postexercise mixed-muscle fractional synthetic rate from the vastus lateralis increased by 49% in the BFR trial (P < 0.05) with no change in the SNP trial (P > 0.05). BFR exercise increased the phosphorylation of mTOR, S6 kinase 1, ribosomal protein S6, ERK1/2, and Mnk1-interacting kinase 1 (P < 0.05) with no changes in mTORC1 signaling in the SNP trial (P > 0.05). We conclude that reactive hyperemia is not a primary mechanism for BFR exercise-induced mTORC1 signaling and MPS. Further research is necessary to elucidate the cellular mechanism(s) responsible for the increase in mTOR signaling, MPS, and hypertrophy following acute and chronic BFR exercise.

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

BACKGROUND: Sarcopenia, 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. METHODS: We 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. RESULTS: Increased 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). CONCLUSIONS: We 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.

Exercise, amino acids, and aging in the control of human muscle protein synthesis.

In this review, we discuss recent research in the field of human skeletal muscle protein metabolism characterizing the acute regulation of mammalian target of rapamycin complex (mTORC) 1 signaling and muscle protein synthesis (MPS) by exercise, amino acid nutrition, and aging. Resistance exercise performed in the fasted state stimulates mixed MPS within 1 h after exercise, which can remain elevated for 48 h. We demonstrate that the activation of mTORC1 signaling (and subsequently enhanced translation initiation) is required for the contraction-induced increase in MPS. In comparison, low-intensity blood flow restriction (BFR) exercise stimulates MPS and mTORC1 signaling to an extent similar to traditional, high-intensity resistance exercise. We also show that mTORC1 signaling is required for the essential amino acid (EAA)-induced increase in MPS. Ingestion of EAAs (or protein) shortly after resistance exercise enhances MPS and mTORC1 signaling compared with resistance exercise or EAAs alone. In older adults, the ability of the skeletal muscle to respond to anabolic stimuli is impaired. For example, in response to an acute bout of resistance exercise, older adults are less able to activate mTORC1 or increase MPS during the first 24 h of postexercise recovery. However, BFR exercise can overcome this impairment. Aging is not associated with a reduced response to EAAs provided the EAA content is sufficient. Therefore, we propose that exercise combined with EAA should be effective not only in improving muscle repair and growth in response to training in athletes, but that strategies such as EAA combined with resistance exercise (or BFR exercise) may be very useful as a countermeasure for sarcopenia and other clinical conditions associated with muscle wasting.