Paul T. Reidy

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.

Muscle protein synthesis and gene expression during recovery from aerobic exercise in the fasted and fed states

The purpose of this investigation was to assess mixed-muscle fractional synthesis rate (FSR) and the expression of genes involved in skeletal muscle remodeling after aerobic exercise in the fasted and fed states. Eight recreationally active males (25 ± 1 yr; Vo(2 max): 52 ± 2 ml·kg(-1)·min(-1)) performed 60-min of cycle ergometry at 72 ± 1% Vo(2 max) on two occasions in a counter-balanced design. Subjects ingested a noncaloric placebo (EX-FAST) or a beverage containing (per kg body wt): 5 kcal, 0.83 g carbohydrate, 0.37 g protein, and 0.03 g fat (EX-FED) immediately and 1 h after exercise. FSR was assessed at rest and following exercise with the use of a l-[ring (2)H(5)]-phenylalanine infusion combined with muscle biopsies at 2 and 6 h postexercise. mRNA expression was assessed at 2 and 6 h postexercise via real-time RT-PCR. FSR was higher (P < 0.05) after exercise in both EX-FAST (0.112 ± 0.010%·h(-1)) and EX-FED (0.129 ± 0.014%·h(-1)) compared with rest (0.071 ± 0.005%·h(-1)). Feeding attenuated the mRNA expression (P < 0.05) of proteolytic factors MuRF-1 (6 h) and calpain-2 (2 and 6 h) postexercise but did not alter FOXO3A, calpain-1, caspase3, or myostatin mRNA expression compared with EX-FAST. Myogenic regulatory factor (MRF4) mRNA was also attenuated (P < 0.05) at 2 and 6 h postexercise in EX-FED compared with EX-FAST. These data demonstrate that a nonexhaustive bout of aerobic exercise stimulates skeletal muscle FSR in the fasted state and that feeding does not measurably enhance FSR between 2 and 6 h after aerobic exercise. Additionally, postexercise nutrient intake attenuates the expression of factors involved in the ubiquitin-proteosome and Ca(2+)-dependent protein degradation pathways. These data provide insight into the role of feeding on muscle protein metabolism during recovery from aerobic exercise.

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.

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.

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.

Effect of age on basal muscle protein synthesis and mTORC1 signaling in a large cohort of young and older men and women

The rate of muscle loss with aging is higher in men than women. However, women have smaller muscles throughout the adult life. Protein content is a major determinant of skeletal muscle size. This study was designed to determine if age and sex differentially impact basal muscle protein synthesis and mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling. We performed a secondary data analysis on a cohort of 215 healthy, non-obese (BMI<30kg·m(-2)) young (18-40y; 74 men, 52 women) and older (60-87y; 57 men, 32 women) adults. The database contained information on physical characteristics, basal muscle protein fractional synthetic rate (FSR; n=215; stable isotope methodology) and mTORC1 signaling (n=125, Western blotting). FSR and mTORC1 signaling were measured at rest and after an overnight fast. mTORC1 and S6K1 phosphorylation were higher (p<0.05) in older subjects with no sex differences. However, there were no age or sex differences or interaction for muscle FSR (p>0.05). Body mass index, fat free mass, or body fat was not a significant covariate and did not influence the results. We conclude that age and sex do not influence basal muscle protein synthesis. However, basal mTORC1 hyperphosphorylation in the elderly may contribute to insulin resistance and the age-related anabolic resistance of skeletal muscle protein metabolism to nutrition and exercise.

Soy-Dairy Protein Blend and Whey Protein Ingestion After Resistance Exercise Increases Amino Acid Transport and Transporter Expression in Human Skeletal Muscle

Increasing amino acid availability (via infusion or ingestion) at rest or postexercise enhances amino acid transport into human skeletal muscle. It is unknown whether alterations in amino acid availability, from ingesting different dietary proteins, can enhance amino acid transport rates and amino acid transporter (AAT) mRNA expression. We hypothesized that the prolonged hyperaminoacidemia from ingesting a blend of proteins with different digestion rates postexercise would enhance amino acid transport into muscle and AAT expression compared with the ingestion of a rapidly digested protein. In a double-blind, randomized clinical trial, we studied 16 young adults at rest and after acute resistance exercise coupled with postexercise (1 h) ingestion of either a (soy-dairy) protein blend or whey protein. Phenylalanine net balance and transport rate into skeletal muscle were measured using stable isotopic methods in combination with femoral arteriovenous blood sampling and muscle biopsies obtained at rest and 3 and 5 h postexercise. Phenylalanine transport into muscle and mRNA expression of select AATs [system L amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, system A amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, cationic amino acid transporter 1/SLC7A1] increased to a similar extent in both groups (P < 0.05). However, the ingestion of the protein blend resulted in a prolonged and positive net phenylalanine balance during postexercise recovery compared with whey protein (P < 0.05). Postexercise myofibrillar protein synthesis increased similarly between groups. We conclude that, while both protein sources enhanced postexercise AAT expression, transport into muscle, and myofibrillar protein synthesis, postexercise ingestion of a protein blend results in a slightly prolonged net amino acid balance across the leg compared with whey protein.

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.

Resistance Exercise Training Alters Mitochondrial Function in Human Skeletal Muscle.

INTRODUCTION Loss of mitochondrial competency is associated with several chronic illnesses. Therefore, strategies that maintain or increase mitochondrial function will likely be of benefit in numerous clinical settings. Endurance exercise has long been known to increase mitochondrial function in the skeletal muscle. Comparatively little is known regarding the effect of resistance exercise training (RET) on skeletal muscle mitochondrial respiratory function. PURPOSE The purpose of the current study was to determine the effect of chronic resistance training on skeletal muscle mitochondrial respiratory capacity and function. METHODS Here, we studied the effect of a 12-wk RET program on skeletal muscle mitochondrial function in 11 young healthy men. Muscle biopsies were collected before and after the 12-wk training program, and mitochondrial respiratory capacity was determined in permeabilized myofibers by high-resolution respirometry. RESULTS RET increased lean body mass and quadriceps muscle strength by 4% and 15%, respectively (P < 0.001). Coupled mitochondrial respiration supported by complex I, and complex I and II substrates increased by 2- and 1.4-fold, respectively (P < 0.01). The ratio of coupled complex I-supported respiration to maximal respiration increased with RET (P < 0.05), as did complex I protein abundance (P < 0.05), whereas the substrate control ratio for succinate was reduced after RET (P < 0.001). Transcripts responsible for proteins critical to electron transfer and NAD production increased with training (P < 0.05), whereas transcripts involved in mitochondrial biogenesis were unaltered. CONCLUSIONS Collectively, 12 wk of RET resulted in qualitative and quantitative changes in skeletal muscle mitochondrial respiration. This adaptation was accompanied by modest changes in mitochondrial proteins and transcript expression. RET seems to be a means to augment the respiratory capacity and intrinsic function of skeletal muscle mitochondria.

Role of Ingested Amino Acids and Protein in the Promotion of Resistance Exercise–Induced Muscle Protein Anabolism

The goal of this critical review is to comprehensively assess the evidence for the molecular, physiologic, and phenotypic skeletal muscle responses to resistance exercise (RE) combined with the nutritional intervention of protein and/or amino acid (AA) ingestion in young adults. We gathered the literature regarding the translational response in human skeletal muscle to acute exposure to RE and protein/AA supplements and the literature describing the phenotypic skeletal muscle adaptation to RE and nutritional interventions. Supplementation of protein/AAs with RE exhibited clear protein dose-dependent effects on translational regulation (protein synthesis) through mammalian target of rapamycin complex 1 (mTORC1) signaling, which was most apparent through increases in p70 ribosomal protein S6 kinase 1 (S6K1) phosphorylation, compared with postexercise recovery in the fasted or carbohydrate-fed state. These acute findings were critically tested via long-term exposure to RE training (RET) and protein/AA supplementation, and it was determined that a diminishing protein/AA supplement effect occurs over a prolonged exposure stimulus after exercise training. Furthermore, we found that protein/AA supplements, combined with RET, produced a positive, albeit minor, effect on the promotion of lean mass growth (when assessed in >20 participants/treatment); a negligible effect on muscle mass; and a negligible to no additional effect on strength. A potential concern we discovered was that the majority of the exercise training studies were underpowered in their ability to discern effects of protein/AA supplementation. Regardless, even when using optimal methodology and large sample sizes, it is clear that the effect size for protein/AA supplementation is low and likely limited to a subset of individuals because the individual variability is high. With regard to nutritional intakes, total protein intake per day, rather than protein timing or quality, appears to be more of a factor on this effect during long-term exercise interventions. There were no differences in strength or mass/muscle mass on RET outcomes between protein types when a leucine threshold (>2 g/dose) was reached. Future research with larger sample sizes and more homogeneity in design is necessary to understand the underlying adaptations and to better evaluate the individual variability in the muscle-adaptive response to protein/AA supplementation during RET.