Protein dose requirements to maximize skeletal muscle protein synthesis after repeated bouts of resistance exercise in young trained women.

Studies examining the effect of protein (PRO) feeding on post resistance exercise (RE) muscle protein synthesis (MPS) have primarily been performed in men, and little evidence is available regarding the quantity of PRO required to maximally stimulate MPS in trained women following repeated bouts of RE. We therefore quantified acute (4 h and 8 h) and extended (24 h) effects of two bouts of resistance exercise, alongside protein-feeding, in women, and the PRO requirement to maximize MPS. Twenty-four RE trained women (26.6 ± 0.7 years, mean ± SEM) performed two bouts of whole-body RE (3 × 8 repetitions/maneuver at 75% 1-repetition maximum) 4 h apart, with post-exercise ingestion of 15 g, 30 g, or 60 g whey PRO (n = 8/group). Saliva, venous blood, and a vastus lateralis muscle biopsy were taken at 0 h, 4 h, 8 h, and 24 h post-exercise. Plasma leucine and branched chain amino acids were quantified using gas chromatography mass spectrometry (GC-MS) after ingestion of D2 O. Fifteen grams PRO did not alter plasma leucine concentration or myofibrillar synthetic rate (MyoFSR). Thirty and sixty grams PRO increased plasma leucine concentration above baseline (105.5 ± 5.3 µM; 120.2 ± 7.4 µM, respectively) at 4 h (151.5 ± 8.2 µM, p < 0.01; 224.8 ± 16.0 µM, p < 0.001, respectively) and 8 h (176.0 ± 7.3 µM, p < 0.001; 281.7 ± 21.6 µM, p < 0.001, respectively). Ingestion of 30 g PRO increased MyoFSR above baseline (0.068 ± 0.005%/h) from 0 to 4 h (0.140 ± 0.021%/h, p < 0.05), 0 to 8 h (0.121 ± 0.012%/h, p < 0.001), and 0 to 24 h (0.099 ± 0.011%/h, p < 0.01). Ingestion of 60 g PRO increased MyoFSR above baseline (0.063 ± 0.003%/h) from 0 to 4 h (0.109 ± 0.011%/h, p < 0.01), 0 to 8 h (0.093 ± 0.008%/h, p < 0.01), and 0 to 24 h (0.086 ± 0.006%/h, p < 0.01). Post-exercise ingestion of 30 g or 60 g PRO, but not 15 g, acutely increased MyoFSR following two consecutive bouts of RE and extended the anabolic window over 24 h. There was no difference between the 30 g and 60 g responses.

Omega-3 supplementation during unilateral resistance exercise training in older women: A within subject and double-blind placebo-controlled trial.

BACKGROUND & AIMS: The skeletal muscle anabolic effects of n-3 polyunsaturated fatty acids (n-3 PUFA) appear favoured towards women; a property that could be exploited in older women who typically exhibit poor muscle growth responses to resistance exercise training (RET). Here we sought to generate novel insights into the efficacy and mechanisms of n-3 PUFA alongside short-term RET in older women. METHODS: We recruited 16 healthy older women (Placebo n = 8 (PLA): 67±1y, n-3 PUFA n = 8: 64±1y) to a randomised double-blind placebo-controlled trial (n-3 PUFA; 3680 mg/day versus PLA) of 6 weeks fully-supervised progressive unilateral RET (i.e. 6 × 8 reps, 75% 1-RM, 3/wk-1). Strength was assessed by knee extensor 1-RM and isokinetic dynamometry âˆ¼ every 10 d. Thigh fat free mass (TFFM) was measured by DXA at 0/3/6 weeks. Bilateral vastus lateralis (VL) biopsies at 0/2/4/6 weeks with deuterium oxide (D2O) dosing were used to determine MPS responses for 0-2 and 4-6 weeks. Further, fibre cross sectional area (CSA), myonuclei number and satellite cell (SC) number were assessed, alongside muscle anabolic/catabolic signalling via immunoblotting. RESULTS: RET increased 1-RM equally in the trained leg of both groups (+23 ± 5% n-3 PUFA vs. +25 ± 5% PLA (both P < 0.01)) with no significant increase in maximum voluntary contraction (MVC) (+10 ± 6% n-3 PUFA vs. +13 ± 5% PLA). Only the n-3 PUFA group increased TFFM (3774 ± 158 g to 3961 ± 151 g n-3 PUFA (P < 0.05) vs. 3406 ± 201 g to 3561 ± 170 PLA) and type II fibre CSA (3097 ± 339 µm2 to 4329 ± 264 µm2 n-3 PUFA (P < 0.05) vs. 2520 ± 316 µm2 to 3467 ± 303 µm2 in PL) with RET. Myonuclei number increased equally in n-3 PUFA and PLA in both type I and type II fibres, with no change in SC number. N-3 PUFA had no added benefit on muscle protein synthesis (MPS), however, during weeks 4-6 of RET, absolute synthesis rates (ASR) displayed a trend to increase with n-3 PUFA only (5.6 ± 0.3 g d-1 to 7.1 ± 0.5 g d-1 n-3 PUFA (P = 0.09) vs. 5.5 ± 0.5 g d-1 to 6.5 ± 0.5 g d-1 PLA). Further, the n-3 PUFA group displayed greater 4EBP1 activation after acute RE at 6 weeks. CONCLUSION: n3-PUFA enhanced RET gains in muscle mass through type II fibre hypertrophy, with data suggesting a role for MPS rather than via SC recruitment. As such, the present study adds to a literature base illustrating the apparent enhancement of muscle hypertrophy with RET in older women fed adjuvant n3-PUFA.

A collagen hydrolysate/milk protein-blend stimulates muscle anabolism equivalently to an isoenergetic milk protein-blend containing a greater quantity of essential amino acids in older men.

BACKGROUND & AIMS: Nutritional composition is key for skeletal muscle maintenance into older age. Yet the acute effects of collagen protein blended with other protein sources, in relation to skeletal muscle anabolism, are ill-defined. We investigated human muscle protein synthesis (MPS) responses to a 20 g blend of collagen protein hydrolysate + milk protein (CP+MP, 125 ml) oral nutritional supplement (ONS) vs. 20 g non-blended milk protein source (MP, 200 ml) ONS, in older adults. METHODS: Healthy older men (N = 8, 71±1 y, BMI: 27±1 kg·m-2) underwent a randomized trial of 20 g protein, from either a CP+MP blend (Fresubin®3.2 kcal DRINK), or a kcal-matched (higher in essential amino acids (EAA) ONS of MP alone. Vastus lateralis (VL) MPS and plasma AA were determined using stable isotope-tracer mass spectrometry; anabolic signaling was quantified via immuno-blotting in VL biopsies taken at baseline and 2/4 h after ONS feeding. Plasma insulin was measured via enzyme-linked immunosorbent assay (ELISA). Measures were taken at rest, after the feed (FED) and after the feed + exercise (FED-EX) conditions (unilateral leg exercise, 6 × 8, 75% 1-RM). RESULTS: MP resulted in a greater increase in plasma leucine (MP mean: 152 ± 6 µM, CP+MP mean: 113 ± 4 µM (Feed P < 0.001) and EAA (MP mean: 917 ± 25 µM, CP+MP mean: 786 ± 15 µM (Feed P < 0.01) than CP+MP. CP + MP increased plasma glycine (peak 385 ± 57 µM (P < 0.05)), proline (peak 323 ± 29 µM (P < 0.01)) and non-essential amino acids (NEAA) (peak 1621 ± 107 µM (P < 0.01)) with MP showing no increase. Plasma insulin increased in both trials (CP+MP: 58 ± 10 mU/mL (P < 0.01), MP: 42 ± 6 mU/mL (P < 0.01), with peak insulin greater with CP+MP vs. MP (P < 0.01). MPS demonstrated equivalent increases in response to CP+MP and MP under both FED (MP: 0.039 ± 0.005%/h to 0.081 ± 0.014%/h (P < 0.05), CP+MP: 0.042 ± 0.004%/h to 0.085 ± 0.007%/h (P < 0.05)) and FED-EX (MP: 0.039 ± 0.005%/h to 0.093 ± 0.013%/h (P < 0.01), CP+MP: 0.042 ± 0.004%/h to 0.105 ± 0.015%/h, (P < 0.01)) conditions. FED muscle p-mTOR fold-change from baseline increased to a greater extent with CP+MP vs. MP (P < 0.05), whilst FED-EX muscle p-eEF2 fold-change from baseline decreased to a greater extent with CP+MP vs. MP (P < 0.05); otherwise anabolic signaling responses were indistinguishable. CONCLUSION: Fresubin®3.2 kcal DRINK, which contains a 20 g mixed blend of CP+MP, resulted in equivalent MPS responses to MP alone. Fresubin® 3.2 Kcal DRINK may provide a suitable alternative to MP for use in older adults and a convenient way to supplement calories and protein to improve patient adherence and mitigate muscle mass loss.

A double-blind placebo controlled trial into the impacts of HMB supplementation and exercise on free-living muscle protein synthesis, muscle mass and function, in older adults.

Age-related sarcopenia and dynapenia are associated with frailty and metabolic diseases. Resistance exercise training (RET) adjuvant to evidence-based nutritional intervention(s) have been shown as mitigating strategies. Given that ß-hydroxy-ß-methyl-butyrate (HMB) supplementation during RET improves lean body mass in younger humans, and that we have shown that HMB acutely stimulates muscle protein synthesis (MPS) and inhibits breakdown; we hypothesized that chronic supplementation of HMB free acid (HMB-FA) would enhance MPS and muscle mass/function in response to RET in older people. We recruited 16 healthy older men (Placebo (PLA): 68.5 ± 1.0 y, HMB-FA: 67.8 ± 1.15 y) for a randomised double-blind-placebo controlled trial (HMB-FA 3 × 1 g/day vs. PLA) involving a 6-week unilateral progressive RET regime (6 × 8 repetitions, 75% 1-RM, 3 · wk-1). Deuterium oxide (D2O) dosing was performed over the first two weeks (0-2 wk) and last two weeks (4-6 wk) with bilateral vastus lateralis (VL) biopsies at 0-2 and 4-6 wk (each time 75 ± 2 min after a single bout of resistance exercise (RE)) for quantification of early and later MPS responses and post-RE myogenic gene expression. Thigh lean mass (TLM) was measured by DXA, VL thickness and architecture (fibre length and pennation angle) by ultrasound at 0/3/6 wk, and strength by knee extensor 1-RM testing and MVC by isokinetic dynamometry (approx. every 10 days). RET induced strength increases (1-RM) in the exercised leg of both groups (398 ± 22N to 499 ± 30N HMB-FA vs. 396 ± 29N to 510 ± 43N PLA (both P < 0.05)). In addition, maximal voluntary contraction (MVC) also increased (179 ± 12 Nm to 203 ± 12 Nm HMB-FA vs. 185 ± 10 Nm to 217 ± 11 Nm PLA (both P < 0.05); with no group differences. VL muscle thickness increased significantly in the exercised leg in both groups, with no group differences. TLM (by DXA) rose to significance only in the HMB-FA group (by 5.8%-5734 ± 245 g p = 0.015 vs. 3.0% to 5644 ± 323 g P = 0.06 in PLA). MPS remained unchanged in the untrained legs (UT) 0-2 weeks being 1.06 ± 0.08%.d-1 (HMB-FA) and 1.14 ± 0.09%.d-1 (PLA), the trained legs (T) exhibited increased MPS in the HMB-FA group only at 0-2-weeks (1.39 ± 0.10%.d-1, P < 0.05) compared with UT: but was not different at 4-6-weeks: 1.26 ± 0.05%.d-1. However, there were no significant differences in MPS between the HMB-FA and PLA groups at any given time point and no significant treatment interaction observed. We also observed significant inductions of c-Myc gene expression following each acute RE bout, with no group differences. Further, there were no changes in any other muscle atrophy/hypertrophy or myogenic transcription factor genes we measured. RET with adjuvant HMB-FA supplements in free-living healthy older men did not enhance muscle strength or mass greater than that of RET alone (PLA). That said, only HMB-FA increased TLM, supported by early increases in chronic MPS. As such, chronic HMB-FA supplementation may result in long term benefits in older males, however longer and larger studies may be needed to fully determine the potential effects of HMB-FA supplementation; translating to any functional benefit.

Impact of the calcium form of ß-hydroxy-ß-methylbutyrate upon human skeletal muscle protein metabolism.

BACKGROUND & AIMS: ß-hydroxy-ß-methylbutyrate (HMB) is purported as a key nutritional supplement for the preservation of muscle mass in health, disease and as an ergogenic aid in exercise. Of the two available forms of HMB (calcium (Ca-HMB) salt or free acid (FA-HMB)) - differences in plasma bioavailability have been reported. We previously reported that ∼3 g oral FA-HMB increased muscle protein synthesis (MPS) and reduced muscle protein breakdown (MPB). The objective of the present study was to quantify muscle protein metabolism responses to oral Ca-HMB. METHODS: Eight healthy young males received a primed constant infusion of 1,2 13C2 leucine and 2H5 phenylalanine to assess MPS (by tracer incorporation in myofibrils) and MPB (via arterio-venous (A-V) dilution) at baseline and following provision of ∼3 g of Ca-HMB; muscle anabolic (MPS) and catabolic (MPB) signalling was assessed via immunoblotting. RESULTS: Ca-HMB led a significant and rapid (<60 min) peak in plasma HMB concentrations (483.6 ± 14.2 µM, p < 0.0001). This rise in plasma HMB was accompanied by increases in MPS (PA: 0.046 ± 0.004%/h, CaHMB: 0.072 ± 0.004%/h, p < 0001) and suppressions in MPB (PA: 7.6 ± 1.2 µmol Phe per leg min-1, Ca-HMB: 5.2 ± 0.8 µmol Phe per leg min-1, p < 0.01). Increases in the phosphorylation of mTORc1 substrates i.e. p70S6K1 and RPS6 were also observed, with no changes detected in the MPB targets measured. CONCLUSIONS: These findings support the pro-anabolic properties of HMB via mTORc1, and show that despite proposed differences in bioavailability, Ca-HMB provides a comparable stimulation to MPS and suppression of MPB, to FA-HMB, further supporting its use as a pharmaconutrient in the modulation of muscle mass.

A novel D2O tracer method to quantify RNA turnover as a biomarker of de novo ribosomal biogenesis, in vitro, in animal models, and in human skeletal muscle.

Current methods to quantify in vivo RNA dynamics are limited. Here, we developed a novel stable isotope (D2O) methodology to quantify RNA synthesis (i.e., ribosomal biogenesis) in cells, animal models, and humans. First, proliferating C2C12 cells were incubated in D2O-enriched media and myotubes ±50 ng/ml IGF-I. Second, rat quadriceps (untrained, n = 9; 7-wk interval-"like" training, n = 13) were collected after ~3-wk D2O (70 atom %) administration, with body-water enrichment monitored via blood sampling. Finally, 10 (23 ± 1 yr) men consumed 150-ml D2O followed by 50 ml/wk and undertook 6-wk resistance exercise (6 × 8 repetitions, 75% 1-repetition maximum 3/wk) with body-water enrichment monitored by saliva sampling and muscle biopsies (for determination of RNA synthesis) at 0, 3, and 6 wk. Ribose mole percent excess (r-MPE) from purine nucleotides was analyzed via GC-MS/MS. Proliferating C2C12 cell r-MPE exhibited a rise to plateau, whereas IGF-I increased myotube RNA from 76 ± 3 to 123 ± 3 ng/µl and r-MPE by 0.39 ± 0.1% (both P < 0.01). After 3 wk, rat quadriceps r-MPE had increased to 0.25 ± 0.01% (P < 0.01) and was greater with running exercise (0.36 ± 0.02%; P < 0.01). Human muscle r-MPE increased to 0.06 ± 0.01 and 0.13 ± 0.02% at 3/6 wk, respectively, equating to synthesis rates of ~0.8%/day, increasing with resistance exercise to 1.7 ± 0.3%/day (P < 0.01) and 1.2 ± 0.1%/day (P < 0.05) at 3/6 wk, respectively. Therefore, we have developed and physiologically validated a novel technique to explore ribosomal biogenesis in a multimodal fashion.

Skeletal muscle homeostasis and plasticity in youth and ageing: impact of nutrition and exercise.

Skeletal muscles comprise a substantial portion of whole body mass and are integral for locomotion and metabolic health. Increasing age is associated with declines in both muscle mass and function (e.g. strength-related performance, power) with declines in muscle function quantitatively outweighing those in muscle volume. The mechanisms behind these declines are multi-faceted involving both intrinsic age-related metabolic dysregulation and environmental influences such as nutritional and physical activity. Ageing is associated with a degree of 'anabolic resistance' to these key environmental inputs, which likely accelerates the intrinsic processes driving ageing. On this basis, strategies to sensitize and/or promote anabolic responses to nutrition and physical activity are likely to be imperative in alleviating the progression and trajectory of sarcopenia. Both resistance- and aerobic-type exercises are likely to confer functional and health benefits in older age, and a clutch of research suggests that enhancement of anabolic responsiveness to exercise and/or nutrition may be achieved by optimizing modifications of muscle-loading paradigms (workload, volume, blood flow restriction) or nutritional support (e.g. essential amino acid/leucine) patterns. Nonetheless, more work is needed in which a more holistic view in ageing studies is taken into account. This should include improved characterization of older study recruits, that is physical activity/nutritional behaviours, to limit confounding variables influencing whether findings are attributable to age, or other environmental influences. Nonetheless, on balance, ageing is associated with declines in muscle mass and function and a partially related decline in aerobic capacity. There is also good evidence that metabolic flexibility is impaired in older age.