Key points• The branched-chain amino acid (BCAA) leucine acts as both a 'trigger' for the initiation of protein synthesis, and as a substrate for newly synthesized protein.• As a BCAA, leucine can be metabolized within skeletal muscle, leaving open the possibility that leucine metabolites might possess anabolic properties.• One metabolite in particular, β-hydroxy-β-methylbutyrate (HMB), has shown positive effects on lean body mass and strength following exercise, and in disease-related muscle wasting, yet its impact on acute human muscle protein turnover is undefined.• We report here that HMB stimulates muscle protein synthesis to a similar extent to leucine.HMB was also found to decrease muscle protein breakdown.• Our observation that HMB enhances muscle protein anabolism may partly (or wholly) underlie its pre-defined anabolic/anti-catabolic supplemental efficacy in humans.Abstract Maintenance of skeletal muscle mass is contingent upon the dynamic equilibrium (fasted losses-fed gains) in protein turnover. Of all nutrients, the single amino acid leucine (Leu) possesses the most marked anabolic characteristics in acting as a trigger element for the initiation of protein synthesis. While the mechanisms by which Leu is 'sensed' have been the subject of great scrutiny, as a branched-chain amino acid, Leu can be catabolized within muscle, thus posing the possibility that metabolites of Leu could be involved in mediating the anabolic effect(s) of Leu. Our objective was to measure muscle protein anabolism in response to Leu and its metabolite HMB. Using [1,2-13 C 2 ]Leu and [ 2 H 5 ]phenylalanine tracers, and GC-MS/GC-C-IRMS we studied the effect of HMB or Leu alone on MPS (by tracer incorporation into myofibrils), and for HMB we also measured muscle proteolysis (by arteriovenous (A-V) dilution). Orally consumed 3.42 g free-acid (FA-HMB) HMB (providing 2.42 g of pure HMB) exhibited rapid bioavailability in plasma and muscle and, similarly to 3.42 g Leu, stimulated muscle protein synthesis (MPS; HMB +70% vs. Leu +110%). While HMB and Leu both increased anabolic signalling (mechanistic target of rapamycin; mTOR), this was more pronounced with Leu (i.e. p70S6K1 signalling ≤90 min vs. ≤30 min for HMB). HMB consumption also attenuated muscle protein breakdown (MPB; −57%) in an insulin-independent manner. We conclude that exogenous HMB induces acute muscle anabolism (increased MPS and reduced MPB) albeit perhaps via distinct, and/or additional mechanism(s) to Leu.

Key points Resistance exercise training (RET) is one of the most effective strategies for preventing declines in skeletal muscle mass and strength with age.Hypertrophic responses to RET with age are diminished compared to younger individuals.In response to 6 weeks RET, we found blunted hypertrophic responses with age are underpinned by chronic deficits in long‐term muscle protein synthesis.We show this is likely to be the result of multifactorial deficits in anabolic hormones and blunted translational efficiency and capacity.These results provide great insight into age‐related exercise adaptations and provide a platform on which to devise appropriate nutritional and exercise interventions on a longer term basis. AbstractAgeing is associated with impaired hypertrophic responses to resistance exercise training (RET). Here we investigated the aetiology of ‘anabolic resistance’ in older humans. Twenty healthy male individuals, 10 younger (Y; 23 ± 1 years) and 10 older (O; 69 ± 3 years), performed 6 weeks unilateral RET (6 × 8 repetitions, 75% of one repetition maximum (1‐RM), 3 times per week). After baseline bilateral vastus lateralis (VL) muscle biopsies, subjects consumed 150 ml D2O (70 atom%; thereafter 50 ml week−1), further bilateral VL muscle biopsies were taken at 3 and 6 weeks to quantify muscle protein synthesis (MPS) via gas chromatography–pyrolysis–isotope ratio mass spectrometry. After RET, 1‐RM increased in Y (+35 ± 4%) and O (+25 ± 3%; P < 0.01), while MVC increased in Y (+21 ± 5%; P < 0.01) but not O (+6 ± 3%; not significant (NS)). In comparison to Y, O displayed blunted RET‐induced increases in muscle thickness (at 3 and 6 weeks, respectively, Y: +8 ± 1% and +11 ± 2%, P < 0.01; O: +2.6 ± 1% and +3.5 ± 2%, NS). While ‘basal’ longer term MPS was identical between Y and O (∼1.35 ± 0.1% day−1), MPS increased in response to RET only in Y (3 weeks, Y: 1.61 ± 0.1% day−1; O: 1.49 ± 0.1% day−1). Consistent with this, O exhibited inferior ribosomal biogenesis (RNA:DNA ratio and c‐MYC induction: Y: +4 ± 2 fold change; O: +1.9 ± 1 fold change), translational efficiency (S6K1 phosphorylation, Y: +10 ± 4 fold change; O: +4 ± 2 fold change) and anabolic hormone milieu (testosterone, Y: 367 ± 19; O: 274 ± 19 ng dl−1 (all P < 0.05). Anabolic resistance is thus multifactorial.

BackgroundDiagnostics of the human ageing process may help predict future healthcare needs or guide preventative measures for tackling diseases of older age. We take a transcriptomics approach to build the first reproducible multi-tissue RNA expression signature by gene-chip profiling tissue from sedentary normal subjects who reached 65 years of age in good health.ResultsOne hundred and fifty probe-sets form an accurate classifier of young versus older muscle tissue and this healthy ageing RNA classifier performed consistently in independent cohorts of human muscle, skin and brain tissue (n = 594, AUC = 0.83–0.96) and thus represents a biomarker for biological age. Using the Uppsala Longitudinal Study of Adult Men birth-cohort (n = 108) we demonstrate that the RNA classifier is insensitive to confounding lifestyle biomarkers, while greater gene score at age 70 years is independently associated with better renal function at age 82 years and longevity. The gene score is ‘up-regulated’ in healthy human hippocampus with age, and when applied to blood RNA profiles from two large independent age-matched dementia case–control data sets (n = 717) the healthy controls have significantly greater gene scores than those with cognitive impairment. Alone, or when combined with our previously described prototype Alzheimer disease (AD) RNA ‘disease signature’, the healthy ageing RNA classifier is diagnostic for AD.ConclusionsWe identify a novel and statistically robust multi-tissue RNA signature of human healthy ageing that can act as a diagnostic of future health, using only a peripheral blood sample. This RNA signature has great potential to assist research aimed at finding treatments for and/or management of AD and other ageing-related conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0750-x) contains supplementary material, which is available to authorized users.

Physical activity and molecular ageing presumably interact to precipitate musculoskeletal decline in humans with age. Herein, we have delineated molecular networks for these two major components of sarcopenic risk using multiple independent clinical cohorts. We generated genome-wide transcript profiles from individuals (n = 44) who then undertook 20 weeks of supervised resistance-exercise training (RET). Expectedly, our subjects exhibited a marked range of hypertrophic responses (3% to +28%), and when applying Ingenuity Pathway Analysis (IPA) up-stream analysis to ∼580 genes that co-varied with gain in lean mass, we identified rapamycin (mTOR) signaling associating with growth (P = 1.4×10−30). Paradoxically, those displaying most hypertrophy exhibited an inhibited mTOR activation signature, including the striking down-regulation of 70 rRNAs. Differential analysis found networks mimicking developmental processes (activated all-trans-retinoic acid (ATRA, Z-score = 4.5; P = 6×10−13) and inhibited aryl-hydrocarbon receptor signaling (AhR, Z-score = −2.3; P = 3×10−7)) with RET. Intriguingly, as ATRA and AhR gene-sets were also a feature of endurance exercise training (EET), they appear to represent “generic” physical activity responsive gene-networks. For age, we found that differential gene-expression methods do not produce consistent molecular differences between young versus old individuals. Instead, utilizing two independent cohorts (n = 45 and n = 52), with a continuum of subject ages (18–78 y), the first reproducible set of age-related transcripts in human muscle was identified. This analysis identified ∼500 genes highly enriched in post-transcriptional processes (P = 1×10−6) and with negligible links to the aforementioned generic exercise regulated gene-sets and some overlap with ribosomal genes. The RNA signatures from multiple compounds all targeting serotonin, DNA topoisomerase antagonism, and RXR activation were significantly related to the muscle age-related genes. Finally, a number of specific chromosomal loci, including 1q12 and 13q21, contributed by more than chance to the age-related gene list (P = 0.01–0.005), implying possible epigenetic events. We conclude that human muscle age-related molecular processes appear distinct from the processes regulated by those of physical activity.

The applications of Western/immuno-blotting (WB) techniques have reached multiple layers of the scientific community and are now considered routine procedures in the field of physiology. This is none more so than in relation to skeletal muscle physiology (i.e. resolving the mechanisms underpinning adaptations to exercise). Indeed, the inclusion of WB data is now considered an essential aspect of many such physiological publications to provide mechanistic insight into regulatory processes. Despite this popularity, also due to the ubiquitous and relatively inexpensive availability of WB equipment, the quality of WB in publications and subsequent analysis and interpretation of the data can be variable, perhaps resulting in spurious conclusions. This may be due to poor laboratory technique and/or lack of comprehension of the critical steps involved in WB and what quality control procedures should be in place to ensure robust data generation. The present review aims to provide a detailed description and critique of WB procedures and technicalities, from sample collection through preparation, blotting and detection to analysis of the data collected. We aim to provide the reader with improved expertise to critically conduct, evaluate and troubleshoot the WB process, to produce reproducible and reliable blots.

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.

PJ. Intake of low-dose leucine-rich essential amino acids stimulates muscle anabolism equivalently to bolus whey protein in older women at rest and after exercise. Am J Physiol Endocrinol Metab 308: E1056-E1065, 2015. First published March 31, 2015; doi:10.1152/ajpendo.00481.2014.-Dysregulated anabolic responses to nutrition/exercise may contribute to sarcopenia; however, these characteristics are poorly defined in female populations. We determined the effects of two feeding regimes in older women (66 Ϯ 2.5 yr; n ϭ 8/group): bolus whey protein (WP-20 g) or novel low-dose leucine-enriched essential amino acids (EAA) [LEAA; 3 g (40% leucine)]. Using [ 13 C6]phenylalanine infusions, we quantified muscle (MPS) and albumin (APS) protein synthesis at baseline and in response to both feeding (FED) and feeding plus exercise (FED-EX; 6 ϫ 8 knee extensions at 75% 1-repetition maximum). We also quantified plasma insulin/AA concentrations, whole leg (LBF)/muscle microvascular blood flow (MBF), and muscle anabolic signaling by phosphoimmunoblotting. Plasma insulinemia and EAA/aemia were markedly greater after WP than LEAA (P Ͻ 0.001). Neither LEAA nor WP modified LBF in response to FED or FED-EX, whereas MBF increased to a similar extent in both groups only after FED-EX (P Ͻ 0.05). In response to FED, both WP and LEAA equally stimulated MPS 0 -2 h (P Ͻ 0.05), abating thereafter (0 -4 h, P Ͼ 0.05). In contrast, after FED-EX, MPS increased at 0 -2 h and remained elevated at 0 -4 h (P Ͻ 0.05) with both WP and LEAA. No anabolic signals quantifiably increased after FED, but p70 S6K1 Thr 389 increased after FED-EX (2 h, P Ͻ 0.05). APS increased similarly after WP and LEAA. Older women remain subtly responsive to nutrition Ϯ exercise. Intriguingly though, bolus WP offers no trophic advantage over LEAA. skeletal muscle; blood flow; protein synthesis; aging; amino acids; exercise ILL HEALTH ASSOCIATED WITH AGING represents a major socioeconomic burden, especially given shifting demographics toward a more aged, populous world. In particular, the loss of skeletal muscle mass associated with aging, or sarcopenia, is a major clinical issue. For instance, not only are there established links between low muscle mass and all-cause mortality per se (1), but also, lower skeletal muscle mass associated with sarcopenia leads to increased frailty, risk of falls, sedentarism, poor quality of life, and prevalence of metabolic comorbidities (17, 53).The two major extrinsic influences over muscle mass are nutrition and physical activity. For example, oral intake of protein-based foods containing essential amino acids (EAA) leads to a transient (2-3 h; see Ref.2) stimulation of muscle protein synthesis (MPS) in younger men. This brief increase in MPS above postabsorptive rates serves the purpose of replenishing protein stores lost during fasting, ensuring preservation of muscle protein mass. Similarly, physical activity is a prerequisite for maintenance of a healthy muscle mass. For example, inactivity (6, 20) causes muscle atrophy by inducing "anabo...

Compromised limb blood flow in aging may contribute to the development of sarcopenia, frailty, and the metabolic syndrome. We developed a novel contrast-enhanced ultrasound technique using Sonovue™ to characterize muscle microvasculature responses to an oral feeding stimulus (15 g essential amino acids) in young (∼20 years) and older (∼70 years) men. Intensity-time replenishment curves were made via an ultrasound probe “fixed” over the quadriceps, with intermittent high mechanical index destruction of microbubbles within muscle vasculature. This permitted real-time measures of microvascular blood volume (MBV), microvascular flow velocity (MFV) and their product, microvascular blood flow (MBF). Leg blood flow (LBF) was measured by Doppler and insulin by enzyme-linked immunosorbent assay. Steady-state contrast concentrations needed for comparison between different physiological states were achieved <150 sec from commencing Sonovue™ infusion, and MFV and MBV measurements were completed <120 sec thereafter. Interindividual coefficients of variation in MBV and MFV were 35–40%, (N = 36). Younger men (N = 6) exhibited biphasic vascular responses to feeding with early increases in MBV (+36%, P < 0.008 45 min post feed) reflecting capillary recruitment, and late increases in MFV (+77%, P < 0.008) and MBF (+130%, P < 0.007 195 min post feed) reflecting more proximal vessel dilatation. Early MBV responses were synchronized with peak insulin but not increased LBF, while later changes in MFV and MBF occurred with insulin at post absorptive values but alongside increased LBF. All circulatory responses were absent in old men (N = 7). Thus, impaired postprandial circulation could impact age-related declines in muscle glucose disposal, protein anabolism, and muscle mass.