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.

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.

Sepsis causes muscle atrophy and insulin resistance, but the underlying mechanisms are unclear. Therefore, the present study examined the effects of lipopolysaccharide (LPS)-induced endotoxaemia on the expression of Akt, Forkhead Box O (FOXO) and its downstream targets, to identify any associations between changes in FOXO-dependent processes influencing muscle atrophy and insulin resistance during sepsis. Chronically instrumented male Sprague-Dawley rats received a continuous intravenous infusion of LPS (15 μg kg −1 h −1 ) or saline for 24 h at 0.4 ml h −1 . Animals were terminally anaesthetized and the extensor digitorum longus muscles from both hindlimbs were removed and snap-frozen. Measurements were made of mRNA and protein expression of selected signalling molecules associated with pathways regulating protein synthesis and degradation and carbohydrate metabolism. LPS infusion induced increases in muscle tumour necrosis factor-α (8.9-fold, P < 0.001) and interleukin-6 (8.4-fold, P < 0.01), paralleled by reduced insulin receptor substrate-1 mRNA expression (−0.7-fold, P < 0.01), and decreased Akt1 protein and cytosolic FOXO1 and FOXO3 phosphorylation. These changes were accompanied by significant increases in muscle atrophy F-box mRNA (5.5-fold, P < 0.001) and protein (2-fold, P < 0.05) expression, and pyruvate dehydrogenase kinase 4 mRNA (15-fold, P < 0.001) and protein (1.6-fold, P < 0.05) expression. There was a 29% reduction in the muscle protein : DNA ratio, a 56% reduction in pyruvate dehydrogenase complex (PDC) activity (P < 0.05), and increased glycogen degradation and lactate accumulation. The findings of this study suggest a potential role for Akt/FOXO in the simultaneous impairment of carbohydrate oxidation, at the level of PDC, and up-regulation of muscle protein degradation, in LPS-induced endotoxaemia.

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BackgroundThe andropause is associated with declines in serum testosterone (T), loss of muscle mass (sarcopenia), and frailty. Two major interventions purported to offset sarcopenia are anabolic steroid therapies and resistance exercise training (RET). Nonetheless, the efficacy and physiological and molecular impacts of T therapy adjuvant to short‐term RET remain poorly defined.MethodsEighteen non‐hypogonadal healthy older men, 65–75 years, were assigned in a random double‐blinded fashion to receive, biweekly, either placebo (P, saline, n = 9) or T (Sustanon 250 mg, n = 9) injections over 6 week whole‐body RET (three sets of 8–10 repetitions at 80% one‐repetition maximum). Subjects underwent dual‐energy X‐ray absorptiometry, ultrasound of vastus lateralis (VL) muscle architecture, and knee extensor isometric muscle force tests; VL muscle biopsies were taken to quantify myogenic/anabolic gene expression, anabolic signalling, muscle protein synthesis (D2O), and breakdown (extrapolated).ResultsTestosterone adjuvant to RET augmented total fat‐free mass (P=0.007), legs fat‐free mass (P=0.02), and appendicular fat‐free mass (P=0.001) gains while decreasing total fat mass (P=0.02). Augmentations in VL muscle thickness, fascicle length, and quadriceps cross‐section area with RET occured to a greater extent in T (P < 0.05). Sum strength (P=0.0009) and maximal voluntary contract (e.g. knee extension at 70°) (P=0.002) increased significantly more in the T group. Mechanistically, both muscle protein synthesis rates (T: 2.13 ± 0.21%·day−1 vs. P: 1.34 ± 0.13%·day−1, P=0.0009) and absolute breakdown rates (T: 140.2 ± 15.8 g·day−1 vs. P: 90.2 ± 11.7 g·day−1, P=0.02) were elevated with T therapy, which led to higher net turnover and protein accretion in the T group (T: 8.3 ± 1.4 g·day−1 vs. P: 1.9 ± 1.2 g·day−1, P=0.004). Increases in ribosomal biogenesis (RNA:DNA ratio); mRNA expression relating to T metabolism (androgen receptor: 1.4‐fold; Srd5a1: 1.6‐fold; AKR1C3: 2.1‐fold; and HSD17β3: two‐fold); insulin‐like growth factor (IGF)‐1 signalling [IGF‐1Ea (3.5‐fold) and IGF‐1Ec (three‐fold)] and myogenic regulatory factors; and the activity of anabolic signalling (e.g. mTOR, AKT, and RPS6; P < 0.05) were all up‐regulated with T therapy. Only T up‐regulated mitochondrial citrate synthase activity (P=0.03) and transcription factor A (1.41 ± 0.2‐fold, P=0.0002), in addition to peroxisome proliferator‐activated receptor‐γ co‐activator 1‐α mRNA (1.19 ± 0.21‐fold, P=0.037).ConclusionsAdministration of T adjuvant to RET enhanced skeletal muscle mass and performance, while up‐regulating myogenic gene programming, myocellular translational efficiency and capacity, collectively resulting in higher protein turnover, and net protein accretion. T coupled with RET is an effective short‐term intervention to improve muscle mass/function in older non‐hypogonadal men.

We recently provided evidence suggesting a role for cytokine-mediated inhibition of Akt/Forkhead box O 1 (FOXO1) signalling in the induction of muscle atrophy and impairment of muscle carbohydrate oxidation during lipopolysaccharide (LPS)-induced endotoxaemia in rats. We hypothesized that a low-dose dexamethasone (Dex; anti-inflammatory agent) infusion during endotoxaemia would prevent the LPS-induced impairment of Akt/FOXO1 signalling, and therefore prevent the muscle atrophy and impairment of carbohydrate oxidation. Chronically instrumented Sprague-Dawley rats received a continuous intravenous infusion of LPS (15 μg kg−1 h −1 ), Dex (12.5 μg kg −1 h −1 ), Dex+LPS or saline for 24 h at 0.4 ml h −1 . LPS infusion caused haemodynamic changes consistent with a hyperdynamic circulation and induced increases in muscle tumour necrosis factor-α (TNF-α; 10-fold, P < 0.001), interleukin-6 (IL-6; 14-fold, P < 0.001) and metallothionein-1A (MT-1A; 187-fold, P < 0.001) mRNA expression. Dex co-administration abolished most of the haemodynamic effects of LPS and reduced the increase in muscle TNF-α, IL-6 and MT-1A by 51% (P < 0.01), 85% (P < 0.001) and 58% (P < 0.01), respectively. Dex infusion during endotoxaemia also prevented the LPS-induced 40% reduction in the muscle protein:DNA ratio and decrease in Akt phosphorylation, and partially prevented the reduction in FOXO1 phosphorylation. However, Dex did not prevent the LPS-mediated increase in muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1) mRNA expression, but did significantly reduce the LPS-mediated increase in cathepsin-L mRNA expression and enzyme activity by 43% (P < 0.001) and 53% (P < 0.05), respectively. Furthermore, Dex suppressed LPS-induced pyruvate dehydrogenase kinase 4 (PDK4) mRNA upregulation by ∼50% (P < 0.01), and prevented LPS-mediated muscle glycogen breakdown and lactate accumulation. Thus, low-dose Dex infusion during endotoxaemia prevented muscle atrophy and the impairment of carbohydrate oxidation, potentially through suppression of cytokine-mediated Akt/FOXO inhibition, and blunting of cathepsin-L-mediated lysosomal protein breakdown.

is an attachment complex protein associated with the regulation of muscle mass through as-of-yet unclear mechanisms. We tested whether FAK is functionally important for muscle hypertrophy, with the hypothesis that FAK knockdown (FAK-KD) would impede cell growth associated with a trophic stimulus. C2C12 skeletal muscle cells harboring FAKtargeted (FAK-KD) or scrambled (SCR) shRNA were created using lentiviral transfection techniques. Both FAK-KD and SCR myotubes were incubated for 24 h with IGF-I (10 ng/ml), and additional SCR cells (ϮIGF-1) were incubated with a FAK kinase inhibitor before assay of cell growth. Muscle protein synthesis (MPS) and putative FAK signaling mechanisms (immunoblotting and coimmunoprecipitation) were assessed. IGF-I-induced increases in myotube width (ϩ41 Ϯ 7% vs. non-IGF-I-treated) and total protein (ϩ44 Ϯ 6%) were, after 24 h, attenuated in FAK-KD cells, whereas MPS was suppressed in FAK-KD vs. SCR after 4 h. These blunted responses were associated with attenuated IGF-I-induced FAK Tyr 397 phosphorylation and markedly suppressed phosphorylation of tuberous sclerosis complex 2 (TSC2) and critical downstream mTOR signaling (ribosomal S6 kinase, eIF4F assembly) in FAK shRNA cells (all P Ͻ 0.05 vs. IGF-I-treated SCR cells). However, binding of FAK to TSC2 or its phosphatase Shp-2 was not affected by IGF-I or cell phenotype. Finally, FAK-KD-mediated suppression of cell growth was recapitulated by direct inhibition of FAK kinase activity in SCR cells. We conclude that FAK is required for IGF-I-induced muscle hypertrophy, signaling through a TSC2/mTOR/S6K1-dependent pathway via means requiring the kinase activity of FAK but not altered FAK-TSC2 or FAK-Shp-2 binding.focal adhesion kinase; hypertrophy; insulin-like growth factor-I; tuberous sclerosis complex 2; mammalian target of rapamycin; S6 kinase 1; skeletal muscle ATTACHMENT COMPLEXES, OR FOCAL ADHESION COMPLEXES, are macromolecular structures situated in the sarcolemma of muscle fibers that link the extracellular matrix (ECM) to the cytoplasmic cytoskeleton and consist of a variety of ECM receptors/ integrins and intracellular cytoskeletal and signaling molecules (7,30). Interactions of ECM proteins with integrin receptors stimulate intracellular signaling pathways that are important in cell growth and migration (51), and in adult skeletal muscle, focal adhesion complexes play a crucial part in the transmission of lateral forces during contraction (41). Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that localizes to focal adhesion complexes and represents a key component of integrin-mediated signaling (9). Engagement of integrin receptors induces phosphorylation of FAK at Tyr 397 , which correlates with its activation (8), and a growing body of evidence has associated FAK activation with the hypertrophic response to mechanical stress in skeletal muscle. Indeed, expression patterns of FAK have been reported to be load dependent; i.e., phosphorylation of FAK was lowered following hindlimb suspension in rodents (28) and immo...

Loss of muscle mass and insulin sensitivity are common phenotypic traits of immobilisation and increased inflammatory burden. The suppression of muscle protein synthesis is the primary driver of muscle mass loss in human immobilisation, and includes blunting of post-prandial increases in muscle protein synthesis. However, the mechanistic drivers of this suppression are unresolved. Immobilisation also induces limb insulin resistance in humans, which appears to be attributable to the reduction in muscle contraction per se. Again mechanistic insight is missing such that we do not know how muscle senses its "inactivity status" or whether the proposed drivers of muscle insulin resistance are simply arising as a consequence of immobilisation. A heightened inflammatory state is associated with major and rapid changes in muscle protein turnover and mass, and dampened insulin-stimulated glucose disposal and oxidation in both rodents and humans. A limited amount of research has attempted to elucidate molecular regulators of muscle mass loss and insulin resistance during increased inflammatory burden, but rarely concurrently. Nevertheless, there is evidence that Akt (protein kinase B) signalling and FOXO transcription factors form part of a common signalling pathway in this scenario, such that molecular cross-talk between atrophy and insulin signalling during heightened inflammation is believed to be possible. To conclude, whilst muscle mass loss and insulin resistance are common end-points of immobilisation and increased inflammatory burden, a lack of understanding of the mechanisms responsible for these traits exists such that a substantial gap in understanding of the pathophysiology in humans endures.