Exploring the variability of sarcopenia prevalence in a research population using different disease definitions.

BACKGROUND: Sarcopenia is the progressive loss of muscle mass and function with age. A number of different sarcopenia definitions have been proposed and utilised in research. This study aimed to investigate how the prevalence of sarcopenia in a research cohort of older adults is influenced by the use of independent aspects of these different definitions. METHODS: Data from 255 research participants were compiled. Defining criteria by the European Working Group on Sarcopenia in Older People, the International Working Group on Sarcopenia (IWGS), and the Foundation for the National Institutes of Health were applied. RESULTS: Prevalence of sarcopenia using muscle mass ranged from 4 to 22%. Gait speed and handgrip strength criteria identified 4-34% and 4-16% of participants as sarcopenic, respectively. CONCLUSION: Prevalence of sarcopenia differs substantially depending on the criteria used. Work is required to address the impact of this for sarcopenia research to be usefully translated to inform on clinical practice.

Motor unit dysregulation following 15 days of unilateral lower limb immobilisation.

Disuse atrophy, caused by situations of unloading such as limb immobilisation, causes a rapid yet diverging reduction in skeletal muscle function when compared to muscle mass. While mechanistic insight into the loss of mass is well studied, deterioration of muscle function with a focus towards the neural input to muscle remains underexplored. This study aimed to determine the role of motor unit adaptation in disuse-induced neuromuscular deficits. Ten young, healthy male volunteers underwent 15 days of unilateral lower limb immobilisation with intramuscular electromyography (iEMG) bilaterally recorded from the vastus lateralis (VL) during knee extensor contractions normalised to maximal voluntary contraction (MVC), pre and post disuse. Muscle cross-sectional area was determined by ultrasound. Individual MUs were sampled and analysed for changes in motor unit (MU) discharge and MU potential (MUP) characteristics. VL CSA was reduced by approximately 15% which was exceeded by a two-fold decrease of 31% in muscle strength in the immobilised limb, with no change in either parameter in the non-immobilised limb. Parameters of MUP size were reduced by 11% to 24% with immobilisation, while neuromuscular junction (NMJ) transmission instability remained unchanged, and MU firing rate decreased by 8% to 11% at several contraction levels. All adaptations were observed in the immobilised limb only. These findings highlight impaired neural input following immobilisation reflected by suppressed MU firing rate which may underpin the disproportionate reductions of strength relative to muscle size. KEY POINTS: Muscle mass and function decline rapidly in situations of disuse such as bed rest and limb immobilisation. The reduction in muscle function commonly exceeds that of muscle mass, which may be associated with the dysregulation of neural input to muscle. We have used intramuscular electromyography to sample individual motor unit and near fibre potentials from the vastus lateralis following 15 days of unilateral limb immobilisation. Following disuse, the disproportionate loss of muscle strength when compared to size coincided with suppressed motor unit firing rate. These motor unit adaptations were observed at multiple contraction levels and in the immobilised limb only. Our findings demonstrate neural dysregulation as a key component of functional loss following muscle disuse in humans.

Higher strength gain after hypoxic vs normoxic resistance training despite no changes in muscle thickness and fractional protein synthetic rate.

Acute hypoxia has previously been suggested to potentiate resistance training-induced hypertrophy by activating satellite cell-dependent myogenesis rather than an improvement in protein balance in human. Here, we tested this hypothesis after a 4-week hypoxic vs normoxic resistance training protocol. For that purpose, 19 physically active male subjects were recruited to perform 6 sets of 10 repetitions of a one-leg knee extension exercise at 80% 1-RM 3 times/week for 4 weeks in normoxia (FiO2 : 0.21; n = 9) or in hypoxia (FiO2 : 0.135, n = 10). Blood and skeletal muscle samples were taken before and after the training period. Muscle fractional protein synthetic rate was measured over the whole period by deuterium incorporation into the protein pool and muscle thickness by ultrasound. At the end of the training protocol, the strength gain was higher in the hypoxic vs the normoxic group despite no changes in muscle thickness and in the fractional protein synthetic rate. Only early myogenesis, as assessed by higher MyoD and Myf5 mRNA levels, appeared to be enhanced by hypoxia compared to normoxia. No effects were found on myosin heavy chain expression, markers of oxidative metabolism and lactate transport in the skeletal muscle. Though the present study failed to unravel clearly the mechanisms by which hypoxic resistance training is particularly potent to increase muscle strength, it is important message to keep in mind that this training strategy could be effective for all athletes looking at developing and optimizing their maximal muscle strength.

Transcriptomic links to muscle mass loss and declines in cumulative muscle protein synthesis during short-term disuse in healthy younger humans.

Muscle disuse leads to a rapid decline in muscle mass, with reduced muscle protein synthesis (MPS) considered the primary physiological mechanism. Here, we employed a systems biology approach to uncover molecular networks and key molecular candidates that quantitatively link to the degree of muscle atrophy and/or extent of decline in MPS during short-term disuse in humans. After consuming a bolus dose of deuterium oxide (D2 O; 3 mL.kg-1 ), eight healthy males (22 ± 2 years) underwent 4 days of unilateral lower-limb immobilization. Bilateral muscle biopsies were obtained post-intervention for RNA sequencing and D2 O-derived measurement of MPS, with thigh lean mass quantified using dual-energy X-ray absorptiometry. Application of weighted gene co-expression network analysis identified 15 distinct gene clusters ("modules") with an expression profile regulated by disuse and/or quantitatively connected to disuse-induced muscle mass or MPS changes. Module scans for candidate targets established an experimentally tractable set of candidate regulatory molecules (242 hub genes, 31 transcriptional regulators) associated with disuse-induced maladaptation, many themselves potently tied to disuse-induced reductions in muscle mass and/or MPS and, therefore, strong physiologically relevant candidates. Notably, we implicate a putative role for muscle protein breakdown-related molecular networks in impairing MPS during short-term disuse, and further establish DEPTOR (a potent mTOR inhibitor) as a critical mechanistic candidate of disuse driven MPS suppression in humans. Overall, these findings offer a strong benchmark for accelerating mechanistic understanding of short-term muscle disuse atrophy that may help expedite development of therapeutic interventions.

The mechanisms of skeletal muscle atrophy in response to transient knockdown of the vitamin D receptor in vivo.

KEY POINTS: Reduced vitamin D receptor (VDR) expression prompts skeletal muscle atrophy. Atrophy occurs through catabolic processes, namely the induction of autophagy, while anabolism remains unchanged. In response to VDR-knockdown mitochondrial function and related gene-set expression is impaired. In vitro VDR knockdown induces myogenic dysregulation occurring through impaired differentiation. These results highlight the autonomous role the VDR has within skeletal muscle mass regulation. ABSTRACT: Vitamin D deficiency is estimated to affect ∼40% of the world's population and has been associated with impaired muscle maintenance. Vitamin D exerts its actions through the vitamin D receptor (VDR), the expression of which was recently confirmed in skeletal muscle, and its down-regulation is linked to reduced muscle mass and functional decline. To identify potential mechanisms underlying muscle atrophy, we studied the impact of VDR knockdown (KD) on mature skeletal muscle in vivo, and myogenic regulation in vitro in C2C12 cells. Male Wistar rats underwent in vivo electrotransfer (IVE) to knock down the VDR in hind-limb tibialis anterior (TA) muscle for 10 days. Comprehensive metabolic and physiological analysis was undertaken to define the influence loss of the VDR on muscle fibre composition, protein synthesis, anabolic and catabolic signalling, mitochondrial phenotype and gene expression. Finally, in vitro lentiviral transfection was used to induce sustained VDR-KD in C2C12 cells to analyse myogenic regulation. Muscle VDR-KD elicited atrophy through a reduction in total protein content, resulting in lower myofibre area. Activation of autophagic processes was observed, with no effect upon muscle protein synthesis or anabolic signalling. Furthermore, RNA-sequencing analysis identified systematic down-regulation of multiple mitochondrial respiration-related protein and genesets. Finally, in vitro VDR-knockdown impaired myogenesis (cell cycling, differentiation and myotube formation). Together, these data indicate a fundamental regulatory role of the VDR in the regulation of myogenesis and muscle mass, whereby it acts to maintain muscle mitochondrial function and limit autophagy.

Phenylbutyrate, a branched-chain amino acid keto dehydrogenase activator, promotes branched-chain amino acid metabolism and induces muscle catabolism in C2C12 cells.

NEW FINDINGS: What is the central question of this study? The compound sodium phenylbutyrate (PB) has been shown to promote branched-chain amino acid (BCAA) catabolism, and as such has been proposed as a treatment for disorders with enhanced BCAA levels: does PB induce muscle protein catabolism by forcing BCAA degradation away from muscle protein synthesis and mechanistic target of rapamycin (mTOR) inhibition? What is the main finding and its importance? Accelerated BCAA catabolism using PB resulted in adverse effects related to mTOR signalling and muscle protein metabolism in skeletal muscle cells, which may limit its application in conditions where muscle wasting is a risk. ABSTRACT: The compound sodium phenylbutyrate (PB) has been used for reducing ammonia in patients with urea cycle disorders and proposed as a treatment for disorders with enhanced branched-chain amino acid (BCAA) levels, due to its effects on promoting BCAA catabolism. In skeletal muscle cells, we hypothesised that PB would induce muscle protein catabolism due to forcing BCAA degradation away from muscle protein synthesis and downregulating mechanistic target of rapamycin (mTOR). PB reduced medium BCAA and branched-chain keto acid (BCKA) concentrations, while total cell protein (-21%; P < 0.001 vs. control) and muscle protein synthesis (-25%; P < 0.001 vs. control; assessed by measurement of puromycin incorporation into polypeptides) were decreased with PB. The regulator of anabolic pathways mTOR and its downstream components were impaired with PB treatment. The present results indicate that accelerated BCAA catabolism using PB resulted in adverse effects related to mTOR signalling and muscle protein metabolism, which may limit its application in settings where muscle wasting is a risk.

Metabolomics as an Important Tool for Determining the Mechanisms of Human Skeletal Muscle Deconditioning.

Muscle deconditioning impairs both locomotor function and metabolic health, and is associated with reduced quality life and increased mortality rates. Despite an appreciation of the existence of phenomena such as muscle anabolic resistance, mitophagy, and insulin resistance with age and disease in humans, little is known about the mechanisms responsible for these negative traits. With the complexities surrounding these unknowns and the lack of progress to date in development of effective interventions, there is a need for alternative approaches. Metabolomics is the study of the full array of metabolites within cells or tissues, which collectively constitute the metabolome. As metabolomics allows for the assessment of the cellular metabolic state in response to physiological stimuli, any chronic change in the metabolome is likely to reflect adaptation in the physiological phenotype of an organism. This, therefore, provides a holistic and unbiased approach that could be applied to potentially uncover important novel facets in the pathophysiology of muscle decline in ageing and disease, as well as identifying prognostic markers of those at risk of decline. This review will aim to highlight the current knowledge and potential impact of metabolomics in the study of muscle mass loss and deconditioning in humans and will highlight key areas for future research.

Exploring mechanistic links between extracellular branched-chain amino acids and muscle insulin resistance: an in vitro approach.

Branched-chain amino acids (BCAAs) are essential for critical metabolic processes; however, recent studies have associated elevated plasma BCAA levels with increased risk of insulin resistance. Using skeletal muscle cells, we aimed to determine whether continued exposure of high extracellular BCAA would result in impaired insulin signaling and whether the compound sodium phenylbutyrate (PB), which induces BCAA metabolism, would lower extracellular BCAA, thereby alleviating their potentially inhibitory effects on insulin-mediated signaling. Prolonged exposure of elevated BCAA to cells resulted in impaired insulin receptor substrate 1/AKT signaling and insulin-stimulated glycogen synthesis. PB significantly reduced media BCAA and branched-chain keto acid concentrations and increased phosphorylation of AKT [+2.0 ± 0.1-fold; P < 0.001 versus without (-)PB] and AS160 (+3.2 ± 0.2-fold; P < 0.001 versus -PB); however, insulin-stimulated glycogen synthesis was further reduced upon PB treatment. Continued exposure of high BCAA resulted in impaired intracellular insulin signaling and glycogen synthesis, and while forcing BCAA catabolism using PB resulted in increases in proteins important for regulating glucose uptake, PB did not prevent the impairments in glycogen synthesis with BCAA exposure.

Targeted genotype analyses of GWAS-derived lean body mass and handgrip strength-associated single-nucleotide polymorphisms in elite master athletes.

Recent large genome-wide association studies (GWAS) have independently identified a set of genetic loci associated with lean body mass (LBM) and handgrip strength (HGS). Evaluation of these candidate single-nucleotide polymorphisms (SNPs) may be useful to investigate genetic traits of populations at higher or lower risk of muscle dysfunction. As such, we investigated associations between six SNPs linked to LBM or HGS in a population of elite master athletes (MA) and age-matched controls as a representative population of older individuals with variable maintenance of muscle mass and function. Genomic DNA was isolated from buffy coat samples of 96 individuals [consisting of 48 MA (71 ± 6 yr, age-graded performance 83 ± 9%) and 48 older controls (75 ± 6 yr)]. SNP validation and sample genotyping were conducted using the tetra-primer amplification refractory mutation system (ARMS). For the three SNPs analyzed that were previously associated with LBM (FTO, IRS1, and ADAMTSL3), multinomial logistic regression revealed a significant association of the ADAMTSL3 genotype with %LBM (P < 0.01). For the three HGS-linked SNPs, neither GBF1 nor GLIS1 showed any association with HGS, but for TGFA, multinomial logistic regression revealed a significant association of genotype with HGS (P < 0.05). For ADAMTSL3, there was an enrichment of the effect allele in the MA (P < 0.05, Fisher's exact test). Collectively, of the six SNPs analyzed, ADAMTSL3 and TGFA showed significant associations with LBM and HGS, respectively. The functional relevance of the ADAMTSL3 SNP in body composition and of TGFA in strength may highlight a genetic component of the elite MA phenotype.

An update on nutrient modulation in the management of disease-induced muscle wasting: evidence from human studies.

PURPOSE OF REVIEW: Skeletal muscle has many essential roles in maintaining human health, not only being crucial for locomotion, but further as a metabolically important organ. Muscle wasting in disease (cachexia) is highly prevalent, associated with poor clinical outcomes and is not fully reversible with nutritional interventions. Understanding proteostasis in diseased states is of great importance to design novel, effective nutritional/nutraceutical strategies aimed at alleviating muscle wasting. In this review, we will provide an update on muscle kinetics in disease and the effects of nutritional interventions. RECENT FINDINGS: Whole body and skeletal muscle kinetics are commonly shown to be imbalanced in disease, promoting overall catabolism that underlies the development of cachexia. However, recent advancements in defining the effectiveness of nutritional interventions on muscle anabolism are clouded by heterogenous patient populations and a lack of direct incorporation stable isotope techniques. Current recommendations are focused on combating malnutrition, with increased protein intake (high in EAA) demonstrating promise. SUMMARY: Recent progress in understanding catabolic states in cachexia across disease is minimal. Further, studies investigating muscle-specific protein turnover along with nutritional interventions are scarce. As such, there is a significant requirement for strong RCT's investigating both acute and chronic nutritional interventions and their impact on skeletal muscle in individual disease states.

Contemporary stable isotope tracer approaches: Insights into skeletal muscle metabolism in health and disease.

NEW FINDINGS: What is the topic of this review? This review discusses the application of new stable isotope tracer techniques in understanding the control of skeletal muscle mass. What advances does it highlight? This review highlights current advances in stable isotope tracer techniques through their combination with high-throughput proteomics technologies. ABSTRACT: Beyond its primary locomotory and key structural functions, skeletal muscle provides additional vital roles for maintenance of metabolic health, acting as a storage point for glucose and intramuscular lipids for energy production, alongside being the largest reservoir for amino acids in the body. Therefore, maintenance of muscle mass is key to the promotion of health and well-being across the lifespan and in several disease states. As such, when skeletal muscle is lost, in either clinical (cancer, organ failure etc.) or non-clinical (ageing, inactivity) situations, there are potentially devastating consequences attached, with robust links existing between muscle mass loss and mortality. Great efforts are being made to reverse or slow muscle mass declines in health and disease, through combinations of lifestyle changes and nutritional and/or pharmaceutical intervention. However, despite this comprehensive research effort, the underlying metabolic and molecular mechanisms have yet to be defined properly. However, with the rapid acceleration of analytical developments over recent years, the application of stable isotope tracers to the study of human muscle metabolism is providing unique insights into the mechanisms controlling skeletal muscle loss and allowing more targeted therapeutic strategies to be developed. The aim of this review is to highlight the technical breakthroughs in our understanding of muscle wasting in health and disease and how future directions and developments incorporating 'omics' with stable isotope tracers will allow for a more personalized and stratified therapeutic approach.

The metabolic and molecular mechanisms of hyperammonaemia- and hyperethanolaemia-induced protein catabolism in skeletal muscle cells.

Hyperammonaemia and hyperethanolaemia are thought to be driving factors behind skeletal muscle myopathy in liver disease, that is, cirrhosis. Despite this, the singular and combined impacts of ethanol- and ammonia-induced protein catabolism are poorly defined. As such, we aimed to dissect out the effects of ammonia and ethanol on muscle catabolism. Murine C2C12 myotubes were treated with ammonium acetate (10 mM) and ethanol (100 mM) either alone or in combination for 4 hr and/or 24 hr. Myotube diameter, muscle protein synthesis and anabolic and catabolic signalling pathways were assessed. In separate experiments, cells were cotreated with selected inhibitors of protein breakdown to assess the importance of proteolytic pathways in protein loss with ammonia and ethanol. Ammonia and ethanol in combination resulted in a reduction in myotube width and total protein content, which was greater than the reduction observed with ammonia alone. Both ammonia and ethanol caused reductions in protein synthesis, as assessed by puromycin incorporation. There was also evidence of impairments in regulation of protein translation, and increased protein expression of markers of muscle protein breakdown. Myotube protein loss with ammonia plus ethanol was not affected by autophagy inhibition, but was completely prevented by proteasome inhibition. Thus, combined ammonia and ethanol incubation of C2C12 myotubes exacerbated myotube atrophy and dysregulation of anabolic and catabolic signalling pathways associated with either component individually. Ubiquitin proteasome-mediated protein breakdown appears to play an important role in myotube protein loss with ethanol and ammonia.

Stable isotope tracers and exercise physiology: past, present and future.

Stable isotope tracers have been invaluable assets in physiological research for over 80 years. The application of substrate-specific stable isotope tracers has permitted exquisite insight into amino acid, fatty-acid and carbohydrate metabolic regulation (i.e. incorporation, flux, and oxidation, in a tissue-specific and whole-body fashion) in health, disease and response to acute and chronic exercise. Yet, despite many breakthroughs, there are limitations to 'substrate-specific' stable isotope tracers, which limit physiological insight, e.g. the need for intravenous infusions and restriction to short-term studies (hours) in controlled laboratory settings. In recent years significant interest has developed in alternative stable isotope tracer techniques that overcome these limitations, in particular deuterium oxide (D2 O or heavy water). The unique properties of this tracer mean that through oral administration, the turnover and flux through a number of different substrates (muscle proteins, lipids, glucose, DNA (satellite cells)) can be monitored simultaneously and flexibly (hours/weeks/months) without the need for restrictive experimental control. This makes it uniquely suited for the study of 'real world' human exercise physiology (amongst many other applications). Moreover, using D2 O permits evaluation of turnover of plasma and muscle proteins (e.g. dynamic proteomics) in addition to metabolomics (e.g. fluxomics) to seek molecular underpinnings, e.g. of exercise adaptation. Here, we provide insight into the role of stable isotope tracers, from substrate-specific to novel D2 O approaches, in facilitating our understanding of metabolism. Further novel potential applications of stable isotope tracers are also discussed in the context of integration with the snowballing field of 'omic' technologies.

"Nutraceuticals" in relation to human skeletal muscle and exercise.

Skeletal muscles have a fundamental role in locomotion and whole body metabolism, with muscle mass and quality being linked to improved health and even lifespan. Optimizing nutrition in combination with exercise is considered an established, effective ergogenic practice for athletic performance. Importantly, exercise and nutritional approaches also remain arguably the most effective countermeasure for muscle dysfunction associated with aging and numerous clinical conditions, e.g., cancer cachexia, COPD, and organ failure, via engendering favorable adaptations such as increased muscle mass and oxidative capacity. Therefore, it is important to consider the effects of established and novel effectors of muscle mass, function, and metabolism in relation to nutrition and exercise. To address this gap, in this review, we detail existing evidence surrounding the efficacy of a nonexhaustive list of macronutrient, micronutrient, and "nutraceutical" compounds alone and in combination with exercise in relation to skeletal muscle mass, metabolism (protein and fuel), and exercise performance (i.e., strength and endurance capacity). It has long been established that macronutrients have specific roles and impact upon protein metabolism and exercise performance, (i.e., protein positively influences muscle mass and protein metabolism), whereas carbohydrate and fat intakes can influence fuel metabolism and exercise performance. Regarding novel nutraceuticals, we show that the following ones in particular may have effects in relation to 1) muscle mass/protein metabolism: leucine, hydroxyl ß-methylbutyrate, creatine, vitamin-D, ursolic acid, and phosphatidic acid; and 2) exercise performance: (i.e., strength or endurance capacity): hydroxyl ß-methylbutyrate, carnitine, creatine, nitrates, and ß-alanine.

Mechanistic Links Underlying the Impact of C-Reactive Protein on Muscle Mass in Elderly.

BACKGROUND/AIMS: Mechanisms underlying the relationship between systemic inflammation and age-related decline in muscle mass are poorly defined. The purpose of this work was to investigate the relationship between the systemic inflammatory marker CRP and muscle mass in elderly and to identify mechanisms by which CRP mediates its effects on skeletal muscle, in-vitro. METHODS: Muscle mass and serum CRP level were determined in a cohort of 118 older women (67±1.7 years). Human muscle cells were differentiated into myotubes and were exposed to CRP. The size of myotubes was determined after immunofluorescent staining using troponin. Muscle protein synthesis was assessed using stable isotope tracers and key signalling pathways controlling protein synthesis were determined using western-blotting. RESULTS: We observed an inverse relationship between circulating CRP level and muscle mass (ß= -0.646 (95% CI: -0.888, -0.405) p<0.05) and demonstrated a reduction (p < 0.05) in the size of human myotubes exposed to CRP for 72 h. We next showed that this morphological change was accompanied by a CRP-mediated reduction (p < 0.05) in muscle protein fractional synthetic rate of human myotubes exposed to CRP for 24 h. We also identified a CRP-mediated increased phosphorylation (p<0.05) of regulators of cellular energy stress including AMPK and downstream targets, raptor and ACC-ß, together with decreased phosphorylation of Akt and rpS6, which are important factors controlling protein synthesis. CONCLUSION: This work established for the first time mechanistic links by which chronic elevation of CRP can contribute to age-related decline in muscle function.

A novel puromycin decorporation method to quantify skeletal muscle protein breakdown: A proof-of-concept study.

The precise roles that the major proteolytic pathways play in the regulation of skeletal muscle mass remain incompletely understood, in part due to technical limitations associated with current techniques used to quantify muscle protein breakdown (MPB). We aimed to develop a method to assess MPB in cells, based on loss of puromycin labelling of translated polypeptide chains. Following an initial 24 h incubation period with puromycin (1 µM), loss of puromycin labelling from murine C2C12 myotubes was assessed over 48 h, both in the presence or absence of protein synthesis inhibitor cycloheximide (CHX). To validate the method, loss of puromycin labelling was determined from cells treated with selected compounds known to influence MPB (e.g. serum starvation, Dexamethasone (Dex), tumour necrosis factor alpha (TNF-α) and MG-132)). Reported established (static) markers of MPB were measured following each treatment. Loss of puromycin labelling from cells pre-incubated with puromycin was evident over a 48 h period, both with and without CHX. Treatment with Dex (-14 ± 2% vs. Ctl; P < 0.01), TNF-α (-20 ± 4% vs. Ctl; P < 0.001) and serum starvation (-14 ± 4% vs. Ctl; P < 0.01) caused a greater loss of puromycin labelling than untreated controls, while the proteasome inhibitor MG-132 caused a relatively lower loss of puromycin labelling (+15 ± 8% vs. Ctl; P < 0.05). Thus, we have developed a novel decorporation method for measuring global changes in MPB, validated in vitro using an established muscle cell line. It is anticipated this non isotopic-tracer alternative to measuring MPB will facilitate insight into the mechanisms that regulate muscle mass/MPB both in vitro, and perhaps, in vivo.

Nutrient modulation in the management of disease-induced muscle wasting: evidence from human studies.

PURPOSE OF REVIEW: In addition to being essential for movement, skeletal muscles act as both a store and source of key macronutrients. As such, muscle is an important tissue for whole body homeostasis, undergoing muscle wasting in times of starvation, disease, and stress, for example, to provide energy substrates for other tissues. Yet, muscle wasting is also associated with disability, comorbidities, and mortality. As nutrition is so crucial to maintaining muscle homeostasis 'in health', it has been postulated that muscle wasting in cachexia syndromes may be alleviated by nutritional interventions. This review will highlight recent work in this area in relation to muscle kinetics, the acute metabolic (e.g. dietary protein), and longer-term effects of dietary interventions. RECENT FINDINGS: Whole body and skeletal muscle protein synthesis invariably exhibit deranged kinetics (favouring catabolism) in wasting states; further, many of these conditions harbour blunted anabolic responses to protein nutrition compared with healthy controls. These derangements underlie muscle wasting. Recent trials of essential amino acid and protein-based nutrition have shown some potential for therapeutic benefit. SUMMARY: Nutritional modulation, particularly of dietary amino acids, may have benefits to prevent or attenuate disease-induced muscle wasting. Nonetheless, there remains a lack of recent studies exploring these key concepts to make conclusive recommendations.

Recent developments in deuterium oxide tracer approaches to measure rates of substrate turnover: implications for protein, lipid, and nucleic acid research.

PURPOSE OF REVIEW: Methods that inform on dynamic metabolism that can be applied to clinical populations to understand disease progression and responses to therapeutic interventions are of great importance. This review perspective will highlight recent advances, development, and applications of the multivalent stable isotope tracer deuterium oxide (D2O) to the study of substrate metabolism with particular reference to protein, lipids, and nucleic acids, and how these methods can be readily applied within clinical and pharmaceutical research. RECENT FINDINGS: Advances in the application of D2O techniques now permit the simultaneous dynamic measurement of a range of substrates (i.e. protein, lipid, and nucleic acids, along with the potential for OMICs methodologies) with minimal invasiveness further creating opportunities for long-term 'free living' measures that can be used in clinical settings. These techniques have recently been applied to ageing populations and further in cancer patients revealing altered muscle protein metabolism. Additionally, the efficacy of numerous drugs in improving lipoprotein profiles and controlling cellular proliferation in leukaemia have been revealed. SUMMARY: D2O provides opportunities to create a more holistic picture of in-vivo metabolic phenotypes, providing a unique platform for development in clinical applications, and the emerging field of personalized medicine.

Human skeletal muscle is refractory to the anabolic effects of leucine during the postprandial muscle-full period in older men.

Leucine modulates muscle protein synthesis (MPS), with potential to facilitate accrual/maintenance of muscle mass. Animal models suggest that leucine boluses shortly after meals may prolong MPS and delay onset of a "muscle-full" state. However, the effects of nutrient "top-ups" in humans, and particularly older adults where deficits exist, have not been explored. We determined the effects of a leucine top-up after essential amino acid (EAA) feeding on anabolic signaling, MPS, and muscle energy metabolism in older men. During 13C6-phenylalanine infusion, 16 men (∼70 years) consumed 15 g of EAA with (n=8, FED + LEU) or without (n=8, FED) 3 g of leucine top-up 90 min later. Repeated blood and muscle sampling permitted measurement of fasting and postprandial plasma EAA, insulin, anabolic signaling including mTOR complex 1 (mTORC1) substrates, cellular ATP and phosphorylocreatine, and MPS. Oral EAA achieved rapid insulinemia (12.5 iU·ml-1 25 min post-feed), essential aminoacidemia (3000 µM, 45-65 min post-feed), and activation of mTORC1 signaling. Leucine top-up prolonged plasma EAA (2800 µM, 135 min) and leucine availability (1050 µM, 135 min post-feed). Fasting FSRs of 0.046 and 0.056%·h-1 (FED and FED + LEU respectively) increased to 0.085 and 0.085%·h-1 90-180 min post-feed and returned to basal rates after 180 min in both groups. Phosphorylation of mTORC1 substrates returned to fasting levels 240 min post-feed in both groups. Feeding had limited effect on muscle high-energy phosphates, but did induce eukaryotic elongation factor 2 (eEF2) phosphorylation. We demonstrate the refractoriness of muscle to nutrient-led anabolic stimulation in the postprandial period; thus, leucine supplements should be taken outside of meals, or with meals containing suboptimal protein in terms of either amount or EAA composition.

Synchronous deficits in cumulative muscle protein synthesis and ribosomal biogenesis underlie age-related anabolic resistance to exercise in humans.

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. ABSTRACT: Ageing 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 D2 O (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.