The impact of immobilisation and inflammation on the regulation of muscle mass and insulin resistance: different routes to similar end points

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 however, 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. An 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 (Fig. 1). 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

A novel stable isotope tracer method to simultaneously quantify skeletal muscle protein synthesis and breakdown

Background/Aims: Methodological challenges have been associated with the dynamic measurement of muscle protein breakdown (MPB), as have the measurement of both muscle protein synthesis (MPS) and MPB within the same experiment. Our aim was to use the transmethylation properties of methionine as proof-of-concept to measure rates of MPB via its methylation of histidine within skeletal muscle myofibrillar proteins, whilst simultaneously utilising methionine incorporation into bound protein to measure MPS.Results: During the synthesis measurement period, incorporation of methyl[D3]-13C-methionine into cellular protein in C2C12 myotubes was observed (representative of MPS), alongside an increase in the appearance of methyl[D3]-methylhistidine into the media following methylation of histidine (representative of MPB). For further validation of this approach, fractional synthetic rates (FSR) of muscle protein were increased following treatment of the cells with the anabolic factors insulin-like growth factor-1 (IGF-1) and insulin, while dexamethasone expectedly reduced MPS. Conversely, rates of MPB were reduced with IGF-1 and insulin treatments, whereas dexamethasone accelerated MPB.Conclusions: This is a novel stable isotope tracer approach that permits the dual assessment of muscle cellular protein synthesis and breakdown rates, through the provision of a single methionine amino acid tracer that could be utilised in a wide range of biological settings

The Regulatory Roles of PPARs in Skeletal Muscle Fuel Metabolism and Inflammation: Impact of PPAR Agonism on Muscle in Chronic Disease, Contraction and Sepsis

The peroxisome proliferator-activated receptor (PPAR) family of transcription factors has been demonstrated to play critical roles in regulating fuel selection, energy expenditure and inflammation in skeletal muscle and other tissues. Activation of PPARs, through endogenous fatty acids and fatty acid metabolites or synthetic compounds, has been demonstrated to have lipid-lowering and anti-diabetic actions. This review will aim to provide a comprehensive overview of the functions of PPARs in energy homeostasis, with a focus on the impacts of PPAR agonism on muscle metabolism and function. The dysregulation of energy homeostasis in skeletal muscle is a frequent underlying characteristic of inflammation-related conditions such as sepsis. However, the potential benefits of PPAR agonism on skeletal muscle protein and fuel metabolism under these conditions remains under-investigated and is an area of research opportunity. Thus, the effects of PPARγ agonism on muscle inflammation and protein and carbohydrate metabolism will be highlighted, particularly with its potential relevance in sepsis-related metabolic dysfunction. The impact of PPARδ agonism on muscle mitochondrial function, substrate metabolism and contractile function will also be described

A statistical and biological response to an informatics appraisal of healthy aging gene signatures

Jacob and Speed did not identify even a single example of a ‘150-gene-set’ that was statistically significant at classifying Alzheimer’s disease (AD) samples, or age in independent studies. We attempt to clarify the various misunderstandings

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 i.e. 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 4h and/or 24h. Myotube diameter, muscle protein synthesis and anabolic and catabolic signalling pathways were assessed. In separate experiments, cells were co-treated 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, that 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

Gene-based analysis of angiogenesis, mitochondrial and insulin-related pathways in skeletal muscle of older individuals following nutraceutical supplementation

Cocoa flavanols and fish oil omega-3 fatty acids are two bio-active nutrients that may improve muscle microvascular function, insulin sensitivity and mitochondrial function in older adults. We assessed changes in gene expression of these pathways in muscle from two nutritional intervention studies in older healthy volunteers: (i) 6-weeks daily fish oil supplementation in older females (3.4 g/d; age: 64.4 ± 0.8 y, BMI: 26.2 ± 0.7 kg/m2), and (ii) 7-day daily cocoa flavanol supplementation in older males (1050 mg/d; age: 70.1 ± 0.9 y, BMI: 25.7 ± 0.6 kg/m2). There was a main effect of 6-weeks fish oil supplementation on angiogenesis gene expression, with no overall changes in mitochondrial or insulin signaling genes. 7-day cocoa supplementation elicited changes in extracellular matrix (ECM) related genes. Thus, the effects of fish oil supplementation on vascular remodeling in skeletal muscle, and ECM remodeling with cocoa supplementation have emerged as areas for future study

Peroxisome proliferator-activated receptor γ agonism attenuates endotoxaemia-induced muscle protein loss and lactate accumulation in rats

The peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone (Rosi) appears to provide protection against organ dysfunction during endotoxaemia. We examined the potential benefits of Rosi on skeletal muscle protein maintenance and carbohydrate metabolism during lipopolysaccharide (LPS)-induced endotoxaemia. Sprague-Dawley rats were fed either standard chow (control) or standard chow containing Rosi (8.5±0.1 mg.kg-1.day-1) for two weeks before and during 24 h continuous intravenous infusion of LPS (15 μg.kg-1.h-1) or saline. Rosi blunted LPS-induced increases in muscle tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA by 70% (P<0.05) and 64% (P<0.01), respectively. Furthermore, Rosi suppressed the LPS-induced reduction in phosphorylated AKT and phosphorylated Forkhead box O (FOXO) 1 protein, as well as the upregulation of muscle RING finger 1 (MuRF1; P<0.01) mRNA, and the LPS-induced increase in 20S proteasome activity (P<0.05). Accordingly, LPS reduced the muscle protein:DNA ratio (~30%, P<0.001), which Rosi offset. Increased muscle pyruvate dehydrogenase kinase 4 (PDK4) mRNA (P<0.001) and muscle lactate accumulation (P<0.001) during endotoxaemia were suppressed by Rosi. Thus, pre-treatment with Rosi reduced muscle cytokine accumulation and blunted muscle protein loss and lactate accumulation during endotoxaemia, and at least in part by reducing activation of molecular events known to increase muscle protein breakdown and mitochondrial pyruvate use

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

Biomarkers of browning of white adipose tissue and their regulation during exercise- and diet-induced weight loss

Background: A hypothesis exists whereby an exercise- or dietary-induced negative energy balance reduces human subcutaneous white adipose tissue (scWAT) mass through the formation of brown-like adipocyte (brite) cells. However, the validity of biomarkers of brite formation has not been robustly evaluated in humans, and clinical data that link brite formation and weight loss are sparse. Objectives: We used rosiglitazone and primary adipocytes to stringently evaluate a set of biomarkers for brite formation and determined whether the expression of biomarker genes in scWAT could explain the change in body composition in response to exercise training combined with calorie restriction in obese and overweight women (n = 79). Design: Gene expression was derived from exon DNA microarrays and preadipocytes from obesity-resistant and -sensitive mice treated with rosiglitazone to generate candidate brite biomarkers from a microarray. These biomarkers were evaluated against data derived from scWAT RNA from obese and overweight women before and after supervised exercise 5 d/wk for 16 wk combined with modest calorie restriction (∼0.84 MJ/d). Results: Forty percent of commonly used brite gene biomarkers exhibited an exon or strain-specific regulation. No biomarkers were positively related to weight loss in human scWAT. Greater weight loss was significantly associated with less uncoupling protein 1 expression (P = 0.006, R(2) = 0.09). In a follow-up global analysis, there were 161 genes that covaried with weight loss that were linked to greater CCAAT/enhancer binding protein α activity (z = 2.0, P = 6.6 × 10(−7)), liver X receptor α/β agonism (z = 2.1, P = 2.8 × 10(−7)), and inhibition of leptin-like signaling (z = −2.6, P = 3.9 × 10(−5)). Conclusion: We identify a subset of robust RNA biomarkers for brite formation and show that calorie-restriction–mediated weight loss in women dynamically remodels scWAT to take on a more-white rather than a more-brown adipocyte phenotype