Background Skeletal muscle loss (sarcopenia) is a major clinical complication in alcoholic cirrhosis with no effective therapy. Skeletal muscle autophagic proteolysis and myostatin expression (inhibitor of protein synthesis) are increased in cirrhosis and believed to contribute to anabolic resistance. A prospective study was performed to determine the mechanisms of sarcopenia in alcoholic cirrhosis and potential reversal by leucine. Methods In 6 well-compensated, stable alcoholic cirrhotic patients and 8 controls, serial vastus lateralis muscle biopsies were obtained before and 7h after a single oral BCAA mixture enriched with leucine (BCAA/LEU). Primed-constant infusion of L-[ring-2H5]-phenylalanine was used to quantify whole body protein breakdown (WbPB) and muscle protein fractional synthesis rate (FSR) using liquid chromatography/mass spectrometry. Muscle expression of myostatin, mTOR targets, autophagy markers, protein ubiquitination and intracellular amino acid deficiency sensor, general control of nutrition 2 (GCN2) were quantified by immunoblots and leucine transporter (SLC7A5) and glutamine exchanger (SLC38A2) by real time PCR. Results Following oral administration, plasma BCAA concentrations showed a similar increase in cirrhosis and controls. Skeletal muscle FSR was 9.63±0.36%/h in controls and 9.05±0.68%/h in cirrhotics (p=0.54). Elevated WbPB in cirrhosis was reduced with BCAA/LEU (p=0.01). Fasting skeletal muscle molecular markers showed increased myostatin expression, impaired mTOR signaling and increased autophagy in cirrhosis compared to controls (p<0.01). BCAA/LEU did not alter myostatin expression but mTOR signaling, autophagy measures and GCN2 activation were consistently reversed in cirrhotic muscle (p<0.01). SLC7A5 expression was higher in basal state in cirrhosis than controls (p<0.05) but increased with BCAA/LEU only in controls (p<0.001). Conclusions We demonstrate that impaired mTOR1 signaling and increased autophagy in skeletal muscle of alcoholic cirrhosis patients is acutely reversed by BCAA/LEU.

Ammonia is a cytotoxic metabolite that is removed primarily by hepatic ureagenesis in humans. Hyperammonaemia occurs in advanced hepatic, cardiac and pulmonary disease, and in urea cycle enzyme deficiencies. Increased skeletal muscle ammonia uptake and metabolism are the major mechanism of non-hepatic ammonia disposal. Non-hepatic ammonia disposal occurs in the mitochondria via glutamate synthesis from α-ketoglutarate resulting in cataplerosis. We show skeletal muscle mitochondrial dysfunction during hyperammonaemia in a comprehensive array of human, rodent and cellular models. ATP synthesis, oxygen consumption, generation of reactive oxygen species with oxidative stress, and tricarboxylic acid (TCA) cycle intermediates were quantified. ATP content was lower in the skeletal muscle from cirrhotic patients, hyperammonaemic portacaval anastomosis rat, and C2C12 myotubes compared to appropriate controls. Hyperammonaemia in C2C12 myotubes resulted in impaired intact cell respiration, reduced complex I/NADH oxidase activity and electron leak occurring at complex III of the electron transport chain. Consistently, lower NAD /NADH ratio was observed during hyperammonaemia with reduced TCA cycle intermediates compared to controls. Generation of reactive oxygen species resulted in increased content of skeletal muscle carbonylated proteins and thiobarbituric acid reactive substances during hyperammonaemia. A cell-permeable ester of α-ketoglutarate reversed the low TCA cycle intermediates and ATP content in myotubes during hyperammonaemia. However, the mitochondrial antioxidant MitoTEMPO did not reverse the lower ATP content during hyperammonaemia. We provide for the first time evidence that skeletal muscle hyperammonaemia results in mitochondrial dysfunction and oxidative stress. Use of anaplerotic substrates to reverse ammonia-induced mitochondrial dysfunction is a novel therapeutic approach.

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