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.

Sarcopenia or skeletal muscle loss is a frequent, potentially reversible complication in cirrhosis that adversely affects clinical outcomes. Hyperammonemia is a consistent abnormality in cirrhosis that results in impaired skeletal muscle protein synthesis and breakdown (proteostasis). Despite availability of effective ammonia lowering therapies, whether lowering ammonia restores proteostasis and reverses muscle mass is unknown. Myotube diameter, protein synthesis and molecular responses in C2C12 murine myotubes to withdrawal of ammonium acetate following 24 h exposure to 10mM ammonium acetate were complemented by in vivo studies in the hyperammonemic portacaval anastomosis rat (PCA) and sham operated, pair-fed (SO) Sprague- Dawley rats treated with ammonia lowering therapy by L-ornithine L-aspartate and rifaximin orally for 4 weeks. We observed reduced myotube diameter, impaired protein synthesis and increased autophagy flux in response to hyperammonemia that were partially reversed following 24h and 48h withdrawal of ammonium acetate. Consistently, 4 weeks of ammonia lowering therapy resulted in significant lowering of blood and skeletal muscle ammonia, increase in lean body mass, improved grip strength and higher skeletal muscle mass, diameter and an increase in type II fibers in the treated compared to untreated PCA rats. Increased skeletal muscle myostatin expression, reduced mTORC1 function, and the hyperammonemic stress response including autophagy markers were also reversed in the PCA rats treated with ammonia lowering therapy. Despite significant improvement, molecular and functional readouts were not completely reversed by ammonia lowering measures. Conclusions Ammonia lowering therapy results in improvement in skeletal muscle phenotype, function and molecular perturbations of hyperammonemia. These preclinical studies complement previous studies on ammonia induced skeletal muscle loss and lay the foundation for prolonged ammonia lowering therapy to reverse sarcopenia of cirrhosis.

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.

Background and Aims Increased skeletal muscle ammonia uptake with loss of muscle mass adversely affects clinical outcomes in cirrhosis. Hyperammonemia causes reduced protein synthesis and sarcopenia but the cellular responses to impaired proteostasis and molecular mechanism of L-leucine induced adaptation to ammonia-induced stress were determined. Methods Response to activation of amino acid deficiency sensor, GCN2, in the skeletal muscle from cirrhotic patients and the portacaval anastomosis (PCA) rat were quantified. During hyperammonemia and L-leucine supplementation, protein synthesis, phosphorylation of eIF2α, mTORC1 signaling, L-leucine transport and response to L-leucine supplementation were quantified. Adaptation to cellular stress via ATF4 and its target GADD34 were also determined. Results Activation of the eIF2α kinase GCN2 and impaired mTORC1 signaling were observed in skeletal muscle from cirrhotic patients and PCA rats. Ammonia activated GCN2 mediated eIF2α phosphorylation (eIF2α–P) and impaired mTORC1 signaling that inhibit protein synthesis in myotubes and MEFs. Adaptation to ammonia-induced stress did not involve translational reprogramming by activation transcription factor 4 (ATF4) dependent induction of the eIF2α-P phosphatase subunit GADD34. Instead, ammonia increased expression of the leucine/glutamine exchanger SLC7A5, L-leucine uptake and intracellular L-leucine levels, the latter not being sufficient to rescue the inhibition of protein synthesis, due to potentially enhanced mitochondrial sequestration of L-leucine. L-leucine supplementation rescued protein synthesis inhibition caused by hyperammonemia. Conclusions Response to hyperammonemia is reminiscent of the cellular response to amino acid starvation, but lacks the adaptive ATF4 dependent integrated stress response (ISR). Instead, hyperammonemia-induced L-leucine uptake was an adaptive response to the GCN2-mediated decreased protein synthesis.

The FERM domain containing protein Kindlin-3 has been recognized as a major regulator of integrin function in hematopoietic cells, but its role in neoplasia is totally unknown. We have examined the relationship between Kindlin-3 and breast cancer in mouse models and human tissues. Human breast tumors showed a ∼7-fold elevation in Kindlin-3 mRNA compared with nonneoplastic tissue by quantitative polymerase chain reaction. Kindlin-3 overexpression in a breast cancer cell line increased primary tumor growth and lung metastasis by 2.5- and 3-fold, respectively, when implanted into mice compared with cells expressing vector alone. Mechanistically, the Kindlin-3-overexpressing cells displayed a 2.2-fold increase in vascular endothelial growth factor (VEGF) secretion and enhanced β1 integrin activation. Increased VEGF secretion resulted from enhanced production of Twist, a transcription factor that promotes tumor angiogenesis. Knockdown of Twist diminished VEGF production, and knockdown of β1 integrins diminished Twist and VEGF production by Kindlin-3-overexpressing cells, while nontargeting small interfering RNA had no effect on expression of these gene products. Thus, Kindlin-3 influences breast cancer progression by influencing the crosstalk between β1 integrins and Twist to increase VEGF production. This signaling cascade enhances breast cancer cell invasion and tumor angiogenesis and metastasis.

The recessive lethal mutation flotte lotte (flo) disrupts development of the zebrafish digestive system and other tissues. We show that flo encodes the ortholog of Mel-28/Elys, a highly conserved gene that has been shown to be required for nuclear integrity in worms and nuclear pore complex (NPC) assembly in amphibian and mammalian cells. Maternal elys expression sustains zebrafish flo mutants to larval stages when cells in proliferative tissues that lack nuclear pores undergo cell cycle arrest and apoptosis. p53 mutation rescues apoptosis in the flo retina and optic tectum, but not in the intestine, where the checkpoint kinase Chk2 is activated. Chk2 inhibition and replication stress induced by DNA synthesis inhibitors were lethal to flo larvae. By contrast, flo mutants were not sensitized to agents that cause DNA double strand breaks, thus showing that loss of Elys disrupts responses to selected replication inhibitors. Elys binds Mcm2-7 complexes derived from Xenopus egg extracts. Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine. These in vivo data indicate a role for Elys in Mcm2-chromatin interactions. Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

Genetic analyses in zebrafish identify a novel physical signaling mechanism that drives formation of invadopodia-like structures and promotes cell invasion in vivo.

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