There are ~ 2.5 million cirrhotics in United States with over 27,000 deaths annually. Despite liver transplantation, loss in muscle mass or sarcopenia is the most frequent complication that contributes to adverse clinical outcomes in cirrhosis. However, there are no established therapies as the underlying mechanisms of sarcopenia in cirrhosis are not well understood and therefore the proposed studies are of high clinical significance. In the previous funding cycle, we showed that hyperammonemia (a consistent abnormality in cirrhosis) results in activation p65NFkB mediated increased expression of myostatin that inhibits muscle protein synthesis. Since the NFkB-myostatin pathway is activated in response to ammonia, the key question in the renewal is to address the molecular mechanisms of how ammonia mediates muscle loss. Based on exciting preliminary data generated by studies in human cirrhotics, rodent models of hyperammonemia, cell culture studies in myotubes, we hypothesize that hyperammonemia inhibits skeletal muscle protein synthesis through non-canonical molecular signaling via MyD88 and metabolic stabilization of HIF1?. We plan to address this hypothesis through following aims: First we will identify the mechanism by which hyperammonemia activates NFkB. Our preliminary data shows that in response to hyperammonemia, RhBG, an ammonia transporter interacts with MyD88 (an adaptor protein for toll like receptor pathway). This non-conical interaction of MyD88 with RhBG activates the downstream signaling response as MyD88 silencing resulted in loss of NFkB activation despite hyperammonemia. Using muscle-specific conditional knockout mice along with cellular studies, we will identify the mechanism by which ammonia promotes RhBG-MyD88 interaction leading to signaling responses that activate NFkB. Second, we will determine how hyperammonemia induced myostatin inhibits the critical protein synthesis regulatory molecule, mTORC1 via the myostatin receptor complex (ALK5-ActIIBr). Our preliminary evidence shows that in hyperammonemia, classical AMPK kinases are not activated and that ALK5 functions as a novel AMPK kinase as depletion of ALK5 by knock down results in loss of AMPK activation. Studies using conditional muscle specific AMPK knockout mice will be complemented with cellular studies in myotubes to determine that ActIIBr-ALK5 is a novel AMPK kinase during hyperammonemia and is responsible for inhibiting protein synthesis. Third, we will dissect how metabolic disposal of ammonia in the muscle via mitochondrial metabolite, ? ketoglutarate (?KG) impairs mTORC1 and hinders protein synthesis by stabilizing HIF1?. We will use mice with muscle specific HIF1? deletion and ?KG precursors in our hyperammonemic cell culture model for these studies. Studies in this aim are critical to the idea that the skeletal muscle plays a key role in ammonia detoxification and that metabolic perturbations result in deleterious effects of hyperammonemia. Our novel, integrated metabolic-molecular studies on how ammonia causes muscle loss can be rapidly translated into targeted therapies to reverse sarcopenia in cirrhosis.
Understanding the mechanisms of muscle loss or sarcopenia in cirrhosis will permit development of novel, effective and targeted therapies. A comprehensive array of animal and in vitro cell culture studies will be used to identify a novel non-canonical activation of and signaling by adapter protein, MyD88 by the ammonia transport protein, RhBG. This upstream response activates both myostatin-AMPK and hypoxia inducible factor 1? that converge and inhibit mTORC1, a critical regulator of protein synthesis in the skeletal muscle with resultant sarcopenia.
| Dasarathy, Srinivasan; Hatzoglou, Maria (2018) Hyperammonemia and proteostasis in cirrhosis. Curr Opin Clin Nutr Metab Care 21:30-36 |
| Dasarathy, Srinivasan (2017) Myostatin and beyond in cirrhosis: all roads lead to sarcopenia. J Cachexia Sarcopenia Muscle 8:864-869 |
| Dasarathy, Srinivasan (2017) Are Exercise Benefits in Nonalcoholic Fatty Liver Disease Due to Increased Autophagy? Exerc Sport Sci Rev 45:125 |
| Dasarathy, Jaividhya; McCullough, Arthur J; Dasarathy, Srinivasan (2017) Sarcopenia in Alcoholic Liver Disease: Clinical and Molecular Advances. Alcohol Clin Exp Res 41:1419-1431 |
| Nieuwoudt, Stephan; Mulya, Anny; Fealy, Ciarán E et al. (2017) In vitro contraction protects against palmitate-induced insulin resistance in C2C12 myotubes. Am J Physiol Cell Physiol 313:C575-C583 |
| Dasarathy, Srinivasan (2016) Cause and management of muscle wasting in chronic liver disease. Curr Opin Gastroenterol 32:159-65 |
