Involvement of Branched-Chain Amino Acid (BCAA) metabolism in the development of NAFLD

Abstract

Nonalcoholic Fatty Liver Disease (NAFLD) is a complex set of highly prevalent chronic liver diseases, ranging from simple steatosis (NALF) to more severe conditions, such as steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC) and has as a common and initial event the excessive accumulation of fat in the liver. Interestingly, metabolomic analysis of serum from patients with NAFLD revealed, regardless of disease stage and severity, an increase in serum concentration of branched- chain amino acids (BCAA). The molecular mechanisms that determine the interrelationships between NAFLD and BCAA metabolism are unknown. In this sense, in a preliminary pilot study, we found an increase in serum BCAA content in mice with NAFL (8 weeks), NASH (24 weeks) and HCC (48 weeks) induced by Pten deletion in hepatocytes. Surprisingly, dietary supplementation with the BCAA leucine, through unknown mechanisms, attenuated the development of NAFLD induced by Pten deletion in hepatocytes. Based on these preliminary data, the goal of this Ph.D. proposal is to investigate the mechanisms by which NAFLD impacts BCAA metabolism, as well as to evaluate the impact of dietary supplementation with BCAAs leucine, isoleucine or valine, which are metabolized through a common metabolic pathway, but generate different final metabolites (acetyl-CoA and succinyl-CoA, respectively), in the development of NAFL and progression to NASH and HCC in two distinct murine models: 1- mice with deletion of Pten in hepatocytes, which is a model of NAFLD induced by increased de novo lipogenesis; and 2- mice fed a high-fat diet rich in cholesterol and fructose (DIN) and treated with a low dose of streptozotocin (STZ), which is a model of NAFLD induced by dietary lipids and associated with type 2 diabetes. For this, mice with Pten deletion specifically in hepatocytes and control siblings (protocol 1) and wild-type C57BL6J mice treated with DIN+STZ and controls (protocol 2) will be treated or not with a diet supplemented with either leucine, or isoleucine or valine for 8 weeks. Mice will be evaluated for body weight, food intake, energy expenditure, glucose, insulin and pyruvate tolerances, serum BCAA content and other metabolites such as short-chain fatty acids (acetate, propionate and butyrate), liver mass and content. of lipids, glycogen and proteins, hepatic metabolism of lipids (de novo lipogenesis, triacylglycerol synthesis, VLDL secretion, b-oxidation) and glucose (gluconeogenesis and glycogen synthesis and degradation), mitochondrial morphology and function, inflammation (leukocyte infiltration, content of cytokines and lipid mediators), intracellular signaling of insulin and mTORC1, oxidative stress and fibrosis. In a third protocol, we will investigate the involvement of mTORC1, which is activated by BCAAs, in the changes induced by leucine or valine in the liver. For this, mice with raptor deletion in hepatocytes will be induced to NAFLD with DIN+STZ and treated with diet supplemented with either leucine, or isoleucine or valine for 8 weeks. The evaluated parameters will be the same as in protocols 1 and 2. Finally, in a fourth protocol, primary hepatocytes with or without Pten deletion will be incubated with either leucine, or isoleucine or valine and evaluated for de novo lipogenesis and b- oxidation of fatty acids, signaling of the PI3K-Akt-mTORC1 pathway and mitochondrial respiration. (AU)