Redox Status on Global Metabolism in Hepatocytes and Its Relevance to Non-Alcoholic Steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) has emerged as a global health problem, and has clinical manifestations ranging from steatosis to non-alcoholic steatohepatitis (NASH). Pathobiology of NAFLD and its progression to NASH is complex and incompletely understood. Oxidative stress is an important pathogenic factor. Lipid overload causes excessive mitochondrial electron transport, reactive oxygen species (ROS) generation, and finally cellular damage. Besides, oxidative stress is involved in production of pro-inflammatory cytokines, which is associated with inflammation and fibrosis. We have recently shown that a defect in an antioxidative system of hepatic cells alters their metabolism to response the oxidative stress. Our findings suggest that alteration in redox status of hepatocytes differentially affects the changes in global metabolism and physiological responses upon treatment with stress-inducing agents, such as oxidants and lipid-load. The primary goal of this project is to delineate the relationship between redox status and global metabolism, as well as its relevance to hepatic lipotoxicity and to pathogenesis of NAFLD and NASH. We will focus on 4 aims: First, we will study how the global metabolic network in hepatic cells changes in response to oxidative stress or lipid overload. We will apply the metabolomic and fluxomic analyses to study changes in metabolic pathways as well as metabolic fluxes in hepatic cells treated with oxidants or lipids. Any proteins that are differentially glutathionylated in treated hepatic cells may represent oxidatively-modified enzymes and regulators of metabolism. These proteins will be identified through a proteomic approach. Through these studies, we would gain an insight into the re-programming of metabolism in hepatocytes upon treatment. Second, we will study how the altered redox metabolism of hepatic cells affects their global metabolic responses to oxidants and lipids. Hepatic cell lines which are deficient in enzymes involved in NADPH biosynthesis and reduction will be generated by RNAi (knockdown) approach. Changes in the global metabolism and protein glutathionylation in oxidant- or lipid-treated cells will be studied by metabolomic, fluxomic and proteomic approaches. Third, after we identify the metabolic pathways and regulators, which are differentially changed or modified upon treatment with oxidants or lipids, we will apply a functional approach to validate the roles of these pathways and glutathionylated proteins in cellular responses of hepatic cells. Fourth, we will study how redox status affects re-programming of metabolism in vivo as well as NASH pathogenesis. High-fat-diet fed G6PD-deficient mice will be used as a model to study whether deficiency in an antioxidative enzyme affects global metabolism and NASH pathogenesis. Temporal changes in the metabolite profiles and in glutathionylated protein of liver will be followed, and data will be correlated with pathologic changes. Through this 3 year project, we will have a better understanding of the relationship between redox homeostasis and global metabolism in hepatocytes, and the significance of these factors in NASH pathogenesis. From the viewpoint of public health the metabolites identified through metabolomic approach will help us to better understand the pathobiology of NASH.

Project ID:PC10501-2221
External Project ID:MOST104-2320-B182-017-MY3