Project Summary/Abstract Non-alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease with a worldwide prevalence of 25%, and without a FDA-approved pharmacological therapy. Thiazolidinediones (TZD) are the agonists of peroxisome proliferator-activated receptor γ (PPARγ), and they are a potential therapy for NAFLD. However, PPARγ is a nuclear receptor and well-known steatogenic factor expressed in hepatocytes that contributes to the development of NAFLD by enhancing hepatic de novo lipogenesis (DNL) and fatty acid uptake. We have assessed the role of hepatocyte PPARγ in the development of non-alcoholic steatohepatitis (NASH) with adult-onset hepatocyte-specific Pparg knockout (Pparg∆Hep) mice in our K01 award. Specifically, we have shown that hepatocyte PPARγ contributes to the onset of inflammation and fibrosis in mice with NASH, suggesting a deleterious role of hepatocyte PPARγ on liver health. In addition, loss of hepatocyte PPARγ in Pparg∆Hep mice enhances the anti-steatogenic, anti-inflammatory, and anti-fibrogenic actions of TZD in a model of NASH. Interestingly, our RNAseq and metabolomics data indicated that hepatocyte PPARγ altered the metabolism of methionine in the liver. Methionine plays a key protective role against NAFLD, and the effect of PPARγ on its metabolism may explain why TZD-mediated activation of hepatocyte PPARγ reduced the therapeutic effects of TZD in NASH. In this project, we hypothesize that hepatocyte PPARγ disrupts methionine metabolism and promotes steatosis and liver injury by regulating gene expression and the ability to methylate phosphatidylethanolamine and homocysteine. In order to test this hypothesis, we will identify the genes regulated by PPARγ to control the use of methionine in NASH with chromatin immunoprecipitation sequencing in samples of control and Pparg∆Hep mice treated with TZD (Aim 1). In addition, we will determine if PPARγ directly disrupts the use of methionine in mouse primary hepatocytes of control and Pparg∆Hep mice with NASH using stable isotopes (Aim 2). Overall, we will determine how hepatocyte PPARγ regulates directly methionine metabolism in pathophysiological conditions. The results of this project will help us to develop novel approaches in our R01 proposals that will target specific mechanisms in preclinical models to improve NASH reversion.