Abnormal mitochondrial function is proposed to contribute to the hepatic insulin resistance and steatosis associated with obesity and type 2 diabetes. Recently, diet-induced obesity in mice was found to be associated with reduced rates of hepatic mitophagy, a mitochondrial quality control pathway that regulates selective removal of damaged mitochondria from the cell. Mitophagy signaling and targeting of damaged mitochondria relies on generation of a phosphorylated ubiquitin signaling motif on the outer mitochondrial membrane that is produced through the coordinated activation of the ubiquitin E3 ligase PARKIN and the serine/threonine kinase PINK1. This proposal will test the hypothesis that obesity-associated liver metabolic disease arises in part due to a loss of mitochondrial homeostasis resulting from impaired PINK1-PARKIN-mediated signaling and subsequently reduced mitophagy. The approach to test this hypothesis consists of characterizing a novel mouse line with liver-specific deletion of PARKIN at baseline and during diet-induced obesity, and will include a breadth of in vivo and ex vivo approaches designed to evaluate hepatic mitochondrial oxidative metabolism, reactive oxygen species production, hepatic insulin sensitivity, rates of mitophagic flux, and changes in hepatic insulin signaling and stress signaling pathways. These studies will define how genetic inhibition of PARKIN- mediated mitophagy in liver impacts mitochondrial biology and the pathogenesis of obesity-associated liver metabolic disease. Additionally, key aspects of PINK1-PARKIN-mediated mitophagy signaling will be evaluated in lean and obese mice using a combination of approaches that includes quantitative mass spectrometry- based proteomics, confocal microscopy and mutagenesis followed by in vivo and in vitro functional characterization using primary and stable cell lines and cell free models. These studies will define how nutritional stress in the obese liver contributes to post-translational modifications of mitochondrial proteins that then interfere with recently defined phosphorylation and ubiquitination signaling events that activate the mitophagy pathway. They will provide further insight into how PINK1 and PARKIN dynamics are affected in the obese liver and may account for the reduced mitophagy observed in liver from diet-induced obese mice. Overall, these studies will explore a novel pathway by which nutritional stress in liver may contribute to the pathogenesis of hepatic steatosis, insulin resistance and mitochondrial dysfunction. This new knowledge will ultimately improve our understanding of the biochemical and molecular events that contribute to these diseases, which may lend themselves to the development of effective life style interventions or targeted therapeutics to treat obesity-associated liver disease.
Studies designed in this application will provide new knowledge regarding the pathogenesis of obesity- associated liver disease. They will specifically define key biochemical changes in a pathway that regulates the health of mitochondria within the liver. This new understanding may lend itself to development of life style interventions or therapeutics that lessens the burden of obesity-associated liver disease.
