Non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis (NASH), is the most common liver disease in the United States and it increases the risk for cirrhosis and hepatocellular carcinoma. Prior research shows that dysregulated lipid partitioning in liver mitochondrial oxidative metabolism pathways [i.e., tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and ketogenesis] is fundamental to liver steatosis and oxidative stress that underlie NAFLD. A traditional view of mitochondrial lipid partitioning is that lipids are fated to ketogenesis when the capacity of terminal oxidation (TCA cycle and OXPHOS) is exceeded, but ketogenesis is limited when the TCA cycle and OXPHOS can support terminal oxidation or under states of high hepatic energy demand. However, this dualist view of lipid partitioning fails to describe heterogeneity in mitochondrial function across the NAFLD spectrum and reveals knowledge gaps in our understanding of how liver mitochondria may coordinate lipid catabolism. The objective of this project is to test the innovate premise that ketogenesis actively supports TCA cycle function and that loss of this salutary coupling contributes to NAFLD. This is based on our preliminary data from mouse models of varying NAFLD severity. Initial studies used phosphatidylethanolamine N-methyltransferase (PEMT)-null mice that exhibit NASH owing to reduced phosphatidylcholine, which is characteristic of human NASH. PEMT-null mice showed lower liver NAD+ and molecular indices of terminal oxidation, however, in vivo TCA cycle flux was unaltered and ketogenesis was increased. This phenotype in mice mimics our preliminary data in humans with NASH which showed elevated ketogenesis and preserved TCA cycle flux. Notably, PEMT-null mice fed a high-fat diet and wild type mice fed a ketogenic diet had lower liver nicotinamide N-methyltransferase (NNMT), which may lead to greater NAD+ salvage to facilitate TCA cycle flux that would otherwise be impaired. In addition, knockdown of ketogenic enzyme 3-hydroxymethylglutaryl-CoA synthase 2 (HMGCS2) in mice on a high-fat diet resulted in lower liver ketogenesis (as expected) and NAD+. Our central hypothesis is that ketogenesis enhances TCA cycle flux via acute and chronic NAD+ provision under conditions of excess lipid availability. This hypothesis will be tested via two Specific Aims. Aim 1 will demonstrate that a ketone body-NNMT-NAD+ axis mitigates liver steatosis by promoting TCA cycle flux in NASH. Mice lacking liver HMGCS2 will receive a Gubra Amylin NASH (GAN) diet and will be independently crossed with liver-specific NNMT knockout mice or provided nicotinamide riboside to increase NAD+. Aim 2 will determine that an increased NAD+/NADH ratio supported by ketogenesis facilitates lipid disposal in response to exercise. HMGCS2 and β-hydroxybutyrate dehydrogenase 1 will be deleted in livers of mice fed a GAN diet to impede ketogenesis and NAD+ provision. Acute and chroni...