Summary: PGAM5 in the regulation of hepatic lipid metabolism and carcinogenesis (PI: Andrea Johnston, DVM, PhD). The broad objective of this project is to determine whether changes in mitochondrial metabolism mechanistically influence the transition from non-alcoholic fatty liver disease (NAFLD) to hepatocellular carcinoma (HCC). NAFLD is emerging as a leading cause of HCC. HCC survival outcomes are poor. Defining the bioenergetic pathophysiology of NAFLD and its progression to HCC will identify new biomarkers and therapeutic targets. The mitochondrial membrane protein phosphoglycerate mutase 5 (PGAM5) regulates an array of mitochondrial homeostatic pathways. Recent research shows that depletion of PGAM5 prevents both high fat diet (HFD) induced obesity and the progression of HCC. The proposed research will test the hypothesis that distinct mitochondrial metabolic pathways are responsible for these effects. A liver specific knockout mouse will be generated to determine whether hepatocyte specific loss of PGAM5 will inhibit steatosis in a HFD model. Single cell RNA sequencing of a human HCC cell line will determine if PGAM5 depletion alters gene expression associated with hepatic lipid metabolism. HCC spheroids will be used to characterize how loss of PGAM5 modulates mitochondrial respiration and glycolysis in a steatosis model. Two specific aims are proposed: Aim 1 will determine whether PGAM5 knockout inhibits hepatocellular steatosis. Aim 2 will determine whether PGAM5 deletion alters mitochondrial metabolism in HCC leading to increased reactive oxygen species production.