PROJECT SUMMARY/ABSTRACT Obesity-induced lipotoxicity is the primary pathophysiological defect that predisposes to non-alcoholic fatty liver disease (NAFLD). Because current management options remain limited, identification of new regulatory mechanisms that govern the maladaptive response to overnutrition will serve to identify novel opportunities for pharmacologic intervention. Acyl-CoA thioesterases (Acots) control the cellular utilization of fatty acids by hydrolyzing acyl-CoA into non-esterified fatty acids. Our long-term goal is to define how aberrant Acot activity can be leveraged for therapeutic purposes. The objective of this research is to determine the mechanisms by which Acot9-mediated hydrolysis of acetyl-CoA culminates in metabolic disease. Our preliminary data indicates that Acot9 locates to the inner membrane (IM) of the mitochondria in the liver where it traffics acetyl-CoA towards the citric acid (TCA) cycle. This results in increased hepatic glucose production (HGP) and de novo lipogenesis (DNL) as well as lipotoxicity as evidenced by reactive oxygen species (ROS) and hepatic insulin resistance. In addition to TCA cycle, Acot9 increased acetyl-CoA supply for lysine acetylation of proteins (AcK) by controlling acetyl-CoA bioavailability and by inhibiting the deacetylase sirtuin 3 (Sirt3), which reduces ROS by inhibiting AcK. In contrast to liver, in thermogenic adipose tissue (BAT), cold-induced translocation of Acot9 from IM into mitochondrial matrix suppressed thermogenesis by trafficking acetyl-CoA away from TCA cycle. Our central hypothesis is that obesity-induced activation of Acot9 impairs nutrient homeostasis by promoting lipotoxicity in the liver and by limiting thermogenesis in BAT. The rationale is that the mechanisms of acetyl- CoA trafficking by Acot9 will reveal novel targets for the prevention of lipotoxicity and its pathophysiological consequences. The central hypothesis will be tested in three specific aims: 1) To identify the molecular mechanisms by which hepatic Acot9 promotes hepatic lipotoxicity; 2) To elucidate the mechanisms by which Acot9 in BAT limits thermogenesis; and 3) To determine the mechanisms by which Acot9 controls AcK and ROS in the liver. In Aim 1, the mechanisms of Acot9-induced lipotoxicity, HGP and DNL will be elucidated in mice with liver-specific ablation of Acot9 (Acot9LKO) using lipidomics and metabolomics. Impact on insulin signaling and lipotoxic pathways will be determined in mice and primary hepatocytes. Aim 2 will use mice with BAT-specific ablation of Acot9 for the indirect calorimetry measurements in climate-controlled cages. Clamp, tissue histology and metabolomics will assess the metabolic function of Acot9 in BAT. Cultured brown adipocytes and recombinant Acot9 will be used to determine the mechanism of Acot9 translocation into mitochondrial matrix. Aim 3 will use Acot9LKO/Sirt3–/– double knockout mice to determine the role of Sirt3 in Acot9-mediated regulation of AcK and ROS in...