Non-alcoholic fatty liver disease (NAFLD) is a fast-growing disease with no FDA-approved treatments. Numerous studies have linked NAFLD-associated hepatocyte lipotoxicity with mitochondrial dysfunction during the last few decades. Though the onset of inflammation that drives the progression from NAFL to NASH is considered multifactorial, damage-associated molecular patterns (DAMPs) and reactive oxygen species (ROS) released by injured hepatocytes are essential in activating Kupffer cells and promoting peripheral immune cell infiltration. Sphingolipids are biochemical signaling mediators and can regulate mitochondrial function in metabolic diseases, including NAFLD. Sphingosine-1-phosphate (S1P) is a well-studied molecule with dualistic roles in cell function, dependent on the location of the sphingosine kinase (SphK) isoforms, SphK1 and SphK2. While SphK1- generated cytosolic S1P signaling is thoroughly described in the literature, few studies assess the role of SphK2- derived S1P. SphK2 is localized to the cell nucleus and mitochondria. In the context of NAFLD, nuclear SphK2 is anti-inflammatory by inhibiting histone deacetylases (HDACs) and preventing the transcription of proinflammatory genes. In contrast, the role of SphK2 within the mitochondria is not well defined. Previous in vitro studies evaluated the effect of cytosolic S1P, rather than mitochondrial S1P, on mitochondrial function. While SphK2 function has been assessed in hepatocytes, these studies have only defined short-term effects of SphK2 on hepatic steatosis. Our long-term goal is to understand sphingolipid regulation and define the mechanisms that influence NAFLD disease progression. The central hypothesis of this application is that dysregulation of mitochondrial SphK2 promotes NAFLD disease progression. Two specific aims are proposed to test the hypothesis. Aim 1 examines the role of SphK2 in regulating mitochondrial function in hepatocytes under metabolic stress. We will determine if exposure to palmitic acid alters mitochondrial sphingolipid metabolism in hepatocytes and further assess the impact of SphK2 depletion on mitochondrial complex IV function and assembly in hepatocytes. Aim 2 determines the impact of hepatic SphK2 on modulating the immune response under metabolic stress. First, we will measure the ability of SphK2 depleted hepatocytes to induce cytokine synthesis in Kupffer cells. Second, we will use spectral flow cytometry to determine SphK2- dependent variations on the hepatic immune cell profile in the WDSW model. Lastly, we will use an adenoviral vector system to determine whether overexpressed hepatocyte SphK2 exhibits protective effects in NAFLD pathogenesis. To date, these functions of hepatocyte SphK2 have not been defined in the literature. Completing our proposed study will contribute to our understanding of SphK2 in hepatocyte mitochondrial function and highlight how SphK2 influences inflammation in NAFLD. Our novel findings are essential to fundamental ...