PROJECT SUMMARY NAFLD has become progressively more common in today's society impacting adults and children. High fructose corn syrup consumption is strongly correlated with diabetes, obesity and nonalcoholic fatty liver disease (NAFLD). Fructose consumption is two to three times higher in patients with NAFLD compared to controls and increases the presence of lobular inflammation and fibrosis. The major contributors of lobular inflammation and fibrosis are activated hepatic nonparenchymal cells (NPCs), which consist of hepatic stellate cells (HSCs), and Kupffer cells. Fructose metabolism has been investigated primarily in hepatocytes and is known to induce lipogenesis and oxidative stress. These events in hepatocytes have been shown to impact Kupffer cell function pushing the cells to an inflammatory phenotype. However, the distinct role of fructose metabolism on NPCs phenotype and function in NAFLD is unclear. The broad, long term goals of this project are to identify new mechanism by which fructose excess leads to the development of NAFLD, by uncovering metabolic pathways regulated by fructose that impact NPCs function. Preliminary data demonstrate that primary Kupffer cells and HSCs express KHK and fructose supplementation in vivo, increases tissue inhibitor of metalloproteinase (TIMP1) expression in Kupffer cells compared to diets supplemented with glucose or no monosaccharides in drinking water. In vitro, fructose treatment increases intracellular levels of acetate and formate in macrophages. Based on these observations, the specific aims of this project are: 1) to determine in vitro the mechanism by which fructose drives a profibrogenic phenotype in Kupffer cells and 2) characterize the phenotype and fructose metabolism in Kupffer cells under high fructose consumption during normal and NAFLD liver conditions. Our research design uses both in vivo and in vitro systems, to investigate the proposed aims. We integrate nuclear magnetic resonance and liquid chromatography mass spectrometry to trace 13C fructose in vitro (Aim 1) and in vivo (Aim 2) in Kupffer cells and to identify fructose generated metabolites that regulate fibrogenic gene and protein expression in NPCs. To investigate the role of TIMP-1 and acetate in Kupffer cells, small interfering RNA (siRNA) transfections (Aim 1) will be used in vitro. Data obtained from this proposal are expected to establish a foundation for new mechanisms of carbohydrate metabolism in NPCs and provide critical insight for the potential development of therapeutic targets for the prevention and treatment of NAFLD.