Project Summary Our long-term goal is to understand the pathologic mechanisms underlying insulin resistance-associated metabolic disease, and in particular non-alcoholic fatty liver disease (NAFLD). Although NAFLD affects 1 in 4 Americans and significantly increases the risk of liver cancer and cardiovascular disease, no FDA-approved NAFLD therapies currently exist. Although insulin resistance increases the incidence of NAFLD, the mechanisms linking these pathologies are not completely understood and warrant further study. To identify the key factors mediating insulin resistance-associated NAFLD, we screened diabetic individuals and healthy controls for differences in plasma protein concentrations and hepatic gene expression We found that ceruloplasmin (CP), a liver-secreted copper-binding protein, was increased in the liver and plasma of diabetic patients. These data are consistent with previous findings of high plasma CP concentrations in individuals with diabetes or obesity, and positive correlations of CP with the severity of diabetes, obesity, CVD risk, and mortality. In addition, our preliminary data and data from others show that hepatic CP expression is high in humans and rodents with NAFLD, while their hepatic copper levels are low11-16, consistent with the role of CP to reduce hepatic copper content. However, the functional significance of the high hepatic CP and low hepatic copper associated with insulin resistance and NAFLD is unclear. To address this deficiency, we have characterized mouse models of insulin resistance and hepatocyte-specific CP deletion. These preliminary studies have suggested a previously unrecognized role of hepatic CP and copper in linking insulin resistance and NAFLD. While a HFD decreases hepatic copper content and promotes NAFLD, liver-specific knockdown or knockout of CP (L-CP KO) increases hepatic copper content, alters the expression of genes involved in hepatic lipid metabolism, and ameliorates NAFLD in these insulin resistant mice. We hypothesize that insulin resistance-induced hepatic CP promotes NAFLD by disrupting copper homeostasis and lipid metabolism. In Aim 1, we will determine how insulin resistance induces hepatic CP gene transcription; in Aim 2, we will determine how hepatic CP regulates copper homeostasis and the development of NAFLD; and in Aim 3, we will elucidate the molecular mechanisms by which dysregulation of hepatic copper homeostasis promotes NAFLD. We expect that the completion of the proposed studies will define the role of hepatic CP and copper in metabolic regulation and the development of NAFLD in insulin resistant states, which may suggest new means of treating NAFLD.