Excessive sugar and fat consumption can lead to a range of metabolic abnormalities including non-alcoholic fatty liver disease (NAFLD), hypercholesterolemia, dyslipidemia, obesity, and insulin resistance. Emerging evidence points to a new mechanism linking carbon metabolism and fatty liver disease to the G protein-coupled Protease Activated Receptor-2 (PAR2), a receptor previously known to be involved in inflammation. To provide key support for the potential importance of hepatic PAR2 in humans, in a cross-sectional study of liver specimens and clinical data from 108 NAFLD patients and controls, we found that PAR2 protein expression in hepatocytes was low in control livers and progressively increased in patients with mild NAFLD fibrosis and in NAFLD with higher stages of fibrosis. The high PAR2-expressing NAFLD patient cohort had significantly elevated plasma LDL cholesterol as compared to the low PAR2-expressing cohort. However, a definitive role for PAR2 in metabolism, cholesterol homeostasis, and liver pathology remains unknown. The overarching goal of this grant proposal is to examine the role of PAR2 signaling in the etiology and pathogenesis of fatty liver disease to test our central hypothesis that hepatic PAR2 is a novel contributor of hypertriglyceridemia, obesity, and insulin resistance. PAR2 is expressed by many tissues and cell types and a conventional mouse whole-body knockout (KO) has been used to date. Therefore, it is unclear how much of the KO phenotype or effects of PAR2 inhibition on steatosis, lipid metabolism, and weight loss is mediated only at the level of the hepatocyte and how much is due to loss of PAR2 at other sites of expression. We will use our new hepatocyte-specific PAR2-KO, PAR2∆Hep mice to define and validate the specific involvement of liver PAR2 in these profound metabolic effects in aims 1-3. It is unclear how PAR2 promotes insulin resistance in diet-induced obesity models. Aim 2 will explore a novel mechanism whereby PAR2 activates a Gq-PLC pathway to stimulate calcium flux and CaMKK2 to interfere in insulin activation of AKT-glucose signaling. To explain our preliminary data that global PAR2-deficiency lowers basal plasma glucose, but increases liver glucose and glycogen in both lean and obese mouse models, Aim 3 will test the hypothesis that hepatocyte PAR2 suppresses the major liver glucose transporter GLUT2 through a mechanism potentially involving Gq-MAPK-FoxA3. Aims 1-3 will utilize our pepducin technology developed to allosterically target G-protein coupled receptors on the inside surface of the plasma membrane to help delineate specific PAR2-effector signaling pathways in aberrant liver metabolism and insulin resistance in vitro and in vivo.