Abstract: The severity and incidence of T2DM is directly related to hepatic lipid concentration. Even before -cell failure ensues the severity of non-alcoholic fatty liver disease (NAFLD) is positively associated withhyperinsulinemia and insulin resistance. The hepatic vagal nerve plays a key role in glucose homeostasisaffecting both pancreatic insulin release and insulin sensitivity. Acutely eliminating hepatic afferent signalingstimulates insulin release and decreases skeletal muscle glucose clearance simultaneously resulting inhyperinsulinemia and insulin resistance. Conversely acutely stimulating the hepatic afferent nerve inhibits insulinrelease and improves glucose clearance. Until recently there was no evidence for a hepatokine that signaled tothe vagal nerve to alter glucose homeostasis. We have established that hepatic lipid accumulation dose-dependently increases hepatic production and release of -aminobutyric acid (GABA) an inhibitoryneurotransmitter. Our data proposes that hepatocyte produced GABA stimulates insulin release and decreaseskeletal muscle glucose clearance by altering activity of the hepatic vagal nerve. To establish therapeuticpotential we have shown that liver GABA transaminase knockdown decreases liver GABA release restoringinsulin sensitivity and normo-insulinemia in diet-induced obese mice. Through clinical trials we have highlightedthe translational impact of potentially targeting hepatic GABA signaling. In clinical samples we have shown thathepatic GABA-transaminase mRNA expression is positively correlated with serum insulin and HOMA-IR. Inthese same clinical samples we have shown that glucose disposal during a hyperinsulinemic euglycemic clampis positively associated with mRNA expression of GABA re-uptake transporters and negatively associated withmRNA expression of GABA exporters. We propose 3 Aims focused on our central hypothesis that GABA is ahepatokine that can help explain the link between hepatic lipid accumulation and hyperinsulinemia and insulinresistance in obesity. Aim 1: Assess how obesity lipids diacylglycerol ceramides and downstream signaling affect direction of flux through the GABA shunt and transport of GABA across the plasma membrane. Aim 2: Assess the glucoregulatory response to exacerbating hepatic GABA production in lean mice or limiting hepatic GABA production in obese mice. Aim 3: Assess the glucoregulatory response to knockout (loss) and adenoviral induced overexpression (gain) of hepatic GABA transporters in lean and diet-induced obese mice.Impact: Validation of GABA as a novel hepatokine that affects serum insulin and insulin sensitivity in obesitywill provide new therapeutic targets to treat this disease.