This proposal investigates mechanisms that combine T2D (type 2 diabetes) with NAFLD (non-alcoholic fatty liver disease), which progress during chronic hepatic IR (insulin resistance) to life-threatening NASH (non- alcoholic steatohepatitis). Several studies argue that selective hepatic IR is required to integrate T2D with NAFLD/NASH. Regardless, we find that complete hepatic IR exacerbates NAFLD/NASH and T2D in mice fed the western GAN diet—which models common sugar-sweetened food and beverages associated with T2D and NAFLD/NASH in people. We model complete hepatic IR with ‘LDKO’ mice that lack hepatic Irs1 (insulin receptor substrate 1) and Irs2, which activates FoxO1 mediated transcription to induce hepatic Fst (follistatin) expression and secretion. Circulating hepatic Fst causes WAT (white adipose tissue) IR and uncontrolled lipolysis in LDKO mice. Our human data show that FST is elevated in NAFLD patients and stimulates lipolysis in human adipocytes in vitro. Interestingly, phenotypic clustering identifies an LDKO-like subset of patients with higher liver fat and circulating FST, WAT IR, and reduced lipid storage in gluteal-femoral fat. Our proposal investigates whether hepatic FoxO1 and Fst accelerate progression of NAFLD to NASH during hepatic IR. We use mouse genetics to determine whether inactivation of FoxO1 or Fst in LDKO mice fed the GAN diet can attenuate both NAFLD/NASH trajectory and liver inflammatory gene expression to identify pathways causing NAFLD/NASH during hepatic IR. Re-esterification of circulating fatty acids with hepatic Gro3P (glycerol-3- phosphate)—a fructose metabolite—is a major source of hepatic triacylglycerol in NAFLD patients. We use a simpler high-fructose diet (HFruD) to investigate whether FoxO1 and/or Fst establish NAFLD from hepatic fructose metabolism during complete or selective hepatic IR. To understand whether fructose accelerates NAFLD/NASH during hepatic IR through re-esterification of circulating fatty acid, we investigate LDKO mice fed the HFruD after deletion of hepatic FoxO1 or Fst—or deletion of Khk (Ketohexokinase) to prevent hepatic fructose metabolism. We feed mice [13C]fructose and use LC-MS to determine the incorporation of fructose metabolites into the glycerol or fatty acid moieties of liver or circulating triacylglycerol. In humans and mice, NAFLD might arise from ‘selective insulin resistance’—owing to uncontrolled hepatic glucose output in conjunction with some insulin-stimulated lipogenesis; however, we posit that selective hepatic IR might have the opposite effect and attenuate NAFLD/NASH owing to some inhibition of FoxO1 and Fst. Since chronic nutrient excess suppresses hepatic Irs2, we investigate GAN diet-induced NAFLD/NASH and T2D in mice lacking hepatic Irs2 (LKO2 mice) or Irs1 (LKO1 mice), as well as our novel Irs2tg transgenic mice with durable Irs2 expression in hepatocytes of Cntr or LDKO mice. Completion of our proposal can impact human health by identifying systemic and hep...