ABSTRACT Non-alcoholic fatty liver disease (NAFLD) is linked to insulin resistance, type 2 diabetes mellitus (T2DM) and several other major comorbidities. Hepatic carbohydrate and lipid metabolism is maintained by the balance between anabolic hormones, primarily insulin, and catabolic hormones including glucagon and catecholamines. Our novel findings show that norepinephrine (NE) resistance occurs within a week of high fat diet (HFD) feeding in mice. This proposal seeks to determine the molecular mechanisms leading to the suppression of NE signaling and the effect this has on hepatic metabolism and the pathogenesis of NALFD and T2DM. NE and related hormones act through G-protein coupled receptors linked to Gq/11 and phospholipase C to generate the second messengers inositol 1,4,5 trisphosphate (IP3) and diacylglycerol, which induce cytosolic Ca2+ oscillations in individual hepatocytes. These dynamic Ca2+ signals are integrated in the intact liver by intercellular propagation of Ca2+ waves across entire hepatic lobules, from the periportal (PP) to the pericentral (PC) zones. Glucagon synergizes with Ca2+ to promote PP glycogenolysis and gluconeogenesis and inhibit PC de novo lipogenesis. Our data show that short-term HFD attenuates NE-induced Ca2+ signaling in hepatocytes and impairs the propagation of intralobular Ca2+ waves in intact perfused liver. PP hepatocytes specialize in gluconeogenesis, whereas PC hepatocytes are the primary site of lipogenesis and the first to show steatosis in NAFLD. We propose that this early lesion in Ca2+ signaling is responsible for the loss of Ca2+-dependent inhibition of lipogenesis selectively in PC hepatocytes. The present studies build on the unique expertise of the investigators and several experimental tools developed by the team. The aims to be investigated are: 1. Identify the molecular mechanisms by which HFD suppresses Ca2+ signaling. We will investigate the role of novel Protein Kinase C (nPKC) isoforms in suppressing Ca2+ signaling by negative feedback on hormone- induced IP3 generation. In vivo knockdown and direct manipulation of nPKC isoforms will test the hypothesis that HFD impairs Ca2+ signaling through constitutive activation of nPKC as a consequence of lipid accumulation, giving rise to a self-sustaining cycle of progressive steatosis. 2. Investigate zonal differences in Ca2+ signaling and how they are affected by HFD. We will examine the effect of feeding HFD on the zonal organization of Ca2+ signaling in intact liver perfused with Ca2+-dependent hormones, and how this modifies the synergistic regulation of hepatic metabolism by glucagon. In addition, isolated hepatocytes will be zonally stratified to elucidate mechanisms of Ca2+ signaling dysfunction. 3. Test whether HFD affects zonal regulation of metabolism by Ca2+ to promote pericentral lipogenesis. We will determine the effect of HFD on the zonal regulation of key enzymes of lipid metabolism by Ca2+-linked hormones. We will also investiga...