Metabolic Reprogramming by Protease-activated Receptor 2

过量的糖和脂肪摄入可导致一系列代谢异常，包括非酒精性脂肪肝（NAFLD）、高胆固醇血症、血脂异常、肥胖和胰岛素抵抗。新出现的证据指出了一种新的机制，将碳代谢和脂肪肝疾病与G蛋白偶联蛋白酶激活受体2（PAR 2）联系起来，PAR 2是一种先前已知参与炎症的受体。为了为肝脏PAR 2在人类中的潜在重要性提供关键支持，在对来自108名NAFLD患者和对照的肝脏标本和临床数据的横断面研究中，我们发现肝细胞中的PAR 2蛋白表达在对照肝脏中较低，并且在轻度NAFLD纤维化患者和纤维化阶段较高的NAFLD中逐渐增加。与低PAR 2表达组相比，高PAR 2表达NAFLD患者组具有显著升高的血浆LDL胆固醇。然而，PAR 2在代谢、胆固醇稳态和肝脏病理学中的明确作用仍然未知。这项拨款提案的首要目标是研究PAR 2信号在脂肪肝疾病病因和发病机制中的作用，以验证我们的中心假设，即肝脏PAR 2是高脂血症，肥胖和胰岛素抵抗的新贡献者。PAR 2由许多组织和细胞类型表达，迄今为止已使用常规的小鼠全身敲除（KO）。因此，目前尚不清楚有多少KO表型或PAR 2抑制对脂肪变性、脂质代谢和体重减轻的影响仅在肝细胞水平介导，有多少是由于PAR 2在其他表达位点的损失。我们将使用我们的新的肝细胞特异性PAR 2-KO、PAR 2 β Hep小鼠来定义和验证肝脏PAR 2在目标1-3中的这些深刻代谢效应中的特异性参与。目前还不清楚PAR 2如何促进饮食诱导的肥胖模型中的胰岛素抵抗。目的2将探索PAR 2激活Gq-PLC途径以刺激钙流和CaMKK 2干扰胰岛素激活AKT-葡萄糖信号的新机制。为了解释我们的初步数据，即在瘦和肥胖小鼠模型中，总体PAR 2缺乏降低基础血糖，但增加肝脏葡萄糖和糖原，目的3将检验肝细胞PAR 2通过可能涉及Gq-MAPK-FoxA 3的机制抑制主要肝脏葡萄糖转运蛋白GLUT 2的假设。目的1-3将利用我们开发的肽调节素技术，以变构靶向质膜内表面上的G蛋白偶联受体，以帮助描述体外和体内异常肝脏代谢和胰岛素抵抗中的特异性PAR 2效应物信号传导途径。

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.