Potential Mechanisms of Glucagon-like peptide-1 in Fatty Liver Disease

描述（由申请人提供）：非酒精性脂肪性肝病（NAFLD）和非酒精性脂肪性肝炎（NASH）正迅速成为美国患者寻求胃肠病学建议的最常见原因。胰岛素抵抗是导致肝细胞中甘油三酯净积累的主要病理生理问题。目前治疗脂肪肝的方法有限。胰高血糖素样肽（GLP-1）是一种由小肠L细胞分泌的天然肠道肽。它被二肽基肽酶IV （DPPIV）裂解，因此其生物半衰期短。Exendin-4是一种抗DPPIV切割的同源肽。最近的数据显示，GLP-1及其同源物可改善胰岛素抵抗。这些肽具有多种作用，包括刺激胰腺b细胞的生长和对脂肪细胞的作用。迄今为止，关于GLP-1在肝细胞中的直接作用的数据有限。我们在这项应用中的长期目标是解决GLP-1对肝细胞脂肪代谢的基本生物学作用，并证明GLP-1的有益作用不仅与胰岛素增敏作用有关，而且是直接作用于肝细胞的生物学作用。由于GLP-1蛋白也是厌氧性的，因此我们将为NAFLD和NASH的潜在新颖和安全的治疗方式提供分子基础，因此这种治疗将对这类患者群体有多重益处。对ob/ob小鼠长期给予Exendin-4的初步数据表明，与胰岛素敏感性增强相关的关键参数显着改善，包括体重减轻，肝脏组织学改善和肝脂肪变性的消失，以及硫代巴比妥酸反应性物质（TEARS）的减少，这是氧化应激的替代标志物。在本提案中，我们将验证GLP-1或其合成类似物通过激活其g蛋白偶联受体（GPCR）直接作用于肝细胞以减少肝甘油三酯净储存的假设。为了验证这一假设，人们提出了三个目标。具体目标1：通过研究(i) GLP-1-肝细胞结合特性，（ii） cAMP产生的测量，以及（iii）阐明GLP-1磷酸化的其他肝细胞信号转导蛋白，确定肝脏中GLP-1-肝细胞相互作用的细胞基础。特异性目标2：对GLP-1受体进行RNA干扰，并确定与GLP-1相关的基因谱（有利于肝细胞脂肪酸消耗）是否被逆转；测量GLP-1对肝脂肪酸代谢关键酶的影响。具体目的3：量化GLP-1对甘油三酯从头合成的生化还原，以及对脂肪酸氧化和VLDL分泌的增强。这项工作的完成将为脂肪肝疾病提供潜在的治疗方法，脂肪肝是美国最常见的肝功能异常原因。该建议符合美国公共卫生服务在治疗慢性肝病方面的目标。

DESCRIPTION (provided by applicant): Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are rapidly becoming the commonest reason patients in the United States seek advice from a gastroenterologist. Insulin resistance is the primary pathophysiologic problem that results in a net accumulation of triglycerides in hepatocytes. At present there is limited therapy for fatty liver disease. Glucagon-like peptide (GLP-1) is a naturally occurring gut peptide secreted by the L cells of the small intestine. It is cleaved by dipeptidyl peptidase IV (DPPIV) and thus it biological half-life is short. Exendin-4 is a homologous peptide resistant to DPPIV cleavage. Recent data reveals that GLP-1 and its homologue improve insulin resistance. These peptides have pleotropic effects including stimulating growth of b cells of the pancreas and action on adipocytes. To date there is limited data about a direct role of GLP-1 in hepatocytes. Our long-term goal in this application is to address the fundamental biological actions of GLP-1 on fat metabolism in the hepatocyte and prove that the beneficial effects of GLP-1 are not only related to insulin sensitizing effects but also result from direct biological actions on hepatocytes. We will provide a molecular basis for a potentially novel and safe treatment modality for NAFLD and NASH since GLP-1 proteins are also anorexigenic and consequently such therapy would have multiple benefits for this patient population. Preliminary data in long-term administration of Exendin-4 to ob/ob mice indicate marked improvement in key parameters associated with enhanced insulin sensitivity including weight loss, improved hepatic histology and loss of hepatic steatosis, and reduced thiobarbituric acid reactive substances (TEARS), a surrogate marker of oxidative stress. In this proposal we will test the hypothesis that GLP-1, or its synthetic analogue, acts directly on the hepatocyte to reduce net hepatic triglyceride stores via activation of its G-protein coupled receptor (GPCR). Three aims have been developed to test this hypothesis. Specific Aim 1 :To determine the cellular basis for GLP-1- hepatocyte interactions in the liver by studying (i) GLP-1-hepatocyte binding characteristics, (ii) measurement of cAMP production, and (iii) elucidation of other hepatocyte signal transduction proteins phosphorylated by GLP-1. Specific Aim 2: To employ RNA interference for GLP-1 receptor and determine whether the gene profile associated with GLP-1, which favors hepatocyte fatty acid depletion, is reversed; and, to measure the effect of GLP-1 on key enzymes involved with hepatic fatty acid metabolism. Specific Aim 3: To quantify the biochemical reduction of de novo synthesis of triglycerides and enhanced oxidation of fatty acids and VLDL secretion by GLP-1. The completion of this work will provide potential treatment for fatty liver disease, the most common cause of liver function abnormalities in the United States. This proposal meets the goals of the US Public Health Service in treating chronic liver diseases.