Mechanisms of glucagon-like peptide 1 (GLP-1) in fatty liver disease

描述（由申请人提供）： 非酒精性脂肪肝（NAFLD）正在迅速成为美国退伍军人的主要健康问题。肝脏脂肪通常是无害的;然而，越来越多的文献表明，相当数量的美国人会因肝脏脂肪储存而发生肝损伤，包括肝炎，肝硬化和肝细胞癌（HCC）。我们已经发现，天然存在的激素胰高血糖素样肽1（GLP-1）有能力显着减少人类细胞中的肝细胞脂肪变性。临床相关性：随着GLP-1激动剂的长效合成亚型的出现，此类药物可能是治疗NAFLD的有吸引力的疗法。本提案的目标是关注GLP-1蛋白如何减少肝细胞脂肪储存并防止肝细胞损伤。目的：GLP-1激动剂增强肝细胞处理内质网（ER）应激的能力，降低脂毒性、肝细胞凋亡和NAFLD进展。研究方法和方法-具体目的1：通过在具有GLP-1激动剂的小鼠中的概念验证研究，证明GLP-1激动剂在野生型小鼠的饮食诱导的脂肪肝模型中预防NAFLD进展的特异性和治疗功效。 肝细胞GLP-1 R的缺失突变体（GLP-1 RCre +/flox/flox）。初步工作包括建立1）能够诱导NAFLD和肝细胞胰岛素抵抗的饮食模型;和2）成功繁殖肝脏中GLP-1受体（GLP-1 R）的组织特异性敲除小鼠，通过肝脏mRNA的qPCR确认GLP-1 RCre +/flox/flox。具体目标2：进一步表征人肝细胞GLP-1受体，并阐明与GLP-1激动剂（可减少游离脂肪酸肝细胞储存）潜在肝细胞胰岛素增敏作用相关的信号传导机制。关于脂肪负载肝细胞的初步数据表明，GLP-1受体是G蛋白偶联受体（GPCR），可减少脂肪酸储存并调节与脂肪酸氧化相关的关键基因，从而增强mRNA和蛋白质产生以及酶活性。这些数据表明脂毒性降低。具体目标3：确定GLP-1蛋白如何调节整合的应激反应，包括与应激转录激活相关的三种ER跨膜蛋白的激活、c-同源蛋白（CHOP）的转录后修饰以及与细胞存活和凋亡相关的关键蛋白的表达。初步体外数据表明，游离脂肪酸对人肝细胞具有脂毒性，GLP-1激动剂毒蜥外泌肽-4可抑制人肝细胞凋亡，包括与未处理对照相比减少裂解的半胱天冬酶-3的产生。毒蜥外泌肽-4竞争性拮抗剂未能证明这种保护作用。额外的数据表明，毒蜥外泌肽-4抑制CHOP表达在体外和高脂饮食喂养的小鼠，但在CHOP基因敲除小鼠喂养相同的饮食，脂肪负载的肝细胞被保护免于凋亡死亡。

DESCRIPTION (provided by applicant): Non-alcoholic fatty liver disease (NAFLD) is rapidly becoming a major health concern among US veterans. Liver fat is usually innocuous; however a growing body of literature suggests that a significant number of Americans will develop liver injury from hepatic fat stores including hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). We have found that the naturally occurring hormone glucagon-like peptide 1 (GLP-1) has the capacity to significantly reduce hepatocyte steatosis in human cells. CLINICAL RELEVANCE: With the advent of long-acting synthetic isoforms of GLP-1 agonists such agents may be attractive therapy for treating NAFLD. The goal of this proposal is to focus on how GLP-1 proteins reduce hepatocyte storage of fat, and prevent hepatocyte injury. OBJECTIVE: GLP-1 agonists enhance hepatocyte capacity to handle endoplasmic reticular (ER) stress reducing lipotoxicity, hepatocyte apoptosis, and progression of NAFLD. RESEARCH PLAN AND METHODS - SPECIFIC AIM 1: To demonstrate the specificity and therapeutic efficacy of GLP-1 agonists to prevent progression of NAFLD in a model of diet-induced fatty liver in wild-type mice, and by proof-of-concept studies in mice with a deletion mutant for the hepatocyte GLP-1R (GLP- 1RCre+/flox/flox). Preliminary work includes establishing 1) a dietary model capable of inducing NAFLD and hepatocyte insulin resistance; and 2) successful breeding of a tissue-specific knockout mouse for the GLP-1 receptor (GLP-1R) in the liver, GLP-1RCre+/flox/flox confirmed by qPCR of liver mRNA. SPECIFIC AIM 2: To further characterize the human hepatocyte GLP-1 receptor and to clarify the signaling mechanisms associated with potential hepatocyte insulin-sensitizing effects of GLP-1 agonists which reduce free fatty acid hepatocyte stores. Preliminary data regarding fat-loaded hepatocytes that GLP-1 receptors are G- protein coupled receptors (GPCRs), and can reduce fatty acid stores and modulate key genes associated with fatty acid ¿-oxidation with respect to enhanced mRNA and protein production as well as enzyme activity. These data have implications for decreased lipotoxicity. SPECIFIC AIM 3: To identify how GLP-1 proteins regulate the integrated stress response including activation of the three ER transmembrane proteins associated with stress transcriptional activation, and post-transcriptional modification o c-¿ homologous protein (CHOP) as well as expression of key proteins associated with cell survival and apoptosis. Preliminary in vitro data indicate that free fatty acids are lipotoxic to human hepatocytes and the GLP-1 agonist, exendin-4, suppresses human hepatocyte apoptosis including reducing cleaved caspase-3 production compared to untreated controls. The exendin-4 competitive antagonist fails to demonstrate such protection. Additional data indicate that exendin-4 suppresses CHOP expression both in vitro and in mice fed a high-fat diet, but in CHOP knockout mice fed an identical diet, fat-laden hepatocytes are protected from apoptotic death.