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

描述(由申请人提供)： 非酒精性脂肪性肝病(NAFLD)正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、肝硬变和肝细胞癌(HCC)。我们发现，自然产生的激素类胰高血糖素样肽1(GLP-1)具有显著减轻人类细胞中肝细胞脂肪变性的能力。临床意义：随着长效GLP-1激动剂合成异构体的出现，这类药物可能是治疗NAFLD的有吸引力的治疗方法。这项提案的目的是关注GLP-1蛋白如何减少肝细胞储存脂肪，防止肝细胞损伤。目的：GLP-1激动剂增强肝细胞应对内质网应激的能力，减少脂毒性、肝细胞凋亡和NAFLD的进展。研究计划和方法-特定目的1：证明GLP-1激动剂在饮食诱导的野生型小鼠脂肪肝模型中预防NAFLD进展的特异性和治疗效果，并通过对患有脂肪肝的小鼠进行概念验证研究。 肝细胞GLP-1R(GLP-1RCre+/FLOX/FLOX)缺失突变体。初步工作包括：1)建立一种能够诱导NAFLD和肝细胞胰岛素抵抗的饮食模型；2)成功培育出肝脏中GLP-1受体(GLP-1R)的组织特异性基因敲除小鼠，GLP-1RCre+/FLOX/FLOX经肝脏mRNA定量聚合酶链式反应证实。特异性目的2：进一步确定人肝细胞GLP-1受体的特性，并阐明GLP-1激动剂减少游离脂肪酸肝细胞储备的潜在的肝细胞胰岛素增敏作用的信号机制。关于脂肪负荷的肝细胞的初步数据表明，GLP-1受体是G蛋白偶联受体(GPCRs)，可以减少脂肪酸的储存，并调节与脂肪酸氧化相关的关键基因，从而增加mRNA和蛋白质的产生以及酶的活性。这些数据对降低脂毒性有一定的意义。目的3：研究GLP-1蛋白对应激反应的调节作用，包括激活与应激反应相关的三种内质网跨膜蛋白、转录后修饰同源蛋白(CHOP)以及与细胞存活和凋亡相关的关键蛋白的表达。初步的体外数据表明，游离脂肪酸对人肝细胞具有脂毒性，与未经处理的对照组相比，GLP-1激动剂exendin-4抑制人肝细胞凋亡，包括减少裂解的caspase-3的产生。Exendin-4竞争性拮抗剂未能证明这种保护作用。其他数据表明，exendin-4在体外和喂食高脂饮食的小鼠中都抑制CHOP的表达，但在喂食相同饮食的CHOP基因敲除小鼠中，富含脂肪的肝细胞受到保护，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.