Pannexin 1 channels in diet-induced metabolic syndrome

项目3项目概要 缺乏体育活动沿着西方饮食和生活方式， 肥胖、糖尿病和脂肪肝（NAFLD）。NAFLD被认为是一个独立的风险因素， 心血管疾病，包括多种进展性疾病状况， 脂肪变性、非酒精性脂肪性肝炎（NASH）和肝纤维化，其也与衰老相关， 肝硬化和肝细胞癌的风险增加。NAFLD的进展伴随着 肝纤维化造成严重的健康问题，最终导致肝移植成为最后的手段。治疗 选择是有限的，并且对新的治疗概念存在巨大的需求。驱动机制 NAFLD进展为纤维化在很大程度上是未知的，可能涉及细胞源性危险的多重损伤- 相关分子模式（DAMP），如细胞外ATP及其代谢产物，这意味着 调节肝脏炎症和纤维化。然而，控制ATP释放的机制， 肝细胞未知。我们的初步数据表明，肝细胞表达功能性泛连接蛋白-1 （Panx 1）通道，在脂毒性诱导的半胱天冬酶依赖性裂解时释放ATP。药理 肝细胞中Panx 1的抑制或基因缺失保护小鼠免受饮食和衰老诱导的脂肪变性， 脂肪性肝炎和肝纤维化。基于这些数据，我们将检验Panx 1- 肝细胞依赖性ATP释放通过激活星状细胞促进纤维化，我们将检测 抑制Panx 1通道功能是对抗NASH可行的治疗方法。在具体目标1中， 将研究肝脏Panx 1缺陷（Panx 1fl/fl/Albcre）和病毒（AAV 8）介导的Panx 1 在脂肪肝的病理定义阶段-脂肪变性、NASH和晚期 纤维化，使用喂食高果糖、棕榈酸酯、胆固醇（FPC）饮食5、12或16周的小鼠。病毒介 将使用Panx 1缺陷小鼠中Panx 1肝脏再表达和Panx 1的转基因过表达 随着功能获得的临近。我们将开发新的肝特异性反义寡核苷酸（阿索）介导的 针对Panx 1的治疗方法，将在FPC饮食模型中进行测试，在转基因APOE 3- Leiden/CETP小鼠，代谢综合征的“人源化”小鼠模型，以及衰老诱导的纤维化。在 具体目标2，我们将测试的假设，（a）Panx 1依赖性ATP释放从肝细胞调节 肝星状细胞通过旁分泌作用的纤维化能力，和（B）ATP或其代谢物对照 通过Panx 1依赖性自分泌机制的肝细胞代谢。这些数据将提供 美国新的信息饮食引起的代谢并发症，现在被认为是诱发和 加速因素，心脏代谢综合征的一部分;此外，我们对肝纤维化的新知识也将 会导致心脏纤维化

PROJECT 3 PROJECT SUMMARY Lack of physical activity along with a western diet and lifestyle is accompanied by an increased incidence of obesity, diabetes, and fatty liver disease (NAFLD). NAFLD is considered an independent risk factor for cardiovascular disease and encompasses multiple progressive disease conditions beginning with hepatic steatosis, non-alcoholic steatohepatitis (NASH) and hepatic fibrosis, which is also associated with aging and increased risk for cirrhosis and hepatocellular carcinoma. The progression of NAFLD with accompanying hepatic fibrosis poses a significant health problem, culminating in liver transplant as the last resort. Treatment options are limited and there is a tremendous need for novel therapeutic concepts. The mechanisms driving NAFLD progression to fibrosis are largely unknown and may involve multiple insults by cell-derived danger- associated molecular patterns (DAMPs) such as extracellular ATP and its metabolites, which were implied to regulate hepatic inflammation and fibrosis. However, the mechanisms that control the release of ATP from hepatocytes are not known. Our preliminary data demonstrate that hepatocytes express functional pannexin-1 (Panx1) channels that release ATP upon lipotoxicity-induced caspase-dependent cleavage. Pharmacological inhibition or genetic deletion of Panx1 in hepatocytes protected mice against diet- and aging-induced steatosis, steatohepatitis and hepatic fibrosis. Based on these data, we will examine the hypothesis that Panx1- dependent ATP release from hepatocytes promotes fibrosis via activation of stellate cells and we will test if inhibition of Panx1 channel function is a feasible therapeutic approach to combat NASH. In specific Aim 1 we will investigate the effect of hepatic Panx1 deficiency (Panx1fl/fl/Albcre) and virus (AAV8)- mediated Panx1 ablation during the pathologically defined stages of fatty liver disease - steatosis, NASH, and advanced fibrosis, using mice fed a high fructose, palmitate, cholesterol (FPC) diet for 5, 12 or 16 weeks. Virus-mediated hepatic re-expression of Panx1 in Panx1-deficient mice and transgenic overexpression of Panx1 will be used as gain-of-function approaches. We will develop novel liver-specific antisense oligonucleotide (ASO)-mediated therapeutic approaches directed at Panx1, which will be tested in the FPC diet model, in transgenic APOE3- Leiden/CETP mice, a “humanized” mouse model of metabolic syndrome, and in aging-induced fibrosis. In specific Aim 2 we will test the hypotheses that (a) Panx1-dependent ATP release from hepatocytes regulates fibrotic capacity of hepatic stellate cells via paracrine effects, and (b) ATP or its metabolites controls metabolism of hepatocytes via Panx1-dependent autocrine mechanisms. Collectively, these data will provide us new information on diet-induced metabolic complications that are now considered predisposing and accelerating factors, part of the cardiometabolic syndrome; also, our new knowledge on liver fibrosis will also have implications for cardiac fibrosis.