Pannexin 1 channels in diet-induced metabolic syndrome

项目3项目总结 缺乏体力活动，加上西式饮食和生活方式，伴随着糖尿病发病率的增加。 肥胖、糖尿病和脂肪肝(NAFLD)。非酒精性脂肪肝被认为是一个独立的危险因素 心血管疾病，包括多种进展性疾病，从肝脏开始 脂肪变性、非酒精性脂肪性肝炎(NASH)和肝纤维化，这也与衰老和 增加患肝硬变和肝细胞癌的风险。伴随而来的非酒精性脂肪肝进展 肝纤维化是一个重大的健康问题，最终导致肝脏移植成为最后的手段。治疗 选择有限，对新的治疗概念有极大的需求。驱动机制 NAFLD进展为纤维化在很大程度上是未知的，可能涉及细胞衍生危险的多重侮辱- 相关分子模式(DAMP)，如细胞外ATP及其代谢物，暗示 调节肝脏炎症和纤维化。然而，控制ATP从体内释放的机制 肝细胞尚不清楚。我们的初步数据表明，肝细胞表达功能性的pAnnexin-1。 (Panx1)在脂毒诱导的caspase依赖的切割时释放ATP的通道。药理作用 肝细胞中Panx1基因的抑制或基因缺失可以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Panx1- 肝细胞依赖的ATP释放通过激活星状细胞促进纤维化，我们将测试 抑制Panx1通道功能是对抗NASH的可行治疗方法。在具体目标1中，我们 将研究肝脏Panx1缺陷(Panx1fl/fl/Albcre)和病毒(AAV8)介导的Panx1的影响 脂肪肝病病理阶段的消融治疗--脂肪变性、NASH和晚期 纤维化，使用高果糖、棕榈酸酯、胆固醇(FPC)饮食喂养5、12或16周的小鼠。病毒介导的 Panx1基因缺陷小鼠肝脏Panx1的再表达和转基因Panx1的过表达 随着函数增益的接近。我们将开发新型肝脏特异性反义寡核苷酸(ASO)介导的 针对Panx1的治疗方法，将在FPC饮食模型中进行测试，在转基因APOE3- 莱顿/CETP小鼠，代谢综合征和衰老诱导的纤维化的人源化小鼠模型。在……里面 具体目标2我们将测试以下假设：(A)依赖Panx1的肝细胞释放ATP调节 肝星状细胞通过旁分泌效应的纤维化能力，以及(B)三磷酸腺苷或其代谢产物控制 肝细胞通过依赖Panx1的自分泌机制进行代谢。总而言之，这些数据将提供 美国关于饮食诱导的代谢并发症的新信息，这些并发症现在被认为是易感和 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 对心脏纤维化有影响。

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.