Contributions of Hepatic and Intestinal Pathways to Cholesterol Excretion

7.项目总结 胆固醇反向转运(RCT)是指过量的胆固醇从体内排出的过程。 尸体。RCT的中断会导致多种代谢性疾病，包括动脉粥样硬化、胰岛素 耐药性和非酒精性脂肪性肝病(NAFLD)。ABCG5、ABCG8(G5G8)甾醇 转运蛋白促进肝脏、胆管和肠道途径的类固醇分泌。我们的新数据 这表明G5G8在反对饮食中的胆固醇积累方面是不可或缺的，然而，相对的 肝脏和肠道G5G8在相反的胆固醇积累中的作用尚不清楚。尽管 它是在20多年前发现的，人们对转运蛋白的翻译后调控知之甚少。 我们最近开发了关键的生物试剂和方法，将使研究 内源性G5G8转录后调控。初步实验揭示了几个 惊喜。1)G5G8在体内定位于细胞内，并定位于极化的肝细胞内。2) G5G8在肠道激素FGF15/19刺激下移位到顶区 在活体内。3)异源二聚体形成后，G5G8在溶酶体中降解。我们假设 肝脏G5G8被翻译后调节以促进胆汁胆固醇分泌，但 依赖肠道G5G8来防止饮食中的胆固醇积聚，其机制是 被影响蛋氨酸表型的临床变异所干扰。Aim I将确定分子 G5G8翻译后调控机制的体内外研究我们将审问 WIF-B极化下G5G8杂二聚体的生物合成路线和稳态分布 肝细胞。它在未受刺激的细胞中所处的亚顶端结构将是 已确认身份。胆汁胆固醇分泌刺激时的心尖移位(胆汁酸， FGF15/19等)将在体内进行检测和结果确认。这些研究将被复制到 人G5G8在肝脏特异性G5G8缺陷小鼠中表达。AIM II将确定 肝脏和肠道G5G8对胆固醇排泄的影响。对照组和小鼠，肝脏中缺乏G5G8， 肠道或两个器官都将受到含胆固醇饮食的挑战。血浆和粪便中的甾醇 并分析了RCT的措施。肝脏基因表达和代谢表型包括 将评估胰岛素敏感性和NAFLD的测量。AIM III将调查人类的影响 ABCG5和ABCG8变体对胆汁酸刺激的基础和胆汁胆固醇分泌的影响 新腺相关病毒载体和组织特异性G5G8缺陷小鼠。我们还将调查 这些变异对肥胖相关表型发展的影响 含胆固醇饮食。

7. Project Summary Reverse cholesterol transport (RCT) is the process by which excess cholesterol is eliminated from the body. Disruptions in RCT contribute to a variety of metabolic diseases including atherosclerosis, insulin resistance and non-alcoholic fatty liver disease (NAFLD). The ABCG5 ABCG8 (G5G8) sterol transporter facilitates sterol secretion in the hepatobiliary and transintestinal pathways. Our new data indicate that G5G8 is indispensable in opposing dietary cholesterol accumulation, however, the relative contribution of hepatic vs. intestinal G5G8 in opposition cholesterol accumulation is unknown. Despite its discovery over 20 years ago, little known about the post-translational regulation of the transporter. We've recently developed key biological reagents and approaches that will allow for the investigation of endogenous G5G8 post-transcriptional regulation. Preliminary experiments revealed several surprises. 1) G5G8 is localized to an intracellular compartment in vivo and in polarized hepatocytes. 2) G5G8 translocates to the apical domain in response to stimulation with the intestinal hormone FGF15/19 in vivo. 3) G5G8 is degraded in lysosomes following heterodimer formation. We hypothesize that hepatic G5G8 is post-translationally regulated to promote biliary cholesterol secretion but dependent on intestinal G5G8 to prevent dietary cholesterol accumulation, mechanisms that are disrupted by clinical variants that contribute to MetS phenotypes. Aim I will determine molecular mechanisms of G5G8 post-translational regulation in vitro and in vivo. We will interrogate the biosynthetic itinerary and steady state distribution of the G5G8 heterodimer in WIF-B polarized hepatocytes. The sub-apically located structures in which it resides in unstimulated cells will be identified. Apical translocation in response to stimuli of biliary cholesterol secretion (bile acids, FGF15/19, etc.) will be determined and results confirmed in vivo. These studies will be replicated with human G5G8 expressed in liver-specific G5G8-deficient mice. Aim II will determine the impact of hepatic and intestinal G5G8 on cholesterol excretion. Control and mice and lacking G5G8 in liver, intestine or both organs will be challenged with cholesterol-containing diets. Plasma and fecal sterols and measures of RCT will be analyzed. Hepatic gene expression and metabolic phenotypes including insulin sensitivity and measures of NAFLD will be assessed. Aim III will investigate the impact of human variants of ABCG5 and ABCG8 on basal and bile acid-stimulated biliary cholesterol secretion using novel adenoassociated viral vectors and tissue-specific G5G8-deficieny mice. We will also investigate the effect of these variants on the development of obesity-related phenotypes in response to cholesterol-containing diets.