Contributions of Hepatic and Intestinal Pathways to Cholesterol Excretion

7.项目摘要 胆固醇逆向转运（RCT）是一个过程，通过该过程，过量的胆固醇被排除在体外， 身体RCT的破坏会导致多种代谢性疾病，包括动脉粥样硬化、胰岛素 抵抗和非酒精性脂肪肝（NAFLD）。ABCG 5 ABCG 8（G5 G8）甾醇 转运蛋白促进肝胆和经肠途径中的固醇分泌。我们的新数据 表明G5 G8在对抗膳食胆固醇积累中是不可缺少的，然而， 肝脏与肠道G5 G8在对抗胆固醇蓄积中的作用尚不清楚。尽管 其发现于20多年前，对转运蛋白的翻译后调控知之甚少。 我们最近开发出了关键的生物试剂和方法， 内源性G5 G8转录后调节。初步实验显示， 惊喜1)G5 G8在体内和极化肝细胞中定位于细胞内区室。（二） G5 G8在肠激素FGF 15/19刺激下易位至顶端结构域 in vivo. 3)G5 G8在异二聚体形成后在溶酶体中降解。我们假设 肝脏G5 G8在胆汁分泌后被调节以促进胆汁胆固醇分泌， 依赖于肠道G5 G8来防止饮食胆固醇积累， 被导致MetS表型的临床变异破坏。目的我将确定分子 G5 G8在体外和体内的翻译后调节机制。我们会审问 G5 G8异二聚体在WIF-B极化中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存在的位于亚顶端的结构将被破坏。 鉴定对胆汁胆固醇分泌刺激（胆汁酸， FGF 15/19等）将在体内测定并确认结果。这些研究将重复进行， 在肝脏特异性G5 G8缺陷小鼠中表达的人G5 G8。目标二将决定 肝和肠G5 G8对胆固醇排泄的影响。对照组和小鼠以及肝脏中缺乏G5 G8， 肠或这两个器官将受到含胆固醇饮食的挑战。血浆和粪便固醇 并对随机对照试验的测量结果进行分析。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。目标三将研究人类 ABCG 5和ABCG 8变体对基础和胆汁酸刺激的胆汁胆固醇分泌的影响， 新型腺相关病毒载体和组织特异性G5 G8缺陷小鼠。我们亦会研究 这些变异体对肥胖相关表型发展的影响， 含胆固醇的饮食。

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.