Contributions of Hepatic and Intestinal Pathways to Cholesterol Excretion

肝脏和肠道途径对胆固醇排泄的贡献

基本信息

批准号：
10656625
负责人：
Gregory A Graf
金额：
$ 49.03万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-13 至 2027-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10656625
关键词：
Ablation Alanine Transaminase Apical Atherosclerosis Bile Acids Bile fluid Biliary Biological Cells Cholesterol Chronic Clinical Complex Coupling Data Development Diet Dietary Cholesterol Disease Enzymes Equilibrium Excretory function Fatty Liver Fatty acid glycerol esters Fibroblast Growth Factor Gall Bladder Diseases Gallbladder Gene Expression Glycosylated hemoglobin A Hepatic Hepatobiliary Hepatocyte High Density Lipoproteins Homeostasis Human In Vitro Inflammation Insulin Resistance Intestinal Hormones Intestines Investigation Lipids Liver Lysosomes Measures Metabolic Diseases Metabolic syndrome Modeling Molecular Mus Organ Orthologous Gene Pathway interactions Phenotype Phospholipids Physiological Plasma Post-Transcriptional Regulation Post-Translational Regulation Predisposition Process Proteins Publishing Reagent Recombinant Proteins Regulation Risk Role Signaling Molecule Small Intestines Sterols Stimulus Structure Tissues Triglycerides Variant absorption adeno-associated viral vector adenoviral mediated cardiovascular risk factor cholesterol absorption cholesterol transporters diet-induced obesity dietary experimental study ezetimibe glycemic control improved in vivo inhibitor insulin sensitivity metabolic phenotype non-alcoholic fatty liver disease novel obesity development overexpression prevent response reverse cholesterol transport tool transcriptome sequencing

项目摘要

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.

七、项目概要胆固醇逆向转运（RCT）是将多余胆固醇从体内消除的过程。身体。 RCT 的中断会导致多种代谢疾病，包括动脉粥样硬化、胰岛素抵抗力和非酒精性脂肪肝病（NAFLD）。 ABCG5 ABCG8 (G5G8) 甾醇转运蛋白促进肝胆和肠途径中甾醇的分泌。我们的新数据表明G5G8对于对抗膳食胆固醇积累是不可或缺的，然而，相对肝脏与肠道 G5G8 在对抗胆固醇积累中的作用尚不清楚。尽管 20 多年前就发现了它，但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，可以进行研究内源性 G5G8 转录后调控。初步实验揭示了几个惊喜。 1) G5G8 在体内和极化肝细胞中定位于细胞内区室。 2） G5G8 响应肠道激素 FGF15/19 的刺激而易位至顶端区域体内。 3) G5G8 在异二聚体形成后在溶酶体中被降解。我们假设肝脏 G5G8 受到翻译后调节以促进胆汁胆固醇分泌，但依赖肠道 G5G8 来防止膳食胆固醇积累，其机制是被导致 MetS 表型的临床变异所破坏。目标我将确定分子 G5G8 翻译后调控的体外和体内机制。我们将询问 WIF-B 极化中 G5G8 异二聚体的生物合成行程和稳态分布肝细胞。它位于未刺激细胞中的亚顶端结构将是确定。顶端易位响应胆汁胆固醇分泌（胆汁酸， FGF15/19等）将在体内测定并确认结果。这些研究将被重复人类 G5G8 在肝脏特异性 G5G8 缺陷小鼠中表达。目标 II 将确定影响肝脏和肠道G5G8对胆固醇排泄的影响。对照和小鼠肝脏中缺乏G5G8，肠道或两个器官都会受到含胆固醇饮食的挑战。血浆和粪便甾醇并对RCT的措施进行分析。肝基因表达和代谢表型包括将评估胰岛素敏感性和 NAFLD 的指标。目标 III 将调查人类的影响 ABCG5 和 ABCG8 的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 G5G8 缺陷小鼠。我们也会调查这些变异对肥胖相关表型发展的影响含胆固醇的饮食。