Characterization of Diet1, a hypercholesterolemia resistance gene

高胆固醇血症抗性基因 Diet1 的表征

• 批准号: 8213725
• 负责人: Karen Reue
• 金额: $ 38.12万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-15 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8213725
• 关键词: Accounting; Adipocytes; Algorithms; Amino Acid Sequence; Atherogenic Diet; Atherosclerosis; Bile Acid Biosynthesis Pathway; Bile Acids; Biochemical; Biological Assay; Blood; C57BL/6 Mouse; Candidate Disease Gene; Cholesterol; Cholesterol Homeostasis; Cholic Acids; Chromosomes, Human, Pair 2; Code; DNA Resequencing; Diet; Disease; Eating; Employee Strikes; Energy Metabolism; Epithelial Cells; Excretory function; Exhibits; Exons; FGF21 gene; Fatty Acids; Fibroblast Growth Factor; Gene Expression; Gene Proteins; Genes; Genetic; Genetic Variation; Genotype; Haplotypes; Hepatic; High Density Lipoproteins; Human; Human Genome; In Vitro; Inbred Strain; Inbred Strains Mice; Individual; Intestines; LDL Cholesterol Lipoproteins; Lead; Lesion; Liver; Low Density Lipoprotein Receptor; Low-Density Lipoproteins; Maps; Mediating; Molecular; Mus; Mutation; Nucleotides; Pathway interactions; Peptide Sequence Determination; Phenotype; Physiological; Polymorphic Microsatellite Marker; Population; Proteins; Regulation; Resistance; Risk; Role; Sampling; Secondary to; Signal Transduction; Small Intestines; System; Tertiary Protein Structure; Testing; Thermogenesis; Transgenic Mice; Variant; Wild Type Mouse; base; cardiovascular disorder risk; energy balance; feeding; genetic variant; genome sequencing; hypercholesterolemia; in vivo; lipid metabolism; lipid transport; mouse genome; mouse model; novel; null mutation; population based; positional cloning; protein function; public health relevance; response; trait

DESCRIPTION (provided by applicant): Elevated levels of total and low-density lipoprotein (LDL) cholesterol are associated with increased risk for atherosclerosis. Individuals within the human population exhibit a large range of cholesterol levels, likely determined by the interplay between environmental and genetic factors. Currently known genetic variations account for only a fraction of the total variance of cholesterol levels, suggesting that novel pathways and genes remain to be identified. Using positional cloning, we have identified the Diet1 gene from an inbred mouse strain that is resistant to diet-induced hypercholesterolemia and atherosclerosis. Diet1 encodes a novel protein characterized by repeating MAM and LDL receptor type A domains, and is expressed predominantly in the small intestine. The Diet1 protein sequence is highly conserved between mouse and humans. We therefore hypothesize that the human DIET1 gene is an excellent candidate gene for effects on cholesterol levels and related traits in humans. We propose to characterize Diet1 function at the molecular, cellular and physiological levels. The specific aims are: (1) Determine the cellular role of Diet1 in lipid metabolism. Our studies indicate that Diet1 is expressed in the small intestinal epithelial cells, and we hypothesize that it functions in intracellular bile acid transport. We will determine the cellular compartment(s) in which Diet1 functions, characterize the potential role of Diet1 in cellular lipid transport, and investigate the regulation of DIET1 gene expression. (2) Determine the physiological role of Diet1 in the regulation of cholesterol homeostasis. Diet1 deficient mice exhibit enhanced bile acid excretion, increased bile acid synthesis, and impaired induction of fibroblast growth factor 15 (FGF15), a key intestinal signal for the regulation of hepatic bile acid synthesis. We will test the hypothesis that the alterations in cholesterol homeostasis in Diet1 deficient mice can be attributed to impaired FGF15 regulation by FGF15 replacement in vivo. We will also determine whether enhanced Diet1 expression leads to altered enterohepatic signaling or altered cholesterol homeostasis using a Diet1 transgenic mouse. (3) Determine the mechanism underlying enhanced adaptive thermogenesis in Diet1 deficient mice. Diet1 deficient mice exhibit enhanced basal energy expenditure and adaptive thermogenesis. We hypothesize that increased circulating bile acid levels resulting from Diet1 deficiency stimulate increased fatty acid fuel availability mediated by effects on FGF21. We will test this mechanism by studies in isolated brown adipocytes, and by FGF21 administration in wild-type mice. (4) Identify common and rare variants in human DIET1 and determine association with cholesterol levels. We hypothesize that common or rare DIET1 genetic variants influence cholesterol levels in the human population. We will resequence the DIET1 coding exons from individuals with extreme high and low bile acid and LDL-C levels in a population-based sample of 8000 individuals. We will identify common and rare variants that lead to potential changes in protein function, and test these using functional assays. PUBLIC HEALTH RELEVANCE: We have identified a mutation in a novel gene-Diet1-that confers resistance to high blood cholesterol levels and atherosclerosis in the mouse. Here we will determine how Diet1 functions in the intestine to regulate bile acid synthesis and cholesterol levels using mouse models, and will investigate whether sequence variations in the human Diet1 gene influence cholesterol levels and cardiovascular disease risk in the human population. Results will further elucidate the mechanisms that control cholesterol homeostasis, and may suggest new strategies for protection or treatment of hypercholesterolemia and related disorders.

描述（由申请人提供）：总胆固醇和低密度脂蛋白（LDL）胆固醇水平升高与动脉粥样硬化风险增加相关。人群中的个体表现出很大范围的胆固醇水平，这可能是由环境和遗传因素之间的相互作用决定的。目前已知的遗传变异仅占胆固醇水平总变异的一小部分，这表明新的途径和基因仍有待确定。通过定位克隆，我们从近交系小鼠品系中鉴定出 Diet1 基因，该基因对饮食引起的高胆固醇血症和动脉粥样硬化具有抵抗力。 Diet1 编码一种新型蛋白质，其特征是重复的 MAM 和 A 型 LDL 受体结构域，主要在小肠中表达。 Diet1 蛋白序列在小鼠和人类之间高度保守。因此，我们假设人类 DIET1 基因是影响人类胆固醇水平和相关性状的绝佳候选基因。我们建议在分子、细胞和生理水平上表征 Diet1 功能。具体目标是：（1）确定Diet1在脂质代谢中的细胞作用。我们的研究表明Diet1在小肠上皮细胞中表达，我们假设它在细胞内胆汁酸转运中发挥作用。我们将确定 Diet1 发挥作用的细胞区室，表征 Diet1 在细胞脂质转运中的潜在作用，并研究 DIET1 基因表达的调节。 (2)确定Diet1在调节胆固醇稳态中的生理作用。 Diet1缺陷小鼠表现出胆汁酸排泄增强、胆汁酸合成增加以及成纤维细胞生长因子15（FGF15）诱导受损，FGF15是调节肝胆汁酸合成的关键肠道信号。我们将测试以下假设：Diet1 缺陷小鼠中胆固醇稳态的改变可归因于体内 FGF15 替代导致的 FGF15 调节受损。我们还将使用 Diet1 转基因小鼠确定 Diet1 表达增强是否会导致肠肝信号传导改变或胆固醇稳态改变。 (3) 确定 Diet1 缺陷小鼠适应性产热增强的机制。 Diet1 缺陷小鼠表现出基础能量消耗和适应性产热增强。我们假设 Diet1 缺乏导致循环胆汁酸水平增加，刺激 FGF21 介导的脂肪酸燃料可用性增加。我们将通过对分离的棕色脂肪细胞的研究以及对野生型小鼠施用 FGF21 来测试这一机制。 (4) 识别人类 DIET1 中常见和罕见的变异并确定与胆固醇水平的关联。我们假设常见或罕见的 DIET1 遗传变异会影响人群中的胆固醇水平。我们将对 8000 名个体的人群样本中胆汁酸和 LDL-C 水平极高和极低的个体的 DIET1 编码外显子进行重新测序。我们将识别导致蛋白质功能潜在变化的常见和罕见变异，并使用功能测定来测试这些变异。 公共健康相关性：我们发现了一种新基因 Diet1 的突变，该基因可以使小鼠抵抗高血液胆固醇水平和动脉粥样硬化。在这里，我们将使用小鼠模型确定 Diet1 如何在肠道中发挥作用，调节胆汁酸合成和胆固醇水平，并将研究人类 Diet1 基因的序列变异是否影响人群中的胆固醇水平和心血管疾病风险。结果将进一步阐明控制胆固醇稳态的机制，并可能提出保护或治疗高胆固醇血症和相关疾病的新策略。