Characterization of Diet1, a hypercholesterolemia resistance gene

高胆固醇血症抗性基因 Diet1 的表征

• 批准号: 7862232
• 负责人: Karen Reue
• 金额: $ 38.5万
• 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/7862232
• 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; C57BL/6ByJ 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; 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 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(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有关。人类群体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只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识别。利用定位克隆，我们已经从近交系小鼠中鉴定出Diet1基因，该基因对饮食诱导的高胆固醇血症和动脉粥样硬化具有抵抗力。Diet1编码一种新的蛋白质，其特征是重复MAM和LDLA型受体结构域，主要在小肠表达。Diet1蛋白序列在老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特征的一个很好的候选基因。我们建议从分子、细胞和生理水平来描述Diet1的功能。其具体目的是：(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，我们推测它在细胞内胆汁酸运输中发挥作用。我们将确定Diet1发挥功能的细胞室(S)，表征Diet1在细胞脂质运输中的潜在作用，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缺陷小鼠表现为胆汁酸排泄增加，胆汁酸合成增加，成纤维细胞生长因子15(FGF15)的诱导受损，成纤维细胞生长因子15是调节肝脏胆汁酸合成的关键肠道信号。我们将检验这一假设，即Diet1缺陷小鼠胆固醇稳态的变化可归因于体内FGF15替代对FGF15调节的损害。我们还将使用Diet1转基因小鼠来确定增强的Diet1表达是否会导致肠道-肝脏信号的改变或胆固醇稳态的改变。(3)探讨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增加和适应性产热。我们假设，由于Diet1缺乏导致的循环胆汁酸水平增加，刺激了通过对FGF21的影响而获得的脂肪酸燃料的增加。我们将通过在分离的棕色脂肪细胞中的研究和在野生型小鼠中使用FGF21来测试这一机制。(4)确定人类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基因变异会影响人类人群中的胆固醇水平。我们将对8000名人群样本中胆汁酸和低密度脂蛋白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的变异，并使用功能分析对其进行测试。 与公共卫生相关：我们已经确定了一种新基因Diet1的突变，该突变赋予小鼠对高血胆固醇水平和动脉粥样硬化的抵抗力。在这里，我们将使用小鼠模型确定Diet1在肠道中如何调节胆汁酸合成和胆固醇水平，并将调查人类Diet1基因的序列变异是否影响人类人群的胆固醇水平和心血管疾病风险。研究结果将进一步阐明控制胆固醇稳态的机制，并可能为预防或治疗高胆固醇血症及相关疾病提供新的策略。