Investigating the Effects of Adipocyte-Specific Knockout of Tribbles1 on Plasma Adiponectin Levels and Lipoprotein Metabolism

研究脂肪细胞特异性敲除 Tribbles1 对血浆脂联素水平和脂蛋白代谢的影响

• 批准号: 10017698
• 负责人: Elizabeth Eun-Jun Ha
• 金额: $ 5.05万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10017698
• 关键词: 3T3-L1 Cells; 8q24; ANGPTL4 gene; ATP binding cassette transporter 1; Academic Medical Centers; Adipocytes; Adipose tissue; Biological Assay; Biology; Blood Vessels; Cardiovascular Diseases; Cause of Death; Cessation of life; Cholesterol; Coronary Arteriosclerosis; Data; Development; Diet; Disease model; Engineering; Fatty Acids; Future; Genes; Genetic study; Genotype-Tissue Expression Project; Glycerol; Hepatic; High Density Lipoprotein Cholesterol; Human Genetics; Immunofluorescence Immunologic; Knock-out; Knockout Mice; LDL Cholesterol Lipoproteins; Lipids; Lipolysis; Lipoproteins; Liver; Measurement; Measures; Mediating; Mentors; Metabolic; Metabolic Diseases; Metabolism; Modeling; Molecular and Cellular Biology; Mus; Nonesterified Fatty Acids; Phenotype; Physiology; Plasma; Post-Translational Protein Processing; Proteins; Publishing; Regulation; Role; Scaffolding Protein; Serum; Signal Transduction; Single Nucleotide Polymorphism; Testing; Tissue Sample; Tissues; Translational Research; Triglycerides; United States; Very low density lipoprotein cholesterol; Western Blotting; Western World; adipokines; adiponectin; base; burden of illness; career; disorder risk; effective therapy; experience; fast protein liquid chromatography; genetic regulatory protein; genome wide association study; genomic locus; human data; improved; in vitro Model; in vivo; in vivo Model; lipid metabolism; lipoprotein lipase; metabolic abnormality assessment; mouse model; novel; novel therapeutics; overexpression; particle; perilipin; protein degradation; protein function; protein protein interaction; radiotracer; success; therapeutic target; trait; transcription factor; transcriptome sequencing; ubiquitin-protein ligase; uptake

Despite the success of cholesterol lowering treatments in reducing disease burden, cardiovascular disease and coronary artery disease (CAD) are still the leading causes of death in the western world, highlighting the need for improved therapies. Genome wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) near the Tribbles-1 pseudokinase (TRIB1) gene in the 8q24 chromosomal region that associate with CAD and lipid traits, suggesting a role for TRIB1 in lipid metabolism. A GWAS for adiponectin levels identified significant SNPs in the TRIB1 locus that overlap with the GWAS signal for lipid levels. Combined with the observation that TRIB1 is highly expressed in adipose tissue, this suggests that TRIB1 has a functional role in adipose in regulating lipids. We have generated adipocyte-specific Trib1 knockout mice (Trib1_ASKO), and these mice have increased plasma adiponectin levels and decreased plasma triglycerides and total cholesterol compared to their wild-type (WT) counterparts. This effect is in the opposite direction of the lipid phenotype observed in liver-specific Trib1 knockout mice, which have increased serum triglycerides and cholesterol, suggesting tissue specific roles for Trib1. Trib1 is a scaffold protein that functions through protein-protein interactions, and can promote the degradation of proteins by mediating their interaction with an E3 ubiquitin ligase. Preliminary data suggest that the increased adiponectin levels in Trib1_ASKO mice is mediated by a posttranscriptional mechanism, and aim 1 of this proposal is to determine if Trib1 regulates adiponectin secretion via specific protein-protein interactions. An ex vivo adipocyte model will be used to study adiponectin secretion, and studies of the multimeric state and posttranslational modifications of serum adiponectin from ASKO mice will inform possible regulatory mechanisms of adiponectin secretion. Potential interactions between Trib1 and candidate adiponectin-regulatory proteins will be tested through immunofluorescence, Co-IP, and western blotting. Aim 2 is focused on understanding the role of adipocyte Trib1 in mechanisms of adipose regulation of lipoproteins. Specifically, I will investigate 1) fatty acid uptake via Lpl, 2) lipolysis, and 3) cholesterol efflux from adipocytes. These will be studied through a combination of in vivo assays of lipoprotein clearance (radiolabel-based) and ex vitro studies of lipolysis and cholesterol efflux (radiolabel-based). Additionally, western blots and qPCR of standard regulators of fatty acid uptake, lipolysis, and cholesterol efflux; assays for adipose-specific Lpl activity; and measurements of plasma glycerol and free fatty acids will help determine Trib1's potential regulation of these mechanisms. Differences in lipid regulation will also be studied through FPLC lipoprotein profiling of pooled plasma and lipid profiling of adipose tissue. This project incorporates aspects of human genetics, mouse physiology and modeling of diseases, and molecular/cellular biology, and, with guidance from experienced mentors in the setting of Columbia University Medical Center, will prepare the trainee for a career in translational research.

尽管降低胆固醇治疗在减轻疾病负担方面取得了成功，但心血管疾病 和冠状动脉疾病（CAD）仍然是西方世界死亡的主要原因，这凸显了 需要改进的治疗方法。全基因组关联研究（GWAS）已鉴定出单核苷酸 8q24 染色体区域 Tribbles-1 假激酶 (TRIB1) 基因附近的多态性 (SNP) 与 CAD 和脂质特征相关，表明 TRIB1 在脂质代谢中发挥作用。脂联素的 GWAS 水平鉴定了 TRIB1 基因座中与脂质水平的 GWAS 信号重叠的显着 SNP。 结合TRIB1在脂肪组织中高表达的观察结果，这表明TRIB1具有 脂肪在调节血脂方面的功能作用。我们已经培育出脂肪细胞特异性 Trib1 基因敲除小鼠 (Trib1_ASKO)，这些小鼠的血浆脂联素水平升高，血浆甘油三酯降低 和总胆固醇与野生型（WT）对应物相比。这种效应是相反的方向 在肝脏特异性 Trib1 敲除小鼠中观察到的脂质表型，其血清甘油三酯增加 和胆固醇，表明 Trib1 具有组织特异性作用。 Trib1 是一种支架蛋白，其功能通过 蛋白质与蛋白质之间的相互作用，并且可以通过介导蛋白质与蛋白质的相互作用来促进蛋白质的降解 E3 泛素连接酶。初步数据表明 Trib1_ASKO 小鼠脂联素水平升高是 由转录后机制介导，该提案的目标 1 是确定 Trib1 是否调节 通过特定的蛋白质-蛋白质相互作用分泌脂联素。离体脂肪细胞模型将用于研究 脂联素分泌、血清多聚体状态和翻译后修饰的研究 ASKO 小鼠的脂联素将揭示脂联素分泌的可能调节机制。潜在的 Trib1 和候选脂联素调节蛋白之间的相互作用将通过以下方式进行测试 免疫荧光、Co-IP 和蛋白质印迹。目标 2 侧重于了解脂肪细胞的作用 Trib1 在脂蛋白脂肪调节机制中的作用。具体来说，我将研究 1) 脂肪酸的摄取 Lpl，2) 脂肪分解，3) 脂肪细胞中的胆固醇流出。这些将通过组合进行研究 脂蛋白清除的体内测定（基于放射性标记）以及脂肪分解和胆固醇流出的体外研究 （基于放射性标记）。此外，还对脂肪酸摄取、脂肪分解、 和胆固醇流出；脂肪特异性 Lpl 活性测定；血浆甘油和游离甘油的测量 脂肪酸将有助于确定 Trib1 对这些机制的潜在调节作用。脂质调节的差异 还将通过合并血浆的 FPLC 脂蛋白分析和脂肪组织的脂质分析进行研究。 该项目结合了人类遗传学、小鼠生理学和疾病建模的各个方面，以及 分子/细胞生物学，并在哥伦比亚大学经验丰富的导师的指导下 医学中心将为实习生从事转化研究职业做好准备。