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: 10242138
• 负责人: Elizabeth Eun-Jun Ha
• 金额: $ 5.1万
• 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/10242138
• 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，心血管疾病 和冠状动脉疾病（CAD）仍然是西方世界的主要死亡原因，强调了 需要改善疗法。基因组广泛的关联研究（GWAS）已鉴定出单核苷酸 在8q24染色体区域的Tribbles-1假酶（Trib1）基因附近的多态性（SNP）（SNP） 与CAD和脂质性状辅助，表明Trib1在脂质代谢中的作用。脂联素的GWA 水平确定了在Trib1基因座中与GWAS信号重叠的脂质水平重叠的明显SNP。 再加上Trib1在脂肪组织中高度表达的观察结果，这表明Trib1具有 在调节脂质中脂肪中的功能作用。我们已经产生了脂肪细胞特异性的Trib1敲除小鼠 （Trib1_asko），这些小鼠的血浆脂联素水平增加，血浆甘油三酸酯降低 与野生型（WT）相比，总胆固醇。这种效果朝着相反的方向 在肝特异性部落1基因敲除小鼠中观察到的脂质表型，血清甘油三酸酯增加了 和胆固醇，表明Trib1的组织特异性作用。 Trib1是一种脚手架蛋白，通过 蛋白质 - 蛋白质相互作用，并可以通过介导其与A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小鼠中脂联素水平的升高是 由转录后机制介导的，本提案的目标1是确定Trib1是否在调节 通过特定蛋白质蛋白相互作用的脂联素分泌。离体脂肪细胞模型将用于研究 脂联素分泌，以及对血清多聚体状态和翻译后修饰的研究 来自Asko小鼠的脂联素将为脂联素分泌的可能调节机制提供信息。潜在的 Trib1和候选脂联素调节蛋白之间的相互作用将通过 免疫荧光，co-IP和蛋白质印迹。 AIM 2专注于了解脂肪细胞的作用 脂肪蛋白调节脂肪调节机理的trib1。具体而言，我将研究1）通过 LPL，2）脂解和3）脂肪细胞的胆固醇外排。这些将通过IN组合进行研究 脂蛋白清除率（基于放射性标记）的体内测定和脂肪分解和胆固醇外排的体外研究 （基于Radiolabel）。另外，脂肪酸摄取，脂解，蛋白质印迹和QPCR 和胆固醇外排；脂肪特异性LPL活性的测定；血浆甘油和游离的测量 脂肪酸将有助于确定Trib1对这些机制的潜在调节。脂质调节的差异 还将通过汇总血浆的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，小鼠生理和疾病建模的各个方面，以及 分子/细胞生物学，并在经验丰富的导师的指导下在哥伦比亚大学的环境中 医疗中心将为受训者做好转化研究职业的准备。