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

尽管降低胆固醇治疗在减少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）仍然是西方世界的主要死亡原因， 需要改进的治疗方法。全基因组关联研究（GWAS）已经确定了单核苷酸 Tribbles-1假激酶（TRIB 1）基因附近的8 q24染色体区域的多态性（SNP）， 与CAD和脂质性状相关，表明TRIB 1在脂质代谢中的作用。脂联素的GWAS 水平鉴定了TRIB 1基因座中与脂质水平的GWAS信号重叠的显著SNP。 结合TRIB 1在脂肪组织中高度表达的观察，这表明TRIB 1具有以下作用： 在脂肪中调节脂质的功能性作用。我们已经产生了脂肪细胞特异性Trib 1敲除小鼠， （Trib1_ASKO），并且这些小鼠具有增加的血浆脂联素水平和减少的血浆甘油三酯 和总胆固醇相比，其野生型（WT）对应物。这种影响是在相反的方向， 在肝脏特异性Trib 1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Trib 1的组织特异性作用。Trib 1是一种支架蛋白， 蛋白质-蛋白质相互作用，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小鼠中脂联素水平的增加是由于 由转录后机制介导，本提案的目的1是确定Trib 1是否调节 脂联素通过特异性蛋白质-蛋白质相互作用分泌。将使用离体脂肪细胞模型来研究 脂联素分泌，以及血清脂联素的多聚体状态和翻译后修饰的研究 从ASKO小鼠的脂联素将告知脂联素分泌的可能的调节机制。潜在 Trib 1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。目的2是了解脂肪细胞的作用 Trib 1在脂肪调节脂蛋白机制中的作用。具体来说，我将研究1）脂肪酸摄取，通过 Lpl，2）脂解，和3）脂肪细胞的胆固醇流出。这些将通过以下方式进行研究： 脂蛋白清除的体内试验（基于放射性标记）和脂解和胆固醇流出的体外研究 （基于放射性标记）。此外，蛋白质印迹和脂肪酸摄取，脂解， 和胆固醇流出;脂肪特异性Lpl活性的测定;以及血浆甘油和游离胆固醇的测量。 脂肪酸将有助于确定Trib 1对这些机制的潜在调节作用。血脂调节的差异 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等方面， 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，将准备实习生在转化研究的职业生涯。

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.