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.
Despite the success of cholesterol lowering treatments in reducing disease burden, cardiovascular disease is still the leading cause of death, both worldwide and in the United States, thus highlighting a need for improved therapies. Genetic studies have identified a robust association between the Tribbles-1 (TRIB1) genomic locus and coronary artery disease (CAD), lipid traits, and adiponectin levels, making the Tribbles-1 pseudokinase a worthwhile protein to study in adipose tissue in the context of lipid metabolism and mechanisms of CAD. This proposal seeks to understand how adipose TRIB1 regulates plasma adiponectin levels and lipid metabolism, and the studies proposed here have the potential to uncover novel biology pertaining to lipid regulation by adipose tissue as well as to contribute to advancing the current understanding of TRIB1's role in CAD risk.
