Functional Screening of Candidate Causal Genes for Insulin Resistance in Human Preadipocytes and Adipocytes.

Rationale: Genome-wide association studies (GWAS) have identified genetic loci associated with insulin resistance (IR) but pinpointing the causal genes of a risk locus has been challenging. Objective: To identify candidate causal genes for IR, we screened regional and biologically plausible genes (16 in total) near the top ten IR-loci in risk-relevant cell types, namely preadipocytes and adipocytes. Methods and Results: We generated 16 human Simpson-Golabi-Behmel syndrome preadipocyte knockout lines (SGBS-KO) by lentivirus-mediated CRISPR/Cas9 system. We evaluated each gene knockout by screening IR-relevant phenotypes in the three insulin-sensitizing mechanisms, including adipogenesis, lipid metabolism and insulin signaling. We performed genetic analyses to evaluate whether candidate genes prioritized by our in vitro studies were eQTL genes in human subcutaneous adipose tissue, and were associated with risk of IR, type 2 diabetes (T2D) and cardiovascular diseases (CVD). We further validated the functions of three new adipose IR genes by phenotypic rescue in the SGBS-KO cell lines. Results: Twelve genes, PPARG, IRS-1, FST, PEPD, PDGFC, MAP3K1, GRB14, ARL15, ANKRD55, RSPO3, COBLL1 and LYPLAL1, showed diverse phenotypes in the three insulin-sensitizing mechanisms, and the first seven of these genes could affect all the three mechanisms. Five of six eQTL genes are among the top candidate causal genes and the abnormal expression levels of these genes (IRS-1, GRB14, FST, PEPD and PDGFC) in human SAT could be associated with increased risk of IR, T2D and CVD. Phenotypic rescue of FST, PEPD and PDGFC in the SGBS-KO lines confirmed their function in adipose IR. Conclusions: Twelve genes showed diverse phenotypes indicating differential roles in insulin sensitization, suggesting mechanisms bridging the association of their genomic loci with IR. We prioritized PPARG, IRS-1, GRB14, MAP3K1, FST, PEPD and PDGFC as top candidate genes. Our work points to novel roles for FST, PEPD and PDGFC in adipose tissue, with consequences for cardiometabolic diseases