The prevalence of type 2 diabetes in the United States is projected to double or triple by 2050. We reasoned that the\r\ngenes that modulate insulin production might be new targets for diabetes therapeutics. Therefore, we developed an\r\nsiRNA screening system to identify genes important for the activity of the insulin promoter in beta cells. We created a\r\nsubclone of the MIN6 mouse pancreatic beta cell line that expresses destabilized GFP under the control of a 362 base pair\r\nfragment of the human insulin promoter and the mCherry red fluorescent protein under the control of the constitutively\r\nactive rous sarcoma virus promoter. The ratio of the GFP to mCherry fluorescence of a cell indicates its insulin promoter\r\nactivity. As G protein coupled receptors (GPCRs) have emerged as novel targets for diabetes therapies, we used this cell\r\nline to screen an siRNA library targeting all known mouse GPCRs. We identified several known GPCR regulators of insulin\r\nsecretion as regulators of the insulin promoter. One of the top positive regulators was Gpr27, an orphan GPCR with no\r\nknown role in beta cell function. We show that knockdown of Gpr27 reduces endogenous mouse insulin promoter activity\r\nand glucose stimulated insulin secretion. Furthermore, we show that Pdx1 is important for Gpr27�s effect on the insulin\r\npromoter and insulin secretion. Finally, the over-expression of Gpr27 in 293T cells increases inositol phosphate levels,\r\nwhile knockdown of Gpr27 in MIN6 cells reduces inositol phosphate levels, suggesting this orphan GPCR might couple to\r\nGq/11. In summary, we demonstrate a MIN6-based siRNA screening system that allows rapid identification of novel\r\npositive and negative regulators of the insulin promoter. Using this system, we identify Gpr27 as a positive regulator of\r\ninsulin production.
Loading....