Abstract
The prevalence of type 2 diabetes in the United States is projected to double or triple by 2050. We reasoned that the genes that modulate insulin production might be new targets for diabetes therapeutics. Therefore, we developed an siRNA screening system to identify genes important for the activity of the insulin promoter in beta cells. We created a subclone of the MIN6 mouse pancreatic beta cell line that expresses destabilized GFP under the control of a 362 base pair fragment of the human insulin promoter and the mCherry red fluorescent protein under the control of the constitutively active rous sarcoma virus promoter. The ratio of the GFP to mCherry fluorescence of a cell indicates its insulin promoter activity. As G protein coupled receptors (GPCRs) have emerged as novel targets for diabetes therapies, we used this cell line to screen an siRNA library targeting all known mouse GPCRs. We identified several known GPCR regulators of insulin secretion as regulators of the insulin promoter. One of the top positive regulators was Gpr27, an orphan GPCR with no known role in beta cell function. We show that knockdown of Gpr27 reduces endogenous mouse insulin promoter activity and glucose stimulated insulin secretion. Furthermore, we show that Pdx1 is important for Gpr27's effect on the insulin promoter and insulin secretion. Finally, the over-expression of Gpr27 in 293T cells increases inositol phosphate levels, while knockdown of Gpr27 in MIN6 cells reduces inositol phosphate levels, suggesting this orphan GPCR might couple to Gq/11. In summary, we demonstrate a MIN6-based siRNA screening system that allows rapid identification of novel positive and negative regulators of the insulin promoter. Using this system, we identify Gpr27 as a positive regulator of insulin production.
Highlights
13% of American adults have diabetes and these numbers continue to rise, mostly from an increase in type 2 diabetes [1,2]
Insulin resistance is a cardinal feature of type 2 diabetes, most people with insulin resistance do not develop diabetes because their pancreatic beta cells are able to compensate by increasing insulin production
To generate an internal control reporter, the GFP positive subline was subsequently infected at a low multiplicity of infection (MOI) with a second lentivirus containing mCherry under the control of the constitutive rous sarcoma virus promoter (RSV) (Figure 1A)
Summary
13% of American adults have diabetes and these numbers continue to rise, mostly from an increase in type 2 diabetes [1,2]. Insulin resistance is a cardinal feature of type 2 diabetes, most people with insulin resistance do not develop diabetes because their pancreatic beta cells are able to compensate by increasing insulin production. If insulin production cannot match the increased demand imposed by insulin resistance, hyperglycemia and frank diabetes ensues. Beta cell function further declines in most people with type 2 diabetes, resulting in the eventual failure of oral medications and the necessity of insulin therapy [3]. Improving insulin production and beta cell function is a universal goal of diabetes therapeutics. We constructed a novel screening system to screen for genes important for insulin promoter activity. By screening siRNAs targeting all GPCRs, we identify several GPCRs that regulate insulin promoter activity and characterize Gpr as a novel regulator of insulin production
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
