Characterization of inositol 1,4,5-trisphosphate receptor isoform mRNA expression and regulation in rat pancreatic islets, RINm5F cells and βHC9 cells

Abstract

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+ channel that plays a role in the regulation of insulin secretion. In rat isolated pancreatic islets the expression of types I, II and III InsP3R mRNA was identified by reverse transcriptase-polymerase chain reaction and confirmed by cDNA cloning and sequencing. The islet ratios of types I, II and III InsP3R mRNA to β-actin mRNA were 0.08 ± 0.02, 0.08 ± 0.03 and 0.25 ± 0.04 respectively. Types I, II and III InsP3R mRNA were also expressed in rat (RINm5F) and mouse (βHC9) pancreatic β-cell lines, and rat cerebellum. Type III InsP3R mRNA was quantitatively the most abundant form in rat islets and RINm5F cells. In βHC9 cells, types II and III InsP3R mRNA were expressed at similar levels, and in much greater abundance than type I mRNA. Type III was the least abundant InsP3R mRNA in cerebellum. Culture of βHC9 cells for 5 days at 2-8 and 25 mM glucose, or RINm5F cells for 7 days at 5.5 and 20 mM glucose, resulted in significantly enhanced expression of type III, but not types I and II, InsP3R mRNA in the cells at the higher glucose concentrations. During short-term (0.5-2 h) incubations, βHC9 cell type III InsP3R mRNA levels increased in response to glucose in a time-and concentration-dependent manner. Actinomycin D inhibited the glucose response. α-Ketoisocaproic acid also stimulated βHC9 cell type III InsP3R mRNA expression in a concentration-dependent manner, whereas 2-deoxyglucose and 3- O-methylglucose were without effect. The different levels of expression of mRNA for three InsP3R isoforms in islets and insulinoma cells, and the influence of glucose and α-ketoisocaproic acid on the expression of type III mRNA, suggests that nutrient metabolism plays a role in the regulation of this gene and that the function of InsP3R subtypes may be unique with each playing a distinct role in β3-cell signal transduction and insulin secretion.