RyR2/IRBIT regulates insulin gene transcript, insulin content, and secretion in the insulinoma cell line INS-1

The role of ER Ca(2+) release via ryanodine receptors (RyR) in pancreatic β-cell function is not well defined. Deletion of RyR2 from the rat insulinoma INS-1 (RyR2(KO)) enhanced IP(3) receptor activity stimulated by 7.5 mM glucose, coincident with reduced levels of the protein IP(3) Receptor Binding protein released with Inositol 1,4,5 Trisphosphate (IRBIT). Insulin content, basal (2.5 mM glucose) and 7.5 mM glucose-stimulated insulin secretion were reduced in RyR2(KO) and IRBIT(KO) cells compared to controls. INS2 mRNA levels were reduced in both RyR2(KO) and IRBIT(KO) cells, but INS1 mRNA levels were specifically decreased in RyR2(KO) cells. Nuclear localization of S-adenosylhomocysteinase (AHCY) was increased in RyR2(KO) and IRBIT(KO) cells. DNA methylation of the INS1 and INS2 gene promotor regions was very low, and not different among RyR2(KO), IRBIT(KO), and controls, but exon 2 of the INS1 and INS2 genes was more extensively methylated in RyR2(KO) and IRBIT(KO) cells. Exploratory proteomic analysis revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. These results suggest that RyR2 regulates IRBIT levels and activity in INS-1 cells, and together maintain insulin content and secretion, and regulate the proteome, perhaps via DNA methylation.