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

Abstract

The role of ER Ca2+ release via ryanodine receptors (RyR) in pancreatic {beta}-cell function is not well defined. Deletion of RyR2 from the rat insulinoma INS-1 (RyR2KO) enhanced the Ca2+ integral (AUC) stimulated by 7.5 mM glucose, and rendered it sensitive to block by the IP3 receptor inhibitor xestospongin C, coincident with reduced levels of the protein IP3 Receptor Binding protein released with Inositol 1,4,5 Trisphosphate (IRBIT; aka AHCYL1). Deletion of IRBIT from INS-1 cells (IRBITKO) increased the Ca2+ AUC in response to 7.5 mM glucose and induced xestospongin sensitivity. Insulin content and basal (2.5 mM glucose) and 7.5 mM glucose-stimulated insulin secretion were reduced in RyR2KO cells and more modestly reduced in IRBITKO cells compared to controls. INS2 mRNA levels were reduced in both RyR2KO and IRBITKO cells, but INS1 mRNA levels were specifically decreased in RyR2KO cells. Nuclear localization of S-adenosylhomocysteinase (AHCY) was increased in RyR2KO and IRBITKO cells. DNA methylation of the INS1 and INS2 gene promotor regions was very low, and not different among RyR2KO, IRBITKO, and controls. In contrast, exon 2 of the INS1 and INS2 genes was more extensively methylated in RyR2KO and IRBITKO cells than in controls. Proteomics analysis using LC-MS/MS 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. One sentence SummaryDeletion of RyR2 from INS-1 cells had the unanticipated effect of reducing IRBIT proteins levels, and both RyR2 and IRBIT contribute to maintenance of glucose- stimulated insulin secretion.