Exploring the role of the RyR2/IRBIT signaling axis in pancreatic beta-cell function
Calcium influx into pancreatic beta-cells is required for proper beta-cell growth and function. While the functional significance of calcium influx into the beta-cells is known, the significance of release of calcium from intracellular stores is less understood. Calcium-induced calcium release (CICR) is a process by which calcium influx into the cell through voltage-gated calcium channels activated release of calcium from intracellular stores. The functional significance of CICR is well understood in cardiac and vascular muscle cells in regard to excitation-contraction coupling. However, the functional significance of CICR in beta-cells in not well understood.
To investigate the role of RyR2 in pancreatic beta-cell function, we utilized CRISPR-Cas9 gene editing to delete RyR2 from the rat insulinoma INS-1 cell line. we found that RyR2KO cells displayed an enhanced glucose-stimulated Ca2+ integral (area under the curve; AUC) which was sensitive to inhibition by the IP3R antagonist, xestospongin C. Loss of RyR2 also resulted in a reduction in IRBIT protein levels. Therefore, we deleted IRBIT from INS-1 cells (IRBITKO) and found that IRBITKO cells also displayed an increased Ca2+ AUC in response to glucose stimulation. RyR2 KO and IRBIT KO cells had reduced glucose-stimulated insulin secretion and insulin content. RT-qPCR revealed that INS2 transcript levels were reduced in both RyR2KO and IRBITKO. Nuclear localization of AHCY were increase in both the RyR2KO and IRBITKO cells, corresponding with increased levels of insulin gene methylation. Proteomic analysis revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. Our results suggest that RyR2 and IRBIT activity regulate insulin content, insulin secretion, and regulate the proteome in INS-1 cells
We next sought to assess the consequences on cellular Ca2+ handling in the absence of RyR2 and IRBIT in INS-1 cells. Store-operated Ca2+ entry (SOCE) stimulated with thapsigargin was reduced in RyR2KO cells compared to controls, but this was not different in IRBITKO cells. STIM1 protein levels were not different between the three cell lines. Basal and carbachol stimulated phospholipase C (PLC) activity was reduced specifically in RyR2KO cells and not IRBITKO cells. However, basal PIP2 levels were elevated in both RyR2KO and IRBITKO cells. Insulin secretion stimulated by tolbutamide was reduced in RyR2KO and IRBITKO cells compared to controls, but this was still potentiated by an EPAC-selective cAMP analog in all three cell lines. Cortical f-actin is known to regulate insulin secretion, and levels were markedly reduced in RyR2KO cells compared to control INS-1 cells. Whole-cell Cav channel current density was reduced in RyR2KO cells compared to controls, and Ba2+ current was significantly reduced by PIP2 depletion preferentially in RyR2KO cells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2KO cells compared to controls, and insensitive to the SK channel inhibitor apamin. Taken together, these results suggest that RyR2 plays a critical role in regulating PLC activity and PIP2 levels via regulation of SOCE. RyR2 also regulates beta-cell electrical activity by controlling Cav current density, via regulation of PIP2 levels, and SK channel activation.
Lastly, we investigated the role of PDE subtypes cAMP in INS-1 cells and human islets. We utilized subtype selective inhibitors of PDE1, PDE3 and PDE8 to assess the potential of these PDEs as potential therapeutic targets. We found that PDE1 is the primary subtype in INS-1 cells, whereas PDE3 appears to be required in human pancreatic β-cells by cAMP measurements. PDE1 inhibition potentiated glucose-stimulated to the greatest extent in both INS-1 cells and human islets. PDE1 inhibition potentiated CREB phosphorylation to the greatest extent and was also capable of mitigating lipotoxicity in INS-1 cells. Collectivity, this work highlights the role of cAMP compartmentalized signaling in pancreatic β-cells, and this has drastic effects on pancreatic beta-cell function and survival.
- Doctor of Philosophy
- Medicinal Chemistry and Molecular Pharmacology
- West Lafayette