Quantification of Insulin Vesicle Dynamics, Fusion Events and Calcium Activity in Intact Mouse Islets

Abstract

Proper regulation of glucose homeostasis requires the controlled secretion of insulin and glucagon by β- and α-cells, respectively, in the pancreatic islets. The secretion profiles of insulin and glucagon track in opposition to each other with respect to glucose stimulation. These two hormones work as counterregulatory partners to maintain blood glucose homeostasis and the misregulation of both is implicated in the onset of diabetes. Therefore, it is crucial to understand the mechanistic pathways that drive the regulation of both insulin and glucagon release in pancreatic islets. For this, we adopt a multi-pronged approach to investigate the dynamics and fusion events of insulin vesicles, and intracellular Ca2+ activity in β- and α-cells of mouse pancreatic islets. The dynamics of insulin vesicle motion and release were monitored by 2D confocal and 3D dual-view inverted selective plane illumination microscopy (diSPIM) time-lapse imaging on c-peptide-EGFP labeled MIN6 cells and intact islets of transgenic ins1 promoter driven c-peptide-superfolder GFP (c-peptide-sfGFP) expressing mice under different glucose conditions. insulin vesicle fusion events were also quantified with pH-sensitive Vamp2-pHluorin labeled vesicles in MIN6 cells using total internal reflection fluorescence (TIRF) microscopy. Quantification of α- and β-cell Ca2+ activities under glucose stimulation and various conditions were conducted in intact islets of transgenic GCG-iCre and INS-Cre-GCaMP6 mice, respectively, utilizing confocal and diSPIM imaging. in addition, we examine the effects of Complexin 2, a protein kinase A (PKA)-dependent SNARE binding protein, on the regulation of glucagon secretion, along with α-cell intracellular Ca2+ activity, in Complexin2 knockout mice islets. by stepwise delineation of the various mechanistic pathways, we aim to decipher the intricate coordination between the intrinsic, paracrine and juxtacrine mechanisms within individual islet cells for proper islet function.