Regulation of docking and priming in pancreatic α- and β-cells

Abstract: The secretion of islet hormones from endocrine cells of the pancreas plays vital roles in maintaining glucose homeostasis. Dysfunction of these cells leads to diabetes, a devastating metabolic disorder affecting millions worldwide, but underlying mechanisms remain poorly understood. In hyperglycemic conditions, β-cells secrete insulin, whereas α-cells secrete an increased amount of glucagon in hypoglycemic conditions. Both insulin and glucagon are stored in secretory granules preceding their release by regulated exocytosis. This process involves several steps, including tethering, docking, priming, and finally, a fusion of the granules with the plasma membrane. Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins and phosphoinositides (PIs) drive pancreatic hormone exocytosis and secretion, which follows a biphasic time course. Biphasic secretion is thought to reflect the vastly different release probabilities of individual granules, but direct evidence for this is still lacking.  Therefore, this thesis investigates exocytosis in the two main pancreatic cell types with a particular focus on preceding steps docking and priming, to identify rate-limiting steps in health and type-2 diabetes (T2D). Our data indicated that granule docking is critical for sustained secretion in α- and β-cells. Glucagon granule exocytosis had a U-shaped sensitivity to glucose in both healthy and T2D α-cells. However, T2D α-cells exhibited a marginal decrease in exocytosis, as well as docking, and they were markedly insensitive to somatostatin and insulin. T2D β-cells reduced exocytosis dramatically, and docking was compromised and no longer responsive to glucose, which correlated with reduced insulin secretion and elevated donor HbA1c. These results were further strengthened by the finding that expression of a group of genes that are involved explicitly in granule docking was reduced (by RNAseq of islets from over 200 human donors), and overexpression of the corresponding proteins increased granule docking in human β-cells.We further aimed to study the basis for the recruitment of these proteins to the docking site. Here we tested the hypothesis that highly charged lipids mainly PIs act as a hotspot to interact with SNARE proteins that initiate docking. We showed the homogenous distribution of all PIs markers in the plasma membrane, with no PIs microdomains at the exocytotic site during granule docking. However, rapid and local PI(4,5)P2 signaling at fusion sites was crucial for stabilizing fusion pore by binding to proteins related to the release site. These results suggested a role of PI(4,5)P2 in priming and fusion regulation rather than docking. Overall, this work gives new insights into the mechanisms underlying pancreatic hormone secretion in both healthy and diabetic conditions.