Isolation and Proteomics of the Insulin Secretory Granule

Insulin, a vital hormone for glucose homeostasis is produced by pancreatic beta-cells and when secreted, stimulates the uptake and storage of glucose from the blood. In the pancreas, insulin is stored in vesicles termed insulin secretory granules (ISGs). In Type 2 diabetes (T2D), defects in insulin action results in peripheral insulin resistance and beta-cell compensation, ultimately leading to dysfunctional ISG production and secretion. ISGs are functionally dynamic and many proteins present either on the membrane or in the lumen of the ISG may modulate and affect different stages of ISG trafficking and secretion. Previously, studies have identified few ISG proteins and more recently, proteomics analyses of purified ISGs have uncovered potential novel ISG proteins. This review summarizes the proteins identified in the current ISG proteomes from rat insulinoma INS-1 and INS-1E cell lines. Here, we also discuss techniques of ISG isolation and purification, its challenges and potential future directions.

Differential Effects of Prostaglandin E2Production and Signaling through the Prostaglandin EP3 Receptor on Human Beta-cell Compensation

ObjectiveSignaling through Prostaglandin E3 Receptor (EP3), a G protein-coupled receptor for E series prostaglandins such as prostaglandin E2(PGE2), has been linked to the beta-cell dysfunction and loss of beta-cell mass in type 2 diabetes (T2D). In the beta-cell, EP3 is specifically coupled to the unique cAMP-inhibitory G protein, Gz. Divergent effects of EP3 agonists and antagonists or Gαzloss on beta-cell function, replication, and survival depending on whether islets are isolated from mice or humans in the lean and healthy, type 1 diabetic, or T2D state suggest a divergence in biological effects downstream of EP3/Gαzdependent on the physiological milieu in which the islets reside.MethodsWe determined the expression of a number of genes in the EP3/Gαzsignaling pathway; PGE2production pathway; and the beta-cell metabolic, proliferative, and survival responses to insulin resistance and its corresponding metabolic and inflammatory derangements in a panel of 80 islet preparations from non-diabetic human organ donors spanning a BMI range of approximately 20-45. In a subset of islet preparations, we also performed glucose-stimulated insulin secretion assays with and without the addition of an EP3 agonist, L798,106, and a glucagon-like peptide 1 receptor agonist, exendin-4, allowing us to compare the gene expression profile of each islet preparation with its (1) total islet insulin content (2), functional responses to glucose and incretin hormones, and (3) intrinsic influence of endogenous EP3 signaling in regulating these functional responses. We also transduced two independent islet preparations from three human organ donors with adenoviruses encoding human Gαzor a GFP control in order to determine the impact of Gαzhyperactivity (a mimic of the T2D state) on human islet insulin content and functional response to glucose.ResultsIn contrast to results from islets isolated from T2D mice and human organ donors, where PGE2-mediated EP3 signaling actively contributes to beta-cell dysfunction, PGE2production and EP3 expression appeared positively associated with various measurements of functional beta-cell compensation. While GαzmRNA expression was negatively associated with islet insulin content, that of each of the Gαz-sensitive adenylate cyclase (AC) isoforms were positively associated with BMI and cyclin A1 mRNA expression, suggesting increased expression of AC1, AC5, and AC6 is a compensatory mechanism to augment beta-cell mass. Human islets over-expressing Gαzvia adenoviral transduction had reduced islet insulin content and secretion of insulin in response to stimulatory glucose as a percent of content, consistent with the effects of hyperactivation of Gαzby PGE2/EP3 signaling observed in islets exposed to the T2D physiological milieu.ConclusionsOur work sheds light on critical mechanisms in the human beta-cell compensatory response, before the progression to frank T2D.