Secretory Granules Of Pancreatic Beta Cells Research Articles

Identification of Human Islet Amyloid Polypeptide as a BACE2 Substrate.

Pancreatic amyloid formation by islet amyloid polypeptide (IAPP) is a hallmark pathological feature of type 2 diabetes. IAPP is stored in the secretory granules of pancreatic beta-cells and co-secreted with insulin to maintain glucose homeostasis. IAPP is innocuous under homeostatic conditions but imbalances in production or processing of IAPP may result in homodimer formation leading to the rapid production of cytotoxic oligomers and amyloid fibrils. The consequence is beta-cell dysfunction and the accumulation of proteinaceous plaques in and around pancreatic islets. Beta-site APP-cleaving enzyme 2, BACE2, is an aspartyl protease commonly associated with BACE1, a related homolog responsible for amyloid processing in the brain and strongly implicated in Alzheimer’s disease. Herein, we identify two distinct sites of the mature human IAPP sequence that are susceptible to BACE2-mediated proteolytic activity. The result of proteolysis is modulation of human IAPP fibrillation and human IAPP protein degradation. These results suggest a potential therapeutic role for BACE2 in type 2 diabetes-associated hyperamylinaemia.

The Rab-Binding Protein Noc2 Is Associated with Insulin-Containing Secretory Granules and Is Essential for Pancreatic β-Cell Exocytosis

The small GTPases Rab3 and Rab27 are associated with secretory granules of pancreatic beta-cells and regulate insulin exocytosis. In this study, we investigated the role of Noc2, a potential partner of these two GTPases, in insulin secretion. In the beta-cell line INS-1E wild-type Noc2, Noc265E, and Noc258A, a mutant capable of interacting with Rab27 but not Rab3, colocalized with insulin-containing vesicles. In contrast, two mutants (Noc2138S,141S and Noc2154A,155A,156A) that bind neither Rab3 nor Rab27 did not associate with secretory granules and were uniformly distributed throughout the cell cytoplasm. Overexpression of wild-type Noc2, Noc265E, or Noc258A inhibited hormone secretion elicited by insulin secretagogues. In contrast, overexpression of the mutants not targeted to secretory granules was without effect. Silencing of the Noc2 gene by RNA interference led to a strong impairment in the capacity of INS-1E cells to respond to insulin secretagogues, indicating that appropriate levels of Noc2 are essential for pancreatic beta-cell exocytosis. The defect was already detectable in the early secretory phase (0-10 min) but was particularly evident during the sustained release phase (10-45 min). Protein-protein binding studies revealed that Noc2 is a potential partner of Munc13, a component of the machinery that controls vesicle priming and insulin exocytosis. These data suggest that Noc2 is involved in the recruitment of secretory granules at the plasma membrane possibly via the interaction with Munc13.

Requirement for N-ethylmaleimide-sensitive factor for exocytosis of insulin-containing secretory granules in pancreatic beta-cells.

N-ethylmaleimide-sensitive factor (NSF) has an important role in fusion processes within intracellular compartments and at the plasma membrane, but the exact role of this protein in the exocytotic machinery has not yet been determined. NSF was found to be present in the cytosol of rat pancreatic beta-cells and rat insulinoma INS-1 cells. Capacitance measurements revealed that exocytosis of primed granules was not affected by the presence of a monoclonal antibody against NSF, mAb 2E5, suggesting that NSF is not involved in the fusion process. The antibody markedly decreased rapid refilling of new granules from a reserve pool during a first stimulation. However, slow refilling of primed granules occurred within a 2 min period between the first and second stimulations. We conclude that NSF is required in the exocytotic process in order to obtain a complete exocytotic response. Possible mechanisms by which NSF takes part in this process in insulin-secreting rat beta-cells are discussed.

Involvement of the Rab27 binding protein Slac2c/MyRIP in insulin exocytosis.

Rab27a is a GTPase associated with insulin-containing secretory granules of pancreatic beta-cells. Selective reduction of Rab27a expression by RNA interference did not alter granule distribution and basal secretion but impaired exocytosis triggered by insulin secretagogues. Screening for potential effectors of the GTPase revealed that the Rab27a-binding protein Slac2c/MyRIP is associated with secretory granules of beta-cells. Attenuation of Slac2c/MyRIP expression by RNA interference did not modify basal secretion but severely impaired hormone release in response to secretagogues. Although beta-cells express Myosin-Va, a potential partner of Slac2c/MyRIP, no functional link between the two proteins could be demonstrated. In fact, overexpression of the Myosin-Va binding domain of Slac2c/MyRIP did not affect granule localization and hormone exocytosis. In contrast, overexpression of the actin-binding domain of Slac2c/MyRIP led to a potent inhibition of exocytosis without detectable alteration in granule distribution. This effect was prevented by point mutations that abolish actin binding. Taken together our data suggest that Rab27a and Slac2c/MyRIP are part of a complex mediating the interaction of secretory granules with cortical actin cytoskeleton and participate to the regulation of the final steps of insulin exocytosis.

Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic beta-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy.

The dynamics of exocytosis/endocytosis of insulin secretory granules in pancreatic beta-cells remains to be clarified. In the present study, we visualized and analysed the motion of insulin secretory granules in MIN6 cells using pH-sensitive green fluorescent protein (pHluorin) fused to either insulin or the vesicle membrane protein, phogrin. In order to monitor insulin exocytosis, pHluorin, which is brightly fluorescent at approximately pH 7.4, but not at approximately pH 5.0, was attached to the C-terminus of insulin. To monitor the motion of insulin secretory granules throughout exocytosis/endocytosis, pHluorin was inserted between the third and fourth amino acids after the identified signal-peptide cleavage site of rat phogrin cDNA. Using this method of cDNA construction, pHluorin was located in the vesicle lumen, which may enable discrimination of the unfused acidic secretory granules from the fused neutralized ones. In MIN6 cells expressing insulin-pHluorin, time-lapse confocal laser scanning microscopy (5 or 10 s intervals) revealed the appearance of fluorescent spots by depolarization after stimulation with 50 mM KCl and 22 mM glucose. The number of these spots in the image at the indicated times was counted and found to be consistent with the results of insulin release measured by RIA during the time course. In MIN6 cells expressing phogrin-pHluorin, data showed that fluorescent spots appeared following high KCl stimulation and remained stationary for a while, moved on the plasma membrane and then disappeared. Thus we demonstrate the visualized motion of insulin granule exocytosis/endocytosis using the pH-sensitive marker, pHluorin.

Distinct molecular mechanisms for protein sorting within immature secretory granules of pancreatic beta-cells.

In the beta-cells of pancreatic islets, insulin is stored as the predominant protein within storage granules that undergo regulated exocytosis in response to glucose. By pulse-chase analysis of radiolabeled protein condensation in beta-cells, the formation of insoluble aggregates of regulated secretory protein lags behind the conversion of proinsulin to insulin. Condensation occurs within immature granules (IGs), accounting for passive protein sorting as demonstrated by constitutive-like secretion of newly synthesized C-peptide in stoichiometric excess of insulin (Kuliawat, R., and P. Arvan. J. Cell Biol. 1992. 118:521-529). Experimental manipulation of condensation conditions in vivo reveals a direct relationship between sorting of regulated secretory protein and polymer assembly within IGs. By contrast, entry from the trans-Golgi network into IGs does not appear especially selective for regulated secretory proteins. Specifically, in normal islets, lysosomal enzyme precursors enter the stimulus-dependent secretory pathway with comparable efficiency to that of proinsulin. However, within 2 h after synthesis (the same period during which proinsulin processing occurs), newly synthesized hydrolases are fairly efficiently relocated out of the stimulus-dependent pathway. In tunicamycin-treated islets, while entry of new lysosomal enzymes into the regulated secretory pathway continues unperturbed, exit of nonglycosylated hydrolases from this pathway does not occur. Consequently, the ultimate targeting of nonglycosylated hydrolases in beta-cells is to storage granules rather than lysosomes. These results implicate a post-Golgi mechanism for the active removal of lysosomal hydrolases away from condensed granule contents during the storage process for regulated secretory proteins.

Effects of L-dopa-induced dopamine accumulation on 45Ca2+ efflux and insulin secretion in isolated rat islets.

It has previously been demonstrated in several species that the secretory granules of pancreatic beta-cells have the ability to store substantial amounts of calcium and bioactive amines, such as dopamine and serotonin. Furthermore, evidence for a similar topographical localization for amine and calcium within the periphery of the granules has been obtained. In the present study, a possible interaction between dopamine and calcium on insulin release was investigated. Isolated rat islets were loaded with 45Ca2+ in the presence of theophylline and high glucose and then perifused in a dynamic system where radioactivity and insulin were determined in the effluent. When perifused in a bicarbonate buffer with 2 mmol/l Ca2+ and supplemented with the monoamine oxidase inhibitor pargyline, L-3,4-dihydroxyphenylalanine (L-dopa)-induced dopamine accumulation in the islets brought about a slight and transient increase in 45Ca2+ efflux. This increase was more pronounced and sustained in a Ca2+-deficient buffer or in a Ca2+-deficient buffer supplemented with ethyleneglycolbis(aminoethylether)tetraacetic acid (EGTA). Insulin release was transiently stimulated by islet dopamine accumulation in the Ca2+-deprived media, but not in a medium with 2 mmol/l Ca2+. Glucose-induced insulin release in 2 mmol/l Ca2+ was potentiated by acute dopamine accumulation. The combined effect of glucose stimulation and islet accumulation of dopamine induced a transient insulin release in the Ca2+-deprived media with and without EGTA. This release of insulin was accompanied by an increased 45Ca2+ efflux which was most pronounced in the presence of EGTA. Stimulation with glucose alone, i.e. without addition of L-dopa tended to decrease insulin release and 45Ca2+ efflux in a Ca2+-deficient medium. No effects of L-dopa or L-dopa + glucose were encountered in a Ca2+-deficient buffer when the monoamine oxidase inhibitor pargyline was replaced by the dopa-decarboxylase inhibitor benserazide. The results are interpreted as being a consequence of a complex interaction between the accumulated dopamine and a pool of Ca2+ mainly confined to the secretory granules. This interaction could be followed by a transient increase in cytosolic Ca2+ and a subsequent efflux of Ca2+ out of the cell, eventually accompanied by insulin release. Increasing the cytosolic Ca2+ by acute dopamine accumulation makes the cell more sensitive to a concomitant stimulation with glucose, and the release of insulin is triggered. A long-term dopamine accumulation. On the other hand, might diminish the granular Ca2+ pool to such a level where insulin release is inhibited after stimulation with certain secretagogues.