Dynamin Is Functionally Coupled to Insulin Granule Exocytosis

The insulin granule integral membrane protein marker phogrin-green fluorescent protein was co-localized with insulin in Min6B1 beta-cell secretory granules but did not undergo plasma membrane translocation following glucose stimulation. Surprisingly, although expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis, it had no effect on phogringreen fluorescent protein localization in the basal or secretagogue-stimulated state. By contrast, co-expression of Dyn/K44A with human growth hormone as an insulin secretory marker resulted in a marked inhibition of human growth hormone release by glucose, KCl, and a combination of multiple secretagogues. Moreover, serial pulse depolarization stimulated an increase in cell surface capacitance that was also blocked in cells expressing Dyn/K44A. Similarly, small interference RNA-mediated knockdown of dynamin resulted in marked inhibition of glucose-stimulated insulin secretion. Together, these data suggest the presence of a selective kiss and run mechanism of insulin release. Moreover, these data indicate a coupling between endocytosis and exocytosis in the regulation of beta-cell insulin secretion

The SNARE Protein, Syntaxin1a, Plays an Essential Role in Biphasic Exocytosis of the Incretin Hormone, Glucagon-Like Peptide-1

Exocytosis of the hormone, glucagon-like peptide-1 (GLP-1), by the intestinal L-cell is essential for the incretin effect after nutrient ingestion, and is critical for the actions of dipeptidylpeptidase IV inhibitors that enhance GLP-1 levels in patients with type 2 diabetes. 2-Photon microscopy revealed that exocytosis of GLP-1 is biphasic, with a 1(st) peak at 1-6min and a 2(nd) peak at 7-12min after stimulation with forskolin. Approximately 75% of the exocytotic events were represented by compound granule fusion, and the remainder were accounted for by full fusion of single granules, under basal and stimulated conditions. The core SNARE protein, syntaxin-1a (syn1a), was expressed by murine ileal L-cells. At the single L-cell level, 1(st) phase forskolin-induced exocytosis was reduced to basal (p<0.05) and 2(nd) phase exocytosis was abolished (p<0.05) by syn1a knockout. L-cells from intestinal-epithelial syn1a-deficient mice demonstrated a 63% reduction in forskolin-induced GLP-1 release in vitro (p<0.001), and a 23% reduction in oral glucose-stimulated GLP-1 secretion (p<0.05) in association with impairments in glucose-stimulated insulin release (by 60%, p<0.01) and glucose tolerance (by 20%, p<0.01). Our findings therefore identify an exquisite mechanism of metered secretory output that precisely regulates release of the incretin hormone, GLP-1 and, hence, insulin secretion following a meal

Islet autoimmunity in human type 1 diabetes: initiation and progression from the perspective of the beta cell

Type 1 diabetes results from the poorly understood process of islet autoimmunity, which ultimately leads to the loss of functional pancreatic beta cells. Mounting evidence supports the notion that the activation and evolution of islet autoimmunity in genetically susceptible people is contingent upon early life exposures affecting the islets, especially beta cells. Here, we review some of the recent advances and studies that highlight the roles of these changes as well as antigen presentation and stress response pathways in beta cells in the onset and propagation of the autoimmune process in type 1 diabetes. Future progress in this area holds promise for advancing islet- and beta cell-directed therapies that could be implemented in the early stages of the disease and could be combined with immunotherapies

Spatial and temporal coordination of insulin granule exocytosis in intact human pancreatic islets

Aims/hypothesis: Insulin secretion is widely studied because it plays a central role in glucose homeostasis and diabetes. Processes from insulin granule fusion in beta cells to in vivo insulin secretion have been elucidated, but data at the cellular level do not fully account for several aspects of the macroscopic secretory pattern. Here we investigated how individual secretory events are coordinated spatially and temporally within intact human islets. Methods: We used the fluorescent probe neuropeptide Y (NPY)–pHluorin to visualise insulin granule secretion in isolated intact human islets. Results: We found that individual beta cells respond to increases in glucose concentration by releasing insulin granules in very discrete bursts with periods consistent with in vivo pulsatile insulin secretion. In successive secretory bursts during prolonged exposure to high glucose levels, secretory events progressively localised to preferential release sites, coinciding with the transition to second phase insulin secretion. Granule secretion was very synchronised in neighbouring beta cells, forming discrete regional clusters of activity. Conclusions/interpretation: These results reveal how individual secretory events are coordinated to produce pulsatile insulin secretion from human islets. © 2015, Springer-Verlag Berlin Heidelberg.