Aims/hypothesisWe set out to test the hypothesis that insulin secretion from beta cells is targeted towards the vasculature.MethodsThe spatial location of granule fusion was identified by live-cell two-photon imaging of mouse pancreatic beta cells within intact islets, using sulforhodamine B labelling. Three-dimensional (3D) immunofluorescence of pancreatic slices was used to identify the location of proteins associated with neuronal synapses.ResultsWe demonstrated an asymmetric, non-random, distribution of sites of insulin granule fusion in response to glucose and focal targeting of insulin granule secretion to the beta cell membrane facing the vasculature. 3D immunofluorescence of islets showed that structural proteins, such as liprin, piccolo and Rab2-interacting molecule, normally associated with neuronal presynaptic targeting, were present in beta cells and enriched at the vascular face. In contrast, we found that syntaxin 1A and synaptosomal-associated protein 25 kDa (SNAP25) were relatively evenly distributed across the beta cells.Conclusions/interpretationOur results show that beta cells in situ, within intact islets, are polarised and target insulin secretion. This evidence for an ‘endocrine synapse’ has wide implications for our understanding of stimulus–secretion coupling in healthy islets and in disease.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-014-3252-6) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Aims/hypothesisIn dispersed single beta cells the response of each cell to glucose is heterogeneous. In contrast, within an islet, cell-to-cell communication leads to glucose inducing a more homogeneous response. For example, increases in NAD(P)H and calcium are relatively uniform across the cells of the islet. These data suggest that secretion of insulin from single beta cells within an islet should also be relatively homogeneous. The aim of this study was to test this hypothesis by determining the glucose dependence of single-cell insulin responses within an islet.MethodsTwo-photon microscopy was used to detect the glucose-induced fusion of single insulin granules within beta cells in intact mouse islets.ResultsFirst, we validated our assay and showed that the measures of insulin secretion from whole islets could be explained by the time course and numbers of granule fusion events observed. Subsequent analysis of the patterns of granule fusion showed that cell recruitment is a significant factor, accounting for a fourfold increase from 3 to 20 mmol/l glucose. However, the major factor is the regulation of the numbers of granule fusion events within each cell, which increase ninefold over the range of 3 to 20 mmol/l glucose. Further analysis showed that two types of granule fusion event occur: ‘full fusion’ and ‘kiss and run’. We show that the relative frequency of each type of fusion is independent of glucose concentration and is therefore not a factor in the control of insulin secretion.Conclusions/interpretationWithin an islet, glucose exerts its main effect through increasing the numbers of insulin granule fusion events within a cell.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-013-3019-5) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Aims/hypothesisWe used the db/db mouse to determine the nature of the secretory defect in intact islets.MethodsGlucose tolerance was compared in db/db and wild-type (WT) mice. Isolated islets were used: to measure insulin secretion and calcium in a two-photon assay of single-insulin-granule fusion; and for immunofluorescence of soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAREs).ResultsThe 13–18-week-old db/db mice showed a diabetic phenotype. Isolated db/db islets showed a 77% reduction in insulin secretion induced by 15 mmol/l glucose and reductions in the amplitude and rise-time of the calcium response to glucose. Ionomycin-induced insulin secretion in WT but not db/db islets. Immunofluorescence showed an increase in the levels of the SNAREs synaptosomal-associated protein 25 (SNAP25) and vesicle-associated membrane protein 2 (VAMP2) in db/db islets, but reduced syntaxin-1A. Therefore, db/db islets have both a compromised calcium response to glucose and a compromised secretory response to calcium. Two-photon microscopy of isolated islets determined the number and distribution of insulin granule exocytic events. Compared with WT, db/db islets showed far fewer exocytic events (an 83% decline at 15 mmol/l glucose). This decline was due to a 73% loss of responding cells and, in the remaining responsive cells, a 50% loss of exocytic responses per cell. An assay measuring granule re-acidification showed evidence for more recaptured granules in db/db islets compared with WT.Conclusions/interpretationWe showed that db/db islets had a reduced calcium response to glucose and a reduction in syntaxin-1A. Within the db/db islets, changes were manifest as both a reduction in responding cells and a reduction in fusing insulin granules per cell.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-014-3226-8) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

dependence of exocytosis, (4) significant reduction in exocytosis in the prescence of cytosolic EGTA, (5) spatial exclusion of secretory granules from the cell membrane by the endoplasmic reticulum, and (6) inability of local Ca 2+ responses to trigger exocytosis. These results strongly indicate that the control of exocytosis, triggered by Ca 2+ release from stores, is through the regulation of cytosolic [Ca 2+ ] within a microdomain.

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