Multivesicular exocytosis in rat pancreatic beta cells

Abstract

Aims/hypothesisTo establish the occurrence, modulation and functional significance of compound exocytosis in insulin-secreting beta cells.MethodsExocytosis was monitored in rat beta cells by electrophysiological, biochemical and optical methods. The functional assays were complemented by three-dimensional reconstruction of confocal imaging, transmission and block face scanning electron microscopy to obtain ultrastructural evidence of compound exocytosis.ResultsCompound exocytosis contributed marginally (<5% of events) to exocytosis elicited by glucose/membrane depolarisation alone. However, in beta cells stimulated by a combination of glucose and the muscarinic agonist carbachol, 15–20% of the release events were due to multivesicular exocytosis, but the frequency of exocytosis was not affected. The optical measurements suggest that carbachol should stimulate insulin secretion by ∼40%, similar to the observed enhancement of glucose-induced insulin secretion. The effects of carbachol were mimicked by elevating [Ca2+]i from 0.2 to 2 μmol/l Ca2+. Two-photon sulforhodamine imaging revealed exocytotic events about fivefold larger than single vesicles and that these structures, once formed, could persist for tens of seconds. Cells exposed to carbachol for 30 s contained long (1–2 μm) serpentine-like membrane structures adjacent to the plasma membrane. Three-dimensional electron microscopy confirmed the existence of fused multigranular aggregates within the beta cell, the frequency of which increased about fourfold in response to stimulation with carbachol.Conclusions/interpretationAlthough contributing marginally to glucose-induced insulin secretion, compound exocytosis becomes quantitatively significant under conditions associated with global elevation of cytoplasmic calcium. These findings suggest that compound exocytosis is a major contributor to the augmentation of glucose-induced insulin secretion by muscarinic receptor activation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-011-2400-5) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Bulk-like endocytosis plays an important role in the recycling of insulin granules in pancreatic beta cells

“…More frequently, fused chain of insulin granules were observed in the primary beta cells ( Fig. 2A, arrow), similar to previous reports (Kwan and Gaisano, 2005;Hoppa et al, 2012). They collapse into the plasma membrane and release insulin in a multiple-granular manner ( Fig.…”

“…However, compound exocytosis of prefused insulin granules was reported in rat pancreatic beta cells in 2005 (Kwan and Gaisano, 2005). Recently, compound exocytosis has been further demonstrated to play a important role under conditions associated with the global elevation of cytosolic Ca 2+ (Hoppa et al, 2012), which is the condition that augments the bulk-like endocytosis found in the present study. Our electron microscope data also show examples of compound fusion of several granules to the plasma membrane (Fig.…”

Syntaxin-3 regulates newcomer insulin granule exocytosis and compound fusion in pancreatic beta cells

“…3). Sequential SG-SG fusion and compound exocytosis have been studied in recent years, in various cell types, with multi-photon imaging [30,31] and other imaging modalities [9]. Realising that static EM images could not distinguish these exocytotic events, we employed TIRFM to rigorously and quantitatively assess the spatio-temporal dynamics of these two types of SG-SG fusion: sequential SG-SG fusion and true compound fusion.…”

“…Third, fluorescence intensity between inner and outer red circles (corresponding to grey lines in the bottom graphs) depicting dissipation of SG cargo release is also much larger in the massive SG fusion than that in single SG fusion. We thus considered that these massive fusion events represent genuine compound fusion of several SGs that were already prefused before a lead SG underwent exocytotic fusion with the PM [8,9,28]. Two additional examples of compound exocytosis (left We then examined whether loss of function (siRNA knockdown) or gain of function (genetic overexpression) of SYN-3 could influence these SG-SG fusion events in INS-1 cells.…”

“…It is suggested that, in addition to docked SGs that are purported to form the readily releasable pool (RRP) that accounts for firstphase insulin release, large numbers of SGs, designated 'newcomer SGs', are mobilised from the cell interior to contribute not only to second-phase but also to first-phase release [5][6][7]. Insulin exocytosis also involves compound exocytosis of SGs when potentiated by cAMP-acting glucagon-like peptide 1 (GLP-1) [8] or Ca 2+ -acting carbachol [9]. Of the four syntaxins mediating exocytosis, SYN-1, SYN-2 and SYN-4 are present and localised predominantly to the beta cell PM, whereas SYN-3 is noted to be more abundant in SGs [10].…”