Glucagon-Like Peptide 1 Regulates Sequential and Compound Exocytosis in Pancreatic Islet β-Cells

Abstract

Glucagon-like peptide 1 (GLP-1) has been postulated to potentiate insulin secretion by cAMP-mediated enhancement of mobilization and priming of secretory granules, but the precise exocytic events are unknown. We used epi-fluorescent microscopy of the fluorescent dye FM1-43, which incorporates into the plasma membrane and the exocytosing secretory granules (appearing as plasma membrane hotspots). KCl evoked exocytosis of 1.8 ؎ 0.5 hotspots/rat ␤-cell at the cell periphery, 82% of which are single transient increases of low amplitudes (151 ؎ 7%), suggesting single secretory granule exocytosis; and the remaining 18% are stepwise increases in plasma membrane hotspots with higher amplitudes (170 ؎ 9%), suggesting sequential secretory granule to secretory granule exocytic fusions. Addition of GLP-1 increased the hotspots to 6.0 ؎ 0.7/␤-cell and exhibited a larger number of stepwise (41%) than transient (10%) increases with higher amplitudes of 259 ؎ 19 and 278 ؎ 23%, respectively. More interestingly, GLP-1 also evoked a robust and sustained pattern (49%) with even higher amplitudes of 354 ؎ 18%, which are likely accelerated sequential secretory granule-secretory granule fusions. Electron microscopy studies collaborated with these imaging results, showing that GLP-1 increased the number of docked secretory granules at the plasma membrane and also increased the number of events showing direct contact of oncoming secretory granules with secretory granules undergoing exocytosis. We conclude that the potentiation of insulin secretion by GLP-1 is contributed by the mobilization of more insulin secretory granules to dock at the plasma membrane and the acceleration of sequential secretory granule-secretory granule fusions.

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.…”

“…Moreover, the intermediate organelle generated by bulk-like endocytosis is proposed to be incapable of efficiently generating fusion-competent synaptic vesicles (Hayashi et al, 2008;Heerssen et al, 2008). 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.…”

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

“…Electron microscopy Islets were fixed and sections were examined and photographed with a Hitachi H-7000 transmission electron microscopy (EM) system as described previously [8]. Please refer to the ESM Methods for further details.…”

“…The roles of SYN-2 and SYN-3 in insulin secretion are unknown. As SYN-3 appears to be the only SYN localised to SGs, we can surmise that SYN-3 possibly influences newcomer SG fusion and/or SG-SG fusion [3,8,13]. We thus proceeded to examine this hypothesis.…”

“…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].…”