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

Abstract

Aims/hypothesis The molecular basis of the exocytosis of secretory insulin-containing granules (SGs) during biphasic glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells remains unclear. Syntaxin (SYN)-1A and SYN-4 have been shown to mediate insulin exocytosis. The insulinsecretory function of SYN-3, which is particularly abundant in SGs, is unclear. Methods Mouse pancreatic islets and INS-1 cells were treated with adenovirus carrying Syn-3 (also known as Stx3) or small interfering RNA targeting Syn-3 in order to examine insulin secretion by radioimmunoassay. The localisation and distribution of insulin granules were examined by confocal and electron microscopy. Dynamic single-granule fusion events were assessed using total internal reflection fluorescence microscopy (TIRFM). Results Depletion of endogenous SYN-3 inhibited insulin release. TIRFM showed no change in the number or fusion competence of previously docked SGs but, instead, a marked reduction in the recruitment of newcomer SGs and their subsequent exocytotic fusion during biphasic GSIS. Conversely, overexpression of Syn-3 enhanced both phases of GSIS, owing to the increase in newcomer SGs and, remarkably, to increased SG-SG fusion, which was confirmed by electron microscopy. Conclusions/interpretation In insulin secretion, SYN-3 plays a role in the mediation of newcomer SG exocytosis and SG-SG fusion that contributes to biphasic GSIS.

Syntaxin-4 mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose-stimulated insulin secretion in human pancreatic beta cells

“…Islet perifusion Human islets infected with lenti-control-RFP and lenti-Syn-4-hRNA-RFP for 48 h were subjected to perifusion assays as previously reported [10]. Results are presented as insulin secretion normalised to total islet insulin content.…”

“…Pancreatic islets from Syn-1A (also known as Stx1a) knockout mice exhibited blunted firstphase secretion, which was attributed to loss of ability of previously docked insulin SGs to undergo exocytosis, without perturbation of recruitment and fusion of newcomer SGs, as shown by total internal reflection fluorescence microscopy (TIRFM) of single beta cells [9]. Endogenous Syn-3 depletion by RNA interference inhibited GSIS by impairing recruitment and fusion of newcomer SGs in first and second phases without affecting pre-docked SGs [10]. Conversely, Syn-3 overexpression enhanced biphasic GSIS by increasing exocytosis of newcomer SGs.…”

“…TIRFM illustrated that Syn-4-knockdown (KD) inhibition of first-phase GSIS was attributed to reduction in exocytosis of both pre-docked and newcomer SGs and second-phase inhibition was attributed to reduction in exocytosis of newcomer SGs. Thus, Syn-4 regulation of exocytosis of both pre-docked and newcomer SGs is reminiscent of and functionally redundant to Syn-1A [9] and Syn-3 [10], respectively.…”

4μ8C Inhibits Insulin Secretion Independent of IRE1α RNase Activity

“…At this time point, pre-formed insulin granules can fuse with the plasma membrane upon high glucose stimulation, whereas newly synthesized proinsulin requires a longer time period for secretion (Sugawara et al, 2009;Wang et al, 2011;Xie et al, 2015;Zhu et al, 2013). Our data imply that 4μ8C inhibits insulin exocytosis.…”

“…Therefore, we examined the possibility that 4μ8C blocked insulin exocytosis, the final step of insulin secretion. Insulin exocytosis occurs within 5 min of exposure to a stimulus (Sugawara et al, 2009;Xie et al, 2015;Zhu et al, 2013). We treated pancreatic β-cells with high glucose for 5, 10, 15, and 30 min and examined insulin secretion under 4μ8C (Fig.…”