Syntaxin-3 regulates recruitment and exocytosis of newcomer insulin granules and granule-granule fusion during biphasic glucose-stimulated insulin secretion in pancreatic β-cells
D. Zhu1, E. Koo1, E. Kwan1, Y. Kang1, S. Park1,2, H. Xie1, S. Sugita1,2 & H. Gaisano1
Introduction: The molecular basis of exocytosis of secretory insulin-containing granules (SGs) during biphasic glucose-stimulated insulin secretion (GSIS) of pancreatic β-cells remains unclear. Syntaxins (Syns) Syn-1A and Syn-4 are t-SNARE proteins situated on the plasma membrane that have been well studied to mediate insulin exocytosis. However, Syn-3, peculiarly more abundant in insulin SGs, has unclear function in GSIS.
Methods/Designs: Confocal microscopy confirmed the localization of Syn-3 to insulin SGs in insulin secretory cells. Syn-3 function was assessed by loss of endogenous function by siRNA downregulation of Syn-3 expression, and gain of function by Syn-3 overexpression in INS-1 cell line and rat islet β-cells. Readouts included electron microscopy (EM), insulin secretion by RIA, and visualizing single insulin SG behavior by total reflection fluorescence microscopy (TIRFM).
Results: Depletion of endogenous Syn-3 in INS-1 cells inhibited GSIS by 42%. Time-lapse TIRFM showed no change in the number and fusion competence of docked SGs, and instead, a marked reduction in the recruitment of newcomer SGs to plasma membrane and subsequent slowing of their exocytotic fusion kinetics per se in both fist and second phases encompassing biphasic GSIS. Conversely, overexpression of Syn-3 into mouse islets and INS-1 cells caused enhancement of first and second phases of GLP-1-potentiated GSIS by 2-fold, accounted for by an increase in newcomer SGs, and remarkably, also by increased SG-SG/compound fusion events, the latter confirmed by EM.
Conclusions: Syn-3 in insulin SGs functions to mediate SG-SG fusion and mobilization of newcomer SGs to the plasma membrane, contributing to both first and second phases of GSIS in pancreatic β-cells.
A) Reduction of endogenous Syntaxin-3 levels impairs glucose-stimulated insulin secretion (GSIS). B) Reduction of endogenous Syntaxin 3 levels inhibits recruitment and exocytosis of newcomer insulin granules but with no effect on docked granules. C) Overexpression of Syntaxin 3 enhances GSIS. D) Enhancement of biphasic GSIS by Syntaxin 3 overexpression is due to recruitment and fusion of newcomer insulin granules with plasma membrane.
Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.
Funding: This work was supported, however funding details are unavailable.