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

• Published in: Diabetologia Vol. 58; no. 6; pp. 1250 - 1259
• Main Authors: Xie, Li, Zhu, Dan, Dolai, Subhankar, Liang, Tao, Qin, Tairan, Kang, Youhou, Xie, Huanli, Huang, Ya-Chi, Gaisano, Herbert Y.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-06-2015; Springer Nature B.V
• Subjects: Animals; Biphasic Insulins - blood; Cells, Cultured; Exocytosis; Gene Knockdown Techniques; Glucagon; Glucose; Glucose - metabolism; Human Physiology; Humans; Hypotheses; Insulin - metabolism; Insulin resistance; Insulin Secretion; Insulin-Secreting Cells - cytology; Internal Medicine; Luminescent Proteins - metabolism; Medicine; Medicine & Public Health; Metabolic Diseases; Mice; Mice, Inbred C57BL; Microscopy; Microscopy, Confocal; Munc18 Proteins - metabolism; Patch-Clamp Techniques; Peptides; Proteins; Qa-SNARE Proteins - metabolism; R-SNARE Proteins - metabolism; Recruitment; Red Fluorescent Protein; RNA, Small Interfering - metabolism; Single-Cell Analysis; Vesicle-Associated Membrane Protein 2 - metabolism; Newcomer insulin granules; Syntaxin-4; Human islets; Exocytosis
• ISSN: 0012-186X; 1432-0428
• DOI: 10.1007/s00125-015-3545-4

Aims/hypothesis
Of the four exocytotic syntaxins (Syns), much is now known about the role of Syn-1A (pre-docked secretory granules [SGs]) and Syn-3 (newcomer SGs) in insulin exocytosis. Some work was reported on Syn-4’s role in biphasic glucose-stimulated insulin secretion (GSIS), but its precise role in insulin SG exocytosis remains unclear. In this paper we examine this role in human beta cells.
Methods
Endogenous function of Syn-4 in human islets was assessed by knocking down its expression with lentiviral single hairpin RNA (lenti-shRNA)–RFP. Biphasic GSIS was determined by islet perifusion assay. Single-cell analysis of exocytosis of red fluorescent protein (RFP)-positive beta cells (exhibiting near-total depletion of Syn-4) was by patch clamp capacitance measurements (Cm) and total internal reflection fluorescence microscopy (TIRFM), the latter to further assess single SG behaviour. Co-immunoprecipitations were conducted on INS-1 cells to assess exocytotic complexes.
Results
Syn-4 knockdown (KD) of 77% in human islets caused a concomitant reduction in cognate Munc18c expression (46%) without affecting expression of other exocytotic proteins; this resulted in reduction of GSIS in the first phase (by 42%) and the second phase (by 40%). Cm of RFP-tagged Syn-4-KD beta cells showed severe inhibition in the readily releasable pool (by 71%) and mobilisation from reserve pools (by 63%). TIRFM showed that Syn-4-KD-induced inhibition of first-phase GSIS was attributed to reduction in exocytosis of both pre-docked and newcomer SGs (which undergo minimal residence or docking time at the plasma membrane before fusion). Second-phase inhibition was attributed to reduction in newcomer SGs. Stx-4 co-immunoprecipitated Munc18c, VAMP2 and VAMP8, suggesting that these exocytotic complexes may be involved in exocytosis of pre-docked and newcomer SGs.
Conclusions/interpretation
Syn-4 is involved in distinct molecular machineries that influence exocytosis of both pre-docked and newcomer SGs in a manner functionally redundant to Syn-1A and Syn-3, respectively; this underlies Syn-4’s role in mediating portions of first-phase and second-phase GSIS.