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

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• 作者：Li Xie ; Dan Zhu ; Subhankar Dolai ; Tao Liang ; Tairan Qin ; Youhou Kang…
• 关键词：Exocytosis ; Human islets ; Newcomer insulin granules ; Syntaxin ; 4
• 刊名：Diabetologia
• 出版年：2015
• 出版时间：June 2015
• 年：2015
• 卷：58
• 期：6
• 页码：1250-1259
• 全文大小：1,670 KB
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• 作者单位：Li Xie (1)
  Dan Zhu (1)
  Subhankar Dolai (1)
  Tao Liang (1)
  Tairan Qin (1)
  Youhou Kang (1)
  Huanli Xie (1)
  Ya-Chi Huang (1)
  Herbert Y. Gaisano (1)
  
  1. Department of Medicine, Faculty of Medicine, University of Toronto, Medical Sciences Building, 1 King’s College Circle, Toronto, ON, Canada, M5S 1A8
  
• 刊物类别：Medicine
• 刊物主题：Medicine & Public Health
  Internal Medicine
  Metabolic Diseases
  Human Physiology
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-0428

文摘

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.