title: Syntaxin-4 mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose-stimulated insulin secretion in human pancreatic beta cells.
abstract: OBJECTIVE
  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
  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.
authors.affiliation: Department of Medicine, Faculty of Medicine, University of Toronto, Medical Sciences Building, 1 King's College Circle, Toronto, ON, Canada, M5S 1A8.
authors.forename: Li
authors.initials: L
authors.lastname: Xie
authors.forename: Dan
authors.initials: D
authors.lastname: Zhu
authors.forename: Subhankar
authors.initials: S
authors.lastname: Dolai
authors.forename: Tao
authors.initials: T
authors.lastname: Liang
authors.forename: Tairan
authors.initials: T
authors.lastname: Qin
authors.forename: Youhou
authors.initials: Y
authors.lastname: Kang
authors.forename: Huanli
authors.initials: H
authors.lastname: Xie
authors.forename: Ya-Chi
authors.initials: YC
authors.lastname: Huang
authors.forename: Herbert Y
authors.initials: HY
authors.lastname: Gaisano
mesh_terms: D000818:Animals; D061265:Biphasic Insulins / Q000097:blood*; D002478:Cells, Cultured; D005089:Exocytosis; D055785:Gene Knockdown Techniques; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000166:cytology*; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D051938:Munc18 Proteins / Q000378:metabolism; D018408:Patch-Clamp Techniques; D050765:Qa-SNARE Proteins / Q000378:metabolism*; D050683:R-SNARE Proteins / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D059010:Single-Cell Analysis; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D061265:Biphasic Insulins; D005947:Glucose; D007328:Insulin; D008164:Luminescent Proteins; D051938:Munc18 Proteins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D034741:RNA, Small Interfering; D050988:Vesicle-Associated Membrane Protein 2; D000097573:Red Fluorescent Protein; C494807:VAMP2 protein, human; C112938:VAMP8 protein, human; C587292:syntaxin 4, human