Munc18c depletion selectively impairs the sustained phase of insulin release

Eunjin Oh, Debbie C. Thurmond

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

OBJECTIVE-The Sec1/Munc18 protein Munc18c has been implicated in Syntaxin 4-mediated exocytosis events, although its purpose in exocytosis has remained elusive. Given that Syntaxin 4 functions in the second phase of glucose-stimulated insulin secretion (GSIS), we hypothesized that Munc18c would also be required and sought insight into the possible mecha- nism(s) using the islet β-cell as a model system. RESEARCH DESIGN AND METHODS-Perifusion analyses of isolated Munc18c- (-/+) or Munc18c-depleted (RNAi) mouse islets were used to assess biphasic secretion. Protein interaction studies used subcellular fractions and detergent lysates prepared from MIN6 β-cells to determine the mechanistic role of Munc18c in Syntaxin 4 activation and docking/ fusion of vesicle-associated membrane protein (VAMP)2- containing insulin granules. Electron microscopy was used to gauge changes in granule localization. RESULTS-Munc18c (-/+) islets secreted ∼60% less insulin selectively during second-phase GSIS; RNAi-mediated Munc18c depletion functionally recapitulated this in wild-type and Munc18c (- /+) islets in a gene dosage-dependent manner. Munc18c depletion ablated the glucose-stimulated VAMP2-Syn- taxin 4 association as well as Syntaxin 4 activation, correlating with the deficit in insulin release. Remarkably, Munc18c depletion resulted in aberrant granule localization to the plasma membrane in response to glucose stimulation, consistent with its selective effect on the second phase of secretion. CONCLUSIONS-Collectively, these studies demonstrate an essential positive role for Munc18c in second-phase GSIS and suggest novel roles for Munc18c in granule localization to the plasma membrane as well as in triggering Syntaxin 4 accessibility to VAMP2 at a step preceding vesicle docking/fusion. Diabetes 58:1165-1174, 2009

Original languageEnglish
Pages (from-to)1165-1174
Number of pages10
JournalDiabetes
Volume58
Issue number5
DOIs
StatePublished - May 2009

Fingerprint

Qa-SNARE Proteins
Vesicle-Associated Membrane Protein 2
Insulin
Glucose
Exocytosis
RNA Interference
Munc18 Proteins
Cell Membrane
Gene Dosage
Subcellular Fractions
Islets of Langerhans
Detergents
Electron Microscopy
Research Design

ASJC Scopus subject areas

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism

Cite this

Munc18c depletion selectively impairs the sustained phase of insulin release. / Oh, Eunjin; Thurmond, Debbie C.

In: Diabetes, Vol. 58, No. 5, 05.2009, p. 1165-1174.

Research output: Contribution to journalArticle

Oh, Eunjin ; Thurmond, Debbie C. / Munc18c depletion selectively impairs the sustained phase of insulin release. In: Diabetes. 2009 ; Vol. 58, No. 5. pp. 1165-1174.
@article{683f1328837f4e85b7f9596917d66c8b,
title = "Munc18c depletion selectively impairs the sustained phase of insulin release",
abstract = "OBJECTIVE-The Sec1/Munc18 protein Munc18c has been implicated in Syntaxin 4-mediated exocytosis events, although its purpose in exocytosis has remained elusive. Given that Syntaxin 4 functions in the second phase of glucose-stimulated insulin secretion (GSIS), we hypothesized that Munc18c would also be required and sought insight into the possible mecha- nism(s) using the islet β-cell as a model system. RESEARCH DESIGN AND METHODS-Perifusion analyses of isolated Munc18c- (-/+) or Munc18c-depleted (RNAi) mouse islets were used to assess biphasic secretion. Protein interaction studies used subcellular fractions and detergent lysates prepared from MIN6 β-cells to determine the mechanistic role of Munc18c in Syntaxin 4 activation and docking/ fusion of vesicle-associated membrane protein (VAMP)2- containing insulin granules. Electron microscopy was used to gauge changes in granule localization. RESULTS-Munc18c (-/+) islets secreted ∼60{\%} less insulin selectively during second-phase GSIS; RNAi-mediated Munc18c depletion functionally recapitulated this in wild-type and Munc18c (- /+) islets in a gene dosage-dependent manner. Munc18c depletion ablated the glucose-stimulated VAMP2-Syn- taxin 4 association as well as Syntaxin 4 activation, correlating with the deficit in insulin release. Remarkably, Munc18c depletion resulted in aberrant granule localization to the plasma membrane in response to glucose stimulation, consistent with its selective effect on the second phase of secretion. CONCLUSIONS-Collectively, these studies demonstrate an essential positive role for Munc18c in second-phase GSIS and suggest novel roles for Munc18c in granule localization to the plasma membrane as well as in triggering Syntaxin 4 accessibility to VAMP2 at a step preceding vesicle docking/fusion. Diabetes 58:1165-1174, 2009",
author = "Eunjin Oh and Thurmond, {Debbie C.}",
year = "2009",
month = "5",
doi = "10.2337/db08-1059",
language = "English",
volume = "58",
pages = "1165--1174",
journal = "Diabetes",
issn = "0012-1797",
publisher = "American Diabetes Association Inc.",
number = "5",

}

TY - JOUR

T1 - Munc18c depletion selectively impairs the sustained phase of insulin release

AU - Oh, Eunjin

AU - Thurmond, Debbie C.

PY - 2009/5

Y1 - 2009/5

N2 - OBJECTIVE-The Sec1/Munc18 protein Munc18c has been implicated in Syntaxin 4-mediated exocytosis events, although its purpose in exocytosis has remained elusive. Given that Syntaxin 4 functions in the second phase of glucose-stimulated insulin secretion (GSIS), we hypothesized that Munc18c would also be required and sought insight into the possible mecha- nism(s) using the islet β-cell as a model system. RESEARCH DESIGN AND METHODS-Perifusion analyses of isolated Munc18c- (-/+) or Munc18c-depleted (RNAi) mouse islets were used to assess biphasic secretion. Protein interaction studies used subcellular fractions and detergent lysates prepared from MIN6 β-cells to determine the mechanistic role of Munc18c in Syntaxin 4 activation and docking/ fusion of vesicle-associated membrane protein (VAMP)2- containing insulin granules. Electron microscopy was used to gauge changes in granule localization. RESULTS-Munc18c (-/+) islets secreted ∼60% less insulin selectively during second-phase GSIS; RNAi-mediated Munc18c depletion functionally recapitulated this in wild-type and Munc18c (- /+) islets in a gene dosage-dependent manner. Munc18c depletion ablated the glucose-stimulated VAMP2-Syn- taxin 4 association as well as Syntaxin 4 activation, correlating with the deficit in insulin release. Remarkably, Munc18c depletion resulted in aberrant granule localization to the plasma membrane in response to glucose stimulation, consistent with its selective effect on the second phase of secretion. CONCLUSIONS-Collectively, these studies demonstrate an essential positive role for Munc18c in second-phase GSIS and suggest novel roles for Munc18c in granule localization to the plasma membrane as well as in triggering Syntaxin 4 accessibility to VAMP2 at a step preceding vesicle docking/fusion. Diabetes 58:1165-1174, 2009

AB - OBJECTIVE-The Sec1/Munc18 protein Munc18c has been implicated in Syntaxin 4-mediated exocytosis events, although its purpose in exocytosis has remained elusive. Given that Syntaxin 4 functions in the second phase of glucose-stimulated insulin secretion (GSIS), we hypothesized that Munc18c would also be required and sought insight into the possible mecha- nism(s) using the islet β-cell as a model system. RESEARCH DESIGN AND METHODS-Perifusion analyses of isolated Munc18c- (-/+) or Munc18c-depleted (RNAi) mouse islets were used to assess biphasic secretion. Protein interaction studies used subcellular fractions and detergent lysates prepared from MIN6 β-cells to determine the mechanistic role of Munc18c in Syntaxin 4 activation and docking/ fusion of vesicle-associated membrane protein (VAMP)2- containing insulin granules. Electron microscopy was used to gauge changes in granule localization. RESULTS-Munc18c (-/+) islets secreted ∼60% less insulin selectively during second-phase GSIS; RNAi-mediated Munc18c depletion functionally recapitulated this in wild-type and Munc18c (- /+) islets in a gene dosage-dependent manner. Munc18c depletion ablated the glucose-stimulated VAMP2-Syn- taxin 4 association as well as Syntaxin 4 activation, correlating with the deficit in insulin release. Remarkably, Munc18c depletion resulted in aberrant granule localization to the plasma membrane in response to glucose stimulation, consistent with its selective effect on the second phase of secretion. CONCLUSIONS-Collectively, these studies demonstrate an essential positive role for Munc18c in second-phase GSIS and suggest novel roles for Munc18c in granule localization to the plasma membrane as well as in triggering Syntaxin 4 accessibility to VAMP2 at a step preceding vesicle docking/fusion. Diabetes 58:1165-1174, 2009

UR - http://www.scopus.com/inward/record.url?scp=65549137424&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=65549137424&partnerID=8YFLogxK

U2 - 10.2337/db08-1059

DO - 10.2337/db08-1059

M3 - Article

VL - 58

SP - 1165

EP - 1174

JO - Diabetes

JF - Diabetes

SN - 0012-1797

IS - 5

ER -