Munc18c depletion selectively impairs the sustained phase of insulin release.
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 mechanism(s) using the islet beta-cell as a model system.


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 beta-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 approximately 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-Syntaxin 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.
Oh
Eunjin
E
Department of Biochemistry and Molecular Biology, Center for Diabetes Research, Indiana University School of Medicine, Indianapolis, Indiana, USA.
Thurmond
Debbie C
DC
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics / Q000503:physiopathology*; D005089:Exocytosis; D005260:Female; D020868:Gene Silencing; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000378:metabolism; D007515:Islets of Langerhans / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D051938:Munc18 Proteins / Q000172:deficiency / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection; D050988:Vesicle-Associated Membrane Protein 2 / Q000502:physiology*
D017931:DNA Primers; D007328:Insulin; D051938:Munc18 Proteins; D012333:RNA, Messenger; D050988:Vesicle-Associated Membrane Protein 2