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Stimulus-induced S-nitrosylation of Syntaxin 4 impacts insulin granule exocytosis.

ABSTRACT: Glucose-stimulated insulin release from pancreatic islet ?-cells involves increased levels of reactive oxygen and nitrogen species. Although this is normal, under pathophysiological conditions such as chronic hyperglycemia and inflammation, insulin exocytosis fails, and yet the mechanistic reason for failure is unclear. Hypothesizing that exocytotic proteins might be targets of S-nitrosylation, with their dysfunction under conditions of nitrosative stress serving as a mechanistic basis for insulin secretory dysfunction, we identified the t-SNARE protein Syntaxin 4 as a target of modification by S-nitrosylation. The cellular content of S-nitrosylated Syntaxin 4 peaked acutely, within 5 min of glucose stimulation in both human islets and MIN6 ?-cells, corresponding to the time at which Syntaxin 4 activation was detectable. S-Nitrosylation was mapped to Syntaxin 4 residue Cys(141), located within the Hc domain predicted to increase accessibility for v-SNARE interaction. A C141S-Syntaxin 4 mutant resisted S-nitrosylation induced in vitro by the nitric oxide donor compound S-nitroso-L-glutathione, failed to exhibit glucose-induced activation and VAMP2 binding, and failed to potentiate insulin release akin to that of wild-type Syntaxin 4. Strikingly, S-nitrosylation of Syntaxin 4 could be induced by acute treatment with inflammatory cytokines (TNF?, IL-1?, and IFN?), coordinate with inappropriate Syntaxin 4 activation and insulin release in the absence of the glucose stimulus, consistent with nitrosative stress and dysfunctional exocytosis, preceding the cell dysfunction and death associated with more chronic stimulation (24 h). Taken together, these data indicate a significant role for reactive nitrogen species in the insulin exocytosis mechanism in ?-cells and expose a potential pathophysiological exploitation of this mechanism to underlie dysfunctional exocytosis.

SUBMITTER: Wiseman DA

PROVIDER: S-EPMC3091240 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Stimulus-induced S-nitrosylation of Syntaxin 4 impacts insulin granule exocytosis.

Wiseman Dean A DA Kalwat Michael A MA Thurmond Debbie C DC

The Journal of biological chemistry 20110310 18

Glucose-stimulated insulin release from pancreatic islet β-cells involves increased levels of reactive oxygen and nitrogen species. Although this is normal, under pathophysiological conditions such as chronic hyperglycemia and inflammation, insulin exocytosis fails, and yet the mechanistic reason for failure is unclear. Hypothesizing that exocytotic proteins might be targets of S-nitrosylation, with their dysfunction under conditions of nitrosative stress serving as a mechanistic basis for insul ...[more]

PMID: 21393240

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Project description:Background & aimsPancreatic acinar cells are polarized epithelial cells that store enzymes required for digestion as inactive zymogens, tightly packed at the cell apex. Stimulation of acinar cells causes the zymogen granules to fuse with the apical membrane, and the cells undergo exocytosis to release proteases into the intestinal lumen. Autophagy maintains homeostasis of pancreatic acini. Syntaxin 2 (STX2), an abundant soluble N-ethyl maleimide sensitive factor attachment protein receptor in pancreatic acini, has been reported to mediate apical exocytosis. Using human pancreatic tissues and STX2-knockout (KO) mice, we investigated the functions of STX2 in zymogen granule-mediated exocytosis and autophagy.MethodsWe obtained pancreatic tissues from 5 patients undergoing surgery for pancreatic cancer and prepared 80-μm slices; tissues were exposed to supramaximal cholecystokinin octapeptide (CCK-8) or ethanol and a low concentration of CCK-8 and analyzed by immunoblot and immunofluorescence analyses. STX2-KO mice and syntaxin 2+/+ C57BL6 mice (controls) were given intraperitoneal injections of supramaximal caerulein (a CCK-8 analogue) or fed ethanol and then given a low dose of caerulein to induce acute pancreatitis, or saline (controls); pancreata were isolated and analyzed by histology and immunohistochemistry. Acini were isolated from mice, incubated with CCK-8, and analyzed by immunofluorescence microscopy or used in immunoprecipitation experiments. Exocytosis was quantified using live-cell exocytosis and Ca2+ imaging analyses and based on formation of exocytotic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes. Dysregulations in autophagy were identified using markers, electron and immunofluorescence microscopy, and protease activation assays.ResultsHuman pancreatic tissues and dispersed pancreatic acini from control mice exposed to CCK-8 or ethanol plus CCK-8 were depleted of STX2. STX2-KO developed more severe pancreatitis after administration of supramaximal caerulein or a 6-week ethanol diet compared with control. Acini from STX2-KO mice had increased apical exocytosis after exposure to CCK-8, as well as increased basolateral exocytosis, which led to ectopic release of proteases. These increases in apical and basolateral exocytosis required increased formation of fusogenic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes, mediated by STX3 and STX4. STX2 bound ATG16L1 and prevented it from binding clathrin. Deletion of STX2 from acini increased binding of AT16L1 to clathrin, increasing formation of pre-autophagosomes and inducing autophagy. Induction of autophagy promoted the CCK-8-induced increase in autolysosome formation and the activation of trypsinogen.ConclusionsIn studies of human pancreatic tissues and pancreata from STX2-KO and control mice, we found STX2 to block STX3- and STX4-mediated fusion of zymogen granules with the plasma membrane and exocytosis and prevent binding of ATG16L1 to clathrin, which contributes to induction of autophagy. Exposure of pancreatic tissues to CCK-8 or ethanol depletes acinar cells of STX2, increasing basolateral exocytosis and promoting autophagy induction, leading to activation of trypsinogen.

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Stimulus-induced S-nitrosylation of Syntaxin 4 impacts insulin granule exocytosis.

Publications

Stimulus-induced S-nitrosylation of Syntaxin 4 impacts insulin granule exocytosis.

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