Filamentous actin regulates insulin exocytosis through direct interaction with Syntaxin 4

Glucose-induced insulin exocytosis is coupled to associations between F-actin and SNARE proteins, although the nature and function of these interactions remains unknown. Toward this end we show here that both Syntaxin 1A and Syntaxin 4 associated with F-actin in MIN6 cells and that each interaction was rapidly and transiently diminished by stimulation of cells with d-glucose. Of the two isoforms, only Syntaxin 4 was capable of interacting directly with F-actin in an in vitro sedimentation assay, conferred by the N-terminal 39-112 residues of Syntaxin 4. The 39-112 fragment was capable of selective competitive inhibitory action, disrupting endogenous F-actin-Syntaxin 4 binding in MIN6 cells. Disruption of F-actin-Syntaxin 4 binding correlated with enhanced glucose-stimulated insulin secretion, mediated by increased granule accumulation at the plasma membrane and increased Syntaxin 4 accessibility under basal conditions. However, no increase in basal level Syntaxin 4-VAMP2 association occurred with either latrunculin treatment or expression of the 39-112 fragment. Taken together, these data disclose a new underlying mechanism by which F-actin negatively regulates exocytosis via binding and blocking Syntaxin 4 accessibility, but they also reveal the existence of additional signals and/or steps required to trigger the subsequent docking and fusion steps of exocytosis.     

Transduction of MIN6 beta cells with TAT-syntaxin SNARE motif inhibits insulin exocytosis in biphasic insulin release in a distinct mechanism analyzed by evanescent wave microscopy

To investigate the in vivo interaction of syntaxin-mediated soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) assembly and insulin exocytosis in biphasic release, we examined the dynamics of insulin granule motion such as docking and fusion with the plasma membrane when the syntaxin SNARE motif (H3 domain) was transduced into living MIN6 beta cells. TAT-H3, produced by fusion of the protein transduction domain of human immunodeficiency virus-1 TAT to the syntaxin-H3 domain, was rapidly transduced into the subplasmalemmal region in living MIN6 cells. Immunoblotting analysis followed by immunoprecipitation on TAT-H3-treated MIN6 cells showed that TAT-H3 binds SNAP-25 and VAMP-2 in vivo. Transduction of MIN6 cells with TAT-H3 caused a decrease in both the first and second phase of insulin release. We therefore quantitatively analyzed approaching, docking, and fusing of green fluorescent protein-labeled single insulin granules in TAT-H3-transduced MIN6 cells by evanescent wave microscopy. Under high glucose stimulation, TAT-H3 treatment not only reduced the fusion events from previously docked granules for the first 120 s (first phase of release) but also strongly inhibited the docking and fusion from newly recruited insulin granules after this point (second phase of release). During the second phase of release we observed a marked reduction in the accumulation of newly docked insulin granules; subsequently, fusion events were significantly decreased. TAT-H3 treatment by itself, however, did not alter the number of previously docked granules without stimulation. We conclude that introduction of the H3 domain into MIN6 cells inhibits biphasic insulin release by two mechanisms. 1) In the first phase of insulin release, the H3 domain interferes with previously docked granules to be fused, and 2) in the second phase of insulin release reduced fusion events result from a marked decline of newly docked granules. Thus, syntaxin-mediated SNARE assembly modulates insulin exocytosis in biphasic insulin release in a distinct way.     

A direct interaction between Cdc42 and vesicle-associated membrane protein 2 regulates SNARE-dependent insulin exocytosis

In pancreatic beta cells, insulin granule exocytosis is regulated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein (SNAP) receptor) proteins, and this is coupled to cortical F-actin reorganization via the Rho family GTPase Cdc42 by an unknown mechanism. We investigated interactions among the target SNARE protein Syntaxin 1A and the vesicle-associated membrane SNARE protein (VAMP2) with Cdc42 and compared these structural interactions with their functional importance to glucose-stimulated insulin secretion in MIN6 beta cells. Subcellular fractionation analyses revealed a parallel redistribution of Cdc42 and VAMP2 from the granule fraction to the plasma membrane in response to glucose that temporally corresponded with the glucose-induced activation of Cdc42. Moreover, within these fractions Cdc42 and VAMP2 were found to co-immunoprecipitate under basal and glucose-stimulated conditions, suggesting that they moved as a complex. Furthermore, VAMP2 bound both GST-Cdc42-GTPgammaS and GST-Cdc42-GDP, indicating that the Cdc42-VAMP2 complex could form under both cytosolic GDP-bound Cdc42 and plasma membrane GTP-bound Cdc42 conformational conditions. In vitro binding analyses showed that VAMP2 bound directly to Cdc42 and that a heterotrimeric complex with Syntaxin 1A could also be formed. Deletion analyses of VAMP2 revealed that only the N-terminal 28 residues were required for Cdc42 binding. Expression of this 28-residue VAMP2 peptide in MIN6 beta cells resulted in the specific impairment of glucose-stimulated insulin secretion, indicating a functional importance for the Cdc42-VAMP2 interaction. Taken together, these data suggest a mechanism whereby glucose activates Cdc42 to induce the targeting of intracellular Cdc42-VAMP2-insulin granule complexes to Syntaxin 1A at the plasma membrane.     

Syntaxin 4 facilitates biphasic glucose-stimulated insulin secretion from pancreatic beta-cells

Numerous overexpression studies have recently implicated Syntaxin 4 as an effector of insulin secretion, although its requirement in insulin granule exocytosis is unknown. To address this, islets from Syntaxin 4 heterozygous (-/+) knockout mice were isolated and compared with islets from wild-type mice. Under static incubation conditions, Syntaxin 4 (-/+) islets showed a 60% reduction in glucose-stimulated insulin secretion compared with wild-type islets. Perifusion analyses revealed that Syntaxin 4 (-/+) islets secreted 50% less insulin during the first phase of glucose-stimulated insulin secretion and that this defect could be fully restored by the specific replenishment of recombinant Syntaxin 4. This essential role for Syntaxin 4 in secretion from the islet was localized to the beta-cells because small interfering RNA-mediated depletion of Syntaxin 4 in MIN6 beta-cells abolished glucose-stimulated insulin secretion. Moreover, immunofluorescent confocal microscopy revealed that Syntaxin 4 was principally localized to the beta-cells and not the alpha-cells of the mouse islet. Remarkably, islets isolated from transgenic mice that express 2.4-fold higher levels of Syntaxin 4 relative to wild-type mice secreted approximately 35% more insulin during both phases of insulin secretion, suggesting that increased Syntaxin 4 may be beneficial for enhancing biphasic insulin secretion in a regulated manner. Taken together, these data support the notion that Syntaxin 4-based SNARE complexes are essential for biphasic insulin granule fusion in pancreatic beta-cells.     

Munc18-1 regulates first-phase insulin release by promoting granule docking to multiple syntaxin isoforms

Attenuated levels of the Sec1/Munc18 (SM) protein Munc18-1 in human islet β-cells is coincident with type 2 diabetes, although how Munc18-1 facilitates insulin secretion remains enigmatic. Herein, using conventional Munc18-1(+/-) and β-cell specific Munc18-1(-/-) knock-out mice, we establish that Munc18-1 is required for the first phase of insulin secretion. Conversely, human islets expressing elevated levels of Munc18-1 elicited significant potentiation of only first-phase insulin release. Insulin secretory changes positively correlated with insulin granule number at the plasma membrane: Munc18-1-deficient cells lacked 35% of the normal component of pre-docked insulin secretory granules, whereas cells with elevated levels of Munc18-1 exhibited a ～20% increase in pre-docked granule number. Pre-docked syntaxin 1-based SNARE complexes bound by Munc18-1 were detected in β-cell lysates but, surprisingly, were reduced by elevation of Munc18-1 levels. Paradoxically, elevated Munc18-1 levels coincided with increased binding of syntaxin 4 to VAMP2 at the plasma membrane. Accordingly, syntaxin 4 was a requisite for Munc18-1 potentiation of insulin release. Munc18c, the cognate SM isoform for syntaxin 4, failed to bind SNARE complexes. Given that Munc18-1 does not pair with syntaxin 4, these data suggest a novel indirect role for Munc18-1 in facilitating syntaxin 4-mediated granule pre-docking to support first-phase insulin exocytosis.     

Elevated SNAP-25 is associated with fatty acid-induced impairment of mouse islet function

The role of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in insulin secretion following chronic exposure to non-esterified fatty acids (NEFAs) has not been extensively investigated. Here, we show that synaptosome-associated protein of 25 kDa (SNAP-25) levels were predominantly elevated in the soluble fraction of mouse islets exposed to palmitate. This coincided with an impairment of insulin secretion to glucose and non-glucose secretagogues, consistent with a defect at a distal regulatory step in exocytosis. Removal of palmitate from the media restored both SNAP-25 protein levels and insulin secretion to control levels. We conclude that increased expression of SNAP-25 is associated with NEFA-induced impairment of insulin secretion in mouse islets.     

Mastoparan stimulates GABA release from MIN6 cells: relationship between SNARE proteins and mastoparan action.

We examined the action of mastoparan on beta cell exocytosis. Mastoparan stimulated GABA and insulin release from MIN6 beta cells. On the other hand, mastopraran-induced GABA release was decreased by expressing the tetanus toxin C1 light chain in MIN6 cells. We have then investigated the relationship between SNARE proteins and mastoparan action using adenovirus-mediated gene transfer system. Overexpression of t-SNAREs, syntaxin 1A, and SNAP-25 inhibited the mastoparan-induced insulin release by approximately half-fold of control levels, however, the mastoparan-induced GABA release was not affected by these t-SNAREs overexpression. The overexpression of mutant alpha-SNAP (1-285), which inhibits the wild-type alpha-SNAP function in a dominant negative manner, did not influence either mastoparan-induced GABA or insulin release in spite of its marked inhibition of glucose-stimulated insulin release. Our data indicate that mastoparan stimulates GABA exocytosis via vesicular transport; however, SNARE proteins are differently involved in the exocytosis of insulin and GABA.     

WNK1 is a novel regulator of Munc18c-syntaxin 4 complex formation in soluble NSF attachment protein receptor (SNARE)-mediated vesicle exocytosis

Defects in soluble NSF attachment protein receptor (SNARE)-mediated granule exocytosis occur in islet beta cells, adipocytes, and/or skeletal muscle cells correlate with increased susceptibility to insulin resistance and diabetes. The serine/threonine kinase WNK1 (with no K (lysine)) has recently been implicated in exocytosis and is expressed in all three of these cell types. To search for WNK1 substrates related to exocytosis, we conducted a WNK1 two-hybrid screen, which yielded Munc18c. Munc18c is known to be a key regulator of accessibility of the target membrane (t-SNARE) protein syntaxin 4 to participate in SNARE core complex assembly, although a paucity of Munc18c-binding factors has precluded discovery of its precise functions. To validate WNK1 as a new Munc18c-interacting partner, the direct interaction between WNK1 and Munc18c was confirmed using in vitro binding analysis, and endogenous WNK1-Munc18c complexes were detected in the cytosolic and plasma membrane compartments of the islet beta cell line MIN6. This binding interaction is mediated through the N-terminal 172 residues of Munc18c and the kinase domain residues of WNK1 (residues 159-491). Expression of either of these two minimal interaction domains resulted in inhibition of glucose-stimulated insulin secretion, consistent with a functional importance for the endogenous WNK1-Munc18c complex in exocytosis. Interestingly, Munc18c failed to serve as a WNK1 substrate in kinase activity assays, suggesting that WNK1 functions in SNARE complex assembly outside its role as a kinase. Taken together, these data support a novel role for WNK1 and a new mechanism for the regulation of SNARE complex assembly by WNK1-Munc18c complexes.     

Munc18-1 and Munc18-2 proteins modulate beta-cell Ca2+ sensitivity and kinetics of insulin exocytosis differently

Fast neurotransmission and slower hormone release share the same core fusion machinery consisting of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. In evoked neurotransmission, interactions between SNAREs and the Munc18-1 protein, a member of the Sec1/Munc18 (SM) protein family, are essential for exocytosis, whereas other SM proteins are dispensable. To address if the exclusivity of Munc18-1 demonstrated in neuroexocytosis also applied to fast insulin secretion, we characterized the presence and function of Munc18-1 and its closest homologue Munc18-2 in β-cell stimulus-secretion coupling. We show that pancreatic β-cells express both Munc18-1 and Munc18-2. The two Munc18 homologues exhibit different subcellular localization, and only Munc18-1 redistributes in response to glucose stimulation. However, both Munc18-1 and Munc18-2 augment glucose-stimulated hormone release. Ramp-like photorelease of caged Ca(2+) and high resolution whole-cell patch clamp recordings show that Munc18-1 and Munc18-2 overexpression shift the Ca(2+) sensitivity of the fastest phase of insulin exocytosis differently. In addition, we reveal that Ca(2+) sensitivity of exocytosis in β-cells depends on the phosphorylation status of the Munc18 proteins. Even though Munc18-1 emerges as the key SM-protein determining the Ca(2+) threshold for triggering secretory activity in a stimulated β-cell, Munc18-2 has the ability to increase Ca(2+) sensitivity and thus mediates the release of fusion-competent granules requiring a lower cytoplasmic-free Ca(2+) concentration, [Ca(2+)](i)(.) Hence, Munc18-1 and Munc18-2 display distinct subcellular compartmentalization and can coordinate the insulin exocytotic process differently as a consequence of the actual [Ca(2+)](i).     

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

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 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 β-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 (TNFα, IL-1β, and IFNγ), 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 β-cells and expose a potential pathophysiological exploitation of this mechanism to underlie dysfunctional exocytosis.     

The HNF-1 target collectrin controls insulin exocytosis by SNARE complex formation

Defective glucose-stimulated insulin secretion is the main cause of hyperglycemia in type 2 diabetes mellitus. Mutations in HNF-1 cause a monogenic form of type 2 diabetes, maturity-onset diabetes of the young (MODY), characterized by impaired insulin secretion. Here we report that collectrin, a recently cloned kidney-specific gene of unknown function, is a target of HNF-1 in pancreatic β cells. Expression of collectrin was decreased in the islets of HNF-1 (−/−) mice, but was increased in obese hyperglycemic mice. Overexpression of collectrin in rat insulinoma INS-1 cells or in the β cells of transgenic mice enhanced glucose-stimulated insulin exocytosis, without affecting Ca²⁺ influx. Conversely, suppression of collectrin attenuated insulin secretion. Collectrin bound to SNARE complexes by interacting with snapin, a SNAP-25 binding protein, and facilitated SNARE complex formation. Therefore, collectrin is a regulator of SNARE complex function, which thereby controls insulin exocytosis.     

DPP-4 inhibitor des-F-sitagliptin treatment increased insulin exocytosis from db/db mice β cells

Incretin promotes insulin secretion acutely. Recently, orally-administered DPP-4 inhibitors represent a new class of anti-hyperglycemic agents. Indeed, inhibitors of dipeptidyl peptidase-IV (DPP-4), sitagliptin, has just begun to be widely used as therapeutics for type 2 diabetes. However, the effects of sitagliptin-treatment on insulin exocytosis from single β-cells are yet unknown. We therefore investigated how sitagliptin-treatment in db/db mice affects insulin exocytosis by treating db/db mice with des-F-sitagliptin for 2 weeks. Perfusion studies showed that 2 weeks-sitagliptin treatment potentiated insulin secretion. We then analyzed insulin granule motion and SNARE protein, syntaxin 1, by TIRF imaging system. TIRF imaging of insulin exocytosis showed the increased number of docked insulin granules and increased fusion events from them during first-phase release. In accord with insulin exocytosis data, des-F-sitagliptin-treatment increased the number of syntaxin 1 clusters on the plasma membrane. Thus, our data demonstrated that 2-weeks des-F-sitagliptin-treatment increased the fusion events of insulin granules, probably via increased number of docked insulin granules and that of syntaxin 1 clusters.     

Glucose-stimulated insulin secretion is coupled to the interaction of actin with the t-SNARE (target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein) complex

The actin monomer sequestering agent latrunculin B depolymerized beta-cell cortical actin, which resulted in increased glucose-stimulated insulin secretion in both cultured MIN6 beta-cells and isolated rat islet cells. In perifused islets, latrunculin B treatment increased both first- and second-phase glucose-stimulated insulin secretion without any significant effect on total insulin content. This increase in secretion was independent of calcium regulation because latrunculin B also potentiated calcium-stimulated insulin secretion in permeabilized MIN6 cells. Confocal immunofluorescent microscopy revealed a redistribution of insulin granules to the cell periphery in response to glucose or latrunculin B, which correlated with a reduction in phalloidin staining of cortical actin. Moreover, the t-SNARE [target membrane soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] proteins Syntaxin 1 and SNAP-25 coimmunoprecipitated polymerized actin from unstimulated MIN6 cells. Glucose stimulation transiently decreased the amount of actin coimmunoprecipitated with Syntaxin 1 and SNAP-25, and latrunculin B treatment fully ablated the coimmunoprecipitation. In contrast, the actin stabilizing agent jasplakinolide increased the amount of actin coimmunoprecipitated with the t-SNARE complex and prevented its dissociation upon glucose stimulation. These data suggest a mechanism whereby glucose modulates beta-cell cortical actin organization and disrupts the interaction of polymerized actin with the plasma membrane t-SNARE complex at a distal regulatory step in the exocytosis of insulin granules.     

Role of SNAP-23 in trafficking of H+-ATPase in cultured inner medullary collecting duct cells

The trafficking of H+-ATPase vesicles to the apical membrane of inner medullary collecting duct (IMCD) cells utilizes a mechanism similar to that described in neurosecretory cells involving soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) proteins. Regulated exocytosis of these vesicles is associated with the formation of SNARE complexes. Clostridial neurotoxins that specifically cleave the target (t-) SNARE, syntaxin-1, or the vesicle SNARE, vesicle-associated membrane protein-2, reduce SNARE complex formation, H+-ATPase translocation to the apical membrane, and inhibit H+ secretion. The purpose of these experiments was to characterize the physiological role of a second t-SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-23, a homologue of the neuronal SNAP-25, in regulated exocytosis of H+-ATPase vesicles. Our experiments document that 25-50 nM botulinum toxin (Bot) A or E cleaves rat SNAP-23 and thereby reduces immunodetectable and (35)S-labeled SNAP-23 by >60% within 60 min. Addition of 25 nM BotE to IMCD homogenates reduces the amount of the 20 S-like SNARE complex that can be immunoprecipitated from the homogenate. Treatment of intact IMCD monolayers with BotE reduces the amount of H+-ATPase translocated to the apical membrane by 52 +/- 2% of control and reduces the rate of H+ secretion by 77 +/- 3% after acute cell acidification. We conclude that SNAP-23 is a substrate for botulinum toxin proteolysis and has a critical role in the regulation of H+-ATPase exocytosis and H+ secretion in these renal epithelial cells.     

The tyrosine phosphorylation of Munc18c induces a switch in binding specificity from syntaxin 4 to Doc2beta

Glucose-stimulated insulin secretion is mediated by syntaxin 4-based SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes and the Sec1/Munc18 protein Munc18c. Our laboratory recently reported that Munc18c-syntaxin 4 complexes are further regulated by the competitive binding of the double C2 domain protein Doc2beta to Munc18c, although the underlying mechanism for this is unknown. Because the Doc2beta binding region of Munc18c contained residue Tyr-219 and this residue becomes phosphorylated in response to glucose stimulation, we hypothesized that the mechanism would involve Munc18c phosphorylation. Coimmunoprecipitation analyses using detergent lysates prepared from pervanadate-treated MIN6 beta cells revealed that the tyrosine phosphorylation of Munc18c corresponded to a 60% decrease in Munc18c-syntaxin 4 association with a coordinate 2-fold increase in Munc18c-Doc2beta binding. In vitro binding assays identified syntaxin 4 residues 118-194 as sufficient to confer its interaction with Munc18c; residues 118-194 contain the Hc alpha-helix and flexible linker region controlling transition of syntaxins between closed and open conformations. When overexpressed in MIN6 cells, this Hc-linker region functioned as a competitive inhibitor of endogenous syntaxin 4-Munc18c binding, increased syntaxin 4 binding to VAMP2, and significantly enhanced glucose-stimulated secretion. Molecular modeling of these new interactions yielded the predictions 1) that Tyr-219 of Munc18c remains buried under basal conditions in a conformation that is favorable for interaction with "closed" syntaxin 4 and 2) that stimulation leads to changes in syntaxin 4 contacts to facilitate exposure of Munc18c Tyr-219 for phosphorylation and Doc2beta binding.     

Synaptotagmins bind calcium to release insulin

Plasma insulin levels are determined mainly by the rate of exocytosis of the insulin-containing large dense core vesicles (LDCVs) of pancreatic islet beta-cells. This process involves the recruitment of LDCVs to the plasma membrane, where they are docked by the assembly of multiprotein SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. However, fusion of the two membranes will proceed only in the presence of Ca(2+) ions, implicating a Ca(2+) sensor protein. The synaptotagmin gene family, comprising 15 members, was proposed to act as such Ca(2+) sensor in regulated exocytosis in neurons and neuroendocrine and endocrine cells. Herein, we review the physiological function of the various synaptotagmins with reference to their impact on insulin exocytosis. Cumulating evidence emphasizes the crucial role of synaptotagmin VII and IX as mediators of glucose-induced insulin secretion.     

Combinatorial SNARE complexes modulate the secretion of cytoplasmic granules in human neutrophils

Mobilization of human neutrophil granules is critical for the innate immune response against infection and for the outburst of inflammation. Human neutrophil-specific and tertiary granules are readily exocytosed upon cell activation, whereas azurophilic granules are mainly mobilized to the phagosome. These cytoplasmic granules appear to be under differential secretory control. In this study, we show that combinatorial soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes with vesicle-associated membrane proteins (VAMPs), 23-kDa synaptosome-associated protein (SNAP-23), and syntaxin 4 underlie the differential mobilization of granules in human neutrophils. Specific and tertiary granules contained VAMP-1, VAMP-2, and SNAP-23, whereas the azurophilic granule membranes were enriched in VAMP-1 and VAMP-7. Ultrastructural, coimmunoprecipitation, and functional assays showed that SNARE complexes containing VAMP-1, VAMP-2, and SNAP-23 mediated the rapid exocytosis of specific/tertiary granules, whereas VAMP-1 and VAMP-7 mainly regulated the secretion of azurophilic granules. Plasma membrane syntaxin 4 acted as a general target SNARE for the secretion of the distinct granule populations. These data indicate that at least two SNARE complexes, made up of syntaxin 4/SNAP-23/VAMP-1 and syntaxin 4/SNAP-23/VAMP-2, are involved in the exocytosis of specific and tertiary granules, whereas interactions between syntaxin 4 and VAMP-1/VAMP-7 are involved in the exocytosis of azurophilic granules. Our data indicate that quantitative and qualitative differences in SNARE complex formation lead to the differential mobilization of the distinct cytoplasmic granules in human neutrophils, and a higher capability to form diverse SNARE complexes renders specific/tertiary granules prone to exocytosis.     

Autocrine anti-apoptotic and proliferative effects of insulin in pancreatic beta-cells

Insulin and glucose inhibited apoptosis in the MIN6 insulin-secreting cell line. The protective effect of 25 mM glucose was prevented by an anti-insulin antibody and this antibody-induced increase in apoptosis was reversed by the presence of excess insulin. Glucose stimulated MIN6 cell proliferation and this was inhibited by blockade of insulin secretion, by an anti-insulin antibody and by phosphatidylinositol-3 kinase (PI-3K) inhibition. Furthermore, MIN6 cell proliferation was stimulated by depolarising concentrations of KCl and by insulin itself. These data indicate that insulin secreted by β-cells in response to elevated glucose exerts autocrine effects to protect against apoptosis and stimulate proliferation, and suggest that the insulin signalling cascade, through the PI-3K pathway, may be an effective means of maintaining β-cell mass in diabetes.