Plasma membrane flipping of Syntaxin-2 regulates its inhibitory action on insulin granule exocytosis.

Enhancing pancreatic ß-cell secretion is a primary therapeutic target for type-2 diabetes (T2D). Syntaxin-2 (Stx2) has just been identified to be an inhibitory SNARE for insulin granule exocytosis, holding potential as a treatment for T2D, yet its molecular underpinnings remain unclear. We show that excessive Stx2 recruitment to raft-like granule docking sites at higher binding affinity than pro-fusion syntaxin-1A effectively competes for and inhibits fusogenic SNARE machineries. Depletion of Stx2 in human ß-cells improves insulin secretion by enhancing trans-SNARE complex assembly and cis-SNARE disassembly. Using a genetically-encoded reporter, glucose stimulation is shown to induce Stx2 flipping across the plasma membrane, which relieves its suppression of cytoplasmic fusogenic SNARE complexes to promote insulin secretion. Targeting the flipping efficiency of Stx2 profoundly modulates secretion, which could restore the impaired insulin secretion in diabetes. Here, we show that Stx2 acts to assist this precise tuning of insulin secretion in ß-cells, including in diabetes.

Pancreas-specific SNAP23 depletion prevents pancreatitis by attenuating pathological basolateral exocytosis and formation of trypsin-activating autolysosomes.

Intrapancreatic trypsin activation by dysregulated macroautophagy/autophagy and pathological exocytosis of zymogen granules (ZGs), along with activation of inhibitor of NFKB/NF-κB kinase (IKK) are necessary early cellular events in pancreatitis. How these three pancreatitis events are linked is unclear. We investigated how SNAP23 orchestrates these events leading to pancreatic acinar injury. SNAP23 depletion was by knockdown (SNAP23-KD) effected by adenovirus-shRNA (Ad-SNAP23-shRNA/mCherry) treatment of rodent and human pancreatic slices and in vivo by infusion into rat pancreatic duct. In vitro pancreatitis induction by supraphysiological cholecystokinin (CCK) or ethanol plus low-dose CCK were used to assess SNAP23-KD effects on exocytosis and autophagy. Pancreatitis stimuli resulted in SNAP23 translocation from its native location at the plasma membrane to autophagosomes, where SNAP23 would bind and regulate STX17 (syntaxin17) SNARE complex-mediated autophagosome-lysosome fusion. This SNAP23 relocation was attributed to IKBKB/IKKß-mediated SNAP23 phosphorylation at Ser95 Ser120 in rat and Ser120 in human, which was blocked by IKBKB/IKKß inhibitors, and confirmed by the inability of IKBKB/IKKß phosphorylation-disabled SNAP23 mutant (Ser95A Ser120A) to bind STX17 SNARE complex. SNAP23-KD impaired the assembly of STX4-driven basolateral exocytotic SNARE complex and STX17-driven SNARE complex, causing respective reduction of basolateral exocytosis of ZGs and autolysosome formation, with consequent reduction in trypsinogen activation in both compartments. Consequently, pancreatic SNAP23-KD rats were protected from caerulein and alcoholic pancreatitis. This study revealed the roles of SNAP23 in mediating pathological basolateral exocytosis and IKBKB/IKKß's involvement in autolysosome formation, both where trypsinogen activation would occur to cause pancreatitis. SNAP23 is a strong candidate to target for pancreatitis therapy.Abbreviations: AL: autolysosome; AP: acute pancreatitis; AV: autophagic vacuole; CCK: cholecystokinin; IKBKB/IKKß: inhibitor of nuclear factor kappa B kinase subunit beta; SNAP23: synaptosome associated protein 23; SNARE: soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor; STX: syntaxin; TAP: trypsinogen activation peptide; VAMP: vesicle associated membrane protein; ZG: zymogen granule.

SNAP23 depletion enables more SNAP25/calcium channel excitosome formation to increase insulin exocytosis in type 2 diabetes.

SNAP23 is the ubiquitous SNAP25 isoform that mediates secretion in non-neuronal cells, similar to SNAP25 in neurons. However, some secretory cells like pancreatic islet ß cells contain an abundance of both SNAP25 and SNAP23, where SNAP23 is believed to play a redundant role to SNAP25. We show that SNAP23, when depleted in mouse ß cells in vivo and human ß cells (normal and type 2 diabetes [T2D] patients) in vitro, paradoxically increased biphasic glucose-stimulated insulin secretion corresponding to increased exocytosis of predocked and newcomer insulin granules. Such effects on T2D Goto-Kakizaki rats improved glucose homeostasis that was superior to conventional treatment with sulfonylurea glybenclamide. SNAP23, although fusion competent in slower secretory cells, in the context of ß cells acts as a weak partial fusion agonist or inhibitory SNARE. Here, SNAP23 depletion promotes SNAP25 to bind calcium channels more quickly and longer where granule fusion occurs to increase exocytosis efficiency. ß Cell SNAP23 antagonism is a strategy to treat diabetes.

Kv2.1 clusters on ß-cell plasma membrane act as reservoirs that replenish pools of newcomer insulin granule through their interaction with syntaxin-3.

The voltage-dependent K+ (Kv) channel Kv2.1 is a major delayed rectifier in many secretory cells, including pancreatic ß cells. In addition, Kv2.1 has a direct role in exocytosis at an undefined step, involving SNARE proteins, that is independent of its ion-conducting pore function. Here, we elucidated the precise step in exocytosis. We previously reported that syntaxin-3 (Syn-3) is the key syntaxin that mediates exocytosis of newcomer secretory granules that spend minimal residence time on the plasma membrane before fusion. Using high-resolution total internal reflection fluorescence microscopy, we now show that Kv2.1 forms reservoir clusters on the ß-cell plasma membrane and binds Syn-3 via its C-terminal C1b domain, which recruits newcomer insulin secretory granules into this large reservoir. Upon glucose stimulation, secretory granules were released from this reservoir to replenish the pool of newcomer secretory granules for subsequent fusion, occurring just adjacent to the plasma membrane Kv2.1 clusters. C1b deletion blocked the aforementioned Kv2.1-Syn-3-mediated events and reduced fusion of newcomer secretory granules. These insights have therapeutic implications, as Kv2.1 overexpression in type-2 diabetes rat islets restored biphasic insulin secretion.

Pancreatitis-Induced Depletion of Syntaxin 2 Promotes Autophagy and Increases Basolateral Exocytosis.

BACKGROUND & AIMS: Pancreatic 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. METHODS: We 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. RESULTS: Human 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. CONCLUSIONS: In 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.

Munc18b Increases Insulin Granule Fusion, Restoring Deficient Insulin Secretion in Type-2 Diabetes Human and Goto-Kakizaki Rat Islets with Improvement in Glucose Homeostasis.

Reduced pancreatic islet levels of Munc18a/SNARE complex proteins have been postulated to contribute to the deficient glucose-stimulated insulin secretion (GSIS) in type-2 diabetes (T2D). Whereas much previous work has purported Munc18a/SNARE complex (Syntaxin-1A/VAMP-2/SNAP25) to be primarily involved in predocked secretory granule (SG) fusion, less is known about newcomer SGs that undergo minimal docking time at the plasma membrane before fusion. Newcomer SG fusion has been postulated to involve a distinct SM/SNARE complex (Munc18b/Syntaxin-3/VAMP8/SNAP25), whose levels we find also reduced in islets of T2D humans and T2D Goto-Kakizaki (GK) rats. Munc18b overexpression by adenovirus infection (Ad-Munc18b), by increasing assembly of Munc18b/SNARE complexes, mediated increased fusion of not only newcomer SGs but also predocked SGs in T2D human and GK rat islets, resulting in rescue of the deficient biphasic GSIS. Infusion of Ad-Munc18b into GK rat pancreas led to sustained improvement in glucose homeostasis. However, Munc18b overexpression in normal islets increased only newcomer SG fusion. Therefore, Munc18b could potentially be deployed in human T2D to rescue the deficient GSIS.

Syntaxin 2 Acts as Inhibitory SNARE for Insulin Granule Exocytosis.

Of the four syntaxins specialized for exocytosis, syntaxin (Syn)-2 is the least understood. In this study, we used Syn-2/epimorphin knockout mice to examine the role of Syn-2 in insulin secretory granule (SG) exocytosis. Unexpectedly, Syn-2 knockout mice exhibited paradoxical superior glucose homeostasis resulting from an enhanced insulin secretion. This was confirmed in vitro by pancreatic islet perifusion showing an amplified biphasic glucose-stimulated insulin secretion arising from an increase in size of the readily releasable pool of insulin SGs and enhanced SG pool refilling. The increase in insulin exocytosis was attributed mainly to an enhanced recruitment of the larger pool of newcomer SGs that undergoes no residence time on plasma membrane before fusion and, to a lesser extent, also the predocked SGs. Consistently, Syn-2 depletion resulted in a stimulation-induced increase in abundance of exocytotic complexes we previously demonstrated as mediating the fusion of newcomer SGs (Syn-3/VAMP8/SNAP25/Munc18b) and predocked SGs (Syn-1A/VAMP2/SNAP25/Muncn18a). This work is the first to show in mammals that Syn-2 could function as an inhibitory SNARE protein that, when relieved, could promote exocytosis in pancreatic islet ß-cells. Thus, Syn-2 may serve as a potential target to treat diabetes.

New Roles of Syntaxin-1A in Insulin Granule Exocytosis and Replenishment.

In type-2 diabetes (T2D), severely reduced islet syntaxin-1A (Syn-1A) levels contribute to insulin secretory deficiency. We generated ß-cell-specific Syn-1A-KO (Syn-1A-ßKO) mice to mimic ß-cell Syn-1A deficiency in T2D. Glucose tolerance tests showed that Syn-1A-ßKO mice exhibited blood glucose elevation corresponding to reduced blood insulin levels. Perifusion of Syn-1A-ßKO islets showed impaired first- and second-phase glucose-stimulated insulin secretion (GSIS) resulting from reduction in readily releasable pool and granule pool refilling. To unequivocally determine the ß-cell exocytotic defects caused by Syn-1A deletion, EM and total internal reflection fluorescence microscopy showed that Syn-1A-KO ß-cells had a severe reduction in the number of secretory granules (SGs) docked onto the plasma membrane (PM) at rest and reduced SG recruitment to the PM after glucose stimulation, the latter indicating defects in replenishment of releasable pools required to sustain second-phase GSIS. Whereas reduced predocked SG fusion accounted for reduced first-phase GSIS, selective reduction of exocytosis of short-dock (but not no-dock) newcomer SGs accounted for the reduced second-phase GSIS. These Syn-1A actions on newcomer SGs were partly mediated by Syn-1A interactions with newcomer SG VAMP8.

Synaptotagmin-7 Functions to Replenish Insulin Granules for Exocytosis in Human Islet ß-Cells.

Synaptotagmin (Syt)-7, a major component of the exocytotic machinery in neurons, is also the major Syt in rodent pancreatic ß-cells shown to mediate glucose-stimulated insulin secretion (GSIS). However, Syt-7's precise exocytotic actions in ß-cells remain unknown. We show that Syt-7 is abundant in human ß-cells. Adenovirus-short hairpin RNA knockdown (KD) of Syt-7 in human islets reduced first- and second-phase GSIS attributed to the reduction of exocytosis of predocked and newcomer insulin secretory granules (SGs). Glucose stimulation expectedly induced Syt-7 association in a Ca(2+)-dependent manner with syntaxin-3 and syntaxin-1A soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes known to mediate exocytosis of newcomer and predocked SGs, respectively. However, Syt-7-KD did not disrupt SNARE complex assembly. Instead, electron microscopy analysis showed that Syt-7-KD reduced the recruitment of SGs to the plasma membrane after glucose-stimulated depletion, which could not be rescued by glucagon-like peptide 1 pretreatment. To assess the possibility that this new action of Syt-7 on SG recruitment may involve calmodulin (CaM), pretreatment of islets with CaM blocker calmidazolium showed effects very similar to those of Syt-7-KD. Syt-7 therefore plays a novel more dominant function in the replenishment of releasable SG pools in human ß-cells than its previously purported role in exocytotic fusion per se.

Syntaxin-3 Binds and Regulates Both R- and L-Type Calcium Channels in Insulin-Secreting INS-1 832/13 Cells.

Syntaxin (Syn)-1A mediates exocytosis of predocked insulin-containing secretory granules (SGs) during first-phase glucose-stimulated insulin secretion (GSIS) in part via its interaction with plasma membrane (PM)-bound L-type voltage-gated calcium channels (Cav). In contrast, Syn-3 mediates exocytosis of newcomer SGs that accounts for second-phase GSIS. We now hypothesize that the newcomer SG Syn-3 preferentially binds and modulates R-type Cav opening, which was postulated to mediate second-phase GSIS. Indeed, glucose-stimulation of pancreatic islet ß-cell line INS-1 induced a predominant increase in interaction between Syn-3 and Cavα1 pore-forming subunits of R-type Cav2.3 and to lesser extent L-type Cavs, while confirming the preferential interactions between Syn-1A with L-type (Cav1.2, Cav1.3) Cavs. Consistently, direct binding studies employing heterologous HEK cells confirmed that Syn-3 preferentially binds Cav2.3, whereas Syn-1A prefers L-type Cavs. We then used siRNA knockdown (KD) of Syn-3 in INS-1 to study the endogenous modulatory actions of Syn-3 on Cav channels. Syn-3 KD enhanced Ca2+ currents by 46% attributed mostly to R- and L-type Cavs. Interestingly, while the transmembrane domain of Syn-1A is the putative functional domain modulating Cav activity, it is the cytoplasmic domain of Syn-3 that appears to modulate Cav activity. We conclude that Syn-3 may mimic Syn-1A in the ability to bind and modulate Cavs, but preferring Cav2.3 to perhaps participate in triggering fusion of newcomer insulin SGs during second-phase GSIS.

Munc18c mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose stimulated insulin secretion in human pancreatic beta-cells.

OBJECTIVE: Pancreatic beta-cells express three Munc18 isoforms. Much is known about the roles of Munc18a (pre-docked secretory granules-SGs) and Munc18b (newcomer SGs and SG-SG fusion) in insulin exocytosis. Although shown to influence glucose-stimulated insulin secretion (GSIS) in rodents the precise role of Munc18c in insulin SG exocytosis has not been elucidated. We here examined the role of Munc18c in human pancreatic beta-cells. METHODS: Munc18c-shRNA/RFP lenti-virus (versus control virus) was used to knock down the expression level of Munc18c in human islets or single beta-cells. Insulin secretion and granule exocytosis were measured by performing islets perifusion, single-cell patch clamp capacitance measurements and total internal reflection fluorescence microscopy (TIRFM). RESULTS: Munc18c is most abundant in the cytosol of human beta-cells. Endogenous function of Munc18c was assessed by knocking down (KD) its islet expression by 70% employing lenti-shRNA virus. Munc18c-KD caused reduction in cognate syntaxin-4 islet expression but not in other exocytotic proteins, resulting in the reduction in GSIS in first- (by 42%) and second phases (by 35%). Single cell analyses of RFP-tagged Munc18c-KD beta-cells by patch clamp capacitance measurements showed inhibition in both readily-releasable pool (by 52%) and mobilization from the reserve pool (by 57%). TIRFM to assess single SG behavior showed that Munc18c-KD inhibition of first phase GSIS was attributed to reduction in exocytosis of pre-docked and newcomer SGs, and second phase inhibition attributed entirely to reduction in newcomer SG fusion (SGs that undergo minimal residence or docking time at the plasma membrane before fusion). CONCLUSION: Munc18c is involved in the distinct molecular machineries that affect exocytosis of both predocked and newcomer SG pools that underlie Munc18c's role in first and second phases of GSIS, respectively.

Syntaxin-4 mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose-stimulated insulin secretion in human pancreatic beta cells.

AIMS/HYPOTHESIS: 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/INTERPRETATION: 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.

Phosphatidylinositol 4,5-biphosphate (PIP2) modulates syntaxin-1A binding to sulfonylurea receptor 2A to regulate cardiac ATP-sensitive potassium (KATP) channels.

Cardiac sarcolemmal syntaxin (Syn)-1A interacts with sulfonylurea receptor (SUR) 2A to inhibit ATP-sensitive potassium (KATP) channels. Phosphatidylinositol 4,5-bisphosphate (PIP2), a ubiquitous endogenous inositol phospholipid, known to bind Kir6.2 subunit to open KATP channels, has recently been shown to directly bind Syn-1A in plasma membrane to form Syn-1A clusters. Here, we sought to determine whether the interaction between Syn-1A and PIP2 interferes with the ability of Syn-1A to bind SUR2A and inhibit KATP channel activity. We found that PIP2 dose-dependently reduced SUR2A binding to GST-Syn-1A by in vitro pulldown assays. FRET studies in intact cells using TIRFM revealed that increasing endogenous PIP2 levels led to increased Syn-1A (-EGFP) cluster formation and a severe reduction in availability of Syn-1A molecules to interact with SUR2A (-mCherry) molecules outside the Syn-1A clusters. Correspondingly, electrophysiological studies employing SUR2A/Kir6.2-expressing HEK cells showed that increasing endogenous or exogenous PIP2 diminished the inhibitory effect of Syn-1A on KATP currents. The physiological relevance of these findings was confirmed by ability of exogenous PIP2 to block exogenous Syn-1A inhibition of cardiac KATP currents in inside-out patches of mouse ventricular myocytes. The effect of PIP2 on physical and functional interactions between Syn-1A and KATP channels is specific and not observed with physiologic concentrations of other phospholipids. To unequivocally demonstrate the specificity of PIP2 interaction with Syn-1A and its impact on KATP channel modulation by Syn-1A, we employed a PIP2-insensitive Syn-1A-5RK/A mutant. The Syn-1A-5RK/A mutant retains the ability to interact with SUR2A in both in vitro binding and in vivo FRET assays, although as expected the interaction is no longer disrupted by PIP2. Interestingly, at physiological PIP2 concentrations, Syn-1A-5RK/A inhibited KATP currents to a greater extent than Syn-1A-WT, indicating that the inhibitory effect of Syn-1A on KATP channels is not due to direct competition between Syn-1A and Kir6.2 for PIP2 binding. At high-dose PIP2, however, inhibition of KATP currents by Syn-1A-5RK/A was greatly reduced, likely overridden by the direct activating effect of PIP2 on KATP channels. Finally, depleting endogenous PIP2 with polyphosphoinositide phosphatase synaptojanin-1 known to disperse Syn-1A clusters, freed Syn-1A from Syn-1A clusters to bind SUR2A, causing optimal inhibition of KATP channels. These results taken together led us to conclude that PIP2 affects cardiac KATP channels not only by its actions on the channel directly but also by multi-modal effects of dynamically modulating Syn-1A mobility from Syn-1A clusters and thereby the availability of Syn-1A to inhibit KATP channels via interaction with SUR2A on the plasma membrane.

Phosphatidylinositol 4,5-biphosphate (PIP2) modulates interaction of syntaxin-1A with sulfonylurea receptor 1 to regulate pancreatic ß-cell ATP-sensitive potassium channels.

In ß-cells, syntaxin (Syn)-1A interacts with SUR1 to inhibit ATP-sensitive potassium channels (KATP channels). PIP2 binds the Kir6.2 subunit to open KATP channels. PIP2 also modifies Syn-1A clustering in plasma membrane (PM) that may alter Syn-1A actions on PM proteins like SUR1. Here, we assessed whether the actions of PIP2 on activating KATP channels is contributed by sequestering Syn-1A from binding SUR1. In vitro binding showed that PIP2 dose-dependently disrupted Syn-1A·SUR1 complexes, corroborated by an in vivo Forster resonance energy transfer assay showing disruption of SUR1(-EGFP)/Syn-1A(-mCherry) interaction along with increased Syn-1A cluster formation. Electrophysiological studies of rat ß-cells, INS-1, and SUR1/Kir6.2-expressing HEK293 cells showed that PIP2 dose-dependent activation of KATP currents was uniformly reduced by Syn-1A. To unequivocally distinguish between PIP2 actions on Syn-1A and Kir6.2, we employed several strategies. First, we showed that PIP2-insensitive Syn-1A-5RK/A mutant complex with SUR1 could not be disrupted by PIP2, consequently reducing PIP2 activation of KATP channels. Next, Syn-1A·SUR1 complex modulation of KATP channels could be observed at a physiologically low PIP2 concentration that did not disrupt the Syn-1A·SUR1 complex, compared with higher PIP2 concentrations acting directly on Kir6.2. These effects were specific to PIP2 and not observed with physiologic concentrations of other phospholipids. Finally, depleting endogenous PIP2 with polyphosphoinositide phosphatase synaptojanin-1, known to disperse Syn-1A clusters, freed Syn-1A from Syn-1A clusters to bind SUR1, causing inhibition of KATP channels that could no longer be further inhibited by exogenous Syn-1A. These results taken together indicate that PIP2 affects islet ß-cell KATP channels not only by its actions on Kir6.2 but also by sequestering Syn-1A to modulate Syn-1A availability and its interactions with SUR1 on PM.

Exocyst sec5 regulates exocytosis of newcomer insulin granules underlying biphasic insulin secretion.

The exocyst complex subunit Sec5 is a downstream effector of RalA-GTPase which promotes RalA-exocyst interactions and exocyst assembly, serving to tether secretory granules to docking sites on the plasma membrane. We recently reported that RalA regulates biphasic insulin secretion in pancreatic islet ß cells in part by tethering insulin secretory granules to Ca(2+) channels to assist excitosome assembly. Here, we assessed ß cell exocytosis by patch clamp membrane capacitance measurement and total internal reflection fluorescence microscopy to investigate the role of Sec5 in regulating insulin secretion. Sec5 is present in human and rodent islet ß cells, localized to insulin granules. Sec5 protein depletion in rat INS-1 cells inhibited depolarization-induced release of primed insulin granules from both readily-releasable pool and mobilization from the reserve pool. This reduction in insulin exocytosis was attributed mainly to reduction in recruitment and exocytosis of newcomer insulin granules that undergo minimal docking time at the plasma membrane, but which encompassed a larger portion of biphasic glucose stimulated insulin secretion. Sec5 protein knockdown had little effect on predocked granules, unless vigorously stimulated by KCl depolarization. Taken together, newcomer insulin granules in ß cells are more sensitive than predocked granules to Sec5 regulation.

An exploratory study of the association between KCNB1 rs1051295 and type 2 diabetes and its related traits in Chinese Han population.

Since the KCNB1 encoding Kv2.1 channel accounts for the majority of Kv currents modulating insulin secretion by pancreatic islet beta-cells, we postulated that KCNB1 is a plausible candidate gene for genetic variation contributing to the variable compensatory secretory function of beta-cells in type-2 diabetes (T2D). We conducted two studies, a case-control study and a cross-section study, to investigate the association of common single-nucleotide polymorphisms (SNPs) in KCNB1 with T2D and its linking traits. In the case-control study, we first examined the association of 20 tag SNPs of KCNB1 with T2D in a population with 226 T2D patients and non-diabetic subjects (screening study). We then identified the association in an enlarged population of 412 T2D patients and non-diabetic subjects (replication study). In the cross-sectional study, we investigated the linkage between the candidate SNP rs1051295 and T2D by comparing beta-cell function and insulin sensitivity among rs1051295 genotypes in a general population of 1051 subjects at fasting and after glucose loading (oral glucose tolerance tests, OGTT) in 84 fasting glucose impaired subjects, and several T2D-related traits. We found that among the 19 available tag SNPs, only the KCNB1 rs1051295 was associated with T2D (P = 0.027), with the rs1051295 TT genotype associated with an increased risk of T2D compared with genotypes CC (P = 0.009). At fasting, rs1051295 genotype TT was associated with a 9.8% reduction in insulin sensitivity compared to CC (P = 0.008); along with increased plasma triglycerides (TG) levels (TT/CC: P = 0.046) and increased waist/hip (W/H) ratio (TT/CC: P = 0.013; TT/TC: P = 0.002). OGTT confirmed that genotype TT exhibited reduced insulin sensitivity by 16.3% (P = 0.030) compared with genotype TC+CC in a fasting glucose impaired population. The KCNB1 rs1051295 genotype TT in the Chinese Han population is associated with decreased insulin sensitivity and increased plasma TG and W/H ratio, which together contribute to an increased risk for T2D.

Munc18b is a major mediator of insulin exocytosis in rat pancreatic ß-cells.

Sec1/Munc18 proteins facilitate the formation of trans-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes that mediate fusion of secretory granule (SG) with plasma membrane (PM). The capacity of pancreatic ß-cells to exocytose insulin becomes compromised in diabetes. ß-Cells express three Munc18 isoforms of which the role of Munc18b is unknown. We found that Munc18b depletion in rat islets disabled SNARE complex formation formed by syntaxin (Syn)-2 and Syn-3. Two-photon imaging analysis revealed in Munc18b-depleted ß-cells a 40% reduction in primary exocytosis (SG-PM fusion) and abrogation of almost all sequential SG-SG fusion, together accounting for a 50% reduction in glucose-stimulated insulin secretion (GSIS). In contrast, gain-of-function expression of Munc18b wild-type and, more so, dominant-positive K314L/R315L mutant promoted the assembly of cognate SNARE complexes, which caused potentiation of biphasic GSIS. We found that this was attributed to a more than threefold enhancement of both primary exocytosis and sequential SG-SG fusion, including long-chain fusion (6-8 SGs) not normally (2-3 SG fusion) observed. Thus, Munc18b-mediated exocytosis may be deployed to increase secretory efficiency of SGs in deeper cytosolic layers of ß-cells as well as additional primary exocytosis, which may open new avenues of therapy development for diabetes.

RalA GTPase tethers insulin granules to L- and R-type calcium channels through binding α2 δ-1 subunit.

RalA GTPase has been implicated in the regulated delivery of exocytotic vesicles to the plasma membrane (PM) in mammalian cells. We had reported that RalA regulates biphasic insulin secretion, which we have now determined to be contributed by RalA direct interaction with voltage-gated calcium (Cav ) channels. RalA knockdown (KD) in INS-1 cells and primary rat ß-cells resulted in a reduction in Ca(2+) currents arising specifically from L-(Cav 1.2 and Cav 1.3) and R-type (Cav 2.3) Ca(2+) channels. Restoration of RalA expression in RalA KD cells rescued these defects in Ca(2+) currents. RalA co-immunoprecipitated with the Cav α2 δ-1 auxiliary subunit known to bind the three Cav s. Moreover, the functional molecular interactions between Cav α2 δ-1 and RalA on the PM shown by total internal reflection fluorescent microscopy/FRET analysis could be induced by glucose stimulation. KD of RalA inhibited trafficking of α2 δ-1 to insulin granules without affecting the localization of the other Cav subunits. Furthermore, we confirmed that RalA and α2 δ-1 functionally interact since RalA KD-induced inhibition of Cav currents could not be recovered by RalA when α2 δ-1 was simultaneously knocked down. These data provide a mechanism for RalA function in insulin secretion, whereby RalA binds α2 δ-1 on insulin granules to tether these granules to PM Ca(2+) channels. This acts as a chaperoning step prior to and in preparation for sequential assembly of exocyst and excitosome complexes that mediate biphasic insulin secretion.

Dual role of VAMP8 in regulating insulin exocytosis and islet ß cell growth.

Optimal insulin secretion required to maintain glucose homeostasis is the summation of total pancreatic islet ß cell mass and intrinsic secretory capacity of individual ß cells, which are regulated by distinct mechanisms that could be amplified by glucagon-like-peptide-1 (GLP-1). Because of these actions of GLP-1 on islet ß cells, GLP-1 has been deployed to treat diabetes. We employed SNARE protein VAMP8-null mice to demonstrate that VAMP8 mediates insulin granule recruitment to the plasma membrane, which partly accounts for GLP-1 potentiation of glucose-stimulated insulin secretion. VAMP8-null mice also exhibited increased islet ß cell mass from increased ß cell mitosis, with ß cell proliferative activity greatly amplified by GLP-1. Thus, despite the ß cell exocytotic defect, VAMP8-null mice have an increased total insulin secretory capacity, which improved glucose homeostasis. We conclude that these VAMP8-mediated events partly underlie the therapeutic actions of GLP-1 on insulin secretion and ß cell growth.

Syntaxin-1A inhibits KATP channels by interacting with specific conserved motifs within sulfonylurea receptor 2A.

We previously demonstrated that syntaxin (Syn)-1A is present in the sarcolemma of rat cardiomyocytes and binds sulfonylurea receptor (SUR) 2A nucleotide binding folds (NBFs) to inhibit ATP-sensitive potassium (K(ATP)) channel. Here, we examined for the precise domains within the NBFs of SUR2A that may interact with Syn-1A. Specifically, we tested truncated NBF protein segments encompassing the conserved motifs Walker A (W(A)), signature/Linker (L), and Walker B (W(B)). In vitro binding results indicate that the domains encompassing W(A) and L of NBF-1 and all three conserved motifs of NBF-2 bound Syn-1A. Electrophysiological studies, employing inside-out patch-clamp recordings from SUR2A/Kir6.2 expressing HEK cells and mouse cardiomyocytes, show that W(B) and L of NBF-1 and all three NBF-2 truncated protein segments reduced Syn-1A inhibition of SUR2A/K(ATP) channels. Remarkably, these same NBF-1 and -2 truncated proteins could independently disrupt the intimate FRET interactions of full length SUR2A (-mCherry) and Syn-1A (-EGFP). These results taken together indicate that Syn-1A possibly maintains inhibition of cardiac ventricular K(ATP) channels by binding to large regions of NBF-1 and NBF-2 to stabilize the NBF-1-NBF-2 heterodimer formation and prevent ATP-binding and ATP hydrolysis. Since K(ATP) channels are closely coupled to metabolic states, we postulate that these very intimate Syn-1A-SUR2A interactions are critically important for myocardial protection during stress, in which profound changes in metabolic factors (pH, ATP) could modulate these Syn-1A-SUR2A interactions.