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

Enhancing pancreatic β-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.

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 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 β 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 β cells in vivo and human β 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.

K2.1 clusters on β-cell plasma membrane act as reservoirs that replenish pools of newcomer insulin granule through their interaction with syntaxin-3.

The voltage-dependent K (K) channel K2.1 is a major delayed rectifier in many secretory cells, including pancreatic β cells. In addition, K2.

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.

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.

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.

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 β-cell-specific Syn-1A-KO (Syn-1A-βKO) mice to mimic β-cell Syn-1A deficiency in T2D. Glucose tolerance tests showed that Syn-1A-βKO mice exhibited blood glucose elevation corresponding to reduced blood insulin levels.

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

Synaptotagmin (Syt)-7, a major component of the exocytotic machinery in neurons, is also the major Syt in rodent pancreatic β-cells shown to mediate glucose-stimulated insulin secretion (GSIS). However, Syt-7's precise exocytotic actions in β-cells remain unknown. We show that Syt-7 is abundant in human β-cells.

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.

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.

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.

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.

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

In β-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.

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 β cells in part by tethering insulin secretory granules to Ca(2+) channels to assist excitosome assembly. Here, we assessed β cell exocytosis by patch clamp membrane capacitance measurement and total internal reflection fluorescence microscopy to investigate the role of Sec5 in regulating insulin secretion.

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.

Munc18b is a major mediator of insulin exocytosis in rat pancreatic β-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 β-cells to exocytose insulin becomes compromised in diabetes. β-Cells express three Munc18 isoforms of which the role of Munc18b is unknown.

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 β-cells resulted in a reduction in Ca(2+) currents arising specifically from L-(Cav 1.

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

Optimal insulin secretion required to maintain glucose homeostasis is the summation of total pancreatic islet β cell mass and intrinsic secretory capacity of individual β 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 β 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.

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)).

Syntaxin-1A interacts with distinct domains within nucleotide-binding folds of sulfonylurea receptor 1 to inhibit beta-cell ATP-sensitive potassium channels.

The ATP-sensitive potassium (K(ATP)) channel regulates pancreatic β-cell function by linking metabolic status to electrical activity. Syntaxin-1A (Syn-1A), a SNARE protein mediating exocytotic fusion, binds and inhibits the K(ATP) channel via the nucleotide-binding folds (NBFs) of its sulfonylurea receptor-1 (SUR1) regulatory subunit. In this study, we elucidated the precise regions within the NBFs required for Syn-1A-mediated K(ATP) inhibition, using in vitro binding assays, whole cell patch clamp and FRET assay.

SUMOylation regulates insulin exocytosis downstream of secretory granule docking in rodents and humans.

Objective: The reversible attachment of small ubiquitin-like modifier (SUMO) proteins controls target localization and function. We examined an acute role for the SUMOylation pathway in downstream events mediating insulin secretion.

Research Design And Methods: We studied islets and β-cells from mice and human donors, as well as INS-1 832/13 cells.

ATP modulates interaction of syntaxin-1A with sulfonylurea receptor 1 to regulate pancreatic beta-cell KATP channels.

ATP-sensitive potassium (K(ATP)) channels are regulated by a variety of cytosolic factors (adenine nucleotides, Mg(2+), phospholipids, and pH). We previously reported that K(ATP) channels are also regulated by endogenous membrane-bound SNARE protein syntaxin-1A (Syn-1A), which binds both nucleotide-binding folds of sulfonylurea receptor (SUR)1 and 2A, causing inhibition of K(ATP) channel activity in pancreatic islet β-cells and cardiac myocytes, respectively. In this study, we show that ATP dose-dependently inhibits Syn-1A binding to SUR1 at physiological concentrations, with the addition of Mg(2+) causing a decrease in the ATP-induced inhibitory effect.

Truncation of SNAP-25 reduces the stimulatory action of cAMP on rapid exocytosis in insulin-secreting cells.

Synaptosomal protein of 25 kDa (SNAP-25) is important for Ca(2+)-dependent fusion of large dense core vesicles (LDCVs) in insulin-secreting cells. Exocytosis is further enhanced by cAMP-increasing agents such as glucagon-like peptide-1 (GLP-1), and this augmentation includes interaction with both PKA and cAMP-GEFII. To investigate the coupling between SNAP-25- and cAMP-dependent stimulation of insulin exocytosis, we have used capacitance measurements, protein-binding assays, and Western blot analysis.

Cab45b, a Munc18b-interacting partner, regulates exocytosis in pancreatic beta-cells.

Cab45b is a cytosolic Ca(2+)-binding protein reported to regulate zymogen secretion in pancreatic acini. We now show that Cab45b is also expressed in pancreatic islet beta-cells and interacts there with the Sec1-Munc18 protein Munc18b. We employed patch clamp cell capacitance measurements to show that antibodies against Cab45b inhibited depolarization-evoked membrane capacitance increments, suggesting an impact on beta-cell granule exocytosis, both the readily releasable granule pool and refilling of this pool.