In pancreatic ␤-cells, the predominant voltage-gated Ca 2؉ channel (Ca V 1.2) and K ؉ channel (K V 2.1) are directly coupled to SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor) proteins. These SNARE proteins modulate channel expression and gating and closely associate these channels with the insulin secretory vesicles. We show that K V 2.1 and Ca V 1.2, but not K V 1.4, SUR1, or Kir6.2, target to specialized cholesterol-rich lipid raft domains on ␤-cell plasma membranes. Similarly, the SNARE proteins syntaxin 1A, SNAP-25, and VAMP-2, but not Munc-13-1 or n-Sec1, are associated with lipid rafts. Disruption of the lipid rafts by depleting membrane cholesterol with methyl-␤-cyclodextrin shunts K V 2.1, Ca V 1.2, and SNARE proteins out of lipid rafts. Furthermore, methyl-␤-cyclodextrin inhibits K V 2.1 but not Ca V 1.2 channel activity and enhances single-cell exocytic events and insulin secretion. Membrane compartmentalization of ion channels and SNARE proteins in lipid rafts may be critical for the temporal and spatial coordination of insulin release, forming what has been described as the excitosome complex.In the pancreatic islets of Langerhans, glucose uptake by ␤-cells initiates a cascade of cellular events resulting in insulin secretion. A key response leading to insulin release is the change in transmembrane potential associated with the opening and closing of ion channels. Glucose uptake and metabolism increases the ratio of ATP/ADP, leading to the blockade of ATP-sensitive potassium (K ϩ -ATP) channels. Inhibition of these channels results in cell membrane depolarization and subsequent activation of voltage-gated Ca 2ϩ (Ca V ) 1 channels. Influx of extracellular Ca 2ϩ through Ca V channels causes oscillatory elevations in [Ca 2ϩ ] i , fusion of insulin-containing vesicles with the cell membrane, and insulin release (reviewed in Ref. 1). This entire process is suppressed or terminated by the opening of voltage-gated K ϩ (K V ) channels (2). The integrated process of channel gating is critical for the coordination of insulin release and thus the consequent maintenance of proper plasma glucose levels.Pancreatic ␤-cells and clonal insulinoma cells express four different families of K V channels (K V 1, K V 2, K V 3, K V 4) in variable levels (2-4). K V 2.1 is the most abundant K V channel isoform expressed in both isolated islet ␤-cells and insulinoma cells. To support this notion, the dominant-negative knockout of K V 2.1 channel or pharmacological blockade with a selective K V 2.1 antagonist reduces steady-state outward K V currents by ϳ60 -70% (2, 5). In addition to K V 2.1, other K V channel ␣ subunits are expressed in pancreatic ␤-cells to a lesser extent, including K V 1.4 and K V 1.6, which account for less than 25% of outward K ϩ currents measured in these cells (2). The central role of Ca V channels in insulin secretion is well recognized (1). The predominant Ca V channel in ␤-cells is the L-type channel (long-lasting; Ca V 1.2/␣ 1C-a and Ca V 1.3/␣ 1D ) (6, 7). T...

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. VAMP8-null mice also exhibited increased islet β cell mass from increased β cell mitosis, with β cell proliferative activity greatly amplified by GLP-1. Thus, despite the β 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 β cell growth.

We demonstrated a significant association between frequent RVOT PVCs and LV dysfunction in patients without structural heart disease.

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. 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 β-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 β-cells as well as additional primary exocytosis, which may open new avenues of therapy development for diabetes.

The pancreatic acinus is the functional unit of the exocrine pancreas whose role is to secrete zymogens into the gut lumen for food digestion via apical exocytosis. We previously reported that supramaximal CCK induced apical blockade and redirected exocytosis to ectopic sites on the basolateral plasma membrane (BPM) of this polarized cell, leading to pancreatitis. Basolateral exocytosis was mediated by protein kinase C phosphorylation of BPM Munc18c, causing its displacement into the cytosol and activation of BPMbound Syntaxin-4 to form a SNARE complex. To mimic the conditions of alcoholic pancreatitis, we now examined whether 20 mM alcohol followed by submaximal CCK might mimic supramaximal CCK in inducing these pathologic exocytotic events. We show that a non-secretory but clinically relevant alcohol concentration (20 mM) inhibited submaximal CCK (50 pM)-stimulated amylase secretion by blocking apical exocytosis and redirecting exocytosis to less efficient BPM, indeed mimicking supramaximal CCK (10 nM) stimulation. We further demonstrate that basolateral exocytosis caused by both stimulation protocols is mediated by PKC␣-induced phosphorylation of Munc18c: 1) PKC␣ is activated, which binds and induces phosphorylation of PM-Munc18c at a Thr site, and these events can be inhibited by PKC␣ blockade; 2) PKC␣ inhibition blocks Munc18c displacement from the BPM; 3) PKC␣ inhibition prevents basolateral exocytosis but does not rescue apical exocytosis. We conclude that 20 mM alcohol/submaximal CCK as well supramaximal CCK stimulation can trigger pathologic basolateral exocytosis in pancreatic acinar cells via PKC␣-mediated activation of Munc18c, which enables Syntaxin-4 to become receptive in forming a SNARE complex in the BPM; and we further postulate this to be an underlying mechanism contributing to alcoholic pancreatitis.The pancreatic acinar cell is a highly polarized epithelial cell designed for zymogen granules (ZG) 3 to undergo regulated exocytosis at the apical pole, thereby emptying the digestive zymogens into the gut lumen for food digestion. This well orchestrated exocytic pathway can be altered by supramaximal stimulation with cholecystokinin (CCK), which causes apical blockade and redirection of exocytosis to the basolateral plasma membrane (BPM) surface (1). Specifically, using epifluorescence imaging of the FM1-43 dye in pancreatic acinar cells, we showed real-time visualization of apical exocytosis induced by maximal CCK, and aberrant exocytosis at the BPM caused by supramaximal CCK stimulation (1). We further found that this basolateral exocytosis was consistent with the paradigm of the SNARE Hypothesis (2-5), involving a distinct set of cognate SNARE partners, Syntaxin-4 (Syn-4) and SNAP 23 on the BPM, and VAMP proteins on the ZG (1, 6, 7), and whose interactions were regulated by the SM protein Munc18c (1). Here, Munc18c on the acinar BPM binds Syn-4, and upon supramaximal CCK stimulation, becomes displaced into the cytosol via a PKC-mediated mechanism; which activates Syn-4 to become capable of ...

No abstract

The molecular mechanism of clinical alcohol-induced pancreatitis remains vague. We had reported that experimental high-dose cholecystokinin (CCK)-induced pancreatitis is in part because of excessive aberrant basolateral exocytosis. High-dose CCK caused Munc18c on basolateral plasma membrane (BPM) to dissociate from syntaxin (Syn)-4, activating Syn-4 to complex with plasma membrane (PM)-SNAP-23 and granule-VAMP to mediate basolateral exocytosis. We now hypothesize that alcohol could render the acinar cell BPM conducive to exocytosis by a similar mechanism. Weakly stimulating postprandial doses of alcohol (20-50 mM) inhibited postprandial lowdose CCK-stimulated secretion by blocking physiologic apical exocytosis and redirecting exocytosis to lessefficient basal PM (visualized by FM1-43 fluorescence imaging) and lateral PM sites (electron microscopy). Alcohol or low-dose CCK had no effect on PM-Munc18c, but alcohol preincubation enabled low-dose CCK to displace Munc18c from BPM, leading to SNARE complex assembly in the BPM. Similarly, alcohol diet-fed rats did not exhibit morphologic defects in the pancreas nor affected PMMunc18c behavior, but subsequent intraperitoneal injections of low-dose CCK analog cerulein caused Munc18c displacement from BPM and cytosolic degradation, which contributed to pancreatitis. We conclude that alcohol induces BPM-Munc18c to become receptive to postprandial CCK-induced displacement into the cytosol, a process which facilitates SNARE complex assembly that in turn activates restricted BPM sites to become available for aberrant exocytosis into the interstitial space, where zymogen activation would take place and cause pancreatitis.

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.