Patrick P. L. Lam

Alcohol Redirects CCK-Mediated Apical Exocytosis to the Acinar Basolateral Membrane in Alcoholic Pancreatitis

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 low‐dose CCK‐stimulated secretion by blocking physiologic apical exocytosis and redirecting exocytosis to less‐efficient 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 PM‐Munc18c 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.

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. 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.

Effects of ethanol metabolites on exocytosis of pancreatic acinar cells in rats.

BACKGROUND & AIMS During development of alcoholic pancreatitis, oxidative (acetaldehyde) and nonoxidative metabolites (ethyl palmitate, ethyl oleate), rather than ethanol itself, mediate toxic injury. Exposure of pancreatic acini to ethanol blocks cholecystokinin (CCK)-8-stimulated apical exocytosis and redirects exocytosis to the basolateral plasma membrane, causing interstitial pancreatitis. We examined how each ethanol metabolite contributes to these changes in exocytosis. METHODS Rat pancreatic acini were incubated with concentrations of ethanol associated with alcoholic pancreatitis (20-50 mmol/L) or ethanol metabolites (1-3 mmol/L) and then stimulated with CCK-8. We performed single zymogen granule (ZG) exocytosis assays, Ca(2+) imaging studies, ultrastructural analyses (with electron microscopy), and confocal microscopy to assess the actin cytoskeleton and track the movement of vesicle-associated membrane protein (VAMP)-8-containing ZGs. Coimmunoprecipitation assays were used to identify complexes that contain the distinct combinations of Munc18 and the soluble N-ethylmaleimide sensitive factor attachment protein receptor proteins, which mediate apical (ZG-apical plasma membrane) and basolateral exocytosis and fusion between ZGs (ZG-ZG). RESULTS The ethanol metabolites acetaldehyde, ethyl palmitate, and ethyl oleate reduced CCK-8-stimulated apical exocytosis and formation of apical exocytotic complexes (between Munc18b and Syntaxin-2, synaptosomal-associated protein of 23 kilodaltons [SNAP23], and VAMP2) in rat pancreatic acini. Acetaldehyde and ethyl oleate redirected CCK-8-stimulated exocytosis to the basal and lateral plasma membranes and translocation of VAMP8-containing ZGs toward the basolateral plasma membrane. This process was mediated primarily via formation of basolateral exocytotic complexes (between Munc18c and Syntaxin-4, SNAP23, and VAMP8). Exposure of the acini to acetaldehyde and ethyl oleate followed by CCK-8 stimulation mildly perturbed the actin cytoskeleton and Ca(2+) signaling; exposure to ethyl palmitate severely affected Ca(2+) signaling. Acetaldehyde, like ethanol, promoted fusion between ZGs by the formation of ZG-ZG exocytotic complexes (between Munc18b and Syntaxin-3, SNAP23, and VAMP8), whereas ethyl palmitate and ethyl oleate reduced ZG-ZG fusion and formation of these complexes. CONCLUSIONS The ethanol metabolites acetaldehyde, ethyl palmitate, and ethyl oleate perturb exocytosis processes in cultured rat pancreatic acini (apical blockade, basolateral exocytosis, and fusion between ZGs). Acetaldehyde and, to a lesser degree, ethyl oleate produce many of the same pathologic effects of ethanol on CCK-8-stimulated exocytosis in pancreatic acini.

Deletion of Fas in the pancreatic β-cells leads to enhanced insulin secretion

Fas/Fas ligand belongs to the tumor necrosis factor superfamily of receptors/ligands and is best known for its role in apoptosis. However, recent evidence supports its role in other cellular responses, including proliferation and survival. Although Fas has been implicated as an essential mediator of beta-cell death in the pathogenesis of type 1 diabetes, the essential role of Fas specifically in pancreatic beta-cells has been found to be controversial. Moreover, the role of Fas on beta-cell homeostasis and function is not clear. The objective of this study is to determine the role of Fas specifically in beta-cells under both physiological and diabetes models. Mice with Fas deletion specifically in the beta-cells were generated using the Cre-loxP system. Cre-mediated Fas deletion was under the control of the rat insulin promoter. Absence of Fas in beta-cells leads to complete protection against FasL-induced cell death. However, Fas is not essential in determining beta-cell mass or susceptibility to streptozotocin- or HFD-induced diabetes. Importantly, Fas deletion in beta-cells leads to increased p65 expression, enhanced glucose tolerance, and glucose-stimulated insulin secretion, with increased exocytosis as manifested by increased changes in membrane capacitance and increased expression of Syntaxin1A, VAMP2, and munc18a. Together, our study shows that Fas in the beta-cells indeed plays an essential role in the canonical death receptor-mediated apoptosis but is not essential in regulating beta-cell mass or diabetes development. However, beta-cell Fas is critical in the regulation of glucose homeostasis through regulation of the exocytosis machinery.

When Pacemakers Fail: An Analysis of Clinical Presentation and Risk in 120 Patients with Failed Devices

Although pacemaker recalls are common, the optimal mechanism for risk assessment and triage of patients at risk for sudden loss of device system function is unknown. A retrospective chart review of 120 patients with factory proven failed devices was performed. Logistic regression analysis was used to determine clinical correlates of emergency room versus outpatient clinic presentation at time of device failure. Twenty‐two patients (18%) presented to emergency and 98 (82%) to clinic. Sixty‐three devices had no device output at the time of presentation. Multivariate logistic regression analysis revealed that antiarrhythmic drug use (odds ratio: 7.4, 95% CI: 2.0–28.0), atrioventricular nodal disease as an indication for pacing (odds ratio: 2.8, 95% CI: 1.2–3.0), and female gender (odds ratio: 2.2, 95% CI: 1.0–4.5) were the only significant correlates of emergency room presentations. Pacemaker dependency (escape heart rate < 40 beats/min) did not correlate with location of presentation even though no device output at the time of presentation was associated with emergency room presentation (odds ratio: 2.5, 95% CI: 1.1–5.8). Neither the presence of structural heart disease nor symptoms at the time of device implantation (syncope or presyncope) were correlated with location of presentation upon unexpected device failure. Although there were no deaths in the 120 failed devices studied, there were 26 deaths in the total group of 227 patients with recalled devices that could not be studied. Antiarrhythmic drug use, electrocardiographic pacing indication, and female gender may be more sensitive predictors of emergency room presentation and significant symptoms in the event of unanticipated pacemaker failure. The inability of any retrospective analysis to accurately assess mortality in the setting of pacemaker system failure underscores the need for prospective databases in recall situations.

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

Cab45b is a cytosolic Ca2+-binding protein reported to regulate zymogen secretion in pancreatic acini. We now show that Cab45b is also expressed in pancreatic islet β-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 β-cell granule exocytosis, both the readily releasable granule pool and refilling of this pool. Site-specific mutants in the Cab45b EF-hands were used to dissect the molecular interactions involved in Cab45b function. Mutants in EF-hands 2 and 3 had no detectable effects on interaction of Cab45b with Munc18b and did not affect the depolarization-evoked calcium currents, but remarkably, they facilitated the complex formation of Munc18b with syntaxin-2 and -3. As a result, these two EF-hand mutants inhibited β-cell membrane capacitance increments. This inhibition is mediated via Munc18b because Munc18b silencing with small interfering RNA abolished the effects of these two mutants. The results suggest a mechanism for Cab45b action that involves regulating the dynamic association of Munc18b with SNAREs to impact β-cell granule exocytosis.

Elevation in Intracellular Long-Chain Acyl-Coenzyme A Esters Lead to Reduced β-Cell Excitability via Activation of Adenosine 5′-Triphosphate-Sensitive Potassium Channels

Closure of pancreatic beta-cell ATP-sensitive potassium (K(ATP)) channels links glucose metabolism to electrical activity and insulin secretion. It is now known that saturated, but not polyunsaturated, long-chain acyl-coenyzme A esters (acyl-CoAs) can potently activate K(ATP) channels when superfused directly across excised membrane patches, suggesting a plausible mechanism to account for reduced beta-cell excitability and insulin secretion observed in obesity and type 2 diabetes. However, reduced beta-cell excitability due to elevation of endogenous saturated acyl-CoAs has not been confirmed in intact pancreatic beta-cells. To test this notion directly, endogenous acyl-CoA levels were elevated within primary mouse beta-cells using virally delivered overexpression of long-chain acyl-CoA synthetase-1 (AdACSL-1), and the effects on beta-cell K(ATP) channel activity and cell excitability was assessed using the perforated whole-cell and cell-attached patch-clamp technique. Data indicated a significant increase in K(ATP) channel activity in AdACSL-1-infected beta-cells cultured in medium supplemented with palmitate/oleate but not with the polyunsaturated fat linoleate. No changes in the ATP/ADP ratio were observed in any of the groups. Furthermore, AdACSL-1-infected beta-cells (with palmitate/oleate) showed a significant decrease in electrical responsiveness to glucose and tolbutamide and a hyperpolarized resting membrane potential at 5 mm glucose. These results suggest a direct link between intracellular fatty ester accumulation and K(ATP) channel activation, which may contribute to beta-cell dysfunction in type 2 diabetes.

RalA GTPase Tethers Insulin Granules to L‐ and R‐Type Calcium Channels Through Binding α2δ‐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 Ca2+ currents arising specifically from L‐(Cav1.2 and Cav1.3) and R‐type (Cav2.3) Ca2+ channels. Restoration of RalA expression in RalA KD cells rescued these defects in Ca2+ currents. RalA co‐immunoprecipitated with the Cavα2δ‐1 auxiliary subunit known to bind the three Cavs. Moreover, the functional molecular interactions between Cavα2δ‐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 α2δ‐1 to insulin granules without affecting the localization of the other Cav subunits. Furthermore, we confirmed that RalA and α2δ‐1 functionally interact since RalA KD‐induced inhibition of Cav currents could not be recovered by RalA when α2δ‐1 was simultaneously knocked down. These data provide a mechanism for RalA function in insulin secretion, whereby RalA binds α2δ‐1 on insulin granules to tether these granules to PM Ca2+ 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.

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.