Factors associated with nocturnal and diurnal glycemic variability in patients with type 2 diabetes: a cross-sectional study.

PURPOSE: There is little information on factors that influence the glycemic variability (GV) during the nocturnal and diurnal periods. We aimed to examine the relationship between clinical factors and GV during these two periods. METHODS: This cross-sectional study included 134 patients with type 2 diabetes. 24-h changes in blood glucose were recorded by a continuous glucose monitoring system. Nocturnal and diurnal GV were assessed by standard deviation of blood glucose (SDBG), coefficient of variation (CV), and mean amplitude of glycemic excursions (MAGE), respectively. Robust regression analyses were performed to identify the factors associated with GV. Restricted cubic splines were used to determine dose-response relationship. RESULTS: During the nocturnal period, age and glycemic level at 12:00 A.M. were positively associated with GV, whereas alanine aminotransferase was negatively associated with GV. During the diurnal period, homeostatic model assessment 2-insulin sensitivity (HOMA2-S) was positively associated with GV, whereas insulin secretion-sensitivity index-2 (ISSI2) was negatively associated with GV. Additionally, we found a J-shape association between the glycemic level at 12:00 A.M. and MAGE, with 9.0 mmol/L blood glucose level as a cutoff point. Similar nonlinear associations were found between ISSI2 and SDBG, and between ISSI2 and MAGE, with ISSI2 value of 175 as a cutoff point. CONCLUSION: Factors associated with GV were different between nocturnal and diurnal periods. The cutoff points we found in this study may provide the therapeutic targets for beta-cell function and pre-sleep glycemic level in clinical practice.

Syntaxin-3 regulates newcomer insulin granule exocytosis and compound fusion in pancreatic beta cells.

AIMS/HYPOTHESIS: The molecular basis of the exocytosis of secretory insulin-containing granules (SGs) during biphasic glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells remains unclear. Syntaxin (SYN)-1A and SYN-4 have been shown to mediate insulin exocytosis. The insulin-secretory function of SYN-3, which is particularly abundant in SGs, is unclear. METHODS: Mouse pancreatic islets and INS-1 cells were treated with adenovirus carrying Syn-3 (also known as Stx3) or small interfering RNA targeting Syn-3 in order to examine insulin secretion by radioimmunoassay. The localisation and distribution of insulin granules were examined by confocal and electron microscopy. Dynamic single-granule fusion events were assessed using total internal reflection fluorescence microscopy (TIRFM). RESULTS: Depletion of endogenous SYN-3 inhibited insulin release. TIRFM showed no change in the number or fusion competence of previously docked SGs but, instead, a marked reduction in the recruitment of newcomer SGs and their subsequent exocytotic fusion during biphasic GSIS. Conversely, overexpression of Syn-3 enhanced both phases of GSIS, owing to the increase in newcomer SGs and, remarkably, to increased SG-SG fusion, which was confirmed by electron microscopy. CONCLUSIONS/INTERPRETATION: In insulin secretion, SYN-3 plays a role in the mediation of newcomer SG exocytosis and SG-SG fusion that contributes to biphasic GSIS.

Role of mammalian homologue of Caenorhabditis elegans unc-13-1 (Munc13-1) in the recruitment of newcomer insulin granules in both first and second phases of glucose-stimulated insulin secretion in mouse islets.

AIMS/HYPOTHESIS: We have previously reported that the haplodeficient Munc13-1(+/-) mouse exhibits impaired biphasic glucose-stimulated insulin secretion (GSIS), causing glucose intolerance mimicking type 2 diabetes. Glucagon-like peptide-1 (GLP-1) can bypass these insulin-secretory defects in type 2 diabetes, but the mechanism of exocytotic events mediated by GLP-1 in rescuing insulin secretion is unclear. METHODS: The total internal reflection fluorescence microscopy (TIRFM) technique was used to examine single insulin granule fusion events in mouse islet beta cells. RESULTS: There was no difference in the density of docked granules in the resting state between Munc13-1(+/+) and Munc13-1(+/-) mouse islet beta cells. While exocytosis of previously docked granules in Munc13-1(+/-) beta cells is reduced during high-K(+) stimulation as expected, we now find a reduction in additional exocytosis events that account for the major portion of GSIS, namely two types of newcomer granules, one which has a short docking time (short-dock) and another undergoing no docking before exocytosis (no-dock). As mammalian homologue of Caenorhabditis elegans unc-13-1 (Munc13-1) is a phorbol ester substrate, phorbol ester could partially rescue biphasic GSIS in Munc13-1-deficient beta cells by enhancing recruitment of short-dock newcomer granules for exocytosis. The more effective rescue of biphasic GSIS by GLP-1 than by phorbol was due to increased recruitment of both short-dock and no-dock newcomer granules. CONCLUSIONS/INTERPRETATION: Phorbol ester and GLP-1 potentiation of biphasic GSIS are brought about by recruitment of distinct populations of newcomer granules for exocytosis, which may be mediated by Munc13-1 interaction with syntaxin-SNARE complexes other than that formed by syntaxin-1A.

The voltage-dependent potassium channel subunit Kv2.1 regulates insulin secretion from rodent and human islets independently of its electrical function.

AIMS/HYPOTHESIS: It is thought that the voltage-dependent potassium channel subunit Kv2.1 (Kv2.1) regulates insulin secretion by controlling beta cell electrical excitability. However, this role of Kv2.1 in human insulin secretion has been questioned. Interestingly, Kv2.1 can also regulate exocytosis through direct interaction of its C-terminus with the soluble NSF attachment receptor (SNARE) protein, syntaxin 1A. We hypothesised that this interaction mediates insulin secretion independently of Kv2.1 electrical function. METHODS: Wild-type Kv2.1 or mutants lacking electrical function and syntaxin 1A binding were studied in rodent and human beta cells, and in INS-1 cells. Small intracellular fragments of the channel were used to disrupt native Kv2.1-syntaxin 1A complexes. Single-cell exocytosis and ion channel currents were monitored by patch-clamp electrophysiology. Interaction between Kv2.1, syntaxin 1A and other SNARE proteins was probed by immunoprecipitation. Whole-islet Ca(2+)-responses were monitored by ratiometric Fura red fluorescence and insulin secretion was measured. RESULTS: Upregulation of Kv2.1 directly augmented beta cell exocytosis. This happened independently of channel electrical function, but was dependent on the Kv2.1 C-terminal syntaxin 1A-binding domain. Intracellular fragments of the Kv2.1 C-terminus disrupted native Kv2.1-syntaxin 1A interaction and impaired glucose-stimulated insulin secretion. This was not due to altered ion channel activity or impaired Ca(2+)-responses to glucose, but to reduced SNARE complex formation and Ca(2+)-dependent exocytosis. CONCLUSIONS/INTERPRETATION: Direct interaction between syntaxin 1A and the Kv2.1 C-terminus is required for efficient insulin exocytosis and glucose-stimulated insulin secretion. This demonstrates that native Kv2.1-syntaxin 1A interaction plays a key role in human insulin secretion, which is separate from the channel's electrical function.

Deploying insulin granule-granule fusion to rescue deficient insulin secretion in diabetes.

According to our current understanding of insulin exocytosis, insulin granules dock on the plasma membrane, undergo priming and then wait for calcium-triggered fusion. In this issue of Diabetologia, Hoppa et al (doi 10.1007/s00125-011-2400-5 ) report that cholinergic stimulation induces granule-granule, or multivesicular, fusion to effect more efficient insulin release. Other exocytotic modes of insulin secretion, particularly those induced by incretin stimulation, include orderly granule fusion with granules already fused with the plasma membrane, called sequential exocytosis, and recruitment of newcomer granules to fuse with plasma membrane with minimal time for docking and priming. The molecular machineries that mediate these distinct exocytotic modes of granule-granule fusion and newcomer granules remain undefined, but they could be therapeutically targeted to couple to cholinergic and incretin stimulation to rescue the deficient glucose-stimulated insulin secretion in diabetes.

Beta cell-specific Znt8 deletion in mice causes marked defects in insulin processing, crystallisation and secretion.

AIMS/HYPOTHESIS: Zinc is highly concentrated in pancreatic beta cells, is critical for normal insulin storage and may regulate glucagon secretion from alpha cells. Zinc transport family member 8 (ZnT8) is a zinc efflux transporter that is highly abundant in beta cells. Polymorphisms of ZnT8 (also known as SLC30A8) gene in man are associated with increased risk of type 2 diabetes. While global Znt8 knockout (Znt8KO) mice have been characterised, ZnT8 is also present in other islet cell types and extra-pancreatic tissues. Therefore, it is important to find ways of understanding the role of ZnT8 in beta and alpha cells without the difficulties caused by the confounding effects of ZnT8 in these other tissues. METHODS: We generated mice with beta cell-specific (Znt8BKO) and alpha cell-specific (Znt8AKO) knockout of Znt8, and performed in vivo and in vitro characterisation of the phenotypes to determine the functional and anatomical impact of ZnT8 in these cells. Thus we assessed zinc accumulation, insulin granule morphology, insulin biosynthesis and secretion, and glucose homeostasis. RESULTS: Znt8BKO mice are glucose-intolerant, have reduced beta cell zinc accumulation and atypical insulin granules. They also display reduced first-phase glucose-stimulated insulin secretion, reduced insulin processing enzyme transcripts and increased proinsulin levels. In contrast, Znt8AKO mice show no evident abnormalities in plasma glucagon and glucose homeostasis. CONCLUSIONS/INTERPRETATION: This is the first report of specific beta and alpha cell deletion of Znt8. Our data indicate that while, under the conditions studied, ZnT8 is absolutely essential for proper beta cell function, it is largely dispensable for alpha cell function.

Presence of functional hyperpolarisation-activated cyclic nucleotide-gated channels in clonal alpha cell lines and rat islet alpha cells.

AIMS/HYPOTHESIS: The hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels, discovered initially in cardiac and neuronal cells, mediate the inward pacemaker current (I (f) or I (h)). Recently, we have demonstrated the presence of HCN channels in pancreatic beta cells. Here, we aim to examine the presence and function of HCN channels in glucagon-secreting alpha cells. METHODS: RT-PCR and immunocytochemistry were used to examine the presence of HCN channels in alpha cells. Whole-cell patch-clamp, calcium imaging and glucagon secretion experiments were performed to explore the function of HCN channels in alpha cells. RESULTS: HCN transcripts and proteins were detected in alpha-TC6 cells and dispersed rat alpha cells. Patch-clamp recording showed hyperpolarisation-activated currents in alpha-TC6 cells, which could be blocked by HCN channel inhibitor ZD7288. Glucagon secretion RIA studies demonstrated that at both low and high glucose concentrations (2 and 20 mmol/l), ZD7288 significantly enhanced glucagon secretion in alpha-TC6 and IN-R1-G9 cell lines. Conversely, activation of HCN channels by lamotrigine significantly suppressed glucagon secretion at the low glucose concentration. Calcium imaging studies showed that blockade of HCN channels by ZD7288 significantly increased intracellular calcium in alpha-TC6 cells, while lamotrigine or the Na(+) channel blocker tetrodotoxin suppressed the effect of ZD7288 on intracellular calcium. Furthermore, we found the HCN channel inhibitors ZD7288 and cilobradine both significantly increased glucagon secretion from rat islets. CONCLUSIONS/INTERPRETATION: These results suggest a potential role for HCN channels in regulation of glucagon secretion via modulating Ca(2+) and Na(+) channel activities.

New insights into the molecular mechanisms of priming of insulin exocytosis.

Exocytosis of insulin vesicles in the pancreatic beta-cell involves a sequence of regulated events, whose normal function and efficient adaptation to increased demand are essential for the maintenance of glucose homeostasis. These exocytotic events comprise the trafficking and docking of vesicles to the plasma membrane, followed by fusion triggered by Ca(2+). Recent studies have unravelled post-docking steps mediated by novel factors, which, by their interactions with soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)- and SNARE-associated proteins, confer the docked vesicles fusion competence. These priming steps define the releasable pool of insulin vesicles, which accounts for the first phase of insulin secretion, and controls the rate at which vesicles are replenished for the second phase of secretion. This article aims to summarize what is currently known about the mechanisms that underlie the priming activity of these proteins, focusing on Munc13, a topic to which we have made some recent contributions. Abnormal glucose homeostasis in type 2 diabetes is because of the failure of islet beta-cells to augment insulin secretion sufficiently to compensate for reduced insulin sensitivity. A better understanding of the priming steps may help develop novel approaches to increase insulin secretory capacity and thereby prevent the progression to type 2 diabetes.

L-type Ca(2+) channel expression along feline smooth muscle oesophagus.

Muscle from the proximal smooth muscle (SM) oesophagus of the cat demonstrates contractions of greater amplitude and greater sensitivity to cholinergic stimulation than muscle from the distal SM oesophagus. In the light of the central role of calcium influx in SM contractility, we hypothesized that regional differences in oesophageal contractility may be associated with differential expression of L-type calcium channels (L(Ca)) along the SM oesophagus. L(Ca) expression was compared between proximal and distal regions of the circular SM oesophagus by Western blots. Patch clamp technique was utilized to study L(Ca) currents. Muscle strip studies assessed L(Ca) contribution to contractile activity. The protein expression of L(Ca) and L(Ca) current density was greater in the proximal than distal region. L(Ca) voltage and time-dependent activation and inactivation curves were similar in cells from both regions. Stimulation of muscle strips with acetylcholine (ACh) in the presence of tetrodotoxin resulted in contractions of greater amplitude in the proximal region. The L(Ca) agonist Bay K 8644 caused a greater increase in ACh-induced contraction amplitude in muscle strips from the proximal region. Therefore, regional myogenic differences in L(Ca) expression along the circular SM oesophageal body exist and may contribute to the nature of oesophageal contractions.

Distinct regional expression of SNARE proteins in the feline oesophagus.

Abstract Soluble N-ethylmaleimide-sensitive factors attachment protein receptors (SNAREs), initially found to mediate membrane fusion, have now been shown to also bind and regulate a number of membrane ion channels in neurones and neuroendocrine cells. We recently reported that the SNARE protein SNAP-25 regulates Ca(2+)- activated (K(Ca)) and delays rectifier K(+) channels (K(V)) in oesophageal smooth muscle cells. This raised the possibility that cognate and other SNARE proteins could also be present in the oesophageal smooth muscle cell to regulate these and other functions. Circular muscle tissue sections and single freshly isolated muscle cells from the oesophageal body circular and longitudinal layers, and from lower oesophageal sphincter clasp and sling regions were studied. The subcellular location of SNAP-23, SNAP-25, syntaxins 1 to 4, and vesicle-associated membrane protein (VAMP)-2 were explored using a laser scanning confocal imaging system. Feline oesophageal smooth muscle of all regions examined demonstrated the presence of SNAP-23, SNAP-25, syntaxins 1 to 4, and VAMP-2 on the plasma membrane. The intensity of these syntaxins and SNAP-25/-23 proteins varied between the different muscle groups of the oesophagus. In some regions, some SNARE proteins were also noted in the muscle cell cytoplasm. No differential expression was found for VAMP-2. The differential expression of SNAP-25 and its regulation of K(+) channels indicate the important role of SNAP-25 in regulating the distinct membrane excitability and contractility along the smooth muscle of the oesophagus. This is further contributed by its interactions with the cognate syntaxins, which are also differentially expressed in the muscle groups of the oesophageal body and lower oesophageal sphincter (LOS). These SNARE proteins probably have other functions in the smooth muscle cell, such as regulating vesicular transport processes.

Syntaxin-3 and syntaxin-1A inhibit L-type calcium channel activity, insulin biosynthesis and exocytosis in beta-cell lines.

AIMS/HYPOTHESIS: Syntaxin-1A (Syn-1A) is known to play a negative regulatory role in insulin secretion but the precise mechanisms for its action are not clear. Syn-2, -3 and -4 are also present in islet beta cells but their functions are not known. Here, we investigated the role of these syntaxins in the insulin secretory process. METHODS: We examined the following effects of Syn-1, -2, -3 and -4 expression in insulinoma beta-cell lines. Endogenous insulin secretion was measured by batch radioimmunoassay (RIA) and single cell patch clamp capacitance measurements. The L-type Ca(2+) channel activity was studied by patch clamp electrophysiology. Insulin gene transcription was examined by Northern blotting and measurement of insulin gene promoter activity by the co-expression of cyan fluorescent protein-labelled rat insulin promoter. RESULTS: Syn-1A or -3, but not Syn-2 or -4 overexpression, inhibited K(+)-induced insulin release as determined by RIA (49.7 +/- 5.5 % and 49.1 +/- 6.2 %, respectively) and electrophysiologic membrane capacitance measurements (68.0 +/- 21.0 % and 58.0 +/- 13.2 %, respectively). Overexpressed Syn-1A and -3, but not Syn-2, inhibited Ca(2+) channel current amplitude by 39.5 +/- 11.6 % and 52.7 +/- 6.0 %, respectively. Of note, overexpression of Syn-1A and -3 also reduced single cell (by confocal microscopy) and total cellular endogenous insulin content (by RIA) by 24.8 +/- 4.2 % and 31.8 +/- 3.9 %, respectively. This correlated to a reduction in endogenous insulin mRNA by 24.5 +/- 4.2 % and 25.7 +/- 4.2 %, respectively. This inhibition of insulin biosynthesis is mainly at the level of insulin gene transcription as demonstrated by an inhibition of insulin gene promoter activity (53.3 +/- 9.15 % and 39.0 +/- 6.8 %, respectively). CONCLUSIONS/INTERPRETATION: These results demonstrate that Syn-1A and -3 possess strong inhibitory actions on both insulin exocytosis and insulin biosynthesis whereas Syn-2 and -4 do not inhibit the insulin secretory process.

Supramaximal cholecystokinin displaces Munc18c from the pancreatic acinar basal surface, redirecting apical exocytosis to the basal membrane.

Exocytosis at the apical surface of pancreatic acinar cells occurs in the presence of physiological concentrations of cholecystokinin (CCK) but is inhibited at high concentrations. Here we show that Munc18c is localized predominantly to the basal membranes of acinar cells. Supramaximal but not submaximal CCK stimulation caused Munc18c to dissociate from the plasma membrane, and this displacement was blocked by protein kinase C (PKC) inhibitors. Conversely, whereas the CCK analog CCK-OPE alone failed to displace Munc18c from the membrane, this agent caused Munc18c displacement following minimal PKC activation. To determine the physiological significance of this displacement, we used the fluorescent dye FM1-43 to visualize individual exocytosis events in real-time from rat acinar cells in culture. We showed that supramaximal CCK inhibition of secretion resulted from impaired apical secretion and a redirection of exocytic events to restricted basal membrane sites. In contrast, CCK-OPE evoked apical exocytosis and could only induce basolateral exocytosis following activation of PKC. Infusion of supraphysiological concentrations of CCK in rats, a treatment that induced tissue changes reminiscent of mild acute pancreatitis, likewise resulted in rapid displacement of Munc18c from the basal membrane in vivo.

Ca(2+) influx and cAMP elevation overcame botulinum toxin A but not tetanus toxin inhibition of insulin exocytosis.

Previous reports showed that cleavage of vesicle-associated membrane protein-2 (VAMP-2) and synaptosomal-associated protein of 25 kDa (SNAP-25) by clostridial neurotoxins in permeabilized insulin-secreting beta-cells inhibited Ca(2+)-evoked insulin secretion. In these reports, the soluble N-ethylmaleimide-sensitive factor attachment protein target receptor proteins might have formed complexes, which preclude full accessibility of the putative sites for neurotoxin cleavage. In this work, VAMP-2 and SNAP-25 were effectively cleaved before they formed toxin-insensitive complexes by transient transfection of insulinoma HIT or INS-1 cells with tetanus toxin (TeTx) or botulinum neurotoxin A (BoNT/A), as shown by immunoblotting and immunofluorescence microscopy. This resulted in an inhibition of Ca(2+) (glucose or KCl)-evoked insulin release proportionate to the transfection efficiency (40-50%) and an accumulation of insulin granules. With the use of patch-clamp capacitance measurements, Ca(2+)-evoked exocytosis by membrane depolarization to -10 mV was abolished by TeTx (6% of control) but only moderately inhibited by BoNT/A (30% of control). Depolarization to 0 mV to maximize Ca(2+) influx partially overcame BoNT/A (50% of control) but not TeTx inhibition. Of note, cAMP activation potentiated Ca(2+)-evoked secretion by 129% in control cells but only 55% in BoNT/A-transfected cells and had negligible effects in TeTx-transfected cells. These results indicate that, whereas VAMP-2 is absolutely necessary for insulin exocytosis, the effects of SNAP-25 depletion on exocytosis, perhaps on insulin granule pool priming or mobilization steps, could be partially reversed by higher levels of Ca(2+) or cAMP potentiation.

Cholecystokinin-regulated exocytosis in rat pancreatic acinar cells is inhibited by a C-terminus truncated mutant of SNAP-23.

INTRODUCTION: Exocytosis is thought to result from the fusion of vesicle and plasma membranes caused by the formation of a trans-complex between proteins of the vesicle-associated membrane protein (VAMP) family on the vesicle with members of the syntaxin and synaptosomal-associated protein of 25 kd (SNAP-25) families on the plasma membrane. In the pancreatic acinar cell, synaptosomal-associated protein of 23 kd (SNAP-23) is the major SNAP-25 isoform expressed in pancreatic acinar cells, but its role in acinar cell exocytosis has not been determined. AIMS: To examine the role of SNAP-23 in regulated exocytosis in acinar cells, we subcloned into adenoviral vectors SNAP-23, SNAP-25, and dominant negative mutants in which the C-terminal domains corresponding to the botulinum neurotoxin A cleavage sites are deleted. METHODOLOGY AND RESULTS: High-efficiency infection of rat pancreatic acini in culture with these adenoviruses by subcellular fractionation showed that the overexpressed SNAP-23, SNAP-25, and their truncated mutant proteins were uniformly targeted to the zymogen granules and plasma membrane. To maximally stimulate apical exocytosis from these infected acini, we used the cholecystokinin-phenylethyl ester analog (CCK-OPE), which does not show inhibition of secretion from maximal levels at high doses. CCK-OPE-stimulated amylase release from adenovirus-cytomegalovirus (AdCMV)-SNAP-23 or AdCMV-SNAP-25-infected acini to the same extent as from acini infected with the empty vector. In contrast, CCK-OPE-evoked enzyme secretion from AdCMV-SNAP-23deltaC8- and AdCMV-SNAP-25(1-197)-infected acini were inhibited by 60% and 40%, respectively. The identical targeting of the mutant SNAP-23 and SNAP-25 proteins to the same membrane compartments as SNAP-23 suggests that the inhibition of secretion was a result of their competition against endogenous SNAP-23. This is supported by the fact that this inhibition by the mutant proteins was partially reversed or rescued when the AdCMV-SNAP-25AC8- or AdCMV-SNAP-25(1-197)-infected acini were co-infected with wild-type SNAP-23 or SNAP-25. CONCLUSION: From these results, we conclude that SNAP-23 plays a role in CCK-evoked regulated exocytosis in the acinar cells.

Members of the Kv1 and Kv2 voltage-dependent K(+) channel families regulate insulin secretion.

In pancreatic beta-cells, voltage-dependent K(+) (Kv) channels are potential mediators of repolarization, closure of Ca(2+) channels, and limitation of insulin secretion. The specific Kv channels expressed in beta-cells and their contribution to the delayed rectifier current and regulation of insulin secretion in these cells are unclear. High-level protein expression and mRNA transcripts for Kv1.4, 1.6, and 2.1 were detected in rat islets and insulinoma cells. Inhibition of these channels with tetraethylammonium decreased I(DR) by approximately 85% and enhanced glucose-stimulated insulin secretion by 2- to 4-fold. Adenovirus-mediated expression of a C-terminal truncated Kv2.1 subunit, specifically eliminating Kv2 family currents, reduced delayed rectifier currents in these cells by 60-70% and enhanced glucose-stimulated insulin secretion from rat islets by 60%. Expression of a C-terminal truncated Kv1.4 subunit, abolishing Kv1 channel family currents, reduced delayed rectifier currents by approximately 25% and enhanced glucose-stimulated insulin secretion from rat islets by 40%. This study establishes that Kv2 and 1 channel homologs mediate the majority of repolarizing delayed rectifier current in rat beta-cells and that antagonism of Kv2.1 may prove to be a novel glucose-dependent therapeutic treatment for type 2 diabetes.

A hypothesis: SNARE-ing the mechanisms of regulated exocytosis and pathologic membrane fusions in the pancreatic acinar cell.

The pancreatic acinar cell has been a classic model to study regulated exocytosis occurring at the apical plasma membrane. The acinar cell is also an excellent model with which to study pathologic membrane fusion events, including aberrant zymogen granule fusion with the lysosome and basolateral exocytosis, which are the earliest cellular events of acute pancreatitis. However, despite much effort, little is known about the precise mechanisms that mediate these physiologic and pathologic membrane fusion events until recently. Over the past 5 years, there has been a major advance in the fundamental understanding of vesicle fusion based on the SNARE hypothesis. A basic tenet of the SNARE hypothesis is that the minimal machinery for membrane fusion is a cognate set of v- and t-SNAREs on opposing membranes. A corollary to this hypothesis is that these SNARE proteins are prevented from spontaneous assembly by clamping proteins. Here, the recent developments in the identification of cognate v- and t-SNAREs and clamping proteins are reviewed, which are strategically located to mediate these physiologic exocytic and pathologic fusion events in the pancreatic acinar cell.

Mutations to the third cytoplasmic domain of the glucagon-like peptide 1 (GLP-1) receptor can functionally uncouple GLP-1-stimulated insulin secretion in HIT-T15 cells.

Glucagon-like peptide-1 (GLP-1) is an insulinotropic hormone with powerful antidiabetogenic effects that are thought to be mediated by adenylyl cyclase (AC). Recently, we generated two GLP-1 receptor mutant isoforms (IC3-1 and DM-1) that displayed efficient ligand binding and the ability to promote Ca2+ mobilization from intracellular stores but lacked the ability to couple to AC. In the present study, the wild-type rat GLP-1 receptor (WT-GLP-1 R) or the IC3-1 and DM-1 mutant forms were expressed for the first time in the insulin-producing HIT-T15 cells. Only cells expressing WT-GLP-1 R displayed dramatically elevated GLP-1-induced cAMP responses and elevated insulin secretion. The increase in GLP-1-stimulated secretion in cells expressing WT-GLP-1 R, however, was not accompanied by differences in glucose-stimulated insulin release. Prolonged exposure to GLP-1 (10 nM, 17 h), not only led to an increase in insulin secretion but also increased insulin mRNA levels, but only in cells expressing the WT-GLP-1 R and not the mutant isoforms. Electrophysiological analyses revealed that GLP-1 application enhanced L-type voltage-dependent Ca2+ channel (VDCC) currents > 2-fold and caused a positive shift in VDCC voltage-dependent inactivation in WT-GLP-1R cells only, not control or mutant (DM-1) cells. This action on the Ca2+ current was further enhanced by the VDCC agonist, BAYK8644, suggesting GLP-1 acts via a distinct mechanism dependent on cAMP. These studies demonstrate that the GLP-1 receptor efficiently couples to AC to stimulate insulin secretion and that receptors lacking critical residues in the proximal region of the third intracellular loop can effectively uncouple the receptor from cAMP production, VDCC activity, insulin secretion, and insulin biosynthesis.

Snare protein expression and adenoviral transfection of amphicrine AR42J.

The amphicrine AR42J acinar cell line is an excellent model to study both exocrine and neuroendocrine exocytotic mechanisms. As a first step toward this goal, we determined the specific isoforms of the v- and t-SNARE and Munc18 families expressed in these cells. In addition, we show that dexamethasone-induced differentiation toward the exocrine phenotype causes an upregulation of several of these proteins. AR42J is notoriously difficult to transfect, limiting its usefulness as a model. However, we have now overcome this obstacle by acheiving high efficiency expression of a beta-galactosidase reporter gene and truncated SNAP-25 gene using adenoviral infection techniques. The AR42J cells can now be used to pursue and elucidate the distinct functions of individual SNARE isoforms used in endocrine and exocrine secretion within a single cell line.

Beta-cell hypertrophy in fa/fa rats is associated with basal glucose hypersensitivity and reduced SNARE protein expression.

In normal isolated beta-cells, the response to glucose is heterogeneous and characterized by an increasing number of secretory cells as glucose concentration rises (Pipeleers DG, Kiekens R, Ling Z, Wilikens A, Schuit F: Physiologic relevance of heterogeneity in the pancreatic beta-cell population. Diabetologia 37 (Suppl. 2):S57-S64, 1994). We hypothesized that fasting hyperinsulinemia in obesity might be explained by altered beta-cell heterogeneity of signal transduction mechanisms, possibly involving exocytotic proteins. Insulin secretion from individual beta-cells sorted according to the size of the islet donor (<125 microm, >250 microm, and intermediate diameter) was measured by reverse hemolytic plaque assay. Beta-cells from fa/fa rats were hypertrophied 25-40%, independent of donor islet size. This was accompanied by an increased proportion of secretory cells (recruitment) at 5.5-11.0 mmol/l glucose, increased secretion per cell at 2.8 mmol/l glucose, and decreased insulin content after acute glucose exposure without an increase in secretion per cell. Decreased expression of exocytotic (soluble N-ethylmaleimide-sensitive fusion protein receptor [SNARE]) proteins, vesicle-associated membrane protein isoform 2 (VAMP-2), synaptosomal protein of 25 kDa (SNAP-25), and syntaxin-1 and -2 in fa/fa beta-cells may contribute to the failure to sustain excessive plaque size at higher glucose concentrations. Fasting hyperinsulinemia may be maintained by increased recruitment and an exaggerated secretory response in all fa-derived islet populations. Glucose regulates beta-cell responsiveness in the short term, and these effects may involve altered expression of SNARE proteins.

Truncated SNAP-25 (1-197), like botulinum neurotoxin A, can inhibit insulin secretion from HIT-T15 insulinoma cells.

We and others have previously shown that insulin-secreting cells of the pancreas express high levels of SNAP-25 (synaptosomal-associated protein of 25 kDa), a 206-amino acid t-SNARE (target soluble N-ethylmaleimide-sensitive factor attachment protein receptors) implicated in synaptic vesicle exocytosis. In the present study, we show that SNAP-25 is required for insulin secretion by transient transfection of Botulinum Neurotoxin A (BoNT/A) into insulin-secreting HIT-T15 cells. Transient expression of BoNT/A cleaved the endogenous as well as overexpressed SNAP-25 proteins and caused significant reductions in K+ and glucose-evoked secretion of insulin. To determine whether the inhibition of release was due to the depletion of functional SNAP-25 or the accumulation of proteolytic by-products, we transfected cells with SNAP-25 proteins from which the C-terminal nine amino acids had been deleted to mimic the effects of the toxin. This modified SNAP-25 (amino acids 1-197) remained bound to the plasma membrane but was as effective as the toxin at inhibiting insulin secretion. Microfluorimetry revealed that the inhibition of secretion was due neither to changes in basal cytosolic Ca2+ levels nor in Ca2+ influx evoked by K(+)-mediated plasma membrane depolarization. Electron microscopy revealed that cells transfected with either BoNT/A or truncated SNAP-25 contained significantly higher numbers of insulin granules, many of which clustered close to the plasma membrane. Together, these results demonstrate that functional SNAP-25 proteins are required for insulin secretion and suggest that the inhibitory action of BoNT/A toxin on insulin secretion is in part caused by the production of the plasma membrane-bound cleavage product, which itself interferes with insulin granule docking and fusion.