Impaired counterregulation during hypoglycemia in type 1 diabetes (T1D) is partly attributable to inadequate glucagon secretion. Intra-islet somatostatin (SST) suppression of hypoglycemia-stimulated α-cell glucagon release plays an important role. We hypothesized that hypoglycemia can be prevented in autoimmune T1D by SST receptor type 2 (SSTR2) antagonism of α-cells, which relieve SSTR2 inhibition, thereby increasing glucagon secretion. Diabetic biobreeding diabetes-prone (BBDP) rats mimic insulin-dependent human autoimmune T1D, whereas nondiabetic BBDP rats mimic prediabetes. Diabetic and nondiabetic rats underwent a 3-h infusion of vehicle compared with SSTR2 antagonist (SSTR2a) during insulin-induced hypoglycemia clamped at 3 ± 0.5 mmol/L. Diabetic rats treated with SSTR2a needed little or no glucose infusion compared with untreated rats. We attribute this effect to SSTR2a restoration of the attenuated glucagon response. Direct effects of SSTR2a on α-cells was assessed by resecting the pancreas, which was cut into fine slices and subjected to perifusion to monitor glucagon release. SSTR2a treatment enhanced low-glucose–stimulated glucagon and corticosterone secretion to normal levels in diabetic rats. SSTR2a had similar effects in vivo in nondiabetic rats and promoted glucagon secretion from nondiabetic rat and human pancreas slices. We conclude that SST contributes to impaired glucagon responsiveness to hypoglycemia in autoimmune T1D. SSTR2a treatment can fully restore hypoglycemia-stimulated glucagon release sufficient to attain normoglycemia in both diabetic and prediabetic stages.
AIM: To assess the effect of nitric oxide (NO) on the large conductance potassium channel (BKCa) in isolated circular (CM) and sling (SM) muscle cells and muscle strips from the cat lower esophageal sphincter (LES) to determine its regulation of resting tone and relaxation.
METHODS: Freshly enzymatically-digested and isolated circular smooth muscle cells were prepared from each LES region. To study outward K+ currents, the perforated patch clamp technique was employed. To assess LES resting tone and relaxation, muscle strips were mounted in perfused organ baths.
RESULTS: (1) Electrophysiological recordings from isolated cells: (a) CM was more depolarized than SM (-39.7 ± 0.8mV vs -48.1 ± 1.6 mV, P < 0.001), and maximal outward current was similar (27.1 ± 1.5 pA/pF vs 25.7 ± 2.0 pA/pF, P > 0.05); (b) The NO donor sodium nitroprusside (SNP) increased outward currents only in CM (25.9 ± 1.9 to 46.7 ± 4.2 pA/pF, P < 0.001) but not SM (23.2 ± 3.1 to 27.0 ± 3.4 pA/pF, P > 0.05); (c) SNP added in the presence of the BKCa antagonist iberiotoxin (IbTX) produced no increase in the outward current in CM (17.0 ± 2.8 vs 13.7 ± 2.2, P > 0.05); and (d) L-NNA caused a small insignificant inhibition of outward K+ currents in both muscles; and (2) Muscle strip studies: (a) Blockade of the nerves with tetrodotoxin (TTX), or BKCa with IbTX had no significant effect on resting tone of either muscle; and (b) SNP reduced tone in both muscles, and was unaffected by the presence of TTX or IbTX.
CONCLUSION: Exogenous NO activates BKCa only in CM of the cat. However, as opposed to other species, exogenous NO-induced relaxation is predominantly by a non-BKCa mechanism, and endogenous NO has minimal effect on resting tone.
Circular smooth muscle; Feline; K+ channel; Lower esophageal sphincter; Nitric oxide; Sling; Tone
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.
AIM: To examine the molecular mechanism of exocytosis in the Brunner’s gland acinar cell.
METHODS: We used a submucosal preparation of guinea pig duodenal Brunner’s gland acini to visualize the dilation of the ductal lumen in response to cholinergic stimulus. We correlated this to electron microscopy to determine the extent of exocytosis of the mucin-filled vesicles. We then examined the behavior of SNARE and interacting Munc18 proteins by confocal microscopy.
RESULTS: One and 6 μmol/L carbachol evoked a dose-dependent dilation of Brunner’s gland acini lumen, which correlated to the massive exocytosis of mucin. Munc18c and its cognate SNARE proteins Syntaxin-4 and SNAP-23 were localized to the apical plasma membrane, and upon cholinergic stimulation, Munc18c was displaced into the cytosol leaving Syntaxin-4 and SNAP-23 intact.
CONCLUSION: Physiologic cholinergic stimulation induces Munc18c displacement from the Brunner’s gland acinar apical plasma membrane, which enables apical membrane Syntaxin-4 and SNAP-23 to form a SNARE complex with mucin-filled vesicle SNARE proteins to affect exocytosis.
Apical exocytosis; Brunner’s gland acini; Munc18c; Syntaxin-4; Carbachol
Early-stage type 1 diabetes (T1D) exhibits hyperglucagonemia by undefined cellular mechanisms. Here we characterized α-cell voltage-gated ion channels in a streptozotocin (STZ)-induced diabetes model that lead to increased glucagon secretion mimicking T1D. GYY mice expressing enhanced yellow fluorescence protein in α cells were used to identify α cells within pancreas slices. Mice treated with low-dose STZ exhibited hyperglucagonemia, hyperglycemia, and glucose intolerance, with 71% reduction of β-cell mass. Although α-cell mass of STZ-treated mice remained unchanged, total pancreatic glucagon content was elevated, coinciding with increase in size of glucagon granules. Pancreas tissue slices enabled in situ examination of α-cell electrophysiology. α cells of STZ-treated mice exhibited the following: 1) increased exocytosis (serial depolarization-induced capacitance), 2) enhanced voltage-gated Na+ current density, 3) reduced voltage-gated K+ current density, and 4) increased action potential (AP) amplitude and firing frequency. Hyperglucagonemia in STZ-induced diabetes is thus likely due to increased glucagon content arising from enlarged glucagon granules and increased AP firing frequency and amplitude coinciding with enhanced Na+ and reduced K+ currents. These alterations may prime α cells in STZ-treated mice for more glucagon release per cell in response to low glucose stimulation. Thus, our study provides the first insight that STZ treatment sensitizes release mechanisms of α cells.
Normal release of glucagon from pancreatic islet α-cells promotes glucose mobilization, which counteracts the hypoglycemic actions of insulin, thereby ensuring glucose homeostasis. In treatment of diabetes aimed at rigorously reducing hyperglycemia to avoid chronic complications, the resulting hypoglycemia triggering glucagon release from α-cells is frequently impaired, with ensuing hypoglycemic complications. This review integrates the physiology of glucagon secretion regulating glucose homeostasis in vivo to single α-cell signaling, and how both become perturbed in diabetes. α-cells within the social milieu of the islet micro-organ are regulated not only by intrinsic signaling events but also by paracrine regulation, particularly by adjacent insulin-secreting β-cells and somatostatin-secreting δ-cells. We discuss the intrinsic α-cell signaling events, including glucose sensing and ion channel regulation leading to glucagon secretion. We then discuss the complex crosstalk between the islet cells and the breakdown of this crosstalk in diabetes contributing to the dysregulated glucagon secretion. Whereas, there are many secretory products released by β- and δ-cells that become deficient or excess in diabetes, we discuss the major ones, including the better known insulin and lesser known somatostatin, which act as putative paracrine on/off switches that very finely regulate α-cell secretory responses in health and diabetes. Of note in several type 1 diabetes (T1D) rodent models, blockade of excess somatostatin actions on α-cell could normalize glucagon secretion sufficient to attain normoglycemia in response to hypoglycemic assaults. There has been slow progress in fully elucidating the pathophysiology of the α-cell in diabetes because of the small number of α-cells within an islet and the islet mass becomes severely reduced and inflamed in diabetes. These limitations are just now being surmounted by new approaches.
islet α-cell; glucagon secretion; diabetes; hypoglycemia; somatostatin
AIM: To investigate chronic stress as a susceptibility factor for developing pancreatitis, as well as tumor necrosis factor-α (TNF-α) as a putative sensitizer.
METHODS: Rat pancreatic acini were used to analyze the influence of TNF-α on submaximal (50 pmol/L) cholecystokinin (CCK) stimulation. Chronic restraint (4 h every day for 21 d) was used to evaluate the effects of submaximal (0.2 μg/kg per hour) cerulein stimulation on chronically stressed rats.
RESULTS: In vitro exposure of pancreatic acini to TNF-α disorganized the actin cytoskeleton. This was further increased by TNF-α/CCK treatment, which additionally reduced amylase secretion, and increased trypsin and nuclear factor-κB activities in a protein-kinase-C δ and ε-dependent manner. TNF-α/CCK also enhanced caspases’ activity and lactate dehydrogenase release, induced ATP loss, and augmented the ADP/ATP ratio. In vivo, rats under chronic restraint exhibited elevated serum and pancreatic TNF-α levels. Serum, pancreatic, and lung inflammatory parameters, as well as caspases’activity in pancreatic and lung tissue, were substantially enhanced in stressed/cerulein-treated rats, which also experienced tissues’ ATP loss and greater ADP/ATP ratios. Histological examination revealed that stressed/cerulein-treated animals developed abundant pancreatic and lung edema, hemorrhage and leukocyte infiltrate, and pancreatic necrosis. Pancreatitis severity was greatly decreased by treating animals with an anti-TNF-α-antibody, which diminished all inflammatory parameters, histopathological scores, and apoptotic/necrotic markers in stressed/cerulein-treated rats.
CONCLUSION: In rats, chronic stress increases susceptibility for developing pancreatitis, which involves TNF-α sensitization of pancreatic acinar cells to undergo injury by physiological cerulein stimulation.
Pancreatitis; Stress; Tumor necrosis factor-α
In rodents and humans, alcohol exposure has been shown to predispose the pancreas to cholinergic or viral induction of pancreatitis. We previously developed a rodent model in which exposure to an ethanol (EtOH) diet, followed by carbachol (Cch) stimulation, redirects exocytosis from the apical to the basolateral plasma membrane of acinar cells, resulting in ectopic zymogen enzyme activation and pancreatitis. This redirection of exocytosis involves a soluble NSF attachment receptor (SNARE) complex consisting of syntaxin-4 and synapse-associated protein of 23 kDa (SNAP-23). Here, we investigated the role of the zymogen granule (ZG) SNARE vesicle-associated membrane protein 8 (VAMP8) in mediating basolateral exocytosis. In WT mice, in vitro EtOH exposure or EtOH diet reduced Cch-stimulated amylase release by redirecting apical exocytosis to the basolateral membrane, leading to alcoholic pancreatitis. Further reduction of zymogen secretion, caused by blockade of both apical and basolateral exocytosis and resulting in a more mild induction of alcoholic pancreatitis, was observed in Vamp8–/– mice in response to these treatments. In addition, although ZGs accumulated in Vamp8–/– acinar cells, ZG-ZG fusions were reduced compared with those in WT acinar cells, as visualized by electron microscopy. This reduction in ZG fusion may account for reduced efficiency of apical exocytosis in Vamp8–/– acini. These findings indicate that VAMP8 is the ZG-SNARE that mediates basolateral exocytosis in alcoholic pancreatitis and that VAMP8 is critical for ZG-ZG homotypic fusion.
In acute pancreatitis, initiating cellular events causing acinar cell injury includes co-localization of zymogens with lysosomal hydrolases, leading to premature enzyme activation and pathological exocytosis of zymogens into the interstitial space. This is followed by processes that accentuate cell injury; triggering acute inflammatory mediators, intensifying oxidative stress, compromising the microcirculation and activating a neurogenic feedback. Such localized events then progress to a systemic inflammatory response leading to multiorgan dysfunction syndrome with resulting high morbidity and mortality. The present review discusses some of the most recent insights into each of these cellular processes postulated to cause or propagate the process of acute pancreatitis, and also the role of alcohol and genetics.
Alcoholic pancreatitis; Cholecystokinin; Exocytosis; Pancreatitis
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.
We set out to test the hypothesis that insulin secretion from beta cells is targeted towards the vasculature.
The spatial location of granule fusion was identified by live-cell two-photon imaging of mouse pancreatic beta cells within intact islets, using sulforhodamine B labelling. Three-dimensional (3D) immunofluorescence of pancreatic slices was used to identify the location of proteins associated with neuronal synapses.
We demonstrated an asymmetric, non-random, distribution of sites of insulin granule fusion in response to glucose and focal targeting of insulin granule secretion to the beta cell membrane facing the vasculature. 3D immunofluorescence of islets showed that structural proteins, such as liprin, piccolo and Rab2-interacting molecule, normally associated with neuronal presynaptic targeting, were present in beta cells and enriched at the vascular face. In contrast, we found that syntaxin 1A and synaptosomal-associated protein 25 kDa (SNAP25) were relatively evenly distributed across the beta cells.
Our results show that beta cells in situ, within intact islets, are polarised and target insulin secretion. This evidence for an ‘endocrine synapse’ has wide implications for our understanding of stimulus–secretion coupling in healthy islets and in disease.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-014-3252-6) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
Beta cell; Exocytosis; Insulin; Islets; Secretion; Synapse; Vasculature
Glucagon is important for maintaining euglycemia during fasting/starvation, and abnormal glucagon secretion is associated with type 1 and type 2 diabetes; however, the mechanisms of hypoglycemia-induced glucagon secretion are poorly understood. We previously demonstrated that global deletion of mitochondrial uncoupling protein 2 (UCP2−/−) in mice impaired glucagon secretion from isolated islets. Therefore, UCP2 may contribute to the regulation of hypoglycemia-induced glucagon secretion, which is supported by our current finding that UCP2 expression is increased in nutrient-deprived murine and human islets. Further to this, we created α-cell–specific UCP2 knockout (UCP2AKO) mice, which we used to demonstrate that blood glucose recovery in response to hypoglycemia is impaired owing to attenuated glucagon secretion. UCP2-deleted α-cells have higher levels of intracellular reactive oxygen species (ROS) due to enhanced mitochondrial coupling, which translated into defective stimulus/secretion coupling. The effects of UCP2 deletion were mimicked by the UCP2 inhibitor genipin on both murine and human islets and also by application of exogenous ROS, confirming that changes in oxidative status and electrical activity directly reduce glucagon secretion. Therefore, α-cell UCP2 deletion perturbs the fasting/hypoglycemic glucagon response and shows that UCP2 is necessary for normal α-cell glucose sensing and the maintenance of euglycemia.
We used the db/db mouse to determine the nature of the secretory defect in intact islets.
Glucose tolerance was compared in db/db and wild-type (WT) mice. Isolated islets were used: to measure insulin secretion and calcium in a two-photon assay of single-insulin-granule fusion; and for immunofluorescence of soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAREs).
The 13–18-week-old db/db mice showed a diabetic phenotype. Isolated db/db islets showed a 77% reduction in insulin secretion induced by 15 mmol/l glucose and reductions in the amplitude and rise-time of the calcium response to glucose. Ionomycin-induced insulin secretion in WT but not db/db islets. Immunofluorescence showed an increase in the levels of the SNAREs synaptosomal-associated protein 25 (SNAP25) and vesicle-associated membrane protein 2 (VAMP2) in db/db islets, but reduced syntaxin-1A. Therefore, db/db islets have both a compromised calcium response to glucose and a compromised secretory response to calcium. Two-photon microscopy of isolated islets determined the number and distribution of insulin granule exocytic events. Compared with WT, db/db islets showed far fewer exocytic events (an 83% decline at 15 mmol/l glucose). This decline was due to a 73% loss of responding cells and, in the remaining responsive cells, a 50% loss of exocytic responses per cell. An assay measuring granule re-acidification showed evidence for more recaptured granules in db/db islets compared with WT.
We showed that db/db islets had a reduced calcium response to glucose and a reduction in syntaxin-1A. Within the db/db islets, changes were manifest as both a reduction in responding cells and a reduction in fusing insulin granules per cell.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-014-3226-8) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
db/db; Insulin; Islet secretion
In dispersed single beta cells the response of each cell to glucose is heterogeneous. In contrast, within an islet, cell-to-cell communication leads to glucose inducing a more homogeneous response. For example, increases in NAD(P)H and calcium are relatively uniform across the cells of the islet. These data suggest that secretion of insulin from single beta cells within an islet should also be relatively homogeneous. The aim of this study was to test this hypothesis by determining the glucose dependence of single-cell insulin responses within an islet.
Two-photon microscopy was used to detect the glucose-induced fusion of single insulin granules within beta cells in intact mouse islets.
First, we validated our assay and showed that the measures of insulin secretion from whole islets could be explained by the time course and numbers of granule fusion events observed. Subsequent analysis of the patterns of granule fusion showed that cell recruitment is a significant factor, accounting for a fourfold increase from 3 to 20 mmol/l glucose. However, the major factor is the regulation of the numbers of granule fusion events within each cell, which increase ninefold over the range of 3 to 20 mmol/l glucose. Further analysis showed that two types of granule fusion event occur: ‘full fusion’ and ‘kiss and run’. We show that the relative frequency of each type of fusion is independent of glucose concentration and is therefore not a factor in the control of insulin secretion.
Within an islet, glucose exerts its main effect through increasing the numbers of insulin granule fusion events within a cell.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-013-3019-5) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
Insulin; Islet; 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 Ca2+ 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. Sec5 is present in human and rodent islet β 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 β cells are more sensitive than predocked granules to Sec5 regulation.
Focal adhesion kinase (FAK) acts as an adaptor at the focal contacts serving as a junction between the extracellular matrix and actin cytoskeleton. Actin dynamics is known as a determinant step in insulin secretion. Additionally, FAK has been shown to regulate insulin signaling. To investigate the essential physiological role of FAK in pancreatic β-cells in vivo, we generated a transgenic mouse model using rat insulin promoter (RIP)–driven Cre-loxP recombination system to specifically delete FAK in pancreatic β-cells. These RIPcre+fakfl/fl mice exhibited glucose intolerance without changes in insulin sensitivity. Reduced β-cell viability and proliferation resulting in decreased β-cell mass was observed in these mice, which was associated with attenuated insulin/Akt (also known as protein kinase B) and extracellular signal–related kinase 1/2 signaling and increased caspase 3 activation. FAK-deficient β-cells exhibited impaired insulin secretion with normal glucose sensing and preserved Ca2+ influx in response to glucose, but a reduced number of docked insulin granules and insulin exocytosis were found, which was associated with a decrease in focal proteins, paxillin and talin, and an impairment in actin depolymerization. This study is the first to show in vivo that FAK is critical for pancreatic β-cell viability and function through regulation in insulin signaling, actin dynamics, and granule trafficking.
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. Insulin secretion, intracellular Ca2+, and β-cell exocytosis were monitored after manipulation of the SUMOylation machinery. Granule localization was imaged by total internal reflection fluorescence and electron microscopy; immunoprecipitation and Western blotting were used to examine the soluble NSF attachment receptor (SNARE) complex formation and SUMO1 interaction with synaptotagmin VII.
SUMO1 impairs glucose-stimulated insulin secretion by blunting the β-cell exocytotic response to Ca2+. The effect of SUMO1 to impair insulin secretion and β-cell exocytosis is rapid and does not require altered gene expression or insulin content, is downstream of granule docking at the plasma membrane, and is dependent on SUMO-conjugation because the deSUMOylating enzyme, sentrin/SUMO-specific protease (SENP)-1, rescues exocytosis. SUMO1 coimmunoprecipitates with the Ca2+ sensor synaptotagmin VII, and this is transiently lost upon glucose stimulation. SENP1 overexpression also disrupts the association of SUMO1 with synaptotagmin VII and mimics the effect of glucose to enhance exocytosis. Conversely, SENP1 knockdown impairs exocytosis at stimulatory glucose levels and blunts glucose-dependent insulin secretion from mouse and human islets.
SUMOylation acutely regulates insulin secretion by the direct and reversible inhibition of β-cell exocytosis in response to intracellular Ca2+ elevation. The SUMO protease, SENP1, is required for glucose-dependent insulin secretion.
In mouse models of type 1 and type 2 diabetes, administration of human erythropoietin protects against disease by acting directly on pancreatic β cells.
A common feature among all forms of diabetes mellitus is a functional β-cell mass insufficient to maintain euglycemia; therefore, the promotion of β-cell growth and survival is a fundamental goal for diabetes prevention and treatment. Evidence has suggested that erythropoietin (EPO) exerts cytoprotective effects on nonerythroid cells. However, the influence of EPO on pancreatic β cells and diabetes has not been evaluated to date. In this study, we report that recombinant human EPO treatment can protect against diabetes development in streptozotocin-induced and db/db mouse models of type 1 and type 2 diabetes, respectively. EPO exerts antiapoptotic, proliferative, antiinflammatory, and angiogenic effects within the islets. Using β-cell–specific EPO receptor and JAK2 knockout mice, we show that these effects of EPO result from direct biological effects on β cells and that JAK2 is an essential intracellular mediator. Thus, promotion of EPO signaling in β cells may be a novel therapeutic strategy for diabetes prevention and treatment.
Thioredoxin-interacting protein (TxNIP) is an endogenous inhibitor of thioredoxin, a ubiquitous thiol oxidoreductase, that regulates cellular redox status. Diabetic mice exhibit increased expression of TxNIP in pancreatic islets, and recent studies suggest that TxNIP is a proapoptotic factor in β-cells that may contribute to the development of diabetes. Here, we examined the role of TxNIP deficiency in vivo in the development of insulin-deficient diabetes and whether it impacted on pancreatic β-cell mass and/or insulin secretion. TxNIP-deficient (Hcb-19/TxNIP−/−) mice had lower baseline glycemia, higher circulating insulin concentrations, and higher total pancreatic insulin content and β-cell mass than control mice (C3H). Hcb-19/TxNIP−/− did not develop hyperglycemia when injected with standard multiple low doses of streptozotocin (STZ), in contrast to C3H controls. Surprisingly, although β-cell mass remained higher in Hcb-19/TxNIP−/− mice compared with C3H after STZ exposure, the relative decrease induced by STZ was as great or even greater in the TxNIP-deficient animals. Consistently, cultured pancreatic INS-1 cells transfected with small-interfering RNA against TxNIP were more sensitive to cell death induced by direct exposure to STZ or to the combination of inflammatory cytokines interleukin-1β, interferon-γ, and tumor necrosis factor-α. Furthermore, when corrected for insulin content, isolated pancreatic islets from TxNIP−/− mice exhibited reduced glucose-induced insulin secretion. These data indicate that TxNIP functions as a regulator of β-cell mass and influences insulin secretion. In conclusion, the relative resistance of TxNIP-deficient mice to STZ-induced diabetes appears to be because of an increase in β-cell mass. However, TxNIP deficiency is associated with sensitization to STZ- and cytokine-induced β-cell death, indicating complex regulatory roles of TxNIP under different physiological and pathological conditions.
PMID: 19223654 CAMSID: cams1604
thioredoxin interacting protein; pancreatic β-cell; apoptosis; insulin secretion
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate pacemaker activity in some cardiac cells and neurons. In the present study, we have identified the presence of HCN channels in pancreatic β-cells. We then examined the functional characterization of these channels in β-cells via modulating HCN channel activity genetically and pharmacologically. Voltage-clamp experiments showed that over-expression of HCN2 in rat β-cells significantly increased HCN current (Ih), whereas expression of dominant-negative HCN2 (HCN2-AYA) completely suppressed endogenous Ih. Compared to control β-cells, over-expression of Ih increased insulin secretion at 2.8 mmol/l glucose. However, suppression of Ih did not affect insulin secretion at both 2.8 mmol/l and 11.1 mmol/l glucose. Current-clamp measurements revealed that HCN2 over-expression significantly reduced β-cell membrane input resistance (Rin), and resulted in a less hyperpolarizing membrane response to the currents injected into the cell. Conversely, dominant negative HCN2-AYA expression led to a substantial increase of Rin, which was associated with a more hyperpolarizing membrane response to the currents injected. Remarkably, under low extracellular potassium conditions (2.5mmol/l K+), suppression of Ih resulted in increased membrane hyperpolarization and decreased insulin secretion. We conclude that Ih in β-cells possess the potential to modulate β-cell membrane potential and insulin secretion under hypokalemic conditions.
pancreatic β-cell; HCN channels; insulin secretion; membrane potential; input resistance
Munc18-1 binds to syntaxin-1A via two distinct sites referred to as the “closed” conformation and N terminus binding. The latter has been shown to stimulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated exocytosis, whereas the former is believed to be inhibitory or dispensable. To precisely define the contributions of each binding mode, we have engineered Munc18-1/-2 double knockdown neurosecretory cells and show that not only syntaxin-1A and -1B but also syntaxin-2 and -3 are significantly reduced as a result of Munc18-1 and -2 knockdown. Syntaxin-1 was mislocalized and the regulated secretion was abolished. We next examined the abilities of Munc18-1 mutants to rescue the defective phenotypes. Mutation (K46E/E59K) of Munc18-1 that selectively prevents binding to closed syntaxin-1 was unable to restore syntaxin-1 expression, localization, or secretion. In contrast, mutations (F115E/E132A) of Munc18-1 that selectively impair binding to the syntaxin-1 N terminus could still rescue the defective phenotypes. Our results indicate that Munc18-1 and -2 act in concert to support the expression of a broad range of syntaxins and to deliver syntaxin-1 to the plasma membrane. Our studies also indicate that the binding to the closed conformation of syntaxin is essential for Munc18-1 stimulatory action, whereas the binding to syntaxin N terminus plays a more limited role in neurosecretory cells.
Although Munc18-1 was originally identified as a syntaxin1–interacting protein, the physiological significance of this interaction remains unclear. In fact, recent studies of Munc18-1 mutants have suggested that Munc18-1 plays a critical role for docking of secretory vesicles, independent of syntaxin1 regulation. Here we investigated the role of Munc18-1 in syntaxin1 localization by generating stable neuroendocrine cell lines in which Munc18-1 was strongly down-regulated. In these cells, the secretion capability, as well as the docking of dense-core vesicles, was significantly reduced. More importantly, not only was the expression level of syntaxin1 reduced, but the localization of syntaxin1 at the plasma membrane was also severely perturbed. The mislocalized syntaxin1 resided primarily in the perinuclear region of the cells, in which it was highly colocalized with Secretogranin II, a marker protein for dense-core vesicles. In contrast, the expression level and the plasma membrane localization of SNAP-25 were not affected. Furthermore, the syntaxin1 localization and the secretion capability were restored upon transfection-mediated reintroduction of Munc18-1. Our results indicate that endogenous Munc18-1 plays a critical role for the plasma membrane localization of syntaxin1 in neuroendocrine cells and therefore necessitates the interpretation of Munc18-1 mutant phenotypes to be in terms of mislocalized syntaxin1.
We identified in a yeast two-hybrid screen the EF-hand Ca2+-binding protein Cab45 as an interaction partner of Munc18b. Although the full-length Cab45 resides in Golgi lumen, we characterize a cytosolic splice variant, Cab45b, expressed in pancreatic acini. Cab45b is shown to bind 45Ca2+, and, of its three EF-hand motifs, EF-hand 2 is demonstrated to be crucial for the ion binding. Cab45b is shown to interact with Munc18b in an in vitro assay, and this interaction is enhanced in the presence of Ca2+. In this assay, Cab45b also binds the Munc18a isoform in a Ca2+-dependent manner. The endogenous Cab45b in rat acini coimmunoprecipitates with Munc18b, syntaxin 2, and syntaxin 3, soluble N-ethylmaleimide-sensitive factor attachment protein receptors with key roles in the Ca2+-triggered zymogen secretion. Furthermore, we show that Munc18b bound to syntaxin 3 recruits Cab45b onto the plasma membrane. Importantly, antibodies against Cab45b are shown to inhibit in a specific and dose-dependent manner the Ca2+-induced amylase release from streptolysin-O–permeabilized acini. The present study identifies Cab45b as a novel protein factor involved in the exocytosis of zymogens by pancreatic acini.
β-Cell apoptosis is a key event contributing to the pathogenesis of type 1 diabetes mellitus. In addition to apoptosis being the main mechanism by which β cells are destroyed, β-cell apoptosis has been implicated in the initiation of type 1 diabetes mellitus through antigen cross-presentation mechanisms that lead to β-cell-specific T-cell activation. Caspase-3 is the major effector caspase involved in apoptotic pathways. Despite evidence supporting the importance of β-cell apoptosis in the pathogenesis of type 1 diabetes, the specific role of caspase-3 in this process is unknown. Here, we show that Caspase-3 knockout (Casp3−/−) mice were protected from developing diabetes in a multiple-low-dose streptozotocin autoimmune diabetes model. Lymphocyte infiltration of the pancreatic islets was completely absent in Casp3−/− mice. To determine the role of caspase-3-dependent apoptosis in disease initiation, a defined antigen-T-cell receptor transgenic system, RIP-GP/P14 double-transgenic mice with Casp3 null mutation, was examined. β-cell antigen-specific T-cell activation and proliferation were observed only in the pancreatic draining lymph node of RIP-GP/P14/Casp3+/− mice, but not in mice lacking caspase-3. Together, our findings demonstrate that caspase-3-mediated β-cell apoptosis is a requisite step for T-cell priming, a key initiating event in type 1 diabetes.