Herbert Y. Gaisano

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

Aims/hypothesisZinc 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.MethodsWe 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.ResultsZnt8BKO 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/interpretationThis 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.

Pancreatic GLP-1 receptor activation is sufficient for incretin control of glucose metabolism in mice

Glucagon-like peptide-1 (GLP-1) circulates at low levels and acts as an incretin hormone, potentiating glucose-dependent insulin secretion from islet β cells. GLP-1 also modulates gastric emptying and engages neural circuits in the portal region and CNS that contribute to GLP-1 receptor-dependent (GLP-1R-dependent) regulation of glucose homeostasis. To elucidate the importance of pancreatic GLP-1R signaling for glucose homeostasis, we generated transgenic mice that expressed the human GLP-1R in islets and pancreatic ductal cells (Pdx1-hGLP1R:Glp1r-/- mice). Transgene expression restored GLP-1R-dependent stimulation of cAMP and Akt phosphorylation in isolated islets, conferred GLP-1R-dependent stimulation of β cell proliferation, and was sufficient for restoration of GLP-1-stimulated insulin secretion in perifused islets. Systemic GLP-1R activation with the GLP-1R agonist exendin-4 had no effect on food intake, hindbrain c-fos expression, or gastric emptying but improved glucose tolerance and stimulated insulin secretion in Pdx1-hGLP1R:Glp1r-/- mice. i.c.v. GLP-1R blockade with the antagonist exendin(9-39) impaired glucose tolerance in WT mice but had no effect in Pdx1-hGLP1R:Glp1r-/- mice. Nevertheless, transgenic expression of the pancreatic GLP-1R was sufficient to normalize both oral and i.p. glucose tolerance in Glp1r-/- mice. These findings illustrate that low levels of endogenous GLP-1 secreted from gut endocrine cells are capable of augmenting glucoregulatory activity via pancreatic GLP-1Rs independent of communication with neural pathways.

Erythropoietin protects against diabetes through direct effects on pancreatic β cells

In mouse models of type 1 and type 2 diabetes, administration of human erythropoietin protects against disease by acting directly on pancreatic β cells.

Caspase-3-Dependent β-Cell Apoptosis in the Initiation of Autoimmune Diabetes Mellitus

ABSTRACT 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.

Inhibition of Cholesterol Biosynthesis Impairs Insulin Secretion and Voltage-Gated Calcium Channel Function in Pancreatic β-Cells

Insulin secretion from pancreatic beta-cells is mediated by the opening of voltage-gated Ca2+ channels (CaV) and exocytosis of insulin dense core vesicles facilitated by the secretory soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein machinery. We previously observed that beta-cell exocytosis is sensitive to the acute removal of membrane cholesterol. However, less is known about the chronic changes in endogenous cholesterol and its biosynthesis in regulating beta-cell stimulus-secretion coupling. We examined the effects of inhibiting endogenous beta-cell cholesterol biosynthesis by using the squalene epoxidase inhibitor, NB598. The expression of squalene epoxidase in primary and clonal beta-cells was confirmed by RT-PCR. Cholesterol reduction of 36-52% was observed in MIN6 cells, mouse and human pancreatic islets after a 48-h incubation with 10 mum NB598. A similar reduction in cholesterol was observed in the subcellular compartments of MIN6 cells. We found NB598 significantly inhibited both basal and glucose-stimulated insulin secretion from mouse pancreatic islets. CaV channels were markedly inhibited by NB598. Rapid photolytic release of intracellular caged Ca2+ and simultaneous measurements of the changes in membrane capacitance revealed that NB598 also inhibited exocytosis independently from CaV channels. These effects were reversed by cholesterol repletion. Our results indicate that endogenous cholesterol in pancreatic beta-cells plays a critical role in regulating insulin secretion. Moreover, chronic inhibition of cholesterol biosynthesis regulates the functional activity of CaV channels and insulin secretory granule mobilization and membrane fusion. Dysregulation of cellular cholesterol may cause impairment of beta-cell function, a possible pathogenesis leading to the development of type 2 diabetes.

SNAREing voltage-gated K+ and ATP-sensitive K+ channels : Tuning β-cell excitability with syntaxin-1A and other exocytotic proteins

The three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, syntaxin, SNAP25 (synaptosome-associated protein of 25 kDa), and synaptobrevin, constitute the minimal machinery for exocytosis in secretory cells such as neurons and neuroendocrine cells by forming a series of complexes prior to and during vesicle fusion. It was subsequently found that these SNARE proteins not only participate in vesicle fusion, but also tether with voltage-dependent Ca2+ channels to form an excitosome that precisely regulates calcium entry at the site of exocytosis. In pancreatic islet β-cells, ATP-sensitive K+ (KATP) channel closure by high ATP concentration leads to membrane depolarization, voltage-dependent Ca2+ channel opening, and insulin secretion, whereas subsequent opening of voltage-gated K+ (Kv) channels repolarizes the cell to terminate exocytosis. We have obtained evidence that syntaxin-1A physically interacts with Kv2.1 (the predominant Kv in β-cells) and the sulfonylurea recep...

New Insights Into the Mechanisms of Pancreatitis

from zymogen granules from the apical pole to the basolateral regions of the acinar cell. 3 How this reduction in secretion might relate to the pathogenesis of acute pancreatitis has not been explored fully, but 2 mechanisms have been suggested. First, the retention of activated enzymes, particularly proteases, in the acinar cell might be required to cause disease. Thus, if the barriers to acinar cell secretion can be overcome, the damaging effects of activated enzymes might be averted. The observation that cyclic adenosine monophosphate agonists enhance secretion from the acinar cell under pancreatitis conditions and that these cells are protected from injury is consistent with this hypothesis. 4 In this context, the beneficial effects of secretin in pancreatitis shown in preliminary studies might be due to its stimulation of secretion of intracellular activated enzymes from the acinar cell. Second, the redirection of pancreatic enzyme secretion from the apical to the basolateral domain of the acinar cell may deliver enzymes and enzymes precursors to the interstitial space. 3 Neither mechanism has been fully explored.

Munc13-1 Deficiency Reduces Insulin Secretion and Causes Abnormal Glucose Tolerance

Munc13-1 is a diacylglycerol (DAG) receptor that is essential for synaptic vesicle priming. We recently showed that Munc13-1 is expressed in rodent and human islet β-cells and that its levels are reduced in islets of type 2 diabetic humans and rat models, suggesting that Munc13-1 deficiency contributes to the abnormal insulin secretion in diabetes. To unequivocally demonstrate the role of Munc13-1 in insulin secretion, we studied heterozygous Munc13-1 knockout mice (+/−), which exhibited elevated glucose levels during intraperitoneal glucose tolerance tests with corresponding lower serum insulin levels. Munc13-1+/− mice exhibited normal insulin tolerance, indicating that a primary islet β-cell secretory defect is the major cause of their hyperglycemia. Consistently, glucose-stimulated insulin secretion was reduced 50% in isolated Munc13-1+/− islets and was only partially rescued by phorbol ester potentiation. The corresponding alterations were minor in mice expressing one allele of a Munc13-1 mutant variant, which does not bind DAG (H567K/+). Capacitance measurements of Munc13-1+/− and Munc13-1H567k/+ islet β-cells revealed defects in granule priming, including the initial size and refilling of the releasable pools, which become accentuated by phorbol ester potentiation. We conclude that Munc13-1 plays an important role in glucose-stimulated insulin secretion and that Munc13-1 deficiency in the pancreatic islets as occurs in diabetes can reduce insulin secretion sufficient to cause abnormal glucose homeostasis.

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.

SNAP-23 is located in the basolateral plasma membrane of rat pancreatic acinar cells

The SNARE hypothesis proposes that specificity of exocytosis is regulated by the appropriate interactions between the vesicle (v‐) SNARE and the target membrane (t‐) SNAREs. We show here that pancreatic acinar cells express the SNAP‐25 t‐SNARE homolog SNAP‐23, and find that this t‐SNARE is most highly concentrated on the basolateral plasma membrane while being expressed below detectable levels in endocrine islets within the same tissue. This is the first localization of SNAP‐23 within a polarized tissue and suggests that this t‐SNAREs may interact with syntaxin‐4 to mediate basolateral secretion.