BACKGROUND AND PURPOSEIntroducing the calcineurin inhibitors cyclosporin (CsA) and tacrolimus (Tac) has improved the outcome of organ transplants, but complications such as new onset diabetes mellitus after transplantation (NODAT) decrease survival rates. EXPERIMENTAL APPROACHWe sought, in a beta-cell culture model, to elucidate the pathogenic mechanisms behind NODAT and the relative contribution of the calcineurin inhibitors. INS-1E cells were incubated at basal and stimulatory glucose concentrations, while exposed to pharmacologically relevant doses of CsA, Tac and vehicle for 6 or 24 h. RESULTSTac inhibited basal (P < 0.05), but not glucose-stimulated insulin secretion (GSIS) after 6 h of exposure. After 24 h, both agents inhibited basal and GSIS (P < 0.05). Calcineurin phosphatase activity was decreased by both drugs during all conditions. Apoptosis was only seen with CsA treatment, which also induced a slight suppression of calcineurin and insulin mRNA, as well as increased levels of the sterol receptor element binding protein (SREBP)-1c, a transcription factor thought to suppress genes essential for beta-cell function and induce insulin resistance. Expression levels of nuclear factor of activated T-cells (NFAT)-c1, -c2, -c3 and -c4 were not decreased notably by either drug. CONCLUSIONS AND IMPLICATIONSTac had acute inhibitory effects on basal insulin secretion, but prolonged exposure (24 h) to Tac or CsA revealed similar suppression of insulin secretion. These prolonged effects were mirrored by a total inhibition of calcineurin activity in beta-cells. CsA showed greater inhibition of beta-cell survival and transcriptional markers, essential for beta-cell function. AbbreviationsBax, Bcl-2 associated X protein; Bcl-2, B-cell leukaemia/lymphoma 2; CaN, calcineurin phosphatase; GSIS, glucose-stimulated insulin secretion; IRS-2, insulin receptor substrate 2; NFATc, nuclear factor of activated T-cells cytoplasmic; NODAT, new onset diabetes mellitus after transplantation; PDX1, pancreatic and duodenal homebox factor 1; SREBP-1c, sterol receptor element binding protein 1c

BackgroundIon transporters of the Slc30A- (ZnT-) family regulate zinc fluxes into sub-cellular compartments. β-cells depend on zinc for both insulin crystallization and regulation of cell mass.Methodology/Principal FindingsThis study examined: the effect of glucose and zinc chelation on ZnT gene and protein levels and apoptosis in β-cells and pancreatic islets, the effects of ZnT-3 knock-down on insulin secretion in a β-cell line and ZnT-3 knock-out on glucose metabolism in mice during streptozotocin-induced β-cell stress. In INS-1E cells 2 mM glucose down-regulated ZnT-3 and up-regulated ZnT-5 expression relative to 5 mM. 16 mM glucose increased ZnT-3 and decreased ZnT-8 expression. Zinc chelation by DEDTC lowered INS-1E insulin content and insulin expression. Furthermore, zinc depletion increased ZnT-3- and decreased ZnT-8 gene expression whereas the amount of ZnT-3 protein in the cells was decreased. Zinc depletion and high glucose induced apoptosis and necrosis in INS-1E cells. The most responsive zinc transporter, ZnT-3, was investigated further; by immunohistochemistry and western blotting ZnT-3 was demonstrated in INS-1E cells. 44% knock-down of ZnT-3 by siRNA transfection in INS-1E cells decreased insulin expression and secretion. Streptozotocin-treated mice had higher glucose levels after ZnT-3 knock-out, particularly in overt diabetic animals.Conclusion/SignificanceZinc transporting proteins in β-cells respond to variations in glucose and zinc levels. ZnT-3, which is pivotal in the development of cellular changes as also seen in type 2 diabetes (e.g. amyloidosis in Alzheimer's disease) but not previously described in β-cells, is present in this cell type, up-regulated by glucose in a concentration dependent manner and up-regulated by zinc depletion which by contrast decreased ZnT-3 protein levels. Knock-down of the ZnT-3 gene lowers insulin secretion in vitro and affects in vivo glucose metabolism after streptozotocin treatment.

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E xocytosis is delicately regulated via dynamic protein-protein interactions between different protein components localized to the plasma membrane, the secretory vesicle membrane, and the cytoplasm. According to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) hypothesis (1,2), the vesicular-SNARE vesicleassociated membrane protein (also called synaptobrevin) interacts with the cognate target-SNAREs syntaxin and synaptosomal-associated protein of 25 kDa (SNAP-25) to form a core complex (also called SNARE complex) (1). The assembly of SNARE proteins between two opposing membranes and the formation of a core complex have been shown to be the key events that initiate membrane fusion and predict the specificity of vesicle fusion (1,2). That the compartmental specificity of cellular membrane fusion is encoded in SNARE proteins is further provided by the observation that these proteins have distinct localization in a cell (3). However, almost any combination of several members of vesicular-and target-SNARE proteins can form a SDS-resistant protein complex (4,5), suggesting that the interactions between SNARE proteins cannot provide all information for vesicle targeting. Additional specificity may be provided by other molecules that interact with SNARE proteins. An example of such a protein is the well-conserved syntaxin-binding protein Sec1/mammalian homolog of the Caenorhabditis elegans unc-18 gene (Munc-18). There are several Munc-18 isoforms in mammals, which are believed to support different vesicular trafficking events (rev. in 6). Munc-18-1 holds syntaxin in a closed conformation, thereby preventing the binding of SNAP-25 and vesicle-associated membrane protein to syntaxin (7). Moreover, each Munc-18 protein interacts more or less exclusively with one or two syntaxin isoforms, thereby providing further vesicle-targeting specificity (8 -13).The existence of another syntaxin-binding protein, designated tomosyn (tomo ϭ friend in Japanese, syn ϭ syntaxin), has been reported (14). Besides the original tomosyn protein, which has been named m-tomosyn, two further splice variants of tomosyn, designated big (b) and small (s) tomosyn, have been identified (15). The m-and s-tomosyn variants are mainly expressed in the brain, whereas b-tomosyn is found ubiquitously (15). More recently, two distinct genes that drive the expression of seven tomosyn isoforms in the mammalian brain have been described (16). Tomosyn is capable of dissociating Munc-18 from syntaxin 1 and thereby forming a novel complex with syntaxin 1, and synaptotagmin (14). The COOH-terminal domain of tomosyn spans a SNARE motif that allows tomosyn to form a stable complex with syntaxin 1A and SNAP-25 (17,18). Endogenous expression or overexpression of tomosyn has been shown to cause a reduction of Ca 2ϩ -dependent exocytosis (14,19 -23). The structural basis for the inhibitory role of tomosyn in exocytosis has recently been presented (24).In this study, we have investigated isoform expression and cellular localization of tomo...

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Cytosolic free Ca 2؉ plays an important role in the molecular mechanisms leading to regulated insulin secretion by the pancreatic ␤ cell. A number of Ca 2؉ -binding proteins have been implicated in this process. Here, we define the role of the Ca 2؉ -binding protein neuronal Ca 2؉ sensor-1 (NCS-1) in insulin secretion. In pancreatic ␤ cells, NCS-1 increases exocytosis by promoting the priming of secretory granules for release and increasing the number of granules residing in the readily releasable pool. The effect of NCS-1 on exocytosis is mediated through an increase in phosphatidylinositol (PI) 4-kinase ␤ activity and the generation of phosphoinositides, specifically PI 4-phosphate and PI 4,5-bisphosphate. In turn, PI 4,5-bisphosphate controls exocytosis through the Ca 2؉ -dependent activator protein for secretion present in ␤ cells. Our results provide evidence for an essential role of phosphoinositide synthesis in the regulation of glucose-induced insulin secretion by the pancreatic ␤ cell. We also demonstrate that NCS-1 and its downstream target, PI 4-kinase ␤, are critical players in this process by virtue of their capacity to regulate the release competence of the secretory granules.insulin ͉ phosphoinositides ͉ islet ͉ secretion ͉ Ca 2ϩ -dependent activator protein for secretion N euronal Ca 2ϩ sensor-1 (NCS-1) belongs to the family of EF-hand Ca 2ϩ -binding proteins and is mainly expressed in neuronal and neuroendocrine cells, where it enhances neurotransmission and Ca 2ϩ -dependent exocytosis (1-5). NCS-1 interacts with and regulates the activity of phosphatidylinositol 4-kinase ␤ (PI4K␤) (6-9). The members of the PI4K family catalyze the first step in the synthesis of PI 4,5-bisphosphate [PI(4,5)P 2 ], which has recently emerged as an important regulator of Ca 2ϩ -dependent secretion (10-12). Both a PI transfer protein (13) and a PI4P 5-kinase (14) are required for regulated exocytosis of dense core granules. In addition, the presence of synaptic vesicle and dense core granule-associated PI4K activity is essential for exocytosis (15,16). These data imply that generation of PI(4,5)P 2 is an important step in the event of secretion.In pancreatic ␤ cells, glucose dose-dependently increases ATP levels and decreases ADP levels (16). The resulting rise in the ATP at the expense of ADP is an important regulator of the two major signaling pathways involved in glucose-induced insulin secretion. The first of these pathways uses ATP-sensitive K ϩ channels to couple glucose metabolism with electrical activity, Ca 2ϩ influx, and initiation of insulin secretion. The second pathway is exerted at the level of granule priming and regulates the ␤ cell secretory capacity by modulation of the granules' release competence (17). The ␤ cell contains Ϸ10,000 insulincontaining secretory granules (18). Interestingly, as many as Ϸ5% of the granules are docked below the membrane. The readily releasable pool (RRP), defined by functional measurements, represents a subset (50-100 granules) of the docked pool (19). Granules belongin...

Background: -cells are extremely rich in zinc and zinc homeostasis is regulated by zinc transporter proteins. -cells are sensitive to cytokines, interleukin-1 (IL-1 ) has been associated with -cell dysfunction and -death in both type 1 and type 2 diabetes. This study explores the regulation of zinc transporters following cytokine exposure.

BackgroundZinc is essential for the activities of pancreatic β-cells, especially insulin storage and secretion. Insulin secretion leads to co-release of zinc which contributes to the paracrine communication in the pancreatic islets. Zinc-transporting proteins (zinc-regulated transporter, iron-regulated transporter-like proteins [ZIPs] and zinc transporters [ZnTs]) and metal-buffering proteins (metallothioneins, MTs) tightly regulate intracellular zinc homeostasis. The present study investigated how modulation of cellular zinc availability affects β-cell function using INS-1E cells.ResultsUsing INS-1E cells, we found that zinc supplementation and zinc chelation had significant effects on insulin content and insulin secretion. Supplemental zinc within the physiological concentration range induced insulin secretion. Insulin content was reduced by zinc chelation with N,N,N’,N-tektrakis(2-pyridylmethyl)-ethylenediamine. The changes in intracellular insulin content following exposure to various concentrations of zinc were reflected by changes in the expression patterns of MT-1A, ZnT-8, ZnT-5, and ZnT-3. Furthermore, high zinc concentrations induced cell necrosis while zinc chelation induced apoptosis. Finally, cell proliferation was sensitive to changes in zinc the concentration.ConclusionThese results indicate that the β-cell-like function and survival of INS-1E cells are dependent on the surrounding zinc concentrations. Our results suggest that regulation of zinc homeostasis could represent a pharmacological target.