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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