Sarco(endo)plasmic reticulum ATPase is a molecular partner of Wolfram syndrome 1 protein, which negatively regulates its expression.

Wolfram syndrome is an autosomal recessive disorder characterized by neurodegeneration and diabetes mellitus. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER)-resident transmembrane protein that is involved in the regulation of the unfolded protein response (UPR), intracellular ion homeostasis, cyclic adenosine monophosphate production and regulation of insulin biosynthesis and secretion. In this study, single cell Ca(2+) imaging with fura-2 and direct measurements of free cytosolic ATP concentration ([ATP]CYT) with adenovirally expressed luciferase confirmed a reduced and delayed rise in cytosolic free Ca(2+) concentration ([Ca(2+)]CYT), and additionally, diminished [ATP]CYT rises in response to elevated glucose concentrations in WFS1-depleted MIN6 cells. We also observed that sarco(endo)plasmic reticulum ATPase (SERCA) expression was elevated in several WFS1-depleted cell models and primary islets. We demonstrated a novel interaction between WFS1 and SERCA by co-immunoprecipitation in Cos7 cells and with endogenous proteins in human neuroblastoma cells. This interaction was reduced when cells were treated with the ER stress inducer dithiothreitol. Treatment of WFS1-depleted neuroblastoma cells with the proteasome inhibitor MG132 resulted in reduced accumulation of SERCA levels compared with wild-type cells. Together these results reveal a role for WFS1 in the negative regulation of SERCA and provide further insights into the function of WFS1 in calcium homeostasis.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic ß Cells.

Pancreatic ß cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca(2+) action potentials due to the activation of voltage-dependent Ca(2+) channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca(2+) release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic ß cells. NAADP-regulated Ca(2+) release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca(2+) from the endolysosomal system, resulting in localized Ca(2+) signals. We show here that NAADP-mediated Ca(2+) release from endolysosomal Ca(2+) stores activates inward membrane currents and depolarizes the ß cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca(2+) release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca(2+) signals, and insulin secretion. Our findings implicate NAADP-evoked Ca(2+) release from acidic Ca(2+) storage organelles in stimulus-secretion coupling in ß cells.

Hypoxia lowers SLC30A8/ZnT8 expression and free cytosolic Zn2+ in pancreatic beta cells.

AIMS/HYPOTHESIS: Hypoxic damage complicates islet isolation for transplantation and may contribute to beta cell failure in type 2 diabetes. Polymorphisms in the SLC30A8 gene, encoding the secretory granule zinc transporter 8 (ZnT8), influence type 2 diabetes risk, conceivably by modulating cytosolic Zn(2+) levels. We have therefore explored the role of ZnT8 and cytosolic Zn(2+) in the response to hypoxia of pancreatic islet cells. METHODS: Human, mouse or rat islets were isolated and exposed to varying O2 tensions. Cytosolic free zinc was measured using the adenovirally expressed recombinant targeted zinc probe eCALWY4. Gene expression was measured using quantitative (q)RT-PCR, western (immuno-) blotting or immunocytochemistry. Beta cells were identified by insulin immunoreactivity. RESULTS: Deprivation of O2 (1% vs 5% or 21%) for 24 h lowered free cytosolic Zn(2+) concentrations by ~40% (p < 0.05) and ~30% (p < 0.05) in mouse and human islet cells, respectively. Hypoxia similarly decreased SLC30A8 mRNA expression in islets, and immunoreactivity in beta cells. Implicating lowered ZnT8 levels in the hypoxia-induced fall in cytosolic Zn(2+), genetic ablation of Slc30a8 from mouse islets lowered cytosolic Zn(2+) by ~40% (p < 0.05) and decreased the induction of metallothionein (Mt1, Mt2) genes. Cell survival in the face of hypoxia was enhanced in small islets of older (>12 weeks) Slc30a8 null mice vs controls, but not younger animals. CONCLUSIONS/INTERPRETATION: The response of pancreatic beta cells to hypoxia is characterised by decreased SLC30A8 expression and lowered cytosolic Zn(2+) concentrations. The dependence on ZnT8 of hypoxia-induced changes in cell survival may contribute to the actions of SLC30A8 variants on diabetes risk in humans.

Imaging dynamic insulin release using a fluorescent zinc indicator for monitoring induced exocytotic release (ZIMIR).

Current methods of monitoring insulin secretion lack the required spatial and temporal resolution to adequately map the dynamics of exocytosis of native insulin granules in intact cell populations in three dimensions. Exploiting the fact that insulin granules contain a high level of Zn(2+), and that Zn(2+) is coreleased with insulin during secretion, we have developed a fluorescent, cell surface-targeted zinc indicator for monitoring induced exocytotic release (ZIMIR). ZIMIR displayed a robust fluorescence enhancement on Zn(2+) chelation and bound Zn(2+) with high selectivity against Ca(2+) and Mg(2+). When added to cultured ß cells or intact pancreatic islets at low micromolar concentrations, ZIMIR labeled cells rapidly, noninvasively, and stably, and it reliably reported changes in Zn(2+) concentration near the sites of granule fusion with high sensitivity that correlated well with membrane capacitance measurement. Fluorescence imaging of ZIMIR-labeled ß cells followed the dynamics of exocytotic activity at subcellular resolution, even when using simple epifluorescence microscopy, and located the chief sites of insulin release to intercellular junctions. Moreover, ZIMIR imaging of intact rat islets revealed that Zn(2+)/insulin release occurred largely in small groups of adjacent ß cells, with each forming a "secretory unit." Concurrent imaging of ZIMIR and Fura-2 showed that the amplitude of cytosolic Ca(2+) elevation did not necessarily correlate with insulin secretion activity, suggesting that events downstream of Ca(2+) signaling underlie the cell-cell heterogeneity in insulin release. In addition to studying stimulation-secretion coupling in cells with Zn(2+)-containing granules, ZIMIR may find applications in ß-cell engineering and screening for molecules regulating insulin secretion on high-throughput platforms.

Glucose regulates free cytosolic Zn²âº concentration, Slc39 (ZiP), and metallothionein gene expression in primary pancreatic islet ß-cells.

Zn²âº is an important cofactor for insulin biosynthesis and storage in pancreatic ß-cells. Correspondingly, polymorphisms in the SLC30A8 gene, encoding the secretory granule Zn²âº transporter ZnT8, are associated with type 2 diabetes risk. Using a genetically engineered (FRET)-based sensor (eCALWY-4), we show here that elevated glucose time-dependently increases free cytosolic Zn²âº ([Zn²âº](cyt)) in mouse pancreatic ß-cells. These changes become highly significant (853 ± 96 pm versus 452 ± 42 pm, p < 0.001) after 24 h and are associated with increased expression of the Zn²âº importer family members Slc39a6, Slc39a7, and Slc39a8, and decreased expression of metallothionein 1 and 2. Arguing that altered expression of the above genes is not due to altered [Zn²âº](cyt), elevation of extracellular (and intracellular) [Zn²âº] failed to mimic the effects of high glucose. By contrast, increases in intracellular cAMP prompted by 3-isobutyl-1-methylxanthine and forskolin partially mimicked the effects of glucose on metallothionein, although not ZiP, gene expression. Modulation of intracellular Ca²âº and insulin secretion with pharmacological agents (tolbutamide and diazoxide) suggested a possible role for changes in these parameters in the regulation of Slc39a6 and Slc39a7 but not Slc39a8, nor metallothionein expression. In summary, 1) glucose induces increases in [Zn²âº](cyt), which are then likely to facilitate the processing and/or the storage of insulin and its cocrystallization with Zn²âº, and 2) these increases are associated with elevated expression of zinc importers. Conversely, a chronic increase in [Zn²âº](cyt) following sustained hyperglycemia may contribute to ß-cell dysfunction and death in some forms of diabetes.

Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis.

We developed genetically encoded fluorescence resonance energy transfer (FRET)-based sensors that display a large ratiometric change upon Zn(2+) binding, have affinities that span the pico- to nanomolar range and can readily be targeted to subcellular organelles. Using this sensor toolbox we found that cytosolic Zn(2+) was buffered at 0.4 nM in pancreatic beta cells, and we found substantially higher Zn(2+) concentrations in insulin-containing secretory vesicles.

Ca2+ signalling: a new route to NAADP.

NAADP (nicotinic acid-adenine dinucleotide phosphate) is a derivative of NADP (nicotinamide-adenine dinucleotide phosphate), which differs by the presence of a nicotinic acid instead of a nicotinamide moiety. This small structural difference makes NAADP one of the most powerful second messengers known, able to mobilize intracellular Ca2+ in a wide range of cellular models, ranging from invertebrates to mammals. Despite this, our understanding of NAADP homoeostasis, metabolism and physiological action is still limited. A new report by Vasudevan and colleagues in this issue of the Biochemical Journal provides important new data by describing a new synthetic activity in sperm cells which may turn out to represent the most physiologically relevant route to this second messenger.

Intracellular zinc in insulin secretion and action: a determinant of diabetes risk?

Zinc is an important micronutrient, essential in the diet to avoid a variety of conditions associated with malnutrition such as diarrhoea and alopecia. Lowered circulating levels of zinc are also found in diabetes mellitus, a condition which affects one in twelve of the adult population and whose treatments consume approximately 10 % of healthcare budgets. Zn2+ ions are essential for a huge range of cellular functions and, in the specialised pancreatic ß-cell, for the storage of insulin within the secretory granule. Correspondingly, genetic variants in the SLC30A8 gene, which encodes the diabetes-associated granule-resident Zn2+ transporter ZnT8, are associated with an altered risk of type 2 diabetes. Here, we focus on (i) recent advances in measuring free zinc concentrations dynamically in subcellular compartments, and (ii) studies dissecting the role of intracellular zinc in the control of glucose homeostasis in vitro and in vivo. We discuss the effects on insulin secretion and action of deleting or over-expressing Slc30a8 highly selectively in the pancreatic ß-cell, and the role of zinc in insulin signalling. While modulated by genetic variability, healthy levels of dietary zinc, and hence normal cellular zinc homeostasis, are likely to play an important role in the proper release and action of insulin to maintain glucose homeostasis and lower diabetes risk.

Molecular Genetic Regulation of Slc30a8/ZnT8 Reveals a Positive Association With Glucose Tolerance.

Zinc transporter 8 (ZnT8), encoded by SLC30A8, is chiefly expressed within pancreatic islet cells, where it mediates zinc (Zn(2+)) uptake into secretory granules. Although a common nonsynonymous polymorphism (R325W), which lowers activity, is associated with increased type 2 diabetes (T2D) risk, rare inactivating mutations in SLC30A8 have been reported to protect against T2D. Here, we generate and characterize new mouse models to explore the impact on glucose homeostasis of graded changes in ZnT8 activity in the ß-cell. Firstly, Slc30a8 was deleted highly selectively in these cells using the novel deleter strain, Ins1Cre. The resultant Ins1CreZnT8KO mice displayed significant (P < .05) impairments in glucose tolerance at 10 weeks of age vs littermate controls, and glucose-induced increases in circulating insulin were inhibited in vivo. Although insulin release from Ins1CreZnT8KO islets was normal, Zn(2+) release was severely impaired. Conversely, transgenic ZnT8Tg mice, overexpressing the transporter inducibly in the adult ß-cell using an insulin promoter-dependent Tet-On system, showed significant (P < .01) improvements in glucose tolerance compared with control animals. Glucose-induced insulin secretion from ZnT8Tg islets was severely impaired, whereas Zn(2+) release was significantly enhanced. Our findings demonstrate that glucose homeostasis in the mouse improves as ß-cell ZnT8 activity increases, and remarkably, these changes track Zn(2+) rather than insulin release in vitro. Activation of ZnT8 in ß-cells might therefore provide the basis of a novel approach to treating T2D.

Mitochondrial and ER-targeted eCALWY probes reveal high levels of free Zn2+.

Zinc (Zn2+) ions are increasingly recognized as playing an important role in cellular physiology. Whereas the free Zn2+ concentration in the cytosol has been established to be 0.1-1 nM, the free Zn2+ concentration in subcellular organelles is not well-established. Here, we extend the eCALWY family of genetically encoded Förster Resonance Energy Transfer (FRET) Zn2+ probes to permit measurements in the endo(sarco)plasmic reticulum (ER) and mitochondrial matrix. Deployed in a variety of mammalian cell types, these probes reveal resting mitochondrial free [Zn2+] values of ∼300 pM, somewhat lower than in the cytosol but 3 orders of magnitude higher than recently reported using an alternative FRET-based sensor. By contrast, free ER [Zn2+] was found to be ≥5 nM, which is >5000-fold higher than recently reported but consistent with the proposed role of the ER as a mobilizable Zn2+ store. Treatment of ß-cells or cardiomyocytes with sarco(endo)plasmic reticulum Ca2+-ATPase inhibitors, mobilization of ER Ca2+ after purinergic stimulation with ATP, or manipulation of ER redox, exerted no detectable effects on [Zn2+]ER. These findings question the previously proposed role of Ca2+ in Zn2+ mobilization from the ER and suggest that high ER Zn2+ levels may be an important aspect of cellular homeostasis.

Animal models of GWAS-identified type 2 diabetes genes.

More than 65 loci, encoding up to 500 different genes, have been implicated by genome-wide association studies (GWAS) as conferring an increased risk of developing type 2 diabetes (T2D). Whilst mouse models have in the past been central to understanding the mechanisms through which more penetrant risk genes for T2D, for example, those responsible for neonatal or maturity-onset diabetes of the young, only a few of those identified by GWAS, notably TCF7L2 and ZnT8/SLC30A8, have to date been examined in mouse models. We discuss here the animal models available for the latter genes and provide perspectives for future, higher throughput approaches towards efficiently mining the information provided by human genetics.

Lipotoxicity disrupts incretin-regulated human ß cell connectivity.

Pancreatic ß cell dysfunction is pathognomonic of type 2 diabetes mellitus (T2DM) and is driven by environmental and genetic factors. ß cell responses to glucose and to incretins such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are altered in the disease state. While rodent ß cells act as a coordinated syncytium to drive insulin release, this property is unexplored in human islets. In situ imaging approaches were therefore used to monitor in real time the islet dynamics underlying hormone release. We found that GLP-1 and GIP recruit a highly coordinated subnetwork of ß cells that are targeted by lipotoxicity to suppress insulin secretion. Donor BMI was negatively correlated with subpopulation responses to GLP-1, suggesting that this action of incretin contributes to functional ß cell mass in vivo. Conversely, exposure of mice to a high-fat diet unveiled a role for incretin in maintaining coordinated islet activity, supporting the existence of species-specific strategies to maintain normoglycemia. These findings demonstrate that ß cell connectedness is an inherent property of human islets that is likely to influence incretin-potentiated insulin secretion and may be perturbed by diabetogenic insults to disrupt glucose homeostasis in humans.

The mitochondrial Ca2+ uniporter MCU is essential for glucose-induced ATP increases in pancreatic ß-cells.

Glucose induces insulin release from pancreatic ß-cells by stimulating ATP synthesis, membrane depolarisation and Ca(2+) influx. As well as activating ATP-consuming processes, cytosolic Ca(2+) increases may also potentiate mitochondrial ATP synthesis. Until recently, the ability to study the role of mitochondrial Ca(2+) transport in glucose-stimulated insulin secretion has been hindered by the absence of suitable approaches either to suppress Ca(2+) uptake into these organelles, or to examine the impact on ß-cell excitability. Here, we have combined patch-clamp electrophysiology with simultaneous real-time imaging of compartmentalised changes in Ca(2+) and ATP/ADP ratio in single primary mouse ß-cells, using recombinant targeted (Pericam or Perceval, respectively) as well as entrapped intracellular (Fura-Red), probes. Through shRNA-mediated silencing we show that the recently-identified mitochondrial Ca(2+) uniporter, MCU, is required for depolarisation-induced mitochondrial Ca(2+) increases, and for a sustained increase in cytosolic ATP/ADP ratio. By contrast, silencing of the mitochondrial Na(+)-Ca(2+) exchanger NCLX affected the kinetics of glucose-induced changes in, but not steady state values of, cytosolic ATP/ADP. Exposure to gluco-lipotoxic conditions delayed both mitochondrial Ca(2+) uptake and cytosolic ATP/ADP ratio increases without affecting the expression of either gene. Mitochondrial Ca(2+) accumulation, mediated by MCU and modulated by NCLX, is thus required for normal glucose sensing by pancreatic ß-cells, and becomes defective in conditions mimicking the diabetic milieu.

Insulin storage and glucose homeostasis in mice null for the granule zinc transporter ZnT8 and studies of the type 2 diabetes-associated variants.

OBJECTIVE: Zinc ions are essential for the formation of hexameric insulin and hormone crystallization. A nonsynonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. We describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele. RESEARCH DESIGN AND METHODS: Slc30a8 null mice were generated and backcrossed at least twice onto a C57BL/6J background. Glucose and insulin tolerance were measured by intraperitoneal injection or euglycemic clamp, respectively. Insulin secretion, electrophysiology, imaging, and the generation of adenoviruses encoding the low- (W325) or elevated- (R325) risk ZnT8 alleles were undertaken using standard protocols. RESULTS: ZnT8(-/-) mice displayed age-, sex-, and diet-dependent abnormalities in glucose tolerance, insulin secretion, and body weight. Islets isolated from null mice had reduced granule zinc content and showed age-dependent changes in granule morphology, with markedly fewer dense cores but more rod-like crystals. Glucose-stimulated insulin secretion, granule fusion, and insulin crystal dissolution, assessed by total internal reflection fluorescence microscopy, were unchanged or enhanced in ZnT8(-/-) islets. Insulin processing was normal. Molecular modeling revealed that residue-325 was located at the interface between ZnT8 monomers. Correspondingly, the R325 variant displayed lower apparent Zn(2+) transport activity than W325 ZnT8 by fluorescence-based assay. CONCLUSIONS: ZnT8 is required for normal insulin crystallization and insulin release in vivo but not, remarkably, in vitro. Defects in the former processes in carriers of the R allele may increase type 2 diabetes risks.

Characterization of cyclic adenine dinucleotide phosphate ribose levels in human spermatozoa.

OBJECTIVE: To determine the presence of the Ca2+-releasing pyridine nucleotide derivative, cyclic adenine dinucleotide phosphate ribose (cADPR), in human spermatozoa and to investigate its role in progesterone-induced Ca2+ oscillations in spermatozoa. DESIGN: Biochemical investigation on human spermatozoa from healthy volunteers. SETTING: Healthy volunteers in an academic research environment. PATIENT(S): Ten volunteers. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The cADPR levels. RESULT(S): Human spermatozoa contain micromolar concentrations of cADPR that do not change significantly during sperm capacitation. An active synthetic machinery for cADPR is present in human spermatozoa, whereas degradation activity is minimal. Although progesterone-induced Ca2+ oscillations are dependent on the ryanodine receptor, they are unaffected by cADPR antagonists. CONCLUSION(S): It appears that cADPR does not to play a role in Ca2+ oscillations in spermatozoa, but the presence of high concentrations of cADPR suggests that, instead, it may be introduced into the egg at fertilization and play a role in the Ca2+ transient immediately following sperm-egg fusion.