A game changer for bipolar disorder diagnosis using RNA editing-based biomarkers.

In clinical practice, differentiating Bipolar Disorder (BD) from unipolar depression is a challenge due to the depressive symptoms, which are the core presentations of both disorders. This misdiagnosis during depressive episodes results in a delay in proper treatment and a poor management of their condition. In a first step, using A-to-I RNA editome analysis, we discovered 646 variants (366 genes) differentially edited between depressed patients and healthy volunteers in a discovery cohort of 57 participants. After using stringent criteria and biological pathway analysis, candidate biomarkers from 8 genes were singled out and tested in a validation cohort of 410 participants. Combining the selected biomarkers with a machine learning approach achieved to discriminate depressed patients (n = 267) versus controls (n = 143) with an AUC of 0.930 (CI 95% [0.879-0.982]), a sensitivity of 84.0% and a specificity of 87.1%. In a second step by selecting among the depressed patients those with unipolar depression (n = 160) or BD (n = 95), we identified a combination of 6 biomarkers which allowed a differential diagnosis of bipolar disorder with an AUC of 0.935 and high specificity (Sp = 84.6%) and sensitivity (Se = 90.9%). The association of RNA editing variants modifications with depression subtypes and the use of artificial intelligence allowed developing a new tool to identify, among depressed patients, those suffering from BD. This test will help to reduce the misdiagnosis delay of bipolar patients, leading to an earlier implementation of a proper treatment.

Phosphodiesterase 8A to discriminate in blood samples depressed patients and suicide attempters from healthy controls based on A-to-I RNA editing modifications.

Mental health issues, including major depressive disorder, which can lead to suicidal behavior, are considered by the World Health Organization as a major threat to global health. Alterations in neurotransmitter signaling, e.g., serotonin and glutamate, or inflammatory response have been linked to both MDD and suicide. Phosphodiesterase 8A (PDE8A) gene expression is significantly decreased in the temporal cortex of major depressive disorder (MDD) patients. PDE8A specifically hydrolyzes adenosine 3',5'-cyclic monophosphate (cAMP), which is a key second messenger involved in inflammation, cognition, and chronic antidepressant treatment. Moreover, alterations of RNA editing in PDE8A mRNA has been described in the brain of depressed suicide decedents. Here, we investigated PDE8A A-to-I RNA editing-related modifications in whole blood of depressed patients and suicide attempters compared to age-matched and sex-matched healthy controls. We report significant alterations of RNA editing of PDE8A in the blood of depressed patients and suicide attempters with major depression, for which the suicide attempt took place during the last month before sample collection. The reported RNA editing modifications in whole blood were similar to the changes observed in the brain of suicide decedents. Furthermore, analysis and combinations of different edited isoforms allowed us to discriminate between suicide attempters and control groups. Altogether, our results identify PDE8A as an immune response-related marker whose RNA editing modifications translate from brain to blood, suggesting that monitoring RNA editing in PDE8A in blood samples could help to evaluate depressive state and suicide risk.

Correction: Brain region-specific alterations of RNA editing in PDE8A mRNA in suicide decedents.

Author forgot to attach a supplementary doc file which includes the supplementary methods and supplementary figure legends.

Brain region-specific alterations of RNA editing in PDE8A mRNA in suicide decedents.

Phosphodiesterases (PDE) are key modulators of signal transduction and are involved in inflammatory cell activation, memory and cognition. There is a two-fold decrease in the expression of phosphodiesterase 8A (PDE8A) in the temporal cortex of major depressive disorder (MDD) patients. Here, we studied PDE8A mRNA-editing profile in two architectonically distinct neocortical regions in a clinically well-characterized cohort of age- and sex-matched non-psychiatric drug-free controls and depressed suicide decedents. By using capillary electrophoresis single-stranded conformational polymorphism (CE-SSCP), a previously validated technique to identify A-to-I RNA modifications, we report the full editing profile of PDE8A in the brain, including identification of two novel editing sites. Editing of PDE8A mRNA displayed clear regional difference when comparing dorsolateral prefrontal cortex (BA9) and anterior cingulate cortex (BA24). Furthermore, we report significant intra-regional differences between non-psychiatric control individuals and depressed suicide decedents, which could discriminate the two populations. Taken together, our results (i) highlight the importance of immune/inflammatory markers in major depressive disorder and suicide and (ii) establish a direct relationship between A-to-I RNA modifications of peripheral markers and A-to-I RNA editing-related modifications in brain. This work provides the first immune response-related brain marker for suicide and could pave the way for the identification of a blood-based biomarker that predicts suicidal behavior.

Modeling human pancreatic beta cell dedifferentiation.

OBJECTIVE: Dedifferentiation could explain reduced functional pancreatic ß-cell mass in type 2 diabetes (T2D). METHODS: Here we model human ß-cell dedifferentiation using growth factor stimulation in the human ß-cell line, EndoC-ßH1, and human pancreatic islets. RESULTS: Fibroblast growth factor 2 (FGF2) treatment reduced expression of ß-cell markers, (INS, MAFB, SLC2A2, SLC30A8, and GCK) and activated ectopic expression of MYC, HES1, SOX9, and NEUROG3. FGF2-induced dedifferentiation was time- and dose-dependent and reversible upon wash-out. Furthermore, FGF2 treatment induced expression of TNFRSF11B, a decoy receptor for RANKL and protected ß-cells against RANKL signaling. Finally, analyses of transcriptomic data revealed increased FGF2 expression in ductal, endothelial, and stellate cells in pancreas from T2D patients, whereas FGFR1, SOX,9 and HES1 expression increased in islets from T2D patients. CONCLUSIONS: We thus developed an FGF2-induced model of human ß-cell dedifferentiation, identified new markers of dedifferentiation, and found evidence for increased pancreatic FGF2, FGFR1, and ß-cell dedifferentiation in T2D.

Pancreatic imaging using an antibody fragment targeting the zinc transporter type 8: a direct comparison with radio-iodinated Exendin-4.

AIM: The zinc transporter 8 (ZnT8) has been suggested as a suitable target for non-invasive visualization of the functional pancreatic beta cell mass, due to both its pancreatic beta cell restricted expression and tight involvement in insulin secretion. METHODS: In order to examine the potential of ZnT8 as a surrogate target for beta cell mass, we performed mRNA transcription analysis in pancreatic compartments. A novel ZnT8 targeting antibody fragment Ab31 was radiolabeled with iodine-125, and evaluated by in vitro autoradiography in insulinoma and pancreas as well as by in vivo biodistribution. The evaluation was performed in a direct comparison with radio-iodinated Exendin-4. RESULTS: Transcription of the ZnT8 mRNA was higher in islets of Langerhans compared to exocrine tissue. Ab31 targeted ZnT8 in the cytosol and on the plasma membrane with 108 nM affinity. Ab31 was successfully radiolabeled with iodine-125 with high yield and > 95% purity. [125I]Ab31 binding to insulinoma and pancreas was higher than for [125I]Exendin-4, but could only by partially competed away by 200 nM Ab31 in excess. The in vivo uptake of [125I]Ab31 was higher than [125I]Exendin-4 in most tissues, mainly due to slower clearance from blood. CONCLUSIONS: We report a first-in-class ZnT8 imaging ligand for pancreatic imaging. Development with respect to ligand miniaturization and radionuclide selection is required for further progress. Transcription analysis indicates ZnT8 as a suitable target for visualization of the human endocrine pancreas.

SLC30A8 mutations in type 2 diabetes.

SLC30A8 encodes the secretory granule-resident and largely endocrine pancreas-restricted zinc transporter ZnT8. Interest in this gene product was sparked amongst diabetologists in 2007 when the first genome-wide association study for type 2 diabetes identified polymorphisms in SLC30A8 as affecting disease risk. Thus, the common polymorphism rs13266634 was associated with lowered beta cell function and a 14% increase in diabetes abundance per risk (C) allele. This non-synonymous variant encodes a tryptophan-to-arginine switch at position 325 in the protein's intracellular carboxy-terminal domain, resulting in reduced zinc transport activity and, consequently, decreased intragranular zinc levels. Whereas insulin secretion from isolated islets is most often increased in mice inactivated for Slc30a8, null animals usually show impaired glucose tolerance and lowered circulating insulin. Since Slc30a8 null animals display little, if any, zinc secretion from islets, the lower plasma insulin levels could be explained by increased hepatic clearance as a result of lowered local zinc levels, or less efficient insulin action on target tissues. Despite the emerging consensus on the role of ZnT8 in glucose homeostasis, a recent genetic study in humans has unexpectedly identified loss-of-function SLC30A8 mutants that are associated with protection from diabetes. Here, we attempt to reconcile these apparently contradictory findings, implicating (1) differing degrees of inhibition of ZnT8 activity in carriers of common variants vs rare loss-of-function forms, (2) effects dependent on age or hypoxic beta cell stress. We propose that these variables conspire to affect both the size and the direction of the effect of SLC30A8 risk alleles in man.

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.

Tolbutamide controls glucagon release from mouse islets differently than glucose: involvement of K(ATP) channels from both α-cells and δ-cells.

We evaluated the role of ATP-sensitive K⁺ (K(ATP)) channels, somatostatin, and Zn²âº in the control of glucagon secretion from mouse islets. Switching from 1 to 7 mmol/L glucose inhibited glucagon release. Diazoxide did not reverse the glucagonostatic effect of glucose. Tolbutamide decreased glucagon secretion at 1 mmol/L glucose (G1) but stimulated it at 7 mmol/L glucose (G7). The reduced glucagon secretion produced by high concentrations of tolbutamide or diazoxide, or disruption of K(ATP) channels (Sur1(-/-) mice) at G1 could be inhibited further by G7. Removal of the somatostatin paracrine influence (Sst(-/-) mice or pretreatement with pertussis toxin) strongly increased glucagon release, did not prevent the glucagonostatic effect of G7, and unmasked a marked glucagonotropic effect of tolbutamide. Glucose inhibited glucagon release in the absence of functional K(ATP) channels and somatostatin signaling. Knockout of the Zn²âº transporter ZnT8 (ZnT8(-/-) mice) did not prevent the glucagonostatic effect of glucose. In conclusion, glucose can inhibit glucagon release independently of Zn²âº, K(ATP) channels, and somatostatin. Closure of K(ATP) channels controls glucagon secretion by two mechanisms, a direct stimulation of α-cells and an indirect inhibition via somatostatin released from δ-cells. The net effect on glucagon release results from a balance between both effects.

Zinc, pancreatic islet cell function and diabetes: new insights into an old story.

Zn is an essential trace element, involved in many different cellular processes. A relationship between Zn, pancreatic function and diabetes was suggested almost 70 years ago. To emphasise the importance of Zn in biology, the history of Zn research in the field of diabetes along with a general description of Zn transporter families will be reviewed. The paper will then focus on the effects of Zn on pancreatic ß-cell function, including insulin synthesis and secretion, Zn signalling in the pancreatic islet, the redox functions of Zn and its target genes. The recent association of two 'Zn genes', i.e. metallothionein (MT) and Zn transporter 8 (SLC 30A8), with type 2 diabetes at the genetic level and with insulin secretion in clinical studies offers a potential new way to identify new drug targets to modulate Zn homeostasis directly in ß-cells. The action of Zn for insulin action in its target organs, as Zn signalling in other pancreatic islet cells, will be addressed. Therapeutic Zn-insulin preparations and the influence of Zn and Zn transporters in type 1 diabetes will also be discussed. An extensive review of the literature on the clinical studies using Zn supplementation in the prevention and treatment of both types of diabetes, including complications of the disease, will evaluate the overall beneficial effects of Zn supplementation on blood glucose control, suggesting that Zn might be a candidate ion for diabetes prevention and therapy. Clearly, the story of the links between Zn, pancreatic islet cells and diabetes is only now unfolding, and we are presently only at the first chapter.

Zinc transporters and their role in the pancreatic ß-cell.

Zinc is an essential nutrient with tremendous importance for human health, and zinc deficiency is a severe risk factor for increased mortality and morbidity. As abnormal zinc homeostasis causes diabetes, and because the pancreatic ß-cell contains the highest zinc content of any known cell type, it is of interest to know how zinc fluxes are controlled in ß-cells. The understanding of zinc homeostasis has been boosted by the discovery of multiprotein families of zinc transporters, and one of them - zinc transporter 8 (ZnT8) - is abundantly and specifically expressed in the pancreatic islets of Langerhans. In this review, we discuss the evidence for a physiological role of ZnT8 in the formation of zinc-insulin crystals, the physical form in which most insulin is stored in secretory granules. In addition, we cross-examine this information, collected in genetically modified mouse strains, to the knowledge that genetic variants of the human ZnT8 gene predispose to the onset of type 2 diabetes and that epitopes on the ZnT8 protein trigger autoimmunity in patients with type 1 diabetes. The overall conclusion is that we are still at the dawn of a complete understanding of how zinc homeostasis operates in normal ß-cells and how abnormalities lead to ß-cell dysfunction and diabetes. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2012.00199.x, 2012).

Free zinc ions outside a narrow concentration range are toxic to a variety of cells in vitro.

The zinc(II) ion has recently been implicated in a number of novel functions and pathologies in loci as diverse as the brain, retina, small intestine, prostate, heart, pancreas, and immune system. Zinc ions are a required nutrient but elevated concentrations are known to kill cells in vitro. Paradoxical observations regarding zinc's effects have appeared frequently in the literature, and often their physiological relevance is unclear. We found that for PC-12, HeLa and HT-29 cell lines as well as primary cultures of cardiac myocytes and neurons in vitro in differing media, approximately 5 nmol/L free zinc (pZn = 8.3, where pZn is defined as--log(10) [free Zn(2+)]) produced apparently healthy cells, but 20-fold higher or (in one case) lower concentrations were usually harmful as judged by multiple criteria. These results indicate that (1) the free zinc ion levels of media should be controlled with a metal ion buffer; (2) adding zinc or strong zinc ligands to an insufficiently buffered medium may lead to unpredictably low or high free zinc levels that are often harmful to cells; and (3) it is generally desirable to measure free zinc ion levels due to the presence of contaminating zinc in many biochemicals and unknown buffering capacity of many media.

Prion protein protects against zinc-mediated cytotoxicity by modifying intracellular exchangeable zinc and inducing metallothionein expression.

PrPC contains several octapeptide repeats sequences toward the N-terminus which have binding affinity for divalent metals such as copper, zinc, nickel and manganese. However, the link between PrPC expression and zinc metabolism remains elusive. Here we studied the relationship between PrPC and zinc ions intracellular homeostasis using a cell line expressing a doxycycline-inducible PrPC gene. No significant difference in 65Zn2+ uptake was observed in cells expressing PrPC when compared with control cells. However, PrPC-expressing cells were more resistant to zinc-induced toxicity, suggesting an adaptative mechanism induced by PrPC. Using zinquin-ethyl-ester, a specific fluorophore for vesicular free zinc, we observed a significant re-localization of intracellular exchangeable zinc in vesicles after PrPC expression. Finally, we demonstrated that PrPC expression induces metallothionein (MT) expression, a zinc-upregulated zinc-binding protein. Taken together, these results suggest that PrPC modifies the intracellular localization of zinc rather than the cellular content and induces MT upregulation. These findings are of major importance since zinc deregulation is implicated in several neurodegenerative disorders. It is postulated that in prion diseases the conversion of PrPC to PrPSc may deregulate zinc homeostasis mediated by metallothionein.

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.

Zinc transporter gene expression is regulated by pro-inflammatory cytokines: a potential role for zinc transporters in beta-cell apoptosis?

BACKGROUND: Beta-cells are extremely rich in zinc and zinc homeostasis is regulated by zinc transporter proteins. beta-cells are sensitive to cytokines, interleukin-1beta (IL-1beta) has been associated with beta-cell dysfunction and -death in both type 1 and type 2 diabetes. This study explores the regulation of zinc transporters following cytokine exposure. METHODS: The effects of cytokines IL-1beta, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) on zinc transporter gene expression were measured in INS-1-cells and rat pancreatic islets. Being the more sensitive transporter, we further explored ZnT8 (Slc30A8): the effect of ZnT8 over expression on cytokine induced apoptosis was investigated as well as expression of the insulin gene and two apoptosis associated genes, BAX and BCL2. RESULTS: Our results showed a dynamic response of genes responsible for beta-cell zinc homeostasis to cytokines: IL-1beta down regulated a number of zinc-transporters, most strikingly ZnT8 in both islets and INS-1 cells. The effect was even more pronounced when mixing the cytokines. TNF-alpha had little effect on zinc transporter expression. IFN-gamma down regulated a number of zinc transporters. Insulin expression was down regulated by all cytokines. ZnT8 over expressing cells were more sensitive to IL-1beta induced apoptosis whereas no differences were observed with IFN-gamma, TNF-alpha, or a mixture of cytokines. CONCLUSION: The zinc transporting system in beta-cells is influenced by the exposure to cytokines. Particularly ZnT8, which has been associated with the development of diabetes, seems to be cytokine sensitive.

Investigation of transport mechanisms and regulation of intracellular Zn2+ in pancreatic alpha-cells.

During insulin secretion, pancreatic alpha-cells are exposed to Zn(2+) released from insulin-containing secretory granules. Although maintenance of Zn(2+) homeostasis is critical for cell survival and glucagon secretion, very little is known about Zn(2+)-transporting pathways and the regulation of Zn(2+) in alpha-cells. To examine the effect of Zn(2+) on glucagon secretion and possible mechanisms controlling the intracellular Zn(2+) level ([Zn(2+)](i)), we employed a glucagon-producing cell line (alpha-TC6) and mouse islets where non-beta-cells were identified using islets expressing green fluorescent protein exclusively in beta-cells. In this study, we first confirmed that Zn(2+) treatment resulted in the inhibition of glucagon secretion in alpha-TC6 cells and mouse islets in vitro. The inhibition of secretion was not likely via activation of K(ATP) channels by Zn(2+). We then determined that Zn(2+) was transported into alpha-cells and was able to accumulate under both low and high glucose conditions, as well as upon depolarization of cells with KCl. The nonselective Ca(2+) channel blocker Gd(3+) partially inhibited Zn(2+) influx in alpha-TC cells, whereas the L-type voltage-gated Ca(2+) channel inhibitor nitrendipine failed to block Zn(2+) accumulation. To investigate Zn(2+) transport further, we profiled alpha-cells for Zn(2+) transporter transcripts from the two families that work in opposite directions, SLC39 (ZIP, Zrt/Irt-like protein) and SLC30 (ZnT, Zn(2+) transporter). We observed that Zip1, Zip10, and Zip14 were the most abundantly expressed Zips and ZnT4, ZnT5, and ZnT8 the dominant ZnTs. Because the redox state of cells is also a major regulator of [Zn(2+)](i), we examined the effects of oxidizing agents on Zn(2+) mobilization within alpha-cells. 2,2'-Dithiodipyridine (-SH group oxidant), menadione (superoxide generator), and SIN-1 (3-morpholinosydnonimine) (peroxynitrite generator) all increased [Zn(2+)](i) in alpha-cells. Together these results demonstrate that Zn(2+) inhibits glucagon secretion, and it is transported into alpha-cells in part through Ca(2+) channels. Zn(2+) transporters and the redox state also modulate [Zn(2+)](i).

In vivo expression and functional characterization of the zinc transporter ZnT8 in glucose-induced insulin secretion.

Insulin-secreting pancreatic beta cells are exceptionally rich in zinc. In these cells, zinc is required for zinc-insulin crystallization within secretory vesicles. Secreted zinc has also been proposed to be a paracrine and autocrine modulator of glucagon and insulin secretion in pancreatic alpha and beta cells, respectively. However, little is known about the molecular mechanisms underlying zinc accumulation in insulin-containing vesicles. We previously identified a pancreas-specific zinc transporter, ZnT-8, which colocalized with insulin in cultured beta cells. In this paper we studied its localization in human pancreatic islet cells, and its effect on cellular zinc content and insulin secretion. In human pancreatic islet cells, ZnT-8 was exclusively expressed in insulin-producing beta cells, and colocalized with insulin in these cells. ZnT-8 overexpression stimulated zinc accumulation and increased total intracellular zinc in insulin-secreting INS-1E cells. Furthermore, ZnT-8-overexpressing cells display enhanced glucose-stimulated insulin secretion compared with control cells, only for a high glucose challenge, i.e. >10 mM glucose. Altogether, these data strongly suggest that the zinc transporter ZnT-8 is a key protein for both zinc accumulation and regulation of insulin secretion in pancreatic beta cells.

ZnT-8, a pancreatic beta-cell-specific zinc transporter.

The zinc content in the pancreatic beta cell is among the highest of the body. Zinc appears to be an important metal for insulin-secreting cells as insulin is stored inside secretory vesicles as a solid hexamer bound with two Zn(2+) ions per hexamer. Zinc is also an important component of insulin secretion mechanisms and is likely to modulate the function of neighbouring cells via paracrine/autocrine interactions. Therefore beta cells undoubtedly need very efficient and specialized transporters to accumulate sufficient amounts of zinc in secretion vesicles. We report here the discovery and the characteristics of a new zinc transporter, ZnT-8, belonging to the CDF (Cation Diffusion Facilitator) family and expressed only in pancreatic beta cells. This transporter, localized in secretion vesicles membrane, facilitates the accumulation of zinc from the cytoplasm into intracellular insulin-containing vesicles and is a major component for providing zinc to insulin maturation and/or storage processes in insulin-secreting pancreatic beta cells. We discovered mammalian orthologs (rat, mouse, chimpanzee, and dog) and found these ZnT-8 proteins very similar (98% conserved amino acids) to human ZnT-8, indicating a high conservation during evolution.

Identification and cloning of a beta-cell-specific zinc transporter, ZnT-8, localized into insulin secretory granules.

SLC30A8, a novel member of the zinc transporter (ZnT) family, was identified by searching the human genomic and expressed sequence tag (EST) databases with the amino acid sequence of all known human ZnT. The protein (369 amino acids) predicted from this gene, ZnT-8, contains six transmembrane domains and a histidine-rich loop between transmembrane domains IV and V, like the other ZnT proteins. We demonstrated by RT-PCR on cDNA libraries and human tissue extracts that the ZnT-8 gene is solely transcribed in the pancreas, mainly in the islets of Langerhans. The gene, named SLC30A8, was cloned and sequenced. Confocal immunofluorescence analysis revealed that a ZnT-8-EGFP (enhanced green fluorescent protein) fusion product colocalized with insulin in the secretory pathway granules of the insulin-secreting INS-1 cells. Exposure of the ZnT-8-EGFP stably expressing HeLa cells to 75 micromol/l zinc caused an accumulation of zinc in intracellular vesicles compared with cells expressing EGFP alone. These results identified ZnT-8 as a ZnT specific to the pancreas and expressed in beta-cells. Because ZnT-8 facilitates the accumulation of zinc from the cytoplasm into intracellular vesicles, ZnT-8 may be a major component for providing zinc to insulin maturation and/or storage processes in insulin-secreting pancreatic beta-cells.

Differential regulation of zinc efflux transporters ZnT-1, ZnT-5 and ZnT-7 gene expression by zinc levels: a real-time RT-PCR study.

Intracellular zinc levels are strictly regulated by zinc channels and zinc-binding proteins to maintain cellular zinc-dependent functions. We demonstrated a correlation between extracellular zinc concentration and intracellular exchangeable zinc levels using the fluorescent zinc-specific probes zinquin and zinpyr-1. The effect of extracellular zinc status on the regulation of the two trans-Golgi network directed zinc transporters ZnT-5 and ZnT-7 was next studied by real-time RT-PCR in zinc supplemented or depleted HeLa cells. While sub-toxic extracellular zinc addition strongly induced the efflux transporter ZnT-1 gene expression, consistent with its activation by the transcription factor MTF-1, treated HeLa cells did not display any change in ZnT-5 and ZnT-7 mRNA levels compared to control cells. In contrast, zinc depletion induced by non-toxic doses of the zinc chelator TPEN (N,N,N',N' tetrakis-(2 pyridylmethyl) ethylene diamine) resulted in a up to eight-fold induction of transporters ZnT-5 and ZnT-7 mRNA levels, providing the first evidence of a transcriptional control of these two zinc efflux transporters by zinc deficiency in cultured cells.