Ion 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.
This 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.
Zinc 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.
Zinc 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.
Using 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.
These 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.
Zinc; Insulin; Zinc transporter; Metallothionein; Chelation; TPEN; INS-1E cells; β –cell; Diabetes
We have demonstrated that zinc exposure induces apoptosis in human prostate cancer cells (PC-3) and benign hyperplasia cells (BPH), but not in normal prostate cells (HPR-1). However, the mechanisms underlying the effects of zinc on prostate cancer cell growth and zinc homeostasis remain unclear.
To explore the zinc effect on gene expression profiles in normal (HPR-1) and malignant prostate cells (PC-3), we conducted a time course study of Zn treatment with microarray analysis. Microarray data were evaluated and profiled using computational approach for the primary and secondary data analyses. Final analyses were focused on the genes: 1. highly sensitive to zinc, 2. associated with zinc homeostasis, i.e. metallothioneins (MTs), solute zinc carriers (ZIPs) and zinc exporters (ZnTs), 3. relevant to several oncogenic pathways. Zinc-mediated mRNA levels of MT isotypes were further validated by semi-quantitative RT-PCR.
Results showed that zinc effect on genome-wide expression patterns was cell type specific, and zinc appeared to have mainly down-regulatory effects on thousands of genes (1,953 in HPR-1; 3,534 in PC-3) with a threshold of ±2.5-fold, while fewer genes were up-regulated (872 in HPR-1; 571 in PC-3). The patterns of zinc effect on functional MT genes’ expression provided evidence for the cell-type dependent zinc accumulation and zinc-induced apoptosis in prostate cells. In PC-3 cells, zinc significantly up-regulated the expression of MT-1 isotypes -J and -M, denoted previously as “non-functional” MT genes, and now a depictive molecular structure of MT-1J was proposed. Examination of genes involved in oncogenic pathways indicated that certain genes, e.g. Fos, Akt1, Jak3 and PI3K were highly regulated by zinc with cell type specificity.
This work provided an extensive database on zinc related prostate cancer research. The strategy of data analysis was devoted to find genes highly sensitive to Zn, and the genes associated with zinc accumulation and zinc-induced apoptosis. The results indicate that zinc regulation of gene expression is cell-type specific, and MT genes play important roles in prostate malignancy.
microarray; zinc; prostate malignant cells; gene isoform; metallothionein; zinc transporter
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.
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 Zn2+ transport activity than W325 ZnT8 by fluorescence-based assay.
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.
Global transcriptomic analysis, non-invasive real-time flux, nutritional profiling and metallomics reveal cell-centric response to zinc supplementation/depletion in zebrafish uptake epithelia.
Much remains to be understood about systemic regulation of zinc uptake in vertebrates, and adequate zinc status is far from always achieved in animals or human. In addition to absorbing zinc from the diet, fish are able to take up zinc directly from the water with the gills. This provides an elegant system to study zinc uptake, how it relates to zinc status, and the expression of genes for proteins involved in zinc acquisition. A 21-day experiment was conducted in which zebrafish were acclimated to deficient, control or excess zinc concentrations in the water and feed. Deficient provision of zinc reduced whole body zinc, potassium, sodium and calcium levels whilst zinc concentrations in the uptake epithelia (gills and gut) remained unchanged. Excess levels of zinc caused accumulation of zinc in the gills, intestine and carcass, but impaired whole body iron, sodium and calcium concentrations. Fish subjected to zinc deficiency had, surprisingly, a reduced zinc influx across the gill epithelium, even when tested at a high concentration of zinc in the water. Zinc influx in the excess group was indistinct from the control. Expression of genes for metallothionein-2 (Mt2) and zinc transporters-1, -2, and -8 (Znt1, Znt2, Znt8) in uptake epithelia showed in general a direct relationship with zinc supply, while mRNA for Zip4 was inversely related to zinc supply. Transcripts for the epithelial calcium channel (Ecac/Trpv6) showed time-dependent increased expression in the gills of the deficiency group, and a transient decrease of expression during zinc excess. Transcriptome profiling by microarrays showed that in both gills and intestine, the most markedly affected biological functions were those related to cell growth, proliferation and cancer, closely followed by processes of gene transcription and protein synthesis in general. Whilst changes in zinc supply had profound effects in the intestine on genes associated with uptake and metabolism of macronutrients, many of the unique categories of genes preferentially regulated in the gill could be mapped onto signalling pathways. This included pathways for PPAR/RXR, LXR/RXR, ATM, chemokine, and BMP signalling. Overall, the responses of epithelial tissue to zinc deficiency and excess are best explained by local epithelial homeostasis with no evidence of systemic control.
Senescence, a hallmark of mammalian aging, is associated with the onset and progression of cardiovascular disease. Angiotensin II (Ang II) signaling and zinc homeostasis dysfunction are increased with age and are linked to cardiovascular disease, but the relationship among these processes has not been investigated. We used a model of cellular senescence induced by Ang II in vascular smooth muscle cells (VSMCs) to explore the role of zinc in vascular dysfunction. We found that Ang II-induced senescence is a zinc-dependent pathway mediated by the downregulation of the zinc transporters ZnT3 and ZnT10, which work to reduce cytosolic zinc. Zinc mimics Ang II by increasing reactive oxygen species (ROS), activating NADPH oxidase activity and Akt, and by downregulating ZnT3 and ZnT10 and inducing senescence. Zinc increases Ang II-induced senescence, while the zinc chelator TPEN, as well as overexpression of ZnT3 or ZnT10, decreases ROS and prevents senescence. Using HEK293 cells, we found that ZnT10 localizes in recycling endosomes and transports zinc into vesicles to prevent zinc toxicity. Zinc and ZnT3/ZnT10 downregulation induces senescence by decreasing the expression of catalase. Consistently, ZnT3 and ZnT10 downregulation by siRNA increases ROS while downregulation of catalase by siRNA induces senescence. Zinc, siZnT3 and siZnT10 downregulate catalase by a post-transcriptional mechanism mediated by decreased phosphorylation of ERK1/2. These data demonstrate that zinc homeostasis dysfunction by decreased expression of ZnT3 or ZnT10 promotes senescence and that Ang II-induced senescence is a zinc and ROS-dependent process. Our studies suggest that zinc might also affect other ROS-dependent processes induced by Ang II, such as hypertrophy and migration of smooth muscle cells.
The SLC30A8 gene codes for a pancreatic beta-cell-expressed zinc transporter, ZnT8. A polymorphism in the SLC30A8 gene is associated with susceptibility to type 2 diabetes, although the molecular mechanism through which this phenotype is manifest is incompletely understood. Such polymorphisms may exert their effect via impacting expression level of the gene product. We used an shRNA-mediated approach to reproducibly downregulate ZnT8 mRNA expression by >90% in the INS-1 pancreatic beta cell line. The ZnT8-downregulated cells exhibited diminished uptake of exogenous zinc, as determined using the zinc-sensitive reporter dye, zinquin. ZnT8-downregulated cells showed reduced insulin content and decreased insulin secretion (expressed as percent of total insulin content) in response to hyperglycemic stimulus, as determined by insulin immunoassay. ZnT8-depleted cells also showed fewer dense-core vesicles via electron microscopy. These data indicate that reduced ZnT8 expression in cultured pancreatic beta cells gives rise to a reduced insulin response to hyperglycemia. In addition, although we provide no direct evidence, these data suggest that an SLC30A8 expression-level polymorphism could affect insulin secretion and the glycemic response in vivo.
Chronic low-grade inflammation in type 2 diabetes mellitus (DM) can elicit changes in whole-body zinc metabolism. The interaction among the expression of inflammatory cytokines, zinc transporter and metallothionein (MT) genes in peripheral blood mononuclear cells in type 2 DM remains unclear. In a 12-week randomized controlled trial, the effects of zinc (40 mg/day) supplementation on the gene expression of cytokines, zinc transporters and MT in women with type 2 DM were examined. In the zinc-supplemented group, gene expression of tumour necrosis factor (TNF)-α tended to be upregulated by 27 ± 10 % at week 12 compared to baseline (P = 0.053). TNF-α fold change in the zinc-treated group was higher than in those without zinc supplementation (P < 0.05). No significant changes were observed in the expression or fold change of interleukin (IL)-1β or IL-6. Numerous bivariate relationships were observed between the fold changes of cytokines and zinc transporters, including ZnT7 with IL-1β (P < 0.01), IL-6 (P < 0.01) and TNF-α (P < 0.01). In multiple regression analysis, IL-1β expression was predicted by the expression of all zinc transporters and MT measured at baseline (r2 = 0.495, P < 0.05) and at week 12 (r2 = 0.532, P < 0.03). The current study presents preliminary evidence that zinc supplementation increases cytokine gene expression in type 2 DM. The relationships found among zinc transporters, MT and cytokines suggest close interactions between zinc homeostasis and inflammation.
Electronic supplementary material
The online version of this article (doi:10.1007/s12263-014-0440-4) contains supplementary material, which is available to authorized users.
Inflammation; Cytokines; Zinc transporters; Metallothionein; Gene expression; Type 2 diabetes mellitus
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 β‐cell contains the highest zinc content of any known cell type, it is of interest to know how zinc fluxes are controlled in β‐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 β‐cells and how abnormalities lead to β‐cell dysfunction and diabetes. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2012.00199.x, 2012)
Insulin crystallization; Pancreatic β‐cell; Zinc transporter 8
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 Zn2+ levels. We have therefore explored the role of ZnT8 and cytosolic Zn2+ in the response to hypoxia of pancreatic islet cells.
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.
Deprivation of O2 (1% vs 5% or 21%) for 24 h lowered free cytosolic Zn2+ 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 Zn2+, genetic ablation of Slc30a8 from mouse islets lowered cytosolic Zn2+ 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.
The response of pancreatic beta cells to hypoxia is characterised by decreased SLC30A8 expression and lowered cytosolic Zn2+ 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.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-014-3266-0) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
Hypoxia; Metallothionein; Type 2 diabetes; Zinc; Zinc transporter
In E. coli, ZitB and ZntA are important metal exporters that enhance cell viability under high environmental zinc. To understand their functions in maintaining zinc homeostasis, we applied a novel genetically-encoded fluorescent zinc sensor to monitor the intracellular free zinc changes in wild type, ΔzitB and ΔzntA E. coli cells upon sudden exposure to toxic levels of zinc (“zinc shock”). The intracellular readily exchangeable zinc concentration (or “free” zinc) increases transiently from picomolar to nanomolar levels, accelerating zinc-activated gene transcription. After zinc shock, the zitB mRNA level is constant while the zntA mRNA increases substantially in a zinc-dependent manner. In the ΔzitB E. coli strain the free zinc concentration rises more rapidly after zinc shock compared to wild-type cells while a prolonged accumulation of free zinc is observed in the ΔzntA strain. Based on these results, we propose that ZitB functions as a constitutive, first-line defense against toxic zinc influx, while ZntA is up-regulated to efficiently lower the free zinc concentration. Furthermore, the ZntR-mediated transcription of zntA exhibits an apparent K1/2 for zinc activation in the nanomolar range in vivo, significantly higher than the femtomolar affinity for zinc binding and transcription activation previously measured in vitro. A kinetically-controlled transcription model is sufficient to explain the observed regulation of intracellular free zinc concentration by ZntR and ZntA after zinc shock.
intracellular free zinc; zinc sensor; zntA; zitB; zntR; transcriptional response
The Slc30a8 gene encodes the islet-specific zinc transporter ZnT-8, which provides zinc for insulin-hexamer formation. Polymorphic variants in amino acid 325 of human ZnT-8 are associated with altered susceptibility to type 2 diabetes and ZnT-8 autoantibody epitope specificity changes in type 1 diabetes. To assess the physiological importance of ZnT-8, mice carrying a Slc30a8 exon 3 deletion were analyzed histologically and phenotyped for energy metabolism and pancreatic hormone secretion. No gross anatomical or behavioral changes or differences in body weight were observed between wild type and ZnT-8 −/− mice and ZnT-8 −/− mouse islets were indistinguishable from wild type in terms of their numbers, size and cellular composition. However, total zinc content was markedly reduced in ZnT-8 −/− mouse islets, as evaluated both by Timm’s histochemical staining of pancreatic sections and direct measurements in isolated islets. Blood glucose levels were unchanged in 16 week old, 6 hr fasted animals of either gender, however, plasma insulin concentrations were reduced in both female (~31%) and male (~47%) ZnT-8 −/− mice. Intraperitoneal glucose tolerance tests demonstrated no impairment in glucose clearance in male ZnT-8 −/− mice but glucose-stimulated insulin secretion from isolated islets was reduced ~33% relative to wild type littermates. In summary, Slc30a8 gene deletion is accompanied by a modest impairment in insulin secretion without major alterations in glucose metabolism.
Autoantigen; Glucose; Zinc Transporter; Insulin; Islet; Mouse
Genetic studies suggest that Zn transporters such as ZnT8 play a role in insulin secretion by pancreatic β-cells; however, little is known about the dynamic roles of Zn trafficking pathways on β-cell physiology. To test the acute effects of the inflammatory cytokines interleukin 1β (IL1β) and tumor necrosis factor α (TNFα) on Zn homeostasis, the mRNA expression profile of Zn transporters of the ZnT and ZIP families was examined. Exposure of MIN6 cells or primary murine islets to IL1β or TNFα altered the mRNA expression profile of Zn transporters; most notable was decreased ZnT8 mRNA levels. siRNA-mediated gene knockdown was used to examine the effects of decreased ZnT8 expression in primary dispersed murine islet cells from C57/BL6 mice and MIN6 cells. ZnT8 knockdown in these murine islets led to reduced glucose stimulated insulin secretion without altering the total cellular insulin content or cell viability at normal or supraphysiological Zn concentrations. The labile Zn content determined by flow cytometry after loading with the Zn-specific sensor FluoZin-3 AM was decreased in MIN6 cells following ZnT8 knockdown or IL1β treatment. These results suggest that an acute decrease in ZnT8 levels impairs β-cell function and Zn homeostasis, and may contribute to inflammatory cytokine-induced alterations in β-cell function.
A blood-brain barrier (BBB) model composed of porcine brain capillary endothelial cells (BCEC) was exposed to a moderately excessive zinc environment (50 µmol Zn/L) in cell culture and longitudinal measurements were made of zinc transport kinetics, ZnT-1 (SLC30A1) expression, and changes in the protein concentration of metallothionein (MT), ZnT-1, ZnT-2 (SLC30A2), and Zip1 (SLC39A1). Zinc release by cells of the BBB model was significantly increased after 12–24 h of exposure, but decreased back to control levels after 48–96 h, as indicated by transport across the BBB from both the ablumenal (brain) and lumenal (blood) directions. Expression of ZnT-1, the zinc export protein, increased 169% within 12 h, but was no longer different from controls after 24 h. Likewise, ZnT-1 protein content increased transiently after 12 h of exposure but returned to control levels by 24 h. Capacity for zinc uptake and retention increased from both the lumenal and ablumenal directions within 12–24 h of exposure and remained elevated. MT and ZnT-2 were elevated within 12 h and remained elevated throughout the study. Zip1 was unchanged by the treatment. The BBB’s response to a moderately high zinc environment was dynamic and involved multiple mechanisms. The initial response was to increase the cell’s capacity to sequester zinc with additional MT and increase zinc export with the ZnT-1 protein. But, the longer term strategy involved increasing ZnT-2 transporters, presumably to sequester zinc into intracellular vesicles as a mechanism to protect the brain and maintain brain zinc homeostasis.
zinc homeostasis; zinc toxicity; blood-brain barrier; zinc transporters; metallothionein
The activation process of secretory or membrane-bound zinc enzymes is thought to be a highly coordinated process involving zinc transport, trafficking, transfer and coordination. We have previously shown that secretory and membrane-bound zinc enzymes are activated in the early secretory pathway (ESP) via zinc-loading by the zinc transporter 5 (ZnT5)-ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes). However, how other proteins conducting zinc metabolism affect the activation of these enzymes remains unknown. Here, we investigated this issue by disruption and re-expression of genes known to be involved in cytoplasmic zinc metabolism, using a zinc enzyme, tissue non-specific alkaline phosphatase (TNAP), as a reporter. We found that TNAP activity was significantly reduced in cells deficient in ZnT1, Metallothionein (MT) and ZnT4 genes (ZnT1−/−MT−/−ZnT4−/− cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in ZnT1−/−MT−/−ZnT4−/− cells was not restored when cytosolic zinc levels were normalized to levels comparable with those of wild-type cells, but was reversely restored by extreme zinc supplementation via zinc-loading by the zinc transport complexes. Moreover, the reduced TNAP activity was adequately restored by re-expression of mammalian counterparts of ZnT1, MT and ZnT4, but not by zinc transport-incompetent mutants of ZnT1 and ZnT4. In ZnT1−/−MT−/−ZnT4−/− cells, the secretory pathway normally operates. These findings suggest that cooperative zinc handling of ZnT1, MT and ZnT4 in the cytoplasm is required for full activation of TNAP in the ESP, and present clear evidence that the activation process of zinc enzymes is elaborately controlled.
Non-covalent and covalent homo-oligomerization of membrane proteins regulates their subcellular localization and function. Here, we described a novel oligomerization mechanism affecting solute carrier family 30 member 3/zinc transporter 3 (SLC30A3/ZnT3). Oligomerization was mediated by intermolecular covalent dityrosine bonds. Using mutagenized ZnT3 expressed in PC12 cells, we identified two critical tyrosine residues necessary for dityrosine-mediated ZnT3 oligomerization. ZnT3 carrying the Y372F mutation prevented ZnT3 oligomerization, decreased ZnT3 targeting to synaptic-like microvesicles (SLMVs), and decreased resistance to zinc toxicity. Strikingly, ZnT3 harboring the Y357F mutation behaved as a “gain-of-function” mutant as it displayed increased ZnT3 oligomerization, targeting to SLMVs, and increased resistance to zinc toxicity. Single and double tyrosine ZnT3 mutants indicate that the predominant dimeric species is formed between tyrosine 357 and 372. ZnT3 tyrosine dimerization was detected under normal conditions and it was enhanced by oxidative stress. Covalent species were also detected in other SLC30A zinc transporters localized in different subcellular compartments. These results indicate that covalent tyrosine dimerization of a SLC30A family member modulates its subcellular localization and zinc transport capacity. We propose that dityrosine-dependent membrane protein oligomerization may regulate the function of diverse membrane protein in normal and disease states.
Recent genome-wide association studies demonstrated that common variants of solute carrier family 30 member 8 gene (SLC30A8) increase susceptibility to type 2 diabetes. SLC30A8 encodes zinc transporter-8 (ZnT8), which delivers zinc ion from the cytoplasm into insulin granules. Although it is well known that insulin granules contain high amounts of zinc, the physiological role of secreted zinc remains elusive. In this study, we generated mice with β cell–specific Slc30a8 deficiency (ZnT8KO mice) and demonstrated an unexpected functional linkage between Slc30a8 deletion and hepatic insulin clearance. The ZnT8KO mice had low peripheral blood insulin levels, despite insulin hypersecretion from pancreatic β cells. We also demonstrated that a substantial amount of the hypersecreted insulin was degraded during its first passage through the liver. Consistent with these findings, ZnT8KO mice and human individuals carrying rs13266634, a major risk allele of SLC30A8, exhibited increased insulin clearance, as assessed by c-peptide/insulin ratio. Furthermore, we demonstrated that zinc secreted in concert with insulin suppressed hepatic insulin clearance by inhibiting clathrin-dependent insulin endocytosis. Our results indicate that SLC30A8 regulates hepatic insulin clearance and that genetic dysregulation of this system may play a role in the pathogenesis of type 2 diabetes.
ZnT2 (zinc transporter-2) expression is restricted to tissues with unique zinc requirements such as mammary and prostate glands. We previously determined that ZnT2 plays a major role in zinc export from mammary glands, as women with a mutation in the gene encoding ZnT2 (SLC30A2) had an ~75% reduction in milk zinc concentration. Two distinct human ZnT2 isoforms (~42 and 35 kDa) are predicted to result from alternative splicing of SLC30A2. We examined the localization and function of each ZnT2 isoform, in cells generated to express ZnT2–HA (haemagglutinin) fusion proteins. The 42 kDa isoform was localized primarily to the endosomal/secretory compartment and overexpression resulted in increased zinc vesicularization. In contrast, the 35 kDa isoform is associated with the plasma membrane. Importantly, zinc transport was higher in cells over-expressing each isoform, indicating that both proteins are functional. Endogenous expression of the secretory vesicle-associated ZnT2 isoform predominates in mammary cells and expression is higher in secreting cells, whereas the smaller isoform plays a minor role in zinc export, directly reflecting the secretory function of the mammary gland. Together our data shed further light on the complex integration of cellular zinc transport mechanisms, which may be facilitated by multiple isoforms of specific zinc transporters with unique cellular functions.
exocytotic vesicles; lactation; mammary gland; plasma membrane; zinc transporters; ZnT2 variants
Zinc is an essential trace element in organisms, which serves as a cofactor for hundreds of enzymes that are involved in many pivotal biological processes including growth, development, reproduction and immunity. Therefore, the homeostasis of zinc in the cell is fundamental. The zinc transporter gene family is a large gene family that encodes proteins which regulate the movement of zinc across cellular and intracellular membranes. However, studies on teleost zinc transporters are mainly limited to model species.
We identified a set of 37 zinc transporters in common carp genome, including 17 from SLC30 family (ZnT), and 20 from SLC39 family (ZIP). Phylogenetic and syntenic analysis revealed that most of the zinc transporters are highly conserved, though recent gene duplication and gene losses do exist. Through examining the copy number of zinc transporter genes across several vertebrate genomes, thirteen zinc transporters in common carp are found to have undergone the gene duplications, including SLC30A1, SLC30A2, SLC30A5, SLC30A7, SLC30A9, SLC30A10, SLC39A1, SLC39A3, SLC39A4, SLC39A5, SLC39A6, SLC39A7 and SLC39A9. The expression patterns of all zinc transporters were established in various tissues, including blood, brain, gill, heart, intestine, liver, muscle, skin, spleen and kidney, and showed that most of the zinc transporters were ubiquitously expressed, indicating the critical role of zinc transporters in common carp.
To some extent, examination of gene families with detailed phylogenetic or orthology analysis could verify the authenticity and accuracy of assembly and annotation of the recently published common carp whole genome sequences. The gene families are also considered as a unique source for evolutionary studies. Moreover, the whole set of common carp zinc transporters provides an important genomic resource for future biochemical, toxicological and physiological studies of zinc in teleost.
We examined the effects of the R325W mutation on the three-dimensional (3D) structure of the β-cell-specific Zn2+ (zinc) transporter ZnT-8.
A model of the C-terminal domain of the human ZnT-8 protein was generated by homology modeling based on the known crystal structure of the Escherichia coli (E. coli) zinc transporter YiiP at 3.8 Å resolution.
The homodimer ZnT-8 protein structure exists as a Y-shaped architecture with Arg325 located at the ultimate bottom of this motif at approximately 13.5 Å from the transmembrane domain juncture. The C-terminal domain sequences of the human ZnT-8 protein and the E. coli zinc transporter YiiP share 12.3% identical and 39.5% homologous residues resulting in an overall homology of 51.8%. Validation statistics of the homology model showed a reasonable quality of the model. The C-terminal domain exhibited an αββαβ fold with Arg325 as the penultimate N-terminal residue of the α2-helix. The side chains of both Arg325 and Trp325 point away from the interface with the other monomer, whereas the ε-NH3+ group of Arg325 is predicted to form an ionic interaction with the β-COO- group of Asp326 as well as Asp295. An amino acid alignment of the β2-α2 C-terminal loop domain revealed a variety of neutral amino acids at position 325 of different ZnT-8 proteins.
Our validated homology models predict that both Arg325 and Trp325, amino acids with a helix-forming behavior, and penultimate N-terminal residues in the α2-helix of the C-terminal domain, are shielded by the planar surface of the three cytoplasmic β-strands and hence unable to affect the sensing capacity of the C-terminal domain. Moreover, the amino acid residue at position 325 is too far removed from the docking and transporter parts of ZnT-8 to affect their local protein conformations. These data indicate that the inherited R325W abnormality in SLC30A8 may be tolerated and results in adequate zinc transfer to the correct sites in the pancreatic islet cells and are consistent with the observation that the SLC30A8 gene variant R325W has a low predicted value for future type 2 diabetes at population-based level.
Zinc transporter 8 (ZnT8) is an islet β-cell secretory granule membrane protein recently identified as an autoantibody antigen in type 1 diabetes. The aim of this study was to determine the prevalence and role of antibodies to ZnT8 (ZnT8As) in adult-onset diabetes.
RESEARCH DESIGN AND METHODS
ZnT8As were measured by a radioimmunoprecipitation assay using recombinant ZnT8 COOH-terminal or NH2-terminal proteins in 193 patients with adult-onset autoimmune diabetes having antibodies to either GAD (GADAs) or IA-2 (IA-2As) and in 1,056 antibody-negative patients with type 2 diabetes from the Non Insulin Requiring Autoimmune Diabetes (NIRAD) study.
ZnT8As-COOH were detected in 18.6% patients with autoimmune diabetes and 1.4% with type 2 diabetes. ZnT8As-NH2 were rare. ZnT8As were associated with younger age and a high GADA titer. The use of GADAs, IA-2As, and ZnT8As in combination allowed a stratification of clinical phenotype, with younger age of onset of diabetes and characteristics of more severe insulin deficiency (higher fasting glucose and A1C, lower BMI, total cholesterol, and triglycerides) in patients with all three markers, with progressive attenuation in patients with two, one, and no antibodies (all Ptrend < 0.001). Autoantibody titers, association with high-risk HLA genotypes, and prevalence of thyroid peroxidase antibodies followed the same trend (all P < 0.001).
ZnT8As are detectable in a proportion of patients with adult-onset autoimmune diabetes and seem to be a valuable marker to differentiate clinical phenotypes.
Calcium overload has been implicated as a critical event in glutamate excitotoxicity associated neurodegeneration. Recently, zinc accumulation and its neurotoxic role similar to calcium has been proposed. Earlier, we reported that free chelatable zinc released during hypobaric hypoxia mediates neuronal damage and memory impairment. The molecular mechanism behind hypobaric hypoxia mediated neuronal damage is obscure. The role of free zinc in such neuropathological condition has not been elucidated. In the present study, we investigated the underlying role of free chelatable zinc in hypobaric hypoxia-induced neuronal inflammation and apoptosis resulting in hippocampal damage.
Adult male Balb/c mice were exposed to hypobaric hypoxia and treated with saline or Ca2EDTA (1.25 mM/kg i.p) daily for four days. The effects of Ca2EDTA on apoptosis (caspases activity and DNA fragmentation), pro-inflammatory markers (iNOS, TNF-α and COX-2), NADPH oxidase activity, poly(ADP ribose) polymerase (PARP) activity and expressions of Bax, Bcl-2, HIF-1α, metallothionein-3, ZnT-1 and ZIP-6 were examined in the hippocampal region of brain.
Hypobaric hypoxia resulted in increased expression of metallothionein-3 and zinc transporters (ZnT-1 and ZIP-6). Hypobaric hypoxia elicited an oxidative stress and inflammatory response characterized by elevated NADPH oxidase activity and up-regulation of iNOS, COX-2 and TNF-α. Furthermore, hypobaric hypoxia induced HIF-1α protein expression, PARP activation and apoptosis in the hippocampus. Administration of Ca2EDTA significantly attenuated the hypobaric hypoxia induced oxidative stress, inflammation and apoptosis in the hippocampus.
We propose that hypobaric hypoxia/reperfusion instigates free chelatable zinc imbalance in brain associated with neuroinflammation and neuronal apoptosis. Therefore, zinc chelating strategies which block zinc mediated neuronal damage linked with cerebral hypoxia and other neurodegenerative conditions can be designed in future.
The zinc (Zn++) transporter ZnT8 plays a crucial role in zinc homeostasis. It’s been reported that an acute decrease in ZnT8 levels impairs β cell function and Zn++ homeostasis, which contribute to the pathophysiology of diabetes mellitus (DM). Although ZnT8 expression has been detected in the retinal pigment epithelium (RPE), its expression profile in the retina has yet to be determined. Furthermore, the link between diabetes and ischemic retinopathy is well documented; nevertheless, the molecular mechanism(s) of such link has yet to be defined. Our aims were to; investigate the expression profile of ZnT8 in the retina; address the influence of ischemia on such expression; and evaluate the influence of YC-1; (3-(50-hydroxymethyl-20-furyl)-1-benzyl indazole), a hypoxia inducible factor-1 (HIF-1) inhibitor, on the status of ZnT8 expression. We used real-time RT-PCR, immunohistochemistry, and Western blot in the mouse model of oxygen-induced retinopathy (OIR) and Müller cells to evaluate the effects of ischemia/hypoxia and YC-1 on ZnT8 expression. Our data indicate that ZnT8 was strongly expressed in the outer nuclear layer (ONL), outer plexiform layer (OPL), ganglion cell layer (GCL), and nerve fiber layer (NFL), whereas the photoreceptor layer (PRL), inner nuclear layer (INL) and inner plexiform layer (IPL) showed moderate ZnT8 immunoreactivity. Furthermore, we demonstrate that retinal ischemic insult induces a significant downregulation of ZnT8 at the message and protein levels, YC-1 rescues the injured retina by restoring the ZnT8 to its basal homeostatic levels in the neovascular retinas. Our data indicate that ischemic retinopathy maybe mediated by aberrant Zn++ homeostasis caused by ZnT8 downregulation, whereas YC-1 plays a neuroprotective role against ischemic insult. Therefore, targeting ZnT8 provides a therapeutic strategy to combat neovascular eye diseases.
Epidermodysplasia verruciformis (EV) is a genodermatosis associated with skin cancers that results from a selective susceptibility to related human papillomaviruses (EV HPV). Invalidating mutations in either of two genes (EVER1 and EVER2) with unknown functions cause most EV cases. We report that EVER1 and EVER2 proteins form a complex and interact with the zinc transporter 1 (ZnT-1), as shown by yeast two-hybrid screening, GST pull-down, and immunoprecipitation experiments. In keratinocytes, EVER and ZnT-1 proteins do not influence intracellular zinc concentration, but do affect intracellular zinc distribution. EVER2 was found to inhibit free zinc influx to nucleoli. Keratinocytes with a mutated EVER2 grew faster than wild-type keratinocytes. In transiently and stably transfected HaCaT cells, EVER and ZnT-1 down-regulated transcription factors stimulated by zinc (MTF-1) or cytokines (c-Jun and Elk), as detected with luciferase assays. To get some insight into the control of EV HPV infection, we searched for interaction between EVER and ZnT-1 and oncoproteins of cutaneous (HPV5) and genital (HPV16) genotypes. HPV16 E5 protein binds to EVER and ZnT-1 and blocks their negative regulation. The lack of a functional E5 protein encoded by EV HPV genome may account for host restriction of these viruses.
We examined whether zinc transporter 8 autoantibodies (ZnT8A; arginine ZnT8-RA, tryptophan ZnT8-WA, and glutamine ZnT8-QA variants) differed between immigrant and Swedish patients due to different polymorphisms of SLC30A8, HLA-DQ, or both. Newly diagnosed autoimmune (≥1 islet autoantibody) type 1 diabetic patients (n = 2,964, <18 years, 55% male) were ascertained in the Better Diabetes Diagnosis study. Two subgroups were identified: Swedes (n = 2,160, 73%) and immigrants (non-Swedes; n = 212, 7%). Non-Swedes had less frequent ZnT8-WA (38%) than Swedes (50%), consistent with a lower frequency in the non-Swedes (37%) of SLC30A8 CT+TT (RW+WW) genotypes than in the Swedes (54%). ZnT8-RA (57 and 58%, respectively) did not differ despite a higher frequency of CC (RR) genotypes in non-Swedes (63%) than Swedes (46%). We tested whether this inconsistency was due to HLA-DQ as 2/X (2/2; 2/y; y is anything but 2 or 8), which was a major genotype in non-Swedes (40%) compared with Swedes (14%). In the non-Swedes only, 2/X (2/2; 2/y) was negatively associated with ZnT8-WA and ZnT8-QA but not ZnT8-RA. Molecular simulation showed nonbinding of the relevant ZnT8-R peptide to DQ2, explaining in part a possible lack of tolerance to ZnT8-R. At diagnosis in non-Swedes, the presence of ZnT8-RA rather than ZnT8-WA was likely due to effects of HLA-DQ2 and the SLC30A8 CC (RR) genotypes.