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Zinc is an essential trace element that is involved in many biological processes and in cellular homeostasis. In pancreatic β-cells, zinc is crucial for the synthesis, processing, and secretion of insulin, which plays a key role in glucose homeostasis and which deficiency is the cause of diabetes. The accumulation of zinc in pancreatic cells is regulated by the solute carrier transporter SLC30A8 (or Zinc Transporter 8, ZnT8), which transports zinc from cytoplasm in intracellular vesicles. Allelic variants of SLC30A8 gene have been linked to diabetes. Given the physiological intracellular localization of SLC30A8 in pancreatic β-cells and the ubiquitous endogenous expression of other Zinc transporters in different cell lines that could be used as cellular model for SLC30A8 recombinant over-expression, it is challenging to develop a functional assay to measure SLC30A8 activity. To achieve this goal, we have firstly generated a HEK293 cell line stably overexpressing SLC30A8, where the over-expression favors the partial localization of SLC30A8 on the plasma membrane. Then, we used the combination of this cell model, commercial FluoZin-3 cell permeant zinc dye and live cell imaging approach to follow zinc flux across SLC30A8 over-expressed on plasma membrane, thus developing a novel functional imaging- based assay specific for SLC30A8. Our novel approach can be further explored and optimized, paving the way for future small molecule medium-throughput screening.

Background: Zinc homeostasis is essential for human health and is regulated by several zinc transporters including ZIP and ZnT. ZIP4 is expressed in the small intestine and is important for zinc absorption from the diet. We investigated in the present study the effects of Panax ginseng (P. ginseng) extract on modulating Zip4 expression and cellular zinc levels in mouse Hepa cells. Methods: Hepa cells were transfected with a luciferase reporter plasmid that contains metal-responsive elements, incubated with P. ginseng extract, and luciferase activity was measured. Using 65ZnCl2, zinc uptake in P. ginseng-treated cells was measured. The expression of Zip4 mRNA and protein in Hepa cells was also investigated. Finally, using a luciferase reporter assay system, the effects of several ginsenosides were monitored. Results: The luciferase activity in cells incubated with P. ginseng extract was significantly higher than that of control cells cultured in normal medium. Hepa cells treated with P. ginseng extract exhibited higher zinc uptake. P. ginseng extract induced Zip4 mRNA expression, which resulted in an enhancement of Zip4 protein expression. Furthermore, some ginsenosides, such as ginsenoside Rc and Re, enhanced luciferase activity driven by intracellular zinc levels. Conclusion: P. ginseng extract induced Zip4 expression at the mRNA and protein level and resulted in higher zinc uptake in Hepa cells. Some ginsenosides facilitated zinc influx. On the basis of these results, we suggest a novel effect of P. ginseng on Zip4-mediated zinc influx, which may provide a new strategy for preventing zinc deficiency.

The human zinc transporter ZnT8 (SLC30A8) is expressed primarily in pancreatic β-cells and plays a key function in maintaining the concentration of blood glucose through its role in insulin storage, maturation and secretion. ZnT8 is an autoantigen for Type 1 diabetes (T1D) and is associated with Type 2 diabetes (T2D) through its risk allele that encodes a major non-synonymous single nucleotide polymorphism (SNP) at Arg325. Loss of function mutations improve insulin secretion and are protective against diabetes. Despite its role in diabetes and concomitant potential as a drug target, little is known about the structure or mechanism of ZnT8. To this end, we expressed ZnT8 in Pichia pastoris yeast and Sf9 insect cells. Guided by a rational screen of 96 detergents, we developed a method to solubilize and purify recombinant ZnT8. An in vivo transport assay in Pichia and a liposome-based uptake assay for insect-cell derived ZnT8 showed that the protein is functionally active in both systems. No significant difference in activity was observed between full-length ZnT8 (ZnT8A) and the amino-terminally truncated ZnT8B isoform. A fluorescence-based in vitro transport assay using proteoliposomes indicated that human ZnT8 functions as a Zn2+/H+ antiporter. We also purified E. coli-expressed amino- and carboxy-terminal cytoplasmic domains of ZnT8A. Circular dichroism spectrometry suggested that the amino-terminal domain contains predominantly α-helical structure, and indicated that the carboxy-terminal domain has a mixed α/β structure. Negative-stain electron microscopy and single-particle image analysis yielded a density map of ZnT8B at 20 Å resolution, which revealed that ZnT8 forms a dimer in detergent micelles. Two prominent lobes are ascribed to the transmembrane domains, and the molecular envelope recapitulates that of the bacterial zinc transporter YiiP. These results provide a foundation for higher resolution structural studies and screening experiments to identify compounds that modulate ZnT8 activity.

Plants have evolved a series of zinc (Zn) homeostasis mechanisms to cope with the fluctuating Zn in the environment. How Zn is taken up, translocated and tolerate by tea plant remains unknown. In this study, on the basis of RNA-Sequencing, we isolated a plasma membrane-localized Metal Tolerance Protein (MTP) family member CsMTP4 from Zn-deficient tea plant roots and investigated its role in regulation of Zn homeostasis in tea plant. Heterologous expression of CsMTP4 specifically enhanced the tolerance of transgenic yeast to Zn excess. Moreover, overexpression of CsMTP4 in tea plant hairy roots stimulated Zn uptake under Zn deficiency. In addition, CsMTP4 promoted the growth of transgenic Arabidopsis plants by translocating Zn from roots to shoots under Zn deficiency and conferred the tolerance to Zn excess by enhancing the efflux of Zn from root cells. Transcriptome analysis of the CsMTP4 transgenic Arabidopsis found that the expression of Zn metabolism-related genes were differentially regulated compared with wild-type plants when exposed to Zn deficiency and excess conditions. This study provides a mechanistic understanding of Zn uptake and translocation in plants and a new strategy to improve phytoremediation efficiency., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The present study aimed to evaluate the role of zinc transporter 3 (ZnT3) on multiple sclerosis (MS) pathogenesis. Experimental autoimmune encephalomyelitis (EAE), a disease model of multiple sclerosis, was induced by immunization with myelin oligodendrocyte glycoprotein (MOG35–55) in female mice. Three weeks after the initial immunization, demyelination, immune cell infiltration and blood brain barrier (BBB) disruption in the spinal cord were analyzed. Clinical signs of EAE first appeared on day 11 and reached a peak level on day 19 after the initial immunization. ZnT3 gene deletion profoundly reduced the daily clinical score of EAE. The ZnT3 gene deletion-mediated inhibition of the clinical course of EAE was accompanied by suppression of inflammation and demyelination in the spinal cord. The motor deficit accompanying neuropathological changes associated with EAE were mild in ZnT3 gene deletion mice. This reduction in motor deficit was accompanied by coincident reductions in demyelination and infiltration of encephalitogenic immune cells including CD4+ T cells, CD8+ T cells, CD20+ B cells and F4/80+ microglia in the spinal cord. These results demonstrate that ZnT3 gene deletion inhibits the clinical features and neuropathological changes associated with EAE. ZnT3 gene deletion also remarkably inhibited formation of EAE-associated aberrant synaptic zinc patches, matrix metalloproteinases-9 (MMP-9) activation and BBB disruption. Therefore, amelioration of EAE-induced clinical and neuropathological changes by ZnT3 gene deletion suggests that vesicular zinc may be involved in several steps of MS pathogenesis.

Intracellular zinc overload causes neuronal injury during the course of neurological disorders, whereas mild levels of zinc are beneficial to neurons. Previous reports indicated that non-steroidal anti-inflammatory drugs, including indomethacin and aspirin, can reduce the risk of ischemic stroke. This study found that chronic pretreatment of rats with indomethacin, a non-selective cyclooxygenase inhibitor, provided tolerance to ischemic injuries in an animal model of stroke by eliciting moderate zinc elevation in neurons. Consecutive intraperitoneal injection of indomethacin (3 mg/kg/day for 28 days) led to modest increases in intraneuronal zinc as well as synaptic zinc content, with no significant stimulation of neuronal death. Furthermore, indomethacin induced the expressions of intracellular zinc homeostatic and neuroprotective proteins, rendering the brain resistant against ischemic damages and improving neurological outcomes. However, administration of a zinc-chelator, N,N,N′,N′-tetra(2-picolyl)ethylenediamine (TPEN; 15 mg/kg/day), immediately after indomethacin administration eliminated the beneficial actions of the drug. Therefore, indomethacin preconditioning can modulate intracellular zinc availability, contributing to ischemic tolerance in the brain after stroke.

An effective vaccine to prevent the SARS-CoV-2 causing COVID-19 is yet to be approved. Further there is no drug that is specific to treat COVID-19. A number of antiviral drugs such as Ribavirin, Remdesivir, Lopinavir/ritonavir, Azithromycin and Doxycycline have been recommended or are being used to treat COVID-19 patients. In addition to these drugs, rationale and evidence have been presented to use chloroquine to treat COVID-19, arguably with certain precautions and criticism. In line with the proposed use of chloroquine, Nigella sativa (black seed) could be considered as a natural substitute that contains a number of bioactive components such as thymoquinone, dithymoquinone, thymohydroquinone, and nigellimine. Further benefits to use N . sativa could be augmented by Zn supplement. Notably, Zn has been proven to improve innate and adaptive immunity in the course of any infection, be it by pathogenic virus or bacteria. The effectiveness of the Zn salt supplement could also be enhanced with N . sativa as its major bioactive component might work as ionophore to allow Zn2+ to enter pneumocytes - the target cell for SARSCoV-2. Given those benefits, this review paper describes how N . sativa in combination with Zn could be useful as a complement to COVID-19 treatment., Competing Interests: The author declare that he has no competing interests., (© 2020 Elsevier GmbH. All rights reserved.)

Recent investigations of rodent Tmem163 suggest that it binds to and transports zinc as a dimer, and that alanine mutagenesis of its two species-conserved aspartate (D123A/D127A) residues proposed to bind zinc, perturbs protein function. Direct corroboration, however, is lacking whether it is an influx or efflux transporter in cells. We hypothesized that human TMEM163 is a zinc effluxer based on its predicted protein characteristics. We used cultured human cell lines that either stably or transiently expressed TMEM163, and pre-loaded the cells with zinc to determine transport activity. We found that TMEM163-expressing cells exhibited significant reduction of intracellular zinc levels as evidenced by two zinc-specific fluorescent dyes and radionuclide zinc-65. The specificity of the fluorescence signal was confirmed upon treatment with TPEN, a high-affinity zinc chelator. Multiple sequence alignment and phylogenetic analyses showed that TMEM163 is related to distinct members of the cation diffusion facilitator (CDF) protein family. To further characterize the efflux function of TMEM163, we substituted alanine in two homologous aspartate residues (D124A/D128A) and performed site-directed mutagenesis of several conserved amino acid residues identified as non-synonymous single nucleotide polymorphism (S61R, S95C, S193P, and E286K). We found a significant reduction of zinc efflux upon cellular expression of D124A/D128A or E286K protein variant when compared with wild-type, suggesting that these particular amino acids are important for normal protein function. Taken together, our findings demonstrate that TMEM163 effluxes zinc, and it should now be designated ZNT11 as a new member of the mammalian CDF family of zinc efflux transporters., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: YiiP is a bacterial zinc-for-proton antiporter belonging to the cation diffusion facilitator family. The zinc(II) ions are transported across the cell membrane, from the cytosol to the extracellular space., Methods: We performed atomistic molecular dynamics simulations of the YiiP dimer with zinc(II) ions in solution to elucidate how the metal ions interact with the protein while moving from the cytosol to the transport site., Results: We observed that of the two cavities of the dimer, only one was accessible from the cytosol during transport. Zinc(II) binding to D49 of the transport site triggered a rearrangement of the transmembrane domain that closed the accessible cavity. Finally, we analyzed the free-energy profiles of metal transit in the channel and observed the existence of a high barrier preventing release from the transport site., Conclusions: The observed dynamics is consistent with the dimer-dimer interface forming a stable scaffold against which the rest of the trans-membrane rearranges., General Significance: Zinc(II) transporters are present in all kingdoms of life. The present study highlights structural features that might be of general relevance., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Affective and cognitive processing of nociception contributes to the development of chronic pain and vice versa, pain may precipitate psychopathologic symptoms. We hypothesized a higher risk for the latter with immanent neurologic diseases and studied this potential interrelationship in progranulin-deficient mice, which are a model for frontotemporal dementia, a disease dominated by behavioral abnormalities in humans. Young naïve progranulin deficient mice behaved normal in tests of short-term memory, anxiety, depression and nociception, but after peripheral nerve injury, they showed attention-deficit and depression-like behavior, over-activity, loss of shelter-seeking, reduced impulse control and compulsive feeding behavior, which did not occur in equally injured controls. Hence, only the interaction of 'pain x progranulin deficiency' resulted in the complex phenotype at young age, but neither pain nor progranulin deficiency alone. A deep proteome analysis of the prefrontal cortex and olfactory bulb revealed progranulin-dependent alterations of proteins involved in synaptic transport, including neurotransmitter transporters of the solute carrier superfamily. In particular, progranulin deficiency was associated with a deficiency of nuclear and synaptic zinc transporters (ZnT9/Slc30a9; ZnT3/Slc30a3) with low plasma zinc. Dietary zinc supplementation partly normalized the attention deficit of progranulin-deficient mice, which was in part reminiscent of autism-like and compulsive behavior of synaptic zinc transporter Znt3-knockout mice. Hence, the molecular studies point to defective zinc transport possibly contributing to progranulin-deficiency-associated psychopathology. Translated to humans, our data suggest that neuropathic pain may precipitate cognitive and psychopathological symptoms of an inherent, still silent neurodegenerative disease., (Copyright © 2017. Published by Elsevier B.V.)

Zinc (Zn) is an essential trace element that is vital in a wide range of cellular machineries because of its effect on the expression and activity of various transcription factors and enzymes. Zn deficiency disturbs Zn homeostasis and has pathogenic consequences, including growth retardation and immune impairment in mammals. Zn homeostasis is tightly controlled by the coordinated activity of Zn transporters and metallothioneins, which regulate the distribution, storage, and intracellular and extracellular concentration of Zn. Recent reverse-genetic approaches using Zn transporter-deficient mice have revealed the physiological functions of specific Zn signaling axes (each formed by Zn and a Zn transporter) in various biological programs. In this review, we describe recent discoveries about the role of Zn transporters which facilitate cellular signaling through Zn uptake in physiology and pathogenesis, with particular focus on the influence of Zn signaling in systemic growth and immunity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Hepcidin mediates the hypoferremia of inflammation by inhibiting iron transfer into circulation; however, a regulator for the hypozincemia observed in individuals with acute and chronic inflammatory and infectious diseases is not known., Objective: The objective of this study was to determine the effects of hepcidin on zinc transport in intestinal epithelial cells., Methods: Differentiated human intestinal Caco-2 cells were untreated or treated with 1 μM hepcidin for 3-24 h. Zinc transport was assessed in cells seeded on Transwell inserts. Media from the apical and basolateral chambers were collected, and zinc concentrations were determined using 67 Zn. Labile zinc pools were imaged and quantified in cells loaded with FluoZin-3-AM and expression of metallothionein and the zinc transporters zrt-/irt-like protein (ZIP)4 (SLC39A4), ZIP5 (SLC39A5), ZIP14 (SLC39A14), and zinc transporter 1 (ZnT1) (SLC30A1) was determined. Cells were transfected with SLC40A1- or SLC30A1-specific small interfering RNA to knock down ferroportin and ZnT1 protein, respectively. Cell surface proteins were isolated by cell surface biotinylation and lysosomal and proteasomal degradation was inhibited by treating cells with chloroquine or MG132, respectively., Results: Hepcidin attenuated zinc transport, as cells treated with hepcidin exported 26% less 67 Zn (P < 0.05) into the basolateral chamber and retained 27% more cellular 67 Zn (P < 0.05) than did control cells. Labile zinc decreased, and the mRNA abundance of metallothionein increased by ∼50% in hepcidin-treated cells compared with control cells (P < 0.05). Hepcidin reduced ZnT1 protein by 75% (P < 0.05) compared with control cells. Hepcidin-mediated reductions in zinc export remained in ferroportin knockdown cells compared with untreated controls (P < 0.05), whereas knockdown of ZnT1 inhibited this effect (P ≥ 0.05). Hepcidin significantly reduced biotinylated cell surface ZnT1 compared with control cells (P < 0.05); chloroquine inhibited hepcidin-mediated degradation of ZnT1 (P ≥ 0.05), whereas MG132 had no effect (P < 0.05)., Conclusions: Hepcidin reduces intestinal zinc export by post-translationally downregulating ZnT1 through a lysosomal-mediated degradation pathway, indicating that hepcidin may contribute to the hypozincemia of inflammation and infection., (© 2016 American Society for Nutrition.)

Zinc is an essential nutrient for humans; however, a sensitive biomarker to assess zinc status has not been identified. The objective of this systematic review was to compile and assess studies that determined zinc transporter and/or metallothionein expression in various blood cell types and to determine their reliability and sensitivity to changes in dietary zinc. Sixteen studies were identified that determined the expression of zrt-, irt-like protein (ZIP) 1 [solute carrier family (SLC) 39A1], ZIP3 (SLC39A3), ZIP5 (SLC39A5), ZIP6 (SLC39A6), ZIP7 (SLC39A7), ZIP8 (SLC39A8), ZIP10 (SLC39A10), ZIP14 (SLC39A14), zinc transporter (ZnT)1 (SLC30A1), ZnT2 (SLC30A2), ZnT4 (SLC30A4), ZnT5 (SLC30A5), ZnT6 (SLC30A6), ZnT7 (SLC30A7), ZnT9 (SLC30A9), and/or metallothionein in various blood cells isolated from healthy adult men and women in response to zinc supplementation or depletion. Cell types included leukocytes, peripheral blood mononuclear cells, T lymphocytes, monocytes, and erythrocytes. ZIP1, ZnT1, and metallothionein were the most commonly measured proteins. Changes in ZIP1 and ZnT1 in response to zinc supplementation or depletion were not consistent across studies. Leukocyte metallothionein decreased with zinc depletion (-39% change from baseline, <5 mg Zn/d, n = 2 studies) and increased with zinc supplementation in a dose-dependent manner (35%, 15-22 mg Zn/d, n = 7 studies; 267%, 50 mg Zn/d, n = 2 studies) and at the earliest time points measured; however, no change or delayed response was observed in metallothionein in erythrocytes. A greater percentage of studies demonstrated that metallothionein in leukocyte subtypes was a more reliable (100%, n = 12; 69%, n = 16) and responsive (92%, n = 12; 82%, n = 11) indicator of zinc exposure than was plasma zinc, respectively. In conclusion, current evidence indicates that metallothionein in leukocyte subtypes may be a component in determining zinc status., (© 2016 American Society for Nutrition.)

Background: Lactation failure is common in overweight and obese women; however, the precise mechanism remains unknown., Objective: We tested the hypothesis that obesity-induced inflammation in the mammary gland (MG) redistributes subcellular zinc pools to promote cell death of mammary epithelial cells (MECs) and premature involution., Methods: Female DBA/2J mice were fed a high-fat (obese; 45% kcal from fat, n = 60) or control diet (lean; 10% kcal from fat, n = 50) for 5 wk and bred. MG cytokines and macrophage infiltration were determined by reverse transcriptase-polymerase chain reaction and F4/80 staining, respectively. Zinc concentration was analyzed by atomic absorption spectroscopy, and zinc transporters and markers of endoplasmic reticulum (ER) stress, autophagy, and involution were measured by immunoblot. To confirm effects of inflammation, tumor necrosis factor-α (TNF) or vehicle was injected into adjacent MGs of lean lactating C57BL/6 mice (n = 5) and cultured MECs (HC11 cells) were treated with TNF in vitro., Results: Seventy-seven percent of obese mice failed to lactate (lean: 39%; P < 0.001). Obese mice capable of lactating had greater macrophage infiltration (obese: 135 ± 40.4 macrophages/mm(2); lean: 63.8 ± 8.9 macrophages/mm(2); P < 0.001) and elevated TNF expression (P < 0.05), concurrent with lower zrt- irt-like protein 7 abundance (P < 0.05) and higher ER zinc concentration (obese: 0.36 ± 0.004 μg Zn/mg protein; lean: 0.30 ± 0.02 μg Zn/mg protein; P < 0.05) compared with lean mice. Heat shock protein 5 (HSPA5) expression (P < 0.05) was suppressed in the MG of obese mice, which was consistent with HSPA5 suppression in TNF-injected MGs (P < 0.01) and MECs treated with TNF in vitro (P < 0.01). Moreover, obesity increased lysosomal activity (P < 0.05) and autophagy in the MG, which corresponded to increased zinc transporter 2 abundance and lysosomal zinc concentration compared with lean mice (obese: 0.20 ± 0.02 μg Zn/mg protein; lean: 0.14 ± 0.01 μg Zn/mg protein; P < 0.05). Importantly, MGs of obese mice exhibited markers of apoptosis (P = 0.05) and involution (P < 0.01), which were not observed in lean mice., Conclusions: Diet-induced obesity created a proinflammatory MG microenvironment in mice, which was associated with zinc-mediated ER stress and autophagy and the activation of premature involution., (© 2015 American Society for Nutrition.)