Absence of Slc39a14/Zip14 in mouse pancreatic beta cells results in hyperinsulinemia.

Zinc is an essential component of the insulin protein complex synthesized in ß cells. The intracellular compartmentalization and distribution of zinc are controlled by 24 transmembrane zinc transporters belonging to the ZnT or Zrt/Irt-like protein (ZIP) family. Downregulation of SLC39A14/ZIP14 has been reported in pancreatic islets of patients with type 2 diabetes (T2D) as well as mouse models of high-fat diet (HFD)- or db/db-induced obesity. Our previous studies observed mild hyperinsulinemia in mice with whole body knockout of Slc39a14 (Zip14 KO). Based on our current secondary data analysis from an integrative single-cell RNA-seq dataset of human whole pancreatic tissue, SLC39A14 (coding ZIP14) is the only other zinc transporter expressed abundantly in human ß cells besides well-known zinc transporter SLC30A8 (coding ZnT8). In the present work, using pancreatic ß cell-specific knockout of Slc39a14 (ß-Zip14 KO), we investigated the role of SLC39A14/ZIP14-mediated intracellular zinc trafficking in glucose-stimulated insulin secretion and subsequent metabolic responses. Glucose-stimulated insulin secretion, zinc concentrations, and cellular localization of ZIP14 were assessed using in vivo, ex vivo, and in vitro assays using ß-Zip14 KO, isolated islets, and murine cell line MIN6. Metabolic evaluations were done on both chow- and HFD-fed mice using time-domain nuclear magnetic resonance and a comprehensive laboratory animal monitoring system. ZIP14 localizes on the endoplasmic reticulum regulating intracellular zinc trafficking in ß cells and serves as a negative regulator of glucose-stimulated insulin secretion. Deletion of Zip14 resulted in greater glucose-stimulated insulin secretion, increased energy expenditure, and shifted energy metabolism toward fatty acid utilization. HFD caused ß-Zip14 KO mice to develop greater islet hyperplasia, compensatory hyperinsulinemia, and mild insulin resistance and hyperglycemia. This study provided new insights into the contribution of metal transporter ZIP14-mediated intracellular zinc trafficking in glucose-stimulated insulin secretion and subsequent metabolic responses.NEW & NOTEWORTHY Metal transporter SLC39A14/ZIP14 is downregulated in pancreatic islets of patients with T2D and mouse models of HFD- or db/db-induced obesity. However, the function of ZIP14-mediated intracellular zinc trafficking in ß cells is unknown. Our analyses revealed that SLC39A14 is the only Zn transporter expressed abundantly in human ß cells besides SLC30A8. Within the ß cells, ZIP14 is localized on the endoplasmic reticulum and serves as a negative regulator of insulin secretion, providing a potential therapeutic target for T2D.

Calcium and the Ca-ATPase SPCA1 modulate plasma membrane abundance of ZIP8 and ZIP14 to regulate Mn(II) uptake in brain microvascular endothelial cells.

Manganese (II) accumulation in human brain microvascular endothelial cells is mediated by the metal-ion transporters ZRT IRT-like protein 8 (ZIP8) and ZRT IRT-like protein 14 (ZIP14). The plasma membrane occupancy of ZIP14, in particular, is increased in cells treated with Mn2+, lipopolysaccharide, or IL-6, but the mechanism of this regulation has not been elucidated. The calcium-transporting type 2C member 1 ATPase, SPCA1, is a Golgi-localized Ca2+-uptake transporter thought to support Golgi uptake of Mn2+ also. Here, we show using surface protein biotinylation, indirect immunofluorescence, and GFP-tagged proteins that cytoplasmic Ca2+ regulates ZIP8- and ZIP14-mediated manganese accumulation in human brain microvascular endothelial cells by increasing the plasma membrane localization of these transporters. We demonstrate that RNAi knockdown of SPCA1 expression results in an increase in cytoplasmic Ca2+ levels. In turn, we found increased cytoplasmic Ca2+ enhances membrane-localized ZIP8 and ZIP14 and a subsequent increase in 54Mn2+ uptake. Furthermore, overexpression of WT SPCA1 or a gain-of-function mutant resulted in a decrease in cytoplasmic Ca2+ and 54Mn2+ accumulation. While addition of Ca2+ positively regulated ZIP-mediated 54Mn2+ uptake, we show chelation of Ca2+ diminished manganese transport. In conclusion, the modulation of ZIP8 and ZIP14 membrane cycling by cytoplasmic calcium is a novel finding and provides new insight into the regulation of the uptake of Mn2+ and other divalent metal ions-mediated ZIP metal transporters.

Enterocyte-specific deletion of metal transporter Zip14 (Slc39a14) alters intestinal homeostasis through epigenetic mechanisms.

Zinc has anti-inflammatory properties using mechanisms that are unclear. Zip14 (Slc39a14) is a zinc transporter induced by proinflammatory stimuli and is highly expressed at the basolateral membrane of intestinal epithelial cells (IECs). Enterocyte-specific Zip14 ablation (Zip14ΔIEC) in mice was developed to study the functions of this transporter in enterocytes. This gene deletion led to increased intestinal permeability, increased IL-6 and IFNγ expression, mild endotoxemia, and intestinal dysbiosis. RNA sequencing was used for transcriptome profiling. These analyses revealed differential expression of specific intestinal proinflammatory and tight junction (TJ) genes. Binding of transcription factors, including NF-κß, STAT3, and CDX2, to appropriate promoter sites of these genes supports the differential expression shown with chromatin immunoprecipitation assays. Total histone deacetylase (HDAC), and specifically HDAC3, activities were markedly reduced with Zip14 ablation. Intestinal organoids derived from ΔIEC mice display TJ and cytokine gene dysregulation compared with control mice. Differential expression of specific genes was reversed with zinc supplementation of the organoids. We conclude that zinc-dependent HDAC enzymes acquire zinc ions via Zip14-mediated transport and that intestinal integrity is controlled in part through epigenetic modifications.NEW & NOTEWORTHY We show that enterocyte-specific ablation of zinc transporter Zip14 (Slc39a14) results in selective dysbiosis and differential expression of tight junction proteins, claudin 1 and 2, and specific cytokines associated with intestinal inflammation. HDAC activity and zinc uptake are reduced with Zip14 ablation. Using intestinal organoids, the expression defects of claudin 1 and 2 are resolved through zinc supplementation. These novel results suggest that zinc, an essential micronutrient, influences gene expression through epigenetic mechanisms.

Hepatic and intestinal manganese excretion are both required to regulate brain manganese during elevated manganese exposure.

Manganese (Mn) is essential but neurotoxic at elevated levels. Under physiological conditions, Mn is primarily excreted by the liver, with the intestines playing a secondary role. Recent analyses of tissue-specific Slc30a10 or Slc39a14 knockout mice (SLC30A10 and SLC39A14 are Mn transporters) revealed that, under physiological conditions: 1) excretion of Mn by the liver and intestines is a major pathway that regulates brain Mn; and surprisingly, 2) the intestines compensate for loss of hepatic Mn excretion in controlling brain Mn. The unexpected importance of the intestines in controlling physiological brain Mn led us to determine the role of hepatic and intestinal Mn excretion in regulating brain Mn during elevated Mn exposure. We used liver- or intestine-specific Slc30a10 knockout mice as models to inhibit hepatic or intestinal Mn excretion. Compared with littermates, both knockout strains exhibited similar increases in brain Mn after elevated Mn exposure in early or later life. Thus, unlike physiological conditions, both hepatic and intestinal Mn excretion are required to control brain Mn during elevated Mn exposure. However, brain Mn levels of littermates and both knockout strains exposed to elevated Mn only in early life were normalized in later life. Thus, hepatic and intestinal Mn excretion play compensatory roles in clearing brain Mn accumulated by early life Mn exposure. Finally, neuromotor assays provided evidence consistent with a role for hepatic and intestinal Mn excretion in functionally modulating Mn neurotoxicity during Mn exposure. Put together, these findings substantially enhance understanding of the regulation of brain Mn by excretion.NEW & NOTEWORTHY This article shows that, in contrast with expectations from prior studies and physiological conditions, excretion of manganese by the intestines and liver is equally important in controlling brain manganese during human-relevant manganese exposure. The results provide foundational insights about the interorgan mechanisms that control brain manganese homeostasis at the organism level and have important implications for the development of therapeutics to treat manganese-induced neurological disease.

The Combined Inactivation of Intestinal and Hepatic ZIP14 Exacerbates Manganese Overload in Mice.

ZIP14 is a newly identified manganese transporter with high levels of expression in the small intestine and the liver. Loss-of-function mutations in ZIP14 can lead to systemic manganese overload, which primarily affects the central nervous system, causing neurological disorders. To elucidate the roles of intestinal ZIP14 and hepatic ZIP14 in maintaining systemic manganese homeostasis, we generated mice with single-tissue or two-tissue Zip14 knockout, including intestine-specific (Zip14-In-KO), liver-specific (Zip14-L-KO), and double (intestine and liver) Zip14-knockout (Zip14-DKO) mice. Zip14flox/flox mice were used as the control. Tissue manganese contents in these mice were compared using inductively coupled plasma mass spectrometry (ICP-MS) analysis. We discovered that although the deletion of intestinal ZIP14 only moderately increased systemic manganese loading, the deletion of both intestinal and hepatic ZIP14 greatly exacerbated the body's manganese burden. Our results provide new knowledge to further the understanding of manganese metabolism, and offer important insights into the mechanisms underlying systemic manganese overload caused by the loss of ZIP14.

Restriction of Manganese Intake Prevents the Onset of Brain Manganese Overload in Zip14-/- Mice.

As a newly identified manganese transport protein, ZIP14 is highly expressed in the small intestine and liver, which are the two principal organs involved in regulating systemic manganese homeostasis. Loss of ZIP14 function leads to manganese overload in both humans and mice. Excess manganese in the body primarily affects the central nervous system, resulting in irreversible neurological disorders. Therefore, to prevent the onset of brain manganese accumulation becomes critical. In this study, we used Zip14-/- mice as a model for ZIP14 deficiency and discovered that these mice were born without manganese loading in the brain, but started to hyper-accumulate manganese within 3 weeks after birth. We demonstrated that decreasing manganese intake in Zip14-/- mice was effective in preventing manganese overload that typically occurs in these animals. Our results provide important insight into future studies that are targeted to reduce the onset of manganese accumulation associated with ZIP14 dysfunction in humans.

HIF-1α Dependent Upregulation of ZIP8, ZIP14, and TRPA1 Modify Intracellular Zn2+ Accumulation in Inflammatory Synoviocytes.

Intracellular free zinc ([Zn2+]i) is mobilized in neuronal and non-neuronal cells under physiological and/or pathophysiological conditions; therefore, [Zn2+]i is a component of cellular signal transduction in biological systems. Although several transporters and ion channels that carry Zn2+ have been identified, proteins that are involved in Zn2+ supply into cells and their expression are poorly understood, particularly under inflammatory conditions. Here, we show that the expression of Zn2+ transporters ZIP8 and ZIP14 is increased via the activation of hypoxia-induced factor 1α (HIF-1α) in inflammation, leading to [Zn2+]i accumulation, which intrinsically activates transient receptor potential ankyrin 1 (TRPA1) channel and elevates basal [Zn2+]i. In human fibroblast-like synoviocytes (FLSs), treatment with inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1α), evoked TRPA1-dependent intrinsic Ca2+ oscillations. Assays with fluorescent Zn2+ indicators revealed that the basal [Zn2+]i concentration was significantly higher in TRPA1-expressing HEK cells and inflammatory FLSs. Moreover, TRPA1 activation induced an elevation of [Zn2+]i level in the presence of 1 µM Zn2+ in inflammatory FLSs. Among the 17 out of 24 known Zn2+ transporters, FLSs that were treated with TNF-α and IL-1α exhibited a higher expression of ZIP8 and ZIP14. Their expression levels were augmented by transfection with an active component of nuclear factor-κB P65 and HIF-1α expression vectors, and they could be abolished by pretreatment with the HIF-1α inhibitor echinomycin (Echi). The functional expression of ZIP8 and ZIP14 in HEK cells significantly increased the basal [Zn2+]i level. Taken together, Zn2+ carrier proteins, TRPA1, ZIP8, and ZIP14, induced under HIF-1α mediated inflammation can synergistically change [Zn2+]i in inflammatory FLSs.

The intestinal metal transporter ZIP14 maintains systemic manganese homeostasis.

ZIP14 (encoded by the solute carrier 39 family member 14 (SLC39A14) gene) is a manganese transporter that is abundantly expressed in the liver and small intestine. Loss-of-function mutations in SLC39A14 cause severe hypermanganesemia. Because the liver is regarded as the main regulatory organ involved in manganese homeostasis, impaired hepatic manganese uptake for subsequent biliary excretion has been proposed as the underlying disease mechanism. However, liver-specific Zip14 KO mice exhibit decreased manganese only in the liver and do not develop manganese accumulation in other tissues under normal conditions. This suggests that impaired hepatobiliary excretion is not the primary cause for manganese overload observed in individuals lacking functional ZIP14. We therefore hypothesized that increased intestinal manganese absorption could induce manganese hyperaccumulation when ZIP14 is inactivated. To elucidate the role of ZIP14 in manganese absorption, here we used CaCo-2 Transwell cultures as a model system for intestinal epithelia. The generation of a ZIP14-deficient CaCo-2 cell line enabled the identification of ZIP14 as the major transporter mediating basolateral manganese uptake in enterocytes. Lack of ZIP14 severely impaired basolateral-to-apical (secretory) manganese transport and strongly enhanced manganese transport in the apical-to-basolateral (absorptive) direction. Mechanistic studies provided evidence that ZIP14 restricts manganese transport in the absorptive direction via direct basolateral reuptake of freshly absorbed manganese. In support of such function of intestinal ZIP14 in vivo, manganese levels in the livers and brains of intestine-specific Zip14 KO mice were significantly elevated. Our findings highlight the importance of intestinal ZIP14 in regulating systemic manganese homeostasis.

The solute carriers ZIP8 and ZIP14 regulate manganese accumulation in brain microvascular endothelial cells and control brain manganese levels.

Manganese supports numerous neuronal functions but in excess is neurotoxic. Consequently, regulation of manganese flux at the blood-brain barrier (BBB) is critical to brain homeostasis. However, the molecular pathways supporting the transcellular trafficking of divalent manganese ions within the microvascular capillary endothelial cells (BMVECs) that constitute the BBB have not been examined. In this study, we have determined that ZIP8 and ZIP14 (Zrt- and Irt-like proteins 8 and 14) support Mn2+ uptake by BMVECs and that neither DMT1 nor an endocytosis-dependent pathway play any significant role in Mn2+ uptake. Specifically, siRNA-mediated knockdown of ZIP8 and ZIP14 coincided with a decrease in manganese uptake, and kinetic analyses revealed that manganese uptake depends on pH and bicarbonate and is up-regulated by lipopolysaccharide, all biochemical markers of ZIP8 or ZIP14 activity. Mn2+ uptake also was associated with cell-surface membrane presentation of ZIP8 and ZIP14, as indicated by membrane protein biotinylation. Importantly, surface ZIP8 and ZIP14 biotinylation and Mn2+-uptake experiments together revealed that these transporters support manganese uptake at both the apical, blood and basal, brain sides of BMVECs. This indicated that in the BMVECs of the BBB, these two transporters support a bidirectional Mn2+ flux. We conclude that BMVECs play a critical role in controlling manganese homeostasis in the brain.

Astrocytic expression of ZIP14 (SLC39A14) is part of the inflammatory reaction in chronic neurodegeneration with iron overload.

Neurodegeneration is associated with inflammation and mismanaged iron homeostasis, leading to increased concentration of non-transferrin-bound iron (NTBI) in the brain. NTBI can be taken up by cells expressing Zrt-, Irt-like protein-14 (ZIP14), which is regulated by iron overload and pro-inflammatory cytokines, for example, interleukin-1ß (IL-1ß) and IL-6. Here, we focus on the astrocytic involvement and regulation of ZIP14 in an experimental model of chronic neurodegeneration with inflammation and iron overload. Rats were unilaterally injected with ibotenic acid in striatum resulting in excitotoxicity-induced neuronal loss in substantia nigra pars reticulata (SNpr). ZIP14 expression was measured in SNpr using immunohistochemistry, western blotting, and RT-qPCR. Cultures of primary astrocytes were examined for Zip14 mRNA expression after stimulation with ferric ammonium citrate (FAC), IL-6, or IL-1ß. To study the involvement of ZIP14 in astrocytic iron uptake, uptake of 59 Fe was investigated after treatment with IL-1ß and siRNA-mediated ZIP14 knockdown. In the lesioned SNpr, reactive astrocytes, but not microglia, revealed increased ZIP14 expression with a main confinement to cell bodies and cellular processes. In astrocyte cultures, FAC and IL-1ß stimulation increased Zip14 expression and IL-1ß stimulation increased uptake of 59 Fe. Increased 59 Fe uptake was also observed after siRNA-mediated ZIP14 knockdown suggesting that lowering of ZIP14 impaired the balance between astrocytic uptake and export of iron. We conclude that astrocytes increase ZIP14 expression in response to inflammation and iron exposure and that ZIP14 seems pertinent for iron uptake in astrocytes and plays a role for a balanced astrocytic iron homeostasis.

Maintaining Translational Relevance in Animal Models of Manganese Neurotoxicity.

Manganese is an essential metal, but elevated brain Mn concentrations produce a parkinsonian-like movement disorder in adults and fine motor, attentional, cognitive, and intellectual deficits in children. Human Mn neurotoxicity occurs owing to elevated exposure from occupational or environmental sources, defective excretion (e.g., due to cirrhosis), or loss-of-function mutations in the Mn transporters solute carrier family 30 member 10 or solute carrier family 39 member 14. Animal models are essential to study Mn neurotoxicity, but in order to be translationally relevant, such models should utilize environmentally relevant Mn exposure regimens that reproduce changes in brain Mn concentrations and neurological function evident in human patients. Here, we provide guidelines for Mn exposure in mice, rats, nematodes, and zebrafish so that brain Mn concentrations and neurobehavioral sequelae remain directly relatable to the human phenotype.

The Functions of ZIP8, ZIP14, and ZnT10 in the Regulation of Systemic Manganese Homeostasis.

As an essential nutrient, manganese is required for the regulation of numerous cellular processes, including cell growth, neuronal health, immune cell function, and antioxidant defense. However, excess manganese in the body is toxic and produces symptoms of neurological and behavioral defects, clinically known as manganism. Therefore, manganese balance needs to be tightly controlled. In the past eight years, mutations of genes encoding metal transporters ZIP8 (SLC39A8), ZIP14 (SLC39A14), and ZnT10 (SLC30A10) have been identified to cause dysregulated manganese homeostasis in humans, highlighting the critical roles of these genes in manganese metabolism. This review focuses on the most recent advances in the understanding of physiological functions of these three identified manganese transporters and summarizes the molecular mechanisms underlying how the loss of functions in these genes leads to impaired manganese homeostasis and human diseases.

Reduced hepatic metallothionein expression in first trimester fetuses in response to intrauterine smoking exposure: a consequence of low maternal zinc levels?

STUDY QUESTION: Does maternal smoking in early pregnancy affect metallothionein 1 and 2 (MT1 and MT2) mRNA and protein expression in first trimester placenta or embryonic/fetal liver? SUMMARY ANSWER: In the first trimester, MT protein expression is seen only in liver, where smoking is associated with a significantly reduced expression. WHAT IS KNOWN ALREADY: Zinc homeostasis is altered by smoking. Smoking induces MT in the blood of smokers properly as a result of the cadmium binding capacities of MT. In term placenta MT is present and smoking induces gene and protein expression (MT2 in particular), but the MT presence and response to smoking have never been examined in first trimester placenta or embryonic/fetal tissues. STUDY DESIGN, SIZE, DURATION: Cross sectional study where the presence of MT mRNA and protein was examined at the time of the abortion. The material was collected with informed consent after surgical intervention and frozen immediately. For protein expression analysis, liver tissue originating from smoking exposed n = 10 and unexposed n = 12 pregnancies was used. For mRNA expression analyses, placental tissue originating from smokers n = 19 and non-smokers n = 23 and fetal liver tissue from smoking exposed n = 16 and smoking unexposed pregnancies n = 13, respectively, were used. PARTICIPANTS/MATERIALS, SETTING, METHODS: Tissues were obtained from women who voluntarily and legally chose to terminate their pregnancy between gestational week 6 and 12. Western blot was used to determine the protein expression of MT, and real-time PCR was used to quantify the mRNA expression of MT2A and eight MT1 genes alongside the expression of key placental zinc transporters: zinc transporter protein-1 (ZNT1), Zrt-, Irt-related protein-8 and -14 (ZIP8 and ZIP14). MAIN RESULTS AND THE ROLE OF CHANCE: A significant reduction in the protein expression of MT1/2 in liver tissue (P = 0.023) was found by western blot using antibodies detecting both MT forms. Overall, a similar tendency was observed on the mRNA level although not statistically significant. Protein expression was not present in placenta, but the mRNA regulation suggested a down regulation of MT as well. A suggested mechanism based on the known role of MT in zinc homeostasis could be that the findings reflect reduced levels of easily accessible zinc in the blood of pregnant smokers and hence a reduced MT response in smoking exposed fetal/embryonic tissues. LIMITATIONS AND REASONS FOR CAUTION: Smoking was based on self-reports; however, our previous studies have shown high consistency regarding cotinine residues and smoking status. Passive smoking could interfere but was found mainly among smokers. The number of fetuses was limited, and other factors such as medication and alcohol might affect the findings. Information on alcohol was not consistently obtained, and we cannot exclude that it was more readily obtained from non-users. In the study, alcohol consumption was reported by a limited number (less than 1 out of 5) of women but with more smokers consuming alcohol. However, the alcohol consumption reported was typically limited to one or few times low doses. The interaction between alcohol and smoking is discussed in the paper. Notably we would have liked to measure zinc status to test our hypothesis, but maternal blood samples were not available. WIDER IMPLICATIONS OF THE FINDINGS: Zinc deficiency-in particular severe zinc deficiency-can affect pregnancy outcome and growth. Our findings indicate that zinc homeostasis is also affected in early pregnancy of smokers, and we know from pilot studies that even among women who want to keep their babies, the zinc status is low. Our findings support that zinc supplements should be considered in particular to women who smoke. STUDY FUNDING/COMPETING INTEREST(S): We thank the Department of Biomedicine for providing laboratory facilities and laboratory technicians and the Lundbeck Foundation and Læge Sofus Carl Emil Friis og Hustru Olga Doris Friis Legat for financial support. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Iron uptake by ZIP8 and ZIP14 in human proximal tubular epithelial cells.

In patients with iron overload disorders, increasing number of reports of renal dysfunction and renal iron deposition support an association between increased iron exposure and renal injury. In systemic iron overload, elevated circulating levels of transferrin-bound (TBI) and non-transferrin-bound iron (NTBI) are filtered to the renal proximal tubules, where they may cause injury. However, the mechanisms of tubular iron handling remain elusive. To unravel molecular renal proximal tubular NTBI and TBI handling, human conditionally immortalized proximal tubular epithelial cells (ciPTECs) were incubated with 55Fe as NTBI and fluorescently labeled holo-transferrin as TBI. Ferrous iron importers ZIP8 and ZIP14 were localized in the ciPTEC plasma membrane. Whereas silencing of either ZIP8 or ZIP14 alone did not affect 55Fe uptake, combined silencing significantly reduced 55Fe uptake compared to control (p < 0.05). Furthermore, transferrin receptor 1 (TfR1) and ZIP14, but not ZIP8, colocalized with early endosome antigen 1 (EEA1). TfR1 and ZIP14 also colocalized with uptake of fluorescently labeled transferrin. Furthermore, ZIP14 silencing decreased 55Fe uptake after 55Fe-Transferrin exposure (p < 0.05), suggesting ZIP14 could be involved in early endosomal transport of TBI-derived iron into the cytosol. Our data suggest that human proximal tubular epithelial cells take up TBI and NTBI, where ZIP8 and ZIP14 are both involved in NTBI uptake, but ZIP14, not ZIP8, mediates TBI-derived iron uptake. This knowledge provides more insights in the mechanisms of renal iron handling and suggests that ZIP8 and ZIP14 could be potential targets for limiting renal iron reabsorption and enhancing urinary iron excretion in systemic iron overload disorders.

Effects of lead and lead-melatonin exposure on protein and gene expression of metal transporters, proteins and the copper/zinc ratio in rats.

Human lead (Pb) exposure induces many adverse health effects, including some related to lead accumulation in organs. Although lead bio-distribution in the body has been described, the molecular mechanism underlying distribution and excretion is not well understood. The transport of essential and toxic metals is principally mediated by proteins. How lead affects the expression of metal transporter proteins in the principal metal excretory organs, i.e., the liver and kidney, is unknown. Considering that co-administration of melatonin and lead reduces the toxic effects of lead and lead levels in the blood in vivo, we examined how lead and co-administration of lead and melatonin affect the gene and protein expression of metal transporter proteins (ZIP8, ZIP14, CTR1 and DMT1) in these organs. Rats were exposed intraperitoneally to lead or lead-melatonin. Our results show that Pb exposure induces changes in the protein and gene expression of ZIP8, ZIP14 and CTR1. Alterations in the copper/zinc ratio found in the blood, liver and kidney were likely related to these changes. With DMT1 expression (gene and protein), a positive correlation was found with lead levels in the kidney. Co-administration of melatonin and lead reduced lead-induced DMT1 expression through an unknown mechanism. This effect of melatonin relates to reduced lead levels in the blood and kidney. The metal transport protein function and our results suggest that DMT1 likely contributes to lead accumulation in organs. These data further elucidate the effects of lead on Cu and Zn and the molecular mechanism underlying lead bio-distribution in animals.

The plasma membrane metal-ion transporter ZIP14 contributes to nontransferrin-bound iron uptake by human ß-cells.

The relationship between iron and ß-cell dysfunction has long been recognized as individuals with iron overload display an increased incidence of diabetes. This link is usually attributed to the accumulation of excess iron in ß-cells leading to cellular damage and impaired function. Yet, the molecular mechanism(s) by which human ß-cells take up iron has not been determined. In the present study, we assessed the contribution of the metal-ion transporters ZRT/IRT-like protein 14 and 8 (ZIP14 and ZIP8) and divalent metal-ion transporter-1 (DMT1) to iron uptake by human ß-cells. Iron was provided to the cells as nontransferrin-bound iron (NTBI), which appears in the plasma during iron overload and is a major contributor to tissue iron loading. We found that overexpression of ZIP14 and ZIP8, but not DMT1, resulted in increased NTBI uptake by ßlox5 cells, a human ß-cell line. Conversely, siRNA-mediated knockdown of ZIP14, but not ZIP8, resulted in 50% lower NTBI uptake in ßlox5 cells. In primary human islets, knockdown of ZIP14 also reduced NTBI uptake by 50%. Immunofluorescence analysis of islets from human pancreatic sections localized ZIP14 and DMT1 nearly exclusively to ß-cells. Studies in primary human islets suggest that ZIP14 protein levels do not vary with iron status or treatment with IL-1ß. Collectively, these observations identify ZIP14 as a major contributor to NTBI uptake by ß-cells and suggest differential regulation of ZIP14 in primary human islets compared with other cell types such as hepatocytes.

Iron importers Zip8 and Zip14 are expressed in retina and regulated by retinal iron levels.

Intracellular retinal iron accumulation has been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of irreversible blindness among individuals over the age of 50. Ceruloplasmin/hephaestin double knockout mice (Cp/Heph DKO) and hepcidin knockout mice (Hepc KO) accumulate retinal iron and model some features of AMD. Two canonical pathways govern cellular iron import - transferrin-bound iron import and non-transferrin bound iron import. In Cp/Heph DKO and Hepc KO iron-loaded retinas, transferrin-bound iron import is downregulated. Despite this effort to reduce cellular iron burden, iron continues to accumulate in these retinas in an age-dependent manner. Quantitative RT-PCR and Western analysis were used to quantify the expression of three ferrous iron importers, Dmt1, Zip8, and Zip14, in wild-type (Wt), Cp/Heph DKO, and Hepc KO retinas. Zip8 and Zip14 protein levels were analyzed using Western analysis in mice injected intravitreally with either apo- or holo-transferrin to elucidate one possible mechanism of Zip14 regulation in the retina. Both zip8 and zip14 were expressed in the mouse retina. Paradoxically, protein levels of non-transferrin bound iron importers were upregulated in both Cp/Heph DKO and Hepc KO retinas. Intravitreal holo-transferrin injection decreased Zip 14 protein levels. These data indicate that Zip8 and Zip14 may take up increasing amounts of non-transferrin bound iron in these two mouse models of retinal iron accumulation. Their upregulation in these already iron-loaded retinas suggests a vicious cycle leading to toxicity.

Hepatic mobilization of zinc after an experimental surgery, and its relationship with inflammatory cytokines release, and expression of metallothionein and Zip14 transporter.

OBJECTIVE: To study the relationship between the release of inflammatory cytokines and mobilization of zinc into liver, and the expression of metallothionein and Zip14 transporter after an abdominal surgery in rats. MATERIALS: Thirty-five male Wistar rats were subjected to experimental surgical stress, then the subgroups of five animals were killed at 3, 6, 9, 12, 16, 20 and 24 h. Matched groups without surgery were used as controls. METHODS: Zinc levels were determined by AAS, intracellular zinc by zinquin and dithizone staining. Hepatic metallothionein was assayed by a Cd-saturation method, and IL-1ß, IL-6, and TNF-ß by immunoassays. Zip14 expression was analyzed by real-time RT-PCR, and protein level by immunohistochemistry and Western blot. RESULTS: Experimental surgery produced a hypozincemia, and the increase of hepatic zinc also produced the release of IL-1ß, IL-6 in serum, and the increase of hepatic MT. Histochemistry showed a decrease of free zinc at 3-6 h, but an increase at 9 h (zinquin); meanwhile, total intracellular zinc increased after 9 h (dithizone). RNAm and protein levels of Zip14 were elevated between 6 and 20 h after surgery. CONCLUSION: Biochemical changes described in this work could be part of the APR, and directed to respond to the damage produced during surgical trauma.

Nramp1 and Other Transporters Involved in Metal Withholding during Infection.

During the course of infection, many natural defenses are set up along the boundaries of the host-pathogen interface. Key among these is the host response to withhold metals to restrict the growth of invading microbes. This simple act of nutritional warfare, starving the invader of an essential element, is an effective means of limiting infection. The physiology of metal withholding is often referred to as "nutritional immunity," and the mechanisms of metal transport that contribute to this host response are the focus of this review.

Tissue-Specific Induction of Mouse ZIP8 and ZIP14 Divalent Cation/Bicarbonate Symporters by, and Cytokine Response to, Inflammatory Signals.

Mouse Slc39a8 and Slc39a14 genes encode ZIP8 and ZIP14, respectively, which are ubiquitous divalent cation/(HCO3-)2 symporters responsible for uptake of Zn2+, Fe2+, and Mn2+ into cells. Cd2+ and other toxic nonessential metals can displace essential cations, thereby entering vertebrate cells. Whereas Slc39a8 encodes a single protein, Slc39a14 has 2 exons 4 which, via alternative splicing, give rise to ZIP14A and ZIP14B; why differences exist in cell type-specific expression of ZIP14A and ZIP14B remains unknown. Inflammatory stimuli have been associated with upregulation of ZIP8 and ZIP14, but a systematic study of many tissues simultaneously in a laboratory animal following inflammatory cytokine exposure has not yet been reported. Herein, we show that C57BL/6J male mice--treated intraperitoneally with lipopolysaccharide or the proinflammatory cytokines tumor necrosis factor (TNF) or interleukin-6 (IL6)--exhibited quantatively very different, highly tissue-specific, and markedly time-dependent up- and downregulation of ZIP8, ZIP14A, and ZIP14B messenger RNA (mRNA) levels in 12 tissues. The magnitude of inflammatory response was confirmed by measuring the proinflammatory cytokine TNF, IL6, and interleukin-1ß mRNA levels in the same tissues of these animals. Our data suggest that most if not all tissues use ZIP8, ZIP14A, and/or ZIP14B for Zn2+ uptake, some tissues under basal conditions and others moreso when inflammatory stressors are present; collectively, this might lead to substantial alterations in plasma Zn2+ levels due to Zn2+ redistribution not just in liver but across many vital organs. In the context of cadmium-mediated toxicity, our data suggest that tissues other than liver, kidney, and lung should also be considered.