Tolunay Beker Aydemir

Zinc transporter ZIP8 (SLC39A8) and zinc influence IFN-γ expression in activated human T cells

The zinc transporter ZIP8 is highly expressed in T cells derived from human subjects. T cell ZIP8 expression was markedly up‐regulated upon in vitro activation. T cells collected from human subjects who had received oral zinc supplementation (15 mg/day) had higher expression of the activation marker IFN‐γ upon in vitro activation, indicating a potentiating effect of zinc on T cell activation. Similarly, in vitro zinc treatment of T cells along with activation resulted in increased IFN‐γ expression with a maximum effect at 3.1 μM. Knockdown of ZIP8 in T cells by siRNA decreased ZIP8 levels in nonactivated and activated cells and concomitantly reduced secretion of IFN‐γ and perforin, both signatures of activation. Overexpression of ZIP8 by transient transfection caused T cells to exhibit enhanced activation. Confocal microscopy established that ZIP8 is localized to the lysosome where ZIP8 abundance is increased upon activation. Loss of lysosomal labile zinc in response to activation was measured by flow cytometry using a zinc fluorophore. Zinc between 0.8 and 3.1 μM reduced CN phosphatase activity. CN was also inhibited by the CN inhibitor FK506 and ZIP8 overexpression. The results suggest that zinc at low concentrations, through inhibition of CN, sustains phosphorylation of the transcription factor CREB, yielding greater IFN‐γ expression in T cells. ZIP8, through control of zinc transport from the lysosome, may provide a secondary level of IFN‐γ regulation in T cells.

Zinc transporter ZIP14 functions in hepatic zinc, iron and glucose homeostasis during the innate immune response (endotoxemia).

ZIP14 (slc39A14) is a zinc transporter induced in response to pro-inflammatory stimuli. ZIP14 induction accompanies the reduction in serum zinc (hypozincemia) of acute inflammation. ZIP14 can transport Zn2+ and non-transferrin-bound Fe2+ in vitro. Using a Zip14−/− mouse model we demonstrated that ZIP14 was essential for control of phosphatase PTP1B activity and phosphorylation of c-Met during liver regeneration. In the current studies, a global screening of ZIP transporter gene expression in response to LPS-induced endotoxemia was conducted. Following LPS, Zip14 was the most highly up-regulated Zip transcript in liver, but also in white adipose tissue and muscle. Using ZIP14−/− mice we show that ZIP14 contributes to zinc absorption from the gastrointestinal tract directly or indirectly as zinc absorption was decreased in the KOs. In contrast, Zip14−/− mice absorbed more iron. The Zip14 KO mice did not exhibit hypozincemia following LPS, but do have hypoferremia. Livers of Zip14−/− mice had increased transcript abundance for hepcidin, divalent metal transporter-1, ferritin and transferrin receptor-1 and greater accumulation of iron. The Zip14−/− phenotype included greater body fat, hypoglycemia and higher insulin levels, as well as increased liver glucose and greater phosphorylation of the insulin receptor and increased GLUT2, SREBP-1c and FASN expression. The Zip14 KO mice exhibited decreased circulating IL-6 with increased hepatic SOCS-3 following LPS, suggesting SOCS-3 inhibited insulin signaling which produced the hypoglycemia in this genotype. The results are consistent with ZIP14 ablation yielding abnormal labile zinc pools which lead to increased SOCS-3 production through G-coupled receptor activation and increased cAMP production as well as signaled by increased pSTAT3 via the IL-6 receptor, which inhibits IRS 1/2 phosphorylation. Our data show the role of ZIP14 in the hepatocyte is multi-functional since zinc and iron trafficking are altered in the Zip14−/− mice and their phenotype shows defects in glucose homeostasis.

The zinc transporter Zip14 influences c-Met phosphorylation and hepatocyte proliferation during liver regeneration in mice.

BACKGROUND & AIMS Zinc homeostasis in cells is maintained through tight regulation of zinc influx, efflux, and distribution to intracellular organelles by zinc transporters. The Zrt-Irt-like protein (ZIP) transporters facilitate zinc influx to the cytosol. Expression of the ZIP family member Zip14 can be induced by inflammatory cytokines, which also initiate liver regeneration. Hepatocyte proliferation is required for liver regeneration. Zinc regulates cell proliferation, tissue growth, and many mitogenic signaling pathways; we investigated its role in hepatocytes. METHODS Wild-type and Zip14(-/-) mice that underwent partial hepatectomy (70% of liver removed) were used as models of liver regeneration. We also analyzed AML12 hepatocytes that overexpressed Zip14. Proliferation was assessed with proliferating cell nuclear antigen, CD1, and Ki67 markers and along with assays of zinc content was related to protein tyrosine phosphatase 1B (PTP1B) and extracellular signal-regulated kinase 1/2 signaling. RESULTS Zip14 was up-regulated and hepatic zinc content increased during liver regeneration. Increased hepatic zinc inhibited activity of the phosphatase PTP1B and increased phosphorylation of c-Met, which promoted hepatocyte proliferation. AML12 cells that overexpressed Zip14 increased in zinc content and proliferation; PTP1B was inhibited and phosphorylation of c-Met increased. The increases in hepatic levels of zinc and hepatocyte proliferation that occurred following partial hepatectomy were not observed in Zip14(-/-) mice. CONCLUSIONS The transporter Zip14 mediates hepatic uptake of zinc during liver regeneration and for hepatocyte proliferation. These findings indicate that zinc transporter activity regulates liver tissue growth by sequestering zinc. Reagents that regulate ZIP14 activity might be developed as therapeutics to promote liver regeneration in patients with chronic liver disease.

Influence of ZIP14 (slc39A14) on intestinal zinc processing and barrier function

ZIP14 is a zinc transport protein with high expression in the small intestine and liver. Zip14 is upregulated during endotoxemia and leads to increased liver zinc content and transient hypozinemia. Since body zinc status and inflammation are associated with changes in intestinal permeability, we hypothesized that ZIP14 may influence intestinal permeability. Wild-type (WT) and Zip14 knockout (KO) mice were used to determine ZIP14-associated intestinal zinc metabolism and effects on permeability. Fractionation of plasma membranes revealed that ZIP14 is localized to the basolateral membrane of enterocytes. Studies utilizing (65)Zn administered by subcutaneous injection revealed greater zinc accumulation in the SI of Zip14 KO mice compared with WT mice. Isolation of endosomes confirmed the presence of ZIP14. Quantification of endosomal zinc concentration by FluoZin-3AM fluorescence demonstrated that zinc is trapped in endosomes of Zip14 KO mice. Intestinal permeability assessed both by plasma FITC-dextran following gavage and by serum endotoxin content was greater in Zip14 KO mice. Threonine phosphorylation of the tight junction protein occludin, which is necessary for tight junction assembly, was reduced in KO mice. Claudin 1 and 2, known to have an inverse relationship in regards to tight junction integrity, reflected impaired barrier function in KO jejunum. These data suggest involvement of ZIP14 in providing zinc for a regulatory role needed for maintenance of the intestinal barrier. In conclusion, ZIP14 is a basolaterally localized protein in enterocytes and is involved in endosomal trafficking of zinc and is necessary for proper maintenance of intestinal tight junctions.

Zinc transporter Slc39a14 regulates inflammatory signaling associated with hypertrophic adiposity.

Zinc is a signaling molecule in numerous metabolic pathways, the coordination of which occurs through activity of zinc transporters. The expression of zinc transporter Zip14 (Slc39a14), a zinc importer of the solute carrier 39 family, is stimulated under proinflammatory conditions. Adipose tissue upregulates Zip14 during lipopolysaccharide-induced endotoxemia. A null mutation of Zip14 (KO) revealed that phenotypic changes in adipose include increased cytokine production, increased plasma leptin, hypertrophied adipocytes, and dampened insulin signaling. Adipose tissue from KO mice had increased levels of preadipocyte markers, lower expression of the differentiation marker (PPARγ), and activation of NF-κB and STAT3 pathways. Our overall hypothesis was that ZIP14 would play a role in adipocyte differentiation and inflammatory obesity. Global Zip14 KO causes systemic endotoxemia. The observed metabolic changes in adipose metabolism were reversed when oral antibiotics were administrated, indicating that circulating levels of endotoxin were in part responsible for the adipose phenotype. To evaluate a mechanism, 3T3-L1 cells were differentiated into adipocytes and treated with siRNA to knock down Zip14. These cells had an impaired ability to mobilize zinc, which caused dysregulation of inflammatory pathways (JAK2/STAT3 and NF-κB). The Zip14 deletion may limit the availability of intracellular zinc, yielding the unique phenotype of inflammation coupled with hypertrophy. Taken together, these results suggest that aberrant zinc distribution observed with Zip14 ablation impacts adipose cytokine production and metabolism, ultimately increasing fat deposition when exposed to endotoxin. To our knowledge, this is the first investigation into the mechanistic role of ZIP14 in adipose tissue regulation and metabolism.

Gastric and Colonic Zinc Transporter ZIP11 (Slc39a11) in Mice Responds to Dietary Zinc and Exhibits Nuclear Localization

Zinc transporters have been characterized to further understand the absorption and metabolism of dietary zinc. Our goal was to characterize zinc transporter Slc39a11 (ZIP11) expression and its subcellular localization within cells of the murine gastrointestinal tract of mice and to determine if dietary zinc regulates ZIP11. The greatest ZIP11 expression was in the stomach, cecum, and colon. Both Zip11 mRNA and ZIP11 protein were shown to be downregulated during dietary zinc restriction (<1 mg Zn/kg) in the murine stomach tissue but were unaffected in the colon. Acute repletion with zinc did not restore Zip11 mRNA levels in the stomach. Immunohistochemistry (IHC) revealed high ZIP11 levels in the lower regions of gastric glands and parietal cells of the stomach. IHC analysis of the colon showed a marked ZIP11 abundance within the cytoplasm of the colonic epithelial cells. IHC also showed an increase in ZIP11 expression in the colon during zinc restriction. There is a robust abundance of ZIP11 in the nuclei of cells of both stomach and colon. Our experiments suggest that when dietary zinc intake is compromised, the colon may increase zinc transporter expression to improve the efficiency for absorption via increased expression of specific zinc transporters, including ZIP11 and also zinc transporter Slc39a4. In conclusion, ZIP11 is highly expressed within the murine stomach and colon and appears to be partially regulated by dietary zinc intake within these tissues. ZIP11 may play a specialized role in zinc homeostasis within these tissues, helping to maintain mucosal integrity and function.

Metal Transporter Zip14 ( Slc39a14 ) Deletion in Mice Increases Manganese Deposition and Produces Neurotoxic Signatures and Diminished Motor Activity

Mutations in human ZIP14 have been linked to symptoms of the early onset of Parkinsonism and Dystonia. This phenotype is likely related to excess manganese accumulation in the CNS. The metal transporter ZIP14 (SLC39A14) is viewed primarily as a zinc transporter that is inducible via proinflammatory stimuli. In vitro evidence shows that ZIP14 can also transport manganese. To examine a role for ZIP14 in manganese homeostasis, we used Zip14 knock-out (KO) male and female mice to conduct comparative metabolic, imaging, and functional studies. Manganese accumulation was fourfold to fivefold higher in brains of Zip14 KO mice compared with young adult wild-type mice. There was less accumulation of subcutaneously administered 54Mn in the liver, gallbladder, and gastrointestinal tract of the KO mice, suggesting that manganese elimination is impaired with Zip14 ablation. Impaired elimination creates the opportunity for atypical manganese accumulation in tissues, including the brain. The intensity of MR images from brains of the Zip14 KO mice is indicative of major manganese accumulation. In agreement with excessive manganese accumulation was the impaired motor function observed in the Zip14 KO mice. These results also demonstrate that ZIP14 is not essential for manganese uptake by the brain. Nevertheless, the upregulation of signatures of brain injury observed in the Zip14 KO mice demonstrates that normal ZIP14 function is an essential factor required to prevent manganese-linked neurodegeneration. SIGNIFICANCE STATEMENT Manganese is an essential micronutrient. When acquired in excess, manganese accumulates in tissues of the CNS and is associated with neurodegenerative disease, particularly Parkinson-like syndrome and dystonia. Some members of the ZIP metal transporter family transport manganese. Using mutant mice deficient in the ZIP14 metal transporter, we have discovered that ZIP14 is essential for manganese elimination via the gastrointestinal tract, and a lack of ZIP14 results in manganese accumulation in critical tissues such as the brain, as measured by MRI, and produces signatures of brain injury and impaired motor function. Humans with altered ZIP14 function would lack this gatekeeper function of ZIP14 and therefore would be prone to manganese-related neurological diseases.

Proteomic analysis shows the upregulation of erythrocyte dematin in zinc-restricted human subjects

BACKGROUND Although the importance of adequate zinc intake has been known for decades, the estimated global prevalence of zinc deficiency remains high. This substantiates the need for a specific and sensitive status assessment tool. OBJECTIVE The objective was to evaluate erythrocyte zinc transporters as candidate molecules with the potential of being a biomarker of dietary zinc status in humans. DESIGN A 24-d observational study with acclimation (7 d, 10.4 mg Zn/d), zinc-depletion (10 d, 0.3 mg Zn/d), and zinc-repletion (7 d, 29.5 mg Zn/d) phases was conducted in healthy men (n = 9). Proteomic approaches including Western blot analyses and tandem mass spectrometry were implemented to identify the zinc responsiveness of selected red blood cell membrane proteins. RESULTS Zinc transporter 1 (ZnT1) and Zrt/Irt-like proteins ZIP8 and ZIP10 were detected in human erythrocyte membranes. No effects of short-term dietary zinc depletion were observed on the amounts of these proteins. However, changes in a cytoskeletal protein, dematin, by zinc depletion were identified through the nonspecific signals produced by an anti-ZIP8 antibody. This response was further validated by a dematin-specific antibody and with erythrocytes collected from mice fed a zinc-deficient diet. CONCLUSIONS The presence of ZnT1, ZIP8, and ZIP10 in human red blood cells implicates their role in the regulation of cellular zinc metabolism in the human erythroid system. The zinc responsiveness of membrane dematin suggests its capability to serve as a biomarker for dietary zinc depletion and its involvement in impaired erythroid membrane fragility by zinc restriction. This trial was registered at clinicaltrials.gov as NCT01221129.

Hepatic ZIP14-mediated Zinc Transport Contributes to Endosomal Insulin Receptor Trafficking and Glucose Metabolism

Zinc influences signaling pathways through controlled targeted zinc transport. Zinc transporter Zip14 KO mice display a phenotype that includes impaired intestinal barrier function with low grade chronic inflammation, hyperinsulinemia, and increased body fat, which are signatures of diet-induced diabetes (type 2 diabetes) and obesity in humans. Hyperglycemia in type 2 diabetes and obesity is caused by insulin resistance. Insulin resistance results in inhibition of glucose uptake by liver and other peripheral tissues, principally adipose and muscle and with concurrently higher hepatic glucose production. Therefore, modulation of hepatic glucose metabolism is an important target for antidiabetic treatment approaches. We demonstrate that during glucose uptake, cell surface abundance of zinc transporter ZIP14 and mediated zinc transport increases. Zinc is distributed to multiple sites in hepatocytes through sequential translocation of ZIP14 from plasma membrane to early and late endosomes. Endosomes from Zip14 KO mice were zinc-deficient because activities of the zinc-dependent insulin-degrading proteases insulin-degrading enzyme and cathepsin D were impaired; hence insulin receptor activity increased. Transient increases in cytosolic zinc levels are concurrent with glucose uptake and suppression of glycogen synthesis. In contrast, Zip14 KO mice exhibited greater hepatic glycogen synthesis and impaired gluconeogenesis and glycolysis related to low cytosolic zinc levels. We can conclude that ZIP14-mediated zinc transport contributes to regulation of endosomal insulin receptor activity and glucose homeostasis in hepatocytes. Therefore, modulation of ZIP14 transport activity presents a new target for management of diabetes and other glucose-related disorders.

Hepatic ZIP14-mediated zinc transport is required for adaptation to endoplasmic reticulum stress.

Significance Unresolved endoplasmic reticulum (ER) stress corresponds with various chronic diseases, such as hepatic steatosis and diabetes. Although cellular zinc deficiency has been implicated in causing ER stress, the effect of disturbed zinc homeostasis on hepatic ER stress and a role for zinc during stress are unclear. This study reveals that ER stress increases hepatic zinc accumulation via enhanced expression of metal transporter ZIP14. Unfolded protein response-activated transcription factors ATF4 and ATF6α regulate Zip14 expression in hepatocytes. During ER stress, ZIP14-mediated zinc transport is critical for preventing prolonged apoptotic cell death and steatosis, thus leading to hepatic cellular adaptation to ER stress. These results highlight the importance of normal zinc transport for adaptation to ER stress and to reduce disease risk. Extensive endoplasmic reticulum (ER) stress damages the liver, causing apoptosis and steatosis despite the activation of the unfolded protein response (UPR). Restriction of zinc from cells can induce ER stress, indicating that zinc is essential to maintain normal ER function. However, a role for zinc during hepatic ER stress is largely unknown despite important roles in metabolic disorders, including obesity and nonalcoholic liver disease. We have explored a role for the metal transporter ZIP14 during pharmacologically and high-fat diet–induced ER stress using Zip14−/− (KO) mice, which exhibit impaired hepatic zinc uptake. Here, we report that ZIP14-mediated hepatic zinc uptake is critical for adaptation to ER stress, preventing sustained apoptosis and steatosis. Impaired hepatic zinc uptake in Zip14 KO mice during ER stress coincides with greater expression of proapoptotic proteins. ER stress-induced Zip14 KO mice show greater levels of hepatic steatosis due to higher expression of genes involved in de novo fatty acid synthesis, which are suppressed in ER stress-induced WT mice. During ER stress, the UPR-activated transcription factors ATF4 and ATF6α transcriptionally up-regulate Zip14 expression. We propose ZIP14 mediates zinc transport into hepatocytes to inhibit protein-tyrosine phosphatase 1B (PTP1B) activity, which acts to suppress apoptosis and steatosis associated with hepatic ER stress. Zip14 KO mice showed greater hepatic PTP1B activity during ER stress. These results show the importance of zinc trafficking and functional ZIP14 transporter activity for adaptation to ER stress associated with chronic metabolic disorders.