Inflammation markers predict zinc transporter gene expression in women with type 2 diabetes mellitus

The pathology of type 2 diabetes mellitus (DM) often is associated with underlying states of conditioned zinc deficiency and chronic inflammation. Zinc and omega-3 polyunsaturated fatty acids each exhibit anti-inflammatory effects and may be of therapeutic benefit in the disease. The present randomized, double-blind, placebo-controlled, 12-week trial was designed to investigate the effects of zinc (40 mg/day) and α-linolenic acid (ALA; 2 g/day flaxseed oil) supplementation on markers of inflammation [interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, C-reactive protein (CRP)] and zinc transporter and metallothionein gene expression in 48 postmenopausal women with type 2 DM. No significant effects of zinc or ALA supplementation were observed on inflammatory marker concentrations or fold change in zinc transporter and metallothionein gene expression. Significant increases in plasma zinc concentrations were observed over time in the groups supplemented with zinc alone or combined with ALA (P=.007 and P=.009, respectively). An impact of zinc treatment on zinc transporter gene expression was found; ZnT5 was positively correlated with Zip3 mRNA (P<.001) only in participants receiving zinc, while zinc supplementation abolished the relationship between ZnT5 and Zip10. IL-6 predicted the expression levels and CRP predicted the fold change of the ZnT5, ZnT7, Zip1, Zip7 and Zip10 mRNA cluster (P<.001 and P=.031, respectively). Fold change in the expression of metallothionein mRNA was predicted by TNF-α (P=.022). Associations among inflammatory cytokines and zinc transporter and metallothionein gene expression support an interrelationship between zinc homeostasis and inflammation in type 2 DM.     

ZnT4 provides zinc to zinc-dependent proteins in the trans-Golgi network critical for cell function and Zn export in mammary epithelial cells

Zinc (Zn) transporter 4 (ZnT4) plays a key role in mammary gland Zn metabolism. A mutation in ZnT4 (SLC30A4) that targets the protein for degradation is responsible for the "lethal milk" (lm/lm) mouse phenotype. ZnT4 protein is only detected in the secreting mammary gland, and lm/lm mice have ～35% less Zn in milk, decreased mammary gland size, and decreased milk secretion. However, the precise contribution of ZnT4 is unknown. We used cultured mouse mammary epithelial cells (HC11) and determined that ZnT4 was localized to the trans-Golgi network (TGN) and cell membrane and transported Zn from the cytoplasm. ZnT4-mediated Zn import into the TGN directly contributed to labile Zn accumulation as ZnT4 overexpression increased FluoZin3 fluorescence. Moreover, ZnT4 provided Zn for metallation of galactosyltransferase, a Zn-dependent protein localized within the TGN that is critical for milk secretion, and carbonic anhydrase VI, a Zn-dependent protein secreted from the TGN into milk. We further noted that ZnT4 relocalized to the cell membrane in response to Zn. Together these studies demonstrated that ZnT4 transports Zn into the TGN, which is critical for key secretory functions of the mammary cell.     

Zip1, Zip2, and Zip8 mRNA Expressions Were Associated with Growth Hormone Level During the Growth Hormone Provocation Test in Children with Short Stature

Short stature of children is affected by multiple factors. One of them is growth hormone (GH) deficiency. Growth hormone therapy can increase the final height of children with growth hormone deficiency. Zinc is found to induce dimerization and to enhance the bioactivity of human GH. Two gene families have been identified involved in zinc homeostasis. Previous studies in our laboratory have shown that Zip1, Zip2, Zip6, and ZnT1 mRNA were associated with zinc level in established human breast cancer in nude mice model; Zip8 was significantly lower in zinc-deficient Wistar rats in kidney. In this study, five zinc transporters: Zip1, Zip2, Zip6, Zip8, and ZnT1 were chosen. We aimed to investigate the mRNA expression of zinc transporters and to explore the relationship between zinc transporters and growth hormone in short stature children. Growth hormone provocation test is used to confirm the diagnosis of growth hormone deficiency. Six short children for the test were enrolled. At the same time, 15 sex- and age-matched normal children were enrolled as control. The expression levels of zinc transporters in peripheral blood mononuclear cells were determined by quantitative real-time PCR. Zip1 and Zip2 mRNA expression positively correlated with growth hormone level (r?=?0.5133, P?=?0.0371; r?=?0.6719, P?=?0.0032); Zip8 mRNA expression negatively correlated with growth hormone level (r?=??0.5264, P?=?0.0285) during the test in short stature children. The average expression level of Zip2 was significantly higher and Zip6, Zip8 mRNA levels were significantly lower in short stature children than in health controls at 0 min (P?<?0.05, P?<?0.05).     

Analysis of zinc transporter,<Emphasis Type="Italic"> hZnT4</Emphasis> (<Emphasis Type="Italic">Slc30A4</Emphasis>), gene expression in a mammary gland disorder leading to reduced zinc secretion into milk

Zinc deficiency, causing impaired growth and development, may have a nutritional or genetic basis. We investigated two cases of inherited zinc deficiency found in breast-fed neonates, caused by low levels of zinc in the maternal milk. This condition is different from acrodermatitis enteropathica but has similarities to the "lethal milk" mouse, where low levels of zinc in the milk of lactating dams leads to zinc deficiency in pups. The mouse disorder has been attributed to a defect in the ZnT4 gene. Little is known about the expression of the human orthologue, hZnT4 (Slc30A4). Sequence analysis of cDNA, real-time PCR and Western blot analysis of hZnT4, carried out on control cells and cells from unrelated mothers of two infants with zinc deficiency, showed no differences. The hZnT4 gene was highly expressed in mouthwash buccal cells compared with lymphoblasts and fibroblasts. The hZnT4 protein did not co-localise with intracellular free zinc pools, suggesting that hZnT4 is not involved in transport of zinc into vesicles destined for secretion into milk. This observation, combined with phenotypic differences between the "lethal milk" mouse and the human disorder, suggests that the "lethal milk" mouse is not the corresponding model for the human zinc deficiency condition.     

In Situ Dimerization of Multiple Wild Type and Mutant Zinc Transporters in Live Cells Using Bimolecular Fluorescence Complementation

Zinc transporters (ZnTs) facilitate zinc efflux and zinc compartmentalization, thereby playing a key role in multiple physiological processes and pathological disorders, presumed to be modulated by transporter dimerization. We recently proposed that ZnT2 homodimerization is the underlying basis for the dominant negative effect of a novel heterozygous G87R mutation identified in women producing zinc-deficient milk. To provide direct visual evidence for the in situ dimerization and function of multiple normal and mutant ZnTs, we applied here the bimolecular fluorescence complementation (BiFC) technique, which enables direct visualization of specific protein-protein interactions. BiFC is based upon reconstitution of an intact fluorescent protein including YFP when its two complementary, non-fluorescent N- and C-terminal fragments (termed YN and YC) are brought together by a pair of specifically interacting proteins. Homodimerization of ZnT1, -2, -3, -4, and -7 was revealed by high subcellular fluorescence observed upon co-transfection of non-fluorescent ZnT-YC and ZnT-YN; this homodimer fluorescence localized in the characteristic compartments of each ZnT. The validity of the BiFC assay in ZnT dimerization was further corroborated when high fluorescence was obtained upon co-transfection of ZnT5-YC and ZnT6-YN, which are known to form heterodimers. We further show that BiFC recapitulated the pathogenic role that ZnT mutations play in transient neonatal zinc deficiency. Zinquin, a fluorescent zinc probe applied along with BiFC, revealed the in situ functionality of ZnT dimers. Hence, the current BiFC-Zinquin technique provides the first in situ evidence for the dimerization and function of wild type and mutant ZnTs in live cells.     

Essential Role for Zinc Transporter 2 (ZnT2)-mediated Zinc Transport in Mammary Gland Development and Function during Lactation

The zinc transporter ZnT2 (SLC30A2) imports zinc into vesicles in secreting mammary epithelial cells (MECs) and is critical for zinc efflux into milk during lactation. Recent studies show that ZnT2 also imports zinc into mitochondria and is expressed in the non-lactating mammary gland and non-secreting MECs, highlighting the importance of ZnT2 in general mammary gland biology. In this study we used nulliparous and lactating ZnT2-null mice and characterized the consequences on mammary gland development, function during lactation, and milk composition. We found that ZnT2 was primarily expressed in MECs and to a limited extent in macrophages in the nulliparous mammary gland and loss of ZnT2 impaired mammary expansion during development. Secondly, we found that lactating ZnT2-null mice had substantial defects in mammary gland architecture and MEC function during secretion, including fewer, condensed and disorganized alveoli, impaired Stat5 activation, and unpolarized MECs. Loss of ZnT2 led to reduced milk volume and milk containing less protein, fat, and lactose compared with wild-type littermates, implicating ZnT2 in the regulation of mammary differentiation and optimal milk production during lactation. Together, these results demonstrate that ZnT2-mediated zinc transport is critical for mammary gland function, suggesting that defects in ZnT2 not only reduce milk zinc concentration but may compromise breast health and increase the risk for lactation insufficiency in lactating women.     

Zinc transporter gene expression and glycemic control in post-menopausal women with Type 2 diabetes mellitus

Type 2 diabetes mellitus (DM) is associated often with underlying zinc deficiency and nutritional supplements such as zinc may be of therapeutic benefit in the disease. In a randomized, double-blind, placebo-controlled, 12-week trial in postmenopausal women (n = 48) with Type 2 DM we investigated the effects of supplementation with zinc (40 mg/d) and flaxseed oil (FSO; 2 g/d) on the gene expression of zinc transporters (ZnT1, ZnT5, ZnT6, ZnT7, ZnT8, Zip1, Zip3, Zip7, and Zip10) and metallothionein (MT-1A, and MT-2A), and markers of glycemic control (glucose, insulin, glycosylated hemoglobin [HbA1c]). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. No significant effects of zinc or FSO supplementation were observed on glycemic marker concentrations, HOMA-IR or fold change over 12 weeks in zinc transporter and metallothionein gene expression. In multivariate analysis, the change over 12 weeks in serum glucose concentrations (P = 0.001) and HOMA-IR (P = 0.001) predicted the fold change in Zip10. In secondary analysis, marginal statistical significance was observed with the change in both serum glucose concentrations (P = 0.003) and HOMA-IR (P = 0.007) being predictive of the fold change in ZnT6. ZnT8 mRNA expression was variable; HbA1c levels were higher (P = 0.006) in participants who exhibited ZnT8 expression compared to those who did not. The significant predictive relationships between Zip10, ZnT6, serum glucose and HOMA-IR are preliminary, as is the relationship between HbA1c and ZnT8; nevertheless the observations support an association between Type 2 DM and zinc homeostasis that requires further exploration.     

Increased inflammatory response in aged mice is associated with age-related zinc deficiency and zinc transporter dysregulation

Aging is a complex process associated with physiological changes in numerous organ systems. In particular, aging of the immune system is characterized by progressive dysregulation of immune responses, resulting in increased susceptibility to infectious diseases, impaired vaccination efficacy and systemic low-grade chronic inflammation. Increasing evidence suggest that intracellular zinc homeostasis, regulated by zinc transporter expression, is critically involved in the signaling and activation of immune cells. We hypothesize that epigenetic alterations and nutritional deficits associated with aging may lead to zinc transporter dysregulation, resulting in decreases in cellular zinc levels and enhanced inflammation with age. The goal of this study was to examine the contribution of age-related zinc deficiency and zinc transporter dysregulation on the inflammatory response in immune cells. The effects of zinc deficiency and age on the induction of inflammatory responses were determined using an in vitro cell culture system and an aged mouse model. We showed that zinc deficiency, particularly the reduction in intracellular zinc in immune cells, was associated with increased inflammation with age. Furthermore, reduced Zip 6 expression enhanced proinflammatory response, and age-specific Zip 6 dysregulation correlated with an increase in Zip 6 promoter methylation. Furthermore, restoring zinc status via dietary supplementation reduced aged-associated inflammation. Our data suggested that age-related epigenetic dysregulation in zinc transporter expression may influence cellular zinc levels and contribute to increased susceptibility to inflammation with age.     

Zinc transport by respiratory epithelial cells and interaction with iron homeostasis

Despite recurrent exposure to zinc through inhalation of ambient air pollution particles, relatively little information is known about the homeostasis of this metal in respiratory epithelial cells. We describe zinc uptake and release by respiratory epithelial cells and test the postulate that Zn²⁺ transport interacts with iron homeostasis in these same cells. Zn²⁺ uptake after 4 and 8 h of exposure to zinc sulfate was concentration- and time-dependent. A majority of Zn²⁺ release occurred in the 4 h immediately following cell exposure to ZnSO₄. Regarding metal importers, mRNA for Zip1 and Zip2 showed no change after respiratory epithelial cell exposure to zinc while mRNA for divalent metal transporter (DMT)1 increased. Western blot assay for DMT1 protein supported an elevated expression of this transport protein following zinc exposure. RT-PCR confirmed mRNA for the metal exporters ZnT1 and ZnT4 with the former increasing after ZnSO₄. Cell concentrations of ferritin increased with zinc exposure while oxidative stress, measured as lipid peroxides, was decreased supporting an anti-oxidant function for Zn²⁺. Increased DMT1 expression, following pre-incubations of respiratory epithelial cells with TNF-α, IFN-γ, and endotoxin, was associated with significantly decreased intracellular zinc transport. Finally, incubations of respiratory epithelial cells with both zinc sulfate and ferric ammonium citrate resulted in elevated intracellular concentrations of both metals. We conclude that exposure to zinc increases iron uptake by respiratory epithelial cells. Elevations in cell iron can possibly affect an increased expression of DMT1 and ferritin which function to diminish oxidative stress. Comparable to other metal exposures, changes in iron homeostasis may contribute to the biological effects of zinc in specific cells and tissues.     

Immunohistochemical analysis of ZnT1, 4, 5, 6, and 7 in the mouse gastrointestinal tract.

Expression of five zinc transporters (ZnT1, 4, 5, 6, and 7) of the Slc30 family in the mouse gastrointestinal tract was studied by immunohistochemical analysis. Results demonstrated unique expression patterns, levels, and cellular localization among ZnT proteins in the mouse gastrointestinal tract with some overlapping. ZnT1 was abundantly expressed in the epithelium of the esophagus, duodenum of the small intestine, and cecum of the large intestine. ZnT4 was predominantly detected in the large intestine. ZnT5 was mainly expressed in the parietal cell of the stomach and in the absorptive epithelium of the duodenum and jejunum. ZnT6 was predominantly detected in the chief cell of the stomach, columnar epithelial cells of the jejunum, cecum, colon, and rectum. Lastly, ZnT7 was observed in all epithelia of the mouse gastrointestinal tract with the highest expression in the small intestine. Expression of ZnT proteins in the absorptive epithelial cell of the gastrointestinal tract suggests that ZnT proteins may play important roles in zinc absorption and endogenous zinc secretion.     

Targeting of the mouse Slc39a2 (Zip2) gene reveals highly cell-specific patterns of expression, and unique functions in zinc, iron, and calcium homeostasis

Fourteen members of the Slc39a superfamily of metal ion uptake transporters have been identified in mice and humans, but the physiological functions of most remain obscure. Herein, we created mice with Zip2 (Slc39a2) genes in which the open reading frame was replaced with that of the enhanced green fluorescent protein (EGFP), to study temporal and spatial patterns of Zip2 gene expression and examine the physiological roles of this transporter. Expression of this gene was remarkably cell-type specific and developmentally regulated in pericentral hepatocytes, developing keratinocytes, and a subset of immature dendritic cells in the immune system. In addition, the Zip2 gene was transiently expressed in giant trophoblast cells in the placenta. Although the Zip2 gene was not essential under conditions of normal dietary zinc, it played an important role in adapting to dietary zinc deficiency during pregnancy, and in the homeostasis of iron in the liver as well as iron and calcium in developing embryos. These studies suggest that active expression of the Zip2 gene in these few specific cell types, aforementioned, plays a particularly important role during zinc deficiency. These studies further reveal novel interactions between zinc transporter function and the homeostasis of other essential metals.     

Increased abundance of labile intracellular zinc during cell proliferation was due to increased retention of extracellular zinc in 3T3 cells

Platelet-derived growth factor (PDGF)-, epidermal growth factor (EGF)- and insulin-like growth factor I (IGF-I)-stimulated cell proliferation in 3T3 cells was accompanied by increased abundance of labile intracellular pool of zinc (LIPZ). However, the origin and regulation of this cell proliferation-associated increase in the abundance of LIPZ are unknown. Cellular zinc homeostasis involves zinc transporters and metallothionein. The objectives of this study were to determine whether cell proliferation-associated increase in the abundance of LIPZ was a result of an increased zinc uptake and to assess the involvement of zinc transporters and metallothionein in this cell proliferation-associated increase in the abundance of LIPZ in 3T3 fibroblasts. Zinc transporters assessed included both zinc importer (Zip1) and zinc exporters (ZnT1, ZnT2 and ZnT4). Growth factors increased the abundance of LIPZ while total cellular zinc concentration remained unaffected, demonstrating that LIPZ was responsive to the increased needs for zinc during growth factor-stimulated cell proliferation. Growth factors also increased net zinc retention as indicated by higher 65zinc radioactivity and elevated mRNA levels of Zip1, ZnT1 and ZnT4. Although zinc is essential to cell proliferation, excessive cellular zinc accumulation causes cytotoxicity. Collectively, these observations suggest that increase in the abundance of LIPZ during growth factor-stimulated cell proliferation was due to increased net retention of extracellular zinc, which was apparently achieved through a coordinated up-regulation of the expression of transporters involved in both zinc influx and efflux to ensure adequate supply of zinc to sustain cell proliferation, yet to prevent potential zinc cytotoxicity in 3T3 cells.     

Up-regulation of Slc39A2(Zip2) mRNA in peripheral blood mononuclear cells from patients with pulmonary tuberculosis

Zinc is the most common trace mineral after iron in the human body. In organisms, zinc transporters help zinc influx and efflux from cells. A previous study has reported that Zip2 was up-regulated over 27-fold in human monocytic THP-1 cells, when intracellular zinc was depleted by TPEN. Our study found Zip2 was over-expressed in leukocytes of asthmatic infants, especially those in which the serum zinc level was lower than those in healthy infants. Pulmonary tuberculosis (PTB) patients have significantly low serum zinc levels. Here we investigated whether Zip2 level was changed in the patients with PTB. Zip2 mRNA and protein levels in peripheral blood mononuclear cells (PBMC) from PTB (n ₁ = 23) and healthy controls (n ₂ = 42) were detected by quantitative real-time PCR and western blot, respectively. mRNA expression levels of another four zinc transporters, Zip1, Zip6, Zip8 and ZnT1, were detected by quantitative real-time PCR. Zip2 mRNA level was significantly up-regulated in PTB patients (P = 0.001), and Zip8 mRNA level was significantly down-regulated compared with control individuals (P < 0.001). In contrast, there were no significant changes in mRNA levels of Zip1, Zip6 and ZnT1 in either group (P > 0.05). Zip2 protein expression levels increased in PTB patients compared with control individuals. Our study found that knockdown of ZIP2 with siRNA caused a decrease in Zip2 levels in PBMC of PTB patients, while reducing the expression of INF-γ (P < 0.01) and increasing the expression of IL-6(P < 0.01). These data provide evidence that increased expression of Zip2 gene is closely associated with immunity of PTB patients, suggesting that the Zip2 gene may play a key role in the initial infection control of the human body, by promoting and maintaining the immune response of adaptive T cells.