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The SLC39A family of metal transporters was identified through homologies with the Zrt- and Irt-like (ZIP) proteins from yeast and plants. Of all the ZIP transporters, ZIP14 is arguably the most robustly characterized in terms of function at the integrative level. Mice with a global knockout of Zip14 are viable, thus providing the opportunity to conduct physiologic experiments. In mice, Zip14 expression is highly tissue specific, with the greatest abundance in the jejunum > liver > heart > kidney > white adipose tissue > skeletal muscle > spleen > pancreas. A unique feature of Zip14 is its upregulation by proinflammatory conditions, particularly increased interleukin 6 (IL-6) and nitric oxide. The transcription factors AP-1, ATF4, and ATF6α are involved in Zip14 regulation. ZIP14 does not appear to be zinc-regulated. The Zip14 knockout phenotype shows multiple sites of ZIP14 function, including the liver, adipose tissue, brain, pancreas, and bone. A prominent feature of the Zip14 ablation is a reduction in intestinal barrier function and onset of metabolic endotoxemia. Many aspects of the phenotype are accentuated with age and accompany increased circulating IL-6. Studies with 65Zn, 59Fe [nontransferrin-bound iron (NTBI)] and 54Mn show that ZIP14 transports these metals. At a steady state, the plasma concentrations of zinc, NTBI, and manganese are such that zinc ions are the major substrate available for ZIP14 at the cell surface. Upregulation of ZIP14 accounts for the hypozincemia and hepatic zinc accumulation associated with acute inflammation and sepsis and is required for liver regeneration and resistance to endoplasmic reticulum (ER) stress. Zip14 ablation in mice produces a defect in manganese excretion that leads to excess manganese accumulation in the brain that produces characteristics of Parkinsonism.

Background: Several in vitro studies have shown that zinc deficiency could induce endoplasmic reticulum (ER) stress, resulting in activation of the unfolded protein response., Objective: We aimed to determine whether consumption of a zinc-deficient diet (ZnD) triggers ER stress and to understand the impact of dietary zinc intake on ER stress-induced apoptosis using a mouse model., Methods: Young adult (8-16 wk of age) male mice of strain C57BL/6 were fed either a ZnD (<1 mg/kg diet), or a zinc-adequate diet (ZnA; 30 mg/kg diet). After 2 wk, liver, pancreas, and serum samples were collected and analyzed for indexes of ER stress. In another experiment, mice were fed either a ZnD, a ZnA, or a zinc-supplementation diet (ZnS; 180 mg/kg diet). After 2 wk, vehicle or tunicamycin (TM; 2 mg/kg body weight) was administered to mice to model ER stress. Liver and serum were analyzed for indexes of ER stress to evaluate the effects of zinc status., Results: Mice fed a ZnD did not activate the apoptotic and ER stress markers in the liver or pancreas. During the TM challenge, mice fed a ZnD showed greater C/EBP-homologous protein expression in the liver (3.8-fold, P < 0.01) than did ZnA-fed mice. TM-treated mice fed a ZnD also had greater terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling-positive cells in the liver (2.2-fold, P < 0.05), greater hepatic triglyceride accumulation (1.5-fold, P < 0.05), greater serum alanine aminotransferase activity (1.6-fold, P < 0.05), and greater protein-tyrosine phosphatase 1B activity (1.5-fold, P < 0.05), respectively, than did those fed a ZnA. No significant differences were observed in these parameters between mice fed ZnAs and ZnSs., Conclusions: Consumption of a ZnD per se is not a critical factor for induction of ER stress in mice; however, once ER stress is triggered, adequate dietary zinc intake is required for suppressing apoptotic cell death and further insults in the liver of mice., Competing Interests: 2 Author disclosures: M-H Kim, TB Aydemir, and RJ Cousins, no conflicts of interest., (© 2016 American Society for Nutrition.)

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.

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.

Zinc is essential for normal erythroid cell functions and therefore intracellular zinc homeostasis during erythroid differentiation is tightly regulated. However, a characterization of zinc transporters in erythrocytes has not been conducted. The membrane fraction of mature mouse RBC was screened for zinc transporter expression using western analysis as a first step in the characterization process. ZnT1, Zip8, and Zip10 were detected among the 12 transporter proteins tested. We examined expression of these zinc transporters during erythropoietin (EPO)-induced differentiation of splenic erythroid progenitor cells into reticulocytes. Both Zip8 and Zip10 mRNA increased by 2-6 h after addition of EPO to the cells. In contrast, maximal RNA levels for the zinc transporter ZnT1 and erythroid delta-aminolevulinic acid synthase were only produced by 24 h after EPO. We confirmed these changes in transcript abundance by western analysis. Dietary zinc status influences zinc-dependent functions of RBC. To determine whether the identified zinc transporters respond to dietary zinc status, mice were fed a zinc-deficient or control diet. Incorporation of (65)Zn into erythrocytes in vitro was significantly increased in cells from the zinc-deficient mice. Western analysis and densitometry revealed that erythrocyte Zip10 was upregulated and ZnT1 was downregulated in the zinc-depleted mice. Zip8 was not affected by restricted zinc intake. Collectively, these data suggest that the zinc transporters ZnT1, Zip8, and Zip10 are important for zinc homeostasis in erythrocytes and that ZnT1 and Zip10 respond to the dietary zinc supply.

Differential mRNA display and cDNA array analysis have identified zinc-regulated genes in small intestine, thymus and monocytes. The vast majority of the transcriptome is not influenced by dietary zinc intake, high or low. Of the genes that are zinc regulated, most are involved in signal transduction (particularly influencing the immune response), responses to stress and redox, growth and energy utilization. Among the genes identified are uroguanylin (UG), cholecystokinin, lymphocyte-specific protein tyrosine kinase (LCK), T-cell cytokine receptor, heat shock proteins and the DNA damage repair and recombination protein-23B. Zinc transporters (ZnT) help regulate the supply of this micronutrient to maintain cellular functions. Expression of ZnT-1 and -2 is regulated by dietary zinc in many organs including small intestine and kidney. ZnT-4 is ubiquitously expressed but is refractory to zinc intake. Expression of ZnT-1, -2 and -4 changes markedly during gestation and lactation from highly abundant to undetectable. Each ZnT has an endosomal-like appearance in the tissues examined. Upregulation of ZnT-1 and ZnT-2 by dietary zinc strongly implicates these transporters in zinc acquisition and/or storage for subsequent systemic needs. THP-1 cells were used as a model to examine the response of human cells to changes in zinc status. Based on mRNA quantities, Zip1 and ZnT-5 were the most highly expressed. Zinc depletion of these cells decreased expression of all transporters except Zip2, where expression increased markedly. Collectively, these findings provide a genomic footprint upon which to address the biological and clinical significance of zinc and new avenues for status assessment.

Zinc metabolism is controlled within relatively restricted limits throughout the life cycle. Expression and localization of zinc transporters 1, 2 and 4 during pregnancy and lactation in small intestine, mammary gland and liver of the rat were investigated using Northern analysis, Western blotting and immunohistochemistry. In maternal tissues, zinc transporter 4 was the most widely expressed among these zinc transporters in the tissues examined. In small intestine and liver, zinc transporter 4 increased from levels found during late gestation, but zinc transporter 1 did not. Zinc transporter 2 expression in small intestine was transient, being highest around parturition, and was not detected in liver. Immunohistochemistry revealed unique patterns of zinc transporter localization at different stages of development. In the placenta, zinc transporters 1 and 4 were found concentrated along the villous visceral splanchnopleure. In the mammary gland, zinc transporter 4 was most abundant in cells surrounding the alveolar ducts and oriented to the basement lamina. All three transporters were highly expressed in neonatal small intestine, principally near the apical surface, but zinc transporters 1 and 4 increased in abundance at the basolateral surface during development. Zinc transporter 2 was oriented apically, directly adjacent to the microvilli of enterocytes. Within the intestine, expression of each transporter was limited to enterocytes. These results support a role for these transporters in maintaining an adequate zinc supply derived from the maternal diet for zinc acquisition and use by the fetus and neonate.

Deficient intake of zinc from the diet upregulates both uroguanylin (UG) and inducible nitric oxide synthase (iNOS) expression in rats. Because these changes influence intestinal fluid secretion and intestinal cell pathophysiology, they relate to the incidence of diarrheal disease and its reversal by zinc as well as intestinal inflammation in general. A model of moderate zinc deficiency in rats, which changes molecular indices of zinc deficiency, was used to further explore the effects of the proinflammatory cytokine interleukin (IL)-1alpha and zinc repletion on these changes. IL-1alpha has been shown to have a role in the intestinal inflammation that occurs with bacterial infection. Our results showed a permissive effect of zinc deficiency on both UG and iNOS expression. Specifically, UG expression was responsive to zinc deficiency and IL-1alpha challenge, which were additive when combined, whereas iNOS expression was upregulated by IL-1alpha only during the deficiency. Immunohistochemistry showed that the increase in UG was limited to enterocytes of the upper villus but, in contrast, the increase in iNOS was principally in cells of the lamina propria of IL-1alpha-treated rats. Cells exhibiting UG upregulation did not co-express serotonin. Repletion with zinc reversed upregulation of the iNOS gene within 1 d, whereas UG upregulation required 3-4 d to return to normal. This differential response to repletion suggests that mechanisms of UG and iNOS dysregulation are different. Dysregulation of both genes may contribute to the severity of zinc-responsive diarrheal disease and intestinal inflammatory disease.

The detrimental sequelae of severe zinc deficiency on the thymus and T-lymphocyte compartment of the mammalian immune system have been established, but underlying mechanisms remain unknown. Hypothesizing that the alterations in T-lymphocyte number and function observed during zinc deficiency may result from changes in gene expression, we sought to compare thymic mRNA expression profiles of zinc-deficient and zinc-normal mice utilizing cDNA arrays. For our murine model described herein, 3 wk of dietary zinc deficiency did not perturb food intake or growth rate in young adult, outbred mice, but significantly depressed multiple parameters of zinc status. Furthermore, fluorescence-activated cell sorting (FACS) analysis demonstrated no changes in thymocyte populations expressing the cell surface markers CD3, CD4 or CD8, establishing that observed changes in mRNA abundances were not attributable to different thymocyte populations. Yet notably, at this moderate level of zinc deficiency, cDNA array analysis identified four potentially zinc-regulated mRNAs whose modulation was confirmed independently, twice, using both semiquantitative and real-time quantitative reverse transcription-polymerase chain reaction. Expression of one of these genes (myeloid cell leukemia sequence-1) was depressed, whereas the others [DNA damage repair and recombination protein 23B, the mouse laminin receptor and the lymphocyte-specific protein tyrosine kinase (LCK)] were elevated in the zinc-deficient mice. Further Western analysis demonstrated that the zinc binding protein LCK was elevated in these zinc-deficient mice. Results demonstrate that 3 wk of dietary zinc insufficiency can alter specific thymic mRNA and protein abundances before alterations occur in thymocyte development as detectable by FACS analysis.

Background: Zinc deficiency in humans produces a secretory diarrhea that is corrected by zinc supplementation. In rats, differential mRNA display analysis has shown that intestinal uroguanylin gene expression is increased in zinc deficiency. An endocrine axis involving intestinal uroguanylin and the kidney may exist. Therefore, we conducted this study to examine whether zinc deficiency would affect uroguanylin expression in the kidney of rats., Methods: A purified diet, deficient or adequate in zinc content, was fed to rats. Preprouroguanylin mRNA was localized in kidney by in situ hybridization, and prouroguanylin/uroguanylin peptides were localized in the kidney by immunohistochemistry. Abundance was measured by Western blotting and slot blotting analyses., Results: In situ hybridization demonstrated that preprouroguanylin mRNA-expressing cells were localized in the proximal tubules, being primarily limited to the cortical-medullary junction. Zinc deficiency did not alter the abundance or distribution of the mRNA. Immunohistochemistry, using a uroguanylin peptide-specific, affinity-purified antibody, demonstrated that immunoreactive uroguanylin peptide was localized to the same cells but that the staining was stronger in zinc-deficient rats. Western blotting analysis of kidney extracts showed that there was no difference in abundance of prouroguanylin between zinc adequate and deficient rats. However, slot blotting analysis demonstrated that the abundance of a low molecular weight immunoreactive peptide, presumably uroguanylin, was higher in extracts of zinc-deficient rats., Conclusion: The results suggest that production of prouroguanylin by the kidney, in contrast to the intestine, is not influenced by dietary zinc intake, but that higher amounts of uroguanylin in kidney extracts may reflect renal processing of the hormone obtained from the systemic circulation.

Metallothionein and zinc have been implicated in cellular defense against a number of cytotoxic agents. With respect to the free radical-generating hepatotoxin carbon tetrachloride, conclusions about a defensive role were reached from in vitro studies, in vivo studies using inducers of metallothionein and studies using injections of pharmacological amounts of zinc. Metallothionein knockout (null) and metallothionein transgenic mice are more direct models to examine the effects of metallothionein expression on induced cytotoxicity. Similarly, zinc presented via the diet is a more physiological model than that presented via injection. We examined whether metallothionein-overexpressing mice or metallothionein knockout mice had altered sensitivity to carbon tetrachloride and whether supplemental dietary zinc reduced sensitivity to carbon tetrachloride in these genotypes. Metallothionein knockout mice produced no metallothionein and were unable to sequester additional hepatic zinc in response to elevated dietary zinc. Hepatotoxicity, as measured by serum alanine aminotransferase activity, histological analyses and hepatic thiol levels, was greater in the knockout mice than in controls 12 h after carbon tetrachloride treatment but not at later time points (up to 48 h). In contrast, metallothionein-overexpressing mice produced more metallothionein and sequestered more liver zinc than control mice, but hepatotoxicity was similar between genotypes. Supplemental dietary zinc had no effect on hepatotoxicity with either genotype. These data suggest metallothionein null mice were more susceptible to carbon tetrachloride-induced hepatotoxicity than were control mice. However, neither metallothionein overexpression nor supplemental dietary zinc provided further protection.

Zinc metabolism is well regulated over a wide range of dietary intakes to help maintain cellular zinc-dependent functions. Expression of transporter molecules, which influence zinc influx and efflux across the plasma and intracellular membranes, contributes to this regulation. We have examined in rats the comparative response of zinc transporters 1, 2, and 4 (ZnT-1, ZnT-2 and ZnT-4) to dietary zinc. ZnT-1 and ZnT-4 are expressed ubiquitously, whereas ZnT-2 is limited to small intestine, kidney, placenta and, in some cases, the liver. When zinc intake was low (<1 mg Zn/kg), ZnT-2 mRNA was extremely low in small intestine and kidney compared with an adequate intake (30 mg Zn/kg). ZnT-1 and ZnT-2 mRNAs were markedly greater in both tissues when a supplemental zinc intake (180 mg Zn/kg) was provided. ZnT-4 was refractory to changes in zinc intake. When zinc was provided as a single oral dose (70 mg/kg body), ZnT-1 and ZnT-2 mRNA levels were increased many fold in small intestine, liver and kidney, whereas ZnT-4 gene expression was not changed. The expression of ZnT-1 and ZnT-2 is comparable to zinc-induced changes in metallothionein mRNA levels, suggesting a similar mode of regulation for these genes. The relative differential in regulation by zinc is ZnT-2 > ZnT-1 > ZnT-4. These data provide evidence that, in an animal model, zinc transporter expression is responsive to zinc under physiologically relevant conditions.

Identification of the upregulation of preprouroguanylin mRNA in the rat small intestine during zinc deficiency provides a potential mechanistic link between production of the intestinal hormone uroguanylin and the diarrhea that may accompany zinc deficiency. In the current study, in situ hybridization demonstrated that the number of preprouroguanylin mRNA-expressing cells was significantly higher in zinc-deficient rats than in zinc-adequate rats. Immunohistochemical studies, with a uroguanylin peptide affinity-purified antibody, demonstrated that immunoreactivity was localized to the tips of villi of the duodenum and jejunum in zinc-adequate rats. However, positive cells were scattered throughout the villus of zinc-deficient rats. A subset of cells, perhaps enterochromaffin cells, exhibited the predominant staining, whereas no specific staining was found in goblet cells or lymphocytes of the lamina propria. Western blotting demonstrated that the expression of prouroguanylin in both duodenum and jejunum was elevated by dietary zinc depletion. These results show that dietary zinc deficiency upregulates prouroguanylin in intestinal cells, which is consistent with a role for uroguanylin in the etiology of diarrhea observed in human zinc deficiency.

A specific, sensitive and reliable index for assessment of human zinc status has not been developed, and continues to present a considerable challenge for nutritionists in the trace element field. We have focused on metallothionein (MT) expression as a potential index. A protocol involving 16 men and a 10-d supplementation period plus a 4-d postsupplementation period was used to examine the relative response of MT expression in erythrocytes, monocytes, peripheral blood mononuclear cells (PBMC) and cells from a dried blood spot (DBS). Zinc was supplemented at the current adult male recommended dietary allowance (RDA) of 15 mg. Erythrocyte MT protein, as measured by ELISA, increased gradually to about twofold over the placebo group during zinc supplementation and remained elevated for 4 d postsupplementation. Competitive reverse transcriptase-polymerase chain reaction showed that MT mRNA levels in both monocytes and PBMC increased (up to 4.7- and 2.7-fold, respectively) after 2 d of supplementation, with greater expression in monocytes compared with PBMC. Total RNA extracted from dried blood spots, representing cells from 50 microL of blood, showed a comparable change in MT mRNA upon zinc supplementation. In each leukocyte population isolated, when zinc supplementation was withdrawn, MT mRNA levels decreased. Collectively, these experiments show that, in men, MT gene expression increases during supplementation at the RDA, and that the DBS sampling method will be of value in measuring MT expression in a variety of clinical and survey situations.

The regulation of gene expression by nutrients plays an important role in the overall manifestations of nutritional deficiencies. Insufficient intakes of dietary micronutrients, such as zinc, produce profound effects in multiple organs and tissues. One of the major challenges, however, is to identify genes affected by changes in nutritional status. Differential display of mRNA has proved to be a valuable technique in meeting this challenge. In our ongoing search for genes responsive to dietary zinc, we compared small intestinal mRNA from rats that were fed zinc-deficient or -adequate diets using differential display to generate 3' anchored expressed sequence tags (EST). EST for intestinal mRNAs with altered expression due to zinc deficiency include two peptide hormones, intestinal fatty acid binding protein, intestinal alkaline phosphatase II, a proteasomal ATPase, cis-Golgi p28 and two subunits of the ubiquinone oxidoreductase. The EST for one of the hormones yielded the sequence for the 3' end of an mRNA encoding preprouroguanylin and was used to clone the remaining portion of the rat cDNA via 5' rapid amplification of cDNA ends. Northern blot analysis of RNA from rat intestine demonstrated that preprouroguanylin mRNA was 2.5-fold more abundant during zinc deficiency. Uroguanylin, a natriuretic peptide hormone, is an endogenous ligand for the same guanylate cyclase C that the Escherichia coli heat-stable enterotoxin (STa) binds when it causes secretory diarrhea by activating the cystic fibrosis transmembrane conductance regulator, thus altering fluid balance in the intestine. This suggests a mechanism whereby zinc deficiency could induce uroguanylin levels in the intestine and cause or potentiate diarrhea.

An integration of knowledge concerning regulation of metallothionein expression with research on metallothionein's proposed functions is necessary to delineate how this metalloprotein affects cellular processes, especially zinc metabolism. Metallothionein expression is driven by a number of physiological mediators through several response elements in the metallothionein gene promoter. Cellular accumulation of metallothionein depends on both gene expression and protein degradation. Both depend largely on availability of cellular zinc derived from the dietary zinc supply. Metallothionein expression is related to zinc accumulation in certain organs. Evidence has been produced, which suggests that metallothionein could act in a number of biochemical processes. It may act in zinc trafficking and/or zinc donation to apoproteins, including zinc finger proteins that act in cellular signaling and transcriptional regulation. As a result, metallothionein expression may affect a number of cellular processes including gene expression, apoptosis, proliferation and differentiation. The ability of metallothionein to exchange other metals with zinc in these proteins may explain a role in metal toxicity. Similarly, mobilization of zinc from metallothionein by oxidative stresses may explain its proposed antioxidant function. Apparent good health of metallothionein-deficient mice argues against a critical biological role for metallothionein; however, expression may be critical in times of stress.

Cells maintain zinc concentrations with relatively narrow limits. Nevertheless, physiologically relevant changes in free Zn(II) pools or changes in Zn bound to specific ligands or within vesicles may occur without a major change in total cellular zinc concentrations. The task of maintaining such levels rests in part with zinc transporter proteins. The genes for some putative zinc transporters have recently been cloned. As of this time, most have not been directly shown to transport zinc in functional studies, albeit evidence is strong that they have such a function. Zinc transporter (ZnT)-1 was identified as a rescue agent for cells maintained in very high extracellular zinc conditions; therefore, ZnT-1 has been suggested to function as an exporter. ZnT-1 is expressed in a variety of tissues, including intestine, kidney and liver. Intestinal expression is regional, being much greater in duodenum and jejunum and in villus versus crypt cells. Immunolocalization places ZnT-1 at the basolateral membrane of intestinal enterocytes and epithelial cells of the distal renal tubules. Regulation of ZnT-1 mRNA and ZnT-1 protein does not change markedly with changes in dietary zinc level except when a large single oral zinc supplement is provided. ZnT-1 is induced by transient ischemia of the forebrain. ZnT-2 and ZnT-3 may function in tissue-specific vesicular zinc transport. ZnT-4 is believed to be abundant in mammary gland and may be associated with zinc secretion into milk. A mutation of the ZnT-4 gene may account for the lethal milk (lm) syndrome. The putative zinc transporters identified thus far appear to have characteristics commensurate with functions in integrative zinc acquisition and homeostasis.

A role for metallothionein in intestinal zinc absorption has been the subject of considerable debate. If metallothionein affects zinc absorption, then those factors that induce metallothionein synthesis (e.g., heavy metals, hormones) should alter zinc absorption and homeostasis. The present studies used metallothionein transgenic mice (overexpressing) and metallothionein knockout mice (no expression of metallothionein-1 or metallothionein-2) to examine directly the effects of metallothionein on zinc absorption, independent of secondary effects that could be caused by metallothionein inducers. Zinc absorption was examined by administering a single oral zinc dose (0.5 mmol/kg) by feeding tube to metallothionein transgenic and metallothionein knockout mice and measuring the serum zinc concentration. Two hours after the dose, the serum zinc concentration was 2.3 times higher in metallothionein knockout mice than in their control strain. Conversely, the concentration was elevated only one third as much in the metallothionein transgenic mice as in their controls after the zinc dose. We found that the serum zinc concentration was inversely related to the level of metallothionein protein. The intestinal zinc content was higher in the metallothionein knockout mice, however, suggesting that metallothionein did not reduce zinc absorption by simply sequestering zinc in the mucosa. The expression of the zinc transporter ZnT-1 was directly related to the serum zinc level and was independent of the level of metallothionein. These results further support metallothionein as an important component for reducing the efficiency of zinc absorption at elevated zinc intakes.

Genes that are involved in mammalian zinc transport recently have been cloned. These all predict proteins with multiple membrane spanning regions, and most have a histidine-rich intracellular loop. ZnT-1 was the first cloned and is associated with zinc efflux. It is found in all tissues examined, and, at least in some, ZnT-1 expression is regulated by dietary zinc intake. In enterocytes of the small intestine and renal tubular cells, ZnT-1 is localized to the basolateral membrane, suggesting an orientation that is consistent with zinc absorption/retention. ZnT-2 is also an exporter and may be involved in zinc efflux or uptake into vesicles in intestine, kidney, and testis. ZnT-3 is involved in zinc uptake into vesicles in neurons and possibly in testis. ZnT-4 is also an exporter and is highly expressed in mammary gland and brain. The divalent cation transporter 1 (DCT1) is regulated by iron, but exhibits transport activity for a number of trace elements including zinc. Description of a family of zinc transporters bridges the integrative and reductionist approach to the study of zinc metabolism. Other members of this transporter family may emerge. Many of these may be regulated by zinc, and some may respond to immune challenge, oxidative stress, and competing metals in the dietary supply. Collectively, description of transporters that influence cellular zinc uptake and efflux will provide a clearer understanding of the molecular events that regulate zinc absorption and homeostasis.

The metallothionein gene is transcriptionally regulated by zinc. Consequently, metallothionein has potential for serving as an index of dietary zinc status in humans. To examine this possibility, an enzyme-linked immunoassay (ELISA) based on a sandwich approach that utilizes monoclonal and chicken egg yolk antibodies was used to compare the response of erythrocyte metallothionein protein levels with the response of monocyte metallothionein mRNA levels as measured by competitive reverse transcriptase-polymerase chain reaction (CRT-PCR) during zinc supplementation. Young male subjects participated in an 18-d supplementation study in which zinc was provided at 50 mg/d. Control subjects received a placebo. The zinc supplement resulted in significantly greater erythrocyte metallothionein levels by d 8 of supplementation compared with controls. Monocyte metallothionein mRNA levels were significantly greater than those of controls by d 2 of supplementation. Both remained elevated through d 18. They returned to base line by 8 and 4 d after supplementation, respectively. The plasma zinc concentration was significantly greater than in controls by d 6 and had returned to control levels by d 22 of supplementation. The results presented here show that both monocyte metallothionein mRNA and erythrocyte metallothionein protein concentrations change in human subjects in response to elevated dietary zinc intake and that monocyte metallothionein mRNA responds more rapidly to elevation of dietary zinc status than erythrocyte metallothionein protein. Consequently, both erythrocyte metallothionein and monocyte metallothionein mRNA may prove to be measures useful for assessment of either zinc depletion or the bioavailability of zinc supplements.

Zinc status is difficult to evaluate in humans. Metallothionein gene expression is transcriptionally regulated by dietary zinc and thus could serve as an assessment parameter based on zinc-dependent function. We used semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to establish that MT mRNA is increased in a human monocytic cell line by addition of zinc to the medium. To examine this response in human subjects, a dietary supplement of 50 mg zinc gluconate/d was given for 15 d. Monocytes were purified from venous blood using NycoPrep 1.068. Monocyte purity was determined by flow cytometry using fluorescent anti-human monocyte CD14 antibodies. Total monocyte RNA was extracted and converted to cDNA by reverse transcription. Competitive RT-PCR was used to analyze differences between cDNA levels that are proportional to MT mRNA levels in monocytes from zinc-supplemented and control subjects. RT-PCR oligonucleotide primers were designed to amplify both a 201 bp segment of the human MT cDNA and a 180 bp competitor cDNA template. The 180 bp competitor cDNA template was used for MT cDNA quantitation. The RT-PCR data show that there was a significant increase in monocyte MT mRNA in subjects within 6 d of zinc supplementation, which remained elevated at d 15 of supplementation. In contrast, plasma zinc was greater at d 6 of zinc supplementation, but by d 15 of supplementation, while still elevated, was close to control levels. These data suggest that monocyte MT mRNA levels respond to zinc supplementation and that the response could serve as a more useful assessment variable than plasma zinc for the measurement of zinc status in humans.

Apolipoprotein B (apo B) mRNA editing is a site-specific, post transcriptional cytidine deamination reaction that generates apo B48 in the mammalian small intestine and in the liver of certain animals. This reaction is mediated by an enzyme complex that includes the catalytic subunit apobec-1, a zinc-dependent cytidine deaminase. To determine the importance of zinc status to apo B mRNA editing in vivo, we examined the effects of experimentally induced zinc deficiency in rats upon hepatic and serum lipid levels and several indices of apo B gene expression. Rats were either given unlimited access to or were pair-fed a semipurified zinc-supplemented (30 mg Zn/kg) diet or were fed a zinc-deficient diet (approximately 1 mg Zn/kg) for 17 d. Significant differences were detected in the ratio of serum apo B100/B48 in the unlimited access, zinc-supplemented group compared with either zinc-deficient rats or pair-fed controls. There were no alterations in hepatic triglyceride and cholesterol concentrations, hepatic apo B mRNA abundance or apo B mRNA editing in either the small intestine or liver. Taken together, these data suggest that the altered ratios of serum apo B isomorphs seen in zinc deficiency are not mediated through changes in hepatic or intestinal apo B mRNA editing.

Changes in zinc status in response to folic acid supplementation and the effect of zinc intake on folate utilization were evaluated in 12 men (20-34 y old) consuming a diet containing 3.5 or 14.5 mg zinc/d for two 25-d intervals. Deuterium-labeled folic acid (800 micrograms/d) or a placebo was administered orally during each phase. No differences in plasma zinc, erythrocyte zinc, urinary zinc, erythrocyte metallothionein or serum alkaline phosphatase, due to supplemental folic acid, were detected at either level of zinc intake. Differences in the response to folic acid supplementation, due to the level of zinc intake, were not detected for serum, erythrocyte or urinary (labeled and unlabeled) folate. Within the constraints of this short-term folic acid supplementation study, adverse effects on zinc status were not observed and our data suggest that folic acid utilization was not influenced by level of zinc intake.

The cysteine-rich intestinal protein (CRIP) is a member of a superfamily of proteins containing the LIM motif (a double zinc finger) that has been shown to bind zinc. The role of zinc in the regulation of CRIP was examined in adult rats, cultured intestinal epithelial cells and in a transient transfection system. When adult male rats were fed diets with various amounts of zinc, the amount of ileal CRIP mRNA was only 19% lower in rats fed a zinc-deficient diet (1 mg Zn/kg) and was not different in the zinc-supplemented group (180 mg Zn/kg) compared with the zinc-adequate group (30 mg Zn/kg). In contrast, metallothionein mRNA levels were 76% lower and 80% greater than control levels in the zinc-deficient and zinc-supplemented groups, respectively. Using the chloramphenicol acetyltransferase (CAT) reporter gene, 5'-deletion products of the CRIP genomic promoter were tested for basal and zinc-induced CAT activity in transiently transfected IEC-6 cells. Treatment of the cells with zinc did not alter CAT activity of any construct. These results suggest that CRIP is not directly regulated by zinc in the intestine of rats.

Acute intestinal inflammation was established in rats by intraluminal administration of acetic acid into loops of distal ileum, proximal jejunum or ascending colon. The study included two control groups of intact (untreated) rats and sham-operated (saline-treated) rats for each intestinal segment. A third group of rats received acetic acid. Histological evaluation demonstrated that acetic acid treatment induced a mild inflammatory response. Two days after treatment, zinc absorption was measured using ligated 10-cm loops of each segment in which 65Zn was injected intraluminally. 65Zn absorption by the ileum, jejunum and colon was markedly reduced in those rats in which inflammation was induced by acetic acid. The liver showed the highest uptake of radioisotope, but the relative tissue distribution generally followed the amount of absorption. The surgical procedure itself seemed to reduce zinc absorption. No changes in [3H]leucine absorption were observed between sham-operated and acetic acid-treated controls. There was no significant serosal-->luminal secretion of intramuscularly injected 65Zn in any of the studied segments. Therefore, based upon the data obtained, we conclude that acetic acid-induced intestinal inflammation reduces absorption of zinc by the small and large intestine, and that a surgical procedure (laparotomy) also reduces zinc absorption. The mechanism of this inflammation is such that malabsorption shows some specificity.

The comparative influence of dietary zinc status and recombinant human interleukin-1 alpha (rhIL-1 alpha) and recombinant human interleukin-6 (rhIL-6) on metallothionein (MT) gene expression was examined in rat bone marrow and liver. Growing male rats were fed a diet with 5 (restricted), 30 (control), or 180 (supplemented) mg Zn/kg for 14 d. On d 15, rats were injected with 5 micrograms of rhIL-1 alpha or rhIL-6. Marrow metallothionein responded directly to dietary zinc but did not respond to these cytokines. Significantly less zinc accumulated in marrow from the zinc-restricted rats compared with control or supplemented rats. Analysis of metallothionein isoform mRNA expression showed MT-1 is the primary gene expressed in marrow. A significant interaction between dietary zinc and cytokine treatment was observed in the liver. Hepatic metallothionein induction following both rhIL-1 alpha and rhIL-6 injection was directly related to dietary zinc intake. Expression of hepatic metallothionein isoform mRNAs suggested MT-1 responded to zinc and MT-2 responded to cytokines. These results indicate that metallothionein gene expression in both the marrow and the liver responds to dietary zinc status. In contrast, liver metallothionein expression can be altered by these cytokines, which are known to act on many cell types. Furthermore, these results suggest that bone marrow metallothionein could be of importance in the development of marrow cells.

Male rats were fed a purified diet containing 1 mg Zn/kg to induce zinc deficiency (-Zn). A second set of rats were fed a 30 mg Zn/kg diet, which supplied adequate levels of zinc (+Zn). The zinc-adequate rats were pair-fed to the rats fed the 1 mg Zn/kg diet to eliminate differences in diet intake. After 16 d, the two conditions of dietary zinc status were confirmed by assaying serum zinc concentrations and by Northern analysis for metallothionein mRNA abundance in the kidney. Messenger RNA was purified from small intestine by oligo(dT) chromatography and pooled within conditions. A cDNA plasmid library was constructed from the mRNA derived from the intestines of zinc-deficient rats. The library was then screened by the method of differential hybridization using 32P-labeled first strand cDNA probes derived from the +Zn and -Zn mRNA. After screening 10,000 independent cDNA clones, nine cDNAs were isolated and studied further, corresponding to mRNAs that are down-regulated by zinc deficiency. The ratio of the abundance between the two conditions for the nine mRNAs ranged from 1.5- to sevenfold as determined by Northern analysis of the +Zn and -Zn intestinal mRNAs. Two of these clones seemed to be specific to the intestine, and four others were abundant in the intestine and one or two other tissues. We are using these cDNAs as models to study gene regulation under various conditions of dietary zinc intake and to explore the genes most sensitive to zinc deficiency.

The response of erythrocyte metallothionein to dietary zinc in human subjects was evaluated in a controlled metabolic protocol including standard indices of zinc status. Fifteen male subjects, age 27 +/- 3.6 y, participated in a 90-d, four-phase study consisting of acclimation (7 d; 15 mg Zn/d), treatment (6 wk; either 3.2, 7.2 or 15.2 mg Zn/d), depletion (12 d; 0.55 mg Zn/d) and supplementation (30 d; self-selected diet plus 50 mg Zn/d) phases. During the treatment phase erythrocyte metallothionein decreased in the group fed 3.2 mg Zn/d. Erythrocyte metallothionein decreased during the depletion phase (46 +/- 10%) to below the normal concentration in all groups and increased in the supplementation phase. Plasma zinc concentration decreased in the group fed 3.2 mg Zn/d during the treatment phase relative to the acclimation phase. Erythrocyte zinc decreased in all groups during the depletion phase relative to the treatment phase, and then increased during the supplementation phase. These data suggest that erythrocyte metallothionein can be used as a measure of status in severe zinc depletion and that comparing the change in erythrocyte metallothionein over a 6-wk period can differentiate between low and adequate levels of dietary zinc intake.

The lethal milk (lm) mutation in mice causes Zn deficiency in pups nursed by lm dams. To examine tissue Zn distribution and Zn transport to milk and pups, 65Zn was administered to lactating normal and lm dams. Transport of 65Zn to milk of lm dams was approximately 50% of that transported to milk of normal dams. The lower milk 65Zn resulted in significantly less 65Zn uptake by tissues of the nursing pups. The decrease in 65Zn transport to the milk was accompanied by a significant increase in 65Zn uptake and metallothionein mRNA levels in kidney of the lm dams. The elevated Zn uptake and metallothionein expression was tissue specific and could be a reflection of altered zinc transport from mammary gland to milk. Polyacrylamide gel electrophoresis and Western transfer of mammary gland proteins from lm dams showed that a 30-kDa protein bound more 65Zn in vitro compared with proteins from normal mammary gland. Normal pups nursed by dams of the lm genotype had down-regulated metallothionein expression due to Zn deficiency. The genetic defect in lm mice decreases Zn transport to milk, thus explaining the neonatal Zn deficiency seen in normal mice fostered by lm dams. The greater metallothionein expression in dams of the lm genotype could be a secondary manifestation of altered tissue zinc distribution or a primary effect on a metallothionein regulatory mechanism.

Oral zinc therapy is effective in controlling copper balance in patients with Wilson's disease and blocks the intestinal absorption of copper, as demonstrated by uptake of copper 64 and copper balance measurements. In this study, 64Cu uptake measurements were concomitantly carried out with intestinal biopsies to investigate the relationship of reduced copper absorption to the levels of intestinal metallothionein in patients with Wilson's disease at different stages of zinc therapy. A pronounced increase in intestinal metallothionein levels and a sharp drop in 64Cu absorption were found 4 to 5 days after the initiation of zinc treatment. Conversely, metallothionein levels decreased and 64Cu uptake increased on the discontinuation of zinc therapy. The data indicate that 64Cu absorption varies as a function of intestinal metallothionein level. Intestinal metallothionein levels were found to correlate linearly with urinary zinc levels, which reflect body zinc status. These findings support our hypothesis that intestinal metallothionein induction mediates decreased copper absorption observed during zinc therapy. The suppressive effect of zinc on copper absorption appears to have a half-life of about 11 days.

Dietary zinc may regulate zinc absorption in part via the inhibitory effect of intestinal metallothionein, but the mechanism is unknown. We recently showed that cysteine-rich intestinal protein (CRIP) binds zinc during transmucosal zinc transport, and that CRIP may function as an intracellular zinc carrier. The present experiments examine the interaction of CRIP and metallothionein with zinc to evaluate their potential roles in the mechanism of zinc absorption. Intestinal metallothionein concentrations were lower and zinc absorption rates from isolated intestinal loops were higher in rats fed a low zinc diet compared with those fed a high zinc diet or given parenteral zinc to induce metallothionein synthesis. Zinc status did not affect the apparent CRIP concentration, but markedly altered the distribution of 65Zn in intestinal cytosol as determined by gel filtration HPLC. More 65Zn was associated with CRIP (40 vs. 14%) and less was bound to metallothionein (4 vs. 52-59%) in rats fed the low zinc diet compared with rats of high zinc status. Luminal zinc concentration also affected the distribution of 65Zn in the cytosol. CRIP bound progressively less (from 42 to 25%) of the 65Zn taken up from the lumen as the luminal zinc concentration was increased from 5 to 300 mumol/L. Collectively these data suggest that CRIP is a saturable, intracellular zinc transport protein, and that metallothionein inhibits zinc absorption by binding zinc in competition with CRIP. A hypothetical model for the mechanism of transcellular zinc absorption involving metallothionein and CRIP is presented and discussed.

Regulation of metallothionein gene expression by dietary zinc and the relationship of dietary zinc to nuclear zinc uptake was examined in growing rats. Zinc was fed at 5, 30 or 180 mg/kg, either in pelleted form for a 2-wk period (ad libitum) or for 2 h as a liquefied preparation (1 g in 4 mL). Two hours after the oral dose, the intestine and liver took up more zinc than other tissues. Nuclei purified from liver, kidney and spleen accumulated substantial amounts of zinc and directly reflected the dietary zinc level within the 2-h feeding period. Nuclei from kidney accumulated the largest amount of dietary zinc within 2 h, accounting for up to 6.2% of the total nuclear zinc concentration. Northern analysis demonstrated that metallothionein expression was proportional to dietary zinc intake in some tissues. It was greatest in kidney, followed in descending order by liver, intestine, spleen and heart. Thymus and lung metallothionein mRNA levels were not changed appreciably by dietary zinc intake. Chromatography of extracts from liver nuclei shows that 65Zn introduced into the portal supply is bound to discrete fractions of nuclear proteins. One of these fractions binds both 65Zn and a 32P-labeled oligonucleotide corresponding to the metal regulatory element of the metallothionein promoter. These results demonstrate that significant amounts of zinc from the diet are rapidly taken up by cell nuclei. Furthermore, they suggest that transcriptional regulation of the metallothionein gene and other genes with metal regulatory elements involves a direct interaction between the dietary supply and intranuclear factors that bind zinc.

The effects of dexamethasone and interleukin 1 alpha on intestinal metallothionein gene expression and zinc absorption were studied. Rats given parenteral zinc served as positive controls. A single intraperitoneal or intravenous dose of dexamethasone, interleukin 1 alpha or zinc markedly increased liver metallothionein synthesis 3-9 h after injection. Intestinal metallothionein mRNA and metallothionein protein were not affected by dexamethasone or interleukin 1 alpha, but were markedly increased by parenteral zinc. Absorption of 65Zn from isolated duodenal segments was inversely related to intestinal metallothionein concentration in rats given zinc, but was not affected by dexamethasone or interleukin 1 alpha. Plasma zinc concentrations decreased in rats given dexamethasone or interleukin 1 alpha and increased in those given zinc, but they were not related to 65Zn absorption. Similarly, multiple intraperitoneal administration of either dexamethasone or interleukin 1 alpha, or oral administration of dexamethasone, for 7 d markedly increased liver metallothionein synthesis but did not affect intestinal metallothionein concentration or 65Zn absorption. These results suggest that intestinal metallothionein gene expression and 65Zn absorption are refractory to glucocorticoid hormone and interleukin 1 alpha.

Hepatocytes were cultured with Waymouth's media containing zinc at concentrations of 1 (the endogenous zinc concentration of basal medium), 16 and 48 mumols Zn/L to examine the effects of extracellular zinc on a variety of zinc-related functions. The zinc concentrations were chosen with the intention of simulating zinc-deficient, adequate and excess extracellular conditions. Basal medium had no effect on cell zinc, metallothionein (MT) or MTmRNA for up to 48 h but reduced delta-aminolevulinic acid dehydratase (delta-ALA-D) activity to 75% of the initial level by 3 h. The addition of zinc at 16 or 48 mumols Zn/L during the initial 3 h of culture did not prevent the decrease in delta-ALA-D activity. Reintroducing zinc at concentrations of 16 or 48 mumols Zn/L to hepatocytes after the initial 3 h of culture in basal medium significantly increased cell zinc, MT and MTmRNA levels and fully restored delta-ALA-D activity by 24 h. Medium zinc had no apparent effect on membrane integrity assessed as leakage of lactate dehydrogenase activity into culture media or de novo protein synthesis as examined by two-dimensional gel electrophoresis of 35S-labeled proteins. Hepatocytes cultured in basal medium resisted losses in cell zinc concentration even when EDTA and bovine serum albumin were present in culture medium. Kinetic experiments using 65Zn suggest hepatocytes maintain zinc concentrations by reducing zinc efflux. The ability of hepatocytes cultured in basal (1 mumol Zn/L) medium to maintain cell zinc content and some zinc-dependent functions underscores the difficulty of producing zinc deficiency in primary hepatocyte culture.

Brush border membrane vesicles were used to evaluate the influence of a variety of potential zinc-binding ligands on zinc transport in the rat. Citrate, glutathione (reduced) and picolinate were added to the extravesicular incubation medium at 0.38 mM. Transport was evaluated for up to 60 minutes at an extravesicular zinc concentration of 0.2 mM. Citrate depressed zinc transport at each time point checked. Picolinate depressed transport after 5 minutes of incubation. Glutathione slightly enhanced transport after 60 minutes of incubation. Neither citrate, glutathione nor picolinate had an influence on D-glucose transport. These results suggest that the compounds evaluated do not enhance zinc transport across the brush border surface of the small intestine of the rat.

The effects of zinc chelators on 65Zn uptake, absorption and tissue distribution were determined in rats using ligated duodenal loops. 65Zn was supplied as Zn-methionine complex, ZnCl2, ZnCl2 + L-methionine or ZnCl2 + EDTA. The effect of EDTA was also determined in the presence of phytic acid. Absorption of 65Zn was markedly reduced in rats given Zn-methionine complex or ZnCl2 + EDTA. The 65Zn level in tissues (liver, bone, muscle, skin, kidney, and thymus) of rats given ZnCl2 + EDTA was also significantly reduced compared to that of rats given ZnCl2. Reduced absorption due to phytic acid was not improved by EDTA, although EDTA increased mucosal 65Zn retention. High performance gel filtration chromatography showed six 65Zn-binding peaks in the mucosal cytosol of rats given ZnCl2. A seventh peak attributable to Zn-EDTA was observed in cytosol from rats given ZnCl2 + EDTA. A comparable peak in plasma was not observed. Both EDTA and Zn-methionine complex reduced 65Zn-binding to a low-molecular-weight component of mucosal cytosol that was not metallothionein. The results suggest that Zn-EDTA is transported intact from the lumen into mucosal cells but not across the basolateral membrane. The adverse effects of EDTA and Zn-methionine complex on zinc absorption were associated with reduced 65Zn-binding to a component of mucosal cytosol that may be involved in zinc absorption.

Response of intestinal metallothionein and zinc to dietary zinc intake was investigated in zinc-depleted rats, after a single feeding by stomach tube of a purified diet supplemented with either 125 ppm zinc or less than 1 ppm zinc. Serum zinc concentration was maximal 3 hours post-feeding, then stabilized at lower levels returning to baseline concentration by 18 hours. Mucosal zinc concentration rose to a maximum by 6 hours after feeding and plateaued at slightly lower levels thereafter, due to association of this zinc with metallothionein. The rate of metallothionein synthesis rose 4-fold, 6-9 hours after feeding, coinciding with the maximal contest of translatable metallothionein mRNA concentration. Together, these findings provide strong evidence for a regulatory role of metallothionein in zinc absorption and homeostasis, where dietary zinc influences subcellular events in mucosa affecting regulation of the metallothionein gene. These events include the induction of de novo metallothionein synthesis, preceded by an increase in translatable mRNA specific for this metalloprotein, and followed by a reduction in zinc absorption simultaneous with the binding of absorbed zinc with newly synthesized thionein polypeptides.

The influence of a 7.7-mumole (0.5-mg) dose of parenteral zinc on the synthesis of metallothionein in rat kidney was examined. The amount of zinc bound to metallothionein was maximal 9--15 hours after zinc administration. Pulse labeling with [35S]cystine showed the rate of renal metallothionein was stimulated to a maximum 6 hours after zinc administration and declined thereafter. Total RNA was extracted by the guanidine thiocyanate procedure from kidneys that had been flash frozen in liquid N2. Polyadenylated RNA (mRNA) was isolated by oligo (dT)-cellulose chromatography. The mRNA was translated in a wheat germ system and newly synthesized metallothionein was isolated by activated thiol--Sepharose 4B chromatography. Metallothionein mRNA activity nearly doubled after zinc administration adn was closely correlated with the enhancement in synthetic rate of this protein in kidney. Actinomycin D administered prior to either zinc or cadmium completely blocked both the stimulation of metallothionein synthesis and mRNA activity found in kidney in response to these metals. The results suggest renal metallothionein is induced by zinc or cadmium through a mechanism that requires altered expression of the metallothionein gene(s).

Very little information is available concerning the relationship between metallothionein and disease in humans. Recently, investigators have used the Cd-heme method to measure metallothionein levels in human liver samples obtained from autopsy. This assay, however, is not sensitive enough to measure metallothionein in small tissue biopsy specimens. As an alternative, we report the development of a human metallothionein enzyme-linked immunosorbent assay (ELISA). This assay used high-performance liquid chromatography-purified human metallothionein-1 and purified sheep anti-human metallothionein-1 IgG. A limiting antigen coating concentration of 100 ng/ml and a minimal antibody dilution of 1:4000 were chosen. The sensitivity of the ELISA extended to 300 ng/ml (15 ng). The coefficients of interassay and intraassay variation were 15.4% and 4.2%, respectively. Human livers obtained from autopsy were assayed by this method and the values compared with values obtained by the Cd-heme method. The livers were separated by their autopsy reports into four groups: normal, immunosuppression, cancer, and infection. Livers from the infection group (ELISA 2979 micrograms/gm, Cd-heme 1201 micrograms/gm) contained significantly more metallothionein than those from the normal (ELISA, 1035 micrograms/gm, Cd-heme 245 micrograms/gm) and the immunosuppression (ELISA 1272 micrograms/gm, Cd-heme 221 micrograms/gm) groups (p less than 0.05). The cancer group (ELISA 2415 micrograms/gm) also had significantly elevated liver metallothionein levels. We conclude that this ELISA is sensitive enough for the measurement of tissue samples. Furthermore this assay is comparable to the Cd-heme assay in its ability to reflect metallothionein values among various treatment groups. We postulate that hepatic metallothionein induction is mediated by disease-related mechanisms such as interleukin-1, glucocorticoid secretion, or both.

The temporal response of zinc and copper metabolism to endotoxin administration was examined in Syrian hamsters over a 144-hour period. Serum copper was significantly elevated at 12, 24 and 72 hours after endotoxin, whereas serum zinc was reduced 4-48 hours after treatment. A brief elevation (8 hours) in liver copper concentration and a sustained (72 hours) increase in liver zinc concentration were also observed. The amount of zinc associated with liver metallothionein (MT) progressively increased with time, to a plateau by 24 hours and persisted at the elevated level until 72 hours after endotoxin treatment. In vitro translation of poly (A)+ RNA from liver polyribosomes showed that following endotoxin treatment MTmRNA activity was maximally elevated 6 hours after endotoxin administration and remained elevated 24 and 48 hours thereafter. Slab gel electrophoresis of serum proteins indicated changes in a stainable protein comigrating with purified ceruloplasmin after endotoxin administration. Pooled gingival tissue from endotoxin-treated hamsters demonstrated a consistently elevated copper content 12-144 hours after treatment. Endotoxin isolated from Bacteroides melaninogenicus was more effective in elevating gingival and serum copper and gingival zinc than Escherichia coli endotoxin. It was concluded that endotoxin administration elicits responses that result in enhanced metaollthionein mRNA activity. In addition, Cu and Zn concentrations in serum, liver and gingival tissue are influenced by different endotoxins to different degrees.

Intestinal 65Zn transport and metallothionein levels were examined in rats fed zinc-adequate and zinc-deficient diets and in rats subjected to an overnight fast. 65Zn uptake by intestines perfused with 1.5 microM 65Zn was greater in both zinc-deficient and fasted groups than in the control group. Mucosal retention of 65Zn was also greater in the zinc-deficient group but not in the fasted group. The greater 65Zn uptake in the fasted group was associated with a compartment that readily released 65Zn back into the lumen. Kinetic analysis of the rate of 65Zn transfer to the vascular space (absorption) showed that 65Zn absorption involved approximately 3% of mucosal 65Zn in a 40-min perfusion period. The half-life (t1/2) of this mucosal 65Zn rapid transport pool corresponded directly to changes in intestinal metallothionein levels. Both metallothionein and t1/2 were higher in the fasted group and lower in the zinc-deficient group than in controls. While the rate of 65Zn transport from this rapid transport pool decreased with increasing metallothionein level, the predicted pool size increased when the metallothionein level was elevated by fasting. These results indicate that the rate of zinc absorption is inversely related to intestinal metallothionein levels, but the portion of mucosal 65Zn available for absorption is directly related to intestinal metallothionein.

The effect of zinc on serum glucose, insulin and glucagon as well as liver glycogen was investigated in normal, adrenalectomized (ADX), and diabetic rats. Serum glucose was significantly elevated within 15 minutes after intraperitoneal administration of zinc (25 mumol) but returned to normal limits within 4 hours. Similar effects on serum glucose were noted with orally administered zinc. Significant depletion of hepatic glycogen in zinc-treated rats suggests glycogenolysis was responsible at least in part for the increased blood glucose. Adrenalectomy completely eliminated the hyperglycemic response to this metal, whereas adrenergic blockade with phenoxybenzamine and propranolol was effective in preventing hyperglycemia. The hyperglycemic response to zinc was not eliminated in diabetic rats. Administration of dexamethasone, alone or in combination with zinc, was unable to change serum glucose concentrations in ADX rats. Plasma glucagon was significantly elevated within 15 minutes but was reduced 6 hours after zinc treatment. Insulin was significantly depressed within 30 minutes after administration of zinc and eventually increased over controls by 4 hours after treatment. These data suggest that the hyperglycemic response to zinc depends on a mechanism, requiring an intact adrenal gland, which acts to produce a rapid alteration in blood glucose.

Regulation of ceruloplasmin synthesis by interleukin-1 (IL-1) as influenced by dietary copper status was examined in rats. In copper-sufficient rats, ceruloplasmin oxidase activity did not peak in the serum until at least 24 h after IL-1 was given. The rates of ceruloplasmin synthesis, as measured by pulse labeling with [3H]leucine and immunoprecipitation, peaked 12 h after IL-1 and returned to basal rates by 24 h. Copper had to be given to copper-deficient rats for the IL-1 to induce oxidase activity. This activity was stimulated by copper alone in the deficient rats but not to the same level as with IL-1 plus copper. Copper alone did not increase the synthesis rate. IL-1 alone stimulated the synthesis rate without increasing the oxidase activity in the copper-deficient rats. The induction pattern of ceruloplasmin synthesis by IL-1 was the same in the copper-deficient rats given copper as in copper-sufficient rats. Actinomycin D blocked the stimulation of ceruloplasmin synthesis by IL-1. It was concluded that ceruloplasmin is dependent on copper incorporation for oxidase activity, but its synthesis is induced by IL-1 regardless of dietary copper levels, probably by a mechanism involving transcriptional regulation.