Infection and inflammation produce systemic responses that include hypozincemia and hypoferremia. The latter involves regulation of the iron transporter ferroportin 1 by hepcidin. The mechanism of reduced plasma zinc is not known. Transcripts of the two zinc transporter gene families (ZnT and Zip) were screened for regulation in mouse liver after turpentine-induced inflammation and LPS administration. Zip14 mRNA was the transporter transcript most up-regulated by inflammation and LPS. IL-6 knockout (IL-6 ؊/؊ ) mice did not exhibit either hypozincemia or the induction of Zip14 with turpentine inflammation. However, in IL-6 ؊/؊ mice, LPS produced a milder hypozincemic response but no Zip14 induction. Northern analysis showed Zip14 up-regulation was specific for the liver, with one major transcript. Immunohistochemistry, using an antibody to an extracellular Zip14 epitope, showed both LPS and turpentine increased abundance of Zip14 at the plasma membrane of hepatocytes. IL-6 produced increased expression of Zip14 in primary hepatocytes cultures and localization of the protein to the plasma membrane. Transfection of mZip14 cDNA into human embryonic kidney cells increased zinc uptake as measured by both a fluorescent probe for free Zn 2؉ and 65 Zn accumulation, as well as by metallothionein mRNA induction, all indicating that Zip14 functions as a zinc importer. Zip14 was localized in plasma membrane of the transfected cells. These in vivo and in vitro experiments demonstrate that Zip14 expression is up-regulated through IL-6, and that this zinc transporter most likely plays a major role in the mechanism responsible for hypozincemia that accompanies the acute-phase response to inflammation and infection.endotoxemia ͉ inflammation ͉ hepatic ͉ Slc39a14 ͉ knockout mice

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.

Among the micronutrients required by humans, zinc has particularly divergent modes of action. cDNA microarray and quantitative PCR technologies were used to investigate the zinc responsiveness of known genes that influence zinc homeostasis and to identify, through global screening, genes that may relate to phenotypic outcomes of altered dietary zinc intake. Human monocytic͞ macrophage THP-1 cells were either acutely zinc depleted, using a cell-permeable zinc-specific chelator, or were supplemented with zinc to alter intracellular zinc concentrations. Initially, genes associated with zinc homeostasis were evaluated by quantitative PCR to establish ranges for fold changes in transcript abundance that might be expected with global screening. Zinc transporter-1 and zinc transporter-7 expression increased when cellular zinc increased, whereas Zip-2 expression, the most zinc-responsive gene examined, was markedly increased by zinc depletion. Microarrays composed of Ϸ22,000 elements were used to identify those genes responsive to either zinc depletion, zinc supplementation, or both conditions. Hierarchal clustering and ANOVA revealed that Ϸ5% or 1,045 genes were zinc responsive. Further sorting based on this pattern of the zinc responsiveness of these genes into seven groups revealed that 104 genes were linearly zinc responsive in a positive mode (i.e., increased expression as cellular zinc increases) and 86 genes that were linearly zinc responsive in a negative mode (i.e., decreased expression as cellular zinc increases). Expression of some genes was responsive to only zinc depletion or supplementation. Categorization by function revealed numerous genes needed for host defense were among those identified as zinc responsive, including cytokine receptors and genes associated with amplification of the Th1 immune response.nutrition ͉ genomics ͉ functional genomics ͉ immunology ͉ microarray

An effective measure to assess zinc status of humans has remained elusive, in contrast to iron, where a number of indicators of metabolism͞function are available. Using monocytes, T lymphocytes, and granulocytes isolated by magnetic sorting and dried blood spots (DBS) derived from 50 l of peripheral blood, we evaluated the response of metallothionein (MT), zinc transporter, and cytokine genes to a modest (15 mg of Zn per day) dietary zinc supplement in human subjects. Transcript abundance was measured by quantitative real-time RT-PCR (QRT-PCR). Zinc supplementation increased MT mRNA abundance by up to 2-fold in RNA from leukocyte subsets, and 4-fold in RNA from DBS. Transcript levels for the zinc transporter genes ZnT1 and Zip3 were increased and decreased, respectively, by zinc supplementation. Expression of the ZnT and Zip genes among leukocyte subsets differ by up to 270-fold. Monocytes and granulocytes from supplemented subjects were activated by LPS, whereas T lymphocytes were activated by mimicking antigen presentation. With zinc consumption, TNF-␣ and IL-1␤ expression was greater in activated monocytes and granulocytes, and IFN-␥ mRNA levels were higher in activated T lymphocytes. These studies show that QRT-PCR is a tool to reliably measure transcript abundance for nutritionally responsive genes in human subjects, and that a small sample of whole dried blood, when appropriately collected, can be used as the source of total RNA for QRT-PCR analysis. The results obtained also show that zinc supplementation of human subjects programs specific leukocytic subsets to show enhanced cytokine expression upon activation by stimulators of immunity.granulocytes ͉ monocytes ͉ nutrition ͉ quantitative RT-PCR ͉ T lymphocytes M ethodological advances to assess gene expression have provided a new spectrum of research tools to identify individual genes and groups of genes that produce normal and altered physiology (1, 2). Quantitative real-time RT-PCR (QRT-PCR) provides a highly sensitive and reproducible method for measuring changes in expression of specific genes through transcript sequence detection. Analytical sensitivity of QRT-PCR rests with the fluorometric basis of this technology, which markedly reduces sample size requirements. The latter makes QRT-PCR an attractive method for clinical, survey, or field studies where the amount of sample is limited.Small amounts of blood, spotted onto filter paper and dried, have been used to examine the blood cell levels of two vitamins (folic acid and retinol) for the purpose of nutritional status assessment of human subjects (3, 4). Furthermore, dried blood spots (DBS) extracted from collection cards have routinely been used for DNA amplification for population screening, e.g., genotyping and for DNA archiving (5, 6), but have not been widely used for . Such an approach of sample acquisition could similarly be used to measure transcript abundance for nutritionally regulated genes, and, therefore, be of value for nutrient assessment purposes. In this regard, we were successful...

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.