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. 2004 Oct 5;101(40):14355-60.
doi: 10.1073/pnas.0406216101. Epub 2004 Sep 20.

Responsive transporter genes within the murine intestinal-pancreatic axis form a basis of zinc homeostasis

Juan P Liuzzi  1 , Jeffrey A Bobo, Louis A Lichten, Don A Samuelson, Robert J Cousins
Affiliations

Responsive transporter genes within the murine intestinal-pancreatic axis form a basis of zinc homeostasis

Juan P Liuzzi et al. Proc Natl Acad Sci U S A. .

Abstract

Zn homeostasis in animals is a consequence of avid uptake and retention, except during conditions of limited dietary availability, and/or factors such as parasites, which compete for this micronutrient or compromise retention by the host. Membrane proteins that facilitate Zn transport constitute the SLC30A (ZnT) and SLC39A (Zip) gene families. Because dietary recommendations are based on the balance between intestinal absorption and endogenous losses, we have studied Zn transporter expression of the murine intestinal-pancreatic axis to identify transporters that are likely to be involved in homeostatic control of Zn metabolism. Marked tissue specificity of expression was observed in Zn-depleted vs. Zn-adequate mice. As shown by quantitative PCR, Western blot analysis, and immunohistochemistry, intestinal Zip4 was markedly up-regulated in response to Zn-depletion conditions. The increased abundance of Zip4 is concentrated at the apical membrane of enterocytes. There are 16 ZnT and Zip transporters expressed in pancreas. Only two, ZnT1 and ZnT2 (both cellular Zn exporters), show a progressive down-regulation under Zn-depleted conditions. In Zn-adequate mice, ZnT1 is diffusely distributed in acinar cell cytoplasm and colocalizes with alpha-amylase but is not detected in pancreatic islets. In acinar cells during Zn depletion, ZnT1 is localized to the plasma membrane. Intestinal Zip4 up-regulation by Zn-depletion conditions is dampened in metallothionein knockout mice, suggesting that intracellular Zn pools influence these responses. The results show that Zn transporter expression in the intestinal-pancreatic axis is a component of the homeostatic regulation of this micronutrient.

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Figures

Fig. 1.
Fig. 1.
Q-PCR analysis of ZnT and Zip transcript abundance in mouse small intestine. Each relative abundance was calculated by using 18S rRNA as the normalizer. Values given as means ± SEM (n = 3). Transcript levels in response to low (Zn-, <1 mg/kg) or high (Zn+, 150 mg/kg) Zn diet relative to adequate (ZnN, 30 mg/kg) Zn diet after the mice were fed for 21 days. (A) ZnT transcript levels. MT mRNA is shown as an example of a Zn-responsive gene. (B) Zip transcript levels. *, P < 0.05, compared with ZnN group.
Fig. 2.
Fig. 2.
Intestinal Zip4 expression as a function of Zn depletion. Mice were fed the Zn- diet for up to 21 days. After 7 days, one group of Zn- mice was repleted with Zn by feeding the ZnN (Zn-adequate) diet for 1 day. (A) Relative abundance of Zip4 mRNA is shown compared with levels in ZnN mice at each time point. It was calculated by using 18S rRNA as the normalizer. Values are given as means ± SEM (n = 3). Serum Zn concentrations in response to Zn- depletion and ZnN repletion are shown. (B) Western blot analysis of total intestinal membrane proteins showing change in Zip4 protein abundance. Estimated molecular masses are also shown. Increase in the 39-kDa Zip4 band occurs in Zn- mice with a 60-kDa band, which is constitutively expressed. (C) Both signals were blocked by preincubating antibody with the Zip4 peptide.
Fig. 3.
Fig. 3.
Immunofluorescence microscopy showing changes in Zip4 protein in murine small intestine during Zn depletion, repletion, and supplementation. Sections from specific regions were incubated individually with Zip4 antibody, followed by IgG-Alexa 594 conjugate. Nuclei are counterstained with 4′,6-diamidino-2-phenylindole. Tissue sections were from mice fed as follows: Villus, ZnN (A); Villus, Zn-, 7 days (B); Villus, Zn-, 21 days (C); Villus, ZnN, repleted for 1 day (D); Crypts, ZnN (E); Crypts, Zn-, 7 days (F); and Villus, Zn-, 7 days (after antibody was preincubated with Zip4 peptide) (G).
Fig. 4.
Fig. 4.
Q-PCR analysis of ZnT transcript abundance in mouse pancreas. Each relative abundance was calculated by using 18S rRNA as the normalizer. Values are given as means ± SEM (n = 3). (A) ZnT transcript levels in response to low (Zn-, 1 mg/kg) or high (Zn+, 150 mg/kg) Zn diets relative to the Zn-adequate (ZnN, 30 mg/kg) diet after the mice were fed for 21 days. *, P < 0.05, compared with ZnN group. Pancreatic MT mRNA is shown as an example of a Zn-responsive gene. (B) ZnT1 and ZnT2 transcript levels decrease as a function of Zn depletion over 21 days. After 7 days, one group of mice was repleted with Zn by feeding the ZnN (Zn-adequate) diet for 1 day.
Fig. 5.
Fig. 5.
ZnT1 and ZnT2 expression in isolated mouse pancreatic acinar cells and islets. Mice were fed the ZnN diet. Q-PCR analysis shows abundance of ZnT1 and ZnT2 mRNAs in acinar cells relative to islets. 18S rRNA was the normalizer.
Fig. 6.
Fig. 6.
Immunofluorescence microscopy showing changes in pancreatic ZnT1 and ZnT2 in response to Zn depletion. Mice were fed the ZnN or Zn- diet for 7 days. Sections were incubated individually with ZnT1 or ZnT2 antibody, followed by IgG-Alexa conjugate (red). Secondary antibody for α-amylase was FITC-IgG conjugate (green). Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (blue). Fluorescences generated by ZnT1 or ZnT2 are shown in tissue sections from mice fed as follows: ZnT2, ZnN (A); ZnT2, Zn- (B); ZnT1, ZnN (C); ZnT1, Zn- (D); ZnT1 in acinar cells surrounding islet (arrow) in ZnN (E); α-Amylase in acinar cells (F); Both α-amylase and ZnT antibodies and respective secondary conjugates with the fluorescence emission images merged (G); and ZnT1 and ZnT2 peptides were preincubated with respective antibodies before addition to sections from ZnN mice (H and I).
Fig. 7.
Fig. 7.
Intestinal Zip4 up-regulation by Zn depletion is less in MT knockout mice than wild type. (A) Zip4 mRNA expression and serum Zn concentrations in mice fed the low (Zn-) relative to the adequate (ZnN) ZnN diet when both were fed for 7 days. Values are given as means ± SEM (n = 3). *, P < 0.05, for Zip4 mRNA compared with WT genotype mice. **, P < 0.05, for serum Zn compared with ZnN mice. (B) Western blot analysis of total intestinal membrane proteins showing decrease in Zip4 protein abundance in MT knockout mice.
Fig. 8.
Fig. 8.
Q-PCR analysis of ZnT1 transcript abundance in mouse PBMCs. The cells were isolated from mice that were fed the low (Zn-), adequate (ZnN), or high (Zn+) Zn diet for 7 days. ZnT1 mRNA levels are expressed relative to the ZnN group by using 18S rRNA as the normalizer. Values are given as means ± SEM (n = 3). *, P < 0.05, compared with ZnN group.
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References

    1. Liuzzi, J. P. & Cousins, R. J. (2004) Annu. Rev. Nutr. 24, 151-172. - PubMed
    1. Wastney, M. E., House, W. A., Barnes, R. M. & Subramanian, K. N. (2000) J. Nutr. 130, 1355S-1359S. - PubMed
    1. Panel on Micronutrients, Subcommittees on Upper Reference Levels of Nutrients and of Interpretation and Use of Dietary Reference Intakes, and the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes (2002) in Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc, eds. Food and Nutrition Board (Natl. Acad. Press, Washington, DC), pp. 442-501.
    1. Hoadley, J. E., Leinart, A. S. & Cousins, R. J. (1987) Am. J. Physiol. 252, G825-G831. - PubMed
    1. Cousins, R. J. (1996) in Present Knowledge in Nutrition, eds. Filer, L. J. & Ziegler, E. E. (ILSI, Washington, DC), 7th Ed., pp. 293-306.

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