The Journal of Nutrition Vol. 128 No. 7 July 1998, pp. 1092-1098

Zinc Deficiency Enhances Interleukin-1-Induced Metallothionein-1 Expression in Rats¹

Li Cui, Yoji Takagi, Masafumi Wasa, Yasuhiko Iiboshi, Masahiro Inoue, Jesmine Khan, Kinya Sando, Riichiro Nezu^*, and Akira Okada²

Department of Pediatric Surgery, ^* First Department of Surgery, Osaka University Medical School, Osaka 565, Japan

	ABSTRACT

Abstract Introduction Methods Results Discussion References

This study investigated whether interleukin-1-induced metallothionein gene expression is affected by zinc deficiency. Weaning male rats were fed a zinc-deficient (ZD) diet (2 mg zinc/kg) or a zinc-supplemented diet [50.8 mg zinc/kg; controls for the diet included pair-fed (PF) and ad libitum consumption groups (AL)] for 4 wk. All rats except those that served as controls for interleukin-1 administration, (injected with vehicle and killed at 0 h) were then injected subcutaneously with interleukin-1 (2 × 10⁷ units/kg body wt) and killed at 3, 6, 12, 24 and 72 h after the injection. Compared with AL and/or PF rats, zinc depletion significantly reduced zinc concentrations in plasma and liver but not in kidney or intestine, and significantly reduced hepatic, renal, and intestinal metallothionein-1 mRNA levels analyzed by competitive reverse transcription-polymerase chain reaction (RT-PCR). Interleukin-1 injection reduced plasma zinc concentration and enhanced liver zinc concentration, but did not affect zinc levels in kidney or intestine. Metallothionein-1 mRNA was significantly elevated by interleukin-1 in liver, kidney and intestine of all groups; the levels in liver and kidney of ZD rats 6 h after the injection were significantly higher than those of AL or PF rats. Liver metallothionein protein levels were enhanced after interleukin-1 injection in both AL and ZD rats. Semiquantitative RT-PCR revealed significantly higher hepatic levels of interleukin-1 receptor type-I mRNA in ZD rats than in AL and PF rats but no differences in renal or intestinal tissues among groups before interleukin-1 challenge. In conclusion, zinc deficiency induces upregulation of metallothionein-1 gene expression in response to interleukin-1 challenge in rats.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Metallothioneins (MT³) are cystine-rich, low molecular weight proteins with high affinity for physiological and nonphysiological heavy metals. Their biologic roles are associated with storage of metals such as zinc. MT are also regarded as acute-phase proteins because a wide variety of factors including pathological, physical and psychological stress, can induce their synthesis. The amount of MT in tissues is highly dependent on metal-ion availability and much of the regulation occurs at the level of transcription (Chesters 1992). Regulation of MT gene expression by zinc and other metals has received considerable attention (Cousins 1985). The metal regulatory elements modulate the specific nucleotide sequences in the promoter region of the MT gene. How the synthesis of MT is regulated by cytokines such as interleukin-1 (IL-1), particularly in zinc-deficient status, remains unresolved. The region responsible for regulation by lipopolysaccharide lies upstream from the sequences necessary for heavy metal regulation (Dunn et al. 1987). MT gene expression modulation by certain cytokines, including IL-1, has been reported to be tissue specific (Cousins and Leinart 1988).

We recently reported that zinc-deficient rats had a greater response to IL-1 challenge than zinc-adequate rats, reflected by a more marked and prolonged inducible nitric oxide synthase (iNOS) expression and a greater incidence of diarrhea (Cui et al. 1997).

IL-1 binds to two cell-surface receptors. The type-I receptor mediates the biological effects of IL-1, whereas the type-II receptor binds IL-1 and thereby prevents it from binding to the type-I receptor, but does not deliver a biological signal (Sims et al. 1994). IL-1 binding to its type-I, but not type-II receptor, modulates the acute-phase response (Oldenburg et al. 1995). Whether zinc status affects IL-1-mediated response by altering IL-1 receptor expression has not been explored.

In this study, by using competitive reverse transcription-polymerase chain reaction (RT-PCR) techniques, we investigated the IL-1-induced MT-1 gene transcription in liver, kidney and intestinal tissues of zinc-deficient rats. Although enough tissue was available for Northern blot analysis, we reasoned that the competitive RT-PCR technique, with its higher sensitivity, might be able to detect a small change (Sullivan and Cousins 1997) induced by IL-1 such as in the kidney that Northern blot failed to detect, as reported elsewhere (Cousins and Leinart 1988) and in our preliminary experiment (data not shown). We tested whether zinc status affected systemic zinc redistribution and whether zinc concentrations in the tissues were linked to induction of MT gene expression in response to administration of IL-1. Because most physiological roles of IL-1 are mediated by IL-1 receptor type-I (IL-1RI), the expression of this receptor in tissues of rats with different zinc status was also examined.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals and diets.  All experiments involving animals were conducted in accordance with NIH guidelines (NRC 1985) and all animal experiments were approved by Osaka University Animal Care and Use Committee. Male Sprague-Dawley rats (n = 105) (Charles River, Yokohama, Japan), 3 wk of age, were individually housed in acid-washed, stainless steel cages at 23°C with a 12-h light:dark cycle. The purified diet was based on the AIN-76A formulation as described in detail previously (Cui et al. 1997). The rats were allowed free access to glass-distilled deionized water, fed a semipurified zinc-adequate diet (50.8 mg zinc/kg) for 1 wk to allow acclimation to our laboratory conditions and then divided in to three groups as described previously (Cui et al. 1997). One group was given free access to a zinc-adequate diet (ad libitum consumption group, AL); the second group was given a zinc-deficient (ZD) diet (2 mg zinc/kg); and the third group was pair-fed (PF) the zinc-adequate diet at a level equal to the mean intake of the ZD group. The above diets were fed for 4 wk. Rats and diets were weighed and recorded daily.

Experimental protocol.  After consuming their respective diets for 4 wk, all rats, except those used for basal (time 0) measurements and subjected to injection of vehicle, were subcutaneously injected with IL-1 (2 × 10⁷ units/kg body weight; human recombinant IL-1 was kindly donated by DaiNippon Pharmaceutical, Osaka, Japan). The rats were killed at 0, 3, 6, 12, 24, 48 and 72 h after the injection. There were 35 rats in each group and 5 rats at each time point. Heparinized blood was collected from the abdominal aorta in rats under ethyl ether anesthesia. Tissues were immediately excised. Intestinal samples were removed from the jejunum including the mucus, muscular and serosa, 10 cm in length starting at 2 cm from the ligament of Treitz. Liver, kidney samples and ~2 cm intestine for RNA analysis were processed immediately. Samples of plasma, liver, kidney and the remaining intestine for zinc determination were stored at 20°C until use.

Determination of zinc concentration.  Plasma was digested with 1 mol/L hydrochloric acid (Takagi et al. 1986). Liver, kidney and intestine samples were digested by using concentrated nitric acid (Cui et al. 1996). Zinc content was measured by atomic absorption spectrophotometry (Z-6100 simultaneous multi-element atomic absorption spectrophotometer, Hitachi Instrument, Tokyo Japan).

RNA isolation.  Total RNA was extracted from tissues by using the commercial reagent ISOGEN (NipponGene, Tokyo, Japan) as described previously (Cui et al. 1997). The RNA was dissolved in diethyl pyrocarbonate-treated distilled water. The concentration of RNA was estimated from the absorbance at 260 nm (the ratio at 260/280 was between 1.6 and 1.9).

mRNA analyses.  The expression of MT-1 mRNA was determined by competitive RT-PCR by using the nonhomologous competitor, PCR MIMIC. PCR MIMIC was generated from pBR322 according to the method of Siebert and Larrick (1993). Briefly, pBR322 cDNA was used to construct the MT-1 MIMIC by two rounds of PCR amplification. In the first PCR reaction, a pair of composite primers containing rat MT-1 gene-specific primer sequence at the 5'-end (Fig. 1) were used to amplify a fragment of pBR322 cDNA. A dilution of the first PCR reaction product was then amplified again using only the MT-1 gene-specific primers. Target MT-1 PCR product was obtained by RT-PCR with total RNA extracted from rat liver used as a cDNA standard. The PCR product of MT-1 was confirmed by using a restriction endonuclease Apa L1, which digested the +170 site of MT-1 cDNA. An aliquot of the MIMIC and cDNA standard was electrophoresed simultaneously with a serially diluted HaeIII digest of 174 DNA (GIBCO-BRL Life Technologies, Gaithersburg, MD) on a 2% agarose gel containing ethidium bromide. The intensities of UV-induced fluorescence were analyzed by NIH Image 1.55 software. The quantities of the MIMIC and cDNA standard were calculated as described by Siebert and Larrick (1993).

View larger version (21K): [in this window] [in a new window]   Fig 1. Sequences of gene-specific and composite primers. A pair of 20-mer gene-specific primers amplified a 186-bp sequence between +7 and +261 of rat metallothionein-1 cDNA. The composite primers were designed to construct polymerase chain reaction (PCR) MIMIC with gene-specific 5' and 3' ends. Each composite primer consists of the target gene-specific primer sequence followed by 21 nucleotides that recognize the complementary sequence in tetracycline resistance gene of pBR322 plasmid.

First strand cDNA of samples were synthesized from 1 µg total RNA by using random 9-mer primers and avian myeloblastosis virus reverse transcriptase (Takara Shuzo, Shiga, Japan). Sample cDNA and serially diluted cDNA standards were then added to PCR amplification reactions containing a constant amount of PCR MIMIC [0.1 attomole (attomole = 10¹⁸ mol) for intestine samples, 1 attomole (amol) for liver and kidney samples] according to the following schedule: denaturation, annealing and extension at 94, 60 and 72°C for 1 min, 1 min and 1 min 30 s, respectively, for 25 cycles. PCR products were electrophoresed on 2% agarose gels containing ethidium bromide. The intensities of UV-induced fluorescence were analyzed by NIH Image 1.55 software.

IL-1RI mRNA basal levels (before IL-1 administration) in tissues were analyzed by a semiquantitative RT-PCR using a pair of gene-specific primers (forward: 5'-TCT CAT TGT GCC TCT GCT GT-3'; reverse: 5'-CTG TCC CTC TTG CTG TCA TC-3') with the schedule of denaturation, annealing and extension at 94, 60, and 72°C for 40 s, 1 min and 1 min 30 s, respectively, for 35 cycles. To ensure that equal amounts of reverse-transcribed RNA were added to the PCR reaction, a parallel amplification of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mRNA was performed as an internal reference as described previously (Cui et al. 1997). The ratio of IL-1RI mRNA/GAPDH mRNA intensities was used to evaluate the relative levels and plotted as a percentage of the level in the AL group.

Western blotting analysis.  MT protein in rat liver was analyzed by Western blotting with immunostaining. Liver tissues were homogenized in homogenate buffer (0.25 mol/L sucrose, 10 mmol/L Tris-HCl, pH 7.4, and 5 mmol dithiothreitol) with a tissue/buffer ratio of 1:5 (wt/v). The homogenate was centrifuged at 100,000 × g at 4°C for 1 h. Protein concentration in the supernatant was determined by the method of Lowry et al. (1951) with bovine serum albumin used as a standard. Samples (100 µg protein) were applied to polyacrylamide gels (15% acrylamide in running gel and 3% acrylamide in staking gel) in a discontinuous Tris-buffer system containing 1% SDS according to the method of Laemmli (1970). Gels were subsequently transferred onto Hybond-ECL membranes (Amersham International, Amersham, UK) at 25 V (constant voltage) for 1 h. The membranes were blocked with Block Ace (DaiNippon Pharmaceutical) and incubated for 1 h with E9 (monoclonal mouse anti-metallothionein, Dako, Carpinteria, CA) diluted 1:2000. The blots were then incubated for 1 h with anti-mouse immunoglobulin G HRP conjugate (Promega, Madison, WI) diluted 1:2000. Immunoblots were developed by ECL Western blotting detection system (Amersham International). Horse metallothioneins (Sigma Chemical, St. Louis, MO) were used for positive immunostaining.

Statistical analysis.  Results are expressed as means ± SD. Differences in body weight, food intake and IL-1RI mRNA levels were examined by one-way ANOVA with post hoc testing by Fisher's protected least significant difference (PLSD). Differences in zinc concentrations and levels of MT-1 mRNA between groups and between the time zero and other time points after IL-1 treatment were examined by two-way ANOVA with post hoc testing by Fisher's PLSD. The statistical software Statview-J 4.1 (Abacus Concepts, Berkeley, CA) was used on an Apple Macintosh computer. Differences of P < 0.05 were considered significant.

	RESULTS

Abstract Introduction Methods Results Discussion References

All ZD rats had significantly lower food intakes and body weights than AL rats (Fig. 2) and developed dermatitis and alopecia during the experimental period, suggesting zinc deficiency. PF rats had lower body weight than AL rats, but had no dermatitis or alopecia.

View larger version (13K): [in this window] [in a new window]   Fig 2. Body weight (A) and food intake (B) in ad libitum consumption (AL), pair-fed (PF) and zinc-deficient (ZD) rats. Values are means ± SD, n = 35. aP < 0.001 vs. AL.

Plasma and tissue zinc concentrations.  The basal plasma zinc concentration (0 h) was significantly lower in ZD rats than in PF and AL rats. After IL-1 injection, plasma zinc concentrations decreased with time with reductions of up to 82, 75 and 50% of the basal levels at 6 h in AL, PF and ZD rats, respectively. At 12 h, the values started to increase and recovered to the basal levels at 48 h in all groups, whereas the value at 24 h was significantly less than that at 0 h only in AL rats. The concentration in ZD rats was significantly lower than that in controls at each time point (Table 1).

View this table: [in this window] [in a new window]   Table 1. Plasma zinc concentration in ad libitum consumption (AL), pair-fed (PF) and zinc-deficient (ZD) rats after interleukin-1 administration1

In ZD rats, the basal hepatic zinc concentration was significantly lower than those in AL and PF rats (Table 2). IL-1 injection significantly augmented hepatic zinc concentrations in all groups, with 1.18-, 1.51- and 1.63-fold basal levels at 6 h after the injection in AL, PF and ZD rats, respectively. The maximal zinc levels in the three groups did not differ. No significant differences of basal zinc concentrations were observed among groups in renal and intestinal tissues of the three groups, and the zinc levels were not changed by IL-1 injection (data not shown).

View this table: [in this window] [in a new window]   Table 2. Redistribution of zinc in liver of ad libitum consumption (AL), pair-fed (PF) and zinc-deficient (ZD) rats after interleukin-1 administration1

MT-1 mRNA expression.  The PCR reaction simultaneously amplified the 186-bp product of MT-1 cDNA and 502-bp product of PCR MIMIC (Fig. 3). The 186-bp PCR product was digested by Apa L1 at 37°C for 24 h into two fragments of 164 bp and 22 bp in length, as expected (Fig. 4). Therefore, it was verified that the RT-PCR specifically amplified MT-1 mRNA.

View larger version (23K): [in this window] [in a new window]   Fig 3. Validity of quantitation of metallothionein-1 mRNA by competitive reverse transcription-polymerase chain reaction (RT-PCR). Upper panel, 0.1 attomole (attomole = 1018 mol) of PCR MIMIC mixed with various amounts of target cDNA (metallothionein-1 cDNA) was amplified using gene-specific primers. The amount of target cDNA was 0.01 (lane 1), 0.1 (lane 2), 1 (lane 3), 10 amol (lane 4) and 100 (lane 5). Lane M is a size marker, HaeIII digest of 74 DNA. Lower panel, the ratio of target/MIMIC (intensities of bands) was linearly related to target cDNA amount.

View larger version (65K): [in this window] [in a new window]   Fig 4. Identification of metallothionein-1 (MT-1) polymerase chain reaction (PCR) product. PCR product of MT-1 was confirmed by using a restriction endonuclease Apa L1, which digested +170 site of MT-1 cDNA. The PCR product before () and after (+) Apa L1 digestion (37°C, 24 h) was electrophoresed on a 10% polyacrylamide gel. Apa L1 digested the 186-bp product to two fragments of 164 bp and 22 bp in length, as expected.

Dietary zinc depletion significantly reduced hepatic, renal and intestinal MT-1 mRNA levels (Fig. 5). IL-1 injection significantly elevated MT-1 mRNA levels in the three tissues of all groups. The maximal expression of MT-1 mRNA in ZD rats was 69.2-fold basal level in liver, 7.4-fold in kidney and 120.1-fold in intestine, respectively, whereas that in AL and PF rats was 9.5- and 6.4-fold in liver, 1.5- and 1.7-fold in kidney and 99.8- and 46.2-fold in intestine, respectively. The maximal expression of MT-1 mRNA induced by IL-1 injection was observed at 3 or 6 h after injection in liver and kidney and then recovered to basal levels. The levels at 6 h in liver and kidney of ZD rats were significantly higher than that of AL or PF rats. In intestine, the MT-1 expression stimulated by IL-1 was delayed. The maximal mRNA level was noted at 12 and 24 h in ZD and AL rats, respectively, whereas it was noted at 72 h in PF rats.

View larger version (41K): [in this window] [in a new window]   Fig 5. Interleukin-1-induced alteration of metallothionein-1 mRNA levels in liver, kidney and intestine of rats fed zinc-supplemented or zinc-deficient diets. Total RNA was extracted from liver, kidney and intestine of ad libitum consumption (AL), pair-fed (PF) and zinc-deficient (ZD) rats at the indicated times after subcutaneous injection of interleukin-1 (IL-1, 2 × 107 units/kg body wt). Total RNA (1 µg) was reverse transcribed. The resulting cDNA were mixed with 0.1 or 1 amol polymerase chain reaction (PCR) MIMIC and amplified by PCR with metallothionein-1 gene-specific primers. Reverse transcription (RT)-PCR products were electrophoresed on 2% agarose gels containing ethidium bromide. Bar graph summarizes metallothionein-1 mRNA levels. Values are means ± SD, n = 5. Significant differences: P < 0.05 vs. AL, #P < 0.05 vs. PF at the same time point; *P < 0.05 vs. respective basal level (0 h).

MT production.  Western blotting showed the MT band between 6.5 and 14.3 kDa. The basal level of liver MT was lower in ZD rats than in AL rats. After IL-1 injection, there were elevations of MT level in the liver of both AL and ZD rats, with the maxima at 3-6 h (Fig. 6).

View larger version (12K): [in this window] [in a new window]   Fig 6. Interleukin-1-induced alteration of metallothionein-1 protein levels in liver of rats fed zinc-supplemented or zinc-deficient diets. Total protein was extracted from liver tissues of ad libitum consumption (AL) and zinc-deficient (ZD) rats. Metallothionein protein was analyzed by Western blotting with immunostaining. Each lane was loaded with 80 µg of protein. Lane 1-10: AL and ZD rats at the indicated times after subcutaneous injection of interleukin-1 (2 × 107 units/kg body wt). Lane 11: horse metallothioneins, used as a positive staining. Lane 12: molecular weight markers.

IL-1RI mRNA expression.  After rats consumed the diets for 4 wk, the basal level of hepatic IL-1RI mRNA was significantly higher in ZD rats than in AL or PF rats (Fig. 7). However, no significant differences in the IL-1RI mRNA levels were observed in renal and intestinal tissues (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig 7. Interleukin-1 receptor type-I (IL-1RI) mRNA levels in livers of rats fed zinc-supplemented or zinc-deficient diets. Total RNA was extracted from liver of ad libitum consumption (AL), pair-fed (PF) and zinc-deficient (ZD) rats without interleukin-1 treatment. Total RNA (1 µg) was reverse transcribed and the resulting cDNA were amplified by polymerase chain reaction (PCR) with gene-specific primers for IL-1RI and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). Upper panel, reverse transcription (RT)-PCR products electrophoresed on 2% agarose gels containing ethidium bromide. Lower panel, the ratio of IL-1RI mRNA/GAPDH mRNA intensities was used to evaluate the relative levels and plotted as a percentage of the level in the AL group. Values are means ± SD, n = 5. Significant differences: *P < 0.05 significantly greater than AL and PF.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Zinc deficiency was induced by a 4-wk dietary zinc restriction, which was characterized by a marked reduction of zinc concentration in plasma and liver, and other signs, including reduced food intake and body weight, and development of dermatitis and alopecia. These results were consistent with our previous report (Cui et al. 1996) and those of others (Cousins and Lee-Ambrose 1992).

IL-1 injection caused a zinc redistribution involving a decrease in plasma zinc concentration and an increase in liver zinc concentration, which supported the findings of other researchers (Cousins and Leinart 1988). IL-1-induced maximal zinc accumulation in liver was similar in zinc-deficient and zinc-adequate rats, indicating that the redistribution of zinc from plasma to liver induced by IL-1 was not compromised because of zinc deficiency. No appreciable change in kidney zinc concentration was observed after IL-1 administration, suggesting that kidney is not an available zinc source for liver. Cousins and Leinart (1988) reported that zinc accumulation in liver after administration of IL-1 was associated with loss of zinc from intestine. In this study, we observed that zinc concentration in whole-layer tissue of intestine in all three groups failed to change after injection of IL-1. Whether IL-1 affects zinc concentration of intestinal mucosa remains to be investigated.

Dietary zinc can influence MT gene expression (Cousins and Lee-Ambrose 1992). This study reconfirmed that zinc deficiency reduced MT-1 mRNA levels in liver, kidney and intestine. The results on hepatic MT-1 induction by IL-1 agree with a series of reports (Hempe et al. 1991, Huber and Cousins 1988 and 1993). However, zinc-deficient rats had a more marked response to IL-1; although the level of MT mRNA in liver of ZD rats was lower than that of AL and PF rats before IL-1 administration, it increased to a level that was significantly higher than that of AL rats 6 h after IL-1 administration. The level of MT protein showed a change similar to that of AL rats in response to IL-1 administration. The result is different from that of Huber and Cousins (1988) who found that induction of MT mRNA in the liver was greater in rats fed an adequate diet than in those fed the low zinc diet. In addition, we observed that MT-1 mRNA levels in kidney and intestine rose in response to IL-1 treatment, apparently independently of zinc status. Cousins and Leinart (1988) observed no appreciable changes of MT mRNA in the kidney of rats fed an adequate diet after IL-1 treatment. Hempe et al. (1991) reported that MT mRNA of the intestinal mucosa was not affected by IL-1. The discrepancies between our observations and the above-mentioned studies may be explained as follows. First, competitive RT-PCR allows absolute quantitation of mRNA level (Devaud et al. 1995), allowing detection of a slight increase (50%) of the MT-1 mRNA level in kidney, whereas Northern blot permits only crude quantitation of mRNA (Thur et al. 1996). Although the calculation of MT mRNA level in this study was based on the linear relationship between the ratio of target/MIMIC intensity and the target cDNA amount without establishment of the 1:1 mass ratio between the target and the MIMIC in each target sample, some of the data presented here were obtained by both the conventional quantitative PCR method, in which multiple PCR were conducted on the same sample with serial dilutions of the MIMIC to establish the 1:1 mass ratio, and the current method, demonstrating that the levels were consistent. Second, the conclusion that the abundance of intestinal MT mRNA did not respond to IL-1 treatment was made by examining the mucosa (Hempe et al. 1991), whereas this study investigated changes in MT mRNA in whole layers of the tissue rather than the mucosa only. Third, in this study, the feeding period of ZD diet was 4 wk, which was longer than that of previous reports, indicating that zinc deficiency of rats in this study was more pronounced. In addition, the maximal gene expression of intestinal MT occurred at 24 h in our model (AL group), whereas Hempe et al. (1991) presented only the data at 6 h after IL-1 administration. On the other hand, because MT can be induced by stress, possibly hours after introduction of the stress, and the comparisons in this study were performed between MT-1 mRNA level at zero hour and levels at the determined time points, the enhancement in MT-1 gene expression may result from the combined effect of IL-1 plus stress of the injections, leading to the discrepancy between our data and those reported by Hempe et al. (1991).

Administration of zinc followed by endotoxin treatment increases MT levels in rat liver by 40-fold, whereas simple challenge with zinc or endotoxin increases MT levels 8-12 times (Hernandez et al. 1996), suggesting a synergistic effect between the two inducers. The synergism between zinc and endotoxin in liver MT induction in vivo is hypothetically attributed to zinc levels in the body and the mobilization capacity of zinc, because the effect was abolished in an in vitro experiment (Hernandez et al. 1996). One explanation for this synergism is that zinc stimulation of the MT promoter could provide an enhancement of transcription regulation by other regulatory elements. Our results agree with this hypothesis because an increase in zinc concentration of 63, 51, and 18% in the liver of ZD, PF and AL rats, respectively, corresponded to an increase in their hepatic MT mRNA amount by 69-fold, 6.4-fold and 9.5-fold, respectively, after IL-1 injection. Consistent with change in liver MT mRNA, the amount of liver MT protein was elevated in both AL and ZD rats in response to IL-1 challenge. It is interesting that ZD rats showed a higher level of hepatic IL-1RI mRNA than AL and PF rats before IL-1 administration. Certain cytokines, including interleukin-1, up-regulate transcription of the IL-1 receptor in some tissues (Takii et al. 1992 and 1995). Because dermatitis occurred in all ZD rats during the 4-wk feeding period, ZD rats may have had chronically elevated levels of systemic proinflammatory cytokines. The increased IL-1RI expression may account in part for the higher response of ZD rats to IL-1 in promoting hepatic MT-1 expression, but it is necessary to further demonstrate whether there is an increased density of the receptor on the cell surface.

In the kidney and intestine, IL-1-induced elevation of MT mRNA was independent of tissue zinc levels. In particular, MT mRNA level at maximum was elevated by 7.4-fold and 120-fold of the basal levels, in the kidney and intestine respectively, of ZD rats. Second messengers and crosstalk among transduction pathways involved in MT induction by cytokines have not been well established. Calcium signaling through protein kinase A, protein kinase C and calmodulin-dependent protein kinase has been suggested to play a role (Arizono et al. 1993, Xiong et al. 1992). Arizono et al. (1995) reported that in an in vitro experiment, MT induction after lipopolysaccharide administration was mediated by nitric oxide. Our current study also suggested that nitric oxide might play a role in regulation of MT mRNA induction in intestine because iNOS was maximally expressed 6 h after IL-1 challenge in the intestines of AL, PF and ZD rats, and the expression in ZD rats was significantly higher than that in controls (Cui et al. 1997). Consequently, MT mRNA level reached maximum elevation at 12 h in ZD, 24 h in AL and 72 h in PF rats. However, MT mRNA induction due to production of iNOS does not adequately explain the phenomenon in the kidney. MT mRNA in the kidney of rats was elevated during the 3-6 h after treatment with IL-1, demonstrating a pattern similar to that in the liver but within a rather small range, particularly in the AL and PF rats. Immunochemical localization studies have shown that specific staining for MT protein was confined to the epithelial tubular cells of the nephron and the collecting ducts; this differs from liver in which MT protein staining was found in all hepatocytes (Banerjee et al. 1982, Danielson et al. 1982). On the other hand, macrophages of polymorphonuclear cells in the kidney were up-regulated more in ZD rats than in the controls (Ercan and Bor 1991), indicating that zinc deficiency may change the function of the reticuloendothelial system in the kidney. Further information on the location of MT mRNA in the kidney of ZD rats before and after IL-1 treatment is necessary for the above possibilities to be confirmed.

In conclusion, dietary zinc depletion reduces basal zinc levels and MT-1 expression but does not attenuate IL-1-induced zinc redistribution and MT-1 expression. IL-1 increases MT-1 mRNA levels in the liver, kidney and intestine of both zinc-deficient and zinc-adequate rats. Consistent with the change in MT mRNA, the amount of MT protein was elevated in liver in response to an IL-1 challenge. The response to an IL-1 challenge is greater in zinc-deficient rats.

	FOOTNOTES

Manuscript received 1 July 1997. Initial reviews completed 8 August 1997. Revision accepted 16 March 1998.

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