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Articles

Actions and Interactions of Thyroid Hormone and Zinc Status in Growing Rats^{1 ,2}

Hedley C. Freake^*3, Kristen E. Govoni, Krishna Guda, Chunli Huang^* and Steven A. Zinn

Departments of ^* Nutritional Sciences and Animal Science, University of Connecticut, Storrs, Connecticut 06269-4017

³To whom correspondence should be addressed. E-mail: hedley.freake{at}uconn.edu

	ABSTRACT

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Both thyroid hormone (triiodo-L-thyronine, T₃) and zinc play important roles in growth and development. The T₃ receptor is thought to require zinc to adopt its biologically active conformation. Some of the effects of zinc deficiency, therefore, may be due to loss of zinc from the T₃ receptor and impairment of T₃ action. This possibility was investigated in growing rats by examining the effects of hypothyroidism and hyperthyroidism in zinc-deficient, pair-fed and control rats. Measurement of serum zinc and T₃ confirmed the efficacy of the treatments. Zinc deficiency and hypothyroidism resulted in lower food intake and growth failure, but no interaction was observed between the two treatments. Individual tissue weights were influenced by thyroid status as expected, regardless of zinc status. Both dietary and hormonal treatments influenced serum insulin-like growth factor (IGF)-I in an interactive manner. IGF-I was reduced to a greater extent in zinc-deficient than in pair-fed rats compared with controls. Both hypothyroidism and hyperthyroidism reduced serum IGF-I, and a greater reduction due to hyperthyroidism was apparent in zinc-deficient rats. IGF binding proteins were also influenced by diet and thyroid status. The hepatic expression of mRNA S14 was assessed as a direct index of the nuclear action of T₃, but its response was not influenced by dietary treatment. Although confirming the role of both T₃ and zinc in the regulation of growth and the somatotrophic axis, the growth failure of zinc deficiency does not appear to be due to impaired T₃ function.

KEY WORDS: • thyroid hormone • zinc • growth • rats

	INTRODUCTION

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The biological effects of zinc are remarkably diverse. It is a constituent of >300 enzymes, representing all six classes described by the International Union of Biochemistry (1) . In addition, 30% of cellular zinc is found within the nucleus (2) , and a large number of proteins that play a role in the regulation of gene expression have been either shown or suspected to contain zinc (3) . Zinc deficiency clearly retards growth and inhibits DNA synthesis, although which of the numerous zinc-dependent proteins underlie these effects is not clear (4) . It has been suggested that nuclear zinc-binding proteins may be more susceptible to zinc deficiency than zinc metalloenzymes because they have a lower affinity for the cation (5) .

The biological effects of thyroid hormones are also remarkably diverse (6) . The active hormone is triiodo-L-thyronine (T₃),⁴ derived through peripheral deiodination from thyroxine (T₄), the major secretory product of the thyroid gland. T₃ actions in its target tissues are initiated by binding of the hormone to specific thyroid hormone receptor proteins within the nucleus (7) . There are two T₃ receptor genes ( and ß), and multiple products are derived from these genes, although not all retain the ability to bind hormone. T₃ receptors are closely related to the receptors of other nuclear acting agents, including those for steroid hormones, retinoic acid and vitamin D (8) . Together, they form the steroid/thyroid nuclear receptor superfamily, sharing similar mechanisms of action and considerable sequence homology. T₃ receptors bind to specific sequences in target genes known as thyroid response elements, probably as heterodimers with another member of the nuclear receptor family, the retinoid X receptor, whose ligand is 9-cis retinoic acid (9) . The receptor heterodimer interacts with other recently described transcription factors, corepressors and coactivators to alter the rate of gene transcription in a hormone-dependent manner (10) .

T₃ receptors, in common with other members of the nuclear receptor family, are thought to be included among the nuclear zinc-binding proteins. They all contain nine invariant cysteine residues in the DNA-binding region (8) . For the glucocorticoid receptor, it has been shown that point mutations in this area, which eliminate the possibility of zinc binding, also render the receptor inactive (11) . X-ray absorption spectroscopy has demonstrated that a bacterially expressed fragment of the glucocorticoid receptor, including the DNA-binding domain, contains two zinc atoms per receptor molecule (12) . Evidence for T₃ receptors is much less complete, but if zinc is removed through chelation, T₃ receptors produced from a bacterial expression system lose their ability to bind to DNA (13) . Thus, it appears likely that zinc is required for the biological functioning of the thyroid hormone and related receptors, but it is not clear whether there would be any circumstances in vivo under which zinc would be lost and receptor function impaired.

We investigated this question in a cell culture system, limiting zinc availability with a chelator and testing the response of the cells to T₃ (14 ,15) . Surprisingly, use of the chelator amplified the action of T₃, and this effect was specifically inhibited by additional zinc. In the absence of the chelator, small negative effects of zinc on T₃ action were observed. Although these results contradicted the experimental hypotheses, they do have precedent. Surks et al. (16) and Lu et al. (17) reported that zinc reduced the binding of T₃ to its receptor in various preparations in vitro. Others have suggested that these effects of zinc are actually due to its ability to aggregate T₃ receptors, resulting in an underestimation of binding during the assay procedure (18) . We were unable to detect any effect of zinc addition or removal on nuclear binding of T₃ in GH3 cells (14) , and zinc does not affect T₃ receptor binding in cultured chick embryonic hepatocytes (19) . However, the latter study showed that zinc inhibited T₃-induced expression of fatty acid synthase and malic enzyme, in a dose-dependent fashion. Given no effect of zinc on T₃ binding to its receptor, the explanation for this inhibition was not apparent.

These in vitro data and sequence predictions do not result in a coherent picture and suggest that the question of whether or how zinc affects thyroid hormone function has to be addressed in vivo. We therefore induced hypothyroidism and hyperthyroidism in zinc-deficient, pair-fed and control rats to determine whether zinc status influences the effects of these endocrine manipulations on thyroid-sensitive variables. Because both T₃ and zinc are known to regulate growth and interact with the somatotrophic axis at multiple levels, we focused on these aspects.

	MATERIALS AND METHODS

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Male Sprague-Dawley rats of the Holtzman strain (Harlan Sprague-Dawley, Indianapolis, IN) were purchased at 3–4 wk of age. They were housed in individual stainless steel cages in a climate-controlled animal room (21 ± 2°C) on a 12-h light/dark cycle, with free access to deionized water. Zinc deficiency was induced in 15 rats by feeding a zinc-deficient diet (<1 mg zinc/kg diet), with egg white as protein (No. 115321; Dyets, Bethlehem, PA) (Table 1 ). All other rats (control and pair-fed groups, 15 rats each) were fed an equivalent control diet (No. 115332; Dyets) containing 30 mg zinc/kg diet. The pair-fed group was included to account for the reduced food intake associated with zinc deficiency. Each rat in that group was restricted to the amount consumed by its paired zinc-deficient rat on the previous day, adjusted on the basis of metabolic body weight (0.75). This corrected for a proportionally greater food deprivation in the pair-fed rats, in the event that their weight exceeded that of the zinc-deficient rats. After 5 d, hypothyroidism was induced in five rats from each dietary group by the addition of 0.25 g methimazole/L to the drinking water for 3 wk. The remaining rats were left euthyroid. Two weeks later, five of the euthyroid rats from each diet group were made hyperthyroid (150 µg T₃ · kg body^-1 · d^-1 via intraperitoneal injection) for the final 7 d. Other rats were sham injected. This dosage of T₃ results in a plasma concentration after 24 h that is sufficient to maintain saturation of nuclear T₃ receptors and therefore a constant, maximal response (20 ,21) . Food intake was recorded daily, and body weight was recorded three times per week. Animal protocols were approved by the institutional animal care and use committee.

Rats were killed between 0900 and 1100 h to minimize diurnal variations. After anesthesia with methoxyflurane, blood was taken from the abdominal aorta. Tissues were removed, rinsed with ice cold saline, weighed, frozen on dry ice and stored at -80°C. Blood was allowed to clot, and serum was prepared through centrifugation and stored at -80°C until analysis.

Serum analyses.

For zinc analysis, serum samples were deproteinized with an equal volume of 25% trichloroacetic acid. A standard curve was prepared in parallel using a zinc standard solution (Sigma Chemical Co., St. Louis, MO). Zinc content of the soluble fraction of serums and standards was estimated with a Perkin-Elmer Cetus 2380 atomic absorption spectrophotometer (Norwalk, CT) (22) . Serum T₃ levels were measured by radioimmunoassay with a commercial kit (Coat-a-Count; Diagnostic Products Corporation, Los Angeles, CA). Serum levels of growth hormone (GH) were assessed using an enzyme immunoassay kit (Cayman Chemicals, Ann Arbor, MI). Insulin-like growth factor (IGF)-I was measured by radioimmunoassay (Diagnostic Systems Laboratories, Webster, TX). Serum IGF-binding proteins (IGFBP)-2, -3 and -4 were determined using modifications to the ligand blot methodology of Hossenlopp et al. (23) and Skaar et al. (24) . Briefly, serum samples (1 µL), recombinant IGFBP-3 (8 ng) (Diagnostic Systems Laboratories, Webster, TX) and standard size markers (BioRad, Richmond, CA) were loaded onto a 5% stacking/12% running polyacrylamide gel in a mini-protean II electrophoresis unit (BioRad). Samples were electrophoresed for 30 min at 100 V followed by 1 h at 150 V. Gels were then transferred to nitrocellulose paper at 0.5 amp for 2 h in a transfer tank with a Transphor Power-Lid (Hoeffer Scientific Instruments, San Francisco, CA). Membranes were incubated overnight at 37°C (rotated at 175 rpm) with 1.6 MBq ¹²⁵I-labeled IGF-I/L (Amersham Pharmacia Biotech, Piscataway, NJ) in Tween 20 (1 mL/L in Tris-buffered saline). After washing, the membrane was exposed to a multipurpose phosphor screen (Packard Instrument Company, Meriden, CT) for 5 h, and bound radioactivity on each blot was quantified with a Cyclone Storage Phosphor System (Packard). Images were analyzed with OptiQuant acquisition and analysis software (Packard). Binding proteins were measured as digital light units (DLU)/mm², and the data are expressed as a percentage of the signal of recombinant IGFBP-3.

Tissue analysis.

Hepatic RNA was isolated independently from each rat using acid guanidinium thiocyanate-phenol-chloroform (25) as previously described (26) . S14 mRNA levels were measured by Northern blot analysis (26) with S14 cDNA (kindly supplied by Howard Towle, University of Minnesota). Total RNA was denatured and electrophoresed through a 1% agarose/6% formaldehyde gel, capillary transferred to a reinforced nitrocellulose membrane and affixed by UV cross-linking. Membranes were hybridized overnight at 65°C with [³²P]cRNA probes. After stringent washing (26) , the hybridized signals were detected by autoradiography. The membranes were also hybridized with a ribosomal protein L32 probe to ensure uniformity of loading and to check specificity of the response.

Statistical analysis.

The influences of diet and thyroid status were analyzed in a randomized complete design using the SAS Institute General Linear Models (27) . Data are expressed as mean ± SEM. If the main effects were significant (P < 0.05), then the means for individual treatment groups were compared using the PDIFF tables within SAS. Initially, there were five rats within each treatment group. However, one rat in each thyroid group fed the zinc-deficient diet did not reduce food intake and therefore gained weight at a much faster rate than the treatment mates. These rats, and their pair-fed controls, were excluded from the subsequent analyses. In addition, two rats died, and the serum sample was lost from one other, resulting in their exclusion. Thus, groups contained three to five rats.

	RESULTS

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The zinc-deficient diet resulted in a reduced food intake and a slowing of growth within the 1st wk (Fig. 1 ). This diet also resulted in a cyclic food intake with a periodicity of 4 d. During the 25 d of the experiment, the zinc-deficient rats, as a group, consumed 34% less food than the controls and exhibited 30% of the weight gain of the controls and 59% of the gain of the pair-fed rats (Fig. 1 , Table 2 ). In addition, both hypothyroid (P < 0.001) and hyperthyroid (P < 0.05) rats weighed less than euthyroid rats. There was no significant interaction between thyroid status and zinc status with respect to body weight (P = 0.08).

View larger version (21K): [in this window] [in a new window]   Figure 1. Effect of zinc-deficient diet and thyroid status on body weight in growing rats. Four-week-old rats were assigned to the zinc-deficient (ZnD) or the control (C) diet on d 1. Pair-fed (PF) rats were fed the control diet in the amounts consumed by their paired zinc-deficient rat on the previous day, corrected for metabolic body weight. On d 5, methimazole (0.25 g/L) was added to the drinking water of one third of the rats in each dietary group to induce hypothyroidism (hypo). Beginning on d 18, one third of the rats were injected daily with T3 (150 µg/kg body intraperitoneal) to induce hyperthyroidism (hyper). The remaining rats were left euthyroid (eu). Results shown are the means of three to five rats in each diet/hormone group. Pooled SEM = 13.1. Final body weights and significant differences are shown in Table 2 .

Measurement of serum zinc confirmed the efficacy of the zinc-deficient diet (Table 3 ). Zinc concentrations did not differ between pair-fed and control rats but were about two thirds lower in serums from zinc-deficient rats. Thyroid status also influenced serum zinc (P < 0.01). This was apparent primarily in the pair-fed group, where zinc levels were correlated to thyroid status (Table 3) . Although a similar tendency was noted in the control rats, there were no significant pairwise differences due to thyroid state within that group or in the zinc-deficient rats.

Serum growth hormone was unaffected by either diet or thyroid treatment (Table 3) . Both, however, had significant effects on serum IGF-I (P < 0.001). Zinc deficiency resulted in reduced IGF-I concentrations compared with pair-fed rats (P < 0.001), which in turn were less than controls (P < 0.001). Concentrations of IGF-I in euthyroid rats were greater than those in either hypothyroid or hyperthyroid rats (P < 0.001). Zinc deficiency and pair feeding had greater effects in hyperthyroid rats (20 and 62% of control levels) than in hypothyroid rats (54 and 80%, respectively), and a significant interaction between the two treatments was observed (P < 0.05).

Ligand blot analysis with an IGF-I probe was used to measure the levels of the more abundant IGFBP in serum (Fig. 2 ). There was a significant effect of diet on IGFBP-3 concentrations (P < 0.001), with control levels being greater than those in either zinc-deficient (P < 0.001) or pair-fed (P < 0.05) rats, although these latter two groups did not differ from each other (Table 3) . IGFBP-2 concentrations were also lower in zinc-deficient rats than in controls, with the pair-fed group intermediate between the two. Neither IGFBP-2 nor -3 was influenced by thyroid status. There were significant effects of both diet and thyroid status on IGFBP-4 (P < 0.001). The abundance of IGFBP-4 was less in zinc-deficient (P < 0.001) and pair-fed (P < 0.05) rats compared with controls. Hypothyroid rats had reduced quantities of this binding protein in serum compared with either the euthyroid or hyperthyroid rats (P < 0.001).

View larger version (88K): [in this window] [in a new window]   Figure 2. Effect of zinc deficiency and thyroid status on serum insulin-like growth factor–binding proteins in growing rats. Rats were assigned to the zinc-deficient, control or pair-fed groups and rendered hypothyroid (Hypo), euthyroid (Eu) or hyperthyroid (Hyper). Blood was collected at the end of the experiment and centrifuged to generate serum. Serum was subjected to polyacrylamide gel electrophoresis, transferred to a nylon membrane and hybridized with an [125I]IGF-I probe. Specific binding proteins (BP) were identified by their molecular weight compared with a ladder (BP-3, 38–42 kDa; BP-2, 30 kDa; BP-4, 24 kDa). Autoradiograms from representative rats from each group are shown, and numerical data are given in Table 3 .

View larger version (93K): [in this window] [in a new window]   Figure 3. Effect of zinc-deficient diet and thyroid status on hepatic expression of mRNA- S14 in rats. Rats were assigned to the zinc-deficient, control or pair-fed groups and rendered hypothyroid (Hypo), euthyroid (Eu) or hyperthyroid (Hyper). Total hepatic RNA was extracted from the liver of each rat and subjected to Northern blot analysis using a [32P]cRNA recognizing mRNA-S14. Representative samples from each treatment group are shown. The expected response to thyroid state was not influenced by dietary treatment.

	DISCUSSION

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The protocols used in this study to alter thyroid state and to induce zinc deficiency are fairly standard; the novelty lies in the use of them together to test for interactions. It is worthy of note, looking at the effects of dietary manipulation in the euthyroid rats and of changes in thyroid state in the rats fed the control diet, that these standard protocols produced the desired effects (1 ,6) . Thus, in the euthyroid rats, the zinc-deficient diet resulted in a reduced and cyclic food intake, growth failure and lower serum zinc concentrations. The growth of the euthyroid pair-fed rats was intermediate between that of the zinc-deficient and control rats, whereas their serum zinc levels did not differ from the controls. In rats with free access to a zinc-sufficient diet, hypothyroidism induced by methimazole reduced food intake and growth rates and lowered serum T₃. In rats fed the same diet, the injection of T₃ for the last week of the experiment produced the expected hyperthyroidism, as evidenced by the serum T₃ levels and slowing of the growth rate.

In rats fed the zinc-sufficient diet, in both the pair-fed and control groups, circulating levels of zinc appeared to be reduced in hypothyroid rats and elevated in hyperthyroid rats. The mechanism for this is unclear. There are data that show zinc deficiency reduces circulating T₃ levels in otherwise euthyroid rats (28 ,29) . This tendency was also apparent in this study, although the differences were not significant, perhaps due to the inclusion of the hyperthyroid group in the analysis.

These studies produced no evidence for an interaction between thyroid status and zinc status with respect to the most basic variable: growth. The effects of manipulation of thyroid hormone levels in this fairly extreme way were seen in all three dietary groups. In addition, interactions between thyroid status and dietary treatment were not apparent with individual tissue weights. For example, the cardiac hypertrophy induced by T₃ treatment and the accumulation of brown adipose tissue in both hypothyroid and hyperthyroid rats were equally apparent in zinc-deficient rats and controls. Although interactions did not occur, there were significant effects of zinc deficiency on the relative weights of some tissues. A lack of zinc resulted in reduced white adipose tissue compared with pair-fed rats, suggesting a particular effect in this tissue beyond that of food deprivation. In addition, cardiac mass was greater with zinc deficiency, suggesting the relative conservation of heart tissue with this nutritional stress.

Both zinc and thyroid hormones affect the somatotrophic axis at multiple levels, although the connection between these effects and the overall growth response of the animal is not very clear. In rats, growth hormone production in the pituitary is influenced by T₃, largely due to its well-described transcriptional regulation of the growth hormone gene (30) . In this experiment, we did not detect an effect of thyroid state or zinc on serum GH, but the variability seen within groups emphasizes the pulsatile nature of GH release. Thus, a single measure from each animal is unlikely to provide a reliable estimate of mean circulating concentrations (31) . However, IGF-I levels were reduced by hypothyroidism, consistent with earlier findings, which also showed these effects to occur at the mRNA level (32 33 34) . Effects of the thyroid state on IGF-I appear to be only in part mediated by GH (32 ,33) , although in the total absence of GH, IGF-I does not respond to thyroid hormone (35) . Significant effects of thyroid state on IGFBP were detected only for IGFBP-4 in the current study. T₃ has been shown to stimulate expression of this binding protein in mouse osteoblasts (36) . However, Nanto-Salonen et al. (33) found a magnitude of reduction in IGFBP-3 in hypothyroid rats similar to that seen here and, in parallel, a significant reduction in the corresponding hepatic mRNA levels. In addition, IGFBP-3 levels appear to be positively correlated with the thyroid state in humans (37) . Thus, direct or indirect regulation of IGFBP-3 by thyroid hormones appears likely. Nanto-Salonen et al. (33) also reported that hypothyroidism increased the expression of IGFBP-2 and its hepatic mRNA in 18-d-old rat pups. They did not detect the corresponding protein or mRNA in adult rats, although others have reported a greater abundance of IGFBP-2 mRNA in the liver of 12-wk-old hypothyroid rats (38) . However, it is unclear whether they represent the direct effects of T₃ on IGFBP gene expression or are mediated by changes in GH, IGF-I or some other thyroid-sensitive variable.

Zinc deficiency has been shown to impair GH release in humans (39) , and GH concentrations rose in GH-deficient patients given zinc supplementation (40) . However, the growth failure in zinc deficiency is not simply due to reduced GH production or secretion. Prasad and coworkers (41) could not restore the growth rate of zinc-deficient rats by administering GH. Oner et al. (42) showed that the impairment of skeletal growth seen in young rats with zinc deficiency could not be corrected with GH and that GH was unable to restore circulating IGF-I to concentrations equal to those of zinc-replete rats. The reduction in serum IGF-I with zinc deficiency found in these reports is consistent with earlier reports showing reduced serum IGF-I (42 ,43) and hepatic IGF-I mRNA levels (44 ,45) in this condition. However, the extent to which the reductions in IGF-I are caused by zinc deficiency per se, rather than the associated energy restriction, has been questioned (46) . IGF-I was sensitive to reduced food intake in these studies, but we detected a clear additional effect of zinc deficiency. It has been shown that the chelation of zinc from hepatocytes does not reduce IGF-I mRNA levels, suggesting that any effect of zinc does not occur directly at the hepatic level (47) . In terms of the binding proteins, in each case the zinc-deficient rats had significantly lower concentrations than the controls but were not significantly different from the pair-fed rats. For IGFBP-3 and -4, the pair-fed group level was lower than that of the controls, suggesting that most of the dietary effect was attributable to reduced food intake. These results are quite similar to those reported by Clegg et al. (46) .

The overall hypothesis underlying these experiments was that lack of zinc might impair thyroid hormone signaling by reducing the ability of the thyroid hormone receptor to bind to DNA and thereby influence target gene transcription. It seemed appropriate, therefore, to look directly at a target gene and determine the ability of T₃ to influence its expression in the different dietary groups. The S14 gene was chosen to this end because its response to T₃ has been well characterized at a number of laboratories, including ours (26) . S14 mRNA levels responded in the expected manner to variations in thyroid state. However, these responses were not influenced by either zinc deficiency or pair feeding, thus failing to support the hypothesis. At this stage, we have examined the expression of only one target gene for thyroid hormone within a single tissue. It is possible given the variations in receptor isoforms, response elements and associated nuclear proteins that other T₃ target genes may differ from S14 and be influenced by zinc deprivation. Future studies will address this possibility.

Food deprivation has been shown to decrease expression of mRNA S14, in common with other mRNAs associated with the lipogenic pathway. The more moderate food deprivation, experienced by the pair-fed rats in this experiment, did not influence the levels of mRNA S14 or the response to changing thyroid state. The euthyroid pair-fed rats consumed only 62% of the food eaten by the control euthyroid group, although this value rises to 84% if corrected on the basis of final metabolic body weight. In addition, this food restriction may result in the rapid consumption of the food provided, followed by food deprivation until more is supplied. Thus, at the time of killing, the pair-fed rats may differ metabolically from both zinc-deficient and control rats. In any event, neither the decrement in food intake nor the change in the pattern of its consumption was apparently sufficient to influence mRNA S14 expression in a detectable manner.

The in vivo approach was adopted for this study, despite its complexity, potential for indirect effects and difficulties in interpretation, because experiments in vitro have produced a variety of findings that have not always been consistent. Thus, Miyamoto et al. (13) showed that the removal of zinc from bacterially expressed T₃ receptors impaired their ability to bind to DNA, a finding quite consistent with the accepted model for zinc–nuclear receptor interactions. Others have shown a negative effect of zinc on the ability of T₃ to bind its receptors (16 ,17) , suggesting that zinc might impair thyroid hormone signaling. It appears likely that these latter findings were due to effects of T₃ in vitro that influenced the ability of the assay systems used to measure receptor binding (14 ,18) . In cultured chick embryonic hepatocytes, the addition of zinc reduces the ability of T₃ to induce lipogenic gene expression (19) . Furthermore, we have shown that the removal of zinc with a membrane-impermeable chelator amplifies the ability of T₃ to induce GH mRNA expression in cultured rat pituitary tumor cells (14 ,15) . This effect is specifically reversed by zinc. The mechanisms that underlie these inhibitory effects of zinc are unclear and thus may or may not be specific to the cell culture conditions. The chelation approach used with GH3 cells may be analogous to feeding a zinc-deficient diet because both approaches reduce the amount of zinc available to cells. However, although we did not measure tissue concentrations of zinc, the use of a chelator in vitro is likely to result in more extreme reductions in intracellular zinc than our dietary manipulations. In the current experiments, we were unable to measure GH mRNA levels, and the variability in the data did not permit any statement to be made about the effects on serum GH protein concentrations.

Lukaski et al. (29) showed that zinc-deficient rats had a reduced ability to withstand cold stress. Wada and King (48) reported that marginal zinc deficiency in humans was associated with a reduction in basal metabolic rate. Both findings are consistent with zinc deficiency resulting in impaired thyroid function. The experiments reported here did not uncover effects such as these with respect to growth, the somatotrophic axis or mRNA S14. Although some interactions between the two main effects were found, for the most part these important regulators of growth and development appeared to be acting independently. However, it remains possible that interactions will be found as other indices of thyroid hormone action are examined.

	FOOTNOTES

 
¹ Presented in part at Experimental Biology 2000, San Diego, CA [Govoni, K. E., Freake, H. C., Guda, K. K. & Zinn, S. A. (2000) Effects of thyroid hormone and zinc deficiency on the somatotrophic axis in rats. FASEB J. 14, A89].

² Supported in part by a grant from the University of Connecticut Research Foundation.

⁴ Abbreviations used: GH, growth hormone; IGF, insulin-like growth factor; IGFBP, insulin-like growth factor–binding protein; T₃, triiodo-L-thyronine.

Manuscript received June 26, 2000. Initial review completed July 27, 2000. Revision accepted December 13, 2000.

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