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Endocrine Division, Thyroid Section, Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, 2350, 90035-003 Porto Alegre, Rio Grande do Sul, Brazil
(Requests for offprints should be addressed to A L Maia; Email: almaia{at}ufrgs.br)
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although several factors such as hormones, growth factors, adrenergic agents, environmental and nutritional conditions influence deiodinase activities, these enzymes are mainly regulated by thyroid hormones (Bianco et al. 2002). In response to severe iodine deficiency or hypothyroidism, serum T3 and T4 are reduced, thyroid-stimulating hormone (TSH) is increased, and the peripheral T3 production from T4 is maintained by upregulation of D2 and downregulation of D1 expression. Conversely, in the hyperthyroid state D1 is increased, whereas D2 is decreased. D1 activity is regulated by thyroid hormones almost exclusively at the transcriptional level (Berry et al. 1990, Maia et al. 1995a). In contrast, the control of the D2 expression is more complex, occurring by transcriptional, posttranscriptional, and posttranslational mechanisms (St Germain 1988, Burmeister et al. 1997, Gereben et al. 2002). At transcriptional level, D2 is downregulated by its end product T3, whereas its substrate, T4, controls enzyme activity at posttranslational level.
The C3H/HeJ (C3H) inbred mouse has an inherited D1-deficiency which results in approximately tenfold reduction in hepatic levels of D1 mRNA and activity when compared with the C57BL/6J (C57) inbred strain, which presents higher Dio1 expression (Berry et al. 1993, Schoenmakers et al. 1993). In addition to the inherited D1 deficiency, the C3H mouse exhibits a higher susceptibility to chemically induced hepatocarcinogenesis and lower susceptibility to atherosclerotic plaque formation in response to a high-fat diet and larger spleen, when compared with C57 mice (Paigen et al. 1987, Buchmann et al. 1991, Manning & McDonald 1997). Despite all these known genetic traits, the C3H mouse presents a mild phenotype in that it appears healthy, and reproduction and growth are unimpaired. The reduced D1 activity in C3H mice correlates with a CGT repeat insertion into the 5'-flanking region of the Dio1 gene that seems to impair C3H promoter potency (Maia et al. 1995b). The serum T3 and TSH concentrations in C3H mice are at normal range, whereas total and free T4 levels are elevated when compared with those in C57 mice. The normal serum T3 is partially explained by the reduction in T3 clearance, due to the lower D1 levels, and increased serum T4 concentration that would compensate for the reduced fractional conversion of T4 to T3. However, the expression of D2, another T4-activating enzyme, has not been entirely assessed in D1-deficient mice. Comparative analysis of D2 activity levels between C3H and C57 mice demonstrated that pituitary and brain D2 activity in C3H mice was about 50% lower than that in C57 animals (Berry et al. 1993), which is attributed to the twofold increase in serum T4 concentration. The D2 mRNA expression has not been evaluated in the C3H mouse. In the recently described D1-deficient mouse (D1KO), created by targeted disruption of the Dio1 gene, D2 activity was assessed in pituitary, brain, liver, skin, and thyroid (Schneider et al. 2006). However, D2 expression in BAT, which can be an important source of peripheral T3 under certain circumstances (Silva & Larsen 1985), was not evaluated.
The aim of the present study was to further investigate Dio2 gene expression and regulation in C3H D1-deficient mice.
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Materials and Methods |
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All reagents were of analytical grade and obtained from commercial sources. T4 and T3 were obtained from Sigma Chemical Co. High specific activity [125I]T4 (1500 µCi/µg) was purchased from Amersham Biosciences. Reagents to determine protein concentration were obtained from Bio-Rad Laboratories.
Animals
Male C57BL/6J and C3H/HeJ mice (22–28 g), ~7 weeks old, obtained from Fundação Estadual de Produção e Pesquisa em Saúde (FEPPS, Porto Alegre, RS, Brazil), were housed under controlled lighting and temperature conditions, and fed a commercial diet and water available ad libitum. The animals were maintained in accordance with the guidelines of the Hospital de Clínicas de Porto Alegre Ethics Committee for the Use and Care of Experimental Animals.
In initial studies, the experimental groups (n = 4–6 mice/group) included euthyroid control and euthyroid animals treated with L-T3 (10 µg/animal, i.p. injected, daily) for 3 days before death to induce hyperthyroidism. Subsequently, the time course of thyroid hormone effects on D2 mRNA and activity in mice tissues was determined using shorter periods of T3 administration. Mice were treated with either saline solution or 10 µg L-T3 for 4, 12, 24, and 72 h.
A second series of experiments was conducted to determine the time course of the reduction of D2 activity by maximal doses of T4 alone or in combination with T3 in tissues of C3H and C57 animals. Euthyroid mice were i.p. injected with L-T4 1, 3 or 9 µg/100 g BW alone or a combination of L-T4 3 µg/100 g BW+L-T3 10 µg for 6, 24, and 48 h before death. Mice treated only with vehicle served as euthyroid controls. The chosen doses of administered thyroid hormones were based on previous reports to induce graded thyrotoxicosis (Escobar-Morreale et al. 1997, Schneider et al. 2001). After treatments, mice were euthanized under CO2 and tissues were rapidly removed, frozen in liquid nitrogen, and stored at –70 °C until RNA extraction or homogenization for activity analysis.
Serum hormone measurements
Assays were performed on batched serum samples that had been stored at –20 °C awaiting study completion. Serum total T4 was measured by radioimmunoassay (Diagnostic Products Corporation, Los Angeles, CA, USA) and interassay coefficient of variation was 8%. Serum total T3 was also determined by radioimmunoassay in the first series of experiments (Immunotech, Marseille, France) and by electro-quimioluminescence immunoassay (Roche Diagnostics) in experiments shown in Tables 2
and 3. Interassay coefficients of variation were 9 and 10% respectively.
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Total RNA was isolated using TRIzol reagent (Invitrogen Corp.) according to the manufacturer’s instructions. Samples of total RNA (~30 µg) were examined for the presence of D2 transcripts by northern analysis, using the rat D2 cDNA as probe. Northern blots and radioactive probes were prepared as previously described (Wagner et al. 2003). D2 hybridization signals were quantified by densitometry using Image Master VDS (Pharmacia Biotech). Blots were rehybridized with 18S ribosomal RNA probe and 18S signals were used as a control to normalize for differences in the amount of total RNA in samples. All experiments were repeated twice.
Real-time PCR analysis
RNA was reverse-transcribed with the SuperScript Pre-amplification System for First Strand cDNA Synthesis (Invitrogen, Corp.) using 3 µg total RNA and 100 ng random hexamers. Reactions for the quantification of target mRNAs were performed in an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Warrington, UK) using the SYBR Green PCR Master Mix (Applied Biosystems) and cyclophilin as a housekeeping internal control. Samples were run in duplicate. The cycle conditions were 94 °Cx5 min (Hot Start), 35 cycles of 94 °Cx30 s; 58 °Cx30 s; 72 °Cx45 s and a final 5 min extension period. Initially, standard curves representing five-point serial dilution of mixed cDNAs of the control and experimental groups were analyzed and used as calibrators to determine the relative quantification of product generated in the exponential phase of the amplification curve. Comparable efficiency was observed presenting r2>0.99. Samples were measured by relative quantification (change in expression C3H versus C57 mice; untreated versus treated animals). The data generated by the ABI Prism 7500 system SDS software (Applied Biosystems, Warrington, UK) were then transferred to an Excel spreadsheet and the experimental values corrected by that of the cyclophilin standard. Oligonucleotides for mouse D2 and cyclophilin respectively, were as follows: (5'-TTCTCCAACTGCCTCTTCCTG-3' and 5'-CCCATCAGCGGTCTTCTCC-3'); (5'-GCCGATGACGAGCC CTTG-3' and 5'TGCCGCCAGTGCCATTATG-3').
Deiodinase assays
5'-Deiodinase assays were performed as previously described (Wagner et al. 2003). Briefly, tissues samples were homogenized on ice in buffer containing 1xPE (0.1 M potassium phosphate and 1 mM EDTA), 0.25 M sucrose, and 10 mM dithiothreitol (DTT; pH 6.9). The reaction mixtures containing 100–300 µg tissue protein were incubated in a total volume of 300 µl with ~100 000 c.p.m. [3',5'-125I]T4 purified by LH-20 column chromatography (Pharmacia), 1 nM (D2) or 1 µM (D1) unlabeled T4, 10, or 20 mM DTT, in the presence or absence of 1 mM propylthiouracil in PE buffer at 37 °C for 2 h. Reactions were terminated by the addition of 200 µl horse serum and 100 µl 50% trichloroacetic acid. After centrifugation at 3000 g for 2 min, the free 125I in the supernatant was counted with a gamma-counter. Deiodination was linear with both protein concentration and time, and the quantity of enzyme assayed was adjusted to consume <30% of substrate. Activity is expressed as fentomoles iodide generated/min per mg protein. In determining deiodination activity, the percent iodide generated was multiplied by two to account for the random labeling and deiodination at the 3' and 5' positions in the [3',5'-125I]T4 (Kuiper et al. 2002). All reactions were performed in duplicate.
Statistical analysis
Results are presented as mean ± S.D. of two experiments. Four to six animals were used per group per experiment. Data were log-transformed prior to analysis. Comparisons among groups were assessed by one-way ANOVA followed by Dunnett’s post hoc test. P<0.05 was considered statistically significant.
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Results |
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To confirm that the C3H mice used in this study present the described D1 deficiency, we determined the level of hepatic D1 activity and compared it with that of the C57 mice. D1 activity in the C3H mouse was significantly lower than that observed in the C57 strain (0.08 ± 0.04 vs 0.41 ± 0.12 pmol/min per mg protein respectively). Accordingly, the serum T4 concentration in C3H mice was approximately twice that in C57 animals (6.0 ± 0.9 vs 3.4 ± 0.5 µg/dl), whereas T3 levels were comparable between both mice strains (51.3 ± 22.2 vs 51.5 ± 8.9 ng/dl).
D2 mRNA and activity levels
D2 mRNA and activity were assessed in pituitary, brain, and BAT from C3H and C57 mice. The D2 mRNA levels in C3H tissues were similar to those observed in corresponding tissues in C57 animals. In the C3H mouse, D2 activity in pituitary and brain was approximately half of that found in the C57 strain (Table 1). However, interestingly, the level of D2 activity in BAT was similar between both mice strains.
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Assuming that, in C3H mice, lower D2 activity levels in the brain and pituitary were due to their chronically elevated serum T4 concentration, we wished to test whether higher levels of circulating T4 would reduce BAT D2 activity in these animals. Therefore, we used a number of different T4 doses to induce a graded increase in the level of serum T4 and correlate them with the D2 activity in BAT and also in pituitary. The latter was used as a reference tissue.
The changes in serum T4 concentration of mice injected with different doses of T4 are shown in Fig. 1A. Serum T4 was well above control levels by 24 h after the injection of either 1 or 3 µg T4/100 g BW and reached the highest level by 6 h after animals were injected with 9 µg T4/100 g BW. A subsequent significant decrease in C3H pituitary D2 activity was observed with increasing circulating T4 concentrations (Fig. 1B). On the contrary, no significant changes in BAT D2 activity were observed until serum T4 was approximately threefold higher than C3H control level (Fig. 1C).
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). However, similarly to what has been seen in the C3H mouse, no significant changes on BAT D2 activity was observed even when serum T4 concentration was approximately threefold higher than control level (Table 2). Effect of T3 administration on D2 mRNA and activity levels in C3H and C57 mice
To evaluate the intrinsic responsiveness and regulation of the C3H Dio2 gene by T3, we analyzed the D2 mRNA and activity in brain and BAT of euthyroid mice T3-treated for 3 days. T3 administration resulted in a significant decrease in BAT D2 mRNA level in both strains (Fig. 2A and B), whereas no changes were observed in D2 mRNA concentration in the brain (Fig. 2C and D).
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). The most likely explanation for the observed T3-induced increase in D2 activity was the reduction of serum T4 concentration, due to the decreased thyroid T4 secretion caused by T3-induced inhibition of TSH release. In this case, one could predict that D2 activity levels would increase earlier in tissues of C57 than in C3H animals in response to T3 treatment, since the latter presents higher T4 concentration and reduced fractional clearance by D1 (Table 3). Indeed, BAT D2 activity increased as early as 4 h after T3 administration in the C57 mouse (Fig. 4A and Table 3). D2 activity reached the highest level at 24 h and remained elevated up to 72 h of treatment (Fig. 4A). In contrast, in C3H mice, BAT D2 activity did not change significantly at 4–24 h after T3 administration (Table 2). By 72 h of T3 exposure, BAT D2 activity was similarly higher in both mice strains.
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). Effects of combined T3 plus T4 treatment on D2 activity levels
In order to determine whether T3 stimulation of D2 activity in mice tissues was caused by a T3-induced fall in serum T4 concentration, C57 mice were injected with a combination of T4 plus T3. After 24 h of treatment with the combined doses of 10 µg T3 + 3 µg T4/100 g BW, circulating T3 was approximately fivefold higher than the control, while T4 did not decrease or exceed normal control values (Table 4). In contrast to the observed ~3.5-fold increase in C57 BAT D2 activity after T3 treatment alone, the administration of T3 plus T4 did not change BAT D2 activity significantly (Table 4).
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Discussion |
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The C3H inbred mouse strain has an inherited D1 deficiency. Besides the reduced hepatic and renal D1 activity, the most notable features of the C3H mouse are that, when compared with the C57 mouse, the circulating levels of T4 and rT3 are elevated, while those of TSH and T3 are unchanged. It would therefore be anticipated that in C3H mice the higher serum free T4 concentration would cause a decrease in D2 activity, which was confirmed by twofold lower D2 activity in pituitary and brain (Berry et al. 1993). Here, we demonstrated that the D2 mRNA levels are not different between C3H and C57 mice, providing additional evidence that the reduced D2 activity in C3H mice results from an increased rate of substrate-induced enzyme inactivation. Interestingly, in the recently described D1KO mouse, the pituitary D2 activity is not reduced when compared with wild type animals, despite a significant increase in serum T4 concentration (Schneider et al. 2006). Since serum T4 in the D1KO mice is ~50% increased, while in the C3H is nearly doubled, a possible explanation for this paradoxical observation would be that this relatively smaller elevation in circulating T4 would not be enough to downregulate pituitary D2 activity. Nevertheless, similar increase in serum T4 concentration (~60%) in the C57 mice induces a marked decrease in pituitary D2 activity (Table 2). Thus, it is conceivable, as suggested by Schneider et al.(2006), that some modification in the set point of the feedback system might have occurred during the development of the D1KO mouse.
An unexpected finding of this study was the lack of difference in BAT D2 activity between C3H and C57 mice strains. A major D2 property that characterizes its homeostatic behavior is a short half-life (~40 min) that can be further reduced by exposure to its substrates, T4 or rT3 (Leonard et al. 1984, Silva & Leonard 1985). The cellular mechanism for substrate-induced inactivation of D2 involves ubiquitination, which accelerates enzyme degradation through the ubiquitin-proteasome pathway (Steinsapir et al. 1998, 2000, Gereben et al. 2000). This regulatory feedback loop efficiently controls T3 production and intracellular T3 concentration based on the amount of T4 available. Indeed, as shown here (Fig. 4) and by others (Croteau et al. 1996), decreases in serum T4 level upregulate BAT D2 activity several times. Hence, it was reasonable to anticipated, based on the higher C3H serum T4 level, that BAT D2 activity would be lower in these mice. Experiments using high doses of T4 administration demonstrated that T4-induced D2 downregulation in BAT requires a much higher T4 level than those observed in the C3H mice (approximately four- to fivefold over C57 serum T4 concentration). The relative resistance of BAT D2 activity to serum T4 levels, in the transition from euthyroidism to hyperthyroidism, was further confirmed in C57 mice (Table 2).
In rodents, BAT is a site of complex interactions between the sympathetic nervous system and thyroid hormone, which make difficult to interpret the already intricate D2 regulation in this tissue (Bianco et al. 2005). D2 plays a critical role in BAT adaptive thermogenesis and similarly to the pituitary and other D2-expressing tissues, BAT is quite dependent on D2-generated T3 to supply nuclear T3-receptor (de Jesus et al. 2001). Therefore, a decrease in D2 activity in this tissue may not be advantageous. Indeed, as recently demonstrated in mouse tumor cell line (T
T1 cells), some other physiological mechanisms may interfere with T4-induced D2 degradation (Christoffolete et al. 2006). In these cells, when a range of concentrations of T4 was used, the loss of D2 activity was impaired at a concentration >50 pM. The potential explanation for this phenomenon is that the rate of D2 synthesis in these cells equals the maximal rate of T4-induced D2 degradation, though the authors also considered a possible exhaustion of the ubiquitinating/proteolytic machinery. Although the mechanism whereby BAT D2 activity remains elevated when marked increases in serum T4 level are induced in euthyroid mice has not been determined in this study, it is conceivable that mechanisms similar to those observed in TT1 cells may operate in this tissue. Furthermore, an increased rate of reactivation of ubiquitinated-D2 via von Hippel–Lindau protein-interacting deubiquinating enzyme (VDU)-1,2-mediated deubiquitination could be involved, since it has been shown that this system is very important in regulating the supply of active thyroid hormone in BAT (Curcio-Morelli et al. 2003). According to this line of reasoning, we can expect that in a setting of T4-induced hyperthyroidism, other mechanisms than inhibition of D2 are present in order to prevent BAT thyrotoxicosis. Indeed, previous studies have shown that infusion of high doses of T4 in rats did not decrease BAT D2 activity, whereas BAT T3 concentration remained normal or only slightly elevated (Escobar-Morreale et al. 1997).
The effect of T3 administration on D2 expression was also investigated in D1-deficient mice. Treatment of euthyroid mice with T3 results in a marked decrease in BAT D2 mRNA levels, whereas it did not significantly change cerebral D2 mRNA. As demonstrated in rats, the negative control of D2 expression in hypothyroid brain and pituitary by T3 is probably mediated by the nuclear T3 receptor, although a putative negative TRE in the promoter region of the D2 gene remains to be identified (Burmeister et al. 1997, Kim et al. 1998). The lack of response of cerebral D2 mRNA to T3 treatment is in agreement with a previous study (Croteau et al. 1996) and is probably due to the near complete saturation of T3 receptors in brain of euthyroid mice (Larsen et al. 1981). On the other hand, in cultured rat brown adipocytes, T3 alone was shown to increase D2 mRNA but did not change significantly D2 activity levels (Martinez-deMena et al. 2002), indicating that discrepancies exist regarding the in vivo and in vitro effects of T3 on the regulation of D2 expression in BAT.
In contrast to the inhibitory effect of T3 administration on D2 mRNA expression, D2 activity levels were increased ~2- to 3.5-fold in brain and BAT in both C3H and C57 strains. Consideration of these data led to the hypothesis that induction of D2 activity in T3-treated mice was due to the decrease in serum T4 levels. Indeed, additional experiments demonstrated that the upregulation of D2 activity induced by T3 was offset by a concurrent decrease in serum T4 level and combined T3 plus T4 administration abolished T3-induce D2 increase (Fig. 4 and Table 4). Although these results are quite predictable, they were somewhat unexpected because most studies that evaluated T3 effect on D2 activity were performed in hypothyroid animals and, in this setting, T3 administration decreases D2 mRNA and activity. In agreement with our results, elegant studies performed by Escobar-Morreale et al.(1997) have shown that in thyroidectomized rats, D2 activity returned to normal with T4 infusion, whereas it was increased in animals infused with T3, when compared with the activities found in animals infused with placebo. Taken together, these observations allow two other inferences. First, serum T3 has a minor role in regulating D2 at post-translational level. Second, the T4 downregulation of D2 activity is remarkable, since decreases in serum T4 level upregulate activity several times, regardless of the T3-induced suppression of D2 mRNA synthesis.
T3 treatment also increases D2 activity in the brain of both strains. However, the time course and magnitude in response to T3 was nearly identical in control and D1-deficient mice. Another interesting observation was that the increase in D2 activity occurred later than that observed in the C57 BAT, which might indicate differences in the time required for the exchange of plasma/tissue T4 among the different tissues.
In conclusion, we demonstrated that D1 deficiency does not affect D2 mRNA levels, but differentially affects D2 activity in pituitary, brain, and BAT in the C3H mice. While approximately twofold increase in serum T4 concentration is enough to induce a significant decrease in D2 activity levels in C3H pituitary and brain, the T4-induced D2 downregulation in BAT requires a much higher serum T4 level. These results suggest that other intrinsic mechanisms prevent the loss of D2 activity in this tissue, probably to avoid a decrease in the D2-generated T3 to supply nuclear T3-receptor. Furthermore, we showed that administration of T3 to euthyroid mice causes a tissue-specific modulation of D2 mRNA levels and increases D2 activity in BAT and brain. The latter T3 effect is rapid and marked, and seems to be the result of the decrease in serum T4 levels, rather than a direct effect of thyrotoxicosis.
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Acknowledgements |
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References |
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Received 28 February 2007
Accepted 2 April 2007
Made available online as an Accepted Preprint 3 April 2007
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