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Am J Physiol Endocrinol Metab 285: E1135-E1141, 2003. First published September 9, 2003; doi:10.1152/ajpendo.00506.2002
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Spared bone mass in rats treated with thyroid hormone receptor TR{beta}-selective compound GC-1

Fatima R. S. Freitas,1 Anselmo S. Moriscot,2 Vanda Jorgetti,3 Antonio G. Soares,2 Marisa Passarelli,3 Thomas S. Scanlan,4 Gregory A. Brent,5 Antonio C. Bianco,6 and Cecilia H. A. Gouveia1

Departments of 1Anatomy and 2Histology, Institute of Biomedical Sciences, and 3School of Medicine, University of Sao Paulo, 05508-900 Sao Paulo, Brazil; 4Departments of Pharmaceutical Chemistry and Cellular and Molecular Pharmacology, University of California, San Francisco 94143; 5Molecular Endocrinology Laboratory, Veterans Affairs Greater Los Angeles Healthcare System and Departments of Medicine and Physiology, UCLA School of Medicine, Los Angeles, California 91301; and 6Thyroid Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

Submitted 18 November 2002 ; accepted in final form 29 July 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Thyrotoxicosis is frequently associated with increased bone turnover and decreased bone mass. To investigate the role of thyroid hormone receptor-{beta} (TR{beta}) in mediating the osteopenic effects of triiodothyronine (T3), female adult rats were treated daily (64 days) with GC-1 (1.5 µg/100 g body wt), a TR{beta}-selective thyromimetic compound. Bone mass was studied by dual-energy X-ray absorptiometry of several skeletal sites and histomorphometry of distal femur, and the results were compared with T3-treated (3 µg/100 g body wt) or control animals. As expected, treatment with T3 significantly reduced bone mineral density (BMD) in the lumbar vertebrae (L2-L5), femur, and tibia by 10–15%. In contrast, GC-1 treatment did not affect the BMD in any of the skeletal sites studied. The efficacy of GC-1 treatment was verified by a reduction in serum TSH (–52% vs. control, P < 0.05) and cholesterol (–21% vs. control, P < 0.05). The histomorphometric analysis of the distal femur indicated that T3 but not GC-1 treatment reduced the trabecular volume, thickness, and number. We conclude that chronic, selective activation of the TR{beta} isoform does not result in bone loss typical of T3-induced thyrotoxicosis, suggesting that the TR{beta} isoform is not critical in this process. In addition, our findings suggest that the development of TR-selective T3 analogs that spare bone mass represents a significant improvement toward long-term TSH-suppressive therapy.

thyrotoxicosis; osteopenia; osteoporosis; bone mineral density; bone histomorphometry

THYROID HORMONE (TRIIODOTHYRONINE, T3) has important effects on skeletal development and bone metabolism. Hypothyroidism retards bone growth and slows bone turnover (2), whereas thyrotoxicosis is frequently associated with accelerated bone development and metabolism and decreased bone mass (37). The osteopenic effects of thyroid hormone were first described by von Recklinghausen in 1891 (33), and today overt thyrotoxicosis is considered a risk factor for secondary osteoporosis. Histomorphometric studies show that thyrotoxicosis increases both osteoblastic and osteoclastic activities, but the latter predominates. As a result, bone turnover is accelerated, favoring bone resorption, negative balance of calcium, and bone loss (3, 29). Accordingly, biochemical markers of bone turnover are increased in patients (9) and experimental animals (19) with thyrotoxicosis.

Although the negative effects of overt thyrotoxicosis to bone mass are clear, the mechanisms by which thyroid hormone regulates bone metabolism are uncertain. Whereas thyroid hormone can affect the skeleton indirectly through changes in other hormones, e.g., growth hormone and IGF-I (23), in vitro studies show that T3 also acts directly in bone cells, increasing the expression of several bone-related genes, such as osteocalcin (18, 39), collagen type I (25), and collagenase 3 (32). Primary cultures of skeletal origin have also been used to confirm the direct effects of T3 on bone development and metabolism. As an example, it has been shown that thyroid hormone induces bone resorption in organ cultures of calvaria and long bones (22).

It is generally accepted that most T3 actions are mediated by nuclear receptors (TRs), which are T3-inducible transcriptional factors (24). There are four classical TR isoforms encoded by two genes (13), TR{alpha} and TR{beta}. The TR{beta} gene encodes TR{beta}1 and TR{beta}2, and the TR{alpha} gene encodes TR{alpha}1 and TR{alpha}2 (21). TR{alpha}1, TR{alpha}2, and TR{beta}1 are expressed in osteoblasts (39), osteoclasts (1), and chondroblasts (5).

Animal models in which TR genes were targeted for disruption have not been used to evaluate bone mass in adult animals. Previous such studies have shown, however, that TR{beta} (10) and TR{alpha}1 (38) knockout (KO) mice have normal bone development. Remarkably, the combined absence of both receptors [double-KO mice, TR{alpha}1–/–/TR{beta}–/– (16)] or both TR{alpha} isoforms (TR{alpha}–/– mice) results in a number of bone defects, including delayed bone maturation and ossification and dysgenesis of epiphysial growth plates. These findings suggest a substantial overlap and, therefore, functional redundancy of the TR{alpha}1 and TR{beta} isoforms on bone development.

Recently, a synthetic thyroid hormone analog that is selective for both binding and activation functions for TR{beta} over TR{alpha} was developed (8). In GC-1, the three iodines were replaced with methyl and isopropyl groups, the biaryl ether linkage with a methylene linkage, and the amino acid side chain with an oxyacetic acid side chain. GC-1 binds TR{beta} with the same affinity as T3 but binds TR{alpha}1 with an affinity that is 10 times lower compared with T3. Accordingly, GC-1 presents selective actions in vitro (8) and in vivo (34, 36), such as lowering serum cholesterol and TSH without affecting thyroid-adrenergic synergism and, hence, heart rate or adaptive thermogenesis (34). Selective effects of GC-1 were also demonstrated in the cerebellar development where it partially enhances Purkinje cell maturation without affecting granule cell migration (28). The TR{beta} selectivity of GC-1 makes it an improved tool over the KO animal models for studying the physiological roles of the different TR isoforms without affecting the equilibrium between the two TRs isoforms.

Given the fact that no data are available regarding the role of TR isoforms in mediating T3 effects on bone mass of the adult animal, our goal in the present study was to investigate this by using the TR{beta}-selective T3 agonist GC-1. Our data indicate that chronic, selective activation of the TR{beta} isoform does not result in bone loss typical of T3-induced thyrotoxicosis, suggesting that the TR{beta} isoform is not critical in this process. In addition, our findings suggest that the development of TR-selective T3 analogs that spare bone mass represents a significant improvement toward long-term TSH-suppressive therapy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Animals and drugs. All experimental procedures were performed in accordance with the guidelines of the Standing Committee on Animal Research of the University of Sao Paulo. Eighteen female Wistar rats were obtained from our breeding colony and maintained under controlled conditions of light and temperature (12:12-h dark-light cycle at 25°C). All animals were kept in plastic cages, six per cage, and had free access to food (rat chow containing 2 mg/kg iodine, 1.4% Pi, 0.7% calcium, and 4.5 IU/g vitamin D) and water. At the age of 140.2 ± 0.5 days and weighing 219 ± 4 g, rats were randomly divided into three groups (n = 6 per group): 1) Control, 2) GC-1, and 3)T3. Animals were treated with a dose of T3 that corresponds to approximately 10 times the physiological replacement dose (10x T3). We based our calculations of T3 dosage on previously published data (7), indicating one physiological dose of T3 to be 0.3 µg · 100 g body wt–1 · day–1. Therefore, the animals received 3 µg · 100 g body wt–1 · day–1 of T3. The administered equimolar dose of GC-1 (10x GC-1) was 1.5 µg · 100 g body wt–1 · day–1, calculated from the molecular weights of T3 (mol wt = 651) and GC-1 (mol wt = 328.4). Previous studies have shown that similar schemes of GC-1 treatment are effective in induce end effects of thyroid hormone mediated through TR{beta} (28, 34, 36). Body weights were measured three times a week, and the amount of hormone administered was adjusted according to the changes in body weight to maintain the proper dosage. Animals were treated with T3, GC-1, or saline, injected intraperitoneally every day for 64 days.

Serum parameters. At the end of the experimental period, the animals were killed by decapitation, and the blood of the trunk was collected. The serum was separated by centrifugation and immediately frozen. TSH was measured using an RIA kit specifically designed for rat TSH (Biotrak, Amersham Pharmacia Biotech, Piscataway, NJ). Total thyroxine (T4) and T3 serum levels were measured by commercial RIA kits (RIA-gnost T4 and RIA-gnost T3; CIS Bio International, Gif-sur-Yvette, France). For the T4 and T3 assays, standard curves were built in our laboratory with a pool of charcoal-stripped rat serum. To test whether GC-1 could cross-react in the T4 and T3 RIAs, serum levels of T4 and T3 were measured in hypothyroid rats treated with different doses of GC-1. Serum T4 was undetectable in all animals, and there were no differences in serum levels of T3 between the GC-1-treated animals and the hypothyroid untreated animals, which indicate that T4 and T3 RIAs cannot detect GC-1. Cholesterol was measured by enzymatic colorimetry with a commercial kit (Labtest Diagnostica, Lagoa Santa, MG, Brazil).

Bone densitometry. Bone mineral density (BMD), bone mineral content (BMC), bone area, and tibial and femoral lengths were all measured by dual-energy X-ray absorptiometry (DEXA) using the pDEXA Sabre Bone Densitometer and the pDEXA Sabre software (version 3.9.4; Norland Medical Systems, Fort Atkinson, WI), both especially designed for small animals. The research mode scan option was used for the measurements. Pixel spacing for the scan was set to 0.5 x 0.5 mm, the scan width to 10.5 cm, the scan length to 11.5 cm, and the scan speed to 7 mm/s. Because the scan area of the bone densitometer was not large enough to allow the scan of the total body of the rats of this study, the measurements were performed from the first lumbar vertebra to the hindlimbs. The regions of interest (ROIs) for analysis were 1) hindbody (HB), which includes L2-L6, pelvic bones, hindlimbs, and the first four caudal vertebrae; 2) lumbar vertebrae (L2-L5); and both 3) femurs and 4) tibias. These regions were selected manually during the scan analysis. To consider the different proportions of cortical bone and trabecular bone in different regions of femur and tibia, the BMD of these bones was also analyzed as three segments: proximal and distal regions (each one determined by one-fourth of the total bone length) and diaphysis, the region between the proximal and distal regions (one-half of the total length). To take into account the possibility of bone mass differences between the left and right limbs as a result of functional bilateral asymmetry (11), the BMD of femur and tibia was expressed as the mean of the left and right limbs for each animal. BMC and bone area were measured in the HB. Femoral and tibial lengths from left limbs were measured indirectly by DEXA using the ruler tool provided by the pDEXA Sabre software. For the scans, the animals were anesthetized with a ketamine-xylazine cocktail (10 and 30 mg/kg body wt, respectively) and scanned in the prone position. The animals were scanned at 0 time point (before treatment was started) and 32 and 64 days after. At the end of the treatment period, the left tibia was dissected out, and an ex vivo scan of each tibia was performed using the same scan adjustments used for the in vivo scan. For the scan analysis, the BMD histogram averaging width was set to 0.01 g/cm2 for all bone scans. The precisions ex vivo of the tibia and in vivo of the different ROIs were evaluated by calculating the coefficient of variation (CV = 100 x SD/mean) of six repeated measurements of a dissected tibia of a control rat and of a 3.5-mo-old female Wistar rat weighing 207 g, respectively. The tibia and animal were repositioned after each scan. The CV of the ex vivo BMD of tibia was 1.1%, and the CVs of in vivo BMD of HB, L2-L5, femur, and tibia were 0.8, 1.9, 0.2, and 0.1%, respectively. The CVs of the BMC and area of the HB were 3.1 and 2.2%, respectively. The in vitro precision was also expressed as CV and calculated by measuring the BMD of a phantom with a nominal density of 0.929 g/cm2. This CV was 0.8% throughout the experiment. The performance of the system was assessed and maintained by a quality assurance (QA) test, which includes scanner calibration and phantom scanning. The QA test was carried out on each day that scans were to be performed.

Bone histomorphometry. At the end of the experimental period, the right distal femur was carefully dissected out and processed as previously described (17). Five- and ten-micrometer-thick longitudinal sections of the distal femur were obtained with a K Jung microtome. The 5-µm sections were stained with 0.1% toluidine blue, pH 6.4, and at least two nonconsecutive sections were examined for each sample. Structural parameters of trabecular bone were measured at the distal metaphyses (magnification x250), 195 µm distant from the epiphysial growth plate, in a total of 30 fields, by use of a semiautomatic method (Osteometrics, Atlanta, GA). The histomorphometric indexes evaluated were trabecular volume (BV/TV, %), trabecular thickness (Tb.Th, µm), trabecular separation (Tb.Sp, µm), and trabecular number (Tb.N, mm–1). The histomorphometric indexes were reported according to the standardized nomenclature recommended by the American Society of Bone and Mineral Research (31). All animal data were obtained with blind measurements.

Statistical analysis. In a previous study (17), we found that rat bone mass presents a Gaussian distribution, which allowed us to employ parametric statistical tests for the analysis of BMD. One-way analysis of variance (ANOVA) was used to compare more than two groups and was always followed by the Student-Newman-Keuls test to detect differences between groups. The paired t-test was used for each group to analyze the significance of longitudinal changes in BMD ({Delta}BMD = BMDday32 – BMDday0 or BMDday64 – BMDday0). To analyze the histomorphometric results expressed as percentages, we used the Kruskal-Wallis nonparametric ANOVA test, followed by Dunn's test. For all tests, P < 0.05 was considered statistically significant. All results are expressed as means ± SE. For statistical analysis, we used the GraphPad Instat Software (GraphPad Software; San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
T3- and GC-1-induced systemic biological effects. Compared with control animals, T3 and GC-1 treatments lowered serum cholesterol by 30% (P < 0.01) and 21% (P < 0.01), respectively (Table 1). In T3- and GC-1-treated animals, serum TSH was 64% (P < 0.001) and 52% (P < 0.01) lower than in controls (Table 1). Note that there was no statistically significant difference in relation to serum cholesterol or TSH between GC-1- and T3-treated animals (Table 1). As a result of TSH suppression, serum T4 was undetectable in the T3-treated animals and fell significantly in the GC-1-treated rats (–37% vs. control, P < 0.001). Serum T3 was significantly higher in the T3-treated animals (48%, P < 0.001 vs. control) but was not affected by treatment with GC-1 (Table 1).


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  		Table 1. Serum levels of T3, T4, TSH, and cholesterol

 

At the end of the 9-wk treatment period, all rats gained approximately the same percentage of their initial body weight, 7–10% (P <= 0.001; Fig. 1). The T3-treated animals, however, lost weight and gained significantly less weight compared with the other two groups from days 3 to 32 of the experimental period (Fig. 1). In the GC-1-treated animals, there were similar but less marked changes in body weight during the same time period, but later both T3- and GC-1-treated rats caught up with control animals and no differences were observed.



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  		Fig. 1. Effect of triiodothyronine (T3) and thyroid hormone receptor-{beta}-selective compound GC-1 treatments on body weight. Therapies were started on day 0 and lasted 64 days. All values are expressed as means ± SE; n = 5–6 per group. P < 0.05 (Student-Newman-Keuls test): *all groups were different; # vs. control (CON) and GC-1; + vs. control; {diamondsuit} vs. T3 and GC-1.

 

T3- and GC-1-induced changes in BMD. At the end of the experimental period, the length of the femur and tibia of all animals increased 3.0–6.0%, and no statistical differences were observed among groups (data not shown; P > 0.05 by ANOVA). Treatment with T3 caused bone loss after 32 and 64 days, whereas treatment with GC-1 had no effect (Fig. 2). In the T3-treated animals, there was a significant reduction in BMD at HB, L2-L5, femur, and tibia in a range of –10 to –12% (P < 0.05, by Student-Newman-Keuls test), depending on the skeletal site. Further reductions in BMD were detected after 64 days of treatment with T3, varying between –16 and –22% (P < 0.05). There were no differences in BMD between control and GC-1 groups at 32 or 64 days of treatment in any of these skeletal sites. Similar findings were obtained when changes in BMD were expressed as the difference ({Delta}) between the first to the third scan (BMDday 64 – BMDday 0; Table 2). The only difference was that GC-1 treatment significantly prevented the increase in distal tibia BMD observed in the control animals (Table 2).



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  		Fig. 2. Effect of T3 and GC-1 treatments on bone mineral density (BMD). A: hindbody BMD, including L2-L6, pelvic bones, hindlimbs, and the first 4 caudal vertebrae. B: L2-L5 BMD. C: femur BMD. D: tibia BMD. Femur and tibia BMDs are expressed as the mean between the left and the right bones. Scans were performed at 0 (before start of treatment), 32, and 64 days (both after start of treatment). Values are expressed as means ± SE; n = 6 per group. *P < 0.01 and {Delta}P < 0.05 vs. CON and GC-1 (Student-Newman-Keuls test).

 

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  		Table 2. Effect of 64 days of treatment with T3 or GC-1 on {Delta}BMD

 

To eliminate the influence of soft tissues in the analysis of bone mass by DEXA, we also performed ex vivo bone densitometry. The ex vivo BMD of tibia was ~17% lower than the in vivo BMD for all groups. The ex vivo vs. in vivo correlation coefficient (r2) was 0.84. In agreement with the in vivo findings, the ex vivo analysis shows that, after 64 days of treatment, the tibial BMD of T3 animals (97.5 ± 2.9 mg/cm2) was 14% lower than in control animals (113 ± 4.3 mg/cm2, P < 0.05), but no differences were detected in the GC-1-treated animals (110 ± 6.9 mg/cm2).

T3- and GC-1-induced changes in distal femur trabecular bone. Trabecular bone was negatively affected by thyrotoxicosis (Table 3 and Fig. 3). In the T3-treated animals, BV/TV was 46% lower compared with controls (P < 0.05). The lower BV/TV was associated with reduced Tb.Th (–23% vs. control, P < 0.01) and Tb.N (–32% vs. control, P < 0.01) and was associated with higher Tb.Sp (72% vs. control, P < 0.001). In contrast, there were no differences between control and GC-1 groups for any of those trabecular parameters.


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  		Table 3. Histomorphometric variables of trabecular bone in the distal femur of rats

 


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  		Fig. 3. Distal femur of rats treated with T3 or GC-1 for 64 days. A: control. B: GC-1. C: T3. Light microscopy of toluidine blue-stained distal femur indicates reduced metaphysial trabecular bone in the T3-treated animal (magnification x25).

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
TR isoform-specific effects on bone development have been described by use of models of mice with targeted disruption or mutation in different TR isoforms (10, 12, 15, 16, 30, 38). However, studies on the role of TR isoforms in the maintenance of bone mass in adult animals are lacking. Using a pharmacological tool, the TR{beta}-selective agonist GC-1, we investigated for the first time the role of TR isoforms in mediating T3 effects on adult bone mass. Remarkably, whereas T3-induced thyrotoxicosis caused the expected osteopenia as assessed by DEXA (Table 2 and Fig. 2) and histomorphometry (Table 3 and Fig. 3), treatment with equimolar doses of GC-1 spared all the skeletal sites of bone loss.

Because the effects of GC-1 treatment on bone mass were largely negative, we studied known GC-1-dependent biological markers to document the effectiveness of treatment with this drug. In fact, after ~2 mo on GC-1, serum cholesterol levels were reduced in 21% (30% in the T3-treated animals) and serum TSH in 64% (52% in the T3-treated animals) (Table 1). These findings clearly indicate that the preservation of bone mass in GC-1-treated animals is not a failure of the treatment per se but a specific result of chronic activation of TR{beta}.

The fact that GC-1 did not cause bone loss indicates that TR{beta} does not play a primary role in mediating the effects of thyroid hormone in decreasing bone mass and suggests TR{alpha} as the major TR isoform involved in the T3-dependent osteopenia. Another explanation for the maintenance of bone mass in GC-1-treated animals may be related to a lower expression of TR{beta} in the skeleton. Recently, O'Shea et al. (30) showed that TR{alpha} is the major isoform expressed in the femur and tibia of mice. Alternatively, it is possible that this apparent biological selectivity is due to a lack of GC-1 distribution in the bone tissue. In fact, on the basis of the GC-1 tissue-to-plasma ratio, it has been suggested that the GC-1 tissue distribution is heterogeneous and differs from T3 in certain organs. This difference, for example, could account for the hepatic effects of GC-1, such as cholesterol lowering, without increasing heart rate (36). However, the net GC-1 TR occupation in a given tissue depends on the TR affinity for GC-1 and on the free nuclear GC-1 concentration. Although data on TR affinity are available, measurements of the nuclear free GC-1 concentration are missing, limiting the application of the tissue-to-plasma ratios.

In the present investigation and in other reports (28, 34), there is strong evidence that, within the same tissue, the effects of the GC-1 compound are limited to only some biological effects of T3. For example, in hypothyroid brown adipose tissue, GC-1 treatment restores uncoupling protein-1 concentration without normalizing cell responsiveness to norepinephrine (34). Selective effects of GC-1 were also observed in the brain, where treatment with GC-1 only partially corrected Purkinje cell differentiation but had no effect on granule cell migration (28). Although in the present study GC-1 treatment did not cause bone loss, a similar treatment with GC-1 in young rats resulted in clear effects on the epiphysial growth plate of femur, tibia, and vertebra (14), suggesting that GC-1 is available in the skeleton. All of these findings confirm GC-1 as a useful in vivo probe for studying the physiological roles of different TRs.

Treatment with T3, but not GC-1, caused 16–22% bone loss in all skeletal sites (Table 2 and Fig. 2). In agreement with the effects of T3 and GC-1 on BMD, the histomorphometric analysis of the distal femur showed that trabecular separation was increased and that trabecular volume, thickness, and number were all decreased by T3 but not by GC-1 (Table 3 and Fig. 3). It is interesting that, as opposed to the generalized osteopenic effect of T3, long-term administration of T4 leads to a preferential femoral over vertebral bone loss, regardless of the type of bone, i.e., cortical vs. trabecular bone (17, 27, 35). This might indicate a role of the iodothyronine deiodinases in the regulation of the local thyroid status within the bone tissue (26, 27).

Although TSH was equally reduced in T3- and GC-1-treated animals (Table 1), T3 was more effective in lowering serum T4 than was GC-1, suggesting differential effects of T3 and GC-1 on T4 clearance. A substantial fraction of T4 produced daily is inactivated by inner-ring deiodination via type III deiodinase (D3) and type I deiodinase (D1) (6). Whereas D1 is a gene positively regulated by TR{beta} (4), no data are available regarding D3, but our present findings suggest that this is a TR{alpha}-regulated gene.

In conclusion, chronic treatment with high doses of T3 causes generalized bone loss, whereas an equivalent treatment with GC-1 spares the skeleton from bone loss. Given that GC-1 works predominantly on the TR{beta} and given that GC-1 treatment was without significant effect on BMD, it would be reasonable to conclude that T3 mediates its effect in bone via TR{alpha}. This is supported by the TR{beta}PV mouse (30), which demonstrates that, in the absence of normal TR{beta} and high thyroid hormone levels, the bones are thyrotoxic. Our findings indicate that the development of TR-selective T3 analogs that spare bone mass represents a significant improvement toward long-term TSH-suppressive therapy.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work was supported by a grant from Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (C. H. A. Gouveia).


   ACKNOWLEDGMENTS
 
We express our gratitude to Rodisete A. Bezerra for essential contribution to the preparation of the histological sections.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. H. A. Gouveia, Dept. of Anatomy, Institute of Biomedical Sciences, Univ. of Sao Paulo, Av. Prof. Lineu Prestes, 2415 (Rm 114), Sao Paulo 05508-900, Brazil (E-mail: cgouveia{at}usp.br).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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C. M Villicev, F. R S Freitas, M. S Aoki, C. Taffarel, T. S Scanlan, A. S Moriscot, M. O Ribeiro, A. C Bianco, and C. H A Gouveia
Thyroid hormone receptor {beta}-specific agonist GC-1 increases energy expenditure and prevents fat-mass accumulation in rats
J. Endocrinol., April 1, 2007; 193(1): 21 - 29.
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