Characterization of the Mechanistic Linkages Between Iodothyronine Deiodinase Inhibition and Impaired Thyroid-Mediated Growth and Development in Xenopus laevis Using Iopanoic Acid.

Iodothyronine deiodinases (DIO) are key enzymes that influence tissue-specific thyroid hormone levels during thyroid-mediated amphibian metamorphosis. Within the larger context of evaluating chemicals for thyroid system disrupting potential, chemical activity toward DIOs is being evaluated using high-throughput in vitro screening assays as part of U.S. EPA's ToxCast program. However, existing data gaps preclude any inferences between in vitro chemical inhibition of DIOs and in vivo outcomes relevant to ecological risk assessment. This study aimed to generate targeted data in a laboratory model species (Xenopus laevis) using a model DIO inhibitor, iopanoic acid (IOP), to characterize linkages between in vitro potency, in vivo biochemical responses, and adverse organismal outcomes. In vitro potency of IOP toward DIOs was evaluated using previously developed in vitro screening assays, which showed concentration-dependent inhibition of human DIO1 (IC50: 97 µM) and DIO2 (IC50: 231 µM) but did not inhibit human or X. laevis DIO3 under the assay conditions. In vivo exposure of larval X. laevis to 0, 2.6, 5.3, and 10.5 µM IOP caused thyroid-related biochemical profiles in the thyroid gland and plasma consistent with hyperthyroxinemia but resulted in delayed metamorphosis and significantly reduced growth in the highest 2 exposure concentrations. Independent evaluations of dio gene expression ontogeny, together with existing literature, supported interpretation of IOP-mediated effects resulting in a proposed adverse outcome pathway for DIO2 inhibition leading to altered amphibian metamorphosis. This study highlights the types of mechanistic data needed to move toward predicting in vivo outcomes of regulatory concern from in vitro bioactivity data.

Thyroid hormone deficiency during zebrafish development impairs central nervous system myelination.

Thyroid hormones are messengers that bind to specific nuclear receptors and regulate a wide range of physiological processes in the early stages of vertebrate embryonic development, including neurodevelopment and myelogenesis. We here tested the effects of reduced T3 availability upon the myelination process by treating zebrafish embryos with low concentrations of iopanoic acid (IOP) to block T4 to T3 conversion. Black Gold II staining showed that T3 deficiency reduced the myelin density in the forebrain, midbrain, hindbrain and the spinal cord at 3 and 7 dpf. These observations were confirmed in 3 dpf mbp:egfp transgenic zebrafish, showing that the administration of IOP reduced the fluorescent signal in the brain. T3 rescue treatment restored brain myelination and reversed the changes in myelin-related gene expression induced by IOP exposure. NG2 immunostaining revealed that T3 deficiency reduced the amount of oligodendrocyte precursor cells in 3 dpf IOP-treated larvae. Altogether, the present results show that inhibition of T4 to T3 conversion results in hypomyelination, suggesting that THs are part of the key signaling molecules that control the timing of oligodendrocyte differentiation and myelin synthesis from very early stages of brain development.

The thyroid hormone degrading type 3 deiodinase is the primary deiodinase active in murine epidermis.

INTRODUCTION: Many tissues express thyroid hormone metabolizing deiodinases that both activate and inactivate thyroid hormones through conversion processes. Many believe that the primary role of thyroid hormone deiodinases is the activation of the prohormone thyroxine (T(4)) to the active hormone triiodothyronine because athyreotic humans can be treated with T(4) alone. In our hands a nonspecific deiodinase inhibitor (iopanoic acid [IOP]) decreased cutaneous cell proliferation in vitro, so we hypothesized that topical IOP would inhibit epidermal proliferation in vivo. METHODS: IOP was applied topically to mice. Treatments were applied daily for 1 week. Skin biopsies were either stained for 5-bromo-2-deoxyuridine or flash-frozen to assay for deiodinase activity. RESULTS: Topical IOP resulted in a dose-dependent increase in epidermal proliferation. Assay revealed significant inactivating type 3 deiodinase (Dio3) activity in the epidermis but little or no activating (Dio1 or Dio2) activity. Dio3 activity was decreased 44%±21% in epidermis from mice treated with low-dose IOP and 80%±4% in epidermis from mice treated with high-dose IOP (p<0.001). CONCLUSION: We hypothesize that keratinocytes express Dio3 in vivo to maintain cutaneous health and prevent the skin from becoming hyperproliferative. Our data support the developing recognition that the primary role of thyroid hormone deiodinases in some tissues may be the degradation of thyroid hormone to protect the tissue against thyrotoxicosis.

A thyroid hormone deiodinase inhibitor can decrease cutaneous cell proliferation in vitro.

BACKGROUND: Although cutaneous manifestations associated with thyroid dysfunction are classic, the potential for thyroid hormone or its antagonists to treat dermatological disease has not been explored with rigor. The predominant circulating thyroid hormone is the pro-hormone, thyroxine (T(4)). Skin, like many tissues, expresses thyroid hormone deiodinases to convert T(4) to the active thyroid hormone, triiodothyronine (T(3)). Previously, we determined that T(3) is necessary for optimal growth of keratinocytes and fibroblasts. The first hypothesis of this experiment was that the deiodinase inhibitor iopanoic acid (IOP) could inhibit cutaneous cell proliferation. The second hypothesis of this experiment was that the action of IOP could be attributed to its inhibition of conversion of T(4) to T(3). Although IOP is known to inhibit T(4) to T(3) conversion, the inhibition of cutaneous cell proliferation by IOP might conceivably result from other properties of IOP. METHODS: In separate experiments, primary culture human keratinocytes and dermal fibroblasts were incubated overnight with IOP. RESULTS: Proliferation was inhibited in a dose-dependent manner in both cell lines. Overnight incubation with T(3) restored the proliferation but overnight incubation with T(4) did not. CONCLUSION: The study is the first to demonstrate that IOP inhibits cutaneous cell proliferation and that supplemental T(3) is sufficient to correct at least part of the inhibition caused by IOP. The data suggest that IOP decreases cutaneous cell proliferation by inhibition of intracellular T(4) to T(3) conversion. The data provide indirect evidence of the presence of type 1 or type 2 iodothyronine deiodinase activity in skin cells. The data support our previous hypothesis that T(3) is necessary for normal cutaneous proliferation.

Effects and safety of iopanoic acid in cats administered levothyroxine.

The purpose of this study was to evaluate the safety and effect of iopanoic acid in 13 cats with hyperthyroidism induced by daily subcutaneous administration of 25microg/kg levothyroxine for a period of 42 days. On day 28 of levothyroxine administration, cats were randomly allocated to receive treatment twice daily with a placebo (control group; n=4), 50mg iopanoic acid (low dose group; n=5), or 100mg iopanoic acid (high dose group; n=4) for 14 days. Compared to the control group, T(3) concentrations were significantly decreased in both the low dose and high dose groups on days 35 and 42. T(3) concentrations in the low dose and high dose groups at days 35 and 42 were not different from day -8. The effect of iopanoic acid on clinical signs of hyperthyroidism was less apparent. Further clinical studies evaluating the long-term effect in cats with spontaneous hyperthyroidism are warranted.

Pharmacodynamic effect of iopanoic acid on free T(3) and T(4) levels in amiodarone-induced thyrotoxicosis.

We describe the effects of iopanoic acid on daily levels of free triiodothyronine (FT(3)) and free thyroxine (FT(4)) in a patient with progressive type II amiodarone-induced thyrotoxicosis (AIT) who was undergoing thyroidectomy. The patient was a 59-year-old man who was undergoing amiodarone therapy while awaiting cardiac transplantation; the use of beta blockers and corticosteroids to control the AIT was contraindicated in this patient. Prior to thyroidectomy, the patient was started on iopanoic acid at 1.0 g twice a day; in response to gastrointestinal side effects, the dosage was subsequently reduced to 0.5 g twice a day. The patient responded to iopanoic acid with a rapid decrease in his FT(3) level and slight increase in his FT(4) level. This control of thyrotoxicosis allowed for an uneventful thyroidectomy, which was later followed by successful cardiac transplantation. Based on our findings in this single case, we believe that iopanoic can be used to rapidly lower FT(3) levels and to treat symptoms of thyrotoxicosis in a preoperative setting. We also discuss the different pharmacodynamic effects that iopanoic acid has on FT(3) and FT(4) levels.

Effects of different treatments for hyperthyroidism on the hypothalamic-pituitary-adrenal axis.

To investigate the effects of iopanoic acid (IA) and carbimazole on increased activity of the hypothalamic-pituitary-adrenal (HPA) axis in hyperthyroidism, we studied 14 women with hyperthyroidism caused by Graves' disease (n + 11) or toxic multinodular goitre (n + 3) before and after carbimazole or IA treatment. Seven normal women comprised the control group. Changes in thyroid-stimulating hormone, total and free thyroid hormones, arginine vasopressin (AVP), urinary free cortisol, adrenocorticotrophin (ACTH) and cortisol in response to human corticotrophin-releasing hormone (hCRH; 100 microg, i.v.) were estimated under basal conditions and after treatment with IA (3 g/day; n + 7) for 7 days or carbimazole (30 mg/day; n + 7) for 1 month. A higher ACTH response, with normal cortisol secretion, was observed in hyperthyroid patients in response to hCRH compared with the control group. After 7 days treatment, IA induced a significant reduction in total tri-iodothyronine (T(3)) and free T(3) to normal levels and a stronger ACTH response to hCRH, whereas plasma and urinary cortisol levels remained unchanged. Patients treated with carbimazole showed normalization of thyroid hormone levels, a reduction in basal and stimulated ACTH secretion and higher urinary free cortisol levels compared with pretreatment levels. Neither IA nor carbimazole treatment had any effect on AVP levels in hyperthyroid patients. In conclusion, hyperthyroid patients showed HPA axis hyperactivity of central origin with reduced cortisol responses, which were reversed by carbimazole treatment. The differential effects of IA and carbimazole on HPA function indicate that thyroid hormones have a role in modulation of the HPA axis.

Morphological and biochemical changes in the Harderian gland of hypothyroid rats.

The secretory activity of the Harderian gland (HG) is influenced by both exogenous (such as light and temperature) and endogenous (such as prolactin, thyroid hormones and steroid hormones) factors, which vary among species. In the present study, the effects of hypothyroidism on the rat HG were examined at morphological and biochemical levels. The decrease in cytoplasmic lipoproteic vacuoles and the increase in mucosubstance secretion in the acinar lumina were the most notable histological effects elicited by hypothyroidism. The release of all granules with nuclei and cellular debris suggested the occurrence of holocrine secretion. Electron microscopy revealed in the glandular cells of hypothyroid rat an increased condensation of chromatin in the nuclei, mitochondria with decreased cristae and vacuolisation, decreased glycogen granules, autophagic vacuoles, and lipofuscins in the cytoplasm. TUNEL reaction indicated DNA fragmentation in hypothyroid HG, indicative of an underlying apoptotic process. Translocation of cytochrome c from mitochondria to cytosol strongly supported this hypothesis. In conclusion, these findings indicate that thyroid hormones play a pivotal role in preserving the structural integrity of the rat HG and, hence, its secretory activity.

Unliganded thyroid hormone receptor is essential for Xenopus laevis eye development.

Thyroid hormone receptors generally activate transcription of target genes in the presence of thyroid hormone (T(3)) and repress their transcription in its absence. Here, we investigated the role of unliganded thyroid hormone receptor (TR) during vertebrate development using an amphibian model. Previous studies led to the hypothesis that before production of endogenous T(3), the presence of unliganded receptor is essential for premetamorphic tadpole growth. To test this hypothesis, we generated a Xenopus laevis TR beta mutant construct ineffective for gene repression owing to impaired corepressor NCoR recruitment. Overexpression by germinal transgenesis of the mutant receptor leads to lethality during early development with numerous defects in cranio-facial and eye development. These effects correlate with TR expression profiles at these early stages. Molecular analysis of transgenic mutants reveals perturbed expression of genes involved in eye development. Finally, treatment with iopanoic acid or NH-3, modulators of thyroid hormone action, leads to abnormal eye development. In conclusion, the data reveal a role of unliganded TR in eye development.

Suppression of hypothalamic deiodinase type II activity blunts TRH mRNA decline during fasting.

Fasting is characterized by disrupted thyroid feedback, with suppressed levels of thyroid hormones and paraventricular thyrotropin releasing hormone (TRH). We found that third ventricle administration of the deiodinase inhibitor, iopanoic acid, dose-dependently reduced deiodinase type II (DII) activity selectively in the hypothalamus. This suppression of DII by iopanoic acid during fasting prevented elevated DII activity and blunted the decline in hypothalamic TRH mRNA levels. Because fasting-induced elevation in hypothalamic DII activity is paralleled by increased hypothalamic T3 concentration, our study suggests that T3 formation by DII in the hypothalamus is the cause of disrupted thyroid feedback during fasting.

Effects of tetraiodothyronine and triiodothyronine on hamster cheek pouch microcirculation.

The aim of the present study was to assess the effects of topically applied triiodothyronine (T(3)) and thyroxine (T(4)) on the arterioles of hamster cheek pouch microcirculation in vivo. Microvessels were visualized using a fluorescent microscopy technique. Topical application of T(3) (3.08, 30.8, 61.5, 307, 615, and 6,150 nM/l) consistently induced dose-dependent dilation of arterioles within 2.0 +/- 0.5 min of administration. The application of T(4) (150, 257, 514, and 5,140 nM/l) caused different dose-dependent effects: dilation at the three lower doses within 16 +/- 2 min and rhythmic diameter changes at the highest dose. Aging of hamsters did not alter the arteriolar responses to T(3) and T(4). T(3)-induced dilation was countered by the inhibition of nitric oxide synthase with N(G)-nitro-L-arginine-methyl ester or N(G)-nitro-L-arginine. Iopanoic acid (IPA), which inhibits types I and II 5'-deiodinase, abolished the dilation elicited by 514 nM T(4) but did not affect T(3)-dependent dilation. 6-Propyl-2-thiouracil (PTU), which inhibits type I 5'-deiodinase only, did not affect the dilation induced by T(4). IPA and PTU did not impair arteriolar dilation induced by acetylcholine or sodium nitroprusside. These results indicate that T(3) induces arteriolar dilation, likely through nitric oxide release. The local conversion of T(4) to T(3) appears to be crucial for the dilation induced by T(4).

Thyroid hormone-dependent seasonality in American tree sparrows (Spizella arborea): effects of GC-1, a thyroid receptor beta-selective agonist, and of iopanoic acid, a deiodinase inhibitor.

To explore the role of TH in the control of seasonality [i.e., photoperiodic testicular growth, photorefractoriness, and postnuptial (prebasic) molt] in American tree sparrows (Spizella arborea), we performed experiments in which THX males were simultaneously photostimulated and given TH replacement therapy. In the first experiment, equimolar concentrations (1X = 1.3 nmol) of T4, T3, or GC-1, an iodine-free TRbeta agonist, were administered s.c. daily during the first 21 days of photostimulation. Two additional THX groups received GC-1 at 0.1X or 10X, and THX and THI control groups received vehicle. In the second experiment, T4 or T3, alone or in combination with the deiodinase inhibitor IOP, was injected i.m. twice daily during the first 14 days of photostimulation. In both experiments, end points were testis length and molt score. In the first experiment, THI birds given vehicle and THX birds given T4 replacement therapy exhibited all three components of seasonality. THX birds given T3 or GC-1 (1X or 10X) showed a subdued photoperiodic testicular response, but they did not become photorefractory or initiate molt. THX birds that received 0.1X GC-1 or vehicle exhibited none of the components of seasonality. These data are consistent with the hypothesis that photoperiodic testicular growth, a vernal component of seasonality, is a TRbeta-mediated response and suggest that T4 may activate TRbeta more efficiently than does T3 or GC-1. By contrast, the failure both of T3 and of GC-1, but not of T4, to program photostimulated THX males for photorefractoriness and postnuptial molt suggests that autumnal components of seasonality may be TRalpha-mediated responses solely to T4. In the second experiment, IOP administered alone had no significant impact on seasonality. THX birds that received T4 with or without IOP showed all components of seasonality, whereas birds that received T3 with or without IOP showed only photoperiodic testicular growth. These results challenge the widely held view that T4 is merely a prohormone for T3 and support the emerging view that T4 has intrinsic hormonal activity. Because IOP augmented the photoperiodic testicular response in T3-treated THX birds, T3 may act either independently or co-dependently with T4 in programming vernal seasonal events.

Expression of type II iodothyronine deiodinase marks the time that a tissue responds to thyroid hormone-induced metamorphosis in Xenopus laevis.

The thyroid gland synthesizes thyroxine (T4), which passes through the larval tadpole's circulatory system. The enzyme type II iodothyronine deiodinase (D2) converts thyroxine (T4) to the active hormone 3,5,3'-triiodothyronine (T3) in peripheral tissues. An early response to thyroid hormone (TH) in the Xenopus laevis tadpole is the stimulation of cell division in cells that line the brain ventricles, the lumen of the spinal cord, and the limb buds. These cells express constitutively high levels of D2 mRNA. Exogenous T4 induces early DNA synthesis in brain, spinal cord, and limb buds as efficiently as T3. The deiodinase inhibitor iopanoic acid blocks T4- but not T3-induced cell division. At metamorphic climax, both TH-induced cell division and D2 expression decrease in the brain. Then D2 expression appears in late-responding tissues including the anterior pituitary, the intestine, and the tail where cell division is reduced or absent. Therefore, constitutive expression of D2 occurs in the earliest target tissues of TH that will grow and differentiate, while TH-induced expression of D2 takes place in late-responding tissues that will remodel or die. This pattern of constitutive and induced D2 expression contributes to the timing of metamorphic changes in these tissues.

Perioperative management of the thyrotoxic patient.

Preoperative thyrotoxicosis is a potentially life-threatening condition that requires medical intervention before surgery. Most patients are undergoing thyroidectomy for persistent thyrotoxicosis, usually Graves' disease, either having contraindications to or failing medical therapy. Fewer patients are undergoing nonthyroidal surgery that is likely urgent or emergent. The choice of treatment depends on the time available for preoperative preparation, the severity of the thyrotoxicosis, and the impact of any current or previous therapies. Generally treatment is directed at a combination of targets in the thyroid hormone synthetic, secretory, and peripheral pathway with concurrent treatment to correct any decompensation of normal homeostatic mechanisms. Thionamides are the preferred initial treatment unless contraindicated, but do require several weeks to render a patient euthyroid. beta-Blockers should always be used unless absolutely contraindicated because they improve thyrotoxic symptoms especially of the cardiovascular system. Other agents including iodine and steroids can be used if rapid preparation is required or more severe thyrotoxicosis is present. The goal of therapy is to render the patient as close as possible to clinical and biochemical euthyroidism before surgery. Overall, the morbidity and mortality of adequately prepared patients is low.

The activity of antioxidant enzymes and the content of uncoupling protein-1 in the brown adipose tissue of hypothyroid rats: comparison with effects of iopanoic acid.

The activity of antioxidant enzymes, copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and catalase (CAT), as well as that of the mitochondrial FAD-dependent alpha-glycerophosphate dehydrogenase (alpha-GPD) in the rat interscapular brown adipose tissue (IBAT) were studied after the treatment with methimazole (MMI) for three weeks or with iopanoic acid (IOP) for five days. Besides, the mitochondrial concentration of uncoupling protein-1 (UCP-1) and the activity of catecholamine degrading enzyme monoamine oxidase (MAO) in the IBAT as well as the activity of the catecholamine synthesizing enzyme, dopamine beta-hydroxylase (DBH) in rat serum were examined. Judging by the significantly enhanced level of serum DBH, which is an index of sympathetic activity, and that of IBAT MAO, the increase in MnSOD and CAT activities in the IBAT of hypothyroid (MMI-treated) rats seems to be due to elevated activity of sympathetic nervous system (SNS). However, CuZnSOD activity is not affected by SNS. On the contrary, IOP, which is a potent inhibitor of T4 deiodination into T3 producing "local" hypothyroidism, did not change either SNS activity or activities of IBAT antioxidant enzyme. However, both treatments significantly decreased IBAT UCP-1 content and alpha-GPD activity suggesting that the optimal T3 concentration in the IBAT is necessary for maintaining basal levels of these key mitochondrial parameters.

Clinical review 129: Oral cholecystographic agents and the thyroid.

Oral cholecystographic agents (OCAs) are known to affect thyroid hormone metabolism by acting as potent inhibitors of type I and type II deiodinases, blocking the conversion of T(4) to T(3) and rT(3) to T(2). In addition, iodine released from the drug blocks thyroid gland secretion of thyroid hormone. These properties make OCAs a potentially useful drug therapy in patients with hyperthyroidism and other thyrotoxic disorders. Short-term treatment with OCAs rapidly reduces serum T(3) levels, with a lesser effect on T(4) levels. OCAs are not useful for long-term treatment, which is usually followed by exacerbation of hyperthyroidism with continued use. The lack of significant side effects makes these drugs an excellent short-term option in situations where a rapid clinical improvement is critical.

A test of the hypothesis that T3 is the "seasonality" thyroid hormone in American tree sparrows (Spizella arborea): intracerebroventricular infusion of iopanoic acid, an inhibitor of T3 synthesis and degradation.

This study tested the hypothesis that L-3,5,3'-triiodothyronine (T3) is the bioactive "seasonality" thyroid hormone in American tree sparrows (Spizella (arborea). The experimental approach coupled thyroid hormone replacement therapy after radiothyroidectomy with photostimulation and intracerebroventricular infusion of iopanoic acid, an inhibitor of L-3,5,3'-triiodothyronine synthesis and degradation. Endpoints were testis length, molt score, and hypothalamic content of chicken gonadotropin-releasing hormone 1. The hypothesis predicts that thyroidectomized male tree sparrows moved to long days and given thyroxine in combination with iopanoic acid will lack L-3,5,3'-triiodothyronine and so will not express thyroid hormone-dependent photoperiodic testicular growth (a vernal component of seasonality) and photorefractoriness or postnuptial molt (autumnal components of seasonality). It further predicts that iopanoic acid will enhance the efficacy of L-3,5,3'-triiodothyronine and so will facilitate the expression of seasonality in thyroidectomized males given L-3,5,3'-triiodothyronine replacement therapy. Iopanoic acid had no significant effect on any component of seasonality in thyroid-intact males given vehicle, or in thyroidectomized males given thyroxine or L-3,5,3'-triiodothyronine. Thyroid-intact males, as well as thyroidectomized males infused with thyroxine alone, commonly expressed all components of seasonality. Thyroidectomized males given L-3,5,3'-triiodothyronine alone exhibited photoperiodic testicular growth, but did not become photorefractory or initiate molt. While these results confirm that thyroid hormone acts centrally to program American tree sparrows for seasonality, they do not support the hypothesis that L-3,5,3'-triiodothyronine is the bioactive "seasonality" thyroid hormone, and they challenge the view that thyroxine is merely a prohormone.

Effects of thyroxine on rat brown fat and muscle thermogenesis in the cold.

We studied whether the activation of rat brown adipose tissue (BAT) by cold exposure or by the administration of beta-3-noradrenergic agonist CGP-12177 could be prevented by the inhibition of thyroxine (T4) to triiodothyronine (T3) conversion. Hypothyroid rats were treated with replacement doses of T4, T4 plus iopanoic acid (IA) or T3. Groups of rats were placed at 4 degrees C for 24 h or kept at room temperature. Cold exposure induced a significant increase in guanosine diphosphate (GDP) binding to BAT mitochondrial proteins in T4-treated rats, an effect not abolished by IA. No significant changes were seen in T3-treated rats. In rats maintained at room temperature and injected with CGP-12177, T4 induced a significant rise in GDP binding which was not blocked by IA. T3 also induced a significant increase in binding. The study of mitochondrial oxygen consumption in muscle from cold-exposed rats showed a marked decrease in consumption in T3-treated rats as compared to values in the warm. Normal oxygen consumption was restored with 2-fold doses of T3 replacement, whereas 5-fold doses increased consumption above normal. The data suggest that in states with low or absent T3, T4 can stimulate heat production and preserve normothermia.

Evidence of UCP1-independent regulation of norepinephrine-induced thermogenesis in brown fat.

UNLABELLED: To study the thermal response of interscapular brown fat (IBF) to norepinephrine (NE), urethan-anesthetized rats (1.2 g/kg ip) maintained at 28-30 degrees C received a constant venous infusion of NE (0-2 x 10(4) pmol/min) over a period of 60 min. IBF temperatures (T(IBF)) were recorded with a small thermistor fixed under the IBF pad. Data were plotted against time and expressed as maximal variation (Deltat degrees C). Saline-injected rats showed a decrease in T(IBF) of approximately 0.6 degrees C. NE infusion increased T(IBF) by a maximum of approximately 3.0 degrees C at a dose of 10(4) pmol x min(-1) x 100 g body wt(-1). Surgically thyroidectomized (Tx) rats kept on 0.05% methimazole showed a flat response to NE. Treatment with thyroxine (T(4), 0.8 microg x 100 g(-1) x day(-1)) for 2-15 days normalized mitochondrial UCP1 (Western blotting) and IBF thermal response to NE, whereas iopanoic acid (5 mg x 100 g body wt(-1) x day(-1)) blocked the effects of T(4). Treatment with 3,5, 3'-triiodothyronine (T(3), 0.6 microg x 100 g body wt(-1) x day(-1)) for up to 15 days did not normalize UCP1 levels. However, these animals showed a normal IBF thermal response to NE. Cold exposure for 5 days or feeding a cafeteria diet for 20 days increased UCP1 levels by approximately 3.5-fold. Nevertheless, the IBF thermal response was only greater than that of controls when maximal doses of NE (2 x 10(4) pmol/min and higher) were used. CONCLUSIONS: 1) hypothyroidism is associated with a blunted IBF thermal response to NE; 2) two- to fourfold changes in mitochondrial UCP1 concentration are not necessarily translated into heat production during NE infusion.