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Thyroid hormones, or THs, are well-known regulators of a wide range of biological processes that occur throughout the lifespan of all vertebrates. THs act through genomic mechanisms mediated by thyroid hormone receptors (TRs). The main product of the thyroid gland is thyroxine or T4, which can be further transformed by different biochemical pathways to produce at least 15 active or inactive molecules. T3, a product of T4 outer-ring deiodination, has been recognized as the main bioactive TH. However, growing evidence has shown that other TH derivatives are able to bind to, and/or activate TRs, to induce thyromimetic effects. The compiled data in this review points to at least two of these TR alternative ligands: TRIAC and T2. Taking this into account, non-mammalian models have proven to be advantageous to explore new TH derivatives with potential novel actions, prompting a re-evaluation of the role and mechanism of action of TR alternative ligands that were previously believed to be inactive. The functional implications of these ligands across different vertebrates may require us to reconsider current established notions of thyroid physiology.

Thyroid hormones (THs) induce pleiotropic effects in vertebrates, mainly through the activation or repression of gene expression. These mechanisms involve thyroid hormone binding to thyroid hormone receptors, an event that is followed by the sequential recruitment of coactivator or corepressor proteins, which in turn modify the rate of transcription. In the present study, we looked for specific coregulators recruited by the long isoform of the teleostean thyroid hormone receptor beta 1 (L-Trb1) when bound to the bioactive TH, 3,5-T2 (T2). We found that jun activation domain-binding protein1 (Jab1) interacts with L-Trb1 + T2 complex. Using both the teleostean and human TRB1 isoforms, we characterized the Jab1–TRB1 by yeast two-hybrid, pull-down and transactivation assays. Our results showed that the TRB1–Jab1 interaction was ligand dependent and involved the single Jab1 nuclear receptor box, as well as the ligand-binding and N-terminal domains of TRB1. We also provide evidence of ligand-dependent, dual coregulatory properties of Jab1. Indeed, when T2 is bound to L-Trb1 or hTRB1, Jab1 acts as a coactivator of transcription, whereas it has corepressor activity when interacting with the T3-bound S-Trb1 or hTRB1. These mechanisms could explain some of the pleiotropic actions exerted by THs to regulate diverse biological processes.

T3 and cortisol activate or repress gene expression in virtually every vertebrate cell mainly by interacting with their nuclear hormone receptors. In contrast to the mechanisms for hormone gene activation, the mechanisms involved in gene repression remain elusive. In teleosts, the thyroid hormone receptor beta gene or thrb produces two isoforms of TRβ1 that differ by nine amino acids in the ligand-binding domain of the long-TRβ1, whereas the short-TRβ1 lacks the insert. Previous reports have shown that the genomic effects exerted by 3,5-T2, a product of T3 outer-ring deiodination, are mediated by the long-TRβ1. Furthermore, 3,5-T2 and T3 down-regulate the expression of long-TRβ1 and short-TRβ1, respectively. In contrast, cortisol has been shown to up-regulate the expression of thrb. To understand the molecular mechanisms for thrb modulation by thyroid hormones and cortisol, we used an in silico approach to identify thyroid- and cortisol-response elements within the proximal promoter of thrb from tilapia. We then characterized the identified response elements by EMSA and correlated our observations with the effects of THs and cortisol upon expression of thrb in tilapia. Our data show that 3,5-T2 represses thrb expression and impairs its up-regulation by cortisol possibly through a transrepression mechanism. We propose that for thrb down-regulation, ligands other than T3 are required to orchestrate the pleiotropic effects of thyroid hormones in vertebrates.

Although 3,5,3′-triiodothyronine (T3) is considered to be the primary bioactive thyroid hormone (TH) due to its high affinity for TH nuclear receptors (TRs), new data suggest that 3,5-diiodothyronine (T2) can also regulate transcriptional networks. To determine the functional relevance of these bioactive THs, RNA-seq analysis was conducted in the cerebellum, thalamus-pituitary and liver of tilapia treated with equimolar doses of T2 or T3. We identified a total of 169, 154 and 2863 genes that were TH-responsive (FDR

Thyroid hormone (TH) actions are mediated by triiodothyronine (T3), which acts by binding to the TH receptors (TRs). Since TH exert pleiotropic effects, interest has grown in identifying other possible bioactive thyronines that could explain their diversity of functions. Accordingly, 3,5-diiodothyronine (T2) has been shown to be bioactive. In mammals, T2 regulates mRNA expression of several T3-regulated genes, but doses up to 100-fold greater than those of T3 were required to generate comparable effects. In teleosts, T2 and T3 regulate gene expression in vivo with equivalent potency. Furthermore, in vivo and in vitro studies support the notion that T2 binds to and activates a specific, long TRβ1 isoform that contains a nine amino acid insert at the beginning of the ligand binding domain, whereas T3 can interact also with a different TRβ1 isoform that lacks this insert. Similarly, T2 and T3 differentially regulate long- and short-TRβ1 expression, respectively, strongly suggesting a different signaling pathway for each hormone, at least in the species that express both receptors. In vivo, T2 effectively triggers a burst of body growth in tilapia by interacting with the long TRβ1 isoform, supporting the notion that T2 is physiologically relevant in this species. Current knowledge of T2 effects and action mechanisms lead us to propose that there is an extra level in the thyroid hormone signaling cascade, and that T2 is produced and regulated specifically for this purpose.

From an evolutionary perspective, deiodinases may be considered pivotal players in the emergence and functional diversification of both thyroidal systems (TS) and their iodinated messengers. To better understand the evolutionary pathway and the concomitant functional diversification of vertebrate deiodinases, in the present review we summarized the highlights of the available information regarding this ubiquitous enzymatic component that represents the final, common physiological link of TS. The information reviewed here suggests that deiodination of tyrosine metabolites is an ancient feature of all chordates studied to date and consequently, that it precedes the integration of the TS that characterize vertebrates. Phylogenetic analysis presented here points to D1 as the oldest vertebrate deiodinase and to D2 as the most recent deiodinase gene, a hypothesis that agrees with the notion that D2 is the most specialized and finely regulated member of the family and plays a key role in vertebrate neurogenesis. Thus, deiodinases seem to be major participants in the evolution and functional expansion of the complex regulatory network of TS found in vertebrates.

The pike silverside has high aquaculture potential despite its slow growth. In this study, thyroxine (T4) concentrations (0.13, 1.3 and 13 nM), a control with no hormonal supplement and a negative control containing 4.5 mM methimazole (MMI) were tested to evaluate the growth of this species. Juveniles (0.2 g) were exposed by immersion to these treatments for 8 h every second day for120 days, and growth evaluations were performed monthly over the entire trial period. In addition, tissue samples from each treatment group were assayed for triiodothyronine (T3) and deiodinase type 2 (D2) activity. The survival rates in all T4 groups were high, and a signi¢cant increase in growth was observed (average of 58%). The MMI treatment caused an increase in mortality and a reduction in the ¢nal body weight compared with the control. T4 administration did not aiect the tissue levels of T3, and it decreased muscular D2 activity only after 30 days of exposure. These results demonstrated that low concentrations of T4 in the culture environment could improve the growth of this species without affecting tissue hormone levels. The technique may have useful applications for early-stage aquaculture of this and other economically important species.

Growth hormone (GH), together with thyroid hormones (TH), regulates growth and development, and has critical effects on vertebrate metabolism. In ectotherms, these physiological processes are strongly influenced by environmental temperature. In reptiles, however, little is known about the direct influences of this factor on the somatotropic and thyroid axes. Therefore, the aim of this study was to describe the effects of both acute (48h) and chronic (2weeks) exposure to sub-optimal temperatures (25 and 18°C) upon somatotropic and thyroid axis function of the green iguana, in comparison to the control temperature (30-35°C). We found a significant increase in GH release (2.0-fold at 25°C and 1.9-fold at 18°C) and GH mRNA expression (up to 3.7-fold), mainly under chronic exposure conditions. The serum concentration of insulin-like growth factor-I (IGF-I) was significantly greater after chronic exposure (18.5±2.3 at 25°C; 15.92±3.4 at 18°C; vs. 9.3±1.21ng/ml at 35°C), while hepatic IGF-I mRNA expression increased up to 6.8-fold. Somatotropic axis may be regulated, under acute conditions, by thyrotropin-releasing hormone (TRH) that significantly increased its hypothalamic concentration (1.45 times) and mRNA expression (0.9-fold above control), respectively; and somatostatin (mRNA expression increased 1.0-1.2 times above control); and under chronic treatment, by pituitary adenylate cyclase-activating peptide (PACAP mRNA expression was increased from 0.4 to 0.6 times). Also, it was shown that, under control conditions, injection of TRH stimulated a significant increase in circulating GH. On the other hand, while there was a significant rise in the hypothalamic content of TRH and its mRNA expression, this hormone did not appear to influence the thyroid axis activity, which showed a severe diminution in all conditions of cold exposure, as indicated by the decreases in thyrotropin (TSH) mRNA expression (up to one-eight of the control), serum T4 (from 11.6±1.09 to 5.3±0.58ng/ml, after 2weeks at 18°C) and T3 (from 0.87±0.09 to 0.05±0.01ng/ml, under chronic conditions at 25°C), and Type-2 deiodinase (D2) activity (from 992.5±224 to 213.6±26.4fmolI(125)T4/mgh). The reduction in thyroid activity correlates with the down-regulation of metabolism as suggested by the decrease in the serum glucose and free fatty acid levels. These changes apparently were independent of a possible stress response, at least under acute exposure to both temperatures and in chronic treatment to 25°C, since serum corticosterone had no significant changes in these conditions, while at chronic 18°C exposure, a slight increase (0.38 times above control) was found. Thus, these data suggest that the reptilian somatotropic and thyroid axes have differential responses to cold exposure, and that GH and TRH may play important roles associated to adaptation mechanisms that support temperature acclimation in the green iguana.

Substrate availability has been thought to be a major regulator of the outer-ring deiodinating pathway (ORD) in fish. However, current information strongly suggests that while fish iodothyronine deiodinase type 2 (D2) responds to iodothyronines in the same manner as its mammalian counterpart, fish deiodinase type 1 (D1) exhibits a distinct response. Furthermore, 3,5-T 2 , generally considered to be an inactive product of iodothyronine metabolism, has recently been described as bioactive, but its effects upon D1 and D2 are not yet known. We examined the effect that short-term immersion in T 4 , T 3 , and 3,5-T 2 (0.1 μM; 12 or 24 h) exerts on both D1 and D2 activities and on the levels of expression of D1 and D2 mRNAs in killifish liver. In agreement with previous reports in teleosts, no iodothyronine exerted a significant effect on D1 enzymatic activity. However, all three iodothyronines significantly decreased D2 activity. Furthermore, at 24 h post-immersion T 4 , T 3 , and 3,5-T 2 inhibited both D1 and D2 transcription. Together, the present results confirm the differential effect of iodothyronines upon the hepatic ORD pathway in fish and show that this effect can occur at a transcriptional level. Furthermore, we provide the first evidence that 3,5-T 2 can affect both activity and transcription of hepatic deiodinases in teleosts.

Iodothyronine deiodinases, a ubiquitous family of selenoenzymes, are key tissue-specific regulators of intracellular thyroid hormone availability and signaling. This chapter reviews current information supporting the notion that the altered expression and/or activity of deiodinases contribute to the pathophysiology of diverse clinical disorders. Experimental and clinical evidence establishes an association of polymorphisms in deiodinase genes with mood, affective and cognitive functioning, as well as type 2 diabetes mellitus and lipid metabolism. Similarly, an imbalance in the activating and inactivating deiodinase pathways may promote cell proliferation and/or invasiveness of different types of neoplasms. Although a clear-cut picture has not yet been achieved, emerging data support the notion that the deiodinase-dependent thyroid hormone transcriptional footprint has a profound functional impact in health and disease.

Recent molecular cloning studies in mammals and amphibians have demonstrated that the types I, II, and III deiodinases constitute a family of selenoproteins of critical importance in metabolizing T4 to active (i.e. T3) and inactive (i.e. rT3) metabolites. In several tissues of teleost fish, various deiodinase processes have been described, but the structural and functional characteristics of these enzymes and their relationship to the deiodinases present in higher vertebrates remains uncertain. Using a complementary DNA library derived from the liver of the teleost Fundulus heteroclitus, we have identified a complementary DNA that codes for a deiodinase with functional characteristics virtually identical to those of the mammalian and amphibian type II deiodinase. Sequence analysis demonstrates a high degree of homology at both the nucleotide and predicted amino acid levels between the Fundulus clone and these previously characterized type II enzymes, including the presence of an in-frame TGA codon that codes for selenocysteine. These findings demonstrate that the deiodinase family of selenoproteins has been highly conserved during vertebrate evolution and underscores their importance in the regulation of thyroid hormone action.

Deiodinases are major determinants of thyroid hormone tissue availability and disposal. The knowledge of the expression of these enzymes in lower species is important to understand evolutionary and ontogenetic aspects of thyroid hormone action and metabolism. Here we have studied outer ring deiodination in the trout liver using both reverse T3 (rT3) and T4 as substrates. The use of rT3 disclosed two enzymatic components with the characteristics of mammalian types I and II 5′-deiodinases. The high rT3-Km type I 5′-deiodinase activity (180 nm) has a low cofactor requirement (5 mm dithiothreitol) and is relatively sensitive to propylthiouracil inhibition, whereas the low rT3-Km activity was akin to the outer ring deiodination of T4 in these regards. The use of T4 exhibited only a single type of activity with a low Km (0.63 nm), a relatively high cofactor requirement (25 mm dithiothreitol), and propylthiouracil-resistance. Teleosts constitute a unique example of type II activity expression in the liver of an ...

This study was supported by grants from PAPIIT 208511 and Consejo Nacional de Ciencia y Tecnologia (CONACYT) 166357. A.M. received Fellowship 227955 from CONACYT. Work from the V.L. laboratory is funded by the French Ministry of Research and Technology (Agence Nationale de la Recherche program) and the Ministry of Ecology (Programme National de Recherche sur les Perturbateurs Endocriniens program). A.M. is a doctoral student from Programa de Doctorado en Ciencias Biomedicas, Universidad Nacional Autonoma de Mexico. Endocrine soc Chevy chase; International audience; Several liganded nuclear receptors have alternative ligands acting in a tissue-specific fashion and playing important biological roles. We present evidence that 3,5-diiodothyronine (T-2), a naturally occurring iodothyronine that results from T-3 outer-ring deiodination, is an alternative ligand for thyroid hormone receptor beta 1 (TR beta 1). In tilapia, 2TR beta isoforms differing by 9 amino acids in the ligand-binding domain were cloned. Binding and transactivation studies showed that T-2 activates the human and the long tilapia TR beta 1 isoform, but not the short one. A chimeric human TR beta 1 (hTR beta 1) that contained the 9-amino-acid insert showed no response to T-2, suggesting that the conformation of the hTR beta 1 naturally allows T-2 binding and that other regions of the receptor are implicated in TR activation by T-2. Indeed, further analysis showed that the N terminus is essential for T-2-mediated transactivation but not for that by T-3 in the long and hTR beta 1, suggesting a functional interaction between the N-terminal domain and the insertion in the ligand-binding domain. To establish the functional relevance of T-2-mediated TR beta 1 binding and activation, mRNA expression and its regulation by T-2 and T-3 was evaluated for both isoforms. Our data show that long TR beta 1 expression is 106-fold higher than that of the short isoform, and T-3 and T-2 differentially regulate the expression of these 2 TR beta 1 isoforms in vivo. Taken together, our results prompted a reevaluation of the role and mechanism of action of thyroid hormone metabolites previously believed to be inactive. More generally, we propose that classical liganded receptors are only partially locked to very specific ligands and that alternative ligands may play a role in the tissue-specific action of receptors.

Iodide is a trace element and a key component of thyroid hormones (TH). The availability of this halogen is the rate-limiting step for TH synthesis; therefore, thyroidal iodide uptake and recycling during TH synthesis are of major importance in maintaining an adequate supply. In the rat, the thyroid gland co-expresses a distinctive pair of intrathyroidal deiodinating enzymes: the thyroid iodotyrosine dehalogenase (tDh) and the iodothyronine deiodinase type 1 (ID1). In the present work, we studied the activity of these two dehalogenases in conditions of hypo- and hyperthyroidism as well as during acute and chronic iodide administration in both intact and hypophysectomized (HPX) rats. In order to confirm our observations, we also measured the mRNA levels for both dehalogenases and for the sodium/iodide symporter, the protein responsible for thyroidal iodide uptake. Our results show that triiodothyronine differentially regulates tDh and ID1 enzymatic activities, and that both acute and chronic iodide administration significantly decreases rat tDh and ID1 activities and mRNA levels. Conversely, both enzymatic activities increase when intrathyroidal iodide is pharmacologically depleted in TSH-replaced HPX rats. These results show a regulatory effect by iodide on the intrathyroidal dehalogenating enzymes and suggest that they contribute to the iodide-induced autoregulatory processes involved in the Wolff-Chaikoff effect.

Until recently, 3,5-diiodothyronine (3,5-T2) has been considered an inactive by-product of triiodothyronine (T3) deiodination. However, studies from several laboratories have shown that 3,5-T2has specific, nongenomic effects on mitochondrial oxidative capacity and respiration rate that are distinct from those due to T3. Nevertheless, little is known about the putative genomic effects of 3,5-T2. We have previously shown that hyperthyroidism induced by supraphysiological doses of 3,5-T2inhibits hepatic iodothyronine deiodinase type 2 (D2) activity and lowers mRNA levels in the killifish in the same manner as T3and T4, suggesting a pretranslational effect of 3,5-T2(Garcia-G C, Jeziorski MC, Valverde-R C, Orozco A. Gen Comp Endocrinol 135: 201–209, 2004). The question remains as to whether 3,5-T2would have effects under conditions similar to those that are physiological for T3. To this end, intact killifish were rendered hypothyroid by administering methimazole. Groups of hypothyroid animals simultaneously received 30 nM of either T3, reverse T3, or 3,5-T2. Under these conditions, we expected that, if it were bioactive, 3,5-T2would mimic T3and thus reverse the compensatory upregulation of D2 and tyroid receptor β1 and downregulation of growth hormone that characterize hypothyroidism. Our results demonstrate that 3,5-T2is indeed bioactive, reversing both hepatic D2 and growth hormone responses during a hypothyroidal state. Furthermore, we observed that 3,5-T2and T3recruit two distinct populations of transcription factors to typical palindromic and DR4 thyroid hormone response elements. Taken together, these results add further evidence to support the notion that 3,5-T2is a bioactive iodothyronine.

We review the experimental evidence accumulated within the past decade regarding the physiologic, biochemical, and molecular characterization of iodothyronine deiodinases (IDs) in piscine species. Agnathans, chondrichthyes, and teleosts express the three isotypes of IDs: ID1, ID2, and ID3, which are responsible for the peripheral fine-tuning of thyroid hormone (TH) bioactivity. At the molecular and operational level, fish IDs share properties with their corresponding vertebrate counterparts. However, fish IDs also exhibit discrete features that seem to be distinctive for piscine species. Indeed, teleostean ID1 is conspicuously resistant to propylthiouracil (PTU) inhibition, and its response to thyroidal status differs from that exhibited by other ID1s. Moreover, both the high level of ID2 activity and its expression in the liver of teleosts are unique among vertebrates. The physiologic role of iodothyronine deiodination in functions regulated by TH in fish is not entirely clear. Nevertheless, current experimental evidence suggests that IDs may coordinate and facilitate, in a tissue-specific fashion, the action of iodothyronines and other hormones involved in such processes.

The initial characterization of a thyroid iodotyrosine dehalogenase (tDh), which deiodinates mono-iodotyrosine and di-iodotyrosine, was made almost 50 years ago, but little is known about its catalytic and kinetic properties. A distinct group of dehalogenases, the so-called iodothyronine deiodinases (IDs), that specifically remove iodine atoms from iodothyronines were subsequently discovered and have been extensively characterized. Iodothyronine deiodinase type 1 (ID1) is highly expressed in the rat thyroid gland, but the co-expression in this tissue of the two different dehalogenating enzymes has not yet been clearly defined. This work compares and contrasts the kinetic properties of tDh and ID1 in the rat thyroid gland. Differential affinities for substrates, cofactors and inhibitors distinguish the two activities, and a reaction mechanism for tDh is proposed. The results reported here support the view that the rat thyroid gland has a distinctive set of dehalogenases specialized in iodine metabolism.

Conversion of T4 to T3 is the first step in TH action and deiodinases are the major determinants of TH tissue availability and disposal. We here report the kinetic characterization of the outer-ring deiodinating (ORD) enzymes in the liver, gill and retina of sea water-adapted killifish, by using both rT3 and T4 as substrates. In liver, by using rT3, we detected a high Km (84 nM) and a low Km (1.3 nM) component with kinetic characteristics similar to mammalian deiodinases DI and DII. In contrast, T4-ORD only generated a low Km (0.5 nM) component. As judged by its Vmax (920 fmol 125I/mg per h) this DII enzyme is very abundant, approximately five and 20 times higher than that found in trout liver and hypothyroid rat, respectively. Kinetic analysis in killifish gill showed only one enzymatic component, with a high rT3 Km (430 nM) and a relatively low Vmax (4.3 pmol 125I/mg per h). Our results in killifish retina show the expression of a T4-low Km (0.6 nM) deiodinase with high cofactor requirements akin to the mammalian DII. The Vmax value for this enzyme is 182 fmol 125I/mg per h, five times lower than the one found in killifish liver, but comparable to that in hypothyroid rat pituitary. The biochemical similarities between fish and mammalian deiodinases could reflect their high conservation during vertebrate evolution and thus their importance in the regulation of thyroid hormone action.