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The stereospecific removal of iodine from thyroid hormones is an essential first step for T3 action and is catalyzed by three different deiodinases: D2 and D3 remove iodine only from the outer or inner ring, respectively, whereas D1 catalyzes both pathways. We used in silico predictions from vertebrate deiodinase sequences to identify two domains: the N-terminal variable region (VR) containing the transmembrane, hinge and linker domains, and the conserved or globular region (CR). Given the high sequence and structural identity of the CR among paralogs as well as of the VR among orthologs but not paralogs, we hypothesized that both the catalytic properties and the subcellular localization rely on the VR. We used shark D2 and D3 as templates to build the chimeric enzymes D2VR/D3CR and D3VR/D2CR. Biochemical characterization revealed that D3VR/D2CR has inner-ring deiodination activity and T3 as preferred substrate, whereas D2VR/D3CR showed no deiodinating activity. Also, D2VR/D3CR and D3VR/D2CR reside in the endoplasmic reticulum and plasmatic membrane, respectively, as do their D2 and D3 wild-type counterparts. We conclude that the VR determines the subcellular localization and is critical in defining the catalytic properties and activity of thyroid hormone deiodinases.

Thyroid hormone bioactivity is finely regulated at the cellular level by the peripheral iodothyronine deiodinases (D). The study of thyroid function in fish has been restricted mainly to teleosts, whereas the study and characterization of Ds have been overlooked in chondrichthyes. Here we report the cloning and operational characterization of both the native and the recombinant hepatic type 3 iodothyronine deiodinase in the tropical shark Chiloscyllium punctatum. Native and recombinant sD3 show identical catalytic activities: a strong preference for T3-inner-ring deiodination, a requirement for a high concentration of DTT, a sequential reaction mechanism, and resistance to PTU inhibition. The cloned cDNA contains 1298 nucleotides [excluding the poly(A) tail] and encodes a predicted protein of 259 amino acids. The triplet TGA coding for selenocysteine (Sec) is at position 123. The consensus selenocysteine insertion sequence (SECIS) was identified 228 bp upstream of the poly(A) tail and corresponds to form 2. The deduced amino acid sequence was 77% and 72% identical to other D3 cDNAs in fishes and other vertebrates, respectively. As in the case of other piscivore teleost species, shark expresses hepatic D3 through adulthood. This characteristic may be associated with the alimentary strategy in which the protection from an exogenous overload of thyroid hormones could be of physiological importance for thyroidal homeostasis.

SUMMARY The physiological role played by thyroid hormones (TH) in hydro-osmotic homeostasis in fish remains a controversial issue. Previous studies have shown that in Fundulus heteroclitus (killifish) hypo-osmotic stress increases liver iodothyronine deiodinase type 2 (D2) mRNA and D2 activity. In this study we identified two conserved osmotic response element (ORE) motifs in the promoter region of the killifish D2 gene (FhDio2) and examined their possible role in the transcriptional regulation of FhDio2during hypo-osmotic stress. As assessed by the electrophoretic mobility shift assay, results from in vivo and in vitro experiments demonstrate that exposure to an abrupt hyposmotic challenge triggers in the liver of killifish a strong nuclear recruitment of a putative osmotic response element binding protein (OREBP). This protein–DNA binding is time-dependent, attains a maximum within 2–8 h after the osmotic stress,and is followed by a significant increase in D2 activity. Furthermore,protein–DNA binding and the subsequent elevation in enzyme activity were blocked by the tyrosine kinase inhibitor genistein. Thus, during hypo-osmotic stress, a putative OREBP kinase-activated pathway stimulates FhDio2transcription and enzymatic activity. These data and the fact that D2 is the major enzyme providing local intracellular T3 suggest that TH plays a direct role in osmoregulation in fish, possibly by participating in hepatic ammonia metabolism. This study provides important insight into the physiological role of TH in hydro-osmotic homeostasis in fish.

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.

The presence of a type 1 deiodinase (D1) in the liver of teleosts has been a controversial issue. Recently we characterized the deiodinase activity in rainbow trout and killifish liver and found that the liver of both species co-expresses the two enzymes (D1 and D2) that catalyze the outer ring-deiodinating pathway. We here report the cloning and characterization of an mRNA from the liver of the killifish Fundulus heteroclitus that encodes a D1 (FhD1). The cDNA amplified by RT-PCR from F. heteroclitus liver is 1314 nt long and encodes a protein of 248 aa. It contains a TGA codon in its open reading frame and a selenocysteine insertion sequence in its 3′ untranslated region, consistent with the structure of a selenoenzyme mRNA. The deduced peptide sequence is 73% identical to that encoded by the tilapia D1 cDNA cloned from kidney and 46% identical to the D1s reported in other vertebrates. Northern blot analysis shows that FhD1 mRNA is expressed in F. heteroclitus liver, consistent with prior biochemical evidence for hepatic D1 activity. Furthermore, heterologous expression of the FhD1 cDNA resulted in a protein with properties similar to the D1-like activity in F. heteroclitus liver. The cloned enzyme, like the native species, is relatively insensitive to inhibition by PTU, but mutation of Ser-159 in FhD1 to the Pro residue found in D2 and D3 isoforms increased the sensitivity to PTU. Our results show that, under basal conditions, killifish liver indeed expresses a D1 enzyme that is homologous to mammalian D1s, establishing this as a useful model in which to study the regulation of D1 and D2 concurrently.

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.

The three distinct but related isotypes of the iodothyronine deiodinase family: D1, D2, and D3, have been amply studied in vertebrate homeotherms and to a lesser extent in ectotherms, particularly in reptiles. Here, we report the molecular and kinetic characteristics of both the native and the recombinant hepatic D3 from the pine snake Pituophis deppei (PdD3). The complete PdD3 cDNA (1680 bp) encodes a protein of 287 amino acids (aa), which is the longest type 3 deiodinase so far cloned. PdD3 shares 78% identity with chicken and 71% with its other orthologs. Interestingly, the hinge domain in D3s, including PdD3, is rich in proline. This structural feature is shared with D1s, the other inner-ring deiodinases, and deserves further study. The kinetic characteristics of both native and recombinant PdD3 were similar to those reported for D3 in other vertebrates. True K(m) values for T(3) IRD were 9 and 11 nM for native and recombinant PdD3, respectively. Both exhibited a requirement for a high concentration of cofactor (40 mM DTT), insensitivity to inhibition by PTU (2 mM), and bisubstrate, sequential-type reaction kinetics. In summary, the present data demonstrate that the liver of the adult pine snake P. deppei expresses D3. Furthermore, this is the first report of the cloning and expression of a reptilian D3 cDNA. The finding of hepatic D3 expression in the adult pine snake P. deppei is consistent with results in adult piscine species in which the dietary T(3) content seems to regulate liver deiodinase expression. Thus, our present results support the proposal that hepatic D3 in adult vertebrates plays a sentinel role in avoiding an inappropriate overload of exogenous T(3) secondary to feeding in those species that devour the whole prey.

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.