Ellen Kaptein

Expression of Chicken Hepatic Type I and Type III Iodothyronine Deiodinases during Embryonic Development

In embryonic chicken liver (ECL) two types of iodothyronine deiodinases are expressed: D1 and D3. D1 catalyzes the activation as well as the inactivation of thyroid hormone by outer and inner ring deiodination, respectively. D3 only catalyzes inner ring deiodination. D1 and D3 have been cloned from mammals and amphibians and shown to contain a selenocysteine (Sec) residue. We characterized chicken D1 and D3 complementary DNAs (cDNAs) and studied the expression of hepatic D1 and D3 messenger RNAs (mRNAs) during embryonic development. Oligonucleotides based on two amino acid sequences strongly conserved in the different deiodinases (NFGSCTSecP and YIEEAH) were used for reverse transcription-PCR of poly(A 1 ) RNA isolated from embryonic day 17 (E17) chicken liver, resulting in the amplification of two 117-bp DNA fragments. Screening of an E17 chicken liver cDNA library with these probes led to the isolation of two cDNA clones, ECL1711 and ECL1715. The ECL1711 clone was 1360 bp long and lacked a translation start site. Sequence alignment showed that it shared highest sequence identity with D1s from other vertebrates and that the coding sequence probably lacked the first five nucleotides. An ATG start codon was engineered by site-directed mutagenesis, generating a mutant (ECL1711M) with four additional codons (coding for MGTR). The open reading frame of ECL1711M coded for a 249-amino acid protein showing 58 ‐ 62% identity with mammalian D1s. An in-frame TGA codon was located at position 127, which is translated as Sec in the presence of a Sec insertion sequence (SECIS) identified in the 39-untranslated region. Enzyme activity expressed in COS-1 cells by transfection with ECL1711M showed the same catalytic, substrate, and inhibitor specificities as native chicken D1. The ECL1715 clone was 1366 bp long and also lacked a translation start site. Sequence alignment showed that it was most homologous with D3 from other species and that the coding sequence lacked approximately the first 46 nucleotides. The deduced amino acid sequence showed 62‐72% identity with the D3 sequences from other species, including a putative Sec residue at a corresponding position. The 39-untranslated region of ECL1715 also contained a SECIS element. These results indicate that ECL1711 and ECL1715 are nearfull-length cDNA clones for chicken D1 and D3 selenoproteins, respectively. The ontogeny of D1 and D3 expression in chicken liver was studied between E14 and 1 day after hatching (C1). D1 activity showed a gradual increase from E14 until C1, whereas D1 mRNA level remained relatively constant. D3 activity and mRNA level were highly significantly correlated, showing an increase from E14 to E17 and a strong decrease thereafter. These results suggest that the regulation of chicken hepatic D3 expression during embryonic development occurs predominantly at the pretranslational level. (Endocrinology 138: 5144 ‐5152, 1997)

Characterization of a propylthiouracil-insensitive type I iodothyronine deiodinase

Mammalian type I iodothyronine deiodinase (D1) activates and inactivates thyroid hormone by outer ring deiodination (ORD) and inner ring deiodination (IRD), respectively, and is potently inhibited by propylthiouracil (PTU). Here we describe the cloning and characterization of a complementary DNA encoding a PTU-insensitive D1 from teleost fish (Oreochromis niloticus, tilapia). This complementary DNA codes for a protein of 248 amino acids, including a putative selenocysteine (Sec) residue, encoded by a TGA triplet, at position 126. The 39 untranslated region contains two putative Sec insertion sequence (SECIS) elements. Recombinant enzyme expressed in COS-1 cells catalyzes both ORD of T4 and rT3 and IRD of T3 and T3 sulfate with the same substrate specificity as native tilapia D1 (tD1), i.e. rT3 . . T4 . T3 sulfate . T3. Native and recombinant tD1 show equally low sensitivities to inhibition by PTU, iodoacetate, and gold thioglucose compared with the potent inhibitions observed with mammalian D1s. Because the residue 2 positions downstream from Sec is Pro in tD1 and in all (PTU-insensitive) type II and type III iodothyronine deiodinases but Ser in all PTU-sensitive D1s, we prepared the Pro128Ser mutant of tD1. The mutant enzyme showed strongly decreased ORD and somewhat increased IRD activity, but was still insensitive to PTU. These results provide new information about the structure-activity relationship of D1 concerning two characteristic properties, i.e. catalysis of both ORD and IRD, and inhibition by PTU. (Endocrinology 138: 5153‐5160, 1997)

Increased glucuronidation of thyroid hormone in hexachlorobenzene-treated rats.

Metabolism of thyroid hormones was investigated in WAG/MBL rats that had been exposed to hexachlorobenzene (HCB). Serum thyroxine (T4) levels were lowered by 35.5%, whereas triiodothyronine (T3) levels were not changed. Bile flow, as well as T4 excretion in bile were increased by HCB treatment. Analysis of bile by HPLC revealed a more than 3-fold increase of T4 glucuronide (T4G) and a concomitant reduction of non-conjugated T4. T4 UDP-glucuronyltransferase activity (T4 UDPGT) activity in hepatic microsomes was increased more than 4.5-fold in animals exposed to HCB. p-Nitrophenol (PNP) UDPGT showed a comparable increase by HCB. Both T3 and androsterone UDPGT activities were low in WAG/MBL rats compared with normal Wistar rats. T3 UDPGT activity was increased 2.5-fold by HCB, but androsterone UDPGT activity was unchanged. These results suggest that T4 is a substrate for HCB-inducible PNP UDPGT and T3 for androsterone UDPGT. In the absence of the latter, T3 is also glucuronidated to some extent by PNP UDPGT. Type 1 iodothyronine deiodinase activity was decreased by HCB treatment. It is concluded that decreased T4 levels in serum of animals after exposure to HCB may be due to a combined effect of displacement of T4 from carriers, an increased glucuronidation of T4 and enhanced bile flow.

Structure-activity relationships for thyroid hormone deiodination by mammalian type I iodothyronine deiodinases.

The bioactivity of thyroid hormone is determined to a large extent by the monodeiodination of the prohormone T4 by the hepatic selenoenzyme type I iodothyronine deiodinase (ID1), i.e. by outer ring deiodination (ORD) to the active hormone T3, or by inner ring deiodination (IRD) to the inactive metabolite rT3. ID1 also catalyzes the IRD of T3 and the ORD of rT3, both to T2, as well as the deiodination of different iodothyronine sulfates, e.g. IRD of T3S and ORD of T2S. Previous studies have indicated important differences in catalytic specificity between dog ID1 (dID1) and human ID1 (hID1), in particular with respect to the ORD of rT3. This study was done to investigate the relationship between structure and catalytic function of this enzyme by comparing the deiodination of T4, T3, rT3, T3S, and T2S by native dID1 and hID1 in liver microsomes as well as by recombinant wild-type, chimeric and mutated d/hID1 enzymes expressed in HEK293 cells. With both native and recombinant wild-type enzymes, the substrate ...

Glucuronidation of thyroid hormone by human bilirubin and phenol UDP‐glucuronyltransferase isoenzymes

The glucuronidation of thyroid hormone by UDP‐glucuronyltransferases (UGTs) stably transfected in Chinese hamster V79 lung fibroblasts was investigated. Human bilirubin UGT (HP3) and phenol UGT (HP4) both catalysed the glucuronidation of T4 and rT3, whereas glucuronidation of T3 was not significant. rT3 was the preferred substrate for both isoenzymes, glucuronidation rates being 1.6‐ and 6.4‐times higher than conjugation of T4 by HP3 and HP4 clones, respectively. This is the first identification of thyroid hormone as potential alternative endogenous substrate for bilirubin UGT.

Multiple UDP‐glucuronyltransferases for the glucuronidation of thyroid hormone with preference for 3,3',5'‐triiodothyronine (reverse T3)

We have studied the glucuronidation of the thyroid hormones T4, T3 and rT3 by liver microsomes of Wistar, Gunn and WAG rats. Gunn rats have a defect in the gene coding for bilirubin and phenol UDP‐glucuronyltransferase (UGT) isoenzymes; WAG rats have a genetic defect in androsterone UGT. In normal Wistar rats UGT activity was ≈5‐fold higher for rT3 than for T4 or T3. UGT activities for T4 and rT3, but not for T3, were impaired in Gunn rats. Conversely, UGT activity for T3, but not for T4 or rT3, was impaired in WAG rats. Thus, in rat liver rT3 is glucuronidated much more rapidly than T4 and T3. Our results support the view that T4 and rT3 are glucuronidated by bilirubin and phenol UGTs and T3 by androsterone UGT.

Cloning and Characterization of Type III Iodothyronine Deiodinase from the Fish Oreochromis niloticus

Type III iodothyronine deiodinase (D3) catalyzes the inner ring deiodination (IRD) of T4 and T3 to the inactive metabolites rT3 and 3,3′-diiodothyronine (3,3′-T2), respectively. Here we describe the cloning and characterization of complementary DNA (cDNA) coding for D3 in fish (Oreochromis niloticus, tilapia). This cDNA contains 1478 nucleotides and codes for a protein of 267 amino acids, including a putative selenocysteine (Sec) residue, encoded by a TGA triplet, at position 131. The deduced amino acid sequence shows 57–67% identity with frog, chicken, and mammalian D3, 33–39% identity with frog, fish (Fundulus heteroclitus) and mammalian D2, and 30–35% identity with fish (tilapia), chicken, and mammalian D1. The 3′ UTR contains a putative Sec insertion sequence (SECIS) element. Recombinant tilapia D3 (tD3) expressed in COS-1 cells and native tD3 in tilapia brain microsomes show identical catalytic activities, with a strong preference for IRD of T3 (Km ∼20 nm). IRD of [3,5-125I]T3 by native and recombina...

Development of a Radioimmunoassay for Triiodothyronine Sulfate

This paper is the first description of a radioimmunoassay (RIA) for triiodothyronine sulfate (T3S). Rabbits were immunized against T3S coupled to bovine serum albumin using carbodiimide. All animals produced antibodies to T3S but also even higher titers of T3 antibodies. Ka values for binding of T3 and T3S to these antisera varied between 2 x 10(10) and 8 x 10(10) M-1. One of the antisera (#8193) was selected for use in the T3S RIA because of a high titer of T3S antibodies (final dilution 1:50,000), a high sensitivity to T3S (less than 2.5 fmol/tube), and a low crossreactivity by T3 (0.4%). This RIA provides a tool for the study of the importance of sulfation as a metabolic pathway for T3.

Differential expression and ciprofibrate induction of hepatic UDP-glucuronyltransferases for thyroxine and triiodothyronine in Fischer rats.

In order to gain more insight into the isozymes responsible for the glucuronidation of T 4 and T 3 we investigated, in parallel, the effects of ciprofibrate on p-nitrophenol (PNP) and androsterone UDP-glucuronyltransferases activities

Tissue-Specific Suppression of Thyroid Hormone Signaling in Various Mouse Models of Aging

DNA damage contributes to the process of aging, as underscored by premature aging syndromes caused by defective DNA repair. Thyroid state changes during aging, but underlying mechanisms remain elusive. Since thyroid hormone (TH) is a key regulator of metabolism, changes in TH signaling have widespread effects. Here, we reveal a significant common transcriptomic signature in livers from hypothyroid mice, DNA repair-deficient mice with severe (Csbm/m/Xpa-/-) or intermediate (Ercc1-/Δ-7) progeria and naturally aged mice. A strong induction of TH-inactivating deiodinase D3 and decrease of TH-activating D1 activities are observed in Csbm/m/Xpa-/- livers. Similar findings are noticed in Ercc1-/Δ-7, in naturally aged animals and in wild-type mice exposed to a chronic subtoxic dose of DNA-damaging agents. In contrast, TH signaling in muscle, heart and brain appears unaltered. These data show a strong suppression of TH signaling in specific peripheral organs in premature and normal aging, probably lowering metabolism, while other tissues appear to preserve metabolism. D3-mediated TH inactivation is unexpected, given its expression mainly in fetal tissues. Our studies highlight the importance of DNA damage as the underlying mechanism of changes in thyroid state. Tissue-specific regulation of deiodinase activities, ensuring diminished TH signaling, may contribute importantly to the protective metabolic response in aging.