Deiodinase Inhibitor Research Articles

L-thyroxine directly affects expression of thyroid hormone-sensitive genes: regulatory effect of RXR β

l-thyroxine (T4) has been considered mainly a prohormone, the hormonal action of which is related to its conversion to 3,5,3′-triiodothyronine (T3) in peripheral tissues. In this study we investigated in transient transfection assays whether T4 might directly affect the expression of thyroid hormone (TH) sensitive genes. The reporter construct ME-TRE-TK-CAT or TSH-TRE-TK-CAT containing the nucleotide sequence of the TH response element (TRE) of either malic enzyme (ME) or TSH β genes, was transfected with either TH receptor (TR) α alone or in combination with retinoid X receptor (RXR) β into NIH3T3 cells. Addition of 100 nM T4 to the culture medium in the presence of TR α increased the basal level of ME-TRE-TK-CAT expression by 4.5-fold. T4 action was due to a direct interaction with TR α and not to its conversion to T3, since T4 effect persisted in the presence of 5′-deiodinase inhibitors (propylthiouracil, iopanoic acid) effectively preventing T3 generation, as assessed by the absence of T3 by HPLC in the cellular extracts of transfected cells. In a dose-response study half-maximal stimulation by T4 was achieved at a concentration of 100 nM, whereas 50% of maximal induction was produced by 1 nM T3 and 6 nM triiodothyroacetic acid (TRIAC). Coexpression of RXR β greatly enhanced the transcriptional activity of the ME-TRE-TK-CAT gene when either T3, T4 or TRIAC was added to the culture medium of NIH3T3 cells, but established a hormonal hierarchy in the reporter activation different than that observed in the presence of TR α alone (TRIAC>T3≥T4, instead of T3>TRIAC>T4). T4 at a concentration of 100 nM could activate the TH/TR-dependent down-regulation mediated by the negative TSH-TRE, although at a lower level than that obtained with similar concentrations of T3 (35 and 55% inhibition, respectively). Our results demonstrate that, in addition to the action mediated through its monodeiodination to T3, T4 exerts a direct effect on genes that are either positively or negatively regulated by TH. Moreover, RXR β, forming heterodimers with TRs, appeared to exert a central role in modulating the sensitivity of TH-responsive genes to different iodothyronines.

Maternal to fetal thyroxine transmission in the human term placenta is limited by inner ring deiodination.

Placental deiodination of T4 to rT3 has been proposed as the factor controlling materno-fetal transmission of T4. We investigated T4 transfer in the isolated perfused human placental lobule with and without addition of the deiodinase inhibitor, iopanoic acid. T4 (150 nmol/L) in protein-free medium was added to the maternal circuit. Without iopanoic acid, the appearance of T4 in the fetal circuit was very low, with fetal T4 levels reaching only 4.1 +/- 0.84 pmol/L at 6 h. Levels of rT3 rose progressively in both circuits, reaching 28.8 +/- 5.5 nmol/L in the maternal and 12.4 +/- 3.2 nmol/L in the fetal circuit by 6 h. No T3 could be measured in either circuit. Addition of 0.5 nmol/L iopanoic acid to maternal perfusate, however, resulted in significant reduction in the appearance of rT3 [maternal levels, 0.58 +/- 0.06 nmol/L (2% of control values); fetal levels, 0.33 +/- 0.03 nmol/L (2.7% of control values)] and a major (approximately 2700-fold) increase in T4 appearance in the fetal circuit, with fetal T4 levels reaching 10.1 +/- 3.4 nmol/L at 6 h. These results support the hypothesis that placental inner ring (type III) deiodination is a major factor controlling placental transmission of maternal T4.

Amiodarone-Induced Elevation of Thyroid Stimulating Hormone in Patients Receiving Levothyroxine for Primary Hypothyroidism

Amiodarone, an iodinated antiarrhythmic agent, has complex effects on thyroid hormone physiology. As a potent hepatic 5′-deiodinase inhibitor, the drug decreases thyroxine (T4) conversion to 3,5,3′...

Does a Hidden Pool of Reverse Triiodothyronine (rT3) Production Contribute to Total Thyroxine (T4) Disposal in High T4States in Man?*

A hidden pool of rT3 production represents a source of rT3 that is minimally reflected in circulating rT3 levels. To test for the existence of such a source of rT3 production in man, varying doses of the generalized deiodinase inhibitor iopanoic acid (IA) were administered to four hyperthyroxinemic subjects. The doses employed included low-IA (0.5-g load, then 0.5 g/day for 5 days), mid-IA (1.0-g load, then 1.0 g/day for 5 days), and high-IA (3.0-g load, then 3.0 g/day for 5 days). Each patient received 25 microCi [125I]rT3, iv, in the high T4 state and on day 3 of each IA dosing regimen. Serial blood and urine samples were obtained to determine serum rT3 clearance rates and the urinary thyronine metabolite patterns. Although total serum rT3 values were increased by all IA dosages (P less than 0.001), rT3 was lower with high-IA administration (P less than 0.02) than with low- or mid-IA regimens. Low-IA decreased rT3 clearance to 33 +/- 2 L/day (P less than 0.005), while increasing the daily rT3 production to 76 +/- 8 nmol/day (P less than 0.04) compared to the control values (150 +/- 10 L/day and 53 +/- 8 nmol/day, respectively). Mid-IA also reduced rT3 clearance (23 +/- 4 L/day; P less than 0.005) without changing rT3 production (50 +/- 10 nmol/day), while high-IA reduced both rT3 clearance (21 +/- 2 L/day; P less than 0.005) and production (39 +/- 9 nmol/day; P less than 0.04). Intravenously administered tracer rT3 could not be detected in the urine in the high T4 state, but rT3 could not be detected in the urine in the high T4 state, but was prominent after IA administration. It is concluded that a hidden pool of rT3 production exists in vivo in man. Further, low dose IA serves as a selective inhibitor of liver and kidney deiodinase systems, allowing reflection of this hidden rT3 pool in the blood and urine. It would appear that hypertrophy of this hidden pool of rT3 production occurs in high T4 states and may account for the majority of the unrecognized deiodinative metabolites of T4 generated in hyperthyroxinemia.

5′-Deiodination in Rat Hepatocytes: Effects of Specific Flavonoid Inhibitors*

5'-Deiodination of T4 or rT3 in the presence of flavonoids was studied in freshly isolated suspended rat hepatocytes. Flavonoids, a novel group of synthetic deiodinase inhibitors, were designed to act as T4 antagonists. 5'-Deiodination of the prohormone T4 to the thyromimetically active T3 is an essential first step in controlling thyroid hormone action. Hepatocytes were incubated with either 2 microM T4 or 2 microM [125I]rT3 as 5'-deiodinase substrates in the absence and presence of inhibitors (0.1-100.0 microM). T3 production from T4 was determined by T3 RIA, and [125I]iodide release from [125I]rT3 was alternatively used as a technically more simple and rapid but sensitive deiodinase assay. Aurones and flavones inhibited both T4 5'-deiodination and rT3 5'-deiodination, with half-maximal inhibitor concentrations from 3-45 microM. Aurones were equally potent in both assays. 3-Methyl-flavones, designed as rT3 analogs, were more active by a factor of 3-5 with T4 5'-deiodination than with rT3 5'-deiodination, with the exception of one relatively cell-toxic compound. Hepatocyte viability was controlled by trypan blue dye exclusion as well as by measuring gluconeogenesis from exogenously added 10 mM lactate. Some of the flavonoids inhibited gluconeogenesis at concentrations that had no effect on trypan blue dye exclusion. Flavonoid inhibitors reduce 5'-deiodinase activity in intact hepatocytes in concentrations equimolar to those of substrates. Therefore, synthetic flavonoids may be suitable substances for further study of iodothyronine physiology or, after modification, could be useful as a new class of antiiodothyronine drugs.

Thyroxine, triiodothyronine, and reverse triiodothyronine processing in the cerebellum: autoradiographic studies in adult rats.

Well confirmed evidence has demonstrated that the cerebellum is an important target of thyroid hormone action during development. Moreover, the presence of nuclear receptors and strong 5'-deiodinase activity in cerebella of adult rats have suggested that this region may continue to respond to thyroid hormones during maturity. Recent autoradiographic observations have focused attention on the cerebellar granular layer, in that [125I]T3 administered iv to adult rats was found to be selectively and saturably concentrated there. To determine the specificity of iodothyronine localization in the granular layer, we have now compared film autoradiographic observations made after iv [125I]T4 and iv [125I]rT3 with those found after iv [125I]T3. The results demonstrated that, as in the case of the latter hormone, labeling within the cerebellar cortex after iv [125I]T4 was both selective and saturable. Moreover, except for a lag in time to resolution and a longer retention time, the distribution of cerebellar radioactivity after iv labeled T4 was qualitatively similar to that seen after iv [125I]T3. However, the ability of T4 to become differentially concentrated in the granular layer of cerebellum was absolutely dependent on its ability to be converted intracerebrally to T3. Thus, pretreatment with ipodate, which blocks brain 5'-deiodinase activity and, therefore, the intracerebral formation of T3 from T4, completely prevented cerebellar granular layer labeling after iv [125I]T4 even though it did not interfere with differential labeling of this region by iv delivered [125I]T3. In the same experiments, propylthiouracil, a potent peripheral, but not central, 5'-deiodinase inhibitor, had no qualitative effect on the distribution of either T4 or T3 in cerebellum. By contrast with the results obtained after administering labeled T3 or T4, brain labeling after iv delivered [125I]rT3 was found to be no different from that produced by markers of cerebral blood flow, which rapidly enter and leave the brain without becoming incorporated into brain cells. This was so even during treatment with propylthiouracil and ipodate, both of which markedly prolonged the normally brief residence time of this iodothyronine in serum and brain. Overall, the autoradiographic results served to highlight the importance of the morphological approach for investigating thyroid hormone action and metabolism in brain. They demonstrated that only T3, whether entering as such from the circulation or formed in situ from T4 (but neither T4 itself nor iv administered rT3) was strongly, selectively, and saturably concentrated in the cerebellar granular layer of adult rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolism of triiodothyroacetic acid (TA3) in rat liver. II. Deiodination and conjugation of TA3 by rat hepatocytes and in rats in vivo.

The hepatic metabolism of 3,3',5-triiodothyroacetic acid (TA3), a naturally occurring side-chain analog of T3, was studied in vitro and in vivo. Metabolites were quantified by HPLC after Sephadex LH-20 prepurification of samples obtained after incubation of [125I]TA3 or 3,[3'-125I]diiodothyroacetic acid (3,[3'-125I]TA2) with isolated rat hepatocytes under various conditions or after iv administration of [125I]TA3 to normal or 6-propyl-2-thiouracil (PTU)-treated rats. In protein-free incubations with hepatocytes, TA3 glucuronide (TA3G) and I- were normally the main TA3 products, i.e. 44% and 49%, respectively. In the presence of the type I deiodinase inhibitor PTU, the I- production from added TA3 decreased to 3%, and TA3 sulfate (TA3S) increased from 2-14%. Normally, 3,3'-TA2 was converted to I-, but in the presence of PTU 3,3'-TA2S was produced. In SO4(2-)-depleted cultures incubated with TA3 or 3,3'-TA2, production of I- was diminished, and the glucoronides of the substrates and the deiodinated products were generated. If both sulfation and deiodination were inhibited, TA3 and 3,3'-TA2 were cleared completely via glucuronidation. The metabolism of TA3 and especially 3,3'-TA2 was greatly retarded in cultures with 0.1% BSA. PTU treatment of TA3-injected rats reduced plasma I- levels 6-fold, increased plasma sulfates 2.6-fold, but did not affect plasma TA3 clearance. Biliary excretion of radioactivity until 4 h after [125I]TA3 injection amounted to 55% of the dose in controls vs. 85% in PTU-treated rats. In both groups, an unknown metabolite X was detected in serum and its sulfate conjugate XS in bile. The mean percent distribution of TA3G/TA3S/XS in bile amounted to 70:8:13 in control and 57:22:12 in PTU rats. In conclusion, TA3 is effectively metabolized in rat liver by glucuronidation and subsequent biliary excretion of TA3G, which may explain its rapid in vivo clearance relative to T3. Furthermore, a significant proportion of TA3 is deiodinated by the type I deiodinase, either directly or after prior sulfation.

Effects of inhibition of type I iodothyronine deiodinase and phenol sulfotransferase on the biliary clearance of triiodothyronine in rats.

Recent studies using isolated rat hepatocytes have indicated that the bioactive form of thyroid hormone, T3, is metabolized in liver predominantly by conjugation with glucuronic acid or sulfate. In contrast to T3 itself and the stable glucuronide, T3 sulfate is rapidly degraded by successive deiodination of the tyrosyl and phenolic rings. In the present study we have investigated the biliary excretion of T3 metabolites in male Wistar rats under pentobarbital anesthesia. The animals were injected iv with 1) saline, 2) the deiodinase inhibitor propylthiouracil (PTU; 1 mg/100 g BW), 3) the phenol sulfotransferase inhibitor dichloronitrophenol (2.6 mumol/100 g BW), or 4) a combination of both drugs. After 15 min, 10 muCi [125I]T3 were administered iv, and bile was collected for 30-min periods until 4 h after tracer injection. Secretory products were analyzed by HPLC. In control animals, 22.4% of the dose was excreted in bile mainly in the form of T3 glucuronide. In PTU-treated rats biliary excretion was increased to 36.0% of the dose (P less than .001) due to a dramatic increase in the sulfates of T3 and 3,3'-diiodothyronine. Dichloronitrophenol by itself had no effect on the biliary clearance of T3, but greatly inhibited PTU-induced excretion of sulfates. These results strongly suggest that sulfation and subsequent deiodination is an important pathway of T3 metabolism in vivo.

Inhibition of rat liver iodothyronine deiodinase. Interaction of aurones with the iodothyronine ligand-binding site.

We report that aurone derivatives of plant extracts produce potent, dose-dependent, and ultimately complete inhibition of three different metabolic monodeiodination pathways catalyzed by rat liver microsomal type I iodothyronine deiodinase. These data show that (3'),4',4,6-(tetra)trihydroxyaurones are the most potent naturally occurring plant-derived inhibitors of this deiodinase enzyme (IC50 V 0.5 microM). Lineweaver-Burk analysis using both L-thyroxine (T4) and 3',5',3-triiodothyronine as substrates suggests a cofactor competitive mechanism of inhibition for 4',4,6-trihydroxyaurone which also can displace 125I-L-T4 from binding to thyroxine-binding prealbumin with a potency comparable to its inhibition of T4-5'-deiodinase. Among type I deiodinase inhibitors, cofactor competition has been observed only for propylthiourea. Computer graphic modeling studies were also carried out to explore aurone conformations and to compare them with those of the thyroid hormones. This analysis shows that the aurones can adopt either a planar or an antiskewed conformation, such as observed for 3',5',3-triiodothyronine, the most potent natural deiodinase substrate inhibitor. The thyroxine-binding prealbumin complex was used to model the deiodinase ligand binding site because of the similarity observed between inhibitor binding affinity and enzyme inhibition characteristics. These studies show that the aurones which adopt an antiskewed conformation can interact favorably in the prealbumin binding site. This model of the deiodinase active site can be used to design other deiodinase inhibitors.

Rat liver iodothyronine monodeiodinase. Evaluation of the iodothyronine ligand-binding site.

Ligand binding characteristics of rat liver microsomal type I iodothyronine deiodinase were evaluated by measuring dose-response inhibition and apparent Michaelis-Menten or inhibitor constants of iodothyronine analogues to compete as substrates or inhibitors for the natural substrate L-thyroxine. These data show strong correlations with the binding requirements of hormone analogues to serum thyroxine-binding prealbumin since iodothyronine analogues with a negatively charged side chain, a negative charge or hydrogen bonding function in the 4'-position, tetraiodo ring substitution, and a skewed hormone conformation are structural features shared in common which markedly affect enzyme activity and protein binding affinity. 3,3',5'-Triiodo-L-thyronine is the most potent natural substrate (IC50 = 0.3 microM) and tetraiodothyroacetic acid is the most potent inhibitor (IC50 = 0.2 microM). Both thyroxine (T4)-5'- and T4-5-deiodination pathways are inhibited by these potent analogues, providing further evidence for a single enzyme catalyzing the rat liver microsomal deiodination reactions. These data also show that L-hormone analogues are preferentially deiodinated via the T4-5'-deiodination pathway, whereas D-analogues produce products via the T4-5-deiodination pathway. The thyroxine-binding prealbumin complex was used to model the interaction of thyroid hormones with the deiodinase active site. Computer graphic modeling of the prealbumin complex showed that only those analogues which are potent deiodinase inhibitors or substrates can be accommodated in the hormone binding site. This model suggests the design of functionally specific ligands which can modulate peripheral thyroid hormone metabolism and act as antithyroidal drugs.

ChemInform Abstract: Synthesis of 6‐Anilino‐2‐thiouracils and Their Inhibition of Human Placenta Iodothyronine Deiodinase.

Several 6-anilino-2-thiouracils were synthesized and tested for their ability to inhibit the inner-ring iodothyronine deiodinase from human placenta. The p-ethyl and p-n-butyl analogues were strongly inhibitory to the enzyme and were much more effective than the standard deiodinase inhibitor, 6-propyl-2-thiouracil. The degree of inhibition caused by 6-(p-n-butylanilino)-2-thiouracil was, moreover, unaffected by high concentrations of reducing agent in the enzyme assay. Attempts to prepare 3-alkyl derivatives via S-debenzylation of 2-benzylthio intermediates led to rearrangement to, for example, 3-methyl-5-benzyl-6-amino-2-thiouracil. This compound also strongly inhibited the deiodinase reaction. Preliminary results suggest that these compounds are useful to study in vitro and in vivo metabolism of thyroid hormones and may be clinically useful to enhance the availability of active thyroid hormones to certain organs.

Regulation of conversion of thyroxine to triiodothyronine in perfused rat kidney.

The factors regulating the renal uptake of thyroxine (T4), its conversion to 3,5,3'-triiodothyronine (T3), and the urinary iodothyronine excretion were studied in the perfused rat kidney. Increasing the perfusate free T4 (FT4) concentration from 1 to 11.5 times that of euthyroid rat serum resulted in a linear increase in T4 uptake and T3 production that was not saturated at the highest dose. When FT4 concentrations were increased by decreasing the perfusate albumin concentration from 7.5 to 2.5 g/dl, T4 uptake and T3 production increased in proportion to the FT4 concentration. Propylthiouracil (PTU), a 5'-deiodinase inhibitor, decreased renal T3 production by 60.5% without affecting tissue T4 uptake. In the absence of glomerular filtration, T4 uptake and T3 production were unchanged, indicating that T4 is extracted by the contraluminal surface of the renal tubule. However, probenecid, an inhibitor of contraluminal organic acid uptake, did not decrease but increased T4 uptake and T3 production by increasing the perfusate FT4 fraction in the perfusate. There was no net renal 3,3',5'-triiodothyronine (rT3) production from T4, and degradation and urinary excretion of T3 were negligible. The urinary excretion of T4 and T3 correlated closely with the degree of proteinuria.