Outer Ring Deiodination Of T4 Research Articles

Non-mammalian models reveal the role of alternative ligands for thyroid hormone receptors

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.

A DFT investigation of a bulky biomimetic model catalyzing the 5'-outer ring deiodination of thyroxine.

This paper illustrates the outcomes of a density functional theory investigation aimed at unraveling mechanistic aspects of the 5'-outer ring deiodination process of thyroxine (T4) assisted by the sterically protected organoselenol compound BpqSeH. BpqSeH, which was previously synthesized and tested for its deiodinase activity, is able to afford the active hormone 3,5,3'-tetraiodothyronine (T3) by selective outer-ring deiodination of T4, and to protect the SeH moiety inside the nano-sized molecular cavity from further reactivity, allowing its isolation and characterization. Calculations were also performed including an imidazole ring that, mimicking a His residue in the active site of the original enzyme, plays an crucial role in deprotonating the selenol moiety. Both the suggested enol/keto tautomerization and the previously proven formation of an intermediate whose main characteristic is the presence of a Se⋯I⋯C halogen bond, were examined along the pathway leading to 5'-outer ring deiodination. The calculated potential energy surface showed that neither the pathway encompassing enol/keto tautomerism nor the formation of a halogen bond paving the way to C-I bond breaking and chalcogen-I bond forming is viable. The exergonic formation of the final selenenyl iodide product confirms the stabilization effect of the molecular cavity. Graphical Abstract Computed free energy profile describing the 5'-outer deiodination of thyroxine assisted by the steric hindered organoselenol BpqSH compound. The molecular electrostatic potential map reoported for the INT1 intermediate shows the non-covalent Se-I interaction, due to the attraction between charges of opposite sign, that weakens the C-I bond and prepares the formation of the new Se-I bond.

WHERE IS THE DEIODINASE SELECTIVITY FOR ORD OR IRD LOCATED? A STRUCTURE-FUNCTION APPROACH

WHERE IS THE DEIODINASE SELECTIVITY FOR ORD OR IRD LOCATED? A STRUCTURE-FUNCTION APPROACH

A cDNA for a putative type III deiodinase in the trout ( Oncorhynchus mykiss): Influence of holding conditions and thyroid hormone treatment on its hepatic expression

A putative rainbow trout type III deiodinase (D3) cDNA was amplified by PCR, using primers to evolutionarily conserved sequences. The RACE-derived complete cDNA was then identified by sequence comparison to that in tilapia and other vertebrates. The cDNA coded for a predicted 31,500 kDa protein of 278 amino acids, with a hydrophobic trans-membrane segment and with 80% similarity to tilapia D3 and 39% similarity to rainbow trout type II deiodinase (D2). It also showed a selenocysteine codon at position 141 and a putative SECIS element in the 3′ untranslated end. In the liver, a second form of D3 was found that differed only at this 3′ untranslated region; the coding region was identical in both forms. The D3 mRNA, measured by RT-PCR using primers located within the common, translated portion of the cDNA, was expressed in the brain and, depending on thyroidal status, in liver and kidney. Holding trout for 7 days in static water as opposed to flowing water caused increased plasma T4 levels, decreased hepatic D2 mRNA levels and T4 outer-ring deiodination (ORD) activity and increased D3 mRNA levels and T3 inner-ring (IRD) activity. Trout held in flowing water and fed T3 for 7 days showed increased plasma T3 levels and hepatic D3 mRNA levels and T3 IRD activity but decreased D2 mRNA levels and T4ORD activity. Trout held in static water and exposed to ambient T4 for 7 days showed increased plasma T4 levels and hepatic T3IRD activity but with no significant change in D2 or D3 mRNA levels. We conclude that hepatic D3 mRNA levels and T3IRD activity are enhanced and D2 mRNA levels and T4ORD activity are suppressed by adverse holding conditions or T3 treatment suggesting that the putative D3 cDNA and D2 cDNA represent respectively the genes determining T3IRD and T4ORD activities. However, there were changes in the ratios of mRNA levels to enzyme activity, raising the potential for post-transcriptional regulation and showing that mRNA levels alone may be unreliable indices of deiodinase activity. Post-transcriptional regulation of D3 enzyme activity may be influenced by the observed alternative 3′ and 5′ untranslated regions of the D3 mRNA.

Thyroid function in growth-hormone-transgenic coho salmon (Oncorhynchus kisutch)

We measured growth rate, plasma thyroxine (T4) and 3,5,3'-triiodothyronine (T3) concentrations, and liver and whole-brain T4 and T3 deiodination activities in yearling non-transgenic coho salmon, Oncorhynchus kisutch (Walbaum, 1792), fed a satiation ration (NTS) and in growth-hormone (GH)-transgenic salmon fed for 63 d with either a satiation ration (TS) or pair-fed the satiation ration consumed by NTS fish (TNT). Daily feed intake and specific growth rate for TS fish were significantly enhanced and approximately double those for TNT and NTS fish. There were no differences among groups in plasma T4 concentration or liver T4 outer-ring deiodination activity, but for both TS and TNT fish, plasma T3 concentrations were higher and liver T4 and T3 inner-ring deiodination activities were lower than for NTS fish. Whole-brain deiodination activities did not differ between TS and NTS fish. We conclude that the elevated plasma T3 concentrations of GH-transgenic salmon neither are driven by elevated plasma T4 concentrations nor are they the result of increased hepatic conversion of T4 to T3 by outer-ring deiodination. Instead they can be explained, at least in part, by reduced hepatic degradation of T3 to 3,3'-diiodothyronine by inner-ring deiodination. These changes in T4 and T3 metabolism are tightly linked to the GH-transgenic state and not to food intake or growth rate.

Thyroid Hormone Deiodinases during Embryonic Development of the Saltwater Crocodile (Crocodylus porosus)

All tissues of the embryonic saltwater crocodile (Crocodylus porosus) gradually increased in weight during development except for lung tissue, which had a peak weight of 1.09 g at day 67, thereafter decreasing in weight. The brain was a relatively large organ. Deiodinase activities in liver, kidney, lung, heart, gut, and brain from day 29 to day 77 of development of the saltwater crocodile were investigated. High-Km reverse triiodothyronine (rT3) outer ring deiodination (ORD) activity was present in all tissues except the brain. Activity ranged from 559 ± 51.3 pmol rT3 deiodinated/mg protein/min in the liver at day 77 to below 10 pmol rT3 deiodinated/mg protein/min in gut, lung, and heart tissue. rT3 ORD increased during development in the liver and kidney but decreased in the gut and lung. Activity in the heart was very low (less than 2 pmol rT3 deiodinated/mg protein/min) and did not change during development. Low-Km thyroxine (T4) ORD in liver and kidney tissue had peaks of activity around day 49 of incubation (0.52 and 0.09 fmol T4 deiodinated/mg protein/min, respectively). After day 49, T4 ORD activity in these tissues decreased. T4 ORD activity in gut, lung, and heart was very low (less than 0.04 fmol T4 deiodinated/mg protein/min), with activity in lung increasing slightly during the rest of development. T4 ORD activity in the brain increased toward day 77 (0.14 ± 0.03 fmol T4 deiodinated/mg protein/min), illustrating its importance in local triiodothyronine (T3) production during brain development. T3 inner ring deiodination activity was present only in the embryonic liver and peaked at day 49 (10.1 fmol T3 deiodinated/mg protein/min), after which activity decreased.

Erratum: The effect of handling and blood removal on plasma levels and hepatic deiodination of thyroid hormones in adult male and female rainbow trout, Oncorhynchus mykiss

We studied the effects of handling and blood removal (0.4% of body mass) on the thyroid system of fasted ~1000-g adult male and female rainbow trout, Oncorhynchus mykiss, over a 3-day period. Relative to undisturbed con- trols, bleeding lowered the mean hematocrit (HCT) by 19% and on day 1 increased the hepatic inner-ring deiodination of 3,5,3'-triiodothyronine (T3 )( T 3IRD), but did not alter thyroxine (T4) outer-ring deiodination (T4ORD) or inner-ring deiodination (T4IRD) activity or plasma T4 or T3 levels. Regardless of treatment, hepatic T 4ORD activity and HCT were lower in females than in males, and over the 3 days following bleeding, the plasma T3 level increased and the plasma T4 level decreased progressively for both sexes. We conclude that handling and bleeding induce a temporary

Characterization of Outer Ring Iodothyronine Deiodinases in Tissues of the Saltwater Crocodile (Crocodylus porosus)

The distribution and characterization of outer ring deiodination (ORD) using reverse triiodothyronine (rT3) and thyroxine (T4) as substrates is reported in microsomes of liver, kidney, lung, heart, gut, and brain tissues from juvenile saltwater crocodiles (Crocodylus porosus). In lung and heart only small amounts of rT3 ORD and T4 ORD were detected, while in brain only a small amount of T4 ORD was detected. More detailed characterization studies could be performed on liver, kidney, and gut microsomes. Reverse T3 outer ring deiodination (rT3 ORD) was the predominant activity in liver and kidney microsomes. The properties of crocodile liver and kidney rT3 ORD, such as preference for rT3 as substrate, a dithiothreitol (DTT) requirement of 10 mM, inhibition by propylthiouracil (PTU), and Michaelis–Menten (Km) constant in the micromolar range, correspond to the properties previously reported for a type I deiodinase. The temperature optimum for rT3 ORD was between 30 and 35°. There was also rT3 ORD activity in gut microsomes, along with what appeared to be a type II-like, low-Km deiodinase with a substrate preference for T4. There was also a small amount of T4 ORD activity in liver and kidney microsomes. Liver T4 ORD, like a type II deiodinase, had a preference for T4 as substrate at low substrate concentrations and a DTT requirement of 15 mM and was insensitive to PTU. However, at high substrate concentrations the predominant activity was of the type I deiodinase nature. T4 ORD in liver had an optimal incubation temperature of 30 to 35°. Gut microsomal T4 ORD was also type II-like at low substrate concentrations and type I-like at high substrate concentrations. Gut T4 ORD had an optimal incubation temperature of 25 to 30° and a DTT requirement of 20 mM DTT. Kidney microsomal T4 ORD had the same optimal temperature and DTT requirement as that in gut microsomes; however, there was no competition by low substrate concentrations. These results suggest that ORD in juvenile saltwater crocodile kidney is most likely exclusively catalyzed by a type I-like deiodinase. Liver and gut ORD, in contrast, is catalyzed by two enzymes, with a predominance of a type I-like deiodinase in liver and a type II-like deiodinase in gut. Low-Km T3 IRD activity could not be detected in any tissues of the juvenile saltwater crocodile.

The effect of handling and blood removal on plasma levels and hepatic deiodination of thyroid hormones in adult male and female rainbow trout, Oncorhynchus mykiss

We studied the effects of handling and blood removal (0.4% of body mass) on the thyroid system of fasted ~1000-g adult male and female rainbow trout, Oncorhynchus mykiss, over a 3-day period. Relative to undisturbed controls, bleeding lowered the mean hematocrit (HCT) by 19% and on day 1 increased the hepatic inner-ring deiodination of 3,5,3'-triiodothyronine (T3) (T3IRD), but did not alter thyroxine (T4) outer-ring deiodination (T4ORD) or inner-ring deiodination (T4IRD) activity or plasma T4 or T3 levels. Regardless of treatment, hepatic T4ORD activity and HCT were lower in females than in males, and over the 3 days following bleeding, the plasma T3 level increased and the plasma T4 level decreased progressively for both sexes. We conclude that handling and bleeding induce a temporary increase in hepatic T3 conversion to 3,3'-diiodothyronine with no change in T4 conversion to reverse T3. This is consistent with independent control of T3IRD and T4IRD activities. Furthermore, HCT and hepatic T4ORD activity are lower in adult females, and 10 days after transfer of trout to smaller tanks, plasma T4 and T3 levels may still be adjusting.

Thyroid of Lake Sturgeon, Acipenser fulvescens: II. Deiodination Properties, Distribution, and Effects of Diet, Growth, and a T3 Challenge

The authors studied the properties and tissue distribution of thyroid hormone (TH) deiodination activities measured in vitro at subnanomolar substrate levels for cultured 2-year-old lake sturgeon held at 12 to 15°. We also studied the deiodination responses to an exogenous 3,5,3′-triiodothyronine (T3) challenge and to a diet-induced growth suppression. Thyroxine (T4) outer-ring deiodination (T4ORD), T4 inner-ring deiodination (T4IRD), T3IRD, and 3,3′,5′-triiodothyronine (rT3)ORD activities were evident in liver and intestine. Their properties resembled those of teleosts. T3IRD and T4IRD activities predominated in brain. Low or negligible deiodination in any form occurred in gill, skeletal muscle, kidney, notochord, or immature gonad. Only T4ORD activity was evident in the thyroid, suggesting that it secretes some T3. T3ORD and rT3IRD activities were undetectable in any tissues. Hepatic T4ORD activity varied during the photophase and was highest during late morning. A dietary T3 challenge that doubled plasma T3 levels decreased hepatic T4ORD activity without altering any other deiodination pathways in liver, intestine, or brain. A diet change from trout pellets to ocean zooplankton reduced somatic growth and plasma T3 levels and increased hepatic and intestinal T3IRD activities and hepatic rT3ORD activity but did not alter hepatic or intestinal T4ORD activity. The authors conclude that plasma T3 in lake sturgeon can be derived both from the thyroid and from hepatic (and intestinal) T4ORD activity, which varies with sampling time and downregulates in response to a T3 challenge. However, a reduction in plasma T3 accompanying a change in diet and reduced growth was not due to a decrease in T4ORD activity; rather, it was due to an increase in hepatic and intestinal T3IRD activities. These results suggest a difference in emphasis in thyroidal regulation between sturgeon and certain teleosts.

Deiodination activity in extrathyroidal tissues of the atlantic hagfish, Myxine glutinosa.

We measured low substrate (<1 nM) thyroid hormone (TH) deiodination activities in liver, muscle, intestine, and brain microsomes of Atlantic hagfish fasted for 2 weeks and found extremely low thyroxine (T(4)) outer-ring deiodination (T(4)ORD) and inner-ring deiodination (T(4)IRD) as well as 3,5,3'-triiodothyronine (T(3)) IRD activities. T(3)ORD, 3',5'-triiodothyronine (rT(3)) ORD and rT(3)IRD activities were undetectable. Hagfish deiodinating pathways resembled those of teleosts in requiring a thiol cofactor (dithiothreitol, DTT) and in their inhibition by established deiodinase inhibitors and by TH analogues. However, under optimal pH and DTT conditions intestinal T(4)ORD activity exceeded that of liver about 10-fold. This contrasts with the situation in teleosts but resembles that reported recently in larval and adult lampreys, suggesting the intestine as a primary site of TH deiodination in lower craniates.

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 3' 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.

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 (IDI), i.e. by outer ring deiodination (ORD) to the active hormone T3' or by inner ring deiodination (IRD) to the inactive metabolite rT3. IDI 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 IDI (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 specificity was T3S > T2S approximately rT3 approximately T4 > T3 for dID1, and rT3 > > T2S approximately T3S > T4 approximately T3 for hID1. Whereas ORD of T4 and of T4, T3, and T3S showed relatively little variation between the different d/hID1 constructs, large differences were found for the ORD of rT3 and T2S. Both reactions were favored by the presence of the amino acids G, E and, in particular, F, present in hID1 at positions 45, 46, and 65, instead of the dID1 residues N, G, and L, respectively. However, although ORD of rT3 was not affected by the presence (hID1) or absence (dID1) of the TGMTR(48-52) sequence, the ORD of T2S was markedly inhibited by the presence of this sequence. Therefore, we have identified structural elements in ID1 that have substrate-specific impacts on deiodination. Our results suggest the specific interaction of the mono-substituted inner ring of the substrates rT3 and T2S but not the disubstituted inner ring of T3, T3S, or T4, with the aromatic ring of F65 in Id1, perhaps by pi-pi interactions.

Rainbow trout liver expresses two iodothyronine phenolic ring deiodinase pathways with the characteristics of mammalian types I and II 5'-deiodinases.

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-K(m) 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-K(m) 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 K(m) (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 adult vertebrate. Furthermore, the Vmax of this enzyme is as high as that found in comparable homogenates from hypothyroid mammalian tissues, whereas the Vmax of the type I activity is lower than that of mammalian liver. These findings are in consonance with the peculiar kinetics of T3 in trout liver, kinetics remarkably similar to those of the mammalian pituitary, cerebral cortex, and brown adipose tissue, which also preferentially express type II deiodinase.

Transition from a hatchery to a laboratory environment induces inner-ring monodeiodination pathways for thyroid hormones in liver of rainbow trout, Oncorhynchus mykiss

In laboratory-acclimated rainbow trout (Oncorhynchus mykiss) the main hepatic deiodination pathway for thyroid hormones is L-thyroxine (T4) outer-ring deiodination (T4ORD), which produces biologically active 3,5,3′-triiodo-L-thyronine (T3); T4 inner-ring deiodination (T4IRD) as well as T3ORD and T3IRD activities are low or undetectable. Surprisingly, trout transported 48 h previously from a local hatchery to the laboratory demonstrated not only low T4ORD activity but also significant T4IRD and T3IRD activities. To test if the transition from hatchery to laboratory environment had induced the unexpected inner-ring deiodinations, we measured hepatic deiodinase activities over the same time frame in trout recently transported to the laboratory and also in trout retained undisturbed at the hatchery. Undisturbed hatchery trout showed typical hepatic deiodinase function: T4ORD activity predominated, while T3IRD, T4IRD, and T3ORD activities were basal. However, after 1–3 days in the laboratory, hepatic T4ORD activity was reduced and T4IRD and T3IRD activities were increased. By 5 days, deiodinase activities of laboratory trout reverted to the levels of hatchery trout. We conclude that physical disturbance can temporarily depress thyroidal status by simultaneously decreasing hepatic production of biologically active T3 and inducing degradation of T4 and T3.

T4 depresses olfactory responses to L-alanine and plasma T3 and T3 production in smoltifying Atlantic salmon.

During natural or induced smoltification, Atlantic salmon exhibit spring elevations in olfactory activity and plasma L-thyroxine (T4). To determine whether T4 influences olfactory activity, we administered T4 to late parr in early April and measured olfactory bulb electroencephalogram and olfactory epithelium electroolfactorogram responses to L-alanine nasal stimulation, plasma T4 and 3,5,3'-triiodo-L-thyronine (T3) levels, and hepatic and brain monodeiodination. T4 treatment raised plasma T4 to 15 ng/ml, simulating the smolt T4 surge, and depressed plasma T3 and electroencephalogram responses, without modifying electroolfactorogram responses. Decreased plasma T3 may be explained by inhibition of hepatic T4 outer-ring deiodination, generating T3, and stimulation of inner-ring deiodination, degrading T4 and T3. Brain T4 outer-ring deiodination was also strongly inhibited. In conclusion, creation of a high plasma T4 concentration simulating the natural smolt peak depressed olfactory bulb responses to L-alanine due to the high T4 concentration or the depressed T3 availability in brain induced by T4. T4 may terminate the period of heightened olfactory responsiveness during smoltification.

Thyroid hormone deiodinase systems in salmonids, and their involvement in the regulation of thyroidal status

The trout thyroid secretes L-thyroxine (T4) which undergoes enzymatic deiodination in liver and other tissues. Based on mammalian studies, T4 outer-ring deiodination (ORD) or T4 inner-ring deiodination (IRD) could generate respectively 3,5,3'-triiodo-L-thyronine (T3) or 3,3',5'-T3(rT3), while subsequent T3ORD or T3IRD could generate respectively 3,5-diiodo-L-thyronine (T2) or 3,3'-T2, and rT3ORD or rT3IRD could generate respectively 3,3'-T2 or 3',5'-T2. In practice, T4 in trout undergoes hepatic ORD to produce T3 but negligible IRD to produce rT3, and T3 in turn undergoes negligible ORD but modest IRD to produce 3,3'-T2. T4ORD, which is particularly important in converting T4 to the biologically more potent T3, also occurs in gill, muscle and kidney. At least two isozymes are involved: i) a high-affinity, propylthiouracil (PTU)-sensitive T4ORD which displays ping-pong kinetics, requires thiol as a cofactor, and is present in liver, gill and muscle, and ii) a low-affinity, PTU-insensitive T4ORD with sequential kinetics with a thiol cofactor, and is present in liver and kidney. Receptor-bound T3 is derived primarily from the plasma for kidney, mainly from intracellular sources for gill and about equally from both plasma and intracellular sources for liver. Thus, the high-affinity T4ORD may produce T3 for local intracellular use while the low-affinity 5'-monodeiodinase may produce T3 for systemic use. T4ORD activity responds to nutritional factors and the physiologic state of the fish. Furthermore, T3 administered orally for either 6 weeks or 24h reduces the functional level (Vmax) of hepatic T4ORD, and T3 added to isolated hepatocytes also reduces activity, indicating direct T3 autoregulation of T4ORD to maintain hepatocyte T3 homeostasis. However, T3 administration also induces T4IRD to produce biologically inactive rT3 and induces T3IRD to produce 3,3'-T2. Thus, the trout liver has several iodothyronine deiodinase systems which in a coordinated manner regulate tissue T3 homeostasis in the face of a T3 challenge. It does this by decreasing formation of T3 itself, by diverting T4 substrate to biologically inactive rT3 and by increasing the degradation of T3. These deiodinases differ in many respects from any mammalian counterparts.

Stimulation of iodothyronine outer ring monodeiodinase by dihydrolipoamide.

The naturally occurring dithiol, dihydrolipoamide (DHL), has been found to be 6-10 times as potent as the synthetic dithiol, dithiothreitol, in stimulating iodothyronine outer ring monodeiodinase activity in the rat kidney. In the presence of NADH, the oxidized form is also active in stimulating the enzymatic activity in whole homogenates and in the postnuclear and mitochondrial, but not in the microsomal fraction. The covalently bound lipoamide in the mitochondrial alpha-keto acid dehydrogenase complexes, when reduced with substrates or NADH, was, however, ineffective. The activation of the enzyme by DHL was very similar to that obtained with dithiothreitol in respect to temperature dependence and inhibition by propylthiouracil and by dicoumarol, suggesting that these thiols activate the same enzyme through similar mechanisms. The higher potency of DHL cannot be explained on the basis of its greater lipophilicity or relatively proximal dithiol structure. The occurrence of DHL in mitochondria raises the possibility of a role as a thiol cofactor in enzymatic outer ring deiodination of T4 or rT3, although attempts to stimulate deiodination in mitochondrial preparations by reducing endogenous bound lipoamide were unsuccessful.

Rat placenta is an active site of inner ring deiodination of thyroxine and 3,3',5-triiodothyronine.

Inner ring deiodination of L-T4 and L-T3 by rat placental homogenates resulted in the generation of rT3 from T4 and 3,3'-diiodothyronine and 3'-monoiodothyronine from T3. Dithiothreitol is required in the incubation medium. There was little or no detectable outer ring deiodination of T4 and T3. These findings suggest that placenta enzymatic inner ring monodeiodination of T4 and T3 could prevent the transplacental passage of T4 and T3 from dam to fetus. They also provide an explanation for our previous observations that fetal serum rT3 is partially dependent on maternal thyroid function.

Proposed mechanism for increased thyroxine deiodination in cold-acclimated rats.

Groups of male rats were administered isoproterenol (ISO), thyroxine (T4), or both (ISO + T4) daily for 20 days in an attempt to mimic the effect of cold acclimation on the rate of outer-ring deiodination of T4 to triiodothyronine (T3) by 2,000-g supernatants of homogenates of liver and kidney. The rates of hepatic and renal deiodination of T4 to T3 in an additional group of rats exposed to 4 +/- 1 degree C for 20 days were 53 and 71% higher, respectively, than control. Administration of ISO (100 micrograms . kg-1 . day-1) did not affect the rate of deiodination of T4 to T3 by either hepatic or renal tissue. Administration of T4 (50 micrograms . kg-1 . day-1) resulted in rates of hepatic and renal deiodination of T4 that were 297 and 222% higher, respectively, than control. Administration of ISO + T4 resulted in a rate of conversion of T4 to T3 not significantly different from that observed when T4 was administered alone. Serum concentration of T4 was elevated after administration of both T4 and ISO + T4, whereas serum concentration of T3 was elevated significantly above that of control only in the cold-acclimated group. These results suggest that the increased rate of 5'-monodeiodination of T4 by hepatic and renal homogenates from cold-acclimated rats is not a result of increased beta-adrenergic activity but can be accounted for by the increase in thyroid activity observed in these animals.