Physiological role and regulation of iodothyronine deiodinases: a 2011 update.

T4 is a prohormone secreted by the thyroid. T4 has a long half life in circulation and it is tightly regulated to remain constant in a variety of circumstances. However, the availability of iodothyronine selenodeiodinases allow both the initiation or the cessation of thyroid hormone action and can result in surprisingly acute changes in the intracellular concentration of the active hormone T3, in a tissue- specific and chronologically-determined fashion, in spite of the constant circulating levels of the prohormone. This fine-tuning of thyroid hormone signaling is becoming widely appreciated in the context of situations where the rapid modifications in intracellular T3 concentrations are necessary for developmental changes or tissue repair. Given the increasing availability of genetic models of deiodinase deficiency, new insights into the role of these important enzymes are being recognized. In this review, we have incorporated new information regarding the special role played by these enzymes into our current knowledge of thyroid physiology, emphasizing the clinical significance of these new insights.

Iodide transport: inhibition by agents reacting at the membrane.

Accumulation of iodide by thyroid tissue is inhibited by two phospholipase A-free proteins from cobra venom, filipin, crude phospholipase C, and lysolecithin. The venom proteins decrease K(+) in tissue but do not significantly affect incorporation of phosphorus-32 into phospholipid or stimulation of this process by thyrotropin. However, filipin and crude phospholipase C, like thyrotropin, do increase phospholipid formation.

Pituitary nuclear 3,5,3'-triiodothyronine and thyrotropin secretion: an explanation for the effect of thyroxine.

An excellent correlation was observed between nuclear triiodothyronine (T3) and the ensuing suppression of thyrotropin (TSH) after a single intravenous injection of T3 to thyroidectomized (hypothyroid) rats. At 1 and 2 hours after injection of thyroxine (T4), in amounts equally potent to the administered T3 in terms of acute suppression of TSH, the same quantities of T3 were found in the pituitary nuclei. Virtually no nuclear T4 was present, and plasma T3 was negligible at these short intervals after T4 injection. These results suggest that suppression of TSH release in hypothyroid rats occurs by interaction of T3 with the nuclear receptor of the thyrotroph. After T4 injection, the T3 found in the nucleus is derived from rapid intrapituitary monodeiodination.

Pyrazole-induced thyroid necrosis: a distinct organ lesion.

One oral dose of pyrazole caused necrosis of rat thyroid follicular epithelial cells but spared the parafollicular (C) cells and the parathyroid glands. Serum thyroxine (T4) and triiodothyronine (T3) were significantly decreased on day 3 after pyrazole administration and were immeasurable on day 5. At day 5 the thyroid was enlarged and the concentration of thyroid-stimulating hormone in the serum was increased, indicating an appropriate pituitary response to a primary lesion in the thyroid. Doses of pyrazole which produced no morphologic change in the thyroids also significantly depressed the concentrations of T4 and T3 in the serum.

Cerebral cortex responds rapidly to thyroid hormones.

In rats subjected to thyroidectomy there was a two- to fourfold increase in cerebral cortex iodothyronine 5'-deiodinase activity within 24 hours. This increase was prevented by thyroxine replacement. The increased cortical 5'-deiodinase in chronically hypothyroid rats was normalized within 4 hours by a single intravenous injection of triiodothyronine. These results indicate that the adult central nervous system can give a very rapid biochemical response to thyroid hormone.

Identification of a thyroid hormone receptor that is pituitary-specific.

Three cellular homologs of the v-erbA oncogene were previously identified in the rat; two of them encode high affinity receptors for the thyroid hormone triiodothyronine (T3). A rat complementary DNA clone encoding a T3 receptor form of the ErbA protein, called r-ErbA beta-2, was isolated. The r-ErbA beta-2 protein differs at its amino terminus from the previously described rat protein encoded by c-erbA beta and referred to as r-ErbA beta-1. Unlike the other members of the c-erbA proto-oncogene family, which have a wide tissue distribution, r-erbA beta-2 appears to be expressed only in the anterior pituitary gland. In addition, thyroid hormone downregulates r-erbA beta-2 messenger RNA but not r-erbA beta-1 messenger RNA in a pituitary tumor-derived cell line. The presence of a pituitary-specific form of the thyroid hormone receptor that may be selectively regulated by thyroid hormone could be important for the differential regulation of gene expression by T3 in the pituitary gland.

Salicylate-induced increases in free triiodothyronine in human serum. Evidence of inhibition of triiodothyronine binding to thyroxine-binding globulin and thyroxine-binding prealbumin.

Addition of sodium salicylate to human serum at concentrations often obtained during aspirin therapy causes 100-200% increases in free triiodothyronine (T(3)) and free thyroxine (T(4)) as estimated by ultrafiltration. The increase in free T(3) was unexpected since previous data had suggested that salicylate inhibits binding of T(4) only to thyroxine-binding prealbumin (TBPA) and that T(3) is not bound to this protein. Using ultrafiltration techniques, we demonstrated binding of T(3) to TBPA. The affinity constant for T(3)-TBPA binding appears to be slightly greater than that for albumin-T(3) binding. While salicylate inhibits the binding of T(3) (and T(4)) to TBPA, it can be predicted that little change will be observed in the free T(3) (or free T(4)) without inhibition of thyroid hormone binding to thyroxine-binding globulin (TBG). Using a competitive-binding protein displacement technique, it has been shown that sodium salicylate, like diphenylhydantoin (DPH), inhibits the binding of T(3) and T(4) to TBG. The magnitude of the increase in free T(3) and free T(4) induced by salicylates suggests that interference with TBG binding is its major effect. Aspirin was administered orally to two normal subjects in quantities sufficient to obtain serum salicylate levels of 20-25 mg/100 ml. This resulted in a decrease of 20-30% in total serum T(3) and T(4) levels. This decrease in T(4) levels is similar in magnitude to that previously observed in subjects receiving DPH. Unlike what has been observed with DPH treatment, therapeutic salicylate levels are associated with increases of 50-75% in the unbound fraction of both T(3) and T(4) which persist throughout an 8-10 day treatment period.

Direct immunoassay of triiodothyronine in human serum.

A sensitive and precise radioimmunoassay for the direct measurement of triiodothyronine (T(3)) in human serum has been designed using sodium salicylate to block T(3)-TBG binding. This assay is sufficiently sensitive to quantitate T(3) accurately in 50-100 mul of normal serum and to measure quantities as small as 12.5 pg in 0.2 ml of hypothyroid serum. The T(3) values observed in euthyroid subjects and in patients with various thyroid diseases are as follows: euthyroid (38) 1.10+/-0.25 (SD) ng/ml, hypothyroid (25) 0.39+/-0.21 (SD) ng/ml, and hyperthyroid (24) 5.46+/-4.42 (SD) ng/ml. The levels of T(3) parallel the thyroxine (T(4)) concentration in the sera of these subjects. In eight pregnant women at the time of delivery, T(3) concentrations were in the upper normal range (mean 1.33 ng/ml). The levels of T(3) in cord serum obtained at the time of delivery of these patients (mean 0.53 ng/ml) are distinctly lower and close to the hypothyroid mean. Administration of 10 U of bovine thyroid-stimulating hormone (TSH) to euthyroid subjects causes a two-fold increase in serum T(3) levels within 8 hr. At this time, the increase in serum T(4) concentration is only 41%. In two subjects in whom thyroid secretion was acutely inhibited, one after pituitary surgery and another after thyroidectomy, the serum T(3) fell into the hypothyroid range within 1-2 days. Thus, serum T(3) concentrations appear to be a sensitive index of acute changes in thyroid hormone secretion and should prove to be a useful adjunct to both the clinical and physiological evaluation of thyroid function.

Potential of brown adipose tissue type II thyroxine 5'-deiodinase as a local and systemic source of triiodothyronine in rats.

Previous reports suggest that a type II iodothyronine 5'-deiodinase may become the main enzymatic pathway for extrathyroidal triiodothyronine (T3) generation when the enzyme levels are sufficiently elevated and/or liver and kidney type I 5'-deiodinase activity is depressed. The present studies assessed the potential of brown adipose tissue (BAT) type II 5'-deiodinase to generate T3 for the plasma pool. BAT 5'-deiodination (BAT 5'D) was stimulated by either short- (4 h) or long-term (7 wk) cold exposure (4 degrees C). Long-term cold exposure increased thyroxine (T4) secretion 40-60% and extrathyroidal T3 production three-fold. In cold-adapted rats treated with propylthiouracil (PTU), extrathyroidal T3 production was 10-fold higher than in PTU-treated rats maintained at room temperature. Cold did not stimulate liver or kidney 5'D, but the cold-adapted rats showed a six- to eightfold higher BAT 5'D content. PTU caused greater than 95% inhibition of liver and kidney 5'D, but did not affect BAT 5'D. Thyroidectomized rats maintained on 0.8 micrograms of T4/100 g of body weight (BW) per day were acutely exposed to 4 degrees C. In rats given 10 mg of PTU/100 g of BW, 4 h of cold exposure still caused a 12-fold increase in BAT 5'D, a 2.3-fold increase in plasma T3 production, and a 4.8-fold increment in the locally produced T3 in BAT itself. All these responses were abolished by pretreatment with the alpha 1-antiadrenergic drug prazosin. Regardless of the ambient temperature, liver 5'D activity was greater than 90% inhibited by PTU. These results indicate that BAT can be a major source of plasma T3 under suitable circumstances such as acute or chronic exposure to cold. Furthermore, BAT 5'D activity affects BAT T3 content itself, suggesting that thyroid hormone may have a previously unrecognized role in augmenting the thermogenic response of this tissue to sympathetic stimulation. Such interactions may be especially important during the early neonatal period in humans, a time of marked thermogenic stress.

Type 2 iodothyronine deiodinase in rat pituitary tumor cells is inactivated in proteasomes.

The goal of these studies was to define the rate-limiting steps in the inactivation of type 2 iodothyronine deiodinase (D2). We examined the effects of ATP depletion, a lysosomal protease inhibitor, and an inhibitor of actin polymerization on D2 activity in the presence or absence of cycloheximide or 3,3', 5'-triiodothyronine (reverse T3, rT3) in rat pituitary tumor cells (GH4C1). We also analyzed the effects of the proteasomal proteolysis inhibitor carbobenzoxy- L-leucyl-L-leucyl-L-leucinal (MG132). The half-life of D2 activity in hypothyroid cells was 47 min after cycloheximide and 60 min with rT3 (3 nM). rT3 and cycloheximide were additive, reducing D2 half-life to 20 min. D2 degradation was partially inhibited by ATP depletion, but not by cytochalasin B or chloroquine. Incubation with MG132 alone increased D2 activity by 30-40% for several hours, and completely blocked the cycloheximide- or rT3-induced decrease in D2 activity. These results suggest that D2 is inactivated by proteasomal uptake and that substrate reduces D2 activity by accelerating degradation through this pathway. This is the first demonstration of a critical role for proteasomes in the post-translational regulation of D2 activity.

Triiodothyronine, thyroxine, and iodine in purified thyroglobulin from patients with Graves' disease.

Previous studies have suggested that there is an overproduction of triiodothyronine (T(3)) relative to thyroxine (T(4)) in patients with thyrotoxicosis associated with Graves' disease. To evaluate whether or not an increased ratio of T(3) to T(4) in thyroidal secretion could be contributing to this relative T(3) hyperproduction, T(3), T(4), and iodine were measured in thyroglobulin (Tg) from controls and patients with Graves' disease who had been treated either with propranolol only or with antithyroid drugs plus iodide before surgery. To avoid possible artifacts associated with pulse labeling and chromatography, T(3) and T(4) were determined by radioimmunoassay of Pronase hydrolysates of purified Tg. Results of analyses of Tg from six control patients and seven with Graves' disease, not receiving thiourea drugs or iodide, showed that the iodine content of Graves' disease Tg was not different from normal. Both contained 3.4 residues of T(4)/molecule Tg, but there was 0.39+/-0.08 (mean+/-SD) residue of T(3)/molecule Tg in Graves' Tg as opposed to 0.23+/-0.07 residue T(3) molecule Tg in controls matched for iodine content (P < 0.01). This difference resulted in a significantly lower T(4)/T(3) molar ratio (9+/-2) in Graves' Tg as opposed to control (15+/-2, P < 0.001). In Tg from patients with treated Graves' disease, iodine, T(3), and T(4) were reduced, but the reduction in the latter was more substantial, resulting in a T(4)/T(3) molar ratio of 3.4+/-1. Fractionation of Tg from all groups by RbCl density gradient ultracentrifugation indicated that at physiological levels of Tg iodination, the molar ratio of T(3)/Tg was consistently higher in Graves' disease. The specific mechanism for this difference is not known, but it is not due to iodine deficiency. If T(3) and T(4) are secreted in this altered ratio in patients with Graves' disease, the magnitude of the difference could explain the relative T(3) hyperproduction which is characteristic of this state.

Contributions of plasma triiodothyronine and local thyroxine monodeiodination to triiodothyronine to nuclear triiodothyronine receptor saturation in pituitary, liver, and kidney of hypothyroid rats. Further evidence relating saturation of pituitary nuclear triiodothyronine receptors and the acute inhibition of thyroid-stimulating hormone release.

Injections of triiodothyronine (T(3)) and thyroxine (T(4)) into chronically hypothyroid rats were used to evaluate the contribution of intracellular T(4) to T(3) conversion to nuclear T(3) in pituitary, liver, and kidney, and to correlate the occupancy of pituitary nuclear T(3) receptors with inhibition of thyroid-stimulating hormone (TSH) release. Injection of a combination of 70 ng T(3) and 400 ng T(4)/100 g body wt resulted in plasma T(3) concentrations of 45+/-7 ng/dl (mean+/-SD) and 3.0+/-0.4 mug/dl T(4) 3 h later. At that plasma T(3) level, the contribution of plasma T(3) to the nuclear receptor sites resulted in saturation of 34+/-7% for pituitary, 27+/-5% for liver, and 33+/-2% for kidney. In addition to the T(3) derived from plasma T(3), there was additional T(3) derived from intracellular monodeiodination of T(4) in all three tissues that resulted in total nuclear occupancy (as percent saturation) of 58+/-11% (pituitary), 36+/-8% (liver), and 41+/-11% (kidney), respectively. The percent contribution of T(3) derived from cellular T(4) added 41% of the total nuclear T(3) in the pituitary which was significantly higher than the contribution of this source in the liver (24%) or the kidney (19%). 3 h after intravenous injection of increasing doses of T(3), the plasma T(3) concentration correlated well with both the change in TSH and the nuclear occupancy, suggesting a linear relationship between the integrated nuclear occupancy by T(3) and TSH release rate. The contribution of intrapituitary T(4) to T(3) conversion to nuclear T(3) was accompanied by an appropriate decrease in TSH, supporting the biological relevance of nuclear T(3). Pretreatment of the animals with 6-n-propylthiouracil before T(4) injection decreased neither the nuclear T(3) derived from intrapituitary T(4) nor the subsequent decrease in TSH. These results indicate that intracellular monodeiodination of T(4) contributes substantially to the nuclear T(3) in the pituitary of the hypothyroid rat, and suggest a linear inverse relationship between nuclear receptor occupancy by T(3) in the pituitary and TSH release rate. The data further indicate that T(4) to T(3) monodeiodination is considerably more important as a source of nuclear T(3) in the pituitary than in the liver and kidney. This provides a mechanism whereby the TSH secretion could respond promptly to a decrease in thyroid secretion (predominantly T(4)) before a decrease in plasma T(3) would be expected to lead to significant metabolic hypothyroidism.

Triiodothyronine and thyroxine in hyperthyroidism. Comparison of the acute changes during therapy with antithyroid agents.

In 66 untreated patients with hyperthyroidism, serum triiodothyronine (T(3)) and thyroxine (T(4)) concentrations were measured by immunoassay. The mean T(3) level was 478+/-28 ng/100 ml (all values mean+/-SEM) and the T(4) was 20.6+/-0.6 mug/100 ml. The serum T(4)/T(3) ratio by weight was 48+/-2 as opposed to a value of 71+/-3 in euthyroid adults. There was a significant inverse correlation of the T(4)/T(3) ratios with serum T(3) (r=0.77; P<0.01) but not with serum T(4)(r=0.21). These results suggested that relative overproduction of T(3) is consistently present in patients with hyperthyroidism. To examine the acute effects of various antithyroid agents on serum T(3) and T(4) concentrations, iodide, propylthiouracil (PTU), and methylmercaptoimidazole (MMI) were given alone to mine patients, and serial T(3) and T(4) measurements were made. There was an acute decrease in serum T(3) over the first 5 days in the three iodide and three PTU-treated patients which was greater than that seen in the MMI group. This suggested that PTU and MMI had different effects on T(3) production. To compare the effects of PTU and MMI under conditions in which thyroidal hormone release was minimized, these drugs were given in combination with iodide. The mean daily dosage of PTU was 827 (n=11) and of MMI was 88 (n=8). In the PTU+iodide group, the initial serum T(3) concentration was 586+/-61 ng/100 ml and decreased significantly to 326+/-41 on day 1 and to 248+/-21 on days 2 and 3, respectively, and did not change further on days 4 and 5. In the MMI + iodide group, basal serum T(3) was 645+/-90 ng/100 ml and decreased to 568+/-81, 452+/-73, and 344+/-51 on days 1, 2, and 3, respectively, and did not change thereafter. While the initial T(3) concentrations in serum were not different in the PTU and MMI groups, the T(3) concentrations in the PTU patients were significantly lower on days 1 and 2 and during the apparent plateau period on days 3-5. Serum T(4) concentrations decreased gradually in both groups, from 23.9+/-2.0 mug/100 ml, initially, to 17.5+/-1.6 on day 5 in the PTU group and from 22.0+/-2.6 to 14.6+/-2.0 in the MMI-treated patients. The T(4) values were not significantly different at any time. These changes resulted in increases in the serum T(4)/T(3) ratios in both groups, but these ratios were substantially higher in the patients treated with PTU + iodide. The initial serum T(4)/T(3) ratio was 43+/-3 and increased to 74+/-7 and 88+/-7 on days 1 and 2 in the PTU group, reaching a plateau value of 91+/-7 during days 3-5. Comparable values for MMI-treated patients were 35+/-2, 42+/-3, 52+/-6, and 54+/-3 during the plateau period. Previous investigations have shown that PTU inhibits T(4) deiodination in hyperthyroid patients and decreases T(3) production from T(4) in animals. The greater acute decrease in serum T(3) and the higher serum T(4)/T(3) ratios in the PTU-treated patients seems best explained by an inhibition of peripheral T(3) production by this agent. This conclusion is further supported by a direct relationship between the T(4)/T(3) ratio on days 3-5 and the dose of PTU administered. These results further suggest that both thyroidal and extrathyroidal pathways contribute substantially to the apparent overproduction of T(3) in hyperthyroidism.

Triiodothyronine and thyroxine in the serum and thyroid glands of iodine-deficient rats.

Triiodothyronine (T(3)) and thyroxine (T(4)) were measured by immunoassay in the serum and thyroid hydrolysates of control (group A), mildly iodine-deficient (group B), and severely iodine-deficient rats (group C). These results were correlated with changes in thyroidal weight, (131)I uptake and (127)I content as well as with the distribution of (131)I in Pronase digests of the thyroid. There was a progressive increase in thyroid weight and (131)I uptake at 24 h with decrease in iodine intake. The (127)I content of the thyroids of the group B animals was 44% and that of the group C animals 2% of that in group A. The mean labeled monoiodotyrosine/diiodotyrosine (MIT/DIT) and T(3)/T(4) ratios in group A were 0.42+/-0.07 (SD) and 0.12+/-0.01, 0.59+/-0.06 and 0.11+/-0.03 in group B, and 2.0+/-0.3 and 1.8+/-0.9 in the group thyroid digests.Mean serum T(4) concentration in the control rats was 4.2+/-0.6 (SD) mug T(4)/100 ml, 4.5+/-0.3 mug/100 ml in group B animals, and undectectable (<0.5 mu(4)/100 ml) in group C animals. There was no effect of iodine deficiency on serum T(3) concentrations, which were 44+/-9 (Mean+/-SD) ng/100 ml in A animals, 48+/-6 ng/100 ml n B animals, and 43+/-6 ng/100 ml in the C group. Thyroidal digest T(3) and T(4) concentrations were 39 and 400 ng/mg in group A animals and were reduced to 5 and 1% of this, respectively, in group C. The molar ratio of T(3)/T(4) in the thyroid digests of the groups A and B animals was identical to the ratio of labeled T(3)/T(4) and was slightly less (1.0+/-0.9) than the labeled T(3)/T(4) ratio in the group C animals. The mean ratio of labeled T(4) to labeled T(3) in the serum of the severely iodine-deficient animals 24 h after isotope injection was 11+/-1 (SEM). With previously published values, it was possible to correlate the ratio of labeled T(4)/T(3) in the thyroid digest with the labeled T(4)/T(3) ratio in the serum of each iodine-deficient animal. This analysis suggested that the labeled thyroid hormones in the severely iodine-deficient rat were secreted in the ratio in which they are present in the gland. Kinetic analysis of total iodothyronine turnover indicated that two-thirds of the T(3) utilized per day by the iodine-sufficient rat arises from T(4). If the T(4)-T(3) conversion ratio remains the same in iodine deficiency, then the analysis suggests that about 90% of the T(3) arises directly from the thyroid. Therefore, it would appear that absolute T(3) secretion by the thyroid increases severalfold during iodine deficiency. The fact that serum T(3) remains constant and T(4) decreases to extremely low levels, combined with previous observations that iodine-deficient animals appear to be euthyroid, is compatible with the hypothesis that T(4) in the normal rat serves primarily as a precursor of T(3).

Comparison of iodothyronine 5'-deiodinase and other thyroid-hormone-dependent enzyme activities in the cerebral cortex of hypothyroid neonatal rat. Evidence for adaptation to hypothyroidism.

Recent studies have shown that approximately 75% of the nuclear 3,5,3'-triiodothyronine (T(3)) present in adult rat cerebral cortex (Cx) derives from 5'-deiodination of thyroxine (T(4)) within this tissue. The activity of iodothyronine 5'-deiodinase (I 5'D), the enzyme catalyzing T(4) to T(3) conversion, increases rapidly after thyroidectomy, suggesting that this could be a compensatory response to hypothyroxinemia. To evaluate this possibility during the period of central nervous system maturation, we studied several thyroid hormone-responsive enzymes (aspartic transaminase [AT], succinic dehydrogenase [S.D.], and Na/K ATPase) in the Cx of 2-, 3-, and 4-wk-old rats. The rats were made congenitally hypothyroid by placing 1, 2, 5, and 20 mg methimazole (MMI) in 100 ml of the mothers' drinking water from day 16 of gestation throughout the nursing period and to the litters after weaning. In addition, serum thyroid hormones, I 5'D, and, in some experiments, in vivo T(4) to T(3) conversion in Cx were measured in the same pups. Serum T(4) concentrations varied from <1 to 40 ng/ml and were generally inversely related to maternal MMI dose. Serum T(3) was less affected by MMI than was T(4). At 2 wk, decreases in AT, S.D., and ATPase were present in the 20-mg-MMI group but not in the 5-mg-MMI pups despite low serum T(4) (<10 ng/ml) in the latter. At 3 and 4 wk, both 5- and 20-mg-MMI groups had significant reductions in these cortical enzymes despite a normal serum T(3) in the 5-mg-MMI rats. Cortical I 5'D activity was 10-fold the control value in 5- and 20-mg-MMI animals at 2 wk but increased only three- to fivefold at 3 and 4 wk. I 5'D correlated inversely with serum T(4) (r >/= 0.96) at all ages, but the less marked elevation of this enzyme in 3- and 4-wk-old pups was not accompanied by an increase in serum T(4). Serum T(3) increased or remained the same between 2 and 3 wk. These results suggested that the 10-fold increase in I 5'D at 2 wk protected the 5-mg-MMI group from tissue hypothyroidism, but that the three- to fivefold increase at 3 and 4 wk could not. Injection of approximately 250 ng T(4)/100 g body weight to 2-wk-old, 20-mg-MMI pups (one-sixth the normal T(4) production rate) led to both a 1.8-ng/g cortical tissue increment in cortical T(3) and a significant increase in AT at 24 h, compared with a 0.38-ng/g cortical tissue T(3) increment and no change in AT in euthyroid controls. The larger increment in T(3) of the 20-mg-MMI pups was due in great part to increased fractional T(4) to T(3) conversion. Although the latter resulted in greater serum T(3) concentrations, three-fourths of the newly formed T(3) in the cortex was generated in situ, and it was blocked by iopanoic acid as was the increase in AT. We conclude that 70-80% of the T(3) in the Cx of the neonatal rat is produced locally. Serum T(4) appears to serve both as a precursor for T(3) and as a critical signal for increases in cortical I 5'D. The increased I 5'D can result in normal or near-normal cerebrocortical T(3) concentrations despite marked reductions in serum T(4). This mechanism seems to be particularly effective around 2 wk of age when many thyroid-hormone-dependent maturational changes occur in the rat Cx.

Rapid thyroxine to 3,5,3'-triiodothyronine conversion and nuclear 3,5,3'-triiodothyronine binding in rat cerebral cortex and cerebellum.

Thyroxine (T(4)) to 3,5,3'-triiodothyronine (T(3)) conversion was evaluated in vivo in cerebral cortex, cerebellum, and anterior pituitary of male euthyroid Sprague-Dawley rats. Tracer quantities of (125)I-T(4) and (131)I-T(3) were injected into controls and iopanoic acid-pretreated rats 3 h before isolation of nuclei from these tissues. Specifically-bound nuclear (131)I-T(3), denoted T(3)(T(3)); (125)I-T(3), denoted T(3)(T(4)); and (125)I-T(4) were extracted and identified by chromatography. Plasma iodothyronines were similarly quantitated. In control rats, nuclear T(3)(T(3)) (percent dose per milligram DNA x 10(-4)) was 174+/-31 in cerebral cortex, 50+/-9 in cerebellum, and 932+/-158 in pituitary (all values, mean+/-SEM). Nuclear T(3)(T(4)) (percent dose per milligram DNA x 10(-4)) was 23.3+/-3.3 in cortex, 3.5+/-0.6 in cerebellum, and 39.4+/-6.9 in pituitary. Two-thirds of nuclear T(3)(T(4)) derived from local T(4) to T(3) conversion. Nuclear T(3)(T(4)) in all tissues was reduced to less than 15% of its control value by iopanoic acid treatment and all of the residual nuclear T(3)(T(4)) could be accounted for by plasma T(3)(T(4)). Nuclear T(3)(T(3)) binding was not inhibited by iopanoic acid. These results indicate there is rapid local T(4) to T(3) conversion in rat brain and nuclear binding of the T(3) produced. We have previously found that local T(3)(T(4)) production is the source of approximately 50% of the T(3) in rat anterior pituitary. The present observations that the ratio of locally derived nuclear T(3)(T(4)) to nuclear T(3)(T(3)) is much higher in cerebral cortex (0.1) and cerebellum (0.04) than in anterior pituitary (0.015) suggest that this locally produced T(3)(T(4)) is the predominant source of intracellular T(3) in these portions of rat brain.

Interrelationships among thyroxine, growth hormone, and the sympathetic nervous system in the regulation of 5'-iodothyronine deiodinase in rat brown adipose tissue.

Thyroxine (T4) and reverse triiodothyronine are potent inhibitors of brown adipose T4 5'-deiodinase (BAT 5'D). This effect does not require protein synthesis and is due to an acceleration of the rate of disappearance of the enzyme. Growth hormone (GH) also inhibits BAT 5'D but by a mechanism mediated through a long-lived messenger that correlates with growth rate. This explains the failure of BAT 5'D to increase abruptly after thyroidectomy as does the type II 5'-deiodinase in pituitary and central nervous system or the BAT 5'D itself after hypophysectomy. Although virtually inactive when given acutely, triiodothyronine replacement partially reduces BAT 5'D in hypophysectomized and thyroidectomized (Tx) animals probably as a result of improvement of systemic hypothyroidism and an increase in GH levels in the Tx rats. The fine balance between these inhibitory factors and the stimulatory effects of the sympathetic nervous system suggests an important physiologic role for the enzyme in this tissue.

Serum triiodothyronine and thyroxine in the neonate and the acute increases in these hormones following delivery.

Low triiodothyronine (T(3)) and high normal thyroxine (T(4)) concentrations are present in cord sera from full term infants. To examine this phenomenon further, radioimmunoassay of T(3) and T(4) was carried out in paired maternal and cord sera as well as capillary sera from neonates at different intervals after delivery. Free T(3) and free T(4) concentrations were also estiamted in cord and maternal sera by equilibrium dialysis. In 12 paired specimens, the T(3) concentration in cord sera was significantly lower than the maternal level (51+/-4 vs. 161+/-11 ng/100 ml, mean +/-SE). Mean free T(3) concentration was also lower in the cord samples (0.15+/-0.02 vs. 0.31+/-0.04 ng/100 ml). whereas total and free T(4) concentrations were not significantly different. Umbilical vein and artery samples from 11 neonates did not differ significantly in their T(3) and T(4) concentrations. In seven infants the mean T(3) concentration increased from 51+/-3 ng/100 ml at delivery to 79+/-13 at 15 min and 191+/-16 at 90 min. In four other infants the mean T(3) concentration at 24 and 48 h was not significantly different from the 90 min value of the previous group. Less pronounced changes were observed for T(4) which increased from 12.3+/-2.0 mug/100 ml (mean +/-SE) at delivery to 14.1+/-1.9 at 90 min and appeared to have reached a plateau at approximately twice the cord value by 24-48 h after delivery.The maternal-fetal gradient observed for free T(3) is further evidence of the autonomy of the fetal thyroidpituitary axis. The time course of the abrupt increase in serum T(3) in the neonate suggests that it results from the earlier acute increase in serum TSH which occurs shortly after birth. This suggests that the neonatal thyroid contains significant quantities of T(3). Therefore, unavailability of thyroidal T(3) does not appear to explain the low total and free T(3) concentrations present in the sera of newborns.

Physiological and pharmacological influences on thyroxine to 3,5,3'-triiodothyronine conversion and nuclear 3,5,3'-triiodothyronine binding in rat anterior pituitary.

Our recent in vivo studies have suggested that intrapituitary l-thyroxine (T(4)) to 3,5,3'-triiodo-l-thyronine (T(3)) conversion with subsequent nuclear binding of T(3) is an important pathway by which circulating T(4) can inhibit thyrotropin release. The present studies were performed to evaluate various physiological and pharmacological influences on these two processes in rat anterior pituitary tissue. Intact pituitary fragments were incubated in buffer-1% bovine serum albumin containing 0.14 ng/ml [(131)I]T(3) and 3.8 ng/ml [(125)I]T(4). Nuclei were isolated after 3 h of incubation and the bound iodothyronines identified by paper chromatography. There was 0.3-1% [(125)I]T(3) contaminating the medium [(125)I]T(4), and this did not change during incubation. Nuclear [(125)I]T(4) was not decreased by 650-fold excesses of medium T(3) or T(4), suggesting that it was nonspecifically bound. The ratio of nuclear to medium [(131)I]- and [(125)I]T(3) were expressed as nuclear counts per minute per milligram wet weight of tissue:counts per minute per microliter medium. Intrapituitary T(4) to T(3) conversion was evidenced by the fact that the nuclear:medium (N:M) ratio for [(131)I]T(3) was 0.45+/-0.21, whereas that for [(125)I]T(3) was 2.23+/-1.28 (mean+/-SD, n = 51). A ratio (R), the N:M [(125)I]T(3) divided by the N:M [(131)I]T(3), was used as an index of intrapituitary T(4) to T(3) conversion. Increasing medium T(3) concentrations up to 50 ng/ml caused a progressive decrease in the N:M ratio for both T(3) isotopes, but no change in the value for R, indicating that both competed for the same limited-capacity nuclear receptors. Increasing concentrations of medium T(4) caused no change in the N:M [(131)I]T(3) but did cause a significant decrease in R in three of four experiments. These results suggest saturation of T(4)-5'-monodeiodination occurred at lower T(4) concentrations than saturation of nuclear T(3) binding sites. In hypothyroid rats, the N:M ratios for both [(131)I]T(3) and [(125)I]T(3) were increased (P < 0.005), but R was three-fold higher than in controls (P < 0.005). Animals given 10 mug T(4)/100 g body wt per d for 5 d had significantly decreased N:M ratios for both [(131)I]T(3) and [(125)I]T(3), as well as a decreased value for R. In fasted rats, neither N:M ratio was depressed, although hepatic T(4) to T(3) conversion in the same animals was 50% of control (P < 0.005). Iopanoic acid (13 muM), but not 6-n-propylthiouracil (29 muM), decreased the N:M [(125)I]T(3) with a significant decrease in the value for R (P < 0.025 or less). Neither sodium iodide (6 muM) nor thyrotropin-releasing hormone (7-700 nM) affected the T(3) N:M ratios. These results indicate that intrapituitary T(4) to T(3) conversion is stimulated in hypothyroidism and depressed in T(4)-treated animals, whereas opposite changes occur in hepatic T(4)-5'-monodeiodination. Unlike liver, anterior pituitary T(4)-5'-monodeiodination is not affected by fasting or incubation with 6-n-propyl-2-thiouracil, but T(4) to T(3) conversion is inhibited in both by iopanoic acid. These results indicate that there are important differences between anterior pituitary and other tissues in the regulation of T(4)-5'-monodeiodination.

Evidence for two pathways of iodothyronine 5'-deiodination in rat pituitary that differ in kinetics, propylthiouracil sensitivity, and response to hypothyroidism.

We have studied 5'-deiodination of thyroxine (T(4)) and 3,3',5'-triiodothyronine (rT(3)) in rat pituitary tissue in vitro, with respect to substrate specificity, reaction kinetics, effects of 6-n-propyl-2-thiouracil (PTU), and the time course of effects of thyroid hormone depletion and repletion. Removal of one phenolic iodine or both tyrosyl iodines from the T(4) molecule resulted in compounds that were not deiodinated, but alterations in the alanine side chain had little effect.5'-Deiodination of 2 nM rT(3) by pituitary microsomes from euthyroid rats was inhibited >90% by 1 mM PTU, but was inhibited <10% by 100 nM T(4). The apparent Michaelis constant (K(m)) and maximum velocity (V(max)) for rT(3) at 20 mM dithiothreitol (DTT) were 33 nM and 84 pmol/mg protein per h. This reaction followed ping-pong type reaction kinetics when concentrations of DTT were varied. PTU inhibition was competitive with DTT and uncompetitive with rT(3). In contrast, when pituitary microsomes from hypothyroid rats (21 d postthyroidectomy) were used, deiodination of 2 nM rT(3) was inhibited only 20% by 1 mM PTU and up to 80% by 100 nM T(4). At 20 mM DTT, the apparent K(m) and V(max) in hypothyroid microsomes were 4.7 nM rT(3) and 16 pmol/mg protein per h. T(4) was a competitive inhibitor of PTU-insensitive rT(3) 5'-deiodination (K(i) = 1.3 nM). T(4) 5'-deiodination by hypothyroid microsomes was not affected by PTU, was competitively inhibited by rT(3) (K(i), 1.7 nM), and exhibited sequential type reaction kinetics with DTT as cosubstrate. When T(4) 5'-deiodination was measured in euthyroid and hypothyroid microsomes, respectively, the apparent K(m) and V(max) for T(4) at 20 mM DTT, were 0.9 nM and 0.55 pmol/mg protein per h (euthyroid), and 0.8 nM and 6.9 pmol/mg protein per h (hypothyroid). The T(4) 5'-deiodination rate and the PTU-insensitive, but not total, rT(3) 5'-deiodination rate (i.e. measured in the presence and the absence of 1 mM PTU, respectively) in pituitary homogenates were significantly elevated 24 h after thyroidectomy. PTU-insensitive activity continued to increase until at >/=30 d after thyroidectomy it was 11 times the PTU-insensitive activity in controls. At the latter time, PTU-sensitive rT(3) 5'-deiodinase activity appeared to be decreased. The increase in PTU-insensitive T(4) and rT(3) 5'-deiodination observed 48 h after thyroidectomy was prevented by replacement doses of T(4) or T(3). The PTU-insensitive activity of long term hypothyroid pituitaries was decreased by 71% and >/=84% 4 h after injection of 20 and 200 mug T(3), respectively, with no change in PTU-sensitive rT(3) deiodination. These data show that rat pituitary tissue contains two distinct iodothyronine 5'-deiodinating pathways that differ with respect to substrate specificity, PTU sensitivity, reaction kinetics, and regulation by thyroid hormone. One of these resembles the 5'-deiodinase of liver and kidney, and predominates in euthyroid pituitary tissue in vitro. The other, also found in rat brain, predominates in hypothyroid pituitary tissue, is rapidly responsive to changes in thyroid hormone availability, and, as judged by previous, in vivo studies, appears to account for all the T(3) produced locally in the pituitary and, thereby, 50% of the intracellular T(3) in this tissue.