Monodeiodinating Activity Research Articles

Hypothalamic hormones that release growth hormone stimulate hepatic 5'-monodeiodination activity in the chick embryo.

Plasma GH, tri-iodothyronine (T3), thyroxine (T4) and liver 5'-monodeiodination (5'-D) activity were measured in 18-day-old chick embryos injected with thyrotrophin-releasing hormone (TRH) and human pancreatic growth hormone releasing factor (hpGRF). Injections of 0.1 and 1 microgram TRH and 1.5 micrograms hpGRF increased the concentration of plasma GH while injection of 15 micrograms hpGRF had no effect. Concentrations of plasma T3 were raised after injection of TRH or hpGRF. Injections of TRH but not of hpGRF raised the concentration of plasma T4. The increases in concentration of plasma T3 after injection of TRH or hpGRF were parallelled by increases in liver 5'-D activity. An injection of 0.25 micrograms T4 significantly raised the concentration of T4 in plasma but had no effect on plasma T3 or liver 5'-D activity. It is concluded that the release of chicken GH by TRH or hpGRF is responsible for the observed increase in plasma concentration of T3 and liver 5'-D activity.

Thyrotropic and peripheral activities of thyrotrophin and thyrotrophin-releasing hormone in the chick embryo and adult chicken.

The influence of an intravenous injection of thyrotrophin-releasing hormone (TRH) and bovine thyrotrophin (TSH) on circulating levels of thyroid hormones and the liver 5'-monodeiodination (5'-D) activity is studied in the chick embryo and the adult chicken. In the 18-day-old chick embryo, an injection of 1 microgram TRH and 0.01 I.U. TSH increase plasma concentrations of triiodothyronine (T3) and of thyroxine (T4). TRH, however, preferentially raises plasma levels of T3, resulting in an increased T3 to T4 ratio, whereas TSH preferentially increases T4, resulting in a decreased T3 to T4 ratio. The 5'-D-activity is also stimulated following TRH but not following TSH administration. The increase of reverse T3 (rT3) is much more pronounced following the administration of TSH. In adult chicken an injection of up to 20 micrograms of TRH never increased plasma concentrations of T4, but increases T3 at every dose used together with 5'-D at the 20 micrograms dose. TSH on the other hand never increased T3 or 5'-D, but elevates T4 consistently. It is concluded that TSH is mainly thyrotropic in the chick embryo or adult chicken whereas TRH is responsible for the peripheral conversion of T4 into T3 by stimulating the 5'-D-activity. The involvement of a TRH induced GH release in this peripheral activity is discussed.

Thyroxine inner ring monodeiodinating activity in fetal tissues of the rat.

We studied thyroxine (T4) inner ring monodeiodinating activity (5-MA) in various tissues of fetal, maternal, and adult male rats. Tissue homogenates were incubated with 0.26 microM T4 in 0.1 M phosphate buffer (pH 7.4) containing 10 mM EDTA and 400 mM dithiothreitol (final volume 0.7 ml) for 10 min at 37 degrees C; the 3,3',5'-triiodothyronine (rT3) generated was measured by radioimmunoassay of ethanol extracts of incubation mixture and the result was corrected for rT3 degradation during incubation. Compared to maternal tissues, T4 to rT3 5-MA in the 14-day-old fetus was increased about 70 times in skeletal muscle (mean +/- SEM, velocity, 5.4 +/- 0.9 versus 0.08 +/- 0.01, pmol rT3/h/mg protein); approximately 8 times in intestine (0.72 +/- 0.17 versus 0.09 +/- 0.03);and approximately 4 times in cerebral cortex (19 +/- 0.5 versus 4.5 +/- 0.9), while it was similar in skin (3.2 +/- 0.48 versus 2.6 +/- 0.52). Hepatic T4 5-MA approximated 1.1 +/- 0.63 in the 14-day-old fetus; it could not be measured reliably in maternal or 19-day fetal tissue because of extensive (greater than 90%) degradation of rT3 during incubation. Relative to mother, T4 5-MA in 19-day fetal tissues was increased approximately 30-fold intestine, approximately 20-fold in skeletal muscle, and approximately 6-fold in cerebral cortex while it was similar in skin. The T4 5-MA in maternal rat tissues did not differ significantly from corresponding values in adult male rat, except skin, where it was lower in the mother rat (2.6 +/- 0.52 versus 4.6 +/- 0.61, p less than 0.05). In summary, relative to adult tissues T4 5-MA is exceedingly active in several fetal tissues, most notably in skeletal muscle followed by intestine and cerebral cortex.

Alterations in thyroidal economy in a systemic illness induced by turpentine oil injection to the rat.

Injection of turpentine oil (5 microliter/g, sc) to Sprague Dawley rats was associated with a significant reduction in serum concentration of T4, T3 and TSH that lasted throughout 48 h of study. Dialyzable fraction of T4 and resin uptake of T3 did not change for 10 h after turpentine oil injection, but were increased significantly at 24 h and 48 h. Serum rT3 concentration was decreased significantly at 24 h and 48 h. The pituitary content of TSH in the experimental rat was significantly increased at 24 h after turpentine oil injection, but the TSH-beta subunit content was decreased significantly. Serum TSH response to TRH did not change in experimental rats. Thyroidal radioiodine uptake, serum T4, and serum T3 in experimental rats demonstrated little or no increase in response to exogenous bovine TSH. A thyroid hormone binding inhibitor (THBI) was detected in serum and iodothyronine 5'-monodeiodinating activity was decreased significantly in liver, kidney and heart of rats injected repeatedly with turpentine oil. Oxygen consumption of experimental rats did not change appreciably. Our data suggest that administration of turpentine oil to the rat leads, within a few hours of the injection, to a systemic illness associated with marked changes in thyroidal economy.

Evidence against benefit from replacement doses of thyroid hormones in nonthyroidal illness (NTI): studies using turpentine oil-injected rat

Sprague-Dawley rats were treated with saline or turpentine oil (5 mu 1/g bw sc at 3-day intervals x3) with or without replacement doses of T4 (0.8 g/100 g bw/day ip) or T3(0.3 microgram/100 g bw/day ip). Injection of turpentine oil to the rat consistently caused a significant reduction in serum total T4, total T3, free T4 index and TSH. Despite marked changes in thyroidal economy in experimental rat, iodothyronine 5'-monodeiodinating activity (MA) in the liver, the kidney and the hearth and the hepatic alpha-glycerophosphate dehydrogenase activity were decreased inconsistently and when decreased, the various enzyme activities were not influenced appreciably by treatment with replacement doses of T4 or T3. Cerebral cortical T4 5-MA was normal or increased in the turpentine oil-injected rat. Dermal T4 5-MA was decreased in the turpentine oil-injected rat and replacement doses of thyroid hormones did not normalize it. Urinary excretion of urea nitrogen was normal in the turpentine oil-injected rat and did not change appreciably after treatment with thyroid hormones. Our data suggest that replacement doses of thyroid hormones are not beneficial to a host with altered thyroid economy during a systemic illness.

Seasonal changes of the 5′-monodeiodination activity in kidney and skin homogenates of male Rana ridibunda: Relation to plasma thyroxine (T 4) and testosterone

Plasma concentrations of thyroxine (T 4) and testosterone were studied together with the in vitro 5′-monodeiodination activity in liver, kidney, and skin homogenates in male Rana ridibunda during 12 consecutive months. Plasma T 4 levels increase in February, drop in April, and increase again in May. In July a sharp decrease is seen and the values remain low during the rest of the year. Testosterone plasma levels also exhibit month to month variations. A maximum is found in February and March, and minimal values are measured in August, September, and October. A 5′-monodeiodination activity with a resulting 3,3′,5-triiodothyrinine (T 3) production could be demonstrated in kidney and skin homogenates but not in liver homogenates. Addition of 1,4-dithio- dl-threitol (DTT) and EDTA to the homogenates significantly increases the T 3 production. In kidney homogenates incubated with DTT and EDTA a significant annual variation in the T 3 production is seen. This T 3 production is very high in April, May, and June and is followed by a minimum in July and August. It is concluded that the opposite changes in 5′-monodeiodination activity and plasma T 4 concentrations may indicate a peripheral monodeiodination of T 4 to T 3. During reproduction, which is preceded by a testosterone peak, the increase in the 5′-monodeionation activity may reflect a greater need for T 3.

The influence of methimazole on the thyrotrophic and peripheral activity of thyrotrophin and thyrotrophin-releasing hormone in the chick embryo and growing chicken

Plasma concentrations of thyroxine (T 4) and triiodothyronine (T 3) were profoundly depressed both in chick embryos and growing chickens after methimazole (MMI) treatment. There was no response of T 4 and T 3 levels to TRH or TSH injections in the MMI group, either in embryos or growing chickens. Peroxidase activity measured in the thyroid gland was significantly higher in embryos and growing chickens treated with MMI. However, neither TRH nor TSH affected this activity 2 hr after injection in either control or the MMI-treated group. Hepatic 5′-monodeiodinase activity was significantly stimulated in the MMI-treated groups of embryos and growing chickens but only when additional sulphydryl groups (DTT) were provided. In embryos, monodeiodination activity 2 hr after TSH injection was not significantly different from control values for either DTT-stimulated or unstimulated conditions within the control and MMI-infused groups. However, in both control and MMI-treated embryos monodeiodination activity significantly increased 2 hr after TRH injection. In the growing chickens, monodeiodination activity 2 hr after TRH or TSH injection was not significantly different from control values in either stimulated or unstimulated conditions of each group.

The Effect of Free Radicals on Hepatic 5′-Monodeiodination of Thyroxine and 3,3′, 5′ -Triiodothy ronine*

Free radicals have been implicated in many pathological processes, including ischemia, inflammation, and malignancy. Since a reduction in extrathyroidal outer ring monodeiodination of T4 and rT3 occurs in virtually all systemic illnesses, we have studied the effect of free radicals on iodothyronine (T4 and rT3) 5'-monodeiodinating activity (MA) of liver tissue in vitro. Rat liver microsomes or homogenate were preincubated in Tris buffer for 30 min with a free radical-generating system (FRGS) and then incubated with T4 (2.5 microM) or [125I]rT3 (0.4 nM) and dithiothreitol (DTT; 5-20 mM with T4 and 20-150 mM with [125I]rT3) in the same buffer for 10 or 30 min. T3 generated during incubation was quantified by RIA of ethanol extracts of the incubation mixture. 125I generated from [125I]rT3 was quantified after precipitation of the incubation mixture with trichloroacetic acid or by paper chromatography. Free radicals caused 55% or more reduction in hepatic T4 MA and 44% or more reduction in rT3 MA in various experiments. The inhibition of hepatic rT3 MA after incubation with FRGS persisted despite removal of FRGS and washing of microsomes preincubated with FRGS before studying the MA. However, inclusion of DTT (1-60 mM) during preincubation of tissue with FRGS prevented the FRGS-induced inhibition of rT3 MA. Depletion of the iodothyronine substrate did not occur when FRGS inhibited T4 and rT3 5'-monodeiodination. Free radical scavengers, i.e. superoxide dismutase (600 IU/ml), catalase (300 U/ml), tocopherol (10 mg/ml), thiourea (0.15 M), and tert-butanol (0.15 M), all significantly reduced the inhibition of hepatic rT3 MA caused by FRGS. The FRGS-induced inhibition of hepatic T4 MA was reduced by the same doses of tocopherol, thiourea, and tert-butanol, but not by superoxide dismutase or catalase. Since free radicals may effect tissue damage by lipid peroxidation and since the latter results in generation of malondialdehyde (MDA) as a by-product of the reaction, we studied MDA by its reaction with 2-thiobarbituric acid. Incubation with FRGS caused an approximately 100-fold increase in MDA formation in liver microsomes. Serum MDA was significantly higher in 16 NTI patients than in 8 normal subjects and also higher in turpentine oil-injected rats [an experimental model of nonthyroidal illness (NTI)] than in saline-injected control rats. The data suggest that generation of free radicals may contribute to the reduced extrathyroidal 5'-monodeiodination of T4 and rT3 in NTI.(ABSTRACT TRUNCATED AT 400 WORDS)

Stimulation of thyroidal iodothyronine 5'-monodeiodinase by long-acting thyroid stimulator (LATS).

To evaluate the effect of long-acting thyroid stimulator (LATS) on thyroid iodothyronine monodeiodinating activity, we have studied the in vitro conversion of T4 to T3 by mouse thyroid homogenate comparing tissue from LATS treated (0.1 ml LATS(+) serum, ip, for 3 days) with tissues from LATS(-) Graves' disease patients' serum or normal serum treated controls. Five out of seven LATS(+) sera were shown to stimulate the T4 5'-deiodinase significantly in mouse thyroid. There was no significant correlation between LATS titre and deiodinase activities in the different sera tested. To compare the effect of LATS and TSH (0.2 IU, ip daily), studies were carried out from 12 to 72 h. LATS had a similar latency of 12 h on the stimulation of thyroid deiodinase compared to TSH as reported earlier. However, the conversion activities reached a plateau by 12 h after LATS treatment, while it continued to rise upon daily TSH injection from 24 to 72 h. In addition, TSH caused a marked reduction of thyroid protein and an early peaking in serum T3 and T4 at 12 h, whereas LATS caused no detectable change in thyroid protein and a gradual rise in circulating T3 and T4. The kinetic analysis indicated that LATS-mediated stimulation of T4 5'-deiodinase was, similar to TSH, associated with an increase in maximum velocity (Vmax were 139, 208 and 505 pmol/mg protein/30 min respectively in control, LATS and TSH-treated animals) without a demonstrable change in the apparent Km (approximately 2.0 microM for T4). The present study demonstrated that some LATS-rich sera stimulate thyroid T4 to T3 conversion in mouse. It provides an insight into the mechanism of increased T3 secretion from Graves' thyroid glands.

Effects of hyper- or hypothyroid status on growth, adiposity and levels of growth hormone, somatomedin C and thyroid metabolism in broiler chickens

Relatively few methods involving dietary manipulation of thyroid status have been used to study the effect of that status on growth and body composition as well as on changes in hormone levels and metabolism. The present work examines the influence of either a hyperthyroid status (induced by feeding triiodothyronine: T3 or thyroxine: T4) or a hypothyroid status (induced by feeding methimazole: MMI) on growth, feed consumption and body composition as well as on changes in thyroid hormone levels and metabolism and in hormone levels of the somatotroph axis of broiler chickens. MMI depressed growth but increased fatness. Long-term administration of thyroid hormones decreased both growth and fat deposition, T3 being more effective than T4. The reduced growth of MMI-treated birds was not only related to the hypothyroid state but also to decreased somatomedin C (Sm-C) production, although growth hormone (GH) levels remained high. Thyroid hormone-treated birds showed slightly depressed GH levels but unchanged Sm-C levels. MMI induced an increase in hepatic 5'-monodeiodination activity, while T3 and T4 reduced that activity. The T4 administered was largely eliminated as inactive reverse T3. Both of the statuses (hypothyroid and hyperthyroid) profoundly changed peripheral thyroid hormone metabolism and influenced GH/Sm-C relationships, but in the opposite way. Hormonal changes could be related to the observed changes in growth and adiposity of broiler chickens and illustrate the negative correlation between T3 and body fat.

The inner ring (5-) monodeiodination of thyroxine (T 4) in cerebral cortex during fetal, neonatal, and adult life

Inner ring (−5) monodeiodination of T 4 was studied by incubating T 4 (∼0.26 μmol/L) with rat cerebral cortical homogenate (∼4 mg protein) in the presence of dithiothreitol (up to 400 mmol/L) and quantifying the amount of the product, rT 3, by a specific radioimmunoassay. The production of rT 3 was dependent on duration of incubation (up to 2 hours), amount of tissue protein (up to 8 mg), temperature (optimal at 37 °C) and pH (optimal, 7.0) of the incubation mixture and the concentration of DTT (maximally stimulated at 400 mmol/L). The apparent Km and Vmax of the T 4-inner ring monodeiodinating activity were 36 nmol/L and 1.75 pmol/mg protein/h, respectively. The activity was inhibited by T 3 and 3,5-T 2, but not by 3′5′-T 2, PTU, methimazole, sodium salicylate, or 8-anilino-I-naphthalene sulfonic acid. Ipodate weakly inhibited T 4-to-rT 3 monodeiodination. Hyperthyroidism induced by T 4 (100 μg/d IP × 3 days), T 3 (80 μg/d IP × 3 days) or DIMIT (45 μg/d IP × 3 days) significantly stimulated T 4-to-rT 3 conversion; DIMIT was the most potent agent. Hypothyroidism inhibited T 4-to-rT 3 converting activity in cerebral cortex. Fasting for three days had no appreciable effect on T 4-to-rT 3 conversion in cerebral cortex. Cerebral cortical T 4 5-deiodinase activity in the pregnant rat at term was about 50% of that in the adult nonpregnant rat, whereas that in the fetus was about three-fold higher than that in the nonpregnant adult. The values in the mother increased while those in the newborn decreased gradually to become comparable to those in the adult nonpregnant rat by two weeks after delivery. The various data suggest that cerebral cortical T 4-to-rT 3 converting activity is enzymic in nature and that it is under physiologic regulation.

Characterization of thyrotropin-induced increase in iodothyronine monodeiodinating activity in mice.

To further characterize the effect of TSH administration on thyroid iodothyronine monodeiodinating activity, we have evaluated the in vitro conversion of T4 to T3 (outer ring deiodination) and T3 to 3,3'-diiodothyronine (T2; inner ring deiodination) by mouse thyroid, liver, and kidney homogenates, comparing tissues from TSH-treated mice (0.1-200 mU bovine TSH, ip, for 1-3 days) with tissues from saline-treated controls. The in vitro conversion activity was studied in the presence of 1-20 mM dithiothreitol; most of the studies were carried out at 4 mM. Studies were carried out at optimal pH 6.5 for outer ring and 7.8 for inner ring deiodination. The iodothyronine monodeiodinase in mouse thyroid is similar to the ones in liver and kidney. It is heat labile (inactivated at 56 C for 5 min), inhibited by propylthiouracil (0.2 mM) and ipodate (0.2 mM), and unaffected by methimazole (up to 20 mM), ascorbate (up to 0.1 M) or KI (up to 20 mM). The mean +/- SE baseline rates of T4 to T3 and T3 to T2 conversion were 100 +/- 6.3 and 56.5 +/- 2.9 pmol/mg thyroid protein X 30 min at 37 C, respectively. A significant increase in each conversion activity was found after TSH treatment (0.2 U, ip, daily for 3 days); T4 to T3 conversion rose to 282 +/- 15.4, and T3 to T2 increased to 153 +/- 7.4 pmol/mg thyroid protein (P less than 0.001). A 12.8% increase in thyroid weight was found in the TSH-treated group (P less than 0.03 compared with saline control group). Similar but less marked increased in monodeiodinating activities were seen in the liver. A minimal but significant increase in inner ring monodeiodination with no significant increase in T4 to T3 converting activity was found in kidney, which, in the mouse, has markedly less outer ring deiodinase than liver or thyroid. The iodothyronine monodeiodinating activities did not increase until 12 h in thyroid and 48 h in liver after the first dose of TSH. Significant increases in T4 to T3 and T3 to T2 conversion were seen with doses of TSH as low as 0.1 mU (ip, daily for 3 days), and there was a linear dose-response thereafter. The decay of the increased iodothyronine monodeiodinating activities after a single dose of TSH (0.2 U) appeared to be linear, with a decay t 1/2 of 1.3 days for T4 to T3 conversion and about 1.0 day for T3 to T2 conversion.(ABSTRACT TRUNCATED AT 400 WORDS)

3,5,3′-Triiodothyronine receptors and thyroxine 5′-monodeiodinating activity in thyroid hormone-insensitive Amphibia

Adult anuran Amphibia and neotenous urodeles, such as Necturus maculosus, rarely respond to thyroid hormone (TH). In this study, the possibility was examined that this lack of response is due either to an inability to convert thyroxine (T 4) to 3,5,3′-triiodothyronine (T 3) or to the absence of nuclear TH receptors. Following injection of [ 125I]T 4, significant amounts of [ 125I]T 3, analyzed by chromatography, were detected in the serum and liver of Necturus and Rana catesbeiana frog indicating that both possesses a T 4 5′-monodeiodinating system. Nuclear binding of T 3 was studied in suspensions of purified hepatic nuclei and intact red blood cells (RBC). Analysis of binding data revealed that frog liver nuclei contained two sets of saturable T 3 binding sets with affinities comparable to those of the two sets of sites previously demonstrated in tadpole liver nuclei ( Galton and Schaafsma, 1983). However, the number of sites per nuclei was small compared to tadpole; expressed as pmol/mg DNA, the maximum binding capacity of the high affinity set of sites (MBC 1) was 2.2 ± 0.31 versus 12.9 ± 1.80 and MBC 2 was 24.9 ± 4.5 versus 42.2 ± 54. Receptor number in RBC nuclei was also smaller in frog than tadpole: 90 ± 26 versus 882 ± 56 sites/nucleus ( K d < 10 −11 M in both groups). No comparable high affinity binding sites were detected in Necturus liver, but some sites were found in Necturus RBC. These cells contained 2111 ± 120 sites/nucleus, more than twice the number found in tadpole. When expressed per milligram DNA, however, the MBC was only one eighth of that in the tadpole. These findings are compatible with the view that a low receptor number may account for the lack of response of frog and Necturus to TH.

Phenolic and tyrosyl ring deiodination in thyroxine from rat retina during postnatal development.

To elucidate tetraiodothyronine (T4) metabolism in developing rat retina 5-monodeiodinating and 5'-monodeiodinating activities were studied. T4 was incubated with aliquots of homogenate or crude primary subcellular fractions, and the 3,3',5'-triiodothyronine (rT3) or 3,5,3'-triiodothyronine (T3) produced were measured by radioimmunoassay. Reaction rates were dependent on incubation time, tissue amount, temperature and pH. The optimum pH values were 7.8 and 7.2 respectively for rT3-forming and T3-forming systems. Conversion of T4 to either T3 or rT3 was dependent on dithiothreitol concentration, and the T4-5'-deiodinating activity was inhibited by propylthiouracil. Deiodinase activities were mainly found in the crude microsomes. The retinal 5'-monodeiodination rate of T4 was immeasurably low by the 2nd day and the highest values were reached on 15th day of postnatal development. On the other hand deiodination of the T4 tyrosyl ring shows a progressive decline from birth, and adult values were reached on the 15th day. Data support the hypothesis that, in developing rat thyroxine, phenolic and tyrosyl-ring deiodinase activities are present in the retina and their reciprocal changes may regulate morphological and biochemical cell maturation.

Effect of 3,5,3'-triiodothyronine-induced hyperthyroidism on iodothyronine metabolism in the rat: evidence for tissue differences in metabolic responses.

We studied the effect of T3-induced hyperthyroidism on the outer ring (5' or 3') monodeiodination of T4 (to T3) and 3',5'-diiodothyronine [3',5'-T2; to 3'-monoiodothyronine (3'-T1)] and on the inner ring (3 or 5) monodeiodination of 3,5-T2 (to 3-T1) by various rat tissues. Weight-matched pairs of male Sprague-Dawley rats were given either saline or T3 (20 micrograms/100 g BW daily) ip for 3 days. The metabolism of the iodothyronines was studied on day 4 in homogenates of the tissues in the presence of 25 mM dithiothreitol. Hyperthyroidism was associated with a significant (P less than 0.05) increase in T4 to T3 monodeiodinating activity in the liver (mean, 95%), kidney (mean, 60%), and heart (mean, 153%), but not in skeletal muscle, small intestine, spleen, testis, cerebral cortex, or cerebellum. The monodeiodinating activity converting 3',5'-T2 to 3'-T1 was greatly increased (P less than 0.05) in the heart (mean, 750%), spleen (mean, 462%), and skeletal muscle (mean, 167%), but not in liver, kidney, small intestine, testis, cerebral cortex, or cerebellum. In the case of liver and kidney, however, there was evidence of an activation of 3',5'-T2 monodeiodinating activity, as suggested by a significant increase in the activity in the absence of added dithiothreitol. The monodeiodination of 3,5-T2 to 3-T1 increased significantly only in the cerebral cortex (mean, 525%) and liver (mean, 69%) and not in any other tissue. The time course of the above-mentioned changes in iodothyronine metabolism was studied in groups of rats (five per group) given T3 (20 micrograms 100 g BW-1 day-1) 6-72 h before death. Significant increases in 3',5'-T2 (to 3'-T1) monodeiodination in the heart and 3,5-T2 (to 3-T1) monodeiodination in the cerebral cortex were evident within 6 h of T3 administration. Changes in T4 to T3 monodeiodinating activity in the kidney and liver, however, did not become statistically significant until 24 and 72 h, respectively. The various effects of T3 on the tissues became maximal between 48 and 72 h after the initiation of T3 treatment. Our data suggest that most tissues, including some that have been considered unresponsive to thyroid hormones, e.g. brain and spleen, demonstrate substantial metabolic changes after T3 administration. The tissue responses are variable in degree; in some instances, they are specific for the substrate and type of tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of rat hepatic thyroxine 5'-monodeiodinase by propylthiouracil: relation to site of interaction of thyroxine and glutathione.

When rat liver cytosol, dialyzed free of glutathione, was chromatographed on Sephadex G-100 after incubation with 35S-propylthiouracil, 2 peaks of bound radioactivity were observed, 1 of which contained nearly all the thyroxine 5'-monodeiodinase activity in rat liver cytosol. Binding of propylthiouracil to this peak was inhibited by glutathione but not by thyroxine. Approximately 25% of 35S-propylthiouracil initially bound to the thyroxine 5'-monodeiodinating activity peak remained bound after dialysis, precipitation with trichloroacetic acid, and multipe extractions with ethanol, methanol, and chloroform, suggesting that binding was at least in part covalent. Dialysis studies showed that the presumed covalent binding of 35S-propylthiouracil to the thyroxine 5'-monodeiodinase peak could be inhibited by glutathione, dithioerythritol, and unlabelled propylthiouracil but not by oxidized glutathione or thyroxine. Conversely, thyroxine binding was unaffected by thiol compounds. We studied the kinetics of thyroxine 5'-monodeiodination by radioimmunoassay techniques using rat liver homogenates as source of enzyme and observed the dependence of enzymic reaction upon glutathione (Km = 2.4 mM). Propylthiouracil inhibited the reaction and this inhibition could be overcome with increasing glutathione concentrations. We conclude that the thiol-dependent thyroxine 5'-monodeiodinase is inhibited by propylthiouracil through its covalent binding, probably as mixed disulfide, to s site on the enzyme at which glutathione interacts either as a cosubstrate or reducing agent. This binding site is separate from the site at which thyroxine binds.

Age-related changes of serum 3,3'-diiodothyronine, 3',5'-diiodothyronine, and 3,5-diiodothyronine concentrations in man.

To evaluate the effects of aging on T4 metabolism, serum 3,3'diiodothyronine (3,3'-T2), 3',5'-diiodothyronine (3',5'-T2), and 3,5-diiodothyronine (3,5-T2) as well as T4, T3, and rT3 concentrations were determined by RIAs in 81 normal subjects, aged 24-81 yr. 3,3'-T2, 3',5'-T2, and 3,5-T2 antisera were prepared by immunizing rabbits with the protein conjugate of each diiodothyronine. Since the 3',5'-T2 antiserum cross-reacted with rT3 to the extent of 1.8%, correction was made by subtracting 1.8% of the rT3 value from the measured 3',5'-T2 value. The other 2 antisera had no noticeable cross-reactivity with various thyroid hormone derivatives. Although no significant relation existed between T4 levels and age (r = -0.14; P greater than 0.1) serum T3 showed a significant correlation with age (r = -0.28; P less than 0.05). In contrast, no significant correlation was observed between rT3 and age (r = 0.12; P greater than 0.1). Serum 3,3'-T2, 3',5'-Tw, and 3,5-T2 values (nanograms per dl) all had significant inverse correlations with age, the coefficients of correlation being -0.38 for 3,3'-T2 (P less than 0.01), -0.58 for 3',5'-Tw (P less than 0.01), and -0.47 for 3,5-T2 (P less than 0.01). These findings suggest that sequential monodeiodinating activities in T4 metabolism decrease with increasing age.

Characteristics of outer ring (5'- or 3'-) monodeiodination of 3',5'- and 3,3'-diiodothyronine: evidence suggesting one outer ring monodeiodinase for various iodothyronines.

Previous studies revealing marked similarities in the activities of outer ring monodeiodination of T4 and rT3 by liver or kidney have suggested that these processes are mediated by the same enzyme. It is not known, however, whether the same, similar, or different enzyme (s) is involved in the removal of iodine from the outer ring of other iodothyronines. This study examines the processes of extrathyroidal monodeiodination of 3′5′-diiodothyronine (3′,5′-T2) and 3,3′-T2 in the outer ring to 3′- monoiodothyronine (3′-T1) and 3-T1, respectively, and describes their responses to several manipulations. 3′5′-T2 (∼8 × 108 M) or 3,3′-T2 (∼3 × 107 M) was incubated in 0.1 M Tris buffer (pH 7.4) with various rat tissue homogenates (∼0.13 geq) for 3 min (in the case of 3′,5′-T2) or 30 min (in the case of 3,3′-T2) at 37 C, and the amount of T1 generated (3′-T1 from 3′,5′-T2 and 3-T1 from 3,3′-T2) during incubation was quantified by pecific RIA of ethanol extracts of incubation mixtures. The radioactive ligand in the 3′-T1 RIA was 125I-labeled 3′-T1; in the 3-T1 RIA it was 3-T1-[125I]iodohistamine prepared by conjugation of 3-T1 to [125I]iodohistamine. The liver and kidney produced more T1 from outer ring deiodination of both T2s than various other tissues. Studies using liver homogenate indicated that onodeiodination of both T2s was influenced in a similar manner by the concentration of the homogenate, duration of incubation, substrate (T2) concentration, pH of incubation, and temperature of incubation. The T2-monodeiodinating activities were unaffected by large concentrations (≥1 mM) of potassium iodide, methimazole, and ascorbic acid. However, they were both stimulated by dithiothreitol and inhibited in a dose-dependent manner by (in order of relative potency on a molar basis, maximal to minimal) rT3, ipodate, T4, propylthiouracil, diamide, 8-anilino-l-naphthalene sulfonic acid, and salicylate. The apparent Km values for monodeiodination of 3′5′-T2 (0.5 μM) and 3,3′- T2 (0.45 μM) were also similar, but the Vmax for monodeiodination of 3′5′-T2 (390 pg/mg protein-min) was substantially higher than that of 3,3′-T2 (33 pg/mg protein min). Among the usual subcellular fractions of the liver homogenate, microsomes were more potent and cytosol was less potent in deiodinating both T2s than mitochondria or the starting homogenate. However, the plasma membrane fraction of the homogenate, prepared separately by sucrose density gradient, was the single most active subcellular fraction for both monodeiodinations. Starvation of the rat for 2 or 4 days was associated with a significant reduction in T2-monodeiodinating activity in the liver homogenate. The various data suggest that 1) 3′5′-T2 (to 3′-T1) and 3,3′-T2 (to 3- T1) monodeiodinating activities are enzymatic in nature; 2) these processes are very similar to each other and to T4 and rT3 outer ring monodeiodinating activities in many characteristics, including organ distribution, subcellular location, and their responses to various manipulations; and 3) there may just be one outerring monodeiodinase acting on various iodothyronines.

Reciprocal relation between serum thyrotropin levels and intrapituitary 3,5,3'L-triiodothyronine generating activity from thyroxine in perinatal rats.

Thyroxine (T4) 5'-monodeiodinating activity in rat pituitary was studied, using paper chromatography. Rat anterior pituitary homogenates were incubated with 125I-T4, dithiothreitol and unlabeled L-T4 at 37 degrees C. The incubation mixture was extracted by ethanol and the extracts were subjected to descending paper chromatography. The conversion ratio of T4 to 3, 5, 3'L-triodothyronine (T3) was calculated from the radioactivity of T3 spot to total radioactivity on the paper strip. The T3 amount generated was estimated from the T4 concentration in the incubation mixture and the conversion ratio. The T3 generating activity from T4 increased as the amount of tissue increased. It was temperature-and pH-dependent, and thiol sensitive. These results suggest the enzymatic nature of T4 5'-monodeiodinating activity of the anterior pituitary. A kinetic study revealed low Km for T4 (7.9 +/- 1.6 nM, Mean +/- SE), with Vmax of 68.0 +/- 12.7 fmoles T3/mg protein/min. T4 monodeiodinating activity was consistently, though minimally, detected in fetal rat pituitaries and increased after birth, reaching the maximum at 22 days. It declined thereafter to the young adult level. Serum TSH levels were markedly elevated in fetus. They decreased after birth, reaching the nadir at 22 days, and then increased to young adult levels. Serum T4 and T3 levels were markedly diminished in fetus and gradually increased after birth, reaching the young adult levels at 17 days. Thus, a reciprocal relationship was observed between intrapituitary T3 generating activity from T4 and serum TSH levels in developing rats. It suggests that the conversion of T4 to T3 in the pituitary plays a role in regulating TSH secretion.

Thyroid hormone metabolism in cultured monkey hepatocarcinoma cells. Monodeiodination activity in relation to cell growth.

Thyroid hormone metabolism in cultured monkey hepatocarcinoma cells. Monodeiodination activity in relation to cell growth.