Existence of extrathyroidal conversion inhibitor (IEC) in starved animals and its influence on thyroid hormones deiodination in liver and kidney in vitro

To find out whether an inhibitor of extrathyroidal conversion of iodothyronines is present in sera of starved animals, pig liver and kidney homogenates were incubated with T4, T3 or rT3 and dithiotreitol in the presence of evaporated diethyl ether extracts of sera obtained from fed and starved (1-12 days) rabbits. Sera extracts of short-term (1-4 days) starved rabbits caused a significant inhibition of T4 to T3 conversion (54% on day 3) and T4 to rT3 deiodination (52% on day 2) in liver homogenates. Extracts of sera from long-term (8 and 12 days) starved animals diminished only liver T4 to T3 conversion on day 8 and had no influence on liver T4 to rT3 conversion. 5'-deiodination of rT3 (to 3,3'-T2) in liver was gradually decreased by extracts of sera from animals starved during 2-12 days. Liver rT3-5-deiodination (to 3',5'-T2) was significantly impaired on day 4 and totally depressed by long-term starvation. In vitro T3 to 3,3'-T2 conversion in liver was markedly (59-103%) increased by ether extracts of sera from short-term fasted rabbits and considerably inhibited (62-72%) by long-term fasting. T4 to T3 conversion in kidney was significantly influenced by sera extracts obtained neither from short-term fasted rabbits and considerably inhibited (62-72%) by long-term fasting. T4 to T3 conversion in kidney was significantly influenced by sera extracts obtained neither from short-term nor from long-term fasted rabbits but T4-5-deiodination (to rT3) was reduced by sera extracts of short-term fasted animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

The substrate specificity, tissue specificity and regulation of the 5' deiodination systems in rat liver and kidney tissues

Studies were carried out to compare the 5' deiodination reactions of thyroxine (T4) and 3, 3', 5'-triiodothyronine (rT3) in rat liver and kidney homogenates. The 5'-deiodinase activity was assayed by the 3, 5, 3'-triiodothyronine (T3) produced from T4 or by the 125I-iodide released from 125I-rT3. The two 5' deiodination reactions had similar ranges of optimal pH, incubation temperature, and apparent Km, T4 1.1 and rT3 1.3 microM. However, the apparent Vmax values for T4 and rT3 deiodination reactions were 0.9 and 220 pmol/mg protein/min, respectively. Both reactions were stimulated by thiol reagent but only rT3 deiodination showed complete thiol dependence. The inhibitory effect of 6-propyl-2-thiouracil (PTU) on the 5' deiodination of rT3 was 50 times as great as that of T4. Only the 5' deiodination of rT3 was inhibited by low concentrations of calcium and magnesium. The 5' deiodination reactions in the liver and kidney tissues showed very similar substrate specificity. However, only the hepatic deiodinase activity was reduced to 60-65% of the control value after fasting, whereas the renal 5'-deiodinase activity was unaffected or even enhanced by fasting up to 72 hours. The results showed the existence of a diverse and complex 5' deiodination system in the rat tissues which is comprised of multiple similar but distinct 5'-deiodinase enzymes with respect to their substrate specificity, tissue specificity and regulation.     

Substrate specificity of iodothyronine 5'-deiodinase in rat liver homogenates and its requirements of divalent cations in vitro

Studies were carried out to compare the 5'-deiodination reactions of thyroxine (T4) and 3,3'-5'-triiodothyronine (rT3) in 2.5% rat liver homogenates. The 5'-deiodinase activity was assayed by the 3,5,3'-triiodothyronine (T3) produced from T4 or by 125I-rT3. Under our experimental conditions, the two 5'-monodeiodination reactions resulted in similar apparent KMs: 1.5 microM for T4 and 1.1 microM for rT3. However, the apparent Vmax values of T4 and rT3 deiodination reactions were, respectively, 0.91 and 222 pmol/mg protein/min. Both reactions were stimulated by thiol reagents but only rT3 deiodination showed complete thiol dependence. The inhibitory effect of 6-propyl-2-thiouracil on the 5'-deiodination of rT3 was at least 50 fold greater than that of T4. The divalent ion requirement of the deiodination system was tested with CaCl2, MgCl2, and ZnCl2 at a range of concentrations. Zinc ion appeared to be a potent inhibitor in both T4 and rT3 deiodination systems. Only the 5'-deiodination of rT3 was inhibited slightly by low concentrations of calcium and magnesium ions. Our results suggest that based on their apparently distinct regulation mechanisms, the 5'-monodiodination of T4 and rT3 in rat liver homogenates is likely mediated by more than one enzyme, despite the similarity of observed KMs.     

The relationship between basal metabolic rate (BMR) and concentrations of plasma thyroid hormones in fasting cockerels

A correlation between the Basal Metabolic Rate (BMR) and the level of rT3, and occasionally between BMR and T3 or T4 was found in 12 month fasting cockerels. The birds were fasted for 48 hrs and BMR was measured eight times (before fasting, at 6, 12, 24, 30, 36, and 48 hrs of fasting, and 4 hours after fasting). Blood samples for plasma collection were taken immediately after measuring the BMR. During starvation a decrease in BMR was observed. After refeeding BMR returned to the starting level. The decrease in BMR was accompanied by an increase in rT3 and T4 plasma levels. Between BMR and levels of T4 and rT3 negative coefficients of correlation were observed (r = -0.20 and r = -0.42, respectively). Contrary to this, the T3 level declined and was correlated with BMR (r = 0.62). After refeeding, the T3 level rapidly increased against the control value. Moreover, a high coefficient of correlation (r = -0.39) was found between the level of T3 and rT3. The data show that the reduction in plasma T3 level and increase in the rT3 one during starvation may be due to inhibition of deiodination of T4 to T3, since rT3 is a competitive inhibitor of this reaction. The presented results support the suggestion that in birds T3 is the metabolically active thyroid hormone, and rT3 antagonizes this effect.     

A study of hepatic metabolism of thyroxine in BB/W rats treated with L-thyroxine

The effect of thyroxine (T4) on T4 conversion to triiodothyronine (T3) and reverse T3 (rT3) was studied in BB/W rats. A colony of 38 BB/W rats was obtained and half were treated with thyroxine (T4), 1 mg per liter of drinking water. At 106 days of age the following groups were identified: nondiabetic, no T4 treatment, 8 rats; nondiabetic, T4 treated, 8 rats; diabetic, no T4 treatment, 10 rats; diabetic, T4 treated, 7 rats. All animals with diabetes were treated with insulin. T4 conversion to T3 and rT3 was assessed in liver homogenates in 0.1 M Tris-HCl buffer, pH 7.4, with or without 5 mM dithiothreitol (DDT). Serum T4 and rT3 were significantly elevated in both T4-treated groups (P less than 0.001), while serum T3 was not affected in either. Basal T4 deiodination to T3 by the liver homogenate did not change on treatment with T4; the addition of DTT increased T3 production in the homogenate from T4 treated nondiabetic animals (P less than 0.05). In both nondiabetic and insulin-treated diabetic rats there was no effect of T4 on the rate of rT3 production. Since, in the rat, 30-40% of circulating T3 is a direct contribution of thyroid gland secretion, and that would be absent in our T4-suppressed animals, the normal serum T3 may reflect increased absolute peripheral T3 production from the greater concentration of circulating T4.     

Conversion of thyroxine into 3,5,3'-triiodothyronine and 3,3',5'-triiodothyronine in the young pig

Estimates have been made of the amounts of 3,5,3'-triiodothyrone (T3) and 3,3',5'-triiodothyronine (rT3) derived from peripheral deiodination of thyroxine (T4) in young pigs. Two methods were used. The first depended on the assumption that deiodination occurs at the same rate in normal animals and in thyroidectomized animals on T4 replacement therapy. The second on the assumption that T3 and rT3 are secreted in the same proportions as they occur in thyroglobulin. The first method arguably gives the better estimate which is that 87% of circulating T4 is monodeiodinated to T3 and rT3. Peripheral conversion accounts for 76 and 69% of the circulating T3 and rT3, respectively.     

Phenolic and nonphenolic ring deiodinations of iodothyronines in cultured hepatocarcinoma cell homogenate from monkey.

Iodothyronine monodeiodinase activities in homogenates of cultured monkey hepatocarcinoma cells were measured by the deiodination of [3.5-(125)I]-diiodo-L-thyronine or 3-[3',5'-(125)I]triiodo-L-thyronine (phenolic ring-labeled 'reverse' triiodothyronine). The assay system utilized a small ion-exchange column (AG50W-X4, O.9 X approximately 1 cm) to measure 125I-. Both deiodinases were destroyed by boiling for 1 min. Maximal nonphenolic ring deiodination was observed at pH 7.9 whereas maximal phenolic ring deiodination was at pH 6.3. Both reactions were enhanced strongly by dithiothreitol (0.1-5mM), and slightly by 5 mM beta-mercaptoethanol. Phenolic ring deiodination was strongly inhibited by 0.1 mM propylthiouracil. Nonphenolic ring deiodination was accelerated by EDTA (1.2 MM) and inhibited by Mg(2+) (5mM). Methylmercaptoimidazol and Mg(2+), Ca(2+) and Mn(2+) (0.1-1.0 mM) had little or no effect on either reaction, but Zn(2+) (0.1 mM) strongly inhibited both. Both reactions were inhibited by excess iodothyronine analogues at 10 mM to 10 micron M, and thyroxine was shown to be a competitive inhibitor in both cases. On the basis of relative affinities and inhibitory effects, it appears that the order of affinity for the phenolic ring deiodinase is 3,3',5'-triiodo-L-thyronine(rT3) greater than L-thyroxine(T4) greater than 3,5,3'-triiodo-L-thyronine(T3), whereas for the nonphenolic ring deiodinase the order is T3 greater than T4 greater than rT3. Diiodotyrosine did not affect their deiodination.     

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.     

Monodeiodination of thyroxine to 3, 5, 3'-triiodothyronine and to 3, 3', 5'-triiodothyronine in isolated dog renal cortical tubuli

Monodeiodination of T4 to T3 and rT3 in the intact cells of dog renal tubuli and glomeruli was investigated. The tubuli and glomeruli were obtained by a sieve method. T4 (2 micrograms/ml) was incubated in Tris-HCl buffer, pH 7.5, with renal cells (180 micrograms protein/ml) and 5 mM DTT for 1 h at 37 degrees C and the T3 and rT3 generated during incubation were measured by specific radioimmunoassays. In order of decreasing activity, dog renal cortical tubuli, cortical homogenate, glomeruli and medullary tubuli were capable of converting T4 to T3. Net rT3 production from T4 in cortical tubuli was also greater than that in cortical homogenate. The conversion of T4 to T3 and also to rT3 in cortical tubuli was enzymatic in nature, since the reactions showed dependence on time and protein concentration; instability to heating; temperature and pH optimum. The production of T3 and rT3 from T4 was maximum at pH 6.5 and at pH 9.5, respectively, indicating that two different enzymic systems, a 5- and a 5'-monodeiodinase, might be involved in the deiodination of the tyrosyl and the phenolic ring of T4 in dog kidney.     

The effect of iodothyronines on the conversion of thyroxine into 3,3''-5-tri-iodothyronine in isolated rat renal tubules.

Isolated rat renal tubules prepared by collagenase digestion were used to study the effects of 3,3',5'-tri-iodothyronine ('reverse T3', rT3) and other iodothyronines on the formation of 3,3',5-tri-iodothyronine (T3) from thyroxine (T4). rT3 inhibited the conversion with a dose response over the concentration range 1.5nM-1.5microM. The inhibition was competitive in nature. Both 3,3'-di-iodothyronine and 3',5'-di-iodothyronine also inhibited the production of T3 and T4 in isolated rat renal tubules, but tetraiodothyroacetic acid and 3,5-di-iodothyronine were found to have no effect. These experiments demonstrate in an intact cell system that some naturally occurring iodothyronines have significant effects on T4 deiodination.     

Hepatic conversion of thyroxine to triiodothyronine in obese and lean Zucker rats

The in vitro conversion of thyroxine (T4) to triiodothyronine (T3) was studied in liver homogenates from fed and fasted lean and obese Zucker rats. T3 generation was decreased in fed young (2 month) obese rats as compared to values in fed lean controls. This was not corrected by the addition of dithiothreitol (DTT), suggesting a deficiency in 5'-deiodinase activity in young obese rats. Both lean and obese 2 month old rats responded to a 2 day fast by decreasing hepatic T3 generation as is always observed in other strains of rats. The hepatic conversion rate was not decreased in older (5 month) fed obese rats when compared to age-matched lean controls. Hepatic conversion of T4 to T3 was markedly decreased in 5 month old lean Zucker rats fasted for 4 days. In contrast, a 4 day fast had no effect on the hepatic conversion rate in the 5 month old obese rats. The hepatic conversion rate was assessed in 5 month old obese rats fasted for up to 28 days and hepatic conversion still did not decrease. This paradoxical response of the 5 month old obese rat may provide a new model to further evaluate the control of hepatic T3 generation from T4.     

Placental outer and inner ring monodeiodination of thyroxine and triiodothyronines in the rabbit

Both inner- and outer-ring iodothyronines deiodinating activity was found in homogenates of rabbit placentas. The T4 to rT3 and T3 to 3,3'-T2 deiodinating activity was already high on day 10 before delivery but decreased being about 7 times lowered on day 5. Once the T3 to 3,3'-T2 monodeiodination reached a low and a relatively steady level, the outer ring deiodination of T4 begun, reaching a peak value at about day 3 before term and then fell again. The fetal serum thyroid hormones levels were low, showing no significant variability during the period of observation. The results suggested that in the rabbit, representing animals in which the thyroid gland activity begins early in fetal life, there are two distinct phases of the placental monodeiodinating activity. The first is characterized by a high inner-ring deiodinating activity (yielding rT3) and is followed by the second phase with a high outer-ring deiodinating activity (yielding T3) declining just before term.     

Thyroid status in the obese syndrome of rats

The thyroid function was explored by comparing serum total and free iodothyronine levels in young male genetically obese Zucker rats and in their lean littermates, aged from 6 to 8 weeks old. Total and free thyroxine (T4) and 3,5,3'triiodothyronine (T3) levels were significantly decreased in obese rat serum while total 3,3',5'-triiodothyronine (rT3) remained constant. Radioactive T4 half life is slower in the plasma of obese rats. Peripheral synthesis of T3 from deiodination of T4 is also decreased in obese rat liver homogenate. These modifications produce changes in liver mitochondria oxidative phosphorylation and in marker enzyme activity, which are usually associated with hypothyroidism and hypothalamic disturbances. Genetic obesity probably involves activation of peripheral deiodination of T4 to rT3 which induces biochemical and metabolic changes.     

Effect of thyroid hormones on the activity of hepatic glucose-6-phosphatase in fed and fasted rats

The action of thyroid hormones on hepatic glucose-6-phosphatase was studied in rats. Fed and 24-h fasted rats received T3 (10 micrograms/day) or T4 (25 micrograms/day) 1 h, 1 or 3 days before sacrificing. In addition a group of fed rats was treated with T4 for 7 and 14 days. The glucose-6-phosphatase activity was measured in the isolated microsomes prepared from the liver. The intactness of the microsomal preparation was checked using 2 mM mannose-6-phosphate as a substrate. In fed rats a single injection of T3 or T4 augmented the activities of the translocase and hydrolase components of glucose-6-phosphatase provided that the rats were killed 24 h after the administration of hormone. This effect was more pronounced in animals treated for 3-14 days. As expected, fasting per se increased the activities of both components of the enzyme. Moreover, in fasted rats treatment with T3 and T4 for 3 days further augmented the activities of the translocase and the hydrolase components of glucose-6-phosphatase. In fed animals T3 and T4 increased the latency of the enzyme whereas in fasted animals thyroid hormones increased the activities of the translocase and hydrolase components in parallel, maintaining the level of latency of the enzyme system. Administration of T3 and T4 increased blood glucose level in fasted rats after one day, while in fed rats a significant hyperglycaemia appeared after 7-14 days of treatment. In conclusion, T3 and T4 increase the activities of the translocase and hydrolase components of hepatic glucose-6-phosphatase in fed and fasted rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Iodothyronine content in the pig thyroid gland

An analysis has been carried out on the contents and reciprocal proportions of three principal iodothyronines (T4, T3 and rT3) in the thyroids of fed and fasted piglets of 8-10 wk and in adult pigs. The mean T4 concentration averaged 62.0 +/- nmol/100 mg wet tissue (in adults: 18.5 +/- 4.3 nmol/100 mg tissue); T3, 9.5 +/- 0.9 nmol/100 mg tissue (in adults: 1.58 +/- 0.2 nmol/100 mg tissue); rT3, 3.0 +/- 0.3 nmol/100 mg tissue. The reciprocal ratios of the hormones in the piglets' thyroids were: for T3:T4, 0.150 (in adults, 0.114) and for rT3:T4, 0.050 (in adults, 0.023). Mean T4:T3:rT3 ratio in piglets and adult pigs was 20.5:3.1:1 and 66.1:5.6:1, respectively. The results from all examined iodothyronines, show the higher absolute concentration in piglets' than in adult pigs' thyroid tissue, while the reciprocal proportions of the hormones reveal smaller T4 thyroid contents (comparing with T3 and rT3) in piglets than in adults. No changes of absolute thyroidal contents or reciprocal ratios of the iodothyronines were observed in fed and fasted piglets. In a comparison, the pig thyroid contains more triiodothyronine and a higher ratio T3:T4 than that in some other species.     

Decreased ratio of serum T3:rT3 in patients with hyperthyroidism

In 14 hyperthyroid patient serum T4:rT3 ratio was significantly lower (399 +/- 20) than in the control subjects (572 +/- 20; p less than 0.001). A similar pattern was found for serum T3:rT3 ratio. In the hyperthyroid group the ratio was significantly lower (10.5 +/- 0.5) than in the control group (12.5 +/- 0.6; p less than 0.05). The data suggest that in hyperthyroidism the organism might shift conversion of T4 from biologically active T3 to poorly calorigenic rT3. It seems possible that the proportionately increased generation of rT3 than that of T3 may be a defence mechanism of the body, as it was found in systemic illnesses and starvation.     

Fever induced oxidative stress: the effect on thyroid status and the 5'-monodeiodinase activity, protective role of selenium and vitamin E.

The thyroid hormones metabolism is considerably altered in many pathological processes including fever. Experiments performed on rabbits (n=62) showed that increase in the rectal temperature by 1 degrees C (after turpentine oil sc injections) decreased 5'-monodeiodinase activity, the enzyme responsible for deiodination of thyroxine to the most active thyroid hormone 3,3',5-triiodothyronine (T3), in the liver by 25% and in the kidney by 20%. Triiodothyronines concentration in serum decreased during fever from 1.57+/-0.12 to 0.52+/-0.02 nmolT3/l and from 0.17+/-0.01 to 0.07+/-0.02 nmol rT3/l. The increase in the body temperature intensified lipid peroxidation processes (malondialdehyde level increased from 1.2 times in kidney, and 1.4 times in the liver homogenates to 1.6 times in serum). The antioxidants (vitamin E and selenium) supplementation decreased lipid peroxidation processes during fever and partly restored the 5'-monodeiodinase activity. The present study confirmed our previous observations in vitro that lipid peroxidation (free radical formation) influences the 5'-monodeiodinase activity in tissues and alters the thyroid hormones metabolism.     

The effect of propylthiouracil on thyroid-stimulating hormone-induced alterations in iodothyronine secretion from perfused dog thyroids

The aim of this study was to see whether the inhibitory effect of propylthiouracil on thyroidal secretion of 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3) could be reproduced in intensively stimulated thyroids, and to elucidate whether an increase in the fractional deiodination of thyroxine (T4) to T3 and rT3 during iodothyronine secretion might be responsible for the transient fall in the T4/T3 and T4/rT3 ratios in thyroid secretion seen in the early phase after stimulation of thyroid secretion. For this purpose T4, T3 and rT3 were measured in effluent from isolated dog thyroid lobes perfused in a non-recirculation system using a synthetic hormone free medium. 1 mmol/1 propylthiouracil induced a significant reduction in thyroid-stimulating hormone (TSH) stimulated T3 and rT3 release while the release of T4 was unaffected. This supports our previous conclusion that T4 is partially monodeiodinated to T3 and rT3 during thyroid secretion. Infusion of 1 mmol/l propylthiouracil for 30 min or 3 mmol/l propylthiouracil for 120 min did not abolish the transient fall in effluent T4/T3 and T4/rT3 induced by TSH stimulation. Thus, this phenomenon seems not to depend on intrathyroidal iodothyronine deiodinating processes.     

Potentiation of thyroxine 5-deiodination by aminotriazole

Aminotriazole, a goitrogen, in addition to its known inhibitory effects on the thyroid, demonstrated a unique effect on peripheral deiodination of thyroxine (T4). In contrast to the well-known peripheral effects of goitrogens such as propylthiouracil in inhibiting 5'-deiodinase activity, i.e., to effect a decrease in T4 to triiodothyronine (T3) conversion, aminotriazole had no effect on the 5'-deiodinative pathway. Rather, this goitrogen appeared to stimulate the alternative pathway, viz. T4 5-deiodination, resulting in an increased reverse triiodothyronine (rT3) serum concentration. This was shown in comparisons of serum T4, T3 and rT3 concentrations and serum T3/T4 and rT3/T4 ratios between rats treated with aminotriazole and T4, and rats treated with T4 alone. The finding that aminotriazole may specifically enhance T4 5-deiodination, independently of T4 5'-deiodination, is novel, as this has not been observed in the case of other goitrogens. It is of interest that this goitrogen is devoid of sulphur, which is a prominent constituent of thiourylene compounds which have been noted to affect 5'-deiodination. The potentiating effect of aminotriazole on 5-deiodination of T4 was not attributable to dietary factors.     

Investigation of thyroxine monodeiodinase activity in the liver, kidney and brown adipose tissue of foetal and neonatal rabbit

The maturation of the 5'- and 5-monodeiodinase system in liver, kidney and brown adipose tissue of rabbits, during the foetal period (from 21 days of gestation to birth) and the neonatal period (from birth to 3 weeks of life) was studied. A sudden increase of 5'- and 5-monodeiodinase activity in liver and kidney 3 days before birth was observed, falling to a nadir at day 3 after birth. Foetal and neonatal serum T4, T3 and rT3 concentration were very low and rose progressively with age, reaching adult values at about day 21. In the foetal brown adipose tissue high 5'-monodeiodinase and low 5-monodeiodinase activity was found. The 5'-monodeiodinase decreased during the first days of life whereas the 5-monodeiodinase activity remained at a low stable level until day 3 when the activities of both enzymes increased. The increase of conversion rate of T4 to T3 and rT3 in liver and kidney well correlate with the triiodothyronines concentration in serum from day 3 after birth.