Deiodase activity in hypothyroid rats]

In rats hypothyroidized with methylthiouracil (MTU), methimazol (MMI), or radiothyroidectomy, the extent of deiodination for L-diiodotyrosine (L-DIT) and L-thyroxine (L-T4) was investigated in homogenate supernatants of liver and kidney. Deiodination in liver and kidney for DIT is twice as high as for T4, but the kidney allows only 25% of the liver deiodination activity both for DIT and T4. In the livers of all hypothyroid animals, iodide splitting both from DIT and T4 is highly significantly reduced by one-half compared with controls. In the kidney of all hypothyroid animals, the DIT deiodination is highly significantly lowered in comparison with controls; the T4 deiodination is significantly reduced only in animals treated with MTU and MMI, and is not significantly enhanced in radiothyroidectomized rats. Thus, there is no difference between MTU and MMI in the extent of deiodination for DIT and T4 in the homogenate supernatants of rat liver or kidney.     

Deiodase activity in rats following an overdose of thyroid hormone]

The L-DIT and L-T4 deiodinating activity in supernatants from liver or kidney homogenates of normal rats or rats loaded with T4, T3 or TSH, was investigated. Deiodination of L-DIT occurs in liver supernatants twice as much as in liver, referring to mg of protein of g of tissue, while the deiodination of L-T4 seems rather equal. The over all rate of deiodination, however, reaches in liver for L-DIT the threefold, for L-T4 the fivefold value, as compared with kidney. Short periods of loading do not alter deiodination of L-DIT in liver or kidney at all; but lead to significant elevation of L-T4-deiodination in liver tissue. Higher doses or longer periods of loading cause a significant rise of L-DIT-deiodination in liver and kidney, while the L-T4-deiodination in liver is significantly decreased and in kidney significantly elevated.     

Differential regulation of beta-adrenergic receptor-coupled adenylate cyclase by thyroid hormones in rat liver and heart: possible role of corticosteroids

Thyroid hormone regulation of beta-adrenergic receptor-coupled adenylate cyclase activity was studied in rat liver and heart particulate fractions. Thyroidectomy (Tx) increased isoproterenol-stimulated cAMP accumulation in the liver and decreased it in the heart. Administration of L-thyroxine (L-T4) or L-3,3',5-triiodothyronine (L-T3) reversed these changes in both liver and heart. The changes observed in liver beta-receptor-coupled adenylate cyclase activity after Tx were similar to those reported after adrenalectomy (ADX). Thus the hypothesis was considered that these changes with altered thyroid status are produced indirectly through alteration in adrenal corticosteroids. Hydrocortisone in Tx rats decreased liver isoproterenol-stimulated adenylate cyclase activity but had no significant effect on the heart. Serum corticosterone levels were decreased significantly (by 34%) in Tx rats, as compared to euthyroid rats. Administration of L-T4 to Tx rats doubled the serum corticosterone levels. In Tx-ADX rats, L-T4 had no significant effect on liver beta-receptor-coupled adenylate cyclase. However, L-T4 significantly increased heart beta-receptor-coupled adenylate cyclase in these animals. Dexamethasone, but not deoxycorticosterone, decreased liver isoproterenol-stimulated cAMP accumulation in Tx animals to the same extent as was observed with L-T4 and hydrocortisone. Thus overall the results indicate that in the liver, as opposed to the heart, thyroid hormones regulate beta-adrenergic receptor-coupled adenylate cyclase indirectly through corticosteroids. Glucocorticoid rather than mineralocorticoid activity seems to be responsible for this regulation.     

Influence of chronic treatment with iopanoic acid on thyroxine metabolism in the newborn rat

Parameters of the peripheral metabolism of thyroxine (T4) were studied in the early postnatal period. Iopanoic acid (IOP) was administered to newborn rats that were either euthyroid or rendered hypothyroid in utero by propylthiouracil (PTU) or methimazole (MMI) administration to the mothers during gestation and injected with thyroxine on postnatal days 6 and 7. In euthyroid newborn rats given IOP from postnatal day 6, the plasma T4 level increased (+50%) while the plasma 3,3',5'-triiodothyronine (T3) level slightly decreased (-18%). Peripheral deiodination of T4 was also reduced (about -50%) as estimated by thyroid 125I uptake after injection of 125I (3'-5')L-T4. In the newborn rats rendered hypothyroid in utero and given T4 on postnatal days 6 and 7, IOP treatment started on day 4 decreased the constant rate of elimination (-50%), the distribution volume (-43%) and the metabolic clearance (-74%) of plasma T4. The results were the same in PTU- and MMI-treated newborn rats. The differences between newborn and adult animals under IOP treatment are discussed.     

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.     

The influence of cultivation on (pro-)insulin biosynthesis and secretion of isolated pancreatic islets of C57BL-mice, long-term treated with glibenclamide in vivo

Weanling male wistar rats were fed 4 weeks a standard diet and separated into 2 groups, which received a high fat diet (50% w fat; HFD) or a low fat diet (3% w fat; LFD). These diets were fed 6-8 weeks and the animals then separated into light and heavy animals in each group. T4 deiodination in liver homogenates was investigated in all groups and compared with T4 clearance rate and thyroidal activity of these animals. The HFD-rats showed independently of weight and body fat content significantly higher liver deiodinase activity than LFD-rats. In light and heavy HFD-rats with great differences in body fat content the liver deiodinase activity was equal. T4 deiodination in liver, contrary to the T4 clearance rate, depends on fat content of diet and not of body. The thyroidal radioiodine uptake and PBI-131-values in some weight groups of HFD-rats were significantly higher than in some LFD-weight-groups, but a dependence of the thyroidal activity from fat content of diet or of body was not clearly evident. The results indicate however, that the thyroidal activity is likely not responsible for the increase of liver deiodinase activity after high fat diet. The apparent discrepancy between the results of higher liver T4 deiodination and equal or lower T4 clearance rate or equal T3 serum concentrations is discussed.     

Na-K-dependent ATPase in red cells and thyroid status

The Relationship between ouabain-sensitive ATPase (Na-K ATPase) activity in erythrocytes and the thyroid status was studied in 36 patients with Graves' disease and 58 patients receiving L-thyroxine (T4) replacement therapy. Forty normal children served as control. Total ATPase activity in 4 untreated hypothyroid patients was significantly reduced (11.0 +/- 4.6 vs 17.3 +/- 4.1 micrograms-P/h/mg-protein, P less than 0.01), and Na-K ATPase was undetectable, both of which were normalized after 4 weeks of L-T4 therapy. Na-K ATPase in hyperthyroid patients was also decreased (0.9 +/- 0.8 vs. 4.0 +/- 2.7, P less than 0.01), but was gradually normalized after 3 months of euthyroid state. Clinically euthyroid children treated with L-T4 were divided into 2 groups with regard to Na-K ATPase activity, normal and low. Analysis of the possible factors producing this difference revealed that, in primary hypothyroidism, the factor appeared to be the endogenous T4 level, while in patients with dwarfism, the secretory capacity of TSH or TSH-releasing hormone (TRH) was contributory. Thus Na-K ATPase activity in red cells remains within the normal range after L-T4 replacement in the presence of a severe degree of primary hypothyroidism or in association with secondary or tertiary hypothyroidism. Other factors such as the L-T4 dose, duration of the therapy, serum T4 and T3 concentrations, were not significantly different in the two groups. These results indicate that (1) Na-K ATPase in red cells is decreased in hyper- or hypothyroid state, (2) restoration of normal activity requires 1-3 months of euthyroid period, and (3) it is a sensitive index of peripheral thyroid status over the preceding few months.     

Thioredoxin and glutaredoxin systems of rat liver cytosol are not influenced by thyroid dysfunction

The relation of thyroid hormone status to the function of hepatic cytosolic components activating microsomal reverse triiodothyronine (rT3) 5'-monodeiodination was studied in rats. Hyperthyroidism was induced by administration of thyroxine and hypothyroidism, by thyroidectomy. The DTT-stimulated microsomal rT3 5'-monodeiodination rate was increased by 125% in hyperthyroid rats and reduced to about 30% in hypothyroid rats (when compared with euthyroid animals). Thyroid status was unrelated to NADPH-dependent activation of microsomal 5'-deiodinase by cytosol components or to cytosolic concentrations of thioredoxin and glutaredoxin, which stimulate in vitro microsomal deiodination of thyroid hormone.     

Optimum replacement dose of thyroid hormone assessed by highly sensitive TSH determination in patients with congenital hypothyroidism

Serum thyroid hormone concentrations were measured in 100 samples from 25 patients with congenital hypothyroidism who were clinically well while receiving L-T4 therapy. Thyroxine concentrations were significantly higher than those of controls (p less than 0.01), while triiodothyronine was not significantly different. These samples were divided into four groups according to serum thyroid stimulating hormone concentrations as measured by highly sensitive immunoradiometric assay (IRMA-TSH). Serum thyroid hormone concentrations were compared among groups. The replacement dose of L-T4 and serum thyroid hormone in groups with undetectable IRMA-TSH were significantly higher than those in groups with normal or increased IRMA-TSH. These results show that serum thyroxine concentrations increase in most patients with congenital hypothyroidism on L-T4 therapy. Therefore, thyroxine concentrations above normal are not necessarily of clinical significance if IRMA-TSH is detectable. Undetectable IRMA-TSH might indicate the necessity for a reduction in the L-T4 replacement dose in patients with congenital hypothyroidism.     

Effect of diet and L-thyroxine on deiodination of L-thyroxine in rat liver

Male Wistar rats on 3 or 50% fat diet for 14 weeks were treated during 6 weeks with L-thyroxine (5 or 25 or 50 microgram 100 g body weight/2 times weekly, sc.) and the activity of L-thyroxine-deiodinase was determined in supernatans of liver homogenates. With increasing thyroxine lading the deiodinating activity increases statistically significantly within each diet group. The liver of animals on 50% fat diet deiodinates increasing thyroxine doses to a lesser extent than livers of animals on 3% fat diet. It is discussed that high fat diet influences thyroid function and that thyroxine shares effectiveness and undergoes deiodination also in other tissues, probably in fat tissue, to a higher extent.     

Effect of thyroid hormone therapy on plasma insulin-like growth factor I levels in normal subjects, hypothyroid patients and endemic cretins

The effect of thyroid hormone therapy (L-T4 or L-T3) on plasma immunoreactive insulin-like growth factor I (somatomedin C, Sm-C) concentrations was studied in 8 normal controls, 14 primary hypothyroid subjects and in 7 patients with endemic cretinism. In normals basal levels of Sm-C (1.56 +/- 0.77 U/ml) increased to (2.46 +/- 1.0 U/ml; L-T4) and to (2.9 +/- 0.95 U/ml; L-T3). Plasma Sm-C basal levels were significantly lower in primary hypothyroid subjects (0.81 +/- 0.48 U/ml) and increased to 2.54 +/- 1.43 U/ml (L-T4) and to 2.16 +/- 0.83 U/ml (L-T3). A significant and positive correlation (r = 0.56) was found between Sm-C and serum T4 and T3 concentrations. Plasma Sm-C concentrations in endemic cretinism were initially normal in 4 patients, but low in the remaining 3 (mean +/- SD: 1.18 +/- 0.63 U/ml) and did not increase after 12 months (1.34 +/- 0.61 U/ml) or 18 months (1.01 +/- 0.43 U/ml) of L-T4 and L-T3 therapy. Plasma T4 levels and free T4 increased considerably in EC after therapy with a significant decrease in the previously elevated plasma TSH concentrations. The subnormal response of plasma Sm-C during effective thyroid thyroid hormone therapy could be an additional factor involved in growth failure of endemic cretins.     

Effects of elevated gonadotropin levels on thyroid function in the male rat

In order to determine if thyroid disease in hypogonadal men is the result of chronic elevation of serum gonadotropins, thyroid histology and serum thyroid hormone levels were evaluated in male rats that had been castrated either 2 weeks or 15 months previously. Despite significantly elevating serum LH levels, castration did not affect thyroid structure or function. Serum total T4 levels were reduced with age in both short and long-term castrate animals but returned to the levels seen in young rats when testosterone was replaced. Testosterone replacement also increased free T4 levels in both the young and old castrate rats. Neither age nor testosterone replacement affected serum T3 or TSH levels.     

Influence of thyroid hormone on glycogen metabolism in rat liver.

The livers removed from thyroidectomized and L-T4 supplemented rats were rapidly frozen by Freon-12 chilled with liquid nitrogen, and concentrations of metabolites which affect glycogen synthetase and phosphorylase were determined. Serum and liver glycose levels were not changed in any thyroid functioning. But liver G6P and ATP were increased by thyroidectomy and decreased by L-T4 supplement, while cAMP was increased by the hormone supplement. The "enzyme activity" ratio of glycogen synthetase a to phosphorylase a was increased by thyroidectomy and decreased by L-T4 supplement. The most intimate correlation was observed between the "enzyme activity" ratio and the ratio of the "energy charge" ratio of cAMP among other indices calculated from changes in the metabolite concentrations in the various thyroid functioning. The change in the substrate levels brought about by thyroidectomy and L-T4 supplement appeared to modulate both the enzyme activities which in turn regulate the glycogen metabolism.     

Therapeutic Potential of Date Palm Pollen for Testicular Dysfunction Induced by Thyroid Disorders in Male Rats

Hyper- or hypothyroidism can impair testicular function leading to infertility. The present study was designed to examine the protective effect of date palm pollen (DPP) extract on thyroid disorder-induced testicular dysfunction. Rats were divided into six groups. Group I was normal control. Group II received oral DPP extract (150 mg kg^-1), group III (hyperthyroid group) received intraperitoneal injection of L-thyroxine (L-T4, 300μg kg^-1; i.p.), group IV received L-T4 plus DPP extract, group V (hypothyroid group) received propylthiouracil (PTU, 10 mg kg^-1; i.p.) and group VI received PTU plus DPP extract. All treatments were given every day for 56 days. L-T4 or PTU lowered genital sex organs weight, sperm count and motility, serum levels of luteinizing hormone (LH), follicle stimulating hormone (FSH) and testosterone (T), testicular function markers and activities of testicular 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD). Moreover, L-T4 or PTU increased estradiol (E2) serum level, testicular oxidative stress, DNA damage and apoptotic markers. Morphometric and histopathologic studies backed these observations. Treatment with DPP extract prevented LT4- or PTU induced changes. In addition, supplementation of DPP extract to normal rats augmented sperm count and motility, serum levels of LH, T and E2 paralleled with increased activities of 3β-HSD and 17β-HSD as well as testicular antioxidant status. These results provide evidence that DPP extract may have potential protective effects on testicular dysfunction induced by altered thyroid hormones.     

Identification of essential histidine residues in rat type I iodothyronine deiodinase.

Deiodination is required for conversion of thyroxine, the inactive prohormone secreted by the thyroid gland, to 3,5,3'-triiodothyronine, the biologically active thyroid hormone. The principal enzyme catalyzing this reaction, Type I iodothyronine 5' deiodinase, was shown recently to contain the amino acid, selenocysteine, and site-directed mutagenesis showed that this amino acid confers the biochemical properties characteristic of this enzyme. Previous studies suggest that a histidine residue may also be critical for activity. To further our understanding of the biochemical mechanism of this reaction, we have used in vitro mutagenesis to examine the contribution of each of the 4 histidines in this enzyme to the deiodination process. Two of the histidines (185 and 253) are not involved in deiodination, as their removal had no effect on activity. Mutagenesis of histidine 158 resulted in complete loss of activity, suggesting a role in either protein conformation or catalysis. The most informative results were obtained from the studies of histidine 174. Mutagenesis of this histidine to asparagine or glutamine altered reactivity with substrate and reduced inhibition by diethylpyrocarbonate and rose bengal. These results demonstrate that histidine 174 is critical to function and appears to be involved in binding of hormone.     

Determination of serum thyroxine enantiomers in patients by liquid chromatography with a chiral mobile phase

A chromatographic method for the separation and determination of D- and L-thyroxine enantiomers (D-, and L-T4) in human serum with a chiral ligand ion-exchange system using a chiral mobile phase additive and a silica column was established. An aqueous eluent containing L-proline (L-pro) sufficiently complexed copper II ions and triethylamine (TEA) was used. It was monitored with a UV detector. The separation was completed in 12 min. The method has acceptable sensitivity, precision and accuracy for analysis. The limit of detection and the limit of quantitation for both D- and L-T4 were 0.1 microg/ml and 0.8 microg/ml, respectively. Calibration curves were linear within 1-100 microg/ml; the mean correlation coefficients were r(D-T4)=0.9986 for D-T4 and r(L-T4)=0.9978 for L-T4. T4 enantiomers were separated on baseline under the optimum condition. L-T4 eluted before D-T4. The concentration of D-T4 and L-T4 in 45 thyroid patients serum (hyperthyroid, hypothyroid, thyroidectomy, goitre or thyroiditis) using HPLC was determined, those results showed that D,L-T4 concentration varied in different thyroid patient. Attention should be paid to this result in treating thyroid disease in the clinic.     

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.     

Hormone synthesis and storage in the thyroid of human preterm and term newborns: effect of thyroxine treatment.

Iodine and thyroglobulin concentrations, as well as iodine, T3, T4 and sialic acid contents of thyroglobulin, were measured in thyroid glands collected postmortem from 42 human premature or term newborns and infants. Three groups were considered: very preterm newborns (24-32 postmenstrual weeks, < 5 days postnatal life), preterm and term newborns (34-41 postmenstrual weeks, < 5 days postnatal life) and infants (born at term, postnatal age 1-8 months). Five very preterm and seven preterm newborns received a daily dose of 10 microg/kg L-T4 for at least 3 days. Thyroid weight and sialic acid content of thyroglobulin progressed with maturation. Intrathyroidal concentrations of iodine and thyroglobulin did not increase significantly before the 42nd week of postmenstrual age. The level of thyroglobulin iodination increased during the postnatal life, except in the very preterm neonates. T4 and T3 content of thyroglobulin was directly proportional to its degree of iodination and positively related to its sialic acid content. L-T4 treatment of preterm newborns increased thyroglobulin iodination and T4-T3 content, without increasing thyroglobulin concentration in the thyroid. It was concluded that the storage of thyroglobulin and iodine in the thyroid develops around term birth. This, associated with the resulting rapid theoretical turnover of the intrathyroidal pool of T4 in Tg, could be an important factor of increased risk of neonatal hypothyroxinemia in the premature infants. The L-T4 treatment of preterm newborns does not accelerate the maturational process of the thyroid gland.     

TSH and prolactin secretions in Hashimoto's thyroiditis following withdrawal of thyroid hormone therapy

Changes in the pituitary-thyroid axis in patients with Hashimoto's thyroiditis following withdrawal of thyroid suppressive therapy were analyzed. The group of patients with thyroid adenoma served as control (group I). Patients with Hashimoto's thyroiditis were divided into 2 groups on the basis of serum TSH levels 8 weeks after discontinuing the exogenous thyroid hormone (group II, less than 10 microunits/ml; group III, more than 10 microunits/ml). During treatment with L-T4(200 micrograms/day) or L-T3(50 micrograms/day), there was no significant difference in serum T4-I and T3 levels among the three groups. Following L-T4 withdrawal, basal serum TSH levels were higher at 2 to 8 weeks in groups II and III than in group I. Serum TSH response to TRH was greater at 4 to 8 weeks in groups II and III than in group I. Following L-T3 withdrawal, basal serum TSH levels were higher at 1 and 2 weeks in group II than in group I, while those of group III were consistently higher during the study. Higher TSH responses to TRH were observed at 1 to 8 weeks in groups II and III. Neither basal nor TRH-induced prolactin (PRL) secretion differed significantly among the three groups. We have demonstrated that pituitary TSH secretion in patients with Hashimoto's thyroiditis is affected more by withdrawal of thyroid hormone therapy than in patients with thyroid adenoma. In addition, the present findings suggest a difference between the sensitivity of thyrotrophs and lactotrophs in Hashimoto's thyroiditis after prolonged thyroid therapy is discontinued.     

Long-term effects of malnutrition during lactation on the thyroid function of offspring.

Some studies have shown that the mother's nutritional condition may influence offspring's endocrine function through metabolic imprinting. Recently, we showed that the kind of maternal malnutrition during lactation affects adult body weight of the offspring and it is related to milk composition. We studied lactating rats fed an 8 % protein-restricted diet (PR), a control 23 % protein diet (C), and an energy-restricted diet group (ER). After weaning, all animals received a normal diet until they were 180 days of age. At this time, the animals received a single i. p. injection of (131)I and were sacrificed 2 h after the injection. Total triiodothyronine (TT3) and total thyroxin (TT4) serum concentrations were measured by enzyme immunoassay. The PR group had significantly a higher thyroid (131)I uptake, TT3 serum concentration and in TT4 serum concentration, compared to the controls. The ER group had only significantly higher TT3 serum concentration. These results showed that thyroid function regulation in adulthood may depend on maternal nutritional condition during lactation. Probably, PR group had a high thyroid function, whereas the ER group only had an increase in the deiodination of T4. The hyperthyroidism in the PR group could explain the low body weight observed in those animals.