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This study was conducted to evaluate the effects of single and combined deficiencies of selenium and iodine on selenoenzyme activities in sheep. Twenty-four male lambs were assigned to one of four semisynthetic diets: combined deficient A (Se-I), Se-deficient B (Se-I+), I-deficient C (Se+I-), and basal diet D (Se+I+). Thyroid hormones (T3, T4), thyroid stimulating hormone (TSH), and inorganic iodine (PII) were determined in plasma. Selenium and glutathione peroxidase activity (GSH-Px) were determined in erythrocytes, and tissue samples, including the thyroid, liver, kidney, and brain, were taken for selenoenzyme analysis. Plasma T3, T4, and TSH concentrations were similar in all groups. Type I deiodinase (ID-I) activity in liver and kidney remained unchanged in Se or I deficiency. In contrast, hepatic ID-I activity was increased by 70% in combined Se-I deficiency. Thyroidal cystolic GSHPx (c-GSH-Px) and phospholipid GSH-Px (ph-GSH-Px) activities remained constant in both Se-deficient groups, whereas thyroidal c-GSH-Px activity increased (57%) in I deficiency. Type II deiodinase (ID-II) activity was not detectable in the cerebrum and cerebellum, whereas cerebellum Type III deiodinase (ID-III) activity was decreased in I deficiency and combined Se-I deficiencies. The results of the present study support a sensitive interaction between Se and I deficiencies in sheep thyroid and brain. Furthermore, the lack of thyroidal ID-I activity, the preservation of the thyroidal antioxidant enzymes, and the increases in hepatic ID-I indicate that a compensatory mechanism(s) works toward retaining plasma T3 levels, mostly by de novo synthesis of T3 and peripheral deiodination of T4 in Se- and I-deficient sheep.

The presence or absence of types I, II and III iodothyronine monodeiodinase enzymes (MDI, MDII and MDIII) and their levels of activity in the skin of goats, which were orally dosed for 60 days with 0, 1.1, 2.2, 4.4, 8.8, 17.5, or 35 mg(-1)kg liveweight day(-1)of the anti-thyroid, enzyme-inhibiting drug, propylthiouracil (PTU), were determined. Contrary to our earlier report that PTU did not influence skin MDII activity, the currect more thorough investigation (in terms of numbers of observations and the efficiency of the enzyme extraction procedure) indicated that doses of 1.1.to 17.5 mg kg(-1)liveweight induced a 2 to 3 fold increase (P = 0.01) in MDII activity. However, in three of the four goats treated with 35 mg kg(-1)group, activity was similar to that of control animals. There were no significant differences between treatments in MDIII activity but there was a trend towards lower levels of activity in the goats dosed with 17.5 and 35 mg kg(-1). It is concluded that there is significant MDII and MDIII activity in the skin of goats and that although there is none of the PTU -sensitive MDI enzyme, synthesis of T3 within the skin could nevertheless be modified through increases in MDII activity induced by lower T4 concentrations in the circulation caused by PTU. Changes in pattern of fibre moult induced by treatment with low doses of MD-inhibiting drugs may therefore be achieved through this effect. Since MDII and MDIII enzyme activity may be reduced by high doses of PTU, prolonged treatment with high doses of PTU may have adverse effects on skin tissue., (Copyright 2000 Harcourt Publishers Ltd.)

The trace elements selenium and iodine are both essential for normal thyroid hormone metabolism. To investigate the relationships between these functions, heifers were maintained on iodine-deficient or iodine-sufficient diets from mid pregnancy to term. In these heifers and their offspring the interrelationship between iodine and selenium was apparent with the preferential 10- to 12-fold induction of the selenoenzyme, thyroidal type I, selenium-containing iodothyronine deiodinase activity by iodine deficiency. This was accompanied by two- to four-fold increases in cytosolic glutathione peroxidase activity, probably reflecting increased oxidative activity and metabolism in the thyroid gland in response to iodine deficiency. The above selenoenzyme activities were not affected in liver, kidney, pituitary and brain by iodine deficiency. The results are consistent with a critical role for selenium in both the normal function of cattle thyroid and key enzymes to compensate for the effects of iodine deficiency.

Adequate dietary iodine supplies and thyroid hormones are needed for the development of the central nervous system (CNS) and brown adipose tissue (BAT) function. Decreases in plasma thyroxine (T4) concentrations may increase the requirement for the selenoenzymes types I and II iodothyronine deiodinase (ID-I and ID-II) in the brain and ID-II in BAT to protect against any fall in intracellular 3,3',5 tri-iodothyronine (T3) concentrations in these organs. We have therefore investigated selenoenzyme activity and expression and some developmental markers in brain and BAT of second generation selenium- and iodine-deficient rats. Despite substantial alterations in plasma thyroid hormone concentrations and thyroidal and hepatic selenoprotein expression in selenium and iodine deficiencies, ID-I, cytosolic glutathione peroxidase (cGSHPx) and phospholipid hydroperoxide glutathione peroxidase (phGSHPx) activities and expression remained relatively constant in most brain regions studied. Additionally, brain and pituitary ID-II activities were increased in iodine deficiency regardless of selenium status. This can help maintain tissue T3 concentrations in hypothyroidism. Consistent with this, no significant effects of iodine or selenium deficiency on the development of the brain were observed, as assessed by the activities of marker enzymes. In contrast, BAT from selenium- and iodine deficient rats had impaired thyroid hormone metabolism and less uncoupling protein than in tissue from selenium- and iodine-supplemented animals. Thus, the effects of selenium and iodine deficiency on the brain are limited due to the activation of the compensatory mechanisms but these mechanisms are less effective in BAT.

The stimulation of thyroid hormone synthesis in iodine deficiency may increase the requirement for the selenoproteins which are involved in thyroid hormone synthesis in the thyroid gland. Selenoenzyme activity and expression were investigated in the thyroid and liver of second generation selenium-and/or iodine-deficient rats. Selenium deficiency caused substantial decreases in hepatic selenium-containing type I iodothyronine deiodinase (ID-I) and cytosolic glutathione peroxidase (cGSHPx) activities and mRNA abundances, but phospholipid hydroperoxide glutathione peroxidase (phGSHPx) activity was only 55% of selenium-supplemented control levels, despite the absence of change in its mRNA abundance. Selenoenzyme mRNA concentrations were maintained at control levels in thyroid glands from the selenium-deficient rat pups. Despite this, a differential effect was observed in selenoenzyme activities: ID-I activity was decreased to 61%, cGSHPx activity to 45% and phGSHPx to 29% of that in selenium-adequate controls. In iodine-deficient thyroid glands, mRNA levels were increased 2.2, 5.0 and 2.8 times for ID-I, cGSHPx and phGSHPx respectively. ID-I and cGSHPx enzyme activities were also increased but the activity of phGSHPx was decreased despite the high mRNA abundance. Thyroid selenoprotein mRNA levels were also increased in combined selenium and iodine deficiency but again there were differential effects on enzyme activities, with ID-I activity increased, cGSHPx unchanged and phGSHPx decreased. Thus, iodine deficiency may produce an oxidant stress on the thyroid gland, increasing the requirement for selenium to maintain selenoenzyme activity. When dietary supplies of selenium are limiting, thyroid selenoprotein mRNA levels are increased to compensate for overall lack of the micronutrient. Furthermore, there is a preferential supply of available selenium to ID-I and cGSHPx to allow maintenance of thyroid function.

Regulation of synthesis of the selenoenzymes cytosolic glutathione peroxidase (GSH-Px), phospholipid hydroperoxide glutathione peroxidase (PHGSH-Px) and type-1 iodothyronine 5'-deiodinase (5'IDI) was investigated in liver, thyroid and heart of rats fed on diets containing 0.405, 0.104 (Se-adequate), 0.052, 0.024 or 0.003 mg of Se/kg. Severe Se deficiency (0.003 mg of Se/kg) caused almost total loss of GSH-Px activity and mRNA in liver and heart. 5'IDI activity decreased by 95% in liver and its mRNA by 50%; in the thyroid, activity increased by 15% and mRNA by 95%. PHGSH-Px activity was reduced by 75% in the liver and 60% in the heart but mRNA levels were unchanged; in the thyroid, PHGSH-Px activity was unaffected by Se depletion but its mRNA increased by 52%. Thus there is differential regulation of the three mRNAs and subsequent protein synthesis within and between organs, suggesting both that mechanisms exist to channel Se for synthesis of a particular enzyme and that there is tissue-specific regulation of selenoenzyme mRNAs. During Se depletion, the levels of selenoenzyme mRNA did not necessarily parallel the changes in enzyme activity, suggesting a distinct mechanism for regulating mRNA levels. Nuclear run-off assays with isolated liver nuclei showed severe Se deficiency to have no effect on transcription of the three genes, suggesting that there is post-transcriptional control of the three selenoenzymes, probably involving regulation of mRNA stability.

The effects of TSH and the activation of the cyclic AMP (cAMP) and Ca(2+)-phosphatidylinositol (Ca(2+)-PI) cascades on the activity and expression of the selenoenzyme thyroidal type-I iodothyronine deiodinase (ID-I) have been studied using human thyrocytes grown in primary culture. Stimulation of ID-I activity and expression was obtained with TSH and an analogue of cAMP, 8-bromo-cAMP. In the presence or absence of TSH, the addition of the phorbol ester, phorbol 12-myristate 13-acetate (PMA) together with the calcium ionophore A23187, caused a decrease in ID-I activity; a decrease in ID-I expression was also observed as assessed by cell labelling with [75Se]selenite. PMA alone had no effect on ID-I activity in the presence or absence of TSH. A23187 alone produced a small but significant reduction in ID-I activity, but only in TSH-stimulated cells. These data provide evidence that the expression of thyroidal ID-I is negatively regulated by the Ca(2+)-PI cascade, and positively regulated by the cAMP cascade.

The effects of dietary selenium deficiency on hepatic and thyroidal type I iodothyronine deiodinase (ID-I) and selenium-dependent glutathione peroxidase (GPx) activities have been studied in weanling rats. In selenium-deficient animals hepatic ID-I activity was reduced to 11% of the activity found in the selenium-replete groups, whilst thyroidal ID-I activity increased by 42%. Hepatic and thyroidal GPx activities were also reduced by selenium deficiency to approximately 0.6 and 70%, respectively, of the values found in the selenium-replete animals. We have also studied the effects of thyrotropin (TSH), and selenium supply on the activity of IDI and GPx in human thyrocytes grown in primary culture. When thyrocytes were grown in selenium-deficient (< 1 nmol l-1 Se) medium in the absence of TSH, addition of sodium selenite up to 1000 nmol l-1 had little or no effect on ID-I activity. In the absence of added selenite, TSH addition produced a significant increase in ID-I activity and this stimulation was increased further when selenite was added at concentrations of 50-1000 nmol l-1 with an optimal effect on ID-I activity being observed at a 500 nmol l-1. Selenium content and GPx activity in human thyrocytes grown in selenium-free media (selenium content < 1 nmol l-1) were not significantly lower than the corresponding measurements made in cells grown in media containing selenium at a concentration of 5.4 nmol l-1.(ABSTRACT TRUNCATED AT 250 WORDS)

The capacity to generate the active thyroid hormone 3,3',5-triiodothyronine (T3) from thyroxine (T4) has been assessed in the major adipose tissue depot (perirenal) of newborn goats through measurements of iodothyronine 5'-deiodinase activity. High activity of the enzyme was found in the perirenal adipose tissue. Developmental studies demonstrated that there was a rise, then a fall, in iodothyronine 5'-deiodinase activity (per milligram of protein) over the first week of postnatal life, with a further decline between the second and third weeks. At 3 wk of age, the activity per gram of tissue was only 2% of that of the newborn; however, because of age-related increases in the lipid content of the perirenal adipose tissue, the activity per cell (per microgram of DNA) at 3 wk of age was approximately 10% of the activity in the newborn. Kinetic studies and inhibition characteristics with propylthiouracil, gold thioglucose, and T4 indicated that the iodothyronine 5'-deiodinase in goat perirenal adipose tissue is of the type I form; there was no evidence for the type II enzyme. Affinity labeling of the iodothyronine 5'-deiodinase with bromoacetyl-rT3 or bromoacetyl-T4 was consistent with this view. In contrast to goats, only the type II enzyme is evident in rats and mice. It is concluded that the perirenal (brown) adipose tissue is likely to be a significant source of T3 for other tissues in newborn goats and that there are major species variations in the form of iodothyronine 5'-deiodinase present in brown fat, with the goat exclusively exhibiting type I.

Two types of iodothyronine deiodinase (ID-I and ID-II) catalyse the 5'-deiodination of thyroxine (T4) to produce the biologically active triiodothyronine (T3). Under normal circumstances ID-I in liver and kidney provides the main source of T3 to the circulation, whilst ID-II is largely responsible for local T3 production in the CNS, brown adipose tissue and pituitary. In some circumstances ID-II in brown adipose tissue and ID-I in the thyroid may provide a significant source of plasma T3, and ID-I in the pituitary may be important for local T3 production in this gland. The IDs thus play a pivotal role in controlling the supply of T3 to the nuclear receptors. ID-I is a selenoenzyme and, although ID-II activity is reduced in selenium deficiency, this is a consequence of increased plasma T4 concentration, rather than ID-II activity being directly dependent on selenium. Changes in 5'-deiodination occur in a number of situations such as poor nutrition, illness, iodine and selenium deficiency, and drug therapy. In iodine deficiency these changes appear to have evolved to ensure that the plasma T3 level is maintained and also to provide the brain with a degree of protection from hypothyroxinaemia. Relatively little is known about the importance of selenium deficiency on thyroid function in humans but, in combination with iodine deficiency, selenium deficiency may prove to be a contributing factor in the pathogenesis of myxodematous cretinism. The changes that occur in ID-I and ID-II in illness produce abnormalities in thyroid function tests which, although of no direct clinical significance, may lead to interpretative problems.

Subcutaneous adipose tissue of neonatal lambs has been examined for the presence of markers diagnostic of thermogenic brown fat. Uncoupling protein, uncoupling protein mRNA, and iodothyronine 5'-deiodinase activity were each detected in subcutaneous adipose tissue, as well as in the major internal fat depot (perirenal), of newborn lambs. These brown fat markers were not present, however, in adipose tissue of adult sheep. It is concluded that subcutaneous fat in newborn lambs is functionally 'brown', and similar to the internal fat; subcutaneous and internal adipose tissues follow a similar developmental path--from 'brown' to 'white'.

We have studied the origin of tri-iodothyronine (T3) secreted by human and sheep thyrocytes in primary culture and also the expression of type-I thyroidal iodothyronine deiodinase (ID-I) in the thyroid and liver of man and various other animals. Inhibitors of ID-I reduced T3 secretion from human but not sheep thyrocytes. In contrast, inhibitors of de-novo thyroid hormone synthesis reduced both thyroxine (T4) and T3 production in sheep thyrocytes, but had no effect on the T3 secreted by human thyrocytes. Human thyrocytes did not produce T4 under the culture conditions used, although some endogenous T4 was present in the cells following their isolation. Although thyrotrophin (TSH) stimulated T3 production in both human and sheep thyrocytes, iodine in the form of potassium iodide was only essential for T3 and T4 production by the sheep cells. Although 125I from Na125I was incorporated into T3 and T4 in TSH-stimulated sheep thyrocytes, no 125I incorporation into T3 or T4 was detected in TSH-stimulated human thyrocytes. Using activity measurements and affinity labelling, ID-I was present in the livers of all species studied, but ID-I could not be detected in thyroid tissue from cattle, pigs, sheep, goats, rabbits, deer or llamas. In contrast, thyroid tissue from man, mice, guinea-pigs and rats had significant ID-I activity and expressed an affinity-labelled protein with a molecular mass of approximately 28.1 kDa on SDS-PAGE. These data show that under the culture conditions used, sheep thyrocytes produced T3 by de-novo synthesis, whilst human thyrocytes produced T3 by deiodination of endogenous T4. We conclude that thyroidal ID-I shows marked species difference in its expression and that, in those species which express the enzyme (man, mice, guinea-pigs and rats, in this study), it appears that it may make an important contribution to thyroidal T3 production.

Much research into the functions of selenium in the cell has concentrated on its role in selenium-containing glutathione peroxidases. However, selenium was recently shown to be an essential component of type I iodothyronine 5'-deiodinase in rats, which converts thyroxin to the more biologically active hormone 3,5,3'-triiodothyronine. Thus, selenium-deficient rats have low tissue deiodinase activities and abnormal thyroid hormone metabolism. The discovery of this function for selenium in thyroid hormone metabolism has important implications for the interpretation of the effects of selenium deficiency, especially in individuals with an adequate vitamin E status.

In selenium-deficient rats, peripheral T4 to T3 conversion is markedly decreased due to the loss of the selenoprotein, type I iodothyronine 5'-deiodinase (5'D-I). Despite the marked increase in circulating T4 that results from this loss of 5'D-I, serum T3 concentrations in selenium-deficient rats remain in the normal range. To determine the physiological mechanism(s) that maintains circulating T3 when peripheral T4 to T3 conversion is impaired, we examined the interrelationships between selenium intake and the metabolism of T3 and T4 in the rat. In euthyroid rats, selenium deficiency caused the expected loss of 5'D-I, with a 52% increase in serum T4, which paralleled an increase in the T4 biological half-life. Consistent with the prolonged t1/2 of T4, short term thyroidectomy (48 h) in selenium-deficient rats failed to decrease serum T4 concentrations to the levels observed in short term thyroidectomized, selenium-supplemented rats. Short term thyroidectomy also caused an expected 33% decrease in liver 5'D-I and a 44% increase in brain type II iodothyronine 5'-deiodinase (5'D-II) activities in selenium-supplemented rats. However, in selenium-deficient rats, short term thyroidectomy did not affect 5'D-I or 5'D-II activities. In contrast to the selenium-dependent changes in circulating T4 levels, little or no change in circulating T3 concentrations occurred. There was a 20% increase in the T3 half-life in selenium-deficient rats. The serum T3 sulfate concentration was increased, and T3 deiodination was reciprocally decreased in the selenium-deficient rats. These data suggest that increased T3 sulfate generation in selenium-deficient rats may lead to greater T3 availability through enterohepatic recycling of the iodothyronine and may explain why there are only minor changes in serum T3 concentrations in selenium-deficient rats.

Selenium deficiency in rats is characterized by elevated serum T4 and decreased serum T3 concentrations, and low liver type I (5'D-I) and brain type II (5'D-II) iodothyronine 5'-deiodinase activities. These findings are partially explained by the demonstration that type I 5'D is a selenoprotein; however, 5'D-II does not contain selenium. Since 5'D-II varies inversely with serum T4 concentrations, and serum T4 is elevated in selenium deficiency, the decreased cerebrocortical 5'D-II activity may be secondary to the increased serum T4 levels. To determine the mechanism(s) by which selenium influences 5'D-II activity, we examined the effects of altered selenium intake on brain 5'D-II levels and enzyme turnover in euthyroid and thyroidectomized rats. Rats were fed a selenium-supplemented or selenium-deficient diet for 5 weeks from weaning; half of the animals were also thyroidectomized 3 weeks before death. Selenium deficiency was confirmed by decreased liver and brain glutathione peroxidase activities. In euthyroid rats, selenium deficiency caused a 38% increase in serum T4, and 91% and 39% decreases in 5'D-I and 5'D-II, respectively, compared to those in selenium-supplemented rats. In the thyroidectomized hypothyroid rats, selenium deficiency caused a 60% decrease in 5'D-I, but had no effect on 5'D-II activity, fractional turnover of the enzyme, or the calculated enzyme synthesis rate. The lack of effect of selenium deficiency on 5'D-II levels in hypothyroid rats is consistent with the finding that 5'D-II is not a seleno-enzyme. Thus, the decrease in brain and pituitary 5'D-II activity in selenium-deficient euthyroid rats is due to the T4-dependent increase in the turnover of the enzyme polypeptide.

Selenium deficiency impairs thyroid hormone metabolism by inhibiting the synthesis and activity of the iodothyronine deiodinases, which convert thyroxine (T4) to the more metabolically active 3,3'-5 triiodothyronine (T3). Hepatic type I iodothyronine deiodinase, identified in partially purified cell fractions using affinity labeling with [125I]N-bromoacetyl reverse triiodothyronine, is also labeled with 75Se by in vivo treatment of rats with 75Se-Na2SeO3. Thus, the type I iodothyronine 5'-deiodinase is a selenoenzyme. In rats, concurrent selenium and iodine deficiency produces greater increases in thyroid weight and plasma thyrotrophin than iodine deficiency alone. These results indicate that a concurrent selenium deficiency could be a major determinant of the severity of iodine deficiency.

Selenium deficiency in rats for a period of up to 6 weeks inhibited both the production of 3,3',5-tri-iodothyronine (T3) from thyroxine (T4) (5'-deiodination) and also the catabolism of T3 to 3,3'-di-iodothyronine (5-deiodination) in liver homogenates. The hepatic stores of T3 were decreased by only 8% in selenium deficiency, despite the T3 production rate from T4 being only 7% of the rate found in selenium-supplemented rats. Hepatic glutathione S-transferase (GST) activity was increased in both hypothyroidism and selenium deficiency, but apparently by different mechanisms, since mRNA expression for this family of enzymes was lowered by hypothyroidism and increased in selenium deficiency. It is concluded that, since both T3 production and catabolism are inhibited by selenium deficiency, there is little change in hepatic T3 stores, and therefore the changes in the activity of certain hepatic enzymes, such as GST, that are found in selenium deficiency are not the result of tissue hypothyroidism.

The activity of the type II iodothyronine 5'-deiodinase enzyme in brown adipose tissue has been examined in rats-fed a selenium-deficient diet. Iodothyronine 5'-deiodinase activity was threefold lower in brown adipose tissue of deficient rats than in control animals. The activity of glutathione peroxidase, a biochemical index of selenium deficiency, was also greatly decreased in deficient animals. Cytochrome oxidase activity in brown fat was, however, unaltered by selenium deficiency. Acute exposure to cold (4 degrees C for 18 h) resulted in a substantial increase in iodothyronine 5'-deiodinase activity in brown adipose tissue of control rats, but the stimulatory effect of cold was attenuated in selenium-deficient animals. These results support the concept that the iodothyronine 5'-deiodinases are selenium-dependent enzymes, and indicate that the thermogenic response to cold may be impaired in selenium deficiency.

We determined protein disulphide-isomerase (PDI) and iodothyronine deiodinase (ID-I) activities in liver homogenates from rats subjected to selenium (Se) and/or iodine deficiencies and food restriction. Additionally, the effects of propylthiouracil (PTU) on the enzymes were studied in vivo and in vitro. Selenium deficiency markedly inhibited ID-I activity, but had no significant effects on PDI. Iodine deficiency resulted in a 1.6-fold stimulation in ID-I and a 1.2-fold stimulation in PDI activities. ID-I was much more sensitive than PDI to the inhibitory effects of PTU both in vitro and in vivo. By using a 3,3',5'-tri[125I]iodothyronine affinity label, two major protein bands were identified when hepatic microsomal fractions from Se-sufficient rats were subjected to SDS/PAGE and autoradiography. These bands had molecular masses of 55 and 27.5 kDa, which are similar to those of PDI and ID-I respectively. Selenium deficiency resulted in the loss of the 27.5 kDa band, but did not affect the intensity of the 55 kDa band. These results are consistent with the changes in PDI and ID-I enzyme activities. Previous studies have shown that 75Se may be incorporated in vivo into the 27.5 kDa protein band. This, taken together with our observation that Se is required for the expression of ID-I and the 27.5 kDa protein band, strongly suggests that ID-I is a selenoprotein.

Selenium (Se) deficiency decreased by 8-fold the activity of type 1 iodothyronine 5'-deiodinase (ID-I) in hepatic microsomal fractions from rats. Solubilized hepatic microsomes from rats injected with 75Se-labelled Na2SeO3 4 days before killing were found by chromatography on agarose gels to contain a 75Se-containing fraction with ID-I activity. PAGE of this fraction under reducing conditions, followed by autoradiography, revealed a single 75Se-containing protein (Mr 27,400 +/- 300). This protein could also be labelled with 125I-bromoacetyl reverse tri-iodothyronine, an affinity label for ID-I. The results suggest that hepatic ID-I is a selenoprotein or has an Se-containing subunit essential for activity.