Early changes in thyroid hormone metabolism in the heart, liver, and brown adipose tissue during the induction of low T3 syndrome in streptozotocin-diabetic rats

In order to elucidate the day-by-day development of low T3 syndrome, we made rats diabetic by an injection of streptozotocin. Untreated controls killed at day 0 and rats treated for 8 days with insulin after they had received streptozotocin served as controls. Sub-groups of animals were killed 1, 2, 3, 4 and 8 days after streptozotocin. In serum, heart and liver, T3 was depressed to less than 50% of controls at day 4, whereas the insulin-treated rats differed from controls only as to heart T3. Heart iodothyronine 5'-deiodinase activity was depressed to a minimum at day 3 and depression was not prevented by insulin. Liver iodothyronine 5'-deiodinase activity had not reached a minimum at day 8, and again, insulin treatment did not normalize this parameter. T3 contents and iodothyronine 5'-deiodinase activity in brown adipose tissue did not differ from values in controls at any point of time. Thus, in the rats with low T3 syndrome induced by streptozotocin-diabetes, a lowered iodothyronine 5'-deiodinase activity is not fully inhibited by insulin treatment, whereas the T3 content in the liver is re-established during an observation period of 8 days. A direct toxic effect of streptozotocin seems unlikely as an in vitro study showed no influence of streptozotocin on iodothyronine 5'-deiodinase activity in the liver. The study thus indicates that iodothyronine 5'-deiodinase activity in the heart and liver is depressed in experimental diabetes, despite near optimal regulation of blood glucose, and we suggest that lowered intracellular T3 production could, after some time, result in a hypothyroid state in different tissues.     

Brain cortex reverse triiodothyronine (rT3) and triiodothyronine concentrations under steady state infusions of thyroxine and rT3

T4 and reverse T3 (rT3) can inhibit 5'-deiodinase type II activity in rat brain cortex, pituitary, and brown adipose tissue, raising the possibility that T4 may act in vivo after conversion to rT3. The aim of this study was to measure in hypothyroid (Tx) rats the content of brain cortex rT3 during a constant 7-day infusion of either [125I]T4 alone, corresponding to 12 pmol T4/day X 100 g body weight (BW), or together with 400 pmol T4/day. [125I]T4, rT3, and T3 were extracted from brain cortex, pituitary, kidney, and liver with a combination of adsorption chromatography on Sephadex G-25, HPLC, and immunoprecipitation. [131I]T4, T3, or rT3 were used as internal standards. [125I]rT3 could be detected in brain cortex, liver, and kidney in Tx rats infused with [125I]T4 (12 pmol T4/day X 100 g BW) and in those infused with 400 pmol T4/day X 100 g BW. The highest rT3 concentrations were found in brain cortex, where it represented 6% to 10.5% of the local T4 concentration. During an infusion of 400 pmol T4/day X 100 g BW, brain cortex T3 concentration was 6 times higher in the brain cortex than in serum, and even exceeded that of T4. In Tx rats receiving [125I]T4 alone the brain cortex to serum T3 ratio was 3:1, but the total serum T3 concentration, measured by RIA, was much higher than that due to conversion [0.50 +/- (SE) 0.1 pmol/ml vs. 0.018 +/- 0.002 pmol T3/ml], indicating thyroidal secretion. The effect of the blood-brain barrier on rT3 was measured by infusing [125I]rT3 over 4 days. After killing, rT3 was isolated as above. Approximately 3% of serum rT3 was retrieved from the brain cortex, whereas during the T4 infusion 40-50% of serum rT3 was found demonstrating that brain cortex rT3 is locally produced.     

Increased plasma 3,5,3'-triiodothyronine sulfate in rats with inhibited type I iodothyronine deiodinase activity, as measured by radioimmunoassay

In contrast to the glucuronide conjugate, T3 sulfate (T3S) undergoes rapid deiodinative degradation in the liver and accumulates in rats and rat hepatocyte cultures if type I iodothyronine deiodinase activity is inhibited. We here report the RIA of plasma T3S in rats treated with the antithyroid drugs propylthiouracil (PTU) or methimazole (MMI), of which only PTU inhibits type I deiodinase. Male Wistar rats were treated acutely by ip injection with 1 mg PTU or MMI/100 g BW and subsequently for 4 days by twice daily injections with these drugs together with 0.5 microgram T4 or 0.25 microgram T3/100 g BW. Blood was obtained 4 h after the last injection, and plasma T4, rT3, T3, and T3S were determined by RIA and compared with pretreatment values. Serum concentrations (mean +/- SEM; nanomoles per liter) in untreated rats were: T4, 51 +/- 1; T3, 1.37 +/- 0.03; T3S, 0.09 +/- 0.01; and rT3, 0.03 +/- 0.002. Serum T3 was decreased, and T3S and rT3 were increased by acute PTU treatment [T3, 1.16 +/- 0.05 (P less than 0.01); T3S, 0.33 +/- 0.04 (P less than 0.001); rT3, 0.27 +/- 0.02 (P less than 0.001)], but unaffected by acute MMI treatment (T3, 1.37 +/- 0.05; T3S, 0.09 +/- 0.01; rT3, 0.02 +/- 0.003). In T4-treated rats, serum T3 was decreased and T4, T3S, and rT3 were increased by PTU vs. MMI [T4, 86 +/- 5 vs. 58 +/- 4 (P less than 0.001); T3, 0.51 +/- 0.07 vs. 0.88 +/- 0.06 (P less than 0.001); T3S, 0.38 +/- 0.03 vs. 0.12 +/- 0.01 (P less than 0.001); rT3, 0.86 +/- 0.19 vs. 0.08 +/- 0.01 (P less than 0.005)]. In T3-substituted rats T3S was increased by PTU vs. MMI (1.09 +/- 0.13 vs. 0.25 +/- 0.03; P less than 0.001). The T3S/T3 ratio in the PTU-treated T3 -replaced rats (0.60 +/- 0.09) was in agreement with that determined by HPLC of serum radioactivity in animals that in addition to this treatment also received about 10 microCi [125I]T3 with the last two injections (0.92 +/- 0.13). In conclusion, this investigation demonstrates the feasibility of the measurement of serum T3S by RIA. Our findings confirm previous observations with radioactive isotopes, suggesting that sulfation is an important pathway for the metabolism of T3 in rats. Analogous to rT3, the accumulation of T3S in PTU-treated rats indicates that this conjugate is metabolized predominantly by type I deiodination.     

The rat placenta and the transfer of thyroid hormones from the mother to the fetus. Effects of maternal thyroid status.

We have studied the effects of maternal thyroid status on the effectiveness of the rat placenta near term as a barrier for the transfer of T4 and T3 to the fetus. Dams were given methimazole to minimize the fetal contribution to the T4 and T3 pools, so that the iodothyronines found in the conceptus are ultimately of maternal origin. The dams were infused with saline, or with T4 or T3 at doses ranging from 2.3-27.8 nmol T4 and from 0.77-20.7 nmol T3/100 g BW per day. A group of normal pregnant dams (C) was included. At 21 days of gestation T4, T3, and rT3 were measured by RIA in maternal and fetal plasma, and in maternal and fetal sides of the placenta. The total fetal extrathyroidal T4 and T3 pools were also determined. The dose-related changes in T4, T3, and rT3 levels in the placenta confirm the presence of both inner and outer ring iodothyronine deiodinase activities, and suggest increasing accumulation of the iodothyronines. Despite this, fetal extrathyroidal T4 and T3 increase progressively in T4-infused groups as a function of maternal circulating T4 levels. Fetal extrathyroidal T3 increases progressively in T3-infused groups as a function of maternal plasma T3. There was no evidence that the net maternal contribution of T4 or T3 would be proportionally less when the maternal pools became very high. It was concluded that the rat placenta is only a limited barrier for the transfer of T4 and T3 to the fetus.     

Comparison of kidney and brown adipose tissue iodothyronine 5'-deiodinases

We have examined the influence of assay conditions on the 6-n-propyl-2-thiouracil (PTU) sensitivity of the iodothyronine 5'-deiodinase in brown adipose tissue (BAT) from hypothyroid rats. These results were compared with similar studies of 5'-deiodinase activity in kidney microsomes from euthyroid animals. Even though BAT microsomes contain largely type II (PTU-insensitive) deiodinase activity, the 5'-deiodination of T4 can be inhibited by PTU if the dithiothreitol (DTT) concentration in the assay is reduced to 5 mM or less. The apparent Ki for PTU of BAT microsomes was 4.3 mM at 5.0 mM DTT and 0.41 mM at 0.5 mM DTT. The kinetics of inhibition were noncompetitive. With kidney microsomes, PTU inhibition of rT3 5'-deiodination was both time and enzyme/substrate ratio dependent. For example, using 1 microgram microsomal protein, 2 nM rT3, and 5 mM DTT, the inhibitory effect of PTU was not maximal until 12 min after PTU addition. At stable reaction velocities PTU inhibition was uncompetitive, and the Ki was about 1 microM. Deiodination by kidney microsomes was completely inhibited by 50 microM PTU. Even though it is possible to inhibit the type II 5'-deiodinase activity with high concentrations of PTU (in the presence of low DTT concentrations), the deiodinase in kidney is about 1000-fold more sensitive to PTU. By these criteria the kidney microsome 5'-deiodinase is type I.     

Kinetic evidence suggesting two mechanisms for iodothyronine 5''-deiodination in rat cerebral cortex.

Enzymatic 5'-deiodination of 3,3',5'-triiodothyronine (rT3) and 3,3',5,5'-tetraiodothyronine (thyroxine, T4) was studied in microsomal preparations of rat cerebral cortex. Evidence was obtained for the existence of two thiol-dependent 5'-deiodinase entities. One of these predominates in tissue from euthyroid and long-term hypothyroid rats, is specific for rT3, follows "ping-pong" kinetics with dithiothreitol as the cosubstrate, and is inhibited by propylthiouracil (PrSUra) and iodoacetate. Inhibition by PrSUra is uncompetitive with rT3 and competitive with dithiothreitol. These properties are shared with the 5'-deiodinase activity of liver and kidney. The activity of a second type of 5'-deiodinase is highest in cerebral cortex from short-term hypothyroid rats, prefers T4 to rT3 as the substrate, is insensitive to PrSUra and iodoacetate, and follows "sequential" reaction kinetics. A similar PrSUra-insensitive 5'-deiodinase activity is also found in pituitary but is not detectable in liver and kidney; it seems, therefore, characteristic of tissues in which local T4 to 3,3',5-triiodothyronine (T3) conversion supplies a major portion of the total intracellular T3.     

Ethanol feeding and thyroid hormone monodeiodination

Adult male rats were placed on a 3 week regimen of ethanol (as 20% of total calories) in a nutritionally adequate diet, and controls were matched equicalorically without ethanol. Serum measurements of T4, T3, FT4, rT3, and TSH were performed in both the fed and the fasted state (18 hours). In the fed state, serum hormone measurements did not differ between control and ethanol-treated rats. Overnight fasting had a significant effect in decreasing serum T3 level in both experimental and control rats and in decreasing serum T4 level in ethanol-treated animals; FT4 and rT3 levels were not affected. Fasting also decreased in vitro hepatic T4 to T3 production to an equivalent degree in control and ethanol-treated rats, but did not alter hepatic T4 to rT3 production rates in control animals. In the fed state, hepatic rT3 neogenesis in animals given ethanol declined relative to the levels observed in control fed rats; fasting restored the depressed rT3 neogenesis to the levels noted in the fed state. Because decreased rT3 production in ethanol-treated rats in the fed state could not be explained on the basis of a change in 5'-deiodinase activity, it is suggested that ethanol administered with a nutritionally adequate diet may inhibit hepatic rT3 generation by inhibiting T4(5)-deiodinase.     

Regulation of rat cerebrocortical and adenohypophyseal type II 5'-deiodinase by thyroxine, triiodothyronine, and reverse triiodothyronine

To further understand the regulation of type II iodothyronine 5'-deiodinase (5'D-II) in the central nervous system and pituitary, we examined the response of this enzyme to the acute administration of T4, T3, and rT3 in hypothyroid rats. Enzyme levels were correlated with serum concentrations of T4 and T3 in thyroidectomized rats after acute administration of either iodothyronine and in animals with hypothyroidism of increasing severity induced by methimazole administration. Estimates of the tissue concentrations of the three iodothyronines, nuclear T3, and serum TSH levels were used to assess mechanisms and intrinsic potencies of the three iodothyronines. In four experiments, doses of T4 that reduced 5'D-II activity by 50% (ID50) ranged from 0.18-0.39 micrograms/100 g BW in the cortex and from 0.34-1.05 in the pituitary, whereas the corresponding ID50 values of rT3 were 1.0 and 3.5, and those of T3 were 4.0 and 5.0 micrograms/100 g BW. T3 doses that saturated nuclear receptors and fully suppressed TSH showed only modest suppression of 5'D-II levels in the cortex and pituitary. Based on estimates of the tissue hormone levels resulting in 5'D-II suppression, T4 and rT3 were much more potent than T3 in decreasing 5'D-II. These findings support the concept that the effect of these iodothyronines on 5'D-II is not mediated by the nuclear T3 receptor. The correlation of serum T4 and T3 with enzyme levels after acute injections of T4 or after chronic treatment with methimazole suggested that plasma T4 is probably the main physiological signal regulating 5'D-II. It is conceivable that rT3 produced locally from T4 also plays a role in the regulation of the enzyme.     

Iodothyronine deiodinase activities in FRTL5 cells: predominance of type I 5'-deiodinase

FRTL5 cells, a thyroid follicular cell line derived from normal rat thyroid, has been extensively used as a model system to study various aspects of the physiology of the thyroid epithelium. The capacity of these cells to metabolize iodothyronines and to generate T3 from T4 has not been previously examined. Here we studied the deiodination of T4, T3, and rT3 in homogenates of FRTL5 cells. By far, these homogenates were more potent catalyzing the 5'-deiodination (outer ring) of T4 and rT3 than the inner ring deiodination of T4 or T3. Both the production of rT3 and the degradation of newly formed T3 from T4 were very limited. Thus, when T4 was used as a substrate, T3 and iodide accumulated in a linear fashion with time, and initially the amounts of iodide and T3 were approximately equal. rT3 and 3,3'-diiodothyronine were rapidly deiodinated by these homogenates, with the 3'-deiodination of 3,3'-diiodothyronine occurring at a slower rate than the 5'-deiodination of rT3. The iodothyronine 5'-deiodinase activity corresponded to type I, as indicated by the following: the Km for T4 and T3 was in the micromolar range; rT3 was a better substrate than T4 (maximum velocity = 101 vs. 19 pmol/min.mg protein; Km = 0.83 vs. 3.1 microM, respectively); and the kinetics of inhibition by 6n-propyl-2-thiouracil were uncompetitive and substrate dependent, suggesting ping-pong kinetics. The type I 5'-deiodinase activity of FRTL5 cells was distinctly stimulated by TSH. This stimulation seems to be mediated by cAMP and requires serum as a permissive factor. In conclusion, 1) FRTL5 cells exhibit both inner and outer ring iodothyronine-deiodinating activities; 2) iodothyronine 5'-deiodination is by far more active; 3) the 5'-deiodination has been characterized as type I deiodinase based on substrate preference, enzyme kinetics, and inhibitors; 4) in all respect iodothyronine deiodination by FRTL5 cell homogenates proceeded with marked similarity to that in homogenates or microsomes of thyroid glands from several species; and 5) the FRTL5 type I deiodinase is more active than that reported in thyroid tissue and as active as that reported in liver and kidney, the prototype of type I deiodinase-containing tissues. The present studies indicate that FRTL5 cells are an excellent model system to study cellular and biochemical aspects of the regulation of this enzyme as well as its regulation by TSH and putative serum factors.     

Intracellular calcium and phospholipid turnover are not involved in the inhibition of iodothyronine 5'-deiodinase type II activity by T4

In glial cell cultures, iodothyronine 5'-deiodinase type II is stimulated by dibutyryl cAMP. Serum-free medium increases enzyme activity and prolongs the half-life of the enzyme. T4 and rT3 specifically inhibit this activity. We tested whether enzyme inactivation by T4 was mediated by changes in cytosolic free calcium concentration and/or phospholipid turnover. Intracellular calcium concentration was decreased either by chelation of extracellular calcium or by chelation of extracellular and intracellular calcium. Neither basal hypothyroid 5'-deiodinase activity nor its inactivation by T4 were modified in such experimental conditions, compared with control cells incubated in normal calcium-containing medium. T4 by itself had no effect on the cytosolic free calcium concentration for up to 20 min. Studies on phospholipid turnover included norepinephrine in parallel to T4 as positive stimulation control. While norepinephrine clearly accelerated phosphoinositide turnover, there was no effect of T4 on any phospholipid turnover. These results suggest that neither cytosolic free calcium nor phospholipid turnover is involved in T4-dependent modulation of 5'-deiodinase type II activity in astrocytes in culture.     

Anterior pituitary type II thyroxine 5'-deiodinase activity is not affected by lesions of the hypothalamic paraventricular nucleus which profoundly depress pituitary thyrotropin secretion

Bilateral destruction of the hypothalamic paraventricular nuclei (PVN) produced a profound depression of plasma TSH and the median eminence TRH concentration in hypothyroid rats. Anterior pituitary type II iodothyronine 5'-deiodinase (5'-D) activity was consistently lower but not significantly different in sham- and PVN-lesioned rats. Treatment with suboptimal replacement doses of 0.15 and 0.75 micrograms T4/100 g BW.day produced a graded depression of plasma TSH in the PVN (P less than 0.02), but not in the sham (P greater than 0.8) groups. Adenohypophyseal 5'-D was depressed in both sham and PVN groups by the highest T4 dose. Plasma T4 was much lower in PVN than in sham rats given comparable doses of T4 (P less than 0.001), but plasma T3 was not significantly different. This suggests that an increase in peripheral T4 metabolism was produced by PVN lesions. Our data indicate that changes in adenohypophyseal 5'-D activity are not responsible for the decrease in plasma TSH in PVN-lesioned rats and that neither the PVN nor endogenous TRH plays a significant role in the regulation of anterior pituitary 5'-D activity.     

The role of 3,3',5'-triiodothyronine in the regulation of type II iodothyronine 5'-deiodinase in the rat cerebral cortex

Type II iodothyronine 5'-deiodinase (5'D-II) activity is the source of 75-80% of the cerebral cortex T3 content in euthyroid rats. The activity of this enzyme is increased in hypothyroidism and can be quickly suppressed by T4 and rT3 by mechanisms involving neither protein synthesis nor nuclear T3 receptors. We have examined the possibility that endogenous cerebrocortical rT3 levels play a physiological role in the regulation of this enzyme. Thyroidectomized rats were injected with graded doses of [125I]rT3, and cortex 5'D-II activity and rT3 content were determined at various times thereafter. Enzyme activity was reduced as early as 10 min after the injection of 0.75 microgram rT3/100 g BW, and 18 h after 25 micrograms/100 g BW remained 60% suppressed. Regardless of the time after the injection, 5'D-II activity was inversely related to the rT3 content in the cortex; nearly complete suppression was observed at 0.5 ng rT3/g tissue, 50% at 80 pg/g, and 20-30% at 30 pg/g, the euthyroid level. After the infusion of 0.75 microgram rT3/100 g, maximal inhibition occurred at 10 min, before the rT3 content reached maximum levels, and the 5'D-II activity started to recover after the rT3 level fell below 300 pg/g tissue. After increasing doses of T4 administered to thyroidectomized rats, serum and cerebrocortical T4 concentrations increased in a dose-dependent manner, but the increment in the latter was steeper than that in the former. Serum rT3 increments were also proportional to the dose of T4, but cerebrocortical rT3 increased to a greater extent, as evidenced by a 3-fold increment in the cerebrocortical rT3 to T4 ratio. With 1.6 microgram T4/100 g BW, cerebrocortical rT3 reached approximately 100 pg/g, about 3 times the euthyroid level, suggesting that at this T4 dose, the rT3 formed from T4 accounts for part of the inhibition of 5'D-II. With the half-maximal suppressive dose of T4, cortex T4 was about 400 pg/g, but rT3 was negligible. We conclude that: suppression of cortex 5'D-II by rT3 is rapid and requires the presence of rT3 in the tissue (i.e. no long-lived mediators); intracortical rT3 is about 5 times more potent than T4 in suppressing this enzyme; the cortex of rT3-5'D-II suppression relationships suggest that the euthyroid levels of cortex rT3 may be significant in the modulation of 5'D-II.(ABSTRACT TRUNCATED AT 400 WORDS)     

5'-Deiodination in rat hepatocytes: effects of specific flavonoid inhibitors

5'-Deiodination of T4 or rT3 in the presence of flavonoids was studied in freshly isolated suspended rat hepatocytes. Flavonoids, a novel group of synthetic deiodinase inhibitors, were designed to act as T4 antagonists. 5'-Deiodination of the prohormone T4 to the thyromimetically active T3 is an essential first step in controlling thyroid hormone action. Hepatocytes were incubated with either 2 microM T4 or 2 microM [125I]rT3 as 5'-deiodinase substrates in the absence and presence of inhibitors (0.1-100.0 microM). T3 production from T4 was determined by T3 RIA, and [125I]iodide release from [125I]rT3 was alternatively used as a technically more simple and rapid but sensitive deiodinase assay. Aurones and flavones inhibited both T4 5'-deiodination and rT3 5'-deiodination, with half-maximal inhibitor concentrations from 3-45 microM. Aurones were equally potent in both assays. 3-Methyl-flavones, designed as rT3 analogs, were more active by a factor of 3-5 with T4 5'-deiodination than with rT3 5'-deiodination, with the exception of one relatively cell-toxic compound. Hepatocyte viability was controlled by trypan blue dye exclusion as well as by measuring gluconeogenesis from exogenously added 10 mM lactate. Some of the flavonoids inhibited gluconeogenesis at concentrations that had no effect on trypan blue dye exclusion. Flavonoid inhibitors reduce 5'-deiodinase activity in intact hepatocytes in concentrations equimolar to those of substrates. Therefore, synthetic flavonoids may be suitable substances for further study of iodothyronine physiology or, after modification, could be useful as a new class of antiiodothyronine drugs.     

Thyroxine, triiodothyronine, and reverse triiodothyronine processing in the cerebellum: autoradiographic studies in adult rats

Well confirmed evidence has demonstrated that the cerebellum is an important target of thyroid hormone action during development. Moreover, the presence of nuclear receptors and strong 5'-deiodinase activity in cerebella of adult rats have suggested that this region may continue to respond to thyroid hormones during maturity. Recent autoradiographic observations have focused attention on the cerebellar granular layer, in that [125I]T3 administered iv to adult rats was found to be selectively and saturably concentrated there. To determine the specificity of iodothyronine localization in the granular layer, we have now compared film autoradiographic observations made after iv [125I]T4 and iv [125I]rT3 with those found after iv [125I]T3. The results demonstrated that, as in the case of the latter hormone, labeling within the cerebellar cortex after iv [125I]T4 was both selective and saturable. Moreover, except for a lag in time to resolution and a longer retention time, the distribution of cerebellar radioactivity after iv labeled T4 was qualitatively similar to that seen after iv [125I]T3. However, the ability of T4 to become differentially concentrated in the granular layer of cerebellum was absolutely dependent on its ability to be converted intracerebrally to T3. Thus, pretreatment with ipodate, which blocks brain 5'-deiodinase activity and, therefore, the intracerebral formation of T3 from T4, completely prevented cerebellar granular layer labeling after iv [125I]T4 even though it did not interfere with differential labeling of this region by iv delivered [125I]T3. In the same experiments, propylthiouracil, a potent peripheral, but not central, 5'-deiodinase inhibitor, had no qualitative effect on the distribution of either T4 or T3 in cerebellum. By contrast with the results obtained after administering labeled T3 or T4, brain labeling after iv delivered [125I]rT3 was found to be no different from that produced by markers of cerebral blood flow, which rapidly enter and leave the brain without becoming incorporated into brain cells. This was so even during treatment with propylthiouracil and ipodate, both of which markedly prolonged the normally brief residence time of this iodothyronine in serum and brain. Overall, the autoradiographic results served to highlight the importance of the morphological approach for investigating thyroid hormone action and metabolism in brain. They demonstrated that only T3, whether entering as such from the circulation or formed in situ from T4 (but neither T4 itself nor iv administered rT3) was strongly, selectively, and saturably concentrated in the cerebellar granular layer of adult rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of glucocorticoids in the stress-induced reduction of extrathyroidal 3,5,3'-triiodothyronine generation in rats

Serum concentrations of T4, T3, and rT3 as well as liver and kidney 5'-deiodinase activity, have been examined in rats stressed by restraint. After immobilization, serum concentrations of T3 decreased significantly (6 hr, -33 +/- 1%; 8 h, -42 +/- 3%), while serum rT3 increased (6 h, +55 +/- 3%; 8 h, +75 +/- 5%). In the same or similarly treated animals, there was a time-dependent reduction in T4 5'-deiodinase activity in both liver (4 h, -23 +/- 2%; 8 h, -43 +/- 3%) and kidney (4 h, -18 +/- 1%; 8 h, -42 +/- 3%) homogenates. The reduction in hepatic and renal T3 production was due to reduced enzyme activity and not to reduced substrate availability. In spite of reductions in serum TSH (4 h, -9 +/- 1%; 8 h, -51 +/- 5%), the serum T4 concentration did not fall. The serum concentration of corticosterone reached 30 times the basal level after 8 h of restraint. Either adrenalectomy or metyrapone treatment, followed by replacement with nonstress doses of B, completely prevented the alterations of iodothyronine metabolism induced by restraint. These results indicate that the stress-induced elevation of plasma glucocorticoids plays a key role in the pathogenesis of the low T3 syndrome in this model. The reduction in serum T3 may be accounted for by a reduction in T3 production by liver and kidney, adding support to the concept that these organs are an important source of plasma T3 in the rat.     

3,3',5'-Triiodothyronine inhibits iodothyronine-5'-deiodinating activity induced by 3,5,3'-triiodothyronine at equimolar concentrations in cultured fetal mouse liver

To investigate the rT3 effect on iodothyronine-5'-deiodinating activity (I-5'-DA) in cultured fetal mouse liver, liver on the 19th day of gestation, in which little or no I-5'-DA was detected and, therefore, rT3 was very stable, was cultured in medium containing thyroid hormone-depleted fetal calf serum supplemented with cortisol, insulin, and various concentrations of iodothyronines. After 4-15 days of culture, I-5'-DA in the homogenate was assessed by the amount of iodide released from outer ring-labeled rT3 and expressed as picomoles of 127I- per mg protein/min. I-5'-DA was induced by T3 (10(-9)-10(-8) M) and T4 (10(-7) M). In contrast, rT3 could not induce I-5'-DA at 10(-8)-10(-6) M. Furthermore, rT3 significantly decreased I-5'-DA induced by T3 (10(-8) M) at almost equimolar concentrations (1-5 X 10(-8) M). The inhibitory action of rT3 was reversible and was specific for I-5'-DA; no alteration in malic enzyme activity or intracellular glutathione concentration was detected. The inhibitory effect of rT3 on I-5'-DA was dose dependent, and the enzyme activity decreased with a half-life of 16 h in the presence of 10(-6) M rT3. The inhibitory effect was not due to contaminating rT3 in the liver homogenates. Lineweaver-Burk analysis revealed that the decrease in I-5'-DA was due to a decrease in the maximum velocity, whereas no alteration in Km was detected, suggesting that rT3 decreases the amount of 5'-deiodinase induced by T3. The minimal free rT3 concentration capable of inhibiting the induction of iodothyronine-5'-deiodinase was approximately 10(-10) M, which may be attainable in vivo, as in amnionic fluid. In summary, we have demonstrated that rT3 exerts a strong antagonistic effect against T3 in inducing I-5'-DA in cultured fetal mouse liver.     

Changes in thyroxine monodeiodination in rat liver, kidney and placenta during pregnancy

Monodeiodination of thyroxine (T4) was studied in the liver, kidney and placenta of pregnant rats. Age matched female non-pregnant and pregnant Sprague-Dawley rats on the 7th, 14th, 17th and 21st days of gestation were used. The 800 X g supernatants of tissue homogenates (protein 1 mg/tube) were incubated with 1 microgram of stable T4 in the presence of 5 mM dithiothreitol (DTT) at 37 degrees C for 60 min at pH 7.5. Net triiodothyronine (T3) generation from T4 in rat liver homogenates on the 7th day of gestation was significantly lower than that in the non-pregnant rat. Thereafter it increased, but values on the 14th, 17th and 21st days of gestation were not significantly different from those obtained in the non-pregnant rat. Net renal T3 generation from T4 on the 14th day was significantly lower than that in the non-pregnant rat. It was increased thereafter and the values at the 17th and 21st days of gestation were not significantly different from those in the non-pregnant rat. Net reverse T3 (rT3) generation from T4 in the placenta rose from the 14th to the 17th day and then dropped by the 21st day and the value at the 17th day was significantly higher than those at the 14th and 21st days of gestation. These results indicate that 1) both T4 outer-ring monodeiodination in the pregnant rat liver and kidney, and T4 inner-ring monodeiodination in the placenta show significant variation with the progress of gestation; 2) the time course of the T4 outer-ring monodeiodination in pregnant rat liver and kidney is completely different from T4 inner-ring monodeiodination in the placenta.     

Iodothyronine deiodination in the brain of diabetic rats: influence of thyroid status

Experimental diabetes causes profound alterations in the metabolism of thyroxine (T4), including a decrease in hepatic triiodothyronine (T3) generation from T4 via 5'-deiodination (5'-D). Because 5'-D in brain differs markedly from that in liver, both in enzymatic mechanism and in the response to hypothyroidism, we studied iodothyronine deiodination, in particular T4 to T3 conversion (T4-T3), by incubating 125I T4 with particulate fractions of cerebral cortex (Cx) and cerebellum (Cm) from rats made diabetic by injection of streptozotocin. In nondiabetic thyroidectomized (Tx) rats Cx and Cm T4-T3 activity was increased approximately ten-fold and two-fold, respectively, compared with intact controls. Diabetic Tx rats did not differ from nondiabetic Tx rats in the rate of net T3 production from T4 but the formation of 3,3'-T2 was slightly reduced. Insulin-treated diabetic-Tx rats showed a pattern of T4 metabolism in Cx and Cm virtually identical to that of nondiabetic Tx rats. The rate of T3 degradation, determined in parallel incubations of Cx and Cm with 125I T3, did not differ significantly among the groups, indicating that the observed differences in net T3 production were due to changes in T4 5'-D activity. Intact diabetic rats compared to nondiabetic controls showed no significant changes in T4-T3 either in Cx or in Cm. Administration of T3, 0.8 microgram per 100 g bw per day for 6 days, by constant infusion to intact rats raised T4-T3 in Cx and Cm to levels found in Tx rats. Treatment of intact diabetics with T3 caused qualitatively similar changes, i.e., a hypothyroid response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormonal control of a low Km (type II) iodothyronine 5'-deiodinase in cultured NB41A3 mouse neuroblastoma cells

The central nervous system manifests complex homeostatic mechanisms for the maintenance of thyroid hormone economy. The present studies used the NB41A3 mouse neuroblastoma cell line as a model system to study the hormonal regulation of the enzymatic conversion of T4 to T3 in neural tissue. NB41A3 cells manifested a thiol-dependent 6-n-propyl-2-thiouracil-insensitive iodothyronine 5'-deiodinase (I5'D) with a Km for T4 of approximately 10 nM. I5'D activity was increased 2- to 4-fold in cells grown in thyroid hormone-depleted medium. Exposure of cells in situ to various thyroid hormones resulted in a rapid dose-dependent inhibition of enzyme activity with the following order of potency: rT3 = T4 greater than T3. The potent inhibitory effect of rT3 on I5'D activity could not be attributed to substrate competition with T4 in the reaction assay. The addition of dexamethasone (2 X 10(-7) M) to the culture medium also inhibited I5'D activity by 46 +/- 6% (+/- SE; n = 4 experiments; P less than 0.02), whereas insulin and epinephrine were without effect. In other experiments, saturation analysis using a purified preparation of isolated nuclei from NB41A3 cells demonstrated the presence of saturable, high affinity nuclear binding sites which had a Kd value for T3 of 0.13 +/- 0.05 nM and a maximum binding capacity of 0.13 +/- 0.01 pmol T3/mg DNA. These studies demonstrate that NB41A3 cells have a low Km (type II) I5'D process and nuclear T3-binding sites very similar to those previously described in the rat central nervous system. I5'D activity in this cell line appears to be regulated by multiple serum factors, including thyroid hormones and glucocorticoids. The potent regulatory effect of rT3 and T4 suggests that T3 formation by thyroid hormones in neural tissue is controlled by a unique cellular mechanism independent of the nuclear T3 receptor. Since tissue and plasma concentrations of T4 are considerably higher than those of rT3, the former hormone is likely to be the principal thyroid hormone regulating this enzymatic process.     

Serum concentrations of thyroid hormones and activity of iodothyronine deiodinase in peripheral tissues of the house musk shrew, Suncus murinus

Serum concentrations of thyroid hormones and the properties of iodothyronine deiodinase in tissues of the house musk shrew, Suncus murinus, were examined and compared with those of the rat. Serum concentrations of thyroxine (T4) and 3,3',5'-tri-iodothyronine (rT3) were higher, while the serum concentration of 3,5,3'-tri-iodothyronine (T3) was lower in the shrew than in the rat. Among liver, kidney, skeletal muscle, heart, brown adipose tissue (BAT), spleen, lung, testes and thymus homogenates of the shrew, T(4)5'-deiodinase (5'D) activity was highest in BAT, and rT3 5'D activity was highest in both the liver and BAT. Intermediate or low T3 5-deiodinase activity was noted in all tissues examined. Activity of 5'D for T4 and rT3 in liver and kidney was much lower, while that of BAT was much higher in the shrew than in the rat. Liver and kidney 5'D may be type-I and that of BAT may be type-II in the shrew, judging from its response to 6-n-propyl-2-thiouracil and iopanoic acid and substrate preference. Thus 5'D of the shrew was similar to that of the rat in type, but was different with respect to its activity in some peripheral tissues. This difference may have relevance to the low T3 state of the shrew.