Flavonoid administration immediately displaces thyroxine (T4) from serum transthyretin, increases serum free T4, and decreases serum thyrotropin in the rat

Naturally occurring and synthetic plant flavonoids, such as EMD 21388, are potent inhibitors of thyroid hormone 5'-deiodinase (5'-D) in vitro, but not when given in vivo, since they are tightly bound by serum transthyretin (TTR). EMD 21388 also inhibits the binding of T4 to human, dog, and rat serum TTR in vitro and when administered to rats in vivo. In the present studies the administration of EMD 21388 inhibited the binding of T4 to TTR within 3 min, resulting in a decrease in the serum T4 concentration, an increase in the percentage of serum free T4 assessed by equilibrium dialysis, and an increase in the serum total free T4 concentration. Depending upon the dose of EMD 21388 employed, the serum total free T4 concentration was either elevated for at least 60 min or transiently elevated, returning to normal values by 60 min. Although the total serum T3 concentration was decreased and the percent free T3 increased, these changes were modest, and the serum free T3 concentrations remained normal after EMD 21388 administration. The transient elevations of serum free T4 concentrations 10 and 20 min after the administration of 0.3 mumol EMD 21388/100 g BW resulted in a significant decrease in the serum TSH concentration at 60 min. These observations strongly suggest that the serum free T4 concentration and not T4 bound to serum TTR is biologically available to the pituitary to regulate TSH secretion and/or synthesis. The administration of EMD 21388, which rapidly increases the serum free T4, but not the serum free T3, concentration, will now permit studies of the effect(s) of endogenously elevated serum free T4 concentrations, rather than those after the administration of pharmacological quantities of T3 and T4, on various aspects of the biosynthesis and release of pituitary TSH.     

Thyroxine (T4) transport and distribution in rats treated with EMD 21388, a synthetic flavonoid that displaces T4 from transthyretin.

To test whether plasma transthyretin (TTR) might play a specific direct role in the transfer of T4 from the plasma to tissues, in vivo kinetic studies were performed in control rats and in rats treated with EMD 21388, a synthetic flavonoid that displaces T4 from TTR. The plasma disappearance curves of simultaneously injected [125I]T4 and [131I]albumin were analyzed to determine the rate constant for the transfer of T4 from the extracellular compartment to the rapidly exchangeable intracellular compartment (KE) and the steady state distribution ratio of T4 between the rapidly exchangeable intracellular compartment and the extracellular compartment (Imax/Emin). When rats were injected ip with EMD 21388 (2 mumol/100 g BW), the free T4 fraction in serum increased approximately 8-fold. This was due to displacement of T4 from TTR, as assessed by electrophoresis of serum proteins in the presence of [125I]T4. Concomitantly, both KE and Imax/Emin increased 6-fold in the treated rats. These results fail to confirm a major specific role for TTR in the transfer of T4 from the plasma to tissues. Instead, they are consistent with both the free hormone transport hypothesis and the free hormone hypothesis in this setting.     

Peripheral metabolism of homologous thyrotropin in euthyroid and hypothyroid rats: acute effects of thyrotropin-releasing hormone, triiodothyronine, and thyroxine.

The peripheral metabolism and metabolic clearance rate (MCR) of homologous TSH was studied in euthyroid and hypothyroid rats. Incubation of freshly labeled [125I]iodo-TSH with rat serum revealed a labeled nonimmunoreactive protein in the void volume of a Sephadex G-100 column which could not be detected by conventional chromatographic purification. Removal of this contaminant from the tracer reduced the nonspecific binding in the absence of serum and increased the binding of tracer in the absence of added exogenous TSH. Injection of [125I]iodo-TSH into rats was followed within 15 min by the appearance of at least three labeled protein components. Gel filtration showed that these peaks were trichloroacetic acid (TCA)-precipitable proteins of larger molecular weight than TSH, but not all were precipitable by antibody to rat TSH. The disappearance rate of TCA-precipitable 125I (t1/2 = 28 min) was significantly longer than the disappearance rate of immunoprecipitable 125I (t1/2 = 22 min). The disappearance rate of immunoprecipitable [125I]iodo-TSH was identical to that of injected purified rat TSH and of the TRH-induced TSH increment in euthyroid rats. The disappearance rate os suppressible TSH (after 100 microgram T3) in hypothyroid animals was only slightly longer than the rate of disappearance of immunoprecipitable [125I]iodo-TSH (40 vs. 36 min) in the same rats. The calculated MCR of TSH was slightly lower (P less than 0.05) in hypothyroid rats (18.3 +/- 3.0 ml/h/100 g BW, mean +/- SD) than it was in euthyroid rats (22.6 +/- 2.1). The pituitary TSH concentration in hypothyroid rats was 29 mU/mg wet wt, similar to that of euthyroid animals. These results indicate that the turnover rate of pituitary TSH in hypothyroid rats with serum TSH concentrations of 1400-3000 microunit/ml is 7-14 times/day. Therefore, the significant increase we observed in pituitary TSH concentration 1 h after T4 (1.5 microgram/100 g BW) or T3 (0.15 microgram/100 g BW) administration indicates that the 35% decrease in plasma TSH at this interval is due to inhibition of TSH release, not to inhibition of TSH synthesis.     

Thyroid hormone antibodies and Hashimoto's thyroiditis in mongrel dogs

Abnormally elevated serum T3 concentrations measured by RIA were observed in 19 clinically euthyroid or hypothyroid mongrel dogs. The serum T4 concentrations in these sera were low, normal, or high. Measurement of the intensity of thyroid hormone binding to serum proteins was determined by equilibrium dialysis. A marked decrease in the percent free T3 was observed in these abnormal sera. Polyacrylamide gel electrophoresis, pH 7.4, of normal dog serum enriched with tracer 125I-labeled thyroid hormones demonstrated binding of [125I]T4 to transthyretin, thyroid hormone-binding globulin, and albumin and of [125I]T3 primarily to thyroid hormone-binding globulin. In all abnormal sera, polyacrylamide gel electrophoresis demonstrated strikingly higher binding of T3 to immunoglobulin (Ig). Eleven of 16 abnormal sera had minimal to moderate binding of T4 to Ig. The percent free T4 was lower only in dogs whose sera demonstrated markedly increased binding of T4 to Ig. All abnormal sera tested had positive antithyroglobulin antibodies, consistent with the diagnosis of autoimmune lymphocytic thyroiditis. As in humans, antibodies to thyroid hormones in dogs are more common in the presence of Hashimoto's thyroiditis and should be considered when elevated serum thyroid hormone concentrations are observed in the absence of clinical thyrotoxicosis. When an antibody to only one thyroid hormone is present, a marked discrepancy in the serum concentrations of T3 and T4 will be observed.     

Transient neonatal hyperthyroidism results in hypothyroidism in the adult rat

Adult rats who had neonatal hyperthyroidism (NH) have reduced BW and serum T4, T3, and TSH concentrations. Pituitary TSH responses to TRH administration under basal, T4-suppressed, and propylthiouracil-stimulated conditions suggest that the thyrotroph of these animals is more sensitive to the feedback effects of thyroid hormones. These studies were undertaken to examine, with the use of various thyroid hormone-responsive variables, thyroid status of adult NH rats. NH was induced by 12 daily sc injections of T4 (0.4 microgram/g BW) to neonatal male Sprague-Dawley rats. Adult NH and control rats were then studied at 120 days of age. Adult NH rats had significantly decreased mean BW (P less than 0.001), and serum T4 (P less than 0.005), T3 (P less than 0.001), and TSH (P less than 0.001) concentrations. The percent decreases were 12% for T4, 20% for T3, and 27% for TSH in adult NH rats. Mean pituitary GH concentration and hepatic alpha-glycerophosphate dehydrogenase and malic enzyme activities were significantly decreased in adult NH rats to 54% (P less than 0.005), 52% (P less than 0.025), and 39% (P less than 0.001), respectively, of control values. Mean pituitary TSH concentrations were similar in adult NH and control rats. Mean hepatic T4 5'-deiodinase activity of adult NH rats [200 +/- (SE) 23 fmol T3/min X mg protein] was significantly decreased to 56% of control levels (355 +/- 31 fmol T3/min . mg protein; P less than 0.005). Mean pituitary T4 5'-deiodinase activity of adult NH rats (32.8 +/- 3.3 fmol T3/min . mg protein) was significantly increased compared with that of control rats (21.2 +/- 1.7 fmol T3/min . mg protein; P less than 0.025). These changes are consistent with a hypothyroid state in adult NH rats. The observation of decreased serum TSH concentration and enhanced thyrotroph sensitivity to thyroid hormones in the face of increased pituitary T4 5'-deiodinase activity suggests that increased thyrotroph monodeiodination of T4 may be the central biochemical aberration responsible for the hypothyroid state in adult NH rats.     

Effect of the cardiac inotropic drug, OPC 8212, on pituitary-thyroid function in the rat

3,4-Dihydro-6-[4-(3,4-dimethoxybenzoyl)-1 piperaznyl]-2(1H)-quinolinone (OPC 8212) is a new synthetic quinolinone with potent cardiac inotropic action in man. Long term oral administration of OPC induces goiter and thyroid tumor formation in rats, associated with decreases in serum T4 and increases in serum TSH concentrations. Studies were carried out to explore the mechanisms responsible for these drug induced abnormalities. OPC 8212, administered for 1 week at doses of 500 and 2000 mg/kg.day mixed with the diet, resulted in an increase in thyroid weight, a decrease in circulating T4 and free T4 concentrations and an increase in serum TSH concentrations. OPC decreased the 5'-deiodinase (5'-D) activity in liver homogenates and increased the 5'-D activity in pituitary homogenates, consistent with hypothyroidism. OPC 8212 did not affect thyroid iodine metabolism and hormone synthesis or the binding of T4 to serum binding proteins. The hepatic uptake of 125 I-T4 4 h after T4 administration was significantly increased in OPC 8212 treated rats. The biliary excretion of administered 125 I-T4 was increased in OPC 8212-treated rats and most of the increase was due to an increase in the excretion of T4-glucuronide. Hepatic T4-glucuronyltransferase activity measured in vitro in OPC 8212 treated rats was increased as compared to that of controls. It is concluded that the effect of OPC 8212 on lowering serum T4 with a compensatory rise in TSH leading to goiter formation is due to a drug-induced increase in hepatic T4 disposal. The induction of T4-glucuronyl-transferase appears to play an important role in the increased biliary excretion of T4 in OPC 8212-treated rats.     

Effects of amiodarone and desethylamiodarone on pituitary deiodinase activity and thyrotropin secretion in the rat

The effect of acute administration of amiodarone, its major metabolite desethylamiodarone and iodine in an amount equal to that contained in amiodarone on serum thyroid hormone and thyrotropin (TSH) concentrations and hepatic and pituitary 5' deiodination of thyroxine (T4) in the euthyroid and hypothyroid rat was evaluated. Amiodarone, desethylamiodarone and iodine all caused a decrease in serum T4 and triiodothyronine (T3) concentrations in euthyroid rats, while serum TSH concentrations and pituitary and hepatic 5' deiodinase activities were decreased only in the amiodarone and desethylamiodarone-treated animals. Serum TSH was increased in the iodine treated rats. Amiodarone, but not iodine, decreased serum T3 and TSH concentrations and pituitary and hepatic 5' deiodinase activities in hypothyroid rats. Inhibition of hepatic 5' deiodinase activity was also observed by the addition of amiodarone in vitro in the absence of dithiothreitol (DTT) but not in the presence of DTT. The decrease in the serum T4 concentration observed with amiodarone and desethylamiodarone administration is probably secondary to the inhibitory effect of iodine released from the drugs on thyroidal T4 synthesis and secretion. Iodine inhibition of thyroidal T3 synthesis and secretion, decreased T4 substrate for a peripheral generation of T3 and inhibition of T4 to T3 conversion all contribute to the decrease in serum T3 observed. The decrease in the serum TSH concentration, despite low serum T4 and T3 concentrations and inhibition of pituitary 5' deiodinase, suggest that amiodarone may function as a thyroid hormone agonist in the pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of oestradiol benzoate on the pituitary secretion and peripheral kinetics of thyrotrophin in the rat

Previous works from this laboratory have demonstrated that oestradiol benzoate (EB) in euthyroid male and female rats induced a significant decrease in the pituitary content of TSH while serum levels of this hormone remained normal. The present work studied the effects of EB (25 micrograms/100 g body weight, during 9 days) on the peripheral metabolism of [125I]rTSH and on the pituitary and plasma concentration of TSH in euthyroid and hypothyroid rats. No significant variations were observed in [125I]rTSH kinetics of EB-treated euthyroid rats vs untreated controls: fractional turnover rate 2.8 +/- 0.2 vs 3.0 +/- 0.3%/min, distribution space 6.5 +/- 0.4 vs 6.8 +/- 0.5 ml/100 g body weight, disposal rate 18.4 +/- 2.4 vs 18.1 +/- 1.9 microU/100 g/min and extrapituitary pool 645 +/- 42 vs 614 +/- 43 microU/100 g body weight. Similarly, in hypothyroid rats oestrogens induced no changes in TSH kinetics except for an increase in distribution space (P less than 0.025). However, oestrogens decreased the pituitary pool of TSH (P less than 0.001) in both euthyroid and hypothyroid rats and increased the plasma TSH in hypothyroid animals (P less than 0.01), all vs their respective controls. Neither hypothyroid group had detectable plasma levels of T4 and T3. In summary: 1) the marked decrease of pituitary TSH with normal plasma TSH induced by EB appears unrelated to the peripheral metabolism of TSH, 2) the results from hypothyroid rats suggest that EB stimulates the release of TSH from the pituitary gland.     

Modulation by oestrogen of thyroid hormone effects on thyrotrophin gene expression

The role of oestrogen in the regulation of TSH gene expression is unclear. We have examined the effect of administration of oestrogen in the rat on serum TSH, pituitary TSH content and pituitary cytoplasmic concentrations of mRNA encoding the TSH beta and alpha subunits, thus deriving measures of hormone release and synthesis. In addition, we have examined the effect of oestrogen on the binding of tri-iodothyronine (T3) to nuclear receptors in the anterior pituitary. Administration of oestrogen did not affect serum concentrations of TSH in euthyroid or untreated hypothyroid rats, but did augment the effects of T3 (1 and 2 micrograms) on serum TSH in hypothyroid animals 6 h after injection of T3. No influence of oestrogen or of thyroid status on pituitary content of TSH was seen. A marked increase in the concentrations of TSH beta and alpha mRNA in pituitary cytoplasm was found in hypothyroidism, compared with those in the euthyroid state. No effect of oestrogen on TSH mRNA was seen in euthyroid animals but concentrations of TSH beta and alpha mRNA were lower in hypothyroid animals than in vehicle-treated controls. A stimulatory influence of T3 on TSH mRNA was seen 6 h after injection of T3; this stimulation was absent in oestrogen-treated rats. No effect of oestrogen on the action of T3 was evident 72 h after beginning treatment with T3. In addition to effects on serum TSH and TSH mRNA, an increase in the number of pituitary nuclear receptors for T3 was seen after oestrogen treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Triiodothyronine binding immunoglobulin in a euthyroid man without apparent thyroid disease; its properties and effects on triiodothyronine metabolism

A 54 year old man with markedly elevated serum T3, but without an apparent thyroid disease, was found to have a specific antibody to T3. His serum thyroxine, TBG and TSH were in normal range, but T3-RSU was markedly low. Antibodies to thyroglobulin and microsome were negative. He was judged euthyroid because of a normal basal metabolic rate and a normal thyroidal 123I uptake which was suppressed by T3 administration. When serum was extracted with ethanol prior to assay, serum T3 was found to be in the upper border of normal range. Several experiments revealed the presence of an antibody to T3 in his serum with an affinity constant of 3.3 X 10(9) M-1. The binding capacity of the antibody was 7.6 ng/mg of IgG. The binding of [125I]T3 was almost specific to T3, and potencies of T4 and fT3 in displacing [125I]T3 binding were only 1.0 and 0.3%, respectively, of that of T3. The antibody contained both kappa and lambda chains and was therefore polyclonal. The T3 metabolic clearance rate, which was determined by disappearance of injected [125I]T3 from serum, was lower in this patient (7.44 1/day) than in normal. The T3-production rate was decreased to 14.9 micrograms/day, and serum free T3 concentration as well as urinary T3 excretion rate were also reduced. Since both serum total and free T4 concentrations were normal, the supply of T4 to peripheral tissues would be sufficient to keep this patient in a euthyroid state in spite of the anti-T3 antibody.     

Regulation of thyroxine 5'-deiodinase by thyroid hormones and activators of protein kinase C in GH4C1 cells

The regulation of T4 5'-deiodinase activity was studied in cultured GH4C1 cells. Enzyme activity was measured in cell sonicates as the release of radioiodide from [125I]T4. Enzyme activity was stimulated 2- to 3-fold by hypothyroid serum and activators of protein kinase C, such as TRH and phorbol esters. The hypothyroid serum effect was maximal by 3 h, whereas TRH and phorbol esters required 6 h to achieve a maximal effect. The hypothyroid serum effect was gone within 4 h of returning the cells to control medium. In contrast, the TRH and phorbol ester effects persisted 24-48 h after removal of those agents. Both T4 and rT3 were at least as potent as T3 in blocking the effect of hypothyroid serum. The stimulation of 5'-deiodinase induced by hypothyroid serum was additive with that induced by kinase C activators. Trifluoperazine blocked the effect of TRH and phorbol esters, but not that of hypothyroid serum. It is concluded that stimulation of 5'-deiodinase activity can occur by at least two independent mechanisms: one involving hypothyroidism and another involving activation of protein kinase C. The relative potencies of various iodothyronines for abolishing the hypothyroid effect differ markedly from the relative binding affinities of these agents for the nuclear T3 receptor, suggesting that this thyroid hormone effect may not be mediated by the classical nuclear thyroid hormone receptor.     

The influence of dexamethasone on serum thyrotrophin and thyrotrophin synthesis in the rat

Glucocorticoid hormones suppress circulating concentrations of thyrotrophin (TSH), but their effect on synthesis of TSH in the pituitary gland is unclear. We have examined the influence of the glucocorticoid dexamethasone on serum TSH, pituitary TSH content and TSH beta- and alpha-subunit mRNA concentrations in pituitary cytoplasm in both the euthyroid and hypothyroid rat, and following triiodothyronine (T3) treatment in the hypothyroid rat. The rise in serum TSH in hypothyroidism was attenuated in animals treated with dexamethasone; in addition the suppression of serum TSH 6 h after T3 administration to hypothyroid rats was enhanced by dexamethasone. In contrast to the changes in serum TSH, pituitary TSH content was unaffected by dexamethasone. Furthermore dexamethasone had no significant effect on changes in pituitary cytoplasmic TSH beta- and alpha-subunit mRNA levels with thyroid status. These findings demonstrate that dexamethasone exerts differential effects on serum TSH levels and TSH biosynthesis which contrast with those of thyroid hormones.     

Caloric restriction does not alter thyrotropin secretion in hypothyroidism

The effect of caloric restriction, as a model of nonthyroid illness, on serum thyroid hormone and TSH concentrations in hypothyroid patients was studied to determine if pituitary-thyroid function is altered in such patients, as it is in euthyroid subjects. Serum T4, T3, and TSH concentrations and serum TSH responses to TRH were measured in 5 untreated hypothyroid patients and 10 hypothyroid patients receiving T4 replacement therapy before and after restriction of caloric intake to 500 cal daily for 7 days. In 5 untreated hypothyroid patients, the mean serum T3 concentration declined 17%, from 75 +/- 14 (+/- SE) to 62 +/- 11 ng/dl. The mean basal serum TSH concentrations were 154 +/- 67 (+/- SE) microU/ml before and 161 +/- 75 microU/ml at the end of the period of caloric restriction, and the serum TSH responses to TRH were similar on both occasions. In 10 T4-treated hypothyroid patients, the mean serum T3 concentration declined 35%, from 110 +/- 8 to 71 +/- 8 ng/dl. In this group, mean basal serum TSH concentrations were 17 +/- 5.1 microU/ml before and 18.2 +/- 7.0 microU/ml at the end of the period of caloric restriction, and as in the untreated hypothyroid patients, the serum TSH responses to TRH were similar on both occasions. Mean serum T4 concentrations and serum free T4 index values did not change in either group. These results indicate that caloric restriction in both untreated and T4-treated hypothyroid patients is accompanied by 1) reduced serum T3 concentrations, as it is euthyroid subjects, and 2) no alterations in basal or TRH-stimulated TSH secretion.     

Euthyroid hyperthyroxinemia due to a generalized 5'-deiodinase defect

We studied an 11-yr-old girl with asymptomatic hyperthyroxinemia, who remained euthyroid and healthy for 5 yr of follow-up. Besides having elevated serum T4 concentrations, her serum free T4 concentrations were consistently elevated, as measured by three different methods, including equilibrium dialysis and ultrafiltration. Serum total and free T3 concentrations were in the low normal range, and serum 3,5-diiodothyronine (3,5-T2) levels were low, suggesting reduced 5'-deiodination of both T4 and T3. Serum total and free rT3 and total and free 3', 5'-T2 concentrations were all markedly elevated, whereas serum total and free 3,3'-T2 were low, suggesting unaltered 5-deiodination of T4 to rT3 and of rT3 to 3',5'-T2 in combination with reduced 5'-deiodination of rT3 and 3',5'-T2. The girl had a small diffuse goiter, her serum TSH response to TRH was exaggerated, and thyroid radioiodine uptake was elevated, suggesting slightly increased TSH secretion and, consequently, increased thyroid secretion. Both T3 and T4 administration resulted in suppressed basal as well as TRH-stimulated serum TSH concentrations, and radioiodine uptake was suppressed during T3 administration. Our data suggest reduced activity of several (all?) peripheral 5'-deiodination pathways, including possibly also thyrotroph T4 5'-deiodination. Thus, this girl seems to have a previously unrecognized syndrome of generalized 5'-deiodinase deficiency.     

Central role of brown adipose tissue thyroxine 5'-deiodinase on thyroid hormone-dependent thermogenic response to cold

As judged by the response of uncoupling protein and key enzymes, brown adipose tissue (BAT) is highly dependent upon the local generation of T3 catalyzed by the tissue type II T4 5'-deiodinase (5'-D-II). In hypothyroid rats treated with T3 or T4, the capacity to withstand cold seems better correlated with the normalization of BAT responses than with the liver thyroid status. 5'D-II is activated by cold via sympathetic nervous system (SNS) stimulation, and the activation generates enough T3 to nearly saturate BAT nuclear T3 receptor (NTR) in euthyroid rats. In hypothyroidism, 5'D-II is highly stimulated by the SNS and hypothyroxinemia. In the present studies we have taken advantage of this situation to test 1) the capacity of 5'D-II to maintain nuclear T3 in rats with various degrees of hypothyroxinemia, and 2) the hypothesis that thyroid hormone-dependent BAT-facultative thermogenesis, rather than the effect of thyroid hormone on obligatory thermogenesis (basal metabolic rate), is the basic mechanism by which thyroid hormone confers protection against acute cold exposure. We treated methimazole-blocked rats (undetectable plasma T4 and T3) for a week with either subreplacement doses of T4 (0.5, 1, 2, and 4 micrograms/kg.day) or replacement doses of T4 or T3 (8 or 3 micrograms/kg.day, respectively). Sources and content of BAT nuclear T3 were studied at 25 C and after 48 h at 4 C by labeling the plasmaborne T3 (T3[T3]) with [131I]T3 and the locally generated T3 (T3[T4]) with [125I]T4. Neither the kinetics of nuclear-plasma exchange of T3[T3], the time of appearance of T3[T4] in BAT nuclei, nor NTR maximal binding capacity (0.71 ng T3/mg DNA) was affected by hypothyroidism. Kinetic analyses indicated a maximal BAT NTR occupancy of 40% at euthyroid serum T3 concentrations if T4 is not present. Replacement with T4 normalized both serum T4 and T3, while replacement with T3 normalized serum T3; for all other doses of T4, serum T4 and T3 concentrations were predictably related to the dose. 5'D-II activity decreased with increasing doses of T4, but for each dose of T4, this activity was 2-4 times greater at 4 C than at 25 C. BAT NTR occupancy normalized with 2 micrograms T4/kg in rats maintained at 25 C and with 4 micrograms T4/kg in cold-exposed rats, although in neither condition were serum T4 and T3 normalized nor more than 30% of NTR occupied by plasma T3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Impairment of hypothalamic-pituitary-thyroid function in rats treated with human recombinant tumor necrosis factor-alpha (cachectin)

Tumor necrosis factor-alpha (TNF; cachectin), a peptide secreted from stimulated macrophages, mediates some of the metabolic derangements in inflammatory and neoplastic disorders. To determine whether TNF is responsible for the changes in hypothalamic-pituitary-thyroid (HPT) function in nonthyroid illnesses, we administered synthetic human TNF to male Sprague-Dawley rats. The rats were given TNF or saline (control; both pair fed and nonpair fed) iv (six to eight per group). HPT function was tested 8 h after administration of 200 micrograms TNF/kg BW, 8 h after 5 days of 150 micrograms TNF/kg BW, and 8 h after a 3-day series of 50, 200, and 800 micrograms TNF/kg BW. The single injection of 200 micrograms TNF/kg significantly reduced (all P less than 0.05) serum TSH, T4, free T4, T3, and hypothalamic TRH compared to the corresponding hormone levels in saline-injected control rats. Serum TSH and hypothalamic TRH recovered to normal levels after 5 days of 150 micrograms/kg TNF treatment. With the increasing daily doses of TNF, serum TSH and hypothalamic TRH fell significantly. Hepatic 5'-deiodinase activity was reduced after 1 day of TNF treatment, but increased after the 3-day series of injections. TNF treatment reduced pituitary TSH beta mRNA, but did not affect alpha-subunit mRNA. TNF treatment also reduced thyroid 125I uptake and reduced thyroidal release of T4 and T3 in response to bovine TSH, but did not change the TSH response to TRH. TNF treatment reduced the binding of pituitary TSH to Concanavalin-A, indicating that it alters the glycosylation of TSH. The TSH with reduced affinity for this lectin had reduced biological activity when tested in cultured FRTL-5 rat thyroid cells. In vitro, TNF inhibited 125I uptake by cultured FRTL-5 rat thyroid cells and blocked the stimulation of [3H]thymidine uptake by these cells. The data indicate that TNF acts on the HPT axis at multiple levels and suggest that TNF is one of the mediators responsible for alterations in thyroid function tests in patients with nonthyroidal illnesses.     

Thyrotropin-releasing hormone stimulates growth hormone release from the anterior pituitary of hypothyroid rats in vitro

We have examined the interaction of thyroid hormone and TRH on GH release from rat pituitary monolayer cultures and perifused rat pituitary fragments. TRH (10(-9) and 10(-8)M) consistently stimulated the release of TSH and PRL, but not GH, in pituitary cell cultures of euthyroid male rats. Basal and TRH-stimulated TSH secretion were significantly increased in cells from thyroidectomized rats cultured in medium supplemented with hypothyroid serum, and a dose-related stimulation of GH release by 10(-9)-10(-8) M TRH was observed. The minimum duration of hypothyroidism required to demonstrate the onset of this GH stimulatory effect of TRH was 4 weeks, a period significantly longer than that required to cause intracellular GH depletion, decreased basal secretion of GH, elevated serum TSH, or increased basal secretion of TSH by cultured cells. In vivo T4 replacement of hypothyroid rats (20 micrograms/kg, ip, daily for 4 days) restored serum TSH, intracellular GH, and basal secretion of GH and TSH to normal levels, but suppressed only slightly the stimulatory effect of TRH on GH release. The GH response to TRH was maintained for up to 10 days of T4 replacement. In vitro addition of T3 (10(-6) M) during the 4-day primary culture period significantly stimulated basal GH release, but did not affect the GH response to TRH. A GH stimulatory effect of TRH was also demonstrated in cultured adenohypophyseal cells from rats rendered hypothyroid by oral administration of methimazole for 6 weeks. TRH stimulated GH secretion in perifused [3H]leucine-prelabeled anterior pituitary fragments from euthyroid rats. A 15-min pulse of 10(-8) M TRH stimulated the release of both immunoprecipitable [3H]rat GH and [3H]rat PRL. The GH release response was markedly enhanced in pituitary fragments from hypothyroid rats, and this enhanced response was significantly suppressed by T4 replacement for 4 days. The PRL response to TRH was enhanced to a lesser extent by thyroidectomy and was not affected by T4 replacement. These data suggest the existence of TRH receptors on somatotrophs which are suppressed by normal amounts of thyroid hormones and may provide an explanation for the TRH-stimulated GH secretion observed clinically in primary hypothyroidism.     

Failure to find evidence of immunoglobulin inhibitor(s) of thyroid hormone binding to serum proteins in non-thyroid illnesses

In order to verify the hypothesis of the presence of IgM (or an IgM-like substance) capable of inhibiting thyroid hormone binding to serum proteins and, therefore, capable of enhancing serum free thyroid fractions in non-thyroid illnesses (NTI), we measured TBG and TBPA maximum binding capacities and TBG concentration by an immunoradiometric system in normal pooled sera (NPS) before and after enrichment with purified immunoglobulins (IgM and IgG) from euthyroid NTI sera (free T4, free T3 and TSH levels were normal). A known amount of TBG was diluted 1:2-1:6 with deionized water or with IgM from NPS or from each of 6 NTI sera; the measured values were not different from these expected on theoretical grounds. Likewise, IgM or IgG from normal or from each of 7 NTI sera failed to modify both TBG and TBPA capacities of different NPS, and NTI immunoglobulins showed no binding activity directed to [125I]T4, [125I]T3 or [125I]TBG. In addition, no inhibitory influence of TBG and TBPA capacities was observed when the whole euglobulin fraction obtained by precipitating the same 7 NTI sera by PEG was mixed with NPS. On the other hand, a significant IgM inhibitory effect on the binding of labelled T4 to TBG was found, only when IgM concentrations were experimentally rendered 41 times greater than that requested in the working mixtures. We conclude that no immunoglobulin inhibitor of thyroid hormone binding to transport proteins was evident in the NTI sera investigated.     

Relationship between pituitary and other target organ responsiveness in hypothyroid patients receiving thyroxine replacement

This study was undertaken to compare the sensitivity of the thyrotrophs to that of other tissues to T4 treatment in hypothyroid patients. To do so, we measured serum total and free thyroid hormones and TSH, in addition to several serum markers of peripheral tissue response to thyroid status, in 21 hypothyroid patients treated with 50-micrograms increments of T4 to a maximum of 200 micrograms daily (group I) and in 104 clinically euthyroid patients receiving a long term constant replacement dose (group II). In group I patients, dose-dependent increases (P less than 0.05) in serum glutathione S-transferase, sex hormone-binding globulin, and angiotensin-converting enzyme occurred, whereas serum T4-binding globulin, creatine kinase, and creatinine levels decreased (P less than 0.05). In both patient groups, abnormally high levels of glutathione S-transferase, sex hormone-binding globulin, angiotensin-converting enzyme, alanine aminotransferase, and gamma-glutamyl transferase were found in some patients during treatment. One or more of these biochemical abnormalities suggestive of hyperthyroidism occurred in 15 (71%) group I patients and 27 (26%) group II patients. These were associated with an undetectable serum TSH (less than 0.1 microU/ml) and raised free T4 concentrations in 13, and raised free T3, T4, and T3 concentrations in only 8, 6, and 1 group I patients, respectively. In group II patients, they were more closely associated with an undetectable TSH (67%) or raised free T4 (85%) level than with raised concentrations of free T3 (33%), T4 (26%), or T3 (0%). The use of high sensitivity TSH assays will permit more accurate adjustment of T4 replacement and minimize abnormalities in peripheral tissue biochemistry indicative of overtreatment.     

Serum free thyroid hormones in different degrees of hypothyroidism and in euthyroid autoimmune thyroiditis

Serum total and free T4 and T3, thyroxine-binding globulin (TBG) and TSH, basal and 20, 30 and 60 min after TRH (200 micrograms, iv), were evaluated in 125 hypothyroid patients (38 with severe, 23 with mild, and 64 with subclinical hypothyroidism), in 35 euthyroid subjects with autoimmune thyroiditis, and in 51 healthy controls. T4/TBG and T3/TBG ratios were also calculated. A significant decrease in all indices of thyroid function except for T3 occurred simultaneously with a significant increase in basal and TRH-stimulated TSH levels from healthy subjects to subclinical hypothyroids, from subclinical to mild and from mild to severe hypothyroids; euthyroid patients with autoimmune thyroiditis did not differ from healthy subjects. All severe hypothyroid patients had low T4 as well as free T4 (FT4), free T3 (FT3), T4/TBG and T3/TBG ratios, but among mild and subclinical hypothyroids direct determination of FT4 and FT3 proved to be a better index of thyroid function than determination of T4 and T3 even after correction for TBG levels. FT4 was the most commonly abnormal index (19 of 23 subjects with mild and 14 of 64 with subclinical disease). Regression analysis showed that FT4, T4/TBG ratio, T4, and FT3 had a significant inverse correlation with TSH in hypothyroid patients. Discriminant analysis showed that among the thyroid parameters, FT4 is the variable which discriminates best between control subjects and the 3 groups of hypothyroid patients. These data extend previous reports and in a large series of patients confirm the biological meaning and the clinical value of direct measurement of serum free thyroid hormones in hypothyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)