A model for chronic, intrahypothalamic thyroid hormone administration in rats.

In addition to the direct effects of thyroid hormone (TH) on peripheral organs, recent work showed metabolic effects of TH on the liver and brown adipose tissue via neural pathways originating in the hypothalamic paraventricular and ventromedial nucleus (PVN and VMH). So far, these experiments focused on short-term administration of TH. The aim of this study is to develop a technique for chronic and nucleus-specific intrahypothalamic administration of the biologically active TH tri-iodothyronine (T3). We used beeswax pellets loaded with an amount of T3 based on in vitro experiments showing stable T3 release (∼5 nmol l(-1)) for 32 days. Upon stereotactic bilateral implantation, T3 concentrations were increased 90-fold in the PVN region and 50-fold in the VMH region after placing T3-containing pellets in the rat PVN or VMH for 28 days respectively. Increased local T3 concentrations were reflected by selectively increased mRNA expression of the T3-responsive genes Dio3 and Hr in the PVN or in the VMH. After placement of T3-containing pellets in the PVN, Tshb mRNA was significantly decreased in the pituitary, without altered Trh mRNA in the PVN region. Plasma T3 and T4 concentrations decreased without altered plasma TSH. We observed no changes in pituitary Tshb mRNA, plasma TSH, or plasma TH in rats after placement of T3-containing pellets in the VMH. We developed a method to selectively and chronically deliver T3 to specific hypothalamic nuclei. This will enable future studies on the chronic effects of intrahypothalamic T3 on energy metabolism via the PVN or VMH.

Differential effects of fasting vs food restriction on liver thyroid hormone metabolism in male rats.

A variety of illnesses that leads to profound changes in the hypothalamus-pituitary-thyroid (HPT) are axis collectively known as the nonthyroidal illness syndrome (NTIS). NTIS is characterized by decreased tri-iodothyronine (T3) and thyroxine (T4) and inappropriately low TSH serum concentrations, as well as altered hepatic thyroid hormone (TH) metabolism. Spontaneous caloric restriction often occurs during illness and may contribute to NTIS, but it is currently unknown to what extent. The role of diminished food intake is often studied using experimental fasting models, but partial food restriction might be a more physiologically relevant model. In this comparative study, we characterized hepatic TH metabolism in two models for caloric restriction: 36 h of complete fasting and 21 days of 50% food restriction. Both fasting and food restriction decreased serum T4 concentration, while after 36-h fasting serum T3 also decreased. Fasting decreased hepatic T3 but not T4 concentrations, while food restriction decreased both hepatic T3 and T4 concentrations. Fasting and food restriction both induced an upregulation of liver D3 expression and activity, D1 was not affected. A differential effect was seen in Mct10 mRNA expression, which was upregulated in the fasted rats but not in food-restricted rats. Other metabolic pathways of TH, such as sulfation and UDP-glucuronidation, were also differentially affected. The changes in hepatic TH concentrations were reflected by the expression of T3-responsive genes Fas and Spot14 only in the 36-h fasted rats. In conclusion, limited food intake induced marked changes in hepatic TH metabolism, which are likely to contribute to the changes observed during NTIS.

Fasting-induced changes in hepatic thyroid hormone metabolism in male rats are independent of autonomic nervous input to the liver.

During fasting, profound changes in the regulation of the hypothalamus-pituitary-thyroid axis occur in order to save energy and limit catabolism. In this setting, serum T3 and T4 are decreased without an appropriate TSH and TRH response reflecting central down-regulation of the hypothalamus-pituitary-thyroid axis. Hepatic thyroid hormone (TH) metabolism is also affected by fasting, because type 3 deiodinase (D3) is increased, which is mediated by serum leptin concentrations. A recent study showed that fasting-induced changes in liver TH sulfotransferases (Sults) and uridine 5'-diphospho-glucuronosyltransferase (Ugts) depend on a functional melanocortin system in the hypothalamus. However, the pathways connecting the hypothalamus and the liver that induce these changes are currently unknown. In the present study, we investigated in rats whether the fasting-induced changes in hepatic TH metabolism are regulated by the autonomic nervous system. We selectively cut either the sympathetic or the parasympathetic input to the liver. Serum and liver TH concentrations, deiodinase expression, and activity and Sult and Ugt expression were measured in rats that had been fasted for 36 hours or were fed ad libitum. Fasting decreased serum T3 and T4 concentrations, whereas intrahepatic TH concentrations remained unchanged. D3 expression and activity increased, as was the expression of constitutive androstane receptor, Sult1b1, and Ugt1a1, whereas liver D1 was unaffected. Neither sympathetic nor parasympathetic denervation affected the fasting-induced alterations. We conclude that fasting-induced changes in liver TH metabolism are not regulated via the hepatic autonomic input in a major way and more likely reflect a direct effect of humoral factors on the hepatocyte.

Skeletal muscle deiodinase type 2 regulation during illness in mice.

We have previously shown that skeletal muscle deiodinase type 2 (D2) mRNA (listed as Dio2 in MGI Database) is upregulated in an animal model of acute illness. However, human studies on the expression of muscle D2 during illness report conflicting data. Therefore, we evaluated the expression of skeletal muscle D2 and D2-regulating factors in two mouse models of illness that differ in timing and severity of illness: 1) turpentine-induced inflammation, and 2) Streptococcus pneumoniae infection. During turpentine-induced inflammation, D2 mRNA and activity increased compared to pair-fed controls, most prominently at day 1 and 2, whereas after S. pneumoniae infection D2 mRNA decreased. We evaluated the association of D2 expression with serum thyroid hormones, (de-)ubiquitinating enzymes ubiquitin-specific peptidase 33 and WD repeat and SOCS box-containing 1 (Wsb1), cytokine expression and activation of inflammatory pathways and cAMP pathway. During chronic inflammation the increased muscle D2 expression is associated with the activation of the cAMP pathway. The normalization of D2 5 days after turpentine injection coincides with increased Wsb1 and tumor necrosis factor alpha expression. Muscle interleukin-1beta (Il1b) expression correlated with decreased D2 mRNA expression after S. pneumoniae infection. In conclusion, muscle D2 expression is differentially regulated during illness, probably related to differences in the inflammatory response and type of pathology. D2 mRNA and activity increases in skeletal muscle during the acute phase of chronic inflammation compared to pair-fed controls probably due to activation of the cAMP pathway. In contrast, muscle D2 mRNA decreases 48 h after a severe bacterial infection, which is associated with local Il1b mRNA expression and might also be due to diminished food-intake.

Lacking thyroid hormone receptor beta gene does not influence alterations in peripheral thyroid hormone metabolism during acute illness.

The downregulation of liver deiodinase type 1 (D1) is supposed to be one of the mechanisms behind the decrease in serum tri-iodothyronine (T3) observed during non-thyroidal illness (NTI). Liver D1 mRNA expression is positively regulated by T3, mainly via the thyroid hormone receptor (TR)beta1. One might thus expect that lacking the TRbeta gene would result in diminished downregulation of liver D1 expression and a smaller decrease in serum T3 during illness. In this study, we used TRbeta-/- mice to evaluate the role of TRbeta in lipopolysaccharide (LPS, a bacterial endotoxin)-induced changes in thyroid hormone metabolism. Our results show that the LPS-induced serum T3 and thyroxine and liver D1 decrease takes place despite the absence of TRbeta. Furthermore, we observed basal differences in liver D1 mRNA and activity between TRbeta-/- and wild-type mice and TRbeta-/- males and females, which did not result in differences in serum T3. Serum T3 decreased rapidly after LPS administration, followed by decreased liver D1, indicating that the contribution of liver D1 during NTI may be limited with respect to decreased serum T3 levels. Muscle D2 mRNA did not compensate for the low basal liver D1 observed in TRbeta-/- mice and increased in response to LPS in TRbeta-/- and WT mice. Other (TRbeta independent) mechanisms like decreased thyroidal secretion and decreased binding to thyroid hormone-binding proteins probably play a role in the early decrease in serum T3 observed in this study.

Effect of amiodarone and dronedarone administration in rats on thyroid hormone-dependent gene expression in different cardiac components.

OBJECTIVE: In view of their different actions on thyroid hormone receptor (TR) isoforms we set out to investigate whether amiodarone (AM) and dronedarone (Dron) have different and/or component-specific effects on cardiac gene expression. DESIGN: Rats were treated with AM or Dron and the expression of TRalpha 1, TRalpha 2, TRbeta 1 and several tri-iodothyronine (T3)-regulated genes was studied in different parts of the heart, namely the right atrium (RA), left ventricular wall (LVW) and apex. METHODS: Rats were treated for 14 days with 100 mg/kg body weight AM or Dron. The expression of TRalpha 1, TRalpha 2, TRbeta 1 and T3-regulated genes was studied using real-time PCR and non-radioactive in situ hybridisation. RESULTS: AM and Dron affected TR expression in the RA similarly by decreasing TRalpha 1 and beta 1 expression by about 50%. In the LVW, AM and Dron decreased TRbeta 1 and, interestingly, AM increased TRalpha 1. In the apex, AM also increased TRalpha 2. The changes seen in T3-dependent gene expression are reminiscent of foetal reprogramming. CONCLUSION: Taken together, our results indicate that AM and Dron have similar effects on the expression of TR isoforms in the RA, which could partly contribute to their ability to decrease heart rate. On the other hand, the more profound effect of AM appears on TR- and T3-dependent gene expression in the left ventricle suggests foetal reprogramming.

Amiodarone inhibits thyroidal iodide transport in vitro by a cyclic adenosine 5'-monophosphate- and iodine-independent mechanism.

Thyroid side effects are common in patients treated for cardiac arrhythmias with amiodarone (AM). A major disturbance is inhibited thyroidal radioiodine uptake in AM-induced thyrotoxicosis, which makes 131I therapy ineffective. On the other hand, failure to escape from the Wolff-Chaikoff effect by down-regulation of the sodium/iodide symporter (NIS) is proposed to explain AM-induced hypothyroidism. However, previously no experimental studies on the possible mechanisms have been conducted. We therefore investigated the early effects of AM on thyroidal iodide transport using bicameral chamber cultures of primary pig thyrocytes that reproduce the three tissue compartments (epithelium, lumen, and extrafollicular space) of the gland. AM dose-dependently (1-50 microm) inhibited the TSH-stimulated transepithelial (basal to apical) transport of 125I- by up to 90%. The inhibitory effect was noticed already after 8 h and was further pronounced after 1-4 d, depending on the AM concentration. The intracellularly accumulated 125I- was reduced by perchlorate but not AM, and quantitative real-time RT-PCR revealed no change in the NIS expression in AM-treated cells. Blocking of cAMP degradation with 3-isobutyl-1-methylxanthine or withdrawal of AM reversed AM-induced changes in electrolyte transport but were unable to recover the suppressed 125I- transport. The iodine-free AM analog dronedarone also inhibited 125I- transport to the same extent as AM. The findings indicate that AM blocks thyroidal iodide uptake by reducing the iodide permeability of the apical plasma membrane of the thyroid epithelial cells. The effect is iodine independent and long-lasting and does not involve impaired function of NIS or the TSH receptor/cAMP signaling pathway.

Dronerarone acts as a selective inhibitor of 3,5,3'-triiodothyronine binding to thyroid hormone receptor-alpha1: in vitro and in vivo evidence.

Dronedarone (Dron), without iodine, was developed as an alternative to the iodine-containing antiarrhythmic drug amiodarone (AM). AM acts, via its major metabolite desethylamiodarone, in vitro and in vivo as a thyroid hormone receptor alpha(1) (TRalpha(1)) and TRbeta(1) antagonist. Here we investigate whether Dron and/or its metabolite debutyldronedarone inhibit T(3) binding to TRalpha(1) and TRbeta(1) in vitro and whether dronedarone behaves similarly to amiodarone in vivo. In vitro, Dron had a inhibitory effect of 14% on the binding of T(3) to TRalpha(1), but not on TRbeta(1). Desethylamiodarone inhibited T(3) binding to TRalpha(1) and TRbeta(1) equally. Debutyldronedarone inhibited T(3) binding to TRalpha(1) by 77%, but to TRbeta(1) by only 25%. In vivo, AM increased plasma TSH and rT(3), and decreased T(3). Dron decreased T(4) and T(3), rT(3) did not change, and TSH fell slightly. Plasma total cholesterol was increased by AM, but remained unchanged in Dron-treated animals. TRbeta(1)-dependent liver low density lipoprotein receptor protein and type 1 deiodinase activities decreased in AM-treated, but not in Dron-treated, animals. TRalpha(1)-mediated lengthening of the QTc interval was present in both AM- and Dron-treated animals. The in vitro and in vivo findings suggest that dronedarone via its metabolite debutyldronedarone acts as a TRalpha(1)-selective inhibitor.

A survey of iodine intake and thyroid volume in Dutch schoolchildren: reference values in an iodine-sufficient area and the effect of puberty.

BACKGROUND: Iodine deficiency and endemic goiter have been reported in the past in The Netherlands, especially in the southeast. OBJECTIVE: To evaluate iodine intake and thyroid size in Dutch schoolchildren, contrasting those living in a formerly iodine-deficient region in the east (Doetinchem) with those living in an iodine-sufficient region in the west (Amsterdam area). DESIGN: Cross-sectional survey of 937 Dutch schoolchildren aged 6--18 years, of whom 390 lived in the eastern and 547 in the western part of the country. METHODS: Thyroid size was assessed by inspection and palpation as well as by ultrasound. Iodine intake was evaluated by questionnaires on dietary habits and by measurement of urinary iodine concentration. RESULTS: Eastern and western regions were similar with respect to median urinary iodine concentration (15.7 and 15.3 microg/dl, NS, Mann-Whitney U test), goiter prevalence by inspection and palpation (0.8 and 2.6%, P=0.08, chi-squared test), and thyroid volumes. The P97.5 values of thyroid volumes per age and body surface area group were all lower than the corresponding sex-specific normative WHO reference values. Iodized salt was not used by 45.7% of households. Daily bread consumption was five slices by boys and four slices by girls. Weekly milk consumption was 3 liters by boys and 2 liters by girls. Seafish was consumed once monthly. From these figures we calculated a mean daily iodine intake of 171 microg in boys and 143 microg in girls, in good agreement with the measured median urinary concentration of 16.7 microg/dl in boys and 14.5 microg/dl in girls. The sex difference in iodine excretion is fully accounted for by an extra daily consumption of one slice of bread (20 microg I) and one-seventh of a liter of milk (8.3 microg I) by boys. Thyroid volume increases with age, but a steep increase by 41% was observed in girls between 11 and 12 years, and by 55% in boys between 13 and 14 years, coinciding with peak height velocity. Girls have a larger thyroid volume at the ages of 12 and 13 years, but thyroid volume is larger in boys as of the age of 14 years. CONCLUSIONS: (1) Iodine deficiency disorders no longer exist in The Netherlands. (2) Bread consumption remains the main source of dietary iodine in The Netherlands; the contribution of iodized table salt and seafish is limited. (3) The earlier onset of puberty in girls renders their thyroid volume larger than in boys at the age of 12--13 years, but boys have a larger thyroid volume as of the age of 14 years.

Desethylamiodarone interferes with the binding of co-activator GRIP-1 to the beta 1-thyroid hormone receptor.

Ligand binding to the thyroid hormone nuclear receptor beta1 (TRbeta(1)) is inhibited by desethylamiodarone (DEA), the major metabolite of the widely used anti-arrhythmic drug amiodarone. Gene expression of thyroid hormone (triiodothyronine, T(3))-regulated genes can therefore be affected by amiodarone due to less ligand binding to the receptor. Previous studies have indicated the possibility of still other explanations for the inhibitory effects of amiodarone on T(3)-dependent gene expression, probably via interference with receptor/co-activator and co-repressor complex. The binding site of DEA is postulated to be on the outside surface of the receptor protein overlapping the regions where co-activator and co-repressor bind. Here we show the effect of a drug metabolite on the interaction of TRbeta(1) with the co-activator GRIP-1 (glucocorticoid receptor interacting protein-1). The T(3)-dependent binding of GRIP-1 to the TRbeta(1) is disrupted by DEA. A DEA dose experiment showed that the drug metabolite acts like an antagonist under 'normal' conditions (at 10(-7) M T(3) and 5x10(-6)-->10(-3) M DEA), but as an agonist under extreme conditions (at 0 and 10(-9) M T(3) and >10(-4) M DEA). To our knowledge, these results show for the first time that a metabolite of a drug which was not devised for this purpose can interfere with nuclear receptor/co-activator interaction.

Effect of mutations in the beta1-thyroid hormone receptor on the inhibition of T3 binding by desethylamiodarone.

Desethylamiodarone (DEA) acts as a competitive inhibitor of triiodothyronine (T3) binding to the alpha1-thyroid hormone receptor (TR alpha1) but as a non-competitive inhibitor with respect to TR beta1. To gain insight into the position of the binding site of desethylamiodarone on TR beta1 we investigated the naturally occurring mutants Y321C, R429Q, P453A, P453T and the artificial mutants L421R and E457A in the ligand binding domain of human TR beta1. The IC50 values (in microM) of DEA for P453A (50 +/- 11) and P453T (55 +/- 16) mutant TR beta1 are not different from that for the wild type TR beta1 (56 +/- 15), but the IC50 values of R429Q (32 +/- 7; P<0.001) and E457A (17 +/- 3; P<0.001) are significantly lower than of the wild type. Scatchard plots and Langmuir analyses indicate a non-competitive nature of the inhibition by DEA of T3 binding to all four mutant TR beta1s tested. Mutants P453A and P453T do not influence overall electrostatic potential, and also do not influence the affinity for DEA compared to wild type. Mutant E457A causes a change from a negatively charged amino acid to a hydrophobic amino acid, enhancing the affinity for DEA. Mutant R429Q, located in helix 11, causes an electrostatic potential change from positive to uncharged, also resulting in greater affinity for DEA. We therefore postulate that amino acids R429 and E457 are at or close to the binding site for DEA, and that DEA does not bind in the T3 binding pocket itself, in line with the non-competitive nature of the inhibition of T3 binding to TR beta1 by DEA.

Structure-function relationship of the inhibition of the 3,5,3'-triiodothyronine binding to the alpha1- and beta1-thyroid hormone receptor by amiodarone analogs.

Desethylamiodarone (DEA), the major metabolite of the potent antiarrhythmic drug amiodarone (A), acts as a competitive inhibitor of T3, binding to the alpha1-thyroid hormone receptor (alpha1-T3R), but as a noncompetitive inhibitor with respect to the beta1-T3R. To gain insight into the structure- function relationship of the interaction between A metabolites and T3Rs, we investigated the effects of several A analogs on T3 binding to the alpha1-T3R and beta1-T3R in vitro. The analogs tested were: 1) compounds obtained by deethylation of A, DEA, and desdiethylamiodarone (DDEA); 2) compounds obtained by deiodination of A, monoiodoamiodarone and desdiiodoamiodarone (DDIA); and 3) benzofuran derivatives with various iodination grades, 2-butyl-3-(4-hydroxy-3,5-diiodo-benzoyl)benzofuran (L3373, two iodine atoms), L6424 (L3373 with one iodine atom), and L3372 (L3373, no iodine atoms). IC50, values of inhibition of T3 binding to alpha1-T3R and beta1-T3R, respectively, were as follows (mean +/- SD, expressed x 10(-5) M): DEA, 4.7 +/- 0.9 and 2.7 +/- 1.4 (P < 0.001); DDEA, 3.7 +/- 0.9 and 1.9 +/- 0.3 (P < 0.001); monoiodoamiodarone, more than 20 and more than 20; DDIA, 16.2 +/- 5.6 and 9.1 +/- 2.1 (P < 0.01); L3373, 3.8 +/- 1.0 and 3.6 +/- 0.5 (P = NS); L6424, 11.3 +/- 5.7 and 10 +/- 2.0 (P = NS); and L3372, no inhibition. Scatchard analyses in the presence of DDEA, DDIA, and L3373 demonstrated a dose-dependent decrease in Ka, but no change in the maximum binding capacity (MBC) of T3 binding to alpha1-T3R. Langmuir plots clearly indicated competitive inhibition of T3 binding to alpha1-T3R by DDEA, DDIA, and L3373. In contrast, these three analogs acted differently with respect to the beta1-T3R. DDEA and DDIA decreased both Ka and MBC in Scatchard plots using beta1-T3R, demonstrating noncompetitive inhibition. L3373 decreased dose-dependently Ka, but not MBC, values of T3 binding to the beta1-T3R and clearly acted as a competitive inhibitor. Ki plots indicated that DDEA, DDIA, and L3373 do not interfere significantly with occupied T3Rs. KI (inhibition constant for the unoccupied receptor) plots demonstrated increasing inhibition of the T3 binding to unoccupied receptors with increasing analog concentrations. In summary, 1) removal of one or two ethyl groups of A results in compounds with strong but almost equal potency of inhibiting T3R binding, whereas removal of one or two iodine atoms of A has a lower potency in this respect. The strong inhibitory potency of the benzofuran derivative L3373 (equalling that of the deethylated compounds) is lost upon deiodination. 2) All tested A analogs acted as competitive inhibitors to the alpha1-T3R. The behavior to the beta1-T3R was different; deethylation or deiodination of A resulted in noncompetitive inhibition, whereas L3373 was a competitive inhibitor. The potency of deethylated and deiodinated compounds (but not of the benzofuran derivatives) for inhibiting T3 binding was twice as high for the beta1-T3R as for the alpha1-T3R. 3) All tested A analogs preferentially interfere with T3 binding to unoccupied receptors. The implications of these findings for the structure-activity relationship are the following: 1) the size of the diethyl-substituted nitrogen group and of the two bulky iodine atoms in the A molecule hamper the binding of A at the T3 binding site of T3Rs; and 2) differences in the hormone-binding domain of alpha1- and beta1-T3Rs are likely to account for the competitive or noncompetitive nature of inhibition of T3 binding by A analogs.

Desethylamiodarone is a competitive inhibitor of the binding of thyroid hormone to the thyroid hormone alpha 1-receptor protein.

Desethylamiodarone (DEA), the major metabolite of the potent antiarrythmic drug amiodarone, is a non-competitive inhibitor of the binding of thyroid hormone (T3) to the beta 1-thyroid hormone receptor (T3R). In the present study, we investigated whether DEA acts in a similar way with respect to the alpha 1-T3R. The chicken alpha 1-T3R, expressed in an E. coli system, was incubated in the presence or absence of DEA with [125I]T3 in buffer containing 0.05% Triton X-100, 0.05% BSA and 1% ethanol (v/v) in order to solubilise DEA. DEA, but not amiodarone, inhibited T3 binding in a dose-dependent manner; the IC50 value was 3.5 x 10(-5) M. Scatchard analyses in the presence of DEA demonstrated a dose-dependent decrease in Ka values, but no change in MBC. Lineweaver-Burk plots clearly indicated competitive inhibition by DEA. Pre-incubation of the alpha 1-receptor with DEA decreased maximal [125I]T3 binding, which was independent of the duration of pre-incubation. In conclusion, in contrast to the beta 1-T3R, where DEA acts as a non-competitive inhibitor, we now report as a new finding the competitive action of DEA to the alpha 1-T3R.

Desethylamiodarone is a noncompetitive inhibitor of the binding of thyroid hormone to the thyroid hormone beta 1-receptor protein.

It has been hypothesized that amiodarone (A), a potent antiarrythmic and antianginal drug, induces a local hypothyroid-like condition in extrathyroidal tissues. This might be related to competitive antagonism of A for the thyroid hormone receptor reported in some studies but denied in others. These conflicting results are presumably due to the poor solubility of A in a hydrophilic environment. We, therefore, studied the effect of the drug and its major metabolite, desethylamiodarone (DEA), on the in vitro binding of thyroid hormone (T3) to its receptor protein using the rat beta 1-thyroid hormone receptor expressed in Escherichia coli. A and DEA stayed in solution up to 10(-4) M when 0.05% Triton X-100 was added to the incubation buffer, as evidenced by a recovery of 80-90% for both chemicals, as measured by HPLC. DEA, but not A, had a clear inhibitory effect on the binding of T3 to its receptor (IC50, 1-3 x 10(-5) M). Scatchard analysis in the presence of DEA demonstrated a dose-dependent decrease in the Ka as well as the maximum binding capacity. Lineweaver-Burke analysis indicated noncompetitive inhibition. Plots of the intercepts of Lineweaver-Burke plots vs. DEA concentration were linear (y = 0.334 + 0.098x), giving a Ki of 30 microM for the binding of DEA to the occupied receptor. Plots of the slopes vs. inhibitor concentration were parabolic (y = 3.01 + 0.06x + 0.16x2), indicating a progressively stronger effect of DEA on the unoccupied receptor as concentrations rise. This preference for the unoccupied receptor is reflected in experiments that show a progressive loss of T3 binding when the receptor was incubated for increasing periods with DEA before adding T3. We conclude that DEA is a noncompetitive inhibitor of the binding of T3 to the beta 1-thyroid hormone receptor protein, interacting preferably with the unoccupied T3 receptor.

Competitive inhibition of T3 binding to alpha 1 and beta 1 thyroid hormone receptors by fatty acids.

This study was undertaken to investigate whether fatty acids inhibit the binding of T3 to the alpha 1 and beta 1 form of the thyroid hormone receptor. Fatty acids inhibited the binding of T3 to both receptor proteins isolated from a bacterial expression system. The effectiveness of inhibition depends on the chain length and degree of saturation of the fatty acids. The inhibition of T3 binding to the alpha 1 and beta 1 receptor by oleic acid is competitive in nature; the Ki value was 5.4 10(-6) M for the c-erbA alpha 1 protein and 3.3 10(-6) M for the c-erb beta 1 protein. The findings indicate a direct interaction of fatty acids with T3 receptor proteins.

Localization of c-ERB A proteins in rat liver using monoclonal antibodies.

Monoclonal antibodies were raised against the nuclear thyroid hormone receptors encoded by c-ERB A genes and against a purified nuclear receptor fraction. These antibodies recognize the c-ERB A protein in nuclear extracts from rat liver and are able to compete with thyroid hormone in Scatchard analyses. In sections of rat liver they react with all the hepatocyte nuclei as well as with the cells of the hepatic bile ducts. Comparison with another putative T3 receptor antibody, described previously, showed that distinct 57 kD proteins with a different cellular distribution were recognized.