Action of Specific Thyroid Hormone Receptor α 1 and β 1 Antagonists in the Central and Peripheral Regulation of Thyroid Hormone Metabolism in the Rat

Abstract

Background:

The iodine-containing drug amiodarone (Amio) and its noniodine containing analogue dronedarone (Dron) are potent antiarrhythmic drugs. Previous in vivo and in vitro studies have shown that the major metabolite of Amio, desethylamiodarone, acts as a thyroid hormone receptor (TR) α₁ and β₁ antagonist, whereas the major metabolite of Dron debutyldronedarone acts as a selective TRα₁ antagonist. In the present study, Amio and Dron were used as tools to discriminate between TRα₁ or TRβ₁ regulated genes in central and peripheral thyroid hormone metabolism.

Methods:

Three groups of male rats received either Amio, Dron, or vehicle by daily intragastric administration for 2 weeks. We assessed the effects of treatment on triiodothyronine (T₃) and thyroxine (T₄) plasma and tissue concentrations, deiodinase type 1, 2, and 3 mRNA expressions and activities, and thyroid hormone transporters monocarboxylate transporter 8 (MCT8), monocarboxylate transporter 10 (MCT10), and organic anion transporter 1C1 (OATP1C1).

Results:

Amio treatment decreased serum T₃, while serum T₄ and thyrotropin (TSH) increased compared to Dron-treated and control rats. At the central level of the hypothalamus-pituitary-thyroid axis, Amio treatment decreased hypothalamic thyrotropin releasing hormone (TRH) expression, while increasing pituitary TSHβ and MCT10 mRNA expression. Amio decreased the pituitary D2 activity. By contrast, Dron treatment resulted in decreased hypothalamic TRH mRNA expression only. Upon Amio treatment, liver T₃ concentration decreased substantially compared to Dron and control rats (50%, p<0.01), but liver T₄ concentration was unaffected. In addition, liver D1, mRNA, and activity decreased, while the D3 activity and mRNA increased. Liver MCT8, MCT10, and OATP1C1 mRNA expression were similar between groups.

Conclusion:

Our results suggest an important role for TRα1 in the regulation of hypothalamic TRH mRNA expression, whereas TRβ plays a dominant role in pituitary and liver thyroid hormone metabolism.

Introduction

B oth the iodine-containing drug amiodarone (Amio) and the noniodine containing dronedarone (Dron) are very potent antiarrhythmic drugs and have been shown to be thyroid hormone receptor (TR) antagonists. Amio acts in vitro and in vivo via its major metabolite desethylamiodarone (DEA), which is a TRα₁ and TRβ₁ antagonist, whereas the major metabolite of Dron, debutyldronedarone (DBDron), selectively inhibits triiodothyronine (T₃) binding to TRα₁ (1). In patients as well as experimental animals on long-term Amio treatment, profound changes in serum thyroid hormone levels occur. Serum thyrotropin (TSH) and thyroxine (T₄) increase, whereas serum T₃ decreases. Amio treatment also results in the accumulation of tissue concentrations of Amio and DEA (2,3), thereby altering T₃-dependent gene expression. In rat liver, low-density lipoprotein receptor and deiodinase type 1 (D1) expression, which are both under TRβ₁ control (4) decrease upon Amio-treatment. By contrast, Dron treatment does not affect the expression of these genes (1).

In addition to their role in the liver, TRs are also involved in negative feedback regulation of the hypothalamus–pituitary–thyroid (HPT) axis. The TRβ₂ isoform has been proposed to mediate negative feedback of T₃ on thyrotropin releasing hormone (TRH) gene expression in the hypothalamic paraventricular nucleus (PVN) (5), although in vitro transfection studies showed T₃-dependent inhibition of the hTRH gene promoter by all TRs (6). TRβ₂ is also involved in the regulation of pituitary TSHβ mRNA expression (7).

Amio and Dron are TR-specific antagonists and, therefore, excellent tools to study TR-specific action with regard to HPT axis regulation and, in more general terms, T₃-regulated gene expression without the need to use genetically modified animals. The aim of the present study was to determine the roles of TR isoforms in local thyroid hormone metabolism in the hypothalamus, pituitary, and liver of rats that were orally treated with Amio or Dron for 14 days.

Materials and Methods

Amio and Dron were gifts from Sanofi Pharmaceuticals, Inc. Synthélabo (Montpellier, France). All reagents were of the highest grade possible.

Experimental design

Male Wistar rats (weight 220–260 g) were housed under normal conditions with free access to standard laboratory chow and tap water, and divided in three groups (n=12). The rats received water (controls), an aqueous suspension of Amio 100 mg/kg BW or Dron 100 mg/kg BW by intragastric tube daily for 2 weeks. The dose used has been described as effective and resulted in marked concentrations of the metabolites in various tissues (3) and in the characteristic alteration in serum thyroid hormone and cholesterol levels (1). Blood was collected by cardiac puncture and plasma was stored at −20°C. The liver, pituitary, and hypothalamus (defined rostrally by the optic chiasm, caudally by the mamillary bodies, laterally by the optic tract, and dorsally by the apex of the third ventricle) were isolated and stored immediately in liquid nitrogen. The tissue block containing the hypothalamus was used for dissection of the periventricular area (PE). The PE contains both paraventricular nuclei and the upper part of the ependymal lining of the third ventricle and was obtained by punching the hypothalamus with a hollow needle (diameter 1800 μm) based on anatomical landmarks (the apex of the third ventricle) (8). The PE samples may include (part of) the dorsomedial nucleus which, like the PVN, contains TRH neurons. All animal experiments were approved by the local animal welfare committee of the Academic Medical Center.

Plasma assays

Total T₄ and total T₃ were measured by in-house radioimmunoassays (9). TSH was determined by a chemiluminescent immunoassay, using the Immulite 2000 (Siemens, Munich, Germany). All samples were measured within one run to prevent interassay variation.

RNA isolation and reverse transcription–polymerase chain reaction

Hypothalamic PE, pituitary, and liver mRNA were isolated on the Magna Pure (Roche Molecular Biochemicals, Mannheim, Germany) using the Magna Pure LC mRNA tissue kit. The protocol and buffers supplied with the corresponding kit were applied. cDNAsynthesis was performed using the First Strand cDNA Synthesis Kit for reverse transcription–polymerase chain reaction (RT-PCR) with oligo d(T) primers (Roche). Real-time PCR was performed using the Lightcycler480 (Roche) and the Lightcycler 480 Sybr Green I Master kit (Roche). Primer pair and PCR condition for hypoxanthine phosphoribosyl transferase (HPRT, housekeeping-gene liver) (10) was previously described.

New primer pairs and PCR conditions were developed for deiodinase type 1 (D1), deiodinase type 2 (D2), deiodinase type 3 (D3), preproTRH, TSHβ, organic anion transporter 1C1 (OATP1C1), monocarboxylate transporter 8 (MCT8), monocarboxylate transporter 10 (MCT10), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, housekeeping gene).

Sequences of the new primer pairs and PCR conditions are listed in Table 1. Primers were intron-spanning except for D3, and in that case genomic DNA contamination was tested using a cDNA synthesis reaction without the addition of reverse transcriptase. Samples were corrected for their mRNA content using HPRT (liver) or GAPDH (hypothalamic PE and pituitary) as a housekeeping gene, as both genes do not change upon treatment.

Table 1.

Primer Sequences of the Primer Pairs Used

Gene	Primer 5′-3′	Amplicon length (bp)	Annealing temperature (^oC)
Dio1	F: GAA GTG CAA CGT CTG GGA TT R: CTG CCG AAG TTC AAC ACC A	59	55
Dio2	F: TCC TGG AGC GTT TCT CCT T R: CAT AAG CTA CGT TGG CAT TAT TGT	78	53
Dio3	F: AGC GCA GCA AGA GTA CTT CAG R: CCA TCG TGT CCA GAA CCA G	61	55
TRH	F: TCT GCA GAG TCT CCA CTT R: AGA GCC AGC AGC AAC CAA	59	54
TSHβ	F: TCG TTC TCT TTT CCG TGC TT R: CGG TAT TTC CAC CGT TCT GT	245	53
OATP1C1	F: AGG AAT CCC AGC CCC CGT GT R: ACA CCG TGC CCA AGA GCG TG	158	60
MCT8	F: CCG AAG GTG GCT TCG GCT GG R: TGA TCC GGC AGC CCA AAC GG	234	70
MCT10	F: GAC CGC CTA TGC CGT GTG GG R: CGG CCG AAG AGA AGC CGT CC	174	68
GAPDH	F: TGA ACG GGA AGC TCA CTG G R: TCC ACC ACC CTG TTG CTG TA	306	55

F, forward; R, reverse; Dio, deiodinase; TRH, thyrotropin releasing hormone; TSH, thyrotropin; OATP1C1, organic anion transporter 1C1; MCT, monocarboxylate transporter; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Quantification was performed using the LinReg software (11). The mean efficiency was calculated for each assay, and samples showing a difference in efficiency >0.05 of the mean value were not taken into account. Aberrant PCR-efficiencies occurred randomly and, therefore, did not bias the results.

Deiodinase activity

The deiodinase activity was measured as previously described (12,13). Samples were homogenized on ice in 10 volumes of phosphate-EDTA buffer (0.1 M sodium phosphate, 2 mM EDTA pH 7.2) using a Polytron (Kinematica, Luzern, Switzerland). 50 mM DTT was added to the phosphate-EDTA buffer for D1, D2, and D3 activity measurements. Homogenates were snap-frozen and stored at −80°C until use for D1 and D3, for D2 homogenates were used immediately. The protein concentration was measured with the Bio-Rad protein assay using bovine serum albumin (BSA) as the standard following the manufacturer's instructions (Bio-Rad Laboratories, Veenendaal, The Netherlands).

The liver D1 activity was measured in duplicate, using 50 μL 100–500 times diluted homogenate, incubated in a final volume of 0.1 mL with 100 nM rT₃ with the addition of ∼1×10⁵ cpm [3′5′-¹²⁵I]rT₃ in PED10 (0.1 M sodium phosphate, 2 mM EDTA pH 7.2, 10 mM DTT).

The pituitary D1 and D2 activity were measured in duplicate, using 50 μL homogenate, incubated 90 minutes at 37°C in a final volume of 0.1 mL with 1 nM T₄ with the addition of ∼2×10⁵cpm [3′5′-¹²⁵I]T₄ in phosphate-EDTA buffer/0.5% BSA in the absence and presence of 0.1 mM PTU.

D1 and D2 reactions were stopped by adding 0.1 mL of 5% BSA on ice. The protein-bound iodothyronines were precipitated by the addition of 10% (w/v) trichloroacetic acid. After centrifugation, ¹²⁵I was separated from the supernatant by chromatography on Sephadex LH-20 columns with a bed volume of 0.25 mL, equilibrated, and eluted with 0.1 M HCl. Released ¹²⁵I was counted using the Packard Cobra Auto-Gamma Counting System (Canberra Packard, Zürich, Switzerland) in the eluate. The D1 and D2 activity were expressed as pmol ¹²⁵I released per minute per mg tissue protein. The pituitary D2 activity was calculated from the amount of ¹²⁵I released in the presence of PTU. The pituitary D1 activity was calculated by subtracting the D2 activity from the total activity in the absence of PTU.

The liver D3 activity was measured in duplicate, using 75 μL homogenate, incubated 2 hours at 37°C in a final volume of 0.15 mL with 1 nM T₃ or 500 nM T₃ with the addition of ∼2×10⁵cpm [3′5′-¹²⁵I]T₃ in phosphate-EDTA buffer. Reactions were stopped by adding 0.15 mL ice-cold ethanol. After centrifugation, 0.125 mL of the supernatant was added to 0.125 mL 0.02 M ammonium acetate (pH 4) and 0.1 mL of the mixture was applied to 4.6×250 mm Symmetry C18 column connected to a Waters high-performance liquid chromatography (HPLC) system (Model 600E pump, Model 717 WISP autosampler; Waters, Etten-Leur, The Netherlands). Mobile phase A: 0.02M ammonium acetate (pH4.0), mobile phase B: acetonitril. The column was eluted with a linear gradient (28%–42% B in 15 minutes) at a flow of 1.2 mL/min. The activity of T₃ and T₂ in the eluate was measured on-line with a Radiomatic Flow-one/Beta scintillation detector (Packard, Meriden, CT) using UltimaGold as scintillation fluid (Perkin Elmer, Groningen, The Netherlands). Incubation with 500 nM T₃ saturates D3, therefore, the D3 activity measured with the incubation with 1 nM T₃ minus the incubation with 500 nM T₃ represents the true D3 activity. The D3 activity was expressed as fmol generated 3,3′T₂ per hour per mg tissue.

T₃ and T₄ tissue concentrations

T₃ and T₄ concentrations in liver and hypothalamus were measured using an ultraperformance liquid chromatography (UPLC) tandem mass spectrometry (MS) method as recently reported (14). The method uses stably labeled internal standards for T₃ and T₄ enabling the measurement of T₃ and T₄ in one analytical run. Briefly, 200 mg of tissue were homogenized in methanol using a Magna Lyser (Roche). Liquid/liquid extraction and solid-phase extraction were performed as described by Morreale de Escobar et al. (15). After evaporation to dryness of the eluate, the sample was redissolved in 200 μL 0.1% NH₄OH and placed in the autosampler. Separation was achieved with gradient elution on a RP BEHC18 column, using an Acquity UPLC (Waters, Milford, MA). Detection was performed with a Waters Quattro Premier tandem mass spectrometer operated in a positive electrospray ionization mode. All aspects of system operation and data acquisition were controlled using MassLynx v4.1 software with automated data processing using the QuanLynx Application Manager (Waters).

Statistical analysis

Data are presented as mean±SEM. Variation between Amio, Dron, and control rats was evaluated by analysis of variance (ANOVA) with one grouping factor (treatment) and, if significant, followed by the Tukey's test for pairwise comparisons. p-values in the figures represent the statistical significance tested by ANOVA and symbols represent the differences between groups. In case of abnormal distribution of the data, data were rank transformed before the ANOVA. All analyses were carried out in SPSS 11.5.1 (SPSS, Inc., Chicago, IL). p-values <0.05 were considered statistically significant.

Results

Plasma thyroid hormone levels

Amio treatment resulted in significantly higher plasma T₄ and TSH levels, whereas T₃ was lower relative to control values. Plasma TSH, T₄, and T₃ did not change in the Dron-treated animals (Table 2).

Table 2.

Plasma TSH, T₃, and T₄ Concentration in Rats After 14 Days Treatment

	Controls	Amio 100	Dron 100
Plasma TSH (ng/mL)	1.57±0.21	5.76±1.19^a	1.25±0.14^c
Plasma T₄ (nM)	81±2.3	168±5.8^b	85±3.2^c
Plasma T₃ (nM)	1.29±0.03	0.85±0.05^b	1.25±0.06^c

p<0.005, ^b p<0.001, Amio versus controls.

p<0.001, Amio versus Dron treatment.

Values are the mean±SEM; differences between groups were evaluated using one-way analysis of variance followed by Tukey's test for pairwise comparisons (plasma values; n=12).

Amio, amiodarone; Dron, dronedarone; T₃, triiodothyronine; T₄, thyroxine.

Hypothalamic thyroid hormone metabolism

The tissue block containing the hypothalamus was used for dissection of the PE, which consists of both PVNs and the upper part of the ependymal lining of the third ventricle. TRH mRNA expressionin the PE was decreased in both Amio- and Dron-treated rats compared to controls (p<0.05 and p<0.01, respectively) (Fig. 1). The observed TRH decrease was negatively correlated to serum T₄ (r=−0.51; p<0.05), but not to serum T₃ concentrations. The mRNA expression of deiodinases and transporters (D2, D3, MCT8, MCT10, and OATP1C1) in the PE did not change upon Amio or Dron treatment compared to saline-treated rats (Fig. 1). T₃ and T₄ concentrations were measured in the whole hypothalamic block and were similar in Amio-, Dron-, and saline-treated rats (Fig. 2).

FIG. 1.

mRNA expression in the hypothalamic periventricular area (PE) by (A) thyrotropin releasing hormone (TRH), (B) deiodinase type 2 (D2), and (C) D3 mRNA expression. mRNA expression of hypothalamic PE thyroid hormone transporters: (D) monocarboxylate transporter (MCT) 8, (E) MCT10, and (F) organic anion transporter 1C1 (OATP1C1). Mean values and SEM of mRNA expression relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH, a housekeeping gene) are depicted. □, control rats (C, n=6); , amiodarone (Amio)-treated rats (n=5); ■, dronedarone (Dron)-treated rats (n=5). p-value indicates difference between the groups by one-way analysis of variance (ANOVA). Differences between specific groups are indicated by *p<0.05, **p<0.01. NS, not significant.

FIG. 2.

Whole hypothalamic block triiodothyronine (T₃) and thyroxine (T₄) concentrations. Mean values and SEM are depicted. □, control rats (C, n=6); , Amio-treated rats (n=5); ■, Dron-treated rats (n=6). No difference between the groups by one-way ANOVA is observed.

Pituitary thyroid hormone metabolism

Pituitary TSHβ mRNA expression was twofold higher in Amio-treated rats (p<0.01) compared to Dron- and saline-treated rats. TSHβ mRNA expression correlated with serum T₄ (r=0.83; p<0.001), negatively with the D2 activity (r=−0.54; p<0.05), but not with serum T₃ concentrations. D1, D2, and D3 (data not shown) mRNA did not change upon Amio or Dron treatment, while the D2 activity was lower in Amio-treated rats compared to Dron-treated rats (p<0.05) (Fig. 3) and correlated negatively with serum T₄ (r=−0.71; p<0.001). The D1 activity was undetectable in the pituitary of Amio-, Dron-, and saline-treated rats. Pituitary MCT8 and OATP1C1 mRNA expression did not differ between the groups, while MCT10 mRNA expression increased upon Amio-treatment (p<0.01) (Fig. 4). MCT10 expression correlated positively with serum T₄ (r=0.69, p<0.01) and negatively with serum T₃ (r=−0.53, p<0.05).

FIG. 3.

Pituitary D2 mRNA expression, D2 activity, D1 mRNA expression, and TSHβ mRNA expression. Mean values and SEM are depicted. mRNA expression is relative to GAPDH, a housekeeping gene. □, control rats (C, n=6); , Amio-treated rats (n=6); ■, Dron-treated rats (n=6). p-value indicates difference between the groups by one-way ANOVA. Differences between specific groups are indicated by *p<0.05, **p<0.01.

FIG. 4.

mRNA expression of pituitary thyroid hormone transporters MCT8, MCT10, and OATP1C1. Mean values and SEM of mRNA expression relative to GAPDH (a housekeeping gene) are depicted. □, control rats (C, n=6); , Amio-treated rats (n=6); ■, Dron-treated rats (n=6). p-value indicates difference between the groups by one-way ANOVA. Difference between specific groups is indicated by **p<0.01.

Liver thyroid hormone metabolism

Liver D1 mRNA and activity decreased substantially upon Amio treatment (p<0.005 and p<0.001, respectively), while both D3 mRNA and activity increased (p<0.05) compared to Dron and saline (Fig. 5). Both D1 mRNA and activity correlated positively with serum T₃ (r=0.71; p<0.01 and r=0.59; p<0.01, respectively), and negatively with serum T₄ (r=−0.64; p<0.01 and r=−0.74; p<0.01, respectively). By contrast, both D3 mRNA and activity correlated negatively with serum T₃ (r=−0.71; p<0.01 and r=−0.56; p<0.01, respectively), while the D3 activity correlated positively with serum T₄ (r=0.52; p<0.01). MCT 8, MCT10, and OATP1C1 mRNA expression did not change upon Amio or Dron treatment (data not shown). The liver T₃ concentration decreased by 50% (p<0.001) upon Amio treatment compared to Dron- and saline-treated rats. In contrast, the liver T₄ concentration was similar between the groups (Fig. 6).

FIG. 5.

Liver D1 mRNA expression, D1 activity, D3 mRNA expression, and D3 activity. Mean values and SEM are depicted. mRNA expression is relative to HPTR, a housekeeping gene. □, control rats (C, mRNA n=6; activity n=8); , Amio-treated rats (mRNA n=6; activity n=8); ■, Dron-treated rats (mRNA n=6; activity n=8). p-value indicates difference between the groups by one-way ANOVA. Differences between specific groups are indicated by *p<0.05, **p<0.01.

FIG. 6.

Liver T₃ and T₄ concentrations. Mean values and SEM are depicted. □, control rats (C, n=8); , Amio-treated rats (n=8); ■, Dron-treated rats (n=8). p-value indicates difference between the groups by one-way ANOVA. Differences between specific groups are indicated by **p<0.01.

Discussion

The aim of this study was to determine the involvement of the TRα₁ or TRβ₁ in Amio- and Dron-induced changes in central and hepatic thyroid hormone metabolism. To this end, we treated rats orally with Amio (TRα/TRβ-specific antagonist) or Dron (TRα-specific antagonist) for 14 days. Our previous in vitro studies have shown that DEA, the major metabolite of Amio, acts as a TRα₁ and β₁ antagonist, whereas DBDron, the major metabolite of Dron, acts as a selective TRα₁ antagonist. Other known Amio-metabolites did not inhibit T₃-TR binding (16). The Amio-model is well-established and results in marked tissue concentrations of the metabolites (3) as well as profound alterations in the serum thyroid hormone and cholesterol levels similar to the changes observed in Amio-treated patients (1). In addition to the expected changes in serum thyroid hormone levels, Amio induced marked changes in the gene expression of hypothalamic, pituitary, and hepatic genes involved in thyroid hormone metabolism, while Dron affects only hypothalamic TRH expression.

Hypothalamic PE

We found decreased TRH mRNA expression in the PE of both Amio- and Dron-treated rats that was independent of changes in serum T₃ as T₃ decreased in Amio-treated rats, but was unaltered in the Dron-treated rats. Our finding is in agreement with a recent study by Rosene et al. reporting decreased TRH expression in the PVN of Amio-treated mice (17). Decreased TRH expression might result from high local T₃ tissue levels as TRH is negatively regulated by T₃, which is assumed to be converted from T₄ by hypothalamic tanycytes expressing D2. However, we found no change in D2 or in D3 mRNA after Amio or Dron treatment as compared with controls. In addition, the expression of thyroid hormone transporters (MCT8, MCT10, and OATP1C1) in the PE was similar after Amio or Dron treatment, suggesting unaltered T₄ and T₃ transport and metabolism. This notion is further supported by the observation that no significant alterations in hypothalamic T₃ and T₄ concentrations were present in Amio or Dron rats compared to controls. Thus, a direct effect of Amio and Dron, or their metabolites, on hypothalamic TRH expression via TRα₁ seems more likely. It has been shown before that Amio treatment for 14 days results in marked accumulation of the major metabolite DEA in the brain (3) and our previous studies showed that both DEA and DBDron, the major metabolite of Dron, inhibit the binding of T₃ to the TRα₁ (1). As TRH decreased upon Amio and Dron treatment and TRH neurons in the rat and human PVN express TRα1 (18,19), our results are in keeping with a role for TRα₁ in positive TRH regulation. In line, positive physiological regulation of TRH has been proposed based on experiments in TRα^0/0 mice. Specifically, the hypersensitivity of negative TRH regulation by T₃ in the PVN of TRα^0/0 mice has been assumed to result from a variety of factors, including overexpression of TRβ isoforms, removal of the dominant negative effect of TRα₂, and a possible repressive effect of TRα1 on TRβ signaling (20). As Dron treatment affects the binding of T₃ to TRα₁ specifically, overexpression of TRβ and/or a dominant negative effect of TRα₂ can be excluded as an explanation for the observed changes in TRH expression in the present experiments. It should be noted, however, that T₃ and T₄ tissue concentration were measured in the whole hypothalamus, which might mask subtle treatment-induced differences in T₃ and T₄ concentrations in specific hypothalamic nuclei, such as the PVN. Another potential bias is that only a proportion of TRH neurons in the tissue block containing the hypothalamic PE consist of hypophysiotropic TRH neurons in the PVN. Therefore, a specific effect of Amio and Dron limited to TRH neurons involved in HPT axis regulation may have been overlooked in our experimental design.

Pituitary

As both Amio and Dron treatment results in decreased TRH expression in the PE, pituitary TSHβ mRNA was expected to be low. However, pituitary TSHβ mRNA expression increased upon Amio treatment, while Dron treatment did not have an effect suggesting that TRH signaling on the pituitary had been overruled by local effects of Amio and Dron in the pituitary. Pituitary TSH expression is negatively regulated by T₃ via the TRβ (21). It has been shown before that treatment of rats with Amio and Dron resulted in decreased heart TRα₁ and TRβ₁ mRNA expression independently of serum T₃ concentrations (22) suggesting that the metabolites of Amio and Dron inhibit T₃–TR binding. By inference, Amio and Dron may have prevented T₃-dependent inhibition of pituitary TSHβ mRNA. Another possibility is that our observation of increased TSHβ mRNA expression in the pituitary is explained by less T₃ produced locally by pituitary D2 (23). To investigate this possibility, we evaluated pituitary expression of the deiodinating enzymes. Pituitary D1, D2, and D3 mRNA expression were unaltered after Amio or Dron treatment, but the pituitary D2 activity decreased after Amio treatment. The decrease may be due either to elevated serum T₄ concentrations (24) or to a direct effect of DEA, the metabolite of Amio (17). An effect via D2 is supported by the observation by other investigators that an Amio-induced TSH increase was absent in D2 KO mice (17). The observed diminished D2 activity—and thus, a lower local T₃ production—may explain why TSHβ mRNA expression increases. Our experimental setup, however, does not allow us to discriminate between direct and indirect effects of Amio on pituitary TSH expression.

Liver

In our previous Amio studies performed in rats, we found that the Amio treatment decreases the liver D1 activity, but not D1 mRNA expression as measured by dot blot hybridization (1,25). In the present study using qPCR, we did find decreased liver D1 mRNA expression and activity upon Amio treatment as compared to Dron and controls rats. DEA has been reported to inhibit the D1 activity very weakly (26). However, we observed a D1 mRNA and activity decrease in the same order of magnitude indicating that the decrease in D1 activity is a result of decreased D1 mRNA expression. Dio1, is a T₃-dependent gene under TRβ₁ control (27). As in the present study, Dron treatment did not alter liver D1 expression; we assume that the effect of Amio on D1 might be due to diminished binding of T₃ to the TRβ_1, subsequently followed by decreased T₃ target gene expression and protein production.

In the brain, D3 mRNA expression has been shown to be positively regulated by T₃ (28) with increased expression during hyperthyroidism and decreased expression during hypothyroidism. In contrast to D3 in the brain, liver D3 may be negatively regulated by T_3. For example, rats and chickens on long-term food restriction display increased the liver D3 activity accompanied by a decrease in serum T₃ levels (29,30). In agreement, we observed increased liver D3 mRNA expression and activity in rats on Amio treatment (low serum T₃) while Dron treatment did not affect liver D3 (unaltered serum T₃). Liver D3 mRNA levels corresponded with the enzyme activity, suggesting regulation of the expression of D3 at the transcriptional level, probably mediated via the TRβ₁. This observation is supported by our previous finding that the illness- induced D3 mRNA decrease is mediated via TRβ₁ (31) and by experiments showing that D3 expression is decreased in GH3 cells transfected with TRβ₁ (32).

Previous studies suggested that Amio affects TH transport across the plasma membrane, resulting in low tissue TH concentrations (33,34). We measured liver T₃ and T₄ concentration by a UPLC tandem MS method (14). Surprisingly, serum TH concentrations were not determining liver TH concentrations; liver T₃ was 50% lower in the Amio-treated group compared to Dron and controls, whereas serum T₃ decreased 35% by Amio treatment. In addition, serum T₄ increased twofold in the Amio group, while liver T₄ did not change. Furthermore, a dominant role for altered TH transport induced by Amio was not supported by the observation that liver MCT8, MCT10, and OATP1C1 mRNA expression did not change upon Amio and Dron treatment. We now hypothesize that the alterations in liver T₃ might result from altered local D1 and D3 expression. A lower liver T₃ concentration may result from decreased D1 and increased D3 expression. Unaltered liver T₄ concentration may be the net result of increased D3 expression, lowering tissue T₄, and increased serum T_4. However, as an in vitro study by de Jong et al. showed diminished T₄, but not T₃ transport into the liver upon Amio (34), we cannot exclude the possibility that diminished T₄ transport contributes to liver T₄ concentrations despite unchanged mRNA expression of the main transporters in the liver.

It has been thought for a long time that high iodine levels, released from Amio, interfere with serum T₃ and T₄ concentrations in patients on Amio treatment via the Wolff Chaikoff effect. Based on more recent findings, including the present study, high serum TSH during Amio treatment may cause increased T₄ production by the thyroid. However, this remains difficult to reconcile with low serum T₃ levels.

Summarized, the present study shows that Amio and Dron treatment have a differential effect on thyroid hormone metabolism in hypothalamus, pituitary, and liver. TRH expression decreases after both Amio and Dron treatment, likely via inhibition of the TRα₁. In contrast, pituitary TSHβ mRNA increases during Amio- treatment, probably due to lower pituitary T₃ as a result of decreased D2 activity. TRβ₁ is selectively affected by Amio treatment explaining the observed changes in the liver as liver D1 mRNA and D1 activity are markedly decreased, while liver D3 increases. In conclusion, using Amio and Dron treatment to antagonize TRα and TRβ specifically, we propose a role for TRα₁ in hypothalamic TRH regulation, while liver D3 is regulated by TRβ₁ signaling.

Footnotes

Acknowledgment

The authors would like to thank Sanofi Recherche (Montpellier, France) for their generous gift of Amio and Dron.

Disclosure Statement

The authors have nothing to disclose.

References

van Beeren

, Jong

, Kaptein

, Visser

, Bakker

, Wiersinga

. 2003. Dronerarone acts as a selective inhibitor of 3,5,3′-triiodothyronine binding to thyroid hormone receptor-alpha1: in vitro and in vivo evidence. Endocrinology, 144:552–558.

Holt

, Tucker

, Jackson

, Storey

. 1983. Amiodarone pharmacokinetics. Am Heart J, 106:840–847.

Plomp

, Wiersinga

, Maes

. 1985. Tissue distribution of amiodarone and desethylamiodarone in rats after repeated oral administration of various amiodarone dosages. Arzneimittelforschung, 35:1805–1810.

Gullberg

, Rudling

, Salto

, Forrest

, Angelin

, Vennstrom

. 2002. Requirement for thyroid hormone receptor beta in T3 regulation of cholesterol metabolism in mice. Mol Endocrinol, 16:1767–1777.

Abel

, Ahima

, Boers

, Elmquist

, Wondisford

. 2001. Critical role for thyroid hormone receptor beta2 in the regulation of paraventricular thyrotropin-releasing hormone neurons. J Clin Invest, 107:1017–1023.

Feng

, Li

, Satoh

, Wilber

. 1994. Ligand (T3) dependent and independent effects of thyroid hormone receptors upon human TRH gene transcription in neuroblastoma cells. Biochem Biophys Res Commun, 200:171–177.

Gauthier

, Chassande

, Plateroti

, Roux

, Legrand

, Pain

, Rousset

, Weiss

, Trouillas

, Samarut

. 1999. Different functions for the thyroid hormone receptors TRalpha and TRbeta in the control of thyroid hormone production and post-natal development. EMBO J, 18:623–631.

Boelen

, Kwakkel

, Chassande

, Fliers

. 2009. Thyroid Hormone Receptor beta mediates acute illness-induced alterations in central thyroid hormone metabolism. J Neuroendocrinol, 21:465–472.

Wiersinga

, Chopra

. 1982. Radioimmunoassay of thyroxine (T4), 3,5,3′-triiodothyronine (T3), 3,3′,5′-triiodothyronine (reverse T3, rT3), and 3,3′-diiodothyronine (T2) Methods Enzymol, 84:272–303.

10.

Sweet

, Leung

, Kang

, Sogaard

, Schulz

, Trajkovic

, Campbell

, Xu

, Liew

. 2001. A novel pathway regulating lipopolysaccharide-induced shock by ST2/T1 via inhibition of Toll-like receptor 4 expression. J Immunol, 166:6633–6639.

11.

Ruijter

, Ramakers

, Hoogaars

, Karlen

, Bakker

, van den Hoff

, Moorman

. 2009. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res, 37:e45.

12.

Kwakkel

, Chassande

, van Beeren

, Wiersinga

, Boelen

. 2008. Lacking thyroid hormone receptor beta gene does not influence alterations in peripheral thyroid hormone metabolism during acute illness. J Endocrinol, 197:151–158.

13.

Peeters

, Wouters

, Kaptein

, van Toor

, Visser

, van den Berghe

. 2003. Reduced activation and increased inactivation of thyroid hormone in tissues of critically ill patients. J Clin Endocrinol Metab, 88:3202–3211.

14.

Ackermans

, Kettelarij-Haas

, Boelen

, Endert

. 2012. Determination of thyroid hormones and their metabolites in tissue using SPE UPLC-tandem MS. Biomed Chromatogr, 26:485–490.

15.

Morreale de

, Pastor

, Obregon

, Escobar del

. 1985. Effects of maternal hypothyroidism on the weight and thyroid hormone content of rat embryonic tissues, before and after onset of fetal thyroid function. Endocrinology, 117:1890–1900.

16.

van Beeren

, Bakker

, Wiersinga

. 1996. Structure-function relationship of the inhibition of the 3,5,3′-triiodothyronine binding to the TRα1 and TRβ1 by amiodarone analogues. Endocrinology, 137:2807–2814.

17.

Rosene

, Wittmann

, Arrojo e

Drigo

, Singru

, Lechan

, Bianco

. 2010. Inhibition of the type 2 iodothyronine deiodinase underlies the elevated plasma TSH associated with amiodarone treatment. Endocrinology, 151:5961–5970.

18.

Lechan

, Qi

, Jackson

, Mahdavi

. 1994. Identification of thyroid hormone receptor isoforms in thyrotropin-releasing hormone neurons of the hypothalamic paraventricular nucleus. Endocrinology, 135:92–100.

19.

Alkemade

, Vuijst

, Unmehopa

, Bakker

, Vennstrom

, Wiersinga

, Swaab

, Fliers

. 2005. Thyroid hormone receptor expression in the human hypothalamus and anterior pituitary. J Clin Endocrinol Metab, 90:904–912.

20.

Dupre

, Guissouma

, Flamant

, Seugnet

, Scanlan

, Baxter

, Samarut

, Demeneix

, Becker

. 2004. Both thyroid hormone receptor (TR)beta 1 and TR beta 2 isoforms contribute to the regulation of hypothalamic thyrotropin-releasing hormone. Endocrinology, 145:2337–2345.

21.

Weiss

, Forrest

, Pohlenz

, Cua

, Curran

, Refetoff

. 1997. Thyrotropin regulation by thyroid hormone in thyroid hormone receptor beta-deficient mice. Endocrinology, 138:3624–3629.

22.

Stoykov

, van Beeren

, Moorman

, Christoffels

, Wiersinga

, Bakker

. 2007. Effect of amiodarone and dronedarone administration in rats on thyroid hormone-dependent gene expression in different cardiac components. Eur J Endocrinol, 156:695–702.

23.

Schneider

, Fiering

, Pallud

, Parlow

, St Germain

, Galton

. 2001. Targeted disruption of the type 2 selenodeiodinase gene (DIO2) results in a phenotype of pituitary resistance to T4. Mol Endocrinol, 15:2137–2148.

24.

Christoffolete

, Ribeiro

, Singru

, Fekete

, da Silva

, Gordon

, Huang

, Crescenzi

, Harney

, Ridgway

, Larsen

, Lechan

, Bianco

. 2006. Atypical expression of type 2 iodothyronine deiodinase in thyrotrophs explains the thyroxine-mediated pituitary thyrotropin feedback mechanism. Endocrinology, 147:1735–1743.

25.

Hudig

, Bakker

, Wiersinga

. 1994. Amiodarone-induced hypercholesterolemia is associated with a decrease in liver LDL receptor mRNA. FEBS Lett, 341:86–90.

26.

, Stieger

, Grassi

, Altorfer

, Follath

. 2000. Structure-effect relationships of amiodarone analogues on the inhibition of thyroxine deiodination. Eur J Clin Pharmacol, 55:807–814.

27.

Amma

, Campos-Barros

, Wang

, Vennstrom

, Forrest

. 2001. Distinct tissue-specific roles for thyroid hormone receptors beta and alpha1 in regulation of type 1 deiodinase expression. Mol Endocrinol, 15:467–475.

28.

St Germain

, Schwartzman

, Croteau

, Kanamori

, Wang

, Brown

, Galton

. 1994. A thyroid hormone-regulated gene in Xenopus laevis encodes a type III iodothyronine 5-deiodinase. Proc Natl Acad Sci USA, 91:11282.

29.

Darras

, Cokelaere

, Dewil

, Arnouts

, Decuypere

, Kuhn

. 1995. Partial food restriction increases hepatic inner ring deiodinating activity in the chicken and the rat. Gen Comp Endocrinol, 100:334–338.

30.

van der Geyten

, van

, Sanders

, Visser

, Kuhn

, Darras

. 1999. Regulation of thyroid hormone metabolism during fasting and refeeding in chicken. Gen Comp Endocrinol, 116:272–280.

31.

Kwakkel

, Chassande

, van Beeren

, Fliers

, Wiersinga

, Boelen

. 2010. Thyroid hormone receptor {alpha} modulates lipopolysaccharide-induced changes in peripheral thyroid hormone metabolism. Endocrinology, 151:1959–1969.

32.

Barca-Mayo

, Liao

, Alonso

, Di

, Hernandez

, Refetoff

, Weiss

. 2011. Thyroid hormone receptor alpha and regulation of type 3 deiodinase. Mol Endocrinol, 25:575–583.

33.

Schroder-van der Elst

, van der Heide

. 1990. Thyroxine, 3,5,3′-triiodothyronine, and 3,3′,5′-triiodothyronine concentrations in several tissues of the rat: effects of amiodarone and desethylamiodarone on thyroid hormone metabolism [corrected] Endocrinology, 127:1656–1664.

34.

, Docter

, van der Hoek

, Krenning

, van der Heide

, Quero

, Plaisier

, Vos

, Hennemann

. 1994. Different effects of amiodarone on transport of T4 and T3 into the perfused rat liver. Am J Physiol, 266:E44–E49.

Action of Specific Thyroid Hormone Receptor α 1 and β 1 Antagonists in the Central and Peripheral Regulation of Thyroid Hormone Metabolism in the Rat

Abstract

Background:

Methods:

Results:

Conclusion:

Introduction

Materials and Methods

Experimental design

Plasma assays

RNA isolation and reverse transcription–polymerase chain reaction

Deiodinase activity

T3 and T4 tissue concentrations

Statistical analysis

Results

Plasma thyroid hormone levels

Hypothalamic thyroid hormone metabolism

Pituitary thyroid hormone metabolism

Liver thyroid hormone metabolism

Discussion

Hypothalamic PE

Pituitary

Liver

Footnotes

Acknowledgment

Disclosure Statement

References

T₃ and T₄ tissue concentrations