3-Iodothyronamine Decreases Expression of Genes Involved in Iodide Metabolism in Mouse Thyroids and Inhibits Iodide Uptake in PCCL3 Thyrocytes

Repeated 3-T₁AM treatment of mice

Male C57BL/6J mice, aged three to four months old, were held at 21–22°C and a 12 h/12 h light/dark cycle in single cages with ad libitum access to water and food. All procedures were carried out in accordance with the European Community Council Directives (86/609/EEC) and with approval by Stockholm's Norra Djurförsöksetiska Nämnd.

Mice were treated with 5 mg/kg of 3-T₁AM (dissolved in 60% dimethyl sulfoxide [DMSO] and 40% physiological saline, pH 7.4) by i.p. injection of 5 μL/g body weight once daily for seven days, as previously described (28). Control mice were sham injected with vehicle (60% DMSO and 40% saline) under the same conditions. Body weight, food and water intake were measured daily. The animals were sacrificed at day 8 (24 h after the last injection). Their organs were collected, snap frozen, and stored at −80°C until further processing.

RNA extraction from mouse tissue and quantitative real-time polymerase chain reaction

RNA was extracted from frozen tissues with the Aurum Total RNA Mini Kit (BioRad, Munich, Germany) according to the manufacturer's instructions, including a DNaseI digestion. A total of 250 ng of RNA from the thyroid or 500 ng of RNA from all other tissues were reverse transcribed to cDNA using the iScript cDNA Synthesis Kit (BioRad). Real-time quantitative polymerase chain reaction (qPCR) was carried out with the CFX Connect Real-Time PCR Detection System (BioRad) using ABsolute qPCR SYBR Green Fluorescein (Thermo Scientific, Waltham, MA). Specific primers were designed to be separated by at least one intron in the corresponding genomic DNA with the NCBI Primer-BLAST online tool (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/thy). The quantity of each gene of interest was normalized to hypoxanthine-guanine phosphoribosyltransferase (Hprt) 1 mRNA and 18S RNA. Fold inductions were calculated with respect to primer efficiency (E) via the E^(−ΔΔCt) method using the following equation to include both reference genes: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$n - fold \ induction = \frac { E^ { \Delta Ct \ goi } } { \sqrt { E^ { \Delta Ct \ Hprt1 } \times E^ { \Delta Ct \ 18S } } } $$ \end{document} (29). Specificity and size of the qPCR products were verified on 2% agarose gels.

Measurement of TH concentrations in mouse sera

Total TH serum concentrations were measured with competitive radioimmunoassays (no. 4525 for total T3 and no. 4524 for total T4) from DRG Instruments GmbH (Marburg, Germany) according to the manufacturer's instructions, as described before (30). The coefficient of variation was ≤5% for both assays.

Culture of PCCL3 thyrocytes

Non-transformed rat thyroid PCCL3 cells (26,27) were grown at 37°C, 5% CO₂, and a humidified atmosphere in Coon's F12 cell culture medium (Biochrom, Berlin, Germany) supplemented with 5% fetal bovine serum (FBS; Biochrom), 1 mIU/mL of bovine TSH, and five hormones (H5; i.e., 10 μg/mL of insulin, 5 μg/mL of transferrin, 10 nM of hydrocortisone, 20 ng/mL of glycyl-histidyl-lysine, and 10 ng/mL of somatostatin; Sigma–Aldrich, St. Louis, MO). Cells were grown without TSH and FBS to deplete them from TSH and TH(-metabolites) and to increase their responsiveness for the following stimulation in cell viability assays, qPCR analyses, Western blot, iodide uptake determination, and measurements of cellular energy metabolism. After seeding, the cells were grown for 24 hours in medium containing FBS, TSH, and H5. When the cells were fully attached to the cell culture dish, TSH depletion was initiated by using medium that only contained FBS and H5. Eighteen hours before the actual experiment, the cells were depleted from FBS by culturing them in medium that only contained H5. A prolonged depletion protocol was applied for a subset of experiments testing iodide uptake and Dio1 activity. In this case, TSH and FBS depletion were prolonged for 24 hours compared with the standard protocol.

Viability test of PCCL3 cells

TSH- and FBS-depleted PCCL3 cells in 96-well plates were stimulated with 0.1 mIU/mL or 0.5 mIU/mL of TSH ±1 μM of 3-T₁AM, iodine-free thyronamine (T₀AM), or 3-iodothyroacetic acid (3-TA₁). Thyronamines and 3-TA₁ were replenished after 24 h. After 48 h, 20 μL of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) stock solution (5 mg/mL) were added, and the cells were further incubated for 1.5 h at 37°C. Thereafter, cells were lysed in 12% HCl in isopropanol, and the optical density of the lysates containing the oxidized MTT dye was measured at 595 nM.

Measurements of TH metabolites by liquid chromatography–tandem mass spectrometry

PCCL3 cells were grown in 12-well plates for 24 h. After another 24 h under FBS depletion, cells were incubated ±1 μM of 3-T₁AM in Krebs Ringer buffer (119 mM of NaCl, 4.74 mM of KCl, 1.19 mM of KH₂PO₄, 25 mM of NaHCO₃, 2.54 mM of CaCl₂, 1.19 mM of MgCl₂, 10 mM of Hepes, and 11 mM of glucose; pH 7.4) for up to 180 min. For enzyme inhibition experiments, cells were pre-incubated with 100 μM of the monoamine oxidase inhibitor iproniazid, 10 μM of the Dio1 inhibitor 6n-propyl-2-thiouracil (PTU; Sigma–Aldrich), or solvent for 30 min before 3-T₁AM was added to the medium. PTU was present during the whole duration of 3-T₁AM incubation. Thyronamines and thyroacetic acids were concentrated from the conditioned cell culture supernatants by liquid/liquid extraction and measured by liquid chromatography–tandem mass spectrometry (LC-MS/MS) as previously described (31).

RNA extraction from cells and real-time qPCR

PCCL3 cells were grown in six-well plates and were TSH and FBS depleted as described above. Subsequently, cells were stimulated with 0.1 mIU/mL of TSH ± 3-T₁AM for 3 h. RNA extraction, cDNA synthesis, and qPCR analysis were performed as described above. Hprt1 served as reference gene, and n-fold inductions were calculated with the 2^(–ΔΔCt) method corrected for primer efficiency (E). Primer sequences are listed in Supplementary Table S2.

Western blot analysis

FBS- and TSH-depleted PCCL3 cells were stimulated with 0.5 mIU/mL of TSH ±1 μM of 3-T₁AM for 48 h. 3-T₁AM was refreshed after 24 h. At the end of the incubation time, cells were snap frozen and stored at −20°C until further processing. Cells were lysed using a homogenization buffer (20 mM of Hepes, 1 mM of EDTA, 250 mM of saccharose; pH 7.4). Homogenates were centrifuged at 13,000 g for 15 min at 4°C, and pellets containing the membrane fraction were resuspended in homogenization buffer. Protein concentration in the enriched membrane fraction was determined by the method of Bradford with Coomassie Brilliant blue G-250 dye (BioRad) (32). Twenty-five micrograms of protein were separated by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare, Little Chalfont, United Kingdom). Membranes were probed with antibodies against the sodium iodide symporter (NIS) diluted 1:10,000 (EUD410; Acris, Herford, Germany) and to β-Actin diluted 1:2000 (600-401-886; Rockland, Limerick, PA) overnight at 4°C followed by incubation with a horseradish peroxidase (HRP) conjugated secondary goat anti-rabbit antibody (P0448; Dako, Glostrup, Denmark). The specific immunosignals were visualized by chemiluminescence using ECL (GE Healthcare) as HRP substrate and films to detect the light reaction.

Nonradioactive measurement of iodide uptake

PCCL3 were depleted as described above and stimulated with 0.1 mIU/mL of TSH ± co-stimulation with 1 μM of 3-T₁AM/T₀AM/3-TA₁ for 48 h. The co-stimulants were refreshed after 24 h. Cells were washed with 1 × phosphate-buffered saline and subsequently incubated with 10 μM of NaI (Sigma–Aldrich) for 20 min at 37°C/5% CO₂. Intracellular iodide content was measured using the Sandell–Kolthoff reaction, as previously described (33,34), and corrected for nonspecific iodide as well as uptake of iodinated compounds by using 100 μM of perchlorate controls for every individual treatment condition.

Nonradioactive measurement of Dio1 activity

PCCL3 were depleted as described above and stimulated with 0.1 mIU/mL of TSH ±1 μM of 3-T₁AM. Dio1 activity was measured with a nonradioactive iodide release assay, as previously described (35). In brief, PCCL3 cells were homogenized, and approximately 10 μg of protein was incubated with 10 μM of 3,3′,5′-triiodothyronine (rT3) (Sigma–Aldrich) for 2 h at 37°C. Afterwards, the iodide released from rT3 was measured with the Sandell–Kolthoff reaction. Specific Dio1 activity was calculated as pmol iodide/(min·mg protein) with the use of a NaI standard curve.

Measurement of cellular energy metabolism

A Seahorse XF^e96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA) was used to measure glucose breakdown as extracellular acidification rate (ECAR) and mitochondrial respiration as oxygen consumption rate (OCR) in intact cells simultaneously (36). To this aim, PCCL3 cells were grown in Seahorse 96-well cell culture plates and were TSH- and FBS-depleted as described above. Before the Seahorse assay, cells were incubated for 60 min in a CO₂-free incubator at 37°C with 1 mIU/mL of TSH ±1 μM of 3-T₁AM in assay medium (D5030; Sigma–Aldrich), supplemented with 11 mM of glucose, 2 mM of L-glutamine, and H5. After 12 min of temperature equilibration in the Seahorse analyzer, measurements of OCR and ECAR were performed in cycles of 3 min mixing and 3 min measurement steps. The basal ECAR and OCR values were defined as the third measurement of each individual Seahorse assay (37). After completion of the Seahorse measurement, the protein content of every well was determined with the Micro BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer's instruction. ECAR and OCR values of each well were normalized to the corresponding amount of protein.

Calcium imaging

PCCL3 cells were grown on glass cover slips for 24 h in medium with FBS, H5, and 1 mIU/mL of TSH. On the day of the experiment, cells were pre-incubated with 2 μM of fura-2/AM (Tocris Bioscience, Bristol, United Kingdom) for 15–45 min and subsequently treated with 3-T₁AM ±20 μM of the TRPM8 blocker 4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazine-carboxamide (BCTC; Tocris Bioscience) onstage on an inverted microscope, which was combined with a digital imaging system with UV excitation capability (TILL Photonics, Munich, Germany), as previously described (38). Fura-2 fluorescence was alternately excited at 340 and 380 nm. Emission was measured at 510 nm. The fluorescence ratio f_340nm/f_380nm served as a relative index of changes in cytosolic free calcium. Before the experiments, stable control baseline levels were obtained for 8–10 min. The control traces are designated with open circles in the figures.

Measurement of intracellular cAMP accumulation

PCCL3 cells were grown in 96-well plates. After TSH and FBS depletion, the cells were stimulated with 0.1–1 mIU/mL of TSH, 0.1–1 μM of 3-T₁AM, or TSH +3-T₁AM for 45 min in a HEPES-buffered solution that contained 1 mM of 3-isobutyl-1-methylxanthine (IBMX; Sigma–Aldrich) to inhibit phosphodiesterases (PDE). After cell lysis, the cAMP concentration was measured with a competitive immunoassay using the AlphaScreen technology (PerkinElmer Life Science, Boston, MA), as previously described (39).

Statistical analyses

GraphPad Prism v5 was used for all statistical analyses. Results are depicted as means ± standard error of the mean of the indicated number of mice or independent in vitro experiments (biological replicates). For comparison of two groups, an unpaired Student's t-test or a Mann–Whitney test was used. For comparison of more than two groups, one-way analysis of variance was applied, and if significant differences between groups were found, Dunnett's post-test was used.

Body weight and food intake in mice are unchanged after seven days of repeated 3-T₁AM treatment

To test whether 3-T₁AM influenced thyroid function and the HPT axis, male C57BL/6 mice were treated for seven days with 5 mg/kg of 3-T₁AM or vehicle i.p. daily. No significant differences between the treatment groups were observed for food and water intake or for body weight at any time during the study (Table 1).

Repeated 3-T₁AM treatment decreases expression of distinct TH synthesis genes without affecting the HPT axis in male mice

After repeated treatment with 3-T₁AM, thyroidal mRNA concentrations of Nis, thyroglobulin (Tg), and pendrin (Pds) were significantly decreased in comparison with sham-injected mice (Fig. 1A). In contrast, the thyroidal expression of other genes involved in TH synthesis (TSH receptor [Tshr], thyroperoxidase [Tpo], dual oxidase 2 [Duox2], and Dio1) was not altered (Supplementary Fig. S1). In order to investigate if the described changes in mRNA expression were direct effects of 3-T₁AM on the mouse thyroid, the study analyzed whether the HPT axis was affected in the treatment model. Serum total T3 and T4 concentrations were not different between treatment groups (Fig. 1B), and the expression of the hepatic T3 responsive genes Dio1 and Spot 14 was not altered by 3-T₁AM treatment (Fig. 1C). In line with this, the evaluation of β TSH (Tshb), TRH receptor (Trhr), TRH degrading enzyme (Trhde), TH receptor β (Thrb), and Dio2 transcripts showed no differences between pituitaries of 3-T₁AM- and sham-treated mice (Fig. 1C). This indicates a direct effect of 3-T₁AM on the thyroid gland. However, it cannot be excluded that a compensatory adaptation of the HPT axis had already occurred after the treatment period of seven days.

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FIG. 1.

Effects of repeated 3-iodothyronamine (3-T₁AM) treatment on the thyroid gland and the hypothalamic–pituitary–thyroid (HPT) axis. Thyroidal mRNA expression of sodium iodide symporter (Nis), thyroglobulin (Tg), and pendrin (Pds) was significantly decreased in 3-T₁AM-treated mice compared with the control group, as determined by real-time quantitative polymerase chain reaction (qPCR; sham n = 8, 3-T₁AM n = 7) (A). Unaltered expression of TH-responsive genes in the pituitary (sham n = 10, 3-T₁AM n = 9) and the liver (sham n = 6, 3-T₁AM n = 6) (C), and total thyroid hormone (TH) serum concentrations (sham n = 8, 3-T₁AM n = 8) (B) showed no involvement of the HPT axis. Bars represent means ± standard error of the mean (SEM). **p < 0.01, Mann–Whitney test.

3-T₁AM exerts direct effects on thyrocytes in vitro

In order to analyze whether 3-T₁AM could directly act on isolated thyrocytes, PCCL3 cells were chosen as an established in vitro model. This cell line is fully differentiated and dependent on TSH for growth (26,27).

3-T₁AM, T₀AM, and 3-TA₁ do not affect the viability of PCCL3 cells

As expected, stimulation of PCCL3 cells with 0.1 mIU/mL and 0.5 mIU/mL of TSH alone led to an increase in viability compared with cells grown in the absence of TSH, as measured by a MTT test. In contrast, the test revealed no changes in viability of cells incubated for 48 h with TSH +1 μM of 3-T₁AM/T₀AM/3-TA₁ compared to cells that were only incubated with TSH (Supplementary Fig. S2).

3-T₁AM accumulates in PCCL3 cells and is enzymatically metabolized to T₀AM and 3-TA₁

3-T₁AM is metabolized to T₀AM and/or 3-TA₁ by deiodination and oxidative deamination, respectively, in different in vivo and in vitro models (chemical structures are shown in Fig. 2A) (40 –42). To analyze the capacity of PCCL3 cells for 3-T₁AM metabolism, first the expression of candidate enzymes for 3-T₁AM metabolism was screened for using real-time qPCR. Figure 2B shows the specific bands representing qPCR products for Dio1 and monoamine oxidase A (Mao A), indicating that PCCL3 cells express two classes of enzymes that are known to be involved in 3-T₁AM metabolism (40,43). No qPCR product from PCCL3 cDNA was obtained for Mao B. In contrast, real-time qPCR with the same Mao B primers in a rat liver cDNA positive control yielded a specific product of the expected size (data not shown).

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FIG. 2.

Uptake and metabolism of 3-T₁AM by PCCL3 cells. Structures of 3-T₁AM and its putative downstream metabolites, with arrows indicating the enzymes catalyzing the reactions (A). Real-time qPCR products of transcripts of 3-T₁AM metabolizing enzymes from PCCL3 cDNA separated on 2% agarose gels (B). 3-T₁AM and metabolites extracted from conditioned PCCL3 cell culture supernatants after incubation with 1 μM of 3-T₁AM ± the Mao inhibitor iproniazid (C) or the Dio1 inhibitor 6n-propyl-2-thiouracil (D) measured by liquid chromatography–tandem mass spectrometry, n = 3.

Based on this transcript pattern of PCCL3 cells, the functional expression of Mao A and Dio1 was analyzed by incubating PCCL3 cells with the substrate 1 μM of 3-T₁AM ± the Mao inhibitor iproniazid or PTU as specific Dio1 inhibitor. After 60 and 180 min of incubation with 3-T₁AM, the expected products T₀AM and 3-TA₁ were detectable in culture supernatants (Fig. 2C and D). After pre-incubation with iproniazid, 3-TA₁ was no longer detectable in the supernatants, indicating an involvement of Mao A in the formation of 3-TA₁ from 3-T₁AM (Fig. 2C). Likewise, if 3-T₁AM incubation was performed in the presence of PTU, no T₀AM was detected in the conditioned media, providing evidence for a contribution of Dio1 in the deiodination of 3-T₁AM in PCCL3 cells (Fig. 2D).

3-T₁AM decreases TSH-dependent NIS expression in PCCL3 cells

Co-incubation with 3-T₁AM attenuated the TSH-dependent expression of Nis mRNA by approximately 50%, starting at a concentration of 100 nM (Fig. 3A). Other genes involved in TH synthesis, namely Tg, Tpo, Dio1, Pds, and Tshr, were not significantly altered in this experimental setting (data not shown). To assess whether the effect on the Nis gene was also manifested by protein translation, cells were stimulated for 48 h with 0.5 mIU/mL of TSH to allow for full NIS synthesis, post-translational modification, and integration into the plasma membrane (44). Co-incubation with 1 μM of 3-T₁AM recapitulated the mRNA results and led to an approximately 40% reduction in TSH-dependent Nis protein expression (Fig. 3B and C).

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FIG. 3.

3-T₁AM decreases thyrotropin (TSH)-dependent Nis expression and function in PCCL3 cells. 3-T₁AM decreased the TSH-dependent Nis mRNA expression after 3 h of incubation, n = 3; *p < 0.05, **p < 0.01, one-way analysis of variance (ANOVA) followed by Dunnett's post-test (A). Nis Western blot band density (normalized to β-actin) after 48 h of TSH ±3-T₁AM incubation shows a 3-T₁AM-induced decrease in Nis expression, n = 5; ***p < 0.001, unpaired t-test with Welch's correction (B). Representative Western blot bands for Nis and β-actin from the same experiment (C). TSH-dependent iodide uptake was decreased by 3-T₁AM and T₀AM after 48 h of incubation, depending on the preceding depletion protocol (D, E). (D) n = 3. (E) 3-T₁AM n = 3, T₀AM/3-TA₁ n = 2 in quadruplicates; ***p < 0.001, one-way ANOVA followed by Dunnett's post-test.

3-T₁AM decreases TSH-dependent NIS-mediated iodide uptake in PCCL3 cells

In order to analyze whether the inhibitory effect of 3-T₁AM on TSH-dependent Nis mRNA and protein expression had a functional relevance, iodide uptake was measured (33). TSH incubation for 48 h substantially increased perchlorate-sensitive iodide uptake by PCCL3 cells (Supplementary Fig. S3). Using the same depletion protocol as for mRNA and protein analyses, no change in TSH-dependent iodide uptake by 3-T₁AM co-incubation was observed (Fig. 3D). Therefore, a prolonged depletion protocol was applied. Cells were depleted from TSH for four days and grown for 42 h without FBS before the TSH ±3-T₁AM co-incubation. Under these experimental conditions, 3-T₁AM significantly decreased TSH-dependent iodide uptake in PCCL3 cells by 50% (Fig. 3E). Since PCCL3 cells metabolize 3-T₁AM to T₀AM and 3-TA₁ (Fig. 2C and D), it was hypothesized that the different depletion protocols induced alterations in enzyme expression or activity profiles and therefore also in 3-T₁AM metabolism. For this reason, PCCL3 cells grown under each depletion protocol were stimulated with TSH ±3-T₁AM metabolites T₀AM and 3-TA₁ for 48 h. 3-TA₁ co-incubation did not alter TSH-dependent iodide uptake under any depletion condition. In contrast, T₀AM co-incubation led to a 50% decrease in TSH-induced iodide uptake under both depletion protocols. This indicates that in this context, T₀AM may represent an active metabolite of 3-T₁AM. Therefore, the question was raised whether the deiodinating metabolism of 3-T₁AM and consequently the formation of T₀AM were increased after prolonged compared with standard depletion. Determination of Dio1 activity revealed that Dio1 activity in TSH +3-T₁AM-treated cells tended to be higher after the prolonged depletion than it was in equally stimulated cells after the standard depletion (Fig. 4).

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FIG. 4.

Effect of TSH and FBS depletion on Dio1 activity in PCCL3 cells. Dio1 activity in PCCL3 cells treated with 0.1 mIU/mL of TSH +1 μM of 3-T₁AM after the standard and the prolonged depletion protocol, n = 3. No significant difference was observed between the two treatment groups (p > 0.05, unpaired t-test).

3-T₁AM affects glucose utilization in PCCL3 cells after acute stimulation

Since 3-T₁AM was previously shown to alter glucose utilization in animal models (20,23), the modulation of TSH-dependent glucose breakdown by 3-T₁AM in PCCL3 cells was analyzed using the Seahorse XFe96 analyzer. Acute incubation of depleted cells with 1 mIU/mL of TSH significantly increased ECAR of PCCL3 cells (Fig. 5A). Co-stimulation with 1 μM of 3-T₁AM decreased the TSH-dependent ECAR by approximately 30% (Fig. 5B). The oxygen consumption rate of the same cells, indicative for mitochondrial respiration, measured simultaneously was affected neither by TSH stimulation alone nor in co-stimulation with 3-T₁AM under the described experimental conditions.

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FIG. 5.

Basal ECAR measured in PCCL3 cells with the Seahorse Analyzer XFe96. TSH increased basal ECAR in PCCL3 cells within 60 min, n = 4; **p < 0.01, one-way ANOVA followed by Dunnett's post-test (A). 3-T₁AM decreased the TSH-dependent ECAR. ECAR, basal extracellular acidification rate in glucose containing fetal bovine serum–free medium, normalized to protein content per well. TSH-dependent ECAR was calculated as the difference of ECAR in presence and absence of TSH, n = 4; *p < 0.05, unpaired t-test (B).

3-T₁AM increases cytosolic free Ca²⁺ in PCCL3 cells

To elucidate the mechanism mediating the thyrocyte-specific effects of 3-T₁AM, first both TSH-dependent signaling cascades were analyzed. Basal or TSH-dependent cAMP signaling was not modified by 3-T₁AM in this experimental setting (Supplementary Fig. S4). No evidence for an effect of 3-T₁AM on inositol 1,4,5-trisphosphate (IP3) formation was obtained. As 3-T₁AM is a strong modulator of intracellular Ca²⁺ signaling via the transient receptor potential channel melastatin 8 (TRPM8, menthol receptor) in different cell types (38,45), the thyrocyte-specific calcium response to 3-T₁AM was tested by using the calcium indicator fura-2 and fluorescence imaging. The application of 1 μM of 3-T₁AM on fura-2-loaded PCCL3 cells led to a significant and quick increase in cytosolic free Ca²⁺ (Fig. 6A). Specifically, the f_340nm/f_380nm ratio increased from 1.201 ± 0.004 to 1.243 ± 0.005 after application of 1 μM of 3-T₁AM (250 sec; p < 0.01; Fig. 6A and C), whereas the TRPM8 channel blocker BCTC (20 μM) clearly suppressed this effect (1.206 ± 0.005; p < 0.005; Fig. 6B and C). This indicates that the 3-T₁AM-induced Ca²⁺ increase is due to Ca²⁺ channel activation such as TRPM8.

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FIG. 6.

Effect of 3-T₁AM on cytosolic free calcium in PCCL3 cells. The traces show intracellular Ca²⁺ measurements with 3-T₁AM (n = 5; filled circles) and without 3-T₁AM (n = 4; open circles), demonstrating a control Ca²⁺ baseline (A). The experiment was repeated in the presence of the TRPM8 blocker BCTC. The trace shows intracellular Ca²⁺ measurements with 3-T₁AM in the presence of BCTC (n = 7) (B). Data are means ± SEM. A summary of the experiments with 3-T₁AM and BCTC are shown in panel (C). The 3-T₁AM-induced Ca²⁺ increase could be clearly suppressed by BCTC. Bars represent means ± SEM. *p < 0.05 and **p < 0.01, significant differences between control (Ca²⁺ base levels) and 3-T₁AM-induced Ca²⁺ increase, paired t-test. ^## p < 0.05 and ^### p < 0.005, significant differences of 3-T₁AM-induced Ca²⁺ response with and without BCTC, unpaired t-test.

In vivo study

As the interference of a single dose of 50 mg/kg of 3-T₁AM was shown to suppress the HPT axis in rats (23), the 10-fold lower dose of 5 mg/kg of 3-T₁AM was chosen in the repeated treatment setup. The mice were injected with this amount of 3-T₁AM or vehicle daily for seven days. Measurements with an in-house chemiluminescence immunoassay revealed that the 3-T₁AM treatment procedure significantly increased 3-T₁AM serum concentrations in the treated mice compared with the endogenous levels in the sham group (sham 17.4 ± 1.4 nM, n = 5; 3-T₁AM 67.7 ± 7.3 nM, n = 6; p = 0.0043). This is only a fourfold increase. Therefore, the applied dose was considered physiological rather than pharmacological. While the expression of the Tshr and several other thyroidal transcripts were unaffected 24 hours after the last injection, mRNA expression of Nis, Tg, and Pds were downregulated by the repeated 3-T₁AM treatment. NIS is the basolateral iodide symporter of thyrocytes, and accumulates iodide in the thyroid gland, which is the prerequisite for TH synthesis (47). The significance of iodide uptake into the thyroid gland is illustrated by the occurrence of thyroid disorders due to inadequate iodide supply (48). The TH synthesis and storage protein TG represents the major component of the follicular colloid. It provides tyrosine residues that are iodinated and form the TH precursors monoiodotyrosine (MIT) and diiodotyronine (DIT), and is therefore an important part of the TH synthesis system (49). PDS, the protein encoded by the Pendred syndrome gene, is an apical iodide transporter candidate in thyrocytes (50,51). Its importance is reflected by the goiter development in Pendred syndrome that is most likely caused by defective iodine organification due to reduced iodide efflux into the follicular lumen (52). Therefore, an inhibitory effect on the expression of Nis, Tg, and Pds as caused by 3-T₁AM in this study may have the potential to interfere with TH homeostasis. The expression of T3-responsive genes in the pituitary that are important for the regulation of the HPT feedback axis (Tshb, Trhr, Trhde, Dio2, Thrb), as well as T3-responsive Dio1 and Thrsp (Spot 14) in the liver, were not altered by 3-T₁AM treatment. These findings indicate that the HPT axis was not affected in the model, and provide evidence for a direct effect of 3-T₁AM on the TH synthesis machinery in thyrocytes. Nevertheless, it cannot entirely be excluded that a compensatory adaptation of the HPT axis has already occurred after the treatment period of seven days. Despite the inhibited gene expression of Nis, Pds, and Tg, total TH serum concentrations were not altered after the seven-day treatment with 3-T₁AM. A likely explanation might be that the duration of seven days of 3-T₁AM treatment was not sufficient to deplete thyroidal Tg and TH stores (53,54).

In vitro studies

In order to comply with the 3R principle of animal ethics and before engaging in prolonged in vivo animal studies, advantage was taken of a more simplified in vitro thyrocyte model. To dissect the direct effects of 3-T₁AM on thyrocytes in an isolated system at the molecular level, the PCCL3 thyrocyte cell line was chosen as an established in vitro model. These cells are highly differentiated and retain a non-transformed phenotype very close to in vivo thyrocytes (27). 3-T₁AM is detectable in serum (18) and was shown to be bound to apolipoprotein B-100 (55). For this reason, PCCL3 cells were deprived from FBS before the 3-T₁AM stimulation in order to start with a well-defined 3-T₁AM concentration in the experimental setup. A decrease in the TSH-dependent mRNA and protein expression of NIS was observed after co-stimulation with 3-T₁AM, which is partly in line with the in vivo data. This supports the concept of a direct inhibitory action of 3-T₁AM on thyrocytes at doses that do not yet interfere with serum TH concentration or HPT feedback adaptation.

The functional TSH-dependent iodide uptake in PCCL3 cells was also decreased by 3-T₁AM. Notably, this decrease only occurred after the cells had undergone a prolonged depletion protocol in which FBS and TSH depletion was prolonged for another day compared with the standard depletion protocol (3 days TSH free, 18 h FBS free). The in vitro setup revealed 3-T₁AM metabolism to T₀AM and 3-TA₁ by Dio1 and Mao A, respectively. Both 3-T₁AM-derived metabolites were shown to possess biological activity themselves in previous studies by others (16,23,56). In several single-dose application studies in rodents, T₀AM was shown to exert similar but partially weaker effects than were exerted by 3-T₁AM, for example in terms of cardiac output, body temperature, and plasma TH levels (16,23). No data on long-term effects of T₀AM have been published yet. In contrast, 3-TA₁ does not provoke cardiac or thermoregulatory changes in mice after acute or repeated treatment over seven days (57). The biological action of 3-TA₁ known so far mainly comprises neurological effects via interaction with the histaminergic system (56,58). It was hypothesized that one of the 3-T₁AM metabolites might actually contribute to the observed decrease in iodide uptake in PCCL3 cells. Furthermore, the different depletion protocols that were applied might have altered the extent of the 3-T₁AM metabolism to T₀AM or 3-TA₁. While 3-TA₁ was inactive regarding modulation of iodide uptake, a strong decrease in TSH-dependent iodide uptake by T₀AM treatment was observed. This occurred regardless of the preceding depletion protocol, and indicates that T₀AM (at least partially) mediates the observed effects of 3-T₁AM incubation. This assumption is supported by the trend toward higher Dio1 activity in TSH ±3-T₁AM-treated cells following the prolonged depletion compared with the standard protocol. The direct quantification of increased T₀AM formation corresponding to the Dio1 activity can hardly be assessed in this experiment. This is probably due to further metabolism of T₀AM as well as 3-TA₁ to iodine-free thyroacetic acid TA₀ (41). The source of TA₀ cannot easily be determined yet by the currently available LC-MS/MS method.

To analyze whether 3-T₁AM has effects on the energy metabolism of thyrocytes, mitochondrial respiration (OCR) and extracellular acidification (ECAR) were measured with the Seahorse Bioanalyzer. The TSH-dependent increase in glucose breakdown measured as ECAR was attenuated by acute 3-T₁AM incubation. This is in line with the previously reported trend of high doses of 3-T₁AM to decrease glucose utilization (20). It was shown that the pentose phosphate cycle plays an important role in the energy metabolism of thyrocytes and that it is TSH dependent (59). Further studies will be needed to clarify whether the measured ECAR is due to glycolysis or metabolism in the pentose phosphate pathway in the PCCL3 model. A reduced uptake of glucose is a possible explanation for the observed decline in ECAR by 3-T₁AM stimulation. In the related FRTL5 thyrocyte model, treatment with 100 μM of 3-T₁AM was shown to decrease glucose uptake within 60 min (41).

In order to elucidate the mechanism underlying the 3-T₁AM-induced effects on thyrocytes, this study first tested whether 3-T₁AM modifies signaling pathways downstream of the TSH receptor. TSH, the major regulator of thyroid function, is known to increase concentrations of the second messengers cAMP and IP3 in thyrocytes (60,61). So far, there is no evidence for an effect of 3-T₁AM on IP3 formation in PCCL3 cells. Additionally, the TSH-dependent activation of the cAMP signaling pathway was not altered by 3-T₁AM under conditions comparable to those resulting in functional and metabolic effects. Although it cannot be excluded that the site of action of 3-T₁AM is further downstream from that of the TSH-mediated increase in the second messenger concentration, the present data support the concept that the 3-T₁AM effects described above are independent of TSH signaling. Interestingly, a 3-T₁AM-dependent BCTC-sensitive Ca²⁺ increase in PCCL3 was observed, which is in line with previous publications in ocular cells (38,45). This result suggests an involvement of Ca²⁺ channels such as TRPM8 in 3-T₁AM-induced Ca²⁺ rise, since BCTC is known to be a TRPM8 blocker (62,63). Several lines of evidence could connect the increase in Ca²⁺ to the present results, and might be tested in future studies to explore Ca²⁺-induced signaling mechanisms in more detail. First, there is a subgroup of PDE that is activated by Ca²⁺ (64,65). PDE activation by 3-T₁AM via Ca²⁺ could decrease intracellular cAMP. Of note, this would not be recorded by the cAMP assay in this study because it was performed in the presence of the PDE inhibitor IBMX. Second, the activation of protein kinase C, which is a downstream target of increasing Ca²⁺ concentration, is known to inhibit iodide metabolism in thyrocytes (66). Third, a complex cross-talk between calcium and cAMP signaling has been reported for thyrocytes (66,67). Therefore, the modulation of Ca²⁺ signals might be a promising hypothesis to be tested to explain the 3-T₁AM effects in thyrocytes.

Taken together, the in vivo and in vitro data suggest that 3-T₁AM can directly act on thyrocytes to inhibit iodide transport and the TH synthesis machinery without involvement of the HPT feedback axis. A non-classical Ca²⁺ signal-associated mechanism might contribute to the observed effects.