Objectives: Thyronamines are decarboxylated and deiodinated metabolites of thyroid hormones (THs). Of all possible thyronamine variants, only 3-iodothyronamine (3-T1AM) and iodine-free thyronamine (T0AM) have been detected in vivo. While intensive research has been done on the (patho-)physiological action of 3-T1AM, the role of T0AM has been studied less intensively. Study Design: We determined whether a single pharmacological dose (50 mg/kg, i.p.) or repeated administration (5 mg/kg/day, i.p., for 7 days) of T0AM affects metabolism, cardiovascular function, or thermoregulation in male C57BL/6J mice. Since selenium (Se) is important for proper TH function and Se metabolism is affected by TH, we additionally analyzed Se concentrations in liver, serum, and kidney using total reflection X-ray analysis. Results: A single injection of T0AM had no effect on heart rate, temperature, or activity as assessed by radio telemetry. Likewise, daily administration of T0AM did not alter body weight, food or water intake, heart rate, blood pressure, brown adipose tissue thermogenesis, or body temperature, and no significant differences in hepatic glycogen content or mRNA expression of genes involved in cardiovascular function or metabolic control were determined. Also, the X-ray analysis of Se concentrations revealed no significant changes. However, hepatic T0AM was significantly increased in the treated animals. Conclusions: In summary, our data demonstrate that T0AM elicits no obvious metabolic, cardiovascular, or thermoregulatory activities in mice. As T0AM does also not interfere with TH or Se metabolism, we conclude that the deiodination of 3-T1AM to T0AM constitutes an efficient inactivation mechanism, terminating the actions of the more powerful precursor.

Thyronamines (TAM) are endogenous molecules derived from thyroid hormones (THs) by deiodination and decarboxylation [1-3], which exhibit antagonizing properties compared to classical TH actions [4]. So far only the two TAM derivatives 3-iodothyronamine (3-T1AM) and the iodine-free thyronamine (T0AM) have been detected in vivo, e.g., in the brain, heart, liver, and blood of male C57BL/6J mice [1]. Nevertheless, the effects of T0AM are much less studied compared to 3-T1AM.

The biosynthesis of T0AM occurs mainly from 3-T1AM via inner ring deiodination by deiodinase 3 (Dio3) [2], but the recently described inhibition of T0AM synthesis by 6-propyl-2-thiouracil indicates an additional involvement of Dio1 [5]. T0AM neither binds to nor activates nuclear TH receptors [1]; however, T0AM may activate the trace amine-associated receptor 1, an amine-activated G protein-coupled receptor located in the cell membrane [1]. From a physiological angle, it has been reported that a single intraperitoneal injection of synthetic T0AM (10–100 mg/kg body weight) into C57BL/6J mice caused a dose-dependent reversible drop in rectal body temperature within 60 min [1]. Concerning cardiac effects, conflicting data have been published. For instance, in a perfused, de-innervated rat heart, T0AM rapidly decreased the cardiac output with no observable changes in heart rate, suggesting a negative inotropic effect [1], whereas the application of T0AM (3–30 mg/kg body weight) to anesthetized rats significantly decreased the heart rate [6]. Intravenous injection of T0AM into anesthetized dogs even increased the cardiac output [6, 7], and using an isolated guinea pig atria model, T0AM (2.9 nM to 9.5 μM) showed positive inotropic effects, evoking no changes in the beat frequency [6]. This indicates that the potential cardiac effects of T0AM are only partially understood and may depend on the dosage, model system, and species analyzed.

Therefore, our study aims to compare the effects of acute and long-term administrations of a pharmacological T0AM dose on metabolism, cardiovascular function, and thermoregulation as well as thyroid and selenium (Se) status in male C57BL/6J mice.

Animal Husbandry

Male C57BL/6J mice (3–4 months) were single housed (21−22°C, 12-h light/dark cycle) and had ad libitum access to standard diet and water. Animal care procedures were in accordance with the guidelines set by the European Community Council directives (86/609/EEC) and approved by Stockholm’s Norra Djurförsöksetiska Nämnd.

Reagents and Drugs

Chemical synthesis of high-purity 3-T1AM and T0AM was performed by Dr. R. Smits (ABX Advanced Biochemical Compounds, Radeberg, Germany). Compounds were dissolved in 60% dimethyl sulfoxide (DMSO) and 40% phosphate-buffered saline (PBS, pH 7.4).

Study 1: Radio Telemetry Measurement after a Single Injection of T0AM

To study the acute effects of a single intraperitoneal injection of 50 mg/kg T0AM (5 μL/g body weight) or vehicle, implantable radio transmitters (Mini Mitter Respironics, Bend, OR, USA) were used to determine the core body temperature, heart rate, and activity of conscious and freely moving mice. Transmitters were implanted as described previously [8-10] and animals recovered for 7 days prior to recording. The validity of the setup to detect changes in heart rate and body temperature was tested previously using 3-T1AM [10, 11]. Parameters were recorded every 30 s and analyzed by calculating the average for every 5 min. The heart rate was normalized to a 30 min baseline measurement prior to the injection. Statistical analysis of radio telemetry data was performed using a paired, nonparametric 2-tailed Wilcoxon t test to compare the mean heart rate change or the area under the curve (AUC) for the first 60 min after injection.

Study 2: Assessment of Cardiovascular and Thermoregulatory Parameters after Repeated Administration of T0AM

The experimental setup involved 7 days of baseline measurement followed by 7 days of daily intraperitoneal injections with 5 mg/kg T0AM (5 μL/g body weight) or vehicle. Throughout this time daily recordings of body weight and food and water intake were performed, and noninvasive measurements of cardiac parameters using a tail-cuff system on a platform at 37°C were conducted (SC1000; Hatteras Instruments, USA) [10]. Surface temperature of the inner ear, interscapular brown adipose tissue (iBAT), and tail was assessed using an infrared camera (T335; FLIR Systems Termisk Systemteknik, Linköping, Sweden; ±0.05°C sensitivity) [10]. Rectal temperature under anesthesia and heart weight were measured 24 h after the last injection, and tissue samples were collected. Statistical analysis was performed using an unpaired nonparametric, 2-tailed Mann-Whitney t test.

Study 3: Repeated Administration of 3-T1AM

Mice were treated intraperitoneally with 5 mg/kg 3-T1AM (5 μL/g body weight) or vehicle daily for 7 days. Parts of this experiment have been published before [5, 12]. Tissue samples were collected for trace element analysis 24 h after the last injection.

Total T4 and T3 ELISA for Mouse Serum Analysis

Serum total T4 (TT4; EIA 1781; DRG Instruments GmbH, Marburg, Germany) and total T3 (TT3; DNOVO53; NovaTec Immundiagnostica GmbH, Dietzenbach, Germany) were determined by commercial ELISA kits according to the manufacturers’ instructions.

TH and TAM Analysis in Liver

Hepatic THs and TAMs were extracted from 80 mg of mouse liver as previously described [2, 13] followed by liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis as published [14]. A brief description of the method can be found in the online supplementary material (for all online suppl. material, see www.karger.com/doi/10.1159/000481856).

Real-Time Quantitative PCR

Real-time quantitative PCR (qPCR) was performed on snap-frozen tissue samples from thyroid, pituitary, liver, kidney, and iBAT as described in the online supplementary material. Primer sequences are published or available on request [5, 9]. For the statistical analysis, significant outliers were determined using a Grubbs test (GraphPad software) and excluded. Statistical testing was performed using a multiple t test and controlled with the Sidak-Bonferroni correction for multiple comparisons for each tissue. For pulmonary angiotensin-1-converting enzyme (Ace) an unpaired, nonparametric, 2-tailed Mann-Whitney t test was used.

Glycogen Measurement in Liver Tissue

Glycogen content in the liver was determined as described previously [15] with minor modifications described in the online supplementary material.

Trace Element Analysis in Serum and Tissue Samples

Se concentrations were determined by total reflection X-ray fluorescence spectroscopy [16-18]. The method was validated with a human serum standard (Seronorm, Billingstad, Norway). Briefly, serum samples were diluted 1: 1 in a gallium- containing solvent for standardization. Tissue samples (liver and kidney) of 10–20 mg were digested in 4× volume of 65% nitric acid for 1 h and diluted with gallium-containing solvent in a 1: 2 ratio before analysis. The analysis was performed in duplicate, and the results of each sample differed by <20%. In every measurement run, a human control serum was included as control.

Statistical Analysis

GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, CA, USA) was used to analyze the data. All data are presented as mean ± standard error of the mean (SEM) and statistical significance was defined as * p < 0.05, ** p < 0.01, and *** p < 0.001.

T0AM and Metabolic Parameters

The analysis of metabolic parameters after repeated T0AM administration revealed no significant effect of T0AM on body weight (Fig. 1a) or on absolute food or water intake (Fig. 1b) comparing the mean of the last 3 treatment days. When the liver was analyzed in detail, no significant difference in hepatic glycogen after T0AM administration (Fig. 1c) was observed. Hepatic pyruvate kinase (Pyrk) mRNA expression (the rate-limiting enzyme for glycolysis) as well as hepatic phosphoenolpyruvate carboxykinase (Pepck) mRNA (the rate-limiting enzyme in gluconeogenesis) remained largely unchanged. Calculating the ratio of Pepck/Pyrk for each animal individually revealed no significant shift upon T0AM treatment, indicating that the glucose utilization of the liver remains unchanged (Fig. 1d, e). Both genes have been shown to be unchanged after 3-T1AM, the precursor of T0AM, treatment [12].

Fig. 1.

T0AM and metabolic endpoints. Body weight (a) and food and water intake (b) after repeated administration of T0AM (5 mg/kg, i.p., daily for 7 days); dashed lines indicate the baseline measurements before treatment. c Determination of liver glycogen in control (white) and T0AM-treated animals (black, n = 6). d Relative mRNA expression of metabolic genes in liver as assessed by quantitative real-time PCR and normalized against a set of unregulated housekeeping genes (18S, Actb, Hprt1) in control (white, n = 6) and T0AM-treated animals (black, n = 5). e Ratio of Pepck and Pyrk gene expression levels to evaluate the metabolic state of the liver. All values are mean ± SEM and statistical testing was performed using an unpaired, nonparametric, 2-tailed Mann-Whitney t test or a multiple t test controlled with the Sidak-Bonferroni correction for multiple comparisons. Actb, β-actin; Hprt1, hypoxanthine-guanine phosphoribosyltransferase 1; Pepck, phosphoenolpyruvate carboxykinase; Pyrk, pyruvate kinase.

Fig. 1.

T0AM and metabolic endpoints. Body weight (a) and food and water intake (b) after repeated administration of T0AM (5 mg/kg, i.p., daily for 7 days); dashed lines indicate the baseline measurements before treatment. c Determination of liver glycogen in control (white) and T0AM-treated animals (black, n = 6). d Relative mRNA expression of metabolic genes in liver as assessed by quantitative real-time PCR and normalized against a set of unregulated housekeeping genes (18S, Actb, Hprt1) in control (white, n = 6) and T0AM-treated animals (black, n = 5). e Ratio of Pepck and Pyrk gene expression levels to evaluate the metabolic state of the liver. All values are mean ± SEM and statistical testing was performed using an unpaired, nonparametric, 2-tailed Mann-Whitney t test or a multiple t test controlled with the Sidak-Bonferroni correction for multiple comparisons. Actb, β-actin; Hprt1, hypoxanthine-guanine phosphoribosyltransferase 1; Pepck, phosphoenolpyruvate carboxykinase; Pyrk, pyruvate kinase.

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T0AM and Cardiovascular Function

To characterize acute effects of a single dose of 50 mg/kg T0AM on cardiac function, we used a radio telemetry system. The results revealed no obvious alterations in heart rate change (Fig. 2a) and no significant differences in mean change of heart rate between T0AM and sham-injected animals (Fig. 2b; paired, nonparametric, 2-tailed Wilcoxon t test; p = 0.844). A similar result was found for the locomotor activity (Fig. 2c, d; paired, nonparametric, 2-tailed Wilcoxon t test; p = 0.9453). The analysis of the cardiovascular system after repeated application of 5 mg/kg T0AM showed no effects, as determined by unaltered heart rate (Fig. 2e) as well as systolic, diastolic, or mean arterial blood pressure (Fig. 2f). These findings were supported by an unaltered heart weight (Fig. 2g, h) after 7 days of treatment. A set of genes known to regulate cardiovascular functions were analyzed in the heart, kidney, lung, and liver by qPCR (Fig. 2i) but were unaltered as well, with the exception of pulmonary Ace (unpaired, nonparametric, 2-tailed Mann-Whitney t test; p = 0.0173).

Fig. 2.

Cardiac endpoints after T0AM-treatment. a Profiles of heart rate change over three hours as measured by radio telemetry (normalized to 30 min baseline measurement prior to the injection) in conscious and freely moving control (white) and T0AM-treated (50 mg/kg, i.p., gray) animals (n = 8–9). b Calculated mean change of heart rate over 60 min after injection in the two groups (paired, nonparametric 2-tailed Wilcoxon t test; p = 0.844). c Three-hour profiles of animal locomotor activity as measured by radio telemetry after injection, in conscious and freely moving control (white) and T0AM-treated (50 mg/kg, i.p., gray) animals (n = 8–9). d Calculated mean AUC of locomotor activity over 60 min after injection in the two groups (paired, nonparametric, 2-tailed Wilcoxon t test; p = 0.9453). No significant effect was observed after 7 days of daily treatment (5 mg/kg or vehicle, i.p.) on heart rate (e) and blood pressure (systolic, diastolic and mean arterial blood pressure; f); dashed lines indicate the baseline measurements before treatment. Absolute heart weight (g) and heart weight in relation to body weight (h) in control (white) and T0AM-treated (black) animals (n = 6 per group). i Analysis of mRNA expression of genes involved in cardiac function and blood pressure regulation as assessed by real-time PCR normalized to a set of unregulated housekeeping genes (heart: 18S, Actb, Hprt1; kidney: Hprt1; lung: 18S, Actb, Hprt1, Ppia; liver: 18S, Actb, Ppia) comparing control (white, n = 5–6) and T0AM-treated (black, n = 5–6) mice. All values are mean ± SEM and statistical testing was performed using a paired, nonparametric, 2-tailed Wilcoxon t test or an unpaired, nonparametric, 2-tailed Mann-Whitney t test (lung Ace; p = 0.0173), or a multiple t test controlled with the Sidak-Bonferroni correction for multiple comparisons for each tissue; * p < 0.05. Ace, angiotensin-I-converting enzyme; Actb, β-actin; Adrβ2, adrenergic receptor beta 2; Angt, angiotensinogen; Chrm2, cholinergic receptor muscarinic 2; cnt, counts; Dio2, type II deiodinase; Hcn2, hyperpolarization-activated cyclic nucleotide-gated K+2 channel; Hprt1, hypoxanthine-guanine phosphoribosyltransferase 1; MAP, mean arterial blood pressure; Ppia, peptidylprolyl isomerase A.

Fig. 2.

Cardiac endpoints after T0AM-treatment. a Profiles of heart rate change over three hours as measured by radio telemetry (normalized to 30 min baseline measurement prior to the injection) in conscious and freely moving control (white) and T0AM-treated (50 mg/kg, i.p., gray) animals (n = 8–9). b Calculated mean change of heart rate over 60 min after injection in the two groups (paired, nonparametric 2-tailed Wilcoxon t test; p = 0.844). c Three-hour profiles of animal locomotor activity as measured by radio telemetry after injection, in conscious and freely moving control (white) and T0AM-treated (50 mg/kg, i.p., gray) animals (n = 8–9). d Calculated mean AUC of locomotor activity over 60 min after injection in the two groups (paired, nonparametric, 2-tailed Wilcoxon t test; p = 0.9453). No significant effect was observed after 7 days of daily treatment (5 mg/kg or vehicle, i.p.) on heart rate (e) and blood pressure (systolic, diastolic and mean arterial blood pressure; f); dashed lines indicate the baseline measurements before treatment. Absolute heart weight (g) and heart weight in relation to body weight (h) in control (white) and T0AM-treated (black) animals (n = 6 per group). i Analysis of mRNA expression of genes involved in cardiac function and blood pressure regulation as assessed by real-time PCR normalized to a set of unregulated housekeeping genes (heart: 18S, Actb, Hprt1; kidney: Hprt1; lung: 18S, Actb, Hprt1, Ppia; liver: 18S, Actb, Ppia) comparing control (white, n = 5–6) and T0AM-treated (black, n = 5–6) mice. All values are mean ± SEM and statistical testing was performed using a paired, nonparametric, 2-tailed Wilcoxon t test or an unpaired, nonparametric, 2-tailed Mann-Whitney t test (lung Ace; p = 0.0173), or a multiple t test controlled with the Sidak-Bonferroni correction for multiple comparisons for each tissue; * p < 0.05. Ace, angiotensin-I-converting enzyme; Actb, β-actin; Adrβ2, adrenergic receptor beta 2; Angt, angiotensinogen; Chrm2, cholinergic receptor muscarinic 2; cnt, counts; Dio2, type II deiodinase; Hcn2, hyperpolarization-activated cyclic nucleotide-gated K+2 channel; Hprt1, hypoxanthine-guanine phosphoribosyltransferase 1; MAP, mean arterial blood pressure; Ppia, peptidylprolyl isomerase A.

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T0AM and Thermoregulation

Using radio telemetry, our measurement revealed that a single application of T0AM (50 mg/kg, i.p.) did not significantly affect body temperature over the time course of the experiment (Fig. 3a; repeated-measurement 2-way ANOVA for T0AM effect; p = 0.3225, and Fig. 3b, paired, nonparametric, 2-tailed Wilcoxon t test; p = 0.195). Likewise, repeated T0AM injection showed no significant effect on thermoregulation as studied by noninvasive infrared thermography. Daily inner ear temperature (Fig. 3c, d), temperature of the skin overlaying the iBAT (Fig. 3e, f), tail base temperature (Fig. 3g, h), and rectal temperature measured on the last day (Fig. 3i) were similar between T0AM-treated and sham-treated animals. Concurring with these in vivo findings, T0AM treatment did not alter the expression of genes involved in thermoregulation in iBAT (Fig. 3j).

Fig. 3.

Thermoregulatory parameters after T0AM-treatment. a Three-hour profiles of body temperature as measured by radio telemetry in conscious and freely moving control (white) and T0AM -treated (50 mg/kg, i.p., gray) animals (n = 8–9, repeated measurement 2-way ANOVA for T0AM effect p = 0.3225). b Calculated mean AUC of body temperature over 60 min after injection in the groups (paired, nonparametric, 2-tailed Wilcoxon t test; p = 0.195). Representative infrared thermography images of inner ear (c), iBAT (e) and tail base (g) of the repeated treatment (control vs. 5 mg/kg T0AM, i.p., daily for 7 days) experiment. Quantified average maximum temperature of the respective areas under both conditions (d, f, h; n = 6 per group). i Rectal temperature in anesthetized animals (n = 6 per group) after 7 days of T0AM or sham treatment. j mRNA expression of thermoregulatory genes in iBAT as assessed by real-time PCR normalized against a set of unregulated housekeeper genes (Actb, Hprt1, Ppia) comparing control (white) and T0AM-treated (black, n = 6 per group) mice. All values are mean ± SEM and statistical testing was performed using a paired, nonparametric 2-tailed Wilcoxon t test or an unpaired, nonparametric, 2-tailed Mann-Whitney t test, or a multiple t test controlled with the Sidak-Bonferroni correction for multiple comparisons. Acc1, acetyl-CoA carboxylase 1; Acc2, acetyl-CoA carboxylase 2; Actb, β-actin; Adrβ3, adrenergic receptor beta 3; Dio2, type II deiodinase; Hprt1, hypoxanthine-guanine phosphoribosyltransferase 1; iBAT, interscapular brown adipose tissue; Mcd, malonyl-CoA decarboxylase; Ppia, peptidylprolyl isomerase A; Ucp1, uncoupling protein 1.

Fig. 3.

Thermoregulatory parameters after T0AM-treatment. a Three-hour profiles of body temperature as measured by radio telemetry in conscious and freely moving control (white) and T0AM -treated (50 mg/kg, i.p., gray) animals (n = 8–9, repeated measurement 2-way ANOVA for T0AM effect p = 0.3225). b Calculated mean AUC of body temperature over 60 min after injection in the groups (paired, nonparametric, 2-tailed Wilcoxon t test; p = 0.195). Representative infrared thermography images of inner ear (c), iBAT (e) and tail base (g) of the repeated treatment (control vs. 5 mg/kg T0AM, i.p., daily for 7 days) experiment. Quantified average maximum temperature of the respective areas under both conditions (d, f, h; n = 6 per group). i Rectal temperature in anesthetized animals (n = 6 per group) after 7 days of T0AM or sham treatment. j mRNA expression of thermoregulatory genes in iBAT as assessed by real-time PCR normalized against a set of unregulated housekeeper genes (Actb, Hprt1, Ppia) comparing control (white) and T0AM-treated (black, n = 6 per group) mice. All values are mean ± SEM and statistical testing was performed using a paired, nonparametric 2-tailed Wilcoxon t test or an unpaired, nonparametric, 2-tailed Mann-Whitney t test, or a multiple t test controlled with the Sidak-Bonferroni correction for multiple comparisons. Acc1, acetyl-CoA carboxylase 1; Acc2, acetyl-CoA carboxylase 2; Actb, β-actin; Adrβ3, adrenergic receptor beta 3; Dio2, type II deiodinase; Hprt1, hypoxanthine-guanine phosphoribosyltransferase 1; iBAT, interscapular brown adipose tissue; Mcd, malonyl-CoA decarboxylase; Ppia, peptidylprolyl isomerase A; Ucp1, uncoupling protein 1.

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T0AM and TH Homeostasis

To determine whether repeated administration of T0AM interferes with peripheral TH metabolism or TH biosynthesis, we measured TH concentrations in serum. TT4 (Fig. 4a) and TT3 (Fig. 4b) serum concentrations as well as the TT3/TT4 ratio (Fig. 4c) were not different between T0AM-treated and control mice, suggesting a euthyroid state in both groups. This was in line with the unaltered expression of genes involved in TH synthesis in the thyroid (Fig. 4d). The evaluation of the pituitary T3 responsive genes TSHβ (Tshb), TRH receptor 1 (Trhr1), TRH degrading enzyme (Trhde), type 2 deiodinase (Dio2), and TH receptor β (Thrb) showed no differences between T0AM-treated and sham-treated mice (Fig. 4e), indicating no effect of T0AM on the hypothalamus-pituitary-thyroid axis.

Fig. 4.

T0AM and thyroid status. Total T4 (a), total T3 (b), and ratio of total T3/total T4 (c) concentrations in serum of controls (white) compared to T0AM-treated animals (black, n = 6 per group). d Thyroid mRNA expression of genes involved in thyroid hormone biosynthesis (control = 5; T0AM = 6; housekeeping genes: 18S, Actb, Hprt1). e Unaltered expression of thyroid hormone responsive genes in pituitary (control = 4; T0AM = 3–4; housekeeping genes: 18S, Actb, Hprt1). f Expression level of renal Dio1 mRNA (n = 6 per group; housekeeping gene: Hprt1). g Expression levels of hepatic thyroid hormone responsive genes (control = 6; T0AM = 5; housekeeping genes: 18S, Actb, Ppia). LC-MC/MC analyses of total T4 (h), total T3 (i) and ratio of total T3/total T4 (j) concentrations in liver. k While T0AM is not detected in the liver of control mice, it is detected in the treatment group 24 h after the last injection. All values are mean ± SEM and statistical testing was performed using an unpaired, nonparametric, 2-tailed Mann-Whitney t test or a multiple t test controlled with the Sidak-Bonferroni correction for multiple comparisons. Actb, β-actin; Ano1, anoctamin 1; Dio1/2, type I / II deiodinase; Duox1/2, dual oxidase 1/2; n.d., not detected; Hprt1, hypoxanthine-guanine phosphoribosyltransferase 1; Mct8, monocarboxylate transporter 8; Nis, sodium/iodide symporter; Pds, pendrin; Ppia, peptidylprolyl isomerase A; SecS, selenocysteine t-RNA synthase; Tg, thyroglobulin; Tpo, thyroid peroxidase; Thrb, TH receptor β; Trhde, thyrotropin-releasing hormone-degrading enzyme; Trhr1, TRH receptor1; Tshβ, β-subunit of thyroid stimulating hormone; Tshr, thyroid stimulating hormone receptor; TT3, total T3; TT4, total T4; Vegfa, vascular endothelial growth factor A.

Fig. 4.

T0AM and thyroid status. Total T4 (a), total T3 (b), and ratio of total T3/total T4 (c) concentrations in serum of controls (white) compared to T0AM-treated animals (black, n = 6 per group). d Thyroid mRNA expression of genes involved in thyroid hormone biosynthesis (control = 5; T0AM = 6; housekeeping genes: 18S, Actb, Hprt1). e Unaltered expression of thyroid hormone responsive genes in pituitary (control = 4; T0AM = 3–4; housekeeping genes: 18S, Actb, Hprt1). f Expression level of renal Dio1 mRNA (n = 6 per group; housekeeping gene: Hprt1). g Expression levels of hepatic thyroid hormone responsive genes (control = 6; T0AM = 5; housekeeping genes: 18S, Actb, Ppia). LC-MC/MC analyses of total T4 (h), total T3 (i) and ratio of total T3/total T4 (j) concentrations in liver. k While T0AM is not detected in the liver of control mice, it is detected in the treatment group 24 h after the last injection. All values are mean ± SEM and statistical testing was performed using an unpaired, nonparametric, 2-tailed Mann-Whitney t test or a multiple t test controlled with the Sidak-Bonferroni correction for multiple comparisons. Actb, β-actin; Ano1, anoctamin 1; Dio1/2, type I / II deiodinase; Duox1/2, dual oxidase 1/2; n.d., not detected; Hprt1, hypoxanthine-guanine phosphoribosyltransferase 1; Mct8, monocarboxylate transporter 8; Nis, sodium/iodide symporter; Pds, pendrin; Ppia, peptidylprolyl isomerase A; SecS, selenocysteine t-RNA synthase; Tg, thyroglobulin; Tpo, thyroid peroxidase; Thrb, TH receptor β; Trhde, thyrotropin-releasing hormone-degrading enzyme; Trhr1, TRH receptor1; Tshβ, β-subunit of thyroid stimulating hormone; Tshr, thyroid stimulating hormone receptor; TT3, total T3; TT4, total T4; Vegfa, vascular endothelial growth factor A.

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Furthermore, mRNA levels of the hepatic TH target genes Spot14 and selenocysteine t-RNA synthase (SecS) were unchanged as well as hepatic and renal Dio1 mRNA, implying an unaffected TH metabolism (Fig. 4f, g). To test this directly, we determined liver concentrations of TT4 and TT3 using LC-MS/MS. In line with serum, no significant differences in hepatic TT4 (Fig. 4h), TT3 (Fig. 4i), or the ratio of TT3/TT4 (Fig. 4j) were observed. To prove that the administration of T0AM was successful, we also analyzed hepatic T0AM levels in both treatment groups (Fig. 4k). While endogenous T0AM was not detected in the control group, T0AM was detected in the treated mice, showing that even 24 h after the last injection, the compound was still present in pmol/mg amounts in the liver.

T0AM and Se Status

To further target the role of T0AM treatment for the gene expression of Se-containing enzymes, we analyzed the concentration of the trace element Se in serum (Fig. 5a), liver (Fig. 5b) and kidney (Fig. 5c). However, no significant differences were found in the tissues tested when compared to control-injected mice, which was in good agreement with unaltered mRNA expression of hepatic (Fig. 5d) and renal (Fig. 5e) genes involved in Se biosynthesis, metabolism, storage, and transport.

Fig. 5.

T0AM and selenium status. Selenium concentrations in serum (a), liver (b), and kidney (c) in control (white) and T0AM-treated (black, 5 mg/kg, i.p., daily for 7 days) animals as assessed by total reflection X-ray analysis (n = 6 per group). Quantification of mRNA expression of genes involved in selenium storage, secretion and metabolism in liver (control = 5–6; T0AM = 5; housekeeping genes: 18S, Actb, Ppia; d) and kidney (control = 5–6; T0AM = 6; housekeeping gene: Hprt1; e). All values are mean ± SEM and statistical testing was performed using an unpaired, nonparametric 2-tailed Mann-Whitney t test or a multiple t test controlled with the Sidak-Bonferroni correction for multiple comparisons. Actb, β-actin; Hprt1, hypoxanthine-guanine phosphoribosyltransferase 1; Sepp, selenoprotein P; GPx1/3, glutathione peroxidase 1/3; Scly, selenocysteine lyase; Pstk, phosphoseryl-tRNA kinase; Ppia, peptidylprolyl isomerase A; SelH, selenoprotein H; SelW, selenoprotein W; Sephs2, selenophosphate-synthetase 2; Sebp1, selenium-binding protein 1.

Fig. 5.

T0AM and selenium status. Selenium concentrations in serum (a), liver (b), and kidney (c) in control (white) and T0AM-treated (black, 5 mg/kg, i.p., daily for 7 days) animals as assessed by total reflection X-ray analysis (n = 6 per group). Quantification of mRNA expression of genes involved in selenium storage, secretion and metabolism in liver (control = 5–6; T0AM = 5; housekeeping genes: 18S, Actb, Ppia; d) and kidney (control = 5–6; T0AM = 6; housekeeping gene: Hprt1; e). All values are mean ± SEM and statistical testing was performed using an unpaired, nonparametric 2-tailed Mann-Whitney t test or a multiple t test controlled with the Sidak-Bonferroni correction for multiple comparisons. Actb, β-actin; Hprt1, hypoxanthine-guanine phosphoribosyltransferase 1; Sepp, selenoprotein P; GPx1/3, glutathione peroxidase 1/3; Scly, selenocysteine lyase; Pstk, phosphoseryl-tRNA kinase; Ppia, peptidylprolyl isomerase A; SelH, selenoprotein H; SelW, selenoprotein W; Sephs2, selenophosphate-synthetase 2; Sebp1, selenium-binding protein 1.

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To examine whether 3-T1AM, the precursor of T0AM, exerts any effects on the status of the trace element Se, we analyzed the serum, liver, and kidney of male C57BL/6J mice that were injected with 3-T1AM (5 mg/kg/day, 7 days, i.p.) or vehicle [5, 12]. No effect of 3-T1AM treatment was observed on Se concentration in the tissues tested (Fig. 6a–c).

Fig. 6.

3-T1AM and selenium status. Selenium concentrations in serum (a), liver (b), and kidney (c) in control (white) and 3-T1AM-treated (black, 5 mg/kg, i.p., daily for 7 days) animals as assessed by total reflection X-ray analysis (n = 6 per group). All values are mean ± SEM and statistical testing was performed using an unpaired, nonparametric, 2-tailed Mann-Whitney t test.

Fig. 6.

3-T1AM and selenium status. Selenium concentrations in serum (a), liver (b), and kidney (c) in control (white) and 3-T1AM-treated (black, 5 mg/kg, i.p., daily for 7 days) animals as assessed by total reflection X-ray analysis (n = 6 per group). All values are mean ± SEM and statistical testing was performed using an unpaired, nonparametric, 2-tailed Mann-Whitney t test.

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THs undergo a cascade of modifications in vivo, transforming them into a large number of metabolites [2, 19-23] (e.g., 3-T1AM). Due to the contrary effects of 3-T1AM compared to THs, it was speculated that TH derivatives may contribute to the fine-tuning of TH signaling [4] and could be involved in the pharmacological effects observed after 3-T1AM administration.

Recently, we reported that 3-iodothyroacetic acid is an inactive product of 3-T1AM metabolism [10]. Hereby, we provided evidence that the ethylamine side chain is indispensable for the rapid cardiac and thermogenic effects of 3-T1AM. In vitro data show that 3-T1AM is also a substrate for Dio1 and Dio3 enzymes, resulting in the noniodinated T0AM [2, 5]. Therefore, we were interested whether deiodination could also constitute an inactivation mechanism for 3-T1AM. Our in vivo experiments show that T0AM lacks the metabolic, cardiovascular, and thermoregulatory properties of 3-T1AM, as it did not induce bradycardia and anapyrexia in mice upon single or repeated administration. Furthermore, T0AM did not interfere with TH homeostasis since TH-regulated genes as well as serum and liver TH concentrations remained unaffected.

Mittag et al. [18, 24] have shown that hepatic trace element metabolism (e.g., Se) is modulated by T3. Thus, we aimed to test whether these effects might at least be partially mediated via TAMs by the administration of T0AM and 3-T1AM. Our analyses have demonstrated that repeated administration of T0AM had no significant effect on Se status in serum, livers, and kidneys or on the major storage, metabolism, and transport proteins of the trace element. Hence, T0AM displays only a very limited biological activity in our paradigm compared to the reports on the physiologically more active 3-T1AM [1, 25-27].

The results presented in this study indicate that T0AM lacks a significant metabolic, cardiovascular, or thermoregulatory activity after single or repeated administration. Only for pulmonary Ace was a modest increase upon repeated T0AM administration observed, which however did not result in altered blood pressure. We thus speculate that the tissue-specific deiodination of 3-T1AM is an important deactivation mechanism in addition to the oxidation of the amine side chain, suggesting another relevant gate-keeper function of Dio1 and Dio3 besides TH metabolism, i.e., the efficient inactivation of 3-T1AM function.

We thank the staff of the CMB animal facility, Dr. Keith Richards for LC-MS/MS analysis, and Raymond Monk and Gabriele Boehm for fruitful discussions and technical assistance.

This work was supported by grants from the Deutsche Forsch-ungsgemeinschaft (SPP1629 Thyroid Trans Act HO 5096/1-1 and HO 5096/2-1 to C.S.H., KO 922/16-2 and 922/17-2 to J.K., and Mi1242/2-1 and Mi1242/3-1 to J.M., as well as GRK 1957 ‘Adipocyte-Brain Crosstalk’ to L.H. and J.M.), the Karolinska Institutet Foundation (to C.S.H. and J.M.), and the Swedish Research Council (to J.M.).

The authors have no conflicts of interest to declare.

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