In vivo Effects of Repeated Thyronamine Administration in Male C57BL/6J Mice

Abstract

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.

Keywords: 3-Iodothyronamine; Brown adipose tissue; Heart rate; Metabolism; Thermoregulation; Thyroid hormone; Trace elements.

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. 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. 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. 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. 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. 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.