Abstract
Thyroid hormone (TH) metabolism and cellular TH action are influenced by ageing. To investigate the response to thyroxine (T4) overtreatment, a kinetic study was conducted in young and aged mice with chronic hyperthyroidism and hormone withdrawal. Five and 22 months old male mice were treated with T4 or PBS over 5 weeks, followed by observation for up to 12 days. Serial analysis was performed for thyroid function parameters, transcript levels of TH target genes, deiodinase type 1 (DIO1) activity as well as serum lipids at 12, 24, 72, 144, 216, and 288 h after cessation of T4 administration. Higher FT3 concentrations and higher renal DIO1 activities were noted in aged mice 12 h after T4 withdrawal and marked thyroid-stimulating hormone elevation was found in aged mice after 12 days compared to respective controls. A biphasic expression pattern occurred for TH target genes in all organs and a hypothyroid organ state was observed at the end of the study, despite normalization of TH serum concentrations after 72 h. In line with this, mirror-image kinetics were detected for serum cholesterol and triglycerides in aged and young mice. Recovery from TH overtreatment in mice involves short- and medium-term adaption of TH metabolism on systemic and organ levels. Increased renal DIO1 activity may contribute to higher T3 concentrations and prolonged thyrotoxicosis followed by hypothyroidism in an aged-mouse organism. Translation of these findings in the clinical setting seems warranted and may lead to better management of hyperthyroidism and prevention of T4 overtreatment in aged patients.
Introduction
Ageing is associated with an elevated risk for thyroid dysfunction (Hollowell et al. 2002, Pearce et al. 2016), and an excess of thyroid hormones (TH) (hyperthyroidism) is particularly harmful in elderly patients (Grossman et al. 2016). Besides endogenous causes of hyperthyroidism, for example, toxic multinodular goiter or Graves’ disease, exogenous hyperthyroidism may arise from overtreatment of hypothyroidism with thyroxine (T4). This unwanted not infrequent complication of TH substitution has been observed in several epidemiological studies (Taylor et al. 2019) and has over the past years stimulated a more vigorous decision-finding on who should receive TH treatment and how it should be monitored (Wiersinga et al. 2012, Jonklaas et al. 2014, Ross et al. 2016). In case of inadvertent TH overtreatment, usual practice is to stop T4 medication immediately and to resume TH substitution at lower doses later. However, so far no studies have investigated whether T4 withdrawal after TH overtreatment has a different impact on serum and organ status in an aged organism. In the latter context, we and others have previously shown that age modulates the biological phenotypes of chronic hyper- and hypothyroidism in a tissue-specific manner in mice, not automatically reflected by TH serum concentrations (Engels et al. 2019, Kerp et al. 2020). Furthermore, we have recently shown that both systemic and organ responses to acute T4 challenge are highly age-dependent (Rakov et al. 2019). In the present study, we asked whether an adjustment to correction of T4 overtreatment is different in young and aged mice and how it may affect TH metabolism and TH action in tissues. Our findings show a hitherto underappreciated age impact on systemic and organ TH response with partially incomplete recovery on organ level after T4 excess, despite normalization of TH serum concentrations.
Material and methods
Animals
Male C57Bl/6N mice were purchased from Taconic, Denmark. Two age cohorts were investigated: 38 were 5 months old (young cohort) and 29 were 22 months old mice (aged cohort). Animals were single-housed in temperature- (23 ± 1°C) and light-controlled (inverse 12 h light:12 h darkness cycle) conditions. Food and water were provided ad libitum. All animal experiments were performed in accordance with the German regulations for Laboratory Animal Science (GVSOLAS) and the European Health Law of the Federation of Laboratory Animal Science Associations (FELASA). The protocols for animal studies were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz, Nordrhein-Westfalen (LANUV-NRW), Germany.
Treatment and organ collection
Mice were randomly divided into control groups (n = 7 for 5 and 22 months) or T4 groups sacrificed at different time points (12, 24, 72, 144, 216, and 288 h) after last T4 treatment (n = 5–6 each time point for 5 months, n = 3–5 each time point for 22 months). T4 mice were injected intraperitoneally (i.p.) for 5 weeks for every 48 h at the same time with 1 μg/g body weight T4 as described previously (Engels et al. 2016, Rakov et al. 2016). Control mice received i.p. injections of 150 µL PBS every 48 h for 5 weeks. At indicated time points after the last injection, mice were deeply anesthetized by an i.p. injection of 200 µL ketamine/xylazine mixture (150 µL of 100 mg/mL Ketamine (Beta-pharm, Vechta, Germany) and 50 µL of 20 mg/mL xylazine (Ceva, Düsseldorf, Germany). Final blood was obtained by heart puncture. For tissue collection, mice were perfused with heparinized saline through a needle placed in the left heart ventricle. Tissues were shock-frozen in liquid nitrogen and stored at −80°C until further processing.
Serum thyroid hormone and TSH measurements
TT4, FT4, and fT3 concentrations in serum of mice were measured as previously described using commercial ELISA kits according to the manufacturer’s instructions (DRG Instruments GmbH, Marburg, Germany) and serum samples with known TH concentrations as standards (provided by company). TSH serum concentration was determined using the Milliplex Map mouse pituitary magnetic bead panel (EMD Millipore Corporation, Billerica, USA; MPTMAG-49K) on the Luminex 200 system (Thermo Fisher) (Rakov et al. 2019).
Serum lipid profiles
Total cholesterol concentrations in serum were measured using an enzymatic cholesterol quantitation kit (Sigma–Aldrich (MAK043)) and total triglyceride concentrations were investigated using an enzymatic serum triglyceride determination kit (Sigma–Aldrich (TR0100)), according to the manufacturer’s instructions.
Quantitative real-time PCR
Total RNA from liver, kidney, and heart was isolated using the RNeasy Kit according to the manufacturer’s instruction (Qiagen) and stored at −80°C as previously described (Rakov et al. 2019). RNA was reverse transcribed into cDNA with SuperScriptIII (Life Technologies) and hexamer primers. Quantitative real-time PCR (qRT-PCR) was performed using Roche SYBR Green I master mix (Roche). Primers were designed to be intron-spanning to exclude genomic DNA signals (sequences provided in Supplementary Table 1, see section on supplementary materials given at the end of this article). In compliance with the MIQE guidelines for RT-PCR (Bustin et al. 2009), we used a set of 2–3 reference genes per tissue to assure accurate normalization and calculation (liver: 18S (18S rRNA), Ppia (peptidylprolyl isomerase A, cyclophilin A); heart: Polr2a (polymerase RNA II), Gapdh (glyceraldehyde-3-phosphate dehydrogenase; kidney: Ppia, Gapdh and Actb (actin beta). Analysis and calculation of the fold-change in gene expression were done on Ct-values ≤ 35 using the efficiency-corrected ΔΔCt method (Pfaffl 2001).
Hepatic and renal deiodinase 1 activity
Liver and kidney protein samples were prepared by mincing and sonification, adjusted to a defined protein concentration (2.5 µg/µL). In the case of lower protein concentration, the calculation of activity was adjusted by the individual protein yield of the sample. Dio1 activity was assayed as previously described (Renko et al. 2012). In brief, a 50 µL reaction mixture containing 40 μg of liver or kidney microsomal proteins, 1 mM 6-n-propyl-2-thio-uracil (PTU) for background controls, was mixed with 50 μL of freshly prepared substrate mix (20 μM rT3 (Sigma–Aldrich), 0.2 M KPO4 (pH 6.8), 2 mM ethylenediaminetetraacetic acid, and 80 mM dithiothreitol). The enzyme reaction lasted for 1 or 2 h at 37 °C, for liver and kidney samples, respectively. After centrifugation (4°C, 15, 000 g, 5 min), the supernatant was used for quantification of released iodide. Dowex W50-X2 resin columns were used for the separation of intact rT3 and the deiodinated breakdown products from the released iodide. The iodide content was determined by the Sandell–Kolthoff reaction, using cerium solution (25 mM (NH4)4Ce(SO4)4 and 0.5 M H2SO4) and arsenite solution (25 mM NaAsO2, 0.8 M NaCl, and 0.5 M H2SO4). The changes in absorption (OD at 415 nm) were determined at the reaction starting point and after 20 min. All protein samples were assayed in duplicate on two separate plates, and mean values were taken for further calculation. The tubes that contained PTU were used for background subtraction. Approximation of the enzyme activity was performed by interpolation from a separately measured iodide standard curve.
Statistical analysis
All data are shown as mean ± s.d. ors.e.m. as indicated. Statistical analysis was performed using GraphPad Prism 6 Software. One or two-way ANOVA followed Dunnett’s or Tukey’s post hoc analysis was applied as indicated. Values of *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.0001 were considered statistically significant.
Results
Male mice of 5 and 22 months of age with exogenously induced chronic hyperthyroidism were investigated for short- and medium-term effects on systemic and organ adaption after stopping T4 treatment. Changes in circulating TH and TSH concentrations, TH target gene expression in heart, kidney, and liver, renal and hepatic DIO1 activities, and serum lipid profiles were determined in young and aged mice and were compared to age-matched controls.
Changes in systemic thyroid hormone state after T4 withdrawal
Age- and time-dependent changes in thyroid function were assessed by serial analysis of TT4, FT4, FT3, and TSH serum concentrations (Fig. 1A, B, C and D). In control animals, no significant difference in serum TH status was noted, although TSH concentrations tended to be higher in aged compared to young mice. As expected, T4 treatment over 5 weeks resulted in significantly increased TT4, FT4, and FT3 serum concentrations, reaching ~5-fold (TT4), ~12-fold (FT4), and ~4–5-fold (FT3) of controls. Twelve hours after the last T4 injection, FT3 concentrations were higher in aged compared to young mice (Fig. 1C). At 72 h, TH concentrations in all T4 treated mice had reached values of control animals and remained stable thereafter with no differences between aged-matched T4 treated and control mice (Fig. 1A, B and C). TSH concentrations remained below the detection limit until 72 h after T4 withdrawal, followed by a steep increase thereafter. At 12 days after cessation of T4 treatment, TSH concentrations in young mice had returned to levels of controls, while a sustained marked TSH elevation was found in aged animals (Fig. 1D).
Thyroid function parameters during recovery from T4 overtreatment in young and old mice compared to age-matched PBS treated controls (ctrl). TT4 (A), FT4 (B), and FT3 (C) serum concentrations declined rapidly over 72 h in young and aged mice. Immediately after T4 withdrawal, FT3 serum concentrations were significantly higher in aged compared to young mice. TSH (D) was suppressed until 72 h in both age groups, after which a marked and sustained increase in TSH concentrations occurred suggesting a transient rise in aged animals. Data are shown as boxplots, min to max with individual values, two-way ANOVA and Tukey’s post-hoc analysis, *P < 0.05, **P < 0.01, ***P < 0.001, # P < 0.0001. Symbols above bars represent significant differences compared to the respective control group, and symbols above half-tick down lines represent age differences.
Citation: Journal of Molecular Endocrinology 67, 4; 10.1530/JME-21-0109
Thyroid function parameters during recovery from T4 overtreatment in young and old mice compared to age-matched PBS treated controls (ctrl). TT4 (A), FT4 (B), and FT3 (C) serum concentrations declined rapidly over 72 h in young and aged mice. Immediately after T4 withdrawal, FT3 serum concentrations were significantly higher in aged compared to young mice. TSH (D) was suppressed until 72 h in both age groups, after which a marked and sustained increase in TSH concentrations occurred suggesting a transient rise in aged animals. Data are shown as boxplots, min to max with individual values, two-way ANOVA and Tukey’s post-hoc analysis, *P < 0.05, **P < 0.01, ***P < 0.001, # P < 0.0001. Symbols above bars represent significant differences compared to the respective control group, and symbols above half-tick down lines represent age differences.
Citation: Journal of Molecular Endocrinology 67, 4; 10.1530/JME-21-0109
Thyroid function parameters during recovery from T4 overtreatment in young and old mice compared to age-matched PBS treated controls (ctrl). TT4 (A), FT4 (B), and FT3 (C) serum concentrations declined rapidly over 72 h in young and aged mice. Immediately after T4 withdrawal, FT3 serum concentrations were significantly higher in aged compared to young mice. TSH (D) was suppressed until 72 h in both age groups, after which a marked and sustained increase in TSH concentrations occurred suggesting a transient rise in aged animals. Data are shown as boxplots, min to max with individual values, two-way ANOVA and Tukey’s post-hoc analysis, *P < 0.05, **P < 0.01, ***P < 0.001, # P < 0.0001. Symbols above bars represent significant differences compared to the respective control group, and symbols above half-tick down lines represent age differences.
Citation: Journal of Molecular Endocrinology 67, 4; 10.1530/JME-21-0109
Impact of T4 withdrawal on TH action in target organs heart, kidney, and liver
Heart
Transcript levels of deiodinases Dio2 and Dio3, that finetune cardiac T3 concentrations in the normal and diseased heart (Janssen et al. 2013), the monocarboxylate transporter 8 (Mct8), and prominent cardiac TH responsive genes myosin heavy chain 6 and 7 (Myh6 and Myh7) were quantified in mouse hearts after T4 excess and compared to expression levels of age-matched controls. Dio2 expression was constant at all time points in young mice after T4 withdrawal, while in hearts of aged mice, reduced Dio2 expression was found at 24 h (Fig. 2A and B) compared to age-matched controls. In contrast, cardiac Dio3 transcript levels were below the detection limit at all time points and in both T4 treatment groups and controls (data not shown). Repression of Myh7 and overexpression of Myh6 confirmed a sustained thyrotoxic state of mouse hearts after T4 withdrawal, whereas a hypothyroid organ state was evident with downregulation of Myh6 and upregulation of Myh7 expression at 144 h. Downregulation of Myh6 persisted until 288 h after cessation of T4 (Fig. 2C, D, E and F) and was most prominent in hearts of aged mice. Mct8 expression was not affected by T4 withdrawal (Fig. 2G and H).
Serial analysis of putative TH responsive gene expression in the heart during recovery from T4 overtreatment in young and old mice compared to age-matched PBS-treated controls (ctrl). Transcript levels of Dio2 (A and B), Myh6 (C and D), Myh7 (E and F), and Mct8 (G and H) were determined by quantitative real-time PCR. At the end of the study, pronounced downregulation of Myh6 was found in old hearts while Myh6 expression had normalized in young hearts. Dio2 and Mct8 expression was not affected. Boxplots, min and max with individual values, two-way ANOVA and Dunnett’s post-hoc analysis, *P < 0.05, **P < 0.01, #P < 0.0001. Symbols above bars represent significant differences compared to the respective control group.
Citation: Journal of Molecular Endocrinology 67, 4; 10.1530/JME-21-0109
Serial analysis of putative TH responsive gene expression in the heart during recovery from T4 overtreatment in young and old mice compared to age-matched PBS-treated controls (ctrl). Transcript levels of Dio2 (A and B), Myh6 (C and D), Myh7 (E and F), and Mct8 (G and H) were determined by quantitative real-time PCR. At the end of the study, pronounced downregulation of Myh6 was found in old hearts while Myh6 expression had normalized in young hearts. Dio2 and Mct8 expression was not affected. Boxplots, min and max with individual values, two-way ANOVA and Dunnett’s post-hoc analysis, *P < 0.05, **P < 0.01, #P < 0.0001. Symbols above bars represent significant differences compared to the respective control group.
Citation: Journal of Molecular Endocrinology 67, 4; 10.1530/JME-21-0109
Serial analysis of putative TH responsive gene expression in the heart during recovery from T4 overtreatment in young and old mice compared to age-matched PBS-treated controls (ctrl). Transcript levels of Dio2 (A and B), Myh6 (C and D), Myh7 (E and F), and Mct8 (G and H) were determined by quantitative real-time PCR. At the end of the study, pronounced downregulation of Myh6 was found in old hearts while Myh6 expression had normalized in young hearts. Dio2 and Mct8 expression was not affected. Boxplots, min and max with individual values, two-way ANOVA and Dunnett’s post-hoc analysis, *P < 0.05, **P < 0.01, #P < 0.0001. Symbols above bars represent significant differences compared to the respective control group.
Citation: Journal of Molecular Endocrinology 67, 4; 10.1530/JME-21-0109
Kidney
A biphasic response to T4 withdrawal – with only minor age impact – was also noted in the kidneys. In young mice, Dio1 transcript levels increased until 72 h and then normalized to levels of controls, while Dio1 transcript levels remained suppressed in aged mice at the end of the study (Fig. 3A and B). No age differences were noted in the biphasic expression kinetics of TH-dependent 1-alpha hydroxylase (Cyp27b1). Mct8 and Mct10 expression (Fig. 3C, D, E, F, G and H) was not affected by T4 withdrawal.
Serial analysis of putative TH target gene expression in kidneys during recovery from T4 overtreatment in young and old mice compared to age-matched PBS-treated controls (ctrl). Transcript levels of Dio1 (A and B), Cyp27b1 (C and D), Mct8 (E and F), and Mct10 (G and H) were investigated by quantitative real-time PCR. Dio1 expression increased until 72 h and subsequently normalized in young mice, while Dio1 expression in kidneys of old mice was suppressed at the end of the study. Similar kinetics was found for Cyp27b1, while Mct8 and Mct10 expression was not affected. Boxplots, min and max with individual values, two-way ANOVA and Dunnett’s post-hoc analysis, *P < 0.05, **P < 0.01, #P < 0.0001. Symbols above bars represent significant differences compared to the respective control group.
Citation: Journal of Molecular Endocrinology 67, 4; 10.1530/JME-21-0109
Serial analysis of putative TH target gene expression in kidneys during recovery from T4 overtreatment in young and old mice compared to age-matched PBS-treated controls (ctrl). Transcript levels of Dio1 (A and B), Cyp27b1 (C and D), Mct8 (E and F), and Mct10 (G and H) were investigated by quantitative real-time PCR. Dio1 expression increased until 72 h and subsequently normalized in young mice, while Dio1 expression in kidneys of old mice was suppressed at the end of the study. Similar kinetics was found for Cyp27b1, while Mct8 and Mct10 expression was not affected. Boxplots, min and max with individual values, two-way ANOVA and Dunnett’s post-hoc analysis, *P < 0.05, **P < 0.01, #P < 0.0001. Symbols above bars represent significant differences compared to the respective control group.
Citation: Journal of Molecular Endocrinology 67, 4; 10.1530/JME-21-0109
Serial analysis of putative TH target gene expression in kidneys during recovery from T4 overtreatment in young and old mice compared to age-matched PBS-treated controls (ctrl). Transcript levels of Dio1 (A and B), Cyp27b1 (C and D), Mct8 (E and F), and Mct10 (G and H) were investigated by quantitative real-time PCR. Dio1 expression increased until 72 h and subsequently normalized in young mice, while Dio1 expression in kidneys of old mice was suppressed at the end of the study. Similar kinetics was found for Cyp27b1, while Mct8 and Mct10 expression was not affected. Boxplots, min and max with individual values, two-way ANOVA and Dunnett’s post-hoc analysis, *P < 0.05, **P < 0.01, #P < 0.0001. Symbols above bars represent significant differences compared to the respective control group.
Citation: Journal of Molecular Endocrinology 67, 4; 10.1530/JME-21-0109
Liver
Kinetics of all four investigated TH target genes Dio1, Tbg, Trhde, and Mct8 showed a biphasic organ response to T4 withdrawal with a thyrotoxic liver state until 72 h, followed by a hypothyroid state in both young and aged mice (Fig. 4A, B, C, D, E, F, G and H). At the end of the study, expression levels of Dio1 and Trhde mice had not completely normalized to controls suggesting an incomplete recovery of TH action in the liver. Thereby changes in Dio1 expression were more pronounced in the livers of aged mice (Fig. 4A and B), while changes in Trhde expression were more pronounced in young mice (Fig. 4E and F).
Serial analysis of TH responsive gene expression in the liver during recovery from T4 overtreatment of young and old mice compared to age-matched PBS-treated controls (ctrl). Transcript levels of Dio1 (A and B), Tbg (C and D), Trhde (E and F), and Mct8 (G and H) were analyzed by quantitative real-time PCR at indicated time points. At the end of the study, hepatic Dio1 expression had not recovered to control levels in young and was even more pronounced in aged mice. Boxplots, min and max with individual values, two-way ANOVA and Dunnett’s post-hoc analysis, *P < 0.05, **P < 0.01, #P < 0.0001. Symbols above bars represent significant differences compared to the respective control group.
Citation: Journal of Molecular Endocrinology 67, 4; 10.1530/JME-21-0109
Serial analysis of TH responsive gene expression in the liver during recovery from T4 overtreatment of young and old mice compared to age-matched PBS-treated controls (ctrl). Transcript levels of Dio1 (A and B), Tbg (C and D), Trhde (E and F), and Mct8 (G and H) were analyzed by quantitative real-time PCR at indicated time points. At the end of the study, hepatic Dio1 expression had not recovered to control levels in young and was even more pronounced in aged mice. Boxplots, min and max with individual values, two-way ANOVA and Dunnett’s post-hoc analysis, *P < 0.05, **P < 0.01, #P < 0.0001. Symbols above bars represent significant differences compared to the respective control group.
Citation: Journal of Molecular Endocrinology 67, 4; 10.1530/JME-21-0109
Serial analysis of TH responsive gene expression in the liver during recovery from T4 overtreatment of young and old mice compared to age-matched PBS-treated controls (ctrl). Transcript levels of Dio1 (A and B), Tbg (C and D), Trhde (E and F), and Mct8 (G and H) were analyzed by quantitative real-time PCR at indicated time points. At the end of the study, hepatic Dio1 expression had not recovered to control levels in young and was even more pronounced in aged mice. Boxplots, min and max with individual values, two-way ANOVA and Dunnett’s post-hoc analysis, *P < 0.05, **P < 0.01, #P < 0.0001. Symbols above bars represent significant differences compared to the respective control group.
Citation: Journal of Molecular Endocrinology 67, 4; 10.1530/JME-21-0109
Impact of T4 withdrawal on renal and hepatic DIO1 activity
Renal and hepatic DIO1 activities not only determine organ-specific TH concentrations but may also contribute to circulating TH concentrations under thyrotoxic conditions (van der Spek et al. 2017). In our study, renal DIO1 activity was increased 24 h after T4 withdrawal in young mice (Fig. 5A), whereas in old animals, DIO1 activity was found to be elevated from 12 to 144 h compared to controls (Fig. 5B). At 216 h and thereafter, no difference in renal DIO1 activity was found between T4-treated mice and age-matched controls. A similar response pattern to T4 withdrawal was observed for liver DIO1 activity in both age groups. However, at the end of the study, hepatic DIO1 activity in T4-treated mice had not normalized but decreased below the level of age-matched controls (Fig. 5C and D).
Serial analysis of renal (A and B) and hepatic (C and D) deiodinase type 1 activities during recovery from T4 overtreatment in kidney and liver of young and old mice compared to age-matched PBS-treated controls (ctrl). Prolonged elevation of renal DIO1 activity immediately after T4 withdrawal and up to 72 h was found in aged animals. No age differences in DIO1 activity were noted in livers, however, at the end of the study, DIO1 activities were reduced in the liver in all T4-treated animals compared to age-matched controls. Boxplots, min and max with individual values, one-way ANOVA and Dunnet’s post-hoc analysis, **P < 0.01, ***P < 0.001, #P < 0.0001. Symbols above bars represent significant differences compared to the respective control group.
Citation: Journal of Molecular Endocrinology 67, 4; 10.1530/JME-21-0109
Serial analysis of renal (A and B) and hepatic (C and D) deiodinase type 1 activities during recovery from T4 overtreatment in kidney and liver of young and old mice compared to age-matched PBS-treated controls (ctrl). Prolonged elevation of renal DIO1 activity immediately after T4 withdrawal and up to 72 h was found in aged animals. No age differences in DIO1 activity were noted in livers, however, at the end of the study, DIO1 activities were reduced in the liver in all T4-treated animals compared to age-matched controls. Boxplots, min and max with individual values, one-way ANOVA and Dunnet’s post-hoc analysis, **P < 0.01, ***P < 0.001, #P < 0.0001. Symbols above bars represent significant differences compared to the respective control group.
Citation: Journal of Molecular Endocrinology 67, 4; 10.1530/JME-21-0109
Serial analysis of renal (A and B) and hepatic (C and D) deiodinase type 1 activities during recovery from T4 overtreatment in kidney and liver of young and old mice compared to age-matched PBS-treated controls (ctrl). Prolonged elevation of renal DIO1 activity immediately after T4 withdrawal and up to 72 h was found in aged animals. No age differences in DIO1 activity were noted in livers, however, at the end of the study, DIO1 activities were reduced in the liver in all T4-treated animals compared to age-matched controls. Boxplots, min and max with individual values, one-way ANOVA and Dunnet’s post-hoc analysis, **P < 0.01, ***P < 0.001, #P < 0.0001. Symbols above bars represent significant differences compared to the respective control group.
Citation: Journal of Molecular Endocrinology 67, 4; 10.1530/JME-21-0109
Changes in serum lipid profile after T4 withdrawal
T4 withdrawal after chronic T4 treatment had a reciprocal impact on serum lipid profiles in aged and young mice (Fig. 6A). At 12 h after the last T4 injection, serum cholesterol concentrations were significantly higher in aged compared to young mice. By 72 h, cholesterol concentrations had normalized to controls in both T4 treatment groups followed by a steep increase in serum cholesterol concentrations reaching 180% in young and 130% in aged animals, compared to age-matched controls at 144 h. At the end of the study, serum cholesterol concentrations were below controls in aged and above control levels in young T4-treated mice.
Serial analysis of lipid profiles in young and old mice compared to age-matched PBS-treated controls (ctrl) after T4 withdrawal. Cholesterol (A) and triglycerides (Tg; B) serum concentrations were determined. Immediately after T4 withdrawal, elevated cholesterol concentrations were found in old mice while lower cholesterol levels were noted at the end of the study. A mirror image pattern of changes in cholesterol concentrations occurred in young mice. Lower Tg concentrations were found in aged mice throughout the entire study period. Data are shown as boxplots, min and max with individual values, two-way ANOVA and Tukey’s post-hoc analysis, *P < 0.05, **P < 0.01, ***P < 0.001. Symbols above bars represent significant differences compared to the respective control group, and symbols above half-tick down lines represent age differences.
Citation: Journal of Molecular Endocrinology 67, 4; 10.1530/JME-21-0109
Serial analysis of lipid profiles in young and old mice compared to age-matched PBS-treated controls (ctrl) after T4 withdrawal. Cholesterol (A) and triglycerides (Tg; B) serum concentrations were determined. Immediately after T4 withdrawal, elevated cholesterol concentrations were found in old mice while lower cholesterol levels were noted at the end of the study. A mirror image pattern of changes in cholesterol concentrations occurred in young mice. Lower Tg concentrations were found in aged mice throughout the entire study period. Data are shown as boxplots, min and max with individual values, two-way ANOVA and Tukey’s post-hoc analysis, *P < 0.05, **P < 0.01, ***P < 0.001. Symbols above bars represent significant differences compared to the respective control group, and symbols above half-tick down lines represent age differences.
Citation: Journal of Molecular Endocrinology 67, 4; 10.1530/JME-21-0109
Serial analysis of lipid profiles in young and old mice compared to age-matched PBS-treated controls (ctrl) after T4 withdrawal. Cholesterol (A) and triglycerides (Tg; B) serum concentrations were determined. Immediately after T4 withdrawal, elevated cholesterol concentrations were found in old mice while lower cholesterol levels were noted at the end of the study. A mirror image pattern of changes in cholesterol concentrations occurred in young mice. Lower Tg concentrations were found in aged mice throughout the entire study period. Data are shown as boxplots, min and max with individual values, two-way ANOVA and Tukey’s post-hoc analysis, *P < 0.05, **P < 0.01, ***P < 0.001. Symbols above bars represent significant differences compared to the respective control group, and symbols above half-tick down lines represent age differences.
Citation: Journal of Molecular Endocrinology 67, 4; 10.1530/JME-21-0109
For kinetics of serum triglycerides, a mirror-image pattern was observed for aged and young T4-treated mice with significantly lower triglyceride concentrations in aged compared to young mice at the end of the study (Fig. 6B).
Expression analysis of genes involved in hepatic lipid and cholesterol metabolism showed a biphasic response for Scd1, while Ldlr and Srebp2 transcript levels were not altered after T4 withdrawal in the livers of young and aged mice (Supplementary Fig. 1).
Discussion
Various clinical studies have raised the awareness for age-specific reference intervals for TH and TSH serum concentrations (Waring et al. 2012, Vadiveloo et al. 2013, Stott et al. 2017, Duntas 2018, Barbesino 2019). Even more importantly, accumulating reports of adverse effects of T4 overtreatment in older patients with subclinical or overt hypothyroidism justify the need for further studies on how correction of T4 overtreatment might affect an aged organism. To address how TH excess and its withdrawal impact on TH action systemically and organ-specifically in the short- and medium-term effects, we subjected young and aged mice to 5 weeks of chronic T4 treatment and followed them after stopping treatment over 12 days.
Prolonged TH excess is followed by systemic hypothyroidism in aged mice after TH overtreatment
Serial analysis of thyroid function parameters after cessation of T4 treatment revealed two important findings with respect to ageing. First, in the early phase of recovery from TH excess, higher TH serum concentrations in particular for FT3 were obvious in 22 months old mice and hence could portend prolonged risk of TH excess to an aged organism. Secondly, at the end of the study, markedly elevated TSH concentrations were found in aged but not young mice despite normalization of serum TH concentrations within 3 days after stopping treatment in both age cohorts, similar to observations in patients after T4 withdrawal (Carlwe et al. 2013).
Ageing has been shown to afflict the hypothalamic–pituitary–thyroid (HPT) axis on several levels (Donda et al. 1987, 1989, Reymond et al. 1992) for example, in TSH secretion, TSH bioactivity, or reduced TSH receptor expression in the aged thyroid gland (Persani 1998). Thyroids of old rodents have different structure and function and do not respond to TSH as sensitive as those of young mice (Studer et al. 1978, Tamura & Fujita 1981, Reymond et al. 1992). In chronically hypothyroid mice, we have previously confirmed lower TSH bioactivity in aged compared to young animals (Engels et al. 2019), using a cell-based RIA (Pohlenz et al. 1999). As TH serum concentrations were comparable for both age groups after 72 h of T4 withdrawal, the sustained higher TSH concentrations could, in principle, result from reduced TSH bioactivity in older animals. In fact, TSH reflects local HPT state, and kinetics of readjustment of TH status results in peripheral changes of gene expression in different target organs due to the local role of deiodinases at this site of the feedback axis. However, by comparison with age-matched controls and supported by changes in organ TH action discussed below, we suggest that the observed age difference in thyroid function parameters represents systemic hypothyroidism due to prolonged recovery time of the HPT from chronic TH excess in an aged organism.
Increased renal DIO1 activity contributes to prolonged TH excess in aged mice after TH overtreatment
Previously we reported an age-dependent delay in hepatic DIO1 activity response upon single T4 injection associated with lower T3 serum and tissue concentrations (Rakov et al. 2019). In the present study, DIO1 activity in the liver was not different between young and aged mice under T4 withdrawal, hence a compensatory effect due to chronic TH excess is likely. DIO1 activity in the liver and kidney has been described to contribute to a minor extent to circulating T3 concentrations under euthyroid conditions in mice (Streckfuss et al. 2005, Schneider et al. 2006, van der Spek et al. 2017). However, under chronic hyperthyroidism, DIO1 may exert a counteracting function and may limit the availability of circulating T3 as shown in a human study (Abuid & Larsen 1974) and in a fish model (Van der Geyten et al. 2005). In our study, DIO1 activities in the liver resembled hepatic Dio1 mRNA expression. Interestingly, DIO1 knockout mice showed increased fecal but reduced urinary TH clearance upon TH excess, suggesting a protective function of renal DIO1 in states of high TH availability (Streckfuss et al. 2005, Schneider et al. 2006). In the present study, renal DIO1 activity was much higher in aged compared to young mice, suggesting increased TH metabolism in kidneys of aged mice, with – at least in aged animals – a possible renal contribution to higher FT3 serum concentrations after TH overtreatment. Thus, we hypothesize that renal DIO1 activity in young mice may be switched on/off faster, while in old mice, DIO1 response to changes in TH serum concentrations may be slower. However, one limitation of our study is the lack of data on rT3 serum concentrations, which might have helped to explain the TH alterations, as rT3 might be increased under T4 treatment and rapidly disappears by DIO1 activity. In addition, the role of DIO3 during T4 excess and recovery could not be solved and might have also influenced organ-specific recovery similarly to its impact during acute or chronic illness (Boelen et al. 2011).
TH action in target organs remains compromised after TH overtreatment despite normalization of TH serum concentrations
Although TT4, FT4, and FT3 serum concentrations had normalized within 72 h after T4 withdrawal, prolonged effects on cardiac, renal, and hepatic organ TH action were observed. On the one hand, the heart was chosen because of the link between thyroid dysfunction and increased cardiac morbidity especially in aged patients (Asvold et al. 2015, Pearce et al. 2016, Jabbar et al. 2017). On the other hand, the liver and kidney play an important role in TH metabolism and fine-tuning of systemic TH serum concentrations in different conditions and ages (Streckfuss et al. 2005, Schomburg et al. 2007, Visser et al. 2016, Rakov et al. 2019, Kerp et al. 2020).
In our study and based on TH target gene expression, a biphasic response was found in the heart, kidney, and liver and was characterized by a thyrotoxic tissue state until 72 h after TH withdrawal followed by a subsequent hypothyroid state with incomplete normalization of organ TH action at the end of the study. Particularly in the heart, a prolonged hypothyroid organ state was obvious. Thereby Myh6 expression changes were more pronounced in aged, while Myh7 expression changes were more pronounced in young mice at 12 days. This is an interesting finding, as both genes are directly transcriptionally regulated by TH (Razvi et al. 2018), but particularly Myh6 expression change does not correlate to serum T3 concentrations. A possible biological relevance of this finding has been suggested in a previous study, where bradycardia and altered cardiac gene expression occurred 14 days after T4 withdrawal in T4-treated young mice (Hoefig et al. 2016). Whether this effect is due to a direct cardiac or an indirect effect of TH on the autonomous system (Herrmann et al. 2020) remains to be clarified. Functional consequences of altered Myh6 vs Myh7 expression in hearts of aged vs young mice remain to be determined and could reflect age-dependent differences in responses to, for example, myosin heavy-chain-associated lncRNA transcript (Mhrt), which has been previously described in this context (Han et al. 2014, Forini et al. 2020). However, the normalization of Myh6 expression in young mice after repression at 144 h and 216 h indicates that although serum T3 concentration is within the reference range, cardiac TH metabolism is under local control, not assessable by serum measurements.
A similar trend as in the heart was found for renal TH target gene expression, suggesting a hypothyroid kidney state 12 days after T4 withdrawal with marked Dio1 suppression in aged mice. Furthermore, in the liver, expression levels of Dio1 and Trhde still indicated a hypothyroid liver state at 12 days, that is, the end of the study. These findings are in concordance with a previous study by Ohba et al. showing incomplete hepatic recovery from chronic T3 treatment in young mice at 10 days of TH withdrawal (Ohba et al. 2016). In their study, expression of approximately 10% of investigated hepatic TH target genes had not returned to control levels and the authors suggested that these sustained changes might contribute to the persistence of metabolic and physiological abnormalities observed clinically in some patients despite normalized TH serum concentrations (Ohba et al. 2016).
Adaption to TH overtreatment involves age-specific changes in lipid profiles
TH impact mobilization, degradation of lipids, and de novo synthesis of fatty acids in the liver (Damiano et al. 2017, Sinha et al. 2019). Furthermore, TH increase bile acid flow, resulting in depletion of hepatic cholesterol and enhancement of cholesterol uptake from the circulation to the liver (Duntas & Brenta 2018). As expected, decreased cholesterol serum concentrations were found upon chronic T4 treatment in young mice but this did not apply to aged mice. Interestingly, after normalization to control level, a drastic transient increase in serum cholesterol occurred in young mice, while the adaptive changes were much subtle in aged mice. Furthermore, a mirror-image pattern was found for kinetics in triglyceride serum concentrations after T4 withdrawal showing normalization in young but not in aged mice, which exhibited significantly lower triglyceride concentrations than young animals at the end of the study. These observed changes in lipid profiles underpin both an age impact and a prolonged recovery of organ TH action from TH overtreatment.
In summary, recovery from TH overtreatment involves adaptation in TH metabolism and TH action with some age differences on systemic and organ level and incomplete normalization of the heart, kidney, and hepatic thyroid hormone status over 12 days in the mouse organism. Thus, although TH serum concentrations normalize fast, once the experimental source of T4 excess is eliminated, TH serum status does not reflect euthyroidism. These findings may have a significant impact on hyperthyroid patients treated with antithyroid drugs, as these patients observe excessive short-term weight gain despite normal serum thyroid function tests and may also affect longer-term susceptibility to other health conditions.
Shortcomings of our study are the restriction to 12 days of observation and the lack of functional correlates of TH action in the heart and kidney, while for the liver, the identified changes in serum lipid profiles are in line with incomplete recovery from TH overtreatment in the aged liver. To our best knowledge, this is the first serial analysis of an age impact on systemic and organ response to TH overtreatment.
Our findings raise awareness that side effects of T4 overtreatment extend beyond the time points of elimination of TH excess and normalization of serum TH concentrations, aspects which should be addressed in future studies.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/JME-21-0109.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was funded by DFG FU 356/7-2 to D F and MO 1018/2-2 to L C M in the framework of SPP 1629 THYROID TRANS ACT and in projects P10 and P18 as part of SFB 296 LOCOTACT.
Acknvowledgements
The authors are grateful to K Engels, E Werbenko, A Jaeger, K Strumann, and S Kwapik for their dedicated technical support. Furthermore, the authors are thankful to Prof Dr. S Brandau and K Bruderek (Department of Otorhinolaryngology, University Hospital Essen) for providing the Luminex 200 system for TSH measurement.
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