Phänotypische Charakterisierung von geschlechtsabhängigen Merkmalen einer Hyperthyreose und Hypothyreose im Mausmodell

Schon lange ist eine erhöhte Prävalenz von Frauen für Schilddrüsen- (SD) Funktionsstörungen wie einer Über- oder Unterfunktion bekannt. Allerdings sind die Auswirkungen einer SD-Fehlfunktion im menschlichen Organismus kaum geschlechtsspezifisch charakterisiert. Zudem steigt die Rate an SD-Funktionsstörungen mit dem Alter an und auch hierbei ist bislang nur unzureichend bekannt, ob eine Über- oder Unterfunktion bei älteren Frauen im Vergleich zu Männern ähnliche Konsequenzen hat im Hinblick auf z.B. das kardiovaskuläre System, den Energiestoffwechsel oder das Verhalten. Um diese klinischen Fragestellungen in einem in vivo Mausmodell nachzustellen, wurden zunächst Protokolle für eine chronische Überfunktion in männlichen und weiblichen Tieren etabliert. Im Anschluss erfolgte eine Charakterisierung von geschlechtsspezifischen Eigenschaften einer Über- und Unterfunktion in verschiedenen Lebensabschnitten bei jungen, erwachsenen und alten Mäusen sowie Analysen zur organspezifischen SD-Hormon (SDH) Wirkung. Im Vergleich der jungen Tiere zeigte sich, dass das Geschlecht eine wichtige Rolle bei SDH-abhängigen Veränderungen des Körpergewichts, der Körpertemperatur, der Futter- und Wasseraufnahme, den SDH-Serumkonzentrationen und neuro-muskulären Eigenschaften spielt. Im Vergleich von erwachsenen und älteren Tieren wurde sowohl eine gleichbleibende (für Körpertemperatur, SDH-Serum-konzentrationen, Aktivität) als auch verstärkte (für Körpergewicht, Muskelfunktion) geschlechtsabhängige SDH-Wirkung beobachtet. Interessanterweise ließen sich in allen drei Altersgruppen keine Unterschiede zwischen Männchen und Weibchen in der Herzfrequenz bei einer Über- und Unterfunktion beobachten. In der Analyse der organspezifischen SDH-Wirkung zeigte sich, dass die SDH-abhängige Genexpression im Herzen die geringsten geschlechtsspezifischen Unterschiede aufweist, während im braunen Fettgewebe und noch deutlicher in der Leber ausgeprägte geschlechts- und SDH-spezifische Transkriptänderungen auftraten. In weiteren Analysen konnte eine geschlechts- und SDH-abhängige Regulation von Claudin-1 in der Leber nachgewiesen werden, welche wahrscheinlich eine Relevanz für die Gallensäure Homeostase hat und einen möglichen Pathomechanismus für geschlechtsabhängige Cholelithiasis bei SD-Funktionsstörungen darstellen könnte. Zusammenfassend sind dies die ersten Arbeiten, welche geschlechtsspezifische Aspekte einer SD-Über- oder Unterfunktion in verschiedenen Lebensaltern im Mausmodell abbilden. Diese Ergebnisse dienen als Grundlage, um die molekularen Mechanismen auf organischer und Zellebene zu verstehen. Dies kann helfen, translational zukünftig am Menschen Komorbiditäten einhergehend mir einer SD-Fehlfunktion bei Männern und Frauen besser vorzubeugen und zu therapieren.It is well known, that the clinical situation of thyroid dysfunctions (TDs), hyper- and hypothyroidism have an increased preponderance in women than men. In contrast, a sex-specific outcome of hyper- and hypothyroidism has not been characterized yet. Moreover, the prevalence of TDs increase with age, with unknown sex-specific consequences for the cardiovascular system, energy metabolism and behaviour. To mimic the clinical situation, first an in vivo mouse model for chronic hyperthyroidism in male and female mice was established, followed by induction of chronic hyper- and hypothyroidism of both sexes in young, adult and old aged mice. This allowed a comprehensive characterization of the interplay between sex and age on phenotypical traits of TD at different life stages and enabled the analysis of organ-specific thyroid hormone (TH) effects. Metabolic parameters of body weight change, food and water intake, TH serum concentrations, heart rate, body temperature, muscle function and activity were assessed. In young mice a distinct sex impact on TH-dependent alterations was identified for body weight, body temperature, nutrient intake, TH serum concentrations and neuromuscular features. In comparison to adult and old age groups sex-effects were persistent (for body temperature, TH serum concentrations, activity) and exaggerated (for body weight and muscle function), whereas no TH-dependent differences between male and female mice were noted for heart rate at any age. Molecular investigations showed less sex influence on TH-dependent gene expression in heart, whereas in brown adipose tissue and even more significant alterations in liver were detected. Further analysis of a sex and TH-dependent regulation of Claudin-1 in livers of mice indicated relevance in an altered biliary homeostasis and a possible pathophysiological role for TH-dependent change of sex-dependency in cholelithiasis. To conclude, these studies allow a comprehensive analysis of sex influence on phenotypical and molecular traits of hyper- and hypothyroidism at different life stages in mice for the first time and enable the subsequent analysis of the underlying mechanisms in an organ and cell-specific manner. This will help to improve our understanding of possible sex impact on complications of TD in women and men and implicate a sex-specific prevention and treatment of patients

Differences in Mouse Hepatic Thyroid Hormone Transporter Expression with Age and Hyperthyroidism

Background: Clinical features of thyroid dysfunction vary with age, and an oligosymptomatic presentation of hyperthyroidism is frequently observed in the elderly. This suggests age modulation of thyroid hormone (TH) action, which may occur, for example, by alterations in TH production, metabolism and/or TH action in target organs. Objectives: In this paper, we address possible changes in TH transporter expression in liver tissues as a mechanism of age-dependent variation in TH action. Methods: Chronic hyperthyroidism was induced in 4- and 20-month-old C57BL6/NTac male mice (n = 8-10) by intraperitoneal injections of 1 µg/g body weight L-thyroxine (T4) every 48 h over 7 weeks. Control animals were injected with PBS. Total RNA was isolated from liver samples for analysis of the TH transporter and TH-responsive gene expression. TH concentrations were determined in mice sera. Results: Baseline serum free T4 (fT4) concentrations were significantly higher in euthyroid young compared to old mice. T4 treatment increased total T4, fT4 and free triiodothyronine to comparable concentrations in young and old mice. In the euthyroid state, TH transporter expression was significantly higher in old than in young mice, except for Mct8 and Oatp1a1 expression levels. Hyperthyroidism resulted in upregulation of Mct10, Lat1 and Lat2 in liver tissue, while Oatp1a1, Oatp1b2 and Oatp1a4 expression was downregulated. This effect was preserved in old animals. Conclusion: Here, we show age-dependent differences in TH transporter mRNA expression in the euthyroid and hyperthyroid state of mice focusing on the liver as a classical TH target organ

Sex-specific phenotypes of hyperthyroidism and hypothyroidism in mice

Background Thyroid dysfunction is more common in the female population, however, the impact of sex on disease characteristics has rarely been addressed. Using a murine model, we asked whether sex has an influence on phenotypes, thyroid hormone status, and thyroid hormone tissue response in hyper- and hypothyroidism. Methods Hypo- and hyperthyroidism were induced in 5 -month-old female and male wildtype C57BL/6N mice, by LoI/MMI/ClO4 − or T4 i.p. treatment over 7 weeks, and control animals underwent sham treatment (N = 8 animals/sex/treatment). Animals were investigated for impact of sex on body weight, food and water intake, body temperature, heart rate, behaviour (locomotor activity, motor coordination, and strength), liver function, serum thyroid hormone status, and cellular TH effects on gene expression in brown adipose tissue, heart, and liver. Results Male and female mice showed significant differences in behavioural, functional, metabolic, biochemical, and molecular traits of hyper- and hypothyroidism. Hyperthyroidism resulted in increased locomotor activity in female mice but decreased muscle strength and motor coordination preferably in male animals. Hypothyroidism led to increased water intake in male but not female mice and significantly higher serum cholesterol in male mice. Natural sex differences in body temperature, body weight gain, food and water intake were preserved under hyperthyroid conditions. In contrast, natural sex differences in heart rate disappeared with TH excess and deprivation. The variations of hyper- or hypothyroid traits of male and female mice were not explained by classical T3/T4 serum state. TH serum concentrations were significantly increased in female mice under hyperthyroidism, but no sex differences were found under eu- or hypothyroid conditions. Interestingly, analysis of expression of TH target genes and TH transporters revealed little sex dependency in heart, while sex differences in target genes were present in liver and brown adipose tissue in line with altered functional and metabolic traits of hyper- and hypothyroidism. Conclusions These data demonstrate that the phenotypes of hypo- and hyperthyroidism differ between male and female mice and indicate that sex is an important modifier of phenotypic manifestations

Sex-specific phenotypes of hyperthyroidism and hypothyroidism in aged mice

Abstract Background Sex and age play a role in the prevalence of thyroid dysfunction (TD), but their interrelationship for manifestation of hyper- and hypothyroidism is still not well understood. Using a murine model, we asked whether sex impacts the phenotypes of hyper- and hypothyroidism at two life stages. Methods Hyper- and hypothyroidism were induced by i.p. T4 or MMI/ClO4-/LoI treatment over 7 weeks in 12- and 20-months-old female and male C57BL/6N mice. Control animals underwent PBS treatment (n = 7–11 animals/sex/treatment). Animals were investigated for impact of sex on body weight, food and water intake, body temperature, heart rate, behaviour (locomotor activity, motor coordination and strength) and serum thyroid hormone (TH) status. Results Distinct sex impact was found in eu- and hyperthyroid mice, while phenotypic traits of hypothyroidism were similar in male and female mice. No sex difference was found in TH status of euthyroid mice; however, T4 treatment resulted in twofold higher TT4, FT4 and FT3 serum concentrations in adult and old females compared to male animals. Hyperthyroid females consistently showed higher locomotor activity and better coordination but more impairment of muscle function by TH excess at adult age. Importantly and in contrast to male mice, adult and old hyperthyroid female mice showed increased body weight. Higher body temperature in female mice was confirmed in all age groups. No sex impact was found on heart rate irrespective of TH status in adult and old mice. Conclusions By comparison of male and female mice with TD at two life stages, we found that sex modulates TH action in an organ- and function-specific manner. Sex differences were more pronounced under hyperthyroid conditions. Importantly, sex-specific differences in features of TD in adult and old mice were not conclusively explained by serum TH status in mice

Additional file 3: Figure S2. of Sex-specific phenotypes of hyperthyroidism and hypothyroidism in aged mice

Water consumption in adult and old groups of male and female mice, under control, TH excess or deprivation. Water intake was related to BW weekly in (A-C) adult and (D-F) old male and female mice under euthyroid condition, T4, or MMI/ClO4−/LoI treatment. Sex difference was observed in control groups (for adult: F(8,108) = 23.26 for time, F(1,108) = 936.2 for sex effect, F(8,108) = 5.017 for interaction, p < 0.001; for old: F(8,134) = 16.38 for time, F(1,134) = 219.4 for sex effect, F(8,134) = 1.788 for interaction, p = 0.0847), and was reversed under hyperthyroid adult (F(8,119) = 42.04 for time, F(1,119) = 0.1882 for sex effect, F(8,119) = 13.24 for interaction, p < 0.001) and hypothyroid adult and old age (for adult: F (8,126) = 15.27 for time, F (1,126) = 560.8 for sex effect, F (8,126) = 73.19 for interaction, p < 0.001; for old: F (8,156) = 4.373 for time, F (1,156) = 106.0 for sex effect, F (8,156) = 9.991 for interaction, p < 0.001). Data are presented as mean ± SD, n = 7–11 animals/sex/treatment, two-way ANOVA followed by Bonferroni post hoc analysis, *p < 0.05, **p < 0.01, ***p < 0.001. (TIFF 775 kb

Additional file 1: Table S1-S3. of Sex-specific phenotypes of hyperthyroidism and hypothyroidism in aged mice

Statistical analysis of TT4, FT4, FT3 and TSH serum measurements in adult and old mice of both sexes. Two-way ANOVA followed by Bonferroni post hoc analysis was applied. Sex dependency was obvious as shown for Δ(mean female-mean male) values. Table S2 Statistical analysis of body temperature measurements in adult and old mice of both sexes. Two-way ANOVA followed by Bonferroni post hoc analysis was applied for hyper- and hypothyroid conditions. Average mean values of body temperature are shown as Δ(female-male). Table S3 Sex differences for area under curve (AUC) analysis of repeated body weight, food and water intake measurements. Two-way ANOVA followed by Bonferroni post hoc analysis was applied to AUC (±SEM) values, calculated by GraphPad Prism 7. (DOCX 18 kb

Additional file 2: Figure S1. of Sex-specific phenotypes of hyperthyroidism and hypothyroidism in aged mice

Food intake behaviour during experimental procedure influenced by sex, age and TH condition. Food intake was related to BW weekly in (A-C) adult and (D-F) old male and female mice. Sex dependency was noted for euthyroid groups (for adult: F(8,108) = 24.92 for time, F(1,108) = 430.6 for sex effect, F(8,108) = 9.204 for interaction, p < 0.001; for old: F(8,134) = 15.75 for time, F(1,134) = 51.5 for sex effect, F(8,134) = 1.783 for interaction, p = 0.0857), which disappeared by TH excess and deprivation. Data are presented as mean ± SD, n = 7-11 animals/sex/treatment, 2-way ANOVA followed by Bonferroni post hoc analysis, *p < 0.05, **p < 0.01, ***p < 0.001. (TIFF 743 kb

Additional file 1: Table S1. of Sex-specific phenotypes of hyperthyroidism and hypothyroidism in mice

Oligonucleotides used for amplification of house-keeping genes, TH responsive genes, and TH transporters by real-time PCR. (DOCX 28 kb