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 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. 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

DataSheet_1_Lipocalin 2 – mutation screen and serum levels in patients with anorexia nervosa or obesity and in lean individuals.xlsx

ContextThe bone-derived adipokine lipocalin-2 is relevant for body weight regulation by stimulating the leptin-melanocortin pathway.ObjectiveWe aimed to (i) detect variants in the lipocalin-2 gene (LCN2) which are relevant for body weight regulation and/or anorexia nervosa (AN); (ii) describe and characterize the impact of LCN2 and MC4R variants on circulating lipocalin-2 level.MethodsSanger sequencing of the coding region of LCN2 in 284 children and adolescents with severe obesity or 287 patients with anorexia nervosa. In-silico analyses to evaluate functional implications of detected LCN2 variants. TaqMan assays for rare non-synonymous variants (NSVs) in additional independent study groups. Serum levels of lipocalin-2 were measured by ELISA in 35 females with NSVs in either LCN2 or MC4R, and 33 matched controls without NSVs in the two genes.ResultsFourteen LCN2-variants (five NSVs) were detected. LCN2-p.Leu6Pro and p.Gly9Val located in the highly conserved signal peptide region may induce functional consequences. The secondary structure change of lipocalin-2 due to LCN2-p.Val89Ile may decrease solubility and results in a low lipocalin-2 level in a heterozygotes carrier (female recovered from AN). Lean individuals had lower lipocalin-2 levels compared to patients with obesity (p = 0.033).ConclusionLipocalin-2 levels are positively associated with body mass index (BMI). Single LCN2-variants might have a profound effect on lipocalin-2 levels.</p

Image_1_Lipocalin 2 – mutation screen and serum levels in patients with anorexia nervosa or obesity and in lean individuals.pdf

ContextThe bone-derived adipokine lipocalin-2 is relevant for body weight regulation by stimulating the leptin-melanocortin pathway.ObjectiveWe aimed to (i) detect variants in the lipocalin-2 gene (LCN2) which are relevant for body weight regulation and/or anorexia nervosa (AN); (ii) describe and characterize the impact of LCN2 and MC4R variants on circulating lipocalin-2 level.MethodsSanger sequencing of the coding region of LCN2 in 284 children and adolescents with severe obesity or 287 patients with anorexia nervosa. In-silico analyses to evaluate functional implications of detected LCN2 variants. TaqMan assays for rare non-synonymous variants (NSVs) in additional independent study groups. Serum levels of lipocalin-2 were measured by ELISA in 35 females with NSVs in either LCN2 or MC4R, and 33 matched controls without NSVs in the two genes.ResultsFourteen LCN2-variants (five NSVs) were detected. LCN2-p.Leu6Pro and p.Gly9Val located in the highly conserved signal peptide region may induce functional consequences. The secondary structure change of lipocalin-2 due to LCN2-p.Val89Ile may decrease solubility and results in a low lipocalin-2 level in a heterozygotes carrier (female recovered from AN). Lean individuals had lower lipocalin-2 levels compared to patients with obesity (p = 0.033).ConclusionLipocalin-2 levels are positively associated with body mass index (BMI). Single LCN2-variants might have a profound effect on lipocalin-2 levels.</p

DataSheet_2_Lipocalin 2 – mutation screen and serum levels in patients with anorexia nervosa or obesity and in lean individuals.docx

ContextThe bone-derived adipokine lipocalin-2 is relevant for body weight regulation by stimulating the leptin-melanocortin pathway.ObjectiveWe aimed to (i) detect variants in the lipocalin-2 gene (LCN2) which are relevant for body weight regulation and/or anorexia nervosa (AN); (ii) describe and characterize the impact of LCN2 and MC4R variants on circulating lipocalin-2 level.MethodsSanger sequencing of the coding region of LCN2 in 284 children and adolescents with severe obesity or 287 patients with anorexia nervosa. In-silico analyses to evaluate functional implications of detected LCN2 variants. TaqMan assays for rare non-synonymous variants (NSVs) in additional independent study groups. Serum levels of lipocalin-2 were measured by ELISA in 35 females with NSVs in either LCN2 or MC4R, and 33 matched controls without NSVs in the two genes.ResultsFourteen LCN2-variants (five NSVs) were detected. LCN2-p.Leu6Pro and p.Gly9Val located in the highly conserved signal peptide region may induce functional consequences. The secondary structure change of lipocalin-2 due to LCN2-p.Val89Ile may decrease solubility and results in a low lipocalin-2 level in a heterozygotes carrier (female recovered from AN). Lean individuals had lower lipocalin-2 levels compared to patients with obesity (p = 0.033).ConclusionLipocalin-2 levels are positively associated with body mass index (BMI). Single LCN2-variants might have a profound effect on lipocalin-2 levels.</p