ERα and lipolysis.

<p>A: Ex-vivo lipolysis assay in murine gonadal-AT explants from wild-type (WT) and estrogen receptor alpha knock out mice (KO) expressed as percent of WT FFA-release after stimulation with forskolin. Bonferroni posttest showed a significant difference between WT and KO in females [n = 4−5 mice/group, two-way ANOVA]. <b>B</b>: Analysis of ERα mRNA expression in gonadal-AT from female/male mice before/after weight reduction. Data are presented as <i>x</i>-fold of females (DIO) [n = 9−10 mice/group, two-way ANOVA]. The black/dark grey columns and symbols represent male mice; white/light grey: females. DIO: before weight reduction, –20%: after weight reduction. *p≤0.05 DIO vs. -20% or WT vs. KO.</p

Animal model.

<p>A: Scheme of the feeding protocol to induce body weight changes (DIO =  diet-induced obesity). <b>B</b>: Original BW data of female/male mice throughout the feeding protocol.</p

Weight maintenance and regain.

<p>A: Stability of body weight of female/male mice during 16 days of adaptive feeding. Shown are the means ±SEM of body weight, measured daily. [n = 10 mice/group] <b>B</b>: During adaptive feeding the amount of food was individually adapted to maintain the target weight over 16 days. Shown are the mean amount of given food ±SEM normalized to the BW of female/male mice [n = 10 mice/group, two-way ANOVA with repeated measures] (factor interaction: p_sex/time<0.001). <b>C</b>: Sex-specific differences during weight regain expressed as percent change of body weight before re-feeding. Shown is the BW-development in female and male mice during 6 weeks ad libitum re-feeding. [n = 10 mice/group, two-way ANOVA with repeated measures] (factor interaction: p_sex/time<0.05). Black symbols represent male mice; white: females. *p≤0.05; **p≤0.01; # p<0.001 vs. other sex.</p

Weight reduction phase.

<p>A: Loss of BW in female/male mice during restricted feeding, expressed as percent of DIO-BW. Body weight target: −20% of DIO-BW, for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037794#s2" target="_blank">method</a> section [n = 10 mice/group, two-way ANOVA with repeated measures] (factor interaction: p_sex/time<0.001). <b>B</b>: Change of lean-mass specific EE measured in female/male mice before weight loss and during caloric restriction. Shown is the mean over 23 h measurement [n = 10 mice/group, two-way ANOVA] (factor interaction: p_{sex/weight loss}<0.001). <b>C</b>: Total locomotor activity of female/male mice before weight loss and during CR. Shown is total activity during 23 h monitoring [n = 10 mice/group, two-way ANOVA]. <b>D</b>: Analysis of body composition (lean and fat mass) in female/male mice calculated as percent reduction (delta: DIO-mass and mass after weight reduction). Lean and fat mass were analyzed separately [n = 10 mice/group, unpaired <i>t</i>-test]. <b>E</b>: Gonadal fat mass as percent of total fat mass in female/male mice before/after weight reduction [n = 10 mice/group, two-way ANOVA] (factor interaction: p_{sex/weight loss}<0.05). <b>F</b>: Liver triglycerides measured in female/male mice before/after weight reduction [n = 8 mice/group, two-way ANOVA]. Black and dark grey columns/symbols represent male mice; white and light grey = female mice. DIO = diet-induced obesity or before weight reduction; restriction: during restrictive feeding phase; −20%: after weight reduction, at target weight. *p≤0.05; **p≤0.01; # p≤0.001 DIO vs. −20% or vs. other sex.</p

Lipolytic activity.

<p>A: Ex-vivo lipolysis assay in murine gonadal adipose tissue explants as a marker of fat-tissue specific lipolytic activity, measured as release of FFA after stimulation with forskolin. Shown is the release of FFA in female/male mice before/after weight reduction [n = 7 mice/group, two-way ANOVA] <b>B</b>: Serum concentration of free glycerol in female/male mice before/during weight loss (day 3 of CR) [n = 10 mice/group, two-way ANOVA] ] (factor interaction: p_{sex/weight loss}<0.05). <b>C</b>: Mean RER during day time (6 a.m.–6 p.m.) in females/males measured during weight loss [n = 10 mice/group, unpaired <i>t</i>-test]. <b>D</b>: Analysis of ATGL, HSL, and LPL mRNA expression in gonadal-AT from female/male mice after weight reduction. Data are presented as <i>x</i>-fold expression of females [n = 8−10 mice/group, unpaired <i>t</i>-test, genes were analyzed separately]. Black columns and symbols represent male mice; white: females. DIO: before weight reduction; restriction: during restrictive feeding phase; -20%: after weight reduction, at target weight. *p≤0.05; **p≤0.01 DIO vs. −20% or vs. other sex.</p

Weight gain phase.

<p>A: Body weight development of female/male mice showed a sex-specific difference in 15 weeks of HFD-feeding. Shown are the means ±SEM of body weight, measured weekly [n = 20 mice/group, two-way ANOVA with repeated measures] (factor interaction: p_sex/time<0.001). <b>B</b>: Relative weight gain in female/male mice after 15 weeks of HFD feeding expressed as percent change from initial weight [n = 10 mice/group, unpaired <i>t</i>-test]. <b>C</b>: Total locomotor activity of female/male mice in 23 h, measured in a metabolic cage system (TSE Systems) at week 4 of HFD feeding [n = 10 mice/group, unpaired <i>t</i>-test]. <b>D</b>: EE of female/male mice normalized to lean body mass, measured at week 4 of HFD feeding, expressed as means ±SEM per hour [n = 10 mice/group, two-way ANOVA with repeated measures] (factor interaction: p_sex/time<0.001). <b>E</b>: Food efficiency (weight gain[g]/[g]food intake) calculated over 15 weeks of HFD-feeding [n = 10 mice/group, two-way ANOVA with repeated measures] (factor interaction: p_sex/time<0.001). Black columns and symbols represent male mice, white =  female. *p≤0.05, **p≤0.01, # p≤0.001 vs. other sex.</p