Sex differences in sympathetic innervation and browning of white adipose tissue of mice

Fig. 1. CL treatment induces brown adipocyte maker UCP1 expression in gWAT female specifically. a, b Immunoblot (a) and quantitative PCR (b) analysis of UCP1 expression in gWAT of male and female mice treated with CL for 3 days and untreated control mice. Two-way ANOVA revealed significant main effects of sex in UCP1 expression (a: p = 0.012, b: p = 0.032) and significant interaction of sex and treatment (a: p = 0.012, b: p = 0.033). Significant differences between male and female were determined by post hoc pairwise comparison with Bonferroni correction (p = 0.012, mean ± SEM; n = 4, **p < 0.01). c Representative images of UCP1 immunostaining in paraffin sections of gWAT from male and female mice treated with CL for 3 days and untreated control mice. Nuclei were counterstained with DAPI. Size bar = 20 μm

Fig. 2. CL treatment induces expression of brown adipocyte markers in gWAT female specifically. a,b Immunoblot analysis of mitochondrial proteins involved in oxidative phosphorylation. Two-way ANOVA revealed significant main effects of sex in mitochondrial proteins (ATP5A: p = 0.013, UQCRC2: p = 0.034, NDUFB8: p = 0.004) and significant interaction of sex and treatment (NDUFB8: p = 0.0054). Significant differences between male and female were determined by post hoc pairwise comparison with Bonferroni correction (mean ± SEM; n = 6, *p < 0.05, **p < 0.01). c qPCR analysis of brown adipocyte markers and genes involved in mitochondrial FFA oxidation in gWAT of male and female mice treated with CL for 3 days and untreated control mice. Two-way ANOVA revealed significant main effects of sex in brown adipocyte markers (Ppargc1a: p = 0.042, Cox8b: p = 0.011, Dio2: p <0. 0001) and significant interaction of sex and treatment (Ppargc1a: p = 0.008, Cox8b: p = 0.013, Dio2: p = 0. 001). Significant differences between male and female were determined by post hoc pairwise comparison with Bonferroni correction (mean ± SEM; n = 4, **p < 0.01). d Mitochondrial respiration in gWAT of male and female mice…

Fig. 3. Induction of brown adipocyte phenotype in subcutaneous adipose tissue by CL treatment. a, b Immunoblot analysis of UCP1 protein expression and quantification. Mean ± SEM; n = 4, two-way ANOVA, interaction of sex and treatment (p = 0.9094) and effect of sex (p = 0.8949) were not significant. Significant main effect of CL treatment (p < 0.0001). c Mitochondrial respiration in brown adipose tissue and iWAT of male and female mice treated with CL for 3 days (mean ± SEM; n = 4) as determined by reduction of the electron acceptor dye TTC. d qPCR analysis of brown adipocyte markers and genes involved in mitochondrial FFA oxidation in iWAT of male and female mice treated with CL for 3 days and untreated control mice. (mean ± SEM; n = 4)

Fig. 4. Sex differences in the effects of β3-adrenergic receptor activation on lipolysis. a, b Sex differences in the effects of β3-adrenergic receptor activation on glycerol (a) and free fatty acid levels (b) in serum. Mice were treated with CL316,243 up to 5 days, and glycerol and FFA levels in serum were measured. (Mean ± SEM; n = 4, *p < 0.05). c, d Immunoblot analysis of p-HSL, HSL, and ATGL in gWAT of mice treated with CL for 4 h or 3 days and untreated controls. Two-way ANOVA revealed significant main effects of sex in p-HSL (4 h: p = 0.014, 4 h: p = 0.040). Significant differences between male and female were determined by post hoc pairwise comparison with Bonferroni correction (mean ± SEM; n = 4, *p<0.05, **p < 0.01). e qPCR analysis of Adrb3 expression in gWAT of mice treated with CL for 3 days and untreated controls. Two-way ANOVA revealed significant main effects of sex in Adrb3 expression (p < 0.001) and significant interaction of sex and treatment (p < 0.001). Significant differences between male and female were determined by post hoc pairwise comparison with Bonferroni correction (mean ± SEM; n = 4, ***p < 0.001)

Fig. 5. Sympathetic innervation levels in gWAT and iWAT of male and female mice. a, b Immunoblot analysis of tyrosine hydroxylase (TH) protein expression in gWAT and iWAT of mice treated with CL for 3 days and untreated controls. Two-way ANOVA revealed significant main effects of sex in TH protein expression (p = 0.0006). Significant differences between male and female were determined by post hoc pairwise comparison with Bonferroni correction (mean ± SEM; n = 4, *p < 0.05) (c, d). Representative images of TH and PLIN1 staining in paraffin sections of gWAT and iWAT from mice treated with CL for 3 days and untreated controls. Nuclei were counterstained with DAPI. Size bar = 20 μm

Fig. 6. Sex differences in NGF and BDNF expression in gWAT of weanling mice and during β3-adrenergic receptor activation. a qPCR analysis of NGF and BDNF expression in gWAT of weanling mice (3 weeks of age), 6-week-old mice treated with CL for 3 days (6W-CL), and untreated control mice (6W). b qPCR analysis of NGF and BDNF expression in SVC and adipocytes obtained from gWAT of female mice treated with CL for 3 days and untreated controls. c qPCR analysis of NGF and BDNF expression in differentiated adipocyte from PDGFRα progenitors obtained from gWAT of mice. Primary cultured adipocytes were treated with 17β-estradiol (10 nM) or vehicle in the presence or absence of 10 μM isoproterenol for 1 day (mean ± SEM; n = 4, *p < 0.05, **p < 0.01)

Fig. 7. 4-Vinylcyclohexene diepoxide induced ovarian failure reduced TH levels and CL-induced browning of gWAT of female mice. a Immunoblot analysis of UCP1 and TH protein in gWAT of vehicle- and VCD-treated mice after 3 days of CL treatment and control mice. Two-way ANOVA revealed significant main effects of VCD treatment in TH (p = 0.0037) and UCP1 expression (p = 0.015). Significant differences between controls and VCD-treated female mice were determined by post hoc pairwise comparison with Bonferroni correction (mean ± SEM; n = 4, *p < 0.05, **p < 0.01) (b). Basal and oligomycin- and rotenone-induced oxygen consumption rate of gWAT obtained from vehicle- and VCD-treated mice treated with CL for 3 days. c. qPCR analysis of NGF and BDNF expression in gWAT of vehicle- and VCD-treated mice after 3 days of CL treatment, and control mice. Two-way ANOVA revealed significant main effects of VCD treatment in BDNF expression (p = 0.0079). Significant differences between controls and VCD-treated female mice were determined by post hoc pairwise comparison with Bonferroni correction (mean ± SEM; n = 4, *p < 0.05)