Metabolic effects of late-onset estradiol replacement in high-fat-fed ovariectomized mice

Abstract

Background: Decreased estrogen levels in postmenopausal women negatively impact metabolic health. It is known that estradiol (E2) replacement can reverse this condition. However, there is no consensus on whether the effects mediated by E2 depend on the starting time of E2 replacement after menopause. We aimed to investigate the effects of different onset E2 treatments on glucose tolerance and metabolic parameters in high-fat-fed ovariectomized mice.

Material and methods: Eight-week-old female C57BL/6J mice were divided into three groups: SHAM, OVX, and E2, to evaluate three different time points of E2 replacement after ovariectomy: early (after 4 weeks), intermediate (after 12 weeks), and late replacement (after 20 weeks). E2 groups received treatment through subcutaneous pellets.

Results: E2 replacement improved the parameters analyzed independently of the time since ovariectomy, reducing body weight gain and fat mass, as well as increasing the percentage of lean mass. Glucose intolerance, fasting insulin, HOMA-IR, and cholesterol levels were also reduced after treatment with E2. In the liver, there was a decrease in triacylglycerol (TAG) deposition, with no difference in the expression of SREBP1 and ERα proteins. In the muscle, there was a decrease in TAG deposition. In periuterine adipose tissue, there was an increase in the expression of SREBP1, FASN, and SCD, with no difference in the expression of ERα.

Conclusions: Our findings reinforce the critical role of E2 in regulating both glucose and lipid metabolism and indicate that E2 action on metabolic health was not dependent on time since ovariectomy for the parameters analyzed.

Keywords: Estradiol replacement; Insulin resistance; Lipid metabolism; Menopause; Ovariectomy.

Fig. 1

wt gain curve data over the weeks of treatment (A, D and G), initial and final weight (B, E and H) and body delta, obtained by the difference between the initial and final weight (C, F and I). In A-C, data from SHAM (n = 9), OVX (n = 10) and E2 (n = 12) group 4 wk. In D-F, SHAM (n = 9), OVX (n = 10) and E2 (n = 12) from group 12 wk. In G-I, SHAM (n = 10), OVX (n = 11) and E2 (n = 12) from the 20 wk group. A, B, D, E, G and H: Significance between groups was determined by two-way ANOVA followed by Tukey's multiple comparisons test. C, F and I: Significance between groups was determined by one-way ANOVA followed by Tukey's multiple comparisons test. Data presented as mean ± standard error of the mean (SEM). The minimum acceptable significance level was p < 0.05, with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig. 2

Lean mass and body fat data obtained by body composition analysis using magnetic resonance imaging. The data presents absolute weight of lean mass (A, E and I), percentage of lean mass in relation to body weight (B, F and J), absolute weight of body fat (C, G and K) and percentage of fat in relation to body weight (D, H and L). In A-D, data from SHAM (n = 4), OVX (n = 6) and E2 (n = 3) group 4 wk. In E-H, SHAM (n = 10), OVX (n = 10) and E2 (n = 12) from group 12 wk. In I-L, SHAM (n = 10), OVX (n = 11) and E2 (n = 12) from the 20 wk group. Significance between groups was determined by one-way ANOVA followed by Tukey's multiple comparisons test. Data presented as mean ± standard error of the mean (SEM). The minimum acceptable significance level was p < 0.05, with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig. 3

Data obtained during the glucose tolerance test (GTT). The data presents a curve of blood glucose variation over time (A, F and K), area over the curve (B, G and L), basal blood glucose (C, H and M), basal insulin (D, I and N), and HOMA-IR (E, J and O). In 2A-2E, data from SHAM (n = 9), OVX (n = 10) and E2 (n = 11) group 4 wk. In F-J, SHAM (n = 10), OVX (n = 10) and E2 (n = 12) from group 12 wk. In K-O, SHAM (n = 10), OVX (n = 11) and E2 (n = 12) from the 20 wk group. A, F and K: Significance between groups was determined by two-way ANOVA followed by Tukey's multiple comparisons test. B-E, G-J, L–O: Significance between groups was determined by one-way ANOVA followed by Tukey's multiple comparisons test. Data presented as mean ± standard error of the mean (SEM). The minimum acceptable significance level was p < 0.05, with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Fig. 4

The data shows the triglycerides concentration in the liver (A, C and E) and muscle tissue (B, D and F). In 1A-1B, data from SHAM (n = 9–10), OVX (n = 9–10) and E2 (n = 12) group 4 wk. In C-D, SHAM (n = 10), OVX (n = 10) and E2 (n = 10) from group 12 wk. In E-F, SHAM (n = 8–10), OVX (n = 10) and E2 (n = 10–12) from the 20 wk group. Significance between groups was determined by one-way ANOVA followed by Tukey's multiple comparisons test. Data presented as mean ± standard error of the mean (SEM). The minimum acceptable significance level was p < 0.05, with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig. 5

Total area of lipid droplets in the liver. In A and B, quantification of the Oil Red O area and representative image of liver tissue, respectively, from groups 4 wk SHAM (n = 2), OVX (n = 3), E2 (n = 3). In C and D, quantification of the Oil Red O area and representative image of liver tissue, respectively, from groups 12 wk SHAM (n = 2), OVX (n = 2), E2 (n = 2). In E and F, quantification of the Oil Red O area and representative image of liver tissue, respectively, from groups 20 wk SHAM (n = 3), OVX (n = 3), E2 (n = 3). In A, C and E, significance between groups was determined by one-way ANOVA followed by Tukey's multiple comparison test. Data presented as mean ± standard error of the mean (SEM). The minimum acceptable significance level was p < 0.05, with ∗p < 0.05.

Fig. 6

Plasma total cholesterol measurement. In A, data from SHAM (n = 10), OVX (n = 11) and E2 (n = 11) group 4 wk. In B, SHAM (n = 10), OVX (n = 10) and E2 (n = 9) from group 12 wk. In C, SHAM (n = 10), OVX (n = 9) and E2 (n = 11) from group 20 wk. Significance between groups was determined by one-way ANOVA followed by Tukey's multiple comparisons test. Data presented as mean ± standard error of the mean (SEM). The minimum acceptable significance level was p < 0.05, with ∗p < 0.05, ∗∗p < 0.01.

Fig. 7

Analysis of protein expression in periuterine adipose tissue by Western Blotting of group 20 wk SHAM (n = 4), OVX (n = 5) and E2 (n = 5). In A, quantification of ERα protein expression. In B, quantification of SREBP1 protein expression. In C, quantification of FASN protein expression. In D, quantification of SCD protein expression. In E, Western blot image of ERα, SREBP1, FASN, SCD and GAPDH,. Significance between groups was determined by one-way ANOVA followed by Tukey's multiple comparisons test. Data presented as mean ± standard error of the mean (SEM). The minimum acceptable significance level was p < 0.05, with ∗p < 0.05 and ∗∗p < 0.01.

Fig. 8

Analysis of protein expression in the liver by Western Blotting of group 20 wk SHAM (n = 4), OVX (n = 5) and E2 (n = 5). In A, quantification of ERα protein expression. In B, quantification of SREBP1 protein expression. In C, Western blot image of ERα, SREBP1 and GAPDH. Significance between groups was determined by one-way ANOVA followed by Tukey's multiple comparisons test. Data presented as mean ± standard error of the mean (SEM). The minimum acceptable significance level was p < 0.05.