Prevention of obesity and insulin resistance by estrogens requires ERα activation function-2 (ERαAF-2), whereas ERαAF-1 is dispensable

Abstract

The beneficial metabolic actions of estrogen-based therapies are mainly mediated by estrogen receptor α (ERα), a nuclear receptor that regulates gene transcription through two activation functions (AFs): AF-1 and AF-2. Using mouse models deleted electively for ERαAF-1 (ERαAF-1°) or ERαAF-2 (ERαAF-2°), we determined their respective roles in the actions of estrogens on body composition and glucose homeostasis in response to either a normal diet or a high-fat diet (HFD). ERαAF-2° males and females developed accelerated weight gain, massive adiposity, severe insulin resistance, and glucose intolerance--quite reminiscent of the phenotype observed in mice deleted for the entire ERα protein (ERα(-/-)). In striking contrast, ERαAF-1° and wild-type (wt) mice shared a similar metabolic phenotype. Accordingly, 17β-estradiol administration regulated key metabolic genes in insulin-sensitive tissues and conferred a strong protection against HFD-induced metabolic disturbances in wt and ERαAF-1° ovariectomized mice, whereas these actions were totally abrogated in ERαAF-2° and ERα(-/-) mice. Thus, whereas both AFs have been previously shown to contribute to endometrial and breast cancer cell proliferation, the protective effect of estrogens against obesity and insulin resistance depends on ERαAF-2 but not ERαAF-1, thereby delineating new options for selective modulation of ERα.

FIG. 1.

ERαAF-2° mice develop obesity and glucose intolerance under NCD. ERα^−/−, ERαAF-1°, and ERαAF-2° female and male mice and their wt littermates were maintained on an NCD until they were killed at 7 months of age. A: Body weight gain during the follow-up period. B: Subcutaneous (SC), perigonadic (Pg), and mesenteric (Mes) adipose tissue distribution at sacrifice. C: Adipocyte mean area in perigonadic deposits (µm²). D: HOMA-IR. E: Plasma glucose (mg/dL) during intraperitoneal glucose tolerance test. The inset represents the area under the curve for each group. Data are means ± SEM (n = 7–14 animals per group). One-way repeated ANOVA (A and E) or one-way ANOVA (B, C, and D) statistical analyses: *P < 0.05, **P < 0.01, and ***P < 0.001 for ERα^−/−, ERαAF-1°, or ERαAF-2° vs. wt mice.

FIG. 2.

ERαAF-2 deficiency worsens the obese and insulin-resistant phenotype induced by HFD. Four-week-old ERα^−/−, ERαAF-1°, and ERαAF-2° female mice and their wt littermates were fed with HFD for 3 months. A: Mice appearance at time they were killed. B: HFD-induced body weight gain. C: Daily food intake. D: Subcutaneous (SC), perigonadic (Pg), and mesenteric (Mes) adipose tissue distribution. E: Representative photomicrographs of perigonadic adipose tissue sections (hematoxylin-eosin staining; original magnification ×250). F: Adipocyte mean area in perigonadic deposits (µm²). G: Fasting plasma insulin levels (pg/mL). H: Fasting plasma leptin levels (ng/mL). I: Fasting plasma resistin levels (ng/mL). J: Fasting plasma adiponectin levels (pg/mL). K: Intraperitoneal insulin tolerance test (0.6 mU/kg). The inset represents the area under the curve (AUC) for each group. L: Intraperitoneal glucose tolerance test (1 g/kg). The inset represents the AUC for each group. Data are means ± SEM (n = 6–15 animals per group). One-way repeated ANOVA (B, K, and L) or one-way ANOVA (C, D, F–J) statistical analyses: *P < 0.05, **P < 0.01, and ***P < 0.001 for ERα^−/−, ERαAF-1°, or ERαAF-2° vs. wt mice.

FIG. 3.

Either ERαAF-1 or ERαAF-2 deficiency alters uterine weight and sex steroid plasma levels. Wet uterus weight at sacrifice in wt and mutant mice fed with NCD (A) and HFD (B). C: Plasma 17β-estradiol concentration in HFD-fed wt and mutant mice. D: Plasma testosterone concentration in HFD-fed wt and mutant mice. Data are means ± SEM (n = 5–6 per group). One-way ANOVA statistical analysis: *P < 0.05, **P < 0.01, and ***P < 0.001 for ERα^−/−, ERαAF-1°, or ERαAF-2° vs. wt mice.

FIG. 4.

Exogenous estradiol prevents HFD-induced adiposity in ERαAF-1°, but not in ERαAF-2°, ovariectomized female mice. Four-week-old wt, ERα ^−/−, ERαAF-1°, and ERαAF-2° female mice were ovariectomized and received either 17β-estradiol (ovx+E2) or placebo (ovx) subcutaneous administration for a 3-month period and were concomitantly fed with HFD. A: Body weight gain during the treatment period. B: Body composition analysis (magnetic resonance technology). C: Subcutaneous (SC), perigonadic (Pg), and mesenteric (Mes) adipose tissue distribution. D: Representative photomicrograph of perigonadic adipose tissue sections (hematoxylin-eosin staining; original magnification ×250). E: Mean adipocyte area in perigonadic deposits (µm²). Data are means ± SEM (n = 5–13 animals per group). Student t test statistical analysis: *P < 0.05, **P < 0.01, and ***P < 0.001 for E2-treated vs. placebo-treated mice.

FIG. 5.

Exogenous E2 enhances energy expenditure in ERαAF-1°, but not ERαAF-2°, ovariectomized female mice. Four-week-old wt, ERα ^−/−, ERαAF-1°, and ERαAF-2° female mice were ovariectomized, received either 17β-estradiol (ovx+E2) or placebo (ovx) subcutaneous administration for a 3-month period, and were concomitantly fed with HFD. A: Daily profile of respiratory quotient measured in metabolic chambers. B: Mean values of respiratory quotient in each group according to the day (light) and night (dark) periods. C: Total activity measured in metabolic chambers during the day (light) and night (dark) periods. Data are means ± SEM (n = 5–6 per group). Two-way ANOVA statistical analyses: *P < 0.05, **P < 0.01, and ***P < 0.001.

FIG. 6.

Prevention of HFD-induced insulin resistance and hyperglycemia by estradiol is abolished in ERαAF-2°, but fully maintained in ERαAF-1°, ovariectomized mice. Four-week-old wt, ERα ^−/−, ERαAF-1°, and ERαAF-2° female mice were ovariectomized, received either 17β-estradiol (ovx+E2) or placebo (ovx) subcutaneous administration for a 3-month period, and were concomitantly fed with HFD. A: Fasting plasma insulin levels (pg/mL). B: Glucose infusion rate (GIR) during hyperinsulinemic-euglycemic clamp procedure. C: Intraperitoneal glucose tolerance test (1 g/kg). The inset represents the area under the curve for each group. U.A., arbitrary unit. Data are means ± SEM (n = 6–16 animals per group). Student t test statistical analyses: **P < 0.01 and ***P < 0.001 for E2-treated vs. placebo-treated mice.

FIG. 7.

Regulation of metabolic gene expression by estradiol is abrogated in peripheral tissues from ERαAF-2° mice. Four-week-old wt, ERα ^−/−, ERαAF-1°, and ERαAF-2° female mice were ovariectomized, received either 17β-estradiol (ovx+E2) or placebo (ovx) subcutaneous administration for a 3-month period, and were concomitantly fed with HFD. Quantification of mRNA levels (relative to Hprt), expressed in terms of fold change relative to wt ovx mice, from selected metabolic genes in the liver (A) and subcutaneous adipose tissue (B). Data are means ± SEM (n = 4–6 animals per group). Student t test statistical analyses: *P < 0.05, **P < 0.01, and ***P < 0.001 for E2-treated vs. placebo-treated mice.

FIG. 8.

Regulation of gene expression in isolated hepatocytes and mature adipocytes requires ERαAF-2, but not ERαAF-1, activation. Cells were isolated from 10-week-old wt, ERα^−/−, ERαAF-1°, and ERαAF-2° mice, previously ovariectomized at 4 weeks of age. These cells were cultured for 24 h with 17β-estradiol (10⁻⁸ mol/L) or vehicle (DMSO). A: Relative gene expression of Stat3, Srebp-1c, Scd1, and Fas in primary isolated hepatocytes. B: Relative gene expression of Fas, Acc-1, Acc-2, and perilipin in primary isolated mature adipocytes. Data are means ± SEM (n = 3–4 individual animals per group from three separate experiments). Student t test statistical analyses: *P < 0.05, **P < 0.01, and ***P < 0.001 for cells stimulated with E2 vs. vehicle.