17α-Estradiol Alleviates Age-related Metabolic and Inflammatory Dysfunction in Male Mice Without Inducing Feminization

Abstract

Aging is associated with visceral adiposity, metabolic disorders, and chronic low-grade inflammation. 17α-estradiol (17α-E2), a naturally occurring enantiomer of 17β-estradiol (17β-E2), extends life span in male mice through unresolved mechanisms. We tested whether 17α-E2 could alleviate age-related metabolic dysfunction and inflammation. 17α-E2 reduced body mass, visceral adiposity, and ectopic lipid deposition without decreasing lean mass. These declines were associated with reductions in energy intake due to the activation of hypothalamic anorexigenic pathways and direct effects of 17α-E2 on nutrient-sensing pathways in visceral adipose tissue. 17α-E2 did not alter energy expenditure or excretion. Fasting glucose, insulin, and glycosylated hemoglobin were also reduced by 17α-E2, and hyperinsulinemic-euglycemic clamps revealed improvements in peripheral glucose disposal and hepatic glucose production. Inflammatory mediators in visceral adipose tissue and the circulation were reduced by 17α-E2. 17α-E2 increased AMPKα and reduced mTOR complex 1 activity in visceral adipose tissue but not in liver or quadriceps muscle, which is in contrast to the generalized systemic effects of caloric restriction. These beneficial phenotypic changes occurred in the absence of feminization or cardiac dysfunction, two commonly observed deleterious effects of exogenous estrogen administration. Thus, 17α-E2 holds potential as a novel therapeutic for alleviating age-related metabolic dysfunction through tissue-specific effects.

Keywords: 17α-Estradiol; Adipose tissue; Hypothalamus; Inflammation; Metabolism.

Figure 1.

17α-E2 reduces body mass and adiposity. Weekly changes in (A) total body mass, (B) fat mass, and (C) lean mass in CON (blue) and 17α-E2 (red) treatment groups from Study 1. Representative cross-sectional images of visceral (pink) and subcutaneous (gray) adiposity in the lumbar region of (D) CON and (E) 17α-E2 treatment groups at Week 12 of Study 1. (F) Quantitative analysis of regional adiposity in the lumbar region from CON and 17α-E2 treatment groups from Study 1. (G) Plasma leptin and (H) plasma leptin normalized to total, lean, and fat mass from CON and 17α-E2 treatment groups from Study 1. (I) Plasma adiponectin and (J) plasma adiponectin normalized to total, lean, and fat mass from CON and 17α-E2 treatment groups from Study 1. Daily changes in (K) total body mass and weekly changes in (L) lean:fat mass ratios in CON (yellow), 17α-E2 (orange), and calorie restriction (green) treatment groups from Study 2. All data are expressed as mean ± SEM of 7–10 mice per treatment group and were analyzed by repeated measures analysis of variance (A–C, K, L) or Student’s t test (F–J). *p < .05. ^# p < .01. ^$ p < .005.

Figure 2.

17α-E2 reduces energy intake but not expenditure or excretion. (A) Oxygen consumption, (B) energy expenditure, and (C) voluntary activity during Weeks 0 and 12 from CON (blue) and 17α-E2 (red) treatment groups from Study 1. (D) Oxygen consumption, (E) energy expenditure, and (F) voluntary activity during Week 4 from CON (yellow), 17α-E2 (orange), and calorie restriction (CR; green) treatment groups from Study 2. (G) Fecal energy excretion during Weeks 0 and 5 from CON, 17α-E2, and CR treatment groups from Study 2. (I) Daily calorie intake per week and total calories consumed from CON, 17α-E2, and CR treatment groups from Study 2. (J) Daily calorie intake per week normalized to body mass and total calories consumed normalized to body mass from CON, 17α-E2, and CR treatment groups from Study 2. (K) Anorexigenic gene expression in the hypothalamus from CON, 17α-E2, and CR treatment groups from Study 2. All data are expressed as mean ± SEM of 7–10 mice per treatment group and were analyzed by Student’s t test (A–C) or one-way analysis of variance (D–K). ^♦ p < .05 (different from nonlabeled groups).

Figure 3.

17α-E2 reduces inflammation in white adipose tissue (WAT) and the circulation. Pro-inflammatory gene expression in (A) subcutaneous (iWAT) and (B) visceral (eWAT) WAT from CON (blue) and 17α-E2 (red) treatment groups from Study 1. Representative images of SA-βgal⁺ in iWAT from (C) CON and (D) 17α-E2 treatment groups from Study 1. (E) Percent SA-βgal⁺ in iWAT from CON and 17α-E2 treatment groups from Study 1. (F) SA-mRNA expression in iWAT from CON and 17α-E2 treatment groups from Study 1. Representative images of SA-βgal⁺ in eWAT from (G) CON and (H) 17α-E2 treatment groups from Study 1. (I) Percent SA-βgal⁺ in eWAT from CON and 17α-E2 treatment groups from Study 1. (J) SA-mRNA expression in eWAT from CON and 17α-E2 treatment groups from Study 1. (K) Plasma pro-inflammatory cytokines and chemokines in CON and 17α-E2 treatment groups at Week 15 of Study 1. Arrows indicate SA-βgal⁺ activity in representative samples. All data are expressed as mean ± SEM of 8–10 mice per treatment group and were analyzed by Student’s t test. *p < .05. ^# p < .01. ^$ p < .005. Scale bars: 50 µm.

Figure 4.

17α-E2 reverses age-related lipid redistribution and improves glucose homeostasis. Representative images of total hepatic lipid content from (A) CON and (B) 17α-E2 treatment groups from Study 1. Triglyceride accumulation in (C) liver and (D) quadriceps from CON (blue) and 17α-E2 (red) treatment groups from Study 1. (E) Fasting glucose at Weeks 0 and 15 in CON and 17α-E2 treatment groups from Study 1. (F) Fasting insulin and (G) Glycosylated hemoglobin (HbA1c) at Week 15 in CON and 17α-E2 treatment groups from Study 1. (H) Glucose tolerance at Week 10 in CON and 17α-E2 treatment groups from Study 1. (I) Glucose tolerance area under the curve at Week 10 in CON and 17α-E2 treatment groups from Study 1. (J) Fasting glucose at Weeks 0, 2, and 4 in CON (yellow), 17α-E2 (orange), and calorie restriction (CR; green) treatment groups from Study 2. (K) HbA1c at Weeks 0 and 5 in CON, 17α-E2, and CR treatment groups from Study 2. All data are expressed as mean ± SEM of 8–10 mice per treatment group and were analyzed by Student’s t test (C–G), repeated measures analysis of variance (ANOVA; H), or one-way ANOVA (J–K). *p < .05. ^# p < .01. ^$ p < .005. ^♦ p < .05 (different from nonlabeled groups); ^♠ p < .05 (different from nonlabeled and ♦-labeled groups). Scale bars: 200 µm.

Figure 5.

17α-E2 improves systemic insulin sensitivity. (A) Glucose and (B) insulin under basal and clamped conditions from CON (blue) and 17α-E2 (red) treatment groups from Study 1. (C) Real-time and (D) average glucose infusion rates (GIRs) from CON and 17α-E2 treatment groups from Study 1. (E) Whole-body glucose disposal, (F) endogenous glucose production (EGP) under basal and clamped conditions, and (G) hyperinsulinemia-induced percent EGP suppression from CON and 17α-E2 treatment groups from Study 1. (H) Tissue-specific disposal of 2-deoxyglucose during hyperinsulinemia from CON and 17α-E2 treatment groups from Study 1. (I) Circulating free fatty acids (FFAs) under basal and clamped conditions and (J) percent lipolysis suppression during hyperinsulinemia from CON and 17α-E2 treatment groups from Study 1. All data are expressed as mean ± SEM of 7 mice per treatment group and were analyzed by Student’s t test (A, B, D–J) or repeated-measures analysis of variance (c). *p < .05. ^$ p < .005.

Figure 6.

17α-E2 selectively activates AMPKα and suppresses mTOR complex 1. Representative Western blots of p-AMPKα, AMPKα, p-mTOR, mTOR, p-S6, S6, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from (A) visceral adipose (eWAT), (B) liver, and (C) quadriceps from CON and 17α-E2 treatment groups from Study 1. Densitometry results for p-AMPKα:AMPKα, p-mTOR:mTOR, and p-S6:S6 ratios from (D) visceral (eWAT), (E) liver, and (F) quadriceps from CON (blue) and 17α-E2 (red) treatment groups from Study 1. All data are expressed as mean ± SEM of 7–10 mice per treatment group and were analyzed by Student’s t test (D–F). ^$ p < .005.

Figure 7.

17α-E2 does not feminize male mice. (A) Gonadal, (B) seminiferous tubule, and (C) breast mass during necropsy from CON (blue) and 17α-E2 (red) treatment groups from Study 1. Plasma (D) testosterone, (E) 17β-E2, and (F) 17α-E2 in CON and 17α-E2 treatment groups at Week 15 of Study 1. (G) ERα and (H) ERβ mRNA expression in liver, quadriceps, subcutaneous and visceral WAT from CON and 17α-E2 treatment groups from Study 1. All data are expressed as mean ± SEM of 8–10 mice per treatment group and were analyzed by Student’s t test. *p < .05. ^$ p < .005.