Hepatic Estrogen Receptor Alpha Overexpression Protects Against Hepatic Insulin Resistance and MASLD

Abstract

Background/Objectives: Metabolic dysfunction-associated steatotic liver disease (MASLD) is associated with cardiometabolic risk. Although studies have shown that estradiol positively contributes to energy metabolism via estrogen receptor alpha (ERα), its role specifically in the liver is not defined. Therefore, this study aimed to evaluate the effects of ERα overexpression, specifically in the liver in mice fed a high-fat diet (HFD). Methods: Male C57BL/6J mice were divided into four groups, vehicle fed with regular chow (RC) (RC-Vehicle); vehicle fed an HFD (HFD-Vehicle); AAV-treated fed with RC (RC-AAV); and AAV-treated fed an HFD (HFD-AAV), for 6 weeks (8-10 mice per group). AAV was administered intravenously to induce ERα overexpression. Results: We demonstrate that overexpression of ERα in RC-fed mice reduces body fat (28%). These mice show increased oxygen consumption in cultured primary hepatocytes, both in basal (19%) and maximal respiration (34%). In HFD-fed mice, we showed a decrease in hepatic TAG content (43%) associated with improved hepatic insulin sensitivity (145%). Conclusions: From this perspective, our results prove that hepatic ERα signaling is responsible for some of the metabolic protective effects of estrogen in mice. Overexpression of ERα improves hepatocyte mitochondrial function, consequently reducing hepatic lipid accumulation and protecting animals from hepatic steatosis and hepatic insulin resistance. Further investigations will be needed to determine the exact molecular mechanism by which ERα improves hepatic metabolic health.

Keywords: MASLD; estrogen signaling; hepatic insulin resistance; insulin action.

Figure 1

AAV administration increases ERα expression in the liver and not in other tissues. Analysis of ERα overexpression in different tissues using RT-qPCR and Western blotting. (A) Esr1 mRNA expression in the liver of RC-fed mice. (B) Esr1 mRNA expression in the liver of mice fed HFD. (C) ERα protein expression in the liver of mice fed RC. (D) ERα protein expression in the liver of mice fed HFD. (E) ERα protein qualitative analysis in mice fed RC and HFD, using ImageJ. (F) ERα protein expression in the muscle of mice fed RC. (G) ERα protein expression in the muscle of mice fed HFD. (H) ERα protein expression in the brown adipose tissue of mice fed RC. (I) ERα protein expression in the brown adipose tissue of mice fed HFD. (J) ERα protein qualitative analysis in the muscle of mice fed RC and HFD, using ImageJ. (K) ERα protein qualitative analysis in WAT in mice fed RC and HFD, using ImageJ. Data are represented as means ± SEM (n = 5–10), with differences considered significant when p < 0.05. In A–D and F–I analysis, T test was performed, significance level p < 0.05. (*** p < 0.001); (**** p < 0.0001).

Figure 2

Overexpression of ERα in the liver improves the body composition of mice fed RC. Analysis of the body composition of mice fed RC and HFD. (A) Comparative analysis of weight before and after virus inoculation in mice fed RC. (B) Comparative analysis of weight before and after virus inoculation in mice fed an HFD. (C) Percentual changing in body weight of mice. (D) Lean mass of mice fed RC. (E) Lean mass of mice fed HFD. (F) Total fat of mice fed RC. (G) Total fat of mice fed HFD. (H) Retroperitoneal fat through magnetic resonance imaging of RC-fed mice. (I) Retroperitoneal fat through magnetic resonance imaging of mice fed HFD. (J) Perigonadal fat through magnetic resonance imaging of RC-fed mice. (K) Perigonadal fat on MRI scans of mice fed HFD. Data are represented as means ± SEM (n = 5–10), with differences considered significant when p < 0.05. In A and B, a two-way ANOVA with Bonferroni’s post-hoc was performed. For C–J analysis, T test was performed, significance level p < 0.05. (* p < 0.05); (** p < 0.01); (**** p < 0.0001).

Figure 3

Overexpression of ERα is involved in glucose metabolism in mice fed an HFD. Evaluation of glucose metabolism using ipGTT. (A) Fasting glycemia of mice fed RC. (B) Fasting glycemia of mice fed an HFD. (C) Fasting insulin in mice fed RC. (D) Fasting insulin in mice fed an HFD. (E) GTT curve analysis. (F) Area under the curve of mice fed RC. (G) Area under the curve of mice fed HFD. Data are represented as means ± SEM (n = 5–10), with differences considered significant when p < 0.05. In A–D, F, and G analysis, T test was performed, significance level p < 0.05. For E, a two-way ANOVA with Bonferroni’s post-hoc was performed. (* p < 0.05).

Figure 4

ERα overexpression regulates insulin sensitivity. (A) The glucose infusion rate (GIR) and plasma glucose curve during clamp. (B) Average of GIR during the last 40 min of the clamp. (C) Whole-body glucose disappearance during clamp. (D) Basal endogenous glucose production (EGP) and clamp EGP. (E) Suppression percentage of EGP. (F) Basal and clamp non-esterified fatty acids (NEFAs). (G) Insulin-stimulated NEFA suppression during the clamp. Data are represented as means ± SEM (n = 5–10), with differences considered significant when p < 0.05. In A and F, a two-way ANOVA with Bonferroni’s post-hoc was performed. For B–E and G analysis, T test was performed, significance level p < 0.05. (** p < 0.01).

Figure 5

ERα overexpression regulates hepatic lipid content. Analysis of quantitative TAG dosage in the liver of mice fed RC and an HFD. (A) TAG content in the liver of mice fed RC. (B) TAG content in the liver of mice fed an HFD. (C) Quantitative analysis of lipid droplets by Oil Red O in mice fed an HFD. (D) Representative image of the liver, stained with Oil Red O. Data are represented as means ± SEM (n = 5–10), with differences considered significant when p < 0.05. In A, B, and C analysis, T test was performed, significance level p < 0.05. (* p < 0.05).

Figure 6

Overexpression of ERα increases cellular respiratory capacity. Analysis of mitochondrial function in animals fed RC and an HFD. (A) Basal O2 consumption rates (OCR) in isolated hepatocytes. (B) Maximum O2 consumption rates (OCR) in isolated hepatocytes. (C) VDAC1 protein expression in mice fed RC. (D) VDAC1 protein expression in mice fed an HFD. (E) VDAC1 protein qualitative analysis using ImageJ. (F) Total OXPHOS protein expression in mice fed RC. (G) Total OXPHOS protein expression in mice fed an HFD. (H) Total OXPHOS protein qualitative images from mice fed RC. (I) Total OXPHOS protein qualitative images from mice fed an HFD. Data are represented as means ± SEM (n = 5–10), with differences considered significant when p < 0.05. In A–B and D–E analysis T test was performed, significance level p < 0.05. (* p < 0.05); (*** p < 0.001).