Hepatic estrogen receptor alpha drives masculinization in post-menopausal women with metabolic dysfunction-associated steatotic liver disease

Abstract

Background & aims: The loss of ovarian functions defining menopause leads to profound metabolic changes and heightens the risk of developing metabolic dysfunction-associated steatotic liver disease (MASLD). Although estrogens primarily act on the female liver through estrogen receptor alpha (ERα), the specific contribution of impaired ERα signaling in triggering MASLD after menopause remains unclear.

Methods: To address this gap in knowledge, we compared the liver transcriptomes of sham-operated (SHAM) and ovariectomized (OVX) control and liver ERα knockout (LERKO) female mice by performing RNA-Seq analysis.

Results: OVX led to 1426 differentially expressed genes (DEGs) in the liver of control mice compared to 245 DEGs in LERKO mice. Gene ontology analysis revealed a distinct ovariectomy-induced modulation of the liver transcriptome in LERKO compared with controls, indicating that hepatic ERα is functional and necessary for the complete reprogramming of liver metabolism in response to estrogen depletion. Additionally, we observed an ovariectomy-dependent induction of male-biased genes, especially in the liver of control females, pointing to hepatic ERα involvement in the masculinization of the liver after estrogen loss. To investigate the translational relevance of such findings, we assessed liver samples from a cohort of 60 severely obese individuals (51 women; 9 men). Notably, a shift of the liver transcriptome toward a male-like profile was also observed only in obese women with MASLD (n = 43), especially in women ≥51 years old (15/15), suggesting that masculinization of the female liver contributes to MASLD development in obese women.

Conclusions: These results highlight the role of hepatic ERα in driving masculinization of the liver transcriptome following menopause, pointing to this receptor as a potential pharmacological target for preventing MASLD in post-menopausal women.

Impact and implications: Despite the increased risk of developing MASLD after menopause, the specific contribution of impaired hepatic estrogen signaling in driving MASLD in females has not been a major research focus, and, thus, has limited the development of tailored strategies that address the specific mechanisms underlying MASLD in post-menopausal women. This study reveals the functional role of hepatic ERα in mediating liver metabolic changes in response to estrogens loss, leading to a shift in the liver transcriptome towards a male-like profile. In women with obesity, this shift is associated with the development of MASLD. These findings underscore the potential of targeting hepatic ERα as a promising approach for developing effective, sex-specific treatments to preserve liver health and prevent or limit the development and progression of MASLD in post-menopausal women.

Keywords: Estrogens; Female; Liver metabolic programming; Menopause.

Graphical abstract

Fig. 1

The lack of hepatic ERα partially mimics changes in liver transcriptome observed in ovariectomized female mice. Distribution of genes upregulated (red) and downregulated (blue) by ovariectomy (A) or by lack of hepatic ERα (B) measured by RNA-Seq in the liver of CTRL SHAM, CTRL OVX, and LERKO SHAM (n = 4). (C) Venn diagram showing differentially expressed genes found in the comparisons between CTRL OVX/CTRL SHAM (grey) or LERKO SHAM/CTRL SHAM (orange). Heatmaps, clusters and Gene Ontology analysis of functional networks significantly upregulated (D, E) or downregulated (F, G) in CTRL OVX as well as LERKO SHAM with respect to CTRL SHAM mice.

Fig. 2

Lack of estrogens reprograms liver metabolism in a mouse model of menopause. Heatmap and cluster (A), Gene Ontology (GO) analysis of functional networks (B), and most enriched motifs (C) associated with genes upregulated in CTRL OVX but not LERKO SHAM with respect to CTRL SHAM. (D) Pparα mRNA measured in the liver of CTRL SHAM, CTRL OVX and LERKO SHAM. Data from RNA-Seq analysis are represented as mean ± SEM (n=4). ∗∗p <0.01 CTRL OVX vs. CTRL SHAM; +p <0.05 LERKO SHAM vs. CTRL SHAM by one-way ANOVA followed by Bonferroni’s post hoc test. Heatmap, cluster (E) and GO analysis of functional networks (F) associated with genes downregulated in CTRL OVX but not LERKO SHAM with respect to CTRL SHAM.

Fig. 3

Hepatic ERα profoundly impacts on ovariectomy-induced changes in liver transcriptome. GO analysis of functional networks significantly upregulated in LERKO (A) or uniquely in CTRL (B) mice following ovariectomy. (C) Venn diagram showing the overlapping among genes differentially regulated by ovariectomy in CTRL (grey) or LERKO (orange). GO analysis of functional networks significantly downregulated in LERKO (D) or uniquely in CTRL (E) mice by ovariectomy.

Fig. 4

Hepatic ERα mediates liver transcriptome reprogramming towards a male-like profile after ovariectomy. (A) Volcano plot of differentially expressed genes (DEGs) in the liver identified in LERKO OVX and CTRL OVX mice. Venn diagram showing the overlap between DEGs in CTRL OVX/SHAM (B), LERKO OVX/SHAM (C), and OVX LERKO/CTRL (D) with well-known sex-biased genes. (E) Venn diagram showing the overlay among DEGs in OVX LERKO/CTRL, sex-biased genes and genes known to be altered in STAT5 KO mice. (F) Heatmap reporting the expression of selected genes in CTRL OVX/SHAM, OVX LERKO/CTRL, wild-type males vs. females (WT MAL/FEM), STAT5 KO males vs. wild-type males (STAT5 KO/WT MAL), and STAT5 KO females vs. wild-type females (STAT5 KO/WT FEM). Male-biased and female-biased genes are shown in blue and red, respectively.

Fig. 5

MASLD in women is associated with a male-like liver transcriptome profile. mRNA levels of genes measured in the liver of women <51 years (open red bars), women <51 years with MASLD (red bars), women ≥51 years with MASLD (grey bars), and men with MASLD (blue bars). Data from RNA-Seq are represented as mean ± SEM (n = 8–25). ∗p <0.05, ∗∗p <0.01 and ∗∗∗p <0.001 vs. women <51 years by one-way ANOVA followed by Bonferroni’s post hoc test. Abbreviations: MASLD, metabolic dysfunction-associated steatotic liver disease; CD36, CD36 molecule; LPIN2, lipin 2; SULT1E, SULT1A1, SULT2A1, sulfotransferases; HSD17B2, hydroxysteroid 17-beta dehydrogenase 2; AGXT, alanine-glyoxylate aminotransferase; AMT, aminomethyltransferase; SLC13A3 and SLC22A5 (Slc22a8 homolog), solute carrier family members; CYP2A6 (Cyp2a5 homolog), CYP4A11 (Cyp4a12b homolog), CYP4A22 (Cyp4a12a homolog), CYP17A1, CYP7B1, cytochrome P450 family members; COL27A1, collagen type XXVII alpha 1 chain; SERPINA1 and SERPINA3, serpin family members.