Pre– and Post–Sexual Maturity Liver-specific ERα Knockout Does Not Impact Hepatic Mitochondrial Function

**Background:** Premenopausal women and female rodents are protected against hepatic steatosis and exhibit superior hepatic mitochondrial function (higher respiration, lower H2O2 emission) compared to males. Estrogen action is believed to mediate this protection, but the specific role of hepatic estrogen receptor alpha (ERα) in mitochondrial function and steatosis susceptibility remains unclear. Previous studies using conventional (embryonic) liver-specific ERα knockout models have yielded conflicting results, and no studies have examined hepatic mitochondrial respiratory function in the context of liver-specific ERα deletion.

**Methods:** The authors validated an inducible liver-specific ERα knockout (LERKO) model using adeno-associated virus (AAV) Cre injection in ERα^f/f^ female mice, with controls receiving AAV-GFP. Experiment 1 (n=4/group, LFD) confirmed receptor and tissue specificity. Experiment 2 (n=10-12/group) examined LERKO effects on hepatic mitochondria and steatosis after 4 weeks of high-fat diet (HFD, 45% kcal fat). Experiment 3 (n=8-11/group) tested whether timing of LERKO induction—at sexual immaturity (4 weeks old) vs. sexual maturity (8-10 weeks old)—altered outcomes after 8 weeks of HFD. Outcomes included body composition (EchoMRI), hepatic triglycerides (biochemical assay), mitochondrial respiration and H2O2 flux (Oroboros O2k-FluoRespirometer), OXPHOS protein expression (Western blot), RNA sequencing, and bile acid profiling (LC-MS/MS).

**Key Results:** AAV Cre injection reduced liver ERα mRNA by 69% (P<0.01) and protein by ~58% (P=0.001) without affecting ERβ or muscle ERα, confirming specificity. Across all experiments, LERKO mice showed no differences from controls in body weight change, fat mass change, or energy intake. Hepatic triglyceride content did not differ between LERKO and control mice on either LFD or HFD. Mitochondrial respiration (Basal, State 3, State 3S, Uncoupled) and H2O2 emission were similar between genotypes under both pyruvate and palmitoyl-CoA substrate conditions. The coupling control ratio and OXPHOS protein expression were also unchanged. Timing of LERKO induction (pre- vs. post-sexual maturity) did not alter any mitochondrial or steatosis outcomes. RNA sequencing revealed 121 differentially expressed genes (DEGs) in immature LERKO and 485 DEGs in mature LERKO mice (unadjusted P<0.05, fold change ±1.5). Pathway analysis showed that mature LERKO mice had enrichment in extracellular matrix, hormone stimulus, and insulin signaling pathways, while immature LERKO mice showed changes in mitochondrial and ROS metabolism genes. Hepatic bile acid analysis showed similar total bile acids between groups, but LERKO mice had significantly lower cholic acid (CA) in both cohorts (P<0.05), and immature LERKO mice also had lower taurolithocholic acid (T-LCA, P<0.05).

**Clinical Implications:** This study demonstrates that hepatic ERα is not required for the female protective phenotype against HFD-induced hepatic steatosis or for the sexual dimorphism observed in hepatic mitochondrial function. The findings suggest that estrogen-mediated metabolic protection likely involves extrahepatic ERα signaling, other estrogen receptors (ERβ, GPER1), or non-genomic estrogen actions. The transcriptomic changes observed—particularly in bile acid and cholesterol metabolism genes—indicate that hepatic ERα does regulate specific metabolic pathways, but these changes were insufficient to alter steatosis or mitochondrial outcomes under the conditions tested. These results have implications for understanding the mechanisms behind the increased NAFLD risk in postmenopausal women and highlight the need to investigate alternative estrogen signaling pathways as therapeutic targets.