Estradiol treatment or modest exercise improves hepatic health and mitochondrial outcomes in female mice following ovariectomy

Abstract

We recently reported that compared with males, female mice have increased hepatic mitochondrial respiratory capacity and are protected against high-fat diet-induced steatosis. Here, we sought to determine the role of estrogen in hepatic mitochondrial function, steatosis, and bile acid metabolism in female mice and investigate potential benefits of exercise in the absence or presence of estrogen via ovariectomy (OVX). Female C57BL mice (n = 6 per group) were randomly assigned to sham surgery (sham), ovariectomy (OVX), or OVX plus estradiol replacement therapy (OVX + Est). Half of the mice in each treatment group were sedentary (SED) or had access to voluntary wheel running (VWR). All mice were fed a high-fat diet (HFD) and were housed at thermoneutral temperatures. We assessed isolated hepatic mitochondrial respiratory capacity using the Oroboros O2k with both pyruvate and palmitoylcarnitine as substrates. As expected, OVX mice presented with greater hepatic steatosis, weight gain, and fat mass gain compared with sham and OVX + Est animals. Hepatic mitochondrial coupling (basal/state 3 respiration) with pyruvate was impaired following OVX, but both VWR and estradiol treatment rescued coupling to levels greater than or equal to sham animals. Estradiol and exercise also had different effects on liver electron transport chain protein expression depending on OVX status. Markers of bile acid metabolism and excretion were also impaired by ovariectomy but rescued with estradiol add-back. Together our data suggest that estrogen depletion impairs hepatic mitochondrial function and liver health, and that estradiol replacement and modest exercise can aid in rescuing this phenotype.NEW & NOTEWORTHY OVX induces hepatic steatosis in sedentary mice which can be prevented by modest physical activity (VWR) and/or estradiol treatment. Estrogen impacts hepatic mitochondrial coupling in a substrate-specific manner. OVX mice have impaired fecal bile acid excretion, which was rescued with estradiol treatment.

Keywords: fatty liver; menopause; metabolism.

Graphical abstract

Figure 1.

Experimental timeline. OVX, ovariectomy; VWR, voluntary wheel running.

Figure 2.

Hepatic steatosis. To determine steatosis, hepatic triglyceride (TAG) content was measured with a biochemical assay and normalized to liver tissue weight (A) and liver sections were stained with H&E (B). Liver TAG data are presented as means ± SE (n = 5–6 mice per group). T, V: main effect of treatment or VWR; T × V: interaction of treatment and VWR from ANOVA. Lowercase t and v denote significant LSD post hoc for treatment and VWR, respectively. All P < 0.05. H&E, hematoxylin and eosin; LSD, least significant difference; OVX, ovariectomy; SED, sedentary; VWR, voluntary wheel running.

Figure 3.

Markers of liver health. Markers of oxidative stress, 4HNE (A), SOD2 (B), and CAT (C), were measured in liver whole homogenate via Western blotting and RT-PCR, respectively. Other indicators of liver health, COL1A1 (D) and EMR1 (E), were evaluated via RT-PCR. Data are presented as means ± SE (n = 5–6 mice per group). T: main effect of treatment; T × V: interaction of treatment and VWR. Lowercase t and v denote significant LSD post hoc for treatment and VWR, respectively from ANOVA. All P < 0.05. CAT, catalase; COL1A1, collagen type I alpha 1 chain; EMR1, adhesion G protein-coupled receptor E1; Est, estradiol; LSD, least significant difference; OVX, ovariectomy; PPIB, cyclophilin B; RT-PCR, reverse transcription-polymerase chain reaction; SOD2, superoxide dismutase 2; VWR, voluntary wheel running; 4HNE, 4‐hydroxynonenal.

Figure 4.

Mitochondrial respiration and coupling with pyruvate. Mitochondrial respiratory capacity was measured in isolated hepatic mitochondria using pyruvate as the main starting substrate at basal (A), ADP-stimulated (state 3; B), ADP-stimulated plus succinate (state 3S; C), and uncoupled (D) respiratory states. All respiration was normalized to mitochondrial protein in the O2k chamber measured via BCA assay. Coupling control ratio was calculated as basal respiration divided by ADP-stimulated respiration (E). Data are presented as means ± SE (n = 6 mice per group). T and V: main effect of treatment or VWR; T × V: interaction of treatment and VWR from ANOVA. Lowercase t and v denote significant LSD post hoc for treatment and VWR, respectively. All P < 0.05. BCA, bicinchoninic acid; Est, estradiol; LSD, least significant difference; OVX, ovariectomy; VWR, voluntary wheel running.

Figure 5.

Mitochondrial respiration and coupling with PC. Isolated hepatic mitochondrial respiratory capacity was measured with an Oroboros O2k-Fluo-Respirometer using l-palmitoylcarnitine as the main starting substrate at basal (A), ADP-stimulated (state 3; B), ADP-stimulated plus succinate (state 3S; C), and uncoupled (D) respiratory states. All respiration was normalized to mitochondrial protein via BCA assay. Coupling control ratio was calculated as basal respiration divided by ADP-stimulated respiration (E) to infer coupling. Data are presented as means ± SE (n = 6 mice per group). V: main effect of VWR from ANOVA when averaged across all treatment groups. All P < 0.05. BCA, bicinchoninic acid; Est, estradiol; OVX, ovariectomy; PC, l-palmitoylcarnitine; VWR, voluntary wheel running.

Figure 6.

Electron transport system protein complexes. Quantified protein expression of oxidative phosphorylation complexes in liver whole homogenate was determined via Western blotting and normalized to total protein (A–E). A representative blot is shown in F. Data are presented as means ± SE (n = 6 mice per group). T and V: main effect of treatment or VWR; T × V: interaction of treatment and VWR from ANOVA. Lowercase t and v denote significant LSD post hoc for treatment and VWR, respectively, when averaged across all other factors. All P < 0.05. Est, estradiol; LSD, least significant difference; OVX, ovariectomy; VWR, voluntary wheel running.

Figure 7.

Markers of mitochondrial biogenesis. Quantified mRNA expression of genes involved in mitochondrial biogenesis—peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1a) (A), nuclear respiratory factor 1 (NRF1) (B), and transcription factor A, mitochondrial (TFAM) (C), normalized to the housekeeping gene Cyclophilin B (PPIB). Data are presented as means ± SE (n = 6 mice per group). T: main effect of treatment from ANOVA; lowercase t: significant LSD post hoc for treatment when averaged across all other factors. All P < 0.05 or equal to 0.05 where noted. Est, estradiol; LSD, least significant difference; mRNA, messenger RNA; OVX, ovariectomy; PPIB, cyclophilin B; VWR, voluntary wheel running.

Figure 8.

Cholesterol and bile acid metabolism. ACLY (A) and HMGCR (B), genes involved in cholesterol metabolism, and CPY7A1 (C) and NR1H4 (D), genes involved in bile acid metabolism, were measured with RT-PCR and normalized to the housekeeping gene PPIB. Bile acids were extracted from feces (E), quantified using a commercially available bile acid assay, and normalized to fecal weight used in the extraction. Data are presented as means ± SE (n = 6 mice per group). Capital T and lowercase t reflect a main effect from ANOVA or significant LSD post hoc for treatment, respectively. Significant values are P < 0.05 or equal to 0.05 where noted. ACLY, ATP ctrate lyase; CPY7A1, Cytochrome P450 Family 7 Subfamily A Member 1; Est, estradiol; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; LSD, least significant difference; NR1H4, nuclear receptor subfamily 1 group H member 4 (FXR, farnesoid X receptor); mOVX, ovariectomy; PPIB, cyclophilin B; RT-PCR, reverse transcription-polymerase chain reaction; VWR, voluntary wheel running.

Figure 9.

Summary of main findings. Up and down arrows designate the directionality of the statistically significant difference (all P < 0.05) between groups due to either treatment (sham, OVX, OVX + Est) or VWR. A horizontal line designates no significant differences. BAs, bile acids; BW, body weight; Est, estradiol; OVX, ovariectomy; PYR, potassium-pyruvate; TAGs, triglycerides; VWR, voluntary wheel running.