Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease that progresses from simple steatosis to steatosis combined with inflammation, referred to as nonalcoholic steatohepatitis, to fibrosis, cirrhosis, and hepatocellular carcinoma ⁽¹⁾. NAFLD is characterized by the accumulation of fat in the liver without excessive alcohol consumption and is an important public health issue affecting approximately 25% of the world population ⁽²⁾. Overall, approximately 5% of NAFLD patients develop cirrhosis over an average of 7.6 years, and 1.7% die from cirrhosis-related complications ⁽³⁾.

The rising rates of overweight and obesity in menopausal women are important public health concerns ⁽⁴⁾. Postmenopausal women tend to have higher total body fat mass, fat percentage, and central fat accumulation compared with men and premenopausal women ^(5-7). These differences can be attributed in part to the decline in the circulating estrogen levels ⁽⁸⁾. The onset of menopause is associated with declining estrogen levels, decreased energy expenditure, and fat oxidation, all of which are accompanied by increases in total body fat and visceral adipose tissue mass ^(9,10).

The growth of adipose tissue occurs through hypertrophy and hyperplasia and also depends on adipose tissue angiogenesis, the formation of new blood vessels from pre-existing vessels ⁽¹¹⁾. The inhibition of adipose tissue vascularization can suppress white adipose tissue expansion and inhibit the development of obesity ⁽¹²⁾. Endogenous angiogenesis inhibitors, such as angiostatin and endostatin, can decrease body weight in obese mice ⁽¹³⁾. One of the most well-established signaling molecules in angiogenesis is vascular endothelial growth factor A (VEGF-A) and its receptors. VEGF-A promotes the growth, survival, and proliferation of endothelial cells ⁽¹⁴⁾. Therefore, we investigated the effects of 17β-estradiol on obesity and NAFLD in obese female ovariectomized (OVX) C57BL/6J mice, a mouse model of obese postmenopausal women, and the involvement of angiogenesis and VEGF-A in this process.

1. Animal treatments

Eight-week-old C57BL/6J female mice (n = 8/group) were purchased from Central Lab Animal. Mice were OVX and then divided into three groups. First group was received a low-fat diet (LFD, 13 kcal% fat, Research Diets). Second group was fed a high-fat diet (HFD, 45 kcal% fat, Research Diets). Third group was given an HFD and subcutaneously implanted with 17β-estradiol with 60-day release (0.05 mg/pellet; Innovative Research of America) (HFD-E). Body weights were measured three times a week. Food intake was calculated by measuring the amounts of food consumed by mice throughout the treatment period. On the last day of the study, 8-hour-fasted mice were sacrificed by cervical dislocation. Blood was collected by heart puncture and serum was separated and stored at –80℃ until analyses. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol, and triglycerides were quantitated using a blood chemical analyzer (Cobas 8000, c502, Roche). All mouse experiments were approved by the Institutional Animal Care and Use Committees of Mokwon University (permit number: NVRQS AEC-24), and followed National Research Council Guidelines.

2. Histological analysis

The liver tissues were fixed in 10% formalin for at least 1 day and embedded in paraffin block. Tissue sections (5 μm) were cut and stained with hematoxylin-eosin (HE) and Masson’s trichrome for microscopic examination. Stained sections were analyzed under an Olympus BH2-RFCA fluorescence microscope (Olympus) and estimated with an image analysis system (ImageJ software).

3. Immunohistochemistry

The liver and parametrial adipose tissues were fixed in 10% formalin and embedded in paraffin block. After epitope retrieval, sections (3-μm thick) were stained with an anti-CD68 (1:200 dilution; ab955; Abcam) primary antibody and a biotinylated secondary antibody (Vector Laboratories). Sections were then counterstained with Mayer’s hematoxylin. Immunostained sections were determined using ImageJ software. Blood vessel staining was performed using a blood vessel staining kit (Millipore). Parametrial adipose tissue sections were incubated with an anti-von Willebrand factor (vWF) antibody as a primary antibody and a biotinylated secondary antibody. Blood vessel density was estimated by ImageJ software analysis system and normalized with the number of adipocytes.

4. Quantitative real-time polymerase chain reaction

Total RNA from visceral adipose tissues were isolated using Trizol reagent (GeneAll Biotechnology). Complementary DNA (cDNA) was synthesized from 2 μg total RNA using the Topscript^TM DryMIX RT kit (Enzynomics). The genes of interest were amplified from the synthesized cDNA using AccuPower 2X GreenStar^TM qPCR Master Mix (Bioneer) and a Exicycler^TM 96 Real Time Quantative Thermal Block machine (Bioneer). The primer sequences were as follows: VEGF-A (forward: 5’-CGAGATAGAGTACA TCTTCAAGCC-3', reverse: 5'-TCATCGTTACAGCAGCCTGC-3') and β-actin (forward: 5'-TGGAATCCTGTGGCATCCAT-3', reverse: 5'-TGGTACCACCAGACAGCACTG-3') (Genotech). Polymerase chain reaction was performed using the following conditions: 1 cycle of 95℃ for 5 minutes, followed by 40 cycles of 95℃ for 10 seconds and 55℃ for 20 seconds. VEGF-A mRNA levels were normalized using β-actin.

5. Statistical analysis

Values were expressed as mean ± standard deviation. Statistical analysis was performed using analysis of variance followed by Turkey’s post-hoc tests. Statistical significance was defined as P < 0.05.

1. 17β-estradiol reduces body weight and total adipose tissue mass in obese OVX mice

After 8 weeks, the average body weight of HFD mice (30.19 ± 3.33 g) was 36% higher than that of LFD mice (22.20 ± 0.97 g) (P < 0.05; Fig. 1A). The average body weight of HFD-E mice; 24.76 ± 1.32 g) was 18% lower than that of HFD mice (P < 0.05). Similar to the body weight changes, HFD-E decreased total adipose tissue mass compared with HFD. 17β-estradiol reduced adipose tissue weight in HFD mice by 98% (P < 0.05; Fig. 1B). Representative photographs of visceral adipose tissue are shown in Fig. 1C. Liver weights of HFD mice were not significantly different compared with LFD mice (Fig. 1D). In addition, 17β-estradiol supplementation also did not change liver weights. The livers of HFD mice were yellowish-red in color compared with those of LFD mice, but 17β-estradiol treatment of HFD mice restored the red color of the livers to the level of LFD mice (Fig. 1E). Food intake was significantly increased in HFD mice compared to LFD mice, but there was no difference between HFD and HFD-E mice (data not shown).

Fig. 1. Body weight, total adipose tissue mass, liver weight, and liver gross morphology. Female ovariectomized mice (n = 8/group) were fed an LFD, an HFD, or an HFD-E for 8 weeks. (A) Body weights at the end of the treatment period are significantly different between the HFD group and the LFD or HFD-E (P < 0.05) groups. (B) Total adipose tissue mass. (C) Photographs of visceral adipose tissue. (D) Liver weights. (E) Gross morphology of the liver. All values are expressed as the mean ± standard deviation. LFD, low-fat diet; HFD, high-fat diet; HFD-E, HFD plus 17β-estradiol. #P < 0.05 compared with LFD. *P < 0.05 compared with HFD.

2. 17β-estradiol lowers serum ALT levels in obese OVX mice

Serum ALT concentrations were 220% higher in HFD mice than in LFD mice (P < 0.05; Fig. 2A), and 17β-estradiol treatment significantly decreased ALT levels by 59% in HFD-E mice compared with HFD mice (P < 0.05). Similarly, AST concentrations were 93% higher in HFD mice compared with LFD mice (P < 0.05; Fig. 2B), but 17β-estradiol administration showed a trend towards decreasing serum AST levels in HFD-E mice compared with HFD mice. Circulating total cholesterol levels were increased by 19% in HFD mice compared with LFD mice (P < 0.05; Fig. 2C). However, 17β-estradiol did not affect these levels. Serum triglyceride levels were not significantly different between HFD and HFD-E mice (Fig. 2D).

Fig. 2. Circulating levels of ALT, AST, total cholesterol, and triglycerides. Female ovariectomized mice (n = 8/group) were fed an LFD, an HFD, or an HFD-E for 8 weeks. Serum levels of (A) ALT, (B) AST, (C) total cholesterol, and (D) triglycerides. All values are expressed as the mean ± standard deviation. ALT, alanine aminotransferase; AST, aspartate aminotransferase; LFD, low-fat diet; HFD, high-fat diet; HFD-E, HFD plus 17β-estradiol. ^#P < 0.05 compared with LFD. *P < 0.05 compared with HFD.

3. 17β-estradiol attenuates hepatic lipid accumulation in obese OVX mice

An analysis of the HE-stained liver sections showed that triglyceride accumulation was 63% greater in HFD mice compared with LFD mice (P < 0.05; Fig. 3A, 3B), whereas lipid droplets were reduced by 74% in HFD-E mice compared with HFD mice (P < 0.05).

Fig. 3. Hepatic steatosis. Female ovariectomized mice (n = 8/group) were fed an LFD, an HFD, or an HFD-E for 8 weeks. (A) Hematoxylin-eosin-stained liver sections (original magnification ×100). (B) Relative lipid droplet area. All values are expressed as mean ± standard deviation. LFD, low-fat diet; HFD, high-fat diet; HFD-E, HFD plus 17β-estradiol. #P < 0.05 compared with LFD. *P < 0.05 compared with HFD.

4. 17β-estradiol decreases CD68-positive cells and inflammatory foci in obese OVX mice

To test the effects of 17β-estradiol on hepatic inflammation, liver sections were stained with an antibody against CD68, a macrophage marker. The number of CD68-positive cells was 307% higher in HFD mice than in LFD mice (P < 0.05; Fig. 4A, 4B) and 83% lower in HFD-E mice compared with HFD mice (P < 0.05). Lobular inflammatory foci were increased by 350% in HFD mice compared with LFD mice (P < 0.05; Fig. 4C, 4D), and HFD-E reduced the inflammatory foci by 50% compared with HFD alone (P < 0.05).

Fig. 4. Liver CD68-positive cells and inflammatory foci. Female ovariectomized mice (n = 8/group) were fed an LFD, an HFD, or an HFD-E for 8 weeks. (A) Liver sections stained with an antibody against CD68 are shown in brown color (original magnification ×100). (B) Relative CD68-positive area. (C) Hematoxylin-eosin-stained liver sections. Dark arrows indicate inflammatory foci (original magnification ×200). (D) Lobular inflammation score. All values are expressed as mean ± standard deviation. LFD, low-fat diet; HFD, high-fat diet; HFD-E, HFD plus 17β-estradiol. #P < 0.05 compared with LFD. *P < 0.05 compared with HFD.

5. 17β-estradiol suppresses liver collagen accumulation in obese OVX mice

Masson’s trichrome–stained liver sections revealed 163% higher collagen levels in HFD mice than in LFD mice (P < 0.05; Fig. 5A, 5B). By contrast, hepatic collagen levels were 64% lower in HFD-E mice than in HFD mice (P < 0.05).

Fig. 5. Hepatic collagen accumulation. Female ovariectomized mice (n = 8/group) were fed an LFD, an HFD, or an HFD-E for 8 weeks. (A) Masson’s trichrome-stained liver sections (original magnification ×100). (B) Relative collagen area. All values are expressed as mean ± standard deviation. LFD, low-fat diet; HFD, high-fat diet; HFD-E, HFD plus 17β-estradiol. #P < 0.05 compared with LFD. *P < 0.05 compared with HFD.

6. 17β-estradiol suppresses angiogenesis and VEGF-A expression in visceral adipose tissue of obese OVX mice

To determine the effects of 17β-estradiol on blood vessel density in visceral adipose tissue, adipose tissue sections were stained with an antibody against vWF, an endothelial cell marker. Blood vessel densities were 71% higher in HFD mice than LFD mice (P < 0.05; Fig. 6A, 6B) and 21% lower in HFD-E mice than in HFD mice (P < 0.05). To examine the effects of 17β-estradiol on VEGF-A expression, VEGF-A mRNA levels were measured in adipose tissue. VEGF-A mRNA levels were 25% lower in HFD mice than in LFD mice (P < 0.05; Fig. 6C) and 30% lower in HFD-E mice than in HFD mice (P < 0.05).

Fig. 6. Blood vessel density and VEGF-A mRNA expression in visceral adipose tissue. Female ovariectomized mice (n = 8/group) were fed an LFD, an HFD, or an HFD-E for 8 weeks. (A) Visceral adipose tissue sections stained with an antibody against von Willebrand factor are shown in dark brown color (original magnification ×100). (B) Quantification of blood vessel density. (C) VEGF-A mRNA levels. All values are expressed as the mean ± standard deviation. VEGF-A, vascular endothelial growth factor A; LFD, low-fat diet; HFD, high-fat diet; HFD-E, HFD plus 17β-estradiol. #P < 0.05 compared with LFD. *P < 0.05 compared with HFD.

The prevalence of NAFLD is rising steeply alongside obesity. Postmenopausal women in particular have a higher risk of obesity and NAFLD compared with premenopausal women due to estrogen deficiency and also experience faster progression and greater severity of NAFLD ⁽¹⁵⁾. There are three primary forms of estrogen: estrone, estradiol, and estriol. In the female reproductive years, the most prevalent and potent circulating estrogen is 17β-estradiol. Therefore, we investigated the effects of 17β-estradiol on obesity and NAFLD in HFD–fed obese female OVX C57BL/6J mice, a mouse model of obese postmenopausal women, and examined the involvement of angiogenesis and VEGF-A in this process.

After 8 weeks of HFD feeding, the mice showed significantly greater body weight and total adipose tissue mass compared with LFD mice. In addition, serum levels of the liver damage markers ALT and AST were higher in HFD mice compared with LFD mice. However, HFD-E mice showed reduced body weight, adipose tissue mass, and serum ALT levels compared with HFD mice. Kim et al. ⁽¹⁶⁾ reported that an 8-week 17β-estradiol treatment reduced body weight and adipose tissue weight in HFD–fed OVX C57BL/6J mice. These results suggest that 17β-estradiol may improve obesity and liver damage.

An HFD is likely to promote the development of NAFLD by causing the accumulation of lipids in the liver ⁽¹⁷⁾. Hepatic fat accumulation may arise due to a combination of factors such as heightened fat synthesis, increased fat delivery, reduced fat export, and decreased fat oxidation ⁽¹⁸⁾. We observed hepatic triglyceride accumulation in HFD mice ^(19,20). By contrast, 17β-estradiol treatment decreased hepatic lipid accumulation in HFD-E mice compared with HFD mice. These results are consistent with the notion that 17β-estradiol inhibits hepatic steatosis in obese OVX mice ⁽¹⁶⁾.

To investigate the effect of 17β-estradiol on HFD-induced hepatic inflammation, liver sections were stained with HE and immunostained with an antibody against CD68, a macrophage marker ^(21,22). The HE-stained sections showed increased inflammatory foci in the livers of HFD mice compared with LFD mice. By contrast, 17β-estradiol decreased hepatocyte inflammatory foci in HFD-E mice compared with HFD mice. 17β-estradiol treatment also reduced the number of CD68-positive cells in HFD mice. These results suggest that 17β-estradiol reduces CD68-positive macrophages and inflammatory foci, resulting in decreased hepatic inflammation in obese OVX mice.

Liver fibrosis results from chronic liver inflammation and is characterized by the excessive accumulation of extracellular matrix proteins, including collagen ^(17,23,24). Approximately 25%–40% of patients with nonalcoholic steatohepatitis develop progressive liver fibrosis ^(25,26). Hepatic collagen levels were determined using Masson’s trichrome ⁽²⁷⁾. The HFD led to an increase in liver collagen levels compared with the LFD. By contrast, treatment with 17β-estradiol reduced hepatic collagen accumulation in HFD mice. These findings suggest that 17β-estradiol can reduce hepatic fibrosis in HFD–fed OVX female mice.

Angiogenesis is the creation of new blood vessels from existing blood vessels. Angiogenesis normally occurs during crucial stages of embryonic development, wound healing, and menstruation ⁽²⁸⁾. Dysregulated angiogenesis is associated with various diseases, such as cancer, rheumatoid arthritis, psoriasis, and proliferative retinopathy. The growth and expansion of adipose tissue also depend on the formation of new blood vessels to support the supply of oxygen and nutrients to the adipocytes ⁽¹²⁾. As adipose tissue enlarges, an increased vascular network is needed to maintain its metabolic demands. Angiogenesis stimulators and inhibitors therefore affect the adipose tissue expansion and may regulate obesity and metabolic disorders ^(13,29). To investigate the effect of 17β-estradiol on angiogenesis in the adipose tissue of HFD-induced obese OVX mice, blood vessel density was examined using an antibody against vWF, an endothelial cell marker. Immunostaining revealed an increase in the blood vessel density of visceral adipose tissue in HFD mice compared with LFD mice. By contrast, mice treated with 17β-estradiol exhibited reduced blood vessel density compared with HFD mice. These results suggest that 17β-estradiol reduces adipose tissue angiogenesis in obese OVX mice.

VEGF-A serves as both a mitogen and a survival factor for vascular endothelial cells, playing a crucial role in promoting the motility of vascular endothelial cells and monocytes ^(30,31). The expression of VEGF-A is regulated by several factors in various tissues and conditions, such as hypoxia; different cytokines, growth factors, and hormones; and, notably, oncogenes and tumor suppressor genes in tumors ⁽³²⁾. Because these diverse regulators of VEGF-A collectively contribute to the process of angiogenesis, we investigated the effects of 17β-estradiol on VEGF-A mRNA expression in adipose tissue, where angiogenesis is most prominent. We found that 17β-estradiol treatment reduced VEGF-A mRNA levels in HFD-E mice compared with HFD mice. In contrast to our results, a previous study reported that VEGF-A mRNA and protein levels were reduced in the adipose tissue of estrogen receptor 1 knockout female mice compared with wild-type mice ⁽³³⁾, suggesting that estrogen enhances VEGF-A expression in adipose tissue of female mice. However, Fatima et al. ⁽³³⁾ studied lean female mice, whereas we investigated the adipose tissues of HFD–fed obese OVX female mice, which may explain our differing results.

In conclusion, our data suggest that 17β-estradiol reduces body and fat weights in obese OVX mice, an animal model of obese postmenopausal women. It also inhibits hepatic steatosis, inflammation, fibrosis, and serum ALT levels. Moreover, 17β-estradiol decreases angiogenesis and VEGF-A mRNA expression in adipose tissue. These findings suggest that 17β-estradiol may prevent obesity and NAFLD in obese OVX female mice in part by regulating adipose tissue angiogenesis and VEGF-A.

This study was financially supported by the research year fund of Mokwon University in 2024.

No potential conflict of interest relevant to this article was reported.

None.

Conceptualization: all authors. Data curation: all authors. Formal analysis: all authors. Funding acquisition: MY. Investigation: all authors. Project administration: MY. Resources: MY. Validation: all authors. Visualization: SJ. Writing – original draft: all authors. Writing – review and editing: all authors.