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Early prenatal undernutrition leads to metabolic dysfunction-associated steatotic liver disease in a rat model of menopause

Published online by Cambridge University Press:  24 October 2025

Tomoko Kimura Open the ORCID record for Tomoko Kimura [Opens in a new window] ,
Kodai Hino ,
Minoru Kuroida  and
Jun Udagawa
Tomoko Kimura*
Affiliation:
Department of Physical Therapy, Faculty of Health Sciences, Kyoto Tachibana University, Kyoto, Japan Division of Anatomy and Cell Biology, Department of Anatomy, Shiga University of Medical Science, Otsu, Japan
Kodai Hino
Affiliation:
Division of Anatomy and Cell Biology, Department of Anatomy, Shiga University of Medical Science, Otsu, Japan
Minoru Kuroida
Affiliation:
Division of Anatomy and Cell Biology, Department of Anatomy, Shiga University of Medical Science, Otsu, Japan
Jun Udagawa
Affiliation:
Division of Anatomy and Cell Biology, Department of Anatomy, Shiga University of Medical Science, Otsu, Japan
*
Corresponding author: Tomoko Kimura; Email: kimura-to@tachibana-u.ac.jp
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Abstract

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has increased. MASLD notably increases after menopause in women owing to the drastic reduction in estrogen, which regulates lipid metabolism. While prenatal undernutrition leads to hepatic steatosis after birth, whether prenatal undernutrition affects the onset of postmenopausal MASLD remains unknown. Therefore, we examined the impact of early prenatal undernutrition on the predisposition to postmenopausal MASLD in a rat model of menopause. Pregnant female rats were assigned to the control (CNTL) group, while the undernourished (UN) group was fed 40% of the diet of the control group. Furthermore, both groups were assigned to the ovariectomized (CNTL-OVX/UN-OVX) and sham-operated (CNTL-Sham/UN-Sham) groups at 12 weeks of age. Two-way analysis of variance revealed significant main effects of ovariectomy and prenatal undernutrition on body weight and hepatic triglyceride content. Triglycerides accumulated in the liver at 12 and 24 weeks after ovariectomy, while hepatic steatosis was histologically observed at 24 weeks after ovariectomy in UN-OVX rats. Hepatic gene expression analyses showed an interaction effect between prenatal undernutrition × ovariectomy in ESR1 expression; however, PPARα, RXRα, RARα, Raldh1, and Raldh3 expression was not affected by prenatal undernutrition and ovariectomy. These results suggest that early prenatal undernutrition predisposes postmenopausal women to MASLD by uncovering aberrant estrogen signaling, which may be influenced by estrogen reduction.

Keywords

Prenatal undernutritionMASLDobesitypostmenopausal

Information

Type
Original Article
Information
Journal of Developmental Origins of Health and Disease , Volume 16 , 2025 , e39
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Copyright
© The Author(s), 2025. Published by Cambridge University Press in association with The International Society for Developmental Origins of Health and Disease (DOHaD)

Introduction

Metabolic dysfunction-associated liver steatotic disease (MASLD) is a metabolic liver injury in which triglycerides (TG) accumulate in the hepatocytes.Reference Scorletti and Carr1 MASLD is a leading cause of liver-related morbidity and mortality and is typically accompanied by obesity.Reference Li, Yang, Ye, Xu, Wu and Wang2 The prevalence of MASLD is increasing globally,Reference Younossi, Golabi, Paik, Henry, Van Dongen and Henry3 and is higher in men than in premenopausal women; however, the prevalence of MASLD is comparable in women after menopause,Reference Lonardo, Nascimbeni and Ballestri4 as estrogen is crucial for regulating lipogenesis and fatty acid (FA) oxidation.Reference Eng, Forlano, Tan, Manousou, Dhillo and Izzi-Engbeaya5 By contrast, maternal undernutrition during pregnancy leads to metabolic syndromes, including obesity and hepatic steatosis.Reference Li, Reynolds, Segovia, Gray and Vickers6,Reference Fall and Kumaran7 The risks of obesity, dyslipidemia, and type 2 diabetes are high not only in individuals with low birth weight who experience rapid postnatal growth but also in normal birth weight offspring subjected to early pregnancy exposure to famine.Reference Fall and Kumaran7 In the Great Chinese Famine study, Zhen et al. reported that women prenatally exposed to famine displayed higher risks of mild, moderate, and severe steatosis.Reference Zheng, Ren, Gong, Long, Luo and Wang8 In animal experiments, a maternal low-protein diet (9% casein) throughout early (gestational days (GD) 0–7), mid (GD 8–14), and late (GD 15–22) gestation could lead to hepatic steatosis without obesity in rat offspring at 18 months, but not at 9 months of age.Reference Erhuma, Salter, Sculley, Langley-Evans and Bennett9 In addition, a severe low-protein diet (4% protein) from 14 days of gestation to delivery led to both obesity and hepatic steatosis in the rat offspring at age 91 days.Reference de Oliveira, Gomes and Miranda10 These reports suggest that prenatal undernutrition during early, mid-, or late gestation may be involved in the development of premenopausal hepatic steatosis. Meanwhile, a high-fat diet after weaning or 5% sucrose administration from 12 months of age exacerbated hepatic steatosis in the offspring of dams fed a low-protein diet between 11.5 and 17.5 days of gestation or throughout gestation, respectively.Reference Nicolás-Toledo, Cervantes-Rodríguez and Cuevas-Romero11–Reference Muramatsu-Kato, Itoh and Kohmura-Kobayashi13 Thus, prenatal undernutrition can be the first hit, while high-fat or high-carbohydrate intake after weaning can be the second hit in the multiple-hit theory of hepatic steatosis. Similarly, the risk of postmenopausal MASLD may be influenced by prenatal undernutrition; however, it remains unclear whether this increases the prevalence and incidence of postmenopausal MASLD.

In this study, we hypothesized that early prenatal undernutrition, which does not alter birth weight, predisposes offspring to postmenopausal MASLD. To verify this hypothesis, changes in body weight and hepatic lipid accumulation were examined in a rat model of menopause subjected to early prenatal undernutrition. Changes in glucose and fatty metabolism, including de novo lipogenesis, βoxidation of fatty acids, and export of hepatic lipids, are involved in the pathogenesis of MASLD.Reference Habibullah, Jemmieh, Ouda, Haider, Malki and Elzouki14 Gene expressions related to lipid metabolism, i.e., the hepatic estrogen receptor (ER), peroxisome proliferator-activated receptor (PPAR), retinoid X receptor (RXRs), retinoic acid receptor α (RARα), and retinaldehyde dehydrogenase 1 and 3 (RALDH1 and 3), were examined as well as serum biochemical analysis owing to the following reasons: (i) ER signaling reduces lipid synthesis and absorption, and promotes FA oxidation,Reference Chen, Wang, Huang and Zhang16 and hepatic ER signaling may be affected by prenatal undernutrition since ER expression was altered in the peripheral organ such as ovary, with reduced serum E2 level at age of 10 months,Reference Khorram, Keen-Rinehart, Chuang, Ross and Desai15 (ii) PPAR regulates FA oxidation metabolism and energy consumption, and modulates lipid storage in the liver,Reference Habibullah, Jemmieh, Ouda, Haider, Malki and Elzouki14,Reference Qiu, Zhang, Zeng and Zhu17 (iii) RXRs heterodimerizes with PPARs and plays a role in regulating hepatic steatosis progression,Reference Puengel, Liu, Guillot, Heymann, Tacke and Peiseler18 (iv) retinoic acid is a key regulator of lipid metabolism in the liver,Reference Saeed, Dullaart, Schreuder, Blokzijl and Faber19 and RARα plays an important role in preventing hepatic steatosis,Reference Cassim Bawa, Xu and Gopoju20 and (vi) retinaldehyde dehydrogenase converts retinal to retinoic acid, and the hepatic expression of RALDH 1 and 3 is elevated in patients with non-alcoholic liver disease.Reference Saeed, Dullaart, Schreuder, Blokzijl and Faber19

Methods

Animals and study design

All experimental procedures and protocols were reviewed and approved by the animal ethics committee of Shiga University of Medical Science (#2018–5–4). Nine–week-old male and eight-week-old female Wistar rats with body weights (BW) of 250–280 and 160–190 g, respectively, were purchased from CLEA Japan, Inc. (Tokyo, Japan) and were housed at room temperature (20–25°C) under 40–60% relative humidity with a 12–h light/dark cycle (lights on at 08:00) and free access to food and tap water. Female rats were housed overnight with one male. The gestational day (G) 0.0 was defined as 0:00 on the day when a vaginal plug was observed. Pregnant rats were randomly assigned to control (CNTL; n = 14) or undernourished (UN; n = 13) groups. Rats in the UN group were fed 40% of the average amount of daily food intake of the rats of the same gestational day in the control group, which were fed a standard AIN-93G diet (Supplementary Table S1) ad libitum from G 5.5 to 11.5 as blastocyst implantation occurs around G 5.5 and liver specification and hepatoblast differentiation are observed during the period of undernutrition.Reference Mu, Xu and Zhong21 After delivery, pups were randomly culled to eight pups per litter on postnatal day (P) 4, and the dams were fed CE-2 (CLEA Japan, Inc., Tokyo, Japan; Supplementary Table S1) during lactation. Offspring were weaned on P21 and fed CE-2. The BWs of the female offspring from the 24 dams were measured weekly until 36 weeks of age. Female rats underwent ovariectomy (OVX) or sham surgery at 12 weeks of age under deep anesthesia using isoflurane (5% of concentration for induction and 2.5% for maintenance) in both control and UN groups; therefore, rats were assigned to a control sham surgery group (CNTL-Sham: n = 22 from 10 dams), control OVX group (CNTL-OVX: n = 20 from 10 dams), undernourished sham surgery group (UN-Sham: n = 20 from 9 dams), and undernourished OVX group (UN-OVX: n = 19 from 9 dams). Rats were anesthetized and euthanized with pentobarbital (60 mg/kg) at 12 weeks of age before surgery (CNTL: n = 5 from 4 dams, UN: n = 9 from 3 dams), 24 weeks of age, i.e., 12 weeks after surgery (CNTL-Sham: n = 11 from 5 dams, CNTL-OVX: n = 9 from 5 dams, UN-Sham: n = 11 from 5 dams, UN-OVX: n = 9 from 5 dams), and 36 weeks of age, i.e., 24 weeks after surgery (CNTL-Sham: n = 11 from 5 dams, CNTL-OVX: n = 11 from 5 dams, UN-Sham: n = 10 from 4 dams, UN-OVX: n = 9 from 4 dams). Blood samples were collected from the left ventricle after fasting for 12 h and used for biochemical examinations. The livers were frozen for histological and biochemical examinations, and gene expression analysis was performed after the livers, spleens, kidneys, and adrenals were weighed.

Body and organ weights

Body weight was measured at birth (CNTL: n = 50, 10 dams; UN: n = 43, 9 dams). After culling the neonates, female rats (CNTL: n = 44 from 10 dams; UN: n = 37 from 9 dams) were weighed once a week until 10 weeks of age. Subsequently, the females that underwent surgery were weighed at 12 weeks of age (before the surgery) and once a week until 24 weeks after the surgery (36 weeks). The liver, spleen, kidney, and adrenal weights were measured at 12, 24, and 36 weeks of age. In addition, the organ and body weight ratio was calculated at the same age.

Blood tests

After fasting for 12 h, blood was collected from the tail vein at 0 (before the surgery) and 2, 4, 6, 9, 12, and 24 weeks after the surgery, followed by glucose level measurement using a glucose meter (Accu-Chek Aviva Nano, Roche DC Japan, Inc., Tokyo, Japan) in the CNTL-Sham (n = 11 from 5 dams), CNTL-OVX (n = 9 from 5 dams), UN-Sham (n = 11 from 5 dams), and UN-OVX (n = 9 from 5 dams) groups. Blood collected from the left ventricle at 12, 24, and 36 weeks of age was allowed to stand for 30 min, followed by centrifugation at 1200 ×g for 10 min. Serum was used for biochemical tests, i.e., blood glucose, aspartate transaminase (AST), and alanine transaminase (ALT) activities, as well as the concentration of the triglyceride (TG), total cholesterol (T-CHO), low- and high-density lipoprotein cholesterols (LDL-CHO and HDL-CHO), free cholesterol (F-CHO), esterified cholesterol (E-CHO), and non-esterified fatty acids (NEFA), in the laboratory testing company (Oriental Bio Service, Kyoto, Japan).

Lipids in the liver

The frozen livers were homogenized with a pestle, centrifuged at 15,000 × g for 10 min, and the supernatant was collected. The TG and T-CHO levels were determined following the instructions of the LabAssay Triglyceride Kit and LabAssay Cholesterol Kit (Wako, Osaka, Japan), respectively. The absorbance at 600 nm of the samples reacted with chromogen reagent was measured using the spectrophotometer (V-570 DS, JASCO, Tokyo, Japan). To measure the protein content, we used a BCA protein assay kit (NACALAI TESQUE, Kyoto, Japan), and the protein concentration was determined by measuring the absorbance of the samples at 562 nm. The amounts of TG and T-CHO per milligram of protein were calculated and compared among the four groups.

Histological analysis of the liver

For lipid accumulation analysis, the liver’s cryosections were cut to a thickness of 10 μm and fixed with 4% paraformaldehyde/PBS solution for 30 min. Lipid droplets and nuclei were stained with Oil Red O and Hematoxylin, respectively, and the ratio of the area of lipid droplets stained red to the total area was measured using image acquisition software (NIS-Elements, Nikon, Tokyo, Japan). The livers of the same samples were also fixed with 4% paraformaldehyde, embedded in paraffin, and cut into serial sections (5 μm). These paraffin sections were stained with hematoxylin-eosin (HE) to verify hepatocyte lipid accumulation and assess inflammation. Furthermore, the paraffin sections were stained with Sirius red to assess hepatic fibrosis.

Gene expression analyses

The gene expressions of the hepatic estrogen receptor (ER), peroxisome proliferator-activated receptor (PPAR), retinoid X receptor (RXRs), retinoic acid receptor α (RARα), retinaldehyde dehydrogenase 1 and 3 (RALDH1 and 3) were determined by real-time PCR analyses. Data with multiple melting curves were excluded from the analysis. The primer sequences used for the real-time PCR analyses are listed in Supplementary Table S2. Total RNA was isolated from the livers using Sepasol-RNA I Super G (NACALAI TESQUE, Kyoto, Japan), converted to cDNA via reverse transcription using PrimeScript RT Master Mix (Takara Bio, Inc., Shiga, Japan), and subjected to real-time PCR analysis using a LightCycler 480 system (Roche Diagnostics GmbH) with SYBR Premix Ex Taq II polymerase (Takara Bio, Inc.). Real-time PCR was performed in duplicate, and comparative Ct values of target genes were normalized to those of B2m and compared among the CNTL-sham, CNTL-OVX, UN-Sham, and UN-OVX groups.

Statistics

All statistical analyses were conducted using JMP Pro 17.0 (SAS Institute Inc., Cary, NC, USA). Data are presented as the mean ± Standard Error. Student’s t-test was used to compare the averages of the UN and CNTL groups regarding BW, organ weights, blood tests, and hepatic accumulation of lipids before surgery. Differences in those measures and hepatic gene expressions among CNTL-OVX, CNTL-Sham, UN-OVX, and UN-Sham groups after surgery were assessed by two-way analysis of variance (ANOVA) with main effects of nutrition (normal vs. undernutrition) and surgery (OVX vs. sham), followed by Tukey’s honestly significant difference (HSD) test, and with an interaction effect between maternal nutrition and surgery. Statistical significance was set at p < 0.05 for all tests, considered significant.

Results

Impact of early prenatal undernutrition on obesity in menopausal model rats

There was no significant difference in BW between the offspring in the CNTL and UN groups before surgery, even at birth (Fig. 1a). Two-way ANOVA revealed significant main effects of ovariectomy on BW from 14 weeks of age and those of prenatal nutrition on BW from 23 weeks of age. However, prenatal nutrition had no significant effects on BW until 13 weeks of age, 1 week after surgery (Supplementary Tables S3–S4). Obesity was more severe in UN-OVX rats than in CNTL-OVX, CNTL-Sham, and UN-Sham rats at 36 weeks of age (Fig. 1b, Fig. 2).

Figure 1.

Changes in offspring body weight. (a) Body weights of CNTL and UN groups before surgery, and (b) body weights of CNTL-Sham, CNTL-VX, UN-Sham, and UN-OVX groups after surgery were measured once a week. CNTL: control group, CNTL-OVX: ovariectomized control group, UN: prenatal undernourished group, UN-OVX: ovariectomized prenatal undernourished group. Values are presented as means ± SEMs. *P < 0.05 CNTL-OVX and UN-OVX vs CNTL-Sham, §P < 0.05 UN-OVX vs UN-Sham, †P < 0.05 UN-OVX vs CNTL-OVX by Tukey’s HSD test.

Figure 2.

Obesity led by prenatal undernutrition and ovariectomy. Dorsal views are shown in the representative rats of the CNTL (a), UN (b), CNTL-OVX (c), and UN-OVX (d) groups 24 weeks after surgery. Scale bar: 5 cm. CNTL: control group, CNTL-OVX: ovariectomized control group, UN: prenatal undernourished group, UN-OVX: ovariectomized prenatal undernourished group.

Impact of early prenatal undernutrition and ovariectomy on organ weights

The average weight of bilateral kidneys was significantly reduced in the UN offspring than in CNTL offspring at 12 weeks of age (Student’s t-test) (Fig. 3a). Two-way ANOVA revealed significant main effects of ovariectomy on the liver, spleen, and adrenal weights at 12 weeks after surgery (Fig. 3 b–d, Supplementary Table S5). Further, ovariectomy had a significant main effect only on the spleen weight 24 weeks after surgery (Fig. 3, Supplementary Table S6). The spleen was heavier in CNTL- and UN-OVX than in CNTL- and UN-Shams, respectively, at 24 weeks after ovariectomy by Tukey’s HSD test (Fig. 3c).

Figure 3.

Organ weights of the offspring. The liver weights (a), the spleen weights (b), and the average weights of bilateral kidneys (c) and adrenals (d) are shown. CNTL: control group, CNTL-OVX: ovariectomized control group, UN: prenatal undernourished group, UN-OVX: ovariectomized prenatal undernourished group. Values are presented as means ± SEMs. ‡‡P < 0.01 vs CNTL by Student’s t-test before surgery. *P < 0.05 CNTL-OVX and UN-OVX vs CNTL-Sham, §P < 0.05 UN-OVX vs UN-Sham by Tukey’s HSD test after surgery.

Change in blood glucose and lipid levels by prenatal undernutrition and ovariectomy

The fasting blood glucose (FBG) concentration was higher in the UN-OVX group than in the other 2 weeks after OVX by Tukey’s HSD test (Fig. 4). FBG levels were also higher in the UN-OVX group than in the CNTL- and UN-Sham groups 4 weeks after surgery and in the UN-Sham group 9 weeks after surgery (Fig. 4). Two-way ANOVA revealed the main effects of maternal nutrition on FBG at 2 weeks after surgery (Supplementary Table S7); however, no significant difference was noted in FBG levels between UN and CNTL offspring before surgery (Student’s t-test), with no main effects of maternal nutrition on FBG at 4 or more weeks after surgery (two-way ANOVA) (Fig 5, Supplementary Table S7–S9). By contrast, two-way ANOVA revealed the main effects of ovariectomy on FBG at 2, 4, and 9 weeks after surgery (Supplementary Table S7). Two-way ANOVA also revealed main effects of maternal nutrition on T-CHO, F-CHO, NEFA, and NEFA/TG and those of ovariectomy on ALT, T-CHO, F-CHO, E-CHO, LDL-C, and HDL-C at 12 weeks after surgery (Supplementary Table S8), while two-way ANOVA revealed main effects of only ovariectomy on T-CHO, E-CHO, and HDL-C at 24 weeks after surgery (Supplementary Table S9). Ovariectomy increased these levels at 12 and 24 weeks after surgery, whereas T-CHO and F-CHO were reduced by undernutrition at 12 weeks after surgery (Figs. 6 and 7, Supplementary Tables S8 and S9).

Figure 4.

Fasting blood glucose until 9 weeks after surgery. Fasting blood glucose from the tail vein was measured until 9 weeks after surgery using a glucometer. CNTL: control group, CNTL-OVX: ovariectomized control group, UN: prenatal undernourished group, UN-OVX: ovariectomized prenatal undernourished group. Values are presented as means ± SEMs. *P < 0.05 vs CNTL-Sham, §P < 0.05 vs UN-Sham, †P < 0.05 vs CNTL-OVX by Tukey’s HSD test.

Figure 5.

Blood examination before surgery. Fasting blood glucose (FBG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (T-CHO), free cholesterol (F-CHO), esterified cholesterol (E-CHO), triglycerides (TG), non-esterified fatty acids (NEFA), NEFA/TG, LDL cholesterol, (LDL-C), HDL cholesterol (HDL-C), and LDL/HDL were measured in the serum of the offspring aged 12 weeks. Values are presented as means ± SEMs. *P < 0.05; Tukey’s HSD test.

Figure 6.

Blood examination 12 weeks after surgery. Fasting blood glucose (FBG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (T-CHO), free cholesterol (F-CHO), esterified cholesterol (E-CHO), triglycerides (TG), non-esterified fatty acids (NEFA), NEFA/TG, LDL cholesterol, (LDL-C), HDL cholesterol (HDL-C), and LDL/HDL in the serum were measured. Values are presented as means ± SEMs. *P < 0.05 CNTL-OVX vs CNTL-Sham, §P < 0.05 UN-OVX vs UN-Sham by Tukey’s HSD test.

Figure 7.

Blood examination 24 weeks after surgery. Fasting blood glucose (FBG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (T-CHO), free cholesterol (F-CHO), esterified cholesterol (E-CHO), triglycerides (TG), non-esterified fatty acids (NEFA), NEFA/TG, LDL cholesterol, (LDL-C), HDL cholesterol (HDL-C), and LDL/HDL in the serum were measured. Values are presented as means ± SEMs.

Hepatic lipid accumulation and inflammation in the menopausal model subjected to early prenatal undernutrition

TG (g/g protein) and T-CHO (g/g protein) levels in the liver did not differ between the UN and CNTL groups before OVX (Fig 8); however, we observed main effects of maternal nutrition and ovariectomy on hepatic TG at 12 and 24 weeks after surgery, and those of maternal nutrition on T-CHO at 24 weeks after surgery in two-way ANOVA (Supplementary Table S10). Furthermore, two-way ANOVA revealed a significant interaction between maternal nutrition and surgery (Supplementary Table S10). Significant simple effects were noted between sham-operated and ovariectomized UN offspring and between CNTL and UN offspring with ovariectomy at 12 weeks and 24 weeks after surgery (Supplementary Table S11). The TG content in the liver was significantly higher in the UN-OVX group than in the CNTL-Sham, UN-Sham, and CNTL-OVX groups, and the T-CHO content in the liver was significantly increased in the UN-OVX group than in the CNTL-Sham group 24 weeks after surgery, as per Tukey’s HSD test (Fig 8). Histological examination using HE and Oil Red O staining showed lipid accumulation in the hepatocytes of the UN-OVX group 24 weeks after OVX; however, inflammatory cell infiltration and fibrosis were not observed in the livers of all groups (Fig. 9). These results indicate that early prenatal undernutrition aggravates lipid accumulation but does not promote inflammation and fibrosis in the liver in a rat model of menopause.

Figure 8.

Hepatic TG and T-CHO. Triglycerides (TG) and total cholesterol (T-CHO) were measured in the offspring’s liver. Values are presented as means ± SEMs. *P < 0.05 CNTL-OVX vs CNTL-Sham, §P < 0.05 UN-OVX vs UN-Sham, †P < 0.05 UN-OVX vs CNTL-OVX by Tukey’s HSD test.

Figure 9.

Lipid accumulation and fibrosis in the liver section of the offspring 24 weeks after surgery. Liver sections were stained by hematoxylin and eosin (a–h), Oil Red O (i–p), and Picrosirius Red (q–t)staining. Images surrounded by a box (a–d) in HE and those in Oil Red O staining (i–l) were magnified in e–h and m–p, respectively. Arrowheads in the hepatic sections indicated lipid droplets with HE staining. Lipid droplets are stained red with Oil Red O (i–p). Scale bar: 100 μm (a–d, i–l, and q–t), 10 μm (e–h and m–p). CNTL: control group, CNTL-OVX: ovariectomized control group, UN: prenatal undernourished group, UN-OVX: ovariectomized prenatal undernourished group.

Prenatal undernutrition alters hepatic gene expression patterns after ovariectomy

Two-way ANOVA revealed a significant interaction between maternal nutrition and surgery in ESR1 expression in the liver (Supplementary Table S12). Significant simple effects were noted between sham-operated and ovariectomized rats with prenatal undernutrition and between CNTL and UN offspring with ovariectomy at 24 weeks after surgery (Supplementary Table S13). ESR1 expression was higher in UN-OVX rats than in UN-Sham rats by the Tukey’s HSD test (Fig. 10a). By contrast, ESR1 expression was not different between the CNTL-OVX and other groups but was significantly higher in the CNTL-Sham group than in the UN-Sham group (Fig. 10a). RXR, RAR, Raldh1, and Raldh3 gene expressions were not significantly different (Fig. 10b–e).

Figure 10.

Gene expression in the liver. ESR1, PPARα, RXRα, RARα, Raldh1, and Raldh3 were examined by qPCR. CNTL: control group, CNTL-OVX: ovariectomized control group, UN: prenatal undernourished group, UN-OVX: ovariectomized prenatal undernourished group. Values are presented as means ± SEMs. #P < 0.05 CNTL-Sham vs UN-Sham, §P < 0.05 UN-OVX vs UN-Sham.

Discussion

Early prenatal undernutrition may aggravate postmenopausal obesity and hepatic steatosis.

Ovariectomy in rats increases body weight and fat accumulation; however, E2 recovers from these conditions.Reference Mahboobifard, Pourgholami and Jorjani22 Maternal food restriction to 30% of daily intake of controls throughout pregnancy reduced the birth weight of offspring and body weight at 4 months of age in the offspring fed high-fat diet (HFD) from weaning; however, HFD increased the perirenal white adipose tissue.Reference Lecoutre, Montel and Vallez23 In another study, maternal caloric restriction to 60% of that of controls from gestational days 11.5 to 17.5 did not reduce the body weight of the offspring fed a normal chow diet after weaning at 9 weeks of age, and even at 17 and 22 weeks of age after being fed HFD for 8 and 13 weeks, respectively.Reference Muramatsu-Kato, Itoh and Kohmura-Kobayashi13 In addition, our previous study showed that early prenatal undernutrition did not affect the body weight of female offspring at birth or 16 weeks of age.Reference Kimura, Hino and Kono24 These reports suggest that early- or mid-prenatal undernutrition, by itself, does not affect the body weight of rodents in adulthood, even when rodents are fed HFD after birth.

By contrast, our study revealed that prenatal undernutrition induced severe obesity in a rat menopausal model and suggests that early prenatal undernutrition can be the first hit of postmenopausal severe obesity. However, ovariectomy is the critical hit of obesity. Meanwhile, previous studies have reported that HFD induces hepatic steatosis in offspring subjected to prenatal undernourishment in rodents.Reference Muramatsu-Kato, Itoh and Kohmura-Kobayashi13,Reference Lecoutre, Montel and Vallez23 Our study demonstrated that hepatic TG accumulation was promoted by early prenatal undernutrition and ovariectomy. Therefore, hepatic steatosis, that is the accumulation of TG in hepatocytes,Reference Ress and Kaser25 was prominent only in early prenatally undernourished rats after ovariectomy. This result indicates that early prenatal undernutrition, as well as severe obesity, was the first hit of postmenopausal hepatic steatosis, which modifies metabolic programming during the prenatal period. Early prenatal undernutrition is suggested to be the first critical factor, in line with our hypothesis. In contrast, estrogen reduction, led by menopause, may be another critical factor in the multiple-hit theory of postmenopausal MASLD.Reference Habibullah, Jemmieh, Ouda, Haider, Malki and Elzouki14

Possible mechanism of postmenopausal MASLD in rats

Previous studies have shown that ovariectomy induces increased food intake and body weight, although female rats have not reduced energy expenditure.Reference Giles, Jackman, Johnson, Schedin, Houser and MacLean26–Reference Richard, Rochon and Deshaies29 In contrast, undernutrition during early- and mid-gestation (GD days 0–7 and GD 8–14, respectively) did not change the intake of standard diet, and the deposits of the subcutaneous, perirenal, and gonadal regions in the female adult offspring aged 22 weeks.Reference Bellinger, Sculley and Langley-Evans30,Reference Erhuma, Bellinger, Langley-Evans and Bennett31 These reports suggest that severe obesity observed in the prenatally undernourished OVX rats in our study is partially attributable to increased food consumption due to ovariectomy. Meanwhile, female rats receiving an early- or mid-prenatal low-protein diet (LPE or LPM) showed higher serum TG, T-CHO, and hepatic TG at 18 months, even though obesity and increased food intake were not observed.Reference Erhuma, Salter, Sculley, Langley-Evans and Bennett9 In contrast, early prenatal undernutrition decreased serum T-CHO 12 weeks after surgery. Yet, it increased hepatic TG 12 and 24 weeks after surgery, while ovariectomy induced an increase in serum T-CHO and LDL-C and hepatic TG at 12 and 24 weeks after surgery in our study. These changes in cholesterol metabolism suggest that ovariectomy affects hepatic steatosis as the development of MASLD is attributed to increased cholesterol uptake and synthesis and other factors such as increased hepatic fatty acid uptake and synthesis, decreased fat oxidation, and increased bile acid synthesis.Reference Feng, Zhang, Yang, Zhang and Xing32 Among cholesterols, elevated LDL-C levels possess hepatotoxic effects and exacerbate liver inflammation and fibrosis; however, the LDL-C/HDL-C ratio, which has been identified as a significant predictor of MASLD in humans,Reference Li, Liu, Ma and Guo33 was elevated by neither prenatal undernutrition nor ovariectomy in our study; therefore, elevated total cholesterol rather than elevated LDL-C may be associated with the onset of MASLD. Conversely, hepatic TG accumulation mainly resulted from increased free fatty acid (FFA) release from adipose tissue and enhanced de novo FFA synthesis in the liver. Erhuma et al. reported that LPE offspring aged 9 months, but not 18 months, exhibited an increased adipose tissue expression of SREBP-1c, which preferentially activates transcription of genes required for fatty acids and TG synthesis,Reference DeBose-Boyd and Ye34 such as fatty acid synthase (FAS) and acetyl-CoA carboxylase-1 (ACC1). Elevation of FAS and ACC1 gene expressions was accompanied by an elevated expression of SREBP-1c in this LPE offspring.Reference Erhuma, Salter, Sculley, Langley-Evans and Bennett9 By contrast, hepatic SREBP-1c, FAS, and ACC1 expressions were reduced at 9 months in the same offspring. However, they were switched to be elevated at 18 months.Reference Erhuma, Salter, Sculley, Langley-Evans and Bennett9 This study suggests that fatty acids from adipocytes and those synthesized in the liver are attributed to MASLD. Our study showed that NEFA in the serum was elevated by prenatal undernutrition in female rats at 24 weeks of age. High serum NEFA released from adipocytes around 24 weeks of age due to prenatal undernutrition may develop MASLD, as suggested by a previous report, which revealed that approximately 59% of liver TG arose from serum NEFAs in comparison to 26.1% and 19.4% from de novo lipogenesis and the diet, respectively, in patients with MASLD. High serum cholesterol following ovariectomy and high serum NEFA levels resulting from prenatal undernutrition may synergistically contribute to the development of MASLD in UN-OVX rat offspring.

From the perspective of the multiple-hit theory,Reference Hao and Chai35,Reference Rong, Zou and Ran36 the development of MASLD is influenced by several other factors, including dietary habits, oxidative stress, inflammation, insulin resistance, gut microbiota, and genetic susceptibility.Reference Hao and Chai35,Reference Rong, Zou and Ran36 Our histological examination revealed no inflammation in the steatotic liver of prenatally undernourished and ovariectomized rat; however, our findings suggest the possibility for the insulin resistance to be related to the early onset of MASLD in prenatal undernourished OVX rats because prenatal undernutrition elevated FBG levels at 14 weeks of age (2 weeks after surgery) and ovariectomy that induces estrogen reduction elevated FBG levels until 9 weeks after surgery (21 weeks of age). Previous reports have suggested that estrogen reduction by ovariectomy leads to impaired insulin sensitivity and glucose homeostasis;Reference Kumagai, Holmäng and Björntorp37 therefore, our results suggest the possibility that increased vulnerability of glucose tolerance to prenatal undernutrition and impaired insulin sensitivity due to rapid estrogen depletion by ovariectomy resulted in the early onset of MASLD in UN-OVX rats. Furthermore, our gene expression analysis revealed that early prenatal undernutrition and ovariectomy synergistically increased ESR1 expression. However, the lack of estrogen by ovariectomy alone tended to reduce ESR1 expression, as reported in a previous study.Reference Della Torre38 Some clinical studies have shown, via histopathological examination, that ERα expression was more frequently expressed in the biopsy specimens of the liver tissue from the patients with non-alcoholic fatty liver disease compared to healthy controls.Reference Elbel, Lavine and Downes39,Reference Choi, Kim and Joo40 Elevated Erα expression of the prenatally undernourished OVX rats may reflect the pathological status of MASLD. Estrogens promote subcutaneous fat distribution, inhibit adipose tissue lipolysis, and reduce the uptake of fatty acids (FA) into the liver through ERα stimulation.Reference Della Torre38 Estrogens also limit dietary-induced de novo lipogenesis, facilitate the export of lipids as VLDL/TG, and promote β-oxidation of FA in hepatocytesReference Della Torre38; therefore, estrogen deficiency is associated with the progression of MASLD.Reference Eng, Forlano, Tan, Manousou, Dhillo and Izzi-Engbeaya5 A recent study showed that systemic ER activation alleviated hepatic lipid accumulation by suppressing the expression of TEA domain transcription factor 1 (TEAD1) gene expression.Reference Sommerauer, Gallardo-Dodd and Savva41 Thus, modulation of estrogen signaling is one target to reduce the risk of postmenopausal MASLD enhanced by prenatal undernutrition. Another study suggested impaired retinoid acid signaling promoted hepatic lipid accumulation; however, mRNA expression of Raldh 1 and Raldh 3, which convert retinol to retinoic acid and are elevated in one of the models of fatty liver disease (leptin-deficient ob/ob mice),Reference Saeed, Bartuzi and Heegsma42 did not change in UN-OVX rats in our study. With no difference in RARα and RXRα expression, prenatal undernutrition may not affect the retinoic acid pathway.

Additionally, prenatal undernutrition and ovariectomy may not modify the PPARα pathway because they did not affect the expression of Pparα and RXRα in our study. Taken together, prenatal undernutrition may make hepatocytes vulnerable to a drastic reduction in estrogen and predispose them to hepatic steatosis through alterations in estrogen signaling. Wang et al. revealed that maternal undernutrition modified estrogen receptor signaling pathways in pig fetuses, and the gene modifications in these pathways were associated with changes in the miRNA induced by maternal undernutrition, as determined by microRNA and mRNA sequencing analyses.Reference Wang, Man, Wang, Odle, Maltecca and Lin43 Altered epigenetic modification in estrogen receptor signaling may affect the onset of postmenopausal MASLD in prenatal undernourished rats.

Limitations

Among the mechanisms that induce a predisposition to postmenopausal MASLD, other pathways examined in our study, such as SRBEP-1c and carbohydrate response element binding protein related to the lipogenesis,Reference Hao and Chai35 PNPLA2 related to the lipolysis of lipid droplets, and PPARβ/γ, may contribute to its pathogenesis.Reference Qiu, Zhang, Zeng and Zhu17 Moreover, the modification of the gut microbiota by prenatal undernutrition may be associated with MASLD.Reference Ohtani, Kamiya and Kawada44,Reference Wankhade, Zhong and Kang45 Further studies are warranted to elucidate the detailed mechanism by which prenatal undernutrition induces a predisposition to postmenopausal MASLD.

Supplementary material

The supplementary material for this article can be found at https://doi.org/10.1017/S204017442510024X.

Acknowledgements

We would like to thank Editage (www.editage.jp) for English language editing. The authors are entirely responsible for the scientific content of the paper.

Financial support

This study was supported by the Japan Society for the Promotion of Science KAKENHI (20K19727).

Competing interests

None.

Ethical standard

The animal study protocol was approved by the Animal Care and Use Committee of Shiga University of Medical Science (2018-5-4).

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Figure 0

Figure 1. Changes in offspring body weight. (a) Body weights of CNTL and UN groups before surgery, and (b) body weights of CNTL-Sham, CNTL-VX, UN-Sham, and UN-OVX groups after surgery were measured once a week. CNTL: control group, CNTL-OVX: ovariectomized control group, UN: prenatal undernourished group, UN-OVX: ovariectomized prenatal undernourished group. Values are presented as means ± SEMs. *P < 0.05 CNTL-OVX and UN-OVX vs CNTL-Sham, §P < 0.05 UN-OVX vs UN-Sham, †P < 0.05 UN-OVX vs CNTL-OVX by Tukey’s HSD test.

Figure 1

Figure 2. Obesity led by prenatal undernutrition and ovariectomy. Dorsal views are shown in the representative rats of the CNTL (a), UN (b), CNTL-OVX (c), and UN-OVX (d) groups 24 weeks after surgery. Scale bar: 5 cm. CNTL: control group, CNTL-OVX: ovariectomized control group, UN: prenatal undernourished group, UN-OVX: ovariectomized prenatal undernourished group.

Figure 2

Figure 3. Organ weights of the offspring. The liver weights (a), the spleen weights (b), and the average weights of bilateral kidneys (c) and adrenals (d) are shown. CNTL: control group, CNTL-OVX: ovariectomized control group, UN: prenatal undernourished group, UN-OVX: ovariectomized prenatal undernourished group. Values are presented as means ± SEMs. ‡‡P < 0.01 vs CNTL by Student’s t-test before surgery. *P < 0.05 CNTL-OVX and UN-OVX vs CNTL-Sham, §P < 0.05 UN-OVX vs UN-Sham by Tukey’s HSD test after surgery.

Figure 3

Figure 4. Fasting blood glucose until 9 weeks after surgery. Fasting blood glucose from the tail vein was measured until 9 weeks after surgery using a glucometer. CNTL: control group, CNTL-OVX: ovariectomized control group, UN: prenatal undernourished group, UN-OVX: ovariectomized prenatal undernourished group. Values are presented as means ± SEMs. *P < 0.05 vs CNTL-Sham, §P < 0.05 vs UN-Sham, †P < 0.05 vs CNTL-OVX by Tukey’s HSD test.

Figure 4

Figure 5. Blood examination before surgery. Fasting blood glucose (FBG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (T-CHO), free cholesterol (F-CHO), esterified cholesterol (E-CHO), triglycerides (TG), non-esterified fatty acids (NEFA), NEFA/TG, LDL cholesterol, (LDL-C), HDL cholesterol (HDL-C), and LDL/HDL were measured in the serum of the offspring aged 12 weeks. Values are presented as means ± SEMs. *P < 0.05; Tukey’s HSD test.

Figure 5

Figure 6. Blood examination 12 weeks after surgery. Fasting blood glucose (FBG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (T-CHO), free cholesterol (F-CHO), esterified cholesterol (E-CHO), triglycerides (TG), non-esterified fatty acids (NEFA), NEFA/TG, LDL cholesterol, (LDL-C), HDL cholesterol (HDL-C), and LDL/HDL in the serum were measured. Values are presented as means ± SEMs. *P < 0.05 CNTL-OVX vs CNTL-Sham, §P < 0.05 UN-OVX vs UN-Sham by Tukey’s HSD test.

Figure 6

Figure 7. Blood examination 24 weeks after surgery. Fasting blood glucose (FBG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (T-CHO), free cholesterol (F-CHO), esterified cholesterol (E-CHO), triglycerides (TG), non-esterified fatty acids (NEFA), NEFA/TG, LDL cholesterol, (LDL-C), HDL cholesterol (HDL-C), and LDL/HDL in the serum were measured. Values are presented as means ± SEMs.

Figure 7

Figure 8. Hepatic TG and T-CHO. Triglycerides (TG) and total cholesterol (T-CHO) were measured in the offspring’s liver. Values are presented as means ± SEMs. *P < 0.05 CNTL-OVX vs CNTL-Sham, §P < 0.05 UN-OVX vs UN-Sham, †P < 0.05 UN-OVX vs CNTL-OVX by Tukey’s HSD test.

Figure 8

Figure 9. Lipid accumulation and fibrosis in the liver section of the offspring 24 weeks after surgery. Liver sections were stained by hematoxylin and eosin (a–h), Oil Red O (i–p), and Picrosirius Red (q–t)staining. Images surrounded by a box (a–d) in HE and those in Oil Red O staining (i–l) were magnified in e–h and m–p, respectively. Arrowheads in the hepatic sections indicated lipid droplets with HE staining. Lipid droplets are stained red with Oil Red O (i–p). Scale bar: 100 μm (a–d, i–l, and q–t), 10 μm (e–h and m–p). CNTL: control group, CNTL-OVX: ovariectomized control group, UN: prenatal undernourished group, UN-OVX: ovariectomized prenatal undernourished group.

Figure 9

Figure 10. Gene expression in the liver. ESR1, PPARα, RXRα, RARα, Raldh1, and Raldh3 were examined by qPCR. CNTL: control group, CNTL-OVX: ovariectomized control group, UN: prenatal undernourished group, UN-OVX: ovariectomized prenatal undernourished group. Values are presented as means ± SEMs. #P < 0.05 CNTL-Sham vs UN-Sham, §P < 0.05 UN-OVX vs UN-Sham.

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