The Journal of Toxicological Sciences
Online ISSN : 1880-3989
Print ISSN : 0388-1350
ISSN-L : 0388-1350
Original Article
Resveratrol reverses the programmed high-susceptibility to non-alcoholic fatty liver disease by targeting the hepatic SIRT1-SREBP1c pathway in prenatal ethanol-exposed rat offspring
Lang ShenLijia XieLi ChenLi LiuXiao ShiXiaocong WangDonghong ChenHui WangSong QuanZhijian Wang
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Supplementary material

2021 Volume 46 Issue 9 Pages 413-423

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Abstract

An increased susceptibility to non-alcoholic fatty liver disease (NAFLD) in female rat offspring that experienced prenatal ethanol exposure (PEE) has been previously demonstrated. The present study further investigated the potential mechanism. Based on the results from both fetal and adult studies of offspring rats that experienced PEE (4 g/kg/day), the fetal weight, serum glucose and triglyceride levels decreased significantly and hepatocellular ultra-structure was altered. Fetal livers exhibited inhibited expression and activity of sirtuin 1 (SIRT1), enhanced expression of lipogenic genes: sterol regulatory element binding protein 1c (SREBP1c), fatty acid synthase (FASN), acetyl-coenzyme A carboxylase α (ACCα), stearyl-coenzyme A desaturase 1 (SCD1). In adult offspring fed with high-fat diet, the PEE offspring revealed obviously catch-up growth, increased food intake, elevated serum metabolic phenotypes, suppressed hepatic SIRT1-SREBP1c pathway, and formation of NAFLD. Resveratrol (the chemical activator of SIRT1) could remarkably reverse the serum metabolic phenotypes and alleviate the hepatocyte steatosis in relation to the PEE offspring through activating the hepatic SIRT1-SREBP1c pathway. Therefore, increased susceptibility to diet-induced NAFLD in PEE offspring appears to be mediated by intrauterine programming of hepatic lipogenesis via the SIRT1-SREBP1c pathway. This altered programming effect could partially be reversed by resveratrol intervention after birth in PEE offspring rats.

INTRODUCTION

Non-alcoholic fatty liver diseases (NAFLD) has prevalence as high as 20–40% in the general population and accounts for 75% incidence of obesity or diabetes in Western countries (Cusi et al., 2017; Polyzos et al., 2019). Metabolic homeostasis disruption is most likely to be responsible for this global epidemic of fatty liver diseases, and most cases of fatty liver diseases are commonly associated with metabolic syndromes (MetS), such as hyperlipidemia and insulin resistance, or other metabolic diseases like obesity and type 2 diabetes mellitus (Eckel et al., 2005).

NAFLD is considered to be the hepatic manifestation of the MetS (Bellentani, 2017). Numerous studies have confirmed that MetS have a fetal developmental origin-intrauterine growth restriction (IUGR) (Seferovic et al., 2015), and animal experiments and population-based studies confirmed that NAFLD has been included among persistent IUGR-dependent metabolic dysfunctions (Alisi et al., 2012; Cao et al., 2012; Nobili et al., 2008). Our previous study demonstrated the intrauterine origin of an increased susceptibility to NAFLD in female IUGR rat offspring induced by prenatal ethanol exposure (PEE), which may be related to the intrauterine programming of hepatic glucose and lipid metabolic function. This altered programming enhanced fetal hepatic lipogenesis and reduced lipid output in utero, which sustained into the postnatal phase and reappeared in adulthood with the introduction of a high-fat diet (HFD), thereby aggravating hepatic lipid accumulation and causing NAFLD (Shen et al., 2014).

Sirtuins are a group of highly conserved NAD+ dependent histone and protein deacetylases and/or ADP-ribosyl transferases, and are involved in numerous biological processes (Finkel et al., 2009; Li and Kazgan, 2011; Mei et al., 2016; Vachharajani et al., 2016; Wang et al., 2016). Accumulating evidence indicates that sirtuins play important roles in regulating the metabolic processes related to the fatty liver diseases. Especially SIRT1, the most extensively studied member of the sirtuin family, is highly involved in NAFLD (Colak et al., 2011; Ding et al., 2017; Nassir and Ibdah, 2016). Recent studies demonstrated that SIRT1 plays a central beneficial role in controlling hepatic lipid metabolism and protects against HFD-induced hepatic steatosis primarily through regulating lipogenesis (Ding et al., 2017). SREBP-1c is a specific DNA binding transcription factor that is critically involved in lipid synthesis regulation by binding to the promoter regions of its target lipogenic genes (FASN, ACCα, SCD1) (Ferré and Foufelle, 2010). SIRT1 has been reported to attenuate the transcriptional activity of SREBP-1c, promote the ubiquitination and proteasomal degradation, and decrease its stability and occupancy at the lipogenic genes (Andrade et al., 2014; Ponugoti et al., 2010; Walker and Näär, 2012; You et al., 2008). Resveratrol, the chemical activator of SIRT1, can protect the liver against HFD-induced hepatic steatosis by inhibiting the expression of SREBP-1c and related hepatic lipogenic gene and protein profile (Andrade et al., 2014). Collectively, the SIRT1-SREBP1c pathway might mainly regulate HFD-induced hepatic steatosis and NAFLD. However, whether the SIRT1-SREBP1c pathway has potential association with fetal-originated NAFLD, if such association is derived from intrauterine metabolic programming, still remains exclusive.

Our previous research has explained that the intrauterine programming of hepatic glucose and lipid metabolic function may underly the intrauterine origin of an increased susceptibility to NAFLD in female IUGR rat offspring induced by PEE. The present study was designed to further identify that this intrauterine programming effect is potentially mediated by hepatic SIRT1-SREBP1c signaling, and resveratrol could reverse this programmed effect in female IUGR rat offspring fed with HFD.

MATERIALS AND METHODS

Materials

Ethanol (analytical pure grade and chromatographic pure grade) was obtained from Zhen Xin Co., Ltd. (Shanghai, China) and Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). Resveratrol was obtained from SIGMA Chemical, (Pool, Dorset, UK). Isoflurane was purchased from Baxter Healthcare Co. (Deerfield, IL, USA). Total cholesterol (TCH) and TG assay kits were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Reverse transcription and quantitative PCR (Q-PCR) kits were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). The oligonucleotide primers for rat Q-PCR genes (PAGE purification) were synthesized by Sangon Biotech Co., Ltd. Other chemicals and agents were of analytical grade.

Animals and treatment

Specific-pathogen-free (SPF) Wistar rats (200–240 g) were used. All animal experiment procedures were approved by and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Chinese Animal Welfare Committee. The designed experiments included 2 separate parts, the fetal offspring, and the postnatal offspring fed with HFD. Each experiment was independent of each other, including breeding, processing and detecting. The experiment of fetal rats was conducted as previously described (Shen et al., 2014).

For postnatal offspring NAFLD model, only female offspring were used, following our previously established PEE-induced NAFLD model. Pregnant rats were transferred to individual cages and randomly divided into control and ethanol groups (n = 8 for each group). The ethanol group (PEE) was given ethanol 4 g/kg·day by gavage administration from gestational day (GD) 11 to GD20, while the control group was given the same volume of distilled water. Briefly, the pregnant rats of control and PEE were kept until normal delivery (GD21), and on postnatal day 1 (PD1) the numbers of pups were normalized to 8 pups per litter to assure adequate and standardized nutrition until weaning (postnatal week 4, PW4). After weaning, one female pup per litter was randomly selected from control and PEE group, and then the control offspring rats were randomly divided into control and resveratrol groups, while the PEE offspring rats into PEE and PEE + resveratrol group. All 4 groups of the offspring rats were fed with HFD (providing 18.9% kcal from protein, 61.7% kcal from carbohydrate and 19.4% kcal from fat) at libitum. The offspring rats in resveratrol and PEE + resveratrol group were given resveratrol 50 mg/kg body weight/day by gavage administration daily, while the control and PEE group were given distilled water for drinking. The body weight was measured weekly and the corresponding growth rate was calculated as previously described (Shen et al., 2014). In brief, 8 randomly selected pregnant rats with 10–14 live fetuses from each group were anesthetized with isoflurane and euthanized on GD20. The female fetuses were quickly removed to weigh, and IUGR was diagnosed when the body weight of an animal was two standard deviations lower than the mean body weight of the control group. Serum samples from each littermate were pooled together and immediately frozen at −80°C for metabolic phenotype analyses. One fetal liver from each group was randomly selected and routine fixed for histological and ultra-structural examination, and the rest of the fetal livers from each group were immediately frozen stored at −80°C for gene expression analyses. At PW24, after recording the food intake for three days and the rectal temperature at 23:00, the offspring rat was anesthetized with isoflurane and decapitated. Serum was prepared and stored at −80°C for metabolic phenotype analyses. The liver was dissected, randomly selected, partly fixed in 4% paraformaldehyde solution for histological examination, and the rest immediately frozen to store at −80°C for the subsequent analyses.

Analysis of blood samples

Glucose, TG, TCH and FFA were detected with the biochemical assay by commercial kits following the manufacturer’s protocol. Data acquisition and analysis were performed using standard software supplied by the manufacturer. The blood ethanol concentration was detected by gas chromatography–mass spectrometry (GC–MS) as previously described (Shen et al., 2014).

Histological measurement

For light microscopic examination, hematoxylin and eosin (HE) staining of liver was achieved by standard procedures. The sections were observed and photographed with an Olympus AH-2 light microscope (Olympus, Tokyo, Japan). Five HE sections of each group were selected and five random fields of each section were scored under the microscope. The grading and staging of NAFLD were scored according to the system reported by Kleiner et al. (2005), which was calculated by the sum of steatosis (0–3), lobular inflammation (0–2), hepatocellular ballooning (0–2), and fibrosis (0–4).

The hepatocyte detection by transmission electron microscopy was achieved by standard procedures and observed with a Hitachi H600 transmission electron microscope (Hitachi, Co., Tokyo, Japan). Digital images were acquired directly by a computer.

RNA extraction, reverse transcription, and quantitative real-time PCR

Detailed protocols for total RNA extraction, RT, and real-time PCR and the primer sequences for genes were reported in our previous study (Shen et al., 2014). In brief, total RNA was isolated from fetal tissues using Trizol reagent (obtained from Invitrogen Co., Ltd.; Carlsbad, CA, USA) according to the manufacturer’s protocol. The tissue samples isolated from the littermates were pooled for homogenization. The isolated RNA was stored at −80°C in aliquots that were only thawed only once. A total of 1 μg of purified RNA was reverse transcribed into cDNA using a First Strand cDNA Synthesis Kit (TransGen Biotech Co., Ltd., Beijing, China). All of the cDNA sequences were obtained from the NCBI Entrez nucleotide databases, and the primers were designed using Premier 5.0 (PREMIER Biosoft International, Palo Alto, CA, USA). The sequences of each of the designed primers were queried using the NCBI BLAST database for homology comparison to determine the final primers used in the study.

SIRT1 activity analysis

SIRT1 activity was measured using a SIRT1 fluorometric assay kit (BIOMOL, Plymouth Meeting, PA, USA) following the manufacturer’s protocol, as previously described in another study (Gurd et al., 2011). Briefly, twenty-five microliters of nuclear extract was incubated with 15 μL of Fluor de Lys-SIRT1 substrate (100 μM) and NAD+ (100 μM) for 30 min at 37°C. The reaction was stopped by the addition of 50 mL of developer reagent and nicotinamide (2 mM), and the fluorescence was subsequently monitored for 30 min at 360 nm (excitation) and 460 nm (emission). The change in fluorescence [arbitrary fluorescence units (AFU)] per minute was normalized to the amount of total liver (mg wet weight) used for the nuclear extraction procedure.

Statistical analysis

SPSS 22 (SPSS Science Inc., Chicago, IL, USA) was used for data analysis. Quantitative data were expressed as the mean ± S.E.M. and assessed by one-way ANOVA followed by Bonferroni post-test. The mean weights for each litter were calculated and used for statistical analyses. For enumeration data, the IUGR rate was presented as group mean values of the litter proportions, and the t-test and chi-squared test were used as appropriate. The Kleiner score was evaluated with Mann–Whitney U test. Statistical significance was defined as P < 0.05.

RESULTS

In utero

Changes in body weight and serum metabolic phenotypes

As shown in Table 1, the average concentrations of serum ethanol (mM) by PEE (4 g/kg·day) were 82.3 in pregnant rats and 57.6 in fetal rats, respectively. PEE significantly decreased the body weights and increased the IUGR rate of the fetus (P < 0.01). The serum levels of glucose and triglyceride were notably decreased in PEE fetus (P < 0.05), and serum FFA level dramatically increased in PEE fetus (P < 0.05). But the level of total cholesterol remained unchanged.

Table 1. Changes in body weight, IUGR rate and serum metabolic phenotypes in female fetal rats of PEE.
Parameters Control PEE P value
Ethanol (MB, mM) - 82.25 ± 18.74 /
Ethanol (FB, mM) - 57.62 ± 9.81 /
Weight (g) 4.12 ± 0.67 3.22 ± 0.91 0.01
IUGR rate (%) 11.21 82.56 0.00
Glucose (mM) 2.01 ± 0.54 1.58 ± 0.66 0.04
Triglyceride (mM) 0.82 ± 0.17 0.55 ± 0.12 0.04
Total cholesterol (mM) 0.53 ± 0.15 0.48 ± 0.21 0.55
FFA (mM) 0.36 ± 0.07 0.42 ± 0.12 0.03

Mean ± S.E.M. except for IUGR rate (Mean), n = 8 for weight and IUGR rate, n = 5 for serum phenotype (serum was randomly merged because of meagre fetal blood). IUGR: intrauterine growth retardation; PEE: prenatal ethanol exposure; MB: maternal blood; FB: fetal blood; FFA: free fat acid.

Histological and hepatocellular ultrastructure changes

Histological observation revealed a reduced cellularity of parenchyma cells in the fetal liver of PEE group (Fig. 1D) relative to the control (Fig. 1A), mainly characterized as a feature of the vacuolization phenotype in the parenchyma cells. As well, the PEE group exhibited increased intracellular vacuoles (red arrows) in hepatocytes compared to the control (black arrows). Ultrastructural observation by transmission electron microscopy revealed that, in the parenchyma cells from PEE (Fig. 1F), the structure of mitochondrial was damaged due to swelling deformation, and cristae mitochondria were fractured, as well as hyperplasia of smooth endoplasmic reticulum. Moreover, a mass of large glycogen granules had accumulated in the cytoplasm of hepatic parenchyma cells in the PEE group.

Fig. 1

Hepatic histological and hepatocellular ultrastructure changes in female fetal rats with prenatal ethanol exposure (PEE). A: control (H&E, × 100); B: control (H&E, × 400); C: control (TEM, × 15,000); D: PEE (H&E, × 100); E:PEE (H&E, × 400); F: PEE (TEM, × 15,000). Hematoxylin and eosin (H&E) staining of PEE liver exhibited reduced cellularity and increased vacuolar hepatic parenchyma cells (showed by arrows). Transmission electron microscopy (TEM) of hepatocyte from PEE exhibited swollen mitochondria (M), hyperplasia of smooth endoplasmic reticulum (E), and accumulated glycogen granule (G).

Alteration of activity of SIRT1 and gene expression of hepatic SIRT1-SREBP1c pathway

The alteration of activity of hepatic SIRT1, and the gene expression of the hepatic SIRT1-SREBP1c pathway were shown in Fig. 2. Compared to the control, PEE not only significantly decreased the SIRT1 activity in fetal liver (Fig. 2A, P < 0.01), but also inhibited the gene expression of hepatic SIRT1 as well (Fig. 2B, P < 0.01). As a result, the downstream transcriptional factor SREBP1c was up-regulated by PEE (Fig. 2B, P < 0.01), and the target lipogenic genes FASN and ACCα were also up-regulated relative to the control (Fig. 2B, P < 0.01). But the expression of SCD1 remains unchanged in fetal liver of PEE group relative to the control (Fig. 2B). All those results revealed that PEE could dramatically inhibit the expression and activity of SIRT1 in fetal liver, then up-regulate the transcriptional activity of key lipogenic factor-SREBP1c, thus stimulating the lipid synthesis through the lipogenic genes (FASN, ACCα).

Fig. 2

Effects of prenatal ethanol exposure (PEE) on the activity of hepatic SIRT1 and gene expression of SIRT1-SREBP1c pathway in female fetal rats with intrauterine growth retardation. A: SIRT1 activity; B: gene expression of SIRT1-SREBP1c pathway. SIRT1: sirtuin 1; SREBP1c: sterol regulatory element binding protein 1c; SCD1: stearoyl-CoA desaturase-1; ACCα: acetyl-coenzyme A carboxylase α; FASN: fatty acid synthase; Mean ± S.E.M., n = 8 fetus from 8 pregnant rats. *P < 0.05, **P < 0.01 vs. control.

Adult offspring

Changes in body weight, rate of body weight gain, food intake and rectal temperature in female adult offspring rats with HFD

As shown in Fig. 3, compared to the control, the PEE group and the PEE + resveratrol group both showed significantly lower body weights in offspring rats at PW1 (P < 0.05, Fig. 3A). After weaning, under induction by high-fat diet, the body weight of the PEE group and the PEE + resveratrol group weighed close to that of the control group from PW4 onwards (Fig. 3A). From PW 16 to PW24, the body weights of PEE group were higher than that of the control, and the PEE + resveratrol group showed decreased body weight relative to the PEE group. The body weight of the resveratrol group remained closely similar to the control group (Fig. 3A). As for the body weight growth rate, the PEE group and the PEE + resveratrol group began significantly increasing by PW12 versus to that of the control (P < 0.01, Fig. 3B). From PW 16 on, the growth rate of the PEE + resveratrol group was significantly lower than that of the PEE group (P < 0.05, Fig. 3B). The rectal temperature of all groups remained unchanged at PW24 (Fig. 3C), when the food intake increased in the PEE group relative to that of the control, and the PEE + resveratrol group showed a decreased food intake compared with the PEE group. These results suggested that PEE adult offspring could undergo catch-up growth induced by high-fat diet, and the resveratrol might slow down the catch-up growth trend in the offspring of PEE.

Fig. 3

Effects of resveratrol on body weights, food intake and rectal temperature in female adult offspring rats with HFD. A: body weights; B: body weight gain rate; C: food intake and rectal temperature. PEE: prenatal ethanol exposure. Mean ± S.E.M., n = 8 offspring from 8 pregnant rats. Body weight and the corresponding growth rate were assessed by repeated measures ANOVA. *P < 0.05, **P < 0.01 vs. Control; #P < 0.05 vs. PEE.

Changes of serum metabolic phenotypes in female adult offspring rats with HFD

The changes of serum metabolic phenotypes in female adult offspring rats fed with HFD were as shown in Fig. 4. All the serum levels of glucose, triglyceride, total cholesterol and free fat acid were notably increased in the PEE group compared to that of the control (P < 0.05, P < 0.01, Fig. 4A–4D), while PEE + resveratrol group showed decreased serum levels of glucose, triglyceride, total cholesterol and free fat acid relative to that of the PEE group (P < 0.05, Fig. 4A–4D). Resveratol alone can alleviate the serum levels of triglyceride and free fat acid compared to that of the control group (P < 0.05, Fig. 4B, 4D), but PEE + resveratrol group revealed higher levels of serum triglyceride and free fat acid (P < 0.05, Fig. 4B, 4D).

Fig. 4

Effects of resveratrol on serum metabolic phenotypes in female adult offspring rats with HFD. A: glucose; B: triglyceride; C: total cholesterol; D: free fat acid. PEE: prenatal ethanol exposure. Mean ± S.E.M., n = 8 offspring from 8 pregnant rats. *P < 0.05, **P < 0.01 vs. Control; #P < 0.05 vs. PEE; &P < 0.05 vs. Resverrol.

Liver histological analysis

Liver histological results by H&E staining showed that, compared with the widespread micro-vesicular steatosis of hepatocytes in the control group (Fig. 5A, 5E), the PEE group exhibited prevalently (> 66%) macro-vesicular steatosis in hepatocytes (Fig. 5B, 5F), with an average Kleiner score of 3.1 (P < 0.01, Fig. 3I), which demonstrated the formation of NAFLD in PEE adult offspring. The resveratrol group mainly showed scattered hepatocyte micro-vesicular steatosis and a decreased Kleiner score (P < 0.05, Fig. 3I). Compared with that of the PEE group, PEE + resveratrol group revealed a significant reduction of hepatocyte steatosis no matter micro-vesicular or macro-vesicular steatosis, accordingly resulting in a notably decreased Kleiner score of liver (P < 0.01, Fig. 3I).

Fig. 5

Effects of resveratrol on hepatic histopathology and Kleiner scoring of female adult offspring rats with HFD. (H&E, 100 ×, 400 ×). A/E: Control (H&E, 100 ×, 400 ×); B/F: prenatal ethanol exposure (PEE). (H&E, 100 ×, 400 ×); C/G: resveratrol (H&E, 100 ×, 400 ×); D/H: PEE + resveratrol (H&E, 100 ×, 400 ×); I: Kleiner score. Liver tissue of the control group showed micro-vesicular steatosis in hepatocytes, enlarged sinusoidal space and sparse lipid droplets, while the PEE group exhibited macro-vesicular steatosis and characterized by extensive accumulation of large lipid droplets. The resveratrol group revealed reduced micro-vesicular steatosis, and in the PEE + resveratrol group, hepatic lipid droplets were significantly reduced, as well as the micro-vesicular steatosis in hepatocytes . Mean ± S.E.M., n = 5 from 5 pregnant rats. *P < 0.05, **P < 0.01 vs. control. ##P < 0.01 vs. PEE.

Changes of SIRT1 activity and gene expression in hepatic SIRT1-SREBP1c pathway

As shown in Fig. 6, both the activity and gene expression of hepatic SIRT1 were significantly decreased in the PEE group relative to that of the control in liver tissue (Fig. 6A, 6B, P < 0.01). The downstream transcriptional factor SREBP1c was up-regulated by PEE (Fig. 6C, P < 0.01). Accordingly, the target lipogenic genes FASN and SCD1 were up-regulated relative to the control (Fig. 6E, 6F, P < 0.01). The expression of ACCα showed an increasing trend but no statistical significance (Fig. 6D). Compared to the PEE group, the PEE + resveratrol group revealed increased activity and gene expression of hepatic SIRT1, and decreased expression of downstream gene SREBP1c, SCD1 and FASN (Fig. 6A-6F, P < 0.05, P < 0.01). The resveratrol group showed an increased activity of hepatic SIRT1 and a decreased gene expression of FASN relative to that of the control group (Fig. 6A, 6F, P < 0.05).

Fig. 6

Effects of resveratrol on the activity of hepatic SIRT1 and gene expression of SIRT1-SREBP1c pathway in female adult offspring rats with HFD. A: SIRT1 activity; B–F: gene expression of SIRT1-SREBP1c pathway. SIRT1: sirtuin 1; SREBP1c: sterol regulatory element binding protein 1c; SCD1: stearoyl-CoA desaturase-1; ACCα: acetyl-coenzyme A carboxylase α; FASN: fatty acid synthase; Mean ± S.E.M., n = 8 offspring from 8 pregnant rats. *P < 0.05, **P < 0.01 vs. Control; #P < 0.05, #P <0.01 vs. PEE; &P < 0.05 vs. Resveratrol.

DISCUSSION

NAFLD and hepatic SIRT1-SREBP1c pathway

Nowadays, NAFLD has been a kind of serious health issue worldwide. The main reasons causing NAFLD are due to the popular high-fat/calorie diet. NAFLD initially develops from excessive TG deposition as lipid droplets in hepatocytes, which is primarily caused by imbalance of lipid homeostasis between TG/fatty acids acquisition and removal, such as increased uptake, enhanced de novo lipogenesis, impaired fatty acids β-oxidation, and/or decreased lipid export in liver (Cohen et al., 2011). Under HFD, dietary intensive fats uptaken are delivered into blood circulation as TG-rich chylomicrons and FFA, and about 20% of them are delivered into the liver by hepatic lipid uptake (Cohen et al., 2011; Ding et al., 2017). In the present study, numerous macrovesicular steatoses induced by HFD could be observed in the PEE offspring rats (Fig. 5B, 5F). The assessment of the Kleiner score of the PEE group was significantly higher than that of the control, indicating that PEE had an effect on stimulating hepatic lipid accumulation and NAFLD formation (Kleiner et al., 2005). Our previous studies had shown that PEE offspring rats developed a high susceptibility to NAFLD under HFD. While under a normal diet, PEE offspring rats showed a pattern of partial catch-up growth, but the NAFLD phenotype was not so typical (Shen et al., 2014). The present result is consistent with those of our previous study, indicating that the animal model of high-susceptibility to NAFLD in PEE offspring rats has been successfully established (Shen et al., 2014).

Recently, SIRT1 has been regarded to play key roles in controlling hepatic lipid metabolism as well as protecting against HFD-induced hepatic steatosis primarily through regulating lipogenesis (Ding et al., 2017). SIRT1 down-regulates the transcriptional activity of SREBP-1c, reduces its DNA binding activity of its target lipogenic genes (FASN, ACCα, SCD1), and decreases its stability and occupancy at the lipogenic genes (Andrade et al., 2014; Ponugoti et al., 2010; Walker and Näär, 2012; Walker et al., 2010). Various studies report that the SIRT1-SREBP1c pathway is crucial in regulating HFD-induced hepatic steatosis (Colak et al., 2014; Ponugoti et al., 2010; Zhang et al., 2016). In the present study, we found that both micro-vesicular and macro-vesicular steatosis could be observed in hepatocytes of the PEE fetus and adult offspring, accompanied with significantly altered gene expression and activity of the hepatic SIRT1-SREBP1c pathway, indicating that the hepatic SIRT1-SREBP1c pathway might regulate the lipogenesis process in hepatocytes of PEE fetus and adult offspring.

Hepatic SIRT1-SREBP1c pathway alteration mediated the intrauterine programming of high-susceptibility of NAFLD induced by PEE

Our previous research has explained that the intrauterine programming of hepatic glucose and lipid metabolic function may underly the intrauterine origin of an increased susceptibility to NAFLD in female IUGR rat offspring induced by PEE (Shen et al., 2014). In this study, we reproduced the animal model of high-susceptibility to NAFLD in PEE offspring rats, we found that PEE could induce developmental abnormalities in the structure and function of fetal liver, resulting in enhanced hepatic lipogenesis and lipid accumulation, and an alteration of gene expression and activity of hepatic SIRT1-SREBP1c pathway. Remarkably, these changes of activity and gene expression, concomitant with the hepatic histological alteration, can reappear in the adulthood when the offspring of PEE were induced by HFD correspondingly. Those consistent prenatal and postnatal changes revealed an intrauterine programming effect of high-susceptibility of NAFLD induced by PEE. These results are consistent with those of previous studies using other models of IUGR (Cao et al., 2012; Yamada et al., 2011).

Furthermore, we explored that hepatic SIRT1-SREBP1c pathway alteration might mediate the intrauterine programming of high-susceptibility of NAFLD induced by PEE. The activity and gene expression of hepatic SIRT1 were dramatically inhibited in the fetal liver induced by PEE (Fig. 2A), with the levels of serum ethanol in the pregnant rats and the fetuses were 82.3 and 57.6 mM respectively (Table 1). In this case, it was reported that alcohol consumption could result in decreased NAD+/NADH ratio as well as SIRT1 activity in hepatocytes (You et al., 2015). As a result, the downstream transcriptional factor SREBP1c and target lipogenic genes FASN and ACCα were all up-regulated by PEE (Fig. 2B), causing enhanced de novo lipogenesis and lipid accumulation in fetal hepatocytes (Fig. 1E). Moreover, reduced SIRT1 activity was concomitant with dietary energy/nutrition overload status such as HFD and/or high-calorie diet conditions (Jang et al., 2019). In the present study, under HFD feeding, the PEE offspring rats showed remarkable increase of food intake (Fig. 3C), obviously catch-up growth (Fig. 3B), accompanied with decreased activity and gene expression of SIRT1 as well as up-regulation of SREBP1c and target lipogenic genes FASN and SCD1 in the liver tissue (Fig. 6). Interestingly, all these changes in activity and gene expression of the SIRT1-SREBP1c pathway can reappear in adulthood when the offspring rats of PEE were fed with HFD, the consistent alteration before and after birth revealed an intrauterine programming effect on hepatic lipid metabolism could potentiate the high-susceptibility to NAFLD in the adult.

Resveratrol reversed the high-susceptibility of NAFLD by targeting the hepatic SIRT1-SREBP1c pathway in PEE rat offspring

Emerging evidence had showed that resveratrol, a kind of chemical activator of SIRT1, could remarkably decrease SREBP-1c and its related hepatic lipogenesis, thereby protecting liver against HFD-induced hepatic steatosis (Andrade et al., 2014). Whether or not this potential therapeutic natural polyphenolic compound could protect the PEE offspring from HFD-induced fetal-originated NAFLD, has not been reported yet. Because of the unknown safety and potential teratogenicity of resveratrol administration during pregnancy, we intervened the offspring rats of PEE with resveratrol from PW4 to adulthood fed with HFD. We found that resveratrol could notably inhibit the nutrition intake and growth rate of the PEE offspring rats, resulting in a significant decrease on body weight of adult offspring in relation to the PEE control group (Fig. 3). Similar trends can be observed in the resveratrol group relative to the control group fed with HFD, suggesting that resveratrol had protective effects on both PEE and HFD. It is reported that SIRT1 serves as important metabolic sensor which couples alcohol, HFD and increased food intake with corresponding lipid/energy homeostasis signaling (Ding et al., 2017).

Furthermore, the serum metabolic phenotypes associated with NAFLD were also obviously alleviated by resveratrol in the adult offspring of PEE fed with HFD (Fig. 4). The serum levels of glucose, TG, TCH and FFA were all reversed by resveratrol intervention to a level similar to the control group, consequently leading to a significant reduction of hepatocyte steatosis and notably decreased Kleiner score of the liver (Fig. 5) in relation to that of the PEE group. Also, resveratrol itself can reduce the serum level of metabolic phenotype of TG and FFA, as well as the degree of hepatocyte steatosis, when compared with that of the control group. These results were partially consistent with another HFD-induced hepatic steatosis mice model (Andrade et al., 2014), indicating that resveratrol not only plays essential and protective roles against HFD-induced hepatic steatosis, but also has a beneficial effect in protecting the PEE offspring from the HFD-induced fetal-originated NAFLD. Many studies also showed that resveratrol could protect IUGR offspring from diet-induced metabolic syndrome through exerting beneficial effects on hepatic injury and metabolic alterations (Cheng et al., 2021; Dolinsky et al., 2011).

Moreover, resveratrol intervention dramatically reversed the activity and gene expression of hepatic SIRT1-SREBP1c pathway in the PEE offspring to a level similar to that of the control group (Fig. 6). For the normal offspring, resveratrol can stimulate the activity of hepatic SIRT1, and inhibit the gene expression of FASN in the liver, thus reducing the lipid accumulation in hepatocytes in relation to the control group. Extensive in vivo studies in many mammal models had demonstrated its protective functions against fatty liver diseases since resveratrol was identified as direct SIRT1 activator for the first time (Howitz et al., 2003), one of the main mechanisms was via inhibiting hepatic lipogenesis mediated by the SIRT1-SREBP1c pathway (Shang et al., 2008).

In conclusion, the present study originally demonstrated the protective effects of SIRT1 activator resveratrol on the high-susceptibility to NAFLD in female IUGR offspring rats induced by PEE, which is most likely mediated by targeting the intrauterine programmed hepatic SIRT1-SREBP1c pathway. Based on our main results, for the first time, we observed the beneficial protecting role of resveratrol intervention on the progression of fetal-originated NAFLD, also providing the basis for resveratrol serve as promising therapeutic approaches for the treatment in the future.

ACKNOWLEDGMENTS

This work was supported by grants from The National Key Research and Development Program of China (Grant No: 2018YFC1004402), The Clinical Research Special fund of Chinese Medical Association (Grant No:17020370706), The PhD Start-up Fund of Natural Science Foundation of Guangdong Province (Grant No: 2015A030310095).

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES
 
© 2021 The Japanese Society of Toxicology
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