Animal experiments were performed at the Laboratory Animal Center of Zhejiang University (Hangzhou, China). All animal experimental procedures were approved by the Animal Ethics Committee of Zhejiang University, School of Medicine.

Briefly, 24 Sprague Dawley rats (16 male and 8 female; SLRC Laboratory Animal Co., Ltd., Shanghai, China) were housed under standard conditions (room temperature, 20–22°C; humidity, 40–60%). After 1 week of acclimation, male and female rats were mated overnight, and the presence of sperm in a vaginal smear was designated as gestational day 1. Pregnant rats were arbitrarily divided into two groups: The control group continued to receive an ad libitum chow diet; the second group was subjected to food restriction and received 50% of their usual daily intake until parturition. The pregnant rats delivered spontaneously, and the litter size was randomly culled to eight per mother at birth, in order to assure uniformity of litter size between the SGA and appropriate for gestational age (AGA) litters. All female offspring, as well as male offsprings that did not meeting the criteria for AGA and SGA, were culled. The criteria for AGA were as follows: Offspring of normal intake and birth weight between mean ± standard deviation (SD). The criteria for SGA were as follows: Offspring of food-restricted mothers and birth weight <-2 SD of the AGA group. Following parturition, mothers from the food restricted group were sacrificed by the administration of 20 ml chloral hydrate (J&I Biological, Shanghai, China). The pups were cross-fostered from food-restricted mothers to ad libitum-fed mothers. Both groups were given ad libitum access to food. At 1 day and 3 weeks of age, the rats were sacrificed by anesthesia (chloral hydrate; dose, 2 ml 1-day-old rats and 10 ml for 3-week-old rats). The ad libitum mothers were sacrificed by the administration of 20 ml chloral hydrate. Liver tissue samples were harvested and snap-frozen in liquid nitrogen for subsequent processing.

Total hepatic TG content was determined using a GPO-PAP enzymatic assay (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, liver tissue (30–50 mg) was homogenized in ethanol (9 ml ethanol, 1 g liver sample), the mixture was microfuged at 664 × g for 10 min at 4°C, and the supernatant was transferred to new tubes. The reaction system was prepared according to the manufacturer's instructions. The GPO-PAP enzymatic assay protocol includes the following steps: First, the lipids are broken down via the hydrolysis of triglycerides into glycerol and FFAs. Then the glycerol is converted to glycerol 3-phosphate via adenosine triphosphate and glycerol kinase, and is further converted to dihydroxyacetone phosphate and hydrogen peroxide via glycerophosphate oxidase. Under the effect of peroxidase, red quinones are produced when hydrogen peroxide meets 4-amino-antipyrine and 4-chlorophenol. The color degree of quionoes is propotional to triglyceride concentration. Following a 5 min incubation at 37°C, the final mixtures in 96-cell plates were rapidly quantified at 500 nm using a micro-plate reader (Varioskan Flash 3001, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Quantification of TG content was based on the following calculation: {[Sample optical density (OD) value-blank OD value]/(calibration OD value-blank OD value)} × calibration concentration. Finally, data were expressed as mg/g of liver.

Briefly, total RNA was isolated from the liver tissue samples using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). High-Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to reverse transcribe 2 µg total RNA using the following program: 25°C for 10 min, 37°C for 120 min, 85°C for 5 min, and a 4°C hold. To measure the relative mRNA expression levels, RT-qPCR was performed using an Applied Biosystems StepOnePlus™ Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR cycling conditions were as follows: 94°C for 10 min, followed by 40 cycles at 94°C for 20 sec and 60°C for 1 min. RT-qPCR was performed using SYBR^® Select Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) All reactions were performed in triplicate. The relative mRNA expression levels were calculated using the 2^−ΔΔCq method (20). The primer sequences were as follows: LXR-α, forward 5′-GAGAGCATCACCTTCCTCAAG-3′, reverse 5′-TCATGGATCTGGAGAACTCAAAG-3′; LPL, forward 5′-ACAGGTGCAATTCCAAGGAG-3′, reverse 5′-CTTTCAGCCACTGTGCCATA-3′; and glyceraldehyde 3-phosphate dehydrogenase (GAP DH), forward 5′-GACAACTTTGGCATCGTGGA-3′ and reverse: 5′-ATGCAGGGATGATGTTCTGG-3′. Primers were purchased from Thermo Fisher Scientific, Inc.

The liver tissue samples were homogenized in lysis buffer (Beyotime Institute of Biotechnology, Hangzhou, China) and centrifuged at 12,000 × g for 10 min at 4°C. The protein concentration was determined using a Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein (80 µg) were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and were electroblotted onto a polyvinylidene difluoride membrane (0.2 µm pore size; EMD Millipore, Billerica, MA, USA). The membrane was blocked in 5% non-fatmilk for 2 h at room temperature. Following blocking, the membrane was incubated at 4°C with anti-LXR-α [dilution 1:3,000; cat. no. ab41902; Abcam (Hong Kong) Ltd., Hong Kong, China), anti-LPL (dilution 1:200; cat. no. sc-32885; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-GAPDH antibodies (dilution 1:5,000; cat. no. 5174; Cell Signaling Technology, Inc., Danvers, MA USA) for 12 h. The blots were analyzed by ImageJ version 1.39 software (National Institutes of Health, Bethesda, MD, USA). Subsequently, the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) (HRP-labeled (cat. no., A0208; dilution 1:2,000; Beyotime Institute of Biotechnology) and HRP-labeled goat anti-mouse IgG secondary antibodies (cat. no., A0216; dilution 1:2,000; Beyotime Institute of Biotechnology) for 2 h at room temperature. Signals were detected using enhanced chemiluminescence, according to the manufacturer's protocol (SuperSignal chemiluminescent substrates; Pierce; Thermo Fisher Scientific, Inc.).

The ChIP assay was performed according to the manufacturer's protocol (EZ-ChIP kit; EMD Millipore). Liver tissue samples (40 mg) were fixed in 1% formaldehyde for 10 min at room temperature. Cross-linking was terminated by the addition of glycine (1 M). Following two washes with cold phosphate-buffered saline, the liver tissue was resuspended in 1 ml SDS lysis buffer supplemented with 5 µl 1X protease inhibitor cocktail II. The lysates were aliquoted to 300–400 µl per microfuge tube and were sonicated on ice, in order to shear the DNA to a length between 200 and 1,000 bp. The sheared crosslinked chromatin was diluted 10-fold in dilution buffer containing protease inhibitor cocktail II. The chromatin solution was pre-cleared with 60 µl protein G agarose at 4°C for 1 h with rotation. The chromatin solutions were then microfuged at 4,000 × g for 1 min at 4°C to pellet agarose, and the supernatant was placed in new tubes, with 10 µl removed as input. The supernatant fractions were incubated overnight on a rocking platform with antibodies against acetylated histone H3K9 [4 µg; cat. no. ab10812; Abcam (Hong Kong) Ltd.], acetylated histone H3K14 [4 µg, cat. no. ab52946; Abcam (Hong Kong) Ltd.] and ChIP-grade LXR-α [5 µg; cat. no. ab41902; Abcam (Hong Kong) Ltd.] at 4°C. Subsequently, 60 µl protein G agarose was added to the tubes, which were incubated on a rocking platform for 1 h at 4°C. Following centrifugation at 4,000 × g at 4°C for 1 min, the agarose beads containing the immunoprecipitated complexes were washed sequentially in Low Salt Immune Complex Wash Buffer, High Salt Immune Complex Wash Buffer, LiCI Immune Complex Wash Buffer, and 2X TE buffer. The immune complexes were eluted twice with 200 µl elution buffer (10 µl 20% SDS, 20 µl 1 M NaHCO₃, 170 µl sterile distilled water) at room temperature. Elution buffer (200 µl) was also added to the input tubes. Subsequently, 8 µl 5 M NaCl was added to the elute and the cross-linking of the immunoprecipitated chromatin complexes and input controls were reversed by heating at 65°C for 5 h. Following treatment with Proteinase K, Tris-HCl and EDTA for 2 h at 45°C, the DNA was purified, according to the protocol of the manufacturer of the EZ-CHIP kit (EMD Millipore, Billerica, MA, USA).

The putative LXR-binding site in the promoter region of LPL was determined using MatInspector Software (http://www.genomatix.de/online_help/help_matinspector/matinspector_help.html). Primers were purchased from Invitrogen (Thermo Fisher Scientific, Inc.) and were as follows: Forward (5′-ATTCTCCACCTTGTCCCTTTG-3′) and reverse (5′-GCTTGATTCCCAGAACCCAC-3′) primers that amplify −3438 to −3423 promoter regions encompassing the rat LPL LXRE site (GAGGCC_DR4_GAGGGC), and primers (promoter A1, forward 5′-TCTGCTTTGCTGCTGGAACT-3′, reverse 5′-AGACGAAACGACGACCTTGA-3′; promoter A2, forward 5′-CACTGTAACGAGGCTCAACG-3′, reverse 5′-GTGACATTGCTCCGAGTTGC-3′; and promoter A3, forward 5′-GAGGCAGAAAGTCATGGTCAAATA-3′ and reverse 5′-CTCCGTCTTTCAGTACCAGTTTAT-3′) surrounding the promoter were used to examine the acetylation status of acetylation of histone H3 (K9, K14) at the promoter of LPL. For negative controls, ChIP assays were performed using an immunoglobulin G antibody (1 µg; part of the EZ-ChIP kit) to determine the immuno-specificity of the antibodies for the LPL promoter. The DNA samples from the input, unbound and bound fractions were determined by qPCR, according to the previously described protocol. The relative abundance of the immunoprecipitated chromatin, as compared with the input chromatin was determined using the 2^−ΔΔCq method (20).

SPSS 18.0 (SPSS, Inc., Chicago, IL, USA) was used for data analysis. All data are expressed as the mean ± standard error of the mean. Statistical significance was calculated using Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

At 1 day of age, no differences were evident in hepatic TG levels between the AGA and SGA male rats (AGA, 17.39±3.25 mg/g; SGA, 18.68±2.05 mg/g; P>0.05) (Fig. 1). At 3 weeks of age, the hepatic TG levels were increased in the SGA male rats (AGA, 5.18±0.63 mg/g; SGA, 7.73±0.91 mg/g; P<0.05), as compared with the AGA rats.

The hepatic mRNA expression patterns in both groups are presented in Fig. 2. At 1 day of age, there was no difference in the mRNA expression levels of LXR-α and LPL between the AGA and SGA male rats (Fig. 2A and C). However, at 3 weeks of age, the SGA male rats had significantly increased mRNA expression levels of LXR-α and LPL, as compared with the AGA rats (P<0.05 and P<0.01, respectively) (Fig. 2B and D).

To obtain further information regarding the differences in protein expression, western blotting was performed (Fig. 3). The protein expression levels of LXR-α and LPL were increased (P<0.05) in the SGA male rats, as compared with the AGA rats at 3 weeks of age (Fig. 3B and D); however, no differences were detected between the SGA and AGA male rats at 1 day of age (Fig. 3A and C).

To determine whether there were alterations in the recruitment of LXR-α to the promoter regions of LPL containing a well-characterized LXRE site, ChIP analyses were performed with antibodies specific for LXR-α. Negative controls demonstrated that the immunoprecipitations were specific for the indicated antibodies. At 3 weeks of age, the SGA male rats exhibited a marked increase in the binding of LXR-α to the promoter regions of LPL, as compared with in the AGA rats (P<0.05) (Fig. 4).

ChIP was further used to investigate whether chromatin remodeling could be a factor influencing the observed increase in LPL mRNA and protein expression levels in 3-week-old male SGA rats. Three sites (A1: −87 to +177; A2: −136 to −533 and A3: −644 to −896) along the LPL promoter were analyzed. The hepatic levels of acetylated histone H3K9 in the LPL promoter A1, A2 and A3 regions of SGA rats were increased 1.71-fold, 2.73-fold and 2.50-fold, respectively (P<0.05), as compared with the AGA rats (Fig. 5A). The hepatic levels of acetylated histone H3K14 in the LPL promoter A1, A2 and A3 regions of SGA rats were increased 1.69-fold, 1.86-fold and 2.67-fold, respectively (P<0.05), as compared with the AGA rats (Fig 5B).