Cell culture and treatment
Hepatocytes were isolated from three 14-day-old Tianfu Meat Goose from the Experimental Farm for Waterfowl Breeding at Sichuan Agricultural University (Sichuan, China) using a modification of the "two-step procedure" described by Seglen [15] . Goose primary hepatocytes were isolated and cultured in dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (PBS). The culture conditions were 37 ℃ with 5% CO2 after 24 h. After an additional 24 h, the cells were separately treated with serum-free media supplemented with 30mmoL/L glucose or fructose or sucros and incubated for 24 h. Cell viability determination was shown in Supplement materias S-Figure 1. The cells were collected for follow-up study. Each experiments was performed at least in triplicate.
Concentration measurement of TG and VLDL
The extracellular VLDL concentration in the supernatant was measured using a chicken VLDL ELISA kit (GBD, USA). The concentration of VLDL in the samples was determined by comparing the optical density (OD) value at 450 nm of the samples to the standard curve. After cultured cell treatment, the culture media was collected for detecting extracellular TG concentration. Cell samples used to measure intracellular TG concentration were collected. The TG levels were quantified using a triglyceride GPO-POD assay kit (Biosinc, China). Measurements will be in accordance with the manufacturer's protocol. All assays were performed in triplicate.
Oil Red O staining
Briefly, after the treatments with goose primary hepatocytes, staining of intracellular lipids was performed using Oil Red O (Sigma) according to the manufacturer instructions. Oil Red O staining images were taken using a light microscope (Olympus Optical, Tokyo, Japan) at 200× magnification. For quantification of lipid accumulation, the Oil Red O-positive cells were extracted using 100% isopropanol for 10 min. The absorbance of the extracted dye was analyzed at a wavelength of 510 nM (BIO-RAD, USA).
Measurement of protein content in culture cells
Protein content of fatty acid synthetase (FAS), acetyl-CoA carboxylase (ACCα), carnitine palmitoyltransferase 1 (CPT1), microsomal triglyceride transfer protein (MTP) and apolipoprotein B (APOB) in culture cells was measured using ELISA kit (GBD, USA). Further measurements will be in accordance with the manufacturer's protocol. All assays were performed in triplicate.
Isolation of total RNA and real-time RT-PCR
Cultured cells total RNA was extracted using extraction kit (TRIzol Reagent) (Invitrogen, USA), and then RNA was transcribed into cDNA via reverse-transcription using the Primer Script TM RT system kit for real-time PCR (TaKaRa, Japan) as described by the manufacturer. The fluorescence quantitative PCR was performed on the CFX 96 instrument (Bio-Rad, USA), using a Takara ExTaq RT-PCR kit and SYBR Green as the detection dye (Takara, Japan); qRT-PCR reaction system contained the newly generated cDNA template (1.0 µL), SYBR Premix Ex Taq TM (6.0 µL), sterile water (4.0 µL), upstream primers of target genes (0.5 µL) and downstream primers of target genes (0.5 µL). After initital denaturation at 95˚C for 5 min, 40 cycles were carried out: 95˚C for 10 sec, 60˚C for 20 sec, 72˚C for 15 sec and 72˚C extension for 10 min. Fluorescence quantitative PCR Primers (BGI, Beijing, China) designed according to the goose gene sequences in current experiment were summarized in S-Table 1. Fold change in the expression of target gene was analyzed using the 2-ΔΔCt method [16]. β-actin and 18S used as the internal reference gene. Each test include 3 biological samples and each sample was analyzed in triplicate.
Protein Analysis by western blotting
Following the incubation with the diferent treatments, SDS buffer was used to extract total proteins from the harvested cells which were washed twice and collected in ice-cold PBS. The untreated cells were used as control. Equal amounts of total proteins (100 μg/lane) were separated by SDS-PAGE gel (6%) electrophoresis and transferred to a PVDF membrane. After blocking with a mixture of 5% skimmed milk/Tris-buffered saline Tween 20 (TBST), the membranes were incubated overnight at 4˚C with the primary antibody rabbit against sterol regulatory element-binding proteins-1 (SREBP1) carnitine palmitoyltransferase (CPT1A), MTP antibodies (1:1,000; Beijing Biosynthesis Biotechnology, China); antobody information was listed in Supplement materials S-Table2. Following three consecutive washes in TBST (0.05%), the membranes were incubated with the goat anti-rabbit horseradish peroxidase-conjugated IgG at 1:2000 (Beijing Biosynthesis Biotechnology, China) for another 2 h at room temperature. The results were normalized to α-Tubulin (Beijing Biosynthesis Biotechnology, China) protein levels. Protein expression levels were finally visualized using enhanced chemiluminescence (ECL) reagents (Beyotime Institute of Biotechnology, China).
Birds and Experiment Design and Sampling
One hundred newborn male Tianfu Meat Geese were raised in Experimental Farm for Waterfowl Breeding at Sichuan Agricultural University (Ya’an, China). When 13 weeks old, the geese of each breed were randomly separated into five groups (control group, corn overfeeding group, glucose overfeeding group, fructose overfeeding group, sucrose overfeeding group), each group consisted of 20 geese, the grouping situation and overfeeding dietary component was shown in Table 1; the geese of control group were normally fed. The overfeeding procedure and diet regimes were performed as previously described [17]. All geese were slaughtered when 16 weeks old. After 12 hours of fasting, the body weight of geese was weighed before slaughter. After 12 hours of fasting, the body weight of geese was weighed before slaughter. Ten mL of blood were collected from wing vein, and then the geese were killed. The serum was separated by blood centrifugation at 4 ℃ for 4000 r/min for 10 min, then kept at -20 °C for follow-up detection. After slaughter, the liver was separated and weighed immediately. Six geese of each group were anesthetized with intraperitoneal injection of sodium pentobarbital (60 mg/kg), and then killed; the liver was collected immediately. The livers were separated into two parts respectively. A part of the liver tissue was frozen in liquid nitrogen immediately, and then kept at -80 °C for transcriptome sequencing and long-chain fatty acid determination. Other part of liver was washed in ice-cold saline (0.9% NaCl; 4 °C) and fixed in 4% formaldehyde-phosphate buffer for histomorphology determination.
Biochemical Index Examinations of serum
Ten individuals blood samples were selected randomly from each group, serum biochemical indices were quantified in whole serum. The assay kits that detected total protein (TP), total cholesterol (T-CHO), albumin (ALB), very low-density lipoprotein (VLDL), very high-density lipoprotein (VHDL), TG, blood glucose, insulin, alanine aminotransferase (ALT), aspertate aminotransferase (AST), uric acid (UA) were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China);
Histomorphology Examinations
According to the methods of previous study [18], the cross-sections from the middle of liver were preserved in 4% formaldehyde-phosphate buffer were prepared using standard paraffin embedding techniques, sectioned (5 µm) and stained with hematoxylin and eosin (HE), and sealed by neutral resin size thereafter, and then examined by microscope photography system (Olympus, Tokyo, Japan), each slice was observed and 5 visual fields were randomly selected at 40× magnifications.
Long -chain fatty acid of foie gras determination
According to the methods described as previous expriment [19], gas chromatography (GC) was used to detected the foie gras fatty acids; GC analysis conditions: HP-FFAP capillary column, 29.5 mm × 320μm (diameter) × 0.25 (thickness); chromatographic column temperature programmed: 160 ℃ retained 1 min, up to 220 ℃ in 5℃ / min, then retained 8 min; carrier gas is nitrogen; total flow velocity: 70 mL/min. The derection were performed by Qingdao Sci-tech Innovation Co., Ltd (Qingdao, Shangdong, China).
Transcriptome Sequencing and analysis
A total amount of 1μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina NexSeq500 (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v4-cBot-HS (Illumia). After cluster generation, the library preparations were sequenced on an Illumina platform and paired-end reads were generated. The sequencing were performed by Baimike biological Technology Co., LTD (Beijing, China).
Raw reads of fastq format were processed through in-house perl scripts. Clean reads were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw reads. Hisat2 tools soft were used to map with reference genome of geese (A. cygnoides) reference genome (assembly Ans Cyg_PRJNA183603_ v1.0, https://www.ncbi.nlm.nih.gov/genome/31397?genome_assembly_id=229313). Differential expression analysis of five groups was performed using the DEseq. The resulting P values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P-value < 0.05 found by DEseq were assigned as differentially expressed. We used KOBAS software to test the statistical enrichment of the differentially expressed genes (DEGs) in KEGG pathways. The sequences of the DEGs was blast to the genome of a related species (the protein protein interaction of which exists in the STRING database: http://string-db.org/) to get the predicted PPI of these DEGs. Transcriptome analysis was performed via Baimike biocloud platform (Baimike biological Technology Co., LTD, Bejing, China).
Statistical analysis
By using SAS 9.13 package (SAS Institute Inc, Cary, NC), the comparisons of multiple groups were analyzed by GLM, and the means were assessed for significant differences using the SNK-q test. All data were presented as means ± standard deviation (SD) and showed with graphs created with GraphPad Prism 5.0 software (GraphPad Prism Software, Inc.). We considered P < 0.05 as statistically significant.