oleic acid (OA) were obtained from Sigma-Aldrich, Inc. (St. Louis, USA). Fructose were purchased from Biotopped Co.Ltd. (Beijing, China). Liraglutide(LG, purity ≥ 98%)were purchased from Meilun Biotechnology Co.Ltd. (Dalian, China). FFA-free Bovine Serum Albumin (BSA) were purchased from BioDee Biotechnology Co.Ltd. (Beijing, China).
For cell culture, RPMI1640, Hank's Balanced Salt Solution (HBSS) were obtained from Macgene Technology Co.Ltd. (Beijing, China). Fetal bovine serum, penicillin, and streptomycin were purchased from Gibco Life Technologies (Grand Island, NY, USA). Pancreatin、Enhanced BCA Protein Assay Kit and RIPA buffer were purchased from Beyotime Biotechnology Inc. (Beijing, China). Total cholesterol assay kit, Triglyceride assay kit and Glucose Assay Kit were purchased from Nanjing Jiancheng Bioengineering Institute. All other chemicals used were of analytical grade.
2.2 Cell culture and treatment
HepG2 (3111C0001CCC000035) were purchased from Institute of Basic Medicine, Chinese Academy of Medical Sciences (Beijing, China), grown in DMEM (containing 10% NCS, 100 µg mL− 1 streptomycin and 100 U mL− 1 penicillin) and incubated at 37 °C with 5% CO2 /95% air atmosphere.
L02 (3111C0001CCC000035) were purchased from Biological Technology Co.Ltd. (Shanghai, China), grown in RPMI 1640 (containing 10% FBS, 100 µg mL− 1 streptomycin and 100 U mL− 1 penicillin) and the culture conditions are same as HepG2.
L02 cells were treated with RPMI1640 medium containing 0.1% BSA for 24 hours as Control (C), or 100 µmol of OA and 15 mmol of fructose (FFAs) for 24 hours as IR model (M), or FFAs and 100 nmol LG for 24 hours as LG intervention (LG), or 100 nmol siRNA and FFAs and LG (100 nmol) for 24 hours as siGLP-1R and negative control (NC).
2.3 Small interfering RNA (siRNA) transient knockdown in L02
The L02 cells were cultured at stationary phase for 24 h, and transfected with 100 nmol siRNA targeting GLP-1R (gene ID: 2740),with a negative siRNA (NC) as control. The transfection efficacy was determined by mRNA analysis. The siRNA (siGLP-1R & NC) was mixed with the riboFECT™ CP transfection reagent. Then, siRNA was delivered into the L02 (~ 30% confluent density) and cultured in normal medium for 24 h. At the same time of transfection, the cells were cotreated with FFAs and LG (100 nmol). Then༌cell lysates were collected to analyze mRNA and protein levels. All siRNAs and transfection reagent were purchased from RiboBio Co. Ltd. (Guangzhou, China).
2.4 Glucose uptake and intracellular glycogen content
Glucose uptake and intracellular glycogen content were measured in different groups. Briefly, L02 were exposed to the treatments for 24 h. Afterward, the medium was removed, and the L02 were washed with HBSS. Subsequently, the medium was changed to RPMI1640 containing 2 g/L glucose for an additional 24 h. Finally, the glucose concentrations in the supernatants of each well were measured by a glucose assay kit and normalized to the total cellular protein. The intracellular glycogen contents under the treatments were measured using the glycogen assay kit and normalized to the total protein. All kits used are from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
2.5 Intracellular TG, TC and supernatant TG levels
After treatments for 24 h, L02 were washed with HBSS and lysed with RIPA buffer. TG, and TC levels were detected using corresponding determination kits and normalized to the total cellular protein. All kits used are from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
2.6 Oil Red O staining
After treatments for 24 h, L02 were washed with HBSS buffer to remove the residual culture medium. And then fixed with 4% polyformaldehyde for 30 min, followed by staining with Oil Red O solution for 10 min, at room temperature(Liu et al., 2017). The stained lipid droplets were visualized and photographed by using CKX41 microscope (CellSens, Olympus, Japan)
2.7 Intracellular AMP/ATP levels
L02 were seeded in 6-well plates. After the same treatment for 24 h, cells were washed with HBSS and lysed with RIPA buffer. The ATP (adenosine triphosphate)、AMP(Adenosine monophosphate) levels were detected by ELISA kits (Inselisa Biotechnology Co. Ltd, Hubei, China) according to the manufacturer’s instructions, and the results were normalized to the total cellular protein.
2.8 Real-time Quantitative PCR
Total RNA was extracted using TransZol Up Plus RNA Kit (Beijing, China). About 1 µg of total RNA was reverse transcribed using TransScript®Green One-Step qRT-PCR SuperMix Kit (Beijing, China). The preparation of 20 µL reaction system,includes: 1 µg RNA template, 0.5 µL forward and reverse primers, 10 µL 2 × TransScript® Tip green qPCR SuperMix, 0.5 µL TransScript® One-Step RT/RI Enzyme Mix and some enzyme free water. SYBR fluorescent dye was used to detect the relative expression of the selected genes. Real-time quantitation was performed on the CFX Connect real-time PCR instrument (Bio-Red, USA). The thermal cycling conditions for PCR were 45℃ for 5 min, 94℃for 30 s, 40 cycles of 94℃ for 5 s;65℃ for 1 min. All measurements were performed in triplicate, and the data were analyzed with Bio-Rad CFX Manager (California, USA). All PCR data were normalized to the glyceraldehyde-3-phosphate dehydrogenase expression. The primers used are all designed on NCBI official website, and the sequence of the gene primers were as follows: GLP-1R, forward 5′- CGATGGCCCAGTCCTGAAC-3′ and reverse 5′-GGACACAGTGGCACCCTGG-3′; phosphoenolpyruvate carboxykinase (PEPCK), forward 5′-AGCTGTGCCAGCCAGAGTAT-3′ and reverse 5′-ATGACCGTCTTGCTTTCGAT-3′; glucose 6 phosphatase (G6Pase), forward 5′-AAAGATAAAGCCGACCTACAGA-3′ and reverse 5′-GGACGAGGGAGGCTACAATA-3′; carnitine palmitoyl transferase 1(CPT-1), forward 5′-GCATGATCGCAAAGATCAGT-3′ and reverse 5′-TGGTAGGAGAGCAGCACCTT-3′; acetyl CoA carboxylase (ACC), forward 5′-GCATGATCGCAAAGATCAGT-3′ and reverse 5′-TGGTAGGAGAGCAGCACCTT-3′; fatty acid synthase(FAS), forward 5′-GCAAGCTGAAGGACCTGTCT-3′ and reverse 5′-AATCTGGGTTGATGCCTCCG-3′; sterol response element binding protein-1c(SREBP-1c), forward 5′-TCTGACAGCCATGAAGACAGAC-3′ and reverse 5′-ATAGGCAGCTTCTCCGCATCTA-3′; GAPDH, forward 5′-GACCCGTGCTGCTTTCTTGA-3′ and reverse 5′-GGGTGGAGTCGTACTGGAAC-3′.
2.9 Western blotting
After protein content determination, equal amounts of proteins from cell lysates were loaded onto an 8% SDS-PAGE gel after denaturation with SDS loading buffer and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk for 2 h at room temperature. Afterward, antibodies of anti-PI3K (p110α), anti-Akt, anti-pAkt, anti-GSK3β, anti-pGSK3β, anti-FOXO1, anti-pFOXO1, anti-AMP-activated protein kinase (AMPK), anti-pAMPK and anti-PPARα (Cell Signaling Technology, Inc.), and anti-beta actin were added overnight at room temperature. The membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The protein bands were captured and analyzed by ChemiScope (ChemiScope 6000 Touch ༆ChemiAnalysis).
2.10 Statistical analysis
Statistical analysis was performed by one-way ANOVA analysis of variance using Origin 8.5 software (Origin Lab Corp., USA). All data are presented as the mean values ± standard deviation (SD). Statistical significance was set at P < 0.05.