Materials
Agrimonia Pilosa Ledeb obtained from Western Medicine City (Chongqing, China). The extraction process represented in reference [20]. FC had a level of total flavonoids with a value of 316.53 ± 6.37 mg/g and was composed of quercetin, quercitrin, vitexin, isovitexin, hyperoside, rutin, kaempferol, tiliroside, apigenin, catechinic acid, taxifolin and digitoflavone-7-O-glucoside mainly. The powder was stored at 4°C.
3T3-L1 pre-adipocytes were purchased from ATCC. C57BL/6 mice were purchased from Children's Hospital of Chongqing Medical University (China). The low-fat diet (LFD, 19% protein, 4% fat, 67% carbohydrate, and 10% of calories from fat) and high-fat diet (HFD, 24% protein, 24% fat, 41% carbohydrate, and 45% calories from fat) were purchased from Shanghai SLAC Laboratory Animal CO. LTD. (Shanghai, China). DMEM, penicillin-streptomycin and FBS were purchased from HyClone. Bovine calf serum (BCS) was purchased from Gibco and BSA from Equitech-Bio. Paraformaldehyde, Oil Red O, 3-isobutylmethylxanthine (IBMX), dexamethasone (DEX) and insulin were purchased from Sigma-Aldrich (St Louis, MO). 2-(N-[7-Nitrobenz-2-oxa-1, 3-diazol-4-yl)Amino]-2-Deoxyglucose (2-NBDG) was purchased from Invitrogen. Glucose oxidase assay, ROS assay kit, total superoxide dismutase (SOD) assay kit with WST-1 and lipid peroxidation malondialdehyde (MDA) assay kit were purchased from Beyotime (Jiangsu, China). Trizol reagent was purchased from Invitrogen and reverse transcription system from Takara. SYBR Green was obtained from BIO-RAD. ELISA kits for insulin, IL-1β, IL-6 and TNF-a were purchased from 4A Biotech (China). The antibodies of β-actin, p-IRS-1, GLUT-4, PPARγ, Nrf2, p-IκBα, NF-κB p65, t-/p-JNK 1/2, PI3K and p-Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cell culture and adipocyte differentiation
3T3-L1 pre-adipocytes were induced to adipocytes based on the protocol [21]. Cells maintained in DMEM (25.0 mmol/L glucose) supplemented with 10% BCS and 1% penicillin-streptomycin. At day 0, after reaching confluence, treating with differentiation medium: DMEM (25.0 mmol/L glucose) supplemented with 0.5 mM IBMX, 1 μg/ml insulin, 0.25 μM DEX and 10% FBS for 48 h. For the next 48 h, treating with DMEM (25.0 mmol/L glucose) supplemented with 1 μg/ml insulin and 10% FBS. Then treating with DMEM (25.0 mmol/L glucose and 10% FBS) for 6 days until more than 90% of cells were differentiation. After differentiation at 11th day, 3T3-L1 adipocytes could be used for Oil Red O staining or induced to establish IR model subsequently.
Establishing 3T3-L1 adipocytes IR model
The model was established as described previously with some modification (Figure S1) [22]. After differentiation, the medium was replaced by DMEM (5.5 mmol/L glucose) with 1% BSA for 12 h. To establish IR model, the differentiated adipocytes were divided into LG (DMEM containing 5.5 mmol/L glucose, 1% BSA and 10-9 mol/L insulin), Piog (10 μM pioglitazone and DMEM containing 25.0 mmol/L glucose, 1% BSA and 10-6 mol/ L insulin), IR model groups which were divided into HG (DMEM containing 25.0 mmol/L glucose, 1% BSA and 10-6 mol/ L insulin) and FC (25 μg/ml or 100 μg/ml or 400 μg/ml FC with DMEM containing 25.0 mmol/L glucose, 1% BSA and 10-6 mol/ L insulin). By glucose oxidase assay, the glucose concentration was analyzed last 24 hours according to the instruction. After 24 hours, adipocytes were collected for further studies.
Animals and drug treatments
All procedures were in accordance with the institutional guidelines for animal experimentation and approved by the Institutional Animal Care and Use Committee of Children's Hospital of Chongqing Medical University. C57BL/6 mice (6 week) were housed under artificial lighting for 12 h/day and standard temperature (22°C–24°C). Mice were fed with LFD or HFD last 16 weeks. In the following 4 weeks, HFD mice were administered with Piog (5 mg/kg/day) or FC (5 mg/kg/day) or same volume of PBS by intragastric gavage each day. Body weight and food intake were recorded each day. After 4 weeks of drug treatment, the fed serum insulin was detected by ELISA kit, blood samples and white adipose tissue (WAT) were recovered and stored at -80°C.
Glucose uptake assay
2-NBDG, a fluorescent glucose analogue, was used to monitor the glucose uptake with the method described previously [23]. 3T3-L1 pre-adipocytes (8×103/well) were plated in 96-well plate before differentiation and establishing the IR model. Each group was divided into 3 wells: 100 μL KRB, 100 μL 50 μM 2-NBDG, 100 μL 50 μM 2-NBDG (Containing 100 nM insulin). These wells were incubated in 37°C for 30 min and detected the fluorescence intensity with the excitation wavelength at 488 nm and emission wavelength at 520 nm. Results of every 3 wells in one treating expressed as Fa, Fb, Fc. The calculation formula was (Fc-Fb)/(Fb-Fa).
Intra-peritoneal glucose tolerance tests (iGTTs)
iGTTs of C57BL/6 mice were performed after fasted overnight at the end of drug treatment. At 2 g/kg, 20% glucose solution was injected, and then the blood glucose concentrations at 0, 15, 30, 60 and 120 min were measured using a glucometer (SureStep OneTouch, USA).
Detecting intracellular ROS, activity of SOD, content of MDA and inflammatory cytokines
The fluorescence intensity of DCF-DA in adipocytes was detected by flow cytometry. The activity of SOD was determined by WST-1 method at 450 nM. The intracellular content of MDA was determined at 532 nM. Both of them were detected by purchased kits with ELIASA.
The secretion of IL-1β, IL-6 and TNF-a were detected by ELISA according to the manufacturer’s protocol.
RNA preparation and Real-Time PCR
Isolating total RNA by Trizol reagent and 1 μg of each sample was reverse-transcribed to cDNA by the reverse transcription system. According to the manufacturer’s protocol, Real-Time PCR with iQTM SYBR Green supermix was performed. Each reaction was performed in triplicate and GAPDH was used as the internal control gene. The primers were shown in Table 1.
Table 1 Adipocyte specific primers for Real-Time PCR
Gene
|
Forward Primer
|
Reverse Primer
|
GAPDH
|
CAA GGT CAT CCA TGA CAA CTT TG
|
GGC CAT CCA CAG TCT TCT GG
|
IRS-1
|
ACC ATA ACC AGA GTG CCA AAG T
|
CGA GTA GGT GCT GAG AAG GTC T
|
GLUT4
|
CAT GGC TGT CGC TGG TTT C
|
AAA CCC ATG CCG ACA ATG A
|
JNK
|
ATG GAT TTG GAG GAA CGA ACT A
|
GAA GAC GAT GGA TGC TGA GAG
|
Nrf2
|
TCT TCC ATT TAC GGA GAC CCA
|
GAT TCA CGC ATA GGA GCA CTG
|
NF-κB
|
ATT CCG CTA TGT GTG TGA AGG
|
TGT GAC CAA CTG AAC GAT AAC C
|
IL-6
|
CTG CAA GAG ACT TCC ATC CAG TT
|
AAG TAG GGA AGG CCG TGG TT
|
TNF-a
|
CTG AGG TCA ATC TGC CCA AGT AC
|
CTT CAC AGA GCA ATG ACT CCA AAG
|
Abbreviations: IRS-1, insulin receptor substrate-1; JNK, c-Jun N-terminal kinase; Nrf2, NF-E2-related factor 2.
Western blot
Proteins were extracted from cell or tissue lysis and the concentration was detected by BCA assay kit. Standard protocols were used. The bands were visualized by ECL and intensities were quantified using image analyzing software (Quantity One, Bio-rad).
Statistical analysis
All data are presented as means ± SEM. A paired-samples T test was used for two groups and one-way ANOVA for more than two groups by SPSS 19.0 software. Difference with a value of p<0.05 were considered significant statistically. All experiments were repeated at least 3 times.