3T3-L1 mouse fibroblast preadipocytes were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were grown to confluence in DMEM (WelGENE, Daegu, Korea) and were supplemented with 10% bovine calf serum (BCS) (WelGENE, Daegu, Korea), 100 units/mL penicillin, and 100 µg/mL streptomycin (WelGENE, Daegu, Korea) to make a regular medium (RM) [17]. The medium was replaced every 2 days and incubated in 5% CO₂ atmosphere at 37℃ until the medium reached confluence state. When cells reached 70 - 80% confluence, cells were rinsed twice with phosphate buffered saline solution (PBS) and treated with trypsin-EDTA (WelGENE, Daegu, Korea). The separated cells were then sub-incubated with the medium being replaced every 2 days.

To differentiate 3T3-L1 preadipocytes into adipocytes, 10% fetal bovine serum (FBS) (WelGENE, Daegu, Korea), 0.5 mmol/L isobuthylmethylxanthine (IBMX), 10 µg/mL insulin, 1 µmol/L dexamethasone (Dex), 100 units/mL penicillin, and 100 µg/mL streptomycin was added to DMEM to make Differential Media (DM), which was used to induce differentiation [18]. Two days after differentiation, 10% FBS, 10 µg/mL insulin, 100 units/mL penicillin, and 100 µg/mL streptomycin was added to DMEM to make post differentiation media (PM). Adipocytes were incubated in PM, which maintains the adipocyte state.

MTT assay was conducted to determine the effects of resveratrol on adipocyte proliferation [19]. 3T3-L1 preadipocytes were incubated with DM to induce differentiation, and then PM, treated with 0, 10, 20, and 40 µmol/L of resveratrol, was added to mature adipocytes. Cell viability was estimated by adding 1mL of MTT solution (1 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and by then incubating 37℃, 5% CO₂ atmosphere for 3 h. After incubation of 0, 2, 4, and 6 days, each MTT solution was diluted with 0.5 mL of isopropyl alcohol and absorbance rate was measured at 490 nm with spectrophotometer (Tecan, Austria GmbH, USA).

After induction of differentiation, cells were stained with oil-red O solution using adipogenesis assay kit (Chemicon International Inc., Temecula, USA) [20]. Cells were washed twice with 100 µL PBS, and then 50 µL of oil-red O solution was added and they were incubated at room temperature for 15 min. After incubation, the stained oil droplets were dissolved in isopropanol and were measured for absorbance at 450 nm using a spectrophotometer (Tecan, Austria GmbH, USA).

Intracellular triglyceride accumulation was measured with a commercially available triglyceride assay kit (Wako chemicals GmbH, Neuss, Germany) [21]. Cells were treated with 0, 10, 20, and 40 µmol/L of resveratrol and were rinsed twice with PBS. Cells were resuspended in 137 mmol/L NaCl, 20 mmol/L Tris-Cl, 1% triton X-100, 10% glycerol, 1 mM/L sodium orthovanadate, 1 mmol/L PMSF, 20 µg/mL aprotinin, 10 µg/mL antipain, 10 µg/mL leupeptin, and 80 µg/mL benzamidine HCl. Cells were then lysed by sonification at 4℃ and centrifuged at 12,800 rpm for 5 min. After centrifugation, the aliquot was mixed with an equal volume of chloroform and was then centrifuged again. The chloroform layer was carefully dried and resuspended in a 1% Triton X-100. The Sample was treated with 300 µL of color reagent and was incubated at 37℃ in a 5% CO₂ incubator for 5 min. Absorbance was then measured at 600 nm using spectrophotometer (Tecan, Austria GmbH, USA).

GPDH activity was measured with GPDH activity kit (Takara Bio Inc., Japan) [22]. Cells were plated at a density of 1 × 10⁴ cells/mL in 96 well plate. Cells were treated with 0, 10, 20, and 40 µmol/L of resveratrol every two days. At six days after differentiation induction, cells were rinsed with PBS and enzyme extraction buffer was added to the cells. Cells were then diluted with a solution containing 0.1 mol/L 2-mercaptoethanol. The change in absorbance density was measured at 340 nm, 30℃ every minute for 10 min using a spectrophotometer (Tecan, Austria GmbH, USA). GPDH activity was calculated as the amount of enzyme used to consume 1 µmol/L of substrate in one min at 30℃, converting this unit to mg protein/mL. Protein in the supernatant was quantitatively measured with a protein assay kit (BioRad, Richmond, CA, USA).

Cell lysates were performed before western blotting analysis [22]. The harvested cells were transferred to 50 mL tubes on ice, centrifuged for 3 min, and the supernatant was discarded. Cells were then rinsed with ice-cold PBS buffer. The protein of the sample were separated using gel electrophoresis employed with polyacrylamide gels and with buffers loaded with 4-20% gradient sodium dodecyl sulfate (SDS) (SDS-PAGE) [23]. It was then transferred overnight to immobilon TM^p membrane (Millipore, Bedford, MA, USA) at 4℃. Blocking of non-specific binding was achieved by placing the membrane in 10 g/L Bovine serum albumin (BSA) or 50 g/L milk in TBST (Tris-buffered saline (20 mmol/L Tris-Cl, and 150 mmol/L of NaCl), and 1 g/L Tween 20, pH7.5). After blocking, proteins in membrane were then immunoblotted with antibodies to PPARγ (Abcam, Cambridge, England), C/EBPα, C/EBPβ (Santa Cruz Biotechnology, Santacruz, USA), and FABP4 (Abcam, Cambridge, England) [24-28]. After rinsing off the membrane with TBST, anti-mouse 1 g horseradish peroxidase/TBST or anti-rabbit 1 g horseradish peroxidase/TBST (Amersham, Buckinghamshire, England) were incubated with the membrane. After a final wash with TBST, the western blot analysis was performed by using the enhanced Chemiluminescence method, and the Super Signal West Dura Extended Duration Substrate (Piece, Rockford, IL). The density of each band image was analyzed by Image J Launcher (Provided by NCBI), which evaluates the relative amount of protein staining and quantifies the results in terms of optical density. Protein expression level of the control group was set as 100% and the results to each resveratrol treatment groups were compared with the control group.

MMPs activity was determined as previously described [29]. After cells were incubated with various concentrations of resveratrol, 0, 10, 20, and 40 µmol/L, for 3 days and centrifuged for 10 min at 3,000 rpm, the supernatants were collected and MMP activity was determined by electrophoresis on a polyacrylamide gel containing 1% gelatin. The gel was shaken twice for 30 min under renaturation buffer (Triton X-100 2.5% in water (v/v)) and also under developing buffer (0.5 mmol/L Tris, Tris-HCl, NaCl, CaCl2, Brij), and incubated for 17 h at 37℃. The gel was stained for 30 min with 0.25% Commassie blue and then the activated parts of each band under the dyed gel was scanned with digitization by image program Image J Launcher (provide by NCBI). The MMPs were detected as clear bands against a blue background of undegraded substrate.

This research was conducted 3 times independently, and the SAS program was used to calculate the average and the standard error of each experimental group. After running an ANOVA test, a Duncan's multiple range test was conducted at α = 0.05 to verify its significance.

According to MTT assay at 0, 2, 4, and 6 days, resveratrol did not show significance on cell proliferation at 0 and 2 days after incubation. However, cell proliferation was found to be decreased significantly in a dose-dependent manner of resveratrol treatments after 4 days of incubation; When the non-treatment group of resveratrol was said to express as standard 100%, cell proliferation was decreased by 77.2%, 68.3%, and 52.1%, at the 4^th day of incubation, and by 84.2%, 65.3%, and 29.6% at the 6^th days of incubation, with increasing resveratrol concentrations of 10, 20, and 40 µmol/L (P < 0.05) (Fig. 1).

In the oil-red O dye staining, two days after resveratrol treatments of 10, 20, and 40 µmol/L, lipid accumulation in adipocyte was significantly inhibited by 93.8%, 92.4%, and 91.5% respectively, compared to the control (P < 0.05) (Fig. 2). At four days of incubation, adipocyte differentiation was determined to be 90.6% with 10 µmol/L of resveratrol and 61.4% with 40 µmol/L resveratrol, respectively. The resveratrol treatments at day 6 caused significant differences in adipocyte differentiation, with 90.6% differentiation for 10 µmol/L resveratrol treatments and 59.7% for 40 µmol/L treatments.

Triglyceride concentrations were very slightly, but significantly decreased by 99%, 98.8%, and 97.7% according to resveratrol treatments with 10, 20, and 40 µmol/L, respectively, compared to control (100%) that contained no resveratrol (P < 0.05) (Fig. 3A).

When GPDH activity in 3T3-L1 treated with resveratrol at 10, 20, and 40 µmol/L were measured, incubation at day 6 caused significantly decreases of GPDH activity in a dose-dependent manner by 63%, 59.9%, and 25.1% with resveratrol treatments of 10, 20, and 40 µmol/L respectively (P < 0.05) (Fig. 3B).

The protein expression of C/EBPβ, which induces initial stages of differentiation, decreased significantly in a dose-dependent manner to 78% 56%, and 30% compared to the control after resveratrol treatments of 10, 20, and 40 µmol/L respectively, (P < 0.05) (Fig. 4A). The protein expression of PPARγ, which induces the middle stages of differentiation, was significantly reduced by 57% and 15% with resveratrol treatments of 20 and 40 µmol/L, respectively (P < 0.05) (Fig. 4B) while the protein expression of C/EBPα was also significantly decreased by 74% and 38% with resveratrol treatment of 20 and 40 µmol/L (P < 0.05) (Fig. 4C). The protein expression of FABP4, which induces final stages of differentiation, was decreased significantly by 88%, 72%, and 44% with the aforementioned increased resveratrol concentrations (P < 0.05) (Fig. 4D).

The activity of MMP-2 was decreased significantly 84%, 70%, and 63% after different resveratrol treatment concentrations of 10, 20, and 40 µmol/L, respectively (P < 0.05) (Fig. 5A). MMP-9 activity was also decreased to 74%, 62%, and 39% that of the control, in a dose-dependent manner (P < 0.05) (Fig. 5B).