Nutr Res Pract.  2012 Dec;6(6):499-504.

Resveratrol inhibits the protein expression of transcription factors related adipocyte differentiation and the activity of matrix metalloproteinase in mouse fibroblast 3T3-L1 preadipocytes

• Kang NE
• Ha AW
• Kim JY
• Kim WK

Abstract

This study attempted to investigate the effects of resveratrol on the differentiation of adipocytes. After cells were treated with various concentrations of resveratrol (0, 10, 20, and 40 micromol/L), adipocyte proliferation, the protein expression of transcription factors, and MMPs' activities were determined. Cell proliferation was inhibited more within 4 days of incubation (P < 0.05), and lipid accumulation in adipocyte was significantly inhibited by 93.8%, 92.4% and 91.5%, respectively, after two days of 10, 20, and 40 micromol/L resveratrol treatment (P < 0.05). Six days of incubation with the three resveratrol concentrations caused a significantly decreases of 63%, 59.9%, and 25.1% GPDH activity as a dose-dependent response. The triglyceride concentration also decreased significantly with the increase of resveratrol concentration (P < 0.05). The protein expression of CCAAT/enhancer-binding protein (C/EBPbeta) was decreased significantly by 56% and 30% while PPARgamma was significantly reduced by 57% and 15% with resveratrol treatments of 20 and 40 micromol/L, respectively (P < 0.05). The protein expression of C/EBPalpha was decreased by 83%, 74%, and 38% to increased dosage levels, with significance determined for this decrease from 20 micromol/L of resveratrol. The protein expression of fatty acid binding protein (FABP4) was decreased significantly by 88%, 72%, and 46% with the increase of resveratrol concentration. The activity of MMP-2 was decreased significantly by 84%, 70%, and 63% while MMP-9 activity was decreased significantly by 74%, 62%, and 39% with the increased resveratrol concentrations of 10, 20, and 40 micromol/L, respectively (P < 0.05).

Keyword

Adipocyte; resveratrol; transcription factor; matrix metalloproteinase; differentiation

MeSH Terms

Adipocytes
Animals
Carrier Proteins
Cell Proliferation
Fibroblasts
Mice
PPAR gamma
Stilbenes
Transcription Factors
Carrier Proteins
PPAR gamma
Stilbenes
Transcription Factors

Figure

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  Fig. 1 Effect of resveratrol on cell proliferation in 3T3-L1 cells. 3T3-L1 cells were plated at a density of 1.5 × 104 cells/mLin 24 well plates with DMEM supplemented with 10% BCS for 2 days, the monolayers were differentiated induction with DMEM supplemented with 10% FBS, 10 µg/mLinsulin, 1 µmol/L Dex, 0.5 mmol/L IBMX for 2 days. After differentiation induction, the monolayer was incubated in post differentiation medium with 0, 10, 20, and 40 µmol/L resveratrol. Viable cell numbers were estimated by MTT assay. Each bar represents the mean ± SE. Comparison among different concentrations of resveratrol that yielded significant differences (P < 0.05) are indicated by different letters above each bar.

• 

  Fig. 2 Effect of resveratrol on quantification of lipid content in 3T3-L1 cells. 3T3-L1 cells were plated at a density of 1 × 104 cells/mLin a 96 well plate with DMEM supplemented with 10% BCS for 2 days, the monolayers were differentiation induction with DMEM supplemented with 10% FBS, 10 µg/mLinsulin, 1 µmol/L Dex, 0.5 mmol/L IBMX for 2 days. After differentiation induction, the monolayer was incubated in post differentiation medium with 0, 10, 20, and 40 µmol/L resveratrol. Lipid accumulation was estimated by the Oil red O staining. Each bar represents the mean ± SE. Comparison among different concentrations of resveratrol that yielded significant differences (P < 0.05) are indicated by different letters above each bar.

• 

  Fig. 3 Effect of resveratrol on triglyceride and GPDH activity in 3T3-L1 cells. 3T3-L1 cells were plated at a density of 2.0 × 104 cells/mLin a 6 well plate with DMEM supplemented with 10% BCS for 2 days, the monolayers were applied to differentiation induction with DMEM supplemented with 10% FBS, 10 µg/mLinsulin, 1 µmol/L Dex, 0.5 mmol/L IBMX for 2 days. After differentiation induction, the monolayer was incubated in post differentiation medium with 0, 10, 20, and 40 µmol/L resveratrol. Triglyceride accumulation was estimated by the commercial triglyceride assay kit (A). GPDH activity was estimated by a commercial GPDH activity assay kit (B). Each bar represents the mean ± SE. Comparison among different concentrations of resveratrol that yielded significant differences (P < 0.05) are indicated by different letters above each bar.

• 

  Fig. 4 Effect of resveratrol on the protein expression of transcription factors in 3T3-L1 cells. 3T3-L1 cells were plated at a density of 2.5 × 104 cells/dish with DMEM supplemented with 10% BCS for 2 days, the monolayers were applied to differentiation induction with DMEM supplemented with 10% FBS, 10 µg/mLinsulin, 1 µmol/L Dex, 0.5 mmol/L IBMX for 2 day. After differentiation induction, the monolayers were incubated in post differentiation medium with 0, 10, 20, and 40 µmol/L resveratrol. Chemiluminescent detection and quantitative analysis of western blots were performed for three independent experiments. The protein expression of C/EBPβ (A), PPARγ (B), C/EBPα (C), and FABP (D) are shown above. Each bar represents the mean ± SE. Comparison among different concentrations of resveratrol that yielded significant differences (P < 0.05) are indicated by different letters above each bar.

• 

  Fig. 5 Effect of resveratrol on MMP 2 activity in 3T3-L1 cells. 3T3-L1 cells were plated at a density of 2.0 × 104 cells/dish with DMEM supplemented with 10% BCS for 2 days, the monolayers were differentiation induction with DMEM supplemented with 10% FBS, 10 µg/mLinsulin, 1 µmol/L Dex, 0.5 mmol/L IBMX for 2 days. After differentiation induction, the monolayer was incubated in post differentiation medium with 0, 10, 20, and 40 µmol/L resveratrol. The media were collected and concentrated for zymography. MMP-2 bands (A) and MMP-9 bands (B), which were representative of three independent experiments, are shown. Each bar represents the mean ± SE. Comparison among different concentrations of resveratrol that yielded significant differences (P < 0.05) are indicated by different letters above each bar.

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