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Abstract
Purpose
Several studies have proven that EGCG, the primary green tea catechin, and glucosamine-6-phosphate (PGlc) reduce triglyceride contents in 3T3-L1 adipocytes. The objective of this study is to evaluate the combination effect of EGCG and PGlc on decline of accumulated fat in differentiated 3T3-L1 adipocytes. Methods: EGCG and PGlc were administered for 6 day for differentiation of 3T3-L1 adipocytes. Cell viability was measured using the CCK assay kit. In addition, TG accumulation in culture 3T3-L1 adipocytes was investigated by Oil Red O staining. We examined the expression level of several genes and proteins associated with adipogenesis and lipolysis using real-time RT-PCR and Western blot analysis. A flow cytometer Calibar was used to assess the effect of EGCG and PGluco on cell-cycle progression of differentiating 3T3-L1 cells. Results: Intracelluar lipid accumulation was significantly decreased by combination treatment with EGCG 60 μM and PGlc 200 μg/m compared with control and EGCG treatment alone. In addition, use of combination treatment resulted in directly decreased expression of PPARγ, C/EBPα, and SREBP1. In addition, it inhibited adipocyte differentiation and adipogenesis through downstream regulation of adipogenic target genes such as FAS, ACSL1, and LPL, and the inhibitory action of EGCG and PGlc was found to inhibit the mitotic clonal expansion (MCE) process as evidenced by impaired cell cycle entry into S phase and the S to G2/M phase transition of confluent cells and levels of cell cycle regulating proteins such as cyclin A and CDK2. Conclusion: Combination treatment of EGCG and PGlc inhibited adipocyte differentiation through decreased expression of genes related to adipogenesis and adipogenic and cell cycle arrest in early stage of adipocyte differentiation.
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Fig. 1.
A: Structure of EGCG and Glucosamine 6-phosphate. B: The change of cell viability in 3T3-L1 preadipocyte in condition of EGCG of serial concentrations. Value represent the mean ± SEM. ∗∗: p < 0.001, vs. EGCG 0 μM.
Fig. 2.
Effect of EGCG & Glucosamine 6-phosphate on lipid accumulation in 3T3-L1 cells. A: Cell were subjected to Oil Red O staining B, C: These cells were then subjected to quantitative analysis of interacelluar lipid accumulation. Value represent the mean ± SEM. ∗∗: p < 0.001, vs. control. †: p < 0.05, E40 vs. E60. ‡: p < 0.05, E60 vs. E60P200. E: EGCG, P: Glucosamine 6-phosphate.
Fig. 3.
Effect of EGCG & Glucosamine 6-phosphate on adipogenesis transcription factors. PPARγ expression in 3T3-L1 cells as examined by real time PCR (A) and western blot (B) analysis. C/EBPα and SREBP1 expression in 3T3-L1 cells as examined by real time PCR (C). Confluent 3T3-L1 preadipocytes in medium with or without differentiation concentrations of GCG & Glucosamine 6-phosphate for 6 days (from day 0 to day 6) were differentiated into adipocytes. Value represent the mean ± SEM. Means with different letters (a-f) at each mRNA are significantly different (p < 0.05) by Duncan's multiple range test. Control: fully differentiated control adipocytes, E: with EGCG, P: Glucosamine 6-phosphate.
Fig. 4.
Effect of EGCG & Glucosamine 6-phosphate on FAS, ACSL1 and LPL expression (A) and HSL and perilipin expression (B) as examined by real time PCR analysis. Confluent 3T3-L1 preadipocytes in medium with or without differentiation concentrations of GCG & Glucosamine 6-phosphate for 6 days (from day 0 to day 6) were differentiated into adipocytes. Value represent the mean ± SEM. Means with different letters (a-d) at each mRNA are significantly different (p < 0.05) by Duncan's multiple range test. Control: fully differentiated control adipocytes, E: with EGCG, P: Glucosamine 6-phosphate
Fig. 5.
Mitotic clonal expansion during the 3T3-L1 preadipocyte differentiation induction process. 3T3-L1 preadipocyte differentiation was induced with the standard induction protocol. FACS analysis for DNA content of 3T3-L1 preadipocytes during mitotic clonal ex-panssion after the differentiation induction. A: Stained with PI for DNA histograms generated with flow cytomatric analysis. B: Cell cycle phase distribution plotted over time follow in MDI exposure. Dotted lines through phase intersections approximate the kinetics of phase transitions. M1: subG1 phase, M2: G0/G1 phase, M3: S phase, M4: G2/M phase.
Fig. 6.
Cell cycle analysis of EGCG and glucosamine-6-phosphate treated 3T3-L1 cell during th MCE process of adipocyte differentiation. Two day postconfluent 3T3-L1 cell were indicated with the adipogenic cocktail in the presence or absence of various concentration and alone or combination of EGCG and glucosamine-6-phosphate. After 18hr of treatment, differentiating cell were stained with propidium iodine (PI). These cells were then subjected to flow cytometric cell-cycle analysis (A) and quantitative analysis of cells in different phases in cell cycle (B). Data were analyzed using CellQuest Pro software. (C) The protein levels of cyclinA and CDK2 in these cells were detected by immunoblot assay using their specific antibody, β-actin was used as a loading control. M1: subG1 phase, M2: G0/G1 phase, M3: S phase, M4: G2/M phase. Control: fully differentiated control adipocytes, P: with Glucosamine 6-phosphate.
Table 1.
Primer sequences used for RT-PCR
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