Epigallocatechin-3-gallate Inhibits Adipogenesis Through Down-regulation Of FAS And PPARγ Expression Mediated By PI3K-AKT Signaling In 3T3-L1 Cells

Background and objective: It is well known that adipocytes have multiple physiological functions including maintaining energy balance and secreting adipocytokines.However,due to excessive energy intake,leading to fat cell hypertrophy and hyperplasia.In addition,adipocyte hypertrophy and hyperplasia,especially visceral adipocytes,will play a variety of pathological roles,including obesity,insulin resistance and hypertensive metabolic syndrome.It is generally believed that adipocytes are derived from preadipocytes,known as adipocyte differentiation,i.e.,adipogenesis.In adipogenesis,adipocytes undergo continuous phenotypic,morphological and functional changes.Preadipocytes can differentiate into adipocytes under the appropriate environmental and gene expression.Thus,inhibition of preadipocyte differentiation may be a therapeutic strategy for obesity and its associated diseases.(EGCG),the main component of green tea(tea tree),has a wide range of biological activities,including anti-inflammatory,anti-oxidation,anti-infection and anti-cancer.However,little is known about its anti-adipogenic activity and potential mechanisms.The aim of this study was to investigate the effect of EGCG on the differentiation of 3T3-L1 adipocytes and to explore its possible mechanism.Method:(1)EGCG effects on cell viability: the preadipocytes into five groups,each with different concentrations of EGCG(0,10,50,100μM),through the MTT assay to assess EGCG differentiation of 3T3-L1 cells survive(2)Determination of the optimal concentration of EGCG: The preadipocytes were divided into five groups,EGCG(0,10,50,100 and 200μM)were added to different concentrations of EGCG respectively,and the differentiation of 3T3-L1 adipocytes was observed by oil red O staining Impact.(3)Determination of the optimal induction time of EGCG: the 3T3-L1 cells induced by EGCG optimum concentration differentiation 2,4,6,8 days,oil red O staining of lipid droplets form,immunoblot analysis was used to detect peroxidase(PPARγ)and FAS expression were detected by immunohistochemistry.(4)The mechanism of EGCG inhibition of adipogenesis by PI3K-AKT signal: 7 groups were as follows: normal + DMSO group;Vehicle group;(3)EGCG group;(4)normal + activator group(10μM SC-3036 or 0.5μM M SC-79);the other group of 3T3-L1 cells was induced for 8 days at the optimal EGCG concentration(100ug)EGCG + activator(10 μM M SC-3036 or 0.5 μM M SC-79).Subsequently,the cell viability was examined by MTT assay,and the cell morphology was observed under an optical microscope by Oil Red O staining.Finally,quantitative real-time PCR and immunoblot analysis were performed to detect mRNA and protein levels,including peroxisome proliferator-activated receptor γ(PPARγ),fatty acid synthase(FAS),phosphatidylinositol 3-kinase(PI3K)AKT and p-AKT.Results:(1)MTT assay showed that cell proliferation and viability did not change significantly at 10,50,and 100μM,while cell viability was significantly decreased at 200 μM,suggesting that 100 μM might be the optimal concentration of EGCG.(2)Oil red O staining results showed that lipid droplets of 3T3-L1 adipocytes decreased with the increase of EGCG concentration,and PPARγ and FAS were significantly decreased at mRNA and protein levels in a concentration-dependent manner,Indicating that the inhibition of 3T3-L1 preadipocyte differentiation was related to the concentration of EGCG,and 100μM was the best inhibitory concentration.(3)With the extension of EGCG treatment,lipid droplets began to decrease on day 2 for 8 days.In addition,the descending value reached the maximum on the 8th day.Similar results were observed from the expression levels of PPAR and FAS.It was shown that 8 days was the best induction time of EGCG.(4)The activation of SC-3036 or SC-79 was reversed,indicating that the inhibition of EGCG was mediated by PI3K-AKT signaling down-regulating the expression of FAS and PPARγ.Subsequently,we attempted to observe the effect of EGCG on the expression of PI3K-AKT during adipocyte differentiation.The expression level of PI3 K decreased in a dose-dependent manner and the expression level of AKT appeared to be constant,but those AKTs were also decreased in a concentration-dependent manner,EGCG can inhibit the activation of AKT in adipocytes rather than the expression of AKT.The results revealed that EGCG anti-adipogenesis and its basic mechanism,in the green tea chemicals for obese patients to provide a new prevention and treatment programs.