Effect Of Resveratrol On Adipokines Expression In 3t3-l1 Preadipocytes Adipogenic Differentiation And The Induction Of Tnf-¦Á

Objective:(1)To determine the effect of resveratrol,a Sirt1 activator,on adipogenesis of 3T3-L1 preadipocyte.(2)To investigate the effect of resveratrol on tumor necrosis factor-α(TNF-α)induced adipokines expression in adipocytes.Methods:(1)The 3T3-L1 post-confluent preadipocytes and lipid-filled adipocytes were incubated with resveratrol(0 to 100μM)for up to 48 hours.Viability was determined using the CCK8 cell proliferation assay. Post-confluent preadipocytes were incubated with resveratrol for up to 6 days during maturation,adipogenesis was quantified by measuring lipid content using triglyceride assay kit.The cells were also stained with Oil Red O for visual confirmation of effects on lipid accumulation.Mature 3T3-L1 adipocytes were incubated with resveratrol or TNF-αfor 2h, lipolysis was quantified by measuring glycerine content in supernatant. (2)Differentiated 3T3-L1 adipocytes were pretreated with or without various concentrations of resveratrol for 6 h,and then were stimulated for 12 or 24h by the addition of 10ng/ml TNF-α.The mRNA expression levels of monocyte chemoattractant protein-1(MCP-1),interleukin-6 (IL-6)and adiponectin were detected by quantitative RT-PCR.The protein content of MCP-1,IL-6 and adiponectin in conditioned medium were measured by ELISA.(3)HEK293 cells were transfected with firefly luciferase reporter construct,pSV-βgal plasmid and expression vector (pCMV-IκBαor pcDNA-Sirt1).Twenty four hours after transfection, cells were untreated or treated with 10ng/ml TNF-αfor 6 h.The luciferase andβ-galactosidase activities were measured.(4)3T3-L1 adipocytes were preincubated for 6 h with different concentrations of resveratrol(10-50μM),followed by lh incubation with 10ng/ml TNF-α. Nuclear extracts were prepared and then assayed for nuclear factor-κB (NF-κB)activation.(5)HEK293 cells were transfected with pcDNA-Sirt1 or pcDNA3.1 vector.Twenty four hours after transfection, cells were treated with or without TNF-α(10 ng/ml)for 1 h,and nuclear extracts were prepared and then assayed for NF-κB activation by electrophoretic gel mobility shift assay(EMSA).Results:(1)In post-confluent preadipocytes and mature adipocytes, resveratrol(≥75μM)caused a time-related and dose-dependent decrease in viability.Resveratrol also inhibited adipogenesis of 3T3-L1 preadipocytes.Resveratrol(50μM)mediated inhibition of adipocyte differentiation occurred during the early,intermediate stages of the differentiation process.Resveratrol(10～50μM)did not increase the release of glycerol into the culture medium compared with control 3T3-L1 cells.(2)Resveratrol could attenuate the TNF-αinduced IL-6 and MCP-1 expression and secretion in a dose-dependent manner,and partially recovered adiponectin expression and secretion which was suppressed by the addition of TNF-αin a dose-dependent fashion.(3) Overexpression of IκBαinhibited TNF-α-induced IL-6 and MCP-1 promoter activities.(4)Resveratrol elicited dose-dependent inhibitory effects on TNF-α-induced DNA binding activity of the NF-κB complex. (5)Overexpression of Sirt1 inhibited TNF-α-induced NF-κB-dependent transcription activities,though no effect on TNF-α-induced DNA binding activity of the NF-κB complex was found.Conclusions:(1)Resveratrol can decrease fat cell mass by direct inhibition on cell viability and adipogenesis in 3T3-L1 cells.(2) Resveratrol attenuates the TNF-αinduced adipokines expression and secretion in 3T3-L1 adipocyte by direct repression of NF-κB binding activity,and indirect inhibition of NF-κB dependent transcription activity through activation of Sirt1.