A Study On Regulation Of CAA To The Differentiation And Lipid Metabolism In 3T3-L1 Adipocytes

ObjectiveOur research team had found that cajanonic acid A(CAA), a new stibene, has an effect on glucose/lipid-decreasing. In ghe present study, we elucidated the further mechanism underling the effect of CAA on adipocytes differentiation and lipid metabolism in the mouse 3T3-L1 adipocytes.Methods(1)Effect of CAA on the proliferation of 3T3-L1 preadipocytes. The 3T3-L1 preadipocytes were treated with different concentrations of CAA for 48h and the cell viability was determined by CCK8 assay. (2)Effect of CAA on 3T3-L1 preadipocytes differentiation.3T3-L1 preadipocytes were induced for differentiation in the presence or absence of various concentrations of CAA. The cell differentiation degree and lipid content were monitored by Oil Red 0 Staining method. (3)Effect of CAA on lipid metabolism. Mature adipocytes were treated with CAA in different dosages for 48h, then total lipids, triacylglycerol, free fatty acid and glycerol were measured. (4)Regulatory effect of CAA on adipocyte differentiation-controlling genes. During the adipogenesis, from zeroth to eigth days, total RNA were isolated and the mRNA levels of differentiation transcription factors and their target genes were quantitatively analyzed by real-time fluorescent quantitative polymearase-chain reaction (RTFQ-PCR). (5) Effect of CAA on JAK2/STAT3 signaling pathway. After mature 3T3-L1 adipocytes were incubated with CAA for 48h, total RNA and proteins were extracted. The expression and phosphorylation levels of JAK2, STAT3 and associated proteins were individually detected by RTFQ-PCR and Western-blot. (6)Effect of CAA on AMPK signaling pathway. Total RNA and proteins were extracted from mature 3T3-L1 adipocytes treated with CAA for 48h. The expression and phosphorylation levels of AMPK and its primary downstream target genes were individually measured by RTFQ-PCR and Western-blot method.Results(1)In proliferation assay. CAA of 25-200 μmol/Lexhibited no remarkable influence on the multiplication of 3T3-L1 preadipocytes(P>0.05). (2)In adipogenic differentiation assay, CAA at different concentrations obviously inhibited the differentiation of cells(P<0.05, P<0.01). Compared with the control group, the differentiation rates of cells treated with CAA (50,100 ,150μmol/L) dropped to 74.48%,46.47%,19.71% respectively. (3) CAA significantly reduced the synthesis of total lipids and trighyceride and the release of glycerol and free fatty acid in mature adipocytes(P<0.05, P<0.01). Compared with the control group, the depositon of total lipids and trighyceride sharply declined by 32%,24%, and the release of glycerol and FFA reduced by 32% and 30%, in 100 μmol/L CAA group. (4)CAA remarkablely down-regulated mRNA levels of the genes encoding differenation transcription factors(C/EBPa, ADD1 and PPARy). And the transcriptional profiles of these factor’s target genes comprising LPL, AP2, FAS, ACC, Adiponectin and Resistin were also quantitiatively assayed by RTFQ-PCR. As results, the expression levels of all tested genes were extremely lower by CAA, in a dose-dependent manner(P<0.05, P<0.01). (5)After CAA treatment, the expression patterns of JAK2 and STAT3 illustrated no difference at mRNA and protein level.But there were significant decrease in JAK2 & STAT3 phosph-orylation. Accompanied by decreased activity of JAK2 & STAT3, the expression of PPARy, both at mRNA and protein level, were down-regulated. (6)CAA actived the AMPK by increasing the phosphorylation of AMPK and inactivated ACC via down-regulating the mRNA level and up-regulating the phosphorylation of ACC. And as the results of AMPK signal pathway activating, there were abvious inhibitory effects in the expressions of the key enzymes involving lipogenesis (FAS), fatty acid uptake(LPL) and lipolysis(LPL) (P<0.05, P<0.01). However, the expressions of fatty oxidization relating genes(CPT-1 and ACOX) were promoted by AMPK activation(P<0.05, P<0.01). (7)Furher research found that, CAA(100(μimol/L) obviously stimulated the secretion of leptin(P<0.01).Conelusion(1)CAA observably inhibited the adipogenesis of 3T3-L1 pradipocytes ,suppressed the lipids synthesis in 3T3-L1 adipocytes, and reduced the release of free fatty acid. (2)CAA suppressed the adipogenic differentiation of 3T3-L1 preadipocytes by down-regulating the expression differentiation transcription genes and their garget genes. (3)CAA may inhibit the expres-sion of PPARy by deactivating the JAK2/STAT3 signaling pathway. (4)CAA may activate the AMPK signaling pathway to regulate the ipid metabolism by stimulating the secretion of leptin.