Mechanism Of Regulation On Differentiation In 3T3-L1 Preadipocytes By Chlorogenic Acid

Obesity is a serious and growing health hazard.With the research on biological effects of polyphenols and its metabolites in advancing,more and more evidence has shown that dietary polyphenols could improve metabolic homeostasis and reduce body weight.Chlorogenic acid,a phenolic compound,is one of the most abundant polyphenols in the human diet.It has been proven to have a wide range of biological activities.Our previous studies showed that chlorogenic acid,as one of the main phenolic substances,may be an important active component of lotus seed polyphenols to play a role in weight loss and lipid reduction.Therefore,in this study,we further analyzed the influence of chlorogenic acid on adipocyte differentiation,and studied the key genes and lipids in the regulation of chlorogenic acid on 3T3-L1 preadipocyte differentiation through transcriptomes and lipids,and further using Phospho-Kinase Array to identify the relevant signaling pathways.Specific research contents and results are as follows:(1)To investigate the effect of chlorogenic acid on the differentiation of 3T3-L1 cells under adipocyte differentiation induction.The results showed that after chlorogenic acid intervention,the cell lipid droplets decreased and the intracellular triglyceride(TG)content decreased significantly,which indicated that chlorogenic acid could effectively inhibit the adipocyte differentiation.Meanwhile,the basic oxygen consumption and maximum respiratory capacity of cells were increased with chlorogenic acid intervention,which indicated that chlorogenic acid increased the respiratory rate of 3T3-L1 cells.(2)To investigate the transcriptome changes induced by chlorogenic acid intervention during induction differentiation of 3T3-L1 cells.483 genes(20 μM chlorogenic acid treatment group)and 783 differentially expressed genes(40 μM chlorogenic acid treatment group)in3T3-L1 cells after chlorogenic acid treatment were differentially expressed.KEGG Pathway enrichment analysis showed that differentially expressed genes were closely related to PPAR signaling pathway and AMPK signaling pathway.It is suggested that chlorogenic acid may regulate the differentiation process of 3T3-L1 cells and may be associated with reducing the expression of PPARγ and its related genes(SLC2A4,CD36,FASN,Scd1,Scd2,Scd3,Lpin1 and RXRγ);decreasing the expression of adipogenic genes(FABP4 and Adipoq);inhibiting the expression of TG uptake,synthesis and storage related genes(LPL,Acsl1 and PCK1).Meanwhile,the results showed that the adipogenesis of 3T3-L1 cells showed a dose-dependent manner with chlorogenic acid.(3)To study the changes of cell lipids induced by chlorogenic acid during the induction of 3T3-L1 adipocyte differentiation.The results showed that the lipids of 3T3-L1 adipocytes changed significantly after chlorogenic acid intervention,especially the changes of glycerol(TG,DG),glycerol phospholipids(PC,PE,PS)and sphingolipids(Cer,SM).These results suggested that the regulation of chlorogenic acid on the differentiation process of 3T3-L1 cells might be related to the decrease of glycerol content and the increase of phospholipid content.At the same time,the increase of phospholipids content suggested that chlorogenic acid might enhance the respiration rate of 3T3-L1 cells by promoting mitochondrial function.The change of sphingolipids may be closely related to the increase of fatty acid content in adipocytes by chlorogenic acid.(4)The mechanism of chlorogenic acid regulation on 3T3-L1 adipocyte differentiation was analyzed by integrated transcriptomics and lipidomics.The correlation analysis of the two omics suggested that the synthesis of TG and DG may be related to a series of genes,including PPARγ-associated genes(e.g.Lpin1,Arid5 a,Scd1,Apmap,Insig1,CD36,Rgcc,Timp4,Cdkn2c)and other adipogenic genes(e.g.Lipe,Prlr,Ghrh,Kbtbd11,Acat2,Mrap,Acsl1,Aoc3,Lgals12,G0s2,Klf15,Cfd,Adipoq,Gpat3,Angptl4,Brca1,Apln,Nr1h3).The expression changes of other lipid regulatory genes(such as FAM107 A,Ubd,Hsd11b1)may be lead to the increased lipid content of PC,PE,and PS.Furthermore,the expression changes of differentially expressed genes(Acat2,Acsl1,Insig1,Apmap)were verified by q RT-PCR.These genes showed an obvious downward trend after the intervention of chlorogenic acid,which was roughly the same as the results of the transcriptome,indicating that the sequencing results of the transcriptome were reliable.(5)The effect of chlorogenic acid on the key signaling pathway of3T3-L1 adipocyte differentiation was further studied by phosphokinase chip.The results showed that the phosphorylation of CREB,p38α and STAT5A/B in 3T3-L1 cells decreased after chlorogenic acid intervention.Combined with transcriptomic and lipid analysis,chlorogenic acid regulated the synthesis of TG and DG and improved the lipid metabolism,which may be related to the decreased expression of PPARγ through CREB,p38α and STAT5A/B pathways in early and middle differentiation.In late differentiation,chlorogenic acid may regulate the expression of latedifferentiation genes FABP4 and FASN by decreasing CREB phosphorylation,thus inhibiting lipogenesis.At the same time,chlorogenic acid may enhance cell respiration rate by regulating p38α pathway,affecting the expression of mitochondria-related genes such as FAM107 A,Ubd and Hsd11b1,and thereby increasing the lipid content of PC,PE and PS.