Proteomic Analysis Of The Insulin-Resistant HepG2 Cell Model Treated By Drugs

ObjectiveTo screen the express-altered proteins before or after effecting of sensitizer of ecdysterone or rosiglitazone on HepG2 cell model of insulin resistance by the strategy of comparative proteomics,which may approach new proves for exploring the target of sensitizer.MethodsHepG2 cells were incubated with 5×10-7mol/L insulin for 16 hours to set up insulin resistant cell model, whose formation was assessed by detecting the ability of uptaking glucose with GOD-POD assay;Then the cell model was treated by ecdysterone or rosiglitazone for 24 h, and proteins of all groups before and after treatment were extracted by lysis buffers. The express-altered proteins were screened by 2-DE technique,some of which were analyzed by MALDI-TOF-MS mass spectrometry and MS-Fit database.Results1. The insulin resistant HepG2 cell model was established successfully by high concentration of insulin in vitro culture: the glucose content in group of insulin resistant HepG2, in which the glucose content was reduced obviously after treated by rosiglitazone,was obviously higher than control group.2. 36 express-altered protein spots of insulin resistant HepG2 cells before and after treated by rosiglitazone were screened by 2-DE technique, in which 22 ones up-regulated and the others down-regulated . While there were 53 express-altered protein spots before and after treated by ecdysterone, in which 35 ones up-regulated and the others down-regulated.3. 6 express-altered protein of insulin resistant HepG2 cells before and after treated by rosiglitazone (In which 3 proteins up-regulated) were analyzed with MALDI-TOF-MS mass spectrometry and identified with MS-Fit database. They were Lipin-1,ANG-4,Chondroitin sulfate glucuronyltransferase,CPT-I,C3/C5 convertase and HSP-70. And 6 express-altered protein of insulin resistant HepG2 cells before and after treated by ecdysterone (In which 3 proteins were up-regulated) were Tyrosine-protein kinase BTK,Phosphatidylinositol-binding clathrin assembly protein,LIM domain-binding protein 2,Phosphoenolpyruvate carboxykinase,Proteasome subunit alpha type 5 and Zinc finger protein 483.Conclusion1. The insulin resistant HepG2 cell models were established successfully by high concentration of insulin in vitro culture.2. The ideal 2-DE maps of proteome of insulin resistant HepG2 cells, which were treated and untreated by drugs, were obtained clearly.3. There were large differences of 6 verified express-altered proteins between rosiglitazone and ecdysterone before and after intervention, which may be explained by a new mechanism of ecdysterone's improving insulin resistance, compared to rosiglitazone.