Effects Of The Expression Of Toll-like Receptor 4 On The Signal Pathway Of AKT In 3T3-L1 Cells

Objective:toll-like receptor 4 plays an important role in the innate immune system. Recently growing evidences suggest that inflammation is involved in the mechanism of diabetes and one of the AKT signal pathway is involved in the insulin signal pathway.The aim of this experiment is to determine whether TLR4 activating has effects on the AKT signal pathway in adipocytes.Then we explore the relation between toll-like receptor 4(TLR4) gene Asp299Gly,Thr399Ile polymorphisms and peroxisome proliferator-activated receptorγ(PPARγ) gene Pro12Ala polymorphism and type 2 diabetes mellitus(T2DM) in Tianjin.Methods:1.3T3-L1 preadipocytes are incubated in Dulbecco's modified Eagle's medium (DMEM).The inducing reagents are administered to induce the preadipocytes to differentiate to adipocytes when 3T3-L1 cells were grown to confluence.2.Adipocytes are characterized by staining the cells with oil red O.3.Adipocytes are treated with lipopolysaccharides for one hour,then we extract the total cellular RNA and detect the expression of toll-like receptor 4 by real-time polymerase chain reaction.4.Then we extract total cellular proteins and detect the expression of p-AKT with western blot method.5.The genome DNA of 365 type 2 diabetes mellitus and 95 normal controls are extracted respectively,then we use polymerase chain reaction(PCR) technology to amply the objective fragment.6.Denaturing high performance liquid chromatography(DHPLC) is used to detect toll-like receptor 4(TLR4) gene Asp299Gly,Thr399Ile polymorphisms and peroxisome proliferator-activated receptorγ(PPARγ) gene Pro12Ala polymorphism.Results:1.The differentiating percentage of 3T3-L1 preadipocytes is quite high after induced.When induced for 7-8 days,over 90 percent of preadipocytes are differentiated to adipocytes.2.The result of oil red O staining is positive.Most of the adipocytes are stained to red and lots of lipid droplets appear in the cytoplasm.3.When treated with lipopolysaccharides for 30 minutes,the mRNA expression of TLR4 increases significantly(p＜0.05).4.The expression of p-AKT decreases(p＜0.05) after lipopolysaccharides is administered to adipocytes.5.The genotypes of TLR4 gene Asp299Gly,Thr399Ile mutations are not found in both T2DM and control groups.6.The genotype frequencies(P/A) of PPARγPro12Ala polymorphism in T2DM and control groups were 0.074 and 0.032 espectively and there was no correlation between the Pro12Ala polymorphisms and T2DM.Conclusion:1.The differentiating percentage of 3T3-L1 preadipocytes is quite high after induced and can be over 90%.2.After treated with lipopolysaccharides,the mRNA expression of TLR4 increases significantly and the activity of p-AKT decreases.This indicates that the expression of TLR4 suppresses AKT signal pathway in adipocytes.3.The genotypes of TLR4 gene Asp299Gly,Thr399Ile mutations are not found and this polymorphisms are not associated with type 2 diabetes mellitus in Tianjin.4.PPARγgene Pro12Ala polymorphism is not associated with type 2 diabetes mellitus in Tianjin.5.DHPLC is an efficient method for the screening of gene mutation,and can be used to detect the predisposing genes of type 2 diabetes mellitus,then to predict the susceptibility of type 2 diabetes mellitus.