Effect Of Visfatin On FoxO1 In Insulin Resistant L6 Cells

Objective: To constructing L6 cell insulin resistance model,up-regulating visfatin expression and detecting the protein expression of PI3K/Akt and its downstream signaling factor Fox O1,Further investigate the effect of visfatin on skeletal muscle insulin resistance.Methods: The L6 cell insulin resistance model was induced by establishing DMEM medium and high concentration of phosphatidic acid(PA):The cells were divided into control group,PA group,insulin group,and PA + insulin group.250 ?M PA was added to the PA group;100 n M insulin was added to the insulin group;250 ?M PA + 100 n M insulin was added to the PA + insulin group;the culture supernatants of each group were collected after 0,6,12,and 24 hours;The differentiation of L6 cells was confirmed by immunofluorescence assay;Determination of glucose content in cell culture supernatant by glucose peroxidase method to verify the establishment of insulin resistance model;The visfatin overexpressing adenoviral vector was administered to the skeletal muscle cell model of insulin resistance.The expression level of visfatin protein in L6 cells was detected by Western blot to verify the successful construction of the virus.Transfected visfatin Sh RNA and PC RNA into the insulin-resistant skeletal muscle cell model.Divide cells into PC group,PC+PA group,visfatin group,visfatin+PA group.Four groups were added to 100 n M Insulin,PC+PA group and visfatin+PA group and then added to 250 ?M PA.After 24 hours,collected the culture supernatant.Determined the glucose content in supernatant and the content of visfatin by glucose peroxidase method;The protein expression of PI3 K,Akt kinase phosphorylation and the protein expression of Fox O1,a key molecule regulating insulin sensitivity downstream,were detected by western blot.Results: 1.Validation of insulin resistance model: there was no significant difference in the glucose content in the supernatant between the PA group and the control group at 0,6,12,and 24 hours.(P>0.05);There was no significant difference in the glucose content in the supernatant of the the insulin+PA group and the insulin group for 0 hours(P>0.05).The insulin group and the insulin+PA group treated for 6 hours,the glucose content in the supernatant of the insulin+PA group was significantly higher than that of the insulin group(P<0.05).The insulin group and the insulin+PA group treated for 12 hours,the glucose content in the supernatant of the insulin+PA group was significantly higher than that of the insulin group(P<0.05).The insulin group and the insulin+PA group treated for 24 hours,the glucose content in the supernatant of the insulin+PA group was significantly higher than that of the insulin group(P<0.01).2.Verification of visfatin overexpressing adenovirus: The normal group compared with the NC group,the expression level of visfatin protein in the overexpressed visfatin group was significantly higher than that in the normal control group(P<0.001).3.Overexpression of visfatin on glucose: the PC+PA group compared with PC group,the glucose content in the PC+PA group was significantly increased(P<0.001);The visfatin group compared with PC group,the concentration of glucose in the supernatant was significantly lower in the visfatin group than in the PC group(P<0.001);The visfatin+PA group compared with the visfatin group,the glucose content in the supernatant of the visfatin+PA group was significantly increased(P<0.001).4.Overexpression of visfatin on the PI3K/Akt pathway: compared the expression of PI3 K in the visfatin group and the PC group,visfatin+PA group and PC+PA group,the difference was not statistically significant(P>0.05);Compared the expression of p-Akt in the visfatin group and PC group,the p-Akt expression levels in the visfatin group was higher than that in the PC group(P<0.01);Compared the expression of p-Akt in the visfatin+PA group and the PC+PA group,the p-Akt expression levels in the visfatin+PA group was higher than that in the PC+PA group(P<0.001);Compared the Fox O1 expression levels in visfatin group and PC group,the Fox O1 content in the visfatin group was significantly lower than that in the PC group(P<0.001);Compared the Fox O1 expression levels in the visfatin+PA group and the PC+PA group,the Fox O1 expression levels in the visfatin+PA group was lower than that of PC+PA group(P<0.01).Conclusion: In PA-induced L6 cells,overexpression of visfatin regulates the activity of the PI3K/Akt pathway and down-regulates the protein expression of Fox O1.It is significantly promotes glucose uptake by L6 cells and improves insulin resistance.