Effect Of Adipose Tissue At Different Sites On The Hepatic Insulin Signaling Pathway

Objective:To explore the effects and differences of different regional adipose tissues on critical proteins of hepatic insulin IRS/PI3K/AKT signaling pathway.Methods:Human cultured HepG2 cells were divided into HepG2 alone,HepG2+VAT co-culture,HepG2+SAT co-culture,and HepG2+ TritonX-100 treatment group,with 2 replicates in each group.In the co-culture group,Transwell chamber was used to co-culture with the collected visceral adipose tissue(VAT)or subcutaneous adipose tissue(SAT)with human HepG2 cells in vivo.After treatment,the LDH levels of supernatant of each group to determine the bioactivity of HepG2 cells.Two replicates of HepG2 alone culture group,HepG2+VAT co-culture group,and HepG2+SAT co-culture group were treated in insulin-free or insulin-containing culture medium for 10 min,namely:Control group,Control+insulin group,VAT group,VAT+insulin group,SAT group,and SAT+insulin group.The expression levels of IRS2,p-IRS2(S731),PI3K-p85,Akt2 and p-Akt2(S473)in HepG2 cells were measured by Western blot using the total lysates.Results:(1)IRS2:①There was a significant difference in IRS2 protein expression of HepG2 cells among the Control group,VAT group and SAT group,namely Control group>SAT group>VAT group(P<0.001).②There was a significant difference in IRS2 protein expression of HepG2 cells among the Control+insulin group,VAT+insulin group,and SAT+insulin group,namely Control+insulin group>SAT+insulin group>VAT+insulin group(P<0.05).③Compared with Control group,the expression of IRS2 protein in HepG2 cells of Control+insulin group significantly increased(P<0.05).④Compared with VAT group,the expression of IRS2 protein in HepG2 cells of VAT+insulin group significantly increased(P<0.05).⑤Compared with SAT group,the expression of IRS2 protein in HepG2 cells of SAT+insulin group significantly increased(P<0.05).(2)p-IRS2(S731):①There was a significant difference in p-IRS2(S731)protein expression of HepG2 cells among the Control group,VAT group and SAT group,namely Control group<SAT group<VAT group(P<0.05).②Compared with Control+insulin group,the expression of p-IRS2(S731)protein in HepG2 cells of VAT+insulin and SAT+insulin group significantly increased(P<0.05).But the difference between VAT+insulin and SAT+insulin for p-IRS2(S731)was not significant(P>0.05).③Compared with Control group,the expression of p-IRS2(S731)protein in HepG2 cells of Control+insulin group significantly decreased(P<0.05).④Compared with VAT group,the expression of p-IRS2(S731)protein in HepG2 cells of VAT+insulin group significantly decreased(P<0.05).⑤Compared with SAT group,the expression of p-IRS2(S731)protein in HepG2 cells of SAT+insulin group significantly decreased(P<0.05).(3)PI3K-p85:①There was a significant difference in PI3K-p85 protein expression of HepG2 cells among the Control group,VAT group and SAT group,namely Control group>SAT group>VAT group(P<0.001).②There was a significant difference in PI3K-p85 protein expression of HepG2 cells among the Control+insulin group,VAT+insulin group,and SAT+insulin group,namely Control+insulin group>SAT+insulin group>VAT+insulin group(P<0.001).③Compared with Control group,the expression of PI3K-p85 protein in HepG2 cells of Control+insulin group significantly increased(P<0.001).④Compared with VAT group,the expression of PI3K-p85 protein in HepG2 cells of VAT+insulin group significantly increased(P<0.05).⑤Compared with SAT group,the expression of PI3K-p85 protein in HepG2 cells of SAT+insulin group significantly increased(P<0.05).(4)Akt2:①There was no significant difference in Akt2 protein expression of HepG2 cells among the Control group,VAT group and SAT group(P>0.05).②There was no significant difference in Akt2 protein expression of HepG2 cells among the Control+insulin group,VAT+insluin group,and SAT+inslin group(P>0.05).③There was no significant difference in Akt2 protein expression of HepG2 cells between the Control group and Control+insulin group(P>0.05).④There was no significant difference in Akt2 protein expression of HepG2 cells between the VAT group and VAT+insulin group(P>0.05).⑤There was no significant difference in Akt2 protein expression of HepG2 cells between the SAT group and SAT+insulin group(P>0.05).(5)p-Akt2(S473):①There was a significant difference in p-Akt2(S473)protein expression of HepG2 cells among the Control group,VAT group and SAT group,namely Control group>SAT group>VAT group(P<0.001).②There was a significant difference in p-Akt2(S473)protein expression of HepG2 cells among the Control+insulin group,VAT+insulin group,and SAT+insulin group,namely Control+insulin group>SAT+insulin group>VAT+insulin group(P<0.05).③Compared with Control group,the expression of p-Akt2(S473)protein in HepG2 cells of Control+insulin group significantly increased(P<0.05).④Compared with VAT group,the expression of p-Akt2(S473)protein in HepG2 cells of VAT+insulin group significantly increased(P<0.05).⑤Compared with SAT group,the expression of p-Akt2(S473)protein in HepG2 cells of SAT+insulin group significantly increased(P<0.05).(6)Compared with the Control group,the changes of IRS2,p-IRS2(S731),PI3K-p85,Akt2 and p-Akt2(S473)protein expression in HepG2 cells of the VAT group showed statistical significance.That is,the changes of p-IRS2(S731)protein expression was greater than IRS2,PI3K-p85,p-Akt2(S473)protein,and the changes of Akt2 protein expression was less than IRS2,PI3K-p85,p-Akt2(S473)protein(P<0.05).(7)Compared with the Control group,the changes of IRS2,p-IRS2(S731),PI3K-p85,Akt2 and p-Akt2(S473)protein expression in HepG2 cells of SAT group showed statistical significance.That is,the changes of p-IRS2(S731)protein expression was greater than IRS2,PI3K-p85,p-Akt2(S473)protein,and the changes of Akt2 protein expression was less than IRS2,PI3K-p85,p-Akt2(S473)protein(P<0.05).(8)Compared with Control+insulin group,the changes of IRS2,p-IRS2(S731),PI3K-p85,Akt2 and p-Akt2(S473)protein expression in HepG2 cells of VAT+insulin group showed statistical significance.That is,the changes of p-IRS2(S731)protein expression was greater than IRS2,PI3K-p85,p-Akt2(S473)protein,and the changes of Akt2 protein expression was less than IRS2,PI3K-p85,p-Akt2(S473)protein(P<0.05).(9)Compared with Control+insulin group,the changes of IRS2,p-IRS2(S731),PI3K-p85,Akt2 and p-Akt2(S473)protein expression in HepG2 cells of SAT+insulin group showed statistical significance.That is,the changes of p-IRS2(S731)protein expression was greater than IRS2,PI3K-p85,p-Akt2(S473)protein,and the changes of Akt2 protein expression was less than IRS2,PI3K-p85,p-Akt2(S473)protein(P<0.05).Conclusions:(1)After co-culture with VAT or SAT,the expression of IRS2,PI3K-p85 and p-Akt2(S473)protein in HepG2 cells decreased significantly,while the expression of p-IRS2(S731)protein increased significantly,suggesting that both VAT and SAT could affect the insulin signaling transduction of hepatic cells.(2)Compared with HepG2 cells co-cultured with SAT,the changes of IRS2,PI3K-p85,p-IRS2(S731)and p-Akt2(S473)protein in HepG2 cells co-cultured with VAT were greater,suggesting that different regional of adipose tissue have different effects on insulin sensitivity of hepatocyte.Namely,the effect of VAT on hepatic insulin resistance was more obvious than that of SAT.(3)After co-culture with VAT or SAT,the expression of p-IRS2(S731)protein in HepG2 cells showed the greatest change,suggesting that p-IRS2(S731)protein is the main participant in VAT and SAT induced hepatic insulin resistance.(4)After co-culture with VAT or SAT,there was no significant change in Akt2 protein expression in HepG2 cells,which did not suggest that Akt2 protein involves in VAT and SAT mediated hepatic insulin resistance.