A Preliminary Study Of The Roles And Mechanisms Of PRDX1 And TRAF1 In Hepatic Insulin Resistance

ObjectiveTo research whether peroxiredoxin 1(PRDX1)and TNF receptor-associated factor 1(TRAF1)are involved in the occurrence of hepatic insulin resistance,and further to explore and clarify their roles as well as molecular mechanisms underlying hepatic insulin resistance,in order to provide a theoretical evidence for new drug design and therapeutic targets in T2 DM.Methods(1)The setup of insulin resistance model: cell model was established using HepG2 cells incubated by palmitate and animal model using C57 BL / 6J mice induced by feeding high-fat forage.The insulin resistance models were recognized with Western blot by detecting the variations of p-AKT and P-GSK3? expression.The variation of PRDX1 and TRAF1 expression were respectively detected by Western Blot and qRT-PCR in both the models.(2)HepG2 Cells were respectively transfected with PRDX1 siRNA or a non-specific siRNA,Western Blot was used to detect the expression variation of PRDX1,p-AKT,p-GSK3?,and qRT-PCR used to detect the gene expression variation of PEPCK and G6 Pase in transfected cells.The glucose uptake of HepG2 cells was detected by glucose kit.(3)HepG2 Cells were respectively transfected with Flag-PRDX1 or Flag plasmid,Western Blot was used to detect the variation of PRDX1,p-AKT,p-GSK3? expression,and qRT-PCR used to detect the gene expression variation of PEPCK and G6 Pase in transfected cells.Oil red O staining indicated intracellular lipid droplets aggregation,and glucose kit was for glucose uptake in HepG2 cells.(4)HepG2 Cells were treated with p38 inhibitor and then transfected with PRDX1 siRNA plasmid,and the protein expression changes of PRDX1,p-AKT,p-GSK3?,p-P38 was detected by Western Blot.(5)HepG2 Cells were respectively transfected with TRAF1 siRNA or a non-specific siRNA,the protein express changes of TRAF1,p-AKT were detected by Western Blot,qRT-PCR was used to detect the gene expression changes of PEPCK and G6 Pase.Oil Red O staining indicated intracellular lipid droplets aggregation,and glucose kit was for glucose uptake in HepG2 cells.(6)In insulin resistant HepG2 cells,the expression levels of P-P65 and I?B? were detected by Western Blot,and the levels of TNF-? and IL-6 were detected by ELISA.After HepG2 Cells were transfected with TRAF1 siRNA and a non-specific siRNA,protein changes of P-P65 and I?B? were detected by Western Blot,and the changes of TNF-? and IL-6 detected by ELISA.Results(1)Expression of PRDX1 and TRAF1 were increased both in HepG2 cells and mice insulin resistance model.(2)In PA-induced insulin resistance HepG2 cells:1)With the knocking down PRDX1 expression,the p-AKT and p-GSK3? protein expression increased,the PEPCK and G6 Pase gene expression decreased,and glucose uptake rate increased.2)With the PRDX1 over-expressing,the p-AKT and p-GSK3? expression obviously decreased,the PEPCK and G6 Pase expression obviously increased,the accumulation of lipid droplets increased,and glucose uptake rate decreased.3)Knocking down PRDX1 expression caused decrease of the p-p38 expression,and no changes of p-JNK expression,but PRDX1 over-expressing caused increase of the p-p38 expression;the inhibition of p38 MAPK could reverse the PRDX1-induced decrease of p-AKT and p-GSK3? expression to some extent.4)With knocking down TRAF1 expression,the p-AKT protein expression increased,the PEPCK,G6 Pase expression dereased,the accumulation of lipid droplets decreased,and glucose uptake rate increased.5)In PA-induced insulin resistance HepG2 cells,the level of P-P65 was up-regulated,I?B? down-regulated,and the levels of TNF-? and IL-6 were up-regulated.With knocking down TRAF1 expression,the level of P-P65 expression decreased,the I?B? increasecd,and the TNF-? and IL-6 decreased.Conclusions(1)The PRDX1 and TRAF1 expression are up-regulated both in HepG2 cells and mice insulin resistance model.(2)The PRDX1 and TRAF1 may depress insulin signaling pathway to some degree,and may be involved in insulin resistance.(3)About the roles of PRDX1 and TRAF1 as well as their molecular mechanisms on hepatic insulin resistance,the PRDX1 may go through p38 MAPK signaling pathway,but the TRAF1 through NF-?B signaling pathway to regulate insulin signaling and involve hepatic insulin resistance.