Inhibition Of Poldip2 Expression Leads To Metabolic Abnormalities Of Glucose In The Hepatic Of Type 2 Diabetic Mice

Objective Polymerase delta-interacting protein 2?poldip2?is an enzyme encoded by the poldip2 gene in humans.It increases NADPH oxidase 4?NOX4?activity..We believe that an increase in poldip2 in kkay mice may promote Hydrogen Peroxide production and alter insulin signaling pathways in the liver,thereby improving the level of Glucose metabolism.The aim of this study was to investigate the potential benefits of poldip2 in improving blood glucose levels and in inhibiting gluconeogenesis in the liver of KKAy mice.Methods KKAy mice were randomly divided into phosphate buffered saline?PBS?group,green fluorescent protein?GFP?group and poldelip2 group.C57BL/6J mice served as a normal control group.Western blot was used to investigate the protein expression of Poldip2 in KKAy mice and the differences of protein expression levels of downstream proteins such as protein kinase A?Akt?,Forkhead transcription factor1?FoxO1?and phosphoenolpyruvate carboxykinase?PEPCK?and glucose-6-phosphatase?G6Pase?,The levels of mRNA expression of PEPCK and G6Pase,the key enzymes for gluconeogenesis,were further verified by RT-PCR.Biochemical kits were also used to verify the differences of the general biochemical markers in diabetic mice and in the mice in normal control group.Results?1?The body weight of KKAy mice increased significantly compared with C57BL/6J mice in normal control group?P<0.05?.Compared with C57BL/6J mice in normal control group,The fasting blood glucose levels in KKAy mice were increased,the difference was statistically significant?P<0.05?.After one week intervention,there was no significant change in body weight in the four groups.However,the level of fasting plasma glucose in the poldip2 group was significantly improved after intervention?P<0.05?,and there was a significant difference in the level of H₂O₂ between the four groups?F=95.17,P<0.05?.Compared with the normal control group,the levels of H₂O₂ in PBS group and GFP group were significantly lowered?P<0.05?,while H₂O₂ in Poidip2 group were not significantly changed.Insulin levels in PBS and GFP groups were significantly increased?P<0.001?compared with those in C57BL/6J mice in the normal control group.Insulin levels in the Poldip2 group were significantly decreased?P<0.001?compared to GFP and PBS mice.?2?The protein expressions of Poldip2 in PBS group and GFP group were significantly inhibited?P<0.05?,respectively,compared with C57BL/6J in normal control group.Poldip2 expressions in Poldip2 group were significantly higher than those in PBS group and GFP group?P<0.05?.Compared with normal control group,Catalase protein expression in mice in PBS group and GFP group was significantly decreased?P<0.05?,but its activity in poldip2 group was not significantly changed compared with that in normal control group.Compared with C57BL/6J mice in normal control group,the ratio of phosphorylated PTEN?p-PTEN?to PTEN in PBS group and GFP group was significantly lowered?P<0.05?.Compared with PBS group and GFP group,the ratio of p-PTEN to PTEN in Poldip2 group was increased to normal level.?3?Compared with the normal control group,the Rictor activities in PBS group and GFP group were decreased significantly?P<0.05?,but there was no significant change in Poldip2 group.There was no significant change in total Akt protein expression in the other three groups compared with C57BL/6J mice in normal control group,but serine473 phosphorylation of Akt?p-Akt473?and The phosphorylation of threonine308?p-Akt308?was significantly decreased?P<0.05?,and the levels of p-Akt473 and p-Akt308 in Poldip2 were increased to normal levels.The ratio of phosphorylated Forkhead transcription factor1?p-FoxO1?to Forkhead transcription factor1?FoxO1?in PBS and GFP groups was significantly lower than that in control C57BL/6J mice?P<0.05?,but the ratio was not significantly changed in Poldip2 group.?4?Compared with C57BL/6J mice in normal control group,the expression levels of PEPCK and G6Pase in PBS group and GFP group were significantly increased?P<0.05?.Compared with PBS group and GFP group,the levels of PEPCK and G6Pase in Poldip2group were decreased significantly?P<0.05?.?5?Compared with C57BL/6J group,the mRNA expression of PEPCK and G6Pase in PBS group and GFP group was significantly increased?P<0.05?,but the mRNA expression of PEPCK and G6Pase was not significantly changed in Poldip2 group.Compared with PBS group and GFP group,PEPCK and G6Pase mRNA expression levels in Poldip2 group were significantly decreased?P<0.05?.The results of RT-PCR were consistent with Western blot.Conclusion The results of western blot and RT-PCR showed that Poldip2 in KKAy mice liver was significantly reduced compared with the normal control group.We using the adenovirus tail vein injection of Poldip2 into KKAy mice,Western blot and RT-PCR methods showed that Poldip2 could improve glucose metabolism in type2diabetic mice by increasing Poldip2 expression and thereby activating NOX4 to produce hydrogen peroxide?H₂O₂?,which could inhibit PTEN activity,as a result,improving insulin signaling pathway and glucose metabolism.