Expression Of Responsive Protein Of Endoplasmic Reticulum In Hippocampus Of GK Rats And Metformin Intervention

Objective: Sensor proteins in endoplasmic reticulum(ER)are type-1 ER tansmembrane protein kinase(IRE1),PER-like ER kinase(PERK),and activating transcription factor-6(ATF6).They are the sign of the unfolded protein response(UPS),which suggests that endoplasmic reticulum stress(ERS)occurs.Hippocampus is one of the tissues of the brain limbic system which it is central to learning and memory.The aim of this study was to investigate the expression of sensor proteins of ER in the hippocampus of type 2 diabetes mellitus(T2DM),and to explore the relationship between ERS and T2 DM.Meanwhile to observe effects of metformin on cognitive function and ERS,the cognitive function of the rat was assayed by using Morris water maze(MWM)test.Method: Goto-Kakizaki(GK)rats with random blood glucose ? 11.1 mmol/l were selected and randomly divided into the following groups: diabetic model group(DM group,n=10)and metformin group(MF group,n=10,ig,85mg/kg/d).The homogenous male Wistar rats were served as normal control group(NC group,n=10).NC and DM groups received the same dose of normal saline.After 8 weeks of continuous medication,oral glucose to tolerance test(OGTT)was done,then area under curve(AUC)of blood glucose at all OGTT time points were calculated.And C-Peptide(C-P)was detected,then homeostasis model assessment-insulin resistance(HOMA-IR)of rats was calculated.Through the orientation navigation experiment and space exploration experiment of MWM test behavior of rats.The expression of ATF6,IRE1 and PREK was analyzed by immunohistochemistry and western blot.SPSS21.0 for Windows software was used for the result analyses.The data were tested by one-way ANOVA.Multiple comparisons between groups were compared with LSD or Dunnett's T3 analysis of variance.Values of P<0.05 were considered significant.Result:1 Changes of weight and FBG in rats after metformin intervention for 8 weeks: The body weight of DM group and MF group were lower than NC group(all P<0.01),but there was no statistically significant on body weight between DM group and MF group(P>0.05).Compared with NC group,DM group and MF group were higher FBG(P<0.01);compared with DM group,MF group was lower FBG(P<0.01)(Shown in Table1 and Fig.1).2 Results of metformin on OGTT and AUC in rats after intervention for 8 weeks: Compared with NC group,the blood glucose values of DM group were higher at all OGTT time points and AUC was significantly increased(all P<0.01).Compared with DM group,the blood glucose values of MF group were lower at all OGTT time points and AUC was significantly decreased(P<0.05,P<0.01)(Shown in Table2 and Fig.2).3 Results of metformin on C-P and HOMA-IR in rats after intervention for 8 weeks: Compared with NC group,the level of C-P in DM group was decreased(P<0.05)and HOMA-IR was significantly increased(P<0.01).Compared with DM group,the level of C-P in DM group was increased(P<0.05)and HOMA-IR was significantly decreased(P<0.01)(Shown in Table3 and Fig.3).4 Effects of metformin on spatial learning ability and spatial memory ability in rats: Compared with NC group,the rats of DM group had obvious learning and memory disorder,and Morris water maze navigation test latency was prolonged,the spatial probe test in the platform quadrant dwell time significantly reduced(all P<0.01);compared with DM group,the rats of MF group showed improvement in learning and memory ability,and Morris water maze navigation test latency was shortened,the spatial probe test in the platform quadrant dwell time significantly prolonged(all P<0.01)(Shown in Table 4 and Fig.4).5 Immunohistochemical results: The immunohistochemical positive products were mainly located in the cytoplasm of the cells,which were brown yellow granules in different shades(Shown in Fig.5-7).Compared with NC group,DM group and MF group had higher relative amount of ATF6;compared with DM group,MF group had lower relative amount of ATF6(all P<0.01).Compared with NC group,DM group and MF group had higher relative amount of IRE1 and PREK(all P<0.01);the difference of the relative amount of IRE1 and PREK between DM group and MF group was not statistically significant(all P>0.05)(Shown in Fig.8).6 Western blot results: Integral of optical density(IOD)of the interest proteins was measured,and the ratio of the IOD of the interest proteins to ?-actin was calculated.The ratio was statistically analyzed as the relative expression level.Compared with NC group,DM group and MF group had higher relative expression levels of ATF6;compared with DM group,MF group had lower relative expression levels of ATF6(all P<0.01).Compared with NC group,DM group and MF group had higher relative expression levels of IRE1 and PREK(all P<0.01);the difference of the relative expression levels of IRE1 and PREK between DM group and MF group was not statistically significant(all P>0.05)(Shown in Fig.9-10).Conclusion:1 Endoplasmic reticulum stress occurs in the hippocampus of T2 DM rats.2 T2 DM rats could the insulin resistance and cognitive decline.3 Metformin might improve insulin resistance and has a protective effect on T2 DM induced cognitive decline.4 Metformin might be through reducing the expression of ATF6 to inhibit the excessive ERS in hippocampus,thereby reducing the damage to cognitive function.