The Mechanism Of O-glcnacylation-mediated Glucose Toxicity In Type 2 Diabetes Mellitus-associated Cognitive Dysfunction

Part I Correlation study between O-GlcNAcylation,tau phosphorylation levels and mild cognitive impairment in the peripheral blood of type 2 diabetic patientsBackground: O-GlcNAcylation is a common post-translational modification whose substrate is derived from the hexosamine biosynthetic pathway in glucose metabolism.Aberrant O-GlcNAc modification has been implicated in type 2 diabetes mellitus(T2DM),as well as the pathogenesis of neurodegenerative diseases via competition with tau phosphorylation at serine or threonine sites.Therefore,O-GlcNAcylation may play an important role in the development of diabetes-associated cognitive dysfunction(DACD).Objectives: To investigate the association among global O-GlcNAcylation,tau phosphorylation levels and mild cognitive impairment(MCI)in the whole blood of T2 DM patients.Methods: This study used a case-control design method and enrolled 48 subjects with T2 DM.According to the Montreal Cognitive Assessment(MoCA)score,48 T2 DM subjects were divided into 24 with MCI and 24 with normal cognition.Sociodemographic,clinical characteristics and cognitive performances were extensively assessed.Global O-GlcNAcylation and tau phosphorylation levels in the whole blood were also determined using Western blot.Results: 1.Compared with cognitively normal controls,fasting blood glucose(FBG)and glycosylated hemoglobin(HbA1c)of MCI subjects were significantly increased,while fasting C-peptide and serum uric acid were significantly decreased(all p<0.05).2.T2 DM with MCI subjects also displayed decreased O-GlcNAcylation level,while increased total tau and phosphorylated tau levels at Ser396,Ser404,Thr212 and Thr231 sites(all p<0.05).In order to reflect the combined effect,ratios of O-GlcNAcylation to tau phosphorylation levels were all significantly decreased in MCI subjects(all p<0.05).3.Further bianary logistic regression analysis revealed that higher HbA1 c was an independent risk factor,while increased O-GlcNAc/p-T212 was an independent protective factor for MCI in T2 DM patients(OR=2.452,95%CI 1.061-5.668,p=0.036;OR=0.028,95%CI 0.002-0.388,p=0.008,respectively).4.With regard to each cognitive domain,O-GlcNAc/p-T212 was positively correlated with the score of Auditory Verbal Learning Test-delayed recall after adjusting for FBG and HbA1 c levels(r=0.377,p=0.010).Conclusions: Our study suggests that decreased ratio of O-GlcNAcylation to tau phosphorylation at Thr212 site in the whole blood is associated with MCI,especially memory dysfunction in T2 DM subjects.Part II Role of O-Glc NAcylation and tau phosphorylation in type 2 diabetes mellitus-associated cognitive dysfunction and intervention study of glimepirideBackground: Chronic hyperglycemia is the most important clinical manifestation of T2 DM,and also one of the independent risk factors for DACD.Our previous studies showed that there is an imbalance of O-Glc NAcylation and tau phosphorylation in the peripheral blood of patients with DACD.However,O-Glc NAcylation and tau phosphorylation levels in the brain remain unclear.To choose an effective mean to prevent its imbalance may provide a new target for DACD.Objectives: To observe O-Glc NAcylation and tau phosphorylation levels in the brain of DACD mice model,and to observe whether glimepiride can improve the cognitive function of diabetic mice,and the possible roles of glimepiride on O-Glc NAcylation and tau phosphorylation.Methods: KK-Ay mice were fed with special feed to construct an animal model of DACD,and C57BL/6 mice were used as controls.Some mice were sacrificed at 16 weeks of age,and some mice were given glimepiride(low dose: 1mg/(kg·d);high dose: 2mg/(kg·d))gavage for twelve weeks.The control group was given an equal volume of vehicle gavage.Morris water maze(MWM)and passive avoidance test(PAT)were used to detect learning and memory functions of mice.The morphological changes in brain tissue of diabetic mice were detected by HE staining and Nissl staining.Immunofluorescence staining was used to detect the location of O-Glc NAcylation,and its expression level with tau phosphorylation in the mouse hippocampus.Western blot was used to detect O-Glc NAcylation and tau phosphorylation levels in mouse hippocampus.Results: 1.The body weight,fasting blood glucose(FBG),and fasting insulin levels of KK-Ay mice and C57BL/6 mice at 16 weeks of age were significantly different.FBG level was significantly decreased in a dose-dependent manner after glimepiride intervention.2.Compared with C57BL/6 mice,the results of MWM and PAT showed that the escape latency and path length to search the platform of 16-week old KK-Ay mice were significantly longer,the percentage of time spent in target quadrant and frequency of crossing platform area were significantly decreased;the step through latency(STL)was significantly shorten,and error times(ETs)were significantly increased.After glimepiride intervention,the escape latency and path length to search the platform of KK-Ay mice were significantly shortened,the percentage of time spent in target quadrant and frequency of crossing platform area were significantly prolonged;the STL was significantly prolonged,and the ETs were significantly reduced.3.HE staining and Nissl staining showed that some cells were loosely arranged or structure destroyed,and the number of Nissl body was reduced in the hippocampus,especially CA3 region of 16-week old KK-Ay mice.After glimepiride intervention,the loosely arranged cells or destroyed structure were partially improved,and the number of Nissl body was increased in the hippocampal CA3 region of KK-Ay mice.4.Immunofluorescence staining showed that O-Glc NAcylation mainly occured in the hippocampal neurons.The number of neuronal cells modified by O-Glc NAcylation and O-Glc NAcylation level were decreased,but tau phosphorylation levels were increased in the hippocampal CA3 region of 16-week old KK-Ay mice.The above-mentioned phenomenons were partly improved after glimepiride intervention.5.The levels of O-Glc NAcylation and its key enzyme O-Glc NAc transferase(OGT)were significantly decreased,and the levels of tau protein phosphorylated at Ser396,Ser404,Thr212 and Thr231 sites,and O-Glc NAcase(OGA)were significantly increased in the hippocampus of 16-week old KK-Ay mice than those of C57BL/6 mice.After glimepiride treatment,O-Glc NAcylation level was increased,and tau protein phosphorylation levels at Ser396 and Thr212 sites were decreased,accompanied by elevated OGT level.Conclusions: Learning and memory dysfunction in T2 DM mice may be associated with chronic hyperglycemia induced-imbalance in O-Glc NAcylation and tau phosphorylation levels.Improving glucose toxicity may up-regulate O-Glc NAcylation and down-regulate tau phosphorylation levels,thus improve learning and memory function in T2 DM mice.Part III Mechanism researches of high glucose toxicity-induced injury of HT22 cellBackground: Tau protein hyperphosphorylation is one of the most important hypotheses of DACD.Our previous studies demonstrated that there is an imbalance between O-Glc NAcylation and tau phosphorylation levels in the hippocampus of DACD mice model.O-Glc NAcylation and tau phosphorylation levels were partially reversed,and the learning and memory function was alleviated after glucose-lowering intervention.However,the mechanisms that how glucose-lowering intervention improves DACD is not clear.Objectives: To investigate the mechanism of high glucose toxicity-induced injury of mouse hippocampal neuron cells(HT22 cell line).Methods: HT22 cells were used as cell models,and high glucose and AGEs intervention was used as a model of high glucose toxicity.CCK8 was used to detect the effect of different glucose or AGEs intervention concentrations and intervention time on the viability of HT22 cells.After OGT plasmid transfection and glimepiride intervention were performed,the m RNA expression level of OGT was detected by real-time quantitative PCR,and the levels of O-Glc NAcylation,tau phosphorylation and OGT protein were detected by Western blot.Results: 1.High glucose(HG)can reduce the viability of HT22 cells in a concentrationand time-dependent manner.In this study,75 m M glucose for 48 h were used to intervene HT22 cells as a model of high glucose toxicity.2.Compared with the control group,O-Glc NAcylation level was increased,and tau phosphorylation levels at Ser396,Ser404,Thr212 and Thr231 sites were also increased in the HG group,accompanied by decrease in OGT m RNA and protein expression levels.3.AGEs treatment can reduce the viability of HT22 cells in a concentration-and time-dependent manner.In this study,400 μg/m L AGEs for 48 h were used to intervene HT22 cells as a model of high glucose toxicity.4.Compared with the control group,O-Glc NAcylation level was decreased and tau phosphorylation levels at Ser396,Ser404,Thr212 and Thr231 sites were increased in the AGEs treatment group,accompanied by decrease in OGT m RNA and protein expression levels.5.OGT plasmid transfection reversed AGEs-induced decreased O-Glc NAcylation level,and increased tau protein phosphorylation levels at Ser396,Ser404,Thr212 and Thr231 sites.6.After glimepiride intervention,OGT m RNA and protein expression levels were increasd,O-Glc NAcylation level was increased,and tau phosphorylation level at Thr212 site was decreased.After OSMI-1 inhibits the expression levels of OGT m RNA and protein,the effects of glimepiride on increasing O-Glc NAcylation and decreasing tau phosphorylation level were partially antagonized.Conclusions: High glucose toxicity can lead to decreased O-Glc NAcylation levels and increased tau phosphorylation levels via decreased OGT level,thus lead to DACD.Glimepiride also can increase O-Glc NAcylation by up-regulating OGT level,then decrease tau phosphorylation levels,thus exert neuroprotective effects.