The Mechanism Of Renin–Angiotensin System In Type 2 Diabetes Mellitus-Associated Cognitive Impairment

Part I Association of ACE level,activity and I/D gene polymorphism in the peripheral blood with mild cognitive impairment in type 2 diabetes mellitusBackground:Angiotensin-converting enzyme(ACE)is one of the key components of the renin–angiotensin system(RAS).ACE is involved in the development of type 2 diabetes mellitus(T2DM)and Alzheimer's disease(AD)respectively.T2 DM can be complicated by different degrees of cognitive dysfunction,including mild cognitive impairment(MCI)and dementia.MCI is a transitional stage between normal aging and dementia.Objectives:This study aimed to investigate the association of ACE level,activity and ACE I/D gene polymorphism in the peripheral blood with mild cognitive impairment in type 2 diabetes mellitus.Methods:A total of 210 T2 DM patients were enrolled.They were divided into 116 MCI group and 94 healthy-cognition controls,according to the Montreal Cognitive Assessment(Mo CA)scores.Sociodemographic,clinical characteristics and cognitive performances were extensively assessed.The serum ACE level and ACE activity were measured via enzyme-linked immunosorbent assay and ultraviolet spectrophotography respectively.The single-nucleotide polymorphisms of Insertion/deletion(I/D)gene of ACE were detected by the polymerase chain reaction-restriction fragment length polymorphism method.Results:1.Compared to the healthy-cognition controls,fasting blood glucose(FBG),glycosylated hemoglobin(Hb A1c),the serum ACE level and ACE activity in T2DM-MCI groups were significantly increased,while fasting C-peptide levels were significantly decreased(all p<0.05).2.Among the T2DM-MCI patients,serum ACE activity was positively correlated with the Mo CA score(r =-0.242,p = 0.009)and logical memory test score(LMT)(r =-0.286,p = 0.002).3.Among the T2DM-MCI patients,the multiple stepwise regression analysis indicated that ACE activity was a risk factor for LMT scores(? =-0.186,p = 0.035),after adjusting for age,education level,FBG,duration of diabetes,duration of hypertension,and blood lipid levels.4.There were no significant difference in the genotype or allele distribution of ACE I/D polymorphism between the T2DM-MCI groups and the controls.Among the T2DM-MCI patients,the serum ACE levels and ACE activity in the DD group were higher than that in the ID and II groups(all p < 0.05).Conclusions:The serum ACE level and activity of MCI patients were both higher than those of normal cognitive function among T2 DM patients.The increased serum ACE activity was associated with cognitive impairment among T2 DM patients,especially memory impairment.In addition,further studies on a large population size are needed to test whether the D-allele of the ACE gene polymorphism may be a candidate gene for memory impairment in T2 DM.Part II The role of RAS in cognitive impairment of KK-Ay miceBackground:Chronic hyperglycemia is one of the major clinical manifestations of Type 2 Diabetes(T2DM).Chronic hyperglycemia could activate renin-angiotensin system(RAS)in vivo.The activation of RAS in the brain can lead to further development of cognitive impairment,but the specific molecular mechanism is complex and unclear.The brain amyloid hypothesis is one of several major pathogenesis hypotheses of cognitive dysfunction in T2 DM.Amyloid beta(A?)is produced from the cleavage of ?-amyloid precursor protein(APP)by ? and ? secretases.Our previous results found that the level and activity of angiotensin converting enzymes(ACE)in the peripheral blood of T2 DM patients with mild cognitive impairment were increased.The serum ACE activity was associated with cognitive impairment in T2 DM patients,especially memory impairment.Objectives:On the basis of our clinical studies,we further studied the pathogenesis of RAS in T2 DM associated cognitive impairment through animal experiments,and observed the protein levels of ACE-Ang II-AT1 R axis,APP and A? metabolism in the brain of T2 DM mice with cognitive impairment.We also aimed to investigate the intervention effects of Captopril,Losartan or PD123319 in T2 DM mice with cognitive impairment.Materials and methods:8 weeks KK-Ay mice fed with special feed were selected as the experimental group,and 8 weeks C57BL/6 mice were as the control group.The two groups of mice were sacrificed at 8 weeks and 20 weeks.The intervention group(20 weeks KK-Ay mice)were respectively administrated Captopril(5 mg/kg/d),Losartan(10 mg/kg/d)or PD123319(10 mg/kg/d)every day for 8 weeks.The control group(20 weeks KK-Ay mice)received 0.9% Na Cl every day for 8 weeks.The mice were sacrificed at 28 weeks.The weight and fasting blood glucose(FBG)were measured weekly.Before the mice were sacrificed,the blood was collected from the epicanthus vein to measure the serum insulin level and to calculate the level of homeostatic model assessment of insulin resistance(HOMA-IR).Morris water maze test and dark-avoidance test were used to compare the learning and memory functions in different groups.HE staining and Nissl staining were performed to observe the changes of neuromorphology and Nissl bodies in the hippocampus of mouse brain tissue.Immunofluorescence staining was used to detect the expression of p-JNK and p-APP in the hippocampus of mouse brain tissue.Western blot was used to measure the protein levels of ACE,Angiotensin II Type 1 Receptor(AT1R),AT2 R,c-Jun N-terminal kinase(JNK),p-JNK,APP,and p-APP Thr 668 in mouse hippocampus.ACE activity,Angiotensin II(Ang II),Amyloid ? 40(A?40)and A?42 levels in hippocampus homogenate were also detected.Results:1.20-week-old KK-Ay mice exhibited higher FBG,insulin and HOMA-IR levels than those of 20-week-old C57BL/6 mice(P < 0.05).The FBG of 20-week-old KK-Ay mice were ? 11.1 mmol/L,which met the diagnostic criteria of T2 DM.After 8 weeks of intervention with Captopril,Losartan or PD123319 respectively in KK-Ay mice,there were no significant differences in FBG,insulin and HOMA-IR levels,compared with the 0.9% Na Cl control group(P > 0.05).2.Morris water maze test results showed that 20-week-old KK-Ay mice had longer incubation period to find the platform and decreased numbers to cross the target platform than the control group(P < 0.05).The dark-avoidance test results showed that 20-week-old KK-Ay mice had shorter avoid dark incubation period(I.e.incubation period to enter the dark area by mistake)and increased number of errors than the control group(P < 0.05).Compared with the 0.9% Na Cl control group,Captopril group and Losartan group both showed that the incubation period to find the platform was shortened and the number to cross the target platform was increased in the Morris water maze test(P < 0.05);the avoid dark incubation period was increased and the number of errors was decreased in the dark-avoidance test(P < 0.05).However,there were no significant differences in the above indexes between the PD123319 group and the control group(P > 0.05).3.HE staining and Nissl staining showed that some cells in the hippocampus of 20-week-old KK-Ay mice were disorderly arranged and structure destroyed,and the number of Nissl bodies was decreased,especially in CA1 regions,compared to the control group.Compared with the 0.9% Na Cl control group,Captopril group and Losartan group both showed that the disorderly arranged and structure destroyed cells in CA1 regions of hippocampus were partially improved,and the number of Nissl bodies was increased.However,there were no obvious changes in PD123319 group compared with the control group.4.Immunofluorescence staining showed that the expression of p-JNK and p-APP Thr 668 was stronger in hippocampal CA1 region of 20-week-old KK-Ay mice compared with 20-week-old C57BL/6.After intervention of KK-Ay mice with Captopril or Losartan,the expression of pJNK and p-APP Thr 668 was partly weakened.However,there were no obvious changes in PD123319 group compared with the control group.5.Compared with 20-week-old C57BL/6 mice,20-week-old KK-Ay mice had significantly higher ACE activity,Ang II,ACE,AT1 R,p-JNK,p-APP Thr 668,A?40 and A?42 levels in hippocampus(P < 0.05).There were no significant changes in AT2 R,total JNK and total APP levels(P > 0.05).Compared to the control group,Captopril group showed that hippocampal tissue ACE activity and Ang II levels were reduced,accompanied by decreased p-JNK,p-APP Thr 668,A?40 and A?42 levels(P < 0.05).Losartan group showed that hippocampal tissue AT1 R levels were reduced,accompanied by decreased p-JNK,p-APP Thr 668,A?40 and A?42 levels(P < 0.05).PD123319 group showed that hippocampal tissue AT2 R levels were decreased(P < 0.05),but the levels of p-JNK,p-APP Thr 668,A?40 and A?42 did not significantly change(P > 0.05).Conclusion:The impairment of learning and memory function in T2 DM mice may be associated with chronic hyperglycemia induced-activation of ACE-Ang II-AT1 R axis,the phosphorylation of JNK and APP Thr668,and the regulation of A? metabolism in the brain.After intervention with Captopril or Losartan respectively,the inhibition of ACE-Ang II-AT1 R axis reduced the levels of p-JNK,p-APP Thr 668,A?40 and A?42 in brian,thus improved the learning and memory function of T2 DM mice with cognitive impairment.Part III Mechanism research of RAS in high glucose induced HT22 cell damageBackground:The brain amyloid hypothesis is one of the major hypotheses of cognitive impairment in Type 2 Diabetes Mellitus(T2DM).The activation of renin-angiotensin system(RAS)can participate in the metabolism of amyloid beta(A?).A? is produced from the cleavage of ?-amyloid precursor protein(APP)by ? and ? secretases.The c-Jun N-terminal kinase(JNK)is required to activate the threonine 668(Thr 668)site of APP when the ? secretase cleaves the carboxylterminal fragment formed by the cleavage of APP by the ? secretase.Our animal experiments found that the ACE-Ang ?-AT1 R axis were activated in the brain of T2 DM cognitive impairment mice,accompanied by the increased levels of p-JNK,p-APP Thr 668,A?40 and A?42.After the intervention with Captopril or Losartan respectively,the levels of p-JNK,p-APP Thr 668,A?40 and A?42 were decreased,and the impairment of learning and memory function in mice was alleviated.However,at the cellular level,the mechanism of RAS in high glucoseinduced injury of mouse hippocampal neuronal cells(HT22 cell line)is not yet clear.Objective:To explore the mechanism of RAS in high glucose induced HT22 cell damage.Methods:HT22 cells were used as the cell model,and high glucose intervention was used as the high glucose toxicity model.CCK8 kit was used to detect the effects of different glucose concentrations and different incubation times on the viability of HT22 cells.The intervention with Captopril(1 ?mol/L),Losartan(1 ?mol/L),PD123319(1 ?mol/L),or SP600125(5 ?mol/L)were performed respectively to high glucose induced HT22 cells.The m RNA expression levels of ACE,AT1 R,and AT2 R were detected by real-time quantitative PCR.The protein levels of ACE,AT1 R,AT2R,p-JNK,JNK,p-APP Thr 668 and APP were detected by Western blot.The levels of Ang?,A?40 and A?42 were detected by enzyme-linked immunosorbent assay(ELISA).Result:1.High glucose intervention affected HT22 cells viability in a concentration-and timedependent manner.In this study,we used 75 m M glucose for 48 h to intervene in HT22 cells as a model of high glucose toxicity.2.Compared with the control group(25m M glucose),the m RNA and protein levels of ACE and AT1 R,and the expression levels of Ang ?,p-JNK,p-APP Thr 668,A?40 and A?42 were increased in high glucose(75m M glucose)group in HT22 cells(P < 0.05).3.Compared with the control group(75 mM glucose),the intervention with Captopril group showed decreased Ang ? level in the cell supernatant,decreasd levels of ACE m RNA and protein,accompanied with the decreased levels of p-JNK,p-APP Thr 668,A?40 and A?42(P < 0.05).Losartan group showed decreased AT1 R m RNA and protein levels,accompanied with the decreased levels of p-JNK,p-APP Thr 668,A?40 and A?42(P < 0.05).PD123319 showed that the levels of AT2 R m RNA and protein were decreased(P < 0.05),but the levels of p-JNK,p-APP Thr 668,A?40 and A?42 were not significantly changed(P > 0.05).4.Compared with the control group(75 m M glucose + DMSO),SP600125 group showed that p-JNK level was decreased,and the levels of p-APP Thr 668,A?40 and A?42 were decreased(P < 0.05).However,the levels of ACE,AT1 R,AT2R m RNA and protein expression were not significantly changed(P > 0.05).Conclusion:High glucose toxicity can up-regulate the ACE-Ang ?-AT1 R axis in neuronal cells,increase the expression levels of p-JNK,p-APP Thr 668,A?40 and A?42.After the intervention of Captopril or Losartan respectively,the inhibition of ACE-Ang II-AT1 R axis may downregulate p-APP Thr 668,A?40 and A?42 levels by inhibiting the phosphorylation levels of JNK,thus exerts a neuroprotective effect.