Abnormal Glucose Metabolism Induced By STZ And MG Leads To AD-Like Lesions And Its Underlying Mechanisms

Background: The incidence of Type 2 Diabetes(T2DM)and Alzheimer’s Disease(AD)is increasing year by year worldwide,and there are some common pathological features between T2DM and AD.For example,hyperphosphorylation of tau protein and insulin resistance in brain have previously been described in patients with T2DM and patients with AD.Hyperphosphorylation of tau protein is one of the two major pathological features of AD,and is correlated with impaired learning and memory ability of patients.Brain insulin resistance could lead to damaged insulin signaling transduction,triggering a variety of metabolic disorders including glucose metabolism,cognitive and emotional processes dysfunction.More and more evidences show that activation of Mammalian Target of Rapamycin(mTOR)is one of the mechanisms of insulin resistance,and activation of mTOR has been observed in brain of AD patients.mTOR has two functionally distinct forms,mTORC1 and mTORC2.Activation of mTORC1 could lead to neuronal glucose deficiency,with consequent decreased glucose metabolism,impaired ATP production and dysfunction and/or death.Therefore,inhibition of mTORC1 might have effect on regulating activity of enzymes related to glucose metabolism,lead to improved learning and memory ability.Objective: To investigate intraperitoneally injected of rapamycin,a specific inhibitor of mTORC1,has beneficial effects to mice with glucose metabolism disorder(with brain insulin resistance)and mice with brain insulin resistance and the underlying molecular mechanisms.Methods: We used two treatments to male wild type C57BL/6 mice(8～10 weeks old)in order to build two different STZ-induced mouse models.In the first way,mice were divided into three groups: CON group,received vehicle solution intraperitoneally;STZ group,received streptozotocin 50 mg/kg intraperitoneally for 5 consecutive days and vehicle solution;and STZ+RAP group,after STZ treatment,received rapamycin 2mg/kg intraperitoneally for 14 consecutive days beginning 2 weeks after STZ treatment.The second way also divided the mice into three groups:(Cannula was embedded in the lateral ventricle of all mice,after a week to recover,drugs were administered through cannula.)a CSF-ICV group,received vehicle solution intracerebroventricularly and intraperitoneally;STZ-ICV group,received STZ 3mg/kg intracerebroventricularly for twice(48h apart)and vehicle solution intraperitoneally;STZ-ICV+RAP group,after STZ treatment,received rapamycin 2mg/kg intraperitoneally for 14 consecutive days.The Morris Water Maze was taken to assess spatial learning and memory ability of mice.Western blotting,Dot blotting and immunohistochemistry were used to investigate the expression of tau protein and the changes in the pattern of insulin signaling in hippocampus of mice.SH-SY5 Y cells were cultured with 30 mM glucose and treated with rapamycin,with or without GSK-3β inhibitor LiCl,AKT inhibitor MK2206,then detected the changes of each enzyme.Results: 1.Rapamycin had no effect on peripheral blood glucose and body weight,but it could improve insulin resistance in brain of two different STZ-induced mice: It was observed that the mice injected STZ intraperitoneally(STZ group)had a significant increase in blood glucose,and a slight decrease in body weight.The mice injected STZ intracerebroventricularly(STZ-ICV group)had no change in blood glucose and body weight.Rapamycin treatment neither had effect on blood glucose and body weight in STZ group,nor in STZ-ICV group.The level of IRS-1 in the hippocampus of STZ group was significantly reduced,STZ-ICV group showed increased level of p S307-IRS-1 and decreased level of IGF-1R.After the injection of rapamycin,brain insulin resistance could be improved in two different STZ-induced mouse models.2.Rapamycin reversed impaired insulin signal pattern,tau hyperphosphorylation and damaged learning and memory ability in two different STZ-induced mouse models:After STZ injected intraperitoneally,tau phosphorylation levels in hippocampus of STZ group were significantly higher than that of CON group,meanwhile levels of pS2448-mTOR,mTOR and pT389-p70s6 elevated,levels of pT172-AMPK,pS9-GSK-3β and pS473-AKT decreased,and level of AGEs significantly enhanced.STZ group showed impaired learning and memory ability in Morris water maze.After STZ injection into the lateral ventricle,the levels of pS2448-mTOR,pT389-p70s6 and AGEs were increased,expression of pT172-AMPK,pS9-GSK-3β and pS473-AKT were suppressed in the hippocampus of STZ-ICV group,and spatial learning and memory ability impaired.Rapamycin injection could reverse all these changes in two different STZ-induced mouse models.3.Rapamycin rescued tau hyperphosphorylation in high glucose induced SH-SY5 Y cells through feedback regulation of the p70s6/AKT/AMPK/GSK-3β pathway: After treated SH-SY5 Y cells with high glucose and inhibitor,we observed that rapamycin could significantly reverse tau phosphorylation caused by high glucose treatment.Treated with the GSK-3β inhibitor LiCl further reduced tau phosphorylation,and exposure to the AKT inhibitor MK2206 abolished rapamycin-induced tau dephosphorylation under HG and rapamycin conditions.At the same time,LiCl treatment did not affect the levels of pS2448-mTOR and pT389-p70s6.pS9-GSK-3βand pT172-AMPK increased further following rapamycin treatment,but pS473-AKT was not affected.The levels of pS2448-mTOR and pT389-p70s6 were not changed in the cells treated with MK2206 compared with HG and rapamycin.MK2206 treatment almost completely abolished pS473-AKT and significantly inhibited p S9-GSK-3β,but did not influence pT172-AMPK.Conclusions:1.The inhibition of mTORC1 by rapamycin in two different STZ-induced mouse models had no effect on blood glucose and body weight,but could improve brain insulin resistance and decrease AGEs.2.The inhibition of mTORC1 in multiple models could reduce the phosphorylation of tau protein by feedback regulation of the p70s6/AKT/AMPK/GSK-3β pathway.3.The inhibition of mTORC1 can improve the spatial learning and memory ability in two different STZ-treated mice.Background: The main pathological features of Alzheimer’s Disease(AD)are senile plaques formed by accumulation of extracellular Aβ and neuronal fiber tangles formed by tau protein accumulation.AD patients have a variety of metabolic abnormalities in brain,including abnormal glucose metabolism.Increasement of methylglgoxal(MG)is one of the indicators of abnormal glucose metabolism,and is also an important percursor of AGEs.Some studies found that levels of MG and AGEs in brains of AD patients enhanced significantly and were positive related to degree of cognitive deficit.MG and AGEs could induce tau phosphorylation,inflammation,brain insulin resistance,etc.It is not clear whether the long-term systemic MG increasement would elevate tau phosphorylation in brain of animals.AMPK is a kinase that regulates energy metabolism.Increased activity of AMPK could improve learning and memory ability,reduce inflammation,and restore BBB permeability in mice.Therefore,it is important to investigate the glycated effects of MG on AMPK,and whether glycation of AMPK could aggravate learning and memory damage and brain inflammation in mice.Objective: 1.To establish an AD-like mouse model through long-term treatment of MG in C57BL/6 mice.2.Explore the molecular mechanism of MG induced AD-like pathology: 1)Whether MG could damage the learning and memory ability and induce tau phosphorylation in mice.2)To investigate the influence of MG treatment on AMPK in mice.3)Whether MG could exacerbate inflammation in brain of mice.Methods: 2-month-old male C57 mice were randomly divided into two groups: NS group,injected normal saline intraperitoneally for 28 consecutive days;MG group,injected MG 60 mg/kg intraperitoneally for 28 consecutive days.After injection,the open field test,elevated plus maze and Morris water maze were taken to analyze the emotional state and spatial learning and memory ability of mice.Western blotting,Dot blotting were taken to examine the levels of tau protein,AMPK and AGEs in hippocampus of mice,RT-PCR were used to detect the hippocampal mRNA expression of inflammatory cytokines,immunohistochemical staining and immunofluorescent staining were used to analysis microglia,astrocyte,integrity of blood brain barrier and involvement of acquired immune system,CO-IP were used to detect glycation of AMPK in hippocampus of mice.BV2 cells were treated with different concentration of MG(0 mM,0.5 mM,1 mM,2.5 mM),the expressions of inflammatory factors and AMPK/STAT1 pathway in BV2 cells were detected by RT-PCR and Western blotting.Results:1.We found that in the open field test,the center duration of MG group was higher than that in NS group,and there was no difference in the corner duration.In the elevated plus maze,the duration of stay in the open arm and close arm of NS and MG group showed no difference.In Morris water maze,MG group showed impaired spatial learning and memory ability.Meanwhile,tau phosphorylation in hippocampus of MG group was significantly higher than that of NS group.2.Compared with NS group,phosphorylation level of Thr172 site,which is an activity-dependent site of AMPK,was decreased,the expression of AGEs enhanced,the binding of AMPK and AGEs increased in hippocampus of MG group.3.The mRNA expression of pro-inflammatory factor IL-6 elevated,mRNA expression of anti-inflammatory factor TGF-β1 decreased in hippocampus of MG group.Compared to NS group,microglia in MG group exhibited enhanced cell body,lessened process length and number of endpoints,and astrocytes showed no difference between two groups.Treated BV2 cells with different concentration of MG,we observed increased m RNA expression of IL-6,reduced AMPK activity and activated transcription factor STAT1 under MG concentration of 2.5m M.In addition,MG group showed progressive Occludin and Ig G infiltration,indicated the blood brain barrier was damaged.Positive staining of CD8+T cells was observed in the hippocampal area and cortex in MG group,and there was no CD4+T cells infiltrated into the brain parenchyma in both groups.Conclusions:1.MG impaired spatial learning and memory ability of mice,and caused AD-like lesions in the hippocampus of mice.2.MG induced inhibition of AMPK activity and increased glycation of AMPK in the hippocampus of mice.3.MG leaded to increased inflammation factors,activated microglia,increased blood brain barrier permeability,peripheral immune cells infiltrating brain parenchyma in the hippocampus of mice.