Effect Of Semaglutide Ameliorates Glucose Metabolism Function In The Alzheimer’s Disease Model Via The GLP-1R/SIRT1/GLUT4 Pathway

Objective:Disorders of glucose metabolism in the brain affect neurodegenerative diseases including Alzheimer’s disease（AD）.A large number of studies have shown a close association between AD and type 2 diabetes.Many reports have found that glucagon-like peptide-1 receptor agonist（GLP-1RA）can improve the cognitive behavior and pathological characteristics of AD patients and animals.The mechanism may be to improve the function of glucose metabolism in the brain.However,the molecular mechanism by which GLP-1RA improves glucose metabolism in AD patients remains unclear.Therefore,this study attempted to use bioinformatics technology to predict the potential targets of novel long-acting GLP-1RA（Semaglutide）and AD,and explore the possible molecular mechanisms in APP/PS1/Tau（3xTg）transgenic mouse models and HT22 cells of mouse hippocampal neurons based on the selected important differential genes.It provides an important basis for the clinical treatment of GLP-1RA for AD.Methods:1.Acquisition of differential genes and key genes:The differential genes of AD and Semaglutide were screened from Dis Ge NET database and Pubchem online database,and the intersection was used to obtain colocalization targets.The protein interaction network of colocalization targets was constructed through STRING online database.The cyto Hubba plugin in Cytoscape software was then used to screen key genes based on their level of linkage.2.Cell experiment:HT22 cells of mouse hippocampal neurons were divided into four groups:HT22+PBS（Control）,HT22+Aβ1-42+PBS（Aβ）,HT22+Aβ1-42+Semaglutide（Aβ+SEM）,HT22+Aβ1-42+Semaglutide+EX527（Aβ）+SEM+EX527).The activity and apoptosis of cells in each group were observed.The glucose metabolism capacity was determined indirectly by absorbing the remaining medium and observing the glucose content in the medium.Subsequently,the expression of SIRT1 and GLUT4 and the translocation of GLUT4 were observed.（1）Culture of HT22 cell lines of mouse hippocampal neurons:DMEM high-glucose medium was used for culture in an incubator at 37℃and 5%CO₂,and 10%fetal bovine serum and 50 U/ml penicycin mixture were added for co-culture;Change the medium every other day.When the cells were 80-90%full at the bottom of the flask,digestion was terminated with complete medium after 2 min digestion with trypsin at 37℃.（2）Observe cell activity and apoptosis:CCK8 and apoptosis kit were used to detect HT22 cells in each group,and observe cell activity and apoptosis respectively.（3）Detection of glucose content in the cultured medium:GOD-POD kit was used to detect the remaining glucose content in the cell cultured medium to indirectly judge the glucose metabolism capacity of cells.（4）Observe the expression of SIRT1 and GLUT4:Immunofluorescence and Western blot were used to observe the expression of SIRT1 and GLUT4 proteins.（5）Observe the translocation of GLUT4:Immunofluorescence technology was used to observe the expression and distribution of GLUT4 in cytoplasm and cell membrane.3.In vivo experiment:12-month-old male 3xTg mice and C57B6/129 wild-type mice（WT,F1）were divided into four groups:WT+Saline（WT）,3xTg+Saline（3xTg）,3xTg+Semaglutide（3xTg+SEM）,3xTg+Semaglutide+EX527（3xTg+SEM+EX527）,15mice in each group;Semaglutide was intraperitoneally injected every other day for one month.（1）Dynamic monitoring of peripheral blood glucose of mice:During administration,the peripheral blood glucose levels of mice were dynamically monitored at four time points（before administration,the fifth,10th and 15th administration days）.（2）PET-CT was used to observe the glucose content in the brain of mice:¹⁸F deoxyglucose(¹⁸F-FDG)was injected into the tail vein,and the distribution of ¹⁸F-FDG in the brain of living mice was read by PET-CT scanning,and the SUV value was calculated.（3）Open field test:the movement track of mice in open field was recorded,and the total distance of movement and the percentage of staying in the center area were observed.（4）New object recognition test:mice were allowed to freely explore two objects with the same shape and color for 10 min,and after 6 h of rest,one of the objects was changed to one with different shape and color.The mouse movements were recorded and the new object recognition index（NOI）was calculated.（5）Y Maze:Mice were placed in the middle region of Y maze and moved freely for 8min without interference.The movement track and arm entry order of mice were recorded,and the correct rate of spontaneous alternation of mice was calculated and analyzed.（6）To observe the expression of SIRT1 and GLUT4 in the hippocampus of mice:immunofluorescence was used to observe the co-localization and protein expression of SIRT1 and GLUT4.Results:（1）In the bioinformatics section,3374 genes associated with AD were screened from Dis Ge NET database,and 101 potential targets of Semaglutide were obtained.Then R language was used to screen out 23 colocalizing target genes of AD and Semaglutide,among which SIRT1 was the most significant key gene.（2）Compared with the control group,the cell activity of Semaglutide（0 n M）+Aβgroup was significantly decreased（P<0.01）.Semaglutide inhibited the ameliorative activity at40 n M,80 n M,100 n M and 200 n M.But the improvement of 100 n M Semaglutide was the best（P<0.05）.（3）The percentage of advanced cells in HT22+Aβgroup was significantly higher than that in Control group（P<0.01）.Compared with HT22+Aβgroup,the percentage of advanced cells in HT22+Aβ+SEM group was significantly decreased（P<0.01）,and this effect was inhibited by the use of SIRT1 inhibitor EX527（P<0.01）.（4）The protein expression of SIRT1 was lower in HT22 cells pretreated with Aβ1-42（P<0.001）.Compared with Aβgroup,SIRT1 expression in Aβ+SEM group was significantly up-regulated（P<0.01）.It was found that Semaglutide also increased the expression of GLUT4 in cells simultaneously（P<0.05）and was blocked by EX527（P<0.01）,which promoted the translocation-to-cell membrane activity of GLUT4.（5）The residual glucose content in Aβgroup was significantly higher than that in Control group（P<0.01）,the residual glucose content in Aβ+SEM group was significantly lower than that in Aβgroup（P<0.01）,and the residual glucose content in Aβ+SEM+EX527group was significantly higher than that in Aβ+SEM group（P<0.01）.（6）The blood glucose of mice in 3xTg group was significantly higher than that in WT group（P<0.001）.Semaglutide could reduce the blood glucose of mice in 3xTg group（P<0.01）,while EX527 could inhibit the effect of Semaglutide（P<0.01）,resulting in the increase of blood glucose in mice.（7）This study analyzed the standard uptake value（SUV）of ¹⁸F-FDG in PET-CT results,and the SUV in the brain of mice in 3xTg group was lower than that in WT group（P<0.01）.The SUV of 3xTg+Semaglutide group was higher than that of 3xTg group（P<0.05）.The number of SUVs in the brain decreased after EX527 treatment（P<0.01）.（8）In open field test,Semaglutide could not significantly improve the motor ability and free exploration ability of mice,and there was no change after inhibiting SIRT1 by EX527（P>0.05）.（9）In the new object test,the total time of exploring the two objects was not significantly different among the four groups in the familiar stage（P>0.05）.At the test stage,the NOI index of mice in 3xTg group was significantly lower than that in WT group（P<0.01）,the NOI index of mice in SEM group was higher than that in 3xTg group（P<0.05）,and the NOI index of mice in SEM+EX527 group was lower than that in SEM group（P<0.05）.（10）In Y maze test,there was no statistical difference in the total number of arm insertion among all groups（P>0.05）;The correct rate of spontaneous alternations in3xTg group was lower than that in WT group（P<0.05）,SEM group was higher than that in 3xTg group（P<0.05）,and SEM+EX527 group was lower than that in SEM group（P<0.05）.（11）The expression levels of SIRT1 and GLUT4 were decreased in the 3xTg group.Semaglutide could promote the expression of SIRT1 and GLUT4 in the SEM group,while the expression level of GLUT4 was decreased after inhibition of SIRT1.CA3 was found to be an active region of glucose metabolism in hippocampus.Conclusion:Semaglutide alleviates the dysfunction of glucose metabolism in AD animals and cell models,and improves the cognitive and memory ability of AD animals,which is regulated by GLP-1R/SIRT1/GLUT4 pathway.Semaglutide is being tested in Phase III clinical trials in patients with AD,and this study provides important evidence on how the drug may improve glucose metabolism dysfunction in the brain.