The Effects And Molecular Mechanism Of Pancreatic β-cell Knockout On β-cell Function And Glucose Metabolism In Mice

Background and Objectives:Type 2 diabetes mellitus（T2DM,Type 2 diabetes mellitus）is a metabolic disease caused by insulin resistance（IR）andβ-cell dysfunction.Investigating the role ofβ-cell dysfunction in the progression of diabetes is of great significance to elucidate the pathogenesis of diabetes.O-GlcNAcylation is a post-translational modification of proteins,which is dynamically regulated by O-GlcNAc transferase（OGT）and O-GlcNAcase（OGA）.OGT is responsible for the introduction of O-GlcNAc to Ser and Thr sites of proteins,while OGA is responsible for the hydrolysis of O-GlcNAc on proteins.The O-GlcNAcylation of protein can function as nutrient-sensor by sensing the flux of hexosamine biosynthesis pathway（HBP）.Glucose metabolism can increase the content of O-GlcNAc modified substrate UDP-GlcNAc,which in turn promotes the O-GlcNAcylation of proteins.Previous studies have shown that OGT loss leads toβ-cell failure and diabetes,but OGT overexpression failed to improveβ-cell dysfunction and glucose metabolism damage.The role of OGA depletion-mediated hyperglycosylation of O-GlcNAc in pancreaticβ-cells remains unclear.Therefore,in this study,the mouse model of isletβ-cell OGA-specific knockout was constructed,and the model was used for high-sugar drinking（HGD）to explore the effect and mechanism of OGA deletion on mouseβ-cell function and glucose and lipid metabolism.Methods:1.Construction and verification ofβ-cell OGA-specific knockout mice.Ins2-Cre mice were successfully constructed using gene targeting technology and Cre-Lox P system.Then OGA-βKO mice were screened by mouse tail genotyping and verified by immunohistochemical staining（IHC）.2.Detection of glucose metabolism phenotypeThe effect ofβ-cell OGA knockout on glucose metabolism in normal-chow diet mice were investigated by blood glucose monitoring and body weight detection,intraperitoneal glucose tolerance test,multi-factor detection of suspension chips,and Tunel staining.In addition,high-glucose drinking water（HGD）mouse model was constructed,and the effect of OGA knockout under metabolic stress was further explored by blood glucose monitoring,body weight,intraperitoneal glucose tolerance,intraperitoneal insulin tolerance,mouse fat weighing,and body fat rate detection.3.Detection of energy metabolism in OGA-βKO miceThe effect of isletβ-cell OGA deletion on energy metabolism in mice was evaluated by metabolic cage monitoring technology,real-time monitoring of oxygen consumption（VO2）,carbon dioxide release（VCO2）,respiratory quotient（RER）and heat production（Heat）of mice at different time periods（Light,Dark）and under different treatment（Fed,Fasted）conditions.4.Construction and functional verification of OGA knockoutβ-cellThe OGA knockdownβ-cell were constructed by si RNA technology,and the effect of OGA deletion onβ-cell was verified by detecting the m RNA levels of insulin and insulin transcription factors（PDX-1,Maf A,Neuro D1）in the OGA knockdownβcell line.5.Detection of drp1 expressionIt is speculated thatβ-cell damage caused by OGA deletion may be related to DRP1-mediated disturbance of mitochondrial dynamics in recent literatures.Terefore,we investigate the effect of OGA deletion on the expression of DRP1 protein inβ-cell using western blot,and then preliminarily explore the influence of DRP1-mediated mitochondrial dynamics disorder regulated by OGA on pancreaticβ-cell function.Results:1.OGA-βKO mice were successfully constructed and verified.There was no significant difference between fasting and random blood glucose in OGA-βKO mice.Glucose tolerance of OGA-βKO mice was significantly impaired（P<0.01）.Serum insulin was decreased in OGA-βKO mice（P<0.01）,serum insulin polypeptide,leptin and resistin（P<0.05）.Apoptosis of islet cells was increased in OGA-βKO mice.2.As for the high-sugar drinking model,monitoration of blood glucose of OGA-βKO mice was not significantly different with OGA-βflox mice.But body fat percentage of OGA-βKO mice was increased（P<0.001）.HGD mice also showed strong gender differences.OGA-βKO female mice had higher body weight（P<0.05）and peripheral fat content（P<0.001）,lower O₂ consumption,CO₂ release,RER and heat production（P<0.001）,and more severe impairment of glucose tolerance（P<0.0001）.While OGA-βKO male mice showed that body weigh,blood glucose,O₂ consumption,CO₂ release,RER,heat production and peripheral fat content was not significant differences with OGA-βflox mice.Middle age OGA-βKO male mice showed impaired glucose tolerance.But old-age OGA-βKO male mice showed restored glucose tolerance and impaired insulin tolerance.It is suggerst that the metabolic disorder degree of OGA-βKO mice was more significant under high-suger drinking modle.3.The MIN6-OGA-KO and INS1-OGA-KO cell lines were successfully constructed and validated.The m RNA levels of insulin and insulin transcription factor Neuro D1 in MIN6-OGA-KO cells were decreased（P<0.05）,and the m RNA levels of insulin and insulin transcription factors PDX-1 and Neuro D1 in INS1-OGA-KO cells were decreased（P<0.05）.The above results suggested that the deletion of OGA can cause the damage ofβ-cell function.In addition,the expression of mitochondrial DRP1 increased after OGA knockout in MIN6 cells,suggesting that OGA deletion can promote the recruitment of DRP1to mitochondria.Conclusion:1.β-cell OGA specific knockout can induce mice glucose metabolism disorder by promotingβ-cell apoptosis,reducingβ-cell insulin secretion and inpaired glucose tolerance.In addition,OGA-βKO female mice glucose tolerance was impaired more severe,suggesting that OGA is more important for maintaining the normal function ofβ-cells and glucose homeostasis in female mice.2.OGA-βKO mice showed more severe glucose tolerance impairment under HGD,suggesting that OGA deletion ofβ-cell aggravates glucose metabolism disorder caused by HGD.In addition,under the condition of HGD,β-cell OGA knockout female mice had more significant impairment of glucose tolerance,and higher body weight,peripheral fat content,body fat rate,and more serious damage to glucose metabolism,suggesting that OGA mediated O-GlcNAc homeostasis plays a more important role in the regulation ofβ-cell and glucose metabolism in female mice.3.β-cell OGA depletion in vitro could result in the decreased the m RNA expression of insulin and insulin transcription factor and promoted DRP1 recruitment to mitochondria.