O-GlcNAcylationn Mediates The Disappearance Of Insulin Cardioprotection On Myocardial Ischemia/Reperfusion Injury In Obesity And Its Underlying Mechanism

Background:Myocardial ischemia/reperfusion(I/R)injury is a very serious problem in reperfusion therapy for acute myocardial infarction(AMI).It not only causes high incidence rate,but also significantly worsens the prognosis of reperfusion therapy.At present,a variety of treatment strategies,like insulin,have been proved to improve reperfusion injury,but some of cardioprotective strategies is significantly diminished in obese.O-GlcNAcylation is an important protein post-translational modification.Many studies suggest that obesity can induce hyper-O-GlcNAcylation,increase the infarct size and decrease the survival rate after myocardial reperfusion.Therefore,we speculate that hyper-O-GlcNAcylation may be the key factor for the disappearance of cardioprotective of these strategies in obesity.Objective:In this study,insulin was taken as an example to explore the key role of hyper-O-GlcNAcylation in the disappearance of insulin cardioprotection in obesity and its underlying mechanism.Method:Through spontaneously obese db/db mice and the insulin resistance H9c2 cells model induced by palmitic acid to explore the level of O-GlcNAcylation in obesity.Establish the mouse myocardial I/R model and H9c2 cells H/R model,then induce O-GlcNAcylation by glucosamine to explore the role of hyper-O-GlcNAcylation in myocardial I/R injury:evaluate the myocardial infarction area by TTC-Evans blue staining,electrophysiological monitoring and assess the arrhythmia score by Lambeth Convention(Ⅱ),detect the cell viability by CCK-8 assay and determine the biomarkers of myocardial injury and oxidative stress after reperfusion or reoxygenation to explore the effect of hyper-O-GlcNAcylation on the cardioprotective of insulin at animal and cell levels.Results:1.Compared with db/m mice,db/db mice showed significant hyperglycemia(5.95±0.75 vs24.45±7.65 mmol/L,P<0.0001),hypertriglyceride(72.1±6.5 vs 167.5±11.2 mg/dl,P<0.0001),hyper non-esterified fatty acids(658.9±84.5 vs 1501±237.3 mmol/L,P<0.0001)and hyper-O-GlcNAcylation(P<0.01).Consistent with the animal experiment,cell experiment showed the hyper-O-GlcNAcylation of palmitic acid treatment group compared with the control group(P<0.01);In addition,CCK-8 assay showed that the cardioprotection of insulin was significantly disappeared after palmitic acid treatment(P<0.05).2.In animal experiment,compared with I/R+Ins group,O-GlcNAcylation level(P<0.05),myocardial infarct size(P<0.01),caspase-3 expression(P<0.01),LDH activity was increased(P<0.001)and Bcl2/Bax was decreased(P<0.05)in I/R+Ins+GlcN group.In cell experiment,compared with H/R+Ins group,H/R+Ins+GlcN group had lower cell activity(P<0.01)and higher LDH activity(P<0.001).3.In animal experiment,compared with I/R+Ins group,I/R+Ins+GlcN group had higher arrhythmia score after reperfusion(P<0.01),the number of episode of PVC and duration of PVC was increased,with no significant difference(P<0.08,P<0.07).4.In animal experiment,compared with I/R+Ins group,the content of MDA was increased(P<0.001,P<0.06)and the activity of SOD was decreased(P<0.05,P<0.05)in serum and myocardial tissue of I/R+Ins+GlcN group.5.Compared with db/m mice,the levels of O-GlcNAcylation(P<0.01)and the Akt basic phosphorylation(P<0.01)were increased in db/db mice.Meanwhile,in animal experiment,compared with the control group,the levels of O-GlcNAcylation(P<0.0001)and the Akt basic phosphorylation(P<0.05)were also increased after glucosamine pretreatment.In addition,compared with I/R+Ins group,the O-GlcNAcylation level was increased(P<0.05)and the Akt phosphorylation level was decreased(P<0.001)in I/R+Ins+GlcN group.6.In cell experiment,compared with PA group,the O-GlcNAcylation level was decreased in PA+DON group(P<0.01),and compared with H/R+PA+Ins group,the phosphorylation level of Akt and cell activity in H/R+PA+DON+Ins group were increased(P<0.01,P<0.01).Conclusion:1.The level of O-GlcNAcylation in spontaneously obese db/db mice and insulin resistance H9c2 cells model induced by palmitic was significantly increased.Meanwhile,palmitic treatment significantly attenuated the cardioprotective of insulin,suggesting that hyper-O-GlcNAcylation may mediate the disappearance of insulin cardioprotective in obese mice.2.Hyper-O-GlcNAcylationn induced by glucosamine significantly attenuated insulin cardioprotection against ischemia/reperfusion injury,which is related to the glucosamine induced hyper-O-GlcNAcylation increase the basic phosphorylation level of Akt and decrease the insulin-stimulated Akt phosphorylation.