Anti-inflammatory Effect Of Insulin In The Ischemic/reperfused Heart: Role Of Akt And ENOS Phosphorylation

Background and objectivesIntensive insulin therapy has been demonstrated to decrease the overall mortality and prevent the incidence of multi-organ failure in the critically ill patients. Experimental evidence revealed that insulin attenuates the systemic inflammatory response in rats subjected to thermal trauma or endotoxin. A recent clinical study reported that insulin decreases plasma levels of high-sensitivity C-reactive protein (CRP) and serum amyloid A (SAA), thus exerting an anti-inflammatory effect in patients with acute myocardial infarction (AMI). These observations have implicated that insulin functions as an anti-inflammatory molecule although the underlying mechanisms remain elusive. Tumor necrosis factor (TNF)-α, an important and potent pro-inflammatory cytokine, triggers the production of other pro-inflammatory cytokines and expression of adhesion molecules. Elevated levels of serum TNF-αhave been reported in patients with bacterial or viral infection-induced inflammation, ischemia/reperfusion injury, chronic heart failure and viral myocarditis. However, whether insulin decreases inflammatory response, especially TNF-αinduction, in myocardial ischemia/reperfusion (MI/R) injury has not been identified.Nitric oxide (NO) is reported exert an anti-inflammatory effect through suppressing adhesion molecule expression and cytokine releases. Ample evidence has implicated a critical role for NO in the pathogenesis of heart diseases in critically ill patients. Among all three NO synthase isozymes, endothelial NO synthase (eNOS) is reported to play a compensatory protective role but is downregulated in sepsis. Results from our laboratory as well as others have demonstrated that insulin facilitates eNOS phosphorylation and consequently NO release through a phosphatidylinositol 3′-kinase (PI3K)-Akt-dependent pathway. However, whether the insulin-stimulated eNOS phosphorylation and subsequent NO release play a role in the anti-inflammatory effect of insulin has not been established.Inflammatory response and cytokine elaboration are particularly active after myocardial infarction and contribute to cardiac function and eventual host outcome. Cytokines such as TNF-αreleased after myocardial ischemic injury can acutely regulate myocyte survival or deaths and trigger subsequent cellular inflammatory response. On the other hand, insulin has been demonstrated to protect injured myocardium and improve clinical outcome in endotoxic shock, stroke and AMI. However, it is not known whether the insulin-elicited anti-inflammatory property contributes to the cardioprotective and prosurvival effects of insulin in the critically ill. Therefore, the objectives of the present study are:1. To determine whether treatment with insulin may inhibit TNF-αinduction and reduce acute inflammatory response in MI/R.2. If so, to investigate the possible mechanism(s) involved in insulin-induced TNF-αreduction in MI/R.3. To elucidate whether the anti-inflammatory effect afforded by insulin may contribute to its cardioprotection in MI/R.Methods 1. Anesthetized rats were subjected to MI/R (30 min/3 h) and were randomized to receive one of the following treatments: (1) Sham; (2) Vehicle (saline); (3) GIK (glucose: 250 g/L, insulin: 60 U/L, potassium: 80 mmol/L, intravenous infusion at 4 ml/kg/h for 3 h, beginning 5 min before reperfusion); (4) GK.2. At the end of reperfusion, blood glucose by a glucosemeter and plasma insulin concentration by a commercial radioimmunoassay kit were measured.3. Serum TNF-αand IL-10 were detected using the enzyme-linked immunosorbent assay (ELISA) kits. Serum creatine kinase (CK) activity, myocardial myeloperoxidase (MPO) activity and TNF-αproduction were also deteced at the end of reperfusion.4. Myocardial infarct size was determined by means of a double-staining technique and a digital imaging system after reperfusion.5. In vitro study was performed on cultured cardiomyocytes subjected to simulated ischemia/reperfusion (SI/R, 2 h/4 h) and randomly exposed to one of the following treatments: vehicle, insulin (10-7 mol/L), insulin plus the PI3K inhibitor wortmannin (10 nmol/L), insulin plus the eNOS inhibitor L-NAME (100μmol/L), neutralizing anti-TNF-αIgG or nonimmune IgG (5μg/ml) and insulin plus neutralizing anti-TNF-αIgG.6. At the end of experiment, cell viability was determined by the trypan blue exclusion assay, culture medium was collected for measurement of TNF-αand total nitric oxide (NOx) production, and fluorescein isothiocyanate-conjugated (FITC)-Annexin V and propidium iodide (PI) were used to identify apoptotic cells.7. Akt and eNOS expression and phosphorylation were determined by western blot at the end of experiment. Results1. Compared with the vehicle group, both GIK and GK treatments increased plasma insulin concentration at the end of 3 h reperfusion. Infusion of GK significantly increased blood glucose concentration but there was no difference between GIK and vehicle groups.2. MI/R (30 min/3 h) resulted in myocardial injury as evidenced by increased infarct size and serum CK activity. Administration of GIK during reperfusion significantly reduced both infarct size (37.6±2.2% vs.49.5±2.8% in Vehicle, P<0.05, n=8) and CK activity (P<0.01, n=8) compared with the vehicle group. However, no difference between GK and vehicle groups was noted.3. Compared with the vehicle group, treatment with GIK significantly decreased serum pro-inflammatory cytokine TNF-αand increased anti-inflammatory cytokine IL-10 levels after reperfusion while treatment with GK had no effect on either TNF-αor IL-10 level.4. Ischemia/reperfusion (30 min/3 h) caused elevated myocardial TNF-αlevel and increased MPO activity. Compared with the vehicle group, treatment with GIK but not with GK significantly reduced MI/R-induced elevation in both TNF-αlevel (19.73±1.45 vs. 28.31±2.31 pg/mg protein in Vehicle, P<0.01, n=8) and MPO activity (P<0.01, n=8).5. Simulated ischemia (2h) and reperfusion (4h) resulted in elevated TNF-αinduction in cultured cardiomyocytes. Compared with the vehicle group, insulin treatment reduced TNF-αinduction and resulted in a 2.6-fold increase in Akt phosphorylation, a 2.1-fold increase in eNOS phosphorylation and 53.4% increase in NO production. Inhibition of insulin-stimulated NO production by either wortmannin or L-NAME blocked TNF-αreduction afforded by insulin. 6. Simulated ischemia (2 h) and reperfusion (4 h) decreased cell viability and increased apoptosis among cardiomyocytes compared with the normal culture condition. Treatment with insulin or neutralizing anti-TNF-αIgG but not with nonimmune IgG increased cell viability and decreased the number of apoptotic cells. Moreover, insulin plus neutralizing anti-TNF-αIgG treatment did not further alter cell viability and apoptosis compared with the insulin-treated group (both P>0.05).ConclusionsWe have demonstrated for the first time that insulin reduces ischemia/reperfusion-induced TNF-αproduction in cardiomyocytes through an Akt-activated and eNOS-NO dependent pathway, which resuts in cardioprotection in MI/R. The anti-inflammatory property elicited by insulin may contribute to its cardioprotective and prosurvival effects in AMI and critical illness.