Original article
Insulin inhibits leukocyte–endothelium adherence via an Akt-NO-dependent mechanism in myocardial ischemia/reperfusion

https://doi.org/10.1016/j.yjmcc.2009.07.010Get rights and content

Abstract

Clinical evidence indicates that intensive insulin therapy during critical illness protects the endothelium and contributes to prevention of organ failure and death but the mechanisms involved remain unclear. This study was designed to test the hypothesis that insulin inhibits adherence of polymorphonuclear leukocytes (PMNs) to endothelial cells in myocardial ischemia/reperfusion (MI/R) and to investigate the underlying mechanisms. Anesthetized rabbits were subjected to MI/R (45 min/4 h) and randomly received saline, glucose-insulin-potassium (GIK) or GK respectively (2 mL/kg/h, i.v.). In vitro study was performed on cultured endothelial cells subjected to simulated ischemia/reperfusion. In vivo treatment with GIK but not GK attenuated myocardial injury as evidenced by reduced plasma creatine kinase activity, myocardial apoptosis and infarct size in MI/R rabbits compared with the saline group. Interestingly, GIK but not GK significantly decreased coronary endothelial expression of P-selectin and intercellular adhesion molecule-1 (ICAM-1), inhibited adherence of PMNs to coronary endothelium (107.7 ± 7.4 vs. 155.0 ± 9.2 PMNs/mm2 in saline group, n = 8, P < 0.01), and therefore decreased myocardial PMNs accumulation. In cultured endothelial cells subjected to simulated ischemia/reperfusion, insulin (10 7 M) increased Akt activity and eNOS phosphorylation with subsequent NO production, and concurrently exerted an anti-adhesive effect as manifested by reduced endothelial P-selectin and ICAM-1 surface expression and PMNs adherence (13.7 ± 1.3% vs. 22.2 ± 1.9% in vehicle, n = 9, P < 0.01), all of which are abolished by the specific Akt inhibitor. Furthermore, inhibition of insulin-stimulated NO production using either the selective eNOS inhibitor cavtratin or the NOS inhibitor L-NAME blocked the anti-adhesive effect of insulin. These results demonstrate that insulin reduces endothelial P-selectin and ICAM-1 expression, and thus inhibits leukocyte–endothelium adherence in MI/R rabbit hearts. The anti-adhesive property by insulin may be mediated by the Akt-mediated and NO-dependent pathway.

Introduction

Recently, insulin as an anti-inflammatory agent has been reevaluated by specialists in cardiovascular diseases and critically illness. Intensive insulin therapy was found to protect the endothelium of critically ill patients and therefore contributed to prevention of organ failure and death [1]. In patients with acute myocardial infarction (AMI), insulin treatment decreased plasma levels of high-sensitivity C-reactive protein and serum amyloid A [2]. Our previous study has demonstrated that insulin inhibits tumor necrosis factor-α (TNF-α) production in myocardial ischemia/reperfusion (MI/R) [3]. Induced by pro-inflammatory cytokines such as TNF-α, adherence of polymorphonuclear leukocytes (PMNs) to endothelial cells is an early and requisite event in ischemia/reperfusion-induced inflammatory injury. However, whether insulin may inhibit PMNs adherence to endothelial cells in MI/R remains unknown.

The initial interaction between PMNs and endothelial cells is mediated by P-selectin and E-selectin expressed on the endothelial cell surface and L-selectin and P-selectin glycoprotein ligand-1 located on the PMNs surface. This selectin-mediated interaction results in the rolling of PMNs along the vessel wall and upregulation of β2-integrin adhesion molecules on the PMNs surface. Upregulated CD11/CD18 adhesion molecules then bind to their counterreceptors on endothelial cells, primarily intercellular adhesion molecule-1 (ICAM-1), resulting in their firm adhesion and transmigration through the vascular wall. Although abundant evidence has shown that adhesion molecules expression by coronary endothelial cells is marked upregulated following MI/R [4], [5], whether insulin may decrease endothelial adhesion molecules expression in MI/R has not been determined.

Nitric oxide (NO) is reported to decrease endothelial ICAM-1 mRNA and surface expression, which results in reduction in PMNs adhesion to endothelium stimulated by TNF-α [6]. Upregulation of endothelial nitric oxide synthesis (eNOS) protects against inflammatory damage involving suppression of vascular cell adhesion molecule expression and thus preventing excessive leukocytes tissue infiltration following ischemia/reperfusion [7]. 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 [8], [9]. Nonetheless, it has not been established whether insulin may inhibit endothelial adhesion molecules expression through increasing NO release.

Therefore, in the present study, we investigated (1) whether insulin may decrease endothelial adhesion molecules expression, inhibit leukocyte–endothelium adherence and thus reduce myocardial injury in MI/R; (2) and if so, the possible mechanism(s) involved in insulin's anti-adhesive effect in MI/R.

Section snippets

Experiment protocol

Male New Zealand white rabbits weighing 2.5–3.0 kg at the beginning of the experiments were used. The rabbits were allowed access to food and water, and maintained at 23 ± 2 °C under a cycle of 12-h light/12-h darkness. This study was performed in accordance with the National Institutes of Health guidelines for the use of experimental animals, and all animal protocols were approved by the Fourth Military Medical University Committee on Animal Care.

Rabbits were anesthetized with sodium

Insulin reduced myocardial injury after ischemia/reperfusion

Blood glucose concentration was determined throughout the experiment (Fig. 1). Infusion of GK during reperfusion significantly increased blood glucose concentration (n = 8, P < 0.01 vs. vehicle) in MI/R rabbits but there was no difference between the GIK-treated and vehicle-treated groups at the end of 4 h reperfusion.

As shown in Fig. 2, 45 min of ischemia and 4 h of reperfusion resulted in significant myocardial injury as evidenced by increased plasma CK activity (A), myocardial infarct size (B)

Discussion

Two novel findings have been made in the present study. First, we have provided in vivo direct evidence that insulin exerts an anti-adhesive effect as manifested by reduced expression of coronary endothelial P-selectin and ICAM-1 and decreased adherence of PMNs to endothelium, and thus attenuates PMNs accumulation in ischemic/reperfused rabbit hearts. Second, using cultured endothelial cells subjected to simulated ischemia/reperfusion, we have demonstrated for the first time that the

Acknowledgments

This study was supported by grants from National Science Fund for Outstanding Young Investigators (No. 30625033), National Natural Science Foundation of China (NSFC, No. 30800447, 30800376, 30728023), and National Basic Research Program of China (973 Program, No. 2007CB512106).

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    These authors contributed equally to this work.

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