Activation of IKKβ by glucose is necessary and sufficient to impair insulin signaling and nitric oxide production in endothelial cells

Abstract

Hyperglycemic impairment of nitric oxide (NO) production by endothelial cells is implicated in the effect of diabetes to increase cardiovascular disease risk, but the molecular basis for this effect is unknown. In skeletal muscle, diabetes induces activation of inhibitor kappaB kinase (IKKβ), a key cellular mediator of the response to inflammatory stimuli, and this impairs insulin signal transduction via the insulin receptor substrate-phosphatidylinositol 3-OH kinase (IRS-1/PI3-kinase) pathway. Since activation of endothelial nitric oxide synthase (eNOS) is dependent on IRS-1/PI3-kinase signaling, we hypothesized that activation of IKKβ may contribute to the effect of glucose to impair NO production. Here, we show that exposure of bovine aortic endothelial cells to high glucose (25 mM) for 24 h impaired insulin-mediated tyrosine phosphorylation of IRS-1, serine phosphorylation of Akt, activation of eNOS, and production of NO. High glucose treatment also activated IKKβ, and pretreatment with aspirin, a pharmacological inhibitor of IKKβ, prevented both glucose-induced IKKβ activation and the effect of high glucose to impair insulin-mediated NO production. These adverse responses to glucose were also blocked by selective inhibition of IKKβ signaling via overexpression of a kinase-inactive form of the enzyme. Conversely, overexpression of wild-type IKKβ recapitulated the deleterious effect of high glucose on insulin-mediated activation of eNOS. These data demonstrate that activation of IKKβ plays a critical and novel role to mediate the deleterious effects of high glucose on endothelial cell function.

Introduction

Insulin-resistant states such as obesity and diabetes are associated with endothelial dysfunction that, in turn, contributes to the increased risk of vascular disease associated with these conditions. The molecular mechanism linking insulin resistance and endothelial dysfunction is not known. Endothelial cells express insulin receptors and, like other insulin-responsive tissues, insulin activates the insulin receptor substrate-1/phosphatidylinositol 3-kinase (IRS-1/PI3-kinase) pathway in these cells, suggesting that endothelial cells are targets for the action of insulin [1], [2]. Unlike other insulin-responsive cell types, however, a prominent feature of the response of endothelial cells to insulin is the activation of endothelial nitric oxide synthase (eNOS) and consequent increase of NO production. Since conditions that impair endothelial NO production, including diabetes, obesity and insulin resistance, can promote atherosclerosis, efforts to clarify the cellular mechanisms responsible for endothelial dysfunction in these conditions are a high priority.

Following insulin binding, the insulin receptor undergoes tyrosine autophosphorylation, which leads to tyrosine phosphorylation of IRS-1 and subsequent activation of PI3-kinase. The resultant generation of phosphatidylinosital triphosphate (PIP-3) [3], ultimately leads to activation of several serine kinases including Akt and subsequently, of eNOS by phosphorylation of ser1179 [4]. Since IRS-1/PI3-kinase signaling appears to be required for activation of eNOS by insulin or by shear stress [5], impaired signaling via this pathway is hypothesized to induce a global impairment of NO production in endothelial cells. Based on these considerations, we hypothesized that reduced IRS-1/PI3-kinase signal transduction plays a prominent role to mediate the deleterious effects of diabetes on endothelial cell function.

In skeletal muscle, activation of inhibitor kappaB kinase (IKKβ), a key cellular mediator of the response to inflammatory stimuli, is associated with impaired IRS-1/PI3-kinase signaling and is implicated in the pathogenesis of insulin resistance. Specifically, diabetes-induced increases in the levels of free fatty acids (FFA) and tumor necrosis factor (TNF-α) have been shown to increase serine phosphorylation of IRS-1 in skeletal muscle via the activation of IKKβ [6], [7]. This in turn impairs insulin-dependent tyrosine phosphorylation of IRS-1 and PI3-kinase activation, and hence attenuates downstream cellular responses to insulin. Combined with evidence that pharmacological inhibition of IKKβ by salicylates improves insulin sensitivity in rodent models of diabetes, activation of IKKβ appears to constitute a critical link between cellular inflammatory pathways and insulin resistance.

In the current work, we investigated the hypothesis that high glucose levels impair insulin signal transduction in endothelial cells via a mechanism involving IKKβ activation. Here we show that after incubation for 24 h in media containing 25 mM of glucose, IKKβ activity is increased in bovine aortic endothelial cells (BAEC) and that this effect is associated with impaired insulin stimulation of both IRS-1/PI3-kinase and NO production. Our finding that pretreatment with aspirin, a pharmacologic inhibitor of IKKβ, reverses this impairment of insulin-dependent NO production implicates IKKβ as a mediator of this response. More direct evidence in support of this conclusion stems from our finding that expressing a dominant negative construct of IKKβ protects BAEC from the deleterious effects of high glucose, while overexpression of a wild-type IKKβ recapitulates the effects of high glucose on signaling via IRS-1/pAkt/peNOS. Taken together, these findings support the hypothesis that activation of IKKβ mediates the negative effects of high glucose on insulin signal transduction and NO production by endothelial cells.