Insulin resistance is characterized by excessive endothelial cell generation of potentially cytotoxic concentrations of reactive oxygen species. We examined the role of NADPH oxidase (Nox) and specifically Nox2 isoform in superoxide generation in two complementary in vivo models of human insulin resistance (endothelial specific and whole body). Using three complementary methods to measure superoxide, we demonstrated higher levels of superoxide in insulin-resistant endothelial cells, which could be pharmacologically inhibited both acutely and chronically, using the Nox inhibitor gp91ds-tat. Similarly, insulin resistance–induced impairment of endothelial-mediated vasorelaxation could also be reversed using gp91ds-tat. siRNA-mediated knockdown of Nox2, which was specifically elevated in insulin-resistant endothelial cells, significantly reduced superoxide levels. Double transgenic mice with endothelial-specific insulin resistance and deletion of Nox2 showed reduced superoxide production and improved vascular function. This study identifies Nox2 as the central molecule in insulin resistance–mediated oxidative stress and vascular dysfunction. It also establishes pharmacological inhibition of Nox2 as a novel therapeutic target in insulin resistance–related vascular disease.
OBJECTIVEIn mice, haploinsufficiency of the IGF-1 receptor (IGF-1R+/−), at a whole-body level, increases resistance to inflammation and oxidative stress, but the underlying mechanisms are unclear. We hypothesized that by forming insulin-resistant heterodimers composed of one IGF-1Rαβ and one insulin receptor (IR), IRαβ complex in endothelial cells (ECs), IGF-1R reduces free IR, which reduces EC insulin sensitivity and generation of the antioxidant/anti-inflammatory signaling radical nitric oxide (NO).RESEARCH DESIGN AND METHODSUsing a number of complementary gene-modified mice with reduced IGF-1R at a whole-body level and specifically in EC, and complementary studies in EC in vitro, we examined the effect of changing IGF-1R/IR stoichiometry on EC insulin sensitivity and NO bioavailability.RESULTSIGF-1R+/− mice had enhanced insulin-mediated glucose lowering. Aortas from these mice were hypocontractile to phenylephrine (PE) and had increased basal NO generation and augmented insulin-mediated NO release from EC. To dissect EC from whole-body effects we generated mice with EC-specific knockdown of IGF-1R. Aortas from these mice were also hypocontractile to PE and had increased basal NO generation. Whole-body and EC deletion of IGF-1R reduced hybrid receptor formation. By reducing IGF-1R in IR-haploinsufficient mice we reduced hybrid formation, restored insulin-mediated vasorelaxation in aorta, and insulin stimulated NO release in EC. Complementary studies in human umbilical vein EC in which IGF-1R was reduced using siRNA confirmed that reducing IGF-1R has favorable effects on NO bioavailability and EC insulin sensitivity.CONCLUSIONSThese data demonstrate that IGF-1R is a critical negative regulator of insulin sensitivity and NO bioavailability in the endothelium.
Rationale: In the endothelium, insulin stimulates endothelial nitric oxide synthase (eNOS) to generate the anti-atherosclerotic signalling radical NO. Insulin resistant type 2 diabetes is associated with reduced NO availability and accelerated atherosclerosis. The effect of enhancing endothelial insulin sensitivity on NO availability is unclear. Objective: To answer this question we generated a mouse with endothelial cell (EC)-specific over-expression of the human insulin receptor (hIRECO) using the Tie2 promoter-enhancer. Methods and Results: hIRECO demonstrated significant endothelial dysfunction measured by blunted endothelium-dependent vasorelaxation to acetylcholine which was normalized by a specific Nox2 NADPH oxidase inhibitor. Insulin-stimulated phosphorylation of Akt was increased in hIRECO EC as was Nox2 NADPH oxidase-dependent generation of superoxide, whereas insulin and shear stress-stimulated eNOS activation were blunted. Phosphorylation at the inhibitory residue Y657 of eNOS and expression of PYK2 which phosphorylates this residue were significantly higher in hIRECO EC. Inhibition of PYK2 improved insulin and shear-induced eNOS activation in hIRECO EC. Conclusions: Enhancing insulin sensitivity specifically in EC leads to a paradoxical decline in endothelial function, mediated by increased tyrosine phosphorylation of eNOS and excess Nox2 derived superoxide. Increased EC insulin sensitivity leads to a pro-atherosclerotic imbalance between NO and superoxide. Inhibition of PYK2 restores insulin-and shearinduced NO production. This study demonstrates for the first time that increased endothelial insulin sensitivity leads to a pro-atherosclerotic imbalance between NO and superoxide.Key Words: Insulin; Insulin Resistance; Endothelial Dysfunction; PYK2; Reactive oxygen Species; Superoxide Non-standard Abbreviations and Acronyms: NO-nitric oxide, hIR-human Insulin Receptor; hIRECO-endothelial cell (EC)-specific over-expression of the human insulin receptor; EC-endothelial cells; PEC-pulmonary endothelial cells; PYK2-proline-rich tyrosine kinase 2; Akt-protein kinase B; ACh-acetylcholine; PE-phenylephrine; SNP-sodium nitroprusside; eNOS-endothelial nitric oxide synthase.3
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