Selective Enhancement of Insulin Sensitivity in the Endothelium In Vivo Reveals a Novel Proatherosclerotic Signaling Loop

Abstract

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

Endothelial SHIP2 Suppresses Nox2 NADPH Oxidase–Dependent Vascular Oxidative Stress, Endothelial Dysfunction, and Systemic Insulin Resistance

“…Plasma insulin concentrations were determined by ELISA (CrystalChem). Free fatty acids and triglycerides were measured in fasting plasma using colorimetric assays (Abcam) (12).…”

“…The mean reading with catalase was subtracted from the mean without catalase, and this value was plotted on a simultaneously prepared H 2 O 2 standard curve. Dry tissue mass was used for normalization (12).…”

“…We used lucigenin (5 mmol/L), enhanced chemiluminescence to measure NADPH-dependent superoxide production in pulmonary ECs, as previously described (12). All experiments were performed in triplicate.…”

Response by Viswambharan and Kearney to Letter Regarding Article, “Selective Enhancement of Insulin Sensitivity in the Endothelium In Vivo Reveals a Novel Proatherosclerotic Signaling Loop”

“…1 Rask-Madsen et al have made a major and sustained contribution to contemporary understanding of insulin signaling in endothelial cells. [2][3][4] In the case of the seminal report of King et al 5,6 describing intracellular selectivity of insulin resistance in endothelial cells from obese rodents, they without doubt changed the way we think about insulin signaling in the endothelium in obesity and type 2 diabetes mellitus.…”

Letter by Rask-Madsen et al Regarding Article, “Selective Enhancement of Insulin Sensitivity in the Endothelium In Vivo Reveals a Novel Proatherosclerotic Signaling Loop”

“…In a paper published online on December 5, Viswambharan et al presents interesting data on atherosclerosis in mice with overexpression of the insulin receptor in vascular endothelial cells [1]. Their work adds to a growing literature on the importance of insulin signaling in endothelial cells with impact on blood flow regulation, whole-body metabolism, angiogenesis and atherogenesis.…”