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Background— Apolipoprotein CIII (apoCIII) is a component of some triglyceride-rich very-low-density and low-density lipoprotein and is elevated in dyslipidemia with insulin resistance and the metabolic syndrome. We previously reported that apoCIII directly activates proinflammatory and atherogenic signaling in vascular endothelial cells through protein kinase C-β (PKCβ). Because PKCβ impairs the response of vascular endothelial cells to insulin, we tested the hypothesis that apoCIII affects insulin signaling in vascular endothelial cells and its function in vitro and in vivo. Methods and Results— ApoCIII inhibited insulin-induced tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), decreasing phosphatidylinositol 3-kinase (PI3K)/Akt activation in human umbilical vein endothelial cells. These effects of apoCIII led to reduced endothelial nitric oxide synthase (eNOS) activation and NO release into the media. ApoCIII activated PKCβ in human umbilical vein endothelial cells, resulting in IRS-1 dysfunction via serine phosphorylation. ApoCIII also activated mitogen-activated protein kinase through PKCβ. The impaired insulin signaling was restored by PKCβ inhibitor or MEK1 inhibitor. ApoCIII-rich very-low-density lipoprotein and apoCIII impaired insulin signaling in the aorta of C57BL/6J mice and in human umbilical vein endothelial cells, which was recovered by PKCβ inhibitor. They also inhibited endothelium-dependent relaxation of the aortas of C57BL/6J mice. In summary, apoCIII in very-low-density lipoprotein impaired insulin stimulation of NO production by vascular endothelium and induced endothelial dysfunction in vivo. This adverse effect of apoCIII was mediated by its activation of PKCβ, which inhibits the IRS-1/PI3K/Akt/eNOS pathway. Conclusion— Our results suggest that apoCIII is a crucial link between dyslipidemia and insulin resistance in vascular endothelial cells with consequential deleterious effects on their atheroprotective functions.

Objective Apolipoprotein (apo) CIII, a prominent component of atherogenic dyslipidemia, is elevated in patients with type2 diabetes and insulin resistance, and independently predicts cardiovascular disease (CVD). Endothelial dysfunction is an important component of the pathophysiology of atherosclerosis, and is associated with insulin resistance. We previously reported that apoCIII has a direct effect on vascular endothelium, activating endothelial cells to recruit monocytes through PKCβ and NF-κB-dependent mechanisms. In the present study, we investigated the effect of apoCIII on insulin-dependent endothelial nitric oxide synthase (eNOS) function in endothelial cells and in the aortas of C57BL/6J mice. Methods and Results ApoCIII treatment (100μg/ml, 30 minutes) inhibited insulin-induced ISR-1/phosphatidylinositol 3-kinase (PI3K)/Akt activation in human umbilical vein endothelial cells (HUVECs). Furthermore, apoCIII reduced insulin-stimulated eNOS activation and NO release into the media. ApoCIII induced PKCβII activation in HUVECs, resulting in IRS-1 phosphorylation at pSer 616 and ERK activation. Impaired insulin-stimulated eNOS activation and NO production were restored by PKCβ inhibitor and MEK1 inhibitor to a lesser extent. In addition, treatment of C57BL/6J mice with apoCIII resulted in impaired insulin-dependent activation of PI3K/Akt/eNOS pathway in the aorta. Pretreatment with PKCβ inhibitor attenuated inhibitory effects of apoCIII. ApoCIII resides on VLDL fraction in the plasma, especially in the postprandial state. Injection of apoCIII-rich VLDL, but not apoCIII-deficient VLDL, also impaired these processes. Conclusion Our data suggest that apoCIII impairs insulin stimulation of NO production by vascular endothelium. This adverse effect of apoCIII is mediated by its activation of PKCβII which inhibits the IRS-1/PI3K/Akt/eNOS pathway. These results indicate that apoCIII not only modulates lipoprotein metabolism, but also may directly contribute to the development of diabetic complications through endothelial dysfunction. Finally, our results may point to a novel mechanism that links dyslipidemia, insulin resistance and endothelial dysfunction.