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Objective-Free fatty acids (FFA) are commonly elevated in diabetes and obesity and have been shown to impair nitric oxide (NO) production by endothelial cells. However, the signaling pathways responsible for FFA impairment of NO production in endothelial cells have not been characterized. Insulin receptor substrate-1 (IRS-1) regulation is critical for activation of endothelial nitric oxide synthase (eNOS) in response to stimulation by insulin or fluid shear stress. Methods and Results-We demonstrate that insulin-mediated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt, eNOS, and NO production are significantly inhibited by treatment of bovine aortic endothelial cells with 100 mol/L FFA composed of palmitic acid for 3 hours before stimulation with 100 nM insulin. This FFA preparation also increases, in a dose-dependent manner, IKK␤ activity, which regulates activation of NF-B, a transcriptional factor associated with inflammation. Similarly, elevation of other common FFA such as oleic and linoleic acid also induce IKK␤ activation and inhibit insulin-mediated eNOS activation. Overexpression of a kinase inactive form of IKK␤ blocks the ability of FFA to inhibit insulin-dependent NO production, whereas overexpression of wild-type IKK␤ recapitulates the effect of FFA on insulin-dependent NO production. Conclusions-Elevated levels of common FFA found in human serum activate IKK␤ in endothelial cells leading to reduced NO production, and thus may serve to link pathways involved in inflammation and endothelial dysfunction. Key Words: diabetes Ⅲ endothelial dysfunction Ⅲ endothelial nitric oxide synthase Ⅲ free fatty acids Ⅲ IKK␤ Ⅲ nitric oxide E ndothelial dysfunction is a hallmark of diabetic vascular disease and can be described as impairment in the generation and function of nitric oxide (NO) as a vasodilator and vascular homeostatic agent. Insulin's physiological action in the vasculature promotes vasodilation through increased NO production and resultant enhanced blood flow may couple metabolic and hemodynamic homeostasis. Insulin increases NO production in endothelial cells through an IRS-1 and phosphatidylinositol 3-kinase (PI3-kinase)-dependent pathway that results in phosphorylation of endothelial nitric oxide synthase (eNOS) by Akt in a calcium-independent manner. 1,2 Similarities have been demonstrated between insulin signaling in endothelial cells and in classic insulin-responsive cells such as skeletal muscle cells, hepatocytes, and adipocytes. Mechanisms of impaired insulin signaling in these better-characterized insulin responsive cells are likely to be relevant to endothelial dysfunction in diabetes that is not well understood. See page 889Metabolic abnormalities found in diabetes and obesity include increases in the circulating levels of cytokines such as tumor necrosis factor-␣ (TNF-␣) and metabolites such as free fatty acids (FFAs), diacylglycerol, and fatty acyl-coenzyme A. Resistance of target tissues to the effects of insulin has been attributed to alteration(s) in cellular resp...

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This stud3 addressed the et'l~cts of a commonplace stressful e~ent on interferon production and natural killer C NK) cell actl'-itx and numbers. The quantit.~ of interferons (IFN) produced b.~ concana~alin A stimulated leukoc.~tes obtained from 40 medical students during examinations ~as significantl.~ Io~er when compared ~ ith IFN levels produced by peripheral blood leukocytes IPBLs) taken 6 ~eeks earlier Ibaseline). In addition, three different assa.~s measuring NK cells also sho~'-ed slgnilicant decrements during examinations ,.,.hen compared with baseline samples. These assa.~s included la) I.~ sis of MOLT-4 target cells, Ib) percentage of anti-Leu-7 § INK) cells, and Ic) percentage of large granular I.vmphoc.~ tes. Self-report data documented the significantly greater d~stress associated with examinations in comparison x~ ith baseline samples. The data ha~e implications for immunosuppressive disorders and stress-associated illnesses.