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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The mechanism that regulates luteolytic PGF2 alpha secretion as stimulated by oxytocin is thought to involve induction of the inositol (1,4,5)-trisphosphate-diacylglycerol second messenger system, which mobilizes intracellular calcium and activates protein kinase C. In Experiment 1, endometrial explants taken from heifers on d 18.5 to 19.5 postestrus had increased PGF2 alpha secretion after treatment with 1 microM calcium ionophore A23187 to increase intracellular calcium, 100 nM phorbol 12-myristate 13-acetate to activate protein kinase C, and 100 nM oxytocin. The stimulatory effects of oxytocin and calcium ionophore A23187 plus phorbol 12-myristate 13-acetate did not differ from each other. In Experiment 2, endometrial explants taken from cows on d 18.5 to 19.5 postestrus had increased PGF2 alpha secretion after treatment with 0.2 and 2 microM thapsigargin to mobilize intracellular calcium that was sensitive to inositol (1,4,5)-trisphosphate. Secretion of PGF2 alpha was also increased by 100 nM oxytocin and was influenced by the interaction of thapsigargin and oxytocin such that 100 nM oxytocin did not further increase the secretion of PGF2 alpha in the presence of 2 microM thapsigargin. In Experiment 3, 100 nM oxytocin stimulated greater production of inositol trisphosphate and total inositol phosphates in the endometrium of cyclic cows than in the endometrium of pregnant cows on d 16.5 to 17.0 postestrus, although luteolysis was not yet initiated in the cyclic cows. These results are consistent with the hypothesis that the activation of the inositol (1,4,5)-trisphosphate-diacylglycerol second messenger system by oxytocin is involved in the stimulation of PGF2 alpha secretion from the endometrium during late diestrus in cows.