Free fatty acids induce endothelial dysfunction and activate protein kinase C and nuclear factor-κB pathway in rat aorta Hua Li a , Hongliang Li a , Yige Bao b , Xiangxun Zhang c , Yerong Yu a, ⁎ a Department of Endocrinology and Metabolism, West China Hospital, Sichuan University, Chengdu City, Sichuan Province, PR China b West China Medical School, Sichuan University, Chengdu City, Sichuan Province, PR China c Lab. of Endocrinology and Metabolism, West China Hospital, Sichuan University, Chengdu City, Sichuan Province, PR China abstract article info Article history: Received 23 February 2010 Received in revised form 6 June 2010 Accepted 4 July 2010 Available online 6 August 2010 Keywords: Free fatty acids Endothelial dysfunction Protein kinase C Nuclear factor-κB Background: Insulin resistance is associated with an inappropriate elevation of plasma free fatty acids (FFAs) and endothelial dysfunction. In this study, we asked if elevated circulating FFA levels led to impaired insulin signaling and endothelial dysfunction in-vivo via activation of PKC-mediated inflammatory pathways. Methods: Sprague–Dawley (S–D) rats were infused with 1) 20% intralipid + heparin (FFA group) or 2) saline alone (Control group) for 6 h. The intact aorta thoracica and aorta abdominalis were then removed. Aortic rings were isolated and evaluated for endothelial-dependent and non-dependent relaxation in an organ bath. The activities of eNOS and PKC were measured in endothelial homogenates prepared from endothelial cells harvested from the aorta. The expression levels of insulin signaling molecules IRS-1, Akt, eNOS, ERK1/ ERK2, PKC-α, NFκB-p65 subunit and IκB-α in rat aortic endothelium were determined by immunohis- tochemistry and Western blot. Results: Elevation of FFAs resulted in a 35.9% reduction in the response to acetylcholine (p b 0.01), a 26% decline in plasma NOx levels (p b 0.05), a 53% decrease in eNOS activity and a 34 ± 9% inhibition in IRS-1 tyrosine phosphorylation (p b 0.05). We also found a 46% decrease in Akt phosphorylation and a 36% decrease in eNOS phosphorylation. FFA-induced endothelial insulin resistance was associated with 82% increase in total membrane-associated PKC activity, a 1.7-fold increase in total PKC-α protein, 1.29-fold decrease in IκB-α expression levels and 1.47-fold increase in NF-κB p65 subunit expression in rat aortic endothelium. Conclusion: The molecular mechanisms underlying FFA-induced endothelial insulin resistance and eNOS inhibition may provide an important link implicating the PKC and IκB-α/NF-κB pathways in FFA-mediated inhibition of vascular insulin signaling. © 2010 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Cardiovascular diseases are the leading cause of morbidity and mortality in patients with insulin resistance syndrome. A number of disorders associated with endothelial dysfunction, including diabetes, obesity, hypertension, coronary heart disease, and atherosclerosis, are characterized by insulin resistance [1]. Conversely, endothelial dysfunc- tion is often present in patients with insulin resistance. Understanding the mechanisms of endothelial dysfunction in insulin-resistant patients is important for the development of novel therapeutic strategies to reduce the morbidity and mortality of cardiovascular disease. Endothelial dysfunction has been described as an impairment in the generation of nitric oxide (NO) and its function as a vasodilator and vascular homeostatic agent. Insulin enhances vasculature via the activation and expression of endothelial nitric oxide synthase (eNOS), and thus NO bioavailability [2,3]. Insulin resistance has been reported to induce alterations in insulin signaling within the endothelium, which could account for reduced NO production. Reduced NO action could, in turn, be instrumental in accelerating the process of atherosclerotic disease. Insulin signaling in endothelial cells has been shown to be similar to that seen in classic insulin-responsive cells such as skeletal muscle cells, hepatocytes, and adipocytes [4]. Mechanisms of impaired insulin signaling in these better-character- ized insulin-responsive cells are therefore likely to help dissect insulin resistance associated endothelial dysfunction, which is not well understood. Elevation in circulating free fatty acids (FFAs) is a characteristic metabolic abnormality of the insulin-resistant state. The skeletal muscle, liver and endothelium of insulin-resistant patients are constantly exposed to high concentrations of FFAs, which are known to interfere with insulin signaling in the phosphatidylinositol 3-kinase (PI3-kinase) pathway via mechanisms that involve en- hanced oxidative stress, activation of the proinflammatory NF-κB International Journal of Cardiology 152 (2011) 218–224 ⁎ Corresponding author. #37 Guo Xue Road, Chengdu, China. 610041, Department of Endocrinology and metabolism, West China Hospital. Tel.: + 86 28 85422357; fax: + 86 28 85423459. E-mail address: yerongyu@scu.edu.cn (Y. Yu). 0167-5273/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2010.07.019 Contents lists available at ScienceDirect International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard