Activation of the AMP-Activated Protein Kinase (AMPK) by Nitrated Lipids in Endothelial Cells Yong Wu1,2, Yunzhou Dong 1 , Ping Song 1 , Ming-Hui Zou 1 * 1 Section of Molecular Medicine, Department of Medicine, Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America, 2 Department of Biochemistry, University of California, Riverside, Riverside, California, United States of America Abstract The AMP-activated protein kinase (AMPK) is an important regulator of endothelial metabolic and functional homeostasis. Here, we examined the regulation of AMPK by nitrated oleic acid (OA-NO2) and investigated the implications in endothelial function. Treatment of bovine aortic endothelial cells (BAECs) with OA-NO2 induced a significant increase in both AMPK- Thr172 phosphorylation and AMPK activity as well as upregulation of heme oxygenase (HO)-1 and hypoxia-inducible factor (HIF)-1a. Pharmacologic inhibition or genetic ablation of HO-1 or HIF-1a abolished OA-NO2-induced AMPK phosphorylation. OA-NO2 induced a dramatic increase in extracellular signal-regulated kinase (ERK)1/2 phosphorylation that was abrogated by the HO-1 inhibitor, zinc deuteroporphyrin IX 2,4-bis-ethylene glycol (ZnBG). Inhibition of ERK1/2 using UO126 or PD98059 reduced but did not abolish OA-NO2-induced HIF-1a upregulation, suggesting that OA-NO2/HO-1-initiated HIF-1a induction is partially dependent on ERK1/2 activity. In addition, OA-NO2 enhanced endothelial intracellular Ca2+, an effect that was inhibited by the HIF-1a inhibitor, YC-1, and by HIF-1a siRNA. These results implicate the involvement of HIF-1a. Experiments using the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor STO-609, the selective CaMKII inhibitor KN-93, and an isoform-specific siRNA demonstrated that OA-NO2-induced AMPK phosphorylation was dependent on CaMKKb. Together, these results demonstrate that OA-NO2 activates AMPK in endothelial cells via an HO-1–dependent mechanism that increases HIF-1a protein expression and Ca2+/CaMKKb activation. Citation: Wu Y, Dong Y, Song P, Zou M-H (2012) Activation of the AMP-Activated Protein Kinase (AMPK) by Nitrated Lipids in Endothelial Cells. PLoS ONE 7(2): e31056. doi:10.1371/journal.pone.0031056 Editor: Aimin Xu, University of Hong Kong, China Received October 19, 2011; Accepted December 31, 2011; Published February 17, 2012 Copyright: 2012 Wu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health Grants, a grant from the Juvenile Diabetes Research Foundation, a grant from the Oklahoma Center for the Advancement of Science and Technology (OCAST), a grant-in-aid from the American Diabetes Association, and funds from the Travis Endowed Chair in Endocrinology at the University of Oklahoma Health Science Center (all to Dr. Zou). Dr. M.H. Zou is a receipient of National Established Investigator Award of American Heart Association. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ming-hui-zou@ouhsc.edu Introduction Accumulating evidence indicates that reactive nitrogen species (RNS), which are inflammatory oxidants, mediate diverse physiologic and pathologic processes in cardiovascular, pulmo- nary, and neurodegenerative diseases [1]. RNS, such as perox- ynitrite (ONOO2), nitrogen-dioxide radical, and nitronium ion, which are formed from nitric oxide (NO), are important factors in complications of obesity and diabetes [2]. RNS react with unsaturated fatty acids to form relatively stable nitrated products (nitroalkene derivatives), including the abundant and clinically important nitrated oleic and linoleic acids [3]. Although this process occurs via various mechanisms, the common denominator is a proclivity for homolytic addition of nitrogen dioxide (NNO2) to the double bond, yielding an array of regio- and stereoisomers [4]. Nitrated unsaturated fatty acids (NO2-UFAs) represent a conver- gence of lipid and NO signaling and have emerged as a novel class of endogenously produced vascular signaling molecules [5]. The beneficial effects of NO2-UFAs include cGMP-dependent vessel relaxation, inhibition of inflammatory cell function, adaptive and anti-inflammatory cell responses, induction of heme oxygenase-1 (HO-1) expression, inhibition of nuclear factor (NF)-kB, and activation of peroxisome proliferator activated receptor (PPAR) and Keap1/Nrf2 [6]. Plasma free and esterified nitrated oleic acid (OA-NO2) concentrations have been reported to be 619652 and 3026369 nM, respectively, and these levels are approximately 50% greater than the level of nitrated linoleic acid (LNO2). The combined blood levels of the free and esterified fatty acid derivatives exceeds 1 mM [4]. The plasma levels of NO2-UFA derivatives in hyperlipidemic patients are elevated relative to levels in normolipidemic subjects [7]. In addition, an increase in oxidative stress in hypercholesterolemia [8] may contribute to the formation of nitrated species in the vascular wall [9]. Thus, nitrated lipids in the plasma can be used as an indicator of the chain-breaking antioxidant role of NO in lipid oxidation [10] and/or provide a footprint for the presence of oxidants/nitrating agents in the vascular system. We postulate that the presence of these nitrated products in vivo promote cardiovascular homeostasis and compensate for impaired vascular endothelial function in the context of dyslipidemia, obesity, and diabetes. The molecular target(s) and mechanisms underlying the vascular-protective and anti-inflammatory effects of NO2-UFAs remain poorly defined. The serine/threonine kinase, AMP-activated protein kinase (AMPK), is a member of the Snf1/AMPK protein kinase family that is found in all eukaryotes [11]. This kinase is thought to act as a cellular energy sensor by stimulating ATP-producing catabolic pathways and inhibiting ATP-consuming anabolic pathways [12]. PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e31056