AMPK is comprised of three subunits: a catalytic a-subunit and regulatory b- and c-subunits. Activation of AMPK requires the phosphorylation of Thr172 in the activation loop of the a-subunit by an upstream AMPK kinase (AMPKK) [13]. Interestingly, the first AMPKK to be identified was LKB1, a tumor suppressor that is mutated in humans with Peutz-Jegher syndrome [14]. Patients with this syndrome have an increased risk of developing carcinomas of the colon, stomach, and pancreas. Recently, calcium (Ca2+) calmodulin-dependent kinase kinase (CaMKK) [15] was identified as an upstream AMPKK. CaMKK is activated by a rise in intracellular Ca2+ concentrations ([Ca2+]i), and this kinase phosphorylates and activates AMPK in an AMP-indepen- dent manner [15]. Therefore, in addition to responding to an increase in the AMP-to-ATP ratio, AMPK may also be activated by a rise in [Ca2+]i in response to nutrients, drugs, or physiological stimuli. While the AMPK pathway is traditionally thought to be a regulator of metabolism, recent studies have demonstrated that AMPK may also act to maintain endothelial function [16]. AMPK exerts pleiotropic effects that are believed to be beneficial for endothelial function. These effects, which are ultimately anti- atherogenic, include induction of the endothelial nitric oxide synthase (eNOS)/NO pathway and result in an increase in NO bioavailability, suppression of endothelial ROS production following exposure to deleterious stimuli, such as hyperglycemia or high free fatty acids (FFAs), and modulation of vascular tone (see review [16]). AMPK also possesses anti-apoptotic and anti- inflammatory activities [17]. Since many of the metabolic changes and endothelial-protective effects attributable to NO2-UFAs are similar to those observed in response to AMPK activation, we hypothesized that AMPK activation is an important mediator of NO2-UFA activity. As such, AMPK activation may explain the pleiotropic beneficial effects of NO2-UFAs on the cardiovascular system in obesity and diabetes. Whether NO2-UFAs activate AMPK and, if so, by what mechanism(s) has yet to be determined. In the present study, we examined the effects of NO2-UFAs on the AMPK upstream kinases, specifically LKB1 and CaMKKb. We also investigated whether NO2-UFAs modulate the eNOS/NO pathway, which is known to be an indicator of endothelial function and an important property of AMPK in cardiovasculature. We report that treatment with NO2-UFAs induces CaMKKb-dependent AMPK activation through an HO-1/HIF-1a/Ca2+ pathway in vitro and show that NO2-UFAs promote p-eNOS and NO production via activation of the AMPK pathway in endothelial cells. Results OA-NO2 induces HO-1 protein and AMPK phosphorylation/activation in bovine aortic endothelial cells (BAECs) To investigate activation of AMPK by OA-NO2 in endothelial cells, we treated confluent BAECs with different concentrations of OA-NO2 for 2–16 h. AMPK activation was indirectly assessed by western blot analysis of AMPK Thr172 phosphorylation, which is essential for AMPK activity [18]. As shown in Figure 1A and 1B, incubation of BAECs with OA-NO2 (0.5–2.5 mM for 16 h, or 2.5 mM for 2–16 h) resulted in a dose- and time-dependent increase in AMPKa phosphorylation at Thr172. AMPK Thr172 phosphorylation in BAECs gradually increased beginning 6 h after incubation with 2.5 mM OA-NO2 and reached peak levels after 16 h. This increased AMPK phosphorylation was associated with elevated AMPK activity, as measured by the SAMS peptide assay (Figure 1C). OA-NO2 treatment did not alter total AMPK levels, suggesting that OA-NO2-induced phosphorylation of AMPK was AMPK-Dependent Angiogenesis by Nitrated Lipids not due to altered expression of these proteins. Since OA-NO2 activated AMPK in both BAECs and human umbilical vein endothelial cells (HUVECs) at similar potencies, we performed the majority of our experiments with BAECs. The changes in AMPK phosphorylation were mirrored by changes in HO-1 protein expression, as evaluated by western blot analysis. Exposure of BAECs to a range of OA-NO2 concentra- tions resulted in an upregulation of HO-1 that was initially observed at 4 h with peak induction at 16 h. HO-1 induction and AMPK activation were specifically due to the nitroalkene moiety of OA-NO2, because oleic acid (OA) did not induce HO-1 protein expression or AMPK phosphorylation (Figure 1D). At a concentration of 2.5 mM, OA-NO2 potently activated AMPK and induced HO-1 expression without causing cellular toxicity. On the basis of these results, we chose to stimulate BAECs with 2.5 mM OA-NO2 for 16 h in subsequent experiments. To determine whether OA-NO2-induced HO-1 expression was mediated by AMPK, we used an adenovirus encoding a dominant- negative mutant form of AMPKa (Ad-DN-AMPK) to suppress AMPK activity. AICAR (5-aminoimidazole-4-carboxamide-1-b- D-ribofuranoside) and metformin, two well characterized AMPK activators, were used as positive controls for AMPK phosphory- lation. OA-NO2 significantly elevated AMPK phosphorylation (Figure 1E). As expected, Ad-DN-AMPK effectively suppressed AMPK phosphorylation but failed to abolish HO-1 induction by OA-NO2, suggesting that AMPK does not act upstream of HO-1 production in this system. Activation of AMPK by OA-NO2 does not require LKB1 Two recent studies [14,19] showed that LKB1 acts as an upstream AMPK kinase. To evaluate the role of LKB1 in OA- NO2-induced AMPK activation, we investigated whether OA- NO2 altered LKB1 Ser 428 phosphorylation, which is essential for LKB1 activity and LKB1-dependent AMPK activation. As shown in Figure 2A, OA-NO2 did not alter the levels of LKB1 Ser428 phosphorylation. We next determined whether OA-NO2 activated AMPK in Hela-S3 cells, an LKB1-deficient cell line. Similar to the results obtained in BAECs, OA-NO2 (2.5 mM) induced AMPK phos- phorylation on Thr172 in Hela-S3 cells (,3-fold, p,0.05; Figure 2B). In parallel with AMPK phosphorylation, OA-NO2 dramatically increased phosphorylation of the known AMPK substrate, acetyl-CoA carboxylase (ACC), at Ser79 (,2.4-fold, p,0.05), providing an additional indication that AMPK was activated in the absence of LKB1. To further explore the role of LKB1, we investigated the effect of siRNA-mediated LKB1 downregulation on AMPK activation induced by OA-NO2. As shown in Figure 2C, OA-NO2-induced AMPK phosphorylation was not significantly altered in cells pre-treated with LKB1 siRNA. The suppression of LKB1 protein expression by LKB1 siRNA (,60%) was confirmed by western analysis. Taken together, these results strongly suggest that OA-NO2-induced phosphorylation of AMPK Thr172 is independent of its upstream kinase, LKB1. Induction of HIF-1a by OA-NO2 is dependent on HO-1 Hypoxia-inducible factor (HIF)-1 is a dimeric protein complex that plays an integral role in the body’s response to hypoxia [20]. Like AMPK, HIF-1 is one of the primary genes involved in the homeostatic process that leads to increased vascularization in hypoxic areas, such as those within localized ischemia and in tumors [20]. HIF-1 and AMPK represent two main cellular pathways involved in coping with hypoxic stress and protecting cells against energy depletion and tissue reperfusion injury in times of metabolic crisis [21]. In addition, both HIF-1 and HO-1 are PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e31056