CaMKK is the upstream AMPKK that mediates OA-NO₂-induced AMPK activation.

<p><b>A</b>) HUVECs were treated with STO-609 (1 µM) or KN-93 (3 µM) for 1 h followed by incubation with OA-NO₂ for 16 h. AMPK and eNOS phosphorylation and protein expression were assayed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031056#s4" target="_blank">Materials and Methods</a>. The blot is representative of three blots obtained from three independent experiments. <i>Lower panels</i>: summary data (*<i>p</i><0.05 <i>vs.</i> control; ^#<i>p</i><0.05 <i>vs.</i> OA-NO₂ group; <i>n</i> = 3). <b>B</b>) HUVECs were incubated with CaMKKβ-specific siRNA or control siRNA for 48 h and then treated with OA-NO₂ for 16 h. After treatment, cell lysates were analyzed for AMPK and eNOS phosphorylation and protein levels. <i>Lower panels</i>: summary data (*<i>p</i><0.05 <i>vs.</i> control; ^#<i>p</i><0.05 <i>vs.</i> OA-NO₂ group; <i>n</i> = 3). <b>C</b>) HUVECs were pre-treated with YC-1 (30 µM) for 30 min followed by incubation with OA-NO₂ for 16 h. An immunoblot of AMPK precipitated with an anti-CaMKK antibody is shown. The blot is representative of three blots obtained from three independent experiments. <i>Lower panels</i>: summary data (*<i>p</i><0.05 <i>vs.</i> control; ^#<i>p</i><0.05 <i>vs.</i> OA-NO₂ group; <i>n</i> = 3).</p

Activation of AMPK by OA-NO₂ does not require LKB1.

<p><b>A</b>) Phosphorylation of LKB1 Ser428 was not affected by OA-NO₂ in BAECs. Confluent BAECs were exposed to 2.5 µM OA-NO₂ for 16 h, and phosphorylated LKB1-Ser428 was detected by a phospho-specific antibody in western blots. The blot is a representative of three blots obtained from three independent experiments. <i>Lower panels</i>: summary data (<i>n</i> = 3). <b>B</b>) LKB1 is not required for AMPK activation by OA-NO₂. Confluent LKB1-deficient Hela-S3 cells were exposed to 2.5 µM OA-NO₂ for 16 h, and then AMPK and ACC phosphorylation were assayed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031056#s4" target="_blank">Materials and Methods</a>. The blot is representative of three blots obtained from three independent experiments. <i>Lower panels</i>: summary data (*<i>p</i><0.05 <i>vs.</i> control; <i>n</i> = 3). <b>C</b>) LKB1 siRNA did not abolish OA-NO₂-stimulated AMPK activation in HUVECs. HUVECs were incubated with LKB1-specific siRNA or control siRNA for 48 h and then treated with OA-NO₂ or vehicle for 16 h. After treatment, cell lysates were analyzed for LKB1 protein levels and AMPK phosphorylation at Thr172. <i>Lower panels</i>: summary data (*<i>p</i><0.05 <i>vs.</i> control; <i>n</i> = 3).</p

Induction of HO-1 protein and AMPK activation by OA-NO₂.

<p><b>A</b>) BAECs were incubated with OA-NO₂ at the indicated concentrations or with BSA (vehicle) for 16 h, and western blot analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031056#s4" target="_blank">Materials and Methods</a> to detect HO-1 protein expression and AMPK phosphorylation at Thr172. The blot is representative of those obtained from three separate experiments. Corresponding densitometric analyses of phosphorylated AMPK and ACC are shown. *<i>p</i><0.05 <i>vs.</i> control. <b>B</b>) BAECs were incubated with 2.5 µM OA-NO₂ for the indicated times, and western blotting was performed as above. The blot is representative of three blots obtained from three separate experiments. *<i>p</i><0.05 <i>vs.</i> corresponding control. <b>C</b>) Confluent BAECs were exposed to vehicle or OA-NO2 (2.5 µM) for 16 h. AMPKα was immunoprecipitated from cell lysates (1 mg) with a specific antibody. AMPK activity was assayed by ³²P-ATP incorporation into the SAMS peptide. *<i>p</i><0.05 <i>vs.</i> control. <b>D</b>) BAECs were incubated with the indicated concentrations of OA for 16 h. Western blotting was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031056#s4" target="_blank">Materials and Methods</a>. <b>E</b>) BAECs were infected with Ad-DN-AMPK (MOI = 50) or Ad-GFP (control). Infected and non-infected cells were treated with 2.5 µM OA-NO₂ for 16 h. AICAR and metformin were used as positive controls. The blot is representative of three blots obtained from three separate experiments.</p

OA-NO₂-induced HIF-1α is dependent on HO-1.

<p><b>A</b>) HUVECs were treated with 1 µM ZnBG for 30 min followed by incubation with OA-NO₂ for the indicated times. AMPK protein and phosphorylation levels and HIF-1α protein expression were assayed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031056#s4" target="_blank">Materials and Methods</a>. The blot is representative of three blots obtained from three independent experiments. <b>B</b>) HUVECs were incubated with HO-1-specific siRNA or control siRNA for 48 h and then treated with OA-NO₂ or vehicle for 16 h. After treatment, cell lysates were analyzed for HIF-1α and HO-1 protein levels and AMPK phosphorylation at Thr172. <i>Lower panels</i>: summary data (*<i>p</i><0.05 <i>vs.</i> control; ^#<i>p</i><0.05 <i>vs.</i> OA-NO₂ group; <i>n</i> = 3).</p

AMPK mediates OA-NO₂-induced eNOS phosphorylation and NO production in BAECs.

<p>BAECs were treated with (<b>A</b>) different concentrations of OA-NO₂ for 16 h or (<b>B</b>) 2.5 µmol/L OA-NO₂ for the indicated times. Lysates were analyzed by western blot for the indicated proteins. The blot is representative of three blots obtained from three separate experiments. <b>C</b>) Western blot of phosphorylated AMPK and eNOS in OA-NO₂-stimulated BAECs infected with Ad-DN-AMPK (MOI = 50). Non-infected cells or cells infected with Ad-GFP served as controls. For <b>A–C</b>, the corresponding densitometric analyses are shown. *<i>p</i><0.05 <i>vs.</i> control; ^#<i>p</i><0.05 <i>vs.</i> GFP with OA-NO₂-treated group. <b>D</b>) NO release by OA-NO₂-stimulated BAECs infected with Ad-DN-AMPK (MOI = 50) or Ad-GFP (control). *<i>p</i><0.05 <i>vs.</i> non-transfected, no OA-NO₂ group; ^#<i>p</i><0.05 <i>vs.</i> OA-NO₂-treated, Ad-GFP group. <b>E</b>) AMPK activity corresponding to <b>C</b> and <b>D</b> above. *<i>p</i><0.05 <i>vs.</i> no OA-NO₂ treatment, Ad-GFP group; ^#<i>p</i><0.05 <i>vs.</i> OA-NO₂-treated, Ad-GFP group. <b>F</b>) The proposed signaling pathway involved in AMPK/eNOS activation in response to OA-NO2 treatment in endothelial cells.</p

AMPK mediates EPA-induced eNOS phosphorylation and NO production in BAEC.

<p>BAEC were treated with (<b>A</b>) EPA 25 µmol/L for the indicated times or (<b>B</b>) varying concentrations of EPA for 24 h. Lysates were analyzed by western blot for the indicated proteins. The blot is a representative of four blots obtained from four separate experiments. <b>C</b>) Western blot of phosphorylated AMPK and eNOS in EPA-stimulated BAEC infected with adenoviruses encoding GFP or Ad-DN-AMPK. <b>D</b>) Phosphorylation of Akt in EPA-stimulated BAEC. The data in C and D represent results of 3 separate experiments. For A–D, corresponding densitometric analyses are shown. *<i>P</i><0.05 <i>vs.</i> control. <b>E</b>) NO release in EPA-stimulated BAEC. BAEC were treated with compound C (AMPK inhibitor) (20 µmol/L), DMSO (vehicle), or _L-NAME (NOS inhibitor) (0.1 mM) for 30 min prior to stimulation with EPA. <i>n</i> = 4 for each treatment group. *<i>P</i><0.05 <i>vs</i>. control; ^#<i>P</i><0.05 <i>vs</i>. EPA. <b>F</b>) NO release by EPA-stimulated BAEC infected with Ad-DN-AMPK (50 multiplicities of infection) or Ad-GFP (control). *<i>P</i><0.05 <i>vs</i>. non EPA-treated, Ad-GFP group; ^#<i>P</i><0.05 <i>vs</i>. EPA-treated, Ad-GFP group. For A and B, the corresponding AMPK activity is shown in the lower panel.</p

EPA activates AMPK in BAEC.

<p>BAEC were treated with (<b>A</b>) 25 µmol/L EPA for the indicated times or (<b>B</b>) varying concentrations of EPA for 24 h. Lysates (80 µg) were analyzed by western blot for the indicated proteins. The blot is a representative of four blots obtained from four separate experiments. Corresponding densitometric analyses of phosphorylated AMPK and ACC are shown. *, <i>P</i><0.05 <i>vs.</i> control groups. <b>C</b>) Confluent BAECs were treated with vehicle or EPA (25 µmol/L) for 24 hours. AMPK activity was assayed using the SAMS peptide as a substrate. Data presented are means ± SD from 3 independent experiments. <i>P</i><0.05 <i>vs.</i> vehicle.</p

EPA upregulates UCP-2 and activates AMPK in mice.

<p><b>A</b>) Western blot analysis of phosphorylated AMPK, ACC, and eNOS in the thoracic aorta of mice receiving EPA or DMSO (vehicle) in their drinking water for 16 weeks (<i>n</i> = 3 for each group). Corresponding densitometric analyses are shown. *<i>P</i><0.05 <i>vs.</i> control. <b>B</b>) Western blot analysis of eNOS phosphorylation in wild type (WT), AMPKα1 knock out (KO), and AMPKα2 KO mice receiving EPA for 16 weeks (<i>n</i> = 3 for each group). The relative p-eNOS/eNOS ratios are shown. *<i>P</i><0.05 <i>vs.</i> non-treated WT; ^#<i>P</i><0.05 <i>vs.</i> EPA-treated WT. *<i>P</i><0.05 <i>vs.</i> control; **<i>P</i><0.01 <i>vs.</i> control.</p

Both AMPK <i>α1</i> and NO are required for EPA-enhanced endothelium-dependent vasodilatation <i>in ex vivo</i> or <i>in vivo</i>.

<p><b>A</b>) Endothelium-dependent relaxation of the aortic rings in response to acetylcholine (Ach) from wild type or Apo-E^−/− mice. Aortic rings were pretreated ± compound C, then incubated with EPA or AICAR (<i>n</i> = 4 for each group). Each data point represents relaxation expressed as a percentage of the value obtained for phenylephrine-preconstricted aorta. *<i>P</i><0.05 <i>vs.</i> wild type; **<i>P</i><0.01 <i>vs.</i> wild type; ^#<i>P</i><0.05 <i>vs.</i> Apo-E^−/−; <i>P</i><0.05 <i>vs.</i> Apo-E^−/− + EPA. <b>B</b>) AMPKα1 and eNOS are required for EPA-induced amelioration of endothelium function. Aortic rings extracted from Apo-E KO, Apo-E/AMPKα1 dual KO or eNOS KO mice were incubated with or without EPA (25 µM) for 24 h in EBM. The endothelium-dependent relaxation were assayed by the addition of acetylcholine at concentrations indicated (<i>n</i> = 4 for each group). *P<0.05; **P<0.01 vs. Apo-E^−/− mice.</p

EPA stimulates UCP-2 expression via a PPARγ-mediated pathway in BAEC.

<p><b>A, B</b>) Western blot analysis of UCP-2 in EPA-stimulated BAEC pretreated with 10 µmol/L Wy14643 (PPARα agonist), <u>10 µmol/L</u> rosiglitazone (PPARγ agonist), 10 µmol/L MK886 (PPARα antagonist), or 10 µmol/L GW9662 (PPARγ antagonist). *<i>P</i><0.05 <i>vs.</i> control; **<i>P</i><0.01 <i>vs.</i> control; ^#<i>P</i><0.05 <i>vs.</i> EPA. <b>C</b>) Western blot analysis of AMPK, UCP-2, and PPARγ in EPA-stimulated HUVEC transfected with PPARγ siRNA or scrambled siRNA for 48 h. *<i>P</i><0.05 <i>vs.</i> control; ^#<i>P</i><0.05 <i>vs.</i> scrambled siRNA + EPA. For A–C, the blot is a representative of four blots obtained from four separate experiments, and the corresponding densitometric analyses are shown.</p