Title: Rosiglitazone Stimulates Nitric Oxide Synthesis in Human Aortic Endothelial Cells via AMP-activated Protein Kinase*
Abstract: The thiazolidinedione anti-diabetic drugs increase activation of endothelial nitric-oxide (NO) synthase by phosphorylation at Ser-1177 and increase NO bioavailability, yet the molecular mechanisms that underlie this remain poorly characterized. Several protein kinases, including AMP-activated protein kinase, have been demonstrated to phosphorylate endothelial NO synthase at Ser-1177. In the current study we determined the role of AMP-activated protein kinase in rosiglitazone-stimulated NO synthesis. Stimulation of human aortic endothelial cells with rosiglitazone resulted in the time- and dose-dependent stimulation of AMP-activated protein kinase activity and NO production with concomitant phosphorylation of endothelial NO synthase at Ser-1177. Rosiglitazone stimulated an increase in the ADP/ATP ratio in endothelial cells, and LKB1 was essential for rosiglitazone-stimulated AMPK activity in HeLa cells. Infection of endothelial cells with a virus encoding a dominant negative AMP-activated protein kinase mutant abrogated rosiglitazone-stimulated Ser-1177 phosphorylation and NO production. Furthermore, the stimulation of AMP-activated protein kinase and NO synthesis by rosiglitazone was unaffected by the peroxisome proliferator-activated receptor-γ inhibitor GW9662. These studies demonstrate that rosiglitazone is able to acutely stimulate NO synthesis in cultured endothelial cells by an AMP-activated protein kinase-dependent mechanism, likely to be mediated by LKB1. The thiazolidinedione anti-diabetic drugs increase activation of endothelial nitric-oxide (NO) synthase by phosphorylation at Ser-1177 and increase NO bioavailability, yet the molecular mechanisms that underlie this remain poorly characterized. Several protein kinases, including AMP-activated protein kinase, have been demonstrated to phosphorylate endothelial NO synthase at Ser-1177. In the current study we determined the role of AMP-activated protein kinase in rosiglitazone-stimulated NO synthesis. Stimulation of human aortic endothelial cells with rosiglitazone resulted in the time- and dose-dependent stimulation of AMP-activated protein kinase activity and NO production with concomitant phosphorylation of endothelial NO synthase at Ser-1177. Rosiglitazone stimulated an increase in the ADP/ATP ratio in endothelial cells, and LKB1 was essential for rosiglitazone-stimulated AMPK activity in HeLa cells. Infection of endothelial cells with a virus encoding a dominant negative AMP-activated protein kinase mutant abrogated rosiglitazone-stimulated Ser-1177 phosphorylation and NO production. Furthermore, the stimulation of AMP-activated protein kinase and NO synthesis by rosiglitazone was unaffected by the peroxisome proliferator-activated receptor-γ inhibitor GW9662. These studies demonstrate that rosiglitazone is able to acutely stimulate NO synthesis in cultured endothelial cells by an AMP-activated protein kinase-dependent mechanism, likely to be mediated by LKB1. Endothelium-derived nitric oxide (NO), 2The abbreviations used are: NO, nitric oxide; eNOS, endothelial NO synthase; PPARγ, peroxisome proliferator-activated receptor γ; AMPK, AMP-activated protein kinase; HAEC, human aortic endothelial cell; TNF, tumor necrosis factor; HPLC, high performance liquid chromatography; HUVEC, human umbilical vein endothelial cell; LKB1-WT, wild type LKB1; LKB1-KD, kinase inactive LKB1; Ad.Null, control adenovirus; Ad.α1DN, adenovirus expressing dominant negative AMPKα1; CaMKK, Ca2+/calmodulin-dependent protein kinase kinase; ACC, acetyl CoA carboxylase; l-NAME, N (G)-nitro-l-arginine methyl ester. 2The abbreviations used are: NO, nitric oxide; eNOS, endothelial NO synthase; PPARγ, peroxisome proliferator-activated receptor γ; AMPK, AMP-activated protein kinase; HAEC, human aortic endothelial cell; TNF, tumor necrosis factor; HPLC, high performance liquid chromatography; HUVEC, human umbilical vein endothelial cell; LKB1-WT, wild type LKB1; LKB1-KD, kinase inactive LKB1; Ad.Null, control adenovirus; Ad.α1DN, adenovirus expressing dominant negative AMPKα1; CaMKK, Ca2+/calmodulin-dependent protein kinase kinase; ACC, acetyl CoA carboxylase; l-NAME, N (G)-nitro-l-arginine methyl ester. synthesized by endothelial NO synthase (eNOS), is a key regulator of vascular function (1Ignarro L.J. Napoli C. Curr. Diab. Rep. 1995; 5: 17-23Crossref Scopus (65) Google Scholar). Endothelial-derived NO promotes vasodilatation and inhibits platelet aggregation, leukocyte adherence, and vascular smooth muscle proliferation, thereby having a profound influence on blood flow, vascular remodeling, and angiogenesis (1Ignarro L.J. Napoli C. Curr. Diab. Rep. 1995; 5: 17-23Crossref Scopus (65) Google Scholar). Endothelial dysfunction, defined by reduced NO bioavailability, is a key early mechanism in the development of atheromatous vascular disease (2Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19061) Google Scholar, 3Reddy K.G. Nair R.N. Sheehan H.M. Hodgson J.M. J. Am. Coll. Cardiol. 1994; 23: 833-843Crossref PubMed Scopus (394) Google Scholar, 4Laight D.W. Carrier M.J. Anggard E.E. Cardiovasc. Res. 2000; 47: 457-469Crossref PubMed Scopus (223) Google Scholar).Type 2 diabetes is associated with a greatly increased risk of atheromatous vascular disease, and vascular endothelial dysfunction has been demonstrated in type 2 diabetic patients (4Laight D.W. Carrier M.J. Anggard E.E. Cardiovasc. Res. 2000; 47: 457-469Crossref PubMed Scopus (223) Google Scholar, 5Williams S.B. Cusco J.A. Roddy M.A. Johnstone M.T. Creager M.A. J. Am. Coll. Cardiol. 1996; 27: 564-574Google Scholar, 6Ting H.H. Timimi F.K. Boles K.S. Creager S.J. Ganz P. Creager M.A. J. Clin. Investig. 1996; 97: 22-28Crossref PubMed Scopus (772) Google Scholar). There is a growing body of evidence to suggest that the thiazolidinedione class of anti-diabetic drugs improve endothelial function, as assessed by endothelium-dependent vasodilatation in patients with type 2 diabetes or insulin resistance (7Pistrosch F. Passauer J. Fischer S. Fuecker K. Hanefeld M. Gross P. Diabetes Care. 2004; 27: 484-490Crossref PubMed Scopus (208) Google Scholar, 8Wang T.D. Chen W.J. Lin J.W. Chen M.F. Lee Y.T. Am. J. Cardiol. 2004; 93: 362-365Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 9Natali A. Baldeweg S. Toschi E. Capaldo B. Barbaro D. Gastaldelli A. Yudkin J.S. Ferrannini E. Diabetes Care. 2004; 27: 1349-1357Crossref PubMed Scopus (164) Google Scholar, 10Satoh N. Ogawa Y. Usui T. Tagami T. Kono S. Uesugi H. Sugiyama H. Sugawara A. Yamada K. Shimatsu A. Kuzuya H. Nakao K. Diabetes Care. 2003; 26: 2493-2499Crossref PubMed Scopus (285) Google Scholar). The hypoglycemic effects of thiazolidinediones are mediated by the transcription factor peroxisome proliferator-activated receptor-γ (PPARγ) (11Semple R.K. Chatterjee V.K. O'Rahilly S. J. Clin. Investig. 2006; 116: 581-589Crossref PubMed Scopus (667) Google Scholar), but recent work suggests that the improvement of endothelial function by thiazolidinediones is independent of the effect on glycemia (7Pistrosch F. Passauer J. Fischer S. Fuecker K. Hanefeld M. Gross P. Diabetes Care. 2004; 27: 484-490Crossref PubMed Scopus (208) Google Scholar, 10Satoh N. Ogawa Y. Usui T. Tagami T. Kono S. Uesugi H. Sugiyama H. Sugawara A. Yamada K. Shimatsu A. Kuzuya H. Nakao K. Diabetes Care. 2003; 26: 2493-2499Crossref PubMed Scopus (285) Google Scholar). Therefore, the mechanism of action by which thiazolidinediones improve vascular endothelial function remains uncertain.Recent studies have suggested that prolonged exposure to thiazolidinediones directly improves NO bioavailability in endothelial cells and increases phosphorylation of eNOS at Ser-1177 (12Calnek D. Mazzella L. Roser S. Roman J. Hart C.M. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 52-57Crossref PubMed Scopus (282) Google Scholar, 13Cho D.H. Choi Y.J. Jo S.A. Jo I. J. Biol. Chem. 2004; 279: 2499-2506Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 14Polikandriotis J.A. Mazzella L.J. Rupnow H.L. Hart C.M. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1810-1816Crossref PubMed Scopus (161) Google Scholar, 15Kim K.Y. Cheon H.G. J. Biol. Chem. 2006; 281: 13503-13512Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Phosphorylation of eNOS at Ser-1177 stimulates NO synthesis, and several protein kinases have been demonstrated to phosphorylate eNOS Ser-1177 in endothelial cells, including protein kinase B (also known as Akt) and AMP-activated protein kinase (AMPK) (16Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2208) Google Scholar, 17Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3011) Google Scholar, 18Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (710) Google Scholar, 19Morrow V.A. Foufelle F. Connell J.M.C. Petrie J.R. Gould G.W. Salt I.P. J. Biol. Chem. 2003; 278: 31629-31639Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 20Fryer L.G. Parbu-Patel A. Carling D. J. Biol. Chem. 2002; 277: 25226-25232Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar), but the protein kinase and signaling mechanism responsible for phosphorylation of eNOS in response to thiazolidinediones is as yet undetermined. As thiazolidinediones have been demonstrated to rapidly and chronically activate AMPK in muscle, liver, and adipose (20Fryer L.G. Parbu-Patel A. Carling D. J. Biol. Chem. 2002; 277: 25226-25232Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 21Saha A.K. Avilucea P.R. Ye J.M. Assifi M.M. Kraegen E.W. Ruderman N.B. Biochem. Biophys. Res. Commun. 2004; 314: 580-585Crossref PubMed Scopus (194) Google Scholar, 22Konrad D. Rudich A. Bilan P.J. Patel N. Richardson C. Witters L.A. Klip A. Diabetologia. 2005; 48: 954-966Crossref PubMed Scopus (97) Google Scholar, 23Lebrasseur N.K. Kelly M. Tsao T.S. Farmer S.R. Saha A.K. Ruderman N.B. Tomas E. Am. J. Physiol. 2006; 291: E175-E181Crossref PubMed Scopus (239) Google Scholar), it is possible that activation of AMPK may mediate eNOS phosphorylation and NO synthesis in response to thiazolidinediones in endothelial cells.In the current study, we tested the hypothesis that AMPK-mediated phosphorylation of eNOS was responsible for rosiglitazone-stimulated NO production in cultured human aortic endothelial cells (HAECs). We demonstrate that rosiglitazone rapidly stimulates eNOS phosphorylation at Ser-1177 and NO synthesis in an AMPK-dependent, PPARγ-independent manner. We propose that this mechanism underlies, at least in part, the rapid effects of thiazolidinediones on vascular function.EXPERIMENTAL PROCEDURESMaterials—Cryopreserved HAECs and cell culture medium were obtained from TCS Cellworks (Botolph Claydon, Buckinghamshire, UK). U937 cells were obtained from Dr. T. Palmer, University of Glasgow, Glasgow, UK. HeLa cells stably expressing wild type LKB1 (LKB1-WT) or kinase-inactive LKB1 (LKB1-KD) have been described elsewhere (24Sapkota G.P. Deak M. Kieloch A. Morrice N. Goodarzi A.A. Smythe C. Shiloh Y. Lees-Miller S.P. Alessi D.R. Biochem. J. 2002; 368: 507-516Crossref PubMed Scopus (91) Google Scholar) and were kindly provided by Prof. D. Alessi, University of Dundee, Dundee, UK. Isoform-specific sheep anti-AMPK and anti-LKB1 antibodies have been described elsewhere (25Woods A. Salt I. Scott J. Hardie D.G. Carling D. FEBS Lett. 1996; 397: 347-351Crossref PubMed Scopus (230) Google Scholar, 26Sakamoto K. McCarthy A. Smith D. Green K.A. Hardie D.G. Ashworth A. Alessi D.R. EMBO J. 2005; 24: 1810-1820Crossref PubMed Scopus (438) Google Scholar) and were a generous gift from Prof. D. G. Hardie, University of Dundee, Dundee, UK. Rosiglitazone was kindly provided by GlaxoSmithKline (Stevenage, UK). Pioglitazone, troglitazone, and GW9662 were obtained from Axxora UK Ltd. (Nottingham, UK). Tumor necrosis factor α (TNFα) and primers specific to human CD36 and 18 S ribosomal RNA were obtained from Sigma-Aldrich. All other reagents were from sources described previously (19Morrow V.A. Foufelle F. Connell J.M.C. Petrie J.R. Gould G.W. Salt I.P. J. Biol. Chem. 2003; 278: 31629-31639Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 27Reihill J.R. Ewart M.A. Hardie D.G. Salt I.P. Biochem. Biophys. Res. Commun. 2007; 354: 1084-1088Crossref PubMed Scopus (78) Google Scholar).Cell Culture—HAECs were grown in large vessel endothelial cell medium at 37 °C in 5% CO2 and used for experiments between passages 3 and 6 as described previously (19Morrow V.A. Foufelle F. Connell J.M.C. Petrie J.R. Gould G.W. Salt I.P. J. Biol. Chem. 2003; 278: 31629-31639Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 27Reihill J.R. Ewart M.A. Hardie D.G. Salt I.P. Biochem. Biophys. Res. Commun. 2007; 354: 1084-1088Crossref PubMed Scopus (78) Google Scholar). U937 pro-monocytic cells were cultured in RPMI 1640, supplemented with 10% (v/v) fetal calf serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2 mm l-glutamine at 37 °C in 5% CO2. HeLa cells stably expressing LKB1-WT or LKB1-KD were cultured as described previously (24Sapkota G.P. Deak M. Kieloch A. Morrice N. Goodarzi A.A. Smythe C. Shiloh Y. Lees-Miller S.P. Alessi D.R. Biochem. J. 2002; 368: 507-516Crossref PubMed Scopus (91) Google Scholar).Evaluation of NO Production—Cells cultured in 12-well plates were incubated in serum-free large vessel cell medium for 3–4 h. The cells were then preincubated for 1 h at 37 °C in 0.5 ml/well Krebs Ringer HEPES (KRH) buffer (119 mm NaCl, 20 mm HEPES-NaOH, pH 7.4, 5 mm NaHCO3, 4.7 mm KCl, 1.3 mm CaCl2, 1.2 mm MgSO4, 1 mm NaH2PO4, 0.1 mm l-arginine, 5 mm glucose). The medium was removed and replaced with fresh KRH buffer (0.5 ml/well) in the presence of various concentrations of test substances. After incubation for various durations, aliquots of medium were removed and analyzed using a Sievers 280A NO analyzer as described previously (19Morrow V.A. Foufelle F. Connell J.M.C. Petrie J.R. Gould G.W. Salt I.P. J. Biol. Chem. 2003; 278: 31629-31639Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 27Reihill J.R. Ewart M.A. Hardie D.G. Salt I.P. Biochem. Biophys. Res. Commun. 2007; 354: 1084-1088Crossref PubMed Scopus (78) Google Scholar). The appropriate control experiments were performed in the presence of the eNOS inhibitor, N (G)-nitro-l-arginine methyl ester (l-NAME, 0.1 mm). Data are presented as l-NAME-sensitive NO synthesis.Preparation of Adenoviruses and Infection of HAECs—Control (Ad.Null) and dominant negative AMPK adenoviruses (Ad.α1DN) were propagated and purified as described previously (19Morrow V.A. Foufelle F. Connell J.M.C. Petrie J.R. Gould G.W. Salt I.P. J. Biol. Chem. 2003; 278: 31629-31639Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 27Reihill J.R. Ewart M.A. Hardie D.G. Salt I.P. Biochem. Biophys. Res. Commun. 2007; 354: 1084-1088Crossref PubMed Scopus (78) Google Scholar). HAECs were infected with 10 plaque-forming units/cell adenovirus in complete medium and the cells cultured for 48 h prior to experimentation. Under these conditions after infection with a green fluorescent protein (GFP)-expressing virus, the majority (>95%) of HAECs expressed GFP (19Morrow V.A. Foufelle F. Connell J.M.C. Petrie J.R. Gould G.W. Salt I.P. J. Biol. Chem. 2003; 278: 31629-31639Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar).Preparation of HAEC Lysates—Cells were incubated in serum-free medium for 3 h prior to preincubation for 1 h at 37 °C in 5 ml of KRH buffer. The medium was replaced with 5 ml of fresh KRH buffer containing test substances and incubated for various durations at 37 °C. The medium was removed, and 0.5 ml of ice-cold lysis buffer (50 mm Tris-HCl, pH 7.4, at 4 °C, 50 mm NaF, 5 mm Na4P2O7, 1 mm Na3VO4, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.1 mm benzamidine, 0.1 mm fluoride, 5 μg/ml soybean trypsin inhibitor, 1% (v/v) Triton X-100, 250 mm mannitol) added. The cell extract was scraped off and transferred to a microcentrifuge tube. Extracts were vortex-mixed and centrifuged (14,000 × g, 3 min, 4 °C). Supernatants were snap-frozen in liquid N2 and stored at –80 °C before use.Immunoprecipitation and Assay of AMPK—AMPK was immunoprecipitated from lysates and assayed using the SAMS substrate peptide as described previously (19Morrow V.A. Foufelle F. Connell J.M.C. Petrie J.R. Gould G.W. Salt I.P. J. Biol. Chem. 2003; 278: 31629-31639Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 27Reihill J.R. Ewart M.A. Hardie D.G. Salt I.P. Biochem. Biophys. Res. Commun. 2007; 354: 1084-1088Crossref PubMed Scopus (78) Google Scholar). Protein concentration was determined by the method of Bradford (28Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213164) Google Scholar).Immunoprecipitation and Assay of LKB1—HAEC lysates (0.1 mg) were added to 5 μg of sheep anti-LKB1 antibody and mixed overnight at 4 °C. Protein G-Sepharose (5 μl of 50% slurry) was added and the volume adjusted to 300 μl with lysis buffer and mixed for 4 h at 4 °C. The mixture was centrifuged (14,000 × g, 30 s, 4 °C) and the pellet washed three times in 50 mm HEPES-NaOH, pH 7.4, 1% (v/v) Triton X-100. Recombinant AMPKα1-(1–312) containing the kinase domain (0.6 μg) constructed and expressed as described previously (29Scott J.W. Norman D.G. Hawley S.A. Kontogiannis L. Hardie D.G. J. Mol. Biol. 2002; 317: 309-323Crossref PubMed Scopus (138) Google Scholar) was incubated with the LKB1 immunoprecipitate in a total volume of 25 μl of assay buffer (50 mm HEPES-NaOH, pH 7.4, 1 mm dithiothreitol, 0.1 mm ATP, 5 mm MgCl2) at 37 °C with shaking for 30 min. The mixture was centrifuged (14,000 × g, 30 s, 4 °C) and the supernatant subsequently assayed for AMPK activity using the SAMS peptide as described previously (19Morrow V.A. Foufelle F. Connell J.M.C. Petrie J.R. Gould G.W. Salt I.P. J. Biol. Chem. 2003; 278: 31629-31639Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 27Reihill J.R. Ewart M.A. Hardie D.G. Salt I.P. Biochem. Biophys. Res. Commun. 2007; 354: 1084-1088Crossref PubMed Scopus (78) Google Scholar).Monocyte Adhesion Assay—HAECs were grown to confluence on 24-well tissue culture plates and infected with recombinant AMPK adenoviruses, if desired, for 24 h at 20 plaque-forming units/cell. After treatment as indicated, the medium was aspirated and HAEC monolayers washed thoroughly with serum-free RPMI 1640 and overlaid with 1 × 105 U937 cells/well in serum-free RPMI 1640. The cells were allowed to adhere for 1 h at 37 °C, the medium removed, and monolayers washed three times (1 ml/well serum-free Dulbecco's modified Eagle's medium) to remove non-adherent U937 cells. Cells were fixed in 0.5 ml/well 4% (w/v) paraformaldehyde in 5% (w/v) sucrose/phosphate-buffered saline, pH 7.2, and the number of adhered U937 cells per field of confluent HAECs counted on a Zeiss Axiovert 135 microscope with a ×20 objective.Nucleotide Extraction and Analysis—Neutralized perchloric acid extracts were prepared as described previously (30Salt I.P. Johnson G. Ashcroft S.J.H. Hardie D.G. Biochem. J. 1998; 335: 533-539Crossref PubMed Scopus (332) Google Scholar). Nucleotides were separated by HPLC using a variation of the method of Uesugi et al. (31Uesugi T. Sano K. Uesawa Y. Ikegami Y. Mohri K. J. Chromatogr. B Biomed. Sci. Appl. 1997; 703: 63-74Crossref PubMed Scopus (53) Google Scholar). Briefly, nucleotides were separated on a stainless steel column packed with octadecylsilane attached to a Varian Prostar HPLC system equilibrated with 0.1 m triethylamine phosphate buffer (pH 8) and methanol (96:4, v/v). Elution was monitored at A259. The elution positions of ADP and ATP were determined using standard solutions.Analysis of mRNA Expression—HAECs were incubated in serum-free large vessel cell medium for 4 h prior to preincubation for 1 h at 37 °C in KRH buffer. The medium was removed and cells incubated in the presence or absence of 10 μm GW9662 in KRH buffer for 1 h. Rosiglitazone (10 μm) was subsequently added as indicated and HAECs incubated for a further 2 h. Total RNA was prepared from cells using an RNeasy kit (Qiagen) according to the manufacturer's instructions and reverse transcribed to cDNA using Moloney murine leukemia virus reverse transcriptase (Finnzymes, Espoo, Finland). Primers specific for human CD36 (forward, 5′-CTGTGACCGGAACTGTGGGCT-3′, and reverse, 5′-GAAGATGGCACCATTGGGCTG-3′) and 18 S ribosomal RNA (forward, 5′-AAACGGCTACCACATCCAAG-3′, and reverse, 5′-CGCTCCCAAGATCCAACTAC-3′) were used to amplify the cDNA by reverse transcription PCR.Statistics—Unless stated otherwise, results are expressed as the mean ± S.E. Statistically significant differences were determined using a two-tailed Student's t test, with p < 0.05 as significant.RESULTSWe examined the ability of rosiglitazone to modulate NO production and AMPK activity in HAECs. Stimulation of HAECs with rosiglitazone (200 μm) stimulated the rate of NO synthesis within 30 min and reached a maximum 2.1-fold increase at 60 min (Fig. 1A). The increase in the rate of NO synthesis was sustained for 24 h. Under identical conditions, AMPK activity was maximally stimulated 3.5-fold within 30 min and activation was sustained for 24 h (Fig. 1A). Stimulation of NO by rosiglitazone was dose-dependent (Fig. 1B) such that NO synthesis was significantly stimulated by 2 μm rosiglitazone and was stimulated maximally by 20 μm rosiglitazone (2.1-fold). Stimulation of AMPK activity was also dose-dependent, such that 2 μm rosiglitazone significantly stimulated AMPK activity, reaching a maximum 2.4-fold increase in AMPK activity at 200 μm (Fig. 1B). The principal therapeutic actions of the thiazolidinediones are thought to be alterations in gene expression mediated by PPARγ (12Calnek D. Mazzella L. Roser S. Roman J. Hart C.M. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 52-57Crossref PubMed Scopus (282) Google Scholar). We therefore determined whether AMPK activation by rosiglitazone was downstream of PPARγ activation. Preincubation of HAECs with the PPARγ inhibitor GW9662 (5 μm) was without effect on both basal or rosiglitazone-stimulated AMPK activity and NO synthesis (Fig. 1B). Preincubation of HAECs with GW9662 did, however, prevent rosiglitazone (10 μm, 2 h)-stimulated expression of CD36 mRNA, indicating that GW9662 effectively inhibits PPARγ-mediated transcription in HAECs under these conditions (supplemental Fig. S1).Activation of AMPK requires phosphorylation at Thr-172 by an AMPK kinase. Two AMPK kinases have been isolated to date, LKB1 and Ca2+/calmodulin-dependent kinase kinase (CaMKK) (32Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28Crossref PubMed Google Scholar, 33Woods A. Johnstone S.R. Dickerson K. Leiper F.C. Fryer L.G.D. Neumann D. Schlattner U. Wallimann T. Carlson M. Carling D. Curr. Biol. 2003; 13: 2004-2008Abstract Full Text Full Text PDF PubMed Scopus (1313) Google Scholar, 34Hawley S.A. Pan D.A. Mustard K.J. Ross L. Bain J. Edelman A.M. Frenguelli B.G. Hardie D.G. Cell Metab. 2005; 2: 9-19Abstract Full Text Full Text PDF PubMed Scopus (1254) Google Scholar, 35Woods A. Dickerson K. Heath R. Hong S.P. Momcilovic M. Johnstone S.R. Carlson M. Carling D. Cell Metab. 2005; 2: 21-33Abstract Full Text Full Text PDF PubMed Scopus (1049) Google Scholar). It has been proposed that LKB1 activity is constitutive, such that AMP binding to AMPK inhibits dephosphorylation at Thr-172, permitting phosphorylation and activation by LKB1 (36Sanders M.J. Grondin P.O. Hegarty B.D. Snowden M.A. Carling D. Biochem. J. 2007; 403: 139-148Crossref PubMed Scopus (517) Google Scholar). Using a phospho-Thr-172-specific anti-AMPK antibody, we demonstrated that rosiglitazone also stimulates phosphorylation of AMPK at Thr-172 in a time- and concentration-dependent manner (Fig. 1C), in close agreement with the AMPK assay data (Fig. 1, A and B). We next determined whether LKB1 activity was necessary for rosiglitazone-stimulated AMPK activity. HeLa cells do not express endogenous LKB1; therefore we determined the effect of rosiglitazone on AMPK activity and AMPK Thr-172 phosphorylation in HeLa cells stably expressing wild type (LKB1-WT) or kinase-inactive mutant LKB1 (LKB1-KD) (24Sapkota G.P. Deak M. Kieloch A. Morrice N. Goodarzi A.A. Smythe C. Shiloh Y. Lees-Miller S.P. Alessi D.R. Biochem. J. 2002; 368: 507-516Crossref PubMed Scopus (91) Google Scholar). Rosiglitazone-stimulated AMPK activity and AMPK Thr-172 phosphorylation were apparent within 15 min in cells expressing LKB1-WT, but no effect of rosiglitazone was apparent in HeLa cells expressing LKB1-KD (Fig. 2, A and B). Furthermore, we measured the concentrations of AMP, ADP, and ATP in extracts from rosiglitazone-stimulated HAECs by HPLC. Using this method, the AMP concentration was too low to measure accurately, but the ADP/ATP ratio was rapidly and significantly stimulated by 20 μm rosiglitazone and was stimulated maximally by 200 μm rosiglitazone (1.26-fold, Fig. 2C). This effect of rosiglitazone was sustained for 24 h as the ADP/ATP ratio increased from 0.12 ± 0.01 under basal conditions to 0.185 ± 0.005 after 24 h of incubation with 100 μm rosiglitazone. To determine whether rosiglitazone was able to directly increase LKB1 activity (independent of adenine nucleotide ratios), we determined the activity of LKB1 after stimulation of HAECs with rosiglitazone. Rosiglitazone had no significant effect on LKB1 activity as assessed by its ability to activate recombinant AMPK kinase domain. Basal LKB1 activity was 1.51 ± 0.06 nmol 32P-incorporated/min/mg protein. In the presence of rosiglitazone, LKB1 activity was 1.62 ± 0.51 nmol/min/mg.FIGURE 2The effect of rosiglitazone on LKB1-mediated AMPK activity and ADP/ATP. HeLa cells stably expressing LKB1-WT or LKB1-KD were treated with 1 μg/ml tetracycline 24 h prior to incubation with 100 μm rosiglitazone for the indicated durations and AMPK assayed (A) or AMPK Thr-172 phosphorylation assessed in cell lysates (B). C, nucleotide extracts were prepared from HAECs incubated with the indicated concentrations of rosiglitazone for 1 h. ADP and ATP were separated and quantified by HPLC. Data shown represent the AMPK activity ± S.E. Representative immunoblots are from three independent experiments, and ADP/ATP ratio is ± S.E. *, p < 0.05 relative to value in absence of rosiglitazone.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We have recently demonstrated that HAECs express CaMKK and that CaMKK mediates vascular endothelial growth factor-stimulated AMPK activation in HAECs (27Reihill J.R. Ewart M.A. Hardie D.G. Salt I.P. Biochem. Biophys. Res. Commun. 2007; 354: 1084-1088Crossref PubMed Scopus (78) Google Scholar). Preincubation of cells with the CaMKK inhibitor STO-609 had no significant effect on rosiglitazone-stimulated AMPK activity, AMPK Thr-172 phosphorylation, or phosphorylation of the AMPK substrate acetyl CoA carboxylase (ACC), yet completely inhibited vascular endothelial growth factor-stimulated AMPK activity (Fig. 3).FIGURE 3STO-609 has no effect on rosiglitazone-stimulated AMPK activity. AMPK activity (A) and ACC phosphorylation and AMPK Thr-172 phosphorylation (B) were determined in lysates prepared from HAECs incubated with 100 μm rosiglitazone for 1 h or 10 ng/ml vascular endothelial growth factor (VEGF) for 5 min after preincubation in the presence or absence of 25 μm STO-609 for 40 min. Data shown represent the mean % basal ± S.E. AMPK activity, and representative immunoblots are from three independent experiments. *, p < 0.05 relative to value in absence of STO-609.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether AMPK activation was required for rosiglitazone-stimulated NO synthesis, HAECs were infected with control (Ad.Null) adenoviruses or adenoviruses expressing dominant negative AMPK (Ad.α1DN) prior to incubation with rosiglitazone (200 μm) for 1 or 24 h, and NO production was assessed. HAECs infected with Ad.α1DN exhibited significantly attenuated rosiglitazone (1 h)-stimulated NO production (Fig. 4A) compared with control virus-infected cells. In HAECs incubated with rosiglitazone for 24 h there was a non-significant reduction in NO synthesis in cells infected with Ad.α1DN compared with control virus-infected cells. Insulin has previously been demonstrated to stimulate NO synthesis by protein kinase B-mediated phosphorylation and activation of eNOS, independent of AMPK (19Morrow V.A. Foufelle F. Connell J.M.C. Petrie J.R. Gould G.W. Salt I.P. J. Biol. Chem. 2003; 278: 31629-31639Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 37Montagnani M. Chen H. Barr V.A. Quon M.J. J. Biol. Chem. 2001; 276: 30392-30398Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). Infection of HAECs had no effect on insulin-stimulated (1 μm, 10 min) NO synthesis, indicating that the effect of infection with Ad.α1DN did not result in the nonspecific down-regulation of NO synthesis in an AMPK-independent manner (Fig. 4A). Infection with Ad.α1DN markedly attenuated rosiglitazone-stimulated AMPK activity as assessed by ACC phosphorylation and was without effect on eNOS expression (Fig. 4B).FIGURE 4R