Adiponectin Stimulates Production of Nitric Oxide in Vascular Endothelial Cells
2003; Elsevier BV; Volume: 278; Issue: 45 Linguagem: Inglês
10.1074/jbc.m307878200
ISSN1083-351X
AutoresHui Chen, Monica Montagnani, Tohru Funahashi, Iichiro Shimomura, Michael J. Quon,
Tópico(s)Adipose Tissue and Metabolism
ResumoAdiponectin is secreted by adipose cells and mimics many metabolic actions of insulin. However, mechanisms by which adiponectin acts are poorly understood. The vascular action of insulin to stimulate endothelial production of nitric oxide (NO), leading to vasodilation and increased blood flow is an important component of insulin-stimulated whole body glucose utilization. Therefore, we hypothesized that adiponectin may also stimulate production of NO in endothelium. Bovine aortic endothelial cells in primary culture loaded with the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2 DA) were treated with lysophosphatidic acid (LPA) (a calcium-releasing agonist) or adiponectin (10 μg/ml bacterially produced full-length adiponectin). LPA treatment increased production of NO by ∼4-fold. Interestingly, adiponectin treatment significantly increased production of NO by ∼3-fold. Preincubation of cells with wortmannin (phosphatidylinositol 3-kinase inhibitor) blocked only adiponectin- but not LPA-mediated production of NO. Using phospho-specific antibodies, we observed that either adiponectin or insulin treatment (but not LPA treatment) caused phosphorylation of both Akt at Ser473 and endothelial nitric-oxide synthase (eNOS) at Ser1179 that was inhibitable by wortmannin. We next transfected bovine aortic endothelial cells with dominant-inhibitory mutants of Akt (Akt-AAA) or AMP-activated protein kinase (AMPK) (AMPKK45R). Neither mutant affected production of NO in response to LPA treatment. Importantly, only AMPKK45R, but not Akt-AAA, caused a significant partial inhibition of NO production in response to adiponectin. Moreover, AMPK-K45R inhibited phosphorylation of eNOS at Ser1179 in response to adiponectin but not in response to insulin. We conclude that adiponectin has novel vascular actions to directly stimulate production of NO in endothelial cells using phosphatidylinositol 3-kinase-dependent pathways involving phosphorylation of eNOS at Ser1179 by AMPK. Thus, the effects of adiponectin to augment metabolic actions of insulin in vivo may be due, in part, to vasodilator actions of adiponectin. Adiponectin is secreted by adipose cells and mimics many metabolic actions of insulin. However, mechanisms by which adiponectin acts are poorly understood. The vascular action of insulin to stimulate endothelial production of nitric oxide (NO), leading to vasodilation and increased blood flow is an important component of insulin-stimulated whole body glucose utilization. Therefore, we hypothesized that adiponectin may also stimulate production of NO in endothelium. Bovine aortic endothelial cells in primary culture loaded with the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2 DA) were treated with lysophosphatidic acid (LPA) (a calcium-releasing agonist) or adiponectin (10 μg/ml bacterially produced full-length adiponectin). LPA treatment increased production of NO by ∼4-fold. Interestingly, adiponectin treatment significantly increased production of NO by ∼3-fold. Preincubation of cells with wortmannin (phosphatidylinositol 3-kinase inhibitor) blocked only adiponectin- but not LPA-mediated production of NO. Using phospho-specific antibodies, we observed that either adiponectin or insulin treatment (but not LPA treatment) caused phosphorylation of both Akt at Ser473 and endothelial nitric-oxide synthase (eNOS) at Ser1179 that was inhibitable by wortmannin. We next transfected bovine aortic endothelial cells with dominant-inhibitory mutants of Akt (Akt-AAA) or AMP-activated protein kinase (AMPK) (AMPKK45R). Neither mutant affected production of NO in response to LPA treatment. Importantly, only AMPKK45R, but not Akt-AAA, caused a significant partial inhibition of NO production in response to adiponectin. Moreover, AMPK-K45R inhibited phosphorylation of eNOS at Ser1179 in response to adiponectin but not in response to insulin. We conclude that adiponectin has novel vascular actions to directly stimulate production of NO in endothelial cells using phosphatidylinositol 3-kinase-dependent pathways involving phosphorylation of eNOS at Ser1179 by AMPK. Thus, the effects of adiponectin to augment metabolic actions of insulin in vivo may be due, in part, to vasodilator actions of adiponectin. Adiponectin is one of a number of proteins secreted by adipose cells (e.g. tumor necrosis factor-α, IL-6, leptin, resistin) that may couple regulation of insulin sensitivity with energy metabolism and serve to link obesity with insulin resistance (1Scherer P.E. Williams S. Fogliano M. Baldini G. Lodish H.F. 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Nature. 2003; 423: 762-769Crossref PubMed Scopus (2721) Google Scholar), and at least some of the biological actions of adiponectin are mediated through activation of AMPK 1The abbreviations used are: AMPK, AMP-activated protein kinase; NO, nitric oxide; eNOS, endothelial nitric-oxide synthase; iNOS, inducible nitric-oxide synthase; BAEC, bovine aortic endothelial cells; PI, phosphatidylinositol; MANOVA, multiple analysis of variance; LPA, lysophosphatidic acid; WT, wild type; RFP, red fluorescent protein; DAF-2 DA, 4,5-diaminofluorescein diacetate. (13Yamauchi T. Kamon J. Minokoshi Y. Ito Y. Waki H. Uchida S. Yamashita S. Noda M. Kita S. Ueki K. Eto K. Akanuma Y. Froguel P. Foufelle F. Ferre P. Carling D. Kimura S. Nagai R. Kahn B.B. Kadowaki T. Nat. Med. 2002; 8: 1288-1295Crossref PubMed Scopus (3537) Google Scholar, 25Tomas E. Tsao T.S. Saha A.K. Murrey H.E. Zhang Cc C. Itani S.I. Lodish H.F. Ruderman N.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16309-16313Crossref PubMed Scopus (860) Google Scholar). Insulin has important vascular actions to stimulate production of nitric oxide (NO) in endothelium, leading to increased blood flow that contributes significantly to insulin-mediated glucose uptake (26Baron A.D. Quon M.J. Reaven G. Laws A. Insulin Resistance. Humana Press Inc., Totowa, NJ1999: 247-263Crossref Google Scholar, 27Zeng G. Quon M.J. J. Clin. Invest. 1996; 98: 894-898Crossref PubMed Scopus (669) Google Scholar). Insulin signaling pathways in vascular endothelium regulating production of NO share striking similarities with metabolic insulin signaling pathways in skeletal muscle and adipose tissue (27Zeng G. Quon M.J. J. Clin. Invest. 1996; 98: 894-898Crossref PubMed Scopus (669) Google Scholar, 28Zeng G. Nystrom F.H. Ravichandran L.V. Cong L. Kirby M. Mostowski H. Quon M.J. Circulation. 2000; 101: 1539-1545Crossref PubMed Scopus (671) Google Scholar, 29Montagnani M. Chen H. Barr V.A. Quon M.J. J. Biol. Chem. 2001; 276: 30392-30398Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 30Montagnani M. Ravichandran L.V. Chen H. Esposito D.L. Quon M.J. Mol. Endocrinol. 2002; 16: 1931-1942Crossref PubMed Scopus (204) Google Scholar, 31Montagnani M. Quon M.J. Diabetes Obes. Metab. 2000; 2: 285-292Crossref PubMed Scopus (94) Google Scholar). Therefore, we hypothesized that adiponectin may exert some of its insulinomimetic actions by stimulating phosphorylation and activation of eNOS in vascular endothelium, resulting in increased production of NO. Demonstrating a novel role for adiponectin in eNOS activation may be helpful for explaining both metabolic and anti-atherogenic properties of adiponectin. This may also give insight into the molecular basis of the relationships among insulin resistance, obesity, atherosclerosis, and other vascular complications of diabetes. Purification of Recombinant Adiponectin—Recombinant full-length human adiponectin protein was produced in bacteria and purified as described previously (6Arita Y. Kihara S. Ouchi N. Takahashi M. Maeda K. Miyagawa J. Hotta K. Shimomura I. Nakamura T. Miyaoka K. Kuriyama H. Nishida M. Yamashita S. Okubo K. Matsubara K. Muraguchi M. Ohmoto Y. Funahashi T. Matsuzawa Y. Biochem. Biophys. Res. Commun. 1999; 257: 79-83Crossref PubMed Scopus (4133) Google Scholar). Plasmid Constructs—The plasmid constructs were as follows: pCIS2-RFP, cDNA for red fluorescent protein was subcloned into pCIS2 expression vector as described (29Montagnani M. Chen H. Barr V.A. Quon M.J. J. Biol. Chem. 2001; 276: 30392-30398Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar); Akt-AAA, pCIS2 expression vector containing cDNA for dominant-inhibitory mutant of mouse Akt containing substitutions K179A, T308A, and S473A as described (29Montagnani M. Chen H. Barr V.A. Quon M.J. J. Biol. Chem. 2001; 276: 30392-30398Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar); eNOS-WT and eNOS-S1179A, pCIS2 expression vectors containing cDNA for wild-type and mutant bovine eNOS as described (29Montagnani M. Chen H. Barr V.A. Quon M.J. J. Biol. Chem. 2001; 276: 30392-30398Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar); AMPK-WT and AMPK-K45R, pcDNA3 expression vectors containing cDNA for wild-type and mutant rat AMPKα2 (generous gift from M. Birnbaum). Cell Culture and Transfection—Bovine aortic endothelial cells (BAEC) in primary culture (Cell Applications; San Diego, CA) were grown in EGM-2 as described (29Montagnani M. Chen H. Barr V.A. Quon M.J. J. Biol. Chem. 2001; 276: 30392-30398Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar) and used between passages 3 and 5. Transient transfections were performed using LipofectAMINE Plus (Invitrogen) according to the manufacturer's protocol. For immunoblotting experiments, BAEC were serum-starved overnight with EBM-A (red phenol-free endothelial basal medium from Clonetics Corp. supplemented with 1% platelet-deprived horse serum (Sigma)) prior to initiation of experiments. For measurement of NO production, BAEC were serum-starved for 2 h in EBM-A medium supplemented with 1% platelet-deprived horse serum. NIH-3T3IR cells (NIH-3T3 fibroblasts stably transfected with human insulin receptors) were cultured as described (32Quon M.J. Cama A. Taylor S.I. Biochemistry. 1992; 31: 9947-9954Crossref PubMed Scopus (24) Google Scholar). Measurement of NO Production in Living Cells—Production of NO was assessed using the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2 DA, Calbiochem) as described (29Montagnani M. Chen H. Barr V.A. Quon M.J. J. Biol. Chem. 2001; 276: 30392-30398Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). Briefly, BAEC grown at 95% confluence were serum-starved for 2 h in EBM-A. l-Arginine (100 μm) was added 1 h prior to each study. Cells were loaded with DAF-2 DA (final concentration 5 μm, 20 min, 37°C) and then rinsed three times, kept in the dark, and maintained at 37°C with a warming stage (Bioptechs, Inc.) on a Zeiss Axiovert S100 TV inverted microscope (Carl Zeiss Inc.; Thornwood, NY). Cells were then treated sequentially with lysophosphatidic acid (LPA, 5 μm) or insulin (250 nm) and adiponectin (10 μg/ml). In some experiments, wortmannin (100 nm) was added 30 min before loading with DAF-2 DA. In other experiments, BAEC were co-transfected first with RFP and either Akt-AAA or AMPK-K45R. Green fluorescence intensity was quantified using IP Labs software (Scanalytics Inc.; Fairfax, VA). Data for each experiment were normalized to a reference image of the basal state. Immunoblotting—BAEC or NIH-3T3IR cells transiently transfected with various plasmids as indicated in the figure legends were serum-starved overnight and treated with either insulin (100 nm, 5 min) or adiponectin (10 μg/ml, 5 min). In some experiments, wortmannin (100 nm) was added to cells 1 h before treating with insulin or adiponectin. Cell lysates were prepared using 300 μl of lysis buffer (100 mm NaCl, 20 mm Hepes, pH 7.9, 1% Triton X-100, 1 mm Na3VO4, 4 mm sodium pyrophosphate, 10 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 10 mm NaF, and the complete protease inhibitor mixture (Roche Applied Science). Samples (50 μg of total protein) were separated by 8% SDS-PAGE and immunoblotted with antibodies against eNOS (Transduction Laboratories; Lexington, KY), Akt (Upstate Biotechnology, Inc.; Lake Placid, NY), phospho-eNOSS1177 (Cell Signaling Technology; Beverly, MA), phospho-AktS473 (Cell Signaling Technology), AMPK (Upstate Biotechnology, Inc.), or phospho-AMPKT172 (Cell Signaling Technology) according to standard methods. Blots were quantified by scanning densitometry (Amersham Biosciences). Statistics—Paired t tests were used where appropriate. For comparison between various time courses of NO production, multiple analysis of variance (MANOVA) was employed. p values less than 0.05 were considered to represent statistical significance. Adiponectin-stimulated Production of NO in BAEC Requires PI 3-Kinase—To determine whether adiponectin can stimulate production of NO in vascular endothelial cells, we employed our previously established method using the NO-specific fluorescent dye DAF-2 DA to assess NO production in BAEC in primary culture (29Montagnani M. Chen H. Barr V.A. Quon M.J. J. Biol. Chem. 2001; 276: 30392-30398Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 30Montagnani M. Ravichandran L.V. Chen H. Esposito D.L. Quon M.J. Mol. Endocrinol. 2002; 16: 1931-1942Crossref PubMed Scopus (204) Google Scholar). The classical mechanism for activation of eNOS involves increased levels of intracellular calcium. Therefore, we used LPA (a phospholipid growth factor that stimulates release of intracellular Ca2+) as a positive control for the production of NO in BAEC. As reported previously (29Montagnani M. Chen H. Barr V.A. Quon M.J. J. Biol. Chem. 2001; 276: 30392-30398Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 30Montagnani M. Ravichandran L.V. Chen H. Esposito D.L. Quon M.J. Mol. Endocrinol. 2002; 16: 1931-1942Crossref PubMed Scopus (204) Google Scholar), LPA treatment of BAEC caused a rapid, ∼4-fold increase in NO production (Fig. 1, A and B, closed circles). Interestingly, when these same cells were subsequently treated with adiponectin (10 μg/ml bacterially produced adiponectin), we observed a significant ∼3-fold increase in production of NO with a distinct time course (Fig. 1, A and B, closed triangles). Similar results were obtained when the order of LPA and adiponectin treatment was reversed (data not shown). Thus, adiponectin has novel vascular actions to acutely stimulate production of NO in vascular endothelium. Moreover, when BAEC were preincubated with the PI 3-kinase inhibitor wortmannin, the production of NO in response to LPA was unaffected, but the action of adiponectin to stimulate NO was completely blocked (Fig. 1C). Therefore, similar to insulin (27Zeng G. Quon M.J. J. Clin. Invest. 1996; 98: 894-898Crossref PubMed Scopus (669) Google Scholar, 28Zeng G. Nystrom F.H. Ravichandran L.V. Cong L. Kirby M. Mostowski H. Quon M.J. Circulation. 2000; 101: 1539-1545Crossref PubMed Scopus (671) Google Scholar, 29Montagnani M. Chen H. Barr V.A. Quon M.J. J. Biol. Chem. 2001; 276: 30392-30398Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar), the ability of adiponectin to stimulate production of NO in endothelium requires PI 3-kinase activity. Adiponectin Phosphorylates Akt and eNOS in a PI 3-Kinase-dependent Manner—The activation of eNOS in response to insulin involves a calcium-independent, phosphorylation-dependent mechanism requiring phosphorylation and activation of Akt that then directly phosphorylates eNOS at Ser1179, leading to activation of eNOS (29Montagnani M. Chen H. Barr V.A. Quon M.J. J. Biol. Chem. 2001; 276: 30392-30398Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 30Montagnani M. Ravichandran L.V. Chen H. Esposito D.L. Quon M.J. Mol. Endocrinol. 2002; 16: 1931-1942Crossref PubMed Scopus (204) Google Scholar). Since the production of NO in response to adiponectin depends on PI 3-kinase (Fig. 1), we next inquired whether adiponectin treatment of endothelial cells results in phosphorylation of Akt and eNOS. As expected, insulin stimulated a significant increase in phosphorylation of Akt at Ser473 and eNOS at Ser1179 in BAEC that was blocked by pretreatment with wortmannin (Fig. 2). Interestingly, adiponectin treatment of BAEC also resulted in phosphorylation of Akt at Ser473 and eNOS at Ser1179 at levels that were similar to those elicited by insulin. Moreover, both Akt and eNOS phosphorylation in response to adiponectin was blocked by wortmannin pretreatment. Thus, similar to insulin (29Montagnani M. Chen H. Barr V.A. Quon M.J. J. Biol. Chem. 2001; 276: 30392-30398Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar), adiponectin stimulates phosphorylation of both Akt and eNOS in a PI 3-kinase-dependent manner. Role for AMPK but Not Akt in Adiponectin-stimulated Production of NO—Both Akt and AMPK are capable of phosphorylating eNOS at Ser1179 (33Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3080) Google Scholar, 34Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. 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Diabetes. 2003; 52: 1355-1363Crossref PubMed Scopus (395) Google Scholar), we used dominant-inhibitory mutants of Akt and AMPK to explore the roles of these serine kinases in production of NO in response to adiponectin in endothelial cells. We transiently co-transfected BAEC with Akt-AAA and pCIS2-RFP, loaded the cells with DAF-2 DA, and stimulated the cells with LPA and adiponectin (Fig. 3A). Transfected cells were distinguished from non-transfected cells in the same field by their expression of RFP. As demonstrated previously (29Montagnani M. Chen H. Barr V.A. Quon M.J. J. Biol. Chem. 2001; 276: 30392-30398Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar), LPA-stimulated production of NO was not affected by expression of Akt-AAA. That is, the time courses for production of NO in response to LPA in cells transfected with Akt-AAA (Fig. 3A, open circles) and untransfected cells (Fig. 3A, closed circles) from the same experimental preparation were comparable (p > 0.69). Similarly, expression of Akt-AAA in BAEC did not affect adiponectin-stimulated production of NO so that the time course for production of NO in response to adiponectin in cells transfected with Akt-AAA (Fig. 3A, open triangles) and untransfected cells (Fig. 3A, closed triangles) were comparable (p > 0.56). Thus, although Akt is phosphorylated in response to adiponectin in BAEC (Fig. 2, A and B), Akt does not appear to play a role in adiponectin-stimulated production of NO in endothelial cells. We next transiently co-transfected BAEC with AMPK-K45R and pCIS2-RFP, loaded the cells with DAF-2 DA, and stimulated the cells with insulin and adiponectin (Fig. 3B). Insulin-stimulated production of NO in cells transfected with AMPKK45R (Fig. 3B, open circles) was comparable with that in untransfected cells (Fig. 3A, closed circles; p > 0.37). Stimulating transfected and untransfected cells with LPA gave similar results (data not shown). By contrast, expression of AMPKK45R in BAEC partially, but significantly, inhibited adiponectin-stimulated production of NO when compared with untransfected cells in the same dish (Fig. 3B, open and closed triangles, respectively; p < 0.02). Taken together, these results suggest that adiponectin-stimulated production of NO does not require Akt but depends, in part, on activation of AMPK. Adiponectin-stimulated Phosphorylation of eNOS Is Mediated by AMPK—Adiponectin stimulates phosphorylation of eNOS at Ser1179 (an AMPK phosphorylation site) (Fig. 2, C and D), and production of NO in response to adiponectin depends, in part, on AMPK (Fig. 3B). Therefore, we next tested whether AMPK is necessary for the ability of adiponectin to stimulate phosphorylation of eNOS. NIH-3T3IR cells transiently co-transfected with expression vectors for eNOS and either wild-type AMPK or AMPK-K45R were treated with adiponectin or insulin. Cell lysates from each group were immunoblotted with antibodies against phospho-eNOSS1179, eNOS, and AMPK (Fig. 4A). Control cells transfected with an empty expression vector did not have detectable levels of endogenous eNOS but showed low levels of endogenous AMPK (Fig. 4A, lane 1). As expected, both adiponectin and insulin stimulation significantly increased phosphorylation of eNOS at Ser1179 in cells co-transfected with eNOS and wild-type AMPK (Fig. 4, A and B, lanes 3 and 4). Interestingly, in cells co-transfected with eNOS and the dominant-inhibitory mutant AMPK-K45R, phosphorylation of eNOS in response to adiponectin was significantly inhibited (Fig. 4, lane 3 versus lane 5; p < 0.03), whereas the response to insulin was unaffected (Fig. 4, A and B, lane 4 versus lane 6; p > 0.50). 5-Aminoimidazole-4-carboxamide-1-β-d-riboside, a chemical activator of AMPK, also significantly increased eNOS phosphorylation in cells co-transfected with eNOS and wild-type AMPK (Fig. 4, A and B, lane 7). In related experiments, we co-transfected NIH-3T3IR cells with eNOS and wild-type AMPK and treated cells with adiponectin or insulin in the absence and presence of wortmannin (Fig. 4C). When cell lysates were immunoblotted with a phospho-specific antibody against AMPKT172, we observed that adiponectin, but not insulin, stimulated phosphorylation of AMPK. Moreover, the phosphorylation of AMPK in response to adiponectin was inhibited by wortmannin pretreatment. Taken together, these results provide additional support for the role of AMPK in phosphorylation and activation of eNOS in response to adiponectin in a PI 3-kinase-dependent manner. Since the discovery of adiponectin (1Scherer P.E. Williams S. Fogliano M. Baldini G. Lodish H.F. J. Biol. Chem. 1995; 270: 26746-26749Abstract Full Text Full Text PDF PubMed Scopus (2791) Google Scholar,
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