Adiponectin Blocks Interleukin-18-mediated Endothelial Cell Death via APPL1-dependent AMP-activated Protein Kinase (AMPK) Activation and IKK/NF-κB/PTEN Suppression*

The adipocyte-derived cytokine adiponectin is known to exert anti-inflammatory and anti-apoptotic effects. In patients with atherosclerotic cardiovascular disease, circulating levels of adiponectin correlate inversely with those of the proinflammatory, proapoptotic cytokine interleukin (IL)-18. The opposing actions of IL-18 and adiponectin on both cell survival and inflammation led us to investigate whether adiponectin signaling antagonizes IL-18-mediated endothelial cell death and to identify the underlying molecular mechanisms. Treatment with IL-18 suppressed Akt phosphorylation and its associated kinase activity, induced IκB kinase (IKK)-NF-κB-dependent PTEN activation, and promoted endothelial cell death. Pretreatment with adiponectin stimulated APPL1-dependent AMPK activation, reversed Akt inhibition in a phosphatidylinositol 3-kinase-dependent manner, blocked IKK-NF-κB-PTEN signaling, reduced caspase-3 activity, blocked Bax translocation, and inhibited endothelial cell death. The cytoprotective effect of adiponectin signaling was recapitulated by treatment with the pharmacological AMPK activator 5-aminoimidazole-4-carboxamide-1-β-riboside. Collectively, these results demonstrated that adiponectin reverses IL-18-mediated endothelial cell death through an AMPK-associated mechanism, which may thus have therapeutic potential for diminishing IL-18-dependent vascular injury and inflammation.

The vascular endothelium plays a critical role in the maintenance of normal vascular function and homeostasis (1). In the healthy vessel, endothelial cells provide an anti-thrombotic and anti-inflammatory barrier to the circulating components of the blood. Dysfunction, injury, or death of vascular endothelial cells contributes to the development and progression of inflammatory vascular diseases such as atherosclerosis (1). In particular, apoptotic endothelial cells are observed within atherosclerotic plaques, and the endothelial turnover rate is accelerated in atherosclerotic-prone regions (2). Further, activated macrophages and endothelial cells specifically express potent cytokines that enhance inflammation by suppression of endothelial nitric-oxide synthase (eNOS), 2 induction of adhesion molecules, promotion of platelet adhesion, and recruitment of immune cells to the site of injury (3,4). Cytokine-induced endothelial cell dysfunction and death has also been shown to correlate with plaque instability, rupture, and thrombus formation (5,6).
Aberrant expression of the proinflammatory cytokine, interleukin-18 plays a causal role in numerous autoimmune disorders and inflammatory diseases. For example, systemic levels of IL-18 are increased in acute coronary syndromes (7,8), and correlate directly with intima-media thickening (9,10). Additionally, the expression of both IL-18 and its receptor is increased in atherosclerotic lesions (11). Since the expression of the IL-18 receptor by the vascular endothelium within atherosclerotic lesions is increased, both systemic and locally produced IL-18 may contribute to endothelial cell dysfunction and death.
Adiponectin, also known as Acrp30 (adipocyte complementrelated protein-30 kDa), adipoQ (gene encoding adiponectin), apM1 (AdiPose most abundant gene transcript 1), and GBP28 (gelatin-binding protein of 28 kDa), is an anti-inflammatory cytokine synthesized and secreted primarily by adipocytes (12). It is present in plasma in quantities that range from 3 to 30 g/ml. It exists in oligomeric forms of low, medium, and high molecular weight. Although the anti-inflammatory effects of the low and intermediate molecular weight forms have not been completely defined, the high molecular weight form has been shown to exert both anti-inflammatory and anti-apoptotic effects (13). In addition, a globular form, derived by the proteolytic cleavage of the whole molecule and containing the C-terminal globular domain, has also been shown to exert potent cytoprotective effects (14). Although found in high levels in the plasma under normal physiological conditions, adiponectin is significantly decreased in obesity, obesity-linked insulin resistance, type 2 diabetes, and metabolic syndrome (12,15,16). In addition, hypoadiponectinemia is characteristic of chronic inflammatory diseases such as coronary artery disease (17). However, secretion of other adipokines such as leptin, tumor necrosis factor ␣ (TNF-␣), and nerve growth factor are not affected under these conditions and in fact are increased, suggesting a specific association between chronic inflammation and hypoadiponectinemia (18,19).
Adiponectin exerts its biological effects via binding to two structurally and functionally distinct, G protein-coupled, seven-transmembrane receptors, adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) (20). Unlike other G protein-coupled receptors, the N termini of both receptors are cytoplasmic, whereas their C termini are extracellular (20). Like adiponectin, both AdipoR1 and AdipoR2 are down-regulated in obesity, obesity-linked insulin resistance, and diabetes (21), plainly indicating that these chronic inflammatory states are associated with reduced expression of both adiponectin and its cell surface receptors. A wide variety of cell types constitutively express adiponectin receptors, including the two key cellular lineages involved in vascular disease, endothelial cells and smooth muscle cells (20). Adiponectin inhibits endothelial cell dysfunction through stimulation of endothelial nitric oxide (NO) and endothelium-dependent vasodilation, suppression of oxidative stress, and inhibition of cytokine, chemokine, and adhesion molecule expression (22). The cytoprotective action of adiponectin signaling is further demonstrated by adiponectin-deficient mice maintained on a high-fat diet, which remarkably exhibit reduced endothelium-dependent vasodilation (23). Similarly, endothelial dysfunction in obese mice is significantly ameliorated following adenovirus-mediated overexpression of full-length adiponectin (24). In addition, adiponectin is reported to promote endothelial cell survival and growth (25).

Materials-Recombinant
Cell Culture-Nontransformed human cardiac microvascular endothelial cells were obtained from ScienCell Research Laboratories (San Diego, CA) and cultured as described previously (26,27). Briefly, the cells were grown in endothelial cell medium (ECM) supplied by the manufacturer and supplemented with 5% serum (complete medium). At 70 -80% confluence, the complete medium was replaced with serum-free medium containing 0.5% bovine serum albumin, and after overnight incubation, recombinant human (rh) IL-18 (R&D Systems) was added for the indicated time periods. The specificity of IL-18 was verified by preincubating IL-18 with anti-IL-18-neutralizing antibodies for 1 h at 37°C and for 14 h at 4°C before the addition of IL-18. Cells were harvested, snap-frozen, and stored at Ϫ80°C.
NF-B Activation-Determination of NF-B activation and its subunit composition in nuclear protein extracts was performed as described previously using a TransAM TM NFB transcription factor ELISA (26). The purity of nuclear extracts was verified by immunoblotting using anti-lamin A/C and tubulin antibodies. Activation of NF-B was confirmed by reporter gene assays. An adenoviral NF-B-luciferase reporter construct (Ad.NFB-Luc; generously provided by John F. Engelhardt, University of Iowa, Iowa City, IA), containing the luciferase gene driven by four tandem copies of the NFB consensus binding sequence fused to a TATA-like promoter from the herpes simplex virus-thymidine kinase gene, was used as described previously (28). The empty adenoviral construct (Ad.MCS-Luc; Vector Biolabs, Philadelphia, PA) served as a base-line control.
mRNA Analysis-DNA-free total RNA was prepared using the RNAqueous-4PCR kit (Ambion). RNA quality was assessed by capillary electrophoresis using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). All RNA samples used for quantitative PCR had RNA integrity numbers greater than 9 (scale ϭ 1-10) as assigned by default parameters of the Expert 2100 Bioanalyzer software package (v2.02). PTEN mRNA expression was analyzed by reverse transcription followed by real-time quantitative PCR using SYBR Green as the detection fluorophore and the following primers: 5Ј-CGGCAGCATCAAATGTTTCAG-3Ј and 5Ј-AACTGGCAG-GTAGAAGGCAACTC-3Ј (32). ␤-Actin mRNA, which served as the invariant control, was amplified using the following primers: sense, 5Ј-TCCTTCCTGGGCATGGAG-3Ј; antisense 5Ј-AGGAGGAGCAATGATCTTGATCTT-3Ј. Samples run without the reverse transcriptase step served as negative controls and gave no signal. Each sample was tested in triplicate.
Protein Analysis-Isolation of whole cell homogenates, electrophoresis, electroblotting, and immunoblot analysis using chemiluminescent detection (ECL) were carried out essentially as described (26 -28). Mitochondrial and cytoplasmic fractions were prepared as described previously (33) using the Mitochondrial Fractionation Kit (Active Motif).
The AMPK activity assay was performed as described previously (30). In brief, EC were treated with adiponectin (30 g/ml) or AICAR (1 mM). The cells were then lysed in buffer A (50 mM HEPES (pH 7.4), 1% Triton, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1 mM dithiothreitol, phosphatase inhibitor mixture, and protease inhibitor mixture). The cell debris was removed by centrifugation at 13,000 rpm for 5 min at 4 C. The supernatant was then adjusted to 10% polyethylene glycol 8000 and incubated for 45 min at 4°C on a rotator. Precipitated proteins were pelleted by centrifugation at 18,000 ϫ g for 15 min and were resuspended in buffer A. Aliquots were assayed for AMPK activity using synthetic substrate for AMPK (SAMS) peptide, corresponding to amino acids 74 -88 of acetyl-coenzyme A carboxylase, in the presence of 100 M AMP. IKK activity was quantified by an in vitro kinase assay that evaluates the ability of immunoprecipitated IKK␤ to phosphorylate GST-IB fusion protein in vitro. The assay was carried out essentially as described by Fan et al. (34). In brief, IL-18treated (for 1 h) EC were washed in ice-cold phosphate-buffered saline and lysed in 1 ml of ice-cold radioimmune precipitation assay buffer (0.15 M NaCl, 50 mM Tris (pH 7.2), 1% deoxycholate, 1% Triton X-100, 0.1% SDS) followed by centrifugation at 10,000 rpm for 10 min at 4°C. Protein concentrations were determined using the Micro BCA TM protein assay kit. 500 g of protein was immunoprecipitated with anti-IKK␤ antibodies and protein A-agarose beads. 1 g of GST-IB fusion protein was then added to washed protein A pellets in the presence of 10 l of kinase buffer (40 mM HEPES, 1 mM ␤-glycerophosphate, 1 mM nitrophenolphosphate, 1 mM Na 3 VO 4 , 10 mM MgCl 2 , 2 mM dithiothreitol, 0.3 mM cold ATP, and 10 Ci of [␥-32 P]ATP) and incubated at 30°C for 30 min. The reaction was terminated by the addition of protein-loading buffer (with SDS) and boiled at 98°C for 5 min. Samples were then centrifuged to remove the agarose beads, and the supernatant was loaded onto a 10% SDS-polyacrylamide gel. After electrophoresis, proteins were transferred to nitrocellulose membrane and exposed to x-ray film (34).
Capase-3 activity was quantified using a highly sensitive assay obtained from Promega (CaspACE TM assay system, Col-orimetric). EC were pretreated with adiponectin for 1 h followed by incubation with IL-18 for 8 h. Cleared cell lysates (5 g) were analyzed for caspase-3 activity according to the manufacturer's instructions.
Cell Death Detection-Following 48 h incubation in ECM plus 0.5% bovine serum albumin, EC were treated with IL-18 (100 ng/ml) for 24 h. Cell viability was assessed by a microplatebased MTT cell viability assay, and viability was expressed as percentage of MTT absorbance at 562/650 nm in the absence of IL-18 (26). Cell death was confirmed by a photometric enzyme immunoassay (cell death detection ELISA PLUS kit, catalog No. 11920685001, Roche Applied Science) (26). The assay is based on the quantitative sandwich enzyme immunoassay principle using mouse monoclonal antibodies directed against DNA and histones. This allows the specific determination of mono-and oligonucleosomes in the cytoplasmic fraction of cell lysates. The samples were placed into a streptavidin-coated microplate and incubated with a mixture of anti-histone-biotin and anti-DNA-peroxidase. During the incubation, nucleosomes were captured via their histone component by the anti-histone-biotin antibody while binding to the streptavidin-coated microplate. Simultaneously, anti-DNA-peroxidase binds to the DNA part of the nucleosomes. After removal of the unbound antibodies, the amount of peroxidase retained in the immunocomplex was photometrically determined with ABTS (2,2Ј-azinodi-(3 ethylbenzthiazolinesulfonic acid)) as the substrate.
Statistical Analysis-Results are expressed as means Ϯ S.E. For statistical analysis we used ANOVA followed by an appropriate post hoc multiple comparison test (Tukey method). Data were considered statistically significant at p Ͻ 0.05.

IL-18 Is a Potent Inducer of Endothelial Cell Death-
We have previously reported that IL-18 is a potent proapoptotic cytokine (26 -28, 33). Consistent with these findings, IL-18-treated endothelial cells in this study showed significant cell death (p Ͻ 0.001 versus untreated, n ϭ 12) by the MTT cell viability assay (Fig. 1A). Further, this effect was blunted by treatment with neutralizing antibodies against IL-18 or IL-18R␣ and with the IL-18-binding protein/Fc fragment chimera (IL-18BP/Fc). Serving as respective controls, normal mouse IgG, goat IgG, and Fc failed to modulate IL-18-mediated EC death. Results obtained by the MTT assay were further confirmed by an ELISA that quantifies mono-and oligonucleosomal fragmented DNA in the cytoplasm. IL-18 significantly increased EC death, and as described above, pretreatment with neutralizing IL-18 or IL-18R␣ antibodies and IL-18BP/Fc chimera blunted this response (Fig. 1B). These results reconfirm that binding of IL-18 to its cognate receptor rapidly and potently induces endothelial cell apoptosis (Fig. 1).
IL-18 Suppresses Akt Activity but Stimulates IKK-dependent NF-B Activation in Endothelial Cells-Akt, or protein kinase B, is a serine-threonine kinase that is critical for fundamental cellular processes including cell survival and proliferation (35). Because IL-18 induces significant cell death in endothelial cells (Fig. 1), we investigated its effects on Akt activation. IL-18 treatment suppressed both Akt phosphorylation ( Fig. 2A) and associated kinase activity (Fig. 2B) in EC. Activation of NF-B is known to potentiate either cell death or survival in a cell typeand stimulus-specific manner. Our previous studies of IL-18 actions on the vascular endothelium have demonstrated that IL-18 induces endothelial cell death via NF-B activation (26,27,33). Because IKK is a key upstream regulator of NF-B activation, we first investigated whether IL-18 treatment induces IKK activity. The results in Fig. 2B show that IL-18 induced IKK␤ activity and the adenoviral transduction of dnIKK␤ blunted this effect (Fig. 2C). Pretreatment with SC-514, an IKK␤-specific inhibitor, significantly attenuated IL-18-mediated IKK␤ activity (Fig. 2D). Further, an ELISA revealed activation of NF-B following IL-18 treatment, comprising both the p50 and p65 subunits (Fig. 2E). The purity of the nuclear protein extracts was verified by immunoblotting, which demonstrated positive signals for lamin A/C but not for tubulin (Fig.  2F). Confirming our ELISA results (Fig. 2D), the reporter assays also demonstrated activation of NF-B by IL-18 (Fig. 2F), and Quiescent EC were treated with neutralizing mouse anti-IL-18 or goat anti-IL-18R␣ antibodies or IL-18BP/Fc chimera (10 g/ml for 1 h) prior to the addition of rhIL-18 (100 ng/ml for 24 h). Normal mouse IgG1, goat IgG, and Fc served as the respective controls. Cell viability was assessed by the MTT assay, and the results were expressed as -fold reduction from untreated cells (n ϭ 12/group). *, p Ͻ 0.001 versus untreated; †, p Ͻ 0.01 versus IL-18. B, IL-18-mediated EC death was confirmed by ELISA. EC treated as described in A were analyzed for cell death by quantifying mono-and oligonucleosomal fragmented DNA in the cytoplasmic extracts by ELISA (n ϭ 12/group). *, p Ͻ 0.001 versus untreated; †, p Ͻ 0.001 versus IL-18 by ANOVA. adenoviral transduction of dnIKK␤ or treatment SC-514 blunted IL-18-mediated NF-B activation as evidenced by a significant reduction in nuclear p65 levels (Fig. 2G). Together, these results demonstrate that IL-18 suppresses Akt but stimulates NF-B in an IKK-dependent manner (Fig. 2).
Adiponectin Blocks IL-18-mediated Endothelial Cell Death-Adiponectin, an adipocyte-derived cytokine, is abundant in blood plasma, and via adiponectin receptors 1 and 2 (20), it exerts both anti-inflammatory and anti-apoptotic effects (12). In light of the role of adiponectin as a survival factor, we investigated whether adiponectin modulates IL-18-mediated endothelial cell death. Results in Fig. 4A show that indeed treatment with adiponectin significantly attenuated IL-18-mediated endothelial cell death in a dose-dependent manner. As maximal inhibition was obtained at 30 g/ml, all subsequent experiments were performed using adiponectin at this concentration. Further, adiponectin blunted IL-18-induced caspase-3 activation (Fig. 4B) and Bax translocation (Fig. 4C). Together, these results demonstrate that adiponectin blocks IL-18-mediated EC death (Fig. 4).
Adiponectin Inhibits IL-18-mediated Endothelial Cell Death via APPL1-dependent AMPK Activation-AMP kinase is an evolutionarily conserved serine/threonine kinase and functions mainly as a sensor of cellular energy status (38,39). It is regulated by various stressors and plays a role in cell death and cell survival and growth (38,39). Therefore, we investigated whether adiponectin blunts endothelial cell death via AMPK activation. Indeed, our results show that adiponectin induces AMPK phosphorylation in a time-dependent manner (Fig.  5A) and that kinase activity (Fig. 5B), and adiponectin-mediated AMPK activation is inhibited by compound C (Fig. 5C). Recently, the adapter molecule APPL1 has been shown to link adiponectin receptors to downstream signaling molecules (29,40). Therefore, we investigated whether APPL1 knockdown blunts adiponectin-mediated AMPK activation. In fact, knockdown of APPL1 significantly attenuated adiponectin-mediated AMPK phosphorylation (Fig. 5D), indicating that APPL1 is upstream of AMPK. Further, our results show that adenoviral transduction of dnAMPK, kinase-dead AMPK, and the AMPK inhibitor compound C reversed the prosurvival effects of adiponectin (Fig. 5E) as did AMPK␣1 knockdown ( Fig. 5F; knockdown of AMPK␣1 was confirmed by immunoblotting, righthand panel). Similarly, knockdown of APPL1 reversed the prosurvival effects of adiponectin in IL-18-treated endothelial cells (Fig. 5G). Together, these results demonstrate that adi-

FIGURE 2. IL-18 suppresses Akt activity and stimulates IKK-dependent NF-B activation. A, IL-18 suppresses Akt phosphorylation. Quiescent EC
were treated with rhIL-18 (100 ng/ml for 1 h). Total and phospho-Akt levels in cell lysates were analyzed by immunoblotting using activation-specific antibodies. B, IL-18 suppresses Akt kinase activity. Quiescent EC treated as described in A were analyzed for Akt kinase activity by an immune complex kinase assay using GSK as a substrate. The specificity of IL-18 was determined by incubating EC with IL-18-neutralizing antibodies (10 g/ml for 1 h; lane 3) prior to IL-18 addition. A representative of three independent experiments is shown. C, IL-18 induces IB kinase activity. Quiescent EC treated as described in A were analyzed for IKK kinase activity by an in vitro kinase assay using GST-IB fusion protein as a substrate. A representative of three independent experiments is shown. D, SC-514 blunts IL-18-mediated IKK activation. Quiescent EC were treated with the IKK␤-specific inhibitor SC-514 (100 M in DMSO for 1 h) prior to IL-18 addition. DMSO served as a control. IKK activity was analyzed as in C. E, IL-18 activates NF-B. Quiescent EC treated as described in A were analyzed for NF-B p50 and p65 subunits in the nuclear protein extracts by ELISA (n ϭ 12). *, p Ͻ 0.001 versus the respective untreated cells (n ϭ 12). F, the purity of nuclear extract was confirmed by immunoblotting using anti-lamin A/C and tubulin antibodies (n ϭ 3). G, IL-18-mediated NF-B activation was confirmed by reporter assay. EC transduced with adenoviral NFB reporter vector (Ad.NFB-Luc) were treated with IL-18 for 12 h. Ad.MCS-Luc served as a control. Ad.␤-gal served as an internal control. *, p Ͻ 0.001 versus the respective untreated cells (n ϭ 12). H, IL-18 induces NF-B activation in IKK-dependent manner. EC either transduced with Ad.dnIKK␤ (24 h) or pretreated with SC-514 (1 h) were treated with rhIL-18 for 12 h. Ad.GFP and DMSO served as the respective controls. Nuclear extracts were analyzed for NFBp65 by ELISA. *, p Ͻ 0.001 versus respective untreated cells; †, p Ͻ 0.01 versus IL-18 by ANOVA (n ϭ 12/group). ponectin signals via APPL1 and AMPK and blocks IL-18-mediated EC death via APPL1-AMPK signaling (Fig. 5).

DISCUSSION
Collectively, these studies demonstrate that adiponectin signaling in endothelial cells induces AMPK activation, reverses IL-18-mediated suppression of Akt activation, inhibits IKK-NF-B-dependent PTEN induction, and significantly attenuates IL-18-mediated endothelial cell death. Further, pretreatment of endothelial cells with AICAR, a pharmacological AMPK activator, recapitulated the prosurvival effects of adiponectin stimulation. These results indicate that both adiponectin and AICAR have therapeutic potential for blocking IL-18 signaling, thereby lessening IL-18-dependent vascular injury and inflammation.
Our investigations further demonstrated that adiponectin and AICAR both induce AMPK activation in endothelial cells. AMPK is a multimeric serine/threonine kinase, consisting of ␣-, ␤-, and ␥-subunits, which acts fundamentally as a sensor of cellular energy status (38,39). A wide range of environmental stressors cause reduction in intracellular ATP levels and increases in cellular AMP levels, which serve to activate AMPK via multiple mechanisms including phosphorylation of the ␣-subunit on Thr 172 by upstream kinases (38,39). Activated AMPK then shuts off anabolic pathways while simultaneously activating catabolic pathways. Although AMPK signaling is intricately tied to energy metabolism and homeostasis, recent reports suggest that it is also critical for various physiological processes including inflammation, proliferation, and death (38,39). The experimental results of the present study establish AMPK as a primary mediator of the anti-apoptotic actions of adiponectin on endothelial cells. Demonstrating that adiponectin signaling involves AMPK activation, adiponectin treatment of endothelial cells induced AMPK phosphorylation in a timedependent manner. Firmly establishing the involvement of AMPK, in EC survival, treatment with the AMPK-specific inhibitor compound C, silencing AMPK␣1 expression by siRNA, and adenoviral transduction of both dominant negative and kinase-dead AMPK constructs impaired the ability of adiponectin to protect against IL-18-induced endothelial cell FIGURE 5. Adiponectin blocks IL-18-mediated EC death via AMPK activation. A, adiponectin-mediated AMPK activation is inhibited by compound C. Quiescent EC were treated with adiponectin (30 g/ml) for the indicated time periods. AMPK activation was analyzed by immunoblotting using activationspecific antibodies. A representative of three independent experiments is shown. B, adiponectin stimulates AMP kinase activity. Quiescent EC treated as described in A were analyzed for AMPK activity using an in vitro assay as described under "Experimental Procedures." *, p Ͻ 0.001 versus respective untreated cells (n ϭ 6/group). C, adiponectin-mediated AMPK phosphorylation is attenuated by compound C. Quiescent EC were treated with the AMPK inhibitor compound C (40 M in DMSO for 1 h) prior to adiponectin (30 g/ml for 30 min). AMPK activation was analyzed as described A. A representative of three independent experiments is shown. D, knockdown of APPL1 blunts adiponectin-mediated AMPK activation. EC treated with APPL1 siRNA (100 nM for 48 h) were treated with adiponectin for 30 min. death. Thus, distinct pharmacological and molecular approaches lead to the common conclusion that the activation of AMP kinase activity is required for the anti-apoptotic actions of adiponectin.
APPL1 is an adaptor protein containing a pleckstrin homology domain, a phosphotyrosine-binding domain, and a leucine zipper motif (42), which links adiponectin receptors to downstream signaling molecules including AMPK (29,40). Correspondingly, our results now demonstrate that siRNA-mediated silencing of APPL1 expression significantly attenuates adiponectin-mediated AMPK phosphorylation, indicating that APPL1 is upstream of AMPK. Knockdown of APPL1 also blunted the prosurvival effects of adiponectin on IL-18-mediated endothelial cell death, suggesting that adiponectin exerts its cell survival effects via APPL1-AMPK signaling. In a yeast two-hybrid study, adiponectin was found to bind to the extracellular C-terminal domain of AdipoR1, whereas APPL1 was shown to interact with the cytoplasmic N-terminal domain. Although APPL1 is known to associate specifically with AdipoR1 (40), APPL1 has recently been reported to physically associate with AdipoR2. In human umbilical vein endothelial cells (HUVEC), Cheng et al. (29) have demonstrated that adiponectin treatment induces eNOS activation and NO generation in an APPL1dependent manner. Using GST pulldown assays, these authors describe that APPL1 physically associates with both AdipoR1 and AdipoR2 and that their knockdown blunted adiponectin-mediated eNOS activation and NO generation (29). Both eNOS and eNOSmediated NO play critical roles in vasodilation and vascular homeostasis (4). Together, these observations indicate that APPL1 is the immediate downstream signaling molecule for both adiponectin receptors and that APPL1 is an essential adaptor protein in adiponectin signaling, critical for the prosurvival effects of adiponectin stimulation.
Our results also show that adiponectin restores IL-18-mediated suppression in Akt levels and activity. Adiponectin induced PI3K activation, and adenoviral transduction of dnPI3Kp85 blunted adiponectinmediated Akt phosphorylation, indicating that adiponectin signals via PI3K to restore Akt levels in endothelial cells. Whether PI3K is downstream of APPL1 is not clear. However, Mitsuuchi et al. (42) have reported interaction between APPL1 and the PI3K catalytic subunit p110␣, which is generally thought to mediate PI3K signaling for G protein-coupled receptors. The authors concluded that APPL1 acts as an adaptor, tethering inactive AKT2 to p110␣ in the cytoplasm and thus hastens their recruitment to the cell membrane following mitogenic stimulation (42). APPL1 has also been shown to associate with and activate Akt. Recently, Saito et al. (43) demonstrated that APPL1 physically associates with Akt, and that siRNA-or small hairpin RNA (shRNA)-mediated APPL1 knockdown suppresses Akt phosphorylation, suggesting that the interaction between APPL1 and Akt is necessary for insulin-stimulated Glut4 translocation in 3T3-L1 adipocytes. Ouchi  (n ϭ 3). C, dnPI3K reverses adiponectin-mediated Akt activation. EC transduced with adenoviral dnPI3Kp85 (100 m.o.i. for 24 h) were treated with adiponectin followed by IL-18 addition. Total and phospho-Akt levels were assessed as described in B. D, AMPK␣1 knockdown reverses adiponectin-mediated Akt activation. EC treated with AMPK␣1 siRNA for 48 h were treated with adiponectin followed by IL-18 addition. Total and phospho-Akt levels were assessed as described in B. E, adiponectin reverses IL-18-induced IKK activity. Quiescent EC were treated with adiponectin for 1 h prior to IL-18 addition. IKK activity was analyzed by an in vitro kinase assay (n ϭ 3). Tubulin served as a loading control. F, AMPK␣1 knockdown reverses adiponectin-mediated IKK suppression. EC treated with AMPK␣1 siRNA for 48 h were treated with adiponectin followed by IL-18 addition. IKK activity was assessed as described in E. G, adiponectin. blunts IL-18-mediated NF-B activation. EC treated with AMPK␣1 siRNA for 48 h were treated with adiponectin followed by IL-18 addition for 2 h. Nuclear protein was extracted and analyzed for NFBp65 levels by ELISA. *, p Ͻ 0.001 versus untreated cells; †, p Ͻ 0.01 versus IL-18; §, p Ͻ 0.05 versus IL-18ϩadiponectin. H, adiponectin reverses IL-18-mediated PTEN induction. Quiescent EC were treated with adiponectin for 1 h prior to IL-18 addition. PTEN levels were analyzed by immunoblotting. Tubulin served as a loading control. A representative of three independent experiments is shown. I, knockdown of AMPK␣1 reverses the cell survival effects of adiponectin. EC treated with IL-18 as described in D, but for 24 h, were analyzed for cell death by quantifying mono-and oligonucleosomal fragmented DNA in the cytoplasmic extracts by ELISA (n ϭ 12/group). *, p Ͻ 0.001 versus untreated cells; †, p Ͻ 0.001 versus IL-18; §, p Ͻ 0.005 versus IL-18ϩControl siRNA by ANOVA. et al. (44) previously demonstrated that adiponectin stimulates the growth of new blood vessels by promoting cross-talk between AMPK and Akt signaling in endothelial cells. In addition, AMPK and Akt cross-talk induces eNOS activation by directly phosphorylating eNOS at Ser 1179 (29). In the present study, we showed that knockdown of AMPK attenuated adiponectin-mediated reversal in IL-18-dependent Akt suppression, suggesting that AMPK is upstream of Akt and that adiponectin blocks endothelial cell death by restoring levels and activity of Akt.
Activation of Akt is known to transmit prosurvival signals within the cell (35). We previously demonstrated that IL-18 up-regulates the expression of numerous proapoptotic molecules, including Bax and Bcl-X s , and that IL-18 induces activation of caspase-8 and -3 in endothelial cells (33). Here we show that treatment with adiponectin inhibited IL-18-mediated Bax translocation and caspase-3 activation. Supporting these results, Kobayashi et al. (13) have reported that adiponectin suppresses apoptosis of serum-starved HUVEC via AMPK activation and caspase-3 inhibition. We have also shown that adiponectin exerts its cell survival effects in endothelial cells by significantly attenuating IL-18-induced IKK activity, IKK-dependent NF-B activation, and PTEN expression. Recently, Wu et al. (45) reported that adiponectin inhibits TNF-␣ or high glucose-induced IKK activation and IB-␣ degradation in HUVEC. They demonstrated that activation of AMPK rather than cAMP/cAMP-dependent protein kinase signaling was more effective in suppressing high glucose-induced IKK activity; however, both the AMPK and cAMP/cAMP-dependent protein kinase pathways were equally effective in suppressing TNF-␣-induced IKK activation (45). Although our present studies clearly implicate AMPK in adiponectin-mediated endothelial cell survival, the undefined role of the cAMP/cAMP-dependent protein kinase pathway in adiponectin-mediated reversal of IL-18-dependent endothelial cell death is currently under investigation.
Our results have also demonstrated that the pharmacological AMPK activator AICAR recapitulates the prosurvival effects of adiponectin in endothelial cells. Similar to adiponectin, AICAR blocked IL-18-mediated cell death via AMPK/Akt signaling. AICAR also blunted IL-18-mediated IKK activation as well as IKK-NF-B-dependent PTEN induction, confirming that AMPK activation blocks IL-18 signaling and IL-18-mediated endothelial cell death. Interestingly, AICAR has recently been shown to block palmitate-induced endothelial cell apoptosis through AMPK activation and suppression of reactive oxygen species generation, suggesting that, in addition to its anti-apoptotic effects, AICAR also exerts antioxidant effects (46) similar to adiponectin treatment (47).
AMPK is a key member of the intracellular signal transduction cascade elicited by the anti-diabetic drug metformin, as well as by fenofibrate, which lowers triglycerides and cholesterol. Metformin blocks cytokine-induced vascular endothelial cell death via AMPK activation and suppression of IKK and NF-B activation induced by TNF and IL-1 in HUVEC (48). Because IL-18 induces pancreatic ␤-cell death leading to destructive insulitis (49) and pancreatic islets produce functional IL-18, it is likely that these AMPK activators may also FIGURE 7. AICAR inhibits EC death. A, AICAR induces AMPK phosphorylation. Quiescent EC were treated with AICAR (1 mM for 1 h). AMPK activation was analyzed by immunoblotting using activation-specific antibodies (n ϭ 3). B, AICAR stimulates AMP kinase activity. Quiescent EC treated with AICAR as described in A were analyzed for AMP kinase activity by an in vitro kinase assay as described under "Experimental Procedures." *, p Ͻ 0.01 versus untreated cells; †, p Ͻ 0.05 versus IL-18 (n ϭ 12). C, AICAR blunts IL-18-induced IKK activity. Quiescent EC were treated with AICAR for 1 h prior to IL-18 addition. IKK activity was analyzed by an in vitro kinase assay (n ϭ 3). D, dnAMPK reverses adiponectin-mediated IKK suppression. EC transduced with dnAMPK were treated with adiponectin followed by IL-18. IKK activity was assessed as described in C (n ϭ 3). E, AICAR reverses IL-18-mediated NF-B activation. Quiescent EC were treated with AICAR for 1 h prior to IL-18 addition. Nuclear protein was extracted and analyzed for NFBp65 levels by ELISA. *, p Ͻ 0.001 versus untreated cells; †, p Ͻ 0.01 versus IL-18 (n ϭ 12). F, AICAR reverses IL-18-mediated Akt suppression. Quiescent EC were treated with AICAR for 1 h prior to IL-18 addition. Total and phospho-Akt levels were assessed by immunoblotting (n ϭ 3). G, AICAR reverses IL-18-mediated PTEN induction. Quiescent EC were treated with AICAR for 1 h prior to IL-18 addition. PTEN levels were analyzed by immunoblotting. Tubulin served as a loading control. H, AICAR attenuates IL-18-mediated EC death. Quiescent EC were treated with AICAR prior to IL-18 addition for 24. EC death was assessed by ELISA. *, p Ͻ 0.001 versus untreated cells; †, p Ͻ 0.001 versus IL-18 (n ϭ 12/group). I, schema showing signaling mechanisms involved in IL-18-mediated EC death and those targeted by adiponectin and AICAR. exert cytoprotective effects in these and other cell types by blunting IL-18 expression and antagonizing its apoptotic effects. As atherosclerosis and other chronic inflammatory conditions are associated with elevated IL-18 expression and decreased adiponectin, our results suggest that AMPK activation by either adiponectin or AICAR can reverse IL-18-mediated endothelial cell death and thus may have therapeutic potential in attenuating IL-18 signaling and IL-18-dependent vascular injury and inflammation.