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Adiponectin suppresses IB kinase activation induced by tumor necrosis factor- or high glucose in endothelial cells: role of cAMP and AMP kinase signaling

Division of Endocrinology, Diabetes, and Metabolic Diseases, Department of Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania

Submitted 20 February 2007 ; accepted in final form 5 October 2007

	ABSTRACT

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Adiponectin is a protein secreted from adipocytes that exhibits salutary effects in the vascular endothelium by signaling mechanisms that are not well understood. In obesity-related disease states and type 2 diabetes, circulating substances, including tumor necrosis factor- (TNF) and high glucose, activate IB kinase (IKK)β and reduce the abundance of its substrate, inhibitor of B (IB), leading to nuclear translocation of the transcription factor NF-B and stimulation of an inflammatory signaling cascade closely associated with endothelial dysfunction. The present study demonstrates that the globular domain of adiponectin (gAd) potently suppresses the activation of IKKβ by either TNF or high glucose in human umbilical vein endothelial cells and ameliorates the associated loss of IB protein. Interestingly, activation of AMP kinase was substantially more effective than cAMP signaling in suppressing high glucose-induced IKKβ activity, whereas both pathways were comparably active in suppressing the TNF-induced increase in IKKβ. Both cAMP/protein kinase A signaling and activation of the AMP kinase pathway played a role in the suppression by gAd of TNF- and high glucose-mediated IKKβ activation. These findings support an important role for adiponectin in anti-inflammatory signaling in the endothelium and also imply that multiple pathways are involved in the cellular effects of adiponectin.

inflammation; endothelial dysfunction; insulin resistance; NF-B

Inflammatory signaling in the vascular endothelium, triggered by circulating cytokines such as tumor necrosis factor- (TNF) in visceral obesity or by hyperglycemia in diabetes mellitus, has been implicated in pathological endothelial cell activation and early vascular events in atherogenesis (2, 26). The mediator NF-B generates a programmed nuclear transcriptional cascade that is a major source of the inflammatory response in endothelial cells (8). NF-B is regulated through its protein interactions with the inhibitor of B (IB) inhibitory proteins, which, on cellular stimulation, are rapidly phosphorylated on serine, ubiquitinated, and degraded in the proteosome, releasing NF-B to function as a nuclear transcription factor (1, 25). Cytokines activate NF-B by inducing IB phosphorylation via IB kinase (IKK). IKK is a complex of at least three subunits: and β, which are kinase enzymes, and , which has a regulatory role. IKKβ, in particular, has been shown to play a prominent role in mediating cellular insulin resistance resulting from cytokine stimulation (28). High glucose has also been implicated in inflammatory signaling in endothelial cells via activation of NF-B (9, 24). Recent work has also shown that high glucose may act upstream of NF-B at the level of IKKβ activity in bovine aortic endothelial cells, resulting in impaired insulin-stimulated production of nitric oxide (NO) (11).

Prior work has shown that adiponectin has several important signaling effects in the endothelium, including enhancing NO generation, reducing reactive oxygen species (ROS) generation, and blocking inflammatory signaling cascades (7). Adiponectin inhibited TNF-induced expression of the adhesion molecules VCAM-1, E-selectin, and ICAM-1 on the surface of endothelial cells and reduced TNF-induced adhesion of monocytic THP-1 cells to cultured endothelial cells (20). Adiponectin has also been shown by two groups to suppress NF-B activation induced by TNF without affecting TNF-mediated activation of several MAP kinases, stress-activated kinases, and Akt (12, 21). Although the cellular mechanisms used by adiponectin signal transduction in the endothelium have not been fully characterized, they appear to involve multiple pathways, in particular, those mediated by 5'-AMP-activated protein kinase (AMP kinase) and cAMP/protein kinase A (PKA) signaling (7).

In the present study, we evaluated whether adiponectin suppressed the activation of the NF-B cascade as an upstream site involving inhibition of the enzyme activity of IKKβ. Both TNF- and high glucose-stimulated IKKβ activation and the potential involvement of cAMP and PKA signaling were examined using a cultured human umbilical vein endothelial cell (HUVEC) model.

	MATERIALS AND METHODS

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Materials. The pTrcHisA vector and Escherichia coli TOP10 strain cells were obtained from Invitrogen (Carlsbad, CA). Acticlean Etox column was from Sterogene Bioseparations (Carlsbad, CA). The Limulus Amebocyte Lysate Pyrogen Plus detection kit was from BioWhittaker (Walkersville, MD). HUVECs were from Cell Applications (San Diego, CA), and endothelial basal medium-2 (EBM-2) and growth factors were obtained from Cambrex BioScience (Walkersville, MD). The AMP kinase inhibitor compound C {6-[4-(2-piperidin-L-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyyrazolo[1,5-a] pyrim idine} was kindly provided by Merck Research Laboratories (Rahway, NJ). Glutathione S-transferase (GST)-IB protein was from Santa Cruz Biotechnology (Santa Cruz, CA). IKKβ and IB rabbit polyclonal antibody and antibody to AMP kinase (1 + 2) and PKA(c-) rabbit polyclonal antibody were from Cell Signaling Technology (Danvers, MA). [-³²P]ATP and the cAMP Biotrak enzyme immunoassay (EIA) system were from GE Healthcare (Piscataway, NJ). AMP kinase-1 rabbit monoclonal antibody, SAMS substrate peptide, PKA assay kit, small interfering RNA (siRNA) SMARTpool AMP kinase-1, siRNA SMARTpool PKA, siRNA nonspecific control pool, siIMPORTER siRNA, and the plasmid DNA transfection reagent were obtained from Millipore/Upstate (Lake Placid, NY). Protein A agarose beads were from Pierce (Rockford, IL). Enhanced chemiluminescence (ECL) reagents were from Perkin-Elmer Life Sciences (Boston, MA). Horseradish peroxidase-conjugated secondary antibodies were obtained from GE Healthcare (Piscataway, NJ). Magnesium/ATP cocktail and IKK substrate peptide were from Upstate Biotechnology (Lake Placid, NY). Bio-Safe Coomassie Stain solution was from Bio-Rad (Hercules, CA). 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), Rp-adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMP), 2',3'-dideoxyadenosine (ddAdo), and other reagents were obtained from Sigma-Aldrich (St. Louis, MO).

Recombinant adiponectin protein. The recombinant globular domain of human adiponectin was subcloned into the pTrcHisA bacterial expression vector and expressed as an NH₂-terminal (his)₆-tagged fusion protein in E. coli TOP10 strain by induction with isopropyl-β-thiogalactopyranoside. The protein was purified under native conditions and was applied to an Acticlean Etox column (Sterogene Bioseparations) to remove endotoxin contamination, as we described previously (30).

Cell culture and treatment. HUVECs before passage 4 were cultured to 80% confluence on six-well plates with growth medium EBM-2 supplemented with endothelial cell growth factors (Clonetics) and 2% FBS. After a washing with PBS, cells were made quiescent in human endothelial serum-free medium (SFM) with 5 mmol/l glucose and 1% BSA, with no growth factor supplement for 3 h. Cells were treated at 37°C with the indicated concentration of globular adiponectin for 3 h before treatment with the indicated concentration of TNF for 5 min or high glucose for 24 h. Where indicated, cells were also treated with ddAdo (100 µM) or Rp-cAMP (10 µM) for 20 min before the addition of globular domain of adiponectin (gAd); forskolin (2 µM), AICAR (2 mM), or compound C (10 µM) was added during the last 20 min of incubation before cell lysis.

Immunoprecipitation and IKK kinase assay using GST-IB protein as substrate. Kinase assays were performed as previously described (6, 14) using substrate protein according to the reagent manufacturer's instructions with minor modifications. Briefly, cells were lysed with ice-cold deoxygenated buffer including 50 mM HEPES, pH 7.4, 150 mM NaCl, 1% (vol/vol) Triton X-100, 5 mM EDTA, 5 mM EGTA, 20 mM Na pyrophorphate, 20 mM NaF, 1 mM MgCl₂, 10% (vol/vol) glycerol, 1 mM Na orthovanadate, 1 mM β-glycerophosphate, 0.5 mM dithiothreitol, and a proteinase inhibitor cocktail (Sigma). The cell lysate was sonicated twice for 10 s each on ice and centrifuged at 13,000 rpm for 5 min at 4°C. The protein concentration was estimated using Bio-Rad protein dye reagent as described by the manufacturer.

Aliquots containing 400 µg of total protein from the cell lysate were immunoprecipitated with 2 µl of IKKβ polyclonal antibody overnight at 4°C and then incubated with 35 µl of protein A agarose beads for 2 h at 4°C. The protein A agarose beads, antibody, and IKK protein complex was washed four times using enzyme dilution buffer (20 mM MOPS, pH 7.5, 1 mM EDTA, 5% glycerol, 0.1% β-mercaptoethanol, 1 mg/ml BSA) for three of the washes and kinase reaction buffer (8 mM MOPS, pH 7.0, 0.2 mM EDTA) once. The kinase reaction was performed using IKK protein coupled to protein A beads with 1 µg of GST-IB protein, 10 µCi of [-³²P]ATP in magnesium/ATP cocktail at 30°C for 30 min. The reaction was ended by addition of 4x Laemmli protein sample buffer, and samples were boiled at 100°C for 5 min. After centrifugation at 4,000 rpm for 2 min, supernatant was loaded to 10% polyacrylamide gel for SDS-PAGE. The gel was fixed in solution with 10% glacial acetic acid and 20% methanol, stained in Bio-Safe Coomassie Stain solution, and dried on Whatman 3MM paper at 60°C for 2 h. The dried gel was covered by X-ray film in a dark room and exposed at –70°C for 1–2 days. Radioactive signal was quantified on an ImageStation 440CF (Kodak, Rochester, NY).

Immunoprecipitation and IKK kinase assay using IKK peptide as substrate. Cell lysate preparation and immunoprecipitation were the same as above. The kinase reaction was performed using IKK protein coupled to protein A beads with 200 µM IKK substrate peptide (KKKKERLLDDRHDSGLDSMKDEE) and 10 µCi of [-³²P]ATP in magnesium/ATP cocktail at 30°C for 30 min. IKK protein was separated from reaction mixture by centrifugation at 4,000 rpm for 2 min to end the kinase reaction. Aliquots of 20 µl of supernatant were spotted onto the center of 2 x 2-cm Whatman P81 paper. The P81 squares were washed three times with 0.75% (vol/vol) phosphoric acid and once with acetone to eliminate unlabeled binding and then transferred to a vial containing 5 ml of scintillation cocktail. The radioactivity was then counted in a β-counter. IKKβ kinase activity was expressed as the amount of substrate peptide phosphorylation relative to control.

Western blotting. Protein immunoblotting was performed essentially as previously reported (17). Twenty-five to fifty micrograms of protein were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes. Primary antibody immunoblotting was performed following the manufacturer's instructions. After incubation with horseradish peroxidase-conjugated secondary antibodies, proteins were visualized by ECL exposure to X-ray film. Immunoblotting signals were quantitated using an ImageStation 440 (Kodak).

Measurement of cellular cAMP content. HUVECs were cultured on 24-well plates and were treated when cells reached 80–90% confluency. An aliquot of 100 µl of cell lysate was used for each cAMP measurement. Intracellular cAMP content was measured using a cAMP Biotrak EIA system (GE Healthcare) according to the manufacturer's instructions.

Immunoprecipitation and PKA activity assay. Aliquots containing 400 µg of cell lysate protein were incubated with 2.0 µg of anti-PKAc- polyclonal rabbit antibody overnight at 4°C and then incubated with 30 µl of protein A agarose beads for 2 h at 4°C. Enzyme activity was assayed using a PKA assay kit and the instructions provided by the manufacturer (Upstate, Lake Placid, NY). Briefly, agarose beads were washed four times with assay dilution buffer (ADB) (20 mM MOPS, pH 7.2, 125 mM β-glycerophosphate, 25 mM EGTA, 5 mM sodium orthovanadate, and 5 mM dithiothreitol) and then incubated with 10 µl of ADB, 5 µl of 20 µM cAMP, 5 µl of kemptide, 10 µl of inhibitor cocktail (10 µl of the inhibitor peptide to the negative controls), and 10 µCi of [-³²P]ATP in magnesium/ATP cocktail for 10 min at 30°C. An aliquot of 25 µl was blotted on the P81 paper square, which was then washed three times with 0.75% (vol/vol) phosphoric acid and once with acetone. The P81 square was transferred to a vial containing 5 ml of scintillation cocktail. The radioactivity was then counted in a β-counter. PKA kinase activity was expressed as picomoles of ³²P incorporated into the substrate kemptide (LRRASLG) per milligram protein per minute.

Immunoprecipitation and AMP kinase enzyme assay. Immunoprecipitation followed by AMP kinase enzyme assay were described as before (30). Briefly, aliquots containing 200 µg of cell lysate protein were incubated with 1.0 µg of anti-AMP kinase 1 + 2 polyclonal rabbit antibody overnight at 4°C and then incubated with 30 µl of protein A agarose beads for 2 h at 4°C. The agarose was washed four times with AMP kinase reaction buffer (20 mM HEPES-NaOH, pH 7.2, 0.4 mM dithiothreitol with 300 µmol of AMP) and then incubated with 20 µl of AMP kinase reaction buffer, 10 µl of SAMS substrate peptide with final concentration of 80 µM, and 10 µCi of [-³²P]ATP mixed with 75 mM magnesium chloride and 500 µmol of unlabeled ATP for 15 min at 30°C. Aliquots of 35 µl were spotted onto the center of a 2-cm square of Whatman P81 paper. The P81 squares were washed three times with 0.75% (vol/vol) phosphoric acid and once with acetone and then transferred to a vial containing 5 ml of scintillation cocktail. The radioactivity was then counted in a β-counter. AMP kinase activity was expressed as picomoles of ³²P incorporated into the substrate SAMS peptide (HMRSAMSGLHLVKRR) per milligram protein per minute.

siRNA-mediated knockdown of AMP kinase-1 and PKA. siRNA duplex oligonucleotides were based on the human cDNAs encoding AMP kinase-1 and PKA. Four pooled selected AMP kinase-1- or PKA-specific siRNA duplexes, nonsilencing control siRNA, and siIMPORTER siRNA transfection reagent were used according to the manufacturer's instructions (Millipore/Upstate). HUVECs were plated on six-well plates before transfection and were 50% confluent when siRNA was applied to each well with a final concentration of 25 nM. Where indicated, cells were treated with gAd (3 µg/ml) for 3 h before being treated with 10 ng/ml TNF for 5 min or 25 mM glucose for 24 h before immunoblots and IKKβ activity assays were performed.

Statistical analyses. Quantitative data are presented as means ± SE for three to five experiments. Statistical analysis was based on Student's t-test for comparison of two groups. A P value <0.05 was used to determine statistical significance.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
IKKβ substrate peptide phosphorylation. TNF activates the proinflammatory IKKβ/NF-B signaling pathway in various cell types and contributes to insulin resistance and endothelial dysfunction. In the present work, we focused on the stimulation of IKKβ enzyme activity by TNF or high glucose in cultured HUVECs and the potential effect of the recombinant gAd to suppress this IKKβ activation. IKKβ activity was measured by two independent assay methods. Initially, cell lysates were adsorbed by a specific IKKβ antibody followed by a kinase assay using [³²P]ATP and a specific peptide substrate (Fig. 1). Treatment with gAd (3 µg/ml for 3 h) did not significantly affect basal IKKβ activity, whereas treatment with 5 or 15 ng/ml TNF for 5 min increased IKKβ activity 2.0- and 2.5-fold, respectively (P < 0.001). Prior cell treatment with gAd for 3 h completely inhibited the subsequent activation of IKKβ by stimulation with 5 ng/ml TNF and significantly reduced the effect of 15 ng/ml TNF by 44% (P < 0.001).

View larger version (23K): [in this window] [in a new window]   Fig. 1. Effect of adiponectin (globular domain of adiponectin; gAd) on TNF-induced IKKβ activity in human umbilical vein endothelial cells (HUVECs) using IKK substrate peptide. HUVECs were cultured as described in MATERIALS AND METHODS. After pretreatment with 3 µg/ml gAd for 3 h, cells were treated with TNF at the indicated concentrations for 5 min. Cells were lysed, and protein samples were immunoprecipitated with IKKβ antibody followed by assay using the IKK substrate peptide. dpm, Disintegrations per minute. Data are expressed as means ± SE. *P < 0.001 vs. control; **P < 0.001 vs. the respective TNF-stimulated samples.

Phosphorylation of IB fusion protein by IKKβ.

View larger version (37K): [in this window] [in a new window]   Fig. 2. Effect of adiponectin (gAd) on TNF-induced IKKβ activity in HUVECs using glutathione S-transferase (GST)-inhibitor of B (IB) protein as IKK substrate. Cell culture was as described in MATERIALS AND METHODS, and the treatment with gAd and TNF was as described in the legend to Fig. 1, except for the use here of 7.5 ng/ml TNF. Following cell lysate immunoprecipitation with IKKβ antibody, the protein kinase activity was assayed by phosphorylation of GST-IB substrate as described in MATERIALS AND METHODS. Top: representative radiograph of phosphorylated GST-IB protein and stained control for protein loading. Bottom: data quantitation. Data are expressed as means ± SE. *P < 0.05 vs. control.

Cellular IB protein mass assay.

View larger version (44K): [in this window] [in a new window]   Fig. 3. Effect of adiponectin (gAd) on TNF-induced degradation of IB in HUVECs. HUVECs were cultured and treated as indicated in MATERIALS AND METHODS. Cells were preincubated for 3 h without or with adiponectin (3 µg/ml) and then treated where indicated with 15 ng/ml TNF for 5 min. Cell lysate samples were separated on a 10% polyacrylamide gel for SDS-PAGE and transferred to nitrocellulose membranes; Western blot with IB antibody was performed using secondary antibody conjugated with horseradish peroxidase. Proteins were visualized by enhanced chemiluminescence, and the signals were quantitated using an ImageStation 440 (Eastman Kodak). Data are expressed as means ± SE. *P < 0.05 vs. control.

View larger version (45K): [in this window] [in a new window]   Fig. 4. Effect of various inhibitors and effectors on TNF-induced activation and adiponectin (gAd) suppression of TNF-induced activation of IKKβ in HUVECs. HUVECs were cultured as described in MATERIALS AND METHODS. Where indicated, cells were preincubated with adiponectin (3 µg/ml) for 3 h and then treated with the reagents shown for 20 min and TNF (15 ng/ml) for 5 min before snap-freezing of the cells and lysis into the buffer described in MATERIALS AND METHODS. Following immunoprecipitation with IKKβ antibody, the protein kinase activity was assayed by phosphorylation of GST-IB substrate as described in MATERIALS AND METHODS. For definitions of ddAdo, Rp-cAMP, AICAR, and compound C, see MATERIALS AND METHODS. Data are expressed as means ± SE. #P < 0.01 vs. TNF treated; **P < 0.01 vs. control; *P < 0.05 vs. gAd and TNF treated.

High glucose-induced activation of IKKβ. High glucose has been shown to initiate an inflammatory signaling cascade in endothelial cells, including the activation of IKKβ (11). Incubation of HUVECs in medium containing 25 mM glucose for 24 h increased IKKβ activity by 1.8-fold (Fig. 5). Prior treatment with gAd (3 µg/ml) completely inhibited the increase in IKKβ activity stimulated by high glucose conditions.

View larger version (47K): [in this window] [in a new window]   Fig. 5. Effect of various inhibitors and effectors on high glucose-induced activation and adiponectin (gAd) suppression of high glucose-induced activation of IKKβ in HUVECs. These studies were performed as described in the legend to Fig. 4, except that in place of TNF stimulation, HUVECs were incubated in 25 mM glucose medium (HG) for 24 h before the addition of adiponectin (3 µg/ml) for 3 h with or without the reagents shown for 20 min before cell lysis. Following immunoprecipitation with IKKβ antibody, the protein kinase activity was assayed by phosphorylation of GST-IB substrate as described in MATERIALS AND METHODS. Data are expressed as means ± SE. #P < 0.01 vs. high glucose-treated; **P < 0.01 vs. control; *P < 0.05 vs. gAd and high glucose treated.

gAd increases cellular cAMP levels under TNF stimulation or in high glucose conditions. To provide further mechanistic evidence for a role of cAMP/PKA signaling in the endothelial action of gAd, we first measured cellular cAMP levels under conditions identical to those used for measuring IKKβ activity. TNF stimulation had no effect on cAMP levels in the HUVECs (Fig. 6A). However, pretreatment with gAd for 3 h before treatment with TNF for 5 min significantly increased the cAMP level by 29% (P < 0.05), to a degree similar to the 46% increase observed following treatment with the adenylyl cyclase activator forskolin. gAd appears to signal via adenylyl cyclase, since the increase in cellular cAMP content was abrogated by the cyclase inhibitor ddAdo.

View larger version (29K): [in this window] [in a new window]   Fig. 6. Effect of TNF, high glucose, adiponectin (gAd), ddAdo, and forskolin on cAMP content in HUVECs. A: cells were stimulated with or without gAd for 3 h before 10 ng/ml TNF for the last 5 min of incubation. Where indicated, cells were treated with ddAdo for 20 min before the addition of gAd; forskolin was added during the last 20 min of incubation. B: cells were treated similarly but with 25 mM glucose (HG) for 24 h. Before lysis, cells were stimulated with or without gAd for 3 h and with or without ddAdo for 20 min before the addition of 25 mM glucose; where indicated, forskolin was added during the last 20 min of incubation. Cells were lysed, and 100 µl of the cleared lysate were used for cAMP assay as described in MATERIALS AND METHODS. Data are expressed as means ± SE; n = 4. *P < 0.05 vs. control.

HUVEC PKA activity is increased by gAd under TNF stimulation or in high glucose conditions. The activation of PKA enzyme activity by gAd was consistent with the observed increases in cAMP described in Fig. 6. Cellular PKA activity was measured in immunoprecipitates using a radiolabeled peptide substrate assay (Fig. 7). In parallel with the cAMP levels shown above, TNF itself did not alter cellular PKA activity, which was significantly increased 49 and 66% by gAd treatment without and with stimulation by TNF, respectively (both P < 0.05; Fig. 7A). Treatment with the direct PKA inhibitor Rp-cAMP suppressed gAd stimulation of PKA by 90% under conditions of TNF stimulation, and the adenylyl cyclase inhibitor ddAdo completely blocked gAd stimulation of PKA.

View larger version (34K): [in this window] [in a new window]   Fig. 7. Effect of TNF, high glucose, gAd, and other agents on PKA activity in HUVECs. A: cells were stimulated with or without gAd for 3 h before 10 ng/ml TNF for the last 5 min of incubation. Where indicated, cells were treated with ddAdo or Rp-cAMP for 20 min before the addition of gAd; forskolin was added during the last 20 min of incubation. B: cells were treated with 25 mM glucose (HG) for 24 h. Before lysis, cells were stimulated where indicated with gAd for 3 h; cells were then treated with ddAdo or Rp-cAMP for 20 min before the addition of 25 mM glucose. Cells were lysed, and PKA activity was assayed as described in MATERIALS AND METHODS. Data are expressed as means ± SE; n = 3. *P < 0.05 vs. control; #P < 0.05 vs. gAd and TNF or high glucose conditions.

AMP kinase activity is increased in HUVECs by gAd under TNF stimulation or in high glucose conditions. We also measured the activity of cellular AMP kinase 1 + 2 isoforms using a radiolabeled peptide substrate assay in immunoprecipitates following gAd stimulation in cells also treated by TNF or high glucose (Fig. 8). TNF itself did not alter cellular AMP kinase activity, which was significantly increased 48 and 58% by gAd treatment without and with stimulation by TNF, respectively (both P < 0.05; Fig. 8A). Treatment with the direct AMP kinase inhibitor compound C significantly reduced the gAd stimulation of AMP kinase by 71% under conditions of TNF stimulation. The effect of gAd on AMP kinase activity was also mimicked by the AMP kinase activator AICAR.

View larger version (34K): [in this window] [in a new window]   Fig. 8. Effect of TNF, high glucose, gAd, and other agents on AMP kinase 1 + 2 activity in HUVECs. A: cells were stimulated with or without gAd for 3 h before 10 ng/ml TNF for the last 5 min of incubation. Where indicated, cells were treated with compound C or AICAR for 20 min before TNF stimulation. B: cells were treated with 25 mM glucose (HG) for 24 h. Before lysis, cells were stimulated where indicated with gAd for 3 h; compound C or AICAR was added during the last 20 min of incubation. Cells were lysed, and AMP kinase 1 + 2 activity was assayed as described in MATERIALS AND METHODS. Data are expressed as means ± SE; n = 3. *P < 0.05 vs. control; #P < 0.05 vs. gAd and TNF or high glucose conditions.

RNAi-mediated knockdown of AMP kinase-1 and PKA and effect on gAd suppression of IKKβ activity in HUVECs. With the use of siRNA transfection, the cellular mass of AMP kinase-1 and the -catalytic subunit of PKA were reduced by 89 and 86%, respectively (Fig. 9). Under these conditions, we tested the effect of reduction of these signaling kinases on the action of gAd in HUVECs treated with TNF or high glucose (Fig. 10). After transfection with control siRNA, TNF increased IKKβ activity by 2.4-fold, which was suppressed 27% by treatment with gAd. Following knockdown of PKA, TNF increased IKKβ by 48%, but gAd suppression of this action of TNF was completely blocked. After transfection for knockdown of AMP kinase-1, TNF stimulated IKKβ activity by 53%, and gAd significantly reduced the effect of TNF by 30% (P < 0.05).

View larger version (34K): [in this window] [in a new window]   Fig. 9. siRNA-mediated knockdown of AMP kinase-1 and PKA. HUVECs were cultured on 6-well plates and transfected with siRNA at 50% confluency. At 24 h posttransfection, aliquots of 25 µg of cell lysate protein were used for immunoblot analysis for AMP kinase-1 and PKA as described in MATERIALS AND METHODS.

View larger version (34K): [in this window] [in a new window]   Fig. 10. Effect of knockdown of PKA and AMP kinase-1 on TNF-, high glucose-, and adiponectin (gAd)-mediated IKKβ activity in HUVECs. siRNA-mediated knockdown of PKA and AMP kinase-1 in HUVECs is described in MATERIALS AND METHODS. At 24 h posttransfection, cells made quiescent were pretreated with gAd for 3 h and then treated with 10 ng/ml TNF for 5 min (A) and 25 mM glucose for 24 h (B). Cells were lysed, and IKKβ activity was assayed using IKK peptide as substrate. Data are expressed as means ± SE; n = 3. *P < 0.05 vs. control; #P < 0.05 vs. TNF or high glucose conditions.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This study provides new evidence that adiponectin suppresses inflammatory signal generation triggered by both TNF and high glucose at the level of IKKβ enzyme activation in endothelial cells. In addition, we found that both AMP kinase and cAMP signaling play a role in the effects of adiponectin to block the rise in IKKβ activity induced by both TNF and high glucose. Interestingly, activation of adenylate cyclase suppressed the activation of IKKβ induced by TNF but was relatively ineffective in opposing the effects of high glucose to activate IKKβ. Nevertheless, the cAMP pathway and AMP kinase signaling were both implicated in the effect of gAd on IKKβ activity induced by either TNF or high glucose concentrations. These results are of interest, since they support an important role for adiponectin in anti-inflammatory signaling in the vasculature and also imply that multiple pathways are involved in the cellular effects of adiponectin.

Prior work in the vasculature has shown that adiponectin suppresses the characteristic pleiotropic proinflammatory activation response pattern in endothelial cells that includes stimulation of the NF-B pathway, upregulation of cell adhesion molecules, and diminished endothelial NO generation and bioavailability (15, 18). We have also found that a major endothelial effect of adiponectin is the suppression of ROS generation in response to treatment with oxidized LDL or high glucose conditions (19, 23). However, there is controversy as to whether the NF-B cascade is activated by ROS, especially at the level of IKKβ, since its enzyme activity has been shown to be oxidatively inhibited by H₂O₂ (14). Thus the mechanism of adiponectin suppression of inflammatory NF-B signaling has not been well characterized.

The effects of adiponectin in metabolically responsive liver, skeletal muscle, and adipose cells to enhance insulin action are closely integrated with the pleiotropic enzyme AMP kinase (10, 27, 30). AMP kinase is also activated by adiponectin in endothelial cells and has been shown to be involved in adiponectin enhancement of endothelial NO availability (3, 13, 22). In addition, adiponectin signaling to NO generation was shown to be linked, at least in part, to phosphatidylinositol 3'-kinase activation (3) and formation of a complex involving endothelial nitric oxide synthase, heat shock protein-90, and Akt (31). Adiponectin effects on angiogenesis were also found to be dependent on adiponectin-stimulated phosphorylation of both AMP kinase and Akt (22). AMP kinase appears to be upstream of Akt, since disruption of AMP kinase activation inhibited adiponectin-induced Akt phosphorylation (22).

In addition to AMP kinase, evidence has been accumulating to support an important role for a cAMP/PKA-linked pathway in adiponectin endothelial signaling. Ouchi et al. (21) initially reported that the inhibitory effect of adiponectin on TNF signaling in endothelial cells was accompanied by cAMP accumulation and blocked by an inhibitor of either adenylate cyclase or PKA. The inhibitory effect of adiponectin on TNF-induced IL-8 synthesis in endothelial cells was shown to be associated with increased intracellular cAMP levels and PKA activity and blocked by PKA inhibition (12).

In our studies of adiponectin suppression of ROS generation induced by high glucose in endothelial cells, adiponectin increased cellular cAMP content, and inhibition of PKA blocked the antioxidant effect of adiponectin (23). Increasing endothelial cell cAMP with forskolin or dibutyryl cAMP also suppressed glucose-induced ROS production. In murine peritoneal macrophages, adiponectin was recently shown to increase cAMP and PKA activity and reduce leptin-induced TNF production by blocking ERK1/2 and p38 MAPK phosphorylation (32). Thus the cAMP/PKA pathway is a major signaling pathway that appears to mediate at least some of the beneficial actions of adiponectin to counter the adverse effects of TNF or high glucose in endothelial and potentially other vascular or circulating cell types.

Additional work will be necessary to help define the regulation of the upstream mechanisms of inflammatory endothelial signaling via IKK activation, which clearly plays a pivotal role in this process. Recent studies have provided new insight into mechanisms of TNF-stimulated IKK activation by protein interactions including ubiquitination of receptor interacting protein-1 and polyubiquitin binding by NF-B essential modulator (5, 16, 29). To date, the mechanism by which high glucose mediates activation of IKK remains poorly understood (11). It will be of interest to determine how common signaling pathways modulated by adiponectin affect the activation of IKKβ by the divergent upstream mediators TNF and high glucose.

	GRANTS

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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-563018 and DK-71360 and a research grant from the American Diabetes Association to B. J. Goldstein.

	FOOTNOTES

 

Address for reprint requests and other correspondence: B. J. Goldstein, Division of Endocrinology, Diabetes and Metabolic Diseases, Dept. of Medicine, Jefferson Medical College, Suite 320 Curtis Bldg., 1015 Walnut St., Philadelphia, PA 19107 (e-mail: Barry.Goldstein{at}jefferson.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

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