Diabetes Care
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Diabetes Care 29:139-141, 2006
DOI: 10.2337/diacare.29.01.06.dc05-1364
© 2006 by the American Diabetes Association
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Pathophysiology/Complications
Brief Report

Globular Adiponectin Activates Nuclear Factor–{kappa}B in Vascular Endothelial Cells, Which in Turn Induces Expression of Proinflammatory and Adhesion Molecule Genes

Yoshiyuki Hattori, MD, PHD, Sachiko Hattori, MD, PHD and Kikuo Kasai, MD, PHD

From the Department of Endocrinology and Metabolism, Dokkyo University School of Medicine, Mibu, Japan

Address correspondence and reprint requests to Yoshiyuki Hattori, MD, PhD, Department of Endocrinology and Metabolism, Dokkyo University School of Medicine, Mibu, Tochigi, 321-0293, Japan. E-mail: yhattori{at}dokkyomed.ac.jp

Abbreviations: HMW, high molecular weight • HUVEC, human umbilical vein endothelial cell • NF-{kappa}B, nuclear factor {kappa}B • TNF, tumor necrosis factor


   INTRODUCTION
TOP
 INTRODUCTION
RESEARCH DESIGN AND METHODS
 RESULTS
 CONCLUSIONS
 References
 
Adiponectin is present in serum in trimer, hexamer, or high–molecular weight (HMW) form. A proteolytic cleavage product of adiponectin, known as globular adiponectin, has also been found to circulate in human plasma (1). The biological activities of these isoforms is controversial. It has been shown that recombinant globular adiponectin is pharmacologically active and induces free fatty acid oxidation in incubated mouse muscle and cultured muscle cells (1). Yamauchi et al. (2) have reported significantly greater potency of globular adiponectin in reversing insulin resistance than uncleaved adiponectin. However, it has recently been proposed that the ratio of HMW to total adiponectin, but not the absolute amounts of adiponectin, determines insulin sensitivity in humans and rodents (3). Clinical data also confirmed that patients with type 2 diabetes and coronary heart diseases have a selective reduction in HMW adiponectin (3,4). The experimental and clinical data collectively suggest that the oligomeric complex distribution of adiponectin is critical for the antidiabetes and antiatherogenic activity of this hormone (4).


   RESEARCH DESIGN AND METHODS
TOP
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
RESULTS
 CONCLUSIONS
 References
 
We thus questioned how globular adiponectin alters the gene expression in human umbilical vein endothelial cells (HUVECs). First, we confirmed the adiponectin receptor 1 gene expression in HUVECs, which have a high affinity for globular adiponectin (5). Microarray analysis of cDNA from the RNA prepared from HUVECs nontreated or treated with globular adiponectin (10 µg/ml) (PeproTech, London, U.K.) for 8 h was performed using Affymetrix GeneChip Expression Analysis (containing oligonucleotide probe sets for ~8,500 human genes). We then examined whether globular adiponectin induces nuclear factor {kappa}B (NF-{kappa}B) activation. To study NF-{kappa}B activation, mouse SVEC4 cells (vascular endothelial cells, SV40 transformed) were stably transfected with a cis-reporter plasmid containing the luciferase reporter gene linked to five repeats of NF-{kappa}B binding sites (pNF{kappa}B-Luc; Stratagene, La Jolla, CA). Several clones were selected for analysis of NF-{kappa}B activation.


   RESULTS
TOP
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
CONCLUSIONS
 References
 
We found that globular adiponectin increased the expression of 187 genes, including intercellular adhesion molecule-1 (3.1-fold), E-selectin (5.3-fold), vascular cell adhesion molecule-1 (7.1-fold), and monocyte chemoattractant protein-1 (2.4-fold) (Fig. 1 indicates experimental-to-basal signal ratio). Unexpected increase in the expression of those genes in HUVECs was confirmed by real-time PCR. Interestingly, the expression of such genes as intercellular adhesion molecule-1, E-selectin, vacular adhesion molecule-1, and monocyte chemoattractant protein-1 is dependent on NF-{kappa}B activation. Induction of the expression of those genes by globular adiponectin was significantly inhibited by the NF-{kappa}B inhibitor BAY11-7082, suggesting that globular adiponectin activates NF-{kappa}B. We therefore examined whether globular adiponectin really induces NF-{kappa}B activity in cells. It was observed that globular adiponectin dose-dependently activated NF-{kappa}B–mediated gene transcription. NF-{kappa}B–dependent transactivation increased by 13.6-fold relative to unstimulated levels in globular adiponectin–treated cells at concentrations of 10 µg/ml. SDS-PAGE confirmed no oligomerization of recombinant globular adiponectin, which we used in this study. In contrast, full-length adiponectin forms monomer and several oligomerization states. Full-length adiponectin was totally ineffective to activate NF-{kappa}B–mediated gene transcription as it was. Heat-denaturing converts the oligomers of adiponectin to monomer. Even after monomerizatoin of full-length adiponectin by heat denaturing, it was also ineffective in activating NF-{kappa}B. To determine the relative strength of NF-{kappa}B activation by globular adiponectin, we compared induction of NF-{kappa}B–mediated reporter gene expression between globular adiponectin and tumor necrosis factor (TNF)-{alpha}, lipopolysaccharide, and interleukin-1ß, known inducers of NF-{kappa}B in endothelial cells. TNF{alpha}, lipopolysaccharide, and interleukin-1ß potently stimulated NF-{kappa}B–mediated gene transcription in endothelial cells, and globular adiponectin showed comparable potency at 10 µg/ml. We then examined the pretreatment of the cells with globular adiponectin on TNF{alpha}-induced NF-{kappa}B–mediated gene transcription, since hyporesponsiveness or desensitization to TNF{alpha} might be caused by pretreatment with globular adiponectin. Cells were treated with various concentrations of globular adiponectin for 16 h and then stimulated with TNF{alpha} to induce NF-{kappa}B activation. TNF{alpha}-induced NF-{kappa}B–mediated gene transcription was suppressed by globular adiponecting pretreatment in a dose-dependent manner.


Figure 1
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  		Figure 1— A: The effects of globular adiponectin on NF-{kappa}B–dependent transcriptional activity. Quiescent cells (transfected with pNF{kappa}B-Luc) were left untreated or were treated with various concentrations (0.1–10 µg/ml) of globular adiponectin. After 2 h, cells were lysed, and luciferase activity was measured. B: The effects of globular adiponectin and control agents on NF-{kappa}B–dependent transcriptional activity. Quiescent cells (transfected with pNF{kappa}B-Luc) were left untreated (Cont) or were treated with globular adiponectin (gAD: 3 or 10 µg/ml) or TNF{alpha} (10 ng/ml), lipopolysaccharide (LPS) (10 µg/ml), interleukin-1ß (10 ng/ml). After 2 h, cells were lysed, and luciferase activities were measured. C: The effect of globular adiponectin pretreatment on TNF{alpha}-induced NF-{kappa}B–dependent transcriptional activity. Cells (transfected with pNF{kappa} B-Luc) were treated with various concentrations of globular adiponectin for 16 h and then treated with TNF{alpha} for 2 h. Then, cells were lysed, and luciferase activities were measured. Data are mean ± SE of triplicate observations. *P < 0.05 and **P < 0.01 compared with control.

 

   CONCLUSIONS
TOP
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 CONCLUSIONS
References
 
In the present study, we have demonstrated that globular adiponectin is a potent stimulator of NF-{kappa}B activation. Recently, Waki et al. (6) showed that adiponectin can be cleaved by leukocyte elastase secreted from activated monocytes and/or neutrophils and that this cleavage could be a possible mechanism for the generation of the globular fragment of adiponectin in plasma. The inducible transcription factor NF-{kappa}B regulates the expression of a variety of genes involved in the inflammatory and proliferative responses of cells, and recent studies strongly indicate that it is involved in the pathogenesis of atherosclerosis (7). Although the pathophysiological importance of adiponectin cleavage by leukocyte elastase in vivo remains to be determined, the present results suggest that adiponectin cleavage in inflammatory sites may facilitate the atherogenic process. Atherosclerosis is now recognized as an inflammatory process, and our study suggests that globular adiponectin may play a role in atherogenesis rather than exerting antiatherogenic effect.


   Footnotes
 
A table elsewhere in this issue shows conventional and Système International (SI) units and conversion factors for many substances.

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

Received for publication July 22, 2005. Accepted for publication September 18, 2005.


   References
TOP
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 CONCLUSIONS
 References
 

  1. Fruebis J, Tsao TS, Javorschi S, Ebbets-Reed D, Erickson MR, Yen FT, Bihain BE, Lodish HF: Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice. Proc Natl Acad Sci U S A 98:2005–2010, 2001[Abstract/Free Full Text]
  2. Yamauchi T, Kamon J, Waki H, Imai Y, Shimozawa N, Hioki K, Uchida S, Ito Y, Takakuwa K, Matsui J, Takata M, Eto K, Terauchi Y, Komeda K, Tsunoda M, Murakami K, Ohnishi Y, Naitoh T, Yamamura K, Ueyama Y, Froguel P, Kimura S, Nagai R, Kadowaki T: Globular adiponectin protected ob/ob mice from diabetes and ApoE-deficient mice from atherosclerosis. J Biol Chem 278:2461–2468, 2003[Abstract/Free Full Text]
  3. Pajvani UB, Hawkins M, Combs TP, Rajala MW, Doebber T, Berger JP, Wagner JA, Wu M, Knopps A, Xiang AH, Utzschneider KM, Kahn SE, Olefsky JM, Buchanan TA, Scherer PE: Complex distribution, not absolute amount of adiponectin, correlates with thiazolidinedione-mediated improvement in insulin sensitivity. J Biol Chem 279:12152–12162, 2004[Abstract/Free Full Text]
  4. Kobayashi H, Ouchi N, Kihara S, Walsh K, Kumada M, Abe Y, Funahashi T, Matsuzawa Y: Selective suppression of endothelial cell apoptosis by the high molecular weight form of adiponectin. Circ Res 94:e27–31, 2004[Abstract/Free Full Text]
  5. Yamauchi T, Kamon J, Ito Y, Tsuchida A, Yokomizo T, Kita S, Sugiyama T, Miyagishi M, Hara K, Tsunoda M, Murakami K, Ohteki T, Uchida S, Takekawa S, Waki H, Tsuno NH, Shibata Y, Terauchi Y, Froguel P, Tobe K, Koyasu S, Taira K, Kitamura T, Shimizu T, Nagai R, Kadowaki T: Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature 423:762–769, 2003[Medline]
  6. Waki H, Yamauchi T, Kamon J, Kita S, Ito Y, Hada Y, Uchida S, Tsuchida A, Takekawa S, Kadowaki T: Generation of globular fragment of adiponectin by leukocyte elastase secreted by monocytic cell line THP-1. Endocrinology 146:790–796, 2005[Abstract/Free Full Text]
  7. Collins T, Cybulsky MI: NF-kappaB: pivotal mediator or innocent bystander in atherogenesis? J Clin Invest 107:255–264, 2001[Medline]

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