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
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 κB (NF-κB) activation. To study NF-κB 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-κB binding sites (pNFκB-Luc; Stratagene, La Jolla, CA). Several clones were selected for analysis of NF-κB activation.
RESULTS
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-κB activation. Induction of the expression of those genes by globular adiponectin was significantly inhibited by the NF-κB inhibitor BAY11-7082, suggesting that globular adiponectin activates NF-κB. We therefore examined whether globular adiponectin really induces NF-κB activity in cells. It was observed that globular adiponectin dose-dependently activated NF-κB–mediated gene transcription. NF-κB–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-κB–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-κB. To determine the relative strength of NF-κB activation by globular adiponectin, we compared induction of NF-κB–mediated reporter gene expression between globular adiponectin and tumor necrosis factor (TNF)-α, lipopolysaccharide, and interleukin-1β, known inducers of NF-κB in endothelial cells. TNFα, lipopolysaccharide, and interleukin-1β potently stimulated NF-κB–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 TNFα-induced NF-κB–mediated gene transcription, since hyporesponsiveness or desensitization to TNFα might be caused by pretreatment with globular adiponectin. Cells were treated with various concentrations of globular adiponectin for 16 h and then stimulated with TNFα to induce NF-κB activation. TNFα-induced NF-κB–mediated gene transcription was suppressed by globular adiponecting pretreatment in a dose-dependent manner.
CONCLUSIONS
In the present study, we have demonstrated that globular adiponectin is a potent stimulator of NF-κB 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-κB 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.
A: The effects of globular adiponectin on NF-κB–dependent transcriptional activity. Quiescent cells (transfected with pNFκB-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-κB–dependent transcriptional activity. Quiescent cells (transfected with pNFκB-Luc) were left untreated (Cont) or were treated with globular adiponectin (gAD: 3 or 10 μg/ml) or TNFα (10 ng/ml), lipopolysaccharide (LPS) (10 μg/ml), interleukin-1β (10 ng/ml). After 2 h, cells were lysed, and luciferase activities were measured. C: The effect of globular adiponectin pretreatment on TNFα-induced NF-κB–dependent transcriptional activity. Cells (transfected with pNFκ B-Luc) were treated with various concentrations of globular adiponectin for 16 h and then treated with TNFα 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.
A: The effects of globular adiponectin on NF-κB–dependent transcriptional activity. Quiescent cells (transfected with pNFκB-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-κB–dependent transcriptional activity. Quiescent cells (transfected with pNFκB-Luc) were left untreated (Cont) or were treated with globular adiponectin (gAD: 3 or 10 μg/ml) or TNFα (10 ng/ml), lipopolysaccharide (LPS) (10 μg/ml), interleukin-1β (10 ng/ml). After 2 h, cells were lysed, and luciferase activities were measured. C: The effect of globular adiponectin pretreatment on TNFα-induced NF-κB–dependent transcriptional activity. Cells (transfected with pNFκ B-Luc) were treated with various concentrations of globular adiponectin for 16 h and then treated with TNFα 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.
References
A table elsewhere in this issue shows conventional and Système International (SI) units and conversion factors for many substances.
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