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Selective suppression of endothelial cell apoptosis by the high molecular weight form of adiponectin - PubMed

  • ️Thu Jan 01 2004

Selective suppression of endothelial cell apoptosis by the high molecular weight form of adiponectin

Hideki Kobayashi et al. Circ Res. 2004.

Abstract

Adiponectin is an adipocyte-derived, antiatherogenic protein that is present in serum as three isoforms. Total adiponectin levels are decreased in obese or diabetic humans or animal models. This study was designed to elucidate the relative isoform distribution of adiponectin in human disease states and identify the active form of adiponectin toward vascular endothelial cells. The percentage of high molecular weight form (HMW) per total adiponectin was significantly lower in patients with coronary artery disease than control subjects, whereas the hexamer form was similar and the trimer form was significantly higher. During weight reduction in obese subjects, the HMW form increased and the trimer and hexamer forms decreased. Recombinant adiponectin dose-dependently suppressed apoptosis and caspase-3 activity in human umbilical vein endothelial cells (HUVECs). Transduction with dominant-negative AMP-activated protein kinase (AMPK) abolished the suppressive effect of adiponectin on HUVECs. Gel filtration chromatography was used to separate the adiponectin isoforms, and the antiapoptotic effect toward HUVECs was only observed with the HMW form. These data suggest that HMW adiponectin specifically confers the vascular-protective activities of this adipocytokine. The full text of this article is available online at http://circres.ahajournals.org.

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Figures

Figure 1
Figure 1

Oligometric state of adiponectin in human plasma. A, Representative elution profiles of adiponectin in plasma from the same individual before and after weight reduction. Plasma was fractionated by gel filtration chromatography, and the concentration of adiponectin in each 1-mL fraction was determined by ELISA. B, Average changes of each form of adiponectin per total adiponectin between before and after weight reduction (n=6). C, Percentage of each form of adiponectin per total adiponectin in the patients with CAD or control subjects (n=8 each). Plasma was fractionated by gel filtration chromatography, and the concentration of adiponectin in each fraction was determined by ELISA. **P<0.01.

Figure 2
Figure 2

Effects of adiponectin on HUVEC viability. A, Effect of adiponectin on endothelial cell death induced by serum starvation. Cell viability was quantified with MTS-based assay. Heat indicates heat-digested adiponectin treated with proteinase K. B, Effect of adiponectin on endothelial cell apoptosis induced by serum deprivation. Pyknotic nuclei were quantified. C, Effect of adiponectin on caspase-3 activation induced by serum starvation. D, Role of AMPK in the regulation of adiponectin-induced reduction of endothelial cell death. HUVECs were transduced with an adenoviral vector expressing dominant-negative AMPK (dn-AMPK) or an adenoviral vector expressing GFP (Control). After 24-hour infection, cells were treated with adiponectin (30 µg/mL) or vehicle under serum deprivation conditions. Cell viability was quantified with MTS-based assay. E, Recombinant mouse adiponectin was separated by gel filtration chromatography into trimer (Tri.), hexamer (Hex.), and higher molecular weight form (HMW) fractions. Representative profile is shown. F, Western blot analysis of the adiponectin fractions. G, Effects of 3 different oligometric forms of adiponectin on endothelial cell death induced by serum starvation. Cell viability was quantified by MTS-based assay. Data are mean±SE of 4 experiments. *P<0.05, **P<0.01 vs each control. H, Effects of 3 different oligometric forms of adiponectin on AMPK phosphorylation. Cells were treated with each form of adiponectin (10 µg/mL) or vehicle for 15 minutes. Representative blots are shown. Relative phosphorylation levels of AMPK were quantified using NIH image program. Immunoblots were normalized to total loaded protein. *P<0.01 vs control.

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