Abstract 28: Adiponectin Stimulates Cholesterol Efflux Efficiently in Human THP-1 Macrophages and Modulates HDL-apoA-I Biogenesis

Abstract

Introduction: Adiponectin (APN) is an anti-inflammatory and anti-atherogenic adipokine that is strongly correlated with circulating HDL levels. However, its role in macrophage lipid metabolism, a crucial process in atherogenesis, remains poorly investigated. We examined the effect of APN on cholesterol efflux from human THP-1 macrophages, elucidated its kinetics, and investigated its role in HDL biogenesis. Methods: APN dose-dependent (0.1 to 60 μM) and time-dependent (0.5 to 24 hours) cholesterol efflux studies were performed in 3 [H]-cholesterol labeled human THP-1 macrophages in the presence of apoA-I. Following efflux studies, the HDL fractions within media were concentrated (10kDa cut-off filter) and subjected to analytical FPLC and 2D-PAGGE technique to reveal HDL species. Results: APN stimulated ABCA1-mediated cholesterol efflux in a dose-dependent and time-dependent manner. Kinetics analysis revealed that increased molar doses of APN and apoA-I had similar Km efficiency of cholesterol efflux but greater velocity ( Km =3.24±0.71 μM, Vmax =4.90±0.07 efflux/6h) when compared to apoA-I alone ( Km =3.33±0.57 μM, Vmax =3.83±0.24 efflux/6h). Importantly, once APN was tested against a fixed dose of apoA-I (10 μg/mL), it promoted cholesterol efflux with Km = 0.17±0.06 μM. This was associated with a 75.7% decrease in intracellular free cholesterol in THP-1 cells in the presence of APN and apoA-I when compared to apoA-I alone (P<0.01). APN alone had no effect on the level of residual efflux (reached a level of 1%). The FPLC cholesterol profiles demonstrated that in the presence of APN and apoA-I there was increased lipidated nascent HDL (nHDL) during the process of cholesterol efflux, compared to apoA-I alone. This was associated with increased size of nHDL-apoA-1 pre-β and α species via 2D-PAGGE analyses. By immunoblotting for apoA-I and APN, APN oligomers exhibited a molecular weight range of 9 to 20 nm, appearing within the size range of nHDL-apoA-I. Conclusion: In addition to promoting macrophage cholesterol efflux in vitro , APN can modulate HDL-apoA-I biogenesis, by increasing the generation of nHDL particles. These findings suggest that APN may be of potential therapeutic value in the modulation of HDL’s protective role in atherosclerosis.