FIGURE 3.

AICARs enhance HDL-mediated cholesterol efflux in macrophages. A, macrophages were loaded with OxLDL (50 μg/ml) alone or in the presence of different concentrations of AICAR for 24 h. These cells were then washed with PBS and incubated with HDL (20 μg/ml) as an acceptor for a further 16 h. Determination of cholesterol efflux from the cells was performed as described under “Experimental Procedures.” The results are represented as the mean ± S.E. of three individual experiments. B, [³H]cholesterol-loaded macrophages were treated with AICAR (0.5–2.0 mm) and subsequently incubated with RPMI 1640 medium with HDL (20 μg/ml) as indicated. HDL-induced [³H]cholesterol efflux was measured as described. Values are expressed relative to the control, set as 1. Results are the mean ± S.E. of triplicate determinations, representative of three independent experiments. *, p < 0.05, or **, p < 0.01 compared with control. C and D, cholesterol-loaded macrophages were transfected with DN-AMPKα (C) or pretreated with the compound C (D) (10 mm). The cells were then incubated with AICAR (1 mm) for a further 24 h. HDL-mediated cholesterol efflux was determined as under “Experimental Procedures.” Results are represented as mean ± S.E. of three individual experiments. **, p < 0.01. E and F, macrophages were transfected with ABCG1 siRNA or a nonspecific control siRNA. E, Western blot analyses using antibody against ABCG1 and β-actin were performed to test transfection efficiency. Representative blots from three independent assays are presented. F, 24 h after transfection, the cells were further incubated with OxLDL (50 μg/ml) alone or in the presence of AICAR (1 mm) for 24 h. Macrophages were then washed with PBS and incubated with or without HDL (20 μg/ml) for 16 h. Results are represented as mean ± S.E. of three individual experiments. **, p < 0.01.