Fig. 2.

Role of ABCA1 in nascent HDL particle formation and cholesterol efflux in primary hepatocytes. Primary hepatocytes from liver-specific ABCA1 knockout mice and C57BL/6 control mice were treated with or without LXR agonist T0901317 (5 µM) for 16 h and then treated with or without glyburide (500 µM) for 1 h. Cells were then incubated in medium with or without glyburide with 20 µg/ml human apoA-I for 16 h. A: ABCA1 expression was determined by Western blot analysis using anti-human/mouse ABCA1, and anti-mouse β-actin as loading control. B: Hepatocytes were labeled with 0.2 µCi/ml [³H] cholesterol for 48 h. Cells were treated with T0901317 and glyburide as described in (A). Following incubation with 20 µg/ml human apoA-I for 16 h, radioactivity in the medium and cells was determined. Cholesterol efflux was calculated as the percentage of counts in the medium relative to the total counts in the medium and cells together. Values shown were the mean ± SEM of triplicate determinations. C: ApoA-I lipidation was assessed by separating cell culture medium (5 µl) by nondenaturing GGE and immunoblotting with anti-human apoA-I. Sizes of nascent HDL particles, determined as set out in Materials and Methods are indicated on the right. Results are representative of two experiments. Significance at ***: P < 0.001.