In vivo reverse cholesterol transport assay

The macrophage-to-feces in vivo RCT assay was performed according to previously described methods (18). Wild-type C57BL/6 and LDLr−/− mice were administered either control or Angptl3 ASO at 50 mg/kg/wk for 6 weeks. After the ASO treatment phase, mice were intraperitoneally injected with ³H-cholesterol-labeled J774A macrophages (approximately 4.79 mil dpm/5.26 mil cells/mouse). In a separate experiment, CETP tg, LDLr−/− mice were administered either control ASO or Angptl3 ASO at 15 mg/kg/wk for 6 weeks. After ASO treatment, mice were intraperitoneally injected with ³H-cholesterol-labeled macrophages (approximately 2.52 mil dpm/6.5 mil cells/mouse). For both studies, mice were singly housed in wire-bottom cages for 72 h, plasma samples were taken at 24 and 48 h, and feces were collected over the entire 72 h period. After 72 h, mice were sacrificed, and terminal plasma samples were collected along with liver samples. A 20 μl aliquot of plasma from each time point/mouse was counted for dpm by liquid scintillation counting (LSC). Liver tissues were extracted according to previously described methods (19). The isolated lipid extracts were dried under N₂ before resuspension in the scintillation cocktail and counted by LSC. Finally, the amount of radiolabeled fecal cholesterol and bile acid was assayed according to previously published methods (18, 20).