Volume 140, Issue 3, Pages 1043-1051 (March 2011) Biliary Sterol Secretion Is Required for Functional In Vivo Reverse Cholesterol Transport in Mice  Niels Nijstad, Thomas Gautier, François Briand, Daniel J. Rader, Uwe J.F. Tietge  Gastroenterology  Volume 140, Issue 3, Pages 1043-1051 (March 2011) DOI: 10.1053/j.gastro.2010.11.055 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 BDL almost completely abolishes macrophage RCT in vivo. After BDL (n = 5) or sham surgery (Con, n = 6) mice received intraperitoneal injections of 3H-cholesterol–labeled macrophage foam cells. Blood samples were taken at 6 and 24 hours after injection and feces were collected continuously for 24 hours. (A) Time course of 3H-cholesterol recovery within plasma. (B) Cholesterol (left panel) and 3H-cholesterol (right panel) distribution over the different lipoprotein subclasses determined by fast protein liquid chromatography analysis of pooled plasma samples 24 hours after injection of 3H-cholesterol–loaded macrophages. (C) 3H-tracer within liver and (D) 3H-tracer content within fecal neutral sterols (NS, left panel) and bile acids (BA, right panel) of the respective groups of mice 24 hours after injection of 3H-cholesterol–loaded macrophages. The 3H-cholesterol data are expressed as the percentage of tracer relative to the total cpm injected. All data are given as means ± standard error of the mean. ***P < .001. VLDL, very low density lipoprotein; LDL, low-density lipoprotein. Gastroenterology 2011 140, 1043-1051DOI: (10.1053/j.gastro.2010.11.055) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Severely reduced biliary cholesterol secretion owing to Abcb4 deficiency decreases macrophage RCT in vivo and functional biliary cholesterol secretion is required to mediate the increasing effect of LXR activation on RCT. Respective groups of wild-type FVB or Abcb4 knockout (KO) mice were administered for 8 days either chow (Con) or chow containing the LXR agonist T0901317 (T09) and then injected intraperitoneally with 3H-cholesterol–labeled macrophage foam cells. Treatments were continued for 48 hours, and during this period blood samples were taken at 6, 24, and 48 hours. Feces were collected continuously for 48 hours. (A) Time course of 3H-cholesterol recovery within plasma. Filled triangles, control wild-type mice; open triangles, LXR agonist-treated wild-type mice; filled circles, control Abcb4 knockout mice; open circles, LXR agonist-treated Abcb4 knockout mice. (B) Cholesterol distribution over the different lipoprotein subclasses determined by fast protein liquid chromatography analysis of pooled plasma samples in wild-type (left panel) and Abcb4 knockout (right panel) mice without (diamonds) or with (squares) LXR agonist administration. (C) 3H-tracer within liver. (D) 3H-tracer recovery within the bile acids (BA, left panel) and the cholesterol fractions (Chol, right panel) of gallbladder bile obtained at 48 hours. (E) 3H-cholesterol within small intestine. (F) 3H-tracer content within fecal neutral sterols (NS, left panel) and bile acids (BA, right panel) of the respective groups of mice 48 hours after injection of 3H-cholesterol–loaded macrophages. The 3H-cholesterol data are expressed as the percentage of tracer relative to the total cpm injected. All data are given as means ± standard error of the mean. *P < .05, **P < .01, ***P < .001 compared with the respective wild-type group, #P < .05, ##P < .01, ###P < .001 compared with the respective chow diet group. VLDL, very low density lipoprotein; LDL, low-density lipoprotein. Gastroenterology 2011 140, 1043-1051DOI: (10.1053/j.gastro.2010.11.055) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 HDL kinetic experiments performed in wild-type, Sr-bI knockout (KO), and Abcb4 knockout mice receiving either chow (Con) or chow supplemented with the LXR agonist T0901317 (T09). HDL was labeled with 3H-cholesteryl ether as described in the Materials and Methods section and injected into the different mouse models (n = 4–6 mice per group). Blood samples were taken at different time points over 48 hours, plasma counts were determined and tracer disappearance curves were constructed and modeled using the SAAM II program. At 48 hours tracer uptake into tissues was assessed. (A) FCRs calculated from the respective plasma disappearance curves. (B) Liver uptake of 3H-cholesteryl ether. (C) Uptake of 3H-cholesteryl ether by the small intestine. Data are given as means ± standard error of the mean. #P < .05, ###P < .001 compared with the respective wild-type group. *P < .05 compared with the respective chow diet group. Gastroenterology 2011 140, 1043-1051DOI: (10.1053/j.gastro.2010.11.055) Copyright © 2011 AGA Institute Terms and Conditions