Liver X receptor-mediated activation of reverse cholesterol transport from macrophages to feces in vivo requires ABCG 5 / G 8

Liver X receptor (LXR) agonists increase both total fecal sterol excretion and macrophage-specific reverse cholesterol transport (RCT) in vivo. In this study, we assessed the effects of ABCG5/G8 deficiency as well as those of LXR agonist-induction of RCT from macrophages to feces in vivo. A [3H]cholesterol-labeled macrophage cell line was injected intraperitoneally into ABCG5/G8-deficient (G5/G82/2), heterozygous (G5G81/2), and wild-type G5/ G81/1 mice. G5/G82/2mice presented increased radiolabeled HDL-bound [3H]cholesterol 24 h after the label injection. However, the magnitude of macrophage-derived [3H]cholesterol in liver and feces did not differ between groups. A separate experiment was conducted in G5G81/1 and G5G82/2 mice treated with or without the LXR agonist T0901317. Treatment with T0901317 increased liver ABCG5/G8 expression, which was associated with a 2-fold increase in macrophage-derived [3H]cholesterol in feces of G5/G81/1 mice. However, T0901317 treatment had no effect on fecal [3H]cholesterol excretion in G5G82/2 mice. Additionally, LXR activation stimulated the fecal excretion of labeled cholesterol after an intravenous injection of HDL[3H]cholesteryl oleate in G5/G81/1 mice, but failed to enhance fecal [3H]cholesterol in G5/G82/2 mice. Our data provide direct in vivo evidence of the crucial role of ABCG5 and ABCG8 in LXR-mediated induction of macrophagespecific RCT. —Calpe-Berdiel, L. , N. Rotllan, C. Fiévet, R. Roig, F. Blanco-Vaca, and J. C. Escolà-Gil. Liver X receptormediated activation of reverse cholesterol transport from macrophages to feces in vivo requires ABCG5/G8. J. Lipid Res. 2008. 49: 1904–1911. Supplementary key words HDL • LXR agonist • mice The classic reverse cholesterol transport (RCT) pathway involves cholesterol efflux from peripheral tissues to HDL, which is taken up by the liver and finally excreted into bile and feces. Liver X receptor (LXR) a is a nuclear receptor activated by oxysterols (1) that upregulates expression of a number of target genes critical for RCT, including ABCA1, ABCG1, ABCG5, ABCG8, and murine cytochrome P450 family 7 subfamily A polypeptide 1 (CYP7A1) (2–6). LXRa is highly expressed in the liver and at lower levels in the adrenal glands, intestine, adipose tissue, macrophages, lung, and kidney, whereas LXRb is ubiquitously expressed (7). ABCG5 and ABCG8 heterodimerize into a functional complex (ABCG5/G8) that is crucial for hepatobiliary and intestinal sterol excretion. Genetic deficiency of ABCG5/ G8 in humans causes sitosterolemia (8, 9), which is characterized by plant sterol accumulation in plasma and tissues due to increased intestinal absorption and decreased biliary secretion of sterols (10, 11). It is difficult at present to quantify RCT in humans (12, 13). A new approach was developed to measure RCT from labeled cholesterol incorporated into macrophages and injected into mice liver and feces (14). Follow-up studies demonstrated that the rate of RCT from macrophages to feces may not always be inferred from studies of total RCT (14–17). This is an important point in the context of current efforts to better understand HDL antiatherogenic functions as well as to develop a new test for predicting those functions. Analysis of ABCG5/G8 function in RCT is, thereThis work was funded by Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Red FIS RD06/0015, and Grant FIS PI06/0551. N.R. is a predoctoral fellow of Fondo de Investigaciones Sanitarias (05/00221). J.C. E-G. is a Ramón y Cajal researcher, funded by the Ministerio de Educación y Ciencia. Manuscript received 17 October 2007 and in revised form 6 February 2008 and in re-revised form 31 March 2008. Published, JLR Papers in Press, May 28, 2008. DOI 10.1194/jlr.M700470-JLR200 1 L. Calpe-Berdiel and N. Rotllan contributed equally to this work. 2 To whom correspondence should be addressed. e-mail: fblancova@santpau.es (F.B-V.); jescola@santpau.es ( J.C.E-G.) Copyright © 2008 by the American Society for Biochemistry and Molecular Biology, Inc. 1904 Journal of Lipid Research Volume 49, 2008 This article is available online at http://www.jlr. org by gest, on A uust 5, 2017 w w w . j.org D ow nladed fom fore, of interest. Overexpression of ABCG5/G8 in transgenic mice and pharmacological activation of LXR result in increased hepatobiliary excretion of cholesterol and increased fecal neutral sterol excretion (18–22), and this action is dependent on ABCG5/G8 (19). Inactivation of both genes simultaneously has the opposite effects on sterol hepatobiliary excretion (23). Importantly, overexpression of both human ABCG5 and -G8 in transgenic mice also reduces intestinal cholesterol absorption (18), although the opposite effect was not observed in ABCG5/G8 doubleknockout mice (19, 23, 24). Synthetic LXR ligands stimulate cholesterol efflux from culturedmacrophages (6, 25), inhibit atherosclerosis progression, and even promote atherosclerosis regression in mice (26–28). This effect has been shown to depend on the presence ofmacrophage LXR (28). On the other hand, the synthetic LXR agonist GW3965 promotes specific RCT from macrophages to feces in vivo despite having little effect on HDL cholesterol levels (29). Considering that ABCG5/G8 is not expressed in macrophages, the results of these studies support the concept that LXR activation may modulate this macrophage-specific RCT pathway by increasing biliary cholesterol excretion, presumably through upregulation of ABCG5 and ABCG8 mRNA expression (19). However, the contribution of ABCG5/G8 heterodimer to RCT from macrophages to feces in vivo remains to be directly established (12, 30). In the present study, we investigated the effects of ABCG5/G8 deficiency as well as those of LXR agonist induction of RCT from a macrophage cell line to feces in vivo. MATERIALS AND METHODS