Tissue-specific liver X receptor activation promotes macrophage reverse cholesterol transport in vivo

Abstract

Objective: We previously reported that a systemic liver X receptor (LXR) agonist promoted macrophage reverse-cholesterol transport (mRCT) in vivo. Because LXR are expressed in multiple tissues involved in RCT (macrophages, liver, intestine), we analyzed the effect of tissue-specific LXR agonism on mRCT.

Methods and results: In initial studies, the systemic LXR agonist GW3965 failed to promote mRCT in a setting in which LXR was expressed in macrophages but not in liver or intestine. To evaluate the effect of LXR activation specifically in small intestine on mRCT, wild-type mice were treated with either intestinal-specific LXR agonist (GW6340) or systemic LXR agonist (GW3965). Both GW3965 and GW6340 significantly promoted excretion of [(3)H]-sterol in feces by 162% and 52%, respectively. To evaluate the requirement for macrophage LXR activation, we assessed the ability of GW3965 to promote mRCT in wild-type mice using primary macrophages deficient in LXR alpha/beta vs wild-type macrophages. Whereas GW3965 treatment promoted fecal excretion compared with vehicle, its overall ability to promote mRCT was significantly attenuated using LXR alpha/beta knockout macrophages.

Conclusions: We demonstrate that intestinal-specific LXR agonism promotes macrophage RCT in vivo and that macrophage LXR itself plays an important, but not predominant, role in promoting RCT in response to an LXR agonist.

Figure 1

Wild-type (WT) and LXR DKO(KO) mice were treated with GW3965 or vehicle for 10 days and RCT study was performed with J774 macrophages as described in Methods (n=6/each group). (A) [³H]-cholesterol in plasma after macrophages injection in wild-type mice. (B) [³H]-cholesterol in plasma after macrophages injection in LXR DKO mice. (C) [³H]-cholesterol in liver. (D) [³H]-tracer distribution in feces. *p<0.05. Data are expressed as mean ± SD.

Figure 2

Gene expression analysis in Liver (A) and intestine (B) of wild-type mice determined by real-time PCR analysis. Animals were treated with vehicle, GW3965, or GW6340 by oral gavage for 12 days. Mice were sacrificed after 4 h gavage and fasting (n=5/each group). Data are expressed as fold change ± SD vs vehicle treated mice and normalized to beta-actin mRNA. *p<0.05,**p<0.01 vs vehicle group. §p<0.05, §§p<0.01 vs GW6340 group.

Figure 3

Intestine-specific LXR agonist promotes RCT in vivo. Wild-type mice (n=5/each group) were treated with indicated drug for 10 days and RCT study was performed with J774 macrophages as described in Methods. (A) FPLC analysis of cholesterol mass profile of pooled 48-hours plasma from wild-type mice treated with indicated drug. (B) FPLC analysis of [³H]-cholesterol profile of pooled 48-hours plasma from wild-type mice treated with indicated drug. (C) [³H]-cholesterol in plasma after macrophages injection. (D) [³H]-cholesterol in liver. (E) [³H]-tracer distribution in feces. *p<0.05, **p<0.01 vs vehicle group. §p<0.05, §§p<0.01 vs GW6340 group. Data are expressed as mean ± SD.

Figure 4

Wild-type mice were treated with either GW3965 or vehicle for 10 days and RCT study was performed with BMMs from either wild-type mice (WT-BMM) or LXR DKO mice (KO-BMM) as described in Methods (n=6/each group). (A) [³H]-cholesterol in plasma after macrophages injection in wild-type mice. *p<0.05, **p<0.01 vs vehicle group. (B) [³H]-cholesterol in plasma after macrophages injection in LXR DKO mice. (C) [³H]-cholesterol in liver. (D) [³H]-tracer distribution in feces. *p<0.05, **p<0.01. Data are expressed as mean ± SD.

Figure 5

Wild-type mice were fed the experimental diets for 2 weeks and HDL turnover studies were performed as described in Methods (n=6/each group). (A) The change of [³H]-HDL-cholesteryl-ester in plasma. (B) [³H]-cholesterol in liver. (C) [³H]-tracer distribution in feces. **P<0.01 vs. control group. Data are expressed as mean ± SD.