Liver X receptor activation promotes macrophage-to-feces reverse cholesterol transport in a dyslipidemic hamster model

Abstract

Liver X receptor (LXR) activation promotes reverse cholesterol transport (RCT) in rodents but has major side effects (increased triglycerides and LDL-cholesterol levels) in species expressing cholesteryl ester transfer protein (CETP). In the face of dyslipidemia, it remains unclear whether LXR activation stimulates RCT in CETP species. We therefore used a hamster model made dyslipidemic with a 0.3% cholesterol diet and treated with vehicle or LXR agonist GW3965 (30 mg/kg bid) over 10 days. To investigate RCT, radiolabeled (3)H-cholesterol macrophages or (3)H-cholesteryl oleate-HDL were then injected to measure plasma and feces radioactivity over 72 or 48 h, respectively. The cholesterol-enriched diet increased VLDL-triglycerides and total cholesterol levels in all lipoprotein fractions and strongly increased liver lipids. Overall, GW3965 failed to improve both dyslipidemia and liver steatosis. However, after (3)H-cholesterol labeled macrophage injection, GW3965 treatment significantly increased the (3)H-tracer appearance by 30% in plasma over 72 h, while fecal (3)H-cholesterol excretion increased by 156% (P < 0.001). After (3)H-cholesteryl oleate-HDL injection, GW3965 increased HDL-derived cholesterol fecal excretion by 64% (P < 0.01 vs. vehicle), while plasma fractional catabolic rate remained unchanged. Despite no beneficial effect on dyslipidemia, LXR activation promotes macrophage-to-feces RCT in dyslipidemic hamsters. These results emphasize the use of species with a more human-like lipoprotein metabolism for drug profiling.

Fig. 1.

Impact of cholesterol enriched diet on plasma and hepatic lipid content. Distribution of cholesterol (A) and triglycerides (B) in the plasma of overnight fasted hamsters after a 4 week chow diet or chow + 0.3% cholesterol analyzed by gel filtration chromatography (pooled plasma). (C) Hepatic total cholesterol, triglycerides, and fatty acid levels per gram wet weight in hamsters fed a 4 week chow (open bars) or chow + 0.3% cholesterol (solid bars) diet. Values are means ± SEM (n = 8 per group, *** P < 0.001 vs. chow fed hamsters).

Fig. 2.

Effect of GW3965 LXR agonist on plasma and liver lipid content. Hamsters on a chow + 0.3% cholesterol diet were orally dosed twice daily for 10 days with vehicle (0.5% hydroxypropyl methylcellulose) or GW3965 (30 mg/kg). Distribution of total cholesterol (A) and triglycerides (B) in the plasma of nonfasted hamsters treated or not with GW3965 were analyzed (6 h postgavage, pooled plasma). (C) Hepatic total cholesterol, triglycerides, and fatty acids levels per gram wet weight in hamsters treated with vehicle (solid bars) or with GW3965 (gray bars) were estimated. Values are means ± SEM (n = 8 per group).

Fig. 3.

Impact of LXR agonist on gene expression in liver and intestine. Hamsters on a chow + 0.3% cholesterol diet were orally dosed twice daily for 10 days with vehicle (0.5% hydroxypropyl methylcellulose) or GW3965 (30 mg/kg). Gene expression was measured by real-time quantitative PCR in liver (A) and intestine (B) in both vehicle (solid bars) and GW3965 (gray bars) groups. Values are means ± SEM (n = 8 per group,* P < 0.05; *** P < 0.001 vs. vehicle).

Fig. 4.

Effects of GW3965 LXR agonist on macrophage-to-feces RCT. Hamsters on a chow + 0.3% cholesterol diet were orally treated twice daily for 7 days with vehicle (0.5% hydroxypropyl methylcellulose) or GW3965 (30 mg/kg) and then injected with ³H-cholesterol labeled macrophages. Animals were treated with vehicle or GW3965 during the 72 h experiment. Time course of ³H-cholesterol distribution in plasma of hamsters of each group was established (A) after injection of radiolabeled macrophages. Liver (B) and fecal (C) ³H-tracer recovery in hamsters of vehicle group (solid bars) and GW3965 group (gray bars) were also monitored. Data are expressed as percent of dpm injected ± SEM (n = 6 per group, * P < 0.05; ** P < 0.01; *** P < 0.001 vs. vehicle).

Fig. 5.

Effects of GW3965 LXR agonist on HDL-cholesteryl esters kinetics and HDL-derived fecal cholesterol excretion. Hamsters on a chow + 0.3% cholesterol diet were orally treated twice daily for 8 days with vehicle (0.5% hydroxypropyl methylcellulose) or GW3965 (30 mg/kg) and then injected with ³H-cholesteryl oleate labeled HDL. Animals were treated with vehicle or GW3965 during the 48 h experiment. After injection of radiolabeled HDL, plasma decay curves (A) of vehicle- (solid plots) and GW3965-treated hamsters (gray plots) were analyzed and fitted curves (dashed line) and FCR were calculated by SAAMII software. Fecal ³H tracer recovery (B) were detected in hamsters treated with vehicle (solid bars) or GW3965 LXR agonist (gray bars). Values are means ± SEM (n = 6 per group, ** P < 0.01 vs. vehicle).