Anacetrapib promotes reverse cholesterol transport and bulk cholesterol excretion in Syrian golden hamsters

Abstract

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) and triglyceride between HDL and apoB-containing lipoproteins. Anacetrapib (ANA), a reversible inhibitor of CETP, raises HDL cholesterol (HDL-C) and lowers LDL cholesterol in dyslipidemic patients; however, the effects of ANA on cholesterol/lipoprotein metabolism in a dyslipidemic hamster model have not been demonstrated. To test whether ANA (60 mg/kg/day, 2 weeks) promoted reverse cholesterol transport (RCT), ³H-cholesterol-loaded macrophages were injected and (3)H-tracer levels were measured in HDL, liver, and feces. Compared to controls, ANA inhibited CETP (94%) and increased HDL-C (47%). ³H-tracer in HDL increased by 69% in hamsters treated with ANA, suggesting increased cholesterol efflux from macrophages to HDL. ³H-tracer in fecal cholesterol and bile acids increased by 90% and 57%, respectively, indicating increased macrophage-to-feces RCT. Mass spectrometry analysis of HDL from ANA-treated hamsters revealed an increase in free unlabeled cholesterol and CE. Furthermore, bulk cholesterol and cholic acid were increased in feces from ANA-treated hamsters. Using two independent approaches to assess cholesterol metabolism, the current study demonstrates that CETP inhibition with ANA promotes macrophage-to-feces RCT and results in increased fecal cholesterol/bile acid excretion, further supporting its development as a novel lipid therapy for the treatment of dyslipidemia and atherosclerotic vascular disease.

Fig.1.

CETP inhibition with ANA (60 mg/kg, 2 weeks) in dyslipidemic hamsters increases plasma HDL-cholesterol but not nonHDL-cholesterol. Lipids were measured as described in Methods. ANA treatment (solid bars) was associated with (A) increased total cholesterol, (B) increased HDL-cholesterol, (C) no change in nonHDL-cholesterol, and (D) reduced triglyceride. ***P < 0.001, *P < 0.05 versus control. Open bars, control; solid bars, ANA treated; n = 24 per group.

Fig.2.

Effects of ANA treatment (60 mg/kg, 2 weeks) of dyslipidemic hamsters on macrophage-to-feces RCT. A: ³H-tracer recovery in plasma; B: ³H-tracer recovery in HDL fraction of plasma; C: ³H-tracer recovery in liver tissue; D: ³H-tracer recovery in liver cholesterol and bile acid fraction; E: liver cholesterol and bile acid mass (unlabeled); F: ³H-tracer recovery in fecal cholesterol and bile acid fraction. ***P < 0.001, ** P < 0.01, *P < 0.05 versus control. Open bars, control; solid bars, ANA treated; n = 12 per group.

Fig.3.

Effects of ANA treatment (60 mg/kg, 2 weeks) of dyslipidemic hamsters on HDL-cholesteryl ester kinetics. A: Time course of ³H-tracer recovery in plasma; B: fractional catabolic rate of ³H-tracer in plasma; C: time course of ³H-tracer recovery in HDL fraction of plasma; D: fractional catabolic rate of ³H-tracer in HDL fraction. ***P < 0.001, **P < 0.01 versus control. Open bars, control; solid bars, ANA treated; n = 12 per group.

Fig.4.

Changes in HDL cholesterol composition with anacetrapib treatment. A: Increase in HDL total cholesterol is more prominent in large HDL fraction; B: majority of increase driven by free cholesterol and cholesteryl linoleate (18:2). *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle.

Fig.5.

Lack of change in LDL cholesterol distribution with anacetrapib treatment. A: Distribution of cholesterol in LDL subfractions, and (B) major cholesterol species present in LDL.

Fig.6.

Anacetrapib treatment increases (A) fecal cholesterol and (B) fecal cholic acid excretion. * P < 0.05 versus day 0.

Fig.7.

Anacetrapib treatment does not affect cholesterol absorption. A: Fecal D₆-cholesterol excretion over the time-course of 48 hr for vehicle and ezetimibe treated hamsters. B: Plasma levels of D₆-cholesterol for vehicle and ezetimibe treated hamsters. C: Fecal D₆-cholesterol excretion over the time-course of 48 hr for vehicle and anacetrapib treated hamsters. D: Plasma levels of D₆-cholesterol for vehicle and anacetrapib treated hamsters. **P < 0.01 ezetimibe 24 h versus vehicle 24 h; *P < 0.05 ezetimibe 48 h versus vehicle 48 h.

Fig.8.

Cholesterol efflux was increased in HDL from anacetrapib-treated hamsters. A: SR-BI mediated cholesterol efflux. B: Cholesterol efflux ± ABCA1 upregulation with cAMP. C: ABCG1 mediated cholesterol efflux. * P < 0.05, ** P < 0.01, *** P < 0.001 versus vehicle.