Cholesterol Absorption From the Intestine Is a Major Determinant of Reverse Cholesterol Transport From Peripheral Tissue Macrophages
Objective— We examined the effect of ezetimibe, a cholesterol absorption (CA) inhibitor, and genetic determinants of CA on reverse cholesterol transport (RCT) from subcutaneously injected macrophages using a new dual isotope label technique.
Methods and Results— Treatment of C57BL/6J mice with ezetimibe decreased dietary CA by 86% and increased RCT from peripheral tissue macrophages (PTM) by 6-fold (P<0.0001). Moreover, congenic 14DKK mice with a modest 41% decrease in dietary CA displayed a 67% increase in RCT from PTM (P<0.007).
Conclusions— These findings indicate that pharmacological and genetic modifiers of cholesterol absorption are major determinants of reverse cholesterol transport from peripheral tissue macrophages.
- cholesterol absorption
- cholesterol efflux
- reverse cholesterol transport
Reverse cholesterol transport (RCT) is a multi-step process that involves the efflux of cholesterol from peripheral tissue macrophages (PTM) for ultimate excretion in the feces. In the initial step, the high-density lipoprotein (HDL) particle promotes cholesterol efflux from macrophages for delivery to the liver. Subsequently, HDL cholesterol is excreted into bile and the intestinal lumen where biliary cholesterol is partly reabsorbed, whereas the remainder is excreted into feces. Rader and colleagues provided the first direct in vivo evidence for transport of cholesterol from PTM into feces by intraperitoneal injection of [3H]-cholesterol-labeled macrophages and detection of tracer in plasma, liver, bile, and feces.1 The presence of [3H]-cholesterol in feces suggests that the intestines may play a role in modifying RCT from PTM. Herein we used pharmacological and genetic approaches to show that cholesterol absorption (CA) from the intestine plays a major role in determining the RCT from PTM.
Detailed descriptions of all methods used as well as the generation and characterization of 14DKK congenic animals are available in the supplemental materials (available online at http://atvb.ahajournals.org).
Time Course Excretion of Fecal [14C]-Cholesterol and [3H]-β-Sitostanol From PTM
Animals were injected subcutaneously with dual-labeled macrophages, and feces were collected daily for 48 hours. As shown in Figure 1, fecal excretion of the [14C]-cholesterol label significantly increased, whereas that of the [3H]-β-sitostanol label did not change from day 1 to day 2.
Treatment With Ezetimibe and the Congenic 14DKK Interval Suppress the Absorption of Dietary Sterols
Feeding for 10 days with 0.005% ezetimibe largely suppressed the absorption of dietary sterols, as indicated by a decrease of 86%, 94%, and 91% in dietary CA, plasma campesterol, and plasma sitosterol levels, respectively. When compared to ezetimibe treatment, congenic 14DKK animals were characterized by only a modest decrease in dietary sterol absorption with 41%, 17%, and 27% decrease in CA, plasma campesterol, and plasma sitosterol levels, respectively (Table).
Treatment With Ezetimibe and the Congenic 14DKK Interval Increase the RCT From PTM
As shown in Figure 2A, in chow-fed animals the 48-hour fecal excretion of [14C]-cholesterol label from PTM amounted to 3.1±0.7%, whereas in ezetimibe treated animals the excretion was 6-fold higher reaching 19.4±6.6%. Concordant with these findings, as shown in Figure 2B, in C57BL/6J control animals the 48-hour fecal excretion of [14C]-cholesterol label from PTM amounted to 4.3±1.8% whereas in 14DKK congenics the excretion was 67% higher reaching 7.2±2.0%. In contrast, neither ezetimibe treatment nor the 14DKK interval displayed an effect on 24-hour and 48-hour plasma levels of [14C]-cholesterol and [3H]-β-sitostanol, or on liver content of the [14C]-cholesterol and [3H]-β-sitostanol labels (Table).
Treatment with bile acid sequestrants and partial ileal bypass, 2 interventions that decrease the absorption of cholesterol, are associated with decreased mortality from atherosclerotic cardiovascular disease.2,3 Similarly, atheroprotective effects were reported in apolipoprotein E null mice treated with ezetimibe.4 Herein we show that potent pharmacological inhibition and moderate genetic suppression of CA markedly increased RCT from PTM. These findings raise the possibility that the atheroprotective effects reported for CA inhibitors may arise not only from reductions in plasma LDL cholesterol levels but also by impacting on net transfer of cholesterol from peripheral cells, such as macrophages in the arterial wall, for ultimate elimination in the feces.
We thank Melanie Bodine and Dave Schmitt for expert technical assistance.
Sources of Funding
This study was supported by grants from the American Heart Association (0755295B) and the National Institutes of Health (P01HL087018, P01 HL076491, and P01 HL077107).
Original received February 27, 2008; final version accepted April 7, 2008.
Zhang Y, Zanotti I, Reilly MP, Glick JM, Rothblat GH, Rader DJ. Overexpression of apolipoprotein A-I promotes reverse transport of cholesterol from macrophages to feces in vivo. Circulation. 2003; 108: 661–663.
Buchwald H, Varco RL, Matts JP, Long JM, Fitch LL, Campbell GS, Pearce MB, Yellin AE, Edmiston WA, Smink RD Jr, et al. Effect of partial ileal bypass surgery on mortality and morbidity from coronary heart disease in patients with hypercholesterolemia. Report of the Program on the Surgical Control of the Hyperlipidemias (POSCH). N Engl J Med. 1990; 323: 946–955.
Davis HR Jr, Compton DS, Hoos L, Tetzloff G. Ezetimibe, a potent cholesterol absorption inhibitor, inhibits the development of atherosclerosis in ApoE knockout mice. Arterioscler Thromb Vasc Biol. 2001; 21: 2032–2038.