Stimulation of cholesterol excretion by the liver X receptor agonist requires ATP-binding cassette transporters G5 and G8

Liver X receptor (LXR) is a nuclear receptor that plays a crucial role in orchestrating the trafficking of sterols between tissues. Treatment of mice with a potent and specific LXR agonist, T0901317, is associated with increased biliary cholesterol secretion, decreased fractional cholesterol absorption, and increased fecal neutral sterol excretion. Here we show that expression of two target genes of LXRalpha, the ATP-binding cassette (ABC) transporters Abcg5 and Abcg8, is required for both the increase in sterol excretion and the decrease in fractional cholesterol absorption associated with LXR agonist treatment. Mice expressing no ABCG5 and ABCG8 (G5G8(-/-) mice) and their littermate controls were treated for 7 days with T0901317. In wild type animals, treatment with the LXR agonist resulted in a 3-fold increase in biliary cholesterol concentrations, a 25% reduction in fractional cholesterol absorption, and a 4-fold elevation in fecal neutral sterol excretion. In contrast, the LXR agonist did not significantly affect biliary cholesterol levels, fractional cholesterol absorption, or neutral fecal sterol excretion in the G5G8(-/-) mice. Thus Abcg5 and Abcg8 are required for LXR agonist-associated changes in dietary and biliary sterol trafficking. These results establish a central role for ABCG5 and ABCG8 in promoting cholesterol excretion in vivo.     

Direct evidence in vivo of impaired macrophage-specific reverse cholesterol transport in ATP-binding cassette transporter A1-deficient mice

The ATP-binding cassette transporter A1 (ABCA1) is a key regulator of high-density lipoprotein (HDL) metabolism. There is strong evidence that ABCA1 is a key regulator of reverse cholesterol transport (RCT). However, this could not be proved in vivo since hepatobiliary cholesterol transport was unchanged in ABCA1-deficient mice (ABCA1-/-). We used ABCA1-/- mice to test the hypothesis that ABCA1 is a critical determinant of macrophage-specific RCT. Although this cell-specific RCT only accounts for a tiny part of total RCT, it is widely accepted that it may have a major impact on atherosclerosis susceptibility. [(3)H]cholesterol-labeled endogenous macrophages were injected intraperitoneally into wild-type ABCA1+/+, ABCA1+/- and ABCA1-/- mice maintained on a chow diet. A direct relationship was observed between ABCA1 gene dose and plasma [(3)H]cholesterol at 24 and 48 h after the injection of tracer into the mice. Forty-eight hours after this injection, ABCA1-/- mice had significantly reduced [(3)H]cholesterol in liver (2.8-fold), small intestine enterocytes (1.7-fold) and feces (2-fold). To our knowledge, this is the first direct in vivo quantitative evidence that ABCA1 is a critical determinant of macrophage-specific RCT.     

Increased hepatobiliary and fecal cholesterol excretion upon activation of the liver X receptor is independent of ABCA1

The ATP-binding cassette transporter ABCA1 is essential for high density lipoprotein (HDL) formation and considered rate-controlling for reverse cholesterol transport. Expression of the Abca1 gene is under control of the liver X receptor (LXR). We have evaluated effects of LXR activation by the synthetic agonist T0901317 on hepatic and intestinal cholesterol metabolism in C57BL/6J and DBA/1 wild-type mice and in ABCA1-deficient DBA/1 mice. In wild-type mice, T0901317 increased expression of Abca1 in liver and intestine, which was associated with an approximately 60% rise in HDL. Biliary cholesterol excretion rose 2.7-fold upon treatment, and fecal neutral sterol output was increased by 150-300%. Plasma cholesterol levels also increased in treated Abca1(-/-) mice (+120%), but exclusively in very low density lipoprotein-sized fractions. Despite the absence of HDL, hepatobiliary cholesterol output was stimulated upon LXR activation in Abca1(-/-) mice, leading to a 250% increase in the biliary cholesterol/phospholipid ratio. Most importantly, fecal neutral sterol loss was induced to a similar extent (+300%) by the LXR agonist in DBA/1 wild-type and Abca1(-/-) mice. Expression of Abcg5 and Abcg8, recently implicated in biliary excretion of cholesterol and its intestinal absorption, was induced in T0901317-treated mice. Thus, activation of LXR in mice leads to enhanced hepatobiliary cholesterol secretion and fecal neutral sterol loss independent of (ABCA1-mediated) elevation of HDL and the presence of ABCA1 in liver and intestine.     

The LXR agonist T0901317 promotes the reverse cholesterol transport from macrophages by increasing plasma efflux potential

The liver X receptors (LXRs) have been shown to affect lipoprotein plasma profile, lipid metabolism, and reverse cholesterol transport (RCT). In the present study, we investigated whether a short-term administration of the synthetic LXR agonist T0901317 (T0) to mice may affect RCT by modulating the capacity of plasma to promote cellular lipid efflux. Consistent with previous data, the pharmacological treatment of mice caused a significant increase of macrophage-derived [3H]cholesterol content in plasma, liver, and feces and resulted in improved capacity of plasma to promote cellular cholesterol release through passive diffusion and scavenger receptor class B type I (SR-BI)-mediated mechanisms. Differently, plasma from treated mice possessed similar or reduced capacity to drive lipid efflux via ABCA1. Consistent with these data, the analysis of plasma HDL fractions revealed that T0 caused the formation of larger, lipid-enriched particles. These results suggest that T0 promotes in vivo RCT from macrophages at least in part by inducing an enrichment of those HDL subclasses that increase plasma capacity to promote cholesterol efflux by passive diffusion and SR-BI-mediated mechanisms.     

Activation of the Liver X Receptor Stimulates Trans-intestinal Excretion of Plasma Cholesterol

Recent studies have indicated that direct intestinal secretion of plasma cholesterol significantly contributes to fecal neutral sterol loss in mice. The physiological relevance of this novel route, which represents a part of the reverse cholesterol transport pathway, has not been directly established in vivo as yet. We have developed a method to quantify the fractional and absolute contributions of several cholesterol fluxes to total fecal neutral sterol loss in vivo in mice, by assessing the kinetics of orally and intravenously administered stable isotopically labeled cholesterol combined with an isotopic approach to assess the fate of de novo synthesized cholesterol. Our results show that trans-intestinal cholesterol excretion significantly contributes to removal of blood-derived free cholesterol in C57Bl6/J mice (33% of 231 μmol/kg/day) and that pharmacological activation of LXR with T0901317 strongly stimulates this pathway (63% of 706 μmol/kg/day). Trans-intestinal cholesterol excretion is impaired in mice lacking Abcg5 (−4%), suggesting that the cholesterol transporting Abcg5/Abcg8 heterodimer is involved in this pathway. Our data demonstrate that intestinal excretion represents a quantitatively important route for fecal removal of neutral sterols independent of biliary secretion in mice. This pathway is sensitive to pharmacological activation of the LXR system. These data support the concept that the intestine substantially contributes to reverse cholesterol transport.Reverse cholesterol transport (RCT)³ is defined as the flux of excess cholesterol from peripheral tissues toward the liver followed by biliary secretion and subsequent disposal via the feces (1). Accumulation of cholesterol in macrophages in the vessel wall is considered a primary event in the development of atherosclerosis and, therefore, removal of excess cholesterol from these cells is of crucial importance for prevention and/or treatment of atherosclerotic cardiovascular diseases. It is generally accepted that HDL is the obligate transport vehicle in RCT and that plasma HDL levels reflect the capacity to accommodate this flux. In line herewith, HDL-raising therapies are currently considered as a promising strategy for prevention and treatment of atherosclerotic cardiovascular diseases (2). In the “classical” scenario, the liver has a central role in RCT (3). Biliary secretion of free cholesterol, facilitated by the heterodimeric ABC-transporter ABCG5/ABCG8 (4), and hepatic conversion of cholesterol into bile acids followed by fecal excretion are referred to as the main routes for quantitatively important elimination of cholesterol from the body. Fecal excretion of sterols is stimulated upon whole body activation of the liver X receptor (LXR, NR1H2/3), a member of the nuclear receptor family for which oxysterols have been identified as natural ligands (5). LXR regulates expression of several genes involved in RCT and activation of LXR by synthetic agonists leads to elevated plasma HDL-cholesterol levels, increased hepatobiliary cholesterol secretion, reduced fractional intestinal cholesterol absorption and increased fecal sterol loss (6). LXR is thus considered an attractive target for therapeutic strategies aimed at stimulation of RCT, which, however, will require approaches to circumvent potential detrimental consequences of LXR activation such as induction of lipogenesis.Recent studies indicate that the classical concept of RCT may require reconsideration. Studies in apoA-I-deficient mice revealed that the magnitude of the centripetal cholesterol flux from the periphery to the liver is not related to the concentration of HDL-cholesterol or apoA-I in plasma (7). Furthermore, Abca1^−/− mice that completely lack plasma HDL show unaffected rates of hepatobiliary cholesterol secretion and fecal sterol loss (8). Additionally, mice lacking both Abcg5 and Abcg8 do not show a reduction in fecal neutral sterol excretion to the extent expected on the basis of their strongly reduced hepatobiliary cholesterol secretion (9). Recent studies by Plösch et al. (6) have revealed that increased fecal neutral sterol loss upon general LXR activation cannot be attributed to the increased hepatobiliary cholesterol secretion only, suggesting a major contribution of the intestine in excretion of cholesterol. This potential role of the intestine in cholesterol removal from the body has been corroborated by Kruit et al. (10), who showed that fecal sterol loss is not affected in Mdr2^−/− (Abcb4^−/−) mice that have a dramatic reduction in biliary cholesterol secretion (11). Moreover, intravenously administered [³H]cholesterol could be recovered in the neutral sterol fraction of the feces in these mice and fecal excretion of neutral sterols was stimulated upon treatment with an LXR agonist (10). However, the exact quantitative contribution of the direct intestinal pathway under physiological conditions has not directly been determined so far. Very recently, intestinal perfusion studies in mice revealed that, in the presence of mixed micelles as cholesterol acceptors in the intestinal lumen, murine enterocytes indeed have a high capacity to secrete cholesterol via a specific process that is most active in the proximal part of the small intestine (12). In addition, it was shown that direct trans-intestinal cholesterol excretion (TICE) could be stimulated by a high fat diet. The existence of a non-biliary route for fecal neutral sterol excretion is further supported by very recent studies by Brown et al. (13) in mice with targeted deletion of hepatic ACAT2.The present study provides insight into the relative and absolute contributions of several cholesterol fluxes relevant to total fecal sterol loss in mice, making use of a panel of stable isotope tracers. Our results show that TICE is a major route for removal of blood-derived free cholesterol and that pharmacological LXR activation strongly stimulates this arm of the reverse cholesterol transport pathway.     

Liver X receptor agonist T0901317 reduces athero-sclerotic lesions in apoE-/- mice by up-regulating NPC1 expression

In this study, we studied the effect of liver X receptor (LXR) agonist T0901317 on Niemann-Pick C1 protein (NPC1) expression in apoE-/- mice. Male apoE-/- mice were randomized into 4 groups, baseline group (n=10), control group (n=14), treatment group (n=14) and prevention group (n=14). All of the mice were fed with a high-fat/high-cholesterol (HFHC) diet containing 15% fat and 0.25% cholesterol. The baseline group treated with vehicle was sacrificed after 8 weeks of the diet. The control group and the prevention group were treated with either vehicle or T0901317 daily by oral gavage for 14 weeks. The treatment group was treated with vehicle for 8 weeks, and then was treated with the agonist T0901317 for additional 6 weeks. Gene and protein expression was analyzed by real-time quantitative PCR, immunohistochemistry and Western blotting, respectively. Plasma lipid concentrations were measured by commercially enzymatic methods. We used RNA interference technology to silence NPC1 gene expression in THP-1 macrophage-derived foam cells and then detected the effect of LXR agonist T0901317 on cholesterol efflux. Plasma triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and apoA-I concentrations were markedly increased in T0901317-treated groups. T0901317 treatment reduced the aortic atherosclerotic lesion area by 64.2% in the prevention group and 58.3% in the treatment group. LXR agonist treatment increased NPC1 mRNA expression and protein levels in the small intestine, liver and aorta of apoE-/- mice. Compared with the normal cells, cholesterol efflux of siRNA THP-1 macrophage-derived foam cells was significantly decreased, whereas cholesterol efflux of LXR agonist T0901317-treated THP-1 macrophage-derived foam cells was significantly increased. Our results suggest that LXR agonist T0901317 inhibits atherosclerosis development in apoE-/- mice, which is related to up-regulating NPC1 expression.     

Hepatic lipase- and endothelial lipase-deficiency in mice promotes macrophage-to-feces RCT and HDL antioxidant properties

Hepatic lipase (HL) and endothelial lipase (EL) are negative regulators of plasma HDL cholesterol (HDLc) levels and presumably could affect two main HDL atheroprotective functions, macrophage-to-feces reverse cholesterol transport (RCT) and HDL antioxidant properties. In this study, we assessed the effects of both HL and EL deficiency on macrophage-specific RCT process and HDL ability to protect against LDL oxidation. HL- and EL-deficient and wild-type mice were injected intraperitoneally with [³H]cholesterol-labeled mouse macrophages, after which the appearance of [³H]cholesterol in plasma, liver, and feces was determined. The degree of HDL oxidation and the protection of oxidative modification of LDL co-incubated with HDL were evaluated by measuring conjugated diene kinetics. Plasma levels of HDLc, HDL phospholipids, apoA-I, and platelet-activated factor acetyl-hydrolase were increased in both HL- and EL-deficient mice. These genetically modified mice displayed increased levels of radiolabeled, HDL-bound [³H]cholesterol 48 h after the label injection. The magnitude of macrophage-derived [³H]cholesterol in feces was also increased in both the HL- and EL-deficient mice. HDL from the HL- and EL-deficient mice was less prone to oxidation and had a higher ability to protect LDL from oxidation, compared with the HDL derived from the wild-type mice. These changes were correlated with plasma apoA-I and apoA-I/HDL total protein levels. In conclusion, targeted inactivation of both HL and EL in mice promoted macrophage-to-feces RCT and enhanced HDL antioxidant properties.     

Induction of intestinal ATP-binding cassette transporters by a phytosterol-derived liver X receptor agonist

The nuclear receptors liver X receptor (LXR) alpha and LXRbeta serve as oxysterol receptors and regulate the expression of genes involved in lipid metabolism. LXR activation induces the expression of ATP-binding cassette (ABC) transporters, such as ABCG5 and ABCG8, which inhibit intestinal absorption of cholesterol and phytosterols. Although several synthetic LXR agonists have been generated, these compounds have limited clinical application, because they cause hypertriglycemia by inducing the expression of lipogenic genes in the liver. We synthesized derivatives of phytosterols and found some of them to act as LXR agonists. Among them, YT-32 [(22E)-ergost-22-ene-1alpha,3beta-diol], which is related to ergosterol and brassicasterol, is the most potent LXR agonist. YT-32 directly bound to LXRalpha and LXRbeta and induced the interaction of LXRalpha with cofactors, such as steroid receptor coactivator-1, as effectively as the natural ligands, 22(R)-hydroxycholesterol and 24(S),25-epoxycholesterol. Although the nonsteroidal synthetic LXR agonist T0901317 induced the expression of intestinal ABC transporters and liver lipogenic genes, oral administration of YT-32 selectively activated intestinal ABC transporters in mice. Unlike T0901317 treatment, YT-32 inhibited intestinal cholesterol absorption without increasing plasma triglyceride levels. The phytosterol-derived LXR agonist YT-32 might selectively modulate intestinal cholesterol metabolism.     

Liver X receptor agonist T0901317 reduces atherosclerotic lesions in <Emphasis Type="Italic">apoE</Emphasis><Superscript>-/-</Superscript> mice by up-regulating <Emphasis Type="Italic">NPC1</Emphasis> expression

In this study, we studied the effect of liver X receptor (LXR) agonist T0901317 on Niemann-Pick C1 protein (NPC1) expression in apoE ^-/- mice. Male apoE ^-/- mice were randomized into 4 groups, baseline group (n=10), control group (n=14), treatment group (n=14) and prevention group (n=14). All of the mice were fed with a high-fat/high-cholesterol (HFHC) diet containing 15% fat and 0.25% cholesterol. The baseline group treated with vehicle was sacrificed after 8 weeks of the diet. The control group and the prevention group were treated with either vehicle or T0901317 daily by oral gavage for 14 weeks. The treatment group was treated with vehicle for 8 weeks, and then was treated with the agonist T0901317 for additional 6 weeks. Gene and protein expression was analyzed by real-time quantitative PCR, immunohistochemistry and Western blotting, respectively. Plasma lipid concentrations were measured by commercially enzymatic methods. We used RNA interference technology to silence NPC1 gene expression in THP-1 macrophage-derived foam cells and then detected the effect of LXR agonist T0901317 on cholesterol efflux. Plasma triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and apoA-I concentrations were markedly increased in T0901317-treated groups. T0901317 treatment reduced the aortic atherosclerotic lesion area by 64.2% in the prevention group and 58.3% in the treatment group. LXR agonist treatment increased NPC1 mRNA expression and protein levels in the small intestine, liver and aorta of apoE ^-/- mice. Compared with the normal cells, cholesterol efflux of siRNA THP-1 macrophage-derived foam cells was significantly decreased, whereas cholesterol efflux of LXR agonist T0901317-treated THP-1 macrophage-derived foam cells was significantly increased. Our results suggest that LXR agonist T0901317 inhibits atherosclerosis development in apoE ^-/- mice, which is related to up-regulating NPC1 expression.     

Hepatic ABCG5 and ABCG8 overexpression increases hepatobiliary sterol transport but does not alter aortic atherosclerosis in transgenic mice

The individual roles of hepatic versus intestinal ABCG5 and ABCG8 in sterol transport have not yet been investigated. To determine the specific contribution of liver ABCG5/G8 to sterol transport and atherosclerosis, we generated transgenic mice that overexpress human ABCG5 and ABCG8 in the liver but not intestine (liver G5/G8-Tg) in three different genetic backgrounds: C57Bl/6, apoE-KO, and low density lipoprotein receptor (LDLr)-KO. Hepatic overexpression of ABCG5/G8 enhanced hepatobiliary secretion of cholesterol and plant sterols by 1.5-2-fold, increased the amount of intestinal cholesterol available for absorption and fecal excretion by up to 27%, and decreased the accumulation of plant sterols in plasma by approximately 25%. However, it did not alter fractional intestinal cholesterol absorption, fecal neutral sterol excretion, hepatic cholesterol concentrations, or hepatic cholesterol synthesis. Consequently, overexpression of ABCG5/G8 in only the liver had no effect on the plasma lipid profile, including cholesterol, HDL-C, and non-HDL-C, or on the development of proximal aortic atherosclerosis in C57Bl/6, apoE-KO, or LDLr-KO mice. Thus, liver ABCG5/G8 facilitate the secretion of liver sterols into bile and serve as an alternative mechanism, independent of intestinal ABCG5/G8, to protect against the accumulation of dietary plant sterols in plasma. However, in the absence of changes in fractional intestinal cholesterol absorption, increased secretion of sterols into bile induced by hepatic overexpression of ABCG5/G8 was not sufficient to alter hepatic cholesterol balance, enhance cholesterol removal from the body or to alter atherogenic risk in liver G5/G8-Tg mice. These findings demonstrate that overexpression of ABCG5/G8 in the liver profoundly alters hepatic but not intestinal sterol transport, identifying distinct roles for liver and intestinal ABCG5/G8 in modulating sterol metabolism.     

Genetic inactivation of NPC1L1 protects against sitosterolemia in mice lacking ABCG5/ABCG8

Mice lacking Niemann-Pick C1-Like 1 (NPC1L1) (NPC1L1(-/-)mice) exhibit a defect in intestinal absorption of cholesterol and phytosterols. However, wild-type (WT) mice do not efficiently absorb and accumulate phytosterols either. Cell-based studies show that NPC1L1 is a much weaker transporter for phytosterols than cholesterol. In this study, we examined the role of NPC1L1 in phytosterol and cholesterol trafficking in mice lacking ATP-binding cassette (ABC) transporters G5 and G8 (G5/G8(-/-) mice). G5/G8(-/-) mice develop sitosterolemia, a genetic disorder characterized by the accumulation of phytosterols in blood and tissues. We found that mice lacking ABCG5/G8 and NPC1L1 [triple knockout (TKO) mice] did not accumulate phytosterols in plasma and the liver. TKO mice, like G5/G8(-/-) mice, still had a defect in hepatobiliary cholesterol secretion, which was consistent with TKO versus NPC1L1(-/-) mice exhibiting a 52% reduction in fecal cholesterol excretion. Because fractional cholesterol absorption was reduced similarly in NPC1L1(-/-) and TKO mice, by subtracting fecal cholesterol excretion in TKO mice from NPC1L1(-/-) mice, we estimated that a 25g NPC1L1(-/-) mouse may secrete about 4 mumol of cholesterol daily via the G5/G8 pathway. In conclusion, NPC1L1 is essential for phytosterols to enter the body in mice.     

Abcg5/Abcg8-independent pathways contribute to hepatobiliary cholesterol secretion in mice

The ATP-binding cassette (ABC) half-transporters ABCG5 and ABCG8 heterodimerize into a functional complex that mediates the secretion of plant sterols and cholesterol by hepatocytes into bile and their apical efflux from enterocytes. We addressed the putative rate-controlling role of Abcg5/Abcg8 in hepatobiliary cholesterol excretion in mice during (maximal) stimulation of this process. Despite similar bile salt (BS) excretion rates, basal total sterol and phospholipid (PL) output rates were reduced by 82% and 35%, respectively, in chow-fed Abcg5(-/-) mice compared with wild-type mice. When mice were infused with the hydrophilic BS tauroursodeoxycholate, similar relative increases in bile flow, BS output, PL output, and total sterol output were observed in wild-type, Abcg5(+/-), and Abcg5(-/-) mice. Maximal cholesterol and PL output rates in Abcg5(-/-) mice were only 15% and 69%, respectively, of wild-type values. An infusion of increasing amounts of the hydrophobic BS taurodeoxycholate increased cholesterol excretion by 3.0- and 2.4-fold in wild-type and Abcg5(-/-) mice but rapidly induced cholestasis in Abcg5(-/-) mice. Treatment with the liver X receptor (LXR) agonist T0901317 increased the maximal sterol excretion capacity in wild-type mice (fourfold), concomitant with the induction of Abcg5/Abcg8 expression, but not in Abcg5(-/-) mice. In a separate study, mice were fed chow containing 1% (wt/wt) cholesterol. As expected, hepatic expression of Abcg5 and Abcg8 was strongly induced (fivefold and fourfold) in wild-type but not LXR-alpha-deficient (Lxra(-/-)) mice. Surprisingly, hepatobiliary cholesterol excretion was increased to the same extent, i.e., 2.2-fold in wild-type mice and 2.0-fold in Lxra(-/-) mice, upon cholesterol feeding. Our data confirm that Abcg5, as part of the Abcg5/Abcg8 heterodimer, strongly controls hepatobiliary cholesterol secretion in mice. However, our data demonstrate that Abcg5/Abcg8 heterodimer-independent, inducible routes exist that can significantly contribute to total hepatobiliary cholesterol output.     

Hepatic overexpression of cholesteryl ester hydrolase enhances cholesterol elimination and in vivo reverse cholesterol transport

Neutral cholesteryl ester hydrolase (CEH)-mediated hydrolysis of cellular cholesteryl esters (CEs) is required not only to generate free cholesterol (FC) for efflux from macrophages but also to release FC from lipoprotein-delivered CE in the liver for bile acid synthesis or direct secretion into the bile. We hypothesized that hepatic expression of CEH would regulate the hydrolysis of lipoprotein-derived CE and enhance reverse cholesterol transport (RCT). Adenoviral-mediated CEH overexpression led to a significant increase in bile acid output. To assess the role of hepatic CEH in promoting flux of cholesterol from macrophages to feces, cholesterol-loaded and [(3)H]cholesterol-labeled J774 macrophages were injected intraperitoneally into mice and the appearance of [(3)H]cholesterol in gallbladder bile and feces over 48 h was quantified. Mice overexpressing CEH had significantly higher [(3)H]cholesterol radiolabel in bile and feces, and it was associated with bile acids. This CEH-mediated increased movement of [(3)H]cholesterol from macrophages to bile acids and feces was significantly attenuated in SR-BI(-/-) mice. These studies demonstrate that similar to macrophage CEH that rate-limits the first step, hepatic CEH regulates the last step of RCT by promoting the flux of cholesterol entering the liver via SR-BI and increasing hepatic bile acid output.     

LXR/RXR activation enhances basolateral efflux of cholesterol in CaCo-2 cells

Regulation of gene expression of ATP-binding cassette transporter (ABC)A1 and ABCG1 by liver X receptor/retinoid X receptor (LXR/RXR) ligands was investigated in the human intestinal cell line CaCo-2. Neither the RXR ligand, 9-cis retinoic acid, nor the natural LXR ligand 22-hydroxycholesterol alone altered ABCA1 mRNA levels. When added together, ABCA1 and ABCG1 mRNA levels were increased 3- and 7-fold, respectively. T0901317, a synthetic non-sterol LXR agonist, increased ABCA1 and ABCG1 gene expression 11- and 6-fold, respectively. ABCA1 mass was increased by LXR/RXR activation. T0901317 or 9-cis retinoic acid and 22-hydroxycholesterol increased cholesterol efflux from basolateral but not apical membranes. Cholesterol efflux was increased by the LXR/RXR ligands to apolipoprotein (apo)A-I or HDL but not to taurocholate/phosphatidylcholine micelles. Actinomycin D prevented the increase in ABCA1 and ABCG1 mRNA levels and the increase in cholesterol efflux induced by the ligands. Glyburide, an inhibitor of ABCA1 activity, attenuated the increase in basolateral cholesterol efflux induced by T0901317. LXR/RXR activation decreased the esterification and secretion of cholesterol esters derived from plasma membranes. Thus, in CaCo-2 cells, LXR/RXR activation increases gene expression of ABCA1 and ABCG1 and the basolateral efflux of cholesterol, suggesting that ABCA1 plays an important role in intestinal HDL production and cholesterol absorption.     

Hepatic ABCG5/G8 overexpression reduces apoB-lipoproteins and atherosclerosis when cholesterol absorption is inhibited

We previously reported that liver-specific overexpression of ABCG5/G8 in mice is not atheroprotective, suggesting that increased biliary cholesterol secretion must be coupled with decreased intestinal cholesterol absorption to increase net sterol loss from the body and reduce atherosclerosis. To evaluate this hypothesis, we fed low density lipoprotein receptor-knockout (LDLr-KO) control and ABCG5/G8-transgenic (ABCG5/G8-Tg)xLDLr-KO mice, which overexpress ABCG5/G8 only in liver, a Western diet containing ezetimibe to reduce intestinal cholesterol absorption. On this dietary regimen, liver-specific ABCG5/G8 overexpression increased hepatobiliary cholesterol concentration and secretion rates (1.5-fold and 1.9-fold, respectively), resulting in 1.6-fold increased fecal cholesterol excretion, decreased hepatic cholesterol, and increased (4.4-fold) de novo hepatic cholesterol synthesis versus LDLr-KO mice. Plasma lipids decreased (total cholesterol, 32%; cholesteryl ester, 32%; free cholesterol, 30%), mostly as a result of reduced non-high density lipoprotein-cholesterol and apolipoprotein B (apoB; 36% and 25%, respectively). ApoB-containing lipoproteins were smaller and lipid-depleted in ABCG5/G8-TgxLDLr-KO mice. Kinetic studies revealed similar 125I-apoB intermediate density lipoprotein/LDL fractional catabolic rates, but apoB production rates were decreased 37% in ABCG5/G8-TgxLDLr-KO mice. Proximal aortic atherosclerosis decreased by 52% (male) and 59% (female) in ABCG5/G8-TgxLDLr-KO versus LDLr-KO mice fed the Western/ezetimibe diet. Thus, increased biliary secretion, resulting from hepatic ABCG5/G8 overexpression, reduces atherogenic risk in LDLr-KO mice fed a Western diet containing ezetimibe. These findings identify distinct roles for liver and intestinal ABCG5/G8 in modulating sterol metabolism and atherosclerosis.     

The combination of ezetimibe and ursodiol promotes fecal sterol excretion and reveals a G5G8-independent pathway for cholesterol elimination

Previous studies suggest an interdependent relationship between liver and intestine for cholesterol elimination from the body. We hypothesized that a combination of ursodiol (Urso) and ezetimibe (EZ) could increase biliary secretion and reduce cholesterol reabsorption, respectively, to promote cholesterol excretion. Treatment with Urso increased hepatic ABCG5 ABCG8 (G5G8) protein and both biliary and fecal sterols in a dose-dependent manner. To determine whether the drug combination (Urso-EZ) further increased cholesterol excretion, mice were treated with Urso alone or in combination with two doses of EZ. EZ produced an additive and dose-dependent increase in fecal neutral sterol (FNS) elimination in the presence of Urso. Finally, we sequentially treated wide-type and G5G8-deficient mice with Urso and Urso-EZ to determine the extent to which these effects were G5G8 dependent. Although biliary and FNS were invariably lower in G5G8 KO mice, the relative increase in FNS following treatment with Urso alone or the Urso-EZ combination was not affected by genotype. In conclusion, Urso increases G5G8, biliary cholesterol secretion, and FNS and acts additively with EZ to promote fecal sterol excretion. However, the stimulatory effect of these agents was not G5G8 dependent.     

Endotoxin down-regulates ABCG5 and ABCG8 in mouse liver and ABCA1 and ABCG1 in J774 murine macrophages: differential role of LXR

Several of the ATP binding cassette (ABC) transporters have recently been shown to play important roles in reverse cholesterol transport (RCT) and prevention of atherosclerosis. In the liver, ABCG5 and ABCG8 have been proposed to efflux sterols into the bile for excretion. ABCG5 and ABCG8 also limit absorption of dietary cholesterol and plant sterols in the intestine. In macrophages, ABCA1 and ABCG1 mediate cholesterol removal from these cells to HDL. Many of these ABC transporters are regulated by the liver X receptor (LXR). We have previously shown that endotoxin (lipopolysaccharide) down-regulates LXR in rodent liver. In the present study, we examined the in vivo and in vitro regulation of these ABC transporters by endotoxin. We found that endotoxin significantly decreased mRNA levels of ABCG5 and ABCG8 in the liver, but not in the small intestine. When endotoxin or cytokines (tumor necrosis factor and interleukin-1) were incubated with J774 murine macrophages, the mRNA levels of ABCA1 were decreased. This effect was rapid and sustained, and was associated with a reduction in ABCA1 protein levels. Endotoxin and cytokines also decreased ABCG1 mRNA levels in J774 cells. Although LXR is a positive regulator of ABCA1 and ABCG1, we did not observe a reduction in protein levels of LXR or in binding of nuclear proteins to an LXR response element in J774 cells. The decrease in ABCG5 and ABCG8 levels in the liver as well as a reduction in ABCA1 and ABCG1 in macrophages during the host response to infection and inflammation coupled with other previously described changes in the RCT pathway may aggravate atherosclerosis.     

Zymosan-mediated inflammation impairs in vivo reverse cholesterol transport

Inflammation has been proposed to impair HDL function and reverse cholesterol transport (RCT). We investigated the effects of inflammation mediated by zymosan, a yeast glucan, on multiple steps along the RCT pathway in vivo and ex vivo. Acute inflammation with 70 mg/kg zymosan impaired RCT to plasma, liver, and feces similarly by 17-22% (P < 0.05), with no additional block at the liver. Hepatic gene expression further demonstrated no change in ABCG5, ABCB4, and ABCB11 expression but a decline in ABCG8 mRNA (32% P < 0.05). Plasma from zymosan-treated mice had a 21% decrease in cholesterol acceptor ability (P < 0.01) and a 35% decrease in ABCA1-specific efflux capacity (P < 0.01) in vitro. Zymosan treatment also decreased HDL levels and led to HDL remodeling with increased incorporation of serum amyloid A. In addition, cholesterol efflux from cultured macrophages declined with zymosan treatment in a dose dependent manner. Taken together, our results suggest that zymosan impairs in vivo RCT primarily by decreasing macrophage-derived cholesterol entering the plasma, with minimal additional blocks downstream. Our study supports the notion that RCT impairment is one of the mechanisms for the increased atherosclerotic burden observed in inflammatory conditions.