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.

Cholesterol is an essential compound in all eukaryotic organisms. It serves as a precursor of steroids such as corticosteroids, sex hormones, vitamin D, and bile acids and serves as an essential structural component of cell membranes (1). Choles-terol homeostasis is maintained by de novo synthesis, absorption from diet, catabolism to bile acids, and excretion into bile. Dysregulation of this mechanism leads to hypercholesterolemia and atherosclerosis, resulting in life-threatening coronary and cerebrovascular disease (2).
The liver X receptors (LXRs) 1 are oxysterol-activated nuclear receptor family transcription factors that regulate the expression of genes involved in cholesterol metabolism (3). LXR␣ (NR1H3) is mainly localized in liver, adipose tissue, intestine, kidney, and macrophages, whereas LXR␤ (NR1H2) is ubiquitously expressed. In the rodent liver, LXR␣ induces transcription of cholesterol 7␣-hydroxylase (CYP7A), the rate-limiting enzyme in the bile acid synthesis. Treatment with LXR agonists prevents the overaccumulation of sterols in the intestine and macrophages by the induction of ATP-binding cassette (ABC) transporters involved in sterol efflux along with acceptor proteins such as apoprotein E (4). In macrophages, activation of LXRs stimulates transcription of the ABC transporters, ABCA1 and ABCG1. Synthetic LXR ligand administration reduced the development of atherosclerosis in mice deficient in low density lipoprotein receptor or apoprotein E (5). Mice with macrophages lacking both LXR␣ and LXR␤ developed more atherosclerotic lesions than mice with wild-type macrophages (6). In the small intestine, ABCA1, ABCG5, and ABCG8 are induced by LXR activation, which results in reduced sterol absorption (7,8). These studies suggest that LXRs play an important role in the regulation of cholesterol homeostasis.
In addition to their role in maintaining cholesterol homeostasis, LXRs also regulate fatty acid metabolism. Administration of synthetic LXR agonists to mice induced expression of fatty acid biosynthetic genes, such as acetyl CoA carboxylase, fatty acid synthase (FAS), and stearyl CoA desaturase-1, in liver and increased plasma triglyceride and phospholipid levels (9). LXRs regulate expression of sterol regulatory element-binding protein-1c (SREBP-1c), a transcription factor that regulates the expression of genes involved in fatty acid biosynthesis (10). LXRs also act on the promoters of FAS and angiopoietin-like protein 3, a liver-specific secretory protein, which inactivates lipoprotein lipase and increases plasma triglyceride levels (11)(12)(13)(14). Because LXRs have both antiatherogenic and lipogenic activities, the development of LXR agonists that could regulate cholesterol metabolism without adverse hypertriglycemia-inducing effects is of great pharmacological interest.
Phytosterols are naturally occurring sterols and are the plant equivalents of mammalian cholesterol. The effects of phytosterols in reducing blood cholesterol have been demonstrated for several decades, although the precise mechanism has not been elucidated (15). Recently, ABCG5 and ABCG8 have been proposed to function as transporters for cholesterol and phytosterols (16 -18). The findings that the expression of ABCG5 and ABCG8 is induced by LXR activation (8) and that the treatment of intestinal cells with phytosterols increases the expression of LXR target genes (19) suggest that phytosterols, or their metabolites, act as LXR ligands and influence cholesterol metabolism. At present, no phytosterol has been identified as a LXR ligand. In this work, we examined the effects of several phytosterols and their derivatives on the activity of LXRs and found that a compound related to ergosterol and brassicasterol is a potent agonist for LXRs.
Cell Culture and Cotransfection Assay-Human embryonic kidney 293 cells were cultured in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum and antibiotic-antimycotic (Nacalai) at 37°C in a humidified atmosphere of 5% CO 2 in air. Transfections were performed by the calcium phosphate coprecipitation assay as described previously (23,26). Eight h after transfection, test compounds were added. Cells were harvested after 16 to 24 h for luciferase and ␤-galactosidase activity using a luminometer (Molecular Devices, Sunnyvale, CA). DNA cotransfection experiments involved utilization of 50 ng of reporter plasmid, 20 ng of pCMX-␤-galactosidase, 15 ng of each receptor and/or cofactor expression plasmid, and pGEM carrier DNA to give 150 ng of DNA total per well of a 96-well plate. Luciferase data were normalized to an internal ␤-galactosidase control and represent the mean (ϮS.D.) of triplicate assays. 2 Y. Tachibana, unpublished data. Glutathione S-transferase (GST) Pull-down Assays-Nuclear receptor interaction domain of SRC-1 (amino acids 601-771) was cloned into the GST fusion vector pGEX-4T1 (Amersham Biosciences). GST-SRC-1 fusion protein was expressed in BL21 DE3 cells (Promega, Madison, WI). 35 S-Labeled LXRs were generated using the TNT Quick Coupled Transcription/Translation System (Promega). GST pull-down assays were performed as reported previously (26,27). Approximately 1 g of GST-SRC-1 was bound in glutathione-Sepharose beads (Amersham Biosciences) and equilibrated in binding buffer (20 mM Tris-HCl, pH 8, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40). Bound GST proteins were then incubated with labeled LXR and test ligand for 1.5 h at 4°C. After binding, beads were washed three times with washing buffer (20 mM Hepes, pH 7.7, 50 mM KCl, 20% glycerol, 0.1% Nonidet P-40), resuspended in SDS-PAGE sample buffer, and loaded onto a 10% SDS-polyacrylamide gel. After electrophoresis, bound proteins were visualized by autoradiography and quantified utilizing BAS2500 system (Fujifilm, Tokyo, Japan).
Animal Studies-C57BL/6J mice were obtained from Charles River (Yokohama, Japan) and housed in a room under controlled temperature (23 Ϯ 1°C) and humidity (45-65%) and had free access to water and chow (Oriental Yeast, Tokyo). Experiments were conducted when the mice (males) were between 8 and 9 weeks of age. Mice were treated orally with YT-32, YT-33, or T0901317 in a polyethylene glycol/Tween 80 (4/1) formulation or vehicle alone. Mice were analyzed 12 h after treatment under non-fasting conditions. Plasma triglyceride levels were determined with Triglycerides E-test Wako (Wako).
To determine whether YT-32 binds directly to LXR␣, GST pull-down analysis was performed. We generated a fusion protein of GST and the nuclear receptor-interacting domain of SRC-1 and evaluated the ligand-dependent interaction between GST-SRC-1 and isotope-labeled hLXR␣. Although LXR␣ did not bind to SRC-1 in the absence of ligand, YT-32 and T0901317 induced the association of LXR␣ with SRC-1 in a concentration-dependent manner (Fig. 3). These interactions were not observed when the AF-2 domain of LXR␣ was deleted (Fig. 3). YT-32 also induced the interaction of hLXR␤ with SRC-1 as effectively as T0901317 (Fig. 3). The data demonstrate that YT-32 activates LXRs by direct binding.
Structure-function Relationship of YT-32 on LXRs-YT-32 is a sterol compound with a saturated cholesterol structure and the same side chain as ergosterol and brassicasterol (see Fig. 1 and Fig. 4A). YT-33 differs from YT-32 only in the absence of the 1␣-hydroxyl group. Interestingly, whereas YT-32 is a potent ligand for LXR␣, YT-33 completely lacks agonist activity, indicating that the presence of 1␣-hydroxyl group is necessary for LXR␣ activation by YT-32 (Fig. 1A). To further examine the structure-function relationship between YT-32 and LXR␣, we synthesized several YT-32 derivatives containing the 1␣-hydroxyl group (Fig. 4A). YT-32 is a 22-ergosten with two hydroxyl groups at 1␣ and 3␤ positions. Although YT-56 and YT-57 have the same hydroxyl groups and side chain configuration as YT-32, they are 5,22-dien and 5,7,22-trien, respectively. YT-59 has a saturated cholesterol ring structure like YT-32 but also has a saturated bond at C22. We compared dose-response curves of these compounds on full-length hLXR␣ (Fig. 4B). Among these compounds, YT-32 was the most potent LXR␣ activator (effective concentration for 50% maximal activation, EC 50 ϭ 0.41 M), followed in rank order by YT-59 (EC 50 ϭ 1.3 M) and YT-56 (EC 50 ϭ 15 M). YT-57 had no activity on LXR␣. Interestingly, YT-32 and YT-59 were able to activate LXR␣ more effectively than the natural oxysterol, 24(S),25epoxycholesterol (EC 50 ϭ 0.81 M). The data indicate that saturated cholesterol structure is important for LXR␣ activation, because activity decreases as saturation decreases (YT-32 Ͼ YT-56 Ͼ YT-57). Comparing YT-32 with YT-59, saturation at C22 slightly impairs LXR␣ activation. We next compared the effects of YT-32 and its related compounds on LXR␤ (Fig. 4C) were not effective LXR␤ agonists. Taken together, these data indicate that the 1␣-hydroxyl group and saturated ring structure are important for LXR␣ and LXR␤ activation.

Phytosterol Derivative Acting as LXR Agonist 36096
YT-32 Induces Intestinal ABC Transporter Genes-To investigate the in vivo effects of YT-32, we administered YT-32 orally to mice and examined the mRNA expression of target genes including ABC transporters in intestine and liver. YT-32 and T0901317 induced the expression of ABCA1, ABCG5, and ABCG8 in the intestine (Fig. 5A). The level of LXR␣ and LXR␤ expression in the intestine were not changed by treatment with these compounds. As reported previously (8,9), T0901317 induced the expression of ABCG5, ABCG8, SREBP-1c, and FAS but did not significantly induce that of ABCA1 in the liver. In contrast to the effects in intestine, YT-32 did not effectively increase the liver mRNA levels of LXR target genes (Fig. 5B). Expression of the LXR␣ and LXR␤ genes in the liver was not changed after treatment with YT-32 or T0901317 (data not shown). YT-33, which lacks the 1␣-hydroxyl group present in YT-32 and does not activate LXRs (see Figs. 1 and 4), did not change the expression of ABCA1, ABCG5, ABCG8, LXR␣, or LXR␤ in the intestine (Fig. 5C). Therefore, treatment with YT-32 induced the expression of intestinal ABC transporters but had slight or no observable effect on the expression of liver lipogenic genes.
YT-32 Inhibits Intestinal Cholesterol Absorption without Inducing Hypertriglycemia-The effects of YT-32 on intestinal cholesterol absorption and plasma triglyceride levels were examined. Mice were treated with YT-32, T0901317, or YT-33 for 12 h, and isotope-labeled cholesterol was administrated via gavage. As reported previously (33), plasma radioactivity increased gradually. Pretreatment with T0901317 and YT-32 decreased cholesterol absorption by 14 and 19%, respectively, 12 h after cholesterol administration, whereas YT-33 pretreatment did not change cholesterol absorption (Fig. 6A). The data strongly suggest that the cholesterol absorption-decreasing effect of YT-32 is mediated through LXRs. Treatment of mice with T0901317 increased plasma triglyceride as reported previously (Fig. 6B) (9). At a dose sufficient to effectively induce expression of ABCG5 and ABCG8 and inhibit intestinal cholesterol absorption, YT-32 did not alter plasma triglyceride levels (Fig. 6B). Treatment with YT-33 did not affect plasma triglycerides. Taken together, the LXR agonist YT-32 inhibited cholesterol absorption without inducing hypertriglycemia. DISCUSSION LXRs were originally identified as orphan members of the nuclear receptor superfamily and were later found to be receptors for endogenous oxysterols such as 22(R)-hydroxycholesterol and 24(S), 25-epoxycholesterol (29, 31). The distribution of these natural ligands is tissue-specific. 24(S),25-Epoxycholesterol is formed in the liver via a shunt in the cholesterol biosynthetic pathway, whereas 22(R)-hydroxycholesterol is a transient intermediate in steroid hormone synthesis in the adrenal cortex. The limited systemic distribution of these oxysterols suggests the presence of other physiological LXR ligands. The finding that cholesterol-treated macrophages express the LXR-responsive ABCA1 and ABCG1 genes and utilize the enzyme CYP27 to produce 27-hydroxycholesterol and cholestenoic acid suggests that 27-hydroxycholesterol may serve as an endogenous ligand for LXRs (34). Cholestenoic acid was also reported to be an LXR␣ agonist (35). Cholesterol feeding increased the intestinal expression of ABCG5 and ABCG8 in wild-type mice but not in LXR␣/␤ null mice (8). Treatment with cholesterol or sitostanol increased the expression of the ABCA1 gene in intestinal cells (19). These findings suggest that LXRs are activated by metabolites of cholesterol and phytosterols in the intestine. However, the metabolic conversion of dietary sterols, such as cholesterol and phytosterols, has not been elucidated. In the present work, we show that an ergosterol derivative is a potent agonist for LXRs and induces the expression of ABC transporters in the mouse intestine. Our findings strongly suggest that the cholesterol-lowering effect of phytosterols is at least partly because of conversion of these compounds to LXR agonists, which activate the expression of sterol efflux transporters such as ABCG5 and ABCG8.
Several synthetic LXR agonists have been shown to modulate LXR regulation of cholesterol metabolism (9,36,37). Unfortunately, potent LXR agonists cause undesirable increases in liver and plasma triglyceride levels, because of the activation of LXR-responsive genes in the liver (SREBP-1c, FAS, and angiopoietin-like protein 3), in addition to their beneficial effects on cholesterol metabolism (10 -13). SREBP-1c activates transcription of the major genes involved in fatty acid synthesis (acetyl CoA carboxylase, FAS, stearyl CoA desaturase-1, and glycerol-3phosphate acyltransferase), and SREBP-1c transgenic mice show markedly increased liver triglycerides (38). Therefore, the development of selective modulators that act specifically on cholesterol metabolism without adverse lipogenic effects is required to realize the therapeutic potential of LXR pharmacophores. Mice were given isotope-labeled cholesterol 12 h after treatment with YT-32, T0901317, or YT-33. Blood was drawn from retroorbital sinus using microcapillary tubes 4, 8, and 12 h after administration of isotope-labeled cholesterol, and plasma radioactivity was measured. B, effects of YT-32, T0901317, and YT-33 on plasma triglyceride levels. Mice (n ϭ 3) were orally administrated with 250 mg/kg of YT-32, 10 mg/kg of T0901317, or 250 mg/kg of YT-33, and blood was drawn for plasma triglyceride measurement 12 h after treatment. T0901317 induced hypertriglycemia, but YT-32 and YT-33 did not. The values represent means Ϯ S.E. *, p Ͻ 0.05 compared with the vehicle control.
We found that YT-32, a derivative of ergosterol or brassicasterol, activated LXR␣ more effectively than the known oxysterol agonists 24(S),25-epoxycholesterol and 22(R)-hydroxycholesterol and that it induced the expression of ABC transporter genes in the intestine. Interestingly, the induction of LXRregulated genes in the liver was only modest or not observed. This selective action of YT-32 may be because of its sterol-based structure. YT-32 acts as a potent LXR agonist and induces the sterol exporting ABC transporters such as ABCA1, ABCG5, and ABCG8. ABCG5 and ABCG8 are expressed exclusively in liver and small intestine and are localized to the apical (canalicular) membrane of cells (39). Overexpression of these genes results in an ϳ50% reduction in absorption of dietary cholesterol and a dramatic increase in the biliary secretion of sterols (17). On the other hand, disruption of Abcg5 and Abcg8 in mice causes increased absorption of dietary phytosterols and a decrease in biliary cholesterol concentration (18). Therefore, ABCG5 and ABCG8 play an important role in biliary cholesterol secretion and the regulation of intestinal cholesterol absorption. Repa et al. (7) reported that ABCA1 expression was increased in the intestines of mice treated with a synthetic LXR agonist and suggested that ABCA1 catalyzed efflux causes the associated decrease in cholesterol absorption. However, the role of ABCA1 in intestinal cholesterol absorption remains to be clarified (2). ABCA1 was dominantly expressed on the basolateral surface of intestinal cells (40), and Plosch et al. (41) reported that the effect of the LXR agonist on fecal sterol loss was observed in Abca1 null mice, as well as wild-type mice. Recently (42), ABCG5 and ABCG8 were demonstrated to mediate the LXR agonist-mediated reduction of cholesterol absorption. YT-32 may reduce the intestinal absorption of dietary sterols, including cholesterol and phytosterols, by inducing the expression ABCG5 and ABCG8, which might excrete the compound from the basolateral membranes of the mucosal cells. Some fraction of YT-32 may be incorporated in the blood circulation where it could be available to stimulate the reverse cholesterol transport on atherosclerotic lesions. Alternatively, YT-32 may be secreted into the bile by ABCG5 and ABCG8 before it would be able to induce liver lipogenic genes. In contrast to YT-32, synthetic nonsteroidal agonists such as T0901317 do not seem to be substrates of the ABC transporters and cause increased serum and liver triglyceride levels by inducing lipogenic genes in the liver (9). Another possibility is that tissue selectivity of YT-32 is mediated by another mechanism as is the case for selective estrogen receptor modulators. The differential cofactor recruitment shown in Fig. 2D may contribute to tissue-specific agonist activity. Further analysis is required to elucidate the mechanism of tissue-specific LXR activation by YT-32. Therefore, our data suggest that LXR agonists based on the structure of natural sterols should be useful to agents in therapeutically modulating cholesterol metabolism.