Hypolipidemic effects of selective liver X receptor alpha agonists

Steroids 66 (2001) 673– 681

Rapid communication

Hypolipidemic effects of selective liver X receptor alpha agonists Ching Song, Shutsung Liao* The Ben May Institute for Cancer Research, Department of Biochemistry and Molecular Biology, the Tang Center for Herbal Medicine Research, 5841 South Maryland Avenue, Chicago, Illinois, 60637, USA Received 27 March 2001; received in revised form 10 May 2001; accepted 17 May 2001

Abstract Recently, a number of nuclear receptors have been identified as key regulators of cholesterol homeostasis. Two of these, liver X receptor alpha (LXR␣) (NR1H3) [1] and ubiquitous receptor (UR) (NR1H2) [1], appear to be involved in cholesterol reverse transport and disposal. LXR␣ null gene mice fail to adapt metabolically to high-cholesterol diets. We have recently shown that some 6␣-hydroxylated bile acid analogs are selective activators of LXR␣. In this report, we show that these orally administered LXR␣ agonists have an overall hypolipidemic effect in hypercholesterolemic rats, mice and hamsters, which indicates that in these animal models, endogenous LXR␣ agonist is a limiting factor for induction of cholesterol disposal. Furthermore, in animals, these 6␣-hydroxylated bile acid analogs exhibit a unique pharmacokinetic profile and do not increase the serum triglyceride level; therefore, they may represent a novel class of therapeutic agents for cholesterol management. © 2001 Elsevier Science Inc. All rights reserved. Keywords: LXR; Nuclear receptors; Steroids; Cholesterol; Hypercholesterolemia; Atherosclerosis

1. Introduction Atherosclerosis is a progressive disease characterized by the accumulation of lipids and fibrous elements in the arteries. It is the underlying cause of heart disease and stroke, which are responsible for half of all deaths in westernized society. Alterations in cholesterol metabolism have been directly linked to the development of atherosclerotic plaques. For instance, elevated LDL cholesterol levels due to LDL receptor gene null mutations result in early coronary heart disease. Inhibitors of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, a rate limiting enzyme involved in cholesterol biosynthesis can cause up-regulation of hepatic LDL receptor, which subsequently results in decreased LDL and increased HDL. In clinical trials, HMG-CoA reductase inhibitors lower the recurrence of heart disease and related mortality (reviewed in reference [2]). A number of nuclear receptors have been identified as key regulators of cholesterol homeostasis. The liver X receptors (LXR) bind to oxidized forms of cholesterol and transactivate gene expression [3– 8]. Studies with mice lack* Corresponding author. Tel.: ⫹1-773-702-6999; fax: ⫹1-773-7026260. E-mail address: [email protected] (S. Liao).

ing LXR have revealed a physiological role for LXR␣ [9]. These mice fail to adapt metabolically when challenged with high-cholesterol diets and accumulate cholesterol in the liver. A number of genes related to cholesterol homeostasis have been identified as direct target genes of LXR. These include a cholesterol efflux transporter ATPbinding cassette 1 (ABCA1) [10 –12] and ABCG1 [13], cholesterol 7␣-hydroxylase [14], cholesteryl ester transfer protein (CETP) [15], and lipoprotein ApoE [16]. Sterol Regulatory Element-Binding Protein 1c (SREBP-1c) is also a direct target gene of LXRs [17]. Interestingly, deficiency in ABCA1 or ApoE genes causes premature atherosclerosis in man. There are a number of oxidized forms of cholesterol that bind to LXR as ligands [14,18 –20]. Among them, we have shown that cholestenoic acid (a metabolite of 27hydroxycholesterol) can function as a signaling molecule for regulation of lipid metabolism via LXR␣ [21]. The half-maximum effective dose (ED50) of cholestenoic acid required to transactivate gene expression is 200 nM. This is consistent with a role as a signaling molecule, as its human serum concentration ranges from 300 to 500 nM [22]. The level of serum cholestenoic acid is also correlated with serum cholesterol levels. Therefore, enzymatically oxidized cholesterol metabolites such as cholestenoic acid may act as cholesterol sensors that promote reverse transport of cholesterol from peripheral tissues to

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2. Materials and methods 2.1. Chemical synthesis

Fig. 1. Effect of Hypocholamide on reporter gene transactivation by LXR␣ or UR. HEK 293 cells co-transfected with LXR␣ (A) or UR (B) expression vectors and a synthetic DR-4 luciferase reporter gene were treated with increasing concentrations of 22R-hydroxycholesterol (open circles) or Hypocholamide (closed circles). C, HEK 293 cells were transfected with a luciferase reporter gene mSREBP-1/-550/pGL2 [26], which contained nucleotides from -550 to ⫹1 of mouse SREBP-1 promoter, and LXR␣ (closed diamonds) or UR (open diamonds) and treated with increasing concentrations of Hypocholamide. D. Structure of LXR agonists. RLU, relative light unit.

the liver and synthesis of bile acids for cholesterol disposal. The importance of the sterol 27-hydroxylase gene (CYP27) in cholesterol metabolism in man has been demonstrated. Deficiency in the CYP27 gene product that catalyzes the synthesis of cholestenoic acid causes cerebrotendinous xanthomatosis (CTX) and induces premature atherosclerosis [23]. There is much evidence supporting a role for oxidative stress in the development of atherosclerosis. We have recently identified some auto-oxidized cholesterol sulfates as LXR antagonists [24] and demonstrated that these sulfates can stimulate cholesterol synthesis and induce apoptosis in macrophages and smooth muscle cells in vitro. These sulfates, therefore, may be pathogenic factors responsible for cholesterol accumulation in vivo. Cholestenoic acid in blood is rapidly cleared by liver and may not be adequate to neutralize the LXR antagonists. Exogenous LXR agonists that can counteract the activity of these antagonists may promote cholesterol catabolism and thus prevent cholesterol accumulation in tissues and circulation. To test this hypothesis, we have screened for LXR activators and evaluated some of these compounds for their efficacy in modulating cholesterol homeostasis in rats, mice and hamsters.

Steroid acids (Steraloids or Sigma) (1 mmol) were dissolved in 2–10 ml of dimethylformamide, to which amines (Sigma-Aldrich) (3 mmol) were added, followed by addition of diethyl cyanophosphonate (2 mmol) (Aldrich-Sigma) and 2 ml of triethylamine. The reaction mixtures were stirred at temperartures from 20 to 80°C for 12–16 h. The reaction was stopped by pouring the reaction mixture onto ice. The amide products were extracted with ethyl acetate. The free acids and amines were washed off with acidic or basic aqueous solutions. The amides were purified using flash chromatography. The structure of 3␣, 6␣-dihydroxy-5␤-cholanoic acid-Nmethyl-N-methoxy-24-amide (Hypocholamide) (Fig. 1D) was confirmed by NMR spectrometry and X-ray refractory analysis of single crystals. The radio-labeled [3- 3 H]Hypocholamide was synthesized by reducing N-methyl-N-methoxy-3-keto-6 ␣ -hydroxy-5 ␤ -cholanoic acid-24-amide using NaB3H4 (Amersham, 40 Ci/mmol) and purified by flash chromatography. Purity of synthesized compounds was verified by thin layer chromatography and structures were confirmed using proton and 13 C magnetic resonance spectrometry. The detailed synthesis and properties of the steroids used will be published elsewhere. 2.2. Transactivation assay Human embryonic kidney (HEK) 293 cells or 293 cells stably expressing human UDP-glucuronosyltransferase (UGT) 2B7 [25] were seeded into 48-well culture plates at 2 ⫻ 105 cells per well in DMEM supplemented with 10% fetal bovine serum. After 24 h, cells were transfected by a calcium phosphate co-precipitation method with 250 ng pGL3/UREluc [21], 40 ng pSG5/hRXR␣, 40 ng pSG5/rUR or CMX/hLXR␣ [18], 10 ng pSG5/hGrip1 [26], 0.4 ng CMV/R-luc (transfection normalization reporter, Promega) and 250 ng carrier DNA per well. In some experiments, another reporter gene, mSREBP-1/-550/pGL2 was used [27]. After another 12–24 h, cells were washed with PBS and re-fed with DMEM supplemented with 4% lipid-deficient fetal bovine serum. Chemicals dissolved in ethanol were added in duplicate to the medium so that the final concentration of alcohol was 0.2%. After 24 – 48 h, cells were harvested and luciferase activity was measured with a commercial kit (Promega Dual luciferase II) on a Monolight luminometer (Beckton Dickenson). Each experiment was repeated at least twice to assure reproducibility. 2.3. Animal studies All animals were fed a commercial chow diet (Harlan Teklad 7001) which contains 24% protein, 4% fat and

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Table 1 Effects of 2, 2, 2-trifluoro ethyl-3␣, 6␣-dihydroxy-5␤-cholan-24-amide on diet-induced hypercholestolemic rats Group

Control Treatment P value (t-test)

Body weight (g)

Serum total cholesterol (mg/dl)

Liver weight (g)

Initial

Final

Change

Initial

Final

Change

Final

162 ⫾ 5.7 160 ⫾ 5.3 0.61

205 ⫾ 9.1 200 ⫾ 6.7 0.23

43 ⫾ 4.9 39 ⫾ 4.5 0.15

115 ⫾ 4.10 115 ⫾ 11.4 0.98

525 ⫾ 54.0 434 ⫾ 91.8 0.04

410 ⫾ 52.6 319 ⫾ 94.4 0.05

14.2 ⫾ 0.84 13.8 ⫾ 1.44 0.57

Male 50-day old Sprague-Dawley rats were used. Both control and treatment group were fed ad libitum a chow diet supplemented with 2% cholesterol and 1% cholic acid for 7 days. The treatment group during the period received diet supplemented with 0.03% 2, 2, 2-trifluoro ethyl-3␣, 6␣-dihydroxy-5␤cholan-24-amide. Overnight fasted animals were used for bodyweight measurement and to collect blood. The average food consumption was 20 –25 g/animal/day. The average feces produced was 9 g/animal/day. There was no statistical difference between control and treatment group for food and water consumption and feces production. The dose for 2, 2, 2-trifluoro ethyl-3␣, 6␣-dihydroxy-5␤-cholan-24-amide in treatment group was about 35– 40 mg/kg/day. Values are expressed as mean ⫹ SD (n ⫽ 7).

5% crude fiber, 0.02% cholesterol or a chow diet supplemented with cholesterol (1–2%) where indicated. Blood from rats and hamsters was collected from overnight fasted animals; blood from mice was collected from 4 h fasted animals. The animals had free access to food and water. Various LXR agonists were added to drinking water containing 0.25% hydroxypropyl-␤-cyclodextrin (HPCD) (Arcos Organic). Control groups received 0.25% HPCD only. Water consumption in control and treatment groups differed by less than 10%. 2.4. Analysis of cholesterol, triglycerides and bile acids Serum cholesterol and triglycerides were measured enzymatically using commercial kits (Sigma). HDL cholesterol was isolated as described [28] and quantified enzymatically. Liver cholesterol and triglycerides were isolated as described [29]. Fecal bile acids were reduced with sodium borohydride, and then extracted as previously described [30]. Bile acids were quantified using a commercial kit (Sigma). 2.5. Pharmacokinetic analysis Two male hamsters were individually housed in metabolic cages. Animals had free access to food and water during the experimental period. One hamster received a single intraperitoneal (i.p.) dose of Hypocholamide (0.4 mg Hypocholamide, 2 ⫻ 107 cpm [3-3H]Hypocholamide (7 Ci/mmol), 20 mg HPCD in 0.05 ml water). The other received a single dose of Hypocholamide by gavage (5 mg Hypocholamide, 2 ⫻ 107 cpm [3-3H]Hypocholamide, 200 mg HPCD in 0.5 ml water). The amount of radioactivity in urine, collected daily for 8 days, was determined by liquid scintillation counting. Feces were extracted with 75% ethanol and radioactivity in the supernatant was measured. Rats were used for the serum clearance study, from which tail blood was collected for analysis.

3. Results 3.1. Effect of 2, 2, 2-trifluoro ethyl-3␣, 6␣-dihydroxy-5␤cholanoic acid 24-amide on diet-induced hypercholesterolemic rats In a previous communication, we reported that certain 6␣-hydroxylated bile acids and their analogs were selective LXR␣ activators [31]. We identified 2, 2, 2-trifluoro ethyl-3␣, 6␣-dihydroxy-5␤-cholanoic acid 24-amide as a selective LXR␣ agonist. This compound has an ED50 of 1 ␮M for LXR␣ and 5 ␮M for UR in vitro. As shown in Table 1, the compound has a hypocholesterolemic effect in male Spraque-Dawley rats. The ED50 of this compound is, however, relatively high and it is desirable to obtain compounds that are more effective at lower concentrations. In addition, the C24 amide bond that appears to be important for the activity may be cleaved in the intestine by bacterial enzymes, such as cholyglycine hydrolase, and form less active hyodeoxycholic acid before being absorbed. We therefore synthesized various 24amide derivatives of hyodeoxycholic acid for their ability to activate LXR␣. 3.2. Identification of LXR␣ selective agonists Using a reporter gene transfection assay, one compound, 3␣, 6␣-dihydroxy-5␤-cholanoic acid-N-methylN-methoxy-24-amide that we named ‘Hypocholamide’ (Fig. 1D) was found to be more active than 2, 2, 2-trifluoro ethyl-3␣, 6␣-dihydroxy-5␤-cholanoic acid 24amide. Hypocholamide was also more potent for LXR␣ than for UR. Hypocholamide has an ED50 of 200 nM on LXR␣ (Fig. 1A) and 1 ␮M on UR (Fig. 1B) for activation of a co-transfected synthetic DR-4 reporter gene. When a natural promoter was used in the reporter gene assay, as shown in Fig. 1C, the ED50s for LXR␣ and UR were 60 nM and 300 nM, respectively. Hypocholamide was inactive on FXR at concentrations up to 20 ␮M (data not shown). Another important advantage of using Hypo-

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Table 2 Effect of Hypocholamide on Non-Swiss Albino mice fed a high cholesterol diet Hypocholamide

0 mg/kg/day 50 mg/kg/day P value, t-test

Serum total cholesterol (mg/dl)

Serum triglycerides (mg/dl)

Liver total (mg/g)

Feces (mg/g)

Male

Female

Male

Female

Cholesterol

Triglycerides

Bile acids

3␤-sterols

98 ⫾ 16 (n ⫽ 6) 109 ⫾ 20 (n ⫽ 7) 0.26

75 ⫾ 11(n ⫽ 6) 71 ⫾ 11 (n ⫽ 8) 0.48

100 ⫾ 25 77 ⫾ 26 0.16

71 ⫾ 16 56 ⫾ 20 0.15

15.4 ⫾ 0.8 3.9 ⫾ 3.2 0.001

39 ⫾ 3.6 37 ⫾ 3.6 0.463

0.69 ⫾ 0.08 1.02 ⫾ 0.27 0.018

16 ⫾ 0.2 19 ⫾ 0.5 0.001

Three-month old mice were maintained on a chow diet supplemented with 1% cholesterol for 7 days. Treatment group received Hypocholamide dissolved in drinking water. Animals were fasted for 4 h before blood collection. Data is presented as mean ⫾ SD.

cholamide is that Hypocholamide has a N-methyl amide, which is resistant to the bacterial cholyglycine hydrolase [32]. Therefore, we used Hypocholamide for further experiments. 3.3. Effect of Hypocholamide on mice fed a highcholesterol diet Non-Swiss Albino mice (Harlan), 3-month old, were fed a chow diet meal supplemented with 1% cholesterol for 7 days. The control group received drinking water containing vehicle (0.25% HPCD) whereas the Hypocholamide group received drinking water containing the vehicle and Hypocholamide. As shown in Table 2, cholesterol feeding did not change circulating cholesterol levels but increased cholesterol levels in liver. Hypocholamide administration prevented the increases in cholesterol in liver. Although increased fecal bile acid secretion was observed in treated animals, some of the fecal bile acids might be derived from Hypocholamide. The level of triglycerides in serum and liver were not affected by Hypocholamide treatment. Hypocholamide was also administered orally to male C57BL/6J mice (Jackson Laboratory) which are susceptible to development of atherosclerosis. As shown in Table 3, Hypocholamide dose-dependently lowered serum cholesterol levels but did not increase serum triglycerides levels significantly during the experimental period. It has been shown that dietary cholic acid lowers plasma level of mouse apolipoprotein A-I (ApoA-I) [33]. We therefore analyzed the serum level of ApoA-I in these mice. As shown in Fig.

2, ApoA-I in serum was elevated upon Hypocholamide treatment in a dose-dependent manner. 3.4. Effect of Hypocholamide on diet-induced hypercholesterolemic hamsters The bile acid and circulating cholesterol profiles of hamsters but not rats or mice are similar to that of humans. In addition, the major cholesterol carrier in human and hamster serum is LDL whereas, in rats and mice, HDL is the major carrier of cholesterol in blood. We therefore used hamsters to evaluate the effect of Hypocholamide on cholesterol and triglyceride profiles. Oral administration of Hypocholamide to hamsters on a regular chow diet at doses up to 200 mg/kg/day for 2 weeks did not change the serum cholesterol or triglyceride levels (data not shown). On the other hand, when Hypocholamide was administered to hamsters fed a chow diet supplemented with 1% cholesterol, it prevented increases in serum cholesterol and cholesteryl ester in liver (Table 4, Fig. 3). The serum triglyceride level in Hypocholamide-treated animals was significantly higher than vehicletreated animals but was about the same level in control animals fed a regular chow diet and within the normal range of triglyceride levels reported in the literature [34]. The decreased level of triglycerides in the vehicle-treated group was probably due to the massive accumulation of cholesteryl esters in the liver. Northern blot analysis revealed that Hypocholamide did not induce SREBP-1 mRNA in liver of hamsters (data not shown).

Table 3 Effect of Hypocholamide on C57BL/6J male mice fed a high-cholesterol diet Hypocholamide

0 mg/kg/day (n ⫽ 5) 25 mg/kg/day (n ⫽ 5) 50 mg/kg/day (n ⫽ 5) 100 mg/kg/day (n ⫽ 5)

1% Cholesterol

Atherogenic diet

Cholesterol (mg/dl)

Triglycerides (mg/dl)

Cholesterol (mg/dl)

Triglycerides (mg/dl)

87.8 ⫾ 14.1 67.0 ⫾ 8.8* 59.6 ⫾ 8.6* 64.8 ⫾ 6.9*

91.5 ⫾ 18.8 79.2 ⫾ 5.6 78.9 ⫾ 6.6 129.4 ⫾ 19.6

145.6 ⫾ 19.8 109.5 ⫾ 17.7* 123 ⫾ 14.4* 95.2 ⫾ 18.6*

29.1 ⫾ 3.1 28.2 ⫾ 3.8 30.9 ⫾ 2.0 28.8 ⫾ 4.2

Two-month old mice were fed a chow diet meal supplemented with 1% cholesterol for 10 weeks, then fed an atherogenic diet containing a chow diet supplemented with 1.25% cholesterol/0.5% cholic acid/12% corn oil for another 5 weeks. Hypocholamide dissolved in 0.25% HPCD which was used as the sole water source for the entire period of experiment. Data was presented as mean ⫾ SD. * P value ⬍ 0.05, compared to the vehicle group; t-test.

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Fig. 2. Western blot analysis of serum ApoA-I from C57BL/6J mice treated with Hypocholamide. ApoA-I in serum samples from individual animals in various dose groups of Hypocholamide were separated by SDS-PAGE and detected with an anti-ApoA-I antibody, which was visualized with ECL and measured using a digital-image/quantitation system.

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shown in Fig. 5A, orally administered Hypocholamide was well-absorbed into the blood and had a serum half-life of 6 days. Clearance of Hypocholamide was mainly through the renal system, since 90% of excreted radioactivity was found in the urine, while bile excretion in feces accounted for the remaining 10%. Radioactivity in urine was extracted and analyzed using reverse-phase HPLC. The results suggested that one-half of the radioactivity was in the form of Hypocholamide, the other half was in water-soluble form, presumably glucuronidated or sulfated metabolites of Hypocholamide. In rats, the half time of Hypocholamide serum clearance is 5 days (Fig. 5B). When serum from animals treated orally with Hypocholamide was added to a reporter gene transfection assay, LXR␣ agonist activity was detected. Serum from the control animals not treated with Hypocholamide was not as active (Fig. 5C) suggesting that Hypocholamide remains active for a considerable time in blood.

3.5. Effect of Hypocholamide on ApoE null mice

3.7. Conjugation of Hypocholamide by hepatic enzymes

ApoE is an important apolipoprotein responsible for clearance of VLDL and IDL particles. Even on a lowcholesterol diet, mice with ApoE null genes have a high serum cholesterol level due to accumulation of VLDL/IDL cholesterol [35,36]. ApoE-deficient mice synthesize more cholesterol in peripheral tissues and accumulate slightly more free cholesterol (50% more) as well as significantly more triacylglycerol (3 times more) in the liver [37]. We tested the effect of Hypocholamide on this non-diet-induced hypercholesterolemic animal model. As shown in Fig. 4, oral administration of Hypocholamide lowered serum cholesterol levels without raising the triglyceride level. The body weight gain between vehicle-treated and Hypocholamide-treated groups were similar during the period of treatment, indicating no significant changes in food intake by Hypocholamide treatment.

Since Hypocholamide-fed hamsters had LXR␣ stimulating activity in circulation but failed to up-regulate serum triglyceride levels, we suspected that Hypocholamide may be inactivated in the liver, the organ responsible for circulating triglycerides. Previous studies on metabolites of hyodeoxycholic acid (a 6␣-hydroxylated bile acid) in humans revealed that hyodeoxycholic acid underwent modification in the liver, intestines and kidneys, mainly to glucuronide conjugates at the 3 and 6 positions [38]. We therefore tested the effects of glucuronidation of LXR␣ agonists on LXR-mediated gene transactivation. As shown in Fig. 6A, glucuronidation of hyodeoxycholic acid methyl ester, a LXR␣ agonist, resulted in complete loss of ability to transactivate LXRmediated gene expression, presumably caused by loss of binding affinity toward LXR␣. Glucuronidation, therefore, deactivates 6␣-hydroxylated bile acid analog-based LXR␣ agonists. At least two enzymes, UGT2B7 [39] and UGT2B4 [40], are responsible for glucuronidation of 3␣, 6␣-hydroxylated bile acids. We tested whether UGT2B7 could conjugate Hypocholamide. When HEK 293 cells stably expressing

3.6. Pharmacokinetic analysis of Hypocholamide in hamsters We used radio-labeled Hypocholamide to analyze the pharmacokinetic profile of Hypocholamide in hamsters. As Table 4 Effect of Hypocholamide on hamsters fed a high cholesterol diet Group

A B C

Hypocholamide

Initial level (n ⫽ 12) 0 mg/kg/day (n ⫽ 6) 100 mg/kg/day (n ⫽ 6)

Serum lipids (mg/dl)

Liver lipids (mg/g tissue)

Total cholesterol

HDL

Triglycerides

Cholesterol

Triglycerides

100 ⫾ 20 201 ⫾ 45 105 ⫾ 15*

– 49 ⫾ 4 57 ⫾ 18

224 ⫾ 17 114 ⫾ 34 267 ⫾ 107*

– 21.2 ⫾ 2.6 1.6 ⫾ 0.7

– 3.2 ⫾ 0.9 2.3 ⫾ 0.2

Male golden Syrian hamsters weighing 80 –100 g were maintained on a chow diet supplemented with 1% cholesterol for 35 days. Animals were fasted overnight before blood collection. The control group (B) was given drinking water containing 0.25% HPCD whereas the treatment group (c) were given Hypocholamide dissolved in drinking water containing 0.25% HPCD. Data is presented as mean ⫾ SD. n, number of animals in each group. * P ⬍ 0.05 compared with group B.

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Fig. 3. Morphology and histology of livers from Hypocholamide-treated (100 mg/kg/day) versus carrier-treated hamsters on a chow diet supplemented with 1% cholesterol for 35 days. Liver sections from carrier-treated animals (A, C) or Hypocholamide-treated animals (B, D) were prepared for histology and stained with oil red O and hematoxylin (A, B) or hematoxylin and eosin (C, D). E. Gross morphology of livers from a carrier-treated hamster (left) or a Hypocholamide-treated animal (right) (see Table 4 for experimental details).

UGT2B7 [25] were used in the reporter gene assay, the ED50 of Hypocholamide to transactivate LXR␣-mediated reporter gene expression increased to 1 ␮M, indicating that it was less active than when UGT2B7 was absent (Fig. 6B). A similar effect was also observed when LXR␣ was replaced with UR (data not shown). When a mouse SREBP-1c promoter reporter gene was used in the 293 cells stably expressing UGT2B7, Hypocholamide but not a ⌬5-oxysterol ligand has decreased potency to activate LXR␣ (Fig. 6C). These results suggest that in tissues expressing a high level of UGT2B7, like the liver, Hypocholamide undergoes conjugation and loses its ability to stimulate LXR␣ transactivation activity.

4. Discussion In this study, we used LXR␣ selective activators such as Hypocholamide to investigate the function of LXR␣ in the regulation of cholesterol metabolism in vivo. The overall hypocholesterolemic effects of Hypocholamide observed in this study are in agreement with the conclusion of genetic analysis using LXR null mice; the analysis suggests that LXR␣ regulates cholesterol metabolism by promoting cholesterol transport and disposal [9]. Furthermore, our result in this communication indicates that under some diet-induced or genetic hypercholesterolemic conditions, LXR␣ agonists are the limiting factors in vivo, since exogenous adminis-

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Fig. 4. Effect of Hypocholamide on ApoE null gene mice. Male 2-month old ApoE null gene mice received vehicle (C) or Hypocholamide (H) (100 mg/kg/day) for 30 days. Serum from 4 h-fasted animals was collected and analyzed for total cholesterol and triglycerides. Body weight gain during the treatment time was shown. Data are shown as mean ⫾ SD (n ⫽ 10). *, P ⬍ 0.0001, t-test.

tration can further stimulate LXR␣ transactivity and reduce cholesterol levels in circulation and tissues. This observation has profound implication in the potential clinical usage of LXR␣ agonists. Recently, a non-steroidal LXR agonist, T0901317 (Fig. 1D), was identified by medicinal-chemical screening [41]. This compound increased both serum HDL cholesterol and triglyceride levels when administered orally in mice, and raised serum triglyceride levels in hamsters. In contrast to TO901317, Hypocholamide reduced the cholesterol level in serum or the liver without increasing serum triglycerides. The reasons for the difference are at present unknown. One possible reason is that Hypocholamide is a LXR␣ selective activator, while T0901317 is a dual activator that activates both LXR␣ and UR. Another possible reason is that Hypocholamide is easily deactivated by glucuronidation in the liver. SREBP-1c in the liver may be responsible for upregulation of serum triglycerides and is a direct target gene of LXR␣ and UR [17]. If Hypocholamide is deactivated in liver and does not induce SREBP-1c expression through LXRs, serum triglycerides shall not be elevated. In earlier studies, hyodeoxycholic acid was evaluated in cholesterol gallstone prevention and dissolution due to its hydrophilic physiochemical properties [42– 44]. The renal clearance of this bile acid and lack of accumulation in

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Fig. 5. A. Excretion of radioactivity in hamsters after oral (closed circles) or i.p. administration (open circles) of [3-3H]Hypocholamide. Y axis, % of the radioactivity remaining in the organism calculated by difference. X axis, time in days. B. Radioactivity remaining in the serum of three adult female (open symbols) or two adult male (closed symbols) Harlan Sprague-Dawley rats after i.p. administration of [3-3H]Hypocholamide. C. Effect of hamster serum on a synthetic DR-4 reporter gene transactivation by LXR␣ or UR. Normal, 5% pooled normal hamster serum; Control, 5% hamster serum fed 1% cholesterol; Treated, 5% hamster serum fed 1% cholesterol plus 100 mg/kg/day Hypocholamide. Error bars, SD from 6 animals. RLU, relative light unit. Cells were treated with serum for 12 h before analysis. *, P value 0.03; **, P value 0.25, t-test compared with Control group. Cpm, counts per minute.

human bile made it unfavorable when compared to ursocholic acid for treatment of gallstones. Hyodeoxycholic acid, when administered orally at doses over 100 mg/kg/day, had hypocholesterolemic effect in rats and hamsters [45– 47]. At present we cannot exclude the possibility that some effects of Hypocholamide are due to its physiochemical properties and interaction with molecules other than LXR␣. However, the doses of Hypocholamide used in our animal studies were in the range of 20 –100 mg/kg/day, making it unlikely that its physiochemical properties were primarily responsible for its hypocholesterolemic effects in vivo. In contrast to hyodeoxycholic acid, which is mainly accumulated and secreted in bile in hamsters, Hypocholamide remains in serum and is cleared through renal systems. The ability of Hypocholamide to lower total serum cholesterol level in ApoE-null mice on a chow diet is interesting. In this animal model, the elevated cholesterol levels in serum are due to increased de novo cholesterol synthesis in peripheral tissues [37]. Other agonists of related nuclear receptors, e.g. PPAR␣, PPAR␥ and RXR were tested in the ApoE knock-out mice model. Interestingly, they reduced atherosclerosis without lowering the serum cholesterol level [48]. Although both LXR and RXR agonists have been

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strategy to treat atherosclerosis. However, activation of LXRs by a LXR␣/UR dual agonist such as T0901317 may result in increased serum triglyceride levels. Since elevated triglyceride levels are an independent risk factor for atherosclerosis [50], T0901317 may not be an ideal drug. In contrast, Hypocholamide, which is both organ and receptor subtype-selective, may represent a new class of LXR␣ agonists for modulation of cholesterol profile. The effects of Hypocholamide and their derivatives on atherosclerosis models, such as ApoE-null mice, should be studied.

Acknowledgments

Fig. 6. A. Effect of Hyodeoxycholic acid methyl ester (HM), 3␣-glucuronidated HM (HM3G) and 6␣-glucuronidated HM (HM6G) on a DR-4 reporter gene transactivation by LXR␣. Compound concentration: 100 nM (open bars), 1 ␮M (shaded bars) and 10 ␮M (closed bars). B, HEK 293 cells (open circles) or 293 cells stably expressing UGT2B7 (closed circles) were transfected with a DR-4 reporter gene and LXR␣ and treated with increasing concentrations of Hypocholamide. C, HEK 293 cells stably expressing UGT2B7 were transfected with a mSREBP-1/-550/pGL2 luciferase reporter plasmid [26], and LXR␣ and treated with increasing concentration of N, N-dimethyl-3␤-hydroxy-5-cholesten-24-amide (open diamonds) or Hypocholamide (closed diamonds). RLU, relative light unit.

shown to inhibit cholesterol absorption in mice, possibly by inducing intestinal ABCA1 expression through LXR/RXR heterodimers [12], only LXR␣ agonist Hypocholamide and RXR agonist LG100364 reduced the serum cholesterol level in the ApoE-deficient mice, suggesting that in this animal model, besides intestinal ABCA1, additional LXR␣ target genes may also be responsible for the hypocholesterolemic effect by Hypocholamide administration. In ApoE null gene mice, Hypocholamide was able to lower serum cholesterol by 40%, indicating that Hypocholamide can lower cholesterol levels in an ApoE-independent manner. The human ApoE gene is polymorphic, with 3 common alleles, designated as E2, E3 and E4. Many population studies have shown that the presence of the E4 allele is associated with elevations in LDL cholesterol. One report estimates that, in the general population, the ApoE alleles may account for up to 14% of the normal inter-individual variation in plasma total and LDL cholesterol [49]. It is possible that LXR␣ selective agonists like Hypocholamide can lower elevated plasma cholesterol levels in individuals with the E4 allele. Recently, we reported that certain oxysterol sulfates are LXR antagonists [24]. These compounds may be present in atherosclerotic lesions and thus play a role in atherogenesis. Exogenous administration of tissue-selective LXR␣ agonists may counteract the pathologic effect of these autooxidized forms of cholesterol and offer a new therapeutic

Authors would like to thank Drs. Richard A. Hiipakka and Junichi Fukuchi and Mr. Andrew L. Ko for technical assistance; Dr. Takashi Iida for hyodeoxycholic acid methyl ester 3-and 6-glucuronides, Dr. Thomas R. Tephly for HEK293 cells stably transfected with UGT2B7 expression genes, Dr. Hitoshi Shimano for mSREBP-1/-550/pGL2 reporter plasmid, and Dr. Godfrey Getz for in-depth discussion and advice.

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