Le modulateur sélectif des récepteurs des œstrogènes (SERM) Acolbifène (ACOL) prévient le cancer en exerçant une action anti-œstrogénique puissante sur les glandes mammaires et l’utérus. Cependant, il démontre des effets bénéfiques semblables aux œstrogènes sur le métabolisme énergétique et lipidique chez la rate. Le composé diminue la prise alimentaire et diminue fortement la cholestérolémie chez des rats nourris avec une diète sans cholestérol, possiblement via une augmentation de l’expression du récepteur d’épuration classe B type 1 (SR-B1) et du récepteur des lipoprotéines de faible densité (LDLr). Parce que ces récepteurs sont modulés à la baisse par le cholestérol alimentaire, cette étude visait à établir si l’ACOL conserve son efficacité à réduire la cholestérolémie chez des rates nourries avec une diète enrichie de cholestérol, et de déterminer si son effet anorexique y contribue. Les rats femelles ont été nourries avec une diète purifiée dépourvue de cholestérol (diète référence) ou enrichie de 2% de cholestérol (C-diète), et étaient non traitées, ou traitées à l’ACOL tous les jours, ou non traitées et appariées pour l’ingesta des rates traitées avec l’ACOL. La C-diète n’a pas modulé la consommation de nourriture ni le gain de poids et de masse grasse. L’ACOL a diminué la prise alimentaire (16%) et le gain de poids (45%, principalement du gras) similairement chez les deux cohortes alimentaires. L’ACOL, mais pas les appariés pour l’ingesta, a diminué la cholestérolémie de 33% chez les rates nourries avec la diète référence. Tel qu’attendu, la C-diète a augmenté le cholestérol plasmatique de près de 4 fois supérieurs. Une telle augmentation a été largement empêchée par l’ACOL, mais pas avec l’appariement pour l’ingesta. Le cholestérol a été diminué par l’ACOL principalement dans la fraction HDL chez les rates nourries avec la diète référence, mais seulement dans la fraction non HDL chez celles nourries avec la C-diète. Ni l’ACOL ni l’appariement pour l’ingesta n’a affecté la grande augmentation du cholestérol hépatique induite par la C-diète. L’ACOL, mais pas l’appariement pour l’ingesta, a augmenté la protéine SR-B1 et l’ARNm du LDLr dans le foie des rates nourries avec la diète référence. Les deux récepteurs ont été fortement diminués par la C-diète, et cette diminution a été complètement abolie par l’ACOL et l’appariement pour l’ingesta. Ces résultats démontrent que l’ACOL exerce une puissante action hypocholestérolémique, indépendante de la diminution de la prise alimentaire, et qu’une telle action est conservée chez des rates nourries avec une diète induisant l’hypercholestérolémie, possiblement parce que le SERM conserve sa capacité à moduler les récepteurs hépatiques clés.

Keywords: Anti-estrogen compound, Blood cholesterol, EM-652, Scavenger receptor B1, LDL receptor

There is increasing interest in the development of compounds that antagonize the effects of female sex steroids in the prevention and treatment of estrogen-dependent cancers. Several such compounds, including a variety of selective estrogen receptor modulators (SERMs), have been developed in the past years, some which display, along with their strong anti-estrogenic action in cancer-prone tissues, interesting estrogen-like actions on bone mineral density, body fat accretion, and the plasma lipid profile^1-5. Of particular interest in this context is Acolbifene (EM-652, ACOL), a fourth generation SERM with potent anti-carcinogenic properties in the mammary gland and uterus that has been shown to reduce bone mineral mass loss and body fat accretion, as well as to improve the lipoprotein profile in rodent models⁶.

ACOL has been consistently found to dramatically reduce the amount of circulating cholesterol in rats^7,8. The fact that in these studies rats were fed a cholesterol-free diet strongly points to mechanisms related to cholesterol clearance from the circulation. In congruence with this concept, we have recently found (C. Lemieux et al., unpublished observations) that ACOL upregulates the abundance of two key hepatic receptors responsible for a large fraction of cholesterol uptake by the liver: the scavenger receptor, class B, type 1 (SR-B1), which selectively takes up cholesterol from HDL^9-11 and plays a central role in reverse cholesterol transport, and the LDL receptor (LDLr), which internalizes mostly VLDL remnants and LDL particles^12-14. ACOL was not found to affect enzymes of cholesterol synthesis, further supporting the involvement of the clearance-related receptors in its hypocholesterolemic action.

As stated above, the hypocholesterolemic effect of ACOL was observed in rats fed a purified high carbohydrate diet virtually devoid of cholesterol^7,8. The downregulation of the LDLr by dietary cholesterol is very well established^15,16, and a recent study has demonstrated an identical effect of dietary cholesterol on the SR-B1¹⁷. These considerations raise the obvious question of whether ACOL retains its capacity to upregulate these receptors and lower cholesterolemia in the setting of diet-induced hypercholesterolemia. In addition, estrogen and SERMs are well known to affect whole body energy balance^18-20. In rats, ACOL exerts an estrogen-like effect on food intake, the reduction of which dampens adipose tissue accretion⁸. Although such negative energy balance is more likely to impact triacylglycerol rather than cholesterol metabolism, there is no a priori reason to reject the possibility that the anorectic effect of ACOL may participate in its hypocholesterolemic action.

The present study was designed to address both of these central issues related to the potency of ACOL to reduce cholesterolemia and to its mechanisms of action. Female rats were fed a purified diet without cholesterol or the same diet to which 2% cholesterol was added. Each of these two dietary cohorts were subjected to ACOL treatment, or alternatively to an imposed reduction in food intake so as to match that of the ACOL-treated animals. The paradigm allowed the assessment of the ability of ACOL to counteract the effects of dietary cholesterol, as well as that of the contribution of changes in ingestive behavior to its hypocholesterolemic action.

Animals and treatments . Forty-two female Sprague-Dawley rats initially weighing 175-200 g were purchased from Charles River Laboratories (St-Constant, Quebec, Canada) and housed individually in stainless steel cages in a room kept at 23 ± 1°C with a 12-h light:12-h dark cycle (lights on at 19:00). The animals were cared for and handled in conformity with the Canadian Guide for the Care and Use of Laboratory Animals, and the protocol was approved by our institutional animal care committee. The animals were acclimated to their environment for one week and had ad libitum access to tap water and to a nonpurified rodent chow diet (Charles River Rodent Diet no. 5075, Ralston Products, WoodStock, Canada). Half of the rats were then fed a purified diet that provided 50% energy as carbohydrate, 30% as fat, and 20% as protein, the composition of which was as follows (in g per 100 g of diet): corn starch, 26.5; sucrose, 26.5; soy oil, 4.0; beef tallow, 10.0; casein, 21.5; DL-methionine, 0.4; vitamin mix (Teklad no. 40,060, Teklad Test Diets, Madison, WI), 1.1; mineral mix (AIN-76, ICN Biochemicals, Montreal, Quebec, Canada), 5.0; cellulose (Alphacel, ICN Biochemicals), 5.0. The other half of the animals were given the same diet to which 2% (w/w) cholesterol (ICN Biochemicals, Montreal, Quebec, Canada) was added. Seven animals in each of the two dietary cohorts were assigned to the placebo (vehicle) group whereas another 7 animals were treated with ACOL. The compound was given once daily by oral gavage at a dose of 2.5 mg/kg in a total volume of 0.5 ml of a 0.4% aqueous solution of methylcellulose. The remaining 7 animals in each dietary cohort were left untreated and were pair-fed to the ACOL-treated group in the following manner: each rat was randomly paired to one of the ACOL-treated animal, and the amount of food provided to the rat was adjusted to the amount of food ingested the previous day by its ACOL-treated counterpart. To avoid intake of the restricted amount of food in one single large meal, two thirds of the food was provided to the pair-fed rats at the beginning of the dark, active period, and the remaining third was given at the beginning of the lighted period. The placebo and pair-fed groups were given daily the methylcellulose vehicle by gavage. Six groups of 7 animals each were thus constituted according to a 2 × 3 factorial design. Treatment was carried out for a period of 21 days. Food intake and body weight were monitored everyday. Because previous studies have shown that ACOL impacts SR-B1 abundance in a nutritional state-dependent fashion, rats were studied in the fasted state. Thus, the day before the completion of the study, food was removed after the last vehicle or ACOL gavage at 21:00, and rats were killed by decapitation the following day between 09:00 and 11:00.

Blood and tissue collection . At the time of killing, blood was collected from the neck wound and was immediately centrifuged at 1,500×g, 4°C for 15 min. Serum was stored at –80°C for later biochemical measurements. A sample of liver was immediately frozen in liquid nitrogen and stored at –80°C until later determination of lipid content. Retroperitoneal and inguinal white adipose tissues (WAT) were excised and weighed.

Serum/tissue measurements . The HDL fraction was isolated by precipitation of apolipoprotein B-containing lipoproteins with sodium phosphotungstate-magnesium chloride. Total and HDL-cholesterol were quantified using a reagent kit from WAKO Diagnostics (Richmond, VA). Non-HDL CHOL was obtained by difference. Frozen liver samples were thawed, total lipids were extracted according to the method of Folch et al²¹, solubilized in isopropanol, and liver cholesterol concentration was quantified in these lipid extracts using the above mentioned reagent kits.

Immunoassay of SR-B1 . To extract the SR-B1 protein, a liver sample (~50 mg) was homogenized in buffer A containing 20 mM Tris-HCl (pH 7.5), 2 mM MgCl₂, 0.2 M sucrose, 5 mM phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, 20 µg/mL aprotinin and 5 µg/mL pepsatin A. The crude extract was centrifuged at 10000×g for 10 min at 4°C to remove tissue debris. The supernatant was then ultracentrifuged at 100000×g for 45 min at 4°C. The pelleted membrane fraction was then resuspended in buffer B containing 62.5 mM Tris-HCl (pH 6.8), 2% (v/v) SDS, 10% (v/v) glycerol and 5% (v/v) β-mercaptoethanol and frozen at –80°C until further processing. Protein concentration of the liver extracts was determined by the method of Lowry et al²². Five µg of SR-B1 protein per lane were loaded on 7.5% polyacrylamide gels. Protein separation by electrophoresis was performed according to the method of Laemmli²³ under non-reducing conditions. The protein bands were transferred onto PVDF membranes (2 h, 100 V at 4°C). The membranes were then washed and blocked with 5% skim milk in washing buffer containing TBS, 0.02% (v/v) Tween 20 and 0.04% (v/v) Igepal CA-630 for 1 h. The membranes were washed and incubated overnight at 4°C in 1% skim milk in washing buffer containing 1:1500 anti-SR-B1 (Novus Biologicals Inc., Littleton, CO, USA). The membranes were washed and then incubated for 1 h at room T° in 1% skim milk in washing buffer containing 1:10000 horseradish peroxidase-linked antirabbit IgG (Amersham Biosciences UK Ltd, Little Chalfont, Buckinghamshire, England). After a final wash, the membranes were developed with chemiluminescent agents ECL+ (Amersham Biosciences) and exposed to autoradiographic film (Kodak BioMax MR film).

Liver total RNA isolation and analysis of LDLr mRNA. Total RNA was prepared from liver using the Trizol RNA extraction method. RNA concentration was estimated from absorbance at 260 nm and RNA was reverse transcribed using the Expand reverse transcriptase (Roche Diagnostics, Laval, Quebec, Canada). The expression level of mRNA was quantitated using quantitative fluorescent real-time PCR (Corbett Research, New South Wales, Australia). Amplification and detection of the target mRNA was performed with Platinum Taq polymerase and the intercalating dye Sybr-Green I. The primers, designed using the Vector NTI program and synthesized by Invitrogen (Burlington, Ontario, Canada), were the following the for LDLr: 5’ Primer (5’-3’) TGG ACC CTT TCT CTC GGA AC; 3’ Primer (5’-3’): AAG GCT GTG GGT TCC ATA GG. The levels of LDLr mRNA were normalized to the amount of L27 mRNA (a gene not affected by treatments) detected in each sample, and results are expressed as gene/L27 mRNA.

Statistical analysis . Data are expressed as means ± SEM, and were analyzed by factorial analysis of variance (ANOVA) with 2 factors: Diet, with 2 levels (Cholesterol-free, Cholesterol 2%), and a factor termed ‘Drug treatment’ for convenience, with 3 levels (Placebo, ACOL, Pair-fed). Main and interactive effects of treatments were determined and are reported within ANOVA tables. Pairwise comparisons were also carried out to locate individual between-group differences using Fisher’s Post hoc Least Squares Difference test. Differences reaching the confidence level of P < 0.05 were considered significant.

Treatment effects on serum total, HDL- and non-HDL-cholesterol are depicted in Fig. 1. Diet and ACOL treatment interacted upon total and lipoprotein cholesterol, due to the fact that the drug did not exert the same effect, or did so but to a different extent, depending on diet. As expected, the C-diet greatly increased (almost 4-fold) serum total cholesterol concentration (Fig. 1A), which in turn was reduced by ACOL treatment, much more so in the C-fed rats (–56%) than in those fed the reference diet (–33%). Pair feeding did not affect cholesterolemia. The C-diet reduced HDL-cholesterol in intact and pair-fed rats to approximately one third of the levels found in rats fed the diet without cholesterol, whereas HDL-cholesterol was unchanged by diet in ACOL-treated rats (Fig 1B). Conversely, the C-diet dramatically increased non-HDL cholesterol (20-fold), and ACOL treatment, which was without significant effect in rats fed the reference diet, dampened the diet-induced elevation in non-HDL cholesterol to less than half of that seen in both intact and pair-fed rats (Fig. 1C). In the cohort fed the reference diet, the ACOL-induced reduction in total cholesterol was almost entirely due to a reduction in HDL-cholesterol, whereas the decrease in non-HDL cholesterol (Fig. 1C) totally explained the drug-induced fall in cholesterol in C-fed animals.

As shown in Fig. 2A, the C-diet vastly increased liver cholesterol concentration (22-fold). In fact, in the placebo groups, the C-diet increased total liver cholesterol content from 15 to 440 mg cholesterol, which explained 28% of the increase in liver weight caused by the C-diet. Neither ACOL treatment nor pair feeding affected liver cholesterol concentration. The concentration of the SR-B1 protein was greatly reduced by the C-diet, to 20% of the levels found in the reference diet (Fig. 2B). ACOL, but not pair feeding, strongly tended ( P = 0.06) to increase liver SR-B1 concentration in rats fed the reference diet, whereas the drug totally prevented the C-diet-induced fall in receptor level, as did pair feeding. Serum HDL-cholesterol concentration was inversely correlated with that of liver SR-B1 in rats fed the reference diet (r = –0.70, P =0.003), but not in those fed the C-diet (r = 0.30, not significant). Fig. 3C depicts treatment effects on liver levels of LDLr mRNA. In rats fed the reference diet, ACOL increased LDLr mRNA 32% whereas pair feeding remained without effect. The C-diet exerted an overall lowering effect on LDLr mRNA, and ACOL did not affect the latter in rats fed this diet.

The two major goals of the present study were to assess whether the reduction in food intake elicited by treatment with the SERM ACOL played a role in its hypocholesterolemic action, and whether the latter was maintained in diet-induced hypercholesterolemia, which downregulates two hepatic receptors that are believed to mediate the ACOL-induced reduction in circulating cholesterol levels. The findings demonstrate that ACOL reduces cholesterol independently of changes in food intake, and that the SERM maintains its hypocholesterolemic potential under cholesterol feeding.

The study confirms previous findings regarding the effects of ACOL on body weight, food intake and fat deposition^7,8. The decrease in body weight, mainly in the form of fat stores, was associated with a reduction in food intake. The drug therefore shares some actions on energy and fat metabolism with natural estrogen and other antiestrogens^8,18.

In the cohort fed the cholesterol-free, reference diet, the hypocholesterolemic action of ACOL described previously^7,8,24 was confirmed. Importantly, the 16% decrease in food intake did not contribute to the ACOL-induced reduction in cholesterolemia, the latter being identical in pair- and ad libitum-fed untreated rats. ACOL therefore acts upon cholesterol metabolism through mechanisms that are independent from ingestive behavior. In previous studies, ACOL was shown to reduce both HDL- and non-HDL-cholesterol fractions. A similar trend was noted in the present study, however the 26% reduction in non-HDL-cholesterol did not reach significance. As expected, because HDL transports more than two-thirds of circulating cholesterol in the rat, its decrease explained most of the hypocholesterolemic effect of ACOL in animals fed the reference diet.

Further confirming a previous study (C. Lemieux et al., in revision), cholesterol lowering in rats fed the reference diet was associated with an increase in hepatic SR-B1 protein concentration and LDLr mRNA level. Most of the reduction in cholesterol occurred in the HDL fraction, which correlated strongly with liver SR-B1 concentration. The ACOL-induced increase in SR-B1 protein levels was more variable and somewhat less robust than in our previous study (C. Lemieux et al., in revision), perhaps because of the higher lipid content of the diet used in the present study. LDLr mRNA was not strongly correlated with any of the cholesterol fraction, however it may have contributed to the small, nonsignificant reduction in the non-HDL cholesterol fraction, as well as to the clearance of apoE-containing HDL present in the rat. Interestingly, pharmacological amounts of natural estrogen (mostly estradiol) strongly upregulate liver LDLr expression, which explains most of its hypocholesterolemic effect^25-30, but at the same time the hormone completely blunts SR-B1 expression^17,31,32, in clear contrast with ACOL. The reasons why ACOL exerts either pro- or anti-estrogenic actions in a target-dependent fashion is likely linked to conformational determinants of the ligand-receptor complex that differentially impact the recruitment of nuclear transcriptional factors⁶. Although the involvement of the SR-B1 and LDLr in the cholesterol-lowering action of ACOL awaits direct confirmation, the fact that ACOL does not appear to impact hepatic cholesterol synthesis (C. Lemieux et al., in revision) and that it is very efficient even in the absence of dietary cholesterol strongly argue in favor of a clearance-related mechanism of action.

The cholesterol-enriched diet brought about the expected shift in cholesterol lipoprotein distribution, the bulk of which was found in the non-HDL fraction, along with a decrease in the absolute amount of cholesterol transported by HDL. It is well established that dietary cholesterol exerts these actions by favoring the hepatic assembly and secretion of cholesterol-enriched VLDL particles, with a concomitant increase in intravascular LDL formation, which result in decreased formation of HDL; from a modulation of apolipoprotein production by dietary cholesterol^33-35. The reduction in SR-B1 abundance and the stable LDLr mRNA expression levels in cholesterol-fed rats are also congruent with the established effects of dietary cholesterol^17,36. Notably, ACOL defeated the diet-induced reduction in SR-B1 levels, maintaining the receptor at levels observed in rats fed the reference diet. Unexpectedly, pair feeding also maintained SR-B1 at reference levels, despite the fact that dietary cholesterol intake was reduced by only one fifth. Although there is no obvious explanation for this phenomenon, because SR-B1 has been found to be modulated by food ingestion (C. Lemieux et al., in revision), it can be suggested that the long-term food intake pattern of the pair-fed animals (two daily large meals rather than continuous feeding during the active dark period) may have played a role therein. It should be noted that the absolute liver cholesterol content was not associated with the ACOL-mediated modulation of SR-B1 levels.

The respective contribution of the SR-B1 and LDLr to the cholesterol-lowering action of ACOL remains to be determined through assessment of liver cholesterol uptake from specific lipoprotein fractions as well as gene invalidation strategies. The present results however suggest that in rats fed a cholesterol-free diet, the large reduction in cholesterol associated with the HDL fraction is linked to SR-B1 abundance, whereas in cholesterol-fed rats, because of the large diet-induced decrease in HDL-cholesterol, SR-B1 does not further impact this fraction even if ACOL (and pair feeding) prevents its reduction. In cholesterol-fed rats, all of the ACOL-induced reduction in cholesterol was found in the non-HDL fraction. Surprisingly, the SERM did not increase LDLr mRNA levels in these rats, as may have been expected if the receptor was involved in the cholesterol-lowering action of ACOL. In another study with ovariectomized rats fed a diet identical to that used here, ACOL was found to increase LDLr protein levels (C. Lemieux and Y. Deshaies, unpublished observations), which were not determined in the present study, and that such increase occurred in the face of unchanged LDLr mRNA abundance. It is therefore possible that ACOL may modulate the LDLr posttranslationally in rats fed cholesterol. Such posttranslational modulation of the LDLr has been reported in other conditions³⁶. Alternatively, preliminary studies suggest that ACOL may modulate the expression of the cholesterol transporters ABCG5/8 (C. Lemieux and Y. Deshaies, unpublished observations), which may in turn impact its absorption by the intestine. Further investigation of these major modulators of cholesterolemia are clearly needed to identify the precise mechanisms of the hypocholesterolemic action of ACOL in cholesterol-fed animals.

The consequences of the HDL-cholesterol-lowering action of ACOL in rats fed a cholesterol-free diet must be considered in the context of the fundamental differences that exist between human and rodent lipoprotein metabolism. As stated above, HDL is the major cholesterol carrier in the rat, and a robust lowering of total cholesterol seldom occurs without a decrease in this fraction. Gene manipulation studies point to clear beneficial consequences of high SR-B1 and reverse cholesterol transport activities⁸. Therefore, in the context of human physiology, the ACOL-mediated increase in liver SR-B1 abundance, along with the increase in the LDLr, would both be considered as positive effects in terms of cardiovascular risk associated with lipoprotein metabolism.

In summary, the present findings demonstrate that ACOL exerts a potent hypocholesterolemic action, independently from concomitant reductions in food intake, and that such action is maintained in rats with diet-induced hypercholesterolemia, possibly because the SERM upregulates key hepatic lipoprotein receptors and defeats their downregulation by dietary cholesterol.

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Figure 1. Serum total (A), HDL- (B), and non-HDL (C) cholesterol concentrations in rats fed a purified diet or the same diet containing 2% cholesterol, treated or not with Acolbifene, or untreated and pair-fed to Acolbifene-treated rats for 4 weeks. Bars represent the means ± SEM of 6-7 animals. See footnote to Table 1 for significance of ANOVA table and symbols.

Figure 2. Liver cholesterol concentration (A), SR-B1 protein (B), and LDL receptor mRNA (relative to L27 mRNA, C) in rats fed a purified diet or the same diet containing 2% cholesterol, treated or not with Acolbifene, or untreated and pair-fed to Acolbifene-treated rats for 4 weeks. Bars represent the means ± SEM of 5-7 animals. See footnote to Table 1 for significance of ANOVA table and symbols.

Figure 1

Figure 2