Introduction

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Cardiovascular disease represents the main cause of death in the United States, and the relation between cardiovascular disease and the amount of serum cholesterol and low-density lipoprotein (LDL) cholesterol has been well established (Anderson et al., 1987; Verschuren et al., 1995). Large-scale clinical trials have also shown that lowering serum LDL cholesterol decreases cardiovascular mortality rates (Pedersen et al., 1995; Shepherd et al., 1995).

The importance of the liver LDL receptor (LDL-R) in maintaining normal LDL cholesterol levels has been well documented since the breakthrough studies of Goldstein and Brown (Brown and Goldstein, 1986; Goldstein and Brown, 1974). Although the types of agents used to lower cholesterol in the clinical trials differ, they have generally been shown to raise LDL-R in animal models (Ma et al., 1986) and in humans (Kervinen et al., 1993) secondary to enzyme inhibition (Matsunaga et al., 1994) or increased bile acid excretion (Einarsson et al., 1991). Because the LDL-R is repressed and largely saturated in hypercholesterolemia (Brown and Goldstein, 1986), it would be advantageous to develop a compound that derepressed the liver LDL-R as its primary effect. Such a compound would be expected to affect LDL cholesterol more directly than do currently available pharmacological interventions.

LDL-R transcription is repressed by a sterol regulatory element (SRE) and mediated through sterol-activated transcription factors (Yokoyama et al., 1993). This system can be modulated in cell culture by 25-hydroxycholesterol (Brown and Goldstein, 1975; Metherall et al., 1989). We expected that compounds that could overcome the 25-hyroxycholesterol-mediated repression of LDL-R expression in cell systems would act to elevate LDL-R in hypercholesterolemia. Although the SRE is also involved in the up-regulation of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase and HMG-CoA synthetase (Molowa and Cimis, 1989), these are modulated by other negative regulatory pathways that would be expected to maintain cholesterol synthesis at a normal rate (Straka and Panini, 1995; Hampton et al., 1996).

Based on these considerations, a primary screen was established using Chinese hamster ovary cells stably transfected with a gene construct in which the DNA coding for the promoter and regulatory control elements for the LDL-R gene was fused to a firefly luciferase reporter gene (Lin et al., 1995). Compounds were sought that increased LDL-R promoter activity under repressed conditions in the presence of 25-hydroxycholesterol. Secondary assays were performed to eliminate compounds that might have general promoter up-regulation activity.

We describe in this report the effects of LY295427 [(3,4,5)-4-(2-propenylcholestan-3-ol)] (Fig. 1), discovered through this screen, on LDL-R gene transcription and on cholesterol homeostasis in normal and hypercholesterolemic hamsters, in which the effects of added cholesterol and fat on the kinetics of hepatic LDL-R-mediated LDL clearance are well characterized (Spady and Dietschy, 1988; Horton et al., 1993).

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Chemical structure of LY295427 [(3,4,5)-4-(2-propenylcholestan-3-ol)].

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animals. All experiments were performed in accordance with Eli Lilly and Company animal care and use policies. Male Syrian hamsters (Charles River Laboratories) weighing 110 to 130 g were housed in temperature- and humidity-regulated rooms with a 14-h light/10-h dark cycle. Except where noted, they were maintained on a high-fat diet consisting of Purina 5001 rodent chow (Purina Mills, Richmond, IN), supplemented with 10% coconut oil and 0.12% cholesterol for a minimum of 2 weeks before the start of experiments (Woollett et al., 1992). Animals were allowed free access to water and diet with test compounds dry mixed into the diet on a weight basis.

Determination of Serum Lipids. Hamsters were bled (400 µl) from the suborbital sinus while under light CO₂ anesthesia 1 to 2 h after the beginning of the light cycle, and serum was separated by brief centrifugation (16,000g, 1 min, Eppendorf model 5414). Total cholesterol and triglyceride levels were determined in serum using enzymatic assay kits (290319 and 701094; Boehringer-Mannheim, Indianapolis, IN) modified for use in 96-well plates (10 µl of sample, 100 µl of cholesterol reagent, or 5 µl of sample, 200 µl of triglyceride reagent, both with 30-min incubation at room temperature). High-density lipoprotein (HDL) cholesterol was determined similarly after removal of non-HDL cholesterol through magnesium phosphotungstate precipitation (543004; Boehringer-Mannheim) (Weingand and Daggy, 1990). Serum from hypercholesterolemic hamsters was diluted with an equal volume of 0.9% NaCl before the removal of non-HDL cholesterol and before triglyceride determination. LY295427 added to the cholesterol assay at concentrations up to its solubility limit (equivalent to 150 mg/dl serum) did not interfere.

Determination of Hepatic Lipids. Livers obtained at the time of sacrifice were frozen in liquid nitrogen and maintained at 75°C until analysis. Neutral lipids were extracted from liver samples (1 g) with chloroform/methanol (2:1, v/v) (Folch et al., 1957). After evaporation of the chloroform from the lipid extract under N₂, samples were resuspended (200 µl minimum) in chloroform/methanol (4:1, v/v). A mixed standard consisting of cholesterol, triolein, and cholesteryl oleate (0.5 mg each/ml) was prepared and used to identify bands. High-performance thin-layer chromatography Kieselgel 60 silica plates (EM Separations, Gibbstown, NJ) were washed in the mobile phase of hexane/heptane/diethyl ether/acetic acid (63:18.5:18.5:18.5:1, v/v) (Schmitz et al., 1984) and activated for 15 min in a 110°C oven. Samples (3 µl) and standards were applied in bands (6 mm) using a Linomat IV TLC Sample Applicator (Camag Scientific, Wilmington, NC). The plates were developed twice (2 min, 10 min) in a saturated chamber with fresh mobile phase. The lipids were quantified after fluorescence induction in hexane/ethanol/sulfuric acid (64:35:1, v/v) (Kurantz et al., 1991) using a Camag TLC Scanner II (Camag Scientific, Wilmington, NC).

Measurement of Serum Concentration of LY295427. Serum (1 ml) was loaded onto a previously conditioned (15 ml water) 1-ml C18 Bond-Elut solid-phase extraction column (Analytichem, Harbor City, CA) and eluted with hexane (3 × 1.5 ml). The eluant was dried and reconstituted in ethanol (40 µl) for chromatography. Components were separated from 3-µl samples on a Varian 3400 gas chromatograph (Walnut Creek, CA) equipped with a J & W Scientific 8 m × 0.25 mm DB-Wax capillary column (Folsom, CA.). The injector temperature was set at 350°C. The initial column temperature was 200°C for 1 min and then ramped at a linear rate of 50°C/min to 280°C. The final oven temperature of 280°C was maintained for 4.5 min. Helium carrier gas was set at 4 psi. The chromatograph was interfaced to a Nermag R 30-10 triple-quadrupole mass spectrometer (Delsi Instruments Inc., Houston TX). Negative-ion methane chemical ionization was used to ionize and detect the analyte. Parameters were optimized to yield the greatest signal for the [M-H] ion of LY295427 (m/z 427). Source pressure was optimal at 6 × 10² mm Hg. Ion source temperature was 200°C, electron energy was 95.5 eV, filament current was 0.240 mA, and the multiplier was 0.66 kV. The 2-butylene derivative of LY295427 (m/z 441) was used as an internal standard. This method could detect 50 ng of LY295427/ml serum with a signal-to-noise ratio of 5:1 and was linear to 2000 ng of LY295427/ml serum.

Cholesterol Absorption Studies. Hypercholesterolemic hamsters were presorted into groups of 12 animals with equal mean serum cholesterol levels and were fed either the control hypercholesterolemic diet or that diet containing various amounts (w/w) of -sitosterol (0.4%, 0.6%, 0.8%, or 1.0%) or 0.2% LY295427. After 2 weeks on the test diets, the mean serum cholesterol level in the 1.0% -sitosterol group was virtually identical to that of the 0.2% LY295427 group. These doses correspond to approximately 100 mg of LY295427/kg/day and 500 mg of -sitosterol/kg/day. Cholesterol absorption in 10 hamsters from each of these two groups and from 10 control hypercholesterolemic hamsters was determined as described by Turley et al. (1994), where the ratio of ³H to ¹⁴C in serum was measured 72 h after the administration of ¹⁴C-cholesterol (by oral gavage) and ³H-cholesterol (by i.v. injection). The ³H-cholesterol in Intralipid was injected into the exposed jugular vein with the animal under ketamine HCl (Ketaset; 80 mg/kg)/xylazine (Rompun; 16 mg/kg) anesthesia.

Measurement of LDL-R mRNA by S1-Nuclease Protection Assay. Total RNA was isolated from liver samples (1 g) by a guanidinium thiocyanate-phenol-chloroform single-step extraction (Chomczynki and Sacchi, 1987) (RNA Isolation Kit; Stratagene, La Jolla, CA) and quantified by the orcinol method (Kabat and Mayer, 1961). The probe template contained a genomic DNA fragment (HincII/HaeIII) of 210 bp inserted into the SmaI site of the M13 mp19 vector. The DNA insert corresponds to exon 2 (123 nucleotides) of the hamster LDL-R gene (gift and personal communication, D. W. Russell, University of Texas). A single-stranded ³²P-labeled cDNA probe was synthesized and hybridized to mRNA with minor modification of the method described by Williams et al. (1986). Single-stranded template DNA (4 µg) was annealed to a universal sequencing primer of 17 nucleotides (56 ng) and extended in the presence of 50 µM [-³²P]dCTP (400 Ci/mmol); 0.25 mM concentration each of dTTP, dATP, and dGTP; and the Klenow fragment of Escherichia coli DNA polymerase I. The extended product was digested with HindIII, and the resulting probe (~2.8 × 10⁶ cpm/fmol) was purified by 7 M urea/5% polyacrylamide gel electrophoresis (Integrated Separation Systems, Natick, MA) and crush-elution of the radioactive DNA band (300 bases) (Maniatis et al., 1982). The single-stranded ³²P probe (200 pg) was hybridized at 68°C for 60 h with various amounts of total cellular RNA (0, 6, 25, 100, and 200 µg) in triplicate and digested with 100 units of S1 nuclease for 2 h at 45°C. The nuclease-resistant hybrids were collected by trichloroacetic acid precipitation, and radioactivity was measured by scintillation counting. A set of samples without RNA was used to determine the S1 nuclease-resistant background of the probe. This background value was subtracted from the experimental samples. Another set of samples without RNA were assayed and not treated with S1 nuclease; these samples were used to confirm the amount of probe used in the assay. The slopes of straight lines fitted to the LDL-R mRNA curve were taken to reflect the cpm of protected probe per microgram of total RNA.

¹⁴C-Acetate Incorporation into Cellular Lipids. HepG2 cells were grown to confluence on 100-mm² plates using 7.5 ml of medium [Dulbecco's modified Eagle's medium/Ham's F-12 nutrient mixture (3:1, v/v), 0.5% BSA]. LY295427, lovastatin, and triparanol were added in 7.5 ml of fresh medium, and cells were incubated for 24 h at 37°C. ¹⁴C-Acetate (15 nCi/7.5 µl) and 1 mM sodium acetate (7.5 µl) were added, and the cells were incubated at 37°C for 4 h. Cells were washed and harvested by scraping and centrifugation (16,000g, 1 min). Pelleted cells were resuspended in water (0.5 ml), aliquots (20 µl) of the suspension were taken for protein determination (Bradford, 1976), the remainder was disrupted by sonication, and neutral lipids were extracted with chloroform/methanol (2:1, v/v) (Folch et al., 1957). Dried extracts were further extracted and saponified (Boogaard et al., 1987). Resuspension volumes (200 µl minimum) of chloroform/methanol (4:1, v/v) were normalized for protein in each sample. High performance thin-layer chromatographic RP-18 plates were washed in the mobile phase consisting of acetonitrile/chloroform (2:1, v/v) and activated for 15 min in a 110°C oven. A mixed standard composed of cholesterol, desmosterol, lanosterol, squalene, farnesol, geraniol, and two presqualene epioxides, 2,3-squalene epoxide and 2,3,22,23-squalene diepoxide, was prepared. Sample and standards (5 µl) were applied in 6-mm bands, and plates were developed in the fresh mobile phase (20 min). The plates were stained with 5% phosphomolybdic acid in ethanol to visualize standards, and ¹⁴C-acetate incorporation into cholesterol and its precursors was detected with autoradiography (Kodak X-OMAT-AR film for 5 days at 70°C).

Reagents. Lovastatin was from Merck, Sharp & Dohme Research Laboratories (Rahway, NJ). Triparanol was from Marion Merrill Dow Inc. (Kansas City, MO). -Sitosterol was from Eli Lilly & Co. (Indianapolis, IN). Ketaset and Rompun were from Baker Veterinarian Supplies (Indianapolis, IN). MCT Oil (medium-chain triglycerides) was from Mead Johnson (Evansville, IN). Intralipid (20%) was from Kabi Pharmacia (Clayton, NC). Na⁺ [2-¹⁴C]acetate, [4-¹⁴C]cholesterol, [1,2-³H]cholesterol, and [-³²P]dCTP (400 Ci/mmol) were from DuPont NEN (Boston, MA). M13 sequencing primer (20) 17-mer was from New England Biolabs (Beverly, MA). The Klenow fragment of E. coli DNA polymerase I, HindIII restriction endonuclease, and unlabeled deoxynucleotides were from Promega (Madison, WI). LY295427 [(3,4,5)-4-(2-propenyl)cholestan-3-ol)] (Fig. 1), (3,4,5)-4-(2-butenyl)cholestan-3-ol (mass spectroscopy standard) (Lin et al., 1995), 2,3-squalene epoxide, and 2,3,22,23-squalene diepoxide (Dr. John Schauss) were synthesized at Lilly Research Laboratories. All other reagents were of the highest grade available commercially.

	Results

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In normocholesterolemic hamsters, 2-week administration of the HMG-CoA reductase inhibitor lovastatin (50 mg/kg/day) lowered the serum cholesterol by 70% (Fig. 2A). In contrast, LY295427 had no effect on serum cholesterol at either 50 or 500 mg/kg/day (Fig. 2B). The distinct difference in the effects of the two compounds was also seen in HDL cholesterol; lovastatin decreased HDL cholesterol levels by 71%, whereas LY295427 was without effect. Thus, in contrast to lovastatin, LY295427 did not alter either total serum cholesterol or HDL cholesterol in chow-fed, normocholesterolemic hamsters.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Differing effects of lovastatin and of LY295427 on serum and HDL cholesterol in normocholesterolemic hamsters. Animals were dosed for 2 weeks with either lovastatin or LY295427 incorporated into Purina Rodent Chow 5001, without added coconut oil or cholesterol. Serum and HDL was prepared and cholesterol was assayed as described in the text. Results are mean ± S.E.M. In experiment A, there were eight control animals and six treated animals. In experiment B, there were six animals in each group. *p < .05 relative to control, Dunnett's test.

To examine the effect of LY295427 under conditions of hyperlipemia, hamsters were fed a diet enriched with 10% coconut oil and 0.12% cholesterol for 2 weeks before the administration of LY295427. As anticipated, the serum cholesterol levels produced by the coconut-cholesterol feeding were about 2.5-fold higher than unenriched diet values (Fig. 3). LY295427 produced dose-dependent decreases in serum cholesterol in hypercholesterolemic animals (Fig. 3). The effect was time dependent, reaching a plateau between 14 and 21 days at the two highest doses. Serum cholesterol in the animals given 25, 50, and 100 mg LY295427/kg/day was decreased by 38%, 56%, and 69%, respectively, compared with controls after 29 days. From these data, the ED₅₀ value for LY295427 was estimated to be 40 mg/kg. At the highest dose tested, serum cholesterol was normalized to values observed in normocholesterolemic hamsters (approximately 150 mg/dl). Thus, unlike the results in normocholesterolemic animals, LY295427 markedly reduced serum cholesterol in hypercholesterolemic hamsters.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Time course for the effect of LY295427 on serum cholesterol in hypercholesterolemic hamsters. Hypercholesterolemic hamsters were given LY295427 in their diet at 0%, 0.02%, 0.05%, 0.1%, and 0.2% for 29 days, with samples taken at each of times indicated. Average daily doses were calculated from daily food consumption. Results are mean ± S.E.M., six animals per group. *p < .05 relative to same day control, Dunnett's test.

The relationship between serum levels of LY295427 and serum cholesterol was examined in hypercholesterolemic animals dosed for 2 weeks. As the dose of LY295427 increased, the serum levels of the compound increased similarly (Fig. 4A), showing that the compound is well absorbed. Furthermore, serum cholesterol levels were highly correlated with serum levels of LY295427 (Fig. 4B). These data suggest that the hypocholesterolemic effect of LY295427 is primarily mediated by drug that is bioavailable as a result of absorption from the gastrointestinal tract.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   A, serum LY295427 in hamsters fed differing amounts of compound. LY295427 was measured in the serum of hypercholesterolemic hamsters given the compound in their diet at 0.05%, 0.1%, and 0.2% for 2 weeks. Average daily doses were calculated from daily food consumption. B, relationship between serum cholesterol and serum LY295427. Serum cholesterol for the individual animals in A is plotted versus their serum LY29547 level.

The effects of LY295427 and the HMG-CoA reductase inhibitor lovastatin on serum lipids were compared in hypercholesterolemic hamsters, and the results are presented in Fig. 5. In these animals, the serum cholesterol lowering by lovastatin resulted from reductions in cholesterol in both the HDL (Fig. 5C) and non-HDL (Fig. 5B) lipoprotein fractions. The effect of lovastatin on serum triglyceride levels was variable and did not appear to be dose dependent (Fig. 5D). Although a dramatic reduction of serum triglyceride was observed at the highest dose of lovastatin (45 mg/kg/day), this may have been due in part to secondary effects of lovastatin as demonstrated by a significant decrease in body weight gain relative to the control group (data not shown).

View larger version (46K): [in this window] [in a new window]   Fig. 5.   Comparison of the effects of lovastatin and LY295427 on serum lipids in hypercholesterolemic hamsters. Hypercholesterolemic animals were dosed for 2 weeks with either lovastatin or LY295427 as described in the text. Results are mean ± S.E.M., eight animals per group. *p < .05 relative to control, Dunnett's test.

Although less potent than lovastatin, LY295427 also decreased serum cholesterol in a dose-dependent manner (Fig. 5E). However, in contrast to lovastatin, the effect of LY295427 was confined to the non-HDL fraction (Fig. 5G). Although there was a modest tendency for LY295424 to increase HDL cholesterol levels, no significant change in HDL cholesterol was seen (Fig. 5F). Also in contrast to lovastatin, there was a dose-dependent decrease in serum triglyceride with LY295427 (Fig. 5H). Furthermore, there was no significant effect on body weight even at the highest dose of LY295427 (data not shown).

The effect of LY295427 and lovastatin on LDL-R mRNA levels was examined in livers from hypercholesterolemic hamsters (Fig. 6). LDL-R mRNA was measured using the S1 nuclease protection assay at doses of lovastatin (5 mg/kg/day) and LY29547 (120 mg/kg/day) that produced comparable reductions in serum cholesterol (64% and 57% reductions, respectively). Both LY295427 and lovastatin significantly increased LDL-R mRNA levels by 60% to 100% relative to control. Furthermore, there was no significant difference in mRNA levels between the two treatments. Thus, there was a comparable increase in LDL-R mRNA levels for a similar decrease in serum cholesterol with both compounds.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Comparison of the effect of lovastatin and LY295427 on hepatic LDL-R mRNA levels in hypercholesterolemic hamsters. Hypercholesterolemic hamsters were given LY295427 or lovastatin in diet for 2 weeks. Hepatic LDL-R mRNA was determined by the S1 nuclease protection assay as detailed in the text. Inset, slope of the line from the S1 nuclease protection assay compared with corresponding change in serum cholesterol in the same animals. Six animals per group, *p < .05 relative to control, Student's t test.

The effects of LY295427 on cholesterol content in livers from drug-treated hypercholesterolemic hamsters were also examined. LY295427 dose dependently lowered total hepatic cholesterol (Fig. 7). This effect was largely confined to cholesterol ester, with little change in the free cholesterol levels. The reduction of liver cholesterol by LY295427 was extensive, with the concentration of hepatic cholesterol ester falling to 6% of the control value at the highest dose used in this experiment, essentially normalizing hepatic cholesterol to chow-fed levels.

View larger version (39K): [in this window] [in a new window]   Fig. 7.   The effect of LY295427 on hepatic cholesterol in hypercholesterolemic hamsters. Hypercholesterolemic hamsters were given LY295427 in their diet at 0%, 0.02%, 0.05%, 0.1%, and 0.2% for 2 weeks. Average daily doses were calculated from food consumption. Results are mean ± S.E.M., six animals per group. *p < .05 relative to control, Dunnett's test.

To determine whether effects on cholesterol absorption might contribute to the reduction in serum cholesterol produced by LY295427, cholesterol absorption was compared with the absorption inhibitor, -sitosterol, at equivalent cholesterol-lowering doses. To establish equivalent doses, groups of hypercholesterolemic hamsters were treated with the dietary administration of 0.2% LY295427 or of different concentrations of -sitosterol (0.4%, 0.6%, 0.8%, or 1.0%) for 2 weeks. The group treated with 1.0% -sitosterol had serum cholesterol levels virtually identical to those of the group treated with 0.2% LY295427 (170.0 ± 5.6 versus 171.1 ± 4.4 mg/dl). Cholesterol absorption was subsequently determined in 10 animals from each of these two groups and in 10 control hypercholesterolemic animals (serum cholesterol, 258.4 ± 14.3 mg/dl) (Fig. 8). The animals in the two treated groups showed a decrease of 32% and 34% in serum cholesterol relative to control for the -sitosterol and LY295427 groups, respectively. Treatment with -sitosterol significantly lowered cholesterol absorption (26%), whereas only a nonsignificant trend to effect was observed with LY295427. This finding demonstrates that the hypocholesterolemic effect of LY295427 cannot be due solely to an effect on cholesterol absorption. Although in a previous experiment, a nonsignificant tendency for increased HDL occurred (Fig. 5G), in this experiment, LY295427 significantly increased HDL by 23%, whereas -sitosterol had no effect on HDL (Fig. 8B).

View larger version (55K): [in this window] [in a new window]   Fig. 8.   Comparison of the effect of equivalent cholesterol-lowering doses of -sitosterol and LY295427 on cholesterol absorption. A, cholesterol absorption was measured as detailed in the text using hypercholesterolemic hamsters titrated to the same serum cholesterol level. B, serum HDL cholesterol levels in the animals in A. Results are mean ± S.E.M., 10 animals per group, *p < .05 relative to control, Tukey-Kramer test.

Because some of these results could be a consequence of the inhibition of cholesterol synthesis, we compared the effects of LY295427 on the incorporation of ¹⁴C-labeled acetate into cholesterol in HepG2 cells with those of lovastatin and triparanol, an inhibitor of desmosterol-24-reductase. As anticipated, after 24 h of treatment with lovastatin, there was a clear reduction in the incorporation of label into cholesterol (Fig. 9). A similar reduction was seen with triparanol, accompanied by the appearance of labeled intermediates at the position of desmosterol and close to the position of squalene. In contrast, even at 100 µM (the limit of solubility in this system), LY295427 had no effect on acetate incorporation into cholesterol. Furthermore, there was no evidence of the formation of pools of intermediates.

View larger version (58K): [in this window] [in a new window]   Fig. 9.   Comparison of the effect of lovastatin, triparanol, and LY295427 on sterol biosynthesis. Incorporation of 14C-acetate into cellular lipids was followed in HepG2 cells for 4 h as detailed in the text. Lane 1, control; 2, lovastatin, 10 µM; 3, triparanol, 1 µM; 4, LY295427, 10 µM; 5, LY295427, 100 µM; and 6, position of nonradioactive standards.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The major finding in this study was that LY295427, shown previously to reverse 25-hydroxycholesterol-mediated repression of LDL-R in vitro (Lin et al., 1995), is an effective cholesterol-lowering agent in hamsters. This effect was highly dependent on conditions predisposing to hypercholesterolemia (e.g., dietary fat administration). When LY295427 was administered to hamsters receiving normal laboratory chow diet, there was no discernible effect on serum lipids, which is in sharp contrast to the hypocholesterolemic action of lovastatin under these conditions. These data reinforce the mechanistic distinction between LY295427 and lovastatin, the latter of which inhibits cholesterol biosynthesis as the underlying mechanism for increasing LDL-R (Ma et al., 1986; Cosgrove et al., 1993). Thus, lack of efficacy of LY295427 in chow-fed hamsters is consistent with the finding that LY295427 acts to reverse the repression of LDL-R and would not be expected to be active when LDL-R is not repressed.

When hamsters are fed a diet enriched in both cholesterol and coconut oil, the LDL-R and LDL fractional catabolic rate are both decreased (Spady and Dietschy, 1988; Spady et al., 1993). Using a diet similar to ours, Horton et al. (1993) demonstrated a decrease in LDL-R mRNA to 38% of chow-fed control values. When we examined LY295427 in cholesterol-coconut oil-fed hamsters, there was a significant, dose-dependent lowering of serum cholesterol by LY295427, which is in sharp contrast to the results in chow-fed hamsters. This decrease was confined to the non-HDL lipoproteins, as would be expected for a compound increasing the number of LDL-R, thus increasing the fractional catabolic rate of LDL (Spady and Dietschy, 1988; Himber et al., 1995). In addition, LY295427 was found in the serum and the serum LY295427 inversely correlated with the serum cholesterol levels, strongly suggesting that LY295427 acts metabolically rather than by interfering with the absorption of fat or cholesterol from the diet (see below). Furthermore, the lowered serum cholesterol was accompanied by an increase in liver LDL-R mRNA of the same magnitude as that induced by lovastatin for the same reduction of serum cholesterol. Although LDL-R was not directly measured in these experiments, LDL-R mRNA has been shown to correlate with LDL-R in a variety of dietary manipulations (Horton et al., 1993).

The decreased serum cholesterol observed with LY295427 in the cholesterol-coconut oil-fed hamsters could not be attributed to accumulation in the liver because the increased hepatic cholesterol ester detected with cholesterol-coconut oil feeding was completely reversed with LY295427. This suggests either that LY295427 is interfering with intestinal absorption of the added cholesterol/fat or that it is increasing cholesterol elimination from the animal. In support of the latter possibility, we observed in preliminary studies an increase in the concentration of bile acids in bile from hypercholesterolemic hamsters treated with LY295427 (data not shown).

Concerning possible effects of LY295427 on cholesterol absorption, we used the dual-isotope method (Turley et al., 1994) to quantify this parameter in drug-treated, hypercholesterolemic hamsters. Because LY295427 has a structure related to that of cholesterol and certain compounds structurally similar to cholesterol are known to be poorly absorbed and to interfere with cholesterol absorption (Wilson and Rudel, 1994; Jones et al., 1997), it was indeed possible that LY295427 might interfere with the absorption of cholesterol from the intestinal lumen. Cholesterol absorption was examined in two groups of hypercholesterolemic hamsters in which serum cholesterol had been reduced to the same extent by LY295427 or by -sitosterol. We found that under these conditions, there was a significant decrease in the percentage of dietary cholesterol absorbed when -sitosterol was present but no significant decrease with LY295427. Furthermore, there was an increase in HDL cholesterol with LY295427 in this experiment, but no change was observed with -sitosterol. The difference in Results between -sitosterol and LY295427 suggests that they are acting via different mechanisms and that changes in cholesterol absorption cannot account for the effects of LY295427. Nevertheless, we cannot rule out a possible contribution of cholesterol absorption inhibition to the overall effects of LY295427.

It is possible that LY295427 could also have a direct effect on cholesterol biosynthesis and thus indirectly affect LDL-R (Ma et al., 1986). Although LY295427 did not directly inhibit HMG-CoA reductase (data not shown), it might inhibit other enzymes in the biosynthetic pathway. When this was explored by comparing the effect of LY295427 on the incorporation of labeled acetate into cholesterol in HepG2 cells, there was no evidence of decreased cholesterol production or of accumulation of synthetic intermediates as there was with the enzyme inhibitors lovastatin and triparanol. Consequently, LY295427 does not appear to act by interfering with cholesterol biosynthesis.

Sterols have been shown to regulate the expression of genes containing SRE by protecting the membrane-bound transcription factors, SRE-binding proteins (SREBP-1 and SREBP-2), from proteolysis through the action of the SREBP activation protein (Brown and Goldstein, 1997). In sterol-depleted situations, the 125-kDa SREBP precursor bound to endoplasmic reticulum or nuclear membrane is cleaved in two proteolytic steps into a soluble 68-kDa fragment that moves into the nucleus, where it stimulates transcription of SRE-containing genes, including HMG-CoA reductase, HMG-CoA synthase, and the LDL-R. Sheng et al. (1995) have shown that in livers of hamsters fed cholesterol-rich diets, the amount of SREBP-1c found in the nucleus decreased without any changes in the amount of its membrane-bound precursor or of its mRNA. This was accompanied by a moderate decrease in the amount of LDL-R mRNA and larger reductions in the amount of mRNA for HMG-CoA reductase and for HMG-CoA synthase. Cell mutants with defects in the proteolytic processing of SREBP precursor are auxotropic for cholesterol, failing to synthesize cholesterol and express LDL-R (Sakai et al., 1996). It is possible that LY295427 may act by interfering with the sterol protection of SREBP proteolysis because it was originally identified by the derepression of a 25-hydroxycholesterol repressed expression system. Bowling et al. (1996) have shown that LY295427 can enhance the binding of 25-hyroxycholesterol to a number of cytosolic proteins, although not to the classic oxysterol binding protein. Conceivably, the LY295427-enhanced binding of 25-hydroxycholesterol, or other oxysterols, to proteins not involved in sterol synthesis or regulation could deplete the pool of oxysterol sufficiently to allow increased proteolytic processing and release of active SREBP, followed by increased transcription of LDL-R gene, leading to increased synthesis of LDL-R. Further experiments are required to demonstrate this possibility.

In summary, LY295427 was shown to be an effective cholesterol-lowering agent in hypercholesterolemic hamsters. This effect was accompanied by an increase in liver LDL-R and could not be accounted for by inhibition of the cholesterol biosynthetic pathway or by interference with intestinal cholesterol absorption. These results are in good agreement with previous studies in which LY295427 derepressed LDL-R in cells treated with 25-hydroxycholesterol. Although delineation of the precise in vivo mechanism awaits further investigation, the results of these studies support the hypothesis that cholesterol lowering by LY295427 is mediated by depression of LDL-R.