Inhibition of cholesterol esterification by DuP 128 decreases hepatic apolipoprotein B secretion in vivo: effect of dietary fat and cholesterol

Biochimica et Biophysica Acta 1393 (1998) 63^79

Inhibition of cholesterol esteri¢cation by DuP 128 decreases hepatic apolipoprotein B secretion in vivo: e¡ect of dietary fat and cholesterol John R. Burnett a , Lisa J. Wilcox a , Dawn E. Telford a , Sandra J. Kleinstiver a , P. Hugh R. Barrett b , Murray W. Hu¡ a; * a

The Departments of Medicine and Biochemistry and The John P. Robarts Research Institute, 4^16, University of Western Ontario, 100 Perth Drive, London, Ont. N6A 5K8, Canada b The Departments of Bioengineering and Medicine, University of Washington, Seattle, WA 98915, USA Received 6 February 1998; accepted 28 April 1998

Abstract To further test the hypothesis that newly synthesized cholesteryl esters regulate hepatic apolipoprotein B (apoB) secretion into plasma, apoB kinetic studies were carried out in seven control miniature pigs and in seven animals after 21 days intravenous administration of the acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor DuP 128 (2.2 mg/kg/day). Pigs were fed a fat (34% of calories; polyunsaturated/monounsaturated/saturated ratio, 1:1:1) and cholesterol (400 mg/day; 0.1%; 0.2 mg/kcal) containing pig chow based diet. DuP 128 significantly reduced total plasma triglyceride and very low density lipoprotein (VLDL) triglyceride concentrations by 36 and 31%, respectively (P 6 0.05). Autologous 131 I-VLDL and 125 I-LDL were injected simultaneously into each pig and apoB kinetic data was analyzed using multicompartmental analysis (SAAM II). The VLDL apoB pool size decreased by 26% (0.443 vs. 0.599 mg/kg; P 6 0.001) which was due entirely to a 28% reduction in VLDL apoB production or secretion rate (1.831 vs. 2.548 mg/kg/h; P = 0.006). The fractional catabolic rate (FCR) for VLDL apoB was unchanged. The LDL apoB pool size and production rate were unaffected by DuP 128 treatment. Hepatic microsomal ACAT activity decreased by 51% (0.44 vs. 0.90 nmol/min/mg ; P 6 0.001). Although an increase in hepatic free cholesterol and subsequent decrease in both LDL receptor expression and LDL apoB FCR might be expected, this did not occur. The concentration of hepatic free cholesterol decreased 12% (P = 0.008) and the LDL apoB FCR were unaffected by DuP 128 treatment. In addition, DuP 128 treatment did not alter the concentration of hepatic triglyceride or the activity of diacylglycerol acyltransferase, indicating a lack of effect of DuP 128 on hepatic triglyceride metabolism. In our previous studies, DuP 128 treatment of miniature pigs fed a low fat, cholesterol free diet, decreased VLDL apoB secretion by 65% resulting in a reduction in plasma apoB of 60%. We conclude that in miniature pigs fed a high fat, cholesterol containing diet, the inhibition of hepatic cholesteryl ester synthesis by DuP 128 decreases apoB secretion into plasma, but the effect is attenuated relative to a low fat, cholesterol free diet. ß 1998 Elsevier Science B.V. All rights reserved.

Abbreviations: apo, apolipoprotein; ACAT, acyl coenzyme A:cholesterol acyltransferase; DGAT, diacylglycerol acyltransferase; DNA, deoxyribonucleic acid; ER, endoplasmic reticulum; FCR, fractional catabolic rate; HFC, high fat, cholesterol containing; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; HPGC, high performance gel chromatography; IDL, intermediate density lipoprotein ; LDL, low density lipoprotein; LFCF, low fat, cholesterol free; mRNA, messenger ribonucleic acid; MTP, microsomal triglyceride transfer protein; TRL, triglyceride rich lipoprotein; VLDL, very low density lipoprotein * Corresponding author. Fax: +1 (519) 663-3789; E-mail: mhu¡@julian.uwo.ca 0005-2760 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 5 - 2 7 6 0 ( 9 8 ) 0 0 0 5 9 - 9

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Keywords: Acyl coenzyme A:cholesterol acyltransferase inhibitor; DuP 128; Apolipoprotein B metabolism; Kinetics; Cholesteryl ester; mRNA

1. Introduction An elevated plasma concentration of apolipoprotein B (apoB) containing lipoproteins is an important risk factor for the development of atherosclerosis [1,2]. ApoB kinetic studies in humans have demonstrated that the hepatic overproduction of these lipoproteins is characteristic of many forms of hyperlipidemia including hypertriglyceridemia, combined hyperlipidemia and Type III hyperlipidemia [3^9]. However, the mechanisms involved in the regulation of the assembly and secretion of apoB containing lipoproteins remain incompletely understood. Secretion of apoB into the circulation requires lipoprotein formation; a complex process requiring the coordinated synthesis and assembly of apoB, triglyceride, free and esteri¢ed cholesterol, and phospholipids. This process involves (1) apoB mRNA transcription; (2) protein translation; (3) translocation of apoB across the endoplasmic reticulum (ER) membrane; and either (4) association of apoB with core and surface lipid facilitated by the microsomal triglyceride transfer protein (MTP), transport through the secretory pathway and secretion into plasma; or (5) intracellular degradation [10]. Lipid availability is important in the posttranscriptional regulation of apoB secretion [11,12]. This concept was highlighted by the discovery that MTP was absent from the liver in subjects with abetalipoproteinemia, resulting in a complete inhibition of apoB containing lipoprotein assembly [13,14]. ApoB is synthesized in the rough ER [15] and contains several hydrophobic domains [16] that facilitate association with lipid [17]. MTP is postulated to mediate the transfer of triglyceride, cholesteryl ester and phospholipid to the apoB molecule [18]. Further to its role in mediating delivery of core lipid to apoB, more recent evidence indicates that MTP is capable of facilitating apoB translocation across the ER membrane [19,20]. Nevertheless, failure to associate with lipid results in intracellular apoB degradation [21^26]. It is possible that the transfer rate of triglyceride and cholesteryl ester to apoB may determine whether very low density lipoprotein (VLDL) or low

density lipoprotein (LDL) like particles are secreted [27]. The mechanisms involved in the regulation of the synthesis and secretion of triglyceride rich lipoproteins, by neutral lipid availability, have recently been reviewed [12,27,28]. There is accumulating evidence that triglyceride availability can regulate apoB secretion [29^34]; however, the importance of cholesterol remains controversial [10,35]. Studies in the human hepatoblastoma cell line, HepG2, have shown that modulation of cellular cholesterol and/or cholesteryl ester fail to alter the rate of apoB secretion [10]. Contrasting results, obtained in the same cell line, demonstrated that modulation of 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase or acyl-coenzyme A:cholesterol acyltransferase (ACAT) decreased oleate stimulated apoB secretion [10]. Recent studies have shown that newly synthesized as well as preformed cholesteryl ester can stimulate apoB secretion in HepG2 cells [36]. Data from other in vitro models support the concept of a regulatory role for cellular cholesterol [26,37,38]. Additional evidence that cholesterol is important in regulating the hepatic secretion of apoB containing lipoproteins has come from in vivo apoB kinetic studies in animals and humans in which treatment with HMG-CoA reductase inhibitors decreases their secretion into plasma [10]. The ACAT enzyme appears to be located in the rough ER [39,40], the site of apoB synthesis, whereas triglyceride synthesis occurs mainly in the smooth ER [41,42]. This would suggest that newly synthesized cholesteryl ester may be required for apoB secretion [36,43^46]. In vivo evidence that hepatic apoB secretion is dependent on cholesteryl ester synthesis catalyzed by ACAT has been provided by studies in several animal models [10]. The treatment of both rats and rabbits with ACAT inhibitors lowers the concentrations of cholesterol and apoB in plasma [45^48]. This e¡ect is observed mainly in animals fed diets containing high amounts of fat and cholesterol, making it di¤cult to determine if the plasma cholesterol lowering e¡ect is due to hepatic or intestinal ACAT inhibition [45^48]. Recently,

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Meiner et al. [49], using gene-targeting techniques in mice demonstrated that disruption of the ACAT gene decreased cholesteryl ester concentrations in the adrenals and peritoneal macrophages. However, in these ACAT de¢cient mice, hepatic cholesterol esteri¢cation was not a¡ected, suggesting the possible existence of multiple esteri¢cation enzymes. ApoB kinetic studies, from this laboratory, have demonstrated that the intravenous (i.v.) administration of DuP 128 to miniature pigs fed a low fat, cholesterol free (LFCF) diet decreases the hepatic secretion of VLDL apoB into plasma by 65% [50]. Studies by Carr et al. [51] using perfused monkey livers support this concept. Addition of ACAT inhibitors to the liver perfusate of animals fed a diet containing fat (35% of energy) and cholesterol (0.8 mg/ kcal) decreased hepatic apoB secretion by a mean 29%; an e¡ect highly correlated with cholesteryl ester secretion [51]. Inhibition of intestinal ACAT following oral doses of ACAT inhibitors in fat and cholesterol fed rodent models decreases mucosal cholesteryl esteri¢cation, absorption, chylomicron formation and secretion [45,47,48,52^55]. A decreased delivery of chylomicron remnants would be expected to decrease the hepatic cholesterol and/or cholesteryl ester pool. In order to test directly the hypothesis that hepatic ACAT inhibition decreases apoB secretion in vivo, while minimizing e¡ects secondary to intestinal ACAT inhibition, our previous studies were carried out in pigs fed a low fat (9% of calories), cholesterol free diet [50]. In addition, the non-competitive ACAT inhibitor DuP 128 was given i.v. to maximize hepatic uptake. However, an important question remained to be answered. Would hepatic ACAT inhibition in vivo decrease hepatic apoB secretion in pigs fed diets containing physiologically relevant concentrations of fat and cholesterol? As reviewed by Grundy and Denke [56], a number of animal and human studies have demonstrated that dietary cholesterol and dietary fat and fat saturation can a¡ect fasting plasma lipid and lipoprotein concentrations. In perfused liver studies in African green monkeys, Carr et al. [57] demonstrated that compared to a chow diet, a fat and cholesterol containing diet increased hepatic apoB secretion 1.6^2-fold. In further studies in monkeys, by the same investigators, addition of ACAT inhibitors to the liver perfu-

65

sate resulted in a 21^43% decrease in hepatic apoB secretion [51]. These results suggested that a fat and cholesterol containing diet may attenuate the e¡ect of ACAT inhibition on hepatic apoB secretion. Therefore, the present studies were carried out to determine the e¡ect of i.v. administration of DuP 128 on hepatic VLDL apoB secretion into plasma in miniature pigs fed a high fat (34% of energy) and cholesterol (400 mg/day; 0.1%; 0.2 mg/kcal) containing diet. In order to assess the relative contribution of hepatic vs. intestinal ACAT inhibition on plasma lipid concentrations, the postprandial plasma responses to an oral fat load were also determined. 2. Materials and methods 2.1. Animals and diets Miniature pigs weighing 22.3 þ 0.7 kg were obtained from a local supplier (Hyde Park Farms, Hyde Park, Ont., Canada). After being acclimatized for one week, animals were maintained on the experimental diet for 21 days before, and during the lipoprotein turnover studies. One week prior to the turnover study, an indwelling silicone elastomer (Silastic) catheter (1.96 mm internal diameter) was surgically implanted in an external jugular vein [58]. Iso£urane USP (Abbott Laboratories Ltd.) was used as the anesthetic and ketamine USP (Vetrepharm Canada Inc.) as the preanesthetic. Catheters that were kept patent by ¢lling with 7% EDTANa2 , allowed for ease of sample injection, as well as blood sampling throughout each turnover study in unrestrained, unanesthetized animals. Pigs were studied in pairs, with each pair being same sex littermates. Seven animals received the ACAT inhibitor DuP 128 (DuPont Merck Pharmaceutical Co., Wilmington, DE, USA), at a dose of 50 mg/day, whereas seven control animals received the vehicle alone. The Animal Care Committee of the University of Western Ontario approved the experimental protocol. DuP 128, NP-2,4-di£uorophenyl-N-[5-[(4,5-diphenyl-1H-imidazol-2-yl)thio]pentyl]-N-heptylurea is a non-competitive inhibitor of ACAT [59] whose chemical structure and in vitro IC50 of 2.5 nmol/l for pig liver microsomal ACAT activity have been reported previously [50]. The drug (50 mg) was dis-

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solved in 1.5 ml of 95% ethanol, then 0.5 ml of propylene glycol (British Drug House, BDH), 2 ml of polyethylene glycol 400 (BDH) and 1 ml of sterile water were added and the resulting mixture vortexed. The dose was given i.v. as a bolus into the indwelling catheter. The drug was given at 9 A.M. each day before the daily food ration. Each animal received a 590 g ration of diet (Bronze Hog Grower, ShurGain, St Marys, Ont., Canada) supplemented with lard, butter, and sa¥ower oil (1:0.6:0.2) generating a ¢nal polyunsaturated/monounsaturated/saturated fatty acid ratio of 1:1:1. Cholesterol (Fisher Scienti¢c, Ottawa, Canada) was added to the diet to a ¢nal concentration of 0.1% (0.2 mg/kcal). This diet provided 34% of calories as fat, 49% as carbohydrate, and 17% as protein. 2.2. Lipoprotein turnover studies Lipoprotein turnover studies were performed essentially as described previously [50,60,61]. VLDL (Sf 20^400) and LDL (Sf 0^12), isolated from plasma (100^150 ml) obtained after a 24-h fast were radiolabeled with 131 I and 125 I, respectively. All labeled lipoproteins were autologous. Radiolabeling was performed using the iodine monochloride technique [61]. Of the total VLDL radioactivity, 6 2% was free iodine, 26^38% was bound to lipid and 24^37% of the protein-bound label was bound to apoB. Of the total LDL radioactivity, 6 1% was free iodine, 5^10% was bound to lipid and 80^90% of the protein-bound label was bound to apoB. After a 24-h fast, each animal received 20 WCi of 131 I-VLDL apoB and 15 WCi of 125 I-LDL apoB given as a bolus through the indwelling catheter. The protocols for blood sampling, lipoprotein isolation, apoB precipitation and speci¢c activity determination have been described in detail previously [50]. The plasma concentration of apoB in each lipoprotein fraction was determined by subtracting the protein value of the ¢rst precipitation supernatant from the total protein concentration [62]. 2.3. Kinetic analysis The turnover data were analyzed using the multicompartmental modeling program SAAM II (SAAM Institute, Seattle, WA, USA) running on a Pentium

based personal computer. The model chosen to describe the data was the same as that previously reported by us for miniature pigs [50]. In brief, this model was simultaneously ¢t to the two sets of tracer data for all lipoprotein fractions, therefore permitting the integration of all tracer data into a single model. Data from both lipoprotein tracers supported di¡erent aspects of the model structure. The model structure, the assumptions made in developing the model and the constraints applied to the model were the same as those previously reported [50]. The model allows for the direct removal of apoB from the two largest and most rapidly turning over compartments of VLDL as well as conversion of VLDL to IDL and LDL. A plasma (compartment 10) and extravascular compartment characterize the LDL section of the model and it was assumed that irreversible loss of LDL occurred only from the plasma compartment. Direct input of apoB into the plasma LDL compartment (compartment 10) was required to ¢t the experimental data. For each study, aliquots of plasma (taken at t = 0) were spiked with a small aliquot of the 131 I-VLDL injected dose. Each spiked sample was then processed with the other plasma samples to determine the amount of radioactivity in apoB in the VLDL, IDL and LDL fractions. The mean distribution of 131 I-apoB radioactivity in the spiked samples was 93.3 þ 1.1%, 5.0 þ 1.2% and 0.93 þ 0.3% in the VLDL, IDL and LDL fractions, respectively. On the basis of the distribution of radioactivity in the spiked samples, the initial conditions (the initial amount of radioactivity in each fraction) were incorporated into the compartmental model. This takes into account (1) any contamination due to incomplete separation by ultracentrifugation and/or (2) any alteration in the reinjected VLDL resulting in an increase in density during the in vitro preparation. 2.4. Liver lipids, liver and intestine ACAT and DGAT activities Liver lipids were extracted using the method of Folch et al. [63], from 1.0-g sections of liver obtained at sacri¢ce that had been stored at 380³C. Hepatic free cholesterol, cholesteryl ester and triglyceride were determined as described previously [50]. Liver and intestine ACAT and diacylglycerol acyltransfer-

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ase (DGAT) activities were determined as described previously [50]. In brief, liver and small intestine samples obtained at necropsy were immediately frozen in liquid nitrogen and stored at 380³C until analyzed. Microsomes were isolated from tissue homogenates and ACAT and DGAT activities were determined according to the methods of Billheimer et al. [64] and Rustan et al. [65], respectively. 2.5. RNase protection assay for liver and intestine apoB mRNA abundances Liver and small intestine samples obtained at necropsy were immediately frozen in liquid nitrogen and stored at 380³C until analysis. Total RNA was isolated using Trizol reagent (Gibco BRL, Gaithersburg, MD, USA). The integrity of the RNA isolated was veri¢ed after agarose gel electrophoresis (1.2%; 2.2 mol/l formaldehyde) by the appearance of the 18S and 28S RNA bands. RNA content was determined by measuring the absorbance at 260 nm. Pig speci¢c cDNA for apoB, cloned into Bluescript plasmid (kindly provided by Dr. Alan D. Attie, University of Wisconsin-Madison, Madison, WI, USA), served as a template to synthesize an antisense RNA probe. This riboprobe was then used to measure apoB mRNA in a modi¢cation of the RNase protection solution hybridization assay of Azrolan and Breslow [66]. This assay uses standard RNA that allows precise quantitation of speci¢c gene transcripts. In brief, a [32 P] RNA probe was synthesized using an in vitro transcription system (Promega, Madison, WI, USA) as per the manufacturer's instructions. Unlabeled cRNA corresponding to the sense DNA strand was prepared for use as a hybridization standard. Riboprobe (150 pg; 2^3U108 cpm/ Wg) and either total or standard cRNA (10^150 pg) were hybridized overnight at 63³C in 40 Wl of hybridization bu¡er (80% formamide, 40 mmol/l HEPES, pH 6.7, 0.4 mol/l NaCl, 1 mmol/l EDTA) with 10 Wg of yeast tRNA. Three hundred Wl of digestion bu¡er (0.3 mol/l NaCl, 10 mmol/l Tris-HCl, pH 7.4, 5 mmol/l EDTA) containing RNase A and RNase T1 (Boehringer Mannheim, Mannheim, Germany) were added to each sample and incubated at 34³C for 1 h. After incubation, 500 Wl of 20% cold trichloroacetic acid and 100 Wg of herring sperm DNA were added to each sample. Samples were

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placed on ice for 15 min then ¢ltered using glass ¢ber ¢lters. Filters were washed with 7% trichloroacetic acid, dried, and 8 ml of scintillation £uid added before counting. mRNA abundance was quantitated by reference to the curve generated using the transcribed hybridization standard. The sample and standard RNA were assayed in triplicate and the hybridization was linear to 120 pg of cRNA. Samples were analyzed in a single batch and within batch coe¤cients of variations of 6 10% were achieved. 2.6. Oral fat tolerance test After a 24-h fast, pigs were fed the high fat, cholesterol containing (HFC) diet described above, in an amount calculated to provide 2 g of fat/kg body weight. This test meal was supplemented with 50 000 IU of retinol (Vitamin A capsules USP, Novopharm Ltd., Toronto, Ont., Canada) and consumed within 10 min. The animals were not fed for the 8 h of study, but had free access to drinking water. Venous blood samples (20 ml) were drawn at 0 (before the test meal), 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, and 8 h and collected into tubes containing EDTA-Na2 . Samples were kept on ice prior to the isolation of plasma lipoproteins and protected from light during processing. Plasma was obtained by centrifugation at 1000Ug for 25 min at 4³C. The isolated plasma underwent preparative ultracentrifugation at d = 1.006 g/ml in a Beckman 50.4 Ti rotor at 35 500 rpm at 12³C for 16 h. Triglyceride rich protein (TRL) fractions (d 6 1.006 g/ml; Sf s 20) were isolated by tube slicing, and each plasma and TRL fraction was analyzed for triglyceride, cholesterol and retinyl ester concentrations. 2.7. Retinyl ester analysis Retinyl ester (retinyl palmitate and retinyl stearate) concentrations were determined in total plasma and in the TRL fraction by a modi¢cation of the high performance liquid chromatography (HPLC) method of Weintraub et al. [67]. Extractions and analyses were carried out with HPLC grade solvents and under subdued light. Retinyl acetate was added to the samples as an internal standard and retinyl esters extracted with a mixture of ethanol/hexane/water (1:5:0.5). The hexane layer was evaporated under

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Table 1 Plasma lipid and lipoprotein concentrations in control and DuP 128 treated miniature pigs Triglyceridea (mmol/l)

Cholesterol (mmol/l)

ApoBb (mg/l)

Total

VLDL

Total

VLDLc

LDLd

HDLe

VLDLf

Control 1 2 3 4 5 6 7 Mean S.E.M.

0.35 0.87 0.29 1.09 0.40 0.57 0.26 0.58 0.11

0.18 0.33 0.16 0.38 0.22 0.48 0.19 0.30 0.04

3.717 4.316 4.098 2.862 2.952 2.945 2.666 3.365 0.251

0.036 0.050 0.060 0.095 0.068 0.088 0.042 0.063 0.008

2.260 2.769 2.474 1.212 1.243 1.528 1.555 1.863 0.237

1.421 1.497 1.564 1.555 1.641 1.329 1.069 1.439 0.073

13.9 14.5 11.5 13.5 10.7 18.2 17.6 14.3 1.1

227.2 229.2 184.3 250.9 163.9 191.2 203.5 207.2 11.4

DuP 128 1 2 3 4 5 6 7 Mean S.E.M. P

0.18 0.74 0.17 0.69 0.21 0.36 0.26 0.37 0.09 0.008

0.10 0.37 0.09 0.26 0.13 0.34 0.18 0.21 0.04 0.037

3.019 2.855 2.465 2.641 3.723 2.590 2.965 2.894 0.158 NS

0.033 0.063 0.037 0.070 0.042 0.021 0.030 0.042 0.007 NS

1.711 1.400 1.530 1.275 2.091 1.899 1.522 1.633 0.108 NS

1.275 1.392 0.898 1.296 1.590 0.670 1.413 1.219 0.121 NS

9.6 11.9 8.5 9.6 7.7 13.7 12.8 10.5 0.9 6 0.001

182.9 199.4 210.0 178.1 199.5 184.1 231.4 197.9 7.0 NS

LDL

a

Each lipid value is a mean of three determinations from each animal. ApoB indicates apolipoprotein B; VLDL, very low density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein. c VLDL cholesterol was determined after ultracentrifugation at d 6 1.006 g/ml. d LDL cholesterol was calculated as total cholesterol minus the sum of VLDL cholesterol and HDL cholesterol. e HDL cholesterol was determined after precipitation of the apoB containing lipoproteins from plasma. f VLDL (d 6 1.006 g/ml) and LDL (d = 1.019^1.063 g/ml) apoB are the mean of all samples obtained during the kinetic study in the respective lipoprotein fractions separated by ultracentrifugation. b

nitrogen. The samples were redissolved in ethanol and separated on a 5-Wm Hypersil C18 (7.5U0.32 cm) column. Pump A consisted of 88% methanol in water, whereas pump B consisted of 75% methanol and 25% isopropanol. 100% pump A was used for the ¢rst 4 min, reducing to 20% pump A to 9 min, and further reducing to 0% pump A to 12 min, at a £ow rate of 0.6 ml/min. The absorbance of the eluent was measured at 325 nm and the retinyl ester concentrations quantitated by ratio of peak height to that of the internal standard. Areas under the retinyl ester curves were calculated.

mined by the high performance gel chromatographic (HPGC) method of Kieft et al. [68]. In brief, lipoprotein separations were performed on a Superose 6HR FPLC column, determined primarily by the size of the lipoproteins. After HPGC, on-line lipoprotein cholesterol distribution was determined. Lipoprotein cholesterol was determined from independent total cholesterol measurements and the percent area distribution of cholesterol fractions by HPGC. Peak retention times were used to estimate and compare the relative sizes of the major lipoprotein particles.

2.8. Plasma cholesterol distribution

2.9. Analyses

Plasma cholesterol lipoprotein pro¢les were deter-

Total cholesterol and triglyceride, VLDL choles-

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Table 2 Metabolic parameters of VLDL apoB metabolism in control and DuP 128 treated miniature pigs VLDLa pool sizeb (mg/kg) Control 1 2 3 4 5 6 7 Mean S.E.M. DuP 128 1 2 3 4 5 6 7 Mean S.E.M. P

VLDL FCRc (h31 )

VLDL total production (mg/kg/h)

0.584 0.609 0.483 0.565 0.449 0.764 0.739 0.599 0.045

4.161 3.847 5.361 4.194 4.260 4.233 3.996 4.293 0.186

2.429 2.343 2.590 2.369 1.914 3.236 2.954 2.548 0.164

0.087 0.097 0.146 0.129 0.335 0.103 0.177 0.153 0.033

4 4 6 6 18 3 6 7 2

2.077 2.245 2.443 2.239 1.579 3.133 2.776 2.356 0.189

86 96 94 95 83 97 94 92 2

0.403 0.500 0.357 0.403 0.323 0.575 0.538 0.443 0.036 6 0.001

3.272 5.163 5.287 3.643 3.831 3.626 4.163 4.141 0.298 NS

1.319 2.580 1.888 1.469 1.239 2.086 2.238 1.831 0.191 0.006

0.164 0.084 0.403 0.288 0.120 0.258 0.170 0.212 0.042 NS

12 3 21 20 10 12 8 12 2 NS

1.154 2.469 1.484 1.181 1.112 1.828 2.068 1.614 0.198 0.008

88 96 79 80 90 88 92 87 2 NS

VLDL conversion to LDL VLDL direct removal (mg/kg/h)

(%)

(mg/kg/h)

(%)

a VLDL indicates very low density lipoprotein; apoB, apolipoprotein B; FCR, fractional catabolic rate; LDL, low density lipoprotein; and IDL, intermediate density lipoprotein. b Pool size refers to the plasma VLDL apoB concentration multiplied by 0.042, making the assumption that in the pig there are 42 ml plasma per kilogram body weight. c FCR is determined by U(5)/VLDL apoB pool size, where U(5) is the production rate of VLDL apoB [50].

terol and triglyceride, and HDL concentrations in the plasma were determined. VLDL was obtained after ultracentrifugation at d 6 1.006 g/ml, and HDL was obtained after precipitation of other lipoproteins by dextran sulfate-magnesium chloride. LDL was calculated by di¡erence. Total cholesterol, triglyceride, free cholesterol, and phospholipid were determined by enzymatic, colorimetric assays using reagents obtained from Boehringer Mannheim GmbH, Germany. Dietary fatty acid composition was determined by gas chromatography with a 2-m column (SP 2230, liquid phase; Supelco, Toronto, Canada) on a Varian 6000 gas chromatograph. Tests for statistical signi¢cance of di¡erences in lipid, apoprotein concentrations and the kinetic parameters during each treatment phase were compared by paired t-test [69]. Comparison of the e¡ect of DuP 128 on metabolic parameters in pigs fed an LFCF

diet [50] to those obtained in the present study were analyzed by unpaired t-test. A P value 6 0.05 was considered statistically signi¢cant. 3. Results The e¡ect of the ACAT inhibitor DuP 128 on plasma and lipoprotein lipid concentrations in pigs fed a HFC diet are shown in Table 1. Total plasma and VLDL triglyceride concentrations were signi¢cantly reduced by 36% (P = 0.008) and 31% (P = 0.037), respectively. VLDL apoB concentrations were reduced 26% (P 6 0.001). The ACAT inhibitor did not signi¢cantly a¡ect total cholesterol, VLDL cholesterol, LDL cholesterol, HDL cholesterol, or LDL apoB concentrations. Each control and DuP 128 treated pig was simul-

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Table 3 Metabolic parameters of LDL apoB metabolism in control and DuP 128 treated miniature pigs LDLa pool sizeb (mg/kg)

LDL FCRc (h31 )

LDL total production (mg/kg/h)

LDL direct productiond

Control 1 2 3 4 5 6 7 Mean S.E.M.

9.54 9.63 7.74 10.54 6.88 8.03 8.55 8.70 0.48

0.042 0.031 0.038 0.039 0.043 0.060 0.049 0.043 0.003

0.404 0.303 0.291 0.415 0.297 0.480 0.421 0.373 0.028

0.317 0.205 0.146 0.286 0.000 0.377 0.244 0.244 0.047

78 68 50 69 0 79 58 57 10

2.746 2.548 2.735 2.655 1.914 3.613 2.773 2.773 0.201

DuP 128 1 2 3 4 5 6 7 Mean S.E.M. P

7.68 8.38 8.82 7.48 8.38 7.73 9.72 8.31 0.30 NS

0.038 0.037 0.063 0.039 0.045 0.053 0.036 0.045 0.010 NS

0.295 0.312 0.554 0.292 0.379 0.413 0.346 0.370 0.035 NS

0.131 0.228 0.151 0.004 0.259 0.154 0.176 0.158 0.031 NS

44 73 27 2 68 37 51 43 9 NS

1.450 2.809 2.038 1.473 1.498 2.241 2.414 1.989 0.202 0.011

(mg/kg/h)

(%)

Total ApoB productione (mg/kg/h)

a

LDL indicates low density lipoprotein; apoB, apolipoprotein B; and FCR, fractional catabolic rate. Pool size refers to the plasma LDL apoB concentration multiplied by 0.042, making the assumption that in the pig there are 42 ml plasma per kilogram body weight. c FCR is the rate constant k(0,10) determined from the model [50]. d Direct production (direct synthesis) is the production of apoB directly into compartment 10, U(10). e Production of apoB into the plasma compartment calculated as VLDL apoB production plus LDL apoB direct production. b

taneously injected with autologous 131 I-VLDL and 125 I-LDL. The kinetic parameters of apoB were determined from the analysis of all speci¢c activity data using the model described previously [50]. The kinetic parameters are summarized in Table 2,3. The i.v. administration of DuP 128 signi¢cantly reduced the pool size of VLDL apoB by 26% (P 6 0.001) as shown in Table 2. This was due, primarily, to a signi¢cant 28% reduction in VLDL apoB production rate (P = 0.006), as the fractional catabolic rate (FCR) was una¡ected. The amount of VLDL apoB converted to LDL apoB was increased by 38%. Although not statistically signi¢cant, conversion was increased in four of seven animals studied. The decreased total production rate of VLDL apoB was associated with a 32% (P = 0.008) decrease in the amount of VLDL apoB leaving the circulation directly, without conversion to IDL or

LDL apoB. Neither the percent of VLDL apoB £ux converted to LDL nor the percent removed directly was a¡ected by DuP 128 treatment. The FCRs for VLDL apoB direct catabolism [k(0,1), k(0,2) and k(0,3)] and for IDL direct catabolism [(k(0,4) and k(0,5)] were not signi¢cantly affected by DuP 128 treatment (data not shown). The model allowed us to determine the production or £ux of VLDL apoB that was converted to LDL without being transported through the plasma IDL pool, as well as the amount of VLDL apoB converted to LDL via the IDL fraction. VLDL apoB production converted to LDL directly was unchanged, whereas the VLDL apoB converted to LDL via IDL was increased by 57% (P = NS) with DuP 128 treatment (data not shown). DuP 128 had no e¡ect on the LDL apoB pool size or on LDL apoB production rate. Although the

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Fig. 1. Cholesterol distribution pro¢les for one representative pair of pigs as determined by high performance gel chromatography [68]. The elution peaks for VLDL, LDL and HDL are indicated. The mean þ S.E.M. retention times for the seven control pigs were: VLDL, 12.56 þ 0.01; LDL, 16.34 þ 0.09; and HDL, 21.99 þ 0.04 min. Values for the seven DuP 128 treated animals were: VLDL, 12.56 þ 0.01; LDL, 16.50 þ 0.09 (P = 0.016); and HDL, 22.04 þ 0.05 min.

amount of apoB synthesized directly was decreased by 30% (P = NS), and the percent of total LDL apoB production as direct synthesis was reduced by 24% (P = NS), this was o¡set by a 38% increase in conversion of VLDL apoB to LDL apoB (P = NS). Total apoB production into plasma, calculated as the sum of VLDL apoB production plus LDL apoB direct production decreased signi¢cantly by 28% (P = 0.011). DuP 128 treatment had no e¡ect on LDL apoB FCR. From plasma samples obtained during the kinetic study, VLDL (d 6 1.006 g/ml) was analyzed for lipids and protein. As shown in Table 4, the percent composition did not change for any of the parameters measured. The surface/core lipid ratio increased with treatment, suggesting a tendency to smaller particles, however, this increase was not signi¢cant. Plasma cholesterol distribution among plasma lipoprotein classes, as assessed by HPGC, showed no major changes (Fig. 1). The percent of total cholesterol in the LDL fraction increased 11% (P = 0.055) with DuP 128 treatment. Although the size distribution of the major lipoprotein classes, as assessed by HPGC, showed no major changes, a small but signi¢cant increase in the peak LDL retention time was observed, suggesting a smaller particle was produced in the DuP 128 treated animals. In three control pigs and three animals treated with DuP 128, an oral fat tolerance test was carried out to determine if the i.v. administration of DuP 128 inhibited intestinal ACAT, such that postprandial lipid and retinyl ester pro¢les were a¡ected. Fig.

71

2 shows changes in TRL triglyceride and TRL retinyl esters with time after a fat meal. In both groups, TRL triglyceride reached peak plasma concentrations at 2 h after ingestion of the fat meal and declined thereafter up to 8 h. Peak plasma TRL concentrations of retinyl esters also occurred at 2 h and declined thereafter. There were no apparent di¡erences in the pro¢les between the control and DuP 128 treated pigs for TRL triglyceride and TRL retinyl esters (Fig. 2) or plasma triglyceride and plasma retinyl esters (data not shown). No signi¢cant changes from baseline for plasma and TRL cholesterol concentrations occurred in either group. DuP 128 treatment did not signi¢cantly a¡ect the areas under the concentration vs. time curves for plasma and TRL triglyceride and retinyl esters. This suggests that the decrease in hepatic VLDL apoB secretion was directly due to hepatic ACAT inhibition and was not secondary to intestinal ACAT inhibition. The pigs were killed 24 h after the last i.v. dose of DuP 128 was administered. Segments of liver and small intestine were removed and frozen at 380³C until analyzed for cholesterol and triglyceride content and ACAT and DGAT activities. The results shown in Table 5 indicate that the concentration of liver free cholesterol was reduced by 12% (P = 0.008) by treatment with DuP 128. Neither hepatic cholesteryl ester, which represented approximately 12% of the total liver cholesterol, nor triglyceride content were Table 4 Percent composition of VLDL isolated from control and DuP 128 treated miniature pigs VLDLa;b Triglyceride Free cholesterol Cholesteryl ester Phospholipid Protein TG/CEc Surface/cored a

Control

DuP 128

64.8 3.7 6.1 12.2 13.2 12.5 0.23

63.1 4.1 6.9 13.3 12.6 11.4 0.26

VLDL indicates very low density lipoprotein. Values are percent of total lipoprotein and are means of determinations on VLDL from seven control and seven DuP 128 treated animals. c Ratios are weight ratios. d Surface/core is the ratio of phospholipid+free cholesterol/triglyceride (TG)+cholesterol ester (CE). b

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Fig. 2. Line graphs showing triglyceride rich lipoprotein (TRL) triglyceride (A) and TRL retinyl ester concentrations (B) after an oral fat load (2 g fat/kg body weight; 50 000 IU retinol). Data points represent the mean of determinations from three control and three DuP 128 treated pigs. (b) Indicates DuP 128; (a) control. TRL triglyceride and retinyl ester concentrations were determined as described in Section 2.

altered by DuP 128. The ACAT activity measured in hepatic microsomes was inhibited 51% by DuP 128 treatment. Values for DuP 128 treated animals were signi¢cantly lower (0.44 þ 0.05 vs. 0.90 þ 0.05 nmol of cholesterol ester produced/min/mg; P 6 0.001). In some samples, ACAT activity was also measured in whole liver homogenates, as well as in microsomes that had been supplemented with exogenous cholesterol in order to optimize assay conditions. The same relative inhibition of activity by DuP 128 was observed (data not shown). ACAT activity was also measured in microsomes prepared from segments of small intestine, however, no statistically signi¢cant di¡erences were observed. Hepatic microsomal DGAT activity was not a¡ected by DuP 128 treatment (data not shown). The mRNA abundance for liver and intestine apoB was analyzed using a sensitive RNase protec-

tion solution hybridization assay. Hepatic mRNA abundance in the DuP 128 treated animals was not di¡erent from that of control animals (117 þ 8 vs. 111 þ 8 pg mRNA/Wg of total RNA, P = NS). Similarly, DuP 128 had no e¡ect on intestinal apoB mRNA abundance (84 þ 7 vs. 80 þ 6 pg mRNA/Wg total RNA, P = NS). The present experiments were carried out in pigs fed a HFC diet, whereas our previous studies were carried out in pigs fed an LFCF diet [50]. A comparison of the e¡ect of hepatic ACAT inhibition under the two dietary conditions is shown in Fig. 3A. Compared to the LFCF diet, the HFC diet increased plasma and VLDL triglyceride concentrations by 88% (P = 0.053) and 33% (P = NS), respectively. DuP 128 treatment decreased plasma triglyceride concentrations by a similar percentage (30^35%) in each dietary group. However, VLDL triglyceride concentrations decreased by 31% and 45% on the HFC and LFCF diets, respectively, with DuP 128 treatment. Compared to the LFCF diet, the HFC diet increased total and LDL cholesterol 45% (P = 0.004) and 70% (P = 0.014), respectively. The HFC diet increased the LDL cholesterol/LDL apoB ratio by 35% compared with the LFCF diet. DuP 128 treatment decreased LDL cholesterol 26% in the LFCF group, whereas the DuP 128 induced decrease was only 10% in the HFC group. The 26% reduction in VLDL apoB pool size in the HFC group treated with DuP 128 was less than the 60% reduction in the LFCF group (Fig. 3B). DuP 128 treatment had no e¡ect on either VLDL apoB Table 5 Liver cholesterol, cholesteryl ester, and triglyceride concentrations in control and DuP 128 treated miniature pigs Liver lipidsa;b (mg/g wet weight) c

Free cholesterol Cholesteryl ester Triglyceride a

Control

DuP 128

P

2.50 þ 0.17 0.35 þ 0.04 2.92 þ 0.44

2.19 þ 0.19 0.40 þ 0.04 2.84 þ 0.42

0.008 NS NS

Values are the mean þ S.E.M. of determinations control and seven DuP 128 treated animals. b Livers were removed approximately 24 h after the DuP 128. Sections from several lobes of liver were frozen at 380³C until analysis. c Liver cholesterol and triglyceride concentrations mined as described in Section 2.

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from seven last dose of excised and were deter-

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amount of VLDL apoB converted to LDL apoB was reduced 20% (P = NS) by the HFC diet compared to LFCF diet. In contrast, DuP 128 treatment increased the conversion of VLDL apoB to LDL apoB by 106% in the HFC group compared with the LFCF group (P = 0.058). The e¡ect of DuP 128 treatment on direct removal was similar to its e¡ect on VLDL apoB total production. The DuP 128 induced reduction in direct removal was attenuated in the HFC group resulting in a 32% reduction compared to a 65% reduction in the LFCF group. LDL apoB pool size was unaltered in the HFC group treated with DuP 128, whereas a 19% reduction was found in the LFCF group (P = 0.010). DuP 128 treatment had no e¡ect on either LDL apoB FCR, total LDL apoB production rate or direct LDL apoB production rate in either dietary group. Fig. 3C shows the e¡ect of the HFC diet on total liver lipid concentrations. The HFC diet increased cholesteryl ester, free cholesterol and triglyceride by 97% (P = 0.009), 42% (P = 0.008) and 90% (P = 0.03), respectively. DuP 128 treatment had no signi¢cant e¡ect on hepatic lipids within each dietary group. 4. Discussion

Fig. 3. Comparison of the e¡ects of the ACAT inhibitor, DuP 128 on: A: plasma and lipoprotein lipids; B: kinetic parameters of VLDL apoB metabolism; and C: hepatic lipids, in pigs fed a low fat, cholesterol free (LFCF) diet to that of a high fat, cholesterol containing (HFC) diet. TG indicates triglyceride ; VLDL, very low density lipoprotein; C, cholesterol; FCR, fractional catabolic rate; LDL, low density lipoprotein; Cont, control animals; DuP, DuP 128 treated animals. Values represent the mean þ S.E.M. for six control and six DuP 128 treated pigs fed the LFCF diet, and seven control and seven DuP 128 treated animals fed the HFC diet. Pool size units, mg/kg; FCR, h31 ; other kinetic parameters, mg/kg/h. a: Indicates signi¢cantly di¡erent from LFCF control animals; b: signi¢cantly di¡erent from LFCF DuP 128 treated animals. Data for the LFCF diet have been reported previously [50].

FCR in either dietary group. The attenuated a¡ect of DuP 128 on VLDL apoB concentrations in the HFC group was due to a smaller percent reduction in the VLDL apoB production rate (28% vs. 65%). The

The experiments described in this paper were designed to test the hypothesis that, in vivo, hepatic synthesis of cholesteryl ester by ACAT contributes to the regulation of secretion of apoB containing lipoproteins. Speci¢cally, we wanted to determine if hepatic ACAT inhibition would decrease apoB secretion and therefore modulate plasma lipids in pigs fed diets containing physiologically relevant concentrations of fat and cholesterol. In vivo apoB kinetic studies revealed that the i.v. administration of the ACAT inhibitor, DuP 128, inhibited VLDL apoB secretion into plasma by 28% resulting in a 26% decrease in VLDL apoB concentrations. VLDL apoB FCRs were unchanged. Conversion of VLDL apoB to LDL apoB was increased by 38% and VLDL apoB direct removal was signi¢cantly decreased by 32%. DuP 128 had no e¡ect on plasma cholesterol, LDL cholesterol, LDL apoB concentrations, or on any kinetic parameter of LDL apoB metabolism. In addition, these experiments showed that the e¡ect of DuP 128 on VLDL apoB secretion was almost en-

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tirely due to hepatic ACAT inhibition. Postprandial lipid metabolism, as assessed by the changes in plasma triglyceride, cholesterol, and retinyl esters following an oral fat challenge, was una¡ected by i.v. administration of the ACAT inhibitor. Taken together with our previous apoB kinetic studies in pigs fed LFCF diets [50], this study further supports the concept that hepatic assembly and secretion of apoB containing lipoproteins is dependent on the ACAT catalyzed synthesis of cholesteryl esters. Inhibition of hepatic ACAT, in vivo, reduces the number of apoB containing lipoproteins secreted, suggesting a coordinate regulation of cholesterol esteri¢cation and apoB secretion. The concept that newly synthesized cholesteryl ester participates in the regulation of hepatic apoB secretion, in vivo, is consistent with in vitro studies in HepG2 cells [12,36,37,43,44,70], other cultured primary hepatocytes [26,37,38], and in vivo studies in small animal models in which ACAT inhibitors decrease plasma cholesterol concentrations [45,46]. Direct evidence demonstrating a role for cholesteryl esters has been provided by studies of Carr et al. [51] in perfused monkey livers demonstrating that three ACAT inhibitors, CI-976, CP-113818 and PD138142-15, decrease hepatic cholesteryl ester and apoB secretion into the perfusate. The e¡ect of ACAT inhibitors on atherogenesis may be indirect and involve inhibition of lipoprotein assembly and secretion from both the intestine and liver, as well as preventing foam cell formation secondary to the direct inhibition of cholesterol esteri¢cation in cells of the arterial wall [45,46,48, 52,54,64,71,72]. Much of the focus of the e¡ect of ACAT inhibitors in regulating plasma lipids has been in rodent models fed diets high in fat and cholesterol. The mechanism for plasma cholesterol lowering has been ascribed to an inhibition of cholesterol absorption, secondary to enterocyte ACAT inhibition, since most ACAT inhibitors have low systemic availability [45,46,48,52,54,72]. In human subjects, oral administration of the ACAT inhibitor DuP 128 had only small e¡ects on cholesterol absorption and plasma cholesterol concentrations [73]. In the present experiments, pigs were fed diets containing 34% of energy as fat and 0.2 mg/kcal (0.1%) of cholesterol. The DuP 128 was given i.v. in order to, ¢rstly, maximize hepatic uptake and secondly,

minimize the e¡ect on the intestine. Using this protocol, intestinal ACAT activity was una¡ected, and there was no e¡ect on postprandial triglyceride or retinyl ester metabolism after an oral fat load (Fig. 2). This allowed us to conclude that the decrease in hepatic VLDL apoB secretion was due to hepatic ACAT inhibition and not secondary to an intestinal e¡ect. These results, in this large animal model, suggest that if systemically available ACAT inhibitors decreased both hepatic and intestinal cholesterol esteri¢cation, hepatic ACAT inhibition would still make a signi¢cant contribution to plasma lipid modi¢cation. This is consistent with ¢ndings in rats that changes in plasma lipids by the ACAT inhibitor CI-976 were related to both hepatic and intestinal ACAT inhibition [45]. The reduction in hepatic apoB secretion in the present study is less than that observed in our previous study using a LFCF diet (28% vs. 65%). The reason for this is not clear, but may be related to the extent of hepatic ACAT inhibition. Hepatic ACAT activity was inhibited by 51% with the HFC diet, whereas in pigs fed the LFCF diet, a 68% inhibition was observed. In the perfused liver studies of Carr et al. [51], monkeys were fed diets containing 35^42% of energy as fat and 8 mg/kcal of cholesterol. The maximal inhibition of hepatic ACAT achieved was 35%, which resulted in a mean decrease in apoB secretion of 29%; values similar to those we observed, in vivo, in the present study. These ¢ndings are consistent with the concept that a minimal level of hepatic cholesterol ester synthesis is required to support apoB secretion. We speculate that the ACAT inhibitor, DuP 128, reduced hepatic cholesteryl ester synthesis below the threshold level required for regulation of apoB secretion. Comparison of pigs fed the LFCF diet and the HFC diet indicates that DuP 128 reduced the hepatic ACAT activity below this threshold and that the greater the inhibition, the greater the reduction in apoB secretion. The mechanism whereby newly synthesized cholesteryl ester regulates apoB secretion is not known. ApoB mRNA abundance was not a¡ected by the ACAT inhibitor indicating a lack of regulation at the level of apoB transcription. Studies in HepG2 cells indicate that apoB becomes cotranslationally associated with the rough ER [15,74]. Since ACAT is located in the rough ER [39,40], some newly syn-

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thesized core cholesteryl ester and triglyceride may become associated with apoB [15,74], facilitated by MTP [13,18,75]. Subsequently, most of the primary neutral lipid core, mainly triglyceride, is added after movement of the particle to the smooth ER, the main site of triglyceride synthesis [15,74,76]. Nascent lipoproteins, released into the ER lumen, are then transported through the secretory pathway. Therefore, a regulatory role for ACAT derived cholesteryl esters may be at the initial stages of particle formation. Failure of apoB to associate with su¤cient cholesteryl ester may target apoB for intracellular degradation, as suggested by studies in primary rabbit hepatocytes [26]. The decrease in hepatic VLDL apoB secretion, in vivo, observed in the present studies is unlikely to be due to a direct inhibition of triglyceride or phospholipid biosynthesis. Studies have shown that triglyceride [29^34] and phospholipid synthesis [77] are required for hepatic apoB secretion. However, previous studies have shown that DuP 128 has no e¡ect on either triglyceride or phospholipid biosynthesis [50]. The reduction in VLDL apoB production did not translate into a reduction in either the conversion of VLDL apoB to LDL, or the LDL apoB pool size. Both the conversion of VLDL apoB to LDL apoB and percent of VLDL £ux converted to LDL were increased. This did not appear to be due to VLDL particle composition or size distribution as shown in Table 4 and Fig. 1. A decrease in hepatic LDL receptor expression is associated with increased conversion of VLDL to LDL [78]. However, DuP 128 treatment did not alter either the LDL apoB FCR (largely determined by LDL receptor expression) or the VLDL apoB FCR. It is possible that the activities of lipoprotein lipase and/or hepatic lipase, both of which regulate VLDL conversion [60], were increased by DuP 128; however, the activities of these enzymes were not measured in the present study. The lack of an e¡ect of DuP 128 on LDL apoB concentrations was not due to a concomitant decrease in LDL clearance. Theoretically, the inhibition of ACAT would result in an increase in hepatic free cholesterol in a regulatory pool such that hepatic LDL receptor expression would be decreased. As in our previous studies [50], DuP 128 had no e¡ect on LDL apoB FCR, and the hepatic free cholesterol

75

concentration decreased with DuP 128 treatment rather than increased. This is consistent with the idea that the ACAT substrate pool is not tightly coupled to the free cholesterol pool that regulates LDL receptor expression [79^81]. Hepatic ACAT inhibition did not result in a decrease in liver cholesteryl ester. As discussed previously [50], newly synthesized cholesteryl esters destined for association with apoB and subsequent secretion do not appear to be tightly coupled to the cytoplasmic storage pool, unless the £ux of liver cholesterol is high. Although liver cholesteryl ester was elevated two-fold in the present study, compared to pigs fed the LFCF diet (Fig. 3C), the moderate level of dietary cholesterol and extent of ACAT inhibition (51%) may have been insu¤cient to be re£ected in a decrease in the total hepatic cholesteryl ester concentration. Compared to the LFCF diet used in our previous studies, the HFC diet resulted in increased plasma total and VLDL triglyceride concentrations, despite a reduced secretion rate of VLDL apoB into plasma and decreased plasma VLDL apoB concentrations. Therefore, more triglyceride per particle (30^40%) was secreted from the liver of the pigs fed the HFC diet. The reason for this response is not understood; however, high carbohydrate diets have been shown to increase VLDL triglyceride and VLDL apoB concentrations [82^84]. In some studies VLDL apoB secretion increased [84], but not in others [85]. Hepatic triglyceride content was higher in pigs fed the HFC diet, which might be expected to increase rather than decrease hepatic VLDL apoB secretion. The greater hepatic secretion of VLDL apoB in the LFCF fed animals was unrelated to hepatic cholesteryl ester concentration or hepatic ACAT activity as suggested by studies in monkeys [51]. Compared to pigs fed the HFC diet, the cholesteryl ester content was lower and ACAT activity was unchanged. Nevertheless, DuP 128 decreased hepatic VLDL apoB secretion in both groups of pigs, although as discussed above, the a¡ect in the HFC group was attenuated. Studies in other animal models have shown that increased dietary fat and cholesterol reduce LDL receptor expression [86,87]; however, the LDL apoB FCR in our studies was not altered by the HFC diet. Compared to the LFCF diet, the HFC diet signi¢cantly increased plasma LDL apoB concentra-

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tions by 26%; however, total apoB production was not signi¢cantly changed. The signi¢cant increase in LDL cholesterol by 70% in the HFC group re£ected an increased LDL cholesterol/apoB ratio of 35%. There was a 20% decrease in the amount of VLDL apoB £ux converted to LDL and a 52% increase in the amount of LDL synthesized directly; however, neither were statistically signi¢cant. In summary, this paper provides further in vivo kinetic evidence that hepatic ACAT plays a regulatory role in apoB secretion into plasma in the miniature pig. Speci¢cally, inhibition of hepatic ACAT activity decreases apoB secretion in pigs fed physiologically relevant fat and cholesterol containing diets, although the e¡ect is attenuated relative to a low fat, cholesterol free diet. The mechanism for this attenuated e¡ect will require further investigation. Acknowledgements

[3]

[4]

[5]

[6]

[7]

We thank Belinda Fireman and Debra Cromley for their expert technical assistance and Kim Wood for performing the surgeries. We are grateful to Dr. Peter J. Gillies, DuPont-Merck Pharmaceutical Co. for supplying DuP 128, and Dr. Charles L. Bisgaier for performing the plasma cholesterol lipoprotein distribution analyses. This work is supported by grants from the Heart and Stroke Foundation of Ontario (T-3371), and the National Institutes of Health (NHLBI HL49110 and NCRR RR02176). J.R.B. is a recipient of a Heart and Stroke Foundation of Canada Research Fellowship, L.J.W. is a recipient of a Medical Research Council of Canada Studentship, and M.W.H. is a Career Investigator of the Heart and Stroke Foundation of Ontario. References

[8]

[9]

[10]

[11]

[12]

[13]

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