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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2589-2600

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2589-2600.)
© 1997 American Heart Association, Inc.

Articles

Inhibition of HMG-CoA Reductase by Atorvastatin Decreases Both VLDL and LDL Apolipoprotein B Production in Miniature Pigs

John R. Burnett; Lisa J. Wilcox; Dawn E. Telford; Sandra J. Kleinstiver; P. Hugh R. Barrett; Roger S. Newton; ; Murray W. Huff

From the Departments of Medicine and Biochemistry and The John P. Robarts Research Institute, University of Western Ontario, London, Ontario, Canada; the Departments of Bioengineering and Medicine, University of Washington, Seattle (P.H.R.B.); and Parke-Davis Pharmaceutical Research, Warner Lambert Co, Ann Arbor, Mich (R.S.N.).

Correspondence to Murray W. Huff, The John P. Robarts Research Institute, 4-16, University of Western Ontario, 100 Perth Dr, London, Ontario N6A 5K8, Canada. E-mail mhuff{at}julian.uwo.ca


*    Abstract
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Abstract In the present studies, the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor atorvastatin was used to test the hypothesis that inhibition of cholesterol biosynthesis in vivo with a consequent reduction in the availability of hepatic cholesterol for lipoprotein synthesis, would (1) reduce very low density lipoprotein (VLDL) apolipoprotein B (apoB) secretion into the plasma, (2) reduce the conversion of VLDL apoB to LDL apoB, and (3) reduce LDL apoB direct synthesis. ApoB kinetic studies were carried out in six control miniature pigs and in six animals after 21 days of administration of atorvastatin (3 mg/kg per day). Pigs were fed a fat- (34% of calories; polyunsaturated to monounsaturated to saturated ratio, 1:1:1) and cholesterol- (400 mg/d cholesterol; 0.1%; 0.2 mg/kcal) containing pig chow–based diet. Atorvastatin treatment significantly reduced plasma total cholesterol, LDL cholesterol, total triglyceride, and VLDL triglyceride concentrations by 16%, 31%, 19%, and 28%, respectively (P<.01). Autologous 131I-VLDL, 125I-LDL, and [3H]leucine were injected simultaneously into each pig, and apoB kinetic data were analyzed using multicompartmental analysis (SAAM II). The VLDL apoB pool size decreased by 29% (0.46 versus 0.65 mg/kg; P=.002), which was entirely due to a 34% reduction in the VLDL apoB production rate (PR) (1.43 versus 2.19 mg/kg per hour; P=.027). The fractional catabolic rate (FCR) was unchanged. The LDL apoB pool size decreased by 30% (4.74 versus 6.75 mg/kg; P=.0004), which was due to a 22% reduction in the LDL apoB PR (0.236 versus 0.301 mg/kg per hour; P=.004), since the FCR was unchanged. The reduction in LDL apoB PR was primarily due to a 34% decrease in conversion of VLDL apoB to LDL apoB; however, this reduction was not statistically significant (P=.114). Hepatic apoB mRNA abundance quantitated by RNase protection assay was decreased by 13% in the atorvastatin-treated animals (P=.003). Hepatic and intestinal LDL receptor mRNA abundances were not affected. We conclude that inhibition of hepatic HMG-CoA reductase by atorvastatin reduces both VLDL and LDL apoB concentrations, primarily by decreasing apoB secretion into the plasma and not by an increase in hepatic LDL receptor expression. This decrease in apoB secretion may, in part, be due to a reduction in apoB mRNA abundance.


Key Words: mRNA • HMG-CoA reductase inhibitor • atorvastatin • apolipoprotein B metabolism • kinetics


*    Introduction
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Hepatic apoB100 is required for the synthesis and secretion of VLDL.1 VLDL and its metabolic products, IDL and LDL, contain apoB100 as their major protein constituent.2 In addition to its structural role, apoB100 is a ligand for LDL receptor–mediated endocytosis of LDL.3 Elevated plasma concentrations of apoB-containing lipoproteins are key risk factors for the development of atherosclerosis. Therefore, it is important to understand the mechanisms regulating their secretion into and removal from the circulation.

The role of cholesterol biosynthesis in the regulation of apoB-containing lipoprotein metabolism is now beginning to be understood. HMG-CoA reductase, an ER protein, catalyzes the rate-limiting, reductive deacylation of HMG-CoA to mevalonate3 4 in cholesterol biosynthesis. Cholesterol and, likely, other oxysterols can inhibit the activity of HMG-CoA reductase.5 6 A class of lipid-lowering compounds, the HMG-CoA reductase inhibitors, have been shown to interfere with the rate-limiting step in cholesterol biosynthesis.7 These compounds are potent LDL cholesterol-lowering agents; however, relative to the LDL response, their ability to reduce TG concentrations is modest. HMG-CoA reductase inhibitors lower plasma LDL cholesterol by one or a combination of the following mechanisms: (1) enhanced catabolism by upregulation of hepatic LDL receptors, (2) production of an LDL particle that is a better ligand for the LDL receptor, (3) reduced production of LDL apoB due to either decreased secretion of LDL directly into the plasma or decreased conversion of VLDL to LDL. Decreased conversion may be due to either reduced secretion of hepatic VLDL or increased removal of VLDL (and IDL) from the plasma by upregulated LDL receptors.

In vivo evidence for reduced LDL apoB production following treatment with HMG-CoA reductase inhibitors has been reported in experimental animals and in human subjects with a variety of hyperlipidemias, as recently reviewed by Thompson et al.8 Other studies have reported no change in LDL apoB production. These differences may reflect the type and dose of statin administered, the methods used to examine apoB kinetics, or possibly subject variability. It has been proposed that HMG-CoA reductase inhibitors lower plasma VLDL by inhibition of VLDL apoB synthesis. The results of kinetics studies in humans, however, are inconsistent. Vega et al,9 using pravastatin, demonstrated decreased VLDL apoB production in subjects with primary moderate hypercholesterolemia and relatively normal TG concentrations. These findings were consistent with those of Arad et al10 11 using lovastatin in subjects with combined hyperlipidemia. In contrast, Gaw et al,12 using simvastatin or colestipol plus simvastatin,13 found no effect on VLDL apoB parameters in subjects with primary moderate hypercholesterolemia. A direct correlation between the rate of cholesterol synthesis and the rate of VLDL apoB secretion has been demonstrated in normolipidemic subjects by stable isotopic techniques.14 Recently in heterozygous FH subjects and using the same techniques, Watts et al15 found that simvastatin treatment decreased the VLDL apoB absolute secretion rate. This reduction correlated with the change in plasma LDL cholesterol but not with those of TG or mevalonic acid. The effect of HMG-CoA reductase inhibitors on VLDL metabolism in animals is also inconsistent. Work from our laboratory (in miniature pigs) showed that lovastatin reduced direct LDL synthesis. No significant change in VLDL apoB metabolic parameters were observed.16 17 The pig shows many aspects of apoB metabolism that are similar to those in nonhuman primates and humans.16 17 18 Simvastatin had no effect on plasma or VLDL TG in FH swine.19 In guinea pigs, treatment with lovastatin had no effect on VLDL or LDL apoB production but did increase hepatic expression of LDL receptors.20 In contrast, using isolated, perfused rat livers, Khan et al21 demonstrated a decrease in VLDL secretion after treatment with lovastatin.

Atorvastatin is a new, synthetic, chiral, tissue-selective inhibitor of HMG-CoA reductase.22 23 Clinical trials using atorvastatin in humans have demonstrated marked plasma LDL cholesterol and TG reductions.24 25 26 The mechanism for these significant lipid-lowering effects is unknown. LDL apoB kinetic studies in casein-fed rabbits treated with atorvastatin have shown a decrease in LDL apoB pool size due to a reduction in LDL apoB PR.27 However, whether cholesterol synthesis inhibition by atorvastatin affects the assembly and secretion of apoB-containing lipoproteins is unknown.

Under normal circumstances, sufficient cholesterol is produced for apoB-containing lipoprotein synthesis and assembly. The inhibition of HMG-CoA reductase by a potent HMG-CoA reductase inhibitor should significantly limit the availability of FC and may also reduce cholesteryl ester for incorporation into VLDL. In the present studies, the HMG-CoA reductase inhibitor atorvastatin was used to test the hypothesis that inhibition of cholesterol biosynthesis in vivo, with a consequent reduction in the availability of hepatic cholesterol for lipoprotein synthesis, will (1) reduce VLDL apoB secretion into the plasma, (2) reduce the conversion of VLDL apoB to LDL apoB, and (3) reduce LDL apoB direct synthesis.


*    Methods
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Animals and Diets
Miniature pigs weighing between 22 and 25 kg were obtained from a local supplier (Premier Quality Genetics Inc, West Lorne, Ontario). After being acclimatized for 1 week, the 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.16 Isoflurane USP (Abbott Laboratories Ltd) was used as the anesthetic and ketamine USP (Vetrepharm Canada Inc) as the preanesthetic. Catheters that were kept patent by being filled with 7% disodium EDTA allowed for ease of sample injection as well as blood sampling throughout each turnover study in unrestrained, unanesthetized animals. The experimental protocol was approved by the Animal Care Committee of the University of Western Ontario.

Pigs were studied in pairs, with each pair being same-sex littermates. Six animals received the HMG-CoA reductase inhibitor atorvastatin (Parke-Davis, Ann Arbor, Mich) at a dose of {approx}3 mg/kg body weight per day (80 mg/d) for 21 days prior to the turnover study. This dose resulted in a 25% to 30% reduction in LDL cholesterol in a dose-finding study (data not shown). The dose given is equivalent to the maximum therapeutic dose used in humans but is {approx}2.6-fold greater than that used in humans when body weight is taken into consideration.

Atorvastatin is a synthetic inhibitor of HMG-CoA reductase with an in vitro IC50 of 7.5 nmol/L for rat liver microsomal HMG-CoA reductase activity.28 The chemical structure of atorvastatin has been reported previously.29 Atorvastatin (calcium salt) was placed in gelatin capsules and, to ensure ingestion, was administered by hand before the daily feeding. The six control animals received a placebo capsule. The atorvastatin was given at 9 AM each day. Each animal received a 590-g ration of diet (B.W.S. Hog Grower, B-W Feed and Seed Ltd) supplemented with lard, unsalted butter, and safflower oil (1:0.6:0.2), thereby generating a final polyunsaturated to monounsaturated to saturated fatty acid ratio of 1:1:1. Cholesterol (Fisher Scientific) was added to the diet to a final concentration of 0.1% (0.2 mg/kcal). This diet provided 34% of calories as fat, 49% as carbohydrate, and 17% as protein.

Lipoprotein Turnover Studies
Lipoprotein turnover studies were performed essentially as described previously,16 18 30 with the addition of an intravenous bolus of [3H]leucine. VLDL (Sf 20 to 400) and LDL (Sf 0 to 12) were isolated from plasma (100 to 150 mL) obtained after a 24-hour fast and subsequently radiolabeled with 131I and 125I, respectively. All labeled lipoproteins were autologous. Radiolabeling was performed using the iodine monochloride technique.18 Lipoproteins were sterilized by the addition of gentamicin sulfate (100 mg/mL) and checked for pyrogenicity and sterility. Of the total VLDL radioactivity, <2% was free iodine, 19% to 32% was bound to lipid, and 25% to 38% of the protein-bound label was bound to apoB. Of the total LDL radioactivity, <1% was free iodine, 14% to 27% was bound to lipid, and 80% to 90% of the protein-bound label was bound to apoB. After a 24-hour fast, each animal received 20 µCi 131I-VLDL apoB, 15 µCi 125I-LDL apoB, and 2.5 mCi L-[4,5-3H]leucine (Amersham Canada Ltd; specific activity, 155 Ci/mmol) given as a bolus by the indwelling catheter. After injection, blood samples (20 mL) were collected into tubes containing disodium EDTA at 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 12, 24, 30, 36, 48, 72, 96, and 120 hours. The pigs received no food but had free access to water until the 12-hour sample on day 1 and 36-hour sample on day 2, at which time they received half their daily ration and either atorvastatin or placebo. Animals were given their full daily ration and either atorvastatin or placebo after the 48-, 72-, 96-, and 120-hour samples.

As described previously,16 18 VLDL (d<1.006 g/mL), IDL (d=1.006 to 1.019 g/mL), and LDL (d=1.019 to 1.063 g/mL) were separated from plasma. ApoB was isolated from each lipoprotein fraction by isopropanol precipitation. For the 0- to 24-hour samples, the washed apoB pellet was dissolved in 620 µL of 0.1N NaOH. A 250-µL aliquot was counted in a gamma counter (LKB Compugamma) and then assayed for protein content by a modified Lowry procedure to calculate the 131I specific activities. To determine 3H counts, a further 250-µL aliquot was counted in a beta counter (Beckman LS 3801 liquid scintillation system) 8 weeks later, after the 131I counts had decayed to background levels. The plasma concentration of apoB in each lipoprotein was determined by subtracting the protein value of the first precipitation supernatant from the total protein concentration and by a sensitive ELISA. For the ELISA, affinity-purified polyclonal antiserum prepared in rabbits against pig LDL was used as the capture antibody, and an affinity-purified polyclonal apoB antibody conjugated to peroxidase (The Binding Site) was used for detection. o-Phenylenediamine dihydrochloride (Sigma-Aldrich) was used as a substrate for color development, and the absorbance at 450 nm was read using an ELISA plate reader. A pig purified LDL standard (d=1.030 to 1.050 g/mL) was used to calibrate the assay. Samples were diluted prior to analysis. Two controls analyzed with each batch gave between-batch and within-batch coefficients of variation of <5% for both concentrations. VLDL, IDL, and LDL apoB concentrations were constant over the sampling time period.16

Leucine concentrations were determined in deproteinized plasma isolated by a modification31 of the method of Hamilton. In brief, 0.5 mL sodium citrate buffer (pH 2.2, 0.2N Na), 1.0 mL of 9% sulfosalicylic acid, and norleucine (internal standard) were added to 0.5 mL of plasma and centrifuged at 37 000g for 10 minutes. After the supernatant was removed, the precipitate was washed with 0.5 mL of sodium citrate buffer and centrifuged at 37 000g for 10 minutes. The supernatants were combined. Amino acid analyses of the deproteinized plasma were performed on a single-column amino acid analyzer (model 119C, Beckman). The plasma leucine concentration was quantitated by the ratio of peak height to that of the internal standard. The radioactivity of an aliquot of deproteinized plasma was measured to determine the plasma leucine specific activity.

Kinetic Analysis
The turnover data were analyzed by using the multicompartmental modeling program SAAM II (SAAM Institute) running on an Pentium-based personal computer. The model chosen to describe the data is shown in Fig 1Down. This model was simultaneously fit to the three sets of tracer data for all lipoprotein fractions. This approach permitted the integration of all tracer data into a single model. Because of the different methods by which the two iodinated tracers and [3H]leucine tracee were introduced into the system, the information contained in each data set helped support different aspects of the model structure. Fig 1Down shows the compartments of the model and the pathways that connect the compartments within and between lipoprotein fractions. The model includes compartments to describe the kinetics of leucine. Although a single compartment is represented, a forcing function described by the sum of three exponentials was used to account for plasma leucine kinetics. The forcing function was used to drive the appearance of leucine tracer into the different lipoprotein fractions. Two delay compartments were included in the model to account for the time required for the synthesis and secretion of apoB into plasma. Plasma VLDL apoB kinetic data are characterized by compartments 1, 2, and 3. The sum of the specific radioactivities in each of these compartments equals the measured apoB specific activity of the VLDL fraction. Upon reinjection of the 131I-labeled VLDL, it was assumed that the apoB radioactivity distributed among the three compartments in proportion to their pool sizes; hence, the apoB specific radioactivity in compartments 1, 2, and 3 were equal at the time of reinjection. Compartment 1, which represented most of the mass of the VLDL apoB fraction, turned over rapidly and accounted for the initial fall of the 131I-VLDL apoB specific radioactivity curve. Compartment 2 turned over more slowly than compartment 1 by one order of magnitude. Compartment 3, which turned over more slowly than compartment 2, described the terminal slope of the 131I-VLDL apoB specific radioactivity curve. The terminal slope of the [3H]VLDL apoB radioactivity data, which was slower than that of 131I-VLDL, represents the kinetics associated with the recycling of the amino acid precursor leucine. Plasma IDL apoB specific activity is characterized by two compartments, 4 and 5. Although the majority of IDL apoB was derived from VLDL, direct IDL apoB input was required for optimal model fit for all three tracers. The percentage derived directly was quantitatively small (<2%), and no significant differences were observed between the treatment groups. The LDL section of the model is characterized by a plasma compartment, designated here as compartment 6, and an extravascular compartment, compartment 7. Although this assumes that LDL apoB is kinetically homogeneous, it is not possible to address the question of LDL heterogeneity without isolating LDL subfractions and/or collecting urine radioactivity data. 125I-urine radioactivity was collected in a previous study32 ; however, due to high lipid labeling, the urine radioactivity data could not be used. It was assumed that at the time of reinjection, all of the 125I-LDL tracer was introduced into the plasma compartment. In addition, it was assumed that the irreversible loss of LDL apoB occurred only from compartment 6.



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  		Figure 1. Diagram of the multicompartmental kinetic model used for analysis of apoB metabolism in VLDL, IDL, and LDL. The VLDL fraction is described by compartments 1, 2, and 3, the IDL fraction by compartments 4 and 5, and the LDL fraction by compartment 6 that is connected to an extravascular exchange compartment 7. A forcing function, described by the sum of three exponentials, was used to account for plasma leucine kinetics, represented as compartment 8. The forcing function was used to drive the appearance of leucine tracer into the different lipoprotein fractions. Compartment 9 represents a delay compartment included in the model to account for the time required for the synthesis and secretion of apoB into plasma. Arrows connecting the compartments describe the paths by which material moves from one compartment to another.

Three pathways from the synthesis and secretion compartment are included in the model. These pathways represent the secretion of apoB directly into VLDL, IDL, and LDL fractions. The model allows for the direct removal of apoB from plasma compartments 1, 2, 3, 4, 5, and 6: movement from compartment 1 to 2 (VLDL), directly to 4 (IDL), and directly to 6 (LDL); movement from compartment 2 to 3 (VLDL), to 4 (IDL), and to 6 (LDL); and movement from 4 to 5 (IDL) and to 6 (LDL). Because compartments 3 and 5 turned over slowly ({approx}0.1 h-1) and represented only small portions of the VLDL and IDL fractions, it was assumed that the material in these compartments was directly removed from the plasma. In each of the studies, the terminal slopes of the VLDL and IDL specific-activity curves were parallel; therefore, rate constants k(0,1) and k(0,3) were constrained to be equal.

During the course of the study, aliquots of plasma (taken at time t=0) were spiked with a small aliquot of the 131I-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. For these studies, the mean distribution of 131I apoB radioactivity in the spiked samples was 94.5±1.5%, 4.1±1.4%, and 1.5±0.6% 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.

Liver Lipids
Liver lipids were extracted using the method of Folch et al33 from 1.0-g sections of liver obtained at sacrifice that had been stored at -80°C. 1{alpha},2{alpha}(n)-[3H]Cholesteryl oleate and glycerol tri[1-14C]oleate (Amersham Canada Ltd) were added to assess recovery. TG, FC, and TC were quantitated in liver lipid extracts by using a modification of the method of Carr et al.34 In brief, a 200 µg/mL triolein standard was prepared in isopropanol, and 1-, 2-, 4-, 8-, 16-, 32-, and 64-µg standard solutions were made. A 200 µg/mL cholesterol standard was prepared in isopropanol, and 1-, 2-, 4-, 8-, 16-, and 32-µg standard solutions were made. Standards were dried under N2 and 400 µL of chloroform added. Samples and standards were dried under N2 and 500 µL of a 1% Triton X-100 solution in chloroform added. Each tube was rolled, thus ensuring that the Triton X-100/chloroform mixture had contacted all surfaces touched by lipid. The samples and standards were capped with a marble, left at room temperature for 1 hour, and dried under N2. Fifty microliters of deionized water was added and each tube rolled before incubation at 37°C for 15 minutes. TG, TC, and FC were determined by enzymatic, colorimetric assays using reagents obtained from Boehringer Mannheim.

RNase Protection Assay for Liver and Intestine ApoB and LDL Receptor mRNAs
Liver and small-intestine samples obtained at necropsy were immediately frozen in liquid N2 and stored at -80°C until analysis. Total RNA was isolated using Trizol reagent (GIBCO BRL). The integrity of the RNA isolated was verified 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-specific cDNA's for apoB and the LDL receptor, cloned into Bluescript plasmid (kindly provided by Dr Alan D. Attie, University of Wisconsin-Madison), served as templates to synthesize antisense RNA probes. These riboprobes were then used to measure apoB and LDL receptor mRNA in a modification of the RNase protection solution hybridization assay of Azrolan and Breslow.35 This assay uses standard RNA that allows precise quantitation of specific gene transcripts. In brief, a 32P-labeled RNA probe was synthesized using an in vitro transcription system (Promega) 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 to 3x108 cpm/µg) and either sample or standard cRNA (10 to 150 pg) were hybridized overnight at 63°C in 40 µL of hybridization buffer (80% formamide; 40 mmol/L HEPES, pH 6.7; 0.4 mol/L NaCl; 1 mmol/L EDTA) with 10 µg of yeast tRNA. Three hundred microliters of digestion buffer (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) were added to each sample and incubated at 34°C for 1 hour. After incubation, 500 µL of 20% cold trichloroacetic acid and 100 µg of herring sperm DNA were added to each sample. Samples were placed on ice for 15 minutes and then filtered using glass fiber filters. Filters were washed with 7% trichloroacetic acid and dried, and 8 mL of scintillation fluid was added before counting. mRNA abundance was quantitated by reference to the curve generated by using the transcribed hybridization standard. The sample and standard RNAs were assayed in triplicate, and hybridization was linear to 120 pg of cRNA. Samples were analyzed in one batch, and within-batch coefficients of variation of <10% were achieved.

Plasma Cholesterol Distribution
Plasma cholesterol lipoprotein profiles were determined by the HPGC method of Kieft et al.36 In brief, lipoprotein separations were performed on a Superose 6HR FPLC column, determined primarily by size of the lipoproteins. After HPGC, on-line lipoprotein cholesterol distribution was determined. Lipoprotein cholesterol was determined from independent TC determinations and the percent area distribution of the cholesterol fractions by HPGC. Peak retention times were used to estimate and compare the relative sizes of the lipoprotein particles.

Analyses
TC, TG, VLDL cholesterol and TG, and HDL cholesterol concentrations in the plasma were determined. VLDL was obtained after ultracentrifugation at d<1.006 g/mL, and HDL was obtained after precipitation of other lipoproteins by dextran sulfate–MgCl2. LDL was calculated by difference. TC, TG, FC, and phospholipid were determined by enzymatic, colorimetric assays by using reagents obtained from Boehringer Mannheim. Dietary fatty acid composition was determined by gas chromatography with a 2-m column (SP 2230, liquid phase; Supelco) on a Varian 6000 gas chromatograph. Tests for statistical significance of differences were compared by paired t test.37 A value P<.05 was considered significant.


*    Results
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 *Results
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The effects of atorvastatin on plasma and lipoprotein lipid concentrations are shown in Table 1Down. Total plasma and VLDL TG concentrations were significantly reduced by 19% (P=.009) and 28% (P=.005), respectively. Total plasma cholesterol and LDL cholesterol concentrations were significantly reduced by 16% (P=.005) and 31% (P=.006), respectively. VLDL cholesterol concentrations decreased by 23%; however, this reduction was not significant (P=.07). The HDL cholesterol concentrations were unaltered by the HMG-CoA reductase inhibitor. Atorvastatin significantly reduced VLDL apoB concentrations by 28% (P=.002) and LDL apoB by 30% (P=.0005).


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  		Table 1. Plasma Lipid and Lipoprotein Concentrations in Control and Atorvastatin-Treated Miniature Pigs

Autologous 131I-VLDL, 125I-LDL, and [3H]leucine were simultaneously injected into each control and atorvastatin-treated pig. The kinetic parameters of apoB were determined from the simultaneous analysis of all the specific-activity data by using the model shown in Fig 1Up. The kinetic parameters are summarized in Tables 2Down and 3Down. A fit of the model to the apoB-specific radioactivity curves for 131I-VLDL, 131I-IDL, and 131I-LDL for a representative pair of animals are shown in Fig 2Down. A fit of the model to the apoB-specific radioactivity curves for [3H]VLDL, [3H]IDL, and [3H]LDL for the same animals are shown in Fig 3Down. In this model, {approx}90% of apoB enters the circulation as VLDL, 5% of the VLDL apoB flux is converted to LDL (either through the IDL pool or directly without passing through plasma IDL), and 10% of apoB enters the circulation directly as LDL.


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  		Table 2. Metabolic Parameters of VLDL ApoB Metabolism in Control and Atorvastatin-Treated Miniature Pigs


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  		Table 3. Metabolic Parameters of LDL ApoB Metabolism in Control and Atorvastatin-Treated Miniature Pigs



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  		Figure 2. Line graph showing apoB specific activity–time curves for VLDL ({Delta}), IDL ({bullet}), and LDL ({blacksquare}) after injection of radiolabeled 131I-VLDL. Data points represent the observed data, and the lines are the best fit generated by the kinetic model. A, Control pig; B, an atorvastatin-treated pig.



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  		Figure 3. Line graph showing apoB specific activity–time curves for VLDL ({Delta}), IDL ({bullet}), and LDL ({blacksquare}) after injection of radiolabeled [3H]leucine. Data points represent the observed data, and the lines are the best fit generated by the kinetic model. A, Control pig; B, an atorvastatin-treated pig.

The administration of atorvastatin decreased the VLDL apoB pool size by 29% (P=.002, Table 2Up). This was primarily due to a significant 34% (P=.027) reduction in the VLDL apoB PR, since the FCR was unchanged. The amount of VLDL apoB converted to LDL apoB decreased by 34%. Although not statistically significant (P=.114), conversion was reduced in five of six animals studied. The flux of apoB cleared without conversion to IDL or LDL was significantly reduced by 34% (P=.025). Neither the percentage of VLDL apoB flux converted to LDL nor the percent cleared directly was affected by atorvastatin 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 significantly affected by atorvastatin treatment (data not shown). The model allowed us to determine the production or flux 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 did not change; however, VLDL apoB converted to LDL via IDL was decreased by 36% with atorvastatin treatment (data not shown; P=.109).

Atorvastatin significantly decreased the LDL apoB pool size by 30% (P=.0004) (Table 3Up). This was primarily due to a significant 22% (P=.004) reduction in the LDL apoB PR, which was accounted for by the combination of a 34% decrease in LDL apoB derived from VLDL apoB catabolism (P=.114) (Table 2Up) and a 13% decrease in LDL apoB direct synthesis (Table 3Up). Although not statistically significant, (P=.115), LDL direct synthesis decreased in five of six animals studied. Total apoB production into plasma, calculated as the sum of VLDL apoB production plus LDL apoB direct production, decreased significantly by 34% (P=.027). Atorvastatin treatment resulted in a nonsignificant 11% increase in the LDL apoB FCR (P=.095). The minimal change in the LDL apoB FCR is further demonstrated by plotting apoB–specific activity curves for LDL after injection of radiolabeled 125I-LDL as a percentage of the initial specific activity value (Fig 4Down).



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  		Figure 4. Line graph showing apoB specific activity–time curves for LDL after injection of radiolabeled 125I-LDL plotted as a percentage of the initial specific activity value. Data points represent the observed data, and the lines are the best fit generated by the kinetic model. Results shown are the mean±SEM for all animals in each group. {blacksquare} Indicates atorvastatin; {square}, control. The error bars are not visible because they are smaller than the symbols in the figure.

VLDL and LDL were analyzed for lipid and protein composition; however, the percent composition was not altered for any of the parameters measured (Table 4Down). Plasma cholesterol distribution among the lipoprotein classes, as assessed by HPGC, showed no major changes (Fig 5Down). VLDL cholesterol was significantly decreased by 21% in the atorvastatin-treated pigs; however, the peak retention time was unchanged. Although the percent LDL cholesterol was unaltered, a small but significant increase in the peak retention time was observed, suggesting that a smaller particle was produced in the atorvastatin-treated animals.


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  		Table 4. Percent Composition of VLDL and LDL Isolated From Control and Atorvastatin-Treated Miniature Pigs



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  		Figure 5. Cholesterol distribution profiles for one representative pair of pigs, as determined by HPGC.36 The elution peaks for VLDL, LDL, and HDL are indicated. The mean±SEM retention times for five control pigs were VLDL, 12.50±0.01; LDL, 16.71±0.06; and HDL, 21.74±0.06 minutes. Values for the five atorvastatin-treated animals were VLDL, 12.54±0.01; LDL, 16.81±0.07 (P=.032); and HDL, 21.85±0.10 minutes.

Approximately 24 hours after the last dose of ator-vastatin was administered, the pigs were killed and sections of liver and small intestine were removed and stored at -80°C prior to analyses. Hepatic FC, cholesteryl ester, and TG concentrations were unaltered by atorvastatin treatment (Table 5Down). Hepatic apoB mRNA abundance was decreased by 13% in the atorvastatin-treated animals (P=.003), whereas intestinal apoB mRNA abundance was unchanged (Table 6Down). Hepatic and intestinal LDL receptor mRNA abundances were unaltered, consistent with the unchanged LDL apoB FCR.


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  		Table 5. Liver Cholesterol, Cholesteryl Ester, and TG Concentrations in Control and Atorvastatin-Treated Miniature Pigs


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  		Table 6. ApoB and LDL Receptor mRNA Abundances in Control and Atorvastatin-Treated Miniature Pigs


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Debate continues over which neutral lipid (cholesterol, cholesteryl ester, or TG) is most important in the regulation of hepatic apoB secretion.8 38 39 The regulation of the synthesis and secretion of TG-rich lipoproteins by lipid availability has recently been reviewed.38 40 41 The current experiments were designed to test the hypothesis in vivo that the potent inhibition of cholesterol biosynthesis by the HMG-CoA reductase inhibitor atorvastatin would decrease apoB secretion and result in reduced plasma concentrations of apoB-containing lipoproteins.

The present experiments were carried out in miniature pigs fed a fat- and cholesterol-containing diet. The apoB kinetic studies employed radioiodinated VLDL and LDL as exogenous tracers and 3[H]leucine to endogenously label apoB to combine the advantages of both kinetics approaches.42 The results clearly demonstrate that atorvastatin significantly reduces the secretion of apoB-containing lipoproteins into plasma. The major findings were as follows: (1) VLDL apoB secretion rate decreased by 36%, resulting in a 29% reduction in VLDL apoB pool size; (2) LDL apoB PR was significantly decreased by 22%, resulting in a 30% reduction in the LDL apoB pool size; (3) the conversion of VLDL apoB to LDL apoB and direct LDL apoB synthesis were decreased by 34% and 13%, respectively; (4) total apoB secretion decreased by 34%; (5) VLDL and LDL apoB FCRs were unchanged; (6) hepatic and intestinal LDL receptor mRNA abundances were consistent with the unchanged LDL apoB FCR; and (7) hepatic apoB mRNA abundance was significantly decreased by 13%.

Cholesterol metabolism in the liver is complex, with close regulation of both the compartmentalization and cellular concentrations of cholesterol.43 Several functional pools of cholesterol are present in the hepatocyte, including a metabolically active pool, an ACAT substrate pool, and a cholesteryl ester pool.44 The relative contribution of these different pools to the regulation of apoB metabolism, however, remains unclear. Rates of cholesterol biosynthesis, LDL receptor expression, and cholesterol esterification respond rapidly to cellular concentrations of FC.43 The inhibition of HMG-CoA reductase by atorvastatin would be expected to decrease the hepatic FC concentration of the ER. We found no decrease in the hepatic concentrations of either FC or esterified cholesterol. Although microsomal cholesterol was not measured in the present study, it is possible that microsomal cholesterol in a putative regulatory pool44 45 is decreased by atorvastatin treatment. We would suggest that the primary action of atorvastatin is through reduced microsomal cholesterol availability for either lipoprotein surface or as a substrate for ACAT, thereby reducing cholesteryl ester availability for lipoprotein core formation. This is consistent with the idea that the rate of cholesterol synthesis, not total hepatocyte cholesterol content, regulates apoB secretion.15 Our results are consistent with a recent report by Conde et al,46 who demonstrated, using Triton WR-1339 to block VLDL catabolism, that apoB secretion was inhibited by up to 70% in atorvastatin-treated guinea pigs. At doses of atorvastatin similar to those used in the present study (3 mg/kg per day), no significant changes were observed in either hepatic FC or cholesteryl ester, whereas microsomal FC was significantly decreased by 30%.

A number of in vitro studies using HepG2 cells have investigated the role of cholesterol and cholesteryl ester in apoB assembly, synthesis, and secretion. In some studies, as recently reviewed by Ginsberg,39 the modulation of cellular cholesterol and/or cholesteryl ester failed to alter the rate of apoB secretion, whereas contrasting results using the same cell line demonstrated decreased oleate-stimulated apoB secretion with HMG-CoA reductase inhibition. Reasons for this discrepancy are not known but may be due to the use of HepG2 cells. HepG2 cells are defective in mobilizing TG for lipoprotein assembly47 and differ from primary hepatocytes in their response to HMG-CoA reductase inhibitors.48 Other in vitro and ex vivo models support the concept that apoB secretion is regulated by cholesterol synthesis. The addition of pravastatin to LDL-loaded isolated rabbit hepatocytes decreased apoB secretion.49 Using isolated perfused rat livers, Khan et al21 demonstrated a decrease in VLDL secretion after treatment with lovastatin.

The results of the present study are consistent with other in vivo studies, in that modulation of apoB synthesis and secretion appears to be an important mechanism whereby HMG-CoA reductase inhibitors lower the plasma concentration of apoB-containing lipoproteins.9 10 11 15 16 17 32 Earlier in vivo apoB kinetic studies conducted in miniature pigs from this laboratory (using lovastatin) demonstrated that the significant reduction in LDL apoB was due to a decrease in LDL apoB production; primarily LDL apoB direct synthesis.16 17 Similarly in the present study, the primary response to atorvastatin was a decrease in LDL apoB production. Both a decrease in direct LDL apoB production and conversion of VLDL apoB to LDL apoB contributed to this reduction in LDL apoB PR. The latter was due to a marked decrease in VLDL apoB production by atorvastatin. The reason why our previous studies only affected LDL direct synthesis is not readily apparent but may be related to differences in diet, the kinetic models employed, and/or the dose and potency of the HMG-CoA reductase inhibitor used. In contrast to the present study, during our experiments with lovastatin, the animals were fed a low-fat, cholesterol-free diet and multicompartmental modeling was not used. In addition, the lovastatin dose was 1.2 mg/kg body weight per day. It is possible that lovastatin did not achieve sufficient inhibition of reductase to decrease the regulatory cholesterol pool involved in VLDL assembly and secretion.

The marked reduction in VLDL apoB secretion in the present study may be related to a more sustained inhibition of HMG-CoA reductase. In a preliminary report, Naoumova et al50 demonstrated that in FH homozygotes, plasma mevalonate concentrations (an indirect measure of cholesterol biosynthesis) were decreased significantly longer following a single dose of atorvastatin compared with a similar dose of simvastatin. Alternatively, atorvastatin could influence TG and/or phospholipid synthesis in addition to its effect on cholesterol synthesis. Inhibition of TG51 and phospholipid synthesis52 in cultured hepatocytes have been shown to decrease apoB secretion.

The decreased conversion of VLDL apoB to LDL apoB in the present study would appear to be related to the reduced total VLDL apoB production that results in both a decreased conversion to LDL apoB and decreased direct removal of VLDL apoB. The fact that the percentage of total VLDL apoB production converted to LDL apoB or of that removed directly did not change suggests that no major alterations in VLDL composition or LDL receptor activity were responsible for the decreased conversion. This conclusion is consistent with the lack of effect of atorvastatin on VLDL composition, LDL apoB FCR, and LDL receptor mRNA abundance. A small but significant decrease was noted in the percent of plasma cholesterol carried in VLDL in atorvastatin-treated animals; however, VLDL particle size was unaffected.

It is commonly believed that the mechanism whereby HMG-CoA reductase inhibitors decrease LDL cholesterol concentrations is by enhanced catabolism via upregulation of hepatic LDL receptors. Hepatic cholesterol synthesis inhibition would be expected to decrease hepatic cholesterol concentrations, thereby increasing the expression of LDL receptors. Plasma LDL cholesterol and its precursors would decrease consequent to the resulting increase in apoB FCR. Consistent with our previous findings using lovastatin,16 17 no significant effect was seen in the LDL apoB FCR in atorvastatin-treated animals. In addition, the FCR for the direct catabolism of VLDL apoB and IDL apoB was unchanged by atorvastatin treatment. One possible explanation for the lack of effect on LDL apoB FCR would be the synthesis of an LDL particle of altered composition such that it interacts less efficiently with upregulated LDL receptors.53 Other explanations may include (1) the preferential removal of an LDL subfraction with a rapid turnover rate or (2) the use of the single-plasma-pool analysis of the LDL apoB kinetic data. No significant effects were noted on percent composition of plasma LDL for any of the parameters measured or on LDL receptor gene expression in the atorvastatin-treated animals. These results are consistent with the concept that atorvastatin reduces a newly synthesized pool of cholesterol that is required for lipoprotein assembly; a pool not in complete equilibrium with the LDL receptor regulatory pool.54 It is possible that enhanced LDL clearance may become apparent at higher doses of atorvastatin. In guinea pigs, treatment with 20 mg/kg per day of atorvastatin enhanced hepatic microsomal LDL receptor number by 29%.46

How the reduced rate of cholesterol synthesis by atorvastatin regulates the assembly and secretion of apoB-containing lipoproteins within the hepatocyte is not known. Potential regulatory elements for gene transcription exist in both the upstream region and the second intron of the apoB gene.55 56 The relatively stable apoB mRNA concentrations found after modification by a number of factors that result in changes to apoB secretion57 58 59 60 61 are consistent with posttranslational regulation, whereby apoB constitutively synthesized in excess of its requirement for lipid transport is degraded within the cell.62 There is, however, some evidence for the regulation of hepatic apoB mRNA by exogenous VLDL,51 61 amino acids,63 and cholesterol.64 The alterations in apoB mRNA abundance reported in vitro tend to be small, and the physiological significance of these changes remains unclear. More recently, studies by Selby and Yao65 on transfected rat hepatoma (McA-RH777) cell lines expressing recombinant human apoB suggest that mRNA concentrations may influence apoB synthesis and secretion. We found a significant 13% decrease in hepatic apoB mRNA abundance with atorvastatin treatment.

The rate of cholesterol synthesis may affect rates of apoB degradation and/or translocation of apoB across the ER membrane. It has been suggested that the degree of apoB degradation66 67 and rate of translocation determine the fate of newly synthesized apoB.68 A preliminary report in HepG2 cells suggests that atorvastatin increases the rate of intracellular degradation and decreases apoB translocation.69 Whether a similar mechanism(s) occurs in vivo is unknown.

It is not known whether other genes involved in apoB secretion, such as those for ACAT or MTP, or any changes in their enzymatic activities are affected by atorvastatin treatment. Inhibitors of HMG-CoA reductase have been shown to decrease the activity of ACAT, preventing the esterification of newly synthesized cholesterol.70 71 The concentration of FC substrate in the cell regulates enzymatic activity of ACAT,3 and inhibition of newly synthesized cholesteryl esters catalyzed by ACAT has been shown to decrease apoB secretion in cell culture models69 72 and perfused monkey livers.73 ApoB kinetic studies in miniature pigs demonstrated that the ACAT inhibitor DuP 128 significantly decreased VLDL apoB secretion.30 In monocytes,74 mice,75 and rabbits,76 ACAT expression is regulated by cholesterol availability on at least two levels: (1) mRNA abundance and (2) enzyme activity. Therefore, any intervention that perturbs the ACAT FC substrate pool, such as atorvastatin, may modulate the expression and/or activity of ACAT and hence apoB secretion. In guinea pigs treated with atorvastatin, hepatic microsomal ACAT activity was significantly decreased, which correlated with a reduction in microsomal FC content.46

MTP is postulated to mediate the transfer of TGs and cholesteryl ester to the apoB molecule,77 and recent evidence indicates that MTP is capable of facilitating apoB translocation in addition to its role in mediating delivery of core lipid to apoB.78 79 The promoter for the MTP large subunit contains a modified sterol-response element, and there is evidence in HepG2 cells that MTP expression is modulated by exogenous cholesterol.80 81 Although we did not measure MTP activity in the present study, the reduced apoB secretion by atorvastatin treatment may be the result of decreased MTP expression and/or activity, secondary to reduced cholesterol synthesis.

In conclusion, the inhibition of HMG-CoA reductase by atorvastatin decreases both VLDL and LDL apoB concentrations, primarily by decreasing apoB secretion into the plasma. No evidence was found for the upregulation of LDL receptors. We postulate that atorvastatin reduces cholesterol and possibly cholesteryl ester synthesis below a critical level required for apoB translocation, lipoprotein assembly, and secretion, thereby stimulating the rate of apoB degradation. The decrease in apoB secretion may, in part, be due to a reduction in apoB mRNA abundance, suggesting additional regulation at the level of apoB gene transcription.


*    Selected Abbreviations and Acronyms
 
ACAT = acyl coenzyme A:cholesterol acyltransferase
apo = apolipoprotein
ELISA = enzyme-linked immunosorbent assay
ER = endoplasmic reticulum
FC = free cholesterol
FCR = fractional catabolic rate
FH = familial hypercholesterolemia
HMG-CoA = 3-hydroxy-3-methylglutaryl coenzyme A
HPGC = high-performance gel chromatography
IDL = intermediate density lipoprotein
MTP = microsomal triglyceride transfer protein
PR = production rate
TC = total cholesterol
TG = triglyceride


*    Acknowledgments
 
This work was supported by grants from the Heart and Stroke Foundation of Ontario (T-2577 to M.W.H.), the National Institutes of Health (NHLBI HL49110 and NCRR RR02176 to P.H.R.B.), and Parke-Davis (to M.W.H.). 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. We thank Kim Wood for performing the surgeries, Dr Charles L. Bisgaier for performing the plasma cholesterol lipoprotein distribution analyses, and Tim Welke for technical assistance.


*    Footnotes
 
Preliminary reports of this work were presented at the XII International Symposium on Drugs Affecting Lipid Metabolism, Houston, Tex, 1995, and at the American Heart Association 69th Scientific Sessions, New Orleans, La, 1996, and printed in abstract form in Circulation 1996; 94:(suppl I):I-632.

Received April 17, 1997; accepted July 15, 1997.


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 up arrowMethods
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