The Effects of Simvastatin and Cholestyramine, Alone and in Combination, on Hepatic Cholesterol Metabolism in the Male Rat John H. Shand and David W. West* Hannah Research Institute, Ayr, KA6 5HL, United Kingdom

ABSTRACT: The influence of dietary simvastatin, cholestyramine, and the combination of simvastatin plus cholestyramine on hepatic cholesterol metabolism has been investigated in male rats. Recovery from the effects of the drugs was also investigated by refeeding normal chow for 24 h. Both drugs, alone and in combination, increased 3-hydroxy-3-methylglutaryI-CoA (HMG-CoA) reductase activity in vitro, but activity returned toward control values, after drug withdrawal. AcyI-CoA:cholesterol acyltransferase (ACAT) was significantly reduced (P < 0.001) by simvastatin (-75%), cholestyramine (-71%), and by the drug combination (-81%), due both to a decrease in microsomal cholesterol and to nonsubstrate-dependent modulation of enzyme activity. Refeeding control diet increased ACAT activity but not to control levels. The enhanced activity arose partly from higher microsomal cholesterol and partly from increases in total enzyme activity. Cytosolic neutral cholesteryl ester hydrolase (CEH) activity was substantially elevated by simvastatin (3-fold) and by the drug combination (6-fold), whereas the effect of cholestyramine was smaller (1.5-fold). Normal chow for 24 h only partially returned cytosolic CEH activity to control values. Microsomal CEH activity was increased by simvastatin, alone and in combination with cholestyramine (1.4 to 1.7-fold), and was also enhanced, in the cholestyramine-treated animals, following drug withdrawal. Removal of simvastatin did not allow recovery of this enzyme activity, while withdrawal of the drug combination led to values 29% below controls. The results indicate that in the rat, simvastatin and cholestyramine alter both ACAT and CEH activity, as well as inhibiting

maintenance of whole-body sterol balance, since net cholesterol excretion occurs almost exclusively through the hepatobiliary tract. In most species, inhibition of the rate-determining step in hepatic de novo cholesterol synthesis, the reductive cleavage of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to mevalonate, catalyzed by HMG-CoA reductase, leads to an increase in low density lipoprotein (LDL) receptor numbers and a consequent decrease in plasma LDL cholesterol (2,3). Hence, drug-induced inhibition of HMG-CoA reductase activity is one of the major strategies for dealing with hypercholesterolemia in patients (4-6). Among the drugs that have been clinically approved for this purpose are the statins, lovastatin, simvastatin, and pravastatin, which are specific competitive inhibitors of the enzyme (3-5). However, it is becoming apparent that the mechanism of action of these HMG-CoA reductase inhibitors is more complex than simply the inhibition of cholesterol synthesis, with consequent up-regulation of LDL receptors and increased catabolism of LDL and other apo-B-cont~ining lipoproteins (6). In particular, evidence has been accumulating that one effect of these drugs is to stimulate the induction of HMG-CoA reductase in the cell to restore cholesterol synthesis (7,8). However, cells do not accumulate large quantities of this compound, and cholesterol, excess to the immediate requirement of the cell, is deposited in the form of cytoplasmic lipid HMG-CoA reductase activity. droplets containing insoluble cholesterol-fatty acid esters. Lipids 30, 917-926 (1995). These are formed from long-chain acyl-CoA derivatives and cholesterol in a reaction catalyzed by acyl CoA:cholesterol The maintenance of cholesterol balance in the intact liver in- acyltransferase (ACAT, EC 2.3.1.26) (9). These esters are not volves the regulation of several pathways of cholesterol in- inert but can be rehydrolyzed to release the cholesterol for celflux and effiux, including the uptake of chylomicron and very lular use. Thus, the regulation of hepatic CE metabolism is low density lipoprotein (VLDL) remnants, the secretion of mediated, in part, by the balance between this esterification by cholesterol and bile acids into bile, and the synthesis and se- ACAT and hydrolysis of the resultant CEs, by a neutral chocretion of lipoproteins as well as storage as cholesteryl esters lesteryl ester hydrolase (CEH, EC 3.1.1.13) (10,11). Never(CEs) (1). In addition, the liver is critically involved in the theless, it has been suggested that ACAT plays the major role in this regulation, by providing a defense against excess free *To whomcorrespondenceshouldbe addressed. Abbreviations:ACAT.AcyI-CoA:cholesterylacyltransferase;CE, choles- cholesterol accumulation (12) with CEH, which includes both teryl ester; CEH, cholesterylesterhydrolase;HDL,high densitylipoprotein; cytosolic (soluble) and membrane-associated fractions (11,13) HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA;LDL. low density lipoproplaying only a passive role, and having no importance in maintein; TAG, triacylglycerols;TRIS, tris(hydroxymethyl)-aminomethane; taining free cholesteryl levels in the hepatocyte (12). VLDL, verylow densitylipoprotein. Copyright 9 1995 by AOCS Press

917

Lipids, Vol. 30, no. 10 (1995)

918

J.l~l. SHAND AND D.W. WEST

Although the mechanisms controlling the activity of these two opposing enzymes are unknown at present, recent evidence has suggested that the statins have major effects on ACAT activity. In the cells of the intestinal wall, ACAT has been implicated in the process of cholesterol absorption from the gut (14-16), and Ishida et al. (17,18) found that simvastatin increased fecal excretion of sterol in rabbits fed cholesterol, but not in rabbits fed on normal chow. Kam et al. (19) found similar results when the ACAT inhibitor 58-035 (Sandoz, Inc., East Hanover, NJ) was used to inhibit the enzyme activity in human intestinal CaCo-2 cells (supplied by Drs. E. Schaefer and J. Ordovas, Tufts University School of Medicine, N J). Since cholesterol feeding increases the ACAT levels in intestinal cells, it was postulated that simvastatin was inhibiting the activity of this enzyme. In a study of VLDL-lipid secretion in perfused livers of rats given iovastatin, Khan et al. (20) found that, while hepatic concentrations of free cholesterol were unaltered, the CE content was markedly reduced along with VLDL-lipid production. Lovastatin was also found to inhibit ACAT in, and to decrease the basal secretion of, newly synthesized CE and triacylglycerols (TAG) from Caco-2 cells (21). These authors postulated that this effect of lovastatin on lipid secretion was due to its inhibition of ACAT. They suggested, furthermore, that the results of Kahn et al. (20) also could be explained on the basis that lovastatin was regulating CE content and VLDL secretion by its inhibition of ACAT activity and not by its effect on newly synthesized cholesterol, as suggested by Reimann et al. (22). Further supporting evidence was provided by Kasim et al. (23) who found that lovastatin-treated obese Zucker rats also showed a decline in VLDL secretion and CE content with no change in free cholesterol. Since the substrate of ACAT was freely available, they again raised the possibility that lovastatin may suppress the activity of ACAT. In contrast, recent investigations have shown that ACAT activity in human liver (24) and in mononuclear leucocyte microsomes (25) was not inhibited by pravastatin, whereas it was increased in diabetic patients following simvastatin treatment (26). Furthermore, Ishida et ak (27) demonstrated that simvastatin had a direct inhibitory effect on ACAT in isolated intestinal microsomes although the parent lactone form of the drug rather than the open ~-hydroxy form was inhibitory in this system. In contrast, the free hydroxy acid, which is rapidly formed in the liver and in the plasma of rats, is the inhibitor of HMG-CoA reductase in tissues. Thus, there is controversy concerning the effect of this drug on ACAT, and although simvastatin is reported to have reduced hepatic ACAT in cholesterol-fed rabbits in a preliminary experiment (18), there appears to have been no systematic investigation of the inhibition of hepatic ACAT by the statins. Cholestyramine is a second type of drug that is used to control plasma cholesterol and LDL levels. This resin sequesters bile acids in the gut, preventing their recirculation to the liver (28,29), one consequence of which is increased diversion of free cholesterol in the liver toward bile acid syn-

Lipids, Vol. 30, no. 10 (1995)

thesis. The liver compensates by producing more cholesterol, via induction of HMG-CoA reductase (30-32), which then tends to blunt the effectiveness of this agent in reducing plasma cholesterol. Because of this effect, cholestyramine often has been combined with the inhibitors of HMG-CoA reductase in treatment of hypercholesterolemia. We therefore fed both simvastatin and cholestyramine, alone and in combination, to male rats and investigated the effect of the drugs on a number of parameters of hepatic cholesterol metabolism. Since removal of simvastatin leads to a rapid reversal of the induction of HMG-CoA reductase (33), we also investigated the effect of refeeding these animals with control diet for a short period. The results indicate that, while these drug treatments influence HMG-CoA reductase activity as expected, they also reduce hepatic ACAT activity and at the same time induce a compensatory increase in the activity of the neutral CEHs in the cell. Refeeding the rats with control diet for 24 h returns HMG-CoA reductase to control levels, but the changes in ACAT and CEH activity require a longer period to overcome the effects of the drugs. These variations in the activities of the cholesterol metabolizing enzymes have major effects on the balance between cholesterol and CE in the cell and plasma. MATERIALS AND METHODS Materials. [9,10(n)-3H]Oleic acid (370 GBq/mmol), [l-14C]-

oleic acid (2 Gbq/mmol), cholesteryl [ 1-14C] oleate (2 Gbq/ mmol), and DL-3-hydroxy-3-methyl[3-1ac]glutaric acid (2 Gbq/mmol) were all obtained frown Amersham (Little Chalfont, United Kingdom). RS-[5-3H]Mevalonolactone (750 Gbq/mmol) was purchased from DuPont (Stevenage, United Kingdom). Cholesteryl [9,10(n)-3H]oleate, [9,10(n)-3H] oleoyl CoA, and 3-hydroxy-3-methyl[3-14C]glutaryI-CoA were synthesized as described previously (34-36). Dithiothreitol was purchased from Calbiochem (Nottingham, United Kingdom). All other biochemicals were from Sigma (Poole, United Kingdom). A n i m a l s a n d diets. Male Wistar rats, weighing about 120-140 g at the start of the experiment, were kept on a constant light/dark cycle (lights on 0800-2000 h) and were fed for five days on a crushed CRM(X) breeding diet (Special Diet Services, Manea, Cambridgeshire, United Kingdom) containing 2.4% (w/w) fat and less than 0.01% (w/w) cholesterol. Rats were split into groups and for five days were fed either the control diet, or that same diet supplemented with cholestyramine (2%), simvastatin (0. 1%), or a combination of cholestyramine (2%) and simvastatin (0. I%). At the end of the five-day period, the controls and one-half of the drugtreated rats were killed while the remainder of the treated rats were returned to the control diet for 24 h before they were killed. Simvastatin was incorporated by mixing an acetone solution with the crushed diet then allowing the solvent to evaporate for at least 48 h. The control diet was treated in the same way but with acetone alone, and cholestyramine resin was mixed dry into this treated control diet. The rats were fed

SIMVASTATIN, CHOLESTYRAMINE, AND CHOLESTEROL METABOLISM

at 0830 at the rate of 30 g per rat per day and were weighed at daily intervals. At the end of the experimental period, the animals were killed by cervical dislocation at 0930, i.e., 25 h after their last feed, and the livers were excised, weighed, and frozen in liquid nitrogen. Blood samples were centrifuged immediately (10,000 • g) in heparinized tubes and the clear plasmas stored at-20~ Tissue processing. Livers were homogenized as described previously (34,37) in 4 vol of ice-cold buffer consisting of 50 mM 2-(N-morpholino)ethanesulfonic acid, 50 mM tris(hydroxymethyl)-aminomethane (TRIS)/HCI buffer, pH 7.2, containing 1 mM ethylenediaminetetraacetic acid, I mM ethyleneglycol-bis(2-aminoethyl)tetraacetic acid, 1 mM dithiothreitol, and proteinase inhibitors (1 Iag/mL each of pepstatin A, leupeptin, and antipain). Microsomal and cytosolic fractions were then prepared according to the method of Shand et al. (34,37) and stored in aliquots at -80 ~ C. Enzyme assays. The activity of ACAT was assayed in microsomes (150 pg protein) by measuring the rate of conversion of [9,10(n)-3H]oleoyl CoA to cholesteryl [9,10(n)-3H]oleate as previously described using endogenous cholesterol as the other substrate (35). In addition, parallel assays were performed in the presence of exogenous cholesterol (200 lag/mL) added as a suspension of liposomes, prepared as described by Batzri and Korn (38), using cholesterol and phosphatidylcholine in a molar ratio of 1:2. All assays were preincubated for 30 min before addition of the oleoyl CoA substrate, and incubation time was limited to 5 min to ensure linearity. Cholesteryl [1-14C]oleate was used as an internal standard to correct for recovery. The activity of neutral CEH was measured, essentially according to Shand and West (34), using 50 lag of either microsomal or cytosolic protein in a total volume of 200 laL. The reaction was initiated by the addition of an ethanolic solution of cholesteryl [9,10(n)-3H]oleate (20 nmol, I • 106 dpm) and terminated after 45 min. [l-14C]Oleic acid was then added as a recovery standard, and the rate of hydrolysis of the substrate was measured by estimation of the released [9,10(n)-3H]oleic acid. The 'total' activity of HMG-CoA reductase was determined using 100 pg of microsomal protein in a final volume of 150 laL according to the method described by Shand and West (36). The reaction was initiated by addition of 3-hydroxy-3-methyl[3-14C]glutaryl-CoA (22 nmol, 2 x 105 dpm) and terminated after 15 min at 37~ by addition of concentrated HCI (10 laL). A recovery standard of RS-[5-3Hlmeval onolactone (30 lamol, 1.5 • l 0 4 dpm) was added and the reaction product separated by thin-layer chromatography. Assay of the neutral CEH inhibitor protein. The liver cytosols were assayed for inhibitory activity against rat mammary gland microsomal neutral CEH essentially as described previously (34) using 30--40 lag of cytosolic protein. The quantity of cytosolic protein required to produce 50% inhibition of the neutral CEH was then calculated. Controls, without mammary gland microsomes, were incubated at the same time to allow correction for the activity of cytosolic neutral CEH.

919

Estimation of cholesterol and CEs. Lipids, extracted (39) from the liver homogenates and microsomal suspensions, were redissolved in ethanol while those extracted from plasma lipids were resuspended, by sonication, in buffer conraining 50 mM TRIS, pH 7.9, 100 mM KCI, 20 mM KF, and 0.05% (wt/vol) Triton X-100. Total and free cholesterol were then assayed by the fluorometric method of Gamble et al. (40) and esterified cholesterol estimated by the difference. Estimation of TAG. TAG were estimated directly in the liver homogenates and in the aqueous suspensions of the extracted plasma lipids by a modification of the method of McGowan et al. (41) using assay kit number 339-10 as supplied by Sigma. Protein and DNA measurements. Protein concentrations were assayed by the dye-binding method of Bradford (42) and DNA as described by Labarca and Paigen (43). RESULTS

Diet-induced effects of cholestyramine. Animals fed the cholestyramine diet gained the same weight as controls but exhibited a small (12%) reduction in liver weight. This diet led to a significant increase in liver TAG, whether expressed on a cellular (i.e., related to DNA concentration) or a total liver basis (+23% and +33% respectively, Table 1). CE levels were unaffected whereas cholesterol levels were reduced (-9%, P < 0.01) in cellular terms (Table I). Drug withdrawal led to increases in all of these parameters within 24 h. In contrast, the diet containing cholestyramine had little effect on plasma lipid levels although drug withdrawal led to a 32% drop in plasma cholesterol levels (Table 2). HMG-CoA reductase activity was increased (1.6-fold) by the cholestyramine treatment (Table 4), as were both microsomal (Table 4) and cytosolic (Table 3) CEH activities, although the increases were much less than was observed with the simvastatin treatment (I.I- and 1.5-fold, respectively, Fig. 1) correlating with a significant decrease (-27%, P < 0.01) in the activity of the cytosolic inhibitor of CEH (Table 3). Refeeding normal diet returned HMG-CoA reductase, cytosolic CEH, and inhibitor activity toward normal values but increased the microsomal CEH still further (to 200% of controls). A significant reduction in ACAT activity (-71%, P < 0.001, Table 4) was also observed in cholestyraminetreated animals, and this also was observed when the assay was repeated in the presence of exogenously supplied cholesterol. Following drug withdrawal, ACAT activity was significantly raised both when measured with endogenous cholesterol (+54%, P < 0.001) and with saturating cholesterol concentrations (+18%, P < 0.05), but not sufficiently to return it to the control value. The evidence from the scatter diagram relating ACAT activity to microsomal cholesterol content (Fig. 2) suggests that this enhancement was not due to an increase in the level of microsomal cholesterol. Diet-induced effects of simvastatin. Animals treated with simvastatin gained considerably less weight during the period of the experiment, and this was reflected in a significant re-

Lipids, Vol. 30, no. 10 (1995)

920

I.H. SHANI.) A N D D.W. WESq

TABLE 1 The Effect of Treatment with Simvastatin and Cholestyramine, Alone and in Combination, on Liver Lipid Levels a Triacylglycerol Treatment

mg/mg D N A

(C)

Cholesterol

rag/total liver

pg/mg D N A

Cholesteryl esters (CE)

rag/total liver

iJg/mg D N A

Ratio

mg/total liver

CE/C in total liver

Control (10)

1.9 • 0.1

80.9 • 5.2

315.6 • 8.5

13.9 _~ 0.5

200.2 • 7.1

8.9 • 0.5

0.63 • 0.03

Cholestyramine (6)

2.3 • 0.1 c

107.6 • 4.4 c

286.6 + 3.8 c

13.2 • 0.5

187.7 • 3.6

8.7 • 0.4

0.66 • 0.02

Cholestyramine + 24-h control diet (6)

2.6 • 0.1 d

111.7•

371.3 • 7.6 d

16.1 _~ 0.9

266.0•

Simvastatin (6)

1.9 • 0.1

70.7 • 5.0

211.7 • 15.3 a

5imvastatin + 24-h control diet (6)

2.1 •

88.4•

246.3•

Simvastatin + cholestyramine (10)

2.0•

86.5•

318.3•

Simvastatin + cholestyramine + 24-h control diet (10)

2.0•

97.8•

b

338.4•

d

b

7.7 • 0.56

11.5•

0.72__0.02

152.6 • 7.0 d

5.4 • 0.2 d

0.73 • 0.03

10.0_~ 0.5 c'e

199.4•

8.1 •

0.81 :t0.03 c

13.6•

220.9•

9.5•

0.68+0.03

16.1 •

b,e

e

3 5 4 . 7 • 13.5 d'f 1 6 . 7 •

d

1.05•

d'f

aLiver triacylglycerol levels were determined directly in the tissue homogenates, whereas liver cholesterol and cholesteryl esters were measured by means of a fluorometric assay in lipid extracts, dissolved in ethanol, as described in the Materials and Methods section. The results are the means • SEM for the number of animals given in parentheses. Significant differences from control values (b,c,d) and between treatments and treatments plus 24 h on control diet (e,h were determined using Student's t-test.bP < 0.05. c, e p < 0.01. d,fp< 0.001.

duction (7.1 _+0.1 g to 5.8 _+0.2 g) in their liver weights. They regained much of their lost body and liver weight after refeeding normal diet. The drug had little effect on the levels of TAG in the plasma (Table 2) and the liver (Table i), but significantly reduced the cholesterol and the CE content of both plasma and liver, as well as that measured in the microsomal fraction of the liver (Table 5). Drug withdrawal allowed the microsomal and the plasma levels of both lipids to return to normal, whereas hepatic free cholesterol levels remained depressed. HMG--CoA reductase activity was markedly increased by simvastatin treatment (Table 4) as was CEH activity, both microsomal (Table 4) and cytosolic (Table 3). The proportionate increase for the cytosolic CEH was, nevertheless, greater than that for the microsomal CEH (3.0- and 1.7-fold respectively,

Fig. I). In contrast, the activity of the inhibitor of CEH was unaffected (Table 3), whereas ACAT activity, measured both in the presence of endogenous and exogenous cholesterol, was dramatically reduced (-75%, Table 4). Feeding normal chow for 24 h allowed the activity of HMG-CoA reductase to decline toward control values but did not cause any further alteration in the elevated activities of the two CEHs. ACAT activity measured under conditions of saturating cholesterol was unaltered following drug withdrawal, whereas it was increased in the presence of endogenous cholesterol. The scatter diagram (Fig. 2) indicates that this stimulation in ACAT was solely due to increased availability of substrate.

Diet-induced effects of cholestyramine plus simvastatin. The livers, obtained from the animals fed these combined drugs,

TABLE 2 The Effect of Treatment with Cholestyramine, Alone and in Combination with Simvastatin, on Plasma Lipid Levels a Triacylglycerol (mg/100 mL)

Cholesterol (C) (mg/]00 mL)

Cholesterol ester (CE) (mg/lO0 mL)

Control (6)

47.2 + 6.5

17.6 + 1.4

22.7 + 1.5

1.31 + 0.08

Cholestyramine (6)

53.7 • 10.4

16.8 • 1.7

19.2 _+ 1.4

1.16 • 0.08

Treatment

Ratio CE/C

Cholestyramine + 24-h control diet (6)

57.9 • 3.0

12.0 • 0.3 c

2 2 . 6 + 1.1

1.87•

Simvastatin (6)

48.5 • 3.2

10.2 • 0.5 a

16.5 • 0.8 (:

1.63 • 0.07

Simvastatin + 24-h control diet (7)

4 7 . 8 • 1.1

15.1 •

2 2 . 7 • 1.4

1.51 •

Simvastatin + cholestyramine (10)

28.4 • 2.7 b

24.8 + 1.8 c

20.7 • 1.5

0.88 • 0.07

Simvastatin + cholestyramine + 24-h control diet (10)

35.5 • 5.2

13.3 + 1.1 b,f

14.7 • 1.1 d'e

1.16 • 0.05

aplasma triacylglycerols and total cholesterol were estimated in lipid extracts of plasma resuspended in buffer as described in the Materials and Methods section. The results are the means + SEM for the number of animals given in parentheses. Significant differences from control values (b,c,d) and between treatment and treatment plus 24 h on control diet (e,t~ were determined using Student's t-test. b p < 0.05. c, ep < 0.01. d,fp < 0.001.

Lipids, Vol. 30, no. 10 (1995)

SIMVASTATIN, CHOLESTYRAMINE, A N D CHOLESTEROL METABOLISM

921

TABLE 3 The Effects of Treatment with Simvastatin and Cholestyramine, Alone and in Combination, on the Activities of the Cytosolic Neutral Cholesteryl Ester Hydrolase and the Cytosolic Protein Inhibitor of Neutral Cholesteryl Ester Hydrolasea Cytosolic CEH

"True" cytosolic CEH activity (allowing for inhibitor activity)

Cytosolic CEH inhibitor activity

% inhibition pmol/min/ mg protein

nmol/min/ total liver

by 50 l.tg protein

50% inhibitory units/total liver (xl 03)

pmol/min/ mg protein

nmol/min/ total liver

Control (8)

13.2 _+ 0.6

11.6 _+ 0.8

47.8 _+ 1.5

16.4 _+ 1.1

24.5 _+ 0.5

21.7 • 1.4

Chotestyramine (6)

20.2 _+ 1.4 d

17.6 _+ 1.2 c

34.9 • 3.4 c

12.1 _+ 1.1 b

31.2 + 1.9 c

27.1 -+ 2.1

Treatment

Cholestyramine + 24-h control diet (6)

14.9•

13.5_+2.3

51.3_+4.9

18.7_+1.7

31.9•

28.7_+4.8

Simvastatin (6)

52.4 _+2.8 ~

32.7 _+2.4 ~

44.3 • 1.9

11.0 • 0.7 c

95.2 _+6.6 d

59.2 _+ 5.0 d

Simvastatin + 24-h control diet (6)

39.7_+1.9 d'e

28.6-+1.96

46.5_+3.7

11.7_+2.4

76.4_+6.3 d

55.8+6.1 a

Simvastatin + cholestyramine (8)

77.4 -+ 4.3 d

61.0 _+4.2 d

37.8 _+ 1.6 c

12.1 • 0.8 c

124.9 _+ 6.8 d

98.6 _+6.2 a

Simvastatin + cholestyramine + 24-h control diet (8)

42.0 _+ 2.46't

44.8 _+ 1.46

45.5 • 2.2

19.3 _+ 0 . 6 b

79.2 _+ 7.2 a

83.5 • 4.9 d

aCytosolic neutral cholesteryl ester hydrolase (CEH) activity was determined using 50 pg of cytosolic protein incubated with cholesteryl [9,10-3H]oleate for 45 rain. The CEH inhibitor protein was assayed by determination of the CEH activity in rat mammary gland microsomes (100 pg protein) in the absence and presence of 30-50 pg of cytosolic protein. The activity in whole liver is expressed as 50% inhibitory units where one unit is the amount (pg) required to inhibit the mammary microsomal CEH by 50%. From an estimate of the percentage inhibition of the CEH activity due to 50 pg, the "true" cytosolic CEH activities could be recalculated to allow for the effect of the inhibitory protein. The results are the means -+ SEM for the number of animals given in parentheses. Significant differences from control values (b,c,d) and between treatments and treatments plus 24 h on control diet (e,t) were determined using Student's t-test, bp < 0.05. c,ep < 0.01. d,fp < 0.001.

weighed 25% less than either controls or animals similarly treated but refed control diet for 24 h after drug withdrawal. Plasma TAG were reduced by the cholestyramine plus simvastatin treatment (-40%, Table 2), whereas hepatic TAG were unaltered (Table 1). Refeeding normal diet for 24 h did not restore plasma TAG to normal levels but did lead to a significant increase (+21%, P < 0.05) in the levels of hepatic TAG.

Hepatic free cholesterol levels were unaffected by this drug combination (Table 1) while cholesterol levels in the plasma (Table 2) and in the microsomes (Table 5) reacted in opposition, being increased in the plasma (+41%) but reduced in the microsomes (-16%). CE levels in the plasma and in the whole liver were not significantly affected by this treatment, whereas the microsomal content of esterified cholesterol was

TABLE 4 The Effect of Treatment with Simvastatin and Cholestyramine, Alone and in Combination, on the Activities of the Microsomal Enzymes AcyI-CoA:Cholesterol Acyltransferase, HMG-CoA Reductase, and Neutral Cholesteryl Ester Hydrolasea Acyl-CoA:cholesterol acyltransferase Endogenous cholesterol Treatment

pmol/minl mg protein

HMG--CoA reductase nmollminl total liver

pmollminl mg protein

nmollmin/ total liver

35.2 • 3.2

399.4 _+ 50.4

37.5 _+4.1

137.6+7.1

12.9+0.5

644.1 • 52.2 c

57.4 _+ 5.5 b

152.1 +7.1

13.5_+0.7

Control

368.9 • 25.4

558.1 • 8.9

Cholestyramine

106.3 • 6.7 d

221.9 _+ 10.7 d

Cholestyramine + 24-h control diet

1 6 3 . 7 + 7 . 3 d'/

2 6 2 . 5 + 1 1 . 6 d'g 2 0 . 6 •

Simvastatin Simvastatin + 24-h control diet Simvastatin + cholestyramine Simvastatin + cholestyramine + 24-h control diet (4)

93.6•

d

261.4+19.5 d

150.9 • 23.6 d'g 251.3 _+26.5 d 70.0•

d

Cholesteryl ester hydrolase

pmollmin/ mg protein

150.3•

d,/

247.9 • 30.0 b'h 378.6 • 31.6 d

9.5 _+0.8 d c

347.1 •

i

15.8•

8.1 + 0 . 6 d 1 4 8 7 . 7 + 8 4 . 8 d 13.5 _+2.0 d 7.4•

22.1 • 1.8 c

387.0 • 56.5 i

d'i

130.6_+6.8 d

678.6 • 105.7 b

b 1683.5•

pmollmin/ mg protein

d

61.2 _+ 10.1 b 174.0+21.1 d 33.9_+ 3.4 i

nmollmin/ total liver

182.9 • 4.0 d'i 22.8 + 1.5 d'i 236.1 _+5.6 d't 20.8 _+0.8 d 233.7 _+8.1d

20.9 _+ 1.0 d

186.1 + 15.8b'f19.3 + 2.7 b 98.0 • 4.4 c'g

8.9 • 0.3 d

aAcyl-CoA:cholesterol acyltransferase was assayed using 150 pg microsomal protein incubated with [9,10-3H]oleoyl CoA for 5 min. Activity was measured using the cholesterol endogenous to the microsomes and in the presence of saturating amounts of exogenous cholesterol added as cholesterohphosphatidylcholine liposomes as described in the Materials and Methods section. 3-Hydroxy-3-methylglutaryl (HMG)~CoA reductase activity was measured by incubation of 100 lag of protein with 3-hydroxy-3-methyl[3- 14 C]-CoA for I 5 min. Neutral cholesteryl ester hydrolase activity was estimated by incubating 50 pg microsomal protein with cholesteryl [9,10-3H]oleate for 45 min. The results are the means 4- SEM for six animals except where indicated by the number in parentheses. Significant differences from control values (b,c,d), between treatments (e,/} and between treatments and treatments plus 24 h on control diet (g,h,i) were determined using Student's t-test, b,gp< 0.05. ep< 0.02. c,hp< 0.01. dZip< 0.001.

Lipids, Vol. 30, no. 10 (1995)

922

J.H. SHAND AND D.W. W[ST

[ • microsomal " •

100

CEH

1108.2) (115.7)

I ~ " - I cytosolic CEH

"1"

9LM 1- 80

(80.0) 133.3)

~o o ,..

(40.61 F.~:

wl

60

40

V/I tD U

Treatment (50.9) Control (8) Cholestyramine (6)

I

v/1 v/1 v/1 v~ v/t

no 20

(76.7)

H

~d VA

g'2

.N~

-g

c

+

9

E~ ~8

z~

"~

o ~

.E_ ~

m ~

"~,+

significantly lowered (-17%, P < 0.001). Withdrawal of the drugs led, within 24 h, to an increase in the cholesterol level in the whole liver (+16%) but reversed the increase in plasma free cholesterol (to 24% below control) without affecting microsomal levels. In contrast, hepatic and microsomal CE content was significantly increased (+77 and +18%, respectively)

500

a.

oD 400

E

~**

r .i

E o

300

* []

E a. 200

O

,I

[]

8~

.i

o ,(

100

I-,< O ,<

0

~ 9 ~Aimnan

A e ,4i

IIN

I

I

I

I

I

I

15

20

2s

30

3s

40

Microsomal Cholesterol (pglmg protein) FIG. 2. Scatter diagram relating acyI-CoA:cholesterol acyltransferase (ACAT) activities to microsomal contents of cholesterol. Controls (,k,), and animals treated with simvastatin (rT,i), cholestyramine (O,O) and a combination of the two drugs (A,A). Open symbols represent drug treatments, filled symbols represent drug-treated animals refed control diet for 24 h.

Lipids, Vol. 30, no. 10 11995)

31.8 + 0.9 24.1 + 0.76

19.6 + 0.6 16.2 + 0.7 c

Cholestyramine + 24-h control diet (6)

21.6 + 0.76

13.6 + 0.46

0.63 + 0.01

Simvastatin (6)

27.6 • 1.1 b

13.0 • 0.56

0.47 + 0.026

Simvastatin + 24-h control diet (6)

33.4 + 0.7 e

20.4 + 0.7 f

0.61 • 0.02

26.6 + 0.9 d

16.1 •

0.61 •

26.1 •

2 3 . 0 + 0 . 7 cJ

(10)

'* |

FIG. 1. Histogram showing the relative proportions of the total cholesteryl ester hydrolase (CEH) activity in the cytosolic and in the microsoreal fractions of the livers (nmol/min/total liver) following drug treatment. Cytosolic CEH activity was calculated making allowance for the activity of the inhibitory protein of CEH in the appropriate cytosols; sim, simvastatin; cholestyr, cholestyramine.

o o

Cholesterol (C) Cholesteryl esters (CE) Ratio (pg/mg protein) (pg/mg protein) CE/C 0.60 + 0.02 0.67 + 0.02

Simvastatin + cholestyramine

g§

.E

TABLE 5 The Effects of Treatment with Simvastatin and Cholestyramine, Alone and in Combination, on the Microsomal Contents of Cholesterol and Cholesteryl Estersa

Simvastatin + cholestyramine + 2 4 - h c o n t r o l diet (10)

d

d

0.88 • 0.03 d

aCholesterol and cholesteryl esters were determined by means of a fluorometric assay on the extracted microsomal lipids in ethanolic solution, rhe results are the means • SEM for the number of animals given in parentheses. Significant differences from control values (b,c,d) and between treatments and treatments plus 24 h on control diet (eft) were determined using Student's t-test, bp< 0.05. c,ep< 0.01. d,fp< 0.001.

following reversion to the normal diet, and this was reflected in a decrease (-35%) in the CE of the plasma. Feeding both drugs had major effects on the activities of the enzymes of cholesterol metabolism. HMG-CoA reductase was higher (4.2-fold), and ACAT activity, measured both with and without exogenous cholesterol, was significantly reduced (-73 and -81%, respectively, P < 0.001 ), both effects being greater than was observed with simvastatin alone (Table 4). Refeeding control diet for 24 h returned HMG-CoA reductase activities to that of the controls and markedly increased ACAT activities (3.5-fold), which nevertheless remained substantially below control levels. The data in Figure 2 indicates that the enhanced ACAT activity, following drug withdrawal, occurred through a combination of nonsubstrate modulation of enzyme activity, together with an increase in substrate availability. Cytosolic CEH was dramatically increased (5.9-fold) by this combined drug treatment (Table 3), and this was correlated with a significant reduction (-21%, P < 0.01) in the activity of the cytosolic inhibitory protein. Microsomal CEH was also increased, although to a lesser extent (l.4-fold, Table 4), as was emphasized in Figure 1. Withdrawal of the drugs reduced microsomal CEH to below control values, but cytosolic CEH remained substantially elevated (3.8-fold). Allowance for the effect of the cytosolic inhibitor of CEH activity made a quantitative difference but did not alter the relative effects of the drugs (Table 3). It did, however, emphasize that the relative proportions of cytosolic to microsomal activity were markedly higher in the refed animals compared with the drugtreated animals (9.4: I and 5. I : I, respectively, Fig. 1).

SIMVASTATIN, CHOLESTYRAMINE,AND CHOLESTEROLMETABOLISM DISCUSSION

The results obtained in the present investigation confirm that the effects of the cholesterol-lowering drugs simvastatin and cholestyramine, alone and in combination, on hepatic cholesterol metabolism are much more complex than simply the inhibition of HMG-CoA reductase with a secondary increase in LDL receptors (4). They clearly cause major variations in the hepatic activities of ACAT and the neutral CEHs which lead to changes in the levels of cholesterol, CEs, and TAG, both in the liver and the plasma of these animals. Moreover, it is clear both from previously published results (44-46) and from the data presented here that the variations in CEH activity, arising from the different dietary conditions, may have an equal importance with ACAT in maintaining intracellular cholesterol levels. Cholestyramine administration, by promoting fecal loss of bile salts (28,29), creates a cellular need for free cholesterol to replace cholesterol utilized for bile salt synthesis (30) which is apparently met by induction of microsomal HMG-CoA reductase activity (30-32; Table 4) and by inhibition of its degradation (47). However, our data indicate that this free cholesterol requirement also could be partially met by a decrease in CE formation, through a reduction in ACAT activity (Table 4), or an increase in CE hydrolysis, via enhancement of CEH activity (Table 4). The significant variation in ACAT was in agreement with previous results (12), whereas the significant increase in cytosolic, but not microsomal, CEH activity contrasted with previous reports (12,48). The change in A C A T appeared to occur partially from a decline in the amount of microsomal cholesterol (Table 5) and partially from a decline in either the amount, or the activity, of the enzyme as indicated by the 50% loss of activity in the presence of saturating concentrations of cholesterol (Table 4). Consideration of the scatter diagram relating ACAT activity to microsomal cholesterol content (Fig. 2) also would suggest that increases in the amount or activity of the enzyme, rather than substrate availability, was the explanation for the enhanced ACAT activity observed when simvastatin was withdrawn. The net result of these changes in enzyme activity was a significant decrease in microsomal cholesterol and CE content although the total hepatic content of these sterols was unaltered. However, at the present stage, it is not possible to apportion these effects between the various enzymes involved. The major reason for the variation between our results and those previously published (12) in relation to CEH activity arises from the different conditions that were used in the assay. The hepatic cytosol contains a protein inhibitor of this enzyme activity (34) for which allowance can be made by performing the assay with small (50 lag) amounts of protein. Stone et aL (12) employed much larger quantities of cytosolic protein, and under the conditions of their assay, it can be assumed that less than 10% of the CEH activity was measurable. Hence, variations in this enzyme activity, due to dietary manipulation, could not be observed.

923

Simvastatin administration also stimulates the induction of HMG-CoA reductase (47) but, at the same time, leads to a decrease in the activity of ACAT, thus allowing unesterified cholesterol to enter the free cholesterol pool (Table 4). A reduction in hepatic ACAT activity in rats due to simvastatin has not been reported previously, although such a reduction has been proposed to explain a selective decrease in hepatic CE content (23), and lovastatin has been shown to inhibit ACAT activity in intestinal cells (18,21). Microsomal cholesterol was, however, only reduced by 14% below controls, suggesting that the decline (-75%) in ACAT activity was only partially due to decreased substrate availability and was predominantly due to a nonsubstrate modulation of enzyme activity. This interpretation was supported by the 53% decrease in ACAT activity observed in the presence of saturating concentrations of cholesterol. Perhaps more significantly in this situation, both microsomal and cytosolic CEH activities were substantially increased (Tables 3 and 4). The combined effect of reduced ACAT and increased CEH was a significant decline in both total and microsomal CE. Nevertheless, in spite of these compensatory variations in HMG-CoA reductase, ACAT, and CEH, hepatic cholesterol content was still markedly reduced below control values. This may result from increased disposal of cholesterol via the biliary route since administration of an HMG-CoA reductase inhibitor, both with (49) and without (50) the addition of a second drug (AOMA, a copolymer of maleic acid and an tx-olefin of 18 carbon chains; Monsanto, St. Louis, MO) to block intestinal cholesterol absorption, prompted an increased output of cholesterol to bile. Moreover, since increased biliary cholesterol secretion also has been reported to occur when ACAT activity was inhibited by progesterone (51,52), the reduction in ACAT observed in the present investigation would also be consistent with increased biliary sterol excretion. The rapid decline in HMG-CoA reductase activity following withdrawal of the simvastatin correlates with the known short half life of this enzyme (47). However, simvastatin is eliminated from the plasma even more quickly (5), and hence, one effect of withdrawal of the drug is a large overshoot in cholesterol production (33). This expanded pool of newly synthesized cholesterol can enrich the hepatic microsomal membrane fraction, part of which can be esterified by ACAT, as indicated by the increased enzyme activity in this fraction (Table 4). Moreover, since the total ACAT activity, measured under saturating substrate conditions, was unaltered, it can be inferred that the observed enhancement of ACAT activity was solely due to the increased substrate availability (Fig. 2) rather than any change in other factors capable of modulating enzyme activity, such as covalent modification (53) or variation in the amount of enzyme protein. Since total ACAT activity was not increased, there appeared to be a limit to the proportion of the excess cholesterol that could be converted to the ester, and this was further constrained by the high CEH activity, which showed negligible response to withdrawal of simvastatin. The inability to further increase the hepatic stores of CEs could help to explain the marked in-

Lipids, Vol. 30, no. 10 (1995)

924

I.H. SHAND AND D.W. WFST

crease in the rate of biliary sterol secretion reported by Bilhartz et al. (33). The marked induction of H M G - C o A reductase activity which results from the diet containing simvastatin and cholestyramine (7,8,30-32) was confirmed by the data presented here. The reduction in ACAT activity following this treatment, which was even greater than that observed in response to the individual drugs, ensured that newly synthesized cholesterol arising from the induced HMG--CoA reductase was not converted to the ester. This strategy was supported by the greater than fivefold increase in cytosolic CEH activity, the net result of which should have been increased free cholesterol levels in the cell. However, in spite of these changes in ACAT and CEH, hepatic cholesterol and CE remained at the same level as in untreated animals, and microsomal CE was only slightly reduced. Major changes had, nevertheless, occurred in the levels of plasma cholesterol, which were substantially elevated, suggesting that increased export of cholesterol to the plasma may have contributed to this maintenance of intracellular sterol levels. A rise in serum cholesterol levels has been observed in rats fed lovastatin for seven days (50), and an increase in high-density lipoprotein (HDL) cholesterol was reported in rats in which hepatic ACAT activity had been inhibited (54). In contrast, Mitchell et al. (55) reported a reduction in plasma cholesterol in rats fed this drug combination. The difference between our results and those of Mitchell et al. (55) could be explained by the fact that their animals were fasted overnight, a period when they normally would be consuming the bulk of their food, whereas the animals in our study and in that reported in (50) had access to food up to the time of slaughter. Nevertheless, both Mitchell et al. (55) and ourselves observed a fall in plasma TG. They attributed this partly to decreased secretion of VLDL, arising from a decline in the hepatic content of mRNA species coding for apo-B, and partly to upregulation of the B/E receptor, leading to increased clearance. Interestingly, a fall in plasma TAG was also recorded (54) when hepatic ACAT was inhibited, and others have shown that inhibition of ACAT selectively decreased the plasma content of apo-B containing lipoproteins and shifted the distribution of cholesterol to HDL (56). Thus, the fact that ACAT activity was inhibited by both simvastatin and cholestyramine also would support the suggestion of reduced TAG secretion. As expected, H M G - C o A reductase activity was reduced to control levels following withdrawal of the drugs but, in contrast to the situation with simvastatin alone, total ACAT activity, i.e., measured with saturating substrate, was markedly increased. In these animals, ACAT would have esterified a greater proportion of the expanded pool of free cholesterol, and this, together with the reduced CEH activity present in the microsomal fraction, resulted in a significant increase in microsomal CEs. Nevertheless, the data indicate that, although cytosolic CEH activity remained substantially elevated, there was an accumulation of both cholesterol and CE in the liver. This correlated with significant reductions in

Lipids, Vol. 30, no. 10 (1995)

the plasma level of these sterols, which could arise from either decreased secretion of lipoproteins and/or increased clearance from the circulation. The increase in the plasma levels of TAG after drug withdrawal would argue against a further reduction in VLDL secretion, particularly as there was an accumulation of hepatic CEs, which have been implicated as a requirement for VLDL secrction (51). Increased clearance is the more likely cause, arising from an upregulation of HDL receptors, since cholesterol loading of cells, that can utilize HDL particles, has been shown to have such effects on plasma sterol levels (57). Furthermore, although this drug combination has been shown to decrease hepatic TAG lipase activity, it resulted in increased levels of the mRNA species coding from this lipase (58). A rapid rise in hepatic TAG lipase levels on drug withdrawal also could contribute to the lowered plasma sterol levels observed. The cause of the variations in enzyme activity are unknown at present, although available evidence would argue against direct action of the drugs on the enzymes. Cholestyramine remains in the gut and does not enter the circulation, and although the lactone form of simvastatin has been reported to directly inhibit intestinal ACAT, in vitro, the open [3-hydroxy acid form of the drug was ineffective (27). Since the lactone is rapidly converted into the hydroxy acid in both rat plasma (5) and liver (59-61), it is unlikely that much of the lactone form exists within the circulation. The results presented here are important since they demonstrate that manipulation of cholesterol synthesis in the rat hepatocyte has major effects on all of the enzymes involved in hepatic cholesterol metabolism, not just on the rate-determining enzyme of cholesterol synthesis. Whether such changes also occur in humans is a subject worthy of investigation.

ACKNOWLEDGMENTS The authors would like to acknowledge the generous donation of simvastatin by Dr. M. Mark (Karl Thomae GMBH, Biberach, Germany). Financial support was supplied by the Scottish Office Agriculture and Fisheries Department.

REFERENCES !. Turley, S.D., and Dietschy, J.M. (1982) in The Liver." Biology and Pathobiology (Arias, I., Popper, H., Schachter, D., and Shafritz, D.A., eds.) pp. 467--492, Raven Press, New York. 2. Goldstein, J.L., and Brown, M.S. (1990) Regulation of the Mevalonate Pathway, Nature 343, 425-430. 3. Davignon, J., Montigny, M., and Dufour, R. (1992) HMG-CoA Reductase Inhibitors: A Look Back and a Look Forward, Can. J. Cardiol. 8, 843-864. 4. Feussner, G. (1994) HMG CoA Reductase Inhibitors, Curr. Opin. Lipidol. 5, 59-68. 5. Blum, C.B. (I 994) Comparison of Properties of Four lnhibitors of 3-Hydroxy-3-MethylglutaryI-Coenzyme A Reductase, Am. J. CardioL 73, 3D-I ID. 6. Hunninghake, D.B. (1992) HMG CoA Reductase Inhibtors, Curr. Opin. Lipidol. 3. 22-28. 7. Chin, D.J., Luskey, K.L., Anderson, R.G.W., Faust, J.R., Goldstein, J.L., and Brown, M.S. (1982) Appearance of Crystalloid Endoplasmic Reticulum in Compactin-Resistant Chinese Hampster Cells With a 500-Fold Increase in 3-Hydroxy-3-Methylglu-

SIMVASTATIN, CHOLESTYRAMINE,AND CHOI ESTEROIMETABOLISM

taryl Coenzyme A Reductase, Proc, Natl. Acad. Sci. USA. 79, 1185-1189. 8. Roitelman, J., and Shechter, I. (1986) Altered Kinetic Properties of Rat Liver 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Following Dietary Manipulations, J. Biol. Chem. 261, 5061-5066. 9. Chang, T.Y., and Doolittle, G.M. (1983) Acyl Coenzyme A:Cholesterol O-Acyltransferase, in The Enzymes, 3rd edn. (Boyer, P., ed.) Vol. 16, pp. 523-539, Academic Press, New York, 10. Dekin, D., and Goodman, D.S. (1962) The Hydrolysis of LongChain Fatty Acid Esters with Rat Liver Enzymes, J. Biol. Chem. 237, 3649-3656. 11. Nilsson, A. (1976) Hydrolysis of Chyle Cholesterol Esters With Ceil-Free Preparations of Rat Liver, Biochim. Biophys. Acta 450, 379-389. 12. Stone, B.G., Evans, C.D., Fadden, R.J., and Scheiber, D. (1989) Regulation of Hepatic Cholesterol Ester Hydrolase and AcylCoenzyme A:Cholesterol Acyltransferase in the Rat, J. Lipid Res. 30, 1681-1690. 13. Erickson, S.K., Lear, S.R., and McCreery, M.J. (1994) Functional Sizes of Hepatic Enzymes of Cholesteryl Ester Metabolism Determined by Radiation Inactivation, J. Lipid Res. 35, 763-769. 14. Heider, J.G., Pickens, C.E., and Kelly, L.A. (1983) Role of AcyI-Coenzyme A:Cholesteroi Acyltransferase in Cholesterol Absorption and Its Inhibition by 57-I i 8 in the Rabbit, J. Lipid Res. 24, 1127-1134. 15. Clark, S.B., and Tercyak, A.M. (1984) Reduced Cholesterol Transmucosal Transport in Rats with Inhibited Mucosal Acyl CoA:Cholesterol Acyltransferase and Normal Pancreatic Function, Z Lipid Res. 25, 148-159. 16. Williams, R.J., McCarthy, A.D., and Sutherland, C.D. (1989) Esterification and Absorption of Cholesterol: In Vitro and in Vivo Observations in the Rat, Biochim. Biophys. Acta 1003, 213-216. 17. Ishida, S., Sato, A., Iizuka, Y., Sawasaki, Y., Aizawa, A., and Kamei, T. (1988), Effects of MK-733, an Inhibitor of 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase, on Absorption and Excretion of [3H]Cholesterol in Rabbits, Biochim. Biophys. Acta 963, 35-41. 18. Ishida, F., Sato, A., lizuka, Y., Kitani, K., Sawasaki, Y., and Kamei, T. (1989) Effects of MK-733 (Simvastatin), an Inhibitor of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase, on Intestinal Acylcoenzyme A:Cholesterol Acyltransferase Activity in Rabbits, Biochim. Biophys. Acta 1004, ! 17-123. 19. Kam, N.T.P., Albright, E., Mathur, S.N., and Field, F.J. (1989) Inhibition of Acyl CoenzymeA:Cholesterol Acyltransferase activity in CaCo-2 Cells Results in Intracellular Triglyceride Accumulation, J. Lipid Res. 30, 371-377. 20. Kahn, B., Wilcox, H.G., and Heimberg, M. (1989), Cholesterol Is Required for Secretion of Very-Low-Density Lipoprotein by Rat Liver, Biochem. J. 259, 807-816. 21. Kam, N.T.P., Albright, E., Mathur, S.N., and Field, F.J. (I 990)Effect of Lovastatin on AcyI-CoA:Cholesterol O-Acyltransferase (ACAT) Activity and the Basolateral-Membrane Secretion of Newly Synthesized Lipids by CaCo-2 Cells, Biochem. J. 272, 427-433. 22. Reimann, F.M., Herold, G., Grosshans, I., Rogler, G., Fellermann, K., and Stange, E.F. (1992) Regulation of Cholesterol Metabolism and Low-Density Lipoprotein Binding in Human Intestinal CaCo-2 Cells, Digestion 51, 10-17. 23. Kasim, S.E., LeBoeuf, R.C., Khilnani, S., Tailapaka, L., Dayananda, D., and Jen, K-L.C. (1992) Mechanisms of Triglyceride-Lowering Effect of an HMG-CoA Reductase Inhibitor in a Hypertriglyceridemic Animal Model, the Zucker Obese Rat, J. Lipid Res. 33, 1-7.

925

24. Reinher, E., Rudling, M., Stahlberg, D., Berglund, L., Ewerth, S., Bjorkhem, I., Einarsson, K., and Angelin, B. (1990) Influence of Pravastatin, a Specific Inhibitor of HMG-CoA Reductase, on Hepatic Metabolism of Cholesterol, N. Eng. J. Med. 323, 224--228. 25. Owens, D., Collins, P., Johnson, A., Tighe, O., Robinson, K., and Tomkin, G.H. (1991) Hypercholesterolaemia: Simvastatin and Pravastatin Alter Cholesterol Metabolism by Different Mechanisms, Biochim. Biophys. Acta 1082, 303-309. 26. Owens, D., Stinson, J., Collins, P., Johnson, A., and Tomkin, G.H (199 I) Improvement in the Regulation of Cellular Cholesterologenesis in Diabetes: the Effect of Reduction in Serum Cholesterol by Simvastatin, Diabetic Med. 8, 151-156. 27. lshida, F., Sato, A., Iizuka, Y., and Kamei, T. (1989) Inhibition of Acyl Coenzyme A:Cholesterol Acyltransferase by 3-1tydroxy-3-Methylglutaryl Coenzyme A Reductase, Chem. Pharm. Bull. 37, 1635-1636. 28. Goldfarb, S., and Pitot, H.C. (1972) Stimulatory Effect of Dietary Lipid and Cholestyramine on Hepatic HMG-CoA Reductase, J. Lipid Res. 13, 797-801. 29. Hardgrave, J.E., Heller, R.A., Herrera, M.G., and Scallen, T.J. (1979) Immunotitration of HMG-CoA Reductase in Various Physiological States, Proc. Natl. Acad. Sci. USA. 76, 3834-3838. 30. Spady, D.K., Turley, S.D., and Dietschy, J.M. (1985) Rates of Low Density Lipoprotein Uptake and Cholesterol Synthesis are Regulated Independently in the Liver, J. Lipid Res. 26, 465-472. 31. Goldstein, J.L., and Brown, M.S. (1984) Progress in Understanding the LDL Receptor and HMG-CoA Reductase, Two Membrane Proteins That Regulate Plasma Cholesterol, J. Lipid Res. 25, 1450-1461. 32. Tanaka, R.D., Edwards, P.A., Lan, S-F., Knoppel, E.M., and Fogelman, A.M. (1982) The Effect of Cholestyramine and Mevinolin on the Cholesterol Cycle of Rat Hepatic 3-Hydroxy-3methylglutaryl Coenzyme A Reductase, J. Lipid Res. 23, 1026-1031. 33. Bilhartz, L.E., Spady, D.K., and Dietschy, J.M. (1989) Inappropriate Hepatic Cholesterol Synthesis Expands the Cellular Pool of Sterol Available for Recruitment by Bile Acids in the Rat, J. Clin. Invest. 84, 1181-1187. 34. Shand, J.H., and West, D.W. (19.92) Characterization of a Cytosolic Protein in Rat Liver Inhibiting Neutral Cholesteryl Ester Hydrolase, Lipids 27, 406-412. 35. Shand, J.H., and West, D.W. (1991) Acyl-CoA:Cholesterol Acyltransferase Activity in the Rat Mammary Gland: Variation During Pregnancy and Lactation, Lipids 26, 150-154. 36. Shand, J.H., and West, D.W. (1991) Effects of an Antiserum to Rat Growth Hormone and Bromocriptine on Cholesterol-metabolizing Enzymes in the Lactating Rat Mammary Gland, J. Endocrinol 128, 287-295. 37. Shand, J.H., West, D.W., McCartney, R.J., Noble, R.C., and Speake, B.K. (1993) The Esterification of Cholesterol in the Yolk Sac Membrane of the Chick Embryo, Lipids 28, 621-625. 38. Batzri, S., and Korn, E.D. (1973) Single Bilayer Liposomes Prepared Without Sonication, Biochim. Biophys. Acta 298, 1015-1019. 39. Folch, J., Lees, M., and Sioane-Stanley, G.N. (1957) A Simple Method for the Isolation and Purification of Total Lipids from Animal Tissues, J. Biol. Chem. 226, 497-509. 40. Gamble, W., Vaughan, M., Kruth, H.S., and Avigan, J. (1978) Procedure for Determination of Free and Total Cholesterol in Micro- or Nanogram Amounts for Studies With Cultured Cells, J. Lipid Res. 19, 1068-1070. 41. McGowan, M.W., Artiss, J.D., Strandbergh, D.R., and Zak, B. (1983) Enzymic Colorimetry of Lecithin and Sphingomyelin in Aqueous Solution, Clin. Chem. 29, 538-542. 42. Bradford, M.M. (1976) A Rapid and Sensitive Method for the

Lipids, Vol. 30, no. 10 (1995)

926

43. 44.

45. 46.

47.

48.

49.

50. 51. 52.

53.

J.H. SHAND AND D.W. WEST

Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding, Anal. Biochem. 72, 248-254. Labarca, C., and Paigen, K. (1980) A Simple, Rapid and Sensitive DNA Assay Procedure, Anal. Biochem. 102, 344-352. Ghosh, S., Kounnas, M.Z., and Grogan, W.M. (1990) Separation and Differential Activation of Rat Liver Cytosolic Cholesteryl Ester Hydrolase, Trigylceride Lipase and Retinyl Palmitate Hydrolase by Cholestyramine and Protein Kinases, Lipids 25, 221-225. Shand, J.H., and West, D.W. (1994) The Effects of Clofibrate and Bezafibrate on Cholesterol Metabolism in the Liver of the Male Rat, Lipids 29, 747-752 Shand, J.H., and West, D.W. (1995) The Effects of Probucol and Clofibrate Alone and in Combination on Hepatic Cholesterol Metabolism in the Male Rat, Biochim. Biophys. Acta 1255, 123-130. Edwards, P.A., Lan, S-F., and Fogelman, A.M. (1983) Alterations in the Rates of Synthesis and Degradation of Rat Liver 3Hydroxy-3-Methylglutaryl Coenzyme A Reductase Produced by Cholestyramine and Mevinolin, J. Biol. Chem. 258, 10219-10222. Erickson, S.K., Shrewsbury, M.A., Brooks, C., and Meyer, D.J. (1980) Rat Liver Acyl-Coenzyme A:Cholesterol Acyltransferase: Its Regulation in Vivo and Some of Its Properties in Vitro, J. Lipid Res. 21,930-941. Turley, S.D., and Dietschy, J.M. (1984) Modulation of the Stimulatory Effect of Pregnenolone-16ct-carbonitrile on Biliary Cholesterol Output in the Rat by Manipulation of the Rate of Hepatic Cholesterol Synthesis, Gastroenterology 87, 284-292. Yamauchi, S., Linscheer, W.G., and Beach, D.H. (1991) Increase in Serum and Bile Cholesterol and HMG-CoA Reductase by Lovastatin in Rats, Am J. Physiol. 23, G625--G-630. Nervi, F., Bronfman, M., Allalon, W., Depiereux, E., and Del Pozo, R. (1984) Regulation of Biliary Cholesterol Secretion in the Rat, J. Clin. Invest. 74, 2226-2237. Stone, B.G., Erickson, S.K., Craig, W.Y., and Cooper, A.D. (1985) Regulation of Rat Biliary Cholesterol Secretion by Agents That Alter Intrahepatic Cholesterol Metabolism, J. Clin. Invest. 76, 1773-1781. Suckling, K.E., Strange, E.F., and Dietschy, J.M. (1983) Dual Modulation of Hepatic and Intestinal Acyl Coenzyme A:Cho-

Lipids, Vol. 30, no. 10 (1995)

54.

55.

56.

57.

58.

59.

60.

61.

lesterol Acyltransferase Activity by (De-)Phosphorylation and Substrate Supply in Vitro, FEBS Lett. 151, 111-116. Bell, F.P., Gammill, R.B., and St. John, L.C. (1992) U-73482: A Novel ACAT Inhibitor That Elevates HDL-Cholesterol, Lowers Plasma Triglyceride anf Facilitates Hepatic Cholesterol Mobilization in the Rat, Atherosclerosis 92, 115-122. Mitchell, A., Fidge, N., and Griffiths, P. (1993) The Effect of the HMG-CoA Reductase Inhibitor Simvastatin and of Cholestyramine on Hepatic Apolipoprotein mRNA levels in the Rat, Biochim. Biophys. Acta 1167, 9-14. Carr, T.P., Parks, J.S., and Rudei, L.L. (1992) Hepatic ACAT Activity in African Green Monkeys Is Highly Correlated to Plasma LDL Cholesteryl Ester Enrichment and Coronary Artery Atherosclerosis, Arteriosclerosis 12, 1274-1283. Svirldov, D.D., Pavlov, M.Y., Safonova, I.G., Repin, V.S., and Smirnov, V.N. (1990) Inhibition of Cholesterol Synthesis and Esterification Regulates High Density Lipoprotein Interaction with Isolated Epithelial Cells of Human Intestine, J. Lipid Res. 31, 1821-1830. Benhiza, F., Lagrange, D., Malewiak, M-I., and Griglio, S. (1994) In Vivo Regulation of Hepatic Lipase Activity and mRNA Levels by Diets Which Modify Cholesterol Influx to the Liver, Biochim. Biophys. Acta 1211, 181-188. Vyas, K.P., Karl, P.H., Prakash, S.R., and Duggan, D.E. (1990) Biotransformation of Lovastatin II. In Vivo Metabolism by Rat and Mouse Liver Microsomes and Involvement of Cytochrome P-450 in Dehydrogenation of Lovastatin, Drug Metab. Dispos. 18, 218-222. Vickers, S., Duncan, C.A., Vyas, K.P., Karl, P.H., Arison, B., Prakash, S.R.. Ramjit, H.G., Pitzenberger, S.M., Stokker, G., and Duggan, D.E. (1990) In Vitro and In vivo Biotransformation of Simvastatin, an Inhibitor of HMG CoA Reductase, Drug Metab. Dispos. 18, 476-483. Parker, R.A., Clark, R.W., Sit, S-Y., Lanier, T.L., Grosso, R.A., and Wright, J.J.K. (1990) Selective Inhibition of Cholesterol Synthesis in Liver versus Extrahepatic Tissues by HMG-CoA Reductase Inhibitors, J. Lipid Res. 31, 1271-1282.

[Received January 24, 1995, and in final revised form May 24, 1995; Revision accepted June 27, 1995]