Opposite effects on cholesterol metabolism and their mechanisms induced by dietary oleic acid and palmitic acid in hamsters

BB ELSEVIER

Biochimica et Biophysica Acta 1258 (1995) 251-256

Biochi ~mic~a et BiophysicaA~ta

Opposite effects on cholesterol metabolism and their mechanisms induced by dietary oleic acid and palmitic acid in hamsters Hitoshi Kurushima, Kozo Hayashi *, Tetsuji Shingu, Yoshio Kuga, Harumi Ohtani, Yoshifumi Okura, Kouichi Tanaka, Yuji Yasunobu, Katsuhiko Nomura, Goro Kajiyama First Department of Internal Medicine, Hiroshima Unil~ersity School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan

Received 2 March 1995; accepted 16 May 1995

Abstract The effects of dietary oleic acid on cholesterol metabolism were investigated and compared with those of palmitic acid in hamsters. Addition of 5% oleic acid to a 0.1% cholesterol-supplemented diet decreased plasma total cholesterol, very low density lipoprotein (VLDL) cholesterol, and low density lipoprotein (LDL) cholesterol, increased hepatic LDL receptor activity, and decreased plasma cholesteryl ester transfer protein (CETP) activity in comparison with 0.1% cholesterol alone. In contrast, addition of 5% palmitic acid to a 0.1% cholesterol-supplemented diet increased total cholesterol and LDL-cholesterol, increased plasma CETP activity, and suppressed hepatic LDL receptor activity to a greater extent than 0.1% cholesterol alone. Neither oleic acid nor palmitic acid altered hepatic microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity, but oleic acid increased hepatic microsomal cholesterol 7c~-hydroxylase activity. These results suggest that dietary oleic acid inhibits the increases in total, VLDL-, and LDL-cholesterol induced by dietary cholesterol by preventing both LDL receptor suppression and increased CETP activity, whereas dietary palmitic acid augments the cholesterol-induced increases in total and LDL-cholesterol by both further suppression of LDL receptor activity and further stimulation of CETP activity. Keywords: Diet; Oleic acid; Palmitic acid; LDL receptor; Cholesteryl ester transfer protein; Cholesterol 7c~-hydroxylase

1. Introduction

It is well known that dietary fatty acids influence plasma cholesterol concentrations and may play an important role in the development of atherosclerotic disease [1-11]. Polyunsaturated fatty acids are known to reduce plasma total cholesterol and LDL-cholesterol when they replace saturated fatty acids in the diet. There are several explanations about the mechanisms by which dietary fatty acids affect plasma cholesterol concentrations such as changes in lipoprotein composition [12], in LDL production [13], in VLDL-cholesterol secretion from the liver, and in hepatic LDL receptor activity [14-19]. Dietary saturated fatty acids are known to influence plasma choles-

Abbreviations: CE, cholesteryl ester; CETP, cholesteryl ester transfer protein; EDTA, ethylenediaminetetraacetic acid; HDL, high density lipoprotein; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; LDL, low density lipoprotein; NADPH, /3-nicotinamide adenine dinucleotide phosphate (reduced form); VLDL, very low density lipoprotein * Corresponding author. Fax: + 81 82 2575194. 0005-2760/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 5 - 2 7 6 0 ( 9 5 ) 0 0 1 2 2 - 0

terol through the suppression of hepatic LDL receptor activity [15-19], or the acceleration of VLDL secretion from the liver [14], and dietary polyunsaturated fatty acids by preventing the suppression of hepatic LDL receptor activity [14,16]. Monounsaturated fatty acids had been historically known to have no effect on plasma cholesterol concentrations [20], but recently it was reported that oleic acid (monounsaturated fatty acid) reduced plasma total cholesterol and LDL-cholesterol levels without the accompanying decrease in high density lipoprotein (HDL) cholesterol induced by polyunsaturated fatty acids [7]. Moreover, an epidemiological study showed that death rates from coronary heart disease were low in countries such as Greece and southern Italy, where the traditional diet was high in olive oil, which was rich in oleic acid [21]. However, the mechanisms by which dietary oleic acid affects plasma cholesterol are not clear. In male Golden Syrian hamsters, we investigated the effects of dietary oleic acid on cholesterol metabolism in comparison with those of dietary palmitic acid, and measured the activities of hepatic LDL receptor, HMG-CoA

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H. Kurushima et al. / Biochimica et Biophysica Acta 1258 (1995) 251-256

reductase, plasma CETP, which facilitates the transfer of cholesteryl esters and triacylglycerols between lipoprotein particles and plays an important role in determining cholesterol distribution among lipoprotein particles [2228], and hepatic cholesterol 7c~-hydroxylase, which catalyzes the rate-limiting step in the conversion of cholesterol into bile acids [29,30].

Havel et al. [31]. The lipoproteins were dialyzed at 4°C against a solution of 0.15 M NaC1 and 0.2 mM EDTA (pH 7.4). Lipoproteins in d > 1.125 plasma were labeled with [4-J4C]cholesterol as described previously [25]. [14C]Cholesteryl ester (CE) HDL 3 was isolated by ultracentrifugation in KBr at d = 1.21 g / m l , harvested by slicing the tube, and dialyzed against a solution of 0.15 M NaCI and 0.2 mM EDTA (pH 7.4).

2. Materials and methods

2.4. Analytical procedures

2.1. Chemicals

Hamster plasma was isolated from blood collected into tubes containing 0.15% EDTA (w/v). VLDL ( d < 1.006 g/ml), LDL (1.019 < d < 1.063 g / m l ) and HDL (1.063 < d < 1.210 g / m l ) were isolated by a modification of the method described by Havel et al. [31]. Cholesterol concentration of each lipoprotein fraction was assayed enzymatically with TC kit-K obtained from Nippon Shoji [33].

All reagents were of the highest purity commercially available. Glucose-6-phosphate, /3-nicotinamide adenine dinucleotide phosphate (reduced form), glucose-6-phosphate dehydrogenase, D,L-3-hydroxy-3-methylglutarylcoenzyme A, isocitrate dehydrogenase, leupeptin, and pepstatin were purchased from Sigma. Cholesterol oxidase and Scintisol 500 were obtained from Wako. D,L-Hydroxymethyl[3- lac]glutaryl_coenzyme A, R,S-[5- 3H(N)]meva_ lonolactone, and [4-14C]cholesterol were obtained from NEN. Sodium [ 125I]iodine was obtained from Amersham.

2.2. Animals and diets Male Golden Syrian hamsters, each weighing 80 g, were housed in standard cages for 4 weeks in natural lighting conditions before use. The standard diet, obtained from Oriental Kobo, contained 0.05% cholesterol, 0.57% linoleic acid, 0.30% oleic acid, 0.16% palmitic acid, and 0.12% other fatty acids. The experimental diets were prepared by adding cholesterol with or without fatty acids to the standard diet. Animals were fed a standard diet alone (n = 8) or diets supplemented with 0.1% cholesterol (n = 6), 0.1% cholesterol plus 5% oleic acid (n = 6), or 0.1% cholesterol plus 5% palmitic acid (n = 6). All experimental diets were fed ad libitum for 4 weeks, and all experiments were started at 9:00 a.m. after a 12-h fast.

2.3. Preparation of lipoproteins Hamster plasma was isolated from blood collected into tubes containing 0.15% EDTA ( w / v ) from normolipidemic fasting hamsters fed a standard diet. LDL (1.019 < density ( d ) < 1.063) was isolated by a modification of the procedure of Havel et al. [31] with sequential ultracentrifugation in KBr. The lipoproteins were dialyzed at 4°C against a solution of 0.15 M NaC1 and 0.2 mM EDTA (pH 7.4). LDL was radiolabeled with 125I by the iodine monochloride procedure as described previously [32]. Human plasma was isolated from blood collected into tubes containing 0.15% EDTA ( w / v ) from normolipidemic fasting healthy volunteers. VLDL plus LDL ( d < i.063 plasma) and d > 1.125 plasma for the CETP assay were isolated by a modification of the procedure of

2.5. Preparation of liL,er microsomes Liver microsomes were prepared by a modification of the method described previously [34,35]. Pieces of each liver (approx. 3 g) were weighed and placed in cold homogenization medium (10:1, v / w ) containing 0.25 M sucrose, 1 mM NaEDTA, 1 /xg/ml leupeptin and pepstatin, and 50 mM potassium phosphate (pH 7.4). The liver was homogenized at 4°C in a Dounce homogenizer. Each homogenate was centrifuged for 12 min at 8500 × g at 4°C, and the supernatant fraction was then centrifuged for 60 min at 100000 × g at 4°C. The pellets were resuspended in the same homogenization medium and then centrifuged for 60 min at 100000 × g at 4°C.

2.6. Assay of hepatic LDL receptor actiuit3, Liver membranes were prepared from hamsters, and hepatic LDL receptor activity was determined as described previously [19,36]. In this study, each reaction mixture contained 10 /xg of sample protein in a final volume of 100 /xl.

2.7. Assay of hepatic microsomal HMG-CoA reductase activi~ Liver microsomes were prepared, and HMG-CoA reductase activity was determined as described previously [35,37].

2.8. Assay of hepatic microsomal cholesterol 7ce-hydroxylase acti~'i~., Cholesterol 7ce-hydroxylase activity was measured by using microsomal cholesterol as a substrate with a modification of the method described by Okuda et al. [38,39]. Approx. 500 /xg of microsomes were incubated at 37°C

H. Kurushima et al. / Biochimica et Biophysica Acta 1258 (1995) 251-256 with 0.1 M potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA, 20 mM cysteamine-HCI, 5 mM MgCI 2, 5 mM sodium isocitrate, 0.1 unit of isocitrate dehydrogenase, and 0.5 mM NADPH in a final volume of 0.25 ml. The reaction was started by the addition of NADPH. After 20 min, 30 /~1 of 5% ( w / v ) sodium cholate and 5 /xl of cholesterol oxidase (276 u n i t s / m l ) dissolved in 10 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol, 1 mM NaEDTA, and 1 mM cysteamine-HCl were added to the reaction mixture, and the mixture was incubated for another 20 rain. The reaction was terminated by adding 0.3 ml of methanol, and the mixture was extracted with 3 ml of petroleum ether. The extract was evaporated to dryness and analyzed by high-performance liquid chromatography with a Zolbax Sil column (4.6 × 250 ram) (Shimadzu, Kyoto) and a mixture of n-hexane and isopropanol (9:!, v / v ) . Authentic 7a-hydroxycholesterol (0.2 nmol) was used for calibration, and 7/3-hydroxycholesterol (0.125 nmol) was used as an internal standard.

2.9. Assay of plasma cholesteryl ester transfer protein ( CETP) activity

253

activities in animals fed diets supplemented with cholesterol alone or cholesterol plus fatty acids were expressed as percentage of control.

2.10. Determination of microsomal protein Microsomal protein was determined by the method of Lowry et al. [40], with bovine serum albumin as a standard.

2.11. Statistical analysis Statistical analyses were carried out by Kruskal-Wallis and Scheffe's statistical methods. A level of P < 0.05 was accepted as statistically significant.

3. Results

3.1. Effects of dietary cholesterol and fatty acids on plasma cholesterol concentrations

CETP activity was determined as the percentage of labeled substrate transferred in 6 h at 37°C from human [14C]CE-HDL3 to human VLDL plus LDL [25]. Each assay tube contained [14C]CE-HDL3 (50 /zg cholesterol) (about 300000 dpm of [14C]) as the donor, unlabeled VLDL plus LDL (500 /.tg cholesterol) as the acceptor, and 5 0 / z l of hamster plasma or blank buffer (10 mM Tris, 150 mM NaC1, pH 7.4) in a final volume of 600 /zl. The tubes were incubated for 6 h at 37°C in a shaking water bath. After incubation, the tubes were chilled on ice for 30 min to stop the reaction, and the donor and acceptor lipoproteins were separated by heparin-MnCl 2 precipitation. Aliquots of the supernatant were transferred to scintillation vials, each containing 20 ml of scintillant (Scintisol 500). The samples were counted in a Liquid Scintillation System, LSC-3500 (Aloka). The percentage of transfer of labeled substrate was determined, the mean CETP activity in animals fed a standard diet was set to 100, and CETP

Plasma cholesterol concentrations of animals after feeding for 4 weeks are given in Table 1. The addition of 0.1% cholesterol to the standard diet significantly increased plasma total cholesterol, VLDL-cholesterol, and LDLcholesterol in comparison with a standard diet. The addition of 5% palmitic acid to a 0.1% cholesterol-supplemented diet further increased plasma total cholesterol and L D L - c h o l e s t e r o l . W h e n c o m p a r e d with a 0.1% cholesterol-supplemented diet, the addition of 5% oleic acid significantly decreased plasma total cholesterol, VLDL-cholesterol, and LDL-cholesterol, but did not affect HDL-cholesterol. The substitution of oleic acid for palmitic acid significantly decreased plasma total cholesterol, VLDL-cholesterol, and LDL-cholesterol.

3.2. Effects of dietary cholesterol and fatty acids on hepatic LDL receptor activity Hepatic LDL receptor activities are shown in Fig. I. The data are plotted according to a modification of the

Table 1 Effects of dietary cholesterol and fatty acids on plasma cholesterol concentrations Diet

n

Total cholesterol (mg/dl)

VLDL cholesterol(mg/dl)

LDL cholesterol(mg/dl)

HDL cholesterol(mg/dl)

Standard diet Standard diet + 0.1% cholesterol Standard diet + 0.1% cholesterol + 5% oleic acid Standard diet + 0.1% cholesterol + 5% palmiticacid

8 6 6 6

111.9_+14.3 180.4+ 19.4 ~ 143.5-+ 22.1 ~'~ 222.0_+20.6 ~x,e

23.7___ 12.0 49.2 -+ 20.2 b 27.7 -+ 22.1 d 69.7 _+ 15.2 ~.e

26.4+ 16.4 45.7 -+ 8.6 b 28.8 _+5.5 d 74.5 ± 20.1 a.c.~

61.9_+ 16.2 86.3 _+ 18.6 ~ 84.5 _+8.9 ~ 77.0 + 29.9

Groups of animals were fed a standard diet alone or diets supplemented with 0.1% cholesterol, 0.1% cholesterol+5% oleic acid, or 0.1% cholesterol + 5% palmitic acid. Each value represents the mean _+S.D. ~' Significantlydifferent from standard diet at P < 0.01. b Significantlydifferent from standard diet at P < 0.05. ' Significantlydifferent from standard diet + 0.1% cholesterol at P < 0.01. d Significantlydifferent from standard diet + 0.1% cholesterol at P < 0.05. Significantlydifferent from standard diet + 0.1% cholesterol + 5% oleic acid at P < 0.01.

H. Kurushima et al. / Biochimica et Biophysica Acta 1258 (1995) 251-256

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(ng protein / lag s a m p l e protein ) Fig. I. Effects o f dietary cholesterol and fatty acids on hepatic L D L receptor activity. G r o u p s o f animals were led a standard diet alone ((2)) or diets supplemented with 0.1% cholesterol ( Q ) , 0.1% cholesterol + 5% oleic acid (El), or 0.1% c h o l e s t e r o l + 5 % palmitic acid ( B ) , Hepatic L D L receptor activities were d e t e r m i n e d as described in Section 2. The data are plotted a c c o r d i n g to a modification o f the method described by Scatchard. B o u n d / F r e e represents the a m o u n t of specially b o u n d L D L divided by the concentration of u n b o u n d L D L in the reaction mixture.

method described by Scatchard [41]. In comparison with a standard diet, the addition of 0.1% cholesterol with or without 5% oleic acid or 5% palmitic acid did not affect the affinity of the receptors for LDL ( K d, 10 /zg/ml). The addition of 0. ! % dietary cholesterol to a standard diet did not affect hepatic LDL receptor activity (Bm~×, 0.64 versus 0.70 ng of LDL-protein bound / / x g of sample protein). The addition of 5% dietary oleic acid to a 0.1% cholesterol-supplemented diet increased hepatic LDL receptor activity about 1.5 times (Bm, x, 1.06 ng of LDL-protein bound / / z g of sample protein), while the addition of 5% palmitic acid suppressed LDL receptor activity (B . . . . 0.38 ng of LDL-protein bound / / z g of sample protein). These results suggest that hepatic LDL receptor activity is suppressed by 0.05-0.15% ( w / w ) dietary cholesterol and that the addition of oleic acid prevents this suppression of hepatic LDL receptor activity, whereas the addition of palmitic acid augments the suppression of hepatic LDL receptor activity. 3.3. Effects of dieta O, cholesterol and fato' acids on hepatic microsomal HMG-CoA reductase actic'i O, Hepatic microsomal HMG-CoA reductase activities are shown in Fig. 2. The addition of 0.1% cholesterol to the standard diet significantly decreased microsomal HMGCoA reductase activity, and the further addition of 5% oleic acid or palmitic acid did not modify this decrease.

C+P

Fig. 2. Effects of dietary cholesterol and fatty acids on hepatic microsoreal H M G - C o A reductase activity. G r o u p s of animals were fed a standard diet alone (St) (n = 8) or diets supplemented with 0.1% cholesterol (Ch) ( n = 6 ) , 0.1% c h o l e s t e r o l + 5 % oleic acid ( C + O ) ( n = 6 ) , or 0.1% cholesterol + 5% palmitic acid (C + P) (n = 6). H M G - C o A reductase activities were determined as described in Section 2. ~' Significantly different at P < 0.01.

3.4. Effects q[dietar), cholesterol and fat O, acids on plasma cholester3,1 ester tran~fer protein (CETP) acticitv Plasma CETP activities are given in Table 2. In comparison with the standard diet, the addition of 0.1% cholesterol significantly increased CETP activity. In comparison with a 0.1% cholesterol-supplemented diet, the addition of 5% oleic acid significantly decreased CETP activity, while the addition of 5% palmitic acid further increased CETP activity. 3.5. Effects of dieta~ cholesterol and fatty acids oli hepatic' microsomal cholesterol 7c~-hydroxylase actit,i O, Hepatic microsomal cholesterol 7u-hydroxylase activities are shown in Fig. 3. The addition of 0.1% dietary Table 2 Effects of dietary cholesterol and fatty acids on cholesteryl ester transfer protein (CETP) activity Diet

n

C E T P activity (cA' control)

Standard diet Standard diet + 0.1% cholesterol Standard diet + 0.1% cholesterol + 5c~ oleic acid

8 6 6

100.0 _+6.5 148.2 -- 20.6 " 120.5 -- 12. I h.~

Standard diet + 0. I c/~ cholesterol + 5'~/~ palmitic acid

6

195.7 _+_13.7 ~"'J

G r o u p s of animals were fed a standard diet alone or diets supplemented with 0.1% cholesterol, 0.1% cholesterol + 5 % oleic acid, or 0.1% cholesterol + 5'~ palmitic acid. C E T P activities were determined as described in Section 2. The mean C E T P activity in animals fed a standard diet was set to 100, and C E T P activities in animals fed diets supplemented with cholesterol alone or cholesterol plus fatty acids were expressed as percent control. Each value represents the mean 4- S.D. J Significantly different from standard diet at P < 0.01. b Significantly different f r o m standard diet at P < 0.05. Significantly different f r o m standard diet + 0. 1% cholesterol at P < 0.01. d Significantly different from standard diet + 0. 1% cholesterol + 5% oleic acid at P < 0.01.

H. Kurushima et al. / Biochimica et Biophysica Acta 1258 (1995) 251-256

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Fig. 3. Effects of dietary cholesterol and fatty acids on hepatic mlcrosomal cholesterol 7a-hydroxylase activity. Groups of animals were fed a standard diet alone (St) (n = 8) or diets supplemented with 0.1% cholesterol (Ch) (n =6), 0.1% cholesterol+5% oleic acid ( C + O ) (n =6), or 0.1% cholesterol + 5% palmitic acid (C + P) (n = 6). Cholesterol 7cr-hydroxylase activities were determined as described in Section 2. a Significantly different at P < 0.01.

cholesterol with or without palmitic acid had no influence on cholesterol 7ce-hydroxylase activity, while the addition of dietary oleic acid caused a significant increase in this activity.

4. Discussion The male hamster is an useful model of human lipoprotein metabolism, because the level of plasma LDLcholesterol and the rate of hepatic sterol synthesis are similar to those in humans [8,15,16]. Therefore, using male hamsters as models, we investigated the mechanisms of the cholesterol-lowering effect of dietary oleic acid and compared them with those of the effect of dietary palmitic acid on cholesterol metabolism. The changes in plasma cholesterol concentrations that we observed after feeding cholesterol and fatty acids to the hamsters were generally in accordance with published data [6-8,14-19]. Increases in plasma total cholesterol and LDL-cholesterol in animals fed a diet supplemented with 0.1% cholesterol were accelerated by the addition of 5% palmitic acid (Table 1). The addition of 5% palmitic acid augmented the suppression of hepatic LDL receptor activity in comparison with cholesterol alone (Fig. 1), as reported previously [15-19]. Plasma CETP activity, which is known to be increased by an atherogenic diet [42-44], was further increased by 5% palmitic acid over that seen with a cholesterol-supplemented diet (Table 2). These results suggest that dietary palmitic acid augments the effect of dietary cholesterol on plasma total cholesterol and LDLcholesterol levels by causing a further suppression of LDL receptor activity and a greater increase in CETP activity. Five percent dietary oleic acid significantly decreased plasma total cholesterol, VLDL-cholesterol, and LDLcholesterol as compared with cholesterol alone, and the substitution of oleic acid for palmitic acid significantly

255

decreased plasma total cholesterol, VLDL-cholesterol and LDL-cholesterol (Table 1). Oleic acid prevented the suppression of hepatic LDL receptor activity seen with a standard diet or a cholesterol-supplemented diet (Fig. 1), and similar results were also obtained with dietary linoleic acid [14]. Moreover, the increase in plasma CETP activity induced by dietary cholesterol was prevented by oleic acid (Table 2). Thus, dietary oleic acid diminishes the increases in plasma total cholesterol. VLDL-cholesterol, and LDLcholesterol induced by dietary cholesterol by preventing suppressed LDL receptor activity and increased CETP activity. Five percent dietary oleic acid also significantly increased hepatic microsomal cholesterol 7a-hydroxylase activity (Fig. 3), which may regulate hepatic LDL receptor activity by decreasing regulatory cholesterol pool for LDL receptor expression. Because hepatic microsomal HMG-CoA reductase activity was significantly suppressed by the addition of 0.1% cholesterol to the standard diet and was not modified by the further addition of 5% palmitic acid or oleic acid (Fig. 2), HMG-CoA reductase activity seems unlikely to determine differences in plasma cholesterol concentrations among animals fed fatty acid-supplemented diets. However, hepatic LDL receptor activity was increased or suppressed by the addition of oleic acid or palmitic acid, respectively, suggesting that dietary fatty acids regulate hepatic LDL receptor activity and HMG-CoA reductase activity independently. These data are consistent with those reported previously [14,45,46]. Our data show that dietary oleic acid and palmitic acid have opposite effects on plasma cholesterol levels through regulation of hepatic LDL receptor activity, hepatic microsomal cholesterol 7c~-hydroxylase activity, and plasma CETP activity. Our data on plasma cholesterol concentrations and hepatic LDL receptors indicate that oleic acid is anti-atherogenic and palmitic acid is atherogenic. However, the effects of changes in plasma CETP activities induced by dietary cholesterol and fatty acids on atherosclerosis are unclear, because the contribution of CETP to atherogenicity is complex. Plasma CETP participates in the 'reverse cholesterol transport system' [22-28]. If this system inhibits atherogenicity, the increase in CETP induced by dietary palmitic acid may be a compensatory change which inhibits the progression of atherosclerosis induced by increased plasma cholesterol levels. However, when LDL receptors are suppressed sufficiently by dietary cholesterol and palmitic acid as shown in this study, an increase in CETP activity may accelerate the increase in LDL-cholesterol and exacerbate atherogenicity. The suppression of CETP activity induced by oleic acid would then seem to inhibit the reverse cholesterol transport system. Is this atherogenic or anti-atherogenic? Although there is no clear answer to this question, CETP may be a potentially atherogenic factor, because species with low plasma cholesteryl ester transfer activity, such as rats, are more resistant to atherosclerosis than those with high

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activity, s u c h as rabbits, m o n k e y s , a n d h u m a n s [47]. T h u s , the i n f l u e n c e o f C E T P o n a t h e r o g e n i c i t y , w h i c h is regulated b y dietary c h o l e s t e r o l a n d fatty acids as s h o w n in this study, m u s t b e c o n s i d e r e d t o g e t h e r w i t h o t h e r m e t a b o l i c c h a n g e s s u c h as t h o s e in h e p a t i c L D L receptors. In a d d i t i o n to t h e i r effects o n c h o l e s t e r o l m e t a b o l i s m , m o n o u n s a t u r a t e d fatty acids m a y also i n h i b i t the p r o g r e s sion o f a t h e r o s c l e r o s i s b y d i r e c t effects on lipid p e r o x i d a tion, b y a l t e r i n g the t e n d e n c y for t h r o m b o s i s , or b y influe n c i n g the v e s s e l h y p e r - r e a c t i v i t y as d e s c r i b e d p r e v i o u s l y [ 4 8 - 5 1 ] . T h u s , the m e c h a n i s m b y w h i c h dietary oleic acid e x e r t s its a n t i - a t h e r o g e n i c effect, as s u g g e s t e d b y an epid e m i o l o g i c a l s t u d y [21], is a c o m p l e x p r o b l e m a n d m u s t b e clarified with further investigation.

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