Effect of cholesteryl ester transfer protein (CETP) expression on diet-induced hyperlipidemias in transgenic rats

Atherosclerosis 178 (2005) 279–286

Effect of cholesteryl ester transfer protein (CETP) expression on diet-induced hyperlipidemias in transgenic rats Zoulika Zaka,c , Thomas Gautiera , Laure Dumonta , David Massona , Val´erie Deckerta , Linda Duverneuila , Jean-Paul Pais De Barrosa , Naig Le Guerna , Martina Schneidera , Philippe Moulinb , Alain Bataillardc , Laurent Lagrosta,∗ a

Laboratoire de Biochimie des Lipoprot´eines, INSERM U498, Facult´e de M´edecine, BP 87900, Dijon Cedex 21033, France b Service d’Endocrinologie-U-11, 69500 Bron, France c D´ epartement de Physiologie et de Pharmacologie Clinique, CNRS FRE 2678, Facult´e de Pharmacie 69373, Lyon, France Received 6 April 2004; received in revised form 19 August 2004; accepted 11 October 2004 Available online 13 December 2004

Abstract Objective: In order to determine the influence of the lipid status on the ability of cholesteryl ester transfer protein (CETP) to modify the plasma lipoprotein profile, the effect of hypercholesterolemia versus hypertriglyceridemia were compared in wild-type and CETP-transgenic (CETPTg) rats expressing CETP at a constant level. Methods and results: Wild-type and CETPTg rats were fed either a chow diet, a high fat/high cholesterol (HF/HC) diet, or a sucrose diet. As compared to wild-type rats, CETPTg rats fed the standard chow exhibited lower high-density lipoproteins (HDL)–cholesterol concentration (−65%, p < 0.01), but similar non-HDL–cholesterol concentrations. Both wild-type and CETPTg rats fed the HF/HC diet displayed pronounced increases in total and non-HDL–cholesterol levels, with no influence of CETP expression in this case. In contrast, the sucrose diet produced significant changes only in CETPTg rats which then exhibited a 82% increase in non-HDL–cholesterol in addition to a 80% reduction in HDL cholesterol when compared to sucrose-fed, wild-type rats (p < 0.01 in both cases). The triglyceride to cholesterol ratio in very low-density lipoprotein (VLDL) was 10-fold lower in ‘HF/HC’ rats than in ‘chow’ and ‘sucrose’ rats (p < 0.005 and p < 0.01, respectively), and VLDL from ‘HF/HC’ animals were proven to constitute poor cholesteryl ester acceptors. Conclusions: CETP expression modified dramatically the lipoprotein phenotype in ‘sucrose’ rats but not in ‘HF/HC’ rats. These observations suggest that a CETP inhibitor treatment is susceptible to produce profound changes in hypertriglyceridemia or combined hyperlipidemia. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Transgenic rats; CETP; Diet; Lipoprotein profile; Hypertriglyceridemia; Hypercholesterolemia; VLDL

1. Introduction Earlier studies in cholesteryl ester transfer protein (CETP)-deficient patients demonstrated that CETP is a key factor in determining the distribution of plasma cholesterol Abbreviations: CETP, cholesteryl ester transfer protein; CETPTg rats, CETP-transgenic rats; HDL, high density lipoprotein; LDL, low density lipoprotein; non-HDL, non-high-density lipoprotein; VLDL, very lowdensity lipoprotein; HF/HC, high fat/high cholesterol diet ∗ Corresponding author. E-mail address: [email protected] (L. Lagrost). 0021-9150/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2004.10.006

between the high-density lipoproteins (HDL) and the apoBcontaining lipoproteins, i.e. very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) [1,2]. Recently, pharmacological inhibition of CETP in human subjects was shown to reproduce, at least in part the CETP-deficient syndrome that is characterized mainly by a substantial rise in plasma HDL cholesterol levels [3,4]. Human observations are in agreement with animal studies that were conducted by the means of either anti-CETP immunotherapy, antisense oligonucleotides or specific pharmacological inhibitors [5–8]. Overall, it appears that CETP inhibition may constitute

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a new strategy in the prevention and treatment of dyslipidemia in high-risk populations [9]. CETP is a plasma factor that promotes the heteroexchange of neutral lipids, i.e. cholesteryl esters and triglycerides between circulating lipoproteins leading in particular to the net mass transfer of cholesterol from the antiatherogenic HDL to the proatherogenic apolipoprotein B-containing lipoproteins (VLDL, and then LDL) [9,10]. The lipid transfer process catalyzed by CETP is complex in nature, and it is mainly dependent on the concentration of CETP in plasma as well as on both the quantity and the neutral lipid composition of lipoprotein particles [10]. Beyond rapid exchanges that are known to occur between nearly equilibrated cholesteryl ester-rich LDL and HDL, net mass transfers in plasma proceed by heteroexchanges between non-equilibrated pools, involving in particular cholesteryl ester-rich HDL donors and triglyceride-rich apoB-containing acceptors. It results that the effect of CETP activity on lipoprotein structure and composition may vary from one abnormal lipoprotein phenotype to another, depending on whether hyperlipidemia is characterized by the elevation of either cholesteryl ester-rich lipoprotein acceptors or triglyceride-rich lipoprotein acceptors. Rat is a CETP-deficient species with relatively low atherogenic potential, contrasting with the high CETP/high risk profile of other species including rabbit, monkey and human [11,12]. In the present studies, the influence of hypertriglyceridemia versus hypercholesterolemia on the CETPmediated lipoprotein changes was monitored in a comprehensive manner by using a new line of CETP-transgenic rats fed either normal chow, HF/HC or sucrose diets. Whereas distinct diets allowed to introduce selective changes in the plasma lipoprotein profile, they did not promote significant alteration in the expression of the CETP transgene that was placed under the control of the metalothionein promoter. As compared to wild-type rats, CETPTg rats displayed specific changes in the plasma lipoprotein profile, and the impact of CETP was largely dependent on the type of dyslipidemia induced by dietary manipulation. These observations indicate that, beyond plasma CETP levels, the concentration and composition of plasma lipoprotein substrates are major determinants of the CETP-mediated changes. In particular, the present studies suggest that the initial levels of triglyceriderich lipoprotein acceptors, in addition to CETP mass levels should be considered in priority when predicting the effect of CETP inhibition on the plasma lipoprotein profile.

2. Methods

using a new line of CETP-transgenic Fisher rats that was recently created and characterized in our laboratory [13]. CETP gene was placed under the control of the metalothionein promoter. Significant plasma CETP levels occurred spontaneously in the CETPTg rat line in the absence of Zn treatment, and they were compatible with physiological ranges previously reported in CETP-deficient, normo- or dyslipidemic human subjects [14–16]. The present metabolic studies were conducted with control and CETPTg rats at 4–6 months of age. Basal lipid parameters, i.e. total plasma cholesterol and triglyceride concentrations, were compared in subgroups of control and CETPTg rats, each one containing 8–10 animals. FPLC lipoprotein distribution data, as well as lipid transfer activities were obtained in independent experiments with four to five animals in each subgroup. Animals were maintained in controlled conditions of temperature (21 ± 1 ◦ C), humidity (60 ± 10%), lighting (12-h cycle, 8 a.m. to 8 p.m.), and they had free access to food and water. Heparanized blood samples were collected by jugular vein puncture at 8 a.m., and a 12-h fasting period preceded (fasted animals) or not (non-fasted animals) blood collection. 2.2. Diets Three different diets were used in the current studies in order to produce controlled changes in the plasma lipoprotein profile. ‘Chow’ rats were fed a regular rodent chow diet (A03, UAR France). ‘High fat/high cholesterol’ (HF/HC) rats were fed a diet containing 1.25% (w/w) cholesterol, 7.5% (w/w) fat and 0.5% (w/w) sodium cholate for 24 weeks. ‘Sucrose’ rats were fed the regular rodent chow diet in combination with sucrose that was provided in drinking water (10% sucrose, w/v) for 3 weeks. 2.3. Plasma samples Fasting blood samples were collected into heparincontaining tubes by jugular vein puncture, under a slight 1% isoflurane anesthesia. Plasma was obtained by low speed centrifugation and stored at −80 ◦ C until analysis. 2.4. Plasma lipid and protein analyses All assays were performed on a Victor2 1420 Multilabel Counter (Wallac). Total cholesterol and triglycerides concentrations were measured by the enzymatic method using Cholesterol 100 reagent and Triglycerides 100 reagent, respectively (ABX Diagnostics). Proteins were measured using bicinchoninic acid reagent (Protein Assay Reagent, Pierce).

2.1. Animals 2.5. Fractionation of plasma lipoproteins All the animal studies were conducted in accordance with institutional guidelines (national agreement number: A21.231.006). They were approved by the Ethical Committee of the animal house of the University of Burgundy (protocol number #6102). The present studies were conducted by

For compositional analysis, individual plasma samples were injected on a Superose 6 HR 10/30 column (Amersham Biosciences) that was connected to a fast protein liquid chromatography (FPLC) system (Amersham Biosciences).

Z. Zak et al. / Atherosclerosis 178 (2005) 279–286

Lipoproteins were eluted at a constant, 0.3 ml/min flow rate with Tris-buffered saline containing 1 mM EDTA and 0.02% sodium azide. The gel filtration column was calibrated with globular protein standards of known Stoke’s diameter (Gel Filtration Calibration Kit, Pharmacia). Total cholesterol and triglyceride concentrations were assayed in individual, 0.3 ml fractions. As shown in previous studies with the same column, human VLDL were contained in fractions 4–11, human IDL + LDL were contained in fractions 12–23, and human HDL eluted in fractions 24–44 [13]. In the case of Fisher rat plasma, no significant amounts of LDL were detected in FPLC fractions 12–23; some of them, however, were shown to contain significant amounts of large-size HDL1, as characterized by their high apolipoproteinE content, and their typical electrophoretic migration in polyacrylamide gradient gels [13]. As a consequence, bimodal rat FPLC profiles corresponded to non-HDL (4–17) and HDL (17–44) fractions. For cholesteryl ester transfer studies, plasma VLDL were ultracentrifugally isolated as the d < 1.006 g/ml plasma fraction with one 1.5 h, 120,000 rpm spin in a TLA100 rotor in a TLX ultracentrifuge (Beckman). 2.6. CETP enzyme-linked immunosorbent assay CETP mass concentration in CETPTg rat plasma was measured by using a competitive ELISA adapted to a Victor2 1420 Multilabel Counter (Wallac), as previously described [15]. Anti-CETP TP2 antibodies were purchased from Ottawa Heart Institute (Ottawa, Canada). 2.7. Net mass transfer of cholesterol in incubated plasma The net mass transfer of total cholesterol from HDL to endogenous VLDL/LDL was measured during incubation at 37 ◦ C in a metabolic shaker in the presence of 1.5 mM iodoacetate to inhibit plasma LCAT. Aliquots of plasma from control and CETPTg rats were removed before or after a 6-h incubation at 37 ◦ C, and they were then chilled on ice. Lipoproteins were subsequently fractionated as described above on a Superose 6 HR 10/30 column (Amersham Biosciences) and they were assayed for cholesterol as described above. 2.8. Cholesteryl ester transfer activity with isolated VLDL The ability of isolated VLDL to exchange cholesteryl esters with synthetic liposomes in the presence of purified CETP [15] was determined with a commercially available fluorescence assay (Roar Biochemical, New York, U.S.A.). Briefly, synthetic liposome donors enriched with nitrobenzoxadiazol (NBD)-labeled cholesteryl esters were incubated for 3 h at 37 ◦ C in the presence of either ‘chow’, ‘sucrose’, or ‘HF/HC’ VLDL acceptors (protein, 0.75 ␮g) in the presence of human CETP (3 ␮g) in a final volume of 250 ␮l. Incubations were conducted in duplicate in 96-well microplates, and changes in fluorescence were monitored

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by using a Victor2 1420 Multilabel Counter (Perkin-Elmer Life Sciences), with 465 nm excitation and 535 nm emission wavelengths. The amounts of NBD-cholesteryl esters transferred were calculated by using a standard curve plotting fluorescence intensity and concentrations of NBD-cholesteryl esters dispersed in isopropanol. 2.9. Statistical analysis Statistical analysis was performed using the StatView 4.5 software. Data are mean ± S.E.M. The significance between data means was determined by using the non-parametric Mann–Whitney U-test.

3. Experimental results 3.1. Effect of dietary manipulation on plasma lipids Wild-type and CETPTg rats were given either a standard chow diet (‘chow’ group), a high fat/high cholesterol diet (‘HF/HC’ group), or a high sucrose diet (‘sucrose’ group). As shown in Table 1, dietary manipulations produced alterations in the plasma levels of total cholesterol and triglycerides in both wild-type and CETPTg rats after an overnight fast. The HF/HC diet produced significant, 2.3- and 2.7-fold rises in the mean plasma concentration of total cholesterol as compared to ‘chow’ and ‘sucrose’ wild-type rats, respectively, with no effect on plasma triglycerides. In CETPTg rats, HF/HC diet induced also a marked increase in plasma cholesterol concentration as compared to ‘chow’ and ‘sucrose’ CETPTg rats (3.5- and 4.8-fold rises, respectively; Table 1). Feeding the high sucrose diet for 3 weeks did not produce significant alterations in the plasma levels of both cholesterol and triglycTable 1 Lipid levels and CETP mass from fasted wild-type and CETPTg rats

Wild-type rats Total cholesterol (g/l) Triglycerides (g/l) CETPTg rats Total cholesterol (g/l) Triglycerides (g/l) CETP mass (mg/l)

Chow group

HF/HC group

Sucrose group

n=9

n=8

n = 10

0.94 ± 0.05

2.17 ± 0.34*

0.79 ± 0.05‡, §

1.72 ± 0.31

1.55 ± 0.16

1.59 ± 0.15

n=9

n = 10

n=9

0.66 ± 0.11||

2.31 ± 0.20*, ||

0.48 ± 0.05, ***

1.88 ± 0.17

2.28 ± 0.26

3.18 ± 0.30†, #

3.16 ± 0.51

3.46 ± 0.50

3.02 ± 0.69

Plasma lipids and CETP mass were determined as described under Section 2. Values are expressed in g/l for lipids and in mg/l for CETP mass, and they are mean ± S.E.M. Significantly different from the chow group (Mann–Whitney test): * p < 0.0005; † p < 0.005 and ‡ p < 0.05, HF/HC group (Mann–Whitney test): § p < 0.0005; p < 0.005 and wild-type rats (Mann–Whitney test): # p < 0.001; ** p < 0.01, || p < 0.05.

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Table 2 Cholesterol and triglyceride contents of non-HDL and HDL fractions isolated from wild-type and CETPTg rats in ‘chow’, ‘HF/HC’ and ‘sucrose’ groups Triglycerides (g/l)

Chow group Wild-type (n = 5) CETPTg (n = 5) p HF/HC group Wild-type (n = 5) CETPTg (n = 5) p Sucrose group Wild-type (n = 5) CETPTg (n = 5) p

Cholesterol (g/l)

Cholesterol ratio (non-HDL to HDL)

Non-HDL

HDL

Non-HDL

HDL

1.76 ± 0.34 1.26 ± 0.12

0.11 ± 0.02 0.15 ± 0.01

0.12 ± 0.01 0.12 ± 0.01

0.69 ± 0.06 0.24 ± 0.04

0.18 ± 0.02 0.46 ± 0.09

NS

NS

NS

0.01

0.005

1.07 ± 0.11 1.29 ± 0.16

0.23 ± 0.04 0.23 ± 0.02

1.42 ± 0.30 1.79 ± 0.24

0.75 ± 0.09 0.57 ± 0.05

1.89 ± 0.28 3.20 ± 0.51

NS

NS

NS

NS

NS

1.58 ± 0.20 3.37 ± 0.50

0.11 ± 0.01 0.19 ± 0.01

0.12 ± 0.01 0.22 ± 0.15

0.71 ± 0.06 0.11 ± 0.02

0.18 ± 0.03 1.85 ± 0.35

0.01

0.01

0.01

0.01

0.01

Lipid contents were determined as described under Section 2. Values are expressed in grams of lipid per litre of plasma, and they are mean ± S.E.M. of five rats.

erides in fasted wild-type rats. However, ‘sucrose’ wild-type rats were previously shown to display transient hypertriglyceridemia in the postprandial state [17,18], and an elevation of plasma triglyceride levels was confirmed after sucrose in both non-fasted control rats (+77%; p < 0.02) and non-fasted CETPTg rats (+73%; p < 0.005) as compared to chow-fed counterparts. In contrast to wild-type rats, a significant 69% increase in the plasma triglyceride level could still be observed in fasted ‘sucrose’ CETPTg rats as compared to fasted ‘chow’ CETPTg counterparts (Table 1). 3.2. Effect of the HF/HC diet versus the sucrose diet on plasma lipoprotein profiles in wild-type and CETPTg rats The effect of CETP expression on the composition of individual plasma lipoprotein fractions was determined in animals fed either the chow diet, the HF/HC diet, or the sucrose diet. Plasmas from wild-type and CETPTg rats were passed through a gel permeation chromatography column, and the cholesterol and triglyceride contents of non-HDL and HDL fractions from wild-type and CETPTg rats were compared under the three distinct dietary conditions. As shown in Fig. 1, upper panel, CETPTg rats fed the chow diet displayed a marked reduction in HDL, mostly in the large, HDL1containing fractions. This was characterized by a 65% reduction in the mean HDL cholesterol level in CETPTg plasmas as compared to wild-type plasmas (Table 2). In contrast, the cholesterol content of the CETPTg non-HDL fraction was unchanged as compared to the wild-type non-HDL fraction (Fig. 1, upper panel; Table 2). When animals were fed the HF/HC diet, a marked, at least 10-fold increase in the cholesterol content of the non-HDL fraction was observed as compared to animals fed the chow diet, with a similar magnitude whether CETP was expressed or not (Fig. 1, middle panel; Table 2). In these animals, CETP expression did not promote significant changes in the HDL fraction, nei-

ther in term of composition nor in terms of size distribution. In both wild-type and CETPTg rats fed the HF/HC diet, HDL appeared as an homogenous population of intermediate size, in either group. The only discrete change mediated by CETP expression in the hypercholesterolemic context was characterized by a slight, non-significant tendency towards an increased non-HDL to HDL cholesterol ratio in CETPTg rats as compared to control rats (Table 2). When animals were fed the sucrose diet, CETP expression led to a drastic redistribution of cholesterol between HDL and non-HDL fractions. Only in this context, CETPTg rats displayed concomitant and opposite changes in the cholesterol content of HDL and non-HDL fractions. Indeed, a marked 80% reduction in HDL cholesterol was accompanied by a simultaneous 82% increase in non-HDL cholesterol (Fig. 1, lower panel; Table 2). In additional support of the greatest effect of CETP expression in ‘sucrose’ rats, the mean 10-fold rise in nonHDL to HDL cholesterol ratio in CETPTg rats compared to wild-type rats was clearly higher than the corresponding 2.5and 1.7-fold rises observed in CETPTg animals fed the chow and HF/HC diets, respectively (Table 2). CETP expression did not result in detectable changes in plasma triglycerides, unless when expressed in ‘sucrose’ animals, with significant 2.1- and 1.7-fold rises in triglyceride contents of non-HDL and HDL fractions of ‘sucrose’ CETPTg rats, respectively (Fig. 2 and Table 2). 3.3. Characterization of the composition of lipoprotein cholesterol acceptors Despite a clear tendency towards the elevation of nonHDL cholesterol levels in both ‘HF/HC’ CETPTg rats and ‘sucrose’ CETPTg rats as compared to ‘chow’ CETPTg rats (Fig. 1), detailed analysis of VLDL revealed marked differences. As shown in Fig. 3, VLDL from the ‘HF/HC’ CETPTg animals contained fewer triglycerides than VLDL from the

Z. Zak et al. / Atherosclerosis 178 (2005) 279–286

Fig. 1. Cholesterol distribution in plasma lipoproteins from wild-type and CETPTg rats. Rats were fed a standard rodent chow diet (upper panel), a HF/HC diet (middle panel) or a high sucrose diet (lower panel). Lipoproteins were separated by gel permeation FPLC chromatography, and cholesterol was assayed in eluted fractions as described in Section 2. Each point is the mean ± S.E.M. of five rats in each group.

‘sucrose’ CETPTg animals, with an even more spectacular decrease in the triglyceride to cholesterol ratio in the former case. Given that the extent of net mass transfers of cholesteryl esters through the CETP-mediated transfer reaction is known to be tightly dependent on the neutral lipid composition of the lipoprotein donors and acceptors, we hypothesized that VLDL from the ‘HF/HC’ CETPTg group might be dysfunctional. In support of the latter view, no net mass redistribution of cholesterol occurred in ‘HF/HC’ plasma during a 6-h incubation at 37 ◦ C (Fig. 4E). In contrast, incubation of fasting plasma from either ‘chow’ CETPTg rats or ‘sucrose’ CETPTg rats led to significant decreases in the cholesterol content of HDL fractions (Fig. 4D and F), indicating a net flux of cholesterol from HDL towards apoB-containing lipoproteins. In contrast, we found no changes in the HDL cholesterol concentration in plasmas of wild-type rats whether they were fed either the chow, the HF/HC or the sucrose diet (Fig. 4A–C). When expressed as percentage of the initial HDL cholesterol concentration and as compared to the incubated ‘chow’ CETPTg rat plasmas, the transfer of HDL cholesterol to the VLDL/LDL fraction was significantly re-

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Fig. 2. Triglyceride distribution in plasma lipoproteins from wild-type and CETPTg rats. Rats were fed a standard rodent chow diet (upper panel), a HF/HC diet (middle panel) or a high sucrose diet (lower panel). Lipoproteins were separated by gel permeation FPLC chromatography, and triglycerides were assayed in eluted fractions as described in Section 2. Each point is the mean ± S.E.M. of five rats in each group.

duced in the ‘HF/HC’ CETPTg rats, whereas it was significantly increased in the ‘sucrose’ CETPTg rats (Fig. 5).

3.4. Comparative ability of VLDL from ‘chow’, ‘HF/HC’ or ‘sucrose’ plasmas to acquire cholesteryl esters As shown in Fig. 6, in vitro incubation of labeled liposomes with isolated VLDL in the presence of purified CETP revealed that ‘HF/HC’ VLDL are poorer substrates in the CETP-mediated neutral lipid exchange process as compared to ‘chow’ VLDL and ‘sucrose’ VLDL. The maximal ability of ‘sucrose’ VLDL to accommodate labeled cholesteryl esters, corresponding to the plateau value of the lipid transfer curve was significantly higher with the ‘sucrose’ VLDL as compared to ‘chow’ or ‘HF/HC’ counterparts (Fig. 6). In addition, the velocity of cholesteryl ester exchanges, as assessed by the initial NBD-cholesteryl ester transfer rate was significantly lower with ‘HF/HC’ VLDL (2.58 ± 0.19 pmol/min) than with ‘chow’ VLDL (4.37 ± 0.32 pmol/min) and ‘sucrose’ VLDL (4.01 ± 0.30 pmol/min), respectively (p < 0.05 in both cases).

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Fig. 3. Characterization of VLDL acceptors in ‘chow’, ‘HF/HC’ and ‘sucrose’ rat plasmas. The triglyceride concentration (A) and the triglyceride to cholesterol ratio (B) were determined in VLDL-containing fractions obtained by FPLC. Data are mean ± S.E.M. of five ‘chow’ CETPTg rats, five ‘HF/HC’ CETPTg rats and five ‘sucrose’ CETPTg rats. * p < 0.02; † p < 0.01 significantly different from ‘sucrose’ CETPTg rats. ‡ p < 0.005; § p < 0.01 significantly different from ‘HF/HC’ CETPTg rats.

4. Discussion The key question of the influence of the concentration and lipid composition of plasma lipoproteins on the consequences of CETP expression versus CETP deficiency on the lipoprotein phenotype was addressed in the present study. To do so, we used an original CETPTg rat model [13], and lipoprotein alterations were induced by dietary manipulation. Wild-type rat is a CETP-deficient species with a lipoprotein phenotype resembling in many ways the hyperalphalipoproteinemic profile of homozygous CETP-deficient subjects [1]. As described in recent studies [13], CETP expression in rats fed a standard chow leads to the selective disappearance of large size HDL1 that are present in CETP-deficient homozygous subjects, but absent from normal subjects or CETPdeficient heterozygotes [19,20]. Controlled changes in the plasma lipoprotein profile were obtained further in the present study through dietary manipulation in CETPTg rats, which express active CETP at a constant level. As reported recently [13], the expression of active CETP in transgenic rats fed the chow diet (‘chow’ animals) was associated with marked alterations in the lipoprotein profile. Changes were characterized mainly by significant reductions in the cholesterol content and mean size of HDL. In particular, a selective reduction of large size HDL1 was observed,

Fig. 4. HDL cholesterol levels in incubated plasma of ‘chow’ rats (A and D; n = 5), ‘HF/HC’ rats (B and E; n = 5), and ‘sucrose’ rats (C and F; n = 5). Plasma was incubated for 6 h at 37 ◦ C in the presence of 1.5 mM iodoacetate. The cholesterol content of HDL was measured as described in Section 2. Data are mean ± S.E.M. * p < 0.02; † p < 0.05 significantly different from ‘time 0 h’.

confirming a peculiar sensitivity of this lipoprotein subclass to CETP action [13]. When wild-type and CETPTg rat were fed the sucrose diet (‘sucrose’ animals), a significant rise in plasma triglyceride levels was observed during the postprandial phase in both groups, whereas a long lasting hypertriglyceridemia, still observable after an overnight fast was obtained only in CETPTg animals. Similarly, a worsening

Fig. 5. Relative transfer of cholesterol from HDL to endogenous non-HDL (A) and CETP mass (B) in ‘chow’, ‘HF/HC’, and ‘sucrose’ CETPTg rats. Plasma from ‘chow’ CETPTg rats (n = 5), ‘HF/HC’ CETPTg rats (n = 5), and ‘sucrose’ CETPTg rats (n = 5) was incubated for 6 h at 37 ◦ C in the presence of 1.5 mM iodoacetate (see Fig. 4, transfer activity was calculated as percentage of the initial cholesterol content of the plasma HDL fraction at time 0. * p < 0.05 significantly different from ‘chow’ CETPTg rats. † p < 0.01 significantly different from ‘HF/HC’ CETPTg rats.

Z. Zak et al. / Atherosclerosis 178 (2005) 279–286

Fig. 6. Time course of the transfer of cholesteryl ester transfer towards ‘chow’, ‘sucrose’ and ‘HF/HC’ VLDL. The d < 1.006 g/ml fraction from either chow (n = 4; opened circles), sucrose (n = 4; hatched circles), or HF/HC (n = 5; closed circles) groups rats was incubated at 37 ◦ C in the presence of liposomes donors and purified CETP. * p < 0.05 significantly different from ‘chow’ CETPTg rats. † p < 0.05 significantly different from ‘HF/HC’ CETPTg rats.

of hypertriglyceridemia was previously reported in CETPTg mice crossbred with either apoE knocked out mice [21], apoE knocked out mice/apoAI transgenic mice [21], or apoCIII/LDL receptor knocked out mice [22]. CETP expression in transgenic mouse models can modify both the fractional catabolic rate and the production rate of triglycerides [23], and the hypertriglyceridemic effect of CETP was explained both in terms of transfer of triglycerides from postprandial chylomicrons into smaller VLDL, IDL, and LDL that are less favorable lipolytic substrates [24], and in terms of accumulation of VLDL as the result of the CETP-mediated downregulation of hepatic apoB/E receptors [25]. In the present studies, sucrose feeding compared to chow diet did not result in significant changes in plasma lipoprotein parameters in fasted wild-type rats. In contrast, sucrose feeding was characterized by the accumulation of triglyceride-rich VLDL in fasted CETPTg rats, together with a significant increase in the triglyceride content of HDL. As compared to ‘chow’ CETPTg rats, hypertriglyceridemia in CETPTg animals produced more dramatic changes in the plasma lipoprotein profile. The significant decrease in the cholesterol content of HDL observed in ‘chow’ CETPTg rats tended to be even more dramatic in ‘sucrose’ CETPTg rats, and a significant, two-fold rise in VLDL cholesterol levels was only observed in the latter group. These observations indicate that the impact of CETP on plasma lipoprotein metabolism is considerably worsened when triglyceride-rich lipoprotein acceptors tend to accumulate. Together with the decrease in the size and cholesterol content of HDL, the increase in non-HDL cholesterol levels in ‘sucrose’ rats expressing CETP led to a considerable rise in the non-HDL to HDL cholesterol ratio as compared to wild-type counterparts. It appears therefore that, at least in this rat model CETP constitutes a leading factor in determin-

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ing plasma lipoprotein parameters. This statement is further sustained by previous studies in which hypertriglyceridemiamediated changes in HDL distribution in apoCIII transgenic mice were observed only upon coexpression of CETP [26]. In another part of the present study, the effect of CETP on plasma lipoprotein parameters was determined in an hypercholesterolemic context that was achieved by feeding wild-type and CETPTg rats a high fat/high cholesterol diet (‘HF/HC’ animals). ‘HF/HC’ CETPTg rats did not show selective alterations as compared to ‘HF/HC’ wild-type rats, despite considerable hypercholesterolemia-mediated rearrangements in both groups. These observations were in direct contrast with data obtained in ‘sucrose’ rats despite similar plasma CETP concentration in either group. Comparative data analysis after distinct dietary manipulations come in favour of a prominent effect of the metabolic context, in particular lipoprotein composition in determining the ability of CETP to modify the plasma lipoprotein profile in an animal model expressing a human-like CETP activity at a constant level. Comparative analysis of ‘HF/HC’ VLDL and ‘sucrose’ VLDL revealed marked differences in their triglyceride content, and even more dramatically in their triglyceride to cholesterol ratio. In particular, ‘HF/HC’ VLDL contained fewer triglyceride molecules, with a 10-fold lower triglyceride to cholesterol ratio than ‘sucrose’ VLDL. Secondly, the lack of significant changes in the cholesterol content of HDL after incubation of ‘HF/HC’ plasma contrasted clearly with the net, 40% reduction in the cholesterol content of incubated ‘sucrose’ HDL. In fact, ‘sucrose’ VLDL were quite comparable to the triglyceride-rich, cholesterol-poor VLDL that were shown to constitute the major determinants of the transfer of cholesteryl esters out of the cholesterol-rich lipoproteins in earlier studies [27,28]. Finally, in ‘HF/HC’ CETPTg rats the ability of isolated VLDL to act as cholesteryl ester acceptors in the lipid transfer process was reduced as compared to ‘chow’ and ‘sucrose’ VLDL, as assessed by significant reductions in both the initial transfer rate and the maximal transfer value corresponding to the plateau of the lipid transfer curves. These observations bring new support to the relative impairment of the cholesteryl ester transfer reaction by elevated levels of cholesterol-rich VLDL [29,30]. In conclusion, the amount of VLDL acceptors, and even more importantly their triglyceride to cholesterol ratio are among the best predictors of the impact of CETP, and as a consequence of CETP inhibitors on the plasma lipoprotein profile in an original model of CETPTg rats. Whether the effect of a CETP inhibitor treatment is drastic in hypertriglyceridemia or combined hyperlipidemia will deserve peculiar attention in future randomized trials.

Acknowledgments This research was supported by an International HDL Research Awards Program grant to Laurent Lagrost. This study was supported by the Institut National de la Sant´e et de

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Z. Zak et al. / Atherosclerosis 178 (2005) 279–286

la Recherche M´edicale, the Conseil R´egional Rhˆone-Alpes, the Conseil R´egional de Bourgogne, and the Fondation de France. Z. Zak is the recipient of a fellowship from the Laboratoires Fournier, and the Nouvelle Soci´et´e Franc¸aise d’Ath´eroscl´erose (NSFA).

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