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* This work was supported by the Université de Bourgogne, the Conseil Régional de Bourgogne, the INSERM, and the Fondation de France.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Transgenic mice expressing human cholesteryl ester transfer protein (HuCETPTg mice) were crossed with apolipoprotein CI-knocked out (apoCI-KO) mice. Although total cholesterol levels tended to be reduced as the result of CETP expression in HuCETPTg heterozygotes compared with C57BL6 control mice (−13%, not significant), a more pronounced decrease (−28%, p < 0.05) was observed when human CETP was expressed in an apoCI-deficient background (HuCETPTg/apoCI-KO mice). Gel permeation chromatography analysis revealed a significant, 6.1-fold rise (p < 0.05) in the cholesteryl ester content of very low density lipoproteins in HuCETPTg/apoCI-KO mice compared with control mice, whereas the 2.7-fold increase in HuCETPTg mice did not reach the significance level in these experiments. Approximately 50% decreases in the cholesteryl ester content and cholesteryl ester to triglyceride ratio of high density lipoproteins (HDL) were observed in HuCETPTg/apoCI-KO mice compared with controls (p < 0.05 in both cases), with intermediate −20% changes in HuCETPTg mice. The cholesteryl ester depletion of HDL was accompanied with a significant reduction in their mean apparent diameter (8.68 ± 0.04 nm in HuCETPTg/apoCI-KO miceversus 8.83 ± 0.02 nm in control mice;p < 0.05), again with intermediate values in HuCETPTg mice (8.77 ± 0.04 nm). In vitro purified apoCI was able to inhibit cholesteryl ester exchange when added to either total plasma or reconstituted HDL-free mixtures, and coincidently, the specific activity of CETP was significantly increased in the apoCI-deficient state (173 ± 75 pmol/μg/h in HuCETPTg/apoCI-KO mice versus 72 ± 19 pmol/μg/h in HuCETPTg, p < 0.05). Finally, HDL from apoCI-KO mice were shown to interact more readily with purified CETP than control HDL that differ only by their apoCI content. Overall, the present observations provide direct support for a potent specific inhibition of CETP by plasma apoCI in vivo.
cholesteryl ester (CE) transfer protein
high density lipoprotein
low density lipoprotein
transgenic mouse to human CETP
apoCI-deficient mouse expressing human CETP
The cholesteryl ester transfer protein (CETP)1 promotes the exchange of neutral lipid species, i.e. cholesteryl esters and triglycerides between plasma lipoproteins (
). In vivostudies demonstrated that the CETP-mediated bidirectional transfers of neutral lipids may influence the atherogenicity of the plasma lipoprotein profile, raising a substantial interest in studying the regulation of plasma CETP activity (
). The CETP-mediated lipid transfer reaction is a complex process that is influenced by a number of plasma modulators, among them the concentration of active CETP as well as the structure, the lipid composition, and the relative proportions of lipoprotein donors and acceptors (
). In addition, studies from several laboratories support the existence of a protein inhibitor of CETP activity in plasma from distinct vertebrate species, including human. This putative inhibitor might account, at least in part, for the substantial alterations in the CETP-specific activity as observed between plasma samples from distinct subgroups of patients (
), and no direct indication of the extent of plasma CETP inhibition by apoF has been yet provided in vivo. Several years ago, apoCI was suggested as a possible CETP inhibitor in comparative in vitro studies (
). In contrast to other putative inhibitors of CETP activity, we demonstrated that apoCI as a specific CETP inhibitor meets in vitro most of the following required criteria. 1) ApoCI inhibitory activity is specifically localized in HDL; 2) it constitutes a potent inhibitor of CETP, and unlike other putative apolipoprotein modulators (
), with the exclusion of activating potential; 3) a complete blockade of CETP can be reached with moderate inhibitor doses; 4) apoCI is active not only as a purified protein but also as a component of the HDL protein moiety; 5) substantial increment in CETP activity can be obtained in the presence of anti-apoCI antibodies in incubation media containing purified CETP and lipoprotein substrates; 6) and immunopurified, apoCI-free HDLs are better substrates for CETP than apoCI-containing particles (
The assessment of the physiological relevance of CETP inhibition constitutes a key step along the quest of a specific protein inhibitor, and this was addressed in the present study in apoCI-knocked out mice (
), and the effects of apoCI deficiency on plasma cholesteryl ester transfer activity and plasma lipoprotein parameters were determined.
Plasma lipid parameters and CETP mass levels in various mouse lines are presented in Table I. In the present studies, the expression of human CETP alone (HuCETPTg mice) did not promote significant alterations in total plasma lipid levels as compared with control mice, although in agreement with previous studies (
), a tendency toward a decrease in total cholesterol levels was observed (Table I). When plasma CETP was expressed in an apoCI-deficient context (HuCETPTg/apoCI-KO mice), decreases in both total cholesterol and cholesteryl ester levels became highly significant when compared with controls (−28% and −45%, respectively; p < 0.05 in both cases) (Table I). The permissive effect of apoCI deficiency was clearly apparent when HuCETPTg/apoCI-KO mice were compared with HuCETPTg mice, with significant −17% and −39% decreases in total cholesterol and cholesteryl ester levels in the former group, respectively (p < 0.05 in both cases). The latter observations could not be explained by differences in CETP expression, with no significant difference in the plasma CETP mass levels between the two groups (2.9 ± 0.9 mg/liter in HuCETPTg/apoCI0 miceversus 3.6 ± 0.7 mg/liter in HuCETPTg mice; not significant) (Table I).
Table IPlasma lipid and CETP mass concentrations in control, apoCI-KO, HuCETPTg, and HuCETPTg/apoCI-KO mice
To bring more insights into the alterations of the plasma lipoproteins in distinct mouse lines, individual plasma samples of control mice, apoCI-KO mice, HuCETPTg mice, and HuCETPTg/apoCI-KO mice were fractionated by gel permeation chromatography, and cholesteryl ester and triglyceride profiles were obtained (Fig.1; Table II). Although plasma cholesteryl esters were localized mainly in HDL (fractions 30–45) in all the mouse lines studied, HDL cholesteryl ester levels were markedly reduced in HuCETPTg/apoCI-KO mice, with the difference reaching the significance levels when HuCETPTg/apoCI-KO mice were compared with control mice (mean 53% decrease, p < 0.05) (Table II). Conversely, the combination of apoCI deficiency and CETP expression produced a marked, 6.1-fold rise in the cholesteryl ester content of VLDL (fractions 5–15) in HuCETPTg/apoCI-KO mice compared with control mice (p < 0.05; Table II). A significant rise in the cholesteryl ester content of LDL (fractions 16–29) was observed in mice expressing human CETP compared with control mice (Table II). Overall, as compared with control mice, the redistribution of cholesteryl esters led to a marked 3.3-fold rise in the VLDL + LDL to HDL cholesteryl ester ratio in HuCETPTg/apoCI-KO mice (p < 0.05), with only a 1.8-fold rise in HuCETPTg mice (not significant) (Fig. 2). Although no significant alterations in the triglyceride content of individual lipoprotein fractions were observed when comparing the distribution profiles from distinct mouse lines, the cholesteryl ester to triglyceride ratio of HDL was decreased by 57% in HuCETPTg/apoCI-KO mice compared with control mice (p < 0.05) (Fig.3). Alterations in the lipid composition of the plasma lipoproteins from HuCETPTg/apoCI-KO mice were associated with significant changes in their size. Indeed, analysis of total lipoproteins by native polyacrylamide gradient gel electrophoresis revealed a significant reduction in the mean apparent diameter of HDL from HuCETPTg/apoCI-KO mice compared with HDL from both control and apoCI-KO mice (Fig. 4, p< 0.05 in both cases). HDL from HuCETPTg mice were of intermediate size as compared with HDL from either control or HuCETPTg/apoCI-KO mice (Fig. 4).
Table IICholesteryl ester and triglyceride contents of VLDL, LDL, and HDL fractions isolated from control, apoCI-KO, HuCETPTg, and HuCETPTg/apoCI-KO mice
To determine further whether the above changes in the size and composition of plasma lipoproteins related to alterations in plasma neutral lipid transfer activity, CETP activity was measured in various plasma samples as the rate of transfer of fluorescent cholesteryl esters from exogenous, labeled liposomes to endogenous plasma lipoproteins. As expected from previous interspecies comparisons (
), the mouse is a CETP-deficient animal, and neither control (not shown) nor apoCI-KO mice displayed detectable cholesteryl ester transfer activity (Fig. 5). Plasma from HuCETPTg mice displayed substantial cholesteryl ester transfer activity, with an initial cholesteryl ester transfer rate of 62 pmol/h (Fig. 5, inset). Despite similar CETP mass concentrations in HuCETPTg and HuCETPTg/apoCI-KO mice, CETP activity was markedly increased in the latter animals, with a mean 84% rise in the initial cholesteryl ester transfer rate compared with HuCETPTg mice (p < 0.05). As a consequence, the specific activity of human CETP, as calculated as the ratio of cholesteryl ester transfer rate to CETP mass concentration, was remarkably higher in HuCETPTg/apoCI-KO mice than in HuCETPTg mice (173 ± 75versus 72 ± 19 pmol/μg/h; p < 0.05). In vitro, the rate of cholesteryl ester transfer in total plasma from HuCETPTg/apoCI-KO mice could be progressively decreased in the presence of increasing concentrations of purified apoCI, with the exclusion of any activating potential (Fig.6). Finally, and in further support of a direct inhibition of plasma CETP activity by apoCI, the rate of transfer of labeled cholesteryl esters was decreased by purified apoCI when added to reconstituted mixtures made of apolipoprotein-free liposome donors and apoB-containing LDL acceptors (Fig.7).
All together the above results suggest that plasma apoCI may constitute a potent regulator of CETP activity in total plasma. To give further insights into the biological relevance of lipoprotein-associated apoCI as a specific inhibitor of CETP activity, HDL were isolated by ultracentrifuge from control and apoCI-KO mouse plasma, and their ability to interact with purified human CETP was compared. As shown in Fig. 8, the apolipoprotein composition of HDL from both sources were similar, with the exception of apoCI that was absent from HDL from apoCI-KO mice. As shown in Fig.9, apoCI deficiency was characterized by a much better ability of isolated HDL to act as a lipoprotein substrate in the lipid transfer assay, with a significant 61% rise in the initial transfer rate measured with apoCI-KO HDL compared with control HDL (p < 0.05).
Recent studies identified a number of pharmacological compounds as potential CETP inhibitors; among them, at least one disulfide derivative (JTT-705) was proven to be efficient in blocking both plasma cholesteryl ester transfer activity and atherosclerosis progression in the rabbit (
), these observations gave rise to an intensive quest for CETP inhibitors that might represent relevant tools for the treatment of dyslipidemia and the prevention of atherosclerosis in future clinical practice. Besides the quest for new, pharmacological compounds, the presence of a physiological inhibitor of CETP in plasma remains a matter of debate. In our hands, apolipoprotein CI arosein vitro as a relevant candidate, meeting most of the required criteria as a potent regulator of CETP activity (
). However, one important question, i.e. the physiological relevance and the consequences of the modulation of plasma CETP activity by apoCIin vivo remained unanswered. The present in vivostudy provides the first, direct evidence in favor of the key role of apoCI as the physiological regulator of plasma cholesteryl ester transfer activity.
Human plasma apolipoprotein CI is a small, exchangeable apolipoprotein with two amphipathic α-helices that are involved in lipid binding (
). Although the precise physiological function of apoCI remains a matter of debate, transgenic mice overexpressing apoCI show a clear phenotype with severe hyperlipidemia that is due to the inhibition of the hepatic uptake of apoB-containing lipoproteins (
) is constantly associated with specific disorders of the lipoprotein profile. The lack of a specific phenotype in apoCI-KO mice fed a standard diet suggests either that apoCI has no direct implication in lipoprotein metabolism or, most likely, that the wild-type mouse is not appropriate to the determination of the physiological function of apoCI. In the present study, the effect of apoCI deficiency on lipoprotein metabolism was addressed in transgenic mice expressing the human CETP gene (HuCETPTg mice) under the control of its natural flanking regions (
), the expression of moderate CETP levels in mouse heterozygotes tended to produce a rise in the cholesteryl ester content of apoB-containing lipoproteins but produced a drop in the cholesteryl ester content of HDL. Whereas the lack of a significant effect of apoCI deficiency on plasma lipid parameters was confirmed in the present work, the CETP-mediated redistribution of cholesteryl esters from HDL to apoB-containing lipoproteins was magnified when both CETP expression and apoCI-deficiency were combined, with a nearly 2-fold rise in the VLDL + LDL to HDL cholesteryl ester ratio in HuCETPTg/apoCI-KO mice compared with the HuCETPTg counterparts. The marked reduction in the cholesteryl ester content of HDL from HuCETPTg/apoCI-KO animals accounted for the significant reduction in total plasma cholesterol level in this line. CETP actually proceeds by a heteroexchange of cholesteryl esters and triglycerides between non-equilibrated pools, i.e. triglyceride-rich apoB-containing lipoproteinsversus cholesteryl ester-rich HDL (
). Accordingly, the heteroexchange of neutral lipid species produced a significant, 2-fold drop in the cholesteryl ester to triglyceride ratio in HDL from HuCETPTg/apoCI-KO mice compared with controls, and a weaker tendency was observed with the HuCETPTg line. Unlike cholesteryl esters, triglycerides of the lipoprotein core are continuously and efficiently hydrolyzed in the plasma compartment through the activity of endothelial lipases. Triglyceride hydrolysis, in conjunction with CETP-mediated neutral lipid exchange is actually a key component of the metabolic process that leads to the emergence of core-depleted, small-sized lipoproteins (
). As a direct consequence of greater neutral lipid exchanges in HuCETPTg/apoCI-KO mice, we observed an effect on the size distribution of HDL, with a significant reduction in the mean apparent diameter of HDL from HuCETPTg/apoCI-KO mice compared with control mice, again with an intermediate effect in HuCETPTg mice.
It is worthy to note that the hyperlipidemic response to high fat feeding is exacerbated in apoCI-KO mice (
and the present study), it may be hypothesized that apoCI deficiency also contributes to the lipid transfer reaction indirectly through its effect on the clearance of cholesteryl ester acceptors. Although the latter hypothesis is rather improbable in apoCI-KO mice fed a standard diet, with no significant lipoprotein alterations in this case (24, 25, present study), it was important to ascertain the real impact of apoCI deficiency on plasma CETP activity in HuCETPTg versusHuCETPTg/apoCI-KO mice. The latter point led us to demonstrate that the specific activity of plasma CETP is significantly increased as the result of apoCI withdrawal in HuCETPTg/apoCI-KO mice. Conversely, as observed in vitro, the rates of cholesteryl ester transfer in total plasma from HuCETPTg/apoCI-KO mice as well as in reconstituted mixtures could be progressively decreased in the presence of purified apoCI. The direct effect of apoCI as a physiological regulator of CETP was further confirmed by the more efficient interaction of CETP with HDL from apoCI-KO mice than with HDL from control mice despite identical size, lipid composition, and apoAI content of the particles from both sources. Interestingly, the ability of plasma HDL to inhibit CETP activity was also shown to disappear as the result of human apoAI overexpression in transgenic mice in previous studies (
). With regard to the putative role of apoCI as an LCAT cofactor, a non-significant tendency toward a reduction in HDL cholesteryl ester levels was observed in apoCI-KO mice as compared with control mice (Table II). However, it is worthy to note that the physiological relevance of this function, if any, is likely to be out of all proportion to the potent activating potential of apoAI, which has longer been recognized as the physiological cofactor of LCAT (
)) was reported in apoCI-KO mice in previous studies. With regard to the role of apoCI in the catabolism of triglyceride-rich lipoproteins, clear evidence appeared only when apoCI-KO mice were fed a high fat diet, but not when they were fed a standard diet (
) as given in the present study. This suggests that the regulation of lipoprotein clearance may not constitute the primary function of apoCIin vivo. To our knowledge, the inhibition of CETP by apoCI as described here provides the first evidence for a direct and clear role of apoCI in normolipidemic mice. This new function of apoCI may well have been missed in previous studies of apoCI-KO (
In conclusion, the present study indicates that the cholesteryl ester transfer activity in total plasma might be largely dependent on the presence of a specific inhibitor, i.e. apoCI. Given that some alterations in the plasma cholesteryl ester transfer activity in dyslipidemic patients cannot be explained by abnormalities in the plasma concentration of active CETP (