High Cholesteryl Ester Transfer Protein Research Articles

Cholesteryl ester transfer protein expressed in lecithin cholesterol acyltransferase-deficient mice.

Regulation of plasma cholesteryl ester transfer protein (CETP) concentration was studied in lecithin-cholesterol acyltransferase (LCAT)-knockout mice. LCAT-knockout mice were cross-bred with CETP transgenic mice. The offspring (n=63) were classified for LCAT genotype and plasma CETP levels (no CETP, low CETP, and high CETP). High density lipoprotein (HDL) decreased as LCAT decreased in each CETP-level group. In the lcat(+/+) and lcat(+/-) mice, plasma CETP varied from 0 to 30 micro g/mL, whereas it was <10 micro g/mL in the lcat(-/-) mice. HDL cholesterol and phospholipid decreased and HDL triglyceride and apolipoprotein B increased in CETP in the lcat(+/+) and lcat(+/-) mice, whereas there was no difference in HDL between low and high CETP. An effect of CETP on HDL was not detected in the lcat(-/-) mice because of the absence of mature HDL. Genomic DNA and mRNA of CETP were correlated and were similar in the lcat(-/-) and lcat(+/+) mice. Plasma CETP was correlated with its genomic DNA and mRNA, but the slope of the increase was much lower in the lcat(-/-) mice. Whereas plasma CETP mostly associates with HDL in the lcat(+/+) mouse, it is found free in the lcat(-/-) mouse. Plasma CETP is posttranscriptionally downregulated in the lcat(-/-) mice, presumably by its extremely low HDL.

A novel cholesteryl ester transfer protein promoter polymorphism (−971G/A) associated with plasma high-density lipoprotein cholesterol levels: Interaction with the TaqIB and −629C/A polymorphisms

The plasma cholesteryl ester transfer protein (CETP) plays a key role in reverse cholesterol transport (RCT) by mediating the transfer of cholesteryl ester (CE) from high-density lipoprotein (HDL) to atherogenic ApoB-containing lipoproteins, including VLDL, IDL and LDL. We describe a new polymorphism located at position −971 in the human CETP gene promoter, which corresponds to a G/A substitution at a potential AvaI restriction site. The relationship between the −971G/A polymorphism, plasma lipid parameters and plasma CETP concentration was evaluated in the Etude Cas-Témoins de l'Infarctus du Myocarde (control–myocardial infarction cases) cohort, and revealed that the −971G/A polymorphism (A allele frequency: 0.491) was significantly associated with both plasma high-density lipoprotein cholesterol (HDL-C) levels and CETP concentration ( P=0.006 and 0.009, respectively). Subjects with genotype −971GG displayed both low HDL-C levels and high plasma CETP concentration, while genotype −971AA subjects displayed the inverse relationship. Evaluation of potential interactions between the −971G/A and the −629C/A or TaqIB polymorphisms demonstrated that the −971G/A polymorphism interacts significantly with the functional −629C/A site and the TaqIB polymorphism with respect to plasma HDL-C levels ( P=0.0014 and 0.012, respectively), but does not affect plasma CETP concentration. These results clearly suggest that the interaction between the 971G/A polymorphism and either the −629C/A or the TaqIB polymorphism on plasma CETP concentration is different than that implicated in HDL-C levels. Transient transfection of HepG2 cells revealed that the −971G/A polymorphism did not modulate transcriptional activity of the human CETP gene promoter. The −971G/A promoter polymorphism therefore constitutes a non-functional marker. Furthermore, the observed effects of the −971G/A polymorphism on both plasma CETP concentration and HDL-C levels are due to functional variants in linkage disequilibrium with it. Our findings strongly suggest the existence of as yet unidentified functional polymorphisms in the CETP gene promoter that could explain the association between specific polymorphisms of the CETP gene and both plasma HDL-C and CETP concentrations.

The relationship between plasma high density lipoprotein cholesterol levels and cholesteryl ester transfer protein activity in six species of healthy experimental animals.

Plasma high-density lipoprotein cholesterol (HDL-C) concentrations are regulated by plasma cholesteryl ester transfer protein (CETP) in humans. The aim of this study was to ascertain the relationship between plasma HDL-C and plasma CETP activities in mouse, rat, dog, hamster, rabbit and monkey. In this study, the plasma HDL-C levels were highest in dogs and lowest in rabbits among the six species. Plasma CETP activities were higher in hamsters, rabbits and monkeys compared to mice, rats and dogs. The present study shows that there are species differences in HDL-C and CETP activity in six species of healthy experimental animals, with the six species being separated into two types. The first type showed a high HDL-C/TC ratio with low or absent CETP activity, and included mouse, rat and dog, whereas the second type showed a low HDL-C/TC ratio and high CETP activity, and included hamster, rabbit and monkey. The present study also shows that there is a strong relationship between plasma HDL-C levels and CETP activity in high CETP activity animals and that the relationship between the HDL-C/TC ratio and CETP activity is an important factor in all animals, regardless of CETP activity level.

Plasma cholesteryl ester transfer protein activity is high in infants and is not affected by thyroid hormones

We investigated the effects of thyroid dysfunction on cholesteryl ester transfer protein (CETP) by studying plasma CETP activity in hypothyroid infants before and after they were rendered euthyroid by L-thyroxine (LT4) replacement therapy. To exclude environmental factors possibly affecting plasma CETP activity, we selected hypothyroid infants to study plasma CETP activity. Plasma CETP activity was measured as the rate of radiolabeled cholesteryl ester transfer from high-density lipoprotein (HDL) to serum apolipoprotein B (apo B)—containing lipoproteins in plasma from 14 hypothyroid infants before and 2 months after LT4 replacement, 23 normal infants, and 61 normal adults. Relationships between CETP and thyroid hormones were examined separately in the 14 hypothyroid infants and 32 euthyroid infants, including the 14 above-described hypothyroid infants and an additional 18 treated hypothyroid infants. Serum levels of thyrotropin (TSH), free thyroxine (FT4), and free triiodothyronine (FT3) were also determined on an autoanalyzer system in our hospital. In contrast to previous reports, we found no differences in plasma CETP activity between hypothyroid infants and age-matched normal infants. LT4 substitution did not cause any changes in plasma CETP activity after therapy. Plasma CETP activity showed no correlation with serum TSH, FT4, and FT3 levels. Both hypothyroid and normal infants were found to have significantly higher plasma CETP activity than normal adults. From these results, we conclude that in infants thyroid hormones do not affect plasma CETP activity, and normal infants have high plasma CETP activity, compared with normal adults.

The capacity of various non-esterified fatty acids to suppress lipid transfer inhibitor protein activity is related to their perturbation of the lipoprotein surface

Lipid transfer inhibitor protein (LTIP) regulates cholesteryl ester transfer protein (CETP) activity by selectively impeding lipid transfer events involving low density lipoproteins (LDLs). We previously demonstrated that LTIP activity is suppressed in a dose-dependent manner by sodium oleate and that its activity can be blocked by physiological levels of free fatty acids [R.E. Morton, D.J. Greene, Arterioscler. Thromb. Vasc. Biol. 17 (1997)]. These data further suggested that palmitate has greater LTIP suppressive activity than oleate. In this report we define the ability of the major non-esterified fatty acids (NEFAs) in plasma to modulate LTIP activity. The greater suppression of LTIP activity by palmitate compared to oleate noted above was also seen in lipid transfer assays with various lipoprotein substrates and in the presence of albumin, showing that the relative effects of these two NEFAs are independent of assay conditions. To assess the effect of other NEFAs on LTIP activity, pure NEFAs were added to assays containing 3H-cholesteryl ester labeled LDLs, unlabeled high density lipoproteins (HDLs) and CETP±LTIP. Whereas myristate, palmitate, stearate, oleate and linoleate stimulated CETP activity to varying extents, all NEFAs suppressed LTIP activity. Among these NEFAs, LTIP suppressive activity was greatest for the long-chain saturated and monounsaturated NEFAs. In contrast, linoleate and myristate were poor inhibitors of LTIP activity. The effects of increasing amounts of a given NEFA on LTIP activity correlated well with the increase in LDL negative charge induced by that NEFA, yet this relationship was unique for each NEFA, especially stearate. Notably, as measured by fluorescence anisotropy, the suppression of LTIP was highly and negatively correlated with the decreased order in the molecular packing of lipoprotein surface phospholipids caused by all NEFAs. Long-chain, saturated and monounsaturated NEFAs appear to be most effective in this regard partly because of their preferential association with LDLs where LTIP inhibition likely takes place. We hypothesize that NEFAs suppress LTIP activity by perturbing the surface properties of LDLs and counteracting the heightened molecular packing normally caused by LTIP. Diets rich in long-chain saturated and monounsaturated fatty acids may lead to a greater suppression of LTIP activity in vivo, which would allow LDLs to participate more actively in CETP-mediated lipid transfer reactions.

Cholesteryl ester transfer protein gene polymorphisms are associated with carotid atherosclerosis in men.

The cholesteryl ester transfer protein (CETP) is involved in the reverse cholesterol transport and is therefore a candidate gene for atherosclerosis. The prevalences of the I405V and the R451Q polymorphisms were studied in a population sample of 515 men and women. Genotypes were determined by PCR and carotid atherosclerosis by ultrasonography as the mean intima-media thickness (IMT) of the carotid arteries. The Q451 allele was associated with significantly lower intima media thickness in men (P = 0.001). The Q451 allele was, in our earlier study, associated with high plasma CETP activity in men. The VV405 genotype was associated with lower plasma CETP activity compared with the II405 genotype (P < 0.01 for the difference). In the general linear model general factorial procedure the interaction between alcohol consumption and the I405V genotype on IMT was significant (P = 0.013) in men, and when the interaction term was taken into the model the I405V genotype also significantly affected IMT (P = 0.008). The VV405 genotype seems to be most harmful for men with the highest alcohol consumption. We describe two polymorphisms of the CETP gene associated with intima media thickness in men. A significant interaction was found between alcohol consumption and the I405V genotype on IMT.

High pre β1-HDL levels in hypercholesterolemia are maintained by probucol but reduced by a low-cholesterol diet

Previous study has shown that pre β1-HDL levels increase in hypercholesterolemia, or high cholesteryl ester transfer protein (CETP) activity. To determine how pre β1-HDL levels change after treatment with probucol or by following a low-cholesterol diet, we randomly assigned 24 hypercholesterolemic patients to either the probucol (P), or low-cholesterol diet group (D), and measured pre β1-HDL levels before and after treatments using native two-dimensional gel electrophoresis. We also examined 12 subjects with normolipidemia (N). At baseline, pre β1-HDL levels were higher in P ( P<0.05) and D ( P<0.05) than in N (9.2±4.3, 10.4±5.5, and 5.9±2.3 mg/dl apo A-I). After a 4-week treatment, pre β1-HDL levels were still high in P (10.5±4.2 mg/dl apo A-I, N.S.), but reduced in D (7.7±3.0 mg/dl apo A-I, P<0.001). Δpre β1-HDL (Y) was positively correlated with ΔCETP mass (X) in P ( y=7.83 x−1.93; r=0.584, P<0.05). In summary, high pre β1-HDL levels in hypercholesterolemia are maintained by probucol but reduced by a low-cholesterol diet. These findings suggest that pre β1-HDL levels may be regulated by cholesterol and CETP levels.

R451Q mutation in the cholesteryl ester transfer protein (CETP) gene is associated with high plasma CETP activity

Cholesteryl ester transfer protein (CETP), as a candidate gene for dyslipoproteinemia and coronary heart disease, was studied in 105 men with low plasma concentrations of high density lipoprotein cholesterol (HDL-C) and established coronary heart disease as well as in 515 randomly selected men and women. A one-nucleotide substitution (G to A) in exon 15, which changes arginine (451) to glutamine in CETP protein, was detected by PCR-SSCP and direct sequencing and screened in the population sample by a simple PCR-based restriction assay. In the random population sample the allele frequency of the R451Q mutation was 1.9%. Men heterozygous for the R451Q mutation ( n=7) had 27% higher CETP activity than age-, body mass index-, smoking- and alcohol consumption-matched controls with normal genotype ( n=21; P=0.003). Women heterozygous for the R451Q mutation ( n=7) had 16% lower total cholesterol compared to matched controls ( n=21; P=0.07), but no such difference was detected in men. In the random population sample the correlation between plasma total cholesterol level and CETP activity was 0.19 ( P=0.044), both in men and women. When women with total cholesterol over 5.2 mmol/l were excluded from analysis, heterozygotes ( n=4) had plasma CETP activity of 113 nmol/h/ml plasma, whereas those of normal genotype ( n=12) had 103 nmol/h/ml plasma, but this difference was not statistically significant. Women heterozygous for the R451Q mutation and consuming less than 10 g alcohol a week had 23% lower HDL-C compared to women with the normal genotype ( P=0.032). In conclusion, we describe a mutation in the CETP gene associated with high plasma CETP activity in men and with low total cholesterol in women. Further studies are needed to evaluate the effect of mutation on the risk of coronary heart disease.

The novel compound NO-1886 elevates plasma high-density lipoprotein cholesterol levels in hamsters and rabbits by increasing lipoprotein lipase without any effect on cholesteryl ester transfer protein activity

Lipoprotein lipase (LPL) and cholesteryl ester transfer protein (CETP) are determinants of high-density lipoprotein (HDL) cholesterol concentrations in plasma. We have previously reported that NO-1886, by increasing LPL activity, causes elevation of HDL cholesterol levels in rats. In the present study, we studied the effect of NO-1886 on CETP activity in experimental animals. Since previous reports suggest that rats may lack CETP, we examined hamsters and rabbits, as well as rats. We found that NO-1886 increased LPL activity, resulting in elevation of plasma HDL cholesterol in all three animals. We confirmed that rats lack CETP and that both hamsters and rabbits have high CETP activity. NO-1886 had no effect on CETP activity in hamsters and rabbits. These results demonstrate that the compound NO-1886 elevates HDL cholesterol in experimental animals by selectively increasing LPL activity without any effect on CETP. Animals with low CETP and high LPL activities appear to be more sensitive to NO-1886 than those with high CETP and low LPL activities.

Heterogeneity at the CETP gene locus. Influence on plasma CETP concentrations and HDL cholesterol levels.

This study was designed to investigate the association(s) between heterogeneity at the cholesteryl ester transfer protein (CETP) gene locus, CETP plasma concentrations, and HDL cholesterol levels. Healthy men with the lowest, median, and highest deciles of HDL cholesterol were selected from a large population database. We accounted for factors that are known to influence HDL cholesterol levels, such as smoking, exercise, body mass index, alcohol consumption, and blood pressure. Plasma CETP concentrations were measured, and we determined the allele frequency distribution of six CETP DNA polymorphisms. The group with low HDL cholesterol exhibited a significant increase in CETP concentration compared with both the median and high HDL cholesterol groups, whereas CETP concentrations did not differ among the groups with median and high HDL cholesterol. The allele frequency distributions of the TaqIB (intron 1), Msp I (intron 8), and Rsa I (exon 14) polymorphisms differed significantly between the groups with low and high HDL cholesterol. Further analysis revealed that the Msp I polymorphism had a 1.5-fold larger impact on CETP concentration than the TaqIB polymorphism and a fivefold larger impact than the Rsa I polymorphism. In conclusion, we demonstrated that heterogeneity at the CETP gene locus is correlated with CETP plasma concentrations and HDL cholesterol levels. More specifically, our data indicate the presence of a strong association between common variants of the CETP gene, high plasma CETP concentrations, and consequently hypoalphalipoproteinemia in healthy white men.

24] Quantification of cholesteryl ester transfer protein: Activity and immunochemical assay

The assay for cholesteryl ester transfer protein (CETP) activity common to most publications consists of incubating [ 3 H] high-density lipoprotein (HDL) (1-10 μg cholesterol) and unlabeled low-density lipoprotein (LDL) (1-10μg cholesterol) at various donor-to-acceptor ratios from 1:1 to 1:10, with a source of CETP activity for 2 to 24 hr. Aliquots are removed from the reaction mixture at timed intervals or at the end of the incubation period and the LDL fraction is precipitated by the addition of heparin/MnCl 2 .The amount of CE transferred from HDL to LDL is measured as the difference between the amount of [ 3 H]CE radioactivity, remaining in the supernatant HDL fraction and the amount of [ 3 H]CE HDL in a control reaction lacking CETP. This chapter describes a rapid, high sample capacity CETP activity assay, utilizing the Packard (Downer Grove, IL) topCount 96-well plate liquid scintillation counter and Millipore (Bedford, MA) multiScreen filtration plates that permit direct measurement of CETP-mediated transfer of cholesteryl ester (CE) into LDL, by counting the radioactivity in the precipitated LDL. In this filter plate-based CETP activity assay, higher or lower levels of CETP activity directly correspond to higher or lower amounts of labeled CE incorporated into the precipitated LDL.

Bimodal Distribution of Cholesteryl Ester Transfer Protein Activities in Normotriglyceridemic Men With Low HDL Cholesterol Concentrations

Increased plasma activities of cholesteryl ester transfer protein (CETP) theoretically could lower HDL cholesterol levels due to enhanced transfer of cholesteryl esters from HDL to apo B-containing lipoproteins. To determine whether high CETP activities are associated with isolated hypoalphalipoproteinemia, CETP activities were measured in 109 adult men with HDL cholesterol < 35 mg/dL, plasma triglycerides < 200 mg/dL, and LDL cholesterol < 160 mg/dL; the results were compared with those of 50 normolipidemic (HDL cholesterol > 40 mg/dL) male subjects. CETP activities were assayed in vitro and expressed as the percent of [3H]cholesteryl ester transferred from HDL3 to LDL during a 16-hour incubation. In addition, postheparin plasma activities of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) were determined in 71 patients with a low HDL cholesterol level. Distributions of CETP activities were unimodal in control subjects (mean +/- SD, 23.1 +/- 5.0%), but they were bimodal in the low-HDL patients. Among the latter, 27 patients had elevated CETP activities (40.8 +/- 4.6%), whereas 82 patients had CETP activities that overlapped the normal range (26.14 +/- 7.6%). Low-HDL patients with normal CETP activities had 20% lower LPL activities (P = .01), 25% higher HTGL activities (P = .03), and 63% lower LPL/HTGL ratios (P < .001) than those of low-HDL patients with increased CETP activity. Furthermore, mean LPL and HTGL activities in the low-HDL patients with elevated CETP activities were in the normal range. Another important distinction between the two subgroups with low HDL was that the subgroup with high CETP activity had only a 30% prevalence of coronary heart disease compared with a 70% prevalence in the subgroup with normal CETP activity (P < .01). These findings suggest that elevated CETP activity may be a significant factor in causing low HDL cholesterol levels in a distinct subgroup of normolipidemic patients with low HDL cholesterol levels.

The role of cholesteryl ester transfer protein in primate apolipoprotein A-I metabolism. Insights from studies with transgenic mice.

To assess the effects of cholesteryl ester transfer protein (CETP) on the primate lipoprotein profile, a transgenic mouse expressing cynomolgus monkey CETP was developed. The C57BL/6 mouse was used, and four lines expressing the primate CETP were established. The level of CETP activity in the plasma of the transgenic mice ranged from values similar to those obtained for the monkey to levels approximately sixfold higher than that in the normal monkey. When all of the lines were taken into consideration, there was a strong (r = -0.81 or higher, p less than 0.01) negative correlation between plasma CETP activity and total plasma cholesterol, plasma apolipoprotein (apo) A-I levels, and plasma apo A-I to apo B ratio. There was a strong positive correlation (r = 0.77) between plasma CETP activity and plasma apo B levels. The size of the apo A-I-containing lipoproteins was significantly reduced in mice with high plasma CETP activity, and that reduction in size was due to the absence of the larger (HDL1 and HDL2) apo A-I-containing particles in the plasma. When the transgenic mice were fed a high-fat, high-cholesterol diet, the effects of the diet on lipoprotein profile were more prominent in the CETP transgenic mice than the controls. The CETP transgenic mice had, for example, substantially higher plasma cholesterol and plasma apo B levels (p less than 0.01), and the apo B-containing lipoproteins were generally larger than those in the nontransgenic C57BL/6 mice consuming the same diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that the nonparenchymal cells of the liver are the principal source of cholesteryl ester transfer protein in primates

Previous studies showed that 90% or more of the cholesteryl ester transfer protein (CETP) mRNA is contained in the liver of cynomolgus monkeys. The purpose of this study was to determine if the parenchymal cells (hepatocytes) were the hepatic cell type that contained that mRNA. The parenchymal and nonparenchymal cells were separated by standard methods, and the CETP, apoA-I, apoB, and apoE mRNA content of the preparation determined at each step in the purification process. ApoA-I and apoB are produced only in the parenchymal cells; apoE is produced by both cell types. The mRNA measurements showed that the CETP mRNA: apoA-I mRNA and the CETP mRNA: apoB mRNA ratios were more than 2500-fold greater in the nonparenchymal cell preparation than in the starting material, and that the purified parenchymal cell fraction was virtually devoid of CETP mRNA. In situ hybridization studies showed that, whereas the apoA-I mRNA signal was evenly distributed over the tissue section, the CETP mRNA signal was associated with the hepatic sinusoids, suggesting that it was the hepatic sinusoidal cells that were principally responsible for the high CETP mRNA levels in the liver. We conclude that the nonparenchymal cells are the principal source of CETP in the cynomolgus monkey.

Elevated Cholesteryl Ester Transfer Protein Activity in IDDM Men Who Smoke: Possible Factor for Unfavorable Lipoprotein Profile

To determine the effect of cigarette smoking on the activity of cholesteryl ester transfer protein (CETP) and high-density (HDL), low-density (LDL), and very-low-density (VLDL) lipoproteins in insulin-dependent diabetic (IDDM) men with microvascular complications. We performed a case-control study in a referral-based diabetes clinic on a sequential sample of 9 cigarette-smoking and 12 nonsmoking IDDM men with microvascular complications and 12 nonsmoking control men. CETP activity was determined in each serum with an isotope assay with exogenous cholesteryl ester-labeled LDL and HDL. The method is independent of the endogenous lipoprotein present in serum. The HDL-cholesterol (VLDL and LDL) ratio was lower in the smoking diabetic men than in the other groups (P less than 0.05 vs. the nonsmoking diabetic men and P less than 0.01 vs. the control subjects). CETP activity was 70% higher in the smoking diabetic men than in the control subjects (P less than 0.01) and 30% higher than in the nonsmoking diabetic men (P less than 0.05). The HDL-cholesterol (VLDL and LDL) ratio and the apolipoprotein A-I-B ratio were inversely correlated to CETP activity in the diabetic patients (r = -0.52, P less than 0.02 and r = -0.45, P less than 0.05, respectively). CETP activity is increased in cigarette-smoking IDDM men with microvascular complications. High CETP activity may contribute to the unfavorable lipoprotein profile in these patients.