Contribution of cholesteryl ester transfer protein and lecithin:cholesterol acyltransferase to HDL size distribution

High-density lipoproteins (HDL) includes a heterogeneous class of lipoproteins grouped into various subclasses that seem to have different antiatherogenic function. Cholesteryl ester transfer protein (CETP) and lecithin cholesterol acyltransferase (LCAT) play an active role in HDL remodeling. This study was designed to define the role of CETP and LCAT activities on HDL-cholesterol (HDL-C) plasma levels and HDL size distribution, as determined by nondenaturating polyacrylamide gradient gel electrophoresis in 47 clinically healthy Mexican individuals without personal and family history of coronary heart disease. Surprisingly, plasma activities of CETP (29+/-4.1% of transfer) and LCAT (4.8+/-2.2% of esterification) did not correlate either with HDL-C plasma levels or with any other lipid parameter, indicating the poor contribution of these proteins to the lipid profile. The CETP activity showed a negative correlation with small HDL3b (r = -0476, P < 0.05), whereas LCAT was positively associated with this HDL subclass (r = 0.466, P < 0.05). The LCAT showed a negative correlation with large HDL2a (r = - 0.674, P < 0.005). Nevertheless, when the LCAT/CETP ratio was calculated, we observed that the higher the ratio, the greater the relative proportion of small HDL3b (r = 0.551, P < 0.05) and HDL3c (r = 0.477, P < 0.05). These results suggest that the balance of LCAT and CETP activities have a great impact in the plasma HDL size distribution.     

Epistatic effect of cholesteryl ester transfer protein and hepatic lipase on serum high-density lipoprotein cholesterol levels

OBJECTIVES: Polymorphisms in the hepatic lipase (LIPC -514C > T) and cholesteryl ester transfer protein (CETP I405V) genes affect high-density lipoprotein cholesterol (HDL-c) levels, but their relationship with cardiovascular disease and their combined effect is unclear. The objectives of the current study were to characterize the effect of the hepatic lipase variant, and its interaction with the CETP variant, in terms of cholesterol levels, atherosclerosis, and risk of myocardial infarction (MI). DESIGN: The study was conducted in the Rotterdam Study, a large single-center prospective cohort study in people aged 55 yr and older. Lipid levels were analyzed using linear regression models, and risk of MI was assessed with Cox proportional hazards models. RESULTS: The hepatic lipase variant was associated with an increase in serum HDL-c levels of 0.11 mmol/liter in both genders, whereas an increased risk of MI was observed only in men [hazard ratio, 1.32 (95% confidence interval, 1.05-1.66) for CT vs. CC and 1.75 (95% confidence interval, 1.39-2.20) for TT vs. CC]. This effect was independent of serum HDL-c. LIPC -514C > T interacted with CETP I405V with respect to serum HDL-c concentrations. Those homozygous for both mutations saw a marked elevation in HDL-c levels (0.29 mmol/liter, P(interaction) = 0.05). These increased HDL-c levels, however, were not inversely associated with atherosclerosis or MI risk. CONCLUSIONS: LIPC genotype affects HDL-c levels and risk of MI in males. The interaction of this variant with CETP on HDL-c levels helps elucidate the underlying mechanisms and suggests that the beneficial effects of CETP inhibition may vary in particular subgroups.     

Secretion of prebeta HDL increases with the suppression of cholesteryl ester transfer protein in Hep G2 cells.

Prebeta HDL are small, protein rich lipoproteins that are predominantly composed of apo A-I, without apo A-II. Prebeta HDL are secreted from the liver as nascent HDL and/or are produced in the incubated plasma by cholesteryl ester transfer protein (CETP). However, the role of CETP in the secretion of HDL from the liver has yet to be determined. In the present study, we examined the effect of the suppression of hepatic CETP by antisense oligodeoxynucleotides (ODNs) against CETP targeted to the liver on the secretion of apo A-I using a Hep G2 cell culture. The ODNs against CETP were coupled to asialoglycoprotein (ASOR) carrier molecules, which serve as an important method for the regulation of liver gene expression. Hep G2 cells were cultured in DMEM supplemented with 10 FBS. After 2 days, the medium was changed to DMEM with EGF and the cells were divided into three groups. The control group received saline, while the sense group was mixed with the sense ODNs complex and the antisense group was mixed with the antisense ODNs complex, respectively, for 2 days. Both the hepatic CETP mRNA and the CETP mass in the medium in the antisense group decreased significantly more than in the sense and the control groups (CETP mass: 1.697 + /- 0.410 ng/mg cell protein vs. 2.367 + /- 0.22 and 2.360 + /- 0.139, n = 3 in each determination). In contrast, both the hepatic apo A-I mRNA and the apo A-I mass in the medium in the antisense group were significantly higher than those in the sense and the control groups (apo A-I mass; 1.877 + /- 0.215 micro/mg cell protein vs. 1.213 + /- 0.282 and 1.097 + /- 0.144, n = 3 in each determination). The increase in apo A-I was mainly due to the increase in prebeta apo A-I. These findings may partly explain why HDL and apo A-I increase in patients with CETP deficiency, while also indicating the possibility that the original level of prebeta HDL is sufficient in such patients.     

Increasing high-density lipoprotein cholesterol in dyslipidemia by cholesteryl ester transfer protein inhibition: an update for clinicians

Reduced HDL cholesterol may be a risk factor comparable in importance to increased LDL cholesterol. Interventions that raise HDL are antiatherosclerotic, presumably through acceleration of reverse cholesterol transport and by antioxidant and antiinflammatory effects. In the hypercholesterolemic rabbit, HDL levels can be increased by >50% by inhibition of cholesteryl ester transfer protein (CETP), a molecule that plays a central role in HDL metabolism. This HDL-raising effect is antiatherosclerotic in moderately severe hyperlipidemia but appears to be ineffective in the presence of severe hypertriglyceridemia. In humans, mutations resulting in CETP inhibition have been associated with both reduced and increased risk of atherosclerosis. Proposed explanations for these apparently disparate observations are that the antiatherosclerotic effect of CETP inhibition varies with either the metabolic milieu or the degree of CETP inhibition. We now have pharmacological inhibitors of CETP that are capable of increasing HDL by as much as 50% to 100% in humans. The importance of this development is that reduced HDL is a risk factor independent of LDL and that these new agents alter HDL by a magnitude comparable to that of statins on LDL. Clinical trials, now beginning, will need to identify the patient subsets in which CETP inhibition may be more or less effective.     

Modulation of HDL metabolism by probucol in complete cholesteryl ester transfer protein deficiency

Probucol, an antioxidative and hypolipidemic agent, has been postulated to increase reverse cholesterol transport by enhancing cholesteryl ester transfer protein (CETP) activity. However, its clinical implication in CETP deficient patients has not been fully defined. To characterize the effects of probucol in the absence of CETP, we evaluated the changes in lipid profile, lipid peroxidation, and paraoxonase 1 (PON1) activity in two complete CETP deficient patients, caused by treatment with probucol.When the patients were not receiving probucol, low-density lipoprotein (LDL) particles were smaller and high-density lipoprotein (HDL) particles were larger in these patients than in controls. Treatment with probucol (500 mg/day) resulted in the decrease in the levels of HDL-C and apolipoprotein (apo) A-I up to 22%. The size of HDL particles became smaller. LDL cholesterol concentration did not change in one patient, while it decreased by 47% in the other. PON1 activity/HDL-C, which was about 40% lower in the patients before treatment than in controls with the matching PON1 genotype, increased by 30% during the treatment. Lag time for LDL and HDL in both cases became prolonged more than 1.8 times after administration of probucol.This study demonstrated for the first time that probucol reduces HDL-C even in humans with complete CETP deficiency. Probucol treatment in these patients was also associated with protection of lipoproteins against oxidative stress, suggesting a clinical benefit of this drug even in such a state.     

Vitamin E supplementation increases the resistance of both LDL and HDL to oxidation and increases cholesteryl ester transfer activity

There is increasing evidence that lipid peroxidation and oxidative modification of low density lipoprotein (LDL) is important in atherogenesis. Evidence that antioxidant therapy decreases mortality is, however, inconclusive. We have examined the effects of vitamin E on the susceptibility of LDL and high density lipoprotein (HDL) to oxidation, and on cholesteryl ester heteroexchange in an in vitro system using autologous serum lipoproteins. Vitamin E in doses of 200 and 400 mg/day were administered orally to 21 healthy volunteers (12 females and nine males) aged between 23 and 50 years, and to 16 healthy volunteers (eight females and eight males) aged between 22 and 51 years for 50 days, respectively. Fasting serum lipoproteins, susceptibility of lipoproteins to oxidation and cholesteryl ester transfer activity (CETA) were measured before and after vitamin E supplementation. Serum lipoprotein and lipid concentrations did not change significantly in either group. The LDL-conjugated diene (CD) lag phase during incubation with Cu(2+) was increased by 157% (110-232%) (median (interquartile range)) (P<0.05) on vitamin E (200 mg/day) and by 235% (185-259%) (P<0.0001) on 400 mg/day. The lag phases for LDL-lipid peroxide (LPO) generation were also significantly increased by 146% (122-192%) (P<0.005) and 177% (101-267%) (P<0.005), respectively. The HDL-CD lag phase also increased on both doses 140% (115-169%) (P<0.005) and 171% (122-192%) (P<0.005), as did the HDL-LPO lag phase by 123% (104-153%) (P<0.05) on 200 mg/day and 240% (97-360%) (P<0.005) on 400 mg daily. Cholesteryl ester transfer activity from HDL to very low and low density lipoproteins significantly increased from 12. 7+/-2.6 (mean+/-SEM) to 16+/-3.4 nmol/ml/h (P<0.05) on 200 mg/daily and 10.4+/-2.0 to 19.2+/-3.3 nmol/ml/h (P<0.005) on vitamin E, 400mg day. Thus, vitamin E (200 and 400mg daily) significantly decreased the susceptibility of LDL and HDL to oxidation in vitro. However, the increase in CETA resembled that reported with another antioxidant, probucol. Some evidence has suggested that increased CETA is potentially deleterious and it might therefore counteract beneficial effects of vitamin E or probucol on the susceptibility of lipoproteins to oxidation.     

Consumption of French-press coffee raises cholesteryl ester transfer protein activity levels before LDL cholesterol in normolipidaemic subjects

OBJECTIVES: To determine the long-term effects of unfiltered coffee consumption on the activity levels of cholesteryl ester transfer protein (CETP), phospholipid transfer protein (PLTP) and lecithin:cholesterol acyltransferase (LCAT) and to assess a possible role of CETP activity levels in the rise in serum LDL cholesterol. SUBJECTS AND DESIGN: Forty-six healthy normolipidaemic subjects consumed 0.9 L of either French-press or filtered coffee for 24 weeks. Fasting blood samples were obtained after 0, 2, 12 and 24 weeks of intervention and after and 12 weeks of follow-up. MAIN OUTCOME MEASURES: Serum activity levels of CETP, PLTP and LCAT. RESULTS: Relative to baseline, French-press coffee significantly increased average CETP activity by 12% after 2 weeks, by 18% after 12 weeks, and by 9% after 24 weeks. PLTP activity was significantly increased by 10% after 12 and 24 weeks. LCAT activity was significantly decreased by 6% after 12 weeks and by 7% after 24 weeks. The increase in CETP clearly preceded the increase in LDL cholesterol, but not the increase in total triglycerides. However, consumption of French-press coffee caused a persistent rise in CETP activity, whereas the rise in serum triglycerides was transient. CONCLUSIONS: Consumption of cafestol and kahweol cause a long-term increase in CETP as well as PLTP activity; the increase in CETP activity may contribute to the rise in LDL cholesterol.     

The effect of cholesteryl ester transfer protein overexpression and inhibition on reverse cholesterol transport

AIMS: Cholesteryl ester transfer protein (CETP) has a well-established role in lipoprotein metabolism, but the effect of its overexpression or inhibition on the efficiency of reverse cholesterol transport (RCT) is unclear. METHODS AND RESULTS: Neither overexpression of CETP nor treatment with CETP inhibitor Torcetrapib of RAW 264.7 macrophages or HepG2 hepatocytes affected cholesterol efflux in vitro. Overexpression of CETP or treatment with Torcetrapib, respectively, stimulated or inhibited HDL cholesteryl ester uptake by HepG2 but not by RAW 264.7 cells. When RAW 264.7 cells transfected with CETP or ATP binding cassette transporter A1 (ABCA1) were injected intraperitoneally into mice, cholesterol egress from macrophages was elevated for ABCA1- but not for CETP-transfected macrophages. Systemic expression of CETP in mice by adenoviral infection stimulated egress of cholesterol to plasma and liver without affecting HDL levels. Treatment with Torcetrapib did not affect appearance of macrophage cholesterol in plasma and liver, but inhibited its excretion into feces. Treatment of hamsters with Torcetrapib led to elevation of HDL cholesterol, an increase in the capacity of plasma to support cholesterol efflux, and increased egress of cholesterol from macrophages to plasma and feces in vivo. CONCLUSION: Both increased (mice study) and decreased (hamster study) CETP activity could result in enhanced RCT.     

Inhibition of cholesteryl ester transfer protein increases serum apolipoprotein (apo) A-I levels by increasing the synthesis of apo A-I in rabbits

BACKGROUND: Inhibition of cholesteryl ester transfer protein (CETP) is an effective way to increase HDL levels in animals and humans. The effects of a CETP inhibitor, JTT-705, on the in vivo kinetics of apolipoprotein (apo) A-I and apo A-I gene expression in the liver and intestine were investigated. METHODS: Japanese White rabbits were randomly fed normal rabbit chow LRC-4 (n=10, control) or a food admixture of LRC-4 and 0.75% JTT-705 (n=10, treated) for 7 months. An in vivo kinetics study of apo A-I was performed by injecting rabbit 125I-apo A-I, and apo A-I mRNA levels were quantified by RT-PCR. RESULTS: JTT-705 significantly inhibited CETP activities, increased serum levels of HDL-cholesterol (C), HDL2-C, HDL-phospholipid, and apo A-I, and decreased HDL-triglyceride levels. The synthetic rate of apo A-I was higher in the treated rabbits than in control rabbits (13.7 +/- 2.6 versus 9.5 +/- 1.3 mg/kg per day, P < 0.05), while the fractional catabolic rate was similar in the two groups. JTT-705 increased apo A-I mRNA levels in the liver without affecting those in the intestine. CONCLUSION: Inhibition of CETP activity by JTT-705 increases HDL levels by increasing the synthesis of apo A-I, suggesting that it could be a promising therapeutic approach for atherosclerosis.     

High HDL cholesterol does not protect against coronary artery disease when associated with combined cholesteryl ester transfer protein and hepatic lipase gene variants

Cholesteryl ester transfer protein (CETP) and hepatic lipase (HL) are two HDL modifying proteins that have both pro- and anti-atherogenic properties. We hypothesized that CETP and HL synergistically affect HDL cholesterol and atherosclerotic risk. To test our hypothesis, we analysed the genotype frequencies of CETP Taq1B (rs708272) and LIPC-514C/T (rs1800588) polymorphisms in male coronary artery disease patients (CAD; n = 792) and non-symptomatic controls (n = 539). Cases and controls had similar allele frequencies, but the occurrence of the combined genotypes differed (p = 0.027). In CAD patients, 1.3% had the CETP-B2B2/LIPC-TT genotype, with only 0.2% in controls (p = 0.033). The presence of the CETP lowering B2 allele and the HL lowering LIPC-T allele synergistically increased HDL cholesterol from 0.87 ± 0.19 mmol/L in the B1B1/CC (n = 183) to 1.21 ± 0.25 mmol/L in the B2B2/TT carriers (n = 10). The B1B1/CC carriers had an increased CAD risk (OR 1.4; p = 0.025). Despite their high HDL cholesterol, the B2B2/TT individuals also had an increased CAD risk (OR 3.7; p = 0.033). In a 2-year follow up, the loss of coronary artery lumen diameter in these patients was higher than in all other patients combined (0.34 ± 0.70 versus 0.10 ± 0.29 mm; p = 0.044). We conclude that a high HDL cholesterol does not protect against coronary artery disease when associated with combined CETP- and HL-lowering gene variants.     

An increased coronary risk is paradoxically associated with common cholesteryl ester transfer protein gene variations that relate to higher high-density lipoprotein cholesterol: a population-based study

BACKGROUND: Several cholesteryl ester transfer protein (CETP) polymorphisms affect high-density lipoprotein (HDL) cholesterol, but the impact of CETP gene variants on incident coronary disease in the general population is uncertain after correction for their effect on HDL cholesterol. DESIGN: We determined relationships between the CETP -629C-->A promoter (n = 8141), the TaqIB (n = 8289), and the I405V (n = 8265) polymorphisms, serum lipids, C-reactive protein, and clinical factors with incident coronary heart disease (defined as death from or hospitalization for myocardial infarction, ischemic heart disease, or coronary intervention) during a median of 4.94 yr follow-up. SUBJECTS: A predominantly Caucasian general population was studied. RESULTS: HDL cholesterol was 0.08 mmol/liter higher in -629A carriers than in -629CC homozygotes (P < 0.001). The unadjusted coronary hazard was 1.26 [95% confidence interval (CI), 0.95-1.68; P = 0.11] in A carriers compared with CC homozygotes and increased to 1.46 (95% CI, 1.10-1.95; P = 0.01) after adjustment for HDL cholesterol. This effect remained after additional adjustment for apolipoprotein A-I, triglycerides, C-reactive protein, age, and gender. Likewise, the HDL-cholesterol-adjusted hazard ratio was also higher in AA than in CC homozygotes (hazard ratio, 1.72; 95% CI, 1.22-2.42; P < 0.01). Similar findings were obtained with the TaqIB polymorphism. The 405V allele was weakly associated with incident coronary heart disease after HDL cholesterol adjustment (P = 0.09). CONCLUSIONS: A common CETP promoter polymorphism, which beneficially contributes to higher HDL cholesterol, is paradoxically associated with increased incidence of coronary disease in the general population. Thus, CETP gene variation may affect coronary risk apart from the level of HDL cholesterol.     

Estimation of HDL cholesteryl ester kinetic parameters in the cebus monkey, an animal species with high plasma cholesteryl ester transfer activity

We studied the kinetic parameters of high density lipoprotein (HDL) cholesteryl esters in the cebus monkey, an animal species with high plasma cholesteryl ester transfer activity. HDL were radiolabeled with cholesteryl [1-14C]oleate and intravenously administered to 4 cebus monkeys. The calculated fractional catabolic rate (FCR) of the HDL cholesteryl esters was 0.081 +/- 0.002 (mean +/- SD) h-1 and the calculated residence time was 12.3 +/- 0.3 h. The production or disposal rate of plasma HDL cholesteryl esters was 34.3 +/- 4.5 mumol/h. The radiolabeled cholesteryl esters were rapidly transferred from the HDL to the very low and low density lipoproteins (VLDL + LDL) and the amount of tracer in the VLDL + LDL had already reached a maximum at 3.5 +/- 0.7 h after tracer administration. The estimated fraction of VLDL + LDL cholesteryl esters derived from the HDL was 0.77 +/- 0.06. We also used radiolabeled [1,2-3H(N)]cholesteryl palmityl ether to trace HDL cholesteryl esters, but the ether tracer was more slowly cleared from the plasma and less readily transferred between plasma lipoproteins than the ester tracer.     

Acute exposure to a high cholesterol diet attenuates myocardial ischemia-reperfusion injury in cholesteryl ester transfer protein mice

BACKGROUND: Previous experiments have demonstrated that acute exposure to a high-cholesterol diet (HCD) increases the severity of myocardial infarction in animals. Recent results suggest that the process is modulated by multiple genes and their interactions with circulating cholesterol. DESIGN: In the present study cholesteryl-ester-transfer-protein (CETP) transgenic mice were generated and fed a normal rodent-chow diet, HCD for 1 week, or a HCD for 6 weeks in order to define the role of CETP in myocardial infarction after acute exposure to a HCD. METHODS: Cholesterol levels in mice of all groups were measured. Separate groups of mice were exposed to 30 min of in-vivo occlusion of coronary artery and 2 h of reperfusion. We assessed the sizes of the ischemic zone and infarct using Evans blue and 2,3,5-triphenyltetrazolium chloride. RESULTS: The extent of infarction (percentage infarct/area at risk) was significantly less (P < 0.05) after 1 week of a HCD (18.7 +/- 7.0%) than those for the normal diet group (51.4 +/- 5.5%) and the group fed a HCD for 6 weeks (44.4 +/- 5.2%). Additionally, there was significantly less infiltration of neutrophils into the ischemic-reperfused mouse hearts for mice fed a HCD for 1 week. Levels of reduced and oxidized glutathione in the hearts of CETP mice were measured for separate groups of animals. The reduced:oxidized-glutathione ratio was significantly (P < 0.01) lower for mice fed a HCD for 1 week (1.5 +/- 0.1) than it was for mice fed a normal diet (3.6 +/- 0.3) and a HCD for 6 weeks (3.3 +/- 0.2). CONCLUSIONS: These data suggest that activity of CETP in hypercholesterolemic mice has an acute effect on size of infarct after 1 week of a HCD. This suggests that CETP induces tolerance of ischemia in the mice fed a HCD via mild oxidative stress.