Cholesteryl ester transfer protein and atherosclerosis

Plasma cholesteryl ester transfer protein facilitates the transfer of cholesteryl ester from HDL to apolipoprotein B-containing lipoproteins. Its significance in atherosclerosis has been debated in studies of human population genetics and transgenic mice. The current review will focus on human plasma cholesteryl ester transfer protein research, including TaqIB, 1405V, and D442G polymorphisms. Plasma cholesteryl ester transfer protein has a dual effect on atherosclerosis, depending on the metabolic background. In hypercholesterolaemia or combined hyperlipidaemia, plasma cholesteryl ester transfer protein may be pro-atherogenic and could be a therapeutic target.     

Cholesteryl ester transfer protein expression is down-regulated in hyperinsulinemic transgenic mice

Cholesteryl ester transfer protein (CETP) mediates cholesteryl ester (CE) and triglyceride redistribution among plasma lipoproteins. In this work, we investigated whether varying levels of insulin regulate the CETP expression in vivo. Insulin deficiency [streptozotocin (STZ) injection], and hyperinsulinemia (insulin injections, 14 days) were induced in transgenic mice expressing a human CETP minigene flanked by its natural regulatory sequences. Glucose supplementation was provided to the hyperinsulinemic group (INS+GLUC) and to an extra group of mice (GLUC). In the STZ group, endogenous CE transfer rate, plasma CETP, and hepatic CETP mRNA levels were enhanced 3.0-, 1.5-, and 2.5-fold, respectively, as compared with controls. Insulin replacement in STZ mice normalized their glycemia and liver mRNA levels. Higher plasma CETP levels were observed in GLUC mice, which were decreased in INS+GLUC mice. Hepatic CETP mRNA was not altered in GLUC mice and was reduced by one-third in INS+GLUC mice. These results show that: 1) STZ treatment increases CETP plasma levels and liver mRNA expression; 2) diet glucose supplementation increases plasma CETP levels but does not change liver mRNA abundance; and 3) daily insulin injections blunt the glucose-stimulated CETP expression by reducing its liver mRNA levels. These data suggest that insulin down-regulates CETP gene expression.     

Cholesteryl ester transfer protein: the controversial relation to atherosclerosis and emerging new biological roles

Cholesteryl ester transfer protein (CETP) exerts a profound impact on high-density lipoprotein (HDL) metabolism and, consequently, on the risk of atherosclerosis development and cardiovascular mortality. Here, we review the complex relationship between CETP and atherosclerosis based upon the experimental, clinical, and epidemiological studies. In addition, we discuss the recent findings that expand the functions of CETP to new areas of interest such as Alzheimer's disease, inflammation, and obesity.     

Cholesteryl ester transfer protein, high density lipoprotein and arterial disease

The reported relationships between cholesteryl ester transfer protein, high density lipoproteins and arterial disease are confusing and conflicting. Several papers published during the review period add substantially to the evidence base regarding the atherogenicity (or anti-atherogenicity) of cholesteryl ester transfer protein, although none clearly resolves the continuing conflict. These new papers are presented against the backdrop of the previous state of knowledge.     

Cholesteryl ester transfer protein inhibition as a strategy to reduce cardiovascular risk

Human and rabbit plasma contain a cholesteryl ester transfer protein (CETP) that promotes net mass transfers of cholesteryl esters from high density lipoproteins (HDL) to other plasma lipoprotein fractions. As predicted, inhibition of CETP in both humans and rabbits increases the concentration of cholesterol in the potentially protective HDL fraction, while decreasing it in potentially proatherogenic non-HDL fractions. Inhibition of CETP in rabbits also inhibits the development of diet-induced atherosclerosis. However, use of the CETP inhibitor torcetrapib in humans did not reduce atheroma in three imaging trials and caused an excess of deaths and cardiovascular events in a large clinical outcome trial. The precise explanation for the harm caused by torcetrapib is unknown but may relate to documented, potentially harmful effects unrelated to inhibition of CETP. More recently, a trial using the weak CETP inhibitor dalcetrapib, which raises HDL levels less effectively than torcetrapib and does not lower non-HDL lipoprotein levels, was terminated early for reasons of futility. There was no evidence that dalcetrapib caused harm in that trial. Despite these setbacks, the hypothesis that CETP inhibitors will be antiatherogenic in humans is still being tested in studies with anacetrapib and evacetrapib, two CETP inhibitors that are much more potent than dalcetrapib and that do not share the off-target adverse effects of torcetrapib.     

Cholesteryl ester transfer protein deficiency causes slow egg embryonation of Schistosoma japonicum

It was investigated whether proteasome activity was implicated in susceptibility of human vascular smooth muscle cells (VSMCs) to Fas-mediated death. Human fetal aorta smooth muscle cells were treated with agonistic anti-Fas antibody (CH11) and proteasome inhibitors (MG115 or MG132) and then cell death was determined by morphology, viability, and DNA fragmentation. The present study reports that: (a) crosslinking of Fas receptor with anti-Fas antibody in the presence of proteasome inhibitor-induced death and DNA degradation in human VSMCs that were blocked by caspases inhibitor z-DEVD.fmk; (b) cotreatment with anti-Fas antibody and proteasome inhibitor activated caspase-3; (c) proteasome inhibitors did not influence expression of procaspase-8, procaspase-3, c-FLIP, and Bcl-2; and (d) proteasome inhibitors up-regulated Fas and FADD. The data indicate that proteasome activity is important in survival of VSMCs and provide the first evidence that proteasome is involved in Fas signal transduction. The present study proposes novel mechanism(s) by which VSMCs become susceptible to FasL.     

Cholesteryl ester transfer protein biosynthesis and cellular cholesterol homeostasis are tightly interconnected

Cholesteryl ester transfer protein (CETP) mediates triglyceride and cholesteryl ester (CE) transfer between lipoproteins, and its activity is strongly modulated by dietary cholesterol. To better understand the regulation of CETP synthesis and the relationship between CETP levels and cellular lipid metabolism, we selected the SW872 adipocytic cell line as a model. These cells secrete CETP in a time-dependent manner at levels exceeding those observed for Caco-2 or HepG2 cells. The addition of LDL, 25OH-cholesterol, oleic acid, or acetylated LDL to SW872 cells increased CETP secretion (activity and mass) up to 6-fold. In contrast, CETP production was decreased by almost 60% after treatment with lipoprotein-deficient serum or beta-cyclodextrin. These effects, which were paralleled by changes in CETP mRNA, show that CETP biosynthesis in SW872 cells directly correlates with cellular lipid status. To investigate a possible, reciprocal relationship between CETP expression and cellular lipid homeostasis, CETP biosynthesis in SW872 cells was suppressed with CETP antisense oligonucleotides. Antisense oligonucleotides reduced CETP secretion (activity and mass) by 60% compared with sense-treated cells. When CETP synthesis was suppressed for 24 h, triglyceride synthesis was unchanged, but cholesterol biosynthesis was reduced by 20%, and acetate incorporation into CE increased 31%. After 3 days of suppressed CETP synthesis, acetate incorporation into the CE pool increased 3-fold over control. This mirrored a similar increase in CE mass. The efflux of free cholesterol to HDL was the same in sense and antisense-treated cells; however, HDL-induced CE hydrolysis in antisense-treated cells was diminished 2-fold even though neutral CE hydrolase activity was unchanged. Thus, CETP-compromised SW872 cells display a phenotype characterized by inefficient mobilization of CE stores leading to CE accumulation. These results strongly suggest that CETP expression levels contribute to normal cholesterol homeostasis in adipocytic cells. Overall, these studies demonstrate that lipid homeostasis and CETP expression are tightly coupled.     

Cholesteryl ester transfer protein. Size of the functional unit determined by radiation inactivation

Radiation inactivation was used to determine the functional Mr of cholesteryl ester transfer protein (CETP) in rabbit plasma from control and irradiated animals. This technique reveals the size of the functional unit required to carry out the transfer function. The functional Mr was calculated to be 70 000 +/- 3000 (mean +/- SD) for both control and irradiated rabbits. This result is in accordance with the Mr obtained by a completely different method, namely SDS-polyacrylamide gel electrophoresis of a partially purified (110-fold) rabbit CETP. The pI of this CETP was found by isoelectric focusing to be equal to 5.95. The results suggest that the functional unit of this enzyme is the monomer.     

Cholesteryl ester transfer protein inhibition, high-density lipoprotein metabolism and heart disease risk reduction

PURPOSE OF REVIEW: Cholesteryl ester transfer protein (CETP) inhibitors (JTT-705 and torcetrapib) are currently in clinical testing, and significantly raise high-density lipoprotein (HDL) cholesterol levels. Low HDL cholesterol is a significant independent predictor of coronary heart disease (CHD) and HDL raising has been associated with coronary heart disease risk reduction, but there is debate about whether CETP inhibition will reduce coronary heart disease risk. RECENT FINDINGS: It has been documented in transgenic mouse models that apolipoprotein (apo) C-I inhibits CETP, and that high mono-unsaturated fat diets prevent the normal stimulation of CETP activity by dietary cholesterol. In rabbits, torcetrapib markedly decreases clearance of HDL cholesteryl ester via an indirect pathway, but has no effect on total plasma cholesteryl ester clearance. In humans, torcetrapib raises HDL apoA-I by modestly decreasing its fractional catabolic rate, while having a very profound effect on raising HDL cholesterol and large alpha-1 migrating HDL particles by more than 50%, with no effect on fecal cholesterol excretion. When JTT-705 at 600 mg/day was given to hypercholesterolemic patients already on pravastatin 40 mg/day, the combination was well tolerated and increases in HDL cholesterol of 28% were noted. SUMMARY: In our view, CETP inhibitors in combination with statins will be profoundly beneficial in reducing human atherosclerosis, primarily because they normalize HDL particles and prevent the transfer of cholesteryl ester from HDL to atherogenic lipoproteins.     

Cholesteryl ester transfer protein activity and atherogenic parameters in rabbits supplemented with cholesterol and garlic powder

The current study was conducted to examine the effect of garlic supplementation on CETP activity, along with its anti-atherosclerotic effect in cholesterol-fed rabbits. Rabbits were fed a 1% cholesterol diet for 12 weeks, including a 1% garlic powder supplement. The garlic-supplemented group exhibited significantly lower CETP activity than the control group during the experimental period (P < 0.05). Among the atherogenic parameters, the total cholesterol, TG, LDL-C, VLDL-C, and atherogenic index were all significantly lower in the garlic group than in the control group during the experimental period (P < 0.05), whereas the HDL-C concentration was significantly higher in the garlic group than in the control group after 12 weeks (P < 0.05). Atherosclerotic lesion area in the aorta arch was also significantly lower in the garlic group (P < 0.05). In the morphological examination, the garlic-supplemented group exhibited far fewer fat droplet deposits than the control group. Furthermore, the garlic supplement also lowered the aortic and hepatic cholesterol, and triglyceride. Accordingly, the current results suggest that garlic exerts hypocholesterolemic and/or antiatherogenic and that plasma CETP activity might be a risk marker related with atherogenesis. As such, the inhibition of CETP activity may delay the progression of atherosclerosis, thereby supporting the atherogenicity of CETP and the inhibitory activity of garlic supplementation against CETP.     

Cholesteryl ester exchange protein in human plasma isolation and characterization.

A protein catalyzing the exchange of cholesteryl esters among the lipoproteins was found in human plasma. A rapid method for assaying this activity was developed based on the transfer of radioactive cholesteryl esters from low density lipoprotein with MnCl2 in the presence of phosphate. Fractionation of plasma through a combination of ammonium sulfate precipitation, ultracentrifugation at p = 1.25, and chromatography on Phenyl-Sepharose, CM-cellulose, and concanavalin A-Sepharose, yielded a preparation purified 3500-fold compared to the starting plasma. The exchange protein was found to be a glycoprotein with an isoelectric point of 5 and apparent molecular weight of 80 000. On the basis of these properties and its immunological characteristics the exchange protein was judged to be distinct from any of the known apolipoproteins. This protein could also be separated from plasma phosphatidylcholine cholesterol acyl-transferase on DEAE-cellulose. The exchange protein did not appear to influence cholesterol esterification in lipoproteins by phosphatidylcholine cholesterol acyl-transferase, and the latter had no effect on the transfer of low density lipoprotein cholesteryl esters to high density lipoprotein. The exchange protein did not esterify cholesterol or hydrolyze cholesteryl esters in lipoproteins.     

Cholesteryl ester transfer protein (CETP) expression enhances HDL cholesteryl ester liver delivery, which is independent of scavenger receptor BI, LDL receptor related protein and possibly LDL receptor

Cholesteryl ester transfer protein (CETP) is a hydrophobic plasma glycoprotein that mediates the transfer and exchange of cholesteryl ester (CE) and triglyceride (TG) between plasma lipoproteins, and also plays an important role in HDL metabolism. Previous studies have indicated that, compared to wild type mice, human CETP transgenic mice had significantly lower plasma HDL CE levels, which was associated with enhancement of HDL CE uptake by the liver. However, the mechanism of this process is still unknown. To evaluate the possibility that this might be directly mediated by CETP, we utilized CETP transgenic (CETPTg) mice with liver scavenger receptor BI (SR-BI) deficiency [i.e., PDZK1 gene knockout (PDZK1O)], and with receptor associated protein (RAP) overexpression, to block LDL receptor-related protein (LRP) and LDL receptor (LDLR). We found that (1) CETPTg/PDZK1O mice have significantly lower HDL-C than that of PDZK1 KO mice (36%, p<0.01); (2) CETPTg and CETPTg/PDZK1O mice have same HDL-C levels; (3) CETPTg/PDZK1O/RAP mice had significant lower plasma HDL-C levels than that of PDZK1O/RAP ones (50%, p<0.001); (4) there is no incremental transfer of HDL CE radioactivity to the apoB-containing lipoprotein fraction in mice expressing CETP; and (5) CETPTg/PDZK1O/RAP mice had significant higher plasma and liver [(3)H]CEt-HDL turnover rates than that of PDZK1O/RAP ones (50% and 53%, p<0.01, respectively). These results suggest that CETP expression in mouse increases direct removal of HDL CE in the liver and this process is independent of SR-BI, LRP, and possibly LDLR.     

Cholesteryl ester transfer protein is secreted by Hep G2 cells and contains asparagine-linked carbohydrate and sialic acid

A cholesteryl ester transfer protein (CETP) of apparent Mr 74,000 has recently been purified from human plasma. Cholesteryl ester transfer activity was found to accumulate in the medium of cultured Hep G2 cells. The transfer activity was removed by immunoprecipitation with specific antibodies to the plasma CETP. Sodium dodecyl sulfate gel electrophoresis of immunoprecipitates prepared from the medium of cells pulsed with [35S]methionine revealed a broad specific band of protein of Mr 72,000 to 76,000; by contrast, immunoprecipitates of cellular homogenates showed a sharp specific band of Mr 58,000. The Mr 72,000 to 76,000 band disappears, concomitant with the appearance of lower Mr products, upon neuraminidase or glycopeptidase F treatment of medium immunoprecipitates or of purified CETP. The results indicate that liver cells have the capacity to synthesize and secrete CETP. The CETP peptide acquires asparagine-linked carbohydrate and sialic acid during intracellular processing.     

Cholesteryl ester transfer protein gene haplotypes, plasma high-density lipoprotein levels and the risk of coronary heart disease

High-density lipoprotein cholesterol (HDL-C) is a known inverse predictor of coronary heart disease (CHD) and is thus a potential therapeutic target. Cholesteryl ester transfer protein (CETP) is a key protein in HDL-C metabolism such that elevated CETP activity is associated with lower HDL-C. Currently available HDL-C raising drugs are relatively ineffective and evidence suggesting the role of CETP in HDL-C levels has promoted the development of CETP inhibitors as potential therapeutic agents for CHD. We investigated three SNPs in the CETP gene in two cross-sectional community-based populations (n = 1,574 and 1,109) and a population of 556 CHD patients to determine if reduced CETP activity due to genetic variations in the CETP gene would increase HDL-C levels and reduce the risk of CHD. CETP genotypes and haplotypes were tested for association with lipid levels, CETP activity and risk of CHD. Multivariate analysis showed the common AAB2 haplotype defined by the G-2708A, C-629A and TaqIB polymorphisms, was consistently associated with reduced CETP activity and increased HDL-C levels. A mean increase in HDL-C levels of 0.16–0.24 mmol/l was observed in individuals with two copies of the AAB2 haplotype relative to non AAB2 carriers across all three populations (P < 0.001). A case-control study of males indicated no association between single SNPs or haplotypes and the risk of CHD. These results suggest that raising HDL-C via CETP inhibition may not alter risk of CHD. Randomized control trials are needed to determine whether CETP inhibition will in reality reduce risk of CHD by raising HDL-C. Pamela A. McCaskie and John P. Beilby contributed equally to this work.     

Cholesteryl ester accumulation and accelerated cholesterol absorption in intestine-specific hormone sensitive lipase-null mice

Hormone sensitive lipase (HSL) regulates the hydrolysis of acylglycerols and cholesteryl esters (CE) in various cells and organs, including enterocytes of the small intestine. The physiological role of this enzyme in enterocytes, however, stayed elusive. In the present study we generated mice lacking HSL exclusively in the small intestine (HSLiKO) to investigate the impact of HSL deficiency on intestinal lipid metabolism and the consequences on whole body lipid homeostasis. Chow diet-fed HSLiKO mice showed unchanged plasma lipid concentrations. In addition, feeding with high fat/high cholesterol (HF/HC) diet led to unaltered triglyceride but increased plasma cholesterol concentrations and CE accumulation in the small intestine. The same effect was observed after an acute cholesterol load. Gavaging of radioactively labeled cholesterol resulted in increased abundance of radioactivity in plasma, liver and small intestine of HSLiKO mice 4h post-gavaging. However, cholesterol absorption determined by the fecal dual-isotope ratio method revealed no significant difference, suggesting that HSLiKO mice take up the same amount of cholesterol but in an accelerated manner. mRNA expression levels of genes involved in intestinal cholesterol transport and esterification were unchanged but we observed downregulation of HMG-CoA reductase and synthase and consequently less intestinal cholesterol biosynthesis. Taken together our study demonstrates that the lack of intestinal HSL leads to CE accumulation in the small intestine, accelerated cholesterol absorption and decreased cholesterol biosynthesis, indicating that HSL plays an important role in intestinal cholesterol homeostasis.     

Differential expression of cholesteryl ester transfer protein in the liver and plasma of fasted and fed transgenic mice

Because cholesteryl ester transfer protein (CETP) is considered a potential target in the treatment of atherosclerosis, several reports have focused on the regulation of this enzyme, and there is evidence that insulin may be a regulatory factor. The present study examines the differential expression of the human CETP gene between physiologic conditions that are accompanied by low (fasted) and high (fed) insulin levels. CETP expression was examined in plasma and tissues of transgenic mice expressing the human CETP minigene after 12 hours of fasting (n = 20) or ad libitum feeding (n = 20) with normal mouse chow. Plasma cholesteryl ester transfer activity (CETA) was 20% higher in fed than in fasted mice, reflecting higher levels of CETP (P < 0.05). This observation was accompanied by higher liver mRNA in fed mice (100%, P < 0.05), as determined by ribonuclease protection assays, as well as by higher CETA (23%, P < 0.05) and CETP mass (29%, P < 0.05) in the particulate fraction of liver homogenates. These parameters of liver CETP expression correlated well with each other, as well as with plasma CETA. CETP in the liver particulate fraction was found as a doublet (approximately 70 and 65 kDa), which resolved to a single band (approximately 60 kDa) upon deglycosylation. No differences in CETP expression were observed in pooled adipose tissue samples from fed and fasted mice. Insulin and glucose were not related to any plasma or tissue parameter of CETP expression. In summary, the concerted, differential expression of CETP in the liver of fed and fasted transgenic mice appears to contribute to higher plasma CETP levels in fed mice, but the precise role of insulin and glucose in regulating CETP expression under fasted and fed conditions needs to be defined.     

Cholesteryl ester and apolipoprotein E transfer between human high density lipoproteins and chylomicrons

The transfer of cholesteryl esters and apolipoprotein E has been studied between plasma HDL and chylomicrons isolated either from ascitic fluid or from the plasma of a patient with type V hyperlipoproteinemia. Whereas apolipoprotein E transfer was rapid and occurred at low temperature, cholesteryl ester transfer was suppressed at 4 degrees C. Apolipoprotein E transfer did not depend upon the presence of cholesteryl ester transfer protein and was in fact inhibited by the partially purified preparation of this protein. Apolipoprotein E transfer was not increased by reduction with dithiothreitol. The transfer of cholesteryl esters increased sharply at a chylomicron to HDL ratio of cholesteryl ester above 1/10, a value which may be of physiological significance at the peak of postprandial lipemia. At this ratio, the transfer of apolipoprotein E was minimal and increased only at ratios above 2/1. From these results, it is concluded that there is no connection between apolipoprotein E and cholesteryl ester transfer from HDL to chylomicrons. It is, therefore, proposed that whereas chylomicron apolipoprotein E is acquired rapidly and mostly in the lymphatic system, the concentration of chylomicron cholesteryl esters increases significantly and independently in the circulation.