Cholesteryl ester transfer protein (CETP) plays an important role in reverse cholesterol transport, with decreased CETP activity increasing HDL levels. Formation of an alternative splice form lacking exon 9 (Δ9-CETP) has been associated with two single nucleotide polymorphisms (SNPs) in high linkage disequilibrium with each other, namely rs9930761 T>C located in intron 8 in a putative splicing branch site and rs5883 C>T in a possible exonic splicing enhancer (ESE) site in exon 9. To assess the relative effect of rs9930761 and rs5883 on splicing, mini-gene constructs spanning CETP exons 8 to 10, carrying all four possible allele combinations, were transfected into HEK293 and HepG2 cells. The minor T allele of rs5883 enhanced splicing significantly in both cell lines whereas the minor C allele of rs9930761 did not. In combination, the two alleles did not yield greater splicing than the rs5883 T allele alone in HepG2 cells. These results indicate that the genetic effect on CETP splicing is largely attributable to rs5883. We also confirm that Δ9-CETP protein is expressed in the liver but fails to circulate in the blood.
Cholesteryl ester transfer protein; coronary artery disease; statin; alternative splicing; CETP levels in liver and plasma
The role of cholesteryl ester transfer protein (CETP) in the metabolism of HDL cholesterol (HDL-C) is well studied but still controversial. More recently, GWAS and metaanalyses reported the association of a promoter variant (rs3764261) with HDL-C in Caucasians and other ethnic groups. In this study, we have examined the role of genetic variation in the promoter region of CETP with HDL-C, CETP activity, coronary artery disease (CAD), CAD risk factors, and the interaction of genetic factors with environment in a unique diabetic cohort of Asian Indian Sikhs.
Methods and Results
We genotyped four variants; three tagSNPs from promoter (rs3764261, rs12447924, rs4783961) and one intronic variant (rs708272 Taq1B) on 2,431 individuals from the Sikh Diabetes Study. Two variants (rs3764261 and rs708272) exhibited a strong associations with HDL-C in both normo-glycemic (NG) controls (β= 0.12; p= 9.35 ×10−7 for rs3764261; β= 0.10, p= 0.002 for rs708272) and diabetic cases (β= 0.07, p= 0.016 for rs3764261; β= 0.08, p= 0.005 for rs708272) with increased levels among minor homozygous ‘AA’ carriers. In addition, the same ‘A’ allele carriers in rs376426 showed a significant decrease in systolic blood pressure (β= −0.08, p= 0.002) in NG controls. Haplotype analysis of rs3764261, rs12447924, rs4783961, and rs708272 further revealed a significant association of ‘ATAA’ haplotype with increased HDL-C (β= 2.71, p= 6.38 ×10−5) and ‘CTAG’ haplotype with decreased HDL–C levels (β= −1.78, p= 2.5×10−2). Although there was no direct association of CETP activity and CETP polymorphisms, low CETP activity was associated with increased risk to CAD (age, BMI and gender adjusted odds ratio 2.2 95% CI (1.4–3.4, p= 0.001) in this study. Our data revealed a strong interaction of rs3764261 and rs708272 for affecting the association between CETP activity and HDL–C levels; p= 2.2 × 10−6, and p= 4.4 × 10−4, respectively.
Our results, in conjunction with earlier reports confirm low CETP activity to be associated with higher CAD risk. Although there was no direct association of CETP activity with CETP polymorphisms, our findings revealed a significant interaction between CETP SNPs and CETP activity for affecting HDL-C levels. These results urge a deeper evaluation of the individual genetic variation in the CETP before implementing pharmaceutical intervention of blocking CETP for preventing CAD events.
The plasma cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters from HDL to other lipoproteins and is a key regulated component of reverse cholesterol transport. Dietary hypercholesterolemia results in increased hepatic CETP gene transcription and higher plasma CETP levels. To investigate the mechanisms by which the liver senses hypercholesterolemia, mice containing a natural flanking region CETP transgene (NFR-CETP transgene) were bred with apo E or LDL receptor gene knockout mice (E0 or LDLr0 mice). Compared to NFR-CETP transgenic (Tg) mice with intact apo E genes, in NFR-CETP Tg/E0 mice there was an eightfold induction of plasma CETP levels and a parallel increase in hepatic CETP mRNA levels. Other sterol-responsive genes (LDL receptor and hydroxymethyl glutaryl CoA reductase) also showed evidence of altered regulation with decreased abundance of their mRNAs in the E0 background. A similar induction of plasma CETP and hepatic CETP mRNA levels resulted from breeding the NFR-CETP transgene into the LDL receptor gene knockout background. When placed on a high cholesterol diet, there was a further increase in CETP levels in both E0 and LDLr0 backgrounds. In CETP Tg, CETP Tg/E0, and CETP Tg/LDLr0 mice on different diets, plasma CETP and CETP mRNA levels were highly correlated with plasma cholesterol levels. The results indicate that hepatic CETP gene expression is driven by a mechanism which senses changes in plasma cholesterol levels independent of apo E and LDL receptors. Hepatic sterol-sensitive genes have mechanisms to sense hypercholesterolemia that do not require classical receptor-mediated lipoprotein uptake.
The cholesteryl ester transport protein (CETP) plays a key role in high-density lipoprotein (HDL) metabolism. Genetic variants that alter CETP activity and concentration may cause significant alterations in HDL-cholesterol (HDL-C) concentration; however, controversies remain about whether these genetic variants are associated with atherosclerosis. We genotyped the CETP R451Q, A373P, -629C/A, Taq1B, and -2505C/A polymorphisms in a cohort of Caucasian, Chinese, African-American, and Hispanic individuals within the Multi-Ethnic Study of Atherosclerosis. Genotypes were examined in relationship to HDL-C, CETP activity, CETP concentration, and three measures of subclinical cardiovascular disease (CVD): coronary artery calcium (CAC) measured by fast CT scanning, and carotid intimal-medial thickness (IMT) and carotid artery plaque, measured by ultrasonography. Carriers of the 451Q and 373P alleles have significantly higher CETP concentration (22.4% and 19.5%, respectively; p<0.001) and activity (13.1% and 9.4%, respectively; p<0.01) and lower HDL-C (5.6% and 6.0%, respectively; p<0.05). The minor alleles of the R451Q and A373P polymorphisms are associated with the presence of CAC, even after adjusting for CVD risk factors and HDL-C (p=0.006 and p=0.01, respectively). The R451Q polymorphism is also associated with presence of carotid artery plaque (p=0.036). Neither polymorphism is associated with common or internal carotid IMT. We confirmed that the -629A, Taq1B B2, and -2505A alleles are significantly associated with lower CETP concentration (20.8%, 25.0%, and 23.7%, respectively; p<0.001) and activity (14.8%, 19.8%, and 18.4%, respectively; p<0.001) and higher HDL-C concentration (9.7%, 11.5%, and 10.4%, respectively; p<0.01). However, we did not find any associations between these non-coding polymorphisms and subclinical CVD.
CETP; CVD; HDL; MESA
Low HDL-C levels are associated with atherosclerosis and non-alcoholic steatohepatitis, and increased levels may reduce the risk of these diseases. Inhibition of cholesteryl ester transfer protein (CETP) activity is considered a promising strategy for increasing HDL-C levels. Since CETP is a self-antigen with low immunogenicity, we developed a novel CETP vaccine (Fc-CETP6) to overcome the low immunogenicity of CETP and for long-term inhibition of CETP activity. The vaccine consists of a rabbit IgG Fc domain for antigen delivery to antigen-presenting cells fused to a linear array of 6 repeats of a CETP epitope to efficiently activate B cells. Rabbits were fed a high fat/cholesterol (HFC) diet to induce atherosclerosis and NASH, and immunized with Fc-CETP6 vaccine. The Fc-CETP6 vaccine successfully elicited anti-CETP antibodies and lowered plasma CETP activity. The levels of plasma HDL-C and ApoA-I were higher, and plasma ox-LDL lower, in the Fc-CETP6-immunized rabbits as compared to the unimmunized HFC diet-fed rabbits. Pathological analyses revealed less lipid accumulation and inflammation in the aorta and liver of the Fc-CETP6-immunized rabbits. These results show that the Fc-CETP6 vaccine efficiently elicited antibodies against CETP and reduced susceptibility to both atherosclerosis and steatohepatitis induced by the HFC diet. Our findings suggest that the Fc-CETP6 vaccine may improve atherosclerosis and NASH and has high potential for clinical use.
Genetic determinants of HDL cholesterol (HDL-C) levels in the general population are poorly understood. We previously described plasma cholesteryl ester transfer protein (CETP) deficiency due to an intron 14 G(+1)-to-A mutation(Int14 A) in several families with very high HDL-C levels in Japan. Subjects with HDL-C > or = 100 mg/dl (n = 130) were screened by PCR single strand conformational polymorphism analysis of the CETP gene. Two other mutations were identified by DNA sequencing or primer-mediated restriction map modification of PCR products: a novel intron 14 splice donor site mutation caused by a T insertion at position +3 from the exon14/intron14 boundary (Int14 T) and a missense mutation (Asp442 to Gly) within exon 15 (D442G). The Int14 T mutation was only found in one family. However, the D442G and Int14 A mutations were highly prevalent in subjects with HDL-C > or = 60 mg/dl, with combined allele frequencies of 9%, 12%, 21% and 43% for HDL-C 60-79, 80-99, 100-119, and > or = 120 mg/dl, respectively. Furthermore, prevalences of the D442G and Int14 A mutations were extremely high in a general sample of Japanese men (n = 236), with heterozygote frequencies of 7% and 2%, respectively. These two mutations accounted for about 10% of the total variance of HDL-C in this population. The phenotype in a genetic compound heterozygote (Int14 T and Int14 A) was similar to that of Int14 A homozygotes (no detectable CETP and markedly increased HDL-C), indicating that the Int14 T produces a null allele. In four D442G homozygotes, mean HDL-C levels (86 +/- 26 mg/dl) were lower than in Int14 A homozygotes (158 +/- 35 mg/dl), reflecting residual CETP activity in plasma. In 47 D442G heterozygotes, mean HDL-C levels were 91 +/- 23 mg/dl, similar to the level in D442G homozygotes, and significantly greater than mean HDL-C levels in Int14 A heterozygotes (69 +/- 15 mg/dl). Thus, the D442G mutation acts differently to the null mutations with weaker effects on HDL in the homozygous state and stronger effects in the heterozygotes, suggesting dominant expression of a partially defective allele. CETP deficiency, reflecting two prevalent mutations (D442G and Int14 A), is the first example of a genetic deficiency state which is sufficiently common to explain a significant fraction of the variation in HDL-C in the general population.
The plasma cholesteryl ester transfer protein (CETP) mediates the exchange of HDL cholesteryl esters (CE) and VLDL triglycerides leading to catabolism of HDL. There is some evidence that HDL ameliorates the toxicity of LPS, and LPS is known to influence several enzymes affecting HDL metabolism. Therefore, the effects of LPS on CETP and plasma lipoproteins were examined in human CETP transgenic mice. Administration of LPS to mice expressing a CETP transgene linked to its natural flanking sequences (NFR-CETP Tg) resulted in a rapid marked decrease in hepatic CETP mRNA and plasma CETP concentration. Corticosteroid injection produced a similar decrease in hepatic CETP mRNA and adrenalectomy abolished this response to LPS. LPS caused disproportionate reductions in plasma CETP activity compared to mass, and was found to be a potent inhibitor of CETP activity when added directly to plasma. LPS was injected into mice expressing (A) a human apoA-I transgene, (B) apoA-I and NFR-CETP transgenes, or (C) apoA-I and LPS-inducible metallothionein promoter-driven CETP transgenes, producing (A) minimal changes in HDL cholesterol, (B) decreased plasma CETP and increased HDL cholesterol, and (C) increased plasma CETP and decreased HDL cholesterol. Thus, LPS administration produces a profound decrease in hepatic CETP mRNA, primarily as a result of adrenal corticosteroid release. The decrease in plasma CETP activity after LPS administration may reflect both this effect as well as a direct interaction between CETP and LPS. The decrease of CETP in response to LPS has major effects on HDL levels, and may represent an adaptive response to preserve or increase HDL and thereby modify the response to LPS.
CETP is a plasma protein that modulates atherosclerosis risk through its HDL-cholesterol reducing action. The aim of this work was to examine the effect of the PPARα agonist, ciprofibrate, on the CETP gene expression, in the presence and absence of apolipoprotein (apo) CIII induced hypertriglyceridemia, and its impact on the HDL metabolism.
Mice expressing apo CIII and/or CETP and non-transgenic littermates (CIII, CIII/CETP, CETP, non-Tg) were treated with ciprofibrate during 3 weeks. Drug treatment reduced plasma triglycerides (30-43%) and non-esterified fatty acids (19-47%) levels. Cholesterol (chol) distribution in plasma lipoprotein responses to ciprofibrate treatment was dependent on the genotypes. Treated CIII expressing mice presented elevation in VLDL-chol and reduction in HDL-chol. Treated CETP expressing mice responded with reduction in LDL-chol whereas in non-Tg mice the LDL-chol increased. In addition, ciprofibrate increased plasma post heparin lipoprotein lipase activity (1.3-2.1 fold) in all groups but hepatic lipase activity decreased in treated CETP and non-Tg mice. Plasma CETP activity and liver CETP mRNA levels were significantly increased in treated CIII/CETP and CETP mice (30-100%). Kinetic studies with 3H-cholesteryl ether (CEt) labelled HDL showed a 50% reduction in the 3H-CEt found in the LDL fraction in ciprofibrate treated compared to non-treated CETP mice. This means that 3H-CEt transferred from HDL to LDL was more efficiently removed from the plasma in the fibrate treated mice. Accordingly, the amount of 3H-CEt recovered in the liver 6 hours after HDL injection was increased by 35%.
Together these data showed that the PPARα agonist ciprofibrate stimulates CETP gene expression and changes the cholesterol flow through the reverse cholesterol transport, increasing plasma cholesterol removal through LDL.
In almost 30 years since the introduction of HMG-CoA reductase inhibitors (statins), no other class of lipid modulators has entered the market. Elevation of high-density lipoprotein-cholesterol (HDL-C) via inhibiting cholesteryl ester transfer protein (CETP) is an attractive strategy for reducing the risk of cardiovascular events in high-risk patients. Triglyceride and cholesteryl ester (CE) transfer between lipoproteins is mediated by CETP; thus inhibition of this pathway increases the concentration of HDL-C. Torcetrapib was the first CETP inhibitor evaluated in Phase 3 clinical trials. Because of off-target effects, torcetrapib raised blood pressure and increased the concentration of serum aldosterone leading to higher cardiovascular events and mortality. Torcetrapib showed positive effects on the cardiovascular risk especially in patients with a greater increase in HDL-C and Apolipoprotein A-1 (apoA-1) levels.
The Phase 3 clinical trial of dalcetrapib, the second CETP inhibitor that has entered clinical development, was terminated because of ineffectiveness. Dalcetrapib is a CETP modulator that elevated HDL-C level but did not reduce the concentration of low-density lipoprotein cholesterol (LDL-C). Both heterotypic and homotypic CE transfer between lipoproteins are mediated by some CETP inhibitors including torcetrapib, anacetrapib and evacetrapib while dalcetrapib only affect the heterotypic CE transfer. Dalcetrapib has a chemical structure that is distinct from other CETP inhibitors with smaller molecular weight and lack of trifluoride moieties. Dalcetrapib is a pro-drug that must be hydrolyzed to a pharmacologically active thiol form.
Two other CETP inhibitors, anacetrapib and evacetrapib, are currently undergoing evaluation in Phase 3 clinical trial. Both molecules have shown beneficial effects by increasing HDL-C and decreasing LDL-C concentration. The success of anacetrapib and evacetrapib will remain to be confirmed upon the completion of Phase 3 clinical trials in 2017 and 2015, respectively.
Generally, the concentration of HDL-C has been considered as biomarker for the activity of CETP inhibitors. However, it is not clear whether a fundamental relationship exist between HDL-C and the risk of coronary artery diseases (CAD). The most crucial role for HDL-C is cholesterol efflux capacity in which HDL can reverse transport cholesterol from foam cells in atherosclerotic plaques. In view of the heterogeneity in HDL-C particle size, charge, and composition, the mere concentration of HDL-C may not be a good surrogate marker for HDL functionality. Recent clinical studies reported that increased HDL-C functionality inversely correlate with the development of atherosclerotic plaque. Future development of CETP inhibitors may therefore benefit from the use of biomarkers that better predict HDL functionality.
Inhibition of cholesteryl ester transfer protein (CETP) lowers plasma low-density lipoprotein cholesterol concentration and raises high-density lipoprotein (HDL) cholesterol, suggesting it might prevent cardiovascular disease (CVD). From the outset, however, the concept has been controversial owing to uncertainty about its effects on HDL function and reverse cholesterol transport (RCT). Although there has long been good evidence that CETP inhibition reduces atherosclerosis in rabbits, the first information on CETP as a CVD risk factor in a prospectively followed cohort was not published until after the first Phase 3 trial of a CETP inhibitor had begun. The worrying finding that CVD incidence was related inversely to plasma CETP has since been reproduced in each of five further prospective cohort studies. Similar results were obtained in subjects on or off statin therapy, for first and second CVD events, and for mortality as well as CVD morbidity. Additionally, two recent studies have found alleles of the
CETP gene that lower hepatic CETP secretion to be associated with an increased risk of myocardial infarction. Meanwhile,
CETP gene transfer in mice was found to increase RCT from peripheral macrophages
in vivo, and human plasma with high CETP activity was shown to have a greater capacity to remove cholesterol from cultured cells than plasma with low activity. This mounting evidence for a protective function of CETP has been given remarkably little attention, and indeed was not mentioned in several recent reviews. It appears to show that CETP inhibition does not test the HDL hypothesis as originally hoped, and raises a pressing ethical issue regarding two Phase 3 trials of inhibitors, involving more than forty thousand subjects, which are currently in progress. As the weight of evidence now clearly supports an adverse effect of CETP inhibition on CVD, an urgent review is needed to determine if these trials should be discontinued.
Although the relationship between cholesteryl ester transfer protein (CETP) and cholesterol metabolism has been characterized in recent years, the effect of CETP genetic variants associated with coronary artery disease (CAD) is still unclear. Therefore, we investigated the association between CETP gene polymorphism and levels of lipid in patients with CAD.
Materials and Methods
We conducted a case-control study that included 194 unrelated subjects who underwent coronary angiography for suspected ischemic heart disease. This group was divided into 96 patients with angiographically documented CAD and 98 subjects (individuals matched for age and gender) without angiographically documented CAD (CAD-free subjects), all of whom were studied to examine the genotypic distribution of the CETP gene polymorphism in CAD. Genotyping was performed via polymerase chain reaction.
Of the 96 patients with CAD, 38 (40%) were B1B1, 42 (44%) B1B2 and 16 (16%) B2B2, compared with the control subjects, of which 35 (36%) were B1B1, 44 (45%) B1B2 and 19 (19%) B2B2. There were no significant differences between patients with CAD and control subjects in the distribution of the CETP gene polymorphism. Patients with the B1B1 genotype had lower high-density lipoprotein-cholesterol (HDL-C) and higher triglyceride (TG) levels than patients with the B2B2 genotype (p<0.05). In addition, among control subjects HDL-C levels were significantly higher in subjects with the B2B2 genotype than in subjects with the B1B1 genotype (p<0.01).
Our results suggest that genetic variations of the CTEP gene may be responsible for low HDL-C levels but may not be considered as a risk factor for CAD in the Turkish population.
Coronary artery disease; Coronary angiography; Cholesteryl ester transfer protein; Genetic
Coronary atherosclerosis, the most common form of coronary artery disease (CAD), is characterized by accumulation of lipid in the walls of coronary arteries. Recent data from clinical trials have showed that high-density lipoprotein cholesterol (HDL-C) has causal role in the pathogenesis and development of coronary atherosclerosis. Cholesteryl ester transfer protein (CETP) is an important regulator of plasma HDL-C. Several genetic mutations in the CETP gene were found to be associated with HDL-C levels. The aim of the present study is to evaluate the association of HDL-C-related CETP polymorphisms and risk of coronary atherosclerosis.
We investigated the association of seven single nucleotide polymorphisms (SNP) (rs1800775, rs708272, rs5882, rs1532624, rs1864163, rs7499892, and rs9989419) in the CETP gene with the risk of coronary atherosclerosis and levels of HDL-C in a case–control study in China. Included in the study were 420 patients with coronary atherosclerosis and 424 healthy controls. SNP genotyping was performed by TaqMan allelic discrimination assay and serum lipid levels were measured by standard laboratory methods.
Carriers of the AA and GA + AA genotypes of rs708272 had significant lower risks of coronary atherosclerosis (OR = 0.55, 95% CI: 0.36-0.85, p = 0.003; OR = 0.67, 95% CI: 0.50-0.90, p = 0.007, respectively) compared to those with GG genotype. These relations remained significant after adjustment for confounding effects of age, smoking, diabetes and hypertension. The rs1800775 polymorphism was significantly associated with serum levels of HDL-C in healthy controls (p = 0.04). Besides, rs708272 was in close linkage disequilibrium (LD) with rs1800775 in this study.
Our findings indicated that CETP rs708272 may be associated with the risk of coronary atherosclerosis and rs1800775 may influence serum HDL-C levels in healthy controls in Chinese.
Coronary atherosclerosis; CETP; Genetic mutation; HDL-C
This study was undertaken to determine potential tissue sources of plasma cholesteryl ester transfer protein (CETP), and to assess the influence of CETP on lipoprotein concentrations and atherosclerosis. In a group of 28 cynomolgus monkeys fed high fat, high cholesterol diets, plasma CETP concentration was strongly correlated with the abundance of CETP mRNA in liver and in adipose tissue, and with the output of CETP in liver perfusates. Plasma CETP concentration showed a strong inverse correlation with HDL cholesterol concentrations (r = -0.62, P less than 0.001) and a positive correlation with LDL cholesterol concentration (r = 0.54, P less than 0.005) and molecular weight (r = 0.57, P less than 0.001). The extent of coronary artery atherosclerosis was positively correlated with LDL cholesterol concentration and molecular weight, and with plasma CETP concentration. Thus, in monkeys fed an atherogenic diet, individual variation in CETP mRNA abundance in liver and adipose tissue probably plays a major role in the determination of plasma CETP levels. In plasma, CETP influences the distribution of cholesteryl esters between LDL and HDL, and CETP concentration appears to be a key determinant of the relative atherogenicity of the plasma lipoproteins.
Cholesteryl ester transfer protein (CETP) mediates the reciprocal transfer of neutral lipids (cholesteryl esters, triglycerides) and phospholipids between different lipoprotein fractions in human blood plasma. A novel molecular agent known as anacetrapib has been shown to inhibit CETP activity and thereby raise high density lipoprotein (HDL)-cholesterol and decrease low density lipoprotein (LDL)-cholesterol, thus rendering CETP inhibition an attractive target to prevent and treat the development of various cardiovascular diseases. Our objective in this work is to use atomistic molecular dynamics simulations to shed light on the inhibitory mechanism of anacetrapib and unlock the interactions between the drug and CETP. The results show an evident affinity of anacetrapib towards the concave surface of CETP, and especially towards the region of the N-terminal tunnel opening. The primary binding site of anacetrapib turns out to reside in the tunnel inside CETP, near the residues surrounding the N-terminal opening. Free energy calculations show that when anacetrapib resides in this area, it hinders the ability of cholesteryl ester to diffuse out from CETP. The simulations further bring out the ability of anacetrapib to regulate the structure-function relationships of phospholipids and helix X, the latter representing the structural region of CETP important to the process of neutral lipid exchange with lipoproteins. Altogether, the simulations propose CETP inhibition to be realized when anacetrapib is transferred into the lipid binding pocket. The novel insight gained in this study has potential use in the development of new molecular agents capable of preventing the progression of cardiovascular diseases.
Cardiovascular disease is a leading cause of morbidity and mortality in Western societies. One of the most encouraging treatment methods to prevent the generation and progression of cardiovascular disease is the elevation of high density lipoprotein (HDL) levels in circulation, as high HDL levels have been found to correlate negatively with the risk of cardiovascular disease. HDL elevation is attainable through inhibition of cholesteryl ester transfer protein (CETP). A novel molecular agent, anacetrapib, fulfills the requirements with an acceptable side-effect profile. In this study, our objective is to gain more detailed information regarding the interactions between CETP and anacetrapib in order to unlock the inhibitory mechanism of the drug that has, to date, remained unclear. Our results point out the primary binding site of anacetrapib and highlight the ability of the drug to regulate the structure-function relationship of those structural regions of CETP that are considered important in CETP inhibition. Our findings could be exploited in the development of new and more efficient molecular agents against cardiovascular disease.
Cardiovascular risk factors and atherosclerosis precursors were examined in 365 Turkish children and adolescents. Study participants were recruited at five different state schools. We tested single and multi-locus effects of six polymorphisms from five candidate genes, chosen based on prior known association with lipid levels in adults, for association with low (≤10th percentile) high density lipoprotein cholesterol (HDL-C) and high (≥90th percentile) triglycerides (TG), and the related continuous outcomes. We observed an association between CETP variant rs708272 and low HDL-C (allelic p=0.020, genotypic p=0.046), which was supported by an independent analysis, PRAT (PRAT control p=0.027). Sex-stratified logistic regression analysis showed that the B2 allele of rs708272 decreased odds of being in the lower tenth percentile of HDL-C measurements (OR=0.36, p=0.02) in girls; this direction of effect was also seen in boys but was not significant (OR=0.64, p=0.21). Logistic regression analysis also revealed that the T allele of rs6257 (SHBG) decreased odds of being in the top tenth percentile of TG measurements in boys (OR=0.43, p=0.03). Analysis of lipid levels as a continuous trait revealed a significant association between rs708272 (CETP) and LDL-C levels in males (p=0.02) with the B2B2 genotype group having the lowest mean LDL-C; the same direction of effect was also seen in females (p=0.05). An effect was also seen between rs708272 and HDL-C levels in girls (p=0.01), with the B2B2 genotype having the highest mean HDL-C levels. Multi-locus analysis, using quantitative multifactor dimensionality reduction (qMDR) identified the previously mentioned CETP variant as the best single locus model, and overall model, for predicting HDL-C levels in children. This study provides evidence for association between CETP and low HDL-C phenotype in children, but the results appear to be weaker in children than previous results in adults and may also be subject to gender effects.
The role of cholesteryl ester transfer protein (CETP) in the development of atherosclerosis is still open to debate. In the ILLUMINATE trial inhibition of CETP in patients with high cardiovascular risk was associated with increased high density lipoprotein levels but increased risk of cardiovascular morbidity and mortality. Here, we present a prospective observational study of patients referred to coronary angiography in which CETP was examined in relation to morbidity and mortality.
Methods and Results
CETP concentration was determined in 3256 participants of the Ludwigshafen Risk and Cardiovascular Health (LURIC) study who were referred to coronary angiography at baseline between 1997 and 2000. Median follow-up time was 7.75 years. Primary and secondary endpoints were cardiovascular and all-cause mortality, respectively. CETP levels were higher in women and lower in smokers, in diabetic patients, and in patients with unstable coronary artery disease (CAD), respectively. In addition, CETP levels were correlated negatively with high-sensitivity C-reactive protein and IL-6. After adjustment for age, sex, medication, CAD status, cardiovascular risk factors, and diabetes mellitus, the hazard ratio for death in the lowest CETP quartile was 1.33 (1.07-1.65, p=0.011) compared to patients in the highest CETP quartile. Corresponding hazard ratios for death in the second and third CETP quartile were 1.17 (0.92-1.48, p=0.19) and 1.10 (0.86-1.39, p=0.46), respectively.
We interpret our data to suggest that low endogenous CETP plasma levels per se are associated with increased cardiovascular and all-cause mortality challenging the rationale of pharmacological CETP inhibition.
atherosclerosis; lipoproteins; mortality; coronary disease; risk factors
Cholesteryl ester transfer protein (CETP) shuttles lipids between lipoproteins, culminating in cholesteryl ester delivery to liver and increased secretion of cholesterol as bile. Since gut bile acids promote insulin sensitivity, we aimed to define if CETP improves insulin sensitivity with high-fat feeding. CETP and nontransgenic mice of both sexes became obese. Female but not male CETP mice had increased ileal bile acid levels versus nontransgenic littermates. CETP expression protected female mice from insulin resistance but had a minimal effect in males. In liver, female CETP mice showed activation of bile acid-sensitive pathways including Erk1/2 phosphorylation and Fxr and Shp gene expression. In muscle, CETP females showed increased glycolysis, increased mRNA for Dio2, and increased Akt phosphorylation, known effects of bile acid signaling. These results suggest that CETP can ameliorate insulin resistance associated with obesity in female mice, an effect that correlates with increased gut bile acids and known bile-signaling pathways.
Insulin resistance; Obesity; Cholesterol; Bile; Glucose; Sex-differences
Plasma high density lipoprotein (HDL) levels are strongly genetically determined and show a general inverse relationship with coronary heart disease (CHD). The cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters from HDL to other lipoproteins and is a key participant in the reverse transport of cholesterol from the periphery to the liver. A high prevalence of two different CETP gene mutations (D442G, 5.1%; intron 14G:A, 0.5%), was found in 3,469 men of Japanese ancestry in the Honolulu Heart Program and mutations were associated with decreased CETP (-35%) and increased HDL chol levels (+10% for D442G). However, the overall prevalence of definite CHD was 21% in men with mutations and 16% in men without mutations. The relative risk (RR) of CHD was 1.43 in men with mutations (P < .05); after adjustment for CHD risk factors, the RR was 1.55 (P = .02); after additional adjustment for HDL levels, the RR was 1.68 (P = .008). Similar RR values were obtained for the D442G mutation alone. Increased CHD in men with mutations was primarily observed for HDL chol 41-60 mg/dl; for HDL chol > 60 mg/dl men with and without mutations had low CHD prevalence. Thus, genetic CETP deficiency appears to be an independent risk factor for CHD, primarily due to increased CHD prevalence in men with the D442G mutation and HDL cholesterol between 41 and 60 mg/dl. The findings suggest that both HDL concentration and the dynamics of cholesterol transport through HDL (i.e., reverse cholesterol transport) determine the anti-atherogenicity of the HDL fraction.
Cholesteryl ester transfer protein (CETP) transports cholesteryl esters, triglycerides, and phospholipids between different lipoprotein fractions in blood plasma. The inhibition of CETP has been shown to be a sound strategy to prevent and treat the development of coronary heart disease. We employed molecular dynamics simulations to unravel the mechanisms associated with the CETP-mediated lipid exchange. To this end we used both atomistic and coarse-grained models whose results were consistent with each other. We found CETP to bind to the surface of high density lipoprotein (HDL) -like lipid droplets through its charged and tryptophan residues. Upon binding, CETP rapidly (in about 10 ns) induced the formation of a small hydrophobic patch to the phospholipid surface of the droplet, opening a route from the core of the lipid droplet to the binding pocket of CETP. This was followed by a conformational change of helix X of CETP to an open state, in which we found the accessibility of cholesteryl esters to the C-terminal tunnel opening of CETP to increase. Furthermore, in the absence of helix X, cholesteryl esters rapidly diffused into CETP through the C-terminal opening. The results provide compelling evidence that helix X acts as a lid which conducts lipid exchange by alternating the open and closed states. The findings have potential for the design of novel molecular agents to inhibit the activity of CETP.
Coronary heart disease is a major cause of death in the Western societies. One of the most promising interventions to prevent and slow down the progress of coronary heart disease is the elevation of high density lipoprotein (HDL) levels in circulation. Animal models together with early clinical studies have shown that the inhibition of cholesteryl ester transfer protein (CETP) is a promising strategy to achieve higher HDL levels. However, drugs with acceptable side-effects for CETP-inhibition do not yet exist, although the next generation CETP inhibitor (anacetrapib) has great potential in this regard. In this study, our objective is to gain more detailed information regarding the interactions of CETP with lipoprotein particles. We show how the CETP-lipoprotein complex is formed and how lipid exchange between CETP and lipoprotein particles takes place. Our findings help to understand in a mechanistic way how CETP-mediated lipid exchange occurs and how it could be exploited in the design of new and more efficient molecular agents against coronary heart disease.
To investigate roles of inflammation and a cholesteryl ester transfer protein (CETP) polymorphism potentially related to recent findings demonstrating coronary risk with increasing HDL cholesterol (HDL-C).
Methods and Results
A novel graphical exploratory data analysis tool allowed examination of coronary risk in postinfarction patients relating to HDL-C and C-reactive protein (CRP). Results demonstrated a high-risk subgroup defined by high HDL-C and CRP exhibiting larger HDL particles and lower lipoprotein-associated phospholipaseA2 (Lp-PLA2) levels than lower-risk patients. Subgroup CETP-associated risk was probed using a functional CETP polymorphism (TaqIB, rs708272). Multivariable modeling revealed in the high-risk subgroup greater risk for B2 allele-carriers (less CETP activity) versus B1 homozygotes (hazard ratio 2.41, 95% CI 1.04-5.60, p=0.041). Within the high-risk subgroup, B2 allele-carriers had higher serum amyloid A levels than B1 homozygotes. Evidence is also presented demonstrating CETP genotypic differences in HDL subfraction distributions regarding nonHDL-C and Lp-PLA2 potentially relating to impaired HDL remodeling.
Postinfarction patients with high HDL-C and CRP levels demonstrate increased risk for recurrent events. Future studies should aim at characterizing altered HDL particles from such patients and elucidating mechanistic details related to inflammation and HDL particle remodeling. Such patients should be considered in drug trials involving raising HDL-C.
Atherosclerosis; cardiovascular diseases; inflammation; cholesteryl ester transfer protein; TaqIB
Genes coding for proteins involved in lipid metabolism and, in women, menopausal status are independently associated with high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c) levels. We examined whether the association between common functional genetic polymorphisms of apolipoprotein E (apoE Cys112Arg and Arg158Cys) gene and LDL-c levels, as well as the associations between the cholesteryl ester transfer protein (CETP TaqIB), hepatic lipase (LIPC C-514T), and lipoprotein lipase (LPL Ser447Stop) genes and HDL-c levels are significantly modified by menopausal status. Plasma lipid concentrations, genotype, and menopausal status were assessed across four examinations in a sample of Caucasian and African-American women (n=4652 to 4876) who were aged 45-64 years at baseline from the Atherosclerosis Risk in Communities Study. The association between LDL-c levels and the apoE gene, and HDL-c levels and the LIPC and LPL genes were not modified by menopausal status. The only statistically significant gene by menopause interaction was with the CETP gene on HDL-c concentrations (p=0.04). However, the significant CETP gene by menopause interaction was possibly due to chance because of multiple testing. Postmenopausal women who were carriers of the A allele of the CETP gene had approximately 0.7 mg/dL lower HDL-c levels than pre-/perimenopausal counterparts, whereas the opposite pattern of HDL-c (0.4 mg/dL higher HDL-c postmenopausally) was observed for the GG genotype. Overall, our data suggest that the decrease in endogenous estrogen as a result of menopause may independently affect lipoprotein concentration, but does not alter the effect on plasma lipids of some common genetic polymorphisms that regulate lipoprotein metabolism.
LDL; HDL; apolipoprotein E; cholesteryl ester transfer protein; hepatic lipase; lipoprotein lipase; menopause
Hyperlipidaemia is a major risk factor for coronary artery disease (CAD) and cholesteryl ester transfer protein (CETP) gene polymorphisms are known to be associated with lipid profiles.
In this study, we investigated the association of two polymorphisms in the CETP, Taq1B (rs708272) and -629C > A (rs1800775), with CAD and lipid levels HDL-C in 662 CAD + cases and 927 controls from the Singapore population comprising Chinese, Malays and Indians.
TaqB2 frequency was significantly lowest in the Malays (0.43) followed by Chinese (0.47) and highest in the Indians (0.56) in the controls. The B2 allele frequency was significantly lower in the Chinese CAD + cases compared to the controls (p = 0.002). The absence of the B2 allele was associated with CAD with an OR 2.0 (95% CI 1.2 to 3.4) after adjustment for the confounding effects of age, smoking, BMI, gender, hypertension, dyslipidemia and diabetes mellitus. The B2 allele was significantly associated with higher plasma HDL-C levels in the Chinese men after adjusting for confounders. Associations with plasma apoA1 levels were significant only in the Chinese men for Taq1B and -629C > A. In addition, the Taq1B polymorphism was only associated with plasma Apo B and Lp(a) in the Malay men. Significant associations were only found in non-smoking subjects with BMI <50th percentile. In this study, the LD coefficients between the Taq1B and -629C > A polymorphisms seemed to be weak.
The absence the Taq1B2 allele was associated with CAD in the Chinese population only and the minor allele of the Taq1B polymorphism of the CETP gene was significantly associated with higher plasma HDL-C levels in Chinese men.
Cholesteryl ester transfer protein; -629C > A polymorphism; TaqB1 polymorphism; HDL-cholesterol; Coronary artery disease
Recent trial data have challenged the hypothesis that cholesteryl ester transfer protein (CETP) and high-density lipoprotein cholesterol (HDLC) have causal roles in atherothrombosis. One method to evaluate this issue is to examine whether polymorphisms in the CETP gene that impact on HDLC levels also impact on the future development of myocardial infarction.
Methods and Results
In a prospective cohort of 18,245 initially healthy American women, we examined over 350,000 singe nucleotide polymorphisms (SNPs) first to identify loci associated with HDLC and then to evaluate whether significant SNPs within these loci also impact upon rates of incident myocardial infarction during an average 10-year follow-up period. Nine loci on 9 chromosomes had one or more SNPs associated with HDLC at genomewide statistical significance (P<5×10−8). However, only SNPs near or in the CETP gene at 16q13 were associated with both HDLC and risk of incident myocardial infarction (198 events). For example, SNP rs708272 in the CETP gene was associated with a per-allele increase in HDLC levels of 3.1 mg/dL and a concordant 24 percent lower risk of future myocardial infarction (age-adjusted HR 0.76, 95%CI 0.62–0.94), consistent with recent meta-analysis. Independent and again concordant effects on HDLC and incident myocardial infarction were also observed at the CETP locus for rs4329913 and rs7202364. Adjustment for HDLC attenuated did not eliminate these effects.
In this prospective cohort of initially healthy women, SNPs at the CETP locus impact upon future risk of myocardial infarction, supporting a causal role for CETP in atherothrombosis, possibly through an HDLC mediated pathway.
HDL cholesterol; myocardial infarction; atherosclerosis; genetic association
A polymorphism of the CETP gene (CETP/TaqIB) with two alleles B1 (60%) and B2 (40%) has been investigated in relation to lipid variables and the risk of myocardial infarction in a large case-control study (ECTIM) of men aged 25-64. No association was observed between the polymorphism and LDL or VLDL related lipid variables. Conversely, B2 carriers had reduced levels of plasma CETP (P < 0.0001) and increased levels of HDL cholesterol (P < 0.0001) and of other HDL related lipid variables. The effects of the polymorphism on plasma CETP and HDL cholesterol were independent, suggesting the presence of at least two functional variants linked to B2. A search for these variants on the coding sequence of the CETP gene failed to identify them. The effect of B2 on plasma HDL cholesterol was absent in subjects drinking < 25 grams/d of alcohol but increased commensurably, with higher values of alcohol consumption (interaction: P < 0.0001). A similar interaction was not observed for plasma CETP. The odds-ratio for myocardial infarction of B2 homozygotes decreased from 1.0 in nondrinkers to 0.34 in those drinking 75 grams/d or more. These results provide the first demonstration of a gene-environment interaction affecting HDL cholesterol levels and coronary heart disease risk.
Endotoxin alters the metabolism of lipoproteins, including that of high density lipoprotein (HDL). Cholesteryl ester transfer protein (CETP) facilitates exchange of HDL cholesterol for very low density lipoprotein (VLDL) triglyceride, leading to catabolism of HDL. We investigated the effects of endotoxin and cytokines on CETP in Syrian hamsters. Endotoxin induced a rapid and progressive decrease in serum CETP levels, by 48 h CETP had decreased to < 20% of control levels. Endotoxin also decreased CETP mRNA and protein levels in adipose tissue, heart, and muscle, the tissues with highest levels of CETP mRNA, providing a plausible mechanism for the endotoxin-induced decrease in circulating CETP. Dexamethasone did not mimic the effects of endotoxin on CETP, but the combination of tumor necrosis factor and interleukin-1 did, indicating that these cytokines may in part mediate the effects of endotoxin on CETP. The endotoxin-induced decrease in CETP may help maintain HDL cholesterol levels during infection and inflammation when increased triglyceride levels could drive the exchange of HDL cholesteryl ester for VLDL triglyceride. Maintaining circulating HDL may be important because HDL protects against the toxic effects of endotoxin and provides cholesterol for peripheral cells involved in the immune response and tissue repair.