Genetic analysis of fluvastatin response and dyslipidemia in renal transplant recipients

The Assessment of Lescol in Renal Transplantation clinical trial demonstrated the efficacy of fluvastatin in reducing cardiovascular (CV) disease in renal transplant recipients. The study included a voluntary pharmacogenetic component, enrolling 1,404 patients, which allowed association testing of baseline measures and longitudinal analysis of the 707 fluvastatin-treated and 697 placebo-treated individuals. A candidate gene approach, examining 42 polymorphisms in 18 genes, was used to test for association between selected polymorphisms and major adverse cardiac events, graft failure, change in LDL and HDL cholesterol, and baseline LDL and HDL cholesterol. Reported associations between cholesteryl ester transfer protein (CETP) and baseline HDL cholesterol were replicated, with four previously implicated single nucleotide polymorphisms significantly associated in males and one in females; tests of reported associations between CETP and CV disease yielded varying results. We found no evidence for genetic factors affecting fluvastatin response. Polymorphisms in 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) previously reported to affect the efficacy of pravastatin did not show a similar effect on the reduction of LDL cholesterol by fluvastatin.     

Influence of dietary fatty acid composition on the relationship between CETP activity and plasma lipoproteins in monkeys.

CETP activity, measured as transfer of cholesteryl ester from exogenous HDL to exogenous VLDL and LDL, reflecting CETP mass as determined by ELISA, was documented in three groups of St. Kitts vervet monkeys fed diets enriched in saturated (Sat), monounsaturated (Mono), or n-6 polyunsaturated (Poly) fatty acids. CETP activity was not different when comparing the three dietary fats. However, CETP activity was significantly higher when cholesterol was added to each of the diets. Significant positive associations between CETP activity and VLDL and LDL cholesterol concentrations were found whereas significant negative associations were seen between CETP activity and HDL cholesterol in each of the diet groups. The strength of these associations was highest in the Sat group. Cholesteryl ester (CE) fatty acid composition of lipoproteins varied widely among diet groups, with the more polyunsaturated CE of the Poly group being associated with a higher rate of CE transfer to endogenous acceptor apolipoprotein B-containing lipoproteins. Finally, only the Sat diet group showed significant positive correlations of CETP activity with LDL particle diameter (r = 0.76), cholesteryl ester percentage (r = 0.67), and a strong negative correlation (r = -0.86) with LDL receptor function, estimated as the difference between native and methylated LDL turnover rates. We speculate that strong associations between CETP and LDL metabolism may explain, at least in part, the increased atherogenicity of dietary saturated fat.     

Structural and biophysical insight into cholesteryl ester-transfer protein

CETP (cholesteryl ester-transfer protein) is essential for neutral lipid transfer between HDL (high-density lipoprotein) and LDL (low-density lipoprotein) and plays a critical role in the reverse cholesterol transfer pathway. In clinical trials, CETP inhibitors increase HDL levels and reduce LDL levels, and therefore may be used as a potential treatment for atherosclerosis. In this review, we cover the analysis of CETP structure and provide insights into CETP-mediated lipid transfer based on a collection of structural and biophysical data.     

Familial cholesteryl ester transfer protein deficiency is associated with triglyceride-rich low density lipoproteins containing cholesteryl esters of probable intracellular origin

The net transfer of core lipids between lipoproteins is facilitated by cholesteryl ester transfer protein (CETP). We have recently documented CETP deficiency in a family with hyperalphalipoproteinemia, due to a CETP gene splicing defect. The purpose of the present study was to characterize the plasma lipoproteins within the low density lipoprotein (LDL) density range and also the cholesteryl ester fatty acid distribution amongst lipoproteins in CETP-deficient subjects. In CETP deficiency, the conventional LDL density range contained both an apoE-rich enlarged high density lipoprotein (HDL) (resembling HDLc), and also apoB-containing lipoproteins. Native gradient gel electrophoresis revealed clear speciation of LDL subclasses, including a distinct population larger in size than normal LDL. Anti-apoB affinity-purified LDL from the CETP-deficient subjects were shown to contain an elevated triglyceride to cholesteryl ester ratio, and also a high ratio of cholesteryl oleate to cholesteryl linoleate, compared to their own HDL or to LDL from normal subjects. Addition of purified CETP to CETP-deficient plasma results in equilibration of very low density lipoprotein (VLDL) cholesteryl esters with those of HDL. These data suggest that, in CETP-deficient humans, the cholesteryl esters of VLDL and its catabolic product, LDL, originate predominantly from intracellular acyl-CoA:cholesterol acyltransferase (ACAT). The CETP plays a role in the normal formation of LDL, removing triglyceride and transferring LCAT-derived cholesteryl esters into LDL precursors.     

Concordant association of lipid gene variation with a combined HDL/LDL-cholesterol phenotype in two European populations

OBJECTIVE: SNP/phenotype associations are difficult to validate. This comparative study demonstrates significant contribution of candidate genes to the variation of a complex cholesterol phenotype, measured in two general populations by a gene-based approach. METHODS: Independent samples of normolipidemic subjects from two Caucasian populations (371 Swiss and 157 Germans) were selected for a case-control-study (high LDL/low HDL versus low LDL/high HDL) with SNP genotypes as independent factors. We examined locus-specific common SNPs that densely cover the genomic regions of 10 lipid genes. RESULTS: Genotype effects were concordant in both ethnic samples, showing that APOE, ABCA1, CETP, and to a lesser degree LDLR, LIPC, and PLTP explained a substantial part of the genetic variation, whereas LPL was associated in only one sample. APOA1, LCAT, and SRB1 exerted no measurable influence. CONCLUSION: This comparison showed that sets of common SNPs representing candidate regions reproducibly validate significant linkage disequilibrium association with a complex metabolic trait.     

Cholesterol ester transfer protein gene is associated with high-density lipoprotein cholesterol levels in Korean population

High-density lipoprotein (HDL) cholesterol levels are associated with decreased risk of coronary artery disease. Several genome-wide association studies (GWAS) for HDL cholesterol levels have implicated cholesterol ester transfer protein (CETP) as possibly causal. We tested for the association between single nucleotide polymorphisms (SNPs) in CETP gene and HDL cholesterol levels in Korean population. A total of 979 subjects in Seoul City were genotyped using a genome-wide marker panel for a discovery study. Another 2,277 subjects in Bundang-Gu in Korea were used for a replication study with selected markers. In the discovery phase, the top SNP associated with mean HDL cholesterol levels was rs6499861 in the CETP gene on chromosome 16 (p=1.18×10^?6 in the Seoul City sample, p=8.91×10^?3 in the Bundang-Gu sample). Another SNP (rs6499863) in the CETP gene was also among the top five SNPs associated with HDL cholesterol levels (p=3.83×10^?5 in the Seoul City sample, p=3.29×10^?3 in the Bundang-Gu sample). SNP rs1800775 was also associated with HDL cholesterol levels (p=4.86×10^?4 in meta-analysis results of 3256 samples). This study clearly demonstrates that genetic variants in CETP influence HDL cholesterol levels in Korean adults.     

A gene score of nine LDL and HDL regulating genes is associated with fluvastatin-induced cholesterol changes in women

While conventional pharmacogenetic studies have considered single gene effects, we tested if a genetic score of nine LDL- and HDL-associated single nucleotide polymorphisms, previously shown to predict cardiovascular disease, is related to fluvastatin-induced lipid change. In patients with asymptomatic plaque in the right carotid artery, thus candidates for statin therapy, we related score LDL [APOB(rs693), APOE(rs4420638), HMGCR(rs12654264), LDLR(rs1529729), and PCSK9(rs11591147)] and score HDL [ABCA1(rs3890182), CETP(rs1800775), LIPC(rs1800588), and LPL(rs328)] as well as the combined score LDL+HDL to fluvastatin-induced LDL reduction (± metoprolol) (n = 395) and HDL increase (n = 187) following 1 year of fluvastatin treatment. In women, an increasing number of unfavorable alleles (i.e., alleles conferring higher LDL and lower HDL) of score LDL+HDL (P = 0.037) and of score LDL (P = 0.023) was associated with less pronounced fluvastatin-induced LDL reduction. Furthermore, in women, both score LDL+HDL (P = 0.001) and score HDL (P = 0.022) were directly correlated with more pronounced fluvastatin-induced HDL increase, explaining 5.9–11.6% of the variance in treatment response in women. There were no such associations in men. This suggests that a gene score based on variation in nine different LDL- and HDL-associated genes is of importance for the magnitude of fluvastatin HDL increase in women with asymptomatic plaque in the carotid artery.     

CETP does not affect triglyceride production or clearance in APOE*3-Leiden mice

The cholesteryl ester transfer protein (CETP) facilitates the bidirectional transfer of cholesteryl esters and triglycerides (TG) between HDL and (V)LDL. By shifting cholesterol in plasma from HDL to (V)LDL in exchange for VLDL-TG, CETP aggravates atherosclerosis in hyperlipidemic APOE*3-Leiden (E3L) mice. The aim of this study was to investigate the role of CETP in TG metabolism and high-fat diet-induced obesity by using E3L mice with and without the expression of the human CETP gene. On chow, plasma lipid levels were comparable between both male and female E3L and E3L.CETP mice. Further mechanistic studies were performed using male mice. CETP expression increased the level of TG in HDL. CETP did not affect the postprandial plasma TG response or the hepatic VLDL-TG and VLDL-apolipoprotein B production rate. Moreover, CETP did not affect the plasma TG clearance rate or organ-specific TG uptake after infusion of VLDL-like emulsion particles. In line with the absence of an effect of CETP on tissue-specific TG uptake, CETP also did not affect weight gain in response to a high-fat diet. In conclusion, the CETP-induced increase of TG in the HDL fraction of E3L mice is not associated with changes in the production of TG or with tissue-specific clearance of TG from the plasma.     

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.     

Effect of CETP on the plasma lipoprotein profile in four strains of transgenic mouse

The plasma cholesteryl ester transfer protein (CETP) plays a central role in high-density lipoprotein (HDL) metabolism and reverse cholesterol transport. There are conflicting views regarding whether or not excessive CETP activity is one of the risk factors of atherosclerosis. To study how much effect CETP can have on the profiles of plasma lipoproteins in vivo, we produced four strains of transgenic mouse that expressed different levels of human CETP gene. We analyzed seven groups of mice that had different levels of CETP expression. The cholesterol level of HDL, chylomicron (CM) and VLDL, intermediate density lipoprotein (IDL) and LDL were proportionally changed in association with plasma CETP concentrations (2.9 +/- 0.6 to 37.4 +/- 1.7 microg/ml) in an allelic dose-dependent manner. We further characterized one of the transgenic strains, CETP-4, by optimizing the experimental condition for the mouse model of atherosclerosis, and found that it would be useful for the development of therapeutics against atherosclerosis.     

LDL particle size in familial combined hyperlipidemia: effects of serum lipids, lipoprotein-modifying enzymes, and lipid transfer proteins

Small, dense LDL particles are typical for FCHL. Intravascular lipid exchange and net transfer among HDL, LDL, and triglyceride-rich lipoproteins as well as lipolysis in the VLDL-IDL-LDL cascade regulate properties of LDL. We investigated postheparin plasma activities of hepatic lipase (HL) and LPL, and plasma activities of CETP and phospholipid transfer protein (PLTP) in 191 individuals from 37 Finnish FCHL families. LDL peak particle diameter (LDL size) was measured with 2-10% gradient polyacrylamide gel electrophoresis. LDL size was significantly smaller in affected FCHL family members (n = 68) as compared with nonaffected FCHL family members (n = 78) or spouses (n = 45) (25.3 +/- 1.5 nm, 26.8 +/- 1.2 nm, and 26.6 +/- 1.2 nm, respectively, P < 0.001 for both). In affected FCHL family members, serum triglycerides were the strongest correlate for LDL size (r = -0.71, P < 0.001). In univariate correlation analysis LDL size was not associated with HL, LPL, CETP, and PLTP activities. In multivariate stepwise regression analysis, however, serum triglycerides, CETP activity, HL activity, and HDL cholesterol were significant predictors of LDL size in affected FCHL subjects (adjusted r (2) = 0.642).We conclude that serum triglyceride concentration is strongly correlated with LDL size in affected FCHL subjects. After adjustment for serum triglycerides, HL and CETP activities are associated with LDL size in FCHL.     

Gene-environment interactions of CETP gene variation in a high cardiovascular risk Mediterranean population

Genome-wide association studies show that cholesteryl ester transfer protein (CETP) single nucleotide polymorphisms (SNPs) are more strongly associated with HDL cholesterol (HDL-C) concentrations than any other loci across the genome. However, gene-environment interactions for clinical applications are still largely unknown. We studied gene-environment interactions between CETP SNPs and dietary fat intake, adherence to the Mediterranean diet, alcohol consumption, smoking, obesity, and diabetes on HDL-C in 4,210 high cardiovascular risk subjects from a Mediterranean population. We focused on the −4,502C>T and the TaqIB SNPs in partial linkage disequilibrium (D''= 0.88; P < 0.001). They were independently associated with higher HDL-C (P < 0.001); this clinically relevant association was greater when their diplotype was considered (14% higher in TT/B2B2 vs. CC/B1B1). No gene-gene interaction was observed. We also analyzed the association of these SNPs with blood pressure, and no clinically relevant associations were detected. No statistically significant interactions of these SNPs with obesity, diabetes, and smoking in determining HDL-C concentrations were found. Likewise, alcohol, dietary fat, and adherence to the Mediterranean diet did not statistically interact with the CETP variants (independently or as diplotype) in determining HDL-C. In conclusion, the strong association of the CETP SNPs and HDL-C was not statistically modified by diet or by the other environmental factors.     

Lipid transfer inhibitor protein defines the participation of high density lipoprotein subfractions in lipid transfer reactions mediated by cholesterol ester transfer protein (CETP)

Cholesterol ester transfer protein (CETP) moves triglyceride (TG) and cholesteryl ester (CE) between lipoproteins. CETP has no apparent preference for high (HDL) or low (LDL) density lipoprotein as lipid donor to very low density lipoprotein (VLDL), and the preference for HDL observed in plasma is due to suppression of LDL transfers by lipid transfer inhibitor protein (LTIP). Given the heterogeneity of HDL, and a demonstrated ability of HDL subfractions to bind LTIP, we examined whether LTIP might also control CETP-facilitated lipid flux among HDL subfractions. CETP-mediated CE transfers from [3H]CE VLDL to various lipoproteins, combined on an equal phospholipid basis, ranged 2-fold and followed the order: HDL3 > LDL > HDL2. LTIP inhibited VLDL to HDL2 transfer at one-half the rate of VLDL to LDL. In contrast, VLDL to HDL3 transfer was stimulated, resulting in a CETP preference for HDL3 that was 3-fold greater than that for LDL or HDL2. Long-term mass transfer experiments confirmed these findings and further established that the previously observed stimulation of CETP activity on HDL by LTIP is due solely to its stimulation of transfer activity on HDL3. TG enrichment of HDL2, which occurs during the HDL cycle, inhibited CETP activity by approximately 2-fold and LTIP activity was blocked almost completely. This suggests that LTIP keeps lipid transfer activity on HDL2 low and constant regardless of its TG enrichment status. Overall, these results show that LTIP tailors CETP-mediated remodeling of HDL3 and HDL2 particles in subclass-specific ways, strongly implicating LTIP as a regulator of HDL metabolism.     

Low density lipoproteins develop resistance to oxidative modification due to inhibition of cholesteryl ester transfer protein by a monoclonal antibody

Although numerous studies have investigated the relationship between cholesteryl ester transfer protein (CETP) and high density lipoprotein (HDL) remodeling, the relationship between CETP and low density lipoproteins (LDL) is still not fully understood. In the present study, we examined the effect of the inhibition of CETP on both LDL oxidation and the uptake of the oxidized LDL, which were made from LDL under condition of CETP inhibition, by macrophages using a monoclonal antibody (mAb) to CETP in incubated plasma. The 6-h incubation of plasma derived from healthy, fasting human subjects led to the transfer of cholesteryl ester (CE) from HDL to VLDL and LDL, and of triglycerides (TG) from VLDL to HDL and LDL. These net mass transfers of neutral lipids among the lipoproteins were eliminated by the mAb. The incubation of plasma either with or without the mAb did not affect the phospholipid compositions in any lipoproteins. As a result, the LDL fractionated from the plasma incubated with the mAb contained significantly less CE and TG in comparison to the LDL fractionated from the plasma incubated without the mAb. The percentage of fatty acid composition of LDL did not differ among the unincubated plasma, the plasma incubated with the mAb, and that incubated without the mAb. When LDL were oxidized with CuSO4, the LDL fractionated from the plasma incubated with the mAb were significantly resistant to the oxidative modification determined by measuring the amount of TBARS and by continuously monitoring the formation of the conjugated dienes, in comparison to the LDL fractionated from the plasma incubated without the mAb. The accumulation of cholesteryl ester of oxidized LDL, which had been oxidized for 2 h with CuSO4, in J774.1 cells also decreased significantly in the LDL fractionated from the plasma incubated with mAb in comparison to the LDL fractionated from the plasma incubated without the mAb. These results indicate that CETP inhibition reduces the composition of CE and TG in LDL and makes the LDL resistant to oxidation. In addition, the uptake of the oxidized LDL, which was made from the LDL under condition of CETP inhibition, by macrophages also decreased.     

Effects of various non-esterified fatty acids on the transfer of cholesteryl esters from HDL to LDL induced by the cholesteryl ester transfer protein

The effects of saturated, monounsaturated and polyunsaturated non-esterified fatty acids on the rate of transfer of radiolabeled cholesteryl esters from high density lipoproteins (HDL) to low density lipoproteins (LDL), induced by the cholesteryl ester transfer protein (CETP), have been studied. Human high-density lipoproteins-subfraction 3 (HDL3) containing radiolabeled cholesteryl esters were incubated with LDL at 37 degrees C with or without CETP and in the absence or in the presence of non-esterified fatty acids. Less than 6% of the total radioactivity was recovered in the LDL fraction after incubation of HDL3, and LDL for 3 h at 37 degrees C in the absence of CETP, regardless of whether or not non-esterified fatty acids were added. The addition of CETP to the incubation mixture induced a time-dependent redistribution of radiolabeled cholesteryl esters from HDL3 to LDL. Non-esterified fatty acids were found to alter the rate of transfer of cholesteryl esters induced by CETP. While short chain saturated non-esterified fatty acids (caprylic and capric acids) had no effect on the rate of transfer of cholesteryl esters, the medium and long chain ones (lauric, myristic, palmitic and stearic acids) significantly increased the CETP-mediated transfers from HDL3 to LDL. At low concentrations, unsaturated fatty acids also stimulated the CETP-mediated redistribution of radiolabeled cholesteryl esters from HDL3 to LDL. As the concentration of either oleic, linoleic or arachidonic acids increased to higher levels, a significant proportion of fatty acids remained unassociated with lipoprotein particles. Under these circumstances the transfer process was inhibited. These results show that non-esterified fatty acids can modulate the CETP-mediated transfer of cholesteryl esters from HDL to LDL and that this effect is dependent on both the length and the degree of unsaturation of their monomeric carbon chain.     

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.     

The interaction of cholesteryl ester transfer protein and unesterified fatty acids promotes a reduction in the particle size of high-density lipoproteins

Purified human cholesteryl ester transfer protein (CETP) has been found, under certain conditions, to promote changes to the particle size distribution of high-density lipoproteins (HDL) which are comparable to those attributed to a putative HDL conversion factor. When preparations of either the conversion factor or CETP are incubated with HDL3 in the presence of very-low-density lipoproteins (VLDL) or low-density lipoproteins (LDL), the HDL3 are converted to very small particles. The possibility that the conversion factor may be identical to CETP was supported by two observations: (1) CETP was found to be the main protein constituent of preparations of the conversion factor and (2) an antibody to CETP not only abolished the cholesteryl ester transfer activity of the conversion factor preparations but also inhibited changes to HDL particle size. In additional studies, the changes to HDL particle size promoted by purified CETP were inhibited by the presence of fatty-acid-free bovine serum albumin; by contrast, albumin had no effect on the cholesteryl ester transfer activity of the CETP. The possibility that albumin may inhibit changes to HDL particle size by removing unesterified fatty acids from either the lipoproteins or CETP was tested by adding exogenous unesterified fatty acids to the incubations. In incubations of HDL with either VLDL or LDL, sodium oleate had no effect on HDL particle size. However, when CETP was also present in the incubation mixtures the capacity of CETP to reduce the particle size of HDL was greatly enhanced by the addition of sodium oleate. It is concluded that the changes in HDL particle size which were previously attributed to an HDL conversion factor can be explained in terms of the interacting effects of CETP and unesterified fatty acids.     

Mechanism of the cholesteryl ester transfer protein-mediated uptake of high density lipoprotein cholesteryl esters by Hep G2 cells

Plasma cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters (CE) between lipoproteins and was reported to also directly mediate the uptake of high density lipoprotein (HDL) CE by human Hep G2 cells and fibroblasts. The present study investigates that uptake and its relationship to a pathway for "selective uptake" of HDL CE that does not require CETP. HDL3 labeled in both the CE and apoprotein moieties was incubated with Hep G2 cells. During 4-h incubations, CE tracer was selectively taken up from doubly labeled HDL3 in excess of apoA-I tracer, and added CETP did not modify that uptake. However, during 18-20-h incubations, CETP stimulated the uptake of CE tracer more than 4-fold without modifying the uptake of apoA-I tracer. This suggested that secreted products, perhaps lipoproteins, might be required for the CETP effect. Four inhibitors of lipoprotein uptake via low density lipoprotein (LDL) receptors (heparin, monensin, an antibody against the LDL receptor, and antibodies against the receptor binding domains of apoB and apoE) effectively blocked the CETP stimulation of CE tracer uptake. Heparin caused an increase in CE tracer in a d less than 1.063 g/ml fraction of the medium that more than accounted for the heparin blockade of CETP-stimulated CE uptake. CETP did not affect the uptake of doubly labeled HDL3 by human fibroblasts, even at twice plasma levels of activity, and heparin did not modify uptake of HDL3 tracers. Thus the CETP effect on Hep G2 cells can be accounted for by transfer of HDL CE to secreted lipoproteins which are then retaken up, and there is no evidence for a direct effect of CETP on cellular uptake of HDL CE.