Apolipoprotein composition of HDL in cholesteryl ester transfer protein deficiency

Our purpose was to compare HDL subpopulations, as determined by nondenaturing two-dimensional gel electrophoresis followed by immunoblotting for apolipoprotein A-I (apoA-I), apoA-II, apoA-IV, apoCs, and apoE in heterozygous, compound heterozygous, and homozygous subjects for cholesteryl ester transfer protein (CETP) deficiency and controls. Heterozygotes, compound heterozygotes, and homozygotes had CETP masses that were 30, 63, and more than 90% lower and HDL-cholesterol values that were 64, 168, and 203% higher than those in controls, respectively. Heterozygotes had approximately 50% lower pre-beta-1 and more than 2-fold higher levels of alpha-1 and pre-alpha-1 particles than controls. Three of the five heterozygotes' alpha-1 particles also contained apoA-II, which was not seen in controls. Compound heterozygotes and homozygotes had very large particles not observed in controls and heterozygotes. These particles contained apoA-I, apoA-II, apoCs, and apoE. However, these subjects did not have decreased pre-beta-1 levels. Our data indicate that CETP deficiency results in the formation of very large HDL particles containing all of the major HDL apolipoproteins except for apoA-IV. We hypothesize that the HDL subpopulation profile of heterozygous CETP-deficient patients, especially those with high levels of alpha-1 containing apoA-I but no apoA-II, represent an improved anti-atherogenic state, although this might not be the case for compound heterozygotes and homozygotes with very large, undifferentiated HDL particles.     

VLDL-bound lipoprotein lipase facilitates the cholesteryl ester transfer protein-mediated transfer of cholesteryl esters from HDL to VLDL

In recent years, it has been established that lipoprotein lipase (LPL) is partly associated with circulating lipoproteins. This report describes the effects of physiological amounts of very low density lipoprotein (VLDL)-bound LPL on the cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester transfer (CET) from high density lipoprotein (HDL) to VLDL. Three patients with severe LPL deficiency exhibited a strong decrease in net mass CET that was more than 80% lower than that of common hypertriglyceridemic subjects. Recombination experiments showed that this was due to an abnormal behavior of the VLDL fraction. Replacement of the latter by normal VLDL totally normalized net mass CET. We therefore prepared VLDL containing controlled amounts of bound LPL that we used as CE acceptors in experiments involving unidirectional radioisotopic CET measurements. These were carried out either in the absence or in the presence of inhibitors of LPL lipolytic activity. When LPL-induced lipolysis was totally blocked, the stimulating effect of the enzyme on the CETP-dependent CET was only reduced by about 50%, showing that it did not entirely result from its lipolytic action. These data were dependent upon neither the type of LPL inhibitor (E600 or THL) nor the source of CETP (delipidated plasma or partially purified CETP). Thus, in addition to the well-known stimulating effect of LPL-dependent lipolysis on CET, our work demonstrates that physiological amounts of VLDL-bound LPL may facilitate CET through a mechanism partially independent of its lipolytic activity.     

Role of cholesteryl ester transfer protein in selective uptake of high density lipoprotein cholesteryl esters by adipocytes

Previous reports attributed cholesteryl ester transfer protein (CETP)-mediated HDL cholesteryl ester (CE) selective uptake to the CETP-mediated transfer of CE from HDL to newly secreted apolipoprotein B-containing lipoproteins, which are then internalized by the LDL receptor (LDL-R). CETP has also been implicated in the remodeling of HDL, which renders it a better substrate for selective uptake by scavenger receptor class B type I (SR-BI). However, CETP-mediated selective uptake of HDL3-derived CE was not diminished in LDL-R null adipocytes, SR-BI null adipocytes, or in the presence of the receptor-associated protein. We found that monensin treatment or energy depletion of the SW872 liposarcoma cells with 2-deoxyglucose and NaN3 had no effect on CETP-mediated selective uptake, demonstrating that endocytosis is not required. This is supported by data indicating that CETP transfers CE into a compartment from which it can be extracted by unlabeled HDL. CETP could also mediate the selective uptake of HDL3-derived triacylglycerol (TG) and phospholipid (PL). The CETP-specific kinetics for TG and CE uptake were similar, and both reached saturation at approximately 5 microg/ml HDL. In contrast, CETP-specific PL uptake did not attain saturation at 5 microg/ml HDL and was approximately 6-fold greater than the uptake of CE. We propose two possible mechanisms to account for the role of CETP in selective uptake.     

Transfer of cholesteryl ester into high density lipoprotein by cholesteryl ester transfer protein: effect of HDL lipid and apoprotein content

Recombinant high density lipoprotein (rHDL) particles were prepared by cosonication of purified lipids and human apoproteins and incubated with partly purified cholesteryl ester transfer protein (CETP) and low density lipoprotein (LDL) containing [3H]cholesteryl ester. Increasing the triglyceride content relative to cholesteryl ester in rHDL significantly decreased the ability of the particles to accept cholesteryl esters transferred by CETP. Kinetic analysis of the data was performed to numerically define the maximum velocity of lipid transfer, Tmax, and the HDL concentration required for half maximal velocity, KH. Increases in rHDL-triglyceride content were shown to result in a significant reduction in the Tmax without a major change in KH. When the free cholesterol content was increased relative to phospholipid, the ability of the particles to accept cholesteryl esters was also decreased in a similar manner. Conversely, rHDL prepared from purified apoprotein A-I, A-II, or mixtures of both, had significantly elevated Tmax and KH values for their interaction with CETP. The results suggest that increases in triglyceride or free cholesterol content of an rHDL particle decrease the catalytic ability of CETP by noncompetitive inhibition. In addition, some component(s) of HDL apoproteins, other than A-I or A-II, were shown to uncompetitively inhibit the activity of CETP, by modifying both Tmax and the KH for the reaction. This study has shown that altered HDL composition may have marked effects on the transfer and equilibration of cholesteryl esters within the HDL pool.     

Reduced high density lipoprotein cholesterol in human cholesteryl ester transfer protein transgenic mice

The human cholesteryl ester transfer protein (CETP) facilitates the exchange of neutral lipids among lipoproteins. In order to evaluate the effects of increased plasma CETP on lipoprotein levels, a human CETP minigene was placed under the control of the mouse metallothionein-I promoter and used to develop transgenic mice. Integration of the human CETP transgene into the mouse genome resulted in the production of active plasma CETP. Zinc induction of CETP transgene expression caused depression of serum cholesterol due to a significant reduction of high density lipoprotein cholesterol. There was no change in total cholesterol content in very low and low density lipoproteins. However, there was a decrease in the free cholesterol/cholesteryl ester ratio in plasma and in all lipoprotein fractions of transgenic mouse plasma, suggesting stimulation of plasma cholesterol esterification. The results suggest that high levels of plasma CETP activity may be a cause of reduced high density lipoproteins in humans.     

Enhanced cholesteryl ester transfer protein activities and abnormalities of high density lipoproteins in familial hypercholesterolemia.

Cholesteryl ester transfer protein may play a role in the cholesteryl ester metabolism between high density lipoproteins (HDL) and apolipoprotein B-containing lipoproteins. To investigate relationship between HDL and cholesteryl ester transfer protein (CETP) activity in the development of atherosclerosis, the present study has focused on CETP activity in the patients with familial hypercholesterolemia (GH). HDL-C and HDL-C/apo A-I mass ratio in heterozygous FH were lower than those in normolipidemic controls. There was a 2-fold increase in total CETP activity in incubated FH serum compared with normolipidemic controls. Assays for CETP activity in the lipoprotein deficient serum (d greater than 1.215 g/ml) were carried out by measuring the transfer of radioactive cholesteryl ester from HDL (1.125 less than d less than 1.21 g/ml) to LDL (1.019 less than d less than 1.060 g/ml). CETP activities in heterozygous FH (79 +/- 4 nmol/ml/h) was significantly higher than those in normolipidemic controls (54 +/- 6 nmol/ml/h). The increased total cholesteryl ester transfer mainly results from increased CETP activity in the d greater than 1.215 g/ml, possibly reflecting an increase in CETP mass in serum. Increased CETP activity in the d greater than 1.215 g/ml was correlated positively with IDL-cholesterol/triglyceride mass ratio (r = 0.496, p less than 0.01), and negatively with HDL-cholesterol/apo A-I mass ratio (r = -0.334, p less than 0.05). These results indicate that the enhanced CETP activities may contribute to increase risk for developing atherosclerosis in FH by changing the distribution of cholesteryl ester in serum lipoproteins.     

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.     

Reconstitution of the lipoprotein cholesteryl ester transfer process using isolated rat ovary plasma membranes.

Steroidogenic cells are able to utilize lipoprotein-derived cholesteryl esters for steroidogenesis without internalizing intact lipoproteins. In the current report, we provide evidence that an early step in this process may be the selective extraction of cholesteryl esters at the cell (plasma membrane) surface. We have used a highly purified plasma membrane preparation from rat luteinized ovaries for incubation with rat- and human-derived high density (HDL) and low density (LDL) lipoproteins. The lipoproteins were modified with residualizing [125I]apoprotein or [3H]cholesteryl ester markers. Following trypsin treatment to remove intact surface-bound apoprotein particles, the membranes were analyzed for transferred radioactive labels. The results show that all the lipoproteins tested could serve as cholesteryl ester donors. Although far more [3H]cholesteryl ester than [125I]apoprotein radioactivity was transferred to plasma membranes in each case, and varied with the ligand used, the total (net) mass of cholesteryl ester transferred was comparable with the different lipoproteins. These data were confirmed using direct chemical methodology. Transfer was found to be specific for cholesteryl esters or ethers and did not involve other lipoprotein core lipids tested. Endomembranes from the same tissue could not substitute for plasma membranes as the primary cholesteryl ester acceptor. These results provide evidence that a reconstituted lipoprotein-plasma membrane system can simulate the cholesteryl ester extraction process described in situ and suggest uses for this methodology in future experiments designed to understand the transfer process.     

Genetic variations in the cholesteryl ester transfer protein gene and high density lipoprotein cholesterol levels in Taiwanese Chinese

This study analyzed the association of the I14A mutation, the D442G mutation, and the TaqIB polymorphism of the cholesteryl ester transfer protein (CETP) gene in 718 Chinese individuals with high-density lipoprotein cholesterol levels (HDL-C) living in Taiwan. The analysis revealed that the I14A mutation was not present in any of the 110 subjects with HDL-C levels above 60 mg/dl. By contrast, the D442G mutation was present in 48 of the 718 (6.7%) subjects tested. Significantly higher HDL-C levels were noted for bearers of the D442G mutation compared with non-bearers; however, this association was weaker for males and for subjects carrying the TaqIB1 allele. The TaqIB2 allele was also associated with higher HDL-C levels. From multivariate analysis, independent associations were demonstrated for the TaqIB2 polymorphism and the D442G mutation, and elevated HDL-C levels. For obese subjects, however, the presence of the TaqIB2 or D442G allele was not associated with increased HDL-C levels. For subjects with triglycerides at a concentration greater than 150 mg/dl, the association of both alleles with HDL-C levels was also diminished. Thus, genetic variation at the CETP gene locus may account for a significant proportion of the difference in HDL-C levels; however, it seems reasonable to suggest that the effects of the allele interact with genetic variations expressed within the sample population, and with sex, obesity, and plasma triglyceride levels.     

Human low density lipoprotein subfractions separated by gradient gel electrophoresis: composition, distribution, and alterations induced by cholesteryl ester transfer protein

Low density lipoprotein (LDL) subfractions were studied in sera from 208 normolipidemic, 22 hypercholesterolemic, and 33 hypertriglyceridemic subjects. Whole serum without preliminary ultracentrifugation was submitted to electrophoresis in a nondenaturing polyacrylamide gel. Three main LDL patterns were observed in normolipidemic sera: type 1, characterized by the presence of only one major band; type 2, characterized by the presence of two close major bands; and type 3, where LDL were more dispersed and presented at least three distinct bands. Type 1 was more frequent in men (43%) than in women (19%). The tendency for a higher potential coronary disease risk profile sera, namely higher triglyceride level, higher very low density lipoprotein + LDL fraction and lower high density lipoprotein (HDL) fraction, was type 3 less than type 2 less than type 1. The LDL patterns found in hypercholesterolemic sera were of type 1. Hypertriglyceridemic sera were characterized by the presence of a major band of small size. Separated LDL subfractions were collected by electroelution and analyzed for composition. In all subspecies, the mass ratio of core to surface components was constant as well as the molar ratio of the two lipid surface components, phospholipids and free cholesterol. Surface lipid to apolipoprotein B ratio, cholesteryl ester to triglyceride ratio, and cholesteryl ester to apoB ratio increased with particle size increment. Incubation of LDL with HDL and purified cholesteryl ester transfer protein induced a transfer of lipids, mainly cholesteryl esters and phospholipids, to LDL and an increase of the sizes of LDL subfractions. This suggests that lipid transfers from HDL to LDL might be a process of intravascular LDL remodeling and a factor of LDL polymorphism.     

Tetrazole and ester substituted tetrahydoquinoxalines as potent cholesteryl ester transfer protein inhibitors

Cholesteryl ester transfer protein is a plasma glycoprotein that transfers cholesterol ester between lipoprotein particles. Inhibition of this protein, in vitro and in vivo, produces an increase in plasma high density lipoprotein cholesterol (HDL-C). This communication will describe the SAR and synthesis of a series of substituted tetrahydroquinoxaline CETP inhibitors from early mu lead to advanced enantiomerically pure analogs.     

Monoclonal antibodies to the Mr 74,000 cholesteryl ester transfer protein neutralize all of the cholesteryl ester and triglyceride transfer activities in human plasma

A cholesteryl ester transfer protein (CETP) of apparent Mr 74,000 has recently been purified from human plasma. Three monoclonal neutralizing antibodies to the CETP were obtained by immunizing mice with purified CETP. The antibodies, each recognizing a similar epitope on CETP, caused parallel and complete immunotitration of plasma cholesteryl ester and triglyceride transfer activities but only partial inhibition of phospholipid transfer activity. Monoclonal immunoaffinity chromatography of plasma or its fractions showed complete removal of cholesteryl ester and triglyceride transfer activities but incomplete removal of phospholipid transfer activity. Sodium dodecyl sulfate gel electrophoresis and immunoblotting of the immunoaffinity-retained fractions showed that only the Mr 74,000 protein was immunoreactive. The results suggest that the previously characterized CETP accounts for all of the cholesteryl ester and triglyceride transfer activity in human plasma but only part of the phospholipid transfer activity.     

Biochemical characterization of cholesteryl ester transfer protein inhibitors

Cholesteryl ester transfer protein (CETP) has been identified as a novel target for increasing HDL cholesterol levels. In this report, we describe the biochemical characterization of anacetrapib, a potent inhibitor of CETP. To better understand the mechanism by which anacetrapib inhibits CETP activity, its biochemical properties were compared with CETP inhibitors from distinct structural classes, including torcetrapib and dalcetrapib. Anacetrapib and torcetrapib inhibited CETP-mediated cholesteryl ester and triglyceride transfer with similar potencies, whereas dalcetrapib was a significantly less potent inhibitor. Inhibition of CETP by both anacetrapib and torcetrapib was not time dependent, whereas the potency of dalcetrapib significantly increased with extended preincubation. Anacetrapib, torcetrapib, and dalcetrapib compete with one another for binding CETP; however anacetrapib binds reversibly and dalcetrapib covalently to CETP. In addition, dalcetrapib was found to covalently label both human and mouse plasma proteins. Each CETP inhibitor induced tight binding of CETP to HDL, indicating that these inhibitors promote the formation of a complex between CETP and HDL, resulting in inhibition of CETP activity.     

Establishment of anti-human cholesteryl ester transfer protein monoclonal antibodies and radioimmunoassaying of the level of cholesteryl ester transfer protein in human plasma.

Plasma cholesteryl ester transfer protein (CETP) facilitates the net transfer and exchange of cholesteryl ester (CE), triglyceride (TG), and phospholipids between lipoproteins. A series of monoclonal antibodies (mAbs) against human CETP was obtained, comprising mAbs either inhibiting or not inhibiting these transfer activities. One mAb (LT-J1) inhibited the transfer activity of TG almost completely, but not that of CE, indicating that CE and TG binding sites on the CETP molecule may be distinct from each other, and that this mAb may specifically recognize the TG binding site. A radioimmunoassay system for determining the level of CETP was also established using these mAbs, and the plasma CETP levels in 20 normolipemic Japanese adults were found to range from 2.1 to 2.7 mg/liter.     

Rat adrenal uptake and metabolism of high density lipoprotein cholesteryl ester

Metabolism of high density lipoprotein (HDL) cholesteryl ester (CE) by cultured rat adrenal cells was studied. Addition of [3H]CE-HDL to cells pretreated with adrenocorticotrophin in lipoprotein poor media resulted in a time- and concentration-dependent accumulation of [3H]cholesteryl ester and production of [3H]cholesterol and [3H]corticosterone. HDL-CE metabolism could be described as the sum of a high affinity ([ HDL-cholesterol]1/2 max = 16 micrograms/ml) and low affinity ([ HDL-cholesterol]1/2 max greater than 70 micrograms/ml) process. [3H]Cholesterol was found both intracellularly and in the media. Accumulation of [3H]cholesteryl ester could not be attributed to uptake and re-esterification of unesterified cholesterol since addition of Sandoz 58-035, an inhibitor of acyl coenzyme A:cholesterol acyltransferase, did not prevent ester accumulation. Moreover, addition of chloroquine did not inhibit cholesteryl ester hydrolysis indicating that hydrolysis was not lysosomally mediated. Aminoglutethimide prevented conversion of [3H]CE-HDL to steroid hormones but did not inhibit [3H]cholesteryl ester uptake. Cellular accumulation of [3H] cholesteryl ester exceeded accumulation of 125I-apoproteins 5-fold at 1 h and 35-fold at 24 h indicating selective uptake of cholesteryl ester moiety. We conclude that rat adrenal cells possess a mechanism for selective uptake of HDL cholesteryl esters which provides substrate for steroidogenesis. These results constitute the first direct demonstration that cholesteryl esters in HDL can be used as steroidogenic substrate by the rat adrenal cortex.     

Pros and cons of inhibiting cholesteryl ester transfer protein

Whether or not it is desirable to inhibit cholesteryl ester transfer protein (CETP) has been an important question for over fifteen years since genetic CETP deficiency was found. Recently, some epidemiological studies which have been reported in Japan as well as Western countries help to clarify the atherogenicity of human subjects with mutations or polymorphisms in the CETP gene. In addition, some experimental atherosclerosis studies, in which CETP was inhibited in rabbits with different approaches, have been reported. There was a considerable difference in the atherogenicity of human CETP deficiency and CETP-inhibited rabbits. In this review, we summarize the recent advances in this field as well as discussing the significance of CETP in reverse cholesterol transport, a major protective system against atherosclerosis.     

Human plasma cholesteryl ester transfer protein enhances the transfer of cholesteryl ester from high density lipoproteins into cultured HepG2 cells

The role of human plasma cholesteryl ester transfer protein (CETP) in the cellular uptake of high density lipoprotein (HDL) cholesteryl ester (CE) was studied in a liver tumor cell line (HepG2). When HepG2 cells were incubated with [3H]cholesteryl ester-labeled HDL3 in the presence of increasing concentrations of CETP there was a progressive increase in cell-associated radioactivity to levels that were 2.8 times control. The CETP-dependent uptake of HDL-CE was found to be saturated by increasing concentrations of both CETP and HDL. The CETP-dependent uptake of CE radioactivity increased continuously during an 18-h incubation. In contrast to the effect on cholesteryl ester, CETP failed to enhance HDL protein cell association or degradation. Enhanced uptake of HDL cholesteryl ester was shown for the d greater than 1.21 g/ml fraction of human plasma, partially purified CETP, and CETP purified to homogeneity, but not for the d greater than 1.21 g/ml fraction of rat plasma which lacks cholesteryl ester transfer activity. HDL cholesteryl ester entering the cell under the influence of CETP was largely degraded to free cholesterol by a process inhibitable by chloroquine. CETP enhanced uptake of HDL [3H]CE in cultured smooth muscle cells and to a lesser extent in fibroblasts but did not significantly influence uptake in endothelial cells or J774 macrophages. These experiments show that, in addition to its known role in enhancing the exchange of CE between lipoproteins, plasma CETP can facilitate the in vitro selective transfer of CE from HDL into certain cells.     

Hydrolysis of cholesteryl ester in low density lipoprotein converts this lipoprotein to a liposome.

Previously, we isolated and characterized unique liposomal-like, cholesterol-rich lipid particles that accumulate in human atherosclerotic lesions. Human plasma low density lipoprotein (LDL) has a molar ratio of total cholesterol to phospholipid (3:1) similar to that of this lesion cholesterol-rich lipid particle. However, LDL is enriched in cholesteryl ester while the lesion lipid particle is enriched in unesterified cholesterol. To examine a possible precursor-product relationship between LDL and the lesion lipid particle, we hydrolyzed the cholesteryl ester core of LDL with cholesterol esterase. Cholesteryl ester hydrolysis occurred only after LDL was treated with trypsin. Trypsin pretreatment was not required for cholesteryl ester hydrolysis of LDL oxidized with copper, a treatment that also degrades apolipoprotein B, the major protein moiety in LDL. In contrast to greater than 90% hydrolysis of cholesteryl ester in trypsin-cholesterol esterase-treated or copper-oxidized LDL, there was only 18% hydrolysis of cholesteryl ester in similarly treated high density lipoprotein. With a limited 10-min hydrolysis of LDL cholesteryl ester, LDL-sized particles and newly formed larger flattened films or discs were present. With complete hydrolysis of LDL cholesteryl ester, LDL particles converted to complex multilamellar, liposomal-like, structures with sizes approximately five times larger than native LDL. These liposomal-like particles derived from LDL were chemically and structurally similar to unesterified cholesterol-rich lipid particles that accumulate in atherosclerotic lesions.