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 (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.     

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.     

PXR agonism decreases plasma HDL levels in ApoE⁎3-Leiden.CETP mice

Pregnane X receptor (PXR) agonism has been shown to affect multiple steps in both the synthesis and catabolism of HDL, but its integrated effect on HDL metabolism in vivo remains unclear. The aim of this study was to evaluate the net effect of PXR agonism on HDL metabolism in ApoE?3-Leiden (E3L) and E3L.CETP mice, well-established models for human-like lipoprotein metabolism. Female mice were fed a diet with increasing amounts of the potent PXR agonist 5-pregnen-3β-ol-20-one-16α-carbonitrile (PCN). In E3L and E3L.CETP mice, PCN increased liver lipids as well as plasma cholesterol and triglycerides. However, whereas PCN increased cholesterol contained in large HDL-1 particles in E3L mice, it dose-dependently decreased HDL-cholesterol in E3L.CETP mice, indicating that CETP expression dominates the effect of PCN on HDL metabolism. Analysis of the hepatic expression of genes involved in HDL metabolism showed that PCN decreased expression of genes involved in HDL synthesis (Abca1, Apoa1), maturation (Lcat, Pltp) and clearance (Sr-b1). The HDL-increasing effect of PCN, observed in E3L mice, is likely caused by a marked decrease in hepatic SR-BI protein expression, and completely reversed by CETP expression. We conclude that chronic PXR agonism dose-dependently reduces plasma HDL-cholesterol in the presence of CETP.     

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.     

Remodeling of HDL by CETP in vivo and by CETP and hepatic lipase in vitro results in enhanced uptake of HDL CE by cells expressing scavenger receptor B-I.

The transport of HDL cholesteryl esters (CE) from plasma to the liver involves a direct uptake pathway, mediated by hepatic scavenger receptor B-I (SR-BI), and an indirect pathway, involving the exchange of HDL CE for triglycerides (TG) of TG-rich lipoproteins by cholesteryl ester transfer protein (CETP). We carried out HDL CE turnover studies in mice expressing human CETP and/or human lecithin:cholesterol acyltransferase (LCAT) transgenes on a background of human apoA-I expression. The fractional clearance of HDL CE by the liver was delayed by LCAT transgene, while the CETP transgene increased it. However, there was no incremental transfer of HDL CE radioactivity to the TG-rich lipoprotein fraction in mice expressing CETP, suggesting increased direct removal of HDL CE in the liver. To evaluate the possibility that this might be mediated by SR-BI, HDL isolated from plasma of the different groups of transgenic mice was incubated with SR-BI transfected or control CHO cells. HDL isolated from mice expressing CETP showed a 2- to 4-fold increase in SR-BI-mediated HDL CE uptake, compared to HDL from mice lacking CETP. The addition of pure CETP to HDL in cell culture did not lead to increased selective uptake of HDL CE by cells. However, when human HDL was enriched with TG by incubation with TG-rich lipoproteins in the presence of CETP, then treated with hepatic lipase, there was a significant enhancement of HDL CE uptake. Thus, the remodeling of human HDL by CETP, involving CE;-TG interchange, followed by the action of hepatic lipase (HL), leads to the enhanced uptake of HDL CE by cellular SR-BI.These observations suggest that in animals such as humans in which both the selective uptake and CETP pathways are active, the two pathways could operate in a synergistic fashion to enhance reverse cholesterol transport.     

Impact of LDL apheresis on atheroprotective reverse cholesterol transport pathway in familial hypercholesterolemia

In familial hypercholesterolemia (FH), low HDL cholesterol (HDL-C) levels are associated with functional alterations of HDL particles that reduce their capacity to mediate the reverse cholesterol transport (RCT) pathway. The objective of this study was to evaluate the consequences of LDL apheresis on the efficacy of the RCT pathway in FH patients. LDL apheresis markedly reduced abnormal accelerated cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) transfer from HDL to LDL, thus reducing their CE content. Equally, we observed a major decrease (-53%; P < 0.0001) in pre-β1-HDL levels. The capacity of whole plasma to mediate free cholesterol efflux from human macrophages was reduced (-15%; P < 0.02) following LDL apheresis. Such reduction resulted from a marked decrease in the ABCA1-dependent efflux (-71%; P < 0.0001) in the scavenger receptor class B type I-dependent efflux (-21%; P < 0.0001) and in the ABCG1-dependent pathway (-15%; P < 0.04). However, HDL particles isolated from FH patients before and after LDL apheresis displayed a similar capacity to mediate cellular free cholesterol efflux or to deliver CE to hepatic cells. We demonstrate that rapid removal of circulating lipoprotein particles by LDL apheresis transitorily reduces RCT. However, LDL apheresis is without impact on the intrinsic ability of HDL particles to promote either cellular free cholesterol efflux from macrophages or to deliver CE to hepatic cells.     

Induction of the phospholipid transfer protein gene accounts for the high density lipoprotein enlargement in mice treated with fenofibrate

Fibrate treatment in mice is known to modulate high density lipoprotein (HDL) metabolism by regulating apolipoprotein (apo)AI and apoAII gene expression. In addition to alterations in plasma HDL levels, fibrates induce the emergence of large, cholesteryl ester-rich HDL in treated transgenic mice expressing human apoAI (HuAITg). The mechanisms of these changes may not be restricted to the modulation of apolipoprotein gene expression, and the aim of the present study was to determine whether the expression of factors known to affect HDL metabolism (i.e. phospholipid transfer protein (PLTP), lecithin:cholesterol acyltransferase, and hepatic lipase) are modified in fenofibrate-treated mice. Significant rises in plasma PLTP activity were observed after 2 weeks of fenofibrate treatment in both wild-type and HuAITg mice. Simultaneously, hepatic PLTP mRNA levels increased in a dose-dependent fashion. In contrast to PLTP, lecithin:cholesterol acyltransferase mRNA levels in HuAITg mice were not significantly modified by fenofibrate despite a significant decrease in plasma cholesterol esterification activity. Fenofibrate did not induce any change in hepatic lipase activity. Fenofibrate significantly increased HDL size, an effect that was more pronounced in HuAITg mice than in wild-type mice. This effect in wild-type mice was completely abolished in PLTP-deficient mice. Finally, fenofibrate treatment did not influence PLTP activity or hepatic mRNA in peroxisome proliferator-activated receptor-alpha-deficient mice. It is concluded that 1) fenofibrate treatment increases plasma phospholipid transfer activity as the result of up-regulation of PLTP gene expression through a peroxisome proliferator-activated receptor-alpha-dependent mechanism, and 2) increased plasma PLTP levels account for the marked enlargement of HDL in fenofibrate-treated mice.     

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.     

Postprandial chylomicrons: potent vehicles for transporting cholesterol from endogenous LDL+HDL and cell membranes to the liver via LCAT and CETP

We examined whether postprandial (PP) chylomicrons (CMs) can serve as vehicles for transporting cholesterol from endogenous cholesterol-rich lipoprotein (LDL+HDL) fractions and cell membranes to the liver via lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) activities. During incubation of fresh fasting and PP plasma containing [(3)H]cholesteryl ester (CE)-labeled LDL+HDL, both CMs and VLDL served as acceptors of [(3)H]CE or cholesterol from LDL+HDL. The presence of CMs in PP plasma suppressed the ability of VLDL to accept [(3)H]CE from LDL+HDL. In reconstituted plasma containing an equivalent amount of triglycerides from isolated VLDL or CMs, a CM particle was about 40 times more potent than a VLDL particle in accepting [(3)H]CE or cholesterol from LDL+HDLs. When incubated with red blood cells (RBCs) as a source for cell membrane cholesterol, the cholesterol content of CMs, VLDL, LDL, and HDL in PP plasma increased by 485%, 74%, 13%, and 30%, respectively, via LCAT and CETP activities. The presence of CMs in plasma suppressed the ability of endogenous lipoproteins to accept cholesterol from RBCs. Our data suggest that PP CMs may play an important role in promoting reverse cholesterol transport in vivo by serving as the preferred ultimate vehicle for transporting cholesterol released from cell membranes to the liver via LCAT and CETP.     

Cholesteryl ester transfer protein corrects dysfunctional high density lipoproteins and reduces aortic atherosclerosis in lecithin cholesterol acyltransferase transgenic mice

Expression of human lecithin cholesterol acyltransferase (LCAT) in mice (LCAT-Tg) leads to increased high density lipoprotein (HDL) cholesterol levels but paradoxically, enhanced atherosclerosis. We have hypothesized that the absence of cholesteryl ester transfer protein (CETP) in LCAT-Tg mice facilitates the accumulation of dysfunctional HDL leading to impaired reverse cholesterol transport and the development of a pro-atherogenic state. To test this hypothesis we cross-bred LCAT-Tg with CETP-Tg mice. On both regular chow and high fat, high cholesterol diets, expression of CETP in LCAT-Tg mice reduced total cholesterol (-39% and -13%, respectively; p < 0.05), reflecting a decrease in HDL cholesterol levels. CETP normalized both the plasma clearance of [(3)H]cholesteryl esters ([(3)H]CE) from HDL (fractional catabolic rate in days(-1): LCAT-Tg = 3.7 +/- 0.34, LCATxCETP-Tg = 6.1 +/- 0.16, and controls = 6.4 +/- 0.16) as well as the liver uptake of [(3)H]CE from HDL (LCAT-Tg = 36%, LCATxCETP-Tg = 65%, and controls = 63%) in LCAT-Tg mice. On the pro-atherogenic diet the mean aortic lesion area was reduced by 41% in LCATxCETP-Tg (21.2 +/- 2.0 micrometer(2) x 10(3)) compared with LCAT-Tg mice (35.7 +/- 2.0 micrometer(2) x 10(3); p < 0.001). Adenovirus-mediated expression of scavenger receptor class B (SR-BI) failed to normalize the plasma clearance and liver uptake of [(3)H]CE from LCAT-Tg HDL. Thus, the ability of SR-BI to facilitate the selective uptake of CE from LCAT-Tg HDL is impaired, indicating a potential mechanism leading to impaired reverse cholesterol transport and atherosclerosis in these animals. We conclude that CETP expression reduces atherosclerosis in LCAT-Tg mice by restoring the functional properties of LCAT-Tg mouse HDL and promoting the hepatic uptake of HDL-CE. These findings provide definitive in vivo evidence supporting the proposed anti-atherogenic role of CETP in facilitating HDL-mediated reverse cholesterol transport and demonstrate that CETP expression is beneficial in pro-atherogenic states that result from impaired reverse cholesterol transport.     

Cholesteryl ester transfer protein expression attenuates atherosclerosis in ovariectomized mice

Reduced estrogen levels result in loss of protection from coronary heart disease in postmenopausal women. Enhanced and diminished atherosclerosis have been associated with plasma levels of cholesteryl ester transfer protein (CETP); however, little is known about the role of CETP-ovarian hormone interactions in atherogenesis. We assessed the severity of diet-induced atherosclerosis in ovariectomized (OV) CETP transgenic mice crossbred with LDL receptor knockout mice. Compared with OV CETP expressing ((+)), OV CETP non-expressing ((-)) mice had higher plasma levels of total, VLDL-, LDL-, and HDL-cholesterol, as well as higher antibodies titers against oxidized LDL. The mean aortic lesion area was 2-fold larger in OV CETP(-) than in OV CETP(+) mice (147 +/- 90 vs. 73 +/- 42 x 10(3) micro m(2), respectively). Estrogen therapy in OV mice blunted the CETP dependent differences in plasma lipoproteins, oxLDL antibodies, and atherosclerosis severity. Macrophages from OV CETP(+) mice took up less labeled cholesteryl ether (CEt) from acetyl-LDL than macrophages from OV CETP(-) mice. Estrogen replacement induced a further reduction in CEt uptake and an elevation in HDL mediated cholesterol efflux from pre-loaded OV CETP(+) as compared with OV CETP(-) macrophages. These findings support the proposed anti-atherogenic role of CETP in specific metabolic settings.     

Stereospecific inhibition of CETP by chiral N,N-disubstituted trifluoro-3-amino-2-propanols

Chiral N,N-disubstituted trifluoro-3-amino-2-propanols represent a recently discovered class of compounds that inhibit the neutral lipid transfer activity of cholesteryl ester transfer protein (CETP). These compounds all contain a single chiral center that is essential for inhibitory activity. (R,S)SC-744, which is composed of a mixture of the two enantiomers, inhibits CETP-mediated transfer of [(3)H]cholesteryl ester ([(3)H]CE) from HDL donor particles to LDL acceptor particles with an IC(50) = 200 nM when assayed using a reconstituted system in buffer and with an IC(50) = 6 microM when assayed in plasma. Upon isolation of the enantiomers, it was found that the (R,+) enantiomer, SC-795, was about 10-fold more potent than the mixture, and that the (S,-) enantiomer, SC-794, did not have significant inhibitory activity (IC(50) > 0.8 microM). All of the activity of the (S,-)SC-794 enantiomer could be accounted for by contamination of this sample with a residual 2% of the highly potent (R,+) enantiomer, SC-795. The IC(50) of (R,+)SC-795, 20 nM, approached the concentration of CETP (8 nM) in the buffer assay. These chiral N,N-disubstituted trifluoro-3-amino-2-propanols were found to associate with both LDL and HDL, but did not disrupt overall lipoprotein structure. They did not affect the on or off rates of CETP binding to HDL disk particles. Inhibition was highly specific since the activities of phospholipid transfer protein and lecithin cholesterol acyl transferase were not affected. Competition experiments showed that the more potent enantiomer (R)SC-795 prevented cholesteryl ester binding to CETP, and direct binding experiments demonstrated that this inhibitor bound to CETP with high affinity and specificity. It is estimated, based on the relative concentrations of inhibitor and lipid in the transfer assay, that (R)SC-795 binds approximately 5000-fold more efficiently to CETP than the natural ligand, cholesteryl ester. We conclude that these chiral N,N-disubstituted trifluoro-3-amino-2-propanol compounds do not affect lipoprotein structure or CETP-lipoprotein recognition, but inhibit lipid transfer by binding to CETP reversibly and stereospecifically at a site that competes with neutral lipid binding.     

Modulating cholesteryl ester transfer protein activity maintains efficient pre-��-HDL formation and increases reverse cholesterol transport

The mechanism by which cholesteryl ester transfer protein (CETP) activity affects HDL metabolism was investigated using agents that selectively target CETP (dalcetrapib, torcetrapib, anacetrapib). In contrast with torcetrapib and anacetrapib, dalcetrapib requires cysteine 13 to decrease CETP activity, measured as transfer of cholesteryl ester (CE) from HDL to LDL, and does not affect transfer of CE from HDL3 to HDL2. Only dalcetrapib induced a conformational change in CETP, when added to human plasma in vitro, also observed in vivo and correlated with CETP activity. CETP-induced pre-β-HDL formation in vitro in human plasma was unchanged by dalcetrapib ≤3 µM and increased at 10 µM. A dose-dependent inhibition of pre-β-HDL formation by torcetrapib and anacetrapib (0.1 to 10 µM) suggested that dalcetrapib modulates CETP activity. In hamsters injected with [³H]cholesterol-labeled autologous macrophages, and given dalcetrapib (100 mg twice daily), torcetrapib [30 mg once daily (QD)], or anacetrapib (30 mg QD), only dalcetrapib significantly increased fecal elimination of both [³H]neutral sterols and [³H]bile acids, whereas all compounds increased plasma HDL-[³H]cholesterol. These data suggest that modulation of CETP activity by dalcetrapib does not inhibit CETP-induced pre-β-HDL formation, which may be required to increase reverse cholesterol transport.     

Leukocyte-derived hepatic lipase increases HDL and decreases en face aortic atherosclerosis in LDLr-/- mice expressing CETP

In addition to hepatic expression, cholesteryl ester transfer protein (CETP) and hepatic lipase (HL) are expressed by human macrophages. The combined actions of these proteins have profound effects on HDL structure and function. It is not known how these HDL changes influence atherosclerosis. To elucidate the role of leukocyte-derived HL on atherosclerosis in a background of CETP expression, we studied low density lipoprotein receptor-deficient mice expressing human CETP (CETPtgLDLr(-/-)) with a leukocyte-derived HL deficiency (HL(-/-) BM). HL(-/-) bone marrow (BM), CETPtgLDLr(-/-) mice were generated via bone marrow transplantation. Wild-type bone marrow was transplanted into CETPtgLDLr(-/-) mice to generate HL(+/+) BM, CETPtgLDLr(-/-) controls. The chimeras were fed a high-fat, high-cholesterol diet for 14 weeks to promote atherosclerosis. In female HL(-/-) BM, CETPtgLDLr(-/-) mice plasma HDL-cholesterol concentration during high-fat feeding was decreased 27% when compared with HL(+/+) BM, CETPtgLDLr(-/-) mice (P < 0.05), and this was associated with a 96% increase in en face aortic atherosclerosis (P < 0.05). In male CETPtgLDLr(-/-) mice, leukocyte-derived HL deficiency was associated with a 16% decrease in plasma HDL-cholesterol concentration and a 25% increase in aortic atherosclerosis. Thus, leukocyte-derived HL in CETPtgLDLr(-/-) mice has an atheroprotective role that may involve increased HDL levels.     

Atherosclerosis is enhanced by testosterone deficiency and attenuated by CETP expression in transgenic mice

In this work, we investigated the impact of testosterone deficiency and cholesteryl ester transfer protein (CETP) expression on lipoprotein metabolism and diet-induced atherosclerosis. CETP transgenic mice and nontransgenic (nTg) littermates were studied 4 weeks after bilateral orchidectomy or sham operation. Castrated mice had an increase in the LDL fraction (+36% for CETP and +79% for nTg mice), whereas the HDL fraction was reduced (-30% for CETP and -11% for nTg mice). Castrated mice presented 1.7-fold higher titers of anti-oxidized LDL (Ox-LDL) antibodies than sham-operated controls. Plasma levels of CETP, lipoprotein lipase, and hepatic lipase were not changed by castration. Kinetic studies showed no differences in VLDL secretion rate, VLDL-LDL conversion rate, or number of LDL and HDL receptors. Competition experiments showed lower affinity of LDL from castrated mice for tissue receptors. Diet-induced atherosclerosis studies showed that testosterone deficiency increased by 100%, and CETP expression reduced by 44%, the size of aortic lesion area in castrated mice. In summary, testosterone deficiency increased plasma levels of apolipoprotein B-containing lipoproteins (apoB-LPs) and anti-OxLDL antibodies, decreased LDL receptor affinity, and doubled the size of diet-induced atherosclerotic lesions. The expression of CETP led to a milder increase of apoB-LPs and reduced atherosclerotic lesion size in testosterone-deficient mice.     

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.     

Anacetrapib reduces (V)LDL cholesterol by inhibition of CETP activity and reduction of plasma PCSK9

Recently, we showed in APOE*3-Leiden cholesteryl ester transfer protein (E3L.CETP) mice that anacetrapib attenuated atherosclerosis development by reducing (V)LDL cholesterol [(V)LDL-C] rather than by raising HDL cholesterol. Here, we investigated the mechanism by which anacetrapib reduces (V)LDL-C and whether this effect was dependent on the inhibition of CETP. E3L.CETP mice were fed a Western-type diet alone or supplemented with anacetrapib (30 mg/kg body weight per day). Microarray analyses of livers revealed downregulation of the cholesterol biosynthesis pathway (P < 0.001) and predicted downregulation of pathways controlled by sterol regulatory element-binding proteins 1 and 2 (z-scores −2.56 and −2.90, respectively; both P < 0.001). These data suggest increased supply of cholesterol to the liver. We found that hepatic proprotein convertase subtilisin/kexin type 9 (Pcsk9) expression was decreased (−28%, P < 0.01), accompanied by decreased plasma PCSK9 levels (−47%, P < 0.001) and increased hepatic LDL receptor (LDLr) content (+64%, P < 0.01). Consistent with this, anacetrapib increased the clearance and hepatic uptake (+25%, P < 0.001) of [¹⁴C]cholesteryl oleate-labeled VLDL-mimicking particles. In E3L mice that do not express CETP, anacetrapib still decreased (V)LDL-C and plasma PCSK9 levels, indicating that these effects were independent of CETP inhibition. We conclude that anacetrapib reduces (V)LDL-C by two mechanisms: 1) inhibition of CETP activity, resulting in remodeled VLDL particles that are more susceptible to hepatic uptake; and 2) a CETP-independent reduction of plasma PCSK9 levels that has the potential to increase LDLr-mediated hepatic remnant clearance.     

Scavenger receptor class B type I-mediated uptake of serum cholesterol is essential for optimal adrenal glucocorticoid production

Impaired scavenger receptor class B type I (SR-BI)-mediated uptake of HDL-cholesterol esters (HDL-CE) induces adrenal insufficiency in mice. Humans contain an alternative route of HDL-CE clearance, namely through the transfer by cholesteryl ester transfer protein (CETP) to apolipoprotein B lipoproteins for subsequent uptake via the LDL receptor. In this study, we determined whether CETP can compensate for loss of adrenal SR-BI. Transgenic expression of human CETP (CETP Tg) in SR-BI knockout (KO) mice increased adrenal HDL-CE clearance from 33–58% of the control value. SR-BI KO/CETP Tg and SR-BI KO mice displayed adrenal hypertrophy due to equally high plasma adrenocorticotropic hormone levels. Adrenal cholesterol levels and plasma corticosterone levels were 38–52% decreased in SR-BI KO mice with and without CETP expression. SR-BI KO/CETP Tg mice also failed to increase their corticosterone level after lipopolysaccharide challenge, leading to an identical >4-fold increased tumor necrosis factor-α response compared with controls. These data indicate that uptake of CE via other routes than SR-BI is not sufficient to generate the cholesterol pool needed for optimal adrenal steroidogenesis. In conclusion, we have shown that CETP-mediated transfer of HDL-CE is not able to reverse adrenal insufficiency in SR-BI knockout mice. Thus, SR-BI-mediated uptake of serum cholesterol is essential for optimal adrenal function.     

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.