Regulation of reconstituted high density lipoprotein structure and remodeling by apolipoprotein E

Apolipoprotein E (apoE) enters the plasma as a component of discoidal HDL and is subsequently incorporated into spherical HDL, most of which contain apoE as the sole apolipoprotein. This study investigates the regulation, origins, and structure of spherical, apoE-containing HDLs and their remodeling by cholesteryl ester transfer protein (CETP). When the ability of discoidal reconstituted high density lipoprotein (rHDL) containing apoE2 [(E2)rHDL], apoE3 [(E3)rHDL], or apoE4 [(E4)rHDL] as the sole apolipoprotein to act as substrates for LCAT were compared with that of discoidal rHDL containing apoA-I [(A-I)rHDL], the rate of cholesterol esterification was (A-I)rHDL > (E2)rHDL approximately (E3)rHDL > (E4)rHDL. LCAT also had a higher affinity for discoidal (A-I)rHDL than for the apoE-containing rHDL. When the discoidal rHDLs were incubated with LCAT and LDL, the resulting spherical (E2)rHDL, (E3)rHDL, and (E4)rHDL were larger than, and structurally distinct from, spherical (A-I)rHDL. Incubation of the apoE-containing spherical rHDL with CETP and Intralipid(R) generated large fusion products without the dissociation of apoE, whereas the spherical (A-I)rHDLs were remodeled into small particles with the formation of lipid-poor apoA-I. In conclusion, i) apoE activates LCAT less efficiently than apoA-I; ii) apoE-containing spherical rHDLs are structurally distinct from spherical (A-I)rHDL; and iii) the CETP-mediated remodeling of apoE-containing spherical rHDL differs from that of spherical (A-I)rHDL.     

Synthesis of reconstituted high density lipoprotein (rHDL) containing apoA-I and apoC-III: the functional role of apoC-III in rHDL

Apolipoprotein (apo) C-III is a marker protein of triacylglycerol (TG)-rich lipoproteins and high-density lipoproteins (HDL), and has been proposed as a risk factor of coronary heart disease. To compare the physiologic role of reconstituted HDL (rHDL) with or without apoC-III, we synthesized rHDL with molar ratios of apoA-I:apoC-III of 1:0, 1:0.5, 1:1, and 1:2. Increasing the apoC-III content in rHDL produced smaller rHDL particles with a lower number of apoA-I molecules. Furthermore, increasing the molar ratio of apoC-III in rHDL enhanced the surfactant-like properties and the ability to lyse dimyristoyl phosphatidylcholine. Furthermore, rHDL containing apoC-III was found to be more resistant to particle rearrangement in the presence of low-density lipoprotein (LDL) than rHDL that contained apoA-I alone. In addition, the lecithin:cholesterol acyltransferase (LCAT) activation ability was reduced as the apoC-III content of the rHDL increased; however, the CE transfer ability was not decreased by the increase of apoC-III. Finally, rHDL containing apoC-III aggravated the production of MDA in cell culture media, which led to increased cellular uptake of LDL. Thus, the addition of apoC-III to rHDL induced changes in the structural and functional properties of the rHDL, especially in particle size and rearrangement and LCAT activation. These alterations may lead to beneficial functions of HDL, which is involved in anti-atherogenic properties in the circulation.     

Adipose tissue-specific CETP expression in mice: impact on plasma lipoprotein metabolism

Adipose tissue appears to be a highly conserved site of cholesteryl ester transfer protein (CETP) expression across species. To investigate the impact of adipose CETP expression on lipid metabolism, we created adipose tissue-specific CETP transgenic (CETPTg) mice. CETP mRNA is predominantly expressed in adipose tissue. Plasma CETP mass and activity are readily detectable in CETPTg mice but not in controls. Plasma lipoprotein analysis shows marked reductions in HDL cholesterol and phospholipids, increases non-HDL lipids, decreases apolipoprotein A-I (apoA-I), and increases apoB. Unexpectedly, CETPTg adipocytes are significantly smaller than those in control mice (44%), triglyceride and cholesterol in adipose tissue were significantly decreased compared with controls (50% and 37%, respectively), and phospholipids showed no significant changes. To study the mechanism, we measured peroxisome proliferator-activated receptor gamma, sterol-regulatory element binding protein-1c, LPL, and hormone-sensitive lipase (HSL) in aP2-CETPTg adipose tissue and controls and found that, except for HSL, all mRNA levels are significantly decreased in the transgenic mice compared with controls (26, 33, and 22%). In conclusion, adipose tissue CETP makes a major contribution to CETP in the circulation, reduces HDL, and increases non-HDL cholesterol levels. Moreover, adipose tissue CETP expression changes triglyceride and cholesterol content and the size of adipocytes.     

Effects of CETP inhibition on triglyceride-rich lipoprotein composition and apoB-48 metabolism

Cholesteryl ester transfer protein (CETP) facilitates the transfer of HDL cholesteryl ester to triglyceride-rich lipoproteins (TRL). This study aimed to determine the effects of CETP inhibition with torcetrapib on TRL composition and apoB-48 metabolism. Study subjects with low HDL cholesterol (<40 mg/dl), either untreated (n = 9) or receiving atorvastatin 20 mg daily (n = 9), received placebo for 4 weeks, followed by torcetrapib 120 mg once daily for the next 4 weeks. A subset of the subjects not treated with atorvastatin participated in a third phase (n = 6), in which they received torcetrapib 120 mg twice daily for an additional 4 weeks. At the end of each phase, all subjects received a primed-constant infusion of [5,5,5-(2)H(3)]L-leucine, while in the constantly fed state, to determine the kinetics of TRL apoB-48 and TRL composition. Relative to placebo, torcetrapib markedly reduced TRL CE levels in all groups (≥-69%; P < 0.005). ApoB-48 pool size (PS) and production rate (PR) decreased in the nonatorvastatin once daily (PS: -49%, P = 0.007; PR: -49%, P = 0.005) and twice daily (PS: -30%, P = 0.01; PR: -27%, P = 0.13) cohorts. In the atorvastatin cohort, apoB-48 PS and PR, which were already lowered by atorvastatin, did not change with torcetrapib. Our findings indicate that CETP inhibition reduced plasma apoB-48 concentrations by reducing apoB-48 production but did not have this effect in subjects already treated with atorvastatin.     

Atherogenic, enlarged, and dysfunctional HDL in human PLTP/apoA-I double transgenic mice

In low density lipoprotein receptor (LDLR)-deficient mice, overexpression of human plasma phospholipid transfer protein (PLTP) results in increased atherosclerosis. PLTP strongly decreases HDL levels and might alter the antiatherogenic properties of HDL particles. To study the potential interaction between human PLTP and apolipoprotein A-I (apoA-I), double transgenic animals (hPLTPtg/hApoAItg) were compared with hApoAItg mice. PLTP activity was increased 4.5-fold. Plasma total cholesterol and phospholipid were decreased. Average HDL size (analyzed by gel filtration) increased strongly, hPLTPtg/hApoAItg mice having very large, LDL-sized, HDL particles. Also, after density gradient ultracentrifugation, a substantial part of the apoA-I-containing lipoproteins in hPLTPtg/hApoAItg mice was found in the LDL density range. In cholesterol efflux studies from macrophages, HDL isolated from hPLTPtg/hApoAItg mice was less efficient than HDL isolated from hApoAItg mice. Furthermore, it was found that the largest subfraction of the HDL particles present in hPLTPtg/hApoAItg mice was markedly inferior as a cholesterol acceptor, as no labeled cholesterol was transferred to this fraction. In an LDLR-deficient background, the human PLTP-expressing mouse line showed a 2.2-fold increased atherosclerotic lesion area. These data demonstrate that the action of human PLTP in the presence of human apoA-I results in the formation of a dysfunctional HDL subfraction, which is less efficient in the uptake of cholesterol from cholesterol-laden macrophages.     

Obesity and altered glucose metabolism impact HDL composition in CETP transgenic mice: a role for ovarian hormones

Mechanisms underlying changes in HDL composition caused by obesity are poorly defined, partly because mice lack expression of cholesteryl ester transfer protein (CETP), which shuttles triglyceride and cholesteryl ester between lipoproteins. Because menopause is associated with weight gain, altered glucose metabolism, and changes in HDL, we tested the effect of feeding a high-fat diet (HFD) and ovariectomy (OVX) on glucose metabolism and HDL composition in CETP transgenic mice. After OVX, female CETP-expressing mice had accelerated weight gain with HFD-feeding and impaired glucose tolerance by hyperglycemic clamp techniques, compared with OVX mice fed a low-fat diet (LFD). Sham-operated mice (SHAM) did not show HFD-induced weight gain and had less glucose intolerance than OVX mice. Using shotgun HDL proteomics, HFD-feeding in OVX mice had a large effect on HDL composition, including increased levels of apoA2, apoA4, apoC2, and apoC3, proteins involved in TG metabolism. These changes were associated with decreased hepatic expression of SR-B1, ABCA1, and LDL receptor, proteins involved in modulating the lipid content of HDL. In SHAM mice, there were minimal changes in HDL composition with HFD feeding. These studies suggest that the absence of ovarian hormones negatively influences the response to high-fat feeding in terms of glucose tolerance and HDL composition. CETP-expressing mice may represent a useful model to define how metabolic changes affect HDL composition and function.     

Changes in lipoprotein subfraction concentration and composition in healthy individuals treated with the CETP inhibitor anacetrapib

We investigated the effects of the cholesteryl ester (CE) transfer protein inhibitor anacetrapib (ANA) on plasma lipids, lipoprotein subfraction concentrations, and lipoprotein composition in 30 healthy individuals. Participants (n = 30) were randomized to ANA 20 mg/day, 150 mg/day, or placebo for 2 weeks. Changes in concentration of lipoprotein subfractions were assessed using ion mobility, and compositional analyses were performed on fractions separated by density gradient ultracentrifugation. ANA 150 mg/day versus placebo resulted in significant decreases in LDL-cholesterol (26%) and apo B (29%) and increases in HDL-cholesterol (82%). Concentrations of medium and small VLDL, large intermediate density lipoprotein (IDL), and medium and small LDL (LDL2a, 2b, and 3a) decreased whereas levels of very small and dense LDL4b were increased. There was enrichment of triglycerides and reduction of CE in VLDL, IDL, and the densest LDL fraction. Levels of large buoyant HDL particles were substantially increased, and there was enrichment of CE, apo AI, and apoCIII, but not apoAII or apoE, in the mid-HDL density range. Changes in lipoprotein subfraction concentrations and composition with ANA 20 mg/day were similar to those for ANA 150 mg/day but were generally smaller in magnitude. The impact of these changes on cardiovascular risk remains to be determined.     

Activation of the constitutive androstane receptor decreases HDL in wild-type and human apoA-I transgenic mice

The nuclear hormone receptor constitutive androstane receptor (CAR, NR1I3) regulates detoxification of xenobiotics and endogenous molecules, and has been shown to be involved in the metabolism of hepatic bile acids and cholesterol. The goal of this study was to address potential effects of CAR on the metabolism of HDL particles, key components in the reverse transport of cholesterol to the liver. Wild-type (WT) mice, transgenic mice expressing human apolipoprotein A-I (HuAITg), and CAR-deficient (CAR(-/-)) mice were treated with the specific CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). CAR activation decreased HDL cholesterol and plasma apolipoprotein A-I (apoA-I) levels in both WT and HuAITg mice, but not CAR(-/-) mice. Both mouse apoA-I and human apoA-I were decreased by more than 40% after TCPOBOP treatment, and kinetic studies revealed that the production rate of HDL is reduced in TCPOBOP-treated WT mice. In transient transfections, TCPOBOP-activated CAR decreased the activity of the human apoA-I promoter. Although loss of CAR function did not alter HDL levels in normal chow-fed mice, HDL cholesterol, apoA-I concentration, and apoA-I mRNA levels were increased in CAR(-/-) mice relative to WT mice when both were fed a high-fat diet. We conclude that CAR activation in mice induces a pronounced decrease in circulating levels of plasma HDL, at least in part through downregulation of apoA-I gene expression.     

Bone marrow-derived HL mitigates bone marrow-derived CETP-mediated decreases in HDL in mice globally deficient in HL and the LDLr

The objective of this study was to determine the combined effects of HL and cholesteryl ester transfer protein (CETP), derived exclusively from bone marrow (BM), on plasma lipids and atherosclerosis in high-fat-fed, atherosclerosis-prone mice. We transferred BM expressing these proteins into male and female double-knockout HL-deficient, LDL receptor-deficient mice (HL^−/−LDLr^−/−). Four BM chimeras were generated, where BM-derived cells expressed 1) HL but not CETP, 2) CETP and HL, 3) CETP but not HL, or 4) neither CETP nor HL. After high-fat feeding, plasma HDL-cholesterol (HDL-C) was decreased in mice with BM expressing CETP but not HL (17 ± 4 and 19 ± 3 mg/dl, female and male mice, respectively) compared with mice with BM expressing neither CETP nor HL (87 ± 3 and 95 ± 4 mg/dl, female and male mice, respectively, P < 0.001 for both sexes). In female mice, the presence of BM-derived HL mitigated this CETP-mediated decrease in HDL-C. BM-derived CETP decreased the cholesterol component of HDL particles and increased plasma cholesterol. BM-derived HL mitigated these effects of CETP. Atherosclerosis was not significantly different between BM chimeras. These results suggest that BM-derived HL mitigates the HDL-lowering, HDL-modulating, and cholesterol-raising effects of BM-derived CETP and warrant further studies to characterize the functional properties of these protein interactions.     

CETP genotype and changes in lipid levels in response to weight-loss diet intervention in the POUNDS LOST and DIRECT randomized trials

Little is known about whether cholesteryl ester transfer protein (CETP) genetic variation may modify the effect of weight-loss diets varying in fat content on changes in lipid levels. We analyzed the interaction between the CETP variant rs3764261 and dietary interventions on changes in lipid levels among 732 overweight/obese adults from a 2 year randomized weight-loss trial [Preventing Overweight Using Novel Dietary Strategies (POUNDS LOST)], and replicated the findings in 171 overweight/obese adults from an independent 2 year weight-loss trial [Dietary Intervention Randomized Controlled Trial (DIRECT)]. In the POUNDS LOST, participants with the CETP rs3764261 CC genotype on the high-fat diet had larger increases in HDL cholesterol (P = 0.001) and decreases in triglycerides (P = 0.007) than those on the low-fat diet at 6 months, while no significant difference between these two diets was observed among participants carrying other genotypes. The gene-diet interactions on changes in HDL-cholesterol and tri­glyc­erides were replicated in the DIRECT (pooled P for interaction ≤ 0.01). Similar results on trajectory of changes in HDL cholesterol and triglycerides over the 2 year intervention were observed in both trials. Our study provides replicable evidence that individuals with the CETP rs3764261 CC genotype might derive greater effects on raising HDL cholesterol and lowering triglycerides by choosing a low-carbohydrate/high-fat weight-loss diet instead of a low-fat diet.     

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.     

High level expression of human apolipoprotein A-I in transgenic rats raises total serum high density lipoprotein cholesterol and lowers rat apolipoprotein A-I

To examine the consequences of increased apolipoprotein A-I production on cholesterol and lipoprotein metabolism, we have produced two lines of transgenic rats; one expressing moderate and one very high levels of human apolipoprotein A-I. The rats were produced by microinjection of a 13 kbp DNA fragment containing the human apolipoprotein A-I gene plus 10 kbp of its 5′ flanking sequence and 1 kbp of its 3′ flanking sequence. Both lines of transgenic rats express human apolipoprotein A-I mRNA in liver and human apolipoprotein A-I in plasma. Sera from these rats contain significantly higher levels of total apolipoprotein A-I, high density lipoprotein cholesterol and phospholipid than sera from non-transgenic littermates. Transgenic rats expressing high levels of human apolipoprotein A-I have reduced levels of serum rat apolipoprotein A-I suggesting a mechanism exists to down-regulate apolipoprotein A-I production. These transgenic rats provide a unique animal model to examine the effects of increased apolipoprotein A-I production on lipid and lipoprotein metabolism.     

Hypertriglyceridemia is associated with prebeta-HDL concentrations in subjects with familial low HDL

Prebeta-HDL particles act as the primary acceptors of cellular cholesterol in reverse cholesterol transport (RCT). An impairment of RCT may be the reason for the increased risk of coronary heart disease (CHD) in subjects with familial low HDL. We studied the levels of serum prebeta-HDL and the major regulating factors of HDL metabolism in 67 subjects with familial low HDL and in 64 normolipidemic subjects. We report that the subjects with familial low HDL had markedly reduced prebeta-HDL concentrations compared with the normolipidemic subjects (17.4 +/- 7.2 vs. 23.4 +/- 7.8 mg apolipoprotein A-I/dl; P < 0.001). A positive correlation was observed between prebeta-HDL concentration and serum triglyceride (TG) level (r = 0.334, P = 0.006). In addition, serum TG level was found to be the strongest predictor of prebeta-HDL concentration in subjects with familial low HDL. The activities of cholesteryl ester transfer protein and hepatic lipase were markedly increased in subjects with familial low HDL without a significant correlation to prebeta-HDL concentration. Our results support the hypothesis that impaired RCT is one mechanism behind the increased risk for CHD in subjects with familial low HDL.     

Inhibition of CETP activity by torcetrapib reduces susceptibility to diet-induced atherosclerosis in New Zealand White rabbits

Cholesteryl ester transfer protein (CETP) inhibitors increase high density lipoprotein-cholesterol (HDL-C) in animals and humans, but whether CETP inhibition will be antiatherogenic is still uncertain. We tested the CETP inhibitor torcetrapib in rabbits fed an atherogenic diet at a dose sufficient to increase HDL-C by at least 3-fold (207 +/- 32 vs. 57 +/- 6 mg/dl in controls at 16 weeks). CETP activity was inhibited by 70-80% throughout the study. Non-HDL-C increased in both groups, but there was no difference apparent by the study's end. At 16 weeks, aortic atherosclerosis was 60% lower in torcetrapib-treated animals (16.4 +/- 3.4% vs. 39.8 +/- 5.4% in controls) and aortic cholesterol content was reduced proportionally. Sera from a separate group of rabbits administered torcetrapib effluxed 48% more cholesterol from Fu5AH cells than did sera from control animals, possibly explaining the reduced aortic cholesterol content. Regression analyses indicated that lesion area in the torcetrapib-treated group was strongly correlated with the ratio of total plasma cholesterol to HDL-C but not with changes in other lipid or lipoprotein levels. CETP inhibition with torcetrapib retards atherosclerosis in rabbits, and the reduced lesion area is associated with increased levels of HDL-C.     

Antisense oligonucleotide inhibition of cholesteryl ester transfer protein enhances RCT in hyperlipidemic,CETP transgenic,LDLr-/- mice

Due to their ability to promote positive effects across all of the lipoprotein classes, cholesteryl ester transfer protein (CETP) inhibitors are currently being developed as therapeutic agents for cardiovascular disease. In these studies, we compared an antisense oligonucleotide (ASO) inhibitor of CETP to the CETP small molecule inhibitor anacetrapib. In hyperlipidemic CETP transgenic (tg) mice, both drugs provided comparable reductions in total plasma cholesterol, decreases in CETP activity, and increases in HDL cholesterol. However, only mice treated with the antisense inhibitor showed an enhanced effect on macrophage reverse cholesterol transport, presumably due to differences in HDL apolipoprotein composition and decreases in plasma triglyceride. Additionally, the ASO-mediated reductions in CETP mRNA were associated with less accumulation of aortic cholesterol. These preliminary findings suggest that CETP ASOs may represent an alternative means to inhibit that target and to support their continued development as a treatment for cardiovascular disease in man.     

Mechanism of inhibition defines CETP activity: a mathematical model for CETP in vitro

Because cholesteryl ester transfer protein (CETP) inhibition is a potential HDL-raising therapy, interest has been raised in the mechanisms and consequences of CETP activity. To explore these mechanisms and the dynamics of CETP in vitro, a mechanistic mathematical model was developed based upon the shuttle mechanism for lipid transfer. Model parameters were estimated from eight published experimental datasets, and the resulting model captures observed dynamics of CETP in vitro. Simulations suggest the shuttle mechanism yields behaviors consistent with experimental observations. Three key findings predicted from model simulations are: 1) net CE transfer activity from HDL to VLDL and LDL can be significantly altered by changing the balance of homoexchange versus heteroexchange of neutral lipids via CETP; 2) lipemia-induced increases in CETP activity are more likely caused by increases in lipoprotein particle size than particle number; and 3) the inhibition mechanisms of the CETP inhibitors torcetrapib and JTT-705 are significantly more potent than a classic competitive inhibition mechanism with the irreversible binding mechanism having the most robust response. In summary, the model provides a plausible representation of CETP activity in vitro, corroborates strong evidence for the shuttle hypothesis, and provides new insights into the consequences of CETP activity and inhibition on lipoproteins.     

CETP expression reverses the reconstituted HDL-induced increase in VLDL

Human data suggest that reconstituted HDL (rHDL) infusion can induce atherosclerosis regression. Studies in mice indicated that rHDL infusion adversely affects VLDL levels, but this effect is less apparent in humans. This discrepancy may be explained by the fact that humans, in contrast to mice, express cholesteryl ester transfer protein (CETP). The aim of this study was to investigate the role of CETP in the effects of rHDL on VLDL metabolism by using APOE*3-Leiden (E3L) mice, a well-established model for human-like lipoprotein metabolism. At 1 h after injection, rHDL increased plasma VLDL-C and TG in E3L mice, but not in E3L mice cross-bred onto a human CETP background (E3L.CETP mice). This initial raise in VLDL, caused by competition between rHDL and VLDL for LPL-mediated TG hydrolysis, was thus prevented by CETP. At 24 h after injection, rHDL caused a second increase in VLDL-C and TG in E3L mice, whereas rHDL had even decreased VLDL in E3L.CETP mice. This secondary raise in VLDL was due to increased hepatic VLDL-TG production. Collectively, we conclude that CETP protects against the rHDL-induced increase in VLDL. We anticipate that studies evaluating the anti-atherosclerotic efficacy of rHDL in mice that are naturally deficient for CETP should be interpreted with caution, and that treatment of atherogenic dyslipidemia by rHDL should not be combined with agents that aggressively reduce CETP activity.     

Three-dimensional models of HDL apoA-I: implications for its assembly and function

The purpose of this review is to highlight recent advances toward the refinement of a three-dimensional structure for lipid-bound apolipoprotein A-I (apoA-I) on recombinant HDL. Recently, X-ray crystallography has yielded a new structure for full-length, lipid-free apoA-I. Although this approach has not yet been successful in solving the three-dimensional structure of lipid-bound apoA-I, analysis of the X-ray structures has been of immense help in the interpretation of structural data obtained from other methods that yield structural information. Recent studies emphasize the use of mass spectrometry to unambiguously identify cross-linked peptides or to quantify solvent accessibility using hydrogen-deuterium exchange. The combination of mass spectrometry, molecular modeling, molecular dynamic analysis, and small-angle X-ray diffraction has provided additional structural information on apoA-I folding that complements previous approaches.