Role of Lp A-I and Lp A-I/A-II in Cholesteryl Ester Transfer Protein–Mediated Neutral Lipid Transfer
Studies in Normal Subjects and in Hypertriglyceridemic Patients Before and After Fenofibrate Therapy
The two major subclasses of HDL contain apo A-I only (Lp A-I) or both apo A-I and apo A-II (Lp A-I/A-II). We have carried out experiments to quantify the participation of Lp A-I and Lp A-I/A-II in the neutral lipid transfer reaction in normal and hypertriglyceridemic subjects. Thirteen hypertriglyceridemic subjects were studied before and after fenofibrate therapy. Fenofibrate treatment resulted in decreases in total cholesterol, triglycerides (TG), and VLDL cholesterol of 19%, 48%, and 70%, respectively, and a 28% increase in HDL cholesterol, with no significant change in the proportion of Lp A-I and Lp A-I/A-II particles. The abundance of cholesteryl ester transfer protein (CETP) mRNA in peripheral adipose tissue decreased with treatment in four of five patients studied; however, no change occurred in plasma CETP mass. Using an isotopic transfer assay, we demonstrated that both Lp A-I and Lp A-I/A-II participated in the CE transfer reaction, with no change after fenofibrate therapy. This finding suggests that the marked increase in HDL cholesterol during fenofibrate therapy is due to normalization of plasma TG and hence decreased opportunity for mass transfer of lipid between HDL and TG-rich proteins in vivo. In this population of hypertriglyceridemic subjects, CETP was distributed in both the Lp A-I and Lp A-I/A-II subfractions of HDL, with preferential association with the smaller Lp A-I pool. In contrast, in nine normal subjects studied, negligible amounts of CETP were associated with Lp A-I/A-II. Nonetheless, the Lp A-I/A-II fraction of HDL contributed significantly to total CE mass transfer in normolipidemic plasma. Lp A-I/A-II is an efficient donor for CE transfer to TG-rich lipoproteins, and its low affinity for CETP may in fact facilitate neutral lipid transfer either by a shuttle mechanism or by formation of a ternary complex.
- Received May 24, 1995.
- Revision received April 5, 1996.
The two major subclasses of HDL are Lp A-I and Lp A-I/A-II. Lp A-I has been proposed to be the major protective HDL fraction in plasma on the basis of case-control studies of subjects with and without coronary heart disease1 and the observation that Lp A-I but not Lp A-I/A-II promotes cholesterol efflux in experiments using a mouse preadipocyte cell line.2 The catabolism of apo A-I on Lp A-I particles is more rapid than that of apo A-I on Lp A-I/A-II particles,3 suggesting that these two particles are metabolized differently.
CETP mediates the heteroexchange of neutral lipid between apo A-I– and apo B–containing lipoproteins, resulting in net transfer of CE to VLDL remnants and LDL. Cheung and colleagues4 have used immunoaffinity chromatography to demonstrate that in normolipidemic subjects, CETP associates with the Lp A-I fraction of HDL rather than Lp A-I/A-II. In previous studies, we demonstrated that probucol significantly increases plasma levels of CETP and that this increase in CETP mass and activity is associated with a selective decrease in Lp A-I. We have hypothesized that CETP may preferentially promote CE transfer from Lp A-I particles.5
Fenofibrate is a fibric acid derivative that has been shown to increase plasma concentrations of HDL-C. In this study, the effects of fenofibrate therapy on plasma lipoproteins, adipose tissue CETP mRNA levels, plasma CETP mass, and in vitro isotopic transfer activity to Lp A-I and Lp A-I/A-II particles in hypertriglyceridemic subjects have been examined. We found no change in the plasma level of CETP or CE transfer activity with treatment. In further studies in normolipidemic subjects, we determined the ability of Lp A-I and Lp A-I/A-II to transfer CE to VLDL in a CE mass transfer assay. The results demonstrate that CETP distribution in Lp A-I and Lp A-I/A-II differs in normal and hypertriglyceridemic subjects and that both Lp A-I and Lp A-I/A-II participate in the CETP-mediated lipid transfer reaction.
Study Population and Study Design
The study was approved by the Institutional Review Board of the Ottawa Civic Hospital. Informed, written consent was obtained from all subjects.
Hypertriglyceridemic Subjects Before and After Fenofibrate Therapy
Thirteen hypertriglyceridemic patients (Table 1⇓) observed in the lipid clinic at the University of Ottawa Heart Institute were studied after 6 weeks of dietary stabilization on an American Heart Association Step I, alcohol-free, weight-maintenance diet (control phase) and after 12 weeks of treatment with fenofibrate (300 mg/d in divided doses with meals) while maintaining dietary stabilization.
EDTA plasma was obtained from nine healthy normolipidemic fasting subjects maintained on an American Heart Association Step I diet (30% calories as total fat, with <10% as saturated fat and <300 mg cholesterol per day) (Table 2⇓).
Plasma Lipoproteins, Apolipoproteins, and CETP
Plasma lipoproteins were determined after ultracentrifugation of fresh plasma at d=1.006 to remove VLDL. Apo B–containing lipoproteins were precipitated using high-molecular-weight dextran sulphate/magnesium chloride,6 and lipoprotein lipids were determined using a Technicon RA-1000 autoanalyzer. The lipid clinic laboratory is standardized with the Centers for Disease Control Lipid Standardization Program (CDC, Atlanta, Ga). On fresh plasma, apo A-I, apo B, and Lp A-I were determined by using Sebia gels (Sebia, Inc); Lp A-I/A-II was measured by differential immunoassay.7 CETP mass was measured in fresh plasma, and all CETP samples were remeasured in a single run using in-plasma aliquots stored at −70°C until assay by solid-phase radioimmunoassay with the monoclonal antibody TP2 as described previously.8
Preparation of Lp A-I
The immunoaffinity column was prepared as described9 using anti–apo A-II monoclonal antibodies prepared by SERLIA (Institut Pasteur, Lille, France). HDL was prepared by ultracentrifugation at d=1.063. Since ultracentrifugation liberates the majority of HDL-associated CETP, the d>1.063 fraction was then incubated at 37°C for 20 minutes with gentle mixing. The effect of this procedure on CETP reassociation with HDL was determined by gel filtration with Superose 6 and 12 columns in tandem. For studies in hypertriglyceridemic subjects before and after fenofibrate therapy, Lp A-I was obtained by passing the d>1.063 fraction on the anti–A-II immunoaffinity column. For studies with the plasma of normolipidemic subjects, immunoaffinity columns were prepared with either anti–apo A-II or irrelevant antibody, anti-ANF:2H2 (control). HDL (d>1.063) from each normolipidemic subject was isolated by ultracentrifugation, and aliquots were passed on each of the immunoaffinity columns to obtain fractions consisting of control HDL passed on an irrelevant immunoaffinity column and the Lp A-I subfraction of HDL plus other d>1.063 proteins not associated with apo A-II. Absence of apo A-II after passage on the anti–apo A-II column was verified by a Western blot of the Lp A-I fraction with anti–apo A-II.
CE Transfer Activity in Hypertriglyceridemic Plasma Before and After Fenofibrate Therapy
CE transfer assays were carried out in 9 randomly chosen hypertriglyceridemic subjects before and after fenofibrate therapy. Since the transfer studies required passage of fresh plasma on an immunoaffinity column, only 3 subjects could be studied at each visit. Subjects were seen for each study visit on three separate days in groups of 4 to 5. This procedure permitted transfer studies to be done for only 9 of the 13 study subjects. A constant amount of LDL labeled with [3H]CE10 was used as a donor particle, and intact HDL (d>1.063) or Lp A-I derived from 1.0 mL of the subjects' plasma was used as the recipient particle. Since the study lasted for 12 weeks, it was necessary to perform transfer activity assays separately for control and treatment conditions. The same donor for LDL preparation was used for studies carried out on different days, and the same normal controls were included to verify that similar values were measured. The coefficient of variation (CV) for the assay was 15%. All assays were carried out in duplicate. Briefly, [3H]CE-LDL and the patients' HDL or Lp A-I were incubated for 6 hours at 37°C. LDL was precipitated using heparin/Mn, and transfer activity was calculated as the percent of [3H]CE transferred from LDL to the HDL fraction per hour.11 For transfer assays using the d>1.063 or the Lp A-I subfraction, the amount of LDL was kept constant, since the study hypothesis was that Lp A-I/A-II did not participate in the CETP-mediated lipid transfer reaction.
Mass Transfer of CE From HDL Fractions to VLDL in Normolipidemic Plasma
Mass transfer of CE from each HDL fraction to a single control VLDL preparation (prepared from a normal donor and concentrated twofold: 0.6 mmol/L CE and 2.6 mmol/L TG) was determined as follows. For each subject, 500 μL of each concentrated sample of (1) native HDL (3.16±0.7 mmol/L CE), derived from 1.0 mL plasma, (2) control HDL (passed on irrelevant immunoaffinity column), and (3) the Lp A-I subfraction of HDL (1.4±0.4 mmol/L CE), also derived from 1.0 mL plasma, was individually mixed with 500 μL of the control VLDL. Samples were sealed under nitrogen and incubated at 37°C for 6.5 hours, with gentle mixing. The reaction was terminated by placing the sample on ice, and the changes in CE and TG content of VLDL and HDL fractions, reisolated by ultracentrifugation, were determined by chemical and enzymatic methods. Studies were carried out in duplicate (CV≤10%). Mass transfer of CE was calculated as nanomoles CE transferred from HDL to VLDL and expressed as (1) total CE transfer per hour, (2) CE transferred per micromole CE present in the donor particle, and (3) CE transferred per microgram CETP present for each of (1) native HDL, (2) control HDL, and (3) the Lp A-I subfraction of HDL. The contribution of Lp A-I/A-II to mass transfer was determined as the difference between CE mass transfer of each control HDL and the Lp A-I subfraction of the same HDL.
CETP mRNA Levels in Peripheral Adipose Tissue
CETP mRNA levels in peripheral adipose tissue were determined in five hypertriglyceridemic subjects who agreed to undergo liposuction biopsies of periumbilical subcutaneous adipose tissue just before the start of fenofibrate treatment and on the last day of the 12 weeks of therapy. Total cellular RNA was extracted from adipose tissue by using the acidic guanidinium thiocyanate extraction as described by Chomczynski and Sacchi.12 Diethylpyrocarbonate-treated water and RNase-free solutions and glassware were used throughout. The abundance of CETP mRNA was determined by a solution hybridization ribonuclease protection assay.5 Using conditions described previously,13 50 μg of test total RNA from human adipose tissue was hybridized to a human antisense RNA probe prepared from the human CETP cDNA (166-bp fragment, nucleotides 727-892; provided by Dr Alan Tall) subcloned into pBluescript KS+. After 16 to 18 hours of hybridization at 48°C, samples were digested by RNase T2 for 2 hours at 30°C, and [32P]RNA-RNA hybrids were analyzed on 5% polyacrylamide-urea sequencing gels. Protected fragments were visualized by autoradiography and quantitated by densitometry. RNA mass was determined by comparison with a standard curve of CETP mRNA hybridized simultaneously. For this purpose, sense strand RNA was synthesized by in vitro transcription and its mass quantitated precisely by standard methods using [3H]NTP incorporation.
Data were analyzed using the SAS statistical program (version 6.03).14 Before-and-after values for the fenofibrate-treated subjects were compared by using Student's paired t test. Pearson's correlation coefficients were used to describe relationships between mass transfer activity and specific CETP and lipid variables.
Subjects and Compliance
A total of 13 hypertriglyceridemic subjects (lipids described in Tables 1 and 2⇑⇑) (6 men, 7 women, aged 35 to 65 years) treated with fenofibrate completed the study. Two additional subjects withdrew from the study for personal reasons. Three patients reported minor gastrointestinal side effects. Compliance with fenofibrate treatment was >90% by pill count for all patients. Other medications were maintained at stable dosages throughout both parts of the study. All patients maintained body weight within 1 kg, and dietary compliance, as determined at three dietitian visits, was good. Normolipidemic subjects studied (4 women, 5 men, aged 25 to 45) had HDL-C concentrations of 1.48±0.36 mmol/L; Lp A-I, 42.2±11.1 mg/dL; Lp A-I/A-II, 133.3±15.4 mg/dL; and plasma CETP, 1.84±0.28 μg/mL (mean±SD).
Plasma Lipoprotein Changes in Response to Fenofibrate
Plasma lipoproteins at baseline and after fenofibrate therapy are shown in Table 3⇓. Plasma cholesterol decreased by 19%, TG by 48%, and VLDL-C by 70%, resulting in a marked improvement in the VLDL-C:TG ratio (−61%). Marked improvements also occurred in LDL-C (−29%) and in apo B (−11%).
HDL-C increased by 28%, but there were no significant changes in plasma apo A-I, Lp A-I, or Lp A-I/A-II, demonstrating that fenofibrate therapy alters the cholesterol but not the protein composition of HDL (Table 3⇑).
Effects of Ultracentrifugation and Reequilibration of d=1.063 Infranatant on Lipoprotein Association of CETP
CETP is loosely associated with HDL, and the majority is liberated from HDL during ultracentrifugation. To reequilibrate the CETP in the d>1.063 pellet with HDL, the entire d>1.063 fraction was incubated at 37°C for 20 minutes, with gentle mixing. As shown in Fig 1⇓, the plasma lipoprotein distribution of CETP following this procedure was identical to that of fresh plasma.
Effects of Fenofibrate on Plasma CETP Mass and on CE Transfer Activity In Vitro
In the population of hyperlipidemic subjects, CETP was associated with both retained (Lp A-I/A-II; 31% of total CETP) and unretained (Lp A-I and free CETP; 69% of CETP) fractions by anti–apo A-II immunoaffinity chromatography. Fenofibrate did not alter plasma CETP mass or CETP association with Lp A-I/A-II. The effects of fenofibrate therapy on the ability of either Lp A-I or Lp A-I/A-II to serve as a recipient particle for CE transferred from LDL in vitro was not significant, and there was no significant decrease in the percentage of LDL-CE transferred to Lp A-I before and after fenofibrate treatment (Table 4⇓). The HDL-CE:LDL-CE ratios in different experiments varied between 0.07 and 0.20, and isotopic transfer activity has been shown to be linear over this range.15 When in vitro isotopic transfer of labeled CE from LDL to each of Lp A-I and Lp A-I/A-II was studied, the results demonstrated that either fraction could participate in the neutral lipid transfer reaction (Table 4⇓).
Distribution of CETP in Plasma Fractions of Normolipidemic Subjects
The lipoprotein distribution of CETP in normolipidemic fasting plasma is shown in Table 5⇓ and confirms that CETP is found in the d>1.063 fraction after ultracentrifugation. Negligible loss of CETP appeared to result from passage of the d>1.063 plasma fraction on a control (anti-ANF) immunoaffinity column. In contrast to the studies with hypertriglyceridemic subjects, the vast majority of plasma CETP eluted with Lp A-I after passage of HDL on an anti–apo A-II immunoaffinity column, suggesting that CETP has a low affinity for apo A-II–containing particles in normolipidemic plasma. No significant correlations were found between plasma concentrations of CETP and either HDL-C, Lp A-I, Lp A-I/A-II, or the ratio of Lp A-I to Lp A-I/A-II.
Mass Transfer of CE From HDL Fractions to VLDL in Normolipidemic Plasma
The above results obtained on HDL subfractions from hypertriglyceridemic patients suggested that both Lp A-I and Lp A-I/A-II could participate in the isotopic transfer of labeled CE in LDL to HDL. Since CETP was found to be associated with Lp A-I/A-II in hyperlipidemic plasma, whereas there is relatively little CETP directly associated with normolipidemic Lp A-I/A-II, we wished to determine whether or not the Lp A-I/A-II fraction of normal plasma accounted for a fraction of the total mass transfer of CE from HDL to apo B–containing lipoproteins. We first studied the determinants of mass transfer of neutral lipid from native HDL (d>1.063) to a control normotriglyceridemic VLDL. These studies demonstrated that, in normal plasma, the mass transfer of CE was dependent on the CETP concentration of the donor particle (r=.81) and on the CE content of the donor HDL (r=.85). When corrected for the CE content of the donor HDL, mass transfer remained significantly correlated with the CETP content of the donor HDL (r=.87).
The mass transfer of CE from HDL fractions to VLDL is illustrated in Table 6⇓. The total mass transfer of CE from HDL to VLDL was twice as great as the mass transfer from the Lp A-I fraction of the HDL to VLDL. These results demonstrate that Lp A-I is not the sole participant in the CETP-mediated neutral lipid transfer reaction and that Lp A-I/A-II must also contribute significantly to the pool of CE transferred from HDL to VLDL. The contribution of Lp A-I/A-II to mass transfer was determined by difference (Table 6⇓).
Effect of Fenofibrate on Adipose Tissue CETP mRNA Abundance
After 12 weeks of fenofibrate therapy, the levels of CETP mRNA in peripheral adipose tissue in five subjects studied decreased by 48%. This change was only marginally significant (P=.09) due to the small number of subjects studied (Fig 2⇓).
These studies demonstrate that fenofibrate treatment effectively lowers plasma total cholesterol and TG and markedly reduces the cholesterol:TG ratio in VLDL. These changes in apo B–containing lipoproteins are likely to be associated with reduced atherogenic potential.16 Therapy resulted in a 28% increase in HDL-C, with no significant change in the proportion of Lp A-I and Lp A-I/A-II particles. There was also no change in apo A-I concentration in plasma. These findings concur with those of Knopp et al17 and suggest that fenofibrate treatment modifies the cholesterol content but not the protein composition of HDL. In contrast, Fruchart and colleagues18 reported a 14% increase in plasma apo A-I and a smaller (5%) increase in apo A-II in response to fenofibrate treatment in dysbetalipoproteinemia. Thus, the effects of fenofibrate on HDL composition may vary in different lipoprotein phenotypes.
Peripheral adipose tissue is an important site of CETP synthesis in humans. We have demonstrated that the abundance of CETP mRNA in human adipose tissue is directly related to the size of the membrane cholesterol pool and that cholesterol loading of peripheral adipocytes increases CETP mRNA concentrations.19 Since fenofibrate reduces the plasma concentration of TG-rich lipoproteins, which are one vehicle for the delivery of CE to adipose tissue,15 the reduction in adipose CETP mRNA noted in four of five subjects studied may have been due to changes in an adipocyte pool of cholesterol that is regulatory for CETP gene expression. In other studies, we have demonstrated that there is a moderate correlation between human adipose tissue CETP mRNA and plasma levels of CETP (r=.64, P=.007) in 15 subjects. In this study, the change in adipose tissue CETP mRNA was not reflected in a change in plasma CETP. Liver is another important site of CETP synthesis, but we were not able to obtain and measure hepatic tissue CETP mRNA. Similarly, isotopic CE transfer activity measured in vitro by using labeled LDL-CE as a donor particle did not change after fenofibrate therapy. However, the percentage of CE transferred to Lp A-I/A-II versus Lp A-I increased and may represent fenofibrate-induced changes in the composition of these particles.
In previous studies, we have demonstrated that there is a strong correlation between plasma CETP mass and isotopic CE transfer activity in normal (r=.91) and hyperlipoproteinemic subjects (r=.72; P<.001).20 Neutral lipid transfer in vivo is a function of plasma CETP mass and the concentration and lipid and apolipoprotein composition of both apo A-I– and apo B–containing lipoproteins.21 This suggests that the marked increase in HDL-C after fenofibrate therapy may be due to normalization of plasma VLDL-TG concentrations and hence decreased mass transfer of lipid between apo A-I– and apo B–containing lipoproteins.22
Previous studies have confirmed that the major portion of plasma CETP is associated with HDL.4 8 Morton23 demonstrated that CETP binds with similar affinity to VLDL, LDL, and HDL, but the higher molar concentration of HDL can explain the predominant distribution of CETP with these particles. Francone and colleagues24 found that CETP was associated with apo A-I particles of a broad size range, but more than a third of CETP signal was present in pre-β3 particles. The CETP/lipoprotein association is, however, dependent on weak hydrophobic binding between epitopes on the carboxyl end of CETP and each of neutral lipid and phospholipid.25 26 Free CETP or lipid-associated CETP diffuses readily in an aqueous medium and has affinity for all lipoproteins, enabling participation of diverse lipid-carrying particles in the neutral lipid transfer reaction.
It is also apparent that CETP has a greater avidity for Lp A-I than Lp A-I/A-II particles in normal subjects. Our present results, obtained by using immunoaffinity chromatography, are in accord with those of Cheung and colleagues4 and show that in normal subjects, the vast majority of CETP in HDL is not associated with apo A-II–containing particles. In contrast, the present studies have demonstrated that in hypertriglyceridemic subjects, a third of plasma CETP is found in the Lp A-I/A-II fraction of HDL. Similarly, Moulin et al27 recently reported that a significant amount of CETP is retained with Lp A-I/A-II by immunoaffinity chromatography in hyperlipidemic but not normolipidemic plasma.
Our original hypothesis was that the Lp A-I but not the Lp A-I/A-II fraction of HDL participates in the CETP-mediated neutral lipid transfer reaction with apo B lipoproteins. Thus, we anticipated that the removal of Lp A-I/A-II from HDL by immunoaffinity chromatography would not alter mass transfer or transfer activity. Using these conditions, whether the studies were carried out with Lp A-I or the entire HDL fraction, the donor:acceptor ratio for each subject would have been constant if only the Lp A-I fraction participated in the lipid transfer reaction. Our results demonstrate that this is not the case, since total CE transfer from LDL to HDL was greater when the entire HDL fraction was used as opposed to the Lp A-I subfraction only. Thus, we can conclude that, contrary to our original hypothesis, both Lp A-I and Lp A-I/A-II participate as recipient particles for isotopic CE transfer from LDL. Rye and colleagues28 have shown that incorporation of apo A-II into recombinant Lp A-I particles reduces the ability of exogenous CETP to convert recombinant HDL into smaller but not larger particles. This observation is compatible with the hypothesis that apo A-II stabilizes the HDL particle but does not inhibit interaction with CETP. Similarly, in studies using normolipidemic plasma, we have demonstrated that Lp A-I/A-II accounts for a significant amount of CE transferred to a control VLDL in a mass transfer assay.
Although the majority of CETP in normal plasma is associated with Lp A-I, Lp A-I/A-II clearly participates to a similar extent in the CETP-mediated neutral lipid transfer reaction, suggesting that CETP dissociates readily from its major carrier lipoprotein, Lp A-I. We have recently carried out CETP kinetic studies in the rabbit and have demonstrated that there is extremely rapid equilibrium between the loosely HDL-associated and non–lipoprotein-associated pools of CETP in plasma.29 Studies by Ohnishi et al30 and Epps and colleagues31 demonstrate that there is rapid equilibration (<3 hours) of CE and TG between lipid microemulsions in the presence of CETP. Their data suggest that CETP interacts with lipoproteins via a dissociation/diffusion/adsorption/exchange mechanism and that the association/dissociation of CETP with Lp A-I or Lp A-I/A-II and other lipoproteins is rapid. Although a minority of plasma CETP is associated with normolipidemic Lp A-I/A-II, this particle remains an efficient donor for CE transfer to TG-rich lipoproteins, perhaps because its low affinity for CETP enhances the transfer rate either by a shuttle mechanism or by formation of a ternary complex (Fig 3⇓).
Although these studies demonstrate clearly that both Lp A-I and Lp A-I/A-II participate in neutral lipid transfer, further investigation with reconstituted systems will be needed to understand the interactions of phospholipid transfer protein and CETP-inhibitor protein with CETP in the transfer of neutral lipids from these two classes of lipoprotein.
Selected Abbreviations and Acronyms
|CETP||=||CE transfer protein|
|HDL-C, LDL-C, and VLDL-C||=||HDL, LDL, and VLDL cholesterol, respectively|
|Lp A-I||=||HDL containing apo A-I only|
|Lp A-I/-II||=||HDL containing apo A-I and A-II|
This study was supported by the Laboratoires Fournier, the Heart & Stroke Foundation of Canada (A2394), and the Medical Research Council of Canada (PG27). Human CETP cDNA (166-bp fragment, nucleotides 727-892) was generously provided by Dr Alan Tall.
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