Metabolism of Very-Low-Density Lipoprotein and Low-Density Lipoprotein Containing Apolipoprotein C-III and Not Other Small Apolipoproteins
Objective— We aimed to clarify the influence of apolipoprotein C-III (apoCIII) on human apolipoprotein B metabolism.
Methods and Results— We studied the kinetics of 4 very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL) types containing: (1) otherApos−CIII−: none of apoCIII, apoAII, apoCI, apoCII, or apoE; (2) otherApos+CIII−: no apoCIII but at least one of the others; (3) otherApos−CIII+: apoCIII, but not any others; and (4) otherApos+CIII+: apoCIII and at least one other. VLDL and IDL otherApos−CIII+ and otherApos−CIII− had similar rates of lipolytic conversion to smaller particles. However, light LDL otherApos−CIII+ compared with otherApos−CIII− had much faster conversion to dense LDL as did light LDL otherApos+CIII+ compared with otherApos+CIII−. VLDL and IDL otherApos−CIII+ had minimal direct removal from circulation, whereas VLDL and IDL otherApos+CIII−, rich in apoE, showed fast clearance. Lipoproteins in fraction otherApos+CIII+ also rich in apoE had very low clearance.
Conclusions— The results suggest that apoCIII strongly inhibits hepatic uptake of VLDL and IDL overriding the opposite influence of apoE when both are present. The presence of apoCIII on dense VLDL is not associated with slow conversion to IDL, a lipoprotein lipase-dependent process; but when on light LDL, apoCIII is associated with enhanced conversion to dense LDL, a process involving hepatic lipase.
ApoCIII is a small apolipoprotein, synthesized mainly in the liver, which circulates in plasma associated with apoB containing lipoproteins and high-density lipoprotein.1 In case–control studies with angiographic or clinical end points and prospective observational studies, plasma concentrations of lipoproteins with apoCIII are strong independent risk factors for cardiovascular disease.1 Humans with genetic deficiency of apoCIII have lower triglyceride (TG) and low-density lipoprotein (LDL) cholesterol levels and reduced atherosclerosis.2
The prevailing idea about the function of apoCIII is that it is an antagonist to apoCII and apoE, impairing intravascular lipolysis by lipoprotein lipase and liver clearance of apoB lipoproteins. This concept is supported by in vitro evidence showing that apoCIII noncompetitively inhibits lipoprotein lipase (LPL)3,4 and by the markedly accelerated catabolism of triglyceride-rich lipoproteins (TRL) in human subjects with a genetic deficiency of apoCIII.5 Also, apoCIII strongly inhibits the in vitro binding of apoB lipoproteins to the hepatic LDL receptor.6 Nevertheless, kinetic studies in humans do not support the idea of apoCIII as a LPL inhibitor in vivo, because very-low-density lipoprotein (VLDL) with apoCIII show faster, not slower, lipolytic conversion rates to smaller lipoproteins than particles without it.7
A great part of the complexity in elucidating the true effect of apoCIII in vivo stems from the fact that apoB lipoproteins with apoCIII also contain many molecules of other small apolipoproteins (ie, apoAII, apoCI, apoCII, apoE),7–9 each one having its own effects on lipolysis or receptor binding. Regarding lipolysis, human apoAII has been shown to directly inhibit LPL in mice10; and apoCI transgenic mice exhibit impaired LDL-dependent TRL lipolysis11; similarly, studies in rats have found that apoE inhibits LPL in vivo in a dose-dependent fashion.12 On the other hand, apoCII is a well-known cofactor of LPL.13 Concerning hepatic uptake, apoCI inhibits the binding of lipoproteins to the VLDL receptor,14 apoCII partially inhibits binding of apoB to the LDL receptor,15 and apoE is a major ligand for the LDL receptor16 promoting hepatic removal of lipoproteins.
To welcome apoCIII as a target for intervention in cardiovascular disease prevention, it is fundamental to understand its real impact on lipoprotein metabolism in vivo without the confounding introduced by other apolipoproteins. The way we approached this problem was by isolating the population of apoB lipoproteins with apoCIII and without any of the usual accompanying apolipoproteins and studying their metabolic behavior using kinetic modeling techniques. As a reference, we compared these results with those for apoB lipoproteins without any of the small apolipoproteins including apoCIII. We also studied the metabolism of apoCIII-containing lipoproteins in the context of other apolipoproteins, apoAII, CI, CII, and E.
We studied 7 men and 5 women with a mean (±SD) age of 50±11 years, body mass index of 28±4 kg/m2, fasting TG of 1.6±1.0 mmol/L, LDL cholesterol of 2.9±0.7 mmol/L, and high-density lipoprotein cholesterol of 1.2±0.3 mmol/L (Supplemental Table I, please see http://atvb.ahajournals.org). Exclusion criteria included secondary hyperlipidemia; APO E2/E2, E4/E4, and E2/E4 genotypes; and use of medications that affect lipid metabolism. The study was approved by the Human Subjects Committees at Harvard School of Public Health and Brigham and Women’s Hospital. All participants gave informed consent. Samples from the same participants in the same dietary and kinetic protocol, separated according to content of apoE and apoCIII, have been the subject of a previous publication.17
All study subjects underwent a diet rich in monounsaturated fat for 3 weeks before the infusion protocol. The diet was 37% fat (8% saturated, 24% monounsaturated fat, 5% polyunsaturated fat), 48% carbohydrate, and 15% protein; had 250 mg per day of cholesterol; and was provided to the participants as outpatients. Subjects were also asked not to consume alcohol. Energy intake was adjusted to keep body weight constant during the study period.
Study participants received a priming dose of 4.2 μmol/kg [D3]l-leucine (Tracer Technologies, Cambridge, Mass) followed by a constant infusion of [D3]l-leucine at 4.8 μmol/kg−1/h−1 for 14 hours. A bolus injection of 7.1 μmol/kg [D5]l-phenylalanine was administered at the same time, like in previous studies.7,17 Blood was collected every 30 minutes in the first 2 hours and hourly thereafter for a total of 14 hours. Small hourly meals with the same nutrient composition as the outpatient diet but low in leucine and phenylalanine were consumed beginning 3 hours before and continuing during the tracer infusion, as used previously.17
Separation of Lipoproteins
Plasma from each time point was incubated with a Sepharose 4B resin coupled to antiapoAII, antiapoCI, antiapoCII, and antiapoE immunoglobulins (Academy Biomedical, Houston, Texas). The unbound fraction (otherApos−) was collected by gravity flow and the bound fraction (otherApos+) was eluted with 3 mol/L NaSCN. Both bound and unbound fractions were then incubated with antiapoCIII immunoaffinity resin, and the bound and unbound fractions were collected using the same procedures.
This generated 4 immunofractions containing lipoproteins with: (1) none of the studied apolipoproteins (otherApos−CIII−); (2) one or more of apoAII, apoCI, apoCII, or apoE but no apoCIII (otherApos+CIII−); (3) apoCIII and not the others (otherApos−CIII+); and (4) apoCIII and at least one of the others (otherApos+CIII+). The main analyses compared fractions (otherApos−CIII− and otherApos−CIII+) to explore the impact of apoCIII.
The efficiency of the immunoaffinity separation (percentage of ligand removed from plasma by the resin) was 91% for apoAII, 97% for apoCI, 88% for apoCII, 99% for apoCIII, and 92% for apoE. The 4 immunofractions underwent ultracentrifugation for the separation of light (Sf: 60 to 400) and dense (Sf: 20 to 60) VLDL, intermediate-density lipoprotein (IDL; 1.006 to 1.025 g/mL), and light (1.025 to 1.032 g/mL) and dense (1.032 to 1.050 g/mL) LDL as described previously.8 Lipids were measured using enzymatic methods and concentrations of apolipoproteins using sandwich enzyme-linked immunosorbent assays. Intra-assay coefficients of variation for lipid and apolipoprotein measurements were <5% and interassay coefficients of variation were <10%.7,8
ApoB isolation, hydrolysis, derivatization, and measurement of tracer enrichment were performed as previously described.7 Plasma volume (L) was assumed to be 4.4% of body weight (kg). ApoB masses were measured by mass spectrometry and were adjusted to enzyme-linked immunosorbent assay measurements of apoB in each fraction.
A multicompartment model was constructed using SAAM II software (SAAM Institute, Seattle, Wash; Figure 1). Besides direct secretion and removal, and stepwise delipidation to denser types, some conversion pathways implying a change in apolipoprotein composition were required to best fit the data (Supplemental Figure I). When evaluating potential conversion pathways, we first excluded pathways whose tracer/tracee (T/T) curves or sizes precluded a precursor-product relationship masses (Supplemental Figure II). Qualified pathways were added to the model one by one and fitted to the data; pathways with zero or negligible rates were eliminated. The model structure was established with the mean T/T data of all subjects, and then each participant’s data were fitted individually to obtain the parameter values. Both [D5]l-phenylalanine and [D3]l-leucine T/T data were included in the same model, and the data were solved simultaneously. When fitted separately, the [D3]l-leucine primed infusion and [D5]l-phenylalanine bolus yielded similar model parameters. By fitting them simultaneously, the coefficients of variation of model parameters were reduced due to the extra precision provided by the use of more data for the estimation of each parameter. When there was a difference in the goodness of fit between the 2 isotopes for a given pool, the parameters predicted by [D3]l-leucine T/T data were privileged, because we considered that the larger dose of this isotope made the corresponding data more stable. In general, the model generated excellent fits to T/T data for both tracers (Figure 2) and apoB masses (Supplemental Figure III).
The results are presented as means±SD unless otherwise specified. Comparisons of rate means between lipoprotein fractions were done using the Mann-Whitney-Wilcoxon test. A probability value <0.05 was considered statistically significant; all reported probability values given are 2-sided.
Plasma total apoB masses ranged between 2331 and 2428 mg. We found apoB lipoproteins of all densities in the 4 immunofractions of all study participants, except for otherApos−CIII+ light VLDL, which was below the detection limit of the apoB enzyme-linked immunosorbent assay, 0.0015 mg/dL, comprising a maximum of 0.5% of total light VLDL apoB mass. Most apoB was detected in lipoproteins without any of the small apolipoproteins. Lipoproteins in the otherApos−CIII− fraction represented 70% to 80% of light VLDL, 40% to 50% of dense VLDL, 60% to 80% of IDL, 80% to 90% of light LDL, and >95% of dense LDL.
Fifty-five percent of all apoB secretion went into the otherApos−CIII− fraction, 21% into the otherApos+CIII+ fraction, 14% into the otherApos+CIII− fraction, and 9.8% into the otherApos−CIII+ fraction (Figure 1; Supplemental Figure IV). On average, 26% of apoB was secreted as light VLDL, 31% as dense VLDL, 12% as IDL, 6.9% as light LDL, and 24% as dense LDL. The single lipoprotein most abundantly secreted was otherApos−CIII− LDL, representing 21% of apoB secretion.
ApoB Lipoproteins in the OtherApos−CIII+ Fraction Undergo Faster Conversion to Smaller Particles and Little Direct Clearance
Both VLDL and IDL particles from the predominant otherApos−CIII− fraction and those in the otherApos−CIII+ fraction were characterized by minimal direct removal and almost complete lipolytic conversion to dense LDL before being withdrawn from circulation.
Compared with their otherApos−CIII− counterparts, otherApos−CIII+ dense VLDL had a similar rate of lipolytic conversion to IDL (mean 10.4 pools/day for otherApos-CIII+, 10.1 pools/day for otherApos−CIII−; P=0.46; Figure 3). OtherApos−CIII+ and otherApos−CIII− IDL particles also exhibited similar rates of lipolytic conversion in all patients (mean, 3.7 versus 4.6 respectively; Figure 1). Contrastingly, otherApos−CIII+ light LDL consistently had a faster rate of lipolytic conversion to dense LDL (range, 1.9- to 3.8-fold among study participants) compared with otherApos−CIII− light LDL (mean, 2.5 pools/day for otherApos−CIII+, 1.3 pools/day for otherApos−CIII−; P=0.021; Figure 3). Approximately half (mean, 48%) of otherApos−CIII+ light LDL particles were converted to otherApos−CIII+ dense LDL particles, whereas the other half lost their apoCIII during lipolysis and were converted into otherApos−CIII− dense LDL (Figure 1B). Pool sizes and kinetic parameters shown in Figure 1 are also shown in tabular form in Supplemental Table II.
ApoCIII in Lipoproteins Is Strongly Associated With Metabolic Rates Even in the Presence of Other Apolipoproteins
VLDL and IDL in the otherApos+CIII+ fraction had very low clearance like otherApos−CIII+. Again, similar to otherApos−CIII+, otherApos+CIII+ light LDL had fast conversion to dense LDL (otherApos+CIII+: 2.9 versus otherApos+CIII−: 0.85 pools/day, P<0.001). Thus, the presence of apoCIII, in both contexts with or without other small apolipoproteins, was associated with similar differences in apoB lipoprotein metabolism compared with cognate lipoproteins that did not have apoCIII. Among the 4 lipoprotein types based on apolipoprotein content, otherApos+CIII+ had the fastest lipolytic conversion from IDL down to dense LDL. Conversely, otherApos+CIII− VLDL and IDL had the slowest lipolytic conversion and the fastest clearance from plasma.
Plasma Triglycerides Were Associated With Changes in the Sources of LDL
The predominant source of LDL in participants with plasma TG below the group median (1.35 mmol/L) was direct liver secretion (56%), whereas in those with TG above the median, it was lipolysis of TRL particles (approximately 70%). The relative contribution of otherApos−CIII+ TRL to total LDL formation went from 4.6% in subjects below the TG median to 9.6% in those above it, and a similar trend was observed for otherApos+CIII+ TRL (Figure 4).
Triglyceride and Apolipoprotein Content of the VLDL, IDL, and LDL Types
These results are presented in Supplemental Figure V and VI.
Our results help clarify the influence of apoCIII on lipoprotein metabolism in vivo in humans.
First, we found that otherApos−CIII+ light LDL clearly evolve to dense LDL faster than cognate particles without apoCIII. This observation is not due to assumptions or technique of kinetic modeling, because the faster turnover of otherApos−CIII+ particles is clearly evident from the bolus tracer enrichment curves showing faster enrichment, higher peak enrichments, and faster disappearance. Light LDL in the otherApos−CIII+ fraction had a lower TG content than their otherApos−CIII− counterparts (Supplemental Figure V), so it seems unlikely that their faster lipolytic conversion is a substrate effect. It is possible that apoCIII is an accelerator of the lipolysis of LDL by hepatic lipase under physiological in vivo conditions. However, apoCIII inhibits postheparin hepatic lipase activity in vitro.18 Channeling of light LDL to dense LDL rather than to clearance from plasma may be an additional mechanism for the association of apoCIII containing LDL with cardiovascular risk.19
We confirmed prior results regarding a shift in the sources of circulating LDL particles with higher plasma TG.7 In participants with plasma TG below the median, direct hepatic secretion was the main contributor to total plasma LDL, whereas lipolysis of TRL was in those above the median. This change involved TRL with apoCIII, contributing 8% of total plasma LDL formation in the lower plasma TG half versus 17% in the higher one. In another clinical setting of high secretion of TRL, postmenopausal estrogen treatment, production of dense LDL from TRL is increased, whereas secretion of dense LDL by the liver is proportionately reduced.20
Our results do not confirm prior findings in vitro6 and from genetic deficiency models5,21,22 supporting an inhibitory action of apoCIII on lipolysis of VLDL. If apoCIII had an important inhibitory action on LPL, in vivo, the otherApos-CIII+ fraction of dense VLDL should have had slow conversion to IDL, a process mediated by LPL.23 This was not the case.
Several lines of evidence suggest that apoCIII has little effect on LPL activity in vivo: human apoCIII transgenic mice exhibit normal tissue24 and plasma postheparin25 LPL activities, and their VLDL, although greatly enriched in human apoCIII, are hydrolyzed normally by LPL in vitro.26 A limitation to this interpretation is that our separation technique depleted markedly but not completely apoCII (a lipoprotein lipase activator) in the VLDL otherApos−CIII+ fraction, so it is possible that the absence of differences between conversion rates of VLDL otherApos−CIII− and otherApos−CIII+ to IDL reflects mutually canceling effects from apoCII and apoCIII. Notwithstanding, the molar contents of apoCIII in otherApos−CIII+ VLDL were 2.5- to 3-fold higher than those of apoCII (Supplemental Figure VI). Furthermore, the conversion rates of light VLDL to dense VLDL to IDL are much faster for otherApos+CIII+ than otherApos+CIII− despite slightly lower contents of apoCII. Hence, if apoCIII had a strong inhibitory influence on VLDL lipolysis, it would most likely be evident in either of these 2 settings.
Lipoproteins in the otherApos+CIII− fraction were rich in apoE and apoCII and displayed fast clearance from circulation, consistent with the essential role of apoE on clearance.27 Fast clearance of otherApos+CIII− lipoproteins, which had much more apoCI and apoCII than apoE, also suggests that apoCI and apoCII may not substantially inhibit apoB lipoprotein uptake in humans.
OtherApos+CIII+ particles showed very little direct clearance, indicating that the impact of apoCIII on removal prevailed over that of other apolipoproteins in the same particle, particularly apoE. These findings strongly support apoCIII as a potent inhibitor of apoB lipoprotein uptake dominating the effects of apoE that often accompany it.
An interesting observation is that a major proportion of lipoproteins in the otherApos+CIII+ fraction underwent virtually complete shedding of their small apolipoproteins, becoming otherApos−CIII− (light VLDL 99%, dense VLDL 31%, light LDL 64%, and dense LDL 100%). However, complete loss of apoCIII occurred less frequently for otherApos−CIII+; percentages of conversion to otherApos−CIII− were: dense VLDL 0%, IDL 7%, and light LDL 52%. This suggests that the detachment of small apolipoproteins from the lipoprotein surface is more likely to occur when lipolysis happens in the presence of other apolipoproteins besides apoCIII.
In the present study, the major type of light LDL, otherApos−CIII−, did not have direct clearance from plasma nor did the other minor LDL types. This result is consistent with our previous report that separated apoB lipoproteins according to content of apoE and apoCIII and had the same subjects fed the same postprandial diet.17 In the previous report, the major type of light LDL, E-CIII−, also did not have direct clearance. In contrast, 28% of light LDL that contained apoE and apoCIII was directly cleared from the circulation. This light LDL fraction with apoE and apoCIII was contained in otherApos+CIII+, although its influence would have been diluted by light LDL that did not have apoE among the “other apos.” In additional studies, we found that the presence of apoE in light LDL is an important determinant of its direct clearance from plasma.7 In contrast, in the same subjects studied in the fasting state, we identified direct clearance of light LDL E-CIII−, 35% of total flux, and a larger direct clearance of apoE containing light LDL, 60%. We also found significant direct clearance of light LDL in postmenopausal women studied in the fasting state, more so when they were treated with estrogen, which increases LDL receptors.20 We hypothesize that direct clearance of light LDL is suppressed in postprandial conditions that include cholesterol intake, like in the present study, perhaps by decreased LDL receptors.
The interchangeability of apoCIII among lipoproteins has been cited as a potential problem in lipoprotein kinetic studies involving apoCIII. There is evidence supporting both separate exchangeable and nonexchangeable pools of apoCIII in VLDL28,29 and quick exchange to equilibrium producing a single homogeneous pool.30 Two considerations favor the idea of a limited transfer of apoCIII among lipoproteins. First, we and others have found that apoCIII occurs only in a portion of apoB lipoproteins.7,8,31–34 If apoCIII were freely exchangeable among VLDL particles, all VLDL would have some apoCIII and there would be no VLDL in the apoCIII(−) fractions. Second, apoCIII containing VLDLs have on average 50 to 100 copies of apoCIII per particle, and they coexist in circulation with particles free of any apoCIII. Thus, the apoCIII lipoprotein fractions in this study are very likely to represent true separate pools.
There are 2 main limitations to our results. First, we cannot completely rule out the influence of other compositional differences associated with the isolated presence of apoCIII on the observed results. The issue of potential functional interactions between apoCIII and other characteristics of the lipoproteins is still not well developed and deserves further study. Second, it is possible that the results of our kinetic model apply only to this nutritional condition, that is, after ingesting a monounsaturated fat-rich diet for 3 weeks, and the postprandial steady-state on the same diet. In this regard, a diet rich in monounsaturated fat compared with carbohydrates increases the fractional catabolic rate of VLDL with apoCIII,17 and the postprandial state may affect the clearance of all types of light LDL. However, having a unified nutritional background allowed us to remove extraneous variation in lipoprotein metabolism between individuals due to differences in baseline diet.
In conclusion, the findings strongly implicate the presence of apoCIII to impair liver clearance of all apoB lipoproteins as they circulate in vivo, leading to formation of LDL. Accelerated conversion of light LDL with apoCIII to dense LDL raises the possibility that apoCIII positively modulates the action of hepatic lipase, contributing to an increase in concentration of plasma dense LDL. These adverse actions of apoCIII on apoB lipoprotein metabolism support the concept of apoCIII as a target for preventive or therapeutic interventions in cardiovascular disease.
We are grateful to the volunteers who participated in this study. We also express thanks to Sue Wong-Lee and Jake Humphries for their technical assistance.
Sources of Funding
This work was supported by Grants R01 HL-34980, R01 HL-56210, and RR-02635 from the National Heart, Lung, and Blood Institute, National Institutes of Health (Bethesda, Md).
Received July 9, 2009; revision accepted October 22, 2009.
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