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. Author manuscript; available in PMC: 2011 Feb 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2009 Nov 12;30(2):239. doi: 10.1161/ATVBAHA.109.197830

Metabolism of VLDL and LDL containing apolipoprotein C-III and not other small apolipoproteins – R2

Carlos O Mendivil 1,2, Chunyu Zheng 1, Jeremy Furtado 1, Julian Lel 1, Frank M Sacks 1
PMCID: PMC2818784  NIHMSID: NIHMS166785  PMID: 19910636

Abstract

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 four VLDL, IDL and 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; (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 to OtherApos−CIII− had much faster conversion to dense LDL, as did light LDL OtherApos+CIII+ compared to OtherApos+CIII−. VLDL and IDL OtherApos−CIII+ had minimal direct removal from circulation, while 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.

Keywords: Apolipoprotein CIII, kinetics, apolipoprotein B100, low density lipoproteins, metabolism

ApoCIII is a small apolipoprotein, synthesized mainly in the liver, which circulates in plasma associated with apoB containing lipoproteins and HDL (1). In case-control studies with angiographic or clinical endpoints and prospective observational studies, plasma concentrations of lipoproteins with apoCIII are strong independent risk factors for cardiovascular disease (CVD) (1). Humans with genetic deficiency of apoCIII have lower triglyceride (TG) and 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, since 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 (i.e. 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 mice (10); and apoCI transgenic mice exhibit impaired LPL dependent TRL lipolysis (11); 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 receptor (16); promoting hepatic removal of lipoproteins.

In order to welcome apoCIII as a target for intervention in CVD 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 and kinetic modeling techniques. As a reference, we compared these results to 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.

Subjects and Methods

Subjects

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 mg/dL, and HDL cholesterol of 1.2+/−0.3 mg/dL (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).

Dietary protocol

All study subjects underwent a diet rich in monounsaturated fat (MUFA) for 3 weeks before the infusion protocol. The diet was 37% fat (8% saturated, 24% MUFA, 5% polyunsaturated [PUFA]), 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.

Tracer infusion

Study participants received a priming dose of 4.2 micromol/kg [D3]L-Leucine (Tracer Technologies, Cambridge, MA), followed by a constant infusion of [D3]L-Leucine at 4.8 micromol*kg-1*h-1 for 14 h. A bolus injection of 7.1 micromol/kg [D5] L-Phenylalanine was administered at the same time, as in previous studies (7,17). Blood was collected every 30 min in the first 2h and hourly thereafter for a total of 14 h. 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 anti-apoAII, anti-apoCI, anti-apoCII and anti-apoE immunoglobulins (Academy Biomedical, Houston, TX). The unbound fraction (OtherApos−) was collected by gravity flow and the bound fraction (OtherApos+) was eluted with 3M NaSCN. Both bound and unbound fractions were then incubated with anti-apoCIII immunoaffinity resin, and the bound and unbound fractions collected using the same procedures.

This generated four 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 four immunofractions underwent ultracentrifugation for the separation of light (Sf: 60–400) and dense (Sf: 20–60) VLDL, IDL (1.006–1.025 g/mL), and light (1.025–1.032 g/mL), and dense (1.032–1.050 g/mL) LDL as described previously (8). Lipids were measured using enzymatic methods, and concentrations of apolipoproteins using sandwich enzyme-linked immunosorbent assays (ELISA). Intraassay CVs for lipid and apolipoprotein measurements were <5%, and interassay CVs were <10% (7,8).

Kinetic analysis

ApoB isolation, hydrolysis, derivitization 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 ELISA measurements of apoB in each fraction.

A multicompartment model was constructed using SAAM II software (SAAM Institute, Seattle WA) (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, please see http://atvb.ahajournals.org). 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, please see http://atvb.ahajournals.org). 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 coefficient 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 two isotopes for a given pool, the parameters predicted by [D3]L-leucine T/T data were privileged, since 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, please see http://atvb.ahajournals.org).

Figure 1.

Figure 1

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ApoB metabolism in the study participants. Oval boxes represent apoB lipoprotein fractions separated by apolipoprotein composition and density, and numbers inside indicate pool sizes estimated by the model (mg). The measured pool sizes are shown in Supplemental Table II and Supplemental Figure III. The large square box in the left represents the liver; arrows out of this box represent direct liver secretion. Arrows out of lipoprotein compartments represent conversion to more dense lipoproteins and direct removal from plasma. Panel A. Numbers above or next to the arrows represent rate constants ± SD. Panel B. Percentages in bold next to the arrows indicate the percentage of total liver secretion into each fraction ± SD; percentages above the arrows indicate the relative proportion of flux out of each compartment. OtherApos−CIII−, lipoproteins without apoAII, apoCI, apoCII, apoCIII or apoE; OtherApos+CIII−, lipoproteins with apoAII or apoCI or apoCII or apoE and without apoCIII; OtherApos−CIII+, lipoproteins with no apoAII, no apoCI, no apoCII and no apoE but with apoCIII; OtherApos+CIII+, lipoproteins with apoCIII and at least one of the others.

Figure 2.

Figure 2

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Tracer-tracee ratios of D3-leucine (A) and D5-phenylalanine (B) in VLDL, IDL, and LDL subfractions in the study participants. Data points represent average leucine and phenylalanine tracer-tracee ratios. Lines represent model-derived curves fitted to the data. Phenylalanine data are presented on a logarithmic scale. See Figure 1 for explanation of lipoprotein fractions.

Statistical analysis

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 p- value <0.05 was considered statistically significant, all reported p-values given are 2 sided.

Results

Plasma total apoB masses ranged between 2331 and 2428 mg. We found apoB lipoproteins of all densities in the four immunofractions of all study participants, except for OtherApos−CIII+ light VLDL which was below the detection limit of the apoB ELISA, 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 70–80% of light VLDL, 40–50% of dense VLDL, 60–80% of IDL, 80–90% of light LDL, and more than 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 and Supplemental Figure V). 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 to their OtherApos−CIII− counterparts, OtherApos−CIII+ dense VLDL had a similar rate of lipolytic conversion to IDL (mean 10.4 pools/d for OtherApos−CIII+, 10.1 pools/d 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 vs 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 to OtherApos−CIII− light LDL (mean 2.5 pools/d for OtherApos−CIII+, 1.3 pools/d for OtherApos−CIII−; p=0.021) (Figure 3). About half (mean 48%) of OtherApos−CIII+ light LDL particles were converted to OtherApos−CIII+ dense LDL particles, while 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.

Figure 3.

Figure 3

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Rate of lipolytic catabolism of dense VLDL to IDL, and of light LDL to dense LDL according to apolipoprotein composition. Bars represent the average rate among study participants, error bars represent SD.

ApoCIII in lipoproteins is strongly associated with metabolic rates even in 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 vs. OtherApos+CIII−: 0.85 pools per 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 to 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 (about 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).

Figure 4.

Figure 4

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Proportion of total plasma LDL coming from direct liver secretion and lipolysis of VLDL and IDL with apoCIII in participants with fasting plasma triglycerides below or above the group median (1.35 mmol/L).

Triglyceride and apolipoprotein content of the VLDL, IDL and LDL types

These results are presented in Supplemental Figures IV and VI (please see http://atvb.ahajournals.org).

Discussion

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, since 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 IV, please see http://atvb.ahajournals.org), 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, while 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 vitro (6) and from genetic deficiency models (5,21,22) 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 tissue (24) and plasma postheparin (25) 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, please see http://atvb.ahajournals.org). 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 two 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 accompanies 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%, 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, as 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 VLDL (28,29) 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 50–100 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 two 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, i.e. after ingesting a MUFA-rich diet for three weeks, and the postprandial steady-state on the same diet. In this regard, a diet rich in MUFA compared to carbohydrate 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.

Supplementary Material

Supp1

Acknowledgments

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).

The authors are grateful to the volunteers who participated in this study. The authors also express thanks to Sue Wong-Lee and Jake Humphries, for their technical assistance.

Footnotes

Disclosures

The authors have no conflicts of interest to disclose.

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