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Originally published In Press as doi:10.1194/jlr.M400301-JLR200 on December 1, 2004

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Journal of Lipid Research, Vol. 46, 297-306, February 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Severe hypertriglyceridemia in human APOC1 transgenic mice is caused by apoC-I-induced inhibition of LPL

Jimmy F. P. Berbée1,*,{dagger}, Caroline C. van der Hoogt*,{dagger}, Deepa Sundararaman*,{dagger}, Louis M. Havekes*,{dagger},§ and Patrick C. N. Rensen*,{dagger}

* Netherlands Organization for Applied Scientific Research-Prevention and Health, Gaubius Laboratory, 2301 CE Leiden, The Netherlands
{dagger} Departments of General Internal Medicine, Leiden University Medical Center, 2300 RC Leiden, The Netherlands
§ Cardiology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands

Published, JLR Papers in Press, December 1, 2004. DOI 10.1194/jlr.M400301-JLR200

1 To whom correspondence should be addressed. e-mail: jfp.berbee{at}pg.tno.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Studies in humans and mice have shown that increased expression of apolipoprotein C-I (apoC-I) results in combined hyperlipidemia with a more pronounced effect on triglycerides (TGs) compared with total cholesterol (TC). The aim of this study was to elucidate the main reason for this effect using human apoC-I-expressing (APOC1) mice. Moderate plasma human apoC-I levels (i.e., 4-fold higher than human levels) caused a 12-fold increase in TG, along with a 2-fold increase in TC, mainly confined to VLDL. Cross-breeding of APOC1 mice on an apoE-deficient background resulted in a marked 55-fold increase in TG, confirming that the apoC-I-induced hyperlipidemia cannot merely be attributed to blockade of apoE-recognizing hepatic lipoprotein receptors. The plasma half-life of [3H]TG-VLDL-mimicking particles was 2-fold increased in APOC1 mice, suggesting that apoC-I reduces the lipolytic conversion of VLDL. Although total postheparin plasma LPL activity was not lower in APOC1 mice compared with controls, apoC-I was able to dose-dependently inhibit the LPL-mediated lipolysis of [3H]TG-VLDL-mimicking particles in vitro with a 60% efficiency compared with the main endogenous LPL inhibitor apoC-III. Finally, purified apoC-I impaired the clearance of [3H]TG-VLDL-mimicking particles independent of apoE-mediated hepatic uptake in lactoferrin-treated mice.

Therefore, we conclude that apoC-I is a potent inhibitor of LPL-mediated TG-lipolysis.

Abbreviations: apoC-I, apolipoprotein C-I; APOC1, human apolipoprotein C-I-encoding gene; apoe–/–, apolipoprotein E-deficient mice; CETP, cholesteryl ester transfer protein; CO, cholesteryl oleate; FC, free cholesterol; FPLC, fast-performance liquid chromatography; LDLr, low density lipoprotein receptor; LRP, low density lipoprotein receptor-related protein; TC, total cholesterol; TG, triglyceride; TO, triolein; VLDLr, very low density lipoprotein receptor

Supplementary key words fatty acids • lipid metabolism • triglycerides • apolipoprotein C-I • very low density lipoprotein • lipoprotein lipase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The human apolipoprotein C-I (apoC-I)-encoding gene APOC1 is part of the APOE/APOC1/APOC2 gene cluster (1). APOC1 is primarily expressed in the liver but also in the lung, skin, spleen, adipose tissue, and brain (2). ApoC-I is secreted as a 6.6 kDa protein into the plasma, where it is present at a relatively high concentration of ~10 mg/dl (3), and is mainly bound to chylomicrons, VLDLs, and HDLs (4). Although human studies to date have not revealed any polymorphism in the APOC1 gene leading to functional apoC-I variants, an HpaI polymorphism in the promotor region has been described that leads to 57% increased expression of the APOC1 gene (5). Interestingly, HpaI carriers display increased plasma triglyceride (TG) levels, which are independent of total cholesterol (TC) levels (6). To gain more insight into the function of apoC-I in lipoprotein metabolism, we and others have generated mice that either lack endogenous apoC-I (7, 8) or express the human APOC1 gene (9, 10). Although apoC-I-deficient mice did not show a phenotype with respect to plasma lipid levels (7), APOC1 transgenic mice showed an APOC1 dose-dependent increase in plasma levels of TG, TC, and FFAs. The most prominent increasing effect of APOC1 was observed on TG levels and could be attributed to severely increased levels of VLDLs (9, 11).

Early reports have postulated that apoC-I may function by modulation of the activity of plasma enzymes involved in lipid metabolism and modulation of TG-rich lipoprotein (remnant) binding and uptake by hepatic receptors. In vitro studies have shown that apoC-I may interfere with VLDL metabolism by partial activation of LCAT (12), inhibition of LPL (13), and inhibition of HL (14). Recently, Conde-Knape et al. (15) confirmed such an HL-modulating function of apoC-I in vitro and suggested that HL modulation may contribute to the hypertriglyceridemic phenotype of APOC1 transgenic mice. Strikingly, HL-deficient mice do not show any sign of disturbed TG metabolism (16–18). In addition, LCAT transgenic mice do not show increased VLDLs (19), suggesting that potential LCAT-activating properties of apoC-I do not contribute to the phenotype of APOC1 mice.

Besides modulation of plasma enzymes, apoC-I has also been reported to interfere with the apoE-dependent hepatic uptake of lipoprotein remnants by the LDL receptor (LDLr) and LDLr-related protein (LRP). In the isolated rat liver perfusion model, it was demonstrated that addition of human apoC-I inhibits the catabolism of chylomicrons and TG-rich emulsions (20, 21). Subsequently, it was shown that apoC-I can interfere with the apoE-mediated uptake of VLDLs by the LDLr (22) and LRP (23), possibly related to apoC-I-induced masking of the receptor binding domain of apoE (22) or displacement of apoE from the lipoprotein particle (23). More recent studies from our group (9, 24) have suggested that the inhibiting properties of apoC-I toward the LRP may exceed those toward the LDLr, because apoC-I-associated hyperlipidemia was severely aggravated on an LDLr-deficient background (9). In addition, it was shown that transfection of LDLr-deficient APOC1 mice with a recombinant adenovirus encoding the 39 kDa receptor-associated protein further increased plasma TG levels 4-fold. However, although these studies certainly suggest a modulating role of apoC-I with respect to hepatic lipoprotein recognition, it remains remarkable that complete blockade of the apoE-mediated lipoprotein clearance in apoE-deficient mice hardly affects plasma TG levels (16, 25, 26), whereas TG levels are severely increased by APOC1 expression, indicating that apoC-I has a profound additional effect.

Because the proposed functions of apoC-I cannot explain the severe hypertriglyceridemic phenotype of APOC1 mice, the aim of the present study was to elucidate the main mechanism underlying the apoC-I-induced combined hyperlipidemia in APOC1 mice. We demonstrate that apoC-I is a potent inhibitor of LPL, which can explain the combined hyperlipidemia observed in APOC1 transgenic mice in both the presence and absence of endogenous apoE.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic animals
Transgenic APOC1 mice with hemizygous expression of the human APOC1 gene were generated previously as described (7, 9) and backcrossed at least 10 times to the C57Bl/6 background. APOC1 mice were intercrossed with apoE-deficient (apoe–/–) mice (C57Bl/6 background) that were originally obtained from Jackson Laboratories (Bar Harbor, ME) to generate mice hemizygous for the APOC1 gene on an apoE-deficient background (apoe–/–APOC1). After initial characterization of both male and female mice, 10–12 week old male APOC1 and apoe–/–APOC1 mice were used for subsequent experiments, with wild-type and apoe–/– littermates as controls. Mice were housed under standard conditions in conventional cages and were fed ad libitum with regular chow (Ssniff, Soest, Germany). Experiments were performed after 4 h of fasting at 1:00 PM with food withdrawn at 9:00 AM, unless stated otherwise.

Plasma lipid and lipoprotein analysis
In all experiments, blood was collected from the tail vein into chilled paraoxon (Sigma, St. Louis, MO)-coated capillary tubes to prevent ongoing in vitro lipolysis (27), unless indicated otherwise. These tubes were placed on ice and centrifuged at 4°C, and the plasma obtained was assayed for TC, TG (without free glycerol), and FFA using the commercially available enzymatic kits 236691 (Roche Molecular Biochemicals, Indianapolis, IN), 337-B (GPO-Trinder kit; Sigma), and NEFA-C (Wako Chemicals, Neuss, Germany), respectively. For determination of the plasma lipoprotein distribution by fast-performance liquid chromatography (FPLC), 50 µl of pooled plasma from 10 mice per group was injected onto a Superose 6 column (Äkta System; Amersham Pharmacia Biotech, Piscataway, NJ), and eluted at a constant flow rate of 50 µl/min with PBS, 1 mM EDTA (Sigma), pH 7.4. Fractions of 50 µl were collected and assayed for TC and TG as described above. Human apoC-I was quantified by ELISA as described below.

VLDL isolation and characterization
Fasted mice were killed by cervical dislocation, and blood was drawn from the retro-orbital vain into Microvette® CB 1000 Z capillaries (Sarstedt, Nümbrecht, Germany). Sera were collected after centrifugation at 4°C and pooled from 10 mice. VLDLs were isolated by flotation (d < 1.006 g/ml) after ultracentrifugation in a SW 40 Ti rotor (Beckman Instruments, Geneva, Switzerland) at 40,000 rpm during 18 h at 4°C. The VLDL fractions were assayed for TG and TC as described above and for free cholesterol (FC) and phospholipids using the commercially available analytical kits 274-47109 and 990-54009 (Wako Chemicals), respectively. Cholesteryl esters were calculated by subtracting the molar concentration of FC from the molar concentration of TC and corrected for the presence of the fatty acid. Protein was determined by the method of Lowry et al. (28), with BSA as a standard. VLDL particle size was determined by photon correlation spectroscopy using a Zetasizer 3000 HSA (Malvern Instruments, Malvern, UK) at 25°C with a 90° angle between the laser and the detector.

Human apoC-I ELISA
Plasma human apoC-I concentrations were determined using a human apoC-I-specific sandwich ELISA. To this, a polyclonal goat anti-human apoC-I antibody (Academy Biomedical Co., Houston, TX) was coated overnight onto Costar medium binding plates (Corning, Inc., New York, NY) (dilution 1:104) at 4°C and incubated with diluted mouse plasma (dilution 1:106 to 1:107) or FPLC fractions (1:104) for 2 h at 4°C. Subsequently, HRP-conjugated polyclonal goat anti-human apoC-I antibody (dilution 1:500; Academy Biomedical Co.) was added and incubated for 2 h at room temperature, and HRP was detected by incubation with tetramethylbenzidine (Organon Teknika, Boxtel, The Netherlands) for 20 min at room temperature. Plasma from wild-type mice spiked with human apoC-I (Labconsult, Brussels, Belgium) was used as a standard.

Intestinal TG absorption
APOC1 mice and wild-type littermates were fasted overnight and injected intravenously with 500 mg of Triton WR-1339 (Sigma) per kilogram of body weight as a 10% (v/v) solution in sterile saline to block LPL-mediated TG hydrolysis (29). Subsequently, mice were given an intragastric load of glycerol tri[9,10 (n)-3H]oleate (10 µCi; Amersham, Buckinghamshire, UK) in 200 µl of olive oil. Blood samples were drawn before and at the indicated times after olive oil administration by tail bleeding. Lipids were extracted from plasma according to the method of Bligh and Dyer (30), and TG was separated from the other lipid components by thin-layer chromatography on Kieselgel 60 F254 plates (Merck, Darmstadt, Germany) using hexane-diethyl ether-acetic acid (83:16:1, v/v/v) as eluents. The radioactivity in the TG fraction was determined by scintillation counting (Packard Instruments, Dowers Grove, IL) according to Voshol et al. (31).

Hepatic VLDL-TG production
Mice were fasted, anesthetized by intraperitoneal injection of Domitor (0.5 mg/kg; Pfizer, New York, NY), Dormicum (5 mg/kg; Roche Netherlands, Mijdrecht, The Netherlands), and fentanyl (0.05 mg/kg; Janssen-Cilag B.V., Tilburg, The Netherlands), and injected via the tail vein with 500 mg of Triton WR-1339 per kilogram of body weight (32). Blood samples were drawn at 1, 30, 60, 90, and 120 min after administration, and plasma TG levels were measured as described above.

Preparation and in vivo clearance of VLDL-like TG-rich emulsion particles
The preparation and characterization of 80 nm protein-free VLDL-like emulsion particles have previously been described (33). Briefly, emulsion particles were prepared by sonication from 100 mg of total lipid at an egg yolk phosphatidylcholine (Lipoid, Ludwigshafen, Germany)-triolein-lysophosphatidylcholine-cholesteryl oleate-cholesterol (all from Sigma) weight ratio of 22.7:70:2.3:3.0:2.0 in the presence of either 75 µCi of [3H]TO or 150 µCi of [1{alpha},2{alpha}(n)-3H]cholesteryl oleate ([3H]CO; Amersham) using a Soniprep 150 (MSE Scientific Instruments, Crawley, UK) at 10 µm output. The lipid composition of the emulsions was determined as described above. Emulsions were stored at 4°C under argon and were used within 7 days. To study the in vivo serum clearance of the radiolabeled emulsions, mice were anesthetized as described above and the abdomens were opened. The emulsion (100 µg of TG), preincubated (30 min at 37°C) with or without human apoC-I (50 µg), was injected intravenously via the inferior vena cava. Where indicated, mice received a preinjection of bovine lactoferrin (70 mg/kg; Serva, Heidelberg, Germany) at 1 min before injection of the radiolabeled emulsion. Blood samples (<50 µl) were taken via the inferior vena cava at the indicated times, and the radioactivity in serum was counted as described above. The total plasma volumes of the mice were calculated from the equation V (ml) = 0.04706 x body weight (g), as determined from 125I-BSA clearance studies as previously described (34).

Plasma LPL level assay
Fasted APOC1 mice and wild-type littermates were injected via the tail vein with heparin (0.1 unit/g; Leo Pharmaceutical Products B.V., Weesp, The Netherlands), and blood was collected after 10 min. The plasma was snap-frozen and stored at –80°C until analysis of the total LPL activity as modified from Zechner (35). In short, a TG substrate mixture containing triolein (TO; 4.6 mg/ml), [3H]TO (2.5 µCi/ml), essentially fatty acid-free BSA (20 mg/ml; Sigma), Triton X-100 (0.1%; Sigma), and heat-inactivated (30 min at 56°C) human serum (20%) in 0.1 M Tris-HCl, pH 8.6, was generated by six sonication periods of 1 min using a Soniprep 150 at 7 µm output, with 1 min intervals between on ice. Ten microliters of postheparin plasma was added to 0.2 ml of substrate mixture and incubated for 30 min at 37°C in the presence or absence of 1 M NaCl, which completely inhibits LPL activity, to estimate both the LPL and HL levels. The reaction was stopped by the addition of 3.25 ml of heptane-methanol-chloroform (1:1.28:1.37, v/v/v), and 1 ml of 0.1 M K2CO3 in saturated H3BO3 (pH 10.5) was added. To quantify the [3H]oleate generated, 0.5 ml of the aqueous phase obtained after vigorous mixing (15 s) and centrifugation (15 min at 3,600 rpm) was counted in 4.5 ml of Ultima Gold (Packard Bioscience, Meriden, CT). The LPL activity was calculated as the fraction of total triacylglycerol hydrolase activity inhibited by 1 M NaCl and expressed as the amount of FFA released per hour per milliliter of plasma.

In vitro LPL activity assay
The effect of apolipoproteins on the TG hydrolysis of VLDL-like emulsion particles was determined as described (36). [3H]TO-labeled emulsion particles (0.5 mg TG/ml) were preincubated with apoC-I, apoC-III (Labconsult), apoA-I (Calbiochem, San Diego, CA), or recombinant apoA-V (37) at the indicated TG/protein weight ratios (30 min at 37°C). Subsequently, the protein-enriched particles were incubated with LPL in the presence of essentially fatty acid-free BSA (60 mg/ml) and heat-inactivated human serum (5%) in 0.1 M Tris-HCl, pH 8.5. At the indicated times, 50 µl samples from a 400 µl total incubation volume were added to 1.5 ml of methanol-chloroform-heptane-oleic acid (1,410:1,250:1,000:1, v/v/v/v) and 0.5 ml of 0.2 N NaOH to terminate lipolysis. Generated [3H]oleate was counted as described above and expressed as a percentage of the total [3H]activity added.

Statistical analysis
Statistical differences with respect to in vivo serum half-lives were determined using a two-way main-effects ANOVA. All other data were analyzed using nonparametric Mann-Whitney U tests. P < 0.05 was regarded as significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of APOC1 on plasma apoC-I and lipid levels
Table 1 summarizes the plasma human apoC-I and lipid levels in fasted male APOC1 mice and wild-type littermates on chow diet. APOC1 mice had ~4-fold higher plasma levels of human apoC-I compared with humans (3), and this was accompanied by severe combined hyperlipidemia. The enhancing effect of APOC1 expression on TG (12-fold) was much more pronounced than that on TC (2.1-fold) and FFA (1.5-fold), in agreement with our previous reports (9, 11). Similar effects of human apoC-I expression were observed in females compared with males (data not shown). Lipoprotein fractionation by FPLC showed that the plasma human apoC-I was primarily distributed toward HDLs and VLDLs (Fig. 1A) . The increase in both plasma TG and TC as a result of APOC1 expression could be mainly attributed to highly increased levels observed in VLDLs and mildly increased levels in intermediate density lipoproteins/LDLs, whereas the neutral lipid levels of the HDL fraction were hardly affected (Fig. 1B, C).


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  		TABLE 1. Effect of APOC1 on plasma lipid levels in wild-type and apoe–/– mice

 


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  		Fig. 1. Effect of the human apolipoprotein C-I (apoC-I)-expressing gene APOC1 on fast-performance liquid chromatography (FPLC) profiles of human apoC-I and lipids. Plasma of male APOC1 (closed circles) and wild-type (open circles) mice (n = 10) was pooled and size-fractionated by FPLC on a Superose 6 column. The individual fractions were analyzed for human apoC-I (A), triglyceride (TG; B), and total cholesterol (C). IDL, intermediate density lipoprotein; SA, Serum Albumin.

 
Effect of APOC1 in apoE-deficient mice
Although apoC-I has been postulated to inhibit the apoE-dependent hepatic uptake of TG-rich lipoprotein remnants, apoe–/– mice show only minor increases of plasma TG. To investigate the effects of APOC1 in the absence of endogenous apoE, APOC1 mice were intercrossed with apoe–/– mice to generate apoe–/–APOC1 mice. Plasma human apoC-I levels in apoe–/–APOC1 mice were ~4-fold higher compared with APOC1 mice and severely further aggravated the hyperlipidemia observed in apoe–/– littermates (Table 1). As on a wild-type background, APOC1 expression on an apoe–/– background had a much more pronounced effect on TG (55-fold) than on TC (3.2-fold) and FFA (3.2-fold). Again, similar data were observed in females compared with males (data not shown).

Effect of APOC1 on VLDL composition
To investigate whether the effect of APOC1 expression on plasma lipid levels was accompanied by a change in VLDL composition and/or size, VLDLs were isolated from apoe–/–APOC1 mice and their apoe–/– littermates, and their relative lipid compositions were determined (Table 2). The composition of VLDLs from wild-type mice could not be determined accurately because of low circulating levels (Fig. 1). VLDLs of apoe–/–APOC1 mice were predominantly enriched in TG, compared with VLDLs from apoe–/– littermates, and had a higher core lipid (TG + cholesteryl ester) to surface lipid (FC + phospholipid) ratio (2.7 vs. 2.4, respectively), indicative of larger VLDL particles. Indeed, VLDL particle size analysis confirmed that APOC1 expression markedly increased VLDL size compared with control littermates on both the wild-type background (average size, 72.9 vs. 44.4 nm, respectively) and the apoe–/– background (average size, 64.5 vs. 50.6 nm, respectively).


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  		TABLE 2. Effect of APOC1 on VLDL lipid composition

 
Effect of APOC1 on intestinal TG absorption and hepatic VLDL-TG production
To further address the mechanism(s) underlying the hypertriglyceridemia in APOC1 mice, we determined whether the intestinal TG absorption and/or the hepatic VLDL-TG production rate were enhanced in these mice. First, the intestinal TG absorption was studied by intravenously injecting Triton WR-1339 to block LPL-mediated TG-hydrolysis (29), after which an intragastric load of olive oil containing [3H]TO was given. As shown in Fig. 2 , no differences were observed between APOC1 and wild-type mice with respect to the appearance of radioactivity in plasma TG, indicating that apoC-I expression does not enhance TG absorption from the intestinal lumen.



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  		Fig. 2. Effect of APOC1 on intestinal lipid absorption. Overnight-fasted APOC1 (closed circles) and wild-type (WT; open circles) mice were injected intravenously with Triton WR-1339 (500 mg/kg) and subsequently given an intragastric load of [3H]triolein (TO) in 200 µl of olive oil. Blood samples were drawn at the indicated times, and lipids were extracted from plasma. Lipids were separated by thin-layer chromatography, and the radioactivity in the TG fraction was determined by scintillation counting. Values are means ± SD (n = 7).

 
The hepatic VLDL-TG production rate was measured by determining plasma TG levels at the indicated times after intravenous Triton WR-1339 injection (Fig. 3) . Whereas the TG levels in the APOC1 mice were higher compared with the wild-type littermates at the start of the experiment (4.9 ± 2.1 mM vs. 0.42 ± 0.08 mM, respectively; Fig. 3A), the relative increase in TG was similar for both types of mice (7.4 ± 0.9 mM/h vs. 6.6 ± 0.8 mM/h, respectively). Likewise, we found no difference in the relative increase in TG levels in apoe–/–APOC1 compared with apoe–/– mice (3.3 ± 1.4 mM/h vs. 3.1 ± 0.7 mM/h, respectively; Fig. 3B), indicating that apoC-I expression does not affect the hepatic VLDL-TG production rate either.



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  		Fig. 3. Effect of APOC1 on hepatic VLDL-TG production. Triton WR-1339 (500 mg/kg) was injected (time 0) into fasted APOC1 (closed circles) and wild-type (WT; open circles) mice (A) and in apolipoprotein E-deficient (apoe–/–) and apoe–/–APOC1 mice (B). Plasma TG levels were determined at 1, 30, 60, 90, and 120 min after injection. Values represent means ± SD (n = 6).

 
Effect of APOC1 on in vivo clearance of VLDL-like emulsion particles
To investigate whether an impaired lipolytic processing of TG-rich lipoproteins may contribute to the hypertriglyceridemia observed in APOC1 mice, mice were injected with [3H]TO-labeled protein-free VLDL-like emulsion particles, which have previously been shown to mimic the metabolic behavior of TG-rich lipoproteins (33, 38). As shown in Fig. 4 , the clearance of [3H]TO was markedly decreased in APOC1 mice compared with their wild-type littermates, as evident from a 2-fold increased serum half-life of [3H]TO (7.9 ± 2.1 min vs. 4.0 ± 0.3 min, respectively; P < 0.05). This observation indicates that APOC1 expression impairs TG clearance, which may result from inhibition of the LPL-mediated VLDL-TG hydrolysis.



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  		Fig. 4. Effect of APOC1 on serum clearance of VLDL-like emulsion particles in vivo. [3H]TO-labeled emulsion particles (100 µg of TG) were injected via the inferior vena cava into anesthetized APOC1 (closed circles) and wild-type (WT; open circles) mice. Blood samples were taken at the indicated times, and 3H activity was determined in serum. Values are means ± SD (n = 3). * P < 0.05, ** P < 0.01.

 
Effect of APOC1 on plasma LPL levels
An impaired clearance of VLDL-TG in APOC1 mice can be attributable to either decreased expression of LPL and/or apoC-I-induced inhibition of the activity of LPL. Therefore, we first determined plasma lipase levels in postheparin plasma by incubation with a [3H]TO-containing substrate mixture (Fig. 5) . Whereas the postheparin HL level was only slightly increased in APOC1 mice compared with wild-type littermates (12.8 ± 1.2 µmol FFA/h/ml vs. 11.3 ± 1.0 µmol FFA/h/ml, respectively; P < 0.05), the postheparin LPL level was even 1.8-fold increased in APOC1 mice compared with wild-type mice (40.7 ± 6.1 µmol FFA/h/ml vs. 22.5 ± 2.2 µmol FFA/h/ml, respectively; P < 0.01). Therefore, the impaired lipolytic conversion of VLDL in APOC1 mice cannot be attributable to decreased levels of LPL.



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  		Fig. 5. Effect of APOC1 on plasma HL and LPL levels in vivo. Fasted APOC1 (closed bars) and wild-type (WT; open bars) mice were injected intravenously with heparin (0.1 unit/g). Plasma, collected at 10 min after injection, was incubated (30 min at 37°C) with a [3H]TO-containing substrate mixture in the absence or presence of 1 M NaCl to estimate both the LPL and HL activity. Generated [3H]oleate was extracted and determined as described. Values represent means ± SD (n = 8). * P < 0.05, ** P < 0.01.

 
Effect of apoC-I on LPL activity
To investigate whether the apoC-I-related impaired lipolytic conversion of VLDL can be attributable to a direct inhibiting effect of apoC-I on LPL activity, protein-free VLDL-like emulsion particles were enriched with increasing concentrations of purified human apoC-I and subsequently incubated with LPL. The well-established endogenous LPL inhibitor apoC-III (39, 40) was used as a control. ApoC-I and apoC-III were compared on a mass basis, as they are also present in human plasma at similar mass concentrations (i.e., 10 and 13 mg/dl) (3). ApoC-I appeared to dose-dependently inhibit the TG hydrolysis rate (Fig. 6A) . At a TG/protein weight ratio of 50:10, apoC-I and apoC-III inhibited the triacylglycerol hydrolase activity of LPL by 33% and 55%, respectively (Fig. 6B). In contrast, apoA-I did not affect lipolysis, and apoA-V even dose-dependently increased the lipolysis rate up to 1.5-fold at a TG/apoA-V weight ratio of 50:3 (data not shown), in agreement with our previous observations (36, 37).



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  		Fig. 6. Effect of apoC-I on LPL-mediated hydrolysis of VLDL-like emulsion TGs. A: [3H]TO-labeled protein-free emulsion particles were preincubated (30 min at 37°C) in the absence (open circles) and presence of apoC-I at TG/apoC-I weight ratios of 50:3 (open triangles), 50:5 (closed triangles), and 50:10 (open squares). At the indicated times after addition of LPL, generated [3H]oleate was extracted and quantified. B: The effect of apoC-I (closed circles) on LPL-mediated TG hydrolysis was compared with that of apoC-III (open circles) and is depicted as a percentage of the TG hydrolysis rate in the absence of protein.

 
Effect of apoC-I enrichment of VLDL-like emulsion particles on in vivo clearance
To assess whether apoC-I can directly inhibit lipolysis in vivo, the effect of apoC-I protein was determined on the plasma decay of [3H]TO-labeled protein-free VLDL-like emulsion particles in wild-type mice. To focus on the effects of apoC-I on peripheral lipolysis rather than on liver uptake, mice were preinjected with lactoferrin. Lactoferrin has previously been shown to block the interaction of chylomicrons and emulsion particles with the liver in vivo (33, 41), which we confirmed using [3H]CO-labeled protein-free VLDL-like emulsion particles (results not shown). As depicted in Fig. 7 , preincubation of emulsion particles with apoC-I markedly delayed the clearance of [3H]TO, as is evident from a 1.9-fold increased serum half-life (17.6 ± 5.7 min vs. 9.2 ± 3.7 min, respectively; P < 0.05), indicating that apoC-I can indeed inhibit lipolytic TG conversion in vivo.



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  		Fig. 7. Effect of human apoC-I enrichment of VLDL-like emulsion particles on their serum clearance in lactoferrin-treated mice. [3H]TO-labeled emulsion particles (100 µg of TG) were preincubated without (open circles) or with (closed circles) human apoC-I (50 µg) for 30 min at 37°C and injected via the inferior vena cava into anesthetized lactoferrin-treated wild-type mice. Blood samples were taken at the indicated times, and 3H activity was determined in serum. Values are means ± SEM (n = 3). * P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Studies in both humans (6) and mice (9, 10) have shown that increased expression of apoC-I results in combined hyperlipidemia, with a more pronounced enhancing effect on TG compared with TC. Because a variety of effects on lipid metabolism have been attributed to apoC-I, including activatory effects (e.g., LCAT) and inhibitory effects [e.g., HL, cholesteryl ester transfer protein (CETP), intestinal absorption, and apoE-dependent recognition by LRP, LDLr, and VLDL receptor (VLDLr)], the aim of the present study was to elucidate the main mechanism underlying the apoC-I-related hypertriglyceridemia using APOC1 transgenic mice. We demonstrated that at moderate plasma human apoC-I levels [i.e., 4-fold higher than those found in humans (3)], the 12-fold increase in plasma TG levels was mainly attributable to inhibition of the lipolytic processing of VLDLs.

The effects of apoC-I on lipid metabolism were mainly confined to VLDL metabolism, leaving HDL metabolism (which crucially involves both CETP and LCAT) unaffected. Analysis of the HDL protein constituents for CETP-modulating properties showed that apoC-I is a very potent and highly selective inhibitor of CETP (42). In addition, Gautier et al. (43) have shown that cross-breeding of human CETP transgenic mice with apoC-I-deficient mice resulted in higher CETP activity in vivo. Although apoC-I thus appears to be a physiologically relevant inhibitor of CETP, this function of apoC-I cannot contribute to the phenotype of APOC1 mice, because mice do not express CETP (44). Activation of LCAT should be expected to lead to increased HDL size and HDL lipids, as was observed in mice and rabbits that overexpress LCAT (19, 45). Because both the cholesterol level and the size of HDL are not affected by apoC-I expression in APOC1 mice, potential LCAT-activating properties of apoC-I (12) do not appear to be relevant for determining HDL levels in mice.

ApoC-I expression thus predominantly affects VLDL-TG metabolism, which can result from i) increased intestinal TG absorption, ii) increased VLDL-TG production, and/or iii) disturbed lipolytic conversion and/or hepatic clearance of VLDL. Previously, we have reported that mice deficient for apoC-I showed a significantly lower intestinal lipid absorption compared with wild-type mice (34). However, no changes in intestinal lipid absorption were observed in APOC1 mice compared with wild-type littermates, which can be related to a relatively low expression of human apoC-I in the intestine. Previously, we have shown that apoE-deficient mice show a decreased VLDL-TG secretion rate (26), and we confirm this observation in our present study. Although human apoC-I is highly expressed in the liver, we did not detect any effect of human apoC-I expression on hepatic VLDL-TG production rate on either a wild-type or an apoE-deficient background, which is in agreement with our previous studies showing that apoC-I deficiency did not alter the hepatic VLDL production rate (7). Apparently, expression of human apoC-I cannot compensate for the decreased VLDL-TG production in apoE-deficient mice. Collectively, the hypertriglyceridemia in APOC1 mice is not caused by either an effect on intestinal TG absorption or hepatic TG production.

Next, we evaluated the effect of apoC-I expression on apoE-dependent VLDL uptake by the liver. ApoC-I has been shown to inhibit the apoE-mediated binding of TG-rich lipoprotein remnants by hepatic lipoprotein receptors (i.e., LDLr and LRP) (22, 23, 46), although Quarfordt, Michalopoulos, and Schirmer (21) reported that the apoC-I-mediated inhibition of the uptake of TG-rich emulsions by cultured hepatocytes was (at least partly) independent on the presence of apoE. Indeed, we have shown that APOC1 expression in mice can interfere with the hepatic interaction of VLDLs primarily via LRP (9, 24). However, the contribution of this effect to the APOC1-induced severe hypertriglyceridemia can be questioned, because complete blockade of the apoE-dependent hepatic lipoprotein clearance in apoe–/– mice only mildly affects plasma TG levels (25), whereas APOC1 expression increases plasma TG as much as 12-fold. In addition, we now show that APOC1 expression on an apoe–/– background further dramatically increased TG levels, showing that the hypertriglyceridemic effects of apoC-I can be independent of the presence of apoE. Taken together, these data indicate that the hypertriglyceridemia observed in APOC1 mice also cannot be explained by the inhibition of apoE-mediated hepatic remnant uptake.

Finally, we evaluated the possibility that the lipolytic conversion of VLDLs may be impaired in APOC1 mice, because such a mechanism may explain the dramatic accumulation of plasma TGs in primarily VLDLs. In addition, decreased plasma TG hydrolysis may also explain the increased VLDL particle size observed in APOC1-expressing mice on both the wild-type and apoe–/– backgrounds and the observed impaired clearance of VLDL-like emulsion particles upon intravenous administration.

Recently, Conde-Knape et al. (15) described the cross-breeding of their human apoC-I-expressing mouse strain with apoe–/– mice, which resulted in a comparable, albeit more modest, hypertriglyceridemic phenotype as our apoe–/– APOC1 mice, and they suggested that the hypertriglyceridemia in these mice was attributable to inhibition of HL-mediated TG hydrolysis. However, HL deficiency or overexpression in mice and rabbits predominantly affects plasma HDL-TC levels, with only mild effects (if any) on TG levels on both wild-type and apoe–/– backgrounds (16–18, 47). Furthermore, HL has a much lower preference for TG compared with LPL (48), and HL is known to primarily mediate the conversion of intermediate density lipoprotein to LDL and of HDL2 to HDL3 (49), whereas both our studies and those of Conde-Knape et al. (15) indicate that APOC1-expressing mice merely have a disturbed VLDL metabolism. Therefore, although a potential inhibiting effect of apoC-I on the activity of HL in vivo cannot be ruled out, and it may add to the observed hypercholesterolemia, it does not contribute to the severe hypertriglyceridemia observed in APOC1 mice.

Thus, impairment of LPL remains the most likely mechanism to explain the hypertriglyceridemic phenotype of APOC1 mice. Although the APOC1 mice showed increased LPL levels in postheparin plasma, we indeed found that apoC-I is very effective in attenuating the LPL activity in vitro, with 60% efficiency on a mass basis compared with the well-known endogenous LPL inhibitor apoC-III (34, 50). Our observations confirm previous in vitro studies by Havel et al. (13), who showed that apoC-I and apoC-III were equally effective on a mass basis with respect to inhibition of the apoC-II-stimulated LPL-mediated TG hydrolysis. In fact, the LPL inhibitory properties of apoC-I and apoC-III are specific for these apolipoproteins, because addition of the negative control apoA-I (36) had no effect on the LPL activity, and the recently identified LPL stimulator apoA-V (37) enhanced the LPL activity in this assay. Importantly, the TG/apoC-I ratios applied in the in vitro assay at which apoC-I inhibited LPL (50:3 to 5:10, w/w) were similar to those found in both APOC1 mice (50:6) and apoe–/–APOC1 mice (50:3), indicating that the LPL inhibitory properties observed in vitro are relevant for the in vivo situation. Indeed, preincubation of VLDL-like emulsion particles with apoC-I inhibited the liver-independent serum clearance of emulsion TG, as was demonstrated in lactoferrin-treated mice. Concomitantly, the uptake of TG-derived fatty acids by white adipose tissue was 1.8-fold decreased (data not shown). Given the fact that apoC-I readily exchanges between lipoproteins, a part of the injected emulsion-associated apoC-I will presumably rapidly redistribute toward endogenous lipoproteins, which will even lead to underestimation of the inhibiting effect of apoC-I on emulsion TG clearance. In a previous study from our group in which VLDL clearance was assessed in functionally hepatectomized APOC1 mice on a low fat/low-cholesterol diet, we also found a tendency toward a decreased VLDL-TG lipolysis rate in APOC1 mice (i.e., 32%), although a statistically significant difference was not reached under the applied experimental conditions (9).

The phenotype of APOC1 mice closely resembles that of human apoC-III-expressing APOC3 mice with respect to the predominant increase of VLDL-TG levels (51). In addition, both APOC1 mice and APOC3 mice (52) show a modest increase in plasma cholesterol levels. In fact, the relative increase in TG compared with cholesterol induced by apoC-I expression (i.e., 5.8) is similar to that induced by apoC-III expression (i.e., 5.5) (52). Indeed, it has been established that LPL activity strongly determines plasma TG levels. Overexpression of LPL in mice markedly reduces plasma VLDL-TG levels (53, 54), whereas heterozygous deficiency of LPL results in the accumulation of plasma VLDL-TG (55). As in APOC1 and APOC3 mice, the effects of LPL modulation on plasma TG exceeded those on TC.

Inhibition of the lipolytic conversion of TG-rich lipoproteins in APOC1 mice can fully account for our previous observation that APOC1 protects against the development of obesity on a genetically obese leptin-deficient (ob/ob) background (56) by impeding the disposition of LPL-liberated fatty acids into adipose tissue. Likewise, we recently observed that deletion of the main endogenous LPL inhibitor apoC-III in apoc3–/– mice markedly aggravates diet-induced obesity as related to increased adipose tissue stores (our unpublished observations). Interestingly, we have reported that VLDLr-deficient (vldlr–/–) mice are protected from diet-induced obesity on both wild-type and ob/ob backgrounds (57). Subsequently, Yagyu et al. (58) have shown that vldlr–/– mice have reduced LPL activity as related to the LPL chaperone function of the VLDLr (59), which may partially explain their resistance to obesity. In addition, the VLDLr may also be involved in LPL-mediated lipolysis by bridging of lipoproteins to the endothelial surface, thereby facilitating the LPL-particle interaction. Because we have firmly established that apoC-I strongly inhibits the interaction of VLDL with the VLDLr (24), a concurring VLDLr-inhibiting effect of apoC-I may further hamper the LPL-mediated VLDL-TG hydrolysis in vivo as observed in APOC1 mice.

In conclusion, we have demonstrated that the hypertriglyceridemic effect of moderate human apoC-I expression in mice is the consequence of an impaired lipolytic conversion of VLDL-TG. This effect probably results from a direct inhibiting effect of apoC-I on LPL activity, although a concomitant inhibiting effect of apoC-I on VLDL binding to the VLDLr, which facilitates lipolysis, cannot be excluded. The mechanism by which apoC-I inhibits LPL activity is currently under investigation.


   ACKNOWLEDGMENTS
 
This work was performed in the framework of the Leiden Center for Cardiovascular Research, Leiden University Medical Center-Netherlands Organization for Applied Scientific Research and was supported by the Leiden University Medical Center (Gisela Thier Fellowship to P.C.N.R.) and the Netherlands Organization for Scientific Research (Netherlands Organization for Scientific Research-Research Institute of Diseases in the Elderly Grant 014-90-001, Netherlands Organization for Scientific Research Grant 908-02-097, and Netherlands Organization for Scientific Research-VIDI Grant 917-36-351).

Manuscript received August 9, 2004 and in revised form October 27, 2004.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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