Enhanced Conversion of Triglyceride-Rich Lipoproteins and Increased Low-Density Lipoprotein Removal in LPLS447X Carriers
Objective— Lipoprotein lipase (LPL) exerts 2 principal actions, comprising enzymatic hydrolysis of triglyceride-rich lipoproteins (TRLs) and nonenzymatic ligand capacity for enhancing lipoprotein removal. The common LPLS447X variant has been associated with cardiovascular protection, for which the mechanism is unknown. We therefore evaluated enzymatic and nonenzymatic consequences of this LPL variant on TRL metabolism.
Methods and Results— TRL apolipoprotein B100 (apoB100) metabolism was determined in 5 homozygous LPLS447X carriers and 5 controls. Subjects were continuously fed and received infusion of stable isotope l-[1-13C]-valine. Results were analyzed by SAAMII modeling. Also, preheparin and postheparin LPL concentration and activity were measured. Compared with controls, carriers presented increased very low–density lipoprotein 1 (VLDL1) to VLDL2 apoB100 flux (P=0.04), increased VLDL2 to intermediate-density lipoprotein (IDL) apoB100 flux (P=0.02), increased IDL to low-density lipoprotein (LDL) apoB100 flux (P=0.049), as well as an increased LDL clearance (P=0.04). Additionally, IDL apoB100 synthesis was attenuated (P=0.05). Preheparin LPL concentration was 4-fold higher compared with controls (P=0.01), and a correlation was observed between preheparin LPL concentration and LDL clearance (r2=0.92; P=0.01).
Conclusions— Enhanced TRL conversion and enhanced LDL removal combined with increased preheparin LPL concentration suggest increased enzymatic consequences as well as increased nonenzymatic consequences of LPL in LPLS447X carriers, which might both contribute to the cardiovascular benefit of this LPL variant.
Evidence has accumulated to show that triglyceride-rich lipoproteins (TRLs) contribute to atherogenesis.1,2 The TRL pool ranges from very large particles consisting of chylomicrons (CMs) and very low–density lipoprotein 1 (VLDL1) to smaller particles like VLDL2 and intermediate-density lipoproteins (IDLs). VLDL1 and VLDL2 contribute to lipid accumulation in human macrophages and promote foam cell formation.3,4 Lipoprotein lipase (LPL) plays a crucial role in TRL delipidation because this enzyme largely determines the conversion of large TRL (VLDL1) via smaller triglyceride (TG)-depleted TRL (VLDL2 and IDL) toward LDL. In addition, LPL exerts a ligand function, facilitating nonenzymatic removal of lipoprotein particles.5–7
Most LPL gene variants have been associated with an increased cardiovascular disease (CVD) risk and result in partial loss of function.8,9 In contrast, the frequent LPLS447X variant, present in 20% of the population, is associated with decreased TG and increased high-density lipoprotein cholesterol (HDL-C).8 Data on the LPL concentration and activity in this variant have been conflicting.8,10,11 Overall, this LPL variant is associated with a lower incidence of CVD.8 However, the precise mechanisms remain to be determined.
Increased turnover of large VLDL to low-density lipoprotein (LDL), with ensuing enhanced clearance of LDL, could be a potential mechanism for cardiovascular protection in LPLS447X carriers. To test this hypothesis, we evaluated apolipoprotein B100 (apoB100) kinetics in homozygous LPLS447X carriers in the fed state by infusion of the stable isotope l-[1-13C]-valine.
Materials and Methods
Five male homozygous LPLS447X carriers were selected from a database at the Academic Medical Center in Amsterdam and matched to controls with respect to age, body mass index (BMI), smoking habits, lipid levels, and use of alcohol. None of the subjects had signs of CVD, nor did they exhibit E2/E2 or E4/E4 genotype. All participants gave written informed consent. The study protocol was approved by the institutional review board of the Academic Medical Center. The study conforms to the principles outlined in the Declaration of Helsinki.
LPL genotyping was performed as described previously.12 Of 2000 DNA samples, 6 males were found to be homozygous carriers of the LPLS447X variant, 5 of whom were willing to participate.
The protocol for infusion of labeled valine has been described previously.13 All participants entered the research ward on the evening before the study. At 3:00 am (t=0), baseline blood sampling was performed. Subsequently, a shake, equivalent to one twentieth of their daily food intake, was ingested every hour until 9:00 pm. This shake consisted of 14% of calories as protein, 44% as carbohydrates, 42% fat (17% saturated, 17% monounsaturated, and 8% polyunsaturated), and 90 mg cholesterol per 1000 kcal. Five hours after baseline blood sampling, intravenous catheters were placed in both forearms, and a priming dose of 17 μmol/kg l-[1-13C]-valine was given intravenously, followed by continuous infusion of 15 μmol/kg per hour for 13 hours. Blood samples were obtained from the contralateral arm at baseline and after 5, 5.5, 6, 7, 8, 9, 10, 11, 12, 14, 16, and 18 hours.
Isotope and Chemicals
l-[1-13C]-valine (isotope mole fraction >0.99; MassTrace) was dissolved in sterile 0.9% saline and sterilized through a 0.22-μm filter.14 Density solutions were made with kaline bromide (KBr) in 0.9% NaCl.
Blood for lipid analysis was drawn in EDTA-coated tubes. Plasma was isolated by centrifugation and stored at −80°C. Baseline total cholesterol was measured by standard enzymatic methods (CHOD-PAP; Roche Diagnostics). Baseline HDL-C was measured in the supernatant fraction after precipitation of apoB-containing lipoproteins with dextran sulfate and magnesium chloride. TG and free fatty acids (FFAs) were measured using commercially available kits (Triglyceride GPO-trinder, Sigma Diagnostics Inc.; NEFA-C, Wako Chemicals). LDL-C was calculated using the Friedewald formula.
Measurement of LPL Concentration and Activity
Blood for preheparin and postheparin LPL concentration and activity was collected in heparin-containing tubes before and 15 minutes after an intravenous injection of heparin (50 IU/kg body weight) ≥1 month before the study. LPL activity was analyzed as published previously.15 LPL concentrations were measured using a commercially available kit (Markit-M LPL; Dainippon Pharmaceutical Co).
LPL Analysis in ApoB and Non-ApoB Fractions
ApoB-containing lipoproteins were precipitated with a solution of magnesium chloride, dextran sulfate, and magnetic beads (Polymedco). Preheparin plasma (200 μL) was mixed with 40 μL of the beads solution. The supernatant was collected and the pellet was resuspended in 150 μL LPL stabilizer solution from an LPL ELISA kit (Markit-M), after which the beads were separated using a magnet. LPL concentration was subsequently measured in the supernatant and in the resuspended apoB-containing pellet.
Isolation and Analysis of ApoB100-Containing Lipoproteins
Isolation of apoB100-containing lipoproteins was performed as described previously.14 In short, CM, VLDL1, VLDL2, IDL, and LDL were isolated using a discontinuous salt gradient by cumulative ultracentrifugation (UC; Beckman Ultracentrifuge Sw41 Ti rotor; Beckman Instruments). After 3 subsequent UC spins, the CM fraction (32 minutes, 40 700 rpm, 4°C, acceleration 5, brake 5), the VLDL1 fraction (3 hours and 28 minutes) and the VLDL2, the IDL, the LDL, and the bottom fraction (HDL+lipoprotein-deficient plasma) were collected after 17 hours. After sample collection of the first and the second UC spin, the UC tubes were refilled using 1 mL D=1.006 g/mL KBr. All fractions were stored at −20°C. Fractional apoB100 concentrations were determined using a nephelometric assay (Dade Behring).
ApoB100 Isolation From Lipoproteins and Determination of 13C-Valine Enrichment
ApoB100 was isolated from lipoproteins, and determination of 13C-valine enrichment was performed as described previously.13,14
ApoB100 production was measured as the rate of incorporation of 13C-enriched valine into circulating VLDL1, VLDL2, IDL, and LDL apoB100, and best data fit in a 6-compartment model was determined using the SAAM II software (Simulation Analysis and Modeling; version 1.1.1.; SAAM Institute) as described previously and shown in the Figure.14,16 Plasma volumes were calculated from body surface area. The plasma pool was calculated by multiplying the plasma volume by the plasma apoB concentration. It was assumed that during the study, each subject remains in steady state with respect to apoB100 metabolism, during which fractional catabolic rate (FCR) equals fractional synthetic rate. The direct catabolism in VLDL1 apoB100 was assumed to be identical to the direct catabolism of VLDL2 apoB100 to reduce the number of unknown variables.
All data are presented as mean values±SD. All data were analyzed with SPSS (version 12.0.1; SPSS Inc.) and were compared using an independent samples t test. Correlations were performed by linear regression. Statistical significance was accepted at a level of P<0.05.
Baseline characteristics of carriers and controls are shown in Table 1. Age, BMI, blood pressure, waist/hip-ratio, and lipids were not significantly different. Also, fasting apoB100, glucose, insulin, and calculated plasma volumes were comparable. Nonfasting lipids, plasma apoB, and apoB100 in the different lipoprotein fractions and FFAs were not significantly different between groups (data not shown). During feeding, plasma TG increased significantly compared with baseline (P<0.01) without differences between groups (data not shown).
Rate constants are listed in supplemental Table I (available online at http://atvb.ahajournals.org).
VLDL1 ApoB100 Metabolism
VLDL1 apoB100 pool size was not significantly different between groups (Table 2). VLDL1 apoB100 synthesis, VLDL1 apoB100 production rate, and transfer of VLDL1 apoB100 were not significantly different between both groups. Interestingly, the flux of VLDL1 to VLDL2 apoB100 was 2.4-fold increased in carriers (P=0.04). No significant differences were found for direct catabolism, total FCR, and direct clearance of VLDL1 apoB100.
VLDL2 ApoB100 Metabolism
VLDL2 apoB100 pool size, VLDL2 apoB100 synthesis, VLDL2 apoB100 production rate, and transfer of VLDL2 apoB100 were similar in both groups (Table 3). The flux of VLDL2 to IDL apoB100 was 1.7-fold higher in carriers (P=0.02). No significant differences were found in direct catabolism, total FCR, and direct clearance of VLDL2 apoB100.
IDL ApoB100 Metabolism
IDL apoB100 pool size, IDL apoB100 production rate, transfer of IDL apoB100 to LDL apoB100, direct catabolism, total FCR, and direct clearance were not significantly different (Table 4). The flux of IDL to LDL apoB100 was 1.6-fold higher in carriers (P=0.049). IDL apoB100 synthesis was lower in carriers (P=0.05).
LDL ApoB100 Metabolism
No significant differences were found for LDL apoB100 pool size, synthesis, production rate, or total FCR between both groups (Table 5). Interestingly, carriers exhibited a 1.4-fold increase in direct clearance of LDL apoB100 (P=0.03).
In carriers, preheparin LPL concentration was increased 4-fold (carriers 104.6±38.3 versus controls 25.4±9.3 ng/mL; P=0.01). Preheparin LPL activity was below the detection limit in both groups. Postheparin LPL concentration (carriers 367±90 versus controls 369±150 ng/mL) as well as postheparin LPL activity (carriers 235.2±45.0 versus controls 196.3±76.1 mU/mL) were similar between groups (P=0.97 and P=0.29, respectively).
In carriers, preheparin LPL concentration correlated with direct LDL apoB100 clearance (r2=0.92; P=0.01). No correlation was found between preheparin LPL and LDL clearance in the controls (P=0.27).
Preheparin LPL and Lipoproteins
To verify whether preheparin LPL was associated with apoB-containing lipoproteins, we quantified LPL in whole plasma, in apoB-depleted plasma, and in a resuspended pellet that resulted from precipitated apoB-containing lipoproteins. In LPLS447X carriers and controls, 71±7% and 60±15% of the total serum LPL was found in the apoB-containing fraction. Compared with controls, the LPLS447X carriers presented an absolute higher amount of LPL (from 200 μL plasma) in the apoB-containing fraction (carriers 8.9±2.5 versus controls 2.8±0.6 ng; P<0.01) as well as in the non-apoB fraction (carriers 3.8±1.6 versus controls 2.3±2.1 ng; P=0.25; Figure I, available online at http://atvb.ahajournals.org). In separate control experiments, we confirmed the presence of LPL in the apoB fraction using an alternative method to separate non–apoB- and apoB-containing lipoproteins (MgCl2 and phosphotungstic acid; data not shown).
In the present study, we show for the first time that LPLS447X carriers exhibit enhanced TRL conversion and increased LDL apoB100 clearance. Also, carriers exhibited a 4-fold increase in preheparin LPL concentration, which was strongly associated with LDL apoB100 clearance. These data suggest enhanced hydrolytic activity as well as increased ligand capacity of LPL in carriers of the LPLS447X variant.
The delipidation cascade reflects TRL conversion attributable to LPL-mediated TG hydrolysis.17 To date, analysis of TRL hydrolysis capacity has concentrated on measurement of postheparin LPL activity. Carriers in the present study exhibited identical postheparin LPL activity compared with controls, all of which were comparable to previously reported activities.18 Of note, postheparin testing only reflects activity of the total LPL pool, of which only part is physiologically active.17 Hence, postheparin values do not reflect the actual in vivo LPL-mediated lipolytic capacity. In the present study, preheparin LPL activity was below the detection limit. After a previous report showing impaired VLDL handling in an LPL variant characterized by attenuated LPL activity,8,9 we now show enhanced conversion of TRL in LPLS447X carriers implying increased lipolytic capacity. Such an increase in LPLS447X carriers can be explained by increased lipolytic activity of the LPL dimer or by enhanced LPL–TRL binding mediating facilitated enzymatic conversion in carriers. The LPLS447X protein lacks 2 amino acids at the terminal carboxyl part, which preserves the binding capacity of the LPL protein to lipoproteins and also to heparan sulfate proteoglycans.
In addition to increased TRL conversion, the carriers also exhibited enhanced LDL apoB100 clearance. This LPL-mediated LDL removal has been shown to be largely LDL receptor reliant, whereas only a minor portion occurs receptor independent.19,20 Carriers were characterized by a 4-fold increase in preheparin LPL concentration.21 Because LPL has been reported previously to be present on LDL, the increased preheparin LPL concentration in carriers might contribute to the enhanced clearance of LDL.21 The latter is underscored by the correlation between preheparin LPL concentration and LDL apoB100 clearance in the current study. Subsequently, we sought for evidence that LPL in serum is indeed associated with apoB-containing lipoproteins. To this purpose, we measured LPL concentration in apoB and non-apoB plasma fractions prepared from plasma of LPLS447X carriers and controls. Our results show that a large portion of plasma LPL can be traced in the apoB fraction. These findings concur with those reported by Olivecrona, who showed that preheparin LPL is predominantly present in the apoB fraction.21 In line with the 4-fold increase of LPL concentration in plasma of LPLS447X carriers, a 3- to 4-fold increase in LPL content was also observed in the apoB fraction in LPLS447X carriers. Collectively, these data lend further support to a potential role for LPL in mediating increased LDL removal in LPLS447X carriers.
Several aspects of our study deserve closer attention. First, we have based our conclusions on data derived from a small group of homozygous carriers (n=5). Still, for kinetic studies, a small sample size is not uncommon, even ranging from 2 to 5 subjects in total.22–25 Second, the increased VLDL1 to VLDL2 flux in carriers may have been affected by high values for direct catabolism resulting in low values for transfer in subject 2. This outlying value is a consequence of the concept that no parameter should be artificially fixed in the model.26 However, on reanalysis of the data by changing k1 and k3 in subject 2 (k1=k3=0), we observed no changes to our initial conclusions. Third, the variation in direct catabolism (variation 0.0 to 7.9 pools per day) as well as the variation in LDL apoB100 synthesis (0 to 348 mg per day) in our study appears quite large. However, such large variations are in line with other apoB100 kinetic studies and are therefore likely to reflect biological reality.26,27 Finally, whereas increased turnover of TRL is generally associated with increased HDL and reduced TG levels,8 lipid profiles in the present study were not significantly different between carriers and controls. This apparent discrepancy has several likely explanations, including baseline matching for lipid levels between carriers and controls as well as the limited study size.28–30
In the present study, we show that homozygous LPLS447X carriers exhibit enhanced TRL conversion as well as increased LDL removal. Combined with increased concentration of LPL in the preheparin plasma, our data suggest increased in vivo enzymatic consequences as well as increased nonenzymatic consequences of LPL in LPLS447X carriers. Both mechanisms might contribute to the cardiovascular protection that is associated with the LPLS447X variant.
We thank Mariëtte Ackermans (Laboratory of Endocrinology and Radiochemistry, Department of Clinical Chemistry, Academic Medical Center, Amsterdam, The Netherlands) for measuring the valine enrichment in the LDL fractions. Jose de Boer is thanked for measurement of the valine enrichment in VLDL1 apoB100, VLDL2 apoB100, and IDL apoB100. We also thank Frans Hoek (Department of Clinical Chemistry, Academic Medical Center, Amsterdam, The Netherlands) for extensive measurement of the apoB100 analyses. Florianne de Ruijter is thanked for her contributions to the design of the study as well as laboratory methods.
- Received April 7, 2005.
- Accepted September 7, 2005.
Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis. Circulation. 2002; 105: 1135.
Merkel M, Heeren J, Dudeck W, Rinninger F, Radner H, Breslow JL, Goldberg IJ, Zechner R, Greten H. Inactive lipoprotein lipase (LPL) alone increases selective cholesterol ester uptake in vivo, whereas in the presence of active LPL it also increases triglyceride hydrolysis and whole particle lipoprotein uptake. J Biol Chem. 2002; 277: 7405–7411.
Casaroli-Marano RP, Garcia R, Vilella E, Olivecrona G, Reina M, Vilaro S. Binding and intracellular trafficking of lipoprotein lipase and triacylglycerol-rich lipoproteins by liver cells. J Lipid Res. 1998; 39: 789–806.
Wittrup HH, Tybjaerg-Hansen A, Nordestgaard BG. Lipoprotein lipase mutations, plasma lipids and lipoproteins, and risk of ischemic heart disease. A meta-analysis. Circulation. 1999; 99: 2901–2907.
Mero N, Suurinkeroinen L, Syvanne M, Knudsen P, Yki-Jarvinen H, Taskinen MR. Delayed clearance of postprandial large TG-rich particles in normolipidemic carriers of LPL Asn291Ser gene variant. J Lipid Res. 1999; 40: 1663–1670.
Kozaki K, Gotoda T, Kawamura M, Shimano H, Yazaki Y, Ouchi Y, Orimo H, Yamada N. Mutational analysis of human lipoprotein lipase by carboxy-terminal truncation. J Lipid Res. 1993; 34: 1765–1772.
Kuivenhoven JA, Groenemeyer BE, Boer JMA, Reymer PWA, Berghuis R, Bruin T, Jansen H, Seidell JC, Kastelein JJP. Ser447stop mutation in lipoprotein lipase is associated with elevated HDL cholesterol levels in normolipidemic males. Arterioscler Thromb Vasc Biol. 1997; 17: 595–599.
de Sain-van der Velden M, Rabelink TJ, Gadellaa MM, Elzinga H, Reijngoud DJ, Kuipers F, Stellaard F. In vivo determination of very-low-density lipoprotein-apolipoprotein B100 secretion rates in humans with a low dose of-[1–13C] valine and isotope ratio mass spectrometry. Anal Biochem. 1998; 265: 308–312.
Prinsen BHCM, Romijn JA, Bisschop PH, de Barse MMJ, Barrett PH, Ackermans M, Berger R, Rabelink TJ, de Sain-van der Velden M. Endogenous cholesterol synthesis is associated with VLDL-2 apoB-100 production in healthy humans. J Lipid Res. 2003; 44: 1341–1348.
Kastelein JJ, Jukema JW, Zwinderman AH, Clee S, van Boven AJ, Jansen H, Rabelink TJ, Peters RJ, Lie KI, Liu G, Bruschke AV, Hayden MR. Lipoprotein lipase activity is associated with severity of angina pectoris. REGRESS Study Group. Circulation. 2000; 102: 1629–1633.
Prinsen BHCM, Rabelink TJ, Romijn JA, Bisschop PH, de Barse MMJ, de Boer J, van Haeften TW, Barrett PH, Berger R, de Sain-van der Velden M. A broad-based metabolic approach to study VLDL apoB100 metabolism in patients with ESRD and patients treated with peritoneal dialysis. Kidney Int. 2004; 65: 1064–1075.
Pruneta V, Autran D, Ponsin G, Marcais C, Duvillard L, Verges B, Berthezene F, Moulin P. Ex vivo measurement of lipoprotein lipase-dependent very low density lipoprotein (VLDL)-triglyceride hydrolysis in human VLDL: an alternative to the postheparin assay of lipoprotein lipase activity? J Clin Endocrinol Metab. 2001; 86: 797–803.
Garenc C, Perusse L, Gagnon J, Chagnon YC, Bergeron J, Despres JP, Province MA, Leon AS, Skinner JS, Wilmore JH, Rao DC, Bouchard C. Linkage and association studies of the lipoprotein lipase gene with postheparin plasma lipase activities, body fat, and plasma lipid and lipoprotein concentrations: the HERITAGE Family Study. Metabolism. 2000; 49: 432–439.
Mulder M, Lombardi P, Jansen H, van Berkel TJ, Frants RR, Havekes LM. Low density lipoprotein receptor internalizes low density and very low density lipoproteins that are bound to heparan sulfate proteoglycans via lipoprotein lipase. J Biol Chem. 1993; 268: 9369–9375.
Rumsey SC, Obunike JC, Arad Y, Deckelbaum RJ, Goldberg IJ. Lipoprotein lipase-mediated uptake and degradation of low density lipoproteins by fibroblasts and macrophages. J Clin Invest. 1992; 90: 1504–1512.
Vilella E, Joven J, Fernandez M, Vilaro S, Brunzell JD, Olivecrona T, Bengtsson-Olivecrona G. Lipoprotein lipase in human plasma is mainly inactive and associated with cholesterol-rich lipoproteins. J Lipid Res. 1993; 34: 1555–1564.
Welty FK, Lichtenstein AH, Barrett PH, Dolnikowski GG, Ordovas JM, Schaefer EJ. Production of apolipoprotein B-67 in apolipoprotein B-67/B-100 heterozygotes: technical problems associated with leucine contamination in stable isotope studies. J Lipid Res. 1997; 38: 1535–1543.
Ouguerram K, Magot T, Zair Y, Marchini JS, Charbonnel B, Laouenan H, Krempf M. Effect of atorvastatin on apolipoprotein B100 containing lipoprotein metabolism in type-2 diabetes. J Pharmacol Exp Ther. 2003; 306: 332–337.
Batista MC, Welty FK, Diffenderfer MR, Sarnak MJ, Schaefer EJ, Lamon-Fava S, Asztalos BF, Dolnikowski GG, Brousseau ME, Marsh JB. Apolipoprotein A-I, B-100, and B-48 metabolism in subjects with chronic kidney disease, obesity, and the metabolic syndrome. Metabolism. 2004; 53: 1255–1261.
Welty FK, Lichtenstein AH, Barrett PH, Dolnikowski GG, Ordovas JM, Schaefer EJ. Decreased production and increased catabolism of apolipoprotein B-100 in apolipoprotein B-67/B-100 heterozygotes. Arterioscler Thromb Vasc Biol. 1997; 17: 881–888.
Groenemeijer BE, Hallman MD, Reymer PW, Gagne E, Kuivenhoven JA, Bruin T, Jansen H, Lie KI, Bruschke AV, Boerwinkle E, Hayden MR, Kastelein JJ. Genetic variant showing a positive interaction with beta-blocking agents with a beneficial influence on lipoprotein lipase activity, HDL cholesterol, and triglyceride levels in coronary artery disease patients. The Ser447-stop substitution in the lipoprotein lipase gene. REGRESS Study Group. Circulation. 1997; 95: 2628–2635.
Lee J, Tan CS, Chia KS, Tan CE, Chew SK, Ordovas JM, Tai ES. The lipoprotein lipase S447X polymorphism and plasma lipids: interactions with APOE polymorphisms, smoking, and alcohol consumption. J Lipid Res. 2004; 45: 1132–1139.