Wednesday October 1, 2003: Poster Session Lipid and lipoprotein metabolism 229 WEDNESDAY Methods: Forty subjects (25 males and 15 females) were recruited from the Glasgow Royal Infirmary, Scotland and from the University of Wollongong, Australia. Fasted blood samples were obtained and plasma was obtained after centrifugation at 3000rpm, 4°C for 15 min. Sequential ultracentrifugation was used to isolate IDL (1.006g/ml-1.030g/ml) using a 50.4Ti rotor at 35,000rpm at 15C in a Beckman L7 55 ultracentrifuge. CS was coupled to CNBr activated Sepharose 6MB using a method provided by Amersham Pharmacia Biotech. Approximately 1mg protein of IDL was allowed to interact with CS for 30 minutes at 4C. The unbound IDL particles were eluted using a low salt buffer whilst the bound IDL particles were eluted using 1M sodium chloride. On average 70 per cent of IDL bound to CS ranging from 56-92 per cent. Total IDL, unbound IDL and bound IDL particles were analysed for lipid and apolipoprotein composition using standard commercially available kits and their particle size was assessed by gradient gel electrophoresis. Results: The unbound IDL particles were triglyceride rich (40% versus 33%, p<0.01), whereas the bound IDL particles were cholesterol rich (21% versus 14%, p<0.01). The unbound particles were significantly larger than the bound particles (30nm versus 28nm diameter respectively, p<0.01). The unbound particles contain at least double the amount of apo C-II and apo C-III per IDL particle compared with the bound IDL particles. Conclusion: There are specific IDL particles that bind to CS in vitro, these being the cholesterol rich IDL particles. It remains to be determined if these cholesterol rich IDL particles are potentially more atherogenic than the triglyceride rich IDL particles. 3P-0762 The difference of the location of Apo-B on the surface of naturally occurring modified low density lipoproteins A. Orekhova 1 , I. Suprun 2 , I. Sobenin 3 , O. Panasenko 1 , A. Orekhov 4 . 1 Research Institute of Physico-Chemical Medicine; 2 Cardiovascular Research Center; 3 Institute of Experimental Cardiology; 4 Institute for Atherosclerosis Center, Russia Earlier we have found and isolated from the blood of coronary atherosclerosis patients a subfraction of naturally occurring modified low density lipoprotein (nomLDL) that can stimulate the accumulation of cholesteryl esters in the cells cultured from unaffected human aortic intima. NomLDL is able to interact with scavenger receptor, while the binding to apoB-receptor is impaired. Additionally, nomLDL are capable to aggregate spontaneously, thus triggering increased phagocytosis of aggregated lipoprotein. We have supposed that the difference between native and nomLDL with the respect to ability to induce intracellular lipid deposition may be due to improper location of apo-B on the surface of nomLDL. In this study we have compared the ability of native and nomLDL to interact with anti-apoB monoclonal antibodies using ELISA tech- nique. It has been shown that nomLDL binds to 2E3, 2G1 and 7C2 monoclonal antibodies more effectively than native LDL. On the other hand, the binding of nomLDL to 2G8 and 6E2 antibodies was decreased as compared to native LDL. There was no difference between native and nomLDL in the binding to 5F8, 4C11 and 3G4 antibodies. The results of this study demonstrate that certain epitopes of apoB are located on lipoprotein surface in native and nomLDL in a different way, thus allowing to explain possible mechanisms of intracellular lipid accumulation caused by nomLDL that may be realized through interaction with scavenger receptor, asialoglycoprotein receptor, cell surface proteoglycans as well as though spontaneous aggregation followed by uncontrolled phagocytosis. 3P-0763 Apolipoprotein CI is a potent inhibitor of lipoprotein lipase in vitro and in vivo P. Rensen 1,2 , J. Berbée 1,2 , C. Van der Hoogt 1,2 , G. Gerritsen 3 , A. Van der Zee 3 , D. Sundararaman 1 , K. Willems van Dijk 2,3 , L. Havekes 1,2,4 . 1 TNO-PG, 2 Dept. General Internal Medicine, 3 Dept. Human Genetics, 4 Dept. Cardiology, LUMC, Netherlands Background: ApoCI is an important modulator of plasma lipid metabolism. Human apoCI-overexpressing transgenic (APOC1) mice show VLDL-specific 12-fold increased plasma TG levels (3.9±0.8 mM) and 2-fold increased cholesterol (TC) levels (4.3±0.6 mM) as compared to wild-type mice. Aim: Since the proposed functions of apoCI (inhibition of hepatic lipase and binding of TG-rich remnants to hepatic apoE-receptors) cannot account for the severe hyperTG observed in APOC1 mice, the aim of this study was to elucidate the mechanism underlying apoCI-induced hyperlipidemia. Results: Apoe-/-APOC1 mice were generated to evaluate the effects of APOC1 in absence of apoE. The high plasma apoCI levels (>150 mg/dL) induced a dramatic VLDL-specific 46-fold increase in TG (23±9 mM) and an additional 2-fold increase in TC (29±6 mM) as compared to apoe-/- mice, indicating that the apoCI-induced hyperlipidemia is not just caused by blocking apoE-mediated receptor binding. Accordingly, adenovirus-mediated moderate APOC1 expression in apoe-/- mice (25±6 mg/dL) increased TG levels 4-fold, leaving TC levels unaffected. Also, the hyperTG in the APOC1 transgenic mouse models was not caused by an increased intestinal [ 3 H]TG absorption or hepatic VLDL production. Therefore, the effect of apoCI on LPL was evaluated. Whereas total post-heparin LPL activity was not different between apoe-/-APOC1 and apoe-/- mice (30.2±10.1 vs 32.9±7.7 μmol FFA/h/ml), apoCI dose-dependently inhibited the LPL-mediated lipolysis of [ 3 H]TG-labeled protein-free VLDL-mimicking TG-rich emulsions in vitro (35% at TG:apoCI = 5:1, w/w) with similar efficacy as the well-established LPL-inhibitor apoCIII. Conclusion: ApoCI is a potent inhibitor of LPL-mediated TG hydrolysis. The apoCI-induced combined hyperlipidemia in mice is thus primarily caused by an impaired LPL-mediated lipolytic conversion rather than by a defective hepatic clearance of TG-rich lipoproteins. 3P-0764 Apolipoprotein AV expression in mice ameliorates the hyperlipidemia associated with APOE2 and APOE3Leiden by stimulating LPL-mediated VLDL-triglyceride hydrolysis P. Rensen 2,3 , F. Schaap 1 , P. Voshol 2,4 , H. Van der Vliet 1 , R. Chamuleau 1 , L. Havekes 2,3,5 , B. Groen 1 , K. Willems van Dijk 3,6 . 1 AMC Liver Center, Amsterdam; 2 TNO-PG; 3 Dept. General Internal Medicine, 4 Dept. Endo- crinology, 5 Dept. Cardiology, 6 Dept. Human Genetics, LUMC, Netherlands Background: The recently discovered apolipoprotein apoAV clearly affects plasma TG levels via a hereto-unknown mechanism. In mouse models, over- expression of apoAV induced hypoTG and apoAV-deficiency was associated with hyperTG. Aim: To further elucidate the role of apoAV in lipoprotein metabolism by adenovirus-mediated transfer of the human apoAV gene in hyperlipidemic transgenic mouse models. Results: Injection of Ad-APOA5 (1E9 pfu) in APOE2-knockin mice de- creased plasma TG levels by 85% as compared to Ad-mock injected mice (0.2±0.03 vs 1.2±0.4 mM). Surprisingly, plasma total cholesterol (TC) was also decreased by 65% (2.9±0.5 vs 8.6±1.2 mM). Similarly, after injection of Ad-APOA5 (1E9 pfu) in APOE3Leiden transgenic mice, plasma TG levels were reduced by 65% (0.5±0.2 vs 1.4±0.2 mM) concomitant with a 50% reduction of plasma TC levels (0.9±0.2 vs 1.9±0.1 mM). This hypolipidemic effect of apoAV was not caused by reducing the VLDL-TG production rate, comparing Ad-APOA5 to Ad-mock injection in both mouse models, as deter- mined using Triton WR1339. These data suggest that apoAV acts by enhancing the rate of TG-disposal from lipoproteins. Therefore, we determined the effect of purified apoAV on bovine LPL-mediated TG-hydrolysis from [ 3 H]TG- labeled VLDL-like emulsions in vitro. Whereas apoCIII dose-dependently decreased the rate of LPL-mediated TG-hydrolysis (56% at TG:apoCIII = 50:5, w/w), and apoAI had no effect, apoAV significantly enhanced the rate of LPL mediated TG-hydrolysis (45% at TG:apoAV = 50:3, w/w). Conclusion: ApoAV enhances the LPL-mediated lipolytic conversion of TG-rich lipoproteins, thereby stimulating remnant formation and hepatic clearance in hyperlipidemic mice. Since this combined lipid-lowering effect of apoAV is clearly dominant over the decreased LDL-receptor binding affinity of both apoE2 and apoE3Leiden, apoAV may represent a novel therapeutic tool in hyperlipidemia. 3P-0765 The VLDL receptor plays a crucial role in postprandial lipoprotein metabolism P. Rensen 1,2 , J. Goudriaan 1 , B. Teusink 1 , K. Willems van Dijk 2,3 , L. Havekes 1,2,4 , P. Voshol 1,5 . 1 TNO-PG, 2 Dept. General Internal Medicine, 3 Dept. Human Genetics, 4 Dept. Cardiology, 5 Dept. Endocrinology, LUMC, Netherlands Background: The VLDL receptor (VLDLr) has previously been implicated to play a role in the metabolism of VLDL by facilitating the delivery of LPL-released VLDL-triglyceride (TG)-derived fatty acids (FA) to adipose tissue. However, fasted vldlr-/- mice have not shown any phenotype regarding plasma lipoprotein and lipid levels, indicating that such a role of the VLDLr in VLDL metabolism is only minor. Aim: To investigate whether the VLDLr does play a prominent role in postprandial lipoprotein metabolism. Results: Vldlr-/- mice did not show altered 4h-fasted plasma TG levels as compared to vldlr+/+ mice (0.44±0.20 vs 0.27±0.06; P>0.05). However, TG XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan