© 1995 American Heart Association, Inc.
Articles |
The 17th Annual Meeting of the European Lipoprotein Club
Correspondence to Dr A.F.H. Stalenhoef, Department of Medicine, Division of General Internal Medicine, University Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
Key Words: meeting summary • European Lipoproteins Club
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Introduction |
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 Top Introduction |
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The European Lipoprotein Club met September 12-15, 1994, in Tutzing, Germany. There were 92 participants from 12 European countries and the United States.
Dr John Wetterau of Bristol-Myers Squibb Pharmaceutical Research Institute opened the meeting with a State of the Art Lecture entitled "The Microsomal Triglyceride Transfer Protein: Characterization and Role in the Assembly of VLDL and Chylomicrons." The microsomal triglyceride transfer protein (MTP) is a soluble lipid transfer protein found in the lumen of microsomes of liver and intestine. This heterodimer is composed of the multifunctional protein disulfide isomerase and a unique large subunit of molecular weight 97 000. In vitro, MTP catalyzes the transport of triglyceride, cholesteryl ester, and a variety of phospholipids between membranes. When one compares the fractional transfer rates, MTP appears to selectively bind and transport hydrophobic neutral lipid. However, the assays used to characterize MTP have at least 100-fold more phospholipid than neutral lipid, so in these assays, MTP actually transports more phospholipid than neutral lipid.
Abetalipoproteinemia is an autosomal recessive disease in which the subjects have a defect in the assembly and secretion of VLDL and chylomicrons. Recent studies by Dr Wetterau of abetalipoproteinemic subjects showed that MTP activity and the unique large subunit were absent in all four unrelated subjects studied. Frame shift, nonsense, and point mutations that explain the absence of MTP were found in these four subjects. These studies indicated that an absence of MTP function causes abetalipoproteinemia and that MTP is required for the assembly of apoB–containing lipoproteins.
Hela cells normally do not synthesize and secrete apoB–containing lipoproteins. When Hela cells were stably transfected with MTP, they were able to efficiently synthesize and secrete apoB-53 particles with a buoyant density of HDL or LDL. Both the level of apoB secretion and the density of the particles formed were regulated by lipid addition to the growth media.
MTP mRNA and protein levels were found to be regulated by dietary manipulation in hamsters. A high-fat diet elevated MTP large-subunit mRNA levels in liver and intestine to about 125% and 50% above control levels, respectively, while a high-sucrose diet elevated mRNA levels 50% in the liver only. When animals from four different dietary conditions were combined, strong positive correlations between intestinal MTP mRNA levels and plasma lipid levels were observed. This suggests that MTP may play a role in regulating plasma lipoprotein levels or that common factors may regulate MTP message and plasma lipid levels.
To determine the factors that may be controlling MTP message levels in vivo, the regulation of MTP mRNA levels was found to be decreased by physiological concentrations of insulin. However, MTP protein has a long half-life in HepG2 cells (more than 4 days), so the acute changes in MTP mRNA levels had no effect on MTP activity levels.
The first session of the meeting entitled "Lipoproteins-Arterial Wall Interactions" began with an excellent lecture by Dr David Hajjar on the role of the cytokine network in LDL cholesterol trafficking in arterial cells. It is apparent that cells participating in the inflammatory response associated with atherogenesis interact not only with components of the developing lesion but also with each other. His talk therefore focused on the mechanisms by which cell-derived soluble mediators such as cytokines, growth factors, and eicosanoids regulate vascular cholesterol metabolism and modulate foam cell development. By use of an experimental design incorporating cocultures of endothelial cells and smooth muscle cells separated by a filter, endothelial cell modulation of cholesterol metabolism in smooth muscle cells (SMC) could be studied. The presence of endothelial cells was found to significantly reduce the amount of free cholesterol in SMC and of both cholesterol and cholesterol ester in cholesterol ester–enriched cells. The question then arose as to whether soluble mediators were modulating the delivery of cholesterol and cholesterol ester to vascular cells. Expression of tumor necrosis factor (TNF) and interleukin-1 (IL-1) has been shown to be increased in atherosclerotic tissue. Both cytokines were found to promote the binding and uptake of LDL into SMC. The mechanisms by which these cytokines modulate the LDL receptor appear to operate at the level of LDL receptor gene transcription through the sterol regulatory element. Growth factors such as platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) also increase LDL-receptor expression; the effect is more rapid than that seen with the cytokines.
Cytokines were also shown to be mediators of intracellular cholesterol metabolism. Thus, TNF and IL-1 increased the activities of acid cholesterol ester hydrolase and neutral cholesterol ester hydrolase, thereby promoting the efflux of cholesterol from the cell. The eicosanoids PGI2 and 12-HETE elevate cholesterol ester hydrolase in the cell by elevating intracellular levels of cAMP. Cytokines such as TNF and IL-1 stimulate endothelial cell eicosanoid biosynthesis by induction of cyclooxygenase and stimulate cAMP production in the cell. The cytokine network therefore appears to be closely linked to eicosanoid pathways, and the interaction of the two systems will have a significant impact on cholesterol trafficking in the arterial vessel wall.
The following talk by Dr L. Nielsen considered the effect of angiotensin II, noradrenaline, and the angiotensin-converting inhibitor enalapril on the transfer of LDL into the arterial wall in rabbit and pig models. Intravascular infusion of angiotensinogen II caused an initial increase in blood pressure. This was followed by a return to normal after 2 hours, despite the continued infusion of angiotensinogen II. The normalized influx of LDL was significantly greater during angiotensinogen II infusion at high blood pressure than during infusion at normal blood pressure or during saline infusions. When noradrenaline was used to increase blood pressure to a level similar to that seen in angiotensin II–treated rabbits, the normalized influx of LDL was also elevated. On the other hand, when the production of endogenous angiotensin II was inhibited with enalapril, the normalized influx of LDL was similar to that seen in a placebo group, despite a lower blood pressure in the enalapril-treated animals. Dr Nielsen concluded that the angiotensin II–mediated increase in the normalized influx of LDL into the arterial wall was due largely to the concomitant increase in blood pressure rather than a direct effect on endothelial permeability.
Dr M. Soma began by briefly reviewing the role of mevalonate and its nonsterol products such as geranylgeraniol and farnesol in the control of cellular proliferation. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors competitively inhibit intracellular synthesis of mevalonate and have been shown to inhibit arterial myocyte proliferation in vitro. Dr Soma then described an in vivo study aimed at determining the effect of statins on neointimal formation in the carotid arteries of normocholesterolemic rabbits. Intimal thickening was induced by inserting a flexible extra-arterial collar around the common carotid artery. Although treatment with various statins did not alter plasma cholesterol concentrations, all statins tested showed less neointimal formation than control animals. He concluded that the inhibition of carotid intimal myocyte proliferation by statins is independent of their effect on cholesterol and is presumably due to local inhibition of mevalonate synthesis.
The next speaker, Dr W. Hendricks, described her experiments to determine the influence of lipoprotein lipase (LPL) on the binding and uptake of both native and oxidized LDL and VLDL by J774 macrophages. LPL caused a 10-fold increase in the binding and uptake of both VLDL and LDL. Preincubation of cells with LDL revealed that this uptake was at least partly mediated through the LDL receptor. Uptake of moderately oxidized LDL was also stimulated by LPL through a mechanism that was independent of the LDL receptor. Although LPL could bind to strongly oxidized LDL and acetyl-LDL, the uptake of these lipoprotein particles by J774 cells was not increased in the presence of LPL.
Dr W. Jaross discussed the induction of lipid accumulation in macrophages through secretory phospholipase A2. This enzyme, present in inflammatory tissue and exudates, is secreted by a number of cells, including platelets, granulocytes, chondrocytes, and SMC. By use of four different monoclonal antibodies specific for phospholipase A2, this enzyme could be detected in foam cells present in atherosclerotic lesions. No expression of the enzyme was observed in normal arteries. Incubation of isolated HDL and LDL with phospholipase A2 led to an increase in the net negative charge of both lipoproteins. With spin labels and electron resonance spectroscopy, it could be demonstrated that these modified lipoprotein particles displayed decreased surface fluidity. Exposure of mouse peritoneal macrophages to phospholipase-modified LDL caused a dramatic increase in intracellular free and esterified cholesterol content as well as an increase in acyl:coenzyme A cholesterol acyltransferase activity.
The isoprenoid requirement for acetyl-LDL degradation and cholesterol accumulation in macrophages was the topic of the presentation by Dr F. Bernini. Statins have been shown to reduce the acetyl-LDL–induced cholesterol esterification and accumulation in macrophages, an effect that is not related to a direct inhibition of ACAT activity. In the present study, he observed a dose-dependent inhibition of 125I-acetyl-LDL in mouse peritoneal macrophages by fluvastatin and a parallel decrease in the ability of this modified lipoprotein to induce cholesterol esterification and accumulation. When macrophages were incubated with 3H-cholesteryl ester–labeled acetyl-LDL in the presence of fluvastatin, this drug was found to reduce the cellular content of 3H-cholesteryl ester, with a concomitant increase of cholesteryl ester in the incubation medium. The effects of fluvastatin could be fully overcome by the addition of mevalonate or geranylgeraniol but not farnesol or squalene.
Dr L. Calabresi presented data on the structural and
functional properties of the apolipoprotein A-IMilano
(A-IM), a natural variant of apoA-I. A-IM is characterized by a
cysteine for arginine substitution that leads to the formation of the
disulfide-linked homodimer A-IM/A-IM. This dimer was purified from the
plasma of carriers and a recombinant form of the protein was also
produced through expression of the cDNA in Escherichia coli.
The A-IM/A-IM dimer showed a significantly higher degree of 
-helices
than normal A-I. Reconstituted HDL (rHDL) containing
phosphatidylcholine and A-IM/A-IM was more effective in promoting the
efflux of cholesterol from cholesterol-loaded mouse peritoneal
macrophages than the corresponding rHDL containing apoA-I. In an in
vivo study, rHDL containing A-IM/A-IM was shown to inhibit SMC
proliferation and intimal thickening in the rabbit model described by
Dr Soma.
Dr V. Tertov discussed the metabolism of native and desialylated LDL by human intimal cells from both normal intima and atherosclerotic lesions. Uptake of desialylated LDL was more avid than that of normal LDL in both types of cells. Competition experiments with a monoclonal antibody specific for the LDL receptor and with acetyl-LDL revealed that desialylated LDL could bind to both the LDL receptor and the scavenger receptor. However, degradation of the modified LDL was 1.5- to 5-fold lower than that of native LDL, suggesting decreased lysosomal degradation of the modified lipoprotein. Furthermore, the activities of lysosomal proteinases were decreased in atherosclerotic cells compared with normal intimal cells. He speculated that increased uptake and decreased lysosomal degradation of desialylated LDL may be major factors in the accumulation of lipid in human aortic SMC.
The evening program began with a stimulating presentation from the second invited speaker of this session, Dr German Camejo, on the interaction of lipoproteins with the intima extracellular matrix and the contribution of this process to lipoprotein deposition during atherogenesis. He pointed out that as early as 1949, Mogens-Faber et al reported on an association between intimal mucopolysaccharides and cholesterol deposition that was confirmed in several later publications. His talk focused on the structural basis for such an interaction, the structural alterations of LDL induced by association with arterial proteoglycans, and the consequence of such alterations for the interaction of LDL with cells.
By use of synthetic segments of apoB-100 and frontal elution affinity chromatography, the hydrophilic nine-residue amino acid RLTRKRGLK was found to effectively compete the interaction of LDL with chondroitin 6-sulfate. A secondary model peptide with hydrophobic tails was then constructed that could bind to lipid vesicles and lipoproteins. The association with lipid substantially enhanced the affinity of this peptide for chondroitin 6-sulfate, demonstrating the importance of the lipid environment for inducing the proper secondary structure of this apoB-100 segment. From his results, Dr Camejo hypothesized that two basic apoB-100 segments may function cooperatively in the binding of LDL to both the receptor and extracellular proteoglycans.
Dr Camejo next considered the consequences of the binding of LDL to arterial proteoglycans. Macrophages rapidly internalize proteoglycan-bound LDL. By use of arterial chondroitin sulfate proteoglycans, four subclasses of LDL with different structural properties and capacity to interact with human monocyte–derived macrophages could be identified. The LDL subclass with the highest affinity for proteoglycan and whose complexes showed the most avid uptake had the highest isoelectric point and the lowest ratio of surface to core lipid. The binding of LDL to arterial proteoglycans or glycosaminoglycans also rendered this lipoprotein more susceptible to copper-induced oxidation than native LDL.
In the final part of his presentation, Dr Camejo presented data on the binding of LDL to chondroitin sulfate–rich proteoglycans that are secreted by quiescent and proliferating human arterial SMC. Measured with a gel mobility shift assay, LDL had a threefold higher apparent affinity constant for the proteoglycans from proliferating cells than for those from resting cells. This increased affinity was apparently related to the higher relative proportion of proteoglycans with longer glycosaminoglycan chains that are secreted by proliferating cells compared with quiescent cells. Such a mechanism could lead to an increased uptake of LDL in atherosclerotic lesions that contain proteoglycans derived from actively proliferating SMC.
Dr C. Banfi then described her experiments into the regulation of plasminogen activator inhibitor type-1 (PAI-1) by acetyl-LDL in human umbilical vein endothelial cells (HUVEC). Immunoprecipitation studies showed that PAI-1 levels in the condition medium from HUVEC and in the extracellular matrix were increased after incubation of the cells with acetyl-LDL. An induction of the 2.2- and 3.4-kb mRNA transcripts for PAI-1 was also observed under these conditions. Fucoidan, a competitive ligand for the scavenger receptor, in contrast to chondroitin sulfate, a noncompetitive compound, could reduce PAI-1 levels in acetyl-LDL–stimulated cells. PAI-1 levels were not affected by enrichment of the cells with nonlipoprotein cholesterol or triglyceride. She concluded that activation of the scavenger receptor in HUVEC by acetyl-LDL is one possible mechanism for induction of PAI-1 synthesis.
The detection of cholesteryl ester transfer protein (CETP) in the human arterial wall was the theme of the talk by Dr P. Moulin. Previous experiments had shown that CETP could be synthesized by extrahepatic tissues such as adipose tissue and muscle. With indirect immunofluorescence and a monoclonal antibody (mab TP2), CETP immunoreactivity could be detected in early lesions from coronary arteries. The protein was also identified by Western blotting after immunoprecipitation of arterial wall homogenates with mab TP2. All aortic samples tested exhibited a 74-kD CETP band. CETP immunoreactivity was located in the SMC of the media. However, no CETP expression could be detected in primary or secondary cell cultures.
The following talk by Dr A. von Eckardstein considered the influence of
apoE polymorphism on the release and reverse cholesterol transport of
cell-derived cholesterol. In addition to pre-ß1-LpA-I, 
-LpE, a
lipoprotein containing apoE as its only apolipoprotein and with
-mobility on electrophoresis, could be identified as a further
lipoprotein capable of accepting cell-derived cholesterol and
transferring it to other lipoproteins. Plasmas from probands with
different apoE phenotypes were pulse-chase incubated with fibroblasts
loaded with 3H-cholesterol; then the lipoproteins were
separated and their radioactive content determined. No -LpE was
found in the E4/4, E4/2, and E2/2 probands. The release of cholesterol
from cells into plasma was on the order of E3/3 >E4/4
E2/2.
Individuals with E3 would therefore appear to have an enhanced ability
for the efflux of cholesterol from cells compared with E4 and E2.
Dr J. Pedreno described studies undertaken to investigate the nature of the platelet LDL receptor by determining its proteolytic susceptibility. In contrast to fibroblast LDL receptor, the platelet LDL receptor was resistant to mild proteolysis with either chymotrypsin or trypsin. Harsher treatment with chymotrypsin also did not alter the binding characteristics of LDL to the platelet LDL receptor, whereas trypsin pretreatment caused a dose-dependent enhancement of LDL binding. This was due to a twofold increase in the number of binding sites. The platelet LDL receptor was also able to recognize both native and oxidized LDL. These data demonstrate that the platelet LDL receptor exhibits characteristic properties that are different than those of the "classic" LDL receptor.
The program of the second session focused on the interaction of postprandial lipoproteins with peripheral cells. In his invited lecture, Dr G. Ailhaud introduced the adipocyte as a model "peripheral cell." He convincingly demonstrated that free fatty acids (FFA) fulfill an important regulatory role in the process of adipocyte differentiation. FFA regulate the transcription of a whole set of genes considered differentiation markers. Most importantly, FFA also induce the expression of a transacting factor called fatty acid–activated receptor, or FAAR protein. This nuclear receptor is highly homologous to human NUC 1 and can, in its activated form, initiate the differentiation of fibroblasts to adipocytes in transfection experiments.
The second invited speaker, Dr U. Beisiegel, reviewed the state of the art regarding LPL as a ligand for cell surface receptors. Dr Beisiegel emphasized that LPL represents a strong ligand for the LDL receptor–related protein (LRP). Additionally, however, LPL might also enhance the apoE3-mediated uptake of lipoproteins by the LDL receptor. Such a cooperative process might be important in the efficient clearance of chylomicron remnants and other triglyceride-rich lipoproteins from circulation. Dr Beisiegel also proposed that as reported for LPL, hepatic triglyceride lipase can act as a ligand for LRP, which may be involved in the uptake of chylomicron remnants by the liver.
Dr J. Glieman and colleagues were able to locate the LRP binding region on LPL between amino acids 378 and 423, having produced a whole series of LPL peptide fragments using CNBr fragmentation of the enzyme or prokaryotic expression systems of LPL fusion proteins. Although oligopeptides spanning this region either bind to LRP or inhibit LPL-LRP interaction in competition experiments, they do not interact with lipoproteins. This suggests that the LRP binding region and the lipid binding domain of LPL are located at separate positions.
Dr W. Schneider presented the chicken oocyte as a model cell for
efficient lipid and lipoprotein uptake. To achieve the enormous
accumulation of lipids and other yolk precursors, chicken oocytes
express a variety of lipoprotein receptors that are distinct from
receptors found on somatic cells. These oocyte receptors belong to the
ever-growing family of LRP genes and include a 95-kD receptor
(homologous to the human VLDL receptor) and an oocyte-specific LRP-like
receptor that is different from the somatic LRP. Both oocyte-specific
receptors are not regulated by sterols. They not only facilitate the
efficient uptake of lipoproteins but also are involved in the uptake of
other yolk precursors such as vitellogenin, retinol binding protein,
and -2-macroglobulin. These results emphasize the functional
diversity of members of the LDL receptor gene family.
Dr M. Gåfvels presented his results on the structure and function of the VLDL receptor. This protein is highly homologous among humans, rabbits, and mice and is closely related to the LDL receptor. It is expressed in many cell types but not in liver cells, can bind apoE-containing lipoproteins, and in contrast to the LDL receptor, is not regulated by sterols. Interestingly, the VLDL receptor was detected on SMC of atherosclerotic plaques, where it is colocalized with factor VIII.
Dr S. Levastel presented her work on the modulation of the binding of LpB to the LDL receptor by lipids and apolipoproteins. Affinity-purified LpB with no other apolipoproteins present besides apoB was enriched with triglycerides and/or apoCI, apoCII, apoCIII, and apoE by in vitro incubation experiments. Decreased binding of LpB to cultured cells was observed in the presence of excess triglycerides or additional apoC peptides. ApoCIII had the most pronounced inhibitory effect. The addition of apoE to LpB did not increase lipoprotein binding at 4°C but markedly increased its degradation at 37°C.
The last presentation of the second session was made by Dr R. Savonen on the role of LPL in the uptake of chylomicrons and ß-VLDL in the perfused rat liver. He demonstrated a strong contribution of LPL for uptake of these lipoproteins by the liver. Using specific inhibitors to inactivate the lipolytic activity of the enzyme, Dr Savonen demonstrated that LPL-mediated lipoprotein uptake by the liver is independent of its enzymatic activity.
The third session, "Determinants of Postprandial Lipoprotein Metabolism," began with an excellent overview by Dr Anders Hamsten entitled "The Atherogenic Chylomicron Remnant Hypothesis Revisited" about the relation between triglyceride-rich lipoproteins, alimentary lipemia, and coronary artery disease (CAD). Recent studies in his laboratory demonstrated a relation between the content of cholesteryl ester molecules in small VLDL particles and the global coronary atherosclerosis score in young male post–myocardial infarction patients with hypertriglyceridemia (Tornvall et al. Circulation. 1993;88:2180-2189). His group has also demonstrated that the amount of small chylomicron remnants (Svedberg flotation rate [Sf] 20 to 60 apoB-48) after a standard mixed meal (50 g/m2, glucose, egg yolk) was distinctly related to the rate of progression of coronary lesions, assessed angiographically with a time interval of around 5 years, whereas no relation was found with larger remnant particles (Karpe et al. Atherosclerosis. 1994;106:83-94).
Dr Hamsten discussed the effects of postprandial lipemia (induced by a mixed-meal type of oral fat tolerance test or intravenous infusion of 10% Intralipid) on the levels of apoB-48– and apoB-100–containing lipoproteins in different Sf fractions. In the Sf fraction of 60 to 400, he observed a rise in both apoB-100 and apoB-48 in normotriglyceridemic and hypertriglyceridemic subjects, whereas in the Sf fraction of 20 to 60, a rise was seen in apoB-48 only in normolipidemic subjects and not in hypertriglyceridemic patients (Karpe et al. J Clin Invest. 1993;91:748-759). A large increase in VLDL–apoB-100 after a fat-containing meal (80% of the increment in triglyceride-rich lipoprotein particle number) was also reported by Schneeman et al (Proc Natl Acad Sci U S A. 1993;90:2069-2073). The infusion of Intralipid during 60 minutes produced a consistent rise in large VLDL–apoB-100, while there was a (less homogeneous) decrease in small VLDL particles, depending on the basal levels. From these studies, Dr Hamsten concluded that the rise in VLDL during alimentary lipemia is secondary to competition by chylomicrons for the same lipolytic pathway and that chylomicrons and their remnants may be implicated in atherosclerosis by impeding the normal lipolytic degradation of VLDL.
In the next part of his talk, Dr Hamsten focused on the relation between the clearance of triglyceride-rich lipoproteins and the plasma level of HDL. He stressed the strong inverse correlation between fasting plasma HDL (in particular, HDL2b subfraction) and the larger Sf fraction of 60 to 400 during postprandial lipemia. HDL could thus be seen as a surrogate marker for a disturbed triglyceride clearance, although this inverse relation between HDL and postprandial lipemia is not always present.
Finally, Dr Hamsten discussed the influence of alimentary lipemia on LDL heterogeneity. To investigate the major metabolic determinants of the LDL subfraction distribution, LDL subfractions were separated by density ultracentrifugation in a study of 32 men with angiographically assessed premature CAD; the response of triglyceride-rich lipoproteins to the standard mixed meal was determined and related to LDL subfraction distribution and different enzyme activities. The response of triglyceride-rich lipoproteins to the oral fat load was positively correlated with the dense LDL apoB concentration (Karpe et al. Atherosclerosis. 1993;98:33-49).
In the next presentation, Dr S. Humphries described the influence of a common genetic variation in the apoB signal peptide on postprandial lipemia. In collaboration with the Stockholm group, young male survivors of a myocardial infarction (normotriglyceridemic and hypertriglyceridemic) and healthy control subjects underwent the standard mixed-meal type of fat tolerance test; the postprandial areas under the curve for lipids, apoB-48, and apoB-100 in the Sf fractions of 20 to 60 and 60 to 400 were examined by genotypes. Individuals with one or more SP-24 alleles of the apoB SP length polymorphism had a 28% smaller mean area under the curve (P=.06) for large chylomicron remnants and a 23% smaller mean area under the curve (P=.01) for large VLDLs compared with individuals homozygous for the wild-type SP-27 allele. This was due to a slower increase of these lipoproteins in the early postprandial phase in carriers of the SP-24 allele (6.5 mg · L-1 · h-1 versus 15 mg · L-1 · h-1 in homozygotes for the SP-27 allele), suggesting that length variation in the apoB for SP influences the rate of secretion of postprandial lipoproteins. These results lend support to yeast expression studies of fusion proteins of the apoB SP isoforms and a yeast secretory protein; compared with the SP-27 common isoform, the SP-24 deletion isoform was inefficient in ensuring maintenance of the growing apoB peptide with the endoplasmic reticulum membranes and was secretion-defective (Sturley et al. J Biol Chem. 1994;269:21670-21675).
Dr A. van Tol described the effects of an oral fat load on HDL lipids in normolipidemic men with angiographically assessed CAD (70% stenosis) in comparison with normal control subjects (<20% stenosis). Control subjects showed no change in HDL cholesteryl esters, while HDL cholesterol esters decreased in the CAD patients. HDL triglycerides and HDL phospholipids increased in both groups, but the postprandial increase in HDL phospholipids and HDL cholesteryl esters was delayed for about 4 hours in the CAD patients. From these results, Dr Van Tol suggested that either the surface fragments of chylomicrons and VLDL may be atherogenic if present in high concentrations for a prolonged time during the postprandial phase or impaired fusion of the surface fragments with HDL might result in less antiatherogenic HDL.
Dr N. Mero investigated the effect of the fat composition of the fat meal on the postprandial response. Three different fat loads were compared at a 1-week interval in eight healthy normolipidemic men with the apoE 3/3 phenotype: mixed meal (63 g fat, polyunsaturated/saturated=0.08, 490 mg cholesterol), soybean oil (63 g fat, polyunsaturated/saturated=3.9, 300 mg cholesterol), cream fat load (63 g fat, polyunsaturated/saturated=0.05, 450 mg cholesterol). Vitamin A was included in each meal. Postprandial responses (area under the curve) of triglycerides and cholesterol showed no statistical differences between the three fat loads either in total plasma or in Sf fractions >400, 60 to 400, and 20 to 60. The areas under the curve of retinyl palmitate in total plasma, chylomicron, and Sf fraction of 60 to 400 were significantly higher after soybean oil than after mixed meal or cream loads, indicating that the composition of the fat in the test meal influences the clearance of remnant particles.
The next two presentations examined the value of retinyl esters as a marker of postprandial metabolism of intestinally derived lipoproteins. Dr F. Karpe described studies in which detailed analysis was performed of the quantitative distribution of apoB-48, apoB-100, and retinyl palmitate in various Sf fractions after the mixed-meal type of oral fat load supplemented with retinyl palmitate in 27 normolipidemic men. The peak level of retinyl palmitate was delayed compared with the peak plasma concentration of apoB-48. The largest increase in apoB-48 was found in the Sf 60 to 400 fraction. The highest concentration of retinyl palmitate (2000 to 4000 molecules) was found in the Sf fraction >400 at 3, 6, and 9 hours. Part of the retinyl palmitate molecules were present in the remnant fractions at 3 and 6 hours, indicating direct secretion of retinyl palmitate in this fraction.
Dr T. de Bruin also studied the exchange of retinyl palmitate between lipoproteins in the postprandial state in normolipidemic and hyperlipidemic subjects. He found limited transfer to lipoproteins with higher density, depending on the type of hyperlipidemia, and argued that retinyl palmitate could be used as a marker, albeit not quantitatively, of the total pool of postprandial intestinally derived lipoproteins. In his studies, a strong correlation was found between the removal of retinyl palmitate from the chylomicron fraction (defined as Sf fraction >1000) and the amount of apoC-III and apoE, whereas this correlation with the remnant fraction (Sf fraction <1000) was much weaker.
Dr E. Windler discussed the regulation of the hepatic uptake of chylomicron remnants in the rat. Removal of small chylomicron remnants by perfused rat livers correlated closely with the degree of suppression of the LDL-receptor mRNA, while the removal of large chylomicrons did not. The size of the remnants also determined the sensitivity of hepatic uptake to pretreatment with heparinase, suggesting a differential binding to heparan sulfates. Despite a similar rate of hepatic removal, small chylomicron remnants reached endosomes earlier than large chylomicron remnants.
Dr Marja-Riitta Taskinen gave a comprehensive review in the second part of this session about the metabolic and genetic factors in the regulation of postprandial lipemia. The magnitude and duration of postprandial lipemia are regulated by metabolic factors and disorders and genetic, body, and lifestyle factors. The central role of LPL in the processing of alimentary triglyceride-rich particles is supported by the fact that there is a strong inverse correlation between postheparin LPL activity and the incremental response of plasma triglyceride after oral fat load in healthy normolipidemic men. The critical role of LPL is further supported by data from studies in LPL-deficient patients, who show a clear elevation of both triglyceride and retinyl palmitate up to 24 hours after an oral fat load (Sprecher et al. J Clin Invest. 1991;88:986-994).
Dr Taskinen also stressed, as discussed earlier, the recent substantial evidence that the increase of postprandial lipoprotein particles is due primarily to those containing apoB-100, and a minor part of the particles represent apoB-48 particles. The preferential clearance of chylomicrons by LPL apparently results in the accumulation of endogenous VLDL particles in the postprandial state. Why chylomicrons are better substrates for LPL than endogenous VLDL is unclear. An open question is, which is the most atherogenic particle in the postprandial state, large or small remnants or endogenous VLDL particles?
Dr Taskinen discussed next the plasma concentrations of fasting triglyceride as a major component that predicts the postprandial response of triglyceride. Her data indicated that although there was a close positive correlation between fasting triglyceride and area under the curve of postprandial triglyceride, the incremental response of triglyceride is less tightly correlated with fasting triglyceride. The clear scattering of values becomes apparent when the fasting triglyceride level exceeds 2.0 mmol/L. Recently, Schrezenmeir (Ann N Y Acad Sci. 1993;683:302-314) reported that peak triglyceride values after an oral fat load displayed bimodal frequency distribution and the two subsets were clearly separated. The study cohort included 117 healthy male volunteers with plasma triglyceride levels below 2.3 mmol/L. The cutoff point of fasting triglyceride level for the normal versus high-level responders averaged 1.7 mmol/L. Fasting triglycerides had a poor sensitivity for predicting the response among high-level responders. Interestingly, not only did the two groups differ with respect to fasting triglycerides, but the high-level responders had also higher fasting insulin levels. Of special interest is that the frequencies of small dense LDL and small dense HDL become more prevalent when fasting triglycerides exceed 1.7 to 2.0 mol/L. From this, it can be concluded that even mild hypertriglyceridemia, where triglyceride levels are within the upper normal range, is associated with metabolic consequences that are potentially highly atherogenic. Dr Taskinen next described her studies in male patients with non–insulin-dependent diabetes mellitus (NIDDM) with mild hypertriglyceridemia (fasting triglyceride, 2.0±1.3 mmol/L). These subjects had markedly higher postprandial responses of both plasma triglyceride and retinyl palmitate than nondiabetic control subjects (Syvänne et al. J Lipid Res. 1994;35:15-26). Interestingly, the most pronounced differences in both triglyceride and retinyl palmitate were observed in Sf fraction of 60 to 400 between NIDDM patients and control subjects. Likewise, NIDDM patients with angiographically defined CAD had also exaggerated postprandial lipemia. Of note was that postprandial lipemia did not distinguish for CAD in the NIDDM cohort. In contrast to healthy subjects, Dr Taskinen observed no correlation between postprandial triglyceride response and postheparin plasma LPL in NIDDM patients. These data suggest that postprandial lipemia in NIDDM is due to dual defects: inefficient lipolysis of chylomicrons and impaired clearance of remnant particles.
Dr Taskinen concluded her presentation by discussing the candidate genes proposed to modulate the postprandial response: apoE polymorphism, LPL gene, apoB and apoC-III genes, and a newcomer, FABP-2 (fatty acid binding protein), in the intestinal wall. Recently, numerous mutations of the LPL gene have been characterized. The phenotypic expression of LPL heterozygotes is highly variable, and the lipoprotein profile is frequently normal. Miesenböck et al (J Clin Invest. 1993;91:448-455) reported that carriers of the LPL mutation in codon 88 had fat intolerance, and the fat load test can be used to reveal the carrier state among family members. The practical relevance of this important observation is to use an oral fat load to screen for carriers in high-risk groups, and this is currently done in subjects who are carriers for LPL mutation at residue 291, which seems to be more frequent than other LPL mutations.
Dr M. Bergö reported on the development and application of LPL activity and mass measurements, as well as the separation of active dimeric and inactive monomeric forms in tissue extracts. Although no significant changes in LPL mass and activity could be seen in the heart of rats in the fed and fasted state, adipose tissue LPL activity decreased by 70% to 80% after an overnight fast, and mass decreased only by 20% to 30% owing to the formation of fewer dimeric forms. One mechanism of monomer-dimer formation might be glycosylation.
Dr S. Coppack studied local clearance of triglycerides and LPL activity release in normal male volunteers in the fasting and postprandial state, taking advantage of arteriovenous differences across subcutaneous abdominal adipose and deep forearm muscle tissue. Evidence was obtained for interregional differences in the release of LPL activities. Insulin stimulated adipose tissue LPL and inhibited muscle LPL activity, but local triglyceride uptake did not parallel local LPL changes. The regulatory mechanisms are not yet understood.
Dr S. Levak-Frank showed that tissue-specific overexpression of LPL in skeletal muscle and heart of transgenic mice leads to decreases in plasma triglyceride levels. Depending on the degree of overexpression, weight loss, myopathy, and death occurred as a result of fiber degeneration, glycogen storage, and the proliferation of mitochondria and peroxisomes in these tissues. The lipolysis-mediated myopathy identifies LPL as a crucial gatekeeper for fatty acids in muscle.
Dr M. Jong investigated the effects of overexpression of the human apoC-I gene together with the apoE3–Leiden gene in transgenic mice compared with animals overexpressing only apoE3–Leiden. On a low-fat diet, female mice overexpressing apoC-I also showed increases in plasma triglycerides. The role of apoC-I in triglyceride metabolism has not yet been determined.
Dr B. Nordestgaard presented data on the
Gly188-
Glu mutation in the LPL gene in 1000 patients
with chronic heart disease (CHD) and in 7000 subjects from the general
population, the Copenhagen City Heart Study. Among the CHD patients,
the mutation occurred more frequently (0.3%) compared with 0.06% in
the general population and was associated with higher triglyceride and
lower HDL cholesterol levels. Although not all carriers of the mutation
presented with CHD, the mutation seems to predispose to the
disease.
Dr H. Funke reported on the identification of four different mutations in the CETP gene. One single nucleotide insertion in codon 38 of the CETP gene was found to be associated with familial hyperalphalipoproteinemia. Two missense mutations in codons 373 and 451 were identified to be cis-located. A phenotypic consequence of such a characterized allele was not reported. A frequent missense mutation in codon 405 was found to be associated with significant changes in plasma HDL in three different ethnic groups.
Dr. R. Fisher investigated patients with familial combined
hyperlipidemia by single-stranded conformational polymorphisms
for mutations in the LPL gene and identified 3 with Asp9-Asn and 3
with Asn291-Ser. Carriers of these mutations have fasting plasma
triglyceride levels 20% to 25% above noncarriers, and these effects
are greater in "patient" groups and are compounded by obesity.
In a group of 749 healthy control subjects from the United
Kingdom divided according to tertiles of body mass index, carriers of
either of the two mutations in the upper two tertiles of body mass
index had significantly higher plasma triglycerides than noncarriers.
Maybe an overproduction of chylomicrons or VLDL in obesity overwhelms
partial LPL deficiency in these carriers.
The 18th Annual Meeting of the European Lipoprotein Club is scheduled for September 11-14, 1995, in Tutzing, Germany. It will begin with a state-of-the-art lecture on "Apolipoproteins and Neurological Disorders," followed by three sessions that will examine (1) innovative animal models for lipoprotein research (eg, transgenics and knockouts toward gene therapy), (2) fatty acid metabolism (eg, regulation and clinical implications), and (3) novel functions of apolipoproteins and lipoprotein receptors.
The European Lipoprotein Club Organizing Committee consists of Katriina Aalto-Setala, University of Helsinki, Finland; Bo Angelin, Karolinska Institute, Huddinge, Sweden; Victor Armstrong, Georg-August Universität, Göttingen, FRG; Gerd Assmann, Westfalische Wilhelms Universität, Münster, FRG; David Bowyer, University of Cambridge, England; Guido Franceschini, Institute of Pharmacological Sciences, Milan, Italy; Steve Humphries, Rayne Institute, London, England; Luis Masana, Facultat de Medicina, Reus, Spain; Maryvonne Rosseneu, General Hospital St Jan, Bruges, Belgium; Anton Stalenhoef, University Hospital Nijmegen, The Netherlands; Armin Steinmetz, Medizinische Klinik, Philipps-Universität, Marburg, Germany; and Rudolf Zechner, University of Graz, Austria.
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