Apolipoprotein E mediates binding of normal very low density lipoprotein to heparin but is not required for high affinity receptor binding

The relationship between the cholesteryl ester content of normal human very low density lipoprotein (VLDL) and its ability to bind to apolipoprotein E (apoE), heparin, and the low density lipoprotein (LDL) receptor have been compared. Plasma VLDL were separated by heparin affinity chromatography into two fractions: one with apoE and one without. Both fractions had the same cholesteryl ester content relative to apolipoprotein B (apoB). LDL, on the other hand, had a greater cholesteryl ester content. VLDL were modified by lipolysis to express the ability to bind apoE (Ishikawa, Y., Fielding, C. J., and Fielding, P. E. (1988) J. Biol. Chem. 263, 2744-2749). Lipolyzed VLDL with or without apoE were compared for their ability to bind to heparin or the up-regulated fibroblast LDL receptor. Lipolyzed VLDL bound with the same affinity to the receptor whether or not the particles contained apoE. ApoB, not apoE, appears then to be the important ligand for normal VLDL. On the other hand, modified VLDL without apoE, even though binding to the LDL receptor, did not bind to heparin. These data suggest that apoE mediates heparin binding in normal VLDL, that apoB mediates receptor binding, and that the cholesteryl ester content of VLDL is not a factor in the induction of the ability to bind apoE.     

Low density lipoprotein uptake: holoparticle and cholesteryl ester selective uptake.

Low density lipoproteins (LDL) contain apolipoprotein B-100 and are cholesteryl ester-rich, triglyceride-poor macromolecules, arising from the lipolysis of very low density lipoproteins. This review will describe the receptors responsible for uptake of whole LDL particles (holoparticle uptake), and the selective uptake of LDL cholesteryl ester. The LDL-receptor mediates the internalization of whole LDL through an endosomal-lysosomal pathway, leading to complete degradation of LDL. Increasing LDL-receptor expression by pharmacological intervention efficiently reduces blood LDL concentrations. The lipolysis stimulated receptor and LDL-receptor related protein may also lead to complete degradation of LDL in presence of free fatty acids and apolipoprotein E- or lipase-LDL complexes, respectively. Selective uptake of LDL cholesteryl ester has been demonstrated in the liver, especially in rodents and humans. This activity brings five times more LDL cholesteryl ester than the LDL-receptor to human hepatoma cells, suggesting that it is a physiologically significant pathway. The lipoprotein binding site of HepG2 cells mediates this process and recognizes all lipoprotein classes. Scavenger receptor class B type I and CD36, which mediate the selective uptake of high density lipoprotein cholesteryl ester, are potentially involved in LDL cholesteryl ester selective uptake, since they both bind LDL with high affinity. It is not known whether they are identical to the uncloned lipoprotein binding site and if the selective uptake of LDL cholesteryl ester produces a less atherogenic particle. If this is verified, pharmacological up-regulation of LDL cholesteryl ester selective uptake may become another therapeutic approach for reducing blood LDL-cholesterol levels and the risk of atherosclerosis.     

Hydrolysis of cholesteryl ester in low density lipoprotein converts this lipoprotein to a liposome.

Previously, we isolated and characterized unique liposomal-like, cholesterol-rich lipid particles that accumulate in human atherosclerotic lesions. Human plasma low density lipoprotein (LDL) has a molar ratio of total cholesterol to phospholipid (3:1) similar to that of this lesion cholesterol-rich lipid particle. However, LDL is enriched in cholesteryl ester while the lesion lipid particle is enriched in unesterified cholesterol. To examine a possible precursor-product relationship between LDL and the lesion lipid particle, we hydrolyzed the cholesteryl ester core of LDL with cholesterol esterase. Cholesteryl ester hydrolysis occurred only after LDL was treated with trypsin. Trypsin pretreatment was not required for cholesteryl ester hydrolysis of LDL oxidized with copper, a treatment that also degrades apolipoprotein B, the major protein moiety in LDL. In contrast to greater than 90% hydrolysis of cholesteryl ester in trypsin-cholesterol esterase-treated or copper-oxidized LDL, there was only 18% hydrolysis of cholesteryl ester in similarly treated high density lipoprotein. With a limited 10-min hydrolysis of LDL cholesteryl ester, LDL-sized particles and newly formed larger flattened films or discs were present. With complete hydrolysis of LDL cholesteryl ester, LDL particles converted to complex multilamellar, liposomal-like, structures with sizes approximately five times larger than native LDL. These liposomal-like particles derived from LDL were chemically and structurally similar to unesterified cholesterol-rich lipid particles that accumulate in atherosclerotic lesions.     

Cholesteryl ester enrichment of plasma low-density lipoproteins in hamsters fed cereal-based diets containing cholesterol

Male Syrian hamsters were fed 0.02, 0.03, or 0.05% cholesterol to test the hypothesis that moderate cholesterol intake increases the cholesteryl ester content of the plasma low-density lipoproteins (LDL). Dietary cholesterol levels of 0.02%-0.05% were chosen to reflect typical human intakes of cholesterol. Hamsters were fed ad libitum a cereal-based diet (modified NIH-07 open formula) for 15 weeks. Increasing dietary cholesterol from 0.02% to 0.05% resulted in significantly increased plasma LDL and high-density lipoprotein cholesterol concentration, increased liver cholesterol concentration, and increased total aorta cholesterol content. The cholesteryl ester content of plasma LDL was determined as the molar ratio of cholesteryl ester to apolipoprotein B and to surface lipid (i.e., phospholipid + free cholesterol). Increasing dietary cholesterol from 0.02% to 0.05% resulted in significantly increased cholesteryl ester content of LDL particles. Furthermore, cholesteryl ester content of LDL was directly associated with increased total aorta cholesterol, whereas a linear relationship between plasma LDL cholesterol concentration and aorta cholesterol was not observed. Thus, the data suggest that LDL cholesteryl ester content may be an important atherogenic feature of plasma LDL.     

Unique structural properties of apolipoprotein B in low-density lipoproteins produced by several human hepatoma-derived cell lines

Previous work has shown that low-density lipoproteins (LDL) secreted by hepatoma-derived cell lines have an unusual composition compared to plasma LDL; rather than cholesteryl ester, the hepatoma cell-secreted LDL have a triacylglycerol core. We have found that they also have an increased negative charge, as judged by agarose electrophoresis. Since apolipoprotein B is a glycoprotein containing carbohydrate chains terminated with negatively charged sialic acid residues, we examined whether increased glycosylation of the apolipoprotein B from three hepatoma cell lines (Hep G2, Hep 3B and Huh 7) might account for the differences in LDL charge. The weight percent carbohydrate for Hep G2, Hep 3B and Huh 7 LDL-protein (1.1 +/- 0.2; 1.7 +/- 0.8; 0.4 +/- 0.1) was found to be extremely low compared with the 2.8-9% range we found for plasma LDL-protein, while the amount of LDL-lipid associated carbohydrate from hepatoma LDL was similar to that we found in plasma LDL. Furthermore, desialation of hepatoma cell-secreted LDL with neuraminidase did not normalize the negative charge to that of neuraminidase-treated plasma LDL. Western blots of thrombin proteolytic fragments indicated that, in addition to the T1-T4 fragments seen in plasma apolipoprotein B, apolipoprotein B of hepatoma-derived LDL produced four to five new fragments (T5-T9), suggesting increased exposure of proteolytic sites. Western blotting of the new fragments with antibodies specific for known apolipoprotein B sequences suggests that many of the new cleavage sites cluster in or near the putative LDL receptor recognition site.     

LDL受体对清道夫受体活性的影响

应用经ＰＭＡ诱导衍生的ＴＨＰ－１巨噬细胞为模型,以单克隆抗体Ｃ７Ｂ封闭ｏｘＬＤＬ上的ＬＤＬ受体结合位点,结果发现,正常细胞在摄取ｏｘＬＤＬ时ＬＤＬ受体与清道夫受体起协同作用;但Ｃ７Ｂ作用于蓄积了脂质的ＴＨＰ－１巨噬细胞时,对细胞脂质蓄积程度无明显影响,清道夫受体活性不但不降低反而有所升高,说明由于脂质蓄积ＬＤＬ受体的作用减弱．     

Effect of fish oil versus lard diets on the chemical and physical properties of low density lipoproteins of nonhuman primates

Twenty-four adult male African green grivet monkeys were fed diets containing 42% of calories as lard or menhaden oil and 0.76 mg of cholesterol/kcal for a period of 8 months. Plasma samples from fasting animals were then taken and low density lipoproteins (LDL) were isolated by ultracentrifugation and agarose column chromatography. The LDL were analyzed chemically, and physical properties of the particles were studied by differential scanning calorimetry. The fish oil group had significantly smaller LDL (2.91 vs. 3.43 g/mumol), which contained fewer molecules per particle of all lipid constituents, except triglyceride, compared to the lard-fed animals. The fish oil-fed group had 15% of the total cholesteryl esters as n-3 fatty acyl species and the number of n-3, but not n-6, cholesteryl esters per LDL particle was proportional to LDL size. The numbers of saturated and monounsaturated cholesteryl ester species per LDL particle were highly correlated with LDL size for both diet groups. The LDL of the fish oil group had broad reversible thermotropic transitions that were 12-13 degrees C lower than those of the lard group. These transitions were indicative of order-disorder transitions of the LDL core cholesteryl esters. The peak transition temperature of LDL of the lard group was proportional to the ratio of saturated and monounsaturated to polyunsaturated cholesteryl ester species (CEFA ratio). However, the much lower peak transition temperature of the LDL of the fish oil group was not related to the CEFA ratio nor to the triglyceride content of the particles, but rather, to the n-3 cholesteryl ester content of the particles. Studies of cholesteryl ester model systems demonstrated that relatively small amounts of n-3 cholesteryl esters (less than 15% of total cholesteryl ester) could result in a lowering of the peak transition temperature of cholesteryl linoleate similar to that seen for intact LDL. We conclude that n-3 cholesteryl esters in small quantities have a marked disordering effect on the core cholesteryl esters of LDL, resulting in a striking depression of LDL transition temperature. In addition, we conclude that n-3 cholesteryl esters are preferentially utilized relative to n-6 cholesteryl esters to increase the number of cholesteryl esters per LDL particle with LDL enlargement in fish oil-fed animals.     

Catabolism of HDL1 cholesteryl ester in the rat. Effect of ethinyl estradiol treatment.

The present study was performed in control and ethinyl estradiol-treated rats in order to determine the mechanisms involved in the catabolism of HDL1 cholesteryl ester. Ligand blottings on liver membranes showed that purified HDL1, containing about 70% apolipoprotein E and 10% apolipoprotein AI, bind to the LDL receptor (130 kDa) and not to HB2 (100 kDa) or SR-BI (82 kDa), candidate HDL receptors. Immunoblots showed that the treatment increased the hepatic level of the LDL receptor five- to ten-fold, strongly decreased that of SRBI and did not change that of HB2. An in vivo kinetic study showed that the turnover of HDL1 cholesteryl ester is more rapid in treated than control rats. The liver participation (60%) in this clearance was not modified by the treatment. Therefore, it can be concluded that the catabolism of HDL1 cholesteryl ester, in control as in treated rats, is essentially ensured by the uptake of entire particles in the hepatocytes via LDL receptors.     

Effects of hypoxia on cholesterol metabolism in human monocyte-derived macrophages

We assessed the metabolism of low density lipoprotein (LDL) of human monocyte-derived macrophages under hypoxia. The specific binding and association of 125I-labeled LDL (125I-LDL) were not changed under hypoxia compared to normoxia. However, the degradation of 125I-LDL under hypoxia decreased to 60%. The rate of cholesterol esterification under hypoxia was 2-fold greater on incubation with LDL or 25-hydroxycholesterol. The cellular cholesteryl ester content was also greater under hypoxia on incubation with LDL. Secretion of apolipoprotein E into the medium was not altered under hypoxia, suggesting that apolipoprotein E independent cholesterol efflux may be reduced under hypoxia. Thus, hypoxia affects the intracellular metabolism of LDL, stimulates cholesterol esterification, and enhances cholesteryl ester accumulation in macrophages. Hypoxia is one of the important factors modifying the cellular lipid metabolism in arterial wall.     

High-density lipoprotein particle uptake and selective uptake of high-density lipoprotein-associated cholesteryl esters by J774 macrophages

High-density lipoprotein (HDL) cholesteryl esters are taken up by fibroblasts via HDL particle uptake and via selective uptake, i.e., cholesteryl ester uptake independent of HDL particle uptake. In the present study we investigated HDL selective uptake and HDL particle uptake by J774 macrophages. HDL3 (d = 1.125-1.21 g/ml) was labeled with intracellularly trapped tracers: 125I-labeled N-methyltyramine-cellobiose-apo A-I (125I-NMTC-apo A-I) to trace apolipoprotein A-I (apo A-I) and [3H]cholesteryl oleyl ether to trace cholesteryl esters. J774 macrophages, incubated at 37 degrees C in medium containing doubly labeled HDL3, took up 125I-NMTC-apo A-I, indicating HDL3 particle uptake (102.7 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein). Apparent HDL3 uptake according to the uptake of [3H]cholesteryl oleyl ether (470.4 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein) was in significant excess on 125I-NMTC-apo A-I uptake, i.e., J774 macrophages demonstrated selective uptake of HDL3 cholesteryl esters. To investigate regulation of HDL3 uptake, cell cholesterol was modified by preincubation with low-density lipoprotein (LDL) or acetylated LDL (acetyl-LDL). Afterwards, uptake of doubly labeled HDL3, LDL (apo B,E) receptor activity or cholesterol mass were determined. Preincubation with LDL or acetyl-LDL increased cell cholesterol up to approx. 3.5-fold over basal levels. Increased cell cholesterol had no effect on HDL3 particle uptake. In contrast, LDL- and acetyl-LDL-loading decreased selective uptake (apparent uptake 606 vs. 366 ng HDL3 protein/mg cell protein per 4 h in unloaded versus acetyl-LDL-loaded cells at 20 micrograms HDL3 protein/ml). In parallel with decreased selective uptake, specific 125I-LDL degradation was down-regulated. Using heparin as well as excess unlabeled LDL, it was shown that HDL3 uptake is independent of LDL (apo B,E) receptors. In summary, J774 macrophages take up HDL3 particles. In addition, J774 cells also selectively take up HDL3-associated cholesteryl esters. HDL3 selective uptake, but not HDL3 particle uptake, can be regulated.     

The interaction of human apoB-containing lipoproteins with mouse peritoneal macrophages: a comparison of Lp(a) with LDL

Cholesteryl ester accumulation in macrophages and foam cell formation is believed to play an important role in atherogenesis. The effect of Lp(a) on the incorporation of [14C]oleate into cholesteryl esters was studied in mouse peritoneal macrophages. In view of the physico-chemical similarities between Lp(a) and LDL, the results were compared with those obtained with LDL. Native Lp(a) and LDL did not stimulate cholesteryl ester formation. Incubation of macrophages with Lp(a)- or LDL-dextran sulfate complexes caused a significant increase in cholesteryl ester formation. A similar effect was observed when Lp(a) or LDL were incubated with macrophages in the presence of antibodies directed against the specific Lp(a) apoprotein or against LpB. Treatment of Lp(a) with acetic anhydride or malondialdehyde (MDA) was followed by precipitation of most of the lipoprotein. Therefore, these modifications were not suitable to study the uptake of modified Lp(a) by macrophages. Studies with acetyl-LDL or MDA-treated LDL caused the well-known stimulation of [14C]oleate incorporation into cholesteryl esters. Thus, the modification of Lp(a) by sulfated polysaccharides or by treatment with antibodies yields similar cholesteryl ester deposition in mouse peritoneal macrophages as observed with modified LDL. This might be one mechanism by which Lp(a) exerts its atherogenicity.     

Studies on the substrate specificity of human and pig lecithin: cholesterol acyltransferase: role of low-density lipoproteins

The substrate properties of low-density lipoprotein (LDL) fractions from human and pig plasma and of lipoprotein a [Lp(a)] upon incubation with either pig or human lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) were investigated and compared with those of pig high-density lipoproteins (HDL) or human HDL-3. The cholesterol esterification using purified native pig LDL-1, human LDL, or Lp(a) as a substrate was approximately 36-42% that of pig HDL or human HDL-3, while cholesteryl ester formation with pig LDL-2 was 41-47%. No significant difference was found in the substrate activity between pig HDL and human HDL-3, and between human LDL and Lp(a), respectively. After depletion of pig LDL-1, pig LDL-2, and human LDL from apolipoprotein A-I (apoA-I), cholesteryl ester formation decreased to about 22-28% of the value found with pig HDL. Depletion of human LDL from apolipoprotein E (apoE) did not result in significantly different esterification rates in comparison to native LDL. Total removal of non-apoB proteins from human LDL resulted in esterification rates of approximately 10-15% that of HDL. Readdition of apoA-I to all these LDL fractions produced solely in apoA-I-depleted LDL fractions an increase of cholesteryl ester formation, whereas in those LDL fractions that were additionally depleted from apoE and/or from apoC polypeptides, a further decrease in the esterification rate occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conversion of human low density lipoprotein into a very low density lipoprotein-like particle in vitro.

The lipid substrate specificity of Manduca sexta lipid transfer particle (LTP) was examined in in vitro lipid transfer assays employing high density lipophorin and human low density lipoprotein (LDL) as donor/acceptor substrates. Unesterified cholesterol was found to exchange spontaneously between these substrate lipoproteins, and the extent of transfer/exchange was not affected by LTP. By contrast, transfer of labeled phosphatidylcholine and cholesteryl ester was dependent on LTP in a concentration-dependent manner. Facilitated phosphatidylcholine transfer occurred at a faster rate than facilitated cholesteryl ester transfer; this observation suggests that either LTP may have an inherent preference for polar lipids or the accessibility of specific lipids in the donor substrate particle influences their rate of transfer. The capacity of LDL to accept exogenous lipid from lipophorin was investigated by increasing the high density lipophorin:LDL ratio in transfer assays. At a 3:1 (protein) ratio in the presence of LTP, LDL became turbid (and aggregated LDL were observed by electron microscopy) indicating LDL has a finite capacity to accept exogenous lipid while maintaining an overall stable structure. When either isolated human non B very low density lipoprotein (VLDL) apoproteins or insect apolipophorin III (apoLp-III) were included in transfer experiments, the sample did not become turbid although lipid transfer proceeded to the same extent as in the absence of added apolipoprotein. The reduction in sample turbidity caused by exogenous apolipoprotein occurred in a concentration-dependent manner, suggesting that these proteins associate with the surface of LDL and stabilize the increment of lipid/water interface created by LTP-mediated net lipid transfer. The association of apolipoprotein with the surface of modified LDL was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and scanning densitometry revealed that apoLp-III bound to the surface of LDL in a 1:14 apoB:apoLp-III molar ratio. Electron microscopy showed that apoLp-III-stabilized modified LDL particles have a larger diameter (29.2 +/- 2.6 nm) than that of control LDL (22.7 +/- 1.9 nm), consistent with the observed changes in particle density, lipid, and apolipoprotein content. Thus LTP-catalyzed vectorial lipid transfer can be used to introduce significant modifications into isolated LDL particles and provides a novel mechanism whereby VLDL-LDL interrelationships can be studied.     

Increases in the particle size of high-density lipoproteins induced by purified lecithin: cholesterol acyltransferase: effect of low-density lipoproteins

Homogeneous subpopulations of human high-density lipoproteins subfraction-3 (HDL3) have been incubated at 37 degrees C with purified lecithin: cholesterol acyltransferase, human serum albumin and varying concentrations of human low-density lipoproteins (LDL). Changes in HDL particle size and composition during these incubations were monitored. Incubation of HDL3a (particle radius 4.3 nm) in the absence of LDL resulted in an esterification of more than 70% of the HDL free cholesterol after 24 h of incubation. This, however, was sufficient to increase the HDL cholesteryl ester by less than 10% and was not accompanied by any change in particle size. When this mixture was incubated in the presence of progressively increasing concentrations of LDL, which donated free cholesterol to the HDL, the molar rate of production of cholesteryl ester was much greater; at the highest LDL concentration HDL cholesteryl ester content was almost doubled after 24 h and there was an increase in the HDL particle size up to the HDL2 range. In the case of HDL3b (radius 3.9 nm), there were again only minimal changes in particle size in incubations not containing LDL. In the presence of the highest concentration of LDL tested, however, the particles were again enlarged into the HDL2 size range after 24 h incubation. These HDL2-like particles were markedly enriched with cholesteryl ester but depleted of phospholipid and free cholesterol when compared with native HDL2. Furthermore, the ratio of apolipoprotein A-I to apolipoprotein A-II resembled that in the parent-HDL3 and was very much lower than that in native HDL2. It has been concluded that purified lecithin: cholesterol acyltransferase is capable of increasing the size of HDL3 towards that of HDL2 but that other factors must operate in vivo to modulate the chemical composition of the enlarged particles.     

LDL and HDL enriched in triglyceride promote abnormal cholesterol transport

Hypertriglyceridemia induces multiple changes in lipoprotein composition. Here we investigate how one of these modifications, triglyceride (TG) enrichment, affects HDL and LDL function when this alteration occurs under conditions in which more polar components can naturally re-equilibrate. TG-enriched lipoproteins were produced by co-incubating VLDL, LDL, and HDL with cholesteryl ester (CE) transfer protein. The resulting 2.5-fold increase in TG/CE ratio did not measurably alter the apoprotein composition of LDL or HDL, or modify LDL size. HDL mean diameter increased slightly from 9.1 to 9.4 nm. Modified LDL was internalized by fibroblasts normally, but its protein was degraded much less efficiently. This likely reflects an aberrant apolipoprotein B (apoB) conformation, as suggested by its resistance to V8 protease digestion and altered LDL electrophoretic mobility. TG-enriched LDL ineffectively down-regulated cholesterol biosynthesis compared with control LDL at the same protein concentration, but was equivalent in sterol regulation when compared on a cholesterol basis. TG-enriched HDL promoted greater net cholesterol efflux from cholesterol-loaded J774 cells. However, cholesterol associated with TG-enriched HDL was inefficiently esterified by lecithin:cholesterol acyltransferase, and TG-enriched HDLs were poor donors of CE to HepG2 hepatocytes by selective uptake. We conclude that TG-enrichment, in the absence of other significant alterations in lipoprotein composition, is sufficient to alter both cholesterol delivery and removal mechanisms. Some of these abnormalities may contribute to increased coronary disease in hypertriglyceridemia.     

Covalent binding of oxidized cholesteryl esters to protein: implications for oxidative modification of low density lipoprotein and atherosclerosis

It has been proposed that plasma low density lipoproteins (LDL) undergo oxidative modification before they can produce foam cells in atherosclerosis. The oxidation of LDL generates a variety of reactive aldehydic products, which covalently bind to the LDL apolipoprotein B-100 (apoB). In the present study, to investigate the mechanisms contributing to the modification of LDL, we analyzed oxidized cholesteryl esters generated during the autoxidation of LDL and characterized their covalent binding to the lysine residues of LDL apoB. In addition, we raised a monoclonal antibody specific to a lysine-bound oxidized cholesteryl ester and determined its production in human atherosclerotic lesions. The peroxidation of LDL with Cu2+ produced 9-oxononanoylcholesterol (9-ONC) and 5-oxovaleroylcholesterol as the major oxidized cholesteryl esters. We observed that the levels of 9-ONC and 5-oxovaleroylcholesterol peaked at 12 h and significantly decreased thereafter. The reduction of the core aldehyde levels was accompanied by (i) the formation of free 7-ketocholesterol and 7-ketocholesteryl ester core aldehydes and (ii) an increase in the amounts of apoB-bound cholesterol and 7-ketocholesterol, suggesting that the cholesteryl ester core aldehydes were further converted to their 7-ketocholesterol- and apoB-bound derivatives. To detect the protein-bound 9-ONC, we raised the monoclonal antibody 2A81, directed against 9-ONC-modified protein, and found that it extensively recognized protein-bound cholesteryl ester core aldehydes. Agarose gel electrophoresis followed by immunoblot analysis of the oxidized LDL clearly demonstrated the formation of antigenic structures. Furthermore, immunohistochemical analysis of the atherosclerotic lesions from the human aorta showed that immunoreactive materials with mAb 2A81 were indeed present in the lesions, in which the intense immunoreactivity was mainly located in the macrophage-derived foam cells and the thickening neointima of the arterial walls. The results of this study suggest that the binding of cholesteryl ester core aldehydes to LDL might represent the process common to the oxidative modification of lipoproteins.     

Influence of dietary fatty acid composition on the relationship between CETP activity and plasma lipoproteins in monkeys.

CETP activity, measured as transfer of cholesteryl ester from exogenous HDL to exogenous VLDL and LDL, reflecting CETP mass as determined by ELISA, was documented in three groups of St. Kitts vervet monkeys fed diets enriched in saturated (Sat), monounsaturated (Mono), or n-6 polyunsaturated (Poly) fatty acids. CETP activity was not different when comparing the three dietary fats. However, CETP activity was significantly higher when cholesterol was added to each of the diets. Significant positive associations between CETP activity and VLDL and LDL cholesterol concentrations were found whereas significant negative associations were seen between CETP activity and HDL cholesterol in each of the diet groups. The strength of these associations was highest in the Sat group. Cholesteryl ester (CE) fatty acid composition of lipoproteins varied widely among diet groups, with the more polyunsaturated CE of the Poly group being associated with a higher rate of CE transfer to endogenous acceptor apolipoprotein B-containing lipoproteins. Finally, only the Sat diet group showed significant positive correlations of CETP activity with LDL particle diameter (r = 0.76), cholesteryl ester percentage (r = 0.67), and a strong negative correlation (r = -0.86) with LDL receptor function, estimated as the difference between native and methylated LDL turnover rates. We speculate that strong associations between CETP and LDL metabolism may explain, at least in part, the increased atherogenicity of dietary saturated fat.     

Angiotensin II stimulates receptor-mediated uptake of LDL by bovine adrenal cortical cells in primary culture

Bovine adrenal cells were isolated from the subcapsular region of the gland to obtain cultures enriched in cells of the zona glomerulosa. The cells kept in primary cultures were shown to respond to angiotensin II and adrenocorticorticotropin (ACTH) by a significant increase in aldosterone production. These primary adrenal cultures were used to study the effect of angiotensin II on LDL metabolism. Addition of angiotensin II for 48 h to the culture medium resulted in a 200-300% increase in LDL metabolism, and the lowest effective concentration was 10(-8) -10(-9) M. The angiotensin II effect became evident after 12-16 h of incubation. To compare the metabolism of the 125I-labeled protein moiety to that of cholesteryl ester of LDL, the lipoprotein was labeled also with cholesteryl linoleyl ether, a nonhydrolyzable analog of cholesteryl ester. Under basal conditions and in the presence of angiotensin II or ACTH the ratio of [3H]cholesteryl linoleyl ether to 125I indicate some preferential uptake of the cholesteryl ester moiety. Stimulation of specific LDL binding at 4 degrees C and LDL metabolism at 37 degrees C by 10(-7) M angiotensin II occurred at all concentrations of LDL studied. Linearization of the kinetic data showed that angiotensin II increased the LDL receptor number significantly but not the affinity of the LDL receptor for its ligand. The present findings indicate that in analogy to ACTH, angiotensin II can influence receptor-mediated uptake of LDL by adrenal cortical cells. It remains to be shown whether the angiotensin II effect on LDL metabolism is limited to adrenal cells or will affect other cells which express the angiotensin II receptor.     

Uptake of rat plasma HDL subfractions labeled with [3H]cholesteryl linoleyl ether or with 125I by cultured rat hepatocytes and adrenal cells

Rat plasma low- and high-density lipoproteins were labeled with [3H]cholesteryl linoleyl ether and isolated by rate-zonal ultracentrifugation into apolipoprotein B-containing LDL, apolipoprotein E-containing HDL1 and apolipoprotein E-poor HDL2. These fractions were incubated with cultured rat hepatocytes and comparable amounts of all lipoproteins were taken up by the cells. Rat HDL was isolated at d 1.085-1.21 g/ml and apolipoprotein E-free HDL was prepared by heparin Sepharose chromatography. The original HDL and the apolipoprotein E-free HDL were labeled with 125I or with [3H]cholesteryl linoleyl ether and incubated with rat hepatocytes or adrenal cells in culture. The uptake of apolipoprotein E-free [3H]cholesterol linoleyl ether HDL by the cultured hepatocytes was 20-40% more than that of the original HDL. Comparison of uptake of cholesteryl ester moiety (represented by uptake of [3H]cholesteryl linoleyl ether) and of protein moiety (represented by metabolism of 125I-labeled protein) was carried out using both original and apolipoprotein E-free HDL. In experiments in which low concentrations of HDL were used, the ratio of 3H/125I exceeded 1.0. In cultured adrenal cells, the uptake of [3H]cholesteryl linoleyl ether-labeled HDL was stimulated 3-6-fold by 1 X 10(-7) M ACTH, while the uptake of 125I-labeled HDL increased about 2-fold. The ratio of 3H/125I representing cellular uptake was 2-3 and increased to 5 in ACTH-treated cells. The present results indicate that in cultured rat hepatocytes the uptake of homologous HDL does not depend on the presence of apolipoprotein E. Evidence was also presented for an uptake of cholesteryl ester independent of protein uptake in cultured rat adrenal cells and to a lesser extent in rat hepatocytes.