The formation of LDL: mechanisms and regulation.
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Animals↗
Biomedical subjects
R J Havel
Publications and source records attributed to R J Havel.
Metabolism of apolipoproteins B-48 and B-100 of triglyceride-rich lipoproteins in normal and lipoprotein lipase-deficient humans.
The metabolism of apolipoproteins B-48 and B-100 (apo B-48 and B-100) in large triglyceride-rich lipoproteins (300 to 1500 A in diameter) has been compared in three normal subjects and two subjects with genetically determined deficiency of lipoprotein lipase. The triglyceride-rich lipoproteins were obtained from a lipoprotein lipase-deficient donor 4 hr after a fat-rich meal in order to obtain chylomicrons (containing apo B-48) and very low density lipoproteins (VLDL) (containing apo B-100), whose properties had not been modified by the action of this enzyme. The triglyceride-rich lipoproteins were labeled with 125I and injected intravenously into recipients who had fasted overnight. In normal recipients, most of the apo B-48 was removed from the blood within 15 min, and most of the apo B-100 was removed within 30 min. In the lipoprotein lipase-deficient recipients, most of the injected apo B-100 remained in the blood for more than 8 hr; removal of apo B-48 was only slightly more rapid. In all subjects, only trace amounts of either protein were found in lipoproteins more dense than 1.006 g/ml. The results indicate that (i) the removal of the apo B of both chylomicrons and large VLDL from the blood is dependent upon the hydrolysis of their component triglycerides by lipoprotein lipase, and (ii) little or no apo B-48 of chylomicrons or apo B-100 of large VLDL is converted appreciably to low density lipoproteins (LDL). Our results suggest that the reported variability of the conversion of VLDL to LDL may be related to the size and composition of the particles secreted from the liver. The rapid production of remnant particles that are removed efficiently by the liver may minimize the opportunity for further reactions leading to the formation of LDL.
Apolipoprotein B-100↗
Effect of chloroquine on low-density lipoprotein catabolic pathway in rat hepatocytes.
Injection of 125I-low-density lipoprotein (LDL) into estradiol-treated rats substantially increases the volume of the multivesicular body compartment in hepatocytes. When these animals were additionally given chloroquine 60 and 120 min before injection of 125I-LDL, the amount of 125I retained by the liver increased threefold and the amount of label in multivesicular bodies increased fivefold, as determined by quantitative analysis of autoradiograms. Lamellar inclusions that appeared after chloroquine also contained a substantial fraction of 125I, whereas the fraction of 125I in secondary lysosomes was reduced. From these data it is concluded that the multivesicular bodies comprise an intermediate endocytic compartment, the conversion of which to secondary lysosomes is impeded by chloroquine.
Animals↗
Impaired receptor-mediated catabolism of low density lipoproteins in fasted rabbits.
Plasma cholesterol levels of New Zealand white rabbits, fasted for 9 days, increased 4-fold owing to elevated levels of low density lipoproteins and intermediate density lipoproteins. Estimates of the turnover of radioiodinated low density lipoproteins and methyl-low density lipoproteins using the Matthews model showed that clearance of low density lipoprotein by receptor-dependent pathways was reduced by 80%. Receptor-independent removal of low density lipoprotein was unchanged. The absolute catabolic rate of low density lipoprotein was not affected by fasting. EDTA-sensitive binding of 125I-labeled low density lipoproteins was selectively lost from liver membranes isolated from fasted rabbits. These results are consistent with the hypothesis that the hypercholesterolemia of fasted rabbits is the result of down-regulation of the hepatic low density lipoprotein receptor.
Animals↗
Uptake and processing of remnants of chylomicrons and very low density lipoproteins by rat liver.
In the rat, chylomicron remnants and very low density lipoprotein (VLDL) remnants are taken up into the liver by high affinity processes and appear to undergo degradation by lysosomes. The relationship of this catabolic process to the known pathways of uptake and degradation of low density lipoproteins (LDL) and the involvement of nonparenchymal cells are addressed in these studies. We have utilized both light and electron microscopic radioautography to determine whether the pathway of intracellular transport and catabolism resembles that established for LDL in hepatocytes. Radioiodinated plasma VLDL remnants and lymph chylomicron remnants were injected into femoral veins of rats and the livers were fixed by perfusion 3 to 30 minutes later. Quantitative light microscopic radioautography showed little or no accumulation of grains over Kupffer cells. Electromicroscopic radioautography confirmed these observations and, in addition, demonstrated that very few grains were associated with endothelial cells. The processing of the remnant particles closely resembled that of LDL. Following an initial association of grains with the parenchymal cell plasma membrane, frequently in regions in close proximity to clathrin-coated endocytic pits, the grains were found in endocytic vesicles just beneath the plasma membrane. By 15 minutes the grains were found over multivesicular bodies located in the Golgi-lysosome region of the cell. Thirty minutes after injection, radioautographic grains began to be associated with secondary lysosomes. These data indicate no significant role for nonparenchymal cells in the internalization and subsequent degradation of triglyceride-rich lipoproteins, and provide evidence that the processing of remnants as well as LDL follows the classical pathway of receptor-mediated endocytosis.
Animals↗
Verapamil suppresses atherosclerosis in cholesterol-fed rabbits.
The effect of verapamil, a drug that reduces the concentration of intracellular calcium, on atherogenesis was evaluated in rabbits fed a cholesterol-rich diet for 10 weeks. Ten rabbits received oral verapamil, 8 mg/kg daily; eight received the same oral dose and 0.5 mg/kg daily subcutaneously; nine received oral lanthanum, 35 mg/kg daily, and nine were controls. Over the 10 week period, all groups had average serum cholesterol levels greater than 1,500 mg/dl (normal = 90 +/- 63 mg/dl). At the end of the experiment, the aortas were removed, opened and stained for lipid with Sudan IV. The extent of atherosclerosis was determined by planimetry. The group receiving oral and parenteral verapamil had significantly less atherosclerosis (25 +/- 26% of total intimal area; mean +/- standard deviation), as compared with the controls (73 +/- 24%). Reduction of atherosclerosis with oral verapamil (51 +/- 22%) and lanthanum (59 +/- 31) was not statistically significant. Indexes of contractility in isolated right ventricular papillary muscles (developed tension at maximal length [Lmax] and maximal velocity of shortening [Vmax]) were reduced in the group treated with oral and parenteral verapamil, but not in the others. It is concluded that verapamil suppresses the development of atherosclerosis in rabbits fed a cholesterol-rich diet.
Adenosine Triphosphatases↗
Secretion of lipoproteins from the liver of normal and Watanabe heritable hyperlipidemic rabbits.
We compared the rate of accumulation of lipoproteins in perfusates of isolated livers from normal New Zealand White rabbits and Watanabe heritable hyperlipidemic (WHHL) rabbits, in which a gene mutation has produced a virtually complete deficiency of low density lipoprotein (LDL) receptors. The rate of accumulation of apolipoprotein B-100 did not differ in perfusates of livers from normal and mutant animals and little or no apolipoprotein B-48 was detected. In both groups, virtually all apolipoprotein B accumulated in very low density lipoprotein (VLDL). Experiments in which [3H]lysine was added to the perfusates showed that the apolipoprotein B that accumulated in VLDL was newly synthesized by the liver whereas the small amount of apolipoprotein B found in lipoproteins of higher density appeared to be washed out of extravascular spaces during perfusion. Perfusate VLDL from both groups contained more triglycerides and less cholesteryl esters than their counterparts from blood plasma. As compared with perfusate VLDL from normal livers, those from livers of WHHL rabbits were enriched in cholesteryl esters. Experiments in which Triton WR-1339 was injected into the blood of intact rabbits confirmed the observations with perfused livers. Previous studies have shown that the extent to which VLDL is converted to LDL is increased several-fold in WHHL rabbits. Taken together with our present results, which fail to provide evidence for increased secretion of apolipoprotein B or de novo secretion of lipoproteins other than VLDL that contain apolipoprotein B, it can be concluded that overproduction of LDL in rabbits lacking LDL receptors is solely the result of altered metabolism of VLDL.
Animals↗
Cholesterol net transport, esterification, and transfer in human hyperlipidemic plasma.
Cholesterol esterification, cholesteryl ester transfer between lipoproteins, and cholesterol transport between lipoproteins and cultured cells have been measured in the plasma of 22 patients with primary hyperlipidemia and 10 normolipidemic subjects. In hyperbetalipoproteinemia, increase in plasma low density lipoprotein levels was associated with a reduction of cholesteryl ester transfer rates, and with a reversal of the normal direction of sterol transport between fibroblasts and their plasma culture medium. Instead of net transport from cells to medium there was a net uptake of sterol from plasma by the cells, despite a level of plasma lecithin/cholesterol acyltransferase activity that was within the normal range. In dysbetalipoproteinemia, esterification rates were increased above normal levels, but cholesteryl ester transfer was reduced and the direction of sterol transport between the cells and plasma medium was reversed, as in the hyperbetalipoproteinemic group. In hypertriglyceridemia, those subjects with cardiovascular disease showed a metabolic pattern similar to the hyperbetalipoproteinemic group. The subjects in this group without symptoms of cardiovascular disease showed a normal direction of sterol transport, normal or raised rates of cholesteryl ester transfer between lipoproteins, and an increased rate of sterol esterification in plasma that decreased towards normal levels as plasma triglyceride levels decreased. Despite their quite distinct metabolic patterns there was no consistent difference between the two hypertriglyceridemic groups in triglyceride or cholesterol levels, very low density lipoprotein composition, or electrophoretic or isoelectric focussing patterns. All hypertriglyceridemic subjects with documented cardiovascular disease showed reversed cell-plasma sterol transport and all subjects without such disease showed a normal direction of cell-plasma sterol transport. The results of this study indicate major and reproducible abnormalities in plasma cholesterol metabolism in several groups of subjects with genetically distinct hyperlipidemias, who are at risk for atherosclerotic vascular disease. The possible predictive value of sterol metabolic measurements in the analysis of cardiovascular disease is discussed.
Adult↗
Atypical familial dysbetalipoproteinemia associated with apolipoprotein phenotype E3/3.
Familial dysbetalipoproteinemia has been reported to be associated uniquely with an apolipoprotein E phenotype (E2/2) that occurs in approximately 1% of all persons. We have observed the typical clinical and biochemical characteristics of this disorder in five members of a family, in all of whom the apolipoprotein E phenotype, as determined by isoelectric focusing electrophoresis, is E3/3. The disorder is present in three generations of the family: the proband, her mother, and three of the proband's five children. The proband's husband, father of all five children, also has apolipoprotein E phenotype E3/3, as do his two unaffected children. As in normal persons with phenotype E3/3, the apolipoprotein E of affected members appears to have a single residue of cysteine. When incorporated with egg lecithin into discoidal complexes, the apolipoprotein E from affected members was taken up normally into perfused livers of estradiol-treated rats, in which a high level of LDL receptors is expressed. When isoelectric focusing electrophoresis was carried out over a narrow range of pH (5-7), each of the apolipoprotein E isoforms of affected members was observed as a doublet, even after reduction of dimers of the protein with 2-mercaptoethanol and treatment with neuraminidase to minimize the content of sialylated forms of the protein. Doublets were also observed in the apolipoprotein E-2 of patients with classical dysbetalipoproteinemia, but only in the affected members of the family with atypical dysbetalipoproteinemia were the components of the doublets equally prominent. As in classical dysbetalipoproteinemia, both apolipoprotein B-100 and B-48 were present in the very low density lipoprotein fraction of plasma obtained in the postabsorptive state, indicating that remnantlike lipoproteins of both hepatic and intestinal origin accumulate. This observation, together with available evidence on the structural and functional heterogeneity of human apolipoprotein E, lead us to suggest that the disorder in this family is caused by one or two structurally abnormal forms of apolipoprotein E that contain a single residue of cysteine.
Adolescent↗
Cholesteryl ester accumulation in mouse peritoneal macrophages induced by beta-migrating very low density lipoproteins from patients with atypical dysbetalipoproteinemia.
The d < 1.006 lipoproteins of patients in a kindred with atypical dysbetalipoproteinemia induced marked cholesteryl ester accumulation in mouse peritoneal macrophages. The affected family members had severe hypercholesterolemia and hypertriglyceridemia, xanthomatosis, premature vascular disease, the apo-E3/3 phenotype, and a predominance of cholesterol-rich beta-very low density lipoproteins (beta-VLDL) in the d < 1.006 fraction. When incubated with mouse peritoneal macrophages, the d < 1.006 lipoproteins or beta-VLDL from the affected family members stimulated cholesteryl [(14)C]oleate synthesis 15- to 30-fold above that caused by normal, control d < 1.006 lipoproteins (VLDL). The ability of the beta-VLDL to stimulate macrophage cholesteryl ester accumulation was greatly reduced as a consequence of treatment with hypolipidemic agents, which specifically reduced the concentration of beta-VLDL. Two important differences were noted in a comparison of the beta-VLDL from these atypical dysbetalipoproteinemic subjects with that of classic E2/2 dysbetalipoproteinemics: (a) the beta-VLDL from the atypical subjects were severalfold more active in stimulating cholesteryl ester accumulation in macrophages, and (b) both the intestinal and hepatic beta-VLDL from the atypical subjects were active. The triglyceriderich, alpha(2)-migrating VLDL from the affected family members constituted <10% of the d < 1.006 fraction and were similar to normal VLDL in that they did not stimulate cholesteryl ester synthesis in the macrophages. Several lines of evidence indicate that the macrophage accumulation of cholesteryl esters was induced by a receptor-mediated uptake process and that the beta-VLDL were bound by a specific beta-VLDL receptor. First, the uptake and degradation of the lipoproteins and the induction of cholesteryl ester formation displayed qualities of high affinity, saturable kinetics. Second, the uptake and degradation process was inhibited when the lysyl residues of the beta-VLDL apoproteins were modified by reductive methylation. Third, the beta-VLDL from the affected subjects competed with diet-induced canine (125)I-beta-VLDL for the same cell surface receptors, but did not compete with chemically modified low density lipoproteins. Finally, the receptor-mediated uptake of these beta-VLDL resulted in lysosomal degradation of the lipoproteins, which could be prevented by incubating the cells with chloroquine. Normal, triglyceride-rich VLDL were also degraded when incubated with the macrophages, but they were not degraded by the same receptor-mediated process responsible for the degradation of the beta-VLDL of the patients. The degradation of the VLDL was not abolished by reductive methylation of the lipoproteins or by treatment of the cells with choloroquine. These studies demonstrate that the beta-VLDL from subjects with atypical dysbetalipoproteinemia are taken up by macrophages via the same receptor-mediated process responsible for the uptake of diet induced beta-VLDL. The accelerated vascular disease seen in these patients may be the result of high concentrations of beta-VLDL capable of binding to and delivering large quantities of cholesterol to macrophages and converting them into cells resembling the foam cells of atherosclerotic lesions.
Adolescent↗
Dietary regulation of plasma lipoprotein metabolism in humans.
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Adult↗
Sex-related differences in the concentrations of apolipoprotein E in human blood plasma and plasma lipoproteins.
Apolipoprotein E (apoE) and lipoprotein cholesterol and triglycerides were measured in blood serum of 272 persons randomly selected from a large industrial population in northern California. Serum apoE level increased linearly by 0.013 mg/dl with each 1 mg/dl increase in very low density lipoprotein (VLDL) triglycerides. This estimate was independent of sex and the use of sex hormones by women. Compositional studies of isolated apoVLDL in 156 hypertriglyceridemic men and 162 normotriglyceridemic persons of both sexes from the same population also indicated that the content of apoE was independent of VLDL level, sex, and hormone use. The estimate of the relationship between serum apoE and VLDL-triglycerides derived from these compositional studies was comparable to that derived by regression analysis. Regression analysis also indicated that only 10-20% of the apoE in the serum of the average person is in the VLDL fraction. Serum apoE levels were 1.4 mg/dl higher in women than in men with the same VLDL-triglyceride level and 1.8 mg/dl lower in women using contraceptive drugs than in nonusers of like age and VLDL-triglyceride level.
Adult↗
Metabolism of apolipoprotein E in plasma high density lipoproteins from normal and cholesterol-fed rats.
High density lipoproteins of rat blood plasma were labeled in vitro with radioiodinated apolipoprotein E and biologically with [3H]cholesteryl esters. These two components, present in high density lipoproteins separated from serum of normal or cholesterol-fed rats by molecular sieve chromatography, were removed slowly from perfused livers and the labeled apolipoprotein E was also removed slowly from the blood of intact rats. However, when labeled serum was subjected to ultracentrifugation at a density of 1.21 g/ml before the floating apolipoprotein E-labeled high density lipoproteins were separated by chromatography, the labeled protein was rapidly removed from the blood of intact rats by uptake into the liver. About one-half of the labeled apolipoprotein E associated with high density lipoproteins was dissociated during ultracentrifugation, but most of it reassociated with the these lipoproteins when the floating lipoproteins were remixed with the sedimented serum proteins. The apolipoprotein E in such reassociated high density lipoproteins was removed from the blood of intact rats at the slow rate observed when the high density lipoproteins were separated chromatographically from whole serum. About 90% of the labeled apolipoprotein E in uncentrifuged or centrifuged high density lipoproteins was shown by affinity chromatography to be associated with particles containing apolipoprotein A-I. Rapid hepatic uptake of apolipoprotein E in centrifuged high density lipoproteins may result from an altered conformation of the apolipoprotein E on the particle surface.
Animals↗
Purification of canine post-heparin hepatic lipase.
A method for the purification of canine hepatic lipase from post-heparin hepatic venous blood plasma was developed and found applicable to mixed venous post-heparin plasma. The method employs sequential (NH4)2SO4 fractionation, heparin-Sepharose chromatography at pH 8.8 and, finally, adsorption to antiserum prepared against dog pre-heparin plasma. The lipase was purified 10,000-fold. The specific activity assayed with Intralipid as substrate was 840 mumol free fatty acid h-1 . mg-1. Enzyme recovery was 20%. Upon electrophoresis of the purified lipase in polyacrylamide gel containing SDS, a major protein-staining band with an apparent molecular weight of 60,000 was consistently found. This component accounted for 85-90% of the protein applied to the gel, and by amino acid analysis appeared to be distinct from canine antithrombin III, a protein thought to contaminate hepatic lipase purified by earlier methods.
Amino Acids↗
Approach to the patient with hyperlipidemia.
To understand the sometimes complex set of disturbances that frequently affect the expression of hyperlipidemia in a patient, it is necessary to be familiar with the operation of the lipoprotein-lipid transport system. After a brief survey of this system, the author classifies the major primary and secondary disorders, discusses the salient features of the primary disorders, summarizes screening and special diagnostic tests, and offers some general principles on the treatment of primary hyperlipidemia.
Adolescent↗
Familial dysbetalipoproteinemia. New aspects of pathogenesis and diagnosis.
New aspects in the pathogenesis and diagnosis of familial dysbetalipoproteinemia are discussed, including the clarification of the chemical basis of the polymorphism of apoprotein E, the allelic nature of the primary isoforms of the protein, the relationship of the abnormality of apoprotein E to the accumulation of remnant lipoproteins in dysbetalipoproteinemia and in persons carrying the trait for abnormal apoprotein E, and the pathogenesis of hyperlipidemia in this disorder. The related clinical features of dysbetalipoproteinemia are included in the discussion.
Apolipoproteins↗
Apolipoprotein B (B-48) of rat chylomicrons is not a precursor of the apolipoprotein of low density lipoproteins.
We have found that, in chylomicrons from intestinal lymph fistulas in the rat, the sole molecular species of apolipoprotein B (apo B) is B-48. This protein is analogous to the B-48 apoprotein of human chylomicrons. In contrast, preparations of chylomicrons from blood serum are known to contain species of B apolipoproteins of higher molecular weight, presumably due to the presence of hepatogenous lipoproteins. We studied the removal of 125I-labeled apo B-48 of intestinal lymph chylomicrons from blood plasma of rats. The removal of [14C]cholesteryl esters of biologically labeled chylomicrons was unaffected by radioiodination. The labeled cholesteryl esters and apo B-48 disappeared rapidly from the density (P) less than 1.006 g/ml fraction of plasma. In contrast to the apo B of very low density lipoproteins (1.019 less then p less then 1.063 g/ml) after 1 hr. No 125I was found in the B-100 or B-95 apolipoproteins at any time. We conclude that, unlike the species of apo B found uniquely in hepatogenous very low density lipoproteins, the apo B-48 protein of chylomicrons is not a precursor of the B apoprotein of low density lipoproteins.
Animals↗
Hepatic uptake of chylomicron remnants in WHHL rabbits: a mechanism genetically distinct from the low density lipoprotein receptor.
Homozygous Watanabe hereditary hyperlipidemic (WHHL) rabbits have a near-complete deficiency of low density lipoprotein (LDL) receptors in liver and other tissues. As a result, these rabbits clear LDL from plasma at an abnormally slow rate. In the current studies we show that WHHL rabbits clear chylomicrons from plasma at a normal rate. Chylomicrons are cleared by a two-step process: (i) hydrolysis of triglycerides in extrahepatic tissues to yield cholesteryl ester-rich remnant particles and (ii) rapid uptake of the remnants by liver. Normal and WHHL rabbits were given intravenous injections of rat chylomicrons labeled either in the lipid portion with [3H]cholesterol and [14C]palmitate or in the protein portion with [125]iodine. All radiolabeled components were removed from plasma at comparable rates in normal and WHHL rabbits. Comparable amounts of radioactivity accumulated in livers of animals from both genotypes. In vitro assays showed that liver membranes from WHHL rabbits were markedly deficient in the binding of 125I-labeled chylomicron remnants as well as 125I-labeled LDL, implying that chylomicron remnants can bind to the hepatic LDL receptor. We conclude that the rabbit liver normally has at least two genetically distinct lipoprotein uptake mechanisms, both of which recognize chylomicron remnants: (i) the LDL receptor and (ii) a specific chylomicron remnant uptake mechanism that is not measured adequately by current in vitro membrane binding assays. WHHL rabbits possess a normal chylomicron remnant uptake mechanism that allows them to clear chylomicrons from plasma at a rapid rate despite their genetic deficiency of LDL receptors.
Animals↗