Enhanced degradation of trypsin-treated low density lipoprotein by fibroblasts from a patient with homozygous familial hypercholesterolemia.

When 125I-labeled native low density lipoprotein was incubated with skin fibroblasts from a patient with homozygous familial hypercholesterolemia, the observed rate of degradation of the protein moiety was less than 5% the rate observed with normal fibroblasts, in agreement with previous studies. When the low density lipoprotein had been first treated with trypsin, with release of about 20% of the protein, its degradation by the patient's fibroblasts was markedly increased 8-20-fold. In contrast, the rate of degradation of the trypsin-treated lipoprotein by normal fibroblasts was, if anything, slightly reduced. In neither the normal cells nor the patient's cells was binding to the cell surface appreciably altered by trypsin treatment of the lipoprotein. Prior incubation with cholesterol and 7-ketocholesterol reduced binding of trypsin-treated low density lipoprotein to normal cells by 67% but did not affect its binding to the patient's cells. The results show that the structural modifications induced by trypsin do not interfere with binding of low density lipoprotein to its normal high affinity receptor nor its degradation by normal cells. However, the modified lipoprotein is much more readily internalized and degraded by cells from the patient with homozygous familial hypercholesterolemia.     

Stability of the apoprotein of human serum low density lipoprotein: absence of endogenous endopeptidase activity.

Protease activity, inhibitable by DFP, has been reported in purified preparations of low density lipoprotein from human serum. Such activity could introduce serious error in studies of structure of its subunit proteins. In this study low density lipoprotein was prepared and stored in an effective antimicrobial medium. The protein moiety was examined for evidence of proteolysis by electrophoresis with sodium dodecyl sulfate, gel permeation chromatography, solubility in tetramethylurea, and measurement of amino and carboxyl termini. No evidence of proteolytic cleavage of the apoprotein was found indicating that low density lipoprotein lacks endogenous protease activity.     

39-kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

A 39-kDa protein of unknown function has previously been reported to copurify with the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor. In this study we demonstrate that a recombinant 39-kDa fusion protein can reversibly bind to the 515-kDa subunit of the LRP/alpha 2-macroglobulin receptor. This interaction inhibits the binding and uptake of the receptor's two known ligands: 1) beta-migrating very low density lipoproteins activated by enrichment with apoprotein E and 2) alpha 2-macroglobulin activated by incubation with plasma proteases or methylamine. A potential in vivo role of the 39-kDa protein is to modulate the uptake of apoE-enriched lipoproteins and activated alpha 2-macroglobulin in hepatic and extrahepatic tissues.     

Studies on the composition of pig serum lipoproteins. Isolation and characterization of different apoproteins.

1. Different lipoprotein density fractions from pig serum were isolated by phosphotungstate precipitation followed by purification in the preparative ultra-centrifuge. 2. The protein part of very low density lipoproteins was composed of approximately 52 percent lipoprotein B apoprotein and the rest of lipoprotein C II apoprotein and other as yet unidentified peptides. 3. The protein moiety of low density lipoproteins consisted primarily of lipoprotein B apoprotein (over 95 percent); the amino acid compositions of lipoprotein B apoprotein of very low and low density lipoproteins were practically identical. 4. The predominant polypeptide of pig serum high density lipoproteins exhibited an amino acid composition and a molecular weight very similar to human liprotein A I apoprotein. In contrast to human lipoprotein A I apoprotein, the apoprotein from pigs was found to release leucine first followed by alanine, threonine, and lysine upon incubation with carboxypeptidase A. 5. In pig serum the major lipoprotein C apoprotein was found to be a polypeptide similar in amino acid composition to lipoprotein C II apoprotein from human serum. The molecular weight of this polypeptide is approximately 8000. Incubation experiments with carboxypeptidase A indicate serine to be the most likely C-terminal amino acid.     

Distinction in the mode of receptor-mediated endocytosis between high density lipoprotein and acetylated high density lipoprotein: evidence for high density lipoprotein receptor-mediated cholesterol transfer

The interactions of high density lipoprotein (HDL) and acetylated high density lipoprotein (acetyl-HDL) with isolated rat sinusoidal liver cells have been investigated. Cellular binding of 125I-acetyl-HDL at 0 degrees C demonstrated the presence of a specific, saturable membrane-associated receptor. This receptor was affected neither by formaldehyde-treated albumin nor by low density lipoprotein modified either by acetylation or malondialdehyde, ligands known to undergo receptor-mediated endocytosis by the cells, indicating that the receptor for acetyl-HDL constitutes a distinct class among the scavenger receptors for chemically modified proteins. Parallel binding experiments using 125I-HDL also revealed the presence on these cells of a receptor for unmodified HDL. The ligand specificities of these two receptors were similar to each other except that the acetyl-HDL receptor was sensitive to polyanions such as dextran sulfate and fucoidin. Interaction of HDL with the cells at 37 degrees C was totally different from that of acetyl-HDL. Cellular binding of HDL was not accompanied by subsequent intracellular degradation of its apoprotein moiety, whereas its cholesterol moiety was significantly transferred to the cells. In contrast, acetyl-HDL was endocytosed and underwent lysosomal degradation as a holoparticle. This shift in receptor-recognition from the HDL receptor to the acetyl-HDL receptor was accomplished by acetylation of approximately 8% of the total lysine residues of HDL apoprotein. This unique difference in endocytic behavior between HDL and acetyl-HDL suggests a potential link of the HDL receptor to HDL-mediated cholesterol transfer in sinusoidal liver cells.     

The effects of hyperlipidemia on lipoprotein metabolism in squirrel monkeys and rabbits.

We studied the metabolism of different classes of lipoprotein in squirrel monkeys and rabbits. Lipoproteins were labeled in vivo in donor animals with (3H)leucine and (3H)cholesterol. The rate of disappearance from plasma of recipient squirrel monkeys of the protein moiety of the very low density lipoproteins was rapid, that of high density lipoproteins slow, and the rate for low density lipoproteins was intermediate. The fractional turnover of the apoprotein of low density lipoproteins was slightly reduced in hyperlipidemic monkeys, but the absolute rates of synthesis and catabolism were increased. Hyperdipidemia in rabbits resulted in a dramatic reduction in the fractional catabolic rate of low density lipoprotein apoprotein. Hyperlipidemia in the donors of biosynthetic low density lipoproteins also influenced the rates of catabolism in rabbits. We showed the cycloheximide that although there was recycling of (3H)leucine into other proteins, the reutilization of leucine from low density lipoproteins for nascent low density lipoproteins was not significant. In most tissues the ratio of cholesterol:protein radioactivity was much greater than that for plasma 24 h after administration of labeled low density lipoproteins, but the ratios for aortic intima plus inner media and for plasma low density lipoproteins were similar. The presence of atherosclerosis resulted in a large increase in the apparent uptake of low density lipoproteins by the aortas of rabbits and monkeys.     

Metabolomic analysis of the response of growing pigs to dietary l-arginine supplementation

Arginine plays an important role regulating nutrient metabolism, but the underlying mechanisms are largely unknown. This study was conducted to determine the effect of dietary arginine supplementation on the metabolome in serum of growing pigs using (1)H nuclear magnetic resonance spectroscopy. Sixteen 120-day-old pigs (48 +/- 1 kg) were randomly assigned to one of two groups, representing supplementation with 0 or 1.0% L: -arginine to corn- and soybean meal-based diets. Serum was collected after a 46-day period of treatment. Dietary arginine supplementation decreased fat deposition and increased protein accretion in the body. Principal component analysis showed that serum concentrations of low density lipoprotein, very low density lipoprotein, and urea were lower, but concentrations of creatinine, tricarboxylic acid cycle metabolites, ornithine, lysine and tyrosine were greater in arginine-supplemented than in control pigs. Additionally, the arginine treatment affected serum concentrations of nitrogenous and lipid signaling molecules (glycerophosphorylcholine and myo-inositol) and intestinal bacterial metabolites (formate, ethanol, methylamine, dimethylamine, acetate, and propionate). These novel findings suggest that dietary arginine supplementation alters the catabolism of fat and amino acids in the whole body, enhances protein synthesis in skeletal muscle, and modulates intestinal microbial metabolism in growing pigs.     

Role of parenchymal and non-parenchymal rat liver cells in the uptake of cholesterolester-labeled serum lipoproteins.

Very low density lipoprotein (VLDL)-remnants, prepared by extrahepatic circulation of VLDL, labeled biosynthetically in the cholesterol (ester) moiety, were injected intravenously into rats in order to determine the relative contribution of parenchymal and non-parenchymal liver cells to the hepatic uptake of VLDL-remnant cholesterol (esters). 82.7% of the injected radioactivity is present in liver, measured 30 min after injection. The non-parenchymal liver cells contain 3.1±0.1 times the amount of radioactivity per mg cell protein as compared to parenchymal cells. The hepatic uptake of biosynthetically labeled (screened) low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterolesters amounts to 26.8% and 24.4% of the injected dose, measured 6 h after injection. The non-parenchymal cells contain 4.3±0.8 and 4.1±0.7 times the amount of radioactivity per mg cell protein as compared to parenchymal cells for LDL and HDL, respectively. It is concluded that in addition to parenchymal cells, the non-parenchymal cells play an important role in the hepatic uptake of cholesterolesters from VLDL-remnants, LDL and HDL.     

Defective catabolism of low-density lipoprotein by fibroblasts from patients with I-cell disease.

Skin fibroblast cultures from patients with I-cell disease (mucolipidosis II) are characterized by multiple lysosomal enzyme deficiencies The present studies deal with the consequences of these deficiencies with respect to the metabolism of plasma low-density lipoproteins. Degradation of the protein moiety was defective in I-cells compared with control cells, but the binding and internalization of low density lipoprotein were much less affected. Measurements of low-density lipoprotein degradation in homogenates demonstrated directly for the first time a deficiency of acid proteinase activity in I-cell fibroblasts. Comparison of results in 6-h incubations with those in 24-h incubations showed accumulation of intracellular low-density lipoprotein in I-cell fibroblasts and an accelerating rate of degradation, possibly attributable to intracellular accumulation of low-density lipoprotein substrate. The significance of these findings with respect to low-density lipoprotein metabolism in vivo is discussed.     

Chloroquine-induced interference with degradation of serum lipoproteins in rat liver, studied in vivo and in vitro.

The effect of chloroquine, an inhibitor of certain lysosomal enzymes including cathepsin B (EC 3.4.22.1), on the degradation of serum lipoproteins in rat liver was studied in vivo and in liver homogenates. Chloroquine had no effect on the clearance from the circulation of 125I-labeled rat or human very low density lipoproteins or human low density lipoproteins. Pretreatment with chloroquine for 3 h, resulted in a 2-2.5 fold increase in 125i-labeled very low density lipoprotein recovered in the liver 45 min after injection of the homologous and heterologous lipoproteins. This effect was evident on both the 125I-labeled protein and 125I-labeled lipid moiety. 30 min after the injection of [3H]-cholesterol linoleate-labeled very low density lipoproteins, 70% of the injected label was recovered in the liver, both in control and chloroquine-treated rats. Since the perl and 20% in the experimental group, it was concluded that chloroquine interferes with the hydrolysis of [3H]cholesterol linoleate. Following injection of 125I-labeled human low density lipoproteins only 4% of the injected lipoprotein was recovered in the liver of control rats and not more than 10% after chloroquine treatment, when about 50% had been cleared from the circulation. Hence, while very low density lipoprotein protein and cholesterol ester are catabolized in the liver, the catabolism of low density lipoproteins occurs mainly in extra-hepatic tissues. Using post-nuclear liver suprnatant, optimal degradation of various serum lipoproteins was found at pH 4.4, and chloroquine inhibited their degradation. Degradation of very low density and low density lipoproteins was completely inhibited at 0.05 M chloroquine, while less pronounced inhibition was seen with high density lipoproteins, apolipoproteins and apolipoprotein AI. These results indicate that liver acid hydrolases in vivo participate in the degradation of serum lipoproteins. Cathepsin B is apparently responsible for the degradation of aplipoprotein B, while other cathepsins might also be active in the degradation of this and the other apolipoproteins.     

Musca domestica larval lipoprotein

A larval specific high-density lipoprotein (HDL) has been isolated from Musca domestica hemolymph by a combination of density gradient and glycerol gradient ultracentrifugations. The larval lipoprotein has a density of 1.134 g/ml and is formed by at least four apoproteins with molecular weights equal to 26,000, 23,000, 21,000, and 20,000. This lipoprotein contains large amounts of hydrocarbons and phospholipids and minor amounts of diacylglycerols and cholesterol. The larval lipoprotein is completely distinct from lipophorin in regard to apoprotein composition, lipid moiety, physiological pattern, and immunological reactions. Larval lipoprotein is accumulated until the end of the feeding period. During the pupal molt this protein is utilized and is no longer detected after 2 days of pupal stadium. The results obtained imply a possible role of this protein in the puparia and/or pupal cuticle formation. Judging from the properties shown, the Musca domestica larval lipoprotein is a completely new type of insect lipoprotein.     

Uptake and degradation of low density lipoprotein by swine arterial smoot muscle cells with inhibition of cholesterol biosynthesis.

We have previously proposed on the basis of studies in hepatectomized animals that low density lipoproteins are degraded at a significant rate by peripheral tissues. To test the capacity of one peripheral cell type to catabolize low density lipoprotein, cultures of swine aortic smooth muscle cells were incubated with homologous 125I-labeled low density lipoprotein and uptake and degradation measured. Degradation of 125I-labeled low density lipoprotein to products soluble in trichloroacetic acid showed an initial lag period of 1--2 h after which the rate increased and remained linear for the following 15 h. Rates of degradation increased sharply with low density lipoprotein concentration over the lower range (from 0--25 mug protein/ml) and then more slowly up to the highest concentration tested, 300 mug protein/ml. Even at very low concentrations, 1 mug low density lipoprotein protein/ml (less than 10% of the plasma low density lipoprotein concentration), the in vitro degradation rate (per kg of smooth muscle cells) exceeded the in vivo degradation rate (per kg of total body weight). To the extent that smooth muscle cells are representative of other peripheral cells, the results support the proposal that peripheral degradation of low density lipoprotein apoprotein may be quantitatively important. The rate of incorporation of labeled acetate into sterols was suppressed in cells incubated with whole serum, low density and very low density lipoproteins, or suspensions of free cholesterol. In this respect, the results were similar to those observed in human skin fibroblasts studied concurrently. However, high density lipoprotein inhibited sterol synthesis by about 25% in swine smooth muscle cells while it had no effect in human skin fibroblasts.     

Location and interactions of phospholipid and cholesterol in human low density lipoprotein from 31P nuclear magnetic resonance.

The major phospholipids, phosphatidylcholine and spingomyelin, of low density lipoprotein (LDL) are accessible to small amounts of Pr3+, suggesting that the head groups of all mobile phospholipids are on the surface of the particle in contact with the aqueous medium. The major source of the nuclear Overhauser effect enhancement of 31P resonances is the N-methyl proton of the choline moiety, indicating close N-methyl phosphate group interactions, probably similar to those found previously in phospholipid vesicles. This behavior of the phospholipid head groups in LDL is similar to that in small vesicles without cholesterol, suggesting that in LDL most of the cholesterol is not associated with mobile, surface phospholipids. In contrast to LDL, where the presence of a large protein immobilizes some phospholipid head groups, immobilization does not occur in high density lipoprotein, consistent with occurrence of smaller peptides in the latter.     

Lipid peroxide and transition metals are required for the toxicity of oxidized low density lipoprotein to cultured endothelial cells

The toxicity of oxidized low density lipoprotein (Ox-LDL) to cultured vascular endothelial cells was investigated. The modification of low density lipoprotein (LDL) by copper led to the production of thiobarbituric acid-reacting substance (TBARS) and lipid hydroperoxide (LPO). TBARS was distributed not only in lipoprotein, but also in the aqueous phase, whereas LPO was observed only in the lipoprotein particle. During the incubation of LDL with copper, the copper bound to lipoprotein and formed a complex. The toxicity of products resulting from the oxidation of LDL to endothelial cells was recognized in Ox-LDL particles, not in the aqueous phase. Following dialysis of Ox-LDL against EDTA, copper which had bound to the Ox-LDL particle was released and the toxicity of Ox-LDL disappeared. The addition of copper to the dialyzed Ox-LDL restored the cytotoxicity. To a lesser extent this effect was also observed with the addition of iron. A study of the time-course of LDL oxidation showed that the toxicity of Ox-LDL depends upon the level of LPO, not upon the content of TBARS, the extent of negative charge or the protein adduct of aldehydes. These results demonstrate that transition metal is required for Ox-LDL toxicity and that the toxic moiety of the products resulting from LDL oxidation is LPO associated with the Ox-LDL particle.     

Determination of B protein of low density lipoprotein directly in plasma.

Quantitation of the apoprotein constituents of lipoproteins has extended our knowledge of plasma lipid transport. Previously, B protein content of low density lipoprotein could be measured by radial immunodiffusion only after ultracentrifugation. However, if performed in 1.5% agarose gel with standards and measured at 18 hr rather than at equilibrium, low density lipoprotein B protein can be measured directly in plasma, eliminating the need to separate very low density lipoprotein.     

Expression of Recombinant LDLR–EGFP Fusion Protein in HEK-293 Cells as a Promising Tool to Assess the Effect of <Emphasis Type="Italic">LDLR</Emphasis> Gene Mutations

Mutations in the low density lipoprotein receptor gene (LDLR) frequently impair folding and intracellular traffic of the receptor protein, resulting in the development of a monogenic disorder, familial hypercholesterolemia (FH). Identification of novel LDLR mutations requires confirmation of their functional importance in distinguishing pathogenic mutations from neutral changes in the aminoacid sequence. To elaborate a system for evaluation of the effect of mutation on the folding and intracellular transport of the LDLR, as well as its ability to bind low density lipoprotein (LDL), we constructed a plasmid containing LDLR cDNA and the gene of enhanced green fluorescent protein (EGFP). Confocal microscopy has shown that, upon transient transfection of HEK293 cells with the plasmid, the recombinant fusion protein LDLR–EGFP is transported onto the cellular membrane and binds labeled LDL. This construct will be further modified by site-directed mutagenesis to reproduce the LDLR missense mutations most common in the population of northwest Russia so as to study the subcellular localization and function of the modified chimeric protein.     

Metabolism of lipoproteins in nonhuman primates. Studies on the origin of low density lipoprotein apoprotein in the plasma of the squirrel monkey.

The plasma of squirrel monkeys contains extremely low levels of very low density lipoproteins. The delipidated apoproteins from the different lipoprotein density classes of this species show a heterogeneity similar to that of man and the rat. The biosynthesis of the apoproteins of squirrel monkey lipoproteins was studied in fasted normal and Triton WR1339-treated animals. After intravenous injection of [3-H] leucine, maximal labeling of very low density lipoproteins occurred after 1 h, intermediate density lipoproteins (d 1.006--1.019) in 2 h, and low density lipoproteins after 3 h. At all times, however, low density lipoproteins had the greatest percentage of radioactivity. Polyacrylamide gel electrophoresis revealed that the apoprotein B moiety of very low density and intermediate density lipoproteins contained 62% and 81% of the total radioactivity in these lipoproteins whereas the fast-migrating peptides were minimally labeled. In monkeys injected with Triton WR1339, 70--80% of the radioactivity incorporated into d smaller than 1.063 lipoproteins was in very low density lipoproteins with only 10--15% in intermediate and low density lipoproteins. After injection of 3-H-labeled very low density lipoproteins and [14-C] leucine into Triton-treated monkeys, catabolism of 3-H-labeled very low density lipoprotein to intermediate and low density lipoproteins was small and was significantly less than corresponding values for the incorporation of [14-C] leucine. Thus, breakdown of very low density lipoproteins could not account for all the labeled apoprotein B present in the intermediate and low density lipoprotein fractions. The results indicate that most, but not all, of the newly synthesized apoprotein B enters plasma in very low density lipoproteins and that the low concentrations of this lipoprotein in squirrel monkey plasma are a consequence of its rapid turnover.     

Lipoprotein interconversions in an insect, Manduca sexta. Evidence for a lipid transfer factor in the hemolymph

Hemolymph lipoproteins (lipophorins) of adult Manduca sexta are disinct from larval forms in density, lipid content, composition, and the presence of a third, low molecular weight apoprotein. Generally, only one lipoprotein species exists in M. sexta hemolymph during any given life stage. Progression through the life cycle results in alterations of existing lipoproteins to produce new forms, without new protein synthesis. The observed alterations in lipoprotein density could result from facilitated lipid transfer in insect hemolymph. An in vitro assay of facilitated lipid transfer was developed which employs a high density lipophorin from the wandering larva (density = 1.18 g/ml) as acceptor and adult low density lipophorin (density = 1.03 g/ml) as donor. Adult lipophorin-deficient hemolymph was shown to catalyze a time-dependent equilibration of the starting lipoproteins to produce a new intermediate lipophorin, Lp-I. Hydrodynamic experiments on the donor, acceptor, and product lipoproteins excluded fusion as the mechanism whereby Lp-I is produced. Thus, it is concluded that Lp-I results from facilitated net lipid transfer from low to high density lipoprotein. Furthermore, experiments conducted with radioiodinated donor and radioiodinated acceptor lipoproteins demonstrated that apoprotein exchange does not occur during the lipid transfer reaction. When donor lipoprotein was labeled in the lipid moiety with carbon-14, evidence of diacylglycerol and phospholipid exchange was obtained. Partial characterization of the lipid transfer factor revealed a relationship between incubation time, donor concentration, acceptor concentration, lipophorin-deficient hemolymph concentration, and transfer activity, as measured by Lp-I production. It is concluded that lipophorin-deficient hemolymph contains one or more factor(s) that catalyze net lipid transfer as well as diacylglycerol and phospholipid exchange between lipophorins to produce a single form at equilibrium.     

Interaction of swine lipoproteins with the low density lipoprotein receptor in human fibroblasts.

HDLc, a cholesterol-rich lipoprotein that accumulates in the plasma of cholesterol-fed swine, was shown to resemble functionally human and swine low density lipoprotein in its ability to bind to the low density lipoprotein receptor in monolayers of cultured human fibroblasts. This binding occurred even though HDLc lacked detectable apoprotein B, which is the major protein of low density lipoprotein. After it was bound to the low density lipoprotein receptor, HDLc, like human and swine low density lipoprotein, delivered its cholesterol to the cells, and this, in turn, caused a suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, an activation of the cholesterol-esterifying system, and a net accumulation of free and esterified cholesterol within the cells. Swine HDLc, like human high density lipoprotein, did not bind to the low density lipoprotein receptor nor did it elicit any of the subsequent metabolic events. HDLc, like human low density lipoprotein, was incapable of producing a metabolic effect in fibroblasts derived from a subject with the homozygous form of familial hypercholesterolemia, which lack low density lipoprotein receptors. These results indicate that two lipoproteins that have been associated with athersclerosis--low density lipoprotein in humans and HDLc in cholesterol-fed swine--both can cause the accumulation of cholesterol and cholesteryl esters within cells through an interaction with the low density lipoprotein receptor.