Hepatic metabolism of oxidatively modified apo E-free high-density lipoproteins.

AIMS/BACKGROUND: The metabolism of rat apo E-free high-density lipoproteins (HDL) was contrasted with oxidatively modified apo E-free high-density lipoproteins (OX-HDL) in the rat hepatoma cell, Fu5AH. RESULTS: When 10-100 microg/ml [125I]-HDL or [125I]-OX-HDL were incubated with cells for 4 h at 37 degrees C, cellular uptake of oxidized lipoproteins was twice control. In contrast, protein degradation was equal. [125I]-HDL or [125I]-OX-HDL were incubated with the cells for 4 h followed by a 4 h chase with unlabeled HDL and OX-HDL, respectively. In these experiments, 80% of [125I]-HDL was resecreted from the cell within 30 min while 50% of [125I]-OX-HDL was retained by the cell after 2 h. Electron microscopy was used to determine if the OX-HDL was retained in lysosomes. Cells were incubated with gold-labeled OX-HDL, and lysosomes were stained with acid phosphatase. Gold-labeled OX-HDL was abundant in intracellular vesicles that were not reactive to acid phosphatase. However, vesicles with a high content of OX-HDL frequently stained positively for 3,3'-diaminobenzidine, a stain that reacts with catalase and is used to detect peroxisomes. CONCLUSIONS: The present evidence indicates that the cellular metabolism of OX-HDL is different from that of unmodified HDL.

A role for retrosomes in intracellular cholesterol transport from endosomes to the plasma membrane.

The recycling component (retrosome) of the endocytic pathway was evaluated as a potential vehicle for the recycling of lipoprotein-derived cholesterol and the maintenance of a high concentration of free cholesterol in plasma membranes. Receptor-to-ligand ratios were established in three distinct endosomal compartments using a recycling receptor (apolipoprotein B/E) to confirm isolated retrosomes as recycling vesicles. Compositional studies showed that retrosomes have twice the free cholesterol in their limiting membranes as do the endosomal compartments from which they derive. Furthermore, of the three isolated endosomal fractions, retrosomes showed the highest ratio of free to esterified cholesterol derived from injected very low density lipoprotein as well as the highest free-to-esterified cholesterol mass ratio overall, confirming endosomal cholesteryl ester hydrolysis and sorting. Endosomal neutral cholesterol esterase was identified by immunoblot, whereas electron microscopy employing membrane cholesterol-specific filipin revealed a high concentration of cholesterol in appendages that appear to be the formative stage of retrosomal biogenesis.

Influence of dietary fat on the effect of endotoxin on murine hepatic peroxisomes.

This study investigates the effects of either a high-fat diet or endotoxin on peroxisome metabolism as assessed by measuring catalase activity, catalase mass, and peroxisomal beta-oxidation. Three mouse strains C3H/HeJ, BALB/c, and C57BL/6J were fed either a low-fat or a high-fat diet and injected intraperitoneally with 1 microg Escherichia coli lipopolysaccharide. These parameters were not different in C3H/HeJ mice fed a high-fat diet compared with controls fed a low-fat diet. Total liver catalase activity and peroxisomal beta-oxidation were higher in BALB/c mice fed high-fat diet compared with low-fat controls. Total liver catalase activity, catalase mass and peroxisomal beta-oxidation increased to the greatest extent in C57BL/6J mice fed a high-fat diet. Endotoxin treatment did not alter any of the parameters in mice fed a low-fat diet. Among mice fed a high-fat diet, endotoxin did not affect hepatic catalase or peroxisomal beta-oxidation in the C3H/HeJ mice, decreased catalase activity in BALB/c mice (28%), and greatly decreased both catalase (66%) and peroxisomal beta-oxidation (69%) in C57BL/6J mice. The decrease in catalase activity in C57BL/6J mice was apparently because of an inactivation of the enzyme as determined by the activity/mass ratio. Thus, endotoxin is showed to inhibit both catalase and peroxisomal beta-oxidation in mice and the sensitivity to endotoxin is greatest in C57BL/6J mice fed a high-fat diet.

Evidence that a neutral cholesteryl ester hydrolase is responsible for the extralysosomal hydrolysis of high-density lipoprotein cholesteryl ester in rat hepatoma cells (Fu5AH).

Diethylumbelliferyl phosphate (UBP) has been shown to inhibit the neutral cholesteryl ester hydrolase activity responsible for hydrolysis of cellular lipid droplet cholesteryl ester (Harrison et al., 1990). The potential for (UBP) to inhibit uptake and hydrolysis of high density lipoprotein (HDL) cholesteryl ester was studied in Fu5AH hepatoma cells, a model for HDL cholesterol delivery. Coincubation of 3H-cholesteryl ester labeled HDL with UBP resulted in a 72% decrease in the cellular free cholesterol/cholesteryl ester (FC/CE) isotope ratio, indicating an inhibition in the conversion of cholesteryl ester to free cholesterol. Total cellular 3H-CE uptake was modestly (27%) but significantly decreased by UBP. Pulse-chase experiments (15 min. pulse and 7 min. chase) were used to study the hydrolysis of HDL 3H-CE in subcellular fractions separated by percoll gradients. The conversion of 3H-CE to 3H-FC could be demonstrated in fractions that comigrated with the plasma membrane/endosome fractions but were well separated from lysosomes. Neutral cholesteryl ester hydrolase activity was detected in those same fractions. These results suggest that an extralysosomal pathway is operating in the metabolism of HDL cholesterol and its delivery to hepatoma cells.

Triacylglycerol hydrolysis in isolated hepatic endosomes.

Three endosomal compartments including the compartment for uncoupling receptor and ligand (CURL), multivesicular bodies (MVB), and a putative recycling fraction (retrosomes) were isolated from rat liver homogenates fifteen minutes after a bolus injection of very low density lipoprotein (VLDL) was delivered into a femoral vein. Assays for enzyme markers indicate a minimal contamination with either lysosomes or Golgi. The increase in specific activity of the radiolabeled ligand (VLDL) during the isolation procedure from homogenate to MVB, demonstrates a 200-250-fold purification of this organelle. All three fractions have the ability to catabolize triacylglycerol substrate both as triolein and as VLDL triacylglycerol. Furthermore, incubation of isolated endosomes following injection of endogenously labeled VLDL demonstrate their ability to hydrolyze VLDL triacylglycerol in situ. Three distinct lipolytic pH optima were found at pH 5.5, 7.1, and 8.6. The effects of serum, MgCl2, CaCl2, NaCl, sodium dodecyl sulfate, bile acids, and antibody to hepatic triacylglycerol lipase on the individual endosome fractions demonstrated distinct lipolytic activities in the different compartments. Results indicate that both an endosomal neutral lipase as well as hepatic triacylglycerol lipase make a significant contribution to lipolytic processing of endocytosed lipoproteins prior to their resecretion of further processing in hepatic lysosomes.

Evidence for extralysosomal hydrolysis of high-density lipoprotein cholesteryl esters in rat hepatoma cells (Fu5AH): a model for delivery of high-density lipoprotein cholesterol.

Rat hepatoma cells (Fu5AH) were studied as a model for the net delivery of apoE-free high-density lipoprotein (HDL) cholesterol to a cell. Incubating cells with HDL results in 1) a decrease in both media-free cholesterol and cholesteryl ester concentration; 2) decreased cell sterol synthesis; and 3) increased cell cholesteryl ester synthesis. HDL cholesteryl ester uptake is increased when cells are incubated for 18 hr in cholesterol poor media. Coincubation of 3H-cholesteryl ester-labeled low-density lipoprotein (LDL) with 50 microM chloroquine or 25 microM monensin results in a decrease in the cellular free cholesterol/cholesteryl ester (FC/CE) isotope ratio, indicating an inhibition in the conversion of cholesteryl ester to free cholesterol. In contrast, chloroquine and monensin do not alter the cellular FC/CE isotope ratio for 3H-CE HDL. This evidence indicates that acidic lysosomal cholesteryl ester hydrolase does not account for the hydrolysis of HDL-CE. Free cholesterol generated from 3H-cholesteryl ester of both LDL and HDL is reesterified intracellularly. At higher HDL concentrations (above 50 micrograms/ml) HDL cholesteryl ester hydrolysis is sensitive to chloroquine. We propose that an extralysosomal pathway is operating in the metabolism of HDL cholesterol and that at higher HDL concentrations a lysosomal pathway may be functioning in addition to an extralysosomal pathway.

Metabolism of apoE-free high density lipoproteins in rat hepatoma cells: evidence for a retroendocytic pathway.

The cellular metabolism of apoE-free HDL (HDL) was studied in rat hepatoma cells (FU5AH). Cells incubated with HDL showed a dose-dependent decreased incorporation of [14C]acetate into cell sterol, indicating a net cholesterol delivery to the cells. HDL was localized both at the cell surface and inside the cell. This conclusion was drawn from both the association of 125I-labeled HDL with the cells under different experimental conditions and morphological evidence based on the association of colloidal gold-labeled HDL with the cells. Up to 63% of the 125I-labeled HDL protein initially inside the cell was subsequently recovered in the media as trichloroacetic acid precipitable (TCA-ppt) protein after a 30-min, 37 degrees C chase with a 100-fold concentration of unlabeled HDL. About 27% of the TCA-ppt apoprotein originally inside the cell was recovered as TCA-soluble material. Thus, we conclude that of the HDL apoprotein taken up by the cells, the majority is resecreted by a retroendocytosis pathway. The quantity of HDL apoprotein reappearing in the media was stimulated by the presence of unlabeled HDL in the media, while the amount of TCA-soluble material produced was not. Retroendocytosis of HDL was inhibited at 0 degree C and by the presence of 10 mM NaCN, 20 mM 2-deoxy-D-glucose in the media. Thus, the pathway appears to be both temperature- and energy-sensitive. HDL resecreted by the cell were depleted of cholesteryl ester and showed an altered size distribution, indicative of lipoprotein catabolism and remodeling. This study provides evidence for the existence of an endocytosis-retroendocytosis pathway for HDL apoproteins in a rat hepatoma cell and for the possibility that the endocytosis-retroendocytosis pathway may be involved in lipid delivery to the cell.

A single high-cholesterol, high-fat meal preferentially increases low molecular weight apolipoprotein B concentration in rat plasma.

Rats were fed either rat chow (control), chow + 20% olive oil (olive oil), or chow + 20% olive oil + 2% cholesterol (olive oil/cholesterol) as a single meal to study the short-term effects of fat and the above combination of fat/cholesterol-containing diets on plasma apoB concentration and its influence on the distribution of apoB subspecies. Rats were given their meals and allowed to consume them ad libitum until they were killed, 3 hr or 9 hr afterwards. Three hours after feeding, serum triglyceride concentrations were increased to the same extent in both the olive oil and olive oil/cholesterol-fed rats as compared with concentrations in control rats, but serum apoB concentrations did not differ among the groups. Nine hours after feeding, serum triglyceride concentrations were still equally elevated in both experimental groups; however, in the olive oil/cholesterol-fed rats, total serum apoB as well as total serum cholesterol were increased above both the control and olive oil groups. In addition, the d less than 1.21 g/ml lipoprotein apoBl/apoBh ratio of the olive oil/cholesterol-fed rats was greatly increased at 9 hr, whereas apoBl/apoBh ratio in the d less than 1.21 g/ml fraction of the olive oil group was unchanged, despite the increase in plasma triglyceride concentration. In the olive oil/cholesterol-fed rats at 9 hr, cholesterol, total apoB, apoBl, and apoBh of both VLDL and IDL were greater than in the control or olive oil rats. In d less than 1.21 g/ml lipoproteins, VLDL, and IDL, the increases in apoBl concentrations were of a greater magnitude than the increases in apoBh.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of apolipoprotein A-IV between the lipoprotein and the lipoprotein-free fractions of rat plasma: possible role of lecithin:cholesterol acyltransferase.

Plasma samples were incubated under various conditions to study the effect of in vitro incubation on apolipoprotein A-IV distribution between the lipoprotein and lipoprotein-free fractions. When plasma was fractionated immediately after bleeding, apolipoprotein A-IV was present in equal concentrations in the lipoprotein and lipoprotein-free fractions. After a 4-hr, 37 degrees C incubation, greater than 90% of total plasma apolipoprotein A-IV was present in the lipoprotein fraction and the percentage of plasma cholesterol present as cholesteryl ester increased from 58% to 74%. When plasma was incubated for 4 hr at 37 degrees C in the presence of 1.5 mM 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), greater than 90% of total plasma apoA-IV was present in the lipoprotein-free fraction, whereas plasma cholesteryl ester concentration did not change. Incubating heat-inactivated plasma for 4 hr also resulted in the redistribution of apolipoprotein A-IV from the lipoprotein fraction to the lipoprotein-free fraction, concurrent with no change in cholesterol esterification. When heat-inactivated plasma was incubated in the presence of a purified lecithin:cholesterol acyltransferase preparation, cholesterol esterification was restored and apolipoprotein A-IV was redistributed from the lipoprotein-free fraction to the lipoprotein fraction in such a manner that greater than 90% was present in the lipoprotein fraction. No changes in apolipoprotein A-I and apolipoprotein E distributions were found under any of the above conditions. Thus, the in vitro plasma incubations show that apolipoprotein A-IV can move bidirectionally between lipoprotein and lipoprotein-free fractions; the direction of this movement depends on the condition of the incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of serum apolipoprotein E metabolism: role of chylomicron metabolism.

The mechanism by which high-fat, high-cholesterol diets lower serum apolipoprotein E (apoE) concentration was studied in rats by feeding a single high-fat, high-cholesterol meal and by intravenously infusing chylomicrons containing low and high amounts of cholesterol. Serum apoE concentrations were unchanged 9 hours after an olive oil meal but were decreased by 35% after an olive oil/cholesterol-enriched meal. The decrease in serum apoE concentrations with the olive oil/cholesterol meal was accompanied by a decrease in apoE concentration in the high density lipoprotein fraction. Three hours after the intravenous infusion of cholesterol-enriched chylomicrons, serum apoE concentrations decreased 40%, whereas serum apoE concentrations decreased by only 10% when chylomicrons with low cholesterol concentrations or saline were infused. It is concluded that the metabolism of cholesterol-enriched chylomicrons results in an increased removal of serum apoE and that the cholesterol content of chylomicrons is a determinant of serum apoE removal.

Effect of cholesterol feeding on apo B and apo E concentrations and distributions in euthyroid and hypothyroid rats.

Lipoprotein profiles in experimental hypercholesterolemia were studied using euthyroid and hypothyroid cholesterol-fed rats. In both groups, serum cholesterol concentration increased, but to a lesser extent in the cholesterol-fed euthyroid rats, with similar changes in distribution among lipoprotein fractions in both groups. Agarose electrophoresis of plasma and individual lipoprotein fractions showed that beta-VLDL and HDLc were present in the hypothyroid, cholesterol-fed rats. In the euthyroid and hypothyroid cholesterol-fed rats, serum apo B concentrations increased three-fold and five-fold, respectively. This reflected increases of apo B in very low density and intermediate density lipoproteins. In the euthyroid cholesterol-fed rat, serum apo E decreased 50%, while the serum apo E concentration was not significantly changed in hypothyroid cholesterol-fed rats. In both the euthyroid and the hypothyroid cholesterol-fed rats, apo E decreased in high density lipoproteins and increased in lower density lipoproteins. We observed qualitative and quantitative differences between hypothyroid and euthyroid cholesterol-fed rats. The major qualitative differences were the appearance of beta migrating very low density lipoproteins (beta-VLDL) and HDLc in the hypothyroid cholesterol-fed rat, and a decrease of serum apo E concentrations in the euthyroid cholesterol-fed rats. Changes in serum cholesterol and apo B concentrations and the distribution of cholesterol, apo B, and apo E among the lipoprotein fractions were similar in direction in both groups, but greater in magnitude in the hypothyroid versus euthyroid cholesterol-fed rats. These data demonstrate that hypothyroidism should be considered when evaluating apolipoprotein changes in hypercholesterolemic animal models.