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Selective uptake of cholesteryl esters from apolipoprotein-E-free high-density lipoproteins by rat parenchymal cells in vivo is efficiently coupled to bile acid synthesis.

ABSTRACT: [3H]Cholesteryl ester-labelled human high-density lipoprotein (HDL) was injected into rats and its decay, intrahepatic cellular distribution and the kinetics of biliary secretion were determined. At 10 min after injection the hepatic uptake of cholesteryl esters from HDL was 3-fold higher as compared with the apolipoprotein. Selective uptake was exerted only by parenchymal cells (5.6-fold more cholesteryl esters than apolipoprotein) and not by liver endothelial or Kupffer cells. The kinetics of biliary secretion of processed cholesteryl esters initially associated with HDL or low-density lipoprotein (LDL) were compared in unrestrained rats, equipped with permanent catheters in bile duct, duodenum and heart. At 72 h after injection of [3H]cholesteryl oleate-labelled HDL, 51.0 +/- 2.5% of the injected dose was recovered as bile acids, which is about twice as high as the secretion of biliary radioactivity after injection of [3H]cholesteryl oleate-labelled LDL. Oestradiol treatment stimulated only liver uptake of LDL cholesteryl esters, and resulted in a 2-fold higher liver uptake than with HDL. However, the rate of radioactive bile acid formation from [3H]cholesteryl oleate-labelled HDL was still more rapid than for LDL. It is concluded that the selective uptake pathway for cholesteryl esters from HDL in parenchymal cells is more efficiently coupled to the formation of bile acids than is the cholesteryl ester uptake from LDL. This efficient coupling may facilitate the role of HDL in reverse cholesterol transport.

SUBMITTER: Pieters MN

PROVIDER: S-EPMC1130554 | biostudies-other | 1991 Dec

REPOSITORIES: biostudies-other

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Project description:Oxidation of low-density lipoprotein (LDL) leads initially to the formation of LDL-associated cholesteryl ester hydroperoxides (CEOOH). LDL-associated CEOOH can be transferred to high-density lipoprotein (HDL), and HDL-associated CEOOH are rapidly reduced to the corresponding hydroxides (CEOH) by an intrinsic peroxidase-like activity. We have now performed in vivo experiments to quantify the clearance rates and to identify the uptake sites of HDL-associated [3H]Ch18:2-OH in rats. Upon injection into rats, HDL-associated [3H]Ch18:2-OH is removed more rapidly from the circulation than HDL-associated [3H]Ch18:2. Two minutes after administration of [3H]Ch18:2-OH-HDL, 19.6 +/- 2.6% (S.E.M.; n = 4) of the label was taken up by the liver as compared with 2.4 +/- 0.25% (S.E.M.; n = 4) for [3H]Ch18:2-HDL. Organ distribution studies indicated that only the liver and adrenals exhibited preferential uptake of [3H]Ch18:2-OH as compared with [3H]Ch18:2, with the liver as the major site of uptake. A cell-separation procedure, employed 10 min after injection of [3H]Ch18:2-OH-HDL or [3H]Ch18:2-HDL, demonstrated that within the liver only parenchymal cells take up HDL-CE by the selective uptake pathway. Selective uptake by parenchymal cells of [3H]Ch18:2-OH was 3-fold higher than that of [3H]Ch18:2, while Kupffer and endothelial cell uptake of the lipid tracers reflected HDL holoparticle uptake (as analysed with iodinated versus cholesteryl ester-labelled HDL). The efficient uptake of [3H]Ch18:2-OH by parenchymal cells was coupled to a 3-fold increase in rate of radioactive bile acid secretion from [3H]Ch18:2-OH-HDL as compared with [3H]Ch18:2-HDL. In vitro studies with freshly isolated parenchymal cells showed that the association of [3H]Ch18:2-OH-HDL at 37 degrees C exceeded [3H]Ch18:2-HDL uptake almost 4-fold. Our results indicate that HDL-associated CEOH are efficiently and selectively removed from the blood circulation by the liver in vivo. The selective liver uptake is specifically exerted by parenchymal cells and coupled to a rapid biliary secretion pathway. The liver uptake and biliary secretion route may allow HDL to function as an efficient protection system against potentially atherogenic CEOOH.

Project description:1. Human total HDL (high-density lipoprotein), HDL2 and HDL3 were labelled in vitro by incubation with lipoprotein-deficient serum (LPDS) which contained either [3H]cholesteryl oleate or [14C]cholesterol under different conditions. The lipoproteins were then subfractionated by heparin-Sepharose column chromatography, and three subfractions (A, B and C) were successively eluted from each preparation of HDL, HDL2 and HDL3. When the labelling was done at 37 degrees C for 17 h, the subfractions were homogeneously labelled with [3H]cholesteryl oleate. However, when it was performed for only 30 min at 4 degrees C, the subfractions showed marked differences in the 3H specific radioactivity, which was much higher in the C fractions than in the others. 2. 3H-labelled HDL2 and HDL3 subfractions behaved differently under the precipitant action of heparin-Mn2+; fraction C (the richest in apolipoprotein E) produced the largest amount of radioactive and chemical precipitate. More 3H radioactivity, but not the cholesterol, was precipitated from HDL2 or HDL3 by the reagent, demonstrating that 3H-labelled HDL2 and HDL3 behave like their fraction C, which becomes labelled to the highest specific radioactivity despite having the smallest mass. 3. The incubation of 3H-labelled HDL subfractions with human LPDS and very-low-density lipoprotein (VLDL) at 37 degrees C increased the quantity of 3H radioactivity that was precipitated, in proportion to the amount of VLDL present in the media. These changes were attributable to the action of cholesterol ester transfer protein, since they did not occur at 4 degrees C or when human LPDS was replaced with rat LPDS. 4. Kinetics of the transfer of HDL [3H]cholesteryl oleate to VLDL showed a greater apparent Vmax for fractions A than for fractions B from either HDL2 or HDL3, whereas the apparent Km values were very similar, which suggest that this transfer process is influenced by the apoprotein composition of the donor lipoprotein.

Project description:We have observed recently that high-density lipoproteins (HDL) are the predominant carriers of cholesteryl ester hydroperoxides (CEOOH), the major class of lipid hydroperoxides detectable at nanomolar concentrations in the plasma of healthy fasting humans. The present study investigates the effect of such very low levels of CEOOH in apolipoprotein E-free HDL3 on lipoprotein particle metabolism and 'selective uptake' of its CE by human Hep G2 cells. Minimal oxidation with aqueous peroxyl radicals had a negligible effect on the binding, internalization and degradation of 125I-labelled HDL3. In contrast, with an increasing degree of radical-mediated oxidation of labelled HDL3, [3H]cholesteryl linoleate ([3H]Ch18:2) was taken up at an increasingly greater rate than were 125I-apoproteins. When [3H]cholesteryl linoleate hydroperoxide ([3H]Ch18:2-OOH was incorporated into unoxidized HDL3 by exchange from donor liposomes, it was taken up at a more than 8-fold higher rate than was incorporated [3H]Ch18:2. The same degree of preferential uptake of oxidized CE was observed when HDL3 was used that was doubly labelled with [3H]Ch18:2-OOH and cholesteryl [14C]oleate ([14C]Ch18:1). In both situations, uptake of [3H]Ch18:2-OOH exceeded that of 125I-apolipoprotein A-I some 40-fold. This increased selective uptake of [3H]Ch18:2-OOH from very mildly oxidized HDL3 was accompanied by a parallel increase in the intracellular levels of labelled free cholesterol. In contrast, lipid hydroperoxides were not detectable within Hep G2 cells, suggesting efficient detoxification of CEOOH by these cells. Neither the increased selective uptake of Ch18:2-OOHs nor the levels of intracellular free cholesterol were influenced by the presence of 50 microM chloroquine, suggesting extralysosomal hydrolysis of oxidized CEs. These results show that the selective uptake of HDL CEOOH by Hep G2 cells is more efficient than that of unoxidized CE, and support a protective role for rapid selective uptake in the removal of circulating HDL CEOOH.

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Selective uptake of cholesteryl esters from apolipoprotein-E-free high-density lipoproteins by rat parenchymal cells in vivo is efficiently coupled to bile acid synthesis.

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