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Greater selective uptake by Hep G2 cells of high-density lipoprotein cholesteryl ester hydroperoxides than of unoxidized cholesteryl esters.

ABSTRACT: 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.

SUBMITTER: Sattler W

PROVIDER: S-EPMC1134528 | biostudies-other | 1993 Sep

REPOSITORIES: biostudies-other

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Project description:Cholesteryl esters (CEs) are the water-insoluble transport and storage form of cholesterol. For both transport and storage, phospholipids and proteins embrace the CEs to form an amphipathic monolayer that surrounds the CEs. CEs are transported extracellularly in lipoproteins and are stored intracellularly as cytoplasmic lipid droplets. To clarify the molecular phenomena related to the above structures, we conducted atomistic molecular-dynamics simulations for a spherical, approximately high density lipoprotein sized lipid droplet comprised of palmitoyl-oleoyl-phosphatidylcholine (POPC) and cholesteryl oleate (CO) molecules. An additional simulation was conducted for a lamellar lipid trilayer consisting of the same lipid constituents. The density profiles showed that COs were located in the core of the spherical droplet. In trilayer simulations, CO molecules were also in the core and formed two denser strata. This is remarkable because the intra- and intermolecular behaviors of the COs were similar to previous findings from bulk COs in the fluid phase. In accordance with previous experimental studies, the solubility of COs in the POPC monolayers was found to be low. The orientation distribution of the sterol moiety with respect to the normal of the system was found to be broad, with mainly isotropic or slightly parallel orientations observed deep in the core of the lipid droplet or the trilayer, respectively. In both systems, the orientation of the sterol moiety changed to perpendicular with respect to the normal close to the phopsholipid monolayers. Of interest, within the POPC monolayers, the intramolecular conformation of the COs varied from the previously proposed horseshoe-like conformation to a more extended one. From a metabolic point of view, the observed solubilization of CEs into the phospholipid monolayers, and the conformation of CEs in the phospholipid monolayers are likely to be important regulatory factors of CE transport and hydrolysis.

Project description:The relationship between the serum factor(s)-mediated induction of low-density lipoprotein (LDL) receptor activity and changes in cellular cholesterol metabolism was examined in the human hepatoma cell line Hep-G2. Relative to incubation with serum-free media [Eagle's minimal essential medium (MEM) control], short-term (less than 8 h) incubation with medium containing 15% of either calf serum (MEM + serum) or the d greater than 1.25 fraction of calf serum (MEM + d greater than 1.25) produced a time- and concentration-dependent increase in the uptake of 125I-LDL. Immunoblotting with anti-(LDL receptor) antibodies demonstrated that this was correlated with a 2-fold increase in the amount of the mature 136,000 Da LDL receptor protein in detergent-solubilized Hep-G2 cell membranes. Incubation with MEM + serum, but not MEM + d greater than 1.25, increased the efflux of radiolabelled cholesterol from Hep-G2 cells. However, the induction of 125I-LDL uptake by MEM + d greater than 1.25 (2.3-fold) and MEM + serum (2.2-fold) was virtually identical. Addition of the d less than 1.063 lipoproteins of calf serum to MEM + d greater than 1.25 at their original or three times their serum concentration decreased the induction of 125I-LDL uptake by MEM + d greater than 1.25 by only 20-30%. Together, these results suggest that the stimulation of 125I-LDL uptake was not due to the presence of high-density lipoprotein, the absence of LDL or the stimulation of cholesterol efflux. MEM + serum stimulated 125I-LDL uptake in cells cholesterol-loaded by incubation with rat very-low-density lipoprotein with beta electrophoretic mobility (beta-VLDL). Compared to incubation with the MEM control, either MEM + serum or MEM + d greater than 1.25 produced time-dependent increases in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase which also occurred in cholesterol-loaded cells. However, cholesterol biosynthesis, whether measured from 3H2O, [14C]acetate or [3H]mevalonic acid, was not increased. Incubation with MEM + serum or MEM + d greater than 1.25 did not affect [3H]oleate incorporation into cellular cholesteryl esters, hydrolysis of intracellular [3H]cholesteryl esters or the cellular mass of unesterified or esterified cholesterol. Incubation with MEM + serum or MEM + d greater than 1.25 produced a transient increase in the level of LDL receptor mRNA, reaching a maximum of 5-10-fold by 2 h and decreasing to near baseline levels by 4 h. Actinomycin D blocked the serum-factor-mediated induction of LDL receptor mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)

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Greater selective uptake by Hep G2 cells of high-density lipoprotein cholesteryl ester hydroperoxides than of unoxidized cholesteryl esters.

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