Distribution of exogenous 25-hydroxycholesterol in Hep G2 cells between two different pools.

Binding of [26,27-(3)H]25-hydroxycholesterol (25HC) to human hepatoma Hep G2 cells was saturated within 120 min. Two intracellular pools of 25HC were identified in a pulse-chase experiment: (i) an exchangeable pool which was in dynamic equilibrium with 25HC in the medium (t(1/2) of reversible exchange 15 min) and (ii) an unexchangeable pool which remained in cells during incubation in medium containing LPDS. 25HC from the exchangeable pool inhibits cholesterol biosynthesis, decreases the HMG CoA reductase mRNA level and stimulates cholesterol acylation. 25HC from the unexchangeable pool was partially bound to cytosolic proteins and apparently utilized for metabolic transformation. Incubation of Hep G2 cells with [26,27-(3)H]25HC in the presence of a 30-fold molar excess of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one was found to cause (i) 2-fold decrease in the binding of [26,27-(3)H]25HC to cytosolic proteins (sedimentation constant of radioactive complex was 4-5 S) and (ii) the 35% inhibition of 25HC transformation to polar metabolites.

The study of nonspecific internalization of cholesteryl esters by the Hep G2 hepatoma cells.

The cholesteryl oleate-POPC dispersions (1:3, mol/mol, mean particle size 110+/-20 nm) were taken up by the human hepatoma line Hep G2 cells via endocytosis. Internalization of the cholesteryl oleate-POPC dispersions by Hep G2 cells was dependent on the incubation time and dispersion concentration. At the cholesteryl oleate concentration 100 microM, its total uptake and internalization were found to be 1.5 nmol and 0.8 nmol per 1 mg of cell protein/24 h, respectively. Intracellular cleavage of the cholesteryl oleate incorporated in dispersions resulted in accumulation of free cholesterol capable of being released into the medium and metabolized to water-soluble polar products, presumably bile acids; oleic acid released is, apparently, involved in biosynthesis of triacylglycerides. The low-density lipoprotein receptor is not involved in internalization of lipid dispersions, and the presence of the cholesteryl oleate-POPC dispersions has no effect on the receptor-dependent internalization of cholesteryl esters of the low-density lipoproteins. The obtained data allow us to consider nonspecific internalization of cholesteryl esters by hepatocytes as a substantial part of the nonpolar lipid clearance.

Effect of 3-substituted Delta8(14)-15-ketosterols on cholesterol metabolism in hepatoma Hep G2 cells.

The effects of 3-substituted Delta8(14)-15-ketosterols--3beta-(2-hydroxyethoxy)-, 3beta-(2-propenyloxy)-, 3beta-[2(R,S),2,3-oxidopropyloxy]-, 3beta-[2(R,S),2,3-dihydroxypropyloxy]-, 3beta-(2-oxoethoxy)-, 3beta-[2(R,S),2-acetoxy-3-acetamidopropyloxy]-, and 3beta-[2(R,S), 2-hydroxy-3-acetamidopropyloxy]-5alpha-cholest-8(14)-en-15-o nes--on cholesterol metabolism were studied in human hepatoma Hep G2 cells. 3beta-(2-Propenyloxy)-, 3beta-(2-oxoethoxy)-, and 3beta-[2(R,S),2, 3-oxidopropyloxy]-5alpha-cholest-8(14)-en-15-ones inhibited cholesterol biosynthesis without any effect on triglyceride biosynthesis, while 3beta-[2(R,S),2-acetoxy-3-acetamidopropyloxy]- and 3beta-[2(R,S), 2-hydroxy-3-acetamidopropyloxy]-5alpha-cholest-8(14)-en-15-o nes inhibited both cholesterol biosynthesis and triglyceride biosynthesis at concentrations exceeding 10 microM. 3beta-[2(R,S),2, 3-Dihydroxypropyloxy]-5alpha-cholest-8(14)-en-15-one, effectively inhibiting cholesterol biosynthesis, was found also to be toxic in Hep G2 cells at micromolar concentrations. 3beta-[2(R,S),2, 3-Oxidopropyloxy]-5alpha-cholest-8(14)-en-15-one effectively inhibited cholesterol acylation. All the tested compounds decreased the HMG-CoA reductase mRNA level at concentrations exceeding 10 microM; however, they did not affect the LDL receptor mRNA level. Among the compounds tested, only 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one decreased the uptake and internalization of LDL-associated cholesteryl esters, being as effective as 25-hydroxycholesterol.

HMG CoA reductase and LDL receptor genes are regulated differently by 15-ketosterols in Hep G2 cells.

The ability of two 15-ketosubstituted sterols, 5alpha-cholest-8(14)-en-3beta-ol-15-one and 3beta-(2-hydroxyethoxy)-5alpha-cholest-8(14)-en-15-one, to alter the mRNA levels of 3-hydroxy-3-methylglutaryl-CoA reductase, low density lipoprotein receptor, and oxysterol binding protein was studied and compared with the effects of 25-hydroxycholesterol in Hep G2 cells. All three oxysterols decreased the level of HMG CoA reductase mRNA at concentrations of 10-30 microM, although 25-hydroxycholesterol was effective at concentrations of 1-3 microM. 25-Hydroxycholesterol lowered the level of LDL receptor mRNA more efficiently after 8 hours than after 24 hours of incubation, whereas 15-ketosterols did not decrease the mRNA level of the LDL receptor. The transcriptions of HMG CoA reductase and LDL receptor genes are therefore independently regulated by 15-ketosterols in Hep G2 cells. In addition, the level of the oxysterol binding protein mRNA is not affected by oxysterols in Hep G2 cells.