Cholesterol and LDL receptors in ubiquinone treated primary hepatocytes and HepG2 cells -

Abstract

Cholesterol is a vital component of cell membranes and a precursor of many biomolecules: cholesterol esters, steroid hormones and bile acids. Its synthesis involves the intermediate mevalonic acid (MVA) which serves also as a precursor for isoprenoids, dolichol and ubiquinone (UQ). UQ is a lipid soluble molecule that mediates the transfer of electrons in the respiratory chain. The role of its antioxidant property in enhancing mitochondrial function and in reducing LDL oxidation has been reported. In this study, we investigate the possible role of UQ in regulating MVA pathway and subsequently cholesterol level. Two types of ubiquinones were used UQ1 and the naturally occurring UQ10. Isolated rat primary rat hepatocytes and HepG2 were pre-treated with UQ1 (25 and 50 µM) and UQ10 (50µM) followed by addition of 2-14C MVA (for 24 hours), bypassing thus the HMGCoA reductase catalyzed step. Lipids were then extracted (Bligh and Dyer), evaporated, and separated by Thin Layer Chromatography using Heptane-Ethyl acetate as solvent system. The radioactivity incorporated into each of cholesterol, lanosterol, squalene and UQ10 was counted (Cpm) and were normalized to inorganic phosphate. Expression of LDL receptor in UQ1 and UQ10 treated cells was also examined using immuno-staining and western blotting. The effect of different inhibitors of MVA pathway in the presence or absence of UQ1-UQ10 was also examined. These include HMGCoA Reductase (Statin-50µM); Farnesyl-geranylgeranyl transferase (FPT-I-47µM); 4-hydroxybenzoate polyprenyl transferase (Bacitracin-50µM). Our results showed a significant decrease in the radioactivity incorporated into cholesterol in both UQ1 and UQ10 treated cells, whereas it increased in lanosterol and squalene. UQ1 exhibited a more potent effect than UQ10 while it increased the incorporation of radioactivity in UQ10. Level of labeled cholesterol did not change in mevastatin treated primary hepatocytes while it decreased in HepG2 treated cells. Mevastatin co-treatment wi