2-3-oxidosqualene and 2-3-22-23-dioxidosqualene

2,3-Oxidosqualene is an intermediate in cholesterol biosynthesis and 2,3:22,23-dioxidosqualene act as the substrate for an alternative pathway that produces 24(S),25-epoxycholesterol which effects cholesterol homeostasis. In light of our previous findings concerning the biological effects of certain epoxidated all-trans-polyisoprenes, the effects of squalene carrying epoxy moieties on the second and third isoprene residues were investigated here. In cultures of HepG2 cells both monoepoxides of squalene and one of their hydrolytic products inhibited cholesterol synthesis and stimulated the synthesis of coenzyme Q (CoQ). Upon prolonged treatment the cholesterol content of these cells and its labeling with [(3)H]mevalonate were reduced, while the amount and labeling of CoQ increased. Injection of the squalene monoepoxides into mice once daily for 6days elevated the level of CoQ in their blood, but did not change the cholesterol level. The same effects were observed upon treatment of apoE-deficient mice and diabetic GK-rats. This treatment increased the hepatic level of CoQ10 in mice, but the amount of CoQ9, which is the major form, was unaffected. The presence of the active compounds in the blood was supported by the finding that cholesterol synthesis in the white blood cells was inhibited. Since the ratio of CoQ9/CoQ10 varies depending on the experimental conditions, the cells were titrated with substrate and inhibitors, leading to the conclusion that the intracellular isopentenyl-PP pool is a regulator of this ratio. Our present findings indicate that oxidosqualenes may be useful for stimulating both the synthesis and level of CoQ both in vitro and in vivo.

Topics: Animals; Bridged Bicyclo Compounds, Heterocyclic; Cholesterol; Diabetes Mellitus, Experimental; Etidronic Acid; Hemiterpenes; Hep G2 Cells; Humans; Lovastatin; Male; Mevalonic Acid; Mice; Mice, Inbred C57BL; Mice, Knockout; Organophosphorus Compounds; Rats; Rats, Wistar; Risedronic Acid; Squalene; Tricarboxylic Acids; Ubiquinone

The inhibition of 2,3-oxidosqualene-lanosterol cyclase (EC 5.4.99.7) (OSC) by new azasqualene derivatives, mimicking the proC-8 and proC-20 carbocationic high-energy intermediates of the cyclization of 2,3-oxidosqualene to lanosterol, was studied using pig liver microsomes, partially purified preparations of OSC, and yeast microsomes. The azasqualene derivatives tested were: 6E- and 6Z-10aza-10,11-dihydrosqualene-2,3-epoxide 17 and 18, 19-aza-18,19,22,23-tetrahydrosqualene-2,3-epoxide 19 and its corresponding N-oxide 20, and 19-aza-18,19,22,23-tetrahydrosqualene 21. The compounds 17 and 19 (i.e. the derivatives bearing the 2,3-epoxide ring and the same geometrical configuration as the OSC substrate) were effective inhibitors, as shown by the Ki obtained using partially purified OSC: 2.67 microM and 2.14 microM, respectively. Compound 18, having an incorrect configuration and the 19-aza derivative 21, lacking the 2,3-epoxide ring, were poor inhibitors, with IC50 of 44 microM and 70 microM, respectively. Compound 21 was a competitive inhibitor of OSC, whereas 17 and 19 were noncompetitive inhibitors, and showed a biphasic time-dependent inactivation of OSC, their apparent binding constants being 250 microM and 213 microM, respectively. The inhibition of sterol biosynthesis was studied using human hepatoma HepG2 cells. The incorporation of [14C] acetate in the C27 sterols was reduced by 50% by 0.55 microM 17, 0.22 microM 19, and 0.45 microM 21, whereas 2 microM 18 did not affect sterol biosynthesis. In the presence of 17, 19 and 21, only the intermediate metabolites 2,3-oxidosqualene and 2,3,22,23-dioxidosqualene accumulated, demonstrating a very specific inhibition of OSC.

Topics: Animals; Epoxy Compounds; Humans; Intramolecular Transferases; Isomerases; Kinetics; Microsomes, Liver; Rats; Squalene; Stereoisomerism; Sterols; Swine; Tumor Cells, Cultured

Partially purified squalene epoxidase (SE) from pig liver converts 2,3-oxidosqualene (SO) to 2,3;22,23-squalene dioxide (SDO) with approximately one-half the efficiency of the epoxidation of squalene at pH 7.4. The SO to SDO conversion is independent of pH and shows an absolute requirement for exogenous FAD. Two noncompetitive SE inhibitors show selectivity in blocking squalene (Sq) to SO versus SO to SDO epoxidations. Finally, SDO inhibits the activity of crude pig liver oxidosqualene cyclase (OSC) with an IC50 of 16 microM. Thus, SE inhibitors not only reduce the production of SO from Sq, but also strongly suppress "leakage" of SO to the potentially harmful SDO in vitro.

Topics: Animals; Flavin-Adenine Dinucleotide; Kinetics; Liver; Oxygenases; Squalene; Squalene Monooxygenase; Swine

Inhibition of sterol uptake in Saccharomyces cerevisiae sterol auxotroph FY3 (alpha hem1 erg7 ura) by delta-aminolevulinic acid (ALA) is dependent on the ability of the organism to synthesize heme from ALA. Sterol-depleted cells not exposed to ALA or strain PFY3 cells, with a double heme mutation, exposed to ALA did not exhibit inhibition of sterol uptake. Addition of ALA to sterol-depleted FY3 stimulated production of a high endogenous concentration of 2,3-oxidosqualene (25.55 micrograms mg-1 [dry weight]) at 24 h, whereas FY3 not exposed to ALA or PFY3 exposed to ALA did not accumulate 2,3-oxidosqualene. The high concentration of 2,3-oxidosqualene in FY3 with ALA decreased, and 2,3;22,23-dioxidosqualene increased to a very high level. The elevation of 2,3-oxidosqualene by ALA was correlated with a fivefold increase in the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC 1.1.1.34). The enhanced activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase was prevented by cycloheximide but not chloramphenicol and was dependent on a fermentative energy source. Inhibition of sterol uptake could not be attributed to 2,3-oxidosqualene or 2,3;22,23-dioxidosqualene but was due to a nonsaturating level of ergosterol produced as a consequence of heme competency through a leaky erg7 mutation.

Topics: Aminolevulinic Acid; Biological Transport; Cholesterol; Ergosterol; Heme; Hydroxymethylglutaryl CoA Reductases; Lipids; Mutation; Saccharomyces cerevisiae; Squalene; Sterols