Oxysterol regulators of 3-hydroxy-3-methylglutaryl-CoA reductase in liver. Effect of dietary cholesterol.

Hepatic regulatory oxysterols were analyzed to determine which oxysterols were present in livers of mice fed a cholesterol-free diet and whether repression of 3-hydroxy-3-methylglutaryl-CoA reductase following cholesterol feeding was accompanied by an increase in one or more oxysterols. Analysis of free and esterified sterols from mice fed a cholesterol-free diet resulted in the identification and quantitation of six regulatory oxysterols: 24-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol, 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, and 7-ketocholesterol. Following the addition of cholesterol to the diet for 1 or 2 nights, hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity declined and the levels of oxysterols, especially those of the side-chain-hydroxylated sterols, increased. Total 3-hydroxy-3-methylglutaryl-CoA reductase repressor units attributable to identified free oxysterols increased 2.5- and 6-fold after 1 and 2 nights, respectively, of cholesterol feeding. The amounts of esterified 24-, 25-, and 26-hydroxycholesterol also increased, with the increase in esterified 24-hydroxycholesterol being the greatest. The 24-hydroxycholesterol was predominantly the 24S epimer and the 26-hydroxycholesterol was predominantly the 25R epimer, indicating enzymatic catalysis of their formation. The observed correlation between increased levels of regulatory oxysterols and repression of 3-hydroxy-3-methylglutaryl-CoA reductase in cholesterol-fed mice is consistent with a hypothesis that intracellular oxysterol metabolites regulate the level of the reductase.

Influence of lipid environment on insulin binding in cultured hepatoma cells.

The influence of alterations of plasma membrane physico-chemical properties on insulin binding have been characterized in an insulin-sensitive rat hepatoma cell line adapted to grow for several generations in culture medium enriched with linoleic acid (18:2) or with 25-hydroxycholesterol. The cells took up 18:2 and 25-hydroxycholesterol added to the culture medium, without exhibiting any sign of intolerance or intoxication. These compounds respectively increased and decreased membrane fluidity at 37 degrees C. The cells demonstrated extensive changes in insulin binding parameters in response to experimental modifications of their membrane lipid composition. When determined at 4 degrees C, insulin receptors were present in the control cells at 136,000 sites/cell but this fell to 111,000 (P less than 0.05) in cells enriched in 18:2, and rose to 176,000 (P less than 0.001) in hydroxysterol-grown cells. According to a two-site model, the main effect of 18:2 was a significant increase of the number of high-affinity sites with a concomitant decrease of low-affinity sites. The hydroxysterol had the opposite effects on these parameters. The high-affinity insulin binding capacity of the hepatoma cells was affected by lipid supplementation in a similar way, whether it was determined at 4 degrees C or at 37 degrees C. Assuming a negative cooperativity model, 18:2 enhanced the degree of negative cooperativity among the sites, while 25-hydroxycholesterol reduced it. The time-course of insulin-induced receptor down-regulation was accelerated in the cells enriched in polyunsaturated fatty acids, but reduced in cells exposed to 25-hydroxycholesterol. These insulin-binding alterations cannot be directly related to modifications of cellular growth rate, receptor internalization or membrane fluidity per se, and are discussed as being more likely due to membrane lipid composition than to overall cell metabolism modifications.

Membrane properties of oxysterols. Interfacial orientation, influence on membrane permeability and redistribution between membranes.

The membrane properties of cholesterol auto-oxidation products, 7-ketocholesterol, 7 beta-hydroxycholesterol, 7 alpha-hydroxycholesterol and 25-hydroxycholesterol were examined. Monolayer studies show that these oxysterols are perpendicularly orientated at the interphase. Only 7 beta-hydroxycholesterol and 7 alpha-hydroxycholesterol are tilted at low surface pressures. In mixed monolayers with dioleoylphosphatidylcholine, 7-ketocholesterol, 7 beta-hydroxycholesterol and 7 alpha-hydroxycholesterol show a condensing effect in this order, although to a lesser extent that that observed for cholesterol. In liposomes these oxysterols also reduce glucose permeability and in the same order as their condensing effect. On the other hand 25-hydroxycholesterol shows no condensing effect in monomolecular layers whereas glucose permeability in liposomes is enormously increased. The permeability increase is already maximal at 2.5 mol% 25-hydroxycholesterol. Differential scanning calorimetry experiments reveal that all four oxysterols tested reduce the heat content of the gel----liquid-crystalline phase transition. It is concluded that 7-ketocholesterol, 7 beta-hydroxycholesterol and 7 alpha-hydroxycholesterol have a cholesterol like effect, although less efficient than cholesterol, whereas 25-hydroxycholesterol showing no condensing effect acts as a spacer molecule. Packing defects in the hydrophobic core of the bilayer due to the presence of the C-25 hydroxyl group are believed to cause the permeability increase. The transfer of radiolabelled (oxy)sterols from the monolayer to lipoproteins or vesicles in the subphase was studied. The transfer rate increases in the following order 7-ketocholesterol, 7 beta-hydroxycholesterol, 7 alpha-hydroxycholesterol, 25-hydroxycholesterol. The difference in rate between 7-ketocholesterol and 25-hydroxycholesterol is 20-fold. A higher rate of transfer is observed in the presence of high density lipoproteins and small unilamellar vesicles. A transfer rate for cholesterol is hardly measurable under these conditions. The transfer measured is consistent with the involvement of a water-soluble intermediate.