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PubMed1996

Effects of cyclosporin on cholesterol 27-hydroxylation and LDL receptor activity in HepG2 cells.

The hypothesis that mitochondrial sterol 27-hydroxylase plays a role in the sterol-mediated down-regulation of LDL receptor activity was evaluated in HepG2 cells. 27-Hydroxycholesterol was found to be more potent at suppressing LDL receptor activity than cholesterol (IC50 values of 8 mu M and 142 mu M for 27-hydroxycholesterol and cholesterol, respectively) when the sterols were delivered to cells from 2-hydroxypropyl-beta-cyclodextrin (beta-CD)-solubilized solutions. Cyclosporin, an immunosuppressant which has been shown to inhibit the 27-hydroxylation of sterols, was used to assess whether the formation of endogenous 27-hydroxycholesterol was required for the cholesterol-induced suppression of LDL receptor activity. Cyclosporin dose-dependently inhibited the 27-hydroxylation of cholesterol by HepG2 mitochondria (Ki = 0.25 mu M) and HepG2 cell cultures (IC50 = 1 mu M). At 1 mu M, cyclosporin had no effect on LDL receptor activity, and did not prevent the suppression of LDL receptor activity caused by: 1) the addition of beta-CD-solubilized cholesterol, 2) the receptor-mediated uptake of beta-VLDL, or 3) the inhibition of cholesterol esterification. In contrast, 10 mu M cyclosporin was found to inhibit the esterification of cholesterol and to increase the cellular level of free cholesterol resulting in suppression of LDL receptor activity. These results suggest that if mitochondrial sterol 27-hydroxylase plays a role in the regulation of LDL receptor activity, it is not through the formation of potent regulatory oxysterols, but through its effects on the availability and/or size of the free cholesterol pool regulating LDL receptor activity.

Biological Transport↗
PubMed1976

The use of [7alpha-3H]- and [7alpha, 7beta-3H]cholesterol in the enzymic assay of cholesterol 7alpha-hydroxylase.

A tritium release method is described for following the enzymic conversion of cholesterol to 7alpha-hydroxycholesterol. Incubations of rat liver subcellular preparations (containing microsomes) with [7alpha-3H]cholesterol or [7alpha,7beta-3H]cholesterol release the labeled hydrogen in the 7alpha position as 3H2O which, after counting, allows for the determination of the fraction of exogenous cholesterol converted to 7alpha-hydroxycholesterol. These findings document those recently reported by Van Cantfort, Renson, and Gielen (1975. Eur J. Biochem. 55:23). Analysis of incubation mixtures containing both [4-14C]cholesterol and either [7alpha-3H] or [7alpha,7beta-3H]cholesterol demonstrate that one atom of hydrogen (from the 7alpha position) is incorporated into H2O for every molecule of exogenous cholesterol that is converted to 7alpha-hydroxycholesterol. In the case of [7alpha-3H]cholesterol no label is retained by the product. With [7alpha,7beta-3H]cholesterol, one atom is released as 3H2O and one is retained by the product in the 7beta position. Microsomal incubations with [7alpha,7beta-3H]cholesterol were performed, followed by the acetylation of the steroid fractions with [14C]acetic anhydride. If intermixing of exogenous with endogenous cholesterol were complete during the enzymic reaction, one would expect the 3H: 14C ratio of the isolated cholesterol acetate to be four times that observed in the 7alpha-acetoxycholesterol acetate. Average values of 4.23 in one series and 4.03 in a second series indicate that intermixing was sufficiently complete to use the tritium release method as an indicator of mass conversion.

Animals↗
PubMed1994

Comparison of the effects of cholesterol and oxysterols on phospholipid bilayer microheterogeneity: a study of fluorescence lifetime distributions.

The homogenizing effect of cholesterol and its oxidative derivatives, 7-ketocholesterol, cholesterol 5 alpha, 6 alpha-epoxide and 25-hydroxycholesterol, in liquid-crystalline 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC) bilayer vesicles was studied using the fluorescence lifetimes of 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-p hosphocholine (DPH-PC). The phase and modulation data were fitted either to discrete exponential models or to models characterized by continuous distributional lifetimes. Among all the models tested, it was found that the best one to account for the experimental data was the unimodal Lorentzian distribution. Thus, the DPH-PC lifetime was adequately described by a distributional center and a full width at half-maximum, for DOPC vesicles these values being 6.23 and 0.48 ns, respectively. Increasing the concentration of cholesterol, 7-ketocholesterol, or cholesterol 5 alpha, 6 alpha-epoxide from 0 to 30 mol% resulted in an increase of the lifetime center (e.g., 7.16 ns at 30 mol% cholesterol) and a decrease of the distributional width (e.g., 0.05 ns at 30 mol% cholesterol). On the other hand, up to 30 mol% of 25-hydroxycholesterol incorporated into the bilayer vesicles showed little influence on both lifetime parameters. Our results support the use of lifetime distributional width to evaluate membrane heterogeneity and suggest that oxysterols, depending on their molecular structural particulars, may exert cholesterol-like homogenizing effect in membranes.

Cholesterol↗
PubMed1993

Differing effects of three oxysterols on low density lipoprotein metabolism in HepG2 cells.

25R)-26-Hydroxycholesterol, 25-hydroxycholesterol, and 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one, all extraordinarily potent suppressors of sterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in mammalian cells, have been studied with respect to their effects on the metabolism of low density lipoproteins (LDL) by human hepatocarcinoma (HepG2) cells. The three oxysterols differed markedly in their effects on LDL metabolism, as measured by the combination of cell-associated plus degraded 125I-LDL. The 26-hydroxysterol, at concentrations from 0.1 microM to 75 microM, lowered LDL metabolism. In contrast, the 25-hydroxysterol and the 15-ketosterol, at concentrations from 0.05 microM to 2.5 microM, caused an increase in LDL metabolism. At higher concentrations of these oxysterols, LDL metabolism was suppressed. However, upon increasing the concentration of the 15-ketosterol further to 75 microM, an extraordinary 9-fold increase in LDL metabolism was observed. In contrast to their effects on LDL metabolism, the 25-hydroxysterol and the 15-ketosterol caused simple concentration-dependent decreases in the levels of HMG-CoA reductase activity under the same conditions.

Carcinoma, Hepatocellular↗
PubMed2002

A Link between cholesterol levels and phenobarbital induction of cytochromes P450.

Squalestatin1 (SQ1), a potent inhibitor of squalene synthase produced a dose-dependent induction of cytochromes P450 CYP2H1 and CYP3A37 mRNAs in chicken hepatoma cells. The effect of SQ1 was completely reversed by 25-hydroxycholesterol. Bile acids elicited an induction of CYP3A37 and CYP2H1 mRNA. Bile acids also reduced the phenobarbital induction of CYP2H1 but not of CYP3A37 mRNA. The effects of SQ1 and its reversal by 25-hydroxycholesterol and the effects of bile acids were reproduced in reporter gene assays with a phenobarbital-responsive enhancer unit of CYP2H1. These data suggest that an endogenous molecule related to cholesterol homeostasis regulates induction of drug-inducible CYPs.

Animals↗
PubMed1998

Dietary protein restriction stress in the domestic turkey (Meleagris gallopavo) induces hypofunction and remodeling of adrenal steroidogenic tissue.

In the present study, we investigated the influence of dietary protein restriction stress on adrenal steroidogenic function of the domestic turkey. Immature male turkeys (2 weeks old) were fed isocaloric synthetic diets containing either 28% (control) or 8% (restriction) soy protein for 4 weeks. Trunk plasma was processed for the determination of adrenocorticotropin (ACTH), corticosterone, aldosterone, and total 3, 5, 3'-triiodothyronine (T3). In addition, adrenal glands were processed for the isolation of defined, density-separable, adrenal steroidogenic cell subpopulations: three low-density adrenal steroidogenic cell subpopulations [LDAC-1 (rho = 1.0350-1.0490 g/ml). LDAC-2 (rho = 1.0490-1.0570 g/ml), and LDAC 3 (rho = 1.0370-1.0585 g/ml)] and a high-density subpopulation [HDAC (rho = 1.0590-1.0720 g/ml)], and the steroidogenic function of these cell subpopulations was evaluated. Protein restriction did not influence plasma ACTH However, it increased relative adrenal weight (mg/100 g body wt) (+37.8%) and plasma corticosterone (+317%). By contrast, it depressed plasma aldosterone (-51.2%). In addition, it caused a modest depression in plasma T3 (-25.9%). At the cellular level, protein restriction induced panhypofunction. Basal corticosteroid (aldosterone and corticosterone) production values of LDAC-1, -2, and -3 and HDAC from protein-restricted birds were, respectively, 42.9, 47.9, 30.8, and 57.5% less than those of corresponding cell subpopulations from control birds. In addition, maximal corticosteroid production values of LDAC-1, -2, and -3 and HDAC from protein-restricted birds, in response to ACTH, angiotensin II (AngII), and 25-hydroxycholesterol support, were depressed by 56.8, 55.1, 22.7, and 42.9%, respectively. Interestingly, LDAC-3 was relatively refractory to the influence of this stressor. By contrast, there was the lack of a concentration-dependent aldosterone response of LDAC-1 and -2 to AngII with protein restriction. This was not due to a failure in cell function since aldosterone responses of these cell subpopulations to ACTH and to 25-hydroxycholesterol support were apparent. In addition, the concentration of AngII receptors of cell subpopulations from protein-restricted turkeys, if anything, was greater than that of cell subpopulations from control turkeys. Protein restriction also altered the cell subpopulation composition of the adrenal gland: compared to control, it decreased the proportion of LDAC-2 by 42.3% and increased the proportion of LDAC-3 and HDAC by 68.7 and 302%, respectively. Thus, dietary protein restriction induces adrenal steroidogenic hypofunction in turkeys. In addition, the present study suggests that this nutritional stressor induces marked remodeling of the steroidogenic tissue in the turkey adrenal gland.

Adrenal Glands↗
PubMed1980

Identification of steroids by chemical ionization mass spectrometry.

Chemical ionization (CI) mass spectra of various natural and synthetic steroids have been studied using methane, isobutane, ammonia, trideuterioammonia and hydroxy anion as reagent gases. The CI spectra of steroids give simple and well characterized ions, which provide information about molicular weight as well as functionalities in the molecules. Trideuterioammonia exchanges rapidly with active hydrogens (e.g., OH, SH, COOH, NH2) in steroid molecules in the CI reaction and thus provides a convenient means of active hydrogen determination by mass spectrometry, Application of various CI processes to the analysis of steroids and conjugates have been made. Low levels of hydroxycholesterols in biological samples and in cholesterol autoxidation products were identified by the 4 ion patterns, [M+NH4]+, [M-OH+NH3]+, [M-0H]+ and [M-H2 O-OH]+, in ammonia CI. The position of hydroxy functions in the cholesterol side chain can be identified from the methane CI of hydroxycholesterol trimethylsilyl (TMS) derivatives. Sterol carboxylic esters can be identified as the ammonium adduct ion of the intact molecule, [M+NH4]+, in ammonia CI. Isobutane and hydroxy anion CI spectra of the steroid esters give abundant ion fragments of both steroids and carboxylic acid moieties. Identification of free bile acids and steroid glycosides without derivatization is also feasible with the CI process when ammonia is used as reagent gas.

Bile Acids and Salts↗
PubMed1994

Inhibition of cytolytic T lymphocyte activity by oxysterols.

The objective of this study was to investigate the effects of oxysterols (OS), namely 5 alpha-hydroxy-6-ketocholestanol, 6-ketocholestanol and 25-hydroxycholesterol, on specific cell-mediated cytotoxicity by C57BL/6 spleen cells against P815-X2 (a DBA/2 mastocytoma) target cells. Cytolytic T lymphocytes (CTL) were generated by intraperitoneally injecting C57BL/6 mice with P815-X2 tumor cells 10 d prior to the cytotoxicity experiments. Preincubation of CTL with 10(-5) M 5 alpha-hydroxy-6-ketocholestanol and 6-ketocholestanol for 45 min in lipoprotein-depleted medium resulted in an inhibition of cytolytic activity (73 and 43%, respectively) as measured by 4-h 51Cr release. At a concentration of 5 x 10(-6) M, 5 alpha-hydroxy-6-ketocholestanol inhibited CTL activity by 65%, whereas 6-ketocholestanol did not elicit any inhibition. By contrast, 25-hydroxycholesterol did not inhibit CTL at either concentration, although it is known to be a potent inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway. When CTL were preincubated with OS in lipoprotein-replete medium, there was no inhibition of CTL activity at the respective concentrations. The results suggest that the inhibition of CTL activity upon short-term incubation with OS is not due to the inhibition of cholesterol synthesis, but may be due to the insertion of OS into the plasma membrane to replace cholesterol and alteration of membrane physical properties.

Animals↗
PubMed1993

Parameters influencing cholesterol oxidation.

The purpose of this study was to investigate the effects of temperature, oxidation time, presence of water, pH, type of buffer and form of substrate used on cholesterol oxidation. Microcrystalline cholesterol films, both solid and melted, and aqueous suspensions of film fragments were used as substrates. Use of dispersing agents was avoided. Quantitative analysis of the unaltered substrate and the products of its autoxidation was carried out by gas chromatography over the course of oxidation. Solid cholesterol films were found to be resistant to autoxidation in the dry state. However, when heated at 125 degrees C, a sudden increase in oxidation rate occurred at a point coinciding with the visible melting followed by a plateau of the oxidation rate. All of the autoxidation products formed underwent further decomposition. Film fragments of cholesterol oxidized at a faster rate in aqueous suspensions than when oxidized in the dry state. In aqueous suspensions, the differences in the resistance of cholesterol to oxidation were not significant within the pH range 6.0-7.4, except for the early stages of oxidation. The 7-ketocholesterol/7-hydroxycholesterol ratio dropped significantly with increasing pH. However, at all pH levels tested, this ratio remained relatively constant during the 6 h of heating. While the 7 beta-hydroxycholesterol/7 alpha-hydroxycholesterol ratio was not affected by pH in the range of 6.0-7.4, at pH 7.4 a high preference was observed for the cholesterol beta-epoxide over its alpha-isomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol↗
PubMed1990

Oxysterol mediated changes in fatty acid distribution and lipid synthesis in cultured bovine aortic smooth muscle cells.

We studied the actions of oxysterols on fatty acid distribution and lipid synthesis in cultured bovine aortic smooth muscle cells. Cultures were labeled with [1-14C]arachidonate or [1-14C]oleate. During a 24-hr incubation, 25- or 22R-hydroxycholesterol enhanced the incorporation of label into triglycerides, concomitant with a reduction in the labeling of phospholipids. Cholestantriol or 20-hydroxycholesterol had the opposite effects. They caused a higher incorporation of radiolabel into phospholipids and a reduction of labeling of triacylglycerols. Similar changes were seen in cells labeled with [1-14C]acetate. Therefore, we conclude that oxysterols can promote changes in the distribution of fatty acids between neutral lipids and phospholipids through mechanisms that still need to be clarified.

Acetates↗
PubMed2003

Programming of initial steps in bile acid synthesis by breast-feeding vs. formula-feeding in the baboon.

We tested the hypothesis that breast- vs. formula-feeding differentially affects the enzymatic activity of three sterol hydroxylases critical in the initial steps of bile acid formation. Thirty baboons were either breast-fed or formula-fed for the first 14 wk of life before weaning to baboon chow. At 14 and 34 wk of age, liver biopsies were assayed for cholesterol 7alpha-hydroxylase (CYP7A1), 27-hydroxycholesterol-7alpha-hydroxylase (CYP7B1), and cholesterol 27-hydroxylase (CYP27A1). We also determined the kinetics of 3H-27-hydroxycholesterol (27-OHC) turnover in vivo at both ages. At 14 wk of age, hepatic CYP7A1 activity was low but sevenfold higher among formula-fed vs. breast-fed baboons. By 34 wk, CYP7A1 activity had increased nearly 10-fold in both infant diet groups, and the sevenfold difference in CYP7A1 between previously breast- and formula-fed animals persisted. There were no differences in CYP7B1 activities between infant diet groups at either 14 or 34 wk of age although the activity increased in both groups by about 50% from 14 to 34 wk. CYP27A1 activity also increased between 14 and 34 wk of age, and, compared with CYP7A1, relatively small differences in CYP27A1 activity due to infant diet were observed at each age. Plasma 27-OHC turnover had a half-time of 2-4 min. We had previously reported that after weaning, the total bile acid synthesis rate was higher among baboons that were formula-fed than among breast-fed animals. The present results suggest that this difference is most likely due to significantly higher CYP7A1 activity among formula-fed vs. breast-fed animals.

Animals↗
PubMed1997

Absorption of dietary cholesterol oxidation products and incorporation into rat lymph chylomicrons.

Cholesterol oxidation products (oxysterols) induce macrophage lipid loading and accumulate in early arterial fatty streaks. The origin of lesion oxysterols has not been elucidated. The absorption of oxysterols from the diet and transport to the arterial wall by postprandial lipoprotein remnants may be a significant source. This study aimed to investigate the extent of oxysterol absorption and the effect on chylomicron composition. Cholesterol was heat-treated, causing 30% oxidation; the major oxidation products were 7 beta-hydroxycholesterol, 7-keto-cholesterol, 5 alpha,6 alpha-epoxycholesterol, and 5 beta,6 beta-epoxycholesterol. Conscious lymph-cannulated rats were given a bolus gastric infusion of 50 mg oxidized cholesterol or 50 mg purified cholesterol in a vehicle of triglyceride. In the rats given the oxidized cholesterol, 6% of the oxysterol load was absorbed and incorporated into lymph chylomicrons. Rats given pure cholesterol had no increase in oxysterols above baseline levels. The incorporation of oxysterols into lymph chylomicrons differed over time with 7 beta-hydroxycholesterol, having peak absorption at 3 h, followed by 7-ketocholesterol at 4 h and 5 alpha,6 alpha-epoxy-cholesterol at 5 h. The absorption of oxysterols in animals given the oxidized cholesterol gastric infusate was associated with lymph chylomicron compositional changes at 2-4 h. The oxidized cholesterol-treated group had a twofold increase in the cholesterol (890 +/- 84 micrograms vs. 440 +/- 83 microgram at 3 h) and triglyceride content (19.76 +/- 3.4 micrograms vs. 8.49 +/- 3.8 micrograms at 3 h). This led to a doubling of chylomicron size over this postprandial period, with particles having a mean diameter of 294 nm in the oxidized cholesterol-treated animals, compared to 179 nm in the purified cholesterol group. In conclusion, dietary oxysterols appear to influence postprandial lipoprotein particle size and composition. These changes may have effects on the clearance of chylomicrons from plasma, arterial delivery of oxysterols, and possible deposition in arterial lesions.

Animals↗
PubMed1986

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in human fibroblasts by reversible phosphorylation: modulation of enzymatic activity by low density lipoprotein, sterols, and mevalonolactone.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase exists in interconvertible active and inactive forms in cultured fibroblasts from normal and familial hypercholesterolemic subjects. The inactive form can be activated by endogenous or added phosphoprotein phosphatase. Active or partially active HMG-CoA reductase in cell extracts was inactivated by a ATP-Mg-dependent reductase kinase. Incubation of phosphorylated (inactive) HMG-CoA reductase with purified phosphoprotein phosphatase was associated with dephosphorylation (reactivation) and complete restoration of HMG-CoA reductase activity. Low density lipoprotein, 25-hydroxycholesterol, 7-ketocholesterol, and mevalonolactone suppressed HMG-CoA reductase activity by a short-term mechanism involving reversible phosphorylation. 25-Hydroxycholesterol, which enters cells without the requirement of low density lipoprotein-receptor binding, inhibited the HMG-CoA reductase activity in familial hypercholesterolemic cells by reversible phosphorylation. Measurement of the short-term effects of inhibitors on the rate of cholesterol synthesis from radiolabeled acetate revealed that HMG-CoA reductase phosphorylation was responsible for rapid suppression of sterol synthesis. Reductase kinase activity of cultured fibroblasts was also affected by reversible phosphorylation. The active (phosphorylated) reductase kinase can be inactivated by dephosphorylation with phosphatase. Inactive reductase kinase can be reactivated by phosphorylation with ATP-Mg and a second protein kinase from rat liver, designated reductase kinase kinase. Reductase kinase kinase activity has been shown to be present in the extracts of cultured fibroblasts. The combined results represent the initial demonstration of a short-term regulation of HMG-CoA reductase activity and cholesterol synthesis in normal and receptor-negative cultured fibroblasts involving reversible phosphorylation of both HMG-CoA reductase and reductase kinase.

AMP-Activated Protein Kinases↗
PubMed1980

Resistance to streptolysin O in mammalian cells treated with oxygenated derivatives of cholesterol. Cholesterol content of resistant cells and recovery of streptolysin O sensitivity.

Cultures of L cells and HeLa cells were made resistant to the cytolytic toxin, streptolysin O, by incubating them in the presence of 20 alpha-hydroxycholesterol or 25-hydroxycholesterol. Such cells were also found to be more resistant to the cytotoxic effects of saponin and digitonin, agents known to interact with membrane cholesterol. Sterol synthesis in L cells that had been treated with either of the oxygenated derivatives of cholesterol was reduced by almost 90%, and the free cholesterol content of streptolysin O-resistant HeLa and L cells fell to approx. 50% of control cell levels. Significant recovery of sensitivity to streptolysin O occurred in about 6 h when refractory L cells were incubated in serum or cholesterol. Partial recovery was observed when the cultures were incubated for 24 h in mevalonate or lipid-depleted serum. The results provide further support for the role of membrane cholesterol in the cytotoxic action of streptolysin O on mammalian cells.

Bacterial Proteins↗
PubMed1992

Increased cholesterol synthesis in Chinese hamster ovary cells deficient in peroxisomes.

In a previous study we have shown that Chinese hamster ovary (CHO) cells deficient in intact peroxisomes, lack the nonspecific lipid transfer protein (nsL-TP; sterol carrier protein 2) (van Heusden, G.P.H., Bos, K., Raetz, C.R.H. and Wirtz, K.W.A. (1990) J. Biol. Chem. 265, 4105-4110). The consequences of the absence of peroxisomes and of nsL-TP on intracellular cholesterol metabolism have been investigated in two peroxisome-deficient CHO cell lines (CHO-82 and CHO-78). Compared with wild-type cells (CHO-K1), the incorporation of [3H]acetate into cholesterol was 3-fold higher in the CHO-82 cells and 2-fold higher in the CHO-78 cells. In agreement with an increased synthesis of cholesterol, a 2-3-fold higher 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was measured in both mutant cell lines. On the other hand, addition of low density lipoprotein (LDL), mevalonate (30 mM) or 25-hydroxycholesterol (2 micrograms/ml) to cells grown in lipoprotein-deficient serum, demonstrated that in both mutant cell lines the down-regulation of HMG-CoA reductase and of cholesterol synthesis were comparable to that in wild-type cells. These results strongly suggest that, in addition to down-regulation by LDL-derived cholesterol, mevalonate and 25-hydroxycholesterol, HMG-CoA reductase activity is under control of peroxisomes and/or nsL-TP.

Acetates↗
PubMed1994

The effect of dietary 7-ketocholesterol, inhibitor of sterol synthesis, on hepatic microsomal cholesterol 7 alpha-hydroxylase activity in rat.

A group of oxygenated sterols has been identified as physiological regulators of hepatic cholesterol biosynthesis. However, the regulatory effects of these oxysterols on cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis, is not clearly elucidated. We administered 0.1% 7-ketocholesterol (15 mg/day), a strong inhibitor of sterol synthesis, to rats orally for 6 days. Then, the levels of accumulated oxysterols in liver microsomes and microsomal 7 alpha-hydroxylase activity were determined. The results were compared to those in the groups of rats treated with either control diet or diets containing 0.1 or 1% cholesterol, 0.1% butylated hydroxytoluene, 3% cholestyramine or 1% taurocholate. 7-Ketocholesterol feeding resulted in significant increase of both 7-ketocholesterol and 7 beta-hydroxycholesterol in microsomal fraction (449.4 +/- 36.8 and 438.2 +/- 46.8 ng/mg protein, respectively; mean +/- S.E.). Hepatic microsomal 7 alpha-hydroxylase activity in the rats fed 7-ketocholesterol was significantly elevated as compared with those of control rats; 44.70 +/- 5.97 vs. 16.57 +/- 2.46 pmol/min per mg protein. Addition of BHT to 7-ketocholesterol reduced the accumulation of 7 beta-hydroxycholesterol, and the stimulatory effect of 7-ketocholesterol on 7 alpha-hydroxylase activity was suppressed. Our results demonstrate that oxysterols do not inhibit but rather stimulate hepatic microsomal 7 alpha-hydroxylase.

Animals↗
PubMed1984

Antagonist action of cholesterol towards the toxicity of hydroxysterols on cultured hepatoma cells.

The cytostatic and cytolytic action of 22R - hydroxydesmosterol on hepatoma cells cultured in a medium containing 10% newborn-calf serum can be reversed within certain concentration limits by adding cholesterol to the culture medium. In contrast, under the same conditions, the cytotoxicity of 7 beta -hydroxycholesterol could not be reversed, whatever the concentrations of cholesterol added. However, in a lipoprotein-poor and in a chemically defined medium, the cytolytic action of both hydroxysterols can be reversed by adding cholesterol, but growth inhibition cannot be suppressed. This demonstrates the importance of serum lipids and lipoproteins for the toxicity of the hydroxysterols and for the antagonistic effect of cholesterol. Our results suggest that the action mechanisms of 7 beta-hydroxycholesterol and 22R - hydroxydesmosterol on HTC hepatoma cells are not fully identical.

Animals↗
PubMed1992

Inhibition of NK cell-mediated cytotoxicity by oxysterols.

Some of the oxidation products of cholesterol (oxysterols) have profound effects on plasma membrane structure and function. The present studies were undertaken to determine the effects of oxysterols on NK cell-mediated cytotoxicity. When mouse spleen cells were preincubated with certain oxysterols, NK cell cytotoxicity was inhibited without loss of effector cell viability. The strongest inhibition was observed with oxysterols that are oxidized at the C-5, C-6, or C-7 positions of the sterol nucleus. Among these, 7 beta-hydroxycholesterol caused more inhibition than 7 alpha-hydroxycholesterol suggesting that the spatial orientation of the hydroxyl group in the beta-position results in a greater perturbation in plasma membrane structure than that oriented in the alpha-position. In contrast, oxysterols that are oxidized at the C-20 and C-25 positions that are located on the C-17 acyl chain had little or no inhibitory effect, suggesting that oxidation in the cholesterol nucleus which is situated closer to the phospholipid headgroups at the lipid bilayer-aqueous interface results in a more profound effect on the plasma membrane physical structure. These results suggest that the lytic function of NK cell is sensitive to alterations in the physical state of its plasma membrane induced by oxysterols.

Animals↗