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PubMed1990

Differential regulation of hepatic triglyceride lipase and 3-hydroxy-3-methylglutaryl-CoA reductase gene expression in a human hepatoma cell line, HepG2.

The level of hepatic triglyceride lipase (H-TGL) synthesis and secretion was examined in response to changes in cholesterol biosynthesis in the human hepatoma cell line HepG2. Cells were first fed a lipoprotein-deficient serum-supplemented medium to eliminate exogenous cholesterol. Mevinolin, a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, was then added at a concentration (37 microM) which inhibited cholesterol biosynthesis by greater than 85% and decreased total cell cholesterol from 36.1 to 27.4 micrograms/ml of cell protein. Mevinolin treatment caused a 4.9 +/- 0.8-fold increase in the amount of H-TGL activity secreted into the medium, a 1.8 +/- 0.4-fold rise in H-TGL-specific mRNA, and a concurrent 14-fold increase in HMG-CoA reductase mRNA. Addition of 1 mM mevalonic acid to normal or mevinolin-treated cells raised the cellular cholesterol content and decreased the amount of secreted H-TGL activity to levels below control values. Mevalonic acid also prevented mevinolin-induction of H-TGL and HMG-CoA reductase mRNA, suggesting a common regulatory step for H-TGL and HMG-CoA reductase. Exposure of cells to mevinolin and 25-hydroxycholesterol together resulted in a marked repression of HMG-CoA reductase mRNA levels, whereas these conditions further enhanced the secretion of H-TGL activity and the expression of H-TGL mRNA. These results demonstrate a differential role for 25-hydroxycholesterol in the regulation of H-TGL and HMG-CoA reductase expression.

Carcinoma, Hepatocellular↗
PubMed1990

Substrate stimulation of 7 alpha-hydroxylase, an enzyme located in the cholesterol-poor endoplasmic reticulum.

We examined the role of cholesterol in altering the activity of the microsomal cytochrome P-450 enzyme, cholesterol-NADPH:oxygen oxidoreductase (cholesterol 7 alpha-hydroxylase). Liposomes were used to deliver cholesterol to hepatic microsomes. Formation of 7 alpha-hydroxycholesterol was quantitated by isotope dilution/gas chromatography-mass spectrometry. As the liposomal cholesterol/phospholipid molar ratio increased, 7 alpha-hydroxylase activity increased, whereas the activity of another microsomal cytochrome P-450 enzyme, ethylmorphine N-demethylase, decreased. To determine if the degree of stimulation was affected by the endogenous activity (without liposomes), microsomes, from rats fed chow alone or chow containing cholestyramine, taurocholate, or cholesterol were challenged with cholesterol-enriched liposomes. The degree of stimulation was dependent upon the endogenous activity: cholestyramine-fed much greater than cholesterol = chow control greater than taurocholate-fed. To determine if cholesterol stimulates 7 alpha-hydroxylase by increasing membrane viscosity, microsomes were incubated with liposomes having the same cholesterol/phospholipid molar ratio as microsomes, but different viscosities. Dipalmitoylphosphatidylcholine (high viscosity) liposomes increased microsomal viscosity and decreased 7 alpha-hydroxylase activity. In contrast, dioleoylphosphatidylcholine (low viscosity) liposomes decreased microsomal viscosity and increased enzyme activity. Since greater viscosity inhibits 7 alpha-hydroxylase, cholesterol cannot stimulate the enzyme by increasing membrane viscosity. The data suggest that cholesterol stimulates production of 7 alpha-hydroxycholesterol by providing substrate.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1987

Effects of cholesterol sulfate on lipid metabolism in cultured human keratinocytes and fibroblasts.

Effects of cholesterol sulfate on acetate incorporation into lipid fractions were examined in normal human fibroblast and keratinocyte cultures. Inhibition of sterologenesis in normal fibroblast cultures by cholesterol sulfate was less profound than that produced by either lipoprotein-containing serum or 25-hydroxycholesterol. Cholesterol sulfate also inhibited sterologenesis in low density lipoprotein receptor-deficient fibroblasts and inhibited both sterologenesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in keratinocytes. Cholesterol sulfate increased incorporation of acetate into fatty acid-containing lipids in preconfluent cultures of both cell types in lipoprotein-depleted media. Similar effects were not observed either in response to lipoprotein-containing serum or 25-hydroxycholesterol. Cholesterol sulfate had no effect on oleic acid incorporation into diglycerides, triglycerides, or phospholipid fractions; neither did it inhibit acid lipase activity; nor did it inhibit fatty acid oxidation, indicating that cholesterol sulfate does not inhibit catabolism of acyl lipids. Because cholesterol sulfate had similar effects on fatty acid metabolism in steroid sulfatase-deficient fibroblasts lines, desulfation to cholesterol is not a prerequisite. Cholesterol sulfate did not significantly affect incorporation of oleic acid into sterol esters in fibroblast cultures, but in contrast, inhibited sterol esterification in keratinocyte cultures. These data suggest a novel role for cholesterol sulfate as a modulator of cellular lipid biosynthesis.

Acetates↗
PubMed1988

Low density lipoprotein receptor degradation is influenced by a mediator protein(s) with a rapid turnover rate, but is unaffected by receptor up- or down-regulation.

Treatment of cultured human skin fibroblasts with cycloheximide retarded the down-regulation of low density lipoprotein (LDL) receptor activity caused by 25-hydroxycholesterol. The rate of LDL receptor degradation, measured directly by means of [35S]methionine pulse-chase experiments, was also markedly inhibited by cycloheximide (or puromycin), suggesting that continuous synthesis of a short-lived mediator protein(s) was necessary for normal LDL receptor turnover. In the absence of cycloheximide, both the up- and down-regulation of LDL receptor activity took place with a half-time of approximately 12 hr. Pulse-chase measurements with [35S]methionine yielded a receptor half-life (t1/2) of 11.7 +/- 2.2 hr (n = 10) in up-regulated cells; the t1/2 in the partially down-regulated state was similar. The presence of LDL or 25-hydroxycholesterol did not alter this degradation rate. Regulation of LDL receptor activity under these various culture conditions therefore probably occurred solely as a result of changes in the rate of receptor synthesis. The cycloheximide-sensitive factor(s) that influences receptor turnover apparently did not play a regulatory role in the up- or down-regulation of the LDL receptor.

Antibodies↗
PubMed1988

Isolation and characterization of Chinese hamster ovary cell mutants deficient in acyl-coenzyme A:cholesterol acyltransferase activity.

A protocol has been developed for isolating cholesterol ester-deficient cells from the Chinese hamster ovary cell clone 25-RA. This cell line previously was shown to be partially resistant to suppression of cholesterogenic enzyme activities by 25-hydroxycholesterol and to accumulate a large amount of intracellular cholesterol ester when grown in medium containing 10% fetal calf serum (Chang, T. Y., and Limanek, J. S. (1980) J. Biol. Chem. 255, 7787-7795). The higher cholesterol ester content of 25-RA is due to an increase in the rate of cholesterol biosynthesis and low density lipoprotein receptor activity compared to wild-type Chinese hamster ovary cells, and not due to an abnormal acyl-CoA:cholesterol acyltransferase enzyme. The procedure to isolate cholesterol ester-deficient mutants utilizes amphotericin B, a polyene antibiotic known to bind to cholesterol and to form pore complexes in membranes. After incubation in cholesterol-free medium plus an inhibitor of endogenous cholesterol biosynthesis, 25-RA cells were found to be 50-500 times more sensitive to amphotericin B killing than were mutant cells containing reduced amounts of cholesterol ester. Twelve amphotericin B-resistant mutants were isolated which retained the 25-hydroxycholesterol-resistant phenotype. These mutants did not exhibit the perinuclear lipid droplets characteristic of 25-RA cells, and lipid analysis revealed a large (up to 40-fold) reduction in cellular cholesterol ester. The acyl-CoA:cholesterol acyltransferase activities of these cholesterol ester-deficient mutants were markedly lower than 25-RA when assayed in intact cells or in an in vitro reconstitution assay. The tightest mutant characterized, AC29, was found to have less than 1% of the parental acyl-CoA:cholesterol acyltransferase activity. These mutants all have reduced rates of sterol synthesis and lower low density lipoprotein receptor activity compared to 25-RA, probably as a consequence of their reduced enzyme activities. Cell fusion experiments revealed that the phenotypes of all the mutants examined are not dominant and that the mutants all belong to the same complementation group. We conclude that these mutants contain a lesion in the gene encoding acyl-CoA:cholesterol acyltransferase or in a gene encoding a factor needed for enzyme production.

Amphotericin B↗
PubMed1986

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol biosynthesis by oxylanosterols.

Treatment of rat intestinal epithelial cell cultures with the oxidosqualene cyclase inhibitor, 3 beta-[2-(diethylamino)-ethoxy]androst-5-en-17-one (U18666A), resulted in an accumulation of squalene 2,3:22,23-dioxide (SDO). When U18666A was withdrawn and the cells were treated with the sterol 14 alpha-demethylase inhibitor, ketoconazole, SDO was metabolized to a product identified as 24(S),25-epoxylanosterol. To test the biological effects and cellular metabolism of this compound, we prepared 24(RS),25-epoxylanosterol by chemical synthesis. The epimeric mixture of 24,25-epoxylanosterols could be resolved by high performance liquid chromatography on a wide-pore, non-endcapped, reverse phase column. Both epimers were effective suppressors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity of IEC-6 cells. The suppressive action of the natural epimer, 24(S),25-epoxylanosterol, but not that of 24(R),25-epoxylanosterol could be completely prevented by ketoconazole. IEC-6 cells could efficiently metabolize biosynthetic 24(S),25-epoxy[3H]anosterol mainly to the known reductase-suppressor 24(S),25-epoxycholesterol. This metabolism was substantially reduced by ketoconazole. These data support the conclusion that 24(S),25-epoxylanosterol per se is not a suppressor of HMG-CoA reductase activity but is a precursor to a regulatory oxysterol(s). It has recently been reported that 25-hydroxycholesterol can occur naturally in cultured cells in amounts sufficient to effect regulation of HMG-CoA reductase (Saucier et al. 1985. J. Biol. Chem. 260: 14571-14579). In order to investigate the biological effects of possible precursors of 25-hydroxycholesterol, we chemically synthesized 25-hydroxylanosterol and 25-hydroxylanostene-3-one. Both oxylanosterol derivatives suppressed cellular sterol synthesis at the level of HMG-CoA reductase. U18666A had the unusual effect of potentiating the inhibitory effect of 25-hydroxylanostene-3-one but did not influence the effect of other oxylanosterols. All the oxylanosterols, with the exception of 25-hydroxylanostene-3-one, enhanced intracellular esterification of cholesterol. The foregoing observations support consideration of oxylanosterols as playing an important role in the biological formation of regulatory oxysterols that modulate sterol biosynthesis at the level of HMG-CoA reductase.

Androstenes↗
PubMed1987

Regulation of sterol biosynthesis and of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in cultured cells by progesterone.

Treatment of rat intestinal epithelial cells in culture (IEC-6) with progesterone (10 micrograms/ml) caused a strong inhibition of cholesterol biosynthesis as indicated by a decreased incorporation of radiolabel from [3H]acetate. This inhibition was accompanied by an accumulation of radioactivity in an intermediate which coeluted with authentic desmosterol upon high performance liquid chromatography (HPLC). In addition, treatment of cells with progesterone caused lesser accumulation of radiolabel in products with retention times (RT) of 7.9 and 13.5 min on reverse-phase HPLC. The RT-13.5 compound was tentatively identified as cholesta-5,7,24-trien-3 beta-ol based on its relative retention and on its conversion to cholesterol upon incubation with untreated cells. The RT-7.9 compound was identified as 24 (S),25-epoxycholesterol (S-EC) based on its coelution with authentic S-EC and by its conversion to 25-hydroxycholesterol upon reduction with LiAlH4. Incubation of IEC-6 cells with chemically prepared S-EC resulted in dose-dependent suppression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity within 6 h (I50 = 0.3 microM). Pretreatment of cells with progesterone prevented this suppressive effect. No suppression of reductase activity was observed in progesterone-treated cells in spite of obvious accumulation of S-EC in amounts sufficient to effect regulation; instead, a 2-3-fold increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity occurred within a 24-h period. Following the removal of progesterone from the culture medium, reductase activity declined rapidly over the next 6 h. However, IEC-6 cells could not metabolize S-EC, derived either endogenously or exogenously, during a similar time frame; nor did progesterone affect the uptake of exogenous S-EC by IEC-6 cells. These results show that although progesterone treatment of cultured cells promotes the synthesis of a natural oxysterol suppressor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, the continued presence of progesterone prevents the regulatory action of S-EC. The unique nature of this interference is high-lighted by the observation that progesterone could not prevent the suppression of reductase activity by either 25-hydroxycholesterol or mevalonolactone.

Animals↗
PubMed1985

Biosynthesis of cholestanol from bile acid intermediates in the rabbit and the rat.

Biliary 7 alpha-hydroxy-4-cholesten-3-one (an intermediate in bile acid biosynthesis) may be 7 alpha-dehydroxylated in the gut and further metabolized to cholestanol (Skrede, S., and Björkhem, I. (1982) J. Biol. Chem. 257, 8363-8367). We have now evaluated the quantitative importance of pathway(s) to cholestanol with 7 alpha-hydroxylated C27 steroids as intermediates. After feeding conventionally fed rabbits or rats or germ-free rats with [7 alpha-3H]cholesterol and [4-14C]cholesterol, tissue cholestanol could be isolated with about a 20% lower 3H/14C ratio than present in cholesterol. We conclude that there is a pathway to cholestanol involving 7 alpha-hydroxylated intermediates. Intestinal microorganisms are not essential for this pathway, which accounts for at most 20% of the cholestanol formed in these species. In bile fistula rats, there was also a significant conversion of intraperitoneally injected [7 beta-3H]7 alpha-hydroxycholesterol and [4-14C]7 alpha-hydroxy-4-cholesten-3-one into cholestanol. The enzymes involved in the 7 alpha-hydroxylation/dehydroxylation pathway for the biosynthesis of cholestanol are probably located in the liver. Both 7 alpha-hydroxycholesterol and 7 alpha-hydroxy-4-cholesten-3-one may be intermediates.

Animals↗
PubMed1985

Regulation of low density lipoprotein receptor synthesis in cultured luteinized human granulosa cells by human chorionic gonadotropin and 8-bromo-cyclic AMP.

We investigated the regulation of synthesis of low density lipoprotein (LDL) receptor in cultured luteinized human granulosa cells using a monoclonal antibody recognizing the human LDL receptor (IgG-C7). Cells cultured under serum-free conditions were treated with human chorionic gonadotropin (hCG) or 8-bromo-cAMP alone or in combination with aminoglutethimide (to block conversion of cholesterol to steroid hormones) and 5-cholesten-3 beta, 25-diol (25-hydroxycholesterol, a potent suppressor of LDL receptor expression in human fibroblasts) and pulse-labeled with [35S]methionine. A labeled protein immunoisolated with IgG-C7 was identified as the mature LDL receptor in 7.5% sodium dodecyl sulfate-polyacrylamide gels on the basis of an apparent molecular mass of 160 kDa, absence of the protein from immunoisolates prepared with a monoclonal antibody against an irrelevant antigen, and an apparent decrease in molecular weight of the mature receptor upon treatment with neuraminidase or electrophoresis under nonreducing conditions. hCG and 8-bromo-cAMP consistently increased the incorporation of radioactivity into the mature LDL receptor by 2-6-fold. The effect of hCG on LDL receptor synthesis was observed with as little as 10 mIU of hCG/ml and was apparent within 2 h of addition of the hormone. A combination of 25-hydroxycholesterol and aminoglutethimide resulted in a 60% suppression of label incorporation into mature LDL receptor compared to untreated cells. This would suggest some regulation of LDL receptor synthesis by negative feedback of sterol. However, both hCG and 8-bromo-cAMP increased label incorporation into the LDL receptor in the face of these agents. We conclude that in human granulosa cells, hCG, through the intermediacy of cAMP, rapidly increases LDL receptor synthesis by a mechanism which is, at least in part, independent of alterations in cellular cholesterol balance.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1985

Effects of oxygenated derivatives of cholesterol on cholesterol uptake by cultured aortic smooth muscle cells.

The cytotoxicity of oxygenated derivatives of cholesterol on the cultured cells may not only be due to their potent inhibition of cholesterol biosynthesis, but also could be related to their capability for inhibiting cholesterol uptake from exogenous sources, particularly in arterial cells, which synthesize cholesterol at a very slow rate. In cultured aortic smooth muscle cells, the most potent inhibitor of cholesterol uptake was cholestane-3 beta,5 alpha,6 beta triol, which at 100 micrograms/ml medium reduced uptake of cholesterol to 10% of the control. The next most potent inhibitors were 5 alpha,6 alpha- epoxycholesterol and 25-hydroxycholesterol, which reduced uptake of cholesterol to 60%. 7 alpha- and 7 beta-hydroxycholesterol and 7-ketocholesterol inhibited cholesterol uptake to 30-50%. A consequence of their inhibitory effects on cholesterol uptake could be a depletion of cell membrane cholesterol, resulting in alterations in membrane structure and function and eventually in cell death.

Animals↗
PubMed1983

Inhibition of adrenocortical cytochrome P-450scc by (20R)-20-phenyl-5-pregnene-3 beta,20-diol.

The effects of the cholesterol analogue, (20R)-20-phenyl-5-pregnene-3 beta,20-diol (20-PPD), on the catalytic and spectral properties of purified bovine adrenocortical cytochrome P-450scc were investigated. In contrast to results with cholesterol and (20R)-20-hydroxycholesterol, no conversion of 20-PPD to pregnenolone could be detected; instead, 20-PPD was found to be a potent inhibitor of cytochrome P-450scc. Kinetic analyses showed that the inhibition is reversible and competitive with respect to cholesterol with an apparent Ki = 30nM. Spectral binding studies with ferricytochrome P-450scc showed that 20-PPD formed a 1:1 complex with the enzyme, having an absorption spectrum similar to that produced by (20R)-20-hydroxycholesterol. These results indicate that 20-PPD binds with very high affinity to the substrate site on cytochrome P-450scc. The finding that the phenyl side chain is readily accommodated suggests the presence in this site of an open pocket which may be normally occupied by C-22 to C-27 of the cholesterol side chain.

Adrenal Cortex↗
PubMed1983

beta-sitosterol: esterification by intestinal acylcoenzyme A: cholesterol acyltransferase (ACAT) and its effect on cholesterol esterification.

Rabbits were fed either 10% coconut oil, 10% coconut oil and 1% beta-sitosterol, 10% coconut oil and 1% cholesterol, or 10% coconut oil and 1% beta-sitosterol plus 1% cholesterol for 4 weeks. Microsomal membranes from intestines of animals fed the 1% beta-sitosterol diet had 48% less cholesterol and were enriched twofold in beta-sitosterol compared to membranes from animals fed the coconut oil diet alone. Acylcoenzyme A:cholesterol acyltransferase (ACAT) activity in jejunum and ileum was decreased significantly in animals fed the plant sterol alone. In membranes from animals fed 1% beta-sitosterol and 1% cholesterol, beta-sitosterol content increased 50% whereas cholesterol was modestly decreased compared to their controls fed only cholesterol. Intestinal ACAT was unchanged in the animals fed both sterols when compared to their controls. beta-Sitosterol esterification was determined by incubating intestinal microsomal membranes with either [(14)C]beta-sitosterol-albumin emulsion or [(14)C]beta-sitosterol:dipalmitoyl phosphatidylcholine (DPPC) liposomes to radiolabel the endogenous sterol pool. Oleoyl-CoA was then added. The CoA-dependent esterification rate of beta-sitosterol was very slow compared to that of cholesterol using both techniques. An increased amount of endogenous microsomal beta-sitosterol, which occurs in animals fed 1% beta-sitosterol, did not interfere with the stimulation of ACAT activity secondary to cholesterol enrichment of the membranes. Enriching microsomal membranes three- to five-fold with beta-sitosterol did not affect ACAT activity. Freshly isolated intestinal cells were incubated for 1 hour with [(3)H]oleic acid and beta-sitosterol:DPPC or 25-hydroxycholesterol:DPPC. Incorporation of oleic acid into cholesteryl esters did not change in the presence of beta-sitosterol but increased fourfold after the addition of 25-hydroxycholesterol. We conclude that the CoA-dependent esterification rate of cholesterol is at least 60 times greater than that of beta-sitosterol. Membrane beta-sitosterol does not interfere with nor compete with cholesterol esterification. Inadequate esterification of this plant sterol may play a role in the poor absorption of beta-sitosterol by the gut.-Field, F. J., and S. N. Mathur. beta-Sitosterol: esterification by intestinal acylcoenzyme A:cholesterol acyltransferase (ACAT) and its effect on cholesterol esterification.

Acyl Coenzyme A↗
PubMed1983

Amplification of the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not for the 53-kDa protein, in UT-1 cells.

32P-labeled cDNA probes were used to study levels of genomic DNA and regulation of mRNA for 3-hydroxy-3-methylglutaryl coenzyme A reductase in UT-1 cells, a clone of compactin-resistant Chinese hamster ovary cells that have a 100-1000-fold increase in the amount of reductase protein. Similar measurements were made for the 53-kDa protein, a cytosolic protein of unknown function that is also expressed at high levels in UT-1 cells. The number of copies of the gene for reductase was increased by 15-fold in UT-1 cells as compared to the parental Chinese hamster ovary cells, as judged from Southern gel analysis of restriction endonuclease-digested genomic DNA. In contrast, there was no detectable increase in the number of gene copies for the 53-kDa protein. The amount of cytoplasmic mRNA for both proteins was markedly elevated in UT-1 cells, as determined by filter hybridization studies using 32P-labeled cDNA probes. The amount of mRNA for both reductase and the 53-kDa protein declined in parallel after addition of low density lipoprotein, 25-hydroxycholesterol, or mevalonate to the culture medium. The decline in reductase mRNA was associated with a marked decrease in the rate of [3H]uridine incorporation into hybridizable cytoplasmic mRNA. When UT-1 cells were grown for 3-4 months in the absence of compactin, the level of reductase mRNA and enzymatic activity decreased markedly, but the number of copies of the reductase gene did not decline. When the compactin-withdrawn cells were rechallenged with compactin, high levels of reductase mRNA and enzymatic activity promptly returned. We conclude that the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not for the 53-kDa protein, has been stably amplified in UT-1 cells. Despite this differential gene amplification, the levels of cytoplasmic mRNA for both gene products are markedly elevated, and both are reduced in parallel by either sterols (low density lipoprotein-cholesterol or 25-hydroxycholesterol) or mevalonate, the product of the reductase-catalyzed reaction.

Animals↗
PubMedDate not supplied

[Content of squalene and sterols in tissues of the hemopoietic system in normal rats and in experimental leukemia].

Contents of scvalene, sterols and their derivatives were studied in lymphoid tissues as well as in liver and kidney tissues of normal rats and the animals with Shvetz experimental leukosis. Amount of cholesterol and of its precursor scvalene was distinctly decreased simultaneously with an increase in total content of nonsaponified substances in thymus, spleen, bone marrow and liver tissue of rats with experimental leukosis. Scvalene was increased in kidney tissue. Simultaneous accumulation of hydroxycholesterol derivatives, among which 7-beta-hydroxycholesterol was identified, was observed in these tissues under conditions of leukosis. Role of cholesterol hydroxyderivatives in biosynthesis of sterols is discussed.

Animals↗
PubMed1994

Bile acid synthesis in HepG2 cells: effect of cyclosporin.

The hypothesis that cyclosporin specifically affects the pathway of bile acid synthesis that begins with 27-hydroxylation of cholesterol was evaluated in HepG2 cells, which synthesize chenodeoxycholic acid and cholic acid from endogenous 7 alpha-hydroxycholesterol. At a concentration in the medium of 8.3 microM cyclosporin, the proportion of cholic acid increased from 29 +/- 7% to 44 +/- 6% (P < 0.001) with no major change in total bile acid production. Chenodeoxycholic acid synthesis was enhanced by the addition of either 7 alpha-hydroxycholesterol or 5 beta-cholestane-3 alpha,7 alpha-diol to the medium and cholic acid synthesis was enhanced by the addition of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol to the medium. Cyclosporin significantly inhibited only enhanced chenodeoxycholic acid synthesis, indicating a selective interference in mitochondrial side chain oxidation of less polar intermediates in bile acid synthesis derived from either initial 7 alpha- or initial 27-hydroxylation of cholesterol. The increase in the proportion of cholic acid that occurs in the presence of cyclosporin mimics that occurring in genetically determined sterol 27-hydroxylase deficiency (cerebrotendinous xanthomatosis). Cyclosporin is useful for dissecting the subcellular pathways of bile acid synthesis.

Bile Acids and Salts↗
PubMed1996

Free and esterified oxysterol: formation during copper-oxidation of low density lipoprotein and uptake by macrophages.

We have defined the lipid composition of copper-oxidized LDL (Cu-oxLDL) and a macrophage-foam cell model generated by the uptake of this modified lipoprotein. An HPLC method previously developed by our group for the measurement of lipid oxidation products of LDL was extended to permit the analysis of an array of 7-ketocholesteryl esters. Gas chromatography was used for the quantitation of oxysterols (free and esterified) in Cu-oxLDL and their subsequent uptake by macrophages. LDL (1.0 mg protein/ml) was oxidized using Cu(II) (20 microM) for up to 48 h at 37 degrees C. Resident mouse peritoneal macrophages were incubated with 24 h Cu-oxLDL (50 micrograms/ml) for 24 h. In 24 h Cu-oxLDL, cholesterol comprised approximately 50% of total sterols, 7-ketocholesterol comprised approximately 30% with five other oxysterols comprising the remainder (7 alpha- and 7 beta-hydroxycholesterol, cholesterol alpha- and beta-epoxides, and 6 beta-hydroxycholesterol). Macrophages that were incubated with 24 h Cu-oxLDL displayed a profile of oxysterols remarkably similar to that of 24 h Cu-oxLDL itself. The majority of cholesteryl esters and 7-ketocholesteryl esters in Cu-oxLDL and in Cu-oxLDL-loaded macrophages contained fatty acyl chains which are presumed oxidized. This work represents a comprehensive survey of free and esterified oxysterols in Cu-oxLDL and Cu-oxLDL-loaded macrophages and provides a basis for exploring how oxysterols are metabolized by macrophages and authentic human foam cells, and how, in turn, these oxysterols influence cellular metabolism.

Animals↗
PubMed1997

Sterol 27-hydroxylase: expression in human arterial endothelium.

Human endothelium obtained from both the aorta and the pulmonary artery has been evaluated for the presence of the messenger RNA coding for the expression of sterol 27-hydroxylase. Unique oligomers were designed to detect the mRNA by reverse transcription followed by the polymerase chain reaction. The amplified product was sequenced and was found to be identical to the published sequence for nucleotides 491 to 802 of the human sterol 27-hydroxylase cDNA. Northern blot analysis confirmed the presence of 27-hydroxylase mRNA in pulmonary artery and aortic endothelium. As part of these studies, enzymatic activity was assayed in cultured arterial endothelium using cholesterol as a substrate and isotope ratio gas-liquid chromatography-mass spectrometry to identify the metabolites, 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid, in the medium. Localization of sterol 27-hydroxylase to vascular endothelium indicates intracellular production of the biologically active metabolite 27-hydroxycholesterol.

Aorta↗
PubMed2000

7beta-hydroxysterol is cytotoxic to neonatal rat astrocytes in primary culture when cAMP levels are increased.

We have shown previously that 7beta-hydroxycholesterol (7betaOHCH) and 7beta-hydroxycholesteryl-3-oleate (7betaOHCH- 3-OL) are potent inhibitors of lesion-induced astrogliosis in the rat cortex or spinal cord; these substances reduce reactive astrocyte proliferation and hypertrophy. In this study, we employed cultured newborn rat astrocytes with increased cAMP levels as an in vitro model of reactive astrocytes. Treatment with either dibutyryl-cAMP (dbcAMP) or isoproterenol resulted in morphologic differentiation of astrocytes which became fibrous. Concomitant incubation with 30 microM 7betaOHCH and dbcAMP (or isoproterenol) provoked the cells to retract and was cytotoxic. When the beta-adrenergic receptor-mediated cAMP increase was abolished by propranolol, the 7betaOHCH cytotoxicity was inhibited. Immunocytochemical labelling for glial fibrillary acidic protein (GFAP) and beta-tubulin and electron microscopy suggested that intermediate filament and microtubular organizations were modified by 7betaOHCH. Analysis of the activity of cAMP-dependent protein kinase (PKA) in astrocytes treated with dbcAMP and 7betaOHCH showed a rapid and marked inhibition of the phosphotransferase activity which lasted for 24 hr. We suggest that this culture system provides an experimental system to study the molecular mechanisms involved in the effect of oxysterols on astrocytic hypertrophy. The cytotoxicity of 7betaOHCH seems to be mediated by inhibition of PKA, which phosphorylates intermediate filaments and the transcription factor cyclic AMP responsive element binding.

Adrenergic beta-Agonists↗