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PubMed1990

Simultaneous quantification of several cholesterol autoxidation and monohydroxylation products by isotope-dilution mass spectrometry.

An assay based on isotope-dilution mass spectrometry with deuterium-labeled internal standards was developed for simultaneous quantification of cholest-5-ene-3 beta,7 alpha-diol (7 alpha-hydroxycholesterol), cholest-5 beta,6 beta-epoxy-3 beta-ol (cholesterol-5 beta,6 beta-epoxide), cholest-5 alpha,6 alpha-epoxy-3 beta-ol (cholesterol-5 alpha,6 alpha-epoxide), cholest-5-en-7-one-3 beta-ol (7-oxocholesterol), cholestane-3 beta,5 alpha,6 beta-triol, cholest-5-ene-3 beta,25-diol (25-hydroxycholesterol), and cholest-5-ene-3 beta,26-diol (26-hydroxycholesterol) in one single serum sample. Recovery experiments and replicate analyses showed that the assay was sufficiently sensitive, accurate, and precise. The concentrations of the listed compounds in sera from 19 healthy subjects were determined and are presented.

Adolescent↗

PubMed1989

Cholesterogenesis and cell division in phytohemagglutinin-stimulated human lymphocytes: a comparative study with several inhibitors.

It has been shown that when lymphocytes are stimulated by phytohemagglutinin the expected stimulation of DNA synthesis is preceded by stimulation of cholesterol synthesis. This confirms the existence of a relation between cell division and cholesterol synthesis. We studied the effect on cell division of six inhibitors of cholesterol biosynthesis, previously shown to interfere with different steps of the process: 7 beta-hydroxycholesterol, 25-hydroxycholesterol, lanost-7-en-3 beta, 32-diol, mevinolin, propiconazole, dodecylimidazole. Since experiments were performed in the presence of a high percentage of human serum, which provided cells with exogenous cholesterol via the LDL-receptor pathway, our investigation was focused on the role of newly synthesized cholesterol. The biosynthesis was evaluated by labeling cells with [14C]sodium acetate; to take into account variations of cell permeability to sodium acetate, the results were expressed as the percentage of total cellularly incorporated radioactivity transformed into cholesterol, after separation from all other labeled metabolites. These data were compared with the percentage of transformation into nonsaponifiable lipids, which varied in parallel with HMG-CoA reductase activity, as confirmed by direct enzymatic measurement. Cell division was assessed by simultaneous measurements of three parameters: thymidine incorporation into DNA, cell proliferation and cellular protein content. All the effectors strongly inhibited the conversion of labeled acetate into cholesterol, but cell division was not inhibited by two of them: propiconazole and 7 beta-hydroxycholesterol. These compounds only slightly inhibited the synthesis of nonsaponifiable lipids, which mainly consisted of methylsterols resulting from a blockage of lanosterol demethylation. Thus, it can be concluded that the nonsaponifiable metabolite essential for cell growth is not newly synthesized cholesterol. It was also found that inhibitors affected cell division only when they were added to the culture medium before the decline of cholesterol synthesis stimulation.

Anticholesteremic Agents↗

PubMed1976

Different effects of cycloheximide and chloramphenicol on corticosterone production by isolated rat adrenal cells.

Cycloheximide and chloramphenicol both inhibit the stimulating effect of adenocorticotropic hormone (ACTH) on adrenal steroid production. To test whether these inhibitors had andy effect on adrenal steroid production, independent fromthe mechanism of action of ACTH we investigated their effect on the conversion of 25-hydroxycholesterol into corticosterone in isolated rat adrenal cells. Cycloheximide, both in the absence and in the presence of ACTH, had no effect on this conversion. Chloramphenicol inhibited the conversion of 25-hydroxycholesterol into corticosterone whether ACTH has no direct efeect on the cholesterol side-chain cleaving system. The inhibition by chloramphenicol of the ACTH-stimulated steroid production is at least partly due to inhibition of one or more of the processes involved in the conversion of 25-hydroxycholesterol into corticosterone.

Adrenal Glands↗

PubMed1991

Capillary gas chromatography for the assessment of cholesterol oxides in the heart.

Recent studies indicated the presence of cholesterol oxides in biological tissues such as heart under pathophysiological conditions. Currently, no suitable method is available to separate and quantitate these oxides in biological tissues. This study was undertaken to develop a method suitable for the quantification of cholesterol oxides in heart. Since under normal conditions cholesterol oxidation does not occur, we exposed isolated perfused rat heart to oxygen-derived free radicals, which are known to oxidize cholesterol in heart. Free radical treated hearts were homogenized and lipids extracted by conventional techniques. The extracted lipids were saponified, and non-saponified portions were extracted with ether. Silyl derivatives of the extracted products were subjected to capillary gas chromatography. One microlitre of the sample was injected onto an SE 54 Supelco capillary column and run by stepwise temperature programming from 230 to 260 degrees C at a rate of 20 degrees C min-1, and then from 260 to 290 degrees C at 6 degrees C min-1. 5-Androsten-3 beta-ol-17-one was used as internal standard. The separated cholesterol oxides were identified by comparing with authentic standards. This method was suitable to separate 7 alpha-hydroxycholesterol, dihydroxycholesterol, 7 beta-hydroxycholesterol, 20 alpha-hydroxycholesterol, cholesterol-5 alpha,6 alpha-epoxide, and 5-cholestene in heart. Since some of these oxides are cytotoxic to the heart, identification of these oxides should be important to understand the pathophysiology of myocardial disease and for the successful therapy to prevent them.

Animals↗

PubMed1990

Cytochrome P-450scc-catalyzed production of progesterone from 22R-hydroxycholest-4-en-3-one by way of 20,22-dihydroxycholest-4-en-3-one.

Transient accumulation of a dihydroxylated steroid was found when 22R-hydroxycholest-4-en-3-one was used as the substrate for a reconstituted cholesterol side-chain cleavage system derived from bovine adrenocortical mitochondria. The indications were that the accumulated steroid was an intermediate in the cytochrome P-450scc-catalyzed reaction. The retention time of the accumulated intermediate was identical with that of authentic 20,22-dihydroxycholest-4-en-3-one on HPLC. When 22R-hydroxycholesterol and 22R-hydroxycholest-4-en-3-one were incubated simultaneously, the total amount of reaction products was essentially the same as that observed with 22R-hydroxycholest-4-en-3-one alone. Under the conditions employed, the apparent turnover number of cytochrome P-450scc for 22R-hydroxycholesterol was calculated to be 77 nmol/min/nmol P-450 from the amount of pregnenolone formed, whereas the apparent turnover number for 22R-hydroxycholest-4-en-3-one was 64 nmol/min/nmol P-450 with respect to the intermediate formation and 77 nmol/min/nmol P-450 with respect to the progesterone formation. The apparent turnover number for 20,22-dihydroxycholest-4-en-3-one was about 125 nmol/min/nmol P-450, which was not significantly different from that of 20,22-dihydroxycholesterol. The apparent Km for 22R-hydroxycholesterol was about 20 microM and those for 22R-hydroxycholest-4-en-3-one and 20,22-dihydroxycholest-4-en-3-one were 50 and 40 microM, respectively. Thus, 22R-hydroxycholest-4-en-3-one was efficiently metabolized to progesterone by way of 20,22-dihydroxycholest-4-en-3-one by cytochrome P-450scc.

Adrenal Glands↗

PubMed2006

Regulation of cholesterol 25-hydroxylase expression by vitamin D3 metabolites in human prostate stromal cells.

Vitamin D3 plays an important role in the control of cell proliferation and differentiation. Cholesterol 25-hydroxylase (CH25H) is an enzyme converting cholesterol into 25-hydroxycholesterol. Vitamin D3 as well as 25-hydroxycholesterol has been shown to inhibit cell growth and induce cell apoptosis. Here we show that 10 nM 1alpha,25(OH)2D3 and 500 nM 25OHD3 upregulate CH25H mRNA expression in human primary prostate stromal cells (P29SN). Protein synthesis inhibitor cycloheximide does not block 1alpha,25(OH)2D3 mediated upregulation of CH25H mRNA. Transcription inhibitor actinomycin D blocks basal level as well as 1alpha,25(OH)2D3 induced CH25H mRNA expression. 1alpha,25(OH)2D3 has no effect on CH25H mRNA stability. 25-Hydroxycholesterol significantly decreased the P29SN cell number. A CH25H enzyme inhibitor, desmosterol, increases basal cell number but has no significant effect on vitamin D3 treated cells. Our data suggest that ch25h could be a vitamin D3 target gene and may partly mediate anti-proliferative action of vitamin D3 in human primary prostate stromal cells.

Apoptosis↗

PubMed2006

Oxysterol mixtures, in atheroma-relevant proportions, display synergistic and proapoptotic effects.

Apoptotic cells in atheroma lesions may contribute to plaque development and instability. Oxysterols constitute the major toxic component in oxLDL and are present in mixed forms in human atheroma lesions. However, the cellular effects of oxysterols have been mostly studied individually. In the present study, we investigated the cytotoxic effects of 7beta-hydroxycholesterol (7betaOH), 7-ketocholesterol (7keto), 25-hydroxycholesterol (25OH), and 27-hydroxycholesterol (27OH) on U937 monocytic cells, both individually and in atheroma-relevant mixtures mimicking the oxysterol composition reported in human atheroma lesions. Apoptosis and necrosis were studied by examining cell morphology, phosphatidylserine exposure, caspase activation, and the terminal dUTP nick end-labeling technique. Cellular reactive oxygen species and total amount of reduced thiols were measured by using fluorescence probes and 5,5'-dithiobis-(2-nitrobenzoic acid), respectively. We found that 7betaOH and 7keto induced caspase activation, ROS production, cellular thiol depletion, permeabilization of lysosomal and mitochondrial membranes, and cell death. 25OH and 27OH did not cause any of the above alterations, whereas 7betaOH and 7keto exerted synergistic toxic effects. Although single 25OH or 27OH exhibited quenching effects on both 7betaOH- and 7keto-induced cell death, the combination of all four oxysterols in atheroma-relevant proportions was proapoptotic. Our findings indicate that the major oxysterols accumulated in human atheroma are proapoptotic and may contribute to atherosclerotic lesion development.

Apoptosis↗

PubMed2000

Cholesterol-rich diets have different effects on lipid peroxidation, cholesterol oxides, and antioxidant enzymes in rats and rabbits.

The objective of this study was to compare the effect of cholesterol feeding of rats and rabbits. The levels of lipid peroxidation products and oxysterols in the plasma of the two species plus the antioxidant enzyme activities in the liver and erythrocytes were measured to explain their different susceptibilities to atherosclerosis. Our study showed that rats are less susceptible than are rabbits to the atherogenic effect of a cholesterol-rich diet because of differences in lipid peroxidation products as well as antioxidant enzymes activities in their livers. In rabbits, cholesterol feeding produced severe hypercholesterolemia (43-fold increase) and increased plasma and liver lipid peroxidation. Total as well as the individual oxysterol contents of 7alpha-, 7beta-hydroxycholesterol, alpha-epoxy, beta-epoxycholesterol, cholestanetriol, 7-keto, and 27-hydroxycholesterol significantly increased in the plasma of hypercholesterolemic (HC) rabbits. Erythrocyte glutathione peroxidase (GSH-Px) activity significantly decreased whereas catalase activity significantly increased in HC rabbits. In rats cholesterol feeding increased the plasma cholesterol only twofold and had no effect on plasma or liver lipid peroxidation. Only 7alpha- and 7beta-hydroxycholesterol increased and no change was observed in any of the antioxidant enzymes activity in the erythrocytes. Although cholesterol feeding caused a 10-fold increase of liver cholesterol as ester in both rats and rabbits, the antioxidant enzyme GSH-Px and catalase activities in the liver significantly increased in rats but significantly decreased in rabbits. The increase of GSH-Px and catalase activities in the liver of cholesterol fed rats could have a protective role against oxidation, thus preventing the formation of lipid peroxidation and oxysterols.

Journal Article↗

PubMed2001

High and low responding strains of laboratory opossums differ in sterol 27-hydroxylase and acyl-coenzyme A:cholesterol acyltransferase activities on a high cholesterol diet.

Two partially inbred strains of laboratory opossums exhibit extremely high or low levels of VLDL+LDL cholesterol concentrations, respectively, when challenged with a high cholesterol and high fat diet. The present studies were conducted to determine whether the high and low responding strains differ in activities of important enzymes that have been shown to affect lipemic responsiveness to diet. We measured plasma 27-hydroxycholesterol and hepatic activities of 27-hydroxylase and 7alpha-hydroxylase in high and low responding opossums while consuming the basal diet and cholesterol-enriched diets. Plasma 27-hydroxycholesterol concentration and 27-hydroxylase activity in liver did not differ between groups on the basal diet, but both were significantly higher in low responders than in high responders on the cholesterol-enriched diet with unsaturated fat (10.79 +/- 0.56 in low vs. 7.31 +/- 0.50 &mgr;g/dl in high responders for 27-hydroxycholesterol and 14.14 +/- 0.79 in low vs. 10.07 +/- 0.80 pmol/mg protein/min in high responders for 27-hydroxylase activity). On the other hand, 7alpha-hydroxylase activity was significantly higher in high responding opossums (75.72 +/- 6.81 pmol/mg protein/min) than in low responding opossums (51.39 +/- 6.18 pmol/mg protein/min) on the basal diet, but it did not differ on the high cholesterol and high fat diet. We measured hepatic ACAT and extrahepatic hepatic 27-hydroxylase activities in high and low responding opossums on the cholesterol enriched diet. Hepatic ACAT activity was significantly higher in high responding opossums (137.00 +/- 18.33 pmol/mg protein/min) than in low responding opossums (47.67 +/- 2.71 pmol/mg protein/min), whereas extrahepatic 27-hydroxylase activity was higher in low responding opossums (33.00 +/- 2.10 pmol/mg protein/min in lungs and 3.69 +/- 0.20 in kidneys) than in high responding opossums (21.17 +/- 1.54 pmol/mg protein/min in lungs and 2.82 +/- 0.31 in kidneys). We also compared the composition of bile between high and low responders. The concentration of taurine conjugates of cholic acid in bile of both groups was similar, but concentration of taurine conjugates of chenodeoxycholic acid in bile of low responding animals was higher than in high responding animals (124.9 +/- 17.3 in low vs. 59.2 +/- 13.2 &mgr;mol/ml in high responders). The results of these studies suggest two enzymes may affect the lipemic response to diet in laboratory opossums: sterol 27-hydroxylase and ACAT. Each of these enzymes may influence diet-induced hyperlipidemia at a different step of lipoprotein metabolism.

Journal Article↗

PubMed1988

8-bromoadenosine cyclic 3',5'-phosphate rapidly increases 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA in human granulosa cells: role of cellular sterol balance in controlling the response to tropic stimulation.

Exposure of cultured human granulosa cells to 8-bromoadenosine cyclic 3',5'-phosphate (8-bromo-cAMP) resulted in a rapid increase in the content of the mRNA for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in the de novo synthesis of cholesterol. HMG-CoA reductase mRNA levels increased within 2 h of stimulation and remained elevated for at least 6 h. Treatment of granulosa cells with 25-hydroxycholesterol, a soluble cholesterol analogue, in combination with aminoglutethimide to block conversion of cellular sterols to pregnenolone, resulted in suppression of HMG-CoA reductase mRNA. When cells were stimulated with 8-bromo-cAMP in the presence of 25-hydroxycholesterol and aminoglutethimide, the increase in HMG-CoA reductase mRNA provoked by the tropic agent was markedly attenuated. This indicates that 8-bromo-cAMP raises HMG-CoA reductase mRNA levels indirectly by accelerating steroidogenesis and depleting cellular sterol pools, thus relieving sterol-mediated negative feedback of HMG-CoA reductase gene expression. 25-Hydroxycholesterol in the presence of aminoglutethimide suppressed low-density lipoprotein (LDL) receptor mRNA, but 8-bromo-cAMP effected a significant stimulation of LDL receptor mRNA levels when added with hydroxysterol and aminoglutethimide. These findings reveal differential regulation of HMG-CoA reductase and LDL receptor mRNAs in the presence of sterol negative feedback.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1966

Substrate and inhibitor specificity of the cholesterol oxidase in bovine adrenal cortex.

1. Cholesteryl 3beta-sulphate is oxidized in vitro by preparations of bovine adrenal-cortex mitochondria to pregnenolone sulphate and isocaproic acid (4-methyl-pentanoic acid) without hydrolysis of the ester linkage. 2. Free cholesterol is the preferred substrate for adrenal-cortex cholesterol oxidase; the apparent K(m) for cholesteryl sulphate is 500mum and for free cholesterol 50mum under the same conditions. 3. Cholesteryl 3beta-acetate is hydrolysed by bovine adrenal-cortex mitochondria in vitro to free cholesterol, which is subsequently oxidized to more polar steroids and isocaproic acid. Evidence was obtained that other cholesterol esters behave similarly. Cholesterol esters may thus act as precursors of steroid hormones. 4. Cholest-4-en-3-one is only poorly oxidized to isocaproic acid and more polar steroids and thus is probably not a significant precursor of steroid hormones. 5. Cholesteryl esters inhibit the oxidation of cholesterol competitively (K(i) for cholesteryl phosphate 28mum, for cholesteryl sulphate 110mum, for cholesteryl acetate 65mum) but pregnenolone esters do not inhibit this system. 6. Pregnenolone and 20alpha-hydroxycholesterol (both metabolites of cholesterol in this system) inhibit the oxidation of cholesterol non-competitively. K(i) for pregnenolone is 130mum and K(i) for 20alpha-hydroxycholesterol is 17mum. 7. 25-Oxo-27-norcholesterol inhibits cholesterol oxidation non-competitively (K(i)16mum). A number of other Delta(5)-3beta-hydroxy steroids inhibit cholesterol oxidation and evidence was obtained that the 3beta-hydroxyl group was necessary for inhibitory activity. 8. Pregnenolone, 20alpha-hydroxycholesterol and 25-oxo-27-norcholesterol inhibit oxidation of cholesteryl sulphate by this system but their sulphates do not. 9. 3beta-Hydroxychol-5-enoic acid, 3alpha-hydroxy-5beta-cholanic acid and 3beta-hydroxy-22,23-bisnorchol-5-enoic acid stimulated formation of isocaproic acid from cholesterol. 10. No evidence was obtained that phosphorylation or sulphation are obligatory steps in cholesterol oxidation by adrenal-cortex mitochondria. 11. The cholesteryl 3beta-sulphate sulphatase of bovine adrenal cortex was found mostly in the microsomal fraction and was inhibited by inorganic phosphate.

Journal Article↗

PubMed2001

Plant sterols regulate rat vascular smooth muscle cell growth and prostacyclin release in culture.

Cardiovascular disease (CVD) is the leading cause of death in the USA and other industrialized countries. A large number of epidemiological studies have established a direct correlation between diet and the development and progression of atherosclerosis. Several studies have shown the incidence of CVD to be lower in populations consuming a predominantly plant-based diet, as compared to meat-based diets. Besides being low in fat and cholesterol, vegetarian and Asian diets contain a large variety of phytochemicals, which may function in the body. For example, phytosterols (PS) are plant sterols that interfere with the absorption of cholesterol from the intestine when present in adequate amounts. Although PS may also function at a cellular level in the body, there are few studies examining the action of PS on cells involved in atherosclerosis. The purpose of this study was to examine the effect of dietary PS on vascular smooth muscle cell (VSMC) growth and function, since VSMC play a central role in the development of atherosclerosis. VSMC were treated with 16 microM cholesterol, 25-hydroxycholesterol, campesterol and beta-sitosterol (SIT) using an ethanol as a vehicle. Cell growth was determined by cell counting and cell proliferation by DNA synthesis, which was measured by [(3)H]-thymidine incorporation. Cholesterol supplementation had no effect on cell growth and proliferation. 25-Hydroxycholesterol decreased cell growth by 68% and DNA synthesis by 99%. SIT was found to inhibit VSMC growth more effectively than campesterol. Of the two PS, campesterol decreased cell growth by 16% and SIT decreased cell growth by 30%. DNA synthesis was decreased 25% by SIT supplementation but was not influenced by campesterol or cholesterol supplementation. Cholesterol, campesterol and SIT were not cytotoxic to VSMC and did not significantly alter cell viability. 25-Hydroxycholesterol, however, was cytotoxic and decreased cell viability by 45% as determined by lactate dehydrogenase release and a trypan blue dye exclusion test. De novo cholesterol synthesis was decreased 28% by campesterol, 49% by SIT and 23% by cholesterol. Beta-sitosterol exhibited a greater effect on cholesterol synthesis than campesterol or cholesterol supplementation. Measurement of cell sterol content demonstrated incorporation of PS into VSMC at the expense of cholesterol. Campesterol decreased VSMC cholesterol content by 36%, representing 40% of the total sterol content following treatment. Beta-sitosterol decreased VSMC cholesterol by 41% following supplementation and represented 49% of the total sterol amount. Cholesterol treatment did not alter the cholesterol content of the cells. Prostacyclin production was significantly altered by PS treatment. Basal prostacyclin release was increased 43% by campesterol and 81% by SIT. A23187 stimulated prostacyclin release was increased 25% by campesterol and 54% by SIT. SIT supplementation induced a greater effect on prostacyclin release from VSMC than cholesterol or campesterol supplementation. The in vitro results presented here suggest that dietary PS, especially SIT, may offer protection from the VSMC hyperproliferation found in atherosclerosis. Further in vivo research is needed to support these observations.

Animals↗

PubMed1996

27-hydroxylated low density lipoprotein (LDL) cholesterol can be converted to 7alpha,27-dihydroxy-4-cholesten-3-one (cytosterone) before suppressing cholesterol production in normal human fibroblasts. Evidence that an altered metabolism of ldl cholesterol can underlie a defective feedback control in malignant cells.

The formation of oxysterols in cultured human fibroblasts and their physiological roles as intracellular regulators of cholesterol production have been investigated. In the presence of low density lipoproteins (LDL), normal fibroblasts converted LDL cholesterol to 27hydroxycholesterol, which was further metabolized to 7alpha, 27-dihydroxycholesterol, 7alpha,27-dihydroxy-4-cholesten-3-one, and 7alpha-hydroxy-3-oxo-4-cholestenoic acid. Autooxidation products of cholesterol contaminating the lipoproteins were also metabolized in the cells. 7alpha-Hydroxycholesterol was converted to 7alpha-hydroxy-4-cholesten-3-one prior to 27-hydroxylation and further oxidation to 7alpha-hydroxy-3-oxo-4-cholestenoic acid. 7beta-Hydroxycholesterol and 7-oxocholesterol were 27-hydroxylated and then oxidized to C27-acids. Oxidation of the 7beta-hydroxy group also occurred. 25-Hydroxycholesterol was 7alpha-hydroxylated and further oxidized to 7alpha,25-dihydroxy-4-cholesten-3-one. 25-Hydroxylation of sterols was observed only under specific conditions. In contrast, only small amounts of oxysterols were formed in virus-transformed human fibroblasts when incubated with lipoproteins. This was due to very low activities of the 27- and 7alpha-hydroxylating enzymes. The rate of oxidation at C-3 was also decreased moderately. A defective suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase by LDL and autooxidation products of cholesterol observed in the transformed fibroblasts could be caused by the deficiencies of the sterol-metabolizing enzymes, since these cells responded normally to the sterol metabolites 7alpha,27-dihydroxy-4-cholesten-3-one, 7alpha, 25-dihydroxy-4-cholesten-3-one, and 27-hydroxy-7-oxo-cholesterol. These metabolites, which all possessed an oxo group with a conjugated double bond in the steroid nucleus and a hydroxyl group in the side chain, did not seem to require further metabolism in order to be active. An impaired response to LDL was also seen in other human tumor cells, including breast carcinoma, colonic carcinoma, and malignant melanoma cells. Common to all the malignant cells was an intracellular shortage of 7alpha, 27-dihydroxy-4-cholesten-3-one caused by a decreased formation or an increased metabolism.

Cell Line, Transformed↗

PubMed2000

Human white/murine ABC8 mRNA levels are highly induced in lipid-loaded macrophages. A transcriptional role for specific oxysterols.

To identify genes that are transcriptionally activated when human macrophages accumulate excess lipids, we employed the mRNA differential display technique using RNA isolated from human monocyte-macrophages incubated in the absence or presence of acetylated low density lipoprotein and sterols (cholesterol and 25-hydroxycholesterol). These studies identified a mRNA whose levels were highly induced in lipid-loaded macrophages. The mRNA encoded the human White protein, a member of the ATP-binding cassette (ABC) transporter superfamily of proteins. The mRNA levels of ABC8, the murine homolog of the human white gene, were also induced when a murine macrophage cell line, RAW264.7, was incubated with acetylated low density lipoprotein and sterols. Additional studies demonstrated that white/ABC8 mRNA levels were induced by specific oxysterols that included 25-, 20(S)-, and 22(R)-hydroxycholesterol, and by a retinoid X receptor-specific ligand. Furthermore, the oxysterol-mediated induction of ABC8 expression in mouse peritoneal macrophages was dependent on the presence of the nuclear oxysterol receptors, liver X receptors (LXRs). Macrophages derived from mice lacking both LXRalpha and LXRbeta failed to up-regulate the expression of ABC8 following incubation with 22(R)-hydroxycholesterol. Oxysterol-dependent induction of white/ABC8 mRNA was blocked by actinomycin D but not by cycloheximide treatment of cells. We conclude that the white and ABC8 genes are primary response genes that are transcriptionally activated by specific oxysterols and that this induction is mediated by the LXR subfamily of nuclear hormone receptors. These data strongly support the hypothesis that white/ABC8 has a role in cellular sterol homeostasis.

ATP-Binding Cassette Transporters↗

PubMed2002

Induction of the cholesterol transporter ABCA1 in central nervous system cells by liver X receptor agonists increases secreted Abeta levels.

The expression, function, and regulation of the cholesterol efflux molecule, ABCA1, has been extensively examined in peripheral tissues but only poorly studied in the brain. Brain cholesterol metabolism is of interest because several lines of evidence suggest that elevated cholesterol increases the risk of Alzheimer's disease. We found a largely neuronal expression of ABCA1 in normal rat brain by in situ hybridization. ABCA1 message was dramatically up-regulated in neurons and glia in areas of damage by hippocampal AMPA lesion after 3-7 days. Immunoblot analysis demonstrated ABCA1 protein in cultured neuronal and glial cells, and expression was induced by ligands of the nuclear hormone receptors of the retinoid X receptor and liver X receptor family. ABCA1 was induced by treatment with retinoic acid and several oxysterols, including 22(R)-hydroxycholesterol and 24-hydroxycholesterol. Expression of an ABCA1-green fluorescent protein construct in neuroblastoma cells demonstrated fluorescence in perinuclear compartments and on the plasma membrane. Because the Abeta peptide is important in Alzheimer's disease pathogenesis, we examined whether ABCA1 induction altered Abeta levels. Treatment of neuroblastoma cells with retinoic acid and 22(R)-hydroxycholesterol caused significant increases in secreted Abeta40 (29%) and Abeta42 (65%). Treatment with a nonsteroidal liver X receptor ligand, TO-901317, similarly increased levels of secreted Abeta40 (25%) and Abeta42 (126%). The increase in secreted Abeta levels was reduced by RNAi blocking of ABCA1 expression. These data suggest that the cholesterol efflux molecule ABCA1 may also be involved in the secretion of the membrane-associated molecule, Abeta.

ATP Binding Cassette Transporter 1↗

PubMed2003

Expression of sterol 27-hydroxylase (CYP27A1) enhances cholesterol efflux.

Cholesterol efflux from CHOP cells transfected with sterol 27-hydroxylase (CYP27A1) was compared with non-transfected and mock-transfected cells. Transfection caused expression of CYP27A1, formation of 27-hydroxycholesterol, and inhibition of cholesterol biosynthesis. Transfection enhanced cholesterol efflux to apolipoprotein A-I or human plasma by 2-3-fold but did not affect the efflux in the absence of acceptor. The analysis of released sterols revealed that 27-hydroxycholesterol represented only a small proportion of sterols, most of which was non-oxidized cholesterol. Time course and dose dependence studies showed that expression of CYP27A1 in CHOP cells mostly affected the efflux of the "fast" cholesterol pool, and relatively more cholesterol was released with low concentrations of an acceptor. Preincubation of non-transfected cells with exogenous 27-hydroxycholesterol (10(-9) and 10(-7) m) led to the stimulation of cholesterol efflux by 24-60%. Expression of CYP27A1 in CHOP cells did not affect ABCA1 expression and abundance of ABCA1 protein. Thus, introduction of CYP27A1 into cells stimulates cholesterol efflux and therefore may increase protection against atherosclerosis.

Animals↗

PubMed2005

Studies on the transcriptional regulation of cholesterol 24-hydroxylase (CYP46A1): marked insensitivity toward different regulatory axes.

Mammalian CNS contains a disproportionally large and remarkably stable pool of cholesterol. Despite an efficient recycling there is some requirement for elimination of brain cholesterol. Conversion of cholesterol into 24S-hydroxycholesterol by the cholesterol 24-hydroxylase (CYP46A1) is the quantitatively most important mechanism. Based on the protein expression and plasma levels of 24S-hydroxycholesterol, CYP46A1 activity appears to be highly stable in adults. Here we have made a structural and functional characterization of the promoter of the human CYP46A1 gene. No canonical TATA or CAAT boxes were found in the promoter region. Moreover this region had a high GC content, a feature often found in genes considered to have a largely housekeeping function. A broad spectrum of regulatory axes using a variety of promoter constructs did not result in a significant transcriptional regulation. Oxidative stress caused a significant increase in transcriptional activity. The possibility of a substrate-dependent transcriptional regulation was explored in vivo in a sterol-deficient mouse model (Dhcr24 null) in which almost all cholesterol had been replaced with desmosterol, which is not a substrate for CYP46A1. Compared with heterozygous littermates there was no statistically significant difference in the mRNA levels of Cyp46a1. During the first 2 weeks of life in the wild-type mouse, however, a significant increase of Cyp46a1 mRNA levels was found, in parallel with an increase in 24S-hydroxycholesterol level and a reduction of cholesterol synthesis. The failure to demonstrate a significant transcriptional regulation under most conditions is discussed in relation to the turnover of brain and neuronal cholesterol.

Alzheimer Disease↗

PubMed1991

Stage of ovarian follicular development associated with the initiation of steroidogenic competence in avian granulosa cells.

Previously described models for avian ovarian steroidogenesis, using mature, 25-40-mm preovulatory follicles as the source of tissues, were based on the assumption that interaction of the granulosa layer, as the predominant source of progesterone, with adjacent theca cells is required for maximal production of C21, C19, and C18 steroids. In the present study, we evaluated the steroidogenic capacity of ovarian cells isolated from less mature, 6-8-mm and 9-12-mm follicles in the chicken ovary (representative of a stage of development 2-3 wk prior to ovulation) to determine at which stage of follicular development granulosa and/or theca cells become steroidogenically competent. Granulosa cells collected from 6-8-mm follicles were found to be virtually incompetent to produce steroids, containing extremely low basal levels of progesterone (12 pg/5 x 10(5) cells) and failing to respond with increased steroid output following a 3-h exposure to ovine LH (oLH; 0.1 and 100 ng/0.5 ml), ovine FSH (oFSH; 100, 500, and 1,000 ng/0.5 ml), 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP; 0.33 and 3.33 mM) or 25-hydroxycholesterol (250 and 2,500 ng/0.5 ml). However, addition of pregnenolone (20 and 200 ng/0.5 ml) to granulosa incubations resulted in significantly increased progesterone levels. Granulosa cells of 6-8-mm follicles also failed to increase cAMP formation in the presence of oLH (10, 100, and 1,000 ng/0.5 ml) and 3-isobutyl-1-methylxanthine (IBMX; 10 microM), but responded to stimulation with 1,000 ng oFSH (4.4-fold increase over basal) or 10 microM forskolin (32-fold increase over basal) in the presence of IBMX. In contrast, granulosa cells isolated from 9-12-mm follicles and incubated for 3 h in vitro were found to contain basal progesterone levels 200-fold higher than those found in granulosa cells of 6-8-mm follicles. Furthermore, granulosa cells of 9-12-mm follicles markedly increased progesterone production following incubation in the presence of oFSH (100-1,000 ng/0.5 ml), 8-bromo-cAMP (0.33 and 3.33 mM), or 25-hydroxycholesterol (250 and 2,500 ng/0.5 ml). However, these granulosa cells remained unresponsive to oLH (0.1, 10, and 100 ng/0.5 ml), failing to increase cAMP accumulation (in the presence of IBMX) and progesterone output. Theca cells of small yellow follicles were found to produce measurable basal levels of progesterone, androstenedione, and estradiol, and levels of each steroid were significantly increased following a 3-h challenge with oLH, 8-bromo-cAMP, 25-hydroxycholesterol, and pregnenolone.(ABSTRACT TRUNCATED AT 400 WORDS)

Androstenedione↗