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PubMed1992

Increased cellular cholesterol upregulates high density lipoprotein binding to rat luteal cells.

Rat luteal cells preferentially utilize cholesterol derived from high density lipoproteins (HDL) as a substrate for steroid hormone synthesis. The uptake of cholesterol from HDL by these cells is in contrast to nonsteroidogenic cells, which export cholesterol to HDL. A previous study demonstrated that HDL binding to luteal cell membranes was increased in conjunction with in vivo cholesterol depletion or cholesterol loading of the ovary induced by pharmacological agents. These results suggest a biphasic regulation of the HDL receptor in luteinized rat ovaries. In the present studies, the in vitro regulation of HDL binding in rat luteal cells by increased intracellular cholesterol was examined. Cultured luteal cells were incubated with increasing doses of low density lipoproteins (LDL) for 2 days after which the cellular sterol content and the effects on progesterone production and HDL binding were measured. As expected, the LDL treatment increased total cellular sterol content in a dose-dependent manner, resulting in a 2.1-fold increase over control at a dose of 1 mg LDL/ml. Increased cellular cholesterol was accompanied by a comparable increase in progesterone secretion. These results suggest that exogenous cholesterol was utilized by these cells. The LDL treatment also increased the binding of HDL to the cells in a dose-dependent manner to a maximum of 2.2-fold over control. The effect of increased cellular sterol on HDL binding was also examined using a more polar cholesterol derivative, 25-hydroxycholesterol. Cells were cultured for 2 days in media containing 0.3-40 micrograms/ml 25-hydroxycholesterol in the presence of 100 micrograms/ml aminoglutethimide, an inhibitor of cholesterol metabolism. The HDL binding to luteal cells exhibited dose-dependent up-regulation by 25-hydroxycholesterol with a 5.8-fold increase in binding at the maximum dose tested. Equilibrium binding studies using cells treated with 10 micrograms/ml 25-hydroxycholesterol revealed a 2.1-fold increase in the number of HDL binding sites on the luteal cells without affecting the binding affinity. From the results of this study, it is concluded that HDL binding in rat luteal cells is up-regulated by an increase in the intracellular cholesterol level.

Aminoglutethimide↗

PubMed1975

Cytochrome P-450 of adrenal mitochondria. Spin states as detected by difference spectroscopy.

Adrenal mitochondrial cytochrome P-450 which functions in cholesterol side chain cleavage (P-450scc) exhibited type I (lambdamax 385, lambdamin 420 nm) and inverse type I (lambdamin 385, lambdamax 420 nm) difference spectra with several steroids. The magnitude and type of response were dependent on the particular steroid and on the extent to which cholesterol was bound to the cytochrome in the intact mitochondrion. the inverse type I difference spectrum induced by 3beta-hydroxy-pregn-5-ene-20-one (pregnenolone) was dependent on the proportion of high spin cholesterol-cytochrome P-450scc complexes. With rat adrenal mitochondria cholest-5-ene-3beta, 20alpha-diol (20alpha-hydroxycholesterol) invariably induced a smaller inverse type I response and, under conditions where cytochrome P-450scc was nearly free of cholesterol, even produced a small type I response. Two distinct steroid binding sites on cytochrome P-450scc were detected by, respectively, the slow type I response to cholest-5-ene-3beta, 25-diol (25-hydroxycholesterol) and the rapid type I response to a subsequent addition of cholest-5-ene-3beta, 20alpha, 22 R-triol (20alpha, 22R-dihydroxycholesterol). The relative proportions of the spectral responses to these steroids were dependent on the previous extent of adrenal activation by adrenocorticotropic hormone (ACTH), because this stimulatory process altered the combination of mitochondrial cholesterol with cytochrome P-450scc. It is proposed that the two steroid binding sites on cytochrome P-450scc interact with steroids in the following way: site I binds cholesterol, 25-hydroxycholesterol, and 20alpha, 22R-dihydroxycholesterol with formation of a partially high spin cytochrome; site II binds both pregnenolone and 20alpha-OH cholesterol resulting in a low spin cytochrome. Interactions between sites I and II are not competitive, and occupancy of site II ensures a low spin state irrespective of the occupancy of site I. A second mode of interaction by 20alpha, 22R-dihydroxycholesterol stabilizes a high spin cytochrome and is competitive with site II binding by 20alpha-hydroxycholesterol or pregnenolone. Formation of a maximally high spin cytochrome follows occupancy by 20alpha, 22R-dihydroxycholesterol at both sites.

Adrenal Glands↗

PubMed1983

Kinetics of O2 and CO Binding to adrenal cytochrome P-450scc. Effect of cholesterol, intermediates, and phosphatidylcholine vesicles.

The kinetics of O2 and CO binding to purified adrenal cytochrome P-450scc has been measured at 25 degrees C by stopped flow spectrophotometry. Measurements were made on the cytochrome in both aqueous buffer and phosphatidylcholine vesicles; little difference in the kinetic constants between the two was observed. 22R-Hydroxycholesterol and 20 alpha,22R-dihydroxycholesterol, intermediates in the cytochrome P-450scc-catalyzed conversion of cholesterol to pregnenolone, markedly influenced rates and equilibria of O2 and CO binding. Binding of the intermediates greatly decreased the association rate of both O2 (80-160-fold) and CO (30-80-fold) relative to cholesterol-bound cytochrome. They had opposite effects on O2 and CO dissociation rates, causing a 5-fold increase in the CO dissociation rate relative to cholesterol and a 150-900-fold decrease in the O2 dissociation rate. Kd values for CO, determined by titration of the reduced cytochrome with CO, increased from 0.3 microM for substrate-depleted cytochrome to 0.7-1.0 microM, 100 microM, and 213-244 microM, respectively, for cholesterol, 22R-hydroxycholesterol, and 20 alpha,22R-dihydroxycholesterol. Conversely, Kd values for O2, determined from the ratio of dissociation and association rate constants, decreased from 23 microM for cholesterol-bound cytochrome to 12 microM and 5 microM for 22R-hydroxycholesterol and 20 alpha,22R-hydroxycholesterol, respectively, indicating a thermodynamic stabilization of the Fe2+ . O2 complex by these intermediates.

Adrenal Cortex↗

PubMed1993

Effect of cholesterol oxides on prostacyclin production and platelet adhesion.

Prostacyclin (PGI2) is synthesized primarily by endothelial cells, is essential for maintenance of vascular integrity, and may play a role in atherogenesis. Human umbilical vein endothelial cells in culture were incubated with either pure cholesterol, 25-hydroxycholesterol, 7-ketocholesterol, cholesterol 5 alpha,6 alpha-epoxide or cholestane-3 beta,5 alpha,6 beta-triol at 10 micrograms/ml culture medium concentration for 12 hours and 24 hours. PGI2 production measured by radioimmunoassay of 6-keto PGF1 alpha, the stable metabolite of PGI2 was inhibited by 39.6%, 27.3%, 40.1% and 31.9% after incubation with 25-hydroxycholesterol, 7-ketocholesterol, cholesterol 5 alpha, 6 alpha-epoxide or cholestane-3 beta,5 alpha,6 beta-triol for 12 hours respectively. Further inhibitory effects were shown after 24 hours of incubation with 25-hydroxycholesterol and 7-ketocholesterol. Platelet adhesion onto endothelial cell monolayers measured by 111In-labeled platelets was enhanced by 104%, 54% and 37% after incubation with cholestane-3 beta, 5 alpha,6 beta-triol, 25-hydroxycholesterol, and 7-ketocholesterol at 10 micrograms/ml concentration for 12 hours respectively. Pure cholesterol at the same concentration had no effect on PGI2 production or platelet adhesion.

Arteriosclerosis↗

PubMedDate not supplied

Toxicity of cholesterol oxidation products to Caco-2 and HepG2 cells: modulatory effects of alpha- and gamma-tocopherol.

Cholesterol can be oxidized to form a variety of cholesterol oxidation products also known as oxysterols. The aims of the present study were to compare the cytotoxic effects of four oxysterols, namely 25-hydroxycholesterol (25-OHC), 7beta-hydroxycholesterol (7beta-OHC), cholesterol-5beta,6beta-epoxide (beta-epox) and cholesterol-5alpha,6alpha-epoxide (alpha-epox), in two human cell culture models. Further, the ability of 10 and 100 micro m alpha- and gamma-tocopherol (alpha-TOC and gamma-TOC, respectively) to protect against oxysterol-induced cytotoxicity was also assessed. Human colonic adenocarcinoma Caco-2 and human hepatoma HepG2 cells were supplemented with increasing concentrations of 25-OHC, 7beta-OHC, beta-epox and alpha-epox (0-25 micro g ml(-1)) for 24, 48 or 96 h. Following 24-h and 48-h exposure, test media were replaced with normal growth media and the cells were maintained for 72 and 48 h, respectively. The 96-h exposure represented a constant challenge to the cells. Cytotoxicity was assessed using the neutral red uptake assay. The concentration of compound that inhibited cell viability by 50% (ic(50) value) was calculated. All four oxysterols investigated induced the greatest cytotoxic effects following 96 h of exposure. 25-Hydroxycholesterol exhibited the greatest cytotoxicity in both cell lines. Both beta-epox and alpha-epox were more toxic to HepG2 cells than to Caco-2 cells after the 48-h exposure. Pretreatment of cells with either alpha- or gamma-TOC did not protect against oxysterol-induced cytotoxicity. The caco-2 cells treated with the high concentration (100 micro m) of gamma-TOC were found to be more susceptible to oxysterol-induced toxicity under the conditions employed in this study.

Antioxidants↗

PubMed2005

Osteogenic oxysterols inhibit the adverse effects of oxidative stress on osteogenic differentiation of marrow stromal cells.

The osteoporosis that occurs with aging is associated with reduced number and activity of osteoblastic cells. Aging, menopause, and osteoporosis are correlated with increased oxidative stress and reduced antioxidant defense mechanisms. We previously demonstrated that oxidative stress induced by a variety of compounds such as xanthine/xanthine oxidase (XXO) and minimally oxidized LDL (MM-LDL) inhibit the osteogenic differentiation of osteoprogenitor cells. Oxysterols are a family of products derived from cholesterol oxidation that have important biological activities. Recently, we reported that a specific oxysterol combination consisting of 22(S)- or 22(R)-hydroxycholesterol and 20(S)-hydroxycholesterol has potent osteogenic properties in vitro when applied to osteoprogenitor cells including M2-10B4 (M2) marrow stromal cells. We now demonstrate that this osteogenic combination of oxysterols prevents the adverse effects of oxidative stress on differentiation of M2 cells into mature osteoblastic cells. XXO and MM-LDL inhibited the osteogenic differentiation of M2 cells, demonstrated by the inhibition of markers of osteogenic differentiation: alkaline phosphatase activity, osteocalcin expression and mineralization. Treatment of M2 cells with osteogenic oxysterol combination 22(S)- and 20(S)-hydroxycholesterol both blocked and reversed the inhibition of osteogenic differentiation produced by XXO and MM-LDL in these cells. The protective effect of the oxysterols against oxidative stress was dependent on cyclooxygenase 1 and was associated with the osteogenic property of the oxysterols. These findings further demonstrate the ability of the osteogenic oxysterols to positively regulate osteogenic differentiation of cells, and suggests that the use of these compounds may be a novel strategy to prevent the adverse effects of oxidative stress on osteogenesis.

Animals↗

PubMed1986

Cholesterol sulfate uptake and outflux in cultured human keratinocytes.

Cholesterol sulfate-cholesterol homeostasis in the epidermis may be important for normal desquamation. Recent evidence from cell-culture studies indicates that cholesterol sulfate inhibits sterologenesis by inhibiting the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. For cholesterol sulfate in the stratum corneum to function as a feedback regulator of epidermal sterologenesis, it must have the capacity enter cells. In these studies, uptake and outflux of cholesterol sulfate was examined in cultured human foreskin keratinocytes, and compared with uptake and outflux of cholesterol and 25-hydroxycholesterol, as well as with sterol uptake by another cell type, the skin fibroblast. The uptake of the free (not lipoprotein-associated) form of all three sterols was not consistent with a receptor-mediated process. Although the cholesterol sulfate uptake was approximately three-fold less than that of 25-hydroxycholesterol, it was nearly seven-fold greater than that of cholesterol itself. Whereas 25-hydroxycholesterol was rapidly washed out of keratinocytes in outflux experiments, both cholesterol and cholesterol sulfate tended to remain cell-associated. These studies demonstrate that cholesterol sulfate is readily taken up by keratinocytes, in which it may be in a position to modulate cellular lipid metabolism.

Cells, Cultured↗

PubMed1994

Modulation of adrenal cell functions by cadmium salts: 2. Sites affected by CdCl2 during unstimulated steroid synthesis.

In previous studies cadmium chloride (CdCl2) nonlethally inhibited Y-1 adrenal mouse adrenal tumour cell 20-dihydroxyprogesterone (20DHP) secretion, affecting unstimulated and stimulated steroidogenic pathway sites differently. We studied CdCl2 effects on unstimulated steroidogenesis using Y-1 cells incubated 0.5 h in medium with or without cadmium (using the concentration that inhibited ACTH-stimulated steroid secretion by 50%). Exogenously added 20-hydroxycholesterol (20OHC), 22(R)-hydroxycholesterol (22OHC), 25-hydroxycholesterol (25OHC), pregnenolone (PREG), or progesterone (PROG) were used to bypass any rate-limited steroidogenic pathway sites that CdCl2 might inhibit. 25OHC is a biologically active nonpathway steroid, while 20OHC, 22OHC, PREG, and PROG are pathway steroids; each increased unstimulated 20DHP secretion nearly 10-fold. Although CdCl2 could not reduce dibutyryl cyclic AMP- (dbcAMP)-stimulated 20DHP secretion significantly, it did significantly reduce basal and 25OHC-induced 20DHP secretion 25% below untreated levels. When 20OHC, 22OHC, PREG, or PROG were incubated with unstimulated Y-1 cells, their synthesis into 20DHP was unaffected by cadmium. dbcAMP bypasses the plasma membrane enzyme complex that synthesizes intracellular cAMP during exogenous ACTH stimulation; dbcAMP was not inhibited by CdCl2. The rate-limited step accelerated by cAMP involves plasma membrane and/or cytoplasmic cholesterol transport to and through outer and inner mitochondrial membranes before the cholesterol is synthesized into pregnenolone by side-chain cleavage enzymes on the inner membrane matrix face. Little is known regarding the mechanisms controlling unstimulated steroidogenesis. Under unstimulated conditions the 25-, 20- and 22(R)-monohydroxyls of cholesterol facilitate plasma membrane, cytoplasm and inner and outer mitochondrial solubility, diffusion and/or transport to bypass rate-limited steps and augment unstimulated steroid synthesis. Since conversion of endogenous mitochondrial cholesterol and 25OHC, but not dbcAMP-mobilized cytoplasmic cholesterol, 20OHC or 22OHC conversion, to 20DHP is inhibited by CdCl2, this suggests that (a) control of mitochondrial cholesterol supplies is independent of the cAMP-regulated mitochondrial steps in the 20DHP steroid synthetic pathway, (b) CdCl2 specifically inhibited endogenous mitochondrial cholesterol and 25OHC utilization, (c) CdCl2 toxicity may affect adrenal, testicular, ovarian, and placental basal steroidogenic functions, and (d) 25OHC may be a useful compound to examine unstimulated steroid synthesis.

Adrenal Glands↗

PubMed1989

HPLC method for quantitation of cholesterol and four of its major oxidation products in muscle and liver tissues.

A fast, sensitive, high performance liquid chromatographic method was developed for the quantitation of cholesterol and four of its major oxidation products: 3 beta-hydroxycholest-5-en-7-one (7-ketocholesterol), cholest-5-ene-3 beta, 7 alpha-diol (7 alpha-hydroxycholesterol), cholest-5-ene-3 beta, 7 beta-diol (7 beta-hydroxycholesterol), and cholest-5-ene-3 beta,25-diol (25-hydroxycholesterol). In this procedure 2:1 chloroform:methanol (v/v) extracts of tissue homogenate were combined, dried over anhydrous Na2SO4, filtered, evaporated to dryness under N2 and dissolved with a mobile phase of either 97:3 or 93:7 hexane:isopropanol (v/v). After membrane filtration and without further purification, aliquots were directly injected onto a 10-microns pore size, 30 X 0.39 cm mu-Porasil normal phase column. The separation of cholesterol and its oxidation products was monitored by a UV detector at 206 and 233 nm. This method was successfully applied to pork muscle as well as mouse liver tissues and was able to detect cholesterol oxidation products (COP) in the ppm range. The identity of the COP was confirmed by mass spectroscopy.

Animals↗

PubMed2005

Effect of pravastatin on plasma sterols and oxysterols in men.

OBJECTIVES: The HMG-CoA reductase inhibitors, or statins, are well established in the prevention and treatment of coronary artery disease, mainly by lowering low-density lipoprotein (LDL) cholesterol levels. These compounds are structurally similar, but differ in their lipophilicity. Several studies have indicated a link between cholesterol and Alzheimer's disease (AD), and there is also epidemiological evidence that statin treatment may decrease the prevalence of dementias. In the present study we wanted to investigate whether pravastatin treatment affects brain cholesterol metabolism. METHODS: A post hoc analysis was performed with plasma material from a clinical trial where 51 healthy men (35+/-4 years) were randomly assigned to receive either pravastatin (40 mg/day) or placebo for 6 months. Cholesterol, its precursor lathosterol, its brain-specific metabolite 24(S)-hydroxycholesterol (24S-OH-chol) and 27-hydroxycholesterol (27-OH-chol) were determined in plasma samples before and after treatment by using gas-liquid chromatography (GC)-flame ionization detection (GC-FID) and GC mass spectrometry (GC-MS). RESULTS: Besides reducing total cholesterol (-20%, P<0.001) and LDL cholesterol (LDL-C; -33%, P<0.001) concentrations, pravastatin treatment resulted in a decrease of the ratio of lathosterol to cholesterol, a surrogate marker of endogenous cholesterol synthesis, by 20% (P<0.05). Absolute concentrations of 24S-OH-chol were not altered, but its ratio to cholesterol slightly increased by 15% (P<0.05). 27-OH-chol concentrations as well as its ratio to cholesterol were both significantly altered due to pravastatin treatment (-7% and +14%, P<0.05 for both, respectively). CONCLUSIONS: The treatment with pravastatin 40 mg once a day for 6 months does not affect brain cholesterol metabolism as judged by plasma concentrations of 24(S)-hydroxycholesterol.

Adult↗

PubMed2005

Polymorphism in neuropeptide Y influences CSF cholesterol levels but is no major risk factor of Alzheimer's disease.

Neuropeptide Y (NPY) is a neurotransmitter expressed in the central nervous system and involved in learning and memory. The NPY L7P polymorphism has been associated with altered cholesterol levels in obese patients. Since altered cholesterol metabolism is also involved in Alzheimer's disease (AD), the effects of two NPY polymorphisms (L7P and IVS1-100 T/G) on CSF and plasma cholesterol and 24S-hydroxycholesterol were investigated in AD patients and non-demented controls. Furtheremore, the effect of both NPY polymorphisms on the risk of AD was studied. The NPY IVS1-100 T/G polymorphism influenced CSF levels of cholesterol, whereas CSF and plasma levels of 24S-hydroxycholesterol and plasma cholesterol were not altered by genotype. NPY L7P polymorphism did not influence CSF or plasma cholesterol or 24S-hydroxycholesterol. Both NPY polymorphisms did not influence the risk of AD. Our data support the observation, that NPY polymorphisms might influence cholesterol metabolism, but might not act as major risk factor in AD.

Aged↗

PubMed2003

Sterol-mediated regulation of hormone-sensitive lipase in 3T3-L1 adipocytes.

We previously reported that intracellular free cholesterol at physiological concentrations regulates the activity of neutral cholesterol esterase (N-CEase) in macrophages. The objective of the present study is to investigate whether the regulation of N-CEase by cholesterol is generally observed in other types of cells such as adipocytes with high activity of hormone-sensitive lipase (HSL), the same gene product as N-CEase. 3T3-L1 adipocytes were cultured with and without cholesterol (1-30 microg/mL) or 25-hydroxycholesterol (0.1-10 microg/mL), and changes in the N-CEase activity, expression of HSL mRNA, and protein were examined. Incubation (24 h) of cells with cholesterol did not change N-CEase activity, but incubation with 25-hydroxycholesterol decreased the activity in a concentration-dependent manner by 24 (24 h) and 54% (36 h). Quantitative reverse transcription-PCR indicated that 25-hydroxycholesterol (10 microg/mL) did not influence expression of HSL mRNA. However, Western blot analysis showed that this sterol reduced HSL protein by 72 (24 h) and by 93% (36 h), respectively. It was concluded that sterol-mediated regulation of HSL/N-CEase occurs not only in macrophages but also in adipocytes, and regulation appears to occur not at a transcriptional level but by a post-transcriptional process. Sterol-mediated proteolysis may be involved in the loss of HSL protein.

3T3-L1 Cells↗

PubMed1988

The effects of alpha-human atrial natriuretic polypeptide on steroidogenesis by fetal zone cells of the human fetal adrenal gland.

The human fetal adrenal gland is primarily composed of fetal zone cells, which exhibit a high rate of steroidogenesis and a rapid growth rate during fetal life. alpha-Human atrial natriuretic polypeptide has been shown to inhibit basal and adrenocorticotropic hormone-stimulated steroidogenesis in the human adult and in some human adrenal adenoma cells. The purpose of this investigation was to determine the effect of atrial natriuretic polypeptide on steroidogenesis by fetal zone cells. Dispersed fetal zone cells were incubated in Krebs-Ringer's medium with adrenocorticotropic hormone, forskolin, 22R-hydroxycholesterol, or dibutyryl cyclic adenosine monophosphate and in the presence of atrial natriuretic polypeptide. The medium was analyzed for content of dehydroepiandrosterone sulfate and cortisol by radioimmunoassay. The addition of adrenocorticotropic hormone, forskolin, 22R-hydroxycholesterol, or dibutyryl cyclic adenosine monophosphate increased the secretion of dehydroepiandrosterone sulfate and cortisol twofold to threefold and twofold to sixfold, respectively, above basal rates. Atrial natriuretic polypeptide significantly inhibited basal dehydroepiandrosterone sulfate and cortisol secretion. When cells were incubated in the presence of adrenocorticotropic hormone, forskolin, 22R-hydroxycholesterol, or dibutyryl cyclic adenosine monophosphate plus atrial natriuretic polypeptide, cortisol secretion was inhibited 50% to 90% and dehydroepiandrosterone sulfate was inhibited 25% to 50%. Atrial natriuretic polypeptide had no effect on the metabolism of progesterone tagged with carbon 14 in fetal zone cells. In conclusion, atrial natriuretic polypeptide inhibited basal and adrenocorticotropic hormone-stimulated steroid secretion by fetal zone cells. Furthermore, these results suggested that the action of atrial natriuretic polypeptide was by inhibition of cholesterol side-chain cleavage or transfer of cholesterol to the mitochondrion.

Adrenal Glands↗

PubMed1991

Expression of functional bovine cholesterol side chain cleavage cytochrome P450 (P450scc) in Escherichia coli.

Escherichia coli expression vectors containing the trc promoter and the complete DNA sequence of either the precursor or the mature form of bovine adrenocortical cholesterol side chain cleavage cytochrome P450 (P450scc) were transformed into E. coli strain JM109 and transcription induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). Immunoreactive cytochrome P450scc was produced using the plasmid containing the mature P450scc sequence but not with the plasmid containing the sequence of the precursor form of P450scc, even though P450scc RNA was detectable in both cases. The mature form of P450scc was detected spectrophotometrically in a reduced CO-difference spectrum in E. coli (40-60 nmol/liter culture). Cholesterol and hydroxylated derivatives (22-hydroxycholesterol and 25-hydroxycholesterol) produce a type 1 substrate-binding spectrum in IPTG-induced, transformed E. coli. The P450scc was found to be associated with the E. coli membrane fraction and the enzymatic activity of side chain cleavage of 25-hydroxycholesterol was reconstituted using solubilized membranes, in the presence of purified bovine adrenocortical adrenodoxin and NADPH-adrenodoxin reductase (turnover number; 15.4 nmol/min/nmol P450). This bacterial expression system provides functional P450scc, in the absence of other forms of P450, which can be used for evaluation of enzymatic and spectral properties of this mitochondrial P450 by site-directed mutagenesis.

Animals↗

PubMed1988

Serum-induced arachidonic acid release and prostaglandin biosynthesis are potentiated by oxygenated sterols in NRK 49F cells.

Fetal calf serum is able to activate arachidonic acid release from phospholipids in NRK 49F cells. We showed that this phenomenon can be potentiated by adding oxysterols to the culture medium. The oxysterol effect was dose-dependent and was not observed in the absence of fetal calf serum. Greater amounts of prostaglandin E2 and prostaglandin F2 alpha were released into the medium in the presence of oxysterols without apparent modification of the cyclooxygenase activity. The most effective oxysterols, in descending order, were the following: calcitriol greater than 7 alpha-hydroxycholesterol greater than 7 beta-hydroxycholesterol greater than 25-hydroxycholesterol. Cholesterol and 7-ketocholesterol were unable to activate phospholipase activity. The mechanism of this activation by oxysterols is still unknown.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1989

Steroidogenesis in Fundulus heteroclitus. I. Production of 17 alpha-hydroxy,20 beta-dihydroprogesterone, testosterone, and 17 beta-estradiol by prematurational follicles in vitro.

In order to understand better the mechanism of gonadotropin action on steroidogenesis in prematurational follicles of Fundulus heteroclitus, follicle synthesis of 17 alpha-hydroxy, 20 beta-dihydroprogesterone (17 alpha-OH,20 beta-DHP), testosterone (T), and 17 beta-estradiol (E2) from a variety of precursors and the maturational response of oocytes were simultaneously followed in vitro. The addition of 25-hydroxycholesterol, pregnenolone, or progesterone to unstimulated follicles increased media 17 alpha-OH,20 beta-DHP, T, and E2, as well as oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner. Inhibition of cholesterol side-chain cleavage by aminoglutethimide blocked 25-hydroxycholesterol-promoted steroid accumulation and GVBD, indicating that 25-hydroxycholesterol does not directly induce GVBD, but rather is metabolized in the follicle to an active steroid (presumably 17 alpha-OH,20 beta-DHP). Likewise, trilostane, an inhibitor of delta 5-3 beta-hydroxysteroid dehydrogenase, blocked pregnenolone action. Both inhibitors also completely abolished steroid accumulation and GVBD promoted by a F. heteroclitus pituitary extract (FPE), but not GVBD induced by exogenous 17 alpha-OH,20 beta-DHP. FPE also significantly depressed T but enhanced E2 production from exogenous precursors. We have concluded from these observations that (1) cholesterol side-chain cleavage and pregnenolone conversion to progesterone are essential for gonadotropin-promoted follicle steroid production and the resulting reinitiation of meiosis by the oocyte, (2) the enzymes necessary for the conversion of cholesterol to 17 alpha-OH,20 beta-DHP, T, and E2 are present in the unstimulated, prematurational follicle, and (3) gonadotropin initiates steroidogenesis by acting at a step prior to the conversion of cholesterol to pregnenolone; it also appears to enhance aromatase activity.

20-alpha-Dihydroprogesterone↗

PubMed1985

Cholesterol oxidation derivatives and arterial endothelial damage.

Three groups of New Zealand male while rabbits were given either 2.5 mg/kg of 25-hydroxycholesterol, cholestane-3 beta, 5 alpha, 6 beta-triol or vehicle only, intravenously. 24 h after treatment, the luminal surfaces of aortae of rabbits receiving 25-hydroxycholesterol were examined by scanning electron microscopy (SEM) and showed numerous balloon-like protrusions and crater-like defects as well as circulating, formed elements adhering on the luminal surface. The luminal surface of aortae of rabbits given cholestane-3 beta, 5 alpha, 6 beta-triol had similar but more frequent lesions when compared with those of the 25-hydroxycholesterol group. Microthrombi were occasionally found. The aortae of the control group had significantly fewer lesions. Transmission electron-microscopic studies showed intracytoplasmic vacuoles and diffuse subendothelial edema in the aortae of the two groups receiving the oxidation derivatives of cholesterol. The balloon-like protrusions and crater-like defects observed by SEM appeared to represent the initial sterol-induced endothelial cell injury. Repeated episodes of arterial injury followed by thrombus formation could eventually lead to atherosclerosis.

Animals↗

PubMed1982

Effect of thyroidectomy on pregnenolone and progesterone biosynthesis in rat adrenal cortex.

The effects of thyroidectomy on pregnenolone and progesterone biosynthesis were investigated in rat adrenal cortex mitochondria and microsomes. The sequential hydroxylations of cholesterol, and the latter's side-chain cleavage, which constitute the limiting step in steroidogenesis, were studied by measuring oxygen consumption rates in the presence of cholesterol derivatives. The addition of (20R)-hydroxycholesterol, (22R)-hydroxycholesterol or (20R,22R)-dihydroxycholesterol stimulated these rates, but addition of cholesterol or (22S)-hydroxycholesterol had little effect. Thyroidectomy significantly reduced oxygen consumption rates in the presence of the sterols by about 30%. Oxygen uptake was small in the presence of respiratory inhibitors; the addition of sterols raised this uptake but subsequent thyroidectomy did not change it. Rat adrenal cortex mitochondria and microsomes converted pregnenolone ino progesterone through 3 beta ol dehydrogenase/delta 4-5 isomerase, in two different successive steps. Values for enzyme activities were 0.18, 0.26 and 0.81 nmol progesterone/min/mg protein for the overall complex, the 3 beta ol dehydrogenase and the delta 4-5 isomerase respectively. All enzyme activities were unchanged by thyroidectomy. Similar results were obtained for corresponding microsomal activities whose values were in the same range. For both microsomes and mitochondria, the dehydrogenase reaction was the limiting step in the enzyme reaction leading to progesterone formation from pregnenolone. The limiting step in corticosteroidogenesis leading to prenenolone formation by an NADPH-dependent step was slowed down by thyroidectomy, probably because the reaction that transfers energy from NADH to NADP was inhibited. The enzyme complex leading to progesterone which involves NAD+ as cofactor, was unchanged by thyroidectomy. Thyroid hormones may therefore affect the availability of the energy mechanisms connected with the proton motive force, since thyroidectomy reduces both the phosphorylative oxidation and energy-dependent hydroxylation reactions involved in steroidogenesis.

Adrenal Cortex↗