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PubMed1991

Cytochrome P450 side-chain cleavage (P450scc) in the hen ovary. I. Regulation of P450scc messenger RNA levels and steroidogenesis in theca cells of developing follicles.

We have recently shown that granulosa cells from hen ovarian follicles, collected at a stage of development 2-3 wk prior to ovulation (e.g. 6-8 mm in diameter) are steroidogenically inactive. Therefore, the hypothesis tested in the present studies was that theca cells from follicles at this stage of development must contain sufficient levels of functional cytochrome P450 side-chain cleavage (P450scc) enzyme to produce the progestin precursor required for the synthesis of androgens and estrogens. Northern blot analysis of total theca RNA collected from 6-8-mm follicles indicated the presence of a single P450scc mRNA transcript of approximately 2 kb whose expression was increased following an 8-h preincubation with 200 ng/ml ovine LH (oLH) or 10 microM forskolin. Western blot analysis of crude mitochondrial protein revealed a band of immunoreactive P450scc protein of approximately 53 kDa that was determined to be capable of converting 25-hydroxycholesterol to pregnenolone in a cell-free system. In the second set of studies, conducted to examine the cellular regulation of steroidogenesis in isolated theca cells of 6-8-mm follicles, theca cells were found to produce measurable basal levels of cAMP, progesterone, androstenedione, and estradiol following a 3-h incubation of 5 x 10(5) cells. Furthermore, significant dose-dependent increases in steroidogenesis were observed in response to oLH (0.2-200 ng/ml), chicken FSH (cFSH; 20-200 ng/ml), cholera toxin (0.002-20 ng/ml), and 8-bromo-cAMP (0.1-3.33 mM). Phorbol 12-myristate 13-acetate (PMA; 10-167 nM) also stimulated dose-dependent increases in basal progesterone, androstenedione, and estradiol production. In addition, while PMA had no effect on oLH (200 ng/ml)-promoted cAMP accumulation, or on oLH (20 ng/ml)- or 8-bromo-cAMP (1 mM)-stimulated progesterone production, it attenuated oLH-induced and 8-bromo-cAMP-induced androstenedione and estradiol accumulation. We conclude that theca cells from 6-8-mm follicles possess mRNA and immunoreactive protein coding for functional P450scc. Furthermore, basal steroidogenesis is increased by both the protein kinase A and protein kinase C pathways, whereas evidence suggests that protein kinase C inhibits LH-induced androstenedione production at a site distal to cAMP and progesterone production, most likely by decreasing C17,20-lyase activity.

Androstenedione↗
PubMed1991

Cytochrome P450 side-chain cleavage (P450scc) in the hen ovary. II. P450scc messenger RNA, immunoreactive protein, and enzyme activity in developing granulosa cells.

Previous studies have indicated that developing avian granulosa cells collected from follicles 2-3 wk prior to ovulation (e.g. 6-8-mm in diameter) are steroidogenically incompetent, apparently due to a lack of functional cytochrome P450 side-chain cleavage (P450scc) enzyme activity. The present studies were designed to test this hypothesis by determining the absence or presence of P450scc messenger RNA, immunoreactive protein, and enzyme activity in granulosa tissue of developing hen ovarian follicles. Additionally, the interactive roles of FSH, the adenylyl cyclase-cAMP system, and the protein kinase C pathway in granulosa cell differentiation were investigated. Granulosa cells collected from developing, 6-8-mm follicles were found to contain extremely low but detectable levels of a single, 2.0-kb P450scc mRNA transcript, as well as immunoreactive P450scc protein (53 kDa). However, this protein was apparently incapable of converting 25-hydroxycholesterol to pregnenolone in a cell-free system. Preincubation of granulosa cells with ovine FSH or forskolin for 24 h rendered the cells capable of converting cholesterol precursor to pregnenolone during a subsequent 3-h incubation. Inclusion of the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), in the preincubation medium blocked the stimulatory actions of FSH and forskolin on the induction of P450scc activity; however, PMA-preincubation did not alter the ability of granulosa cells to convert exogenous pregnenolone to progesterone compared to vehicle-pretreated cells. These data suggest that steroidogenic incompetency in differentiating avian granulosa cells is primarily due to a lack of active P450scc enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Site of macrophage inhibition of luteinizing hormone-stimulated testosterone production by purified leydig cells.

It has been observed that testicular macrophages and testicular macrophage-conditioned medium reduce LH-stimulated, but not basal, testosterone production by purified Leydig cells in vitro. In order to determine how this inhibition occurs, we have examined the effects of testicular macrophages and testicular macrophage-conditioned medium at discrete stages of the steroidogenic pathway. The lesion in steroidogenesis is located at a step beyond cAMP formation, because the addition of dibutyryl cAMP or cholera toxin did not overcome the testicular macrophage-conditioned medium inhibition of LH-stimulated steroidogenesis by Leydig cells. This effect of testicular macrophage-conditioned medium on Leydig cell testosterone production is first observed at 18 h after initiation of culture. However, subsequent additions of 22R-hydroxycholesterol, pregnenolone, dehydroepiandrosterone, or androstenedione to the Leydig cell cultures can overcome the inhibition so that, after a further 6 h of incubation, testosterone production is not significantly different from that of control Leydig cells cultured in the absence of testicular macrophage-conditioned medium. These results suggest that the block in steroidogenesis is beyond cAMP production but prior to the formation of pregnenolone, dehydroepiandrosterone, and androstenedione. Since the medium for these cultures contained lipoprotein, it is possible that the testicular macrophage-conditioned medium metabolizes the lipoprotein, making it unavailable to the Leydig cells. However, our results show that preincubation of lipoprotein with testicular macrophage-conditioned medium does not significantly alter testosterone production by the Leydig cells in the culture. It is concluded that testicular macrophage-conditioned medium affects the transport or availability of cholesterol to mitochondria prior to further steps in the steroidogenic pathway.

Androstenedione↗
PubMed1995

Loss of low-density lipoprotein utilization by regressing porcine luteal cells: effects of protein kinase C activation.

We recently reported a decline in low-density lipoprotein (LDL) uptake by regressing porcine luteal cells that correlated with diminished LDL stimulation of progesterone (P) production. The objectives of the present study were to determine 1) whether loss of LDL utilization is a specific lesion in the steroidogenic pathway in regressing luteal cells and 2) whether in vitro activation of protein kinase C (PKC) in mid-cycle luteal cells acutely suppresses LDL utilization. Dispersed cells (4 x 10(4)/0.2 ml) from mid-(Days 6-10, estrus = Day 0; n = 5) or late-(Days 15-18; n = 7) cycle porcine CL were cultured in Dulbecco's Modified Eagle/F-12 medium supplemented with insulin, transferrin, sodium selenite, and aprotinin for 24 h with human (h) LDL (0-100 micrograms/ml), 22(R)-hydroxycholesterol (22[OH]-C;-0-25 micrograms/ml), or pregnenolone (0-1000 nM). P production by mid-cycle luteal cells was dose-dependently increased (p < 0.05) by LDL (up to 2.8-fold), 22(OH)-C (up to 3.2-fold), and pregnenolone (> 3-fold). In contrast, LDL (10-100 micrograms/ml) failed to stimulate P production by late-cycle luteal cells. But 22(OH)-C (up to 4-fold) and pregnenolone (> 10-fold) were as effective in promoting (p < 0.05) steroidogenesis relative to basal levels in late luteal cells as in mid-cycle cultures. The PKC activator, TPA (12-O-tetradecanoyl-phorbol-13-acetate; 10 ng/ml), inhibited (approximately 20%; p < 0.01) basal and LDL-supported steroidogenesis, but did not suppress 22(OH)-C-stimulated P secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Luteinizing hormone inhibits conversion of pregnenolone to progesterone in luteal cells from rats on day 19 of pregnancy.

We have previously reported that intrabursal ovarian administration of LH at the end of pregnancy in rats induces a decrease in luteal progesterone (P4) synthesis and an increase in P4 metabolism. However, whether this local luteolytic effect of LH is exerted directly on luteal cells or on other structures, such as follicular or stromal cells, to modify luteal function is unknown. The aim of the present study was to determine the effect of LH on isolated luteal cells obtained on Day 19 of pregnancy. Incubation of luteal cells with 1, 10, 100, or 1000 ng/ml of ovine LH (oLH) for 6 h did not modify basal P4 production. The addition to the culture medium of 22(R)-hydroxycholesterol (22R-HC, 10 microgram/ml), a membrane-permeable P4 precursor, or pregnenolone (10(-2) microM) induced a significant increase in P4 accumulation in the medium in relation to the control value. When luteal cells were preincubated for 2 h with oLH, a significant (p < 0.01) reduction in the 22R-HC- or pregnenolone-stimulated P4 accumulation was observed. Incubation of luteal cells with dibutyryl cAMP (1 mM, a cAMP analogue) plus isobutylmethylxanthine (1 mM, a phosphodiesterase inhibitor) also inhibited pregnenolone-stimulated P4 accumulation. Incubation with an inositol triphosphate synthesis inhibitor, neomycin (1 mM), or an inhibitor of intracellular Ca2+ mobilization, (8,9-N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate (1 mM), did not prevent the decrease in pregnenolone-stimulated P4 secretion induced by oLH. It was concluded that the luteolytic action of LH in late pregnancy is due, at least in part, to a direct action on the luteal cells and that an increase in intracellular cAMP level might mediate this effect.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Astrocytes synthesize and secrete the lipophilic ligand carrier apolipoprotein D.

Expression of the lipophilic ligand transporter, apolipoprotein D (apoD) by primary astrocyte cultures derived from neonatal mouse brain was investigated. Western blot analysis of cell lysates and media showed that apoD is constitutively secreted by astrocytes with little intracellular storage. The secreted apoD floated primarily at density 1.063-1.21 g ml-1 upon sequential ultracentrifugation indicating its association with lipids. Treatment of astrocytes with the carboxylic ionophore, monensin, resulted in intracellular retention and decreased secretion of apoD that was of slightly reduced M(r). Progesterone, a steroid hormone that binds to apoD with high affinity (10(-6) mol l-1) and the oxysterol, 25-hydroxycholesterol which is a potent regulator of cellular cholesterol homeostasis in mammalian cells, differentially stimulated apoD, but not apoE secretion. These results show that astrocytes synthesize and constitutively secrete apoD and suggest a physiologic role for this lipocalin in cholesterol metabolism in the nervous system.

Animals↗
PubMed2003

PPAR agonists protect mesangial cells from interleukin 1beta-induced intracellular lipid accumulation by activating the ABCA1 cholesterol efflux pathway.

Previous studies have demonstrated that inflammatory cytokines such as interleukin-1beta (IL-1beta) promote lipid accumulation in human mesangial cells (HMC) by dysregulating the expression of lipoprotein receptors. Intracellular lipid accumulation is governed by both influx and efflux; therefore, the effect of IL-1beta on the efflux of lipid from HMC was investigated. IL-1beta was shown to inhibit (3)H-cholesterol efflux from HMC and increase total intracellular cholesterol concentration, probably as a result of reduced expression of the adenosine triphosphate (ATP) binding cassette A1 (ABCA1), a transporter protein involved in apolipoprotein-A1 (apo-A1)-mediated lipid efflux. To ascertain the molecular mechanisms involved, expression of peroxisome proliferator-activated receptors (PPAR) and liver X receptoralpha (LXRalpha) were examined. IL-1beta (5 ng/ml) reduced PPARalpha, PPARgamma, and LXRalpha mRNA expression. Activation of PPARgamma with the agonist prostaglandin J2 (10 micro M) and of PPARalpha with either bezafibrate (100 micro M) or Wy14643 (100 micro M) both increased LXRalpha and ABCA1 gene expression also and enhanced apoA1-mediated cholesterol efflux from lipid-loaded cells, even in the presence of IL-1beta. A natural ligand of LXRalpha, 25-hydroxycholesterol (25-OHC), had similar effects; when used together with PPAR agonists, an additive effect was observed, indicating co-operation between PPAR and LXRalpha in regulating ABCA1 gene expression. This was supported by the observation that overexpression of either PPARalpha or PPARgamma by transfection enhanced LXRalpha and ABCA1 gene induction by PPAR agonists. Taken together with previous data, it appears that, in addition to increasing lipid uptake, inflammatory cytokines promote intracellular lipid accumulation by inhibiting cholesterol efflux through the PPAR-LXRalpha-ABCA1 pathway. These results suggest potential mechanisms whereby inflammation may exacerbate lipid-mediated cellular injury in the glomerulus and in other tissues and indicate that PPAR agonists may have a protective effect.

ATP-Binding Cassette Transporters↗
PubMed2025

Plasma membrane accessible cholesterol is regulated by ACC1 and lipid droplets.

Proper maintenance of plasma membrane (PM) cholesterol is essential for diverse processes ranging from animal development to pathogen evasion. Despite decades of study, the mechanisms governing cellular cholesterol regulation are incomplete. Using genome-wide screens we find that ACC1, the rate-limiting enzyme in fatty acid biosynthesis, regulates PM cholesterol transport. ACC1 loss causes a ~10-fold increase in PM accessible cholesterol in cells and mice. Mechanistically, we find that ACC1 regulates lipid droplet (LD) catabolism, and LDs are intimately tied to PM accessible cholesterol levels since reductions or elevations in their numbers block or promote cholesterol trafficking, respectively. Furthermore, LDs are required for cholesterol trafficking induced by 25-hydroxycholesterol, a modulator of inflammation and an interferon-stimulated second messenger that protects cells from pathogen invasion. This work identifies an unrecognized role for ACC1 and LDs in cholesterol regulation, which has implications for diseases where LD numbers are altered, from metabolic syndromes to neurodegeneration.

25-hydroxycholesterol↗
PubMed1984

Defective low-density lipoprotein metabolism in cultured, normal, transformed, and malignant keratinocytes.

To obtain more information on differences in cellular behavior during the differentiation process, a number of types of epithelial cells with and without a defect in cornified envelope formation were compared as to the regulation of intracellular cholesterol synthesis by low-density lipoprotein (LDL) and the uptake and degradation of [125I]LDL. The following cells were cultured: normal skin fibroblasts (F), normal (K) and SV 40 transformed (SVK14) keratinocytes, and a number of squamous carcinoma cell (SCC) lines in which the defective terminal differentiation was found to occur in the following order of intensity: SCC-12F2 less than SCC-25 approximately equal to SCC-15 less than SCC-12B2 less than SCC-4. Compared with normal human fibroblasts, most of the cells under study showed a defective response to changes of the extracellular serum LDL concentration. The degree of inducibility of cholesterol synthesis after the cells were deprived of extracellular sources of cholesterol as well as the degree of the LDL-induced suppression of the intracellular cholesterol synthesis in cells preincubated in medium supplemented with lipoprotein-deficient serum decreased in the following order: F greater than SCC-4 greater than SCC-15 approximately equal to SCC-25 greater than SCC-12B2 congruent to SCC-12F2 greater than SVK14 approximately equal to K. A defect in LDL metabolism was found to be responsible for the partial or complete failure of LDL to regulate the cholesterol metabolism, because when sterol was delivered to all cell types in artificial nonlipoprotein form (i.e., as 25-hydroxycholesterol) a marked suppression of cholesterol synthesis was observed. For all SCC lines tested except SCC-12B2 good correlation was found between the degree of LDL-induced suppression of cholesterol synthesis and the decreasing ability of cells to differentiate into squames or cornified envelope-forming cells. The transformation of keratinocytes by SV 40 virus did not lead to any change in the response of the cells to changes in the extracellular LDL concentration since both the normal and the transformed keratinocytes showed the same response to LDL (i.e., no response).

Carcinoma, Squamous Cell↗
PubMed2006

Biliary lipids, cholesterol and bile synthesis: different adaptive mechanisms to dietary cholesterol in lean and obese subjects.

BACKGROUND: Increased biliary cholesterol secretion together with elevated cholesterol synthesis may predispose obese subjects to cholesterol gallstone formation. AIM: To investigate whether processing of dietary cholesterol is altered in obesity, we enrolled eight lean and seven obese subjects in a double-blind crossover study. METHODS: Cholesterol consumption was 300 mg/day on low and 1300 mg/day on high cholesterol diet. After 3 weeks on either diet, hepatic bile was collected to determine biliary lipid secretion, and bile salt composition by high-performance liquid chromatography and cholesterol saturation index was calculated. Cholesterol synthesis was measured employing mass isotopomer distribution analysis. Bile acid synthesis via neutral and acidic pathway was assessed by serum levels of 7alpha-hydroxy-4-cholesten-3-one and 27-hydroxycholesterol. RESULTS: Cholesterol synthesis was increased in obese compared with lean and feedback inhibited only in obese. On low cholesterol diet, cholesterol secretion was doubled in obese but bile acid composition and synthesis was similar between the two groups. After high cholesterol diet, cholesterol saturation index and bile secretion were unchanged. In contrast to obese, lean increased bile acid synthesis only via the acidic pathway. CONCLUSIONS: Dietary cholesterol appears to preferentially induce bile acid synthesis via the acidic pathway in lean, whereas cholesterol synthesis was inhibited in obese. Thus, stable cholesterol saturation index may be achieved by different mechanisms.

Adult↗
PubMed2005

Lipid metabolism and TNF-alpha secretion in response to dietary sterols in human monocyte derived macrophages.

BACKGROUND: The postprandial phase is characterized by the circulation of atherogenic dietary-triacylglycerol rich lipoproteins. Little is known about the modulation of lipid and immune functions in macrophages by these particles or of the role of the oxysterols found in food such as 7beta-hydroxycholesterol and 7-ketocholesterol. MATERIALS AND METHODS: Human macrophages were tested with different concentrations of chylomicron remnant-like particles (CRLP) with or without incorporated oxysterols to study their uptake by the cells, and their effects on cholesteryl ester and triacylglycerol synthesis and the secretion of inflammatory mediators, including tumour necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6) and interleukin 10 (IL-10). RESULTS: Independently of the presence of oxysterols, CRLP caused cholesterol accumulation. However, the dose-dependent increase in [3H]cholesterol internalization by macrophages after incubation with [3H]cholesteryl ester-labelled CRLP was abolished by the presence of oxysterols in the particles. TNF-alpha secretion was decreased and that of IL-10 unaffected by CRLP independently of the presence of oxysterol. Exposure to CRLP containing 7beta-hydroxysterol, but not to CRLP or 7-ketosterol-containing CRLP, reduced IL-6 secretion with respect to cells not exposed to any particles. Because TNF-alpha levels have been related to scavenger receptor expression, we tested the uptake of modified LDL in macrophages exposed to human postprandial triacylglycerol-rich lipoproteins and found it to be markedly increased. CONCLUSIONS: Cholesterol loading as a result of dietary lipids depresses the inflammatory response of macrophages and the presence of 7beta-hydroxysterol may exacerbate this effect. In addition, exposure to dietary lipids enhances scavenger receptor activity in macrophages. These results suggest that changes induced by dietary lipids in human macrophage function are related to an increased propensity of the cells to accumulate lipids during the postprandial phase.

Cells, Cultured↗
PubMed1983

A simple model for studies on the regulation of cholesterol synthesis using freshly isolated hepatocytes.

Rat hepatocytes isolated by the procedure described here showed 3-hydroxy-3-methylglutaryl-CoA reductase activity in the range of that reported for rat liver at the maximum of the circadian cycle, even if they were taken from rats at the time of the minimum. The enzyme was present in cells as both its active dephosphorylated (20 +/- 8%) and the inactive phosphorylated forms. The enzyme activity and the ratio between the two forms were unaltered during 3 h of cell incubation. 25-Hydroxycholesterol (50 microM) induced about 50% inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase activity during 1 h incubation but the relative amount of the two forms was not modified by the sterol. Cells isolated by the described procedure may therefore be a useful tool in studies on the regulation of cholesterol neogenesis, both through the synthesis of the enzyme, which can be shown by measuring the activity after complete dephosphorylation of the enzyme, and via the rapid reversible shift of the inactive to the active form, resulting from the ratio between the two enzyme forms. The latter mechanism for the modulation of cholesterol synthesis cannot be tested in cell cultures because full activation of the enzyme occurs during hepatocyte plating.

Animals↗
PubMed1987

Overaccumulation of 3-hydroxy-3-methylglutaryl-coenzyme-A reductase in a compactin(ML-236B)-resistant mouse cell line with defects in the regulation of its activity.

Using a stepwise selection procedure, we have developed a line of mouse FM3A cells which is resistant to compactin (ML-236B), a competitive inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme-A (HMG-CoA) reductase. This line, designated CR200, expressed a stable phenotype of a greater than 100-fold higher resistance to the inhibitor and a 160-fold higher HMG-CoA reductase activity than did the parental FM3A cell line when grown in the presence of low-density lipoprotein (LDL). In contrast to its action in the parental cells, HMG-CoA reductase in CR200 cells was not suppressed by human LDL. The activity of both cell-surface binding and degradation of 125I-LDL was approximately 10% in CR200 cells as compared with that in parental cells. Affinity of the 125I-LDL binding in CR200 cells was similar to that in the parental cells. In addition, HMG-CoA reductase was not suppressed normally by mevalonate and 25-hydroxycholesterol in CR200 cells; suppression was 50-60% by both compounds under conditions where suppression was about 99% in the parental cells. Sterol synthesis from [3H]mevalonate was normal in the variant cells. These observations demonstrate two defects in CR200 cells: decreased levels of LDL receptor and a defective regulation of HMG-CoA reductase by one or more products derived from mevalonate. When grown without compactin for 21 weeks, the variant cells became slightly sensitive to compactin and concomitantly recovered partly from both defects. However, these phenotypes of CR200 cells were stable up to 15 months when exposed to compactin. These two defects may account for the overaccumulation of HMG-CoA reductase in CR200 cells and thereby for the resistance to compactin.

Animals↗
PubMed1989

Regulation of low-density-lipoprotein-receptor mRNA by insulin in human hepatoma Hep G2 cells.

The low-density-lipoprotein (LDL)-receptor mRNA content of human hepatoma (Hep G2) cells has been estimated from densitometric scans of autoradiograms obtained following the hybridisation of Northern blots of a poly(A)-rich RNA fraction with a 32P-labelled cDNA probe for the LDL-receptor gene. The recovery of beta-actin mRNA was used to correct for losses occurring during the preparation of the poly(A)-rich RNA. The content of LDL-receptor mRNA was reduced when the cells were pre-incubated in medium containing foetal calf serum, 25-hydroxycholesterol, or LDL, compared to that measured in cells which had been pre-incubated in medium containing lipoprotein-deficient serum (LPDS). When insulin (100 mU/ml) was included in pre-incubation medium containing LPDS, the amount of LDL-receptor mRNA increased approximately twofold. The level of beta-actin mRNA was not significantly increased by insulin treatment. Addition of insulin to incubation medium containing LPDS also overcame the suppressive effect of exogenous LDL on the cellular content of mRNA for the LDL receptor. These findings suggest that one action of insulin in these cells may be to promote transcription of the LDL-receptor gene by a mechanism that can override the sterol regulatory pathway.

Autoradiography↗
PubMed1993

Side-chain specificities of human and bovine cytochromes P-450scc.

Cytochrome P-450scc catalyses the conversion of cholesterol to pregnenolone by the sequential hydroxylation of the side chain of cholesterol. This occurs at a single active site and produces 22R-hydroxycholesterol and 22R-20 alpha-dihydroxycholesterol as intermediates. To further define the active site of human and bovine cytochromes P-450scc, we have examined the kinetics of the conversion of structural analogues of cholesterol with modified side chains, to pregnenolone. Analysis of the side-chain cleavage of analogues of cholesterol modified at C22 confirmed the high degree of structural specificity for the 22R position by cytochrome P-450scc, the major effect being on the turnover number (kcat) rather than on binding. The analogues of cholesterol that had a polar group at C24, C25 or C26 had much lower Km values and generally lower kcat values than the non-polar analogues which were tested. Km values of the polar analogues were 3-25-times lower than the Km for cholesterol and kcat values were also much lower than the kcat values for cholesterol, particularly for the human enzyme. The data suggest that the tight binding of the analogues with a hydroxyl or ketone group at C24, C25 or C26 places C20 and C22 in a poor orientation relative to the heme group for hydroxylation to occur. Many of the polar analogues which were tested are postulated regulators of cellular cholesterol metabolism. Several of these analogues are good substrates for bovine and human cytochromes P-450scc at low substrate concentration, as determined from their kcat/Km values. This study also indicates that the active site of cytochrome P-450scc is well conserved between bovine and human cytochromes. However, small species differences are evident since lower kcat values relative to the kcat of cholesterol are observed for some polar side-chain analogues of cholesterol with the human enzyme.

Adrenal Cortex↗
PubMed1991

Cholesterol content but not plasma membrane fluidity influences the susceptibility of L1210 leukemia cells to merocyanine 540-sensitized irradiation.

This paper examines the relationship between lipid composition, plasma membrane fluidity, expression of dye binding sites, and susceptibility to merocyanine 540 (MC540)-sensitized irradiation in L1210 leukemia cells. Reducing the cells' cholesterol content by exchange diffusion with phosphatidylcholine liposomes or by inhibiting its biosynthesis with 25-hydroxycholesterol enhanced plasma membrane fluidity, the expression of dye binding sites, and the cells' susceptibility to MC540-sensitized irradiation. Conversely, if the cholesterol content was enhanced by exchange diffusion with cholesterol:phosphatidylcholine liposomes, the cells' susceptibility to MC540-sensitized irradiation was decreased. However, contrary to expectations, dye-binding was slightly enhanced and plasma membrane fluidity remained unchanged. Growing the cells in fatty acid-supplemented medium had profound effects on their lipid composition. Cells enriched in polyunsaturated fatty acids had more fluid plasma membranes. However, dye-binding was not significantly affected and photosensitivity was slightly reduced. These results suggest that cholesterol is one, but probably not the only, determinant of the expression of cellular dye binding sites and, consequently, the cell's susceptibility to MC540-sensitized irradiation. By contrast, plasma membrane fluidity does not appear to play a major role in the regulation of dye-binding site expression.

Animals↗
PubMed2004

Transcriptional regulation of human CYP27 integrates retinoid, peroxisome proliferator-activated receptor, and liver X receptor signaling in macrophages.

Cholesterol uptake and efflux are key metabolic processes associated with macrophage physiology and atherosclerosis. Peroxisome proliferator-activated receptor gamma (PPARgamma) and liver X receptor alpha (LXRalpha) have been linked to the regulation of these processes. It remains to be identified how activation of these receptors is connected and regulated by endogenous lipid molecules. We identified CYP27, a p450 enzyme, as a link between retinoid, PPARgamma, and LXR signaling. We show that the human CYP27 gene is under coupled regulation by retinoids and ligands of PPARs via a PPAR-retinoic acid receptor response element in its promoter. Induction of the enzyme's expression results in an increased level of 27-hydroxycholesterol and upregulation of LXR-mediated processes. Upregulated CYP27 activity also leads to LXR-independent elimination of CYP27 metabolites as an alternative means of cholesterol efflux. Moreover, human macrophage-rich atherosclerotic lesions have an increased level of retinoid-, PPARgamma-, and LXR-regulated gene expression and also enhanced CYP27 levels. Our findings suggest that nuclear receptor-regulated CYP27 expression is likely to be a key integrator of retinoic acid receptor-PPARgamma-LXR signaling, relying on natural ligands and contributing to lipid metabolism in macrophages.

Animals↗
PubMed1985

Sterols accelerate degradation of hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase encoded by a constitutively expressed cDNA.

A recombinant plasmid containing a full-length cDNA for hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase was introduced by calcium phosphate-mediated transfection into UT-2 cells, a mutant line of Chinese hamster ovary cells that lack 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus require low density lipoprotein-cholesterol and mevalonate for growth. We selected a line of permanently transfected cells, designated TR-36 cells, that expressed high levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus grew in the absence of low density lipoprotein and mevalonate. Constitutive synthesis of reductase mRNA in TR-36 cells was driven by the simian virus 40 early promoter, and therefore the mRNA was not suppressed by sterols, such as 25-hydroxycholesterol or cholesterol derived from low density lipoprotein, which normally suppresses transcription of reductase mRNA when the reductase gene is driven by its own promoter. Although TR-36 cells continued to synthesize large amounts of reductase mRNA and protein in the presence of sterols, reductase activity declined by 50 to 60%. This decline was caused by a twofold increase in the rate of degradation of preformed enzyme molecules. The current data demonstrate that sterols accelerate the degradation of reductase protein independently of any inhibitory effect on the synthesis of the protein.

Animals↗