| For medical practitioners and the general public - Cholesterol Journal Article Catalog. |
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| Action of ciprofibrate in type IIb hyperlipoproteinemia: modulation of the atherogenic lipoprotein phenotype and stimulation of high-density lipoprotein-mediated cellular cholesterol efflux Guerin, M., W. Le Goff, et al. (2003), J Clin Endocrinol Metab 88(8): 3738-46. Abstract: The effects of ciprofibrate (100 mg/d) on apolipoprotein (apo)B- and apoAI-containing lipoprotein subclasses, cholesteryl ester (CE) transfer protein activity, and plasma high-density lipoprotein (HDL)-mediated cellular cholesterol efflux were evaluated in 10 patients displaying type IIB hyperlipidemia. Plasma concentrations of large very low-density lipoprotein (VLDL)-1 (Sf 60-400) and of small VLDL-2 (Sf 20-60) were markedly diminished after fibrate treatment (-40%, P = 0.001; and -25%, P = 0.003, respectively). We observed a reduction (-17%; P = 0.005) in plasma low-density lipoprotein (LDL) levels resulting from significant reductions in concentrations of dense LDL particles (-46%; P < 0.0001). Ciprofibrate induced elevation in plasma total HDL (+13%; P = 0.005) levels; such elevation occurred preferentially in HDL-3 (+22%; P = 0.009). Marked reduction in numbers of atherogenic apoB100-containing particle acceptors was associated with a 25% decrease (P < 0.02) in CE transfer protein-mediated CE transfer from HDL. Finally, a significant fibrate-mediated elevation (+13%; P = 0.01 compared with baseline) in the capacity of plasma from type IIB subjects to mediate free cholesterol efflux from scavenger receptor class B, type I-expressing Fu5AH hepatoma cells was observed. In conclusion, the action of ciprofibrate in type IIB dyslipidemia leads to preferential reduction in particle numbers of atherogenic VLDL-1, VLDL-2, and dense LDL and, concomitantly, to elevation in HDL-3 levels that are associated with stimulation of HDL-mediated cellular free cholesterol efflux through the scavenger receptor class B, type I receptor pathway. |
| Action of lovastatin--an inhibitor of cholesterol biosynthesis on bacterial bioluminescence Baranova, N. A., V. G. Kreier, et al. (1995), Antibiot Khimioter 40(8): 12-6. Abstract: The action of lovastatin, a competing inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase, on bacterial bioluminescence was studied. The lovastatin lactone form and sodium salt of mevinolinic acid inhibited bacterial luciferase in vitro but did not affect bioluminescence of the intact cells of the luminous bacteria. The inhibition was found to be of a competing character in regard to aliphatic aldehyde, the bacterial luciferase substrate. Conditions under which the bioluminescence inhibition was proportional to the lovastatin concentration in the incubation mixture and a bioluminescence method for quantitative determination of the inhibitor were developed. |
| Action of melittin on the DPPC-cholesterol liquid-ordered phase: a solid state 2H-and 31P-NMR study Pott, T. and E. J. Dufourc (1995), Biophys J 68(3): 965-77. Abstract: Solid-state deuterium and phosphorus-31 nuclear magnetic resonance studies of deuterium-labeled beta--2,2',3,4,4',6-2H6-cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine have been undertaken to monitor the action of melittin on model membranes containing 30 mol% cholesterol, both at the molecular and macroscopic level. Cholesterol totally inhibits the toxin-triggered formation of large unilamellar vesicles and strongly restricts the appearance of small discs. The latter remain stable over a wide temperature range (20-60 degrees C) because of an increase in their cholesterol content as the temperature increases. This process is related to a constant disc hydrophobic thickness of approximately 29 A. The system, when not in the form of discs, appears to be composed of very large vesicles on which melittin promotes magnetically induced ellipsoidal deformation. This deformation is the greatest when the maximum of discs is observed. A model to describe both the disc formation and stability is proposed. |
| Activation and cholesterol accumulation of macrophages induced by hypercholesterolemia. A study using a rat peritoneal macrophage model for extravascular in vivo generation of foam cells Fan, J., T. Shimokama, et al. (1994), Pathobiology 62(1): 1-7. Abstract: Intimal infiltration of lipid-filled macrophages (M psi s) is an important event in the pathogenesis of atherosclerosis. To better understand M psi functions, a model for the extravascular in vivo generation of foam cells in rat peritoneal cavities was utilized. Morphologic alterations, intracellular cholesterol accumulation, subpopulation, activation and proliferative properties of M psi from hypercholesterolemic rats (HM psi s) were compared with M psi s from normal rats (NM psi s). HM psi s revealed a significant increase of cholesterol mass in the cytoplasm; 65% of HM psi s were loaded with various amounts of oil-red-O-stainable lipid droplets which were barely identified in NM psi s. Ultrastructurally, accumulated lipid droplets in HM psi s were either membrane-bound or membrane-free in the cytoplasm. Further biochemical analysis revealed that cellular levels of total cholesterol, free cholesterol, and cholesteryl esters in HM psi s were increased 6-, 8-, and 4-fold, respectively. As to the M psi subpopulation, there was a significant increase of Ia-antigen-positive cells in HM psi s (15.8 vs. 8.8%), indicating that these cells were in a state of activation. To investigate the mitotic activity, the proliferative potential of M psi s was determined both in vivo and in vitro using monoclonal anti-bromodeoxyuridine (anti-BrdU) antibody to detect BrdU incorporated into cell DNA. However, both NM psi s and HM psi s showed little proliferation; proliferative indices were less than 2%. This implies that M psi s are barely replicating, and hypercholesterolemia does not stimulate M psi s in this aspect.(ABSTRACT TRUNCATED AT 250 WORDS) |
| Activation of acyl-CoA cholesterol acyltransferase: redistribution in microsomal fragments of cholesterol and its facilitated movement by methyl-beta-cyclodextrin Cheng, D. and C. L. Tipton (1999), Lipids 34(3): 261-8. Abstract: Acyl-CoA cholesterol acyltransferase (ACAT) (EC 2.3.1.26) in the yolk sac membrane of chicken eggs plays an important role in the transport of lipids, which serve as both structural components and as an energy source during embryogenesis. ACAT from the yolk sac membrane of chicken eggs 16 d after fertilization has higher activity and better stability than its mammalian liver counterpart. During our study of the avian enzyme, ACAT was found to be activated up to twofold during storage at 4 degrees C. The activation was investigated, and data suggest that redistribution of cholesterol within microsomal vesicles leads to the increase. Methyl-beta-cyclodextrin (MbetaCD) increases activation an additional twofold, possibly by facilitating the movement of cholesterol within microsomal fragments and allowing redistribution of cholesterol in lipid bilayers to a greater extent. Treatment of microsomes with MbetaCD removes cholesterol from the membranes. Controlled amounts of cholesterol can be restored to the membranes by mixing them with cholesterol-phosphatidylcholine liposomes in the presence of MbetaCD. Under these conditions, the plot of ACAT vs. cholesterol mole fraction in the liposomes is sigmoidal. The finding that MbetaCD can enhance cholesterol transfer between liposomes and microsomes and reduce the limitation of slow movement of nonpolar molecules in aqueous media should make cyclodextrins more useful in in vitro studies of apolar molecule transport between membrane vesicles. |
| Activation of acyl-CoA: cholesterol acyltransferase in rat liver microsomes by 25-hydroxycholesterol Bhuvaneswaran, C., S. Synouri-Vrettakou, et al. (1997), Biochem Pharmacol 53(1): 27-34. Abstract: 25-Hydroxycholesterol stimulated acyl-CoA:cholesterol acyltransferase (ACAT) activity in rat liver microsomes in vitro with half-maximal stimulation at 16.8 microM oxysterol and a maximal activity that was three times that in its absence. The current study was conducted to determine the effect of 25-hydroxycholesterol on rates and extent of intervesicular cholesterol transfers within microsomes and to determine whether this activation of ACAT could be accounted for on the basis of increased cholesterol availability for the enzyme. Cholesterol transfer kinetics were assessed in systems that either enriched or depleted microsomal cholesterol. Incubation of microsomes at 37 degrees C with phosphatidylcholine:cholesterol liposomes or purified plasma membranes resulted in enrichment of microsomal cholesterol. Incubation of microsomes with just phosphatidylcholine liposomes resulted in depletion of cholesterol. The extent of cholesterol enrichment or depletion depended on incubation time and the initial concentration of cholesterol in donor and acceptor vesicles. The rate and extent of cholesterol transfer from liposomes to microsomes were slightly increased when 25-hydroxycholesterol was present during the transfer process. Irrespective of the treatment, 25-hydroxycholesterol continued to stimulate the ACAT activity of the treated microsomes. Microsomes that were enriched or depleted of cholesterol in the absence of 25-hydroxycholesterol yielded as much enzyme activities when assayed in the presence of 25-hydroxycholesterol as with the systems that contained 25-hydroxycholesterol during both the transfer process and enzyme assays. The results suggest that a major part of the activation of microsomal ACAT by 25-hydroxycholesterol is not ascribable to increased substrate availability for the enzyme. |
| Activation of acyl-coenzyme A:cholesterol acyltransferase activity by cholesterol is not due to altered mRNA levels in HepG2 cells Matsuda, H., H. Hakamata, et al. (1996), Biochim Biophys Acta 1301(1-2): 76-84. Abstract: Many studies have shown that sterols can stimulate acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in cells. To elucidate this mechanism, effects of sterol-mediated induction on both the enzyme activity of ACAT and its mRNA levels were studied in human hepatoblastoma cell line, HepG2 cells. When HepG2 cells were loaded with cholesterol and 25-hydroxycholesterol, both the whole-cell ACAT activity and the microsomal ACAT activity were increased by 85.1% and 41.3%. In contrast, cholesterol depletion of HepG2 cells with compactin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, resulted in a decrease in both the whole-cell and the microsomal ACAT activity by 46.4% and 58.3%. Under identical conditions, RT-PCR and Northern blotting analyses revealed that neither cholesterol loading nor cholesterol depletion of HepG2 cells altered the amounts of ACAT mRNA. Moreover, these treatments had no effect on the enzymatic ACAT activity determined by the reconstituted assay in which HepG2 cell homogenate had been supplemented in vitro with a saturating level of exogenous cholesterol. These results indicate that cholesterol-induced up-regulation of ACAT activity in HepG2 cells does not occur at the level of transcription, but rather at a posttranscriptional level. |
| Activation of acyl-coenzyme A:cholesterol acyltransferase by cholesterol or by oxysterol in a cell-free system Cheng, D., C. C. Chang, et al. (1995), J Biol Chem 270(2): 685-95. Abstract: Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the conjugation of long chain fatty acid and cholesterol to form cholesteryl esters. It is an integral membrane protein located in the endoplasmic reticulum. Experiments performed in intact mammalian cells have shown that the rate of cholesteryl ester synthesis in intact cells, as well as the ACAT activity from cell extracts, are greatly activated by the addition of low density lipoprotein (LDL) or oxygenated sterols such as 25-hydroxycholesterol to the growth medium. However, the molecular mechanism(s) by which sterol(s) stimulate the ACAT activity remains to be elucidated. Recently, our laboratory reported the expression cloning of human ACAT cDNA (Chang, C. C. Y., Huh, H. Y., Cadigan, K. M., and Chang, T. Y. 1993) J. Biol. Chem. 268, 20747-20755). In the current study, we report the expression of human ACAT cDNA in insect Sf9 cells. Uninfected Sf9 cells do not express detectable ACAT-like activity. Infecting these cells with recombinant virus containing ACAT cDNA caused these cells to express high levels of ACAT protein and high levels of ACAT activity when assayed in vitro. The catalytic properties of ACAT expressed in these cells were found to be similar to those found in human tissue culture cells. The combination of high level of ACAT protein expression and the low level of cellular cholesterol content in the infected cells have provided us a novel opportunity to establish a simple cell-free system, whereby stimulation of ACAT by sterols can be readily demonstrated. Using this system, we have shown that cholesterol itself can serve as an ACAT activator in vitro, in addition to its role as an ACAT substrate. The current work provides the experimental basis to hypothesize that, inside mammalian cells, cholesterol itself may serve as a physiological regulator of ACAT. |
| Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2 Horton, J. D., I. Shimomura, et al. (1998), J Clin Invest 101(11): 2331-9. Abstract: We produced transgenic mice that express a dominant-positive truncated form of sterol regulatory element-binding protein-2 (SREBP-2) in liver and adipose tissue. The encoded protein lacks the membrane-binding and COOH-terminal regulatory domains, and it is therefore not susceptible to negative regulation by cholesterol. Livers from the transgenic mice showed increases in mRNAs encoding multiple enzymes of cholesterol biosynthesis, the LDL receptor, and fatty acid biosynthesis. The elevations in mRNA for 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase were especially marked (13-fold and 75-fold, respectively). As a result, the transgenic livers showed a 28-fold increase in the rate of cholesterol synthesis and a lesser fourfold increase in fatty acid synthesis, as measured by intraperitoneal injection of 3Hwater. These results contrast with previously reported effects of dominant-positive SREBP-1a, which activated fatty acid synthesis more than cholesterol synthesis. In adipose tissue of the SREBP-2 transgenics, the mRNAs for cholesterol biosynthetic enzymes were elevated, but the mRNAs for fatty acid biosynthetic enzymes were not. We conclude that SREBP-2 is a relatively selective activator of cholesterol synthesis, as opposed to fatty acid synthesis, in liver and adipose tissue of mice. |
| Activation of C-kinase eta through its cholesterol-3-sulfate-dependent phosphorylation by casein kinase I in vitro Okano, M., T. Yokoyama, et al. (2004), Biol Pharm Bull 27(1): 109-12. Abstract: The physiological correlation between casein kinase I (CK-I) and an isoform eta of protein kinase C (C-kinase eta) was investigated in vitro, since it has been reported that (i) cholesterol-3-sulfate (CH-3S) effectively activates C-kinase eta rather than the other isoforms (C-kinase epsilon and C-kinase delta) in vitro; and (ii) CK-I efficiently phosphorylates CH-3S-binding proteins, such as high mobility group protein 1 (HMG1), in the presence of CH-3S in vitro. We found that (i) CK-I phosphorylated Thr in preference to Ser on recombinant human C-kinase isoform eta (rhC-kinase eta) in the presence of CH-3S; (ii) this phosphorylation was selectively inhibited by CK-I-7 (a CK-I inhibitor); and (iii) the activity (phosphorylation of protamine sulfate) of rhC-kinase eta was approx. 3.2-fold stimulated by its full phosphorylation by CK-I in the presence of 3 microM CH-3S. These results suggest that CK-I is a protein kinase responsible for the activation of rhC-kinase eta in the presence of CH-3S in vitro. |
| Activation of IP(3)-protein kinase C-alpha signal transduction pathway precedes the changes of plasma cholesterol, hepatic lipid metabolism and induction of low-density lipoprotein receptor expression in 17-beta-oestradiol-treated rats Marino, M., E. Distefano, et al. (2001), Exp Physiol 86(1): 39-45. Abstract: The intracellular concentration of cholesterol is regulated by the balance between endogenous synthesis and exogenous uptake. Oestrogens have been reported to be involved in the physiological regulation of cellular cholesterol content. Relevant reports have focused on long-term responses and there is a lack of information about the relationship between the timing of the oestrogen effect and the regulation of cholesterol homeostasis. The aim of this work has been to set up a systematic picture of the short-term effects induced by oestrogen on hepatic lipid metabolism in vivo and the involvement of some relevant signal transduction pathways. At intervals after oestrogen administration (30 min to 6 h), oestrogen receptor expression and changes in liver cAMP, IP(3) and protein kinase C-alpha (PKC-alpha) were followed. Changes in the expression of the low density lipoprotein receptor at mRNA and protein levels, and of hydroxy-methyl-glutaryl-CoA reductase activity have been verified. At the same time, the content of hepatic cholesterol, ubiquinone and dolichol and of plasma cholesterol have been determined. Changes of rab 5 and rab 8, small GTP-binding prenylated proteins involved in the transfer of neosynthesised proteins through the cell, have been also checked. In vivo treatment with oestradiol produced no change in cyclic AMP but a rapid increase in IP(3), increased PKC-alpha localisation on the membranes and enhanced expression of the low density lipoprotein receptor in the liver occurred. PKC inhibition completely prevented any increase in low density lipoprotein receptor mRNA in isolated and perfused rat liver. Early changes of ubiquinone and dolichol content and a later reduction in hepatic hydroxy-methyl-glutaryl-CoA reductase activity and plasma cholesterol content were also detectable. A functional role of the IP(3) -protein kinase C-alpha pathway in the induction of the low density lipoprotein receptor is suggested. Experimental Physiology (2001) 86.1, 39-45. |
| Activation of mast cells by incorporation of cholesterol into rafts Baumruker, T., R. Csonga, et al. (2003), Int Immunol 15(10): 1207-18. Abstract: IgE plus antigen-stimulated mast cells degranulate, synthesize leukotrienes and secrete cytokines. According to the coalescence model this process is initiated in specific membrane compartments termed rafts. There, enhanced levels of glycosphingolipids and cholesterol stabilize the interaction of FcepsilonRI and Lyn, and thus facilitate the first steps of signal transduction. Enforced changes in raft architecture by cholesterol deprivation and exogenous application of glycosphingolipids influence these early events by loss of tyrosine kinase activity or receptor-independent signal initiation respectively. Here we show that exogenously added cholesterol accumulates in rafts and activates mast cells. An investigation of the signaling events reveals that in contrast to IgE plus antigen-mediated stimulation, cholesterol triggers p38 mitogen-activated protein kinase and preferentially induces expression of FosB. Consequently, a comparative large-scale microarray analysis demonstrates that a number of IgE plus antigen-induced immediate early genes (peak expression at 30 min after induction) are repressed by cholesterol. These changes further translate into altered expression levels and time kinetics of a number of early genes (peak expression at 2 h after stimulation). As the most prominent example for cholesterol-dependent genes, we identified PAI1 (plasminogen activator inhibitor 1), a protein regarded as a risk factor for atherosclerosis. |
| Activation of microglial poly(ADP-ribose)-polymerase-1 by cholesterol breakdown products during neuroinflammation: a link between demyelination and neuronal damage Diestel, A., O. Aktas, et al. (2003), J Exp Med 198(11): 1729-40. Abstract: Multiple sclerosis (MS) is a chronic demyelinating disease in which it has only recently been suggested that damage to neuronal structures plays a key role. Here, we uncovered a link between the release of lipid breakdown products, found in the brain and cerebrospinal fluid (CSF) of MS patients as well as in experimental autoimmune encephalomyelitis, and neuronal damage mediated by microglial activation. The concentrations of the breakdown product 7-ketocholesterol detected in the CSF of MS patients were capable of inducing neuronal damage via the activation and migration of microglial cells in living brain tissue. 7-ketocholesterol rapidly entered the nucleus and activated poly(ADP-ribose)-polymerase (PARP)-1, followed by the expression of migration-regulating integrins CD11a and intercellular adhesion molecule 1. These findings reveal a novel mechanism linking demyelination and progressive neuronal damage, which might represent an underlying insidious process driving disease beyond a primary white matter phenomenon and rendering the microglial PARP-1 a possible antiinflammatory therapeutic target. |
| Activation of phosphatidylinositol-specific phospholipase C by HDL-associated lysosphingolipid. Involvement in mitogenesis but not in cholesterol efflux Nofer, J. R., M. Fobker, et al. (2000), Biochemistry 39(49): 15199-207. Abstract: Our earlier studies demonstrated that high-density lipoproteins (HDLs) stimulate multiple signaling pathways, including activation of phosphatidylcholine-specific phospholipases C and D (PC-PLs) and phosphatidylinositol-specific phospholipase C (PI-PLC). However, only activation of PC-PLs was linked to the HDL-induced cholesterol efflux. In the study presented here, the role of HDL-induced PI-PLC activation was studied. In human skin fibroblasts, HDL potently induced PI-PLC as inferred from enhanced phosphatidylinositol bisphosphate (PtdInsP(2)) turnover and Ca(2+) mobilization. The major protein component of HDL, apo A-I, did not induce PtdInsP(2) turnover or Ca(2+) mobilization in these cells. Both HDL and apo A-I promoted cellular cholesterol efflux, whereas only HDL induced fibroblast proliferation. Inhibition of PI-PLC with U73122 or blocking intracellular Ca(2+) elevation with Ni(2+) or EGTA markedly reduced the extent of HDL-induced cell proliferation but had no effect on cholesterol efflux. In fibroblasts from patients with Tangier disease which are characterized by defective cholesterol efflux, neither HDL-induced PtdInsP(2) breakdown and Ca(2+) mobilization nor cell proliferation was impaired. HDL-induced fibroblast proliferation, PtdInsP(2) turnover, and Ca(2+) mobilization were fully mimicked by the lipid fraction isolated from HDL. Analysis of this fraction with high-performance liquid chromatography (HPLC) and time-of-flight secondary ion mass spectroscopy (TOF-SIMS) revealed that the PI-PLC-inducing activity is identical with two bioactive lysosphingolipids, namely, lysosulfatide (LSF) and sphingosylphosphorylcholine (SPC). Like native HDL, LSF and SPC induced PtdInsP(2) turnover, Ca(2+) mobilization, and fibroblast proliferation. However, both compounds did not promote cholesterol efflux. In conclusion, two agonist activities are carried by HDL. Apo A-I stimulates phosphatidylcholine breakdown and thereby facilitates cholesterol efflux, whereas LSF and SPC trigger PI-PLC activation and thereby stimulate cell proliferation. |
| Activation of the cholesterol pathway and Ras maturation in response to stress Shack, S., M. Gorospe, et al. (1999), Oncogene 18(44): 6021-8. Abstract: All cells depend on sterols and isoprenoids derived from mevalonate (MVA) for growth, differentiation, and maintenance of homeostatic functions. In plants, environmental insults like heat and sunlight trigger the synthesis of isoprene, also derived from MVA, and this phenomenon has been associated with enhanced tolerance to heat. Here, we show that in human prostate adenocarcinoma PC-3M cells heat shock leads to activation of the MVA pathway. This is characterized by a dose- and time-dependent elevation in 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) activity, enhanced sterol and isoprenoid synthesis, and increased protein prenylation. Furthermore, prenylation and subsequent membrane localization of Ras, a central player in cell signaling, was rapidly induced following heat stress. These effects were dose-dependent, augmented with repeated insults, and were prevented by culturing cells in the presence of lovastatin, a competitive inhibitor of HMGR. Enhanced Ras maturation by heat stress was also associated with a heightened activation of extracellular signal-regulated kinase (ERK), a key mediator of both mitogenic and stress signaling pathways, in response to subsequent growth factor stimulation. Thus, activation of the MVA pathway may constitute an important adaptive host response to stress, and have significant implications to carcinogenesis. |
| Activation of the silent endogenous cholesterol-7-alpha-hydroxylase gene in rat hepatoma cells: a new complementation group having resistance to 25-hydroxycholesterol Leighton, J. K., S. Dueland, et al. (1991), Mol Cell Biol 11(4): 2049-56. Abstract: The oxysterol 25-hydroxycholesterol acts both as a regulatory sterol determining the expression of genes governed by sterol regulatory elements and as a substrate for 7-alpha-hydroxylase, the first and rate-limiting enzyme in the bile acid synthetic pathway. Most wild-type nonhepatic cells are killed by the cytotoxic action of 25-hydroxycholesterol. In contrast, liver cells, which express 7-alpha-hydroxylase activity, are resistant to killing by 25-hydroxycholesterol. We examined the possibility that selection for resistance to 25-hydroxycholesterol might lead to the derivation of a cell line expressing 7-alpha-hydroxylase. A rat hepatoma cell line (7-alpha-hydroxylase minus) was transfected with human DNA and screened for resistance to 25-hydroxycholesterol. Although parental hepatoma cells were all killed within a week, a 25-hydroxycholesterol-resistant cell line (L35 cells) which showed stable expression of 7-alpha-hydroxylase activity and mRNA was obtained. These cells exhibited normal inhibition of cholesterol biosynthesis by 25-hydroxycholesterol. Blocking 7-alpha-hydroxylase activity with ketoconazole also blocked the resistance of L35 cells to 25-hydroxycholesterol. Isolation of microsomes from these cells showed levels of 7-alpha-hydroxylase activity (22.9 pmol/min/mg of protein) that were comparable to the activity (33.2 pmol/min/mg) of microsomes isolated from the livers of rats killed during the high point of the diurnal cycle. Parental cells had no detectable activity. These data show a new complementation group for 25-hydroxycholesterol resistance: expression of 7-alpha-hydroxylase. Dexamethasone increased both the activity and the cellular content of mRNA coding for 7-alpha-hydroxylase. Since dactinomycin blocked the ability of dexamethasone to induce mRNA, active transcription is required. Southern analysis of genomic DNA showed that L35 cells contain the rat (endogenous) gene but not the human gene. Furthermore, the RNA expressed by L35 cells is similar in size to rat RNA and is distinct from the human form of 7-alpha-hydroxylase. The combined data indicate that L35 cells are resistant to 25-hydroxycholesterol because they express 7-alpha-hydroxylase. The mechanism responsible involves activation of the endogenous (silent) gene of the parental rat hepatoma cell. |
| Active cholesterol biosynthesis in cultured aortic smooth muscle cells: evolution during the life-span of the culture Tabacik, C., J. P. Valentin, et al. (1991), Atherosclerosis 86(2-3): 123-37. Abstract: Cultured aortic smooth muscle cells from rabbit, in synthetic and contractile state, are considered good models for studying pathological and normal cells, respectively, during the atherosclerotic process. Cholesterogenic activity was compared in cells which were obtained in both states of the same subculture and incubated with labeled sodium acetate. This activity (expressed as the percentage of total cell radioactivity uptake transformed into cholesterol) was very high in synthetic cells and comparable to that of cancer cells. Cholesterol synthesis was lower in contractile cells and similar to that observed in a nonpathological cultured cell. During the cell life-span (studied in two cultures) cholesterogenic activity initially increased and then slowly decreased, in the two phenotypic states. Near the end of the culture life, cholesterol production drastically decreased, but this was due to a blocking of the last steps, lanosterol demethylation and C27 sterol transformation into cholesterol, rather than to a sharp decrease in the first steps of the cholesterogenic process. Cells in the synthetic and contractile states released newly synthesized lipids which were essentially late precursors of cholesterol, but accumulation of oxy-sterols was not observed. The excretion of metabolites increased with culture aging. |
| Active taurocholic acid flux through hepatoma cells increases the cellular pool of unesterified cholesterol derived from lipoproteins Li, Q., S. Yokoyama, et al. (2002), Biochim Biophys Acta 1580(1): 22-30. Abstract: The effect of bile acid flux on the fate of lipoprotein-derived cholesterol was studied in bile acid-transporting McNtcp.18 hepatoma cells. The intracellular unesterified cholesterol (UC) concentration rose when McNtcp.18 cells grown in the presence of either high density lipoproteins (HDL) or low density lipoproteins (LDL) were incubated with taurocholic acid (TCA). This effect was more pronounced when the exogenous source of cholesterol was HDL. The presence of TCA in the culture medium of McNtcp.18 cells had no discernible effect on the uptake of cholesteryl esters (CE) from either lipoprotein. TCA treatment of cells preincubated with either lipoprotein did not affect cholesterol synthesis but antagonized the stimulation of cholesterol esterification in cells that were incubated with LDL. The CE concentration in cells treated with TCA was decreased, relative to cells not incubated with TCA, suggesting that cellular CE stores were also hydrolyzed. The TCA treatment reduced the amount of total cholesterol released into the medium by the lipoprotein-treated cells, which was coincident with the reduction in the amount of apolipoprotein B in the culture medium. However, the proportion of UC released into the medium by the lipoprotein-treated cells was increased in cells capable of active bile acid transport. The results indicate that active bile acid flux through hepatoma cells increases the cellular pool of UC derived from lipoproteins. The UC released by the cells into the culture medium under this condition may represent cholesterol destined for direct biliary secretion. |
| Active-site topology of bovine cholesterol side-chain cleavage cytochrome P450 (P450scc) and evidence for interaction of tyrosine 94 with the side chain of cholesterol Pikuleva, I. A., R. L. Mackman, et al. (1995), Arch Biochem Biophys 322(1): 189-97. Abstract: Combining site-directed mutagenesis with analysis of the active-site topology of bovine cholesterol side-chain cleavage cytochrome P450scc (P450scc), we have investigated the roles of tyrosine residues 93 and 94 on substrate binding. Four single mutants (Y93A, Y93S, Y94A, and Y94S) and one double mutant (Y93S/Y94S) were examined. The largest increase in Ks was observed for binding of cholesterol and 25-hydroxycholesterol to the Y94S mutant (approximately 5.5-fold), with a smaller increase (< 2.5-fold) for binding of 22-hydroxycholesterol. Mutation of Y94 thus appears to influence the interaction with cholesterol, 25-hydroxycholesterol, and possibly 22-hydroxycholesterol. Y93 is not involved in binding of 22- and 25-hydroxycholesterol but may interact with cholesterol. The active-site topologies of P450scc and its mutants were probed by reaction with three arylhydrazines. The N-arylprotoporphyrin IX regioisomer patterns obtained with phenyl- and 2-naphthylhydrazine indicate that the active site is primarily open above pyrrole ring A and suggest that a region some distance above pyrrole ring D is also open. The single mutations Y93S, Y93A, Y94A, and Y94S do not detectably alter the regioisomer patterns obtained with the phenyl- and 2-naphthyl probes, but a small, reproducible change is observed with the 2-naphthyl probe for the Y93S/Y94S double mutant. The conformational alteration implied by this change could not be detected by titration with 22- and 25-hydroxycholesterol but is detectable by titration with cholesterol. The results indicate that cholesterol binds over pyrrole ring D of the heme in bovine P450scc, strongly suggest that Y94 interacts with the side chain of cholesterol, and provide evidence that the side chains of 22- and 25-hydroxycholesterol bind to a different region of the active site than the side chain of cholesterol. |
| Activities of lipoprotein lipase and hepatic triglyceride lipase in postheparin plasma of patients with low concentrations of HDL cholesterol Blades, B., G. L. Vega, et al. (1993), Arterioscler Thromb 13(8): 1227-35. Abstract: Previous investigations have shown that abnormalities in the postheparin plasma levels of the lipolytic enzymes, lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL), are correlated with variations in plasma high-density lipoprotein cholesterol (HDL-C) levels. The present study was performed to determine correlations between the postheparin plasma activities of these two enzymes and HDL levels in a sizable number of subjects with low HDL-C levels. Two types of low-HDL subjects were investigated: 159 male subjects with low HDL-C (< 40 mg/dL) and normal triglyceride (< 250 mg/dL) levels (the low-HDL group) and 80 male subjects with low HDL-C (< 40 mg/dL) and elevated triglyceride (> or = 250 mg/dL) levels (the low-HDL/high-TG group). Postheparin plasma activities of LPL and HTGL were determined in these two groups, and these levels were compared with those obtained from 51 normolipidemic (normal-HDL) male subjects. Postheparin LPL activities were significantly lower in the low-HDL and low-HDL/high-TG groups (mean +/- SD, 9.9 +/- 2.9 and 10.4 +/- 3.0 mmol/h per liter, respectively; P <.001 for both) compared with the normal-HDL group (12.5 +/- 3.7 mmol/h per liter). Conversely, postheparin HTGL activities were significantly higher in the low-HDL and low-HDL/high-TG groups (39.3 +/- 16.2 and 44.4 +/- 16.7 mmol/h per liter, respectively; P <.001 for both) compared with the normal-HDL group (29.7 +/- 11.3 mmol/h per liter). Consequently, mean LPL/HTGL ratios were markedly lower in the two low-HDL groups compared with the normal-HDL group.(ABSTRACT TRUNCATED AT 250 WORDS) |