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PubMed1987

Low density lipoprotein (LDL)-mediated suppression of cholesterol synthesis and LDL uptake is defective in Niemann-Pick type C fibroblasts.

One characteristic of type C Niemann-Pick (NPC) disease is the substantial intracellular accumulation of unesterified cholesterol. The increased cholesterol content in NPC fibroblasts which are grown in the presence of low density lipoproteins (LDL) has been postulated to be due to a deficiency in cellular cholesterol esterification. We have examined several aspects of LDL metabolism in NPC fibroblasts. We observe that LDL binding, internalization, and lysosomal hydrolysis of LDL cholesteryl esters are normal in NPC cells. As reported by Pentchev et al. (Pentchev, P. G., Comly, M. E., Kruth, H. S., Vanier, M. T., Wenger, D. A., Patel, S., and Brady, R. O. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 8247-8251), we find that LDL does not stimulate cholesterol esterification. However, we also show that LDL does not down-regulate cholesterol synthesis or LDL receptor activity as normal. In NPC cells, these processes are regulated normally by nonlipoprotein effectors, such as 25-hydroxycholesterol or mevalonate. Since NPC cells are not defective in lysosomal hydrolysis of LDL-derived cholesteryl esters, they must exhibit a different defect than Wolman's or cholesteryl ester storage diseases. We conclude that NPC cells are defective specifically in LDL-mediated regulation of cellular cholesterol metabolism. We suggest that the intracellular processing of LDL-derived cholesterol may be defective in NPC fibroblasts.

Cholesterol↗
PubMed1986

Enhancement in the adhesion of tumor cells to endothelial cells by decreased cholesterol synthesis.

Adhesive interactions between tumor cells and the endothelial cells are presumed to be an obligatory step in the metastatic process. Using an in vitro model, we have examined the role of endothelial lipids in the regulation of this interaction. The cholesterol levels of bovine aorta endothelial cell monolayers were inhibited by the addition of compactin, 25-hydroxycholesterol, or 7-ketocholesterol. Metastatic B16 melanoma cells prelabeled with 14C-amino acid mixture were then deposited on this monolayer as a suspension and, at various time intervals, the number of cells adhering to the monolayer was determined. The results indicated that inhibition of cellular cholesterol caused enhancement in cell adhesion. On the other hand, perturbations of the glycosylation in the endothelial cells were without any effect on cell adhesion. The presence of cholesterol dispersion in the growth medium partially reversed the enhancement in cell adhesion caused by the cholesterol inhibitors. Growth in the presence of retinol or dexamethasone (1 microM) also caused enhancement in the adhesiveness of the tumor cells to endothelial cells, possibly because of their effects on cholesterol synthesis. Procaine, a local anesthetic which is known to increase membrane fluidity, also increased the tumor cell-endothelial interaction, suggesting that the membrane fluidity plays an important role in the regulation of cell adhesion.

Animals↗
PubMed1986

Expression of low density lipoprotein receptor in cultured human granulosa cells: regulation by human chorionic gonadotropin, cyclic AMP, and sterol.

Steroidogenic cells utilize lipoprotein-delivered cholesterol as a primary substrate for hormone synthesis. We studied low density lipoprotein (LDL) receptors in cultured human granulosa cells to determine what factors regulate receptor expression. Granulosa cells cultured under serum-free conditions were treated with human chorionic gonadotropin (hCG) for 1.5 to 14 hr. The LDL receptor content of cells increased by approximately twofold within 6 hr of hCG treatment, and the content continued to increase for at least 14 hr, as determined by immunoblotting. The rate of LDL receptor synthesis was also demonstrated to increase within 2.5 to 3.5 hr of hCG treatment by immunoisolation of LDL receptor from cells metabolically labeled with a pulse of [35S]methionine. The cyclic AMP analogue, 8-bromo-cAMP, was also found to increase LDL receptor synthesis. This increased rate of synthesis was shown to be dependent on ongoing RNA synthesis, since actinomycin D abolished hCG- or 8-bromo-cAMP-stimulated LDL receptor synthesis. We also demonstrated that hCG- and 8-bromo-cAMP-mediated regulation of LDL receptor synthesis in granulosa cells supersedes the classical cholesterol-mediated regulation of the receptor described in fibroblasts. Although 25-hydroxycholesterol induced a decrease in LDL receptor content and synthesis within 6 hr, this action was overridden by simultaneous exposure to hCG. Our findings demonstrate the existence of a novel cAMP-mediated mechanism for regulation of LDL receptor synthesis in steroidogenic cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1986

Inhibition of acyl coenzyme A:cholesterol acyl transferase in J774 macrophages enhances down-regulation of the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase and prevents low density lipoprotein-induced cholesterol accumulation.

Cholesteryl ester accumulation in arterial wall macrophages (foam cells) is a prominent feature of atherosclerotic lesions. We have previously shown that J774 macrophages accumulate large amounts of cholesteryl ester when incubated with unmodified low density lipoprotein (LDL) and that this is related to sluggish down-regulation of the J774 LDL receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase. To further explore intracellular cholesterol metabolism and regulatory events in J774 macrophages, we studied the effect of inhibitors of acyl-CoA:cholesterol acyl transferase (ACAT) on the cells' ability to accumulate cholesterol and to down-regulate receptor and reductase. Treatment of J774 cells with LDL in the presence of ACAT inhibitor 58-035 (Sandoz) prevented both cholesteryl ester and total cholesterol accumulation. Furthermore, 58-035 markedly enhanced down-regulation of the J774 LDL receptor and 3-hydroxy-3-methylglutaryl-CoA reductase in the presence of LDL. In dose-response studies, down-regulation of the receptor by 58-035 paralleled its inhibition of ACAT activity. Compound 58-035 also increased the down-regulation of the J774 LDL receptor in the presence of 25-hydroxycholesterol and acetyl-LDL but not in the presence of cholesteryl hemisuccinate, which is not an ACAT substrate. The ability of 58-035 to enhance LDL receptor down-regulation was negated when cells were simultaneously incubated with recombinant high density lipoprotein3 discs, which promote cellular cholesterol efflux. In contrast to the findings with J774 macrophages, down-regulation of the human fibroblast LDL receptor was not enhanced by 58-035. These data suggest that in J774 macrophages, but not in fibroblasts, ACAT competes for a regulatory pool of intracellular cholesterol, contributing to diminished receptor and reductase down-regulation, LDL-cholesterol accumulation, and foam cell formation.

Amides↗
PubMed1985

3-Hydroxy-3-methylglutaryl coenzyme A reductase activity in the human gastrointestinal tract.

Activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) was measured in intestinal mucosa of the human gastrointestinal tract. Activity was highest in gastric mucosa (18.2 pmol per mg per min) and there was a constant low level in the small bowel and colon (approximately 10 pmol per mg per min). Phosphorylation/dephosphorylation modulation of intestinal reductase activity was demonstrated in normal mucosa. Expressed jejunal reductase activity was significantly higher in celiac sprue mucosa and mucosa from defunctionalized intestine of jejunoileal bypass patients. Enzyme activity also increased during 24-hr mucosal organ culture in the absence of exogenous cholesterol. Addition to the culture medium of pure cholesterol or 25-hydroxycholesterol dissolved in a small volume of ethanol suppressed the culture-induced increase to 86 +/- 3% and 69 +/- 5% of paired controls, respectively. This evidence suggests that a moderate degree of feedback regulation of intestinal cholesterol synthesis by luminal sterol occurs in man. Mucosal HMG-CoA reductase activity was also measured in patients with hyperlipoproteinemia. Patients with either predominant hypercholesterolemia or predominant hypertriglyceridemia lipid profiles had "normal" expressed reductase activity, but feedback regulation by free cholesterol could not be demonstrated in either group under these conditions.

Biopsy↗
PubMed1979

Topological studies of cytochromes P-450scc and P-45011 beta in bovine adrenocortical inner mitochondrial membranes. Effects of controlled tryptic digestion.

The topology of the steroid hydroxylase complexes in bovine adrenocortical mitochondria were studied by using controlled digestion with trypsin of purified inner mitochondrial membranes. Inhibition of steroid hydroxylase activity by trypsin was only observed in inner mitochondrial membranes which had been disrupted by various techniques. The steroid hydroxylase activity of intact inner membranes was not inhibited by trypsin. The effect of tryptic digestion was monitored by measuring 11 beta-hydroxylase and cholesterol side chain cleavage activities, as well as cytochrome P-450 reduction. The effect of trypsin on the steroid-induced difference spectra using pregnenolone, 20 alpha-hydroxycholesterol, and deoxycorticosterone was also measured. The results were similar regardless of which procedure was utilized and strongly suggest that both cytochrome P-45011 beta and cytochrome P-450scc are located on the matrix side of the mitochondrial inner membrane.

Adrenal Cortex↗
PubMed1978

Effect of auto-oxidation products from cholesterol on aortic smooth muscle cells: an in vitro study.

Purified cholesterol is quite unstable when stored in air at room temperature. Products of cholesterol auto-oxidation were concentrated from several lots of USP-grade cholesterol by recrystallizing cholesterol from the methanol extract, retaining the mother liquor, and evaporating the residuum to dryness under vacuum. By application of thin-layer chromatography and gas-liquid chromatography, major, individual, oxidation compounds were identified and quantitated. Biological activities of these oxidation compounds were studied by using cultured rabbits' aortic smooth muscle cells. The concentrate of the auto-oxidation products of cholesterol showed remarkable in vitro cytotoxic effects, whereas purified cholesterol at the same concentration produced no toxic effects. The concentrate was further separated into six thin-layer chromatographical fractions. The results showed that 25-hydroxycholesterol and cholestane-3beta, 5alpha, 6beta-triol were probably responsible for the biological toxicity of the concentrate

Aorta↗
PubMed1984

Different forms of the oxysterol-binding protein. Binding kinetics and stability.

Based upon measurements of the sedimentation coefficient and the Stokes radii, three forms of the oxysterol-binding protein were identified. The unliganded binding protein was the largest (7.7 S, Stokes radius = 71.6 A, Mr = 236,000) was relatively asymmetric (f/f0 = 1.7), and was composed of at least three subunits. Binding of 25-hydroxycholesterol was associated with a reduction in the size of the protein (7.5 S, Stokes radius = 50 A, Mr approximately 169,000) and an increase in symmetry (f/f0 = 1.4), due to the loss of a subunit of Mr approximately 67,000. At pH 6 or lower, the Mr = 169,000 sterol-protein complex was altered so that reversible dissociation to give a smaller (4.2 S, Stokes radius = 53 A, Mr = 97,000) more asymmetric (f/f0 = 1.8) sterol-protein complex occurred when it was sedimented in a sucrose gradient buffered at pH 7.4 containing 0.3 M KCl and 2.5 M urea. Irreversible dissociation of the 7.5 S, Mr = 169,000 form to a 4.2 S form occurred spontaneously when the complex in whole cytosol buffered at pH 7.8 was allowed to stand overnight at 0 degree C, or when the partially purified complex was incubated at pH 5.5 at 0 degree C for several days. The partially purified, unliganded binding protein was unstable at 0 degree C (approximately 75% loss of binding activity in 24 h) whereas the liganded protein was stable for 7 days at 0 degree C although irreversible conversion to a 4.2 S form occurred under some conditions. Rates of sterol binding and dissociation were increased in the presence of 2.5 M urea at pH 7.4 or when the pH was lowered to 5.5 Kd values were not greatly altered under the various incubation conditions.

Animals↗
PubMed1984

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by oxysterol by-products of cholesterol biosynthesis. Possible mediators of low density lipoprotein action.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34, reductase) activity was studied in cultured rat intestinal epithelial cells using 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one ( U18666A ), an inhibitor of 2,3- oxidosqualene cyclase (EC 5.4.99.7, cyclase) that causes cellular accumulation of squalene 2,3:22,23-dioxide ( Sexton , R. C., Panini , S.R., Azran , F., and Rudney , H. (1983) Biochemistry 22, 5687-5692). Treatment of cells with U18666A (5-50 ng/ml) caused a progressive inhibition of reductase activity. Further increases in the level of the drug paradoxically lessened the inhibition such that at a level of 1 microgram/ml, no inhibition of enzyme activity was observed. Cellular metabolism of squalene 2,3:22,23-dioxide to compounds with the chromatographic properties of polar sterols led to an inhibition of reductase activity that could be prevented by U18666A (1 microgram/ml). The drug was unable to prevent the inhibition of enzyme activity by 25-hydroxycholesterol or mevalonolactone, but totally abolished the inhibitory action of low density lipoproteins. Pretreatment with U18666A did not affect the ability of cells to degrade either the apoprotein or the cholesteryl ester component of low density lipoproteins. These results suggest that oxysterols derived from squalene 2,3:22,23-dioxide may act as physiological regulators of reductase and raise the possibility that the suppressive action of low density lipoproteins on reductase may be partially or wholly mediated by such endogenous oxysterols generated through incomplete inhibition of the cyclase.

Androstenes↗
PubMed1981

Bile acid synthesis in humans.

Metabolic pathways involved in the conversion of cholesterol to cholic and chenodeoxycholic acids have been investigated in bile fistula patients treated with a number of labeled potential bile acid intermediates. The findings of the present report indicate that the human liver cell has the capacity to synthesize both primary bile acids via multiple routes from cholesterol and 7 alpha-hydroxycholesterol. Evidence has been obtained for the existence of a major pathway to chenodeoxycholic acid via the 26-hydroxylation of 7 alpha-hydroxycholest-4-en-3-one. Cholic acid is synthesized preferentially via pathways from 5 beta-cholestane 3 alpha, 7 alpha-diol and a pathway from cholesterol not involving an initial 7 alpha-hydroxylation.

Bile Acids and Salts↗
PubMed1981

In vivo regulation of canine intestinal 3-hydroxy-3-methylglutaryl coenzyme A reductase by cholesterol, lipoprotein, and fatty acids.

Thirty-Vella-isolated ileal segments in dogs were used to study the regulation of intestinal cholesterol synthesis. This excluded fistula enabled independent in vivo manipulation of luminal and vascular influences on mucosal cells. Segments were studied repeatedly and each animal served as its own control. Cholesterol synthesis rate was assessed by measuring mucosal activity of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. Luminal cholesterol and 25-hydroxycholesterol were shown to reduce HMG CoA reductase activity to 64 +/- 7% and 42 +/- 4%, respectively, of control within 4 hr. Reductase activity in the excluded segment also responded to alterations in serum cholesterol produced by cholesterol or cholestyramine feeding. Similarly, in vitro studies showed that lipoprotein cholesterol inhibited HMG CoA reductase in mucosa from the excluded segment but not in mucosa from intact bowel. In contrast to sterols, fatty acids stimulated HMG CoA reductase activity by luminal contact. These findings suggest that the cholesterol needs of canine intestinal epithelial cells are acutely balanced by absorption and synthesis of cholesterol. Mucosal cells may also utilize lipoprotein cholesterol under certain conditions, perhaps via low density lipoprotein receptors. Fatty acid absorption stimulated cholesterol synthesis in the absence of luminal cholesterol, perhaps to facilitate chylomicron formation.

Animals↗
PubMed1980

Rat liver acyl-coenzyme A:cholesterol acyltransferase: its regulation in vivo and some of its properties in vitro.

To gain insight into the role of the enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT) in cellular cholesterol homeostasis, its regulation in rat liver was investigated both in vivo and in vitro. In vitro assay conditions were optimized and some properties of the microsomal enzyme in vitro was also studied. Arrhenius plots of microsomal ACAT activity showed discontinuities at about 28-29 degrees C and 16-17 degrees C. Detergents above their critical micelle concentrations and organic solvents both inhibited the enzyme. Addition of progesterone or SC 31769, a 7-keto-cholesterol analogue, to microsomes inhibited activity while addition of 25-hydroxycholesterol increased the rate of cholesterol esterification, suggesting that the enzyme is susceptible to both negative and positive modulation by steroids, steroid analogues, or their metabolic products. Increasing the rate of cholesterol biosynthesis had a variable effect on ACAT activity. It was higher at the circadian peak of sterol biosynthesis than at the nadir. Increasing sterol biosynthesis by intragastric administration of mevalonolactone resulted in increased activity. In contrast, increasing the rate of sterol biosynthesis by feeding cholestyramine or administration of Triton WR 1339 had little effect on ACAT. Increasing hepatic cholesterol content by feeding cholesterol, cholate, or an atherogenic diet, fasting or intragastric administration of mevalonolactone all resulted in increased ACAT activity. ACAT activity showed a positive correlation with changes in microsomal free and esterified cholesterol contents. The response of ACAT to changes in hepatic cholesterol concentration in vivo and its response to changes in the rate of cholesterol synthesis support the hypothesis that this enzyme plays an important role in maintenance of hepatic cholesterol homeostasis.

Acyl Coenzyme A↗
PubMed1980

Secretion of very low density lipoproteins enriched in cholesteryl esters by cultured rat hepatocytes during simulation of intracellular cholesterol esterification.

Very low density lipoproteins, newly secreted by cultured rat hepatocytes into a serum-free medium, contain some cholesteryl esters although the percentage of total cholesterol in ester form is less than that in plasma very low density lipoproteins. When acyl CoA:cholesterol acyltransferase activity in hepatocytes was stimulated by the addition of 25-hydroxycholesterol (10 microgram/ml) or mevalonolactone (1 mM), the absolute amount of esterified cholesterol secreted in very low density lipoproteins increased significantly, but the amount of free cholesterol decreased or showed no change. Thus the percentage of very low density lipoprotein cholesterol in ester form increased, in some experiments to as much as 50% of the total. These results provide additional evidence that hepatic acyl CoA:cholesterol acyltransferase plays a role in the generation of some of the cholesteryl ester in newly-secreted lipoproteins. They further suggest that changes in the activity of the enzyme can potentially regulate the fraction of cholesterol secreted in esterified form.

Animals↗
PubMed1993

Apoptosis induced by oxysterols in murine lymphoma cells and in normal thymocytes.

Oxygenated derivatives of cholesterol (oxysterols), a family of naturally occurring compounds, possess marked anti-proliferative and immunosuppressive activities, in particular they have been shown to inhibit T-cell responses to different stimuli. 25-Hydroxycholesterol (25-OHC) and 7 beta,25-dihydroxycholesterol (7.25-OHC) are able to kill not only RDM4 murine lymphoma in vitro, but also, surprisingly, mouse thymocytes after several hours of incubation. In this study, we report that the death of RDM4 and thymocytes induced by oxysterols exhibits the features of apoptosis. This phenomenon was identified by agarose gel electrophoresis of DNA fragments extracted from the cells and quantified by flow cytometric analysis of the DNA fluorescence of propidium iodide-stained cells. Cycloheximide and actinomycin D were found to decrease the number of apoptotic cells and to increase cell viability, indicating a requirement for the synthesis of macromolecules in oxysterol-induced programmed cell death. The pathway by which 25-OHC and 7.25-OHC are able to induce apoptosis in this type of cell and the possible contribution of these compounds to thymus involution during development are discussed.

Animals↗
PubMed1994

Structure-specific inhibition of lysosomal cholesterol transport in macrophages by various steroids.

Cultured mouse peritoneal macrophages effectively take up and metabolize liposomes containing phosphatidylserine and cholesterol, resulting in massive accumulation of cholesteryl esters and triacylglycerols in their cytoplasm (Nishikawa, K., Arai, H. and Inoue, K. (1990) J. Biol. Chem. 265, 5226-5231). With this system, various steroid derivatives were assessed as to their ability to inhibit the cholesteryl ester formation from endocytosed cholesterol in macrophages. Among the steroids tested, one group of steroids having an oxo group at the C17 or C20 position, such as androstenedione, dehydroisoandrosterone, progesterone and pregnenolone, completely inhibited cholesteryl ester formation at 10 microM. Another group of steroids having a hydroxy group at the C17 position, such as testosterone and androstenediol, had a lesser effect; complete inhibition of cholesteryl ester formation was achieved with 100 microM or more. The mechanism underlying the inhibition by the former class of steroids was further studied. These steroids did not block the uptake or lysosomal hydrolysis of liposomes, nor esterification of fatty acyl chains into triacylglycerols. Moreover, dehydroisoandrosterone and pregnenolone, both of which possess a hydroxy group at the C3 position, had essentially no effect on 25-hydroxycholesterol-stimulated esterification of endogenous cellular cholesterol. On the other hand, androstenedione and progesterone, which possess an oxo group at the C3 position, had a mild inhibitory effect on the esterification of endogenous cholesterol. Upon incubation with a steroid having an oxo group at the C17 or C20 position, free cholesterol taken up by macrophages was accumulated in phagolysosomes, as judged from cytochemical study with filipin-cholesterol staining. These results indicate that a series of structurally-related steroids characterized by the presence of an oxo group at the C17 or C20 position inhibit cholesteryl ester formation in macrophages through blocking the intracellular transport of endocytosed cholesterol from lysosomes to endoplasmic reticulum.

Animals↗
PubMed1994

Sterol 27-hydroxylase: high levels of activity in vascular endothelium.

Sterol 27-hydroxylase activity in bovine aortic endothelial (BAE) cells in culture has been compared with that in HepG2 cells and in Chinese hamster ovary (CHO) cells using identical culture conditions. The total enzyme activity of BAE cells (3.0 nmol/72 h per mg cell protein) was comparable with that of HepG2 cells (4.0 nmol/72 h per mg protein) and both values were significantly greater than that in CHO cells (0.002 nmol/72 h per mg protein). The enzyme was identified in the mitochondria extracted from BAE cells by Western blotting using an antibody of proven specificity, and its metabolites 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid were identified by mass spectrum analysis. The presence of the enzyme in endothelium provides a mechanism for preventing accumulation of intracellular cholesterol by initiating a pathway of bile acid synthesis different from that initiated by 7 alpha-hydroxylation of cholesterol in the liver.

Animals↗
PubMed1993

Hypochlorous acid-mediated modification of cholesterol and phospholipid: analysis of reaction products by gas chromatography-mass spectrometry.

Oxidative modification of membrane lipids by hypochlorous acid could be an important element in the mechanism of membrane disruption by activated neutrophils. We have previously shown that hypochlorous acid reacts with unsaturated fatty acids of membrane phospholipids to give fatty acid chlorohydrins (Winterbourn et al. 1992. Arch. Biochem. Biophys. 296: 547-555). In the present study, we examined the reaction of cholesterol in bilayers with an inert phospholipid carrier. Product separation and identification was performed using gas chromatography-mass spectrometry after trimethylsilyl-derivatization. Unlike the reaction of hypochlorous acid with unsaturated fatty acids, no chlorohydrin derivatives were found with cholesterol. Instead, the main oxidation products were identified as the epimeric cholesterol 5,6-epoxides and 4-hydroxycholesterol, while several other hydroxy- and keto-derivatives were also found in smaller amounts. Analysis of the products obtained after reaction of vesicles composed of a mixture of several unsaturated phospholipid species plus cholesterol revealed that the individual fatty acids and cholesterol all exhibit comparable susceptibilities toward hypochlorous acid. Using myeloperoxidase to generate hypochlorous acid, basically the same products and product distribution were obtained. These studies show that unsaturated phospholipids and cholesterol can be profoundly modified by reaction with hypochlorous acid. This warrants further investigation to define the role of lipid modifications in neutrophil-mediated membrane disruption.

Cholesterol↗
PubMed1993

Cholesterol and sphingomyelin syntheses are regulated independently in cultured human intestinal cells, CaCo-2: role of membrane cholesterol and sphingomyelin content.

There is a presumed association between cellular cholesterol and sphingomyelin metabolism. To study this relationship in the intestine, the activity of the rate controlling enzyme of sphingolipid synthesis, serine palmitoyltransferase (SPT), and the biosynthesis of long-chain bases were characterized in cultured human intestinal cells, CaCo-2. Cells were then incubated with substances known to alter cholesterol biosynthesis, and the effect of these mediators on SPT activity and long-chain base synthesis was determined and compared with their effects on HMG-CoA reductase activity and cholesterol synthesis. The polar sterol, 25-hydroxycholesterol, the squalene epoxide inhibitor, U18666A, and the inhibitor of HMG-CoA reductase, lovastatin, all significantly inhibited the synthesis of cholesterol without altering either SPT activity or long-chain base synthesis. Mevalonate, which increased cholesterol production 3-fold, also had no affect on SPT activity or sphingoid base synthesis. Serine, which significantly increased the synthesis of long-chain bases, did not alter cholesterol biosynthesis. Moreover, the suicide inhibitors of SPT, beta-chloroalanine and cycloserine, did not alter cholesterol synthesis while markedly decreasing long chain base synthesis. Cells were incubated with palmitic, oleic, linoleic, and eicosapentaenoic acids. Only palmitic acid, the preferred substrate for SPT, increased the production of long-chain bases. Both palmitic and oleic acids, however, increased the synthesis of cholesterol. Cells enriched in sphingomyelin had higher rates of synthesis of both cholesterol and long-chain bases compared to their controls. In contrast, cholesterol and long-chain base syntheses were significantly decreased in cells enriched in cholesterol. Control cells incubated with phospholipid liposomes alone had higher rates of synthesis of both lipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyltransferases↗