Acyl coenzyme A:cholesterol acyltransferase in neonatal chick brain

An acyl coenzyme A:cholesterol acyltransferase activity which directly incorporates palmitoyl coenzyme A into cholesterol esters using endogenous cholesterol as substrate was demonstrated in microsomal preparations from neonatal chick brain. The enzyme showed, at pH 7.4, about 2-fold greater activity than that observed at pH 5.6. Nearly 10-times higher esterifying activity was found in brain microsomes using palmitoyl coenzyme A than that with palmitic acid. The acyltransferase activity was clearly different from the other cholesterol-esterifying enzymes previously found in brain, which incorporated free fatty acids into cholesterol esters and did not require ATP or coenzyme A as cofactors. Chick brain microsomes also incorporated palmitoyl coenzyme A into phospholipids and triacylglycerols. However, most of the radioactivity from this substrate was found in the fatty acid fraction, due to the presence of an acyl coenzyme A hydrolase activity in the enzyme preparations. Therefore, the formation of palmitate was tested during all the experiments. The brain acyltransferase assay conditions were optimized with respect to protein concentration, incubation time and palmitoyl coenzyme A concentration. Microsomal activity was independent of the presence of dithiothreitol in the incubation medium and microsomes can be stored at -40 degrees C for several weeks without losing activity. Addition of fatty acid-free bovine serum albumin to brain microsomal preparations produced a considerable increase in the acyltransferase activity, while acyl coenzyme A hydrolase was clearly inhibited. Results obtained show the existence in neonatal chick brain of an acyl coenzyme A:cholesterol acyltransferase activity similar to that found in a variety of tissues from different species but not previously reported in brain.     

Acyl coenzyme A: cholesterol acyltransferase in neonatal chick brain

An acyl coenzyme A:cholesterol acyltransferase activity which directly incorporates palmitoyl coenzyme A into cholesterol esters using endogenous cholesterol as substrate was demonstrated in microsomal preparations from neonatal chick brain. The enzyme showed, at pH 7.4, about 2-fold greater activity than that observed at pH 5.6. Nearly 10-times higher esterifying activity was found in brain microsomes using palmitoyl coenzyme A than that with palmitic acid. The acyltransferase activity was clearly different from the other cholesterol-esterifying enzymes previously found in brain, which incorporated free fatty acids into cholesterol esters and did not require ATP or coenzyme A as cofactors. Chick brain microsomes also incorporated palmitoyl coenzyme A into phospholipids and triacylglycerols. However, most of the radioactivity from this substrate was found in the fatty acid fraction, due to the presence of an acyl coenzyme A hydrolase activity in the enzyme preparations. Therefore, the formation of palmitate was tested during all the experiments. The brain acyltransferase assay conditions were optimized with respect to protein concentration, incubation time and palmitoyl coenzyme A concentration. Microsomal activity was independent of the presence of dithiothreitol in the incubation medium and microsomes can be stored at −40°C for several weeks without losing activity. Addition of fatty acid-free bovine serum albumin to brain microsomal preparations produced a considerable increase in the acyltransferase activity, while acyl coenzyme A hydrolase was clearly inhibited. Results obtained show the existence in neonatal chick brain of an acyl coenzyme A:cholesterol acyltransferase activity similar to that found in a variety of tissues from different species but not previously reported in brain.     

Cholesterol metabolism in fibroblasts from rabbits resistant to diet-induced hypercholesterolemia

We have previously described a colony of New Zealand White rabbits that are resistant to hypercholesterolemia when fed a cholesterol-enriched diet. The present studies used skin fibroblasts obtained from normal and hypercholesterolemia-resistant rabbits to investigate cholesterol metabolism and lipid composition in vitro. The lipid compositions of the two cell lines after incubation in either fetal calf serum or lipoprotein-deficient serum were similar. The conversion of radiolabeled acetate into sterol and phospholipids was higher in resistant fibroblasts than in normal fibroblasts. In contrast, incorporation of radiolabeled oleic acid into cholesteryl ester was significantly lower in resistant fibroblasts than in normal cells. In parallel experiments, the 3-hydroxy-3-methylglutaryl coenzyme A reductase activity was higher and acyl-coenzyme A:cholesterol acyltransferase activity was lower in resistant cells compared to normal cells. Furthermore, binding, uptake, and degradation of normal rabbit 125I-labeled LDL (low density lipoproteins) were 30% higher in resistant than in normal fibroblasts. These observations are consistent with results from previous studies of cholesterol metabolism in the liver membranes of these rabbits. The results indicate that extrahepatic cells (such as fibroblasts) from the resistant rabbit exhibit the same altered cholesterol metabolism as that found in the hepatic tissues of these rabbits. These studies suggest that the resistant rabbit may provide an in vivo and in vitro system for studying the mechanisms by which some individuals of a species can minimize the effect of dietary cholesterol on the development of hypercholesterolemia and atherosclerosis.     

Regulation of hepatic cholesterol and lipoprotein metabolism in ethinyl estradiol-treated rats

The regulation of hepatic cholesterol and lipoprotein metabolism was studied in the ethinyl estradiol-treated rat in which low density lipoprotein (LDL) receptors are increased many fold. Cholesterol synthesis was reduced at both its diurnal peak and trough by ethinyl estradiol. The diurnal variation in 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was abolished, whereas that for acyl coenzyme A: cholesterol acyltransferase (ACAT) was retained. LDL receptor number did not vary diurnally. Feeding these animals a cholesterol-rich diet for 48 h suppressed cholesterol synthesis and reductase activities to levels similar to those found in cholesterol-fed control animals, but ACAT activity was unaffected. LDL receptors were reduced about 50%. Intravenously administered cholesterol-rich lipoproteins suppressed HMG-CoA reductase and LDL receptors in 2 h but had a variable effect on ACAT activity. Intragastric administration of mevalonolactone reduced reductase and increased acyltransferase activity but had little effect on LDL receptors when given 2 or 4 h before death. Although animals fed a cholesterol-rich diet before and during ethinyl estradiol treatment became hypocholesterolemic, free and esterified cholesterol concentrations in liver were high as was ACAT activity. HMG-CoA reductase was inhibited to levels found in control animals fed the cholesterol-rich diet. LDL receptors were increased to a level about 50% of that reached in animals receiving a control diet and ethinyl estradiol. These data demonstrate that key enzymes of hepatic cholesterol metabolism and hepatic LDL receptors respond rapidly to cholesterol in the ethinyl estradiol-treated rat. Furthermore, estradiol increases LDL receptor activity several fold in cholesterol-loaded livers.     

Inhibition of cholesterol synthesis and esterification regulates high density lipoprotein interaction with isolated epithelial cells of human small intestine

The effect of two inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, lovastatin and monacolin L, and an inhibitor of acyl coenzyme A:cholesterol acyltransferase (ACAT), Sandoz compound 58-035, on the interaction of 125I-labeled high density lipoprotein-3 (HDL3) with isolated human enterocytes was studied. Both HMG-CoA reductase inhibitors inhibited cholesterol synthesis and 125I-labeled HDL3 binding and degradation by enterocytes; a strong correlation between changes in cholesterol synthesis and interaction of 125I-labeled HDL3 with cells was observed. Lovastatin caused reduction of the apparent number of 125I-labeled HDL3 binding sites without affecting the binding affinity. No changes of cell cholesterol content were observed after incubation of cells with lovastatin. Mevalonic acid reversed the effect of lovastatin on 125I-labeled HDL3 binding. Lovastatin blocked up-regulation of the HDL receptor in response to loading of cells with nonlipoprotein cholesterol and modified cholesterol-induced changes of 125I-labeled HDL3 degradation. Lovastatin also reduced HDL-mediated efflux of endogenously synthesized cholesterol from enterocytes. The ACAT inhibitor caused a modest increase of 125I-labeled HDL3 binding to enterocytes and significantly decreased its degradation; both effects correlated with inhibition of cholesteryl ester synthesis. The results allow us to assume that the intracellular free cholesterol pool may play a key role in regulation of the HDL receptor.     

Effects of treatment with clofibrate, bezafibrate, and ciprofibrate on the metabolism of cholesterol in rat liver microsomes

The effects of treatment of rats with clofibrate, bezafibrate, and ciprofibrate on the hepatic metabolism of cholesterol were studied in rat liver microsomes. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase activity, regulating cholesterol biosynthesis, was unaffected by clofibrate and ciprofibrate and slightly decreased (20%) by bezafibrate. Also cholesterol 7 alpha-hydroxylase activity, governing bile acid biosynthesis, was unaffected by clofibrate and was reduced by 25-30% in the two other groups of rats. A major new finding was that all three fibric acid derivatives reduced ACAT (acyl-coenzyme A:cholesterol acyltransferase) activity, catalyzing the esterification of cholesterol, by 50-70%. The hepatic content of free and esterified cholesterol was determined in the bezafibrate-treated rats. The concentration of microsomal cholesteryl ester was about 60% lower in the treated rats compared to the controls whereas the concentration of total cholesterol was unchanged.     

The effects of 6-azacholest-4-en-3 beta-ol-7-one, an inhibitor of cholesterol 7 alpha-hydroxylase, on cholesterol metabolism and bile acid synthesis in primary cultures of rat hepatocytes

6-Azacholest-4-en-3 beta-ol-7-one (azacholesterol) was shown to be a specific inhibitor of cholesterol 7 alpha-hydroxylase. It inhibited cholesterol hydroxylation by a rat liver microsomal preparation with non-competitive kinetics and a Ki of 4 microM. No evidence was found for a time-dependent inhibition of activity. Azacholesterol did not inhibit acyl-CoA: cholesterol acyltransferase or 3-hydroxy-3-methylglutaryl coenzyme A reductase in rat liver microsomal preparations, or cholesterol esterification and synthesis in primary cultures of rat hepatocytes. The synthesis of bile acids was inhibited by azacholesterol in these cells in a dose-dependent way. When bile acid synthesis was inhibited by azacholesterol, newly-synthesized cholesterol from exogenous mevalonate was secreted by the hepatocyte cultures into the cell culture medium in several-fold excess over control incubations. No changes in the secretion of cholesteryl ester occurred in the presence of azacholesterol. This observation suggests that newly synthesised cholesterol that has entered the substrate pool for hydroxylation is no longer accessible to the substrate pool for esterification. This is further evidence for the compartmentation of cholesterol metabolism in the hepatocyte.     

Plasma fibronectin-binding glycosaminoglycans in the substratum adhesion sites of neural tumor cells

The metabolism of high-density lipoprotein (HDL) in cells of five human cancer cell lines maintained in monolayer culture was investigated. In cells of some of the lines there was evidence of high-affinity binding sites for HDL, whereas in others this could not be demonstrated. However, in one cell line, viz., HEC-B-296 (human endometrial carcinoma), degradation of the protein component of HDL was demonstrated. The proteolytic activity was specific for HDL in so far as human serum albumin was not degraded by these cells. However, this degradative process did not involve internalization of the HDL molecule and degradation was not mediated by lysosomal proteolytic enzymes. HDL, when present in the medium, did not affect the degradation of low-density lipoprotein and low-density lipoprotein did not affect the degradation of HDL. HDL did not affect significantly cholesterol biosynthesis or cholesteryl ester biosynthesis as estimated from the activity of the regulatory enzymes, 3-hydroxy-3-methylglutaryl coenzyme A reductase and acyl-CoA:cholesterol acyltransferase. The degradation of HDL by HEC-B-296 cells was inhibited, to various degrees, when trypsin inhibitor or a protease inhibitor such as leupeptin, was present in the culture medium. It is concluded that degradation of the protein component of HDL by human neoplastic cells of the HEC-B-296 line was the result of activity of a proteolytic enzyme that is present on the external surface of the cells.     

Effect of catecholamines on the hepatic rate-limiting enzymes of cholesterol metabolism in normally fed and cholesterol-fed rabbits

Adrenergic control of liver cholesterol metabolism was studied in the rabbit. The effects of noradrenaline (α₁, α₂, β₂ agonist) and isoprenaline (β₁, β₂ agonist) on 3-hydroxy-3-methylglutaryl coenzyme A reductase, acyl-coenzyme A: cholesterol-o-acyltransferase (cholesterol acyltransferase) and cholesterol 7α-hydroxylase, the rate-limiting enzymes of cholesterol biosynthesis and esterification and bile acid synthesis, respectively, were examined in the normally fed and cholesterol-fed male New Zealand White rabbit. Isoprenaline increased the activities of hydroxymethylglutaryl CoA reductase and cholesterol acyltransferase approx. 12-fold and 5-fold, respectively, in normally fed rabbits. Noradrenaline, by contrast, produced an effect only on hydroxymethylglutaryl CoA reductase, the activity of which was increased 3-fold in these animals. Neither catecholamine had an effect on hydroxymethylglutaryl CoA reductase in the cholesterol-fed rabbit. Isoprenaline decreased the activity of cholesterol acyltransferase by approx. 40% and increased the activity of cholesterol 7α-hydroxylase 2-fold in the cholesterol-fed rabbit compared to cholesterol-fed controls. Noradrenaline had no effect on either cholesterol acyltransferase or cholesterol 7α-hydroxylase in either the normally fed or the cholesterol-fed rabbit. We suggest that β₂-adrenergic stimulation by isoprenaline in the normally fed rabbit may enhance cholesterol synthesis and storage, but that in the cholesterol-fed rabbit, it facilitates the elimination of cholesterol from the body by increasing the rate of bile acid synthesis.     

Selective uptake of cholesteryl ester from low density lipoprotein is involved in HepG2 cell cholesterol homeostasis.

Low density lipoprotein (LDL) can follow either a holoparticle uptake pathway, initiated by the LDL receptor (LDLr), and be completely degraded, or it can deliver its cholesteryl esters (CE) selectively to HepG2 cells. Although high density lipoprotein-CE selective uptake has been shown to be linked to cell cholesterol homeostasis in nonhepatic cells, there is no available information on the effect of LDL-CE selective uptake on hepatic cell cholesterol homeostasis. In order to define the role of the LDL-CE selective uptake pathway in hepatic cell cholesterol homeostasis, we used a cellular model that expresses constitutively a LDLr antisense mRNA and that shows LDLr activity at 31% the normal level (HepG2-all cells). The addition of a specific antibody anti-LDLr (IgG-C7) reduces LDL protein degradation (LDLr activity) to 7%. This cellular model therefore reflects, above all, LDL-CE selective uptake activity when incubated with LDL. The inactivation of LDLr reduces LDL-protein association by 78% and LDL-CE association by only 43%. The LDL-CE selective uptake was not reduced by the inactivation of LDLr. The activities of the various enzymes involved in cell cholesterol homeostasis were measured in normal and LDLr-deficient cells during incubation in the absence or presence of LDL as a cholesterol source. Essentially, 3-hydroxy-3-methylglutaryl coenzyme A reductase and acyl coenzyme A:cholesterol acyltransferase (ACAT) activities responded to LDL in LDLr-deficient cells as well as in normal HepG2 cells. Inhibition of lysosomal hydrolysis with chloroquine abolished the effect measured on ACAT activity in the presence of LDL, suggesting that CE of LDL, but not free cholesterol, maintains cell cholesterol homeostasis. Thus, in HepG2 cells, when LDLr function is virtually abolished, LDL-CE selective uptake is coupled to cell cholesterol homeostasis.     

Stimulation of LDL receptor activity in Hep-G2 cells by a serum factor(s)

The regulation of low-density lipoprotein (LDL) receptor activity in the human hepatoma cell line Hep-G2 by serum components was examined. Incubation of dense monolayers of Hep-G2 cells with fresh medium containing 10% fetal calf serum (FM) produced a time-dependent increase in LDL receptor activity. Uptake and degradation of 125I-LDL was stimulated two- to four-fold, as compared with that of Hep-G2 cells cultured in the same media in which they had been grown to confluence (CM); the maximal 125I-LDL uptake plus degradation increased from 0.2 microgram/mg cell protein/4 h to 0.8 microgram/mg cell protein/4 h. In addition, a two-fold increase in cell surface binding of 125I-LDL to Hep-G2 cells was observed when binding was measured at 4 degrees C. There was no change in the "apparent" Kd. The stimulation of LDL receptor activity was suppressed in a concentration-dependent manner by the addition of cholesterol, as LDL, to the cell medium. In contrast to the stimulation of LDL receptor activity, FM did not affect the uptake or degradation of 125I-asialoorosomucoid. Addition of FM increased the protein content per dish, and DNA synthesis was stimulated approximately five-fold, as measured by [3H]thymidine incorporation into DNA; however, the cell number did not change. Cellular cholesterol biosynthesis was also stimulated by FM; [14C]acetate incorporation into unesterified and esterified cholesterol was increased approximately five-fold. Incubation of Hep-G2 cells with high-density lipoproteins (200 micrograms protein/ml) or albumin (8.0 mg/ml) in the absence of the serum factor did not significantly increase the total processed 125I-LDL. Stimulation of LDL receptor activity was dependent on a heat-stable, nondialyzable serum component that eluted in the inclusion volume of a Sephadex G-75 column. Uptake of 125I-LDL by confluent monolayers of human skin fibroblasts was not changed by incubation with FM or by incubation with Hep-G2 conditioned medium. Taken together, these data demonstrate that LDL receptor activity in Hep-G2 cells is stimulated by a serum component. Furthermore, this serum factor shows some specificity for the LDL receptor pathway in liver-derived Hep-G2 cells.     

Sterol carrier protein2-like activity in rat intestine

A sterol carrier protein2 (SCP2)-like activity has been demonstrated in rat intestinal mucosal homogenates and in isolated intestinal cells from both crypt and villus zones. The results indicate the presence of a protein with similar molecular weight and antigenicity to that of authentic SCP2 purified from rat liver cytosol. Like liver SCP2, mucosal cytosol stimulates pregnenolone production in rat adrenal mitochondria and acyl coenzyme A:cholesterol acyltransferase activity of liver and mucosal microsomes. The distribution of SCP2-like activity as determined by radioimmunoassay indicates high levels in mitochondria and cytosol and relatively lower levels in microsomes and in brush-border membranes. The widespread distribution of SCP2-like protein in the intestine is consistent with potential transfer functions in all phases of cholesterol processing.     

In vitro regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A: cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine

The regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A:cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine was studied in vitro. Preparing intestinal microsomes in the presence of 50 mM NaF caused a 64% decrease in the reductase activity. It had no effect on acyl-CoA:cholesterol acyltransferase activity. Microsomes that were prepared in NaF were incubated with intestinal cytosol, a partially purified phosphatase from cytosol, and Escherichia coli alkaline phosphatase. All three preparations increased 3-hydroxy-3-methylglutaryl-CoA reductase by two- or three-fold suggesting dephosphorylation and 'reactivation' of enzyme activity. Cytosol caused a 78% increase in acyl-CoA:cholesterol acyltransferase activity, but neither the partially purified phosphatase nor the E. coli alkaline phosphatase affected the acyltransferase activity. Microsomes incubated with increasing concentrations of MgCl2 and ATP decreased both the activities of 3-hydroxy-3-methylglutaryl-CoA reductase and acylcoenzyme A:cholesterol acyltransferase in a step-wise fashion. Whereas this inhibitory effect was specific for reductase, the effect on acyl-CoA:cholesterol acyltransferase activity was secondary to the presence of ATP in the assay mixture. The 8500 X g supernatant of intestinal whole homogenate from isolated intestinal cells or scraped mucosa was incubated with MgCl2, ATP and NaF. In microsomes prepared from this supernatant, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase was significantly decreased. Again, no change was observed in the acyltransferase activity. The rate of cholesterol esterification in isolated intestinal cells was not affected by 0.1 mM cAMP or 50 mM NaF. We conclude that under conditions which regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in rabbit intestine by phosphorylation-dephosphorylation, no regulation of acyl-CoA:cholesterol acyltransferase activity is observed.     

Hepatic metabolism and secretion of a cholesterol-enriched lipoprotein fraction

A potentially important source of cholesterol secreted in bile is cholesterol-rich lipoproteins. However, the fate of the cholesterol carried in these lipoproteins after hepatic uptake has not been investigated. We harvested an apoE- and cholesterol-rich lipoprotein fraction (d 1.02-1.06 g/ml) from hypercholesterolemic rats and examined the acute effects of these lipoproteins on hepatic cholesterol metabolism, very low density lipoprotein (VLDL) secretion, and biliary lipid secretion. Administration of a lipoprotein bolus (20 mg of cholesterol) to rats resulted in a significant decrease in 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and a significant increase in acyl-coenzyme A:cholesterol acyltransferase activity over controls at 1 hr. Hepatic cholesteryl ester content increased 400% with no change in hepatic free cholesterol content or biliary cholesterol secretion. These cholesterol-rich lipoproteins delivered in the isolated perfused liver effected a fivefold increase in hepatic VLDL secretion with no change in composition. Therefore, cholesterol-rich lipoproteins do not acutely alter biliary cholesterol secretion. Rather, the majority of the cholesterol delivered to the liver in these lipoproteins is either esterified and stored as cholesteryl ester or resecreted as free and esterified cholesterol in hepatic VLDL.     

Abnormal hepatobiliary and circulating lipid metabolism in the Long-Evans Cinnamon rat model of Wilson's disease

Long-Evans Cinnamon (LEC) rats exhibit a genetic defect in Atp7b gene, which is homologous to the human Wilson's disease gene, resulting in an inability to mobilize copper from the liver. This study was undertaken to gain insight into the relationship between liver copper accumulation and plasma lipid profile, circulating lipoprotein composition, hepatic sterol metabolism and biliary lipid secretion rates in 12-week-old LEC rats compared to control Long-Evans rats. Concomitant with hepatic copper deposition, LEC rats displayed increased content of triglycerides (TGs), free cholesterol (FC) and cholesteryl ester (CE) in the liver. Hepatic concentrations of malondialdehyde (MDA), an index of lipid peroxidation were also significantly elevated in LEC rats (50%). This steatosis was associated with aberrant microsomal apolipoprotein (apo) B-100 and microsomal triglyceride transfer protein (MTP) content, hypotriglyceridemia, hypocholesterolemia and abnormalities in both circulating lipoprotein composition and size. Atypical hepatobiliary sterol metabolism was established by the assessment of the activity of key intracellular enzymes for cholesterol homeostasis, which demonstrated, with respect to controls, a 40% reduction in 3-hydroxy-3-methylglutaryl coenzyme A reductase, a 30% reduction in cholesterol 7alpha-hydroxylase, and a 54% reduction in acyl CoA:cholesterol acyltransferase. During a 6-h biliary drainage, a decline in the bile acid output was recorded and might be linked to the low protein expression of the bile salt export pump (BSEP or ABCB11). Our data emphasize the crucial role of copper balance in hepatic sterol homeostasis and lipoprotein metabolism in LEC rats. Additional studies are needed to delineate the mechanisms of these disorders.     

Two major regulatory steps in cholesterol synthesis by human renal cancer cells.

Two major mechanisms regulating cholesterol biosynthesis exist in a human renal cancer cell line, Caki-1. Caki-1 is a newly established cell line whose characteristics of rapid growth and active cholesterol synthesis qualify it as a potentially valuable tool for elucidation of regulatory mechanism of cholesterol synthesis and transport. In the absence of exogenous cholesterol, cholesterol is the dominant sterol arising from labeled acetate and mevalonate. As expected, in the presence of exogenous cholesterol, the conversion of acetate to cholesterol and the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) is markedly reduced and this inhibition is released when cholesterol is removed from the medium. An unexpected and possibly unique finding is the inhibition of the conversion of mevalonate to cholesterol in the presence of exogenous cholesterol. This second major control process results in the accumulation of squalene and may involve additional late steps in cholesterol biosynthesis or metabolism. The occurrence of two major mechanisms regulating cholesterol synthesis may be a unique property of renal cancer cells or a previously unrecognized characteristic of a variety of cultured cells.     

Multifunctional role for fetuin (fetal protein) in lipid transport.

Recent studies from this laboratory have shown that fetuin 1) is nearly 50-fold more efficient than albumin in incorporating exogenous fatty acids into cultured cells, (JBC, 265: 5883, 1990), and 2) is associated with a lipoprotein-like particle (FASEB J. 3: 2075-2080, 1989). In the present study, this lipid-containing fraction (FLP) was isolated by ultracentrifugation, and its effect on cholesterol efflux from cultured human skin fibroblasts and Hep-G2 cells prelabeled with [14C]cholesterol was investigated. FLP fraction caused a significant efflux of [14C]cholesterol from cells, the same in magnitude as HDL. This effect of fetuin supranatant fraction increased proportionately with concentration and time. Similar results were observed with Hep-G2 cells. This ability to induce efflux of cholesterol was confirmed by a decrease in cholesterol mass of cells after 24 h incubation with FLP. The ultracentrifugal bottom (infranatant) fraction of fetuin (FI) was ineffective in this regard. However, FI was more effective in the incorporation of exogenous fatty acids into cellular triglycerides. These studies suggest that the fetuin molecule is a multifunctional protein (delivery of fatty acids to cells and cholesterol efflux from cells) which may play a role in lipid transport during fetal life.     

In vitro regulation of 3-hydroxy-3-methylglutarylcoenzyme a reductase and acylcoenzyme a: Cholesterol acyltransferase activities by phoshorylation-dephosphorylation in rabbit intestine

The regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A: cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine was studied in vitro. Preparing intestinal microsomes in the presence of 50 mM NaF caused a 64% decrease in the reductase activity. It had no effect on acyl-CoA: cholesterol acyltransferase activity. Microsomes that were prepared in NaF were incubated with intestinal cytosol, a partially purified phosphatase from cytosol, and Escherichia coli alkaline phosphatase. All three preparations increased 3-hydroxy-3-methylglutaryl-CoA reductase by two- or three-fold suggesting dephosphorylation and ‘reactivation’ of enzyme activity. Cytosol caused a 78% increase in acyl-CoA: cholesterol acyltransferase activity, but neither the partially purified phosphatase nor the E. coli alkaline phosphatase affected the acyltransferase activity. Microsomes incubated with increasing concentrations of MgCl₂ and ATP decreased both the activities of 3-hydroxy-3-methylglutaryl-CoA reductase and acylcoenzyme A: cholesterol acyltransferase in a step-wise fashion. Whereas this inhibitory effect was specific for reductase, the effect on acyl-CoA: cholesterol acyltransferase activity was secondary to the presence of ATP in the assay mixture. The 8500×g supernatant of intestinal whole homogenate from isolated intestinal cells or scraped mucosa was incubated with MgCl₂, ATP and NaF. In microsomes prepared from this supernatant, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase was significantly decreased. Again, no change was observed in the acyltransferase activity. The rate of cholesterol esterification in isolated intestinal cells was not affected by 0.1 mM cAMP or 50 mM NaF. We conclude that under conditions which regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in rabbit intestine by phosphorylation-dephosphorylation, no regulation of acyl-CoA: cholesterol acyltransferase activity is observed.     

Role of the cyclic AMP-dependent pathway in free radical-induced cholesterol accumulation in vascular smooth muscle cells

We have previously reported that free radical-treated vascular smooth muscle cells (SMC) lead to cholesterol accumulation in vitro. In the current study, we investigated the effects of oxidative stress on cyclic AMP concentration and cAMP-dependent enzymes involved in cholesterol homeostasis in A7r5 cells. Under our conditions of a mild oxidative stress, namely with no change in cell viability, we found that free radicals, initiated using azobis-amidinopropane dihydrochloride (AAPH), resulted in a dose-dependent decrease in cellular cAMP which was opposed by vitamin E preincubation. Although the addition of adenylate cyclase activators (carbacyclin and forskolin) increased cAMP levels it did not succeed in restoring the AAPH-induced decrease. The oxidative stress-induced increase in activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase and of acyl coenzyme A: cholesterol acyltransferase and the decrease in neutral cholesteryl ester hydrolase activity were suppressed by addition of dibutyryl cAMP. Taken together, these results strongly suggest that free radicals reduce cAMP concentrations by altering cell membrane adenylate cyclase activity. The changes of cAMP-dependent enzymes induced by oxidative stress resulting in cholesterol accumulation might be one of the processes leading to SMC-derived foam cells depicted in atheroma plaque. Moreover, if extrapolated to in vivo, these data may explain in part the beneficial effects of antioxidants in the reduction of cardiovascular diseases.