Supernatant protein factor requires phosphorylation and interaction with Golgi to stimulate cholesterol synthesis in hepatoma cells

Supernatant protein factor (SPF) is a poorly characterized cytosolic protein that stimulates HMG-CoA reductase and squalene monooxygenase in vitro and cholesterol synthesis when expressed in hepatoma cells. The activation of SPF by protein kinases A (PKA) and Cdelta enhances its ability to stimulate these cholesterolgenic enzymes in microsomal preparations. The present studies demonstrate that the ability of SPF to stimulate cholesterol synthesis in cell culture is also modulated by phosphorylation. Addition of dibutyryl-cAMP, a PKA activator, to hepatoma cells expressing SPF increased cholesterol synthesis by 62%, whereas addition of a cell-permeable PKA inhibitor blocked the SPF-mediated increase in cholesterol synthesis. To confirm a role for PKA in the regulation of SPF, substitution of alanine for serine-289 (a putative PKA recognition site) blocked the stimulation of cholesterol synthesis by SPF. Serine-289 is located at the junction of the proposed lipid-binding domain and the carboxyl-terminal Golgi dynamics domain, suggesting that phosphorylation may alter the interaction of these two domains. In a test of this hypothesis, deletion of the Golgi dynamics domain blocked the ability of SPF to stimulate cholesterol synthesis, supporting a role for Golgi in SPF function; this finding was buttressed by the observation that addition of brefeldin A, which disrupts Golgi formation, also abolished the ability of SPF to stimulate cholesterol synthesis. The activation of SPF by PKA suggests that cholesterol synthesis can be rapidly modulated in response to external stimuli by changes in cAMP levels, and that this regulation is dependent on an as yet undefined interaction with Golgi.     

Variations of hepatic cholesterol precursors during altered flows of endogenous and exogenous squalene in the rat

Hepatic and serum levels of cholesterol precursors were analyzed in rats under basal (control) conditions and when cholesterol synthesis was activated by feeding 1% squalene or 5% cholestyramine. Exogenous squalene stimulated the activity of acyl-coenzyme A:cholesterol acyltransferase (ACAT) but strongly inhibited the activity of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase; cholestyramine did not affect ACAT but increased HMG-CoA reductase several-fold, indicating enhanced production of endogenous squalene. Activation of cholesterol synthesis by the two methods markedly increased the hepatic and serum contents of cholesterol precursor sterols. However, the sterol profiles were clearly different. Thus, exogenous squalene raised most significantly (up to 109-fold) free and esterified methyl sterols, and less so (up to 2-fold) demethylated C27 sterols (desmosterol and cholestenols) and also esterified cholesterol. Activation of endogenous squalene production by cholestyramine was associated with a depletion of esterified cholesterol and by a marked, up to 8-fold, increase of the free demethylated sterol precursor levels, whereas the increase of methyl sterols, up to 5-fold, was less conspicuous than during the squalene feeding. The changes were mostly insignificant for esterified sterols. The altered serum sterol profiles were quite similar to those in liver. Serum cholestenols and especially their portion of total serum precursor sterols were closely correlated with the hepatic activity of HMG-CoA reductase.     

Regulation of squalene synthetase in human hepatoma cell line Hep G2 by sterols, and not by mevalonate-derived non-sterols

Incubations of Hep G2 cells for 18 h with human low-density lipoprotein (LDL) resulted in a decrease of squalene synthetase activity, whereas heavy high-density lipoprotein (hHDL) stimulated the activity. Simultaneous addition of LDL abolished the hHDL-induced stimulation, indicating that manipulating the regulatory sterol pool within the cells influenced the enzyme activity. Blocking the endogenous cholesterol synthesis either at the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase site with compactin or at the 2,3-oxidosqualene cyclase site with the inhibitor U18666A gave rise to an elevation of the squalene synthetase activity. Simultaneous addition of mevalonate abolished the compactin-induced increase. However, at total blockade of sterol synthesis by 30 microM U18666A, added compactin and/or mevalonate did not change the enzyme activity further. It was concluded that sterols regulate the squalene synthetase activity, whereas, in contrast with the regulation of the HMG-CoA reductase activity in Hep G2 cells, mevalonate-derived non-sterols did not influence this enzyme.     

Regulation of squalene epoxidase in HepG2 cells

Regulation of squalene epoxidase in the cholesterol biosynthetic pathway was studied in a human hepatoma cell line, HepG2 cells. Since the squalene epoxidase activity in cell homogenates was found to be stimulated by the addition of Triton X-100, enzyme activity was determined in the presence of this detergent. Incubation of HepG2 cells for 18 h with L-654,969, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, increased squalene epoxidase activity dose-dependently. On the other hand, low density lipoprotein (LDL) and 25-hydroxy-cholesterol decreased the enzyme activity. These results demonstrate that squalene epoxidase is regulated by the concentrations of endogenous and exogenous sterols. The affinity of the enzyme for squalene was not changed by treatment with L-654,969. Cytosolic (S105) fractions, prepared from HepG2 cells treated with or without L-654,969, had no effect on microsomal squalene epoxidase activity of HepG2 cells, in contrast to the stimulating effect of S105 fractions from rat liver homogenate. Mevalonate, LDL, and oxysterol treatment abolished the effect of L-654,969. Simultaneous addition of cycloheximide and actinomycin D also prevented enzyme induction in HepG2 cells. From these results, the change in squalene epoxidase activity is thought to be caused by the change in the amount of enzyme protein. It is further suggested that squalene epoxidase activity is suppressed only by sterols, not by nonsterol derivative(s) of mevalonate, in contrast to the regulation of HMG-CoA reductase.     

Isoprenoid synthesis in isolated embryonic Drosophila cells. Sterol-independent regulatory signal molecule is distal to isopentenyl 1-pyrophosphates

Embryonic Drosophila cells (Kc cells) were used to further characterize sterol-independent modulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity. 3-Methyl-3-5-dihydroxyvalerate (mevalonate), 3-fluoromethyl-3,5-dihydroxyvalerate (fluoromevalonate), and 3-ethyl-3,5-dihydroxyvalerate (homomevalonate) were tested as modulators. Although mevalonate caused a rapid, reversible suppression of reductase activity, fluoro- and homomevalonate increased activity; fluoromevalonate was more effective than homomevalonate. Mevalonate, added simultaneously with fluoromevalonate, blocked the analogue's effect on Kc cell reductase activity. However, mevalonate did not suppress an established fluoromevalonate increase in HMG-CoA reductase activity. Fluoromevalonate blocked [1-14C, 5-3H]mevalonate conversion to 14CO2- and 3H-labeled lipids and [3H] mevalonate 5-pyrophosphate accumulated. Neither protein nor RNA synthesis were required for mevalonate-mediated suppression of reductase activity. However, fluoromevalonate's effect on reductase activity required protein synthesis. Furthermore, in the absence of protein synthesis, fluoromevalonate-stabilized Kc cell HMG-CoA reductase activity. We have concluded that mevalonate, fluoromevalonate, homomevalonate, and compactin (mevinolin) modulated HMG-CoA reductase activity because they altered isoprenoid carbon flow to a post-isopentenyl 1-pyrophosphate regulatory, signal molecule.     

Sterol synthesis is up-regulated in cholesterol-loaded pigeon macrophages during induction of cholesterol efflux.

The extent to which cholesterol synthesis is modulated in macrophage foam cells by changes in cholesterol influx and efflux was determined using thioglycollate-elicited peritoneal macrophages from normal and cholesterol-fed White Carneau (WC) and Show Racer (SR) pigeons. In peritoneal macrophages from normocholesterolemic pigeons, sterol synthesis from [(14)C]-acetate was down-regulated by more than 90% following incubation in vitro with beta-VLDL. Sterol synthesis was increased when the cellular free cholesterol concentration was decreased in response to stimulation of cholesterol efflux with apoHDL/phosphatidylcholine vesicles and cyclodextrin. Peritoneal macrophages isolated from hypercholesterolemic pigeons were loaded with cholesterol to levels similar to foam cells from atherosclerotic plaques (375-614 microg/mg cell protein), and had an extremely low rate of sterol synthesis. When cholesterol efflux was stimulated in these cells, sterol synthesis increased 8 to 10-fold, even though the cells remained grossly loaded with cholesterol. Cholesterol efflux also stimulated HMG-CoA reductase activity and LDL receptor expression. This suggests that only a small portion of the total cholesterol pool in macrophage foam cells was responsible for regulation of sterol synthesis, and that cholesterol generated by hydrolysis of cholesteryl esters was directed away from the regulatory pool by efflux from the cells. When the increase in sterol synthesis was blocked with the HMG-CoA reductase inhibitor mevinolin, there was no difference in the cholesterol content of the cells, or in the mass efflux of cholesterol into the culture medium.Thus, under these conditions, the increase in cholesterol synthesis during stimulation of cholesterol efflux does not appear to contribute significantly to the mass of cholesterol in these macrophage foam cells. Whether a similar situation exists in vivo is unknown.     

Posthatching evolution of in vivo mevalonate metabolism in chick liver

The in vivo mevalonate incorporation into total nonsaponifiable lipids by chick liver was minimal after hatching and drastically increased between 1-5 days. The hepatic synthesis of different cholesterol precursors emerged sequentially after hatching. Between 1-5 days increased strongly the conversion of mevalonate into squalene and also the formation of oxygenated lanosterol derivatives from squalene. The conversion of squalene became completely active at day 8. Cholesterol formation from lanosterol derivatives was completely activated between 8-11 days. Results in this paper demonstrate for the first time the accumulation of a fraction of nonsaponifiable lipids identified as lanosterol derivatives and cholesterol precursors formed from [5-14C]mevalonate in experiments carried out in vivo. Postnatal evolution of these oxysterols may explain the great increase of 3-hydroxy-3-methylglutaryl-CoA reductase activity found in chick liver between 5-11 days, simultaneous or posterior to the diminution of the oxygenated cholesterol precursors.     

HMG-CoA reductase inhibitors perturb fatty acid metabolism and induce peroxisomes in keratinocytes.

Topical lovastatin stimulates epidermal fatty acid synthesis in vivo; therefore, studies were undertaken to examine the effects of HMG-CoA reductase inhibitors on fatty acid metabolism in cultured keratinocytes. When exposed to fluindostatin or lovastatin for greater than or equal to 24 h, keratinocytes in serum-free media accumulated nile red-fluorescent lipid droplets. By 72 h, the triacylglycerol and phospholipid content were increased 2.5- and 1.3-fold, respectively. Reductase inhibitors (1-10 microM) increased fatty acid synthesis approximately 1.5-fold; increased synthesis was noted only after greater than 15 h exposure and was distributed among phospholipids and triacylglycerols. Oxidation of [14C]palmitate to CO2 was decreased greater than 50% in inhibitor-treated cultures, and label accumulated in triacylglycerols. Inhibitor-treated keratinocytes exhibited increased numbers of peroxisomes, using diaminobenzidene ultracytochemistry. Peroxisomal hyperplasia was also demonstrated by increased catalase activity (1.5- to 2.5-fold), increased dihydroxyacetone phosphate acyltransferase activity (1.4-fold) and increased peroxisomal (KCN-insensitive) fatty acid oxidation (1.4-fold) in inhibitor-treated cultures. Thus HMG-CoA reductase inhibitors increase fatty acid synthesis, induce triacylglycol and phospholipid accumulation, and induce peroxisomes in cultured keratinocytes. Coincubations with either low density lipoproteins or 25-hydroxycholesterol prevented both the peroxisomal hyperplasia and increased fatty acid synthesis, suggesting that these effects of reductase inhibitors may be linked to their effects on the cholesterol biosynthetic pathway.     

HMG-CoA reductase inhibition causes neurite loss by interfering with geranylgeranylpyrophosphate synthesis

To determine whether neurite outgrowth depends upon the mevalonate pathway, we blocked mevalonate synthesis in nerve growth factor-treated PC12 cells or primary cortical neurones with atorvastatin, a 3-hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, and substituted different intermediates of the mevalonate pathway. We show that HMG-CoA reductase inhibition causes a profound reduction of neurite length, neurite loss and ultimatively cell death in undifferentiated and pre-differentiated PC12 cells and also in rat primary cortical neurones. Geranylgeranylpyrophosphate, but not farnesylpyrophosphate, squalene or cholesterol, completely compensated for the lack of mevalonate. Our data indicate that, under HMG-CoA reductase inhibition, geranylgeranylpyrophosphate rather than farnesylpyrophosphate or cholesterol is critical for neurite outgrowth and/or maintenance. Loss of neurites is an early manifestation of various neurodegenerative disorders, and dysfunction of isoprenylation might play a role in their pathogenesis.     

Regulation of cholesterol synthesis in cultured mouse mammary carcinoma FM3A cells

Mouse mammary carcinoma FM3A cells, which are able to grow in a serum-free medium, have novel characteristics that could be valuable in biochemical and somatic cell genetic studies. In FM3A cells grown in the presence of serum, both sterol synthesis and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the major rate-limiting enzyme in the cholesterol biosynthetic pathway, were strongly suppressed by human low density lipoprotein (LDL). The addition of LDL (50 micrograms protein/ml) resulted in a 50% decrease in the reductase activity within 3 h and a 95% reduction after 24 h. Similarly, over 90% suppression of the reductase activity was obtained by the addition of LDL or mevalonolactone when the cells were grown on a serum-free medium. ML-236B (compactin), a specific inhibitor of HMG-CoA reductase, inhibited sterol synthesis from [14C]acetate by 80% at 1 microM. Reductase activity in FM3A cells was increased by 2.5- to 5-fold when the cells were treated with ML-236B (at 0.26-2.6 microM for 24 h). Thus, in FM3A cells, HMG-CoA reductase activity responded well to LDL, as is observed in human skin fibroblasts. Along with other novel features of this cell line, the present observations indicate that FM3A cells should be useful in biochemical and somatic cell genetic analysis of cholesterol metabolism, especially as regards the regulation of HMG-CoA reductase activity.     

Cholesterol synthesis in cultured skin fibroblasts from patients with Huntington's disease

In view of the proposed membrane defect in Huntington's disease, cultured skin fibroblasts from healthy volunteers and patients with Huntington's disease were compared with respect to their ability to carry out de novo synthesis of cholesterol. At confluency, values for incorporation of [14C]acetate and 3H2O into cholesterol, and activities of HMG-CoA reductase (the rate-limiting enzyme in the cholesterol biosynthetic pathway), did not differ significantly in the Huntington's disease cells compared to the controls. Determinations of total cellular cholesterol gave similar ratios of cholesterol/protein and cholesterol/phospholipid in the Huntington's disease and control fibroblasts. The data suggest that the proposed generalized cell membrane abnormality in Huntington's disease cannot be attributed to a defect in the cholesterol biosynthetic pathway.     

Chloroquine inhibits cyclization of squalene oxide to lanosterol in mammalian cells

Chloroquine inhibits the incorporation of [14C]acetate into sterols at a concentration of 10 microM or more in mouse L cells but has no effect on fatty acid synthesis and CO2 production from the same substrate even at a 10-fold higher concentration of the drug. The site of inhibition is distal to the formation of mevalonate since chloroquine also inhibits [14C]mevalonate metabolism to sterols and does not decrease the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) or the incorporation of [14C]acetate into the total nonsaponifiable lipids. Analyses by thin layer and high pressure liquid chromatography of the nonsaponifiable lipid fraction from cultures incubated with chloroquine show an accumulation of radioactivity in the region of squalene oxide. Identification of the radiolabeled lipid as squalene oxide has been established by: (a) its co-migration with the authentic squalene oxide standard; (b) its conversion into squalene glycol by acid hydrolysis; and (c) its further metabolism to desmosterol when chloroquine is removed from the medium. Addition of chloroquine (12.5-50 microM) to 20,000 X g supernatant fractions of mouse liver homogenates inhibits the incorporation of [14C]mevalonolactone into cholesterol and lanosterol, with corresponding increases of [14C]squalene oxides, in a concentration-dependent manner. It appears, therefore, that chloroquine inhibits the enzymatic step catalyzed by 2,3-oxidosqualene-lanosterol cyclase (EC 5.4.99.7). Incubation of cell cultures with chloroquine (50 microM) arrests cell growth and causes cell death after 1-3 days. However, simultaneous incubation of chloroquine with either cholesterol or lanosterol prevents cell death and permits cell growth. Uptake of chloroquine is not affected by exogenous sterols since intracellular chloroquine concentrations are the same in cells grown with or without added sterols. The cytotoxicity of chloroquine, under our experimental conditions, must, therefore, be due primarily to its inhibition of sterol synthesis. In addition to its well known effect on protein catabolism, chloroquine has been found to inhibit protein synthesis. The significance of these findings concerning the use of chloroquine in studying the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity is discussed.     

Modulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase by azole antimycotics requires lanosterol demethylation, but not 24,25-epoxylanosterol formation

The lanosterol 14 alpha-methyl demethylase inhibitors miconazole and ketoconazole have been used to assess their effects upon cholesterol biosynthesis in cultured Chinese hamster ovary cells. In Chinese hamster ovary cells treated with either agent, an initial accumulation of lanosterol and dihydrolanosterol has been observed. At elevated concentrations, however, ketoconazole, but not miconazole, causes the preferential accumulation of 24,25-epoxylanosterol and squalene 2,3:22,23-dioxide. These metabolites accumulate at the expense of lanosterol, thereby demonstrating a second site of inhibition for ketoconazole in the sterol biosynthetic pathway. Both demethylase inhibitors produced a biphasic modulation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway. The biphasic modulation is characterized by low levels of the drugs suppressing HMG-CoA reductase activity which is restored to either control or above control values at higher drug concentrations. This modulatory effect of the lanosterol demethylase inhibitors upon HMG-CoA reductase was not observed in the lanosterol 14 alpha-methyl demethylase-deficient mutant AR45. Suppression of HMG-CoA reductase activity is shown to be due to a decrease in the amount of enzyme protein consistent with a steroidal regulatory mechanism. Collectively, the results establish that lanosterol 14 alpha-methyl demethylation, but not 24,25-epoxylanosterol formation, is required to suppress HMG-CoA reductase in the manner described by lanosterol demethylase inhibitors.     

Incorporation of mevalonate into dolichol and other isoprenoids during estrogen-induced chick oviduct differentiation

Incorporation of [14C]mevalonate into dolichol and other isoprenoid compounds by chick oviduct explants has been studied. A reliable assay of dolichol biosynthesis employing several chromatographic procedures, including two-dimentional TLC, was developed. Incorporation of [14C]mevalonate into dolichol by oviduct explants was linear for at least 6 h. The effect of estrogen-induced differentiation was studied by incubation of explants obtained from chicks treated for various periods of time with diethylstilbestrol. Mevalonate incorporation into dolichol, when expressed as cpm per g of tissue, was not affected by estrogen treatment, but since the oviduct increased about 100-fold in mass during differentiation, each oviduct synthesizes about 100-fold more dolichol. In most tissues, the major product of mevalonate incorporation is cholesterol. However, although approx. 90% of the non-saponifiable 14C-labeled compounds were in the so-called 'cholesterol fraction', oviduct explants from estrogenized chicks synthesized little, if any, cholesterol. A number of cholesterol biosynthetic intermediates were observed, with compounds comigrating with squalene and lanosterol accounting for about 50% of the total. Since the estrogenized chick has serum cholesterol levels in the range of 800-900 mg/dl, these results suggest that oviduct has secondary control points which allow it to inhibit cholesterol synthesis when mevalonate is used as the precursor. In support of this hypothesis is the observation that explants from untreated chicks can incorporate mevalonate into cholesterol.     

Green and black tea extracts inhibit HMG-CoA reductase and activate AMP kinase to decrease cholesterol synthesis in hepatoma cells

Recent studies have demonstrated that green and black tea consumption can lower serum cholesterol in animals and in man, and suppression of hepatic cholesterol synthesis is suggested to contribute to this effect. To evaluate this hypothesis, we measured cholesterol synthesis in cultured rat hepatoma cells in the presence of green and black tea extracts and selected components. Green and black tea decreased cholesterol synthesis by up to 55% and 78%, respectively, as measured by a 3-h incorporation of radiolabeled acetate. Inhibition was much less evident when radiolabeled mevalonate was used, suggesting that the inhibition was mediated largely at or above the level of HMG-CoA reductase. Both extracts directly inhibited HMG-CoA reductase when added to microsomal preparations, although the extent of inhibition was considerably less than the decrease in cholesterol synthesis observed in whole cells. As HMG-CoA reductase activity also can be decreased by enzyme phosphorylation by AMP kinase, the phosphorylation state of HMG-CoA reductase and AMP kinase, which is activated by phosphorylation, was determined in lysates from cells treated with tea extracts. Both extracts increased AMP-kinase phosphorylation and HMG-CoA reductase phosphorylation by 2.5- to 4-fold, but with different time courses: maximal phosphorylation with green tea was evident within 30 min of treatment, whereas with black tea phosphorylation was slower to develop, with maximal phosphorylation occurring > or =3 hours after treatment. These results suggest that both green and black tea decrease cholesterol synthesis in whole cells by directly inhibiting HMG-CoA reductase and by promoting its inactivation by AMP kinase.     

Isopentenoid synthesis in isolated embryonic Drosophila cells. Possible regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by shunted mevalonate carbon

Our previous studies (Watson, J. A., Havel, C. M., Lobos, D. V., Baker, F. C., and Morrow, C. J. (1985) J. Biol. Chem. 260, 14083-14091) suggested that a matabolite, distal to isopentenyl 1-pyrophospate (IPP), served as a regulatory signal for sterol-independent modulation of Kc cell 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity. This report summarizes efforts to localize the potential source of the post-IPP regulatory signal molecule. We found no direct correlation between mevalonate-mediated suppression of Kc cell HMG-CoA reductase activity and the rates of [1-14C]-, [3-14C]-, [5-14C]-, or [5-3H]mevalonate incorporation into either carbon dioxide, neutral lipids, water, or water-soluble isopentenoid pyrophosphate esters. [1-14C]Mevalonate's rate of conversion to 14CO2 (a measure of total isopentenyl 1-pyrophosphate synthesis) was minimally 5-fold greater than that for neutral isopentenoid lipid synthesis (measured with either [5-3H]-, [3-14C]-, or [5-14C]mevalonate). However, [5-3H]mevalonate's rate of conversion into [3H]H2O (measure of shunted mevalonate carbon) was equivalent or greater than that measured for neutral isopentenoid lipid synthesis. [5-14C]Mevalonate radioactivity was incorporated into macromolecules and n-fatty acids. Kc cell extracts (100,000 X g supernatant fluid) readily oxidized alcohols with the following activity sequence: geraniol = nerol greater than farnesol = dimethylallyl alcohol greater than geranylgeraniol, isopentenyl alcohol, and allyl alcohol. Oxidation required NAD, and ethanol was not a substrate. We conclude that (a) Kc cells shunted a significant fraction (greater than or equal to 40%) of their post-IPP carbon to prenols for oxidative catabolism and (b) that shunted mevalonate carbon may play a significant role in the mevalonate-mediated regulation of Kc cell HMG-CoA reductase activity.