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.     

Lanosterol 14 alpha-methyl demethylase. Isolation and characterization of the third metabolically generated oxidative demethylation intermediate

Conditions have been identified which permit metabolic formation of the third oxidized intermediate in the lanosterol 14 alpha-methyl demethylase reaction cascade. Metabolism of either the immediate precursor substrate 3 beta-hydroxylanost-8-en-32-al or lanost-8-ene-3 beta,32-diol under mixed function oxidase conditions affords formation of the intermediate. It must be emphasized that the intermediate can only be detected if saponification procedures are omitted during sterol isolation. Comparative chemical and biochemical studies of the isolated metabolite with 3 beta,15 alpha-dihydroxylanost-8-en-32-al reveal that the metabolite is not the 15 alpha-hydroxylanosterol aldehyde, a putative demethylase intermediate. The metabolite is efficiently converted to the demethylated delta 8,14-diene sterol in the absence of molecular oxygen or NADPH, thus supporting its identity as the final oxidized intermediate in the lanosterol 14 alpha-methyl demethylase cascade. 1H NMR analysis shows a proton resonance at 7.86 ppm consistent with a formyloxy proton. Mass spectral and infrared analysis of the metabolite clearly establish oxygen insertion into the immediate precursor substrate, 3 beta-hydroxylanost-8-en-32-al. Collectively, the biochemical and chemical characteristics of the metabolite support a structural assignment for the metabolite as 14 alpha-formyloxy-lanost-8-en-3 beta-ol.     

Regulation of hepatic cholesterol biosynthesis. Effects of a cytochrome P-450 inhibitor on the formation and metabolism of oxygenated sterol products of lanosterol.

The involvement of oxygenated cholesterol precursors in the regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was studied by examining the effect of ketoconazole on the metabolism of mevalonic acid, lanosterol and the lanosterol metabolites, lanost-8-ene-3 beta,32-diol,3 beta-hydroxylanost-8-en-32-al and 4,4-dimethylcholesta-8,14-dien-3 beta-ol, in liver subcellular fractions and hepatocyte cultures. Inhibition of cholesterol synthesis from mevalonate by ketoconazole at concentrations up to 30 microM was due exclusively to a suppression of cytochrome P-450LDM (LDM = lanosterol demethylase) activity, resulting in a decreased rate of lanosterol 14 alpha-demethylation. No enzyme after the 14 alpha-demethylase step was affected. When [14C]mevalonate was the cholesterol precursor, inhibition of cytochrome P450LDM was accompanied by the accumulation of several labelled oxygenated sterols, quantitatively the most important of which was the C-32 aldehyde derivative of lanosterol. There was no accumulation of the 24,25-oxide derivative of lanosterol, nor of the C-32 alcohol. Under these conditions the activity of HMG-CoA reductase declined. The C-32 aldehyde accumulated to a far greater extent when lanost-8-ene-3 beta,32-diol rather than mevalonate was used as the cholesterol precursor in the presence of ketoconazole. With both precursors, this accumulation was reversed at higher concentrations of ketoconazole in liver subcellular fractions. A similar reversal was not observed in hepatocyte cultures.     

A mammalian mutant cell lacking detectable lanosterol 14 alpha-methyl demethylase activity

A Chinese hamster ovary cell mutant, AR45, was selected for amphotericin B resistance after treatment with the mutagen ethyl methanesulfonate. The mutant is a cholesterol auxotroph with a deficiency in cholesterol biosynthesis. Whole cell experiments demonstrate that the mutant accumulates the C30 sterols, lanosterol and dihydrolanosterol, under culture conditions which promote active sterol biosynthesis. Metabolic studies show that the C29 sterol demethylation product of lanosterol, but not lanosterol itself, is actively converted to end product cholesterol by whole cells as well as by microsomal preparations derived from the mutant. Detectable amounts of several cytochromes can be observed spectrally in the AR45 demonstrating that it is not a general heme-deficient mutant. Collectively, these results characterize the AR45 mutant cells as being lanosterol 14 alpha-methyl demethylase-deficient. The cell line should prove useful in studying regulation of the demethylase enzyme and the putative endogenous regulatory oxysterol. It should also be a useful tool in the molecular cloning and elucidation of genetic properties of the demethylase.     

Regulation of hepatic lanosterol 14 alpha-demethylase gene expression by dietary cholesterol and cholesterol-lowering agents.

Binding of sterol response element binding protein 1a to sterol response element-1 (SRE-1) in the promoter region of lanosterol 14 alpha-demethylase (14DM) has been demonstrated previously. Decreased 14DM activity has been shown to result in accumulation of the intermediate, 3 beta-hydroxy-lanost-8-en-32-al, a known translational downregulator of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Since it has also been demonstrated that feedback regulation of hepatic HMG-CoA reductase occurs primarily at the level of translation, the effects of dietary cholesterol and cholesterol lowering agents on levels of hepatic 14DM mRNA and immunoreactive protein were investigated. Addition of 1% cholesterol to a chow diet markedly decreased hepatic 14DM mRNA and protein levels in Sprague-Dawley rats. The extent and time course of this decrease in 14DM immunoreactive protein closely paralleled that of HMG-CoA reductase. Supplementation of the diet with the HMG-CoA reductase inhibitor, Lovastatin, to a level of 0.02%, raised 14DM mRNA and protein levels 2- to 3-fold. Addition of 2% Colestipol, a bile acid binding resin, to the chow diet caused smaller increases. The highest level of 14DM protein expression was observed in liver, the major site of feedback regulation of HMG-CoA reductase by cholesterol. Taken together, these observations suggest a critical role for 14DM in the feedback regulation of hepatic HMG-CoA reductase.     

Sterol-independent regulation of 3-hydroxy-3-methylglutaryl-CoA reductase by mevalonate in Chinese hamster ovary cells. Magnitude and specificity

In this paper, we assess the relative degree of regulation of the rate-limiting enzyme of isoprenoid biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, by sterol and nonsterol products of mevalonate by utilizing cultured Chinese hamster ovary cells blocked in sterol synthesis. We also examine the two other enzymes of mevalonate biosynthesis, acetoacetyl-CoA thiolase and HMG-CoA synthase, for regulation by mevalonate supplements. These studies indicate that in proliferating fibroblasts, treatment with mevalonic acid can produce a suppression of HMG-CoA reductase activity similar to magnitude to that caused by oxygenated sterols. In contrast, HMG-CoA synthase and acetoacetyl-CoA thiolase are only weakly regulated by mevalonate when compared with 25-hydroxycholesterol. Furthermore, neither HMG-CoA synthase nor acetoacetyl-CoA thiolase exhibits the multivalent control response by sterol and mevalonate supplements in the absence of endogenous mevalonate synthesis which is characteristic of nonsterol regulation of HMG-CoA reductase. These observations suggest that nonsterol regulation of HMG-CoA reductase is specific to that enzyme in contrast to the pleiotropic regulation of enzymes of sterol biosynthesis observed with oxygenated sterols. In Chinese hamster ovary cells supplemented with mevalonate at concentrations that are inhibitory to reductase activity, at least 80% of the inhibition appears to be mediated by nonsterol products of mevalonate. In addition, feed-back regulation of HMG-CoA reductase by endogenously synthesized nonsterol isoprenoids in the absence of exogenous sterol or mevalonate supplements also produces a 70% inhibition of the enzyme activity.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol biosynthesis by oxylanosterols

Treatment of rat intestinal epithelial cell cultures with the oxidosqualene cyclase inhibitor, 3 beta-[2-(diethylamino)-ethoxy]androst-5-en-17-one (U18666A), resulted in an accumulation of squalene 2,3:22,23-dioxide (SDO). When U18666A was withdrawn and the cells were treated with the sterol 14 alpha-demethylase inhibitor, ketoconazole, SDO was metabolized to a product identified as 24(S),25-epoxylanosterol. To test the biological effects and cellular metabolism of this compound, we prepared 24(RS),25-epoxylanosterol by chemical synthesis. The epimeric mixture of 24,25-epoxylanosterols could be resolved by high performance liquid chromatography on a wide-pore, non-endcapped, reverse phase column. Both epimers were effective suppressors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity of IEC-6 cells. The suppressive action of the natural epimer, 24(S),25-epoxylanosterol, but not that of 24(R),25-epoxylanosterol could be completely prevented by ketoconazole. IEC-6 cells could efficiently metabolize biosynthetic 24(S),25-epoxy[3H]anosterol mainly to the known reductase-suppressor 24(S),25-epoxycholesterol. This metabolism was substantially reduced by ketoconazole. These data support the conclusion that 24(S),25-epoxylanosterol per se is not a suppressor of HMG-CoA reductase activity but is a precursor to a regulatory oxysterol(s). It has recently been reported that 25-hydroxycholesterol can occur naturally in cultured cells in amounts sufficient to effect regulation of HMG-CoA reductase (Saucier et al. 1985. J. Biol. Chem. 260: 14571-14579). In order to investigate the biological effects of possible precursors of 25-hydroxycholesterol, we chemically synthesized 25-hydroxylanosterol and 25-hydroxylanostene-3-one. Both oxylanosterol derivatives suppressed cellular sterol synthesis at the level of HMG-CoA reductase. U18666A had the unusual effect of potentiating the inhibitory effect of 25-hydroxylanostene-3-one but did not influence the effect of other oxylanosterols. All the oxylanosterols, with the exception of 25-hydroxylanostene-3-one, enhanced intracellular esterification of cholesterol. The foregoing observations support consideration of oxylanosterols as playing an important role in the biological formation of regulatory oxysterols that modulate sterol biosynthesis at the level of HMG-CoA reductase.     

Mechanistic studies of lanosterol 14 alpha-methyl demethylase: substrate requirements for the component reactions catalyzed by a single cytochrome P-450 isozyme

Lanosterol 14 alpha-methyl demethylation is a cytochrome P-450-dependent process that proceeds through the oxidative sequence of alcohol, aldehyde followed by decarbonylation with formic acid release. Microsomal metabolism studies shown here indicate that only lanostenols and 32-oxy-lanostenols with unsaturation at either the delta 7 or delta 8 position in the sterol can be demethylated. The 14 alpha-methyl group of either lanostan-3 beta-ol or delta 6 lanostenol is not oxidized to the anticipated C-32 alcohol or aldehyde by the enzyme, nor are the corresponding 32-oxy-lanostanols demethylated when incubated with microsomal preparations. Despite the lack of metabolism, the saturated and delta 6 sterol analogues are effective competitive inhibitors of demethylase activity. Utilizing preferred substrates, comparison of the component reactions of the demethylation sequence shows that both the oxidative function and lyase function are sensitive to common inhibitors and that both activities require NADPH. These findings strongly support the premise that a P-450 isozyme does catalyze each phase of the lanosterol 14 alpha-methyl demethylation sequence. Collectively these results demonstrate the double-bond requirement for both components of the demethylation sequence and suggest that the olefinic electrons at delta 7 or delta 8 but not delta 6 may participate directly during demethylation. This participation may involve stabilizing a transition state intermediate or directing activated oxygen insertion as part of the P-450 monoxygenase mechanism.     

Identification of lanosterol 14 alpha-methyl demethylase in human tissues

Lanosterol 14 alpha-methyl demethylase was investigated in human tissues using a radio-HPLC assay to detect the 4,4-dimethyl-5 alpha-cholesta-8, 14-dien-3 beta-ol (diene) metabolite. The sequence of events leading to the demethylated product in human liver microsomes involves the conversion of the diol to the aldehyde followed by diene formation. Enzyme activity displayed a greater than 10 fold variation among the 9 liver samples studied. Kinetic parameters were determined and shown to differ between two separate liver samples. Addition of inhibitors of yeast lanosterol 14 alpha demethylase, ketoconazole and miconazole, resulted in extensive inhibition of formation of the demethylated metabolite. The enzyme, detected in microsomes isolated from human kidney and lymphocytes, also catalyzed the conversion of dihydrolanosterol to oxylanosterol intermediates and the diene. The presence of this enzyme in microsomes from various human tissues suggests that it may play a role in cellular regulation of cholesterol synthesis.     

Cytochrome P-450-dependent oxidation of lanosterol in cholesterol biosynthesis. Microsomal electron transport and C-32 demethylation

Electron transfer to rat liver microsomal cytochrome P-450 of 14 alpha-methyl group demethylation of 24,25-dihydrolanosterol (C30-sterol) has been studied with a new radio-high-performance liquid chromatography assay. The monooxygenase is dependent upon NADPH plus oxygen, insensitive to CN-, and sensitive to CO. Microsomal oxidation is also sensitive to trypsin digestion, and reactivation is dependent upon the addition of purified, detergent-solubilized cytochrome P-450 reductase. Electron transport of C-32 sterol demethylation can be fully supported by very low concentrations of NADPH (approximately 10 microM) only in the presence of saturating concentrations of NADH (approximately 200 microM) suggesting involvement of cytochrome b5-dependent electron transfer in addition to the NADPH-supported pathway. The cytochrome P-450 of 14 alpha-demethylation has been solubilized with detergents, resolved chromatographically from cytochrome P-450 reductase and cytochrome b5, and fully active C-32 demethylase reconstituted. Incubation of intact microsomes with NADH and very low concentrations of NADPH described above leads to interruption of demethylation without 14 alpha-methyl group elimination. Under these conditions, C-32 oxidation products of the C30-sterol substrate accumulate at the expense of formation of demethylated, C29-sterol products. This enzymic interruption of C-32 demethylation, accumulation of oxygenated C30-sterols, along with subsequent demethylation of the isolated C30-oxysterols under similar oxidative conditions supports the suggestion that 14 alpha-hydroxymethyl and aldehydic sterols are metabolic intermediates of sterol 14 alpha-demethylation. Only very modest inductions of the constitutive cytochrome P-450 isozyme of 14 alpha-methyl sterol oxidase can be obtained with just 2 out of 12 known, potent inducers of mammalian hepatic cytochrome P-450s. Alternatively, administration of complete adjuvant in mineral oil drastically reduces amounts of total microsomal cytochrome P-450 while activity of 14 alpha-methyl sterol oxidase is not affected dramatically. Thus, as much as 2.5-fold enhancement of C-32 oxidase specific activity is obtained when expressed per unit of cytochrome P-450.     

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.     

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in human hepatoma cell line Hep G2. Effects of inhibitors of cholesterol synthesis on enzyme activity.

Incubating Hep G2 cells for 18 h with triparanol, buthiobate and low concentrations (less than 0.5 microM) of U18666A, inhibitors of desmosterol delta 24-reductase, of lanosterol 14 alpha-demethylase and of squalene-2,3-epoxide cyclase (EC 5.4.99.7) respectively, resulted in a decrease of the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase activity. However, U18666A at concentrations higher than 3 microM increased the HMG-CoA reductase activity in a concentration-dependent manner. None of these inhibitors influenced directly the reductase activity in Hep G2 cell homogenates. Analysis by t.l.c. of 14C-labelled non-saponifiable lipids formed from either [14C]acetate or [14C]mevalonate during the cell incubations confirmed the sites of action of the drugs used. Beside the 14C-labelled substrates of the blocked enzymes and 14C-labelled cholesterol, another non-saponifiable lipid fraction was observed, which behaves as polar sterols on t.l.c. This was the case with triparanol and at those concentrations of U18666A that decreased the reductase activity, suggesting that polar sterols may play a role in suppressing the reductase activity. In the presence of 30 microM-U18666A (sterol formation blocked) the increase produced by simultaneously added compactin could be prevented by addition of mevalonate. This indicates the existence of a non-sterol mevalonate-derived effector in addition to a sterol-dependent regulation. LDL (low-density lipoprotein), which was shown to be able to decrease the compactin-induced increase in reductase activity, could not prevent the U18666A-induced increase. On the contrary, LDL enhanced the U18666A effect, showing that the LDL regulation is not merely the result of introducing cholesterol to the cells.     

Differential regulation of low density lipoprotein suppression of HMG-CoA reductase activity in cultured cells by inhibitors of cholesterol biosynthesis

Treatment of rat intestinal epithelial cells (IEC-6 cells) with lanosterol 14 alpha-demethylase inhibitors, ketoconazole and miconazole, had similar effects on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and cholesterol biosynthesis but the drugs differed in their ability to prevent the low density lipoprotein (LDL) suppression of reductase activity. Miconazole, at concentrations that inhibited the metabolism of lanosterol and epoxylanosterol to the same degree as ketoconazole, did not prevent low density lipoprotein action on reductase activity, whereas ketoconazole totally abolished the low density lipoprotein action on reductase activity. Both drugs caused: 1) a biphasic response in reductase activity such that at low concentrations (less than 2 microM) reductase activity was inhibited and at high concentrations (greater than 5 microM) the activity returned to control or higher than control levels; 2) an inhibition of metabolism of lanosterol to cholesterol, and 24(S), 25-epoxylanosterol to 24(S), 25-epoxycholesterol. Neither drug prevented suppression of reductase activity by 25-hydroxylanosterol, 25-hydroxycholesterol, or mevalonolactone added to the medium. Each drug increased the binding, uptake, and degradation of 125I-labeled LDL and inhibited the re-esterification of free cholesterol to cholesteryl oleate and cholesteryl palmitate. The release of free cholesterol from [3H]cholesteryl linoleate LDL could not account for the differential effect of ketoconazole and miconazole on the prevention of low density lipoprotein suppression of reductase activity. The differential effect of the drugs on low density lipoprotein suppression of reductase activity was not unique to IEC-6 cells, but was also observed in several cell lines of different tissue origin such as human skin fibroblast cells (GM-43), human hepatoblastoma cells (HepG2), and Chinese hamster ovary cells (wild type, K-1; 4 alpha-methyl sterol oxidase mutant, 215). These observations suggest that the suppressive action of low density lipoprotein on reductase activity 1) does not require the de novo synthesis of cholesterol, or 24(S), 25-epoxysterols; 2) is not mediated via the same mechanism as that of mevalonolactone; and 3) does not involve cholesteryl reesterification. Ketoconazole blocks a site in the process of LDL suppression of reductase activity that is not affected by miconazole.     

A relationship between the activities of hepatic lanosterol 14 alpha-demethylase and 3-hydroxy-3-methylglutaryl-CoA reductase.

At 1-2 h after intragastric administration of ketoconazole, a cytochrome P-450 inhibitor, to rats, there was a 50-60% decrease in the activity of hepatic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Inhibition reached a maximum at 6-12 h after the drug was given, but after 24 h enzyme activity was stimulated by 60%. The rates of synthesis of hepatic non-saponifiable lipids in vivo showed a similar time-dependent pattern of change. During the first few hours after drug administration, the hepatic cytochrome P-450-dependent metabolism of lanosterol was suppressed in vivo. However, 24 h after treatment, this activity was stimulated, an effect which was also observed by pre-treatment of the rats with the drug for several days. Suppression of hepatic HMG-CoA reductase and lanosterol 14 alpha-demethylase activities was accompanied by a relative increase in the accumulation of labelled polar sterols in the liver in vivo. In the intestine, ketoconazole also resulted in a rapid decline in the rate of synthesis of non-saponifiable lipids and an inhibition of lanosterol 14 alpha-demethylation in vivo. However, in contrast with the liver, there was no stimulation of non-saponifiable lipid synthesis after 24 h.     

Cholesterol Biosynthesis and Its Regulation in Dissociated Cell Cultures of Fetal Rat Brain: Developmental Changes and the Role of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase

Primary cultures of cells dissociated from fetal rat brain were utilized to define the developmental changes in cholesterol biosynthesis and the role of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in the regulation of these changes. Cerebral hemispheres of fetal rats of 15-16 days of gestation were dissociated mechanically into single cells and grown in the surface-adhering system. Cholesterol biosynthesis, studied as the rate of incorporation of [14C]acetate into digitonin-precipitable sterols, was shown to exhibit two distinct increases in synthetic rates, a prominent increase after 6 days in culture and a smaller one after 14 days in culture. Parallel measurements of HMG-CoA reductase activity also demonstrated two discrete increases in enzymatic activity, and the quantitative and temporal aspects of these increases were virtually identical to those for cholesterol synthesis. These data indicate that cholesterol biosynthesis undergoes prominent alterations with maturation and suggest that these alterations are mediated by changes in HMG-CoA reductase activity. The timing of the initial prominent peak in both cholesterol biosynthesis and HMG-CoA reductase activity at 6 days was found to be the same as the timing of the peak in DNA synthesis, determined as the rate of incorporation of [3H]thymidine into DNA. The second, smaller peak in reductase activity and sterol biosynthesis at 14 days occurred at the time of the most rapid rise in activity of the oligodendroglial enzyme, 2':3'-cyclic nucleotide 3'-phosphohydrolase (CNP). These latter observations suggest an intimate relationship of the sterol biosynthetic pathway with cellular proliferation and with oligodendroglial differentiation in developing mammalian brain.     

Activities of 3-hydroxyl-3-methylglutaryl-CoA reductase and acetyl-CoA carboxylase and the rate of mevalonic acid, squalene, sterol and fatty acid biosynthesis from [1-14C]acetyl-CoA and [2-14C]malonyl-CoA in rat liver: effects of Triton WR 1339, starvation and cholesterol diet

The effects of Triton WR 1339, starvation and cholesterol diet on the activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and acetyl-CoA carboxylase and on the rates of mevalonic acid (MVA) biosynthesis from acetyl-CoA and malonyl-CoA in the soluble (140 000 g) and microsomal fractions of rat liver, on the rate of incorporation of these substrates into squalene, cholesterol and lanosterol in the rat liver postmitochondrial fraction and on the rate of fatty acid biosynthesis was studied. The administration of Triton WR 1339 (200 mg per 100 g of body weight twice) stimulated the activity of HMG-CoA reductase and MVA biosynthesis from acetyl-CoA and malonyl-CoA in the intact and solubilized microsomal fractions and had no effect on these parameters in the soluble fraction. Starvation for 36 hrs did not cause inhibition of the reductase activity or MVA biosynthesis from both substrates in the soluble fraction. Alimentary cholesterol significantly increased the activity of HMG-CoA reductase, had no effect on the rate of MVA biosynthesis from acetyl-CoA and stimulated the malonyl-CoA incorporation in to MVA in the soluble fraction. Starvation an alimentary cholesterol inhibited the HMG-CoA reductase activity and MVA biosynthesis from both substrates in the solubilized microsomal fraction. Triton WR 1339 stimulated 4--19-fold the lipid formation in the total unsaponified fraction and its components i.e. squalene, lanosterol, cholesterol, from acetyl-CoA and only insignificantly (1,2--1,7-fold) increased malonyl-CoA incorporation into these compounds. Starvation and alimentary cholesterol repressed lanosterol and cholesterol biosynthesis from acetyl-CoA, decreased malonyl-CoA incorporation into these sterols and had no influence on squalene biosynthesis from the two substrates. Triton WR 1339 and starvation inhibited the acetyl-CoA carboxylase activity, unaffected by alimentary cholesterol. No significant changes in the rate of fatty acid biosynthesis from the substrates were observed. The data obtained provide evidence for the existence of autonomic pathways of MVA biosynthesis localized in the soluble and microsomal fractions of rat liver. The pathway of MVA biosynthesis in the soluble fraction is less sensitive to regulatory factors. Sterol biosynthesis from malonyl-CoA is also more resistant to regulatory effects than sterol biosynthesis from acetyl-CoA. This suggests that HMG-CoA reductase localized in the soluble fraction takes part in MVA and sterol biosynthesis from malonyl-CoA.     

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase and the rate of sterol synthesis diminish in cultures with high cell density

The specific activity of HMG-CoA reductase, the major rate-limiting enzyme in the sterol biosynthetic pathway, declined linearly with increasing cell density in four different lines of mammalian cell cultures. As expected, this caused the rates of sterol synthesis from [14C]acetate to decline in a parallel manner. The decrease in reductase activity in the dense cultures was also correlated with decreased incorporation of [14C]acetate into fatty acids and [3H]thymidine into DNA. In contrast, the activities of two enzymes, NADH dehydrogenase and 5'-nucleotidase, which are not involved in lipid synthesis, were independent of changes in cell density. The simplest explanation for these data is tht HMG-CoA reductase and the synthesis of sterol and fatty acids are regulated in concordance with the rate of cell growth and proliferation.     

Different substrate specificities of lanosterol 14a-demethylase (P-45014DM) of Saccharomyces cerevisiae and rat liver for 24-methylene-24,25-dihydrolanosterol and 24,25-dihydrolanosterol.

The purified lanosterol 14a-demethylase (P-45014DM) of S. cerevisiae catalyzed the 14a-demethylation of 24-methylene-24,25-dihydrolanosterol (24-methylenelanost-8-en-3 beta-ol, 24-methylene-DHL), the natural substrate of the demethylase of filamentous fungi, as well as its natural substrate, lanosterol. Lanosterol 14a-demethylase of rat liver microsomes also catalyzed the 14a-demethylation of 24-methylene-DHL, but the activity was considerably lower than that for lanosterol. The activity of the rat liver enzyme for 24-methylene-DHL was also lower than that for 24,25-dihydrolanosterol (DHL), while the activity of yeast P-45014DM for 24-methylene-DHL was considerably higher than that for DHL. Since 24-substituted sterols are not found in mammals and DHL is not an intermediate of ergosterol biosynthesis by yeast, above-mentioned different substrate specificities between the yeast and the mammalian 14a-demethylases may reflect certain evolutional alteration in their active sites in relation to the difference in their sterol biosynthetic pathways.     

Cholesterol is not synthesized in membranes bearing 3-hydroxy-3-methylglutaryl coenzyme A reductase

We have shown previously that newly synthesized lanosterol and cholesterol in homogenates of cultured human fibroblasts do not have the same equilibrium buoyant density as the 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) in the smooth endoplasmic reticulum (SER) (Lange, Y., and Steck, T. L. (1985) J. Biol. Chem. 260, 15592-15597). This finding suggested two alternative and novel hypotheses: (a) that lanosterol and cholesterol might be transported rapidly from the SER to other internal membranes or (b) that synthesis of the sterols is not associated with the SER, or at least not with that portion of this organelle bearing HMG-CoA reductase. We therefore compared the subcellular distribution of HMG-CoA reductase with that of enzymes which convert lanosterol to cholesterol. The two activities studied were the consumption of exogenous [3H]lanosterol and the conversion of exogenous radiolanosterol to radiocholesterol. Differential centrifugation, rate zonal centrifugation, and equilibrium sucrose gradient centrifugation of rat liver homogenates all showed that these enzyme activities did not comigrate with HMG-CoA reductase. The subcellular distribution of newly synthesized sterols also was examined in cultured human fibroblasts. Cells were incubated with radioactive acetate to label endogenous sterols biosynthetically, homogenized, and spun to equilibrium on sucrose gradients. The buoyant density profiles of radioactive cholesterol and lanosterol both had a peak at 1.12 g/cm3. Digitonin treatment shifted both sterols to higher densities, strong evidence that they resided in cholesterol-rich membranes. Pretreatment of intact cells with cholesterol oxidase, which selectively oxidizes plasma membrane cholesterol, abolished the digitonin shift of lanosterol but not of intracellular cholesterol. These findings provide support for the hypothesis that newly synthesized cholesterol and lanosterol are not in the same membrane.