Incorporation of mevalonate into squalene, lanosterol and cholesterol by different neonatal chick tissues

The role of neonatal chick liver and kidneys in the incorporation of mevalonic acid into squalene, lanosterol and cholesterol was studied. Differences between the synthesizing ability of these and other tissues and the influence of the in vivo or in vitro conditions were also examined. In the in vivo experiments, distribution of radioactivity among the nonsaponifiable lipids was not dependent of the doses of mevalonic acid injected. About 80-95% of radioactivity was recovered as cholesterol in liver and brain, whereas in kidneys this percentage was only about 35%. Squalene and lanosterol were formed by kidneys in a high percentage, higher than in liver and other tissues. 12 hr after mevalonate injection, the percentage of cholesterol formed by kidneys increased until more than 50%. In the in vitro experiments carried out in the presence of 0.045-4.0 mM mevalonate, cholesterol was also the main nonsaponifiable identified, but in a lesser percentage than in vivo. In the same conditions, the incorporation of mevalonic acid by kidneys was maximal into squalene. After in vitro incubations for 2 hr, the percentage of cholesterol in kidneys also increased.     

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.     

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.     

Age-related changes in the mevalonate metabolism in vivo in chick kidneys

The mevalonate incorporation in vivo into total nonsaponifiable lipids by chick kidneys drastically increased after hatching, reaching similar levels to those previously observed in liver. Cholesterol was the major sterol formed from mevalonate from 11 days onward, while a fraction of polar nonsaponifiable lipid(s) was observed as the major compound(s) synthesized at 5-8 days. Relative percentages of squalene, squalene oxide(s) and lanosterol synthesized from mevalonate also increased between 11-18 days after hatching. Results in this paper demonstrate for the first time the accumulation of a fraction of nonsaponifiable lipid(s) identified as lanosterol derivatives and cholesterol precursors formed by kidneys from [5-14C]mevalonate in experiments carried out in vivo, as well as their evolution during postnatal period.     

Quantitative role of different embryonic tissues in mevalonate metabolism by sterol and nonsterol pathways. Relationship with enzyme activities of cholesterogenesis

3-Hydroxy-3-methylglutaryl-CoA reductase, mevalonate kinase, mevalonate-5-phosphate kinase and mevalonate-5-pyrophosphate decarboxylase activities have been determined in brain, liver, intestine and kidneys from 19-day-old chick embryo. Levels of brain reductase and decarboxylase were clearly higher than those found in the other tissues assayed. However, only small differences were observed in the activity of both kinases among the different tissues. Mevalonate metabolism by sterol and nonsterol pathways has been investigated in chick embryo at the same developmental stage. Mevalonate incorporation into total nonsaponifiable lipids was maximal in liver, followed by intestine, brain and kidneys. The shunt pathway of mevalonate not leading to sterols was negligible in both brain and liver, while a clear CO2 production was observed in intestine and kidneys. Sterols running in TLC as lanosterol and cholesterol were the major sterols formed from mevalonate by brain and kidney slices, while squalene and squalene oxide(s) were found to be mainly synthesized by liver slices. Minor differences in the percentage of different sterols were observed in chick embryo intestine. The importance of free and esterified cholesterol accumulation in the different tissues on the inhibition of cholesterogenic activity is discussed.     

Sterol biosynthesis in the echinoderm Asterias rubens.

1. [2(-14)C]Mevalonic acid injected into the echinoderm Asterias rubens (Class Asteroidea) was effectively incorporated into the non-saponifiable lipid. 2. The most extensively labelled compounds were squalene and the 4,4-dimethyl sterols with much lower incorporations into the 4alpha-monomethyl and 4-demethyl sterol fractions. 3. Labelled compounds identified were squalene, lanosterol, 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol and 4alpha-methyl-5alpha-cholest-7-en-3beta-ol; these are all intermediates in sterol biosynthesis. 4. The major sterol in A. rubens, 5alpha-cholest-7-en-3beta-ol, was also labelled showing that this echinoderm is capable of sterol biosynthesis de novo. 5. No evidence was obtained for the incorporation of [2(-14)C]mevalonic acid into the C28 and C29 components of the 4-demethyl sterols or 9beta,19-cyclopropane sterols found in A. rubens and it is assumed that these sterols are of dietary origin. 6. Another starfish Henricia sanguinolenta also incorporated [2(-14)C]mevalonic acid into squalene and lanosterol. 7. Various isolated tissues of A. rubens were all capable of incorporation of [2(-14)C]mevalonic acid into the nonsaponifiable lipid. With the body-wall and stomach tissues radioactivity accumulated in squalene and the 4,4-dimethyl sterols, but with the gonads and pyloric caecae there was a more efficient incorporation of radioactivity into the 4-demethyl sterols, principally 5alpha-cholest-7-en-3beta-ol.     

Mevalonate metabolism by renal tissue in vitro

Previous studies from this laboratory have demonstrated that the kidneys rather than the liver play the major role in the in vivo metabolism of circulating mevalonic acid. Kidneys, however, convert mevalonic acid primarily to the precursors of cholesterol, squalene and lanosterol, rather than to cholesterol. This study was designed to define the specific tissue site within the kidney responsible for mevalonic acid metabolism. Tissue slices from rat and dog renal cortex and medulla and glomeruli and tubules were isolated, and the incorporation of (14)C-labeled mevalonic acid into the nonsaponifiable lipids squalene, lanosterol, and cholesterol was determined in these tissues. The results demonstrate that the renal cortex is the primary site of mevalonic acid metabolism within the kidney and that the glomerulus is responsible for 95% of the mevalonic acid metabolized by the renal cortex. As was the case for the whole kidney, the major metabolites of mevalonate in the glomeruli are squalene and lanosterol.     

Synthesis of sterols by chick brain and liver during embryonic development

The evolution throughout embryonic development of the rate at which acetate was converted into sterols was studied in chick brain and liver. Acetate incorporation (nmol/h/g tissue) was clearly higher in brain than in liver and sharply decreased with the age of embryo. Cholesterol and desmosterol were the major sterols formed from acetate by chick embryo brain, followed by lanosterol and squalene. No desmosterol was found in chick embryo liver, organ where cholesterol was the major sterol synthesized. In brain, the relative percentage of cholesterol increased throughout embryonic development reaching more than 50% at hatching, while the percentage of desmosterol decreased during the same period and represented at hatching only about 10–15% of the total nonsaponifiable fraction. The relative percentages of lanosterol and squalene did not change significantly throughout the period assayed. In liver, the percentage of cholesterol increased until 19 days but sharply decreased at hatching.     

Evidence for rate-limiting steps in sterol synthesis beyond 3-hydroxy-3-methylglutaryl-coenzyme A reductase in human leukocytes.

When human blood leukocytes are incubated with [2-14C]acetate only about 32% of the nonsaponifiable lipid radioactivity is recovered in digitonin-precipitable material. Using thin-layer chromatography and gas-liquid radiochromatography, we have determined that most of the label from [2-14C]acetate in the nonsaponifiable fractions is in lanosterol, squalene and an unidentified sterol. Only 11% of the acetate radioactivity is contained in cholesterol. This distribution does not change when cholesterol synthesis is depressed by the addition of lipoproteins to the medium. These findings are in marked contrast to studies with liver, where most of the nonsaponifiable radioactivity derived from acetate is recovered in digitonin-precipitable sterols. Furthermore, they suggest that rate-limiting steps beyond the 3-hydroxy-3-methylglutaryl coenzyme A reductase reaction exist in the sterol synthesis pathway of human leukocytes.     

Effect of geraniol on fatty-acid and mevalonate metabolism in the human hepatoma cell line Hep G2.

Monoterpenes have multiple pharmacological effects on the metabolism of mevalonate. Geraniol, a dietary monoterpene, has in vitro and in vivo anti-tumor activity against several cell lines. We have studied the effects of geraniol on growth, fatty-acid metabolism, and mevalonate metabolism in the human hepatocarcinoma cell line Hep G2. Up to 100 micromol geraniol/L inhibited the growth rate and 3-hydroxymethylglutaryl coenzyme A reductase (HMG-CoA) reductase activity of these cells. At the same concentrations, it increased the incorporation of cholesterol from the medium in a dose-dependent manner. Geraniol-treated cells incorporated less 14C-acetate into nonsaponifiable lipids, inhibiting its incorporation into cholesterol but not into squalene and lanosterol. This is indicative of an inhibition in cholesterol synthesis at a step between lanosterol and cholesterol, a fact confirmed when cells were incubated with 3H-mevalonate. The incorporation of 3H-mevalonate into protein was also inhibited, whereas its incorporation into fatty acid increased. An inhibition of delta5 desaturase activity was demonstrated by the inhibition of the conversion of 14C-dihomo-gamma-linolenic acid into arachidonic acid. Geraniol has multiple effects on mevalonate and lipid metabolism in Hep G2 cells, affecting cell proliferation. Although mevalonate depletion is not responsible for cellular growth, it affects cholesterogenesis, protein prenylation, and fatty-acid metabolism.     

Regulation of hepatic cholesterogenesis by polar steroids accumulated after cholesterol feeding

The incorporation of mevalonate into nonsaponifiable lipids by chick liverin vivo strongly increased between 1–18 days after hatching. Cholesterol feeding (2%) inhibited this. Synthesis of cholesterol was strongly inhibited, whereas the intermediates isolated by TLC accumulated. Most of the polar nonsaponifiable lipids that accumulated in liver 90 minutes after mevalonate administration to 18-day-old cholesterol-fed chicks were identified as lanosterol derivative. 3-Hydroxy-3-methylglutaryl-CoA reductase activity, as well as acetate and mevalonate incorporation into nonsaponifiable lipids, was inhibited by the presence of these compounds. To our knowledge, this is the first report of such inhibition; this confirms the physiological function of polar steroids in the regulation of cholesterogenesisin vivo.To whom correspondence should be addressed.     

Biosynthesis of squalene and cholesterol by cell-free extracts of adult rat brain

Cell-free extracts of adult rat brain incubated with mevalonic acid-2-(14)C synthesize (14)C-labeled nonsaponifiable fractions consisting largely of squalene-(14)C. If the cofactor concentrations of the incubation medium are adjusted, much of the squalene can be induced to undergo turnover, with a resultant increase in (14)C-labeled digitonin-precipitable sterols, which include a small amount of cholesterol. The synthesis of labeled sterols is markedly increased in the presence of Mg(++) and depressed by nicotinamide. ATP, NADH, GSH, and glucose-6-phosphate are required for optimal synthesis of digitonin-precipitable material but, unlike Mg(++), are not essential. The cofactor-adjusted extracts also synthesize a complex ester mixture containing, in addition to cholesterol-(14)C, several compounds less polar than cholesterol. The biosynthesis of cholesterol in the extracts is a slow process; at least 12 hr of incubation is required for maximal sterol biosynthesis. A complex mixture of hydrocarbons accompanies squalene in the incubated extracts.     

Isoprenoid biosynthesis in the retina. Quantitation of the sterol and dolichol biosynthetic pathways

The isoprenoid pathway provides several important products for retina function. In this study the sterol and dolichol pathways were investigated in retinas from Rana pipiens in order to assess the contribution of de novo synthesis. Levels of 5.9 +/- 2.0 (n = 13) nmol/retina for squalene, 134 +/- 27 (n = 16) nmol/retina for cholesterol, and 0.14 +/- 0.04 (n = 11) nmol/retina for dolichyl phosphate (Dol-P) were determined by high performance liquid chromatography analysis. When whole retinas were incubated with 3H2O, radioactivity was incorporated into compounds which chromatographed on reversed-phase and silica high performance liquid chromatography at the elution positions of squalene, cholesterol, lathosterol, and methyl sterols. From these results, the upper limit for the absolute rate of the sterol pathway was estimated to be 3.4 pmol/h. When retinas were incubated with [3H]acetate, the major labeled product was squalene. The relatively low level of incorporation into cholesterol was apparently due to a substantial pool of squalene which accumulated de novo incorporated [3H]acetate. Dol-P was also labeled with [3H]acetate, and by comparing the ratio of 3H incorporation into Dol-P/squalene with the absolute rate of the sterol pathway, the absolute rate of Dol-P synthesis was determined to be 0.022 pmol/h. Our calculations indicate that the retina does not synthesize sufficient quantities of cholesterol de novo to account for that which is utilized in the biogenesis of rod outer segment membranes.     

Effect of 26-oxygenosterols from Ganoderma lucidum and their activity as cholesterol synthesis inhibitors

Ganoderma lucidum is a medicinal fungus belonging to the Polyporaceae family which has long been known in Japan as Reishi and has been used extensively in traditional Chinese medicine. We report the isolation and identification of the 26-oxygenosterols ganoderol A, ganoderol B, ganoderal A, and ganoderic acid Y and their biological effects on cholesterol synthesis in a human hepatic cell line in vitro. We also investigated the site of inhibition in the cholesterol synthesis pathway. We found that these oxygenated sterols from G. lucidum inhibited cholesterol biosynthesis via conversion of acetate or mevalonate as a precursor of cholesterol. By incorporation of 24,25-dihydro-[24,25-3H2]lanosterol and [3-3H]lathosterol in the presence of ganoderol A, we determined that the point of inhibition of cholesterol synthesis is between lanosterol and lathosterol. These results demonstrate that the lanosterol 14alpha-demethylase, which converts 24,25-dihydrolanosterol to cholesterol, can be inhibited by the 26-oxygenosterols from G. lucidum. These 26-oxygenosterols could lead to novel therapeutic agents that lower blood cholesterol.     

The isolation of 2,3-oxidosqualene from the liver of rats treated with 1-dodecylimidazole, a novel hypocholesterolaemic agent

1. Non-saponifiable lipid from the livers of rats treated with 1-dodecylimidazole contained an unidentified compound that was not present in the livers from untreated animals. 2. Treated rats had lower serum cholesterol concentrations than control rats. 3. 1-Dodecylimidazole, when added to rat liver slices, inhibited the incorporation of [1-(14)C]acetate and [2-(14)C]mevalonate into digitonin-precipitable sterols and resulted in the accumulation of a labelled compound, which was chromatographically identical with the unknown compound described in 1 above. 4. Rats treated with 1-dodecylimidazole incorporated less [(14)C]mevalonate into liver digitonin-precipitable sterols than untreated animals and accumulated the unknown compound as a labelled intermediate. 5. The unknown intermediate had the same chromatographic properties, n.m.r. and mass spectra as authentic 2,3-oxidosqualene. 6. The identity of the intermediate as 2,3-oxidosqualene was further established by showing that it was incorporated into sterols by rat liver homogenates under anaerobic conditions. In addition, incubation of [(14)C]squalene with rat liver homogenates resulted in trapping of the radioactivity by the added intermediate. 7. It is suggested that the hypocholesterolaemic activity of 1-dodecylimidazole results in part from the inhibition of cholesterol biosynthesis at the level of 2,3-oxidosqualene sterol cyclase.     

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.     

Biosynthesis of delta-7-cholesten-3-beta-ol, delta-5,7-cholestadien-3-beta-ol, and delta-5-cholesten-3-beta-ol by guinea pig intestinal mucosa in vitro

Methods were developed for the separation and determination of the various 27-carbon sterols of intestinal mucosa by means of thin-layer chromatography. Scrapings of the mucosa of the small intestine of guinea pig and rat were shown to incorporate isotope from (14)C-labeled acetate and mevalonate into sterols in vitro. For each substrate this activity was lowest in mucosa from the proximal third of the small intestine and greatest in mucosa from the more distal regions of the small intestine. The total 27-carbon sterol content of guinea pig mucosa varied only slightly along the length of the small intestine, but the concentration of cholesterol was highest distally. More than 95% of the radioactivity incorporated from acetate-2-(14)C into 27-carbon sterols by guinea pig mucosa in 4 hr was recovered as lathosterol and 7-dehydrocholesterol; less than 5% was in cholesterol. The specific activities of the 27-carbon sterols were consistent with the concept that synthesis proceeds from lathosterol to 7-dehydrocholesterol to cholesterol.     

Effect of 26-Oxygenosterols from Ganoderma lucidum and Their Activity as Cholesterol Synthesis Inhibitors

Ganoderma lucidum is a medicinal fungus belonging to the Polyporaceae family which has long been known in Japan as Reishi and has been used extensively in traditional Chinese medicine. We report the isolation and identification of the 26-oxygenosterols ganoderol A, ganoderol B, ganoderal A, and ganoderic acid Y and their biological effects on cholesterol synthesis in a human hepatic cell line in vitro. We also investigated the site of inhibition in the cholesterol synthesis pathway. We found that these oxygenated sterols from G. lucidum inhibited cholesterol biosynthesis via conversion of acetate or mevalonate as a precursor of cholesterol. By incorporation of 24,25-dihydro-[24,25-³H₂]lanosterol and [3-³H]lathosterol in the presence of ganoderol A, we determined that the point of inhibition of cholesterol synthesis is between lanosterol and lathosterol. These results demonstrate that the lanosterol 14α-demethylase, which converts 24,25-dihydrolanosterol to cholesterol, can be inhibited by the 26-oxygenosterols from G. lucidum. These 26-oxygenosterols could lead to novel therapeutic agents that lower blood cholesterol.     

The effect of non-ionic detergents and phospholipase A on enzymes involved in adult rat brain sterol biosynthesis from (2-14C)-mevalonic acid in vitro

Incubation of (2-¹⁴C)-mevalonic acid with 3000 × g adult rat brain supernate in the presence of Tween 80 or Triton X-100 produced a marked shift in the distribution of radioactivity among squalene, steryl ester and free sterol when compared to controls. Although the conversion of labeled mevalonate to cholesterol was only slightly influenced, the detergents markedly affected the turnover of squalene and methyl sterol precursors. The major neutral isoprenoid material formed had the general properties of geranylgeraniol on TLC and radioactivity-monitored-GLC. Phospholipase A from $\text{Vipera russelli}$, at an optimal tissue to phospholipase ratio, resulted in a 25% increase in mevalonate incorporation into total neutral isoprenoids. An increase in radioactivity of almost 25% was also seen in the free sterol fraction. Phospholipase A from bee venom did not show this type of increase. No labeled geranylgeraniol-type material was found on incubation with either of the phospholipases.     

Interrelationships of Ubiquinone and Sterol Syntheses in Cultured Cells of Neural Origin

Abstract: Ubiquinone synthesis has been studied in cultured C-6 glial and neuroblastoma cells by utilizing an inhibitor, 3-β-(2-diethylaminoethoxy) androst-5-en-17-one hydrochloride (U18666A), of cholesterol biosynthesis. Exposure of C-6 glial cells to nanomolar quantities of U18666A caused a marked inhibition of total sterol synthesis from [¹⁴C]acetate or [³H]mevalonate within minutes. A 95% inhibition was apparent after a 3-h exposure to 200 ng/ml of U18666A. These observations, together with studies of the incorporation of radioactivity from the two precursors into cholesterol, desmosterol, lanosterol, and squalene, indicated that although the most sensitive site to inhibition by U18666A is desmosterol reduction to cholesterol, a major site of inhibition is demonstrable at a more proximal site, perhaps squalene synthetase. As a consequence of the latter inhibition, exposure of C-6 glial cells to U18666A caused a marked stimulation of incorporation of [¹⁴C]acetate or [³H]mevalonate into ubiquinone. Over a wide range of U18666A concentrations, the increase in ubiquinone synthesis was accompanied by an approximately similar decrease in total sterol synthesis. Whereas in the absence of U18666A only approximately 7% of the radioactivity incorporated from [³H]mevalonate into isoprenoid compounds was found in ubiquinone, in the presence of the drug approximately 90% of incorporated radioactivity was found in ubiquinone. The reciprocal effects of U18666A on ubiquinone and sterol syntheses were apparent also in the neuronal cells. The data thus demonstrate a tight relationship between ubiquinone and sterol biosyntheses in cultured cells of neural origin. In such cells ubiquinone synthesis is exquisitely sensitive to the availability of isoprenoid precursors derived from the cholesterol biosynthetic pathway.