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.     

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.     

Biosynthesis of squalene and sterols by rat aorta

The synthesis of nonsaponifiable compounds from radioactive mevalonate by segments of adult rat aorta was studied in vitro. The labeled products consisted largely of substances with the chromatographic and chemical behavior of squalene, lanosterol, lathosterol, and cholesterol. Even after 3 or 4 hr of incubation, the incorporation of mevalonate into squalene was higher than its incorporation into C(27) sterols; cholesterol contained less than 20% of the radioactivity in the total sterols. Lanosterol was the most highly labeled sterol. The level of radioactivity in lathosterol was comparable to the level in cholesterol. Small amounts of radioactivity were found in other sterols. Material with the same mobility on TLC as 7-dehydrocholesterol had less radioactivity than cholesterol, but more than sterols with the mobility of desmosterol. The results of measurements made after short periods of incubation showed that squalene and lanosterol became labeled before the other nonsaponifiable compounds.     

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.     

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.     

The in vivo incorporation of acetate into different non-saponifiable lipids by neonatal chick tissues

The in vivo incorporation of [l-14C]acetate into non-saponifiable lipids was higher in neonatal chick liver than in intestinal mucosa, brain and kidneys, and proportional to the amount of substrate injected (2-20 mumole). 14CO2 expired in the breath was also proportional to the dose of acetate. Radioactivity from [l-14C]acetate accumulated by liver was maximal 30 min after the injection of acetate and decreased afterwards. Acetate was mainly incorporated into cholesterol by all the tissues assayed, although small percentages of lanosterol and squalene were obtained in liver. In this tissue, distribution of radioactivity was practically independent from the dose of substrate injected while in intestinal mucosa, brain and kidneys the percentage of cholesterol increased with this dose. The time course of the in vivo formation of different non-saponifiable lipids by neonatal chick tissues was also studied. More than 90% of radioactivity in this fraction obtained 15 min after the acetate injection was recovered as cholesterol in liver and kidneys, while in brain and intestinal mucosa this percentage was about 50% at this time, increasing afterwards. A high percentage of lanosterol was found in brain and intestinal mucosa 15 min after the injection of acetate.     

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.     

Studies of the in vivo metabolism of mevalonic acid in the neonatal chick

After 4 hr of the intraperitoneal injection of different doses of (R)-[5-14C]mevalonic acid (MVA), its incorporation into nonsaponifiable and saponifiable lipids was maximal in neonatal chick kidneys and liver, and minimal in brain, spinal cord and skin. Using 14CO2 production from [5-14C]MVA as an index of the shunt pathway not leading to sterols, we have demonstrated for the first time that about 11% of MVA was in vivo metabolized by this pathway in nonmammalian species. Kidneys presented the maximal ability to incorporate MVA into nonsaponifiable and saponifiable lipids at any time considered (15-750 min). The percentage of radioactivity recovered as saponifiable lipids in liver and kidney decreased after 12 hr the injection of MVA. Although the absolute amounts of 14C incorporated in both derivatives were much less in brain, spinal cord and skin than in liver and kidneys, the relative percentages found in the saponifiable fraction were clearly higher in the former tissues, especially in the spinal cord.     

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.     

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.     

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.     

Studies on the diurnal rhythm of mevalonate metabolism by sterol and nonsterol pathways and of mevalonate-activating enzymes

Both in vivo and in vitro incorporation of mevalonic acid into nonsaponifiable lipids by 17-day-old chick liver and kidney did not show diurnal rhythm. Using 14CO2 production from MVA as an index of the shunt pathway not leading to sterols, we have demonstrated for the first time that there is no diurnal rhythm in this pathway. No significant differences were found in the specific activities of mevalonate kinase, mevalonate-5-phosphate kinase and mevalonate-5-pyrophosphate decarboxylase from chick liver and kidney throughout a period of 24 hr, using [1-14C]mevalonate as substrate. The absence of diurnal rhythm in the decarboxylase activity was corroborated by further experiments carried out using [2-14C]mevalonate-5-pyrophosphate as specific substrate of this enzyme.     

The biosynthesis of squalene and sterols by the adipose tissue of rat, sheep and man

1. The subcutaneous and omental adipose tissue of man, the epididymal fat pads of the rat and the fat tail of the Syrian sheep incorporate mevalonic acid into non-saponifiable lipids. 2. Time studies showed that the rates of decarboxylation of mevalonic acid and synthesis of non-saponifiable lipids slightly decline after 20min. but subsequently remain linear for 6hr. 3. About one-half of the incorporated radioactivity in the non-saponifiable lipids was in squalene, 20% in lanosterol and cholesterol, and the remainder in unidentified substances.     

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.     

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.     

Liver mevalonate 5-pyrophosphate decarboxylase is responsible for reduced serum cholesterol in stroke-prone spontaneously hypertensive rat.

Spontaneously hypertensive rat (stroke-prone) (SHRSP) has an interestingly low serum cholesterol level due to a reduced biosynthesis of cholesterol in the liver (Iritani, N., Fukuda, E., Nara, Y., and Yamori, Y. (1977) Atherosclerosis 28, 217-222). In this study, we examined the mechanism underlying the reduction of hepatic cholesterol biosynthesis in the rat. Our initial findings in SHRSP, as compared with normotensive Wistar Kyoto rat (WKY), showed that 1) the incorporation of [14C]acetate into cholesterol in the liver slices was markedly less, 2) 3-hydroxyl-3-methylglutaryl (HMG) CoA reductase activity was not reduced, and 3) the incorporation of [3H]mevalonic acid into both cholesterol and squalene was significantly less. The above initial findings suggested that the reduction in the hepatic cholesterol biosynthesis took place in one or more enzymatic processes starting with mevalonic acid and continuing to squalene. When the incorporation of [3H]mevalonic acid into phosphomevalonate derivatives was studied using an ion exchange column, only the radioactivity incorporated into isopentenyl-pyrophosphate (isopentenyl-PP) was less in SHRSP. Furthermore, the specific activity of diphosphomevalonate (mevalonate-PP) decarboxylase in the liver-soluble fractions was reduced 50% in SHRSP as compared with WKY. Kinetic studies using liver crude extracts indicated a lower Vmax value in SHRSP (SHRSP, 0.47; WKY, 2.05 nmol/min/mg), and an unchanged Km value (SHRSP, 18.2; WKY, 19.6 microM). The activity of mevalonate-PP decarboxylase was also found to be reduced in other tissues, including the brain, testis, small intestine, and cultured vascular smooth muscle cells. From the above observations, we concluded that the lower activity of mevalonate-PP decarboxylase was responsible for the reduced cholesterol biosynthesis in the liver of SHRSP.     

Effect of dietary cholesterol on mevalonate metabolism by sterol and nonsterol pathways

Results in the present communication demonstrate for the first time that the shunt pathway of mevalonate not leading to sterols is regulated by cholesterol feeding in a reverse fashion to the sterol pathway. Mevalonate incorporation into nonsaponifiable lipids by liver slices was inhibited by cholesterol feeding while the shunt pathway was clearly enhanced. Moreover, inhibition of renal sterologenesis by dietary cholesterol is also reported. These changes in the mevalonate metabolism are closely correlated with the increase observed in the esterified cholesterol content in neonatal chick liver and kidneys after 10 days of 2% cholesterol supplementation of the diet.     

Stimulation of liver cholesterol synthesis by actinomycin D

1. An eightfold increase in the incorporation of [2-(14)C]acetate into liver cholesterol in vivo was observed 24hr. after starved rats had been given actinomycin D (0.5mg./kg. of body wt.). Liver cholesterol radioactivity declined faster in the treated animals, suggesting a greater rate of cholesterol turnover. 2. Liver slices from treated animals showed a tenfold increase in the incorporation of [2-(14)C]acetate into cholesterol; conversion into CO(2) and into fatty acids was less markedly increased, and conversion into ketone bodies was not significantly affected. 3. The patterns of conversion into liver cholesterol in vivo of the lactone and the sodium salt of mevalonic acid differed markedly. The former was converted at a faster rate and to a greater extent than the latter. Treatment with actinomycin D increased the conversion of both forms of mevalonic acid into liver cholesterol, but only to a small extent. 4. Stimulation of the incorporation of acetate into cholesterol occurred at 4hr. after the administration of actinomycin D but not at 2hr. The response was abolished by the simultaneous administration of dl-ethionine or puromycin. 5. Pre-feeding with a cholesterol-rich diet greatly diminished the stimulation of conversion of acetate into cholesterol caused by actinomycin D, though it did not completely suppress it. Adrenalectomized animals responded to the drug, but much less markedly. 6. It is concluded that actinomycin D stimulates the synthesis of cholesterol in the liver at a stage in the pathway before mevalonic acid, by a mechanism that probably requires protein synthesis. A likely site would be the beta-hydroxy-beta-methylglutaryl-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Some possible mechanisms by which the drug may lead to increased activity of this enzyme are considered.     

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.     

Cholesterol Inhibition of Pentacyclic Triterpenoid Biosynthesis in Tetrahymena pyriformis*†

SYNOPSIS. Tetrahymena pyriformis synthesizes tetrahymanol and “diplopterol” from acetate, mevalonate or squalene. The formation of these pentacyclic triterpenoid alcohols is inhibited by the addition of cholesterol to the culture fluid of the ciliates. Cholesterol also inhibits the biosynthesis of squalene from acetate or mevalonic acid. The synthesis of other terpene derivatives from acetate and mevalonate continues in the presence of cholesterol, thus suggesting that a major block occurs after “isoprene” formation and before squalene formation. Further, inhibition of squalene conversion to the pentacyclic alcohols by cholesterol has been established.