Metabolism of intracerebrally injected mevalonate in brain of suckling and young adult rats

We have investigated the in vivo metabolism via sterol and nonsterol pathways of intracerebrally injected mevalonate (MVA) in brains from suckling (10-day-old) and young adult (60-day-old) rats. Results of our study indicated that increasing the amounts of MVA injected increased MVA incorporation into all the lipid fractions examined. The incorporation of MVA into nonsaponiable lipids (NSF) and digitonin precipitable sterols (DPS) was similar in brains from adult and suckling rats. In brain tissue from both suckling and young adult rats the synthesis of dolichol from MVA varied with the amounts of MVA injected. Significant amounts of MVA were recovered in phosphorylated and free polyprenols (farnesol and geraniol) in brain tissue from rats of both ages. Also in both groups of animals, the amounts of MVA incorporated in phosphorylated and free farnesol were higher than the amounts recovered in either, phosphorylated or free geraniol. The amounts of MVA incorporated into the prenoic/fatty acid fraction by brain tissue from both suckling and young adult rats were less than 1% of the total MVA incorporated (nonsaponifiable and saponifiable lipids). Incorporation of MVA into the prenoic/fatty acid fraction by brain tissue was higher in suckling than in young adult rats. These data indicate that the brain tissue from suckling and young adult rats do not differ in their capacity to metabolize MVA into squalene and sterols and that in brain, metabolism of MVA by a shunt pathway is minimal. This suggests that in vivo regulation of cholesterol synthesis during brain development must occur at a step(s) in the sterol synthetic pathway prior to mevalonate, and that metabolism of mevalonate by shunt pathway did not play a role in the developmental regulation of brain sterol synthesis. The data also suggest that in both groups of animals the synthesis of squalene by synthetase may in part control brain sterol synthesis and the synthesis of dolichol is regulated by MVA concentration in the tissue.     

Changes in sterol biosynthesis accompanying cessation of glial cell growth in serum-free medium

C-6 glioma cells, grown in medium supplemented with 5% delipidated foetal calf serum, were induced to enter a quiescent state by removing serum from the medium. Within 24h there was a 75–80% decline in the rate of incorporation of [¹⁴C]acetate or ³H₂O into digitonin-precipitable sterols. Experiments with [³H]mevalonolactone as a labelled sterol precursor suggested that the decline in sterol synthesis was regulated primarily at a point in the pathway before the formation of mevalonate. The specific activities of 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA reductase decreased sharply in conjunction with the decline in sterol synthesis in the serum-free cultures; however, the activity of acetoacetyl-CoA thiolase was altered only slightly. The magnitude of the initial decline in reductase activity was not affected when 50-mm-NaF was included in the preincubation and assay buffers to prevent activation of physiologically inactive enzyme. However, after 6h of serum deprivation the decline in 3-hydroxy-3-methylglutaryl-CoA reductase activity was due to a decrease in the amount of latent activity. The sterol concentration in C-6 cells was unchanged after 24h in serum-free medium, although a 20% decrease in the sterol/fatty acid molar ratio occurred as a result of a small increase in the fatty-acid concentration. Incorporation of ³H₂O into fatty acids was inhibited in the serum-deprived glial cells; however, this inhibition developed more slowly and was not as pronounced as the diminution in sterol synthesis. The results suggest that in C-6 glia, which resemble the glial stem cells of the developing brain, the decreased demand for membrane sterols in the quiescent state results in a decline in sterol synthesis, mediated primarily through co-ordinate changes in the activities of 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA reductase.     

Reversal of cerulenin-induced inhibition of phospholipids and sterol synthesis by exogenous fatty acids/sterols in Epidermophyton floccosum

Cerulenin, a specific inhibitor of fatty acids and sterol biosynthesis inhibited the growth of Epidermophyton floccosum, which was reversed when growth medium was supplemented with palmitic acid and sterols. Unsaturated fatty acids partially restored the growth. Cerulenin inhibited both phospholipid and sterol biosynthesis (60-70%) at the minimum inhibitory concentration (0.5 microgram/ml) as demonstrated by [32P]orthophosphoric acid and [14C]acetate incorporation into the respective lipids. Cerulenin-induced inhibition of phospholipid and sterol synthesis was dose dependent up to 0.5 microgram/ml. Exogenously supplied fatty acids and sterols restored the biosynthesis of phospholipids in cerulenin-treated cultures, while that of sterols was enhanced. The biosynthesis of both saturated and unsaturated fatty acids was inhibited by cerulenin.     

Possible role of acetyl-CoA-carboxylase in biosynthesis of mevalonic acid and sterols in rat liver]

Effect of citrate on acetyl-CoA incorporation into mevalonic acid, sterols and fatty acids after preliminary incubation of rat liver extracts under conditions optimal for acetyl-CoA carboxylase activation, was studied. 30 min preincubation with the citrate at 37 degrees C results in a 2--3-fold stimulation of the mevalonic acid biosynthesis from acetyl-CoA in the microsomal and soluble (140 000 g) fraction, and in that of sterols precipitated by digitonin or isolated by TLC in the mitochondria--free fraction. 2-14C-malonyl-CoA incorporation into the mevalonic acid and sterols and biosynthesis of sterols from 2-14C-mevalonic acid were not stimulated under those conditions. A correlation was shown to exist between the activity of acetyl-CoA carboxylase and the rate of acetyl-CoA incorporation into mevalonate and sterols; the activity of beta-hydroxy-beta-methylglutaryl-CoA reductase, limiting the rate of the sterol biosynthesis, was not changed. The stimulating effect of citrate was found to depend on the concentration of acetyl-CoA and NADPH in the medium. The data obtained suggest that the mevalonic acid biosynthesis in rat liver may occur in the presence of acetyl-CoA carboxylase through the formation of malonyl-CoA.     

A mevalonate requirement for maintenance of fatty acid and protein synthesis during hormonally stimulated development of mammary gland in vitro

The effect of compactin on hormonally induced lipogenesis and protein synthesis was studied in vitro in explants of mammary gland from mid-pregnant rabbits. Compactin blocks mevalonate synthesis by the specific inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase, and in this system, culture with 10 microM compactin for 24, 48, and 72 h inhibited incorporation of [1-14C]acetate (but not [2-14C]mevalonate) into sterol by 98, 95, and 86%, respectively. Removal of compactin prior to assay rapidly reversed this effect and was associated with increased tissue 3-hydroxy-3-methylglutaryl-CoA reductase activity. Fatty acid synthesis (measured by incorporation of [1-14C]acetate or [4,5-3H]leucine) and protein synthesis (measured by incorporation of [4,5-3H]leucine) were both inhibited by around 50% after culture with compactin. This inhibition was not rapidly reversed by removal of compactin prior to assay, but it was prevented by inclusion of 1 mM mevalonolactone in the culture medium. After removal of compactin and continued culture in its absence for 24 h with hormones, the normal tissue capacity for fatty acid and protein synthesis was restored, indicating no permanent cell damage. The results suggest a specific requirement for mevalonate (or derived products) for the hormonal maintenance of the increased fatty acid and protein synthesis characteristic of the development of the mammary gland.     

Inhibition by 6-fluoromevalonate demonstrates that mevalonate or one of the mevalonate phosphates is necessary for lymphocyte proliferation

The sterol synthesis inhibitor 6-fluoromevalonate (Fmev) was used to explore the role of mevalonate products in lymphocyte proliferation. Fmev blocks the synthesis of isopentenyl pyrophosphate and all more distal products in the sterol pathway. When cells were cultured in lipoprotein-deficient medium, Fmev (200 microM) completely inhibited mitogen-stimulated human lymphocyte proliferation, quantified by measuring DNA synthesis. The addition of low density lipoprotein (LDL) restored lymphocyte responses to normal, whereas mevalonate was totally ineffective. Similar results were obtained with concentrations of Fmev up to 1 mM. These results contrast with those observed when sterol biosynthesis was blocked with lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. When lymphocyte proliferation was blocked with lovastatin (5 microM), either high concentrations of mevalonate or LDL together with low concentrations of mevalonate was required to restore responses. In contrast, neither LDL nor low concentrations of mevalonate when alone was able to restore lymphocyte DNA synthesis in cultures blocked with 5 microM lovastatin. The effect of Fmev on the capacity of exogenous mevalonate to restore proliferation of lovastatin-blocked lymphocytes was directly examined. Fmev had no effect on the capacity of LDL plus low concentrations of mevalonate to restore DNA synthesis to lovastatin-blocked lymphocytes, indicating that the synthesis of the necessary factor from mevalonate was unaltered by Fmev. Fmev profoundly blocked lymphocyte endogenous sterol synthesis, decreasing incorporation of radiolabeled acetate into digitonin-precipitable sterols by up to 98%. LDL did not alter the capacity of Fmev to block sterol synthesis. The possibility that Fmev allowed shunting of endogenous mevalonate into essential lipid products was assessed by examining the incorporation of radiolabeled mevalonate. Fmev (200 microM) inhibited the incorporation of mevalonate into all lipids, including ubiquinone, dolichol, and other non-sterol lipids by up to 98%, and this was not altered by LDL. Furthermore, Fmev (200 microM) suppressed the incorporation of radiolabeled mevalonate into protein by up to 97%. These data confirm that a product of mevalonate is essential for cell proliferation. However, the results indicate that the required product is directly synthesized from mevalonate or mevalonate phosphates rather than from a more distal isoprenoid metabolite.     

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.     

Action of the antiepileptic drug, valproic acid, on fatty acid oxidation in isolated rat hepatocytes

Valproate at 0.1 to 5 mM strongly inhibited oxidation of 1-(14C)-palmitate in isolated rat hepatocytes. Valproate at the same concentrations markedly decreased ketogenesis from 1 mM oleate. Valproate in a dose up to 5 mM did not significantly affect cellular concentration of ATP but lowered beta-hydroxybutyrate/acetoacetate and lactate/pyruvate ratios which paralleled its effect on ketogenesis. Moreover concomitant acetyl-CoA levels were drastically decreased by valproate. From this it may be concluded that inhibition of fatty acid oxidation by valproate results in reduced production of two carbons units and a drop of NADH/NAD+ ratio in rat hepatocyte. This suggests that valproate seriously interferes with beta-oxidation of physiological long-chain fatty acids.     

Alterations in cholesterol and fatty acid biosynthesis in rat liver homogenates by aryloxy acids (Short Communication)

2,4-Dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid inhibited the incorporation of [2-(14)C]mevalonate into cholesterol and non-saponifiable lipids. Both compounds inhibited the conversion of [1-(14)C]isopentenyl pyrophosphate into cholesterol and the synthesis of cholesterol and fatty acids from [2-(14)C]acetate. There was no inhibition of the conversion of [1-(14)C]mevalonate into CO(2). At low concentrations (0.5mm) of the compounds there was a stimulation of acetate incorporation into fatty acids.     

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.     

Effect of cerulenin on the growth and differentiation of Dictyostelium discoideum.

The growth of Dictyostelium discoideum Ax-2 was inhibited completely by cerulenin at a concentration of 5 mug/ml. This inhibition of growth was found to be due to the inhibition of fatty acid synthesis. Acetate incorporation into a long-chain fatty acid was inhibited completely by cerulenin, and the growth inhibition could be reversed by inclusion of certain saturated fatty acids in the medium. Unsaturated fatty acids and sterols failed to reverse the inhibitory effect. The fatty acid and sterol compositions of cerulenin-treated cells were determined to establish whether the drug could be used to manipulate the organism's lipid composition. Only relatively small manipulations were obtained under the conditions employed in this study. Cerulenin inhibited differentiation but only at high concentrations (150 mug/ml). This inhibition could be reversed by palmitic acid, suggesting that the prime cause of the inhibition was an inhibition of fatty acid synthesis. Thus, it appears that continued fatty acid synthesis is required for the cellular process of differentiation in D. discoideum.     

Specific sites of fatty acid sterol synthesis in isolated skin components

Metabolic studies on isolated mouse skin components were undertaken to determine the specific sites of fatty acid and sterol synthesis. The concentrations of long-chain fatty acids and sterols and the incorporation of radioactivity from acetate-1-(14)C into these lipids are reported for various skin components and intact whole skin. Only fatty acids having chain lengths of 18 carbons or less were produced by the connective tissue cells of the dermis, while fatty acids containing 20 carbons or more, as well as the acids of 18 carbons or less, were synthesized in the upper dermis (papillary reticulum). The upper dermis also produced significant quantities of eicosenoic acid and of an octadecadienoic acid (not linoleic acid), and incorporated labeled acetate into fatty acids containing an odd number of carbons. Removal of the epidermis and adnexa diminished sterol synthesis. However, the upper region of the dermis was capable of synthesizing, from acetate, large quantities of unidentified nonsaponifiable lipids which were neither sterols nor squalene.     

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.     

Effect of α-p-chlorophenoxyisobutyrate on the metabolism of isoprenoid compounds in the rat

1. Feeding of alpha-p-chlorophenoxyisobutyrate (CPIB) to rats increased ubiquinone concentration in the liver but not in other tissues. The increase was progressive with the time of feeding and related to the concentration of CPIB in the diet. 2. Incorporation of [1-(14)C]acetate, but not of [2-(14)C]mevalonate, into sterols in the liver in vivo or by liver slices in vitro was decreased on feeding the rats with CPIB. However, incorporation of mevalonate into ubiquinone increased. 3. CPIB, when added in low concentrations to liver slices, had no effect on isoprene synthesis from acetate; higher concentrations, however, were inhibitory. 4. No activation of ubiquinone synthesis from mevalonate was observed when CPIB was added to the liver slices synthesizing ubiquinone. 5. The increase in ubiquinone in CPIB-fed animals appears to be due to increased synthesis in the initial stages and to decreased catabolism in the later stages. 6. An inverse relationship was found between the concentration of ubiquinone in the liver and the serum sterol concentration in CPIB-fed rats.     

Synthesis of sterols and 5-lipoxygenase products are required for the G1-S phase transition of interleukin-2-dependent lymphocyte proliferation

A murine killer T cell line, G-CTLL 1, whose proliferation depends on the presence of interleukin 2 (IL-2), was used to analyze the mechanism of IL-2 action with respect to sterol synthesis and arachidonate metabolism. De novo sterol synthesis was substantially enhanced much earlier than DNA synthesis, and the rate reached a maximum at 13 hr after the addition of IL-2. Compactin, which is a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase, the enzyme in the rate-limiting step of the sterol synthesis), inhibited the IL-2-induced DNA synthesis. The addition of mevalonate, the product of HMG CoA reductase, prevented the inhibition of DNA synthesis by compactin, suggesting that the supply of a sufficient amount of sterol is an essential prerequisite for IL-2 action. The IL-2-induced DNA synthesis was also inhibited by AA861, a specific inhibitor of arachidonate 5-lipoxygenase, and by other lipoxygenase inhibitors such as nordihydroguaiaretic acid and esculetin. In contrast, indomethacin, an inhibitor of arachidonate cyclooxygenase, had no effect. These findings suggest that synthesis of 5-lipoxygenase products is also a prerequisite. The inhibition of DNA synthesis was effectively inhibited only when compactin or lipoxygenase inhibitors were added early enough to block the synthesis of sterols or 5-lipoxygenase products; addition of the reagents after 3 hr decreased the inhibition with time. Therefore, about 3 hr after the addition of IL-2, several drastic intracellular changes are assumed to begin and to lead to DNA synthesis.     

The inhibition of cytoplasmic acetoacetyl-CoA thiolase by a triyne carbonate (L-660, 631)

The compound L-660, 631 (2-oxo-5-(1-hydroxy-2,4,6-heptatriynyl)-1,3-dioxolane-4 heptanoic acid), a natural product isolated from an Actinomycete culture, was found to inhibit rat liver cytosolic acetoacetyl-CoA thiolase, the first step in the cholesterol biosynthesis pathway, with an IC50 of 1.0 x 10(-8) M. The inhibitor had no effect on other sulfhydryl containing enzymes of lipid synthesis such as HMG-CoA synthase, HMG-CoA reductase, and fatty acid synthase. When tested in cultured human liver Hep G2 cells the compound inhibited the incorporation of 14C-acetate and 14C-octanoate into sterols 56% and 48% respectively at 3 x 10(-6) M with no effect on fatty acid synthesis. No noticeable effect was seen on fatty acid biosynthesis. This strongly suggests that the locus of inhibition of acetate incorporation into sterols found with this compound is the acetoacetyl-CoA thiolase step in the cholesterol biosynthesis pathway.     

Inhibition of a plant sesquiterpene cyclase by mevinolin

The specificity of mevinolin as an inhibitor of sterol and sesquiterpene metabolism in tobacco cell suspension cultures was examined. Exogenous mevinolin inhibited [14C]acetate, but not [3H]mevalonate incorporation into free sterols. In contrast, mevinolin inhibited the incorporation of both [14C]acetate and [3H]mevalonate into capsidiol, an extracellular sesquiterpene. Microsomal 3-hydroxy-3-methylglutaryl Coenzyme A reductase was inhibited greater than 90% by microM mevinolin, while squalene synthetase was insensitive to even 600 microM mevinolin. Sesquiterpene cyclase, the first branch point enzyme specific for sesquiterpene biosynthesis, was inhibited in a dose-dependent manner by mevinolin with a 50% reduction in activity at 100 microM. Kinetic analysis indicated that the mechanism for inhibition was complex with mevinolin acting as both a competitive and noncompetitive inhibitor. The results suggest that the mevinolin inhibition of [3H]mevalonate incorporation into extracellular sesquiterpenes can, in part, be attributed to a secondary, but specific, site of inhibition, the sesquiterpene cyclase.     

The in vitro metabolism of mevalonate by sterol and non-sterol pathways.

The metabolism of mevalonic acid by both sterol and non-sterol pathways has been evaluated in nine tissues of the rat. An in vitro estimation of the non-sterol, or "shunt", pathway of mevalonate metabolism was made possible by determining the conversion of [2-14C]mevalonate or [5-14C]mevalonate to 14CO2 in tissue slices. In confirmation of our previous results, the kidney was found to play a major role in the metabolism of mevalonate to sterols and sterol precursors. The shunt pathway accounted for a significant percentage of the mevalonate metabolized in kidney, ileum, spleen, lung and testes, but was of minor importance or undetectable in liver, brain, skin, and adipose tissue. Kidney, however, proved to be by far the most active tissue site of mevalonate metabolism by the shunt mechanism in that, on an average, renal tissue metabolized (R)-[14C]mevalonate over the non-sterol pathway at a rate that was 21 times that of any other tissue examined. These results indicate that the kidneys are of major importance in the metabolism of mevalonate by each of the known pathways of metabolism of this sterol precursor.     

Acetoacetate is a cholesterogenic precursor for myelinating rat brain and spinal cord. Incorporation of label from [3-14C]acetoacetate, [14C]glucose and 3H2O

Rat pups, 3 weeks old, were injected i.p. with combinations of 3H2O and either [3-14C]acetoacetate or [14C]glucose. 3H/14C incorporation ratios were measured in lipid fractions of homogenates and myelin prepared from whole brain and spinal cord. Spinal cord synthesized at least twice as much fatty acids and 3-fold more sterols than whole brain. Both tissues used acetoacetate preferentially for sterol synthesis, whereas label from [14C]glucose was distributed between fatty acids and sterols in the same way as 3H from 3H2O. The relative contributions of acetoacetate to sterol synthesis in whole tissue and in the purified myelin fraction were about the same, both for the cerebrum and for the spinal cord.     

Coordinate regulation of cholesterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A synthase but not 3-hydroxy-3-methylglutaryl coenzyme A reductase in C-6 glia

The effects on cholesterol biosynthesis of growth of cultured C-6 glial cells in serumfree medium ± supplementation with linoleic or linolenic acid were studied. Markedly higher activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) were observed in cells grown in linoleate- or linolenate-supplemented versus nonsupplemented medium. After 48 h HMG-CoA reductase activities were two-and four-fold higher in cells supplemented with 20 and 100 μm linoleate, respectively. The increase in activity became apparent after 24 h and was marked after 48 h. Rates of incorporation of [¹⁴C]acetate or ³H₂O into sterols did not reflect the changes in reductase activity. Thus, in cells supplemented with 50 μm linoleate for 24 and 48 h rates of incorporation of [¹⁴C]acetate were 75–80% lower than rates in nonsupplemented cells. This difference resulted because over the first 24 h of the experiment a fivefold increase in the rate of sterol synthesis occurred in the nonsupplemented cells, whereas essentially no change occurred in the linoleate-supplemented cells; little further change occurred between 24 and 48 h in the nonsupplemented and the linoleate-supplemented cells. That the difference in sterol synthesis under these experimental conditions could be mediated at the level of HMG-CoA synthase (EC 4.1.3.5) was suggested by two series of findings, i.e., first, similar quantitative and temporal changes in the activity of this enzyme, and, second, no change in the activity of acetoacetyl-CoA thiolase (EC 2.3.1.9) or the incorporation of [¹⁴C]mevalonate into sterols. Thus, the data suggest that HMG-CoA synthase, and not HMG-CoA reductase, may direct the rate of cholesterol biosynthesis under these conditions of serum-free growth ± supplementation with polyunsaturated fatty acid.