DNA and cholesterol biosynthesis in synchronized embryonic rat fibroblasts: II. Effects of sterol biosynthesis inhibitors on cell division

The relationships between cholesterogenesis and cell division were studied by using two inhibitors of hydroxymethylglutaryl-CoA reductase activity — 25-hydroxycholesterol and compactin. The effects of both compounds on DNA synthesis were compared in synchronized rat fibroblasts cultured in a cholesterol-containing medium. Compactin did not inhibit DNA synthesis, except after a long time of contact and at high and almost cytotoxic concentrations. 25-Hydroxycholesterol inhibited DNA synthesis (without cytotoxic effects) after only 9–16 h of contact, depending on the phase of the cell cycle at which this compound was added to the culture medium. Sensitivity of cells to 25-hydroxycholesterol was maximal at the end of the S phase/beginning of the G₂M phase. The rapid effect of 25-hydroxycholesterol on DNA synthesis appears to be separate from the inhibitory effect on sterol or non-sterol mevalonate-derived compound synthesis. Indeed, under our experimental conditions, the suppression of cholesterol biosynthesis is compensated by the presence of cholesterol in the culture medium, as demonstrated by the lack of effect of compactin on DNA synthesis; moreover, addition of mevalonolactone to the culture medium did not reverse the effect of 25-hydroxycholesterol. 25-Hydroxycholesterol could inhibit DNA synthesis by a direct action on the nucleus, after transfer by the intermediary of a specific hydroxysterol-binding protein.     

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.     

Investigations on the Δ23-, Δ24(28)- and Δ25-Sterols of zea mays

The sterols of Zea mays shoots were isolated and characterized by TLC, HPLC, GC/MS and ¹H NMR techniques. In all, 22 4-demethyl sterols were identified and they included trace amounts of the Δ²³-, Δ²⁴- and Δ²⁵-sterols, 24-methylcholesta-5,E-23-dien-3β-ol, 24-methylcholesta-5,Z-23-dien-3β-ol, 24-methylcholesta-5,25-dien-3β-ol, 24-ethylcholesta-5,25-dien-3β-ol and 24-ethylcholesta-5,24-dien-3β-ol. In the 4,4-dimethyl sterol fraction, cycloartenol and 24-methylenecycloartanol were the major sterol components but small amounts of the Δ²³-compound, cyclosadol, and the Δ²⁵-compound, cyclolaudenol, were recognized. These various Δ²³- and Δ²⁵-sterols may have some importance in alternative biosynthetic routes to the major sterols, particularly the 24β-methylcholest-5-en-3β-ol component of the C₂₈-sterols. Radioactivity from both [2-¹⁴C]MVA and [methyl-¹⁴C]methionine was incorporated by Z. mays shoots into the sterol mixture. Although 24-methylene and 24-ethylidene sterols were relatively highly labelled, the various Δ²³- and Δ²⁵-sterols contained much lower levels of radioactivity, which is possibly indicative of their participation in alternative sterol biosynthetic routes. (24R)-24-Ethylcholest-5-en-3β-ol (sitosterol) had a significantly higher specific activity than the 24-methylcholest-5-en-3β-ol indicating that the former is synthesized at a faster rate.     

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.     

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.     

Regulation of cholesterol synthesis in primary rat hepatocyte culture cells. Possible regulatory site at sterol demethylation.

Primary rat hepatocyte culture cells were used to study the acute regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in response to 25-hydroxycholesterol, 3 beta,5 alpha,6 beta-cholestantriol, and mevalonolactone. All three effectors caused a rapid suppression of HMG-CoA reductase activity. 25-Hydroxycholesterol also caused an increase in the ratio of newly synthesized methyl sterols to newly synthesized C27-sterols. Furthermore, in 25-hydroxycholesterol-treated cells, the relative contribution of delta 24-sterol precursors to the nonsaponifiable lipid fraction increased. Di- and trimethyl-diene sterols were the dominant methyl sterols synthesized in the presence of 25-hydroxycholesterol. 3 beta,5 alpha,6 beta-Cholestrantriol (50 microM) also caused a very strong (97%) suppression of sterol demethylation; 4,4-dimethylmonoene sterols were more prominent (23%) in cells treated with 3 beta,5 alpha,6 beta-cholestrantriol, than in cells treated with 25-hydroxycholesterol (2%). The rates of both unesterified and esterified sterol synthesis increased as a function of exogenous mevalonolactone concentration. C27-sterol synthesis was saturated at a concentration of (R)-mevalonolactone which produced only a 33% suppression of HMG-CoA reductase activity. However, there was a direct relationship between the accumulation of methyl sterols and the decrease in HMG-CoA reductase activity. With the aid of triparanol, it was demonstrated that the suppression of HMG-CoA reductase activity by mevalonolactone was linked with the ability of the cells to convert squalene-2,3-epoxide into sterols. The results described in the present article support an important and perhaps necessary relationship between the rate of methyl sterol conversion of C27-sterols and the suppression or inhibition of HMG-Coa reductase in primary hepatocyte culture cells.     

Regulation of sterol biosynthesis in yeast: induction of 3-hydroxy-3-methylglutaryl-CoA reductase by glucose

Anaerobically cultured yeast cells have a very low HMG-CoA reductase activity and a low sterol content. When these cells are transfered to phosphate buffer containing 1.2 % glucose and held under aerobic conditions, the specific activity of the HMG-CoA reductase increases up to sixfold within 8 hrs. The increase in the reductase activity is paralled by an increase in the sterol content. This induction of HMG-CoA reductase in resting yeast cells is inhibited by cycloheximide indicating that a $\text{de novo}$ synthesis of enzyme protein is mediated by glucose under aerobic conditions. It appears that the regulation of sterol synthesis in yeast is closely connected with the aerobic glucose metabolism.     

Sterols of Xanthoria parietina: Evidence for two sterol ‘pools’ and the identification of a novel C,8 triene,ergosta-5,8,22-trien-3β-ol

Sterols extracted from Xanthoria parietina with organic solvents and released by saponification of the residual lichen tissue were analysed by GC-MS. The main components of the solvent-extractable sterols were two C₂₈ trienes and those of the more tightly bound sterols were ergost-5-en-3β-ol and two C₂₉ compounds. The structures of the C₂₈ compounds were shown to be ergosta-5,7,22-trien-3β-ol, Ia (ergosterol) and the previously unreported ergosta-5,8,22-trien-3β-ol, IIa, for which the name lichesterol is proposed. The main C₂₉ sterol was identified as (24R)-24-ethylcholesta-5,22-dien-3β-ol (poriferasterol).     

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

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

Amo 1618 effects on incorporation of 14C-MVA and 14C-acetate into sterols in Nicotiana and Digitalis seedlings and cell-free preparations from Nicotiana

Incorporation of radioactivity from acetate-[¹⁴C] and MVA-[¹⁴C] into sterols and sterol precursors in tobacco was inhibited by Amo 1618; differing patterns of accumulation were obtained with the two precursors, suggesting more than one point of inhibition. This was borne out with cell-free preparations with which it was demonstrated that both HMG-CoA reductase and squalene-2,3-epoxide cyclase were inhibited, the latter more strongly than the former. GLC analysis of gross sterol and hydrocarbon fractions confirmed previous indications that incorporation of radioactivity into individual sterols was inhibited by Amo 1618. Finally, incorporation of MVA-[¹⁴C] into sterols and sterol precursors of Digitalis was significantly altered by the retardant, thus expanding the generality of the relationship between sterol (particularly 4-desmethylsterol) biosynthesis inhibition and retardant effect.     

Free sterols of the red alga Gigartina skottsbergii

The free sterols of the red alga Gigartina skottsbergii have been identified by means of GC and GC/MS analyses. The mixture contained saturated and unsaturated C²⁷, C²⁸ and C²⁹ sterols. The major component was cholest-5-en-3β-ol. Cholesta-5,24-dien-3β-ol (desmosterol) was present in low proportion but no side chain hydroxylated components were detected.     

Inhibitors of sterol biosynthesis. Synthesis and activities of ring C oxygenated sterols

The chemical syntheses of a number of C27 ring C oxygenated sterols have been pursued to permit evaluation of their activity in the inhibition of sterol biosynthesis in cultured mammalian cells. Thus, 5 alpha-cholest-7-ene-3 beta, 11 alpha-diol, 3 alpha-hydroxy-5 alpha-cholest-9(11)-en-12-one, and the previously unreported 11 alpha-hydroxy-5 alpha-cholest-7-en-3-one, 5 alpha-cholest-9(11)-ene-3,12-dione, and 3 beta-hydroxy-5 alpha-cholest-9 (11)-en-12-one have been synthesized. The effects of these compounds on the synthesis of digitonin-precipitable sterols from labeled acetate in mouse L cells and on the levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in the same cells have been investigated and compared with previously published data on other ring C oxygenated sterols. 5 alpha-Cholest-7-ene-3 beta, 11 alpha-diol was shown to be the most potent inhibitor of sterol synthesis.     

Sterol and sesquiterpenoid biosynthesis during a growth cycle of tobacco cell suspension cultures

The accumulation and biosynthesis of sterols and fungal elicitor-inducible sesquiterpenoids by tobacco (Nicotiana tabacum) cell suspension cultures were examined as a function of a 10 day culture cycle. Sterols accumulated concomitantly with fresh weight gain. The rate of sterol biosynthesis, measured as the incorporation rate of [¹⁴C]acetate and [³H]mevalonate, was maximal when the cultures entered into their rapid phase of growth. Changes in squalene synthetase enzyme activity correlated more closely with thein vivo synthesis rate and accumulation of sterols than 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) enzyme activity. Cell cultures entering into the rapid phase of growth also responded maximally to fungal elicitor as measured by the production of capsidiol, an extracellular sesquiterpenoid. However, the rate of sesquiterpenoid biosynthesis, measured as the incorporation rate of [¹⁴C]acetate and [³H]mevalonate, could not be correlated with elicitor-inducible HMGR or sesquiterpene cyclase enzyme activities, nor elicitor-suppressible squalene synthetase enzyme activity.Abbreviations FPP farnesyl diphosphate - HMGR 3-hydroxy-3-methylglutaryl coenzyme A reductase     

Inhibition of sterol biosynthesis by 14α-hydroxymethyl sterols

14α-Hydroxymethyl-5α-cholest-7-en-3β-ol (I) and 14α-hydroxymethyl-5α-cholest-6-en-3β-ol (II) have been prepared by chemical synthesis from 3β-acetoxy-7α,32-epoxy-14α-methyl-5α-cholestane. Compound I, previously shown to be efficiently convertible to cholesterol upon incubation with rat liver homogenate preparations, has been found to be a potent inhibitor of sterol synthesis in animal cells in culture. Compound I caused a 50% reduction of the levels of HMG-CoA reductase activity in cultures of L cells and fetal liver cells at concentrations of 3 × 10^?6 M and 8 × 10^?6 M, respectively. Compound II, the Δ⁶-analogue of I, caused a 50% suppression of the enzyme activity in the two cell types at even lower concentrations, 5 × 10^?7 M and 2 × 10^?6 M, respectively. Concentrations of I and II required to specifically inhibit sterol synthesis from acetate were similar to those required to suppress the levels of HMG-CoA reductase activity.     

Effect of phenylmethylsulfonyl fluoride on sterol biosynthesis in 10,000 x g supernatant fraction of rat liver homogenates

Phenylmethylsulfonyl fluoride (PMSF), a reagent commonly employed for the inhibition of serine proteases, has been found to cause significant inhibition of the incorporation of labeled acetate, but not mevalonate, into nonsaponifiable lipid and digitonin-precipitable sterols in the 10,000 X g supernatant fraction of rat liver homogenate preparations. In two experiments, the extent of inhibition of the synthesis of digitonin-precipitable sterols from acetate by PMSF at 1 mM was 81 and 65%. PMSF inhibited the synthesis of nonsaponifiable lipid from acetate at concentrations as low as 0.1 microM. Preincubation of the 10,000 X g supernatant fraction of rat liver homogenates with PMSF (1 mM) resulted in a significant reduction of the activities of acetate thiokinase and 3-hydroxy-3-methylglutaric acid (HMG)-CoA synthase, but did not affect the activities of acetoacetyl-CoA thiolase. Preincubation of rat liver microsomes with PMSF (1 mM) caused a 50% reduction in the level of HMG-CoA reductase activity. The combined results indicate that major sites of action of PMSF in the inhibition of sterol biosynthesis from labeled acetate appear to be on the activities of acetate thiokinase, HMG-CoA synthase, and HMG-CoA reductase. Another reagent used to inhibit serine proteases, diisopropylfluorophosphate, had (at a concentration of 1 mM) no effect on the activities of cytosolic acetoacetyl-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase.     

Sur le sterol a 26 atomes de carbone de l'algue rouge Rhodymenia palmata

Triterpene alcohols and sterols of the red alga Rhodymenia palmata have been investigated. Cycloartanol, 31-nor-cycloartanol and the C₂₆ sterol 24-dimethylchola-5,22-diene-3β-ol (1) have been identified. Feeding experiments have been performed using 1-¹⁴C-acetate, 5-¹⁴C-mevalonic acid or ¹⁴C-methylmethionine. The C₂₇, C₂₈ and C₂₉ sterols incorporate radioactivities but the C₂₆ sterol is unlabelled after each experiment; its possible origin is discussed.     

Target of inhibition by the anti-lipogenic antibiotic cerulenin of sterol synthesis in yeast

The antibiotic cerulenin inhibited the incorporation of ¹⁴C-acetyl-CoA by 67% at a concentration of 9 × 10^?6 M but not that of ¹⁴C-HMG-CoA into the non-saponifiable fraction in a cell-free extract of Saccharomyces cerevisiae. Cerulenin markedly inhibited the activity of partially purified HMG-CoA synthase. No inhibition of acetoacetyl-CoA thiolase activity was observed in the same preparation of HMG-CoA synthase. Therefore, cerulenin inhibition of overall sterol synthesis may be accounted for by the specific inhibition of HMG-CoA synthase activity.     

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.     

DNA synthesis and 3-hydroxy-3-methylglutaryl CoA reductase activity in PHA stimulated human lymphocytes: A comparative study of the inhibitory effects of some oxysterols with special reference to side chain hydroxylated derivatives

The effects of a series of oxygenated sterols on DNA synthesis and HMG-CoA reductase activity were tested in human lymphocytes. The cells were stimulated by PHA and cultured in cholesterol containing medium. The inhibitory effects of sterols on DNA synthesis were strictly related to the position and the configuration of the hydroxyl on the side chain, to the side chain conformation and integrity and to the structure of the sterol nucleus. The inhibition of HMG-CoA reductase activity was less dependent on these structural features since all the sterols tested were strong inhibitors. In our experimental conditions the inhibition of DNA synthesis was not related to the suppression of the HMG CoA reductase activity. The specificity of the structures required for DNA synthesis inhibition could be explained by the involvement of a specific hydroxysterol binding protein.     

Codisterol and other Δ5-sterols in the seeds of Cucurbita maxima

A substantial amount (ca 18%) of the sterol found in the seeds of Cucurbita maxima had a Δ-bond and consisted of seven components. They were identified as 25(27)-dehydroporiferasterol, clerosterol, isofucosterol, stigmasterol, sitosterol, campesterol and codisterol. The C-24 configuration of each of the sterols was unequivocally established by a ¹H NMR spectral comparison with authentic standards. This is the first time codisterol has been found in a higher plant and also the first time the structures and configurations of the Δ⁵-sterols from a Cucurbitaceae species have been clearly characterized.