Effect of inhibition of sterol delta 14-reductase on accumulation of meiosis-activating sterol and meiotic resumption in cumulus-enclosed mouse oocytes in vitro

Two sterols of the cholesterol biosynthetic pathway induce resumption of meiosis in mouse oocytes in vitro. The sterols, termed meiosis-activating sterols (MAS), have been isolated from human follicular fluid (FF-MAS, 4,4-dimethyl-5 alpha-cholest-8,14,24-triene-3 beta-ol) and from bull testicular tissue (T-MAS, 4,4-dimethyl-5 alpha-cholest-8,24-diene-3 beta-ol). FF-MAS is the first intermediate in the cholesterol biosynthesis from lanosterol and is converted to T-MAS by sterol delta 14-reductase. An inhibitor of delta 7-reductase and delta 14 reductase, AY9944-A-7, causes cells with a constitutive cholesterol biosynthesis to accumulate FF-MAS and possibly other intermediates between lanosterol and cholesterol. The aim of the present study was to evaluate whether AY9944-A-7 added to cultures of cumulus-oocyte complexes (COC) from mice resulted in accumulation of MAS and meiotic maturation. AY9944-A-7 stimulated dose dependently (5-25 mumol l-1) COC to resume meiosis when cultured for 22 h in alpha minimal essential medium (alpha-MEM) containing 4 mmol hypoxanthine l-1, a natural inhibitor of meiotic maturation. In contrast, naked oocytes were not induced to resume meiosis by AY9944-A-7. When cumulus cells were separated from their oocytes and co-cultured, AY9944-A-7 did not affect resumption of meiosis, indicating that intact oocyte-cumulus cell connections are important for AY9944-A-7 to exert its effect on meiosis. Cultures of COC with 10 mumol AY9944-A-7 l-1 in the presence of [3H]mevalonic acid, a natural precursor for steroid synthesis, resulted in accumulation of labelled FF-MAS, which had an 11-fold greater amount of radioactivity incorporated per COC compared with the control culture without AY9944-A-7. In contrast, incorporation of radioactivity into the cholesterol fraction was reduced 30-fold in extracts from the same oocytes. The present findings demonstrate for the first time that COC can synthesize cholesterol from mevalonate and accumulate FF-MAS in the presence of AY9944-A-7. Furthermore, AY9944-A-7 stimulated meiotic maturation dose dependently, indicating that FF-MAS, and possibly other sterol intermediates of the cholesterol synthesis pathway, play a central role in stimulating mouse oocytes to resume meiosis. The results also indicate that oocytes may not synthesize steroids from mevalonate.     

Formation of 7-dehydrocholesterol-containing membrane rafts in vitro and in vivo, with relevance to the Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS) is a recessive disease typified by 7-dehydrocholesterol (7DHC) accumulation and depletion of cholesterol. Because cholesterol is a primary component of detergent-resistant membrane domains ("rafts"), we examined the compatibility of 7DHC with raft formation. Liposomes containing bovine brain phosphatidylcholine, sphingomyelin, cerebrosides, and either cholesterol, 7DHC, or coprostanol (the latter being incompatible with raft formation) were prepared. 7DHC was indistinguishable from cholesterol in its ability to become incorporated into membrane rafts, as judged by physical and chemical criteria, whereas coprostanol did not form rafts. The in vivo compatibility of 7DHC with raft formation was evaluated in brains of rats treated with trans-1,4-bis(2-dichlorobenzylamino-ethyl)cyclohexane dihydrochloride (AY9944), which mimics the SLOS biochemical defect. 7DHC/cholesterol ratios in rafts and whole brains from AY9944-treated rats were similar, indicating comparable efficiency of 7DHC and cholesterol incorporation into brain rafts. In contrast, dolichol (a nonsterol isoprenoid incompatible with raft formation) was greatly depleted in brain rafts relative to whole brain. Although brain raft fractions prepared from AY9944-treated and control rats yielded similar sterol-protein ratios, their gel electrophoresis profiles exhibited multiple differences, suggesting that altered raft sterol composition perturbs raft protein content. These results are discussed in the context of the SLOS phenotype, particularly with regard to the associated central nervous system defects.     

Cholesterol deficit but not accumulation of aberrant sterols is the major cause of the teratogenic activity in the Smith-Lemli-Opitz syndrome animal model

Low cholesterol and high 7-dehydrocholesterol (7DHC) levels are associated with a blockade of Delta7-reductase in the Smith-Lemli-Opitz syndrome (SLOS) and in the animals treated with the inhibitor AY9944. The impact of the cholesterol deficit and of the accumulation of 7DHC on the embryo were investigated in AY9944-treated pregnant rats receiving an enriched cholesterol or 7DHC diet. Sterol profiling was performed under the various nutritional conditions. AY9944 caused a severe decrease in the maternal and embryo cholesterol. The deficit in the embryo was sustained by the embryonic uptake of the inhibitor. A cholesterol-rich diet was efficient in restoring the maternal and embryonic cholesterol and phenotype but a 7DHC-rich diet did not modify the sterol status compared with dams treated with only AY9944. The offspring phenotype remained deleterious whether or not the dams received 7DHC-rich diet. Over 80% of the 7DHC was absorbed, as was cholesterol, which was not quantitatively influenced by AY9944. When cholesterol and 7DHC were simultaneously administered, a competition for intestinal absorption enhanced the lowering cholesterol effect of AY9944.Whether or not the dams received a 7DHC dietary supplement, the offspring's phenotype became normal when the diet was supplemented with cholesterol. Under conditions in which the ratio of cholesterol/7DHC is substantially varied, the normal development of embryos can be achieved as long as the cholesterol is sufficient. The phenotype is reversed in vivo by cholesterol which contrasts with the irreversible effects manifested in vitro by oxidized 7DHC by-products.     

Lovastatin exacerbates atypical absence seizures with only minimal effects on brain sterols

AY-9944 (AY) exacerbates chronic recurrent seizures in rats that are analogous to atypical absence epilepsy in humans. The mechanism by which AY affects the slow spike-and-wave discharges associated with these seizures is not known, but is thought to involve inhibition of cholesterol synthesis. We tested the hypothesis that seizures seen with AY are due to significant reduction in brain cholesterol and/or elevated brain 7-dehydrocholesterol by assessing whether three other cholesterol synthesis inhibitors mimic AY seizures in rats. Effects of AY on brain sterols and spike-and-wave discharge duration were compared with those of two other late-stage cholesterol inhibitors [BM 15.766 (BM) and U18666A (UA)] and to an HMG-CoA reductase (early-stage cholesterol) inhibitor, lovastatin. With BM or UA, prolongation of seizure duration and brain sterol changes was similar to that caused by AY. AY effects on both brain sterols and seizure duration were dose-related. Lovastatin, with or without concurrent AY, mimicked AY seizures but reduced brain cholesterol by <10% and did not significantly change brain 7-dehydrocholesterol. Either lovastatin has a different mechanism of action than these late-stage cholesterol inhibitors or the brain sterol changes are not directly responsible for seizures in this model.     

The effect of the hypocholesteremic drug, AY 9944 on the synthesis of bile salts in rat liver

1. The compound trans-1,4 bis-(2-dichlorobenzylaminomethyl)cyclohexane dihydrochloride (AY9944) blocks cholesterol synthesis at a late stage. This leads to a decrease in cholesterol and accumulation of cholesta-5,7-diene-3-beta-ol (7-dehydrocholesterol) in tissues and plasma. 2. The effect of AY9944 on bile salt synthesis in rat liver was studied. The synthesis of conjugated cholic and chenodeoxycholic acids was measured in hepatocytes isolated from rats 2 h, 24 h and 48 h after administration of a single oral dose of AY9944. Production of the two bile salts was inhibited by 70-80% in hepatocytes from AY9944-treated as compared to untreated animals. 3. When AY9944 was added to the incubation medium in vitro of hepatocytes prepared from untreated rats the synthesis of conjugated cholic and chenodeoxycholic acids was not inhibited during the first hour of incubation, probably because of the presence of endogenous cholesterol. However when hepatocytes from untreated rats were incubated with AY9944 for periods of 2 h or longer, bile salt production was decreased markedly. 4. Bile salt synthesis is stimulated when rats are subjected to total biliary drainage for 24 h. The effect of AY9944 on this stimulation was studied. The content of conjugated cholic and chenodeoxycholic acid in the bile was measured as an indicator of bile salt synthesis. 5. In control animals the rate of secretion of biliary bile salts began to increase after about 24 h of total biliary drainage and reached a maximum after approximately 36 h. A single oral dose of AY9944 given 2 h after the start of total biliary drainage delayed and reduced this response. 6. The results show that inhibition of cholesterol synthesis by AY9944 resulting in the replacement of cholesterol by 7-dehydrocholesterol decreases but does not completely prevent bile salt synthesis.     

Effect of long-term administration of AY-9944, an inhibitor of 7-dehydrocholesterol delta 7-reductase, on serum and tissue lipids in the rat

The effect of long-term administration of AY-9944, a specific inhibitor of cholesterol biosynthesis, was examined in rats maintained on diets with low and high cholesterol and fat content. Sterol and phospholipid levels were determined in the serum, liver, adrenals, lungs, and brain after 6 and 12 months of feeding AY-9944 at several dose levels. In all the tissues examined, the cholesterol content was lowered and the cholesterol was partly replaced by 7-dehydrocholesterol biosynthesized instead of cholesterol in the presence of AY-9944. Cholesterol levels were particularly low in the serum and adrenals, while 7-dehydrocholesterol accumulated in the lungs. The fall in cholesterol and appearance of 7-dehydrocholesterol were reversible. Alterations of this type in the brain indicated that sterol metabolism is active in the adult rat brain. Addition of cholesterol to the diet reduced the effect of the inhibitor by eliminating the liver as a site of sterol synthesis.     

Effects of distal cholesterol biosynthesis inhibitors on cell proliferation and cell cycle progression

Cholesterol is a major lipid component of the plasma membrane in animal cells. In addition to its structural requirement, cholesterol is essential in cell proliferation and other cell processes. The aim of the present study was to elucidate the stringency of the requirement for cholesterol as a regulator of proliferation and cell cycle progression, compared with other sterols of the cholesterol biosynthesis pathway. Human promyelocytic HL-60 cells were cultured in cholesterol-free medium and treated with different distal inhibitors of cholesterol biosynthesis (zaragozic acid, SKF 104976, SR 31747, BM 15766, and AY 9944), which allow the synthesis of isoprenoid derivatives and different sets of sterol intermediates, but not cholesterol. The results showed that only the inhibition of sterol Delta7-reductase was compatible with cell proliferation. Blocking cholesterol biosynthesis upstream of this enzyme resulted in the inhibition of cell proliferation and cell cycle arrest selectively in G2/M phase.     

The use of the Dhcr7 knockout mouse to accurately determine the origin of fetal sterols

Mice with a targeted mutation of 3beta-hydroxysterol Delta(7)-reductase (Dhcr7) that cannot convert 7-dehydrocholesterol to cholesterol were used to identify the origin of fetal sterols. Because their heterozygous mothers synthesize cholesterol normally, virtually all sterols found in a Dhcr7 knockout fetus having a Delta(7) or a Delta(8) double bond must have been synthesized by the fetus itself but any cholesterol had to have come from the mother. Early in gestation, most fetal sterols were of maternal origin, but at approximately E13-14, in situ synthesis became increasingly important, and by birth, 55-60% of liver and lung sterols had been made by the fetus. In contrast, at E10-11, upon formation of the blood-brain barrier, the brain rapidly became the source of almost all of its own sterols (90% at birth). New, rapid, de novo sterol synthesis in brain was confirmed by the observation that concentrations of C24,25-unsaturated sterols were low in the brains of all very young fetuses but increased rapidly beginning at approximately E11-12. Reduced activity of sterol C24,25-reductase (Dhcr24) in brain, suggested by the abundance of C24,25-unsaturated compounds, seems to be the result of suppressed Dhcr24 expression. The early fetal brain also appears to conserve cholesterol by keeping cholesterol 24-hydroxylase expression low until approximately E18.     

Exchange of sterols between myelin and other membranes of developing rat brain

1. The effect of inhibition of cholesterol synthesis by a hypocholesterolaemic drug (AY-9944) was studied in rat brain during development. 2. At 2 weeks after administration of AY-9944 to young rats 7-dehydrocholesterol accounted for half the total sterol of myelin and other subcellular components. 3. At 4 weeks after injection of the drug 7-dehydrocholesterol had disappeared whereas the cholesterol content of myelin had increased by an equivalent amount. Our studies show that purified myelin has low 7-dehydrocholesterol reductase activity and suggest that 7-dehydrocholesterol is largely converted into cholesterol outside the myelin sheath. 4. Resultant cholesterol may be re-incorporated into myelin by an exchange process. 5. The metabolism of sterols in developing brain is discussed.     

A comparison of the behavior of cholesterol and selected derivatives in mixed sterol-phospholipid Langmuir monolayers: a fluorescence microscopy study

Eukaryotic cells require sterols to achieve normal structure and function of their plasma membranes, and deviations from normal sterol composition can perturb these features and compromise cellular and organism viability. The Smith-Lemli-Opitz syndrome (SLOS) is a hereditary metabolic disease involving cholesterol (CHOL) deficiency and abnormal accumulation of the CHOL precursor, 7-dehydrocholesterol (7DHC). In this study, the interactions of CHOL and the related sterols desmosterol (DES) and 7DHC with l-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers were compared. Pressure-area isotherms and fluorescence microscopy were used to study DPPC monolayers containing 0, 10, 20, or 30 mol% sterol. Similar behavior was noted for CHOL- and DES-containing DPPC monolayers with both techniques. However, while 7DHC gave isotherms similar to those obtained with the other sterols, microscopy indicated limited domain formation with DPPC, indicating that 7DHC packs somewhat differently in DPPC membranes compared to CHOL and DES. These results are discussed in relation to SLOS pathobiology.     

Selective sterol accumulation in ABCG5/ABCG8-deficient mice

The ATP binding cassette (ABC) transporters ABCG5 and ABCG8 limit intestinal absorption and promote biliary secretion of neutral sterols. Mutations in either gene cause sitosterolemia, a rare recessive disease in which plasma and tissue levels of several neutral sterols are increased to varying degrees. To determine why patients with sitosterolemia preferentially accumulate noncholesterol sterols, levels of cholesterol and the major plant sterols were compared in plasma, liver, bile, and brain of wild-type and ABCG5/ABCG8-deficient (G5G8(-/-)) mice. The total sterol content of liver and plasma was similar in G5G8(-/-) mice and wild-type animals despite an approximately 30-fold increase in noncholesterol sterol levels in the knockout animals. The relative enrichment of each sterol in the plasma and liver of G5G8(-/-) mice (stigmasterol > sitosterol = cholestanol > bassicasterol > campesterol > cholesterol) reflected its relative enrichment in the bile of wild-type mice. These results indicate that 24-alkylated, Delta22, and 5alpha-reduced sterols are preferentially secreted into bile and that preferential biliary secretion of noncholesterol sterols by ABCG5 and ABCG8 prevents the accumulation of these sterols in normal animals. The mRNA levels for 13 enzymes in the cholesterol biosynthetic pathway were reduced in the livers of the G5G8(-/-) mice, despite a 50% reduction in hepatic cholesterol level. Thus, the accumulation of sterols other than cholesterol is sensed by the cholesterol regulatory machinery.     

THE EFFECT OF HYPOCHOLESTEREMIC AGENTS ON MYELINOGENESIS

Abstract— Three drugs known to inhibit biosynthesis of cholesterol, Clofibrate, 20, 25-diazacholesterol and AY-9944 were administered by stomach intubation to suckling rats. At weaning the rats were killed and subcellular fractions, including myelin, were prepared from the brains and spinal cords and analysed for sterol content. Central nervous tissue fractions from Clofibrate-treated rats showed some decrease in total sterols, but the sterol species were qualitatively normal. AY-9944 given to rats caused high amounts of 7-dehydro-cholesterol to accumulate in all brain and spinal cord fractions with the highest amounts (32–38 percent of total sterols) in myelin. In diazasterol-treated rats desmosterol reached 48 per cent of the sterols of myelin. A group of rats was allowed to survive after the final drug intake (21 days) and their brain and spinal cord sterol content followed up to 60 days. At 30 days the proportion of dehydrocholesterol or desmosterol comprised over half the total myelin sterol. By 60 days of age the 7-dehydrocholesterol had almost completely disappeared from all fractions while substantial amounts of desmosterol were retained in myelin. Myelination was retarded by treatment with AY-9944 and 20, 25-diazasterol, possibly by the limited amount of sterols available. The metabolism of the abnormal myelin constituents in drug-treated animals is discussed in relation to the molecular structure of the myelin membrane.     

Role of cholesterol in the regulation of growth plate chondrogenesis and longitudinal bone growth

Inborn errors of cholesterol synthesis are associated with multiple systemic abnormalities, including skeletal malformations. The regulatory role of cholesterol during embryogenesis appears to be mediated by Shh, a signaling molecule in which activity depends on molecular events involving cholesterol. Based on this evidence, we hypothesized that cholesterol, by modifying the activity of Ihh (another of the Hedgehog family proteins) in the growth plate, regulates longitudinal bone growth. To test this hypothesis, we treated rats with AY 9944, an inhibitor of the final reaction of cholesterol synthesis. After 3 weeks, AY 9944 reduced the cumulative growth, tibial growth, and the tibial growth plate height of the rats. To determine whether cholesterol deficiency affects bone growth directly at the growth plate, we then cultured fetal rat metatarsal bones in the presence of AY 9944. After 4 days, AY 9944 suppressed metatarsal growth and growth plate chondrocyte proliferation and hypertrophy. The inhibitory effect on chondrocyte hypertrophy was confirmed by the AY 9944-mediated decreased expression of collagen X. Lastly, AY 9944 decreased the expression of Ihh in the metatarsal growth plate. We conclude that reduced cholesterol synthesis in the growth plate, possibly by altering the normal activity of Ihh, results in suppressed longitudinal bone growth and growth plate chondrogenesis.     

Cholesterol for Synthesis of Myelin Is Made Locally, Not Imported into Brain

Abstract: We examined whether cholesterol needed for myelin formation is locally synthesized or whether it comes from the circulation. The experimental design was to inject [³H]water and to use incorporation of label into brain cholesterol as a measure of the rate of accumulation of newly synthesized cholesterol in brain. The contribution of the circulation to this labeled cholesterol pool was minimized by repressing liver synthesis of cholesterol with a high cholesterol diet. The rate of accumulation of total cholesterol was calculated from the increasing amounts of sterol in brain regions at successive time intervals during development. Thus, accumulating cholesterol not explained as being newly synthesized (radioactive) could be assumed to have come from the circulation. Long-Evans rats, ranging in age from birth to 35 days, were injected intraperitoneally with [³H]water (0.3–1.0 mCi/g of body weight) and killed 2 h later. The brain was dissected into brainstem, cerebellum, and cerebral hemispheres, and total lipids were extracted. Cholesterol and its precursors were quantified by HPLC. The radioactivity associated with the sterol fractions and the specific activity of body water determined from serum were used to calculate the absolute amount of newly synthesized sterol. The rates of cholesterol synthesis were compared with the rates of accumulation of total cholesterol in each brain region. The rate of accumulation of total sterol (cholesterol and desmosterol) closely followed that of newly synthesized total sterol in all brain regions from the second through the fifth postnatal weeks. Thus, all sterol accumulation in brain during the period of rapid myelination can be explained by local synthesis; neither diet nor production of cholesterol by other organs plays a direct role in supplying cholesterol for myelination in brain.     

AY9944 A-7 promotes meiotic resumption and preimplantation development of prepubertal sheep oocytes maturing in vitro

Follicular fluid meiosis-activating sterol (FF-MAS), an intermediate in the cholesterol biosynthetic pathway, has been identified as a compound that induces the resumption of meiosis in mammalian oocyte. FF-MAS is converted to testis meiosis-activating sterol by a sterol Δ14-reductase. An inhibitor of Δ14-reductase and Δ7-reductase, AY9944 A-7, causes accumulation of FF-MAS by inhibiting its metabolism. The objective of this study was to determine the effects of AY9944 A-7 supplementation to oocyte maturation media on prepubertal sheep oocyte meiotic resumption and subsequent preimplantation development of embryos. Prepubertal sheep oocytes isolated at the germinal vesicle stage from their follicles were cultured with 0, 10, 20, 30, and 40 μM AY9944 A-7 for 24 hours in media with or without a meiotic inhibitor hypoxanthine (Hx, 4 mM). The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown and the first polar body (PBI) extrusion. After maturation for 24 hours, oocytes with PBI were inseminated in vitro, and the percentages developing to the two-cell stage and blastocyst stage were measured as indicators of early embryonic developmental competence. AY9944 A-7 induced maturation of sheep cumulus-oocyte complexes with optimal concentrations of 10 and 20 μM both in Hx-inhibited meiotic maturation and spontaneous maturation, whereas AY9944 A-7 with any concentrations had no significant effect on that of denuded oocytes and split cumulus-oocyte complexes. Furthermore, maturing oocytes treated with either 10 or 20 μM AY9944 A-7 dramatically increased the percentages of ovine embryos developing to the two-cell stage and blastocyst stage. Higher concentrations of AY9944 A-7, 30 and 40 μM, were detrimental to oocytes and led to their degeneration. The present findings indicated for the first time that AY9944 A-7 was not only able to promote meiotic maturation, both Hx-inhibited and spontaneous, but also enhanced preimplantation developmental competence of prepubertal sheep oocytes maturing in vitro.     

Involvement of mitogen-activated protein kinase (MAPK) pathway in LH- and meiosis-activating sterol (MAS)-induced maturation in rat and mouse oocytes

Gonadotropic stimulation of meiotic resumption in mice is dependent upon mitogen-activated protein kinase (MAPK) activation in the somatic compartment of the follicle. By contrast, spontaneous resumption of meiosis is independent of MAPK activation. In view of the suggested role of meiosis-activating sterol (MAS) in oocyte maturation we have (i) compared MAPK activation in rat preovulatory follicles stimulated by LH or by accumulation of endogenous MAS by using an inhibitor of MAS conversion, AY9944; (ii) examined whether stimulation of meiosis by MAS is dependent upon MAPK activation using denuded oocytes (DO) of Mos- null mice (hereafter Mos(-/-)) with oocytes unable to activate MAPK. Rat preovulatory follicles responded to LH or AY9944 stimulation by MAPK activation. Inhibition of MAPK phosphorylation blocked both LH- and AY9944 triggered resumption of meiosis. In mouse cumulus-enclosed oocytes (CEOs) and DOs AY9944 stimulated GVB in wild-type and Mos(-/-) mouse CEOs cultured with hypoxanthine (Hx). Addition of MAS or AY9944 to mouse DOs cultured with Hx induced resumption of meiosis only in wild-type and Mos(+/-) oocytes, but they were ineffective in Mos(-/-) oocytes. The observed sluggish activation of MAPK induced by AY9944 in rat follicle-enclosed oocytes (FEO) may cause the delay in meiotic resumption in response to MAS and AY9944 stimulation. Further, it is incompatible with the suggested role of MAS as an obligatory mediator of LH in the induction of meiotic maturation. MAPK/MOS activation, whether in the somatic compartment or in denuded oocytes, is required for MAS- like LH-, FSH-, or EGF-induced resumption of meiosis.     

Origin of cholesterol in myelin

We review some of the older literature concerning metabolic turnover of cholesterol in the nervous system. The overall picture is that incorporation of radioactive precursors into brain cholesterol is roughly proportional to the rate of myelination and that, once incorporated, radioactive cholesterol is relatively stable metabolically. We outline a strategy for demonstrating the source (local synthesis or uptake from the circulation) of cholesterol in brain. The experimental design involves determining the rate of accumulation of cholesterol this is calculated as the increasing amounts of sterol in brain at successive time intervals during development. The rate of appearance of newly synthesized cholesterol is determined from incorporation of radioactivity from³H₂O (injected i.p. several hours prior to sacrifice) into cholesterol. The radioactivity associated with the sterol fractions and the specific activity of body water determined from the serum can be used to calculate the absolute amount of sterol newly synthesized during the time when³H₂O was present. The results obtained demonstrated that all of the bulk cholesterol accumulating in brain can be accounted for by newly synthesized cholesterol. None of the radioactive cholesterol came from the circulation, since cholesterol feeding suppressed cholesterol biosynthesis in the liver and specific radioactivity of circulating cholesterol was negligible. Thus, almost all cholesterol accumulating in brain during development is locally synthesized. Special issue dedicated to Dr. Marion R. Smith.     

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.     

Dietary intake of plant sterols stably increases plant sterol levels in the murine brain

Plant sterols such as sitosterol and campesterol are frequently administered as cholesterol-lowering supplements in food. Recently, it has been shown in mice that, in contrast to the structurally related cholesterol, circulating plant sterols can enter the brain. We questioned whether the accumulation of plant sterols in murine brain is reversible. After being fed a plant sterol ester-enriched diet for 6 weeks, C57BL/6NCrl mice displayed significantly increased concentrations of plant sterols in serum, liver, and brain by 2- to 3-fold. Blocking intestinal sterol uptake for the next 6 months while feeding the mice with a plant stanol ester-enriched diet resulted in strongly decreased plant sterol levels in serum and liver, without affecting brain plant sterol levels. Relative to plasma concentrations, brain levels of campesterol were higher than sitosterol, suggesting that campesterol traverses the blood-brain barrier more efficiently. In vitro experiments with brain endothelial cell cultures showed that campesterol crossed the blood-brain barrier more efficiently than sitosterol. We conclude that, over a 6-month period, plant sterol accumulation in murine brain is virtually irreversible.     

Regulation of bile acid synthesis. V. Inhibition of conversion of 7-dehydrocholesterol to cholesterol is associated with down-regulation of cholesterol 7 alpha-hydroxylase activity and inhibition of bile acid synthesis.

In the chronic bile fistula rat, the administration of a bolus dose of mevinolinic acid, an inhibitor of HMG-CoA reductase, was followed by rapid down-regulation of cholesterol 7 alpha-hydroxylase activity and a decrease in bile acid synthesis. These observations suggested that either newly synthesized cholesterol or some other metabolite of mevalonate may be involved in the regulation of bile acid synthesis. In order to distinguish between these two alternatives, we carried out experiments in which cholesterol synthesis was blocked by AY9944, a compound that inhibits the conversion of 7-dehydrocholesterol to cholesterol, a last step in the cholesterol biosynthesis pathway. Rats underwent biliary diversion for 72 h at which time they were given intravenously either a bolus dose of AY9944 (1 mg/kg) or control vehicle. At 0 (pre-treatment control), 0.5, 1.5, and 3 h post bolus, livers were harvested and specific activities of cholesterol 7 alpha-hydroxylase were determined. At 1.5, 3, and 6 h post bolus, AY9944 inhibited bile acid synthesis by 19 +/- 6%, 40 +/- 4%, and 41 +/- 6%, respectively, as compared to pretreatment baseline. Cholesterol 7 alpha-hydroxylase activity determined at 0.5, 1.5, and 3 h was decreased by 44 +/- 6%, 44 +/- 2%, and 36 +/- 2%, respectively, as compared to the control value. In in vitro experiments using microsomes from livers of control bile fistula rats, the addition of AY9944 (up to 100 microM) failed to inhibit cholesterol 7 alpha-hydroxylase activity. The results of this study demonstrate that, in the chronic bile fistula rat, acute inhibition of cholesterol synthesis at either early or late steps leads to a rapid down-regulation of cholesterol 7 alpha-hydroxylase activity and decrease in bile acid synthesis.