Lathosterol side chain amides: a new class of human lathosterol oxidase inhibitors

Inhibition of cholesterol biosynthesis offers the opportunity for treatment of cardiovascular diseases. Numerous enzymes are involved in the post-squalene part of this biosynthesis, and selective inhibitors for almost all of the enzymes involved there have been described in literature. The only exception is the enzyme lathosterol oxidase (EC 1.14.21.6), for which up to now no selective inhibitor has been found. Up to date only triarimol has been reported as a weak inhibitor. In this paper we report on lathosterol side chain amides as a new class of selective lathosterol oxidase inhibitors. To study the influence of different sterol amides on inhibition of this enzyme, numerous compounds were prepared and the sterol patterns resulting from incubation of HL 60 cells with these enzyme inhibitors were monitored in a whole cell screening assay by means of GC/MS analysis. Small alkyl residues at the amide nitrogen (hydrogen and methyl) lead to an inhibition of the enzyme Delta24-reductase, the N-ethyl and N-propyl derivatives show a dual action, inhibiting both Delta24-reductase and lathosterol oxidase. Lathosterol-derived amides with larger substituents (butyl, isobutyl, tert-butyl, pentyl) at the amide nitrogen were found to be selective inhibitors of lathosterol oxidase. The corresponding 3beta-acetoxy derivatives showed comparable activities and can be considered as prodrugs, since they are transformed into the 3beta-hydroxy derivatives under the test conditions, as proven by HPLC analysis.     

Evidence for similarities and differences in the biosynthesis of fungal sterols

The sterol composition of two ascomycetous fungi, Saccharomyces cerevisiae and Gibberella fujikuroi, was examined by chromatographic (TLC, GLC, and HPLC) and spectral (MS and 1H-NMR) methods. Of notable importance was that both fungi produced cholesterol and a homologous series of long chain fatty alcohols (C22 to C30). In addition to ergosterol two novel sterols, ergosta-5,7, 9(11), 22-tetraenol and ergosterol endoperoxide, were isolated as minor compounds in growth-arrested cultures of yeast and in mycelia of G. fujikuroi. 24-Ethylidenelanosterol was also detected in mycelia of G. fujikuroi. A shift in sterol biosynthesis was observed by treatment with 24 (RS), 25-epiminolanosterol (an inhibitor of the S-adenosylmethionine C-24 transferase) and by monitoring the sterol composition at various stages of development. The results are interpreted to imply that the genes for 24-desalkyl, e.g., cholesterol, and 24-alkyl sterols, e.g., 24 beta- methyl cholesterol and 24-ethyl cholesterol, are distributed (but not always expressed) generally throughout the fungi but the occurrence of one or another compounds is influenced by the fitness (structure and amount) for specific sterols to act functionally during fungal ontogeny; sterol fitness is coordinated with Darwinian selection pressures.     

Preparation of transition-state analogues of sterol 24-methyl transferase as potential anti-parasitics

There is an urgent need for new drugs to treat leishmaniasis and Chagas disease. One important drug target in these organisms is sterol biosynthesis. In these organisms the main endogenous sterols are ergosta- and stigmata-like compounds in contrast to the situation in mammals, which have cholesterol as the sole sterol. In this paper we discuss the design, synthesis and evaluation of potential transition state analogues of the enzyme Delta24(25)-methyltransferase (24-SMT). This enzyme is essential for the biosynthesis of ergosterol, but not required for the biosynthesis of cholesterol. A series of compounds were successfully synthesised in which mimics of the S-adenosyl methionine co-factor were attached to the sterol nucleus. Compounds were evaluated against recombinant Leishmania major 24-SMT and the parasites L. donovani and Trypanosoma cruzi in vitro, causative organisms of leishmaniasis and Chagas disease, respectively. Some of the compounds showed inhibition of the recombinant Leishmania major 24-SMT and induced growth inhibition of the parasites. Some compounds also showed anti-parasitic activity against L. donovani and T. cruzi, but no inhibition of the enzyme. In addition, some of the compounds had anti-proliferative activity against the bloodstream forms of Trypanosoma brucei rhodesiense, which causes African trypanosomiasis.     

Lanosterol 14 alpha-demethylase (P45014DM): effects of P45014DM inhibitors on sterol biosynthesis downstream of lanosterol

Lanosterol 14 alpha-demethylase (P45014DM) is the cytochrome P450 enzyme complex responsible for an early step in cholesterol biosynthesis, namely the 14 alpha-demethylation of lanosterol. We have synthesized a novel series of steroidal substrate analogues, designed to be specific and potent inhibitors of P45014DM. We describe here the effects of these compounds on sterol biosynthesis downstream from lanosterol, focusing ultimately on their efficacy as inhibitors of cholesterol biosynthesis. Results using a radio-high performance liquid chromatography (HPLC) assay show that in rat liver microsomal preparations, with [24,25-3H]dihydrolanosterol as substrate, the compounds do indeed inhibit the biosynthesis of sterols downstream from lanosterol. A range of inhibitory potencies was observed, and the key enzyme being inhibited was believed to be P45014DM. Inhibitor efficacy was readily correlated with non-metabolized [24,25-3H]dihydrolanosterol, formation of 4,4-dimethyl-cholest-8-en-3 beta-ol, and formation of lathosterol, a sterol believed to be an excellent indicator of whole body cholesterol biosynthesis in humans.     

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.     

Inhibition of cholesterol biosynthesis disrupts lipid raft/caveolae and affects insulin receptor activation in 3T3-L1 preadipocytes

Lipid rafts are plasma membrane microdomains that are highly enriched with cholesterol and sphingolipids and in which various receptors and other proteins involved in signal transduction reside. In the present work, we analyzed the effect of cholesterol biosynthesis inhibition on lipid raft/caveolae composition and functionality and assessed whether sterol precursors of cholesterol could substitute for cholesterol in lipid rafts/caveolae. 3T3-L1 preadipocytes were treated with distal inhibitors of cholesterol biosynthesis or vehicle (control) and then membrane rafts were isolated by sucrose density gradient centrifugation. Inhibition of cholesterol biosynthesis with either SKF 104976, AY 9944, 5,22-cholestadien-3β-ol or triparanol, which inhibit different enzymes on the pathway, led to a marked reduction in cholesterol content and accumulation of different sterol intermediates in both lipid rafts and non-raft domains. These changes in sterol composition were accompanied by disruption of lipid rafts, with redistribution of caveolin-1 and Fyn, impairment of insulin-Akt signaling and the inhibition of insulin-stimulated glucose transport. Cholesterol repletion abrogated the effects of cholesterol biosynthesis inhibitors, reflecting they were specific. Our results show that cholesterol is required for functional raft-dependent insulin signaling.     

α-Onocerin and sterol content of twelve species of Ononis

The sterols and triterpenoids of 12 species of the genus Ononis were analysed by GLC. α-Onocerin was found in all but one of these species, although in some others its concentration was low. In all species examined, sitosterol was the major sterol; stigmasterol, campesterol, cholesterol and the triterpenoids cycloartenol and 24-methylene cycloartanol also occurred. The patterns of α-onocerin and sterols found seem to be consistent with the accepted classification of species within the genus.     

Microsomal enzymes of cholesterol biosynthesis from lanosterol. Characterization, solubilization, and partial purification of NADPH-dependent delta 8,14-steroid 14-reductase

The membrane-bound enzyme of microsomes that catalyzes NADPH-dependent reduction of the 14-double bond of conjugated delta 8,14- and delta 7,14-sterols has been studied both as collected in microsomes from broken cell preparations of rat liver and after solubilization. Optimal incubation conditions for assay of the membrane-bound enzyme have been determined, and properties of the microsomal enzyme have been established with respect to cofactor requirements, kinetics, pH, addition of inhibitors, addition of glycerol phosphatides, and sterol substrate specificity. The 14-reductase is readily solubilized with a mixture of octylglucoside and taurodeoxycholic acid. The solubilized enzyme has been enriched by precipitation with polyethylene glycol and chromatography on DEAE-Sephacel and hydroxylapatite columns. The resulting partially purified enzyme has been obtained free of other microsomal enzymes of cholesterol biosynthesis: 4-methyl sterol oxidase, delta 5,7-sterol 7-reductase, delta 8,24-sterol 24-reductase, 3-ketosteroid reductase, and steroid 8----7-ene isomerase, plus microsomal cytochrome P-450, cytochrome P-450 reductase, cytochrome b5 reductase, and cytochrome b5. The partially purified enzyme is stimulated by addition of phospholipids. All of the properties exhibited by partially purified 14-reductase are consistent with the suggestion that the solubilized and enriched enzyme catalyzes the microsomal reduction of the 14-double bond of the sterol-conjugated dienes. However, presence of the enzyme does not prove that the sterol-conjugated dienes are obligatory precursors of cholesterol.     

黄花蒿培养细胞中青蒿素合成代谢的体外调节

黄花蒿培养细胞通过两步培养积累青蒿素．第１步在含有０．２～０．４ｍｇ／Ｌ６－苄基氨基嘌呤（６－ＢＡ）和３～４ｍｇ／Ｌ吲哚乙酸（ＩＡＡ）的Ｎ６培养基中进行细胞的增殖培养,第２步将培养好的细胞转入含０．２～０．４ｍｇ／Ｌ６－ＢＡ和０．２～０．４ｍｇ／ＬＩＡＡ的改良Ｎ６培养基中进行青蒿素的合成．青蒿素的合成量为１９０μｇ／ｇ干细胞左右．当在第２步培养中加入青蒿素合成前体青蒿酸,青蒿素合成量比仅靠激素诱导提高了３倍多．青蒿素的合成途径是植物固醇合成途径的分支途径,当在青蒿素合成过程即第２步培养中加入固醇生物合成抑制剂双氯苯咪唑和氯化氯胆碱处理,可使代谢向合成青蒿素的方向移动,青蒿素合成量明显提高．经２００ｍｇ／Ｌ氯化氯胆碱处理２ｄ,黄花蒿细胞合成青蒿素量为３７２μｇ／ｇ干细胞;经２０ｍｇ／Ｌ双氯苯咪唑处理４ｄ,黄花蒿细胞合成青蒿素量为１５４０μｇ／ｇ干细胞,比靠激素诱导提高了８倍多,与诱导脱分化细胞的黄花蒿叶中所含的青蒿素（３０００μｇ／ｇ干细胞）处于同一个数量级．以上结果表明：在通过植物激素调节可以合成青蒿素的黄花蒿培养细胞中,缺乏青蒿素合成前体是青蒿素合成量低的重要原因．因此,在青蒿素合成的过程中通过体外调节,     

Bisphosphonates used for the treatment of bone disorders inhibit squalene synthase and cholesterol biosynthesis.

Some bisphosphonates used for the treatment of bone disorders are also potent inhibitors of squalene synthase, a critical enzyme for sterol biosynthesis. Among seven drugs tested, YM 175 (cycloheptylaminomethylene-1,1-bisphosphonic acid) was the most potent inhibitor of rat liver microsomal squalene synthase (Ki = 57 nM) and sterol biosynthesis from [14C]mevalonate in rat liver homogenate (IC50 = 17 nM). EB 1053 (3-(1-pyrolidino)-1-hydroxypropylidene-1,1-bisphosphonic acid) and PHPBP (3-(1-piperidino)-1-hydroxypropylidene-1,1-bisphosphonic acid) were less potent inhibitors in both these assays. Pamidronate and alendronate were poor inhibitors of squalene synthase (IC50 > 10 microM) but were potent inhibitors of sterol biosynthesis from mevalonate (IC50 = 420 and 168 nM, respectively), suggesting that the latter two agents may have inhibited other enzymes involved in the synthesis of farnesyl pyrophosphate from mevalonate. Etidronate and clodronate were inactive in both these assays. YM 175 also inhibited sterol biosynthesis in mouse macrophage J774 cells (IC50 = 64 microM) and in rats, when administered acutely, it inhibited cholesterol biosynthesis in the liver (ED50 = 30 mg/kg, s.c.). Structural modifications on YM 175 to enhance cell permeability may result in a new class of cholesterol-lowering agents.     

Side chain azasteroids and thiasteroids as sterol methyltransferase inhibitors in ergosterol biosynthesis

The synthesis of some novel azasteroids and thiasteroids based on a pregnan nucleus with a Δ7 double bond in two to five steps from the key aldehyde (3S,20S)-20-formylpregn-7-en-3-yl acetate has been disclosed herein. These compounds were evaluated as potential inhibitors of the enzyme Δ24-sterol methyltransferase (24-SMT), which is a key enzyme in the biosynthesis of ergosterol, and for their effects on the growth of the yeast Yarrowia lipolytica. Most of the side chain modified analogues were recognized as 24-SMT inhibitors, and in particular the 23-azasteroids 5f–5i and the 24-azasteroid 11 showed potent antifungal activity. The target enzyme could be identified unambiguously using an improved whole-cell assay combined with GC–MS analysis of the sterol pattern resulting upon incubation with the inhibitors.     

76 The Pneumocystis sam:smt, an atypical fungal enzyme protein

Pneumocystis , an opportunistic fungal protist, causes a type of pneumonia in immunocompromised individuals such as AIDS patients. Rat-derived P. carinii and human-derived P. jiroveci contain a large number of sterols with C-24 alkyl groups. S-Adenosyl-L-methionine:sterol C-24 methyl transferase (SAM:SMT) is the enzyme that transfers methyl groups from SAM to the C-24 position of the sterol side chain. An alkyl group at the C-24 sterol side chain position appears to be essential for the organism to proliferate. Thus SAM:SMT, which is absent in mammals, is an attractive target for chemotherapeutic attack against the pathogen. The P. carinii erg6 gene that codes for SAM:SMT has been sequenced, cloned, and the protein expressed in E. coli . Since bacteria do not synthesize sterols, and do not have SAM:SMT, the P. carinii erg6 gene product expressed in E. coli would only transmethylate exogenously provided sterol substrates. The P. carinii recombinant SAM:SMT is unique because lanosterol, a central intermediate in sterol biosynthesis, is its preferred substrate for enzyme activity. Most SAM:SMT from other organisms do not bind lanosterol and prefer other sterol substrates produced from lanosterol. Furthermore, it appears that this unusual P. carinii SAM:SMT can also methylate cholesterol, which is readily scavenged from the lungs of its rat host. The recombinant enzyme protein is being purified by affinity chromatography techniques, which will be used to obtain definitive structural analyses of the sterol compounds formed by the enzyme reaction using different sterols substrates and allow detailed structural analysis of this unusual SAM:SMT enzyme protein.     

Sterol Composition of the Corn Cyst Nematode,Heterodera zeae,and Corn Roots

Sterols from free sterol and steryl ester fractions from Heterodera zeae and from total lipids of Zea mays roots were analyzed by gas-liquid chromatography (GLC) and by GLC-mass spectrometry. The major free sterols of H. zeae were 24-ethylcholesterol (54.4% of total free sterol), 24-ethylcholesta-5,22-dien-3β-ol (13.3%), 24-methylcholesterol (12.5%), and cholesterol (7.2%). The same four sterols comprised 34.6%, 7.2%, 30.3%, and 18.6%, respectively, of the esterified sterols of H. zeae. Corn root sterols included 46.6% 24-ethylcholesta-5,22-dien-3β-ol, 16.7% methylcholesterol, 16.4% cycloartenol, 12.7% 24-ethylcholesterol, and 0.5% cholesterol. The sterol 24-composition of H. zeae differed greatly from that of the only other cyst nematode previously investigated, Globodera solanacearum.     

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.     

Cholesterol, 7-ketocholesterol and 25-hydroxycholesterol uptake studies and effect on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in human fibroblasts.

In human fibroblasts two oxidized derivatives of cholesterol, 7-ketocholesterol and 25-hydroxycholesterol, but not cholesterol itself, are potent inhibitors of 3-hydroxy-3-methylglutaryl co-enzyme A reductase (mevalonate: NADP+ oxidoreductase (Co-enzyme A acylating), (EC 1.1.1.34), the rate-limiting enzyme in sterol biosynthesis. In addition, these derivatives of cholesterol are effective regulators in cells from homozygous familial hypercholesterolemic individuals. The differences in the inhibitory potencies of the sterols cannot be explained in terms of the amount of uptake into the cell.     

Biosynthetic studies of marine lipids. 35. The demonstration of de novo sterol biosynthesis in sponges using radiolabeled isoprenoid precursors.

1. De novo sterol biosynthesis in the sponges Tethya aurantia and Aplysina fistularis was investigated, using sodium [5,5-3H]-mevalonate, [1-3H]-farnesol and [3-3H]-squalene. [3-3H]-Squalene was found to be the best precursor for demonstrating de novo sterol biosynthesis in a wider range of sponges. 2. By feeding [3-3H]-squalene and using cell-free techniques, the de novo sterol biosynthesis was established in 18 sponges belonging to nine orders. Among these sponges were Axinella polypoides and Axinella verrucosa which had previously been thought to be incapable of de novo sterol biosynthesis based on work with radiolabeled lanosterol, cycloartenol, mevalonate, and acetate. 3. In contrast to earlier assumptions, it is likely that all sponges are capable of de novo sterol biosynthesis.     

The role of sterol carrier protein2 and other hepatic lipid-binding proteins in bile-acid biosynthesis.

The ability of different lipid-binding proteins in liver cytosol to affect enzyme activities in bile-acid biosynthesis was studied in whole microsomes (microsomal fractions) and mitochondria and in purified enzyme systems. Sterol carrier protein2 stimulated the 7 alpha-hydroxylation of cholesterol and the 12 alpha-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol in microsomes and the 26-hydroxylation of cholesterol in mitochondria 2-3-fold. It also stimulated the oxidation of 5-cholestene-3 beta, 7 alpha-diol into 7 alpha-hydroxy-4-cholesten-3-one in microsomes. The stimulatory effect of sterol carrier protein2 was much less with purified cholesterol 7 alpha- and 26-hydroxylase systems than with microsomes and mitochondria. No stimulatory effect of sterol carrier protein2 was observed with purified 12 alpha-hydroxylase and 3 beta-hydroxy-delta 5-C27-steroid oxidoreductase. Sterol carrier protein (fatty-acid-binding protein), 'DEAE-peak I protein' [Dempsey, McCoy, Baker, Dimitriadou-Vafiadou, Lorsbach & Howards (1981) J. Biol. Chem. 256, 1867-1873], ligandin (glutathione transferase B) and serum albumin had no marked stimulatory effects in either crude or in purified systems. The results suggest that sterol carrier protein2 facilitates the introduction of the less-polar substrates in bile-acid biosynthesis to the membrane-bound enzymes in crude systems in vitro. The broad substrate specificity appears, however, not to be consistent with a specific regulatory function for sterol carrier protein2 in bile-acid biosynthesis.     

Isolation and chemical characterization of delta 5,7,24-cholestatrien-3 beta-ol from pig tissues

Although indirect evidence has implicated Delta(5,7,24)-cholestatrien-3-ol as a possible intermediate in cholesterol biosynthesis, this sterol has not previously been isolated from tissues. Administration of two inhibitors of cholesterol biosynthesis to pigs led to the accumulation of Delta(5,7,24)-cholestatrien-3-ol in the tissues, and this sterol was isolated from the lung. Proof of its chemical identity was based upon UV, IR, NMR, circular dichroism, and mass spectra, as well as comparison with synthetic Delta(5,7,24)-cholestatrien-3-ol. A fragment at m/e 143 is particularly prominent in the mass spectrum of Delta(5,7)-sterols, and this fact may prove useful for the detection of this functional group. It is proposed that Delta(5,7,24)-cholestatrien-3-ol may be an intermediate in sterol biosynthesis in both animals and plants.     

Lovastatin treatment inhibits sterol synthesis and induces HMG-CoA reductase activity in mononuclear leukocytes of normal subjects

The mechanism by which competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase decrease serum cholesterol is incompletely understood. The few available data in humans suggest that chronic administration of the competitive inhibitor, lovastatin, decreases serum cholesterol with little or no change in total body sterol synthesis. To further define the effect of lovastatin on cholesterol synthesis in normal subjects, we investigated the effect of a single oral dose of lovastatin and a 4-week treatment period of lovastatin on mononuclear leukocyte (ML) sterol synthesis as a reflection of total body sterol synthesis. In parallel, we measured serum lipid profiles and HMG-CoA reductase activity in ML microsomes that had been washed free of lovastatin. ML sterol synthesis did not significantly decrease (23 +/- 5%, mean +/- SEM) at 3 h after a single 40-mg dose of lovastatin. With a single oral 80-mg dose, ML sterol synthesis decreased by 57 +/- 10% (P less than 0.05) and remained low for the subsequent 6 h. With both doses, total HMG-CoA reductase enzyme activity in microsomes prepared from harvested mononuclear leukocytes was induced 4.8-fold (P less than 0.01) over baseline values. Both the 20-mg bid dose and the 40-mg bid dose of lovastatin administered for a 4-week period decreased serum cholesterol by 25-34%. Lovastatin at 20 mg bid decreased ML sterol synthesis by 23 +/- 6% (P less than 0.02) and increased ML HMG-CoA reductase 3.8 times (P less than 0.001) the baseline values. Twenty four hours after stopping lovastatin, ML sterol synthesis and HMG-CoA reductase enzyme activity had returned to the baseline values. The higher dose of lovastatin (40 mg bid) decreased ML sterol synthesis by 16 +/- 3% (P less than 0.05) and induced HMG-CoA reductase to 53.7 times (P less than 0.01) the baseline value at 4 weeks. Stopping this higher dose effected a rebound in ML sterol synthesis to 140 +/- 11% of baseline (P less than 0.01), while HMG-CoA reductase remained 12.5 times baseline (P less than 0.01) over the next 3 days. No rebound in serum cholesterol was observed. From these data we conclude that in normal subjects lovastatin lowers serum cholesterol with only a modest effect on sterol synthesis. The effect of lovastatin on sterol synthesis in mononuclear leukocytes is tempered by an induction of HMG-CoA reductase enzyme quantity, balancing the enzyme inhibition by lovastatin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of sterol 27-hydroxylase and an alternative pathway of bile acid biosynthesis in primary cultures of rat hepatocytes

In man, hepatic mitochondrial sterol 27-hydroxylase and microsomal cholesterol 7-hydroxylase initiate distinct pathways of bile acid biosynthesis from cholesterol, the “acidic” and “neutral” pathways, respectively. A similar acidic pathway in the rat has been hypothesized, but its quantitative importance and ability to be regulated at the level of sterol 27-hydroxylase are uncertain. In this study, we explored the molecular regulation of sterol 27-hydroxylase and the acidic pathway of bile acid biosynthesis in primary cultures of adult rat hepatocytes. mRNA and protein turnover rates were approximately 10-fold slower for sterol 27-hydroxylase than for cholesterol 7-hydroxylase. Sterol 27-hydroxylase mRNA was not spontaneously expressed in culture. The sole requirement for preserving sterol 27-hydroxylase mRNA at the level of freshly isolated hepatocytes (0 h) after 72 h was the addition of dexamethasone (0.1 μM; > 7-fold induction). Sterol 27-hydroxylase mRNA, mass and specific activity were not affected by thyroxine (1.0 μM), dibutyryl-cAMP (50 μM), nor squalestatin 1 (150 nM-1.0 μM), an inhibitor of cholesterol biosynthesis. Taurocholate (50 μM), however, repressed sterol 27-hydroxylase mRNA levels by 55%. Sterol 27-hydroxylase specific activity in isolated mitochondria was increased > 10-fold by the addition of 2-hydroxypropyl-β-cyclodextrin. Under culture conditions designed to maximally repress cholesterol 7-hydroxylase and bile acid synthesis from the neutral pathway but maintain sterol 27-hydroxylase mRNA and activity near 0 h levels, bile acid synthesis from [¹⁴C]cholesterol remained relatively high and consisted of β-muricholate, the product of chenodeoxycholate in the rat. We conclude that rat liver harbors a quantitatively important alternative pathway of bile acid biosynthesis and that its initiating enzyme, sterol 27-hydroxylase, may be slowly regulated by glucocorticoids and bile acids.