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Liver X receptor (LXR) agonism has theoretical potential for treating NAFLD/NASH, but synthetic agonists induce hyperlipidemia in preclinical models. Desmosterol, which is converted by Δ24-dehydrocholesterol reductase (DHCR24) into cholesterol, is a potent endogenous LXR agonist with anti-inflammatory properties. We aimed to investigate the effects of DHCR24 inhibition on NAFLD/NASH development. Here, by using APOE*3-Leiden. CETP mice, a well-established translational model that develops diet-induced human-like NAFLD/NASH characteristics, we report that SH42, a published DHCR24 inhibitor, markedly increases desmosterol levels in liver and plasma, reduces hepatic lipid content and the steatosis score, and decreases plasma fatty acid and cholesteryl ester concentrations. Flow cytometry showed that SH42 decreases liver inflammation by preventing Kupffer cell activation and monocyte infiltration. LXRα deficiency completely abolishes these beneficial effects of SH42. Together, the inhibition of DHCR24 by SH42 prevents diet-induced hepatic steatosis and inflammation in a strictly LXRα-dependent manner without causing hyperlipidemia. Finally, we also showed that SH42 treatment decreased liver collagen content and plasma alanine transaminase levels in an established NAFLD model. In conclusion, we anticipate that pharmacological DHCR24 inhibition may represent a novel therapeutic strategy for treatment of NAFLD/NASH., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Squalene is a natural bioactive triterpene and an important intermediate in the biosynthesis of sterols. To assess the effect of this compound on the hepatic transcriptome, RNA-sequencing was carried out in two groups of male New Zealand rabbits fed either a diet enriched with 1% sunflower oil or the same diet with 0.5% squalene for 4 weeks. Hepatic lipids, lipid droplet area, squalene, and sterols were also monitored. The Squalene administration downregulated 9 transcripts and upregulated 13 transcripts. The gene ontology of transcripts fitted into the following main categories: transporter of proteins and sterols, lipid metabolism, lipogenesis, anti-inflammatory and anti-cancer properties. When the results were confirmed by RT-qPCR, rabbits receiving squalene displayed significant hepatic expression changes of LOC100344884 ( PNPLA3 ), GCK , TFCP2L1 , ASCL1 , ACSS2 , OST4 , FAM91A1 , MYH6 , LRRC39 , LOC108176846 , GLT1D1 and TREH . A squalene-enriched diet increased hepatic levels of squalene, lanosterol, dihydrolanosterol, lathosterol, zymostenol and desmosterol. Strong correlations were found among specific sterols and some squalene-changed transcripts. Incubation of the murine AML12 hepatic cell line in the presence of lanosterol, dihydrolanosterol, zymostenol and desmosterol reproduced the observed changes in the expressions of Acss2 , Fam91a1 and Pnpla3 . In conclusion, these findings indicate that the squalene and post-squalene metabolites play important roles in hepatic transcriptional changes required to protect the liver against malfunction.

During the last decade, the understanding of the biological functions of cholesterol biosynthesis intermediates has changed significantly. Particularly, the enzyme sterol dehydrocholesterol reductase 24 (DHCR24) has taken center stage as a potential drug target. Inhibition of DHCR24 leads to accumulation of the endogenous, biologically active metabolite cholesta-5,24-dien-3β-ol (desmosterol). Desmosterol is an endogenous agonist of the liver X receptor (LXR). LXR is a master regulator of lipid metabolism and, as such, is involved in numerous pathophysiological processes such as inflammation, atherosclerosis, cancer, diabetes mellitus (DM), multiple sclerosis (MS), nonalcoholic steatohepatitis (NASH), and the progression of viral infections. Up to now, selective pharmacological targeting of LXR without activating the sterol-response element binding proteins (SREBP) and thereby boosting endogenous lipid biosynthesis has not been achieved. In turn, no selective LXR receptor agonists leveraging its beneficial activation have yet reached the clinic. Therefore, using potent and selective inhibitors of DHCR24 leading to an accumulation of endogenous desmosterol is a promising alternative strategy for the selective activation of LXR. Here we summarize the present landscape of novel lead structures for targeting DHCR24, covering steroidal enzyme inhibitors (e.g., 20,25-diazacholesterol, SH42) as well as nonsteroidal scaffolds (e.g., amiodarone, triparanol). Further, we explain the molecular mechanisms of DHCR24 inhibition/LXR activation, discuss possible therapeutic applications, and underpin why DHCR24 is an upcoming promising drug target., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Cholesterol biosynthetic intermediates, such as lanosterol and desmosterol, are emergent immune regulators of macrophages in response to inflammatory stimuli or lipid overloading, respectively. However, the participation of these sterols in regulating macrophage functions in the physiological context of atherosclerosis, an inflammatory disease driven by the accumulation of cholesterol-laden macrophages in the artery wall, has remained elusive. Here, we report that desmosterol, the most abundant cholesterol biosynthetic intermediate in human coronary artery lesions, plays an essential role during atherogenesis, serving as a key molecule integrating cholesterol homeostasis and immune responses in macrophages. Depletion of desmosterol in myeloid cells by overexpression of 3β-hydroxysterol Δ 24 -reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol, promotes the progression of atherosclerosis. Single-cell transcriptomics in isolated CD45 + CD11b + cells from atherosclerotic plaques demonstrate that depletion of desmosterol increases interferon responses and attenuates the expression of antiinflammatory macrophage markers. Lipidomic and transcriptomic analysis of in vivo macrophage foam cells demonstrate that desmosterol is a major endogenous liver X receptor (LXR) ligand involved in LXR/retinoid X receptor (RXR) activation and thus macrophage foam cell formation. Decreased desmosterol accumulation in mitochondria promotes macrophage mitochondrial reactive oxygen species production and NLR family pyrin domain containing 3 (NLRP3)-dependent inflammasome activation. Deficiency of NLRP3 or apoptosis-associated speck-like protein containing a CARD (ASC) rescues the increased inflammasome activity and atherogenesis observed in desmosterol-depleted macrophages. Altogether, these findings underscore the critical function of desmosterol in the atherosclerotic plaque to dampen inflammation by integrating with macrophage cholesterol metabolism and inflammatory activation and protecting from disease progression., Competing Interests: The authors declare no competing interest.

Ram sperm are particularly sensitive to freeze-thawing mainly due to their lipid composition, limiting their use in artificial insemination programs. We evaluated the extent of cholesterol and desmosterol incorporation into ram sperm through incubation with increasing concentrations of methyl-β-cyclodextrin (MβCD)-sterol complexes, and its effect on membrane biophysical properties, membrane lateral organization and cryopreservation outcome. Sterols were effectively incorporated into the sperm membrane at 10 and 25 mM MβCD-sterols, similarly increasing membrane lipid order at physiological temperature and during temperature decrease. Differential ordering effect of sterols in ternary-mixture model membranes revealed a reduced tendency of desmosterol of segregating into ordered domains. Live cell imaging of fluorescent cholesterol showed sterol incorporation and evidenced the presence of sperm sub-populations compatible with different sterol contents and a high concentration of sterol rich-ordered domains mainly at the acrosome plasma membrane. Lateral organization of the plasma membrane, assessed by identification of GM1-related rafts, was preserved after sterol incorporation except when high levels of sterols (25 mM MβCD-desmosterol) were incorporated. Ram sperm incubation with 10 mM MβCD-sterols prior to cryopreservation in a cholesterol-free extender improved sperm quality parameters after cooling and freezing. While treatment with 10 mM MβCD-cholesterol increased sperm motility, membrane integrity and tolerance to osmotic stress after thawing, incorporation of desmosterol increased the ability of ram sperm to overcome osmotic stress. Our research provides evidence on the effective incorporation and biophysical behavior of cholesterol and desmosterol in ram sperm membranes and on their consequences in improving functional parameters of sperm after temperature decrease and freezing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Activation of liver X receptors (LXRs) with synthetic agonists promotes reverse cholesterol transport and protects against atherosclerosis in mouse models. Most synthetic LXR agonists also cause marked hypertriglyceridemia by inducing the expression of sterol regulatory element-binding protein (SREBP)1c and downstream genes that drive fatty acid biosynthesis. Recent studies demonstrated that desmosterol, an intermediate in the cholesterol biosynthetic pathway that suppresses SREBP processing by binding to SCAP, also binds and activates LXRs and is the most abundant LXR ligand in macrophage foam cells. Here we explore the potential of increasing endogenous desmosterol production or mimicking its activity as a means of inducing LXR activity while simultaneously suppressing SREBP1c-induced hypertriglyceridemia. Unexpectedly, while desmosterol strongly activated LXR target genes and suppressed SREBP pathways in mouse and human macrophages, it had almost no activity in mouse or human hepatocytes in vitro. We further demonstrate that sterol-based selective modulators of LXRs have biochemical and transcriptional properties predicted of desmosterol mimetics and selectively regulate LXR function in macrophages in vitro and in vivo. These studies thereby reveal cell-specific discrimination of endogenous and synthetic regulators of LXRs and SREBPs, providing a molecular basis for dissociation of LXR functions in macrophages from those in the liver that lead to hypertriglyceridemia., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

The enzyme Δ 24 -dehydrocholesterol reductase (DHCR24) catalyzes the reduction of the Δ 24 -double bond in the side chain of cholesterol precursors. Recent biochemical investigations fuel the hope that inhibition of DHCR24, resulting in an accumulation of desmosterol, can open new therapeutic options for treating hepatitis C virus infections, certain forms of cancer and atherosclerosis. In turn, there is a high need for selective, potent and non-toxic inhibitors of DHCR24. Previous reports as well as our re-evaluation showed that established DHCR24 inhibitors are not suitable for this purpose. Based on the lathosterol-derived amide MGI-21 (IC 50 823 nM for inhibition of overall cholesterol biosynthesis in HL-60 cells) we performed a systematic variation of the side chain functionality and identified the steroidal 3,22-diols 29 and 30, as well as several esters thereof, as extremely potent (IC 50  < 5 nM), selective, and non-toxic DHCR24 inhibitors. In mice, diester 27 (SH-42) led to a significant increase in plasma desmosterol levels. The new inhibitors described here are valuable tools for investigating the therapeutic potential of DHCR24 inhibition., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Hepatitis C virus (HCV) infection has been clinically associated with serum lipid abnormalities, yet our understanding of the effects of HCV on host lipid metabolism and conversely the function of individual lipids in HCV replication remains incomplete. Using liquid chromatography-mass spectrometry metabolite profiling of the HCV JFH1 cell culture infection model, we identified a significant steady-state accumulation of desmosterol, an immediate precursor to cholesterol. Pharmacological inhibition or RNAi-mediated depletion of DHCR7 significantly reduced steady-state HCV protein expression and viral genomic RNA. Moreover, this effect was reversed when cultures were supplemented with exogenous desmosterol. Together, these observations suggest an intimate connection between HCV replication and desmosterol homeostasis and that the enzymes responsible for synthesis of desmosterol may be novel targets for antiviral design.

Cholesterol is a unique molecule in terms of high level of in-built stringency, fine tuned by natural evolution for its ability to optimize physical properties of higher eukaryotic cell membranes in relation to biological functions. We previously demonstrated the requirement of membrane cholesterol in maintaining the ligand binding activity of the hippocampal serotonin(1A) receptor. In order to test the molecular stringency of the requirement of cholesterol, we depleted cholesterol from native hippocampal membranes followed by replenishment with desmosterol. Desmosterol is an immediate biosynthetic precursor of cholesterol in the Bloch pathway differing only in a double bond at the 24th position in the alkyl side chain. Our results show that replenishment with desmosterol does not restore ligand binding activity of the serotonin(1A) receptor although replenishment with cholesterol led to significant recovery of ligand binding. This is in spite of similar membrane organization (order) in these membranes, as monitored by fluorescence anisotropy measurements. The requirement for restoration of ligand binding activity therefore appears to be more stringent than the requirement for the recovery of overall membrane order. These novel results have potential implications in understanding the interaction of membrane lipids with this important neuronal receptor in diseases such as desmosterolosis.

Cholesterol homoeostasis is critical for cell viability and proliferation. The SREBP (sterol regulatory element-binding protein) pathway is crucial for the maintenance of cholesterol homoeostasis. This pathway is controlled by cholesterol and cholesterol-derived oxysterols. J774 cells cannot convert desmosterol into cholesterol, a defect resulting from the absence of mRNA for sterol-Delta24-reductase. Using J774 cells, we addressed the capacity of desmosterol to replace cholesterol in sustaining cell proliferation and regulating the SREBP pathway. J774 cells were able to grow indefinitely after the virtually total replacement of cholesterol by desmosterol (J774-D cells). Inhibition of sterol biosynthesis with lovastatin suppressed J774-D cell proliferation. Desmosterol prevented this effect, but its analogue, cholest-5,22-trans-dien-3beta-ol, did not. Addition of desmosterol inhibited processing of SREBP-1 and -2 and also reduced the expression of SREBP-targeted genes. As occurs in cholesterol-containing cells, 25-hydroxycholesterol was more potent than desmosterol or cholesterol in suppressing these processes. Moreover, desmosterol addition enhanced the expression of Abca1 and Srebf1c, two LXR (liver X receptor)-targeted genes. To test the ability of endogenously produced desmosterol to regulate gene expression, J774-D cells were pretreated with lovastatin to inhibit sterol biosynthesis. After removal of the inhibitor the expression of SREBP-targeted genes decreased and that of an LXR-targeted gene increased, reaching control levels. Our results demonstrate that the virtually complete replacement of cholesterol by desmosterol is compatible with cell growth and the functioning of the SREBP pathway. In these cells, desmosterol suppresses SREBP processing and targeted gene expression, and it is especially effective activating LXR-targeted genes.

Cholesterol is an apparently indispensable lipid for numerous processes required for cell proliferation. Levels of this molecule are primarily regulated at the transcriptional level by the SREBPs (sterol-regulatory-element-binding proteins) and LXR (liver X receptor). In this issue of the Biochemical Journal, Rodríguez-Acebes et al. show that a cholesterol precursor, desmosterol, can support cell proliferation in the absence of cholesterol in a murine macrophage-like model (J774-D cells). These cells are defective in DHCR24 (sterol-Delta24-reductase, or 3beta-hydroxysterol Delta24-reductase), leading to desmosterol accumulation, and yet sterol homoeostasis appears to be normal with respect to SREBP processing and LXR activation. Other potentially cholesterol-dependent processes which were not the focus of this study are briefly discussed, such as lipid-raft-dependent cell signalling.

We addressed the ability of native, oxidized and acetylated low-density lipoproteins (nLDL, oxLDL and acLDL, respectively) and desmosterol to act as sources of sterol for the proliferation of J774A.1 macrophages. Treatment with 0.5 microM lovastatin and lipoprotein-deficient serum suppressed cell proliferation. This inhibition was effectively prevented by nLDL, but only to a lesser extent by oxLDL. AcLDL, despite its ability to deliver a higher amount of cholesterol to J774 macrophages than the other LDLs, was dependent on mevalonate supply to sustain cell proliferation. Similarly, exogenous desmosterol, which is not converted into cholesterol in J774 cells, required the simultaneous addition of mevalonate to support optimal cell growth. Expression of hydroxymethyl glutaryl coenzyme A reductase mRNA was potently down-regulated by acLDL and exogenous desmosterol, but the effect was weaker with other sterol sources. We conclude that nLDL is more efficient than modified LDL in sustaining macrophage proliferation. Despite the requirement of cholesterol or desmosterol for J774 cell proliferation, excessive provision of either sterol limits mevalonate availability, thus suppressing cell proliferation.

The liver X receptors (LXRs) are ligand-activated transcription factors that regulate the expression of genes controlling lipid metabolism. Oxysterols bind LXRs with high affinity in vitro and are implicated as ligands for the receptor. We showed previously that accumulation of selected dietary sterols, in particular stigmasterol, is associated with activation of LXR in vivo. In the course of the defining of structural features of stigmasterol that confer LXR agonist activity, we determined that the presence of an unsaturated bond in the side chain of the sterol was necessary and sufficient for activity, with the C-24 unsaturated cholesterol precursor sterols desmosterol and zymosterol exerting the largest effects. Desmosterol failed to increase expression of the LXR target gene, ABCA1, in LXRalpha/beta-deficient mouse fibroblasts, but was fully active in cells lacking cholesterol 24-, 25-, and 27-hydroxylase; thus, the effect of desmosterol was LXR-dependent and did not require conversion to a side chain oxysterol. Desmosterol bound to purified LXRalpha and LXRbeta in vitro and supported the recruitment of steroid receptor coactivator 1. Desmosterol also inhibited processing of the sterol response element-binding protein-2 and reduced expression of hydroxymethylglutaryl-CoA reductase. These observations are consistent with specific intermediates in the cholesterol biosynthetic pathway regulating lipid homeostasis through both the LXR and sterol response element-binding protein pathways.

Vitamin D3 plays an important role in the control of cell proliferation and differentiation. Cholesterol 25-hydroxylase (CH25H) is an enzyme converting cholesterol into 25-hydroxycholesterol. Vitamin D3 as well as 25-hydroxycholesterol has been shown to inhibit cell growth and induce cell apoptosis. Here we show that 10 nM 1alpha,25(OH)2D3 and 500 nM 25OHD3 upregulate CH25H mRNA expression in human primary prostate stromal cells (P29SN). Protein synthesis inhibitor cycloheximide does not block 1alpha,25(OH)2D3 mediated upregulation of CH25H mRNA. Transcription inhibitor actinomycin D blocks basal level as well as 1alpha,25(OH)2D3 induced CH25H mRNA expression. 1alpha,25(OH)2D3 has no effect on CH25H mRNA stability. 25-Hydroxycholesterol significantly decreased the P29SN cell number. A CH25H enzyme inhibitor, desmosterol, increases basal cell number but has no significant effect on vitamin D3 treated cells. Our data suggest that ch25h could be a vitamin D3 target gene and may partly mediate anti-proliferative action of vitamin D3 in human primary prostate stromal cells.

Cholesterols are enriched in the brain and can be oxidized to oxysterols by several processes. Oxysterols are transport forms of cholesterols across cell membranes and the blood-brain barrier. Here, to elucidate the roles of oxysterols in brain inflammation, we treated lipopolysaccharide-stimulated rat brain astrocytes with two oxysterols, 7-ketocholesterol and 22(R)-hydroxycholesterol. Both oxysterols suppressed inducible nitric oxide synthase expression and nitric oxide release as well as upstream signaling molecules including interferon-beta, phosphorylated signal transducer and activator of transcription 1/3, and interferon regulatory factor-1. Oxysterols are known as liver X receptor agonists, and inhibitory effects were also observed with synthetic agonists of liver X receptor and retinoid X receptor. Thus, we conclude that it is most likely mediated by liver X receptor/retinoid X receptor heterodimers.

Ejaculated mammalian sperm must undergo a final maturation (capacitation) before they can acrosome-react and fertilize eggs. Loss of cholesterol is an essential step in the capacitation of human sperm. Experimentally maintaining a high level of cholesterol inhibits capacitation, but the mechanism is unknown. The present study investigated the structural features that are required for cholesterol's inhibitory activity. Human sperm also contain much desmosterol, which is lost from sperm during capacitation. Preventing the loss of desmosterol inhibited capacitation (as assessed by acrosomal responsiveness), with an effectiveness approximately equal to cholesterol's inhibitory activity. Other structural analogs were added to the incubation medium to replace sperm cholesterol and desmosterol. Most inhibited capacitation, including those that lacked cholesterol's 3beta-OH group (cholesteryl methyl ether and epicholesterol) and those with modified C17 groups (ergosterol and diosgenin). Two steroids did not inhibit capacitation well. Coprostanol, which has a nonplanar steroid nucleus, had low inhibitory activity that could be explained by an elevated endogenous cholesterol concentration. Epicoprostanol, which has a nonplanar ring structure and a 3alpha-OH group, promoted rather than inhibited capacitation. The inhibitory activity of the analogs was correlated with their ability to promote order of egg phosphatidylcholine as measured by fluorescence anisotropy. In summary, a planar ring structure is required for sterol inhibitory activity, but a 3beta-OH group and a saturated cholesterol-like aliphatic tail on C17 are not required. The present results support the hypothesis that sperm sterols block capacitation by increasing order of phospholipids.

The pivotal role of liver X receptors (LXRs) in the metabolic conversion of cholesterol to bile acids in mice is well established. More recently, the LXRalpha promoter has been shown to be under tight regulation by peroxisome proliferator-activated receptors (PPARs), implying a role for LXRalpha in mediating the interplay between cholesterol and fatty acid metabolism. We have studied the role of LXR in fat cells and demonstrate that LXR is regulated during adipogenesis and augments fat accumulation in mature adipocytes. LXRalpha expression in murine 3T3-L1 adipocytes as well as in human adipocytes was up-regulated in response to PPARgamma agonists. Administration of a PPARgamma agonist to obese Zucker rats also led to increased LXRalpha mRNA expression in adipose tissue in vivo. LXR agonist treatment of differentiating adipocytes led to increased lipid accumulation. An increase of the expression of the LXR target genes, sterol regulatory binding protein-1 and fatty acid synthase, was observed both in vivo and in vitro after treatment with LXR agonists for 24 h. Finally, we demonstrate that fat depots in LXRalpha/beta-deficient mice are smaller than in age-matched wild-type littermates. These findings imply a role for LXR in controlling lipid storage capacity in mature adipocytes and point to an intriguing physiological interplay between LXR and PPARgamma in controlling pathways in lipid handling.

The effects of exogenous isoprenoids were investigated on A549 human lung adenocarcinoma cells. Among the tested isoprenoids, only farnesol and geranylgeraniol induce actin cytoskeleton disorganization, growth inhibition, and apoptosis. In contrast, desmosterol leads only to growth inhibition. We show that all tested isoprenoids are potent inhibitors of HMG CoA reductase activity, the sterols being the most powerful while they induce neither F-actin disorganization nor apoptosis. Thus the molecular mechanisms induced by farnesol and geranylgeraniol appear independent of reductase regulation. Our results point out the specific role of farnesol and geranylgeraniol on actin cytoskeleton organization and apoptosis in adenocarcinoma cells.

Desmosterol (1), 24,25-epoxycholesterol (2), 24-hydroperoxycholesta-5,25-dien-3 beta-ol (3), 25-hydroperoxycholesta-5,23(E)-dien-3 beta-ol (4), cholesta-5,25-diene-3 beta,24-diol (5), and 24,25-epoxy-6 beta-hydroxycholest-4-en-3-one (7) were isolated from the marine red alga Galaxaura marginata; sterols 3, 4, and 7 were isolated for the first time from a natural source. Sterols 3-7 exhibited significant cytotoxicity toward several cancer cell lines.

A number of neutral marine steroids such as desmosterol, campesterol, brassicasterol, gorgosterol, and other trace steroids were isolated from the coelomic fluid of ripe Nereis succinea and checked for biological activity as sex pheromones on swarming specimens of Platynereis dumerilii and Nereis succinea. No significant influence of synthetic gorgosterol or a natural extract of gorgosterol or the other identified steroids on the swarming behavior was observed.

Steroid sulfotransferase activity has been assayed in cytosol extracts obtained from the male hamster reproductive tract. Dehydroisoandrosterone and desmosterol were used as substrates in the presence of phosphoadenosine phosphosulfate-35S as sulfate donor. No significant sulfotransferase activity was found in the testis. In the epididymis, a severalfold increase in activity was found in the tissue from the caput to the caudal regions. A lower but significant activity was detected in the vas deferens. The enzyme appears to be secreted into the luminal fluid while little activity is associated with the spermatozoa. This increase in activity along the epididymis is undoubtedly responsible for the accumulation of sterol sulfates reported previously. In view of the fact that sterol sulfates are potent and specific inhibitors of acrosin, as reported for the porcine and confirmed herein for hamster acrosin, the epididymal production of steroid and sterol sulfates may represent a protective mechanism against the premature release of proteolytic activity within the male reproductive tract.

Several effects of hydroxylated sterols on cell cultures are known. Most of these can be explained by an inhibition of the cholesterol synthesis at the level of the 3-hydroxy-3-methylglutaryl CoA reductase. When studying cholesterol metabolism in rat adrenal cells, an inhibitory action of some sterols on the ACTH-stimulated corticosterone production was observed. The effects of one sterol, 22S-OH-cholesterol, were investigated further. The sterol had no effect on the ACTH-stimulated cyclic AMP production, suggesting an intact receptor-adenylate cyclase complex and cellular membrane. In the presence of ACTH and 22-OH-cholesterol particularly the free cholesterol concentration was elevated; 22S-OH-cholesterol therefore probably exerts its inhibitory effect at a step located after hydrolysis of the cholesterol esters. 22S-OH-cholesterol had no effect on the conversion of exogenous pregnenolone into corticosterone. These results make it probable, that the inhibitory effect of 22S-OH-cholesterol on the ACTH-stimulated corticosterone production is situated at the level of the cholesterol side-chain cleavage.

Four 3 beta-hydroxy-delta 5-steroid sulphates were found to be potent and specific inhibitors of the sperm acrosomal proteinase, acrosin. Two of these acrosin inhibitors, desmosteryl sulphate and cholesteryl sulphate, occur naturally in spermatozoa. Desmosteryl sulphate, an inhibitor of the in-vitro capacitation of hamster spermatozoa, has a Ki of 3.5 x 10(-6) M for the inhibition of acrosin. The mechanism of inhibition of sperm capacitation by sterol sulphates is probably due to their inhibition of acrosin.

The hypocholesterolemic agent 3 beta-(2-diethylaminoethoxy)androst-5-en-17-one . HCl (U18666A) is known to induce experimental epilepsy. The possibility that this drug interferes with cholesteryl ester formation in glioblastoma cells was examined. The incorporation of radioactive oleic acid into cellular cholesteryl ester was drastically and specifically inhibited by U18666A. The inhibitory effect of U18666A persisted in different oleic acid concentrations. Kinetic studies revealed the rapidity of U18666A action. U18666A was found to be ineffective in inhibiting acyl-CoA:cholesterol acyltransferase activity when it was added directly to the cell homogenates. In contrast, the acyltransferase activity was greatly diminished in homogenates derived from U18666A-treated cells. Thus, U18666A appeared to block cellular cholesteryl ester biosynthesis by indirectly inactivating acyl-CoA:cholesterol acyltransferase activity in a cell-dependent manner. The potent inhibition of cholesteryl ester formation by U18666A represents one unique aspect of the drug which might contribute to its ability to induce chronic epileptiform activity.

Reduced streptolysin O, a toxin produced by certain beta-haemolytic streptococci, lyses human erythrocytes. The reaction is inhibited by cholesterol at concentrations of about 1.0mug/ml. Other sterols inhibit the lysin and there is a specific requirement for a 3beta-hydroxyl group. Inhibition was obtained with 3beta-hydroxychol-5-en-24-oic acid, containing a hydrophilic group at C-24. The mode of inhibition is likely to involve attachment to the fixation site of the lysin which attaches the molecule to cell membranes, probably to membrane cholesterol. A second streptolysin site, concerned in the final haemolytic event, may also be involved. Inhibitors of the latter site have not been characterized, other than antibody with specificity for the site.

Previous spin-label and electromyographic experiments with rats fed 20,25-diazacholesterol, an inhibitor of the biosynthetic conversion of desmosterol to cholesterol, demonstrated an increased erythrocyte membrane fluidity and myotonia, a prolonged muscle contraction upon stimulation. The current studies with rats showed normal erythrocyte fluidity in animals fed 20,25-diazacholesterol but maintained on a high-cholesterol diet and no myotonia. Studies of model membrane systems composed of phospholipid vesicles containing desmosterol, cholesterol, or both demonstrated that desmosterol increased membrane lipid fluidity relative to cholesterol, suggesting that in 20,25-diazacholesterol-induced myotonia, in which desmosterol accounts for 85% of the plasma sterol, the increased membrane fluidity previously observed in erythrocytes and sarcolemma in this animal model of human congenital myotonia may be due to desmosterol.

The acyl-CoA: cholesterol acyl transferase (ACAT) reaction in macrophages is a critical step in atherosclerotic foam cell formation, but little is known about the reaction's sterol substrate specificity. In this report we examine the macrophage ACAT reactivity of the shellfish sterol, desmosterol, and other sterols found in man because of shellfish ingestion or in association with the foam cell diseases sitosterolemia and cerebrotendinous xanthomatosis (CTX). We first show that the J774 macrophage, a foam cell model with a hyperactive ACAT pathway, synthesizes desmosterol instead of cholesterol and that both endogenous and exogenous desmosterol are substrates and stimulators of the ACAT reaction in these cells. When exogenous desmosterol was added to human monocyte-derived macrophages, ACAT was stimulated 29- and 4-fold compared with control and cholesterol-treated cells, respectively. Steryl ester mass accumulation in desmosterol-treated human macrophages was 10-fold greater than in control cells and 3-fold greater than in cholesterol-treated cells. Another shellfish sterol, 24-methylene cholesterol, also stimulated ACAT in human macrophages, but most of the xanthomatosis-related sterols did not stimulate ACAT. These data suggest that: (a) the shellfish sterols desmosterol and 24-methylene cholesterol may be atherogenic; and (b) the excessive foam cell formation seen in sitosterolemia and CTX cannot be explained by ACAT hyperreactivity of their associated sterols.

The biochemical basis for the cholesterol-dependent growth phenotype of the NS-1 myeloma cell line has been investigated. In one series of experiments, the growth response of NS-1 cells to several of the intermediates of cholesterol biosynthesis was studied in serum-free medium. The cholesterol precursors, squalene and lanosterol, were totally ineffective in promoting NS-1 cell growth. In contrast, cholesterol precursors downstream from lanosterol, i.e., desmosterol and 7-dehydrocholesterol, completely replaced cholesterol in supporting NS-1 cell growth. In a second series of experiments, NS-1 cells and NS-1-503 cells (a cholesterol growth-independent variant of NS-1 cells) were labelled with [2-14C]acetate and the distributions of radioactivity between cholesterol and its precursors were determined by thin-layer chromatography using two different solvent systems. The major labelled sterol product (greater than 80%) in NS-1 cells after a 24-h exposure to [2-14C]acetate was lanosterol. In contrast, the major labelled sterol product (greater than 95%) in NS-1-503 cells after a 24-h exposure to [2-14C]acetate was cholesterol. Taken together, these results indicate that NS-1 cells are defective in cholesterol biosynthesis and identify the site of lesion as the demethylation of lanosterol to C-29 sterol intermediates.

NS-1 mouse myeloma cells, a cholesterol auxotrophic cell line with a lesion in the cholesterol biosynthetic pathway at the demethylation of lanosterol to C-29 sterol, were depleted of cholesterol by incubation in cholesterol-free medium for 24 to 48 h. The low-density lipoprotein receptor activities in untreated and in cholesterol-depleted cells were then compared. The cholesterol-depleted NS-1 cells consistently exhibited a 75 to 90% reduction in receptor-mediated low-density lipoprotein binding compared to untreated cells. The decline of the low-density lipoprotein binding of cholesterol-free medium-incubated NS-1 cells was prevented by addition of free cholesterol or its biosynthetic intermediate, demosterol, to the medium. The addition of lanosterol, an intermediate upstream to the lesion site in the cholesterol biosynthetic pathway, was completely ineffective. The results indicate that proper membrane cholesterol content is necessary for the maintenance of normal low-density lipoprotein receptor function in NS-1 cells.

The proximate cholesterol precursors lathosterol, 7-dehydrocholesterol and desmosterol supported the growth of NS-1 and X63 mouse myeloma cells. These cells and X63.653 cells are cholesterol auxotrophs, yet each was able to convert [3H]lathosterol to [3H]cholesterol. These results are consistent with the conclusion that cholesterol auxotrophy in these myeloma cells is due to a deficiency in 3-ketosteroid reductase activity. The steroid hormones testosterone, progesterone and hydrocortisone could not replace cholesterol as a medium supplement. These results provide a greater understanding of the cholesterol auxotrophy characteristic of cell lines clonally-derived from the MOPC 21 myeloma tumor, and they provide a rational basis for the use of sterols in defined culture medium for mouse myeloma cells.