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At least 847 records · Page 47Linked to original sources
PubMed2002

Interaction of the Vibrio cholerae cytolysin (VCC) with cholesterol, some cholesterol esters, and cholesterol derivatives: a TEM study.

The Vibrio cholerae cytolysin (VCC) 63-kDa monomer has been shown to interact in aqueous suspension with cholesterol microcystals to produce a ring/pore-like heptameric oligomer approximately 8 nm in outer diameter. Transmission electron microscopy data were produced from cholesterol samples adsorbed to carbon support films, spread across the holes of holey carbon films, and negatively stained with ammonium molybdate. The VCC oligomers initially attach to the edge of the stacked cholesterol bilayers and with increasing time cover the two planar surfaces. VCC oligomers are also released into solution, with some tendency to cluster, possibly via the hydrophobic membrane-spanning domain. At the air/water interface, the VCC oligomers are likely to be selectively oriented with the hydrophobic domain facing the air. Despite some molecular disorder/plasticity within the oligomers, multivariate statistical analysis and rotational self-correlation using IMAGIC-5 strongly suggest the presence of sevenfold rotational symmetry. To correlate the electron microscopy data with on-going biochemical and permeability studies using liposomes of varying lipid composition, the direct interaction of VCC with several cholesterol derivatives and other steroids has been examined. 19-Hydroxycholesterol and 7 beta-hydroxycholesterol both induce VCC oligomerization. beta-Estradiol, which does not possess an aliphatic side chain, also efficiently induces VCC oligomer formation, as does cholesteryl acetate. Cholesteryl stearate and oleate and the C22 (2-trifluoroacetyl)naphthyloxy analogue of cholesterol fail to induce VCC oligomerization, but binding of the monomer to the surface of these steroids does occur. Stigmasterol has little tendency to induce oligomer formation, and oligomers are largely confined to the edge of the bilayers; ergosterol has even less oligomerization ability. Attempts to solubilize and stabilize the VCC oligomers from cholesterol suspensions have been pursued using the neutral surfactant octylglucoside. Although individual solubilized oligomers have been defined which exhibit a characteristic cytolysin channel conformation in the side-on orientation, a tendency remains for the oligomers to cluster via their hydrophobic domains.

Cholesterol↗
PubMedDate not supplied

Use of dominant negative nuclear receptors to study xenobiotic-inducible gene expression in primary cultured hepatocytes.

INTRODUCTION: To determine the feasibility of using dominant negative nuclear receptors to dissect the regulation of inducible gene expression in primary cultured hepatocytes, a series of dominant negative nuclear receptor expression plasmids were designed with truncated AF-2 subdomains. METHODS: Plasmids expressing dominant negative or wild-type constitutive androstane receptor (CAR), pregnane X receptor (PXR), farnesoid X receptor (FXR), liver X receptor (LXR), or peroxisome proliferator-activated receptor alpha (PPARalpha) were transiently cotransfected into primary cultured rat hepatocytes, together with an appropriate reporter plasmid. RESULTS: Treatment with prototypic inducers, 10(-4) M phenobarbital (CAR activator), 10(-5) M pregnenolone 16alpha-carbonitrile (PXR activator), 3x10(-5) M chenodeoxycholate (FXR activator), or 10(-4) M ciprofibrate (PPARalpha activator), significantly activated expression from the corresponding reporter plasmid. Treatment with 22(R)-hydroxycholesterol (LXR activator) only weakly activated the LXR-responsive reporter, while pregnenolone 16alpha-carbonitrile treatment significantly activated this reporter. Cotransfection with wild-type LXRalpha strongly enhanced 22(R)-hydroxycholesterol-inducible expression from the LXR-responsive reporter. Cotransfection of hepatocyte cultures with each of the dominant negative nuclear receptor plasmids significantly inhibited inducible expression of the corresponding reporter while, with one exception (LXRalpha), cotransfection with the wild-type receptor moderately enhanced or had little effect on reporter expression. When each dominant negative nuclear receptor was cross-examined against all inducer-reporter pairs, effects on multiple inducer-reporter pairs were frequently observed. However, in general, only cotransfection with the appropriate dominant negative inhibited inducible reporter expression to a greater extent than did cotransfection with the corresponding wild-type receptor. DISCUSSION: We suggest that the application of dominant negative nuclear receptors has utility in transient transfection studies aimed at discerning the regulatory role of individual nuclear receptor transcription factors in inducible hepatic gene expression, provided that appropriate controls are employed.

Animals↗
PubMed2003

Expression, regulation, and activity of ABCA1 in human cell lines.

Mutations in the ATP-binding cassette transporter A1 (ABCA1) gene cause familial high-density lipoprotein deficiency and Tangier disease. ABCA1 plays a crucial role in active apolipoprotein A-I (apoA-I) lipidation, a key step in reverse cholesterol transport. We compared ABCA1 transcriptional regulation and cholesterol efflux in human skin fibroblasts, monocyte-derived macrophages and hepatocytes (HepG2). 8-Br-cAMP did not increase ABCA1 transcription in these tissues compared to mouse macrophages. We found that ABCA1 is differentially regulated among tissues. While transcription in HepG2 appears to be constitutive, sterols stimulate ABCA1 transcription in fibroblasts and monocyte-derived macrophages. ApoA-I promoted cholesterol efflux in fibroblasts, macrophages, and HepG2. Cholesterol homeostasis in fibroblasts is tightly regulated, and ABCA1 mRNA closely follows the cellular mass of free cholesterol (dose- and time-dependent manner). To further determine the mechanism used by fibroblasts to maintain sterol balance, we used a competitive inhibition approach with geranylgeranyl pyrophosphate (GGPP) to block the LXR induction pathway. GGPP blocked basal, 22-(R)-hydroxycholesterol- and cholesterol-induced ABCA1 expression. Taken together, these results demonstrate that: (1) ABCA1 expression varies among tissues, and (2) cholesterol conversion to hydroxycholesterol is an important mechanism for the maintenance of cholesterol homeostasis in fibroblasts.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2005

Cholesterol binding to cytochrome P450 7A1, a key enzyme in bile acid biosynthesis.

The conversion of cholesterol to 7alpha-hydroxycholesterol catalyzed by cytochrome P450 7A1 (CYP7A1) initiates the major pathway for cholesterol elimination in mammals. In the present work we focused on identification of determinants of the CYP7A1 substrate specificity inside the active site using a homology model with a novel P450-fold, site-directed mutagenesis, and substrate-binding and kinetic studies. Forty-one mutants, encompassing twenty-six amino acid residues, were generated and characterized, and of these, seven residues appear to determine cholesterol binding in the active site. In addition, four cholesterol derivatives were used as active site probes in the wild type and the seven mutant enzymes, and the spectral binding constants and products were analyzed. It was concluded that Asn288 in the I helix plays a key role in the P450-cholesterol contacts by hydrogen bonding to the steroid 3beta-hydroxyl, while Val280 and Ala284 are beside and the Trp283 is above the steroid nucleus orienting the cholesterol molecule. Leu360 and Ala358 between the K helix and the beta1-4 strand and Leu485 in the beta4 sheet-turn appear to define the size of the active site over the heme pyrrole ring A, thus limiting the orientation and size of the substrate at the steroid A ring. Additionally, the A358V mutant was found to form two new products, one being 7beta-hydroxycholesterol. Our data indicate that a tight fit of cholesterol in the enzyme active site is in part responsible for the high efficiency of cholesterol turnover by CYP7A1.

2-Hydroxypropyl-beta-cyclodextrin↗
PubMed2003

Identification and quantification of cholesterol and cholesterol oxidation products in different types of Iberian hams.

Cholesterol and cholesterol oxidation products (COPs) were determined in four different groups of dry-cured Iberian hams, based on the feeding received by pigs and their degree of crossbreeding. After lipid extraction, GC-FID for cholesterol determination and GC-MS to analyze COPs were used. Cholesterol content ranged from 30 to 34 mg/100 g of muscle. Some of the COPs analyzed, such as 7alpha-hydroxycholesterol, 7beta-hydroxycholesterol, and 7-ketocholesterol, were detected in all of the samples. The major cholesterol oxide was 7-ketocholesterol; its concentration ranged from 57 to 71 microg/100 g of muscle. The content of cholesterol and cholesterol oxides in intramuscular lipids of hams was not affected by diet or crossbreeding of Iberian pigs.

Animals↗
PubMed2006

Formation of cholesterol oxidation products in marinated foods during heating.

The objectives of this study were to develop a gas chromatography-mass spectrometry (GC-MS) method to analyze the contents of cholesterol oxidation products (COPs) in marinated eggs, pork, and juice and to compare the effect of heating time and soy sauce or sugar on the formation of COPs. By using a silica cartridge for purification and GC-MS with selected ion monitoring for detection, seven COPs, including 7alpha-hydroxycholesterol, 7beta-hydroxycholesterol, 5,6alpha-epoxycholesterol, 5,6beta-epoxycholesterol, 5alpha-cholestane-3beta, 5,6beta-triol, 5-cholesten-3beta-25-diol, and 7-ketocholesterol, as well as internal standard 5alpha-cholestane, were resolved within 16 min by using a HP-5MS capillary column. During marinating, the levels of most COPs followed an increasing trend with increasing heating time. However, a higher amount of COPs was generated for ground pork as compared to eggs. The incorporation of soy sauce or sugar (1 and 10%) was effective in inhibiting COPs formation, with the latter being more pronounced than the former in both marinated eggs and pork.

Animals↗
PubMed2006

Synthesis of the aglycone of the shark repellent pavoninin-4 using remote functionalization.

[reaction: see text] The aglycone of shark repellent pavoninin-4, (25R)-5alpha-cholestan-3alpha,15alpha,26-triol 26-acetate 1a, was synthesized from (25R)-cholest-5-en-3beta,26-diol 4 (26-hydroxycholesterol) in eight steps in 18% overall yield. Breslow's remote functionalization strategy was used as a key step to introduce the C-15alpha alcohol on a steroid D ring. An efficient synthesis of the 26-hydroxycholesterol from the 16beta hydroxyl steroid, (25R)-cholest-5-ene-3beta,16beta,26-triol (3a), is also reported.

Aminoglycosides↗
PubMed1998

Cholesterol and oxygenated cholesterol concentrations are markedly elevated in peripheral tissue but not in brain from mice with the Niemann-Pick type C phenotype.

Niemann-Pick disease type C (NP-C) is a rare genetic disorder characterized by progressive neurodegeneration, frequent developmental delay and early death. Tissues of affected individuals accumulate large quantities of free cholesterol in lysosomes. Because cytotoxic oxygenated derivatives of cholesterol are known to form readily when cholesterol concentrations are elevated, we searched for these compounds in liver, kidney, spleen and brain from mice with the NP-C phenotype. In order of abundance, we identified 7 alpha- and 7 beta-hydroxycholesterol, 5 alpha, 6 alpha-epoxycholestan-3 beta-ol, 4 beta-hydroxycholesterol, cholest-4-en-3 beta, 7 alpha-diol and cholest-4-en-3 beta, 6 beta-diol in most tissue samples. Cholesterol concentrations in affected mice were increased 3-fold in kidney and 7- to 8-fold in spleen and liver compared to controls (all p < 0.001) but were unchanged in brain. Although oxysterol levels were markedly elevated in nonbrain tissue, the oxysterol and cholesterol concentrations increased proportionally so that oxysterols expressed as percentage of total sterols were the same for all animals (0.34 +/- 0.19% averaged over all organs in affected animals vs 0.40 +/- 0.42% in control mice). In contrast to peripheral tissue, we could not detect any increase in either absolute or relative oxysterol levels in the brains of affected and control mice (49 +/- 61 vs 53 +/- 43 micrograms/g wet weight and 0.45 +/- 0.52 vs 0.47 +/- 0.37%, respectively). Thus, brain sterols are normal in NP-C mice and it is unlikely that an accumulation of cytotoxic oxygenated derivatives of cholesterol could account for the progressive neuropathology seen in the disease.

Animals↗
PubMed1978

Control of sterol metabolism in rat adrenal mitochondria.

Steroidogenesis by adrenal mitochondria from endogenous precursors is stimulated by corticotropin (ACTH) and is sensitive to the protein-synthesis inhibitor cycloheximide. In the present investigation the effect of cycloheximide treatment on the metabolism of a number of analogues of the normal steroidogenic substrate, i.e. cholesterol, by rat adrenal mitochondria was studied. It was observed that the metabolism of analogues such as desmosterol, 26-norcholest-5-en-3beta-ol and 5-cholen-3beta-ol (that is with non-polar alkyl side chains like cholesterol), was sensitive to cycloheximide treatment. By contrast, the metabolism of those analogues with polar groupings on the side chain, i.e., 20alpha-, 24-, 25- and 26-hydroxycholesterols was insensitive to pretreatment with cycloheximide. The binding of added sterol to the cytochrome P-450 component of the mitochondrial sterol desmolase was studied. Similar studies on the equilibration time on addition of exogenous sterols to achieve maximum rates of pregnenolone production were also made. Both studies show that cholesterol, a non-polar sterol, penetrated slowly through the mitochondrial milieu to reach the cytochrome P-450 reaction centre whereas 24- and 26-hydroxycholesterols rapidly attained the enzymic environment. The cycloheximide-sensitive process in sterol metabolism appeared related to the transfer of non-polar sterols such as cholesterol within the mitochondria to a region in close proximity to the enzyme. The importance, and possible mechanism of action, of the cycloheximide-sensitive factor in the control of adrenal steroidogenesis is discussed.

Adrenal Glands↗
PubMed1984

Inhibition of steroid production in Leydig cells by non-steroidal anti-inflammatory and related compounds: evidence for the involvement of lipoxygenase products in steroidogenesis.

The effect of inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism on steroidogenesis in rat testis Leydig cells and rat tumour Leydig cells has been investigated. In the presence of nordihydroguaiaretic acid [NDGA; 4,4'-(2,3- dimethylbutan -1,4- diyl )bis[1,2- benzendiol ]], 5,8,11,14-eicosatetraynoic acid (ETYA), BW 755C [3-amino-1-[3-(trifluoromethyl)phenyl]-2-pyrazoline hydrochloride] and benoxaprofen [ Opren ; 2-(2-p-chlorophenyl- benzoxazol -5-yl)propionic acid)] (which inhibit lipoxygenase activity), but not indomethacin and aspirin (which inhibit cyclo-oxygenase activity), a dose-related inhibition of lutropin (LH)-stimulated testosterone and pregnenolone production was obtained (ID50 values of 2.5, 30, 25 and 30 microM for NDGA, ETYA, BW 755C and benoxaprofen were obtained, respectively). BW 755C and benoxaprofen had no significant effect on LH-stimulated cyclic AMP production except at the highest concentrations examined (330 and 380 microM, respectively), whereas NDGA and ETYA inhibited LH-stimulated cyclic AMP production in a dose-dependent manner (ID50 7.0 and 22 microM respectively). However, NDGA and ETYA also caused a dose-dependent inhibition of dibutyryl cyclic AMP-stimulated testosterone and pregnenolone production. The metabolism of exogenous ( 22R )-hydroxycholesterol or pregnenolone to testosterone by Leydig cells was not inhibited by either NDGA, ETYA or indomethacin. At low concentrations of NDGA and ETYA a significant increase in the conversion of both pregnenolone and ( 22R )-hydroxycholesterol to testosterone was obtained. Studies in which the metabolism of [14C]arachidonic acid by purified rat tumour Leydig cells was investigated indicate that products are formed by tumour Leydig cells that have similar mobilities in a thin layer chromatography system to 5-L-hydroxy-6,8,11,14-eicosatetraenoic acid, 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid and leukotriene B4. The formation of these products was inhibited to varying degrees by NDGA, BW 755C and benoxaprofen but not by aspirin and indomethacin. These studies demonstrate for the first time that inhibition of lipoxygenase activity but not cyclo-oxygenase activity causes an inhibition of LH- and dibutyryl cyclic AMP-stimulated steroid production and suggest a stimulatory role for products of the lipoxygenase pathway of arachidonic acid metabolism in steroidogenesis. The site of this stimulation is apparently distal to the production of cyclic AMP and before the side chain cleavage of cholesterol.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1998

The activity of HMG-CoA reductase and acetyl-CoA carboxylase in human apocrine sweat glands, sebaceous glands, and hair follicles is regulated by phosphorylation and by exogenous cholesterol.

Human apocrine and sebaceous glands function to secrete lipids, predominantly triglycerides, fatty acids, cholesterol and its esters, and, in the sebaceous gland, squalene. The enzymes that catalyze the important regulatory steps in cholesterol and fatty acid biosyntheses, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and acetyl-CoA carboxylase, respectively, were therefore studied in isolated human skin appendages, and their relevant kinetic parameters determined. The enzyme activities that were observed can account for previously described rates of incorporation of radiolabeled substrates into the appropriate lipids by glands in vitro. Reduced enzyme activities following homogenization in the presence of fluoride indicated that both of these enzymes in skin appendages are inactivated by phosphorylation. The activity of the enzyme known to catalyze this phosphorylation, the AMP-activated protein kinase, was also measured. Compactin was shown to inhibit HMG-CoA reductase in homogenates of these appendages. Conversely, incubation of whole sebaceous glands with compactin resulted in the stimulation of enzyme activity, which suggests that these appendages can respond to diminishing cholesterol levels. The effect of exogenous low density lipoprotein and 25-hydroxycholesterol on HMG-CoA reductase activity from skin appendages was investigated. HMG-CoA reductase activity in both apocrine and sebaceous glands was reduced following incubation with either low density lipoprotein or 25-hydroxycholesterol. Low density lipoprotein receptor and lipoprotein lipase mRNA expression was also detected in skin appendages. These results indicate that apocrine and sebaceous glands have the capacity to sequester dietary cholesterol and fatty acids that may have important implications for the understanding of both acne and axillary odor.

Acetyl-CoA Carboxylase↗
PubMed1999

Structural requirements of ligands for the oxysterol liver X receptors LXRalpha and LXRbeta.

LXRalpha and -beta are nuclear receptors that regulate the metabolism of several important lipids, including cholesterol and bile acids. Previously, we have proposed that LXRs regulate these pathways through their interaction with specific, naturally occurring oxysterols, including 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol. Using a ligand binding assay that incorporates scintillation proximity technology to circumvent many of the problems associated with assaying extremely hydrophobic ligands, we now demonstrate that these oxysterols bind directly to LXRs at concentrations that occur in vivo. To characterize further the structural determinants required for potent LXR ligands, we synthesized and tested a series of related compounds for binding to LXRs and activation of transcription. These studies revealed that position-specific monooxidation of the sterol side chain is requisite for LXR high-affinity binding and activation. Enhanced binding and activation can also be achieved through the use of 24-oxo ligands that act as hydrogen bond acceptors in the side chain. In addition, introduction of an oxygen on the sterol B-ring results in a ligand with LXRalpha-subtype selectivity. These results support the hypothesis that naturally occurring oxysterols are physiological ligands for LXRs and show that a rational, structure-based approach can be used to design potent LXR ligands for pharmacologic use.

Cholesterol 7-alpha-Hydroxylase↗
PubMed1997

Coordinate regulation of lipogenesis, the assembly and secretion of apolipoprotein B-containing lipoproteins by sterol response element binding protein 1.

Stable plasmid-driven expression of the liver-specific gene product cholesterol 7alpha-hydroxylase (7alpha-hydroxylase) was used to alter the cellular content of transcriptionally active sterol response element binding protein 1 (SREBP1). As a result of stable expression of 7alpha-hydroxylase, individual single cell clones expressed varying amounts of mature SREBP1 protein. These single cell clones provided an opportunity to identify SREBP1-regulated genes that may influence the assembly and secretion of apoB-containing lipoproteins. Our results show that in McArdle rat hepatoma cells, which normally do not express 7alpha-hydroxylase, plasmid-driven expression of 7alpha-hydroxylase results in the following: 1) a linear relationship between (i) the cellular content of mature SREBP1 and 7alpha-hydroxylase protein, (ii) the relative expression of 7alpha-hydroxylase mRNA and the mRNA's encoding the enzymes regulating fatty acid, i.e. acetyl-CoA carboxylase and sterol synthesis, i.e. HMG-CoA reductase, (iii) the relative expression of 7alpha-hydroxylase mRNA and microsomal triglyceride transfer protein mRNA, a gene product that is essential for the assembly and secretion of apoB-containing lipoproteins; 2) increased synthesis of all lipoprotein lipids (cholesterol, cholesterol esters, triglycerides, and phospholipids); and 3) increased secretion of apoB100 without any change in apoB mRNA. Cells expressing 7alpha-hydroxylase contained significantly less cholesterol (both free and esterified). The increased cellular content of mature SREBP1 and increased secretion of apoB100 were concomitantly reversed by 25-hydroxycholesterol, suggesting that the content of mature SREBP1, known to be decreased by 25-hydroxycholesterol, mediates the changes in the lipoprotein assembly and secretion pathway that are caused by 7alpha-hydroxylase. These data suggest that several steps in the assembly and secretion of apoB-containing lipoproteins by McArdle hepatoma cells may be coordinately linked through the cellular content of mature SREBP1.

Acetyl-CoA Carboxylase↗
PubMed2000

Disruption of the oxysterol 7alpha-hydroxylase gene in mice.

Mice without oxysterol 7alpha-hydroxylase, an enzyme of the alternate bile acid synthesis pathway with a sexually dimorphic expression pattern, were constructed by the introduction of a null mutation at the Cyp7b1 locus. Animals heterozygous (Cyp7b1(+/-)) and homozygous (Cyp7b1(-/-)) for this mutation were grossly indistinguishable from wild-type mice. Plasma and tissue levels of 25- and 27-hydroxycholesterol, two oxysterol substrates of this enzyme with potent regulatory actions in cultured cells, were markedly elevated in Cyp7b1(-/-) knockout animals. Parameters of bile acid metabolism as well as plasma cholesterol and triglyceride levels in male and female Cyp7b1(-/-) mice were normal. The cholesterol contents of major tissues were not altered. In vivo sterol biosynthetic rates were unaffected in multiple tissues with the exception of the male kidney, which showed a approximately 40% decrease in de novo synthesis versus controls. We conclude that the major physiological role of the CYP7B1 oxysterol 7alpha-hydroxylase is to metabolize 25- and 27-hydroxycholesterol and that loss of this enzyme in the liver is compensated for by increases in the synthesis of bile acids by other pathways. A failure to catabolize oxysterols in the male kidney may lead to a decrease in de novo sterol synthesis.

Animals↗
PubMed2000

7-ketocholesterol is an endogenous modulator for the arylhydrocarbon receptor.

We have identified 7-ketocholesterol (7-KC) as an endogenous modulator that inhibits transactivation by the arylhydrocarbon receptor (AhR) through competitive binding against xenobiotic ligands. 7-KC binds AhR and displaces labeled dioxin (2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD)). IC(50) is 5 x 10(-7) m in vivo and 7 x 10(-6) m in vitro. These figures are consistent with its concentration in human blood plasma and tissues. Association with 7-KC prevents AhR binding to DNA. 7-KC blocks the TCDD-mediated transactivation of stably expressed reporter gene constructs in T47-D cells as well as the expression of the endogenous CYP 1A1 gene in HepG2 cells and in primary porcine aortic endothelial cells. Injection of 7-KC to rats blocks the induction of CYP 1A1 messenger RNA and protein in endothelial cells from myocardial blood vessels. The differential sensitivity of mammalian species to toxic effects of AhR ligands, especially dioxin (TCDD), correlates with the expression of 7-hydroxycholesterol dehydrogenase, which synthesizes 7-KC from 7-hydroxycholesterol. The documented involvement of AhR ligands in cardiovascular diseases through lipid peroxidation and endothelium dysfunction can now be examined in the context of displacement of this protective modulator.

Animals↗
PubMed2002

Transport of cholesterol into mitochondria is rate-limiting for bile acid synthesis via the alternative pathway in primary rat hepatocytes.

Bile acid synthesis occurs mainly via two pathways: the "classic" pathway, initiated by microsomal cholesterol 7alpha-hydroxylase (CYP7A1), and an "alternative" (acidic) pathway, initiated by sterol 27-hydroxylase (CYP27). CYP27 is located in the inner mitochondrial membrane, where cholesterol content is very low. We hypothesized that cholesterol transport into mitochondria may be rate-limiting for bile acid synthesis via the "alternative" pathway. Overexpression of the gene encoding steroidogenic acute regulatory (StAR) protein, a known mitochondrial cholesterol transport protein, led to a 5-fold increase in bile acid synthesis. An increase in StAR protein coincided with an increase in bile acid synthesis. CYP27 overexpression increased bile acid synthesis by <2-fold. The rates of bile acid synthesis following a combination of StAR plus CYP27 overexpression were similar to those obtained with StAR alone. TLC analysis of (14)C-labeled bile acids synthesized in cells overexpressing StAR showed a 5-fold increase in muricholic acid; in chloroform-extractable products, a dramatic increase was seen in bile acid biosynthesis intermediates (27- and 7,27-hydroxycholesterol). High-performance liquid chromatography analysis showed that 27-hydroxycholesterol accumulated in the mitochondria of StAR-overexpressing cells only. These findings suggest that cholesterol delivery to the inner mitochondrial membrane is the predominant rate-determining step for bile acid synthesis via the alternative pathway.

Animals↗
PubMed2003

Knockout of the cholesterol 24-hydroxylase gene in mice reveals a brain-specific mechanism of cholesterol turnover.

Most cholesterol turnover takes place in the liver and involves the conversion of cholesterol into soluble and readily excreted bile acids. The synthesis of bile acids is limited to the liver, but several enzymes in the bile acid biosynthetic pathway are expressed in extra-hepatic tissues and there also may contribute to cholesterol turnover. An example of the latter type of enzyme is cholesterol 24-hydroxylase, a cytochrome P450 (CYP46A1) that is expressed at 100-fold higher levels in the brain than in the liver. Cholesterol 24-hydroxylase catalyzes the synthesis of the oxysterol 24(S)-hydroxycholesterol. To assess the relative contribution of the 24-hydroxylation pathway to cholesterol turnover, we performed balance studies in mice lacking the cholesterol 24-hydroxylase gene (Cyp46a1-/- mice). Parameters of hepatic cholesterol and bile acid metabolism in the mutant mice remained unchanged relative to wild type controls. In contrast to the liver, the synthesis of new cholesterol was reduced by approximately 40% in the brain, despite steady-state levels of cholesterol being similar in the knockout mice. These data suggest that the synthesis of new cholesterol and the secretion of 24(S)-hydroxycholesterol are closely coupled and that at least 40% of cholesterol turnover in the brain is dependent on the action of cholesterol 24-hydroxylase. We conclude that cholesterol 24-hydroxylase constitutes a major tissue-specific pathway for cholesterol turnover in the brain.

Alleles↗
PubMed2005

Insig required for sterol-mediated inhibition of Scap/SREBP binding to COPII proteins in vitro.

When added to living cells, sterols such as cholesterol and 25-hydroxycholesterol block the lateral movement of sterol regulatory element-binding proteins (SREBPs) into COPII-coated vesicles on endoplasmic reticulum (ER) membranes and thereby prevent the SREBPs from reaching the Golgi complex for processing to the mature forms that activate cholesterol synthesis. Sorting of SREBPs into COPII vesicles is mediated by Sar1 and the coat proteins Sec23 and Sec24. Here, we explore the mechanism of sterol inhibition in vitro through use of protein pull-down assays. We show that addition of cholesterol or 25-hydroxycholesterol to microsomal membranes in vitro blocks Sar1-dependent binding of the Sec23/24 complex to Scap, the SREBP escort protein. This in vitro inhibition is dependent on the presence of Insig-1, an ER resident protein that is necessary for sterol-mediated inhibition of Scap/SREBP transport in intact cells. Sec23/24 binding to Scap requires the hexapeptide sequence MELADL located in a cytoplasmic loop of Scap. This hexapeptide acts as a sterol-regulated ER sorting signal. These studies define the biochemical parameters responsible for regulated sorting of an ER membrane protein into COPII-coated vesicles.

Amino Acid Sequence↗