In nonhepatic cells, cholesterol 7alpha-hydroxylase induces the expression of genes regulating cholesterol biosynthesis, efflux, and homeostasis.
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CHO cells expressing the liver-specific gene product cholesterol-7alpha-hydroxylase showed a 6-fold increase in the biosynthesis of [(14)C]cholesterol from [(14)C]acetate, as well as increased enzymatic activities of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and squalene synthase. Cells expressing cholesterol-7alpha-hydroxylase contained less sterol response element-binding protein 1 (SREBP1) precursor, whereas the cellular content of mature SREBP1, as well as the mRNAs of cholesterol biosynthetic genes (HMG-CoA reductase and squalene synthase), were all increased approximately 3-fold. Cells expressing cholesterol-7alpha-hydroxylase displayed greater activities of luciferase reporters containing the SREBP-dependent promoter elements derived from HMG-CoA reductase and farnesyl diphosphate synthase, in spite of accumulating significantly more free and esterified cholesterol and 7alpha-hydroxycholesterol. While cells expressing cholesterol-7alpha-hydroxylase displayed increased SREBP-dependent transcription, sterol-mediated repression of SREBP-dependent transcription by LDL-cholesterol and exogenous oxysterols was similar in both cell types. Cells expressing cholesterol-7alpha-hydroxylase displayed greater rates of secretion of cholesterol as well as increased expression of the ABC1 cassette protein mRNA. Adding 25-hydroxycholesterol to the culture medium of both cell types increased the expression of ABC1 cassette protein mRNA. The combined data suggest that in nonhepatic CHO cells multiple regulatory processes sensitive to cellular sterols act independently to coordinately maintain cellular cholesterol homeostasis. (+info)
Dietary psyllium increases expression of ileal apical sodium-dependent bile acid transporter mRNA coordinately with dose-responsive changes in bile acid metabolism in rats.
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Psyllium (PSY), a type of dietary fiber containing mainly soluble components, has been shown to decrease serum cholesterol concentrations in several species; however, mechanisms involved are not clearly defined. Four groups of 10 rats were fed semipurified diets containing 10% dietary fiber from cellulose and/or PSY for 21 d. Increasing levels of PSY were fed (0,3.33, 6.67 and 10% PSY) with the remaining 10% made up with cellulose. Liver cholesterol, cholesterol 7alpha-hydroxylase (CYP7A) activity and mRNA, 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) mRNA, ileal apical sodium-dependent bile acid transporter (ASBT) mRNA, fecal bile acids and total steroids, and intestinal bile acid content were measured. All variables responded in a dose-dependent manner to PSY in the diet. Total liver cholesterol content was significantly reduced in all groups fed PSY compared to cellulose-fed controls [138(a), 105(b), 105(b) and 93(c) micromol (SEM = 4.2) for 0, 3.33, 6.67 and 10% PSY, respectively]. Activity of CYP7A was significantly greater in all groups fed PSY compared to the cellulose-fed controls [6.36(c), 16.92(b), 15.28(b) and 20.37(a) pmol x min(-1) x mg protein(-1) (SEM = 3.19) for 0, 3.33, 6.67 and 10% PSY, respectively]. These differences in CYP7A activity were similar to differences in CYP7A, HMGR and ASBT mRNA levels. Fecal bile acid and total steroid excretion as well as total intestinal bile acids were significantly greater in rats fed PSY-containing diets compared to 0% PSY-fed rats. These results suggest that the reduction in liver cholesterol involves modulating the size and composition of the bile acid pool via regulation of ileal ASBT, CYP7A and HMGR mRNA levels. (+info)
Hepatic LDL receptor mRNA in rats is increased by dietary mushroom (Agaricus bisporus) fiber and sugar beet fiber.
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Plasma cholesterol concentration is reduced by feeding some dietary fibers and mushroom fruit body, but the mechanism is not fully understood. We examined the effects of mushroom (Agaricus bisporus) fiber and sugar beet fiber on serum cholesterol and hepatic LDL receptor mRNA in rats. Rats were fed a cholesterol-free diet with 50 g/kg cellulose powder (CP), 50 g/kg mushroom (Agaricus bisporus) fiber (MSF) or 50 g/kg sugar beet fiber (BF) for 4 wk. There were no significant differences in the body weight, food intake and cecum weight among the groups. The relative liver weight in the CP group was significantly greater than that in the MSF and BF groups. The cecal pH in the CP and MSF groups was significantly higher than that in the BF group. Cecal acetic acid, butyric acid and total short-chain fatty acid (SCFA) concentrations in the BF group were significantly higher than those in the other groups. The serum total cholesterol, VLDL + intermediate density lipoprotein (IDL) + LDL cholesterol concentrations in the CP group were significantly greater than those in the MSF and BF groups. The HDL cholesterol concentration in the MSF group was significantly lower than that in the CP group. The hepatic LDL receptor mRNA level in the MSF and BF groups was significantly higher than that in the CP group. The results of this study demonstrate that mushroom fiber and sugar beet fiber lowered the serum total cholesterol level by enhancement of the hepatic LDL receptor mRNA. (+info)
Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers.
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Several nuclear hormone receptors involved in lipid metabolism form obligate heterodimers with retinoid X receptors (RXRs) and are activated by RXR agonists such as rexinoids. Animals treated with rexinoids exhibited marked changes in cholesterol balance, including inhibition of cholesterol absorption and repressed bile acid synthesis. Studies with receptor-selective agonists revealed that oxysterol receptors (LXRs) and the bile acid receptor (FXR) are the RXR heterodimeric partners that mediate these effects by regulating expression of the reverse cholesterol transporter, ABC1, and the rate-limiting enzyme of bile acid synthesis, CYP7A1, respectively. Thus, these RXR heterodimers serve as key regulators of cholesterol homeostasis by governing reverse cholesterol transport from peripheral tissues, bile acid synthesis in liver, and cholesterol absorption in intestine. (+info)
Role of FXR and FTF in bile acid-mediated suppression of cholesterol 7alpha-hydroxylase transcription.
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Bile acid biosynthesis is subjected to feedback regulation whereby bile acids down-regulate their own synthesis. The major point of this regulation is at the level of cholesterol 7alpha-hydroxylase (7alpha-hydroxylase), which controls bile acid output from the classic pathway. This regulation is at the level of transcription of the gene. Two bile acid response elements have been localized within the 5'-flanking region of the rat gene and these elements overlap three nuclear receptor binding sites for hepatocyte nuclear factor (HNF-4), liver X receptor (LXR) and alpha(1)-fetoprotein transcription factor (FTF). Recently it has been shown that bile acids are physiological ligands for the farnesyl X receptor (FXR), which suggested that FXR could function by binding to one of the three nuclear receptor sites to mediate regulation of 7alpha-hydroxylase transcription by bile acids. In this study we show that FXR is indeed a crucial factor for bile acid-mediated regulation, but that it functions without binding to DNA. Furthermore, we also demonstrate that neither the LXR nor the HNF-4 sites are involved in bile acid-mediated regulation of 7alpha-hydroxylase transcription. Most importantly, we show that the FTF site is essential for regulation of 7alpha-hydroxylase by bile acids, similar to what we have recently demonstrated for another gene of the bile acid biosynthetic pathway, the sterol 12alpha-hydroxylase gene. These studies demonstrate the crucial role of FTF in the expression and regulation of a critical gene in the bile acid biosynthetic pathways. (+info)
Disruption of the sterol 27-hydroxylase gene in mice results in hepatomegaly and hypertriglyceridemia. Reversal by cholic acid feeding.
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Sterol 27-hydroxylase (CYP27) participates in the conversion of cholesterol to bile acids. We examined lipid metabolism in mice lacking the Cyp27 gene. On normal rodent chow, Cyp27(-/-) mice have 40% larger livers, 45% larger adrenals, 2-fold higher hepatic and plasma triacylglycerol concentrations, a 70% higher rate of hepatic fatty acid synthesis, and a 70% increase in the ratio of oleic to stearic acid in the liver versus Cyp27(+/+) controls. In Cyp27(-/-) mice, cholesterol 7alpha-hydroxylase activity is increased 5-fold, but bile acid synthesis and pool size are 47 and 27%, respectively, of those in Cyp27(+/+) mice. Intestinal cholesterol absorption decreases from 54 to 4% in knockout mice, while fecal neutral sterol excretion increases 2.5-fold. A compensatory 2.5-fold increase in whole body cholesterol synthesis occurs in Cyp27(-/-) mice, principally in liver, adrenal, small intestine, lung, and spleen. The mRNA for the cholesterogenic transcription factor sterol regulatory element-binding protein-2 (SREBP-2) and mRNAs for SREBP-2-regulated cholesterol biosynthetic genes are elevated in livers of mutant mice. In addition, the mRNAs encoding the lipogenic transcription factor SREBP-1 and SREBP-1-regulated monounsaturated fatty acid biosynthetic enzymes are also increased. Hepatic synthesis of fatty acids and accumulation of triacylglycerols increases in Cyp27(-/-) mice and is associated with hypertriglyceridemia. Cholic acid feeding reverses hepatomegaly and hypertriglyceridemia but not adrenomegaly in Cyp27(-/-) mice. These studies confirm the importance of CYP27 in bile acid synthesis and they reveal an unexpected function of the enzyme in triacylglycerol metabolism. (+info)
Assay of microsomal oxysterol 7alpha-hydroxylase activity in the hamster liver by a sensitive method: in vitro modulation by oxysterols.
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A method of assaying hepatic cytochrome P-450, oxysterol 7alpha-hydroxylase (CYP7B), was developed by combining the use of 25-[26,27-(3)H]hydroxycholesterol as a substrate and hydroxypropyl-beta-cyclodextrin as a substrate vehicle. When these assay conditions were tested, an undesirable transformation was observed of the reaction product, 7alpha,25-dihydroxycholesterol, into 3-oxo-7alpha,25-dihydroxy-4-cholesten by the activity of 3beta-hydroxy-Delta(5)-C(27) steroid oxydoreductase, a microsomal NAD(+) and NADP(+) dependent enzyme of bile acid metabolism. A great improvement was reached by using a continuous NADPH generating system which constantly re-transforms NADP(+) into NADPH, thus inhibiting this activity. This improved CYP7B assay, comparable to our previously described assay for cholesterol 7alpha-hydroxylase (CYP7A), allowed a 3-fold increase of the apparent enzyme activity. The possibility to simultaneously measure CYP7A and CYP7B activities on the same microsomal preparation was investigated. A marked decrease (-33%) in the CYP7B activity was noticed, while that of CYP7A remained unchanged. The CYP7B activity was observed to be inhibited by cholesterol (-30%) and also by the oxysterols 7alpha-hydroxycholesterol (-21%), 7beta-hydroxycholesterol (-25%) and epicoprostanol (-20%), and by cyclosporin A (-26%). It can be concluded that this sensible and easy to perform CYP7B assay allows to observe, at least in vitro, a modulation of the enzyme activity by oxysterols. (+info)
24-hydroxycholesterol is a substrate for hepatic cholesterol 7alpha-hydroxylase (CYP7A).
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(24S)-Hydroxycholesterol is formed from cholesterol in the brain and is important for cholesterol homeostasis in this organ. Elimination of (24S)-hydroxycholesterol has been suggested to occur in the liver but little is known about the metabolism of this oxysterol. In the present investigation, we report formation of 7alpha, 24-dihydroxycholesterol in pig and human liver. 7alpha-hydroxylase activity toward both isomers of 24-hydroxycholesterol [(24S) and (24R)] was found in a partially purified and reconstituted cholesterol 7alpha-hydroxylase (CYP7A) enzyme fraction from pig liver microsomes. In contrast, a purified enzyme fraction of pig liver oxysterol 7alpha-hydroxylase with high activity toward 27-hydroxycholesterol did not show any detectable activity toward 24-hydroxycholesterol. 7alpha-Hydroxylation of 24-hydroxycholesterol was strongly inhibited by 7-oxocholesterol, a known inhibitor of CYP7A. Human CYP7A, recombinantly expressed in Escherichia coli and in simian COS cells, showed 7alpha-hydroxylase activity toward both cholesterol and the two isomers of 24-hydroxycholesterol, with a preference for the (24S)-isomer. Our results show that 24-hydroxycholesterol is metabolized by CYP7A, an enzyme previously considered to be specific for cholesterol and cholestanol and not active toward oxysterols. Because CYP7A is the rate-limiting enzyme in the major pathway of bile acid biosynthesis, the possibility is discussed that at least part of the 24-hydroxycholesterol is converted into 7alpha-hydroxylated bile acids by the enzymes involved in the normal biosynthesis of bile acids. (+info)