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1.
Metabolism ; 55(3): 292-9, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16483871

ABSTRACT

Plant sterols compete with cholesterol (cholest-5-en-3beta-ol) for intestinal absorption to limit absorption and lower plasma concentrations of cholesterol. Stigmasterol (24-ethyl-cholesta-5,22-dien-3beta-ol; Delta(22) derivative of sitosterol [24-ethyl-cholest-5-en-3beta-ol]), but not campesterol (24-methyl-cholest-5-en-3beta-ol) and sitosterol, is reported to inhibit cholesterol biosynthesis via inhibition of sterol Delta(24)-reductase in human Caco-2 and HL-60 cell lines. We studied the effect of feeding 0.5% stigmasterol on plasma and liver sterols and intestinal cholesterol and sitosterol absorption in 12 wild-type Kyoto (WKY) and 12 Wistar rats. After 3 weeks of feeding, cholesterol and sitosterol absorption was determined in 6 rats from each group by plasma dual-isotope ratio method. After 3 more weeks, plasma and hepatic sterols and hepatic enzyme activities were determined in all rats. After feeding stigmasterol, baseline plasma cholesterol was 1.3 times and plant sterols 3 times greater in WKY compared with Wistar rats. Stigmasterol feeding lowered plasma cholesterol by approximately 11%, whereas plasma campesterol and sitosterol levels were virtually unchanged in both rat strains, and stigmasterol constituted 3.2% of plasma sterols in WKY rats and 1% in Wistar rats. After 6 weeks of feeding, cholesterol and sitosterol absorption decreased 23% and 30%, respectively, in WKY, and 22% and 16%, respectively, in the Wistar rats as compared with untreated rats. The intestinal bacteria in both rat strains metabolized stigmasterol to mainly the 5beta-H stanol (>40%), with only small amounts of 5alpha-H derivative (approximately 1.5%), whereas the C-22 double bond was resistant to bacterial metabolism. Hepatic stigmasterol levels increased from 11 microg/g liver tissue to 104 mug/g in WKY rats and from 5 microg/g liver tissue to 21 microg/g in Wistar rats. 3-Hydroxy-3-methylglutaryl coenzyme A reductase activity was suppressed 4-fold in the WKY and almost 1.8-fold in Wistar rats, cholesterol 7alpha-hydroxylase activity was suppressed 1.6-fold in the WKY and 3.5-fold in Wistar rats, whereas cholesterol 27-hydroxylase activity was unchanged after feeding. In conclusion, stigmasterol, when fed, lowers plasma cholesterol levels, inhibits intestinal cholesterol and plant sterol absorption, and suppresses hepatic cholesterol and classic bile acid synthesis in Wistar as well as WKY rats. However, plasma and hepatic incorporation of stigmasterol is low.


Subject(s)
Cholesterol/blood , Intestinal Absorption/drug effects , Liver/metabolism , Stigmasterol/pharmacology , Animals , Bile Acids and Salts/biosynthesis , Cholesterol/biosynthesis , Cholesterol/metabolism , Liver/drug effects , Male , Rats , Rats, Inbred WKY , Rats, Wistar , Sitosterols/blood , Sterols/blood , Stigmasterol/administration & dosage , Stigmasterol/metabolism
2.
J Natl Cancer Inst ; 97(11): 846-53, 2005 Jun 01.
Article in English | MEDLINE | ID: mdl-15928305

ABSTRACT

BACKGROUND: Ursodeoxycholic acid (UDCA) treatment is associated with a reduced incidence of colonic neoplasia in preclinical models and in patients with conditions associated with an increased risk for colon cancer. We conducted a phase III, double-blind placebo-controlled trial of UDCA to evaluate its ability to prevent colorectal adenoma recurrence. METHODS: We randomly assigned 1285 individuals who had undergone removal of a colorectal adenoma within the past 6 months to daily treatment with UDCA (8-10 mg/kg of body weight; 661 participants) or with placebo (624 participants) for 3 years or until follow-up colonoscopy. Recurrence rates (number of recurrent adenomas per unit time) were compared by use of a Huber-White variance estimator. Proportions of participants with one or more recurrent adenomas were compared with a Pearson chi-square statistic; adjusted odds ratios (ORs) were obtained by logistic regression. All statistical tests were two-sided. RESULTS: We observed a non-statistically significant 12% reduction in the adenoma recurrence rate associated with UDCA treatment, compared with placebo treatment. However, UDCA treatment was associated with a statistically significant reduction (P = .03) in the recurrence of adenomas with high-grade dysplasia (adjusted OR = 0.61, 95% confidence interval = 0.39 to 0.96). We observed no statistically significant differences between UDCA and placebo groups in recurrence with regard to adenoma size, villous histology, or location. CONCLUSIONS: UDCA treatment was associated with a non-statistically significant reduction in total colorectal adenoma recurrence but with a statistically significant 39% reduction in recurrence of adenomas with high-grade dysplasia. Because severely dysplastic lesions have a high risk of progression to invasive colorectal carcinoma, this finding indicates that future chemoprevention trials of UDCA in individuals with such lesions should be considered.


Subject(s)
Adenoma/prevention & control , Antineoplastic Agents/therapeutic use , Colorectal Neoplasms/prevention & control , Neoplasm Recurrence, Local/prevention & control , Ursodeoxycholic Acid/therapeutic use , Adenoma/pathology , Adult , Aged , Antineoplastic Agents/adverse effects , Colorectal Neoplasms/pathology , Double-Blind Method , Female , Humans , Male , Middle Aged , Neoplasm Invasiveness , Treatment Outcome , Ursodeoxycholic Acid/adverse effects
3.
Metabolism ; 54(1): 38-48, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15562378

ABSTRACT

BACKGROUND AND AIMS: Plant sterols are widely distributed in human diet but are poorly absorbed so that their plasma levels are very low. However, when fed in large amounts, they lower plasma cholesterol levels by interfering with cholesterol absorption. We have studied the effect of 4 weeks of feeding a chow diet supplemented with 1% plant sterols [brassicasterol (6.3%), campesterol (28.5%), stigmasterol (15.6%) and sitosterol (49.6%)], with or without SCH 58235 (a derivative of ezetimibe), 30 mg/kg per day, known to suppress intestinal cholesterol absorption, on plasma, tissue, biliary, and fecal sterols in Wistar and wild-type Kyoto (WKY) rats, and their metabolism by intestinal bacteria. METHODS: After 2 weeks of feeding control or experimental diet, rats were given [3alpha-(3)H]sitosterol intravenously and [4-(14)C]sitosterol by mouth, and blood was collected after 1, 2, 3, and 5 days after labeling to determine sitosterol absorption. Feces were collected during the last 3 days and freeze dried. At the end of feeding, bile fistulas were created in 3 rats of each strain and bile was collected for 1 hour. All rats were then sacrificed and plasma and liver were collected for sterol measurements and activities of hepatic HMG-CoA reductase, cholesterol 7alpha-hydroxylase, and cholesterol 27-hydroxylase. RESULTS: Wild-type Kyoto rats were hypercholesterolemic compared to Wistar rats and had increased plant sterols in the plasma. Plasma cholesterol tended to be lower in WKY rats after feeding with plant sterol-enriched diet whereas plant sterol levels rose to approximately 31% of plasma sterols in WKY and 14% in Wistar rats. However, brassicasterol and stigmasterol, with a double bond at C-22, constituted less than 3.5% of total plasma plant sterols. After feeding, biliary plant sterols increased 2.25-fold in Wistar and 1.5-fold in WKY rats, suggesting less hepatic clearance in WKY rats. SCH 58235 feeding significantly increased plasma as well as biliary cholesterol levels in both the untreated and plant sterol-fed WKY rats, and the plasma plant sterols showed a tendency to increase but did not reach significant level. Intestinal bacteria in both rat strains metabolized all plant sterols to mainly the 5beta-H-stanols. However, the C-22 double bond was stable to bacterial degradation. Intestinal absorption of sitosterol and cholesterol was increased 1.5- and 1.3-fold, respectively, in the WKY rats as compared to the Wistar rats, and plant sterol feeding lowered absorption of these sterols in both strains. Absorption of both these sterols was also lowered in SCH 58235-treated rats in both strains and was further lowered when SCH 58235 and plant sterols were simultaneously fed. The activity of the rate-limiting enzyme, HMG-CoA reductase, was increased 1.57-fold in Wistar rats and 1.27-fold in WKY rats that were fed plant sterols as compared to untreated rats. CONCLUSIONS: (1) Plant sterol absorption was increased whereas hepatic elimination of all sterols was diminished in WKY rats accounting for elevated cholesterol and plant sterol levels. (2) The 1% plant sterol-enriched diet tended to lower plasma cholesterol levels whereas SCH 58235 feeding significantly increased plasma cholesterol levels in the WKY rats. (3) Intestinal absorption of sterols with C-22 double bond is diminished and the side-chain double bond is resistant to intestinal bacteria.


Subject(s)
Azetidines/pharmacology , Hypercholesterolemia/metabolism , Intestinal Absorption , Phytosterols/administration & dosage , Phytosterols/pharmacokinetics , Animals , Cholesterol/blood , Diet , Ezetimibe , Hydroxymethylglutaryl CoA Reductases/metabolism , Male , Rats , Rats, Inbred WKY , Rats, Wistar , Species Specificity
4.
J Lipid Res ; 46(2): 287-96, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15576845

ABSTRACT

Cerebrotendinous xanthomatosis (CTX), sterol 27-hydroxylase (CYP27A1) deficiency, is associated with markedly reduced chenodeoxycholic acid (CDCA), the most powerful activating ligand for farnesoid X receptor (FXR). We investigated the effects of reduced CDCA on FXR target genes in humans. Liver specimens from an untreated CTX patient and 10 control subjects were studied. In the patient, hepatic CDCA concentration was markedly reduced but the bile alcohol level exceeded CDCA levels in control subjects (73.5 vs. 37.8 +/- 6.2 nmol/g liver). Cholesterol 7alpha-hydroxylase (CYP7A1) and Na+/taurocholate-cotransporting polypeptide (NTCP) were upregulated 84- and 8-fold, respectively. However, small heterodimer partner (SHP) and bile salt export pump were normally expressed. Marked CYP7A1 induction with normal SHP expression was not explained by the regulation of liver X receptor alpha (LXRalpha) or pregnane X receptor. However, another nuclear receptor, hepatocyte nuclear factor 4alpha (HNF4alpha), was induced 2.9-fold in CTX, which was associated with enhanced mRNA levels of HNF4alpha target genes, CYP7A1, 7alpha-hydroxy-4-cholesten-3-one 12alpha-hydroxylase, CYP27A1, and NTCP. In conclusion, the coordinate regulation of FXR target genes was lost in CTX. The mechanism of the disruption may be explained by a normally stimulated FXR pathway attributable to markedly increased bile alcohols with activation of HNF4alpha caused by reduced bile acids in CTX liver.


Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Xanthomatosis, Cerebrotendinous/genetics , Xanthomatosis, Cerebrotendinous/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 11 , ATP-Binding Cassette Transporters/biosynthesis , Adult , Alcohols/metabolism , Bile Acids and Salts/metabolism , Case-Control Studies , Chenodeoxycholic Acid/metabolism , Cholestanetriol 26-Monooxygenase , Cholestanols/metabolism , Cholesterol 7-alpha-Hydroxylase/biosynthesis , DNA Primers/metabolism , DNA-Binding Proteins/biosynthesis , Female , Gene Expression Regulation , Hepatocyte Nuclear Factor 4 , Humans , Liver/metabolism , Liver X Receptors , Male , Membrane Transport Proteins/biosynthesis , Middle Aged , Models, Biological , Organic Anion Transporters, Sodium-Dependent , Orphan Nuclear Receptors , Phosphoproteins/biosynthesis , Pregnane X Receptor , Protein Binding , RNA, Messenger/metabolism , Receptors, Cytoplasmic and Nuclear/biosynthesis , Receptors, Steroid/biosynthesis , Steroid Hydroxylases/biosynthesis , Symporters , Transcription Factors/biosynthesis , Up-Regulation
5.
J Lipid Res ; 46(1): 76-85, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15520450

ABSTRACT

The CYP27A gene encodes a mitochondrial cytochrome P450 enzyme, sterol 27-hydroxylase, that is expressed in many different tissues and plays an important role in cholesterol and bile acid metabolism. In humans, CYP27A deficiency leads to cerebrotendinous xanthomatosis. To gain insight into the roles of CYP27A in the regulation of cholesterol and bile acid metabolism, cyp27A gene knockout heterozygous, homozygous, and wild-type littermate mice were studied. In contrast to homozygotes, heterozygotes had increased body weight and were mildly hypercholesterolemic, with increased numbers of lipoprotein particles in the low density lipoprotein size range. Cyp7A expression was not increased in heterozygotes but was in homozygotes, suggesting that parts of the homozygous phenotype are secondary to increased cyp7A expression and activity. Homozygotes exhibited pronounced hepatomegaly and dysregulation in hepatic cholesterol, bile acid, and fatty acid metabolism. Hepatic cholesterol synthesis and synthesis of bile acid intermediates were increased; however, side chain cleavage was impaired, leading to decreased bile salt concentrations in gallbladder bile. Expression of Na-taurocholate cotransporting polypeptide, the major sinusoidal bile salt transporter, was increased, and that of bile salt export pump, the major canalicular bile salt transporter, was decreased. Gender played a modifying role in the homozygous response to cyp27A deficiency, with females being generally more severely affected. Thus, both cyp27A genotype and gender affected the regulation of hepatic bile acid, cholesterol, and fatty acid metabolism.


Subject(s)
Bile Acids and Salts/metabolism , Cholesterol/metabolism , Steroid Hydroxylases/physiology , Animals , Cholestanetriol 26-Monooxygenase , Fatty Acids/metabolism , Female , Genotype , Male , Mice , Mice, Knockout , Phenotype , Sex Factors , Steroid Hydroxylases/deficiency , Steroid Hydroxylases/genetics
6.
Metabolism ; 53(7): 927-32, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15254889

ABSTRACT

We investigated the effect of SC-435, a competitive inhibitor of ileal apical sodium-dependent bile acid cotransporter (ASBT) on ileal bile acid absorption and the hepatic nuclear receptor FXR (farnesoid X receptor), which regulates cholesterol 7 alpha-hydroxylase (CYP7A1) activity and mRNA levels. Eighteen New Zealand White (NZW) rabbits were divided into 2 groups: controls (n = 10) and fed SC-435 125 mg/kg/d for 1 week (n = 8). In rabbits treated with SC-435, fecal bile acid outputs increased by more than 8 times, reflecting substantial bile acid malabsorption. Plasma cholesterol levels decreased 26%, while bile acid pool sizes and biliary bile acid outputs did not change after treatment. CYP7A1 activity increased 64% and mRNA rose by 4 times after treatment. The expression of FXR target genes in the liver, short heterodimer partner (SHP) and bile salt export pump (BSEP), decreased 11.6 and 2.6 times, respectively, after treatment, which indicates inactivation of hepatic FXR. However, the mRNA levels of ileal bile acid binding protein (IBABP) did not change significantly, while ileal ASBT mRNA expression increased by 2.4 times after treatment. Rabbits treated with SC-435 developed ileal bile acid malabsorption, which decreased the return of bile acids (FXR ligands) to the liver to inactivate hepatic FXR, which upregulated CYP7A1 and lowered plasma cholesterol levels. Although fecal bile acid malabsorption was substantial, increased bile acid production from hepatic cholesterol kept biliary bile acid outputs intact. Thus, a new balance was reached in the liver, where increased bile acid synthesis compensated for diminished ileal bile acid absorption to maintain the circulating enterohepatic bile acid pool.


Subject(s)
Bile/metabolism , Cholesterol 7-alpha-Hydroxylase/metabolism , Cholesterol/blood , DNA-Binding Proteins/antagonists & inhibitors , Ileum/metabolism , Liver/metabolism , Transcription Factors/antagonists & inhibitors , Animals , Bile Acids and Salts/biosynthesis , Biological Transport, Active/drug effects , Blotting, Northern , Cyclic N-Oxides/pharmacology , Cyclophilins/pharmacology , Feces/chemistry , Male , RNA, Messenger/biosynthesis , Rabbits , Receptors, Cytoplasmic and Nuclear , Reverse Transcriptase Polymerase Chain Reaction , Tropanes/pharmacology
7.
Metabolism ; 53(5): 556-62, 2004 May.
Article in English | MEDLINE | ID: mdl-15131757

ABSTRACT

We compared the effect of treatments with hydrophilic bile acids (ursodeoxycholic and ursocholic acids), cholestyramine, and lovastatin versus chenodeoxycholic acid in 4 patients with cerebrotendinous xanthomatosis (CTX). Bile acids and bile alcohols in plasma, bile, and urine before and after treatment were quantitated by gas-liquid chromatography. Untreated, all patients showed abnormal biliary bile acid composition: cholic acid (72.7%) and chenodeoxycholic acid (6.2%), and polyhydroxylated C(27)-bile alcohols (10.0%), and elevated plasma cholestanol levels. Treatment with hydrophobic chenodeoxycholic acid inhibited abnormal bile acid synthesis (virtual disappearance of C(27)-bile alcohols from plasma, bile, and urine and marked reduction of plasma cholestanol levels). Hydrophilic ursodeoxycholic and ursocholic acids did not inhibit abnormal bile acid synthesis, while cholestyramine increased abnormal bile acid synthesis (continued increased formation of polyhydroxylated C(27)-bile alcohols and further elevation of plasma cholestanol levels). Lovastatin did not affect abnormal bile acid synthesis or reduce plasma cholestanol levels. The results demonstrate that impaired side-chain oxidation in bile acid synthesis due to mutations of Cyp27 results in increased formation of polyhydroxylated C(27)-bile alcohols and cholestanol in CTX. Hydrophobic chenodeoxycholic acid, but not cholestyramine, lovastatin, or hydrophilic 7beta-hydroxy acids, inhibited the abnormal synthetic pathway. The role of chenodeoxycholic acid in downregulating abnormal bile acid synthesis in CTX is emphasized.


Subject(s)
Anticholesteremic Agents/therapeutic use , Chenodeoxycholic Acid/therapeutic use , Xanthomatosis, Cerebrotendinous/drug therapy , Adult , Anticholesteremic Agents/chemistry , Bile/drug effects , Bile/metabolism , Bile Acids and Salts/metabolism , Case-Control Studies , Chenodeoxycholic Acid/chemistry , Cholestanols/blood , Cholestanols/chemistry , Cholestanols/urine , Cholesterol/blood , Cholestyramine Resin/chemistry , Cholestyramine Resin/therapeutic use , Cholic Acids/chemistry , Cholic Acids/therapeutic use , Homozygote , Humans , Lovastatin/chemistry , Lovastatin/therapeutic use , Male , Middle Aged , Ursodeoxycholic Acid/chemistry , Ursodeoxycholic Acid/therapeutic use , Xanthomatosis, Cerebrotendinous/blood , Xanthomatosis, Cerebrotendinous/genetics , Xanthomatosis, Cerebrotendinous/urine
8.
BMC Med ; 2: 5, 2004 Mar 24.
Article in English | MEDLINE | ID: mdl-15040800

ABSTRACT

BACKGROUND: Mutations in either of two genes comprising the STSL locus, ATP-binding cassette (ABC)-transporters ABCG5 (encoding sterolin-1) and ABCG8 (encoding sterolin-2), result in sitosterolemia, a rare autosomal recessive disorder of sterol trafficking characterized by increased plasma plant sterol levels. Based upon the genetics of sitosterolemia, ABCG5/sterolin-1 and ABCG8/sterolin-2 are hypothesized to function as obligate heterodimers. No phenotypic difference has yet been described in humans with complete defects in either ABCG5 or ABCG8. These proteins, based upon the defects in humans, are responsible for regulating dietary sterol entry and biliary sterol secretion. METHODS: In order to mimic the human disease, we created, by a targeted disruption, a mouse model of sitosterolemia resulting in Abcg8/sterolin-2 deficiency alone. Homozygous knockout mice are viable and exhibit sitosterolemia. RESULTS: Mice deficient in Abcg8 have significantly increased plasma and tissue plant sterol levels (sitosterol and campesterol) consistent with sitosterolemia. Interestingly, Abcg5/sterolin-1 was expressed in both liver and intestine in Abcg8/sterolin-2 deficient mice and continued to show an apical expression. Remarkably, Abcg8 deficient mice had an impaired ability to secrete cholesterol into bile, but still maintained the ability to secrete sitosterol. We also report an intermediate phenotype in the heterozygous Abcg8+/- mice that are not sitosterolemic, but have a decreased level of biliary sterol secretion relative to wild-type mice. CONCLUSION: These data indicate that Abcg8/sterolin-2 is necessary for biliary sterol secretion and that loss of Abcg8/sterolin-2 has a more profound effect upon biliary cholesterol secretion than sitosterol. Since biliary sitosterol secretion is preserved, although not elevated in the sitosterolemic mice, this observation suggests that mechanisms other than by Abcg8/sterolin-2 may be responsible for its secretion into bile.


Subject(s)
Cholesterol/analogs & derivatives , Cholesterol/metabolism , Lipoproteins/deficiency , Models, Animal , Mutation/genetics , Sitosterols/blood , ATP Binding Cassette Transporter, Subfamily G, Member 5 , ATP Binding Cassette Transporter, Subfamily G, Member 8 , ATP-Binding Cassette Transporters/genetics , Animals , Bile/metabolism , Bile Acids and Salts/metabolism , Cholesterol/blood , Female , Gene Expression , Intestines/chemistry , Lipoproteins/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Phytosterols/blood
9.
J Lipid Res ; 44(5): 1001-9, 2003 May.
Article in English | MEDLINE | ID: mdl-12588950

ABSTRACT

Cholesterol 7alpha-hydroxylase, a rate-limiting enzyme for bile acid synthesis, has been implicated in genetic susceptibility to atherosclerosis. The gene, CYP7A1, encoding a protein with this activity, is expressed normally only in hepatocytes and is highly regulated. Our cyp7A1 gene knockout mouse colony, as young adults on a chow diet, is hypercholesterolemic. These mice were characterized extensively to understand how cyp7A1 affects lipid and bile acid homeostasis in different tissue compartments and whether gender plays a modifying role. Both male and female cyp7A1-deficient mice had decreased hepatic LDL receptors, unchanged hepatic cholesterol synthesis, increased intestinal cholesterol synthesis and bile acid transporters, and decreased fecal bile acids but increased fecal sterols. In females, cyp7A1 deficiency also caused changes in hepatic fatty acid metabolism, decreased hepatic canalicular bile acid transporter, Bsep, and gallbladder bile composition altered to a lithogenic profile. Taken together, the data suggest that cyp7A1 deficiency results in a proatherogenic phenotype in both genders and leads to a prolithogenic phenotype in females.


Subject(s)
Bile Acids and Salts/metabolism , Cholesterol 7-alpha-Hydroxylase/deficiency , Hypercholesterolemia/genetics , Lipids/blood , Animals , Cholestanetriol 26-Monooxygenase , Cholesterol/blood , Cholesterol/metabolism , Cholesterol 7-alpha-Hydroxylase/genetics , Cholesterol 7-alpha-Hydroxylase/metabolism , Fatty Acids/metabolism , Female , Gallbladder/metabolism , Hypercholesterolemia/blood , Hypercholesterolemia/metabolism , Intestinal Mucosa/metabolism , Liver/enzymology , Liver/metabolism , Male , Mice , Mice, Inbred Strains , Mice, Knockout , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, LDL/metabolism , Steroid Hydroxylases/genetics , Steroid Hydroxylases/metabolism , Sterols/metabolism
10.
Proc Natl Acad Sci U S A ; 100(3): 833-8, 2003 Feb 04.
Article in English | MEDLINE | ID: mdl-12569201

ABSTRACT

The nuclear receptor PXR (pregnane X receptor) is a broad-specificity sensor that recognizes a wide variety of synthetic drugs and xenobiotic agents. On activation by these compounds, PXR coordinately induces a network of transporters, cytochrome P450 enzymes, and other genes that effectively clear xenobiotics from the liver and intestine. Like PXR, the majority of its target genes also possess a broad specificity for exogenous compounds. Thus, PXR is both a sensor and effector in a well integrated and generalized pathway for chemical immunity. Although it is clear that PXR responds to numerous foreign compounds, it is unclear whether it possesses an endogenous ligand. To address this issue, we noted that there is substantial overlap in the substrate specificities of PXR and its critical CYP3A target gene. This prompted us to ask whether endogenous CYP3A substrates also serve as PXR ligands. We demonstrate that 5beta-cholestane-3alpha,7alpha,12alpha-triol (triol), a cholesterol-derived CYP3A substrate, is a potent PXR agonist that effectively induces cyp3a expression in mice. This defines a critical salvage pathway that can be autoinduced to minimize triol accumulation. In contrast, triol can accumulate to very high levels in humans, and unlike mice, these people develop the severe clinical manifestations of cerebrotendinous xanthomatosis. The reason for these dramatic species differences has remained unclear. We now demonstrate that triol fails to activate human PXR or induce the CYP3A-salvage pathway. This explains why humans are more susceptible to sterol accumulation and suggests that synthetic ligands for human PXR could be used to treat cerebrotendinous xanthomatosis and other disorders of cholesterol excess.


Subject(s)
Ethanol/analogs & derivatives , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Steroid/metabolism , Sterols/metabolism , Anesthetics/pharmacology , Animals , Aryl Hydrocarbon Hydroxylases/metabolism , Binding, Competitive , Cell Line , Cells, Cultured , Cholestanols/metabolism , Cholesterol/metabolism , Cytochrome P-450 CYP3A , Dose-Response Relationship, Drug , Ethanol/pharmacology , Female , Gas Chromatography-Mass Spectrometry , Homeostasis , Humans , Ligands , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Oxidoreductases, N-Demethylating/metabolism , Pregnane X Receptor , Protein Structure, Tertiary , Transfection
11.
Proc Natl Acad Sci U S A ; 99(25): 16215-9, 2002 Dec 10.
Article in English | MEDLINE | ID: mdl-12446833

ABSTRACT

Plasma plant sterol levels differ among humans due to genetic and dietary factors. A disease characterized by high plasma plant sterol levels, beta-sitosterolemia, was recently found to be due to mutations at the ABCG5ABCG8 locus. To detect variants at this and other loci, a genetic cross was carried out between two laboratory mouse strains. Parental C57BL6J had almost twice the campesterol and sitosterol levels compared with parental CASARk mice, and F(1) mice had levels halfway between the parentals. An intercross between F(1)s was performed and plasma plant sterol levels measured in 102 male and 99 female F(2) mice. Plasma plant sterols in F(2)s displayed a unimodal distribution, suggesting the effects of several rather a single major gene. In the F(2) mice, a full genome scan revealed significant linkages on chromosomes 14 and 2. With regard to chromosome 14, analysis showed a single peak for linkage at 17 cM with a logarithm of odds (LOD) score of 9.9, designated plasma plant sterol 14 (Plast14). With regard to chromosome 2, analysis showed two significant peaks for linkage at 18 and 65 cMs with LOD scores of 4.1 and 3.65, respectively, designated Plast2a and Plast2b, respectively. Four interactions between loci, predominantly of an additive nature, were also demonstrated, the most significant between Plast14 and Plast2b (LOD 16.44). No significant linkage or gene interaction was detected for the ABCG5ABCG8 locus on chromosome 17. Therefore, other genes besides ABCG5ABCG8 influence plasma plant sterol levels and now become candidates to explain differences in plasma plant sterol levels between humans.


Subject(s)
Cholesterol/analogs & derivatives , Cholesterol/blood , Chromosome Mapping , Phytosterols , Sitosterols/blood , ATP Binding Cassette Transporter, Subfamily G, Member 5 , ATP Binding Cassette Transporter, Subfamily G, Member 8 , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Animals , Biomarkers , Cholesterol/pharmacokinetics , Crosses, Genetic , Dietary Fats/pharmacokinetics , Epistasis, Genetic , Female , Genotype , Lipoproteins/genetics , Lipoproteins/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Sitosterols/pharmacokinetics , Species Specificity
12.
J Biol Chem ; 277(52): 50491-6, 2002 Dec 27.
Article in English | MEDLINE | ID: mdl-12401785

ABSTRACT

We investigated the roles of hydrophobic deoxycholic acid (DCA) and hydrophilic ursocholic acid (UCA) in the regulation of the orphan nuclear farnesoid X receptor (FXR) in vivo. Rabbits with bile fistula drainage (removal of the endogenous bile acid pool), rabbits with bile fistula drainage and replacement with either DCA or UCA, and intact rabbits fed 0.5% cholic acid (CA) (enlarged endogenous bile acid pool) were studied. After bile fistula drainage, cholesterol 7alpha-hydroxylase (CYP7A1) mRNA and activity levels increased, FXR-mediated transcription was decreased, and FXR mRNA and nuclear protein levels declined. Replacing the enterohepatic bile acid pool with DCA restored FXR mRNA and nuclear protein levels and activated FXR-mediated transcription as evidenced by the increased expression of its target genes, SHP and BSEP, and decreased CYP7A1 mRNA level and activity. Replacing the bile acid pool with UCA also restored FXR mRNA and nuclear protein levels but did not activate FXR-mediated transcription, because the SHP mRNA level and CYP7A1 mRNA level and activity were unchanged. Feeding CA to intact rabbits expanded the bile acid pool enriched with the FXR high affinity ligand, DCA. FXR-mediated transcription became activated as shown by increased SHP and BSEP mRNA levels and decreased CYP7A1 mRNA level and activity but did not change FXR mRNA or nuclear protein levels. Thus, both hydrophobic and hydrophilic bile acids are effective in maintaining FXR mRNA and nuclear protein levels. However, the activating ligand (DCA) in the enterohepatic flux is necessary for FXR-mediated transcriptional regulation, which leads to down-regulation of CYP7A1.


Subject(s)
Bile Acids and Salts/physiology , Cholic Acids/physiology , DNA-Binding Proteins/physiology , Deoxycholic Acid/physiology , Transcription Factors/physiology , Animals , Bile Duct Diseases/physiopathology , Bile Ducts/physiology , Cholesterol 7-alpha-Hydroxylase/genetics , Cyclophilins/pharmacology , Fistula , Homeostasis , Male , RNA, Messenger/genetics , Rabbits , Receptors, Cytoplasmic and Nuclear , Transcription, Genetic
13.
Article in English | MEDLINE | ID: mdl-12113981

ABSTRACT

A simple method for the simultaneous gas-liquid chromatographic quantitation of fatty acids, sterols and bile acids from human fecal samples is described. The various compounds are directly converted into the n-butyl ester-trimethylsilyl ether derivatives, without prior isolation from the stool. Under these conditions, fecal bile acid derivatives are well resolved from each other and from those of fecal fatty acids and sterols without overlaps. The method was found to be reproducible and recoveries were similar to those obtained after exhaustive solvent extraction of fecal sterols, fatty acids and bile acids. Optimum derivatization conditions that allowed maximum recovery of fecal components with minimal destruction and application of the method for simultaneous bile acid, fatty acid and sterol analysis in human stool are described.


Subject(s)
Bile Acids and Salts/analysis , Chromatography, Gas/methods , Fatty Acids/analysis , Feces/chemistry , Sterols/analysis , Gas Chromatography-Mass Spectrometry , Humans
14.
J Clin Invest ; 110(1): 109-17, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12093894

ABSTRACT

Bile acid synthesis plays a critical role in the maintenance of mammalian cholesterol homeostasis. The CYP7A1 gene encodes the enzyme cholesterol 7alpha-hydroxylase, which catalyzes the initial step in cholesterol catabolism and bile acid synthesis. We report here a new metabolic disorder presenting with hyperlipidemia caused by a homozygous deletion mutation in CYP7A1. The mutation leads to a frameshift (L413fsX414) that results in loss of the active site and enzyme function. High levels of LDL cholesterol were seen in three homozygous subjects. Analysis of a liver biopsy and stool from one of these subjects revealed double the normal hepatic cholesterol content, a markedly deficient rate of bile acid excretion, and evidence for upregulation of the alternative bile acid pathway. Two male subjects studied had hypertriglyceridemia and premature gallstone disease, and their LDL cholesterol levels were noticeably resistant to 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. One subject also had premature coronary and peripheral vascular disease. Study of the kindred, which is of English and Celtic background, revealed that individuals heterozygous for the mutation are also hyperlipidemic, indicating that this is a codominant disorder.


Subject(s)
Cholesterol 7-alpha-Hydroxylase/deficiency , Cholesterol 7-alpha-Hydroxylase/genetics , Hyperlipoproteinemia Type II/enzymology , Hyperlipoproteinemia Type II/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Base Sequence , Bile Acids and Salts/metabolism , Cholelithiasis/enzymology , Cholelithiasis/genetics , Cholesterol/metabolism , DNA/genetics , Female , Heterozygote , Homozygote , Humans , Hyperlipoproteinemia Type II/metabolism , Hypertriglyceridemia/enzymology , Hypertriglyceridemia/genetics , Lipoproteins, LDL/blood , Liver/metabolism , Male , Middle Aged , Pedigree , Phenotype , Sequence Deletion
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