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Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive disease caused by mutations in the CYP27A1 gene, encoding the sterol 27-hydroxylase. Disruption of the bile acid biosynthesis pathway and accumulation of toxic precursors such as cholestanol cause chronic diarrhea, bilateral juvenile cataracts, tissue deposition of cholestanol and cholesterol (xanthomas), and progressive motor/neuropsychiatric alterations. We have evaluated the therapeutic potential of adeno-associated virus (AAV) vectors expressing CYP27A1 in a CTX mouse model. We found that a vector equipped with a strong liver-specific promoter (albumin enhancer fused with the α1 anti-trypsin promoter) is well tolerated and shows therapeutic effect at relatively low doses (1.5 × 1012 viral genomes [vg]/kg), when less than 20% of hepatocytes overexpress the transgene. This vector restored bile acid metabolism and normalized the concentration of most bile acids in plasma. By contrast, standard treatment (oral chenodeoxycholic acid [CDCA]), while reducing cholestanol, did not normalize bile acid composition in plasma and resulted in supra-physiological levels of CDCA and its derivatives. At the transcriptional level, only the vector was able to avoid the induction of xenobiotic-induced pathways in mouse liver. In conclusion, the overexpression of CYP27A1 in a fraction of hepatocytes using AAV vectors is well tolerated and provides full metabolic restoration in Cyp27a1−/− mice. These features make gene therapy a feasible option for the etiological treatment of CTX patients., Graphical abstract, Cerebrotendinous xanthomatosis (CTX) is caused by mutations in the CYP27A1 gene, which is involved in the biosynthesis of bile acids. We found that a relatively low intravenous dose of an AAV vector expressing this gene under the control of a strong liver-specific promoter restores bile acid metabolism in knockout mice.

Adenosine monophosphate-activated protein kinase (AMPK) plays a pivotal role in the regulation of carbohydrate, lipid, and protein metabolism in animals. In this study, we examined whether any cross talk exists between glucocorticoids and AMPK in the regulation of the liver bile acid biosynthesis pathway. Dexamethasone treatment decreased the growth performance of broiler chickens. The liver mRNA levels of fatty acid transport protein (FATP-1), farnesoid X receptor (FXR), AMPK alpha 1 subunit (AMPKα1), and glucocorticoid receptor were significantly upregulated in DEX-treated broilers; the gene expression of liver cholesterol 7 alpha-hydroxylase (CYP7A1) was significantly downregulated. The protein level of liver CYP7A1 was significantly decreased by DEX treatment at both 24 and 72 h, while the protein level of p-AMPK/ t-AMPK stayed unchanged. In the in vitro cultured hepatocytes, compound C pretreatment blocked the increase in CYP7A1 protein level by DEX and significantly suppressed FATP-1, SREBP-1c, FXR, and CYP7A1 gene expression stimulated by DEX. Compound C treatment significantly reduces the protein level of p-AMPK, and the combination of compound C and DEX significantly reduces the protein level of t-AMPK. Thus, glucocorticoids affected liver AMPK and the bile acid synthesis signal pathway, and AMPK might be involved in the glucocorticoid effect of liver bile acid synthesis.

Parenteral nutrition-associated liver disease (PNALD) is a major complication of long-term parenteral nutrition (PN). The pathogenesis of PNALD remains unclear. We investigated the changes in taxonomic and functional composition of gut microbiota and serum bile acid levels in a rat model of PNALD.Male 4-week-old Sprague Dawley rats received either total parenteral nutrition or standard chow with 0.9% saline for 7 days. The taxonomic composition of cecal microbiota and its functional composition associated with bile acid metabolism were measured.There were differences in taxonomic composition between the two groups. The abundance of the secondary bile acid biosynthesis pathway was higher in the TPN group (p 0.05) with an increase in the percentage of bacteria expressing 7-alpha-hydroxysteroid dehydrogenase (p 0.05). The abundance of enzymes associated with bile salt hydrolase was also higher (p 0.05) in the TPN group. The TPN group showed a distinct bile acid profile characterized by a higher ratio of secondary bile acids to primary bile acids.The alteration of bile acid-associated microbiota may lead to increased secondary bile acid production in a rat model of PNALD.

Ethnopharmacological relevance Danggui Buxue Tang (DBT) has been used to promote hematopoiesis and relieve myelosuppression in China. Antibiotics can cause myelosuppression through gut microbiota disorders. Aim of the study: This study aims to explore the way of DBT to alleviate the metabolic disorder caused by antibiotics. Materials and methods In this study, 16S rRNA sequencing was used to detect the change of gut microbiota, metabolomics to analyze the change of metabolites. Correlation analysis was used to establishment the correlation between gut microbiota and metabolites. PICRUST 2 was used to predict the function of gut microbiota. Results Results showed that eighty-two genera of gut microbiota were affected by antibiotic, while twelve were significantly restored after DBT. Seventy-four potential metabolites were significantly different from the antibiotics and DBT. We found significant recovery by the Bacteroides and Rikenellaceae RC9 after DBT. The metabolic pathways influenced by the antibiotic treatment included primary and secondary bile biosynthesis, etc. The metabolic pathways that could be restored after DBT included the primary and secondary bile acid biosynthesis pathway, etc. Through correlation analysis, we found a correlation between the Bacteroides, Rikenellaceae_RC9_gut_group and other potential differential metabolisms such as those of taurodeoxycholic acid, N-phenylacetyl glycine, etc. The functional prediction showed that the biosynthesis of primary bile acid, secondary bile acid was significantly affected. Conclusions DBT can restore the gut and reverse the metabolic disorder caused by antibiotics through Bacteroides, and it provides a new medical idea regarding the gut microbiota balance.

Cytochrome P450 (P450) 27A1 catalyzes 27-hydroxylation of cholesterol and 25-hydroxylation of vitamin D(3), serving as an important component for the maintenance of lipid homeostasis. In eukaryotic cells P450 27A1 is a membrane-bound protein located on the inner mitochondrial membrane and requires two auxiliary reduction partners, adrenodoxin (Adx) and NADPH-adrenodoxin reductase (Adr), for catalysis in the bile acid biosynthesis pathway. A strategy was developed for the functional coexpression of P450 27A1 with Adr and Adx in a tricistronic fashion (single RNA, three proteins) in Escherichia coli, mimicking the mitochondrial P450 system. Intact bacterial cells coexpressing the P450 vector (pTC27A1) efficiently hydroxylated cholesterol at the 27 position as well as vitamin D(3) at the 25 position when supplemented with glycerol as a carbon source. Thus, E. coli containing pTC27A1 is able to hydroxylate cholesterol in a self-sufficient fashion and is suitable for further applications of protein interaction, drug discovery, and inhibitor evaluation and for the study of other mitochondrial P450s and oxysterol production in microorganisms without a need for membrane reconstitution, membrane simulation by detergents, or purification of the components.

Cholesterol 7 alpha-hydroxylase (CYP7A1) of the bile acid biosynthesis pathway is suppressed by bile acids and inflammatory cytokines. Bile acids are known to induce inflammatory cytokines to activate the mitogen-activated protein kinase/c-Jun N-terminal kinase (JNK) signaling pathway that inhibits CYP7A1 gene transcription. c-Jun has been postulated to mediate bile acid inhibition of CYP7A1. However, the c-Jun target involved in the regulation of CYP7A1 is unknown. Human primary hepatocytes and HepG2 cells were used as models to study chenodeoxycholic acid (CDCA) and interleukin-1 beta (IL-1 beta) regulation of human CYP7A1 gene expression via real-time polymerase chain reaction, reporter assays, co-immunoprecipitation and chromatin immunocipitation (ChIP) assays. IL-1 beta and CDCA reduced CYP7A1 but induced c-Jun messenger RNA expression in human primary hepatocytes. IL-1beta inhibited human CYP7A1 reporter activity via the HNF4 alpha binding site. A JNK-specific inhibitor blocked the inhibitory effect of IL-1 beta on HNF4 alpha expression and CYP7A1 reporter activity. c-Jun inhibited HNF4 alpha and PPARgamma coactivator-1 alpha (PGC-1 alpha) coactivation of CYP7A1 reporter activity, whereas a dominant negative c-Jun did not. Co-immunoprecipitation and ChIP assays revealed that IL-1 beta and CDCA reduced HNF4 alpha bound to the CYP7A1 chromatin, and that c-Jun interacted with HNF4 alpha and blocked HNF4 alpha recruitment of PGC-1 alpha to the CYP7A1 chromatin. In conclusion, IL-1 beta and CDCA inhibit HNF4 alpha but induce c-Jun, which in turn blocks HNF 4 alpha recruitment of PGC-1 alpha to the CYP7A1 chromatin and results in inhibition of CYP7A1 gene transcription. The JNK/c-Jun signaling pathway inhibits bile acid synthesis and protects hepatocytes against the toxic effect of inflammatory agents.

These enzymes play important roles in the biosynthesis of bile acids. They are cholesterol 7alpha-hydroxylase (CYP7A1), the rate limiting enzyme in the classic pathway, sterol 12alpha-hydroxylase (CYP8B1), the key enzyme for synthesis of cholic acid (CA), and sterol 27-hydroxylase (CYP27), the initial enzyme in the alternative pathway. In the present study, the susceptibility of these three enzymes to dietary cholesterol and cholate, and the cholesterol lowering effect of taurine were determined in male C57BL/6 mice and Wistar rats. Both mice and rats were divided into 6 groups: control group (N), high cholesterol diet group (C), high cholesterol and cholate diet group (CB), and their 1% taurine-supplemented groups (NT, CT, CBT, respectively). After animals were fed with the respective diets for one week, the mRNA levels of CYP7A1 increased in the C-group compared with those of the N-group, and decreased in the CB-group compared with those of the C-group in both mice and rats. But the extent of decrease is different between the two species. CYP8B1 was also markedly repressed by cholate in mice, but not in rats. These results are consistent with the changes in serum and liver cholesterol concentrations. Taurine significantly increased CYP7A1 mRNA levels in the CBT-group compared with the CB-group in both animal models, with a subsequent decrease in serum and liver cholesterol levels and increase in fecal bile acid excretion. Up-regulated CYP8B1 was also observed after taurine supplementation in the CBT-group in mice. No increase in CYP7A1 was produced by taurine in the CT-group compared with that of the C-group in mice, although the changes of serum and liver cholesterol and fecal bile acids indicated taurine showed an efficient cholesterol lowering effect. In addition, CYP27 was induced in both C- and CB-groups of rats but not of mice, and no changes were produced by taurine. The overall results suggest that there are differences between mice and rats in susceptibility of the three enzymes to dietary cholesterol and cholate, and taurine induced CYP7A1 to produce its cholesterol-lowering effect only in the presence of cholate in the cholesterol diet.

Cholesterol 7-alpha-hydroxylase (CYP7A1) is the key enzyme that commits cholesterol to the neutral bile acid biosynthesis pathway and is highly regulated. In the current studies, we have uncovered a role for the transcriptional co-activator PGC-1alpha in CYP7A1 gene transcription. PGC-1alpha plays a vital role in adaptive thermogenesis in brown adipose tissue and stimulates genes important to mitochondrial function and oxidative metabolism. It is also involved in the activation of hepatic gluconeogenesic gene expression during fasting. Because the mRNA for CYP7A1 was also induced in mouse liver by fasting, we reasoned that PGC-1alpha might be an important co-activator for CYP7A1. Here we show that PGC-1alpha and CYP7A1 are also co-induced in livers of mice in response to streptozotocin induced diabetes. Additionally, infection of cultured HepG2 cells with a recombinant adenovirus expressing PGC-1alpha directly activates CYP7A1 gene expression and increases bile acid biosynthesis as well. Furthermore, we show that PGC-1alpha activates the CYP7A1 promoter directly in transient transfection assays in cultured cells. Thus, PGC-1alpha is a key activator of CYP7A1 and bile acid biosynthesis and is likely responsible for the fasting and diabetes dependent induction of CYP7A1. PGC-1alpha has already been shown to be a critical activator of several other oxidative processes including adaptive thermogenesis and fatty acid oxidation. Our studies provide further evidence of the fundamental role played by PGC-1alpha in oxidative metabolism and define PGC-1alpha as a link between diabetes and bile acid metabolism.

Bile acids are important physiological agents required for disposal of cholesterol and absorption of vitamins and fats. Bile acids are synthesized from cholesterol in the liver. Enterohepatic circulation of bile acids is very efficient and plays an important physiological role in lipid absorption and secretion, and regulation of bile acid biosynthesis and cholesterol homeostasis. Conversion of cholesterol to bile acids requires 15 different enzymatic steps. Four cytochrome P450 enzymes play important roles in bile acid biosynthesis. The classic bile acid biosynthesis pathway starts with modification of the sterol ring and followed by side chain cleavage reactions to synthesize cholic acid (CA) and chenodeoxycholic acid (CDCA), the primary bile acids in most species. The first and rate-limiting enzyme in this pathway is cholesterol 7alpha -hydroxylase, a microsomal cytochrome P450, CYP7A. Another microsomal cytochrome P450 sterol 12alpha-hydroxylase (CYP12) is required for the synthesis of cholic acid. Mitochondrial cytochrome P450 sterol 27-hydroxylase (CYP27) catalyzes sterol side chain oxidation to convert C27 sterol to C24 bile acids. An alternative bile acid biosynthesis pathway (acidic) has been known for sometime but only recently has attracted much attention. In this pathway, side chain oxidation precedes modification of the sterol ring. Mitochondrial sterol 27-hydroxylase (CYP27) catalyzes the first reaction and followed by 7alpha-hydroxylation catalyzed by a microsomal oxysterol 7alpha-hydroxylase (CYP7B). Recent advances in purification and cloning of these major enzymes in the pathways have led to better understanding the molecular basis of regulation of bile acid synthesis and physiological role of the alternative pathways.

Patients with cholesterol gallstones have a reduced pool of bile acids. This study was undertaken to clarify the mechanism by which bile acid biosynthesis does not increase to supranormal levels to cause a reexpansion of the pool. We investigated the first two steps of the bile acid biosynthesis pathway by assaying the activities of cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in this pathway, and 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase/isomerase, and by measuring the concentrations of 7 alpha-hydroxycholesterol and 7 alpha-hydroxy-4-cholesten-3-one in liver specimens from ten patients with cholesterol gallstones and ten gallstone-free controls. In the patients with gallstones, cholesterol 7 alpha-hydroxylase activity, 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase/isomerase activity, and hepatic 7 alpha-hydroxy-4-cholesten-3-one concentration did not significantly different from levels in controls, but hepatic 7 alpha-hydroxycholesterol concentration was more than twofold that of controls (12.9 +/- 2.6 vs 5.3 +/- 1.2 nmol/g liver, P0.01). The concentration of 7 alpha-hydroxycholesterol positively correlated with the ratio of cholesterol 7 alpha-hydroxylase activity to 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase/isomerase activity (r = 0.93; P0.005) in the gallstone-free controls. In contrast, this correlation disappeared in the patients with gallstones. These results suggest a derangement of the normal 7 alpha-hydroxycholesterol metabolism in the patients with gallstones. The reason for the accumulation of 7 alpha-hydroxycholesterol remains unclear; however, it is possible that, in patients with cholesterol gallstone, the accumulated 7 alpha-hydroxycholesterol causes inappropriate suppression of cholesterol 7 alpha-hydroxylase activity by product inhibition.

Cholesterol 7 alpha-hydroxylase, the rate-determining enzyme in the bile acid biosynthesis pathway, is regulated in a negative feedback manner by hydrophobic bile salts returning to the liver via the portal circulation. The role of cholesterol in the regulation of cholesterol 7 alpha-hydroxylase and the interrelationship between the cholesterol and bile acid biosynthesis pathways remain controversial. The objective of the present study was to define the role of cholesterol in the regulation of cholesterol 7 alpha-hydroxylase and determine the molecular level of its control. In order to avoid intestinal or intravenous administration of cholesterol, we manipulated the flow of cholesterol within the hepatocytes by decreasing cholesterol synthesis with lovastatin in bile fistula rats (bile acid synthesis is up-regulated), or by increasing cholesterol supply by administering mevalonate, a precursor of cholesterol, to rats with intact enterohepatic circulation (bile acid synthesis is normal). In the first series of studies, lovastatin was administered as a single intravenous bolus (10 mg/kg) to rats with chronic bile fistula and to rats with intact enterohepatic circulation (cholesterol and bile acid synthesis is normal). Three hours after lovastatin administration, cholesterol 7 alpha-hydroxylase specific activity, enzyme mass, mRNA, and gene transcriptional activity were decreased by 35%, 32%, 56%, and 34%, respectively, in rats with chronic bile fistula. In rats with intact enterohepatic circulation, lovastatin administration resulted in a similar decrease (34%) of cholesterol 7 alpha-hydroxylase specific activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological activation of the constitutive androstane receptor (CAR) protects the liver during cholestasis. The current study evaluates how activation of CAR influences genes involved in bile acid biosynthesis as a mechanism of hepatoprotection during bile acid-induced liver injury. CAR activators phenobarbital (PB) and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) or corn oil (CO) were administered to C57BL/6 wild-type (WT) and CAR knockout (CAR-null) mice before and during induction of intrahepatic cholestasis using the secondary bile acid, lithocholic acid (LCA). In LCA-treated WT and all the CAR-null groups (excluding controls), histology revealed severe multifocal necrosis. This pathology was absent in WT mice pretreated with PB and TCPOBOP, indicating CAR-dependent hepatoprotection. Decreases in total hepatic bile acids and hepatic monohydroxy, dihydroxy, and trihydroxy bile acids in PB- and TCPOBOP-pretreated WT mice correlated with hepatoprotection. In comparison, concentrations of monohydroxylated and dihydroxylated bile acids were increased in all the treated CAR-null mice compared with CO controls. Along with several other enzymes (Cyp7b1, Cyp27a1, Cyp39a1), Cyp8b1 expression was increased in hepatoprotected mice, which could be suggestive of a shift in the bile acid biosynthesis pathway toward the formation of less toxic bile acids. In CAR-null mice, these changes in gene expression were not different among treatment groups. These results suggest CAR mediates a shift in bile acid biosynthesis toward the formation of less toxic bile acids, as well as a decrease in hepatic bile acid concentrations. We propose that these combined CAR-mediated effects may contribute to the hepatoprotection observed during LCA-induced liver injury.

The sterol storage disorder cerebrotendinous xanthomatosis (CTX) is characterized by abnormal deposition of cholesterol and cholestanol in multiple tissues. Deposition in the central nervous system leads to neurological dysfunction marked by dementia, spinal cord paresis, and cerebellar ataxia. Deposition in other tissues causes tendon xanthomas, premature atherosclerosis, and cataracts. In two unrelated patients with CTX, we have identified different point mutations in the gene (CYP27) encoding sterol 27-hydroxylase, a key enzyme in the bile acid biosynthesis pathway. Transfection of mutant cDNAs into cultured cells results in the synthesis of immunoreactive sterol 27-hydroxylase protein with greatly diminished enzyme activity. We have localized the CYP27 gene to the q33-qter interval of human chromosome 2, and to mouse chromosome 1, in agreement with the autosomal recessive inheritance pattern of CTX. These findings underscore the essential role played by sterols in the central nervous system and suggest that mutations in other sterol metabolizing enzymes may contribute to diseases with neurological manifestations.

Maturity-onset diabetes of the young type 3 (MODY3) is caused by haploinsufficiency of hepatocyte nuclear factor-1alpha (encoded by TCF1). Tcf1-/- mice have type 2 diabetes, dwarfism, renal Fanconi syndrome, hepatic dysfunction and hypercholestrolemia. Here we explore the molecular basis for the hypercholesterolemia using oligonucleotide microchip expression analysis. We demonstrate that Tcf1-/- mice have a defect in bile acid transport, increased bile acid and liver cholesterol synthesis, and impaired HDL metabolism. Tcf1-/- liver has decreased expression of the basolateral membrane bile acid transporters Slc10a1, Slc21a3 and Slc21a5, leading to impaired portal bile acid uptake and elevated plasma bile acid concentrations. In intestine and kidneys, Tcf1-/- mice lack expression of the ileal bile acid transporter (Slc10a2), resulting in increased fecal and urinary bile acid excretion. The Tcf1 protein (also known as HNF-1alpha) also regulates transcription of the gene (Nr1h4) encoding the farnesoid X receptor-1 (Fxr-1), thereby leading to reduced expression of small heterodimer partner-1 (Shp-1) and repression of Cyp7a1, the rate-limiting enzyme in the classic bile acid biosynthesis pathway. In addition, hepatocyte bile acid storage protein is absent from Tcf1-/- mice. Increased plasma cholesterol of Tcf1-/- mice resides predominantly in large, buoyant, high-density lipoprotein (HDL) particles. This is most likely due to reduced activity of the HDL-catabolic enzyme hepatic lipase (Lipc) and increased expression of HDL-cholesterol esterifying enzyme lecithin:cholesterol acyl transferase (Lcat). Our studies demonstrate that Tcf1, in addition to being an important regulator of insulin secretion, is an essential transcriptional regulator of bile acid and HDL-cholesterol metabolism.

Bile acid synthesis is believed to be regulated by bile salts returning to the liver via the portal vein and suppressing cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in the bile acid biosynthesis pathway. In order to characterize the relative effectiveness of bile salts in regulating bile acid synthesis, seven different bile acids were administered (1% w/w in chow) to rats over a 14-day period. Biliary bile salt composition was determined from bile samples obtained prior to killing; in all cases, the fed bile acid became the predominant bile salt in bile. The specific activities of microsomal cholesterol 7 alpha-hydroxylase, HMG-CoA reductase and acylconenzyme A:cholesterol acyltransferase were determined after killing. Hydrophilic bile salts (ursocholic, hyocholic, ursodeoxycholic and hyodeoxycholic) did not inhibit HMG-CoA reductase or cholesterol 7 alpha-hydroxylase activities. By contrast, more hydrophobic bile salts (cholic, chenodeoxycholic and deoxycholic) inhibited the activities of these two enzymes in order of increasing hydrophobicity. Neither hydrophobic nor hydrophilic bile salts inhibited acylcoenzyme A:cholesterol acyltransferase activity. No consistent effect of bile acid feeding on total microsomal cholesterol was observed. Based on the results of these studies, we propose that the hydrophilic-hydrophobic balance of the bile acid pool may play an important role in the regulation of bile acid synthesis. We postulate that the activities of cholesterol 7 alpha-hydroxylase and HMG-CoA reductase may be regulated by hydrophobic bile acid-induced changes in the lipid composition and physicochemical properties (fluidity) of the microsomal membranes to which both of these rate-limiting enzymes are attached.(ABSTRACT TRUNCATED AT 250 WORDS)

Publisher Summary This chapter describes the development of bile acid biogenesis and its significance in cholesterol homeostasis. Epidemiological studies have indicated that cholesterol is a major risk factor for the development of atherosclerosis. Therefore, studies of the development of a cholesterol homeostatic mechanism and potential means of modulating this process in an effort to improve the handling of cholesterol in adult life are of interest. Because bile acid synthesis is the major pathway of cholesterol degradation and elimination from the body, knowledge about the development of the bile acid synthetic process in early life and factors affecting its course are critical. Bile acid synthesis is fairly underdeveloped at birth in most mammals and more investigations regarding the various pathways of bile acid synthesis in fetal and neonatal life are desired. The significance of the atypical bile acid noted in meconium is not clear, and more studies on the effect of maternal factors in influencing fetal and neonatal bile acid synthesis are needed. Evidence is accumulating to suggest that manipulation of the bile acid biosynthesis pathway in early life maybe beneficial in improving the handling of cholesterol in adult life.