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Forkhead transcription factor 3 (FOXA3) has been shown to regulate metabolism and development. Hepatic FOXA3 is reduced in obesity and fatty liver disease. However, the role of hepatic FOXA3 in regulating obesity or steatohepatitis remains to be investigated. In this work, C57BL/6 mice were i.v. injected with AAV8-ALB-FOXA3 or the control virus. The mice were then fed a chow or Western diet for 16 weeks. The role of hepatic FOXA3 in energy metabolism and steatohepatitis was investigated. Plasma bile acid composition and the role of Takeda G protein–coupled receptor 5 (TGR5) in mediating the metabolic effects of FOXA3 were determined. Overexpression of hepatic FOXA3 reduced hepatic steatosis in chow-fed mice and attenuated Western diet–induced obesity and steatohepatitis. FOXA3 induced lipolysis and inhibited hepatic genes involved in bile acid uptake, resulting in elevated plasma bile acids. The beneficial effects of hepatic FOXA3 overexpression on Western diet–induced obesity and steatohepatitis were abolished in Tgr5−/− mice. Our data demonstrate that overexpression of hepatic FOXA3 prevents Western diet–induced obesity and steatohepatitis via activation of TGR5.

Bile acids are detergents derived from cholesterol that function to solubilize dietary lipids, remove cholesterol from the body, and act as nutrient signaling molecules in numerous tissues with functions in the liver and gut being the best understood. Studies in the early 20th century established the structures of bile acids, and by mid-century, the application of gnotobiology to bile acids allowed differentiation of host-derived “primary” bile acids from “secondary” bile acids generated by host-associated microbiota. In 1960, radiolabeling studies in rodent models led to determination of the stereochemistry of the bile acid 7-dehydration reaction. A two-step mechanism was proposed, which we have termed the Samuelsson-Bergström model, to explain the formation of deoxycholic acid. Subsequent studies with humans, rodents, and cell extracts of Clostridium scindens VPI 12708 led to the realization that bile acid 7-dehydroxylation is a result of a multi-step, bifurcating pathway that we have named the Hylemon-Björkhem pathway. Due to the importance of hydrophobic secondary bile acids and the increasing measurement of microbial bai genes encoding the enzymes that produce them in stool metagenome studies, it is important to understand their origin.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease with increased risk in patients with metabolic syndrome. There are no FDA-approved treatments, but FXR agonists have shown promising results in clinical studies for NAFLD management. In addition to FXR, fibroblast growth factor receptor FGFR4 is a key mediator of hepatic bile acid synthesis. Using N-acetylgalactosamine–conjugated siRNA, we knocked down FGFR4 specifically in the liver of mice on chow or high-fat diet and in mouse primary hepatocytes to determine the role of FGFR4 in metabolic processes and hepatic steatosis. Liver-specific FGFR4 silencing increased bile acid production and lowered serum cholesterol. Additionally, we found that high-fat diet–induced liver steatosis and insulin resistance improved following FGFR4 knockdown. These improvements were associated with activation of the FXR-FGF15 axis in intestinal cells, but not in hepatocytes. We conclude that targeting FGFR4 in the liver to activate the intestinal FXR-FGF15 axis may be a promising strategy for the treatment of NAFLD and metabolic dysfunction.

FXR regulates bile acid metabolism, and FXR null (Fxr−/−) mice have elevated bile acid levels and progressive liver injury. The inositol-requiring enzyme 1α/X-box binding protein 1 (XBP1) pathway is a protective unfolded protein response pathway activated in response to endoplasmic reticulum stress. Here, we sought to determine the role of the inositol-requiring enzyme 1α/XBP1 pathway in hepatic bile acid toxicity using the Fxr−/− mouse model. Western blotting and quantitative PCR analysis demonstrated that hepatic XBP1 and other unfolded protein response pathways were activated in 24-week-old Fxr−/− compared with 10-week-old Fxr−/− mice but not in WT mice. To further determine the role of the liver XBP1 activation in older Fxr−/− mice, we generated mice with whole-body FXR and liver-specific XBP1 double KO (DKO, Fxr−/−Xbp1LKO) and Fxr−/−Xbp1fl/fl single KO (SKO) mice and characterized the role of hepatic XBP1 in cholestatic liver injury. Histologic staining demonstrated increased liver injury and fibrosis in DKO compared with SKO mice. RNA sequencing revealed increased gene expression in apoptosis, inflammation, and cell proliferation pathways in DKO mice. The proapoptotic C/EBP-homologous protein pathway and cell cycle marker cyclin D1 were also activated in DKO mice. Furthermore, we found that total hepatic bile acid levels were similar between the two genotypes. At age 60 weeks, all DKO mice and no SKO mice spontaneously developed liver tumors. In conclusion, the hepatic XBP1 pathway is activated in older Fxr−/− mice and has a protective role. The potential interaction between XBP1 and FXR signaling may be important in modulating the hepatocellular cholestatic stress responses.

Bile acids (BAs) play important roles in lipid homeostasis, and BA signaling pathways serve as therapeutic targets for nonalcoholic fatty liver disease (NAFLD). Recently, we generated cytochrome P450, family 2, subfamily C, polypeptide 70 (Cyp2c70−/−) mice with a human-like BA composition lacking mouse-/rat-specific muricholic acids to accelerate translation from mice to humans. We employed this model to assess the consequences of a human-like BA pool on diet-induced obesity and NAFLD development. Male and female Cyp2c70−/− mice and WT littermates were challenged with a 12-week high-fat Western-type diet (WTD) supplemented with 0.25% cholesterol. Cyp2c70 deficiency induced a hydrophobic BA pool with high abundances of chenodeoxycholic acid, particularly in females, because of sex-dependent suppression of sterol 12α-hydroxylase (Cyp8b1). Plasma transaminases were elevated, and hepatic fibrosis was present in Cyp2c70−/− mice, especially in females. Surprisingly, female Cyp2c70−/− mice were resistant to WTD-induced obesity and hepatic steatosis, whereas male Cyp2c70−/− mice showed similar adiposity and moderately reduced steatosis compared with WT controls. Both intestinal cholesterol and FA absorption were reduced in Cyp2c70−/− mice, the latter more strongly in females, despite unaffected biliary BA secretion rates. Intriguingly, the biliary ratio 12α-/non-12α-hydroxylated BAs significantly correlated with FA absorption and hepatic triglyceride content as well as with specific changes in gut microbiome composition. The hydrophobic human-like BA pool in Cyp2c70−/− mice prevents WTD-induced obesity in female mice and NAFLD development in both genders, primarily because of impaired intestinal fat absorption. Our data point to a key role for 12α-hydroxylated BAs in control of intestinal fat absorption and modulation of gut microbiome composition.

ABCB4, also called multidrug-resistant protein 3 (MDR3), is an ATP binding cassette transporter located in the canalicular membrane of hepatocytes that specifically translocates phosphatidylcholine (PC) lipids from the cytoplasmic to the extracellular leaflet. Due to the harsh detergent effect of bile acids, PC lipids provided by ABCB4 are extracted into the bile. While it is well known that bile acids are the major extractor of PC lipids from the membrane into bile, it is unknown whether only PC lipid extraction is improved or whether bile acids also have a direct effect on ABCB4. Using in vitro experiments, we investigated the modulation of ATP hydrolysis of ABC by different bile acids commonly present in humans. We demonstrated that all tested bile acids stimulated ATPase activity except for taurolithocholic acid, which inhibited ATPase activity due to its hydrophobic nature. Additionally, we observed a nearly linear correlation between the critical micelle concentration and maximal stimulation by each bile acid, and that this modulation was maintained in the presence of PC lipids. This study revealed a large effect of 24-nor-ursodeoxycholic acid, suggesting a distinct mode of regulation of ATPase activity compared with other bile acids. In addition, it sheds light on the molecular cross talk of canalicular ABC transporters of the human liver.

Glucagon and insulin have opposing action in governing glucose homeostasis. In type 2 diabetes mellitus (T2DM), plasma glucagon is characteristically elevated, contributing to increased gluconeogenesis and hyperglycemia. Therefore, glucagon receptor (GCGR) antagonism has been proposed as a pharmacologic approach to treat T2DM. In support of this concept, a potent small-molecule GCGR antagonist (GRA), MK-0893, demonstrated dose-dependent efficacy to reduce hyperglycemia, with an HbA1c reduction of 1.5% at the 80 mg dose for 12 weeks in T2DM. However, GRA treatment was associated with dose-dependent elevation of plasma LDL-cholesterol (LDL-c). The current studies investigated the cause for increased LDL-c. We report findings that link MK-0893 with increased glucagon-like peptide 2 and cholesterol absorption. There was not, however, a GRA-related modulation of cholesterol synthesis. These findings were replicated using structurally diverse GRAs. To examine potential pharmacologic mitigation, coadministration of ezetimibe (a potent inhibitor of cholesterol absorption) in mice abrogated the GRA-associated increase of LDL-c. Although the molecular mechanism is unknown, our results provide a novel finding by which glucagon and, hence, GCGR antagonism govern cholesterol metabolism.

Vitamin D receptor (VDR) mediates vitamin D signaling involved in bone metabolism, cellular growth and differentiation, cardiovascular function, and bile acid regulation. Mice with an intestine-specific disruption of VDR (VdrΔIEpC) have abnormal body size, colon structure, and imbalance of bile acid metabolism. Lithocholic acid (LCA), a secondary bile acid that activates VDR, is among the most toxic of the bile acids that when overaccumulated in the liver causes hepatotoxicity. Because cytochrome P450 3A4 (CYP3A4) is a target gene of VDR-involved bile acid metabolism, the role of CYP3A4 in VDR biology and bile acid metabolism was investigated. The CYP3A4 gene was inserted into VdrΔIEpC mice to produce the VdrΔIEpC/3A4 line. LCA was administered to control, transgenic-CYP3A4, VdrΔIEpC, and VdrΔIEpC/3A4 mice, and hepatic toxicity and bile acid levels in the liver, intestine, bile, and urine were measured. VDR deficiency in the intestine of the VdrΔIEpC mice exacerbates LCA-induced hepatotoxicity manifested by increased necrosis and inflammation, due in part to over-accumulation of hepatic bile acids including taurocholic acid and taurodeoxycholic acid. Intestinal expression of CYP3A4 in the VdrΔIEpC/3A4 mouse line reduces LCA-induced hepatotoxicity through elevation of LCA metabolism and detoxification, and suppression of bile acid transporter expression in the small intestine. This study reveals that intestinal CYP3A4 protects against LCA hepatotoxicity.

Cholesteryl ester transfer protein (CETP) activity results in a proatherogenic lipoprotein profile. In cholestatic conditions, farnesoid X receptor (FXR) signaling by bile acids (BA) is activated and plasma HDL cholesterol (HDL-C) levels are low. This study tested the hypothesis that FXR-mediated induction of CETP contributes to this phenotype. Patients with cholestasis and high plasma BA had lower HDL-C levels and higher plasma CETP activity and mass compared with matched controls with low plasma BA (each P < 0.01). BA feeding in APOE3*Leiden transgenic mice expressing the human CETP transgene controlled by its endogenous promoter increased cholesterol within apoB-containing lipoproteins and decreased HDL-C (each P < 0.01), while hepatic CETP mRNA expression and plasma CETP activity and mass increased (each P < 0.01). In vitro studies confirmed that FXR agonists substantially augmented CETP mRNA expression in hepatocytes and macrophages dependent on functional FXR expression (each P < 0.001). These transcriptional effects are likely mediated by an ER8 FXR response element (FXRE) in the first intron. In conclusion, using a translational approach, this study identifies CETP as novel FXR target gene. By increasing CETP expression, FXR activation leads to a proatherogenic lipoprotein profile. These results have clinical relevance, especially when considering FXR agonists as emerging treatment strategy for metabolic disease and atherosclerosis.

Steroidogenic acute regulatory-related lipid transfer (START) domain proteins are involved in the nonvesicular intracellular transport of lipids and sterols. The STARD1 (STARD1 and STARD3) and STARD4 subfamilies (STARD4–6) have an internal cavity large enough to accommodate sterols. To provide a deeper understanding on the structural biology of this domain, the binding of sterols to STARD5, a member of the STARD4 subfamily, was monitored. The SAR by NMR [1H-15N heteronuclear single-quantum coherence (HSQC)] approach, complemented by circular dichroism (CD) and isothermal titration calorimetry (ITC), was used. Titration of STARD5 with cholic (CA) and chenodeoxycholic acid (CDCA), ligands of the farnesoid X receptor (FXR), leads to drastic perturbation of the 1H-15N HSQC spectra and the identification of the residues in contact with those ligands. The most perturbed residues in presence of ligands are lining the internal cavity of the protein. Ka values of 1.8·10−4 M−1 and 6.3·104 M−1 were measured for CA and CDCA, respectively. This is the first report of a START domain protein in complex with a sterol ligand. Our original findings indicate that STARD5 may be involved in the transport of bile acids rather than cholesterol.

The apical Na+-dependent bile salt transporter (ASBT/SLC10A2) is essential for maintaining the enterohepatic circulation of bile salts. It is not known when Slc10a2 evolved as a bile salt transporter or how it adapted to substantial changes in bile salt structure during evolution. We characterized ASBT orthologs from two primitive vertebrates, the lamprey that utilizes early 5α-bile alcohols and the skate that utilizes structurally different 5β-bile alcohols, and compared substrate specificity with ASBT from humans who utilize modern 5β-bile acids. Everted gut sacs of skate but not the more primitive lamprey transported 3H-taurocholic acid (TCA), a modern 5β-bile acid. However, molecular cloning identified ASBT orthologs from both species. Cell-based assays using recombinant ASBT/Asbt's indicate that lamprey Asbt has high affinity for 5α-bile alcohols, low affinity for 5β-bile alcohols, and lacks affinity for TCA, whereas skate Asbt showed high affinity for 5α- and 5β-bile alcohols but low affinity for TCA. In contrast, human ASBT demonstrated high affinity for all three bile salt types. These findings suggest that ASBT evolved from the earliest vertebrates by gaining affinity for modern bile salts while retaining affinity for older bile salts. Also, our results indicate that the bile salt enterohepatic circulation is conserved throughout vertebrate evolution.

Type I diabetes mellitus (T1DM) increases atherosclerotic cardiovascular disease; however, the underlying pathophysiology is still incompletely understood. We investigated whether experimental T1DM impacts HDL-mediated reverse cholesterol transport (RCT). C57BL/6J mice with alloxan-induced T1DM had higher plasma cholesterol levels (P < 0.05), particularly within HDL, and increased hepatic cholesterol content (P < 0.001). T1DM resulted in increased bile flow (2.1-fold; P < 0.05) and biliary secretion of bile acids (BA, 10.5-fold; P < 0.001), phospholipids (4.5-fold; P < 0.001), and cholesterol (5.5-fold; P < 0.05). Hepatic cholesterol synthesis was unaltered, whereas BA synthesis was increased in T1DM (P < 0.001). Mass fecal BA output was significantly higher in T1DM mice (1.5-fold; P < 0.05), fecal neutral sterol excretion did not change due to increased intestinal cholesterol absorption (2.1-fold; P < 0.05). Overall in vivo macrophage-to-feces RCT, using [3H]cholesterol-loaded primary mouse macrophage foam cells, was 20% lower in T1DM (P < 0.05), mainly due to reduced tracer excretion within BA (P < 0.05). In vitro experiments revealed unchanged cholesterol efflux toward T1DM HDL, whereas scavenger receptor class BI-mediated selective uptake from T1DM HDL was lower in vitro and in vivo (HDL kinetic experiments) (P < 0.05), conceivably due to increased glycation of HDL-associated proteins (+65%, P < 0.01). In summary, despite higher mass biliary sterol secretion T1DM impairs macrophage-to-feces RCT, mainly by decreasing hepatic selective uptake, a mechanism conceivably contributing to increased cardiovascular disease in T1DM.

Z-Guggulsterone is a major ingredient in the Indian traditional hypolipidemic remedy guggul. A study in mice has established that its hypolipidemic effect involves the farnesoid X receptor (FXR), presumably by acting as an antagonist of this receptor. It is generally assumed that the antagonism leads to induction of cytochrome P450 7A1 (CYP7A1), the rate-limiting enzyme converting free cholesterol to bile acids. In this study, we tested whether Z-guggulsterone indeed induces human CYP7A1. In addition, the expression of cholesteryl ester hydrolase CES1 and bile salt export pump (BSEP) was monitored. Contrary to the general assumption, Z-guggulsterone did not induce CYP7A1. Instead, this phytosterol significantly induced CES1 and BSEP through transactivation. Z-Guggulsterone underwent metabolism by CYP3A4, and the metabolites greatly increased the induction potency on BSEP but not on CES1. BSEP induction favors cholesterol elimination, whereas CES1 involves both elimination and retention (probably when excessively induced). Interestingly, clinical trials reported the hypolipidemic response rates from 18% to 80% and showed that higher dosages actually increased VLDL cholesterol. Our findings predict that better hypolipidemic outcomes likely occur in individuals who have a relatively higher capacity of metabolizing Z-guggulsterone with moderate CES1 induction, a scenario possibly achieved by lowering the dosing regimens.

Cholesterol 7α-hydroxylase (CYP7A1) encodes for the rate-limiting step in the conversion of cholesterol to bile acids in the liver. In response to acute cholesterol feeding, mice upregulate CYP7A1 via stimulation of the liver X receptor (LXR) α. However, the effect of a chronic high-cholesterol diet on hepatic CYP7A1 expression in mice is unknown. We demonstrate that chronic cholesterol feeding (0.2% or 1.25% w/w cholesterol for 12 weeks) in FVB/NJ mice results in a >60% suppression of hepatic CYP7A1 expression associated with a >2-fold increase in hepatic cholesterol content. In contrast, acute cholesterol feeding induces a >3-fold upregulation of hepatic CYP7A1 expression. We show that chronic, but not acute, cholesterol feeding increases the expression of hepatic inflammatory cytokines, tumor necrosis factor (TNF)α, and interleukin (IL)-1β, which are known to suppress hepatic CYP7A1 expression. Chronic cholesterol feeding also results in activation of the mitogen activated protein (MAP) kinases, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). Furthermore, we demonstrate in vitro that suppression of CYP7A1 by TNFα and IL-1β is dependent on JNK and ERK signaling. We conclude that chronic high-cholesterol feeding suppresses CYP7A1 expression in mice. We propose that chronic cholesterol feeding induces inflammatory cytokine activation and liver damage, which leads to suppression of CYP7A1 via activation of JNK and ERK signaling pathways.

To investigate the effects of abolished cholic acid (CA) synthesis in the ApoE knockout model [apolipoprotein E (apoE) KO],a double-knockout (DKO) mouse model was created by crossbreeding Cyp8b1 knockout mice (Cyp8b1 KO), unable to synthesize the primary bile acid CA, with apoE KO mice. After 5 months of cholesterol feeding, the development of atherosclerotic plaques in the proximal aorta was 50% less in the DKO mice compared with the apoE KO mice. This effect was associated with reduced intestinal cholesterol absorption, decreased levels of apoB-containing lipoproteins in the plasma, enhanced bile acid synthesis, reduced hepatic cholesteryl esters, and decreased hepatic activity of ACAT2. The upregulation of Cyp7a1 in DKO mice seemed primarily caused by reduced expression of the intestinal peptide FGF15. Treatment of DKO mice with the farnesoid X receptor (FXR) agonist GW4064 did not alter the intestinal cholesterol absorption, suggesting that the action of CA in this process is confined mainly to formation of intraluminal micelles and less to its ability to activate the nuclear receptor FXR. Inhibition of CA synthesis may offer a therapeutic strategy for the treatment of hyperlipidemic conditions that lead to atherosclerosis.

Biliary bile salt composition of 677 vertebrate species (103 fish, 130 reptiles, 271 birds, 173 mammals) was determined. Bile salts were of three types: C27 bile alcohols, C27 bile acids, or C24 bile acids, with default hydroxylation at C-3 and C-7. C27 bile alcohols dominated in early evolving fish and amphibians; C27 bile acids, in reptiles and early evolving birds. C24 bile acids were present in all vertebrate classes, often with C27 alcohols or with C27 acids, indicating two evolutionary pathways from C27 bile alcohols to C24 bile acids: a) a ‘direct’ pathway and b) an ‘indirect’ pathway with C27 bile acids as intermediates. Hydroxylation at C-12 occurred in all orders and at C-16 in snakes and birds. Minor hydroxylation sites were C-1, C-2, C-5, C-6, and C-15. Side chain hydroxylation in C27 bile salts occurred at C-22, C-24, C-25, and C-26, and in C24 bile acids, at C-23 (snakes, birds, and pinnipeds). Unexpected was the presence of C27 bile alcohols in four early evolving mammals. Bile salt composition showed significant variation between orders but not between families, genera, or species. Bile salt composition is a biochemical trait providing clues to evolutionary relationships, complementing anatomical and genetic analyses.

In liver and intestine, transporters play a critical role in maintaining the enterohepatic circulation and bile acid homeostasis. Over the past two decades, there has been significant progress toward identifying the individual membrane transporters and unraveling their complex regulation. In the liver, bile acids are efficiently transported across the sinusoidal membrane by the Na+ taurocholate cotransporting polypeptide with assistance by members of the organic anion transporting polypeptide family. The bile acids are then secreted in an ATP-dependent fashion across the canalicular membrane by the bile salt export pump. Following their movement with bile into the lumen of the small intestine, bile acids are almost quantitatively reclaimed in the ileum by the apical sodium-dependent bile acid transporter. The bile acids are shuttled across the enterocyte to the basolateral membrane and effluxed into the portal circulation by the recently indentified heteromeric organic solute transporter, OSTα-OSTβ. In addition to the hepatocyte and enterocyte, subgroups of these bile acid transporters are expressed by the biliary, renal, and colonic epithelium where they contribute to maintaining bile acid homeostasis and play important cytoprotective roles. This article will review our current understanding of the physiological role and regulation of these important carriers.

The nuclear hormone receptor constitutive androstane receptor (CAR, NR1I3) regulates detoxification of xenobiotics and endogenous molecules, and has been shown to be involved in the metabolism of hepatic bile acids and cholesterol. The goal of this study was to address potential effects of CAR on the metabolism of HDL particles, key components in the reverse transport of cholesterol to the liver. Wild-type (WT) mice, transgenic mice expressing human apolipoprotein A-I (HuAITg), and CAR-deficient (CAR−/−) mice were treated with the specific CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). CAR activation decreased HDL cholesterol and plasma apolipoprotein A-I (apoA-I) levels in both WT and HuAITg mice, but not CAR−/− mice. Both mouse apoA-I and human apoA-I were decreased by more than 40% after TCPOBOP treatment, and kinetic studies revealed that the production rate of HDL is reduced in TCPOBOP-treated WT mice. In transient transfections, TCPOBOP-activated CAR decreased the activity of the human apoA-I promoter. Although loss of CAR function did not alter HDL levels in normal chow-fed mice, HDL cholesterol, apoA-I concentration, and apoA-I mRNA levels were increased in CAR−/− mice relative to WT mice when both were fed a high-fat diet. We conclude that CAR activation in mice induces a pronounced decrease in circulating levels of plasma HDL, at least in part through downregulation of apoA-I gene expression.

The metabolic conversion of cholesterol into bile acids in liver is initiated by the rate-limiting cholesterol 7α-hydroxylase (CYP7A1), whereas the bile salt export pump (BSEP) is responsible for the canalicular secretion of bile acids. Liver receptor homolog 1 (LRH-1) is a key transcriptional factor required for the hepatic expression of CYP7A1. We hypothesized that LRH-1 was also involved in the transcriptional regulation of BSEP. In support of our hypothesis, we found that overexpression of LRH-1 induced, whereas knockdown of LRH-1 decreased, BSEP expression. Consistent with its role in transcriptional regulation, LRH-1 dose-dependently transactivated the BSEP promoter. In addition, such transactivation by LRH-1 was required for maximal induction of BSEP expression through the bile acid/farnesoid X receptor (FXR) activation pathway. Bioinformatic and mutational analysis led to the identification of a functional liver receptor homolog 1-responsive element (LRHRE) in the BSEP promoter. Specific binding of LRH-1 to the LRHRE and recruitment of LRH-1 to the BSEP promoter were demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay, respectively. In conclusion, LRH-1 transcriptionally activated the BSEP promoter and functioned as a modulator in bile acid/FXR-mediated BSEP regulation. These results suggest that LRH-1 plays a supporting role to FXR in maintaining hepatic bile acid levels by coordinately regulating CYP7A1 and BSEP for bile acid synthesis and elimination, respectively.

Constitutive expression of a cholesterol-7α-hydroxylase (CYP7A1) transgene in LDL receptor-deficient mice blocked the ability of a cholesterol-enriched diet to increase plasma levels of apolipoprotein B-containing lipoproteins. LDL receptor-deficient mice expressing the CYP7A1 transgene exhibited complete resistance to diet-induced hypercholesterolemia and to the accumulation of cholesterol in the liver. Hepatic mRNA expression of liver X receptor-inducible ABCG5 and ABCG8 was decreased in CYP7A1 transgenic, LDL receptor-deficient mice fed a cholesterol-enriched diet. Thus, increased biliary cholesterol excretion could not account for the maintenance of cholesterol homeostasis. CYP7A1 transgenic, LDL receptor-deficient mice fed the cholesterol-enriched diet exhibited decreased jejunal Niemann-Pick C1-Like 1 protein (NPC1L1) mRNA expression, an important mediator of intestinal cholesterol absorption. A taurocholate-enriched diet also decreased NPC1L1 mRNA expression in a farnesoid X receptor-independent manner. Reduced expression of NPC1L1 mRNA was associated with decreased cholesterol absorption (∼20%; P < 0.05) exhibited by CYP7A1 transgenic LDL receptor-deficient mice fed the cholesterol-enriched diet. The combined data show that enhanced expression of CYP7A1 is an effective means to prevent the accumulation of cholesterol in the liver and of atherogenic apolipoprotein B-containing lipoproteins in plasma.

This study reports the discovery of a novel sulfonated oxysterol found at high levels in the mitochondria and nuclei of primary rat hepatocytes after overexpression of the gene encoding steroidogenic acute regulatory protein (StarD1). Forty-eight hours after infection of primary rat hepatocytes with recombinant adenovirus encoding StarD1, rates of bile acid synthesis increased by 4-fold. Concurrently, [14C]cholesterol metabolites (oxysterols) were increased dramatically in both the mitochondria and nuclei of StarD1-overexpressing cells, but not in culture medium. A water-soluble [14C]oxysterol product was isolated and purified by chemical extraction and reverse-phase HPLC. Enzymatic digestion, HPLC, and tandem mass spectrometry analysis identified the water-soluble oxysterol as 5-cholesten-3β,25-diol 3-sulfonate. Further experiments detected this cholesterol metabolite in the nuclei of normal human liver tissues. Based upon these observations, we hypothesized a new pathway by which cholesterol is metabolized in the mitochondrion.

Bone is the most common site to which breast cancer cells metastasize. We found that osteoblast-like MG63 cells and human bone tissue contain the bile acid salt sodium deoxycholate (DC). MG63 cells take up and accumulate DC from the medium, suggesting that the bone-derived DC originates from serum. DC released from MG63 cells or bone tissue promotes cell survival and induces the migration of metastatic human breast cancer MDA-MB-231 cells. The bile acid receptor farnesoid X receptor (FXR) antagonist Z-guggulsterone prevents the migration of these cells and induces apoptosis. DC increases the gene expression of FXR and induces its translocation to the nucleus of MDA-MB-231 cells. Nuclear translocation of FXR is concurrent with the increase of urokinase-type plasminogen activator (uPA) and the formation of F-actin, two factors critical for the migration of breast cancer cells. Our results suggest a novel mechanism by which DC-induced increase of uPA and binding to the uPA receptor of the same breast cancer cell self-propel its migration and metastasis to the bone.

Secondary bile acids, produced solely by intestinal bacteria, can accumulate to high levels in the enterohepatic circulation of some individuals and may contribute to the pathogenesis of colon cancer, gallstones, and other gastrointestinal (GI) diseases. Bile salt hydrolysis and hydroxy group dehydrogenation reactions are carried out by a broad spectrum of intestinal anaerobic bacteria, whereas bile acid 7-dehydroxylation appears restricted to a limited number of intestinal anaerobes representing a small fraction of the total colonic flora. Microbial enzymes modifying bile salts differ between species with respect to pH optima, enzyme kinetics, substrate specificity, cellular location, and possibly physiological function. Crystallization, site-directed mutagenesis, and comparisons of protein secondary structure have provided insight into the mechanisms of several bile acid-biotransforming enzymatic reactions. Molecular cloning of genes encoding bile salt-modifying enzymes has facilitated the understanding of the genetic organization of these pathways and is a means of developing probes for the detection of bile salt-modifying bacteria. The potential exists for altering the bile acid pool by targeting key enzymes in the 7α/β-dehydroxylation pathway through the development of pharmaceuticals or sequestering bile acids biologically in probiotic bacteria, which may result in their effective removal from the host after excretion.

As previously reported by us, mice with targeted disruption of the CYP8B1 gene (CYP8B1−/−) fail to produce cholic acid (CA), upregulate their bile acid synthesis, reduce the absorption of dietary cholesterol and, after cholesterol feeding, accumulate less liver cholesterol than wild-type (CYP8B1+/+) mice. In the present study, cholesterol-enriched diet (0.5%) or administration of a synthetic liver X receptor (LXR) agonist strongly upregulated CYP7A1 expression in CYP8B1−/− mice, compared to CYP8B1+/+ mice. Cholesterol-fed CYP8B1−/− mice also showed a significant rise in HDL cholesterol and increased levels of liver ABCA1 mRNA. A combined CA (0.25%)/cholesterol (0.5%) diet enhanced absorption of intestinal cholesterol in both groups of mice, increased their liver cholesterol content, and reduced their expression of CYP7A1 mRNA. The ABCG5/G8 liver mRNA was increased in both groups of mice, but cholesterol crystals were only observed in bile from the CYP8B1+/+ mice. The results demonstrate the cholesterol-sparing effects of CA: enhanced absorption and reduced conversion into bile acids. Farnesoid X receptor (FXR)-mediated suppression of CYP7A1 in mice seems to be a predominant mechanism for regulation of bile acid synthesis under normal conditions and, as confirmed, able to override LXR-mediated mechanisms. Interaction between FXR- and LXR-mediated stimuli might also regulate expression of liver ABCG5/G8.

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 7α-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 α (LXRα) or pregnane X receptor. However, another nuclear receptor, hepatocyte nuclear factor 4α (HNF4α), was induced 2.9-fold in CTX, which was associated with enhanced mRNA levels of HNF4α target genes, CYP7A1, 7α-hydroxy-4-cholesten-3-one 12α-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 HNF4α caused by reduced bile acids in CTX liver.

Sitosterolemia is a rare autosomal recessive disorder of lipoprotein metabolism characterized by xanthomas and increased plasma concentrations of plant sterols, such as sitosterol. Causative mutations occur in either the ABCG5 or ABCG8 gene, each of which encodes a sterol half-transporter expressed in the intestine. We report five Canadian subjects with nonsense mutations in these half-transporters: four related Caucasian subjects were homozygous for the ABCG8 S107X mutation, and one unrelated Japanese-Canadian subject was homozygous for a complex insertion/deletion (I/D) mutation in ABCG5 exon 3. A female subject with each mutation was symptomatic with coronary atherosclerosis: a 5-year-old ABCG8 S107X homozygote and a 75-year-old ABCG5 exon 3 I/D homozygote; these represent the extreme ends of the spectrum of vascular involvement in sitosterolemia. The largest reductions in plasma concentrations of sitosterol and LDL-cholesterol were seen with ezetimibe or bile acid sequestrant treatment, and less dramatic reductions were seen with statin drug treatment.These findings extend the range of clinical phenotypes in sitosterolemia caused by nonsense mutations in either ABCG5 or ABCG8.

5α-Cyprinol sulfate was isolated from bile of the Asiatic carp, Cyprinus carpio. 5α-Cyprinol sulfate was surface active and formed micelles; its critical micellization concentration (CMC) in 0.15 M Na+ using the maximum bubble pressure device was 1.5 mM; by dye solubilization, its CMC was ∼4 mM. At concentrations >1 mM, 5α-cyprinol sulfate solubilized monooleylglycerol efficiently (2.1 molecules per mol micellar bile salt). When infused intravenously into the anesthetized rat, 5α-cyprinol sulfate was hemolytic, cholestatic, and toxic. In the isolated rat liver, it underwent little biotransformation and was poorly transported (Tmax ≅ 0.5 μmol/min/kg) as compared with taurocholate. 5α-Cyprinol, its bile alcohol moiety, was oxidized to its corresponding C27 bile acid and to allocholic acid (the latter was then conjugated with taurine); these metabolites were efficiently transported. 5α-Cyprinol sulfate inhibited taurocholate uptake in COS-7 cells transfected with rat asbt, the apical bile salt transporter of the ileal enterocyte. 5α-Cyprinol had limited aqueous solubility (0.3 mM) and was poorly absorbed from the perfused rat jejunum or ileum. Sampling of carp intestinal content indicated that 5α-cyprinol sulfate was present at micellar concentrations, and that it did not undergo hydrolysis during intestinal transit.These studies indicate that 5α-cyprinol sulfate is an excellent digestive detergent and suggest that a micellar phase is present during digestion in cyprinid fish.

Patients with severe malabsorption have abnormal lipid metabolism with low plasma cholesterol and frequently high triglyceride (TG) levels. The mechanisms behind these abnormalities and the respective roles of malabsorption itself and of the parenteral nutrition given to these patients are unclear. We measured endogenous lipids synthesis (cholesterol synthesis and hepatic lipogenesis) and the expression (mRNA concentrations in circulating mononuclear cells) of regulatory genes of cholesterol metabolism in 10 control subjects and 22 patients with severe malabsorption receiving (n = 18) or weaned of parenteral nutrition (n = 4). Patients had low plasma cholesterol (P < 0.01) and raised TG (P < 0.05) levels. Both fractional and absolute cholesterol synthesis (P < 0.001) and hepatic lipogenesis (P < 0.01) were increased. These abnormalities are independent of parenteral nutrition since they were present in patients receiving or weaned of parenteral nutrition. No relation between hepatic lipogenesis and plasma TG levels was found, suggesting that other metabolic abnormalities participated in hypertriglyceridemia. HMG-CoA reductase and LDL receptor mRNA levels were decreased (P < 0.05) in patients on long-term parenteral nutrition. HMG-CoA reductase mRNAs were normal in weaned patients.Severe malabsorption induces large increases of cholesterol synthesis and hepatic lipogenesis independently of the presence of parenteral nutrition. These abnormalities are probably due to the malabsorption of bile acids.

Solubilization of cholesterol in the intestinal lumen by bile acids and the subsequent formation of mixed micelles is an important step in the absorption of cholesterol. We propose that oxidized fatty acids (ox-FA) may mimic bile acids and form mixed micelles with cholesterol much more efficiently, as compared with unoxidized fatty acids, thereby increasing there absorption. In an in vitro assay at concentrations of 1, 5, and 10 mM, oxidized linoleic acid (ox-18:2) increased the solubilization of cholesterol (3.06, 8.16, and 15.46 nmol/ml) in a dose dependent manner compared with a 10 mM unoxidized linoleic acid (unox-18:2 at 0.97 nmol/ml). The uptake of cholesterol solubilized in the presence of ox-18:2 by Caco-2 cells and everted rat intestinal sacs was greater (1.78 and 1.95 nmol/ml respectively) as compared with the cholesterol solubilized in the presence of unox-18:2 (0.29 and 0.61 nmol/ml; P = 0.05). In addition, when LDL receptor deficient mice were fed a high fat diet along with ox-18:2 their plasma cholesterol levels were greater than animals fed the high fat diet alone (1290 mg/dl vs. 1549 mg/dl, P = 0.013).From these results, we suggest that ox-FA, by enhancing the solubilization of luminal cholesterol, increases the uptake of cholesterol that might lead to hypercholesterolemia and atherosclerosis.

Unlike macrophages, the hepatic parenchymal cells express cholesterol-7α-hydroxylase (CYP7A1) which regulates the conversion of cholesterol into bile acids, the major quantitative pathway maintaining cholesterol homeostasis. We examined if CYP7A1 expression in RAW 264.7 macrophages could prevent the accumulation of cholesterol when they were incubated with acetyl-LDL. A single cell clone (designated as 7αRAW cells) that stably expresses rat CYP7A1 displayed similar rates of growth as non-transfected RAW cells. The CYP7A1 enzymatic activity expressed by microsomes obtained from 7αRAW cells was similar to that expressed by microsomes obtained from mouse liver. Incubating non-transfected RAW with increasing amounts of acetyl-LDL caused a parallel accumulation of cholesterol, whereas 7αRAW cells displayed a complete resistance to cholesterol accumulation. 7αRAW cells displayed increased expression of both ABCA1 mRNA (3.1-fold, P < 0.001) and ABCG1 mRNA (2.2-fold, P < 0.01), whereas the expression of scavenger receptor class A mRNA was unchanged. 7 αRAW cells also displayed small but significant increases in the rate of efflux of [3H]cholesterol to both delipidated apolipoprotein A1 and to HDL. Thus, CYP7A1 expression in RAW cultured macrophages prevented the accumulation of cholesterol from acetyl-LDL via both increased metabolism and efflux of cholesterol.—Moore, G. L., and R. A. Davis. Expression of cholesterol-7 α-hydroxylase in murine macrophages prevents cholesterol loading by acetyl-LDL. J. Lipid Res. 2002. 43: 629–635.

During the last three years there have been a plethora of publications on the liver X-activated receptors (LXRα, NR1H3, and LXRβ, NR1H2), the farnesoid X-activated receptor (FXR, NR1H4), and the pregnane X receptor (PXR, NR1I2) and the role these nuclear receptors play in controlling cholesterol, bile acid, lipoprotein and drug metabolism. The current interest in these nuclear receptors is high, in part, because they appear to be promising therapeutic targets for new drugs that have the potential to control lipid homeostasis. In this review we emphasize i) the role of LXR in controlling many aspects of cholesterol and fatty acid metabolism, ii) the expanded role of FXR in regulating genes that control not only bile acid metabolism but also lipoprotein metabolism, and iii) the regulation of bile acid transport/metabolism in response to bile acid-activated PXR.— Edwards, P. A., H. R. Kast, and A. M. Anisfeld. BAREing it all: the adoption of LXR and FXR and their roles in lipid homeostasis. J. Lipid Res. 2002. 43: 2–12.

A genetic mapping strategy was employed to identify chromosomal regions harboring genes that influence the absorption of intestinal cholesterol in the mouse. Analysis of seven inbred strains of male mice (129P3, AKR, BALB/c, C3H/He, C57BL/6, DBA/2, and SJL, all from Jackson Laboratories) revealed substantial differences in their abilities to absorb a bolus of cholesterol delivered by gavage. Crosses between high (AKR, 129) and low (DBA/2, SJL) absorbing strains revealed evidence for the presence of dominant genes that increase and decrease cholesterol absorption. Backcrosses between F1 offspring and parental strains (DBA/2xAKD2F1 and 129xSJL129F1) followed by linkage analyses revealed four quantitative trait loci that influenced cholesterol absorption. Analyses of recombinant inbred strains identified an additional three loci affecting this phenotype. These seven quantitative trait loci, which map to different chromosomes and are termed Cholesterol absorption 1–7 (Chab1–7) loci, together influence the absorption of intestinal cholesterol in mice and are likely to be involved in different steps of this complex pathway.—Schwarz, M., D. L Davis, B. R. Vick, and D. W. Russell. Genetic analysis of intestinal cholesterol absorption in inbred mice.

Genetic linkage analysis in the laboratory mouse identified chromosomal regions containing genes that contribute to cholesterol accumulation in the liver and plasma. Comparisons between five inbred strains of mice obtained from the Jackson Laboratory (DBA/2, AKR, C57BL/6, SJL, and 129P3) revealed a direct correlation between intestinal cholesterol absorption and susceptibility to diet-induced hypercholesterolemia. This correlation was lost in the F1 generation arising from crosses between high- and low-absorbing strains. Linkage analyses in AKxD recombinant inbred strains and 129xSJL129F1 N2 backcross mice identified four quantitative trait loci (QTL) that influenced Liver cholesterol accumulation (Lcho1–4) and one locus that affected Plasma cholesterol accumulation (Pcho1). These loci map to five chromosomes and, with one exception, are different from the seven QTL identified previously that influence intestinal cholesterol absorption. We conclude that a large number of genes affects the amount of cholesterol absorbed in the small intestine and its accumulation in the liver and plasma of inbred mice.—Schwarz, M., D. L. Davis, B. R. Vick, and D. W. Russell. Genetic analysis of cholesterol accumulation in inbred mice.

We examined the effect of hyodeoxycholic acid (HDCA) on plasma cholesterol levels and atherosclerosis in mice. In wild-type C57BL/6 mice, feeding increasing amounts of HDCA resulted in i) progressive decrease in dietary cholesterol absorption, ii) increased concentrations of HDCA in the gallbladder bile, iii) decreased liver cholesterol content, iv) increased liver cholesterol synthesis, and v) increased plasma concentrations of HDCA. In C57BL/6 LDL-receptor knockouts (LDLR-KO) the addition of HDCA to chow and a 0.5% cholesterol diet decreased their total plasma cholesterol levels by 21% and 62%, respectively, because of a decrease in VLDL and LDL cholesterol. Turnover studies showed that HDCA has no effect on VLDL removal from plasma. Furthermore, the addition of HDCA to chow- and 0.5% cholesterol-fed LDLR-KO mice decreased the aortic root atherosclerosis lesion area by 50% and 80%, respectively. Finally, we tested the effect of HDCA on intestinal tumor formation. Feeding C57BL/6 ApcMin mice with HDCA did not affect the number of tumors but decreased the tumor volume in these animals. These results suggest that HDCA might have beneficial effects in the treatment of increased plasma cholesterol levels and atherosclerosis. —Sehayek, E., J. G. Ono, E. M. Duncan, A. K. Batta, G. Salen, S. Shefer, L. B. Neguyen, K. Yang, M. Lipkin, and J. L. Breslow. Hyodeoxycholic acid efficiently suppresses atherosclerosis formation and plasma cholesterol levels in mice.

Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive disorder of bile acid biosynthesis. Clinically, CTX patients present with tendon xanthomas, juvenile cataracts, and progressive neurological dysfunction and can be diagnosed by the detection of elevated plasma cholestanol levels. CTX is caused by mutations affecting the sterol 27-hydroxylase gene (CYP27). CTX has been identified in a number of populations, but seems to have a higher prevalence in the Japanese, Sephardic Jewish, and Italian populations. We have assembled 12 previously unreported pedigrees from the United States. The CYP27 locus had been previously mapped to chromosome 2q33-qter. We performed linkage analyses and found no evidence of genetic heterogeneity. All CTX patients showed segregation with the CYP27 locus, and haplotype analysis and recombinant events allowed us to precisely map CYP27 to chromosome 2q35, between markers D2S1371 and D2S424. Twenty-three mutations were identified from 13 probands analyzed thus far; 11 were compound heterozygotes and 2 had homozygous mutations. Of these, five are novel mutations [Trp100Stop, Pro408Ser, Gln428Stop, a 10-base pair (bp) deletion in exon 1, and a 2-bp deletion in exon 6 of the CYP27 gene]. Three-dimensional structural modeling of sterol 27-hydroxylase showed that, while the majority of the missense mutations disrupt the heme-binding and adrenodoxin-binding domains critical for enzyme activity, two missense mutations (Arg94Trp/Gln and Lys226Arg) are clearly located outside these sites and may identify a potential substrate-binding or other protein contact site. —Lee, M-H., S. Hazard, J. D. Carpten, S. Yi, J. Cohen, G.T. Gerhardt, G. Salen, and S. B. Patel. Fine-mapping, mutation analyses, and structural mapping of cerebrotendinous xanthomatosis in U.S. pedigrees.

2-Methylacyl-CoA racemase is an auxiliary enzyme required for the peroxisomal β-oxidative breakdown of (2R)-pristanic acid and the (25R)-isomer of C27 bile acid intermediates. The enzyme activity is found not only in peroxisomes but also is present in mitochondria of human liver and fibroblasts. The C terminus of the human racemase, a protein of 382 amino acids with a molecular mass of 43,304 daltons as deduced from its cloned cDNA, consists of KASL. Hitherto this sequence has not been recognized as a peroxisomal targeting signal (PTS1). From the in vitro interaction between recombinant racemase and recombinant human PTS1 receptor (Pex5p), and the peroxisomal localization of green fluorescent protein (GFP) fused to the N terminus of full-length racemase or its last six amino acids in tranfected Chinese hamster ovary (CHO) cells, we concluded that ASL is a new PTS1 variant. To be recognized by Pex5p, however, the preceding lysine residue is critical. As shown in another series of transfection experiments with GFP fused to the C terminus of the full-length racemase or racemase with deletions of the N terminus, mitochondrial targeting information is localized between amino acids 22 and 85. Hence, our data show that a single transcript gives rise to a racemase protein containing two topogenic signals, explaining the dual cellular localization of the activity.—Amery, L., M. Fransen, K. De Nys, G. P. Mannaerts, and P. P. Van Veldhoven. Mitochondrial and peroxisomal targeting of 2-methylacyl-CoA racemase in humans. J. Lipid Res. 2000. 41: 1752–1759.

Abstract: In classic cholic acid biosynthesis, a series of ring modifications of cholesterol precede side chain cleavage and yield 5β-cholestane-3α, 7α, 12α-triol. Side chain reactions of the triol then proceed either by the mitochondrial 27-hydroxylation pathway or by the microsomal 25-hydroxylation pathway. We have developed specific and precise assay methods to measure the activities of key enzymes in both pathways, 5β-cholestane-3α, 7α, 12α-triol 25- and 27-hydroxylases and 5β-cholestane-3α, 7α, 12α, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases. The extracts from either the mitochondrial or microsomal incubation mixtures were purified by means of a disposable silica cartridge column, derivatized into trimethylsilyl ethers, and quantified by gas chromatography–mass spectrometry with selected-ion monitoring in a high resolution mode. Compared with the addition of substrates in acetone, those in 2-hydroxypropyl-β-cyclodextrin increased mitochondrial triol 27-hydroxylase activity 132% but decreased activities of the enzymes in microsomal 25-hydroxylation pathway (triol 25-hydroxylase and 5β-cholestane-3α, 7α, 12α, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases) 13–60% in human liver. The enzyme activities in both pathways were generally 2- to 4-times higher in mouse and rabbit livers compared with human liver. In all species, microsomal triol 25-hydroxylase activities were 4- to 11-times larger than mitochondrial triol 27-hydroxylase activities but the activities of tetrol 24S-hydroxylase were similar to triol 27-hydroxylase activities in our assay conditions. The regulation of both pathways in rabbit liver was studied after bile acid synthesis was perturbed. Cholesterol feeding up-regulated enzyme activities involved in both 25- (64–142%) and 27- (77%) hydroxylation pathways, while bile drainage up-regulated only the enzymes in the 25-hydroxylation pathway (178–371%). Using these new assays, we demonstrated that the 25- and 27-hydroxylation pathways for cholic acid biosynthesis are more active in mouse and rabbit than human livers and are separately regulated in rabbit liver. —Honda, A., G. Salen, S. Shefer, Y. Matsuzaki, G. Xu, A. K. Batta, G. S. Tint, and N. Tanaka. Regulation of 25- and 27-hydroxylation side chain cleavage pathways for cholic acid biosynthesis in humans, rabbits, and mice: assay of enzyme activities by high-resolution gas chromatography–mass spectrometry. J. Lipid Res. 2000. 41: 442–451.

We investigated the effect of ileal bile acid transport on the regulation of classic and alternative bile acid synthesis in cholesterol-fed rats and rabbits. Bile acid pool sizes, fecal bile acid outputs (synthesis rates), and the activities of cholesterol 7α-hydroxylase (classic bile acid synthesis) and cholesterol 27-hydroxylase (alternative bile acid synthesis) were related to ileal bile acid transporter expression (ileal apical sodium-dependent bile acid transporter, ASBT). Plasma cholesterol levels rose 2.1-times in rats (98 ± 19 mg/dl) and 31-times (986 ± 188 mg/dl) in rabbits. The bile acid pool size remained constant (55 ± 17 mg vs. 61 ± 18 mg) in rats but doubled (254 ± 46 to 533 ± 53 mg) in rabbits. ASBT protein expression did not change in rats but rose 31% (P < 0.05) in rabbits. Fecal bile acid outputs that reflected bile acid synthesis increased 2- and 2.4-times (P < 0.05) in cholesterol-fed rats and rabbits, respectively. Cholesterol 7α-hydroxylase activity rose 33% (24 ± 2.4 vs. 18 ± 1.6 pmol/mg/min, P < 0.01) and mRNA levels increased 50% (P < 0.01) in rats but decreased 68% and 79%, respectively, in cholesterol-fed rabbits. Cholesterol 27-hydroxylase activity remained unchanged in rats but rose 62% (P < 0.05) in rabbits. Classic bile acid synthesis (cholesterol 7α-hydroxylase) was inhibited in rabbits because an enlarged bile acid pool developed from enhanced ileal bile acid transport. In contrast, in rats, cholesterol 7α-hydroxylase was stimulated but the bile acid pool did not enlarge because ASBT did not change. Therefore, although bile acid synthesis was increased via different pathways in rats and rabbits, enhanced ileal bile acid transport was critical for enlarging the bile acid pool size that exerted feedback regulation on cholesterol 7α-hydroxylase in rabbits. —Xu, G., B. L. Shneider, S. Shefer, L. B. Nguyen, A. K. Batta, G. S. Tint, M. Arrese, S. Thevananther, L. Ma, S. Stengelin, W. Kramer, D. Greenblatt, M. Pcolinsky, and G. Salen. Ileal bile acid transport regulates bile acid pool, synthesis, and plasma cholesterol levels differently in cholesterol-fed rats and rabbits.

In addition to several other enzymes, the short-chain alcohol dehydrogenase superfamily includes a group of peroxisomal multifunctional enzymes involved in fatty acid and cholesterol side-chain β-oxidation. Mammalian peroxisomal multifunctional enzyme type 2 (perMFE-2) is a 2-enoyl-CoA hydratase-2/(R)-3-hydroxyacyl-CoA dehydrogenase. As has been shown previously, perMFE-2 hydrates (24E)-3α,7α,12α-trihydroxy-5β-cholest-24-enoyl-CoA to (24R, 25R)-3α,7α,12α,24ξ-tetrahydroxy-5β-cholestanoyl-CoA, which has been characterized as a physiological intermediate in cholic acid synthesis. Out of four possible stereoisomers of 3α,7α,12α,24ξ-tetrahydroxy-5β-cholestanoyl-CoA, the mammalian perMFE-2 dehydrogenates only the (24R,25R)-isomer. The yeast peroxisomal multifunctional enzyme (MFE) was first described as 2-enoyl-CoA hydratase-2/(R)-3-hydroxyacyl-CoA dehydrogenase. To investigate the stereospecificity of yeast peroxisomal MFE, the two dehydrogenase domains of C. tropicalis MFE were expressed in E. coli as a 65 kDa recombinant protein. This protein catalyzes the dehydrogenation of straight-chain (R)-3-hydroxyacyl-CoAs, but it is devoid of (S)-3-hydroxyacyl-CoA dehydrogenase and 2-enoyl-CoA hydratase activities. The protein dehydrogenates (24R,25R)- and (24R,25S)-isomers of 3α,7α, 12α,24ξ-tetrahydroxy-5β-cholestanoyl-CoA. Interestingly, the protein also shows 17β-estradiol dehydrogenase activity. As a monofunctional (R)-specific 3-hydroxyacyl-CoA dehydrogenase is currently unavailable, this recombinant enzyme can be used to study the stereochemistry of bile acid synthesis. —Qin, Y-M., M. H. Poutanen, and D. K. Novikov. Substrate specificities of peroxisomal members of short-chain alcohol dehydrogenase superfamily: expression and characterization of dehydrogenase part of Candida tropicalis multifunctional enzyme.

To design a reliable 3D QSAR model of the intestinal Na+/bile acid cotransporter, we have used a training set of 17 inhibitors of the rabbit ileal Na+/bile acid cotransporter. The IC50 values of the training set of compounds covered a range of four orders of magnitude for inhibition of [3H]cholyltaurine uptake by CHO cells expressing the rabbit ileal Na+/bile acid cotransporter allowing the generation of a pharmacophore using the CATALYST algorithm. After thorough conformational analysis of each molecule, CATALYST generated a pharmacophore model characterized by five chemical features: one hydrogen bond donor, one hydrogen bond acceptor, and three hydrophobic features. The 3D pharmacophore was enantiospecific and correctly estimated the activities of the members of the training set. The predicted interactions of natural bile acids with the pharmacophore model of the ileal Na+/bile acid cotransporter explain exactly the experimentally found structure–activity relationships for the interaction of bile acids with the ileal Na+/bile acid cotransporter (Kramer et al. 1999. J. Lipid. Res. 40: 1604–1617). The natural bile acid analogues cholyltaurine, chenodeoxycholyltaurine, or deoxycholyltaurine were able to map four of the five features of the pharmacophore model: a) the five-membered ring D and the methyl group at position 18 map one hydrophobic site and the 21-methyl group of the side chain maps a second hydrophobic site; b) one of the α-oriented hydroxyl groups at position 7 or 12 fits the hydrogen bond donor feature; c) the negatively charged side chain acts as hydrogen bond acceptor; and d) the hydroxy group at position 3 does not specifically map any of the five binding features of the pharmacophore model. The 3-hydroxy group of natural bile acids is not essential for interactions with ileal or hepatic Na+/bile acid cotransporters. A modification of the 3-position of a natural bile acid molecule is therefore the preferred position for drug targeting strategies using bile acid transport pathways.—Baringhaus, K-H., H. Matter, S. Stengelin, and W. Kramer. Substrate specificity of the ileal and the hepatic Na+/bile acid cotransporters of the rabbit. II. A reliable 3D QSAR pharmacophore model for the ileal Na+/bile acid cotransporter.

The substrate specificity of the ileal and the hepatic Na+/bile acid cotransporters was determined using brush border membrane vesicles and CHO cell lines permanently expressing the Na+/bile acid cotransporters from rabbit ileum or rabbit liver. The hepatic transporter showed a remarkably broad specificity for interaction with cholephilic compounds in contrast to the ileal system. The anion transport inhibitor diisothiocyanostilbene disulfonate (DIDS) is a strong inhibitor of the hepatic Na+/bile acid cotransporter, but does not show any affinity to its ileal counterpart. Inhibition studies and uptake measurements with about 40 different bile acid analogues differing in the number, position, and stereochemistry of the hydroxyl groups at the steroid nucleus resulted in clear structure–activity relationships for the ileal and hepatic bile acid transporters. The affinity to the ileal and hepatic Na+/bile acid cotransport systems and the uptake rates by cell lines expressing those transporters as well as rabbit ileal brush border membrane vesicles is primarily determined by the substituents on the steroid nucleus. Two hydroxy groups at position 3, 7, or 12 are optimal whereas the presence of three hydroxy groups decreased affinity. Vicinal hydroxy groups at positions 6 and 7 or a shift of the 7-hydroxy group to the 6-position significantly decreased the affinity to the ileal transporter in contrast to the hepatic system. 6-Hydroxylated bile acid derivatives are preferred substrates of the hepatic Na+/bile acid cotransporter. Surprisingly, the 3α-hydroxy group being present in all natural bile acids is not essential for high affinity interaction with the ileal and the hepatic bile acid transporter. The 3α-hydroxy group seems to be necessary for optimal transport of a bile acid across the hepatocyte canalicular membrane. A modification of bile acids at the 3-position therefore conserves the bile acid character thus determining the 3-position of bile acids as the ideal position for drug targeting strategies using bile acid transport pathways.—Kramer, W., S. Stengelin, K-H. Baringhaus, A. Enhsen, H. Heuer, W. Becker, D. Corsiero, F. Girbig, R. Noll, and C. Weyland. Substrate specificity of the ileal and the hepatic Na+/bile acid cotransporters of the rabbit. I. Transport studies with membrane vesicles and cell lines expressing the cloned transporters. J. Lipid Res. 1999. 40: 1604–1617.

A simple method for the gas–liquid chromatographic quantitation of human fecal bile acids and sterols is described where bile acids are subjected to n-butyl ester derivatization, without prior isolation from the stool, followed by trimethylsilylation of the sterols and bile acids. Under these conditions, bile acid derivatives are well resolved from each other and from the trimethylsilyl ether derivatives of fecal sterols and no overlap occurs. The method was shown to be highly reproducible and recoveries were similar to those obtained with other methods used for fecal bile acid analysis. Application of the method for bile acid and sterol analysis in human stool is described.—Batta, A. K., G. Salen, K. R. Rapole, M. Batta, P. Batta, D. Alberts, and D. Earnest. Highly simplified method for gas–liquid chromatographic quantitation of bile acids and sterols in human stool. J. Lipid Res. 1999. 40: 1148–1154.

The final steps in bile acid biosynthesis take place in peroxisomes and involve oxidative cleavage of the side chain of C27-5β-cholestanoic acids leading to the formation of the primary bile acids cholic acid and chenodeoxycholic acid. The enoyl-CoA hydratase and β-hydroxy acyl-CoA dehydrogenase reactions involved in the chain shortening of C27-5β-cholestanoic acids are catalyzed by the recently identified peroxisomal d-bifunctional protein. Deficiencies of d-bifunctional protein lead, among others, to an accumulation of 3α,7α,12α,24-tetrahydroxy-5β-cholest-26-oic acid (varanic acid). The ability to resolve the four C24, C25 diastereomers of varanic acid has, so far, only been carried out on biliary bile acids using p-bromophenacyl derivatives. Here, we describe a sensitive gas chromatography–mass spectrometry (GC/MS) method that enables good separation of the four varanic acid diastereomers by use of 2R-butylester-trimethylsilylether derivatives. This method showed the specific accumulation of (24R,25R)-varanic acid in the serum of a patient with isolated deficiency of the d-3-hydroxy acyl-CoA dehydrogenase part of peroxisomal d-bifunctional protein, whereas this diastereomer was absent in a serum sample from a patient suffering from complete d-bifunctional protein deficiency. In samples from both patients an accumulation of (24S,25S)-varanic acid was observed, most likely due to the action of l-bifunctional protein on Δ24E-THCA-CoA. This GC/MS method is applicable to serum samples, obviating the use of bile fluid, and is a helpful tool in the subclassification of patients with peroxisomal d-bifunctional protein deficiency.—Vreken, P., A. van Rooij, S. Denis, E. G. van Grunsven, D. A. Cuebas, and R. J. A. Wanders. Sensitive analysis of serum 3α,7α,12α,24-tetrahydroxy-5β-cholestan-26-oic acid diastereomers using gas chromatography–mass spectrometry and its application in peroxisomal d-bifunctional protein deficiency. J. Lipid Res. 1998. 39: 2452–2458.

Fecal bile acid excretion is one of the two major routes by which cholesterol is eliminated from the body, fecal cholesterol being the other. During their enterohepatic circulation, bile acids are secreted into the duodenum, pass down the jejunum and into the ileum where more than 95% is reabsorbed by the gut. Bile acids that escape reabsorption in the small intestine are metabolized by microorganisms in the large intestine. The major routes of metabolism are reported to be deconjugation, dehydroxylation, especially at the 7 alpha-hydroxy position, and dehydrogenation of the hydroxyl moieties. There are also some reports that saponifiable metabolites containing mostly deoxycholic acid form a major component of the bile acids found in human feces. We have identified a novel metabolite of cholic acid, 3 alpha-hydroxy polydeoxycholate, in both human and hamster feces that is the major constituent of these saponifiable metabolites. Furthermore, we have shown in hamsters that the animals that excreted more bile acid were excreting the additional bile acid as polydeoxycholate. As expected, there was a negative correlation between bile acid excretion in the feces and plasma cholesterol concentrations in these animals. We speculate that polydeoxycholate is formed in the lower gut of both humans and hamsters and that, by its formation, bile acid will be sequestered in an insoluble form, thus inhibiting its reabsorption by the gut. This process may help to reduce plasma cholesterol concentrations and coronary heart disease in humans.