Expression Of Bile Acid Transporters Research Articles

Intestinal CYP3A4 protects against lithocholic acid-induced hepatotoxicity in intestine-specific VDR-deficient mice

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 (Vdr(ΔIEpC)) 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 Vdr(ΔIEpC) mice to produce the Vdr(ΔIEpC)/3A4 line. LCA was administered to control, transgenic-CYP3A4, Vdr(ΔIEpC), and Vdr(ΔIEpC)/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 Vdr(ΔIEpC) 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 Vdr(ΔIEpC)/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.

Dietary Supplementation of Chardonnay Grape Seed Flour Reduces Plasma Cholesterol Concentration, Hepatic Steatosis, and Abdominal Fat Content in High-Fat Diet-Induced Obese Hamsters

The mechanisms for the hypocholesterolemic and antiobesity effects of grape seed flours derived from white and red winemaking processing were investigated using male Golden Syrian hamsters fed high-fat (HF) diets supplemented with 10% partially defatted grape seed flours from Chardonnay (ChrSd), Cabernet Sauvignon (CabSd), or Syrah (SyrSd) pomace as compared to a HF control diet for 3 weeks. Hamsters fed the ChrSd diet had significantly lowered plasma total-, VLDL-, and LDL-cholesterol concentrations compared to the CabSd, SyrSd, and control diets. The improved plasma cholesterol after ChrSd was correlated with the up-regulation of hepatic genes related to cholesterol (CYP51) and bile acid (CYP7A1) synthesis as well as LDL-cholesterol uptake (LDLR). A reduction of hepatic lipid content was associated with altered expression of the genes related to lipid metabolism. However, fecal total lipid content was not changed. Expression of ileal apical sodium bile acid transporter (ASBT) was not affected by ChrSd, indicating unchanged ileal bile acid reabsorption. The antiobesity effect of the ChrSd diet appears to be related to expression of adipogenesis- and inflammation-related genes in adipose tissue. These findings suggest that flavonoid-rich Chardonnay grape seed flour induced cholesterol-lowering, antiobesity, and anti-inflammatory health benefits and attenuation of hepatic steatosis via regulation of gene expression related to cholesterol, bile acid, and lipid metabolism in liver and adipose tissue.

Effect of chronic renal failure on the hepatic, intestinal, and renal expression of bile acid transporters

Although the kidney is believed to play a minor role in bile acid (BA) excretion, chronic renal failure (CRF) has been reported to be associated with increased serum bile acid levels and alterations in BA homeostasis. The mechanisms for elevated BA levels are poorly understood in both clinical and experimental studies. This study was designed to examine the effects of naturally progressing CRF of longer duration on the hepatic and renal mRNA and protein levels of the BA-synthesizing enzyme Cyp7a1 and the BA transporters Ntcp, Bsep, Mrp3, Ost-α, and Ost-β. Sprague-Dawley rats were randomized to the CRF group (⅚ nephrectomy) or to the sham-operated control group and were analyzed 8 wk after surgery. Results obtained in the CRF rats were compared with those obtained in rats that had undergone uninephrectomy (UNX). The CRF group exhibited significantly increased plasma cholesterol and BA concentrations. Hepatic Cyp7a1 mRNA and protein levels were almost identical in the two groups. Hepatic Mrp3, Ost-α, and Ost-β expression was increased, suggesting increased basolateral efflux of bile acids into the blood. However, no such changes in BA transporter expression were observed in the remnant kidney. In UNX rats, similar changes in plasma BA levels and in the expression of BA transporters were found. We hypothesize that the increase in plasma BA is an early event in the progression of CRF and is caused by increased efflux across the basolateral hepatocyte membrane.

Muricholic bile acids are potent regulators of bile acid synthesis via a positive feedback mechanism

Bile acid (BA) synthesis is regulated by negative feedback end-product inhibition, initiated by farnesoid X receptors (FXRs) in liver and gut. Studies on cholic acid (CA)-free Cyp8b1(-/-) mice have concluded that CA is a potent suppressor of BA synthesis. Cyp8b1(-/-) mice have increased BA synthesis and an enlarged BA pool, a phenotype shared with bile-duct-ligated, antibiotics-administered and with germ-free mice. Studies on such mice have concluded BA synthesis is induced due to reduced hormonal signalling by fibroblast growth factor (FGF)15 from intestine to liver. A mutual finding in these models is that potent FXR-agonistic BAs are reduced. We hypothesized that the absence of the potent FXR agonist deoxycholic acid (DCA) may be important for the induction of BA synthesis in these situations. Two of these models were investigated, antibiotic treatment and Cyp8b1(-/-) mice and their combination. Secondary BA formation was inhibited by ampicillin (AMP) given to wild-type and Cyp8b1(-/-) mice. We then administered CA, chenodeoxycholic acid (CDCA) or DCA to AMP-treated Cyp8b1(-/-) mice. Our data show that the phenotype of AMP-treated wild-type mice resembles that of Cyp8b1(-/-) mice with fourfold induced Cyp7a1 expression, increased intestinal apical sodium-dependent BA transporter expression and increased hepatic BA levels. We also show that reductions in the FXR-agonistic BAs CDCA, CA, DCA or lithocholic acid cannot explain this phenotype; instead, it is likely due to increases in levels of α- and β-muricholic BAs and ursodeoxycholic acid, three FXR-antagonistic BAs. Our findings reveal a potent positive feedback mechanism for regulation of BA synthesis in mice that appears to be sufficient without endocrine effects of FGF15 on Cyp7a1. This mechanism will be fundamental in understanding BA metabolism in both mice and humans.

Su1684 Adenosine Regulates Cholangiocyte IL-6 Secretion via the A2B Receptor

Background. Although the kidney is believed to play a minor role in bile acid (BA) excretion, chronic renal failure (CRF) has been reported to be associated with increased serum bile acid levels and alterations in BA homeostasis. According to a recent report, rats studied 3 weeks after 5/6 nephrectomy and fed a high-protein diet exhibited increased activities of hepatic HMG-CoA reductase (HMG-CoAR) and cholesterol 7 alpha-hydroxylase (Cyp7a1), despite normal corresponding mRNA levels. Design and Methods. This study was designed to examine the effects of naturally progressing CRF of longer duration on the hepatic and renal mRNA and protein levels of the bile acid synthesizing enzyme Cyp7a1 and the bile acid transporters, Ntcp, Bsep, Mrp3, Ost-alpha and OST-beta. Sprague-Dawley rats were randomized to the CRF group (5/6 nephrectomy) or to the sham-operated, placebo-treated normal control group. They were allowed free access to regular rat chow and were analyzed 8 weeks after surgery. Results obtained in CRF rats were compared to those obtained in rats that had undergone uninephrectomy (UNX). Results. The CRF group exhibited significantly increased plasma cholesterol and bile acid concentrations. Hepatic Cyp7a1 mRNA, and protein levels were almost identical in the two groups. Hepatic Mrp3, Ostα and Ost-β expression was increased at both the mRNA and protein levels, suggesting increased basolateral efflux of bile acids into basolateral blood. However, no such changes in bile acid transporter expression were observed in kidney. In UNX rats, similar changes in plasma bile acid levels and in the expression of bile acid transporters were found. We hypothesize, that the increase in plasma bile acids is an early event in the progression of CRF and is caused by increased efflux across the basolateral hepatocyte membrane.

Su1681 Hypoxia Induces Dysregulation of Lipid Metabolism in HEPG2 Cells via Activation of HIF-2a

Background. Although the kidney is believed to play a minor role in bile acid (BA) excretion, chronic renal failure (CRF) has been reported to be associated with increased serum bile acid levels and alterations in BA homeostasis. According to a recent report, rats studied 3 weeks after 5/6 nephrectomy and fed a high-protein diet exhibited increased activities of hepatic HMG-CoA reductase (HMG-CoAR) and cholesterol 7 alpha-hydroxylase (Cyp7a1), despite normal corresponding mRNA levels. Design and Methods. This study was designed to examine the effects of naturally progressing CRF of longer duration on the hepatic and renal mRNA and protein levels of the bile acid synthesizing enzyme Cyp7a1 and the bile acid transporters, Ntcp, Bsep, Mrp3, Ost-alpha and OST-beta. Sprague-Dawley rats were randomized to the CRF group (5/6 nephrectomy) or to the sham-operated, placebo-treated normal control group. They were allowed free access to regular rat chow and were analyzed 8 weeks after surgery. Results obtained in CRF rats were compared to those obtained in rats that had undergone uninephrectomy (UNX). Results. The CRF group exhibited significantly increased plasma cholesterol and bile acid concentrations. Hepatic Cyp7a1 mRNA, and protein levels were almost identical in the two groups. Hepatic Mrp3, Ostα and Ost-β expression was increased at both the mRNA and protein levels, suggesting increased basolateral efflux of bile acids into basolateral blood. However, no such changes in bile acid transporter expression were observed in kidney. In UNX rats, similar changes in plasma bile acid levels and in the expression of bile acid transporters were found. We hypothesize, that the increase in plasma bile acids is an early event in the progression of CRF and is caused by increased efflux across the basolateral hepatocyte membrane.

Mo1803 Ileal Bile Acid Transporter and FGF19 Transcript Expression Related to SEHCAT Retention in Patients With Chronic Diarrhea

Intestinal NPC1L1 transporter is essential for cholesterol absorption and the increase in its expression is associated with high risk for atherosclerosis. NPC1L1 is differentially expressed along the gastrointestinal tract with high levels of expression in the small intestine as compared to colon. Also, NPC1L1 is expressed in villus but not in crypt cells. Recent studies have shown that epigenetic regulation may play a role in controlling the expression of intestinal transport proteins. Whether epigenetic mechanisms such as DNA methylation is involved in determining NPC1L1 expression along the GI tract is not known. Current studies were, therefore, undertaken to investigate the effect of DNA methylation on NPC1L1 gene expression utilizing both in vitro and in vivo models. Intestinal HUTU-80 cells have very low levels of NPC1L1 expression and, thus, were used to investigate the effects of changes in DNA methylation on its expression. HUTU-80 cells were incubated with 10 μM of 5azacytidine, inhibitor of DNA methylation, and NPC1L1 mRNA expression was assessed by real time PCR. As compared to control cells, treatment with 5-azacytidine significantly increased the NPC1L1 expression in a time dependent manner with a maximum induction occurring after 72 h (∼24 fold, P,0.005). We also examined the methylation status of the promoter region of the NPC1L1 gene utilizing methylation specific PCR (MSP) on bisulfite converted genomic DNA. MSP analysis demonstrated that NPC11L1 is highly methylated in HUTU-80 cells consistent with the low level of NPC1L1 mRNA expression. Treatment with 5-azacytidine (10 μM, 72 h) decreased the DNA methylation of NPC1L1 promoter region by 53.13 ± 11.67 % as compared to control cells concomitant with the increase in NPC1L1 expression. DNA methylation is mediated by the activity of DNAmethyltransferases: DNMT1, 3A and 3B. Silencing the expression of both DNMT1 and 3B simultaneously by specific siRNA in HUTU-80 cells significantly increased NPC1L1 mRNA expression by ∼2 fold (P,0.05) as compared to control. Similar to the observed changes in the cell culture model, intraperitoneal injections of 5-azacytidine (10 mg/kg body weight, 3 injections/week for three weeks) to C57BL/6 mice (8 weeks of age) resulted in a significant increase in NPC1L1 expression in the colon ( ∼32 fold, P,0.005) as compared to control mice. In conclusion, our data demonstrate that DNA methylation in the promoter region of NPC1L1 gene appears to be a major mechanism underlying differential expression of NPC1L1 along the GI tract. We speculate that harnessing alterations in DNA methylation by dietary manipulations may prove to be of potential benefit in decreasing cholesterol absorption and preventing atherosclerosis (Supported by NIDDK & VA).

Expression and role of the genes involved in the transport of bile acids in the liver and kidneys in mice

Bile acids are synthesized in the liver from cholesterol. This study investigated the impact and expression of different carriers of bile acid in the liver and kidneys. Eight-week-old male mice were used, which were fed for 15 days and divided into two groups: 15 mice fed with standard diet (control group) and another 15 mice fed with a rich diet of 5% cholesterol (second group). Bile acid dosage was based on their oxidation by 7α hydroxyl-steroid dehydrogenize. The mRNA expression was quantitatively analyzed by the real time of polymerase chain reaction (RT-PCR), and the expression of the renal carrier bile acid protein was analyzed by Western blot. The expression of bile salt export pump involved in the uptake of bile acids in the basolateral membrane of hepatocytes revealed no differences between the two groups of mice. However, the expression of multidrug resistance-associated protein 2 was reduced in mice of the second group. Moreover, the expressions of organic anion transporting polypeptide 4, organic anion transporting polypeptide 1, and sodium taurocholate co-transporting polypeptide (Ntcp) involved in the uptake of bile acids in the apical pole of hepatocytes are suppressed in mice of the second group. The expression of multidrug resistance-associated protein 3 involved in the secretion of bile acids in the apical membrane of hepatocytes revealed no significant differences between the two groups. In mice of the second group, blood concentration of bile acids on the last day was increased. In those mice, the expression of intestinal bile acid transporter was reduced in the kidneys compared with the control mice.

Antibacterial drug treatment causes enhanced levels of hepatic bile acid through alteration of ileal bile acid transporter expression

Antibacterial drug treatment causes enhanced levels of hepatic bile acid through alteration of ileal bile acid transporter expression

Reply to Bile acids in Non-Alcoholic Steato-Hepatitis: inserting nuclear receptors into the circle.

We thank Drs. Gabbi and Gustafsson for their interest in our study on serum metabolomic analysis of nonalcoholic steatohepatitis (NASH) in mice (1). We measured the hepatic contents of taurocholate and tauro-β-muricholate using ultra-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry. However, the hepatic levels of these bile acids were not decreased in mice treated for two weeks with a methionine- and choline-deficient (MCD) diet (Fig. 1). Although changes in expression of the canalicular bile acid transporters were not detected, hepatic bile excretion might be decreased to some degree. When hepatic bile acid levels are altered, the expression of small heterodimer partner (Shp) may be changed accordingly to maintain hepatic bile acid levels. Indeed, the levels of Shp mRNA were decreased by MCD treatment, even in the absence of a significant decrease in hepatic bile acid content (Fig. 2). These findings suggest an abnormal response of transcription factors and their target genes associated with bile acid homeostasis, and thus we propose a disruption of bile acid homeostasis during the course of NASH where pro-inflammatory cytokines and cellular stress may disrupt bile acid homeostasis, causing a vicious cycle. Thus, we are generally in agreement with Drs. Gabbi and Gustafsson's view that clarification of the dynamic changes in bile acid homeostasis and the related transcriptional factors during NASH development might lead to new bile-modulating therapies for NASH.

Intestinal transporters for endogenic and pharmaceutical organic anions: the challenges of deriving in-vitro kinetic parameters for the prediction of clinically relevant drug–drug interactions

This review provides an overview of intestinal human transporters for organic anions and stresses the need for standardization of the various in-vitro methods presently employed in drug-drug interaction (DDI) investigations. Current knowledge on the intestinal expression of the apical sodium-dependent bile acid transporter (ASBT), the breast cancer resistance protein (BCRP), the monocarboxylate transporters (MCT) 1, MCT3-5, the multidrug resistance associated proteins (MRP) 1-6, the organic anion transporting polypetides (OATP) 2B1, 1A2, 3A1 and 4A1, and the organic solute transporter α/β (OSTα/β) has been covered along with an overview of their substrates and inhibitors. Furthermore, the many challenges in predicting clinically relevant DDIs from in-vitro studies have been discussed with focus on intestinal transporters and the various methods for deducting in-vitro parameters for transporters (K(m) /K(i) /IC50, efflux ratio). The applicability of using a cut-off value (estimated based on the intestinal drug concentration divided by the K(i) or IC50) has also been considered. A re-evaluation of the current approaches for the prediction of DDIs is necessary when considering the involvement of other transporters than P-glycoprotein. Moreover, the interplay between various processes that a drug is subject to in-vivo such as translocation by several transporters and dissolution should be considered.

Dietary fish oil regulates gene expression of cholesterol and bile acid transporters in mice

Fish oil rich in n-3 polyunsaturated fatty acids is known to affect hepatic lipid metabolism. Several studies have demonstrated that fish oil may affect the bile acid metabolism as well as lipid metabolism, whereas only scarce data are available. The aim of this study was to investigate the effect of fish oil on the gene expression of the transporters and enzymes related to bile acid as well as lipid metabolism in the liver and small intestine. Seven-week old male C57BL/6 mice were fed diets enriched in 10% soybean oil or 10% fish oil for 4 weeks. After 4 weeks, blood, liver and small intestine were obtained. Hepatic mRNA expression of lipids (Abcg5/8, multidrug resistance gene product 2) and bile acids transporters (bile salt export pump, multidrug resistance associated protein 2 and 3, organic solute transporter α) was induced in fish oil-fed mice. Hepatic Cyp8b1, Cyp27a1 and bile acid CoA : amino acid N-acyltransferase were increased in fish oil-fed mice compared with soybean-oil fed mice. Besides, intestinal cholesterol (Abcg5/8) and bile acid transporters (multidrug resistance associated protein 2 and organic solute transporter α) were induced in fish oil-fed mice. Fish oil induced the expression of cholesterol and bile acid transporters not only in liver but in intestine. The upregulation of Abcg5/g8 by fish oil is caused by an increase in cellular 27-HOC through Cyp27a1 induction. The hepatic induction of bile acid synthesis through Cyp27a1 may upregulate expression of bile acid transporters in both organs.

Nuclear factor-E2-related factor 2 is a major determinant of bile acid homeostasis in the liver and intestine

The transcription factor nuclear factor-E2-related factor 2 (Nrf2) is a key regulator for induction of hepatic detoxification and antioxidant mechanisms, as well as for certain hepatobiliary transporters. To examine the role of Nrf2 in bile acid homeostasis and cholestasis, we assessed the determinants of bile secretion and bile acid synthesis and transport before and after bile duct ligation (BDL) in Nrf2(-/-) mice. Our findings indicate reduced rates of biliary bile acid and GSH excretion, higher levels of intrahepatic bile acids, and decreased expression of regulators of bile acid synthesis, Cyp7a1 and Cyp8b1, in Nrf2(-/-) compared with wild-type control mice. The mRNA expression of the bile acid transporters bile salt export pump (Bsep) and organic solute transporter (Ostα) were increased in the face of impaired expression of the multidrug resistance-associated proteins Mrp3 and Mrp4. Deletion of Nrf2 also decreased ileal apical sodium-dependent bile acid transporter (Asbt) expression, leading to reduced bile acid reabsorption and increased loss of bile acid in feces. Finally, when cholestasis is induced by BDL, liver injury was not different from that in wild-type BDL mice. These Nrf2(-/-) mice also had increased pregnane X receptor (Pxr) and Cyp3a11 mRNA expression in association with enhanced hepatic bile acid hydroxylation. In conclusion, this study finds that Nrf2 plays a major role in the regulation of bile acid homeostasis in the liver and intestine. Deletion of Nrf2 results in a cholestatic phenotype but does not augment liver injury following BDL.

Bile acids via FXR initiate the expression of major transporters involved in the enterohepatic circulation of bile acids in newborn mice

The enterohepatic circulation (EHC) of bile acids (BAs) plays a pivotal role in facilitating lipid absorption. Therefore, initiation of the EHC in newborns is of crucial importance for lipid absorption from milk. The purpose of this study was to determine at what age BA transporters in liver are expressed, and the mechanism for their initiation. Serum and liver samples were collected from C57BL/6 mice at 2 days before birth and various postnatal ages. Messenger RNA assays revealed a dramatic increase at birth in the expression of the BA transporters (Ntcp, Bsep, Mrp4, Ostβ), as well as the phospholipid floppase Mdr2 in mouse liver, with the highest expression at 1 day of age. The mRNA expression of the ileal BA transporters (Ostα and Ostβ) also markedly increased at birth. Meanwhile, taurine-conjugated cholic acid markedly increased in both serum and liver of newborns, correlated with upregulation of the classic pathway of BA biosynthesis in newborn liver. The mRNA levels of the major BA sensors, FXR and PXR, were increased at 1 day of age, and their prototypical target genes were upregulated in liver. The mRNA expression of transporters involved in the EHC of BAs was similar in wild-type and PXR-null mice. In contrast, in FXR-null mice, the "day 1 surge" pattern of Ntcp, Bsep, Ostβ, and Mdr2 was blocked in newborn mouse liver, and the induction of Ostα and Ostβ was also abolished in ileums of FXR-null mice. In conclusion, at birth, BAs from the classic pathway of synthesis trigger the induction of transporters involved in EHC of BAs in mice, through activation of the nuclear receptor FXR.

Intestinal inflammation induces alterations in enterohepatic Fgf15 signalling, changes in hepatic bile acid metabolism and a “toxic bile” composition in DSS-treated mice

Aims: Primary sclerosing cholangitis is a chronic cholestatic liver disease affecting intra- and extrahepatic bile ducts frequently associated with inflammatory bowel disease. The pathogenetic pathway leading from intestinal inflammation to cholestatic liver disease is still poorly understood. Aims: To study alterations in enterohepatic Fgf15 signalling and associated changes in hepatic gene expression and bile composition in murine colitis. Methods: Fgf15 pathway target genes in gut and liver of DSS-treated mice (7d, 2% DSS in drinking water) and control mice were quantified by RT-PCR. Changes of bile acid transporter expression were quantified by RT-PCR and confirmed by western blotting. Fgf15 levels in serum were analyzed by ELISA. Biliary bile acid composition was differentiated by GC/MS analysis. Results: DSS-treated mice showed induction of Fxr expression in ileo-cecal region and concomitant higher concentration of Fgf15 in serum (180% vs. cntrl; p<0.05). In the liver, inhibition of bile acids synthesis by down-regulation of Cyp7a1, regulated by Fgf15 could be observed (40% vs. cntrl; p<0.05). In addition, expression of bile acid transporters such as Bsep, Ntcp, Mrp2, Mrp3 and Mrp4 were significantly down-regulated. Most importantly, bile acid composition in bile was significantly different in treated mice with lower levels of hydrophilic bile acids including α- and ß-muricholic acid and taurodeoxycholic acid (20%, 20% and 50% vs. cntrl; p<0.05). Conclusions: Our results show for the first time a functionally relevant alteration in gut-liver interaction by activated Fgf15 signalling through Fxr activation in ileo-cecal region of DSS-treated mice. As functional consequence of hepatic alterations in bile acid metabolism less cytotoxic hydrophilic bile acids are reduced and may contribute to a more toxic bilary environment by hydrophobic bile acids. The study supports the “toxic bile concept” in the pathogenesis of IBD-associated biliary disease.

Mouse ghrelin‐ O ‐acyltransferase (GOAT) plays a critical role in bile acid reabsorption

Ghrelin is a unique peptide gut hormone that requires post-translational modification to stimulate both feeding and growth hormone release. Ghrelin O-acyltransferase (GOAT) was identified as a specific acyl-transferase for ghrelin, and recent genetic deletion studies of the Goat gene (Goat(-/-)) uncovered the role of ghrelin in the regulation of glucose homeostasis. To further understand the physiological functions of the GOAT/ghrelin system, we have conducted a metabolomic and microarray profile of Goat-null mice, as well as determined Goat expression in different tissues using the lacZ reporter gene. Serum metabolite profile analysis revealed that Goat(-/-) mice exhibited increased secondary bile acids >2.5-fold. This was attributed to increased mRNA and protein expression of the ileal sodium-dependent bile acid transporter (ISBT) in the intestinal and biliary tract. Increased expression of additional solute carrier proteins, including Slc5a12 (>10-fold) were also detected in the small intestine and bile duct. Goat staining was consistently observed in the pituitary glands, stomach, and intestines, and to a lesser extent in the gallbladder and pancreatic duct. This is the first report that the GOAT/ghrelin system regulates bile acid metabolism, and these findings suggest a novel function of GOAT in the regulation of intestinal bile acid reabsorption..

Cytoprotective properties of rifampicin are related to the regulation of detoxification system and bile acid transporter expression during hepatocellular injury induced by hydrophobic bile acids

Rifampicin has been used for the treatment of patients with jaundice and pruritus. This study evaluated the effect of rifampicin on the expression of different detoxification systems and bile acid transporters during in-vivo and in-vitro experimental models of cholestasis. Rifampicin was administered to glycochenodeoxycholic acid (GCDCA)-treated human hepatocytes and bile duct-obstructed rats. Different parameters related to cell death, and the expression of phase I and II drug metabolizing enzymes (DME) and bile acid transporters were determined. The induction of hepatocellular injury induced by cholestasis was associated with a reduction in cytochrome P4503A4 (CYP3A4), CYP7A1, and UDP-glucuronosyltransferase 2B4 (UGT2B4) expression, as well as an increase in import (Na(+)-taurocholate co-transporting polypeptide, NTCP) system expression. The beneficial properties of rifampicin were associated with an increase in DME and export bile acid systems (multidrug resistance-associated protein 4, MRP4, and bile acid export pump to bile duct, BSEP) expression, as well as a reduction in NTCP expression. The beneficial effect of rifampicin in cholestasis is associated with an increase in DME expression involved in toxic, bile acid and cholesterol metabolism, as well as a reduction in the bile acid importing system in hepatocytes.

ABC Transporters, Bile Acids, and Inflammatory Stress in Liver Cancer

The biliary secretion of bile acids is critical for multiple liver functions including digesting fatty nutrients and driving bile flow. When this process is impaired, the accumulating bile acids cause inflammatory liver injury. Multiple ABC transporters in the liver are key players to safeguard the hepatocyte and avoid toxicity due to bile acid over-accumulation. BSEP provides for efficient secretion of bile acids across the canalicular membrane against a steep concentration gradient. MDR3/Mdr2 and ABCG5/G8 secrete phosphatidylcholine and cholesterol, respectively, in coordination with BSEP-mediated bile acid secretion to mask the detergent/toxic effects of bile acids in the bile ductular space. Several lines of evidence indicate that when these critical steps are compromised, bile acid toxicity in vivo leads to inflammatory liver injury and liver cancer. In bsep-/- mice, liver cancer is rare. These mice display greatly increased expression of alternative bile acid transporters, such as Mdr1a/1b, Mrp3 and Mrp4. We believe these alternative transport systems provide an additional safeguard to avoid bile acid overload in liver. Such backup systems appear to be under-utilized in humans, as defects in BSEP and MDR3 lead to severe, often fatal childhood diseases. It is possible, therefore, that targeting ABC transporters and modulating the toxicity of the bile acid pool could be vital interventions to alleviate chronic inflammation and reduce the incidence of liver cancer in high-risk populations. The combination of an alternative ABC transporter with a novel substrate may prove an effective chemo-preventive or therapeutic strategy.

Overexpression of cholesterol 7α-hydroxylase promotes hepatic bile acid synthesis and secretion and maintains cholesterol homeostasis

We reported previously that mice overexpressing cytochrome P450 7a1 (Cyp7a1; Cyp7a1-tg mice) are protected against high fat diet-induced hypercholesterolemia, obesity, and insulin resistance. Here, we investigated the underlying mechanism of bile acid signaling in maintaining cholesterol homeostasis in Cyp7a1-tg mice. Cyp7a1-tg mice had two-fold higher Cyp7a1 activity and bile acid pool than did wild-type mice. Gallbladder bile acid composition changed from predominantly cholic acid (57%) in wild-type to chenodeoxycholic acid (54%) in Cyp7a1-tg mice. Cyp7a1-tg mice had higher biliary and fecal cholesterol and bile acid secretion rates than did wild-type mice. Surprisingly, hepatic de novo cholesterol synthesis was markedly induced in Cyp7a1-tg mice but intestine fractional cholesterol absorption in Cyp7a1-tg mice remained the same as wild-type mice despite the presence of increased intestine bile acids. Interestingly, hepatic but not intestinal expression of several cholesterol (adenosine triphosphate-binding cassette G5/G8 [ABCG5/G8], scavenger receptor class B, member 1) and bile acid (ABCB11) transporters were significantly induced in Cyp7a1-tg mice. Treatment of mouse or human hepatocytes with a farnesoid X receptor (FXR) agonist GW4064 or bile acids induced hepatic Abcg5/g8 expression. A functional FXR binding site was identified in the Abcg5 gene promoter. Study of tissue-specific Fxr knockout mice demonstrated that loss of the Fxr gene in the liver attenuated bile acid induction of hepatic Abcg5/g8 and gallbladder cholesterol content, suggesting a role of FXR in the regulation of cholesterol transport. This study revealed a new mechanism by which increased Cyp7a1 activity expands the hydrophobic bile acid pool, stimulating hepatic cholesterol synthesis and biliary cholesterol secretion without increasing intestinal cholesterol absorption. This study demonstrated that Cyp7a1 plays a critical role in maintaining cholesterol homeostasis and underscores the importance of bile acid signaling in regulating overall cholesterol homeostasis.

Nitric oxide mimics transcriptional and post-translational regulation during α-Tocopherol cytoprotection against glycochenodeoxycholate-induced cell death in hepatocytes

Reactive oxygen species (ROS) and nitric oxide (NO) exert a relevant role during bile acid-induced hepatotoxicity. Whether α-Tocopherol regulates oxidative and nitrosative stress, bile acid transporter expression and their NO-dependent post-translational modifications, and cell death were assessed in vitro and in vivo. α-Tocopherol and/or NO donors (DETA-NONOate or CSNO, and V-PYRRO/NO) were administered to glycochenodeoxycholic acid (GCDCA)-treated cultured human hepatocytes or to bile duct obstructed rats. Cell injury, superoxide anion (O⁻₂) production, as well as inducible nitric oxide synthase (NOS-2), cytochrome P4507A1 (CYP7A1), heme oxygenase-1, (HO-1) and bile acid transporter expression were determined. Cysteine S-nitrosylation and tyrosine nitration of Na(+)-taurocholate co-transporting polypeptide (NTCP), as well as taurocholic acid (TC) uptake were also evaluated. GCDCA-induced cell death was associated with increased (O⁻₂) production, NTCP and HO-1 expression, and with a reduction of CYP7A1 and NOS-2 expression. α-Tocopherol reduced cell death, (O⁻₂) production, CYP7A1, NTCP, and HO-1 expression, as well as increased NOS-2 expression and NO production in GCDCA-treated hepatocytes. α-Tocopherol and NO donors increased NTCP cysteine S-nitrosylation and tyrosine nitration, and reduced TC uptake in hepatocytes. α-Tocopherol and V-PYRRO/NO reduced liver injury and NTCP expression in obstructed rats. The regulation of CYP7A1, NTCP, and HO-1 expression may be relevant for the cytoprotective properties of α-Tocopherol and NO against mitochondrial dysfunction, oxidative stress and cell death in GCDCA-treated hepatocytes. The regulation of NO-dependent post-translational modifications of NTCP by α-Tocopherol and NO donors reduces the uptake of toxic bile acids by hepatocytes.