Intestinal Bile Acid-binding Protein Research Articles

The hypocholesterolemic effect of capsaicinoids in ovariectomized rats fed with a cholesterol-free diet was mediated by inhibition of hepatic cholesterol synthesis

Previous studies showed that capsaicinoid supplementation favorably modifies the plasma lipoprotein profile. The present study investigated the effect of capsaicinoids on plasma lipids and gene expressions of key receptors and enzymes involved in cholesterol metabolism in ovariectomized (OVX) rats. OVX rats were fed with a cholesterol-free diet and orally administered 0 mg kg(-1) bw (OVX-CON), 5 mg kg(-1) bw (OVX-LD), 10 mg kg(-1) bw (OVX-MD), and 15 mg kg(-1) bw (OVX-HD) capsaicinoids daily for 28 days. As the capsaicinoids dose increased, body weight gain and concentrations of plasma triglyceride, total cholesterol, and low-density lipoprotein cholesterol, as well as total lipid accumulation were significantly decreased. In addition, the mRNA levels of hepatic 3-hydroxyl-3-methylglutaryl CoA reductase and cholesterol-7α-hydroxylase were down-regulated, whereas those of transient receptor potential vanilloid type-1, ileal apical sodium-dependent bile acid transporter, and intestinal bile acid binding protein were up-regulated. The excretion of small intestinal bile acid contents and fecal bile acid also decreased. These results suggest that capsaicinoids can prevent ovarian hormone deficiency-induced hypercholesterolemia by inhibiting the hepatic cholesterol synthesis.

Bacterial-dependant up-regulation of intestinal bile acid binding protein and transport is FXR-mediated following ileo-cecal resection

Bacterial-dependant up-regulation of intestinal bile acid binding protein and transport is FXR-mediated following ileo-cecal resection

Bacterial-dependent up-regulation of intestinal bile acid binding protein and transport is FXR-mediated following ileo-cecal resection

Bile acid (BA) reclamation following ileo-cecal resection (ICR) may prevent colonic mucosa from chronic injury. In this study, we hypothesized that in a murine model of ICR the remnant colon would upregulate the cellular machinery necessary for BA reclamation and would do so in an FXR- and bacteria-dependent manner. Conventional (WT), conventional FXR knockout (FXR null) and germ-free (GF) mice were randomized to undergo either ICR or sham operation. The ascending colon was harvested for histology and immunohistochemistry and changes in bile acid homeostatic gene expression determined by real-time polymerase chain reaction (RT-PCR) 7 days following surgery. Following ICR WT mice showed significant increases in the expression of genes regulating bile acid transport including IBABP, Asbt, Ost beta and FGF 15. Increased expression of IBABP and Asbt was confirmed by immunohistochemistry. Induction of bile acid transport genes was absent or attenuated in FXR null and GF mice. Bacterial dependent up regulation of IBABP is FXR mediated in the colon following ICR. Mice lacking microbiota (GF) or FXR are unable to increase the expression of IBABP or FGF 15.

Bacterial-dependent up-regulation of intestinal bile acid binding protein and transport is FXR-mediated following ileo-cecal resection

Bacterial-dependent up-regulation of intestinal bile acid binding protein and transport is FXR-mediated following ileo-cecal resection

The Cholesterol-Raising Factor from Coffee Beans, Cafestol, as an Agonist Ligand for the Farnesoid and Pregnane X Receptors

Cafestol, a diterpene present in unfiltered coffee brews such as Scandinavian boiled, Turkish, and cafetière coffee, is the most potent cholesterol-elevating compound known in the human diet. Several genes involved in cholesterol homeostasis have previously been shown to be targets of cafestol, including cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid biosynthesis. We have examined the mechanism by which cafestol elevates serum lipid levels. Changes in several lipid parameters were observed in cafestol-treated APOE3Leiden mice, including a significant increase in serum triglyceride levels. Microarray analysis of these mice identified alterations in hepatic expression of genes involved in lipid metabolism and detoxification, many of which are regulated by the nuclear hormone receptors farnesoid X receptor (FXR) and pregnane X receptor (PXR). Further studies demonstrate that cafestol is an agonist ligand for FXR and PXR, and that cafestol down-regulates expression of the bile acid homeostatic genes CYP7A1, sterol 12alpha-hydroxylase, and Na(+)-taurocholate cotransporting polypeptide in the liver of wild-type but not FXR null mice. Cafestol did not affect genes known to be up-regulated by FXR in the liver of wild-type mice, but did increase expression of the positive FXR-target genes intestinal bile acid-binding protein and fibroblast growth factor 15 (FGF15) in the intestine. Because FGF15 has recently been shown to function in an enterohepatic regulatory pathway to repress liver expression of bile acid homeostatic genes, its direct induction in the gut may account for indirect effects of cafestol on liver gene expression. PXR-dependent gene regulation of cytochrome P450 3A11 and other targets by cafestol was also only seen in the intestine. Using a double FXR/PXR knockout mouse model, we found that both receptors contribute to the cafestol-dependent induction of intestinal FGF15 gene expression. In conclusion, cafestol acts as an agonist ligand for both FXR and PXR, and this may contribute to its impact on cholesterol homeostasis.

The Farnesoid X Receptor Is Expressed in Breast Cancer and Regulates Apoptosis and Aromatase Expression

Bile acids are present at high concentrations in breast cysts and in the plasma of postmenopausal women with breast cancer. The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that regulates bile acid homeostasis. FXR was detected in normal and tumor breast tissue, with a high level of expression in ductal epithelial cells of normal breast and infiltrating ductal carcinoma cells. FXR was also present in the human breast carcinoma cells, MCF-7 and MDA-MB-468. Activation of FXR by high concentrations of ligands induced MCF-7 and MDA-MB-468 apoptosis. At lower concentrations that had no direct effect on viability, the FXR agonist GW4064 induced expression of mRNA for the FXR target genes, small heterodimer partner (SHP), intestinal bile acid binding protein, and multidrug resistance-associated protein 2 (MRP-2), and repressed the expression of the SHP target gene aromatase. In contrast to MRP-2, mRNA for the breast cancer target genes MDR-3, MRP-1, and solute carrier transporter 7A5 were decreased. Although multidrug resistance transporters were regulated and are known FXR target genes, GW4064 had no effect on the cell death induced by the anticancer drug paclitaxel. Our findings show for the first time that FXR is expressed in breast cancer tissue and has multiple properties that could be used for the treatment of breast cancer.

The bile acid nuclear receptor FXR and the bile acid binding protein IBABP are differently expressed in colon cancer.

Bile acids have been implicated in the development of colorectal cancers. We investigated the expression of the transcription factor regulated by bile acids, farnesoid X receptor (FXR), as well as other components of this pathway in human colorectal tumors and cell lines. The most significant changes were a decrease in FXR mRNA levels in adenomas (5-fold average) and carcinomas (10 fold average) and an increase in peroxisome proliferator activated receptor-gamma (2-fold average). FXR was not expressed in undifferentiated colon adenocarcinoma SW480 cells and metastasis derived SW620 cells. In Caco-2 and HT-29 cells, the level of FXR expression increased with the degree of differentiation. Intestinal bile acid binding protein was activated by chenodeoxycholic acid and the synthetic FXR agonist GW4064 in Caco-2 and HT-29 but not in SW cells unless FXR was transfected. The down-regulation of the nuclear receptor FXR in colon cancer might be of clinical and pharmacological importance.

The farnesoid X receptor controls gene expression in a ligand- and promoter-selective fashion.

Farnesoid X receptor (FXR) is a nuclear receptor for bile acids. Ligand activated-FXR regulates transcription of genes to allow feedback control of bile acid synthesis and secretion. There are five major bile acids in humans. We have previously demonstrated that lithocholate acts as an FXR antagonist, and here we show that the other four bile acids, chenodeoxycholate (CDCA), deoxycholate (DCA), cholate (CA), and ursodeoxycholate (UDCA), act as selective FXR agonists in a gene-specific fashion. In an in vitro coactivator association assay, CDCA fully activated FXR, whereas CA partially activated FXR and DCA and UDCA had negligible activities. Similar results were also obtained from a glutathione S-transferase pull-down assay in which only CDCA and the synthetic FXR agonist GW4064 significantly increased the interaction of SRC-1 with FXR. In FXR transactivation assays with a bile salt export pump (BSEP) promoter-driven luciferase construct, bile acids showed distinct abilities to activate the BSEP promoter: CDCA, DCA, CA, and UDCA increased luciferase activity by 25-, 20-, 18-, and 8-fold, respectively. Consistently, CDCA increased BSEP mRNA by 750-fold in HepG2 cells, whereas DCA, CA, and UDCA induced BSEP mRNA by 250-, 75-, and 15-fold, respectively. Despite the partial induction of BSEP mRNA, CA, DCA, and UDCA effectively repressed expression of cholesterol 7alpha-hydroxylase, another FXR target. We further showed that all four bile acids significantly increased FXR protein, suggesting the existence of an auto-regulatory loop in FXR signaling pathways. In conclusion, these results suggest that the binding of each bile acid results in a different FXR conformations, which in turn differentially regulates expression of individual FXR targets.

Modification of intestinal bile acid-binding protein (I-BABP) expression after exposure to ionising radiation

Modification of intestinal bile acid-binding protein (I-BABP) expression after exposure to ionising radiation

Guggulsterone Is a Farnesoid X Receptor Antagonist in Coactivator Association Assays but Acts to Enhance Transcription of Bile Salt Export Pump

Guggulipid is an extract of the guggul tree Commiphora mukul and has been widely used to treat hyperlipidemia in humans. The plant sterol guggulsterone (GS) is the active agent in this extract. Recent studies have shown that GS can act as an antagonist ligand for farnesoid X receptor (FXR) and decrease expression of bile acid-activated genes. Here we show that GS, although an FXR antagonist in coactivator association assays, enhances FXR agonist-induced transcription of bile salt export pump (BSEP), a major hepatic bile acid transporter. In HepG2 cells, in the presence of an FXR agonist such as chenodeoxycholate or GW4064, GS enhanced endogenous BSEP expression with a maximum induction of 400-500% that induced by an FXR agonist alone. This enhancement was also readily observed in FXR-dependent BSEP promoter activation using a luciferase reporter construct. In addition, GS alone slightly increased BSEP promoter activation in the absence of an FXR agonist. Consistent with the results in HepG2, guggulipid treatment in Fisher rats increased BSEP mRNA. Interestingly, in these animals expression of the orphan nuclear receptor SHP (small heterodimer partner), a known FXR target, was also significantly increased, whereas expression of other FXR targets including cholesterol 7alpha-hydroxylase (Cyp 7a1), sterol 12alpha-hydroxylase (Cyp 8b1), and the intestinal bile acid-binding protein (I-BABP), remained unchanged. Thus, we propose that GS is a selective bile acid receptor modulator that regulates expression of a subset of FXR targets. Guggulipid treatment in rats lowered serum triglyceride and raised serum high density lipoprotein levels. Taken together, these data suggest that guggulsterone defines a novel class of FXR ligands characterized by antagonist activities in coactivator association assays but with the ability to enhance the action of agonists on BSEP expression in vivo.

Bile salt transporters.

Bile salts are the major organic solutes in bile and undergo extensive enterohepatic circulation. Hepatocellular bile salt uptake is mediated predominantly by the Na(+)-taurocholate cotransport proteins Ntcp (rodents) and NTCP (humans) and by the Na(+)-independent organic anion-transporting polypeptides Oatp1, Oatp2, and Oatp4 (rodents) and OATP-C (humans). After diffusion (bound by intracellular bile salt-binding proteins) to the canalicular membrane, monoanionic bile salts are secreted into bile canaliculi by the bile salt export pump Bsep (rodents) or BSEP (humans). Both belong to the ATP-binding cassette (ABC) transporter superfamily. Dianionic conjugated bile salts are secreted into bile by the multidrug-resistance-associated proteins Mrp2/MRP2. In bile ductules, a minor portion of protonated bile acids and monomeric bile salts are reabsorbed by non-ionic diffusion and the apical sodium-dependent bile salt transporter Asbt/ASBT, transported back into the periductular capillary plexus by Mrp3/MRP3 [and/or a truncated form of Asbt (tAsbt)], and subjected to cholehepatic shunting. The major portion of biliary bile salts is aggregated into mixed micelles and transported into the intestine, where they are reabsorbed by apical Oatp3, the apical sodium-dependent bile salt transporter (ASBT), cytosolic intestinal bile acid-binding protein (IBABP), and basolateral Mrp3/MRP3 and tAsbt. Transcriptional and posttranscriptional regulation of these enterohepatic bile salt transporters is closely related to the regulation of lipid and cholesterol homeostasis. Furthermore, defective expression and function of bile salt transporters have been recognized as important causes for various cholestatic liver diseases.

The nuclear receptors FXR and LXRalpha: potential targets for the development of drugs affecting lipid metabolism and neoplastic diseases.

The orphan nuclear receptors FXR and LXRalpha have become challenging targets for the discovery of new therapeutic agents. Bile acids and hydroxysterol intermediates are the respective natural ligands of these two structurally and functionally closely related receptors. Both FXR and LXRalpha; are thought to play a major role in the control of cholesterol catabolism by regulating the expression of cholesterol 7alpha-hydroxylase, the rate limiting enzyme of bile acid synthesis. Reverse cholesterol transport might also be affected by FXR and LXR since they control the expression of PLTP and CETP, two proteins involved in the transfer of phospholipid, cholesterol and cholesteryl esters among plasma lipoproteins. A new class of potent synthetic activators of FXR, the 1,1-bisphosphonate esters, has been discovered which up regulate the Intestinal Bile Acid Binding Protein gene (I-BABP) as demonstrated for chenodeoxycholic acid, however there are no known synthetic activators yet identified for LXRalpha. The evaluation of FXR as a potential target for the development of drugs affecting plasma cholesterol can take advantage of the fact that the activators of FXR (farnesol, bile acids and the 1,1-bisphosphonate esters) have been studied in various in vitro and in vivo models. Administration of chenodeoxycholic acid to animals and man did not result in the increase in plasma cholesterol expected from a decrease in cholesterol 7alpha-hydroxylase expression. Like farnesol, the 1,1-bisphosphonate esters increase the rate of degradation of HMGCoA reductase and have the unexpected property of inducing hypocholesterolemia in normal animals. The natural and synthetic FXR agonists trigger differentiation, inhibit cell proliferation and are potent inducers of apoptosis. The 1,1-bisphosphonate ester SR-45023A (Apomine) is presently being developed as an antineoplastic drug.

Farnesoid X Receptor Responds to Bile Acids and Represses Cholesterol 7α-Hydroxylase Gene (CYP7A1) Transcription

Cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription is repressed by bile acids. The goal of this study is to elucidate the mechanism of CYP7A1 transcription by bile acid-activated farnesoid X receptor (FXR) in its native promoter and cellular context and to identify FXR response elements in the gene. In Chinese hamster ovary cells transfected with retinoid X receptor alpha (RXRalpha)/FXR, only chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) were able to stimulate a heterologous promoter/reporter containing an ecdysone response element. In HepG2 cells, all bile acids (25 microM) were able to repress CYP7A1/luciferase reporter activity, and only CDCA and DCA further repressed reporter activity when cotransfected with RXRalpha/FXR. The concentration of CDCA required to inhibit 50% of reporter activity (IC(50)) was determined to be approximately 25 microM without FXR and 10 microM with FXR. Deletion analysis revealed that the bile acid response element located between nucleotides -148 and -128 was the FXR response element, but RXRalpha/FXR did not bind to this sequence. These results suggest that bile acid-activated FXR exerts its inhibitory effect on CYP7A1 transcription by an indirect mechanism, in contrast to the stimulation and binding of FXR to intestinal bile acid-binding protein gene promoter. Results also reveal that bile acid receptors other than FXR are present in HepG2 cells.

Identification of a Bile Acid-responsive Element in the Human Ileal Bile Acid-binding Protein Gene: INVOLVEMENT OF THE FARNESOID X RECEPTOR/9-cis-RETINOIC ACID RECEPTOR HETERODIMER

Intestinal bile acid-binding protein (I-BABP) is a cytosolic protein that binds bile acids (BAs) with a high affinity. In the small intestine, its expression is restricted to the ileum where it is involved in the enterohepatic circulation of BAs. Using the human enterocyte-like Caco-2 cell line, we have recently shown that BAs increased I-BABP gene expression. To determine whether this regulation occurs in vivo, the effect of BA depletion or supplementation was studied in mice. A dramatic drop in I-BABP mRNA levels was observed in mice treated with the BA-binding resin cholestyramine, whereas an increase was found in animals fed with taurocholic acid. BAs are physiological ligands for the nuclear farnesoid X receptor (FXR). Both FXR and I-BABP are co-expressed along the small intestine and in Caco-2 cells. To determine the role of FXR in the regulation of I-BABP expression, the promoter of the human I-BABP gene was cloned. In Caco-2 cells, cotransfection of FXR and RXRalpha is required to obtain the full transactivation of the I-BABP promoter by BAs. Deletion and mutation analyses demonstrate that the FXR/RXRalpha heterodimer activates transcription through an inverted repeat bile acid responsive element located in position -160/-148 of the human I-BABP promoter. In conclusion, we show that FXR is a physiological BA sensor that is likely to play an essential role in BA homeostasis through the regulation of genes involved in their enterohepatic circulation.

Identification of a nuclear receptor for bile acids.

Bile acids are essential for the solubilization and transport of dietary lipids and are the major products of cholesterol catabolism. Results presented here show that bile acids are physiological ligands for the farnesoid X receptor (FXR), an orphan nuclear receptor. When bound to bile acids, FXR repressed transcription of the gene encoding cholesterol 7alpha-hydroxylase, which is the rate-limiting enzyme in bile acid synthesis, and activated the gene encoding intestinal bile acid-binding protein, which is a candidate bile acid transporter. These results demonstrate a mechanism by which bile acids transcriptionally regulate their biosynthesis and enterohepatic transport.

Regulation of expression of human intestinal bile acid-binding protein in Caco-2 cells

Molecular mechanisms of the bile acid active transport system in the ileal enterocytes remain unknown. We examined whether bile acids affect human enterocyte gene expression of intestinal bile acid-binding protein (I-BABP), a component of this transport system. Differentiated Caco-2 cells were incubated in the presence of human bile, bile acids or other lipids. The level of I-BABP expression was evaluated by Northern and Western blot analyses. A 24 h incubation of Caco-2 cells in a medium containing either bile or bile acids resulted in a remarkable 7.5-fold increase in the I-BABP mRNA level over the control level. Neither cholesterol, palmitic acid, phosphatidylcholine nor cholestyramine treated bile showed any difference in I-BABP mRNA expression from the control. Bile acid treatment increased the level of I-BABP mRNA in Caco-2 cells in a time- and dose-dependent manner. Western blot analysis showed that this induction led to increase in cytosolic I-BABP. Chenodeoxycholic acid and deoxycholic acid showed greater induction effects than other hydrophilic bile acids, including their own glycine conjugates. Pretreatment by actinomycin D or cycloheximide completely inhibited the up-regulation of I-BABP expression by bile acid. Bile acids, especially lipophilic bile acids, increase the I-BABP expression in Caco-2-cells, suggesting that luminal bile acids play an important role in regulating the I-BABP gene expression.

Effect of bile on the intestinal bile-acid binding protein (I-BABP) expression. In vitro and in vivo studies

Enterocytes actively transport bile acids from the ileal lumen to the portal blood. This physiological process greatly contributes to maintaining the bile acid homeostasis. However, little is known about the molecular mechanisms involved in this transport system. The effect of bile on gene expression of the intestinal bile-acid binding protein (I-BABP) expressed in the enterocytes was studied in vivo, using the by-pass method, and in vitro, using organ culture of ileum explants and Caco-2 cell line. The low cytosolic I-BABP concentration and I-BABP mRNA level found in diverted ileum was totally recovered when bile was added in the ileal lumen. Northern blot analysis of the ileal explants revealed a dose-dependent increase in the I-BABP mRNA in the presence of bile. In Caco-2 cells, the I-BABP transcript was dramatically increased in the presence of human bile while it was undectable in the control cultures. These data offer the first evidence that biliary components regulate the I-BABP gene expressed in the enterocytes.

Fatty Acid-Binding Protein, FABP

Although FABPs have been extensively investigated, their function is not finally established. Based upon their tissue expression, binding affinities, regulation, in vitro effects on fatty acid transfer and upon enzymes, it has been postulated that these proteins play an important role in the transport and metabolism of long-chain fatty acid.

Tissue-specific regulation of the expression of rat intestinal bile acid-binding protein

A lipid-binding protein identical to the rat intestinal bile acid-binding protein, termed I-15P, was expressed in steroid hormone-producing tissues such as ovary and adrenal gland, but not testis. In immature rats, I-15P was expressed in intestine but not in ovaries. The expression of I-15P in the ovaries of immature rats was induced to the level in immature rats by gonadotropin treatment. This suggests that the expression of I-15P in the ovaries is controlled by the ovarian cycle. The present results indicate that the expression of I-15P is developmentally and hormonally controlled in a tissue-specific manner.

Molecular cloning, tissue distribution, and expression of a 14-kDa bile acid-binding protein from rat ileal cytosol.

A cDNA clone encoding the major intestinal cytosolic 14-kDa bile acid-binding protein (14-kDa I-BABP) was isolated from a rat ileal lambda gt22A library following immunoscreening using a monospecific antiserum raised against a 14-kDa polypeptide found in the rat ileal cytosol. One clone of 516 bp encoded a 128-amino acid protein with a predicted molecular mass of 14,544 Da. The deduced amino acid sequence of 14-kDa I-BABP showed 100% homology to rat intestinal 15-kDa protein (I-15P) and 72% homology to porcine 15-kDa gastrotropin, whereas comparison of I-BABP to rat 14-kDa fatty acid-binding proteins of liver, intestine, and heart revealed homologies of 44%, 25%, and 28%, respectively. Northern blot analysis revealed a single transcript of approximately 0.5 kb in ileum and ovary; however, the abundance of I-BABP mRNA was much greater in ileum than in ovary. No transcript was seen in RNA extracted from stomach, jejunum, colon, liver, adrenal, brain, heart, kidney, or testis. Transfection of the I-BABP cDNA into COS-7 cells resulted in the expression of a 14-kDa protein that was identical to the ileal cytosolic I-BABP as determined by immunoblotting. Photoaffinity labeling of expressed 14-kDa protein was saturable with respect to increasing concentrations of 7,7-azo[3H]taurocholate (Km, 83.3 microM; Vmax, 6.7 pmol/mg per 5 min). Taurocholate inhibited 7,7-azotaurocholate labeling by > 96% with lesser inhibition by taurochenodeoxycholate (83.1%), chenodeoxycholate (74.6%), cholate (50.5%), and progesterone (38.5%), whereas oleic acid and estradiol did not inhibit binding.