Apical Sodium-dependent Bile Acid Research Articles

Saikosaponin D improves non-alcoholic fatty liver disease via gut microbiota-bile acid metabolism pathway

Non-alcoholic fatty liver disease (NAFLD) is the main cause of chronic liver disease worldwide. Bupleurum is widely used in the treatment of non-alcoholic fatty liver, and saikosaponin D (SSD) is one of the main active components of Bupleurum. The purpose of this study was to investigate the efficacy of SSD in the treatment of NAFLD and to explore the mechanism of SSD in the improvement of NAFLD based on “gut-liver axis”. Our results showed that SSD dose-dependently alleviated high fat diet-induced weight gain in mice, improved insulin sensitivity, and also reduced liver lipid accumulation and injury-related biomarkers aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Further exploration found that SSD inhibited the mRNA expression levels of farnesoid X receptor (Fxr), small heterodimer partner (Shp), recombinant fibroblast growth factor 15 (Fgf15) and apical sodium dependent bile acid transporter (Asbt) in the intestine, suggesting that SSD improved liver lipid metabolism by inhibiting intestinal FXR signaling. SSD can significantly reduce the gut microbiota associated with bile salt hydrolase (BSH) expression, such as Clostridium. Decreased BSH expression reduced the ratio of unconjugated to conjugated bile acids, thereby inhibiting the intestinal FXR. These data demonstrated that SSD ameliorated NAFLD potentially through the gut microbiota-bile acid-intestinal FXR pathway and suggested that SSD is a promising therapeutic agent for the treatment of NAFLD.

FXR-FGF19 signaling in the gut–liver axis is dysregulated in patients with cirrhosis and correlates with impaired intestinal defence

Background and aimsExperimental studies linked dysfunctional Farnesoid X receptor (FXR)-fibroblast growth factor 19 (FGF19) signaling to liver disease. This study investigated key intersections of the FXR-FGF19 pathway along the gut–liver axis and their link to disease severity in patients with cirrhosis.MethodsPatients with cirrhosis undergoing hepatic venous pressure gradient measurement (cohort-I n = 107, including n = 53 with concomitant liver biopsy; n = 5 healthy controls) or colonoscopy with ileum biopsy (cohort-II n = 37; n = 6 controls) were included. Hepatic and intestinal gene expression reflecting FXR activation and intestinal barrier integrity was assessed. Systemic bile acid (BA) and FGF19 levels were measured.ResultsSystemic BA and FGF19 levels correlated significantly (r = 0.461; p < 0.001) and increased with cirrhosis severity. Hepatic SHP expression decreased in patients with cirrhosis (vs. controls; p < 0.001), indicating reduced FXR activation in the liver. Systemic FGF19 (r = −0.512, p < 0.001) and BA (r = −0.487, p < 0.001) levels correlated negatively with hepatic CYP7A1, but not SHP or CYP8B1 expression, suggesting impaired feedback signaling in the liver. In the ileum, expression of FXR, SHP and FGF19 decreased in patients with cirrhosis, and interestingly, intestinal FGF19 expression was not linked to systemic FGF19 levels. Intestinal zonula occludens-1, occludin, and alpha-5-defensin expression in the ileum correlated with SHP and decreased in patients with decompensated cirrhosis as compared to controls.ConclusionsFXR-FGF19 signaling is dysregulated at essential molecular intersections along the gut–liver axis in patients with cirrhosis. Decreased FXR activation in the ileum mucosa was linked to reduced expression of intestinal barrier proteins. These human data call for further mechanistic research on interventions targeting the FXR-FGF19 pathway in patients with cirrhosis.Clinical trial number: NCT03267615Graphical abstractPhysiology of enterohepatic FXR-FGF19 signaling and its regulation in patients with advanced chronic liver disease (ACLD). (FXR) farnesoid X receptor; (FGF19) fibroblast growth factor 19; (BA) bile acids; (c/dACLD) compensated/decompensated advanced chronic liver disease; (FXR) farnesoid X receptor; (SHP) small heterodimer partner; (OST-α/-β) organic solute transporter subunit alpha/beta; (CYP7A1) cholesterol 7 alpha-hydroxylase; (NTCP) Na+-taurocholate cotransporting polypeptide; (CYP8B1) sterol 12-alpha-hydroxylase; (HVPG) hepatic venous pressure gradient; (TJ) tight junctions; (AMP) antimicrobial peptides; (ASBT) Apical Sodium Dependent Bile Acid Transporter; (ZO 1) zonula occludens-1; (OCLN) occluding; (DEFA5) alpha-5-defensin.

Bile acid dysmetabolism in inflammatory bowel diseases

Aim: To summarize the state-of-the-art data on the molecular mechanisms of bile acid (BA) synthesis and absorption, their impaired absorption and receptor-dependent signaling, as well as on the effects of the gut microbiota on BA metabolism in inflammatory bowel diseases (IBD).&#x0D; Key messages: BA malabsorption is one of the relevant mechanisms in the development of diarrhea in IBD. It may occur due to various disorders of the ileum, such as terminal ileitis, ileocolitis or ileocecal resection in Crohn's disease and ileoanal reservoir in ulcerative colitis. Molecular mechanisms of BA malabsorption in IBD are related to a defect in the BA uptake by the apical sodium dependent bile acid transporter (ASBT), as well as to a decrease in the expression of pregnane X receptor (PXR) and farnesoid X receptor (FXR), whose activation by glucocorticoids results in an increase in the BA reabsorption in the ileum and a decrease in hologenic diarrhea. The metabolic profile of luminal BA in IBD is characterized by an increased content of conjugated and 3-OH-sulfated BA and reduced levels of secondary BA. The decrease in the relative abundance of the Lachnospiraceae and Oscillospiraceae spp. in IBD patients leads to a decrease in the efficiency of microbial biotransformation of BA. Changes in the BA metabolic profile in IBD affect the gut microbiota, and impaired interaction with the FXR, PXR, G protein-coupled bile acid receptor (GPBAR1), retinoid-related orphan receptors (RORs) and vitamin D receptor (VDR) results in a pro-inflammatory response and increased intestinal permeability, bacterial translocation, and IBD progression. BA metabolism in IBD-associated primary sclerosing cholangitis (PSC-IBD) is characterized by a significant decrease in the luminal BA pool, and the microbiota composition is remarkable for an increase in the relative abundance of Fusobacterium and Ruminococcus spp., and a decrease in Veillonella, Dorea, Blautia, Lachnospira and Roseburia.&#x0D; Conclusion: Disordered synergistic interplay of BA with intestinal microbiota results in disruption of the ligand-receptor interaction and BA metabolic transformation, which contributes to the activation of the immune system, formation of a vicious circle of chronic inflammation and IBD progression. Further studies into mutual influence of the gut microbiota, BA metabolism and receptor signaling may promote the development of new methods for the diagnosis and treatment of IBD.

Dietary cholesterol drives the development of nonalcoholic steatohepatitis by altering gut microbiota mediated bile acid metabolism in high-fat diet fed mice

Nonalcoholic fatty liver disease (NAFLD) is the most widespread chronic liver disorder globally. Unraveling the pathogenesis of simple fatty liver to nonalcoholic steatohepatitis (NASH) has important clinical significance for improving the prognosis of NAFLD. Here, we explored the role of a high-fat diet alone or combined with high cholesterol in causing NASH progression. Our results demonstrated that high dietary cholesterol intakes accelerate the progression of spontaneous NAFLD and induces liver inflammation in mice. An elevation of hydrophobic unconjugated bile acids cholic acid (CA), deoxycholic acid (DCA), muricholic acid and chenodeoxycholic acid, was observed in high-fat and high-cholesterol diet fed mice. Full-length sequencing of the 16S rDNA gene of gut microbiota revealed a significant increase in the abundance of Bacteroides, Clostridium, and Lactobacillus that possess bile salt hydrolase activity. Furthermore, the relative abundance of these bacterial species was positively correlated with content of unconjugated bile acids in liver. Moreover, the expression of genes related to bile acid reabsorption (organic anion-transporting polypeptides, Na+-taurocholic acid cotransporting polypeptide, apical sodium dependent bile acid transporter and organic solute transporter β) was found to be increased in mice with a high-cholesterol diet. Lastly, we observed that hydrophobic bile acids CA and DCA induce an inflammatory response in free fatty acids-induced steatotic HepG2 cells. In conclusion, high dietary cholesterol promotes the development of NASH by altering gut microbiota composition and abundance and thereby influencing with bile acid metabolism.

Impact of dietary glutamate and glycine on growth and nutrient utilization in rainbow trout (Oncorhynchus mykiss)

A feeding trial was conducted to test the effect of dietary glutamate and glycine supplementation on growth potential, nutritional utilization and digestion, and energy metabolism of rainbow trout (Oncorhynchus mykiss). Four isonitrogenous and isolipidic diets (2 × 2 factorial design) were formulated with glutamate (Glu) and glycine (Gly) as the main factors. The protein source consisted mainly of black soldier fly larvae meal (40% inclusion in all diets) for its low-level content of glutamate and glycine. Triplicate groups of rainbow trout (30 fish/tank with mean initial body weight of 87.5 g ± 0.9) were restrictively fed by hand twice a day for 6 weeks. Dietary Glu and Gly supplementation did not affect feed intake, specific growth rate, or the feed conversion ratio. Whole body protein, lipid, amino acid and fatty acid composition were also unaffected by the supplementation of Glu and Gly in the diets. Dietary Gly supplementation increased the apparent digestibility of amino acids. Furthermore, supplementation with Gly, increased the concentration of serine, glycine, tryptophane, tyrosine and citrulline, while supplementation with Glu in the diets increased the concentration of hydroxy-proline and β-alanine in the serum of rainbow trout. Both dietary Glu and Gly supplementation improved the digestibility of the fatty acids. The expression of genes involved in the hepatic bile acid synthesis (e.g., apical sodium dependent bile acid transporter and organic solute transporter) were affected by supplementation of Gly in the diets. In general, this study showed that dietary supplementation with Gly and Glu improved the nutrient digestibility in rainbow trout.

Novel nitroxide-bile acid conjugates inform substrate requirements for human bile acid transporters

Transport of bile acids within the enterohepatic circulation from the liver to the intestines via the gallbladder and back to the liver via the portal vein plays a critical role in bile acid regulation and homeostasis. Deficiency of fibroblast growth factor 19 (FGF19), a hormone whose role is to suppress de novo hepatic bile acid synthesis to maintain homeostatic levels, results in bile acid diarrhea (BAD). FGF19 also modulates gallbladder motility so that bile acids are concentrated in the gallbladder until postprandial contraction. To assess bile acid transport and diagnose ailments like BAD that are associated with altered bile acid synthesis and transport, we created bile acid conjugates with nitroxide radicals. Because nitroxides are paramagnetic and can promote proton relaxation, we reasoned that these paramagnetic conjugates should act as contrast agents in in vivo magnetic resonance imaging (MRI). We tested substrate capability by assessing the inhibitory potential of these novel agents against taurocholate uptake by the apical sodium dependent bile acid transporter (ASBT) and the Na+/taurocholate cotransporting polypeptide (NTCP). Surprisingly, neither the paramagnetic compounds CA-Px-1 and CA-Px-2, nor their reduced forms, CA-Px-1H and CA-Px-2H, inhibited hASBT- or hNTCP-mediated taurocholate uptake. Therefore, the new conjugates cannot serve as contrast agents for MRI in vivo. However, our findings identify important structural constraints of transportable bile acid conjugates and suggest potential modifications to overcome these limitations.

IBD and Bile Acid Absorption: Focus on Pre-clinical and Clinical Observations.

Inflammatory bowel disease (IBD) causes chronic inflammation affecting the GI tract. It is classified as consisting of Crohn’s Disease (CD) and Ulcerative Colitis (UC). Bile Acid absorption is altered in both pre-clinical models of Inflammatory Bowel Disease (IB) and in human IBD. The bile acid transporter apical sodium dependent bile acid transporter (ASBT) showed decreased expression in rats with TNBS colitis. Decreased ASBT expression has also been described in murine, canine and rabbit models of intestinal inflammation. Human IBD studies have shown that an inflamed ileum can interrupt enterohepatic recirculation of bile acid, which could be due to inflammatory cytokine induced repression of the ASBT promoter. There are different hypotheses as to why ASBT is downregulated during CD. In addition, one study has demonstrated the beneficial effect of a glucocorticoid on ASBT expression, when treating IBD. Our aim in this paper was to systematically review various aspects of bile acid malabsorption in animal models of intestinal inflammation, as well as in IBD.

An Update on Further Progression of NAFLD, NASH with Prospective Therapies Like L-Carnitine (LC), Nicotinamide Ribose (NR) Combination, as well as Apical Sodium Dependent Bile Acids Transporter (ASBT) or Volixibat and Silybin as Alternatives

An Update on Further Progression of NAFLD, NASH with Prospective Therapies Like L-Carnitine (LC), Nicotinamide Ribose (NR) Combination, as well as Apical Sodium Dependent Bile Acids Transporter (ASBT) or Volixibat and Silybin as Alternatives

Neurotensin contributes to pediatric intestinal failure-associated liver disease via regulating intestinal bile acids uptake.

Although the pathogenesis of intestinal failure (IF)-associated liver disease (IFALD) is uncertain, IF-associated cholestasis mediated by the combination of intestinal injury and parenteral nutrition (PN) can lead to disturbed hepatocyte bile acids (BA) homeostasis and cause liver damages. We here show that neurotensin (NT; also known as NTS) concentrations were lower compared to healthy matched controls. Patients with cholestasis [56.1 ng/L (9.7–154.7) vs. 210.4 ng/L (134–400.4), p < .001] had lower serum NT concentrations than others. In patients' ileum, the levels of NT mRNA were positively correlated with the apical sodium dependent bile acid transporter (ASBT) mRNA levels. In mice and in cultured intestinal cells, NT treatments stimulated the expression of ASBT and led to increase BA uptake via NT receptors (NTR1 and NTR3; also known as NTSR1and NTSR3). In conclusion, these findings directly link NT with BA homeostasis, which provide an insight into the complex mechanisms mediating the development of liver disease in pediatric patients with IF.

Epiberberine reduces serum cholesterol in diet-induced dyslipidemia Syrian golden hamsters via network pathways involving cholesterol metabolism

This study aimed to evaluate the cholesterol-lowering effect of epiberberine in dyslipidemia Syrian golden hamsters induced by high fat and high cholesterol (HFHC) diet and its regulation mechanism on some key genes involved in cholesterol metabolism. Hamsters were divided into six groups: normal control group (NC), HFHC group, simvastatin (Sim) and three doses of epiberberine group. The body weight, organs weight and serum lipid levels, as well as total cholesterol (TC) and total bile acids (TBA) levels in liver and feces were determined. Furthermore, the antidyslipidemia effect of epiberberine on key genes involved in cholesterol biosynthesis, uptake, conversion and elimination such as 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), low density lipoprotein receptor (LDL receptor), 7-alpha-hydroxylase (CYP7A1) and apical sodium dependent bile acid transporter (ASBT) were investigated. The results showed that epiberberine at high dosage significantly reduced serum TC, low density lipoprotein cholesterol (LDL-c) and TBA levels by 20.2%, 22.3% and 43.8%, respectively, and increased TBA and TC levels in feces. Epiberberine inhibited HMGCR mRNA and protein expressions and slightly reduced the protein level of ASBT, as well as dramatically up-regulated mRNA and protein expressions of CYP7A1 and LDL receptor. These findings suggested that the antidyslipidemia effects of epiberberine can be achieved via inhibiting the synthesis of cholesterol, promoting the uptake and conversion of TC in liver and increasing the excretion of TC and TBA in feces. Thus, epiberberine should be considered as one of the promising natural drugs for the treatment of dyslipidemia.

Importance of Large Intestine in Regulating Bile Acids and Glucagon-Like Peptide-1 in Germ-Free Mice.

It is known that 1) elevated serum bile acids (BAs) are associated with decreased body weight, 2) elevated glucagon-like peptide-1 (GLP-1) levels can decrease body weight, and 3) germ-free (GF) mice are resistant to diet-induced obesity. The purpose of this study was to test the hypothesis that a lack of intestinal microbiota results in more BAs in the body, resulting in increased BA-mediated transmembrane G protein-coupled receptor 5 (TGR5) signaling and increased serum GLP-1 as a mechanism of resistance of GF mice to diet-induced obesity. GF mice had 2- to 4-fold increased total BAs in the serum, liver, bile, and ileum. Fecal excretion of BAs was 63% less in GF mice. GF mice had decreased secondary BAs and increased taurine-conjugated BAs, as anticipated. Surprisingly, there was an increase in non-12α-OH BAs, namely, β-muricholic acid, ursodeoxycholic acid (UDCA), and their taurine conjugates, in GF mice. Further, in vitro experiments confirmed that UDCA is a primary BA in mice. There were minimal changes in the mRNA of farnesoid X receptor target genes in the ileum (Fibroblast growth factor 15, small heterodimer protein, and ileal bile acid-binding protein), in the liver (small heterodimer protein, liver receptor homolog-1, and cytochrome P450 7a1), and BA transporters (apical sodium dependent bile acid transporter, organic solute transporter α, and organic solute transporter β) in the ileum of GF mice. Surprisingly, there were marked increases in BA transporters in the large intestine. Increased GLP-1 levels and gallbladder size were observed in GF mice, suggesting activation of TGR5 signaling. In summary, the GF condition results in increased expression of BA transporters in the colon, resulting in 1) an increase in total BA concentrations in tissues, 2) a change in BA composition to favor an increase in non-12α-OH BAs, and 3) activation of TGR5 signaling with increased gallbladder size and GLP-1.

PepT1, ASBT-Linked Prodrug Strategy to Improve Oral Bioavailability and Tissue Targeting Distribution.

With the rapid development of molecular biology, various of drug transporters have been discovered in several important organs of the body, which determine intracellular exposure and pharmacokinetic performances of drugs by modulating cellular entry and exit. This article focuses on the design of transporter-linked prodrug to enhance oral bioavailability and to acquire tissue-specific distribution pattern, especially paying attention to peptide transporter 1 (PepT1) and apical sodium dependent bile acid transporter (ASBT). Conjugation of the native promotety to active drug was one of the effective methods to improve membrane permeability and distribution profiles of drugs. In this review, we highlight the transporter-linked prodrug design modality based on PepT1 and ASBT. The biology of transporters, structure-transport relationship, key features of natural substrates and successful examples of transporter-linked prodrugs are overviewed in detail.

Inflammatory bowel disease alters intestinal bile acid transporter expression.

The enterohepatic circulation of bile acids (BAs) critically depends on absorption of BA in the terminal ileum and colon, which can be affected by inflammatory bowel disease (IBD). Diarrhea in IBD is believed to result in part from BA malabsorption (BAM). We explored whether IBD alters mRNA expression of key intestinal BA transporters, BA detoxifying systems, and nuclear receptors that regulate BA transport and detoxification. Using real-time polymerase chain reaction, mucosal biopsy specimens from the terminal ileum in Crohn's disease (CD) patients and from the descending colon in ulcerative colitis (UC) patients were assessed for mRNA expression. Levels were compared with healthy controls. The main ileal BA uptake transporter, the apical sodium dependent bile acid transporter, was downregulated in active CD and UC and in CD in remission. Other significant changes such as repression of breast cancer-related protein and sulphotransferase 2A1 were seen only during active disease. In UC, pancolitis (but not exclusively left-sided colitis) was associated with altered expression of major BA transporters [multidrug resistance-associated protein 3 (MRP3), MRP4, multidrug resistance gene 1, organic solute transporter α/β] and nuclear receptors (pregnane X receptor, vitamin D receptor) in the descending colon. UC pancolitis leads to broad changes and CD ileitis to selective changes in intestinal BA transporter expression. Early medical manipulation of intestinal BA transporters may help prevent BAM.

616 C-FOS Mediated Regulation of the Apical Sodium Dependent Bile Acid Transporter (ASBT) Plays a Critical Role in the Ileal Response to FGF-19

BACKGROUND AND AIMS: In addition to hepatitis B virus, HCV is also an important cause of HCC in Asians; however, it is often overlooked. This study aims to examine baseline characteristics, timing of HCV diagnosis and long-term survival of HCV-related HCC in Asians compared to non-Asian patients. METHODS: We conducted a retrospective cohort study of 798 consecutive Asian (n=220) and non-Asian (n=572) patients with HCV-related HCC who were identified via computer query using ICD-9 diagnosis at a U.S. university medical center between 7/1996 and 6/2012. Individual records were reviewed. RESULTS: Asians were much older (66 [38-88] vs. 56 [31-87] years, P ,0.0001) and more likely to be female (33% vs. 19%, P,0.0001). A significantly larger proportion of Asians were also diagnosed with HCC within only 2 years of HCV diagnosis compared to non-Asians (35% vs. 20%, p=0.001). Non-Asians were more likely to have decompensated liver disease and had higher median Child-Turcotte-Pugh score (6 [5-11] vs. 7 [5-13], P ,0.0001). Asian patients were more likely to undergo liver-directed palliative therapy (46% vs. 28%) and much less likely to be listed for liver transplantation(20% vs. 48%) (P ,0.001), despite similar rates of meeting Milan criteria for liver transplantation (52 vs.58%, P=0.16). Overall, there was a trend for higher median survival rates in Asians compared to non-Asians (30 vs. 21 months, P=0.091). Among those who were listed for liver transplantation, there was no statistically significant difference in survival between Asians and non-Asians in the first 2 years (72% vs. 68%) but there was divergence with Asians having higher survival afterwards (61% vs. 51%) (Figure 1). However, among those undergoing liver-directed palliative therapy, Asians had significantly higher long-term survival compared to non-Asians (5-year survival: 28% vs. 10%, P,0.0001) (Figure 2). On multivariate Cox proportional hazards model also inclusive of age, sex, BCLC staging, Child-Pugh score, meeting Milan criteria, liver transplantation listing, non-Asian ethnicity was an independent predictor for lower overall survival (HR=0.70 [0.52-0.86]). CONCLUSIONS: Despite being older and having a lower rate of liver transplantation listing, Asian HCV/HCC patients had higher median survival compared to non-Asian patients, particularly among those undergoing palliative therapy. Non-Asians were more likely to have decompensated liver disease and non-Asian ethnicity was an independent predictor for poorer long-term survival. Compared to non-Asians, Asians with HCV-related HCCwere also muchmore likely to have delayed HCV diagnosis. Improved strategies in HCV screening in Asians are needed as it may lead to earlier diagnosis and treatment of HCV infection and possible prevention of HCC development in this ethnic population with a disproportionate HCC disease burden.

Structure–Activity Relationship for FDA Approved Drugs As Inhibitors of the Human Sodium Taurocholate Cotransporting Polypeptide (NTCP)

The hepatic bile acid uptake transporter sodium taurocholate cotransporting polypeptide (NTCP) is less well characterized than its ileal paralog, the apical sodium dependent bile acid transporter (ASBT), in terms of drug inhibition requirements. The objectives of this study were (a) to identify FDA approved drugs that inhibit human NTCP, (b) to develop pharmacophore and Bayesian computational models for NTCP inhibition, and (c) to compare NTCP and ASBT transport inhibition requirements. A series of NTCP inhibition studies were performed using FDA approved drugs, in concert with iterative computational model development. Screening studies identified 27 drugs as novel NTCP inhibitors, including irbesartan (Ki = 11.9 μM) and ezetimibe (Ki = 25.0 μM). The common feature pharmacophore indicated that two hydrophobes and one hydrogen bond acceptor were important for inhibition of NTCP. From 72 drugs screened in vitro, a total of 31 drugs inhibited NTCP, while 51 drugs (i.e., more than half) inhibited ASBT. Hence, while there was inhibitor overlap, ASBT unexpectedly was more permissive to drug inhibition than was NTCP, and this may be related to NTCP possessing fewer pharmacophore features. Findings reflected that a combination of computational and in vitro approaches enriched the understanding of these poorly characterized transporters and yielded additional chemical probes for possible drug-transporter interaction determinations.

Design and Characterization of a Novel Fluorinated Magnetic Resonance Imaging Agent for Functional Analysis of Bile Acid Transporter Activity

To synthesize a trifluorinated bile acid that can be used for (19)F magnetic resonance imaging (MRI) of bile acid enterohepatic circulation, characterize its in vitro transporter affinity, stability, and (19)F-MRI signal, and assess its ability to concentrate in the gallbladder of C57BL/6 mice. Target compound CA-lys-TFA was synthesized and tested for affinity toward the apical sodium dependent bile acid transporter (hASBT) and the Na+/taurocholate cotransporting polypeptide (hNTCP). In a pilot study, fasted mice were gavaged with vehicle control, 150mg/kg or 300mg/kg CA-lys-TFA. CA-lys-TFA in gallbladder, liver and plasma at t = 5h was quantified. Additionally, a 24-h time course (24 mice across eight time points) was studied using 50mg/kg CA-lys-TFA. CA-lys-TFA was a potent substrate of hASBT (Kt = 39.4μM, normalized Vmax = 0.853) and hNTCP (Kt = 8.99μM, normalized Vmax = 0.281). (19)F MRI phantom imaging showed linear signal-concentration dependence. In vivo studies showed that rapid accumulation of CA-lys-TFA in the gallbladder was maximal within 4-7h. These findings suggest that CA-lys-TFA, a fluorinated non-radioactive bile acid analogue, has potential for use in MRI to measure in vivo bile acid transport and diagnose bile acid malabsorption and other conditions associated with impaired bile acid transport.

Disruption of Stard10 gene alters the PPARα-mediated bile acid homeostasis

STARD10, a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) protein family, is highly expressed in the liver and has been shown to transfer phosphatidylcholine. Therefore it has been assumed that STARD10 may function in the secretion of phospholipids into the bile. To help elucidate the physiological role of STARD10, we produced Stard10 knockout mice (Stard10−/−) and studied their phenotype. Neither liver content nor biliary secretion of phosphatidylcholine was altered in Stard10−/− mice. Unexpectedly, the biliary secretion of bile acids from the liver and the level of taurine-conjugated bile acids in the bile were significantly higher in Stard10−/− mice than wild type (WT) mice. In contrast, the levels of the secondary bile acids were lower in the liver of Stard10−/− mice, suggesting that the enterohepatic cycling is impaired. STARD10 was also expressed in the gallbladder and small intestine where the expression level of apical sodium dependent bile acid transporter (ASBT) turned out to be markedly lower in Stard10−/− mice than in WT mice when measured under fed condition. Consistent with the above results, the fecal excretion of bile acids was significantly increased in Stard10−/− mice. Interestingly, PPARα-dependent genes responsible for the regulation of bile acid metabolism were down-regulated in the liver of Stard10−/− mice. The loss of STARD10 impaired the PPARα activity and the expression of a PPARα-target gene such as Cyp8b1 in mouse hepatoma cells. These results indicate that STARD10 is involved in regulating bile acid metabolism through the modulation of PPARα-mediated mechanism.

291 Development and Testing of a Novel Fluorinated Bile Acid for Detection of Bile Acid Malabsorption by Magnetic Resonance Imaging (MRI)

Bile Acid Malabsorption (BAM) resulting from bile acid overproduction or reduced transport can increase fecal bile acids and cause chronic diarrhea. BAM is thought to account for up to 30% of patients diagnosed with irritable bowel syndrome (IBS-D). Current methods of diagnosing BAM are limited; it is likely under-diagnosed. Our objective was to develop a non-invasive method of detecting BAM by synthesizing and testing a multi-fluorinated bile acid (MFBA) that can be used for fluorine magnetic resonance imaging (MRI) of bile acid enterohepatic circulation. A prototype MFBA was synthesized by conjugating trifluoroacetyl lysine to cholic acid (19F-CA-lys). This MFBA was tested for transport and inhibition of the apical sodium dependent bile acid transporter (ASBT) using stably transfected MDCK cell monolayers and of Na+/taurocholate cotransporting polypeptide (NTCP) using stably transfected HEK cell monolayers. In Vitro, 19F-CA-lys was a potent inhibitor and substrate of both ASBT (Ki = 20.0±3.9 μM, Kt = 39.4±23.8 μM, normalized Vmax =0.853 ± 0.197) and NTCP (Ki = 2.93±0.60 μM, Kt = 8.99 ± 2.79 μM, normalized Vmax = 0.281±0.052). Stability of 19F-CA-lys (5 μM) was measured after treatment with Caco-2 cell homogenates, rat plasma, rat liver microsomal S9 pools, simulated intestinal fluid with pancreatic enzymes, 1 M HCl, buffers at pH 7.4 and 6.8, and choloylglycine hydrolase (CGH) (N=3 for each condition). Except for CGH, 19F-CA-lys was stable in each condition. After 1 h in CGH, 60.4±4.0% 19F-CA-lys remained compared to 2.4±0.5% naturally-occurring taurocholic acid (P<0.005). To test In Vivo stability and utility of 19F-CA-lys for MRI, we examined its distribution in 8 male C57BL/6 mice (average weight, 25.7 g). After overnight fasting, mice were gavaged with 1:1 polyethylene glycol (PEG):water vehicle control (2 mice), 150 mg/ kg 19F-CA-lys (3 mice) and 300 mg/kg 19F-CA-lys (3 mice). Four hours after dosing, 19FCA-lys in gallbladder, liver and plasma was measured by LC/MS. 19F-CA-lys was not detected in control mice receiving vehicle alone. With 150 and 300 mg/kg 19F-CA-lys, 4 h gallbladder 19F-CA-lys concentrations were 27.0±2.5 and 36.8±8.8 μmol/g, liver concentrations were 0.079±0.035 and 0.057±0.010 μmol/g, and plasma concentrations were 0.444±0.170 and 0.418 ± 0.015 μM, respectively. We calculated that at both doses, 10.8±1.9% (95% CI, 5.915.8%) of administered 19F-CA-lys was present in the gallbladder. We conclude that 19FCA-lys, a novel MFBA, is stable In Vitro and In Vivo, and accumulates in the gallbladder at concentrations that will provide a 19F signal in excess of that we previously determined to be the minimum detectable by 19F MRI (Drug Metab. Disp. 39:736, 2011). These findings suggest that non-radioactive MFBAs have excellent potential to measure In Vivo bile acid transport and serve as clinical imaging tools to diagnosis BAM.

Structural requirements of bile acid transporters: C-3 and C-7 modifications of steroidal hydroxyl groups

The apical sodium dependent bile acid transporter (ASBT) and sodium-taurocholate cotransporting polypeptide (NTCP) are potential prodrug targets, but the structural requirements for these transporters are incompletely defined. The objective of this study was to evaluate the effect of C-3 and C-7 substitution on bile acid interaction with these bile acid transporters. Nineteen bile acid analogs were tested against ASBT and NTCP for binding, as well as translocation. Results indicated that ASBT and NTCP accommodated a wide range of substituents for binding, but all major C-7 modifications resulted in analogs that did not demonstrate active uptake by either ASBT or NTCP. A C-3 modification that was not tolerated at C-7 still afforded translocation via ASBT and NTCP, confirming the relative unacceptability of C-7 modification. Both ASBT and NTCP demonstrated a generally similar binding potency. Results suggest that drug conjugation to the C-3 hydroxyl group, rather than C-7, has potential to lead to a successful prodrug targeting ASBT and NTCP.

Identification of Novel Nonsteroidal Compounds as Substrates or Inhibitors of hASBT

A prodrug approach that employs the human apical sodium dependent bile acid transporter (hASBT) for absorption requires a recognition moiety for hASBT. Bile acids are natural ligands for hASBT, but are hormones with high molecular weight, such that a recognition moiety that is not a bile acid may be advantageous. The objective was to identify nonsteroidal small molecules that could potentially serve as promoieties in the design of prodrugs that target hASBT. Three searches for bile acid analogues were conducted and it involved molecular fingerprints as the computational tools for similarity searching, as well as traditional medicinal chemistry pattern recognition. Sixty-three compounds were tested using a hASBT-Madin-Darby canine kidney cell monolayer model. Twenty-three of these compounds were found to be hASBT inhibitors and represent novel hASBT inhibitors. Three were selected for hASBT uptake studies. Two were substrates, which represent the first reported nonsteroidal substrates of hASBT. Interestingly, each compound lacked a negative charge. These compounds promise to serve as leads to identify hASBT recognition moieties in a prodrug approach to target hASBT to increase drug absorption.