Expression Of Bile Acid Transporters Research Articles

Hepatology Highlights.

Potential conflict of interest: Nothing to report. DR CELLS ROUND UP NEW TUBES Ryan Watkins1 and Sumera I. Ilyas2 Liver injury increases ductular reaction (DR) and contributes to the wound‐healing process, but how DR cells affect the local microenvironment is not fully understood. The slit guidance ligand 2 (SLIT2)/roundabout guidance receptor 1 (ROBO1) axis promotes angiogenesis in select solid tumors, but whether this signaling pathway induces intrahepatic angiogenesis in acute or chronic liver injury is unknown. Using murine liver injury models and human liver specimens, Coll et al. elucidate that SLIT2 secreted by DR cells binds ROBO1 on new endothelial cells and promotes neoangiogenesis in chronic liver injury. Administering mice a diet enriched in 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidin or a choline‐deficient, ethionine‐supplemented (CDE) diet results in DR expansion. This was paralleled by increased neoangiogenesis, which is diminished in mice heterozygous for ROBO1/2. Similar findings were demonstrated in a cohort of patients with alcohol‐associated liver disease (ARLD). Interestingly, SLIT2 serum levels correlated with disease severity in patients with alcohol‐associated hepatitis. In a separate cohort of patients with NAFLD or HBV infected and without cirrhosis, liver DR markers correlated with SLIT2‐ROBO1 signaling, indicating that this response is not specific to ARLD. Altogether, these findings suggest that intrahepatic angiogenesis mediated by DR cell SLIT2 expression in response to chronic liver injury is an important and clinically relevant process in liver repair. (Hepatology 2022;75:353‐69). THE CHOLESTATIC CONUNDRUM Daniel D. Penrice2 and Sumera I. Ilyas2 Patients with intestinal failure related to short bowel syndrome or other causes are often on prolonged parenteral nutrition (PN) and thus are at risk of developing PN‐associated cholestasis (PNAC). PNAC is characterized by persistent hyperbilirubinemia during PN administration. Although the development of PNAC is multifactorial, phytosterols present in the PN solution have been implicated in the process and shown to suppress hepatocyte farnesoid X receptor (FXR) signaling. FXR is activated by bile acids and plays a major regulatory role in the liver. El Kasmi et al. used a murine model of PNAC using the combination of dextran sodium sulfate–induced intestinal injury and PN infusion. In this model, the FXR agonist, GW4064, activated FXR signaling with restoration of bile acid transporter expression and normalization of the serum bile acid profile. Moreover, FXR agonism reduced hepatic macrophage infiltration with a consequent reduction in hepatic inflammation and cholestasis. These findings suggest that FXR agonists added to PN are a potential therapeutic strategy to decrease the incidence of PNAC. (Hepatology 2022;75:252‐66). CHECK THE TIME: IS CCA READY FOR IMMUNOTHERAPY? Caitlin B. Conboy3 and Sumera I. Ilyas2 Cancer initiation and progression are facilitated by an immunosuppressive tumor immune microenvironment (TIME), characterized by suppressive immune cells and immune checkpoint molecules. Immune checkpoint inhibitors (ICI) have transformed the treatment of many cancers, yet their efficacy in cholangiocarcinoma (CCA) is limited to date. Carapeto et al. sought to identify which immune checkpoints are salient in CCA and how their expression correlates with leukocyte infiltration, underlying genetic drivers, and patient survival. The investigators focused on a subset of patients with resected, fluke‐negative, and predominantly HBV/HCV‐negative intrahepatic CCA (N = 96). Using immunohistochemistry profiling, they identified that both immune cell (CD4+/CD8+ T cells, macrophages) and checkpoint molecule expression (lymphocyte activating 3 [LAG3], OX40, programmed cell death 1 [PD‐1], and T‐cell immunoglobulin and mucin‐domain containing 3) were decreased in the tumor center compared to the invasive margin. Only a small subset of patients had a “hot” TIME. In contrast, a signature of low T‐cell infiltration and high PD‐1 or LAG3 expression was associated with poor overall survival. Correlation of immune checkpoints and genetic mutations identified an association of BRCA1‐associated protein 1 loss‐of‐function mutation with increased expression of the immune checkpoint, B7H4. Tumors with a “cold” TIME have been inherently resistant to ICI therapy. However, broad profiling of the CCA TIME may facilitate development of, and predict response to, novel immunotherapy approaches. (Hepatology 2022;75:297‐308). ALCOHOL AND OBESITY ADDITIVELY INCREASE CIRRHOSIS RISK Gustavo Ayares4 and Juan Pablo Arab4 The relationship between alcohol and obesity on the risk of cirrhosis morbidity remains unclear given that some studies have demonstrated a reduction and others have demonstrated an additive or supra‐additive effect. To assess this interaction in a large population‐based study, Innes et al. used data from the UKB (United Kingdom Biobank) study and examined the relationship between body mass index (BMI) and alcohol intake on cirrhosis morbidity, which was defined by first‐time hospital admission for cirrhosis. Incidence of cirrhosis morbidity increased with alcohol intake and BMI independently. Excess cumulative incidence was highest for obese harmful drinkers (1.83%; 95% CI, 1.46–2.20), compared to harmful drinking alone (1.22%; 95% CI, 0.89–1.55), and obesity alone (0.4%; 95% CI, 0.34–0.46). These findings are consistent with previous studies that have demonstrated an additive effect of alcohol and BMI on the risk of liver disease. (Hepatology 2022;75:369‐78).

Histone acetylation of bile acid transporter genes plays a critical role in cirrhosis

Owing to the lack of genetic animal models that adequately recreate key clinical characteristics of cirrhosis, the molecular pathogenesis of cirrhosis has been poorly characterized, and treatments remain limited. Hence, we aimed to better elucidate the pathological mechanisms of cirrhosis using a novel murine model. We report on the first murine genetic model mimicking human cirrhosis induced by hepatocyte-specific elimination of microspherule protein 1 (MCRS1), a member of non-specific lethal (NSL) and INO80 chromatin-modifier complexes. Using this genetic tool with other mouse models, cell culture and human samples, combined with quantitative proteomics, single nuclei/cell RNA sequencing and chromatin immunoprecipitation assays, we investigated mechanisms of cirrhosis. MCRS1 loss in mouse hepatocytes modulates the expression of bile acid (BA) transporters - with a pronounced downregulation of Na+-taurocholate cotransporting polypeptide (NTCP) - concentrating BAs in sinusoids and thereby activating hepatic stellate cells (HSCs) via the farnesoid X receptor (FXR), which is predominantly expressed in human and mouse HSCs. Consistently, re-expression of NTCP in mice reduces cirrhosis, and genetic ablation of FXR in HSCs suppresses fibrotic marks in mice and invitro cell culture. Mechanistically, deletion of a putative SANT domain from MCRS1 evicts histone deacetylase 1 from its histone H3 anchoring sites, increasing histone acetylation of BA transporter genes, modulating their expression and perturbing BA flow. Accordingly, human cirrhosis displays decreased nuclear MCRS1 and NTCP expression. Our data reveal a previously unrecognized function of MCRS1 as a critical histone acetylation regulator, maintaining gene expression and liver homeostasis. MCRS1 loss induces acetylation of BA transporter genes, perturbation of BA flow, and consequently, FXR activation in HSCs. This axis represents a central and universal signaling event in cirrhosis, which has significant implications for cirrhosis treatment. By genetic ablation of MCRS1 in mouse hepatocytes, we generate the first genetic mouse model of cirrhosis that recapitulates human features. Herein, we demonstrate that the activation of the bile acid/FXR axis in liver fibroblasts is key in cirrhosis development.

Sodium salicylate rewires hepatic metabolic pathways in obesity and attenuates IL-1β secretion from adipose tissue: The implications for obesity-impaired reverse cholesterol transport

IntroductionHigh-fat diet (HFD)-induced obesity impairs clearance of cholesterol through the Reverse Cholesterol Transport (RCT) pathway, with downregulation in hepatic expression of cholesterol and bile acid transporters, namely ABCG5/8 and ABCB11, and reduced high-density lipoprotein (HDL) cholesterol efflux capacity (CEC). In the current study, we hypothesized that the development of hepatosteatosis, secondary to adipose-tissue dysfunction, contributes to obesity-impaired RCT and that such effects could be mitigated using the anti-inflammatory drug sodium salicylate (NaS). Materials and methodsC57BL/6J mice, fed HFD ± NaS or low-fat diet (LFD) for 24 weeks, underwent glucose and insulin tolerance testing. The 3H-cholesterol movement from macrophage-to-feces was assessed in vivo. HDL-CEC was determined ex vivo. Cytokine secretion from adipose-derived stromal vascular fraction (SVF) cells was measured ex vivo. Liver and HDL proteins were determined by mass spectrometry and analyzed using Ingenuity Pathway Analysis. ResultsNaS delayed HFD-induced weight gain, abrogated priming of pro-IL-1β in SVFs, attenuated insulin resistance, and prevented steatohepatitis (ectopic fat accumulation in the liver). Prevention of hepatosteatosis coincided with increased expression of PPAR-alpha/beta-oxidation proteins with NaS and reduced expression of LXR/RXR-induced proteins including apolipoproteins. The latter effects were mirrored within the HDL proteome in circulation. Despite remarkable protection shown against steatosis, HFD-induced hypercholesterolemia and repression of the liver-to-bile cholesterol transporter, ABCG5/8, could not be rescued with NaS. Discussions and conclusionsThe cardiometabolic health benefits of NaS may be attributed to the reprogramming of hepatic metabolic pathways to increase fatty acid utilization in the settings of nutritional overabundance. Reduced hepatic cholesterol levels, coupled with reduced LXR/RXR-induced proteins, may underlie the lack of rescue of ABCG5/8 expression with NaS. This remarkable protection against HFD-induced hepatosteatosis did not translate to improvements in cholesterol homeostasis.

Placental Expression of Bile Acid Transporters in Intrahepatic Cholestasis of Pregnancy.

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-related condition characterized by increased maternal circulating bile acids (BAs) having adverse fetal effects. We investigated whether the human placenta expresses specific regulation patterns to prevent fetal exposition to harmful amounts of BAs during ICP. Using real-time quantitative PCR, we screened placentae from healthy pregnancies (n = 12) and corresponding trophoblast cells (n = 3) for the expression of 21 solute carriers and ATP-binding cassette transporter proteins, all acknowledged as BA- and/or cholestasis-related genes. The placental gene expression pattern was compared between healthy women and ICP patients (n = 12 each). Placental SLCO3A1 (OATP3A1) gene expression was significantly altered in ICP compared with controls. The other 20 genes, including SLC10A2 (ASBT) and EPHX1 (EPOX, mEH) reported for the first time in trophoblasts, were comparably abundant in healthy and ICP placentae. ABCG5 was undetectable in all placentae. Placental SLC10A2 (ASBT), SLCO4A1 (OATP4A1), and ABCC2 mRNA levels were positively correlated with BA concentrations in ICP. Placental SLC10A2 (ASBT) mRNA was also correlated with maternal body mass index. We conclude that at the transcriptional level only a limited response of BA transport systems is found under ICP conditions. However, the extent of the transcriptional response may also depend on the severity of the ICP condition and the magnitude by which the maternal BA levels are increased.

Sub-chronic exposure to realgar induces liver injury via upregulating the TXNIP/NLRP3 pathway and disturbing bile acid homeostasis in mice

Ethnopharmacological relevanceRealgar is a traditional Chinese medicine used in China for a long history. Long-time or excessive use of realgar causes liver injury. However, its underlying mechanism is not fully clarified. Aim of the studyIn this study, we investigated the toxic effect of sub-chronic exposure to realgar on mice liver, and further revealed its underlying mechanism focused on the TXNIP/NLRP3 pathway and bile acid homeostasis. Material and methodsMice were divided into control and different doses of sub-chronic realgar exposed groups. Total arsenic levels in the blood and liver were determined by atomic fluorescence spectrometry. The effect of realgar on liver function was evaluated by biochemical analysis and histopathological examination. Assay kits were applied for the measurement of oxidative stress indexes, MPO and plasma inflammatory cytokines. The mRNA and proteins involved in the TXNIP/NLRP3 and NF-κB pathways were determined by RT-qPCR, western blot, Immunofluorescence and Immunohistochemistry. UHPLC/MS/MS was used for the quantitative analysis of bile acids (BAs) in mice plasma, liver and urine. The genes related to BAs metabolism were measured by RT-qPCR. ResultsSub-chronic exposure to realgar led to arsenic accumulation and caused oxidative damage and inflammatory infiltration in mouse liver, finally resulting in liver injury. Realgar treatment activated the NF-κB pathway and significantly upregulated the TXNIP/NLRP3 pathway in mouse liver. Realgar altered the metabolic balance of BAs, which is related to the abnormal expression of BAs transporters and enzymes. ConclusionSub-chronic exposure to realgar caused liver injury in mouse, and the mechanism may involve the upregulation of the TXNIP/NLRP3 pathway and disordered BAs homeostasis.

Combination Treatment of Arazyme and Soy Leaf Extract Attenuates Hyperglycemia and Hepatic Steatosis in High-Fat Diet-Fed C57BL/6J Mice.

Arazyme and extracts of soy leaves (ESLs) are used as ingredients for functional foods; however, their combined administration has not been studied. This study assessed the combined effect of Arazyme and ESLs in high-fat-diet (HFD)-induced obese C57BL/6J mice fed 2 mg/kg Arazyme, 50 mg/kg ESLs, or a combination of 2 mg/kg Arazyme and 50 mg/kg ESLs by oral gavage for 13 weeks. Individually, Arazyme and ESLs had no effect on the HFD-induced phenotypes. The combination of Arazyme and ESLs significantly suppressed body weight gain, improved glucose and insulin tolerance, and suppressed hepatic steatosis by reducing lipid synthesis and enhancing lipid utilization gene expression. Furthermore, the combination significantly reduced HFD-induced plasma bile acid reabsorption by suppressing bile acid transporter expression, including the ATP biding cassette subfamily B member 11 (Abcb11), solute carrier family 10 member 1 (Slc10a1), Slc10a2, Slc51a, and Slc51b in the liver and gut. Moreover, the combination of Arazyme and ESLs significantly prevented HFD-induced islet compensation in the pancreas. These results suggest that the incorporation of Arazyme combined with ESLs reduces HFD-induced body weight, hyperglycemia, and hepatic steatosis by regulating liver–gut bile acid circulation in HFD-fed mice. This combination can markedly reduce treatment doses and enhance their therapeutic effects, thereby reducing therapeutic healthcare costs.

ASBT Vivo‐Morpholino Decreases Hepatic Mast Cell, Fibrosis and Biliary Senescence in Mdr2 ‐/‐ Mice

Background Primary Sclerosing Cholangitis (PSC) is characterized by increased biliary senescence, liver fibrosis, and mast cell (MC) infiltration. Mdr2-/- mice, model of PSC, mimic some features of PSC. PSC features include (i) increased conjugated total bile acid (TBA) levels, (ii) dysregulation of BA synthesis and (iii) higher hepatic expression of the apical sodium bile acid transporter (ASBT), expressed only by cholangiocytes. Hydrophobic BAs enhance cholestasis, biliary proliferation and ASBT expression. MCs infiltrate the liver in PSC and reside near senescent cholangiocytes, increasing biliary damage and TBA levels. ASBT is regulated by farnesoid X receptor (FXR) and blocking MC-FXR decreases biliary damage, fibrosis, and biliary ASBT expression. No information exists to demonstrate if blocking hepatic ASBT inhibits MC activation and subsequent liver damage in Mdr2-/- mice. Methods 12 wk male Mdr2−/− mice were treated with control or ASBT Vivo-Morpholino (2 injections, 12.5 mg oligo/kg BW via tail vein for 1 wk). Liver damage was determined by H&E and serum enzyme levels. Ductular reaction (DR) was evaluated by immunohistochemistry (IHC) for CK-19, Ki-67 and immunofluorescence (IF) for p16, co-stained with CK-19. ASBT expression was measured by qPCR and IF in liver, isolated cholangiocytes and small intestine. MC infiltration was measured by IHC for Tryptase β-2 in liver and intestine. Hepatic fibrosis was determined by Fast Green-Sirius Red staining. TBA levels were measured in serum, liver and feces. BA receptors/transporters were co-stained with CK-19 in small intestine and liver. Results Mdr2−/− mice have increased DR, fibrosis, and serum/hepatic TBA levels, which decrease in Mdr2−/− ASBT Vivo-Morpholino mice. Inhibition of ASBT in Mdr2-/- mice reduced (i) biliary and intestinal ASBT expression and (ii) serum and hepatic CA and CDCA levels. Fecal TBA levels decreased, but CA and CDCA levels increased, following ASBT Vivo-Morpholino. ASBT Vivo-Morpholino treatment reduced hepatic and intestinal MC-infiltration/activation. Conclusion Elevated serum TBA levels seen in PSC lead to increased intestinal BA transport inducing MC infiltration and increased biliary ASBT. Inhibition of biliary ASBT blocks hepatic and intestinal MC recruitment and subsequent DR, hepatic fibrosis and TBA levels. Targeting MCs via ASBT inhibition is a novel strategy for the management of PSC.

Neurotensin differentially regulates bile acid metabolism and intestinal FXR-bile acid transporter axis in response to nutrient abundance.

Studies demonstrate a role for neurotensin (NT) in obesity and related comorbidities. Bile acid (BA) homeostasis alterations are associated with obesity. We determined the effect of NT on BA metabolism in obese and non-obese conditions. Plasma and fecal BA profiles were analyzed by LC-MS/MS in male and female NT+/+ and NT-/- mice fed low-fat (LFD) or high-fat diet (HFD) for 6weeks (early stage of obesity) or greater than 20weeks (late stage of obesity). The nuclear farnesoid X receptor (FXR) and BA transporter mRNA expression were assessed in ileum, mouse enteroids, and human cell lines. HFD decreased plasma primary and secondary BAs in NT+/+ mice; HFD-induced decrease of plasma BAs was improved in NT-deficient mice. In NT+/+ mice, HFD inhibited ileal FXR and BA transporter expression; HFD-decreased expression of FXR and BA transporters was prevented in NT-/- mice. Compared with LFD-fed NT+/+ mice, LFD-fed NT-/- mice had relatively lower levels of ileal FXR and BA transporter expression. Moreover, NT stimulates the expression of FXR and BA transporters in Caco-2 cells; however, stimulated expression of BA transporters was attenuated in NT-/- enteroids. Therefore, we demonstrate that HFD disrupts the BA metabolism and ileal FXR and BA transporter axis which are improved in the absence of NT, suggesting that NT contributes to HFD-induced disruption of BA metabolism and plays an inhibitory role in the regulation of ileal FXR and BA transporter signaling under obese conditions. Conversely, NT positively regulates the expression of ileal FXR and BA transporters under non-obese conditions. Therefore, NT plays a dual role in obese and non-obese conditions, suggesting possible therapeutic strategies for obesity control.

Protective Functions of ZO-2/Tjp2 Expressed in Hepatocytes and Cholangiocytes Against Liver Injury and Cholestasis

Liver tight junctions (TJs) establish tissue barriers that isolate bile from the blood circulation. TJP2/ZO-2-inactivating mutations cause progressive cholestatic liver disease in humans. Because the underlying mechanisms remain elusive, we characterized mice with liver-specific inactivation of Tjp2. Tjp2 was deleted in hepatocytes, cholangiocytes, or both. Effects on the liver were assessed by biochemical analyses of plasma, liver, and bile and by electron microscopy, histology, and immunostaining. TJ barrier permeability was evaluated using fluorescein isothiocyanate-dextran (4 kDa). Cholic acid (CA) diet was used to assess susceptibility to liver injury. Liver-specific deletion of Tjp2 resulted in lower Cldn1 protein levels, minor changes to the TJ, dilated canaliculi, lower microvilli density, and aberrant radixin and bile salt export pump (BSEP) distribution, without an overt increase in TJ permeability. Hepatic Tjp2-defcient mice presented with mild progressive cholestasis with lower expression levels of bile acid transporter Abcb11/Bsep and detoxification enzyme Cyp2b10. A CA diet tolerated by control mice caused severe cholestasis and liver necrosis in Tjp2-deficient animals. 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene ameliorated CA-induced injury by enhancing Cyp2b10 expression, and ursodeoxycholic acid provided partial improvement. Inactivating Tjp2 separately in hepatocytes or cholangiocytes showed only mild CA-induced liver injury. Tjp2 is required for normal cortical distribution of radixin, canalicular volume regulation, and microvilli density. Its inactivation deregulated expression of Cldn1 and key bile acid transporters and detoxification enzymes. The mice provide a novel animal model for cholestatic liver disease caused by TJP2-inactivating mutations in humans.

Enterobacter aerogenes ZDY01 inhibits choline-induced atherosclerosis through CDCA-FXR-FGF15 axis.

Atherosclerosis is the leading cause of cardiovascular diseases worldwide. Trimethylamine N-oxide (TMAO), a metabolite of intestinal flora from dietary quaternary amines, has been shown to be closely related to the development of atherosclerosis. Previous studies have shown that Enterobacter aerogenes ZDY01 significantly reduces the serum levels of TMAO and cecal trimethylamine (TMA) in Balb/c mice; however, its role in the inhibition of choline-induced atherosclerosis in ApoE-/- mice remains unclear. Here, we demonstrated that E. aerogenes ZDY01 inhibited choline-induced atherosclerosis in ApoE-/- mice fed with 1.3% choline by reducing cecal TMA and modulating CDCA-FXR/FGF15 axis. We observed that E. aerogenes ZDY01 decreased the cecal TMA and serum TMAO levels by utilizing cecal TMA as a nutrient, not by changing the expression of hepatic FMO3 and the composition of gut microbiota. Furthermore, E. aerogenes ZDY01 enhanced the expression of bile acid transporters and reduced the cecal CDCA levels, thereby attenuating the FXR/FGF15 pathway, upregulating the expression of Cyp7a1, promoting reverse cholesterol transport. Taken together, E. aerogenes ZDY01 attenuated choline-induced atherosclerosis in ApoE-/- mice by decreasing cecal TMA and promoting reverse cholesterol transport, implying that E. aerogenes ZDY01 treatment might have therapeutic potential in atherosclerosis.

Gut Immunity and Microbiota Dysbiosis Are Associated with Altered Bile Acid Metabolism in LPS-Challenged Piglets.

Bacterial infections are among the major factors that cause stress and intestinal diseases in piglets. Lipopolysaccharide (LPS), a major component of the Gram-negative bacteria outer membrane, is commonly employed for inducing an immune response in normal organisms for convenience. The association between LPS stimulation and gut immunity has been reported. However, the effects of gut immunity on microbial homeostasis and metabolism of host, especially bile acid and lipid metabolism in piglets, remain unclear. Hence, in the current study, we elucidated the effect of gut immunity on microbial balance and host metabolism. Twenty-one-day-old healthy piglets (male) were randomly assigned into the CON and LPS groups. After 4 hours of treatment, related tissues and cecal contents were obtained for further analysis. The obtained results showed that stimulated LPS considerably damaged the morphology of intestinal villi and enhanced the relative expression of proinflammatory cytokines. Besides, LPS partially changed the microbial structure as indicated by β-diversity and increased operational taxonomic units (OTUs) related to Oxalobacter and Ileibacterium. Furthermore, bile acid, a large class of gut microbiota metabolites, was also assessed by many proteins related to the enterohepatic circulation of bile acids. It was also revealed that LPS markedly inhibited the mRNA and protein expression of TGR5 and FXR (bile acid receptors) in the ileum, which expressed negative feedback on bile acid de novo synthesis. Additionally, results indicated upregulated mRNA of genes associated with the production of bile acid in the liver tissues. Moreover, LPS reduced the expression of bile acid transporters in the ileum and liver tissues and further disturbed the normal enterohepatic circulation. Taken together, gut immunity and microbial dysbiosis are associated with altered bile acid metabolism in LPS-challenged piglets, which provided theoretical basis for revealing the potential mechanism of intestinal inflammation in swine and seeking nutrients to resist intestinal damage.

Lactobacillus delbrueckii Interfere With Bile Acid Enterohepatic Circulation to Regulate Cholesterol Metabolism of Growing-Finishing Pigs via Its Bile Salt Hydrolase Activity.

Microbiota-targeted therapies for hypercholesterolemia get more and more attention and are recognized as an effective strategy for preventing and treating cardiovascular disease. The experiment was conducted to investigate the cholesterol-lowering mechanism of Lactobacillus delbrueckii in a pig model. Twelve barrows (38.70 ± 5.33 kg) were randomly allocated to two groups and fed corn–soybean meal diets with either 0% (Con) or 0.1% Lactobacillus delbrueckii (Con + LD) for 28 days. L. delbrueckii–fed pigs had lower serum contents of total cholesterol (TC), total bile acids (TBAs), and triglyceride, but higher fecal TC and TBA excretion. L. delbrueckii treatment increased ileal Lactobacillus abundance and bile acid (BA) deconjugation and affected serum and hepatic BA composition. Dietary L. delbrueckii downregulated the gene expression of ileal apical sodium-dependent bile acid transporter (ASBT) and ileal bile acid binding protein (IBABP), and hepatic farnesoid X receptor (FXR), fibroblast growth factor (FGF19), and small heterodimer partner (SHP), but upregulated hepatic high-density lipoprotein receptor (HDLR), low-density lipoprotein receptor (LDLR), sterol regulatory element binding protein-2 (SREBP-2), and cholesterol-7α hydroxylase (CYP7A1) expression. Our results provided in vivo evidence that L. delbrueckii promote ileal BA deconjugation with subsequent fecal TC and TBA extraction by modifying ileal microbiota composition and induce hepatic BA neosynthesis via regulating gut–liver FXR–FGF19 axis.

Inflammation Drives MicroRNAs to Limit Hepatocyte Bile Acid Transport in Murine Biliary Atresia

BackgroundBiliary atresia (BA) is an inflammatory pediatric cholangiopathy with only surgical means for treatment. Many contributors to bile acid synthesis and transport have previously been reported to be downregulated in patients with BA; yet, the driving factors of the abnormal bile acid synthesis and transport in regard to BA have not been previously studied. Materials and methodsWild type or Ig-α-/- mice were injected with salt solution (control) or rotavirus on day of life 0, and analyses were performed on day of life 14. The mRNA levels of bile acid transporters/nuclear receptors and liver microRNAs (miRNAs) were compared between groups. A mouse hepatocyte cell line was used to examine the effects of innate cytokines on miRNA levels and bile acid transporter/nuclear receptor expression and miRNAs on bile acid transporter/nuclear receptor expression. ResultsBA mice had significantly increased mRNA expression of innate cytokines and miRNAs known to bind bile acid transporters/nuclear receptors (miRNAs -22-5p, -34a-5p, and -222-3p) and decreased mRNA expression of bile acid transporters and nuclear receptors. In vitro, TNF-α and IL-1β decreased BSEP and CYP7A1 while increasing miRNA-34a-5p and miRNA 222-3p. LXR, SHP, CYP7A1, NTCP, and MRP2 were decreased by miRNA-34a-5p, whereas miRNA-222-3p decreased NTCP and MRP4. TNF-α and IL-1β increased expression of miRNAs 34a-5p and 222-3p and these miRNAs then decrease expression of multiple bile acid transporters and nuclear receptors. ConclusionsLoss of bile acid transporters increases hepatotoxicity via bile acid retention. Therapeutic agents that increase bile acid transport or nuclear receptor functioning should be investigated in BA.

1662-P: Lower Expression of Bile Acid Transporters and Fibroblast Growth Factor 19 in Mucosa Biopsies from the Ileocecal Region in Persons with Type 2 Diabetes Compared with Healthy Controls

Background and Aims: Bile acids stimulate the secretion of gluco-regulatory and appetite-regulating gut hormones (including glucagon-like peptide 1 and fibroblast factor 19 (FGF19)) and increase energy expenditure. Changes in postprandial concentrations of circulating bile acids (primarily determined by the apical sodium-dependent bile acid transporter (ASBT) and organic solute transporter α/β (OSTα/β) in the terminal ileum) have been linked to type 2 diabetic pathophysiology. Methods: In a cross-sectional study, 12 persons with type 2 diabetes (T2D) (mean[range] age 51[34-63] years, BMI 26.8[23.7-31.5] kg/m2, HbA1c 48[36-85] mmol/mol) and 12 gender, age and BMI-matched nondiabetic controls (CTRLs) (age 50[41-67] years, BMI 27.1[20.3-30.8] kg/m2, HbA1c 34[29-43] mmol/mol) underwent upper and lower double-balloon enteroscopy with retrieval of mucosal biopsies from every 30 cm along the entire intestinal tract. The biopsies were subjected to full mRNA sequencing and here, the expression of ASBT, OSTα/β and FGF19 is reported. Results: For both groups, robust expression (reads per kilobase million (RPKM) ≥1) of ASBT and OSTα/β was evident in the duodenum and this increased along the small intestine, with highest expression of ASBT in the ileocecal region and highest expression of OSTα/β in terminal ileum. Compared to healthy controls, persons with T2D exhibited significantly lower ASBT and OSTα/β expression in the ileocecal region (P<0.05). Compared to persons with T2D in whom expression of FGF19 was below 1 RPKM in all locations, the control group had robust and significantly greater expression of FGF19 in the terminal ileum (P<0.01). Conclusions: T2D is associated with reduced expression of ASBT, OSTα/β and FGF19 in the terminal ileum and the proximal colon. This may point to a link between dysfunctional enterohepatic circulation of bile acids and type 2 diabetic pathophysiology. Disclosure H.H. Nerild: Employee; Spouse/Partner; Novo Nordisk A/S. A. Brønden: None. A. El Haddouchi: None. T. Jorsal: None. D.P. Sonne: None. J. Jelsing: Board Member; Self; Gubra. Stock/Shareholder; Self; Gubra. K. Rigbolt: Employee; Self; Gubra. T. Vilsbøll: Advisory Panel; Self; AstraZeneca, Mundipharma International, Novo Nordisk A/S, Sun Pharmaceutical Industries Ltd. Consultant; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Lilly Diabetes, Medscape, Merck Sharp & Dohme Corp., Sanofi. F.K. Knop: Advisory Panel; Self; AstraZeneca, Merck Sharp & Dohme Corp., Mundipharma International, Novo Nordisk A/S, Sanofi. Consultant; Self; Carmot Therapeutics, Inc., Eli Lilly and Company, Novo Nordisk A/S. Research Support; Self; AstraZeneca, Gubra, Novo Nordisk A/S, Sanofi, Zealand Pharma A/S. Speaker’s Bureau; Self; AstraZeneca, Lupin Pharmaceuticals, Inc., Merck Sharp & Dohme Corp., Norgine B.V., Novo Nordisk A/S.

Pediatric Cholestatic Liver Disease: Review of Bile Acid Metabolism and Discussion of Current and Emerging Therapies.

Cholestatic liver diseases are a significant cause of morbidity and mortality and the leading indication for pediatric liver transplant. These include diseases such as biliary atresia, Alagille syndrome, progressive intrahepatic cholestasis entities, ductal plate abnormalities including Caroli syndrome and congenital hepatic fibrosis, primary sclerosing cholangitis, bile acid synthesis defects, and certain metabolic disease. Medical management of these patients typically includes supportive care for complications of chronic cholestasis including malnutrition, pruritus, and portal hypertension. However, there are limited effective interventions to prevent progressive liver damage in these diseases, leaving clinicians to ultimately rely on liver transplantation in many cases. Agents such as ursodeoxycholic acid, bile acid sequestrants, and rifampicin have been mainstays of treatment for years with the understanding that they may decrease or alter the composition of the bile acid pool, though clinical response to these medications is frequently insufficient and their effects on disease progression remain limited. Recently, animal and human studies have identified potential new therapeutic targets which may disrupt the enterohepatic circulation of bile acids, alter the expression of bile acid transporters or decrease the production of bile acids. In this article, we will review bile formation, bile acid signaling, and the relevance for current and newer therapies for pediatric cholestasis. We will also highlight further areas of potential targets for medical intervention for pediatric cholestatic liver diseases.

Mast Cell Signaling Regulates Biliary Farnesoid X Receptor and Apical Sodium Bile Acid Transporter Expression During Cholestatic Liver Injury

BackgroundChronic liver diseases and cholangiopathies (targets cholangiocytes) are diagnosed at advanced stages leading to poor prognosis. Mast cells (MCs) infiltrate the liver during damage, release histamine (HA) and act via H1‐H4 HA receptors (HRs). Cholangiocytes and MCs express apical sodium bile acid transporter (ASBT); farnesoid X receptor (FXR) and H2HR. Introduction of MCs into C57BL/6 (WT) and MC deficient (KitW‐sh) mice increases liver damage, biliary proliferation/liver fibrosis, and total bile acid (TBA) levels. We aimed to determine if loss of MC FXR signaling alters biliary FXR and ASBT, injury and hepatic fibrosis via HA/H2HR signaling.MethodsWT or KitW‐sh mice were subjected to tail vein injection of cultured MCs (ATCC®) pretreated with PBS (PBS‐MC) to induce liver damage or the FXR inhibitor, Z‐guggulsterone (10μM, FXR‐MC) and sacrificed after 3 days. H&E was used to determine pathological changes. Ductular reaction was determined by CK‐19 staining. Biliary proliferation and hepatic senescence were evaluated by Ki‐67 and SA‐b‐galactosidase staining. Hepatic fibrosis was determined by Sirius Red staining. Hepatic stellate cell (HSC) presence was determined by immunofluorescence (IF). Biliary senescence was determined by IF and qPCR. Cholangiocyte FXR and ASBT expression was determined by staining and qPCR in cholangiocytes. Senescence associated secretory phenotypes (SASPs) and HA secretion were measured by EIA in serum and cholangiocyte supernatants. H2HR expression was evaluated in isolated cholangiocytes. TBAs were measured in serum and liver tissues. In vitro, cultured MCs were treated with PBS or Z‐guggulsterone (10μM, 48 hr). Treated MC supernatants were placed on cholangiocytes prior to evaluating biliary ASBT and H2HR expression and secretion of HA, TGF‐b1 and IL‐1b by qPCR and EIA, respectively. In human tissue from patients with PSC or PBC, biliary ASBT, FXR and MC infiltration (marked by tryptase) was measured by IHC.ResultsHepatic damage, ductular reaction, biliary senescence, ASBT expression and liver fibrosis were induced in WT and KitW‐sh mice injected with PBS‐MC; whereas, these parameters were reversed in mice injected with FXR‐MCs. HA secretion and H2HR expression increased in serum and cholangiocytes, respectively in WT and KitW‐sh mice injected with PBS‐MC, but were reduced in mice injected with FXR‐MC. Hepatic and serum TBA increased in WT and KitW‐sh mice injected with PBS‐MC and were reduced in FXR‐MC groups. In vitro, stimulation with MC supernatant increased biliary ASBT, FXR, H2HR, HA, TGF‐b1 and IL‐1b expression. These parameters decreased in cholangiocytes treated with FXR‐MC supernatant. In human tissues, upregulation of MCs was coupled with increased biliary FXR and ASBT expression.ConclusionsReintroduction of MCs recapitulates cholestatic liver injury in WT and KitW‐sh mice. Inhibition of MC FXR signaling decreases biliary ASBT expression, liver injury and hepatic fibrosis via downregulation of HA/H2HR signaling. Manipulation of BA transporters, receptors and MC mediators may be a therapeutic route for patients with cholestatic liver damage.Support or Funding InformationNIH NIDDK R01s, VA Merit, IUSOM

The alpha-glucosidase inhibitor miglitol increases hepatic CYP7A1 activity in association with altered short-chain fatty acid production in the gut of obese diabetic mice

PurposeBile acids (BAs) have been shown to contribute to glucose and energy homeostasis. We have recently reported that miglitol, an alpha-glucosidase inhibitor, increases fecal BA excretion and ameliorate insulin resistance and obesity in mice. The aim of this study was to clarify the mechanisms by which miglitol affects BA metabolism. The expression of genes regulating BA metabolism, gut microbiome and short-chain fatty acids (SCFA) were examined. ProceduresNSY mice, representing an obese type 2 diabetic model, were fed with a high-fat diet with or without miglitol for 4 weeks. The expression of BA-related genes in the liver and the lower intestine were measured. Alterations in fecal microbiome, fecal SCFA along with plasma lipid levels were also evaluated. Major findingsMiglitol significantly increased fecal BA secretion and markedly upregulated the mRNA expression, protein levels and enzyme activity of hepatic cholesterol 7α-hydroxylase, a rate-limiting enzyme of BA synthesis. In the intestine, miglitol treatment significantly suppressed the mRNA expression of apical sodium-dependent bile acid transporter and ATP-binding cassette transporter G5 and G8. In fecal microbiome, the prevalence of prevotella was remarkably reduced and that of clostridium subcluster XIVa was increased by miglitol. Miglitol elevated formic and n-butyric acids along with total SCFA concentration in feces, while succinic acid was decreased. There was no change in plasma total cholesterol levels. ConclusionsCollectively, miglitol may affect BA metabolism via enhanced CYP7A1 activity resulting from at least in part the alterations in gut microbiome and SCFA production in obese diabetic mice.

Wnt/β-Catenin Signaling Plays a Protective Role in the Mdr2 Knockout Murine Model of Cholestatic Liver Disease.

The Wnt/β-catenin signaling pathway has a well-described role in liver pathobiology. Its suppression was recently shown to decrease bile acid (BA) synthesis, thus preventing the development of cholestatic liver injury and fibrosis after bile duct ligation (BDL). To generalize these observations, we suppressed β-catenin in Mdr2 knockout (KO) mice, which develop sclerosing cholangitis due to regurgitation of BA from leaky ducts. When β-catenin was knocked down (KD) in KO for 2 weeks, hepatic and biliary injury were exacerbated in comparison to KO given placebo, as shown by serum biochemistry, ductular reaction, inflammation, and fibrosis. Simultaneously, KO/KD livers displayed increased oxidative stress and senescence and an impaired regenerative response. Although the total liver BA levels were similar between KO/KD and KO, there was significant dysregulation of BA transporters and BA detoxification/synthesis enzymes in KO/KD compared with KO alone. Multiphoton intravital microscopy revealed a mixing of blood and bile in the sinusoids, and validated the presence of increased serum BA in KO/KD mice. Although hepatocyte junctions were intact, KO/KD livers had significant canalicular defects, which resulted from loss of hepatocyte polarity. Thus, in contrast to the protective effect of β-catenin KD in BDL model, β-catenin KD in Mdr2 KO aggravated rather than alleviated injury by interfering with expression of BA transporters, hepatocyte polarity, canalicular structure, and the regenerative response. The resulting imbalance between ongoing injury and restitution led to worsening of the Mdr2 KO phenotype, suggesting caution in targeting β-catenin globally for all cholestatic conditions.

Alteration in bile acids profile in Large White pigs during chronic heat exposure.

Bile acids (BAs) are critical for cholesterol homeostasis and new roles in metabolism and endocrinology have been demonstrated recently. It remains unknown whether BA metabolism can be affected by heat stress (HS). The objective of this study was to describe the shifts in serum, hepatic and intestinal BA profiles induced by chronic HS. Twenty-seven Large White pigs weighing 40.8 ± 2.7 kg were assigned to one of the three treatments: a control group (CON, 23 °C), a HS group (33 °C), or a pair-fed group (PF, 23 °C and fed the same amount as HS group) for 21 d. The concentrations of taurine-conjugated BAs (TUDCA and THDCA in serum and TCDCA, TUDCA, THDCA and THCA in liver) were decreased in HS and PF pigs. However, in HS pigs, a reduction in taurine-conjugated BAs (TCBA) correlated with decreased liver genes expression of BA synthesis, conjugation and uptake transport. BA regulated-genes (FXR, TGR5 and FGFR4) in HS pigs and TGR5, FGFR4 and KLβ in PF pigs were down-regulated in liver. In ileum, total BAs and glycoursodeoxycholic acid concentrations were higher in HS pigs than other groups and PF group, respectively (P < 0.05). TCBA (P = 0.01) and tauroursodeoxycholic acid (P < 0.01) were decreased in PF group. BA transporters (OSTα and MRP3) were up-regulated in HS pigs compared with CON and PF pigs, respectively (P < 0.01). In cecum, ursodeoxycholic acid was higher in HS (P = 0.02) group than CON group. The expression of apical sodium-coupled bile acid transporter (P = 0.04) was lower in HS pigs than CON pigs, while OSTβ (P < 0.01) was greater in HS group than PF group. These results suggest that chronic HS suppressed liver activity of synthesis and uptake of TCBA, at least in part, which was independent of reduced feed intake.

Ursodeoxycholic acid accelerates bile acid enterohepatic circulation.

Ursodeoxycholic acid (UDCA) is the first-line treatment for primary biliary cholangitis, but its effects on the enterohepatic circulation of bile acid (BA) have been under-investigated. Therefore, we studied the influence of UDCA on BA enterohepatic circulation in vivo and the mechanisms by which UDCA affects the BA kinetics. Mice were treated with UDCA and other BAs to observe changes in BA pool and BA transporters involved in enterohepatic circulation. Isotope dilution techniques and biochemical analyses were applied to study BA kinetics after oral administration of UDCA, and the mechanism involved. Oral administration of UDCA in mice reduced the overall BA pool and produced a unique BA profile with high-abundance conjugated UDCA species, including tauroursodeoxycholic acid (TUDCA) and GUDCA. We found increased expression of several main BA transporters in the ileum and liver. BA kinetic experiment showed that feeding UDCA shortened cycling time of BA and accelerated BA enterohepatic circulation. Additionally, we found evidence that the effect of UDCA administration on accelerating BA enterohepatic circulation was due to the inhibition of farnesoid X receptor (FXR) signalling in the ileum and FGF15/19 in the liver. Oral administration of UDCA produced a unique BA profile with high-abundance TUDCA and GUDCA and significantly accelerated BA enterohepatic circulation through the inhibition of intestinal FXR signalling and reduced level of FGF15/19, which in turn, induced the expression of BA transporters in the liver. These findings highlight a critical role for UDCA in maintaining the homeostasis of BA enterohepatic circulation in vivo.