Bile Acid Pool Size Research Articles

Gly-β-MCA is a potent anti-cholestasis agent against “human-like” hydrophobic bile acid-induced biliary injury in mice

Cholestasis is a chronic liver disease with limited therapeutic options. Hydrophobic bile acid-induced hepatobiliary injury is a major pathological driver of cholestasis progression. This study investigates the anti-cholestasis efficacy and mechanisms of action of glycine-conjugated β-muricholic acid (Gly-β-MCA). We use female Cyp2c70 KO mice, a rodent cholestasis model that does not produce endogenous muricholic acid (MCA) and exhibits a “human-like” hydrophobic bile acid pool and female-dominant progressive hepatobiliary injury and portal fibrosis. The efficacy of Gly-β-MCA and ursodeoxycholic acid (UDCA), the first line drug for cholestasis, on cholangiopathy and portal fibrosis are compared. At a clinically relevant dose, Gly-β-MCA shows comparable efficacy as UDCA in reducing serum transaminase, portal inflammation and ductular reaction, and better efficacy than UDCA against portal fibrosis. Unlike endogenous bile acids, orally administered Gly-β-MCA is absorbed at low efficiency in the gut and enters the enterohepatic circulation mainly after microbiome-mediated deconjugation, which leads to taurine-conjugated MCA enrichment in bile that alters enterohepatic bile acid pool composition and reduces bile acid pool hydrophobicity. Gly-β-MCA also promotes fecal excretion of endogenous hydrophobic bile acids and decreases total bile acid pool size, while UDCA treatment does not alter total bile acid pool. Furthermore, Gly-β-MCA treatment leads to gut unconjugated MCA enrichment and reduces gut hydrophobic lithocholic acid (LCA) exposure. In contrast, UDCA treatment drives a marked increase of LCA flux through the large intestine. In conclusion, Gly-β-MCA is a potent anti-cholestasis agent with potential clinical application in treating human cholestasis.

IBAT inhibitors in pediatric cholestatic liver diseases: Transformation on the horizon?

Historically, the therapeutic options available to hepatologists managing cholestasis have been limited. Apart from bile acid--binding resins and the choleretic ursodeoxycholic acid, the medical management of cholestasis in children has been predominately focused on managing the complications of cholestasis, namely pruritus, malnutrition, fat-soluble vitamin deficiencies, and portal hypertension. As such, invasive surgical procedures such as biliary diversion and liver transplantation may become the only options for progressive and unremitting cases of cholestasis. Particularly in the pediatric population, where debilitating pruritus is a common indication for a liver transplant, effective anti-cholestatic medications have the potential to prolong native liver survival without the need for biliary diversion. Ileal bile acid transporter (IBAT) inhibitors are a relatively new class of drugs which that target the ileal re-uptake of bile acids, thus interrupting the enterohepatic circulation and reducing the total bile acid pool size and exposure of the liver. Oral, minimally absorbed IBAT inhibitors have been demonstrated to reduce serum bile acid levels and pruritus with a minimal side effect profile in clinical trials in Alagille Ssyndrome and progressive familial intrahepatic cholestasis, leading to FDA and EMA approval. The indications for IBAT inhibitors will likely expand in the coming years as clinical trials in other adult and pediatric cholestatic conditions are ongoing. This review will summarize the published clinical and pre-clinical data on IBAT inhibitors and offer providers guidance on their practical use.

Synthesis, Characterization, and Potential Usefulness in Liver Function Assessment of Novel Bile Acid Derivatives with Near-Infrared Fluorescence (NIRBAD).

Conventional serum markers often fail to accurately detect cholestasis accompanying many liver diseases. Although elevation in serum bile acid (BA) levels sensitively reflects impaired hepatobiliary function, other factors altering BA pool size and enterohepatic circulation can affect these levels. To develop fluorescent probes for extracorporeal noninvasive hepatobiliary function assessment by real-time monitoring methods, 1,3-dipolar cycloaddition reactions were used to conjugate near-infrared (NIR) fluorochromes with azide-functionalized BA derivatives (BAD). The resulting compounds (NIRBADs) were chromatographically (FC and PTLC) purified (>95%) and characterized by fluorimetry, 1H NMR, and HRMS using ESI ionization coupled to quadrupole TOF mass analysis. Transport studies using CHO cells stably expressing the BA carrier NTCP were performed by flow cytometry. Extracorporeal fluorescence was detected in anesthetized rats by high-resolution imaging analysis. Three NIRBADs were synthesized by conjugating alkynocyanine 718 with cholic acid (CA) at the COOH group via an ester (NIRBAD-1) or amide (NIRBAD-3) spacer, or at the 3α-position by a triazole link (NIRBAD-2). NIRBADs were efficiently taken up by cells expressing NTCP, which was inhibited by taurocholic acid (TCA). Following i.v. administration of NIRBAD-3 to rats, liver uptake and consequent release of NIR fluorescence could be extracorporeally monitored. This transient organ-specific handling contrasted with the absence of release to the intestine of alkynocyanine 718 and the lack of hepatotropism observed with other probes, such as indocyanine green. NIRBAD-3 administration did not alter serum biomarkers of hepatic and renal toxicity. NIRBADs can serve as probes to evaluate hepatobiliary function by noninvasive extracorporeal methods.

The supplementation of the multi-strain probiotics WHHPRO™ alleviates high-fat diet-induced metabolic symptoms in rats via gut-liver axis.

Metabolic syndrome (MS) has emerged as one of the major global health concerns, accompanied by a series of related complications, such as obesity and type-2 diabetes. The gut-liver axis (GLA) is a bidirectional communication between the gut and the liver. The GLA alterations have been revealed to be closely associated with the development of MS. Probiotics within Lactobacillus and Bifidobacterium confer beneficial effects on improving MS symptoms. WHHPRO™ is a mixture of four probiotic strains, with potential MS-improving abilities. This study aimed to investigate the effects of WHHPRO™ on MS symptoms using a high-fat diet (HFD) rat model. Oral administration of WHHPRO™ for 12 weeks improved glucose tolerance, blood lipid, body weight, and liver index in HFD rats. WHHPRO™ shaped the gut microbiome composition by increasing the abundance of Lactobacillus and Akkermansia and normalized the reduced SCFA levels in HFD rats. Besides, WHHPRO™ modulated the fecal bile acids (BAs) profile, with decreased levels of T-b-MCA and 12-KDCA and increased levels of LCA and ILCA. Meanwhile, WHHPRO™ increased total unconjugated BAs in feces and liver and reduced the accumulation of total hepatic BA pool size in HFD rats. Moreover, WHHPRO™ reversed the expression of genes associated with impaired BA metabolism signaling in the ileum and liver. Our findings suggest that WHHPRO™ exerted beneficial effects on improving MS symptoms, involving the modulation of the gut microbiome composition, SCFAs, and the FXR-FGF15 signaling along the GLA. Supplementation of WHHPRO™ may serve as a novel strategy for improving MS symptoms.

Bile acids metabolism in the gut-liver axis mediates liver injury during lactation

AimsThe obesity epidemic, especially in pregnant women, linked to a higher risk of liver diseases. Bile acids (BAs) are known to participate in liver metabolism, but this function during obesogenic reproductive process remains largely uncertain. The study aims to identify whether a high-fat diet (HFD) during pregnancy negatively disturbs liver metabolism and the potential role of BAs and gut microbiota (GM)in a sow model. Main methodsReproductive (RP) or non-reproductive (NRP) sows were fed a 15 % HFD containing compound oil. Body condition, blood parameters, and BAs levels/profile during gestation and lactation were monitored. The tissues and colonic GM were collected after euthanasia at the end of lactation. HepG2 hepatocytes were used to test the effects of BAs on liver damage and the mechanism. Key findingsReproductive sows fed an HFD (HF-RP) experienced increased weight loss, and elevated plasma non-esterified fatty acid (NEFA) during lactation, consistent with exacerbated lipolysis, aggravating the risk of liver damage. HF-RP sows exhibited an enlarged BAs pool size and alterations in composition (higher levels of CDCA and LCA species) along with a drastic change in the GM (increased Firmicutes/Bacteroidetes ratio and declined Lactobacillus abundance). Furthermore, the liver FXR-SHP pathway, BAs synthesis and transport underwent adaptive regulation to sustain the BAs homeostasis and hepatic lipid metabolism. CDCA alleviated endoplasmic reticulum (ER) stress induced by palmitic acid via FXR pathway, in HepG2 cells. SignificanceLactation BAs metabolism signal in gut-liver axis coordinated the risk of liver damage induced by exacerbated lipolysis in obesogenic pregnancy.

Targeted bile acids metabolomics in cholesterol gallbladder polyps and gallstones: From analytical method development towards application to clinical samples

Bile acids (BAs) are synthesized by the liver from cholesterol through several complementary pathways and aberrant cholesterol metabolism plays pivotal roles in the pathogeneses of cholesterol gallbladder polyps (CGP) and cholesterol gallstones (CGS). To date, there is neither systematic study on BAs profile of CGP or CGS, nor the relationship between them. To explore the metabolomics profile of plasma BAs in healthy volunteers, CGP and CGS patients, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for simultaneous determination of 42 free and conjugated BAs in human plasma. The developed method was sensitive and reproducible to be applied for the quantification of BAs in the investigation of plasma samples. The results show that, compared to healthy volunteers, CGP and CGS were both characterized by the significant decrease in plasma BAs pool size, furthermore CGP and CGS shared aberrant BAs metabolic characteristics. Chenodeoxycholic acid, glycochenodeoxycholic acid, λ-muricholic acid, deoxycholic acid, and 7-ketolithocholic acid were shared potential markers of these two cholesterol gallbladder diseases. Subsequent analysis showed that clinical characteristics including cysteine, ornithine and body mass index might be closely related to metabolisms of certain BA modules. This work provides metabolomic information for the study of gallbladder diseases and analytical methodologies for clinical target analysis and efficacy evaluation related to BAs in medical institutions.

Glycine-β-Muricholic Acid Improves Liver Fibrosis and Gut Barrier Function by Reducing Bile Acid Pool Size and Hydrophobicity in Male Cyp2c70 Knockout Mice.

Cyp2c70 knockout mice lack the enzyme that produces muricholic acids and show a "human-like" hydrophobic bile acid pool-induced hepatobiliary injury. In this study, we investigated the potential anti-cholestasis effect of glycine-conjugated β muricholic acid (G-β-MCA) in male Cyp2c70 KO mice based on its hydrophilic physiochemical property and signaling property as an farnesoid X receptor (FXR) antagonist. Our results showed that G-β-MCA treatment for 5 weeks alleviated ductular reaction and liver fibrosis and improved gut barrier function. Analysis of bile acid metabolism suggested that exogenously administered G-β-MCA was poorly absorbed in the small intestine and mostly deconjugated in the large intestine and converted to taurine-conjugated MCA (T-MCA) in the liver, leading to T-MCA enrichment in the bile and small intestine. These changes decreased the biliary and intestine bile acid hydrophobicity index. Furthermore, G-β-MCA treatment decreased intestine bile acid absorption via unknown mechanisms, resulting in increased fecal bile acid excretion and a reduction in total bile acid pool size. In conclusion, G-β-MCA treatment reduces the bile acid pool size and hydrophobicity and improves liver fibrosis and gut barrier function in Cyp2c70 KO mice.

Cholesterol 7α-Hydroxylase Activity is Increased by Dietary Modification with Psyllium Hydrocolloid, Pectin, Cholesterol and Cholestyramine in Rats

Sources of dietary fiber known to alter cholesterol metabolism and/or bile acid pool size were fed to rats, and activity of the rate-limiting step in bile acid synthesis, cholesterol 7α-hydroxylase, was measured. In the first experiment, semipurified diets containing 5% cellulose, psyllium hydrocolloid, pectin or oat bran as dietary fiber sources or 2% cholestyramine were fed to groups of 10 male Wistar rats for 4 wk. In the second experiment, groups of six rats were fed diets containing 5% cellulose, rice bran, oat bran or psyllium with and without 0.25% cholesterol. In the first experiment, the activity of cholesterol 7α-hydroxylase (pmol·min-1·mg protein-1) was highest in the cholestyramine-treated group (95.6 ± 3.6), followed by groups fed psyllium (35.5 ± 3.5) or pectin (36.0 ± 4.5), which exhibited more than twice the enzyme activity of groups fed cellulose (16.9 ± 1.9) or oat bran (12.3 ± 2.0). In the second experiment, feeding cholesterol resulted in significantly higher enzyme activity when cellulose (65%), oat bran (118%) and rice bran (60%) were fed, but no difference in activity was observed when cholesterol was added to the psyllium-containing diet. Higher activity of cholesterol 7α-hydroxylase when pectin or psyllium rather than cellulose was fed may explain the almost twofold higher bile acid pool sizes previously reported in response to feeding either of these fibers. These data support the hypothesis that the hypocholesterolemic effect of soluble fibers is modulated through increased synthesis and therefore pool size of bile acids.

Invited Commentary for Disruption of Enterohepatic Circulation of Bile Acids Ameliorates Small Bowel Resection Associated Hepatic Injury

In this issue, Tecos et al [ 1 Tecos ME Steinberger AE Guo J Rubin DC Davidson NO Warner BW Disruption of enterohepatic circulation of bile acids ameliorates small bowel resection associated hepatic injury. J Pediatr Surg. 2023; (in press) Google Scholar ] expand on their previous demonstration that parenteral nutrition (PN)-independent hepatoxicity in a murine small bowel resection (SBR) model is region dependent and may be mitigated by tauroursodeoxycholic acid (TUDCA) supplementation. Interestingly, these results contradict a previous report from their own group as to the impact of proximal vs. distal resection, seemingly due to the presence or absence of the ileocecal valve (ICV) and adjacent bowel, which differed between the two studies [ 2 Barron LK Gayer GP Roberts A Golden JM Aladegbami BG Guo J Erwin CR Warner BW Liver steatosis induced by small bowel resection is prevented by oral vancomycin. Surgery. 2016; 160: 1485-1495 Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar ]. Dr. Warner’s group was the first to report liver injury in a PN-independent murine SBR model [ 3 Chiang JYL Bile acid metabolism and signaling in liver disease and therapy. Liver Res. 2017 Jun; 1: 3-9 Crossref PubMed Scopus (140) Google Scholar ]. In this latest study, they reveal that proximal SBR results in greater oxidative stress in the liver as evidenced by elevated mRNA transcripts for tumor necrosis factor-α, nicotininamide adenine dinucleotide phosphate oxidase, and glutathione synthetase with a greater compensatory antioxidant response consisting of elevated lipocalin 2. Furthermore, distal SBR demonstrated decreased hepatic bile acid content, smaller total bile acid pool size, and significant changes in bile acid composition, such as a decrease in the most insoluble, toxic, and hydrophobic bile acids, as well as an increase in de novo bile acid synthesis of physiologic, less toxic, hydrophilic bile acids. Equally compelling, the hepatotoxicity and injurious bile acid profile observed in proximal SBR was mitigated by exogenous TUDCA supplementation. In addition to these intriguing observations, the authors propose a mechanism for this difference. Distal SBR results in disruption of enterohepatic bile acid recycling, leading to amplified production of more physiologic, less toxic, and more hydrophilic bile acids, resulting in less oxidative stress to the liver and ultimately decreased liver injury. While TUDCA had similar hepatoprotective effects to distal SBR, they were not identical, and thus, the authors suggest it may differ in mechanism of action. Regardless, this study expands on the identification of therapeutic targets for intestinal failure-associated liver disease.

Combining ASBT inhibitor and FGF15 treatments enhances therapeutic efficacy against cholangiopathy in female but not male Cyp2c70 KO mice

Therapeutic reduction of hydrophobic bile acids exposure is considered beneficial in cholestasis. The Cyp2c70 KO mice lack hydrophilic muricholic acids and have a human-like hydrophobic bile acid pool resulting in hepatobiliary injury. This study investigates if combining an apical sodium-dependent bile acid transporter inhibitor GSK2330672 (GSK) and fibroblast growth factor-15 (FGF15) overexpression, via simultaneous inhibition of bile acid synthesis and gut bile acid uptake, achieves enhanced therapeutic efficacy in alleviating hepatobiliary injury in Cyp2c70 KO mice. The effects of GSK, adeno-associated virus (AAV)-FGF15, and the combined treatment on bile acid metabolism and cholangiopathy were compared in Cyp2c70 KO mice. In female Cyp2c70 KO mice with more severe cholangiopathy than male Cyp2c70 KO mice, the combined treatment was more effective in reversing portal inflammation, ductular reaction, and fibrosis than AAV-FGF15, while GSK was largely ineffective. The combined treatment reduced bile acid pool by ∼80% compared to ∼50% reduction by GSK or AAV-FGF15, and enriched tauro-conjugated ursodeoxycholic acid in the bile. Interestingly, the male Cyp2c70 KO mice treated with AAV-FGF15 or GSK showed attenuated cholangiopathy and portal fibrosis but the combined treatment was ineffective despite reducing bile acid pool. Both male and female Cyp2c70 KO mice showed impaired gut barrier integrity. AAV-FGF15 and the combined treatment, but not GSK, reduced gut exposure to lithocholic acid and improved gut barrier function. In conclusion, the combined treatment improved therapeutic efficacy against cholangiopathy than either single treatment in the female but not male Cyp2c70 KO mice by reducing bile acid pool size and hydrophobicity.

Farnesoid X receptor antagonizes macrophage-dependent licensing of effector T lymphocytes and progression of sclerosing cholangitis.

Immune-mediated bile duct epithelial injury and toxicity of retained hydrophobic bile acids drive disease progression in fibrosing cholangiopathies such as biliary atresia or primary sclerosing cholangitis. Emerging therapies include pharmacological agonists to farnesoid X receptor (FXR), the master regulator of hepatic synthesis, excretion, and intestinal reuptake of bile acids. Unraveling the mechanisms of action of pharmacological FXR agonists in the treatment of sclerosing cholangitis (SC), we found that intestinally restricted FXR activation effectively reduced bile acid pool size but did not improve the SC phenotype in MDR2-/- mice. In contrast, systemic FXR activation not only lowered bile acid synthesis but also suppressed proinflammatory cytokine production by liver-infiltrating inflammatory cells and blocked progression of hepatobiliary injury. The hepatoprotective activity was linked to suppressed production of IL1β and TNFα by hepatic macrophages and inhibition of TH1/TH17 lymphocyte polarization. Deletion of FXR in myeloid cells caused aberrant TH1 and TH17 lymphocyte responses in diethoxycarbonyl-1,4-dihydrocollidine-induced SC and rendered these mice resistant to the anti-inflammatory and liver protective effects of systemic FXR agonist treatment. Pharmacological FXR activation reduced IL1β and IFNγ production by liver- and blood-derived mononuclear cells from patients with fibrosing cholangiopathies. In conclusion, we demonstrate FXR to control the macrophage-TH1/17 axis, which is critically important for the progression of SC. Hepatic macrophages are cellular targets of systemic FXR agonist therapy for cholestatic liver disease.

Population pharmacokinetic model to generate mechanistic insights in bile acid homeostasis and drug-induced cholestasis

Bile acids (BA) fulfill a wide range of physiological functions, but are also involved in pathologies, such as cholestasis. Cholestasis is characterized by an intrahepatic accumulation of BAs and subsequent spillage to the systemic circulation. The aim of the present study was to develop physiologically based kinetic (PBK) models that would provide a tool to predict dose-dependent BA accumulation in humans upon treatment with a Bile Salt Export Pump (BSEP) inhibitor. We developed a PBK model describing the BA homeostasis using glycochenodeoxycholic acid as an exemplary BA. Population wide distributions of BSEP abundances were incorporated in the PBK model using Markov Chain Monte Carlo simulations, and alternatively the total amount of BAs was scaled empirically to describe interindividual differences in plasma BA levels. Next, the effects of the BSEP inhibitor bosentan on the BA levels were simulated. The PBK model developed adequately predicted the in vivo BA dynamics. Both the Markov Chain Monte Carlo simulations based on a distribution of BSEP abundances and empirical scaling of the total BA pool readily described the variations within and between data in human volunteers. Bosentan treatment disproportionally increased the maximum BA concentration in individuals with a large total BA pool or low BSEP abundance. Especially individuals having a large total BA pool size and a low BSEP abundance were predicted to be at risk for rapid saturation of BSEP and subsequent intrahepatic BA accumulation. This model provides a first estimate of personalized safe therapeutic external dose levels of compounds with BSEP-inhibitory properties.

Bile acid conjugation deficiency causes hypercholanemia, hyperphagia, islet dysfunction, and gut dysbiosis in mice.

Bile acid‐CoA: amino acid N‐acyltransferase (BAAT) catalyzes bile acid conjugation, the last step in bile acid synthesis. BAAT gene mutation in humans results in hypercholanemia, growth retardation, and fat‐soluble vitamin insufficiency. The current study investigated the physiological function of BAAT in bile acid and lipid metabolism using Baat −/− mice. The bile acid composition and hepatic gene expression were analyzed in 10‐week‐old Baat −/− mice. They were also challenged with a westernized diet (WD) for additional 15 weeks to assess the role of BAAT in bile acid, lipid, and glucose metabolism. Comprehensive lab animal monitoring system and cecal 16S ribosomal RNA gene sequencing were used to evaluate the energy metabolism and microbiome structure of the mice, respectively. In Baat −/− mice, hepatic bile acids were mostly unconjugated and their levels were significantly increased compared with wild‐type mice. Bile acid polyhydroxylation was markedly up‐regulated to detoxify unconjugated bile acid accumulated in Baat −/− mice. Although the level of serum marker of bile acid synthesis, 7α‐hydroxy‐4‐cholesten‐3‐one, was higher in Baat −/− mice, their bile acid pool size was smaller. When fed a WD, the Baat −/− mice showed a compromised body weight gain and impaired insulin secretion. The gut microbiome of Baat −/− mice showed a low level of sulfidogenic bacteria Bilophila. Conclusion: Mouse BAAT is the major taurine‐conjugating enzyme. Its deletion protected the animals from diet‐induced obesity, but caused glucose intolerance. The gut microbiome of the Baat −/− mice was altered to accommodate the unconjugated bile acid pool.

Dietary fat composition shapes bile acid metabolism and severity of liver injury in a pig model of pediatric NAFLD.

The objective of this study was to investigate the effect of dietary fatty acid (FA) composition on bile acid (BA) metabolism in a pig model of NAFLD, by using a multiomics approach combined with histology and serum biochemistry. Thirty 20-day-old Iberian pigs pair-housed in pens were randomly assigned to receive 1 of 3 hypercaloric diets for 10 wk: 1) lard-enriched (LAR; n = 5 pens), 2) olive oil-enriched (OLI; n = 5), and 3) coconut oil-enriched (COC; n = 5). Animals were euthanized on week 10 after blood sampling, and liver, colon, and distal ileum (DI) were collected for histology, metabolomics, and transcriptomics. Data were analyzed by multivariate and univariate statistics. Compared with OLI and LAR, COC increased primary and secondary BAs in liver, plasma, and colon. In addition, both COC and OLI reduced circulating fibroblast growth factor 19, increased hepatic necrosis, composite lesion score, and liver enzymes in serum, and upregulated genes involved in hepatocyte proliferation and DNA repair. The severity of liver disease in COC and OLI pigs was associated with increased levels of phosphatidylcholines, medium-chain triacylglycerides, trimethylamine-N-oxide, and long-chain acylcarnitines in the liver, and the expression of profibrotic markers in DI, but not with changes in the composition or size of BA pool. In conclusion, our results indicate a role of dietary FAs in the regulation of BA metabolism and progression of NAFLD. Interventions that aim to modify the composition of dietary FAs, rather than to regulate BA metabolism or signaling, may be more effective in the treatment of NAFLD.NEW & NOTEWORTHY Bile acid homeostasis and signaling is disrupted in NAFLD and may play a central role in the development of the disease. However, there are no studies addressing the impact of diet on bile acid metabolism in patients with NAFLD. In juvenile Iberian pigs, we show that fatty acid composition in high-fat high-fructose diets affects BA levels in liver, plasma, and colon but these changes were not associated with the severity of the disease.

Farnesoid X receptor activation by the novel agonist TC-100 (3α, 7α, 11β-Trihydroxy-6α-ethyl-5β-cholan-24-oic Acid) preserves the intestinal barrier integrity and promotes intestinal microbial reshaping in a mouse model of obstructed bile acid flow

The intestinal tract hosts the gut microbiota (GM), actively shaping health. Bile acids(BAs) are both digestive and signaling molecules acting as hormones via the activation of farnesoid X receptor (FXR). Obstruction of bile flow initiates a cascade of pathological events ultimately leading to intestinal mucosal injury. Administration of BAs in models of obstructed bile flow counteracts these detrimental effects. Objective of this study was to investigate the effects of the novel FXR agonist 3α, 7α, 11β-Trihydroxy-6α-ethyl-5β-cholan-24-oic Acid (TC-100) on intestinal mucosa integrity and cecal microbiome composition after surgical bile duct ligation (BDL), a rodent model causing bile flow obstruction.Pharmacological FXR activation was accomplished by daily oral gavage with TC-100 for 5 days. 2 days after treatment initiation, BDL was performed. BAs measurement was carried out and the 16S rDNA (V5-V6 hyper-variable regions) extracted from the cecal content was sequenced.TC-100 activates Fxr in the gut-liver axis and this translated into a significant reduction of serum and bile BA pool size with a shift to a more hydrophilic composition, while signs of intestinal mucosal damage were prevented. Firmicutes:Bacteroidota ratio progressively increased from Sham Operated (SO) mice to TC-100-treated mice. LEfSe analysis showed that Verrucomicrobia, and particularly Akkermansia muciniphila (Amuc) increasingly recognized for improving gut homeostasis and immune functions, were strongly associated to TC-100-treated mice. Intriguingly, Amuc abundance was also negatively associated to cholic acid levels.Collectively, these data indicate that intestinal FXR activation by TC-100 prevents early signs of intestinal mucosal damage by modulating BA homeostasis and GM composition.

Intestinal FXR Activation via Transgenic Chimera or Chemical Agonism Prevents Colitis-Associated and Genetically-Induced Colon Cancer.

Simple SummaryDisruption of Bile Acids (BA) regulation with increased BA concentration and modulation or their detergent pro-inflammatory activity has been linked to colorectal cancer (CRC). Farnesoid X Receptor (FXR) is the master regulator of BA homeostasis; FXR is a nuclear receptor that transcriptionally modulates their synthesis, transport and metabolism. In this study, we demonstrated that intestinal FXR activation prevented both inflammation- and genetically-driven colorectal tumorigenesis by modulating BA pool size and composition. This could open new avenues for the therapeutic management of intestinal inflammation and tumorigenesis.The Farnesoid X Receptor (FXR) is the master regulator of Bile Acids (BA) homeostasis orchestrating their synthesis, transport and metabolism. Disruption of BA regulation has been linked to gut-liver axis diseases such as colorectal cancer (CRC). In this study, firstly we examined the role of constitutive activation of intestinal FXR in CRC; then we pre-clinically investigated the therapeutic potential of a diet enriched with a synthetic FXR agonist in two models of CRC (chemically-induced and genetic models). We demonstrated that mice with intestinal constitutive FXR activation are protected from AOM/DSS-induced CRC with a significant reduction of tumor number compared to controls. Furthermore, we evaluated the role of chemical FXR agonism in a DSS model of colitis in wild type (WT) and FXRnull mice. WT mice administered with the FXR activating diet showed less morphological alterations and decreased inflammatory infiltrates compared to controls. The FXR activating diet also protected WT mice from AOM/DSS-induced CRC by reducing tumors’ number and size. Finally, we proved that the FXR activating diet prevented spontaneous CRC in APCMin/+ mice via an FXR-dependent modulation of BA homeostasis. Our results demonstrate that intestinal FXR activation prevented both inflammation- and genetically-driven colorectal tumorigenesis by modulating BA pool size and composition. This could open new avenues for the therapeutic management of intestinal inflammation and tumorigenesis.

Altered Circulating Bile Acid Composition After Cross-Linked Resistant Starch Consumption

ObjectivesRecent studies have shown potential mechanisms underlying the role of bile acid (BA)-gut microbiota axis in human health and diseases. We and others have shown that resistant starch type 4 (RS4) intake modulates gut microbiota and a range of immunometabolic outcomes in humans and mice. Using available samples (#NCT01887964), we retrospectively examined the hitherto unknown effects of RS4-intake on human plasma BA.MethodsIn a placebo-controlled, two-arm crossover trial, 14 adults with metabolic syndrome consumed control and RS4 supplemented (30% v/v in wheat flour) diets, each for 12 weeks separated by a 2-week washout. Stealth organoleptic properties of RS4 allowed double-blinding. Overnight-fasted samples were collected before and after each diet phase. LC/MS with isotope-labeled internal standards and 16S-rDNA sequencing were used for targeted metabolite and microbiome measurements, respectively. Univariate and multivariate data were analyzed with supervised and unsupervised machine learning algorithms in R-platform.ResultsDifferences in food ingredients, meal preparations, macronutrients, and energy intakes were minimal among participants due to following communal kitchen and dining practices (p > 0.05). Being a prebiotic fiber, RS4 increased fiber consumption by 1.5-fold (p < 0.001). Taurocholic acid, glycochenodeoxycholic acid, deoxycholic acid, and glycodeoxycholic acid increased in the RS4 group by 92%, 58%, 77%, and 79% respectively (all, p < 0.05) and enriched species of Bacteroides, Bifidobacteria, Roseburia, Eubacterium, Ruminococcus, and Blautia. Members of these genuses are known to have high bile salt hydrolase activity, an enzyme proposed to have a cholesterol-lowering effect. One such species, Bifidobacterium adolescentis showed RS4 specific association with deoxycholic acid, glycodeoxycholic acid, and taurodeoxycholic acid (all, rho >0.50, p < 0.05). Interestingly, the parent study reported significant cholesterol-lowering effects of RS4.ConclusionsRS4-enriched diet increased fiber consumption and resulted in altered microbiota-dependent secondary BA pool size and composition. Future research may help advance the understanding of the role of prebiotic fibers in BA-microbiota interactions.Funding SourcesNational Institute of Food and Agriculture.

Effects of intestine-specific deletion of fibroblast growth factor 15 on alcoholic liver disease development in mice

Background and aimsAlcoholic liver disease (ALD) is an important and growing cause for the development of chronic liver diseases in the world. Bile acid (BA) levels are increased in patients with ALD and dysregulation of BA homeostasis worsens ALD. BA synthesis is critically regulated by fibroblast growth factor (FGF)15 in mice and FGF19 in humans. FGF15/19 are mainly produced in the ileum and their main function is to suppress BA synthesis in the liver through the activation of fibroblast growth factor receptor 4 (FGFR4) on hepatocytes. The effects of intestine-specific Fgf15 deficiency on the development of ALD were determined in the current study. MethodsEnterocyte-specific Fgf15 knockout mice (Fgf15int−/−) and the established mouse model by chronic and binge ethanol feeding (NIAAA model) were adapted in this study. ResultsThe Fgf15int−/− mice had increased BA pool size, consistent with negative effects of FGF15-FGFR4 signaling on BA synthesis. There were not obviously physical and hepatic histological abnormalities presented in Fgf15int−/− mice compared to wild-type mice. Following alcohol treatment, the Fgf15int−/− mice exhibited a higher degree of liver injury, increased hepatic expression of Cd14, a receptor for lipopolysaccharide expressed in the liver, and increased hepatic lipid levels. We did not observe alterations in the levels of fibrosis in the liver or expression of genes involved in hepatic fibrosis, regardless of genotypes or following the alcohol treatment. ConclusionsFGF15 may prevent hepatic steatosis in the development of ALD in mice, and maintaining FGF19/FGFR4 signaling may be critical in the prevention and/or treatment of ALD in humans in the future.

Maternal obesogenic diet enhances cholestatic liver disease in offspring

Human and animal model data show that maternal obesity promotes nonalcoholic fatty liver disease in offspring and alters bile acid (BA) homeostasis. Here we investigated whether offspring exposed to maternal obesogenic diets exhibited greater cholestatic injury. We fed female C57Bl6 mice conventional chow (CON) or high fat/high sucrose (HF/HS) diet and then bred them with lean males. Offspring were fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) for 2 weeks to induce cholestasis, and a subgroup was then fed CON for an additional 10 days. Additionally, to evaluate the role of the gut microbiome, we fed antibiotic-treated mice cecal contents from CON or HF/HS offspring, followed by DDC for 2 weeks. We found that HF/HS offspring fed DDC exhibited increased fine branching of the bile duct (ductular reaction) and fibrosis but did not differ in BA pool size or intrahepatic BA profile compared to offspring of mice fed CON. We also found that after 10 days recovery, HF/HS offspring exhibited sustained ductular reaction and periportal fibrosis, while lesions in CON offspring were resolved. In addition, cecal microbiome transplant from HF/HS offspring donors worsened ductular reaction, inflammation, and fibrosis in mice fed DDC. Finally, transfer of the microbiome from HF/HS offspring replicated the cholestatic liver injury phenotype. Taken together, we conclude that maternal HF/HS diet predisposes offspring to increased cholestatic injury after DDC feeding and delays recovery after returning to CON diets. These findings highlight the impact of maternal obesogenic diet on hepatobiliary injury and repair pathways during experimental cholestasis.

LKB1 in Intestinal Epithelial Cells Regulates Bile Acid Metabolism by Modulating FGF15/19 Production.

Background & AimsLiver kinase B1 (LKB1) is a master upstream protein kinase involved in nutrient sensing and glucose and lipid metabolism in many tissues; however, its metabolic role in intestinal epithelial cells (IEC) remains unclear. In this study, we investigated the regulatory role of LKB1 on bile acid (BA) homeostasis.MethodsWe generated mice with IEC-specific deletion of LKB1 (LKB1ΔIEC) and analyzed the characteristics of IEC development and BA level. In vitro assays with small interfering RNA, liquid chromatography/mass spectrometry, metagenomics, and RNA-sequencing were used to elucidate the regulatory mechanisms underlying perturbed BA homeostasis.ResultsLKB1 deletion resulted in abnormal differentiation of secretory cell lineages. Unexpectedly, BA pool size increased substantially in LKB1ΔIEC mice. A significant reduction of the farnesoid X receptor (FXR) target genes, including fibroblast growth factor 15/19 (FGF15/19), known to inhibit BA synthesis, was found in the small intestine (SI) ileum of LKB1ΔIEC mice. We observed that LKB1 depletion reduced FGF15/19 protein level in human IECs in vitro. Additionally, a lower abundance of bile salt hydrolase-producing bacteria and elevated levels of FXR antagonist (ie, T-βMCA) were observed in the SI of LKB1ΔIEC mice. Moreover, LKB1ΔIEC mice showed impaired conversion of retinol to retinoic acids in the SI ileum. Subsequently, vitamin A treatment failed to induce FGF15 production. Thus, LKB1ΔIEC mice fed with a high-fat diet showed improved glucose tolerance and increased energy expenditure.ConclusionsLKB1 in IECs manages BA homeostasis by controlling FGF15/19 production.