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A series of extrudates was prepared from oat meal, oat bran and Novelose 330®, differing in concentrations of individual dietary fibre (DF) components, such as β-glucan and resistant starch, as well as total DF. After simulated digestion, the digested DF-rich oat-based extrudates were used to evaluate their physiological effects in vitro. A strong interaction occurred between the digested extrudates and bile acids (BA). The binding of BA increased with increasing proportions of oat bran, total DF, insoluble DF and β-glucan in the extrudates. Dihydroxy-BA was more strongly bound to the extrudates than trihydroxy-BA. Interactions at pH 5.0 were greater than at pH 6.5. During fermentation of digested extrudates with human faecal samples, concentrations of short-chain fatty acid (SCFA) formed and the molar proportion of butyrate increased continuously. Higher SCFA concentrations were found when extrudates contained more oat bran, soluble and insoluble DF and β-glucan. Extrudates, on the basis of oat, have several beneficial nutritional and protective effects in vitro. Therefore, physiological effects occurring in the small and large intestine are also related to the DF composition of the cereal products. [Copyright &y& Elsevier]

In order to investigate the influence of variety on the structure and properties of enzymatically degraded cell wall materials (CWM), carrots of the varieties Major (direct consumption) and Karotan (cultivated for industrial processing) were used to prepare cold water extracted reference materials (RM) as well as materials treated with macerating enzymes (MC) and with liquefying enzymes (LF). Rehydrated samples RM and MC showed a good maintenance of the grown biological structure (tissue particles for RM; single cells for MC), a high porosity, excellent hydration properties as well as remarkable visco-elastic and structural viscous properties, independent of the carrot variety used. However, the cell wall of the variety Major was more strongly degraded by enzymatic treatment than that of Karotan. Thus, the strongly degraded LF of Major showed the lowest values for porosity, hydration properties and rheological parameters of their suspensions. In vitro, the CWM from Karotan also gave in higher concentrations of short-chain fatty acids during its fermentation with human faeces flora and had a better binding capacity of glycochenodeoxycholic acid than the CWM from Major. Such CWM from carrots may be promising as ingredients for the production of functional foods. [Copyright &y& Elsevier]

Background/Aims: To examine the extent and mechanisms of apoptosis in cholestatic liver injury and to explore the role of the transcription factor nuclear factor-kappa B in protection against bile acid-induced apoptosis.Methods: Cholestatic liver injury was induced by bile duct ligation in Wistar rats. Furthermore, primary cultures of rat hepatocytes were exposed to glycochenodeoxycholic acid (GCDCA), tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA) and to cytokines. Apoptosis was determined by TUNEL-staining, active caspase-3 staining, activation of caspase-8, -9 and -3.Results: Limited hepatocyte apoptosis and an increased expression of NF-κB-regulated anti-apoptotic genes A1 and cIAP2 were detected in cholestatic rat livers. Bcl-2 expression was restricted to bile duct epithelium. In contrast to TCDCA and TUDCA, GCDCA induced apoptosis in a Fas-associated protein with death domain (FADD)-independent pathway in hepatocytes. Although bile acids do not activate NF-κB, NF-κB activation by cytokines (induced during cholestasis) protected against GCDCA-induced apoptosis in vitro by upregulating A1 and cIAP2.Conclusions: GCDCA induces apoptosis in a mitochondria-controlled pathway in which caspase-8 is activated in a FADD-independent manner. However, bile acid-induced apoptosis in cholestasis is limited. This could be explained by cytokine-induced activation of NF-κB-regulated anti-apoptotic genes like A1 and cIAP2. [Copyright &y& Elsevier]

Fas-mediated apoptosis of biliary epithelial cells (BECs) is suggested as a main effector process in immune-mediated biliary diseases. Glycochenodeoxycholic acid (GCDCA), a conjugated hydrophobic bile acid, is known to cause apoptosis of hepatocytes via the direct activation of Fas receptor (FasR). In this study, we investigated the apoptotic effect of GCDCA on cultured murine intrahepatic BECs. It was found that GCDCA induced apoptosis of BECs derived from BALB/c mice in a dose- and incubation time-dependent manner. The morphology and TUNEL positivity for the apoptotic process induced by GCDCA were similar to those induced by beauvericin (BV) which is known to cause apoptosis by a direct activation of caspase-3, a member of the caspase family, at its downstream. The GCDCA-induced apoptosis of BECs was accompanied by an up-regulation of FasR, Fas ligand (FasL), and also caspase-3 expression. GCDCA did not induce the apoptosis of BECs derived from C3H · MRL-Faslpr mice possessing a non-functioning FasR, supporting that the GCDCA-induced apoptosis of BECs in BALB/c involves the Fas system. Furthermore, GCDCA induced an increase in interleukin-18 (IL-18) mRNA in BECs and secretion of activated IL-18 from BECs of BALB/c, which might have led to an up-regulation of FasL mRNA and protein expression in BECs followed by FasR/FasL interaction. These results suggest that GCDCA induces apoptosis of BECs through FasR/FasL interaction via an autocrine/paracrine effect, IL-18 is responsible for the expression of FasL in BEC, and the up-regulation of caspase-3 expression is involved in this model. This model could be useful for molecular and genetic studies of Fas-mediated apoptosis of BECs. [Copyright &y& Elsevier]

Background & Aims: Mitochondria are the major organelles for the formation of reactive oxygen species (ROS) in the cell, and mitochondrial dysfunction has been described as a key factor in the pathogenesis of cholestatic liver disease. The methylation-controlled J-protein (MCJ) is a mitochondrial protein that interacts with and represses the function of complex I of the electron transport chain. The relevance of MCJ in the pathology of cholestasis has not yet been explored., Methods: We studied the relationship between MCJ and cholestasis-induced liver injury in liver biopsies from patients with chronic cholestatic liver diseases, and in livers and primary hepatocytes obtained from WT and MCJ-KO mice. Bile duct ligation (BDL) was used as an animal model of cholestasis, and primary hepatocytes were treated with toxic doses of bile acids. We evaluated the effect of MCJ silencing for the treatment of cholestasis-induced liver injury., Results: Elevated levels of MCJ were detected in the liver tissue of patients with chronic cholestatic liver disease when compared with normal liver tissue. Likewise, in mouse models, the hepatic levels of MCJ were increased. After BDL, MCJ-KO animals showed significantly decreased inflammation and apoptosis. In an in vitro model of bile-acid induced toxicity, we observed that the loss of MCJ protected mouse primary hepatocytes from bile acid-induced mitochondrial ROS overproduction and ATP depletion, enabling higher cell viability. Finally, the in vivo inhibition of the MCJ expression, following BDL, showed reduced liver injury and a mitigation of the main cholestatic characteristics., Conclusions: We demonstrated that MCJ is involved in the progression of cholestatic liver injury, and our results identified MCJ as a potential therapeutic target to mitigate the liver injury caused by cholestasis., Lay Summary: In this study, we examine the effect of mitochondrial respiratory chain inhibition by MCJ on bile acid-induced liver toxicity. The loss of MCJ protects hepatocytes against apoptosis, mitochondrial ROS overproduction, and ATP depletion as a result of bile acid toxicity. Our results identify MCJ as a potential therapeutic target to mitigate liver injury in cholestatic liver diseases., Competing Interests: Dr. María Luz Martínez-Chantar advises for Mitotherapeutix LLC. Dr. Javier Crespo reports grants and research support from Gilead Sciences, AbbVie, MSD, and Intercept Pharmaceuticals (all outside the scope of the submitted work). He is a speaker for Gilead Sciences and AbbVie. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2021 The Author(s).)

Background & Aims: Higher serum bile acid levels are associated with an increased risk of cirrhosis and liver-related morbidity and mortality. Herein, we report secondary analyses of aldafermin, an engineered analogue of the gut hormone fibroblast growth factor 19, on the circulating bile acid profile in prospective, phase II studies in patients with metabolic or cholestatic liver disease., Methods: One hundred and seventy-six patients with biopsy-confirmed non-alcoholic steatohepatitis (NASH) and fibrosis and elevated liver fat content (≥8% by magnetic resonance imaging-proton density fat fraction) received 0.3 mg (n = 23), 1 mg (n = 49), 3 mg (n = 49), 6 mg (n = 28) aldafermin or placebo (n = 27) for 12 weeks. Sixty-two patients with primary sclerosing cholangitis (PSC) and elevated alkaline phosphatase (>1.5× upper limit of normal) received 1 mg (n = 21), 3 mg (n = 21) aldafermin or placebo (n = 20) for 12 weeks. Serum samples were collected on day 1 and week 12 for determination of bile acid profile and neoepitope-specific N-terminal pro-peptide of type III collagen (Pro-C3), a direct measure of fibrogenesis., Results: Treatment with aldafermin resulted in significant dose-dependent reductions in serum bile acids. In particular, bile acids with higher hydrophobicity indices, such as deoxycholic acid, lithocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, and glycocholic acid, were markedly lowered by aldafermin in both NASH and PSC populations. Moreover, aldafermin predominantly suppressed the glycine-conjugated bile acids, rather than the taurine-conjugated bile acids. Changes in levels of bile acids correlated with changes in the novel fibrogenesis marker Pro-C3, which detects a neo-epitope of the type III collagen during its formation, in the pooled NASH and PSC populations., Conclusions: Aldafermin markedly reduced major hydrophobic bile acids that have greater detergent activity and cytotoxicity. Our data provide evidence that bile acids may contribute to sustaining a pro-fibrogenic microenvironment in the liver across metabolic and cholestatic liver diseases., Lay Summary: Aldafermin is an analogue of a gut hormone, which is in development as a treatment for patients with chronic liver disease. Herein, we show that aldafermin can potently and robustly suppress the toxic, hydrophobic bile acids irrespective of disease aetiology. The therapeutic strategy utilising aldafermin may be broadly applicable to other chronic gastrointestinal and liver disorders., Clinical Trials Registration: The study is registered at Clinicaltrials.govNCT02443116 and NCT02704364., Competing Interests: A.J.S. reports serving as an unpaid consultant to Intercept, Zydus, Echosense, Immuron, Madrigal, Galectin, Blade, Pliant, Albireo, and AMRA; is a consultant to Gilead, Allergan, Bristol-Myers Squibb, Pfizer, Merck, Galmed, Novartis, Novo Nordisk, Lilly, Siemens, Genentech, Boehringer Ingelhiem, Glympse Bio, Genfit, Coherus, Surrozen, Poxel, 89 Bio, Perspectum, AstraZeneca, Medimmune, and Lipocine; owns stock options in Indalo, Durect, Tiziana, Exhalenz, and Northsea; and is president of Sanyal Bio. L.L., A.M.D., and H.D.L. are employees and stockholders of NGM Biopharmaceuticals. U.B. reports consulting for Novartis, Intercept; grant/research support from Norwegian, American, and South African PSC patient foundations and German DCCV; received lecture fees from Abbvie, Falk Foundation, Gilead, Intercept, Novartis, Roche, Shire, and Zambon; and received research support for investigator-initiated studies from Dr. Falk Pharma and Intercept. S.A.H. reports research support from Conatus, Galectin, Galmed, Genfit, Gilead, Intercept, Madrigal, NGM, Akero, Axcella, Metacrine, Sagimet, Enyo, NorthSea, Genentech, Hepion, Cymabay, and Hightide; and is a consulting adviser for the Chronic Liver Disease Foundation, Cirius, Echosens, Akero, Galectin, Galmed, Genfit, Gilead, Intercept, Madrigal, NGM, Novartis, Perspectum, Metacrine, Medpace, Sagimet, Blade, Viking, Poxel, Axcella, Terns, HistoIndex, Hightide, Hepion, Ridgeline Therapeutics, CiVi, and Prometic. G.M.H. reports consulting for Cymabay, Genfit, Falk, Intercept, GSK, Mirum, Roche, and Pliant. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2021 The Author(s).)

Background & Aims: Plasma bile acids (BAs) have been extensively studied as pathophysiological actors in non-alcoholic steatohepatitis (NASH). However, results from clinical studies are often complicated by the association of NASH with type 2 diabetes (T2D), obesity, and insulin resistance (IR). Here, we sought to dissect the relationship between NASH, T2D, and plasma BA levels in a large patient cohort., Methods: Four groups of patients from the Biological Atlas of Severe Obesity (ABOS) cohort (Clinical Trials number NCT01129297) were included based on the presence or absence of histologically evaluated NASH with or without coincident T2D. Patients were matched for BMI, homeostatic model assessment 2 (HOMA2)-assessed IR, glycated haemoglobin, age, and gender. To study the effect of IR and BMI on the association of plasma BA and NASH, patients from the HEPADIP study were included. In both cohorts, fasting plasma BA concentrations were measured., Results: Plasma BA concentrations were higher in NASH compared with No-NASH patients both in T2D and NoT2D patients from the ABOS cohort. As we previously reported that plasma BA levels were unaltered in NASH patients of the HEPADIP cohort, we assessed the impact of BMI and IR on the association of NASH and BA on the combined BA datasets. Our results revealed that NASH-associated increases in plasma total cholic acid (CA) concentrations depend on the degree of HOMA2-assessed systemic IR, but not on β-cell function nor on BMI., Conclusions: Plasma BA concentrations are elevated only in those NASH patients exhibiting pronounced IR., Lay Summary: Non-alcoholic steatohepatitis (NASH) is a progressive liver disease that frequently occurs in patients with obesity and type 2 diabetes. Reliable markers for the diagnosis of NASH are needed. Plasma bile acids have been proposed as NASH biomarkers. Herein, we found that plasma bile acids are only elevated in patients with NASH when significant insulin resistance is present, limiting their utility as NASH markers., Competing Interests: The authors declare that they have no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2021 The Authors.)

Since metabolic process differs between humans and mice, studies were performed in hamsters, which are generally considered to be a more appropriate animal model for studies of obesity-related metabolic disorders. The modulation of gut microbiota, bile acids and the farnesoid X receptor (FXR) axis is correlated with obesity-induced insulin resistance and hepatic steatosis in mice. However, the interactions among the gut microbiota, bile acids and FXR in metabolic disorders remained largely unexplored in hamsters. In the current study, hamsters fed a 60% high-fat diet (HFD) were administered vehicle or an antibiotic cocktail by gavage twice a week for four weeks. Antibiotic treatment alleviated HFD-induced glucose intolerance, hepatic steatosis and inflammation accompanied with decreased hepatic lipogenesis and elevated thermogenesis in subcutaneous white adipose tissue (sWAT). In the livers of antibiotic-treated hamsters, cytochrome P450 family 7 subfamily B member 1 (CYP7B1) in the alternative bile acid synthesis pathway was upregulated, contributing to a more hydrophilic bile acid profile with increased tauro- β -muricholic acid (T β MCA). The intestinal FXR signaling was suppressed but remained unchanged in the liver. This study is of potential translational significance in determining the role of gut microbiota-mediated bile acid metabolism in modulating diet-induced glucose intolerance and hepatic steatosis in the hamster.

Background/aims: Primary sclerosing cholangitis (PSC) is a chronic, progressive hepatobiliary disorder characterized by extensive fibrosis and stricturing of the intra- and/or extra-hepatic bile ducts: Previous studies have documented low phosphatylcholine (PC) concentrations in PSC bile. The aim of this study was to determine whether low PC levels in bile facilitate toxic bile acid induced injury of biliary tract epithelial cells resulting in enhanced transforming growth factor-beta (TGF-β) expression and increased collagen synthesis by myofibroblasts., Methods: TGF-β mRNA expression was documented in bile duct epithelial cells exposed to varying concentrations of the toxic bile acid; glycochenodeoxycholic acid (GCDCA) ± PC., Results: In these experiments, as well as in co-culture experiments where bile duct epithelial cells were cultured with peripheral blood mononuclear cells and myofibroblasts, TGF-β mRNA expression remained unaltered in the presence or absence of PC. Moreover, collagen type Iα1 mRNA expression by myofibroblasts also remained unaltered., Conclusion: The results of this study do not support the hypothesis that PC deficiency contributes to toxic bile acid-induced bile duct injury and/or myofibroblast activation.