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Background: Cancer cachexia is a multifactorial metabolic syndrome characterized by systemic inflammation and ongoing skeletal muscle loss resulting in weakness, poor quality of life, and decreased survival. Whereas lipid accumulation in skeletal muscle is associated with cancer cachexia as well as the prognosis of cancer patients, surprisingly little is known about the nature of the lipids that accumulate in the muscle during cachexia, and whether this is related to inflammation. We aimed to identify the types and distributions of intramyocellular lipids in patients with and without cancer cachexia., Methods: Rectus abdominis muscle biopsies were collected during surgery of patients with pancreatic ductal adenocarcinoma (n = 10 without cachexia, n = 20 cachectic without inflammation (CRP < 10 mg/L), n = 10 cachectic with inflammation (CRP ≥ 10 mg/L). L3-CT scans were analysed to assess body composition based on validated thresholds in Hounsfield units (HU). Muscle sections were stained with Oil-Red O and H&E to assess general lipid accumulation and atrophy. Untargeted lipidomic analyses were performed on laser-microdissected myotubes using LC-MS/MS. The spatial distribution of intramyocellular lipids with differential abundance between groups was visualized by mass-spectrometry imaging. Genes coding for inflammation markers and enzymes involved in de novo ceramide synthesis were studied by qPCR., Results: Muscle radiation attenuation was lower in cachectic patients with inflammation (median 24.3 [18.6-30.8] HU) as compared with those without inflammation (34.2 [29.3-38.7] HU, P = 0.033) or no cachexia (37.4 [33.9-42.9] HU, P = 0.012). Accordingly, intramyocellular lipid content was lower in non-cachectic patients (1.9 [1.6-2.1]%) as compared with those with cachexia with inflammation (5.5 [4.5-7.3]%, P = 0.002) or without inflammation (4.8 [2.6-6.0]%, P = 0.017). Intramyocellular lipid accumulation was associated with both local IL-6 mRNA levels (r s  = 0.57, P = 0.015) and systemic CRP levels (r s  = 0.49, P = 0.024). Compared with non-cachectic subjects, cachectic patients had a higher relative abundance of intramyocellular glycerophospholipids and a lower relative abundance of glycerolipids. Furthermore, increases in several intramyocellular lipids such as SM(d36:1), PC(34:1), and TG(48:1) were found in cachectic patients with inflammation and correlated with specific cachexia features. Altered intramyocellular lipid species such as PC(34:1), LPC(18:2), and TG(48:1) showed an uneven distribution in muscle sections of cachectic and non-cachectic patients, with areas featuring abundance of these lipids next to areas almost devoid of them., Conclusions: Intramyocellular lipid accumulation in patients with cachexia is associated with both local and systemic inflammation, and characterized by changes in defined lipid species such as glycerolipids and glycerophospholipids., (© 2024 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by Wiley Periodicals LLC.)

Cholangiocarcinoma (CCA), or bile duct cancer, is the second most common liver malignancy, with an increasing incidence in Western countries. The lack of effective treatments associated with the absence of early symptoms highlights the need to search for new therapeutic targets for CCA. Sulfatides (STs), a type of sulfoglycosphingolipids, have been found in the biliary tract, with increased levels in CCA and other types of cancer. STs are involved in protein trafficking and cell adhesion as part of the lipid rafts of the plasma membrane. We aimed to study the role of STs in CCA by the genetic targeting of GAL3ST1, an enzyme involved in ST synthesis. We used the CRISPR-Cas9 system to generate GAL3ST1 -deficient TFK1 cells. GAL3ST1 KO cells showed lower proliferation and clonogenic activity and reduced glycolytic activity compared to TFK1 cells. Polarized TFK1 GAL3ST1 KO cells displayed increased transepithelial resistance and reduced permeability compared to TFK1 wt cells. The loss of GAL3ST1 showed a negative effect on growth in 30 out of 34 biliary tract cancer cell lines from the DepMap database. GAL3ST1 deficiency partially restored epithelial identity and barrier function and reduced proliferative activity in CCA cells. Sulfatide synthesis may provide a novel therapeutic target for CCA.

Background: Bile acids play vital roles in control of lipid-, glucose-, and energy metabolism by activating Takeda G protein-coupled receptor 5 (TGR5) and Farnesoid X receptor (FXR), the latter promoting production of the endocrine-acting fibroblast growth factor 19 (FGF19). Short-term administration of single bile acids has been reported to enhance plasma levels of GLP-1 and to enhance energy expenditure. However, prolonged bile acid supplementation, e.g. of chenodeoxycholic acid (CDCA) for gallstone dissolution, has been reported to have adverse effects., Study Design: In this proof-of-concept study, we assessed the safety and metabolic effects of oral glycine-conjugated deoxycholic acid (GDCA) administration at 10 mg/kg/day using regular and slow-release capsules (mimicking physiological bile acid release) over 30 days in two groups of each 10 healthy lean men respectively., Main Findings: GDCA increased postprandial total bile acid and FGF19 concentrations while suppressing those of the primary bile acids CDCA and cholic acid. Plasma levels of 7α-hydroxy-4-cholesten-3-one were reduced, indicating repressed hepatic bile acid synthesis. There were minimal effects on indices of lipid-, glucose-, and energy metabolism. No serious adverse events were reported during GDCA administration in either capsule types, although 50% of participants showed mild increases in plasma levels of liver transaminases and 80% (regular capsules) and 50% (slow-release capsules) of participants experienced gastrointestinal adverse events., Conclusion: GDCA administration leads to elevated FGF19 levels and effectively inhibits primary bile acid synthesis, supporting therapy compliance and its effectiveness. However, effects on lipid, glucose- and energy metabolism were minimal, indicating that expanding the pool of this relatively hydrophobic bile acid does not impact energy metabolism in healthy subjects., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society.)

Background: Extended liver resection is the only treatment option for perihilar cholangiocarcinoma (pCCA). Bile salts and the gut hormone FGF19, both promoters of liver regeneration (LR), have not been investigated in patients undergoing resection for pCCA. We aimed to evaluate the bile salt-FGF19 axis perioperatively in pCCA and study its effects on LR., Methods: Plasma bile salts, FGF19, and C4 (bile salt synthesis marker) were assessed in patients with pCCA and controls (colorectal liver metastases), before and after resection on postoperative days (PODs) 1, 3, and 7. Hepatic bile salts were determined in intraoperative liver biopsies., Results: Partial liver resection in pCCA elicited a sharp decline in bile salt and FGF19 plasma levels on POD 1 and remained low thereafter, unlike in controls, where bile salts rose gradually. Preoperatively, suppressed C4 in pCCA normalized postoperatively to levels similar to those in the controls. The remnant liver volume and postoperative bilirubin levels were negatively associated with postoperative C4 levels. Furthermore, patients who developed postoperative liver failure had nearly undetectable C4 levels on POD 7. Hepatic bile salts strongly predicted hyperbilirubinemia on POD 7 in both groups. Finally, postoperative bile salt levels on day 7 were an independent predictor of LR., Conclusions: Partial liver resection alters the bile salt-FGF19 axis, but its derailment is unrelated to LR in pCCA. Postoperative monitoring of circulating bile salts and their production may be useful for monitoring LR., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

Ageing changes the impact of nutrition, whereby inflammation has been suggested to play a role in age-related disabilities such as diabetes and cardiovascular disease. The aim of this study was to investigate differences in postprandial bile-acid response and its effect on energy metabolism between young and elderly people. Nine young, healthy men and nine elderly, healthy men underwent a liquid mixed-meal test. Postprandial bile-acid levels, insulin, glucose, GLP-1, C4, FGF19 and lipids were measured. Appetite, body composition, energy expenditure and gut microbiome were also measured. The elderly population showed lower glycine conjugated CDCA and UDCA levels and higher abundances of Ruminiclostridium , Marvinbryantia and Catenibacterium , but lower food intake, decreased fat free mass and increased cholesterol levels. Aging is associated with changes in postprandial bile-acid composition and microbiome, diminished hunger and changes in body composition and lipid levels. Further studies are needed to determine if these changes may contribute to malnutrition and sarcopenia in elderly.

Background: Bile salts of hepatic and microbial origin mediate interorgan cross talk in the gut-liver axis. Here, we assessed whether the newly discovered class of microbial bile salt conjugates (MBSCs) activate the main host bile salt receptors (Takeda G protein-coupled receptor 5 [TGR5] and farnesoid X receptor [FXR]) and enter the human systemic and enterohepatic circulation., Methods: N-amidates of (chenodeoxy) cholic acid and leucine, tyrosine, and phenylalanine were synthesized. Receptor activation was studied in cell-free and cell-based assays. MBSCs were quantified in mesenteric and portal blood and bile of patients undergoing pancreatic surgery., Results: MBSCs were activating ligands of TGR5 as evidenced by recruitment of Gsα protein, activation of a cAMP-driven reporter, and diminution of lipopolysaccharide-induced cytokine release from macrophages. Intestine-enriched and liver-enriched FXR isoforms were both activated by MBSCs, provided that a bile salt importer was present. The affinity of MBSCs for TGR5 and FXR was not superior to host-derived bile salt conjugates. Individual MBSCs were generally not detected (ie, < 2.5 nmol/L) in human mesenteric or portal blood, but Leu-variant and Phe-variant were readily measurable in bile, where MBSCs comprised up to 213 ppm of biliary bile salts., Conclusions: MBSCs activate the cell surface receptor TGR5 and the transcription factor FXR and are substrates for intestinal (apical sodium-dependent bile acid transporter) and hepatic (Na+ taurocholate co-transporting protein) transporters. Their entry into the human circulation is, however, nonsubstantial. Given low systemic levels and a surplus of other equipotent bile salt species, the studied MBSCs are unlikely to have an impact on enterohepatic TGR5/FXR signaling in humans. The origin and function of biliary MBSCs remain to be determined., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

Cerebrotendinous xanthomatosis (CTX) is a rare inherited disease characterized by sterol 27-hydroxylase (CYP27A1) deficiency and, thus, a lack of bile acid synthesis with a marked accumulation of 7α-hydroxylated bile acid precursors. In addition to their renowned lipid-emulgating role, bile acids have been shown to stimulate secretion of the glucose-lowering and satiety-promoting gut hormone glucagon-like peptide 1 (GLP-1). In this paper, we examined postprandial bile acid, glucose, insulin, GLP-1 and fibroblast growth factor 19 (FGF19) plasma profiles in patients with CTX and matched healthy controls. Seven patients and seven age, gender and body mass index matched controls were included and subjected to a 4 h mixed meal test with regular blood sampling. CTX patients withdrew from chenodeoxycholic acid (CDCA) and statin therapy three weeks prior to the test. Postprandial levels of total bile acids were significantly lower in CTX patients and consisted of residual CDCA with low amounts of ursodeoxycholic acid (UDCA). The postprandial plasma glucose peak concentration occurred later in CTX patients compared to controls, and patients' insulin levels remained elevated for a longer time. Postprandial GLP-1 levels were slightly higher in CTX subjects whereas postprandial FGF19 levels were lower in CTX subjects. This novel characterization of CTX patients reveals very low circulating bile acid levels and FGF19 levels, aberrant postprandial glucose and insulin profiles, and elevated postprandial GLP-1 responses.

The gut and the liver are characterized by mutual interactions between both organs, the microbiome, diet and other environmental factors. The sum of these interactions is conceptualized as the gut-liver axis. In this Review we discuss the gut-liver axis, concentrating on the barriers formed by the enterohepatic tissues to restrict gut-derived microorganisms, microbial stimuli and dietary constituents. In addition, we discuss the establishment of barriers in the gut and liver during development and their cooperative function in the adult host. We detail the interplay between microbial and dietary metabolites, the intestinal epithelium, vascular endothelium, the immune system and the various host soluble factors, and how this interplay establishes a homeostatic balance in the healthy gut and liver. Finally, we highlight how this balance is disrupted in diseases of the gut and liver, outline the existing therapeutics and describe the cutting-edge discoveries that could lead to the development of novel treatment approaches., (© 2023. Springer Nature Limited.)

Purpose: Cholangiocarcinoma (CCA) is a malignancy arising from the bile duct epithelium and has a poor outcome. Sulfatides are lipid components of lipid rafts, and are implicated in several cancer types. In the liver, sulfatides are specifically present in the bile ducts. Here, sulfatide abundance and composition were analyzed using mass spectrometry imaging in intrahepatic CCA (iCCA) tumor tissue, and correlated with tumor biology and clinical outcomes., Methods: Sulfatides were analyzed in iCCA (n = 17), hepatocellular carcinoma (HCC, n = 10) and colorectal liver metastasis (CRLM, n = 10) tumor samples, as well as tumor-distal samples (control, n = 16) using mass spectrometry imaging. Levels of sulfatides as well as the relative amount in structural classes were compared between groups, and were correlated with clinical outcomes for iCCA patients., Results: Sulfatide localization was limited to the respective tumor areas and the bile ducts. Sulfatide abundance was similar in iCCA and control tissue, while intensities were notably higher in CRLM in comparison with control (18-fold, P < 0.05) and HCC tissue (47-fold, P < 0.001). Considerable variation in sulfatide abundance was observed in iCCA tumors. A high ratio of unsaturated to saturated sulfatides was associated with reduced disease-free survival (10 vs. 20 months) in iCCA. The sulfatide pattern in HCC deviated from the other groups, with a higher relative abundance of odd- versus even-chain sulfatides., Conclusion: Sulfatides were found in tumor tissue of patients with iCCA, with sulfatide abundance per pixel being similar to bile ducts. In this explorative study, sulfatide abundance was not related to overall survival of iCCA patients. A high ratio of unsaturated to saturated sulfatides was associated with earlier tumor recurrence in patients with iCCA., (© 2022. The Author(s).)

Background: Cancer cachexia is a multifactorial syndrome characterized by body weight loss and systemic inflammation. The characterization of the inflammatory response in patients with cachexia is still limited. Lipocalin-2, a protein abundant in neutrophils, has recently been implicated in appetite suppression in preclinical models of pancreatic cancer cachexia. We hypothesized that lipocalin-2 levels could be associated with neutrophil activation and nutritional status of pancreatic ductal adenocarcinoma (PDAC) patients., Methods: Plasma levels of neutrophil activation markers calprotectin, myeloperoxidase, elastase, and bactericidal/permeability-increasing protein (BPI) were compared between non-cachectic PDAC patients (n=13) and cachectic PDAC patients with high (≥26.9 ng/mL, n =34) or low (<26.9 ng/mL, n =34) circulating lipocalin-2 levels. Patients' nutritional status was assessed by the patient-generated subjective global assessment (PG-SGA) and through body composition analysis using CT-scan slices at the L3 level., Results: Circulating lipocalin-2 levels did not differ between cachectic and non-cachectic PDAC patients (median 26.7 (IQR 19.7-34.8) vs . 24.8 (16.6-29.4) ng/mL, p =0.141). Cachectic patients with high systemic lipocalin-2 levels had higher concentrations of calprotectin, myeloperoxidase, and elastase than non-cachectic patients or cachectic patients with low lipocalin-2 levels (calprotectin: 542.3 (355.8-724.9) vs . 457.5 (213.3-606.9), p =0.448 vs . 366.5 (294.5-478.5) ng/mL, p =0.009; myeloperoxidase: 30.3 (22.1-37.9) vs . 16.3 (12.0-27.5), p =0.021 vs . 20.2 (15.0-29.2) ng/mL, p =0.011; elastase: 137.1 (90.8-253.2) vs . 97.2 (28.8-215.7), p =0.410 vs . 95.0 (72.2-113.6) ng/mL, p =0.006; respectively). The CRP/albumin ratio was also higher in cachectic patients with high lipocalin-2 levels (2.3 (1.3-6.0) as compared to non-cachectic patients (1.0 (0.7-4.2), p =0.041). Lipocalin-2 concentrations correlated with those of calprotectin ( r s =0.36, p <0.001), myeloperoxidase ( r s =0.48, p <0.001), elastase ( r s =0.50, p <0.001), and BPI ( r s =0.22, p =0.048). Whereas no significant correlations with weight loss, BMI, or L3 skeletal muscle index were observed, lipocalin-2 concentrations were associated with subcutaneous adipose tissue index ( r s =-0.25, p =0.034). Moreover, lipocalin-2 tended to be elevated in severely malnourished patients compared with well-nourished patients (27.2 (20.3-37.2) vs . 19.9 (13.4-26.4) ng/mL, p =0.058)., Conclusions: These data suggest that lipocalin-2 levels are associated with neutrophil activation in patients with pancreatic cancer cachexia and that it may contribute to their poor nutritional status., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Deng, Aberle, van Bijnen, van der Kroft, Lenaerts, Neumann, Wiltberger, Schaap, Olde Damink and Rensen.)

Purpose: In the pre-clinical setting, hepatocellular bile salt accumulation impairs liver regeneration following partial hepatectomy. Here, we study the impact of cholestasis on portal vein embolization (PVE)-induced hypertrophy of the future liver remnant (FLR)., Methods: Patients were enrolled with perihilar cholangiocarcinoma (pCCA) or colorectal liver metastases (CRLM) undergoing PVE before a (extended) right hemihepatectomy. Volume of segments II/III was considered FLR and assessed on pre-embolization and post-embolization CT scans. The degree of hypertrophy (DH, percentual increase) and kinetic growth rate (KGR, percentage/week) were used to assess PVE-induced hypertrophy., Results: A total of 50 patients (31 CRLM, 19 pCCA) were included. After PVE, the DH and KGR were similar in patients with CRLM and pCCA (5.2 [3.3-6.9] versus 5.7 [3.2-7.4] %, respectively, p = 0.960 for DH; 1.4 [0.9-2.5] versus 1.9 [1.0-2.4] %/week, respectively, p = 0.742 for KGR). Moreover, pCCA patients with or without hyperbilirubinemia had comparable DH (5.6 [3.0-7.5] versus 5.7 [2.4-7.0] %, respectively, p = 0.806) and KGR (1.7 [1.0-2.4] versus 1.9 [0.8-2.4] %/week, respectively, p = 1.000). For patients with pCCA, unilateral drainage in FLR induced a higher DH than bilateral drainage (6.7 [4.9-7.9] versus 2.7 [1.5-4.2] %, p = 0.012). C-reactive protein before PVE was negatively correlated with DH (ρ = - 0.539, p = 0.038) and KGR (ρ = - 0.532, p = 0.041) in patients with pCCA., Conclusions: There was no influence of cholestasis on FLR hypertrophy in patients undergoing PVE. Bilateral drainage and inflammation appeared to be negatively associated with FLR hypertrophy. Further prospective studies with larger and more homogenous patient cohorts are desirable., (© 2023. The Author(s).)

Bile acids are crucial for the uptake of dietary lipids and can shape the gut-microbiome composition. This latter function is associated with the toxicity of bile acids and can be modulated by bile acid modifying bacteria such as Eggerthella lenta , but the molecular details of the interaction of bacteria depending on bile acid modifications are not well understood. In order to unravel the molecular response to bile acids and their metabolites, we cultivated eight strains from a human intestinal microbiome model alone and in co-culture with Eggerthella lenta in the presence of cholic acid (CA) and deoxycholic acid (DCA). We observed growth inhibition of particularly gram-positive strains such as Clostridium ramosum and the gram-variable Anaerostipes cacae by CA and DCA stress. C. ramosum was alleviated through co-culturing with Eggerthella lenta . We approached effects on the membrane by zeta potential and genotoxic and metabolic effects by (meta)proteomic and metabolomic analyses. Co-culturing with Eggerthella lenta decreased both CA and DCA by the formation of oxidized and epimerized bile acids. Eggerthella lenta also produces microbial bile salt conjugates in a co-cultured species-specific manner. This study highlights how the interaction with other bacteria can influence the functionality of bacteria.

Bile acids are a key mediator of the molecular microbiome-host interaction, and various mass spectrometry-based assays have been developed in the recent decade to quantify a wide range of bile acids. We compare existing methodologies to harmonize them. Methodology for absolute quantification of bile acids from six laboratories in Europe were compared for the quantification of the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) and conjugated products glycocholic acid (GCA) and taurocholic acid (TCA). For the bacterially modified secondary bile acids, the quantification of deoxycholic acid (DCA) and lithocholic acid (LCA) was compared. For the murine bile acids, we used the primary muricholic acids (α-MCA and, β-MCA) and the intestinally produced secondary bile acid muricholic (ω-MCA). The standards were spiked into methanol:water (1:1) mix as well as in human and murine serum at either low concentration range (150-3000 nM) or high concentration range (1500-40,000 nM). The precision was better for higher concentrations. Measurements for the hydrophobic unconjugated bile acids LCA and ω-MCA were the most challenging. The quality assessments were generally very similar, and the comprehensive analyses demonstrated that data from chosen locations can be used for comparisons between studies.

Biotransformation of host bile salts by gut microbes results in generation of secondary bile salt species that have biological and physicochemical properties that are distinct from the parent compounds. There is increased awareness that a bile salt-gut microbiome axis modulates various processes in the host, including innate and adaptive immunity, by interaction of microbial bile salt metabolites with host receptors. Omics and targeted approaches have vastly expanded the number and repertoire of secondary bile salt species. A new class of microbial bile salt metabolites was reported in 2020 and comprises bile salts that are conjugated by microbial enzymes. Amino acids other than those employed by host enzymes (glycine and taurine) are used as substrates in the formation of these microbial bile salt conjugates (MBSCs). Leucocholic acid, phenylalanocholic acid and tyrosocholic acid were the first MBSCs identified in mice and humans. The number of distinct MBSCs is now approaching 50, with variation both at the level of bile salt and amino acid employed for conjugation. Evidence is emerging that MBSC generation is a common feature of human gut bacteria, and initial links with disease states have been reported. In this review, we discuss this intriguing new class of secondary bile salts, with yet enigmatic function.

Duodenal mucosal resurfacing (DMR) is a new endoscopic ablation technique aimed at improving glycemia and metabolic control in patients with type 2 diabetes mellitus (T2DM). DMR appears to improve insulin resistance, which is the root cause of T2DM, but its mechanism of action is largely unknown. Bile acids function as intestinal signaling molecules in glucose and energy metabolism via the activation of farnesoid X receptor and secondary signaling [e.g., via fibroblast growth factor 19 (FGF19)], and are linked to metabolic health. We investigated the effect of DMR and glucagon-like peptide-1 (GLP-1) on postprandial bile acid responses in 16 patients with insulin-dependent T2DM, using mixed meal tests performed at the baseline and 6 mo after the DMR procedure. The combination treatment allowed discontinuation of insulin treatment in 11/16 (69%) of patients while improving glycemic and metabolic health. We found increased postprandial unconjugated bile acid responses (all P < 0.05), an overall increased secondary bile acid response ( P = 0.036) and a higher 12α-hydroxylated:non-12α-hydroxylated ratio ( P < 0.001). Total bile acid concentrations were unaffected by the intervention. Postprandial FGF19 and 7-α-hydroxy-4-cholesten-3-one (C4) concentrations decreased postintervention (both P < 0.01). Our study demonstrates that DMR with GLP-1 modulates the postprandial bile acid response. The alterations in postprandial bile acid responses may be the result of changes in the microbiome, ileal bile acid uptake and improved insulin sensitivity. Controlled studies are needed to elucidate the mechanism linking the combination treatment to metabolic health and bile acids. NEW & NOTEWORTHY Glycemic and metabolic improvements are seen in patients with type 2 diabetes after replacing their insulin therapy with DMR and GLP-1. These changes are accompanied by changes in postprandial bile acid concentrations: increased unconjugated and secondary bile acids.

Background: Generally, food intake occurs in a three-meal per 24 h fashion with in-between meal snacking. As such, most humans spend more than ∼ 12-16 h per day in the postprandial state. It may be reasoned from an evolutionary point of view, that the human body is physiologically habituated to less frequent meals. Metabolic flexibility (i.e., reciprocal changes in carbohydrate and fatty acid oxidation) is a characteristic of metabolic health and is reduced by semi-continuous feeding. The effects of time-restricted feeding (TRF) on metabolic parameters and physical performance in humans are equivocal. Methods: To investigate the effect of TRF on metabolism and physical performance in free-living healthy lean individuals, we compared the effects of eucaloric feeding provided by a single meal (22/2) vs. three meals per day in a randomized crossover study. We included 13 participants of which 11 (5 males/6 females) completed the study: age 31.0 ± 1.7 years, BMI 24.0 ± 0.6 kg/m 2 and fat mass (%) 24.0 ± 0.6 (mean ± SEM). Participants consumed all the calories needed for a stable weight in either three meals (breakfast, lunch and dinner) or one meal per day between 17:00 and 19:00 for 11 days per study period. Results: Eucaloric meal reduction to a single meal per day lowered total body mass (3 meals/day -0.5 ± 0.3 vs. 1 meal/day -1.4 ± 0.3 kg, p = 0.03), fat mass (3 meals/day -0.1 ± 0.2 vs. 1 meal/day -0.7 ± 0.2, p = 0.049) and increased exercise fatty acid oxidation ( p < 0.001) without impairment of aerobic capacity or strength ( p > 0.05). Furthermore, we found lower plasma glucose concentrations during the second half of the day during the one meal per day intervention ( p < 0.05). Conclusion: A single meal per day in the evening lowers body weight and adapts metabolic flexibility during exercise via increased fat oxidation whereas physical performance was not affected., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Meessen, Andresen, van Barneveld, van Riel, Johansen, Kolnes, Kemper, Olde Damink, Schaap, Romijn, Jensen and Soeters.)

Background: Postprandial rise of plasma essential amino acids (EAAs) determines the anabolic effect of dietary protein. Disturbed gastrointestinal function could impair the anabolic response in critically ill patients. Aim was to investigate the postprandial EAA response in critically ill patients and its relation to small-intestinal function., Methods: Twenty-one mechanically ventilated patients and 9 healthy controls received a bolus containing 100 ml of a formula feed (Ensure) and 2 g of 3-O-Methyl-d-glucose (3-OMG) via postpyloric feeding tube. Fasting and postprandial plasma concentrations of EAAs, 3-OMG, total bile salts, and the gut-released hormone fibroblast growth factor 19 (FGF19) were measured over a 4-hour period. Changes over time and between groups were assessed with linear mixed-effects analysis. Early (0-60 minutes) and total postprandial responses are summarized as the incremental area under the curve (iAUC)., Results: At baseline, fasting EAA levels were similar in both groups: 1181 (1055-1276) vs 1150 (1065-1334) μmol·L-1, P = .87. The early postprandial rise in EAA was not apparent in critically ill patients compared with healthy controls (iAUC 60 , -4858 [-6859 to 2886] vs 5406 [3099-16,853] µmol·L -1 ·60 minutes; P = .039). Impaired EAA response did not correlate with impaired 3-OMG response (Spearman ρ 0.32, P = .09). There was a limited increase in total bile salts but no relevant FGF19 response in either group., Conclusion: Postprandial rise of EAA is blunted in critically ill patients and unrelated to glucose absorption measured with 3-OMG. Future studies should aim to delineate governing mechanisms of macronutrient malabsorption., (© 2021 The Authors. Journal of Parenteral and Enteral Nutrition published by Wiley Periodicals LLC on behalf of American Society for Parenteral and Enteral Nutrition.)

Background and Aims: Automated chyme reinfusion (CR) in patients with intestinal failure (IF) and a temporary double enterostomy (TDE) restores intestinal function and protects against liver injury, but the mechanisms are incompletely understood. The aim was to investigate whether the beneficial effects of CR relate to functional recovery of enterohepatic signaling through the bile salt-FGF19 axis., Approach and Results: Blood samples were collected from 12 patients, 3 days before, at start, and 1, 3, 5, and 7 weeks after CR initiation. Plasma FGF19, total bile salts (TBS), 7-α-hydroxy-4-cholesten-3-one (C4; a marker of bile salt synthesis), citrulline (CIT), bile salt composition, liver tests, and nutritional risk indices were determined. Paired small bowel biopsies prior to CR and after 21 days were taken, and genes related to bile salt homeostasis and enterocyte function were assessed. CR induced an increase in plasma FGF19 and decreased C4 levels, indicating restored regulation of bile salt synthesis through endocrine FGF19 action. TBS remained unaltered during CR. Intestinal farnesoid X receptor was up-regulated after 21 days of CR. Secondary and deconjugated bile salt fractions were increased after CR, reflecting restored microbial metabolism of host bile salts. Furthermore, CIT and albumin levels gradually rose after CR, while abnormal serum liver tests normalized after CR, indicating restored intestinal function, improved nutritional status, and amelioration of liver injury. CR increased gene transcripts related to enterocyte number, carbohydrate handling, and bile salt homeostasis. Finally, the reciprocal FGF19/C4 response after 7 days predicted the plasma CIT time course., Conclusions: CR in patients with IF-TDE restored bile salt-FGF19 signaling and improved gut-liver function. Beneficial effects of CR are partly mediated by recovery of the bile salt-FGF19 axis and subsequent homeostatic regulation of bile salt synthesis., (© 2021 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Individuals with hepatic steatosis often display several metabolic abnormalities including insulin resistance and muscle atrophy. Previously, we found that hepatic steatosis results in an altered hepatokine secretion profile, thereby inducing skeletal muscle insulin resistance via inter-organ crosstalk. In this study, we aimed to investigate whether the altered secretion profile in the state of hepatic steatosis also induces skeletal muscle atrophy via effects on muscle protein turnover. To investigate this, eight-week-old male C57BL/6J mice were fed a chow (4.5% fat) or a high-fat diet (HFD; 45% fat) for 12 weeks to induce hepatic steatosis, after which the livers were excised and cut into ~200-µm slices. Slices were cultured to collect secretion products (conditioned medium; CM). Differentiated L6-GLUT4myc myotubes were incubated with chow or HFD CM to measure glucose uptake. Differentiated C2C12 myotubes were incubated with chow or HFD CM to measure protein synthesis and breakdown, and gene expression via RNA sequencing. Furthermore, proteomics analysis was performed in chow and HFD CM. It was found that HFD CM caused insulin resistance in L6-GLUT4myc myotubes compared with chow CM, as indicated by a blunted insulin-stimulated increase in glucose uptake. Furthermore, protein breakdown was increased in C2C12 cells incubated with HFD CM, while there was no effect on protein synthesis. RNA profiling of C2C12 cells indicated that 197 genes were differentially expressed after incubation with HFD CM, compared with chow CM, and pathway analysis showed that pathways related to anatomical structure and function were enriched. Proteomics analysis of the CM showed that 32 proteins were differentially expressed in HFD CM compared with chow CM. Pathway enrichment analysis indicated that these proteins had important functions with respect to insulin-like growth factor transport and uptake, and affect post-translational processes, including protein folding, protein secretion and protein phosphorylation. In conclusion, the results of this study support the hypothesis that secretion products from the liver contribute to the development of muscle atrophy in individuals with hepatic steatosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pasmans, Adriaens, Olinga, Langen, Rensen, Schaap, Olde Damink, Caiment, van Loon, Blaak and Meex.)

The involvement of bile salt-fibroblast growth factor 19 (FGF19) signaling in human liver regeneration (LR) is not well studied. Therefore, we studied aspects of bile salt-FGF19 signaling shortly after liver resection in patients. We compared plasma bile salt and FGF19 levels in arterial, portal and hepatic venous blood, calculated venous-arterial differences (ΔVA), and determined hepatic transcript levels on two intra-operative time points: before (< 1 hour) and immediately after (> 2-3 hours) liver resection (i.e., following surgery). Postoperative bile salt and FGF19 levels were assessed on days 1, 2, and 3. LR was studied by computed tomography (CT)-liver volumetry. Following surgery, the liver, arterial, and portal bile salt levels were elevated ( P  < 0.05). Furthermore, an increased amount of bile salts was released in portal blood and extracted by the remnant liver ( P  < 0.05). Postoperatively, bile salt levels were elevated from day 1 onward ( P  < 0.001). For FGF19, intra-operative or postoperative changes of ΔVA or plasma levels were not observed. The bile salt-homeostatic regulator farnesoid X receptor ( FXR ) was markedly up-regulated following surgery ( P  < 0.001). Cell-cycle re-entry priming factors (interleukin 6 [ IL-6 ], signal transducer and activator of transcription 3 [ STAT3 ], and cJUN ) were up-regulated following surgery and were positively correlated with FXR expression (P  < 0.05). Postoperative hyperbilirubinemia was preceded by postsurgery low FXR and high Na+/Taurocholate cotransporting polypeptide ( NTCP ) expression in the remnant liver coupled with higher liver bile salt content ( P  < 0.05). Finally, bile salt levels on postoperative day 1 were an independent predictor of LR ( P  < 0.05). Conclusion : Systemic, portal, and liver bile salt levels are rapidly elevated after liver resection. Postoperative bile salts were positively associated with liver volume gain. In the studied time frame, FGF19 levels remained unaltered, suggesting that FGF19 plays a minor role in human LR. These findings indicate a more relevant role of bile salts in human LR., (© 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of the American Association for the Study of Liver Diseases.)

Background & Aims: To investigate the acute effects of intravenous vs enteral meal administration on circulating bile acid and gut hormone responses., Methods: In a randomized crossover design, we compared the effects of duodenal (via a nasoduodenal tube) vs parenteral (intravenous) administration over 180 min of identical mixed meals on circulating bile acid and gut hormone concentrations in eight healthy lean men. We analysed the bile acid and gut hormone responses in two periods: the intraprandial period from time point (T) 0 until T180 during meal administration and the postprandial period from T180 until T360, after discontinuation of meal administration., Results: Intravenous meal administration decreased the intraprandial (AUC (μmol/L∗min) duodenal 1469 ± 284 vs intravenous 240 ± 39, p < 0.01) and postprandial bile acid response (985 ± 240 vs 223 ± 5, p < 0.05) and was accompanied by decreased gut hormone responses including glucose-dependent insulinotropic polypeptide, glucagon-like peptide 1, glucagon-like peptide 2 and fibroblast growth factor 19. Furthermore, intravenous meal administration elicited greater glucose concentrations, but similar insulin concentrations compared to enteral administration., Conclusions: Compared to enteral administration, parenteral nutrition results in lower postprandial bile acid and gut hormone responses in healthy lean men. This was accompanied by higher glucose concentrations in the face of similar insulin concentrations exposing a clear incretin effect of enteral mixed meal administration. The alterations in bile acid homeostasis were apparent after only one intravenous meal., Competing Interests: Conflict of interest None., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

: In a previous study, obeticholic acid (OCA) increased liver growth before partial hepatectomy (PHx) in rats through the bile acid receptor farnesoid X-receptor (FXR). In that model, OCA was administered during obstructive cholestasis. However, patients normally undergo PHx several days after biliary drainage. The effects of OCA on liver regeneration were therefore studied in post-cholestatic Wistar rats. Rats underwent sham surgery or reversible bile duct ligation (rBDL), which was relieved after 7 days. PHx was performed one day after restoration of bile flow. Rats received 10 mg/kg OCA per day or were fed vehicle from restoration of bile flow until sacrifice 5 days after PHx. Liver regeneration was comparable between cholestatic and non-cholestatic livers in PHx-subjected rats, which paralleled liver regeneration a human validation cohort. OCA treatment induced ileal Fgf15 mRNA expression but did not enhance post-PHx hepatocyte proliferation through FXR/SHP signaling. OCA treatment neither increased mitosis rates nor recovery of liver weight after PHx but accelerated liver regrowth in rats that had not been subjected to rBDL. OCA did not increase biliary injury. Conclusively, OCA does not induce liver regeneration in post-cholestatic rats and does not exacerbate biliary damage that results from cholestasis. This study challenges the previously reported beneficial effects of OCA in liver regeneration in cholestatic rats.

Brown adipose tissue (BAT) might be a beneficial mediator in the development and treatment of nonalcoholic steatohepatitis (NASH). We aim to evaluate the gene expression of BAT activity-related genes during the development and the dietary and surgical treatment of NASH. BAT was collected from male C57BL/6J mice that received a high fat-high sucrose diet (HF-HSD) or a normal chow diet (NCD) for 4 and 20 weeks (n=8-9 per dietary group and timepoint) and from mice that underwent dietary intervention (return to NCD) (n=8), roux-en-y gastric bypass (RYGB) (n=6), or sham procedure (n=6) after 12 weeks HF-HSD. Expression of BAT genes involved in lipid metabolism (Cd36 and Cpt1b; p<0.05) and energy expenditure (Ucp1 and Ucp3; p<0.05) were significantly increased after 4 weeks HF-HSD compared with NCD, whereas in the occurrence of NASH after 20 weeks HF-HSD no difference was observed. We observed no differences in gene expression regarding lipid metabolism or energy expenditure at 8 weeks after dietary intervention (no NASH) compared with HF-HSD mice (NASH), nor in mice that underwent RYGB compared with SHAM. However, dietary intervention and RYGB both decreased the BAT gene expression of inflammatory cytokines (Il1b, Tnf-α and MCP-1; p<0.05). Gene expression of the batokine neuregulin 4 was significantly decreased after 20 weeks HF-HSD (p<0.05) compared with NCD, but was restored by dietary intervention and RYGB (p<0.05). In conclusion, BAT is hallmarked by dynamic alterations in the gene expression profile during the development of NASH and can be modulated by dietary intervention and bariatric surgery., Competing Interests: The authors declare that they have no conflict of interest., (Georg Thieme Verlag KG Rüdigerstraße 14, 70469 Stuttgart, Germany.)

Acute intake of the wheat bran extract Arabinoxylan-Oligosaccharide (AXOS) modulates the gut microbiota, improves stool characteristics and postprandial glycemia in healthy humans. Yet, little is known on how long-term AXOS intake influences gastrointestinal (GI) functioning, gut microbiota, and metabolic health. In this randomized, placebo-controlled, double-blind study, we evaluated the effects of AXOS intake on GI function and metabolic health in adults with slow GI transit without constipation. Forty-eight normoglycemic adults were included with whole-gut transit time (WGTT) of >35 h receiving either 15 g/day AXOS or placebo (maltodextrin) for 12-wks. The primary outcome was WGTT, and secondary outcomes included stool parameters, gut permeability, short-chain fatty acids (SCFA), microbiota composition, energy expenditure, substrate oxidation, glucose, insulin, lipids, gut hormones, and adipose tissue (AT) function. WGTT was unchanged, but stool consistency softened after AXOS. 12-wks of AXOS intake significantly changed the microbiota by increasing Bifidobacterium and decreasing microbial alpha-diversity. With a good classification accuracy, overall microbiota composition classified responders with decreased WGTT after AXOS. The incretin hormone Glucagon-like protein 1 was reduced after AXOS compared to placebo. Energy expenditure, plasma metabolites, AT parameters, SCFA, and gut permeability were unchanged. In conclusion, intake of wheat bran extract increases fecal Bifidobacterium and softens stool consistency without major effects on energy metabolism in healthy humans with a slow GI transit. We show that overall gut microbiota classified responders with decreased WGTT after AXOS highlighting that GI transit and change thereof were associated with gut microbiota independent of Bifidobacterium . NCT02491125.

Following birth, the neonatal intestine is exposed to maternal and environmental bacteria that successively form a dense and highly dynamic intestinal microbiota. Whereas the effect of exogenous factors has been extensively investigated, endogenous, host-mediated mechanisms have remained largely unexplored. Concomitantly with microbial colonization, the liver undergoes functional transition from a hematopoietic organ to a central organ of metabolic regulation and immune surveillance. The aim of the present study was to analyze the influence of the developing hepatic function and liver metabolism on the early intestinal microbiota. Here, we report on the characterization of the colonization dynamics and liver metabolism in the murine gastrointestinal tract (n = 6-10 per age group) using metabolomic and microbial profiling in combination with multivariate analysis. We observed major age-dependent microbial and metabolic changes and identified bile acids as potent drivers of the early intestinal microbiota maturation. Consistently, oral administration of tauro-cholic acid or β-tauro-murocholic acid to newborn mice (n = 7-14 per group) accelerated postnatal microbiota maturation.

The number of patients diagnosed with chronic bile duct disease is increasing and in most cases these diseases result in chronic ductular scarring, necessitating liver transplantation. The formation of ductular scaring affects liver function; however, scar-generating portal fibroblasts also provide important instructive signals to promote the proliferation and differentiation of biliary epithelial cells. Therefore, understanding whether we can reduce scar formation while maintaining a pro-regenerative microenvironment will be essential in developing treatments for biliary disease. Here, we describe how regenerating biliary epithelial cells express Wnt-Planar Cell Polarity signalling components following bile duct injury and promote the formation of ductular scars by upregulating pro-fibrogenic cytokines and positively regulating collagen-deposition. Inhibiting the production of Wnt-ligands reduces the amount of scar formed around the bile duct, without reducing the development of the pro-regenerative microenvironment required for ductular regeneration, demonstrating that scarring and regeneration can be uncoupled in adult biliary disease and regeneration.

Bile acids, glucagon-like peptide-1 (GLP-1), and fibroblast growth factor 19 (FGF19) play an important role in postprandial metabolism. In this study, we investigated the postprandial bile acid response in plasma and its relation to insulin, GLP-1, and FGF19. First, we investigated the postprandial response to 40-h fast. Then we administered glycine-conjugated deoxycholic acid (gDCA) with the meal. We performed two separate observational randomized crossover studies on healthy, lean men. In experiment 1 : we tested 4-h mixed meal after an overnight fast and a 40-h fast. In experiment 2 , we tested a 4-h mixed meal test with and without gDCA supplementation. Both studies measured postprandial glucose, insulin, bile acids, GLP-1, and FGF19. In experiment 1 , 40 h of fasting induced insulin resistance and increased postprandial GLP-1 and FGF19 concentrations. After an overnight fast, we observed strong correlations between postprandial insulin and gDCA levels at specific time points. In experiment 2 , administration of gDCA increased GLP-1 levels and lowered late postprandial glucose without effect on FGF19. Energy expenditure was not affected by gDCA administration. Unexpectedly, 40 h of fasting increased both GLP-1 and FGF19, where the former appeared bile acid independent and the latter bile acid dependent. Second, a single dose of gDCA increased postprandial GLP-1. Therefore, our data add complexity to the physiological regulation of the enterokines GLP-1 and FGF19 by bile acids.

Critical illness is associated with a disturbed regulation of gastrointestinal hormones resulting in functional and metabolic anomalies. Fibroblast growth factor 19 (FGF19) is an ileum-derived metabolic hormone induced by bile salts upon gallbladder emptying after enteral nutrient stimulation. Our aim was to study the nutrient-stimulated FGF19 response in 24 patients admitted to the intensive care unit (ICU) compared with 12 healthy controls. All subjects received intraduodenal high-lipid nutrient infusion for 120 minutes. Blood was collected every 30 minutes until 1 hour after infusion, and gallbladder emptying was studied by ultrasound. Serum levels of bile salts and FGF19 were assessed. ICU patients had significantly higher fasting bile salt serum levels compared with controls, whereas FGF19 serum levels were similar. In both groups, nutrient infusion elicited substantial bile salt elevations (P < 0.001), peaking at 90 minutes, albeit with a significantly lower peak in the ICU patients (P = 0.029). In controls, FGF19 was significantly elevated relative to baseline from 120 minutes onward (P < 0.001). In ICU patients, the FGF19 response was blunted, as reflected by significantly lower FGF19 elevations at 120, 150, and 180 minutes (P < 0.05) and significantly lower area under the curve (AUC) values compared with controls (P < 0.001). Gallbladder dysmotility was associated with the impaired FGF19 response in critical illness. The gallbladder ejection fraction correlated positively with FGF19 AUC values (ρ = +0.34, P = 0.045). In 10 of 24 ICU patients, gallbladder emptying was disturbed. These patients had significantly lower FGF19 AUC values (P < 0.001). Gallbladder emptying and the FGF19 response were respectively disturbed or absent in patients receiving norepinephrine. Conclusion: The nutrient-stimulated FGF19 response is impaired in ICU patients, which is mechanistically linked to gallbladder dysmotility in critical illness. This may contribute to disturbed liver metabolism in these patients and has potential as a nutritional biomarker., (© 2019 by the American Association for the Study of Liver Diseases.)

Background: Patients with chronic intestinal failure (CIF) often develop cholestatic liver injury, which may lead to liver failure and need for organ transplantation., Objectives: The aim of this study was to investigate whether citrulline (CIT) and the enterokine fibroblast growth factor 19 (FGF19) are associated with chronic cholestasis and survival in adult CIF patients, and to develop a risk score to predict their survival., Methods: We studied 135 adult CIF patients on intravenous supplementation (>3 mo). Associations of plasma CIT and FGF19 with chronic cholestasis and survival were estimated by logistic and Cox regression models. A predictive risk score was developed and validated internally., Results: Patients with chronic cholestasis (17%) had a reduced 5-y survival rate compared with patients without chronic cholestasis (38% and 62%, respectively). In multivariable analysis, low FGF19, low CIT, and female sex were associated with chronic cholestasis. Patients with low rather than high CIT or FGF19 also had reduced 5-y survival rates (29% compared with 69%; 54% compared with 66%, respectively). Risk factors identified in multivariable analysis of survival were low FGF19 (HR: 3.4), low CIT (HR: 3.3), and number of intravenous infusions per week (HR: 1.4). These 3 predictors were incorporated in a risk model of survival termed Model for End-Stage Intestinal Failure (MESIF) (C-statistic 0.78). The 5-y survival rates for patients with MESIF scores of 0 to <20 (n = 47), 20-40 (n = 75), and >40 (n = 13) were 80%, 58%, and 14%, respectively., Conclusions: CIT and FGF19 predict chronic cholestasis and survival in this cohort of adult CIF patients, and the derived MESIF score is associated with their survival. Pending external validation, the MESIF score may help to identify patients for closer clinical monitoring or earlier referral to intestinal transplantation centers., (Copyright © American Society for Nutrition 2019.)

Background and Aims: Placement of the duodenal-jejunal bypass liner (DJBL) leads to rapid weight loss and restoration of insulin sensitivity in a similar fashion to bariatric surgery. Increased systemic bile acid levels are candidate effectors for these effects through postprandial activation of their receptors TGR5 and FXR. We aimed to quantify postprandial bile acid, GLP-1 and FGF19 responses and assess their temporal relation to the weight loss and metabolic and hormonal changes seen after DJBL placement., Methods: We performed mixed meal testing in 17 obese patients with type 2 diabetes mellitus (DM2) directly before, one week after and 6 months after DJBL placement., Results: Both fasting and postprandial bile acid levels were unchanged at 1 week after implantation, and greatly increased 6 months after implantation. The increase consisted of unconjugated bile acid species. 3 hour-postprandial GLP-1 levels increased after 1 week and were sustained, whereas FGF19 levels and postprandial plasma courses were unaffected., Conclusions: DJBL placement leads to profound increases in unconjugated bile acid levels after 6 months, similar to the effects of bariatric surgery. The temporal dissociation between the changes in bile acids, GLP-1 and FGF19 and other gut hormone responses warrant caution about the beneficial role of bile acids after DJBL placement. This observational uncontrolled study emphasizes the need for future controlled studies., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction: Currently, no therapies are available for Zellweger spectrum disorders (ZSDs), a group of genetic metabolic disorders characterised by a deficiency of functional peroxisomes. In a previous study, we showed that oral cholic acid (CA) treatment can suppress bile acid synthesis in ZSD patients and, thereby, decrease plasma levels of toxic C 27 -bile acid intermediates, one of the biochemical abnormalities in these patients. However, no effect on clinically relevant outcome measures could be observed after 9 months of CA treatment. It was noted that, in patients with advanced liver disease, caution is needed because of possible hepatotoxicity., Methods: An extension study of the previously conducted pretest-posttest design study was conducted including 17 patients with a ZSD. All patients received oral CA for an additional period of 12 months, encompassing a total of 21 months of treatment. Multiple clinically relevant parameters and markers for bile acid synthesis were assessed after 15 and 21 months of treatment., Results: Bile acid synthesis was still suppressed after 21 months of CA treatment, accompanied with reduced levels of C 27 -bile acid intermediates in plasma. These levels significantly increased again after discontinuation of CA. No significant changes were found in liver tests, liver elasticity, coagulation parameters, fat-soluble vitamin levels or body weight., Conclusions: Although CA treatment did lead to reduced levels of toxic C 27 -bile acid intermediates in ZSD patients without severe liver fibrosis or cirrhosis, no improvement of clinically relevant parameters was observed after 21 months of treatment. We discuss the implications for CA therapy in ZSD based on these results., (© 2018 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Cholestasis impairs liver regeneration following partial liver resection (PHx). Bile acid receptor farnesoid X-receptor (FXR) is a key mediator of liver regeneration. The effects of FXR agonist obeticholic acid (OCA) on liver (re)growth were therefore studied in cholestatic rats. Animals underwent sham surgery or reversible bile duct ligation (rBDL). PHx with concurrent internal biliary drainage was performed 7 days after rBDL. Animals were untreated or received OCA (10 mg/kg/day) per oral gavage from rBDL until sacrifice. After 7 days of OCA treatment, dry liver weight increased in the rBDL + OCA group, indicating OCA-mediated liver growth. Enhanced proliferation in the rBDL + OCA group prior to PHx concurred with a rise in Ki67-positive hepatocytes, elevated hepatic Ccnd1 and Cdc25b expression, and an induction of intestinal fibroblast growth factor 15 expression. Liver regrowth after PHx was initially stagnant in the rBDL + OCA group, possibly due to hepatomegaly prior to PHx. OCA increased hepatobiliary injury markers during BDL, which was accompanied by upregulation of the bile salt export pump. There were no differences in histological liver injury. In conclusion, OCA induces liver growth in cholestatic rats prior to PHx but exacerbates biliary injury during cholestasis, likely by forced pumping of bile acids into an obstructed biliary tree.

The liver is the primary organ involved in handling of bile salts, a class of amphipathic molecules with signaling activities as well as desired and detrimental detergent actions. To allow in-depth investigation of functions of bile salts in healthy and diseased liver, the spatial distribution of bile salt species within the liver needs to be studied. Therefore, the aim of our study was to determine hepatic bile salt distribution and identify specific lipid markers that define the structural elements of the liver. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) was used to monitor the spatial distribution of bile salts and lipids in liver sections of rat, dog, and patients with unaffected and cholestatic parenchyma. MALDI-MSI in negative ion mode showed the local presence of a variety of bile salts, predominantly taurine-conjugates, as localized patches of varying sizes (representing the bile ducts) throughout the liver tissue. Specific molecular markers were identified for the connective tissue (phosphatidic acids, e.g., [PA (18:0&#95;18:1)-H] - ), the liver parenchyma (phosphatidylinositols, e.g., [PI (18:0&#95;20:4)-H] - ), and the bile ducts (hydroxylated-sulfatides, e.g., [ST-OH (18:1&#95;24:0)-H] - ). One of these sulfatides (at m/ z 906.6339) was found to be uniquely localized in a thin lining on the inside of the bile duct, colocalized with cytokeratins, and encased luminal bile salts. A similar distribution of the aforementioned sulfatide was observed, albeit in constricted ductular structures, in the liver of a patient with a mild clinical phenotype of primary sclerosing cholangitis (PSC). In contrast, sulfatides were virtually absent in the liver of patients with PSC and a severe clinical phenotype, with (atypical) cholanoids (e.g., the bile alcohol 5-cyprinolsulfate) abundant in the extra-ductular space and glyco(cheno)deoxycholic acid-3-sulfate localized to fibrotic connective tissue. The latter two molecular species were able to discriminate between healthy liver tissue ( n = 3) and tissue from PSC patients with a severe clinical phenotype ( n = 3). In conclusion, the distinct structural elements of the mammalian liver are characterized by specific classes of lipids. We propose that (hydroxylated-)sulfatides are specific molecular markers of the bile duct.

Introduction: Endotoxins, in the form of lipopolysaccharides (LPS), are potent inducers of biliary injury. However the mechanism by which injury develops remains unclear. We hypothesized that hepatic macrophages are pivotal in the development of endotoxin-induced biliary injury and that no injury would occur in their absence., Material and Methods: Clodronate liposomes were used to deplete macrophages from the liver. Forty-eight rats were equally divided across six study groups: sham operation (sham), liposome treatment and sham operation (liposomes+sham), 1mg/kg LPS i.p. (LPS), liposome treatment and LPS administration (liposomes+LPS), hepatic ischaemia-reperfusion injury with LPS administration (IRI+LPS) and liposome treatment followed by IRI+LPS (liposomes+IRI+LPS). Following 6h of reperfusion, blood, bile, and liver tissue was collected for further analysis. Small bile duct injury was assessed, serum liver tests were performed and bile composition was evaluated. The permeability of the blood-biliary barrier (BBB) was assessed using intravenously administered horseradish peroxidase (HRP)., Results: The presence of hepatic macrophages was reduced by 90% in LPS and IRI+LPS groups pre-treated with clodronate liposomes (P<0.001). Severe small bile duct injury was not affected by macrophage depletion, and persisted in the liposomes+IRI+LPS group (50% of animals) and liposomes+LPS group (75% of animals). Likewise, BBB impairment persisted following macrophage depletion. LPS-induced elevation of the chemokine Mcp-1 in bile was not affected by macrophage depletion., Conclusions: Depletion of hepatic macrophages did not prevent development of biliary injury following LPS or LPS-enhanced IRI. Cholangiocyte activation rather than macrophage activation may underlie this injury. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background and Aim: Animal studies indicated that systemic ophthalmic acid (OPH) is a biomarker for hepatic glutathione (GSH) homeostasis, an important determinant of liver function. We aimed to clarify whether OPH levels can be used as a read-out for hepatic GSH homeostasis after paracetamol (APAP) challenges during pylorus-preserving pancreaticoduodenectomy (PPPD) or partial hepatectomy (PH)., Methods: Nineteen patients undergoing PPPD (n=7, control group) or PH (n=12) were included. APAP (1000 mg) was administered intravenously before resection (first challenge), and six and twelve hours later, with sequential blood sampling during this period. Arterial, hepatic and portal venous blood samples and liver biopsies were taken on three occasions during the first APAP challenge. Plasma and hepatic OPH and GSH levels were quantified, and venous-arterial differences were calculated to study hepatic release., Results: Systemic GSH levels decreased during the course of the APAP challenge in both surgical groups, without notable change in OPH levels. Hepatic GSH and OPH content was not affected within ˜3 hours after administration of the first APAP dose in patients undergoing PPPD or PH. In this period, net release of OPH by the liver was observed only in patients undergoing PPPD., Conclusion: The drop in circulating GSH levels following APAP administrations, did not result in an increase in plasma OPH in both patients with an intact liver and in those undergoing liver resection. Hepatic content of GSH and OPH was not affected during the first APAP dose. It is uncertain whether hepatic GSH homeostasis was sufficiently challenged in the present study., Relevance for Patients: In the present study, plasma OPH seemed not useful as a marker for GSH depletion because APAP administration during liver surgery did not lead to (immediate) GSH depletion or increased OPH levels. Based on stable levels of hepatic GSH, OPH and thiyl radicals during surgery, standard APAP administration seems to be safe in a postoperative care program with regards to GSH homeostasis in this specific population. However, no general statements can be made on the basis of the current experiment, since GSH homeostasis and susceptibility to xenobiotic toxicity are influenced by several metabolic and genetic factors.

Objective: Environmental factors driving the development of type 1 diabetes (T1D) are still largely unknown. Both animal and human studies have shown an association between altered fecal microbiota composition, impaired production of short-chain fatty acids (SCFA) and T1D onset. However, observational evidence on SCFA and fecal and oral microbiota in adults with longstanding T1D vs healthy controls (HC) is lacking., Research Design and Methods: We included 53 T1D patients without complications or medication and 50 HC matched for age, sex and BMI. Oral and fecal microbiota, fecal and plasma SCFA levels, markers of intestinal inflammation (fecal IgA and calprotectin) and markers of low-grade systemic inflammation were measured., Results: Oral microbiota were markedly different in T1D (eg abundance of Streptococci) compared to HC. Fecal analysis showed decreased butyrate producing species in T1D and less butyryl-CoA transferase genes. Also, plasma levels of acetate and propionate were lower in T1D, with similar fecal SCFA. Finally, fecal strains Christensenella and Subdoligranulum correlated with glycemic control, inflammatory parameters and SCFA., Conclusions: We conclude that T1D patients harbor a different amount of intestinal SCFA (butyrate) producers and different plasma acetate and propionate levels. Future research should disentangle cause and effect and whether supplementation of SCFA-producing bacteria or SCFA alone can have disease-modifying effects in T1D.

Background & Aims: Bile acids (BA) are pleiotropic hormones affecting glucose and lipid metabolism. The physiochemical properties of different BA species affect their enterohepatic dynamics and their affinity for bile acid receptors. The BA pool composition is altered in patients with type 2 diabetes and obesity. In this study we used a 2-week very-low-calorie diet (VLCD) to investigate the effects of weight loss on BA pool composition and postprandial dynamics., Methods: We performed mixed meal tests in obese, insulin resistant subjects before and after the VLCD. We measured postprandial plasma levels of glucose, insulin, BA and the BA-induced enterokine fibroblast growth factor 19 (FGF19)., Results: The VLCD decreased weight by 4.5 ± 2.3 kg (p < 0.0001) within 14 days. Weight loss increased peak postprandial deoxycholate (DCA) levels (median [IQR]: 0.90 [0.90] vs. 1.25 [1.35] μmol/L; p = 0.045*). Other BA species, glucose, insulin and FGF19 levels and prandial excursions were not significantly affected. The VLCD decreased resting and postprandial energy expenditure by 7 and 11% respectively., Conclusions: VLCD induced weight loss increased postprandial DCA peak levels and decreased resting energy expenditure in obese insulin resistant subjects., (Copyright © 2016 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Background: Intestinal failure-associated liver disease (IFALD) is a clinical challenge. The pathophysiol-ogy is multifactorial and remains poorly understood. Disturbed recirculation of bile salts, e.g. due to loss of bile via an enterocutaneous fistula, is considered a major contributing factor. We hypothesize that impaired signaling via the bile salt receptor FXR underlies the development of IFALD. The aim of this study was to investigate whether activation of FXR improves liver homeostasis during chronic loss of bile in rats., Methods: To study consequences of chronic loss of bile, rats underwent external biliary drainage (EBD) or sham surgery for seven days, and the prophylactic potential of the FXR agonist INT-747 was assessed., Results: EBD for 7 days resulted in liver test abnormalities and histological liver damage. Expression of the intestinal FXR target gene Fgf15 was undetectable after EBD, and this was accompanied by an anticipated increase in hepatic Cyp7a1 expression, indicating increased bile salt synthesis. Treatment with INT-747 improved serum biochemistry, reduced loss of bile fluid in drained rats and prevented development of drainage-associated histological liver injury., Conclusions: EBD results in extensive hepatobiliary injury and cholestasis. These data suggest that FXR activation might be a novel therapy in preventing liver dysfunction in patients with intestinal failure., Relevance for Patients: This study demonstrates that chronic loss of bile causes liver injury in rats. Abro-gated recycling of bile salts impairing of enterohepatic bile salt/FXR signaling underlies these pathological changes, as administration of FXR agonist INT747 prevents biliary drainage-induced liver damage. Phar-macological activation of FXR might be a therapeutic strategy to treat disorders accompanied by a per-turbed enterohepatic circulation such as intestinal failure-associated liver disease.

The intestinal microbiota has been implicated in insulin resistance, although evidence regarding causality in humans is scarce. We therefore studied the effect of lean donor (allogenic) versus own (autologous) fecal microbiota transplantation (FMT) to male recipients with the metabolic syndrome. Whereas we did not observe metabolic changes at 18 weeks after FMT, insulin sensitivity at 6 weeks after allogenic FMT was significantly improved, accompanied by altered microbiota composition. We also observed changes in plasma metabolites such as γ-aminobutyric acid and show that metabolic response upon allogenic FMT (defined as improved insulin sensitivity 6 weeks after FMT) is dependent on decreased fecal microbial diversity at baseline. In conclusion, the beneficial effects of lean donor FMT on glucose metabolism are associated with changes in intestinal microbiota and plasma metabolites and can be predicted based on baseline fecal microbiota composition., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background & Aims: Parenteral nutrition (PN), a lifesaving therapy in patients with intestinal failure, has been associated with hepatobiliary complications including steatosis, cholestasis and fibrosis, collectively known as parenteral nutrition-associated liver disease (PNALD). To date, the pathogenesis of PNALD is poorly understood and therapeutic options are limited. Impaired bile salt homeostasis has been proposed to contribute PNALD. The objective of this study was to establish a PNALD model in rats and to evaluate the effects of continuous parenteral nutrition (PN) on bile salt homeostasis., Methods: Rats received either PN via the jugular vein or received normal diet for 3, 7 or 14 days. Serum biochemistry, hepatic triglycerides, circulating bile salts and C4, IL-6 and TNF-alpha, and lipogenic and bile salt homeostatic gene expression in liver and ileum were assessed., Results: PN increased hepatic triglycerides already after 3 days of administration, and resulted in conjugated bilirubin elevation after 7 or more days. This indicates PN-induced steatosis and impaired canalicular secretion of bilirubin, the latter which is in line with reduced hepatic expression of Mrp2 mRNA. There was no histological evidence for liver inflammation after PN administration, and circulating levels of pro-inflammatory cytokines IL-6 and TNF-α, were comparable in all groups. Hepatic expression of Fxr mRNA was decreased after 7 days of PN, without apparent effect on expression of Fxr targets Bsep and Shp. Nonetheless, Cyp7a1 expression was reduced after 7 days of PN, indicative for lowered bile salt synthesis. Circulating levels of C4 (marker of bile salt synthesis) were also decreased after 3, 7 and 14 days of PN. Levels of circulating bile salts were not affected by PN., Conclusions: This study showed that PN in rats caused early mild steatosis and cholestasis, while hepatic and systemic inflammation were not present. The onset of these abnormalities was associated with alterations in bile salt synthesis and transport. This animal model serves as an experimental model to further investigate the pathogenesis of PNALD inflicted by steatosis and cholestasis., (Copyright © 2016 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Drug-induced liver injury remains the most common cause of acute liver failure and a frequently indicated reason for withdrawal of drugs. For the purpose of evaluating the relevance of liver cell models for assessing hepatotoxic risks in intact humans, we here aimed to benchmark 'omics-derived mechanistic data from three in vitro models for parenchymal liver function, intended for the investigation of drug-induced cholestasis, against 'omics data from cholestatic patients. Transcriptomic changes in HepG2 cells, primary mouse hepatocytes and primary human hepatocytes exposed to known cholestatic compounds were analyzed using microarrays. Some of the differentially expressed genes in HepG2 cells were also differentially expressed into the same direction in human cholestasis. The overlap between drug-induced transcriptomic responses in primary mouse hepatocytes and primary human hepatocytes appeared limited and no genes overlapping with in vivo cholestasis were found. Thereupon, a pathway for drug-induced cholestasis was used to map the drug-induced transcriptomic modifications involved in bile salt homeostasis. Indications of an adaptive response to prevent and reduce intracellular bile salt accumulation were observed in vivo as well as in the in vitro liver models. Furthermore, drug-specific changes were found, which may be indicative for their cholestatic properties. Furthermore, connectivity mapping was applied in order to investigate the predictive value of the in vitro models for in vivo cholestasis. This analysis resulted in a positive connection score for most compounds, which may indicate that for identifying cholestatic compounds the focus should be on gene expression signatures rather than on differentially expressed genes., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bile salts are the water-soluble end products of hepatic cholesterol catabolism that are released into the duodenum and solubilize lipids due to their amphipathic structure. Bile salts also act as endogenous ligands for dedicated nuclear receptors that exert a plethora of biological processes, mostly related to metabolism. Bile salts are actively reclaimed in the distal part of the small intestine, released into the portal system, and subsequently extracted by the liver. This enterohepatic cycle is critically dependent on dedicated bile salt transporters. In the intestinal lumen, bile salts exert direct antimicrobial activity based on their detergent property and shape the gut microbiota. Bile salt metabolism by gut microbiota serves as a mechanism to counteract this toxicity and generates bile salt species that are distinct from those of the host. Innate immune cells of the liver play an important role in the early recognition and effector response to invading microbes. Bile salts signal primarily via the membrane receptor TGR5 and the intracellular farnesoid-x receptor, both present in innate immune cells. In this review, the interactions between bile salts, gut microbiota, and hepatic innate immunity are discussed., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: The impact of chemotherapy-associated liver injury (CALI) on postoperative outcome in patients undergoing partial hepatectomy for colorectal liver metastases (CRLM) remains controversial. The objective of this study was to clarify the effect of CALI (sinusoidal dilatation (SD), steatosis and steatohepatitis) on postoperative morbidity and mortality by investigating a large data set from multiple international centres., Methods: PubMed and Embase were searched for studies published between 1 January 2004 and 31 December 2013 with keywords 'chemotherapy', 'liver resection', 'outcome' and 'colorectal metastases' to identify potential collaborating centres. Univariable and multivariable analyses were performed using binary logistic regression models, with results presented as odds ratios (ORs) with 95 per cent confidence intervals., Results: A consolidated database comprising 788 patients who underwent hepatectomy for CRLM in eight centres was obtained. In multivariable analyses, severe SD was associated with increased major morbidity (Dindo-Clavien grade III-V; OR 1·73, 95 per cent c.i. 1·02 to 2·95; P = 0·043). Severe steatosis was associated with decreased liver surgery-specific complications (OR 0·52, 95 per cent c.i. 0·27 to 1·00; P = 0·049), whereas steatohepatitis was linked to an increase in these complications (OR 2·08, 1·18 to 3·66; P = 0·012). Subgroup analysis showed that lobular inflammation was the sole component associated with increased overall morbidity (OR 2·22, 1·48 to 3·34; P = 0·001) and liver surgery-specific complications (OR 3·35, 2·11 to 5·32; P < 0·001). Finally, oxaliplatin treatment was linked to severe SD (OR 2·74, 1·67 to 4·49; P < 0·001)., Conclusion: An increase in postoperative major morbidity and liver surgery-specific complications was observed after partial hepatectomy in patients with severe SD and steatohepatitis. Postoperative liver failure occurred more often in patients with severe SD., (© 2017 BJS Society Ltd Published by John Wiley & Sons Ltd.)