ABSTRACT
Background & Aims: The nuclear receptor farnesoid X receptor (FXR) is a key regulator of hepatic bile acid (BA) and lipid metabolism, inflammation and fibrosis. Here, we aimed to explore the potential of cilofexor (GS-9674), a non-steroidal FXR agonist, as a therapeutic approach for counteracting features of cholestatic liver injury by evaluating its efficacy and mechanisms in the Mdr2/Abcb4 knockout (-/-) mouse model of sclerosing cholangitis. Methods: FVB/N wild-type and Mdr2-/- or BALB/c wild-type and Mdr2-/- mice were treated with 0, 10, 30 or 90 mg/kg cilofexor by gavage every 24 h for 10 weeks. Serum biochemistry, gene expression profile, hydroxyproline content, and picrosirius red and F4/80 immunostaining, were investigated. Bile flow, biliary bicarbonate and BA output, and hepatic BA profile, were assessed. Results: Cilofexor treatment improved serum levels of aspartate aminotransferase, alkaline phosphatase as well as BAs in Mdr2-/- animals. Hepatic fibrosis was improved, as reflected by the reduced picrosirius red-positive area and hydroxyproline content in liver sections of cilofexor-treated Mdr2-/- mice. Intrahepatic BA concentrations were lowered in cilofexor-treated Mdr2-/- mice, while hepatobiliary bile flow and bicarbonate output were increased. Conclusion: Collectively the current data show that cilofexor treatment improves cholestatic liver injury and decreases hepatic fibrosis in the Mdr2-/- mouse model of sclerosing cholangitis. Impact and implications: Treatment with cilofexor, a non-steroidal farnesoid X receptor (FXR) agonist, improved histological features of sclerosing cholangitis, cholestasis and hepatic fibrosis in the Mdr2-/- mouse model. These findings indicate, that pharmacological stimulation of intestinal FXR-mediated gut-liver signaling, via fibroblast growth factor 15 (thereby reducing bile acid synthesis), may be sufficient to attenuate cholestatic liver injury in the Mdr2-/- mouse model of sclerosing cholangitis, thus arguing for potential therapeutic properties of cilofexor in cholestatic liver diseases.
ABSTRACT
BACKGROUND AND AIMS: Schistosoma mansoni infection is one of the worldwide leading causes of liver fibrosis and portal hypertension. The objective of this study was to evaluate whether polyhydroxylated bile acids (BAs), known to protect mice from the development of acquired cholestatic liver injury, counteract S. mansoni-induced inflammation and fibrosis. METHODS: Adult FVB/N wild type (WT) and Abcb11/Bsep-/- mice were infected with either 25 or 50 S. mansoni cercariae. Eight weeks post infection, effects on liver histology, serum biochemistry, gene expression profile of proinflammatory cytokines and fibrotic markers, hepatic hydroxyproline content and FACS analysis were performed. RESULTS: Bsep-/- mice infected with S. mansoni showed significantly less hepatic inflammation and tendentially less fibrosis compared to infected WT mice. Despite elevated alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase levels in infected Bsep-/- mice, inflammatory cells such as M2 macrophages and Mac-2/galectin-3+ cells were reduced in these animals. Accordingly, mRNA-expression levels of anti-inflammatory cytokines (IL-4 and IL-13) were increased in Bsep-/- mice upon infection. Furthermore, infected Bsep-/- mice exhibited decreased hepatic egg load and parasite fecundity, consequently affecting the worm reproduction rate. This outcome could arise from elevated serum BA levels and lower blood pH in Bsep-/- mice. CONCLUSIONS: The loss of Bsep and the resulting changes in bile acid composition and blood pH are associated with the reduction of parasite fecundity, thus attenuating the development of S. mansoni-induced hepatic inflammation and fibrosis.
Subject(s)
Parasites , Schistosomiasis mansoni , Animals , Mice , Bile Acids and Salts/metabolism , Cytokines/metabolism , Fertility , Inflammation/pathology , Liver/pathology , Liver Cirrhosis/prevention & control , Liver Cirrhosis/etiology , Schistosoma mansoni , Schistosomiasis mansoni/complicationsABSTRACT
BACKGROUND & AIMS: Glucagon-like peptide (GLP)-2 may exert antifibrotic effects on hepatic stellate cells (HSCs). Thus, we aimed to test whether application of the GLP-2 analogue teduglutide has hepatoprotective and antifibrotic effects in the Mdr2/Abcb4-/- mouse model of sclerosing cholangitis displaying hepatic inflammation and fibrosis. METHODS: Mdr2-/- mice were injected daily for 4 weeks with teduglutide followed by gene expression profiling (bulk liver; isolated HSCs) and immunohistochemistry. Activated HSCs (LX2 cells) and immortalized human hepatocytes and human intestinal organoids were treated with GLP-2. mRNA profiling by reverse transcription polymerase chain reaction and electrophoretic mobility shift assay using cytosolic and nuclear protein extracts was performed. RESULTS: Hepatic inflammation, fibrosis, and reactive cholangiocyte phenotype were improved in GLP-2-treated Mdr2-/- mice. Primary HSCs isolated from Mdr2-/- mice and LX2 cells exposed to GLP-2 in vitro displayed significantly increased mRNA expression levels of NR4a1/Nur77 (P < .05). Electrophoretic mobility shift assay revealed an increased nuclear NR4a1 binding after GLP-2 treatment in LX2 cells. Moreover, GLP-2 alleviated the Tgfß-mediated reduction of NR4a1 nuclear binding activity. In vivo, GLP-2 treatment of Mdr2-/- mice resulted in increased intrahepatic levels of muricholic acids (accordingly Cyp2c70 mRNA expression was significantly increased), and in reduced mRNA levels of Cyp7a1 and FXR. Serum Fgf15 levels were increased in Mdr2-/- mice treated with GLP-2. Accordingly, GLP-2 treatment of human intestinal organoids activated their FXR-FGF19 signaling axis. CONCLUSIONS: GLP-2 treatment increased NR4a1/Nur77 activation in HSCs, subsequently attenuating their activation. GLP-2 promoted intestinal Fxr-Fgf15/19 signaling resulting in reduced Cyp7a1 and increased Cyp2c70 expression in the liver, contributing to hepatoprotective and antifibrotic effects of GLP-2 in the Mdr2-/- mouse model.
Subject(s)
Hepatic Stellate Cells , Liver Cirrhosis , Mice , Humans , Animals , Hepatic Stellate Cells/metabolism , Mice, Knockout , Liver Cirrhosis/drug therapy , Liver Cirrhosis/metabolism , Disease Models, Animal , RNA, Messenger/metabolism , Inflammation/metabolismABSTRACT
Preventing SARS-CoV-2 infection by modulating viral host receptors, such as angiotensin-converting enzyme 2 (ACE2)1, could represent a new chemoprophylactic approach for COVID-19 that complements vaccination2,3. However, the mechanisms that control the expression of ACE2 remain unclear. Here we show that the farnesoid X receptor (FXR) is a direct regulator of ACE2 transcription in several tissues affected by COVID-19, including the gastrointestinal and respiratory systems. We then use the over-the-counter compound z-guggulsterone and the off-patent drug ursodeoxycholic acid (UDCA) to reduce FXR signalling and downregulate ACE2 in human lung, cholangiocyte and intestinal organoids and in the corresponding tissues in mice and hamsters. We show that the UDCA-mediated downregulation of ACE2 reduces susceptibility to SARS-CoV-2 infection in vitro, in vivo and in human lungs and livers perfused ex situ. Furthermore, we reveal that UDCA reduces the expression of ACE2 in the nasal epithelium in humans. Finally, we identify a correlation between UDCA treatment and positive clinical outcomes after SARS-CoV-2 infection using retrospective registry data, and confirm these findings in an independent validation cohort of recipients of liver transplants. In conclusion, we show that FXR has a role in controlling ACE2 expression and provide evidence that modulation of this pathway could be beneficial for reducing SARS-CoV-2 infection, paving the way for future clinical trials.
Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Receptors, Virus , Ursodeoxycholic Acid , Animals , Humans , Mice , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , COVID-19/prevention & control , Receptors, Virus/genetics , Receptors, Virus/metabolism , Retrospective Studies , SARS-CoV-2/metabolism , COVID-19 Drug Treatment , Cricetinae , Transcription, Genetic , Ursodeoxycholic Acid/pharmacology , Lung/drug effects , Lung/metabolism , Organoids/drug effects , Organoids/metabolism , Liver/drug effects , Liver/metabolism , Nasal Mucosa/drug effects , Nasal Mucosa/metabolism , Registries , Reproducibility of Results , Liver TransplantationABSTRACT
Bile salt export pump (Bsep) (Abcb11)-/- mice are protected from acquired cholestatic injury due to metabolic preconditioning with a hydrophilic bile acid (BA) pool with formation of tetrahydroxylated bile acids (THBAs). We aimed to explore whether loss of Bsep and subsequent elevation of THBA levels may have immunomodulatory effects, thus improving liver injury in the multidrug resistance protein 2 (Mdr2) (Abcb4)-/- mouse. Cholestatic liver injury in Mdr2-/- Bsep-/- double knockout (DKO), Mdr2-/- , Bsep-/- , and wild-type mice was studied for comparison. Mdr2-/- mice were treated with a THBA (3α,6α,7α,12α-Tetrahydroxycholanoic acid). RNA/protein expression of inflammatory/fibrotic markers were investigated. Serum BA-profiling was assessed by ultra-performance liquid chromatography tandem mass spectrometry. Hepatic immune cell profile was quantified by flow cytometric analysis (FACS). In vitro, the THBA effect on chenodeoxycholic acid (CDCA)-induced inflammatory signaling in hepatocyte and cholangiocytes as well as lipopolysaccharide (LPS)/interferon-γ (IFN-γ)-induced macrophage activation was analyzed. In contrast to Mdr2-/- , DKO mice showed no features of sclerosing cholangitis. Sixty-seven percent of serum BAs in DKO mice were polyhydroxylated (mostly THBAs), whereas Mdr2-/- mice did not have these BAs. Compared with Mdr2-/- , DKO animals were protected from hepatic inflammation/fibrosis. THBA feeding in Mdr2-/- mice improved liver injury. FACS analysis in DKO and Mdr2-/- THBA-fed mice showed changes of the hepatic immune cell profile towards an anti-inflammatory pattern. Early growth response 1 (EGR1) protein expression was reduced in DKO and in Mdr2-/- THBA-fed mice compared with Mdr2-/- control mice. In vitro, THBA-reduced CDCA induced EGR1 protein and mRNA expression of inflammatory markers in hepatocytes and cholangiocytes. LPS/IFN-γ-induced macrophage activation was ameliorated by THBA. THBAs repress EGR1-related key pro-inflammatory pathways. Conclusion: THBA and their downstream targets may represent a potential treatment strategy for cholestatic liver diseases.
Subject(s)
Bile Acids and Salts , Cholangitis, Sclerosing , Cholestasis , ATP Binding Cassette Transporter, Subfamily B/genetics , Animals , Bile Acids and Salts/chemistry , Bile Acids and Salts/pharmacology , Bile Ducts/pathology , Cholangitis, Sclerosing/genetics , Cholestasis/complications , Cholestasis/genetics , Disease Models, Animal , Immunomodulation/drug effects , Interferon-gamma , Lipopolysaccharides/pharmacology , Liver Cirrhosis/genetics , Mice , Mice, Knockout , ATP-Binding Cassette Sub-Family B Member 4ABSTRACT
Bile acids (BAs) can regulate their own metabolism and transport as well as other key aspects of metabolic homeostasis via dedicated (nuclear and G protein-coupled) receptors. Disrupted BA transport and homeostasis results in the development of cholestatic disorders and contributes to a wide range of liver diseases, including nonalcoholic fatty liver disease and hepatocellular and cholangiocellular carcinoma. Furthermore, impaired BA homeostasis can also affect the intestine, contributing to the pathogenesis of irritable bowel syndrome, inflammatory bowel disease, and colorectal and oesophageal cancer. Here, we provide a summary of the role of BAs and their disrupted homeostasis in the development of gastrointestinal and hepatic disorders and present novel insights on how targeting BA pathways might contribute to novel treatment strategies for these disorders.
Subject(s)
Cholestasis , Non-alcoholic Fatty Liver Disease , Bile Acids and Salts/metabolism , Cholestasis/metabolism , Homeostasis , Humans , Liver/metabolism , Non-alcoholic Fatty Liver Disease/metabolism , Receptors, G-Protein-CoupledABSTRACT
BACKGROUND AND AIMS: Lipopolysaccharide (LPS) clearance is delayed in cholestatic liver diseases. While compromised clearance by Kupffer cells (KCs) is involved, the role of LPS uptake into hepatocytes and canalicular excretion remains unclear. APPROACH AND RESULTS: Wild-type (WT) and bile salt export pump (Bsep) knockout (KO) mice were challenged i.p. with LPS. Liver injury was assessed by serum biochemistry, histology, molecular inflammation markers, and immune cell infiltration. LPS concentrations were determined in liver tissue and bile. Subcellular kinetics of fluorescently labeled LPS was visualized by intravital two-photon microscopy, and the findings in Bsep KO mice were compared to common bile duct-ligated (BDL) and multidrug resistance protein 2 (Mdr2) KO mice. Changes in gut microbiota composition were evaluated by 16S ribosomal RNA gene amplicon sequencing analysis. Bsep KO mice developed more pronounced LPS-induced liver injury and inflammatory signaling, with subsequently enhanced production of proinflammatory cytokines and aggravated hepatic immune cell infiltration. After LPS administration, its concentrations were higher in liver but lower in bile of Bsep KO compared to WT mice. Intravital imaging of LPS showed a delayed clearance from sinusoidal blood with a basolateral uptake block into hepatocytes and reduced canalicular secretion. Moreover, LPS uptake into KCs was reduced. Similar findings with respect to hepatic LPS clearance were obtained in BDL and Mdr2 KO mice. Pretreatment with the microtubule inhibitor colchicine inhibited biliary excretion of LPS in WT mice, indicating that LPS clearance is microtubule-dependent. Microbiota analysis showed no change of the gut microbiome between WT and Bsep KO mice at baseline but major changes upon LPS challenge in WT mice. CONCLUSIONS: Absence of Bsep and cholestasis in general impair LPS clearance by a basolateral uptake block into hepatocytes and consequently less secretion into canaliculi. Impaired LPS removal aggravates hepatic inflammation in cholestasis.
Subject(s)
Chemical and Drug Induced Liver Injury, Chronic , Cholestasis , ATP Binding Cassette Transporter, Subfamily B, Member 11/metabolism , Animals , Bile Acids and Salts/metabolism , Cholestasis/pathology , Endotoxins , Inflammation/metabolism , Kinetics , Lipopolysaccharides/metabolism , Liver/pathology , Mice , Mice, KnockoutABSTRACT
BACKGROUND AND AIMS: Increased fatty acid (FA) flux from adipose tissue to the liver contributes to the development of NAFLD. Because free FAs are key lipotoxic triggers accelerating disease progression, inhibiting adipose triglyceride lipase (ATGL)/patatin-like phospholipase domain containing 2 (PNPLA2), the main enzyme driving lipolysis, may attenuate steatohepatitis. APPROACH AND RESULTS: Hepatocyte-specific ATGL knockout (ATGL LKO) mice were challenged with methionine-choline-deficient (MCD) or high-fat high-carbohydrate (HFHC) diet. Serum biochemistry, hepatic lipid content and liver histology were assessed. Mechanistically, hepatic gene and protein expression of lipid metabolism, inflammation, fibrosis, apoptosis, and endoplasmic reticulum (ER) stress markers were investigated. DNA binding activity for peroxisome proliferator-activated receptor (PPAR) α and PPARδ was measured. After short hairpin RNA-mediated ATGL knockdown, HepG2 cells were treated with lipopolysaccharide (LPS) or oleic acid:palmitic acid 2:1 (OP21) to explore the direct role of ATGL in inflammation in vitro. On MCD and HFHC challenge, ATGL LKO mice showed reduced PPARα and increased PPARδ DNA binding activity when compared with challenged wild-type (WT) mice. Despite histologically and biochemically pronounced hepatic steatosis, dietary-challenged ATGL LKO mice showed lower hepatic inflammation, reflected by the reduced number of Galectin3/MAC-2 and myeloperoxidase-positive cells and low mRNA expression levels of inflammatory markers (such as IL-1ß and F4/80) when compared with WT mice. In line with this, protein levels of the ER stress markers protein kinase R-like endoplasmic reticulum kinase and inositol-requiring enzyme 1α were reduced in ATGL LKO mice fed with MCD diet. Accordingly, pretreatment of LPS-treated HepG2 cells with the PPARδ agonist GW0742 suppressed mRNA expression of inflammatory markers. Additionally, ATGL knockdown in HepG2 cells attenuated LPS/OP21-induced expression of proinflammatory cytokines and chemokines such as chemokine (C-X-C motif) ligand 5, chemokine (C-C motif) ligand (Ccl) 2, and Ccl5. CONCLUSIONS: Low hepatic lipolysis and increased PPARδ activity in ATGL/PNPLA2 deficiency may counteract hepatic inflammation and ER stress despite increased steatosis. Therefore, lowering hepatocyte lipolysis through ATGL inhibition represents a promising therapeutic strategy for the treatment of steatohepatitis.
Subject(s)
Lipase/metabolism , Lipolysis/immunology , Liver/pathology , Non-alcoholic Fatty Liver Disease/immunology , Adult , Animals , Diet, Carbohydrate Loading/adverse effects , Diet, High-Fat/adverse effects , Disease Models, Animal , Fatty Acids, Nonesterified/metabolism , Female , Hep G2 Cells , Humans , Lipase/genetics , Lipolysis/genetics , Liver/enzymology , Liver/immunology , Male , Mice , Mice, Knockout , Middle Aged , Non-alcoholic Fatty Liver Disease/etiology , Non-alcoholic Fatty Liver Disease/pathologyABSTRACT
BACKGROUND & AIMS: 24-Norursodeoxycholic acid (NorUDCA) is a novel therapeutic bile acid used to treat immune-mediated cholestatic liver diseases, such as primary sclerosing cholangitis (PSC), where dysregulated T cells including CD8+ T cells contribute to hepatobiliary immunopathology. We hypothesized that NorUDCA may directly modulate CD8+ T cell function thus contributing to its therapeutic efficacy. METHODS: NorUDCA's immunomodulatory effects were first studied in Mdr2-/- mice, as a cholestatic model of PSC. To differentiate NorUDCA's immunomodulatory effects on CD8+ T cell function from its anticholestatic actions, we also used a non-cholestatic model of hepatic injury induced by an excessive CD8+ T cell immune response upon acute non-cytolytic lymphocytic choriomeningitis virus (LCMV) infection. Studies included molecular and biochemical approaches, flow cytometry and metabolic assays in murine CD8+ T cells in vitro. Mass spectrometry was used to identify potential CD8+ T cell targets modulated by NorUDCA. The signaling effects of NorUDCA observed in murine cells were validated in circulating T cells from patients with PSC. RESULTS: NorUDCA demonstrated immunomodulatory effects by reducing hepatic innate and adaptive immune cells, including CD8+ T cells in the Mdr2-/- model. In the non-cholestatic model of CD8+ T cell-driven immunopathology induced by acute LCMV infection, NorUDCA ameliorated hepatic injury and systemic inflammation. Mechanistically, NorUDCA demonstrated strong immunomodulatory efficacy in CD8+ T cells affecting lymphoblastogenesis, expansion, glycolysis and mTORC1 signaling. Mass spectrometry identified that NorUDCA regulates CD8+ T cells by targeting mTORC1. NorUDCA's impact on mTORC1 signaling was further confirmed in circulating PSC CD8+ T cells. CONCLUSIONS: NorUDCA has a direct modulatory impact on CD8+ T cells and attenuates excessive CD8+ T cell-driven hepatic immunopathology. These findings are relevant for treatment of immune-mediated liver diseases such as PSC. LAY SUMMARY: Elucidating the mechanisms by which 24-norursodeoxycholic acid (NorUDCA) works for the treatment of immune-mediated liver diseases, such as primary sclerosing cholangitis, is of considerable clinical interest. Herein, we uncovered an unrecognized property of NorUDCA in the immunometabolic regulation of CD8+ T cells, which has therapeutic relevance for immune-mediated liver diseases, including PSC.
Subject(s)
CD8-Positive T-Lymphocytes/metabolism , Inflammation/drug therapy , Liver/drug effects , Ursodeoxycholic Acid/analogs & derivatives , Animals , CD8-Positive T-Lymphocytes/drug effects , Disease Models, Animal , Inflammation/physiopathology , Liver/physiopathology , Mice , Mice, Inbred C57BL , Ursodeoxycholic Acid/pharmacology , Ursodeoxycholic Acid/therapeutic useABSTRACT
Altered lipid metabolic pathways including hydrolysis of triglycerides are key players in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Whether adiponutrin (patatin-like phospholipase domain containing protein-3-PNPLA3) and monoacylglycerol lipase (MGL) synergistically contribute to disease progression remains unclear. We generated double knockout (DKO) mice lacking both Mgl and Pnpla3; DKO mice were compared to Mgl-/- after a challenge by high-fat diet (HFD) for 12 weeks to induce steatosis. Serum biochemistry, liver transaminases as well as histology were analyzed. Fatty acid (FA) profiling was assessed in liver and adipose tissue by gas chromatography. Markers of inflammation and lipid metabolism were analyzed. Bone marrow derived macrophages (BMDMs) were isolated and treated with oleic acid. Combined deficiency of Mgl and Pnpla3 resulted in weight gain on a chow diet; when challenged by HFD, DKO mice showed increased hepatic FA synthesis and diminished beta-oxidation compared to Mgl-/-.DKO mice exhibited more pronounced hepatic steatosis with inflammation and recruitment of immune cells to the liver associated with accumulation of saturated FAs. Primary BMDMs isolated from the DKO mice showed increased inflammatory activities, which could be reversed by oleic acid supplementation. Pnpla3 deficiency aggravates the effects of Mgl deletion on steatosis and inflammation in the liver under HFD challenge.
Subject(s)
Membrane Proteins/deficiency , Monoacylglycerol Lipases/deficiency , Non-alcoholic Fatty Liver Disease/enzymology , Non-alcoholic Fatty Liver Disease/pathology , Weight Gain , Adipose Tissue/metabolism , Adipose Tissue/pathology , Animals , Cells, Cultured , Fatty Acids/metabolism , Humans , Inflammation/pathology , Lipid Metabolism , Liver/pathology , Macrophages/metabolism , Male , Membrane Proteins/metabolism , Mice, Inbred C57BL , Mice, Knockout , Monoacylglycerol Lipases/metabolism , Oleic Acid , Phenotype , U937 CellsABSTRACT
BACKGROUND: Bile acids (BAs) regulate hepatic lipid metabolism and inflammation. Bile salt export pump (BSEP) KO mice are metabolically preconditioned with a hydrophilic BA composition protecting them from cholestasis. We hypothesize that changes in hepatic BA profile and subsequent changes in BA signalling may critically determine the susceptibility to steatohepatitis. METHODS: Wild-type (WT) and BSEP KO mice were challenged with methionine choline-deficient (MCD) diet to induce steatohepatitis. Serum biochemistry, lipid profiling as well as intestinal lipid absorption were assessed. Markers of inflammation, fibrosis, lipid and BA metabolism were analysed. Hepatic and faecal BA profile as well as serum levels of the BA synthesis intermediate 7-hydroxy-4-cholesten-3-one (C4) were also investigated. RESULTS: Bile salt export pump KO MCD-fed mice developed less steatosis but more inflammation than WT mice. Intestinal neutral lipid levels were reduced in BSEP KO mice at baseline and under MCD conditions. Faecal non-esterified fatty acid concentrations at baseline and under MCD diet were markedly elevated in BSEP KO compared to WT mice. Serum liver enzymes and hepatic expression of inflammatory markers were increased in MCD-fed BSEP KO animals. PPARα protein levels were reduced in BSEP KO mice. Accordingly, PPARα downstream targets Fabp1 and Fatp5 were repressed, while NFκB subunits were increased in MCD-fed BSEP KO mice. Farnesoid X receptor (FXR) protein levels were reduced in MCD-fed BSEP KO vs WT mice. Hepatic BA profile revealed elevated levels of TßMCA, exerting FXR antagonistic action, while concentrations of TCA (FXR agonistic function) were reduced. CONCLUSION: Presence of hydroxylated BAs result in increased faecal FA excretion and reduced hepatic lipid accumulation. This aggravates development of MCD diet-induced hepatitis potentially by decreasing FXR and PPARα signalling.
Subject(s)
Fatty Liver , Methionine , ATP Binding Cassette Transporter, Subfamily B, Member 11 , Animals , Bile Acids and Salts , Choline , Diet , Fatty Acid-Binding Proteins , Inflammation , Liver , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
BACKGROUND AND AIMS: Monoacylglycerol lipase (MGL) is the last enzymatic step in triglyceride degradation, hydrolyzing monoglycerides into glycerol and fatty acids (FAs) and converting 2-arachidonoylglycerol into arachidonic acid, thus providing ligands for nuclear receptors as key regulators of hepatic bile acid (BA)/lipid metabolism and inflammation. We aimed to explore the role of MGL in the development of cholestatic liver and bile duct injury in mouse models of sclerosing cholangitis, a disease so far lacking effective pharmacological therapy. APPROACH AND RESULTS: To this aim we analyzed the effects of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding to induce sclerosing cholangitis in wild-type (WT) and knockout (MGL-/- ) mice and tested pharmacological inhibition with JZL184 in the multidrug resistance protein 2 knockout (Mdr2-/- ) mouse model of sclerosing cholangitis. Cholestatic liver injury and fibrosis were assessed by serum biochemistry, liver histology, gene expression, and western blot characterization of BA and FA synthesis/transport. Moreover, intestinal FAs and fecal microbiome were analyzed. Transfection and silencing were performed in Caco2 cells. MGL-/- mice were protected from DDC-induced biliary fibrosis and inflammation with reduced serum liver enzymes and increased FA/BA metabolism and ß-oxidation. Notably, pharmacological (JZL184) inhibition of MGL ameliorated cholestatic injury in DDC-fed WT mice and protected Mdr2-/- mice from spontaneous liver injury, with improved liver enzymes, inflammation, and biliary fibrosis. In vitro experiments confirmed that silencing of MGL decreases prostaglandin E2 accumulation in the intestine and up-regulates peroxisome proliferator-activated receptors alpha and gamma activity, thus reducing inflammation. CONCLUSIONS: Collectively, our study unravels MGL as a metabolic target, demonstrating that MGL inhibition may be considered as potential therapy for sclerosing cholangitis.
Subject(s)
Benzodioxoles/therapeutic use , Cholangitis, Sclerosing/drug therapy , Cholestasis/drug therapy , Enzyme Inhibitors/therapeutic use , Liver Cirrhosis, Biliary/prevention & control , Monoacylglycerol Lipases/antagonists & inhibitors , Piperidines/therapeutic use , ATP Binding Cassette Transporter, Subfamily B/genetics , Animals , Bile Acids and Salts/metabolism , Caco-2 Cells , Cholangitis, Sclerosing/complications , Cholestasis/complications , Disease Models, Animal , Fatty Acids/metabolism , Humans , Liver Cirrhosis, Biliary/etiology , Male , Mice, Inbred C57BL , Mice, Knockout , Pyridines/toxicity , ATP-Binding Cassette Sub-Family B Member 4ABSTRACT
Monoacylglycerol lipase (MGL) is the rate-limiting enzyme in the degradation of monoacylglycerols. To examine the role of MGL in hepatic steatosis, WT and MGL KO (MGL-/-) mice were challenged with a Western diet (WD) over 12 weeks. Lipid metabolism, inflammation, and fibrosis were assessed by serum biochemistry, histology, and gene-expression profiling of liver and adipose depots. Intestinal fat absorption was measured by gas chromatography. Primary adipocyte and 3T3-L1 cells were analyzed by flow cytometry and Western blot. Human hepatocytes were treated with MGL inhibitor JZL184. The absence of MGL protected mice from hepatic steatosis by repressing key lipogenic enzymes in liver (Srebp1c, Pparγ2, and diacylglycerol O-acyltransferase 1), while promoting FA oxidation. Liver inflammation was diminished in MGL-/- mice fed a WD, as evidenced by diminished epidermal growth factor-like module-containing mucin-like hormone receptor-like 1 (F4/80) staining and C-C motif chemokine ligand 2 gene expression, whereas fibrosis remained unchanged. Absence of MGL promoted fat storage in gonadal white adipose tissue (gWAT) with increased lipogenesis and unchanged lipolysis, diminished inflammation in gWAT, and subcutaneous AT. Intestinal fat malabsorption prevented ectopic lipid accumulation in livers of MGL-/- mice fed a WD. In vitro experiments demonstrated increased adipocyte size/lipid content driven by PPARγ. In conclusion, our data uncover that MGL deletion improves some aspects of nonalcoholic fatty liver disease by promoting lipid storage in gWAT and fat malabsorption.
Subject(s)
Adipose Tissue/metabolism , Liver/enzymology , Liver/metabolism , Monoacylglycerol Lipases/metabolism , 3-Hydroxybutyric Acid/blood , 3T3-L1 Cells , Adiponectin/blood , Animals , Blotting, Western , Cells, Cultured , Fatty Acids/blood , Glycerol/blood , Humans , Immunohistochemistry , Insulin/blood , Intestinal Absorption/genetics , Intestinal Absorption/physiology , Lipid Metabolism/genetics , Lipid Metabolism/physiology , Lipolysis/genetics , Lipolysis/physiology , Mice , Mice, Inbred C57BL , Monoacylglycerol Lipases/deficiency , Monoacylglycerol Lipases/genetics , Obesity/genetics , Obesity/metabolism , Oxidation-Reduction , Peroxisome Proliferator-Activated Receptors/metabolism , Triglycerides/bloodABSTRACT
BACKGROUND: Wnt/ß-catenin signaling plays a crucial role in embryogenesis, tissue homeostasis, metabolism and malignant transformation of different organs including the liver. Continuous ß-catenin signaling due to somatic mutations in exon 3 of the Ctnnb1 gene is associated with different liver diseases including cancer and cholestasis. RESULTS: Expression of a degradation resistant form of ß-catenin in hepatocytes resulted in 100% mortality within 31 days after birth. Ctnnb1CAhep mice were characterized by reduced body weight, significantly enlarged livers with hepatocellular fat accumulation around central veins and increased hepatic triglyceride content. Proteomics analysis using whole liver tissue revealed significant deregulation of proteins involved in fat, glucose and mitochondrial energy metabolism, which was also reflected in morphological anomalies of hepatocellular mitochondria. Key enzymes involved in transport and synthesis of fatty acids and cholesterol were significantly deregulated in livers of Ctnnb1CAhep mice. Furthermore, carbohydrate metabolism was substantially disturbed in mutant mice. CONCLUSION: Continuous ß-catenin signaling in hepatocytes results in premature death due to severe disturbances of liver associated metabolic pathways and mitochondrial dysfunction. METHODS: To investigate the influence of permanent ß-catenin signaling on liver biology we analyzed mice with hepatocyte specific expression of a dominant stable form of ß-catenin (Ctnnb1CAhep ) and their WT littermates by serum biochemistry, histology, electron microscopy, mRNA profiling and proteomic analysis of the liver.
ABSTRACT
Accumulation of bile salts (BSs) during cholestasis leads to hepatic and biliary injury, driving inflammatory and fibrotic processes. The Na+ -Taurocholate Cotransporting Polypeptide (NTCP) is the major hepatic uptake transporter of BSs, and can be specifically inhibited by myrcludex B. We hypothesized that inhibition of NTCP dampens cholestatic liver injury. Acute cholestasis was induced in mice by a 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) diet or by bile duct ligation (BDL). Chronic cholestasis was investigated in Atp8b1-G308V and Abcb4/Mdr2 deficient mice. Mice were injected daily with myrcludex B or vehicle. Myrcludex B reduced plasma alkaline phosphatase (ALP) levels in DDC-fed, Atp8b1-G308V and BDL mice by 39%, 27% and 48% respectively. Expression of genes involved in fibrosis, proliferation and inflammation was reduced by myrcludex B treatment in DDC-fed and Atp8b1-G308V mice. NTCP-inhibition increased plasma BS levels from 604±277 to 1746±719 µm in DDC-fed mice, 432±280 to 762±288 µm in Atp8b1-G308V mice and from 522±130 to 3625±378 µm in BDL mice. NTCP-inhibition strongly aggravated weight loss in BDL mice, but not in other cholestatic models studied. NTCP-inhibition reduced biliary BS output in DDC-fed and Atp8b1-G308V mice by â¼50% while phospholipid (PL) output was maintained, resulting in a higher PL/BS ratio. Conversely, liver injury in Abcb4 deficient mice, lacking biliary phospholipid output, was aggravated after myrcludex B treatment. Conclusion: NTCP-inhibition by myrcludex B has hepatoprotective effects, by reducing BS load in hepatocytes and increasing the biliary PL/BS ratio. High micromolar plasma BS levels after NTCP-inhibition were well tolerated. NTCP-inhibition may be beneficial in selected forms of cholestasis. (Hepatology 2018).
Subject(s)
Cholestasis/drug therapy , Lipopeptides/therapeutic use , Organic Anion Transporters, Sodium-Dependent/antagonists & inhibitors , Symporters/antagonists & inhibitors , Animals , Bile Acids and Salts/blood , Cholestasis/blood , Drug Evaluation, Preclinical , Lipopeptides/pharmacology , Male , Mice, Inbred C57BL , Mice, Knockout , Phospholipids/metabolismABSTRACT
The farnesoid X receptor (FXR) and C/EBP homologous protein (CHOP) have critical functions in hepatic lipid metabolism. Here, we aimed to explore a potential relationship between FXR and CHOP. We fed wild-type (WT) and FXR KO mice a MCD diet (model of steatohepatitis) and found that Chop mRNA expression is upregulated in WT but not FXR KO mice. The absence of FXR aggravates hepatic inflammation after MCD feeding. In HepG2 cells, we found that Chop expression is regulated in a FXR/Retinoid X receptor (RXR)-dependent manner. We identified a FXR/RXR-binding site in the human CHOP promoter, demonstrating a highly conserved regulatory pathway. Our study shows that FXR/RXR regulates Chop expression in a mouse model of steatohepatitis, providing novel insights into pathogenesis of this disorder.
Subject(s)
Liver/metabolism , Non-alcoholic Fatty Liver Disease/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Retinoid X Receptors/genetics , Transcription Factor CHOP/genetics , Animals , Binding Sites , Chenodeoxycholic Acid/pharmacology , Diet, High-Fat , Disease Models, Animal , Gene Expression Regulation , Glucose/pharmacology , Hep G2 Cells , Humans , Lipid Metabolism/drug effects , Liver/drug effects , Liver/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Non-alcoholic Fatty Liver Disease/etiology , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/pathology , Promoter Regions, Genetic , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Retinoid X Receptors/metabolism , Signal Transduction , Transcription Factor CHOP/metabolism , Tretinoin/pharmacologyABSTRACT
BACKGROUND & AIMS: Cholestasis is characterized by intrahepatic accumulation of potentially cytotoxic bile acids (BAs) subsequently leading to liver injury with disruption of hepatocellular integrity, inflammation, fibrosis and ultimately liver cirrhosis. Bile salt export pump (BSEP/ABCB11) is the main canalicular BA transporter and therefore the rate limiting step for hepatobiliary BA excretion. In this study we aimed to investigate the role of BSEP/ABCB11 in the development of acquired cholestatic liver and bile duct injury. METHODS: Wild-type (WT) and BSEP knockout (BSEP-/-) mice were subjected to common bile duct ligation (CBDL) or 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) feeding as models for cholestasis with biliary obstruction and bile duct injury. mRNA expression profile, serum biochemistry, liver histology, immunohistochemistry, hepatic hydroxyproline levels and BA composition as well as biliary pressure were assessed. RESULTS: BSEP-/- mice were protected against acquired cholestatic liver injury induced by 7days of CBDL or 4weeks of DDC feeding, as reflected by unchanged serum levels of liver transaminases, alkaline phosphatase and BAs. Notably, BSEP-/- mice were also protected from cholestasis-induced hepatic inflammation and biliary fibrosis. In line with induced BA detoxification/hydroxylation pathways in BSEP-/- mice, polyhydroxylated BAs were increased 4-fold after CBDL and 6-fold after DDC feeding in comparison with cholestatic WT mice. Finally, following CBDL, biliary pressure in WT mice increased up to 47mmH2O but remained below 11mmH2O in BSEP-/- mice. CONCLUSION: Metabolic preconditioning with subsequent changes in BA metabolism favors detoxification of potentially toxic BAs and thereby protects BSEP-/- mice from cholestatic liver and bile duct injury. LAY SUMMARY: Reduced hepatobiliary bile acid transport due to loss of BSEP function leads to increased hydroxylation of bile acids in the liver. Metabolic preconditioning with a hydrophilic bile pool protects the BSEP-/- mice from acquired cholestatic liver disease.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 11/metabolism , Bile Acids and Salts/metabolism , Bile Ducts , Cholestasis, Intrahepatic/metabolism , Hepatocytes/metabolism , Ligation/methods , Therapeutic Occlusion/methods , Animals , Bile Canaliculi , Bile Ducts/physiopathology , Bile Ducts/surgery , Cholestasis, Intrahepatic/prevention & control , MiceABSTRACT
BACKGROUND: The Wnt/ß-catenin signaling pathway plays a crucial role in embryonic development, tissue homeostasis, wound healing and malignant transformation in different organs including the liver. The consequences of continuous ß-catenin signaling in hepatocytes remain elusive. RESULTS: Livers of Ctnnb1CA hep mice were characterized by disturbed liver architecture, proliferating cholangiocytes and biliary type of fibrosis. Serum ALT and bile acid levels were significantly increased in Ctnnb1CA hep mice. The primary bile acid synthesis enzyme Cyp7a1 was increased whereas Cyp27 and Cyp8b1 were reduced in Ctnnb1CA hep mice. Expression of compensatory bile acid transporters including Abcb1, Abcb4, Abcc2 and Abcc4 were significantly increased in Ctnnb1CA hep mice while Ntcp was reduced. Accompanying changes of bile acid transporters favoring excretion of bile acids were observed in intestine and kidneys of Ctnnb1CA hep mice. Additionally, disturbed bile acid regulation through the FXR-FGF15-FGFR4 pathway was observed in mice with activated ß-catenin. MATERIALS AND METHODS: Mice with a loxP-flanked exon 3 of the Ctnnb1 gene were crossed to Albumin-Cre mice to obtain mice with hepatocyte-specific expression of a dominant stable form of ß-catenin (Ctnnb1CA hep mice). Ctnnb1CA hep mice were analyzed by histology, serum biochemistry and mRNA profiling. CONCLUSIONS: Expression of a dominant stable form of ß-catenin in hepatocytes results in severe cholestasis and biliary type fibrosis.