24-Norursodeoxycholic acid ameliorates experimental alcohol-related liver disease and activates hepatic PPARγ.

Background & Aims: Alcohol-related liver disease (ALD) is a global healthcare challenge with limited treatment options. 24-Norursodeoxycholic acid (NorUDCA) is a synthetic bile acid with anti-inflammatory properties in experimental and human cholestatic liver diseases. In the present study, we explored the efficacy of norUDCA in experimental ALD. Methods: NorUDCA was tested in a preventive and therapeutic setting in an experimental ALD model (Lieber-DeCarli diet enriched with ethanol). Liver disease was phenotypically evaluated using histology and biochemical methods, and anti-inflammatory properties and peroxisome proliferator-activated receptor gamma activation by norUDCA were evaluated in cellular model systems. Results: NorUDCA administration ameliorated ethanol-induced liver injury, reduced hepatocyte death, and reduced the expression of hepatic pro-inflammatory cytokines including tumour necrosis factor (Tnf), Il-1ß, Il-6, and Il-10. NorUDCA shifted hepatic macrophages towards an anti-inflammatory M2 phenotype. Further, norUDCA administration altered the composition of the intestinal microbiota, specifically increasing the abundance of Roseburia, Enterobacteriaceae, and Clostridum spp. In a therapeutic model, norUDCA also ameliorated ethanol-induced liver injury. Moreover, norUDCA suppressed lipopolysaccharide-induced IL-6 expression in human peripheral blood mononuclear cells and evoked peroxisome proliferator-activated receptor gamma activation. Conclusions: NorUDCA ameliorated experimental ALD, protected against hepatic inflammation, and affected gut microbial commensalism. NorUDCA could serve as a novel therapeutic agent in the future management of patients with ALD. Impact and implications: Alcohol-related liver disease is a global healthcare concern with limited treatment options. 24-Norursodeoxycholic acid (NorUDCA) is a modified bile acid, which was proven to be effective in human cholestatic liver diseases. In the present study, we found a protective effect of norUDCA in experimental alcoholic liver disease. For patients with ALD, norUDCA could be a potential new treatment option.

GLP-2 Improves Hepatic Inflammation and Fibrosis in Mdr2-/- Mice Via Activation of NR4a1/Nur77 in Hepatic Stellate Cells and Intestinal FXR Signaling.

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.

KIAA1363 affects retinyl ester turnover in cultured murine and human hepatic stellate cells.

Large quantities of vitamin A are stored as retinyl esters (REs) in specialized liver cells, the hepatic stellate cells (HSCs). To date, the enzymes controlling RE degradation in HSCs are poorly understood. In this study, we identified KIAA1363 (also annotated as arylacetamide deacetylase 1 or neutral cholesterol ester hydrolase 1) as a novel RE hydrolase. We show that KIAA1363 is expressed in the liver, mainly in HSCs, and exhibits RE hydrolase activity at neutral pH. Accordingly, addition of the KIAA1363-specific inhibitor JW480 largely reduced RE hydrolase activity in lysates of cultured murine and human HSCs. Furthermore, cell fractionation experiments and confocal microscopy studies showed that KIAA1363 localizes to the endoplasmic reticulum. We demonstrate that overexpression of KIAA1363 in cells led to lower cellular RE content after a retinol loading period. Conversely, pharmacological inhibition or shRNA-mediated silencing of KIAA1363 expression in cultured murine and human HSCs attenuated RE degradation. Together, our data suggest that KIAA1363 affects vitamin A metabolism of HSCs by hydrolyzing REs at the endoplasmic reticulum, thereby counteracting retinol esterification and RE storage in lipid droplets.

24-Norursodeoxycholic acid reshapes immunometabolism in CD8+ T cells and alleviates hepatic inflammation.

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.

The Role of Lipid Sensing Nuclear Receptors (PPARs and LXR) and Metabolic Lipases in Obesity, Diabetes and NAFLD.

Obesity and type 2 diabetes mellitus (T2DM) are metabolic disorders characterized by metabolic inflexibility with multiple pathological organ manifestations, including non-alcoholic fatty liver disease (NAFLD). Nuclear receptors are ligand-dependent transcription factors with a multifaceted role in controlling many metabolic activities, such as regulation of genes involved in lipid and glucose metabolism and modulation of inflammatory genes. The activity of nuclear receptors is key in maintaining metabolic flexibility. Their activity depends on the availability of endogenous ligands, like fatty acids or oxysterols, and their derivatives produced by the catabolic action of metabolic lipases, most of which are under the control of nuclear receptors. For example, adipose triglyceride lipase (ATGL) is activated by peroxisome proliferator-activated receptor γ (PPARγ) and conversely releases fatty acids as ligands for PPARα, therefore, demonstrating the interdependency of nuclear receptors and lipases. The diverse biological functions and importance of nuclear receptors in metabolic syndrome and NAFLD has led to substantial effort to target them therapeutically. This review summarizes recent findings on the roles of lipases and selected nuclear receptors, PPARs, and liver X receptor (LXR) in obesity, diabetes, and NAFLD.

Absence of Adiponutrin (PNPLA3) and Monoacylglycerol Lipase Synergistically Increases Weight Gain and Aggravates Steatohepatitis in Mice.

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.

PNPLA3 I148M Up-Regulates Hedgehog and Yap Signaling in Human Hepatic Stellate Cells.

Liver fibrosis represents the wound healing response to sustained hepatic injury with activation of hepatic stellate cells (HSCs). The I148M variant of the PNPLA3 gene represents a risk factor for development of severe liver fibrosis. Activated HSCs carrying the I148M variant display exacerbated pro-inflammatory and pro-fibrogenic features. We aimed to examine whether the I148M variant may impair Hedgehog and Yap signaling, as key pathways implicated in the control of energy expenditure and maintenance of myofibroblastic traits. First, we show that TGF-ß rapidly up-regulated the PNPLA3 transcript and protein and Yap/Hedgehog target gene expression. In addition, HSCs overexpressing PNPLA3 I148M boosted anaerobic glycolysis, as supported by higher lactate release and decreased phosphorylation of the energy sensor AMPK. These cells displayed higher Yap and Hedgehog signaling, due to accumulation of total Yap protein, Yap promoter activity and increased downstream targets expression, compared to WT cells. HSCs exposed to TGF-ß and leptin rapidly increased total Yap, together with a reduction in its inhibited form, phosphorylated Yap. In line, Yap-specific inhibitor Verteporfin strongly abolished Yap-mediated genes expression, at baseline as well as after TGF-ß and leptin treatments in HSCs with I148M PNPLA3. Finally, Yap transcriptional activity was strongly reduced by a combination of Verteporfin and Rosiglitazone, a PPARγ synthetic agonist. In conclusion, HSCs carrying the PNPLA3 variant show activated Yap/Hedgehog pathways, resulting in altered anaerobic glycolysis and enhanced synthesis of Hedgehog markers and sustained Yap signaling. TGF-ß and leptin exacerbate Yap/Hedgehog-related fibrogenic genes expression, while Yap inhibitors and PPARγ agonists abrogate these effects in PNPLA3 I148M carrying HSCs.

Impact of osteopontin on the development of non-alcoholic liver disease and related hepatocellular carcinoma.

BACKGROUND & AIMS: Osteopontin, a multifunctional protein and inflammatory cytokine, is overexpressed in adipose tissue and liver in obesity and contributes to the induction of adipose tissue inflammation and non-alcoholic fatty liver (NAFL). Studies performed in both mice and humans also point to a potential role for OPN in malignant transformation and tumour growth. To fully understand the role of OPN on the development of NAFL-derived hepatocellular carcinoma (HCC), we applied a non-alcoholic steatohepatitis (NASH)-HCC mouse model on osteopontin-deficient (Spp1-/- ) mice analysing time points of NASH, fibrosis and HCC compared to wild-type mice. METHODS: Two-day-old wild-type and Spp1-/- mice received a low-dose streptozotocin injection in order to induce diabetes, and were fed a high-fat diet starting from week 4. Different cohorts of mice of both genotypes were sacrificed at 8, 12 and 19 weeks of age to evaluate the NASH, fibrosis and HCC phenotypes respectively. RESULTS: Spp1-/- animals showed enhanced hepatic lipid accumulation and aggravated NASH, as also increased hepatocellular apoptosis and accelerated fibrosis. The worse steatotic and fibrotic phenotypes observed in Spp1-/- mice might be driven by enhanced hepatic fatty acid influx through CD36 overexpression and by a pathological accumulation of specific diacylglycerol species during NAFL. Lack of osteopontin lowered systemic inflammation, prevented HCC progression to less differentiated tumours and improved overall survival. CONCLUSIONS: Lack of osteopontin dissociates NASH-fibrosis severity from overall survival and HCC malignant transformation in NAFLD, and is therefore a putative therapeutic target only for advanced chronic liver disease.

Absence of Bsep/Abcb11 attenuates MCD diet-induced hepatic steatosis but aggravates inflammation in mice.

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.

Metabolic regulation of hepatic PNPLA3 expression and severity of liver fibrosis in patients with NASH.

BACKGROUND AND AIMS: The genetic PNPLA3 polymorphism I148M has been extensively associated with higher risk for development and progression of NAFLD towards NASH. METHODS: PNPLA3 and α-SMA expression were quantified in liver biopsies collected from NASH patients (n = 26) with different fibrosis stages and PNPLA3 genotypes. To study the potential mechanisms driving PNPLA3 expression during NASH progression towards fibrosis, hepatocytes and hepatic stellate cells (HSCs) were cultivated in low and high glucose medium. Moreover, hepatocytes were treated with increasing concentrations of palmitic acid alone or in combination with glucose. Conditioned media were collected from challenged hepatocytes to stimulate HSCs. RESULTS: Tissue expression of PNPLA3 was significantly enhanced in biopsies of patients carrying the I148M polymorphism compared to wild type (WT). In NASH biopsies, PNPLA3 significantly correlated with fibrosis stage and α-SMA levels independently of PNPLA3 genotype. In line, PNPLA3 expression was higher in α-SMA positive cells. Low glucose increased PNPLA3 in HSCs, whereas high glucose induced PNPLA3 and de-novo lipogenesis-related genes expression in hepatocytes. Palmitic acid induced fat accumulation and cell stress markers in hepatocytes, which could be counteracted by oleic acid. Conditioned media collected from lipotoxic challenged hepatocytes markedly induced PNPLA3 mRNA and protein levels, fibrogenic and autophagic markers and promoted migration in HSCs. Notably, conditioned media collected from hepatocytes cultivated with both glucose and palmitic acid exacerbated HSCs migration, PNPLA3 and fibrogenic gene expression, promoting release of cytokines from HSCs. CONCLUSIONS: Collectively, our observations uncover the diverse metabolic regulation of PNPLA3 among different hepatic cell populations and support its relation to fibrosis progression.

Monoacylglycerol Lipase Inhibition Protects From Liver Injury in Mouse Models of Sclerosing Cholangitis.

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.

PNPLA3 I148M Variant Impairs Liver X Receptor Signaling and Cholesterol Homeostasis in Human Hepatic Stellate Cells.

The patatin-like phospholipase domain-containing protein 3 (PNPLA3) I148M variant predisposes to hepatic steatosis and progression to advanced liver injury with development of fibrosis, cirrhosis, and cancer. Hepatic stellate cells (HSCs) drive the wound healing response to chronic injury, and lack of liver X receptor (LXR) signaling exacerbates liver fibrogenesis by impairing HSC cholesterol homeostasis. However, the contribution of the I148M variant to this process is still unknown. We analyzed LXR expression and transcriptional activity in primary human HSCs and overexpressing LX-2 cells according to PNPLA3 genotype (wild type [WT] versus I148M). Here we demonstrate that LXRα protein increased whereas LXR target gene expression decreased during in vitro activation of primary human HSCs. Notably, LXRα levels and signaling were reduced in primary I148M HSCs compared to WT, as displayed by decreased expression of LXR target genes. Moreover, reduced expression of cholesterol efflux and enzymes generating oxysterols was associated with higher total and free cholesterol accumulation whereas endogenous cholesterol synthesis and uptake were diminished in I148M HSCs. Luciferase assays on LXR response element confirmed decreased LXR transcriptional activity in I148M HSCs; in contrast the synthetic LXR agonist T0901317 replenished LXR functionality, supported by adenosine triphosphate-binding cassette subfamily A member 1 (ABCA1) induction, and reduced collagen1α1 and chemokine (C-C motif) ligand 5 expression. Conversely, the peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone had only partial effects on the LXR target gene ABCA1, and neither diminished expression of proinflammatory cytokines nor increased de novo lipogenic genes in I148M HSCs. Conclusion: As a consequence of reduced PPARγ activity, HSCs carrying I148M PNPLA3 show impaired LXR signaling, leading to cholesterol accumulation. The use of a specific LXR agonist shows beneficial effects for diminishing sustained HSC activation and development of liver fibrogenesis.

Lack of monoacylglycerol lipase prevents hepatic steatosis by favoring lipid storage in adipose tissue and intestinal malabsorption.

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.

Thyroid hormone receptor ß1 stimulates ABCB4 to increase biliary phosphatidylcholine excretion in mice.

The ATP-binding cassette transporter ABCB4/MDR3 is critical for biliary phosphatidylcholine (PC) excretion at the canalicular membrane of hepatocytes. Defective ABCB4 gene expression and protein function result in various cholestatic liver and bile duct injuries. Thyroid hormone receptor (THR) is a major regulator of hepatic lipid metabolism; we explored its potential role in ABCB4 regulation. Thyroid hormone T3 stimulation to human hepatocyte models showed direct transcriptional activation of ABCB4 in a dose- and time-dependent manner. To determine whether THRß1 (the main THR isoform of the liver) is involved in regulation, we tested THRß1-specific agonists (e.g., GC-1, KB-141); these agonists resulted in greater stimulation than the native hormone. KB-141 activated hepatic ABCB44 expression in mice, which enhanced biliary PC secretion in vivo. We also identified THR response elements 6 kb upstream of the ABCB4 locus that were conserved in humans and mice. Thus, T3-via THRß1 as a novel transcriptional activator regulates ABCB4 to increase ABCB4 protein levels at the canalicular membrane and promote PC secretion into bile. These findings may have important implications for understanding thyroid hormone function as a potential modifier of bile duct homeostasis and provide pharmacologic opportunities to improve liver function in hepatobiliary diseases caused by low ABCB4 expression.

Nuclear Receptor Regulation of Aquaglyceroporins in Metabolic Organs.

Nuclear receptors, such as the farnesoid X receptor (FXR) and the peroxisome proliferator-activated receptors gamma and alpha (PPAR-γ, -α), are major metabolic regulators in adipose tissue and the liver, where they govern lipid, glucose, and bile acid homeostasis, as well as inflammatory cascades. Glycerol and free fatty acids are the end products of lipid droplet catabolism driven by PPARs. Aquaporins (AQPs), a family of 13 small transmembrane proteins, facilitate the shuttling of water, urea, and/or glycerol. The peculiar role of AQPs in glycerol transport makes them pivotal targets in lipid metabolism, especially considering their tissue-specific regulation by the nuclear receptors PPARγ and PPARα. Here, we review the role of nuclear receptors in the regulation of glycerol shuttling in liver and adipose tissue through the function and expression of AQPs.

AQP3 is regulated by PPARγ and JNK in hepatic stellate cells carrying PNPLA3 I148M.

Aquaglyceroporins (AQPs) allow the movement of glycerol that is required for triglyceride formation in hepatic stellate cells (HSC), as key cellular source of fibrogenesis in the liver. The genetic polymorphism I148M of the patatin-like phospholipase domain-containing 3 (PNPLA3) is associated with hepatic steatosis and its progression to steatohepatitis (NASH), fibrosis and cancer. We aimed to explore the role of AQP3 for HSC activation and unveil its potential interactions with PNPLA3. HSC were isolated from human liver, experiments were performed in primary HSC and human HSC line LX2. AQP3 was the only aquaglyceroporin present in HSC and its expression decreased during activation. The PPARγ agonist, rosiglitazone, recovered AQP3 expression also in PNPLA3 I148M carrying HSC. When PNPLA3 was silenced, AQP3 expression increased. In liver sections from patients with NASH, the decreased amount of AQP3 was proportional to the severity of fibrosis and presence of the PNPLA3 I148M variant. In PNPLA3 I148M cells, the blockade of JNK pathway upregulated AQP3 in synergism with PPARγ. In conclusion, we demonstrated profound reduction of AQP3 in HSC carrying the PNPLA3 I148M variant in parallel to decreased PPARγ activation, which could be rescued by rosiglitazone and blockade of JNK.

PNPLA3 expression and its impact on the liver: current perspectives.

A single-nucleotide polymorphism occurring in the sequence of the human patatin-like phospholipase domain-containing 3 gene (PNPLA3), known as I148M variant, is one of the best characterized and deeply investigated variants in several clinical scenarios, because of its tight correlation with increased risk for developing hepatic steatosis and more aggressive part of the disease spectrum, such as nonalcoholic steatohepatitis, advanced fibrosis and cirrhosis. Further, the I148M variant is positively associated with alcoholic liver diseases, chronic hepatitis C-related cirrhosis and hepatocellular carcinoma. The native gene encodes for a protein that has not yet a fully defined role in liver lipid metabolism and, according to recent observations, seems to be divergently regulated among distinct liver cells type, such as hepatic stellate cells. Therefore, the aim of this review is to collect the latest data regarding PNPLA3 expression in human liver and to analyze the impact of its genetic variant in human hepatic pathologies. Moreover, a description of the current biochemical and metabolic data pertaining to PNPLA3 function in both animal models and in vitro studies is summarized to allow a better understanding of the relevant pathophysiological role of this enzyme in the progression of hepatic diseases.

FXR controls CHOP expression in steatohepatitis.

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.

Adiponectin regulates AQP3 via PPARα in human hepatic stellate cells.

Aquaporins (AQPs) are trans-membrane proteins which allow the movement of water and glycerol required by hepatic stellate cells (HSC) for triglyceride formation and lipid storage. Adiponectin (ADPQ) is a hormone produced by the adipose tissue, which is known to increase AQP3 expression. Since ADPQ receptor signals via the nuclear receptor PPAR we aimed to explore the role of this pathway in AQP3 regulation by ADPQ in HSC. AQP3 and CPT1α expression increased only after ADPQ but not rosiglitazone stimulation. In LX2 cells co-transfected with plasmids expressing PPARα or PPARγ coupled to a luciferase reporter gene, only PPARα increased luciferase activity after ADPQ stimulation. Collectively, our findings demonstrate that ADPQ increases AQP3 expression through PPARα-mediated signaling in HSC.

Impact of higher-order heme degradation products on hepatic function and hemodynamics.

BACKGROUND & AIMS: Biliverdin and bilirubin were previously considered end products of heme catabolism; now, however, there is evidence for further degradation to diverse bioactive products. Z-BOX A and Z-BOX B arise upon oxidation with unknown implications for hepatocellular function and integrity. We studied the impact of Z-BOX A and B on hepatic functions and explored their alterations in health and cholestatic conditions. METHODS: Functional implications and mechanisms were investigated in rats, hepatocytic HepG2 and HepaRG cells, human immortalized hepatocytes, and isolated perfused livers. Z-BOX A and B were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in acute and acute-on-chronic liver failure and hereditary unconjugated hyperbilirubinemia. RESULTS: Z-BOX A and B are found in similar amounts in humans and rodents under physiological conditions. Serum concentrations increased ∼20-fold during cholestatic liver failure in humans (p<0.001) and in hereditary deficiency of bilirubin glucuronidation in rats (p<0.001). Pharmacokinetic studies revealed shorter serum half-life of Z-BOX A compared to its regio-isomer Z-BOX B (p=0.035). While both compounds were taken up by hepatocytes, Z-BOX A was enriched ∼100-fold and excreted in bile. Despite their reported vasoconstrictive properties in the brain vasculature, BOXes did not affect portal hemodynamics. Both Z-BOX A and B showed dose-dependent cytotoxicity, affected the glutathione redox state, and differentially modulated activity of Rev-erbα and Rev-erbß. Moreover, BOXes-triggered remodeling of the hepatocellular cytoskeleton. CONCLUSIONS: Our data provide evidence that higher-order heme degradation products, namely Z-BOX A and B, impair hepatocellular integrity and might mediate intra- and extrahepatic cytotoxic effects previously attributed to hyperbilirubinemia. LAY SUMMARY: Degradation of the blood pigment heme yields the bile pigment bilirubin and the oxidation products Z-BOX A and Z-BOX B. Serum concentrations of these bioactive molecules increase in jaundice and can impair liver function and integrity. Amounts of Z-BOX A and Z-BOX B that are observed during liver failure in humans have profound effects on hepatic function when added to cultured liver cells or infused into healthy rats.