HDL cholesterol protects from liver injury in mice with intestinal specific LXRα activation.

BACKGROUND AND AIMS: Liver X receptors (LXRs) exert anti-inflammatory effects even though their hepatic activation is associated with hypertriglyceridemia and hepatic steatosis. Selective induction of LXRs in the gut might provide protective signal(s) in the aberrant wound healing response that induces fibrosis during chronic liver injury, without hypertriglyceridemic and steatogenic effects. METHODS: Mice with intestinal constitutive LXRα activation (iVP16-LXRα) were exposed to intraperitoneal injection of carbon tetrachloride (CCl4 ) for 8 weeks, and in vitro cell models were used to evaluate the beneficial effect of high-density lipoproteins (HDL). RESULTS: After CCl4 treatment, the iVP16-LXRα phenotype showed reduced M1 macrophage infiltration, increased expression M2 macrophage markers, and lower expression of hepatic pro-inflammatory genes. This anti-inflammatory effect in the liver was also associated with decreased expression of hepatic oxidative stress genes and reduced expression of fibrosis markers. iVP16-LXRα exhibited increased reverse cholesterol transport in the gut by ABCA1 expression and consequent enhancement of the levels of circulating HDL and their receptor SRB1 in the liver. No hepatic steatosis development was observed in iVP16-LXRα. In vitro, HDL induced a shift from M1 to M2 phenotype of LPS-stimulated Kupffer cells, decreased TNFα-induced oxidative stress in hepatocytes and reduced NF-kB activity in both cells. SRB1 silencing reduced TNFα gene expression in LPS-stimulated KCs, and NOX-1 and IL-6 in HepG2. CONCLUSIONS: Intestinal activation of LXRα modulates hepatic response to injury by increasing circulating HDL levels and SRB1 expression in the liver, thus suggesting this circuit as potential actionable pathway for therapy.

Lipidomic biomarkers and mechanisms of lipotoxicity in non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) represents the most common form of chronic liver disease worldwide (about 25% of the general population) and 3-5% of patients develop non-alcoholic steatohepatitis (NASH), characterized by hepatocytes damage, inflammation and fibrosis, which increase the risk of developing liver failure, cirrhosis and hepatocellular carcinoma. The pathogenesis of NAFLD, particularly the mechanisms whereby a minority of patients develop a more severe phenotype, is still incompletely understood. In this review we examine the available literature on initial mechanisms of hepatocellular damage and inflammation, deriving from toxic effects of excess lipids. Accumulating data indicate that the total amount of triglycerides stored in the liver cells is not the main determinant of lipotoxicity and that specific lipid classes act as damaging agents. These lipotoxic species affect the cell behavior via multiple mechanisms, including activation of death receptors, endoplasmic reticulum stress, modification of mitochondrial function and oxidative stress. The gut microbiota, which provides signals through the intestine to the liver, is also reported to play a key role in lipotoxicity. Finally, we summarize the most recent lipidomic strategies utilized to explore the liver lipidome and its modifications in the course of NALFD. These include measures of lipid profiles in blood plasma and erythrocyte membranes that can surrogate to some extent lipid investigation in the liver.

Nonalcoholic Fatty Liver Disease: Basic Pathogenetic Mechanisms in the Progression From NAFLD to NASH.

Nonalcoholic fatty liver disease (NAFLD) represents a growing cause of chronic liver injury, especially in western countries, where it is becoming the most frequent indication for liver transplantation. Nonalcoholic fatty liver disease encompasses a spectrum of diseases that from simple steatosis (pure NAFLD) can progress to nonalcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma. The pathogenesis of NAFLD and the mechanisms behind its progression to NASH have been extensively studied. However, although the processes that determine fat accumulation are mostly clear, the mechanisms associated with the progression of the disease are not fully characterized. In predisposed patients, lipid accumulation can promote lipotoxicity and mitochondrial dysfunction, thus triggering hepatocyte death, inflammation and fibrosis. The specific role of different lipids has been identified and free fatty acids as well as free cholesterol have been identified as toxic species. To make the picture more complex, the pathogenesis of NAFLD involves pathological connections between several organs, including the adipose tissue and the gut, with the liver. The "inflamed" adipose tissue plays a key role in the release of toxic lipids, whereas alterations in the gut-liver axis have been associated with the progression from NAFLD to NASH mediated by dysbiosis, alteration of intestinal barrier, and finally bacterial translocation, which can trigger proinflammatory and profibrogenetic pathways, finally leading to cirrhosis development.

Author Correction: Lack of NLRP3-inflammasome leads to gut-liver axis derangement, gut dysbiosis and a worsened phenotype in a mouse model of NAFLD.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Lack of NLRP3-inflammasome leads to gut-liver axis derangement, gut dysbiosis and a worsened phenotype in a mouse model of NAFLD.

Non-Alcoholic Fatty Liver Disease (NAFLD) represents the most common form of chronic liver injury and can progress to cirrhosis and hepatocellular carcinoma. A "multi-hit" theory, involving high fat diet and signals from the gut-liver axis, has been hypothesized. The role of the NLRP3-inflammasome, which senses dangerous signals, is controversial. Nlrp3-/- and wild-type mice were fed a Western-lifestyle diet with fructose in drinking water (HFHC) or a chow diet. Nlrp3-/--HFHC showed higher hepatic expression of PPAR γ2 (that regulates lipid uptake and storage) and triglyceride content, histological score of liver injury and greater adipose tissue inflammation. In Nlrp3-/--HFHC, dysregulation of gut immune response with impaired antimicrobial peptides expression, increased intestinal permeability and the occurrence of a dysbiotic microbiota led to bacterial translocation, associated with higher hepatic expression of TLR4 (an LPS receptor) and TLR9 (a receptor for double-stranded bacterial DNA). After antibiotic treatment, gram-negative species and bacterial translocation were reduced, and adverse effects restored both in liver and adipose tissue. In conclusion, the combination of a Western-lifestyle diet with innate immune dysfunction leads to NAFLD progression, mediated at least in part by dysbiosis and bacterial translocation, thus identifying new specific targets for NAFLD therapy.

Fibronectin Type III Domain-Containing Protein 5 rs3480 A>G Polymorphism, Irisin, and Liver Fibrosis in Patients With Nonalcoholic Fatty Liver Disease.

Context: Contrasting data have been reported on the role of irisin, a novel myokine encoded by the fibronectin type III domain-containing protein 5 (FNDC5) gene, in nonalcoholic fatty liver disease (NAFLD) pathogenesis. We tested in patients with suspected nonalcoholic steatohepatitis (NASH) the association of FNDC5 variants, hepatic expression, and circulating irisin with liver damage (F2 to F4 fibrosis as main outcome). We also investigated whether irisin modulates hepatocellular fat accumulation and stellate cell activation in experimental models. Methods: We considered 593 consecutive patients who underwent liver biopsy for suspected NASH and 192 patients with normal liver enzymes and without steatosis. FNDC5 rs3480 and rs726344 genotypes were assessed by 5' nuclease assays. Hepatic irisin expression was evaluated in mice fed a high-fat diet or treated with CCl4. The effect of irisin was evaluated in fat-laden HepG2 hepatocytes and in hepatic stellate cells (HSCs). Results: In patients at risk for NASH [odds ratio (OR) = 0.64, 95% confidence interval (CI), 0.47 to 0.87; P = 0.005], and more so in the high-risk subgroup of those with impaired fasting glucose/diabetes (OR = 0.44, 95% CI, 0.26 to 0.74; P = 0.002), the rs3480 A>G variant was independently associated with protection from F2 to F4 fibrosis. Irisin is expressed in human activated HSC, where it mediated fibrogenic actions and collagen synthesis, and is overexpressed in NAFLD patients with F2 to F4 fibrosis and CCl4-treated mice. However, Irisin does not affect fat accumulation in HepG2 and is not induced by high-fat-diet-inducing NAFLD. Conclusions: The FNDC5 rs3480 variant is associated with protection from clinically significant fibrosis in patients with NAFLD, while irisin expression is correlated with the severity of NAFLD and may be involved in extracellular matrix deposition. These data suggest that irisin is involved in regulation of hepatic fibrogenesis.

Current Targets for Primary Sclerosing Cholangitis.

Primary sclerosing cholangitis (PSC) is a biliary disease characterized by liver inflammation and death of cholangiocytes which, in turn, drive to fibrosis, cirrhosis and functional alterations of the liver. PSC is also associated with an increased risk of developing cholangiocarcinoma. To date, the etiopathogenesis of PSC is still not completely understood, although a genetic predisposition in association to environmental factors contribute to immune-mediated liver damage. The lack of such knowledge is responsible for the failure of most available therapies. At this time, many studies are evaluating potential approaches that could have a positive impact on the progression of the disease. This review aims to provide a summary of present and past therapeutic approaches for PSC.

HCC development is associated to peripheral insulin resistance in a mouse model of NASH.

UNLABELLED: NAFLD is the most common liver disease worldwide but it is the potential evolution to NASH and eventually to hepatocellular carcinoma (HCC), even in the absence of cirrhosis, that makes NAFLD of such clinical importance. AIM: we aimed to create a mouse model reproducing the pathological spectrum of NAFLD and to investigate the role of possible co-factors in promoting HCC. METHODS: mice were treated with a choline-deficient L-amino-acid-defined-diet (CDAA) or its control (CSAA diet) and subjected to a low-dose i.p. injection of CCl4 or vehicle. Insulin resistance was measured by the euglycemic-hyperinsulinemic clamp method. Steatosis, fibrosis and HCC were evaluated by histological and molecular analysis. RESULTS: CDAA-treated mice showed peripheral insulin resistance at 1 month. At 1-3 months, extensive steatosis and fibrosis were observed in CDAA and CDAA+CCl4 groups. At 6 months, equal increase in steatosis and fibrosis was observed between the two groups, together with the appearance of tumor. At 9 months of treatment, the 100% of CDAA+CCl4 treated mice revealed tumor versus 40% of CDAA mice. Insulin-like Growth Factor-2 (IGF-2) and Osteopontin (SPP-1) were increased in CDAA mice versus CSAA. Furthermore, Immunostaining for p-AKT, p-c-Myc and Glypican-3 revealed increased positivity in the tumors. CONCLUSIONS: the CDAA model promotes the development of HCC from NAFLD-NASH in the presence of insulin resistance but in the absence of cirrhosis. Since this condition is increasingly recognized in humans, our study provides a model that may help understanding mechanisms of carcinogenesis in NAFLD.

PDX-1/Hes-1 interactions determine cholangiocyte proliferative response to injury in rodents: possible implications for sclerosing cholangitis.

BACKGROUND & AIMS: Cholangiocyte proliferation plays a role in the progression of cholangiopathies, in particular in primary sclerosing cholangitis. The mechanisms regulating cholangiocyte proliferation are still undefined. Pancreatic Duodenal Homeobox protein 1 (PDX-1) is expressed by reactive cholangiocytes. In the adult pancreas, PDX-1 regulates the proliferative response to injury of ductal cells. Its effects can be counteracted by Hairy and enhancer of split 1 (Hes-1). We aimed at studying whether PDX-1/Hes-1 interactions regulate cholangiocyte proliferation in response to injury. METHODS: The effect of the loss of PDX-1 on cholangiocyte proliferation was studied in vitro. In vivo PDX-1-heterozygous (+/-) mice were subjected to either DDC feeding (a model of sclerosing cholangitis) or to bile duct ligation (BDL). PDX-1/Hes-1 interactions on cell proliferation were determined by exposure to All-trans Retinoic Acid (At-RA), an inductor of Hes-1. RESULTS: In vitro, cholangiocyte proliferation was undetectable in cells pre-treated with PDX-1 siRNA. In vivo, increases in bile duct mass and collagen deposition observed after DDC feeding or BDL were significantly reduced in PDX-1(+/-) mice. Hes-1 expression is reduced in proliferating cholangiocytes; At-RA induced a dose-dependent increase in Hes-1 and a decrease in PDX-1 expression. At-RA neutralized the increases in PDX-1 expression and cell proliferation, both in vitro and in vivo in DDC mice. PDX-1 is overexpressed and Hes-1 downregulated in cholangiocytes isolated from PSC livers. CONCLUSIONS: Hes-1 downregulation allows PDX-1 to act as a major determinant of cholangiocyte proliferation in response to cholestatic injury. These findings provide novel mechanistic insights into the pathophysiology of cholangiopathies.

The significance of genetics for cholangiocarcinoma development.

Cholangiocarcinoma (CCA) is a rare malignancy of the liver, arising from bile ducts. The incidence is increasing worldwide, but the prognosis has remained dismal and virtually unchanged in the past 30 years. Although several risk factors have been associated with the development of this cancer, none of them are normally identified in most patients. Diagnosis in advanced stages of the disease and limited therapeutic options contribute to poor survival rates. The recent analysis of genetic and epigenetic alterations occurring in CCA has shed new light in the understanding of the molecular mechanisms leading to the malignant transformation of biliary cells. Further studies in this direction may foster new diagnostic, prognostic and therapeutic approaches. This review provides a global overview of recent advances in CCA and describes the most important genetic mutations and epigenetic alterations so far reported in CCA.

Post-load insulin resistance does not predict virological response to treatment of chronic hepatitis C patients without the metabolic syndrome.

BACKGROUND AND AIM: The role of insulin resistance in predicting virological response to therapy of chronic hepatitis C is debated. We assessed the association between basal (defined as homeostasis model assessment of insulin resistance (HOMA-IR)>2) and post-load insulin resistance (as oral glucose insulin sensitivity index<9.8 mg/kg/min) with the rapid and sustained virological responses in chronic hepatitis C. METHODS: Observational prospective study of 124 treatment-naïve patients with chronic hepatitis C not fulfilling the metabolic syndrome criteria, adherent to a standard treatment with pegylated interferon alpha plus ribavirin. RESULTS: Insulin resistance was detected in 50% (by HOMA-IR) and 29% (by oral glucose insulin sensitivity index) of patients. Independent predictors of rapid virologic response were hepatitis C virus (HCV) genotype 2 (odds ratio 5.66; 95% confidence interval 1.88-17.01), HCV genotype 3 (odds ratio 5.23; 95% confidence interval 1.84-14.84) and lower basal ferritin levels (odds ratio 0.99; 95% confidence interval 0.993-0.998). Independent predictors of sustained virologic response were HCV genotype 2 (odds ratio 19.54; 95% confidence interval 2.29-166.41) and HCV genotype 3 (odds ratio 3.24; 95% confidence interval 1.10-9.58). Rapid virologic response was by itself predictive of sustained virologic response (odds ratio 40.90; 95% confidence interval 5.37-311.53). CONCLUSIONS: Insulin resistance, measured by both static and dynamic methods, does not predict rapid or sustained virologic response in chronic hepatitis C patients without the metabolic syndrome.

An oestrogen receptor ß-selective agonist exerts anti-neoplastic effects in experimental intrahepatic cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma cells over-express oestrogen receptor-ß, which displays anti-proliferative and pro-apoptotic effects. AIM: To evaluate the effects of a newly developed and highly selective oestrogen receptor-ß agonist (KB9520) on experimental intrahepatic cholangiocarcinoma. METHODS: In vitro, the effects of KB9520 on apoptosis and proliferation of HuH-28 cells, HuH-28 cells with selective oestrogen receptor-ß silencing (by small interfering RNA), HepG2 cells (oestrogen receptor-α and oestrogen receptor-ß negative) and HepER3 cells (HepG2 cells transformed to stably express oestrogen receptor-α) were evaluated. In vivo, the effects of KB9520 on experimental intrahepatic cholangiocarcinoma, induced by thioacetamide administration were tested. RESULTS: In vitro, KB9520 induced apoptosis and inhibited proliferation of HuH-28 cells. KB9520 effects were absent in cells lacking oestrogen receptor-α and ß (HepG2) and in cells expressing only oestrogen receptor-α (HepER3); its pro-apoptotic effect was impaired in cells where oestrogen receptor-ß expression was decreased by specific small interfering RNA. In vivo, KB9520 inhibited experimental intrahepatic cholangiocarcinoma development in thioacetamide-treated rats and promoted tumour regression in rats where tumour was already established. In treated animals, tumour areas showed reduced proliferation but increased apoptosis. CONCLUSIONS: KB9520 induced apoptosis in cholangiocarcinoma by selectively acting on oestrogen receptor-ß, suggesting that oestrogen receptor-ß selective agonists may be a novel and effective therapeutic option for the medical treatment of intrahepatic cholangiocarcinoma.

Insulin resistance and necroinflammation drives ductular reaction and epithelial-mesenchymal transition in chronic hepatitis C.

OBJECTIVE: To study the mechanism(s) linking insulin resistance (IR) to hepatic fibrosis and the role of the epithelial component in tissue repair and fibrosis in chronic hepatitis C (CHC). DESIGN: Prospective observational study. SETTING: Tertiary care academic centre. PATIENTS: 78 consecutive patients with CHC. MAIN OUTCOME MEASURES: IR, calculated by the oral glucose insulin sensitivity during oral glucose tolerance test; necroinflammatory activity and fibrosis, defined according to Ishak's score; steatosis, graded as 0 (<5% of hepatocytes), 1 (5-33%), 2 (33-66%) and 3 (>66%). To evaluate the role of the epithelial component in tissue repair and fibrosis, the expansion of the ductular reaction (DR) was calculated by keratin-7 (CK7) morphometry. Nuclear expression of Snail, downregulation of E-cadherin and expression of fibroblast specific protein-1 (FSP1) and vimentin by CK7-positive cells were used as markers of epithelial-mesenchymal transition in DR elements. RESULTS: IR, the degree of necroinflammation and expansion of the DR (stratified as reactive ductular cells (RDCs), hepatic progenitor cells and intermediate hepatobiliary cells according to morphological criteria) were all associated with the stage of fibrosis. Nuclear Snail expression, E-cadherin downregulation and vimentin upregulation were observed in RDCs. By dual immunofluorescence for CK7 and FSP1, the number of RDCs undergoing epithelial-mesenchymal transition progressively increased together with the necroinflammatory score. By multivariate analysis, total inflammation and insulin resistance were the only factors significantly predicting the presence of advanced fibrosis (Ishak score ≥3) and the expansion of RDCs. CONCLUSION: This study indicates that IR is associated with the degree of necroinflammatory injury in CHC and contributes to hepatic fibrosis by stimulating the expansion of RDCs that express epithelial-mesenchymal transition markers.

Pancreatic Duodenal Homeobox-1 de novo expression drives cholangiocyte neuroendocrine-like transdifferentiation.

BACKGROUND & AIMS: Reactive cholangiocytes acquire a neuroendocrine-like phenotype, with synthesis and local release of neuropeptides and hormones. The mechanism that drives such phenotypical changes is still undefined. Pancreatic Duodenal Homeobox-1 (PDX-1) is a transcription factor required for pancreatic development, that sustains pancreatic beta-cell response to injury and insulin synthesis. PDX-1 induces neuroendocrine-like transition of pancreatic ductal cells. Cholangiocyte response to injury is modulated by Glucagon-Like Peptide-1 Receptor (GLP-1R), which, in the pancreas, activates PDX-1. We wanted to verify whether PDX-1 plays any role in cholangiocyte neuroendocrine-like transdifferentiation in response to injury. METHODS: PDX-1 expression was assessed in cholangiocytes from normal and one week bile duct ligated (BDL) rats. Changes in PDX-1 expression and activation upon GLP-1R activation were then assayed. The effects of the lack of PDX-1 in cholangiocytes were studied in vitro by siRNA and in vivo by the employment of PDX-1-deficient (+/-) mice. RESULTS: BDL but not normal cholangiocytes express PDX-1. GLP-1R activation elicits, in a PI3K-dependent fashion, PDX-1 expression, together with its nuclear translocation. In vitro, GLP-1R-induced increases in VEGF and IGF-1 mRNA expression were blunted in cells with PDX-1 siRNA. In vivo, the VEGF and IGF-1 mRNA expression in the liver after one week BDL was markedly reduced in PDX-1-deficient mice, together with reduced bile duct mass. CONCLUSIONS: In response to injury, reactive cholangiocytes de novo express PDX-1, the activation of which allows cholangiocytes to synthesize IGF-1 and VEGF. These findings suggest that PDX-1 drives the acquisition of the neuroendocrine-like phenotype by cholangiocytes in response to cholestatic injury.