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Metabolic dysfunction-associated steatohepatitis (MASH) represents a global health threat. MASH pathophysiology involves hepatic lipid accumulation and progression to severe conditions like cirrhosis and, eventually, hepatocellular carcinoma. Fibroblast growth factor (FGF)-19 has emerged as a key regulator of metabolism, offering potential therapeutic avenues for MASH and associated disorders. We evaluated the therapeutic potential of non-mitogenic (NM)-FGF19 mRNA formulated in liver-targeted lipid nanoparticles (NM-FGF19-mRNAs-LNPs) in C57BL/6NTac male mice with diet-induced obesity and MASH (DIO-MASH: 40% kcal fat, 20% kcal fructose, 2% cholesterol). After feeding this diet for 21 weeks, NM-FGF19-mRNAs-LNPs or control (C-mRNA-LNPs) were administered (0.5 mg/kg, i.v.) weekly for another six weeks, in which diet feeding continued. NM-FGF19-mRNAs-LNPs treatment in DIO-MASH mice resulted in reduced body weight, adipose tissue depots, and serum transaminases, along with improved insulin sensitivity. Histological analyses confirmed the reversal of MASH features, including steatosis reduction without worsening fibrosis. NM-FGF19-mRNAs-LNPs reduced total hepatic bile acids (BAs) and changed liver BA composition, markedly influencing cholesterol homeostasis and metabolic pathways as observed in transcriptomic analyses. Extrahepatic effects included the down-regulation of metabolic dysfunction-associated genes in adipose tissue. This study highlights the potential of NM-FGF19-mRNA-LNPs therapy for MASH, addressing both hepatic and systemic metabolic dysregulation. NM-FGF19-mRNA demonstrates efficacy in reducing liver steatosis, improving metabolic parameters, and modulating BA levels and composition. Given the central role played by BA in dietary fat absorption, this effect of NM-FGF19-mRNA may be mechanistically relevant. Our study underscores the high translational potential of mRNA-based therapies in addressing the multifaceted landscape of MASH and associated metabolic perturbations., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Alcoholic hepatitis (AH) is a life-threatening condition characterized by profound hepatocellular dysfunction for which targeted treatments are urgently needed. Identification of molecular drivers is hampered by the lack of suitable animal models. By performing RNA sequencing in livers from patients with different phenotypes of alcohol-related liver disease (ALD), we show that development of AH is characterized by defective activity of liver-enriched transcription factors (LETFs). TGFβ1 is a key upstream transcriptome regulator in AH and induces the use of HNF4α P2 promoter in hepatocytes, which results in defective metabolic and synthetic functions. Gene polymorphisms in LETFs including HNF4α are not associated with the development of AH. In contrast, epigenetic studies show that AH livers have profound changes in DNA methylation state and chromatin remodeling, affecting HNF4α-dependent gene expression. We conclude that targeting TGFβ1 and epigenetic drivers that modulate HNF4α-dependent gene expression could be beneficial to improve hepatocellular function in patients with AH.

Glycine N-methyltransferase (GNMT) is the most abundant methyltransferase in the liver and a master regulator of the transmethylation flux. GNMT downregulation leads to loss of liver function progressing to fibrosis, cirrhosis, and hepatocellular carcinoma. Moreover, GNMT deficiency aggravates cholestasis-induced fibrogenesis. To date, little is known about the mechanisms underlying downregulation of GNMT levels in hepatic fibrosis and cirrhosis. On this basis, microRNAs are epigenetic regulatory elements that play important roles in liver pathology. In this work, we aim to study the regulation of GNMT by microRNAs during liver fibrosis and cirrhosis. Luciferase assay on the 3'UTR-Gnmt was used to confirm in silico analysis showing that GNMT is potentially targeted by the microRNA miR-873-5p. Correlation between GNMT and miR-873-5p in human cholestasis and cirrhosis together with miR-873-5p inhibition in vivo in different mouse models of liver cholestasis and fibrosis [bile duct ligation and Mdr2 (Abcb4) -/- mouse] were then assessed. The analysis of liver tissue from cirrhotic and cholestatic patients, as well as from the animal models, showed that miR-873-5p inversely correlated with the expression of GNMT. Importantly, high circulating miR-873-5p was also detected in cholestastic and cirrhotic patients. Preclinical studies with anti-miR-873-5p treatment in bile duct ligation and Mdr2 -/- mice recovered GNMT levels in association with ameliorated inflammation and fibrosis mainly by counteracting hepatocyte apoptosis and cholangiocyte proliferation. In conclusion, miR-873-5p emerges as a novel marker for liver fibrosis, cholestasis, and cirrhosis and therapeutic approaches based on anti-miR-873-5p may be effective treatments for liver fibrosis and cholestatic liver disease.

At high doses, glucocorticoids (GC) have been associated with enhanced serum bile acids and liver injury. We have evaluated the effect of GC, in the absence of hepatotoxicity, on FXR/FGF91(Fgf15)/FGF21-mediated ileum-liver crosstalk. Rats and mice (wild type and Fxr -/- , Fgf15 -/- and int-Gr -/- strains; the latter with GC receptor (Gr) knockout selective for intestinal epithelial cells), were treated (i.p.) with dexamethasone, prednisolone or budesonide. In both species, high doses of GC caused hepatotoxicity. At a non-hepatotoxic dose, GC induced ileal Fgf15 down-regulation and liver Fgf21 up-regulation, without affecting Fxr expression. Fgf21 mRNA levels correlated with those of several genes involved in glucose and bile acid metabolism. Surprisingly, liver Cyp7a1 was not up-regulated. The expression of factors involved in transcriptional modulation by Fxr and Gr (p300, Drip205, CBP and Smrt) was not affected. Pxr target genes Cyp3a11 and Mrp2 were not up-regulated in liver or intestine. In contrast, the expression of some Pparα target genes in liver (Fgf21, Cyp4a14 and Vanin-1) and intestine (Vanin-1 and Cyp3a11) was altered. In mice with experimental colitis, liver Fgf21 was up-regulated (4.4-fold). HepG2 cells transfection with FGF21 inhibited CYP7A1 promoter (prCYP7A1-Luc2). This was mimicked by pure human FGF21 protein or culture in medium previously conditioned by cells over-expressing FGF21. This response was not abolished by deletion of a putative response element for phosphorylated FGF21 effectors present in prCYP7A1. In conclusion, GC interfere with FXR/FGF19-mediated intestinal control of CYP7A1 expression by the liver and stimulate hepatic secretion of FGF21, which inhibits CYP7A1 promoter through an autocrine mechanism., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The liver is a highly differentiated organ with a central role in metabolism, detoxification and systemic homeostasis. To perform its multiple tasks, liver parenchymal cells, the hepatocytes, express a large complement of enabling genes defining their complex phenotype. This phenotype is progressively acquired during fetal development and needs to be maintained in adulthood to guarantee the individual's survival. Upon injury or loss of functional mass, the liver displays an extraordinary regenerative response, mainly based on the proliferation of hepatocytes which otherwise are long-lived quiescent cells. Increasing observations suggest that loss of hepatocellular differentiation and quiescence underlie liver malfunction in chronic liver disease and pave the way for hepatocellular carcinoma development. Here, we briefly review the essential mechanisms leading to the acquisition of liver maturity. We also identify the key molecular factors involved in the preservation of hepatocellular homeostasis and finally discuss potential strategies to preserve liver identity and function.

Background & Aims: Bile salts inhibit their own production by inducing the nuclear receptor small heterodimer partner (SHP) (encoded by NR0B2), which contributes to repression of the gene encoding cholesterol 7α-hydroxylase (CYP7A1), a key enzyme for the control of bile salt synthesis. On the other hand, bile salts stimulate hepatic synthesis of nitric oxide. We investigated the role of nitric oxide signaling in the control of CYP7A1 expression and the involvement in this process of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which participates in intracellular propagation of nitric oxide signals., Methods: We studied the effects of inhibitors of nitric oxide synthesis (L-NG-nitroarginine methyl ester [L-NAME]) or protein nitrosylation (via dithiothreitol) on bile salt homeostasis in male Wistar rats placed on a cholate-rich diet for 5 days and in cultured primary hepatocytes. S-nitrosylation of GAPDH was assessed using a biotin-switch assay. Interacions of SHP with other proteins and with the Cyp7a1 promoter sequence were studied using immunoprecipitation and chromatin immunoprecipitation (ChIP) assays. We reduced the GAPDH levels in H35 cells with small interfering RNAs. GAPDH nitrosylation was assessed in normal and cholestatic rat and human livers., Results: Rats placed on cholate-rich diets and given L-NAME had increased intrahepatic and biliary levels of bile salts, and deficiency in repression of CYP7A1 (at the messenger RNA and protein levels) in liver tissue, despite preserved induction of SHP. In cultured hepatocytes, L-NAME or dithiothreitol blocked cholate-induced down-regulation of CYP7A1 without impairing SHP up-regulation. In hepatocytes, cholate promoted S-nitrosylation of GAPDH and its translocation to the nucleus, accompanied by S-nitrosylation of histone deacetylase 2 (HDAC2) and Sirtuin 1 (SIRT1), deacetylases that participate, respectively, in the formation of Cyp7a1 and Shp repressor complexes. Knockdown of GAPDH prevented repression of CYP7A1 by cholate, and blocking nuclear transport of nitrosylated GAPDH reduced cholate-induced nitrosylation of HDAC2 and SIRT1; this effect was accompanied by abrogation of Cyp7a1 repression. Cholate induced binding of SHP to HDAC2 and its recruitment to the Cyp7a1 promoter; these processes were inhibited by blocking nitric oxide synthesis. Levels of nitrosylated GAPDH and nitrosylated HDAC2 were increased in cholestatic human and rat livers reflecting increased concentrations of bile salts in these conditions., Conclusions: In rat liver, excess levels of bile salts activate a GAPDH-mediated transnitrosylation cascade that provides feedback inhibition of bile salt synthesis., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The liver displays an outstanding wound healing and regenerative capacity unmatched by any other organ. This reparative response is governed by a complex network of inflammatory mediators, growth factors and metabolites that are set in motion in response to hepatocellular injury. However, when liver injury is chronic, these regenerative mechanisms become dysregulated, facilitating the accumulation of genetic alterations leading to unrestrained cell proliferation and the development of hepatocellular carcinoma (HCC). The epidermal growth factor receptor (EGFR or ErbB1) signaling system has been identified as a key player in all stages of the liver response to injury, from early inflammation and hepatocellular proliferation to fibrogenesis and neoplastic transformation. The EGFR system engages in extensive crosstalk with other signaling pathways, acting as a true signaling hub for other growth factors, cytokines and inflammatory mediators. Here, we briefly review essential aspects of the biology of the EGFR, the other ErbB receptors, and their ligands in liver injury, regeneration and HCC development. Some aspects of the preclinical and clinical experience with EGFR therapeutic targeting in HCC are also discussed.

Notwithstanding numerous evidences implicating toll-like receptor-4 (TLR4) in the pathogenesis of chronic hepatitis C virus (HCV) infection, the localization and level of TLR4 expression in the liver of patients with hepatitis C have never been investigated. We aimed to evaluate, by means of immunohistochemistry and real-time PCR (rt-PCR), hepatic TLR4 expression in patients with chronic HCV infection. Fifty patients who had undergone liver biopsy and 11 patients transplanted because of chronic HCV infection, and 12 controls free of liver disease, were included in the study. Each case was analyzed by immunohistochemistry for TLR4, α-smooth muscle actin and cytokeratin-7 (CK-7), and a subgroup of patients and all controls by rt-PCR for TLR4. Immunohistochemistry for α-smooth muscle actin was used to derive a score of activation of hepatic stellate cells and portal/septal myofibroblasts, while immunohistochemistry for CK-7 was used to evaluate and count hepatic progenitor cells, interlobular bile ducts and intermediate hepatocytes. In patients, the parenchymal elements responsible for the highest TLR4 level of expression were hepatic progenitor cells and biliary epithelial cells of interlobular bile ducts. Double-labeling experiments between anti-TLR4 and anti-CK7, anti-CD133, anti-CD44, anti-neural cell adhesion molecule, anti-epithelial cell adhesion molecule and anti-sex determining region Y-box 9, confirmed these findings. TLR4-positive hepatic progenitor cells and interlobular bile ducts were significantly correlated with the stage of liver disease (P<0.001), the grade of inflammation (P<0.001), and the activity of portal/septal myofibroblasts (P<0.001). rt-PCR study confirmed an increased TLR4 expression in the 26 patients analyzed with respect to controls (P<0.001). TLR4 expression positively correlated with fibrosis (P<0.05) and inflammation (P<0.05). The present results suggest that TLR4 expression by hepatic progenitor cells and biliary epithelial cells contributes to the progression of liver damage in the course of chronic HCV-related infection.

Background/aims: The modulation of the hepatic acute-phase reaction (APR) that occurs during inflammation and liver regeneration is important for allowing normal hepatocellular proliferation and the restoration of homeostasis. Activation of acute-phase protein (APP) gene expression by interleukin-6 (IL-6)-type cytokines is thought to be counteracted by growth factors released during hepatic inflammation and regeneration. The epidermal growth factor receptor (EGFR) ligand amphiregulin (AR) is readily induced by inflammatory signals and plays a nonredundant protective role during liver injury. In this paper, we investigated the role of AR as a modulator of liver APP gene expression., Methods: Expression of APP genes was measured in the livers of AR(+/+) and AR(-/-)mice during inflammation and regeneration and in cultured liver cells treated with AR and oncostatin M (OSM). Crosstalk between AR and OSM signalling was studied., Results: APP genes were overexpressed in the livers of AR(-/-) mice during inflammation and hepatocellular regeneration. In cultured AR-null hepatocytes and human hepatocellular carcinoma (HCC) cells after AR knockdown, APP gene expression is enhanced. AR counteracts OSM-triggered signal transducer and activator of transcription 3 signalling in hepatocytes and attenuates APP gene transcription., Conclusions: Our data support the relevance of EGFR-mediated signalling in the modulation of cytokine-activated pathways. We have identified AR as a key regulator of hepatic APP gene expression during inflammation and liver regeneration.

Activation of the epidermal growth factor receptor (EGFR) plays an important role in liver regeneration and resistance to acute injury. However its chronic activation participates in the progression of liver disease, including fibrogenesis and malignant transformation. Hepatobiliary disease represents a constant feature in the clinically relevant Fechm1pas/Fechm1pas genetic model of erythropoietic protoporphyria (EPP). Similarly, chronic administration of griseofulvin to mice induces pathological changes similar to those found in patients with EPP-associated liver injury. We investigated the hepatic expression of the EGFR and its seven most relevant ligands in Fechm1pas/Fechm1pas mice bred in three different backgrounds, and in griseofulvin-induced protoporphyria. We observed that the expression of amphiregulin, betacellulin and epiregulin was significantly increased in young EPP mice when compared to aged-matched controls in all genetic backgrounds. The expression of these ligands was also tested in older (11 months) BALB/cJ EPP mice, and it was found to remain induced, while that of the EGFR was downregulated. Griseofulvin feeding also increased the expression of amphiregulin, betacellulin and epiregulin. Interestingly, protoporphyrin accumulation in cultured hepatic AML-12 cells readily elicited the expression of these three EGFR ligands. Our findings suggest that protoporphyrin could directly induce the hepatic expression of EGFR ligands, and that their chronic upregulation might participate in the pathogenesis of EPP-associated liver disease.

Unlabelled: The hepatic wound-healing response to chronic noxious stimuli may lead to liver fibrosis, a condition characterized by excessive deposition of extracellular matrix. Fibrogenic cells, including hepatic stellate cells and myofibroblasts, are activated in response to a variety of cytokines, growth factors, and inflammatory mediators. The involvement of members of the epidermal growth factor family in this process has been suggested. Amphiregulin (AR) is an epidermal growth factor receptor (EGFR) ligand specifically induced upon liver injury. Here, we have addressed the in vivo role of AR in experimental liver fibrosis. To this end, liver fibrosis was induced in AR+/+ and AR-/- mice by chronic CCl(4) administration. Histological and molecular markers of hepatic fibrogenesis were measured. Additionally, the response of cultured human and mouse liver fibrogenic cells to AR was evaluated. We observed that AR was expressed in isolated Kupffer cells and liver fibrogenic cells in response to inflamatory stimuli and platelet-derived growth factor, respectively. We demonstrate that the expression of alpha-smooth muscle actin and collagen deposition were markedly reduced in AR-/- mice compared to AR+/+ animals. AR-/- mice also showed reduced expression of tissue inhibitor of metalloproteinases-1 and connective tissue growth factor, two genes that responded to AR treatment in cultured fibrogenic cells. AR also stimulated cell proliferation and exerted a potent antiapoptotic effect on isolated fibrogenic cells., Conclusion: These results indicate that among the different EGFR ligands, AR plays a specific role in liver fibrosis. AR may contribute to the expression of fibrogenic mediators, as well as to the growth and survival of fibrogenic cells. Additionally, our data lend further support to the role of the EGFR system in hepatic fibrogenesis.

Methionine adenosyltransferase (MAT) is an essential enzyme because it catalyzes the formation of S-adenosylmethionine, the main methyl donor. Two MAT-encoding genes (MAT1A, MAT2A) are found in mammals. The latter is expressed in proliferating liver, dedifferentiation and cancer, whereas MAT1A is expressed in adult quiescent hepatocytes. Here, we report studies on the molecular mechanisms controlling the induction of MAT2A in regenerating rat liver and in proliferating hepatocytes. The MAT2A is up-regulated at two discrete moments during liver regeneration, as confirmed by RNApol-ChIP analysis. The first one coincides with hepatocyte priming (i.e. G0-G1 transition), while the second one takes place at the G1-S interface. Electrophoretic mobility shift assays showed that a putative E2F sequence present in MAT2A promoter binds this factor and ChIP assays confirmed that E2F1, E2F3 and E2F4, as well as the pocket protein p130, are bound to the promoter in quiescent liver. MAT2A activation is accompanied by changes in the binding of histone-modifying enzymes to the promoter. Interestingly, p130 is not displaced from MAT2A promoter during hepatocyte priming, but it is in the late expression of the gene at the G1-S transition. Finally, the transcription factor Sp1 seems to play a decisive role in MAT2A induction, as it binds the promoter when the gene is being actively transcribed. In summary, the present work shows that the molecular mechanism of MAT2A expression is different during G0-G1 or G1-S transition and this may be related to the distinct requirements of S-adenosylmethionine during liver regeneration.

Methionine (Met) metabolism involves the sequential formation of S-adenosylmethionine (SAM, the main biological methyl donor), S-adenosylhomocysteine (SAH) and homocysteine (Hcy). Hcy can be remethylated to Met or catabolized through the trans-sulfuration pathway. In mammals, as much as 48% of Met metabolism and up to 85% of all transmethylation reactions occur in the liver. These figures underscore the central role played by this organ in Met metabolism. Maintaining the homeostasis of this metabolic cycle has proved to be essential for the preservation of liver function up to the point of preventing its neoplastic transformation. However, an adequate hepatic metabolism of Met is not only important for the liver parenchymal cell. Evidence has accumulated over the past few years supporting the involvement of Met-derived metabolites in the triggering or attenuation of pathological processes with systemic implications. This is best illustrated by the fact that a deteriorated liver function has emerged as a major factor in the development of hyperhomocysteinemia. Elevated plasma levels of Hcy have been related to several disorders including cardiovascular and cerebrovascular diseases. On the other end, liver damage also leads to deficient SAM synthesis. Among the consequences of impaired SAM synthesis in liver tissue are the enhanced production of pro-inflammatory cytokines and mediators. In this review, we will address the mechanisms and consequences of abnormal Met metabolism in liver injury, the systemic implications of such impairment and finally the potential therapeutic interventions.

Clinically, the Fas and Fas ligand system plays a central role in the development of hepatocyte apoptosis, a process contributing to a broad spectrum of liver diseases. Therefore, the development of therapies aimed at the inhibition of hepatocyte apoptosis is a major issue. Activation of the epidermal growth factor receptor has been shown to convey survival signals to the hepatocyte. To learn about the endogenous response of epidermal growth factor receptor ligands during Fas-mediated liver injury we investigated the expression of epidermal growth factor, transforming growth factor alpha, heparin-binding epidermal growth factor-like growth factor, betacellulin, epiregulin, and amphiregulin in the liver of mice challenged with Fas-agonist antibody. Amphiregulin expression, barely detectable in healthy liver, was significantly up-regulated. Amphiregulin administration abrogated Fas-mediated liver injury in mice and showed direct anti-apoptotic effects in primary hepatocytes. Amphiregulin activated the Akt and signal transducer and activator of transcription-3 survival pathways, and up-regulated Bcl-xL expression. Amphiregulin knock-out mice showed signs of chronic liver damage in the absence of any noxious treatment, and died faster than wild type mice in response to lethal doses of Fas-agonist antibody. In contrast, these mice were more resistant against sublethal liver damage, supporting the hypothesis that chronic liver injury can precondition hepatocytes inducing resistance to subsequent cell death. These results show that amphiregulin is a protective factor induced in response to liver damage and that it may be therapeutic in liver diseases.

Methionine adenosyltransferase (MAT) is an essential enzyme because it catalyzes the formation of S-adenosylmethionine (SAMe), the principal biological methyl donor. Of the two genes that encode MAT, MAT1A is mainly expressed in adult liver and MAT2A is expressed in all extrahepatic tissues. Mice lacking MAT1A have reduced hepatic SAMe content and spontaneously develop hepatocellular carcinoma. The current study examined the influence of chronic hepatic SAMe deficiency on liver regeneration. Despite having higher baseline hepatic staining for proliferating cell nuclear antigen, MAT1A knockout mice had impaired liver regeneration after partial hepatectomy (PH) as determined by bromodeoxyuridine incorporation. This can be explained by an inability to up-regulate cyclin D1 after PH in the knockout mice. Upstream signaling pathways involved in cyclin D1 activation include nuclear factor kappaB (NFkappaB), the c-Jun-N-terminal kinase (JNK), extracellular signal-regulated kinases (ERKs), and signal transducer and activator of transcription-3 (STAT-3). At baseline, JNK and ERK are more activated in the knockouts whereas NFkappaB and STAT-3 are similar to wild-type mice. Following PH, early activation of these pathways occurred, but although they remained increased in wild-type mice, c-jun and ERK phosphorylation fell progressively in the knockouts. Hepatic SAMe levels fell progressively following PH in wild-type mice but remained unchanged in the knockouts. In culture, MAT1A knockout hepatocytes have higher baseline DNA synthesis but failed to respond to the mitogenic effect of hepatocyte growth factor. Taken together, our findings define a critical role for SAMe in ERK signaling and cyclin D1 regulation during regeneration and suggest chronic hepatic SAMe depletion results in loss of responsiveness to mitogenic signals.

S-adenosylmethionine (SAM) is an essential metabolite in all cells. SAM is the most important biological methyl group donor and is a precursor in the synthesis of polyamines. Methionine adenosyltransferase (MAT; EC 2.5.1.6) catalyzes the only known SAM biosynthetic reaction from methionine and ATP. In mammalian tissues, three different forms of MAT (MAT I, MAT III and MAT II) have been identified that are the product of two different genes (MAT1A and MAT2A). Although MAT2A is expressed in all mammalian tissues, the expression of MAT1A is primarily restricted to adult liver. In mammals, up to 85% of all methylation reactions and as much as 48% of methionine metabolism occurs in the liver, which indicates the important role of this organ in the regulation of blood methionine. Recent evidence indicates that not only is SAM the main biological methyl group donor and an intermediate metabolite in methionine catabolism, but it is also an intracellular control switch that regulates essential hepatic functions such as liver regeneration and differentiation as well as the sensitivity of this organ to injury. Therefore, knowledge of factors that regulate the activity of MAT I/III, the specific liver enzyme, is essential to understand how cellular SAM levels are controlled.

Dietary methionine is mainly metabolized in the liver where it is converted into S-adenosylmethionine (AdoMet), the main biologic methyl donor. This reaction is catalyzed by methionine adenosyltransferase I/III (MAT I/III), the product of MAT1A gene, which is exclusively expressed in this organ. It was first observed that serum methionine levels were elevated in experimental models of liver damage and in liver cirrhosis in human beings. Results of further studies showed that this pathological alteration was due to reduced MAT1A gene expression and MAT I/III enzyme inactivation associated with liver injury. Synthesis of AdoMet is essential to all cells in the organism, but it is in the liver where most of the methylation reactions take place. The central role played by AdoMet in cellular function, together with the observation that AdoMet administration reduces liver damage caused by different agents and improves survival of alcohol-dependent patients with cirrhosis, led us to propose that alterations in methionine metabolism could play a role in the onset of liver disease and not just be a consequence of it. In the present work, we review the recent findings that support this hypothesis and highlight the mechanisms behind the hepatoprotective role of AdoMet.

We have previously shown that the administration of low doses of insulin-like growth factor-I (IGF-I) to CCl4-cirrhotic rats improves liver function and reduces fibrosis. To better understand the mechanisms behind the hepatoprotective effects of IGF-I, and to identify those genes whose expression is affected in cirrhosis and after IGF-1 treatment, we have performed differential display of mRNA analysis by means of polymerase chain reaction (PCR) in livers from control and CCl4-cirrhotic rats treated or not with IGF-I. We have identified 16 genes that were up- or down-regulated in the cirrhotic liver. IGF-I treatment partially normalized the expression of eight of these genes, including serine proteinase inhibitors such as serpin-2 and alpha-1-antichymotripsin, alpha-1-acid glycoprotein, and alpha-2u-globulin. Additionally, we show that IGF-I enhanced the regenerative activity in the cirrhotic liver, as determined by the increased expression of the proliferating cell nuclear antigen (PCNA). Finally, IGF-I treatment partially restored the expression of growth hormone receptor (GHR) and the levels of global genomic DNA methylation, which are reduced in human and experimental cirrhosis. Taken together, our observations confirm the hepatoprotective effects of IGF-I, and suggest that this action can be exerted in part through the normalization of liver gene expression, growth hormone (GH) responsiveness and global genomic DNA methylation.

Genome sequence analysis reveals that all organisms synthesize S-adenosylmethionine (AdoMet) and that a large fraction of all genes is AdoMet-dependent methyltransferases. AdoMet-dependent methylation has been shown to be central to many biological processes. Up to 85% of all methylation reactions and as much as 48% of methionine metabolism occur in the liver, which indicates the crucial importance of this organ in the regulation of blood methionine. Of the two mammalian genes (MAT1A, MAT2A) that encode methionine adenosyltransferase (MAT, the enzyme that makes AdoMet), MAT1A is specifically expressed in adult liver. It now appears that growth factors, cytokines, and hormones regulate liver MAT mRNA levels and enzyme activity and that AdoMet should not be viewed only as an intermediate metabolite in methionine catabolism, but also as an intracellular control switch that regulates essential hepatic functions such as regeneration, differentiation, and the sensitivity of this organ to injury. The aim of this review is to integrate these recent findings linking AdoMet with liver growth, differentiation, and injury into a comprehensive model. With the availability of AdoMet as a nutritional supplement and evidence of its beneficial role in various liver diseases, this review offers insight into its mechanism of action.

Background/aims: Hepatocellular availability of S-adenosylmethionine, the principal biological methyl donor, is compromised in situations of liver damage. S-Adenosylmethionine administration alleviates experimental liver injury and increases survival in cirrhotic patients. The mechanisms behind these beneficial effects of S-adenosylmethionine are not completely known. An inflammatory component is common to many of the pathological conditions in which S-adenosylmethionine grants protection to the liver. This notion led us to study the effect of S-adenosylmethionine administration on hepatic nitric oxide synthase-2 induction in response to bacterial lipopolysaccharide and proinflammatory cytokines., Methods: The effect of S-adenosylmethionine on nitric oxide synthase-2 expression was assessed in rats challenged with bacterial lipopolysaccharide and in isolated rat hepatocytes treated with proinflammatory cytokines. Interactions between S-adenosylmethionine and cytokines on nuclear factor kappa B activation and nitric oxide synthase-2 promoter transactivation were studied in isolated rat hepatocytes and HepG2 cells, respectively., Results: S-Adenosylmethionine attenuated the induction of nitric oxide synthase-2 in the liver of lipopolysaccharide-treated rats and in cytokine-treated hepatocytes. S-Adenosylmethionine accelerated the resynthesis of inhibitor kappa B alpha, blunted the activation of nuclear factor kappa B and reduced the transactivation of nitric oxide synthase-2 promoter., Conclusions: Our findings indicate that the hepatoprotective actions of S-adenosylmethionine may be mediated in part through the modulation of nitric oxide production.

Methionine adenosyltransferase (MAT) is an essential enzyme that catalyzes the synthesis of S-adenosylmethionine (AdoMet), the most important biological methyl donor. Liver MAT I/III is the product of the MAT1A gene. Hepatic MAT I/III activity and MAT1A expression are compromised under pathological conditions such as alcoholic liver disease and hepatic cirrhosis, and this gene is silenced upon neoplastic transformation of the liver. In the present work, we evaluated whether MAT1A expression could be targeted by the polycyclic arylhydrocarbon (PAH) 3-methylcholanthrene (3-MC) in rat liver and cultured hepatocytes. MAT1A mRNA levels were reduced by 50% following in vivo administration of 3-MC to adult male rats (100 mg/kg, p.o., 4 days' treatment). This effect was reproduced in a time- and dose-dependent fashion in cultured rat hepatocytes, and was accompanied by the induction of cytochrome P450 1A1 gene expression. This action of 3-MC was mimicked by other PAHs such as benzo[a]pyrene and benzo[e]pyrene, but not by the model arylhydrocarbon receptor (AhR) activator 2,3,7,8-tetrachlorodibenzo-p-dioxin. 3-MC inhibited transcription driven by a MAT1A promoter-reporter construct transfected into rat hepatocytes, but MAT1A mRNA stability was not affected. We recently showed that liver MAT1A expression is induced by AdoMet in cultured hepatocytes. Here, we observed that exogenously added AdoMet prevented the negative effects of 3-MC on MAT1A expression. Taken together, our data demonstrate that liver MAT1A gene expression is targeted by PAHs, independently of AhR activation. The effect of AdoMet may be part of the protective action of this molecule in liver damage.

Methionine metabolism starts with the formation of S-adenosylmethionine (AdoMet), the most important biological methyl donor. This reaction is catalyzed by methionine adenosyltransferase (MAT). MAT is the product of two different genes: MAT1A, which is expressed only in the adult liver, and MAT2A, which is widely distributed, expressed in the fetal liver, and replaces MAT1A in hepatocarcinoma. In the liver, preservation of high expression of MAT1A and low expression of MAT2A is critical for the maintenance of a functional and differentiated organ. Here we describe that in cultured rat hepatocytes MAT1A expression progressively decreased, as described for other liver-specific genes, and MAT2A expression was induced. We find that this switch in gene expression was prevented by adding AdoMet to the culture medium. We also show that in cultured hepatocytes with decreased MAT1A expression AdoMet addition markedly increased MAT1A transcription in a dose-dependent fashion. This effect of AdoMet was mimicked by methionine, and blocked by 3-deazaadenosine and L-ethionine, but not D-ethionine, indicating that the effect was specific and mediated probably by a methylation reaction. These findings identify AdoMet as a key molecule that differentially regulates MAT1A and MAT2A expression and helps to maintain the differentiated status of the hepatocyte.

Methionine adenosyltransferase (MAT) is the enzyme that catalyzes the synthesis of S-adenosylmethionine (AdoMet), the main donor of methyl groups in the cell. In mammals MAT is the product of two genes, MAT1A and MAT2A. MAT1A is expressed only in the mature liver whereas fetal hepatocytes, extrahepatic tissues and liver cancer cells express MAT2A. The mechanisms behind the tissue and differentiation state specific MAT1A expression are not known. In the present work we examined MAT1A promoter methylation status by means of methylation sensitive restriction enzyme analysis. Our data indicate that MAT1A promoter is hypomethylated in liver and hypermethylated in kidney and fetal rat hepatocytes, indicating that this modification is tissue specific and developmentally regulated. Immunoprecipitation of mononucleosomes from liver and kidney tissues with antibodies mainly specific to acetylated histone H4 and subsequent Southern blot analysis with a MAT1A promoter probe demonstrated that MAT1A expression is linked to elevated levels of chromatin acetylation. Early changes in MAT1A methylation are already observed in the precancerous cirrhotic livers from rats, which show reduced MAT1A expression. Human hepatoma cell lines in which MAT1A is not expressed were also hypermethylated at this locus. Finally we demonstrate that MAT1A expression is reactivated in the human hepatoma cell line HepG2 treated with 5-aza-2'-deoxycytidine or the histone deacetylase inhibitor trichostatin, suggesting a role for DNA hypermethylation and histone deacetylation in MAT1A silencing.

Elevated plasma levels of homocysteine have been shown to interfere with normal cell function in a variety of tissues and organs, such as the vascular wall and the liver. However, the molecular mechanisms behind homocysteine effects are not completely understood. In order to better characterize the cellular effects of homocysteine, we have searched for changes in gene expression induced by this amino acid. Our results show that homocysteine is able to induce the expression and synthesis of the tissue inhibitor of metalloproteinases-1 (TIMP-1) in a variety of cell types ranging from vascular smooth muscle cells to hepatocytes, HepG2 cells and hepatic stellate cells. In this latter cell type, homocysteine also stimulated alpha 1(I) procollagen mRNA expression. TIMP-1 induction by homocysteine appears to be mediated by its thiol group. Additionally, we demonstrate that homocysteine is able to promote activating protein-1 (AP-1) binding activity, which has been shown to be critical for TIMP-1 induction. Our findings suggest that homocysteine may alter extracellular matrix homeostasis on diverse tissular backgrounds besides the vascular wall. The liver could be considered as another target for such action of homocysteine. Consequently, the elevated plasma levels of this amino acid found in different pathological or nutritional circumstances may cooperate with other agents, such as ethanol, in the onset of liver fibrosis.

Uncoupling protein 2 (UCP2) expression in liver is restricted to non-parenchymal cells. By means of differential display screening between normal rat liver and H4IIE hepatoma cells we have isolated a cDNA clone encompassing part of UCP2 cDNA. Northern blot analysis revealed that UCP2 is expressed in some hepatocarcinoma cell lines, while it is absent in adult hepatocytes. UCP2 mRNA in H4IIE cells was downregulated when cells were cultured for 36 h in 0.1% serum and its expression was restored upon addition of 10% serum or phorbol esters. Hypomethylation of UCP2 was observed in transformed UCP2 expressing cells. Our results indicate that UCP2 is expressed in some hepatocarcinoma cell lines and that serum components may participate in maintaining elevated UCP2 levels.

Background & Aims: Oxygen supply to the hepatic parenchyma is compromised by long- or short-term ethanol consumption and pathological conditions such as cirrhosis. Impairment in the production of S-adenosyl-L-methionine, the major methylating agent, occurs during hypoxia. In this study, the molecular mechanisms implicated in the regulation of S-adenosyl-L-methionine synthesis by oxygen levels were investigated., Methods: Rat hepatocytes were isolated and cultured under normoxic (21% O2) or hypoxic (3% O2) conditions for different periods. Methionine adenosyltransferase activity, messenger RNA levels, and nuclear transcription were evaluated., Results: Methionine adenosyltransferase was inactivated in hepatocytes kept under low oxygen levels. Hypoxia induced the expression of nitric oxide (NO) synthase, and the inactivation of methionine adenosyltransferase was prevented by the NO synthase inhibitor N(G)-monomethyl-L-arginine methyl ester. Methionine adenosyltransferase messenger RNA levels were down-regulated by hypoxia, through a mechanism that might involve a hemoprotein. Hypoxia dramatically reduced methionine adenosyltransferase gene transcription, and messenger stability was also decreased, although to a lesser extent., Conclusions: We have established the molecular basis for the regulation of methionine adenosyltransferase activity and gene expression by hypoxia. NO-mediated inactivation and transcriptional arrest seem to be the two major pathways by which oxygen levels control hepatic methionine adenosyltransferase, the enzyme necessary for methylation reactions and for the synthesis of polyamines and glutathione.

Although free radicals have been traditionally implicated in cell injury, and associated to pathophysiological processes, recent data implicate them in cell signaling events. Free radicals are naturally occurring oxygen-,nitrogen-and sulfur-derived species with an unpaired electron, such as superoxide, hydroxyl radical or nitric oxide. In order to assess the role of free radicals in cell signaling, we have studies the modulator effect of oxygen and nitrogen active species on liver methionine adenosyltransferase (MAT), a key metabolic enzyme. The presence of 10 cysteine residues per subunit, makes liver MAT a sensitive target for oxidation/nitrosylation. Here we show that purified MAT from rat liver is nitrosylated and oxidized in vitro. Incubation with H202 or the NO donor S-nitrosylated GSH (GSNO), diminish MAT activity in a dose-and time-dependent manner. Furthermore, the inactivation derived from both oxidation and nitrosylation, was reverted by GSH. MAT inactivation originates on the specific and covalent modification of the sulphydryl group of cysteine residue 121. We also studied how free radicals modulate MAT activity in vivo. It was previously shown that MAT activity is strongly dependent on cellular GSH levels. Generation of oxygen and nitrogen active species in rats by injection of LPS, induced a decrease of liver MAT activity. This effect might derive from nitrosylation and/or oxidation of the enzyme. Modulation of liver MAT by NO is further supported by the inactivation of this enzyme observed in experimental models in which NO is produced; such as the administration of NO donors to rats and in hepatocytes cultured in hypoxia, a condition that induces the expression of the inducible nitric oxide synthase (iNOS). Oxidation also controls liver MAT activity in a cell environment as shown in CHO cells stably transfected with rat liver MAT cDNA upon addition of H2O2 to the culture medium. This effect depends upon the generation of the hydroxyl radical. On the basis of the metabolic implications of liver MAT, together with the structural features accounting for the sensitivity of this enzyme to active oxygen and nitrogen species, we propose that modulation of MAT by these agents could be a mechanism to regulate the consumption of ATP in the liver, and thus preserve cellular viability under different stress conditions.

We investigated the modulation of rat liver S-adenosylmethionine (SAM) synthetase in a model of acute sepsis. Our results show that animals treated with bacterial lipopolysaccharide experience a marked decrease in liver SAM synthetase activity. No changes were detected in the hepatic levels of SAM synthetase protein, suggesting that inactivation of the existing enzyme was the cause of the observed activity loss. Lipopolysaccharide treatment resulted in the expression of calcium-independent/cytokine-inducible nitric oxide (NO) synthase in liver and the accumulation in plasma of the NO-derived species nitrite and nitrate. NO implication in the in vivo regulation of SAM synthetase was evaluated in animals treated with the NO donor molecule 3-morpholinosydnonimine. The analysis of liver enzymatic activity, along with protein and messenger RNA levels yielded results similar to those obtained with lipopolysaccharide treatment. To assess directly the sensitivity of SAM synthetase to NO, the rat liver-purified high- and low-molecular weight forms of the enzyme were exposed to various doses of 3-morpholinosydnonimine and other NO donors such as S-nitroso-N-acetylpenicillamine, resulting in a dose-dependent inhibition of enzymatic activity. This effect was reversed by addition of the reducing agents beta-mercaptoethanol and glutathione. Finally, cysteine 121 was identified as the site of molecular interaction between NO and rat liver SAM synthetase that is responsible for the inhibition of the enzyme. To reach this conclusion, the 10 cysteine residues of the enzyme were changed to serine by site-directed mutagenesis, and the effect of NO on the various recombinant enzymes was measured.

The ability of an inositol phosphate-glycan (IPG) to mimic the effects of insulin on regulation of the expression of specific mRNAs was studied in isolated hepatocytes from normal and diabetic rats. Incubation of normal liver cells with IPG (10 microM) during 90 min produced a 5-fold decrease in phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels, which had been previously increased about 10-fold by incubation with 8-bromo-cAMP (0.1 mM). The effect of IPG was dose dependent and could not be reproduced by galactose, glucosamine, or myo-inositol. IPG reduction of PEPCK mRNA is primarily due to a decrease in the rate of transcription of the gene, as judged by nuclear run-on transcription experiments performed in rat hepatoma H4IIE cells. In hepatocytes isolated from diabetic rats, treatment with 5 microM IPG for 15 min caused a 4-fold induction in the expression of alpha 2-microglobulin mRNA concomitantly with a 2.5-fold decrease in the level of PEPCK mRNA. Cleavage of IPG with nitrous acid abolished both the increase and the decrease in specific mRNAs levels. Glycosyl-phosphatidylinositol, the lipid precursor of IPG, did not modify either PEPCK or alpha 2-microglobulin mRNA levels. These data indicate that both positive and negative effects of insulin on the regulation of gene expression are mimicked by IPG.