NO sensitizes rat hepatocytes to proliferation by modifying S-adenosylmethionine levels

BACKGROUND & AIMS: Liver regeneration is a fundamental response of this organ to injury. Hepatocyte proliferation is triggered by growth factors, such as hepatocyte growth factor. However, hepatocytes need to be primed to react to mitogenic signals. It is known that nitrous oxide (NO), generated after partial hepatectomy, plays an important role in hepatocyte growth. Nevertheless, the molecular mechanisms behind this priming event are not completely known. S-adenosylmethionine (AdoMet) synthesis by methionine adenosyltransferase is the first step in methionine metabolism, and NO regulates hepatocyte S-adenosylmethionine levels through specific inhibition of this enzyme. We have studied the modulation of hepatocyte growth factor-induced proliferation by NO through the regulation of S-adenosylmethionine levels. METHODS: Studies were conducted in cultured rat hepatocytes isolated by collagenase perfusion, which triggers NO synthesis. RESULTS: The mitogenic response to hepatocyte growth factor was blunted when inducible NO synthase was inhibited; this process was overcome by the addition of an NO donor. This effect was dependent on methionine concentration in culture medium and intracellular S-adenosylmethionine levels. Accordingly, we found that S-adenosylmethionine inhibits hepatocyte growth factor-induced cyclin D1 and D2 expression, activator protein 1 induction, and hepatocyte proliferation. CONCLUSIONS: Together our findings indicate that NO may switch hepatocytes into a hepatocyte growth factor-responsive state through the down-regulation of S-adenosylmethionine levels

L-methionine availability regulates expression of the methionine adenosyltransferase 2A gene in human hepatocarcinoma cells: role of S-adenosylmethionine

In mammals, methionine adenosyltransferase (MAT), the enzyme responsible for S-adenosylmethionine (AdoMet) synthesis, is encoded by two genes, MAT1A and MAT2A. In liver, MAT1A expression is associated with high AdoMet levels and a differentiated phenotype, whereas MAT2A expression is associated with lower AdoMet levels and a dedifferentiated phenotype. In the current study, we examined regulation of MAT2A gene expression by l-methionine availability using HepG2 cells. In l-methionine-deficient cells, MAT2A gene expression is rapidly induced, and methionine adenosyltransferase activity is increased. Restoration of l-methionine rapidly down-regulates MAT2A mRNA levels; for this effect, l-methionine needs to be converted into AdoMet. This novel action of AdoMet is not mediated through a methyl transfer reaction. MAT2A gene expression was also regulated by 5'-methylthioadenosine, but this was dependent on 5'-methylthioadenosine conversion to methionine through the salvage pathway. The transcription rate of the MAT2A gene remained unchanged during l-methionine starvation; however, its mRNA half-life was significantly increased (from 100 min to more than 3 h). The effect of l-methionine withdrawal on MAT2A mRNA stabilization requires both gene transcription and protein synthesis. We conclude that MAT2A gene expression is modulated as an adaptive response of the cell to l-methionine availability through its conversion to AdoMet

GARBAN: genomic analysis and rapid biological annotation of cDNA microarray and proteomic data

Genomic Analysis and Rapid Biological ANnotation (GARBAN) is a new tool that provides an integrated framework to analyze simultaneously and compare multiple data sets derived from microarray or proteomic experiments. It carries out automated classifications of genes or proteins according to the criteria of the Gene Ontology Consortium at a level of depth defined by the user. Additionally, it performs clustering analysis of all sets based on functional categories or on differential expression levels. GARBAN also provides graphical representations of the biological pathways in which all the genes/proteins participate. AVAILABILITY: http://garban.tecnun.es

Methionine adenosyltransferase II beta subunit gene expression provides a proliferative advantage in human hepatoma

BACKGROUND & AIMS: Of the 2 genes (MAT1A, MAT2A) encoding methionine adenosyltransferase, the enzyme that synthesizes S-adenosylmethionine, MAT1A, is expressed in liver, whereas MAT2A is expressed in extrahepatic tissues. In liver, MAT2A expression associates with growth, dedifferentiation, and cancer. Here, we identified the beta subunit as a regulator of proliferation in human hepatoma cell lines. The beta subunit has been cloned and shown to lower the K(m) of methionine adenosyltransferase II alpha2 (the MAT2A product) for methionine and to render the enzyme more susceptible to S-adenosylmethionine inhibition. METHODS: Methionine adenosyltransferase II alpha2 and beta subunit expression was analyzed in human and rat liver and hepatoma cell lines and their interaction studied in HuH7 cells. beta Subunit expression was up- and down-regulated in human hepatoma cell lines and the effect on DNA synthesis determined. RESULTS: We found that beta subunit is expressed in rat extrahepatic tissues but not in normal liver. In human liver, beta subunit expression associates with cirrhosis and hepatoma. beta Subunit is expressed in most (HepG2, PLC, and Hep3B) but not all (HuH7) hepatoma cell lines. Transfection of beta subunit reduced S-adenosylmethionine content and stimulated DNA synthesis in HuH7 cells, whereas down-regulation of beta subunit expression diminished DNA synthesis in HepG2. The interaction between methionine adenosyltransferase II alpha2 and beta subunit was demonstrated in HuH7 cells. CONCLUSIONS: Our findings indicate that beta subunit associates with cirrhosis and cancer providing a proliferative advantage in hepatoma cells through its interaction with methionine adenosyltransferase II alpha2 and down-regulation of S-adenosylmethionine levels

S-Adenosylmethionine revisited: its essential role in the regulation of liver function

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

S-adenosylmethionine and methylthioadenosine are antiapoptotic in cultured rat hepatocytes but proapoptotic in human hepatoma cells

S-adenosylmethionine (AdoMet) is an essential compound in cellular transmethylation reactions and a precursor of polyamine and glutathione synthesis in the liver. In liver injury, the synthesis of AdoMet is impaired and its availability limited. AdoMet administration attenuates experimental liver damage, improves survival of alcoholic patients with cirrhosis, and prevents experimental hepatocarcinogenesis. Apoptosis contributes to different liver injuries, many of which are protected by AdoMet. The mechanism of AdoMet's hepatoprotective and chemopreventive effects are largely unknown. The effect of AdoMet on okadaic acid (OA)-induced apoptosis was evaluated using primary cultures of rat hepatocytes and human hepatoma cell lines. AdoMet protected rat hepatocytes from OA-induced apoptosis dose dependently. It attenuated mitochondrial cytochrome c release, caspase 3 activation, and poly(ADP-ribose) polymerase cleavage. These effects were independent from AdoMet-dependent glutathione synthesis, and mimicked by 5'-methylthioadenosine (MTA), which is derived from AdoMet. Interestingly, AdoMet and MTA did not protect HuH7 cells from OA-induced apoptosis; conversely both compounds behaved as proapoptotic agents. AdoMet's proapoptotic effect was dose dependent and observed also in HepG2 cells. In conclusion, AdoMet exerts opposing effects on apoptosis in normal versus transformed hepatocytes that could be mediated through its conversion to MTA. These effects may participate in the hepatoprotective and chemopreventive properties of this safe and well-tolerated drug

5'-methylthioadenosine modulates the inflammatory response to endotoxin in mice and in rat hepatocytes

5'-methylthioadenosine (MTA) is a nucleoside generated from S-adenosylmethionine (AdoMet) during polyamine synthesis. Recent evidence indicates that AdoMet modulates in vivo the production of inflammatory mediators. We have evaluated the anti-inflammatory properties of MTA in bacterial lipopolysaccharide (LPS) challenged mice, murine macrophage RAW 264.7 cells, and isolated rat hepatocytes treated with pro-inflammatory cytokines. MTA administration completely prevented LPS-induced lethality. The life-sparing effect of MTA was accompanied by the suppression of circulating tumor necrosis factor-alpha (TNF-alpha), inducible NO synthase (iNOS) expression, and by the stimulation of IL-10 synthesis. These responses to MTA were also observed in LPS-treated RAW 264.7 cells. MTA prevented the transcriptional activation of iNOS by pro-inflammatory cytokines in isolated hepatocytes, and the induction of cyclooxygenase 2 (COX2) in RAW 264.7 cells. MTA inhibited the activation of p38 mitogen-activated protein kinase (MAPK), c-jun phosphorylation, inhibitor kappa B alpha (IkappaBalpha) degradation, and nuclear factor kappaB (NFkappaB) activation, all of which are signaling pathways related to the generation of inflammatory mediators. These effects were independent of the metabolic conversion of MTA into AdoMet and the potential interaction of MTA with the cAMP signaling pathway, central to the anti-inflammatory actions of its structural analog adenosine. In conclusion, these observations demonstrate novel immunomodulatory properties for MTA that may be of value in the management of inflammatory diseases

Regulation of mammalian liver methionine adenosyltransferase

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

Expression of insulin-like growth factor I by activated hepatic stellate cells reduces fibrogenesis and enhances regeneration after liver injury

BACKGROUND/AIM: Hepatic stellate cells (HSCs) express alpha-smooth muscle actin (alphaSMA) and acquire a profibrogenic phenotype upon activation by noxious stimuli. Insulin-like growth I (IGF-I) has been shown to stimulate HSCs proliferation in vitro, but it has been reported to reduce liver damage and fibrogenesis when given to cirrhotic rats. METHODS: The authors used transgenic mice (SMP8-IGF-I) expressing IGF-I under control of alphaSMA promoter to study the influence of IGF-I synthesised by activated HSCs on the recovery from liver injury. RESULTS: The transgene was expressed by HSCs from SMP8-IGF-I mice upon activation in culture and in the livers of these animals after CCl4 challenge. Twenty four hours after administration of CCl4 both transgenic and wild type mice showed similar extensive necrosis and increased levels of serum transaminases. However at 72 hours SMP8-IGF-I mice exhibited lower serum transaminases, reduced hepatic expression of alphaSMA, and improved liver morphology compared with wild type littermates. Remarkably, at this time all eight CCl4 treated wild type mice manifested histological signs of liver necrosis that was severe in six of them, while six out of eight transgenic animals had virtually no necrosis. In SMP8-IGF-I mice robust DNA synthesis occurred earlier than in wild type animals and this was associated with enhanced production of HGF and lower TGFbeta1 mRNA expression in the SMP8-IGF-I group. Moreover, Colalpha1(I) mRNA abundance at 72 hours was reduced in SMP8-IGF-I mice compared with wild type controls. CONCLUSIONS: Targeted overexpression of IGF-I by activated HSCs restricts their activation, attenuates fibrogenesis, and accelerates liver regeneration. These effects appear to be mediated in part by upregulation of HGF and downregulation of TGFbeta1. The data indicate that IGF-I can modulate the cytokine response to liver injury facilitating regeneration and reducing fibrosis