[show abstract][hide abstract] ABSTRACT: Resveratrol is growth suppressive and pro-apoptotic in liver cancer cells. Methionine adenosyltransferase 2B (MAT2B) encodes for two dominant variant proteins V1 and V2 that positively regulate growth and V1 is anti-apoptotic when overexpressed. Interestingly crystal structure analysis of MAT2B protein (MATβ) protomer revealed two resveratrol binding pockets, which raises the question of the role of MAT2B in resveratrol's biological activities. We found that resveratrol induced the expression of MAT2BV1 and V2 in a time and dose-dependent manner by increasing transcription, mRNA and protein stabilization. Following resveratrol treatment, HuR expression increased first, followed by SIRT1 and MAT2B. SIRT1 induction contributes to increased MAT2B transcription while HuR induction increased MAT2B mRNA stability. MATβ interacts with HuR and SIRT1 and resveratrol treatment enhanced these interactions while reducing the interaction between MATβ and MATβ2. Since MATβ lowers the Ki of MATβ2 for S-adenosylmethionine (SAMe), this allowed steady state SAMe level to rise. Interaction between MATβ, SIRT1 and HuR increased stability of these proteins. Induction of MAT2B is a compensatory response to resveratrol as knocking down MAT2BV1 potentiated resveratrol's pro-apoptotic and growth suppressive effects, while the opposite occurred with V1 overexpression. The same effect on growth occurred with MAT2BV2. In conclusion, resveratrol induces HuR, SIRT1 and MAT2B expression; the latter may represent a compensatory response against apoptosis and growth inhibition. However, MATβ induction also facilitates SIRT1 activation, as the interaction stabilizes SIRT1. This complex interplay between MATβ, HuR and SIRT1 has not been previously reported and suggest these proteins may regulate each other's signaling.
Journal of Biological Chemistry 06/2013; · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Methionine adenosyltransferase 1A (MAT1A) and glycine N-methyltransferase (GNMT) are the primary genes involved in hepatic S-adenosylmethionine (SAMe) synthesis and degradation, respectively. Mat1a ablation in mice induces a decrease in hepatic SAMe, activation of lipogenesis, inhibition of triglyceride (TG) release, and steatosis. Gnmt deficient mice, despite showing a large increase in hepatic SAMe, also develop steatosis. We hypothesized that as an adaptive response to hepatic SAMe accumulation, phosphatidylcholine (PC) synthesis via the phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway is stimulated in Gnmt(-/-) mice. We also propose that the excess PC thus generated is catabolized leading to TG synthesis and steatosis via diglyceride (DG) generation. We observed that Gnmt(-/-) mice present with normal hepatic lipogenesis and increased TG release. We also observed that the flux from PE to PC is stimulated in the liver of Gnmt(-/-) mice and that this results in a reduction in PE content and a marked increase in DG and TG. Conversely, reduction of hepatic SAMe following the administration of a methionine deficient diet reverted the flux from PE to PC of Gnmt(-/-) mice to that of wild type animals and normalized DG and TG content preventing the development of steatosis. Gnmt(-/-) mice with an additional deletion of perilipin2, the predominant lipid droplet protein, maintain high SAMe levels, with a concurrent increased flux from PE to PC, but do not develop liver steatosis. Conclusion: These findings indicate that excess SAMe reroutes PE towards PC and TG synthesis, and lipid sequestration. (HEPATOLOGY 2013.).
[show abstract][hide abstract] ABSTRACT: The basic mechanisms underlying promoter DNA hypermethylation in cancer are still largely unknown. It has been proposed that the levels of the methyl donor group in DNA methylation reactions, S-adenosylmethionine (SAMe), might be involved. SAMe levels depend on the glycine-N-methyltransferase (GNMT), a one-carbon group methyltransferase, which catalyzes the conversion of SAMe to S-adenosylhomocysteine in hepatic cells. GNMT has been proposed to display tumor suppressor activity and to be frequently repressed in hepatocellular carcinoma (HCC). In this study, we show that GNMT shows aberrant DNA hypermethylation in some HCC cell lines and primary tumors (20 %). GNMT hypermethylation could contribute to gene repression and its restoration in cell lines displaying hypermethylation-reduced tumor growth in vitro. In agreement, human primary tumors expressing GNMT were of smaller size than tumors showing GNMT hypermethylation. Genome-wide analyses of gene promoter methylation identified 277 genes whose aberrant methylation in HCC was associated with GNMT methylation/expression. The findings in this manuscript indicate that DNA hypermethylation plays an important role in the repression of GNMT in HCC and that loss of GNMT in human HCC could promote the establishment of aberrant DNA methylation patterns at specific gene promoters.
Journal of Molecular Medicine 03/2013; · 4.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: Methionine is an essential amino acid that is metabolized mainly by the liver where it is converted to Sadenosylmethionine (SAMe) by the enzyme methionine adenosyltransferase. Although all mammalian cells synthesize SAMe, the liver is where the bulk of SAMe is generated as it is the organ where about 50% of all dietary methionine is metabolized. SAMe is mainly needed for methylation of a large variety of substrates (DNA, proteins, lipids and many other small molecules) and polyamine synthesis, so if the concentration of SAMe falls below a certain level or rises too much the normal function of the liver will be also affected. There are physiological conditions that can affect the hepatic content of SAMe. Consequently, to control these fluctuations, the rate at which the liver both synthesizes and catabolizes SAMe is tightly regulated. In mice, failure to do this can lead to fatty liver disease and to the development of hepatocellular carcinoma (HCC). Therefore, maintaining SAMe homeostasis may be a therapeutic target in nonalcoholic steatohepatitis, alcoholic- and non-alcoholic liver cirrhosis, and for the chemoprevention of HCC formation.
Annals of hepatology: official journal of the Mexican Association of Hepatology 03/2013; 12(2):183-9. · 1.67 Impact Factor
[show abstract][hide abstract] ABSTRACT: OBJECTIVES:Idiopathic portal hypertension (IPH) is a rare cause of portal hypertension that lacks a specific diagnostic test. Requiring ruling-out other causes of portal hypertension it is frequently misdiagnosed. This study evaluates whether using high-throughput techniques there is a metabolomic profile allowing a noninvasive diagnosis of IPH.METHODS:Thirty-three IPH patients were included. Matched patients with cirrhosis (CH) and healthy volunteers (HV) were included as controls. Metabolomic analysis of plasma samples was performed using UPLC-time-of-flight-mass spectrometry. We computed Student's P-values, corrected by multiple comparison and VIP score (Variable Importance in the Projection). The metabolites were selected with an adjusted Benjamini Hochberg P value <0.05. We use markers with a greater VIP score, to build partial least squares projection to latent structures regression with discriminant analysis (PLS-DA) representative models to discriminate IPH from CH and from HV. The performance of the PLS-DA model was evaluated using R(2) and Q(2) parameter. An additional internal cross-validation was done.RESULTS:PLS-DA analysis showed a clear separation of IPH from CH with a model involving 28 metabolites (Q(2)=0.67, area under the curve (AUC)=0.99) and a clear separation of IPH from healthy subjects with a model including 31 metabolites (Q(2)=0.75, AUC=0.98). After cross-validation, both models showed high rates of sensitivity (94.8 and 97.5), specificity (89.1 and 89.7), and AUC (0.98 and 0.98), reinforcing the strength of our findings.CONCLUSIONS:A metabolomic profile clearly differentiating patients with IPH from CH and healthy subjects has been identified using subsets of 28 and 31 metabolites, respectively. Therefore, metabolomic analysis appears to be a valuable tool for the noninvasive diagnosis of IPH.Am J Gastroenterol advance online publication, 19 February 2013; doi:10.1038/ajg.2013.11.
The American Journal of Gastroenterology 02/2013; · 7.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: BACKGROUND & AIM: The susceptibility to develop nonalcoholic fatty liver disease (NAFLD) has genetic bases, but the associated variants are uncertain. The aim of the present study is to identify genetic variants that could help to prognose and further understand the genetics and development of NAFLD. METHODS: Allele frequencies of 3072 single-nucleotide-polymorphisms (SNPs) in 92 genes were characterized in 69 NAFLD-patients and 217 healthy-individuals. The markers that showed significant allele frequency differences in the pilot-groups were subsequently studied in 451 NAFLD-patients and 304 healthy-controls. Besides, 4414 Type-2-Diabetes-mellitus (T2DM) cases and 4567 controls were genotyped. The liver expression of the associated gene was measured and the effect of its potential role was studied by silencing the gene in-vitro. Whole genome expression, oxidative stress and the consequences of oleic-acid-enriched medium on lipid accumulation in siSLC2A1-THLE2-cells were studied by gene expression analysis, dihydroethidium-staining, BODIPY(®) and quantification of intracellular-triglycerides content, respectively. RESULTS: Several SNPs of SLC2A1 (solute carrier family 2 (facilitated glucose transporter) member 1) showed association with NAFLD but not with T2DM, being the haplotype containing the minor allele of SLC2A1-sequence related to the susceptibility to develop NAFLD. Gene expression analysis demonstrated a significant down-regulation of SLC2A1 in NAFLD-livers. Enrichment functional analyses of the transcriptome profiles drove us to demonstrate that in-vitro silencing of SLC2A1 induces an increased oxidative stress activity and a higher lipid accumulation under oleic acid treatment. CONCLUSIONS: Genetic variants of SLC2A1 are associated with NAFLD and in-vitro down-regulation of this gene promotes lipid accumulation. Moreover, the oxidative response detected in siSLC2A1-THLE2-cells corroborated the antioxidant properties previously related to this gene and linked the most representative clinical characteristics of NAFLD patients: oxidative injury and increased lipid storage. (HEPATOLOGY 2012.).
[show abstract][hide abstract] ABSTRACT: Steatohepatitis (SH) is associated with mitochondrial dysfunction and excessive production of superoxide, which can then be converted into H(2)O(2) by SOD2. Since mitochondrial GSH (mGSH) plays a critical role in H(2)O(2) reduction, we explored the interplay between superoxide, H(2)O(2), and mGSH in nutritional and genetic models of SH, which exhibit mGSH depletion.
We used isolated mitochondria and primary hepatocytes, as well as in vivo SH models showing mGSH depletion to test the consequences of superoxide scavenging.
In isolated mitochondria and primary hepatocytes, superoxide scavenging by SOD mimetics or purified SOD decreased superoxide and peroxynitrite generation but increased H(2)O(2) following mGSH depletion, despite mitochondrial peroxiredoxin/thioredoxin defense. Selective mGSH depletion sensitized hepatocytes to cell death induced by SOD mimetics, and this was prevented by RIP1 kinase inhibition with necrostatin-1 or GSH repletion with GSH ethyl ester (GSHee). Mice fed the methionine-choline deficient (MCD) diet or MAT1A(-/-) mice exhibited reduced SOD2 activity; in vivo treatment with SOD mimetics increased liver damage, inflammation, and fibrosis, despite a decreased superoxide and 3-nitrotyrosine immunoreactivity, effects that were ameliorated by mGSH replenishment with GSHee, but not NAC. As a proof-of-principle of the detrimental role of superoxide scavenging when mGSH was depleted transgenic mice overexpressing SOD2 exhibited enhanced susceptibility to MCD-mediated SH.
These findings underscore a critical role for mGSH in the therapeutic potential of superoxide scavenging in SH, and suggest that the combined approach of superoxide scavenging with mGSH replenishment may be important in SH.
Journal of Hepatology 06/2012; 57(4):852-9. · 9.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: Patients with cirrhosis are at high risk for developing hepatocellular carcinoma (HCC), and their liver tissues have abnormal levels of S-adenosylmethionine (SAMe). Glycine N-methyltransferase (GNMT) catabolizes SAMe, but its expression is down-regulated in HCC cells. Mice that lack GNMT develop fibrosis and hepatomas and have alterations in signaling pathways involved in carcinogenesis. We investigated the role of GNMT in human HCC cell lines and in liver carcinogenesis in mice.
We studied hepatoma cells from GNMT knockout mice and analyzed the roles of liver kinase B1 (LKB1, STK11) signaling via 5'-adenosine monophosphate-activated protein kinase (AMPK) and Ras in regulating proliferation and transformation.
Hepatoma cells from GNMT mice had defects in LKB1 signaling to AMPK, making them resistant to induction of apoptosis by adenosine 3',5'-cyclic monophosphate activation of protein kinase A and calcium/calmodulin-dependent protein kinase kinase 2. Ras-mediated hyperactivation of LKB1 promoted proliferation of GNMT-deficient hepatoma cells and required mitogen-activated protein kinase 2 (ERK) and ribosomal protein S6 kinase polypeptide 2 (p90RSK). Ras activation of LKB1 required expression of RAS guanyl releasing protein 3 (RASGRP3). Reduced levels of GNMT and phosphorylation of AMPKα at Thr172 and increased levels of Ras, LKB1, and RASGRP3 in HCC samples from patients were associated with shorter survival times.
Reduced expression of GNMT in mouse hepatoma cells and human HCC cells appears to increase activity of LKB1 and RAS; activation of RAS signaling to LKB1 and RASGRP3, via ERK and p90RSK, might be involved in liver carcinogenesis and be used as a prognostic marker. Reagents that disrupt this pathway might be developed to treat patients with HCC.
[show abstract][hide abstract] ABSTRACT: RNA-binding proteins (RBPs) play a major role in the control of messenger RNA (mRNA) turnover and translation rates. We examined the role of the RBP, human antigen R (HuR), during cholestatic liver injury and hepatic stellate cell (HSC) activation. HuR silencing attenuated fibrosis development in vivo after BDL, reducing liver damage, oxidative stress, inflammation, and collagen and alpha smooth muscle actin (α-SMA) expression. HuR expression increased in activated HSCs from bile duct ligation mice and during HSC activation in vitro, and HuR silencing markedly reduced HSC activation. HuR regulated platelet-derived growth factor (PDGF)-induced proliferation and migration and controlled the expression of several mRNAs involved in these processes (e.g., Actin, matrix metalloproteinase 9, and cyclin D1 and B1). These functions of HuR were linked to its abundance and cytoplasmic localization, controlled by PDGF, by extracellular signal-regulated kinases (ERK) and phosphatidylinositol 3-kinase activation as well as ERK/LKB1 (liver kinase B1) activation, respectively. More important, we identified the tumor suppressor, LKB1, as a novel downstream target of PDGF-induced ERK activation in HSCs. HuR also controlled transforming growth factor beta (TGF-β)-induced profibrogenic actions by regulating the expression of TGF-β, α-SMA, and p21. This was likely the result of an increased cytoplasmic localization of HuR, controlled by TGF-β-induced p38 mitogen-activated protein kinase activation. Finally, we found that HuR and LKB1 (Ser428) levels were highly expressed in activated HSCs in human cirrhotic samples. Conclusion: Our results show that HuR is important for the pathogenesis of liver fibrosis development in the cholestatic injury model, for HSC activation, and for the response of activated HSC to PDGF and TGF-β. (HEPATOLOGY 2012).
[show abstract][hide abstract] ABSTRACT: An important prerequisite to myelination in peripheral nerves is the establishment of one-to-one relationships between axons and Schwann cells. This patterning event depends on immature Schwann cell proliferation, apoptosis, and morphogenesis, which are governed by coordinated changes in gene expression. Here, we found that the RNA-binding protein human antigen R (HuR) was highly expressed in immature Schwann cells, where genome-wide identification of its target mRNAs in vivo in mouse sciatic nerves using ribonomics showed an enrichment of functionally related genes regulating these processes. HuR coordinately regulated expression of several genes to promote proliferation, apoptosis, and morphogenesis in rat Schwann cells, in response to NRG1, TGFβ, and laminins, three major signals implicated in this patterning event. Strikingly, HuR also binds to several mRNAs encoding myelination-related proteins but, contrary to its typical function, negatively regulated their expression, likely to prevent ectopic myelination during development. These functions of HuR correlated with its abundance and subcellular localization, which were regulated by different signals in Schwann cells.
Journal of Neuroscience 04/2012; 32(14):4944-58. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Glycine N-methyltransferase (GNMT) catabolizes S-adenosylmethionine (SAMe), the main methyl donor of the body. Patients with cirrhosis show attenuated GNMT expression, which is absent in hepatocellular carcinoma (HCC) samples. GNMT(-/-) mice develop spontaneous steatosis that progresses to steatohepatitis, cirrhosis, and HCC. The liver is highly enriched with innate immune cells and plays a key role in the body's host defense and in the regulation of inflammation. Chronic inflammation is the major hallmark of nonalcoholic steatohepatitis (NASH) progression. The aim of our study was to uncover the molecular mechanisms leading to liver chronic inflammation in the absence of GNMT, focusing on the implication of natural killer (NK) / natural killer T (NKT) cells. We found increased expression of T helper (Th)1- over Th2-related cytokines, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-R2/DR5, and several ligands of NK cells in GNMT(-/-) livers. Interestingly, NK cells from GNMT(-/-) mice were spontaneously activated, expressed more TRAIL, and had strong cytotoxic activity, suggesting their contribution to the proinflammatory environment in the liver. Accordingly, NK cells mediated hypersensitivity to concanavalin A (ConA)-mediated hepatitis in GNMT(-/-) mice. Moreover, GNMT(-/-) mice were hypersensitive to endotoxin-mediated liver injury. NK cell depletion and adoptive transfer of TRAIL(-/-) liver-NK cells protected the liver against lipopolysaccharide (LPS) liver damage. CONCLUSION: Our data allow us to conclude that TRAIL-producing NK cells actively contribute to promote a proinflammatory environment at early stages of fatty liver disease, suggesting that this cell compartment may contribute to the progression of NASH.
[show abstract][hide abstract] ABSTRACT: Our understanding of the mechanisms by which nonalcoholic fatty liver disease (NAFLD) progresses from simple steatosis to steatohepatitis (NASH) is still very limited. Despite the growing number of studies linking the disease with altered serum metabolite levels, an obstacle to the development of metabolome-based NAFLD predictors has been the lack of large cohort data from biopsy-proven patients matched for key metabolic features such as obesity. We studied 467 biopsied individuals with normal liver histology (n=90) or diagnosed with NAFLD (steatosis, n=246; NASH, n=131), randomly divided into estimation (80% of all patients) and validation (20% of all patients) groups. Qualitative determinations of 540 serum metabolite variables were performed using ultraperformance liquid chromatography coupled to mass spectrometry (UPLC-MS). The metabolic profile was dependent on patient body-mass index (BMI), suggesting that the NAFLD pathogenesis mechanism may be quite different depending on an individual's level of obesity. A BMI-stratified multivariate model based on the NAFLD serum metabolic profile was used to separate patients with and without NASH. The area under the receiver operating characteristic curve was 0.87 in the estimation and 0.85 in the validation group. The cutoff (0.54) corresponding to maximum average diagnostic accuracy (0.82) predicted NASH with a sensitivity of 0.71 and a specificity of 0.92 (negative/positive predictive values=0.82/0.84). The present data, indicating that a BMI-dependent serum metabolic profile may be able to reliably distinguish NASH from steatosis patients, have significant implications for the development of NASH biomarkers and potential novel targets for therapeutic intervention.
Journal of Proteome Research 02/2012; 11(4):2521-32. · 5.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: S-Adenosylmethionine, abbreviated as SAM, SAMe or AdoMet, is the principal methyl group donor in the mammalian cell and the first step metabolite of the methionine cycle, being synthesized by MAT (methionine adenosyltransferase) from methionine and ATP. About 60 years after its identification, SAMe is admitted as a key hepatic regulator whose level needs to be maintained within a specific range in order to avoid liver damage. Recently, in vitro and in vivo studies have demonstrated the regulatory role of SAMe in HGF (hepatocyte growth factor)-mediated hepatocyte proliferation through a mechanism that implicates the activation of the non-canonical LKB1/AMPK/eNOS cascade and HuR function. Regarding hepatic differentiation, cellular SAMe content varies depending on the status of the cell, being lower in immature than in adult hepatocytes. This finding suggests a SAMe regulatory effect also in this cellular process, which very recently was reported and related to HuR activity. Although in the last years this and other discoveries contributed to throw light into the tangle of regulatory mechanisms that govern this complex process, an overall understanding is still a challenge. For this purpose, the in vitro hepatic differentiation culture systems by using stem cells or fetal hepatoblasts are considered as valuable tools which, in combination with the methods used in current days to elucidate cell signaling pathways, surely will help to clear up this question.
Methods in molecular biology (Clifton, N.J.) 01/2012; 826:133-49.
[show abstract][hide abstract] ABSTRACT: Hu antigen R (HuR) is a central RNA-binding protein regulating cell dedifferentiation, proliferation, and survival, which are well-established hallmarks of cancer. HuR is frequently overexpressed in tumors correlating with tumor malignancy, which is in line with a role for HuR in tumorigenesis. However, the precise mechanism leading to changes in HuR expression remains unclear. In the liver, HuR plays a crucial role in hepatocyte proliferation, differentiation, and transformation. Here, we unraveled a novel mean of regulation of HuR expression in hepatocellular carcinoma (HCC) and colon cancer. HuR levels correlate with the abundance of the oncogene, murine double minute 2 (Mdm2), in human HCC and colon cancer metastases. HuR is stabilized by Mdm2-mediated NEDDylation in at least three lysine residues, ensuring its nuclear localization and protection from degradation. Conclusion: This novel Mdm2/NEDD8/HuR regulatory framework is essential for the malignant transformation of tumor cells, which, in turn, unveils a novel signaling paradigm that is pharmacologically amenable for cancer therapy.
[show abstract][hide abstract] ABSTRACT: Very low-density lipoprotein (VLDL) secretion provides a mechanism to export triglycerides (TG) from the liver to peripheral tissues, maintaining lipid homeostasis. In nonalcoholic fatty liver disease (NAFLD), VLDL secretion disturbances are unclear. Methionine adenosyltransferase (MAT) is responsible for S-adenosylmethionine (SAMe) synthesis and MAT I and III are the products of the MAT1A gene. Deficient MAT I and III activities and SAMe content in the liver have been associated with NAFLD, but whether MAT1A is required for normal VLDL assembly remains unknown. We investigated the role of MAT1A on VLDL assembly in two metabolic contexts: in 3-month-old MAT1A-knockout mice (3-KO), with no signs of liver injury, and in 8-month-old MAT1A-knockout mice (8-KO), harboring nonalcoholic steatohepatitis. In 3-KO mouse liver, there is a potent effect of MAT1A deletion on lipid handling, decreasing mobilization of TG stores, TG secretion in VLDL and phosphatidylcholine synthesis via phosphatidylethanolamine N-methyltransferase. MAT1A deletion also increased VLDL-apolipoprotein B secretion, leading to small, lipid-poor VLDL particles. Administration of SAMe to 3-KO mice for 7 days recovered crucial altered processes in VLDL assembly and features of the secreted lipoproteins. The unfolded protein response was activated in 8-KO mouse liver, in which TG accumulated and the phosphatidylcholine-to-phosphatidylethanolamine ratio was reduced in the endoplasmic reticulum, whereas secretion of TG and apolipoprotein B in VLDL was increased and the VLDL physical characteristics resembled that in 3-KO mice. MAT1A deletion also altered plasma lipid homeostasis, with an increase in lipid transport in low-density lipoprotein subclasses and decrease in high-density lipoprotein subclasses. Conclusion: MAT1A is required for normal VLDL assembly and plasma lipid homeostasis in mice. Impaired VLDL synthesis, mainly due to SAMe deficiency, contributes to NAFLD development in MAT1A-KO mice.
[show abstract][hide abstract] ABSTRACT: Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of hepatic damage in developed countries. For this reason, mouse models of NAFLD have been developed to show progression of the disease because it perfectly resembles the human pathology. Here we show that diagnostic high-frequency ultrasound imaging (US) may be used as an effective method for monitoring the progression of liver disease, from steatosis to hepatocellular carcinoma in the methionine adenosyl transferase and glycine N-methyltransferase-deficient mice models. US reliably detected murine liver lesions associated with NAFLD in the two mice strains tested, with excellent agreement among US images, gross pathology and histological sections. Our results suggest US as a relevant approach for the study of NAFLD in mice, with interesting technical and therapeutic implications.
Ultrasound in medicine & biology 07/2011; 37(7):1161-9. · 2.02 Impact Factor
[show abstract][hide abstract] ABSTRACT: Intercellular communication via GAP Junctions plays an important role in tissue homeostasis, apoptosis, carcinogenesis, cell proliferation and differentiation. Hepatocyte connexins (Cx) 26 and 32 levels are decreased during the de-differentiation process of primary hepatocytes in culture, a situation that is also characterized by a decrease in S-Adenosylmethionine (SAMe) levels. In this current study, we show that SAMe supplementation in cultured hepatocytes every 12h, leads to an up-regulation of Cx26 and 32 mRNA and protein levels and blocks culture-induced Cx43 expression, although it failed to increase Cx26 and 32 membrane localization and GAP junction intracellular communication. SAMe reduced nuclear β-catenin accumulation, which is known to stimulate the TCF/LEF-dependent gene transcription of Cx43. Moreover SAMe-induced reduction in Cx43 and β-catenin was prevented by the proteasome inhibitor MG132, and was not mediated by GSK3 activity. SAMe, and its metabolite 5'-methylthioadenosine (MTA) increased Cx26 mRNA in a process partially mediated by Adenosine A(2A) receptors but independent of PKA. Finally livers from MAT1A knockout mice, characterized by low hepatic SAMe levels, express higher Cx43 and lower Cx26 and 32 protein levels than control mice. These results suggest that SAMe maintains a characteristic expression pattern of the different Cxs in hepatocytes by differentially regulating their levels.
European journal of cell biology 04/2011; 90(4):312-22. · 3.31 Impact Factor
[show abstract][hide abstract] ABSTRACT: The enzyme AMP-activated protein kinase (AMPK) is the main energy sensor in cells and is responsible for controlling the balance of anabolic/catabolic processes under metabolic stress conditions. This metabolic control exerted by AMPK is critical for energy-demanding situations, such as liver regeneration. Immediately after partial hepatectomy (PH), the liver undergoes the priming phase, mediated by the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6, which promote responsiveness of hepatocytes to growth factors, such as hepatocyte growth factor (HGF) and epidermal growth factor, which lead to proliferation. In addition to its metabolic function, AMPK is likely to be a key mediator in both hepatocyte priming and the proliferative phases, induced by TNF-α and HGF, respectively. TNF-α-induced AMPK activation has been shown to be necessary for nuclear factor κappa B (NF-κB)-induced inducible nitric oxide synthase expression and for blocking TNF-α-induced apoptosis. On the other hand, HGF-induced LKB1/AMPK activation has been found to play a critical role in controlling Hu antigen R cytosolic localization and endothelial nitric oxide synthase activation, and consequently Cyclin D1 and Cyclin A expressions, and nitric oxide generation, respectively. During PH, levels of S-adenosylmethionine (SAMe), the principal methyl donor in the liver, have to decrease to allow liver proliferation. Our studies also show that SAMe inhibits hepatocyte proliferation by controlling the hepatocyte's responsiveness to mitogenic signals such as HGF through the inhibition of AMPK activity. In summary, these data highlight the essential role of AMPK in controlling the balance between hepatocyte metabolic adaptations, cell cycle progression and apoptosis during liver regeneration.
Experimental Biology and Medicine 03/2011; 236(4):402-8. · 2.80 Impact Factor
[show abstract][hide abstract] ABSTRACT: LKB1, originally considered a tumor suppressor, plays an important role in hepatocyte proliferation and liver regeneration. Mice lacking the methionine adenosyltransferase (MAT) gene MAT1A exhibit a chronic reduction in hepatic S-adenosylmethionine (SAMe) levels, basal activation of LKB1, and spontaneous development of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). These results are relevant for human health because patients with liver cirrhosis, who are at risk to develop HCC, have a marked reduction in hepatic MAT1A expression and SAMe synthesis. In this study, we isolated a cell line (SAMe-deficient [SAMe-D]) from MAT1A knockout (MAT1A-KO) mouse HCC to examine the role of LKB1 in the development of liver tumors derived from metabolic disorders. We found that LKB1 is required for cell survival in SAMe-D cells. LKB1 regulates Akt-mediated survival independent of phosphoinositide 3-kinase, adenosine monophosphate protein-activated kinase (AMPK), and mammalian target of rapamycin complex (mTORC2). In addition, LKB1 controls the apoptotic response through phosphorylation and retention of p53 in the cytoplasm and the regulation of herpesvirus-associated ubiquitin-specific protease (HAUSP) and Hu antigen R (HuR) nucleocytoplasmic shuttling. We identified HAUSP as a target of HuR. Finally, we observed cytoplasmic staining of p53 and p-LKB1(Ser428) in a NASH-HCC animal model (from MAT1A-KO mice) and in liver biopsies obtained from human HCC derived from both alcoholic steatohepatitis and NASH. CONCLUSION: The SAMe-D cell line is a relevant model of HCC derived from NASH disease in which LKB1 is the principal conductor of a new regulatory mechanism and could be a practical tool for uncovering new therapeutic strategies.