Mercedes Vazquez-Chantada

Loss of the glycine N‐methyltransferase gene leads to steatosis and hepatocellular carcinoma in mice

Glycine N‐methyltransferase (GNMT) is the main enzyme responsible for catabolism of excess hepatic S‐adenosylmethionine (SAMe). GNMT is absent in hepatocellular carcinoma (HCC), messenger RNA (mRNA) levels are significantly lower in livers of patients at risk of developing HCC, and GNMT has been proposed to be a tumor‐susceptibility gene for liver cancer. The identification of several children with liver disease as having mutations of the GNMT gene further suggests that this enzyme plays an important role in liver function. In the current study we studied development of liver pathologies including HCC in GNMT‐knockout (GNMT‐KO) mice. GNMT‐KO mice have elevated serum aminotransferase, methionine, and SAMe levels and develop liver steatosis, fibrosis, and HCC. We found that activation of the Ras and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways was increased in liver tumors from GNMT‐KO mice coincidently with the suppression of the Ras inhibitors Ras‐association domain family/tumor suppressor (RASSF) 1 and 4 and the JAK/STAT inhibitors suppressor of cytokine signaling (SOCS) 1–3 and cytokine‐inducible SH2‐protein. Finally, we found that methylation of RASSF1 and SOCS2 promoters and the binding of trimethylated lysine 27 in histone 3 to these 2 genes was increased in HCC from GNMT‐KO mice. Conclusion: These data demonstrate that loss of GNMT induces aberrant methylation of DNA and histones, resulting in epigenetic modulation of critical carcinogenic pathways in mice. (HEPATOLOGY 2008.)

Sirtuin 1 regulation of developmental genes during differentiation of stem cells

The longevity-promoting NAD+–dependent class III histone deacetylase Sirtuin 1 (SIRT1) is involved in stem cell function by controlling cell fate decision and/or by regulating the p53-dependent expression of NANOG. We show that SIRT1 is down-regulated precisely during human embryonic stem cell differentiation at both mRNA and protein levels and that the decrease in Sirt1 mRNA is mediated by a molecular pathway that involves the RNA-binding protein HuR and the arginine methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). SIRT1 down-regulation leads to reactivation of key developmental genes such as the neuroretinal morphogenesis effectors DLL4, TBX3, and PAX6, which are epigenetically repressed by this histone deacetylase in pluripotent human embryonic stem cells. Our results indicate that SIRT1 is regulated during stem cell differentiation in the context of a yet-unknown epigenetic pathway that controls specific developmental genes in embryonic stem cells.

Evidence for LKB1/AMP-activated protein kinase/ endothelial nitric oxide synthase cascade regulated by hepatocyte growth factor, S-adenosylmethionine, and nitric oxide in hepatocyte proliferation.

S‐adenosylmethionine (SAMe) is involved in numerous complex hepatic processes such as hepatocyte proliferation, death, inflammatory responses, and antioxidant defense. One of the most relevant actions of SAMe is the inhibition of hepatocyte proliferation during liver regeneration. In hepatocytes, SAMe regulates the levels of cytoplasmic HuR, an RNA‐binding protein that increases the half‐life of target messenger RNAs such as cyclin D1 and A2 via inhibition of hepatocyte growth factor (HGF)‐mediated adenosine monophosphate–activated protein kinase (AMPK) phosphorylation. Because AMPK is activated by the tumor suppressor kinase LKB1, and AMPK activates endothelial nitric oxide (NO) synthase (eNOS), and NO synthesis is of great importance for hepatocyte proliferation, we hypothesized that in hepatocytes HGF may induce the phosphorylation of LKB1, AMPK, and eNOS through a process regulated by SAMe, and that this cascade might be crucial for hepatocyte growth. We demonstrate that the proliferative response of hepatocytes involves eNOS phosphorylation via HGF‐mediated LKB1 and AMPK phosphorylation, and that this process is regulated by SAMe and NO. We also show that knockdown of LKB1, AMPK, or eNOS with specific interference RNA (iRNA) inhibits HGF‐mediated hepatocyte proliferation. Finally, we found that the LKB1/AMPK/eNOS cascade is activated during liver regeneration after partial hepatectomy and that this process is impaired in mice treated with SAMe before hepatectomy, in knockout mice deficient in hepatic SAMe, and in eNOS knockout mice. Conclusion: We have identified an LKB1/AMPK/eNOS cascade regulated by HGF, SAMe, and NO that functions as a critical determinant of hepatocyte proliferation during liver regeneration after partial hepatectomy. (HEPATOLOGY 2009;49:608–617.)

S-adenosylmethionine and proliferation: new pathways, new targets.

SAMe (S-adenosylmethionine) is the main methyl donor group in the cell. MAT (methionine adenosyltransferase) is the unique enzyme responsible for the synthesis of SAMe from methionine and ATP, and SAMe is the common point between the three principal metabolic pathways: polyamines, transmethylation and transsulfuration that converge into the methionine cycle. SAMe is now also considered a key regulator of metabolism, proliferation, differentiation, apoptosis and cell death. Recent results show a new signalling pathway implicated in the proliferation of the hepatocyte, where AMPK (AMP-activated protein kinase) and HuR, modulated by SAMe, take place in HGF (hepatocyte growth factor)-mediated cell growth. Abnormalities in methionine metabolism occur in several animal models of alcoholic liver injury, and it is also altered in patients with liver disease. Both high and low levels of SAMe predispose to liver injury. In this regard, knockout mouse models have been developed for the enzymes responsible for SAMe synthesis and catabolism, MAT1A and GNMT (glycine N-methyltransferase) respectively. These knockout mice develop steatosis and HCC (hepatocellular carcinoma), and both models closely replicate the pathologies of human disease, which makes them extremely useful to elucidate the mechanism underlying liver disease. These new findings open a wide range of possibilities to discover novel targets for clinical applications.

Activation of LKB1‐Akt pathway independent of phosphoinositide 3‐kinase plays a critical role in the proliferation of hepatocellular carcinoma from nonalcoholic steatohepatitis

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. (HEPATOLOGY 2010)

Impaired liver regeneration in mice lacking glycine N-methyltransferase†

Hepatic S‐adenosylmethionine (SAMe) is maintained constant by the action of methionine adenosyltransferase I/III (MATI/III), which converts methionine into SAMe and glycine N‐methyltransferase (GNMT), which eliminates excess SAMe to avoid aberrant methylation reactions. During liver regeneration after partial hepatectomy (PH) MATI/III activity is inhibited leading to a decrease in SAMe. This injury‐related reduction in SAMe promotes hepatocyte proliferation because SAMe inhibits hepatocyte DNA synthesis. In MATI/III‐deficient mice, hepatic SAMe is reduced, resulting in uncontrolled hepatocyte growth and impaired liver regeneration. These observations suggest that a reduction in SAMe is crucial for successful liver regeneration. In support of this hypothesis we report that liver regeneration is impaired in GNMT knockout (GNMT‐KO) mice. Liver SAMe is 50‐fold higher in GNMT‐KO mice than in control animals and is maintained constant following PH. Mortality after PH was higher in GNMT‐KO mice than in control animals. In GNMT‐KO mice, nuclear factor κB (NFκB), signal transducer and activator of transcription‐3 (STAT3), inducible nitric oxide synthase (iNOS), cyclin D1, cyclin A, and poly (ADP‐ribose) polymerase were activated at baseline. PH in GNMT‐KO mice was followed by the inactivation of STAT3 phosphorylation and iNOS expression. NFκB, cyclin D1 and cyclin A were not further activated after PH. The LKB1/AMP‐activated protein kinase/endothelial nitric oxide synthase cascade was inhibited, and cytoplasmic HuR translocation was blocked despite preserved induction of DNA synthesis in GNMT‐KO after PH. Furthermore, a previously unexpected relationship between AMPK phosphorylation and NFκB activation was uncovered. Conclusion: These results indicate that multiple signaling pathways are impaired during the liver regenerative response in GNMT‐KO mice, suggesting that GNMT plays a critical role during liver regeneration, promoting hepatocyte viability and normal proliferation. (HEPATOLOGY 2009.)

Solute carrier family 2 member 1 is involved in the development of nonalcoholic fatty liver disease.

Susceptibility to develop nonalcoholic fatty liver disease (NAFLD) has genetic bases, but the associated variants are uncertain. The aim of the present study was to identify genetic variants that could help to prognose and further understand the genetics and development of NAFLD. Allele frequencies of 3,072 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 this, 4,414 type 2 diabetes mellitus (T2DM) cases and 4,567 controls were genotyped. 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 (OS), and the consequences of oleic acid (OA)‐enriched medium on lipid accumulation in siSLC2A1‐THLE2 cells were studied by gene‐expression analysis, dihydroethidium staining, BODIPY, and quantification of intracellular triglyceride content, respectively. 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 transcriptome profiles drove us to demonstrate that in vitro silencing of SLC2A1 induces an increased OS activity and a higher lipid accumulation under OA 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 2013)

SAMe and HuR in Liver Physiology

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.

378 HUMAN SERUM METABOLIC PROFILING REVEALS NOVEL BIOMARKERS ASSOCIATED WITH NAFLD PROGRESSION

alcohol intake, ferritin and transferrin saturation. Sixty-three % of patients with hepatocellular siderosis were positive for at least one of these genetic variants. The beta-thalassemic trait had the highest specificity (93.8%; prevalence 31.2% in those with parenchymal vs. 6.2% in those without parenchymal siderosis) for predominantly parenchymal iron accumulation, and was the only genetic factor independently associated with the fibrosis >1 (OR 2.122; 95%CI 1.280–3.462). The IVS1–24 ferroportin polymorphism and the PiZ and PiS AAT variants did not influence the presence and pattern of siderosis or liver fibrosis. Conclusions: In patients with NAFLD, predominantly hepatocellular iron deposition is often related to genetic factors. Beta-globin mutations predispose to parenchymal iron accumulation and progressive liver fibrosis.