Marta Varela-Rey

Non-alcoholic steatohepatitis and animal models : Understanding the human disease

Non-alcoholic fatty liver disease includes a broad spectrum of liver abnormalities ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), which can progress to cirrhosis and hepatocellular carcinoma. Patients with primary NASH have the metabolic (or insulin resistance) syndrome, condition typically associated with obesity, diabetes, hyperlipidemia and hypertension. To understand the mechanisms implicated in development of NASH, animal models of non-alcoholic fatty liver disease have been generated. These have greatly improved our understanding of some of the aspects of this disease. The challenge now is to identify the common mechanisms between the animal models and humans, which could eventually lead to a better prognosis and development of novel therapeutic strategies.

Schwann cell autophagy, myelinophagy, initiates myelin clearance from injured nerves

Schwann cells degrade myelin after injury by a novel form of selective autophagy, myelinophagy, which is positively regulated by the JNK/c-Jun pathway and is defective in the injured central nervous system.

p38 MAPK mediates the regulation of α1(I) procollagen mRNA levels by TNF-α and TGF-β in a cell line of rat hepatic stellate cells1

The role of members of the mitogen‐activated protein kinase (MAPK) family on tumor necrosis factor α (TNF‐α)‐mediated down‐regulation of col1a1 gene was studied. TNF‐α increased extracellular‐regulated kinase and Jun‐N‐terminal kinase phosphorylation, but these effects were not related to its inhibitory effect on α1(I) procollagen (col1a1) mRNA levels. Phosphorylation of p38 MAPK was decreased in response to TNF‐α, and the specific p38 MAPK inhibitor SB203580 mimicked the effect of TNF‐α on col1a1 mRNA levels. Transforming growth factor β (TGF‐β) increased p38 MAPK phosphorylation and SB203580 prevented the induction of col1a1 mRNA levels by TGF‐β. These results suggest that p38 MAPK plays an important role in regulating the expression of col1a1 in hepatic stellate cells in response to cytokines.

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‐α (TNF‐α), 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 (IκBα) degradation, and nuclear factor κB (NFκB) 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. (HEPATOLOGY 2004;39:1088–1098.)

Murine double minute 2 regulates Hu antigen R stability in human liver and colon cancer through NEDDylation

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. (Hepatology 2012)

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.)

Excess S‐adenosylmethionine reroutes phosphatidylethanolamine towards phosphatidylcholine and triglyceride synthesis

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 by way of 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 by way of 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 wildtype 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;58:1296–1305)

Fatty liver and fibrosis in glycine N-methyltransferase knockout mice is prevented by nicotinamide

Deletion of glycine N‐methyltransferase (GNMT), the main gene involved in liver S‐adenosylmethionine (SAM) catabolism, leads to the hepatic accumulation of this molecule and the development of fatty liver and fibrosis in mice. To demonstrate that the excess of hepatic SAM is the main agent contributing to liver disease in GNMT knockout (KO) mice, we treated 1.5‐month‐old GNMT‐KO mice for 6 weeks with nicotinamide (NAM), a substrate of the enzyme NAM N‐methyltransferase. NAM administration markedly reduced hepatic SAM content, prevented DNA hypermethylation, and normalized the expression of critical genes involved in fatty acid metabolism, oxidative stress, inflammation, cell proliferation, and apoptosis. More importantly, NAM treatment prevented the development of fatty liver and fibrosis in GNMT‐KO mice. Because GNMT expression is down‐regulated in patients with cirrhosis, and because some subjects with GNMT mutations have spontaneous liver disease, the clinical implications of the present findings are obvious, at least with respect to these latter individuals. Because NAM has been used for many years to treat a broad spectrum of diseases (including pellagra and diabetes) without significant side effects, it should be considered in subjects with GNMT mutations. Conclusion: The findings of this study indicate that the anomalous accumulation of SAM in GNMT‐KO mice can be corrected by NAM treatment leading to the normalization of the expression of many genes involved in fatty acid metabolism, oxidative stress, inflammation, cell proliferation, and apoptosis, as well as reversion of the appearance of the pathologic phenotype. (HEPATOLOGY 2010)

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)