Repression of the nuclear receptor small heterodimer partner by steatotic drugs and in advanced nonalcoholic fatty liver disease.

The small heterodimer partner (SHP) (NR0B2) is an atypical nuclear receptor that lacks a DNA-binding domain. It interacts with and inhibits many transcription factors, affecting key metabolic processes, including bile acid, cholesterol, fatty acid, and drug metabolism. Our aim was to determine the influence of steatotic drugs and nonalcoholic fatty liver disease (NAFLD) on SHP expression and investigate the potential mechanisms. SHP was found to be repressed by steatotic drugs (valproate, doxycycline, tetracycline, and cyclosporin A) in cultured hepatic cells and the livers of different animal models of NAFLD: iatrogenic (tetracycline-treated rats), genetic (glycine N-methyltransferase-deficient mice), and nutritional (mice fed a methionine- and choline-deficient diet). Among the different transcription factors investigated, CCAAT-enhancer-binding protein α (C/EBPα) showed the strongest dominant-repressive effect on SHP expression in HepG2 and human hepatocytes. Reporter assays revealed that the inhibitory effect of C/EBPα and steatotic drugs colocalize between -340 and -509 base pair of the SHP promoter, and mutation of a predicted C/EBPα response element at -473 base pair abolished SHP repression by both C/EBPα and drugs. Moreover, inhibition of major stress signaling pathways demonstrated that the mitogen-activated protein kinase kinase 1/2 pathway activates, while the phosphatidylinositol 3 kinase pathway represses SHP in a C/EBP-dependent manner. We conclude that SHP is downregulated by several steatotic drugs and in advanced NAFLD. These conditions can activate signals that target C/EBPα and consequently repress SHP, thus favoring the progression and severity of NAFLD.

S-Adenosylmethionine increases circulating very-low density lipoprotein clearance in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Very-low-density lipoproteins (VLDLs) export lipids from the liver to peripheral tissues and are the precursors of low-density-lipoproteins. Low levels of hepatic S-adenosylmethionine (SAMe) decrease triglyceride (TG) secretion in VLDLs, contributing to hepatosteatosis in methionine adenosyltransferase 1A knockout mice but nothing is known about the effect of SAMe on the circulating VLDL metabolism. We wanted to investigate whether excess SAMe could disrupt VLDL plasma metabolism and unravel the mechanisms involved. METHODS: Glycine N-methyltransferase (GNMT) knockout (KO) mice, GNMT and perilipin-2 (PLIN2) double KO (GNMT-PLIN2-KO) and their respective wild type (WT) controls were used. A high fat diet (HFD) or a methionine deficient diet (MDD) was administrated to exacerbate or recover VLDL metabolism, respectively. Finally, 33 patients with non-alcoholic fatty-liver disease (NAFLD); 11 with hypertriglyceridemia and 22 with normal lipidemia were used in this study. RESULTS: We found that excess SAMe increases the turnover of hepatic TG stores for secretion in VLDL in GNMT-KO mice, a model of NAFLD with high SAMe levels. The disrupted VLDL assembly resulted in the secretion of enlarged, phosphatidylethanolamine-poor, TG- and apoE-enriched VLDL-particles; special features that lead to increased VLDL clearance and decreased serum TG levels. Re-establishing normal SAMe levels restored VLDL secretion, features and metabolism. In NAFLD patients, serum TG levels were lower when hepatic GNMT-protein expression was decreased. CONCLUSIONS: Excess hepatic SAMe levels disrupt VLDL assembly and features and increase circulating VLDL clearance, which will cause increased VLDL-lipid supply to tissues and might contribute to the extrahepatic complications of NAFLD.

Systems biology for hepatologists.

Medicine is expected to benefit from combining usual cellular and molecular studies with high-throughput methods (genomics, transcriptomics, proteomics, and metabolomics). These methods, collectively known as omics, permit the determination of thousands of molecules (variations within genes, RNAs, proteins, metabolites) within a tissue, cell, or biological fluid. The use of these methods is very demanding in terms of the design of the study, acquisition, storage, analysis, and interpretation of the data. When carried out properly, these studies can reveal new etiological pathways, help to identify patients at risk for disease, and predict the response to specific treatments. Here we review these omics methods and mention several applications in hepatology research.

The human liver fatty acid binding protein (FABP1) gene is activated by FOXA1 and PPARα; and repressed by C/EBPα: Implications in FABP1 down-regulation in nonalcoholic fatty liver disease.

Liver fatty acid binding protein (FABP1) prevents lipotoxicity of free fatty acids and regulates fatty acid trafficking and partition. Our objective is to investigate the transcription factors controlling the human FABP1 gene and their regulation in nonalcoholic fatty liver disease (NAFLD). Adenovirus-mediated expression of multiple transcription factors in HepG2 cells and cultured human hepatocytes demonstrated that FOXA1 and PPARα are among the most effective activators of human FABP1, whereas C/EBPα is a major dominant repressor. Moreover, FOXA1 and PPARα induced re-distribution of FABP1 protein and increased cytoplasmic expression. Reporter assays demonstrated that the major basal activity of the human FABP1 promoter locates between -96 and -229bp, where C/EBPα binds to a composite DR1-C/EBP element. Mutation of this element at -123bp diminished basal reporter activity, abolished repression by C/EBPα and reduced transactivation by HNF4α. Moreover, HNF4α gene silencing by shRNA in HepG2 cells caused a significant down-regulation of FABP1 mRNA expression. FOXA1 activated the FABP1 promoter through binding to a cluster of elements between -229 and -592bp, whereas PPARα operated through a conserved proximal element at -59bp. Finally, FABP1, FOXA1 and PPARα were concomitantly repressed in animal models of NAFLD and in human nonalcoholic fatty livers, whereas C/EBPα was induced or did not change. We conclude that human FABP1 has a complex mechanism of regulation where C/EBPα displaces HNF4α and hampers activation by FOXA1 and PPARα. Alteration of expression of these transcription factors in NAFLD leads to FABP1 gen repression and could exacerbate lipotoxicity and disease progression.

Excess S-adenosylmethionine reroutes phosphatidylethanolamine towards phosphatidylcholine and triglyceride synthesis.

UNLABELLED: 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.

S-adenosylmethionine metabolism and liver disease.

Methionine is an essential amino acid that is metabolized mainly by the liver where it is converted to S-adenosylmethionine (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.

Methionine adenosyltransferase 1A gene deletion disrupts hepatic very low-density lipoprotein assembly in mice.

UNLABELLED: 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.

Novel function and intracellular localization of methionine adenosyltransferase 2beta splicing variants.

Human methionine adenosyltransferase 2beta (MAT2beta) encodes for two major splicing variants, V1 and V2, which are differentially expressed in normal tissues. Both variants are induced in human liver cancer and positively regulate growth. The aim of this work was to identify interacting proteins of V1 and V2. His-tagged V1 and V2 were overexpressed in Rosetta pLysS cells, purified, and used in a pulldown assay to identify interacting proteins from human colon cancer cell line RKO cell lysates. The eluted lysates were subjected to Western blot and in solution proteomic analyses. HuR, an mRNA-binding protein known to stabilize the mRNA of several cyclins, was identified to interact with V1 and V2. Immunoprecipitation and Western blotting confirmed their interaction in both liver and colon cancer cells. These variant proteins are located in both nucleus and cytoplasm in liver and colon cancer cells and, when overexpressed, increased the cytoplasmic HuR content. This led to increased expression of cyclin D1 and cyclin A, known targets of HuR. When endogenous expression of V1 or V2 is reduced by small interference RNA, cytoplasmic HuR content fell and the expression of these HuR target genes also decreased. Knockdown of cyclin D1 or cyclin A blunted, whereas knockdown of HuR largely prevented, the ability of V1 or V2 overexpression to induce growth. In conclusion, MAT2beta variants reside mostly in the nucleus and regulate HuR subcellular content to affect cell proliferation.

HuR/methyl-HuR and AUF1 regulate the MAT expressed during liver proliferation, differentiation, and carcinogenesis.

BACKGROUND & AIMS: Hepatic de-differentiation, liver development, and malignant transformation are processes in which the levels of hepatic S-adenosylmethionine are tightly regulated by 2 genes: methionine adenosyltransferase 1A (MAT1A) and methionine adenosyltransferase 2A (MAT2A). MAT1A is expressed in the adult liver, whereas MAT2A expression primarily is extrahepatic and is associated strongly with liver proliferation. The mechanisms that regulate these expression patterns are not completely understood. METHODS: In silico analysis of the 3' untranslated region of MAT1A and MAT2A revealed putative binding sites for the RNA-binding proteins AU-rich RNA binding factor 1 (AUF1) and HuR, respectively. We investigated the posttranscriptional regulation of MAT1A and MAT2A by AUF1, HuR, and methyl-HuR in the aforementioned biological processes. RESULTS: During hepatic de-differentiation, the switch between MAT1A and MAT2A coincided with an increase in HuR and AUF1 expression. S-adenosylmethionine treatment altered this homeostasis by shifting the balance of AUF1 and methyl-HuR/HuR, which was identified as an inhibitor of MAT2A messenger RNA (mRNA) stability. We also observed a similar temporal distribution and a functional link between HuR, methyl-HuR, AUF1, and MAT1A and MAT2A during fetal liver development. Immunofluorescent analysis revealed increased levels of HuR and AUF1, and a decrease in methyl-HuR levels in human livers with hepatocellular carcinoma (HCC). CONCLUSIONS: Our data strongly support a role for AUF1 and HuR/methyl-HuR in liver de-differentiation, development, and human HCC progression through the posttranslational regulation of MAT1A and MAT2A mRNAs.

Liver-specific deletion of prohibitin 1 results in spontaneous liver injury, fibrosis, and hepatocellular carcinoma in mice.

UNLABELLED: Prohibitin 1 (PHB1) is a highly conserved, ubiquitously expressed protein that participates in diverse processes including mitochondrial chaperone, growth and apoptosis. The role of PHB1 in vivo is unclear and whether it is a tumor suppressor is controversial. Mice lacking methionine adenosyltransferase 1A (MAT1A) have reduced PHB1 expression, impaired mitochondrial function, and spontaneously develop hepatocellular carcinoma (HCC). To see if reduced PHB1 expression contributes to the Mat1a knockout (KO) phenotype, we generated liver-specific Phb1 KO mice. Expression was determined at the messenger RNA and protein levels. PHB1 expression in cells was varied by small interfering RNA or overexpression. At 3 weeks, KO mice exhibit biochemical and histologic liver injury. Immunohistochemistry revealed apoptosis, proliferation, oxidative stress, fibrosis, bile duct epithelial metaplasia, hepatocyte dysplasia, and increased staining for stem cell and preneoplastic markers. Mitochondria are swollen and many have no discernible cristae. Differential gene expression revealed that genes associated with proliferation, malignant transformation, and liver fibrosis are highly up-regulated. From 20 weeks on, KO mice have multiple liver nodules and from 35 to 46 weeks, 38% have multifocal HCC. PHB1 protein levels were higher in normal human hepatocytes compared to human HCC cell lines Huh-7 and HepG2. Knockdown of PHB1 in murine nontransformed AML12 cells (normal mouse hepatocyte cell line) raised cyclin D1 expression, increased E2F transcription factor binding to cyclin D1 promoter, and proliferation. The opposite occurred with PHB1 overexpression. Knockdown or overexpression of PHB1 in Huh-7 cells did not affect proliferation significantly or sensitize cells to sorafenib-induced apoptosis. CONCLUSION: Hepatocyte-specific PHB1 deficiency results in marked liver injury, oxidative stress, and fibrosis with development of HCC by 8 months. These results support PHB1 as a tumor suppressor in hepatocytes.

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

UNLABELLED: 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.

Liquid chromatography-mass spectrometry-based parallel metabolic profiling of human and mouse model serum reveals putative biomarkers associated with the progression of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in most western countries. Current NAFLD diagnosis methods (e.g., liver biopsy analysis or imaging techniques) are poorly suited as tests for such a prevalent condition, from both a clinical and financial point of view. The present work aims to demonstrate the potential utility of serum metabolic profiling in defining phenotypic biomarkers that could be useful in NAFLD management. A parallel animal model/human NAFLD exploratory metabolomics approach was employed, using ultra performance liquid chromatography-mass spectrometry (UPLC-MS) to analyze 42 serum samples collected from nondiabetic, morbidly obese, biopsy-proven NAFLD patients, and 17 animals belonging to the glycine N-methyltransferase knockout (GNMT-KO) NAFLD mouse model. Multivariate statistical analysis of the data revealed a series of common biomarkers that were significantly altered in the NAFLD (GNMT-KO) subjects in comparison to their normal liver counterparts (WT). Many of the compounds observed could be associated with biochemical perturbations associated with liver dysfunction (e.g., reduced Creatine) and inflammation (e.g., eicosanoid signaling). This differential metabolic phenotyping approach may have a future role as a supplement for clinical decision making in NAFLD and in the adaption to more individualized treatment protocols.

S-adenosylmethionine regulates apurinic/apyrimidinic endonuclease 1 stability: implication in hepatocarcinogenesis.

BACKGROUND & AIMS: Genomic instability participates in the pathogenesis of hepatocellular carcinoma (HCC). Apurinic/apyrimidinic endonuclease 1 (APEX1) participates in the base excision repair of premutagenic apurinic/apyrimidinic (AP) sites. Mice deficient in methionine adenosyltransferase 1a (Mat1a KO) have chronic hepatic deficiency of S-adenosylmethionine (SAMe) and increased oxidative stress, and develop HCC. We examined livers of Mat1a KO mice for genomic instability and dysregulation of APEX1. METHODS: Studies were conducted using Mat1a KO mice livers and cultured mouse and human hepatocytes. RESULTS: Genomic instability increased in the livers of 1-month-old Mat1a KO mice, compared with wild-type mice, whereas Apex1 mRNA and protein levels were reduced by 20% and 50%, respectively, in Mat1a KO mice of all ages. These changes correlated with increased numbers of AP sites and reduced expression of Bax, Fas, and p21 (all APEX targets). When human and mouse hepatocytes were placed in culture, transcription of MAT1A mRNA decreased whereas that of APEX1 and c-MYC increased. However, the protein levels of APEX1 decreased to 60% of baseline. Addition of 2 mmol/L SAMe prevented increases in APEX1 and c-MYC mRNA levels, as well as decreases in MAT1A expression and cytosolic and nuclear APEX1 protein levels. CONCLUSIONS: By 1 month of age, genomic instability increases in livers of Mat1a KO mice, possibly due to reduced APEX1 levels. Although SAMe inhibits APEX1 transcription, it stabilizes the APEX1 protein. This novel aspect of SAMe on APEX1 regulation might explain the chemopreventive action of SAMe and the reason that chronic SAMe deficiency predisposes to HCC.

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.

UNLABELLED: 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.

Impaired liver regeneration in mice lacking glycine N-methyltransferase.

UNLABELLED: 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 kappaB (NFkappaB), 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. NFkappaB, 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 NFkappaB 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.

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

UNLABELLED: 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.

Metabolomic-based noninvasive serum test to diagnose nonalcoholic steatohepatitis: Results from discovery and validation cohorts.

Nonalcoholic fatty liver disease (NAFLD) is the most common type of chronic liver disease worldwide and includes a broad spectrum of histologic phenotypes, ranging from simple hepatic steatosis or nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH). While liver biopsy is the reference gold standard for NAFLD diagnosis and staging, it has limitations due to its sampling variability, invasive nature, and high cost. Thus, there is a need for noninvasive biomarkers that are robust, reliable, and cost effective. In this study, we measured 540 lipids and amino acids in serum samples from biopsy-proven subjects with normal liver (NL), NAFL, and NASH. Using logistic regression analysis, we identified two panels of triglycerides that could first discriminate between NAFLD and NL and second between NASH and NAFL. These noninvasive tests were compared to blinded histology as a reference standard. We performed these tests in an original cohort of 467 patients with NAFLD (90 NL, 246 NAFL, and 131 NASH) that was subsequently validated in a separate cohort of 192 patients (7 NL, 109 NAFL, 76 NASH). The diagnostic performances of the validated tests showed an area under the receiver operating characteristic curve, sensitivity, and specificity of 0.88 ± 0.05, 0.94, and 0.57, respectively, for the discrimination between NAFLD and NL and 0.79 ± 0.04, 0.70, and 0.81, respectively, for the discrimination between NASH and NAFL. When the analysis was performed excluding patients with glucose levels >136 mg/dL, the area under the receiver operating characteristic curve for the discrimination between NASH and NAFL increased to 0.81 ± 0.04 with sensitivity and specificity of 0.73 and 0.80, respectively. Conclusion: The assessed noninvasive lipidomic serum tests distinguish between NAFLD and NL and between NASH and NAFL with high accuracy. (Hepatology Communications 2018;2:807-820).

Importance of a deficiency in S-adenosyl-L-methionine synthesis in the pathogenesis of liver injury.

One of the features of liver cirrhosis is an abnormal metabolism of methionine--a characteristic that was described more than a half a century ago. Thus, after an oral load of methionine, the rate of clearance of this amino acid from the blood is markedly impaired in cirrhotic patients compared with that in control subjects. Almost 15 y ago we observed that the failure to metabolize methionine in cirrhosis was due to an abnormally low activity of the enzyme methionine adenosyltransferase (EC 2.5.1.6). This enzyme converts methionine, in the presence of ATP, to S-adenosyl-L-methionine (SAMe), the main biological methyl donor. Since then, it has been suspected that a deficiency in hepatic SAMe may contribute to the pathogenesis of the liver in cirrhosis. The studies reviewed here are consistent with this hypothesis.

Non-alcoholic fatty liver disease proteomics.

PURPOSE: Non-alcoholic fatty liver disease (NAFLD) is an important cause of chronic liver injury that has gained concern in clinical hepatology. The principal aim of this study was to find differences in protein expression between patients with NAFLD and healthy controls. EXPERIMENTAL DESIGN: Changes in protein expression of liver samples from each of the three groups of subjects, controls, non-alcoholic steatosis, and non-alcoholic steatohepatitis (NASH), were analyzed by DIGE combined with MALDI TOF/TOF analysis, a proteomic approach that allows to compare hundreds of proteins simultaneously. RESULTS: Forty-three proteins exhibiting significant changes (ratio ≥1.5, p<0.05) were characterized, 22 comparing steatosis samples versus control samples and 21 comparing NASH versus control samples. Ten of these proteins were further analyzed by Western blot in tissue samples to confirm the observed changes of protein expression using DIGE. The proteins validated were further tested in serum samples of different cohorts of patients. CONCLUSIONS AND CLINICAL RELEVANCE: Following this approach we identified two candidate markers, carbamoyl phosphate synthase 1 and 78 kDa glucose-regulated protein, differentially expressed between control and NASH. This proteomics approach demonstrates that DIGE combined with MALDI TOF/TOF and Western blot analysis of tissue and serum samples is a useful approach to identify candidate markers associated with NAFLD, resulting in proteins whose level of expression can be correlated to a disease state.

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.