Redox signaling by glutathione peroxidase 2 links vascular modulation to metabolic plasticity of breast cancer.

In search of redox mechanisms in breast cancer, we uncovered a striking role for glutathione peroxidase 2 (GPx2) in oncogenic signaling and patient survival. GPx2 loss stimulates malignant progression due to reactive oxygen species/hypoxia inducible factor-α (HIF1α)/VEGFA (vascular endothelial growth factor A) signaling, causing poor perfusion and hypoxia, which were reversed by GPx2 reexpression or HIF1α inhibition. Ingenuity Pathway Analysis revealed a link between GPx2 loss, tumor angiogenesis, metabolic modulation, and HIF1α signaling. Single-cell RNA analysis and bioenergetic profiling revealed that GPx2 loss stimulated the Warburg effect in most tumor cell subpopulations, except for one cluster, which was capable of oxidative phosphorylation and glycolysis, as confirmed by coexpression of phosphorylated-AMPK and GLUT1. These findings underscore a unique role for redox signaling by GPx2 dysregulation in breast cancer, underlying tumor heterogeneity, leading to metabolic plasticity and malignant progression.

Developmental Consequences of Defective ATG7-Mediated Autophagy in Humans.

BACKGROUND: Autophagy is the major intracellular degradation route in mammalian cells. Systemic ablation of core autophagy-related (ATG) genes in mice leads to embryonic or perinatal lethality, and conditional models show neurodegeneration. Impaired autophagy has been associated with a range of complex human diseases, yet congenital autophagy disorders are rare. METHODS: We performed a genetic, clinical, and neuroimaging analysis involving five families. Mechanistic investigations were conducted with the use of patient-derived fibroblasts, skeletal muscle-biopsy specimens, mouse embryonic fibroblasts, and yeast. RESULTS: We found deleterious, recessive variants in human ATG7, a core autophagy-related gene encoding a protein that is indispensable to classical degradative autophagy. Twelve patients from five families with distinct ATG7 variants had complex neurodevelopmental disorders with brain, muscle, and endocrine involvement. Patients had abnormalities of the cerebellum and corpus callosum and various degrees of facial dysmorphism. These patients have survived with impaired autophagic flux arising from a diminishment or absence of ATG7 protein. Although autophagic sequestration was markedly reduced, evidence of basal autophagy was readily identified in fibroblasts and skeletal muscle with loss of ATG7. Complementation of different model systems by deleterious ATG7 variants resulted in poor or absent autophagic function as compared with the reintroduction of wild-type ATG7. CONCLUSIONS: We identified several patients with a neurodevelopmental disorder who have survived with a severe loss or complete absence of ATG7, an essential effector enzyme for autophagy without a known functional paralogue. (Funded by the Wellcome Centre for Mitochondrial Research and others.).

Loss of the RNA polymerase III repressor MAF1 confers obesity resistance.

MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1(-/-) mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1(-/-) mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD(+), and is associated with obesity resistance. Consistent with this, NAD(+) levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences.

Macrophage scavenger receptor 1 mediates lipid-induced inflammation in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Obesity-associated inflammation is a key player in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, the role of macrophage scavenger receptor 1 (MSR1, CD204) remains incompletely understood. METHODS: A total of 170 NAFLD liver biopsies were processed for transcriptomic analysis and correlated with clinicopathological features. Msr1-/- and wild-type mice were subjected to a 16-week high-fat and high-cholesterol diet. Mice and ex vivo human liver slices were treated with a monoclonal antibody against MSR1. Genetic susceptibility was assessed using genome-wide association study data from 1,483 patients with NAFLD and 430,101 participants of the UK Biobank. RESULTS: MSR1 expression was associated with the occurrence of hepatic lipid-laden foamy macrophages and correlated with the degree of steatosis and steatohepatitis in patients with NAFLD. Mice lacking Msr1 were protected against diet-induced metabolic disorder, showing fewer hepatic foamy macrophages, less hepatic inflammation, improved dyslipidaemia and glucose tolerance, and altered hepatic lipid metabolism. Upon induction by saturated fatty acids, MSR1 induced a pro-inflammatory response via the JNK signalling pathway. In vitro blockade of the receptor prevented the accumulation of lipids in primary macrophages which inhibited the switch towards a pro-inflammatory phenotype and the release of cytokines such as TNF-ɑ. Targeting MSR1 using monoclonal antibody therapy in an obesity-associated NAFLD mouse model and human liver slices resulted in the prevention of foamy macrophage formation and inflammation. Moreover, we identified that rs41505344, a polymorphism in the upstream transcriptional region of MSR1, was associated with altered serum triglycerides and aspartate aminotransferase levels in a cohort of over 400,000 patients. CONCLUSIONS: Taken together, our data suggest that MSR1 plays a critical role in lipid-induced inflammation and could thus be a potential therapeutic target for the treatment of NAFLD. LAY SUMMARY: Non-alcoholic fatty liver disease (NAFLD) is a chronic disease primarily caused by excessive consumption of fat and sugar combined with a lack of exercise or a sedentary lifestyle. Herein, we show that the macrophage scavenger receptor MSR1, an innate immune receptor, mediates lipid uptake and accumulation in Kupffer cells, resulting in liver inflammation and thereby promoting the progression of NAFLD in humans and mice.

Autophagy, lipophagy and lysosomal lipid storage disorders.

Autophagy is a catabolic process with an essential function in the maintenance of cellular and tissue homeostasis. It is primarily recognised for its role in the degradation of dysfunctional proteins and unwanted organelles, however in recent years the range of autophagy substrates has also been extended to lipids. Degradation of lipids via autophagy is termed lipophagy. The ability of autophagy to contribute to the maintenance of lipo-homeostasis becomes particularly relevant in the context of genetic lysosomal storage disorders where perturbations of autophagic flux have been suggested to contribute to the disease aetiology. Here we review recent discoveries of the molecular mechanisms mediating lipid turnover by the autophagy pathways. We further focus on the relevance of autophagy, and specifically lipophagy, to the disease mechanisms. Moreover, autophagy is also discussed as a potential therapeutic target in several key lysosomal storage disorders.

Methionine and S-adenosylmethionine levels are critical regulators of PP2A activity modulating lipophagy during steatosis.

BACKGROUND & AIMS: Glycine N-methyltransferase (GNMT) expression is decreased in some patients with severe non-alcoholic fatty liver disease. Gnmt deficiency in mice (Gnmt-KO) results in abnormally elevated serum levels of methionine and its metabolite S-adenosylmethionine (SAMe), and this leads to rapid liver steatosis development. Autophagy plays a critical role in lipid catabolism (lipophagy), and defects in autophagy have been related to liver steatosis development. Since methionine and its metabolite SAMe are well known inactivators of autophagy, we aimed to examine whether high levels of both metabolites could block autophagy-mediated lipid catabolism. METHODS: We examined methionine levels in a cohort of 358 serum samples from steatotic patients. We used hepatocytes cultured with methionine and SAMe, and hepatocytes and livers from Gnmt-KO mice. RESULTS: We detected a significant increase in serum methionine levels in steatotic patients. We observed that autophagy and lipophagy were impaired in hepatocytes cultured with high methionine and SAMe, and that Gnmt-KO livers were characterized by an impairment in autophagy functionality, likely caused by defects at the lysosomal level. Elevated levels of methionine and SAMe activated PP2A by methylation, while blocking PP2A activity restored autophagy flux in Gnmt-KO hepatocytes, and in hepatocytes treated with SAMe and methionine. Finally, normalization of methionine and SAMe levels in Gnmt-KO mice using a methionine deficient diet normalized the methylation capacity, PP2A methylation, autophagy, and ameliorated liver steatosis. CONCLUSIONS: These data suggest that elevated levels of methionine and SAMe can inhibit autophagic catabolism of lipids contributing to liver steatosis.

Autophagy and Lipid Droplets in the Liver.

Autophagy is a conserved quality-control pathway that degrades cytoplasmic contents in lysosomes. Autophagy degrades lipid droplets through a process termed lipophagy. Starvation and an acute lipid stimulus increase autophagic sequestration of lipid droplets and their degradation in lysosomes. Accordingly, liver-specific deletion of the autophagy gene Atg7 increases hepatic fat content, mimicking the human condition termed nonalcoholic fatty liver disease. In this review, we provide insights into the molecular regulation of lipophagy, discuss fundamental questions related to the mechanisms by which autophagosomes recognize lipid droplets and how ATG proteins regulate membrane curvature for lipid droplet sequestration, and comment on the possibility of cross talk between lipophagy and cytosolic lipases in lipid mobilization. Finally, we discuss the contribution of lipophagy to the pathophysiology of human fatty liver disease. Understanding how lipophagy clears hepatocellular lipid droplets could provide new ways to prevent fatty liver disease, a major epidemic in developed nations.

Autophagy in Myf5+ progenitors regulates energy and glucose homeostasis through control of brown fat and skeletal muscle development.

Macroautophagy (MA) regulates cellular quality control and energy balance. For example, loss of MA in aP2-positive adipocytes converts white adipose tissue (WAT) into brown adipose tissue (BAT)-like, enhancing BAT function and thereby insulin sensitivity. However, whether MA regulates early BAT development is unknown. We report that deleting Atg7 in myogenic Myf5+ progenitors inhibits MA in Myf5-cell-derived BAT and muscle. Knock out (KO) mice have defective BAT differentiation and function. Surprisingly, their body temperature is higher due to WAT lipolysis-driven increases in fatty acid oxidation in 'Beige' cells in inguinal WAT, BAT and muscle. KO mice also present impaired muscle differentiation, reduced muscle mass and glucose intolerance. Our studies show that ATG7 in Myf5+ progenitors is required to maintain energy and glucose homeostasis through effects on BAT and muscle development. Decreased MA in myogenic progenitors with age and/or overnutrition might contribute to the metabolic defects and sarcopenia observed in these conditions.

Autophagy and aging.

Autophagy is a critical quality control pathway that is conserved across diverse systems ranging from simple unicellular organisms like yeast to more complex systems, for instance mammals. Although, the fundamental role of autophagy is to maintain cellular quality control through lysosomal degradation of unwanted proteins and organelles, recent studies have mapped several new functions of this pathway that range from fuel utilization, cellular differentiation to protection against cell death. Given the importance of this pathway in maintaining cellular homeostasis, it has been considered that compromised autophagy could contribute to several of the commonly observed age-associated pathologies including neurodegeneration, reduction of muscle mass, cardiac malfunction, excessive lipid accumulation in tissues and glucose intolerance. The present chapter describes the two best-characterized autophagy pathways­macroautophagy and chaperone-mediated autophagy, and discusses how changes in these pathways associate with age-associated disorders. Understanding how to maintain "clean cells" by activation of autophagy could be an attractive strategy to maintain healthspan in aged individuals.

Insulin and leptin oscillations license food-entrained browning and metabolic flexibility.

Timed feeding drives adipose browning, although the integrative mechanisms for the same remain unclear. Here, we show that twice-a-night (TAN) feeding generates biphasic oscillations of circulating insulin and leptin, representing their entrainment by timed feeding. Insulin and leptin surges lead to marked cellular, functional, and metabolic remodeling of subcutaneous white adipose tissue (sWAT), resulting in increased energy expenditure. Single-cell RNA-sequencing (scRNA-seq) analyses and flow cytometry demonstrate a role for insulin and leptin surges in innate lymphoid type 2 (ILC2) cell recruitment and sWAT browning, since sWAT depot denervation or loss of leptin or insulin receptor signaling or ILC2 recruitment each dampens TAN feeding-induced sWAT remodeling and energy expenditure. Consistently, recreating insulin and leptin oscillations via once-a-day timed co-injections is sufficient to favorably remodel innervated sWAT. Innervation is necessary for sWAT remodeling, since denervation of sWAT, but not brown adipose tissue (BAT), blocks TAN-induced sWAT remodeling and resolution of inflammation. In sum, reorganization of nutrient-sensitive pathways remodels sWAT and drives the metabolic benefits of timed feeding.

An iron rheostat controls hematopoietic stem cell fate.

Mechanisms governing the maintenance of blood-producing hematopoietic stem and multipotent progenitor cells (HSPCs) are incompletely understood, particularly those regulating fate, ensuring long-term maintenance, and preventing aging-associated stem cell dysfunction. We uncovered a role for transitory free cytoplasmic iron as a rheostat for adult stem cell fate control. We found that HSPCs harbor comparatively small amounts of free iron and show the activation of a conserved molecular response to limited iron-particularly during mitosis. To study the functional and molecular consequences of iron restriction, we developed models allowing for transient iron bioavailability limitation and combined single-molecule RNA quantification, metabolomics, and single-cell transcriptomic analyses with functional studies. Our data reveal that the activation of the limited iron response triggers coordinated metabolic and epigenetic events, establishing stemness-conferring gene regulation. Notably, we find that aging-associated cytoplasmic iron loading reversibly attenuates iron-dependent cell fate control, explicating intervention strategies for dysfunctional aged stem cells.

Hepatoma cells from mice deficient in glycine N-methyltransferase have increased RAS signaling and activation of liver kinase B1.

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Human antigen R contributes to hepatic stellate cell activation and liver fibrosis.

UNLABELLED: 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-ß)-induced profibrogenic actions by regulating the expression of TGF-ß, α-SMA, and p21. This was likely the result of an increased cytoplasmic localization of HuR, controlled by TGF-ß-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-ß.

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

UNLABELLED: 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.

mTORC2-NDRG1-CDC42 axis couples fasting to mitochondrial fission.

Fasting triggers diverse physiological adaptations including increases in circulating fatty acids and mitochondrial respiration to facilitate organismal survival. The mechanisms driving mitochondrial adaptations and respiratory sufficiency during fasting remain incompletely understood. Here we show that fasting or lipid availability stimulates mTORC2 activity. Activation of mTORC2 and phosphorylation of its downstream target NDRG1 at serine 336 sustains mitochondrial fission and respiratory sufficiency. Time-lapse imaging shows that NDRG1, but not the phosphorylation-deficient NDRG1Ser336Ala mutant, engages with mitochondria to facilitate fission in control cells, as well as in those lacking DRP1. Using proteomics, a small interfering RNA screen, and epistasis experiments, we show that mTORC2-phosphorylated NDRG1 cooperates with small GTPase CDC42 and effectors and regulators of CDC42 to orchestrate fission. Accordingly, RictorKO, NDRG1Ser336Ala mutants and Cdc42-deficient cells each display mitochondrial phenotypes reminiscent of fission failure. During nutrient surplus, mTOR complexes perform anabolic functions; however, paradoxical reactivation of mTORC2 during fasting unexpectedly drives mitochondrial fission and respiration.

Glioblastoma Cells Counteract PARP Inhibition through Pro-Survival Induction of Lipid Droplets Synthesis and Utilization.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. Poly (ADP-ribose) polymerase inhibitors (PARPi) represent a new class of anti-neoplastic drugs. In the current study, we have characterized the mechanism by which glioblastoma cells evade the effect of PARPi as anti-tumor agents. We have found that suppression of PARP activity exerts an anti-stemness effect and has a dual impact on autophagy, inducing its activation in the first 24 h (together with down-regulation of the pro-survival mTOR pathway) and preventing autophagosomes fusion to lysosomes at later time-points, in primary glioma cells. In parallel, PARPi triggered the synthesis of lipid droplets (LDs) through ACC-dependent activation of de novo fatty acids (FA) synthesis. Notably, inhibiting ß-oxidation and blocking FA utilization, increased PARPi-induced glioma cell death while treatment with oleic acid (OA) prevented the anti-glioma effect of PARPi. Moreover, LDs fuel glioma cells by inducing pro-survival lipid consumption as confirmed by quantitation of oxygen consumption rates using Seahorse respirometry in presence or absence of OA. In summary, we uncover a novel mechanism by which glioblastoma escapes to anti-tumor agents through metabolic reprogramming, inducing the synthesis and utilization of LDs as a pro-survival strategy in response to PARP inhibition.

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.

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

UNLABELLED: 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.

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.

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.