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.

S-adenosylmethionine limitation induces p38 mitogen-activated protein kinase and triggers cell cycle arrest in G1.

The primary methyl group donor S-adenosylmethionine (SAM) is important for a plethora of cellular pathways including methylation of nucleic acids, proteins, and the 5' cap structure of mRNAs, as well as biosynthesis of phospholipids and polyamines. In addition, because it is the cofactor for chromatin methylation, SAM is an important metabolite for the establishment and maintenance of epigenetic marks. Here, we demonstrate that cells halt proliferation when SAM levels become low. Cell cycle arrest occurs primarily in the G1 phase of the cell cycle and is accompanied by activation of the mitogen-activated protein kinase p38 (MAPK14) and subsequent phosphorylation of MAPK-activated protein kinase-2 (MK2). Surprisingly, Cdk4 activity remains high during cell cycle arrest, whereas Cdk2 activity decreases concomitantly with cyclin E levels. Cell cycle arrest was induced by both pharmacological and genetic manipulation of SAM synthesis through inhibition or downregulation of methionine adenosyltransferase, respectively. Depletion of methionine, the precursor of SAM, from the growth medium induced a similar cell cycle arrest. Unexpectedly, neither methionine depletion nor inhibition of methionine adenosyltransferase significantly affected mTORC1 activity, suggesting that the cellular response to SAM limitation is independent from this major nutrient-sensing pathway. These results demonstrate a G1 cell cycle checkpoint that responds to limiting levels of the principal cellular methyl group donor S-adenosylmethionine. This metabolic checkpoint might play important roles in maintenance of epigenetic stability and general cellular integrity.

The metabolic burden of methyl donor deficiency with focus on the betaine homocysteine methyltransferase pathway.

Methyl groups are important for numerous cellular functions such as DNA methylation, phosphatidylcholine synthesis, and protein synthesis. The methyl group can directly be delivered by dietary methyl donors, including methionine, folate, betaine, and choline. The liver and the muscles appear to be the major organs for methyl group metabolism. Choline can be synthesized from phosphatidylcholine via the cytidine-diphosphate (CDP) pathway. Low dietary choline loweres methionine formation and causes a marked increase in S-adenosylmethionine utilization in the liver. The link between choline, betaine, and energy metabolism in humans indicates novel functions for these nutrients. This function appears to goes beyond the role of the nutrients in gene methylation and epigenetic control. Studies that simulated methyl-deficient diets reported disturbances in energy metabolism and protein synthesis in the liver, fatty liver, or muscle disorders. Changes in plasma concentrations of total homocysteine (tHcy) reflect one aspect of the metabolic consequences of methyl group deficiency or nutrient supplementations. Folic acid supplementation spares betaine as a methyl donor. Betaine is a significant determinant of plasma tHcy, particularly in case of folate deficiency, methionine load, or alcohol consumption. Betaine supplementation has a lowering effect on post-methionine load tHcy. Hypomethylation and tHcy elevation can be attenuated when choline or betaine is available.

S-adenosyl methionine: a natural therapeutic agent effective against multiple hallmarks and risk factors associated with Alzheimer's disease.

Recent preclinical and clinical findings demonstrate that dietary supplementation with S-adenosyl methionine alleviates a variety of risk factors and hallmarks associated with Alzheimer's disease. These findings support and extend prior studies, some of which are decades old, and support the notion that nutritional supplementation may represent an important augmentation for therapy in Alzheimer's disease.

B-vitamin deprivation induces hyperhomocysteinemia and brain S-adenosylhomocysteine, depletes brain S-adenosylmethionine, and enhances PS1 and BACE expression and amyloid-beta deposition in mice.

Etiological and molecular studies on the sporadic form of Alzheimer's disease have yet to determine the underlying mechanisms of neurodegeneration. Hyperhomocysteinemia is associated with Alzheimer's disease, and has been hypothesized to promote neurodegeneration, by inhibiting brain methylation activity. The aim of this work was to determine whether a combined folate, B12 and B6 dietary deficiency, would induce amyloid-beta overproduction, and to study the mechanisms linking vitamin deficiency, hyperhomocysteinemia and amyloidogenesis in TgCRND8 and 129Sv mice. We confirmed that B-vitamin deprivation induces hyperhomocysteinemia and imbalance of S-adenosylmethionine and S-adenosylhomocysteine. This effect was associated with PS1 and BACE up-regulation and amyloid-beta deposition. Finally, we detected intraneuronal amyloid-beta and a slight cognitive impairment in a water maze task at a pre-plaque age, supporting the hypothesis of early pathological function of intracellular amyloid. Collectively, these findings are consistent with the hypothesis that abnormal methylation in association with hyperhomocysteinemia may contribute to Alzheimer's disease.

Molecular profiling of hepatocellular carcinoma in mice with a chronic deficiency of hepatic s-adenosylmethionine: relevance in human liver diseases.

S-adenosylmethionine arises as a central molecule in the preservation of liver homeostasis as a chronic hepatic deficiency results in spontaneous development of steatohepatitis and hepatocellular carcinoma. In the present work, we have attempted a comprehensive analysis of proteins associated with hepatocarcinogenesis in MAT1A knock out mice using a combination of two-dimensional electrophoresis and mass spectrometry, to then apply the resulting information to identify hallmarks of human HCC. Our results suggest the existence of individual-specific factors that might condition the development of preneoplastic lesions. Proteomic analysis allowed the identification of 151 differential proteins in MAT1A-/- mice tumors. Among all differential proteins, 27 changed in at least 50% of the analyzed tumors, and some of these alterations were already detected months before the development of HCC in the KO liver. The expression level of genes coding for 13 of these proteins was markedly decreased in human HCC. Interestingly, seven of these genes were also found to be down-regulated in a pretumoral condition such as cirrhosis, while depletion of only one marker was assessed in less severe liver disorders.

Impaired liver regeneration in mice lacking methionine adenosyltransferase 1A.

Methionine adenosyltransferase (MAT) is an essential enzyme because it catalyzes the formation of S-adenosylmethionine (SAMe), the principal biological methyl donor. Of the two genes that encode MAT, MAT1A is mainly expressed in adult liver and MAT2A is expressed in all extrahepatic tissues. Mice lacking MAT1A have reduced hepatic SAMe content and spontaneously develop hepatocellular carcinoma. The current study examined the influence of chronic hepatic SAMe deficiency on liver regeneration. Despite having higher baseline hepatic staining for proliferating cell nuclear antigen, MAT1A knockout mice had impaired liver regeneration after partial hepatectomy (PH) as determined by bromodeoxyuridine incorporation. This can be explained by an inability to up-regulate cyclin D1 after PH in the knockout mice. Upstream signaling pathways involved in cyclin D1 activation include nuclear factor kappaB (NFkappaB), the c-Jun-N-terminal kinase (JNK), extracellular signal-regulated kinases (ERKs), and signal transducer and activator of transcription-3 (STAT-3). At baseline, JNK and ERK are more activated in the knockouts whereas NFkappaB and STAT-3 are similar to wild-type mice. Following PH, early activation of these pathways occurred, but although they remained increased in wild-type mice, c-jun and ERK phosphorylation fell progressively in the knockouts. Hepatic SAMe levels fell progressively following PH in wild-type mice but remained unchanged in the knockouts. In culture, MAT1A knockout hepatocytes have higher baseline DNA synthesis but failed to respond to the mitogenic effect of hepatocyte growth factor. Taken together, our findings define a critical role for SAMe in ERK signaling and cyclin D1 regulation during regeneration and suggest chronic hepatic SAMe depletion results in loss of responsiveness to mitogenic signals.

Methyl deficiency causes reduction of the methyl-CpG-binding protein, MeCP2, in rat liver.

MeCP2 is a member of a family of proteins [methyl- (cytosine-guanine)CpG-binding proteins] that bind specifically to methylated DNA and induce chromatin remodeling and gene silencing. Dietary deficiency of folate, choline and methionine causes decreased tissue S-adenosylmethionine concentrations (methyl deficiency), global DNA hypomethylation, hepatic steatosis, cirrhosis and ultimately hepatic tumorigenesis in rodents. We investigated the effects of this diet on expression of MeCP2 during pre-neoplastic transformation of liver tissue. After 9 weeks, MeCP2 mRNA level was slightly higher in methyl-deficient rats compared with replete controls, while after 36 weeks, a difference in MeCP2 mRNA level was no longer observed. In contrast, MeCP2 protein level was reduced almost 2-fold in the deficient rats compared with replete controls at both 9 and 36 weeks. Conversely, a second methyl-CpG-binding protein, MBD2, showed increased levels of both message and protein at the two time points. Low MeCP2 protein in the deficient rats was associated with a low level of the co-repressor protein, Sin3a, at 36 weeks. Moreover, a known gene target of MeCP2, the tumor suppressor gene metallothionein-I, was over-expressed in the deficient rat livers at both 9 and 36 weeks, suggesting that reduction in MeCP2 may have functional consequences. Methyl deficiency also caused an increase in the ratio of long to short variants of MeCP2 transcripts. This finding suggests that reduced MeCP2 protein level is the result of a reduced rate of translation. Reduction of MeCP2 protein expression may influence the initiation and/or progression of hepatic cancer induced by methyl deficiency and may provide a useful marker of pre-neoplastic change.

Dose-dependent biotransformation of arsenite in rats--not S-adenosylmethionine depletion impairs arsenic methylation at high dose.

Arsenite (AsIII) is eliminated via excretion and methylation. Monomethylarsonous acid (MMAsIII) is a super toxic metabolite of AsIII, while dimethylarsinic acid produced in the next metabolic step is relatively atoxic. Since the role of methylation in the acute toxicity and elimination of AsIII in vivo is unclear, we have examined the excretion and tissue retention of AsIII and its metabolites in rats exposed to increasing AsIII doses. Rats were injected i.v. with 20, 50 and 125 micromol/kg AsIII and arsenic metabolites in bile, urine and tissues were analysed. The excretion of AsIII increased almost proportionately to the dose, while its concentration in tissues rose more than proportionately. In contrast, the excretion and tissue concentrations of methylated metabolites increased less than the dosage, or they even decreased after injection of the largest dose of AsIII. To elucidate the mechanism of the dose-dependent decrease of methylation, we quantified S-adenosylmethionine (SAME), glutathione (GSH), and adenine nucleotides in the liver of AsIII-injected rats. AsIII decreased the hepatic concentrations of GSH and adenosine 5'-triphosphate (ATP) and the energy charge in a dose-dependent manner, but increased the level of SAME. Thus, impaired methylation after AsIII overdose is not due to SAME shortage, but probably to methyltransferase inhibition. It appears that exhausted elimination capacity of AsIII, rather than MMAsIII produced from AsIII, contributes significantly to the acute toxicity of AsIII. After GSH depletion the retained AsIII can increasingly inhibit SH-enzymes, thus causing ATP depletion and energetic disorder.

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.

S-Adenosyl-L-methionine and alcoholic liver disease in animal models: implications for early intervention in human beings.

In patients with severe alcoholic liver disease (i.e., cirrhosis), a deficiency of S-adenosylmethionine (SAMe) develops as a result of decreased SAMe synthetase activity. Whether a sizeable SAMe depletion occurs already at earlier stages of alcoholic liver disease has been the subject of debate. To address this issue, rats were fed alcohol (or isocaloric carbohydrate) in Lieber-DeCarli liquid diets containing adequate amounts of protein, vitamins, and lipotropic factors, including methionine. Alcohol feeding resulted in hepatic steatosis (without fibrosis) and unchanged SAMe synthetase activity, yet SAMe concentration was already greatly decreased. This most likely resulted from oxidative stress associated with the metabolism of alcohol and the induction of cytochrome P4502E1 (CYP2E1), which generates free radicals. Indeed, the decrease in hepatic SAMe correlated with parameters of oxidative stress, such as increased 4-hydroxynonenal (measured by gas chromatography-mass spectrometry) and diminished glutathione (GSH). Decreased GSH, occurring as a result of excessive GSH consumption caused by the oxidative stress, probably generated by enhanced utilization of SAMe, a precursor of GSH, thereby explaining the depletion of SAMe. In view of the known differences between rodents and primates in the metabolism of lipotropes, my colleagues and I have also studied the interaction between alcohol and SAMe in baboons and found again that, at early stages preceding the development of cirrhosis, there was already a significant lowering of hepatic SAMe concentration, associated with a striking oxidative stress documented by decreased levels and accelerated turnover of GSH. This was associated with increased lipid peroxidation and damage to cellular membranes, including those of the mitochondria, assessed by electron microscopy. Oral administration of SAMe resulted in its hepatic repletion with a corresponding attenuation of the ethanol-induced oxidative stress and liver injury, with significantly less GSH depletion, less increases in plasma aspartate aminotransferase (AST) levels, less leakage of mitochondrial glutamic dehydrogenase into the plasma, and fewer megamitochondria. In conclusion, (1) both in rodents and in non-human primates, significant SAMe depletion occurs already at early stages of alcoholic liver disease, despite the consumption of adequate diets; (2) the decreased hepatic SAMe concentration and the associated liver lesions, including mitochondrial injury, can be corrected with SAMe supplementation; and (3) accordingly, therapeutic administration of SAMe should be the subject of a comprehensive clinical trial to assess its capacity to attenuate early stages of alcoholic liver injury in human beings.

S-Adenosyl-L-methionine and mitochondrial reduced glutathione depletion in alcoholic liver disease.

The pathogenesis of alcohol-induced liver disease is not well understood, and many factors have been described to contribute to the progressive loss of liver functions, including the overgeneration of reactive oxygen species. Mitochondria are specific targets of the toxic effects of ethanol, reflected in the loss of phosphorylative oxidation and defective ATP generation, which underlie one of the hallmarks of the hepatic alterations induced by chronic alcohol intake. Mitochondrial reduced glutathione (GSH), whose primary function is to maintain a competitive functional organelle, becomes depleted by alcohol intake. Furthermore, GSH depletion in hepatocyte mitochondria has been revealed as an important mechanism in the sensitization of liver to alcohol-induced injury. This depletion of the mitochondrial GSH level is determined by an impaired transport of GSH from the cytosol into the mitochondrial matrix owing to a partial inactivation of mitochondrial GSH carrier. The loss of function of this specific mitochondrial transporter is due to the alterations in the physicochemical properties of the inner mitochondrial membrane caused by alcohol. Because of the primary defect in the transport of cytosolic GSH into mitochondria, GSH precursors are inefficient in replenishing the levels of mitochondrial GSH despite significant increase in cytosolic GSH. Supplementation of S-adenosyl-L-methionine (SAM) to rats fed alcohol chronically has been shown to replete the mitochondrial GSH levels because of normalization of the microviscosity of the mitochondrial inner membrane. Because of the instrumental role of GSH in mitochondria in hepatocyte survival against inflammatory cytokines, its repletion by SAM feeding may underlie the potential therapeutic use of this hepatoprotective agent in the treatment of alcohol-induced liver injury.

Spontaneous oxidative stress and liver tumors in mice lacking methionine adenosyltransferase 1A.

In mammals, methionine metabolism occurs mainly in the liver via methionine adenosyltransferase-catalyzed conversion to S-adenosylmethionine. Of the two genes that encode methionine adenosyltransferase(MAT1Aand MAT2A), MAT1A is mainly expressed in adult liver whereas MAT2A is expressed in all extrahepatic tissues. Mice lacking MAT1A have reduced hepatic S-adenosylmethionine content and hyperplasia and spontaneously develop nonalcoholic steatohepatitis. In this study, we examined whether chronic hepatic S-adenosylmethionine deficiency generates oxidative stress and predisposes to injury and malignant transformation. Differential gene expression in MAT1A knockout mice was analyzed following the criteria of the Gene Ontology Consortium. Susceptibility of MAT1A knockout mice to CCl4-induced hepatotoxicity and malignant transformation was determined in 3- and 18-month-old mice, respectively. Analysis of gene expression profiles revealed an abnormal expression of genes involved in the metabolism of lipids and carbohydrates in MAT1A knockout mice, a situation that is reminiscent of that found in diabetes, obesity, and other conditions associated with nonalcoholic steatohepatitis. This aberrant expression of metabolic genes in the knockout mice was associated with hyperglycemia, increased hepatic CYP2E1 and UCP2 expression and triglyceride levels, and reduced hepatic glutathione content. The knockout animals have increased lipid peroxidation and enhanced sensitivity to CCl4-induced liver damage, which was largely due to increased CYP2E1 expression because diallyl sulfide, an inhibitor of CYP2E1, prevented CCl4-induced liver injury. Hepatocellular carcinoma developed in more than half of the knockout mice by 18 months of age. Taken together, our findings define a critical role for S-adenosylmethionine in maintaining normal hepatic function and tumorigenesis of the liver.

Liver diseases by alcohol and hepatitis C: early detection and new insights in pathogenesis lead to improved treatment.

Much progress has been made in the understanding of the pathogenesis of alcoholic liver disease, resulting in improvement of treatment. Therapy must include correction of nutritional deficiencies, while taking into account changes of nutritional requirements. Methionine is normally activated to S-adenosylmethionine (SAMe). However, in liver disease, the corresponding enzyme is depressed. The resulting deficiencies can be attenuated by the administration of SAMe but not by methionine. Similarly, phosphatidylethanolamine methyltransferase activity is depressed, but the lacking phosphatidylcholine (PC) can be administrated as polyenylphosphatidylcholine (PPC). Chronic ethanol consumption increases CYP2E1, resulting in increased generation of toxic acetaldehyde and free radicals, tolerance to ethanol and other drugs, and multiple ethanol-drug interactions. Experimentally, PPC opposes CYP2E1 induction and fibrosis. Alcoholism and hepatitis C infection commonly co-exist, with acceleration of fibrosis, cirrhosis, and hepatocellular carcinoma. PPC is being tested clinically as a corresponding antifibrotic agent. Available antiviral agents are contraindicated in the alcoholic. Anti-inflammatory agents, such as steroids, may be selectively useful. Finally, anticraving agents, such as naltrexone or acamprosate, should be part of therapy.

ALCOHOL: its metabolism and interaction with nutrients.

In the past, alcoholic liver disease was attributed exclusively to dietary deficiencies, but experimental and judicious clinical studies have now established alcohol's hepatotoxicity. Despite an adequate diet, it can contribute to the entire spectrum of liver diseases, mainly by generating oxidative stress through its microsomal metabolism via cytochrome P4502E1 (CYP2E1). It also interferes with nutrient activation, resulting in changes in nutritional requirements. This is exemplified by methionine, one of the essential amino acids for humans, which needs to be activated to S-adenosylmethionine (SAMe), a process impaired by liver disease. Thus, SAMe rather than methionine is the compound that must be supplemented in the presence of significant liver disease. In baboons, SAMe attenuated mitochondrial lesions and replenished glutathione; it also significantly reduced mortality in patients with Child A or B cirrhosis. Similarly, decreased phosphatidylethanolamine methyltransferase activity is associated with alcoholic liver disease, resulting in phosphatidylcholine depletion and serious consequences for the integrity of membranes. This can be offset by polyenylphosphatidylcholine (PPC), a mixture of polyunsaturated phosphatidylcholines comprising dilinoleoylphosphatidylcholine (DLPC), which has high bioavailability. PPC (and DLPC) opposes major toxic effects of alcohol, with down-regulation of CYP2E1 and reduction of oxidative stress, deactivation of hepatic stellate cells, and increased collagenase activity, which in baboons, results in prevention of ethanol-induced septal fibrosis and cirrhosis. Corresponding clinical trials are ongoing.

[Postoperative combined medullary sclerosis revealing Biermer's disease: toxic effect of nitrous oxide]. / Sclérose combinée de la moelle post-opératoire révélant une maladie de Biermer: toxicité du protoxyde d'azote.

Combined medullary sclerosis developed suddenly postoperatively in a patient with unknown Biermer's disease. The neurological lesions were undoubtedly induced by nitrogen protoxide via an inactivation of vitamin B12.

Lack of S-adenosylmethionine results in a cell division defect in Escherichia coli.

The enzyme S-adenosylmethionine (SAM) synthetase, the Escherichia coli metK gene product, produces SAM, the cell's major methyl donor. We show here that SAM synthetase activity is induced by leucine and repressed by Lrp, the leucine-responsive regulatory protein. When SAM synthetase activity falls below a certain critical threshold, the cells produce long filaments with regularly distributed nucleoids. Expression of a plasmid-carried metK gene prevents filamentation and restores normal growth to the metK mutant. This indicates that lack of SAM results in a division defect.

S-adenosylmethionine deficiency and TNF-alpha in lipopolysaccharide-induced hepatic injury.

S-adenosylmethionine (Adomet) is a substrate for de novo synthesis of choline. Adomet deficiency occurs in certain types of liver injury, and the injury is attenuated by exogenous Adomet. Tumor necrosis factor-alpha (TNF-alpha) is also a mediator of these models of hepatotoxicity. We investigated the role of Adomet in lipopolysaccharide (LPS)-induced liver injury in rats made deficient in both Adomet and choline. Rats were maintained on either a methionine-restricted and choline-deficient (MCD) diet or a diet containing sufficient amounts of all nutrients [methionine and choline sufficient (MCS)] and then administered either LPS or saline. MCS-LPS rats had normal liver histology and no change in serum transaminases compared with the MCS-saline control group. MCD-saline rats had hepatosteatosis but no necrosis, and a five- to sevenfold increase in transaminases vs. the MCS-saline group. MCD-LPS rats additionally had hepatonecrosis and a 30- to 50-fold increase in transaminases. Exogenous Adomet administration to MCD-LPS rats corrected the hepatic deficiency of Adomet but not of choline, prevented necrosis but not steatosis, and attenuated transaminases. Serum TNF-alpha was sixfold higher in MCD rats even without LPS challenge and 300-fold higher with LPS challenge. Exogenous Adomet attenuated increased serum TNF-alpha in MCD-LPS rats.