S-adenosylmethionine in treatment of cholestasis after total parenteral nutrition: laboratory investigation and clinical application

Objective: To observe the effects of S-adenosylmethio-nine (SAMe) in the treatment of cholestasis after totalparenteral nutrition (TPN).Methods: Thirty SD rats were randomly divided intocontrol group, hypercalorie group, hypercalorie+SAMegroup, sepsis group and sepsis+SAMe group to com-pare their states of cholestasis. Sixteen patients re-ceived SAMe because of cholestasis after prolongedTPN, and the therapeutic efficacy was observed.Results: Bile flow was obviously decreased and theserum levels of total bile acid and gamma-glutamyltranspeptidase(γ-GT) were markedly increased in thehypercalorie and sepsis groups. Meanwhile, hepatocytefatty degeneration, dilatation of cholangioles, and bilesludge could be seen microscopically. SAMe adminis-tration in the hypercalorie+SAMe and sepsis+SAMegroups could increase the bile flow, decrease theserum levels of total bile acid and γ-GT, reduce thepathological damage to the liver, and clear the bilesludge in the cholangioles. Cholestasis and abnormalliver function were the main manifestations of the 16patients before SAMe administration. After SAMe treat-ment for 3 weeks, serum levels of total bilirubin, al-kaline phosphatase(AKP), γ-GT, alanine aminotrans-ferase(ALT), and aspartate aminotransferase(AST)were obviously decreased, and normalized in the 4thweek.Conclusion: SAMe could prevent and treat cholestasiswithout discontinuation of TPN.

Early treatment efficacy of S-adenosylmethionine in patients with intrahepatic cholestasis: A systematic review

BACKGROUND S-adenosylmethionine(AdoMet)is a metabolically pleiotropic molecule used to treat intrahepatic cholestasis(IHC)and chronic liver diseases.While the efficacy of AdoMet has been demonstrated previously,it has not been systematically investigated within the early weeks of treatment.AIM To systematically review the early treatment efficacy of AdoMet in adult patients with IHC.METHODS Studies reporting the efficacy of intravenous,intramuscular,or oral forms of AdoMet within 8 wk of treatment initiation were considered;three randomized and six non-randomized studies were eligible for inclusion(PROSPERO registration number CRD42018090936).Of the three randomized studies,two were double-blind and placebo-controlled,and one was comparator-controlled with unclear blinding and a relatively high risk of bias.Mean serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),and gamma-glutamyl transferase(γGT)following AdoMet treatment vs placebo,comparator,or baseline were summarized to determine differences in liver enzymes.Changes in patient-reported clinical symptoms of cholestasis were also summarized.RESULTS Both placebo-controlled randomized studies reported significant reductions in serum ALT levels with AdoMet vs placebo within 2 wk.One of these also reported significant ALP reductions,and the other reported significant AST andγGT reductions within 2 wk.The comparator-controlled randomized study,which had a number of notable limitations,reported significant reductions in serum ALT and AST levels with AdoMet vs potassium magnesium aspartate within 4 wk,but not within2 wk.All of the non-randomized studies(4/4)that investigated ALT,AST,ALP and/orγGT reported significant reductions in at least two of these parameters within 2 wk.Of the five studies that evaluated fatigue,reductions were observed within 2 wk in one randomized and two nonrandomized studies.The remaining two non-randomized studies reported improvements in fatigue within 6 and 8 wk.Of the four studies reporting symptoms of depression,two non-randomized studies observed improvements within 2 wk and the other two observed improvements within 17 d and 8 wk.CONCLUSION Data from both randomized and non-randomized studies suggest that AdoMet improves some biochemical liver parameters and symptoms of cholestasis within 2 wk,with further improvements observed in some studies after 4 and 8 wk of treatment.

A Prospective, Open-Label, 12 Week Trial of S-adenosylmethionine in the Symptomatic Treatment of Alzheimer’s Disease

Objective: To determine if treatment with S-adenosylmethionine (SAM-e) might lead to cognitive and behavioral improvement in patients with Alzheimer’s disease (AD). Interventions: We conducted a prospective, open-label study of six subjects who were given oral SAM-e over 12 weeks and measured the effects on cognition and behavior. Outcome measures: Outcome measures of cognition and behavior included the Alzheimer’s Disease Assessment Scale-Cognitive subscale (ADAS-Cog), Mini-Mental State Examination (MMSE), Behave-AD, Clinical Global Impression Scale (CGI), Hamilton Rating Scale for Depression (HAM-D), informant version, Blessed Dementia Scale to assess activities of daily living (ADL), the Physical Self-Maintainance Scale (PSMS), and the Lawton-Brody IADL Scale to measure instrumental activities of daily living. Results: At study completion, two subjects were “moderately improved” and 4 were “minimally improved” on the CGI. Five subjects improved on the ADAS-Cog by an average of 20%. No significant side effects were reported. Conclusions: In this small open label study, SAM-e appeared to have a beneficial effect in patients with AD, but the small subject number didn’t provide enough power to show statistical significance. Controlled trials with adequate statistical power to investigate its utility in AD are warranted.

S-adenosylmethionine Administration Attenuates Low Brain-Derived Neurotrophic Factor Expression Induced by Chronic Cerebrovascular Hypoperfusion or Beta Amyloid Treatment

Chronic cerebrovascular hypoperfusion is a high-risk factor for Alzheimer＇s disease（AD） as it is conducive to beta amyloid（Ab） over-production. Brainderived neurotrophic factor（BDNF） is a member of the neurotrophin family widely expressed in the central nervous system. The structure of the rat BDNF gene is complex, consisting of eight non-coding exons（I–VIII） and one coding exon（IX）. The BDNF gene is transcribed from multiple promoters located upstream of different 50 noncoding exons to produce a heterogeneous population of BDNF m RNAs. S-adenosylmethionine（SAM） produced in the methionine cycle is the primary methyl donor and the precursor of glutathione. In this study, a cerebrovascular hypoperfusion rat model and an Ab intrahippocampal injection rat model were used to explore the expression profiles of all BDNF transcripts in the hippocampus with chronic cerebrovascular hypoperfusion or Ab injection as well as with SAM treatment. We found that the BDNF m RNAs and protein were down-regulated in the hippocampus undergoing chronic cerebrovascular hypoperfusion as well as Ab treatment, and BDNF exons IV and VI played key roles. SAM improved the low BDNF expression following these insults mainly through exons IV and VI.These results suggest that SAM plays a neuroprotective role by increasing the expression of endogenous BDNF and could be a potential target for AD therapy.

Biomarkers and subtypes of deranged lipid metabolism in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease(NAFLD)is a heterogeneous and complex disease that is imprecisely diagnosed by liver biopsy.NAFLD covers a spectrum that ranges from simple steatosis,nonalcoholic steatohepatitis(NASH)with varying degrees of fibrosis,to cirrhosis,which is a major risk factor for hepatocellular carcinoma.Lifestyle and eating habit changes during the last century have made NAFLD the most common liver disease linked to obesity,type 2 diabetes mellitus and dyslipidemia,with a global prevalence of 25%.NAFLD arises when the uptake of fatty acids(FA)and triglycerides(TG)from circulation and de novo lipogenesis saturate the rate of FAβ-oxidation and verylow density lipoprotein(VLDL)-TG export.Deranged lipid metabolism is also associated with NAFLD progression from steatosis to NASH,and therefore,alterations in liver and serum lipidomic signatures are good indicators of the disease’s development and progression.This review focuses on the importance of the classification of NAFLD patients into different subtypes,corresponding to the main alteration(s)in the major pathways that regulate FA homeostasis leading,in each case,to the initiation and progression of NASH.This concept also supports the targeted intervention as a key approach to maximize therapeutic efficacy and opens the door to the development of precise NASH treatments.

Methionine adenosyltransferases in liver cancer

Methionine adenosyltransferases(MATs)are essential enzymes for life as they produce S-adenosylmethionine(SAMe),the biological methyl donor required for a plethora of reactions within the cell.Mammalian systems express two genes,MAT1A and MAT2A,which encode for MATα1 and MATα2,the catalytic subunits of the MAT isoenzymes,respectively.A third gene MAT2B,encodes a regulatory subunit known as MATβwhich controls the activity of MATα2.MAT1A,which is mainly expressed in hepatocytes,maintains the differentiated state of these cells,whilst MAT2A and MAT2B are expressed in extrahepatic tissues as well as non-parenchymal cells of the liver(e.g.,hepatic stellate and Kupffer cells).The biosynthesis of SAMe is impaired in patients with chronic liver disease and liver cancer due to decreased expression and inactivation of MATα1.A switch from MAT1A to MAT2A/MAT2B occurs in multiple liver diseases and during liver growth and dedifferentiation,but this change in the expression pattern of MATs results in reduced hepatic SAMe level.Decades of study have utilized the Mat1a-knockout(KO)mouse that spontaneously develops non-alcoholic steatohepatitis(NASH)and hepatocellular carcinoma(HCC)to elucidate a variety of mechanisms by which MAT proteins dysregulation contributes to liver carcinogenesis.An increasing volume of work indicates that MATs have SAMe-independent functions,distinct interactomes and multiple subcellular localizations.Here we aim to provide an overview of MAT biology including genes,isoenzymes and their regulation to provide the context for understanding consequences of their dysregulation.We will highlight recent breakthroughs in the field and underscore the importance of MAT’s in liver tumorigenesis as well as their potential as targets for cancer therapy.

Oxidative stress modulation in hepatitis C virus infected cells

Hepatitis C virus(HCV) replication is associated with the endoplasmic reticulum, where the virus can induce cellular stress. Oxidative cell damage plays an important role in HCV physiopathology. Oxidative stress is triggered when the concentration of oxygen species in the extracellular or intracellular environment exceeds antioxidant defenses. Cells are protected and modulate oxidative stress through the interplay of intracellular antioxidant agents, mainly glutathione system(GSH) and thioredoxin; and antioxidant enzyme systems such as superoxide dismutase, catalase, GSH peroxidase, and heme oxygenase-1. Also, the use of natural and synthetic antioxidants(vitamin C and E, N-acetylcysteine, glycyrrhizin, polyenylphosphatidyl choline, mitoquinone, quercetin, S-adenosylmethionine and silymarin) has already shown promising results as co-adjuvants in HCV therapy. Despite all the available information, it is not known how different agents with antiviral activity can interfere with the modulation of the cell redox state induced by HCV and decrease viral replication. This review describes an evidence-based consensus on molecular mechanisms involved in HCV replication and their relationship with cell damage induced by oxidative stress generated by the virus itself and cell antiviral machinery. It also describes some molecules that modify the levels of oxidative stress in HCV-infected cells.

Prolonged feeding with guanidinoacetate, a methyl group consumer, exacerbates ethanol-induced liver injury

AIM To investigate the hypothesis that exposure to guanidinoacetate(GAA, a potent methyl-group consumer) either alone or combined with ethanol intake for a prolonged period of time would cause more advanced liver pathology thus identifying methylation defects as the initiator and stimulator for progressive liver damage.METHODS Adult male Wistar rats were fed the control or ethanolLieber De Carli diet in the absence or presence of GAA supplementation. At the end of 6 wk of the feeding regimen, various biochemical and histological analyses were conducted. RESULTS Contrary to our expectations, we observed that GAA treatment alone resulted in a histologically normal liver without evidence of hepatosteatosis despite persistence of some abnormal biochemical parameters. This protection could result from the generation of creatine from the ingested GAA. Ethanol treatment for 6 wk exhibited changes in liver methionine metabolism and persistence of histological and biochemical defects as reported before. Further, when the rats were fed the GAA-supplemented ethanol diet, similar histological and biochemical changes as observed after 2 wk of combined treatment, including inflammation, macroand micro-vesicular steatosis and a marked decrease in the methylation index were noted. In addition, rats on the combined treatment exhibited increased liver toxicity and even early fibrotic changes in a subset of animals in this group. The worsening liver pathology could be related to the profound reduction in the hepatic methylation index, an increased accumulation of GAA and the inability of creatine generated to exert its hepato-protective effects in the setting of ethanol.CONCLUSION To conclude, prolonged exposure to a methyl consumer superimposed on chronic ethanol consumption causes persistent and pronounced liver damage.

PDRG1 at the interface between intermediary metabolism and oncogenesis

PDRG1 is a small oncogenic protein of 133 residues. In normal human tissues, the p53 and DNA damageregulated gene 1(PDRG1) gene exhibits maximal expression in the testis and minimal levels in the liver. Increased expression has been detected in several tumor cells and in response to genotoxic stress. High-throughput studies identified the PDRG1 protein in a variety of macromolecular complexes involved in processes that are altered in cancer cells. For example, this oncogene has been found as part of the RNA polymerase Ⅱ complex, the splicing machinery and nutrient sensing machinery, although its role in these complexes remains unclear. More recently, the PDRG1 protein was found as an interaction target for the catalytic subunits of methionine adenosyltransferases. These enzymes synthesize S-adenosylmethionine, the methyl donor for, among others, epigenetic methylations that occur on the DNA and histones. In fact, downregulation of S-adenosylmethionine synthesis is the first functional effect directly ascribed to PDRG1. The existence of global DNA hypomethylation, together with increased PDRG1 expression, in many tumor cells highlights the importance of this interaction as one of the putative underlying causes for cell transformation. Here, we will review the accumulated knowledge on this oncogene, emphasizing the numerous aspects that remain to be explored.

Prostate Specific Antigen Promoter-Driven Adenovirus-Mediated Expression of Both ODC and AdoMetDC Antisenses Inhibit Prostate Cancer Growth

Objective：To generate recombinant adenovirus that could simultaneously express ornithine decarboxylase（ODC） and S-adenosylmethionine decarboxylase（AdoMetDC） antisenses specifically in prostate cancer cells,and evaluate its inhibitory effect on prostate cancer in vivo.Methods：Fragments of ODC and AdoMetDC genes were generated by PCR,cloned into the pPGL-PSES,and then recombined with pAdEasy-1 vectors in AdEasy-1 cells.Ad-PSES-ODC-AdoMetDCas virus was produced in HEK293 cells.Following transfection with Ad-PSES-ODC-AdoMetDCas,the levels of ODC or AdoMetDC were determined by RT-PCR and western blot assays.The effect of Ad-PSES-ODC-AdoMetDCas treatment on tumor formation and growth was evaluated in xenograft models of prostate cancers in vivo.Results：The plasmid pAdEasy-PSES-ODC-AdoMetDCas was successfully constructed and the recombinant Ad-PSES-ODC-AdoMetDCas adenovirus was produced.Transfection with Ad-PSES-ODC-AdoMetDCas adenovirus significantly inhibited the expression of ODC and AdoMetDC genes specifically in prostate DU145 cells,but not H1299,HT29 and HepG2 cancer cells,and disrupted the ability of DU145 cells to form solid prostate cancer in vivo.Intratumoral treatment with Ad-PSES-ODC-AdoMetDCas adenovirus significantly inhibited the growth of engrafted prostate tumors in vivo.Conclusion：The recombinant Ad-PSES-ODC-AdoMetDCas adenovirus specifically reduces the expression of both ODC and AdoMetDC genes in prostate cells and may be used for treatment of prostate cancers at the clinic.

Cloning and Analysis of Genes SAMS From Glycine soja

S-adenosylmethionine （SAM） plays important role in trans-methyl reactions. Under the condition of drought （30% PEG）, salinity （200 mmol· L^-1 NaCl） and low temperature （4℃）, total RNA was extracted from the leaf and the first strand of cDNA was synthesized with reverse transcription. S-adenosylmethionine synthetase gene （SAMS gene） was amplified by PCR with the first strand cDNA as template and a pair of primers which was based on constructed ESTs sequence. Full-length SAMS gene sequence was obtained by BLAST comparison. According to the analysis, completed sequence of SAMS gene was integrality. The sequence of the SAMS gene was 1 185 bp in length with an opening reading frame （ORF） encoding 394 amino acids. The cDNA sequence showed a significant homology to the SAM genes from Phaseolus lunatus （89%）, Medicago sativa （85%）. A prokaryotic expression vectors based on pET-32b had been constructed and prokaryotic expression was analyzed in order to lay a strong foundation for resist adversity function analysis through situation of genic expression analysis.

Maternally-preset program of apoptosis and caspases involved in execution of the apoptosis at midblastula transition (MBT) but not before in <i>Xenopus laevis</i>embryogenesis

To study gene control mechanisms in Xenopus embryos, we analyzed polyamines, cloned SAMDC (S-adenosylmethionine decarboxylase), a key enzyme of polyamine metabolism, and microinjected its mRNA into Xenopus fertilized eggs. The microinjection induced a large increase in SAMDC activity, exhaustion of the substrate SAM (S-adenosylmethionine), and execution of apoptosis at the stage called midblastula transition (MBT). By tracing GFP (green fluorescence protein)-marked apoptotic cells, we reached a conclusion that the apoptosis provides pre-blastula embryos with a fail-safe mechanism of early development. We analyzed caspase mRNAs and found that caspase-9 and -3 mRNAs are maternal mRNA and activation of caspase-9 is one of the key steps for the execution of the apoptosis. We also found that over- expression of caspase-8, and in addition p53, a tumor suppressor protein, also induces apoptosis at MBT, just like the overexpression of SAMDC and caspase-9 does. The apoptosis induced by p53 was suppressed by Xdm-2, a negative regulator of p53, and by a peptide inhibitor and a dominant-negative type mutant of caspase-9, but not by those of caspase-8. By contrast, apoptosis induced by SAMDC was suppressed by peptide inhibitors and dominant-negative mutants of both caspase-9 and caspase-8, but not by Xdm-2. Unlike caspase-9 mRNA, caspase-8 mRNA was not a maternal mRNA, but newly expressed during cleavage stage (pre-MBT stage) only in embryos overexpressed with SAMDC. In SAMDC-induced apoptotic embryos activities to process procaspase-8 and procaspase-9 appeared, whereas in p53-induced apoptotic embryos only activity to process procaspase-9 appeared. Thus, Xenopus embryos have at least two pathways to execute the maternal program of apoptosis: One induced by SAMDC overexpression through activation of caspase-9 and do novo expression of caspase-8 gene, and the other induced by p53 overexpression through activation of caspase-9 but not caspase-8. In Xenopus embryos, it has long been believed that zygotic genes are silent until MBT, but results obtained with caspase-8 may provide a novel example of gene expression before MBT.

Expression of SAMDC Gene for Enhancing the Shelf Life for Improvement of Fruit Quality Using Biotechnological Approaches into Litchi (Litchi chinensis Sonn.) Cultivars

Polyamines play an important role in plant response to abiotic stress. S-adenosyl-1-methionine decarboxylase (SAMDC) is one of the key regulatory enzymes in the biosynthesis of polyamines. In order to better understand the effect of regulation of polyamine biosynthesis on the shelf life improvement of litchi fruit, SAMDC cDNA isolated from Datura stramonium cloned in pBI121 was introduced into litchi genome by means of Agrobacterium tumefaciens through zygote disc transformation. Transgene and its expression are confirmed by Southern and Northern blot analyses, respectively. Transgenic plants expressing Datura SAMDC produced 1.7- to 2.4-fold higher levels of spermidine and spermine than wildtype plants under normal environmental condition, which indicated that the transgenic litchi presented an enhanced polyamines synthesis compared to wildtype plants. Our results demonstrated clearly that increasing polyamine biosynthesis in plants may be a means of creating improved fruit shelf life germplasm.

Polyamine Accumulation in Transgenic Tomato Enhances the Tolerance to High Temperature Stress

Polyamines play an important role in plant response to abiotic stress. S-adenosyl-l-methionine decarboxylase （SAMDC） is one of the key regulatory enzymes in the biosynthesis of polyamines. In order to better understand the effect of regulation of polyamine biosynthesis on the tolerance of high-temperature stress in tomato, SAMDC cDNA isolated from Saccharomyces cerevisiae was introduced into tomato genome by means of Agrobacterium tumefaciens through leaf disc transformation. Transgene and expression was confirmed by Southern and Northern blot analyses, respectively. Transgenic plants expressing yeast SAMDC produced 1.7- to 2.4-fold higher levels of spermidine and spermine than wildtype plants under high temperature stress, and enhanced antioxidant enzyme activity and the protection of membrane lipid peroxidation was also observed. This subsequently improved the efficiency of CO2 assimilation and protected the plants from high temperature stress, which indicated that the transgenic tomato presented an enhanced tolerance to high temperature stress （38℃） compared with wild-type plants. Our results demonstrated clearly that increasing polyamine biosynthesis in plants may be a means of creating high temperature-tolerant germplasm.

Methionine adenosyltransferases in liver health and diseases

Methionine adenosyltransferases(MATs)are essential for cell survival because they catalyze the biosynthesis of the biological methyl donor S-adenosylmethionine(SAMe)from methionine and adenosine triphosphate(ATP).Mammalian cells express two genes,MAT1A and MAT2A,which encode two MAT catalytic subunits,α1 andα2,respectively.Theα1 subunit organizes into dimers(MATIII)or tetramers(MATI).Theα2 subunit is found in the MATII isoform.A third gene MAT2B,encodes a regulatory subunit b,that regulates the activity of MATII by lowering the inhibition constant(Ki)for SAMe and the Michaelis constant(Km)for methionine.MAT1A expressed mainly in hepatocytes maintains the differentiated state of these cells whereas MAT2A and MAT2B are expressed in non-parenchymal cells of the liver(hepatic stellate cells[HSCs]and Kupffer cells)and extrahepatic tissues.A switch from the liverspecific MAT1A to MAT2A has been observed during conditions of active liver growth and dedifferentiation.Liver injury,fibrosis,and cancer are associated with MAT1A silencing and MAT2A/MAT2B induction.Even though both MAT1A and MAT2A are involved in SAMe biosynthesis,they exhibit distinct molecular interactions in liver cells.This review provides an update on MAT genes and their roles in liver pathologies.

Coculture engineering for efficient production of vanillyl alcohol in Escherichia coli

Vanillyl alcohol is a precursor of vanillin,which is one of the most widely used flavor compounds.Currently,vanillyl alcohol biosynthesis still encounters the problem of low efficiency.In this study,coculture engineering was adopted to improve production efficiency of vanillyl alcohol in E.coli.First,two pathways were compared for biosynthesis of the immediate precursor 3,4-dihydroxybenzyl alcohol in monocultures,and the 3-dehydroshikimate-derived pathway showed higher efficiency than the 4-hydroxybenzoate-derived pathway.To enhance the efficiency of the last methylation step,two strategies were used,and strengthening S-adenosylmethionine(SAM)regeneration showed positive effect while strengthening SAM biosynthesis showed negative effect.Then,the optimized pathway was assembled in a single cell.However,the biosynthetic efficiency was still low,and was not significantly improved by modular optimization of pathway genes.Thus,coculturing engineering strategy was adopted.At the optimal inoculation ratio,the titer reached 328.9 mg/L.Further,gene aroE was knocked out to reduce cell growth and improve 3,4-DHBA biosynthesis of the upstream strain.As a result,the titer was improved to 559.4 mg/L in shake flasks and to 3.89 g/L in fed-batch fermentation.These are the highest reported titers of vanillyl alcohol so far.This work provides an effective strategy for sustainable production of vanillyl alcohol.