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1.
Int J Dev Neurosci ; 78: 198-209, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31476364

RESUMO

Elevated levels of methionine in blood characterize the hypermethioninemia, which may have genetic or non-genetic origin, as for example from high protein diet. Born rats from hypermethioninemic mothers presented cerebral oxidative stress, inhibition of Na+,K+-ATPase, memory deficit and ultrastructure cerebral changes. Melatonin is a hormone involved in circadian rhythm and has antioxidant effects. The aim of this study was to verify the possible neuroprotective effects of melatonin administration in hypermethioninemic pregnant rats on damage to biomolecules (Na+,K+-ATPase, sulfhydryl content and DNA damage index) and behavior (open field, novel object recognition and water maze tasks), as well as its effect on cells morphology by electron microscopy in offspring. Wistar female rats received methionine (2.68 µmol/g body weight) and/or melatonin (10 mg/kg body weight) by subcutaneous injections during entire pregnancy. Control rats received saline. Biochemical analyzes were performed at 21 and 30 days of life of offspring and behavioral analyzes were performed only at 30 days of age in male pups. Results showed that gestational hypermethioninemia diminished Na+,K+-ATPase activity and sulfhydryl content and increased DNA damage at 21 and 30 days of life. Melatonin was able to totally prevent Na+,K+-ATPase activity alteration at 21 days and partially prevent its alteration at 30 days of rats life. Melatonin was unable in to prevent sulfhydryl and DNA damage at two ages. It also improved DNA damage, but not at level of saline animals (controls). Regarding to behavioral tests, data showed that pups exposed to gestational hypermethioninemia decreased reference memory in water maze, spent more time to the center of the open field and did not differentiate the objects in the recognition test. Melatonin was able to prevent the deficit in novel object recognition task. Electron microscopy revealed ultrastructure alterations in neurons of hypermethioninemic at both ages of offspring, whose were prevented by melatonin. These findings suggest that melatonin may be a good neuroprotective to minimize the harmful effects of gestational hypermethioninemia on offspring.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/tratamento farmacológico , Glicina N-Metiltransferase/deficiência , Melatonina/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Complicações na Gravidez/tratamento farmacológico , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Modelos Animais de Doenças , Feminino , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Melatonina/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Gravidez , Ratos , Ratos Wistar , Reconhecimento Psicológico/efeitos dos fármacos
2.
Int J Mol Sci ; 20(17)2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470507

RESUMO

Imbalance of lipid metabolism is a main cause of metabolic syndrome leading to life-threatening metabolic diseases. Angiopoietin-like protein 8 (Angptl8) was recently identified as a liver and adipose tissue-released hormone that is one of the molecules involved in triglyceride metabolism. However, the regulatory mechanism of Angptl8 is largely unknown. A high fat diet (HFD)-fed mouse model, which showed high cholesterol, high triglyceride, and high insulin in the blood, revealed the upregulation of hepatic and plasma Angptl8 and the downregulation of hepatic glycine N-methyltransferase (GNMT). The inverse correlation of hepatic Angptl8 and GNMT expression in the livers of HFD-fed mice was also confirmed in a publicly available microarray dataset. The mechanistic study using primary hepatocytes showed that the Angptl8 expression could be induced by insulin treatment in a dose- and time-dependent manner. Inhibition of PI3K/Akt pathway by the specific inhibitors or the dominant-negative Akt blocked the insulin-induced Angptl8 expression. Moreover, knockout of GNMT promoted the Akt activation as well as the Angptl8 expression. These results suggested that GNMT might be involved in insulin-induced Angptl8 expression in HFD-mediated metabolic syndrome.


Assuntos
Proteínas Semelhantes a Angiopoietina/genética , Dieta Hiperlipídica/efeitos adversos , Regulação da Expressão Gênica/genética , Glicina N-Metiltransferase/genética , Fígado/metabolismo , Síndrome Metabólica/genética , Proteínas Semelhantes a Angiopoietina/sangue , Proteínas Semelhantes a Angiopoietina/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Glicina N-Metiltransferase/sangue , Glicina N-Metiltransferase/metabolismo , Hepatócitos/metabolismo , Insulina/farmacologia , Lipídeos/sangue , Fígado/enzimologia , Masculino , Síndrome Metabólica/etiologia , Síndrome Metabólica/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/genética
3.
J Vasc Res ; 56(5): 230-240, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31307051

RESUMO

OBJECTIVE: The relationship between methionine (Met) and abdominal aortic aneurysm (AAA) has been previously demonstrated, but the mechanisms controlling this association remain unclear. This study investigated the potential contribution of hypermethioninemia (HMet) to the development of AAA. METHODS: A model of AAA was induced by intraluminal porcine pancreatic elastase (PPE) infusion in 60 male Sprague-Dawley rats divided into 4 groups (n = 15 per group). Met was supplied by intragastric administration (1 g/kg body weight/day) from 1 week before surgery until 4 weeks after surgery. The aortic diameter was measured by ultrasound. Aortas were collected 4 weeks after surgery and subjected to biochemical analysis, histological assays, and transmission electron microscopy. RESULTS: After 5 weeks of Met supplementation, HMet increased the dilation ratio of the HMet + PPE group, and hyperhomocysteinemia was also induced in HMet and HMet + PPE rats. Increased matrix metalloproteinase-2 (MMP-2), osteopontin, and interleukin-6 expression was detected in HMet + PPE rats. Furthermore, increased autophagy was detected in the HMet + PPE group. CONCLUSION: This study demonstrates that HMet may exacerbate the formation of AAA due to the increased dilation ratio partially via enhancing MMP-2 and inflammatory responses.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/induzido quimicamente , Aneurisma da Aorta Abdominal/induzido quimicamente , Glicina N-Metiltransferase/deficiência , Metionina , Erros Inatos do Metabolismo dos Aminoácidos/sangue , Animais , Aorta Abdominal/metabolismo , Aorta Abdominal/ultraestrutura , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Dilatação Patológica , Modelos Animais de Doenças , Progressão da Doença , Glicina N-Metiltransferase/sangue , Interleucina-6/metabolismo , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Osteopontina/metabolismo , Elastase Pancreática , Ratos Sprague-Dawley , Fatores de Risco , Fatores de Tempo
4.
Oxid Med Cell Longev ; 2019: 9148535, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31214284

RESUMO

Oxidative stress is detrimental to animals and can depress the growth performance and regulate the gene expression of animals. However, it remains unclear how oxidative stress regulates the expression of long noncoding RNAs (lncRNAs) and mRNAs. Therefore, the purpose of this article was to explore the profiles of lncRNAs and mRNAs in the liver of piglets under oxidative stress. Here, we constructed a piglet oxidative stress model induced by diquat and evaluated the effects of oxidative stress on the growth performance and antioxidant enzyme activity of piglets. We also used RNA-Seq to examine the global expression of lncRNAs and mRNAs in piglets under oxidative stress. The targets of lncRNAs and mRNAs were enriched in gene ontology (GO) terms and signaling pathways. The results show that the growth performance and activities of antioxidant enzymes were decreased in piglets under oxidative stress. Moreover, eight lncRNAs (6 upregulated and 2 downregulated) and 30 mRNAs (8 upregulated and 22 downregulated) were differentially expressed in the oxidative stress group of piglets compared to the negative control group. According to biological processes in enriched GO terms, the oxoacid metabolic process, intramolecular oxidoreductase activity, and oxidation-reduction process play important roles in oxidative stress. Pathway analysis showed that the signaling pathways involved in insulin and glucose metabolism had a close relationship with oxidative stress. Further in vitro experiments showed that the expression of the upregulated gene GNMT was significantly increased in primary porcine hepatocytes after diquat stimulation. In contrast, the level of the downregulated gene GCK was significantly decreased at 12 h in primary porcine hepatocytes after diquat stimulation. Our results expand our knowledge of the lncRNAs and mRNAs transcribed in the livers of piglets under oxidative stress and provide a basis for future research on the molecular mechanisms mediating oxidative stress and tissue damage.


Assuntos
Hepatócitos/metabolismo , Fígado/fisiologia , Estresse Oxidativo , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Animais , Diquat , Regulação da Expressão Gênica , Ontologia Genética , Quinases do Centro Germinativo/genética , Quinases do Centro Germinativo/metabolismo , Glicina N-Metiltransferase/genética , Glicina N-Metiltransferase/metabolismo , Hepatócitos/patologia , Masculino , Análise de Sequência de RNA , Transdução de Sinais , Suínos
5.
World J Gastroenterol ; 25(15): 1890-1898, 2019 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-31057302

RESUMO

BACKGROUND: Exosomes contain proteins, lipids, and biological molecules such as DNA and RNA. Nucleic acids in exosomes are a group of molecules that can act as biomarkers. Currently, there are many reports on exosomal microRNAs, which are ideal biomarkers for the early diagnosis of cancer. However, there are few reports on the role of exosomal microRNAs in the diagnosis and prognosis of hepatocellular carcinoma (HCC). AIM: To understand the mechanism of exosomal microRNA-224 (miR-224) in the development of HCC and evaluate its diagnostic and prognostic value. METHODS: Cell culture and transfection of exosomal miRNA-224, real-time quantitative PCR, luciferase reporter assay, and other methods were used to find new biomarkers related to the development of HCC that can be used to diagnose HCC and predict HCC prognosis. RESULTS: By targeting glycine N-methyltransferase, incubating exosomes with miR-224 mimic resulted in a significant increase in cell proliferation compared to that of the control group, while incubation with the miR-224 inhibitor significantly reduced cell proliferation. The same results were obtained for the cell invasion assay. Serum exosomal miR-224 did have some ability to differentiate patients with HCC from healthy controls, with an area under the curve of 0.910, and HCC patients with higher serum exosomal miR-224 expression had lower overall survival. CONCLUSION: Exosomal miR-224 is a tumor promotor and can be a marker of diagnosis and prognosis of HCC patients, however, its ability to distinguish liver diseases needs further verification.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Glicina N-Metiltransferase/genética , MicroRNAs/metabolismo , Regiões 3' não Traduzidas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Carcinogênese/genética , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/mortalidade , Linhagem Celular Tumoral , Proliferação de Células/genética , Exossomos/metabolismo , Feminino , Glicina N-Metiltransferase/metabolismo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Masculino , MicroRNAs/sangue , MicroRNAs/genética , Pessoa de Meia-Idade , Prognóstico
6.
Mol Metab ; 23: 1-13, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30850319

RESUMO

OBJECTIVE: The loss of liver glycine N-methyltransferase (GNMT) promotes liver steatosis and the transition to hepatocellular carcinoma (HCC). Previous work showed endogenous glucose production is reduced in GNMT-null mice with gluconeogenic precursors being used in alternative biosynthetic pathways that utilize methyl donors and are linked to tumorigenesis. This metabolic programming occurs before the appearance of HCC in GNMT-null mice. The metabolic physiology that sustains liver tumor formation in GNMT-null mice is unknown. The studies presented here tested the hypothesis that nutrient flux pivots from glucose production to pathways that incorporate and metabolize methyl groups in GNMT-null mice with HCC. METHODS: 2H/13C metabolic flux analysis was performed in conscious, unrestrained mice lacking GNMT to quantify glucose formation and associated nutrient fluxes. Molecular analyses of livers from mice lacking GNMT including metabolomic, immunoblotting, and immunochemistry were completed to fully interpret the nutrient fluxes. RESULTS: GNMT knockout (KO) mice showed lower blood glucose that was accompanied by a reduction in liver glycogenolysis and gluconeogenesis. NAD+ was lower and the NAD(P)H-to-NAD(P)+ ratio was higher in livers of KO mice. Indices of NAD+ synthesis and catabolism, pentose phosphate pathway flux, and glutathione synthesis were dysregulated in KO mice. CONCLUSION: Glucose precursor flux away from glucose formation towards pathways that regulate redox status increase in the liver. Moreover, synthesis and scavenging of NAD+ are both impaired resulting in reduced concentrations. This metabolic program blunts an increase in methyl donor availability, however, biosynthetic pathways underlying HCC are activated.


Assuntos
Carcinoma Hepatocelular/metabolismo , Gluconeogênese , Glicina N-Metiltransferase/metabolismo , Homeostase , Neoplasias Hepáticas/metabolismo , Oxirredução , Animais , Metilação de DNA , Fígado Gorduroso/metabolismo , Técnicas de Inativação de Genes , Glucose/metabolismo , Glicina N-Metiltransferase/genética , Fígado/metabolismo , Masculino , Metionina/metabolismo , Camundongos , Camundongos Knockout , NAD/metabolismo
7.
FEBS J ; 286(11): 2135-2154, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30776190

RESUMO

Methylation is an underpinning process of life and provides control for biological processes such as DNA synthesis, cell growth, and apoptosis. Methionine adenosyltransferases (MAT) produce the cellular methyl donor, S-Adenosylmethionine (SAMe). Dysregulation of SAMe level is a relevant event in many diseases, including cancers such as hepatocellular carcinoma and colon cancer. In addition, mutation of Arg264 in MATα1 causes isolated persistent hypermethioninemia, which is characterized by low activity of the enzyme in liver and high level of plasma methionine. In mammals, MATα1/α2 and MATßV1/V2 are the catalytic and the major form of regulatory subunits, respectively. A gating loop comprising residues 113-131 is located beside the active site of catalytic subunits (MATα1/α2) and provides controlled access to the active site. Here, we provide evidence of how the gating loop facilitates the catalysis and define some of the key elements that control the catalytic efficiency. Mutation of several residues of MATα2 including Gln113, Ser114, and Arg264 lead to partial or total loss of enzymatic activity, demonstrating their critical role in catalysis. The enzymatic activity of the mutated enzymes is restored to varying degrees upon complex formation with MATßV1 or MATßV2, endorsing its role as an allosteric regulator of MATα2 in response to the levels of methionine or SAMe. Finally, the protein-protein interacting surface formed in MATα2:MATß complexes is explored to demonstrate that several quinolone-based compounds modulate the activity of MATα2 and its mutants, providing a rational for chemical design/intervention responsive to the level of SAMe in the cellular environment. ENZYMES: Methionine adenosyltransferase (EC.2.5.1.6). DATABASE: Structural data are available in the RCSB PDB database under the PDB ID 6FBN (Q113A), 6FBP (S114A: P221 21 ), 6FBO (S114A: I222), 6FCB (P115G), 6FCD (R264A), 6FAJ (wtMATα2: apo), 6G6R (wtMATα2: holo).


Assuntos
Metionina Adenosiltransferase/química , S-Adenosilmetionina/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Apoptose/genética , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Domínio Catalítico/genética , Proliferação de Células/genética , Metilação de DNA/genética , Replicação do DNA/genética , Regulação Enzimológica da Expressão Gênica/genética , Glicina N-Metiltransferase/deficiência , Glicina N-Metiltransferase/genética , Humanos , Fígado/enzimologia , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Metionina Adenosiltransferase/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Mutação , Domínios e Motivos de Interação entre Proteínas/genética , Quinolonas/metabolismo , S-Adenosilmetionina/química
8.
J Inherit Metab Dis ; 42(1): 128-139, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30740731

RESUMO

PURPOSE: To assess how the current practice of newborn screening (NBS) for homocystinurias compares with published recommendations. METHODS: Twenty-two of 32 NBS programmes from 18 countries screened for at least one form of homocystinuria. Centres provided pseudonymised NBS data from patients with cystathionine beta-synthase deficiency (CBSD, n = 19), methionine adenosyltransferase I/III deficiency (MATI/IIID, n = 28), combined remethylation disorder (cRMD, n = 56) and isolated remethylation disorder (iRMD), including methylenetetrahydrofolate reductase deficiency (MTHFRD) (n = 8). Markers and decision limits were converted to multiples of the median (MoM) to allow comparison between centres. RESULTS: NBS programmes, algorithms and decision limits varied considerably. Only nine centres used the recommended second-tier marker total homocysteine (tHcy). The median decision limits of all centres were ≥ 2.35 for high and ≤ 0.44 MoM for low methionine, ≥ 1.95 for high and ≤ 0.47 MoM for low methionine/phenylalanine, ≥ 2.54 for high propionylcarnitine and ≥ 2.78 MoM for propionylcarnitine/acetylcarnitine. These decision limits alone had a 100%, 100%, 86% and 84% sensitivity for the detection of CBSD, MATI/IIID, iRMD and cRMD, respectively, but failed to detect six individuals with cRMD. To enhance sensitivity and decrease second-tier testing costs, we further adapted these decision limits using the data of 15 000 healthy newborns. CONCLUSIONS: Due to the favorable outcome of early treated patients, NBS for homocystinurias is recommended. To improve NBS, decision limits should be revised considering the population median. Relevant markers should be combined; use of the postanalytical tools offered by the CLIR project (Collaborative Laboratory Integrated Reports, which considers, for example, birth weight and gestational age) is recommended. tHcy and methylmalonic acid should be implemented as second-tier markers.


Assuntos
Homocistinúria/diagnóstico , Acetilcarnitina/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Carnitina/análogos & derivados , Carnitina/metabolismo , Feminino , Glicina N-Metiltransferase/deficiência , Glicina N-Metiltransferase/metabolismo , Homocisteína/metabolismo , Homocistinúria/metabolismo , Humanos , Recém-Nascido , Masculino , Metionina/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Ácido Metilmalônico/metabolismo , Espasticidade Muscular/diagnóstico , Espasticidade Muscular/metabolismo , Triagem Neonatal/métodos , Fenilalanina/metabolismo , Transtornos Psicóticos/diagnóstico , Transtornos Psicóticos/metabolismo
9.
BMC Med Genet ; 20(1): 3, 2019 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-30612563

RESUMO

BACKGROUND: Tandem mass spectrometry (MS MS) and simple fluorometric assays are currently used in newborn screening programs to detect inborn errors of metabolism (IEM). The aim of the study was to evaluate the clinical utility of exome sequencing as a second tier screening method to assist clinical diagnosis of the newborn. METHODS: A novel PCR-exome amplification and re-sequencing (PEARS) assay was designed and used to detect mutations in 122 genes associated with 101 IEM. Newborn bloodspots positive by biochemical testing were analysed by PEARS assay to detect pathogenic mutations relevant to the IEM. RESULTS: In initial validation studies of genomic DNA samples, PEARS assay correctly detected 25 known mutations associated with 17 different IEM. Retrospective gene analysis of newborns with clinical phenylketonuria (PKU), identified compound heterozygote phenylalanine hydroxylase (PAH) gene mutations in eight of nine samples (89%). Prospective analysis of 211 bloodspots correctly identified the two true PKU samples, yielding positive and negative predictive values of 100%. Testing of 8 true positive MS MS samples correctly identified potentially pathogenic compound heterozygote genotypes in 2 cases of citrullinemia type 1 and one case each of methylmalonic acidemia, isobutyryl-CoA dehydrogenase deficiency, short chain acyl-CoA dehydrogenase deficiency and glutaric acid type II and heterozygous genotypes in 2 cases of autosomal dominant methioninemia. Analysis of 11 of 12 false positive MS MS samples for other IEM identified heterozygous carriers in 8 cases for the relevant genes associated with the suspected IEM. In the remaining 3 cases, the test revealed compound heterozygote mutations in other metabolic genes not associated with the suspected IEM, indicating a misinterpretation of the original MS MS data. CONCLUSIONS: The PEARS assay has clinical utility as a rapid and cost effective second-tier test to assist the clinician to accurately diagnose newborns with a suspected IEM.


Assuntos
Exoma/genética , Erros Inatos do Metabolismo/diagnóstico , Erros Inatos do Metabolismo/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Triagem Neonatal/métodos , Sequenciamento Completo do Exoma/métodos , Acil-CoA Desidrogenase/deficiência , Acil-CoA Desidrogenase/genética , Erros Inatos do Metabolismo dos Aminoácidos/genética , Citrulinemia/genética , Aconselhamento Genético , Genótipo , Glutaratos , Glicina N-Metiltransferase/deficiência , Glicina N-Metiltransferase/genética , Heterozigoto , Humanos , Recém-Nascido , Erros Inatos do Metabolismo Lipídico/genética , Masculino , Técnicas de Diagnóstico Molecular/métodos , Mutação , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/diagnóstico , Fenilcetonúrias/genética , Estudos Prospectivos , Estudos Retrospectivos , Espectrometria de Massas em Tandem/métodos
10.
Brain Dev ; 41(4): 382-388, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30389272

RESUMO

Methionine adenosyltransferase I/III (MAT I/III) deficiency is characterized by persistent hypermethioninemia. The clinical manifestations in cases with MAT I/III deficiency vary from a complete lack of symptoms to neurological problems associated with brain demyelination. We experienced a neonatal case with MAT I/III deficiency, in which severe hypermethioninemia was detected during the newborn screening test. The patient gradually showed hyperreflexia, foot clonus, and irritability from the age of 1 month onwards, and his brain magnetic resonance imaging scans showed abnormal signal intensity in the bilateral central tegmental tracts. His neurological manifestations improved after the S-adenosylmethionine (SAMe) treatment, deteriorated after discontinuation of SAMe, and re-improved owing to re-administration of SAMe. He achieved normal neurodevelopment through SAMe and methionine restriction therapy. Lack of SAMe as well as severe hypermethioninemia were thought to contribute towards the clinical psychophysical state. Moreover, impaired MAT I/III activity contributed to the development of neurological disorder from the early neonatal period.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/fisiopatologia , Glicina N-Metiltransferase/deficiência , Metionina Adenosiltransferase/deficiência , Metionina Adenosiltransferase/fisiologia , Encéfalo/metabolismo , Doenças Desmielinizantes/tratamento farmacológico , Humanos , Lactente , Recém-Nascido , Masculino , Metionina/metabolismo , Triagem Neonatal , Doenças do Sistema Nervoso/tratamento farmacológico , Tegmento Pontino/fisiopatologia , S-Adenosilmetionina/uso terapêutico
11.
J Dairy Sci ; 102(1): 866-870, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30391174

RESUMO

The d-isomer of Met cannot be used directly by the mammary gland in dairy cows; instead, it is transformed into l-Met, the proteogenic isomer, in the liver and other extramammary tissues. It remains unclear whether different Met forms and a Met hydroxy analog, 2-hydroxy-4-(methylthio)butanoic acid (HMB), are metabolized and function similarly in the liver. The objective of the present study was to examine the regulation of key genes in Met regeneration, transulfuration, and transmethylation pathways in response to increasing doses of different Met forms. Hepatocytes isolated from 4 calves between 4 and 7 d old were maintained as monolayer cultures for 24 h before addition of treatments. Treatments of (0, 10, 20, 40 µM) d-Met, l-Met, dl-Met, dl-HMB, or a 1:1 mixture of dl-Met and dl-HMB were added to Met-free medium in triplicate. After 24 h, cell lysates were collected for quantification of gene expression by quantitative PCR, and mRNA abundance was normalized to the mean of 3 reference genes. Data were analyzed with PROC MIXED of SAS 9.3 (SAS Institute Inc., Cary, NC). Analyses of covariance confirmed equivalent slopes of Met form, and the final model included form, dose, and random effect of calf within form. Data are reported as least squares means ± standard error. No main effect of Met form was observed for any genes examined. The enzymes encoded by betaine-homocysteine methyltransferase (BHMT) and 5-methyltetrahydrofolate-homocysteine methyltransferase use betaine and 5-methyltetrahydrofolate, respectively, to regenerate Met from homocysteine. Increasing concentration of Met did not alter 5-methyltetrahydrofolate expression, but decreased BHMT expression. Expression of glycine N-methyltransferase, the enzyme that controls transmethylation flux from S-adenosyl-methionine, was not affected by Met concentration. Methionine concentration had no effect on expression of cystathionine ß-synthase, a key enzyme for the transulfuration pathway. The decrease in BHMT expression indicates a decreased need for cellular Met regeneration with increasing Met concentration, independent of Met form. The lack of differences among Met forms on regulating genes examined indicates that all Met forms similarly reduced genes controlling Met regeneration and metabolism in primary bovine hepatocytes.


Assuntos
Ácido Butírico/metabolismo , Bovinos/genética , Hepatócitos/metabolismo , Metionina/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Animais , Animais Recém-Nascidos , Betaína/farmacologia , Betaína-Homocisteína S-Metiltransferase/genética , Betaína-Homocisteína S-Metiltransferase/metabolismo , Ácido Butírico/química , Bovinos/metabolismo , Células Cultivadas , Feminino , Glicina N-Metiltransferase/genética , Glicina N-Metiltransferase/metabolismo , Hepatócitos/enzimologia , Fígado/citologia , Fígado/enzimologia , Fígado/metabolismo , Metionina/química , S-Adenosilmetionina/metabolismo
12.
Int J Oral Maxillofac Surg ; 47(11): 1381-1388, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30318092

RESUMO

The objective of this study was to identify new environmental and genetic risk factors for orofacial clefts that arise during early foetal development. In this retrospective, case-control, mother-child pair study, 172 orofacial clefts cases and 199 healthy controls, and their respective mothers, were genotyped for common variants in relevant genes obtained by text and database mining using STRING 10.0. Exposure to environmental risk factors was evaluated using questionnaires. Variant glycine N-methyltransferase (odds ratio (OR) 2.1, 95% confidence interval (95% CI) 1.0-4.4) and dihydrofolate reductase (OR 2.4, 95% CI 1.3-4.5) genotypes were identified as risk factors for cleft lip with or without cleft palate formation. Furthermore, synergy was detected between variant glycine N-methyltransferase and dihydrofolate reductase genotypes in promoting cleft lip with or without cleft palate formation (OR 7, 95% CI 2-23). This study is novel in finding that common glycine N-methyltransferase variant genotypes increase the risk of cleft lip with or without cleft palate.


Assuntos
Glicina N-Metiltransferase/genética , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Fenda Labial , Fissura Palatina , Feminino , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Estudos Retrospectivos , Fatores de Risco , Eslovênia , Inquéritos e Questionários
13.
Biochemistry ; 57(40): 5775-5779, 2018 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-30226369

RESUMO

Methyl donor balance is critical for epigenetic regulation in cells and is maintained by the so-called methionine cycle proteins that regenerate S-adenosylmethionine (SAM), the universal methyl donor, from homocysteine formed by the activity of methyltransferases. Nnmt is a liver enzyme that methylates nicotinamide, but its role in regulating methyl donor balance in the liver is unclear. In this study, we assessed the effect of altered Nnmt expression on various aspects of methyl donor metabolism in the liver. We found that Nnmt overexpression decreased SAM levels and the SAM/ S-adenosylhomocysteine (SAH) ratio both in vivo and in vitro. Nnmt knockdown did not change methyl donor balance in mouse primary hepatocytes but increased SAM levels and the SAM/SAH ratio when Gnmt, the dominantly expressed methyltransferase in liver, was simultaneously knocked down. Paradoxically, expression of enzymatically deficient Nnmt increased the SAM/SAH ratio, suggesting that Nnmt can regulate methyl donor balance independent of its methyltransferase activity. Proteomics analysis of Nnmt-interacting proteins in the liver identified Bhmt, Mat1a, and Ahcy, all components of the methionine cycle, and functional experiments showed that mutant Nnmt increased the level of remethylation of homocysteine to SAM. In summary, we show that the function of Nnmt in hepatic methyl donor balance is multifactorial. On one hand, Nnmt decreases methyl donor balance, consistent with its activity as a methyltransferase consuming methyl donors. On the other hand, by co-opting the enzymes of the methionine cycle, Nnmt aids the recycling of homocysteine to SAM for another round of methylation.


Assuntos
Glicina N-Metiltransferase/metabolismo , Hepatócitos/enzimologia , Fígado/enzimologia , Nicotinamida N-Metiltransferase/metabolismo , S-Adenosilmetionina/metabolismo , Animais , Técnicas de Silenciamento de Genes , Glicina N-Metiltransferase/genética , Hepatócitos/citologia , Camundongos , Nicotinamida N-Metiltransferase/genética , S-Adenosil-Homocisteína/metabolismo
14.
Cell Death Dis ; 9(10): 958, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30237481

RESUMO

Glycine N-methyltransferase (GNMT) is the most abundant methyltransferase in the liver and a master regulator of the transmethylation flux. GNMT downregulation leads to loss of liver function progressing to fibrosis, cirrhosis, and hepatocellular carcinoma. Moreover, GNMT deficiency aggravates cholestasis-induced fibrogenesis. To date, little is known about the mechanisms underlying downregulation of GNMT levels in hepatic fibrosis and cirrhosis. On this basis, microRNAs are epigenetic regulatory elements that play important roles in liver pathology. In this work, we aim to study the regulation of GNMT by microRNAs during liver fibrosis and cirrhosis. Luciferase assay on the 3'UTR-Gnmt was used to confirm in silico analysis showing that GNMT is potentially targeted by the microRNA miR-873-5p. Correlation between GNMT and miR-873-5p in human cholestasis and cirrhosis together with miR-873-5p inhibition in vivo in different mouse models of liver cholestasis and fibrosis [bile duct ligation and Mdr2 (Abcb4)-/- mouse] were then assessed. The analysis of liver tissue from cirrhotic and cholestatic patients, as well as from the animal models, showed that miR-873-5p inversely correlated with the expression of GNMT. Importantly, high circulating miR-873-5p was also detected in cholestastic and cirrhotic patients. Preclinical studies with anti-miR-873-5p treatment in bile duct ligation and Mdr2-/- mice recovered GNMT levels in association with ameliorated inflammation and fibrosis mainly by counteracting hepatocyte apoptosis and cholangiocyte proliferation. In conclusion, miR-873-5p emerges as a novel marker for liver fibrosis, cholestasis, and cirrhosis and therapeutic approaches based on anti-miR-873-5p may be effective treatments for liver fibrosis and cholestatic liver disease.


Assuntos
Fibrose/metabolismo , Fibrose/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Fígado/metabolismo , MicroRNAs/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Glicina N-Metiltransferase/genética , Glicina N-Metiltransferase/metabolismo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética
15.
Sci Rep ; 8(1): 13802, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30217986

RESUMO

Glycine N-methyltransferase (GNMT) is abundantly expressed in normal livers and plays a protective role against tumor formation. GNMT depletion leads to progression of hepatocellular carcinoma (HCC). In this study, we investigated the activity of ectopic GNMT delivered using recombinant adeno-associated virus (AAV) gene therapy in mouse models of liver cirrhosis and HCC. Injection of AAV serotype 8 (AAV8) vector carrying the GNMT gene (AAV8-GNMT) in Gnmt-/- mice increased GNMT expression and downregulated pro-inflammatory responses, resulting in reduced liver damage and incidence of liver tumors. Moreover, AAV8-GNMT resulted in the amelioration of carbon tetrachloride (CCl4)-induced liver fibrosis in BALB/c mice. We showed that AAV8-GNMT protected hepatocytes from CCl4-induced liver damage.  AAV8-GNMT significantly attenuated the levels of pro-fibrotic markers and increased efficiency of hepatocyte proliferation. These results suggest that correction of hepatic GNMT by gene therapy of AAV8-mediated gene enhancement may provide a potential strategy for preventing and delaying development of liver diseases.


Assuntos
Carcinoma Hepatocelular/terapia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Terapia Genética/métodos , Glicina N-Metiltransferase/biossíntese , Neoplasias Hepáticas/terapia , Animais , Tetracloreto de Carbono/efeitos adversos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células/fisiologia , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Metilação de DNA , Dependovirus/enzimologia , Dependovirus/genética , Progressão da Doença , Regulação para Baixo , Feminino , Glicina N-Metiltransferase/genética , Glicina N-Metiltransferase/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL
16.
Biosci Rep ; 38(5)2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30061177

RESUMO

Uranium tailings (UT) are formed as a byproduct of uranium mining and are of potential risk to living organisms. In the present study, we sought to identify potential biomarkers associated with chronic exposure to low dose rate γ radiation originating from UT. We exposed C57BL/6J mice to 30, 100, or 250 µGy/h of gamma radiation originating from UT samples. Nine animals were included in each treatment group. We observed that the liver central vein was significantly enlarged in mice exposed to dose rates of 100 and 250 µGy/h, when compared with nonirradiated controls. Using proteomic techniques, we identified 18 proteins that were differentially expressed (by a factor of at least 2.5-fold) in exposed animals, when compared with controls. We chose glycine N-methyltransferase (GNMT), glutathione S-transferase A3 (GSTA3), and nucleophosmin (NPM) for further investigations. Our data showed that GNMT (at 100 and 250 µGy/h) and NPM (at 250 µGy/h) were up-regulated, and GSTA3 was down-regulated in all of the irradiated groups, indicating that their expression is modulated by chronic gamma radiation exposure. GNMT, GSTA3, and NPM may therefore prove useful as biomarkers of gamma radiation exposure associated with UT. The mechanisms underlying those changes need to be further studied.


Assuntos
Glutationa Transferase/metabolismo , Glicina N-Metiltransferase/metabolismo , Fígado/efeitos da radiação , Proteínas Nucleares/metabolismo , Urânio , Animais , Biomarcadores/análise , Biologia Computacional/métodos , Relação Dose-Resposta à Radiação , Eletroforese em Gel Bidimensional/métodos , Raios gama/efeitos adversos , Glutationa Transferase/genética , Glicina N-Metiltransferase/genética , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Proteômica/métodos , Exposição à Radiação/efeitos adversos , Exposição à Radiação/análise , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
17.
J Biol Chem ; 293(30): 11944-11954, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-29891549

RESUMO

Glycine N-methyltransferase (GNMT) is the most abundant liver methyltransferase regulating the availability of the biological methyl donor, S-adenosylmethionine (SAM). Moreover, GNMT has been identified to be down-regulated in hepatocellular carcinoma (HCC). Despite its role in regulating SAM levels and association of its down-regulation with liver tumorigenesis, the impact of reduced GNMT on metabolic reprogramming before the manifestation of HCC has not been investigated in detail. Herein, we used 2H/13C metabolic flux analysis in conscious, unrestrained mice to test the hypothesis that the absence of GNMT causes metabolic reprogramming. GNMT-null (KO) mice displayed a reduction in blood glucose that was associated with a decline in both hepatic glycogenolysis and gluconeogenesis. The reduced gluconeogenesis was due to a decrease in liver gluconeogenic precursors, citric acid cycle fluxes, and anaplerosis and cataplerosis. A concurrent elevation in both hepatic SAM and metabolites of SAM utilization pathways was observed in the KO mice. Specifically, the increase in metabolites of SAM utilization pathways indicated that hepatic polyamine synthesis and catabolism, transsulfuration, and de novo lipogenesis pathways were increased in the KO mice. Of note, these pathways utilize substrates that could otherwise be used for gluconeogenesis. Also, this metabolic reprogramming occurs before the well-documented appearance of HCC in GNMT-null mice. Together, these results indicate that GNMT deletion promotes a metabolic shift whereby nutrients are channeled away from glucose formation toward pathways that utilize the elevated SAM.


Assuntos
Carbono/metabolismo , Deleção de Genes , Gluconeogênese , Glicina N-Metiltransferase/genética , Metionina/metabolismo , Animais , Ciclo do Ácido Cítrico , Metabolismo Energético , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Glucose/metabolismo , Glicina N-Metiltransferase/metabolismo , Fígado/metabolismo , Masculino , Análise do Fluxo Metabólico , Camundongos , Camundongos Knockout , S-Adenosilmetionina/metabolismo
18.
Sci Rep ; 8(1): 6960, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29725048

RESUMO

Plants containing aristolochic acids (AA) are nephrotoxins. Glycine N-methyltransferase (GNMT) acts to bind environmental toxins such as benzo(a)pyrene and aflatoxin B1, translocate into nucleus, and alter hepatic metabolism. This study aims to determine the role of GNMT in AA-induced nephropathy. We established an AA nephropathy mouse model and found that AA type I (AAI)-induced nephropathy at a lower concentration in male than in female mice, implying sex differences in AAI resistance. Microarray analysis and AAI-treated mouse models showed that GNMT moderately reduced AAI-induced nephropathy by lowering the upregulated level of NQO1 in male, but significantly improved the nephropathy additionally by increasing Cyp3A44/3A41 in female. The protective effects of GNMT were absent in female GNMT knockout mice, in which re-expression of hepatic GNMT significantly decreased AAI-induced nephropathy. Mechanism-wise, AAI enhanced GNMT nuclear translocation, resulting in GNMT interaction with the promoter region of the genes encoding Nrf2 and CAR/PXR, the transcription factors for NQO1 and CYP3A44/3A41, respectively. Unlike the preference for Nrf2/NQO1 transcriptions at lower levels of GNMT, overexpression of GNMT preferred CAR/PXR/CYP3A44/3A41 transcriptions and alleviated kidney injury upon AAI treatment. In summary, hepatic GNMT protected mice from AAI nephropathy by enhancing CAR/PXR/CYP3A44/3A41 transcriptions and reducing Nrf2/NQO1 transcriptions.


Assuntos
Ácidos Aristolóquicos/efeitos adversos , Sistema Enzimático do Citocromo P-450/genética , Glicina N-Metiltransferase/metabolismo , Nefropatias/induzido quimicamente , NAD(P)H Desidrogenase (Quinona)/genética , Ativação Transcricional , Animais , Regulação para Baixo , Feminino , Glicina N-Metiltransferase/genética , Hepatócitos/metabolismo , Hepatócitos/patologia , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Proteção , Fatores Sexuais , Regulação para Cima
19.
Nat Commun ; 9(1): 540, 2018 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-29416063

RESUMO

Phosphatidylcholines (PC) and S-adenosylmethionine (SAM) are critical determinants of hepatic lipid levels, but how their levels are regulated is unclear. Here, we show that Pemt and Gnmt, key one-carbon cycle genes regulating PC/SAM levels, are downregulated after feeding, leading to decreased PC and increased SAM levels, but these effects are blunted in small heterodimer partner (SHP)-null or FGF15-null mice. Further, aryl hydrocarbon receptor (AhR) is translocated into the nucleus by insulin/PKB signaling in the early fed state and induces Pemt and Gnmt expression. This induction is blocked by FGF15 signaling-activated SHP in the late fed state. Adenoviral-mediated expression of AhR in obese mice increases PC levels and exacerbates steatosis, effects that are blunted by SHP co-expression or Pemt downregulation. PEMT, AHR, and PC levels are elevated in simple steatosis patients, but PC levels are robustly reduced in steatohepatitis-fibrosis patients. This study identifies AhR and SHP as new physiological regulators of PC/SAM levels.


Assuntos
Ciclo do Carbono , Fosfatidilcolinas/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , S-Adenosilmetionina/metabolismo , Animais , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Glicina N-Metiltransferase/genética , Glicina N-Metiltransferase/metabolismo , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fosfatidiletanolamina N-Metiltransferase/genética , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Ligação Proteica/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/genética , Receptores Citoplasmáticos e Nucleares/genética
20.
J Am Chem Soc ; 140(12): 4327-4334, 2018 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-29460630

RESUMO

The origin of enzyme catalysis remains a question of debate despite much intense study. We report a QM/MM theoretical study of the SN2 methyl transfer reaction catalyzed by a glycine N-methyltransferase (GNMT) and three mutants to test whether recent experimental observations of rate-constant reductions and variations in inverse secondary α-3H kinetic isotope effects (KIEs) should be attributed to changes in the methyl donor-acceptor distance (DAD): Is catalysis due to a compression effect? Semiempirical (AM1) and DFT (M06-2X) methods were used to describe the QM subset of atoms, while OPLS-AA and TIP3P classical force fields were used for the protein and water molecules, respectively. The computed activation free energies and KIEs are in good agreement with experimental data, but the mutations do not meaningfully affect the DAD: Compression cannot explain the experimental variations on KIEs. On the contrary, electrostatic properties in the active site correlate with the catalytic activity of wild type and mutants. The plasticity of the enzyme moderates the effects of the mutations, explaining the rather small degree of variation in KIEs and reactivities.


Assuntos
Glicina N-Metiltransferase/metabolismo , Teoria Quântica , Biocatálise , Glicina N-Metiltransferase/química , Glicina N-Metiltransferase/genética , Cinética , Conformação Molecular , Eletricidade Estática
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