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1.
Biochem Biophys Res Commun ; 581: 25-30, 2021 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-34653675

RESUMO

The industrial yeast Pichia pastoris can utilize amino acids as the sole source of carbon. It possesses a post-transcriptional regulatory circuit that governs the synthesis of cytosolic glutamate dehydrogenase 2 (GDH2) and phosphoenolpyruvate carboxykinase (PEPCK), key enzymes of amino acid catabolism. Here, we demonstrate that the post-transcriptional regulatory circuit is activated during carbon starvation resulting in the translation of GDH2 and PEPCK mRNAs. GDH2 and PEPCK synthesis is abrogated in Δatg1 indicating a key role for autophagy or an autophagy-related process. Finally, carbon-starved Δgdh2 and Δpepck exhibit poor survival. This study demonstrates a key role for amino acid catabolism during carbon starvation, a phenomenon hitherto unreported in other yeast species.


Assuntos
Carbono/deficiência , Proteínas Fúngicas/genética , Desidrogenase de Glutamato (NADP+)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , RNA Mensageiro/genética , Saccharomycetales/efeitos dos fármacos , Aminoácidos/metabolismo , Autofagia/genética , Proteínas Relacionadas à Autofagia , Carbono/farmacologia , Proteínas Fúngicas/agonistas , Proteínas Fúngicas/biossíntese , Regulação Fúngica da Expressão Gênica , Desidrogenase de Glutamato (NADP+)/biossíntese , Metabolismo/genética , Viabilidade Microbiana , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Biossíntese de Proteínas , RNA Mensageiro/agonistas , RNA Mensageiro/biossíntese , Saccharomycetales/enzimologia , Saccharomycetales/genética , Saccharomycetales/crescimento & desenvolvimento
2.
Microb Cell Fact ; 20(1): 39, 2021 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-33557832

RESUMO

BACKGROUND: Cyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium. RESULTS: We were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium. CONCLUSIONS: Heterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle.


Assuntos
Proteínas de Bactérias , Glioxilatos/metabolismo , Engenharia Metabólica , Fosfoenolpiruvato Carboxiquinase (ATP) , Ácido Succínico/metabolismo , Synechocystis , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Synechocystis/genética , Synechocystis/metabolismo
3.
Biochem J ; 477(5): 1021-1031, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32096546

RESUMO

Hepatic glucose metabolism signaling downstream of insulin can diverge to multiple pathways including AKT. Genetic studies suggest that AKT is necessary for insulin to suppress gluconeogenesis. To specifically address the role of AKT2, the dominant liver isoform of AKT in the regulation of gluconeogenesis genes, we generated hepatocytes lacking AKT2 (Akt2-/-). We found that, in the absence of insulin signal, AKT2 is required for maintaining the basal level expression of phosphoenolpyruvate carboxyl kinase (PEPCK) and to a lesser extent G6Pase, two key rate-limiting enzymes for gluconeogenesis that support glucose excursion due to pyruvate loading. We further showed that this function of AKT2 is mediated by the phosphorylation of cyclic AMP response element binding (CREB). Phosphorylation of CREB by AKT2 is needed for CREB to induce the expression of PEPCK and likely represents a priming event for unstimulated cells to poise to receive glucagon and other signals. The inhibition of gluconeogenesis by insulin is also dependent on the reduced FOXO1 transcriptional activity at the promoter of PEPCK. When insulin signal is absent, this activity appears to be inhibited by AKT2 in manner that is independent of its phosphorylation by AKT. Together, this action of AKT2 on FOXO1 and CREB to maintain basal gluconeogenesis activity may provide fine-tuning for insulin and glucocorticoid/glucagon to regulate gluconeogenesis in a timely manner to meet metabolic needs.


Assuntos
Regulação Enzimológica da Expressão Gênica , Glucose-6-Fosfatase/biossíntese , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Proteínas Proto-Oncogênicas c-akt/deficiência , Animais , Células Cultivadas , Glucose-6-Fosfatase/genética , Hepatócitos/enzimologia , Camundongos , Camundongos Knockout , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Proteínas Proto-Oncogênicas c-akt/genética
4.
Exp Clin Endocrinol Diabetes ; 127(9): 615-622, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30296791

RESUMO

Diabetes mellitus is a complex metabolic disease around the world that is characterized by hyperglycemia resulting from impaired insulin secretion, insulin action, or both. MicroRNA-29a is an important regulator of insulin signaling and gluconeogenesis pathways through IRS2, PI3K and PEPCK expressions which up regulates in Diabetes. Morin is a substantial bioflavonoid which has insulin mimetic effect, and interacting with nucleic acids and proteins. In this study HepG2 cells, were exposed to high glucose to induce diabetic condition. We have determined whether high glucose stimulation might promotes miR-29a expression level in HepG2 cells and subsequently evaluated the Morin treatment effects on this state. In HepG2 cells, high glucose increases miR-29a expression level and decreases its target genes, IRS2 and PI3K expression, and increases associated downstream gene in gluconeogenic pathway, PEPCK. Morin treatment down regulates miR-29a expression level and improves insulin signaling and glucose metabolism. To confirm the inhibitory effects of Morin on miR-29a, we have transfected cells with mimic and inhibitor-miR-29a. This study for the first time identifies that Morin improves diabetic condition through down regulation of the miR-29a level, and suggest that this new inhibitor of miR-29a may be a useful biomedicine to treat diabetes.


Assuntos
Regulação para Baixo/efeitos dos fármacos , Flavonoides/farmacologia , Hipoglicemiantes/farmacologia , MicroRNAs/biossíntese , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Células Hep G2 , Humanos , Proteínas Substratos do Receptor de Insulina/biossíntese , Fosfatidilinositol 3-Quinases/biossíntese , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese
5.
Biochimie ; 140: 106-116, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28711683

RESUMO

Non-alcoholic fatty liver disease (NAFLD) presents with growing prevalence worldwide, though its pharmacological treatment remains to be established. This study aimed to evaluate the effects of a PPAR-alpha agonist on liver tissue structure, ultrastructure, and metabolism, focusing on gene and protein expression of de novo lipogenesis and gluconeogenesis pathways, in diet-induced obese mice. Male C57BL/6 mice (three months old) received a control diet (C, 10% of lipids, n = 10) or a high-fat diet (HFD, 50% of lipids, n = 10) for ten weeks. These groups were subdivided to receive the treatment (n = 5 per group): C, C-alpha (PPAR-alpha agonist, 2.5 mg/kg/day mixed in the control diet), HFD and HFD-alpha group (PPAR-alpha agonist, 2.5 mg/kg/day mixed in the HFD). The effects were compared with biometrical, biochemical, molecular biology and transmission electron microscopy (TEM) analyses. HFD showed greater body mass (BM) and insulinemia than C, both of which were tackled by the treatment in the HFD-alpha group. Increased hepatic protein expression of glucose-6-phosphatase, CHREBP and gene expression of PEPCK in HFD points to increased gluconeogenesis. Treatment rescued these parameters in the HFD-alpha group, eliciting a reduced hepatic glucose output, confirmed by the smaller GLUT2 expression in HFD-alpha than in HFD. Conversely, favored de novo lipogenesis was found in the HFD group by the increased expression of PPAR-gamma, and its target gene SREBP-1, FAS and GK when compared to C. The treatment yielded a marked reduction in the expression of all lipogenic factors. TEM analyses showed a greater numerical density of mitochondria per area of tissue in treated than in untreated groups, suggesting an increase in beta-oxidation and the consequent NAFLD control. PPAR-alpha activation reduced BM and treated insulin resistance (IR) and NAFLD by increasing the number of mitochondria and reducing hepatic gluconeogenesis and de novo lipogenesis protein and gene expressions in a murine obesity model.


Assuntos
Gorduras na Dieta/efeitos adversos , Fígado/metabolismo , Mitocôndrias Hepáticas/metabolismo , Obesidade/tratamento farmacológico , PPAR alfa/agonistas , Pirimidinas/farmacologia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Gorduras na Dieta/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose-6-Fosfatase/biossíntese , Resistência à Insulina , Lipogênese/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Mitocôndrias Hepáticas/patologia , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Proteínas Nucleares/biossíntese , Obesidade/induzido quimicamente , Obesidade/metabolismo , Obesidade/patologia , PPAR alfa/metabolismo , PPAR gama/biossíntese , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Proteína de Ligação a Elemento Regulador de Esterol 1/biossíntese , Fatores de Transcrição/biossíntese , Receptor fas/biossíntese
6.
J Biol Chem ; 292(25): 10444-10454, 2017 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-28473467

RESUMO

Maintenance of whole-body glucose homeostasis is critical to glycemic function. Genetic variants mapping to chromosome 8p23.1 in genome-wide association studies have been linked to glycemic traits in humans. The gene of known function closest to the mapped region, PPP1R3B (protein phosphatase 1 regulatory subunit 3B), encodes a protein (GL) that regulates glycogen metabolism in the liver. We therefore sought to test the hypothesis that hepatic PPP1R3B is associated with glycemic traits. We generated mice with either liver-specific deletion (Ppp1r3bΔhep ) or liver-specific overexpression of Ppp1r3b The Ppp1r3b deletion significantly reduced glycogen synthase protein abundance, and the remaining protein was predominantly phosphorylated and inactive. As a consequence, glucose incorporation into hepatic glycogen was significantly impaired, total hepatic glycogen content was substantially decreased, and mice lacking hepatic Ppp1r3b had lower fasting plasma glucose than controls. The concomitant loss of liver glycogen impaired whole-body glucose homeostasis and increased hepatic expression of glycolytic enzymes in Ppp1r3bΔhep mice relative to controls in the postprandial state. Eight hours of fasting significantly increased the expression of two critical gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, above the levels in control livers. Conversely, the liver-specific overexpression of Ppp1r3b enhanced hepatic glycogen storage above that of controls and, as a result, delayed the onset of fasting-induced hypoglycemia. Moreover, mice overexpressing hepatic Ppp1r3b upon long-term fasting (12-36 h) were protected from blood ketone-body accumulation, unlike control and Ppp1r3bΔhep mice. These findings indicate a major role for Ppp1r3b in regulating hepatic glycogen stores and whole-body glucose/energy homeostasis.


Assuntos
Glicemia/metabolismo , Metabolismo Energético/fisiologia , Gluconeogênese/fisiologia , Glicogênio/biossíntese , Fígado/metabolismo , Proteína Fosfatase 1/biossíntese , Animais , Glicemia/genética , Jejum/sangue , Regulação Enzimológica da Expressão Gênica/fisiologia , Glucose-6-Fosfatase/biossíntese , Glucose-6-Fosfatase/genética , Glicogênio/genética , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Proteína Fosfatase 1/genética
7.
Oncogene ; 36(25): 3609-3617, 2017 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-28166201

RESUMO

For cancer cells to proliferate, a balance must be built between biomass-forming, glucose-metabolized intermediates and ATP production. How intrinsic glucose carbon flow regulates this balance remains unclear. Here we show that mitochondrial phosphoenolpyruvate carboxykinase (PCK2), the hub molecule linking tricarboxylic acid (TCA) cycle, glycolysis and gluconeogenesis by conversion of mitochondrial oxaloacetate (OAA) to phosphoenolpyruvate, regulates glucose carbon flow direction in stem-like cells that repopulate tumors (tumor-repopulating cells (TRCs)). PCK2 downregulation accelerated biosynthesis and transportation of citrate from mitochondria to the cytosol, leading to cytosolic glucose carbon flow via OAA-malate-pyruvate and acetyl-CoA-fatty acid pathways in TRCs. On the other hand, downregulating PCK2 hindered fumarate carbon flows in TCA cycle, leading to attenuated oxidative phosphorylation. In pathological terms, PCK2 overexpression slowed TRC growth in vitro and impeded tumorigenesis in vivo. Overall, our work unveiled unexpected glucose carbon flows of TRCs in melanoma that have implications for targeting metabolic aspects of melanoma.


Assuntos
Ciclo do Ácido Cítrico , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , Proteínas de Neoplasias/biossíntese , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Melanoma/genética , Melanoma/patologia , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Fosforilação Oxidativa , Fosfoenolpiruvato Carboxiquinase (ATP)/genética
8.
Toxicol Lett ; 263: 34-43, 2016 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-27713024

RESUMO

Trovafloxacin develops severe hepatotoxicity; however, the underlying mechanism of the trovafloxacin-induced liver injury has not been cleared. It has been shown that microRNAs (miRNAs) can be involved in the development of drug-induced liver injuries. We performed a miRNA microarray analysis to identify hepatic miRNAs that were induced or reduced by trovafloxacin in mice. It was demonstrated that miR-877-5p was the most increased miRNA in the mouse liver 24h after the trovafloxacin administration. To investigate the role of miR-877-5p in the liver, we established miR-877-5p-overexpressed HepG2 cells. Microarray analysis detected altered expressions in 2077 (>2-fold) and 1547 (<0.5-fold) genes in the miR-877-5p overexpressing cells compared to the mock cells. Especially, SLCO4C1, PEPCK, MT1M, HIST1H2BM, LGI1, and PLA2G2A were markedly increased or decreased in the miR-877-5p overexpressing cells. We conducted a correlation analysis between the expression levels of miR-877-5p and the six genes in eight miR-877-5p stably-expressed clones. It was shown that the PEPCK expression levels were correlated with miR-877-5p expression levels. PEPCK is associated with development of apoptotic cell death; therefore, the increased miR- 877-5p-induced PEPCK can be a trigger that is involved in the development of trovafloxacin-induced liver injury.


Assuntos
Antibacterianos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/genética , Fluoroquinolonas/toxicidade , MicroRNAs/genética , Naftiridinas/toxicidade , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Análise em Microsséries , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Fosfoenolpiruvato Carboxiquinase (ATP)/genética
9.
Proc Natl Acad Sci U S A ; 113(11): 3084-9, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26903647

RESUMO

Leptin is the primary adipostatic factor in mammals. Produced largely by adipocytes in proportion to total adipose mass, the hormone informs the brain regarding total energy stored as triglycerides in fat cells. The hormone acts on multiple circuits in the brain to regulate food intake, autonomic outflow, and endocrine function to maintain energy balance. In addition to regulating adipose mass, mammalian leptin also plays a role in the regulation of glucose homeostasis and as a gating factor in reproductive competence. Leptin-deficient mice and people exhibit early onset profound hyperphagia and obesity, diabetes, and infertility. Although leptin and the leptin receptor are found in fish, the hormone is not expressed in adipose tissue, but is found in liver and other tissues. Here, we show that adult zebrafish lacking a functional leptin receptor do not exhibit hyperphagia or increased adiposity, and exhibit normal fertility. However, leptin receptor-deficient larvae have increased numbers of ß-cells and increased levels of insulin mRNA. Furthermore, larval zebrafish have been shown to exhibit ß-cell hyperplasia in response to high fat feeding or peripheral insulin resistance, and we show here that leptin receptor is required for this response. Adult zebrafish also have increased levels of insulin mRNA and other alterations in glucose homeostasis. Thus, a role for leptin in the regulation of ß-cell mass and glucose homeostasis appears to be conserved across vertebrates, whereas its role as an adipostatic factor is likely to be a secondary role acquired during the evolution of mammals.


Assuntos
Adiposidade/fisiologia , Glucose/metabolismo , Células Secretoras de Insulina/fisiologia , Leptina/fisiologia , Receptores para Leptina/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Sequência de Aminoácidos , Animais , Tamanho Corporal , Peso Corporal , Contagem de Células , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Gorduras na Dieta , Fertilidade , Teste de Tolerância a Glucose , Glicogenólise , Glicólise , Homeostase , Hiperfagia/genética , Hiperfagia/fisiopatologia , Insulina/metabolismo , Resistência à Insulina , Secreção de Insulina , Larva , Leptina/genética , Fígado/metabolismo , Masculino , Dados de Sequência Molecular , Fenótipo , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores para Leptina/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genética
10.
Oncotarget ; 6(31): 30568-78, 2015 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-26375250

RESUMO

In this study, we tested whether a standardized epigallocatechin-3-gallate (EGCG) rich green tea extract (comprising > 90% EGCG) affects fitness and lifespan as well as parameters of glucose metabolism and energy homeostasis in the fruit fly, Drosophila melanogaster. Following the application of the green tea extract a significant increase in the mean lifespan (+ 3.3 days) and the 50% survival (+ 4.3 days) as well as improved fitness was detected. These effects went along an increased expression of Spargel, the homolog of mammalian PGC1α, which has been reported to affect lifespan in flies. Intriguingly, in flies, treatment with the green tea extract decreased glucose concentrations, which were accompanied by an inhibition of α-amylase and α-glucosidase activity. Computational docking analysis proved the potential of EGCG to dock into the substrate binding pocket of α-amylase and to a greater extent into α-glucosidase. Furthermore, we demonstrate that EGCG downregulates insulin-like peptide 5 and phosphoenolpyruvate carboxykinase, major regulators of glucose metabolism, as well as the Drosophila homolog of leptin, unpaired 2. We propose that a decrease in glucose metabolism in connection with an upregulated expression of Spargel contribute to the better fitness and the extended lifespan in EGCG-treated flies.


Assuntos
Antioxidantes/farmacologia , Catequina/análogos & derivados , Metabolismo Energético/efeitos dos fármacos , Glucose/metabolismo , Longevidade/fisiologia , Animais , Camellia sinensis/metabolismo , Catequina/farmacologia , Proteínas de Drosophila/biossíntese , Drosophila melanogaster/metabolismo , Homeostase/efeitos dos fármacos , Insulina/biossíntese , Longevidade/efeitos dos fármacos , Simulação de Acoplamento Molecular , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Extratos Vegetais/farmacologia , Fator B de Elongação Transcricional Positiva/biossíntese , Proteínas , Fatores de Transcrição/biossíntese , Regulação para Cima , alfa-Amilases/metabolismo , alfa-Glucosidases/metabolismo
11.
PLoS One ; 10(7): e0132113, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26148093

RESUMO

Increased maternal androgen exposure during pregnancy programmes a polycystic ovary syndrome (PCOS)-like condition, with metabolic dysfunction, in adult female offspring. Other in utero exposures associated with the development of insulin resistance, such as intrauterine growth restriction and exposure to prenatal glucocorticoids, are associated with altered fetal gluconeogenesis. We therefore aimed to assess the effect of maternal androgenisation on the expression of PEPCK and G6PC in the ovine fetus. Pregnant Scottish Greyface sheep were treated with twice weekly testosterone propionate (TP; 100mg) or vehicle control from day 62 to day 102 of gestation. At day 90 and day 112 fetal plasma and liver and kidney tissue was collected for analysis. PEPCK and G6PC expression were analysed by quantitative RT-PCR, immunohistochemistry and western blotting. PEPCK and G6PC were localised to fetal hepatocytes but maternal androgens had no effect on female or male fetuses. PEPCK and G6PC were also localised to the renal tubules and renal PEPCK (P<0.01) and G6PC (P = 0.057) were lower in females after prenatal androgenisation with no change in male fetuses. These tissue and sex specific observations could not be explained by alterations in fetal insulin or cortisol. The sexual dimorphism may be related to the increase in circulating estrogen (P<0.01) and testosterone (P<0.001) in females but not males. The tissue specific effects may be related to the increased expression of ESR1 (P<0.01) and AR (P<0.05) in the kidney when compared to the fetal liver. After discontinuation of maternal androgenisation female fetal kidney PEPCK expression normalised. These data further highlight the fetal and sexual dimorphic effects of maternal androgenisation, an antecedent to adult disease and the plasticity of fetal development.


Assuntos
Androgênios/efeitos adversos , Gluconeogênese/efeitos dos fármacos , Rim/embriologia , Síndrome do Ovário Policístico/embriologia , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Propionato de Testosterona/efeitos adversos , Androgênios/farmacologia , Animais , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucose-6-Fosfatase/biossíntese , Hepatócitos/metabolismo , Humanos , Rim/patologia , Masculino , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Síndrome do Ovário Policístico/induzido quimicamente , Síndrome do Ovário Policístico/patologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/patologia , Ovinos , Propionato de Testosterona/farmacologia
12.
Biomed Res Int ; 2015: 414860, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25922835

RESUMO

The aim of this study was to determine whether inhibition of resistin by a synthetic antiresistin RNA (oligonucleotide) oligo ameliorates metabolic and histological abnormalities in nonalcoholic fatty liver disease (NAFLD) induced by high-fat diet (HFD) in mice. The antiresistin RNA oligo and a scrambled control oligo (25 mg/kg of body weight) were i.p. injected to HFD mice. Serum metabolic parameters and hepatic enzymes were measured after 4-week treatment. The treatment significantly reduced epididymal fat and attenuated the elevated serum resistin, cholesterol, triglycerides, glucose, and insulin with an improved glucose tolerance test. Antiresistin RNA oligo also normalized serum AST and ALT levels with improved pathohistology of NAFLD. Immunoblotting and qRT-PCR revealed that decreased protein and mRNA expression of resistin in fat and liver tissues of the treated mice were associated with reduction of adipose TNF-α and IL-6 expression and secretion into circulation. mRNA and protein expression of hepatic phosphoenolpyruvate carboxykinase (PEPCK) and sterol regulatory element-binding protein-1c (SREBP-1c) were also significantly decreased in the treated mice. Our results suggest that resistin may exacerbate NAFLD in metabolic syndrome through upregulating inflammatory cytokines and hepatic PEPCK and SREBP-1c. Antiresistin RNA oligo ameliorated metabolic abnormalities and histopathology of NAFLD through attenuating proinflammatory cytokines.


Assuntos
Hepatopatia Gordurosa não Alcoólica/genética , Obesidade/genética , Oligonucleotídeos/administração & dosagem , Resistina/genética , Animais , Dieta Hiperlipídica , Regulação da Expressão Gênica/genética , Resistência à Insulina/genética , Interleucina-6/biossíntese , Camundongos , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/terapia , Obesidade/sangue , Obesidade/patologia , Obesidade/terapia , Oligonucleotídeos/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , RNA/administração & dosagem , RNA/genética , Resistina/antagonistas & inibidores , Resistina/sangue , Proteína de Ligação a Elemento Regulador de Esterol 1/biossíntese , Fator de Necrose Tumoral alfa/biossíntese
13.
Mol Cell Endocrinol ; 399: 22-31, 2015 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-25179821

RESUMO

Polycystic ovary syndrome (PCOS) is a reproductive and metabolic disorder characterized by hyperandrogenism, ovulatory dysfunction, visceral obesity and insulin resistance. We hypothesized that changes in glucocorticoid metabolism and signaling in the visceral adipose tissue may contribute to disturbances of lipid metabolism in the rat model of PCOS obtained by 5α-dihydrotestosterone (DHT) treatment of prepubertal female Wistar rats. The results confirmed that DHT treatment caused anovulation, obesity and dyslipidemia. Enhanced glucocorticoid prereceptor metabolism, assessed by elevated intracellular corticosterone and increased 11 beta-hydroxysteroid dehydrogenase type 1 mRNA and protein levels, was accompanied by glucocorticoid receptor (GR) nuclear accumulation. In concert with the increased expression of GR-regulated prolipogenic genes (lipin-1, sterol regulatory element binding protein 1, fatty acid synthase, phosphoenolpyruvate carboxykinase), histological analyses revealed hypertrophic adipocytes. The results suggest that glucocorticoids influence lipid metabolism in the visceral adipose tissue in the way that may contribute to pathogenesis of metabolic disturbances associated with PCOS.


Assuntos
Adipócitos/metabolismo , Androgênios/efeitos adversos , Di-Hidrotestosterona/efeitos adversos , Glucocorticoides/metabolismo , Gordura Intra-Abdominal/metabolismo , Síndrome do Ovário Policístico/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/biossíntese , Adipócitos/patologia , Androgênios/farmacologia , Animais , Di-Hidrotestosterona/farmacologia , Ácido Graxo Sintase Tipo I/biossíntese , Feminino , Gordura Intra-Abdominal/patologia , Proteínas Nucleares/biossíntese , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/patologia , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Síndrome do Ovário Policístico/induzido quimicamente , Síndrome do Ovário Policístico/complicações , Síndrome do Ovário Policístico/patologia , Ratos , Ratos Wistar , Receptores de Glucocorticoides/biossíntese , Proteína de Ligação a Elemento Regulador de Esterol 1/biossíntese
14.
FASEB J ; 28(8): 3339-50, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24755741

RESUMO

Reduced AMP kinase (AMPK) activity has been shown to play a key deleterious role in increased hepatic gluconeogenesis in diabetes, but the mechanism whereby this occurs remains unclear. In this article, we document that another AMP-dependent enzyme, AMP deaminase (AMPD) is activated in the liver of diabetic mice, which parallels with a significant reduction in AMPK activity and a significant increase in intracellular glucose accumulation in human HepG2 cells. AMPD activation is induced by a reduction in intracellular phosphate levels, which is characteristic of insulin resistance and diabetic states. Increased gluconeogenesis is mediated by reduced TORC2 phosphorylation at Ser171 by AMPK in these cells, as well as by the up-regulation of the rate-limiting enzymes PEPCK and G6Pc. The mechanism whereby AMPD controls AMPK activation depends on the production of a specific AMP downstream metabolite through AMPD, uric acid. In this regard, humans have higher uric acid levels than most mammals due to a mutation in uricase, the enzyme involved in uric acid degradation in most mammals, that developed during a period of famine in Europe 1.5 × 10(7) yr ago. Here, working with resurrected ancestral uricases obtained from early hominids, we show that their expression on HepG2 cells is enough to blunt gluconeogenesis in parallel with an up-regulation of AMPK activity. These studies identify a key role AMPD and uric acid in mediating hepatic gluconeogenesis in the diabetic state, via a mechanism involving AMPK down-regulation and overexpression of PEPCK and G6Pc. The uricase mutation in the Miocene likely provided a survival advantage to help maintain glucose levels under conditions of near starvation, but today likely has a role in the pathogenesis of diabetes.


Assuntos
AMP Desaminase/fisiologia , Gluconeogênese/fisiologia , Fígado/metabolismo , Inanição/fisiopatologia , Ácido Úrico/metabolismo , AMP Desaminase/antagonistas & inibidores , AMP Desaminase/genética , Proteínas Quinases Ativadas por AMP/fisiologia , Animais , Diabetes Mellitus Experimental/metabolismo , Europa (Continente) , Regulação Enzimológica da Expressão Gênica , Gluconeogênese/efeitos dos fármacos , Glucose-6-Fosfatase/biossíntese , Células Hep G2 , História Antiga , Hominidae/fisiologia , Humanos , Insulina/metabolismo , Resistência à Insulina , Secreção de Insulina , Fígado/enzimologia , Masculino , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Complexos Multiproteicos/fisiologia , Fosfatos/metabolismo , Fosfatos/farmacologia , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Seleção Genética , Organismos Livres de Patógenos Específicos , Inanição/história , Serina-Treonina Quinases TOR/fisiologia , Transdução Genética , Urato Oxidase/genética , Urato Oxidase/história , Urato Oxidase/metabolismo , Ácido Úrico/farmacologia
15.
Enzyme Microb Technol ; 56: 8-14, 2014 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-24564896

RESUMO

Succinate is not the dominant fermentation product from xylose in wild-type Escherichia coli K12. E. coli BA 203 is a lactate dehydrogenase (ldhA), pyruvate formate lyase (pflB), and phosphoenolpyruvate (PEP)-carboxylase (ppc) deletion strain. To increase succinate accumulation and reduce byproduct formation, engineered E. coli BA204, in which ATP-forming PEP-carboxykinase (PEPCK) is overexpressed in BA203, was constructed and produced 2.17-fold higher succinate yield. To further improve the biomass and the consumption rate of xylose, nicotinic acid phosphoribosyltransferase (NAPRTase), a rate limiting enzyme in the synthesis of NAD(H), was also overexpressed. Thus, co-expression of PEPCK and NAPRTase in recombinant E. coli BA209 was investigated. In BA209, the pck gene and the pncB gene each have a trc promoter, hence, both genes are well expressed. During a 72-h anaerobic fermentation in sealed bottles, the total concentration of NAD(H) in BA209 was 1.25-fold higher than that in BA204, and the NADH/NAD+ ratio decreased from 0.28 to 0.11. During the exclusively anaerobic fermentation in a 3-L bioreactor, BA209 consumed 17.1 g L⁻¹ xylose and produced 15.5 g L⁻¹ succinate. Furthermore, anaerobic fermentation of corn stalk hydrolysate contained 30.1 g L⁻¹ xylose, 2.1 g L⁻¹ glucose and 1.5 g L⁻¹ arabinose, it produced a final succinate concentration of 17.2 g L⁻¹ with a yield of 0.94 g g⁻¹ total sugars.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/biossíntese , Escherichia coli K12/enzimologia , Microbiologia Industrial/métodos , Pentosiltransferases/biossíntese , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Succinatos/metabolismo , Anaerobiose , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Reatores Biológicos , Clonagem Molecular , Escherichia coli K12/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Fermentação , Genes Bacterianos , Genes Sintéticos , Hidrólise , Pentosiltransferases/genética , Pentosiltransferases/isolamento & purificação , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/isolamento & purificação , Extratos Vegetais/metabolismo , Brotos de Planta , Succinatos/isolamento & purificação , Resíduos , Xilose/metabolismo , Zea mays
16.
J Biol Chem ; 289(9): 5518-27, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24415752

RESUMO

Liver gluconeogenesis is essential to provide energy to glycolytic tissues during fasting periods. However, aberrant up-regulation of this metabolic pathway contributes to the progression of glucose intolerance in individuals with diabetes. Phosphoenolpyruvate carboxykinase (PEPCK) expression plays a critical role in the modulation of gluconeogenesis. Several pathways contribute to the regulation of PEPCK, including the nuclear receptor Rev-erbα and the histone deacetylase SIRT1. Deleted in breast cancer 1 (DBC1) is a nuclear protein that binds to and regulates both Rev-erbα and SIRT1 and, therefore, is a candidate to participate in the regulation of PEPCK. In this work, we provide evidence that DBC1 regulates glucose metabolism and the expression of PEPCK. We show that DBC1 levels decrease early in the fasting state. Also, DBC1 KO mice display higher gluconeogenesis in a normal and a high-fat diet. DBC1 absence leads to an increase in PEPCK mRNA and protein expression. Conversely, overexpression of DBC1 results in a decrease in PEPCK mRNA and protein levels. DBC1 regulates the levels of Rev-erbα, and manipulation of Rev-erbα activity or levels prevents the effect of DBC1 on PEPCK. In addition, Rev-erbα levels decrease in the first hours of fasting. Finally, knockdown of the deacetylase SIRT1 eliminates the effect of DBC1 knockdown on Rev-erbα levels and PEPCK expression, suggesting that the mechanism of PEPCK regulation is, at least in part, dependent on the activity of this enzyme. Our results point to DBC1 as a novel regulator of gluconeogenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Gluconeogênese/fisiologia , Glucose/biossíntese , Fígado/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Gorduras na Dieta/farmacologia , Jejum/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Glucose/genética , Células Hep G2 , Humanos , Fígado/citologia , Camundongos , Camundongos Knockout , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Sirtuína 1/genética , Sirtuína 1/metabolismo
17.
Stem Cells Dev ; 23(2): 124-31, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24020366

RESUMO

Stem cell-derived hepatocyte-like cells hold great potential for the treatment of liver disease and for drug toxicity screening. The success of these applications hinges on the generation of differentiated cells with high liver specific activities. Many protocols have been developed to guide human embryonic stem cells (hESCs) to differentiate to the hepatic lineage. Here we report cultivation of hESCs as three-dimensional aggregates that enhances their differentiation to hepatocyte-like cells. Differentiation was first carried out in monolayer culture for 20 days. Subsequently cells were allowed to self-aggregate into spheroids. Significantly higher expression of liver-specific transcripts and proteins, including Albumin, phosphoenolpyruvate carboxykinase, and asialoglycoprotein receptor 1 was observed. The differentiated phenotype was sustained for more than 2 weeks in the three-dimensional spheroid culture system, significantly longer than in monolayer culture. Cells in spheroids exhibit morphological and ultrastructural characteristics of primary hepatocytes by scanning and transmission electron microscopy in addition to mature functions, such as biliary excretion of metabolic products and cytochrome P450 activities. This three-dimensional spheroid culture system may be appropriate for generating high quality, functional hepatocyte-like cells from ESCs.


Assuntos
Células-Tronco Embrionárias/metabolismo , Hepatócitos/citologia , Fígado/citologia , Esferoides Celulares/metabolismo , Albuminas/biossíntese , Hidrocarboneto de Aril Hidroxilases/metabolismo , Receptor de Asialoglicoproteína/biossíntese , Diferenciação Celular , Células Cultivadas , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2B6 , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450 , Hepatócitos/metabolismo , Humanos , Oxazinas/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese
18.
J Biol Chem ; 287(50): 41875-87, 2012 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-23074219

RESUMO

Cyclic AMP (cAMP) induces steroidogenic enzyme gene expression and stimulates testosterone production in Leydig cells. Phosphoenolpyruvate carboxykinase (PEPCK) is expressed in Leydig cells, but its role has not been defined. In this study, we found that PEPCK and glucose-6-phosphatase (Glc-6-Pase) are increased significantly following cAMP treatment of mouse Leydig cells. Moreover, cAMP treatment increased recruitment of the cAMP-response element-binding transcription factor and decreased recruitment of the corepressor DAX-1 on the pepck promoter. Furthermore, cAMP induced an increase in ATP that correlated with a decrease in phospho-AMP-activated protein kinase (AMPK). In contrast, knockdown or inhibition of PEPCK decreased ATP and increased phospho-AMPK. Treatment with an AMPK activator or overexpression of the constitutively active form of AMPK inhibited cAMP-induced steroidogenic enzyme promoter activities and gene expression. Liver receptor homolog-1 (LRH-1) was involved in cAMP-induced steroidogenic enzyme gene expression but was inhibited by AMPK activation in Leydig cells. Additionally, inhibition or knockdown of PEPCK and Glc-6-Pase decreased cAMP-mediated induction of steroidogenic enzyme gene expression and steroidogenesis. Finally, pubertal mouse (8-week-old) testes and human chorionic gonadotropin-induced prepubertal mouse testes showed increased PEPCK and Glc-6-Pase gene expression. Taken together, these results suggest that induction of PEPCK and Glc-6-Pase by cAMP plays an important role in Leydig cell steroidogenesis.


Assuntos
Glucose-6-Fosfatase/biossíntese , Células Intersticiais do Testículo/enzimologia , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , AMP Cíclico/genética , AMP Cíclico/metabolismo , Receptor Nuclear Órfão DAX-1/genética , Receptor Nuclear Órfão DAX-1/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Glucose-6-Fosfatase/genética , Células HeLa , Humanos , Células Intersticiais do Testículo/citologia , Masculino , Camundongos , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Regiões Promotoras Genéticas/fisiologia , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo
19.
PLoS One ; 7(7): e41719, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22844518

RESUMO

BACKGROUND: Glyceroneogenesis is an important step in the control of fatty acid re-esterification with PEPCK and PDK4 being identified as key enzymes in this process. We have previously shown that glyceroneogenic enzymes such as PDK4 are rapidly induced in white adipose tissue during exercise. Recent studies have suggested that IL-6 regulates adipose tissue metabolism and gene expression during exercise. Interestingly, IL-6 has been reported to directly decrease PEPCK expression. The purpose of this investigation was to determine the role of IL-6 in modulating the effects of exercise on the expression of glyceroneogenic enzymes in mouse adipose tissue. We hypothesized that the exercise-mediated induction of PDK4 and PEPCK would be greater in adipose tissue from IL-6 deficient mice compared to wild type controls. METHODOLOGY AND PRINCIPLE FINDINGS: Treatment of cultured epididymal adipose tissue (eWAT) with IL-6 (150 ng/ml) increased the phosphorylation of AMPK, ACC and STAT3 and induced SOCS3 mRNA levels while decreasing PEPCK and PDK4 mRNA. AICAR decreased the expression of PDK4 and PEPCK. The activation of AMPK by IL-6 was independent of increases in lipolysis. An acute bout of treadmill running (15 meters/minute, 5% incline, 90 minutes) did not induce SOCS3 or increase phosphorylation of STAT3 in eWAT, indicating that IL-6 signalling was not activated. Exercise-induced increases in PEPCK and PDK4 mRNA expression were attenuated in eWAT from IL-6(-/-) mice in parallel with a greater relative increase in AMPK phosphorylation compared to exercised WT mice. These changes occurred independent of alterations in beta-adrenergic signalling in adipose tissue from IL-6(-/-) mice. CONCLUSIONS AND SIGNIFICANCE: Our findings question the role of IL-6 signalling in adipose tissue during exercise and suggest an indirect effect of this cytokine in the regulation of adipose tissue gene expression during exercise.


Assuntos
Tecido Adiposo/metabolismo , Glicerídeos/biossíntese , Glicerol/metabolismo , Interleucina-6/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Condicionamento Físico Animal , Proteínas Quinases/biossíntese , Proteínas Quinases Ativadas por AMP/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/enzimologia , Animais , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Epididimo/citologia , Interleucina-6/deficiência , Interleucina-6/farmacologia , Masculino , Camundongos , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosforilação/efeitos dos fármacos , Proteínas Quinases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos
20.
Am J Physiol Endocrinol Metab ; 302(4): E409-16, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22127228

RESUMO

The aim of the present study was to examine the effects of pyrrolidine dithiocarbamate (PDTC) on hepatic glycogen synthesis and FoxO1 transcriptional activity in type 2 diabetic rats and the mechanism underlying these effects. Fasting blood glucose and glycogen deposition, together with expressions of two key genes related to gluconeogenesis, were studied in the liver of rats fed a normal diet (NC), high-fat diet (HFD)-induced insulin-resistant rats made type 2 diabetic by a single intraperitoneal injection of streptozotocin (DM), and a DM with intervention of PDTC (DM + PDTC) for 1 wk. The phosphorylation of Akt, GSK-3ß, and FoxO1 was assessed in liver extracts of fasted rats by Western blot, whereas indirect immunofluorescence staining was performed to determine the cellular distribution of FoxO1. The DM rats exhibited obvious increases in fasting blood glucose as well as decreased hepatic glycogen content compared with the NC group. Activation of the Akt/GSK-3ß pathway and inactivating phosphorylation of FoxO1 were reduced greatly in DM rat livers (P < 0.01). By contrast, PDTC treatment protected DM rats against high fasting blood glucose and hepatic glycogen deposition loss. PDTC also elicited an increase in Akt/GSK-3ß signaling and subsequent inactivation and nuclear export of FoxO1 in DM rat livers, which translated into a significant reduction in the expression of two FoxO1 target genes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. This study suggests that PDTC enhances hepatic glycogen synthesis, whereas it reduces FoxO1 transcriptional activity in DM rats.


Assuntos
Antioxidantes/farmacologia , Diabetes Mellitus Tipo 2/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Glicogênio Hepático/biossíntese , Fígado/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Pirrolidinas/farmacologia , Tiocarbamatos/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Glicemia/efeitos dos fármacos , Dieta Hiperlipídica , Jejum/sangue , Gluconeogênese/efeitos dos fármacos , Glucose-6-Fosfatase/biossíntese , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Resistência à Insulina , Fígado/química , Fígado/metabolismo , Masculino , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Fosforilação , Ratos , Ratos Wistar
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