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1.
Blood ; 134(22): 1983-1995, 2019 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-31570489

RESUMO

Host cell competition is a major barrier to engraftment after in utero hematopoietic cell transplantation (IUHCT). Here we describe a cell-engineering strategy using glycogen synthase kinase-3 (GSK3) inhibitor-loaded nanoparticles conjugated to the surface of donor hematopoietic cells to enhance their proliferation kinetics and ability to compete against their fetal host equivalents. With this approach, we achieved remarkable levels of stable, long-term hematopoietic engraftment for up to 24 weeks post-IUHCT. We also show that the salutary effects of the nanoparticle-released GSK3 inhibitor are specific to donor progenitor/stem cells and achieved by a pseudoautocrine mechanism. These results establish that IUHCT of hematopoietic cells decorated with GSK3 inhibitor-loaded nanoparticles can produce therapeutic levels of long-term engraftment and could therefore allow single-step prenatal treatment of congenital hematological disorders.


Assuntos
Comunicação Autócrina , Engenharia Celular , Inibidores Enzimáticos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Sobrevivência de Enxerto/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Nanopartículas/química , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C
2.
J Biol Chem ; 289(14): 9710-29, 2014 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-24523404

RESUMO

Agmatine (AGM), a product of arginine decarboxylation, influences multiple physiologic and metabolic functions. However, the mechanism(s) of action, the impact on whole body gene expression and metabolic pathways, and the potential benefits and risks of long term AGM consumption are still a mystery. Here, we scrutinized the impact of AGM on whole body metabolic profiling and gene expression and assessed a plausible mechanism(s) of AGM action. Studies were performed in rats fed a high fat diet or standard chow. AGM was added to drinking water for 4 or 8 weeks. We used (13)C or (15)N tracers to assess metabolic reactions and fluxes and real time quantitative PCR to determine gene expression. The results demonstrate that AGM elevated the synthesis and tissue level of cAMP. Subsequently, AGM had a widespread impact on gene expression and metabolic profiling including (a) activation of peroxisomal proliferator-activated receptor-α and its coactivator, PGC1α, and (b) increased expression of peroxisomal proliferator-activated receptor-γ and genes regulating thermogenesis, gluconeogenesis, and carnitine biosynthesis and transport. The changes in gene expression were coupled with improved tissue and systemic levels of carnitine and short chain acylcarnitine, increased ß-oxidation but diminished incomplete fatty acid oxidation, decreased fat but increased protein mass, and increased hepatic ureagenesis and gluconeogenesis but decreased glycolysis. These metabolic changes were coupled with reduced weight gain and a curtailment of the hormonal and metabolic derangements associated with high fat diet-induced obesity. The findings suggest that AGM elevated the synthesis and levels of cAMP, thereby mimicking the effects of caloric restriction with respect to metabolic reprogramming.


Assuntos
Agmatina/farmacologia , AMP Cíclico/metabolismo , Ácidos Graxos/metabolismo , Gluconeogênese/efeitos dos fármacos , Fígado/metabolismo , Obesidade/tratamento farmacológico , Agmatina/farmacocinética , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Carnitina/análogos & derivados , Carnitina/metabolismo , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Metaboloma , Obesidade/induzido quimicamente , Obesidade/metabolismo , Oxirredução/efeitos dos fármacos , PPAR gama/biossíntese , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Fatores de Transcrição/biossíntese
3.
J Biol Chem ; 288(6): 3938-51, 2013 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-23266825

RESUMO

Paracrine signaling between pancreatic islet ß-cells and α-cells has been proposed to play a role in regulating glucagon responses to elevated glucose and hypoglycemia. To examine this possibility in human islets, we used a metabolomic approach to trace the responses of amino acids and other potential neurotransmitters to stimulation with [U-(13)C]glucose in both normal individuals and type 2 diabetics. Islets from type 2 diabetics uniformly showed decreased glucose stimulation of insulin secretion and respiratory rate but demonstrated two different patterns of glucagon responses to glucose: one group responded normally to suppression of glucagon by glucose, but the second group was non-responsive. The non-responsive group showed evidence of suppressed islet GABA levels and of GABA shunt activity. In further studies with normal human islets, we found that γ-hydroxybutyrate (GHB), a potent inhibitory neurotransmitter, is generated in ß-cells by an extension of the GABA shunt during glucose stimulation and interacts with α-cell GHB receptors, thus mediating the suppressive effect of glucose on glucagon release. We also identified glycine, acting via α-cell glycine receptors, as the predominant amino acid stimulator of glucagon release. The results suggest that glycine and GHB provide a counterbalancing receptor-based mechanism for controlling α-cell secretory responses to metabolic fuels.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Glucose/metabolismo , Glicina/metabolismo , Células Secretoras de Insulina/metabolismo , Oxibato de Sódio/metabolismo , Adulto , Diabetes Mellitus Tipo 2/patologia , Feminino , Células Secretoras de Glucagon/patologia , Humanos , Células Secretoras de Insulina/patologia , Masculino , Pessoa de Meia-Idade , Receptores de GABA/metabolismo , Receptores de Glicina/metabolismo , Ácido gama-Aminobutírico/metabolismo
4.
J Pediatr ; 165(2): 401-403.e3, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24880889

RESUMO

Identical studies using stable isotopes were performed before and after a 3-day trial of oral N-carbamyl-l-glutamate (NCG) in 5 subjects with late-onset carbamyl phosphate synthetase deficiency. NCG augmented ureagenesis and decreased plasma ammonia in 4 of 5 subjects. There was marked improvement in nitrogen metabolism with long-term NCG administration in 1 subject.


Assuntos
Doença da Deficiência da Carbamoil-Fosfato Sintase I/tratamento farmacológico , Glutamatos/uso terapêutico , Glutamina/sangue , Ureia/metabolismo , Adolescente , Adulto , Amônia/sangue , Doença da Deficiência da Carbamoil-Fosfato Sintase I/sangue , Criança , Pré-Escolar , Feminino , Humanos , Modelos Lineares , Masculino , Espectrometria de Massas , Resultado do Tratamento , Adulto Jovem
5.
Biochem J ; 444(3): 537-51, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22448977

RESUMO

GKAs (glucokinase activators) are promising agents for the therapy of Type 2 diabetes, but little is known about their effects on hepatic intermediary metabolism. We monitored the fate of (13)C-labelled glucose in both a liver perfusion system and isolated hepatocytes. MS and NMR spectroscopy were deployed to measure isotopic enrichment. The results demonstrate that the stimulation of glycolysis by GKA led to numerous changes in hepatic metabolism: (i) augmented flux through the TCA (tricarboxylic acid) cycle, as evidenced by greater incorporation of (13)C into the cycle (anaplerosis) and increased generation of (13)C isotopomers of citrate, glutamate and aspartate (cataplerosis); (ii) lowering of hepatic [Pi] and elevated [ATP], denoting greater phosphorylation potential and energy state; (iii) stimulation of glycogen synthesis from glucose, but inhibition of glycogen synthesis from 3-carbon precursors; (iv) increased synthesis of N-acetylglutamate and consequently augmented ureagenesis; (v) increased synthesis of glutamine, alanine, serine and glycine; and (vi) increased production and outflow of lactate. The present study provides a deeper insight into the hepatic actions of GKAs and uncovers the potential benefits and risks of GKA for treatment of diabetes. GKA improved hepatic bioenergetics, ureagenesis and glycogenesis, but decreased gluconeogenesis with a potential risk of lactic acidosis and fatty liver.


Assuntos
Benzenoacetamidas/farmacologia , Glucoquinase/metabolismo , Hepatócitos/enzimologia , Metabolômica/métodos , Animais , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Ratos , Ratos Sprague-Dawley
6.
J Biol Chem ; 286(25): 22055-68, 2011 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-21540182

RESUMO

We previously reported that isobutylmethylxanthine (IBMX), a derivative of oxypurine, inhibits citrulline synthesis by an as yet unknown mechanism. Here, we demonstrate that IBMX and other oxypurines containing a 2,6-dione group interfere with the binding of glutamate to the active site of N-acetylglutamate synthetase (NAGS), thereby decreasing synthesis of N-acetylglutamate, the obligatory activator of carbamoyl phosphate synthase-1 (CPS1). The result is reduction of citrulline and urea synthesis. Experiments were performed with (15)N-labeled substrates, purified hepatic CPS1, and recombinant mouse NAGS as well as isolated mitochondria. We also used isolated hepatocytes to examine the action of various oxypurines on ureagenesis and to assess the ameliorating affect of N-carbamylglutamate and/or l-arginine on NAGS inhibition. Among various oxypurines tested, only IBMX, xanthine, or uric acid significantly increased the apparent K(m) for glutamate and decreased velocity of NAGS, with little effect on CPS1. The inhibition of NAGS is time- and dose-dependent and leads to decreased formation of the CPS1-N-acetylglutamate complex and consequent inhibition of citrulline and urea synthesis. However, such inhibition was reversed by supplementation with N-carbamylglutamate. The data demonstrate that xanthine and uric acid, both physiologically occurring oxypurines, inhibit the hepatic synthesis of N-acetylglutamate. An important and novel concept emerging from this study is that xanthine and/or uric acid may have a role in the regulation of ureagenesis and, thus, nitrogen homeostasis in normal and disease states.


Assuntos
Aminoácido N-Acetiltransferase/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Fígado/metabolismo , Ureia/metabolismo , Ácido Úrico/farmacologia , Xantina/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , Aminoácido N-Acetiltransferase/isolamento & purificação , Aminoácido N-Acetiltransferase/metabolismo , Animais , Carbamoil-Fosfato Sintase (Amônia)/isolamento & purificação , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Citrulina/biossíntese , Relação Dose-Resposta a Droga , Glutamatos/biossíntese , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Cinética , Fígado/citologia , Fígado/enzimologia , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ratos , Ratos Sprague-Dawley
7.
Proc Natl Acad Sci U S A ; 105(48): 18782-7, 2008 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-19033189

RESUMO

Mammalian cells fuel their growth and proliferation through the catabolism of two main substrates: glucose and glutamine. Most of the remaining metabolites taken up by proliferating cells are not catabolized, but instead are used as building blocks during anabolic macromolecular synthesis. Investigations of phosphoinositol 3-kinase (PI3K) and its downstream effector AKT have confirmed that these oncogenes play a direct role in stimulating glucose uptake and metabolism, rendering the transformed cell addicted to glucose for the maintenance of survival. In contrast, less is known about the regulation of glutamine uptake and metabolism. Here, we report that the transcriptional regulatory properties of the oncogene Myc coordinate the expression of genes necessary for cells to engage in glutamine catabolism that exceeds the cellular requirement for protein and nucleotide biosynthesis. A consequence of this Myc-dependent glutaminolysis is the reprogramming of mitochondrial metabolism to depend on glutamine catabolism to sustain cellular viability and TCA cycle anapleurosis. The ability of Myc-expressing cells to engage in glutaminolysis does not depend on concomitant activation of PI3K or AKT. The stimulation of mitochondrial glutamine metabolism resulted in reduced glucose carbon entering the TCA cycle and a decreased contribution of glucose to the mitochondrial-dependent synthesis of phospholipids. These data suggest that oncogenic levels of Myc induce a transcriptional program that promotes glutaminolysis and triggers cellular addiction to glutamine as a bioenergetic substrate.


Assuntos
Metabolismo Energético/fisiologia , Glutamina/metabolismo , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transcrição Gênica , Animais , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Humanos , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais/fisiologia
8.
Mol Genet Metab ; 100 Suppl 1: S37-41, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20338795

RESUMO

Stable isotopes have been an invaluable adjunct to biomedical research for more than 70years. Indeed, the isotopic approach has revolutionized our understanding of metabolism, revealing it to be an intensely dynamic process characterized by an unending cycle of synthesis and degradation. Isotopic studies have taught us that the urea cycle is intrinsic to such dynamism, since it affords a capacious mechanism by which to eliminate waste nitrogen when rates of protein degradation (or dietary protein intake) are especially high. Isotopes have enabled an appreciation of the degree to which ureagenesis is compromised in patients with urea cycle defects. Indeed, isotopic studies of urea cycle flux correlate well with the severity of cognitive impairment in these patients. Finally, the use of isotopes affords an ideal tool with which to gauge the efficacy of therapeutic interventions to augment residual flux through the cycle.


Assuntos
Marcação por Isótopo/métodos , Ureia/metabolismo , Aminoácido N-Acetiltransferase/deficiência , Aminoácido N-Acetiltransferase/metabolismo , Cloreto de Amônio/administração & dosagem , Cloreto de Amônio/farmacologia , Dióxido de Carbono/metabolismo , Isótopos de Carbono/metabolismo , Humanos , Ureia/sangue
9.
Proc Natl Acad Sci U S A ; 104(49): 19345-50, 2007 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-18032601

RESUMO

Tumor cell proliferation requires rapid synthesis of macromolecules including lipids, proteins, and nucleotides. Many tumor cells exhibit rapid glucose consumption, with most of the glucose-derived carbon being secreted as lactate despite abundant oxygen availability (the Warburg effect). Here, we used 13C NMR spectroscopy to examine the metabolism of glioblastoma cells exhibiting aerobic glycolysis. In these cells, the tricarboxylic acid (TCA) cycle was active but was characterized by an efflux of substrates for use in biosynthetic pathways, particularly fatty acid synthesis. The success of this synthetic activity depends on activation of pathways to generate reductive power (NADPH) and to restore oxaloacetate for continued TCA cycle function (anaplerosis). Surprisingly, both these needs were met by a high rate of glutamine metabolism. First, conversion of glutamine to lactate (glutaminolysis) was rapid enough to produce sufficient NADPH to support fatty acid synthesis. Second, despite substantial mitochondrial pyruvate metabolism, pyruvate carboxylation was suppressed, and anaplerotic oxaloacetate was derived from glutamine. Glutamine catabolism was accompanied by secretion of alanine and ammonia, such that most of the amino groups from glutamine were lost from the cell rather than incorporated into other molecules. These data demonstrate that transformed cells exhibit a high rate of glutamine consumption that cannot be explained by the nitrogen demand imposed by nucleotide synthesis or maintenance of nonessential amino acid pools. Rather, glutamine metabolism provides a carbon source that facilitates the cell's ability to use glucose-derived carbon and TCA cycle intermediates as biosynthetic precursors.


Assuntos
Neoplasias Encefálicas/metabolismo , Transformação Celular Neoplásica/metabolismo , Glioblastoma/metabolismo , Glutamina/metabolismo , Glicólise , Aerobiose , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/patologia , Ciclo do Ácido Cítrico , Glioblastoma/patologia , Glucose/metabolismo , Humanos , Nucleotídeos/biossíntese , Biossíntese de Proteínas
10.
Mol Genet Metab ; 98(4): 325-30, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19660971

RESUMO

We studied the effect on ureagenesis of a single dose of N-carbamylglutamate (NCG) in healthy young adults who received a constant infusion (300 min) of NaH(13)CO(3). Isotope ratio-mass spectrometry was used to measure the appearance of label in [(13)C]urea. At 90 min after initiating the H(13)CO3-infusion each subject took a single dose of NCG (50 mg/kg). In 5/6 studies the administration of NCG increased the formation of [(13)C]urea. Treatment with NCG significantly diminished the concentration of blood alanine, but not that of glutamine or arginine. The blood glucose concentration was unaffected by NCG administration. No untoward side effects were observed. The data indicate that treatment with NCG stimulates ureagenesis and could be useful in clinical settings of acute hyperammonemia of various etiologies.


Assuntos
Glutamatos/administração & dosagem , Glutamatos/farmacologia , Ureia/metabolismo , Adulto , Aminoácidos/sangue , Glicemia/efeitos dos fármacos , Dióxido de Carbono/metabolismo , Isótopos de Carbono , Demografia , Relação Dose-Resposta a Droga , Expiração/efeitos dos fármacos , Feminino , Saúde , Humanos , Infusões Intravenosas , Masculino , Bicarbonato de Sódio/administração & dosagem , Bicarbonato de Sódio/farmacologia , Ureia/sangue , Adulto Jovem
11.
Epilepsia ; 49 Suppl 8: 73-5, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19049594

RESUMO

We hypothesize that one mechanism of the anti-epileptic effect of the ketogenic diet is to alter brain handling of glutamate. According to this formulation, in ketotic brain astrocyte metabolism is more active, resulting in enhanced conversion of glutamate to glutamine. This allows for: (a) more efficient removal of glutamate, the most important excitatory neurotransmitter; and (b) more efficient conversion of glutamine to GABA, the major inhibitory neurotransmitter.


Assuntos
Encéfalo/metabolismo , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Cetose/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Dieta Cetogênica , Humanos
12.
Cancer Res ; 66(15): 7824-31, 2006 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-16885387

RESUMO

The efficacy of ifosfamide (IFO), an antineoplastic drug, is severely limited by a high incidence of nephrotoxicity of unknown etiology. We hypothesized that inhibition of complex I (C-I) by chloroacetaldehyde (CAA), a metabolite of IFO, is the chief cause of nephrotoxicity, and that agmatine (AGM), which we found to augment mitochondrial oxidative phosphorylation and beta-oxidation, would prevent nephrotoxicity. Our model system was isolated mitochondria obtained from the kidney cortex of rats treated with IFO or IFO + AGM. Oxidative phosphorylation was determined with electron donors specific to complexes I, II, III, or IV (C-I, C-II, C-III, or C-IV, respectively). A parallel study was done with (13)C-labeled pyruvate to assess metabolic dysfunction. Ifosfamide treatment significantly inhibited oxidative phosphorylation with only C-I substrates. Inhibition of C-I was associated with a significant elevation of [NADH], depletion of [NAD], and decreased flux through pyruvate dehydrogenase and the TCA cycle. However, administration of AGM with IFO increased [cyclic AMP (cAMP)] and prevented IFO-induced inhibition of C-I. In vitro studies with various metabolites of IFO showed that only CAA inhibited C-I, even with supplementation with 2-mercaptoethane sulfonic acid. Following IFO treatment daily for 5 days with 50 mg/kg, the level of CAA in the renal cortex was approximately 15 micromol/L. Taken together, these observations support the hypothesis that CAA is accumulated in renal cortex and is responsible for nephrotoxicity. AGM may be protective by increasing tissue [cAMP], which phosphorylates NADH:oxidoreductase. The current findings may have an important implication for the prevention of IFO-induced nephrotoxicity and/or mitochondrial diseases secondary to defective C-I.


Assuntos
Antineoplásicos Alquilantes/toxicidade , Ifosfamida/toxicidade , Nefropatias/induzido quimicamente , Nefropatias/prevenção & controle , Acetaldeído/análogos & derivados , Acetaldeído/farmacocinética , Agmatina/farmacologia , Animais , Interações Medicamentosas , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/metabolismo , Ifosfamida/farmacocinética , Córtex Renal/enzimologia , Córtex Renal/metabolismo , Nefropatias/enzimologia , Masculino , Fosforilação Oxidativa/efeitos dos fármacos , Ratos
13.
Sci Transl Med ; 9(416)2017 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-29141885

RESUMO

Gut dysbiosis during inflammatory bowel disease involves alterations in the gut microbiota associated with inflammation of the host gut. We used a combination of shotgun metagenomic sequencing and metabolomics to analyze fecal samples from pediatric patients with Crohn's disease and found an association between disease severity, gut dysbiosis, and bacterial production of free amino acids. Nitrogen flux studies using 15N in mice showed that activity of bacterial urease, an enzyme that releases ammonia by hydrolysis of host urea, led to the transfer of murine host-derived nitrogen to the gut microbiota where it was used for amino acid synthesis. Inoculation of a conventional murine host (pretreated with antibiotics and polyethylene glycol) with commensal Escherichia coli engineered to express urease led to dysbiosis of the gut microbiota, resulting in a predominance of Proteobacteria species. This was associated with a worsening of immune-mediated colitis in these animals. A potential role for altered urease expression and nitrogen flux in the development of gut dysbiosis suggests that bacterial urease may be a potential therapeutic target for inflammatory bowel diseases.


Assuntos
Proteínas de Bactérias/metabolismo , Doença de Crohn/metabolismo , Doença de Crohn/microbiologia , Disbiose/metabolismo , Disbiose/microbiologia , Microbioma Gastrointestinal/fisiologia , Urease/metabolismo , Animais , Humanos , Camundongos
14.
Neurochem Int ; 48(6-7): 650-6, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16510212

RESUMO

The ketogenic diet is an effective treatment for seizures, but the mechanism of action is unknown. It is uncertain whether the anti-epileptic effect presupposes ketosis, or whether the restriction of calories and/or carbohydrate might be sufficient. We found that a relatively brief (24 h) period of low glucose and low calorie intake significantly attenuated the severity of seizures in young Sprague-Dawley rats (50-70 gms) in whom convulsions were induced by administration of pentylenetetrazole (PTZ). The blood glucose concentration was lower in animals that received less dietary glucose, but the brain glucose level did not differ from control blood [3-OH-butyrate] tended to be higher in blood, but not in brain, of animals on a low-glucose intake. The concentration in brain of glutamine increased and that of alanine declined significantly with low-glucose intake. The blood alanine level fell more than that of brain alanine, resulting in a marked increase ( approximately 50%) in the brain:blood ratio for alanine. In contrast, the brain:blood ratio for leucine declined by about 35% in the low-glucose group. When animals received [1-(13)C]glucose, a metabolic precursor of alanine, the appearance of (13)C in alanine and glutamine increased significantly relative to control. The brain:blood ratio for [(13)C]alanine exceeded 1, indicating that the alanine must have been formed in brain and not transported from blood. The elevated brain(alanine):blood(alanine) could mean that a component of the anti-epileptic effect of low carbohydrate intake is release of alanine from brain-to-blood, in the process abetting the disposal of glutamate, excess levels of which in the synaptic cleft would contribute to the development of seizures.


Assuntos
Aminoácidos/metabolismo , Encéfalo/metabolismo , Jejum , Convulsões/metabolismo , Convulsões/prevenção & controle , Alanina/biossíntese , Alanina/sangue , Animais , Isótopos de Carbono , Convulsivantes , Dieta com Restrição de Carboidratos , Ingestão de Energia , Glucose/administração & dosagem , Pentilenotetrazol , Ratos , Ratos Sprague-Dawley , Convulsões/induzido quimicamente , Fatores de Tempo
15.
Biochem J ; 388(Pt 2): 419-25, 2005 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15656789

RESUMO

An important but unresolved question is whether mammalian mitochondria metabolize arginine to agmatine by the ADC (arginine decarboxylase) reaction. 15N-labelled arginine was used as a precursor to address this question and to determine the flux through the ADC reaction in isolated mitochondria obtained from rat liver. In addition, liver perfusion system was used to examine a possible action of insulin, glucagon or cAMP on a flux through the ADC reaction. In mitochondria and liver perfusion, 15N-labelled agmatine was generated from external 15N-labelled arginine. The production of 15N-labelled agmatine was time- and dose-dependent. The time-course of [U-15N4]agmatine formation from 2 mM [U-15N4]arginine was best fitted to a one-phase exponential curve with a production rate of approx. 29 pmol x min(-1) x (mg of protein)(-1). Experiments with an increasing concentration (0- 40 mM) of [guanidino-15N2]arginine showed a Michaelis constant Km for arginine of 46 mM and a Vmax of 3.7 nmol x min(-1) x (mg of protein)(-1) for flux through the ADC reaction. Experiments with broken mitochondria showed little changes in Vmax or Km values, suggesting that mitochondrial arginine uptake had little effect on the observed Vmax or Km values. Experiments with liver perfusion demonstrated that over 95% of the effluent agmatine was derived from perfusate [guanidino-15N2]arginine regardless of the experimental condition. However, the output of 15N-labelled agmatine (nmol x min(-1) x g(-1)) increased by approx. 2-fold (P<0.05) in perfusions with cAMP. The findings of the present study provide compelling evidence that mitochondrial ADC is present in the rat liver, and suggest that cAMP may stimulate flux through this pathway.


Assuntos
Agmatina/metabolismo , Carboxiliases/metabolismo , Fígado/enzimologia , Mitocôndrias Hepáticas/enzimologia , Animais , AMP Cíclico/fisiologia , Glucagon/fisiologia , Técnicas In Vitro , Insulina/fisiologia , Cinética , Masculino , Isótopos de Nitrogênio , Consumo de Oxigênio , Ratos
16.
Neurochem Int ; 47(1-2): 119-28, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15888376

RESUMO

Our objective was to study brain amino acid metabolism in response to ketosis. The underlying hypothesis is that ketosis is associated with a fundamental change of brain amino acid handling and that this alteration is a factor in the anti-epileptic effect of the ketogenic diet. Specifically, we hypothesize that brain converts ketone bodies to acetyl-CoA and that this results in increased flux through the citrate synthetase reaction. As a result, oxaloacetate is consumed and is less available to the aspartate aminotransferase reaction; therefore, less glutamate is converted to aspartate and relatively more glutamate becomes available to the glutamine synthetase and glutamate decarboxylase reactions. We found in a mouse model of ketosis that the concentration of forebrain aspartate was diminished but the concentration of acetyl-CoA was increased. Studies of the incorporation of 13C into glutamate and glutamine with either [1-(13)C]glucose or [2-(13)C]acetate as precursor showed that ketotic brain metabolized relatively less glucose and relatively more acetate. When the ketotic mice were administered both acetate and a nitrogen donor, such as alanine or leucine, they manifested an increased forebrain concentration of glutamine and GABA. These findings supported the hypothesis that in ketosis there is greater production of acetyl-CoA and a consequent alteration in the equilibrium of the aspartate aminotransferase reaction that results in diminished aspartate production and potentially enhanced synthesis of glutamine and GABA.


Assuntos
Acetilcoenzima A/metabolismo , Aminoácidos/biossíntese , Química Encefálica/fisiologia , Encéfalo/metabolismo , Metabolismo Energético/fisiologia , Cetose/metabolismo , Acetatos/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Ácido Aspártico/biossíntese , Radioisótopos de Carbono/farmacocinética , Glucose/metabolismo , Ácido Glutâmico/biossíntese , Glutamina/biossíntese , Masculino , Camundongos , Modelos Animais , Ácido Oxaloacético/metabolismo , Ácido gama-Aminobutírico/biossíntese
17.
Biochem J ; 376(Pt 1): 179-88, 2003 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-12935293

RESUMO

The present study was designed to determine: (i) the role of the reductive amination of alpha-ketoglutarate via the glutamate dehydrogenase reaction in furnishing mitochondrial glutamate and its transamination into aspartate; (ii) the relative incorporation of perfusate 15NH4Cl, [2-15N]glutamine or [5-15N]glutamine into carbamoyl phosphate and aspartate-N and, thereby, [15N]urea isotopomers; and (iii) the extent to which perfusate [15N]aspartate is taken up by the liver and incorporated into [15N]urea. We used a liver-perfusion system containing a physiological mixture of amino acids and ammonia similar to concentrations in vivo, with 15N label only in glutamine, ammonia or aspartate. The results demonstrate that in perfusions with a physiological mixture of amino acids, approx. 45 and 30% of total urea-N output was derived from perfusate ammonia and glutamine-N respectively. Approximately two-thirds of the ammonia utilized for carbamoyl phosphate synthesis was derived from perfusate ammonia and one-third from glutamine. Perfusate [2-15N]glutamine, [5-15N]glutamine or [15N]aspartate provided 24, 10 and 10% respectively of the hepatic aspartate-N pool, whereas perfusate 15NH4Cl provided approx. 37% of aspartate-N utilized for urea synthesis, secondary to the net formation of [15N]glutamate via the glutamate dehydrogenase reaction. The results suggest that the mitochondrial glutamate formed via the reductive amination of alpha-ketoglutarate may have a key role in ammonia detoxification by the following processes: (i) furnishing aspartate-N for ureagenesis; (ii) serving as a scavenger for excess ammonia; and (iii) improving the availability of the mitochondrial [glutamate] for synthesis of N -acetylglutamate. In addition, the current findings suggest that the formation of aspartate via the mitochondrial aspartate aminotransferase reaction may play an important role in the synthesis of cytosolic argininosuccinate.


Assuntos
Ácido Aspártico/metabolismo , Glutamato Desidrogenase/metabolismo , Fígado/enzimologia , Nitrogênio/metabolismo , Ureia/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Amônia/química , Amônia/metabolismo , Animais , Ácido Aspártico/química , Constrição , Congelamento , Glutamato Desidrogenase/fisiologia , Glutamatos/metabolismo , Glutamina/química , Glutamina/metabolismo , Fígado/metabolismo , Masculino , Isótopos de Nitrogênio , Perfusão , Ratos , Ratos Sprague-Dawley
18.
Sci Signal ; 8(384): ra68, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26152695

RESUMO

Nitric oxide (NO) is a signaling intermediate during glutamatergic neurotransmission in the central nervous system (CNS). NO signaling is in part accomplished through cysteine S-nitrosylation, a posttranslational modification by which NO regulates protein function and signaling. In our investigation of the protein targets and functional impact of S-nitrosylation in the CNS under physiological conditions, we identified 269 S-nitrosocysteine residues in 136 proteins in the wild-type mouse brain. The number of sites was significantly reduced in the brains of mice lacking endothelial nitric oxide synthase (eNOS(-/-)) or neuronal nitric oxide synthase (nNOS(-/-)). In particular, nNOS(-/-) animals showed decreased S-nitrosylation of proteins that participate in the glutamate/glutamine cycle, a metabolic process by which synaptic glutamate is recycled or oxidized to provide energy. (15)N-glutamine-based metabolomic profiling and enzymatic activity assays indicated that brain extracts from nNOS(-/-) mice converted less glutamate to glutamine and oxidized more glutamate than those from mice of the other genotypes. GLT1 [also known as EAAT2 (excitatory amino acid transporter 2)], a glutamate transporter in astrocytes, was S-nitrosylated at Cys(373) and Cys(561) in wild-type and eNOS(-/-) mice, but not in nNOS(-/-) mice. A form of rat GLT1 that could not be S-nitrosylated at the equivalent sites had increased glutamate uptake compared to wild-type GLT1 in cells exposed to an S-nitrosylating agent. Thus, NO modulates glutamatergic neurotransmission through the selective, nNOS-dependent S-nitrosylation of proteins that govern glutamate transport and metabolism.


Assuntos
Encéfalo/metabolismo , Cisteína/metabolismo , Ácido Glutâmico/metabolismo , Óxido Nítrico/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Cromatografia Líquida , Cisteína/análogos & derivados , Cisteína/genética , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Glutamina/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Mutação , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Ratos , S-Nitrosotióis/metabolismo , Espectrometria de Massas em Tandem
19.
J Clin Invest ; 125(7): 2841-50, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-26098218

RESUMO

Increasing evidence indicates that the gut microbiota can be altered to ameliorate or prevent disease states, and engineering the gut microbiota to therapeutically modulate host metabolism is an emerging goal of microbiome research. In the intestine, bacterial urease converts host-derived urea to ammonia and carbon dioxide, contributing to hyperammonemia-associated neurotoxicity and encephalopathy in patients with liver disease. Here, we engineered murine gut microbiota to reduce urease activity. Animals were depleted of their preexisting gut microbiota and then inoculated with altered Schaedler flora (ASF), a defined consortium of 8 bacteria with minimal urease gene content. This protocol resulted in establishment of a persistent new community that promoted a long-term reduction in fecal urease activity and ammonia production. Moreover, in a murine model of hepatic injury, ASF transplantation was associated with decreased morbidity and mortality. These results provide proof of concept that inoculation of a prepared host with a defined gut microbiota can lead to durable metabolic changes with therapeutic utility.


Assuntos
Terapia Biológica/métodos , Sistema Digestório/microbiologia , Hiperamonemia/microbiologia , Hiperamonemia/terapia , Microbiota , Amônia/metabolismo , Animais , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bioengenharia , Doença Hepática Induzida por Substâncias e Drogas/terapia , Sistema Digestório/metabolismo , Modelos Animais de Doenças , Fezes/microbiologia , Feminino , Genes Bacterianos , Hiperamonemia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Microbiota/fisiologia , Fatores de Tempo , Urease/genética , Urease/metabolismo
20.
Artigo em Inglês | MEDLINE | ID: mdl-14769486

RESUMO

We do not know the mode of action of the ketogenic diet in controlling epilepsy. One possibility is that the diet alters brain handling of glutamate, the major excitatory neurotransmitter and a probable factor in evoking and perpetuating a convulsion. We have found that brain metabolism of ketone bodies can furnish as much as 30% of glutamate and glutamine carbon. Ketone body metabolism also provides acetyl-CoA to the citrate synthetase reaction, in the process consuming oxaloacetate and thereby diminishing the transamination of glutamate to aspartate, a pathway in which oxaloacetate is a reactant. Relatively more glutamate then is available to the glutamate decarboxylase reaction, which increases brain [GABA]. Ketosis also increases brain [GABA] by increasing brain metabolism of acetate, which glia convert to glutamine. GABA-ergic neurons readily take up the latter amino acid and use it as a precursor to GABA. Ketosis also may be associated with altered amino acid transport at the blood-brain barrier. Specifically, ketosis may favor the release from brain of glutamine, which transporters at the blood-brain barrier exchange for blood leucine. Since brain glutamine is formed in astrocytes from glutamate, the overall effect will be to favor the release of glutamate from the nervous system.


Assuntos
Encéfalo/metabolismo , Dieta , Ácido Glutâmico/metabolismo , Corpos Cetônicos/metabolismo , Convulsões/metabolismo , Convulsões/prevenção & controle , Aminoácidos/metabolismo , Animais , Humanos , Cetose/metabolismo
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