RESUMO
BACKGROUND: The mitochondrial branched-chain aminotransferase (BCATm) is a recently discovered cancer marker with a poorly defined role in tumour progression. METHODS: To understand how a loss of function of BCATm affects cancer, the global knockout mouse BCATmKO was challenged with EL-4 lymphoma under different diet compositions with varying amounts of branched-chain amino acids (BCAAs). Next, the growth and metabolism of EL-4 cells were studied in the presence of different leucine concentrations in the growth medium. RESULTS: BCATmKO mice experienced delayed tumour growth when fed standard rodent chow or a normal BCAA diet. Tumour suppression correlated with 37.6- and 18.9-fold increases in plasma and tumour BCAAs, 37.5% and 30.4% decreases in tumour glutamine and alanine, and a 3.5-fold increase in the phosphorylation of tumour AMPK in BCATmKO mice on standard rodent chow. Similar results were obtained with a normal but not with a choice BCAA diet. CONCLUSIONS: Global deletion of BCATm caused a dramatic build-up of BCAAs, which could not be utilised for energy or amino acid synthesis, ultimately delaying the growth of lymphoma tumours. Furthermore, physiological, but not high, leucine concentrations promoted the growth of EL-4 cells. BCATm and BCAA metabolism were identified as attractive targets for anti-lymphoma therapy.
Assuntos
Linfoma/patologia , Mitocôndrias/metabolismo , Transaminases/genética , Transaminases/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Aminoácidos de Cadeia Ramificada/sangue , Animais , Progressão da Doença , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Transplante de Neoplasias , FosforilaçãoRESUMO
This review aims to provide a historical reference of branched-chain amino acid (BCAA) metabolism and provide a link between peripheral and central nervous system (CNS) metabolism of BCAAs. Leucine, isoleucine, and valine (Leu, Ile, and Val) are unlike most other essential amino acids (AA), being transaminated initially in extrahepatic tissues, and requiring interorgan or intertissue shuttling for complete catabolism. Within the periphery, BCAAs are essential AAs and are required for protein synthesis, and are key nitrogen donors in the form of Glu, Gln, and Ala. Leucine is an activator of the mammalian (or mechanistic) target of rapamycin, the master regulator of cell growth and proliferation. The tissue distribution and activity of the catabolic enzymes in the peripheral tissues as well as neurological effects in Maple Syrup Urine Disease (MSUD) show the BCAAs have a role in the CNS. Interestingly, there are significant differences between murine and human CNS enzyme distribution and activities. In the CNS, BCAAs have roles in neurotransmitter synthesis, protein synthesis, food intake regulation, and are implicated in diseases. MSUD is the most prolific disease associated with BCAA metabolism, affecting the branched-chain α-keto acid dehydrogenase complex (BCKDC). Mutations in the branched-chain aminotransferases (BCATs) and the kinase for BCKDC also result in neurological dysfunction. However, there are many questions of BCAA metabolism in the CNS (as well as the periphery) that remain elusive. We discuss areas of BCAA and BCKA metabolism that have yet to be researched adequately.
Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Encéfalo/metabolismo , Transdução de Sinais/fisiologia , Aminoácidos de Cadeia Ramificada/genética , Animais , Humanos , Leucina/genética , Leucina/metabolismo , Doença da Urina de Xarope de Bordo/genética , Doença da Urina de Xarope de Bordo/metabolismo , Transaminases/genética , Transaminases/metabolismo , Valina/genética , Valina/metabolismoRESUMO
Cytosolic and mitochondrial human branched chain aminotransferase (hBCATc and hBCATm, respectively) play an integral role in brain glutamate metabolism. Regional increased levels of hBCATc in the CA1 and CA4 region of Alzheimer's disease (AD) brain together with increased levels of hBCATm in frontal and temporal cortex of AD brains, suggest a role for these proteins in glutamate excitotoxicity. Glutamate toxicity is a key pathogenic feature of several neurological disorders including epilepsy associated dementia, AD, vascular dementia (VaD) and dementia with Lewy bodies (DLB). To further understand if these increases are specific to AD, the expression profiles of hBCATc and hBCATm were examined in other forms of dementia including DLB and VaD. Similar to AD, levels of hBCATm were significantly increased in the frontal and temporal cortex of VaD cases and in frontal cortex of DLB cases compared to controls, however there were no observed differences in hBCATc between groups in these areas. Moreover, multiple forms of hBCATm were observed that were particular to the disease state relative to matched controls. Real-time PCR revealed similar expression of hBCATm mRNA in frontal and temporal cortex for all cohort comparisons, whereas hBCATc mRNA expression was significantly increased in VaD cases compared to controls. Collectively our results suggest that hBCATm protein expression is significantly increased in the brains of DLB and VaD cases, similar to those reported in AD brain. These findings indicate a more global response to altered glutamate metabolism and suggest common metabolic responses that might reflect shared neurodegenerative mechanisms across several forms of dementia.
Assuntos
Demência Vascular/enzimologia , Regulação Enzimológica da Expressão Gênica , Doença por Corpos de Lewy/enzimologia , Transaminases/biossíntese , Idoso , Idoso de 80 Anos ou mais , Encéfalo/enzimologia , Encéfalo/patologia , Estudos de Coortes , Demência Vascular/genética , Demência Vascular/patologia , Feminino , Humanos , Doença por Corpos de Lewy/genética , Doença por Corpos de Lewy/patologia , Masculino , Antígenos de Histocompatibilidade Menor/biossíntese , Proteínas da Gravidez/biossíntese , Transaminases/genéticaRESUMO
Unlike other amino acids, the branched-chain amino acids (BCAAs) largely bypass first-pass liver degradation due to a lack of hepatocyte expression of the mitochondrial branched-chain aminotransferase (BCATm). This sets up interorgan shuttling of BCAAs and liver-skeletal muscle cooperation in BCAA catabolism. To explore whether complete liver catabolism of BCAAs may impact BCAA shuttling in peripheral tissues, the BCATm gene was stably introduced into mouse liver. Two transgenic mouse lines with low and high hepatocyte expression of the BCATm transgene (LivTg-LE and LivTg-HE) were created and used to measure liver and plasma amino acid concentrations and determine whether the first two BCAA enzymatic steps in liver, skeletal muscle, heart and kidney were impacted. Expression of the hepatic BCATm transgene lowered the concentrations of hepatic BCAAs while enhancing the concentrations of some nonessential amino acids. Extrahepatic BCAA metabolic enzymes and plasma amino acids were largely unaffected, and no growth rate or body composition differences were observed in the transgenic animals as compared to wild-type mice. Feeding the transgenic animals a high-fat diet did not reverse the effect of the BCATm transgene on the hepatic BCAA catabolism, nor did the high-fat diet cause elevation in plasma BCAAs. However, the high-fat-diet-fed BCATm transgenic animals experienced attenuation in the mammalian target of rapamycin (mTOR) pathway in the liver and had impaired blood glucose tolerance. These results suggest that complete liver BCAA metabolism influences the regulation of glucose utilization during diet-induced obesity.
Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Dieta Hiperlipídica , Fígado/metabolismo , Transaminases/genética , Aminoácidos de Cadeia Ramificada/sangue , Animais , Glicemia/metabolismo , Composição Corporal/genética , Feminino , Regulação da Expressão Gênica , Homeostase , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transaminases/metabolismoRESUMO
In connection with the increasing interest in metabolic regulation of the immune response, this review discusses current advances in understanding the role of leucine and leucine metabolism in T lymphocyte (T cell) activation. T cell activation during the development of an immune response depends on metabolic reprogramming to ensure that sufficient nutrients and energy are taken up by the highly proliferating T cells. Leucine has been described as an important essential amino acid and a nutrient signal that activates complex 1 of the mammalian target of rapamycin (mTORC1), which is a critical regulator of T cell proliferation, differentiation, and function. The role of leucine in these processes is further discussed in relation to amino acid transporters, leucine-degrading enzymes, and other metabolites of leucine metabolism. A new model of T cell regulation by leucine is proposed and outlines a chain of events that leads to the activation of mTORC1 in T cells.
Assuntos
Imunidade/fisiologia , Leucina/metabolismo , Ativação Linfocitária/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Transdução de Sinais/fisiologia , Linfócitos T/imunologia , Animais , Humanos , Fatores Imunológicos , Leucina/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Transaminases/metabolismoAssuntos
Neoplasias Encefálicas , Regulação Neoplásica da Expressão Gênica/genética , Glioma , Transaminases/metabolismo , Adulto , Idoso , Análise de Variância , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Estudos de Coortes , Feminino , Glioma/genética , Glioma/metabolismo , Glioma/mortalidade , Humanos , Isocitrato Desidrogenase , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Mutação/genética , Transaminases/genéticaRESUMO
The human cytosolic branched-chain aminotransferase (hBCATc) enzyme is strategically located in glutamatergic neurons, where it is thought to provide approximately 30% of de novo nitrogen for brain glutamate synthesis. In health, glutamate plays a dominant role in facilitating learning and memory. However, in patients with Alzheimer's disease (AD), synaptic levels of glutamate become toxic, resulting in a direct increase in postsynaptic neuronal calcium, causing a cascade of events that contributes to the destruction of neuronal integrity and cell death, pathological features of AD. Our group is the first to map the hBCAT proteins to the human brain, where cell-specific compartmentation indicates key roles for these proteins in regulating glutamate homeostasis. Moreover, increased expression of hBCAT was observed in the brains of patients with AD relative to matched controls. We reflect on the importance of the redox-active CXXC motif, which confers novel roles for the hBCAT proteins, particularly with respect to substrate channeling and protein folding. This implies that, in addition to their role in glutamate metabolism, these proteins have additional functional roles that might impact redox cell signaling. This review discusses how these proteins behave as potential neuroprotectors during periods of oxidative stress. These findings are particularly important because an increase in misfolded proteins, linked to increased oxidative stress, occurs in several neurodegenerative conditions. Together, these studies give an overview of the diverse role that these proteins play in brain metabolism, in which a dysregulation of their expression may contribute to neurodegenerative conditions such as AD.
Assuntos
Encéfalo/enzimologia , Transaminases/metabolismo , HumanosRESUMO
Enhanced fibroblast growth factor 21 (FGF21) production and circulation has been linked to the metabolic adaptation to starvation. Here, we demonstrated that hepatic FGF21 expression is induced by dietary protein restriction, but not energy restriction. Circulating FGF21 was increased 10-fold in mice and rats fed a low-protein (LP) diet. In these animals, liver Fgf21 expression was increased within 24 hours of reduced protein intake. In humans, circulating FGF21 levels increased dramatically following 28 days on a LP diet. LP-induced increases in FGF21 were associated with increased phosphorylation of eukaryotic initiation factor 2α (eIF2α) in the liver, and both baseline and LP-induced serum FGF21 levels were reduced in mice lacking the eIF2α kinase general control nonderepressible 2 (GCN2). Finally, while protein restriction altered food intake, energy expenditure, and body weight gain in WT mice, FGF21-deficient animals did not exhibit these changes in response to a LP diet. These and other data demonstrate that reduced protein intake underlies the increase in circulating FGF21 in response to starvation and a ketogenic diet and that FGF21 is required for behavioral and metabolic responses to protein restriction. FGF21 therefore represents an endocrine signal of protein restriction, which acts to coordinate metabolism and growth during periods of reduced protein intake.
Assuntos
Proteínas Alimentares/administração & dosagem , Fatores de Crescimento de Fibroblastos/fisiologia , Animais , Restrição Calórica , Dieta Cetogênica , Metabolismo Energético , Fatores de Crescimento de Fibroblastos/sangue , Fatores de Crescimento de Fibroblastos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , PPAR alfa/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
The mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC) is negatively regulated by reversible phosphorylation.BCKDC kinase (BDK) inhibitors that augment BCKDC flux have been shown to reduce branched-chain amino acid (BCAA) concentrations in vivo. In the present study, we employed high-throughput screens to identify compound 3,6- dichlorobenzo[b]thiophene-2-carboxylic acid (BT2) as a novel BDK inhibitor (IC(50) = 3.19 µM). BT2 binds to the same site in BDK as other known allosteric BDK inhibitors, including (S)-α-cholorophenylproprionate ((S)-CPP). BT2 binding to BDK triggers helix movements in the N-terminal domain, resulting in the dissociation of BDK from the BCKDC accompanied by accelerated degradation of the released kinase in vivo. BT2 shows excellent pharmacokinetics (terminal T(1/2) = 730 min) and metabolic stability (no degradation in 240 min), which are significantly better than those of (S)-CPP. BT2, its analog 3-chloro-6-fluorobenzo[ b]thiophene-2-carboxylic acid (BT2F), and a prodrug of BT2 (i.e. N-(4-acetamido-1,2,5-oxadiazol-3-yl)-3,6-dichlorobenzo[ b]thiophene-2-carboxamide (BT3)) significantly increase residual BCKDC activity in cultured cells and primary hepatocytes from patients and a mouse model of maple syrup urine disease. Administration of BT2 at 20 mg/kg/day to wild-type mice for 1 week leads to nearly complete dephosphorylation and maximal activation of BCKDC in heart, muscle, kidneys, and liver with reduction in plasma BCAA concentrations. The availability of benzothiophene carboxylate derivatives as stable BDK inhibitors may prove useful for the treatment of metabolic disease caused by elevated BCAA concentrations.
Assuntos
Inibidores Enzimáticos/farmacologia , Hepatócitos/enzimologia , Complexo Cetoglutarato Desidrogenase/antagonistas & inibidores , Complexo Cetoglutarato Desidrogenase/metabolismo , Proteólise/efeitos dos fármacos , Tiofenos/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Estabilidade Enzimática/efeitos dos fármacos , Estabilidade Enzimática/genética , Hepatócitos/patologia , Humanos , Complexo Cetoglutarato Desidrogenase/genética , Camundongos , Camundongos Knockout , Tiofenos/farmacocinéticaRESUMO
Intracerebroventricular injections of leucine are sufficient to suppress food intake, but it remains unclear whether brain leucine signaling represents a physiological signal of protein balance. We tested whether variations in dietary and circulating levels of leucine, or all three branched-chain amino acids (BCAAs), contribute to the detection of reduced dietary protein. Of the essential amino acids (EAAs) tested, only intracerebroventricular injection of leucine (10 µg) was sufficient to suppress food intake. Isocaloric low- (9% protein energy; LP) or normal- (18% protein energy) protein diets induced a divergence in food intake, with an increased consumption of LP beginning on day 2 and persisting throughout the study (P < 0.05). Circulating BCAA levels were reduced the day after LP diet exposure, but levels subsequently increased and normalized by day 4, despite persistent hyperphagia. Brain BCAA levels as measured by microdialysis on day 2 of diet exposure were reduced in LP rats, but this effect was most prominent postprandially. Despite these diet-induced changes in BCAA levels, reducing dietary leucine or total BCAAs independently from total protein was neither necessary nor sufficient to induce hyperphagia, while chronic infusion of EAAs into the brain of LP rats failed to consistently block LP-induced hyperphagia. Collectively, these data suggest that circulating BCAAs are transiently reduced by dietary protein restriction, but variations in dietary or brain BCAAs alone do not explain the hyperphagia induced by a low-protein diet.
Assuntos
Encéfalo/efeitos dos fármacos , Dieta com Restrição de Proteínas , Proteínas Alimentares/farmacologia , Ingestão de Alimentos/efeitos dos fármacos , Leucina/farmacologia , Aminoácidos/metabolismo , Aminoácidos de Cadeia Ramificada/farmacologia , Animais , Encéfalo/metabolismo , Dieta com Restrição de Proteínas/efeitos adversos , Ingestão de Alimentos/fisiologia , Hiperfagia/etiologia , Injeções Intraventriculares , Leucina/administração & dosagem , Masculino , Modelos Animais , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Here we show that expression of the cytosolic branched chain aminotransferase (BCATc) is triggered by the T cell receptor (TCR) of CD4(+) T cells. Induction of BCATc correlates with increased Leu transamination, whereas T cells from the BCATc(-/-) mouse exhibit lower Leu transamination and higher intracellular Leu concentrations than the cells from wild type (WT) mice. Induction of BCATc by TCR in WT cells is prevented by the calcineurin-nuclear factor of activated T cells (NFAT) inhibitor, cyclosporin A (CsA), suggesting that NFAT controls BCATc expression. Leu is a known activator of the mammalian target of rapamycin complex 1 (mTORC1). mTOR is emerging as a critical regulator of T cell activation, differentiation, and metabolism. Activated T cells from BCATc(-/-) mice show increased phosphorylation of mTORC1 downstream targets, S6 and 4EBP-1, indicating higher mTORC1 activation than in T cells from WT mice. Furthermore, T cells from BCATc(-/-) mice display higher rates of glycolysis, glycolytic capacity, and glycolytic reserve when compared with activated WT cells. These findings reveal BCATc as a novel regulator of T cell activation and metabolism and highlight the important role of Leu metabolism in T cells.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Citosol/enzimologia , Glicólise , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Transaminases/metabolismo , Sequência de Aminoácidos , Animais , Linfócitos T CD4-Positivos/imunologia , Deleção de Genes , Regulação da Expressão Gênica , Leucina/metabolismo , Ativação Linfocitária , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T/metabolismo , Transaminases/deficiência , Transaminases/genéticaRESUMO
Here we show that glioblastoma express high levels of branched-chain amino acid transaminase 1 (BCAT1), the enzyme that initiates the catabolism of branched-chain amino acids (BCAAs). Expression of BCAT1 was exclusive to tumors carrying wild-type isocitrate dehydrogenase 1 (IDH1) and IDH2 genes and was highly correlated with methylation patterns in the BCAT1 promoter region. BCAT1 expression was dependent on the concentration of α-ketoglutarate substrate in glioma cell lines and could be suppressed by ectopic overexpression of mutant IDH1 in immortalized human astrocytes, providing a link between IDH1 function and BCAT1 expression. Suppression of BCAT1 in glioma cell lines blocked the excretion of glutamate and led to reduced proliferation and invasiveness in vitro, as well as significant decreases in tumor growth in a glioblastoma xenograft model. These findings suggest a central role for BCAT1 in glioma pathogenesis, making BCAT1 and BCAA metabolism attractive targets for the development of targeted therapeutic approaches to treat patients with glioblastoma.
Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Neoplasias Encefálicas/metabolismo , Proliferação de Células , Glioma/metabolismo , Transaminases/fisiologia , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Feminino , Glioma/genética , Glioma/patologia , Células HEK293 , Humanos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/fisiologia , Metabolismo/genética , Camundongos , Camundongos Nus , Modelos Biológicos , Transaminases/genética , Transaminases/metabolismoRESUMO
The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are elevated in maple syrup urine disease, heart failure, obesity, and type 2 diabetes. BCAA homeostasis is controlled by the mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC), which is negatively regulated by the specific BCKD kinase (BDK). Here, we used structure-based design to develop a BDK inhibitor, (S)-α-chloro-phenylpropionic acid [(S)-CPP]. Crystal structures of the BDK-(S)-CPP complex show that (S)-CPP binds to a unique allosteric site in the N-terminal domain, triggering helix movements in BDK. These conformational changes are communicated to the lipoyl-binding pocket, which nullifies BDK activity by blocking its binding to the BCKDC core. Administration of (S)-CPP to mice leads to the full activation and dephosphorylation of BCKDC with significant reduction in plasma BCAA concentrations. The results buttress the concept of targeting mitochondrial BDK as a pharmacological approach to mitigate BCAA accumulation in metabolic diseases and heart failure.
Assuntos
Proteínas Mitocondriais/química , Inibidores de Proteínas Quinases/química , Proteínas Quinases/química , Estrutura Terciária de Proteína , Regulação Alostérica , Animais , Sítios de Ligação/genética , Cromatografia Líquida , Cristalografia por Raios X , Isoleucina/sangue , Isoleucina/metabolismo , Cinética , Leucina/sangue , Leucina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Estrutura Molecular , Mutação , Fenilpropionatos/química , Fenilpropionatos/metabolismo , Fenilpropionatos/farmacologia , Fosforilação , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Espectrometria de Massas em Tandem , Valina/sangue , Valina/metabolismoRESUMO
The function of MEX3C, the mammalian homolog of Caenorhabditis elegans RNA-binding protein muscle excess 3 (MEX-3), was unknown until our recent report that MEX3C is necessary for normal postnatal growth and enhances the expression of local bone Igf1 expression. Here we report the pivotal role of Mex3c in energy balance regulation. Mex3c mutation caused leanness in both heterozygous and homozygous transgenic mice, as well as a more beneficial blood glucose and lipid profile in homozygous transgenic mice, in both sexes. Although transgenic mice showed normal food intake and fecal lipid excretion, they had increased energy expenditure independent of physical activity. Mutant mice had normal body temperature, Ucp1 expression in brown adipose tissue, and muscle and liver fatty acid oxidation. Mex3c is expressed in neurons and is detectable in the arcuate nucleus, the ventromedial nucleus, and the dorsomedial nucleus of the hypothalamus. Mex3c was not detected in NPY or POMC neurons but was detected in leptin-responsive neurons in the ventromedial nucleus. Mex3c and Leptin double mutant mice were growth retarded and obese and had blood profiles similar to those of ob/ob mice but showed none of the steatosis observed in ob/ob mice. Our data show that Mex3c is involved in energy balance regulation.
Assuntos
Adiposidade/genética , Metabolismo Energético/genética , Mutação , Proteínas de Ligação a RNA/genética , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/metabolismo , Animais , Glicemia/análise , Ingestão de Alimentos , Feminino , Canais Iônicos/biossíntese , Leptina/deficiência , Leptina/genética , Lipídeos/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Mitocondriais/biossíntese , Neurônios/metabolismo , Obesidade/genética , Proteína Desacopladora 1RESUMO
In the brain, metabolism of the essential branched chain amino acids (BCAAs) leucine, isoleucine, and valine, is regulated in part by protein synthesis requirements. Excess BCAAs are catabolized or excreted. The first step in BCAA catabolism is catalyzed by the branched chain aminotransferase (BCAT) isozymes, mitochondrial BCATm and cytosolic BCATc. A product of this reaction, glutamate, is the major excitatory neurotransmitter and precursor of the major inhibitory neurotransmitter γ-aminobutyric acid (GABA). The BCATs are thought to participate in a α-keto-acid nitrogen shuttle that provides nitrogen for synthesis of glutamate from α-ketoglutarate. The branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC) catalyzes the second, irreversible step in BCAA metabolism, which is oxidative decarboxylation of the branched-chain α-keto acid (BCKA) products of the BCAT reaction. Maple Syrup Urine Disease (MSUD) results from genetic defects in BCKDC, which leads to accumulation of toxic levels of BCAAs and BCKAs that result in brain swelling. Immunolocalization of BCATm and BCKDC in rats revealed that BCATm is present in astrocytes in white matter and in neuropil, while BCKDC is expressed only in neurons. BCATm appears uniformly distributed in astrocyte cell bodies throughout the brain. The segregation of BCATm to astrocytes and BCKDC to neurons provides further support for the existence of a BCAA-dependent glial-neuronal nitrogen shuttle since the data show that BCKAs produced by glial BCATm must be exported to neurons. Additionally, the neuronal localization of BCKDC suggests that MSUD is a neuronal defect involving insufficient oxidation of BCKAs, with secondary effects extending beyond the neuron.
RESUMO
Elevation of dietary or brain leucine appears to suppress food intake via a mechanism involving mechanistic target of rapamycin, AMPK, and/or branched chain amino acid (BCAA) metabolism. Mice bearing a deletion of mitochondrial branched chain aminotransferase (BCATm), which is expressed in peripheral tissues (muscle) and brain glia, exhibit marked increases in circulating BCAAs. Here, we test whether this increase alters feeding behavior and brain neuropeptide expression. Circulating and brain levels of BCAAs were increased two- to four-fold in BCATm-deficient mice (KO). KO mice weighed less than controls (25·9 vs 20·4âg, P<0·01), but absolute food intake was relatively unchanged. In contrast to wild-type mice, KO mice preferred a low-BCAA diet to a control diet (P<0·05) but exhibited no change in preference for low- vs high-protein (HP) diets. KO mice also exhibited low leptin levels and increased hypothalamic Npy and Agrp mRNA. Normalization of circulating leptin levels had no effect on either food preference or the increased Npy and Agrp mRNA expression. If BCAAs act as signals of protein status, one would expect reduced food intake, avoidance of dietary protein, and reduction in neuropeptide expression in BCATm-KO mice. Instead, these mice exhibit an increased expression of orexigenic neuropeptides and an avoidance of BCAAs but not HP. These data thus suggest that either BCAAs do not act as physiological signals of protein status or the loss of BCAA metabolism within brain glia impairs the detection of protein balance.
Assuntos
Preferências Alimentares , Hipotálamo/metabolismo , Neuropeptídeos/metabolismo , Transaminases/metabolismo , Animais , Proteínas Alimentares/administração & dosagem , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Transaminases/genéticaRESUMO
Branched-chain amino acids (BCAAs) catabolism follows sequential reactions and their metabolites intersect with other metabolic pathways. The initial enzymes in BCAA metabolism, the mitochondrial branched-chain aminotransferase (BCATm), which deaminates the BCAAs to branched-chain α-keto acids (BCKAs); and the branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC), which oxidatively decarboxylates the BCKAs, are organized in a supramolecular complex termed metabolon. Glutamate dehydrogenase (GDH1) is found in the metabolon in rat tissues. Bovine GDH1 binds to the pyridoxamine 5'-phosphate (PMP)-form of human BCATm (PMP-BCATm) but not to pyridoxal 5'-phosphate (PLP)-BCATm in vitro. This protein interaction facilitates reamination of the α-ketoglutarate (αKG) product of the GDH1 oxidative deamination reaction. Human GDH1 appears to act like bovine GDH1 but human GDH2 does not show the same enhancement of BCKDC enzyme activities. Another metabolic enzyme is also found in the metabolon is pyruvate carboxylase (PC). Kinetic results suggest that PC binds to the E1 decarboxylase of BCKDC but does not effect BCAA catabolism. The protein interaction of BCATm and GDH1 promotes regeneration of PLP-BCATm which then binds to BCKDC resulting in channeling of the BCKA products from BCATm first half reaction to E1 and promoting BCAA oxidation and net nitrogen transfer from BCAAs. The cycling of nitrogen through glutamate via the actions of BCATm and GDH1 releases free ammonia. Formation of ammonia may be important for astrocyte glutamine synthesis in the central nervous system. In peripheral tissue association of BCATm and GDH1 would promote BCAA oxidation at physiologically relevant BCAA concentrations.
Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Metabolismo Energético/fisiologia , Glutamato Desidrogenase/metabolismo , Leucina/metabolismo , Complexos Multienzimáticos/fisiologia , Regulação Alostérica/fisiologia , Animais , Bovinos , Ativação Enzimática/fisiologia , Glutamato Desidrogenase/química , Humanos , Mapas de Interação de Proteínas/fisiologia , RatosRESUMO
The NIPSNAP (4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1) proteins belong to a highly conserved family of proteins of unknown function. We found that NIPSNAP1 binds to the branched-chain alpha-keto acid (BCKA) dehydrogenase enzyme complex, which is disrupted in maple syrup urine disease, a disease of branched-chain amino acid catabolism that results in neurological dysfunction. Phenylketonuric (PKU) and epileptic mice show altered expression of NIPSNAP1 in the brain. Therefore, the distribution and localization of NIPSNAP1 in rat brain was determined. Results show that NIPSNAP1 is expressed exclusively in neurons including pyramidal neurons in the cerebral cortex, Purkinje neurons in the cerebellum and motor neurons in the spinal cord. Dopaminergic neurons in midbrain and noradrenergic neurons in the brainstem, which are affected in PKU, also express NIPSNAP1. NIPSNAP1 is found to be localized in the mitochondrial matrix and can bind dihydrolipoyl-transacylase and -transacetylase components of the BCKA and pyruvate dehydrogenase complexes in vitro. Our data provide the first experimental evidence for a strictly neuronal expression of this mitochondrial protein in the rat nervous system.
Assuntos
Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sistema Nervoso/metabolismo , Neurônios/metabolismo , Proteínas/metabolismo , Animais , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Feminino , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Doença da Urina de Xarope de Bordo/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Mutantes , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas do Tecido Nervoso/genética , Sistema Nervoso/anatomia & histologia , Neurônios/citologia , Fenilcetonúrias/metabolismo , Proteínas/genética , Ratos , Ratos Sprague-DawleyRESUMO
The branched-chain amino acid, leucine, acts as a nutrient signal to stimulate protein synthesis in skeletal muscle of young pigs. However, the chemical structure responsible for this effect has not been identified. We have shown that the other branched-chain amino acids, isoleucine and valine, are not able to stimulate protein synthesis when raised in plasma to levels within the postprandial range. In this study, we evaluated the effect of leucine, alpha-ketoisocaproic acid (KIC), and norleucine infusion (0 or 400 micromol kg(-1) h(-1) for 60 min) on protein synthesis and activation of translation initiation factors in piglets. Infusion of leucine, KIC, and norleucine raised plasma levels of each compound compared with controls. KIC also increased (P < 0.01) and norleucine reduced (P < 0.02) plasma levels of leucine compared with controls. Administration of leucine and KIC resulted in greater (P < 0.006) phosphorylation of eukaryotic initiation factor (eIF) 4E binding protein-1 (4E-BP1) and eIF4G, lower (P < 0.04) abundance of the inactive 4E-BP1.eIF4E complex, and greater (P < 0.05) active eIF4G.eIF4E complex formation in skeletal muscle compared with controls. Protein synthesis in skeletal muscle was greater (P < 0.02) in leucine- and KIC-infused pigs than in those in the control group. Norleucine infusion did not affect muscle protein synthesis or translation initiation factor activation. In liver, neither protein synthesis nor activation of translation initiation factors was affected by treatment. These results suggest that the ability of leucine to act as a nutrient signal to stimulate skeletal muscle protein synthesis is specific for leucine and/or its metabolite, KIC.
Assuntos
Animais Recém-Nascidos/metabolismo , Cetoácidos/farmacologia , Leucina/farmacologia , Proteínas Musculares/biossíntese , Músculo Esquelético/metabolismo , Suínos/metabolismo , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/metabolismo , Aminoácidos/análise , Aminoácidos/sangue , Animais , Fatores de Iniciação em Eucariotos/análise , Fatores de Iniciação em Eucariotos/metabolismo , Insulina/sangue , Músculo Esquelético/efeitos dos fármacos , Norleucina/farmacologiaRESUMO
The catabolic pathway for branched-chain amino acids includes deamination followed by oxidative decarboxylation of the deaminated product branched-chain alpha-keto acids, catalyzed by the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKDC). We found that BCATm binds to the E1 decarboxylase of BCKDC, forming a metabolon that allows channeling of branched-chain alpha-keto acids from BCATm to E1. The protein complex also contains glutamate dehydrogenase (GDH1), 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1, pyruvate carboxylase, and BCKDC kinase. GDH1 binds to the pyridoxamine 5'-phosphate (PMP) form of BCATm (PMP-BCATm) but not to the pyridoxal 5'-phosphate-BCATm and other metabolon proteins. Leucine activates GDH1, and oxidative deamination of glutamate is increased further by addition of PMP-BCATm. Isoleucine and valine are not allosteric activators of GDH1, but in the presence of 5'-phosphate-BCATm, they convert BCATm to PMP-BCATm, stimulating GDH1 activity. Sensitivity to ADP activation of GDH1 was unaffected by PMP-BCATm; however, addition of a 3 or higher molar ratio of PMP-BCATm to GDH1 protected GDH1 from GTP inhibition by 50%. Kinetic results suggest that GDH1 facilitates regeneration of the form of BCATm that binds to E1 decarboxylase of the BCKDC, promotes metabolon formation, branched-chain amino acid oxidation, and cycling of nitrogen through glutamate.