RESUMEN
Maple Syrup Urine Disease (MSUD) is an autosomal recessive inborn error of metabolism (IEM), responsible for the accumulation of the branched-chain amino acids (BCAA) leucine, isoleucine, and valine, in addition to their α-keto acids α-ketoisocaproic acid (KIC), α-keto-ß-methylvaleric acid (KMV), and α-ketoisovaleric acid (KIV) in the plasma and urine of patients. This process occurs due to a partial or total blockage of the dehydrogenase enzyme activity of branched-chain α-keto acids. Oxidative stress and inflammation are conditions commonly observed on IEM, and the inflammatory response may play an essential role in the pathophysiology of MSUD. We aimed to investigate the acute effect of intracerebroventricular (ICV) administration of KIC on inflammatory parameters in young Wistar rats. For this, sixteen 30-day-old male Wistar rats receive ICV microinjection with 8 µmol KIC. Sixty minutes later, the animals were euthanized, and the cerebral cortex, hippocampus, and striatum structures were collected to assess the levels of pro-inflammatory cytokines (INF-γ; TNF-α, IL-1ß). The acute ICV administration of KIC increased INF-γ levels in the cerebral cortex and reduced the levels of INF-γ and TNF-α in the hippocampus. There was no difference in IL-1ß levels. KIC was related to changes in the levels of pro-inflammatory cytokines in the brain of rats. However, the inflammatory mechanisms involved in MSUD are poorly understood. Thus, studies that aim to unravel the neuroinflammation in this pathology are essential to understand the pathophysiology of this IEM.
Asunto(s)
Enfermedad de la Orina de Jarabe de Arce , Factor de Necrosis Tumoral alfa , Ratas , Animales , Masculino , Ratas Wistar , Factor de Necrosis Tumoral alfa/metabolismo , Estrés Oxidativo , Cetoácidos/farmacología , Enfermedad de la Orina de Jarabe de Arce/tratamiento farmacológico , Enfermedad de la Orina de Jarabe de Arce/metabolismo , Aminoácidos de Cadena Ramificada/metabolismoRESUMEN
The venom of the South American rattlesnake Crotalus durissus terrificus causes an irreversible neuromuscular blockade in isolated preparations due to action of the presynaptically-acting heterodimeric phospholipase A2 (PLA2) crotoxin. Some populations of this subspecies contain, in addition to crotoxin, the toxin crotamine, which acts directly on muscle fibers. In this study we used C. d. terrificus venoms with (crot+) or without (crot-) crotamine to test whether Varespladib, a PLA2 inhibitor, is able to abrogate the neuromuscular blockade induced by these venoms comparatively with crotalic antivenom. Mouse phrenic nerve-diaphragm preparations were exposed to venoms previously incubated with two different concentrations of Varepladib or antivenom, or with a mixture of these two agents, before addition to the bath. In another experimental setting, venoms were initially added to the system, followed by the addition of Varespladib or antivenom 10, 30, or 60 min after venom. At the highest concentrations tested, Varespladib and antivenom inhibited the action of the venom >80% and >70%, respectively. With lower concentrations the inhibition of neuromuscular blockade decreased, but when low doses of the two agents were incubated together with the venom, the inhibitory effect improved, underscoring a synergistic phenomenon. When added after venom, Varespladib was able to halt the progression of the neuromuscular blockade even when added at 60 min. Antivenom exhibited a lower ability to inhibit the toxic effect of the venoms in these conditions. In conclusion, the PLA2 inhibitor Varespladib is highly effective at abrogating the neuromuscular blocking activity of crotamine-positive and crotamine-negative C. d. terrificus venoms and seems to act synergistically with antivenom.
Asunto(s)
Antivenenos , Venenos de Crotálidos , Crotoxina , Indoles , Bloqueo Neuromuscular , Enfermedades Neuromusculares , Acetatos/farmacología , Animales , Antivenenos/farmacología , Venenos de Crotálidos/farmacología , Crotoxina/farmacología , Sinergismo Farmacológico , Indoles/farmacología , Cetoácidos/farmacología , Ratones , Fosfolipasas A2RESUMEN
Maple syrup urine disease (MSUD) is an autosomal recessive neurometabolic disorder caused by severe deficiency of branched-chain α-keto acid dehydrogenase complex activity, which catalyzes the oxidative decarboxylation of the branched-chain α-keto acids (BCKA). The metabolic blockage results in tissue accumulation and high urinary excretion of the branched-chain amino acids (BCAA) leucine, isoleucine and valine, as well as alloisoleucine, and their respective BCKA α-ketoisocaproic (α-KIC), α-ketoisovaleric and α-keto-ß-methylvaleric acids. Affected patients usually manifest acute episodes of encephalopathy associated with seizures, coma and life-threatening cerebral edema in the first weeks of life, which is followed by progressive neurological deterioration with motor delay, ataxia, intellectual disability and psychiatric symptoms. The pathophysiology of the brain damage in MSUD has been mainly focused on brain amino acid imbalance leading to deficient cerebral protein and neurotransmitter synthesis. However, the acute episodes of severe neurological symptoms accompanied by large increases of BCKA/BCAA levels suggest neurotoxic actions of these compounds. In this particular, mounting evidence from humans and animal models support an important role of particularly leucine and α-KIC on the pathogenesis of the brain injury in MSUD. In this review we will present the current knowledge of the major mechanisms presumably involved in MSUD neuropathology and highlight the neurotoxic properties of the BCAA and BCKA, disturbing brain bioenergetics and redox homeostasis, besides inducing neuroinflammation. We suggest that these pathomechanisms may contribute to the neurological sequelae of MSUD patients and hopefully allow the design of novel therapeutic strategies, including antioxidant and bioenergetics stimulating drugs targeting the mitochondria.
Asunto(s)
Enfermedad de la Orina de Jarabe de Arce , Síndromes de Neurotoxicidad , Aminoácidos , Aminoácidos de Cadena Ramificada/metabolismo , Animales , Humanos , Cetoácidos/farmacología , Leucina/metabolismo , Enfermedad de la Orina de Jarabe de Arce/metabolismoRESUMEN
In this study, we investigated the action of varespladib (VPL) alone or in combination with a coral snake antivenom (CAV) on the local and systemic effects induced by Micrurus corallinus venom in rats. Adult male Wistar rats were exposed to venom (1.5 mg/kg - i.m.) and immediately treated with CAV (antivenom:venom ratio 1:1.5 'v/w' - i.p.), VPL (0.5 mg/kg - i.p.), or both of these treatments. The animals were monitored for 120 min and then anesthetized to collect blood samples used for haematological and serum biochemical analysis; after euthanasia, skeletal muscle, renal and hepatic tissue samples were collected for histopathological analysis. M. corallinus venom caused local oedema without subcutaneous haemorrhage or apparent necrosis formation, although there was accentuated muscle morphological damage; none of the treatments prevented oedema formation but the combination of CAV and VPL reduced venom-induced myonecrosis. Venom caused neuromuscular paralysis and respiratory impairment in approximately 60 min following envenomation; CAV alone did not prevent the neurotoxic action, whereas VPL alone prevented neurotoxic symptoms developing as did the combination of CAV and VPL. Venom induced significant increase of serum CK and AST release, mostly due to local and systemic myotoxicity, which was partially prevented by the combination of CAV and VPL. The release of hepatotoxic serum biomarkers (LDH and ALP) induced by M. corallinus venom was not prevented by CAV and VPL when individually administered; their combination effectively prevented ALP release. The venom-induced nephrotoxicity (increase in serum creatinine concentration) was prevented by all the treatments. VPL alone or in combination with CAV significantly prevented the venom-induced lymphocytosis. In conclusion, VPL shows to be effective at preventing the neurotoxic, nephrotoxic, and inflammatory activities of M. corallinus venom. In addition, VPL acts synergistically with antivenom to prevent a number of systemic effects caused by M. corallinus venom.
Asunto(s)
Acetatos/farmacología , Serpientes de Coral/fisiología , Venenos Elapídicos/toxicidad , Indoles/farmacología , Cetoácidos/farmacología , Inhibidores de Fosfolipasa A2/farmacología , Animales , Biomarcadores/sangre , Trastornos de la Coagulación Sanguínea/inducido químicamente , Trastornos de la Coagulación Sanguínea/tratamiento farmacológico , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , L-Lactato Deshidrogenasa/sangre , Fármacos Neuroprotectores/farmacología , Fosfolipasas A2/genética , Fosfolipasas A2/metabolismo , Ratas , Ratas WistarRESUMEN
Although phenylalanine (Phe) is known to be neurotoxic in phenylketonuria (PKU), its exact pathogenetic mechanisms of brain damage are still poorly known. Furthermore, much less is known about the role of the Phe derivatives phenylacetic (PAA), phenyllactic (PLA) and phenylpyruvic (PPA) acids that also accumulate in this this disorder on PKU neuropathology. Previous in vitro and in vivo studies have shown that Phe elicits oxidative stress in brain of rodents and that this deleterious process also occurs in peripheral tissues of phenylketonuric patients. In the present study, we investigated whether Phe and its derivatives PAA, PLA and PPA separately or in combination could induce reactive oxygen species (ROS) formation and provoke DNA damage in C6 glial cells. We also tested the role of L-carnitine (L-car), which has been recently considered an antioxidant agent and easily cross the blood brain barrier on the alterations of C6 redox status provoked by Phe and its metabolites. We first observed that cell viability was not changed by Phe and its metabolites. Furthermore, Phe, PAA, PLA and PPA, at concentrations found in plasma of PKU patients, provoked marked DNA damage in the glial cells separately and when combined. Of note, these effects were totally prevented (Phe, PAA and PPA) or attenuated (PLA) by L-car pre-treatment. In addition, a potent ROS formation also induced by Phe and PAA, whereas only moderate increases of ROS were caused by PPA and PLA. Pre-treatment with L-car also prevented Phe- and PAA-induced ROS generation, but not that provoked by PLA and PPA. Thus, our data show that Phe and its major metabolites accumulated in PKU provoke extensive DNA damage in glial cells probably by ROS formation and that L-car may potentially represent an adjuvant therapeutic agent in PKU treatment.
Asunto(s)
Lesiones Encefálicas , Fenilcetonurias , Lesiones Encefálicas/tratamiento farmacológico , Carnitina/farmacología , Carnitina/uso terapéutico , Humanos , Cetoácidos/farmacología , Estrés Oxidativo , Fenilalanina/farmacología , Fenilalanina/uso terapéuticoRESUMEN
BACKGROUND: The treatment for snakebites is early administration of antivenom, which can be highly effective in inhibiting the systemic effects of snake venoms, but is less effective in the treatment of extra-circulatory and local effects. To complement standard-of-care treatments such as antibody-based antivenoms, natural and synthetic small molecules have been proposed for the inhibition of key venom components such as phospholipase A2 (PLA2) and PLA2-like toxins. Varespladib (compound LY315920) is a synthetic molecule developed and clinically tested aiming to block inflammatory cascades of several diseases associated with high PLA2s. Recent studies have demonstrated this molecule is able to potently inhibit snake venom catalytic PLA2 and PLA2-like toxins. METHODS: In vivo and in vitro techniques were used to evaluate the inhibitory effect of varespladib against MjTX-I. X-ray crystallography was used to reveal details of the interaction between these molecules. A new methodology that combines crystallography, mass spectroscopy and phylogenetic data was used to review its primary sequence. RESULTS: Varespladib was able to inhibit the myotoxic and cytotoxic effects of MjTX-I. Structural analysis revealed a particular inhibitory mechanism of MjTX-I when compared to other PLA2-like myotoxin, presenting an oligomeric-independent function. CONCLUSION: Results suggest the effectiveness of varespladib for the inhibition of MjTX-I, in similarity with other PLA2 and PLA2-like toxins. GENERAL SIGNIFICANCE: Varespladib appears to be a promissory molecule in the treatment of local effects led by PLA2 and PLA2-like toxins (oligomeric dependent and independent), indicating that this is a multifunctional or broadly specific inhibitor for different toxins within this superfamily.
Asunto(s)
Acetatos/farmacología , Bothrops/metabolismo , Fosfolipasas A2 Grupo II/antagonistas & inhibidores , Indoles/farmacología , Cetoácidos/farmacología , Fibras Musculares Esqueléticas/efectos de los fármacos , Inhibidores de Fosfolipasa A2/farmacología , Fosfolipasas A2/química , Proteínas de Reptiles/antagonistas & inhibidores , Animales , Cristalografía por Rayos X , Fosfolipasas A2 Grupo II/toxicidad , Ratones , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Proteínas de Reptiles/toxicidadRESUMEN
Maple syrup urine disease (MSUD) is characterized by a deficiency in the mitochondrial branched-chain α-keto acid dehydrogenase complex activity and, consequently, accumulation of the branched-chain amino acids and their respective branched-chain α-keto acids in fluids and the tissue. MSUD clinical symptoms include neurological alterations. KIC is considered one of the significant neurotoxic metabolites since its increased plasma concentrations are associated with neurological symptoms. We evaluated the effect of KIC intracerebroventricular (ICV) injection in hippocampal mitochondria function in rats. We also investigated the impact of KIC in cells' metabolic activity (using MTT assay) and reactive species (RS) production in HT-22 cells. For this, thirty-day-old male rats were bilaterally ICV injected with KIC or aCSF. Thus, 1 hour after the administration, animals were euthanized, and the hippocampus was harvested for measured the activities of mitochondrial respiratory chain enzymes and RS production. Furthermore, HT-22 cells were incubated with KIC (1-10 mM) in 6, 12, and 24 h. Mitochondrial complexes activities were reduced, and the formation of RS was increased in the hippocampus of rats after KIC administration. Moreover, KIC reduced the cells' metabolic ability to reduce MTT and increased RS production in hippocampal neurons. Impairment in hippocampal mitochondrial function seems to be involved in the neurotoxicity induced by KIC.
Asunto(s)
Aminoácidos de Cadena Ramificada/metabolismo , Hipocampo/efectos de los fármacos , Cetoácidos/farmacología , Estrés Oxidativo/efectos de los fármacos , Animales , Línea Celular , Hipocampo/metabolismo , Masculino , Enfermedad de la Orina de Jarabe de Arce/metabolismo , Ratones , Ratas , Ratas WistarRESUMEN
Keto analogues and amino acids (KAAA) supplementation can reduce blood ammonia concentrations in athletes undergoing high-intensity exercise under both ketogenic and thermoneutral conditions. This study evaluated the acute effects of KAAA supplementation on ammonia metabolism during extenuating endurance exercise in rats fed a ketogenic diet. In all, eighty male Fischer rats at 90 d of age were divided into eight groups, and some were trained using a swimming endurance protocol. A ketogenic diet supplemented with keto analogues was administered for 10 d. Administration of the ketogenic diet ended 3 d before the exhaustion test (extenuating endurance exercise). A ketogenic diet plus KAAA supplementation and extenuating endurance exercise (trained ketogenic diet supplemented with KAAA (TKKa)) increased blood ammonia concentrations by approximately 50 % compared with the control diet (trained control diet supplemented with KAAA (TCKa)) and similar training (effect size=1·33; statistical power=0·50). The KAAA supplementation reduced blood urea concentrations by 4 and 18 % in the control and ketogenic diet groups, respectively, compared with the groups fed the same diets without supplementation. The trained groups had 60 % lower blood urate concentrations after TCKa treatment than after TKKa treatment. Our results suggest that KAAA supplementation can reduce blood ammonia concentrations after extenuating endurance exercise in rats fed a balanced diet but not in rats fed a ketogenic diet.
Asunto(s)
Aminoácidos/uso terapéutico , Amoníaco/sangre , Dieta , Suplementos Dietéticos , Hiperamonemia/prevención & control , Cetoácidos/uso terapéutico , Resistencia Física/fisiología , Aminoácidos/farmacología , Animales , Dieta Cetogénica , Hiperamonemia/sangre , Hiperamonemia/etiología , Cetoácidos/farmacología , Masculino , Condicionamiento Físico Animal/fisiología , Ratas Endogámicas F344RESUMEN
Maple Syrup Urine Disease (MSUD) is biochemically characterized by elevated levels of leucine, isoleucine and valine, as well as their corresponding transaminated branched-chain α-keto acids in tissue and biological fluids. Neurological symptoms and cerebral abnormalities, whose mechanisms are still unknown, are typical of this metabolic disorder. In the present study, we evaluated the early effects (1 h after injection) and long-term effects (15 days after injection) of a single intracerebroventricular administration of α-ketoisocaproic acid (KIC) on oxidative stress parameters and cognitive and noncognitive behaviors. Our results showed that KIC induced early and long-term effects; we found an increase in TBARS levels, protein carbonyl content and DNA damage in the hippocampus, striatum and cerebral cortex both one hour and 15 days after KIC administration. Moreover, SOD activity increased in the hippocampus and striatum one hour after injection, whereas after 15 days, SOD activity decreased only in the striatum. On the other hand, KIC significantly decreased CAT activity in the striatum one hour after injection, but 15 days after KIC administration, we found a decrease in CAT activity in the hippocampus and striatum. Finally, we showed that long-term cognitive deficits follow the oxidative damage; KIC induced impaired habituation memory and long-term memory impairment. From the biochemical and behavioral findings, it we presume that KIC provokes oxidative damage, and the persistence of brain oxidative stress is associated with long-term memory impairment and prepulse inhibition.
Asunto(s)
Conducta Animal/efectos de los fármacos , Cognición/efectos de los fármacos , Cetoácidos/administración & dosificación , Cetoácidos/farmacología , Estrés Oxidativo/efectos de los fármacos , Animales , Reacción de Prevención/efectos de los fármacos , Catalasa/metabolismo , Inyecciones Intraventriculares , Masculino , Enfermedad de la Orina de Jarabe de Arce/psicología , Trastornos de la Memoria/inducido químicamente , Trastornos de la Memoria/psicología , Carbonilación Proteica , Ratas , Ratas Wistar , Reflejo de Sobresalto/efectos de los fármacos , Superóxido Dismutasa-1/metabolismo , Natación/psicología , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
Maple syrup urine disease (MSUD) is an inherited aminoacidopathy resulting from dysfunction of the branched-chain keto acid dehydrogenase complex, leading to accumulation of the branched-chain amino acids (BCAA) leucine, isoleucine and valine as well as their corresponding transaminated branched-chain α-ketoacids. This disorder is clinically characterized by ketoacidosis, seizures, coma, psychomotor delay and mental retardation whose pathophysiology is not completely understood. Recent studies have shown that oxidative stress may be involved in neuropathology of MSUD. However, the effect of accumulating α-ketoacids in MSUD on neurotrophic factors has not been investigated. Thus, the objective of the present study was to evaluate the effects of acute intracerebroventricular administration of α-ketoisocaproic acid (KIC) on brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) levels in the brains of young male rats. Ours results showed that intracerebroventricular administration of KIC decreased BDNF levels in hippocampus, striatum and cerebral cortex, without induce a detectable change in pro-BDNF levels. Moreover, NGF levels in the hippocampus were reduced after intracerebroventricular administration of KIC. In conclusion, these data suggest that the effects of KIC on demyelination and memory processes may be mediated by reduced trophic support of BDNF and NGF. Moreover, lower levels of BDNF and NGF are consistent with the hypothesis that a deficit in this neurotrophic factor may contribute to the structural and functional alterations of brain underlying the psychopathology of MSUD, supporting the hypothesis of a neurodegenerative process in MSUD.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Cetoácidos/farmacología , Precursores de Proteínas/metabolismo , Factores de Edad , Animales , Encéfalo/metabolismo , Cetoácidos/administración & dosificación , Masculino , Estrés Oxidativo/efectos de los fármacos , Ratas WistarRESUMEN
During exercise, ammonia levels are related to the appearance of both central and peripheral fatigue. Therefore, controlling the increase in ammonia levels is an important strategy in ameliorating the metabolic response to exercise and in improving athletic performance. Free amino acids can be used as substrates for ATP synthesis that produces ammonia as a side product. Keto analogues act in an opposite way, being used to synthesise amino acids whilst decreasing free ammonia in the blood. Adult male rats were divided into four groups based on receiving either keto analogues associated with amino acids (KAAA) or a placebo and resistance exercise or no exercise. There was an approximately 40% increase in ammonaemia due to KAAA supplementation in resting animals. Exercise increased ammonia levels twofold with respect to the control, with a smaller increase (about 20%) in ammonia levels due to exercise. Exercise itself causes a significant increase in blood urea levels (17%). However, KAAA reduced blood urea levels to 75% of the pre-exercise values. Blood urate levels increased 28% in the KAAA group, independent of exercise. Supplementation increased glucose levels by 10% compared with control animals. Exercise did not change glucose levels in either the control or supplemented groups. Exercise promoted a 57% increase in lactate levels in the control group. Supplementation promoted a twofold exercise-induced increase in blood lactate levels. The present results suggest that an acute supplementation of KAAA can decrease hyperammonaemia induced by exercise.
Asunto(s)
Aminoácidos/farmacología , Cetoácidos/química , Cetoácidos/farmacología , Actividad Motora/fisiología , Condicionamiento Físico Animal/fisiología , Aminoácidos/administración & dosificación , Alimentación Animal , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Dieta , Suplementos Dietéticos , Hiperamonemia , Cetoácidos/administración & dosificación , Ácido Láctico/sangre , Masculino , Ratas , Ratas Wistar , Ácido Úrico/sangreRESUMEN
Accumulation of the branched-chain alpha-keto acids (BCKA), alpha-ketoisocaproic acid (KIC), alpha-keto-beta-methylvaleric acid (KMV) and alpha-ketoisovaleric acid (KIV) and their respective branched-chain alpha-amino acids (BCAA) occurs in tissues and biological fluids of patients affected by the neurometabolic disorder maple syrup urine disease (MSUD). The objective of this study was to verify the effect of the BCKA on S100B release from C6 glioma cells. The cells were exposed to 1, 5 or 10 mM BCKA for different periods and the S100B release was measured afterwards. The results indicated that KIC and KIV, but not KMV, significantly enhanced S100B liberation after 6 h of exposure. Furthermore, the stimulatory effect of the BCKA on S100B release was prevented by coincubation with the energetic substrate creatine and with the N-nitro-l-arginine methyl ester (l-NAME), a nitric oxide synthase inhibitor, indicating that energy deficit and nitric oxide (NO) were probably involved in this effect. Furthermore, the increase of S100B release was prevented by preincubation with the protein kinase inhibitors KN-93 and H-89, indicating that KIC and KIV altered Ca2+/calmodulin (PKCaMII)- and cAMP (PKA)-dependent protein kinases activities, respectively. In contrast, other antioxidants such as glutathione (GSH) and trolox (soluble vitamin E) were not able to prevent KIC- and KIV-induced increase of S100B liberation, suggesting that the alteration of S100B release caused by the BCKA is not mediated by oxidation of sulfydryl or other essential groups of the enzyme as well as by lipid peroxyl radicals. Considering the importance of S100B for brain regulation, it is conceivable that enhanced liberation of this protein by increased levels of BCKA may contribute to the neurodegeneration characteristic of MSUD patients.
Asunto(s)
Encéfalo/metabolismo , Cetoácidos/metabolismo , Enfermedad de la Orina de Jarabe de Arce/complicaciones , Enfermedad de la Orina de Jarabe de Arce/metabolismo , Degeneración Nerviosa/etiología , Degeneración Nerviosa/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Neuroglía/metabolismo , Proteínas S100/metabolismo , Animales , Encéfalo/fisiopatología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular Tumoral , Creatina/metabolismo , Creatina/farmacología , Relación Dosis-Respuesta a Droga , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/fisiología , Inhibidores Enzimáticos/farmacología , Cetoácidos/farmacología , Enfermedad de la Orina de Jarabe de Arce/fisiopatología , Degeneración Nerviosa/fisiopatología , Neuroglía/efectos de los fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo I/metabolismo , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Proteínas Quinasas/efectos de los fármacos , Proteínas Quinasas/metabolismo , Ratas , Subunidad beta de la Proteína de Unión al Calcio S100RESUMEN
Accumulation of the branched-chain alpha-keto acids (BCKA), alpha-ketoisocaproic acid (KIC), alpha-keto-beta-methylvaleric acid (KMV), and alpha-ketoisovaleric acid (KIV) and their respective branched-chain alpha-amino acids (BCAA) in tissues and biological fluids is the biochemical hallmark of patients affected by the neurometabolic disorder known as maple syrup urine disease (MSUD). Considering that brain energy metabolism is possibly altered in MSUD, the objective of this study was to determine creatine kinase (CK) activity, a key enzyme of energy homeostasis, in C6 glioma cells exposed to BCKA. The cells were incubated with 1, 5, or 10 mM BCKA for 3 h and the CK activity measured afterwards. The results indicated that the BCKA significantly inhibited CK activity at all tested concentrations. Furthermore, the inhibition caused by the BCKA on CK activity was totally prevented by preincubation with the energetic substrate creatine and by coincubation with the N-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, indicating that deficit of energy and nitric oxide (NO) are involved in these effects. In contrast, other antioxidants such as glutathione (GSH) and trolox (soluble Vitamin E) were not able to prevent CK inhibition. In addition, we observed that the C6 cells changed their usual rounded morphology when exposed for 3 h to 10 mM BCKA and that creatine and L-NAME prevented these morphological alterations. Considering the importance of CK for brain metabolism homeostasis, it is conceivable that inhibition of this enzyme by increased levels of BCKA may contribute to the neurodegeneration of MSUD patients.
Asunto(s)
Antioxidantes/uso terapéutico , Forma de la Célula/efectos de los fármacos , Creatina Quinasa/metabolismo , Creatina/uso terapéutico , Cetoácidos , Enfermedad de la Orina de Jarabe de Arce , Animales , Antioxidantes/metabolismo , Línea Celular Tumoral , Creatina/metabolismo , Creatina Quinasa/antagonistas & inhibidores , Glioma/metabolismo , Hemiterpenos , Humanos , Cetoácidos/metabolismo , Cetoácidos/farmacología , Enfermedad de la Orina de Jarabe de Arce/tratamiento farmacológico , Enfermedad de la Orina de Jarabe de Arce/metabolismo , RatasRESUMEN
Since the human immunodeficiency virus was identified as etiological agent of the acquired immunodeficiency syndrome, great advances have been accomplished in the therapeutic field leading to reduced morbidity and mortality among infected patients. However, the high mutation rate of the viral genome generates strains resistant to multiple drugs, pointing to the importance of finding new therapeutic targets. Among the HIV structural genes, the POL gene codes for three essential enzymes: reverse transcriptase, protease, and integrase; nineteen of the twenty drugs currently approved by the Food and Drug Administration to treat this viral infection, inhibit the reverse transcriptase and the protease. Although intense research has been carried out in this area during the last 10 years, HIV integrase inhibitors are not yet approved for clinical use; however the fact that presence of this enzyme is a sine qua non for a productive HIV life cycle joined to its unique properties makes it a promissory target for anti-HIV therapy. Many compounds have been claimed to inhibit integrase in vitro; however, few of them have proven to have antiviral activity and low cytotoxicity in cell systems. Diketoacid derivatives are the most promising integrase inhibitors so far reported. Initially discovered independently by Shionogi & Co. and the Merck Research Laboratories, these compounds are highly specific for the integrase with potent antiviral activity in vitro and in vivo, and low cytotoxicity in cell cultures. Some of these compounds have recently entered clinical trials. Due to the high relevance of integrase inhibitors, and specifically of diketoacid derivatives, we review the latest findings and patents in this important field of research.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Inhibidores de Integrasa VIH/uso terapéutico , VIH-1/efectos de los fármacos , Cetoácidos/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida/virología , Animales , Farmacorresistencia Viral , Integrasa de VIH/química , Integrasa de VIH/genética , Inhibidores de Integrasa VIH/síntesis química , Inhibidores de Integrasa VIH/farmacología , VIH-1/genética , VIH-1/crecimiento & desarrollo , Humanos , Cetoácidos/síntesis química , Cetoácidos/farmacología , Patentes como AsuntoRESUMEN
In this study we investigated the involvement of Ca2+ on the effects of alpha-ketoisocaproic acid (KIC), the main metabolite accumulating in maple syrup urine disease (MSUD), on the phosphorylating system associated with the intermediate filament (IF) proteins in slices from cerebral cortex of 9-day-old rats. We first observed that KIC significantly decreased the in vitro phosphorylation of IF proteins in brain slices. KIC-induced dephosphorylation was mediated especially by the protein phosphatase PP2B, a Ca2+-dependent protein phosphatase, but also by PP2A. We also demonstrated the involvement of Ca2+-dependent mechanisms in the KIC effects using the specific L-voltage-dependent Ca2+ channels (L-VDCC) inhibitor nifedipine, the NMDA antagonist DL-AP5 and the intracellular Ca2+ chelator BAPTA-AM. Blockage of Ca2+ channels or chelating intracellular Ca2+ completely prevented the effects of KIC on the phosphorylating system associated to IF proteins. In addition, we verified that KIC increased 45Ca2+ uptake in brain slices after 3 and 30 min incubation. Taken together, our present data indicate that KIC increase intracellular Ca2+ levels, probably promoting the activation of calcineurin. These results might be associated with the increased dephosphorylation of the IF proteins in slices of cerebral cortex of immature rats exposed to KIC at similar concentrations from those found in blood and tissues of patients with MSUD.
Asunto(s)
Calcio/fisiología , Corteza Cerebral/fisiología , Proteínas del Citoesqueleto/fisiología , Cetoácidos/farmacología , Animales , Autorradiografía , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Radioisótopos de Calcio , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Inmunosupresores/farmacología , Técnicas In Vitro , Enfermedad de la Orina de Jarabe de Arce/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Ratas , Ratas Wistar , Tacrolimus/farmacologíaRESUMEN
Maple syrup urine disease (MSUD) is an inherited neurometabolic disorder caused by deficiency of branched-chain alpha-keto acid dehydrogenase complex activity which leads to tissue accumulation of the branched-chain alpha-keto acids (BCKAs) alpha-ketoisocaproic acid (KIC), alpha-ketoisovaleric acid (KIV) and alpha-keto-beta-methylvaleric acid (KMV) and their respective amino acids. Neuropathologic findings characteristic of the disease are cerebral edema and atrophy, whose pathophysiology is poorly known. In the present study, we investigated the in vitro effect of BCKAs on various parameters of oxidative stress, namely chemiluminescence (CL), thiobarbituric acid-reactive substances (TBA-RS), total radical-trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), and the activities of the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) in cerebral cortex of 30-day-old rats. The major effects observed were with KIC, which significantly increased CL and TBA-RS measurements, decreased TRAP and TAR values, and markedly inhibited GPx activity. KMV and KIV increased CL and decreased TRAP and TAR values. In contrast, these compounds did not affect CAT and SOD activities. Taken together, it was shown that: the BCKAs studied stimulated lipid peroxidation and reduced the brain antioxidant defences, suggesting an increased production of free radicals. In case the in vitro effects here detected also occur in vivo in MSUD, it can be presumed that oxidative stress might contribute, at least in part, to the brain damage found in the affected patients.
Asunto(s)
Antioxidantes/metabolismo , Corteza Cerebral/metabolismo , Cetoácidos/metabolismo , Peroxidación de Lípido/fisiología , Enfermedad de la Orina de Jarabe de Arce/metabolismo , Animales , Catalasa/metabolismo , Corteza Cerebral/química , Modelos Animales de Enfermedad , Radicales Libres/metabolismo , Glutatión Peroxidasa/metabolismo , Hemiterpenos , Cetoácidos/farmacología , Peroxidación de Lípido/efectos de los fármacos , Masculino , Estrés Oxidativo/fisiología , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
In this study we investigated the effects of alpha-ketoisovaleric (KIV) and alpha-keto-beta-methylvaleric acids (KMV), metabolites accumulating in the inherited neurometabolic disorder maple syrup urine disease (MSUD), on the in vitro incorporation of 32P into intermediate filament (IF) proteins from cerebral cortex of young rats during development (9-21 days of age) We observed that KMV significantly increased the in vitro incorporation of 32P into the IF proteins studied in cortical slices of 12-day-old rats through the PKA and PKCaMII, with no alteration at the other ages. In contrast, KIV was ineffective in altering the phosphorylating system associated with IF proteins at all ages examined. A similar effect on IF phosphorylation was achieved by incubating cortical slices with gamma-aminobutiric acid (GABA). Furthermore, by using specific GABA antagonists, we verified that KMV induced a stimulatory effect on IF phosphorylation of tissue slices from 12-day-old rats mediated by GABA(A) and GABA(B) receptors. In conclusion, our results indicate the involvement of the GABAergic system in the alterations of IF phosphorylation caused by KMV, one of the branched-chain keto acids accumulating in MSUD.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Filamentos Intermedios/metabolismo , Cetoácidos/farmacología , Factores de Edad , Análisis de Varianza , Animales , Animales Recién Nacidos , Bencilaminas/farmacología , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Electroforesis en Gel de Poliacrilamida/métodos , Inhibidores Enzimáticos/farmacología , Antagonistas del GABA/farmacología , Proteína Ácida Fibrilar de la Glía/metabolismo , Hemiterpenos , Técnicas In Vitro , Isoquinolinas/farmacología , Isótopos de Fósforo/metabolismo , Fosforilación/efectos de los fármacos , Radioquímica/métodos , Ratas , Ratas Wistar , Sulfonamidas/farmacología , Vimentina/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Neurological dysfunction is a common finding in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the neuropathology of brain damage in this disorder are poorly known. In the present study, we investigated the effect of the in vitro effect of the branched chain alpha-keto acids (BCKA) accumulating in MSUD on some parameters of energy metabolism in cerebral cortex of rats. [14CO(2)] production from [14C] acetate, glucose uptake and lactate release from glucose were evaluated by incubating cortical prisms from 30-day-old rats in Krebs-Ringer bicarbonate buffer, pH 7.4, in the absence (controls) or presence of 1-5 mM of alpha-ketoisocaproic acid (KIC), alpha-keto-beta-methylvaleric acid (KMV) or alpha-ketoisovaleric acid (KIV). All keto acids significantly reduced 14CO(2) production by around 40%, in contrast to lactate release and glucose utilization, which were significantly increased by the metabolites by around 42% in cortical prisms. Furthermore, the activity of the respiratory chain complex I-III was significantly inhibited by 60%, whereas the other activities of the electron transport chain, namely complexes II, II-III, III and IV, as well as succinate dehydrogenase were not affected by the keto acids. The results indicate that the major metabolites accumulating in MSUD compromise brain energy metabolism by blocking the respiratory chain. We presume that these findings may be of relevance to the understanding of the pathophysiology of the neurological dysfunction of MSUD patients.
Asunto(s)
Encéfalo/metabolismo , Metabolismo Energético/efectos de los fármacos , Cetoácidos/farmacología , Enfermedad de la Orina de Jarabe de Arce/metabolismo , Animales , Transporte Biológico , Dióxido de Carbono/metabolismo , Modelos Animales de Enfermedad , Glucosa/metabolismo , Hemiterpenos , Humanos , Lactatos/metabolismo , Ratas , Ratas Wistar , Succinato Citocromo c Oxidorreductasa/metabolismoRESUMEN
In this study we investigated the effects of alpha-ketoisocaproic acid (KIC), the main keto acid accumulating in the inherited neurometabolic disorder maple syrup urine disease (MSUD), on the in vitro incorporation of 32P into intermediate filament (IF) proteins from cerebral cortex of rats during development. KIC decreased the in vitro incorporation of 32P into the IF proteins studied up to day 12, had no effect on day 15, and increased this phosphorylation in cortical slices of 17- and 21-day-old rats. A similar effect on IF phosphorylation was achieved along development by incubating cortical slices with glutamate. Furthermore, the altered phosphorylation caused by the presence of KIC in the incubation medium was mediated by the ionotropic receptors NMDA, AMPA and kainate up to day 12 and by NMDA and AMPA in tissue slices from 17- and 21-day-old rats. The results suggest that alterations of IF phosphorylation may be associated with the neuropathology of MSUD.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Filamentos Intermedios/efectos de los fármacos , Cetoácidos/metabolismo , Enfermedad de la Orina de Jarabe de Arce/metabolismo , Neuronas/efectos de los fármacos , Animales , Animales Recién Nacidos , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Proteína Ácida Fibrilar de la Glía/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacología , Proteínas de Filamentos Intermediarios/efectos de los fármacos , Proteínas de Filamentos Intermediarios/metabolismo , Filamentos Intermedios/metabolismo , Cetoácidos/farmacología , Enfermedad de la Orina de Jarabe de Arce/fisiopatología , Neuronas/metabolismo , Técnicas de Cultivo de Órganos , Fosforilación/efectos de los fármacos , Ratas , Ratas Wistar , Receptores AMPA/efectos de los fármacos , Receptores AMPA/metabolismo , Receptores de Glutamato/efectos de los fármacos , Receptores de Glutamato/metabolismo , Receptores de Ácido Kaínico/efectos de los fármacos , Receptores de Ácido Kaínico/metabolismo , Receptores de Glutamato Metabotrópico/efectos de los fármacos , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Vimentina/efectos de los fármacos , Vimentina/metabolismoRESUMEN
In this study we investigated the effects of the branched chain alpha-ketoacids accumulating in maple syrup urine disease (MSUD) on the concentrations of the high molecular weight neurofilament subunit (NF-H) associated with the cytoskeletal fraction of the cerebral cortex of 12-day-old rats. Cortical slices were incubated with alpha-ketoisocaproic acid (KIC), alpha-keto beta-methylvaleric acid (KMV) and alpha-ketoisovaleric acid (KIV) at concentrations ranging from 0.5 to 1.0 mM. The cytoskeletal fraction was extracted and the immunoreactivity for phosphorylated and total NF-H was analyzed by immunoblotting. The in vitro 32P incorporation into NF-H was also determined. Results showed that treatment of tissue slices induced with KMV increased Triton-insoluble phosphorylated NF-H immunoreactivity, with no alteration in total NF-H immunoreactivity. Furthermore, KIC treatment drastically increased the total amount of NF-H, whereas KIV did not change either phosphorylated or total NF-H immunoreactivity. KMV also increased the in vitro 32P incorporation into NF-H, confirming the highly phosphorylated NF-H levels detected in the immunoblot. These findings demonstrate that KIC and KMV alter the dynamic regulation of NF-H assembly in the cytoskeletal fraction. Therefore we may suggest that cytoskeletal disorganization may be one of the factors associated with the neurodegeneration characteristic of MSUD disease.