RESUMEN
Heterozygosity for missense mutations (N88S/S90L) in BSCL2 (Berardinelli-Seip congenital lipodystrophy type 2)/Seipin is associated with a broad spectrum of motoneuron diseases. To understand the underlying mechanisms how the mutations lead to motor neuropathy, we generated transgenic mice with neuron-specific expression of wild-type (tgWT) or N88S/S90L mutant (tgMT) human Seipin. Transgenes led to the broad expression of WT or mutant Seipin in the brain and spinal cord. TgMT, but not tgWT, mice exhibited late-onset altered locomotor activities and gait abnormalities that recapitulate symptoms of seipinopathy patients. We found loss of alpha motor neurons in tgMT spinal cord. Mild endoreticular stress was present in both tgMT and tgWT neurons; however, only tgMT mice exhibited protein aggregates and disrupted Golgi apparatus. Furthermore, autophagosomes were significantly increased, along with elevated light chain 3 (LC3)-II level in tgMT spinal cord, consistent with the activation of autophagy pathway in response to mutant Seipin expression and protein aggregation. These results suggest that induction of autophagy pathway is involved in the cellular response to mutant Seipin in seipinopathy and that motoneuron loss is a key pathogenic process underlying the development of locomotor abnormalities.
Asunto(s)
Subunidades gamma de la Proteína de Unión al GTP/genética , Proteínas de Unión al GTP Heterotriméricas/genética , Enfermedad de la Neurona Motora/genética , Neuronas Motoras/metabolismo , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Aparato de Golgi/patología , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Enfermedad de la Neurona Motora/metabolismo , Enfermedad de la Neurona Motora/patología , Mutación Missense , Degeneración Nerviosa , Médula Espinal/metabolismoRESUMEN
AIMS/HYPOTHESIS: While chronic low-grade inflammation is associated with obesity, acute inflammation reduces food intake and leads to negative energy balance. Although both types of inflammation activate nuclear factor κB (NF-κB) signalling, it remains unclear how NF-κB activation results in opposite physiological responses in the two types of inflammation. The goal of this study was to address this question, and to understand the link between inflammation and leptin signalling. METHODS: We studied the ability of NF-κB to modulate Pomc transcription, and how it impinges on signal transducer and activator of transcription 3 (STAT3)-mediated leptin signalling by using a combination of animal models, biochemical assays and molecular biology. RESULTS: We report that suppression of food intake and physical movement with acute inflammation is not dependent on STAT3 activation in pro-opiomelanocortin (POMC) neurons. Under these conditions, activated NF-κB independently leads to increased Pomc transcription. Electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) experiments reveal that NF-κB v-rel reticuloendotheliosis viral oncogene homologue A (avian) (RELA [also known as p65]) binds to the Pomc promoter region between -138 and -88 bp, which also harbours the trans-acting transcription factor 1 (SP1) binding site. We found significant changes in the methylation pattern at this region and reduced Pomc activation under chronic inflammation induced by a high-fat diet. Furthermore, RELA is unable to bind and activate transcription when the Pomc promoter is methylated. Finally, RELA binds to STAT3 and inhibits STAT3-mediated promoter activity, suggesting that RELA, possibly together with forkhead box-containing protein 1 (FOXO1), may prevent STAT3-mediated leptin activation of the Pomc promoter. CONCLUSIONS/INTERPRETATION: Our study provides a mechanism for the involvement of RELA in the divergent regulation of energy homeostasis in acute and chronic inflammation.
Asunto(s)
Ingestión de Alimentos , Regulación de la Expresión Génica , FN-kappa B/metabolismo , Proopiomelanocortina/genética , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Conducta Animal , Conducta Alimentaria , Inflamación , Leptina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Factor de Transcripción STAT3/metabolismo , Transducción de SeñalRESUMEN
AIMS/HYPOTHESIS: Glucagon-like peptide-1 (GLP-1), a hormone with potent antihyperglycaemic effects, is produced and secreted from highly specialised gut endocrine L-cells. It regulates glucose homeostasis by promoting glucose-dependent insulin secretion, suppressing glucagon secretion and enhancing insulin sensitivity. Similar to islet alpha and beta cells, L-cells are electrically excitable, and express calcium channels and ATP-sensitive potassium channels. GLP-1 is also stored in secretory granules, the exocytosis of which is triggered by increased intracellular calcium levels. Although the calcium dependence of GLP-1 granule exocytosis is well established, the identities of calcium-sensing proteins in GLP-1 secretion remain elusive. Here we tested whether synaptotagmin-7, a calcium sensor in pancreatic alpha and beta cells, regulates GLP-1 secretion. METHODS: We studied GLP-1 secretion using synaptotagmin-7 knockout (KO) mice and GLUTag cells with lentiviral-mediated synaptotagmin-7 silencing. RESULTS: We found that synaptotagmin-7 was co-localised with GLP-1 in intestinal L-cells. GLP-1 secretion was impaired in synaptotagmin-7 KO mice when they were challenged by glucose ingestion. Lentiviral knockdown (KD) of synaptotagmin-7 in GLUTag cells led to similar reductions in GLP-1 secretion, as determined by biochemical assays and by membrane capacitance measurements. Calcium response was not altered in synaptotagmin-7 KD cells. CONCLUSIONS/INTERPRETATION: These results demonstrate that synaptotagmin-7 functions as a positive regulator of GLP-1 secretion in intestinal L-cells and GLUTag cells, consistent with its proposed role as a calcium sensor in GLP-1 secretion.
Asunto(s)
Péptido 1 Similar al Glucagón/metabolismo , Intestinos/citología , Sinaptotagminas/metabolismo , Animales , Western Blotting , Células Cultivadas , Electrofisiología , Inmunohistoquímica , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa , ARN Interferente Pequeño/genética , Sinaptotagminas/genéticaRESUMEN
AIMS/HYPOTHESIS: Type 2 diabetes is caused by relative deficiency of insulin secretion and is associated with dysregulation of glucagon secretion during the late stage of diabetes development. Like insulin secretion from beta cells, glucagon secretion is dependent on calcium signals and a calcium sensing protein, synaptotagmin-7. In this study, we tested the relative contribution of dysregulated glucagon secretion and reduced insulin release in the development of hyperglycaemia and type 2 diabetes by using synaptotagmin-7 knockout (KO) mice, which exhibit glucose intolerance, reduced insulin secretion and nearly abolished Ca(2+)-stimulated glucagon secretion. METHODS: We fed the synaptotagmin-7 KO and control mice with a high-fat diet (HFD) for 14 weeks, and compared their body weight, glucose levels, glucose and insulin tolerance, and insulin and glucagon secretion. RESULTS: On the HFD, synaptotagmin-7 KO mice showed progressive impairment of glucose tolerance and insulin secretion, along with continued maintenance of a low glucagon level. The control mice were less affected in terms of glucose intolerance, and showed enhanced insulin secretion with a concurrent increase in glucagon levels. Unexpectedly, after 14 weeks of HFD feeding, only the control mice displayed resting hyperglycaemia, whereas in synaptotagmin-7 KO mice defective insulin secretion and reduced insulin sensitivity were not sufficient to cause hyperglycaemia in the absence of enhanced glucagon secretion. CONCLUSIONS/INTERPRETATION: Our data uncover a previously overlooked role of dysregulated glucagon secretion in promoting hyperglycaemia and the ensuing diabetes, and strongly suggest maintenance of adequate regulation of glucagon secretion as an important therapeutic target in addition to the preservation of beta cell function and mass in the prevention and treatment of diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Glucagón/metabolismo , Hiperglucemia/metabolismo , Sinaptotagminas/metabolismo , Animales , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/genética , Grasas de la Dieta , Glucógeno/metabolismo , Hiperglucemia/sangre , Hiperglucemia/genética , Masculino , Ratones , Ratones Noqueados , Sinaptotagminas/genéticaRESUMEN
John Newsom-Davis played a crucial role in supporting areas of scientific exploration beyond his own research interests. In particular, he was one of the key players in establishing human neuroimaging in Oxford. Here, we celebrate the role that he played in this endeavour, both in the early days of pulling together funding, and solving practical challenges, and in the following years, when we all appreciated his ongoing encouragement and support.
Asunto(s)
Academias e Institutos , Investigación Biomédica/historia , Investigación Biomédica/organización & administración , Diagnóstico por Imagen , Neuroinmunomodulación , Academias e Institutos/historia , Academias e Institutos/organización & administración , Diagnóstico por Imagen/historia , Diagnóstico por Imagen/métodos , Inglaterra , Historia del Siglo XX , Historia del Siglo XXI , Humanos , FotograbarRESUMEN
The physiological role of mitochondrial uncoupling proteins (UCPs) in heart and skeletal muscle is unknown, as is whether mitochondrial uncoupling of oxidative phosphorylation by fatty acids occurs in vivo. In this study, we found that UCP2 and UCP3 protein content, determined using Western blotting, was increased by 32 and 48%, respectively, in hyperthyroid rat heart mitochondria. Oligomycin-insensitive respiration rate, a measure of mitochondrial uncoupling, was increased in all mitochondria in the presence of palmitate: 36% in controls and 71 and 100% with 0.8 and 0.9 mM palmitate, respectively, in hyperthyroid rat heart mitochondria. In the isolated working heart, 0.4 mM palmitate significantly lowered cardiac output by 36% and cardiac efficiency by 38% in the hyperthyroid rat heart. Thus increased mitochondrial UCPs in the hyperthyroid rat heart were associated with increased uncoupling and decreased myocardial efficiency in the presence of palmitate. In conclusion, a physiological effect of UCPs on fatty acid oxidation has been found in heart at the mitochondrial and whole organ level.
Asunto(s)
Proteínas Portadoras/metabolismo , Hipertiroidismo/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Proteínas Mitocondriales , Miocardio/metabolismo , Palmitatos/farmacología , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Respiración de la Célula/efectos de los fármacos , Respiración de la Célula/fisiología , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/fisiología , Canales Iónicos , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Contracción Miocárdica/fisiología , Consumo de Oxígeno/efectos de los fármacos , Consumo de Oxígeno/fisiología , Proteínas/metabolismo , Ratas , Ratas Wistar , Triyodotironina/farmacología , Proteína Desacopladora 1 , Proteína Desacopladora 2 , Proteína Desacopladora 3RESUMEN
To determine whether the effects of fatty acids on the diabetic heart during ischemia involve altered glycolytic ATP and proton production, we measured energetics and intracellular pH (pH(i)) by using (31)P NMR spectroscopy plus [2-(3)H]glucose uptake in isolated rat hearts. Hearts from 7-wk streptozotocin diabetic and control rats, perfused with buffer containing 11 mM glucose, with or without 1.2 mM palmitate or the ketone bodies, 4 mM beta-hydroxybutyrate plus 1 mM acetoacetate, were subjected to 32 min of low-flow (0.3 ml x g wet wt(-1) x min(-1)) ischemia, followed by 32 min of reperfusion. In control rat hearts, neither palmitate nor ketone bodies altered the recovery of contractile function. Diabetic rat hearts perfused with glucose alone or with ketone bodies, had functional recoveries 50% lower than those of the control hearts, but palmitate restored recovery to control levels. In a parallel group with the functional recoveries, palmitate prevented the 54% faster loss of ATP in the diabetic, glucose-perfused rat hearts during ischemia, but had no effect on the rate of ATP depletion in control hearts. Palmitate decreased total glucose uptake in control rat hearts during low-flow ischemia, from 106 +/- 17 to 52 +/- 12 micromol/g wet wt, but did not alter the total glucose uptake in the diabetic rat hearts, which was 42 +/- 5 micromol/g wet wt. Recovery of contractile function was unrelated to pH(i) during ischemia; the glucose-perfused control and palmitate-perfused diabetic hearts had end-ischemic pH(i) values that were significantly different at 6.36 +/- 0.04 and 6.60 +/- 0.02, respectively, but had similar functional recoveries, whereas the glucose-perfused diabetic hearts had significantly lower functional recoveries, but their pH(i) was 6.49 +/- 0.04. We conclude that fatty acids, but not ketone bodies, protect the diabetic heart by decreasing ATP depletion, with neither having detrimental effects on the normal rat heart during low-flow ischemia.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Ácidos Grasos no Esterificados/farmacología , Cuerpos Cetónicos/farmacología , Isquemia Miocárdica/metabolismo , Animales , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/fisiología , Glucosa/farmacocinética , Hipoglucemiantes/farmacología , Técnicas In Vitro , Insulina/farmacología , Espectroscopía de Resonancia Magnética , Masculino , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/fisiología , Palmitatos/farmacología , Isótopos de Fósforo , Ratas , Ratas WistarRESUMEN
The vascular system exhibits altered growth, calcium responses and metabolism during hypertension. To relate such changes, we compared histological, tension and metabolic responses in the aorta from 32-week-old spontaneously hypertensive rats (SHRs), normotensive Wistar-Kyoto (WKY) rats, and SHRs treated with Verapamil (V) and ACE-inhibitor, Trandolapril (T) as well as a combination of the two treatments (C). Vascular hypertrophy was apparent in the SHRs. Contractile responses induced by 50 mmol/1 KCl and 2.5 mmol/1 Ca2+ were significantly lower in the SHR (64.4 mN/mm2 vs. 49.2 mN/mm2), but an associated increase in Ca2+ -sensitivity (EC50 of extracellular Ca2+ (mumol/1): SHR, 456 vs. WKY, 616) normalised tension generating ability. All treatments led to significant decreases in blood pressure, although only T and C treated animals became normotensive with concomitant normalisation of vascular hypertrophy. An increase in oxygen consumption was apparent in the SHR aorta, which was associated with significant differences in the activities of key metabolic enzymes. Anti-hypertensive treatment normalised many of the metabolic parameters, with the C therapy being the most efficacious. We conclude that the treatment of hypertension by combined therapy leads to a better normalisation of structural, contractile, and metabolic parameters in the SHR, than either treatment alone and that metabolic changes with the pathology are resolved with appropriate therapy.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Antihipertensivos/farmacología , Aorta/efectos de los fármacos , Indoles/farmacología , Músculo Liso Vascular/efectos de los fármacos , Vasodilatadores/farmacología , Verapamilo/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Antihipertensivos/uso terapéutico , Aorta/metabolismo , Aorta/fisiopatología , Hipertensión/tratamiento farmacológico , Hipertensión/metabolismo , Hipertensión/fisiopatología , Indoles/uso terapéutico , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Vasodilatadores/uso terapéutico , Verapamilo/uso terapéuticoRESUMEN
A decrease in the apparent diffusion coefficient (ADC) of water is important in the detection of acute brain disorders, yet it is unknown whether changes in myocardial ADCs hold similar potential. Consequently, in this study a STEAM pulse sequence was modified in order to measure the ADCs of water and the (1)H-NMR detectable metabolites, taurine (an inert marker) and creatine, during perfusion, ischemia, and reperfusion in the isolated rat heart. At the short diffusion time of 50 ms, myocardial ADCs were (1.06 +/- 0. 07) x 10(-3) mm(2)/s for water, (0.29 +/- 0.01) x 10(-3) mm(2)/s for taurine and (0.26 +/- 0.01) x 10(-3) mm(2)/s for creatine. Heart water and taurine ADCs remained constant during ischemia, yet the total creatine ADC increased by 35% owing to the hydrolysis of PCr to creatine. The average cardiomyocyte diameter, calculated from taurine ADC values measured at diffusion times between 50 ms and 1510 ms, was 40 microm in the perfused heart and 27 microm by the end of ischemia. It is concluded that the taurine ADC measured at short diffusion times does not reveal ischemic injury in the heart, but at long diffusion times may be used to calculate changes in myocyte diameter. Magn Reson Med 44:208-214, 2000.
Asunto(s)
Creatinina/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Isquemia Miocárdica/metabolismo , Taurina/metabolismo , Agua/metabolismo , Análisis de Varianza , Animales , Difusión , Masculino , Miocardio/citología , Ratas , Ratas WistarAsunto(s)
Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Animales , Estudios de Casos y Controles , Proteínas del Citoesqueleto/genética , Distrofina/deficiencia , Distrofina/genética , Metabolismo Energético , Glucosa/metabolismo , Cardiopatías/etiología , Humanos , Técnicas In Vitro , Insulina/farmacología , Espectroscopía de Resonancia Magnética , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos mdx , Ratones Transgénicos , Distrofia Muscular Animal/complicaciones , Distrofia Muscular de Duchenne/complicaciones , Daño por Reperfusión Miocárdica/complicaciones , Daño por Reperfusión Miocárdica/metabolismo , Miocardio/metabolismo , Utrofina , Función Ventricular IzquierdaRESUMEN
Just before I became an editor of Biochemical and Biophysical Research Communications in 1977 we published our first paper in this same journal on the study of tiny perfused rat hearts by (31)P NMR. In this article I trace the development of this in vivo NMR approach from the study of small rat and mouse hearts to human investigations. With the advent of molecular genetics the mouse became a key model organism for understanding and characterizing the function of human genes. I illustrate this by some of our recent work on Duchenne and Becker muscular dystrophy where the in vivo biochemical abnormalities observed in the human can be better understood from investigations of the muscle and heart of the murine model for muscular dystrophy, the mdx mouse. In particular, the mdx mouse heart exhibits ECG (conduction) abnormalities similar to that in the human which we associate with the reduction of the neuronal nitric oxide synthase activity compared to controls. We have also demonstrated in the mouse model that the increased sensitivity of the heart to ischemia is associated with a decrease in the insulin-stimulated glucose transport. Imaging techniques involving NMR, visible light, and others will play an increasingly important role in linking genomics to functional "molecular physiology."
Asunto(s)
Genoma Humano , Miocardio/química , Animales , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Ratones Endogámicos mdxRESUMEN
Duchenne muscular dystrophy is a devastating neuromuscular disease caused by lack of the protein, dystrophin, in skeletal muscle and heart, although the biochemical mechanism by which dystrophin loss causes muscle dysfunction is unknown. Here we show that the dystrophin-deficient mdx mouse and a mouse lacking both dystrophin and the dystrophin-related protein, utrophin (dko), have abnormal electrocardiograms (ECGs). In skeletal muscle, dystrophin is normally associated with neuronal nitric oxide synthase (nNOS) at the sarcolemma. Consequently, we have measured NOS isoform activities in hearts from control, mdx and dko mice. In control mouse hearts, eNOS and nNOS activities increased by 120% and 47%, respectively, between 2 and 6 months of age. In mdx mice, myocardial nNOS activity was decreased by 60%, 84% and 80% at 2, 6 and 12 months of age, respectively. Similarly, hearts from dko mice showed a 65% decrease in nNOS activity compared to controls at 2 months of age. Endothelial NOS (eNOS) activity was not affected by dystrophin loss, but inducible NOS (iNOS) activity was seven-fold higher than control in the mdx mouse heart by 12 months of age. We conclude that lack of dystrophin in the mdx mouse results in abnormal ECGs that are associated with decreased myocardial nNOS and increased iNOS activities.
Asunto(s)
Electrocardiografía , Distrofia Muscular Animal/fisiopatología , Distrofia Muscular de Duchenne/enzimología , Distrofia Muscular de Duchenne/fisiopatología , Óxido Nítrico Sintasa/metabolismo , Envejecimiento , Animales , Peso Corporal , Proteínas del Citoesqueleto/genética , Distrofina/genética , Corazón/crecimiento & desarrollo , Corazón/fisiopatología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Endogámicos mdx , Distrofia Muscular Animal/enzimología , Distrofia Muscular Animal/genética , Óxido Nítrico Sintasa de Tipo I , Óxido Nítrico Sintasa de Tipo II , Tamaño de los Órganos , UtrofinaRESUMEN
We investigated the time course of genesis of skeletal muscle dysfunction and sympatho-vagal imbalance after myocardial infarction. We studied 22 normal controls, 22 patients with >6 months stable chronic heart failure and 10 patients after a first massive myocardial infarction at 1-3 weeks (the "early" period), 6-8 weeks ("mid") and 6-9 months ("late") following their infarct. Four patients developed overt heart failure. Forearm muscle metabolism was studied using (31)P magnetic resonance spectroscopy (MRS). Sympatho-vagal balance was assessed by heart rate variability and radiolabelled norepinephrine kinetics. Increased norepinephrine spillover (0.55+/-0.02 v 0.27+/-0.04 mg/min/m(2); P<0.01) and decreased heart rate variability were confined to those post-myocardial infarction patients who subsequently developed heart failure. Resting cardiac output was normal in all the post-myocardial infarction patients, although the response of cardiac output to supine bicycle exercise at the "mid" study point was less in the group who subsequently developed heart failure (9+/-1 v 41+/-8 %; P<0.005). In the MRS studies, there were no detectable differences between those who did or did not develop heart failure. The initial rate of ATP turnover, calculated from initial-exercise changes in pH and phosphocreatine (PCr), was increased in established chronic heart failure, but in the post-myocardial infarction patients a numerically similar increase reached statistical significance only in the early group (19+/-3 v 11+/-1 mM/min; P<0.005). The apparent maximum rate of oxidative ATP synthesis, calculated from post-exercise PCr recovery kinetics, was lower than control in the late post-myocardial infarction and established chronic heart failure groups 34+/-5 v 55+/-4 mM/min; P<0.03 and 38+/-3 v 55+/-4 mM/min; P<0.003, respectively). Skeletal muscle metabolism and autonomic function become abnormal after an extensive myocardial infarction. While skeletal muscle abnormalities are relatively slow to develop and unrelated to the degree of failure, excessive neurohormonal activation and impaired cardiac output response to exercise seem from an early stage to characterize patients who subsequently develop chronic heart failure.
Asunto(s)
Sistema Nervioso Autónomo/fisiopatología , Hemodinámica/fisiología , Músculo Esquelético/metabolismo , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Adulto , Anciano , Enfermedad Crónica , Metabolismo Energético , Prueba de Esfuerzo , Femenino , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Frecuencia Cardíaca , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Persona de Mediana Edad , Norepinefrina/sangre , Consumo de Oxígeno , Valores de Referencia , Factores de TiempoRESUMEN
Extracellular lactate concentration rises following ischaemic stroke in both the infarcted area and in the surrounding ischaemic penumbra. We investigated the effect of lactate accumulation on glucose metabolism in cortical slices from guinea pigs initially by varying superfusion medium to tissue volumes. Stable intracellular K+ concentrations indicated that a decrease in media/ tissue volume did not impair viability of the tissue, but 13C NMR demonstrated that lactate accumulation in the superfusion medium reduced glucose oxidation with inhibition of glial metabolism via pyruvate carboxylase. The concentration of lactate which had accumulated when significant inhibition was observed was approximately 0.85 mM. In independent experiments we found that superfusion of brain slices with lactate at this concentration (even using a 'high-volume' of superfusion fluid) decreased oxygen consumption by 40 +/- 3%. K(-)-induced depolarisation partially reversed this effect. These results suggest that even low extracellular lactate concentrations may depress metabolic rates in inactive and poorly perfused brain tissue in vivo through inhibition of glial metabolism of glucose.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Glucosa/antagonistas & inhibidores , Ácido Láctico/farmacología , Animales , Transporte Biológico , Isótopos de Carbono , Corteza Cerebral/enzimología , Corteza Cerebral/metabolismo , Glucosa/metabolismo , Cobayas , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Oxígeno/metabolismo , Piruvato Carboxilasa/metabolismoRESUMEN
Glycogen in glial cells is the largest store of glucose equivalents in the brain. Here we describe evidence that brain glycogen contributes to aerobic energy metabolism of the guinea pig brain in vivo. Five min after an intra-arterial bolus injection of d-[U-14C]glucose, 28+/-11% of the radioactivity in brain tissue was associated with the glycogen fraction, indicating that a significant proportion of labelled glucose taken up by the brain is converted to glycogen shortly after bolus infusion. Incorporation of 13C-label into lactate generated by brains made ischaemic after d-[1-13C]glucose injection confirms that these glucose equivalents can be mobilised for anaerobic glucose metabolism. Aerobic metabolism was monitored by following the time course of 13C-incorporation into glutamate in guinea pig cortex and cerebellum in vivo. After an intra-arterial bolus injection of d-[1-13C]glucose, glutamate labelling reached a maximum 40-60 min after injection, suggesting that a slowly metabolised pool of labelled glucose equivalents was present. As the concentration of 13C-labelled glucose in blood was shown to decrease below detectable levels within 5 min of bolus injection, this late phase of glutamate labelling must occur with mobilisation of a brain storage pool of labelled glucose equivalents. We interpret this as evidence that glucose equivalents in glycogen may contribute to energy metabolism in the aerobic guinea pig brain.
Asunto(s)
Encéfalo/metabolismo , Glucosa/farmacología , Ácido Glutámico/metabolismo , Glucógeno/metabolismo , Aerobiosis , Animales , Isótopos de Carbono , Cerebelo/metabolismo , Corteza Cerebral/metabolismo , Glucosa/química , Glucosa/metabolismo , Ácido Glutámico/química , Glutamina/metabolismo , Cobayas , Infusiones Intraarteriales , Ácido Láctico/metabolismo , Espectroscopía de Resonancia Magnética , Factores de TiempoRESUMEN
The increased use of creatine by athletes as a dietary supplement to improve their physical performance assumes that increased serum creatine levels will increase intracellular skeletal muscle creatine. Despite this common assumption, skeletal muscle creatine uptake awaits full characterization. Consequently, we have investigated 14C-labelled creatine uptake in isolated, incubated rat soleus (type I) muscle preparations at 37 degrees C. We found that the apparent Km for creatine uptake was 73 microM and the Vmax was 77 nmol h-1 gww-1. Creatine uptake was 82% inhibited by 2 mM beta-guanidinopropionic acid, the structural analogue of creatine. In addition, a decrease in buffer Na+ concentration, from 145 to 25 mM, reduced the rate of 14C-labelled creatine uptake by 77%, indicating that uptake is largely Na+-dependent in soleus muscle. Insulin had no effect on the rate of creatine uptake in vitro. The total creatine content was 34% lower, but the rate of creatine uptake in the presence of 100 microM extracellular creatine was 45% higher, in soleus than in extensor digitorum longus (type II) muscle. However, at 1 mM extracellular creatine, the maximal rate of uptake was not significantly different for the two muscle types, implying that soleus muscle has a lower Km for creatine uptake. We suggest that intracellular creatine levels may play a role in the regulation of skeletal muscle creatine uptake.
Asunto(s)
Creatina/farmacocinética , Insulina/farmacología , Músculo Esquelético/metabolismo , Sodio/farmacología , Animales , Radioisótopos de Carbono , Guanidinas/farmacología , Técnicas In Vitro , Masculino , Músculo Esquelético/efectos de los fármacos , Propionatos/farmacología , Ratas , Ratas WistarRESUMEN
To determine the contribution of changes in extracellular osmolarity to ischemic injury, isolated guinea pig hearts were perfused with hyposmotic (220 mosM) or hyperosmotic (380 mosM) buffer. 31P NMR spectroscopy was used to follow changes in intracellular pH (pHi) and energetics. Hyposmotic buffer decreased myocardial developed pressure by 30 +/- 2% and pHi by 0.02 +/- 0.01 unit, whereas hyperosmotic buffer increased myocardial developed pressure by 34 +/- 1% and pHi by 0.14 +/- 0.01 unit. All hearts recovered to control values on restoration of isosmotic (300 mosM) buffer. The hyperosmolar-induced intracellular alkalosis and developed pressure increase were not prevented by inhibition of Na+/H+ exchange with use of 1 microM HOE-642 but were abolished with use of bicarbonate-free buffers. After 20 min of total global ischemia, hearts perfused with hyposmotic buffer showed significantly greater recoveries of developed pressure, phosphocreatine, and ATP than control hearts, but hearts perfused with hyperosmotic buffer did not recover after ischemia. In conclusion, buffer osmolarities between 220 and 380 mosM alter myocardial pHi and developed pressure but are not deleterious during perfusion. However, buffer osmolarity significantly alters the extent of myocardial ischemic injury.
Asunto(s)
Hidrógeno/metabolismo , Membranas Intracelulares/metabolismo , Contracción Miocárdica/fisiología , Isquemia Miocárdica/patología , Isquemia Miocárdica/fisiopatología , Miocardio/metabolismo , Animales , Bicarbonatos/farmacología , Tampones (Química) , Proteínas Portadoras/metabolismo , Femenino , Cobayas , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Transportadores de Ácidos Monocarboxílicos , Miocardio/patología , Presión Osmótica , Perfusión , Valores de Referencia , Sodio/farmacología , Intercambiadores de Sodio-Hidrógeno/metabolismo , Agua/metabolismoRESUMEN
Myocardial pH has been shown in animal models to be a sensitive indicator of ischemia. In vivo measurement in humans using 31p magnetic resonance spectroscopy is complicated by the overlap of blood 2,3-diphosphoglycerate peaks with the P(i) peak used for pH measurement. A "saturation transfer" method combined with spatial presaturation of skeletal muscle signal is presented which can obtain spectra from the heart free of contamination of 2,3-DPG signal in which intracellular P(i) resonance can be clearly observed. Application to a group of six normal subjects found that the chemical shift of the intracellular inorganic phosphate peak was 4.95+/-0.06 relative to the phosphocreatine peak. This is equivalent to a pH of 7.11+/-0.05.
Asunto(s)
Espectroscopía de Resonancia Magnética , Miocardio/metabolismo , Adenosina Trifosfato/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Líquido Intracelular/metabolismo , Músculo Esquelético/metabolismo , Fantasmas de Imagen , Fosfatos/metabolismo , Fosfocreatina/metabolismo , Isótopos de Fósforo , Tórax/metabolismoRESUMEN
Excitatory amino acids are an important cause of cell death in the hypoxic and ischaemic brain. Neuronal glutamate stores are depleted rapidly in hypoxia, but alanine production rises under such conditions and has been suggested to be a potential precursor of glutamate. To test this hypothesis, we have investigated amino acid metabolism using 13C NMR with superfused guinea pig cortical slices subjected to varying degrees of hypoxia. During severe hypoxia, brain slices metabolising 5 mM [2-(13)C]pyruvate exported [2-(13)C]alanine into the superfusion fluid. The metabolic fate of alanine during normoxia and hypoxia was tested by superfusion of brain slices with 10 mM glucose and 2 mM [2-(13)C,15N]alanine. Metabolism of exogenous alanine leads to the release of aspartate into the superfusion fluid. The pattern of labelling of aspartate indicated that it was synthesised via the glial-specific enzyme pyruvate carboxylase. 13C-labelled glutamate was produced with both normoxia and hypoxia, but concentrations were 30-fold lower than for labelled aspartate. Thus, although substantial amounts of glutamate are not synthesised from alanine in hypoxia, there is significant production of aspartate, which also may have deleterious effects as an excitatory amino acid.