RESUMEN
Loss of retinal ganglion cells (RGCs) is a leading cause of blinding conditions. The purpose of this study was to evaluate the effect of extracellular l-lactate on RGC survival facilitated through lactate metabolism and ATP production. We identified lactate as a preferred energy substrate over glucose in murine RGCs and showed that lactate metabolism and consequently increased ATP production are crucial components in promoting RGC survival during energetic crisis. Lactate was released to the extracellular environment in the presence of glucose and detained intracellularly during glucose deprivation. Lactate uptake and metabolism was unaltered in the presence and absence of glucose. However, the ATP production declined significantly for 24â¯h of glucose deprivation and increased significantly in the presence of lactate. Finally, lactate exposure for 2 and 24â¯h resulted in increased RGC survival during glucose deprivation. In conclusion, the metabolic pathway of lactate in RGCs may be of great future interest to unravel potential pharmaceutical targets, ultimately leading to novel therapies in the prevention of blinding neurodegenerative diseases, for example, glaucoma.
Asunto(s)
Adenosina Trifosfato/biosíntesis , Células Ependimogliales/efectos de los fármacos , Glucosa/farmacología , Ácido Láctico/farmacología , Células Ganglionares de la Retina/efectos de los fármacos , Animales , Animales Recién Nacidos , Transporte Biológico , Supervivencia Celular/efectos de los fármacos , Células Ependimogliales/citología , Células Ependimogliales/metabolismo , Glucosa/deficiencia , Ácido Láctico/biosíntesis , Ratones , Ratones Endogámicos C57BL , Cultivo Primario de Células , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
Müller cells are pivotal in sustaining retinal ganglion cells, and an intact energy metabolism is essential for upholding Müller cell functions. The present study aimed to investigate the impact of lactate on Müller cell survival and function. Primary mice Müller cells and human Müller cell lines (MIO-M1) were treated with or without lactate (10 or 20 mM) for 2 and 24 hours. Simultaneously, Müller cells were incubated with or without 6 mM of glucose. L-lactate exposure increased Müller cell survival independently of the presence of glucose. This effect was abolished by the addition of the monocarboxylate inhibitor 4-cinnamic acid to the treatment media, whereas survival continued to increase in response to addition of D-lactate during glucose restriction. ATP levels decreased over time in MIO-M1 cells and remained stable over time in primary Müller cells. Lactate was preferably metabolized in MIO-M1 cells compared to glucose, and 10 mM of L-Lactate exposure prevented complete glycogen depletion in MIO-M1 cells. Glutamate uptake increased after 2 hours and decreased after 24 hours in glucose-restricted Müller cells compared to cells with glucose supplement. The addition of 10 mM of lactate to the treatment media increased glutamate uptake in glucose supplemented and restricted cells. In conclusion, lactate is a key component in maintaining Müller cell survival and function. Hence, lactate administration may be of great future interest, ultimately leading to novel therapies to rescue retinal ganglion cells.
Asunto(s)
Células Ependimogliales/citología , Células Ependimogliales/metabolismo , Ácido Láctico/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Glucosa/metabolismo , Ácido Glutámico/metabolismo , Glucógeno/metabolismo , Ratones Endogámicos C57BL , Transportadores de Ácidos Monocarboxílicos/metabolismo , Factores de TiempoRESUMEN
A key enzyme in brain glutamate homeostasis is glutamate dehydrogenase (GDH) which links carbohydrate and amino acid metabolism mediating glutamate degradation to CO2 and expanding tricarboxylic acid (TCA) cycle capacity with intermediates, i.e. anaplerosis. Humans express two GDH isoforms, GDH1 and 2, whereas most other mammals express only GDH1. hGDH1 is widely expressed in human brain while hGDH2 is confined to astrocytes. The two isoforms display different enzymatic properties and the nature of these supports that hGDH2 expression in astrocytes potentially increases glutamate oxidation and supports the TCA cycle during energy-demanding processes such as high intensity glutamatergic signaling. However, little is known about how expression of hGDH2 affects the handling of glutamate and TCA cycle metabolism in astrocytes. Therefore, we cultured astrocytes from cerebral cortical tissue of hGDH2-expressing transgenic mice. We measured glutamate uptake and metabolism using [3 H]glutamate, while the effect on metabolic pathways of glutamate and glucose was evaluated by use of 13 C and 14 C substrates and analysis by mass spectrometry and determination of radioactively labeled metabolites including CO2 , respectively. We conclude that hGDH2 expression increases capacity for uptake and oxidative metabolism of glutamate, particularly during increased workload and aglycemia. Additionally, hGDH2 expression increased utilization of branched-chain amino acids (BCAA) during aglycemia and caused a general decrease in oxidative glucose metabolism. We speculate, that expression of hGDH2 allows astrocytes to spare glucose and utilize BCAAs during substrate shortages. These findings support the proposed role of hGDH2 in astrocytes as an important fail-safe during situations of intense glutamatergic activity. GLIA 2017;65:474-488.
Asunto(s)
Astrocitos/metabolismo , Ciclo del Ácido Cítrico/fisiología , Regulación Enzimológica de la Expresión Génica , Glucosa/deficiencia , Glutamato Deshidrogenasa/metabolismo , Ácido Glutámico/metabolismo , Animales , Astrocitos/efectos de los fármacos , Dióxido de Carbono/farmacocinética , Isótopos de Carbono/farmacocinética , Células Cultivadas , Corteza Cerebral/citología , Ciclo del Ácido Cítrico/efectos de los fármacos , Ciclo del Ácido Cítrico/genética , Relación Dosis-Respuesta a Droga , Proteína Ácida Fibrilar de la Glía/metabolismo , Glutamato Deshidrogenasa/genética , Ácido Glutámico/farmacología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Tritio/farmacocinéticaRESUMEN
In retina, like in brain, lactate equilibrates across cell membranes via monocarboxylate transporters and in the extracellular space by diffusion, forming a basis for the action of lactate as a transmitter of metabolic signals. In the present paper, we argue that the lactate receptor GPR81, also known as HCAR1, may contribute importantly to the control of retinal cell functions in health and disease. GPR81, a G-protein coupled receptor, is known to downregulate cAMP both in adipose and nervous tissue. The receptor also acts through other down-stream mechanisms to control functions, such as excitability, metabolism and inflammation. Recent publications predict effects of the lactate receptor on neurodegeneration. Neurodegenerative diseases in retina, where the retinal ganglion cells die, notably glaucoma and diabetic retinopathy, may be linked to disturbed lactate homeostasis. Pilot studies reveal high GPR81 mRNA in retina and indicate GPR81 localization in Müller cells and retinal ganglion cells. Moreover, monocarboxylate transporters are expressed in retinal cells. We envision that lactate receptors and transporters could be useful future targets of novel therapeutic strategies to protect neurons and prevent or counteract glaucoma as well as other retinal diseases.
Asunto(s)
Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Retina/fisiología , Enfermedades de la Retina/metabolismo , Células Ganglionares de la Retina/metabolismo , Animales , Humanos , Retina/patología , Enfermedades de la Retina/patología , Células Ganglionares de la Retina/patologíaRESUMEN
Glucose, the main energy substrate used in the CNS, is continuously supplied by the periphery. Glutamate, the major excitatory neurotransmitter, is foreseen as a complementary energy contributor in the brain. In particular, astrocytes actively take up glutamate and may use it through oxidative glutamate dehydrogenase (GDH) activity. Here, we investigated the significance of glutamate as energy substrate for the brain. Upon glutamate exposure, astrocytes generated ATP in a GDH-dependent way. The observed lack of glutamate oxidation in brain-specific GDH null CnsGlud1(-/-) mice resulted in a central energy-deprivation state with increased ADP/ATP ratios and phospho-AMPK in the hypothalamus. This induced changes in the autonomous nervous system balance, with increased sympathetic activity promoting hepatic glucose production and mobilization of substrates reshaping peripheral energy stores. Our data reveal the importance of glutamate as necessary energy substrate for the brain and the role of central GDH in the regulation of whole-body energy homeostasis.
Asunto(s)
Metabolismo Energético , Ácido Glutámico/metabolismo , Hipotálamo/metabolismo , Receptores de Glutamato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Astrocitos/metabolismo , Células Cultivadas , Glucosa/metabolismo , Glutamato Deshidrogenasa , Hipotálamo/citología , Hígado/metabolismo , Masculino , Ratones , Oxidación-Reducción , Receptores de Glutamato/genéticaRESUMEN
Astrocytes take up glutamate in the synaptic area subsequent to glutamatergic transmission by the aid of high affinity glutamate transporters. Glutamate is converted to glutamine or metabolized to support intermediary metabolism and energy production. Glutamate dehydrogenase (GDH) and aspartate aminotransferase (AAT) catalyze the reversible reaction between glutamate and α-ketoglutarate, which is the initial step for glutamate to enter TCA cycle metabolism. In contrast to GDH, AAT requires a concomitant interconversion of oxaloacetate and aspartate. We have investigated the role of GDH in astrocyte glutamate and glucose metabolism employing siRNA mediated knock down (KD) of GDH in cultured astrocytes using stable and radioactive isotopes for metabolic mapping. An increased level of aspartate was observed upon exposure to [U-(13) C]glutamate in astrocytes exhibiting reduced GDH activity. (13) C Labeling of aspartate and TCA cycle intermediates confirmed that the increased amount of aspartate is associated with elevated TCA cycle flux from α-ketoglutarate to oxaloacetate, i.e. truncated TCA cycle. (13) C Glucose metabolism was elevated in GDH deficient astrocytes as observed by increased de novo synthesis of aspartate via pyruvate carboxylation. In the absence of glucose, lactate production from glutamate via malic enzyme was lower in GDH deficient astrocytes. In conclusions, our studies reveal that metabolism via GDH serves an important anaplerotic role by adding net carbon to the TCA cycle. A reduction in GDH activity seems to cause the astrocytes to up-regulate activity in pathways involved in maintaining the amount of TCA cycle intermediates such as pyruvate carboxylation as well as utilization of alternate substrates such as branched chain amino acids.
Asunto(s)
Astrocitos/enzimología , Ciclo del Ácido Cítrico/fisiología , Glutamato Deshidrogenasa/deficiencia , Animales , Aspartato Aminotransferasas/metabolismo , Ácido Aspártico/metabolismo , Dióxido de Carbono/metabolismo , Células Cultivadas , Corteza Cerebral/metabolismo , Técnicas de Silenciamiento del Gen , Glucosa/metabolismo , Glutamato Deshidrogenasa/genética , Ácido Glutámico/metabolismo , Isoleucina/metabolismo , Ácido Láctico/metabolismo , Ratones , ARN Interferente Pequeño/metabolismo , Ácidos Tricarboxílicos/metabolismoRESUMEN
Cultured astrocytes treated with siRNA to knock down glutamate dehydrogenase (GDH) were used to investigate whether this enzyme is important for the utilization of glutamate as an energy substrate. By incubation of these cells in media containing different concentrations of glutamate (range 100-500 µM) in the presence or in the absence of glucose, the metabolism of these substrates was studied by using tritiated glutamate or 2-deoxyglucose as tracers. In addition, the cellular contents of glutamate and ATP were determined. The astrocytes were able to maintain physiological levels of ATP regardless of the expression level of GDH and the incubation condition, indicating a high degree of flexibility with regard to regulatory mechanisms involved in maintaining an adequate energy level in the cells. Glutamate uptake was found to be increased in these cells when exposed to increasing levels of extracellular glutamate independently of the GDH expression level. Moreover, increased intracellular glutamate content was observed in the GDH-deficient cells after a 2-hr incubation in the presence of 100 µM glutamate. It is significant that GDH-deficient cells exhibited an increased utilization of glucose in the presence of 250 and 500 µM glutamate, monitored as an increase in the accumulation of tritiated 2-deoxyglucose-6-phosphate. These findings underscore the importance of the expression level of GDH for the ability to utilize glutamate as an energy source fueling its own energy-requiring uptake.
Asunto(s)
Astrocitos/enzimología , Glucosa/metabolismo , Glutamato Deshidrogenasa/deficiencia , Ácido Glutámico/metabolismo , Adenosina Trifosfato/metabolismo , Análisis de Varianza , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Relación Dosis-Respuesta a Droga , Líquido Extracelular/efectos de los fármacos , Líquido Extracelular/metabolismo , Glucosa-6-Fosfato/análogos & derivados , Glucosa-6-Fosfato/metabolismo , Ácido Glutámico/farmacología , Ratones , Ratones Endogámicos , ARN Interferente Pequeño/farmacologíaRESUMEN
In pancreatic ß-cells, glutamate dehydrogenase (GDH) modulates insulin secretion, although its function regarding specific secretagogues is unclear. This study investigated the role of GDH using a ß-cell-specific GDH knockout mouse model, called ßGlud1(-/-). The absence of GDH in islets isolated from ßGlud1(-/-) mice resulted in abrogation of insulin release evoked by glutamine combined with 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid or l-leucine. Reintroduction of GDH in ßGlud1(-/-) islets fully restored the secretory response. Regarding glucose stimulation, insulin secretion in islets isolated from ßGlud1(-/-) mice exhibited half of the response measured in control islets. The amplifying pathway, tested at stimulatory glucose concentrations in the presence of KCl and diazoxide, was markedly inhibited in ßGlud1(-/-) islets. On glucose stimulation, net synthesis of glutamate from α-ketoglutarate was impaired in GDH-deficient islets. Accordingly, glucose-induced elevation of glutamate levels observed in control islets was absent in ßGlud1(-/-) islets. Parallel biochemical pathways, namely alanine and aspartate aminotransferases, could not compensate for the lack of GDH. However, the secretory response to glucose was fully restored by the provision of cellular glutamate when ßGlud1(-/-) islets were exposed to dimethyl glutamate. This shows that permissive levels of glutamate are required for the full development of glucose-stimulated insulin secretion and that GDH plays an indispensable role in this process.
Asunto(s)
Glutamato Deshidrogenasa/fisiología , Ácido Glutámico/fisiología , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Alanina Transaminasa/metabolismo , Animales , Aspartato Aminotransferasas/metabolismo , Ácido Aspártico/biosíntesis , Señalización del Calcio , Células Cultivadas , Femenino , Glucosa/fisiología , Glutamato Deshidrogenasa/deficiencia , Glutamato Deshidrogenasa/genética , Ácido Glutámico/biosíntesis , Ácido Glutámico/metabolismo , Glutamina/fisiología , Secreción de Insulina , Células Secretoras de Insulina/enzimología , Leucina/fisiología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Glutamate dehydrogenase (GDH), encoded by GLUD1, participates in the breakdown and synthesis of glutamate, the main excitatory neurotransmitter. In the CNS, besides its primary signaling function, glutamate is also at the crossroad of metabolic and neurotransmitter pathways. Importance of brain GDH was questioned here by generation of CNS-specific GDH-null mice (CnsGlud1(-/-)); which were viable, fertile and without apparent behavioral problems. GDH immunoreactivity as well as enzymatic activity were absent in Cns-Glud1(-/-) brains. Immunohistochemical analyses on brain sections revealed that the pyramidal cells of control animals were positive for GDH, whereas the labeling was absent in hippocampal sections of Cns-Glud1(-/-) mice. Electrophysiological recordings showed that deletion of GDH within the CNS did not alter synaptic transmission in standard conditions. Cns-Glud1(-/-) mice exhibited deficient oxidative catabolism of glutamate in astrocytes, showing that GDH is required for Krebs cycle pathway. As revealed by NMR studies, brain glutamate levels remained unchanged, whereas glutamine levels were increased. This pattern was favored by up-regulation of astrocyte-type glutamate and glutamine transporters and of glutamine synthetase. Present data show that the lack of GDH in the CNS modifies the metabolic handling of glutamate without altering synaptic transmission.
Asunto(s)
Encéfalo/enzimología , Eliminación de Gen , Ácido Glutámico/metabolismo , Receptores de Glutamato/deficiencia , Receptores de Glutamato/genética , Transmisión Sináptica/genética , Animales , Encéfalo/patología , Encéfalo/fisiología , Células Cultivadas , Femenino , Glutamato Deshidrogenasa , Glutamina/metabolismo , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Vías Nerviosas/metabolismo , Vías Nerviosas/patología , Vías Nerviosas/fisiopatología , Técnicas de Cultivo de Órganos , Receptores de Glutamato/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
Glutamate is the most abundant excitatory neurotransmitter in the brain and astrocytes are key players in sustaining glutamate homeostasis. Astrocytes take up the predominant part of glutamate after neurotransmission and metabolism of glutamate is necessary for a continuous efficient removal of glutamate from the synaptic area. Glutamate may either be amidated by glutamine synthetase or oxidatively metabolized in the mitochondria, the latter being at least to some extent initiated by oxidative deamination by glutamate dehydrogenase (GDH). To explore the particular importance of GDH for astrocyte metabolism we have knocked down GDH in cultured cortical astrocytes employing small interfering RNA (siRNA) achieving a reduction of the enzyme activity by approximately 44%. The astrocytes were incubated for 2h in medium containing either 1.0mM [(15)NH(4)(+)] or 100 µM [(15)N]glutamate. For those exposed to [(15)N]glutamate an additional 100 µM was added after 1h. Metabolic mapping was performed from isotope incorporation measured by mass spectrometry into relevant amino acids of cell extracts and media. The contents of the amino acids were measured by HPLC. The (15)N incorporation from [(15)NH(4)(+)] into glutamate, aspartate and alanine was decreased in astrocytes exhibiting reduced GDH activity. However, the reduced GDH activity had no effect on the cellular contents of these amino acids. This supports existing in vivo and in vitro studies that GDH is predominantly working in the direction of oxidative deamination and not reductive amination. In contrast, when exposing the astrocytes to [(15)N]glutamate, the reduced GDH activity led to an increased (15)N incorporation into glutamate, aspartate and alanine and a large increase in the content of glutamate and aspartate. Surprisingly, this accumulation of glutamate and net-synthesis of aspartate were not reflected in any alterations in either the glutamine content or labeling, but a slight increase in mono labeling of glutamine in the medium. We suggest that this extensive net-synthesis of aspartate due to lack of GDH activity is occurring via the concerted action of AAT and the part of TCA cycle operating from α-ketoglutarate to oxaloacetate, i.e. the truncated TCA cycle.
Asunto(s)
Ácido Aspártico/metabolismo , Astrocitos/enzimología , Técnicas de Silenciamiento del Gen , Glutamato Deshidrogenasa/genética , Ácido Glutámico/metabolismo , ARN Interferente Pequeño/genética , Animales , Western Blotting , Células Cultivadas , Cromatografía Líquida de Alta Presión , RatonesRESUMEN
Astrocyte cultures were prepared from cerebral cortex of new-born and 7-day-old mice and additionally, the cultures from new-born animals were passaged as secondary cultures. The cultures were characterized by immunostaining for the astrocyte markers glutamine synthetase (GS), glial fibrillary acidic protein, and the glutamate transporters EAAT1 and EAAT2. The cultures prepared from 7-day-old animals were additionally characterized metabolically using (13)C-labeled glucose and glutamate as well as (15)N-labeled glutamate as substrates. All types of cultures exhibited pronounced immunostaining of the astrocyte marker proteins. The metabolic pattern of the cultures from 7-day-old animals of the labeled substrates was comparable to that seen previously in astrocyte cultures prepared from new-born mouse brain showing pronounced glycolytic and oxidative metabolism of glucose. Glutamate was metabolized both via the GS pathway and oxidatively via the tricarboxylic acid cycle as expected. Additionally, glutamate underwent pronounced transamination to aspartate and alanine and the intracellular pools of alanine and pyruvate exhibited compartmentation. Altogether the results show that cultures prepared from cerebral cortex of 7-day-old mice have metabolic and functional properties indistinguishable from those of classical astrocyte cultures prepared from neocortex of new-born animals. This provides flexibility with regard to preparation and use of these cultures for a variety of purposes.