RESUMEN
BACKGROUND AND AIMS: Nephropathy involves pathophysiological changes to the glomerulus. The primary glomerular endothelial cells (GEnCs) have emerged as an important tool for studying glomerulosclerotic mechanisms and in the screening process for drug-candidates. The success of the studies is dependent on the quality of the cell model. Therefore, we set out to establish an easy, reproducible model of the quiescent endothelial monolayer with the use of commercially available extracellular matrices (ECMs). METHODS: Primary hGEnCs were seeded on various ECMs. Cell adhesion was monitored by an impedance sensing system. The localization of junctional proteins was assessed by immunofluorescence and the barrier function by passage of fluorescent dextrans and magnitude of VEGF response. RESULTS: All ECM matrices except recombinant human laminin 111 (rhLN111) supported comparable cell proliferation. Culturing hGEnCs on rhLN521, rhLN511 or fibronectin resulted in a physiologically relevant barrier to 70kDa dextrans which was 82% tighter than that formed on collagen type IV. Furthermore, only hGEnCs cultured on rhLN521 or rhLN511 showed plasma-membrane localized zonula occludens-1 and vascular endothelial cadherin indicative of proper tight and adherens junctions (AJ). CONCLUSION: We recommend culturing hGEnCs on the mature glomerular basement membrane laminin - rhLN521 - which, as the only commercially available ECM, promotes all of the characteristics of the quiescent hGEnC monolayer: cobblestone morphology, well-defined AJs and physiological perm-selectivity.
Asunto(s)
Células Endoteliales/fisiología , Matriz Extracelular/química , Permeabilidad Capilar , Adhesión Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Materiales Biocompatibles Revestidos , Colágeno Tipo IV/química , Medios de Cultivo , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/citología , Fibronectinas/química , Humanos , Glomérulos Renales/irrigación sanguínea , Laminina/química , Microvasos/citología , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
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
Mammalian AMP-activated protein kinase (AMPK) functions as a metabolic switch. It is composed of 3 different subunits and its activation depends on phosphorylation of a threonine residue (Thr172) in the α-subunit. This phosphorylation can be brought about by 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR) which in the cells is converted to a monophosphorylated nucleotide mimicking the effect of AMP. We show that the preparation of cultured astrocytes used for metabolic studies expresses AMPK, which could be phosphorylated by exposure of the cells to AICAR. The effect of AMPK activation on glutamate metabolism in astrocytes was studied using primary cultures of these cells from mouse cerebral cortex during incubation in media containing 2.5 mM glucose and 100 µM [U-(13)C]glutamate. The metabolism of glutamate including a detailed analysis of its metabolic pathways involving the tricarboxylic acid (TCA) cycle was studied using high-performance liquid chromatography analysis supplemented with gas chromatography-mass spectrometry technology. It was found that AMPK activation had profound effects on the pathways involved in glutamate metabolism since the entrance of the glutamate carbon skeleton into the TCA cycle was reduced. On the other hand, glutamate uptake into the astrocytes as well as its conversion to glutamine catalyzed by glutamine synthetase was not affected by AMPK activation. Interestingly, synthesis and release of citrate, which are hallmarks of astrocytic function, were affected by a reduction of the flux of glutamate derived carbon through the malic enzyme and pyruvate carboxylase catalyzed reactions. Finally, it was found that in the presence of glutamate as an additional substrate, glucose metabolism monitored by the use of tritiated deoxyglucose was unaffected by AMPK activation. Accordingly, the effects of AMPK activation appeared to be specific for certain key processes involved in glutamate metabolism.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Astrocitos/metabolismo , Glutamatos/metabolismo , Proteínas Quinasas Activadas por AMP/biosíntesis , Adenosina Monofosfato/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Astrocitos/enzimología , Células Cultivadas , Ciclo del Ácido Cítrico/efectos de los fármacos , Desoxiglucosa/metabolismo , Activación Enzimática/efectos de los fármacos , Ratones , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , Ribonucleótidos/farmacologíaRESUMEN
Glutamate dehydrogenase (GDH) is a crucial enzyme on the crossroads of amino acid and energy metabolism and it is operating in all domains of life. According to current knowledge GDH is present only in one functional isoform in most animals, including mice. In addition to this housekeeping enzyme (hGDH1 in humans), humans and apes have acquired a second isoform (hGDH2) with a distinct tissue expression profile. In the current study we have cloned both mouse and human GDH constructs containing FLAG and (His)6 small genetically-encoded tags, respectively. The hGDH1 and hGDH2 constructs containing N-terminal (His)6 tags were successfully expressed in Sf9 cells and the recombinant proteins were isolated to ≥95 % purity in a two-step procedure involving ammonium sulfate precipitation and Ni(2+)-based immobilized metal ion affinity chromatography. To explore whether the presence of the FLAG and (His)6 tags affects the cellular localization and functionality of the GDH isoforms, we studied the subcellular distribution of the expressed enzymes as well as their regulation by adenosine diphosphate monopotassium salt (ADP) and guanosine-5'-triphosphate sodium salt (GTP). Through immunoblot analysis of the mitochondrial and cytosolic fraction of the HEK cells expressing the recombinant proteins we found that neither FLAG nor (His)6 tag disturbs the mitochondrial localization of GDH. The addition of the small tags to the N-terminus of the mature mitochondrial mouse GDH1 or human hGDH1 and hGDH2 did not change the ADP activation or GTP inhibition pattern of the proteins as compared to their untagged counterparts. However, the addition of FLAG tag to the C-terminus of the mouse GDH left the recombinant protein fivefold less sensitive to ADP activation. This finding highlights the necessity of the functional characterization of recombinant proteins containing even the smallest available tags.
Asunto(s)
Glutamato Deshidrogenasa/metabolismo , Histidina/metabolismo , Mitocondrias/metabolismo , Oligopéptidos/metabolismo , Adenosina Difosfato/metabolismo , Animales , Línea Celular , Citosol/metabolismo , Glutamato Deshidrogenasa/genética , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Ratones , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Glutamate dehydrogenase (GDH) uses ammonia to reversibly convert α-ketoglutarate to glutamate using NADP(H) and NAD(H) as cofactors. While GDH in most mammals is encoded by a single GLUD1 gene, humans and other primates have acquired a GLUD2 gene with distinct tissue expression profile. The two human isoenzymes (hGDH1 and hGDH2), though highly homologous, differ markedly in their regulatory properties. Here we obtained hGDH1 and hGDH2 in recombinant form and studied their Km for ammonia in the presence of 1.0 mM ADP. The analyses showed that lowering the pH of the buffer (from 8.0 to 7.0) increased the Km for ammonia substantially (hGDH1: from 12.8 ± 1.4 mM to 57.5 ± 1.6 mM; hGDH2: from 14.7 ± 1.6 mM to 62.2 ± 1.7 mM), thus essentially precluding reductive amination. Moreover, lowering the ADP concentration to 0.1 mM not only increased the K0.5 [NH4 (+)] of hGDH2, but also introduced a positive cooperative binding phenomenon in this isoenzyme. Hence, intra-mitochondrial acidification, as occurring in astrocytes during glutamatergic transmission should favor the oxidative deamination of glutamate. Similar considerations apply to the handling of glutamate by the proximal convoluted tubules of the kidney during systemic acidosis. The reverse could apply for conditions of local or systemic hyperammonemia or alkalosis.
Asunto(s)
Adenosina Difosfato/metabolismo , Amoníaco/metabolismo , Glutamato Deshidrogenasa/metabolismo , Baculoviridae/genética , Línea Celular , ADN Complementario/biosíntesis , ADN Complementario/genética , Humanos , Concentración de Iones de Hidrógeno , Isoenzimas/metabolismo , Ácidos Cetoglutáricos/metabolismo , Cinética , Mitocondrias Hepáticas/enzimología , Mitocondrias Hepáticas/metabolismo , NADP/metabolismoRESUMEN
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
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.
Asunto(s)
Astrocitos/citología , Corteza Cerebral/citología , Animales , Astrocitos/enzimología , Astrocitos/metabolismo , Células Cultivadas , Corteza Cerebral/enzimología , Corteza Cerebral/metabolismo , Transportador 1 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Glucosa/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Inmunohistoquímica , RatonesRESUMEN
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
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.