RESUMO
Mobilization of astrocyte glycogen is key for processes such as synaptic plasticity and memory formation but the link between neuronal activity and glycogen breakdown is not fully known. Activation of cytosolic soluble adenylyl cyclase (sAC) in astrocytes has been suggested to link neuronal depolarization and glycogen breakdown partly based on experiments employing pharmacological inhibition of sAC. However, several studies have revealed that sAC located within mitochondria is a central regulator of respiration and oxidative phosphorylation. Thus, pharmacological sAC inhibition is likely to affect both cytosolic and mitochondrial sAC and if bioenergetic readouts are studied, the observed effects are likely to stem from inhibition of mitochondrial rather than cytosolic sAC. Here, we report that a pharmacologically induced inhibition of sAC activity lowers mitochondrial respiration, induces phosphorylation of the metabolic master switch AMP-activated protein kinase (AMPK), and decreases glycogen stores in cultured primary murine astrocytes. From these data and our discussion of the literature, mitochondrial sAC emerges as a key regulator of astrocyte bioenergetics. Lastly, we discuss the challenges of investigating the functional and metabolic roles of cytosolic versus mitochondrial sAC in astrocytes employing the currently available pharmacological tool compounds.
Assuntos
Proteínas Quinases Ativadas por AMP , Inibidores de Adenilil Ciclases , Adenilil Ciclases , Astrócitos , Glicogênio , Proteínas Quinases Ativadas por AMP/metabolismo , Inibidores de Adenilil Ciclases/farmacologia , Adenilil Ciclases/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/enzimologia , Ativação Enzimática/efeitos dos fármacos , Glicogênio/metabolismo , Camundongos , Mitocôndrias/enzimologia , Fosforilação OxidativaRESUMO
Gene expression studies are reported to be influenced by pre-analytical factors that can compromise RNA yield and integrity, which in turn may confound the experimental findings. Here we investigate the impact of four pre-analytical factors on brain-derived RNA: time-before-collection, tissue specimen size, tissue collection method, and RNA isolation method. We report no significant differences in RNA yield or integrity between 20 mg and 60 mg tissue samples collected in either liquid nitrogen or the RNAlater stabilizing solution. Isolation of RNA employing the TRIzol reagent resulted in a higher yield compared to isolation via the QIAcube kit while the latter resulted in RNA of slightly better integrity. Keeping brain tissue samples at room temperature for up to 160 min prior to collection and isolation of RNA resulted in no significant difference in yield or integrity. Our findings have significant practical and financial consequences for clinical genomic departments and other laboratory settings performing large-scale routine RNA expression analysis of brain samples.
Assuntos
Encéfalo/metabolismo , RNA/metabolismo , Animais , Camundongos , RNA/isolamento & purificação , Estabilidade de RNA , Manejo de Espécimes/métodos , Temperatura , Fatores de TempoRESUMO
Synaptic transmission is closely linked to brain energy and neurotransmitter metabolism. However, the extent of brain metabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA), and the relative metabolic contributions of neurons and astrocytes, are yet unknown. The present study was designed to investigate the functional significance of brain GABA metabolism using isolated mouse cerebral cortical slices and slices of neurosurgically resected neocortical human tissue of the temporal lobe. By using dynamic isotope labeling, with [15 N]GABA and [U-13 C]GABA as metabolic substrates, we show that both mouse and human brain slices exhibit a large capacity for GABA metabolism. Both the nitrogen and the carbon backbone of GABA strongly support glutamine synthesis, particularly in the human cerebral cortex, indicative of active astrocytic GABA metabolism. This was further substantiated by pharmacological inhibition of the primary astrocytic GABA transporter subtype 3 (GAT3), by (S)-SNAP-5114 or 1-benzyl-5-chloro-2,3-dihydro-1H-indole-2,3-dione (compound 34), leading to significant reductions in oxidative GABA carbon metabolism. Interestingly, this was not the case when tiagabine was used to specifically inhibit GAT1, which is predominantly found on neurons. Finally, we show that acute GABA exposure does not directly stimulate glycolytic activity nor oxidative metabolism in cultured astrocytes, but can be used as an additional substrate to enhance uncoupled respiration. These results clearly show that GABA is actively metabolized in astrocytes, particularly for the synthesis of glutamine, and challenge the current view that synaptic GABA homeostasis is maintained primarily by presynaptic recycling.
Assuntos
Astrócitos , Animais , Carbono , Córtex Cerebral , Ácido Glutâmico , Glutamina , Camundongos , Neurotransmissores , Ácido gama-AminobutíricoRESUMO
Epilepsy is one of the most common chronic neurological conditions. Today, close to 30 different medications to prevent epileptic seizures are in use; yet, far from all patients become seizure free upon medical treatment. Thus, there is a need for new pharmacological approaches including novel drug targets for the management of epilepsy. Despite the fact that a role for cAMP signaling in epileptogenesis and seizures was first suggested some four decades ago, none of the current medications target the cAMP signaling system. The reasons for this are probably many including limited knowledge of the underlying biology and pathology as well as difficulties in designing selective drugs for the different components of the cAMP signaling system. This review explores selected aspects of cAMP signaling in the context of epileptogenesis and seizures including cAMP response element binding (CREB)-mediated transcriptional regulation. We discuss the therapeutic potential of targeting cAMP signaling in epilepsy and point to an increased knowledge of the A-kinase anchoring protein-based signaling hubs as being of seminal importance for future drug discovery within the field. Further, in terms of targeting CREB, we argue that targeting upstream cAMP signals might be more fruitful than targeting CREB itself. Finally, we point to astrocytes as cellular targets in epilepsy since cAMP signals may regulate astrocytic K+ clearance affecting neuronal excitability.
Assuntos
Anticonvulsivantes/metabolismo , AMP Cíclico/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Epilepsia/metabolismo , Convulsões/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Anticonvulsivantes/administração & dosagem , AMP Cíclico/antagonistas & inibidores , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Sistemas de Liberação de Medicamentos/tendências , Epilepsia/tratamento farmacológico , Humanos , Convulsões/tratamento farmacológico , Transdução de Sinais/fisiologiaRESUMO
The brain contains a fairly low amount of glycogen, mostly located in astrocytes, a fact that has prompted the suggestion that glycogen does not have a significant physiological role in the brain. However, glycogen metabolism in astrocytes is essential for several key physiological processes and is adversely affected in disease. For instance, diminished ability to break down glycogen impinges on learning, and epilepsy, Alzheimer's disease, and type 2 diabetes are all associated with abnormal astrocyte glycogen metabolism. Glycogen metabolism supports astrocytic K+ and neurotransmitter glutamate uptake and subsequent glutamine synthesis-three fundamental steps in excitatory signaling at most brain synapses. Thus, there is abundant evidence for a key role of glycogen in brain function. Here, we summarize the physiological brain functions that depend on glycogen, discuss glycogen metabolism in disease, and investigate how glycogen breakdown is regulated at the cellular and molecular levels.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glicogênio/metabolismo , Doença de Alzheimer/metabolismo , Animais , Cálcio/metabolismo , AMP Cíclico/metabolismo , Glutamina/biossíntese , Glicogênio Fosforilase/metabolismo , Humanos , Isoenzimas/metabolismo , Aprendizagem/fisiologia , Memória/fisiologia , Neurotransmissores/metabolismo , Potássio/metabolismo , Transdução de Sinais , Sono/fisiologiaRESUMO
This review discusses aspects of known and putative compartmentalized 3',5'-cyclic adenosine monophosphate (cAMP) signaling in astrocytes, a cell type that has turned out to be a key player in brain physiology and pathology. cAMP has attracted less attention than Ca2+ in recent years, but could turn out to rival Ca2+ in its potential to drive cellular functions and responses to intra- and extracellular cues. Further, Ca2+ and cAMP are known to engage in extensive crosstalk and cAMP signals often take place within subcellular compartments revolving around multi-protein signaling complexes; however, we know surprisingly little about this in astrocytes. Here, we review aspects of astrocytic cAMP signaling, provide arguments for an increased interest in this subject, suggest possible future research directions within the field, and discuss putative drug targets.
Assuntos
Astrócitos/metabolismo , AMP Cíclico/metabolismo , Animais , HumanosRESUMO
Mitochondria produce the bulk of the ATP in most cells, including brain cells. Regulating this complex machinery to match the energetic needs of the cell is a complicated process that we have yet to understand in its entirety. In this context, 3',5'-cyclic AMP (cAMP) has been suggested to play a seminal role in signaling-metabolism coupling and regulation of mitochondrial ATP production. In cells, cAMP signals may affect mitochondria from the cytosolic side but more recently, a cAMP signal produced within the matrix of mitochondria by soluble adenylyl cyclase (sAC) has been suggested to regulate respiration and thus ATP production. However, little is known about these processes in brain mitochondria, and the effectors of the cAMP signal generated within the matrix are not completely clear since both protein kinase A (PKA) and exchange protein activated by cAMP 1 (EPAC1) have been suggested to be involved. Here, we review the current knowledge and relate it to brain mitochondria. Further, based on measurements of respiration, membrane potential, and ATP production in isolated mouse brain cortical mitochondria we show that inhibitors of sAC, PKA, or EPAC affect mitochondrial function in distinct ways. In conclusion, we suggest that brain mitochondria do regulate their function via sAC-mediated cAMP signals and that both PKA and EPAC could be involved downstream of sAC. Finally, due to the role of faulty mitochondrial function in a range of neurological diseases, we expect that the function of sAC-cAMP-PKA/EPAC signaling in brain mitochondria will likely attract further attention.
Assuntos
Adenilil Ciclases/metabolismo , Córtex Cerebral/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mitocôndrias/metabolismo , Animais , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Consumo de Oxigênio , Transdução de SinaisRESUMO
Astrocytes are crucial cells in the brain that are intimately coupled with neuronal metabolism. A new paper from San Martín et al. provides evidence that physiological levels of the gaseous signal molecule NO can rapidly and reversibly increase astrocyte metabolism of glucose and production of lactate. A proposed neurological coupling-from the potential source of NO, endothelial cells, to the potential beneficiary from the lactate, neurons-prompts new questions regarding the controversial role of lactate in the brain.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Ácido Láctico/metabolismo , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais/fisiologia , Animais , Células Endoteliais/metabolismo , CamundongosRESUMO
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.
Assuntos
Astrócitos/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Regulação Enzimológica da Expressão Gênica , Glucose/deficiência , Glutamato Desidrogenase/metabolismo , Ácido Glutâmico/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Dióxido de Carbono/farmacocinética , Isótopos de Carbono/farmacocinética , Células Cultivadas , Córtex Cerebral/citologia , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ciclo do Ácido Cítrico/genética , Relação Dose-Resposta a Droga , Proteína Glial Fibrilar Ácida/metabolismo , Glutamato Desidrogenase/genética , Ácido Glutâmico/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Desidrogenase do Álcool de Açúcar/metabolismo , Trítio/farmacocinéticaRESUMO
Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate involving the coenzyme NAD+ . Part of the foundation for the proposed shuttling of lactate from astrocytes to neurons during brain activation is the differential distribution of LDH isoenzymes between the two cell types. In this short review, we outline the basic kinetic properties of the LDH isoenzymes expressed in neurons and astrocytes, and argue that the distribution of LDH isoenzymes does not in any way govern directional flow of lactate between the two cellular compartments. The two main points are as follows. First, in line with the general concept of chemical catalysis, enzymes do not influence the thermodynamic equilibrium of a chemical reaction but merely the speed at which equilibrium is obtained. Thus, differential distribution of LDH isoenzymes with different kinetic parameters does not predict which cells are producing and which are consuming lactate. Second, the thermodynamic equilibrium of the reaction is toward the reduced substrate (i.e., lactate), which is reflected in the concentrations measured in brain tissue, suggesting that the reaction is at near-equilibrium at steady state. To conclude, the cellular distribution of LDH isoenzymes is of little if any consequence in determining any directional flow of lactate between neurons and astrocytes. © 2017 Wiley Periodicals, Inc.
Assuntos
Regulação Enzimológica da Expressão Gênica , L-Lactato Desidrogenase/biossíntese , Termodinâmica , Animais , Encéfalo/metabolismo , Glucose/metabolismo , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Cinética , L-Lactato Desidrogenase/genética , Ácido Láctico/metabolismo , Neurônios/metabolismoRESUMO
Synaptosomes prepared from various aged and gene modified experimental animals constitute a valuable model system to study pre-synaptic mechanisms. Synaptosomes were isolated from whole brain and the XFe96 extracellular flux analyzer (Seahorse Bioscience) was used to study mitochondrial respiration and glycolytic rate in presence of different substrates. Mitochondrial function was tested by sequentially exposure of the synaptosomes to the ATP synthase inhibitor, oligomycin, the uncoupler FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone) and the electron transport chain inhibitors rotenone and antimycin A. The synaptosomes exhibited intense respiratory activity using glucose as substrate. The FCCP-dependent respiration was significantly higher with 10 mM glucose compared to 1 mM glucose. Synaptosomes also readily used pyruvate as substrate, which elevated basal respiration, activity-dependent respiration induced by veratridine and the respiratory response to uncoupling compared to that obtained with glucose as substrate. Also lactate was used as substrate by synaptosomes but in contrast to pyruvate, mitochondrial lactate mediated respiration was comparable to respiration using glucose as substrate. Synaptosomal respiration using glutamate and glutamine as substrates was significantly higher compared to basal respiration, whereas oligomycin-dependent and FCCP-induced respiration was lower compared to the responses obtained in the presence of glucose as substrate. We provide evidence that synaptosomes are able to use besides glucose and pyruvate also the substrates lactate, glutamate and glutamine to support their basal respiration. Veratridine was found to increase respiration supported by glucose, pyruvate, lactate and glutamine and FCCP was found to increase respiration supported by glucose, pyruvate and lactate. This was not the case when glutamate was the only energy substrate.
Assuntos
Respiração Celular/fisiologia , Glucose/metabolismo , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Ácido Láctico/metabolismo , Terminações Pré-Sinápticas/metabolismo , Animais , Antimicina A/farmacologia , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Respiração Celular/efeitos dos fármacos , Camundongos , Terminações Pré-Sinápticas/efeitos dos fármacos , Rotenona/farmacologiaRESUMO
Glycogen is the main storage form of glucose in the brain. In contrast with previous beliefs, brain glycogen has recently been shown to play important roles in several brain functions. A fraction of metabolized glucose molecules are being shunted through glycogen before reentering the glycolytic pathway, a phenomenon known as the glycogen shunt. The significance of glycogen in astrocyte energetics is underlined by high activity of the glycogen shunt and the finding that inhibition of glycogen degradation, under some conditions leads to a disproportional increase in glycolytic activity, so-called glycolytic supercompensation. Glycogen phosphorylase, the key enzyme in glycogen degradation, is expressed in two different isoforms in brain, the muscle and the brain isoform. Recent studies have illustrated how these are differently regulated. In the present study, we investigate the role of the two isoforms in glycolytic supercompensation in cultured astrocytes with the expression of either one of the isoforms silenced by siRNA knockdown. When reintroducing glucose to glucose-starved astrocytes, glycolytic activity increased dramatically. Interestingly, the increase was 30% higher in astrocytes not expressing the muscle isoform of glycogen phosphorylase. Based on these results and previously published data we couple the muscle isoform of glycogen phosphorylase to glycolytic supercompensation and glycogen shunt activity, giving insights to the underlying mechanistic of these phenomena.
Assuntos
Astrócitos/metabolismo , Glicogênio Fosforilase/metabolismo , Glicogênio/metabolismo , Glicólise/fisiologia , Músculo Esquelético/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Células Cultivadas , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Glucose/farmacologia , Glicólise/efeitos dos fármacos , CamundongosRESUMO
Glycogen phosphorylase (GP) is activated to degrade glycogen in response to different stimuli, to support both the astrocyte's own metabolic demand and the metabolic needs of neurons. The regulatory mechanism allowing such a glycogenolytic response to distinct triggers remains incompletely understood. In the present study, we used siRNA-mediated differential knockdown of the two isoforms of GP expressed in astrocytes, muscle isoform (GPMM), and brain isoform (GPBB), to analyze isoform-specific regulatory characteristics in a cellular setting. Subsequently, we tested the response of each isoform to phosphorylation, triggered by incubation with norepinephrine (NE), and to AMP, increased by glucose deprivation in cells in which expression of one GP isoform had been silenced. Successful knockdown was demonstrated on the protein level by Western blot, and on a functional level by determination of glycogen content showing an increase in glycogen levels following knockdown of either GPMM or GPBB. NE triggered glycogenolysis within 15 min in control cells and after GPBB knockdown. However, astrocytes in which expression of GPMM had been silenced showed a delay in response to NE, with glycogen levels significantly reduced only after 60 min. In contrast, allosteric activation of GP by AMP, induced by glucose deprivation, seemed to mainly affect GPBB, as only knockdown of GPBB, but not of GPMM, delayed the glycogenolytic response to glucose deprivation. Our results indicate that the two GP isoforms expressed in astrocytes respond to different physiological triggers, therefore conferring distinct metabolic functions of brain glycogen.
Assuntos
Astrócitos/metabolismo , Glicogênio Fosforilase/metabolismo , Glicogênio/metabolismo , Animais , Células Cultivadas , Glucose/metabolismo , Glicogenólise/fisiologia , Isoenzimas/metabolismo , Camundongos , FosforilaçãoRESUMO
The malate-aspartate NADH shuttle (MAS) operates in neurons and other cells to translocate reducing equivalents from the cytosol to the mitochondrial matrix, thus allowing a continued flux through the glycolytic pathway and metabolism of extracellular lactate. Recent discoveries have taught us that MAS is regulated by fluctuations in cytosolic Ca(2+) levels, and that this regulation is required to maintain a tight coupling between neuronal activity and mitochondrial respiration and oxidative phosphorylation. At cytosolic Ca(2+) fluctuations below the threshold of the mitochondrial calcium uniporter, there is a positive correlation between Ca(2+) and MAS activity; however, if cytosolic Ca(2+) increases above the threshold, MAS activity is thought to be reduced by an intricate mechanism. The latter forces the neurons to partly rely on anaerobic glycolysis producing lactate that may be metabolized subsequently, by neurons or other cells. In this review, we will discuss the evidence for Ca(2+)-mediated regulation of MAS that have been uncovered over the last decade or so, together with the need for further verification, and examine the metabolic ramifications for neurons.
Assuntos
Ácido Aspártico/metabolismo , Sinalização do Cálcio/fisiologia , Citosol/metabolismo , Metabolismo Energético/fisiologia , Malatos/metabolismo , NAD/metabolismo , Neurônios/metabolismo , Animais , Humanos , Ácido Láctico/metabolismo , Mitocôndrias/metabolismoRESUMO
The operation of a glutamine-glutamate/GABA cycle in the brain consisting of the transfer of glutamine from astrocytes to neurons and neurotransmitter glutamate or GABA from neurons to astrocytes is a well-known concept. In neurons, glutamine is not only used for energy production and protein synthesis, as in other cells, but is also an essential precursor for biosynthesis of amino acid neurotransmitters. An excellent tool for the study of glutamine transfer from astrocytes to neurons is [(14)C]acetate or [(13)C]acetate and the glial specific enzyme inhibitors, i.e. the glutamine synthetase inhibitor methionine sulfoximine and the tricarboxylic acid cycle (aconitase) inhibitors fluoro-acetate and -citrate. Acetate is metabolized exclusively by glial cells, and [(13)C]acetate is thus capable when used in combination with magnetic resonance spectroscopy or mass spectrometry, to provide information about glutamine transfer. The present review will give information about glutamine trafficking and the tools used to map it as exemplified by discussions of published work employing brain cell cultures as well as intact animals. It will be documented that considerably more glutamine is transferred from astrocytes to glutamatergic than to GABAergic neurons. However, glutamine does have an important role in GABAergic neurons despite their capability of re-utilizing their neurotransmitter by re-uptake.
Assuntos
Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Ácido gama-Aminobutírico/biossínteseRESUMO
We have previously shown that synaptic transmission fails in cultured neurons in the presence of lactate as the sole substrate. Thus, to test the hypothesis that the failure of synaptic transmission is a consequence of insufficient energy supply, ATP levels were monitored employing the ATP biosensor Ateam1.03YEMK. While inducing synaptic activity by subjecting cultured neurons to two 30 s pulses of NMDA (30 µM) with a 4 min interval, changes in relative ATP levels were measured in the presence of lactate (1 mM), glucose (2.5 mM) or the combination of the two. ATP levels reversibly declined following NMDA-induced neurotransmission activity, as indicated by a reversible 10-20 % decrease in the response of the biosensor. The responses were absent when the NMDA receptor antagonist memantine was present. In the presence of lactate alone, the ATP response dropped significantly more than in the presence of glucose following the 2nd pulse of NMDA (approx. 10 vs. 20 %). Further, cytosolic Ca(2+) homeostasis during NMDA-induced synaptic transmission is partially inhibited by verapamil indicating that voltage-gated Ca(2+) channels are activated. Lastly, we showed that cytosolic Ca(2+) homeostasis is supported equally well by both glucose and lactate, and that a pulse of NMDA causes accumulation of Ca(2+) in the mitochondrial matrix. In summary, we have shown that ATP homeostasis during neurotransmission activity in cultured neurons is supported by both glucose and lactate. However, ATP homeostasis seems to be negatively affected by the presence of lactate alone, suggesting that glucose is needed to support neuronal energy metabolism during activation.
Assuntos
Trifosfato de Adenosina/metabolismo , Citosol/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Glutamatos/fisiologia , N-Metilaspartato/farmacologia , Neurônios/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Glucose/metabolismo , Ácido Láctico/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismoRESUMO
Astrocytic glycogen, the only storage form of glucose in the brain, has been shown to play a fundamental role in supporting learning and memory, an effect achieved by providing metabolic support for neurons. We have examined the interplay between glycogenolysis and the bioenergetics of astrocytic Ca(2+) homeostasis, by analyzing interdependency of glycogen and store-operated Ca(2+) entry (SOCE), a mechanism in cellular signaling that maintains high endoplasmatic reticulum (ER) Ca(2+) concentration and thus provides the basis for store-dependent Ca(2+) signaling. We stimulated SOCE in primary cultures of murine cerebellar and cortical astrocytes, and determined glycogen content to investigate the effects of SOCE on glycogen metabolism. By blocking glycogenolysis, we tested energetic dependency of SOCE-related Ca(2+) dynamics on glycogenolytic ATP. Our results show that SOCE triggers astrocytic glycogenolysis. Upon inhibition of adenylate cyclase with 2',5'-dideoxyadenosine, glycogen content was no longer significantly different from that in unstimulated control cells, indicating that SOCE triggers astrocytic glycogenolysis in a cAMP-dependent manner. When glycogenolysis was inhibited in cortical astrocytes by 1,4-dideoxy-1,4-imino-D-arabinitol, the amount of Ca(2+) loaded into ER via sarco/endoplasmic reticulum Ca(2)-ATPase (SERCA) was reduced, which suggests that SERCA pumps preferentially metabolize glycogenolytic ATP. Our study demonstrates SOCE as a novel pathway in stimulating astrocytic glycogenolysis. We also provide first evidence for a new functional role of brain glycogen, in providing local ATP to SERCA, thus establishing the bioenergetic basis for astrocytic Ca(2+) signaling. This mechanism could offer a novel explanation for the impact of glycogen on learning and memory.
Assuntos
Astrócitos/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Glicogenólise/fisiologia , Homeostase/fisiologia , Análise de Variância , Animais , Animais Recém-Nascidos , Arabinose/farmacologia , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/ultraestrutura , Encéfalo/citologia , Células Cultivadas , AMP Cíclico/metabolismo , Citosol/efeitos dos fármacos , Citosol/microbiologia , Didesoxiadenosina/análogos & derivados , Didesoxiadenosina/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Metabolismo Energético , Glicogênio/metabolismo , Glicogenólise/efeitos dos fármacos , Imino Furanoses/farmacologia , Camundongos , Álcoois Açúcares/farmacologiaRESUMO
UNLABELLED: Studies have shown decreased cerebral oxygen metabolism (CMRO(2)) and blood flow (CBF) in patients with cirrhosis with hepatic encephalopathy (HE). It remains unclear, however, whether these disturbances are associated with HE or with cirrhosis itself and how they may relate to arterial blood ammonia concentration and cerebral metabolic rate of blood ammonia (CMRA). We addressed these questions in a paired study design by investigating patients with cirrhosis during and after recovery from an acute episode of HE type C. CMRO(2), CBF, and CMRA were measured by dynamic positron emission tomography (PET)/computed tomography (CT). Ten patients with cirrhosis were studied during an acute episode of HE; nine were reexamined after recovery. Nine patients with cirrhosis with no history of HE served as controls. Mean CMRO(2) increased from 0.73 µmol oxygen/mL brain tissue/min during HE to 0.91 µmol oxygen/mL brain tissue/min after recovery (paired t test; P < 0.05). Mean CBF increased from 0.28 mL blood/mL brain tissue/min during HE to 0.38 mL blood/mL brain tissue/min after recovery (P < 0.05). After recovery from HE, CMRO(2) and CBF were not significantly different from values in the control patients. Arterial blood ammonia concentration decreased 20% after recovery (P < 0.05) and CMRA was unchanged (P > 0.30); both values were higher than in the control patients (both P < 0.05). CONCLUSION: The low values of CMRO(2) and CBF observed during HE increased after recovery from HE and were thus associated with HE rather than the liver disease as such. The changes in CMRO(2) and CBF could not be linked to blood ammonia concentration or CMRA.