Functional significance of brain glycogen in sustaining glutamatergic neurotransmission

The involvement of brain glycogen in sustaining neuronal activity has previously been demonstrated. However, to what extent energy derived from glycogen is consumed by astrocytes themselves or is transferred to the neurons in the form of lactate for oxidative metabolism to proceed is at present unclear. The significance of glycogen in fueling glutamate uptake into astrocytes was specifically addressed in cultured astrocytes. Moreover, the objective was to elucidate whether glycogen derived energy is important for maintaining glutamatergic neurotransmission, induced by repetitive exposure to NMDA in co-cultures of cerebellar neurons and astrocytes. In the astrocytes it was shown that uptake of the glutamate analogue d -[³H]aspartate was impaired when glycogen degradation was inhibited irrespective of the presence of glucose, signifying that energy derived from glycogen degradation is important for the astrocytic compartment. By inhibiting glycogen degradation in co-cultures it was evident that glycogen provides energy to sustain glutamatergic neurotransmission, i.e. release and uptake of glutamate. The relocation of glycogen derived lactate to the neuronal compartment was investigated by employing d -lactate, a competitive substrate for the monocarboxylate transporters. Neurotransmitter release was affected by the presence of d -lactate indicating that glycogen derived energy is important not only in the astrocytic but also in the neuronal compartment.     

Effects of Potassium and Glutamine on Metabolism of Glucose in Astrocytes

The metabolic effects of extracellular glutamine (2.5 mM) or high potassium (25 mM) on glucose metabolism were studied in cultured cerebellar astrocytes. High potassium caused an increased glycolytic flux and an increase in glutamine release. Exposure to glutamine increased glycolytic flux and alanine formation, indicating that glutamine uptake is an energy requiring process. The effects of glutamine and high potassium on glycolytic flux were additive. Formation of metabolites from [1-¹³C]glucose and [2-¹³C]acetate confirmed the effects of glutamine and high potassium on glycolytic metabolism. In the presence of extracellular glutamine, analysis of the ¹³C labeling patterns of citrate and glutamine indicated a decrease in the cycling ratio and/or pyruvate carboxylation and glutamine synthesis from [1-¹³C]glucose did occur, but was decreased. Exposure to high potassium led to extracellular accumulation of acetate, presumably through non-enzymatic decarboxylation of pyruvate.     

Neighborly interactions of metabolically-activated astrocytes in vivo

Metabolic responses of brain cells to a stimulus are governed, in part, by their enzymatic specialization and interrelationships with neighboring cells, and local shifts in functional metabolism during brain activation are likely to be influenced by the neurotransmitter system, subcellular compartmentation, and anatomical structure. Selected examples of functional activation illustrate the complexity of metabolic interactions in working brain and of interpretation of changes in brain lactate levels. The major focus of this article is the disproportionately higher metabolism of glucose compared to oxygen in normoxic brain, a phenomenon that occurs during activation in humans and animals. The glucose utilized in excess of oxygen is not fully explained by accumulation of glucose, lactate, or glycogen in brain or by lactate efflux from brain to blood. Thus, any lactate derived from the excess glucose could not have been stoichiometrically exported to and metabolized by neighboring neurons because oxygen consumption would have otherwise increased and matched that of glucose. Metabolic labeling of tricarboxylic acid cycle-derived amino acids increased during brief sensory stimulation, reflecting a rise in oxidative metabolism. Brain glycogen is mainly in astrocytes, and its level falls throughout the stimulus and early post-activation interval. Glycogenolysis cannot be accounted for by lactate accumulation or oxidation; there must be rapid product clearance. Glycogen restoration is slow and diversion of glucose from oxidative pathways for its re-synthesis could reduce the global O(2)/glucose uptake ratio; astrocytes could downshift this ratio for up to an hour after 5 min stimulus. Morphological studies of astrocytes reveal a paucity of cytoplasm and organelles in the fine processes that surround synapses and form gap junction connections with neighboring astrocytes. Specialized regions of astrocytes, e.g. their endfeet and thin peripheral lamellae, are likely to have compartmentalized metabolic activities. Anatomical constraints imposed upon the fine processes might require preferential utilization of glycolysis to satisfy their energy demands, but rapid lactate clearance would then be essential, since its accumulation would inhibit glycolysis. Gap junctional connections between neighboring astrocytes provide a mechanism for rapid metabolite spreading via the astrocytic syncytium and elimination of by-products. Local structure-function relationships need to be incorporated into experimental models of neuron-astrocyte and astrocyte-astrocyte interactions in working brain.     

Brain glycogen and its role in supporting glutamate and GABA homeostasis in a type 2 diabetes rat model

The number of people suffering from diabetes is hastily increasing and the condition is associated with altered brain glucose homeostasis. Brain glycogen is located in astrocytes and being a carbohydrate reservoir it contributes to glucose homeostasis. Furthermore, glycogen has been indicated to be important for proper neurotransmission under normal conditions. Previous findings from our laboratory suggested that glucose metabolism was reduced in type 2 diabetes, and thus we wanted to investigate more specifically how brain glycogen metabolism contributes to maintain energy status in the type 2 diabetic state. Also, our objective was to elucidate the contribution of glycogen to support neurotransmitter glutamate and GABA homeostasis. A glycogen phosphorylase (GP) inhibitor was administered to Sprague-Dawley (SprD) and Zucker Diabetic Fatty (ZDF) rats in vivo and after one day of treatment [1-13C]glucose was used to monitor metabolism. Brain levels of 13C labeling in glucose, lactate, alanine, glutamate, GABA, glutamine and aspartate were determined. Our results show that inhibition of brain glycogen metabolism reduced the amounts of glutamate in both the control and type 2 diabetes models. The reduction in glutamate was associated with a decrease in the pyruvate carboxylase/pyruvate dehydrogenase ratio in the control but not the type 2 diabetes model. In the type 2 diabetes model GABA levels were increased suggesting that brain glycogen serves a role in maintaining a proper ratio between excitatory and inhibitory neurotransmitters in type 2 diabetes. Both the control and the type 2 diabetic states had a compensatory increase in glucose-derived 13C processed through the TCA cycle following inhibition of glycogen degradation. Finally, it was indicated that the type 2 diabetes model might have an augmented necessity for compensatory upregulation at the glycolytic level.     

Novel model of neuronal bioenergetics: postsynaptic utilization of glucose but not lactate correlates positively with Ca2+ signalling in cultured mouse glutamatergic neurons

We have previously investigated the relative roles of extracellular glucose and lactate as fuels for glutamatergic neurons during synaptic activity. The conclusion from these studies was that cultured glutamatergic neurons utilize glucose rather than lactate during NMDA (N-methyl-d-aspartate)-induced synaptic activity and that lactate alone is not able to support neurotransmitter glutamate homoeostasis. Subsequently, a model was proposed to explain these results at the cellular level. In brief, the intermittent rises in intracellular Ca²⁺ during activation cause influx of Ca²⁺ into the mitochondrial matrix thus activating the tricarboxylic acid cycle dehydrogenases. This will lead to a lower activity of the MASH (malate–aspartate shuttle), which in turn will result in anaerobic glycolysis and lactate production rather than lactate utilization. In the present work, we have investigated the effect of an ionomycin-induced increase in intracellular Ca²⁺ (i.e. independent of synaptic activity) on neuronal energy metabolism employing ¹³C-labelled glucose and lactate and subsequent mass spectrometric analysis of labelling in glutamate, alanine and lactate. The results demonstrate that glucose utilization is positively correlated with intracellular Ca²⁺ whereas lactate utilization is not. This result lends further support for a significant role of glucose in neuronal bioenergetics and that Ca²⁺ signalling may control the switch between glucose and lactate utilization during synaptic activity. Based on the results, we propose a compartmentalized CiMASH (Ca²⁺-induced limitation of the MASH) model that includes intracellular compartmentation of glucose and lactate metabolism. We define pre- and post-synaptic compartments metabolizing glucose and glucose plus lactate respectively in which the latter displays a positive correlation between oxidative metabolism of glucose and Ca²⁺ signalling.     

Complex Glutamate Labeling from [U-13C]glucose or [U-13C]lactate in Co-cultures of Cerebellar Neurons and Astrocytes

Glutamate metabolism was studied in co-cultures of mouse cerebellar neurons (predominantly glutamatergic) and astrocytes. One set of cultures was superfused (90 min) in the presence of either [U-¹³C]glucose (2.5 mM) and lactate (1 mM) or [U-¹³C]lactate (1 mM) and glucose (2.5 mM). Other sets of cultures were incubated in medium containing [U-¹³C]lactate (1 mM) and glucose (2.5 mM) for 4 h. Regardless of the experimental conditions cell extracts were analyzed using mass spectrometry and nuclear magnetic resonance spectroscopy. ¹³C labeling of glutamate was much higher than that of glutamine under all experimental conditions indicating that acetyl-CoA from both lactate and glucose was preferentially metabolized in the neurons. Aspartate labeling was similar to that of glutamate, especially when [U-¹³C]glucose was the substrate. Labeling of glutamate, aspartate and glutamine was lower in the cells incubated with [U-¹³C]lactate. The first part of the pyruvate recycling pathway, pyruvate formation, was detected in singlet and doublet labeling of alanine under all experimental conditions. However, full recycling, detectable in singlet labeling of glutamate in the C-4 position was only quantifiable in the superfused cells both from [U-¹³C]glucose and [U-¹³C]lactate. Lactate and alanine were mostly uniformly labeled and labeling of alanine was the same regardless of the labeled substrate present and higher than that of lactate when superfused in the presence of [U-¹³C]glucose. These results show that metabolism of pyruvate, the precursor for lactate, alanine and acetyl-CoA is highly compartmentalized. Special issue dedicated to John P. Blass.     

Accumulation of high-molecular-weight amylose in Alzheimer's disease brains

Although most of the glucose metabolized in the brain is taken up from the blood, glucose derived from glycogen stores is increasingly implicated in both normal brain function and injury repair. An impaired glucose metabolism is one of the features of Alzheimer's disease (AD) entailing a reduction in glucose transporters and the uptake of glucose as well as alterations in the specific activity of glycolytic enzymes. Here we report that AD brains accumulate amylose, the unbranched alpha(1,4)-linked glucose polymer that is resistant to degradation by glycolytic enzymes. Neutral polysaccharides harvested from postmortem brains were purified with hydrazinolysis, ion exchange, and sizing chromatography and subjected to NMR spectroscopy, GC, GC-MS, and methylation analysis. Five percent of the polysaccharides (50 micro g [0.3 micro mol]/g wet weight brain tissue) consisted of amylose with molecular weights exceeding 600,000 Da. There is no evidence for 1,6-branching, indicating that the polymer is not a form of high-molecular-weight glycogen. By GC analysis, the glucose content of the AD brains was almost three times greater than that of the age-matched control brains. A synthesis of amylose in AD brains at the expense of glycogen would compromise glucose metabolism and enhance neural degeneration.     

The in vivo neuron-to-astrocyte lactate shuttle in human brain: evidence from modeling of measured lactate levels during visual stimulation

Functional magnetic resonance spectroscopy (fMRS) allows the non-invasive measurement of metabolite concentrations in the human brain, including changes induced by variations in neurotransmission activity. However, the limited spatial and temporal resolution of fMRS does not allow specific measurements of metabolites in different cell types. Thus, the analysis of fMRS data in the context of compartmentalized metabolism requires the formulation and application of mathematical models. In the present study we utilized the mathematical model introduced by Simpson et al . (2007) to gain insights into compartmentalized metabolism in vivo from the fMRS data obtained in humans at ultra high magnetic field by Mangia et al . (2007a) . This model simulates brain glucose and lactate levels in a theoretical cortical slice. Using experimentally determined concentrations and catalytic activities for the respective transporter proteins, we calculate inflow and export of glucose and lactate in endothelium, astrocytes, and neurons. We then vary neuronal and astrocytic glucose and lactate utilization capacities until close correspondence is observed between in vivo and simulated glucose and lactate levels. The results of the simulations indicate that, when literature values of glucose transport capacity are utilized, the fMRS data are consistent with export of lactate by neurons and import of lactate by astrocytes, a mechanism that can be referred to as a neuron-to-astrocyte lactate shuttle. A shuttle of lactate from astrocytes to neurons could be simulated, but this required the astrocytic glucose transport capacity to be increased by 12-fold, and required that neurons not respond to activation with increased glycolysis, two conditions that are not supported by current literature.     

Altered glycogen metabolism in cultured astrocytes from mice with chronic glutathione deficit; relevance for neuroenergetics in schizophrenia

Neurodegenerative and psychiatric disorders including Alzheimer's, Parkinson's or Huntington's diseases and schizophrenia have been associated with a deficit in glutathione (GSH). In particular, a polymorphism in the gene of glutamate cysteine ligase modulatory subunit (GCLM) is associated with schizophrenia. GSH is the most important intracellular antioxidant and is necessary for the removal of reactive by-products generated by the utilization of glucose for energy supply. Furthermore, glucose metabolism through the pentose phosphate pathway is a major source of NADPH, the cofactor necessary for the regeneration of reduced glutathione. This study aims at investigating glucose metabolism in cultured astrocytes from GCLM knockout mice, which show decreased GSH levels. No difference in the basal metabolism of glucose was observed between wild-type and knockout cells. In contrast, glycogen levels were lower and its turnover was higher in knockout astrocytes. These changes were accompanied by a decrease in the expression of the genes involved in its synthesis and degradation, including the protein targeting to glycogen. During an oxidative challenge induced by tert-Butylhydroperoxide, wild-type cells increased their glycogen mobilization and glucose uptake. However, knockout astrocytes were unable to mobilize glycogen following the same stress and they could increase their glucose utilization only following a major oxidative insult. Altogether, these results show that glucose metabolism and glycogen utilization are dysregulated in astrocytes showing a chronic deficit in GSH, suggesting that alterations of a fundamental aspect of brain energy metabolism is caused by GSH deficit and may therefore be relevant to metabolic dysfunctions observed in schizophrenia.     

Regulatory mechanisms for glycogenolysis and K uptake in brain astrocytes

Recent advances in brain energy metabolism support the notion that glycogen in astrocytes is necessary for the clearance of neuronally-released K⁺ from the extracellular space. However, how the multiple metabolic pathways involved in K⁺-induced increase in glycogen turnover are regulated is only partly understood. Here we summarize the current knowledge about the mechanisms that control glycogen metabolism during enhanced K⁺ uptake. We also describe the action of the ubiquitous Na⁺/K⁺ ATPase for both ion transport and intracellular signaling cascades, and emphasize its importance in understanding the complex relation between glycogenolysis and K⁺ uptake.     

The effects of isofagomine, a potent glycogen phosphorylase inhibitor, on glycogen metabolism in cultured mouse cortical astrocytes

A novel inhibitor of liver glycogen phosphorylase, isofagomine, was investigated as a possible inhibitor of the enzyme in the brain and in cultured astrocytes. Additionally, the effect of the drug on norepinephrine (NE) induced glycogen degradation in astrocytes was studied. Astrocytes were cultured from mouse cerebral cortex and homogenates were prepared from the cells as well as from mouse brain. Isofagomine dose-dependently inhibited glycogen phosphorylase when measured in the direction of glycogen degradation in both preparations with IC50 values (mean +/- SEM) of 1.0 +/- 0.1 microM and 3.3 +/- 0.5 microM in brain and astrocyte homogenates, respectively. Moreover, isofagomine at a concentration of 400 microM completely prevented NE induced depletion of glycogen stores and the concomitant lactate production in intact astrocytes. It is suggested that this novel glycogen phosphorylase inhibitor may be a valuable tool to investigate the functional importance of glycogen in astrocytes and in the brain.     

Characterization of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) as an inhibitor of brain glycogen shunt activity

The pharmacological properties of 1,4-dideoxy-1,4-imino- d -arabinitol (DAB), a potent inhibitor of glycogen phosphorylase and synthase activity in liver preparations, were characterized in different brain tissue preparations as a prerequisite for using it as a tool to investigate brain glycogen metabolism. Its inhibitory effect on glycogen phosphorylase was studied in homogenates of brain tissue and astrocytes and IC₅₀-values close to 400 nM were found. However, the concentration of DAB needed for inhibition of glycogen shunt activity, i.e. glucose metabolism via glycogen, in intact astrocytes was almost three orders of magnitude higher. Additionally, such complete inhibition required a pre-incubation period, a finding possibly reflecting a limited permeability of the astrocytic membrane. DAB did not affect the accumulation of 2-deoxyglucose-6-phosphate indicating that the transport of DAB is not mediated by the glucose transporter. DAB had no effect on enzymes involving glucose-6-phosphate, i.e. glucose-6-phosphate dehydrogenase, phosphoglucoisomerase and hexokinase. Furthermore, DAB was evaluated in a functional preparation of the isolated mouse optic nerve, in which its presence severely reduced the ability to sustain evoked compound action potentials in the absence of glucose, a condition in which glycogen serves as an important energy substrate. Based on the experimental findings, DAB can be used to evaluate glycogen shunt activity and its functional importance in intact brain tissue and cells at a concentration of 300–1000 μM and a pre-incubation period of 1 h.     

Human brain glycogen content and metabolism: implications on its role in brain energy metabolism

The adult brain relies on glucose for its energy needs and stores it in the form of glycogen, primarily in astrocytes. Animal and culture studies indicate that brain glycogen may support neuronal function when the glucose supply from the blood is inadequate and/or during neuronal activation. However, the concentration of glycogen and rates of its metabolism in the human brain are unknown. We used in vivo localized 13C-NMR spectroscopy to measure glycogen content and turnover in the human brain. Nine healthy volunteers received intravenous infusions of [1-(13)C]glucose for durations ranging from 6 to 50 h, and brain glycogen labeling and washout were measured in the occipital lobe for up to 84 h. The labeling kinetics suggest that turnover is the main mechanism of label incorporation into brain glycogen. Upon fitting a model of glycogen metabolism to the time courses of newly synthesized glycogen, human brain glycogen content was estimated at approximately 3.5 micromol/g, i.e., three- to fourfold higher than free glucose at euglycemia. Turnover of bulk brain glycogen occurred at a rate of 0.16 micromol.g-1.h-1, implying that complete turnover requires 3-5 days. Twenty minutes of visual stimulation (n=5) did not result in detectable glycogen utilization in the visual cortex, as judged from similar [13C]glycogen levels before and after stimulation. We conclude that the brain stores a substantial amount of glycogen relative to free glucose and metabolizes this store very slowly under normal physiology.     

Lactate produced by glycogenolysis in astrocytes regulates memory processing

When administered either systemically or centrally, glucose is a potent enhancer of memory processes. Measures of glucose levels in extracellular fluid in the rat hippocampus during memory tests reveal that these levels are dynamic, decreasing in response to memory tasks and loads; exogenous glucose blocks these decreases and enhances memory. The present experiments test the hypothesis that glucose enhancement of memory is mediated by glycogen storage and then metabolism to lactate in astrocytes, which provide lactate to neurons as an energy substrate. Sensitive bioprobes were used to measure brain glucose and lactate levels in 1-sec samples. Extracellular glucose decreased and lactate increased while rats performed a spatial working memory task. Intrahippocampal infusions of lactate enhanced memory in this task. In addition, pharmacological inhibition of astrocytic glycogenolysis impaired memory and this impairment was reversed by administration of lactate or glucose, both of which can provide lactate to neurons in the absence of glycogenolysis. Pharmacological block of the monocarboxylate transporter responsible for lactate uptake into neurons also impaired memory and this impairment was not reversed by either glucose or lactate. These findings support the view that astrocytes regulate memory formation by controlling the provision of lactate to support neuronal functions.     

Glycogen metabolism in the rat retina

It has been reported that glycogen levels in retina vary with retinal vascularization. However, the electrical activity of isolated retina depends on glucose supply, suggesting that it does not contain energetic reserves. We determined glycogen levels and pyruvate and lactate production under various conditions in isolated retina. Ex vivo retinas from light- and dark-adapted rats showed values of 44 +/- 0.3 and 19.5 +/- 0.4 nmol glucosyl residues/mg protein, respectively. The glycogen content of retinas from light-adapted animals was reduced by 50% when they were transferred to darkness. Glycogen levels were low in retinas incubated in glucose-free media and increased in the presence of glucose. The highest glycogen values were found in media containing 20 mm of glucose. A rapid increase in lactate production was observed in the presence of glucose. Surprisingly, glycogen levels were the lowest and lactate production was also very low in the presence of 30 mm glucose. Our results suggest that glycogen can be used as an immediate accessible energy reserve in retina. We speculate on the possibility that gluconeogenesis may play a protective role by removal of lactic acid.     

α₂-Adrenoceptors activate noradrenaline-mediated glycogen turnover in chick astrocytes

In the brain, glycogen is primarily stored in astrocytes where it is regulated by several hormones/neurotransmitters, including noradrenaline that controls glycogen breakdown (in the short term) and synthesis. Here, we have examined the adrenoceptor (AR) subtype that mediates the glycogenic effect of noradrenaline in chick primary astrocytes by the measurement of glycogen turnover (total (14) C incorporation of glucose into glycogen) following noradrenergic activation. Noradrenaline and insulin increased glycogen turnover in a concentration-dependent manner. The effect of noradrenaline was mimicked by stimulation of α(2) -ARs (and to a lesser degree by β(3) -ARs), but not by stimulation of α(1) -, β(1) -, or β(2) -ARs, and occurred only in astrocytes and not neurons. In chick astrocytes, studies using RT-PCR and radioligand binding showed that α(2A) - and α(2C) -AR mRNA and protein were present. α(2) -AR- or insulin-mediated glycogen turnover was inhibited by phosphatidylinositol-3 kinase inhibitors, and both insulin and clonidine caused phosphorylation of Akt and glycogen synthase kinase-3 in chick astrocytes. α(2) -AR but not insulin-mediated glycogen turnover was inhibited by pertussis toxin pre-treatment indicating involvement of Gi/o proteins. These results show that the increase in glycogen turnover caused by noradrenaline is because of activation of α(2) -ARs that increase glycogen turnover in astrocytes utilizing a Gi/o-PI3K pathway.     

Compartmentation of TCA cycle metabolism in cultured neocortical neurons revealed by 13C MR spectroscopy

Cultured neocortical neurons were incubated in medium containing [U-13C]glucose (0.5 mM) and in some cases unlabeled glutamine (0.5 mM). Subsequently the cells were "superfused" for investigation of the effect of depolarization by 55 mM K+. Cell extracts were analyzed by 13C magnetic resonance spectroscopy and gas chromatography/mass spectrometry to determine incorporation of 13C in glutamate, GABA, aspartate and fumarate. The importance of the tricarboxylic acid (TCA) cycle for conversion of the carbon skeleton of glutamine to GABA was evident from the effect of glutamine on the labeling pattern of GABA and glutamate. Moreover, analysis of the labeling patterns of glutamate in particular indicated a depolarization induced increased oxidative metabolism. This effect was only observed in glutamate and not in neurotransmitter GABA. Based on this a hypothesis of mitochondrial compartmentation may be proposed in which mitochondria associated with neurotransmitter synthesis are distinct from those aimed at energy production and influenced by depolarization. The hypothesis of mitochondrial compartmentation was further supported by the finding that the total percent labeling of fumarate and aspartate differed significantly from each other. This can only be explained by the existence of multiple TCA cycles with different turnover rates.     

Portacaval Anastomosis Results in Altered Neuron-Astrocytic Metabolic Trafficking of Amino Acids: Evidence from 13C-NMR Studies

Abstract: ¹³C-NMR spectroscopy was used to evaluate the dynamic consequences of portacaval anastomosis on neuronal and astrocytic metabolism and metabolic trafficking between neurons and astrocytes. Glutamate is predominantly labeled from [1-¹³C]glucose, whereas [2-¹³C]acetate is more efficient in labeling glutamine, in accordance with its primary metabolism in astrocytes. Alanine and succinate labeling was only observed with [1-¹³C]glucose as precursor. Brain [1-¹³C]glucose metabolism in portacaval-shunted rats was similar to that in sham-operated controls with the exception of labeled glutamine and succinate formation, which was increased in shunted rats. The ¹³C enrichment was, however, decreased owing to an increase in total glutamine and succinate. Using [2-¹³C]acetate, on the other hand, flux of astrocytic label to neurons was severely decreased because label incorporation into glutamate, aspartate, and GABA was decreased following portacaval shunting. The latter amino acids are predominantly localized in neurons. These findings demonstrate that metabolic trafficking of amino acids from astrocytes to neurons is impaired in portacaval-shunted rats.     

Metabolic differences between primary cultures of astrocytes and neurons from cerebellum and cerebral cortex. Effects of fluorocitrate

Astrocytes and neurons cultured from mouse cerebellum and cerebral cortex were analyzed with respect to content and synthesis of amino acids as well as export of metabolites to the culture medium and the response to fluorocitrate, an, inhibitor of aconitase. The intracellular levels of amino acids were similar in the two astrocytic populations. The release of citrate, lactate and glutamine, however, was markedly higher from cerebellar than from cortical astrocytes. Neurons contained higher levels of glutamate, aspartate and GABA than astrocytic cultures. Cortical neurons were especially high in GABA and aspartate, and the level of aspartate increased specifically when the extracellular level of glutamine was elevated. Fluorocitrate inhibited the TCA cycle in the astrocytes, but was less effective in cerebellar neurons. Whereas neurons responded to fluorocitrate with an increase in the formation of lactate, reflecting, glycolysis, astrocytes decreased the formation of lactate in the presence of fluorocitrate, indicating that astrocytes to a high degree synthesize pyruvate and hence lactate from TCA cycle intermediates.     

A possible role of alanine for ammonia transfer between astrocytes and glutamatergic neurons

The metabolism of [U-(13)C]lactate (1 mM) in the presence of unlabeled glucose (2.5 mM) was investigated in glutamatergic cerebellar granule cells, cerebellar astrocytes, and corresponding co-cultures. It was evident that lactate is primarily a neuronal substrate and that lactate produced glycolytically from glucose in astrocytes serves as a substrate in neurons. Alanine was highly enriched with (13)C in the neurons, whereas this was not the case in the astrocytes. Moreover, the cellular content and the amount of alanine released into the medium were higher in neurons than astrocytes. On incubation of the different cell types in medium containing alanine (1 mM), the astrocytes exhibited the highest level of accumulation. Altogether, these results indicate a preferential synthesis and release of alanine in glutamatergic neurons and uptake in cerebellar astrocytes. A new functional role of alanine may be suggested as a carrier of nitrogen from glutamatergic neurons to astrocytes, a transport that may operate to provide ammonia for glutamine synthesis in astrocytes and dispose of ammonia generated by the glutaminase reaction in glutamatergic neurons. Hence, a model of a glutamate-glutamine/lactate-alanine shuttle is presented. To elucidate if this hypothesis is compatible with the pattern of alanine metabolism observed in the astrocytes and neurons from cerebellum, the cells were incubated in a medium containing [(15)N]alanine (1 mM) and [5-(15)N]glutamine (0.5 mM), respectively. Additionally, neurons were incubated with [U-(13)C]glutamine to estimate the magnitude of glutamine conversion to glutamate. Alanine was labeled from [5-(15)N]glutamine to 3.3% and [U-(13)C]glutamate generated from [U-(13)C]glutamine was labeled to 16%. In spite of the modest labeling in alanine, it is clear that nitrogen from ammonia is transferred to alanine via transamination with glutamate formed by reductive amination of alpha-ketoglutarate. With regard to the astrocytic part of the shuttle, glutamine was labeled to 22% in one nitrogen atom whereas 3.2% was labeled in two when astrocytes were incubated in [(15)N]alanine. Moreover, in co-cultures, [U-(13)C]alanine labeled glutamate and glutamine equally, whereas [U-(13)C]lactate preferentially labeled glutamate. Altogether, these results support the role proposed above of alanine as a possible ammonia nitrogen carrier between glutamatergic neurons and surrounding astrocytes and they show that lactate is preferentially metabolized in neurons and alanine in astrocytes.