The central process in energy production is the oxidation of acetyl‐CoA to CO2 by the tricarboxylic acid (TCA, Krebs, citric acid) cycle. However, this cycle functions also as a biosynthetic pathway ...from which intermediates leave to be converted primarily to glutamate, GABA, glutamine and aspartate and to a smaller extent to glucose derivatives and fatty acids in the brain. When TCA cycle ketoacids are removed, they must be replaced to permit the continued function of this essential pathway, by a process termed anaplerosis. Since the TCA cycle cannot act as a carbon sink, anaplerosis must be coupled with cataplerosis; the exit of intermediates from the TCA cycle. The role of anaplerotic reactions for cellular metabolism in the brain has been studied extensively. However, the coupling of this process with cataplerosis and the roles that both pathways play in the regulation of amino acid, glucose, and fatty acid homeostasis have not been emphasized. The concept of a linkage between anaplerosis and cataplerosis should be underscored, because the balance between these two processes is essential. The hypothesis that cataplerosis in the brain is achieved by exporting the lactate generated from the TCA cycle intermediates into the blood and perivascular area is presented. This shifts the generally accepted paradigm of lactate generation as simply derived from glycolysis to that of oxidation and might present an alternative explanation for aerobic glycolysis.
Intermediates leave the tricarboxylic acid cycle and must be replaced by a process termed anaplerosis that must be coupled to cataplerosis. We hypothesize that cataplerosis is achieved by exporting the lactate generated from the cycle into the blood and perivascular area. This shifts the paradigm of lactate generation as solely derived from glycolysis to that of oxidation and might present an alternative explanation for aerobic glycolysis.
Intermediates leave the tricarboxylic acid cycle and must be replaced by a process termed anaplerosis that must be coupled to cataplerosis. We hypothesize that cataplerosis is achieved by exporting the lactate generated from the cycle into the blood and perivascular area. This shifts the paradigm of lactate generation as solely derived from glycolysis to that of oxidation and might present an alternative explanation for aerobic glycolysis.
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Although oligodendrocytes constitute a significant proportion of cells in the central nervous system (CNS), little is known about their intermediary metabolism. We have, therefore, characterized ...metabolic functions of primary oligodendrocyte precursor cell cultures at late stages of differentiation using isotope‐labelled metabolites. We report that differentiated oligodendrocyte lineage cells avidly metabolize glucose in the cytosol and pyruvate derived from glucose in the mitochondria. The labelling patterns of metabolites obtained after incubation with 1,2‐13Cglucose demonstrated that the pentose phosphate pathway (PPP) is highly active in oligodendrocytes (approximately 10% of glucose is metabolized via the PPP as indicated by labelling patterns in phosphoenolpyruvate). Mass spectrometry and magnetic resonance spectroscopy analyses of metabolites after incubation of cells with 1‐13Clactate or 1,2‐13Cglucose, respectively, demonstrated that anaplerotic pyruvate carboxylation, which was thought to be exclusive to astrocytes, is also active in oligodendrocytes. Using 1,2‐13Cacetate, we show that oligodendrocytes convert acetate into acetyl CoA which is metabolized in the tricarboxylic acid cycle. Analysis of labelling patterns of alanine after incubation of cells with 1,2‐13Cacetate and 1,2‐13Cglucose showed catabolic oxidation of malate or oxaloacetate. In conclusion, we report that oligodendrocyte lineage cells at late differentiation stages are metabolically highly active cells that are likely to contribute considerably to the metabolic activity of the CNS. GLIA 2016;64:21–34
Main Points
Oligodendrocytes metabolise glucose via the pentose phosphate pathway to a similar extent as astrocytes.
They have avid mitochondrial metabolism, can carboxylate pyruvate, decarboxylate malate and oxaloacetate and metabolise acetate in the mitochondria.
The malate–aspartate shuttle and the glycerol phosphate shuttle act to transfer reducing equivalents from NADH in the cytosol to the mitochondria since the inner mitochondrial membrane is impermeable ...to NADH and NAD
+. This transfer of reducing equivalents is essential for maintaining a favorable NAD
+/NADH ratio required for the oxidative metabolism of glucose and synthesis of neurotransmitters in brain. There is evidence that both the malate–aspartate shuttle and glycerol phosphate shuttle function in brain; however, there is controversy about the relative importance and cellular localization of these shuttles. The malate–aspartate shuttle is considered the most important shuttle in brain. It is particularly important in neurons and may be extremely low, or even non-existent in brain astrocytes. Several studies provide evidence of glycerol phosphate shuttle activity in brain cells; however, the activity of this shuttle in brain has been questioned. A number of pharmacological tools, including aminooxyacetic acid, β-methyleneaspartate, phenylsuccinate, and 3-nitropropionic acid, have been used to inhibit the four enzymes and two carrier proteins that participate in the malate–aspartate shuttle. Although no drugs completely inhibit the glycerol phosphate shuttle, evidence for the existence of this shuttle is provided by studies using drugs to inhibit the malate–aspartate shuttle. This report evaluates the evidence for each shuttle in brain cells and the drugs that can be used as pharmacological tools to study these shuttles.
Protoplasmic astrocytes are critically important to energy metabolism in the CNS. Our current understanding of the metabolic interactions between neurons and glia is based on studies using cultured ...cells, from which mainly inferential conclusions have been drawn as to the relative roles of neurons and glia in brain metabolism. In this study, we used functional genomics to establish the relative compartmentalization of neuronal and astrocytic metabolic pathways in the adult brain. To this end, fluorescence-activated cell sorting was used to directly isolate neurons and protoplasmic astrocytes from the cortex of adult mice. Microarray analysis showed that astrocytes and neurons each express transcripts predicting individual self-sufficiency in both glycolysis and oxidative metabolism. Surprisingly, most enzymes in the tricarboxylic acid (TCA) cycle were expressed at higher relative levels in astrocytes than in neurons. Mass spectrometric analysis of the TCA cycle intermediates confirmed that freshly isolated adult astrocytes maintained an active TCA cycle, whereas immuno-electron microscopy revealed that fine astrocytic processes encompassing synapses contained a higher density of mitochondria than surrounding cells. These observations indicate that astrocytes exhibit robust oxidative metabolism in the intact adult brain and suggest a prominent contribution of astrocytic metabolism to functional brain imaging, including BOLD (blood-oxygen level-dependent) functional magnetic resonance imaging signals.
BACKGROUND AND PURPOSE—Increased susceptibility to excitotoxicity of the neonatal brain after hypoxia-ischemia (HI) may be caused by limited capacity of astrocytes for glutamate uptake, and ...mitochondrial failure probably plays a key role in the delayed injury cascade. Male infants have poorer outcome than females after HI, possibly linked to differential intermediary metabolism.
METHODS—1-Cglucose and 1,2-Cacetate were injected at zero, 6, and 48 hours after unilateral HI in 7-day-old rats. Intermediary metabolism was analyzed with magnetic resonance spectroscopy.
RESULTS—Mitochondrial metabolism was generally reduced in the ipsilateral hemisphere for ≤6 hours after HI, whereas contralaterally, it was reduced in neurons but not in astrocytes. Transfer of glutamate from neurons to astrocytes was increased in the contralateral, but not in the ipsilateral hemisphere at 0 hour, and reduced bilaterally at 6 hours after HI. The transfer of glutamine from astrocytes to glutamatergic neurons was unaltered in both hemispheres, whereas the transfer of glutamine to GABAergic neurons was increased ipsilaterally at 0 hour. Anaplerosis (astrocytes) was decreased, whereas partial pyruvate recycling (astrocytes) was increased directly after HI. Male pups had lower astrocytic mitochondrial metabolism than females immediately after HI, whereas that of females was reduced longer and encompassed both neurons and astrocytes.
CONCLUSIONS—The prolonged depression in mitochondrial metabolism indicates that mitochondria are vulnerable targets in the delayed injury after neonatal HI. The degree of astrocytic malfunction may be a valid indicator of outcome after hypoxic/HI brain injury and may be linked to the differential outcome in males and females.
Glutamate is the major excitatory neurotransmitter, and is inactivated by cellular uptake catalyzed mostly by the glutamate transporter subtypes GLT‐1 (EAAT2) and GLAST (EAAT1). Astrocytes express ...both GLT‐1 and GLAST, while axon terminals in the neocortex only express GLT‐1. To evaluate the role of GLT‐1 in glutamate homeostasis, we injected GLT‐1 knockout (KO) mice and wild‐type littermates with 1‐13Cglucose and 1,2‐13Cacetate 15 min before euthanization. Metabolite levels were analyzed in extracts from neocortex and cerebellum and 13C labeling in neocortex. Whereas the cerebellum in GLT‐1‐deficient mice had normal levels of glutamate, glutamine, and 13C labeling of metabolites, glutamate level was decreased but labeling from 1‐13C glucose was unchanged in the neocortex. The contribution from pyruvate carboxylation toward labeling of these metabolites was unchanged. Labeling from 1,2‐13C acetate, originating in astrocytes, was decreased in glutamate and glutamine in the neocortex indicating reduced mitochondrial metabolism in astrocytes. The decreased amount of glutamate in the cortex indicates that glutamine transport into neurons is not sufficient to replenish glutamate lost because of neurotransmission and that GLT‐1 plays a role in glutamate homeostasis in the cortex.
Glutamate is the major excitatory neurotransmitter, and is inactivated by uptake via GLT‐1 (EAAT2) and GLAST (EAAT1) transporters, while axon terminals in the neocortex only express GLT‐1. To evaluate the role of GLT‐1 in glutamate homeostasis, we used 1‐13Cglucose and 1,2‐13Cacetate injection and NMR spectroscopy. The results indicate that glutamine transport into neurons is not sufficient to replenish glutamate lost because of neurotransmission and that GLT‐1 plays a role in glutamate homeostasis in the neocortex.
Glutamate is the major excitatory neurotransmitter, and is inactivated by uptake via GLT‐1 (EAAT2) and GLAST (EAAT1) transporters, while axon terminals in the neocortex only express GLT‐1. To evaluate the role of GLT‐1 in glutamate homeostasis, we used 1‐13Cglucose and 1,2‐13Cacetate injection and NMR spectroscopy. The results indicate that glutamine transport into neurons is not sufficient to replenish glutamate lost because of neurotransmission and that GLT‐1 plays a role in glutamate homeostasis in the neocortex.
The process of cell differentiation goes hand‐in‐hand with metabolic adaptations, which are needed to provide energy and new metabolites. Carbon monoxide (CO) is an endogenous cytoprotective molecule ...able to inhibit cell death and improve mitochondrial metabolism. Neuronal differentiation processes were studied using the NT2 cell line, which is derived from human testicular embryonic teratocarcinoma and differentiates into post‐mitotic neurons upon retinoic acid treatment. CO‐releasing molecule A1 (CORM‐A1) was used do deliver CO into cell culture. CO treatment improved NT2 neuronal differentiation and yield, since there were more neurons and the total cell number increased following the differentiation process. CO supplementation enhanced the mitochondrial population in post‐mitotic neurons derived from NT2 cells, as indicated by an increase in mitochondrial DNA. CO treatment during neuronal differentiation increased the extent of the classical metabolic change that occurs during neuronal differentiation, from glycolytic to more oxidative metabolism, by decreasing the ratio of lactate production and glucose consumption. The expression of pyruvate and lactate dehydrogenases was higher, indicating an augmented oxidative metabolism. Moreover, these findings were corroborated by an increased percentage of 13C incorporation from U‐13Cglucose into the tricarboxylic acid cycle metabolites malate and citrate, and also glutamate and aspartate in CO‐treated cells. Finally, under low levels of oxygen (5%), which enhances glycolytic metabolism, some of the enhancing effects of CO on mitochondria were not observed. In conclusion, our data show that CO improves neuronal and mitochondrial yield by stimulation of tricarboxylic acid cycle activity, and thus oxidative metabolism of NT2 cells during the process of neuronal differentiation.
The process of cell differentiation is coupled with metabolic adaptations. Carbon monoxide (CO) is an endogenous cytoprotective gasotransmitter able to prevent cell death and improve mitochondrial metabolism. Herein CO supplementation improved neuronal differentiation yield, by enhancing mitochondrial population and promoting the classical metabolic change that occurs during neuronal differentiation, from glycolytic to oxidative metabolism.
The process of cell differentiation is coupled with metabolic adaptations. Carbon monoxide (CO) is an endogenous cytoprotective gasotransmitter able to prevent cell death and improve mitochondrial metabolism. Herein CO supplementation improved neuronal differentiation yield, by enhancing mitochondrial population and promoting the classical metabolic change that occurs during neuronal differentiation, from glycolytic to oxidative metabolism.
Summary It has been suggested that loss of the astrocytic water channel aquaporin-4 (AQP4) from perivascular endfeet in sclerotic hippocampi contributes to increased seizure propensity in human ...mesial temporal lobe epilepsy (MTLE). Whether this loss occurs prior to or as a consequence of epilepsy development remains to be resolved. In the present study, we investigated whether the expression and distribution of AQP4 was altered prior to (i.e., in the latent phase) or after the onset of chronic epileptic seizures (i.e., in the chronic phase) in the kainate (KA) model of MTLE. Immunogold electron microscopic analysis revealed that AQP4 density in adluminal endfoot membranes was reduced in KA treated rats already in the latent phase, while the AQP4 density in the abluminal endfoot membrane was stable or slightly increased. The decrease in adluminal AQP4 immunogold labeling was accompanied by a reduction in the density of AQP4's anchoring protein alpha-syntrophin. The latent and chronic phases were associated with an upregulation of the M1 isoform of AQP4, as judged by semi-quantitative Western blot analysis. Taken together, the findings in this model suggest that a mislocalization of AQP4 – reflecting a loss of astrocyte polarization – is an integral part of the epileptogenic process.
Glutamine (Gln) is synthesized in astrocytes from glutamate (Glu) and ammonia, whereupon it can be released to be transferred to neurons. This study evaluated the as yet not definitely established ...role of the astrocytic Gln transporters SN1 and SN2 (Slc38a3 and Slc38a5 respectively) in Gln release and metabolic fluxes of glucose and acetate, the canonical precursors of Glu. Cultured neocortical astrocytes were grown in the absence or presence of ammonia (5 mM NH4Cl, 24 h), which deregulates astrocytic metabolism in hyperammonemic encephalopathies. HPLC analyses of cell extracts of SN1/SN2 siRNA‐treated (SN1/SN2−) astrocytes revealed a ~ 3.5‐fold increase in Gln content and doubling of glutathione, aspartate, alanine and glutamate contents, as compared to SN1/SN2+ astrocytes. Uptake and efflux of preloaded 3HGln was likewise significantly decreased in SN1/SN2− astrocytes. The atom percent excess 13C values (given as M + 1) for alanine, aspartate and glutamate were decreased when the SN1/SN2− cells were incubated with 1‐13C glucose, while Gln consumption was not changed. No difference was seen in M + 1 values in SN1/SN2− cells incubated with 2‐13C acetate, which were not treated with ammonia. In SN1/SN2− astrocytes, the increase in Gln content and the decrease in radiolabeled Gln release upon exposure to ammonia were found abrogated, and glutamate labeling from 2‐13Cacetate was decreased as compared to SN1/SN2+ astrocytes. The results underscore a profound role of SN1 and/or SN2 in Gln release from astrocytes under physiological conditions, but less so in ammonia‐overexposed astrocytes, and appear to manifest dependence of astrocytic glucose metabolism to Glu/Gln on unimpaired SN1/SN2− mediated Gln release from astrocytes.
The astrocytic N system transporters SN1 and SN2 show preponderance to mediate glutamine (Gln) efflux. Under hyperammonemic conditions, accumulation of Gln, a direct product of ammonia detoxification, may deregulate astrocytic metabolism and seems to be responsible for astrocytic swelling. This study evaluated not definitely established role of SN1 and SN2 in Gln release and metabolic fluxes of radiolabeled glucose and acetate. Simultaneous silencing of SN1/SN2 transporters increase Gln, glutathione, aspartate, alanine and glutamate contents (Panel B; marked in red) as compare to non‐silenced astrocytes (Panel A). The atom percent excess 13C values (given as M + 1) for alanine, aspartate and glutamate were decreased when the cells with silenced transporters were incubated with 1‐13Cglucose, whereas no difference was seen in M + 1 values when those cells were incubated with 2‐13Cacetate. Ammonia abrogated the increase in Gln content and decrease in radiolabeled Gln release in astrocytes with silenced transporters, but caused a decrease in glutamate labeling from 2‐13Cacetate.
The astrocytic N system transporters SN1 and SN2 show preponderance to mediate glutamine (Gln) efflux. Under hyperammonemic conditions, accumulation of Gln, a direct product of ammonia detoxification, may deregulate astrocytic metabolism and seems to be responsible for astrocytic swelling. This study evaluated not definitely established role of SN1 and SN2 in Gln release and metabolic fluxes of radiolabeled glucose and acetate. Simultaneous silencing of SN1/SN2 transporters increase Gln, glutathione, aspartate, alanine and glutamate contents (Panel B; marked in red) as compare to non‐silenced astrocytes (Panel A). The atom percent excess 13C values (given as M + 1) for alanine, aspartate and glutamate were decreased when the cells with silenced transporters were incubated with 1‐13Cglucose, whereas no difference was seen in M + 1 values when those cells were incubated with 2‐13Cacetate. Ammonia abrogated the increase in Gln content and decrease in radiolabeled Gln release in astrocytes with silenced transporters, but caused a decrease in glutamate labeling from 2‐13Cacetate.
Glucose is the primary energy substrate for the adult mammalian brain. However, lactate produced within the brain might be able to serve this purpose in neurons. In the present study, the relative ...significance of glucose and lactate as substrates to maintain neurotransmitter homeostasis was investigated. Cultured cerebellar (primarily glutamatergic) neurons were superfused in medium containing U-13Cglucose (2.5 mmol/L) and lactate (1 or 5 mmol/L) or glucose (2.5 mmol/L) and U-13Clactate (1 mmol/L), and exposed to pulses of N-methyl-D-aspartate (300 μmol/L), leading to synaptic activity including vesicular release. The incorporation of 13C label into intracellular lactate, alanine, succinate, glutamate, and aspartate was determined by mass spectrometry. The metabolism of U-13Clactate under non-depolarizing conditions was high compared with that of U-13Cglucose; however, it decreased significantly during induced depolarization. In contrast, at both concentrations of extracellular lactate, the metabolism of U-13Cglucose was increased during neuronal depolarization. The role of glucose and lactate as energy substrates during vesicular release as well as transporter-mediated influx and efflux of glutamate was examined using preloaded D-3Haspartate as a glutamate tracer and DL-threo-β-benzyloxyaspartate to inhibit glutamate transporters. The results suggest that glucose is essential to prevent depolarization-induced reversal of the transporter (efflux), whereas vesicular release was unaffected by the choice of substrate. In conclusion, the present study shows that glucose is a necessary substrate to maintain neurotransmitter homeostasis during synaptic activity and that synaptic activity does not induce an upregulation of lactate metabolism in glutamatergic neurons.
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