The astrocyte biochemistry

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Review

The astrocyte biochemistry Débora G. Souzaa,1, Roberto F. Almeidaa,f,1, Diogo O. Souzaa,b, Eduardo R. Zimmera,c,d,e,

⁎,2

a

Graduate Program in Biological Sciences: Biochemistry, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil Department of Biochemistry, UFRGS, Porto Alegre, Brazil Department of Pharmacology, UFRGS, Porto Alegre, Brazil d Graduate Program in Biological Sciences: Pharmacology and Therapeutics, UFRGS, Porto Alegre, Brazil e Brain Institute of Rio Grande do Sul (BraIns), Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Brazil f Exact and Biological Sciences Institute, Biological Sciences Department, Federal University of Ouro Preto, Ouro Preto, Brazil b c

HIGHLIGHTS

a subset of glial cells, are essential for regulating metabolic fuel availability in the brain. • Astrocytes, have at least two sources of glucose: the bloodstream or glycogen, which they convert to lactate for export. • Astrocytes may oxidize fatty acids and produce ATP and ketone bodies with it, depending on the metabolic status. • Astrocytes trough energy-demanding processes, take up glutamate and GABA. • Astrocytes, • Glutamate and GABA can also be ATP sources. ARTICLE INFO

ABSTRACT

Keywords: Astrocyte Biochemistry Glucose Fatty acids Glutamate

Astrocytes are a unique and dynamic subtype of glial cells in the central nervous system (CNS). Understanding their biochemical reactions and their influence in the surrounding cells is extremely important in the neuroscience field. They exert important influence in the neurotransmission, ionic homeostasis and also release neuroactive molecules termed gliotransmitters. Additionally, they metabolize, store and release metabolic substrates to meet high brain energy requirements. In this review, we highlight the main biochemical reactions regarding energy metabolism that take place in astrocytes. Special attention is given to synthesis, storage and catabolism of glucose, release of lactate, oxidation of fatty acids, production of ketone bodies, and metabolism of the main neurotransmitters, glutamate and GABA. The recent findings allow proposing these cells as key players controlling the energetic homeostasis in the CNS.

1. Introduction At this precise moment, uncountable biochemical reactions are happening in your brain when you read and decode this article’s first sentence. The biochemistry of the brain has always focused in the understanding of metabolic routes acknowledging neurons as the main functional cellular entity. However, the brain is a complex system in which several cell types are constantly and coordinately exchanging information/metabolites in a sub-second frequency [1,2]. Thus, biochemical reactions may occur in a refined, controlled and efficient manner to maintain brain homeostasis. In a simplified approach, it is possible to classify brain cells in two main subtypes: neurons or glial

cells [2]. The neuronal role in brain activity has been covered by an extensive literature [3–6]. In the last years, more attention has been given to glial cells. Glial cells are also essential for brain functions, working in a highly coupled and synchronized way with neurons. They work together to assure the acquisition, processing and storage of information, allowing the maintenance of efficient, fast and precise synaptic processes [1,7]. Glial cells have been delighting scientists for a long time as emblematically demonstrated by Santiago Jamón y Cajal’s drawings [8]. Since the end of the nineteenth century, Jamón y Cajal made several important observations that helped to pave the way for a better understanding of glial cells structure and function [9]. Glia is the Greek

Corresponding author at: Department of Pharmacology, 500 Sarmento Leite St, Farroupilha, 90035–003, Porto Alegre, RS, Brazil. E-mail address: [email protected] (E.R. Zimmer). 1 These authors contributed equally. 2 www.zimmer-lab.org. ⁎

https://doi.org/10.1016/j.semcdb.2019.04.002 Received 3 November 2018; Received in revised form 19 March 2019; Accepted 1 April 2019 1084-9521/ © 2019 Published by Elsevier Ltd.

Please cite this article as: Débora G. Souza, et al., Seminars in Cell and Developmental Biology, https://doi.org/10.1016/j.semcdb.2019.04.002

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term equivalent to "glue", intuitively suggesting that these cells are only physically supporting neurons. However, astrocytes are much more talented and efficient then it was first thought [1,10]. Quantifying the precise numbers of neuronal and glial cells in the adult human brain is a challenging task but the development of new techniques is contributing to refining the estimation of overall cell number in the brain and the neuron/glia ratio [11]. It is important to emphasize that several different studies pointed that the neuron/glia ratio in human brain is intensively variable among brain structures and also among sub regions in each structure [12,13]. The firstly proposed human brain 1:10 neuron/glia ratio has been replaced by compelling evidences indicating a 1:1 neuron/glia ratio [11,12,14]. Differences in neuron/glia ratio seem evolutionarily conserved in mammals and do not determine a species cognitive ability. However, the human glia seems more complex and efficient than non-human mammals glia [11]. In addition, it is important to note that in the human cerebral cortex, our most specialized brain region, there are 60.84 billion glial cells and only 16.34 billion neurons [15–17]. Glial cells are divided in three main cell types: oligodendrocytes, astrocytes (both recognized as macroglia) and microglia. These cells are constantly collaborating with neurons and with each other to accurately accomplish functional tasks in the CNS [1,2,18–21]. Astrocytes are the most versatile cells in the CNS, having multiple layers of complexity and the ability to interact with neighboring cell types in the nervous tissue and to modulate their function [19,22,23]. Interestingly, the term astrocyte is derived from “astron”, a Greek word that means star, due to its characteristically stellar shape morphology [24]. The astroglia, another term used to address these cells collectively, are involved in virtually every aspect of the brain’s functioning. Therefore, proper functioning of the CNS is directly related to the proper integrative functioning of astrocytes [25–27]. Such aspects are possible due to the presence of a high number of very thin astrocytic processes that extend from the soma and enwrap synapses and blood vessels. Through this tight communication pathway, they are able to directly interact with neurons and modulate synapses [28,29]. Interestingly, features classically related to other brain cells are currently also being attributed to astrocytes. For example, the creation, modulation and elimination of synapses, were, at first, thought to be exclusive neuronal functions, and nowadays are admittedly controlled by glial cells [10,30–32]. Moreover, astrocytes are involved in important metabolic functions such as maintaining the ion homeostasis, neurotransmitter clearance, energetic metabolism and modulating the interactions among various synapses [33], therefore, crucially impacting learning, behavior and cognition [10,34–36]. Astrocytes play pivotal roles in the formation and maintenance of the blood brain barrier (BBB), closely controlling the input of glucose and water into the CNS [22,37]. In the extracellular context,they manage K+ and neurotransmitters concentration, providing suitable enviroment for synaptic transmission [21,38–42]. In addition, it is known that they contribute with the release of neuroactive molecules, which are now called gliotransmitterslike D-serine, adenosine triphosphate (ATP) and glutamate [23,43]. The astrocyte metabolism is very active, but not only for its own benefit. Several studies already showed the importance of astrocytes supplying precursors for ATP generation to meet neuronal action potential requirements. These energy precursors are derived in great part from glycogen degradation and high rates of the glycolytic pathway in the astrocytic compartment [44,45]. According to their morphology and anatomical location, astrocytes are currently divided into two main subtypes, namely protoplasmatic and fibrous (Fig. 1) [46]. Protoplasmatic astrocytes are present in the gray matter and possess several branches that reach synapses and the vascular endothelium (neurovascular unity) [47]. This subtype is responsible for most of the known astrocyte’s functions. Fibrous astrocytes also reach the endothelium but have fewer branches, are located in the white matter and contact the nodes of Ranvier [46]. It is still not clear the role that this subtype plays in brain function and more studies

are necessary to better elucidate their metabolic contribution to the CNS environment. Through the connection by gap junctions, hundreds of astrocytes might be in constant communication and interchange of substances, working as a syncytial-like structure to ensure the K+ buffering and the detoxification of toxins (metabolic waste) [48,49]. This simplistic classification system will for sure be expanded in the near future since it is intuitive that astrocytes are a very heterogeneous class of cells. Making a parallel, how many neuronal types do we know? In specific situations, neuronal morphology and location do not allow to identify the neuronal phenotype. The brain cells complexity is still far from being precisely elucidated, since we are still deciphering new cellular phenotypes such as the recently discovered "rosehip neuron" [50]. The heterogeneity of astrocytes has now gained more attention but we are still far from having a definitive classification of them. Recently, a great and authoritative review has covered the physiology of astroglia but the information about biochemical routes has been only briefly mentioned [51]. In fact, the information on the astrocyte biochemistry is still widely scattered in more than 10 thousand works [number based on a PubMed (US National Library of Medicine) search using the terms “astrocyte” and “biochemistry” performed on 01/21/ 2019]. In this review, we aim to integrate all this bulk of biochemical evidence and discuss the astrocyte biochemistry, focusing on metabolic routes, metabolites and neuron-glia interaction in the brain. 2. Metabolic substrate for astroglia means metabolic fuel for the brain The morphological location of astrocytes around the endothelic vasculature is a key fact to understand their importance in fuel availability in the brain [37,52,53]. Astrocytes promote the linkage between the endothelial blood flux and neurons, and exert important contributions to the formation and maintenance of the BBB [22,37,54]. The BBB, a functional unit composed by astrocytes, pericytes and endothelial cells, is the regulatory gatekeeper in the CNS and possesses highly selective mechanisms of transport. The BBB selectivity, a coordinate action of endothelial cells and astrocytes, defines which are the peripheral cells, electrolytes and xenobiotics allowed to migrate to the CNS [22,37]. As a part of the BBB, by releasing vasoactive substances through their endfeet, the astrocytes are able to modulate the blood flow in arterioles to increase the oxygen and nutrient offer when the ATP demand is high [55]. At the same time, they are severely strict regarding the substances that will indeed cross the BBB [22,37]. The release of glial nitric oxide and a metabolite of the arachidonic acid have been proposed as some of the glial mediators of parenchymal arteriole vascular tone [56,57]. This allows a very well controlled exchange between blood and CNS cells, which is mediated by transporter proteins with unique features, such as solute carriers present in astrocytic membranes [58–60]. The nutrient offer, specifically glucose and lactate, is also possible due to glycogen granules present in astroglial cytoplasm that undergo glycogenolysis under catabolic stimulus. They also have the ability to generate glucose de novo through the gluconeogenesis pathway, an astrocyte-specific pathway in the CNS, under particular circumstances [61]. This helps to provide the main substrates to other cells, considering that energetic substrates in astrocytes can be released as energy precursors. In this context, the main energy substrates oxidized by astrocytes are glucose, amino acids and fatty acids (FAs) [62–64]. 2.1. Glycogen and gluconeogenesis Glycogen synthesis in astrocytes has been shown to respond to glucose availability and to insulin signaling. Indeed, under insulin stimulus astroglial glycogen synthase is dephosphorylated and active [52,65,66] (Fig. 2). Glycogen storage in astrocytes varies from 3 to 12 μmol/g tissue in humans, which is around a hundred times less than 2

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Fig. 1. Astrocyte morphology. Astrocytes are abundantly distributed in the mammals’ brain. a) Astrocytic marker glial fibrillary acidic protein (GFAP) staining in the CA region of rat hippocampus. b) Fibrous-shaped astrocyte scheme. c) Protoplasmic-shaped astrocyte scheme. d) Immunohistochemistry for GFAP in the human cortex shows star-shaped cells (Image credit: Human Protein Atlas, available from https:// www.proteinatlas.org/ENSG00000131095GFAP/tissue/cerebral+cortex[137]). e) Immunofluorescence for GFAP in the piriform area of the mouse brain (Image credit: Human Protein Atlas, available from https://www.proteinatlas. org/ENSG00000131095-GFAP/tissue/mouse +brain [137]).

the liver storage [66]. However, astroglial glycogenolysis is crucial for brain functioning especially in short periods of higher neuronal activation where the concentration of adenosine monophosphate (AMP) rises acting as an positive allosteric modulator of glycogen phosphorylase [67]. More specifically, there is an increasing bulk of evidence showing the role of astrocytes in supporting axonal function during periods of high energetic demand and providing metabolic support during less availability of glucose [66]. Since glucose is stored in the form of glycogen, the AMP stimulus allows for immediate increase in intracellular glucose concentration [67–69]. In fact, Öz and colleagues have shown that in a moderate hypoglycemic event, glycogen is degraded to meet the glucose need. Interestingly, after the glycaemia reestablishment, the glycogen levels in astrocytic cytoplasm rise higher than normal. One could propose this phenomenon as a compensatory mechanism that contributes to hypoglycemia unawareness observed in a few pathological conditions [70]. Intriguingly, it is speculated that during fasting astrocytes are able to perform gluconeogenesis [71]. Gluconeogenesis is a multistep metabolic pathway that synthesizes glucose from non-carbohydrate precursors, such as pyruvate, lactate, glycerol and amino acids [72] (Fig. 2). Indeed, the gluconeogenic capacity of astrocytes has been suggested as a mechanism to assure adequate glucose concentration in the astroglia under particular conditions such as ischemic stroke and glioma [71]. If glucose produced by astrocytes is capable of flowing to neurons is still a matter of debate. Another key unanswered question is whether glucose can flow from blood through astrocytes to neurons. Both hypotheses cannot be excluded since astrocytes express functional glucose-6-phosphatase [68,71]. A recent study by Müller and colleagues showed that astrocytes are able to sequester glucose in endoplasmic reticulum (ER) compartment through the activity of glucose6-phosphatase to assure that hexokinase is not inhibited by glucose-6phosphate cytosolic concentration and allow glucose transport to perisynaptic site [73,74]. In such way, astrocytes would have two pools of glucose - one stored as glycogen and other transiently stored in the ER. Still, it is important to highlight that no robust evidence of glucose release by astrocytes has been shown so far.

In astrocytes, gluconeogenesis is fed by aspartate, alanine, glutamine and lactate [71]. These cells possess the key gluconeogenic enzymes, pyruvate carboxylase [75], which converts pyruvate to oxaloacetate, and the bifunctional enzyme 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase isoform 3, necessary for reciprocal regulation of glycolysis and gluconeogenesis [71,76]. The contribution of gluconeogenesis for in situ glucose generation in the brain could be marginal, however, more studies are necessary to clarify the astrocytic gluconeogenesis contribution to brain energetic homeostasis in physiological conditions. 2.2. Glucose and lactate It was long thought that only glucose was suitable to satisfy the high brain energy requirements [77,78]. Glucose may be the essential and preferential energetic substrate in the brain, although nowadays it is known that other substrates contribute to meet the high ATP requirement [79,80]. Through the facilitated transport by GLUT1, a glucose specific transporter, astrocytes take up glucose [81], metabolize it for its own use or distribute its derivatives to the other cells. It has been previously shown that the glucose content in astrocytes and neurons is similar, however, the metabolic destination in each cell seems to be different [76,82]. Glycolytic pathway is constantly active in astrocytes with a primary goal of generating pyruvate [78] (Fig. 2). Neurons also metabolize large amounts of glucose but the glycolytic pathway is not favored in these cells. Actually, neurons possess very few amounts of fructose-2,6-biphosphate, which is needed for phosphofructokinase 1 activation, and, therefore, for glycolysis [76,83]. So, in neurons, most glucose flows through the pentose phosphate pathway, generating ribose and reducing power in the form of nicotinamide adenine dinucleotide phosphate hydrogen (NADPH), to meet the biosynthetic pathways and the antioxidant requirements [76]. Based on the aforementioned, a key question arises. Which metabolic substrate would release energy and generate ATP in neurons? In 1994, Pellerin and Magistretti proposed for the first time a coupling mechanism between astrocytic glycolysis and neuronal ATP generation, 3

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Fig. 2. Astrocytes assume different profiles of glucose metabolism in anabolic and catabolic phases. When insulin is present, astrocytes take up glucose and store it as glycogen in the cytosol. Glycolytic pathway is activated by the presence of Fru-2,6-biP. Glycolysis yields pyruvate and, subsequently, lactate for the replenishment of NAD+ pool. Lactate can be released to extracellular compartment. During the catabolic phase, glycogen is degraded under AMP stimulus and glucose can be metabolized. Under specific conditions, astrocytes can produce glucose through gluconeogenesis. GLUT1: glucose transporter 1; Fru-2,6-biP: Fructose-2,6-biphosphate; MCT: monocarboxylate transporter; AA: amino acids; AMP: adenosine monophosphate; UDP: uridine diphosphate.

a model described as the “astrocyte-to-neuron lactate shuttle” (ANLS) hypothesis [84] (for review see [85]). The general idea is that astrocytes oxidize glucose and the end product, pyruvate, is further oxidized in small amounts because, in astrocytes, the pyruvate dehydrogenase complex (PDHC) is inhibited by phosphorylation [86]. Then, pyruvate is converted to lactate through lactate dehydrogenase (which oxidizes NADH and keeps the glycolytic pathway running, favoring the glyceraldehyde 3-phosphate dehydrogenase reaction) and shuttled to neurons through monocarboxylate transporters (MCTs) [58,85,87]. In neurons, lactate is converted back to pyruvate and, subsequently to acetyl-CoA to enter in the tricarboxylic acid (TCA) cycle. This coupling proposed by the ANLS model seems pivotal for long-term potentiation and memory acquisition, reinforcing the undoubted importance of astroglial glucose metabolism for maintaining brain function [88,89].The bulk of evidence favoring the ANLS hypothesis is becoming each day more robust. Nevertheless, mechanisms behind brain energetics remain a highly debated topic, not fully resolved [90,91]. A very interesting discussion about this topic is available at https://physoc.onlinelibrary.wiley.com/ action/downloadSupplement?doi=10.1113%2FJP274944&file= tjp12731-sup-0001_Comments.pdf. So far, three MCT isoforms have been described in the brain: MCT1, MCT2 and MCT4 [58,92,93]. Of note, these isoforms are remarkably

unique in terms of activity, mainly due to their striking differences in KM for energetic substrates, such as lactate and ketone bodies (KBs). Endothelial cells of microvessels express MCT1, as well as ependymocytes and astrocytes [58,92]. MCT1 was reported to have between 3.5 and 6 mM KM for lactate [58,92,93]. MCT4 expression appears to be specific in astrocytes in the CNS [94] and to have the highest KM for lactate, varying from 22.0 to 28.0 mM. – [58,93]. Interestingly, as response to hypoxia, ischemia or inflammation, astrocytes increase MCT4 expression [58]. In astroglial cellular models, MCTs expression varies depending on culture conditions. For instance, in cellular models there is high availability of oxygen, which downregulates MCT4 expression [95]. These important biochemical differences should be considered when using in vitro models for studying cerebral properties. MCT2 is predominantly located in neurons and has the lowest KM for lactate (0.7 mM) [92,96]. MCT2 has an important role on providing energy substrates during synaptic transmission demands [58]. Of note, low KM per se does not provide information about the direction of substrate flux. However, considering the low lactate neuronal production [97], it is likely that MCT2 plays a major role in importing lactate from the extracellular compartment [89]. MCTs specific KM for pyruvate, lactate and KBs (β-hydroxybutyrate - β−OHB - and acetoacetate) are depicted in Table 1. 4

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Table 1 Cellular distribution and affinities (KM values) for different substrates of monocarboxylate transporters (MCTs) in the CNS. Transporter

MCT1 (Astrocytes and endothelial cells – BBB) MCT2 (Neurons) MCT4 (Astrocytes)

Substrate Pyruvate (mM)

Lactate (mM)

L-βOH (mM)

Acetoacetate (mM)

1.0 [58,92,93] 0.08-0.1 [58,92,93,96] 150 -153 [58,92,93,96]

3.5 – 10 [58,92,93,96] 0.5 – 0.75 [58,92,93,96]

11.4 – 12.5 [92,93,107]

5.5 [92,93,107] 0.8 [92,93,107] 216 ± 27 [107]

22.0-28.0 [92,93,96]

2.3. Fatty acids and ketone bodies

1.2 [92,93,107] 824 [107]

comparison of the pace-setting step enzyme in FAs oxidation, carnitine acyltransferase 1 (CAT1), isolated from astrocytes and hepatocytes showed that they exhibited similar properties [112]. Thus, it seems that the regulatory properties and kinetics for FAs oxidation and KBs production are similarly regulated in hepatocytes and astrocytes (for review see [106]). In both cell types, the β-oxidation products and leucine metabolism favor the activity of KBs synthesizing enzymes [108,111,113]. Indeed, specific enzymatic machinery for KBs synthesis present in astrocytes includes the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase and HMG-CoA liase [105,109,114]. The signaling exerted by increased levels in cyclic AMP (cAMP) in astrocytes, a typical fasting signal, is the main stimulus for on-site synthesis of KBs [115]. This signaling pathway leads to phosphorylation and inactivation of the acetyl-CoA carboxylase, which decreases the malonyl-CoA concentration and allows CAT1 to transport FAs inside the mitochondria, where ketogenesis occurs. Importantly, β−OHB, a water-soluble KB, is released as fuel by astrocytes to supply other brain cells [108]. Interestingly, one study investigating the substrate oxidative metabolism in brain cellular models showed that KBs oxidation by neurons and oligodendrocytes is three fold more efficient than by astrocytes [105]. KBs oxidation generates acetyl-CoA and feeds the TCA cycle. Considering the current knowledge, it seems intuitive that the astrocytes have the capacity to take up, synthesize and release β−OHB. Altogether, these observations support that KBs produced by astrocytes can be used as important substrate for neuronal ATP synthesis in situations of impaired glucose homeostasis. Yet, in the adult brain, the importance of astrocytic ketogenesis is elusive. During fasting periods, the increase in KBs levels, generated in situ, could contribute to the switch in energy substrate utilization from glucose to KBs. Based on that, one can speculate that the ANLS could be slowly and transiently adapted to an "astrocyte-to-neuron ketone shuttle" [106]. Of course, more experimental data are needed to corroborate this hypothesis.

The FAs are derived from the adipose tissue, which is the major energy storage site in mammals. They are distributed in the systemic circulation carried by albumin or lipoproteins, where FAs are present in the form of triacylglycerol [98]. The generation of metabolic energy as ATP in the body highly relies on β-oxidation, the FAs degradation pathway [99]. Under normal circumstances, brain cells metabolize alternative substrates but FAs oxidation in cerebral tissue has always been a matter of skepticism. Apparently astrocytes are the only cell type in the brain capable of oxidizing FAs [62]. Still, there are several physiological reasons to believe that sustained FAs oxidation in the brain is harmful [100]. Based on the high FADH2/NADH ratio generated in β-oxidation, one could propose that persistent FAs oxidation in the brain could trigger mitochondrial impairment and oxidative stress, as has been shown in skeletal muscle mitochondria [101–103]. In addition, brain low β-oxidation capacity would be a limiting factor to use these metabolic substrates as major energy suppliers in the brain [100]. Yet, concomitantly to the glycolytic pathway, it is likely that other energy pathways are actively providing energy to the brain [78]. In this context, most of the pyruvate generated by glycolysis cannot be converted to acetyl-CoA in astrocytes since PDHC is constantly phosphorylated, i.e., it is inactive. In neurons, the PDHC activity operates in a maximum rate, which subsequently allows pyruvate conversion to acetyl-CoA, an essential substrate of tricarboxylic acid (TCA) cycle [80,86]. Therefore, in astrocytes, ATP needs should be met, at least partially, by FAs β-oxidation [79,80]. Indeed, it is estimated that approximately 20% of the total energy consumption in adult brain is satisfied by β-oxidation of short and medium chain FAs (Fig. 3) [62]. In a lower rate, long chain FAs can also be metabolized by astrocytes [104,105]. It has been reported that mitochondria isolated from neurons or astrocytes are capable of oxidizing FAs if supplied along with other respiratory components, which mimics substrates offer in vivo [80]. Astrocytes located in the vicinity of synaptic processes are especially rich in mitochondria. They synthesize high amounts of ATP to fuel Na+/K+ ATPase aiming at maintaining Na+ out of the cytosol. This process ensures neurotransmitters uptake by astrocytes – predominantly glutamate – from the synaptic cleft through active co-transport with Na+ [41]. Accordingly, β-oxidation in perisynaptic mitochondria can contribute to generate ATP and maintain astrocytic functions. High rates of β-oxidation in the periphery are the signal for triggering hepatic ketogenesis. It is important to emphasize that the liver supplies most of the KBs oxidized by the brain when circulating glucose is scarce. Particular conditions – including prolonged fasting, uncontrolled diabetes and breastfed newborn babies – increase circulating β−OHB and acetoacetate. In such cases, the brain slowly adapts to the use of KBs to preserve neuronal synaptic function and structural stability [59,106,107]. Based on the evidences above mentioned, one could argue that β-oxidation in astrocytes also leads to on-site production of KBs under these particular conditions. Although the liver is the major source of KBs in the body, astrocytes are the only production source of KBs in the brain [105,108–111]. The

3. Neurotransmitter metabolism Considering the wide range of functions that astroglia performs in the brain, it is necessary to mention the role they play in neurotransmitter metabolism and its implications for energetic substrates management [59,61]. Astrocytes express several transporters, allowing them to modulate synaptic activity by taking up the neurotransmitters glutamate and γ-aminobutyric acid (GABA) from synaptic cleft [41]. Among the astroglial functions, the neurotransmitter management is one of the most remarkable because it associates astroglial cells directly to a position of control in the synapse duration, strength and elimination [116]. Astrocytes account also for the de novo synthesis of glutamate and GABA since they express pyruvate carboxylase and produce these amino acids from the glycolysis end-product and from other amino acids [38,75]. Since both glutamate and GABA may be oxidized, their de novo synthesis is mandatory to replenish the oxidized amount. The role played by astrocytes in glutamate and GABA metabolism is discussed in the following sections (Fig. 4). 5

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Fig. 3. Oxidation of FAs, ketogenesis and differential pathways followed by energy substrates in astrocytes and neurons. a) FAs are metabolized in astrocytes to generate ATP. In fasted state, ketogenesis is favored and astrocytes release ketone bodies for neuronal use. b) In astrocytes, glucose is substrate for the glycolytic pathway, generating pyruvate and, consequently, lactate. Pyruvate is substrate for the anaplerotic reaction that generates oxaloacetate. In neurons, glucose is the substrate for the pentose phosphate pathway, which leads to the generation of ribose and NADPH. The oxidation of FAs and amino acids yields acetyl-CoA and ATP in astrocytes, while catabolic signaling such as cAMP together with high levels of β-oxidation products favor the synthesis of KBs. In neurons, lactate and KBs generate ATP. FA: fatty acid; CAT1: carnitine acyltransferase 1; KB: ketone body; ATP: adenosine triphosphate; MCT: monocarboxylate transporter; NADPH: nicotinamide adenine dinucleotide phosphate hydrogen. Fig. 4. Glutamate and GABA astroglial metabolism. The astrocytic endfeet is part of the tripartite synapse composed also by a presynaptic neuron and a postsynaptic neuron. This functional coupling allows for controlling the strength and duration of the synaptic activity. Astrocytes take up neurotransmitters from the synaptic cleft through their high-affinity transporters (GLT-1 and GLAST transport glutamate; GAT1 and 3 transport GABA). GABA is oxidized and generates ATP, while glutamate may follow several fates.−, More specifically, glutamate can be: the precursor of glutamine or glutathione; released again through transporters such as Xc−; or oxidized in mitochondria. GLT-1: glutamate transporter 1; GLAST: glutamate-aspartate transporter; Xc−: cystine/glutamate antiporter; GABA: γaminobutyric acid; GAT: GABA transporter; ATP: adenosine triphosphate.

3.1. Glutamate and GABA

EAATs have been described in mammals: GLAST (EAAT1), GLT-1 (EAAT2), EAAC1 (EAAT3), EAAT4, and EAAT5 [41]. However, only GLAST and GLT-1 are predominantly found in adult astrocytes [42]. Often this nomenclature causes confusion in many readers. To be specific, GLAST and GLT-1 are the rodent isoforms of the human EAAT1 and EAAT2, respectively. According to the inputs astrocytes are receiving, after being taken up, glutamate can follow four distinct pathways. I) Glutamate-

Glutamate is the main excitatory neurotransmitter in the brain. Extracellularly, its primary destination is the astrocytic compartment. High affinity excitatory amino acid transporters (EAATs) in the astroglial membranes take up glutamate from synaptic cleft, modulating excitatory neurotransmission, which makes astrocytes major coordinators of excitatory synaptic transmission [41,117]. To date, five 6

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glutamine cycle: The enzyme glutamine synthetase (GS) uses ATP to attach one ammonium ion into glutamate – a reaction that occurs exclusively in astrocytes in the CNS [118]. The glutamine release from astrocytes is crucial for allowing rapid neurotransmitter replenishment in neuronal compartment (glutamine is deaminated by glutaminase, regenerating glutamate) [119]. Of particular interest, GS becomes hyperactive in cases of ammonia poisoning, such as in hepatic failure. In this scenario, GS reaction will occur at high rates, which might displace α-ketoglutarate from the TCA cycle [120]. II) Glutathione synthesis: glutamate participates in the reaction catalyzed by glutamate-cysteine ligase, which is the first enzyme in this pathway [121]. Glutathione synthesis in astrocytes is vital for protecting the brain against redox imbalance [122]. Indeed, astrocytes release both glutathione and its precursor to the extracellular compartment [123]. III) The oxidative degradation: glutamate dehydrogenase generates α-ketoglutarate prone to be converted to malate, through the TCA cycle reactions. Then, malate can be exported from the mitochondria to the cytosol where it is metabolized by the malic enzyme generating pyruvate. Through PDHC reaction, pyruvate yields acetyl-CoA, which enters the TCA cycle, culminating in CO2 generation [124]. The pathway above described, together with PDHC very low activity in astrocytes [86], affords low pyruvate complete oxidation. Thus, glutamate taken up by astrocytes can be another source of lactate to feed the neurons [84]. IV) Gliotransmission: glutamate can also be released from astrocytes in the extracellular medium, actively modulating local and long-distance neuronal circuits [125,126]. Transporter proteins such as the cystine/ glutamate antiporter (Xc− system) account for astroglial glutamate release. In this particular case, Xc− system takes up cystine for GSH synthesis. The role of glutamate as a gliotransmitter still remains under investigation [127]. Beyond releasing glutathione and its precursor, astrocytic role in supporting neuronal antioxidant function also encompasses the ascorbate-dehydroascorbate cycle [128]. The ascorbic acid – an important enzymatic co-factor – is able to terminate free radical chain reactions by directly scavenging reactive oxygen and nitrogen species [128,129]. Astrocytes take up dehydroascorbic acid – the oxidized product of ascorbate – from the extracellular compartment and recycle it back to ascorbic acid. The regenerated ascorbic acid can then be used by astrocytes, or alternatively released to the extracellular space to be utilized as an antioxidant defense by adjacent cells (for review, see [130]). GABA is the main inhibitory neurotransmitter in the brain. It has been shown that astrocytes are able to take up GABA from synaptic cleft through the GABA transporters (GAT1, 2 and 3). The GAT3 isoform is exclusively located in astrocytes [131]. Unlike glutamate, GABA taken up by astrocytes will have as primary destination the energy production in mitochondria [63,118,132,133]. GAT3 activity was recently reported to contribute to the control of excitatory signaling by releasing ATP, which is converted extracellularly to adenosine and activates presynaptic adenosine receptors [134]. Interestingly, if astroglial GAT3 is able to modulate glutamate release, it could be a player in excitotoxicity and, consequently, neurodegeneration. Similarly to the glutamate transport, the GABA uptake is an energy-demanding process that co-transports Na+ ion, which is compensated by GABA's entry as αketoglutarate in the TCA cycle [63]. The importance of GABA-glutamate coupling in the CNS is remarkable. More specifically, glutamate generates GABA via glutamate decarboxylase in neurons and GABA generates glutamate in neurons/astrocytes trough a transaminase reaction [63,133,135,136].

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