Respiration-Deficient Astrocytes Survive As Glycolytic Cells In Vivo.

Neurons and glial cells exchange energy-rich metabolites and it has been suggested, originally based on in vitro data, that astrocytes provide lactate to glutamatergic synapses ("lactate shuttle"). Here, we have studied astrocytes that lack mitochondrial respiration in vitro and in vivo A novel mouse mutant (GLASTCreERT2::Cox10flox/flox) was generated, in which the administration of tamoxifen causes mutant astrocytes to fail in the assembly of mitochondrial cytochrome c oxidase (COX). Focusing on cerebellar Bergmann glia (BG) cells, which exhibit the highest rate of Cre-mediated recombination, we found a normal density of viable astrocytes even 1 year after tamoxifen-induced Cox10 gene targeting. Our data show that BG cells, and presumably all astrocytes, can survive by aerobic glycolysis for an extended period of time in the absence of glial pathology or unspecific signs of neurodegeneration.SIGNIFICANCE STATEMENT When astrocytes are placed into culture, they import glucose and release lactate, an energy-rich metabolite readily metabolized by neurons. This observation led to the "glia-to-neuron lactate shuttle hypothesis," but in vivo evidence for this hypothesis is weak. To study astroglial energy metabolism and the directionality of lactate flux, we generated conditional Cox10 mouse mutants lacking mitochondrial respiration in astrocytes, which forces these cells to survive by aerobic glycolysis. Here, we report that these mice are fully viable in the absence of any signs of glial or neuronal loss, suggesting that astrocytes are naturally glycolytic cells.

Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity.

Oligodendrocytes, the myelin-forming glial cells of the central nervous system, maintain long-term axonal integrity. However, the underlying support mechanisms are not understood. Here we identify a metabolic component of axon-glia interactions by generating conditional Cox10 (protoheme IX farnesyltransferase) mutant mice, in which oligodendrocytes and Schwann cells fail to assemble stable mitochondrial cytochrome c oxidase (COX, also known as mitochondrial complex IV). In the peripheral nervous system, Cox10 conditional mutants exhibit severe neuropathy with dysmyelination, abnormal Remak bundles, muscle atrophy and paralysis. Notably, perturbing mitochondrial respiration did not cause glial cell death. In the adult central nervous system, we found no signs of demyelination, axonal degeneration or secondary inflammation. Unlike cultured oligodendrocytes, which are sensitive to COX inhibitors, post-myelination oligodendrocytes survive well in the absence of COX activity. More importantly, by in vivo magnetic resonance spectroscopy, brain lactate concentrations in mutants were increased compared with controls, but were detectable only in mice exposed to volatile anaesthetics. This indicates that aerobic glycolysis products derived from oligodendrocytes are rapidly metabolized within white matter tracts. Because myelinated axons can use lactate when energy-deprived, our findings suggest a model in which axon-glia metabolic coupling serves a physiological function.

Metabolism of [U-(13)C]aspartate by astroglial cultures: nuclear magnetic resonance analysis of the culture media.

In brain the amino acid L-aspartate serves roles as: (1) putative transmitter, (2) protein precursor, (3) donor of atoms for the biosynthesis of pyrimidine and purine bases, and (4) fuel for energy metabolism. Astrocytes dominate aspartate clearance in brain, and in culture they take up aspartate and quickly metabolize it. In brain, only astrocytes were shown to express the enzymes for de novo pyrimidine biosynthesis. To gain more details about the spectrum of metabolites generated from aspartate and subsequently released by cultured astrocytes a (13)C-nuclear magnetic resonance analysis was performed of [U-(13)C]aspartate supplemented incubation media exposed to astroglial cultures. The results show that astrocytes readily metabolize aspartate and release into their culture media (13)C-isotopomers of lactate, glutamine, citrate and alanine. Despite the presence in astroglial cells of two tandem enzymes of pyrimidine biosynthesis and their mRNAs, pyrimidine nucleotide-related heterocyclic compounds such as dihydroorotate and orotate could not be detected in the culture media.

Glial metabolism of valine.

The three essential amino acids, valine, leucine and isoleucine, constitute the group of branched-chain amino acids (BCAAs). BCAAs are rapidly taken up into the brain parenchyma, where they serve several distinct functions including that as fuel material in brain energy metabolism. As one function of astrocytes is considered the production of fuel molecules that support the energy metabolism of adjacent neural cells in brain. Astroglia-rich primary cultures (APC) were shown to rapidly dispose of the BCAAs, including valine, contained in the culture medium. While the metabolisms of leucine and isoleucine by APC have already been studied in detail, some aspects of valine metabolism remained to be determined. Therefore, in the present study an NMR analysis was performed to identify the (13)C-labelled metabolites that are generated by APC during catabolism of [U-(13)C]valine and that are subsequently released into the incubation medium. The results presented show that APC (1) are potently disposing of the valine contained in the incubation medium; (2) are capable of degrading valine to the tricarboxylic acid (TCA) cycle member succinyl-CoA; and (3) release into the extracellular milieu valine catabolites and compounds generated from them such as [U-(13)C]2-oxoisovalerate, [U-(13)C]3-hydroxyisobutyrate, [U-(13)C]2-methylmalonate, [U-(13)C]isobutyrate, and [U-(13)C]propionate as well as several TCA cycle-dependent metabolites including lactate.

Glial metabolism of isoleucine.

Isoleucine, together with leucine and valine, constitutes the group of branched-chain amino acids (BCAAs). BCAAs are transported from the blood into the brain parenchyma, where they can serve several distinct functions. Since brain tissue is known to oxidatively metabolize BCAAs to CO(2), they are considered as fuel material in brain energy metabolism. Also, in the case of leucine, cultured astrocytes have been reported to be able to completely oxidize BCAA. While the metabolism of leucine by astroglia-rich primary culture (APC) has already been studied in detail, the metabolic fates of isoleucine and valine in these cells remained to be identified. Therefore, in the present study an NMR analysis was performed of (13)C-labelled metabolites generated in the catabolism of [U-(13)C]Ile by astrocytes and released by them into the incubation medium. APC potently removed isoleucine from the medium and metabolized it. The major isoleucine metabolites released from APC are 2-oxo-3-methylvalerate, 2-methylbutyrate, 3-hydroxy-2-methylbutyrate and propionate. To a lesser extent, APC generate and release also [2,3-(13)C]glutamine, [4,5-(13)C]glutamine and (13)C-labelled isotopomers of lactate and citrate. These results show that APC can release into the extracellular milieu catabolites and several TCA cycle dependent metabolites resulting from the degradation of isoleucine.

Immunohistochemical localization of glycogen phosphorylase isozymes in the rat gastrointestinal muscle layers and enteric nervous system.

Glycogen represents the major brain energy reserve though its precise functions are still under debate. Glycogen has also been found in different cell types of the enteric nervous system (ENS), the largest and most complex component of the peripheral nervous system. In the present work we have demonstrated, by application of isozyme-specific antibodies, the presence of isozymes of glycogen phosphorylase (GP), one of the major control sites in glycogen metabolism, in the rat ENS. Immunohistochemistry revealed that isoform BB (brain) is the predominant isozyme expressed in enteric glial cells (EGC) and rare neurons of the myenteric and submucosal plexuses. Isoform MM (muscle) appears in cells which are, according to their location and morphology, probably interstitial cells of Cajal (ICC). In addition, both GP isoforms are expressed in longitudinal and circular intestinal smooth muscle layers. As GP BB is mainly regulated by the cellular AMP level, a special function of glycogen in the energy supply of neural gut functions is suggested.

Expression of pyruvate carboxylase in cultured oligodendroglial, microglial and ependymal cells.

The mitochondrial enzyme, pyruvate carboxylase (PC; EC 6.4.1.1) is considered to play a significant role in the intermediary metabolism of neural tissue. PC-catalyzed carboxylation of pyruvate to oxaloacetate is a major anaplerotic reaction in brain. Anaplerosis is essential for homeostasis of the members of the tricarboxylic acid (TCA) cycle. Several biochemical pathways rely on withdrawing TCA cycle members. Prominent among these are biosynthesis of fatty acids and of non-essential amino acids such as aspartate, asparagine, glutamate and glutamine, gluconeogenesis, glycogen synthesis, and regeneration of NADPH. The expression of PC in brain has already been described and assigned to astrocytes. Since pyruvate carboxylase deficiency is associated with malformations of the brain, e.g., inadequate development of the corpus callosum and the lack of myelination, one can hypothesize that PC may be expressed also in glial cells other than astrocytes. Therefore, the expression of PC was investigated in cultured oligodendroglial, microglial, and ependymal cells. As assessed by RT-PCR, all these cultures contain PC mRNA. This mRNA is generated in a transcription process that is regulated by the "distal class" of promoters of the PC gene. The expression of PC among cultured glial cells was studied with a rabbit antiserum by immunoblotting and immunocytochemistry. The results indicate that PC is not only expressed in cultured astroglial cells but also in cultured oligodendrocytes, microglial cells, and ependymocytes. It appears that the intermediary metabolism of these cells includes the anaplerotic action of PC as well as possibly also functions of the enzyme in biosynthetic pathways and the provision of NADPH for defense against reactive oxygen species.

Expression of the brain and muscle isoforms of glycogen phosphorylase in rat heart.

Heart glycogen represents a store of glucosyl residues which are mobilized by the catalysis of glycogen phosphorylase (GP) and are mainly destined to serve as substrates for the generation of ATP. The brain isoform of GP (GP BB) was studied in rat heart in comparison with the muscle isoform (GP MM) to find functional analogies to the brain. Western blotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) experiments revealed that at the protein level, but not at the mRNA level, the content of GP BB is similar in heart and brain. In contrast, GP MM is more abundant in the heart than in the brain. Immunocytochemically GP BB was colocalized with GP MM in cardiomyocytes. GP MM was also detected in interstitial cells identified as fibroblasts. The physiological role of co-expression of GP BB and GP MM in cardiomyocytes and in brain astrocytes is discussed in a comparative way.

Expression of 3-hydroxyisobutyrate dehydrogenase in cultured neural cells.

The branched-chain amino acids (BCAAs)--isoleucine, leucine, and valine--belong to the limited group of substances transported through the blood-brain barrier. One of the functions they are thought to have in brain is to serve as substrates for meeting parenchymal energy demands. Previous studies have shown the ubiquitous expression of a branched-chain alpha-keto acid dehydrogenase among neural cells. This enzyme catalyzes the initial and rate-limiting step in the irreversible degradative pathway for the carbon skeleton of valine and the other two branched-chain amino acids. Unlike the acyl-CoA derivates in the irreversible part of valine catabolism, 3-hydroxyisobutyrate could be expected to be released from cells by transport across the mitochondrial and plasma membranes. This could indeed be demonstrated for cultured astroglial cells. Therefore, to assess the ability of neural cells to make use of this valine-derived carbon skeleton as a metabolic substrate for the generation of energy, we investigated the expression in cultured neural cells of the enzyme processing this hydroxy acid, 3-hydroxyisobutyrate dehydrogenase (HIBDH). To achieve this, HIBDH was purified from bovine liver to serve as antigen for the production of an antiserum. Affinity-purified antibodies against HIBDH specifically recognized the enzyme in liver and brain homogenates. Immunocytochemistry demonstrated the ubiquitous expression of HIBDH among cultured glial (astroglial, oligodendroglial, microglial, and ependymal cells) and neuronal cells. Using an RT-PCR technique, these findings were corroborated by the detection of HIBDH mRNA in these cells. Furthermore, immunofluorescence double-labeling of astroglial cells with antisera against HIBDH and the mitochondrial marker pyruvate dehydrogenase localized HIBDH to mitochondria. The expression of HIBDH in neural cells demonstrates their potential to utilize valine imported into the brain for the generation of energy.

Renal expression of the brain and muscle isoforms of glycogen phosphorylase in different cell types.

Kidney contains glycogen. Glycogen is degraded by glycogen phosphorylase (GP). This enzyme comes in three isoforms, one of which, the brain isozyme (GP BB), is known to occur in kidney. Its pattern of distribution in rat kidney was studied in comparison to that of the muscle isoform (GP MM) with the aim to see if for GP BB and GP MM there were functional similarities in brain and kidney. In immunoblotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) experiments, both isozymes and their respective mRNAs were found in kidney homogenates. GP BB was immunocytochemically detected in collecting ducts which were identified by the marker protein aquaporin-2. GP MM was localized exclusively in interstitial cells of cortex and outer medulla. These cells were identified as fibroblasts by their expression of 5'-ectonucleotidase (cortex) or by their morphology (outer medulla). The physiological role of both isozymes is discussed in respect to local demands of energy and of proteoglycan building blocks.

Somatostatin receptor subtype-2-deficient mice with diet-induced obesity have hyperglycemia, nonfasting hyperglucagonemia, and decreased hepatic glycogen deposition.

Hypersecretion of glucagon contributes to abnormally increased hepatic glucose output in type 2 diabetes. Somatostatin (SST) inhibits murine glucagon secretion from isolated pancreatic islets via somatostatin receptor subtype-2 (sst2). Here, we characterize the role of sst2 in controlling glucose homeostasis in mice with diet-induced obesity. Sst2-deficient (sst2(-/-)) and control mice were fed high-fat diet for 14 wk, and the parameters of glucose homeostasis were monitored. Hepatic glycogen and lipid contents were quantified enzymatically and visualized histomorphologically. Enzymes regulating glycogen and lipid synthesis and breakdown were measured by real-time PCR and/or Western blot. Gluconeogenesis and glycogenolysis were determined from isolated primary hepatocytes and glucagon or insulin secretion from isolated pancreatic islets. Nonfasting glucose, glucagon, and fasting nonesterified fatty acids of sst2(-/-) mice were increased. Inhibition of glucagon secretion from sst2-deficient pancreatic islets by glucose or somatostatin was impaired. Insulin less potently reduced blood glucose concentration in sst2-deficient mice as compared with wild-type mice. Sst2-deficient mice had decreased nonfasting hepatic glycogen and lipid content. The activity/expression of enzymes controlling hepatic glycogen synthesis of sst2(-/-) mice was decreased, whereas enzymes facilitating glycogenolysis and lipolysis were increased. Somatostatin and an sst2-selective agonist decreased glucagon-induced glycogenolysis, without influencing de novo glucose production using cultured primary hepatocytes. This study demonstrates that ablation of sst2 leads to hyperglucagonemia. Increased glucagon concentration is associated with impaired glucose control in sst2(-/-) mice, resulting from decreased hepatic glucose storage, increased glycogen breakdown, and reduced lipid accumulation. Sst2 may constitute a therapeutic target to lower hyperglucagonemia in type 2 diabetes.

Glycogen phosphorylase isozymes and energy metabolism in the rat peripheral nervous system--an immunocytochemical study.

Glycogen represents the major brain energy reserve which is located mainly in astrocytes. Though the role of brain glycogen has drawn increasing attention, little is known about glycogen metabolism in the peripheral nervous system. In the present work, we have demonstrated immunocytochemically the ubiquitous presence of glycogen phosphorylase (GP), one of the major control sites in glycogen metabolism, in the axons of rat spinal and sciatic nerves, but not in Schwann cells. Application of isozyme-specific antibodies revealed the presence of the GP BB (brain) isoform, but not the MM (muscle) isoform. This is in accord with previous results demonstrating the presence of isoform BB, but not MM, in the few GP-containing brain and spinal cord neurons and in vagus nerve axons. In contrast, brain astrocytes express both isoforms. As GP BB is mainly regulated by the cellular AMP level, a special role of glycogen in the energization of the nerve axons is suggested. The cellular locations of hexokinase, pyruvate dehydrogenase and glucose transporters are discussed in respect to possible metabolic roles of glycogen in peripheral nerves.

Immunocytochemical analysis of rat vagus nerve by antibodies against glycogen phosphorylase isozymes.

Glycogen is an endogenous store of glucose equivalents for energy metabolism in many tissues. The brain contains a significant amount of glycogen the role of which as an energy reserve is currently under debate. Apparently little is known concerning a possible role of glycogen in peripheral nerves. We have demonstrated immunocytochemically the presence of glycogen phosphorylase (GP), a key enzyme in glycogen metabolism, in large and small axons of the rat vagus nerve, but not in Schwann cells. Furthermore, the isozyme-specific antibodies applied detected only the presence of the brain isoform BB of GP, but not the muscle isoform MM. This is in agreement with the occurrence of solely the BB isoform in the few brain and spinal cord neurons that contain GP. In contrast, astroglial cells in brain and spinal cord have previously been shown to contain both isoforms. Since GP isozymes are regulated differentially, the expression of isoform BB may provide hints to possible functions of glycogen in the vagus nerve.

Natriuretic peptides, but not nitric oxide donors, elevate levels of cytosolic guanosine 3',5'-cyclic monophosphate in ependymal cells ex vivo.

Atrial natriuretic peptide-(1-28) (ANP), brain natriuretic peptide-(1-32) (BNP) and C-Type natriuretic polypeptide (CNP) occur in the brain, are concentrated in the anteroventral area of the third cerebral ventricle and participate in the regulation of body fluid homeostasis. The ventricles of the mammalian brain are lined by a continuous monolayered epithelium of polyciliated ependymal cells. In the adult rat, the ependymocytes continue to express the intermediate filament vimentin, but do not contain glial fibrillary acidic protein. Ependymal functions are poorly understood, but may extend to osmoregulation and volume sensing. Ependymal cells possess receptors for the natriuretic peptides, and in cell culture respond to them with an increase in their cyclic GMP content. In this study, a cyclic GMP-specific antibody was employed together with an ex vivo brain slice system to assess the ependymal response to ANP, BNP and CNP under close to life-like conditions. While ANP in concentrations of 0.1 nM and 1 nM had no effect, at concentrations of 10nM and 100 nM it increased ependymal cyclic GMP levels in a concentration-dependent manner. The other natriuretic peptides BNP, and CNP, also increased the cyclic GMP content of ependymocytes, while nitric oxide (NO) donors had no effect. However, in contrast to the natriuretic peptides, the NO donors elevated the level of cyclic GMP in the brain parenchyma below the ependymal layer.

Glycogen metabolism in rat ependymal primary cultures: regulation by serotonin.

Ependymal primary cultures are a model for studying ependymal energy metabolism. Intracellular glycogen is built up in the cultures dependent on culture age and the presence of glucose and glutamate. This energy store is mobilized upon glucose withdrawal, stimulation with isoproterenol, forskolin or serotonin and after uncoupling of oxidative phosphorylation from ATP production. Serotonin regulates ependymal glycogen metabolism predominantly via 5-HT receptor (5-HTR) 7, which elicits an increase in the level of ependymal cyclic AMP. Although the most abundant mRNAs for serotonin receptors are those of 5-HTR 2B and 5-HTR 3A, ependymal cells in primary culture do not respond to serotonin with an increase in their concentration of cytosolic calcium ions. The mRNAs of 5-HTRs 1A, 6, 1B, 5B, 7, 1/2C and 5A are also detectable in order of decreasing abundance. The mRNAs for 5-HTRs 1D, 1F, 3B and 4 are absent from the cultured cells. The ability of serotonin to mobilize ependymal glycogen depends on the culture age and the time allowed for glycogen buildup. During glycogen buildup time, glutamate is consumed by the cells. An increased ability of 5-HT to mobilize ependymal glycogen stores is noticed after the depletion of glutamate from the glycogen buildup medium. In ependymal primary cultures, cilia are colocalized with glycogen phosphorylase isozyme BB, while the MM isoform is not expressed. It is known from the literature that an increase in the concentration of cytosolic cAMP in ependymal cells leads to a decrease in ciliary beat frequency. Therefore, the present data point towards a function for ependymal glycogen other than supplying energy for the movement of cilia.

Metabolic and regulatory roles of leucine in neural cells.

Dietary leucine transported into the brain parenchyma serves several functions. Most prominent is the role of leucine as a metabolic precursor of fuel molecules, alpha-ketoisocaproate and ketone bodies. As alternatives to glucose, these compounds are forwarded by the producing astrocytes to the adjacent neural cells. Leucine furthermore participates in the maintenance of the nitrogen balance in the glutamate/glutamine cycle pertinent to the neurotransmitter glutamate. Leucine also serves as a regulator of the activity of some enzymes important for brain energy metabolism. Another role of leucine as an informational molecule is in mTOR signaling that participates in the regulation of food ingestion. The importance of leucine for brain function is stressed by the fact that inborn errors in its metabolism cause metabolic diseases often associated with neuropathological symptoms. In this overview, the current knowledge on the metabolic and regulatory roles of this essential amino acid in neural cells are briefly summarized.

Immunocytochemical analysis of glycogen phosphorylase isozymes in the developing and adult retina of the domestic chicken (Gallus domesticus).

Glycogen is the major energy reserve in neural tissues including the retina. A key-enzyme in glycogen metabolism is glycogen phosphorylase (GP) which exists in three differentially regulated isoforms. By applying isozyme-specific antibodies it could be demonstrated that the GP BB (brain), but not the GP MM (muscle) isoform is expressed in the chicken retina in neuronal and glial (Müller) cells. In the embryonic chicken retina, GP showed a development-dependent expression pattern. Double-labeling experiments with cell type-specific antibodies revealed that GP is expressed in various layers of the retina some of which, e.g., the photoreceptor inner segments, are known to be sites of high energy consumption. This suggests important roles of GP BB, and therefore glycogen, in early differentiation, spontaneous wave generation and in formation and stabilization of synapses.

Thrombin causes the enrichment of rat brain primary cultures with ependymal cells via protease-activated receptor 1.

Ependymal cell culture models from rat have been developed over the last 20 years to facilitate biochemical studies on this least-studied glial cell type. The cell culture protocol calls for the presence of thrombin, which is essential for obtaining a high proportion of multiciliated ependymal cells. The serine protease appears to act via protease-activated receptor 1 to prevent the apoptosis of ependymal precursors and enhance their proliferation without affecting contaminating cells. Unciliated precursors differentiate into polyciliated ependymocytes by passing through a stage of monociliation. The message for protease-activated receptor (PAR) 1 is initially abundant in the cultures, but its level declines as the cells differentiate. Besides PAR 1, signalling through PAR 2 also promotes ciliation in rat brain primary cultures, albeit to a lesser degree than the thrombin receptor. Thrombin and other proteases may be involved in the regulation of ventricular wall development. This action would be mediated mainly by PAR1.

Biosynthesis of Wdr16, a marker protein for kinocilia-bearing cells, starts at the time of kinocilia formation in rat, and wdr16 gene knockdown causes hydrocephalus in zebrafish.

The rat ortholog of the WD40 repeat protein Wdr16 is abundantly expressed in testis and cultured ependymal cells. Low levels are found in lung and brain, respectively, while it is absent from kinocilia-free tissues. In testis and ependymal primary cultures, Wdr16 messenger RNA appears concomitantly with the messages for sperm-associated antigen 6, a kinocilia marker, and for hydin, a protein linked to ciliary function and hydrocephalus. In testis, ependyma and respiratory epithelium, the Wdr16 protein is up-regulated together with kinocilia formation. The wdr16 gene is restricted to genera in possession of kinocilia, and it is strongly conserved during evolution. The human and zebrafish proteins are identical in 62% of their aligned amino acids. On the message level, the zebrafish Wdr16 ortholog was found exclusively in kinocilia-bearing tissues by in situ hybridisation. Gene knockdown in zebrafish embryos by antisense morpholino injection resulted in severe hydrocephalus formation with unaltered ependymal morphology or ciliary beat. Wdr16 can be considered a differentiation marker of kinocilia-bearing cells. In the brain, it appears to be functionally related to water homeostasis or osmoregulation.