The Motor Neuron-Like Cell Line NSC-34 and Its Parent Cell Line N18TG2 Have Glycogen that is Degraded Under Cellular Stress.

Brain glycogen has a long and versatile history: Primarily regarded as an evolutionary remnant, it was then thought of as an unspecific emergency fuel store. A dynamic role for glycogen in normal brain function has been proposed later but exclusively attributed to astrocytes, its main storage site. Neuronal glycogen had long been neglected, but came into focus when sensitive technical methods allowed quantification of glycogen at low concentration range and the detection of glycogen metabolizing enzymes in cells and cell lysates. Recently, an active role of neuronal glycogen and even its contribution to neuronal survival could be demonstrated. We used the neuronal cell lines NSC-34 and N18TG2 and could demonstrate that they express the key-enzymes of glycogen metabolism, glycogen phosphorylase and glycogen synthase and contain glycogen which is mobilized on glucose deprivation and elevated potassium concentrations, but not by hormones stimulating cAMP formation. Conditions of metabolic stress, namely hypoxia, oxidative stress and pH lowering, induce glycogen degradation. Our studies revealed that glycogen can contribute to the energy supply of neuronal cell lines in situations of metabolic stress. These findings shed new light on the so far neglected role of neuronal glycogen. The key-enzyme in glycogen degradation is glycogen phosphorylase. Neurons express only the brain isoform of the enzyme that is supposed to be activated primarily by the allosteric activator AMP and less by covalent phosphorylation via the cAMP cascade. Our results indicate that neuronal glycogen is not degraded upon hormone action but by factors lowering the energy charge of the cells directly.

Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons.

BACKGROUND: Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes. RESULTS: Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves. CONCLUSIONS: We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission.

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 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.

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.

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.

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.

Glycogen phosphorylase isozyme pattern in mammalian retinal Müller (glial) cells and in astrocytes of retina and optic nerve.

Müller cells, the radially oriented dominant macroglial cells of the retina, are known to contain abundant glycogen as well as the key enzyme for its degradation, glycogen phosphorylase (GP), but the expressed isozyme pattern is unknown. To elucidate the isoform expression pattern, specific antisera directed against the brain (BB) and muscle (MM) isoforms of GP were applied to retinal sections, isolated Müller cells, and sections of the optic nerve. We show that Müller cells of rat, rabbit, guinea pig, and mouse retina exclusively express the BB isoform. Astrocytes of rat and rabbit optic nerve, as well as retina express only the BB isoform. In contrast, astrocytes in the brain and spinal cord as well as the epithelial cells of the pars caeca and of the ciliary body express both the BB and MM isoform. This result may indicate some differences in the role of glycogen in retinal macroglia and brain astrocytes, reflecting a local specialization of macroglia in the retina proper.

Immunocytochemical localization of glycogen phosphorylase isozymes in rat nervous tissues by using isozyme-specific antibodies.

Isozyme-specific antibodies were raised against peptides from the low-homology regions of the sequences of rat glycogen phosphorylase BB and MM isozymes by immunization of rabbits and guinea pigs. Immunocytochemical double-labelling experiments on frozen sections of rat nervous tissues were performed to investigate the isozyme localization pattern. Astrocytes throughout the brain and spinal cord expressed both isozymes in perfect co-localization. Ependymal cells only expressed the BB isozyme. Most neurones were not immunoreactive. The rare neurones that contained glycogen phosphorylase only expressed the BB isozyme. Nearly all of these neurones formed part of the afferent somatosensory system. These findings stress the general importance of glycogen in neural energy metabolism and indicate a special role for the glycogen phosphorylase BB isozyme in neurones in the somatosensory system.