Regulation of the pentose phosphate pathway in human astrocytes and gliomas.

Several aspects of the regulation of the pentose phosphate pathway were examined in cultured normal human cortical astrocytes and gliomas of pathological grades I-IV. The generation of radiolabeled CO2 from [1-14C]glucose by the oxidative arm of the pentose phosphate pathway is a saturable process and has a maximum flux rate of 8-9 nmol/hr/mg cell protein. The flux can be blocked by the glycolytic inhibitor iodoacetamide but is unaffected by agents which inhibit oxidative phosphorylation. The magnitude of the pentose phosphate flux is directly related to the glioma grade. Grade IV gliomas (glioblastoma) show a pentose phosphate flux rate of approximately 4% of the total glucose flux. The flux rate can be increased by pharmacological agents which decrease the NADPH/NADP+ ratio. Both the activity and the regulation of glioma glucose-6-phosphate dehydrogenase (G6PDH) are altered in high-grade gliomas. While the affinity constants for cofactors in whole homogenates were not significantly different in glioma or normal astrocyte homogenates, normal astrocytes have a lower Km for glucose-6-phosphate and a G6PDH activity which is 10-fold greater than that of gliomas. NADPH is a powerful regulator of G6PDH activity in the normal astrocytes and in gliomas. At a NADPH/NADP+ ratio of 7:1 the normal astrocyte G6PDH is entirely inhibited, while the glioma enzyme is only 70% inhibited even at a ratio of 20:1. Increased metabolic flux through the oxidative arm of the pentose phosphate pathway is apparently due to an altered form of G6PDH.

Enzymes of glucose metabolism in cultured human gliomas: neoplasia is accompanied by altered hexokinase, phosphofructokinase, and glucose-6-phosphate dehydrogenase levels.

The enzymes of glycolysis and selected enzymes of the pentose phosphate pathways were measured by fluorometric methods in extracts prepared from cultures of normal cortical human astrocytes and from cultures derived from low-grade (II) or high-grade (IV) gliomas. The hexokinase and phosphofructokinase levels of the low-grade glioma-derived line were not significantly different from those of the normal astrocyte cultures. However, the activities of hexokinase and phosphofructokinase were consistently and significantly increased in the high-grade glioma-derived lines. The activity of glucose-6-phosphate dehydrogenase was significantly decreased in all glioma-derived lines and by more than 90% in the high-grade-derived lines. Other enzymes of the glycolytic pathway were not significantly different from those of normal astrocytes, or they showed a variation inconsistently related to the neoplastic state. Glucose flux is not apparently regulated to a significant degree of hexokinase in glioma-derived lines, since the measured Vmax values are in substantial excess over the measured flux rates. Reversible binding of hexokinase to the particulate fraction was observed in both the normal astrocytes cultures and the high-grade glioma-derived lines. A twofold displacement of particulate hexokinase by ATP, ADP, 1-O-methylglucose, sorbitol-6-phosphate, and dibutyryl cyclic AMP was observed in the high-grade glioma-derived lines. The degree of displacement by various agents and the basal ratio of free/bound was not significantly different between the transformers and the nontransformants. The hexokinase from both the gliomas and the normal astrocytes was noncompetitively inhibited by the glucose analogue 2-deoxy-d-glucose. Phosphofructokinase activity is close to the observed glucose flux rates in both the normal astrocyte and the glioma-derived cultures. The phosphofructokinase activity of normal astrocytes is activated twofold or more by ADP, AMP, fructose-2,6-diphosphate, and Pi. However, these same ligands activate phosphofructokinase by less than twofold in a typical high-grade glioma-derived line. ATP, dibutyryl cyclic AMP, and citrate inhibit glioma and normal astrocytic phosphofructokinase, but the magnitude of the inhibition is much less than in the glioma-derived lines.

Glucose metabolism in human gliomas: correspondence of in situ and in vitro metabolic rates and altered energy metabolism.

The rates of disappearance of glucose from the medium of 13 human glioma-derived cell lines and one cultured of normal human cortical astrocytes were determined by fluorometric techniques. High-grade glioma-derived cultures showed a range of glucose consumption between 1 and 5 nmol/min/mg protein. Normal astrocyte cultures and cultures derived from grades I-III gliomas had a glucose consumption rate of 2-3 nmol/min/mg protein. Seven high-grade glioma lines were derived from surgical samples taken from patients who had been scanned by 18F-2-deoxy-d-glucose positron computed tomography. The rate of glucose consumption in these high-grade glioma-derived lines was close to the maximum local cerebral metabolic rate for glucose (LCMRglc) measured in situ in the tumors from which the cultures were derived. In cultured glioma-derived lines, approximately one-half of the glucose consumed was recovered as lactate and pyruvate, suggesting a reliance of glioma cells on aerobic glycolysis. ATP and phosphocreatine (PCr) levels were variable in the glioma-derived lines, and ATP was lower in the glioma-derived lines than in the normal astrocytes. Levels and regulation of glycogen differed significantly among the various glioma-derived cell lines. Glycogen content did not diminish as glucose was consumed, suggesting that glycogen utilization is not tightly regulated by the glucose metabolic rate. These results suggest that human glioma-derived cell cultures (1) adequately reflect the metabolic capacity of gliomas in situ and (2) are significantly altered in several aspects of their glycolytic metabolism.

Regulation of hexokinase in cultured gliomas.

Positron emission computed tomographic (PECT) scanning studies have demonstrated that high grade gliomas exhibit increased 2-[18F]fluoro-2-deoxyglucose (18FDG) uptake compared to cerebral white matter and low grade gliomas. Hexokinase catalyzes the phosphorylation of glucose, as well as 18FDG and 2-deoxyglucose (2DG), thereby "trapping" these slowly metabolized analogues intracellularly. We hypothesize that a similar hexokinase-mediated uptake of glucose and glucose analogues occurs in vitro. Hexokinase activity was assayed in homogenates of tissue-cultured lines derived from high (IV) and low (II) grade gliomas and in fibroblasts derived from skin. With glucose as substrate, the maximal activity (Vmax) in the Grade IV lines was 200% of the activity found in the Grade II line, fibroblasts, and astrocytes; however, the Michaelis substrate affinity constant (Km) bore no relationship to tumor grade. With 2DG as substrate, the Vmax of all cell lines decreased, but the Grade IV lines still tended to have greater activity than the others. The Km values for 2DG were 5 times higher than those for glucose. Hexokinase is found in two subcellular compartments: an active form reversibly bound to mitochondria and a less active, cytosolic form. Up to 20% of the total hexokinase was found in the cytosol in all lines tested. High energy phosphate compounds (ATP, ADP, CTP, and others) displaced mitochondria-bound hexokinase, which increased the cytosolic form by 2-fold in the glioma lines, but fibroblast hexokinase distribution was unaffected. Our results suggest that: (a) high grade gliomas have increased hexokinase activity, which may explain the grade-related differences in 18FDG uptake observed by PECT scanning, and (b) human glioma hexokinases may be regulated by reversible subcellular compartmentation.

Plasma fibronectin in patients with brain tumors.

Fibronectin, a large molecular weight glycoprotein normally found on the surface of many cells, was measured in the plasma of 15 normal volunteers and 75 patients with various kinds of brain tumors or other kinds of neurological disorders. The mean plasma fibronectin in the control group was 253 micrograms/ml. Statistically significant elevations in fibronectin levels were found in patients with progressive high grade astrocytomas (mean = 413 micrograms/ml). The majority of patients with high grade astrocytomas in remission had normal levels of fibronectin (mean = 273 micrograms/ml). Elevations were noted in patients with low grade astrocytomas, meningiomas, and benign nonneoplastic neurological disorders. Plasma fibronectin may be useful diagnostically and therapeutically in identifying and managing some patients with high grade gliomas. However, of greater importance is the observation that fibronectin elevation is the result of a tumor-host interaction.

Brain tumors and plasmin inhibitors.

Four different intracranial neoplasms inhibited purified plasmin in an in vitro assay. The greatest inhibition (80%) was observed with extracts of a parasagittal meningioma that had invaded and completely occluded the sinus. Significant inhibition (greater than 40%) was also observed with extracts prepared from a glioma surgically removed from a patient who had suffered three thromboembolic episodes during the preceding several months. Lesser (less than 30%) inhibition was obtained with extracts from two other patients who had no evidence of thromboembolic complications. This report constitutes the first demonstration of plasmin inhibition activity in association with brain tumors. Further studies will be required to test the hypothesis that certain intracranial tumors may escape host antitumor fibrinolytic activity by producing plasmin inhibitors and that plasmin inhibitors may play a role in the thromboembolic complications frequently seen in patients with intracranial neoplasms.

Effects of time and experience on hippocampal neurochemistry after damage to the CA3 subfield.

Bilateral injections of kainic acid into the hippocampal CA3 subfield destroyed the CA 3 pyramidal cells and produced a behavioral impairment, an inability to solve spatial maze problems. The behavior recovered, however, with daily experience in a maze task, and the rate of recovery was accelerated by additional daily experience. This recovery of function could be the result of compensatory changes in the distribution or function of the various hippocampal pathways. In the present experiment, this possibility was investigated neurochemically. Five putative neurotransmitters or their synthetic enzymes were measured in dissected regions of the hippocampal formation. Both the long-term effects of the lesions and the effects of behavioral training were determined. A number of alterations in hippocampal neurochemical systems were detected. Acute changes due to the lesions included a widespread loss of glutamate, and regionally specific decreases in glutamic acid decarboxylase (GAD) activity and cholecystokinin (CCK) and norepinephrine (NE) concentrations. Long-term changes included a decline in choline acetyltransferase (ChAT) activity throughout the hippocampal formation, and increases in NE in certain regions. Behavioral testing prevented the decline of ChAT activity, and increased the concentrations of GAD and CCK. The neurochemical conditions present at the time when trained rats recovered behavioral function may indicate the crucial conditions for the occurrence of the behavior.

Regulation of glycogen metabolism in primary and transformed astrocytes in vitro.

Glycogen metabolism was studied in primary and Herpesvirus-transformed cultures of neonatal rat brain astrocytes. A small fraction of the glucose consumed was conserved in glycogen in both the primary and the transformed astrocytic cell cultures. After addition of culture medium containing 5.5 mM glucose, glycogen increased to maximal levels within 2.5 h, the approximate time at which half of the medium glucose was consumed, and rapidly declined thereafter in both the primary and transformed astrocytic cultures. Maximum levels of glycogen were apparently related to the cell density of the Herpesvirus-transformed cultures, but primary cultures did not show this behavior. At any given cell density, maximal levels of glycogen were dependent on the concentration of extracellular glucose. Administration of glucose caused a transient activation of glycogen synthase alpha and a rapid inactivation of glycogen phosphorylase alpha.

Regulation of glycogenolysis in transformed astrocytes in vitro.

Cultured astrocytes, transformed by Herpesvirus, were used as a model system to study several aspects of the control of glycogenolysis. Adrenergic agonists such as norepinephrine and isoproterenol caused an immediate and dose-dependent increase in the intracellular levels of cyclic AMP. Concomitant with the initial phase of cyclic AMP increase, conversion of phosphorylase b to a and glycogenolysis were observed. The elevation of cyclic AMP, phosphorylase conversion, and glycogenolysis were simultaneously blocked by beta-adrenergic blockers, but not by alpha-adrenergic blocking agents. Repeated administration of norepinephrine caused an attenuated response in both cyclic AMP accumulation and glycogenolysis. Glycogen degradation is also partially regulated by glucose availability. In the presence of glucose, norepinephrine-induced glycogenolysis is blocked, despite elevations in cyclic AMP. The direct role of glucose is postulated, since glucose analogs mimic the effects of glucose.

Alpha-aminoisobutyric acid uptake in primary cultures of astrocytes.

Homotypically pure cultures of rat brain astrocytes were used to examine some aspects of non-neuronal A-system (alanine preferring) amino acid uptake. The A-system specific probe, alpha-aminoisobutyric acid is transported rapidly, and a steady state distribution ratio of 9-25 is reached after 30 minute incubations. Kinetic estimates derived from uptake progress curves indicated a Km of 1.35 mM and a Vmax of 133 nmol/min/mg protein. Uptake is reduced in the absence of either Na+ or K+. Elevations in extracellular K+, a putative metabolic modulator of neuroglia, did not affect uptake.

Beta-alanine uptake is not a marker for brain astroglia in culture.

The properties of the beta amino acid transport system were examined in cultivated rat brain astrocytes, using the specific probe, beta-alanine. The uptake of beta-alanine is thought to be glial specific. Beta-alanine was not actively transported and the intracellular accumulation was not altered by coincubation with GABA, alanine, glutamate, or methionine. We suggested therefore that beta-alanine is passively taken up by a mechanism distinct from the transport system for GABA.

Potassium modulation of methionine uptake in astrocytes in vitro.

Methionine participates in a large variety of metabolic pathways in brain, and its transport may play an important regulatory role. The properties of methionine uptake were examined in a preparation of neonatal rat brain astrocytes. Uptake is linear for 15 minutes, up to 2.5 microM. At steady state conditions, methionine is concentrated 30-50-fold. Measured methionine homoexchange accounts for a significant fraction of uptake at concentrations greater than 10 microM. We recently reported that methionine uptake is decreased by elevations in extracellular K+. Potassium induced efflux cannot account for this apparent effect; and thus for concentrations less than 2.5 microM, and for short times of incubation, measured rates of methionine uptake represent unidirectional flux. At extracellular concentrations of K+ equal to 6.9 mM, the apparent Vmax of methionine transport is 182 pmol/min/mg protein, and the Km is 1.3 microM. Where K+ is shifted to 11.9 mM, the Km remains unchanged, and the Vmax is reduced by half.

Propagation and histological characterization of a homotypic population of astrocytes derived from neonatal rat brain.

Tissue derived from the cerebra of 3-4 day old rats was minced, trypsinized and placed in tissue culture medium. Cells attained a monolayer in twelve to fourteen days when plated at a density of 10(2) cells/cm2. Selected culturing procedures designed to inhibit neuronal growth while encouraging glial growth yielded monolayers which consisted almost exclusively of astrocytes. Cells were identified using established morphological criteria for brain cells in culture. These observations were supplemented using certain histological and histochemical techniques frequently employed in investigative work for the identification of glial cells, particularly astrocytes. Results of all the above procedures indicate cell cultures which apparently consist exclusively of astrocytes. The preparation of such a homotypic population of normal, viable astroglial cells, derived from an animal universally used in investigative work on the nervous system, is perceived as an important contribution to the future study of glial characteristics and their interactions at morphological and functional levels.

The developmental pattern of homologous and heterologous tRNA methylation in rat brain differential effect of spermidine.

UsingS-adenosyl-L-[Me-(14)C] methionine, rat cerebral cortex methyltransferase activity was determined during the early postnatal period in the absence of addedEscherichia coli tRNA and in its presence. [Me-(14)C] tRNA was purified from both systems and its [Me-(14)C] base composition determined. The endogenous formation of [Me-(14)C] tRNA (homologous tRNA methylation) was totally abolished in the presence of 2.5 mM spermidine, whereasE. coli B tRNA methylation (heterologous methylation) was markedly stimulated. Only [Me-(14)C] 1-methyl guanine and [Me-(14)C]N (2)-methyl guanine were formed by homologous methylation, there being an inverse shift in their relative proportions with age. Heterologous tRNA methylation led, additionally, to the formation of [Me-(14)C]N 2 (2) -dimethyl guanine, 5-methyl cytosine, 1-methyl adenine, 5-methyl uracil, 2-methyl adenine, and 1-methyl hypoxanthine. A comparison of heterologous tRNA methylation between the whole brain cortex (containing nerve and glial cells) and bulk-isolated nerve cell bodies revealed markedly lower proportions of [Me-(14)C]N 2-methyl andN 2 (2) -dimethyl guanine and significantly higher proportions of [Me-(14)C] 1-methyl adenine in the neurons. The present findings suggest (1) that homologous tRNA methylation may provide developing brain cells with continuously changing populations of tRNA and (2) that neurons are enriched in adenine residue-specific tRNA methyltransferases that are highly sensitive to spermidine.