Mary Ellen Pusateri

Diversity of Metabolic Patterns in Human Brain Tumors: Enzymes of Energy Metabolism and Related Metabolites and Cofactors

Abstract: Biopsies from 15 human gliomas, five meningiomas, four Schwannomas, one medulloblastoma, and four normal brain areas were analyzed for 12 enzymes of energy metabolism and 12 related metabolites and cofactors. Samples, 0.01–0.25 μg dry weight, were dissected from freeze‐dried microtome sections to permit all the assays on a given specimen to be made, as far as possible, on nonnecrotic pure tumor tissue from the same region. Great diversity was found with regard to both enzyme activities and metabolite levels among individual tumors, but the following generalities can be made. Activities of hexokinase, phosphorylase, phosphofructokinase, glycerophosphate dehydrogenase, citrate synthase, and malate dehydrogenase levels were usually lower than in brain; glycogen synthase and glucose‐6‐phosphate dehydrogenase were usually higher; and the averages for pyruvate kinase, lactate dehydrogenase, 6‐phosphogluconate dehydrogenase, and β‐hydroxyacyl coenzyme A dehydrogenase were not greatly different from brain. Levels of eight of the 12 enzymes were distinctly lower among the Schwannomas than in the other two groups. Average levels of glucose‐6‐phosphate, lactate, pyruvate, and uridine diphosphoglucose were more than twice those of brain; 6‐phosphogluconate and citrate were about 70% higher than in brain; glucose, glycogen, glycerol‐1‐phosphate, and malate averages ranged from 104% to 127% of brain; and fructose‐1,6‐bisphosphate and glucose‐1,6‐bis‐phosphate levels were on the average 50% and 70% those of brain, respectively.

Enzymatic assays for 2-deoxyglucose and 2-deoxyglucose 6-phosphate☆

Methods for 2-deoxyglucose (2-DG) and 2-deoxyglucose 6-phosphate (DG6P) are described which are based on the fact that DG6P is oxidized by glucose-6-phosphate dehydrogenase (G6PDH), but at a rate 1000-fold slower than for glucose 6-phosphate, whereas hexokinase phosphorylates 2DG and glucose at comparable rates. Therefore, by adding the two enzymes in a suitable order, and in appropriate concentrations, 2DG, glucose, DG6P, and glucose 6-P can all be separately measured. To avoid a side reaction from the use of a high level of G6PDH, when measuring DG6P, glucose is first removed with glucose oxidase plus aldose reductase.

Measurement of 2-deoxyglucose and 2-deoxyglucose 6-phosphate in tissues☆

The enzymatic methods previously described for 2-deoxyglucose (DG) and 2-deoxyglucose 6-phosphate have been refined and adapted to measurements of brain samples ranging from 50 mg wet weight to less than a microgram dry weight. Procedures for preparing such samples for assay are described. Analytical properties of the enzymes employed are given together with means for overcoming their possible short comings. Emphasis is placed on information useful for employing DG to assess rapid changes in glucose metabolism.

Uptake of Exogenous Aspartate into Circumventricular Organs but Not Other Regions of Adult Mouse Brain

Abstract: Adult mice were treated intraperitoneally with aspartate (Asp) at one of several doses (0.47–3.75 mmol/kg) and 30 min later given a subcutaneous Asp injection at the same dose. This treatment regimen resulted in steady state blood Asp elevations, a given dose producing the same degree of elevation at both 30 and 60 min. The lowest and highest doses, respectively, produced fourfold and 55‐fold elevations of serum Asp. In selected circumventricular organ (CVO) regions of brain which lack blood brain barriers, tissue Asp levels rose 1.5 and 3 times above control values following the lowest and highest doses, respectively, whereas tissue Asp remained unchanged in non‐CVO brain regions. Thus, even very moderate Asp dosing causes marked increases in CVO Asp. In order to analyze the pattern of Asp uptake into CVO, Asp was assayed in numerous subdivisions of each CVO, and maps were constructed which reflected microregional concentration differences. The pattern of Asp distribution suggests that Asp enters brain via fenestrated capillaries serving certain portions of CVO and then spreads into adjacent brain tissue. In separate experiments, we administered a single high dose of Asp (15 mmol/kg) to both adult and infant mice and measured Asp in serum and select brain regions 60 min later. Asp concentrations in serum and CVO (but not other brain regions) rose markedly at both ages but the increases were greater in serum and therefore also in CVO of infants. This may explain in part the observation that a given dose of Asp destroys a larger number of CVO neurons in infant than in adult mice. Our findings support a growing body of evidence that CVO are an important communication link between blood and brain.

Enzyme levels in cultured astrocytes, oligodendrocytes and Schwann cells, and neurons from the cerebral cortex and superior cervical ganglia of the rat

Data are presented for 16 enzymes from 8 metabolic systems in cell cultures consisting of approximately 95% astrocytes and 5% oligodendrocytes. Nine of these enzymes were also measured in cultures of oligodendrocytes, Schwann cells, and neurons prepared from both cerebral cortex and superior cervical ganglia. Activities, in mature astrocyte cultures, expressed as percentage of their activity in brain, ranged from 9% for glycerol-3-phosphate dehydrogenase to over 300% for glucose-6-phosphate dehydrogenase. Creatine phosphokinase activity in astrocytes was about the same as in brain, half as high in oligodendrocytes, but 7% or less of the brain level in Schwann cells and superior cervical ganglion neurons and only 16% of brain in cortical neurons. Three enzymes which generate NADPH, the dehydrogenases for glucose-6-phosphate and 6-phosphogluconate, and the NADP-requiring isocitrate dehydrogenase, were present in astrocytes at levels at least twice that of brain. Oligodendrocytes had enzyme levels only 30% to 70% of those of astrocytes. Schwann cells had much higher lactate dehydrogenase and 6-phosphogluconate dehydrogenase activities than oligodendrocytes, but showed a remarkable similarity in enzyme pattern to those of cortical and superior cervical ganglion neurons.

Distribution of Three Enzymes of γ-Aminobutyric Acid Metabolism in Monkey Retina

Abstract: The distributions of glutamate decarboxylase (EC 4.1.1.15), γ‐aminobutyric acid transaminase (EC 2.6.1.19), and succinate semialdehyde dehydrogenase (EC 1.2.1.24) were determined in monkey retina. The decarboxylase was almost restricted to the inner plexiform layer. The transaminase was also highest in this layer, but activities were 40% as high in the adjacent third of the inner nuclear layer and in the ganglion cell and fiber layers. Succinate semialdehyde dehydrogenase was distributed very differently. Although it also showed a peak of activity in the inner plexiform layer, there was a second equal peak in the photoreceptor inner segment layer and a smaller peak in the outer plexiform layer, regions where both γ‐aminobutyric acid transaminase and glutamate decarboxylase were essentially absent.

Glutamate and potassium stimulation of hippocampal slices metabolizing glucose or glucose and pyruvate

Using 2-deoxyglucose phosphorylation as an index of glucose use and concentrations of selected intermediates to monitor metabolic pathways, responses of rat hippocampal slices to glutamate and K+ stimulation were examined. With glutamate, the glucose phosphorylation rate (GPR) increased, and the slices accumulated glutamate at a constant rate, for 10 min. The uptake rate at each glutamate level was matched, approximately, by the increase in GPR at that level, with 4 or 5 glutamate molecules accumulated for every glucose molecule phosphorylated. Phosphocreatine and ATP levels fell abruptly, and lactate rose, probably reflecting neuronal activity, found by others to be very brief in the presence of glutamate. K+ stimulation produced responses of phosphocreatine, ATP and lactate levels and of GPR similar to those due to glutamate. There were also prolonged changes in the levels of other metabolites: with both stimulants glucose 6-phosphate fell, and malate rose. The changes in malate may be the result of the participation of mitochondrial malate dehydrogenase in both citrate cycle and malate shuttle. Citrate and alpha-ketoglutarate rose only with K+. When pyruvate was added to the medium, resting GPR was reduced, but for both stimulants the relative increases in GPR with stimulation were the same as without pyruvate. The changes in metabolic intermediates in response to K+ were like those with glucose alone. But with glutamate, the rise in lactate was greatly diminished, and malate fell instead of rising. Glutamate interference with the transfer of both 3-carbon as well as 4- and 5-carbon intermediates from glia to neurons may explain these results. If so, this interference is greater with pyruvate supplementation than with glucose alone.

Metabolite changes associated with heat shocked avian fibroblast mitochondira

A previous report from our laboratory (Collier et al 1993) showed that the elongated tubules of mitochondria in the cytoplasm of cultured chicken embryo fibroblasts collapsed to irregularly shaped structures surrounding the nuclear membrane after a 1 h heat shock treatment. The normal mitochondrial morphology reappeared upon removal of the thermal stress. We have now determined that several changes occurred in mitochondrial-related metabolites under these same heat shock and recovery conditions. Among these were significant decreases in the levels of fumarate and malate and increases in the amounts of aspartate and glutamate. In contrast, other intermediates of the tri-carboxylic acid cycle were unaltered as were levels of ATP and phosphocreatine. The changes observed might result from heat shock-induced changes in enzyme activities of the mitochondria, from alterations in the membrane-embedded specialized carrier proteins that transport metabolites between cytosol and mitochondria or from a disorganization of the electron-transport system normally coupled to oxidative metabolism. The rapid recovery, however, suggested that these changes were transient and readily reversible.

Distribution of the glucose-1,6-bisphosphate system in brain and retina

The distribution of glucose‐1,6‐bisphosphate (G16P2) synthase was measured in more than 70 regions of mouse brain, and nine layers of monkey retina. Activities in gray areas varied as much as 10‐fold, in a hierarchical manner, from highest in telencephalon. especially the limbic system, to lowest in cerebellum, medulla, and spinal cord. The synthase levels were significantly correlated among different regions with G16P2 itself, as well as with previously published levels of a brain specific IMP‐dependent G16P2 phosphatase. In contrast, neither G16P2 nor either its synthase or phosphatase correlated positively with phosphoglucomutase. and in all regions the G16P2 levels greatly exceeded requirements for activation of this mutase. This strengthens the view that G16P: has some function besides serving as coenzyme for phosphoglucomutase. However, attempts to correlate the “G16P2 system,” as defined by the three coordinately related elements, synthase, phosphatase, and G16P2, with other enzymes of carbohydrate metabolism, or with regional data of Sokoloff et al. [J. Neurochem. 28, 897–916 (1977)] for glucose consumption, were unsuccessful. This leaves open the possibility that brain G16P2 might serve as a phosphate donor for specific nonmetabolic effector proteins.

Distribution in brain and retina of four enzymes of acetyl CoA synthesis in relation to choline acetyl transferase and acetylcholine esterase.

Eleven regions of mouse brain and twelve layers of monkey retina were assayed for choline acetyl transferase (ChAT), acetylcholine esterase (AChE), and 4 enzymes that synthesize acetyl CoA. The purpose was to seek evidence concerning the source of acetyl CoA for acetylcholine generation. In brain ATP citrate lyase was strongly correlated with ChAT as well as AChE (r=0.914 in both cases). Weak, but statistically significant correlation, was observed between ChAT and both cytoplasmic and mitochondrial thiolase, whereas there was a significant negative correlation between ChAT and acetyl thiokinase. In retina ChAT was essentially limited to the inner plexiform and ganglion cell layers, whereas substantial AChE activity extended as well into inner nuclear, outer plexiform and fiber layers, but no further. ATP citrate lyase activity was also highest in the inner four retinal layers, but was not strongly correlated with either ChAT or AChE (r=0.724 and 0.761, respectively). Correlation between ChAT and acetyl thiokinase was at least as strong (r=0.757), and in the six inner layers of retina, the correlation between ChAT and acetylthiokinase was very strong (r=0.932).