LABELLING OF BRAIN PROTEINS AT EARLY PERIODS AFTER SUBCUTANEOUS INJECTION OF A MIXTURE OF [U-14C]GLUCOSE AND [3H]GLUTAMATE

To obtain evidence of the site of conversion of [U-¹⁴C]glucose into glutamate and related amino acids of the brain, a mixture of [U-¹⁴C]glucose and [³H]glutamate was injected subcutaneously into rats. [³H]Glutamate gave rise to several ³H-labelled amino acids in rat liver and blood; only ³H-labelled glutamate, glutamine or γ-aminobutyrate were found in the brain. The specific radioactivity of [³H]glutamine in the brain was higher than that of [³H]glutamate indicating the entry of [³H]glutamate mainly in the ‘small glutamate compartment’. The ¹⁴C-labelling pattern of amino acids in the brain and liver after injection of [U-¹⁴C]glucose was similar to that previously reported (Gaitonde et al., 1965). The specific radioactivity of [¹⁴C]glutamine in the blood and liver after injection of both precursors was greater than that of glutamate between 10 and 60 min after the injection of the precursors. The extent of labelling of alanine and aspartate was greater than that of other amino acids in the blood after injection of [U-¹⁴C]glucose. There was no labelling of brain protein with [³H]glutamate during the 10 min period, but significant label was found at 30 and 60 min. The highest relative incorporation of [¹⁴C]glutamate and [¹⁴C]aspartate in rat brain protein was observed at 5 min after the injection of [U-¹⁴C]glucose. The results have been discussed in the context of transport of glutamine synthesized in the brain and the site of metabolism of [U-¹⁴C]glucose in the brain.     

Decreased labeling of amino acids by inhibition of the utilization of [3H,14C]glucose via the hexosemonophosphate shunt in rat brain in vivo

Treatment of rats with 6-aminonicotinamide showed a small but significant decrease in the labeling of amino acids in the brain after injection of [³H]acetate. The results of these experiments also gave evidence of the presence of [³H]glucose and [³H]lactate, and an increase in [³H]glucose content in the brain of 6-aminonicotinamide treated rats. To apportion the contribution of [³H]glucose formed by gluconeogenesis from [³H]acetate to the labeling of amino acids a method was formulated based on the measurement of radioactivity of amino acids, lactate and free sugars in brain after injection of [6-³H]glucose or [1-³H]glucose relative to that after co-injection of [U-¹⁴C]glucose or [2-¹⁴C]glucose. In contrast to the expected formation of [1, 6-³H]glucose by gluconeogenesis from [³H]acetate,³H-labeled glucose isolated from brain, blood and liver showed the presence of [6-³H]glucose only. The values corrected for the presence of [6-³H]glucose showed that treatment with 6-aminonicotinamide had no effect on the labeling of amino acids by oxidation of [³H]acetate. These findings indicated that a significant decrease in the labeling of amino acids from [U-¹⁴C]glucose reported previously and again confirmed using [1-³H], [6-³H], [2-¹⁴C] or [U-¹⁴C]glucose in the present investigation was not due to the inhibition of the activities of enzymes of the citric acid cycle. These results support the postulated role of the hexosemonophosphate shunt for the utilization of glucose in providing neurotransmitter amino acids glutamate and -aminobutyrate.Dedicated to Professor K. A. C. Elliott on his 80th birthday.     

THE METABOLISM OF LEUCINE BY DEVELOPING RAT BRAIN: EFFECT OF LEUCINE AND 2-OXO-4-METHYLVALERATE ON LIPID SYNTHESIS FROM GLUCOSE AND KETONE BODIES

Abstract— The oxidation of l -[U-¹⁴C]leucine and l -[l-¹⁴C]leucine at varying concentrations from 0.1 to 5mM to CO₂ and the incorporation into cerebral lipids and proteins by brain slices from 1-week old rats were markedly stimulated by glucose. Although the addition of S mM-dl -3-hydroxybutyrate had no effect on the metabolism of [U-¹⁴C]leucine by brain slices from suckling rats, the stimulatory effects of glucose on the metabolism of l -[U-¹⁴C]leucine were markedly reduced in the presence of dl -3-hydroxybutyrate. The stimulatory effect of glucose on leucine oxidation was, however, not observed in adult rat brain. Furthermore, the incorporation of leucine-carbon into cerebral lipids and proteins was also very low in the adult brain. The incorporation of l -[U-¹⁴C]leucine into cerebral lipids by cortex slices was higher during the first 2 postnatal weeks, which then declined to the adult level. During this time span, the oxidation of l -[U-¹⁴C]leucine to CO₂ remained relatively unchanged. The incorporation in vivo of D-3-hydroxy[3-¹⁴C]butyrate into cerebral lipids was markedly decreased by acute hyperleucinemia induced by injecting leucine into 9-day old rats. In in vitro experiments, 5 mM-leucine had no effect on the oxidation of [U-¹⁴C]glucose to CO₂ or its incorporation into lipids by brain slices from 1-week old rats. However, 5 mM-leucine inhibited the oxidation of d -3-hydroxy-[3-¹⁴C]butyrate, [3-¹⁴C]acetoacetate and [1-¹⁴C]acetate to CO₂ by brain slices, but their incorporation into cerebral lipids was not affected by leucine. In contrast 2-oxo-4-methylvalerate, a deaminated metabolite of leucine, markedly inhibited both the oxidation to CO₂ and the incorporation into lipids of labelled glucose, ketone bodies and acetate by cortex slices from 1-week old rats. These findings suggest that the reduction in the incorporation in vivo of d -3-hydroxy[3-¹⁴C]butyrate into cerebral lipids in rats injected with leucine is most likely caused by 2-oxo-4-methylvalerate formed from leucine. Since the concentrations of leucine and 2-oxo-4-methylvalerate in plasma of untreated patients with maple-syrup urine disease are markedly elevated, our findings are compatible with the possibility that an alteration in the metabolism of glucose and ketone bodies in the brain may contribute to the pathophysiology of this disease.     

The effect of inhibition of hexosemonophosphate shunt on the metabolism of glucose and function in rat brain in vivo

Rats treated 4 hr previously with 6-aminonicotinamide showed a twenty-four fold increase of [¹⁴C]phosphogluconate in the adult brain at 30 min after injection of [U-¹⁴C]glucose indicating a blockade of the hexosemonophosphate shunt. There was a significant increase in the¹⁴C-content of glucose and glucose 6-phosphate, and a decrease in that of amino acids. [¹⁴C]Phosphoglycerate content showed no consistent change after 6-aminonicotinamide treatment. The concentration of glucose and glucose 6-phosphate increased significantly without a significant change in the lactate pool in the brain of 6-aminonicotinamide treated rats. The rate of utilization of glucose in the brain of control rats was 0.73 mol/min per g of brain. It decreased by 16% in rats treated with 6-aminonicotinamide; the results suggested that both glycolysis and pyruvate oxidation were affected. The amount of glucose utilized in the brain by the hexosemonophosphate shunt was approximately 0.0093 mol/min per g of brain, i.e. 1.3% of the total rate of utilization of glucose. The observed changes were not due to hypothermia. The rate of glucose utilization was higher in animals exposed to higher ambient temperature and to stress caused by handling. The results were explained by postulating a role for the hexosemonophosphate shunt in providing neurotransmitter amino acids glutamate and -aminobutyrate, and interdependence of brain function and glucose utilization.This paper is dedicated to Dr. Derek Richter on his seventy-fifth birthday.     

The effect of 6-aminonicotinamide on the levels of brain amino acids and glucose,and their labeling with14C after injection of [U-14C] glucose

The brains of rats paralysed at 4 hr after the administration of 6-aminonicotinamide were found to contain decreased levels of glutamate and -aminobutyrate. The glucose content of the brain of the treated rats was several fold higher than in controls. The incorporation of¹⁴C into brain amino acids at 30 min after the injection of [U-¹⁴C]glucose was decreased by 16%: this was attributed to mainly decreased labeling of glutamate and associated amino acids. The results are discussed in the light of previous findings that the administration of 6-aminonicotinamide resulted in the blockade of the direct oxidation of glucose by the pentose phosphate pathway.     

INTERACTION OF LEUCINE, GLUCOSE, AND KETONE METABOLISM IN RAT BRAIN IN VITRO

Brain cortex slices from fed, 48 h and 120 h fasted rats were incubated and ¹⁴CO₂ was measured from (a) [U-¹⁴C]glucose (5 mm ) either alone or in the presence of l -lcucine (0.1 or 1 mm ), and (b) [U-¹⁴C]leucine or [l-¹⁴C]leucine at 0.1 or 1 mm with or without glucose (5 mm ). In other experiments, sodium dl -3-hydroxybutyrate (3-OHB) or acetoacetate (AcAc) at 1 or 5 mm were added in the above incubation mixture. The rate of conversion of [U¹⁴C]glucose to CO₂ was decreased 20% by leucine at 1 mm and 30–50% by 3-OHB at 1 or 5 mm but not by leucine at 0.1 mm . The effects of 3-OHB and of leucine (1 mm ) were not additive. The effects of leucine were similar in the fed and fasted rats. The rate of conversion of [U-¹⁴C]leucine or [l-^,4C]leucine to ¹⁴CO₂ at 0.1 mm and 1.0 mm was increased by glucose (35%) in the fed or fasted rats. Ketone bodies in the absence of glucose had no effect on leucine oxidation. However, the stimulatory effect of glucose on the rate of conversion of leucine to CO₂ was inhibited by 3-OHB at 5 mm . These results suggest that (a) leucine in increased concentrations (1 mm ) may reduce glucose oxidation by brain cortex while itself becoming an oxidative fuel for brain, and (b) leucine oxidation by brain may be influenced by the prevailing glucose and ketone concentrations.     

Cerebral lipid metabolism in experimental hyperphenylalaninaemia: incorporation of 14C-labelled glucose into total lipids

—1. Effects of the administration of phenylalanine to rats on incorporation in vivo or in vitro of [U-¹⁴C]glucose into cerebral lipids were studied during the first 5–10 days of postnatal development. In addition, the effects of added phenylalanine and its deaminated metabolites on incorporation of [U-¹⁴C]glucose by homogenates into lipids of developing rat brain were investigated. Hyperphenylalaninaemia reduced incorporation both in vivo and in vitro of [U-¹⁴C]glucose into cerebral lipids. 2. Phenylalanine or tyrosine added in vitro at concentrations equivalent to those in the brain of the hyperphenylalaninaemic rat (0-1 μmole/ml incubation medium) did not inhibit incorporation of [U-¹⁴C)glucose into lipids, although at much higher concentrations of phenylalanine (36 μumoles/ml incubation medium) slight inhibition (10 per cent) of incorporation of [U-¹⁴C]glucose into lipids was observed. 3. In contrast, the deaminated metabolites in general exerted greater inhibitory effects at lower concentrations. Phenyllactic acid, in comparison to phenylpyruvic and phenyl-acetic acid, was the most potent inhibitor of the incorporation in vitro of [U-¹⁴C]glucose into cerebral lipids. These results indicated that these metabolites of phenylalanine were the more potent inhibitors of cerebral lipid metabolism in immature animals.     

Lipid biosynthesis in anoxic-ischaemic rat brain

The uptake of [U-¹⁴C]glucose and [2-¹⁴C]acetate into lipids was measured in brain slices from anoxic, unilaterally ischaemic, unilaterally anoxic-ischaemic, and control rats. The rate of incorporation was significantly decreased in the brain slices from the treated animals except for the contralateral hemisphere of the unilaterally ischaemic animals. Also, there was no significant difference between the anoxic and the anoxic-ischaemic cerebral hemispheres of the anoxic-ischaemic animals. Fractionation of the total lipid extract demonstrated that the decrease in incorporation was general and not due to any particular class of lipid.     

DEVELOPMENT OF LIPOGENESIS IN RAT BRAIN CORTEX: THE DIFFERENTIAL INCORPORATION OF GLUCOSE AND ACETATE INTO BRAIN LIPIDS IN VITRO

—The oxidation to CO₂ and the incorporation of [U-¹⁴C]glucose and [U-¹⁴C]acetate into lipids by cortex slices from rat brain during the postnatal period were investigated. The oxidation of [U-¹⁴C]glucose was low in 2-day-old rat brain, and increased by about two-fold during the 2nd and 3rd postnatal weeks. The oxidation of [U-¹⁴C]acetate was increased markedly in the second postnatal week, but decreased to rates observed in 2-day-old rat brain at the time of weaning. Both labeled substrates were readily incorporated into non-saponifiable lipids and fatty acids by brain slices from 2-day-old rat. Their rates of incorporation and the days on which maximum rates occurred were different, however, maximum incorporation of [U-¹⁴C]glucose and [U-¹⁴]acetate into lipid fractions being observed on about the 7th and 12th postanatal days, respectively. The metabolic compartmentation in the utilization of these substrates for lipogenesis is suggested. The activities of glucose-6-phosphate dehydrogenase, cytosolic NADP-malate dehydrogenase, cytosolic NADP-isocitrate dehydrogenase, ATP-citrate lyase and acetyl CoA carboxylase were measured in rat brain during the postnatal period. All enzymes followed somewhat different courses of development; the activity of acetyl CoA carboxylase was, however, the lowest among other key enzymes in the biosynthetic pathway, and its developmental pattern paralleled closely the fatty acid synthesis from [U-¹⁴C]glucose. It is suggested that acetyl CoA carboxylase is a rate-limiting step in the synthesis de novo of fatty acids in developing rat brain.     

Effect of thiamine deprivation and thiamine antagonists on the level of gamma-aminobutyric acid and on 2-oxoglutarate metabolism in rat brain

Brain levels of y-aminobutyric acid (GABA), glutamate and 2-oxoglutarate, activities of glutamate decarboxylase GABA-transaminase plus succinic semiaidehyde dehydrogenase and blood levels of glutamate and 2-oxoglutarate were determined in normal, thiamine-deprived, oxythiamine-treated and pyrithiamine-treated rats. Brain GABA levels were significantly reduced in thiamine-deprived and pyrithiamine-treated rats, but the activities of the enzymes of the GABA shunt pathway were not affected. Brain levels of glutamate were decreased and of 2-oxoglutarate increased in all three types of deficiency. This was associated with similar decreases in glutamate and increases in 2-oxoglutarate in the blood in all three deficient groups. Intraventricular injections of 2-[U-¹⁴C] oxoglutarate into the brain in these four groups of rats resulted in some significant differences in distribution of ¹⁴C in various TCA-pathway intermediates and satellite compounds in the brain. Increases in ¹⁴C-label were observed for glutamine and 2-oxoglutarate in all three deficient groups as compared to controls. The ¹⁴C content of succinate, fumarate and aspartate was decreased in the thiamine deprived and PTh-treated groups and [¹⁴C]glutamate was decreased in all three deficient groups. The ¹⁴C content of GABA was not significantly affected.     

Glucose handling by hepatocytes from obese Zucker rats

Hepatocytes isolated from obese Zucker rats showed a significantly higher rate of both [U-¹⁴C]glucose and [U-¹⁴C]lactate incorporation into [¹⁴C]lipid than those from their lean counterparts. This was associated with a marked increase in the lipogenic rate measured by the incorporation of³H₂O into the cell esterified fatty acids. Although there were no changes in the incorporation of the tracer into either [¹⁴C]glycogen or¹⁴CO₂, the [¹⁴C] total uptake was significantly higher in the obese animals. The high rate of [¹⁴C]lipid synthesis from glucose was observed both at 15 and 30 mM substrate concentrations and was linked to an enhanced uptake of the tracer into the cell as measured using the decarboxilation of [1-¹⁴C]glucose in the presence of phenazine methosulphate. The presence of insulin in the incubation medium had no effect on the uptake of glucose by the liver cells. However, the large uptake of glucose by the hepatocytes from the obese animals was not related to an enhanced rate of transport as measured using 3-O-methyl[U-¹⁴C]glucose. The activity of glucose-6-phosphate dehydrogenase together with a higher [1-¹⁴C]glucose/[U-¹⁴C]glucose descarboxylation ratio indicate a predominant very active pentose phosphate pathway which may be responsible for the enhanced glucose uptake observed in the hepatocytes from the obese animals.     

Acetylcholine formation from glucose following acute choline supplementation

The effects of choline administration on acetylcholine metabolism in the central nervous system are controversial. Although choline supplementation may elevate acetylcholine (ACh) content in brain, turnover studies with labelled choline precursors suggest that systemic choline administration either has no effect or actually diminishes brain ACh synthesis. Since choline supplementation elevates brain choline levels, the apparent decreases in previous turnover studies may reflect dilution of the labelled choline precursor pool rather than altered ACh formation. Therefore, brain ACh formation from [U-¹⁴C]glucose was determined after choline supplementation. A two to three fold elevation of brain choline did not alter ACh levels or [U-¹⁴C]glucose incorporation into ACh in the cortex, hippocampus or striatum. Although atropine stimulated ACh formation from [U-¹⁴C]glucose in hippocampus, two to three fold increases in brain choline did not augment ACh synthesis or content in atropine pretreated animals. Atropine depressed brain regional glucose utilization and this effect was not reversed by choline treatment. These results suggest that shorttern elevation of brain choline does not enhance ACh formation from [U-¹⁴C]glucose, and argue against enhanced presynaptic cholinergic function after acute, systemic choline administration.Special issue dedicated to Dr. Louis Sokoloff.     

Ontogenetic Study of the Effects of Energetic Nutrients on Amino Acid Metabolism of Rat Cerebral Cortex

We studied the effect of various energetic nutrients on metabolism of l-[U-¹⁴C]leucine and [1–¹⁴C]glycine in cerebral cortex of rats at different ages. At gestational age, glucose and lactate stimulated protein synthesis from l-[U-¹⁴C]leucine and [1–¹⁴C]glycine and from l-[U-¹⁴C]leucine, respectively; glucose, -OH-butyrate and lactate stimulated lipid synthesis from l-[U-¹⁴C]leucine. At 10 days of age, glucose, mannose, and fructose stimulated protein synthesis, and glucose and mannose stimulated oxidation to CO₂ as well as lipid synthesis from l-[U-¹⁴C]leucine. In adult rats, glucose, mannose, and fructose stimulated protein synthesis from l-[U-¹⁴C]leucine and [1–¹⁴C]glycine; glutamine also markedly decreased the oxidation of l-[U-¹⁴C]leucine and [1–¹⁴C]glycine in 10–day-old and adult rats.     

Measurement of mouse brain glucose utilization in vivo using [U-14C]glucose

The rate of [2-¹⁴C]glucose uptake has been used as an indication of the status of energy consumption by the rat brain, but the cost of this radiolabel can be prohibitive and the surgical manipulation involved in published methods is extensive. A method for measuring glucose utilization in vivo in mouse brain with [U-¹⁴C]glucose is described in this article. Glucose consumption in whole mouse brain obtained with [U-¹⁴C]glucose or [2-¹⁴C]glucose was 0.650±0.022 and 0.716±0.36 nmol/mg/min, respectively. In all instances the rate obtained with the uniformly labeled isotope was somewhat lower than that found with [2-¹⁴C]glucose. The rate of glucose utilization measured with either isotope was significantly depressed in sodium pentobarbital anesthetized mice. The method described here is advantageous because [U-¹⁴C]glucose is substantially less expensive than [2-¹⁴C]glucose and surgical intervention is avoided.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE: DISTRIBUTION IN SUBCELLULAR FRACTIONS AS A FUNCTION OF TIME AFTER INJECTION OF [2-14C]MEVALONIC ACID, SODIUM [2-14C]ACETATE AND [U-14C]GLUCOSE INTO 15-DAY-OLD RATS

The distribution of [¹⁴C]-labelled material into subcellular fractions of 15-day-old rat brain was studied at 2 and 24 h following intraperitoneal and intracerebral injection of [2-¹⁴C]sodium acetate, [U-¹⁴C]glucose and [2-¹⁴C]mevalonic acid respectively. The total quantity of labelled isoprenoids in the brain was, except for glucose, greater when the precursor was administered intracerebrally. The intraperitoneal route was more advantageous in the case of [U-¹⁴C]glucose. The subcellular distribution of both labelled total isoprenoid material and sterol was distinct for each labelled precursor. Intracerebrally injected [U-¹⁴C]glucose at both time periods studied suggested no dominance of labelling in any fraction. After intraperitoneal injection of [U-¹⁴C]glucose the microsomes were more prominently labelled. Both methods of administration of sodium [2-¹⁴C]acetate resulted in heavy labelling of the myelin fraction after 24 h. The total labelled isoprenoids resided mainly in the microsomes 24 h after injection of [2-¹⁴C]mevalonic acid. Labelled sterol was found to be localized more in the myelin and microsomal fractions for all three precursors than was the labelled total isoprenoids. Depending on the type of experiment to be conducted, each of these precursors can give different results, which must be interpreted accordingly.     

Some neurochemical aspects of fluorocitrate intoxication

Abstract— Some metabolic and biochemical effects of fluorocitrate were studied in vivo in rat brain and cat spinal cord. During the preconvulsant and convulsant phases of fluorocitrate poisoning the contents of free glutamate, glutamine and aspartate declined progressively, while that of alanine increased. Incorporation of ¹⁴C from [U-¹⁴C]glucose into these amino acids also decreased, although somewhat more gradually. GABA exhibited a biphasic change, its content rising after an initial decrease while its relative specific activity rose initially and subsequently diminished. Incorporation of ¹⁴C from [U-¹⁴C]glucose and [U-¹⁴C]lysine into neural protein declined sharply. The citric acid content rose markedly in rat brain and cat spinal cord. In rat brain the glycogen content declined but ATP and ammonia contents were unchanged. The significance of these results with respect to energy metabolism and the possible mechanism of the convulsions during fluorocitrate poisoning is discussed.     

Studies on lipogenesis in vivo: Comparison of cholesterol and fatty acid synthesis in rats and mice

1. The importance of fatty acid synthesis as a pathway for the disposal of ingested glucose has been evaluated in rats and mice given a purified diet high in glucose and low in fat. [U-¹⁴C]Glucose was either added to the diet and fed for 24hr. or given by stomach tube as a 250mg. (mice) or 1000mg. (rats) meal. The two methods of isotope administration gave similar results. 2. Under the conditions employed fatty acid synthesis appeared to be a more important pathway for glucose disposal in mice than in rats. In mice 15·3% of ingested [U-¹⁴C]glucose was converted into fatty acid and in rats the corresponding value was 8·6%. In contrast, the conversion of [U-¹⁴C]glucose into cholesterol, as a percentage of dose, was twice as high in rats as in mice. 3. The effect of 20% of corn oil in the diet on the conversion of dietary [U-¹⁴C]glucose into fat was also investigated. Mice given diets containing 1% or 20% of corn oil converted 14·6% or 7·0% respectively of dietary [U-¹⁴C]glucose into fatty acid over a 24hr. period. There was no effect of fat on the incorporation of the isotope into cholesterol. 4. In mice given diets containing 1% or 20% of corn oil approx. 10% and 2% respectively of newly synthesized fatty acids were found in the liver. Hepatic fatty acid synthesis appears to be more sensitive to dietary fat than is extrahepatic synthesis.     

Carbohydrate oxidation by leucoplasts from suspension cultures of soybean (Glycine max L.)

The aim of this work was to discover how leucoplasts from suspension cultures of soybean (Glycine max L.) oxidize hexose monophosphates. Leucoplasts were isolated from protoplast lysates on a continuous gradient of Nycodenz with a yield of 28% and an intactness of 80%. Incubation of the leucoplasts with ¹⁴C-labelled substrates led to ¹⁴CO₂ production, that was dependent upon leucoplast intactness, from [U-¹⁴C]glucose 6-phosphate, [U-¹⁴C]glucose 1-phosphate, [U-¹⁴C] fructose 6-phosphate and [U-¹⁴C]glucose+ATP, but not from [U-¹⁴C]fructose-1,6-bisphosphate or [U-¹⁴C]triose phosphate. The yield from [U-¹⁴C]glucose 6-phosphate was at least four times greater than that from any of the other substrates. When [1-¹⁴C]-, [2-¹⁴C]-, [3,4-¹⁴C]-, and [6-¹⁴C]glucose 6-phosphate were supplied to leucoplasts significant ¹⁴CO₂ production that was dependent upon leucoplast intactness was found only for [1-¹⁴C]glucose 6-phosphate. It is argued that soybean cell leucoplasts oxidize glucose 6-phosphate via the oxidative pentose phosphate pathway with very little recycling, and that in these plastids glycolysis to acetyl CoA is negligible.S.A.C. thanks the Science and Engineering Research Council for a research studentship.     

LIPID COMPOSITION AND METABOLISM OF CULTURED HAMSTER BRAIN ASTROCYTES

Abstract— The lipid composition and metabolism of confluent cultures of cells derived from newborn hamster brain and having morphology characteristic of immature astrocytes or spongioblasts was investigated and compared to that of newborn hamster brain dispersions and cloned glioma cells (C6). The cells displayed stable morphology for at least 30 subcultures; thereafter spontaneous transformation occurred. No appreciable changes were observed in either composition or metabolic characteristics of any major neutral lipid or phospholipid class in successive subcultures or following transformation. The overall lipid composition of the hamster astrocyte cultures closely resembled that of newborn hamster brain, but the phospholipid composition showed substantial differences. The cells contained as a percent of lipid P relatively more ethanolamine plasmalogen, choline plasmalogen and sphingomyelin and somewhat less phosphatidylcholine and phosphatidylethanolamine. The phospholipids of the hamster astrocyte and C6 cells were similar. Of the lipid precursors examined, [U-¹⁴C]glucose was incorporated best into all preparations. C6 glioma cells incorporated both [U-¹⁴C]glucose and [1-¹⁴C]acetate most actively. From 69–88% of ³²P incorporated into hamster astrocyte phospholipids was present in choline phosphoglycerides, whereas the corresonding figure for hamster brain dispersions was 53%. The ratio of specific activities of phosphatidylcholine to phosphatidylinositol was substantially higher in the cultured cells than in the brain preparations. The small pool of choline plasmalogen in the hamster astrocytes usually achieved the highest specific activity of any phospholipid. When [U-¹⁴C]glucose and [1-¹⁴C]acetate were precursors, the bulk of label in the astrocytes appeared in choline phosphoglycerides and triacyglycerol. Our results indicate that the hamster astrocyte cell line as grown expresses distinctive features of lipid composition and metabolism which are nearly constant through many generations.     

EFFECTS OF SODIUM FLUOROACETATE ON THE METABOLISM OF N-ACETYLASPARTATE AND ASPARTATE IN MOUSE BRAIN

A subconvulsant dose of sodium fluoroacetate inhibited the metabolic utilization of intracerebrally-administered N-acetyl-l -[U-¹⁴C]asparticacid and the labelling of glutamine from this precursor in mouse brain, but not the labelling of glutamate or aspartate. A convulsant dose also inhibited the utilization of l -[U-¹⁴C]aspartic acid. When intraperitoneal injection of a convulsant dose of sodium fluoroacetate was followed by intracerebral injection of N-acetyl-l -[U-¹⁴C]asparticacid, the levels of N-acetylaspartate, aspartate and glutamate in brain were lowered, while the glutamine content was increased. The specific radioactivity of glutamine relative to that of glutamate was much lower when these compounds were labelled from l -[U-¹⁴C]aspartic acid than when N-acetyl-l -[U-¹⁴C]aspartic acid was used as the precursor. Intracerebral injection of tracer amounts of l -[U-¹⁴C]aspartic acid reduced the content of N-acetylaspartate in brain and raised the glutamine content. Sodium fluoroacetate had no additional effect on the relative specific radioactivity of glutamine or the content of N-acetylaspartate, aspartate, glutamate or glutamine when l -[U-¹⁴C]aspartic acid was the precursor. We consider the results to be consistent with a selective inhibition both by sodium fluoroacetate and by exogenous aspartic acid of the tricarboxylic acid cycle in brain associated with the biosynthesis of glutamine. We suggest that the activity of this pathway may regulate the metabolism of N-acetylaspartate and aspartate.