Partitioning of CO2 Production Between Glucose and Lactate in Excised Sympathetic Ganglia, with Implications for Brain

Abstract: Chains of lumbar sympathetic ganglia from 15-day-old chicken embryos were incubated for 4 h at 36°C in a bicarbonate-buffered salt solution equilibrated with 5% CO₂-95% O₂. Glucose (1–10 m M ), lactate (1–10 m M ), [U-¹⁴C]glucose, [1-¹⁴C]glucose, [6-¹⁴C]glucose, and [U-¹⁴C]lactate were added as needed. ¹⁴CO₂ output was measured continuously by counting the radioactivity in gas that had passed through the incubation chamber. Lactate reduced the output of CO₂ from [U-¹⁴C]glucose, and glucose reduced that from [U-¹⁴C]lactate. When using uniformly labeled substrates in the presence of 5.5 m M glucose, the output of CO₂ from lactate exceeded that from glucose when the lactate concentration was >2 m M . The combined outputs at each concentration tested were greater than those from either substrate alone. The ¹⁴CO₂ output from [1-¹⁴C]glucose always exceeded that from [6-¹⁴C]glucose, indicating activity of the hexose monophosphate shunt. Lactate reduced both of these outputs, with the maximum difference between them during incubation remaining constant as the lactate concentration was increased, suggesting that lactate may not affect the shunt. Modeling revealed many details of lactate metabolism as a function of its concentration. Addition of a blood-brain barrier to the model suggested that lactate can be a significant metabolite for brain during hyperlactemia, especially at the high levels reached physiologically during exercise.     

THE EFFECT OF DIABETES, INSULIN AND WALLERIAN DEGENERATION ON LEUCINE METABOLISM OF ISOLATED RAT SCIATIC NERVES

Abstract– ¹⁴CO₂ production and ¹⁴C incorporation into proteins was studied in isolated rat sciatic nerves during incubation with 0.1 mM-[1-¹⁴C]leucine. Rats were made diabetic with streptozotocin. Nerves from diabetic rats incubated with glucose oxidized more [¹⁴C]leucine than controls. This difference was abolished in the presence of insulin (1 mU/ml). The effects of diabetes and insulin on leucine oxidation could not be demonstrated in the absence of glucose. Insulin stimulated the incorporation of [¹⁴C] from leucine into proteins by nerves from controls and diabetic rats.
Nerves undergoing Wallerian degeneration showed a marked increase in DNA content and stimulated incorporation of [¹⁴C]leucine into proteins. ¹⁴CO₂ production from leucine proceeded at 75% of the rate observed in intact nerves. Neither insulin nor diabetes affected leucine metabolism in degenerating nerves.
Neither the extracellular space nor the concentration of free amino acids were significantly different in nerves obtained from control and diabetic rats, except for lower glutamine content in the latter.
In vitro leucine metabolism of nerves is affected by diabetes, insulin and the integrity of the axon. The Schwann cell is suggested as a possible site of the observed changes in leucine metabolism.     

Effects of Osmotic Pressure on the Oxidative Metabolism of Leishmania major Promastigotes

Leishmania major promastigotes were washed and resuspended in an iso-osmotic buffer. The rate of oxidation of ¹⁴C-labeled substrates was then measured as a function of osmolality. An acute decrease in osmolality (achieved by adding H₂O to the cell suspension) caused an increase in the rates of ¹⁴CO₂ production from [6-¹⁴C]glucose and, to a lesser extent, from [1, (3)-¹⁴C]glycerol. An acute increase in osmolality (achieved by adding NaCl, KCl, or mannitol) strongly inhibited the rates of ¹⁴CO₂ production from [1-: ¹⁴C]alanine, [1-¹⁴C]glutamate, and [1, (3)-¹⁴C]glycerol. The rates of ¹⁴CO₂ formation from [1-¹⁴C]laurate, [1-¹⁴C]acetate, and [2-¹⁴C]glucose (all of which form [1-¹⁴C]acetyl CoA prior to oxidation) were also inhibited, but less strongly, by increasing osmolality. These data suggest that with increasing osmolality there is an inhibition of mitochondrial oxidative capacity, which could facilitate the increase in alanine pool size that occurs in response to hyper-osmotic stress. Similarly, an increase in oxidative capacity would help prevent a rebuild up of the alanine pool after its rapid loss to the medium in response to hypo-osmotic stress.     

METABOLISM OF GLUCOSE AND FREE AMINO ACIDS IN BRAIN, STUDIED WITH 14C-LABELLED GLUCOSE AND BUTYRATE IN RATS INTOXICATED WITH CARBON DISULPHIDE

Abstract— The effect of 15 h continuous exposure to CS₂ on the metaboliam of glucose and free amino acids in the brain of rats was studied. CS₂ caused a moderate hypoglycaemia. There were also changes in the amounts of some amino acids in the brain. Glutamate and γ-aminobutyrate were lower whereas glutamine was markedly increased. Comparative studies in vivo of the metabolism of [2-¹⁴C]glucose and [1-¹⁴C]butyrate indicated that CS₂ did not affect glycolysis or the incorporation of ¹⁴C from glucose into amino acids except into γ-aminobutyrate which was reduced. Contrary to the findings with [¹⁴C]glucose, CS₂ provoked distinct changes in the labelling of amino acids when [¹⁴C]butyrate was the precursor. The most notable change was a markedly increased incorporation of ¹⁴C into glutamine. Based on the two-compartment model of brain glutamate the experimental findings indicated that CS₂ affected metabolism associated with the 'small' pool of glutamate but had a minimal effect on metabolism associated with the 'large' glutamate pool. The possibility is suggested that the changes observed involved an increased rate of ammonia removal. The low incorporation of ¹⁴C into γ-aminobutyrate from either precursor is consistent with other evidence showing that CS₂ interferes with pyridoxal phosphate-dependent enzymes.     

METABOLISM OF γ-HYDROXY-[1-14C] BUTYRATE BY RAT BRAIN: RELATIONSHIP TO THE KREBS CYCLE AND METABOLIC COMPARTMENTATION OF AMINO ACIDS

Abstract— Ninhydrin decarboxylation experiments were carried out on the labelled amino acids produced following intraventricular injection of either γ-hydroxy-[1-¹⁴C]butyric acid (GHB) or [1-¹⁴C] succinate. The loss of isotope (as ¹⁴CO₂) was similar for both substances. The [1-¹⁴C]GHB metabolites lost 75% of the label and the [1-¹⁴C] succinate metabolites lost 68%. This observation gives support to the hypothesis that the rat brain has the enzymatic capacity to metabolize [1-¹⁴C]GHB to succinate and to amino acids that have the isotope in the carboxylic acid group adjacent to the a-amino group. These results also indicate that the label from [1-¹⁴C]GHB does not enter the Krebs cycle as acetate. The specific activity ratio of radiolabelled glutamine to glutamic acid was determined in order to evaluate which of the two major metabolic compartments preferentially metabolize GHB. It was found that for [1-¹⁴C]GHB this ratio was 4.20 ± 0.18 (S.E. for n = 7) and for [l-¹⁴C]succinate this ratio was 7.71 (average of two trials, 7.74 and 7.69). These results suggest that the compartment thought to be associated with glial cells and synaptosomal structures is largely responsible for the metabolism of GHB. Metabolism as it might relate to the neuropharmacological action of GHB is discussed.     

Analysis of [14C] indole-3-acetic acid metabolites from the primary roots of Zea mays seedlings using reverse-phase high-performance liquid chromatography

Methanolic extracts of Zea mays L. cv. Fronica root segments which had been incubated in [¹⁴C] indole-3-acetie acid were analysed by reverse-phase high-performance liquid chromatography. Metabolism of indole-3-acetic acid was found to be rapid and extensive with at least 11 products apparent after a 2 h incubation. A comparison of metabolites of [1-¹⁴C]– and [2-¹⁴C] IAA, calculations of ¹⁴CO₂ evolution, and data on the polarity of products indicated that decarboxylation had not occurred. An average of 34% of the radioactivity remained associated with the indole-3-acetic acid peak.     

Dual function of pyrimidine metabolism in potato (Solanum tuberosum) plants: pyrimidine salvage and supply of β-alanine to pantothenic acid synthesis

Uridine and cytidine are major nucleosides and are produced as catabolites of pyrimidine nucleotides. To study the metabolic fates and role of these nucleosides in plants, we have performed pulse (2 h) and chase (12 h) experiments with [2-¹⁴C]uridine and [2-¹⁴C]cytidine and determined the activities of some related enzymes using tubers and fully expanded leaves from 10-week-old potato plants ( Solanum tuberosum L.). In tubers, more than 94% of exogenously supplied [2-¹⁴C]uridine and [2-¹⁴C]cytidine was converted to pyrimidine nucleotides and RNA during 2-h pulse, and radioactivity in these salvage products still remained at 12 h after the chase. Little degradation of pyrimidine was found. A similar pyrimidine salvage was operative in leaves, although more than 20% of the radioactivity from [2-¹⁴C]uridine and [2-¹⁴C]cytidine was released as ¹⁴CO₂ during the chase. Enzyme profile data show that uridine/cytidine kinase (EC 2.7.1.48) activity is higher in tubers than in leaves, but uridine nucleosidase (EC 3.2.2.3) activity was higher in leaves. In leaves, radioactivity from [U-¹⁴C]uracil was incorporated into β-ureidopropionic acid, CO₂, β-alanine, pantothenic acid and several common amino acids. Our results suggest two functions of uridine and cytidine metabolism in leaves; these nucleosides are not only substrates for the classical pyrimidine salvage pathways but also starting materials for the biosynthesis of β-alanine. Subsequently, some β-alanine units are utilized for the synthesis of pantothenic acid in potato leaves.     

Effect of Denervation and Reinnervation on Oxidation of [6-14C]Gucose by Rat Skeletal Muscle Homogenates

Abstract: We studied the effects of denervation and reinnervation of the rat extensor digitorum longus muscle (EDL) on the oxidation of [6-¹⁴C]glucose to ¹⁴CO₂. The rate of ¹⁴CO₂ production decreased dramatically following denervation, and the decrease became significant 20 days after nerve section. Prior to day 20, changes apparently reflected the decline of muscle mass. Decreased ¹⁴CO₂ production was due to reduced capacity of the enzymatic system (apparent V_max); there was no change in apparent affinity for glucose (apparent K _m). Mixing experiments revealed that the loss of oxidative capacity following denervation is not caused by production of soluble inhibitors by degenerating muscle. Oxidative metabolism, as measured by ¹⁴CO₂ evolution, recovered during reinnervation. Surprisingly, the specific activity in reinnervated muscles displayed an "overshoot" of approximately 50%, which returned to control by day 60, possibly reflecting increased energy demand by the growing muscle. The time-course of the denervation-mediated change indicates that altered oxidative capacity is secondary to events that initiate denervation changes in muscle. Nevertheless, diminished oxidative capacity may be of considerable metabolic significance in denervated muscle.     

Metabolic Precursors and Compartmentation of Cerebral GABA in Vigabatrin-Treated Rats

Abstract: The metabolic precursors and cerebral compartmentation of the augmented GABA pool induced by vigabatrin, an irreversible inhibitor of GABA transaminase, have been investigated by ¹³C NMR. Adult rats receiving rat chow ad libitum were given either drinking water only or drinking water containing 2.5 g/L vigabatrin for 7 days. Both groups of animals were infused either with [1,2-¹³C₂]acetate (15 µmol/min/100 g body weight), an exclusive precursor of GABA formation through the glial glutamine pathway, or with [1,2-¹³C₂]glucose (15 µmol/min/100 g body weight), a substrate that can produce GABA through the glial glutamine pathway or by direct metabolism in the neurons. The brains were frozen in situ, extracted with perchloric acid, and analyzed by ¹³C NMR. In vigabatrin-treated animals [¹³C]glutamine, a common intermediate for [¹³C]GABA synthesis from glucose or acetate, was accumulated to similar amounts during infusions with [1,2-¹³C₂]glucose or [1,2-¹³C₂]acetate. However, [¹³C]GABA accumulation was sevenfold higher during [1,2-¹³C₂]glucose infusions or twofold higher during [1,2-¹³C₂]acetate infusions. These results show that the direct pathway of GABA formation by neuronal metabolism of glucose predominates over the alternative pathway through glial glutamine. Near-equilibrium relationships of the aminotransferases of GABA and aspartate imply that the observed [¹³C]GABA accumulation occurs initially in the neuronal compartment.     

Portacaval Anastomosis Results in Altered Neuron-Astrocytic Metabolic Trafficking of Amino Acids: Evidence from 13C-NMR Studies

Abstract: ¹³C-NMR spectroscopy was used to evaluate the dynamic consequences of portacaval anastomosis on neuronal and astrocytic metabolism and metabolic trafficking between neurons and astrocytes. Glutamate is predominantly labeled from [1-¹³C]glucose, whereas [2-¹³C]acetate is more efficient in labeling glutamine, in accordance with its primary metabolism in astrocytes. Alanine and succinate labeling was only observed with [1-¹³C]glucose as precursor. Brain [1-¹³C]glucose metabolism in portacaval-shunted rats was similar to that in sham-operated controls with the exception of labeled glutamine and succinate formation, which was increased in shunted rats. The ¹³C enrichment was, however, decreased owing to an increase in total glutamine and succinate. Using [2-¹³C]acetate, on the other hand, flux of astrocytic label to neurons was severely decreased because label incorporation into glutamate, aspartate, and GABA was decreased following portacaval shunting. The latter amino acids are predominantly localized in neurons. These findings demonstrate that metabolic trafficking of amino acids from astrocytes to neurons is impaired in portacaval-shunted rats.     

Effects of In Vivo Hypoxia on Acetylcholine Synthesis by Rat Brain Synaptosomes

Abstract: Synaptosomes from normoxic and hypoxic rats were incubated aerobically in the presence and absence of veratridine. In the absence of veratridine, no significant difference was observed between the two types of preparation regarding either ATP/ADP ratio or ¹⁴CO₂ or [¹⁴C]acetylcholine synthesis from D-[U-¹⁴C]glucose. However, in the presence of veratridine, significant reductions in the output of ¹⁴CO₂ and [¹⁴C]acetylcholine by synaptosomes from hypoxic rats were apparent. It was concluded that irreversible metabolic lesions occur at the synapse as a result of hypoxia, which are apparent only when the metabolism of the preparation is accelerated to a level comparable with the maximal rate occurring in vivo. The presence of such lesions is further evidenced by the significant reductions in ATP/ADP ratio, ¹⁴CO₂ output, and [¹⁴C]acetylcholine synthesis that occur in synaptosomes from hypoxic rats made anoxic in vitro and permitted to recover. Such decreases are not seen when synaptosomes from normoxic rats are similarly treated.     

Brain Carbohydrate Metabolism in Developing Rats During Hypercapnia

Abstract: Brain glucose metabolism was studied in developing rats at ages 10 and 20 days postnatal under normal and hypercapnic conditions. Brains were removed and frozen within 1 s with a freeze-blowing apparatus. Glucose utilization was measured with [2-¹⁴C]glucose and [³H]deoxyglucose as tracers. Metabolites were determined by standard enzymatic techniques. Data from [³H]deoxyglucose phosphorylation indicated that normal brain glucose utilization increased almost threefold between the 10th and 20th postnatal days. From the relative rates of utilization of the two isotopes in the 20-day-old control group, it appeared that about 25% of ¹⁴C label derived from metabolism of [2-¹⁴C]glucose was lost from brain (probably as lactate) rather than entering the Krebs cycle. Under hypercapnic conditions (20% CO₂-21% O₂-59% N₂), rates of glucose utilization by brain were decreased by one-half at both ages and there were progressive decreases in the concentrations of many intermediary metabolites. The bases for concluding that these metabolites were used to supplement glucose as a fuel for respiration, rather than being lost by leakage into blood, are discussed. Despite the differences in brain glucose metabolism between 10-day-old and 20-day-old rats, their responses to hypercapnia are remarkably similar: Rates of glucose utilization are reduced to approximately the same proportion of the original rate by 20% CO₂, and endogenous metabolites (particularly glutamate and lactate) appear to be oxidized as replacement fuels.     

Inhibition of the synthesis of acetylcholine in rat brain slices by (-)-hydroxycitrate and citrate

Abstract: Slices of rat caudate nucleus were incubated in a solution of 123 mM-NaCl, 5 mM-KCl, 1.2 mM-MgCl₂, 1.2 mM-NaH₂PO₄, 25 mM-NaHCO₃, 0.2 mM-choline chloride, 0.058 mM-paraoxon, 1 mM-EGTA, and oxidizable substrates. (−)-Hydroxycitrate, a specific inhibitor of ATP-citrate lyase (EC 4.1.3.8), used at a concentration of 2.5 mM, inhibited the synthesis of acetylcholine (ACh) from [1,5-¹⁴C]citrate by 82–86%, but that from [U-¹⁴C]glucose by only 33%, from [2-¹⁴C]pyruvate by 24% and from [1-¹⁴C-acetyl]carnitine by 8%; the production of ¹⁴CO₂ from these substrates was not substantially changed. The synthesis of ACh from glucose and pyruvate was in hibited also by citrate; 2.5 mM- and 5 mM-citrate diminished it by 43% and 66%, respectively; the production of from [U-¹⁴C]glucose and from [1-¹⁴C]pyruvate was not affected. The mechanism of the inhibitory effect of citrate on the synthesis of ACh is not clear; the possibility is discussed that citrate alters the intracellular milieu in cholinergic neurons by chelating the intracellular Ca²⁺ and decreases the supply of mitochondrial acetyl-CoA to the cytosol. The results with (−)-hydroxycitrate indicate that the cleavage of citrate by ATP-citrate lyase is not responsible for the supply of more than about one-third of the acetyl-CoA which is used for the synthesis of ACh when glucose or pyruvate are the main oxidizable substrates. This proportion may be even smaller, since (−)-hydroxycitrate possibly affects the synthesis of ACh from glucose and pyruvate by a mechanism (unknown) similar to that of citrate, rather than by the inhibition of ATP-citrate lyase.     

Selective Inhibition of the Tricarboxylic Acid Cycle of GABAergic Neurons with 3-Nitropropionic Acid In Vivo

Abstract: The effects of 3-nitropropionic acid (3-NPA), an inhibitor of succinate dehydrogenase, on cerebral metabolism were investigated in mice by NMR spectroscopy. 3-NPA, 180 mg/kg, caused a dramatic buildup of succinate. Succinate was labeled 5.5 times better from [1-¹³C]glucose than from [2-¹³C]acetate, showing a predominantly neuronal accumulation. [1-¹³C]Glucose labeled GABA in the C-2 position only, compatible with inhibition of the tricarboxylic acid (TCA) cycle associated with GABA formation, at the level of succinate dehydrogenase. Aspartate was not labeled by [1-¹³C]glucose in 3-NPA-intoxicated animals. In contrast, [1-¹³C]glucose labeled glutamate in the C-2, C-3, and C-4 positions showing uninhibited cycling of label in the TCA cycle associated with the large, neuronal pool of glutamate. The labeling of glutamine, and hence GABA, from [2-¹³C]acetate showed that the TCA cycle of glial cells was unaffected by 3-NPA and that transfer of glutamine from glia to neurons took place during 3-NPA intoxication. The high ¹³C enrichment of the C-2 position of glutamine from [1-¹³C]glucose showed that pyruvate carboxylation was active in glia during 3-NPA intoxication. These findings suggest that 3-NPA in the initial phase of intoxication fairly selectively inhibited the TCA cycle of GABAergic neurons; whereas the TCA cycle of glia remained uninhibited as did the TCA cycle associated with the large neuronal pool of glutamate, which includes glutamatergic neurons. This may help explain why the caudoputamen, which is especially rich in GABAergic neurons, selectively undergoes degeneration both in humans and animals intoxicated with 3-NPA. Further, the present results may be of relevance for the study of basal ganglia disorders such as Huntington's disease.     

14C-LABELLED AMINO ACIDS AND GLUCOSE IN RAT BRAIN AND LIVER AFTER INJECTION OF [2-14C]PROPIONATE

Abstract— [2-¹⁴C]Propionate injected into rats was metabolized into [¹⁴C]glucose and ¹⁴C-labelled aspartate, glutamate, glutamine and alanine. The results are consistent with the conversion of propionate into succinate and the oxidation of succinate into oxaloacetate, the precursor of labelled amino acids and the substrate for gluconeogenesis.
The ratio of the specific radioactivity of glutamine to glutamate was greater than 1 during the 30 min period in the brain, indicating that propionate taken up by the brain was metabolized mainly in the 'small glutamate compartment' in the brain. The results, therefore, support the previous conclusion (G aitonde , 1975) that the labelling of amino acids by [¹⁴C]propionate formed from [U-¹⁴C>]-threonine in thiamin-deficient rats was metabolized in the 'large glutamate compartment' of the brain.
The specific radioactivity ratio of glutamine to glutamate in the liver was less than 1 during the 10 min period but greater than 1 at 30min. These findings which gave evidence against metabolic compartments of glutamate in the liver, were interpreted as indicative of the entry of blood-borne [¹⁴C]glutamine synthesized in other tissues, e.g. brain. The labelling of amino acids when compared to that after injection of [U-¹⁴C]glucose showed that [2-¹⁴C]propionate was quantitatively a better source of amino acids in the liver. The concentration of some amino acids in the brain and liver was less in the adult than in the young rats, except for alanine and glutathione, where the liver content was more than double that in the adult.     

Glucose Metabolism in the Developing Cerebral Cortex as Detected by 1H{13C} Nuclear Magnetic Resonance Spectroscopy Ex Vivo

Abstract: Metabolism of [1-¹³C]glucose was monitored in superfused cerebral cortex slice preparations from 1-, 2-, and 5-week-old rats using ¹H-observed/¹³C-edited (¹H{¹³C}) NMR spectroscopy. The rate of label incorporation into glutamate C-4 did not differ among the three age groups: 0.52–0.67% of total ¹H NMR-detected glutamate/min. This was rather unexpected, as oxygen uptake proceeded at 1.1 ± 0.1, 1.9 ± 0.1, and 2.0 ± 0.1 µmol/min/g wet weight in brain slices prepared from 1-, 2-, and 5-week-old animals, respectively. Steady-state glutamate C-4 fractional enrichments in the slice preparations were ∼23% in all age groups. In the acid extracts of slices glutamate C-4 enrichments were smaller, however, in 1- and 2-week-old (17.8 ± 1.7 and 16.8 ± 0.8%, respectively) than in 5-week-old rats (22.7 ± 0.7%) after 75 min of incubation with 5 m M [1-¹³C]glucose. We add a new assignment to the ¹H{¹³C} NMR spectroscopy, as acetate C-2 was detected in slice preparations from 5-week-old animals. In the acid extracts of slice preparations acetate C-2 was labeled by ∼30% in 5-week-old rats but by 15% in both 1- and 2-week-old animals, showing that the turnover rate was increased in 5-week-old animals. In the extracts 3–4% of the C-6 of N -acetyl-aspartate (NAA; CH₃ of the acetyl group) contained label as determined by both NMR and mass spectrometry, which indicated that there was no significant labeling to other carbons in NAA. NAA accumulated label from [1-¹³C]glucose but not from [2-¹³C]acetate, and the rate of label incorporation increased by threefold on cerebral maturation.     

GAMMA-HYDROXYBUTYRATE DEGRADATION IN THE BRAIN IN VIVO: NEGLIGIBLE DIRECT CONVERSION TO GABA

Abstract— The metabolism of γ-hydroxybutyrate (GHB) was studied by following the fate of [1-¹⁴C]GHB in mouse brain after an intravenous injection. Cerebral uptake of GHB was rapid and this substance disappeared from brain tissue with a half-life of approx 5 min. Degradation of [1-¹⁴C]GHB took place in the brain since ¹⁴C was incorporated in amino acids associated with the tricarboxylic acid cycle: the labelling pattern was consistent with the oxidation of GHB via succinate through the cycle, rather than with β-oxidation of GHB. Conversion of [¹⁴C]GHB into [¹⁴C]GABA prior to oxidation was negligible, thus it is unlikely that the pharmacological action of GHB would be mediated through GABA formation. [¹⁴C]GHB oxidation also elicited the signs of metabolic compartmentation of the tricarboxylic acid cycle in the brain (glutamine/glutamate specific radioactivity ratio was about 4).     

Metabolism of Glycine in Primary Astroglial Cells: Synthesis of Creatine, Serine, and Glutathione

Abstract: The metabolism of [2-¹³C]glycine in astrogliarich primary cultures obtained from brains of neonatal Wistar rats was investigated using ¹³C NMR spectroscopy. After a 24-h incubation of the cells in a medium containing glucose, glutamate, cysteine, and [2-¹³C]glycine, cell extracts and incubation media were analyzed for ¹³C-labeled compounds. Labeled creatine, serine, and glutathione were identified in the cell extracts. If arginine and methionine were present during the incubation with [2-¹³C]glycine, the amount of de novo synthesized [2-¹³C]creatine was two-fold increased, and in addition, ¹³C-labeled guanidinoacetate was found in cell extracts and in the media after 24 h of incubation. A major part of the [2-¹³C]glycine was utilized for the synthesis of glutathione in astroglial cells. ¹³C-labeled glutathione was found in the cell extracts as well as in the incubation medium. The presence of newly synthesized [2-¹³C]serine, [3-¹³C]serine, and [2,3-¹³C]serine in the cell extracts and the incubation medium proves the capability of astroglial cells to synthesize serine out of glycine and to release serine. Therefore, astroglial cells are able to utilize glycine as a precursor for the synthesis of creatine and serine. This proves that at least one cell type of the brain is able to synthesize creatine. In addition, guanidinoacetate, the intermediate of creatine synthesis, is released by astrocytes and may be used for creatine synthesis by other cells, i.e., neurons.     

Exchange-mediated dilution of brain lactate specific activity: implications for the origin of glutamate dilution and the contributions of glutamine dilution and other pathways

The magnitude of metabolic activation is greatly underestimated in autoradiographic studies using [1- or 6-¹⁴C]glucose compared to parallel assays with [¹⁴C]deoxyglucose indicating that most of the label corresponding to the additional [¹⁴C]glucose consumed during activation compared to rest is quickly released from activated structures. Label could be lost by net release of [¹⁴C]lactate from brain or via lactate exchange between blood and brain. These possibilities were distinguished by comparison of glucose and lactate specific activities in arterial blood and brain before, during, and after generalized sensory stimulation and during spreading cortical depression. Over a wide range of brain lactate concentrations, lactate specific activity was close to the theoretical maximum, i.e. half that of [6-¹⁴C]glucose, indicating that exchange-mediated dilution of lactate is negligible and that efflux of [¹⁴C]lactate probably accounts for most of the label loss. Low lactate dilution also indicates that dilution of glutamate C4 fractional enrichment in [¹³C]glucose studies, currently ascribed predominantly to lactate exchange, arises from other unidentified pathways or factors. Alternative explanations for glutamate dilution (presented in Supporting Information) include poorly labeled amino acid pools and oxidative metabolism of minor substrates in astrocytes to first dilute the astrocytic glutamine pool, followed by dilution of glutamate via glutamate–glutamine cycling.     

Regulation of n-3 and n-6 Fatty Acid Metabolism in Retinal and Cerebral Microvascular Endothelial Cells by High Glucose

Abstract: The present study was undertaken to determine whether polyunsaturated fatty acid metabolism is affected by high glucose levels in cerebral and retinal microvascular endothelial cells. The metabolism of [3-¹⁴C]22:5n-3 and [1-¹⁴C]18:2n-6 was studied in cells previously cultured for 5 days in normal (5 m M ) or high (30 m M ) glucose medium. After incubation of retinal endothelial cells with [3-¹⁴C]22:5n-3 in the high glucose condition, the formation of labeled 24:6n-3 and 22:6n-3 was increased, and that of labeled 24:5n-3 was decreased, compared with the normal glucose condition. The changes were found for fatty acids esterified in cellular lipids and those released into the medium. After incubation with [1-¹⁴C]18:2n-6, levels of all elongation/desaturation products were increased at the expense of the precursor in retinal endothelial cells cultured in high glucose medium. The changes were primarily found for esterified fatty acids, with the release of n-6 fatty acids being minor in both glucose concentrations. By contrast, high glucose levels did not affect the metabolism of [3-¹⁴C]22:5n-3 and [1-¹⁴C]18:2n-6 in cerebral endothelial cells. The changes in metabolic activity of retinal endothelial cells were not reflected in the fatty acid composition. The present data suggest that high glucose can increase the desaturation process in retinal but not cerebral endothelial cells. This may produce some lipid abnormalities in retinal microvasculature and contribute to altered vascular function observed in diabetic retinopathy.