(13)C MR spectroscopy study of lactate as substrate for rat brain

In order to address the question whether lactate in blood can serve as a precursor for cerebral metabolites, fully awake rats were injected intravenously with [U-(13)C]lactate or [U-(13)C]glucose followed 15 min later by decapitation. Incorporation of label from [U-(13)C]glucose was seen mainly in glutamate, GABA, glutamine, aspartate, alanine and lactate. More label was found in glutamate than glutamine, underscoring the predominantly neuronal metabolism of pyruvate from [U-(13)C]glucose. It was estimated that the neuronal metabolism of acetyl CoA from glucose accounts for at least 66% and the glial for no more than 34% of the total glucose consumption. When [U-(13)C]lactate was the precursor, label incorporation was similar to that observed from [U-(13)C]glucose, but much reduced. Plasma analysis revealed the presence of approximately equal amounts of [1,2,3-(13)C]- and [1,2-(13)C]glucose, showing gluconeogenesis from [U-(13)C]lactate. It was thus possible that the labeling seen in the cerebral amino acids originated from labeled glucose, not [U-(13)C]lactate. However, the presence of significantly more label in [U-(13)C]- than in [2,3-(13)C]alanine demonstrated that [U-(13)C]lactate did indeed cross the blood-brain barrier, and was metabolized further in the brain. Furthermore, contributions from pyruvate carboxylase (glial enzyme) were detectable in glutamine, glutamate and GABA, and were comparatively more pronounced in the glucose group. This indicated that relatively more pyruvate from lactate than glucose was metabolized in neurons. Surprisingly, the same amount of lactate was synthesized via the tricarboxylic acid cycle in both groups, indicating transfer of neurotransmitters from the neuronal to the astrocytic compartment, as previous studies have shown that this lactate is synthesized primarily in astrocytes. Taking into consideration that astrocytes take up glutamate more avidly than GABA, it is conceivable that neuronal lactate metabolism was more prominent in glutamatergic neurons.     

GABA alters the metabolic fate of [U-13C]glutamate in cultured cortical astrocytes

The effect of gamma-aminobutyric acid (GABA) on glutamate metabolism was studied by (13)C-nuclear magnetic resonance (NMR) spectroscopy. Cerebral cortical astrocytes were incubated with 0.5 mM [U-(13)C]glutamate and 5 mM glucose in the presence or absence of 0.2 mM GABA for 2 hr. (13)C-labeled glutamate, glutamine, and aspartate were observed in cell extracts, and (13)C-labeled glutamine and lactate were present in the media. Both uniformly labeled glutamate and [1,2,3-(13)C]glutamate derived from the tricarboxylic acid (TCA) cycle were present in the cells. The consumption of [U-(13)C]glutamate and glucose was unchanged in the presence of GABA; however, the formation of [U-(13)C]lactate and [U-(13)C]aspartate from metabolism of [U-(13)C]glutamate was increased in cells incubated with GABA. The total concentration of aspartate was increased to the same extent as the (13)C-labeled aspartate, suggesting increased entry of [U-(13)C]glutamate into the TCA cycle to allow for the transamination of GABA. Although the concentrations of unlabeled glucose and lactate in the media were unchanged in the presence of GABA, the concentration of alanine was decreased, indicating that there was decreased transamination of the unlabeled pyruvate from glucose metabolism. The amount of [U-(13)C]glutamate converted to [U-(13)C]glutamine and [U-(13)C]lactate was increased in the presence of GABA. However, since the overall consumption of [U-(13)C]glutamate was not different, it can be concluded that the amount of [U-(13)C]glutamate used for energy was decreased. This suggests that exogenous GABA could substitute for glutamate as an energy source for astrocytes. The results indicate that the presence of GABA influences the metabolic fate of both glutamate and glucose in astrocytes, suggesting that fluctuations in the concentration of GABA in normal and pathological conditions can alter the compartmentation of glial metabolism in brain.     

Methylmercury has a selective effect on mitochondria in cultured astrocytes in the presence of [U-(13)C]glutamate.

The effect of methylmercury on glutamate metabolism was studied by (13)C magnetic resonance spectroscopy. Cerebral cortical astrocytes were pretreated with methylmercury, either 1 microM for 24 h, or 10 microM for 30 min, and subsequently with 0.5 mM [U-(13)C]glutamate for 2 h. Labeled glutamate, glutamine, aspartate and glutathione were present in cell extracts, and glutamine, aspartate and lactate in the medium of all groups. HPLC analysis of these amino acids showed no changes in concentrations between groups. Surprisingly, the amounts of [U-(13)C]glutamate and unlabeled glucose taken up by the astrocytes were unchanged. Furthermore, the amounts of most metabolites synthesized from [U-(13)C]glutamate were also unchanged in all groups. However, formation of [U-(13)C]lactate was decreased in the 10 microM methylmercury group. This was not observed for labeled aspartate. It is noteworthy that both [U-(13)C]lactate and [U-(13)C]aspartate can only be derived from [U-(13)C]glutamate via mitochondrial metabolism. [U-(13)C]glutamate enters the tricarboxylic acid cycle (located in mitochondria) after conversion to 2-[U-13C]oxoglutarate and [U-(13)C]aspartate is formed from [U-(13)C]oxaloacetate, as is [U-(13)C]lactate. [U-(13)C]lactate can also be formed from [U-(13)C]malate. This differential effect on labeled aspartate and lactate indicates cellular compartmentation and thus selective vulnerability of mitochondria within the astrocytes to the effects of methylmercury. The decreased lactate production from glutamate might be detrimental to surrounding cells since lactate has been shown to be an important substrate for neurons.     

Glutamate decreases pyruvate carboxylase activity and spares glucose as energy substrate in cultured cerebellar astrocytes.

The effects of glutamate on [U-(13)C]glucose metabolism were studied in cerebellar astrocytes using (13)C magnetic resonance spectroscopy. Labeled glutamate, glutamine, aspartate, lactate, and alanine were observed both in the cell extracts and in media, and, additionally, labeled glycogen was detected in the cell extracts. However, only labeled lactate and alanine were quantifiable in the medium in addition to [U-(13)C]glucose. In the presence of unlabeled glutamate, the amount of [U-(13)C]glucose removed from the medium was decreased, indicating that glutamate might spare glucose as an energy substrate and thus decrease the uptake of glucose. Labeled glycogen, [4,5-(13)C]glutamate, [3,4,5-(13)C]glutamate, [3,4-(13)C]aspartate, and [U-(13)C]alanine were increased in the presence of glutamate. However, the increase in the amount of [3,4,5-(13)C]glutamate from the second turn in the tricarboxylic acid (TCA) cycle was less pronounced than that of [4,5-(13)C]glutamate from the first turn in the TCA cycle. This indicates the dilution of label, probably resulting from the synthesis of unlabeled oxaloacetate from glutamate in the TCA cycle. Furthermore, exogenous glutamate had an inhibiting effect on pyruvate carboxylation, presumably by formation of oxaloacetate from 2-oxoglutarate derived from glutamate. It could be shown that glucose is a better substrate for energy production than glutamate; it is, however, less efficient in labeling amino acids than glutamate in cerebellar astrocytes.     

Brain amino acid metabolism and ketosis

The relationship between ketosis and brain amino acid metabolism was studied in mice that consumed a ketogenic diet (>90% of calories as lipid). After 3 days on the diet the blood concentration of 3-OH-butyrate was approximately 5 mmol/l (control = 0.06-0.1 mmol/l). In forebrain and cerebellum the concentration of 3-OH-butyrate was approximately 10-fold higher than control. Brain [citrate] and [lactate] were greater in the ketotic animals. The concentration of whole brain free coenzyme A was lower in ketotic mice. Brain [aspartate] was reduced in forebrain and cerebellum, but [glutamate] and [glutamine] were unchanged. When [(15)N]leucine was administered to follow N metabolism, this labeled amino acid accumulated to a greater extent in the blood and brain of ketotic mice. Total brain aspartate ((14)N + (15)N) was reduced in the ketotic group. The [(15)N]aspartate/[(15)N]glutamate ratio was lower in ketotic animals, consistent with a shift in the equilibrium of the aspartate aminotransferase reaction away from aspartate. Label in [(15)N]GABA and total [(15)N]GABA was increased in ketotic animals. When the ketotic animals were injected with glucose, there was a partial blunting of ketoacidemia within 40 min as well as an increase of brain [aspartate], which was similar to control. When [U-(13)C(6)]glucose was injected, the (13)C label appeared rapidly in brain lactate and in amino acids. Label in brain [U-(13)C(3)]lactate was greater in the ketotic group. The ratio of brain (13)C-amino acid/(13)C-lactate, which reflects the fraction of amino acid carbon that is derived from glucose, was much lower in ketosis, indicating that another carbon source, i.e., ketone bodies, were precursor to aspartate, glutamate, glutamine and GABA.     

13C-Labeled substrates and the cerebral metabolic compartmentalization of acetate and lactate

[1-13C]Glucose, [2-13C]acetate and [3-13C]lactate were infused into male Sprague-Dawley rats (150-170 g) for periods of 3-100 min (n=4 per time) and neocortex extracts were analyzed using 13C-edited 1H magnetic resonance (MR) spectroscopy. The time dependence of the [4-13C]glutamine/[4-13C]glutamate labeling ratio was significantly different for all three substrates infused (p<0.001) and showed that acetate is primarily utilized by glia and lactate by neurons, whereas glucose is ubiquitous. The ratio of second- to first-turn TCA cycle labeling for glutamine was significantly lower for acetate (30-100 min infusion; p<0.02) and greater for lactate (10-30 min; p<0.02) than for glucose infusions, while the C-2/C-4 glutamate labeling ratio was similar for all the three substrates. This indicated that transfer of [2-13C]acetate-derived [4-13C]glutamine to neurons was preferred to reentry of label into the glial TCA cycle and that the neuronal TCA cycle turnover is significantly faster than that for glia. Fitting parameters of a function representing a pseudo-first-order process to the time dependence of labeling demonstrated that GABA labeling reaches steady state faster with glutamine labeled from [2-13C]acetate than with glutamate labeled from [3-13C]lactate. It is concluded that lactate represents a significant improvement over glucose in the study of neuronal metabolism and complements the use of acetate to study glial metabolism and glial/neuronal metabolic relationships.     

Glucose is necessary to maintain neurotransmitter homeostasis during synaptic activity in cultured glutamatergic neurons.

Glucose is the primary energy substrate for the adult mammalian brain. However, lactate produced within the brain might be able to serve this purpose in neurons. In the present study, the relative significance of glucose and lactate as substrates to maintain neurotransmitter homeostasis was investigated. Cultured cerebellar (primarily glutamatergic) neurons were superfused in medium containing [U-13C]glucose (2.5 mmol/L) and lactate (1 or 5 mmol/L) or glucose (2.5 mmol/L) and [U-13C]lactate (1 mmol/L), and exposed to pulses of N-methyl-D-aspartate (300 micromol/L), leading to synaptic activity including vesicular release. The incorporation of 13C label into intracellular lactate, alanine, succinate, glutamate, and aspartate was determined by mass spectrometry. The metabolism of [U-13C]lactate under non-depolarizing conditions was high compared with that of [U-13C]glucose; however, it decreased significantly during induced depolarization. In contrast, at both concentrations of extracellular lactate, the metabolism of [U-13C]glucose was increased during neuronal depolarization. The role of glucose and lactate as energy substrates during vesicular release as well as transporter-mediated influx and efflux of glutamate was examined using preloaded D-[3H]aspartate as a glutamate tracer and DL-threo-beta-benzyloxyaspartate to inhibit glutamate transporters. The results suggest that glucose is essential to prevent depolarization-induced reversal of the transporter (efflux), whereas vesicular release was unaffected by the choice of substrate. In conclusion, the present study shows that glucose is a necessary substrate to maintain neurotransmitter homeostasis during synaptic activity and that synaptic activity does not induce an upregulation of lactate metabolism in glutamatergic neurons.     

Glutamate is preferred over glutamine for intermediary metabolism in cultured cerebellar neurons.

The glutamate-glutamine cycle is thought to be of paramount importance in the mature brain; however, its significance is likely to vary with regional differences in distance between astrocyte and synapse. The present study is aimed at evaluating the role of this cycle in cultures of cerebellar neurons, mainly consisting of glutamatergic granule cells. Cells were incubated in medium containing [U-13C]glutamate or [U-13C]glutamine in the presence and absence of unlabeled glutamine and glutamate, respectively. Cell extracts and media were analyzed using high-performance liquid chromatography (HPLC) and gas chromatography combined with mass spectrometry (GC/MS). Both [U-13C]glutamate and [U-13C]glutamine were shown to be excellent precursors for synthesis of neuroactive amino acids and tricarboxylic acid (TCA) cycle intermediates. Labeling from [U-13C]glutamate was higher than that from [U-13C]glutamine in all metabolites measured. The presence of [U-13C]glutamate plus unlabeled glutamine in the experimental medium led to labeling very similar to that from [U-13C]glutamate alone. However, incubation in medium containing [U-13C]glutamine in the presence of unlabeled glutamate almost abolished labeling of metabolites. Thus, it could be shown that glutamate is the preferred substrate for intermediary metabolism in cerebellar neurons. Label distribution indicating TCA cycle activity showed more prominent cycling from [U-13C]glutamine than from [U-13C]glutamate. Labeling of succinate was lower than that of the other TCA cycle intermediates, indicating an active role of the gamma-amino butyric acid shunt in these cultures. It can be concluded that the cerebellar neurons rely more on reuptake of glutamate than supply of glutamine from astrocytes for glutamate homeostasis.     

13C NMR spectroscopy study of cortical nerve cell cultures exposed to hypoxia

Primary cultures of cerebral cortical GABA-ergic neurons growing on top of a preformed layer of astrocytes (co-cultures) were incubated with [1-¹³C]glucose and exposed to a low oxygen atmosphere (2% O₂) for 17 hr. ¹³C, ¹H, and ¹³P nuclear magnetic resonance (NMR) spectroscopy was performed on perchloric acid (PCA) extracts of cells and of media collected from these cultures. In the control groups incorporation of ¹³C label into glutamine, citrate, and lactate could be demonstrated in both cell extracts and culture media. Labeled GABA and glutamate were only observed in cell extracts. During hypoxia high energy phosphates decreased but lactate production and glucose consumption increased. There was a decreased amount of citrate and glutamine in cell extracts and media of the hypoxic co-cultures. There was a change in distribution of the ¹³C label within the GABA molecule, with an increase of labeling in the C-2 position. This change in ¹³C distribution was not found in glutamine present in the media where it is a precursor for GABA in neurons. Instead a decrease in the corresponding C-4 position was observed. These results suggest that energy depletion during hypoxia leads to reduced export from the astrocytic tricarboxylic acid (TCA) cycle as demonstrated by a decreased amount of citrate and changed distribution of ¹³C in glutamine. The change in the distribution of label in GABA from cell extracts as compared to glutamine in the medium may indicate that neurons are synthesizing GABA using precursors supplied from their own TCA cycle and not from precursors supplied by astrocytes.     

Intracellular compartmentation of pyruvate in primary cultures of cortical neurons as detected by (13)C NMR spectroscopy with multiple (13)C labels.

The intracellular compartmentation of pyruvate in primary cultures of cortical neurons was investigated by high resolution (13)C NMR using mixtures of different pyruvate precursors conveniently labeled with (13)C or unlabeled. Cells were incubated with 1-5 mM (1-(13)C, 1,2-(13)C(2) or U-(13)C(6)) glucose only or with mixtures containing 1.5 mM (1-(13)C or U-(13)C(6)) glucose, 0.25-2.5 mM (2-(13)C or 3-(13)C) pyruvate and 1 mM malate. Extracts from cells and incubation media were analyzed by (13)C NMR to determine the relative contributions of the different precursors to the intracellular pyruvate pool. When ((13)C) glucose was used as the sole substrate fractional (13)C enrichments and (13)C isotopomer populations in lactate and glutamate carbons were compatible with a unique intracellular pool of pyruvate. When mixtures of ((13)C) glucose, ((13)C) pyruvate and malate were used, however, the fractional (13)C enrichments of the C2 and C3 carbons of lactate were higher than those of the C2 and C3 carbons of alanine and depicted a different (13)C isotopomer distribution. Moreover, neurons incubated with 1 mM (1,2-(13)C(2)) glucose and 0.25-5 mM (3-(13)C) pyruvate produced exclusively (3-(13)C) lactate, revealing that extracellular pyruvate is the unique precursor of lactate under these conditions. These results reveal the presence of two different pools of intracellular pyruvate; one derived from extracellular pyruvate, used mainly for lactate and alanine production and one derived from glucose used primarily for oxidation. A red-ox switch using the cytosolic NAD(+)/NADH ratio is proposed to modulate glycolytic flux, controlling which one of the two pyruvate pools is metabolized in the tricarboxylic acid cycle when substrates more oxidized or reduced than glucose are used.     

Pentylenetetrazole affects metabolism of astrocytes in culture

Cortical and cerebellar astrocytes were cultured in medium containing pentylenetetrazole (PTZ), a gamma-aminobutyric acid (GABA)(A) receptor antagonist, for 3 weeks (up to 6 mM) or 2 hr (10 mM). Cells were incubated in medium containing [U-(13)C]glutamate (0.5 mM) and unlabeled glucose (3 mM) for 2 hr and cell extracts and media were analyzed by (13)C magnetic resonance (MR) spectroscopy and high-performance liquid chromatography (HPLC). When cerebellar astrocytes were incubated with PTZ for 2 hr, the amount of glucose removed from the medium and glucose and [U-(13)C]glutamate oxidation were decreased. Metabolism in cortical astrocytes was affected only slightly; amounts of glutathione and aspartate were decreased. When cerebellar and cortical cells were cultured in the presence of PTZ for 3 weeks, the amount of glucose removed from the medium and lactate formed were increased, indicating increased glycolytic activity. Despite the increased intracellular [U-(13)C]glutamate concentration in both types of astrocytes cultured with PTZ, labeled glutamine and glutathione were unchanged, indicating intracellular compartmentation. The amount of cellular protein was decreased at 6 mM PTZ for cerebellar astrocytes and 1 mM for cortical astrocytes, indicating a differential sensitivity to the effects of PTZ. In conclusion, mitochondrial metabolism and glycolysis were decreased by short-term incubation with PTZ in cerebellar astrocytes, whereas long-term incubation affected both types of astrocytes, leading to increased glycolysis.     

Elucidation of the quantitative significance of pyruvate carboxylation in cultured cerebellar neurons and astrocytes.

Pyruvate carboxylation was studied in cerebellar astrocytes and granule neurons. The cells were incubated in medium containing [U-(13)C]glucose (2.5 mM) and [U-(13)C]lactate (1 mM) and varying amounts of 3-nitropropionic acid (3-NPA) plus/minus aspartate. 3-NPA alone clearly stopped tricarboxylic acid (TCA) cycle activity at the succinate dehydrogenase step in both culture types as evidenced by a buildup of succinate. Labeling of aspartate and glutamate was abolished in neurons in the presence of 3-NPA. In astrocytes, however, labeled glutamate and glutamine derived from pyruvate carboxylation was detected. Unchanged glucose and lactate metabolism in the absence of a functioning malate aspartate shuttle indicates the importance of the glycerol-3-phosphate shuttle in brain cells. To compensate for the loss of oxaloacetate in the presence of 3-NPA, unlabeled aspartate (0.25 mM) was added. In this case [1,2-(13)C] and [3,4-(13)C]aspartate were observed in neurons but not in astrocytes. This labeling pattern in aspartate occurs after a full turn of the TCA cycle and thus indicates only partial inhibition by 3-NPA in the neurons when aspartate is present. In astrocytes, however, aspartate derived from uniformly labeled pyruvate was observed clearly indicating pyruvate carboxylation. The present study has unequivocally demonstrated a quantitatively important pyruvate carboxylation in astrocytes but it was not possible to demonstrate the presence of such carboxylation in neurons. Based on the present results it may be safely concluded that neuronal pyruvate carboxylation is unlikely to be of quantitative significance.     

Effect of acute insulin-induced hypoglycemia on fetal versus adult brain fuel utilization, assessed by (13)C MRS isotopomer analysis of [U-(13)C]glucose metabolites

Tight glycemic control during diabetic pregnancy has been shown to significantly reduce the occurrence of congenital malformations and other effects of maternal diabetes on the offspring. However, intensive insulin therapy often causes recurring acute maternal hypoglycemia, which has been found to be harmful to the developing fetus, although the mechanisms involved are not clear. The aim of our work was to study the effect of acute insulin-induced maternal hypoglycemia on glucose metabolism in the fetal brain. To this end, near-term pregnant New Zealand rabbits were rendered hypoglycemic, and [U-(13)C]glucose was infused into maternal circulation. The metabolic fate of the (13)C-labeled glucose was then studied in fetal brain extracts by (13)C NMR isotopomer analysis, together with conventional biochemical assays of glucose and lactate levels in both plasma and brain. For comparison [U-(13)C]glucose was also administered to insulin-induced hypoglycemic young adult rabbits. Our results showed that while plasma glucose levels were significantly reduced (approximately 70%) relative to controls, no changes in cerebral glucose levels could be detected. Lactate levels were found to be significantly decreased in hypoglycemic fetal plasma and brain. No differences in lactate levels between control and hypoglycemic young rabbit plasma and brain were observed. These differences were attributed to the utilization of lactate as an energy substrate in the fetal brain, but not in the adult brain. Higher relative (13)C enrichments of most glucose metabolites, except lactate, in the hypoglycemic fetal and young rabbit brains, observed by (13)C NMR, stem from reduced endogenous plasma glucose pools, thereby diluting the labeled glucose to a lower extent. The relative glucose (or glucose-derived lactate) flux via the pyruvate carboxylase and pyruvate dehydrogenase pathways (PC/PDH ratio) was not altered under hypoglycemic conditions in the fetal brain for both glutamine and glutamate, but significantly increased in the adult brain for both glutamine and glutamate. The presented data indicate the ability of the fetal brain to maintain energy metabolism during acute hypoglycemia, via lactate utilization. The increase in the adult PC/PDH ratio was suggested by us to stem from increased PC activity, in order to replenish TCA cycle intermediates.     

13C isotopomer analysis of glucose and alanine metabolism reveals cytosolic pyruvate compartmentation as part of energy metabolism in astrocytes

After incubation of glial cells with both (13)C-labeled and unlabeled glucose and alanine, (13)C isotopomer analysis indicates two cytosolic pyruvate compartments in astrocytes. One pyruvate pool is in an exchange equilibrium with exogenous alanine and preferentially synthesizes releasable lactate. The second pyruvate pool, which is of glycolytic origin, is more closely related to mitochondrial pyruvate, which is oxidized via tri carbonic acid (TCA) cycle activity. In order to provide 2-oxoglutarate as a substrate for cytosolic alanine aminotransferase, glycolytic activity is increased in the presence of exogenous alanine. Furthermore, in the presence of alanine, glutamate is accumulated in astrocytes without subsequent glutamine synthesis. We suggest that the conversion of alanine to releasable lactate proceeds at the expense of flux of glycolytic pyruvate through lactate dehydrogenase, which is used for ammonia fixation by alanine synthesis in the cytosol and for mitochondrial TCA cycle activity. In addition, an intracellular trafficking occurs between cytosol and mitochondria, by which these two cytosolic pyruvate pools are partly connected. Thus, exogenous alanine modifies astrocytic glucose metabolism for the synthesis of releasable lactate disconnected from glycolysis. The data are discussed in terms of astrocytic energy metabolism and the metabolic trafficking via a putative alanine-lactate shuttle between astrocytes and neurons.     

Effect of exogenous lactate on rat glioma metabolism

Glioma-bearing rats were infused intravenously with a solution containing either [3-(13)C]lactate or both glucose and [3-(13)C]lactate for 20 min or 1 hr. Perchloric acid extracts of healthy and tumoral brain tissues were prepared and analyzed by (13)C- and (1)H-observed (13)C-edited nuclear magnetic resonance (NMR) spectroscopy to determine (13)C-label incorporation into brain tissue and glioma metabolites. Moreover, (13)C enrichments in blood lactate and glucose were determined from (1)H-NMR spectra. In the nontumoral tissue, (13)C labeling of amino acids indicated that [3-(13)C]lactate entered the brain and was metabolized. There was no labeling difference between the contralateral and the ipsilateral hemispheres. Lactate metabolism appeared more specifically neuronal, in agreement with our previous results obtained with normal rat brain (Bouzier et al. [2000] J. Neurochem. 75:480-486). In the glioma tissue, comparison of Ala C3, Glu C4, and Gln C4 labeling indicated that the contributions of blood glutamine and tricarboxylic acid (TCA) cycle to glutamate labeling were about 80% and 20%, respectively, after 1 hr of [3-(13)C]lactate infusion. In contrast, these contributions were about 10% and 90%, respectively, when [1-(13)C]glucose was infused in the absence of lactate. This indicated a major effect of the exogenous lactate on glioma metabolism, which may be due to the following process: The high blood lactate level might hinder the drain of glycolytic lactate produced inside the glioma and thus generate a change in redox potential such that the tumor cells are unable to restore it with oxidative phosphorylation. Thereafter, the high NADH level might inhibit glycolysis and the TCA cycle, and glutamine could become the major carbon source for glutamate labeling.     

Homeostasis of neuroactive amino acids in cultured cerebellar and neocortical neurons is influenced by environmental cues

Neuronal function is highly influenced by the extracellular environment. To study the effect of the milieu on neurons from cerebellum and neocortex, cells from these brain areas were cultured under different conditions. Two sets of cultures, one neocortical and one cerebellar neurons, were maintained in media containing [U-(13)C]glucose for 8 days at initial concentrations of 12 and 28 mM glucose, respectively. Other sets of cultures (8 days in vitro) maintained in a medium containing initially 12 mM glucose were incubated subsequently for 4 hr either by addition of [U-(13)C]glucose to the culture medium (final concentration 3 mM) or by changing to fresh medium containing [U-(13)C]glucose (3 mM) but without glutamine and fetal calf serum. (13)C Nuclear magnetic resonance (NMR) spectra revealed extensive gamma-aminobutyric acid (GABA) synthesis in both cultured neocortical and cerebellar neurons after maintenance in medium containing [U-(13)C]glucose for 8 days, whereas no aspartate labeling was observed in these spectra. Mass spectrometry analysis, however, revealed high labeling intensity of aspartate, which was equal in the two types of neurons. Addition of [U-(13)C]glucose (4 hr) on Day 8 in culture led to a similar extent of labeling of GABA in neocortical and in cerebellar cultures, but the cellular content of GABA was considerably higher in the neocortical neurons. The cellular content of alanine was similar regardless of culture type. Comparing the amount of labeling, however, cerebellar neurons exhibited a higher capacity for alanine synthesis. This is compatible with the fact that cerebellar neurons could ameliorate a low alanine content after culturing in low glucose (12 mM) by a 4-hr incubation in medium containing 3 mM glucose. A low glucose concentration during the culture period and a subsequent medium change were associated with decreases in glutathione and taurine contents. Moreover, glutamate and GABA contents were reduced in cerebellar cultures under either of these conditions. In neocortical neurons, the GABA content was decreased by simultaneous exposure to low glucose and change of medium. These conditions also led to an increase in the aspartate content in both types of cultures, although most pronounced in the neocortical neurons. Further experiments are needed to elucidate these phenomena that underline the impact of extracellular environment on amino acid homeostasis.     

Cerebral metabolism of lactate in vivo: evidence for neuronal pyruvate carboxylation.

The cerebral metabolism of lactate was investigated. Awake mice received [3-13C]lactate or [1-13C]glucose intravenously, and brain and blood extracts were analyzed by 13C nuclear magnetic resonance spectroscopy. The cerebral uptake and metabolism of [3-13C]lactate was 50% that of [1-13C]glucose. [3-13C]Lactate was almost exclusively metabolized by neurons and hardly at all by glia, as revealed by the 13C labeling of glutamate, gamma-aminobutyric acid and glutamine. Injection of [3-13C]lactate led to extensive formation of [2-13C]lactate, which was not seen with [1-13C]glucose, nor has it been seen in previous studies with [2-13C]acetate. This formation probably reflected reversible carboxylation of [3-13C]pyruvate to malate and equilibration with fumarate, because inhibition of succinate dehydrogenase with nitropropionic acid did not block it. Of the [3-13C]lactate that reached the brain, 20% underwent this reaction, which probably involved neuronal mitochondrial malic enzyme. The activities of mitochondrial malic enzyme, fumarase, and lactate dehydrogenase were high enough to account for the formation of [2-13C]lactate in neurons. Neuronal pyruvate carboxylation was confirmed by the higher specific activity of glutamate than of glutamine after intrastriatal injection of [1-14C]pyruvate into anesthetized mice. This procedure also demonstrated equilibration of malate, formed through pyruvate carboxylation, with fumarate. The demonstration of neuronal pyruvate carboxylation demands reconsideration of the metabolic interrelationship between neurons and glia.     

Futile cycling of lactate through the plasma membrane of C6 glioma cells as detected by (13C, 2H) NMR

We report a novel ((13)C, (2)H) nuclear magnetic resonance (NMR) procedure to investigate lactate recycling through the monocarboxylate transporter of the plasma membrane of cells in culture. C6 glioma cells were incubated with [3-(13)C]lactate in Krebs-Henseleit Buffer containing 50% (2)H(2)O (vol/vol) for up to 30 hr. (13)C NMR analysis of aliquots progressively taken from the medium, showed: (1) a linearly decreasing singlet at approximately 20.85 parts per million (ppm; -0.119 micromol/mg protein/hr) derived from the methyl carbon of [3-(13)C]lactate; and (2) an exponentially increasing shifted singlet at approximately 20.74 ppm (0.227 micromol/ mg protein/hr) from the methyl carbon of [3-(13)C, 2-(2)H]lactate. The shifted singlet appears because during its transit through the cytosol, [3-(13)C]lactate generates [3-(13)C, 2-(2)H]lactate in the lactate dehydrogenase (LDH) equilibrium, which may return to the incubation medium through the reversible monocarboxylate carrier. The methyl group of [3-(13)C, 2-(2)H]lactate is shifted -0.11 ppm with respect to that of [3-(13)C]lactate, making it possible to distinguish between both molecules by (13)C NMR. During incubations with 2.5 mM [1-(13)C]glucose and 3.98 mM [U-(13)C(3)]lactate or with 2.5 mM [1-(13)C]glucose and 3.93 mM [2-(13)C]pyruvate, C2-deuterated lactate was produced only from [1-(13)C]glucose or [U-(13)C(3)]lactate, revealing that this deuteration process is redox sensitive. When [1-(13)C]glucose and [U-(13)C(3)]lactate were used as substrates, no significant [3-(13)C]lactate production from [1-(13)C]glucose was detected, suggesting that glycolytic lactate production may be stopped under the high lactate concentrations prevailing under mild hypoxic or ischemic episodes or during cerebral activation.     

Brain energy metabolism in a sub-acute rat model of manganese neurotoxicity: an ex vivo nuclear magnetic resonance study using [1-13C]glucose

Ex vivo high-resolution NMR spectroscopy combined with in vivo injection of [1-13C]glucose was applied to gain insight into the mechanism(s) leading to energy failure in manganese neurotoxicity. In rats treated for 4 days with 50mg/kg MnCl(2) (intraperitoneally, i.p.), the concentration of 13C-labeled lactate increased to 154% compared to control rats. Changes in the absolute amounts of lactate were much less, resulting in increased fractional 13C-enrichments in lactate (indicating relative changes of de novo synthesis from glucose via the glycolytic pathway) to 143% of control values (P < 0.001). Analysis of samples obtained from blood plasma and peripheral organs demonstrate a selective increase of lactate synthesis from [1-13C]glucose in the brain, which is released into the circulation. In parallel, manganese treatment resulted in stimulation of flux through pyruvate dehydrogenase (PDH), leading to accumulation of [4-13C]glutamate, [4-13C]glutamine and [2-13C]GABA to 168, 247 and 144% of control, respectively. The relative flux of glucose through astrocytic pyruvate carboxylase (PC), on the other hand, was impaired by manganese, as evident from a decreased ratio of [2-13C]/[4-13C]glutamate or [2-13C]/[4-13C] glutamine. Consistent with stimulated glucose oxidative metabolism, the fractional 13C-enrichment in [2-13C]acetyl-CoA entering the tricarboxylic acid (TCA) cycle and contributing to glutamate and glutamine synthesis increased to 138 and 156% of control, respectively (P < 0.001). In parallel, the TCA cycling ratio increased to 134% compared to control rats, prior to the label ending up in glutamate. In contrast, glutamine is synthesized mainly during the first TCA cycle turn. The present data provide new evidence in support of changes in brain energy metabolism playing an important role in manganese neurotoxicity. In particular, increased glycolytic flux and lactate synthesis may contribute to the deleterious effects of manganese in the brain. Furthermore, stimulated astrocytic glucose oxidation and glutamine synthesis may be associated with astrocytic pathology and altered astrocytic-neuronal metabolic trafficking in manganese neurotoxicity.     

Glutamine as an energy substrate in cultured neurons during glucose deprivation

During glucose deprivation an increase in aspartate formation from glutamine has been observed in different brain preparations, including synaptosomes and cultured astrocytes. To what extent this reaction, which provides a substantial amount of energy, occurs in different types of neurons is unknown. The present study shows that (14)CO(2) formation from [U-(14)C]glutamine in cerebellar granule neurons, a glutamatergic preparation, increased by 60% during glucose deprivation, indicating enhanced aspartate formation or increased complete oxidative degradation of glutamine. In primary cultures of cerebrocortical interneurons, a GABAergic preparation, the rate of (14)CO(2) production from [U-(14) C] glutamine was four times lower and not stimulated by glucose deprivation. During incubation with glutamine (0.8 mM) as the only metabolic substrate, cerebellar granule cells maintained an oxygen consumption rate of 12 nmol/min/mg protein, corresponding to an aspartate formation of 8 nmol/min/mg protein (three oxidations occur between glutamine and aspartate) or to a total oxidative degradation of 3 nmol/min/mg protein. During glucose deprivation, the rate of aspartate formation increased, and during a 20-min incubation in phosphate-buffered saline it amounted to 3.3 nmol/min/mg protein at 0.2 mM glutamine, which might have been more if measured at 0.8 mM glutamine. These values are consistent with the rate of glutamine utilization calculated based on oxygen consumption and leaves open the possibility that some glutamine is completely degraded oxidatively, as has been shown by other authors based on pyruvate recycling and labeling of lactate from aspartate in cerebellar granule neurons.