Significance A near one-to-one relationship had previously been observed between increments in the fluxes of the glutamate−glutamine neurotransmitter cycle and neuronal glucose oxidation in the tricarboxylic acid (TCA) cycle. This flux relationship was consistent with a hypothesized mechanism involving glycolytic ATP in astrocytes and astrocyte-to-neuron lactate shuttling. Here, 2-fluoro-2-deoxy-d-glucose was used to evaluate the glucose flux through glycolysis and the TCA cycle in nerve terminals isolated from the brains of rats under baseline and high-activity conditions. In a direct contradiction of this hypothesis, the results show that nerve terminals metabolize significant amounts of glucose.

Abstract Previous 13C magnetic resonance spectroscopy experiments have shown that over a wide range of neuronal activity, approximately one molecule of glucose is oxidized for every molecule of glutamate released by neurons and recycled through astrocytic glutamine. The measured kinetics were shown to agree with the stoichiometry of a hypothetical astrocyte-to-neuron lactate shuttle model, which predicted negligible functional neuronal uptake of glucose. To test this model, we measured the uptake and phosphorylation of glucose in nerve terminals isolated from rats infused with the glucose analog, 2-fluoro-2-deoxy-d-glucose (FDG) in vivo. The concentrations of phosphorylated FDG (FDG 6P ), normalized with respect to known neuronal metabolites, were compared in nerve terminals, homogenate, and cortex of anesthetized rats with and without bicuculline-induced seizures. The increase in FDG 6P in nerve terminals agreed well with the increase in cortical neuronal glucose oxidation measured previously under the same conditions in vivo, indicating that direct uptake and oxidation of glucose in nerve terminals is substantial under resting and activated conditions. These results suggest that neuronal glucose-derived pyruvate is the major oxidative fuel for activated neurons, not lactate-derived from astrocytes, contradicting predictions of the original astrocyte-to-neuron lactate shuttle model under the range of study conditions.

Metabolic and neurophysiological research has experimentally related brain energy consumption, in the form of glucose oxidation, to the brain work supporting neuronal firing. Carbon-13 magnetic resonance spectroscopy (MRS) measurements (1, 2) of the associated fluxes in cerebral cortex of anesthetized rats over a range of electrical activity revealed, surprisingly, a near 1:1 relationship (in molar equivalent units) between increments in the glutamate−glutamine neurotransmitter cycle and neuronal glucose oxidation. Subsequent studies of rat and human cerebral cortex have been consistent with this finding (3, 4). The near 1:1 flux relation was consistent with a cellular/molecular model, originally proposed by Pellerin and Magistretti (5), and subsequently expanded to include the glutamate/glutamine cycle (1, 6). Evidence for the astrocyte-to-neuron lactate shuttle (ANLS) model is summarized in ref. 7. In this model (Fig. 1A), glutamate released from neurons is taken up by astrocytes and converted to glutamine using ATP derived from glycolysis. Lactate produced by this process is transferred to neurons where oxidation occurs. This ANLS model predicts a 1:1 relationship between increments in astrocytic glutamate uptake and glycolysis. Glycolytically derived ATP might provide for more rapid clearance of glutamate from the synaptic cleft into astrocyte processes devoid of mitochondria (8).

Fig. 1. Schematic depiction of two neuroenergetics models under consideration to account for the 1:1 flux relationship between increments in V cyc and V TCAn . (A) ANLS-type model (model 1) described by Sibson et al. (1). Above isoelectricity, lactate transfer from astrocytes to neurons (expressed as glucose equivalents) is determined by the rate of the glutamate−glutamine cycle, V cyc . Neuronal glucose phosphorylation was assumed to be equivalent to the isoelectric rate for all activity levels, i.e., in A, . Predicted rates of CMRglc n (P+Ox) for model 1 were calculated using Eq. 1, shown at the bottom of A. In the revised description of model 1 by Hyder et al. (3), glucose phosphorylation in neurons above isoelectricity can occur depending on the magnitude of astroglial oxidation and its dependence on neural activity. (B) Independent-type model (model 2) in which neurons and astrocytes take up and oxidize glucose according to their respective energy needs. Phosphorylated glucose not oxidized within the cell may be effluxed as lactate, V Lac(efflux) , which is shown by dashed lines, reflecting uncertainty of the lactate-releasing neural cells (13, 41). Predicted rates of for model 2 were calculated using Eq. 4, shown at the bottom of B. , rate of glucose oxidation in astrocytes ; , rate of glucose oxidized in neurons at isoelectricity ; , rate of total glucose phosphorylation in astrocytes, which includes oxidative and nonoxidative (net lactate efflux) catabolism; , rate of glucose phosphorylation in astrocytes with oxidation, includes lactate efflux to neurons at the rate V cyc (model 1) or to extracellular fluid (model 2); , rate of total glucose phosphorylation in neurons, which includes oxidative and nonoxidative (lactate efflux) catabolism; , rate of glucose phosphorylation in neurons with oxidation; Gln, glutamine; Glu, glutamate; Lac, lactate; OAA, oxaloacetate; Pyr, pyruvate; αKG, α-ketoglutarate; V PC , pyruvate carboxylase rate in astrocytes; V PDHa , pyruvate dehydrogenase rate in astrocytes; V TCAa , TCA cycle flux in astrocytes; V TCAn , TCA cycle flux in neurons ; , TCA cycle flux in neurons at isoelectricity.

The ANLS hypothesis has been challenged on biochemical, in vivo, in situ, and in vitro experimental and theoretical grounds (9⇓⇓⇓–13), as well as the lack of direct in vivo evidence for oxidation of astroglia-derived lactate by neurons. The experimental data against the ANLS model are described in a recent review by Dienel (13). The present study tested predictions of the ANLS model in anesthetized rats (both at baseline and during seizure-induced activation) by direct measurement in brain nerve terminals of the uptake and phosphorylation of an i.v.-infused glucose analog, 2-fluoro-2-deoxy-d-glucose (FDG). To test the hypothesized model (1, 6) (Fig. 1A), we measured the rate of glucose phosphorylation in nerve terminals isolated from the brains of rats receiving a short-timed infusion of FDG mixed with 13C-labeled glucose. FDG is phosphorylated by hexokinase to fluoro-2-deoxy-glucose-6-phosphate (FDG 6P ), which is metabolized only to a limited extent; thus, FDG 6P accumulates in neural cells at a rate proportional to glucose utilization (14). The FDG and 13C-glucose mixture (1:7) was infused for 8 min followed by a 52-min washout of FDG by infusing only 13C-glucose (Fig. 2A), after which the animals were euthanized and the nerve terminals were isolated. FDG 6P was measured in extracts using 19F NMR and normalized to N-acetylaspartate (NAA) or glutamate plus γ-aminobutyrate (GABA), predominantly neuronal metabolites, measured in the same extracts using 1H-[13C] NMR. Measurements were made both of nerve terminals and of total brain tissue homogenate, the latter consisting of metabolites from all cells, including neurons and astrocytes. Comparisons were made of the normalized FDG 6p formed in brain nerve terminals and homogenate during control conditions and a more stimulated state (seizure) induced by the GABA A receptor antagonist, bicuculline. The normalized FDG flux in nerve terminals was then compared with the cortical flux of glucose oxidation, measured in vivo in a previous study of anesthetized rats (15), as well as with the flux in whole brain homogenate, both of which included neuronal and glial contributions. The present results reveal high levels of neuronal phosphorylation of FDG, suggesting that over a significant activity range, neurons are capable of supporting a substantial fraction of their substrate requirements by direct uptake and phosphorylation of glucose.

Fig. 2. Time courses of blood FDG and glucose concentrations during FDG/[1,6-13C 2 ] glucose infusion (A) and 19F NMR spectra of nerve terminal (NT) (B) and homogenate (C) extracts for control and seizure conditions. FDG was infused for 8 min (solid bar) and [1,6-13C 2 ] glucose for 60 min (open bar) followed by euthanasia and nerve terminal preparation (arrow). α,βFDG 6P , 2-fluoro-2-deoxyglucose-6-phosphate; with resolved C1 α and β anomers.

Discussion Factors Influencing the FDG 6P /NAA Ratio in Nerve Terminals. NAA as a measure of neuronal cytosolic volume. The conclusion that similar amounts of FDG were taken up (on a per cellular cytosol basis) in the nerve terminals and in total neuronal volume assumes that NAA, which is found exclusively in neurons, reflects quantitatively the neuronal cytosolic volume. The concentration of NAA is relatively homogenous (6–8 mM) across multiple rat brain regions (19). Because the rate of intracellular diffusion of NAA greatly exceeds its metabolic turnover, the intraneuronal distribution of NAA is anticipated to be relatively homogenous. For example, based on the apparent diffusion coefficient of NAA of 0.27 µm2/ms determined by MRS (20), mixing of NAA throughout neurons would occur in ∼60 min, whereas the metabolic turnover of NAA is slow [NAA C3 time constant, 13–14 h (21)]. Accuracy with which nerve terminals reflect total neuronal FDG uptake. Surprisingly, the absolute FDG 6P /NAA ratio for the nerve terminals was the same as that for total brain homogenate, which contains contributions of glucose uptake from astroglia and other neural cells not containing NAA. A ∼20–30% higher FDG 6P /NAA would be anticipated for brain homogenate based on estimates of the rate of glial glucose oxidation (3, 22, 23). A possible explanation for this discrepancy is that FDG 6P could be more concentrated in nerve terminals. Evidence for this was previously reported (24, 25) using 14C-2-deoxyglucose autoradiography. Based on considerations of energy budget modeling and mitochondrial density (26), and in vitro measurements (27), the highest neuronal rates of glucose uptake and oxidation may occur in postsynaptic dendrites, spines, and axon collaterals. Thus, differences in the distribution of FDG 6P between presynaptic and postsynaptic neuronal elements may explain the equal FDG 6P /NAA ratios found. Effect of postmortem anaerobic glycolysis. Another factor that could lead to an artifactually high amount of FDG 6P in the nerve terminals would be extensive postmortem anaerobic glycolysis. To minimize this possibility, we designed the FDG infusion so that the maximum FDG uptake would occur during the premortem stage by incorporating a washout period of 52 min. At the end of the washout period, the mole fraction of FDG to glucose in blood was reduced to <2% (Table S1 and Fig. S1), which would insignificantly impact the FDG 6P levels. Effects of FDG on energetics, kinetics, and metabolism beyond FDG 6P . FDG competes with glucose for transport into the brain and, at high doses, can interfere with ATP formation by limiting glucose availability and sequestering inorganic phosphate (P i ) as FDG 6P . The average FDG 6P concentrations, 0.44 μmol·g−1 (control) and 1.1 μmol·g−1 (seizure) in cortex and 4.2 nmol·mg−1 protein (control) and 19.9 nmol·mg−1 protein (seizures) in the nerve terminals, were significantly less than those reported in two previous in vivo studies using bolus i.v. or intra-arterial injections (500 mg/kg) of 2-[6-13C]deoxyglucose (28) or 2-deoxyglucose (29). Deuel et al. (29) observed no significant effects of the FDG infusion on brain levels of ATP, phosphocreatine, or P i , or in the intracerebral pH as measured by 31P MRS, despite using approximately a 10× higher dose of FDG than in the present study. The relatively low brain FDG 6P concentration was also reflected in the absence of other fluorinated phosphorylated metabolites of FDG (27). However, low levels of fluorodeoxymannose-6-P (β, −59.15 ppm; α, −40.85) were detected in the homogenate extract, which may reflect postmortem metabolism. Thus, FDG and FDG 6P levels would not be expected to significantly impact glucose metabolism. A potential source of overestimation of nerve terminal glucose uptake in the seizure condition is that the lumped constant (LC), which corrects for different kinetic properties of FDG versus glucose, may rise steeply when brain glucose concentration falls to <1 μmol·g−1 (12). In awake rats during 1 h of bicuculline seizures (30), glucose levels remained above the critical value of ∼1 μmol·g−1, supporting our use of a single LC value. However, if the decline in glucose is more pronounced in the nerve terminals compared with whole tissue, FDG/glucose ratio would rise, leading to artifactually high rates of FDG phosphorylation relative to glucose. A study of glucose utilization using [2-14C]deoxyglucose (DG) during penicillin-induced seizures (31) found that brain glucose decreased by 20% (to 1.7 μmol·g−1), which corresponded to an increase in LC (and overestimate of CMRglc rate) of ∼10%. Because FDG is transported and phosphorylated faster than 2-DG (LC of 0.71 vs. 0.48) (32), the expected overestimate would be lower, ∼5–7%. Effect of astroglial contamination in the nerve terminal fraction. Another potential source of FDG 6P could arise through contamination of nerve terminals by glial processes or gliosomes (33). The nerve terminals were judged to be relatively pure based on the absence of glutamine (a glial marker), their inability to synthesize 13C labeled glutamine from [U-13C 5 ]glutamate and [1,6-13C 2 ]glucose or [2-13C]acetate in vitro when supplied with these substrates, and relatively low enzymatic activity of glutamine synthetase and a glial marker protein (GFAP) (Fig. S3). Impact of Activity-Dependent Astroglial Glucose Oxidation. In model 1, the glia are approximated as transferring all of the lactate they produce through glycolysis-coupled glutamate uptake to the neuron. However, as discussed in ref. 3, because the astroglia can in principle oxidize the pyruvate resulting from this process, the net transfer of lactate to the neurons may be favored only when the rate of glutamate/glutamine cycling (V cyc ) exceeds the rate of astroglial glucose (pyruvate) oxidation. The magnitude of this effect was estimated by combining our previous data on neuronal glucose oxidation and glutamate cycling under similar conditions of halothane anesthesia and seizures (15) with the reported rates of astroglial glucose oxidation (22, 34, 35). Assuming a glial oxidative activity dependence similar to what has been reported in other conditions (22, 23, 36), and the model proposed in ref. 3, the predicted neuronal FDG uptake during seizures compared with control would rise by at most ∼38%, leading to a predicted seizure-to-control ratio of 1.4 rather than 1 (SI Text, Effects of Astroglial Glucose Oxidation on Predicted Neuronal Glucose Uptake), which still does not agree with the high neuronal uptake of FDG observed. Potential Neuronal Basis of the 1:1 Ratio. The finding of a large fraction of activity-dependent glucose uptake in neurons requires an alternative explanation of the 1:1 relationship. Two alternate mechanisms have been postulated, one involving the coupling of neuronal glycolysis (or glycolytic ATP) to vesicular loading of glutamate (37, 38) and another involving the coupling of neuronal glutamate formed from glutamine to redox movements into mitochondria via the malate aspartate shuttle (39, 40). Glutamate accumulation into synaptic vesicles is driven by a H+ electrochemical gradient produced by a vacuolar (H+)-ATPase, the energetic cost of which was estimated to be ∼0.33 ATP/glutamate (26). Assuming this ATP to be derived entirely from glycolysis would lead to a predicted flux ratio ΔV cyc :ΔCMRglc n (P+Ox) of ∼6:1, well above the observed value of 1:1. In the scheme of Hertz and coworkers (39, 40), the processing of glutamine to transmitter glutamate is indirect, involving mitochondrial formation of α-ketoglutarate from glutamine and with efflux to cytoplasm in exchange with malate. Because there is a fixed stoichiometric relationship between the formation of glycolytically produced NADH from glucose and the transfer of reducing equivalents into mitochondria via malate (1 glucose: 2 NADH: 2 malate), the formation of transmitter glutamate from glutamine will correlate with the exchange-mediated flow of glycolytically produced reducing equivalents from cytoplasm to mitochondria. Because one molecule of glucose provides 2 molecules of NADH in cell cytoplasm by glycolysis, the expected incremental glutamine/glucose flux ratio (ΔV cyc :ΔCMRglc n ) would be 2:1 in the compartment of glutamate transmitter synthesis, i.e., the nerve terminal. This value, however, is twice the value of 1:1 determined in vivo. Noting that the measured value of neuronal TCA cycle flux (denominator in the ratio) includes all neuronal compartments, including those where glutamine metabolism and cycling may be limited, e.g., postsynaptic dendritic sites and/or cell bodies, might explain this discrepancy. Rates of glucose oxidation in glutamatergic postsynaptic/dendritic compartments might be expected to be strongly correlated with presynaptic glutamate release and cycling (V cyc ), consistent with recent experimental (41) and theoretical findings (27). Reconciling the Experimental Evidence: Rest vs. Activation. The finding of high resting neuronal FDG phosphorylation in adult rats is consistent with in vivo findings from high-resolution 2-DG autoradiography (42), reporting approximately equal amounts of glucose utilization by neurons and astrocytes. In addition, an in vivo study using the fluorescent glucose analog, 2-deoxy-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-aminoglucose (2-NBDG), which is phosphorylated by hexokinase to 2-NBDP 6P (43), also found uptake/metabolism in both neurons (hippocampal pyramidal and cerebellar Purkinje cells) and astroglia of adult rats. The in vivo findings, however, are at odds with two recent in vitro studies using 2-NBDG in immature (P10 to P21) cerebellar (44) and hippocampal (45) brain slices, which found less uptake in neurons than in astrocytes, inferring support for the ANLS mechanism. However, the relevance for functional metabolism is problematic because rates of neuronal glucose oxidation and glutamate−glutamine cycling in P10 neocortex are ∼1/3 of the mature cortex (46), and in unstimulated slices, glutamate−glutamine cycling is not detected and oxygen consumption rate is very low (47). Because the quantitative use of 2-NBDG (unlike 2-DG and 2-FDG) and stability of 2-NBDG 6P remains to be thoroughly validated (13), conclusions of cell type-specific glucose utilization (and inferences to unmeasured lactate movements) may be premature. Our results go further than previous studies in showing that with intense activation, glucose phosphorylation is increased in nerve terminals, suggesting that direct glucose uptake and oxidation is a major pathway to satisfy energy demands. As such, our findings do not support the proposal that neuronal glycolysis is inhibited under high-activity conditions (48) or that neurons in vivo lack the ability to increase glycolysis as seen in cultured cells (49). However, our results do not rule out an important role for an ANLS mechanism under certain conditions, e.g., that existing during the initial stages of intense neural activation when net glycogen breakdown occurs (50, 51)), such as for seizure onset (15). Also, our findings do not address whether neurons are a source (10, 52) or a sink (5, 53) of the extracellular lactate rise seen with neural activation (see ref. 13 for a review of the evidence on this topic).

Conclusions The present findings, which indicate that neuronal glycolysis is capable of substantial support of its oxidative needs, are incompatible with an ANLS-type model previously proposed to explain the ∼1:1 relationship observed between ΔV cyc and ΔCMRglc (ox)n , suggesting this relation may arise in neurons. Furthermore, the results demonstrate up-regulation of neuronal glycolysis during neural activation and direct neuronal oxidation of glucose-derived pyruvate; but they do not support astrocytic lactate production strongly coupled with lactate shuttling to neurons to provide a major neuronal fuel. The synaptosome data are consistent with and extend (i.e., by doing the activation in vivo instead of in vitro) studies of synaptosomes from adult rodents, clearly demonstrating their high capacity for increasing glycolysis and oxidative metabolism.

Materials and Methods Adult male Wister rats were prepared under halothane anesthesia (15) in accordance with protocols approved by the Yale Animal Care and Use Committee. FDG and [1,6-13C 2 ]glucose were infused for 8 min, followed by [1,6-13C 2 ]glucose alone (washout) for 60 min. Nerve terminals were prepared from rat forebrain using isotonic media and Ficoll density gradient centrifugation (54). Tissue extracts were prepared using ethanol (15) for high-resolution 19F or 1H-[13C] NMR at 11.7T. The values reported reflect mean ± SD. Statistical significance of differences was assessed by Student t test with level of P < 0.05. Further details appear in SI Text.

Acknowledgments The authors thank Bei Wang for animal preparation and Shirley Wang for immunoblotting. This study was supported by National Institutes of Health Grants R01-DK27121 and R01-DK27121-28S.