Glutamate stimulates glycolysis in astrocytes, a phenomenon that couples astrocytic metabolism with neuronal activity. However, it is not known whether glutamate also affects glucose transporter-1 (GLUT1), the transporter responsible for glucose entry into astrocytes. To address this question, two different real-time single-cell hexose uptake assays were applied to cultured hippocampal astrocytes using confocal epifluorescence microscopy. Glutamate caused a twofold to threefold increase in the zero-trans uptake rates of the fluorescent hexoses 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) and 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (6-NBDG). Galactose uptake, determined by the calcein volumetric assay, was stimulated to a similar extent, confirming the fluorescent hexose data, and also demonstrating that glutamate stimulation is a Vmax effect. Remarkably, glucose transport stimulation developed fully inside 10 sec, which is 100 times faster than acute stimulations of glucose transport in other cell types. Glutamate did not significantly affect the rate of 6-NBDG uptake by GLUT1-expressing epithelial Clone 9 cells, suggesting that an astrocyte-specific factor is required for transport stimulation. We conclude that glucose transport stimulation occurs early during astrocytic activation by glutamate, which provides a novel regulatory node to current models of brain energy metabolism. This mechanism should also be considered for the interpretation of functional imaging data based on hexoses.

Synaptic activity is followed within seconds by a local surge in lactate concentration, a phenomenon that underlies functional magnetic resonance imaging and whose causal mechanisms are unclear, partly because of the limited spatiotemporal resolution of standard measurement techniques. Using a novel Förster resonance energy transfer-based method that allows real-time measurement of the glycolytic rate in single cells, we have studied mouse astrocytes in search for the mechanisms responsible for the lactate surge. Consistent with previous measurements with isotopic 2-deoxyglucose, glutamate was observed to stimulate glycolysis in cultured astrocytes, but the response appeared only after a lag period of several minutes. Na ϩ overloads elicited by engagement of the Na ϩ -glutamate cotransporter with D-aspartate or application of the Na ϩ ionophore gramicidin also failed to stimulate glycolysis in the short term. In marked contrast, K ϩ stimulated astrocytic glycolysis by fourfold within seconds, an effect that was observed at low millimolar concentrations and was also present in organotypic hippocampal slices. After removal of the agonists, the stimulation by K ϩ ended immediately but the stimulation by glutamate persisted unabated for Ͼ20 min. Both stimulations required an active Na ϩ /K ϩ ATPase pump. By showing that small rises in extracellular K ϩ mediate short-term, reversible modulation of astrocytic glycolysis and that glutamate plays a long-term effect and leaves a metabolic trace, these results support the view that astrocytes contribute to the lactate surge that accompanies synaptic activity and underscore the role of these cells in neurometabolic and neurovascular coupling.

While glucose is constantly being "pulled" into the brain by hexokinase, its flux across the blood brain barrier (BBB) is allowed by facilitative carriers of the GLUT family. Starting from the microscopic properties of GLUT carriers, and within the constraints imposed by the available experimental data, chiefly NMR spectroscopy, we have generated a numerical model that reveals several hidden features of glucose transport and metabolism in the brain. The half-saturation constant of glucose uptake into the brain (K(t)) is close to 8 mM. GLUT carriers at the BBB are symmetric, show accelerated-exchange, and a K(m) of zero-trans flux (K(zt)) close to 5 mM, determining a ratio of 3.6 between maximum transport rate and net glucose flux (T(max)/CMR(glc)). In spite of the low transporter occupancy, the model shows that for a stimulated hexokinase to pull more glucose into the brain, the number or activity of GLUT carriers must also increase, particularly at the BBB. The endothelium is therefore predicted to be a key modulated element for the fast control of energy metabolism. In addition, the simulations help to explain why mild hypoglycemia may be asymptomatic and reveal that [glucose](brain) (as measured by NMR) should be much more sensitive than glucose flux (as measured by PET) as an indicator of GLUT1 deficiency. In summary, available data from various sources has been integrated in a predictive model based on the microscopic properties of GLUT carriers.

The glycolytic rate is sensitive to physiological activity, hormones, stress, aging, and malignant transformation. Standard techniques to measure the glycolytic rate are based on radioactive isotopes, are not able to resolve single cells and have poor temporal resolution, limitations that hamper the study of energy metabolism in the brain and other organs. A new method is described in this article, which makes use of a recently developed FRET glucose nanosensor to measure the rate of glycolysis in single cells with high temporal resolution. Used in cultured astrocytes, the method showed for the first time that glycolysis can be activated within seconds by a combination of glutamate and K+, supporting a role for astrocytes in neurometabolic and neurovascular coupling in the brain. It was also possible to make a direct comparison of metabolism in neurons and astrocytes lying in close proximity, paving the way to a high-resolution characterization of brain energy metabolism. Single-cell glycolytic rates were also measured in fibroblasts, adipocytes, myoblasts, and tumor cells, showing higher rates for undifferentiated cells and significant metabolic heterogeneity within cell types. This method should facilitate the investigation of tissue metabolism at the single-cell level and is readily adaptable for high-throughput analysis.

Although it is known that brain activity is fueled by glucose, the identity of the cell type that preferentially metabolizes the sugar remains elusive. To address this question, glucose uptake was studied simultaneously in cultured hippocampal neurons and neighboring astrocytes using a real-time assay based on confocal epifluorescence microscopy and fluorescent glucose analogs. Glutamate, although stimulating glucose transport in astrocytes, strongly inhibited glucose transport in neurons, producing in few seconds a 12-fold increase in the ratio of astrocytic-to-neuronal uptake rate. Neuronal transport inhibition was reversible on removal of the neurotransmitter and displayed an IC 50 of 5 M, suggesting its occurrence at physiological glutamate concentrations. The phenomenon was abolished by CNQX and mimicked by AMPA, demonstrating a role for the cognate subset of ionotropic glutamate receptors. Transport inhibition required extracellular sodium and calcium and was mimicked by veratridine but not by membrane depolarization with high K ϩ or by calcium overloading with ionomycin. Therefore, glutamate inhibits glucose transport via AMPA receptor-mediated sodium entry, whereas calcium entry plays a permissive role. This phenomenon suggests that glutamate redistributes glucose toward astrocytes and away from neurons and represents a novel molecular mechanism that may be important for functional imaging of the brain using positron emission tomography.

In recent years, the use of fluorescent glucose analogs has allowed the study of rapid transport modulation in heterogeneous cell cultures and complex tissues. However, the kinetic behavior of these tracers is not conventional. For instance, the fluorescent glucose analog 6-NBDG permeates the cell 50-100 times slower than glucose but the uptake of 6-NBDG is almost insensitive to glucose, an observation that casts doubts as to the specificity of the uptake pathway. To investigate this apparent anomaly in cultured astrocytes, which are rich in the glucose transporter GLUT1, we first estimated the kinetic parameters of 6-NBDG uptake, which were then incorporated into the kinetic model of GLUT1. The main outcome of the analysis was that 6-NBDG binds to GLUT1 with 300 times higher affinity than glucose, which explains why its uptake is not efficiently displaced by glucose. The high binding affinity of 6-NBDG also explains why cytochalasin B is less effective at inhibiting 6-NBDG uptake than at inhibiting glucose uptake. We conclude that 6-NBDG, used at low concentrations, permeates into astrocytes chiefly through GLUT1, and advise that the exofacial GLUT1 inhibitor 4,6-ethylidine-D-glucose be used, instead of glucose, as the tool of choice to confirm the specificity of 6-NBDG uptake.

Glutamate triggers an acute stimulation of the glucose transporter GLUT1 in cultured astrocytes, a phenomenon thought to facilitate energy delivery to active areas in the brain. Here we have explored the cell signaling mechanisms involved in this response. Half-stimulation of GLUT1 occurred at low micromolar glutamate, thus within the physiological range estimated in brain interstitium. The effect was mimicked by D-aspartate and inhibited by L-threo-beta-benzyloxyaspartate or Na(+) replacement with NMDG(+), showing the participation of the Na(+)-glutamate co-transporter. AMPA and the mGLURI agonist DHPG had no effect. The stimulation of GLUT1 was fully inhibited by ouabain, but independent activation of the Na(+)/K(+) ATPase pump with gramicidin did not affect glucose transport. Simultaneous with the Na(+) rise, glutamate and D-aspartate triggered a Ca(2+)signal, whose inhibition with BAPTA prevented the stimulation of GLUT1. However, an isolated Ca(2+) signal, triggered with endothelin 1, ATP or DHPG, did not affect glucose transport. The stimulation of GLUT1 could finally be mimicked by simultaneous induction of Na(+) and Ca(2+) signals. The requirement for both cations in the stimulation of the astrocytic glucose transporter, may help to explain how glucose metabolism in the brain is strongly activated by glutamate, but not by GABA or by inter-astrocytic signaling.

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