Glutamate triggers rapid glucose transport stimulation in astrocytes as evidenced by real-time confocal microscopy

Abstract

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.

Figure 2.

Glutamate stimulates the uptake of galactose by single hippocampal astrocytes. A, Confocal section of astrocytes loaded with calcein. The cell for which galactose uptake curves are depicted in C is indicated with a white square. Scale bar, 20 μm. B, Calcein fluorescence was measured in cells exposed to KRH plus increasing concentrations of extracellular mannitol (final osmolarities of 285, 345, 400, 510, and 740 mOsm). Relative fluorescence is plotted against relative osmolarity with F_b/F = 0.79 × Osm_b/Osm + 0.22; r = 0.98. C, Uptake was estimated from galactose-induced changes in calcein fluorescence in the cell boxed in A before and 10 min after exposure to 0.5 mm glutamate. Initial rates, calculated by fitting two-parameter single exponential functions to the data, were 0.38 and 0.66 mm/sec for basal and stimulated uptake, respectively.

Figure 1.

Glutamate stimulates the uptake of fluorescent hexoses by single hippocampal astrocytes. A, Cells were stained with antibodies against GFAP (left) or GLUT1 (right) and then imaged by confocal microscopy, as described in Materials and Methods. Scale bars, 20 μm. The inset shows GLUT1 immunodetected in 50 μg of total hippocampal proteins (left) and 50 ng of human erythrocyte membranes (right). B, Intracellular 2-NBDG was measured in two neighboring astrocytes during continuous exposure to 300 μm extracellular sugar. At the time indicated, 0.5 mm glutamate (glu) was added to the cells, resulting in different degrees of stimulation. C, Intracellular 6-NBDG was measured in a single astrocyte during continuous exposure to 300 μm extracellular sugar (filled symbols). At the time indicated, 0.5 mm glutamate was added. Data from 20 nonstimulated control cells are shown for comparison (mean ± SEM).

Figure 3.

Astrocytic glucose transport stimulation occurs within seconds. A, Intracellular calcium was recorded simultaneously with 2-NBDG uptake to correlate the onset of the effect of glutamate (glu) on both parameters. Note that fura-red emission varies reciprocally with intracellular calcium. Linear equations were fitted by nonlinear regression to the data before (open symbols) and after (filled symbols) addition of 0.5 mm glutamate. B, Magnification of A to illustrate the relative positions of the intercept between the regression curves and the first detection of calcium increase. For this astrocyte, the delay was estimated at 14 sec.