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It has been demonstrated that glutamatergic activity induces ascorbic acid (AA) depletion in astrocytes. Additionally, different data indicate that AA may inhibit glucose accumulation in primary cultures of rat hippocampal neurons. Thus, our hypothesis postulates that AA released from the astrocytes during glutamatergic synaptic activity may inhibit glucose uptake by neurons. We observed that cultured neurons express the sodium-vitamin C cotransporter 2 and the facilitative glucose transporters (GLUT) 1 and 3, however, in hippocampal brain slices GLUT3 was the main transporter detected. Functional activity of GLUTs was confirmed by means of kinetic analysis using 2-deoxy-d-glucose. Therefore, we showed that AA, once accumulated inside the cell, inhibits glucose transport in both cortical and hippocampal neurons in culture. Additionally, we showed that astrocytes are not affected by AA. Using hippocampal slices, we observed that upon blockade of monocarboxylate utilization by α-cyano-4-hydroxycinnamate and after glucose deprivation, glucose could rescue neuronal response to electrical stimulation only if AA uptake is prevented. Finally, using a transwell system of separated neuronal and astrocytic cultures, we observed that glutamate can reduce glucose transport in neurons only in presence of AA-loaded astrocytes, suggesting the essential role of astrocyte-released AA in this effect.
In situ hybridization and immunohistochemical analyses have demonstrated a high sodium–vitamin C cotransporter 2 (SVCT2) expression in neurons from different regions of the brain (Tsukaguchi et al. 1999; Astuya et al. 2005; Garcia et al. 2005; Mun et al. 2006). SVCT2 is not expressed in astrocytes; however, it has been postulated that SVCT2 is induced after ischemia or in culture conditions (Berger et al. 2003; Astuya et al. 2005; Garcia et al. 2005). Kinetic analyses have shown that ascorbic acid (AA), the reduced form of vitamin C, is taken up into neurons using SVCT2 (Castro et al. 2001). Additionally, functional data indicate that astrocytes transport mainly dehydroascorbic acid, the oxidized form of vitamin C, using the glucose transporter (GLUT1) (Korkok et al. 2003; Astuya et al. 2005). Thus, astrocytes concentrate vitamin C (after reduction to AA) at extremely high concentrations. Regarding these physiological characteristics, it has been postulated that astrocytes may be involved in vitamin C recycling in the nervous system, avoiding the hydrolysis of dehydroascorbic acid produced by antioxidative protection (Hediger 2002).
In the present study, our hypothesis is that AA released from astrocytes inhibits glucose uptake in neurons during extended glutamatergic synaptic activity. We demonstrated the expression of SVCT2 and GLUT1 and 3 in cultured neurons, however, in brain sections the main GLUT detected in hippocampal CA1 neurons was GLUT3. Additionally, we found that intracellular AA is able to inhibit deoxyglucose (2-DOG) uptake in cortical and hippocampal neurons. Using hippocampal slices treated with α-cyano-4-hydroxycinnamate (4-CIN), we defined that glucose is able to support synaptic activity only when AA uptake is inhibited. Results from transwell system experiments, using astrocyte and neuron primary cultures, suggest that in presence of glutamate, AA is preferably released from astrocytes. Thus, during glutamatergic synaptic activity, neuronal 2-DOG uptake may be inhibited by AA released from astrocytes.
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Figure 7. Ascorbic acid (AA) is able to modulate neuronal glucose transport during glutamatergic neurotransmission. Neurons use glucose to support their synaptic activity only when AA is not intracellularly concentrated (1). AA could be released from nearby astrocytes in response to glutamate stimuli (2). Thus, this modulation would be driven by glutamate and astrocytes surrounding the synaptic cleft. The neuronal effect may be potentiated regarding that glucose transporter (GLUT3; the neuronal GLUT) transports glucose seven times faster than GLUT1, the main glial GLUT (Vannucci et al. 1997). Astrocytes are involved in the recycling of vitamin C. It has been postulated that intracellularly generated AA is released after glutamate stimulation.
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Different data indicate that AA uptake in neurons is a sodium-dependent mechanism. Thus, using RT-PCR, western blot, and immunocytochemistry, we have confirmed the SVCT2 expression in neurons isolated from the brain cortex. Similar results have been shown in brain tissue by immunohistochemistry and in situ hybridization analyses (Tsukaguchi et al. 1999; Astuya et al. 2005; Garcia et al. 2005;Mun et al. 2006). These data indicate that SVCT2 is the main AA transporter expressed by neurons and therefore plays a central role in providing these cells with reduced vitamin C (Fig. 7). Previously, we have studied the kinetic parameters associated to the AA uptake in neurons (Castro et al. 2001), and surprisingly, we observed the presence of two affinity constants, 103 and 8 μmol/L, suggesting a regulatory mechanism associated with the uptake of AA in these cells. Recently, an apparent Km of 113 μmol/L for ascorbate has been established in neuroblastoma cells (May et al. 2006), which is different from the one observed in SVCT2-expressing Xenopus oocytes (Tsukaguchi et al. 1999) but is similar to the higher Km observed in our previous study in cultured neurons (Castro et al. 2001). These data indicate that SVCT2 transporter (apparent Km > 100 μmol/L in neurons) should be responsible for the transport and accumulation of near millimolar concentration of AA inside these cells.
Our experiments to analyze the inhibitory effect of AA on neuronal glucose uptake were performed over a few seconds (15 s). This time was determined in time-course experiments to assure initial velocity conditions. Under these conditions, we observed that intracellular AA is able to inhibit 2-DOG uptake in cultured neurons. AA oxidation was not observed until 60 min of incubation in presence of DTT at 37°C (HPLC determination, data not shown), therefore these results confirm that reduced vitamin C (AA), and not oxidized vitamin C (dehydroascorbic acid, transported by GLUTs) (Nualart et al. 2003; Astuya et al. 2005) is the inhibiting agent in neurons. Finally, we observed that the inhibition of 2-DOG transport by intracellular AA was temperature dependent; however, additional experiments are required to clarify this point.
Patel et al. (2001) observed glucose uptake inhibition in presence of extracellular AA, however, they performed accumulation experiments using incubation times of 30 min, which allows neuronal uptake of AA. In agreement with this, we observed glucose uptake inhibition in cultured neurons pre-loaded with AA during 20 min (data not shown). Moreover, the substrate concentration used by Patel was three orders of magnitude less than the Km for 2-DOG transport. Patel et al. (2001) suppose that AA may directly inhibit the GLUT by reducing sulfhydryl groups in the transporter due to AA being an electron donor. But this supposition is questionable because thermodynamically AA cannot reduce an oxidized thiol.
To directly investigate if an AA effect takes place under synaptically active conditions, we carried out experiments using rat hippocampal slices. In agreement with previous reports using extracellular recordings (Izumi et al. 1997), in presence of a MCT inhibitor (4-CIN, a cellular and mitochondrial monocarboxylate transporter inhibitor), we observed that glucose is not able to support the EPSCs recovery after the deprivation period. However, when AA uptake was inhibited in the recording neuron, by using an anti-SVCT2 antibody, we observed EPSCs recovery after deprivation period. Thus, AA entry to neurons under synaptic activity is able to modulate the use of glucose in neural cells. We were able to observe the EPSCs recovery by using an anti-SVCT2 antibody when loading AA directly into the recording cell. Therefore, the anti-SVCT2 antibody used in the present experiments was able to inhibit SVCT2 function from the intracellular site. In summary, when AA uptake and monocarboxylate uptake in recording neuron is inhibited, glucose is able to support the recovery of synaptic activity. Only when monocarboxylate transport is inhibited, glucose is not able to support the neural activity recovery. Additionally, as it was early observed by Cox and Bachelard (1988), our data indicate that the metabolism of glucose itself, not lactate or pyruvate, is necessary to sustain neuronal function under the conditions studied.
It has been proposed that glutamate directly inhibits hexose transport in neurons co-cultivated with astrocytes (Porras et al. 2004); however, we were unable to detect this glutamate effect. Perhaps the phenomenon being observed actually happens subsequently to glutamate activated release of AA from the astrocytes present in co-cultured cells. In our cultured cortical neurons, glutamate did not affect 2-DOG transport. In the same way, in transwell experiments neuronal 2-DOG transport was not inhibited in the presence of glutamate and unloaded astrocytes. Furthermore, based on our electrophysiology experiments in which AA by itself was able to inhibit the recovery of observed current it is most likely that AA and not glutamate explains the observed results. In conclusion, after glucose deprivation, glucose could rescue the neuronal response to electrical stimulation only if AA uptake is prevented.