Ascorbic acid-dependent GLUT3 inhibition is a critical step for switching neuronal metabolism

Abstract

Intracellular ascorbic acid is able to modulate neuronal glucose utilization between resting and activity periods. We have previously demonstrated that intracellular ascorbic acid inhibits deoxyglucose transport in primary cultures of cortical and hippocampal neurons and in HEK293 cells. The same effect was not seen in astrocytes. Since this observation was valid only for cells expressing glucose transporter 3 (GLUT3), we evaluated the importance of this transporter on the inhibitory effect of ascorbic acid on glucose transport. Intracellular ascorbic acid was able to inhibit (3)H-deoxyglucose transport only in astrocytes expressing GLUT3-EGFP. In C6 glioma cells and primary cultures of cortical neurons, which natively express GLUT3, the same inhibitory effect on (3)H-deoxyglucose transport and fluorescent hexose 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) was observed. Finally, knocking down the native expression of GLUT3 in primary cultured neurons and C6 cells using shRNA was sufficient to abolish the ascorbic acid-dependent inhibitory effect on uptake of glucose analogs. Uptake assays using real-time confocal microscopy demonstrated that ascorbic acid effect abrogation on 2-NBDG uptake in cultured neurons. Therefore, ascorbic acid would seem to function as a metabolic switch inhibiting glucose transport in neurons under glutamatergic synaptic activity through direct or indirect inhibition of GLUT3.

Brain Energy Metabolism in Health and Disease

“…The third mechanism is our own theory. We have demonstrated that neuronal intracellular ascorbic acid inhibits glucose utilization in neurons (Castro et al, 2009) through GLUT3 inhibition (Figure 8, Beltrán et al, 2011). This mechanism is supported by the idea of ascorbic acid recycling in brain (see above, Figure 10, Astuya et al, 2005).…”

Beyond the redox imbalance: Oxidative stress contributes to an impaired GLUT3 modulation in Huntington's disease

“…The glioma cell lines (C6, ATCC ® number CCL 107™) were grown using DMEM-F12 (United States Biological, Swampscott, MA) containing 10% foetal bovine serum (FBS; HyClone, Logan, UT), 50 U/ml penicillin, 50mg/ml streptomycin, 50 ng/ml amphoreticin B, and 2 mM L-glutamine (Gibco Invitrogen Corporation, Grand Island, NY). Primary cultures: Striatal neurons were obtained from 17-day-old embryos of Wistar rats as described previously (Acuña et al, 2013, Beltrán et al, 2011). Adult female rats were anaesthetised using isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether), embryo forebrains were removed and the striatum dissected.…”

“…The fluorescence signal was normalised according to: $$\text{Normalised fluorescence}=\mathrm{normal\Delta }\mathrm{normalF}/\mathrm{normalF}=false(\mathrm{normalF}-{\mathrm{F}}_{0}false)/{\mathrm{F}}_0$$ where F 0 correspond to the mean of fluorescence intensities measured in the first 10 frames. For experiments in the presence of intracellular ascorbic acid, cells were preloaded using a solution of 1 mM ascorbic acid, for 60 min at 37 °C (Beltrán et al, 2011). …”

“…Moreover, glutamatergic synaptic activity stimulates the release of ascorbic acid from glial reservoirs (Wilson, 2000). Neuronal sodium vitamin C co-transporters (SVCT2) allow the uptake of ascorbic acid, which inhibits glucose transport and stimulates lactate uptake in neurons through a GLUT3-dependent mechanism (ascorbic acid metabolic switch: Beltrán et al, 2011, Castro et al, 2009). …”

The Tiny Drosophila Melanogaster for the Biggest Answers in Huntington’s Disease

“…Despite efforts to define the role of glial cells in HD pathogenesis [128,129,130], or the effects that abnormal polyQ repeats have on glia [131], these aspects remain almost unattended in HD. Glial cells express huntingtin constitutively; the aggregation of mutant htt in glia induces locomotor deficits and decreases lifespan in the fly HD model [132,133].…”