Direct evidence for activity-dependent glucose phosphorylation in neurons with implications for the astrocyte-to-neuron lactate shuttle

Abstract

Previous (13)C 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 (FDG6P), 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 FDG6P 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.

Keywords: 2-fluorodeoxyglucose; glutamate-glutamine cycle; neuroenergetics; neuronal glucose phosphorylation; synaptoneurosomes.

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.

Fig. 2.

Time courses of blood FDG and glucose concentrations during FDG/[1,6-¹³C₂] 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 [1,6-¹³C₂] 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.

Fig. 3.

Comparison of seizure-to-control (Sez/Con) ratios for glucose phosphorylation in nerve terminals (NT) and brain homogenate (H) with predictions of the two neuroenergetics models. Values shown for NT and H reflect the Sez/Con ratios of FDG_6P/NAA expressed as mean ± SD (n = 3,3). Predicted rates of neuronal glucose phosphorylation with oxidation, , were calculated using Eqs. 1−3 (model 1) and Eqs. 4 and 5 (model 2) for control and seizure conditions and expressed as a ratio.