Anticonvulsant and antiepileptic actions of 2-deoxy-D-glucose in epilepsy models

Abstract

Objective: Conventional anticonvulsants reduce neuronal excitability through effects on ion channels and synaptic function. Anticonvulsant mechanisms of the ketogenic diet remain incompletely understood. Because carbohydrates are restricted in patients on the ketogenic diet, we evaluated the effects of limiting carbohydrate availability by reducing glycolysis using the glycolytic inhibitor 2-deoxy-D-glucose (2DG) in experimental models of seizures and epilepsy.

Methods: Acute anticonvulsant actions of 2DG were assessed in vitro in rat hippocampal slices perfused with 7.5mM [K(+)](o), 4-aminopyridine, or bicuculline, and in vivo against seizures evoked by 6 Hz stimulation in mice, audiogenic stimulation in Fring's mice, and maximal electroshock and subcutaneous pentylenetetrazol (Metrazol) in rats. Chronic antiepileptic effects of 2DG were evaluated in rats kindled from olfactory bulb or perforant path.

Results: 2DG (10mM) reduced interictal epileptiform bursts induced by 7.5mM [K(+)](o), 4-aminopyridine, and bicuculline, and electrographic seizures induced by high [K(+)](o) in CA3 of hippocampus. 2DG reduced seizures evoked by 6 Hz stimulation in mice (effective dose [ED]50 = 79.7 mg/kg) and audiogenic stimulation in Fring's mice (ED50 = 206.4 mg/kg). 2DG exerted chronic antiepileptic action by increasing afterdischarge thresholds in perforant path (but not olfactory bulb) kindling and caused a twofold slowing in progression of kindled seizures at both stimulation sites. 2DG did not protect against maximal electroshock or Metrazol seizures.

Interpretation: The glycolytic inhibitor 2DG exerts acute anticonvulsant and chronic antiepileptic actions, and has a novel pattern of effectiveness in preclinical screening models. These results identify metabolic regulation as a potential therapeutic target for seizure suppression and modification of epileptogenesis.

FIGURE 1

(A) Chemical structures of glucose, 2DG, and intermediates of the initial steps of glycolysis. Phosphorylation of 2DG yields 2DG-6P, which cannot undergo isomerization by glucose-6P-isomerase to fructose-6P, thereby preventing subsequent steps of glycolysis. (B) Schematic diagram of key steps of glycolysis illustrating the rate-limiting step involving phosphofructokinase which is inhibited by pyruvate, the end-product of the pathway. Oxidation of phosphoenolpyruvate (structure not shown) to pyruvate generates NADH prior to entry into the TCA cycle. Abbreviations: Glu, glucose; G-6-P, glucose-6-phosphate; GPI, glucose-6-phosphate isomerase; 2DG, 2-deoxyglucose; F-6-P, fructose-6-phosphate; NADH, nicotinamide adenine dinucleotide; TCA, tricarboxylic acid.

FIGURE 2

Anticonvulsant effects of removal of glucose and substitution with alternative energy sources lactate or pyruvate on interictal burst firing induced by 7.5 mM [K⁺]_O. (A) Extracellular recording of spontaneous interictal discharges in hippocampal area CA3 (1 to 4 month old rats) in standard ACSF with 10 mM glucose. Faster sweep speed in the inset demonstrates that the discharges consisted of spontaneous extracellular depolarizations with superimposed population spikes. Removal of glucose and substitution with 10 mM lactate reduced the frequency of interictal discharges. Washout of lactate and return to standard ACSF containing 10 mM glucose increased interictal discharges, which were subsequently reduced by removal of glucose and substitution with 10 mM pyruvate, a direct inhibitor of glycolysis. (B) Effects of removal of glucose and isomolar substitution of 10 mM lactate or 10 mM pyruvate as illustrated in (A) from recordings in 21 hippocampal slices from 8 rats. Asterisks indicate significant differences.

FIGURE 3

Actions of 2DG on spontaneous interictal and ictal epileptiform bursts induced in hippocampal area CA3 by 7.5 mM [K⁺]_O. (A) Representative example of reversible effects of 10 mM 2DG in reducing frequency of spontaneous interictal discharges. The inset shows an interictal burst at faster sweep speed. (B) Effects of 30 min of bath application of 10 mM 2DG in 8 hippocampal slices from 5 rats. There was partial washout after return to standard ACSF. (C) Effect of increasing bath concentration of glucose from 10 to 20 mM. After 30 minutes of application of 20 mM glucose, there was no change in interictal burst frequency (n = 5 slices from 3 rats). (D) Examples of spontaneous ictal discharges in CA3 consisting of a prolonged extracellular DC shift with superimposed high frequency spike discharges which were observed in a subset of hippocampal slices exposed to 7.5 mM [K⁺]_O. Slower sweep speed in lower trace demonstrates rhythmicity of the spontaneous ictal discharges. (E) Anticonvulsant effect of 30 min bath application of 10 mM 2DG on ictal discharges (n = 7 hippocampal slices from 5 rats). Interictal data (A, B, C above) are from 1 to 4 month old rats. Ictal data (D, E above) are from 10 to 13 day old rats.

FIGURE 4

Effects of 10 mM 2DG on CA3 interictal bursts induced in hippocampal slices (from 1 to 4 month old rats) by 10 μM bicuculline or 50-100 μM 4AP. (A) Bath application of 10mM 2DG for 30 min reduced interictal burst discharges (middle trace) compared to baseline (upper trace). Bursts persisted after return to normal ACSF (lower trace). (B) Effects of 30 min bath application of 10 mM 2DG on interictal bursts induced by 10 μM bicuculline (n = 9 hippocampal slices from 4 rats). (C) Effects of 30 min bath application of 10 mM 2DG on interictal bursts induced by 50 μM 4AP. Interictal burst frequency increased after washout of 2DG and return to normal ACSF (lower trace). (D). Overall effects of 30 min bath application of 10 mM 2DG on interictal bursts induced by 50-100 μM 4AP (n = 13 hippocampal slices from 6 rats).

FIGURE 5

(A) Effects of 2DG on 6-Hz seizures in mice. Percent of mice protected at a stimulus of 22 mA is shown as a function of intraperitoneal 2DG dose. The ED50 is 79.5 mg/kg. (B) Effects of 2DG on audiogenic seizures in Fring’s mice. Percent of mice protected 2 hrs after 2DG administration is shown as a function of intraperitoneal 2DG dose. The ED50 is 206.4 mg/kg.

FIGURE 6

Effects of 2DG on afterdischarge (AD) threshold in rats experiencing kindled seizures evoked by stimulation of the olfactory bulb and perforant path. 2DG (250 mg/kg i.p.) was administered 30 min prior to delivery of stimulation (see text for details of normalization and stimulation methods). Treatment with 2DG increased the normalized mean AD threshold (filled triangles) in rats receiving perforant path stimulation compared to saline treated controls, which demonstrated a reduction in AD threshold (filled circles, reported previously6). In contrast, there were no effects of 2DG on AD threshold of rats receiving stimulation of the olfactory bulb (open triangles) compared to saline treated controls (open circles).

FIGURE 7

Antiepileptic effects of 2DG (250 mg/kg i.p.) on kindling progression in rats experiencing seizures evoked by perforant path or olfactory bulb stimulation. Number of ADs required to achieve behavioral seizure stages of class III, class IV, and the first to third class V seizures in response to kindling of the olfactory bulb (A) or perforant path (B). There was a ~ 2-fold slowing in the rate of number of AD required to reach each stage in animals treated with 2DG for both olfactory bulb and perforant path stimulation.