These experiments demonstrate that 2DG, a glucose analogue used for decades as the positron emitting tracer 18F-2DG for examination of regional glucose uptake, has acute anticonvulsant and chronic antiepileptic effects in a variety of in vitro and in vivo models of seizures and epilepsy. 2DG reduced the frequency of both interictal epileptiform bursts and ictal electrographic seizures induced by 7.5mM [K+]o in the CA3 region of hippocampal slices, and also reduced interictal epileptiform bursts in CA3 evoked by bath application of 4-AP, a K+ channel antagonist, and bicuculline, a GABAA receptor antagonist.
Inhibition of glycolysis may account for at least some of the reduction in neuronal excitability underlying the acute anticonvulsant action of 2DG in hippocampal slices, since blocking glycolysis by isomolar replacement of glucose with alternative energy sources such as pyruvate or lactate also suppressed epileptic discharges evoked by 7.5 mM [K+]o.
, 2DG demonstrated chronic antiepileptic effects against kindling progression in response to stimulation of the olfactory bulb, confirming a previous observation of action against seizure progression by stimulation of the perforant path.6
2DG also acutely protected against seizures evoked in vivo
by 6 Hz stimulation in mice and against audiogenic seizures in Fring’s mice. It showed only minimal evidence of anticonvulsant activity against seizures evoked by Metrazol and had no protective effects against MES seizures. These results provide evidence that 2DG has acute and chronic anticonvulsant and antiepileptic properties in several preclinical models. The spectrum of 2DG’s effectiveness against seizures evoked by 6 Hz, audiogenic, and kindling stimulation is distinctive relative to other available anticonvulsants. Although the suppression of seizures by 2DG in these models was not complete, many well established anticonvulsants do not confer complete protection or demonstrate effectiveness in all preclinical screening models. Partial but significant protection in various subsets of models implies potential for effectiveness in clinical trials in patients. While none of the currently used screening models have been formally validated as reliable predictors of anticonvulsant action in people, there is consensus that identification of compounds with distinctive profiles of action in the models compared to available drugs is desirable and may be valuable for developing new drugs that are more effective than the currently marketed agents.22 23 24 25
With this unique spectrum of protection in these animal models of epilepsy, 2DG has potential as a therapy for epilepsy with novel mechanisms of action compared to currently available anticonvulsants.
Acute In Vivo Anticonvulsant Actions of 2DG
Protection against minimal clonic seizures evoked by 6 Hz stimulation in mice, regarded as a model of “psychomotor” seizures,16
was observed at 15 min - 1 hr after intraperitoneal administration. Anticonvulsant effects against audiogenic seizures in Fring’s mice, which are generated in part by auditory circuitry in the brainstem, were observed at 1- 2 hr after intraperitoneal administration. Anticonvulsant properties at these short term intervals after in vivo
administration are unlikely to be dependent on altered neuronal gene expression. Furthermore, 2DG had rapid acute onset of anticonvulsant action against interictal and ictal discharges in CA3 within minutes after bath application, suggesting that its acute anticonvulsant effects are likely to be operating at the membrane or synaptic levels, or possibly through alterations in second messenger pathways influenced acutely by metabolic effects of 2DG such as inhibition of glycolysis. As 2DG suppressed interictal and ictal events induced by distinctive mechanisms such as depolarization by elevated [K+
, blockade of potassium channels by 4-AP, and antagonism of GABAA
receptors by bicuculline, its acute anticonvulsant actions appear to be broadly suppressive against a variety of cellular and membrane processes generating network synchronization.
Chronic In Vivo Effects Against Progression of Kindled Seizures
These experiments confirmed that the previously reported antiepileptic effect of 2DG against progression of kindled seizures evoked by perforant path stimulation is also observed when kindled seizures are evoked by stimulation of the olfactory bulb. The rate of progression to the first evoked Class V seizure was approximately doubled when 2DG was administered at 30 min prior to stimulation of either the perforant path or the olfactory bulb, indicating that 2DG interferes with neuronal mechanisms underlying seizure progression in limbic circuitry. As temporal lobe epilepsy is the most common form of intractable drug-resistant epilepsy, 2DG could potentially modify the progressive course of seizure-induced alterations in limbic circuitry in that disorder.
While protective effects of 2DG against progression of kindled seizures were observed with either perforant path or olfactory bulb stimulation, differences were observed as a function of the site of seizure induction. ADT was increased by 2DG in perforant path kindling but not in olfactory bulb kindling. The increase in the ADT in response to perforant path stimulation induced by 2DG is an anticonvulsant effect, as elevation of the ADT indicates that seizure induction requires stimulation of increased intensity.6
The ADT typically decreases as kindling progresses, so the observation of an increase in the ADT in perforant path kindling indicates that 2DG modifies progressive seizure-induced plasticity that is normally a part of the kindling process. However, the anticonvulsant effect against ADT was not observed in the olfactory bulb, indicating that at least some of the effects of 2DG are region-specific. The reasons for this difference are uncertain, but one possibility is that 2DG might have different effects on excitability or seizure-induced plasticity in the distinctive circuitries of the perforant path and olfactory bulb. None of the currently available conventional anticonvulsants demonstrates such regionally specific properties.
Possible Mechanisms Underlying the Acute Anticonvulsant and Chronic Antiepileptic Actions of 2DG
The observations that 2DG acutely protects against seizures evoked by 6 Hz stimulation in mice and audiogenic seizures in Fring’s mice and rapidly suppresses hippocampal interictal epileptiform bursts and electrographic seizures in vitro, suggest that 2DG has acute anticonvulsant properties in addition to its chronic antiepileptic effects against kindling progression. These two actions could involve different cellular and molecular mechanisms.
The chronic antiepileptic effects of 2DG have been associated with repression of BDNF and trkB expression. Conditional knockout of BDNF slowed kindling and conditional knockout of trkB blocked kindling progression.8
The repression by 2DG of seizure-induced increases in BDNF and trkB is mediated by the transcriptional repressor NRSF and its NADH sensitive co-repressor CtBP acting at the promoter regions of BDNF and its receptor, trkB.6
In pathophysiological conditions of high neuronal activity such as seizures, which increase glucose uptake and glycolysis, increases in NADH dissociate CtBP from NRSF and decrease repression, resulting in increased expression of BDNF and trkB. In the presence of 2DG, which reduces NADH levels as a consequence of glycolytic inhibition, the NRSF-CtBP complex maintains repression of BDNF and trkB, and kindling progression is slowed.
The rapid onset of anticonvulsant effects of 2DG suggest that this compound may be acting via different mechanisms, such as effects at the synaptic or membrane levels. Alternatively, the acute anticonvulsant actions of 2DG may be a result of unrecognized metabolic effects associated with glycolytic inhibition. For example, reduction of glycolysis might be accompanied by increases in systemic lipid metabolism and alterations in activity of the Kreb’s cycle and mitochondrial metabolism that could influence neuronal hyperexcitability. 2DG has been shown to increase neuronal resistance to oxidative and metabolic insults in cultured hippocampal neurons26
and to increase epileptic tolerance evoked by cerebral ischemia in mice.27
Glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) maintain GABAA
responses by phosphorylation of the α1 subunits of GABAA
While GAPDH and glycolytic intermediates can influence GABAA
receptors, the suppressive effects of 2DG on burst discharges in vitro
and seizures in vivo
suggest that glycolysis must have other acute effects on cellular and synaptic mechanisms contributing to network synchronization.
2DG, the Ketogenic Diet, and Potential Anticonvulsant Effects of Altering Brain Energy Metabolism
This study was not intended to investigate cellular mechanisms underlying the KD, but rather to mimic some the diet’s seizure suppressive effects through modulation of glycolysis. Nevertheless, some of these findings may have relevance for the anticonvulsant action of the KD. Hypotheses for the KD mechanism of action include a direct or indirect effect of ketosis,29
improvement in neuronal energy reserves, 30, 31
enhancement of GABAergic inhibition,32
alteration of mitochondrial metabolism,33
upregulation of gene transcripts encoding energy metabolism enzymes,34
and effects of lipids on neuronal excitability.35, 36
Clinical evidence suggests that carbohydrate restriction has beneficial effects and that ingestion of even small amounts of carbohydrate can result in recurrence of seizures in patients who are well controlled on the KD.4
The possibility that decreased carbohydrate (glucose) availability plays a role in the effects of the KD has also been considered 37
Glucose is an obligate energy source for the brain under normal conditions, but in situations of low glucose availability (e.g., fasting or the high-fat, low-carbohydrate KD), other substances can provide the brain’s energy requirements. Alternative energy sources that can maintain brain function and synaptic activity include lactate, pyruvate and β-hydroxybutyrate.38-42
Pyruvate is also a potent inhibitor of glycolysis by direct feedback inhibition of phosphofructokinase (PFK), the rate limiting enzyme of glycolysis. In subjects on the KD, fatty acids are converted into ketone bodies (acetoacetate, β-hydroxybutyrate, acetone), and enter the brain via a monocarboxylate transporter.43
The current results and other studies implicate the reduction in carbohydrate availability or metabolism as feasible mechanisms for decreased neuronal excitability and enhanced seizure control.37, 44, 45
The ability of calorie restriction to afford seizure protection in EL mice supports the hypothesis that reduction in carbohydrate metabolism may play a beneficial role in the KD.46
2DG does not model the KD since 2DG does not produce ketosis. However, 2DG and the KD do share some anticonvulsant effects. Both the KD and 2DG reduce audiogenic seizures in Fring’s mice and in the 6 Hz model.47
The KD is effective in MES whereas 2DG is not. 2DG and the KD are both effective in kindling although experimental studies have utilized different protocols. In our experiments, ADT was determined by stimulation of the perforant path or olfactory bulb, followed by i.p. administration of 2DG 30 minutes before each kindling stimulation. Another study used a “rapid” amygdala kindling protocol and monitored ADT in rats already fully kindled on either KD or standard diet.48
While the KD and 2DG are clearly different therapies, these results support carbohydrate restriction as a potential anticonvulsant and antiepileptic strategy.
2DG Uptake, Metabolism and Storage: Implications for Anticonvulsant Development
Glucose is taken up by neurons and glia, both of which possess uptake transporters and glycolytic enzymes that metabolize glucose for energy. 49-51
As an analogue of glucose, 2DG enters cells through glucose transporters and its uptake is preferentially increased in cells with increased energy consumption and metabolic demands. This aspect of 2DG may be advantageous for enhanced delivery of 2DG in the specific regions of the brain that generate seizures. The activity-dependent uptake of 2DG in regions of brain with increased energy metabolism also implies that pharmacodynamic actions (including anticonvulsant actions) and potential toxicity are likely to be nonlinear in relation to serum levels.
Although 2DG-6P does not undergo isomerization and progress through subsequent steps of glycolysis, glycogen becomes radiolabeled after injection of radiolabeled 2DG through isomerization to 2DG-1P and conjugation to UDP-2DG, 2DG glycogen, and 2DG glycoproteins.52
As 2DG is incorporated into glycogen and glycosylated macromolecules presumably stored in astrocytes, it might be released when astrocytic glycogen is metabolized during states of high energy demand such as seizures. The possibility that 2DG may be stored for long periods in glycogen also raises questions about its potential for chronic toxicity.53
However, 2DG has been administrated safely to humans for decades as the PET imaging tracer 18
F-2DG and has been administered to humans in doses as high as 1 gm/kg as adjuvant therapy for cancer,54, 55
suggesting that it has a relatively favorable preliminary toxicity profile for development as an anticonvulsant.
In summary, the experiments reported here demonstrate that 2DG has distinctive acute and chronic anticonvulsant therapeutic effects in preclinical in vivo models of seizures. The rapid onset of anticonvulsant suppressant action against burst discharges evoked in vitro by a variety of induction methods suggests that its anticonvulsant actions may be broad spectrum against a variety of mechanisms of network synchronization. The activity-dependent delivery of 2DG to brain regions with high metabolic activity, as occurs during seizures, further supports the clinical potential of this compound. Experiments are underway to further characterize the dose response and time course of anticonvulsant and antiepileptic actions of 2DG, and its effects on synaptic properties. Future experiments addressing the effects of 2DG on ATP-dependent K+ currents or other ion channels influencing neuronal excitability are of potential interest for understanding how glycolytic metabolism affects seizures and may contribute to the therapeutic actions of the KD.