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Please cite this article in press as: Rho, J.M., Stafstrom, C.E., The ketogenic diet: What has science taught us? Epilepsy

Res. (2011), doi:10.1016/j.eplepsyres.2011.05.021

ARTICLE IN PRESS+Model

EPIRES-4566; No. of Pages8

Epilepsy Research (2011) xxx, xxxxxx

journal homepage: www.elsevier .com/ locate/epi lepsyres

The ketogenic diet: What has science taught us?

Jong M. Rhoa,, Carl E. Stafstromb

a Departments ofPaediatrics and Clinical Neurosciences, Alberta Childrens Hospital, Universityof Calgary, Calgary, Alberta,

Canadab Department ofNeurology, UniversityofWisconsin, Madison, WI, USA

Received 3 February 2011; received in revised form 28 April 2011; accepted 1 May 2011

KEYWORDSEpilepsy;Animal models;Anticonvulsantmechanisms;Ketogenic diet;Metabolism;2-Deoxy-D-glucose

Summary Despite intense and growing interest in studying the mechanisms of ketogenic

diet (KD) action, and recently published studies implicating novel molecular interactions with

metabolic substrates, there nevertheless remains the pragmatic and scientific challenge ofsus-

taining continued research in this field. This is in part a consequence oflimited research funding

and perhaps skepticism regarding the ultimate need to understand underlying mechanisms, par-

ticularly when clinical studies have increasingly validated the efficacy of the KD and its variants.

After a decade and a halfof more concerted laboratory efforts to understand KD mechanisms,

it would be prudent to ask what has all this scientific research really taught us? In this regard,

it is instructive to compare and contrast laboratory research in dietary approaches for epilepsy

with that traditionally used to screen for potential antiepileptic drugs (AEDs). In this review,

lessons learned from AED development are applied to the more recent experimental findings and

approaches attempting to link metabolic changes induced by the KD to neuronal and network

excitability in the brain.

2011 Published by Elsevier B.V.

Introduction

Many reviews of the ketogenic diet (KD) in the modern erabemoan the fact that little has changed with regard to theKD formulation since the diet was originally designed almosta hundred years ago. While some diet modifications offer

effectiveness comparable to the classic KD (e.g., modifiedAtkins diet, low-glycemic index treatment), a validated sci-entific basis remains lacking for epilepsy diet treatments.

Corresponding author at: Division of Paediatric Neurology,Alberta Childrens Hospital, 2888 Shaganappi Trail, NW, Calgary,Alberta T3B 6A8, Canada. Tel.: +1 403 955 2635.

E-mail address: [email protected] (J.M. Rho).

Why should we be concerned, other than to satisfy a sci-entific curiosity? The KD does stop seizures in many casesof refractory childhood epilepsy, so why bother spendingresources and scientific talent to figure out what alreadyworks? There are several reasons. First, as mentioned above,the KD is effective but there has been little progress in mak-

ing its administration more convenient and more palatable(Rho and Sankar, 2008). Second, current KD formulationsare fraught with potentially serious adverse effects and cancause metabolic derangements; these might be amelioratedifmore was known about how the diet works. Third, greaterknowledge about the KDs mechanisms could provide insightsinto the metabolic and physiological basis of normal brainfunction, both in the normal state, and under pathologi-cal conditions of stress and injury, such as seizure activity.

0920-1211/$ see front matter 2011 Published by Elsevier B.V.doi:10.1016/j.eplepsyres.2011.05.021


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2 J.M. Rho, C.E. Stafstrom

Finally, just as the KDs clinical efficacy has recently beenestablished through randomized, blinded trials (Neal et al.,2008; Freeman et al., 2009), an understanding of basicmechanisms would further validate the diet and provide atemplate for future therapeutic improvements. The betterwe understand how the KD suppresses seizures, retards theformation ofthe epileptic state (i.e., epileptogenesis), andimproves the cognition and daily function of its adherents,the more the KD will be a rational choice for our patients.Eventually, various dietary formulations could be developedfor patients with specific etiologies of epilepsy, in particu-lar age ranges, or with certain pharmacogenetic or morespecifically, metabolomic profiles.

This overview is not intended to provide a comprehen-sive treatise on the scientific basis of the KD. The readeris referred to several recent reviews that already providethis information (Stafstrom, 2004; Bough and Rho, 2007;Hartman et al., 2007; Kim and Rho, 2008; Maalouf et al.,2009; Bough and Stafstrom, 2010). Instead, in the presentarticle, we offer an overview ofrecent trends in research onKD mechanisms, to illustrate how science has contributedto and continues to provide a better understanding of the

diet (Schwartzkroin et al., 1999; Stafstrom et al., 2008).We emphasize the key questions that should be asked andthe approaches required to answer them. By analogy, we dis-cuss the process by which investigational antiepileptic drugs(AEDs) are developed and describe how the KD is both similarand unique compared to the standard AEDs used in clinicalpractice.

In a sense, the KD challenges us with a reversedilemma compared to most drugs used in clinical medicine.In the usual situation, a disease or pathology exists and weseek to find a therapy based on the disorders pathophys-iology or underlying molecular dysfunction. Indeed, eventhough the mechanism(s) of action of AEDs remain incom-pletely understood, early investigational studies have often

revealed specific molecular targets or interactions that havebeen linked to seizure genesis for example, voltage-gatedsodium channels. With the KD, we have the opposite situa-tion the therapy is effective, but our goal is to understandits mechanism ofaction. Onthe one hand, this is a fortuitoussituation since we can continue to use the KD empiricallywithout necessarily knowing how it works. On the otherhand, refractory epilepsy, especially in children, continuesto represent an urgent and intractable problem, and tocomprehend the KD in greater detail can only aid in our over-all therapeutic efforts. Furthermore, clinical experiencehas taught us that the number of patients with refractoryepilepsy is not declining, despite all the new AEDs now avail-able (Arroyo et al., 2002; Kwan and Brodie, 2010) hence

the urgent need to think outside the traditional drug screen-ing box (Bialer and White, 2010).

Animal models: a road to understanding or ablind alley?

Are animal models a route to understanding mechanisticcomplexity in the epilepsies or merely a trifle to occupy ide-alistic scientists? It is perhaps a bit of both. Animal modelshave been considered the gold standard for testing poten-tial AEDs. There are animal models for most ofthe common

seizure types, for some epilepsy syndromes (but not all, dueto irreconcilable species differences), and for a few path-ways underlying epileptogenesis an area in dire need offurther study (Gasior et al., 2006). To address the KD specif-ically, what models have been informative? And what arethe caveats to placing too much hope in models? Finally,have experimental systems involving more reduced systems(e.g., cellular electrophysiology on brain slices, cells in cul-ture, etc.) been helpful in unraveling the mechanisms of theKD?

No single laboratory test can fully predict the clinicalutility of an AED, either established or investigational. Tofully evaluate the overall spectrum of clinical activity (i.e.,narrow vs. broad), all investigational AEDs should ideallybe screened in a variety of different seizure and epilepsymodels. And despite efficacy in one more of these mod-els, the ultimate validation must always await the results ofwell-designed prospective clinical trials. Nevertheless, withfew exceptions, the majority ofAEDs currently on the mar-ket today were advanced to clinical assessment based ontheir ability to block evoked seizures in one or more ani-mal seizure or epilepsy models (White, 2003; Gareri et al.,

2005).Over the years, many animal seizure models have beenstudied as potentially predictive seizure models (Table 1).Historically, the maximal electroshock (MES) test, the subcu-taneous pentylenetetrazol (sc PTZ) test, and the electricalkindling model have been the three in vivo model sys-tems most commonly employed in the search for new AEDs(White, 2003). Currently, animal models that more closelyresemble the human epileptic condition are being utilized,including several with known underlying genetic defects(Frankel, 2009). The MES test and the kindling model aretwo highly predictive models that have proven useful inthe characterization of a drugs potential to block general-ized tonicclonic and partial seizures, respectively. Further,

the sc PTZ test has been considered predictive of a drugspotential utility against generalized absence seizures. Thissimplified approach, unfortunately, is imperfect. For exam-ple, barbiturates are effective in the sc PTZ test in animals,yet can provoke or worsen human generalized spike-wavedischarges and associated absence seizures (Sazgar andBourgeois, 2005). This dilemma has been addressed in partthrough the use ofadditional animal models that are perhapsmore predictive than the sc PTZ test for generalized absenceseizures. These include the spike-wave seizures induced bythe chemoconvulsant -butyrolactone, the genetic absenceepileptic rat of Strasbourg (GAERS), and the lethargic (lh/lh)mutant mouse (Van Luijtelaar et al., 2002).

Given its name, it should not be surprising that the ear-

liest animal studies involving the KD paid keen attention tothe acute effects of ketone bodies. Keith first demonstratedthat acetoacetate (ACA) protected against seizures inducedby thujone, a convulsant constituent of many essential oils(Keith, 1933). This intriguing observation was confirmeddecades later by Likhodii and colleagues who found that bothACA and acetone blocked seizures induced by MES and PTZ(Likhodii et al., 2003). These investigators further demon-strated that intraperitoneal injection of acetone resultedin plasma and cerebrospinal fluid (CSF) concentrations con-sistent with doses used to suppress seizures, supporting thedistinct possibility that this volatile agent might mediate the


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The ketogenic diet: What has science taught us? 3

Table 1 Correlation between anticonvulsant efficacy and clinical utility of established and second-generation (in italics) AEDs

in experimental animal models. Note that PB, TGB, and VGB block clonic seizures induced by sc PTZ but are inactive against

generalized absence seizures and may exacerbate spike-wave seizures. Abbreviations: sc, subcutaneous; Hz, Hertz; mA, mil-

liampere; MES, maximal electroshock; PTZ, pentylenetetrazol; BZD, benzodiazepines; CBZ, carbamazepine; ESM, ethosuximide;

FBM, felbamate; GBP, gabapentin; LTG, lamotrigine; LVT, levetiracetam; PB, phenobarbital; PHT, phenytoin; LCM, lacosamide;

PGB, pregabalin; RUF, rufinamide; TGB, tiagabine; TPM, topiramate; VPA, valproic acid; ZNS, zonisamide; VGB, vigabatrin.

Experimental model Clinical seizure type

Tonic and/or clonicgeneralized seizures

Myoclonic/generalizedabsence seizures

Generalizedabsence seizures

Partial seizures

MES (tonic extension) CBZ, PHT, VPA, PB

FBM, GBP, LCM, LTG,

PGB, RUF, TPM, ZNS

sc PTZ (clonic seizures) ESM, VPA, PB, BZD

FBM, GBP, PGB, RUF,

TGB, VGB

Spike-wave discharges ESM, VPA, BZD

LTG, TPM, LVT

Electrical kindling (focal seizures) CBZ, PHT, VPA, PB, BZD

FBM, GBP, LCM, LTG,

TPM, TGB, ZNS, LVT, VGB

Phenytoin-resistant kindled rat LVT, GBP, TPM, FBM, LTG6 Hz (44 mA) VPA

LVT

From: White, H.S., 2010. Epilepsy and disease modification. In: Rho, J.M., Sankar, R., Stafstrom, C.E. (Eds.), Epilepsy: Mechanisms,Models, and Translational Perspectives. CRC Press/Taylor & Francis Group, Boca Raton, Florida, USA, p. 146.

clinical effects of the KD. Curiously, however, there are nostudies clearly demonstrating that the major ketone body,-hydroxybutyrate (BHB), can exert similar effects whenadministered acutely.

Most of the existing KD animal studies have involvedhigh-fat treatments implemented prior to acute provoca-tion with either electrical stimulation or chemoconvulsant

administration in rodents. Further, most animal diets havemodeled the classic long-chain triglyceride (LCT) diet andconform closely to either a 4:1 or approximately 6:1 keto-genic ratio offats to carbohydrates plus protein (by weight).In general, irrespective of the precise dietary formulation,as long as ketosis is seen, protective effects have been seenagainst seizures provoked by numerous methods, includ-ing corneal electroshock, hydration electroshock, MES, PTZ,bicuculline, semicarbizide, kainate, fluorothyl, or in the 6 Hzstimulation model (Appleton and DeVivo, 1974; Hori et al.,1997; Rho et al., 1999; Bough et al., 2000; Thavendiranathanet al., 2000; Bough et al., 2002; Rho et al., 2002; Noh et al.,2003; Zhao et al., 2004; Kwon et al., 2008; Hartman et al.,2008).

Published animal studies, in attempting to mirror clini-cal experience, suggest that dietary effects in controllingexcitability in the brain extend beyond species boundaries,but they do little to enhance our knowledge of underly-ing mechanisms of action. Rather, they raise more issuesand highlight the necessity of developing and studying amore clinically relevant model. One critical limitation is thatthese laboratory investigations have been conducted in nor-mal, not epileptic, rodent brain. What is needed is a chronicmodel of the KD, employing an animal with early-onset,medically refractory epilepsy, that responds to a particu-

lar formulation of a high-fat diet that best recapitulates allofthe essential elements of the human experience.

Cellular electrophysiology

Epilepsy is defined by the occurrence of recurrentspontaneous seizures arising from hyperexcitable and hyper-synchronous brain activity. The essential currency ofneuronal excitability both normal and aberrant is thecomplex array of ion channels (primarily voltage-gated andligand-gated) that determine the firing properties of neuronsand mediate synaptic transmission. Against this increas-ingly complex backdrop, the conventional thinking has beenthat seizure activity arises due to perturbations in thenormal balance between inhibition and excitation in a local-ized region, multiple brain areas (linked in a multi-nodalnetwork), or throughout the whole brain. Recently, how-ever, this paradigm has been shown to be too simplified,with epilepsy mechanisms and therapeutic targets address-

ing heretofore unsuspected physiological functions (synapticvesicles, brain energetics, etc. the latter of particularrelevance to the KD) (Kim and Rho, 2008).

At present, there appears to be at least six impor-tant mechanisms through which the currently availableAEDs exert their anticonvulsant action. These are summa-rized in Table 2. As should be noted, and not surprisingly,the vast majority of molecular targets are ion channelsand transporters that are localized to plasmalemmal mem-branes. While the precise manners in which AEDs exertclinical activity remain unclear, there is a wealth of in vivoand in vitro experimental data supporting this mechanistic


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Table 2 Classification of antiepileptic drug mechanisms.

Modulation of voltage-dependent Na+ or Ca2+ channels leading to a secondary inhibition of neurotransmitter release

(particularly ofglutamate) or to inhibition of intrinsic bursting

Enhancement of GABA-mediated inhibition or other modulatory effects on GABA uptake and metabolism

Inhibition of synaptic excitation mediated by ionotropic glutamate receptors

Modulation of synaptic release, particularly of glutamate, through direct actions on release machinery (SV2A and 2)

Activation of voltage-dependent K+ channels and improved spike-frequency adaptation

Enhanced activation of dendritic hyperpolarization-activated cation (HCN)-channels and suppressed action potential initiationby dendritic inputs

From: White, H.S., Rho, J.M., 2010. Mechanisms of Action of Antiepileptic Drugs. Professional Communications, Inc., New York.

framework. In this light, a fundamental question pertainingto KD effects at the cellular and molecular levels has beenwhether any of the metabolic substrates elaborated by thisnon-pharmacological intervention can interact with ionchannels that regulate neuronal excitability.

Thio and colleagues found that acute application ofBHBor ACA did not affect standard measures of synaptic trans-mission in hippocampus (Thio et al., 2000). Specifically,these ketones did not affect GABAA receptors, ionotropic

glutamate receptors, or voltage-gated sodium channels overa wide concentration range (300M to 10 mM). The fact thatACA was ineffective in their hands was surprising, especiallygiven its clear anticonvulsant effects when administeredin vivo (Rho et al., 2002). These negative results may havebeen influenced in part by the facts that: (1) ketone bodieswere infused acutely, not chronically; (2) experiments wereconducted in normal, not epileptic, brain; and (3) both cul-ture and perfusion media contained glucose, which mighthave countered a ketotic environment.

ATP-sensitive potassium (KATP) channels are logical can-didates for linking metabolic changes to cellular membrane

excitability. KATP channels are a type of inwardly rectifyingpotassium channel (Kir6) that is activated when intracel-lular ATP levels fall. Ma et al. found that ketone bodiesreduced the spontaneous firing of GABAergic neurons in ratsubstantia nigra pars reticulata (SNr), a putative subcorticalseizure gate, by opening KATP channels (Ma et al., 2007).Despite the intuitive appeal and novelty ofthis observation,an inherent discrepancy remains to be reconciled. Earlierstudies had shown that the KD can increase levels of ATP and

other bioenergetic substrates through enhanced mitochon-drial respiration (Cullingford et al., 2002; Sullivan et al.,2004; Bough et al., 2006; Maaloufet al., 2007; Jarrett et al.,2008). Since high ATP levels block KATP channel activity, it isunclear how opening of these channels is achieved by infu-sion of ketone bodies in the SNr. Ma et al. suggested thatthe area subjacent to KATP channels may actually exhibitlower ATP levels than other cellular compartments due toexcessive membrane discharge which consumes ATP (Maet al., 2007).In other words, while ketone bodies might raiseglobal ATP levels, ATP levels near the plasma membrane(where KATP channels are localized) could be substantially

Table 3 Ketogenic diet: clinical correlates and experimental observations.

Clinical correlate Observation in animal models

Seizure type KD is effective in many seizure types

and epilepsy syndromes

KD is effective in models employing a

wide variety of seizure paradigms

Age range Children extract and utilize ketones

from blood more efficiently than

older individuals

Younger animals respond better to

the KD

Calorie restriction Associated with seizure reduction Increases seizure threshold

Diet type Classic and MCT KDs are equally

efficacious

Classic and MCT diets both increase

seizure threshold

Ketosis Ketosis is necessary but not sufficient

for seizure control

A threshold level of ketosis is

necessary but not sufficient to

explain anti-seizure effectsFat Practical concerns limit the ketogenic

ratio; possible role of fat chain length

and degree of saturation (e.g., PUFA)

Better effectiveness with higher

ketogenic ratios; uncertain if type of

fat is a critical variable

Latency to KD effectiveness Seizures may be reduced during the

pre-diet fast or after a latency of

days to weeks

Several days

Reversal ofprotective effect when

KD discontinued

Rapid (hours) Rapid (hours)

Adapted from Stafstrom (2004).Abbreviations: KD, ketogenic diet; MCT, medium chain triglycerides; PUFA, polyunsaturated fatty acid.


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decreased, and as such, KATP channels could be recruited todampen neuronal excitability. Although this is a potentiallyelegant solution to the dilemma posed, there are yet no datadirectly supporting this hypothesis. Taken together, the roleofketone bodies as direct anticonvulsant mediators remainsuncertain, especially since a clear and convincing correla-tion between the degree of ketonemia and seizure controlhas yet to be established (Bough et al., 2000; Gilbert et al.,2000).

Further actions of metabolic substrates have recentlybeen reported. Kawamura and colleagues evaluated theelectrophysiological effects of reduced glucose (a consis-tent finding in patients successfully treated with the KD) importantly, under conditions of adequate or enhanced ATPlevels in CA3 hippocampal pyramidal neurons using whole-cell recording techniques (Kawamura et al., 2010). Theseauthors found that glucose restriction led to ATP releasethrough pannexin hemichannels on CA3 neurons, and thatthe increased extracellular ATP, upon rapid degradation byectonucleotidases to adenosine, resulted in activation ofadenosine A1 receptors which was also shown to be cou-pled to opening of plasmalemmal KATP channels. While this

study revealed a novel mechanism of metabolic autocrineregulation, involving close cooperativity among pannexinhemichannels, adenosine receptors and KATP channels, itis yet uncertain whether adenosine is the key metabolicmediator of the KDs anticonvulsant activity. The con-tribution of adenosine is plausible, however, given thewell-documented role ofthis endogenous purine in suppress-ing cellular excitability (Boison, 2009).

There is yet another intriguing link between metabolicsubstrates and neuronal excitability. Juge and colleaguesrecently reported that ACA inhibits vesicular glutamatetransporters (VGLUTs), which are required for exocytoticrelease of the excitatory neurotransmitter glutamate,specifically by competing with an anion-dependent regula-

tory site on presynaptic vesicles (Juge et al., 2010). Theseauthors demonstrated that ACA decreased the quantal sizeof excitatory neurotransmission at hippocampal synapses,and suppressed glutamate release and seizures evoked bythe convulsant 4-aminopyridine in rats. So, is this a plausi-ble explanation for the acute in vivoeffects ofACA observednearly eight decades earlier? Possibly, but ACA is reportedto have other actions as well, notably effects on mitochon-dria (Maalouf et al., 2007; Bentourkia et al., 2009). Theother caveat is that ACA is highly unstable, and under-goes spontaneous decarboxylation to acetone; further, inthe presence of BHB dehydrogenase, is interconverted tothe major ketone body BHB. Thus, it appears that seeminglystraightforward metabolic substrates are players in a more

complex arena.

Metabolic pathways relevant to KDmechanisms

Key clinical observations regarding KD use provide start-ing points from which to investigate underlying mechanisms(Table 3). In this section, we discuss some recent trends inKD research, focusing on studies of biochemical pathwaysthat could link metabolism with neuronal excitability. Theoriginal formulation ofthe KD was based on the assumption

that the diet mimicked the fasting state, which was knownto reduce seizures. The KD restricts carbohydrate intakeand provides energy from fat breakdown, leading to keto-sis, but the observational leap to exactly how ketosis (orsome other factor) constrains cellular excitability remainsunknown. (The effects of fatty acids on neuronal excitabil-ity, another possible mechanism of KD action, are discussedelsewhere in this volume.)

Another key feature is that ingestion of a small amountofcarbohydrate in a patient on the KD results in rapid loss ofseizure control (Huttenlocher, 1976), leading to the hypoth-esis that carbohydrate restriction could be protective inepilepsy (Greene et al., 2003; Yamada, 2008). By utilizingketones as the energy source, the KD bypasses glycolysis,raising the possibility that glycolytic inhibition itself mightalso protect against seizures.

2-Deoxy-D-glucose (2DG) is a glucose analog differingfrom glucose only by substitution of a single oxygen atom inthe 2-position. 2DG cannot be metabolized and inhibits gly-colysis by blocking the glycolytic enzyme, phosphoglucoseisomerase, thereby preventing the conversion ofglucose-6-phosphate to fructose-6-phosphate. 2DG has been shown to

be a potent anticonvulsant and antiepileptic agent in sev-eral animal models, including kindling, audiogenic seizuresin Frings mice and 6-Hz corneal stimulation (Garriga-Canutet al., 2006; Stafstrom et al., 2009), as well as in vitro in CA3neurons in hippocampal slices exposed to elevated extracel-lular K+, bicuculline, or 4-aminopyridine (Stafstrom et al.,2009) or metabotropic Group 1 agonists (Pan et al., 2008).

Seizure-suppressing effects of2DG are seen both acutelyand chronically. The acute anticonvulsant effects of 2DGin vitro and in vivo against both ictal and interictal activitysuggest that 2DG may exert direct actions at the synapticor membrane levels, but through mechanisms indepen-dent of altered gene expression (Stafstrom et al., 2009).Acute effects of 2DG could be related to rapid-onset

metabolic or electrophysiologicalconsequences ofglycolyticinhibition leading to reduction of network synchroniza-tion. For example, there could be an effect on systemiclipid metabolism, mitochondrial function, or the phospho-rylation state of GABAA receptor subunits (Pumain et al.,2008), each ofwhich can influence neuronal excitability. Inwhole-cell recordings of CA3 neurons in hippocampal slices,acute application of 2DG suppressed spontaneous excita-tory postsynaptic currents (EPSCs) after transient epilepticactivity induced by elevated extracellular potassium ormetabotropic Group 1 agonists, but not in normal slices,implicating an activity-dependence to effects of glycolyticinhibition (Pan et al., 2009). These and other potential acutemechanisms are currently under study.

The chronic antiepileptic effects of 2DG have beenrelated to the molecular regulation of genes for brain-derived neurotrophic factor (BDNF) and its receptor, tyrosinekinase B (trkB). Repression ofboth BDNF and trkB expressionare required for the progression ofkindling (He et al., 2004).2DG suppresses seizure-induced increases in BDNF and trkB,mediated by the transcriptional repressor neuron restrictivesilencing factor (NRSF) and its nicotinamide adenine din-ucleotide (NADH)-sensitive co-repressor carboxy-terminalbinding protein (CtBP). NRSF and CtBP act at the pro-moter regions of BDNF and trkB genes (Garriga-Canut et al.,2006). During seizure activity, when glycolysis and glucose


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Figure 1 Glucose metabolism and points at which interventions could affect neuronal excitability and seizure control Glucose can

be diverted to the pentose phosphate shunt (PPP) via fructose-1,6-diphosphate (FDP). 2-Deoxy-2-glucose (2DG) inhibits glycolysis by

blocking the phosphoglucose isomerase step (see text). The ketogenic diet (KD), via ketone bodies, bypasses glycolysis by providing

acetyl-CoA (ACoA) to the TCA (tricarboxylic acid cycle) after glycolysis. Anaplerotic compounds refill depleted intermediates

from the TCA. Otherabbreviations: -OHB, beta-hydroxybutyrate; AcAc, acetoacetate; PDH, pyruvate dehydrogenase.

production are increased, elevated levels of NADH causesdissociation of CtBP from NRSF, decreasing transcriptionalrepression and resulting in increased expression of BDNFand trkB. In the presence of 2DG, which reduces NADHlevels as a consequence of glycolytic inhibition, the NRSF-CtBP complex maintains repression of BDNF and trkB, andkindling progression is slowed (Garriga-Canut et al., 2006;Huang and McNamara, 2006). These chronic anticonvulsantand antiepileptic effects of2DG, coupled with its favorablesafety profile (Stafstrom et al., 2009), position this com-pound as a viable candidate for clinical trials and raise thepossibility that this agent can modify both seizure suscepti-bility and disease progression.

Continuing the theme that a key KD mechanism mightinvolve alteration of glucose metabolism, another compo-nent of the glycolytic pathway, fructose-1,6-diphosphate(FDP), has been shown to exert acute anticonvulsant activ-ity in several seizure models in adult rats including kainate,pilocarpine, pentylenetetrazole, and kindling (Lian et al.,2007; Ding et al., 2010). Indeed, FDP was more effectiveas an anticonvulsant than 2DG, KD, and valproate in thesestudies. FDP increases glucose flux from glycolysis into thepentose phosphate pathway (PPP). NADPH generated in thePPP reduces glutathione, which has anticonvulsant activ-ity. Therefore, FDP may exert an endogenous anticonvulsant

(and perhaps anti-oxidant) action (Stringer and Xu, 2008),but the precise mechanism by which FDP produces an anti-convulsant effect remains unclear.

Another dietary approach to epilepsy derives from theobservation that seizures cause a deficiency in tricarboxylicacid cycle (TCA) intermediates (especially -ketoglutarateand oxaloacetate), leading to increased excitability andpossibly increased seizures. It has been hypothesized thatrefilling these deficient compounds, a process calledanaplerosis, might oppose seizure generation. One suchanaplerotic compound, triheptanoin, has recently beeninvestigated in both acute and chronic seizure models (Willis

et al., 2010). Mice fed triheptanoin exhibited delayed devel-opment of corneal kindled seizures and triheptanoin feedingincreased PTZ seizure threshold in chronically epileptic micethat had undergone status epilepticus 3 weeks before PTZtesting (Willis et al., 2010). Therefore, like 2DG, anapleroticcompounds alter both acute and chronic seizure suscepti-bility. Anaplerosis represents a novel approach that expandsthe potential metabolic modifications that could be anticon-vulsant or antiepileptic. Together, results from studies of theKD, 2DG, FDP, and anaplerosis suggest that modification ofmetabolic pathways such as glycolysis could be a possiblenovel mechanism for treatment of seizures (Fig. 1).

A final example ofthe involvement of a metabolic path-way that could play a role in KD action is the endogenouspeptide leptin. Leptin is part of the hormonal system thatlimits energy intake and expenditure and is intimatelyinvolved in appetite regulation, but it also exerts modula-tory effects on neuronal excitability and suppresses seizures(Harvey, 2007; Obeid et al., 2010). Administration of lep-tin to rats undergoing either focal or generalized seizuresresulted in shorter, less frequent seizures, possibly throughalteration of AMPA receptor-mediated synaptic transmission(Xu et al., 2008). Since the KD causes a rise in leptin levels(Xu et al., 2008), it is possible that the KDs mechanism, atleast in part, may be related to leptin-associated reduction

in synaptic excitability.

Conclusions

It is often stated that no single mechanism is likely to explainthe clinical effects ofthe KD. We concur with this viewpoint,and certainly, the same could be said of virtually all AEDsused in clinical practice. However, such a conclusion shouldnot dissuade us from attempting to elucidate the multiple,likely interacting mechanisms by which the KD constrainsneuronal hyperexcitability and suppresses seizures. In fact,


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the complexity of the KD mechanism of action should beviewed as a guide and friend, enabling the develop-ment ofnovel therapies based on the intersection betweenmetabolism and neuronal excitability. Importantly, eluci-dation of critical control points in the epileptic cellularnetwork ones that could potentiallybe influenced by shiftsin metabolic activity lays the foundation of an excitingnew direction in epilepsy therapeutics. Science has indeedcome a long way toward unraveling KD mechanisms and weanticipate further progress, to the eventual benefit of thethousands ofchildren suffering from intractable epilepsy.

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