Bough2007 Anticonvulsant Mechanisms of the Ketogenic Diet

8/18/2019 Bough2007 Anticonvulsant Mechanisms of the Ketogenic Diet

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Epilepsia, 48(1):43–58, 2007Blackwell Publishing, Inc.C 2007 International League Against Epilepsy

Anticonvulsant Mechanisms of the Ketogenic Diet

∗Kristopher J. Bough and †Jong M. Rho

∗Center for Drug Evaluation & Research, Food and Drug Administration, Rockville, Maryland;

†Barrow Neurological Institute, Phoenix, Arizona, U.S.A.

Summary: The ketogenic diet (KD) is a broadly effective treat-ment for medically refractory epilepsy. Despite nearly a centuryof use, the mechanisms underlying its clinical efficacy remainunknown. In this review, we present one intersecting view of howthe KD may exertits anticonvulsantactivityagainst the backdropof several seemingly disparate mechanistic theories. We summa-rize key insights gleaned from experimental and clinical studiesof the KD, and focus particular attention on the role that ketonebodies, fatty acids, and limited glucose may play in seizure con-trol. Chronic ketosis is anticipated to modify the tricarboxcylicacid cycle to increase GABA synthesis in brain, limit reactiveoxygen species (ROS) generation, and boost energy productionin brain tissue. Among several direct neuro-inhibitory actions,

polyunsaturated fatty acids increased after KD induce the ex-pression of neuronal uncoupling proteins (UCPs), a collectiveup-regulation of numerous energy metabolism genes, and mito-chondrial biogenesis. These effects further limit ROS generationand increase energy production. As a result of limited glucoseand enhanced oxidativephosphorylation,reduced glycolytic fluxis hypothesized to activate metabolic KATP channels and hyper-polarize neurons and/or glia. Although it is unlikely that a singlemechanism, however well substantiated, will explain all of thediet’s clinical benefits, these diverse, coordinated changes seempoised to stabilize synaptic function and increase the resistanceto seizures throughout the brain. Key Words: Ketogenic diet— Epilepsy—Metabolism—Polyunsaturated fatty acids.

The ketogenic diet (KD) is a high-fat, low-protein, low-

carbohydrate diet that has been employed as a treatment

for medically refractory epilepsy for 86 years. The “clas-

sic” KD is based upon consumption of long-chain satu-rated triglycerides (LCTs) in a 3:1–4:1 ketogenic diet ratio

(KD ratio) of fats to carbohydrates + protein (by weight).

The vast majority of calories (>90%) are derived from fat.

While clinical implementation of the KD has varied from

center to center (Kossoff and McGrogan, 2005), diet treat-

ment generally begins with a period of fasting followed by

gradual increase in calories to a target KD ratio of 3:1–4:1.

This is conducted in the inpatient setting over the course

of several days, where blood glucose, urine ketones, and

several other metabolic variables are closely monitored.

The hallmark feature of KD treatment is the production of

ketone bodies by the liver. Ketone bodies provide an al-

ternative substrate to glucose for energy utilization, and in

developing brain, also constitute essential building blocks

for biosynthesis of cell membranes and lipids.

While the clinical effectiveness of the KD is widely

accepted, surprisingly little is understood about its under-

Accepted October 24, 2006.

Address correspondence and reprint requests to Kristopher J. Boughat FDA – Center for Drug Evaluation & Research, MPN 1 – Room1345, 7520 Standish Place, Rockville, MD 20855, U.S.A. E-mail:[email protected]

doi:10.1111/j.1528-1167.2007.00915.x

lying mechanisms of action. Although some studies sug-

gest that dietary constituents or metabolites have direct

anticonvulsant effects, emerging evidence indicates that

adaptations to chronic administration of the KD result inimproved seizure control. These data suggest that the KD

activates several endogenous metabolic and genetic “pro-

grams” to stabilize and/or enhance cellular metabolism,

and that these fundamental changes help counter neuronal

dysfunction associated with seizure activity.

MECHANISTIC INSIGHTS FROM STUDIES

OF KD EFFICACY

The anticonvulsant efficacy of the KD has been ex-

amined in various acute and chronic animal models of

epilepsy over the years (Stafstrom, 1999). Clinical andexperimental studies have provided key insights into im-

portant treatment-related variables and, when considered

together, these studies have helped direct mechanistic re-

search. Commonalities between clinical and experimen-

tal studies of efficacy are summarized in Table 1 (adapted

from Stafstrom, 2004).

Based on vast clinical experience, almost any diet that

produces ketonemia and/or diminished blood glucose lev-

els can induce an anticonvulsant effect. Ketogenic diets

comprised of either LCTs (Freeman et al., 1998; Vin-

ing et al., 1998) or medium-chain triglycerides (MCTs;

43


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44 K. J. BOUGH AND J. M. RHO

TABLE 1. Translational correlations of ketogenic diet (KD) efficacy

Variable Experimental findings Clinical findings References

Age Young rats and mice P40 at dietonset

Infants, children and adolescents


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ANTICONVULSANT ACTIONS OF KETOGENIC DIET 45

are administered in ratios of ≥3:1 (Freeman et al., 2000),

and higher KD ratios increased both clinical (Dekaban,

1966; Livingston, 1972) and experimental anticonvulsant

efficacy (Bough et al., 2000b). Similar to the KD ratio,

increasing the extent of CR resulted in improved seizure

control in epileptic mice (Greene et al., 2001), irrespec-

tive of the type of diet that was restricted (Eagles et al.,2003). In general, extra calories in the form of carbohy-

drates or proteins translate to additional metabolic sub-

strates for gluconeogenesis and diminished KD efficacy.

Breakthrough seizures are believed to result from overes-

timation and administration of excess calories (Freeman

et al., 2000). As such, CR may share common anticonvul-

sant mechanisms and adjunctively optimize KD efficacy.

In rodents, maximal seizure control develops 1 – 2 weeks

after initiation of a KD (Appleton and DeVivo, 1974; Rho

et al., 1999; Bough et al., 2006). Similarly in humans,

clinical efficacy does not reach its zenith in many patients

until after 2 weeks (Dekaban, 1966; Freeman et al., 2000).

One notable feature of the KD is the rapid occurrence of

breakthrough seizures and loss of ketosis when carbohy-

drates are introduced (e.g., after a child sneaks a cookie;

Huttenlocher, 1976). As a result, the KD must be strictly

enforced in order for efficacy to be maintained. However,a

breakthrough seizure may not necessarily translate to a to-

tal loss of seizure control. Studies have shown that, despite

an abrupt discontinuation of the KD, the increased resis-

tance to seizures waned gradually when switched back to

control (Bough et al., 2006) or even high-carbohydrate,

antiketogenic chow (Appleton and DeVivo, 1974). This

decline in seizure threshold generally occurred over 1 – 2

weeks, mirroring the onset of seizure protection (Apple-ton and DeVivo, 1974; Bough et al., 2006). This indicates

that a critical, minimal level of sustained ketosis is neces-

sary but not sufficient to maintain seizure control. Thus,

it would seem that metabolic adaptations to KDs underlie

their key anticonvulsant actions.

Many studies and anecdotal observations have sug-

gested that the KD is most effective in immature animals

or infants and children (Livingston, 1972; Uhlemann and

Neims, 1972; Otani et al., 1984; Bough et al., 1999b; Rho

et al., 1999). This is perhaps dueto enhanced metabolicca-

pacity, more efficient extraction of ketone bodies from the

blood (Morris, 2005), and/or greater compliance of KDsin the pediatric population. However, a lack of efficacy

in older children or adults may simply reflect noncompli-

ance or dietary intolerance rather than an inadequate re-

sponse physiological (Livingston, 1972). The KD has been

demonstrated to be similarly effective in infants (Nordli

et al., 2001; Kossoff et al., 2002), adolescents (Kinsman

et al., 1992; Mady et al., 2003), and adults (Sirven et al.,

1999; Coppola et al., 2002). Furthermore, experimental

KDs are effective in both young (i.e.,


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glucose restriction might each lead directly or indirectly

to seizure control. While it is possible that any one of

these KD-induced changes is responsible for the anticon-

vulsant action of the KD, available evidence suggests that

improved seizure control, at a minimum, likely requires

all three.

Role of ketone bodies

Beta-hydroxybutyrate (BHB)is the predominant ketone

body measured in the blood, and as such, has been used as

a clinical measure of KD implementation (Fig. 1). Accord-

ingly, nearly all KD studies have attempted to establish a

causative link between ketonemia and anticonvulsant ef-

ficacy. Although robust elevations in plasma BHB levels

have been observed during KD treatment (Bough et al.,

1999b; Thavendiranathan et al., 2000), there is no signif-

icant correlation between plasma BHB levels and seizure

protection. Optimal seizure protection generally lags days

to weeks behind the development of ketonemia, which oc-

curs within hours of KD onset.

Nevertheless, there is some evidence that ketones other

than BHB may possess anticonvulsant properties. When

injected into animals, acetone and its parent acetoacetate

(ACA), prevent acutely provoked seizures. Seminal work

in the 1930s revealed that acute intraperitoneal admin-

istration of acetone or ethyl-acetoacetate protected rab-

bits from thujone-induced seizures (Helmholz and Keith,

1930; Keith, 1933). Thujone is the active constituent of

wormword oil, and is an antagonist of GABAA receptors

(Hold et al., 2000). More recent experimental studies have

shown similar results in rodents. Acetone (Likhodii et al.,

2003) and ACA (Rho et al., 2002) — but not BHB — wereanticonvulsant in a variety of acute and chronic models of

epilepsy, consistent with earlier observations (Helmholz

FIG. 1. Metabolic pathwayshighlighting the production of ketone bodies fatty acids during fasting or treatment with the ketogenic diet (KD).Estimated fasting- or KD-induced concentrations of beta-hydroxybutyrate, acetoacetate, and acetone in blood are listed (large boxes).Measures of beta-hydroxybutyrate levels in blood are most commonly used as the clinical indicator of successful KD treatment. FromLikhodii and Burnham (2004).

and Keith, 1930; Yamashita et al., 1976; Vodickova et al.,

1995). Clinically, acetone levels of up to 1 millimolar

(mM) were detected in the brains of five of seven well-

controlled epileptic patients following KD using mag-

netic resonance spectroscopic techniques (Seymour et al.,

1999).Althoughacetone could notbe detected in twoother

seizure-free patients, the authors concluded that acetonecontributes to the anticonvulsant effect of the KD. Inter-

estingly, the concept that a lipophilic solvent may potently

block seizure activity is not new. The classic example of

this is valproic acid, which was initially used as a solvent

to dissolve investigational anticonvulsant compounds, but

was serendipitously discovered to possess intrinsic anti-

convulsant properties.

Whereas in vivo pharmacodynamic studies have

suggested that both ACA and acetone may act as

anticonvulsant agents, there is no evidence that ketone

bodies can directly modulate synaptic transmission and/or

neuronal excitability. In vitro cellular electrophysiologi-

cal experiments have failed to demonstrate an effect on the

principal ion channels that mediate neuronal excitability

and inhibition. Specifically, neither L-BHB nor ACA were

found to modulate GABAA receptors, AMPA receptors,

or NMDA receptors in both hippocampus and neocortex

of rats (Thio et al., 2000; Donevan et al., 2003). Despite

these negative observations, it remained possible that ke-

tone bodies might affect network activity or synchrony.

However, in field potential recordings conducted in vitro,

Thio et al. (2000) demonstrated clearly that neither ACA

nor BHB modified evoked excitatory postsynaptic poten-

tials (EPSPs) or population spikes in the CA1 subfield of

the hippocampal tissue. In summary, there is no evidencefor direct anticonvulsant effects for either ACA or BHB,

and acetone has yet to be studied in neuronal (CNS) tissue.

Epilepsia, Vol. 48, No. 1, 2007


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This may, in large part, reflect the technical difficulties in

investigating a compound that is highly volatile and can

react with perfusion systems ordinarily used in pharma-

cological in vitro experiments.

Recently, it has been suggested that ACA and/or its

metabolic byproduct, acetone, may activate a novel class

of potassium leak channels known as the two-pore do-main or K2P channels (Vamecq et al., 2005). K2P channels

represent a diverse superfamily of channels that generally

hyperpolarize cell membranes,and regulate membrane ex-

citability both pre- and postsynaptically (Lesage, 2003).

These channels can be modulated by changes in pH, os-

molality, temperature, mechanical pressure, and certain

fatty acids (Franksand Honore, 2004).Links between KD-

induced elevations in ketone bodies (and/or fatty acids, as

discussed below) and K2P channels, however, have yet to

be explored.

In conclusion, although ketone bodies have been shown

to possess anticonvulsant properties in vivo, there is no ev-

idence to date that they mediate directly these effects. It

is clear that some degree of sustained ketosis is required

for clinical efficacy and that efficacy is maximized over a

period of weeks versus days, despite a rapid onset of keto-

sis within hours. Whereas it is plausible that some dietary,

pharmacokinetic factor(s) results in some level of seizure

protection, the approximate 2-week time course for opti-

mal seizure protection suggests a pharmacodynamiceffect

of the KD (e.g., parallel time course for changes in gene

expression, mitochondrial proliferation, up-regulation of

UCPs/transporters, etc) likely underlies the anticonvulsant

nature of the diet. Thus, available data suggest that adap-

tations to, rather than a direct effect of, ketosis underliethe anticonvulsant nature of the KD.

Role of glucose restriction

Whereas most studies have suggested that persistent

ketosis is essential to KD-induced seizure protection, oth-

ers have posited that glucose restriction is the key fea-

ture (Greene et al., 2003). In addition to ketosis, it is

clear that as ketonemia develops, another immediate con-

sequence of CR and/or KDs is a ‘moderate’ reduction

in blood glucose. Does caloric restriction simply act to

limit gluconeogenic substrates that would otherwise re-duce KD ratio and counter efficacy? Or, might glucose re-

striction result in another metabolic adaptation that helps

quell aberrant hyperexcitability? Calorie restriction alone

was sufficient to retard seizure susceptibility in juvenile

and adult epileptic EL mice; and, blood glucose lev-

els were inversely correlated with a decreased risk of

seizures (Greene et al., 2001). Greeneet al.(2003) hypoth-

esized that CR reduces energy production through glycol-

ysis, which limits a neuron’s ability to reach (and main-

tain) high levels of synaptic activity necessary for seizure

genesis.

Others have hypothesized that glucose restriction dur-

ing KD treatment activates ATP-sensitive potassium

(KATP) channels (Schwartzkroin, 1999; Vamecq et al.,

2005). Interestingly, KATP channels are ligand-gated re-

ceptors broadly expressed throughout the central nervous

system, in both neurons and glia (Thomzig et al., 2005).

These channels act as metabolic sensors, linking cellular membrane excitability to fluctuating levels of ADP and

ATP. Activation of this receptor by reduced ATP/ADP ra-

tios opens the channel and leads to membrane hyperpo-

larization. When glucose is limited (e.g., during adminis-

tration of a classic KD, which is typically CR by 25%),

KATP channels might open to hyperpolarize the cell as the

intracellular ATP concentrations fall. Conversely, when

glucose is present andATP concentrations rise, KATP chan-

nels close. As such, KATP channels may provide a mea-

sure of protection against a variety of metabolic stressors

such as hypoxia, ischemia, and hypoglycemia, and are

believed to regulate seizure threshold (Seino and Miki,

2003).

KATP channels are particularly abundant in the substan-

tia nigra (Hicks et al., 1994), a region of the brain thought

to act centrally in the propagation of seizure activity

(Iadarola and Gale, 1982). KATP channels would therefore

be ideally positioned to metabolically regulate the onset

of several different seizure types, as does the KD. There is

growing evidence that KATP channels may critically reg-

ulate seizure activity. Genetically engineered mice that

overexpress the sulfonylurea (SUR) subunit of KATP chan-

nels were significantly more resistant to seizures induced

by kainate, and showed no marked cell loss in hippocam-

pus (Hernandez-Sanchez et al., 2001). Studies of KATPchannel ( Kir6.2−/−) knockout mice suggested that these

channels help determine seizure threshold (Yamada et al.,

2001). Following hypoxic challenge (∼5% O2), knock-

out mice exhibited myoclonic – tonic seizure activity, and,

ultimately, death compared to controls who all recovered

without sequelae.

Despite these observations, there is one important

caveat in implicating KATP channels as mediators of a KD-

induced anticonvulsant effect. Other studies have demon-

strated an increase in energy reserves (specifically, ATP)

after KD treatment (DeVivo et al., 1978; Pan et al., 1999).

These data predict that KATP channels would remainclosed, not open, during diet treatment, and would thus

contribute to neuronal/glial cell membrane depolarization.

Nevertheless, several findings are consistent with the no-

tion that KATP channels are selectively activated during

administration of a low-glucose, high-fat KD. First, K ATPchannels are regulated preferentially via glycolytic energy

sources (Dubinskyet al., 1998). It hasrecentlybeen shown

that the glycolytic enzyme glyceraldehyde 3-phosphate

dehydrogenase (GAPDH) serves as an accessory pro-

tein to KATP channels and regulates directly their activity

(Dhar-Chowdhury et al., 2005; Jovanovic et al., 2005).



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The observed reduction in glycolytic processes af-

ter KD treatment (specifically, the concentration of

fructose-1,6-bisphosphate, the key regulatory enzyme

of glycolysis) is consistent with this notion (DeVivo

et al., 1978; Puchowicz et al., 2005; Melo et al., 2006).

Glycolytic flux may be further limited as a consequence

of elevated ATP (DeVivo et al., 1978; Otani et al., 1984;Pan et al., 1999; Bough et al., 2006) and citrate (Yudkoff

et al., 2001) levels on KD treatment; both ATP and citrate

are feedback inhibitors of glycolysis.

Second, it is hypothesized that the accumulation of free

fatty acids over thecourse of KD administration (Dekaban,

1966) may boost KATP channel activation (Vamecq et al.,

2005). Whereas PUFAs freely cross the BBB, saturated

free fatty acids are transported across the BBB via carrier-

mediated processes (Avellini et al., 1994). Fatty acids

that intercalate within neuronal cell membranes have been

shown to interact potently with KATP channels, specifically

reducing their affinity for (and inhibition by) ATP (Shyng

and Nichols, 1998). Overall, these findings suggest that

the unique nature of low-glucose, high-fat KDs promotes

KATP channel activation, despite observed enhancements

in oxidative energy production.

Recent experiments involving 2-deoxyglucose (2-DG)

provide further support for a glucose-restriction hypothe-

sis of KD action. Two-deoxyglucoseis a glucose analogue,

which inhibits phosphoglucose isomerase and, hence, gly-

colysis. Stafstrom et al. (2005) reported that the addi-

tion of 1 mM 2-DG decreased epileptiform burst fre-

quency to 25 – 80% of baseline in rat hippocampal slices

exposed to elevated extracellular potassium. More signifi-

cantly, the same group also showedthat 2-DG (250 mg/kg,i.p) elevated the after-discharge threshold in olfactory

bulb of perforant-path kindled rats, markedly reduced the

progression of kindling, and limited the expression of

BDNF and its cognate receptor, trkB (Garriga-Canut et al.,

2006).

Interestingly, there are a number of anticonvulsant par-

allels between 2-DG (Stafstrom et al., 2005; Garriga-

Canut et al., 2006) and KD treatment (Bough et al., 2003).

First, both 2-DG and KD elevated electrographic seizure

threshold in vivo; second, both 2-DG and KD potently re-

tarded the progression of epileptogenesisin kindling mod-

els of epilepsy in vivo; and, third, both 2-DG (in vitro)and KD (in vivo) diminished measures of hippocampal

hyperexcitability. These results collectively suggest that

the anticonvulsant actions of KD may work, in large part,

via an inhibition of glycolysis. Importantly, because 2-DG

is fairly well tolerated when administered orally (Pelicano

et al., 2006), this compound may represent a novel treat-

ment strategy for epilepsy.

Role of fatty acids

Polyunsaturated fatty acids (PUFAs) such as docosa-

hexanoic acid (DHA, C22:6ω3), arachidonic acid (AA,

C20:4ω6), or eicosapentanoic acid (EPA, C20:5ω3) are

believed to affect profoundly cardiovascular function and

health (Leaf and Kang, 1996; Nordoy, 1999; Leaf et al.,

2003).In cardiac myocytes, PUFAs inhibited fast, voltage-

gated sodium channels (Xiao et al., 1998) and L-type cal-

cium channels (Xiao et al., 1997). Similar findings have

been observed in neuronal tissue. For example, DHA andEPA diminished neuronal excitability and bursting in hip-

pocampus (Xiao and Li, 1999).

It is not surprising then that PUFAs are becoming an in-

creasingly popular focus of KD research. After KD treat-

ment, specific PUFAs (i.e., AA and DHA) were found to

be elevated in both serum (Cunnane et al., 2002; Fraser

et al., 2003) and brain (Taha et al., 2005) of patients and

animals. Importantly, one report documented that the rise

(or drop) in total fatty acids during KD treatment closely

paralleled clinical improvement (or loss)of seizure control

(Dekaban, 1966). An additional study found that dietary

supplementation with 5 g of (65%) n-3 PUFAs once per

day produced a marked reduction in seizure frequency

and intensity in a few epileptic patients (Schlanger et al.,

2002). These findings suggest that KD-induced elevations

in PUFAs such as DHA and/or AA might act directly to

limit neuronal excitability and dampen seizure activity.

PUFAs could ultimately block seizure activity in a num-

ber of ways (Fig. 2). First, PUFAs may inhibit directly ion

channel activity. Omega-3 (ω-3) PUFAs have been shown

to: (1) inhibit both voltage-gated Na+ and Ca2+ channels,

(2) increase the resistance to bursting induced by bicu-

culline, zero Mg2+, pentylenetetrazole or glutamate, and

(3) prolong the recovery time from inactivation in hip-

pocampal neurons (Vreugdenhil et al., 1996; Xiao and Li,1999; Young et al., 2000). Second, in conjunction with ke-

tone bodies, PUFAs may activate a lipid-sensitive class of

K2P potassium channels (Vamecq et al., 2005). And, third,

PUFAs may enhance the activity of the Na+ /K+-ATPase

(sodium pump). Elevated ω-3 and diminished ω-6 PU-

FAs levels in plasma membranes significantly increased

sodium pump function (Wu et al., 2004). These findings

indicate that elevations in brain levels of PUFAs after KD

treatment (Taha et al., 2005) could help reduce neuronal

hyperexcitability via a variety of direct mechanisms.

Uncoupling proteins

In addition to their direct actions on neuronal ex-

citability, PUFAs may also act indirectly to limit ex-

citotoxicity and neurodegeneration. PUFAs regulate the

expression of numerous genes in brain via transcription

factors such as PPARα (peroxisome proliferator-activated

receptor-α; Sampath and Ntambi, 2004). Through in-

duction of PPARα and its coactivator PGC-1, PUFAs

induce the expression of mitochondrial uncoupling pro-

teins (UCPs) and activate these proteins directly as well

(Jaburek et al., 1999; Diano et al., 2003). Recent evidence



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FIG. 2. Potential pathways through which polyunsaturated fatty acids (PUFAs) may limit hyperexcitability in the brain. Acting directly,PUFAs such as arachidonic acid (AA), docosahexanoic acid (DHA), and/or eicosapentanoic acid (EPA) might inhibit both voltage-gatedNa+ and Ca2+ channels, activate a lipid-sensitive class of K2P potassium channels, and enhance the activity of the Na

+ /K+-ATPase tolimit neuronal excitability and dampen seizure activity. Acting indirectly, PUFAs might induce the expression and activity of uncouplingproteins (UCPs) to diminish reactive oxygen species (ROS), reduce neuronal dysfunction and induce a neuroprotective effect. Finally,PUFAs are expected to activate PPARα and induce a coordinate up-regulation of energy transcripts leading to enhanced energy reserves,stabilized synaptic function and limited hyperexcitability.

suggests that PUFAs are required for mitochondrial UCP

activity (Garlid et al., 2001).

Uncoupling proteins are homodimers that span the in-

ner mitochondrial membrane and allow a proton leak fromthe intermembrane space to the mitochondrial matrix.

There are three major isoforms that have been identified

in the brain, UCP2, UCP4 and UCP5 (a.k.a., BMCP-1

or brain mitochondrial carrier protein-1). UCP proteins

are increasingly implicated in the regulation of neuronal

excitability and survival (Andrews et al., 2005). The un-

coupling effect, albeit of small magnitude, reduces the

proton-motive force, disassociates or ‘uncouples’ elec-

tron transport from ATP production, and indirectly de-

creases the production of reactive oxygen species (ROS).

Although it would seem that increased levels of UCP pro-

teins would diminish cellular energy production, Diano

et al. (2003) showed that chronic overexpression of UCP2

in neuronal tissue increased cellular ATP and ADP levels

by triggering mitochondrial biogenesis. KD appears to do

the same; that is, studies show that the KD induces UCP

expression, stimulates mitochondrial biogenesis, and en-

hances energy production (see also below). Seizures, by

comparison, increase ROS generation and/or mitochon-

drial dysfunction, which can lead to neuronal dysfunction

and excitotoxicity (Layton and Pazdernik, 1999; Kovacs

et al., 2001; Kovacs et al., 2002; Sullivan et al., 2003).

Interestingly, UCP2 is up-regulated after seizures (Diano

et al., 2003). The protective role of UCPs was recently

highlighted by Sullivan et al. (2003) who demonstrated

that dietary enhancement of UCP expression and function

in immature rats protected against kainate-induced exci-totoxicity, most likely by decreasing ROS generation (An-

drews et al., 2005). Further work demonstrated that mice

maintained on a high-fat KD demonstrated an increase in

the hippocampal expression and activity of all three mi-

tochondrial UCPs and exhibited a significant reduction in

ROS generation in mitochondria isolated from the same

brain region (Sullivan et al., 2004). In conjunction with

reports that ketone bodies potently decrease ROS gen-

eration (Veech et al., 2001; Veech, 2004), these reports

suggest that the KD compensates for seizure-induced el-

evations in ROS generation and neuronal dysfunction to

provide a neuroprotective effect.

Energy production

Polyunsaturated fatty acids additionally regulate the

transcription of numerous genes linked to energy

metabolism (Sampath and Ntambi, 2005) through activa-

tion of PPARα, a scenario inwhich the KDis thought to re-

program cellular metabolism (Cullingford, 2004). Indeed,

numerous studies have described changes consistent with

an enhancement in energy production following KD treat-

ment. First, microarray expression studies demonstrated

that KD induces a coordinated up-regulation of several



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dozenmetabolic genesassociated with oxidative phospho-

rylation after KD (Noh et al., 2004; Bough et al., 2006).

Second, KD treatment stimulated mitochondrial biogene-

sis, resulting in a striking 46% increase in the number of

mitochondria in the hilar/dentate gyrus region of rat hip-

pocampus (Bough et al., 2006). And, third, levels of en-

ergy metabolites were increased after KD. Brain glycogenand ATP concentrations were boosted throughout rodent

brain (DeVivo et al., 1978; Otani et al., 1984) andtherewas

an elevation in the phosphocreatine-to-creatine (PCr:Cr)

energy-reserve ratio in both animals (Bough et al., 2006)

and humans (Pan et al., 1999). These findings are consis-

tent with results that show ketones (4 mM BHB + 1 mM

ACA) increased hydraulic work by 14% and improved

energy status in perfused heart tissue (Sato et al., 1995).

Further, there is an overall increased metabolic efficiency

(DeVivo et al., 1978; Bough et al., 2006), decreased res-

piratory quotient (Bough et al., 2000b), and maximal mi-

tochondrial respiratory rate in rodents following the KD

(Sullivan et al., 2004). Collectively, these data provide

compelling evidence that the KD enhances oxidative en-

ergy production by activating a variety of transcriptional,

translational, and biochemical mechanisms in a concerted

fashion.

Metabolic dysfunction has been identified in regions of

hyperexcitability within the brain and is associated with

several epileptic conditions. Impairment of mitochondrial

function has been observed in the seizure foci of both hu-

man and experimental epilepsies (Kunz et al., 2000). Se-

vere metabolic dysfunction occurred in both human and

rat hippocampal tissue during periods of heightened neu-

ronal activity (Kann et al., 2005). Kudin et al. (2002)demonstrated that seizure activity down-regulated mito-

chondrial enzymes involved in oxidative phosphorylation.

In an earlier study, the same group demonstrated a specific

deficiency in complex I activity and mitochondrial ultra-

structural abnormalities within the hippocampal CA3 re-

gion of epileptic tissue resected from 57 human patients

(Kunz et al., 2000). In view of previous studies demon-

strating impaired oxidative phosphorylation capacity in

pilocarpine-treated rats (Kudin et al., 2002) and in patients

with epilepsy (Antozzi et al., 1995; Kunz et al., 2000), a

KD-induced augmentation in oxidative phosphorylation

and energy reserves seems likely to counter energetic de-ficiencies in epileptic tissue, making neuronal tissue more

resilient to aberrant neuronal activity and, in this way, con-

tributing to the diet’s anticonvulsant actions.

Stabilized synaptic function

Intriguing as this argument may be, how exactly would

enhanced energy reserves lead to stabilized synaptic func-

tion and diminished seizures? One possibility is via the

sodium pump. ATP is primarily used to maintain ionic gra-

dients, especially through actions of the transmembrane

sodium pump (Hulbert and Else, 2000). Schwartzkroin

originally hypothesized that KD-induced elevations in

ATP concentrations might enhance and/or prolong the ac-

tivation of theNa+ /K+-ATPase , perhaps via an increase in

the delta-G

of ATP hydrolysis (Veech et al., 2001; Veech,

2004). In neurons, increased sodium pump activity might

hyperpolarize the cell and/or reduce the resting mem-

brane potential to diminish firing probability. EnhancedNa+ /K+-ATPase function in neurons might also preserve

normal neuronal functioning and/or delay a pathological

buildup of high external K+ (Xiong and Stringer, 2000).

In glia, increased activation of the Na+ /K+-ATPase might

slow glial depolarization and allow for prolonged uptake

of extracellular K+ during periods of intense neuronal ac-

tivity (e.g., high-frequency bursting). Increases in neu-

ronal and/or glial action of the sodium pump would be

expected to limit hyperexcitability and increase the resis-

tance to seizures, as is noted after treatment with KD.

Although no studies have tested this sodium-pump

hypothesis directly, a recent report suggests that KD

tissue is more resistant to metabolic stress. When chal-

lenged with mild hypoglycemia, synaptic transmission

within the dentate gyrus was maintained for approxi-

mately 60% longer in tissue from KD-fed animals com-

pared to controls (Bough et al., 2006). These data suggest

that the KD stabilizes synaptic transmission (both excita-

tory and inhibitory) for prolonged periods of time during

metabolic stress such as during seizure activity. Hence,

it seems likely that the KD induces seizure protection

in part by preventing neuronal dysfunction (diminution

of ROS/enhancement of energy reserves) and stabilizing

synaptic transmission (enhancement in energy reserves).

A role for neurotransmitter systems

The noradrenergic hypothesis

One of the more intriguing observations regarding KD

action involves norepinephrine, its receptors and signaling

cascades. In general, increases in noradrenergic tone re-

sult in an anticonvulsant effect. Several lines of evidence

support this view. Norepinephrine (NE) re-uptake in-

hibitors can prevent seizure activity in genetically epilepsy

prone rats (GEPRs; Yan et al., 1993) and pharmacolog-

ical NE agonists are generally, though not always, an-

ticonvulsant (Weinshenker and Szot, 2002); damage tothe locus coeruleus — the principal region of the brain

from which ascending and descending noradrenergic in-

nervation originates — converts occasional seizures into

self-sustaining status epilepticus (SSSE) in rats (Giorgi

et al., 2004); animals are more prone to seizures when NE

is chemically depleted with reserpine (Weinshenker and

Szot, 2002); and, interestingly, there are several reports of

diminished brain levels of NE in several animal models

of epilepsy, including GEPRs, kindled animals, EL mice,

seizure-sensitive Mongolian gerbils, and tottering mice

(Weinshenker and Szot, 2002).



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Of significant interest is the observation that mice lack-

ing the ability to produce NE ( Dbh−/− knockout mice)

do not exhibit an increased resistance to flurothyl seizures

when treated with a KD (Szot et al., 2001). These data

indicate that NE is required for the anticonvulsant effect

of KD, at least in the flurothyl seizure threshold model.

Weinshenker and Szot (2002) additionally reported an ap-proximate twofold increase in NE levels in hippocampus

following a KD, suggesting that KD increases basal re-

lease of NE. These studies indicate the anticonvulsant ac-

tion of KD may result in part from an enhancement in

noradrenergic signaling in the brain.

If the KD enhances NE release as described above, it

may also promote the corelease of anticonvulsant orexi-

genicpeptides such as neuropeptide-Y (NPY)and galanin.

NPY has been shown to inhibit glutamatergic synaptic

transmission and epileptogenesis in vitro (Rhim et al.,

1997; Richichi et al., 2004; Vezzani and Sperk, 2004);

galanin has been shown to limit SSSE (Saar et al., 2002)

and diminish both excitatory synaptic transmission and

ictal activity in vitro (Schlifke et al., 2006). Both neu-

ropeptides are elevated after calorie restriction. However,

there was no evidence for enhanced transcription of either

of these peptides in the brain after KD treatment, suggest-

ing that neither NPY nor galanin contribute significantly

to the anticonvulsant actions of KD (Tabb et al., 2004).

The GABAergic hypothesis

One of the more popular hypotheses for KD action in-

volves γ -aminobutyric acid (GABA), the major inhibitory

neurotransmitter in the mammalian brain. In general, the

KD is most effective against seizures evoked by GABAer-gic antagonists. The KD potently inhibits seizures in-

FIG. 3. Metabolic modifications of glutamate and GABA synthesis as a consequence of diminished glucose and ketosis. In ketosis, beta-hydroxybutyrate and acetoacetate contribute heavily to brain energy needs. A variable fraction of pyruvate (1) is ordinarily converted toacetyl-CoA via pyruvate dehydrogenase. In contrast, all ketone bodies generate acetyl-CoA, which enters the tricarboxcylic acid (TCA)cycle via the citrate synthetase pathway (2). This involves the consumption of oxaloacetate, which is necessary for the transamination ofglutamate to aspartate. Oxaloacetate is then less available as a reactant of the aspartate aminotransferase pathway, which couples theglutamate-aspartate interchange via transamination to the metabolism of glucose through the TCA cycle. Less glutamate is convertedto aspartate and thus, more glutamate is available for synthesis of GABA (3) through glutamic acid decarboxylase (GAD). Adapted fromYudkoff et al. (2004).

duced by pentylenetetrazole, bicuculline, picrotoxin, and

γ -butyrolactone. In contrast, the diet demonstrates lit-

tle if any efficacy in acute seizure models involving ac-

tivation of ionotropic glutamate receptors (e.g., kainic

acid), voltage-dependent sodium channels (e.g., maximal

electroshock [MES]), or glycine receptor inhibition (e.g.,

strychnine; Bough et al., 2002).Yudkoff et al. (2005) have proposed that ketosis in-

duces major shifts in brain amino acid handling favoring

the production of GABA. This results from a reduction

of aspartate relative to glutamate, the precursor to GABA

synthesis, and a shift in the equilibrium of the aspartate

aminotransferase reaction in the ketotic state. As a re-

sult, there is an increase in glutamic acid decarboxylase

(GAD) activity and GABA production (Fig. 3). Elevated

GABA levels would,in turn, be expectedto dampenhyper-

excitability throughout the brain. Several studies support

this possibility. First, KD and CR diet treatments both

increased GAD transcript and protein levels in inferior

and superior colliculi, cerebellar and temporal cortex, and

striatum (the latter, KD only; Cheng et al., 2004). Sec-

ond, both BHB and ACA increased the rate and extent

of GABA formation in synaptosomes (Erecinska et al.,

1996; Yudkoff et al., 1997). And, finally, KD treatment

in vivo modified amino acid levels in a manner consistent

with enhanced GABA production (Yudkoff et al., 2001;

Melo et al., 2006). Although brain levels of glutamate

and GABA have not been consistently elevated in rodents

(DeVivo et al., 1978; Al-Mudallal et al., 1996; Yudkoff

et al., 2001; Bough et al., 2006), two recent clinical stud-

ies report significant increases in GABA levels following

KD treatment (Wang et al., 2003; Dahlin et al., 2005),further substantiating this view.



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In addition to biochemical measures of KD-enhanced

GABAergic inhibition, there is functional evidence as

well. Electrophysiological recordings conducted in vivo

demonstrated that network excitability was diminished in

both KD- and calorie-restricted rats (Bough et al., 2003);

greater stimulus intensities were required to evoke popu-

lation spikesin both CR-and KD-fed animals compared toad libitum controls. Paired-pulse inhibition was increased.

Both CR and KD dietary treatments resulted in greater

paired-pulse inhibition compared to controls at the 30-

ms interpulse interval (Bough et al., 2003), a result con-

sistent with an enhancement in fast, GABAA-mediated

inhibition. Additionally, KD-fed animals exhibited an el-

evated electrographic seizure threshold and an increased

resistance to a modified, 1-day kindling protocol (max-

imal dentate activation). These data suggested that both

KD and calorie-restricted diets limited network excitabil-

ity and elevated seizure threshold via an enhancement of

GABAergic inhibition.

GABAergic interneurons, which at baseline have more

depolarized resting membrane potentials, endure non-

accommodating bursts of neuronal firing and must

metabolically persist (Attwell and Laughlin, 2001), lest

network inhibition becomes compromised. Previous stud-

ies have shown that a KD increases total brain [ATP]

(DeVivo et al., 1978) and PCr/Cr or PCr/ATP energy re-

serve (Pan et al., 1999; Bough et al., 2006). Accordingly,

KD-induced elevations in PCr are likely to play a pivotal

role in maintaining the activity of the Na+ /K+-ATPase

during periods of intense seizure activity, in both gluta-

matergic and GABAergic neurons. In a recent study of

human temporal lobe epilepsy (Williamson et al., 2005),the PCr/ATP ratio correlated with the recovery of the

membrane potential following a stimulus train, which

was inversely correlated with granule cell bursting. Be-

cause creatine kinase is predominantly localized within

GABAergic interneurons (Boero et al., 2003), Boero et al.

concluded that PCr and energy levels are especially crit-

ical to the maintenance of GABAergic inhibitory output.

In this manner, a KD-induced increase in energy reserves

might enhance GABAergic function in particular and im-

prove seizure control.

HOW CAN THE KETOGENICDIET BE OPTIMIZED?

Historically, few guidelines have emerged regarding the

clinical implementation of the KD and its variants – in-

cluding the medium-chain triglyceride (MCT) formula-

tion (Huttenlocher et al., 1971) and more recent options

such as the Atkins diet (Kossoff et al., 2003; Kossoff

et al., 2006). This is largely a result of the fact that, until re-

cently, few KD centers existed throughout the world. Even

with a resurgence of interest in dietary approaches toward

epilepsy treatment in the past decade, there remains a no-

table absence of Class I and II clinical studies. Today, few

question the clinical efficacy of the KD in both young and

older patients (Vining, 1999; Coppola et al., 2002; Mady

et al., 2003), and many successful international centers

have evolved (Kossoff and McGrogan, 2005; Freeman

et al., 2006). However, since we do not fundamentally

know how the KD prevents seizures, there exists as yet norational basis for optimizing the efficacy of the diet, other

than through trial and error.

When examining the accumulated clinical data, it ap-

pears seizure control can be achieved in the majority

of epileptic patients as long as there is a shift from

glycolytic flux to intermediary metabolism (resulting in

measurable ketosis), irrespective of the precise dietary

formulation (Henderson et al., 2006). On the other hand,

the experimental literature suggests that different treat-

ment protocols may result in differential efficacy or even

lack of efficacy, despite significant ketosis (Bough et al.,

2000a; Thavendiranathan et al., 2000; Bough et al., 2002;

Thavendiranathan et al., 2003). Most of the published

studies have been based on acute seizure models, and not

on developmental epilepsy models. Hence, of course, one

must bear in mind that, despite dozens of animal models

of the KD, none recapitulate all of the essential features

in the human epileptic condition (Stafstrom, 1999).

So how can we reach the goal of developing a safer and

more effective KD? The reductionist approach posits that

were we to identify the critical mediator of the diet’s an-

ticonvulsant effect, administration of this substrate alone

would likely yield a similar clinical effect as the tradi-

tional KD, and importantly, spare the patient significant

side-effects that may preclude its use — even in the face of clear clinical efficacy. The closest we have come to this

situation is the recent use of BHB as an oral neuroprotec-

tant. Promising results have already been demonstrated

in Phase I clinical trials (Smith et al., 2005). Neverthe-

less, despite increasing experimental evidence that BHB

and ACA both possess neuroprotective properties (Kashi-

waya et al., 2000; Noh et al., 2006), a direct anticonvulsant

effect of ketone bodies has not yet been demonstrated in

epileptic brain, either animal or human. Intriguing, how-

ever, are animal studies indicating that ACA and acetone

are anticonvulsant in acute seizure models. Yet, there re-

mains a perplexing lack of an acute anticonvulsant effectof the principal ketone body, BHB.

Conversely, if we believe that certain PUFAs, in lieu of

ketone bodies, are direct mediators of an anticonvulsant ef-

fect (Cunnane et al., 2002; Cunnane, 2004), as suggested

by clinical studies (Schlanger et al., 2002; Fraser et al.,

2003; Fuehrlein et al., 2004; Yuen et al., 2005), we may be

closer to distilling the essence of the KD. However, there

is likely no single fatty acid that is necessary and sufficient

for an anticonvulsant effect. And experimentally, while it

has been straightforward to demonstrate the inhibitory ef-

fects of PUFAs on specific voltage-gated ion channels and



11/16


FIG. 4. Hypothetical pathways leading to the anticonvulsant effects of the ketogenic diet (KD). Elevated free fatty acids (FFA) lead tochronic ketosis and increased concentrations of polyunsaturated fatty acids (PUFAs) in the brain. Chronic ketosis is predicted to leadto increased levels of acetone; this might activate K2P channels to hyperpolarize neurons and limit neuronal excitability. Chronic ketosisis also anticipated to modify the tricarboxcylic acid (TCA) cycle. This would increase glutamate and, subsequently, GABA synthesis inbrain. Among several direct inhibitory actions (see also Fig. 2), PUFAs boost the activity of brain-speci fic uncoupling proteins (UCPs).This is expected to limit ROS generation, neuronal dysfunction, and resultant neurodegeneration. Acting via the nuclear transcriptionfactor peroxisome proliferator-activated receptor-α (PPARα), PUFAs would induce the expression of UCPs and coordinately up-regulateseveral dozen genes related to oxidative energy metabolism. PPARα expression is inversely correlated with IL-1β cytokine expression;given the role of IL-1β in hyperexcitability and seizure generation (Vezzani et al., 2000), diminished expression of IL-β cytokines during KDtreatment could lead to improved seizure control. Ultimately, PUFAs would stimulate mitochondrial biogenesis. Mitochondrial biogenesis ispredicted to increase ATP production capacity and enhance energy reserves, leading to stabilized synaptic function and improved seizurecontrol. In particular, an elevated phosphocreatine:creatine (PCr:Cr) energy-reserve ratio is predicted to enhance GABAergic output,

perhaps in conjunction with the ketosis-induced elevated GABA production, leading to diminished hyperexcitability. Reduced glucosecoupled with elevated free fatty acids are proposed to reduce glycolytic flux during KD, which would further be feedback inhibited by highconcentrations of citrate and ATP produced during KD treatment. This would activate metabolic KATP channels. Opening of KATP channelswould hyperpolarize neurons and diminish neuronal excitability to contribute to the anticonvulsant (and perhaps neuroprotective) actionof the KD. Reduced glucose is also expected to downregulate brain-derived neurotrophic factor (BDNF) and trkB signaling in brain. Asactivation of TrkB pathways by BDNF have been shown to promote hyperexcitability and kindling, these potential KD-induced effects wouldbe expected to limit the symptom (seizures) as well as the progression of epilepsy. Boxed variables depict findings taken from KD studies;up (↑) or down (↓) arrows indicate the direction of the relationship between variables as a result of KD treatment.

the resultant diminution of cellular excitability in vitro,

it is not an easy task to demonstrate that ingestion of a

specific fatty acid or fatty acid cocktail, acts directly on

relevant brain receptor targets without first undergoing

beta-oxidation. The collective data, from both animals and

humans, indicate that the critical condition necessary for

achieving seizure control is a metabolic shift toward fatty

acid oxidation from glycolysis, reflected in the variable



12/16


rise in blood/brain ketone levels and a concomitant (mod-

erate) reduction in blood/brain glucose. Fatty acid compo-

sition may not ultimately matter, as long as this important

metabolic shift occurs. And, interestingly, calorie restric-

tion (Greene et al., 2001; Bough et al., 2003; Eagles et al.,

2003; Greene et al., 2003) or intake of 2-DG (Stafstrom

et al., 2005), both of which result in mild hypoglycemia,may be the only requirement for seizure protection, re-

gardless of whether fats are consumed or not.

As we continue to explore putative anticonvulsant

mechanisms of KD action, we are left with many out-

standing clinical questions regarding dietary treatments

for epilepsy. Well-designed, multicenter prospective- and

controlled clinical trials are essential toward developing

the optimum KD. If woven together with pharmacokinetic

and pharmacogenetic investigations, these clinical stud-

ies will not only provide further insights into mechanistic

underpinnings, but will also help differentiate responders

from non-responders and identify patients in whom the

diet is definitively contraindicated. Clinicians would be

given the tools to make evidence-based decisions rather

than rely upon a few case – controlled studies, anecdotal re-

ports of efficacy, or clinical folklore as has been the prac-

tice in the past. Toward this end, information regarding the

impact of pharmacogenetics on epilepsy treatment is now

beginning to emerge (Depondt and Shorvon, 2006; Spurr,

2006), although much less is known regarding the genet-

ically determined variables influencing dietary impact on

brain function, particularly as it relates to the epileptic

brain.

CONCLUSIONS

After nearly a century of clinical use, we still do not

know how the KD works. However, much progress in KD

research has been made in the past decade. Among other

factors, current evidence indicates KD optimizes cellular

metabolism. Endogenous biochemical and genetic ‘pro-

grams’ are switched on in the brain in response to keto-

sis, glucose restriction, and elevated free fatty acids. This

unique metabolic state, if maintained, induces a shift away

from glycolytic energy production (glucose restriction)

toward the production of energy via oxidative phospho-

rylation (beta-oxidation of fatty acids and production of ketone bodies). The reduction in glycolytic energy sup-

ply may activate selectively KATP channels to increase the

resistance to onset of ictal activity. An increase in oxida-

tive phosphorylation coupled with an induction of UCPs

and mitochondrial biogenesis can diminish ROS genera-

tion and increase energy reserves, both of which would

be expected to prevent neuronal dysfunction, seizures and

even neurodegeneration.

It is improbable that one mechanistic target or medi-

ator will produce entirely the seizure protection associ-

ated with the KD. Rather, several factors likely contribute

mechanistically to this broadly efficacious treatment for

epilepsy. The challenge of finding key variables is made

ever more difficultby the intrinsic complexity of metabolic

effects and their resultant actions on neurons, glia and on

the epileptic condition itself. We have reviewed here a

number of seemingly disparate variables that must be sus-

tained for a meaningful anticonvulsant effect to be ren-dered. These interrelationships are summarized in Fig. 4.

The fact that a fundamental modification in diet can have

such profound, therapeutic effects on neurological disease

underscores the importance of elucidating mechanisms of

KD action. Future studies will no doubt provide unique

insights into how diet can affect the brain, both in health

and disease, and likely provide the scientific basis for the

development of potent new treatment strategies for the

epilepsies.

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Bough2007 Anticonvulsant Mechanisms of the Ketogenic Diet