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Epilepsia, 48(1):43–58, 2007 Blackwell 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 century of use, the mechanisms underlying its clinical efficacy remain unknown. In this review, we present one intersecting view of how the KD may exert its anticonvulsant activity against the backdrop of several seemingly disparate mechanistic theories. We summa- rize key insights gleaned from experimental and clinical studies of the KD, and focus particular attention on the role that ketone bodies, fatty acids, and limited glucose may play in seizure con- trol. Chronic ketosis is anticipated to modify the tricarboxcylic acid cycle to increase GABA synthesis in brain, limit reactive oxygen species (ROS) generation, and boost energy production in brain tissue. Among several direct neuro-inhibitory actions,

polyunsaturated fatty acids increased after KD induce the ex- pression of neuronal uncoupling proteins (UCPs), a collective up-regulation of numerous energy metabolism genes, and mito- chondrial biogenesis. These effects further limit ROS generation and increase energy production. As a result of limited glucose and enhanced oxidative phosphorylation, reduced glycolytic flux is hypothesized to activate metabolic K ATP channels and hyper- polarize neurons and/or glia. Although it is unlikely that a single mechanism, however well substantiated, will explain all of the diet’s clinical benefits, these diverse, coordinated changes seem poised to stabilize synaptic function and increase the resistance to 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. Bough at FDA – Center for Drug Evaluation & Research, MPN 1 – Room 1345, 7520 Standish Place, Rockville, MD 20855, U.S.A. E-mail:

Kristopher.Bough@fda.hhs.gov

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 in improved 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 and experimental 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;

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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 diet onset Adult rats and mice >P40 at diet onset

Infants, children and adolescents <19 years of age Some evidence of efficacy in adults

Millichap (1964), Uhlemann and Neims (1972), Appleton and DeVivo (1974), Nakazawa et al. (1983), Kinsman et al. (1992), Hori et al. (1997), Bough et al. (1999b), Muller-Schwarze et al. (1999), Sirven et al. (1999), Nordli et al. (2001), Coppola et al. (2002), Kossoff et al. (2002), Mady et al.

 

(2003)

Seizure type

Effective in a wide variety of acute and chronic seizure models However, protection however can be transient; maximal seizure activity can be prolonged

Equally efficacious in a variety of seizure types

Livingston (1972), Appleton and DeVivo (1974), Mahoney et al. (1983), Otani et al. (1984), Schwartz et al. (1989a), Hori et al. (1997), Freeman et al. (1998), Vining et al. (1998), Rho et al. (1999), Su et al. (2000), Thavendiranathan et al. (2000), Greene et al. (2001), Bough et al. (2002)

Dietary composition

Qualitative differences do not affect outcome Classic LCT = MCT = PUFA fat diet

Qualitative differences do not affect outcome Classic LCT, MCT, Atkins can all diminish seizure frequency

Huttenlocher et al. (1971), Sills et al. (1986), Schwartz et al. (1989b), Freeman et al. (1998), Vining et al. (1998), Mak et al. (1999), Likhodii et al. (2000), Dell et al. (2001), Kossoff et al. (2003), Henderson et al. (2006), Kossoff et al. (2006)

KD ratio

Increase KD ratio = greater KD effect

Increase KD ratio = greater KD effect

Dekaban (1966), Livingston (1972), Bough et al. (1999a), Bough et al. (2000b), Freeman et al. (2000)

Calorie restriction

CR is anticonvulsant CR enhances KD anticonvulsant effect NAL < NCR < KAL < KCR

CR is an integral part of KD regimen (CR associated with seizure control)

Bough et al.(2000a), Bough et al. (2000b), Freeman et al. (2000), Greene et al. (2001), Greene et al.

 

(2003)

Growth rate

Initial transient decline in body weight Followed by, resumption of normal or near-normal growth rate

Decline in weight/height over first few months Followed by resumption of normal or near-normal height/weight gain Very young children do grow poorly

Rho et al. (1999), Freeman et al. (2000), Su et al. (2000), Vining et al. (2002), Peterson et al. (2005), Thio et al. (2006)

Latency to KD effect

12 weeks

Seizures can be reduced within 12 days Fine-tuning is key to success of the diet; at least 12 weeks are required

Livingston (1972), Appleton and DeVivo (1974), Freeman and Vining (1999), Rho et al. (1999), Freeman et al. (2000), Bough et al. (2006)

Reversal of KD effect

Seizure are behaviorally more severe within hours, but threshold returns to baseline over the course of 12

to see if changes are maximally effective Breakthrough seizure activity can occur immediately with ingestion of carbohydrates

Appleton and DeVivo (1974), Huttenlocher (1976), Freeman et al. (2000), Mady et al. (2003), Bough

Gender

weeks Males = females

Males = females

et al. (2006) Millichap (1964), Nakazawa et al. (1983), Schwartz et al. (1989b), Freeman et al. (1998), Bough et al. (2002), Mady et al. (2003)

Abbreviations: P40 age, postnatal day 40; LCT, long-chain triglycerides; MCT, medium-chain triglycerides; PUFA, polyunsaturated fatty acid; Atkins diet, diet high in fats + protein, low in carbohydrates; CR, calorie-restriction; NAL, normal, ad libitum rodent diet; NCR, normal, calorie-restricted (by 15%) rodent diet; KAL, ketogenic, ad libitum diet; KCR, ketogenic, calorie-restricted diet; KD ratio, ratio of [fats / (carbohydrates + proteins)]; KAP, ketogenic/antiketogenic potential. Table adapted from Stafstrom (2004).

Huttenlocher et al., 1971; Sills et al., 1986; Schwartz et al., 1989b; Mak et al., 1999) can control seizures with similar efficacy. Even the high-fat, high-protein, and low- carbohydrate Atkins diet that produced a ketotic state, re- duced seizures in epileptic patients (Kossoff et al., 2003, 2006). Similar effects have been observed experimentally. Ketogenic diets containing myriad types of fats (i.e., with low carbohydrate content) all produced similar levels of

Epilepsia, Vol. 48, No. 1, 2007

seizure control (Likhodii et al., 2000; Dell et al., 2001). Thus, available evidence indicates that dietary composi- tion per se does not appear to affect the anticonvulsant efficacy of the diet, as long as there is a degree of sus- tained ketosis. By comparison, KD ratios and calorie-restriction (CR) appear to be important variables in enabling seizure pro- tection. Seizure control is reportedly optimized when KDs

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 12 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 12 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 due to enhanced metabolic ca- pacity, more efficient extraction of ketone bodies from the blood (Morris, 2005), and/or greater compliance of KDs in 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., <P40 days; Uhle- mann and Neims, 1972; Otani et al., 1984; Bough et al., 1999b) and adult rodents (Appleton and DeVivo, 1974;

Muller-Schwarze et al., 1999). CR, too, is equally anti- convulsant in both juvenile and adult mice (Greene et al., 2001). Thus, increasing evidence suggests the anticonvul- sant effects of KDs do not appear to be age-dependent. Clinical reports indicate that outcome is unrelated to seizure type or frequency (Freeman et al., 1998; Schwartz et al., 1989a; Vining et al., 1998). At least 50% of pa- tients treated with a classical KD will exhibit at least a 50% reduction in seizures (Livingston, 1972; Freeman et al., 1998). Comparatively, anticonvulsant effects of KD in animals are more modest. Rats and mice only demon- strate a 1520% increase in seizure threshold (Appleton and DeVivo, 1974; Bough and Eagles, 1999; Rho et al., 1999; Bough et al., 2000b). Despite efficacy across a vari- ety of acute and chronic seizure models (Hori et al., 1997; Muller-Schwarze et al., 1999; Su et al., 2000), KD-induced anticonvulsant effects have been incomplete (Bough et al., 2002) or of limited duration (Hori et al., 1997). Further, there is little evidence to indicate that KDs diminish sever- ity once a seizure begins. Indeed, many studiesincluding our ownhave shown that CR and/or KDs can even ex- acerbate (maximal) seizures (Mahoney et al., 1983; Otani et al., 1984; Bough et al., 2000a; Thavendiranathan et al., 2000; Bough et al., 2003). If one considers that seizure activity requires large amounts of energy, seizure exacer- bation may be a reflection of enhanced energy reserves after KD treatment, a situation that would allow for pro- longed ictal activity once it begins (discussed below). In summary, the following generalizations can be made about the KD: (1) its anticonvulsant effects appear inde- pendent of dietary formulation, but appear to be strongly linked to the total quantity of calories consumed; (2) the KD must be strictly adhered to, if the anticonvulsant effect is to be maintained; (3) CR may work synergistically with KD to limit seizures and optimize treatment; (4) maximum efficacy is not achieved for several days or weeks after ini- tiation, suggesting that adaptive metabolic and/or genetic programsunderlie KD-induced seizure protection; (5) these adaptations are likely generalized throughout the (epileptic) brain, irrespective of underlying pathology or genetic predisposition to seizures since the KD is an ef- fective treatment for diverse epileptic conditions; and (6) efficacy is independent of gender and age, suggesting that KD treatment produces seizure control via a common set of pathways in all clinical responders.

ANTICONVULSANT MECHANISMS OF THE KETOGENIC DIET

Since the KD was originated over 85 years ago, several major hypotheses have been advanced, but none have been widely accepted. Several key aspects of the KD might ul- timately result in seizure protection. Ketone bodies, free fatty acids (in particular, polyunsaturated fatty acids), or

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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 GABA A 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 BHBwere anticonvulsant in a variety of acute and chronic models of epilepsy, consistent with earlier observations (Helmholz

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). Although acetone could not be detected in two other seizure-free patients, the authors concluded that acetone contributes 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 GABA A 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 evidence for direct anticonvulsant effects for either ACA or BHB, and acetone has yet to be studied in neuronal (CNS) tissue.

46 K. J. BOUGH AND J. M. RHO glucose restriction might each lead directly or indirectly

FIG. 1. Metabolic pathways highlighting 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. From Likhodii and Burnham (2004).

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ANTICONVULSANT ACTIONS OF KETOGENIC DIET 47

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 K 2P channels (Vamecq et al., 2005). K 2P 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 (Franks and Honore, 2004). Links between KD- induced elevations in ketone bodies (and/or fatty acids, as discussed below) and K 2P 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 pharmacodynamic effect 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 underlie the 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 moderatereduction 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). Greene et al. (2003) hypoth- esized that CR reduces energy production through glycol- ysis, which limits a neurons 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 (K ATP ) channels (Schwartzkroin, 1999; Vamecq et al., 2005). Interestingly, K ATP 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%), K ATP channels might open to hyperpolarize the cell as the intracellular ATP concentrations fall. Conversely, when glucose is present and ATP concentrations rise, K ATP chan- nels close. As such, K ATP 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).

K ATP 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). K ATP 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 K ATP channels may critically reg- ulate seizure activity. Genetically engineered mice that overexpress the sulfonylurea (SUR) subunit of K ATP 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 K ATP channel (Kir6.2 / ) knockout mice suggested that these channels help determine seizure threshold (Yamada et al., 2001). Following hypoxic challenge (5% O 2 ), knock- out mice exhibited myoclonictonic seizure activity, and, ultimately, death compared to controls who all recovered without sequelae. Despite these observations, there is one important caveat in implicating K ATP 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 K ATP channels would remain closed, 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 K ATP channels are selectively activated during administration of a low-glucose, high-fat KD. First, K ATP channels are regulated preferentially via glycolytic energy sources (Dubinsky et al., 1998). It has recently been shown that the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serves as an accessory pro- tein to K ATP 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 the course of KD administration (Dekaban, 1966) may boost K ATP 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 K ATP 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 K ATP 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-deoxyglucose is 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 2580% of baseline in rat hippocampal slices exposed to elevated extracellular potassium. More signifi- cantly, the same group also showed that 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 epileptogenesis in 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, C22:6ω 3), arachidonic acid (AA,

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C20:4ω 6), or eicosapentanoic acid (EPA, C20:5ω 3) 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 and EPA 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 Ca 2+ channels, (2) increase the resistance to bursting induced by bicu- culline, zero Mg 2+ , 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 K 2P 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

ANTICONVULSANT ACTIONS OF KETOGENIC DIET

49

ANTICONVULSANT ACTIONS OF KETOGENIC DIET 49 FIG. 2. Potential pathways through which polyunsaturated fatty acids (PUFAs)

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-gated Na + and Ca 2+ channels, activate a lipid-sensitive class of K 2P potassium channels, and enhance the activity of the Na + /K + -ATPase to limit neuronal excitability and dampen seizure activity. Acting indirectly, PUFAs might induce the expression and activity of uncoupling proteins (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 from the 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 uncoupleselec- 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 in which the KD is 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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dozen metabolic genes associated 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 glycogen and ATP concentrations were boosted throughout rodent brain (DeVivo et al., 1978; Otani et al., 1984) and there was 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 diets 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

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originally hypothesized that KD-induced elevations in ATP concentrations might enhance and/or prolong the ac- tivation of the Na + /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. Enhanced Na + /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 to the locus coeruleusthe principal region of the brain from which ascending and descending noradrenergic in- nervation originatesconverts 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).

ANTICONVULSANT ACTIONS OF KETOGENIC DIET 51

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- genic peptides 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-

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 expected to dampen hyper- 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.

ANTICONVULSANT ACTIONS OF KETOGENIC DIET 51 Of significant interest is the observation that mice lack- ing

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 to acetyl-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 of glutamate to aspartate. Oxaloacetate is then less available as a reactant of the aspartate aminotransferase pathway, which couples the glutamate-aspartate interchange via transamination to the metabolism of glucose through the TCA cycle. Less glutamate is converted to aspartate and thus, more glutamate is available for synthesis of GABA (3) through glutamic acid decarboxylase (GAD). Adapted from Yudkoff et al. (2004).

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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 spikes in both CR- and KD-fed animals compared to ad 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, GABA A -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 KETOGENIC DIET 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-

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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 no rational 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 diets 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 useeven 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 effect of 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

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53

ANTICONVULSANT ACTIONS OF KETOGENIC DIET 53 FIG. 4. Hypothetical pathways leading to the anticonvulsant effects of

FIG. 4. Hypothetical pathways leading to the anticonvulsant effects of the ketogenic diet (KD). Elevated free fatty acids (FFA) lead to chronic ketosis and increased concentrations of polyunsaturated fatty acids (PUFAs) in the brain. Chronic ketosis is predicted to lead to increased levels of acetone; this might activate K 2P channels to hyperpolarize neurons and limit neuronal excitability. Chronic ketosis is also anticipated to modify the tricarboxcylic acid (TCA) cycle. This would increase glutamate and, subsequently, GABA synthesis in brain. Among several direct inhibitory actions (see also Fig. 2), PUFAs boost the activity of brain-specic uncoupling proteins (UCPs). This is expected to limit ROS generation, neuronal dysfunction, and resultant neurodegeneration. Acting via the nuclear transcription factor peroxisome proliferator-activated receptor-α (PPARα ), PUFAs would induce the expression of UCPs and coordinately up-regulate several 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 KD treatment could lead to improved seizure control. Ultimately, PUFAs would stimulate mitochondrial biogenesis. Mitochondrial biogenesis is predicted to increase ATP production capacity and enhance energy reserves, leading to stabilized synaptic function and improved seizure control. 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 glucose coupled with elevated free fatty acids are proposed to reduce glycolytic ux during KD, which would further be feedback inhibited by high concentrations of citrate and ATP produced during KD treatment. This would activate metabolic K ATP channels. Opening of K ATP channels would hyperpolarize neurons and diminish neuronal excitability to contribute to the anticonvulsant (and perhaps neuroprotective) action of the KD. Reduced glucose is also expected to downregulate brain-derived neurotrophic factor (BDNF) and trkB signaling in brain. As activation of TrkB pathways by BDNF have been shown to promote hyperexcitability and kindling, these potential KD-induced effects would be expected to limit the symptom (seizures) as well as the progression of epilepsy. Boxed variables depict ndings 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

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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 casecontrolled 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- gramsare 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 K ATP 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

Epilepsia, Vol. 48, No. 1, 2007

mechanistically to this broadly efficacious treatment for epilepsy. The challenge of finding key variables is made ever more difficult by 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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