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The ketogenic diet: What has science taught us?
Epilepsy Research (2012) 100, 210—217
journal homepage: www.elsevier.com/locate/epilepsyres
The ketogenic diet: What has science taught us? Jong M. Rho a,∗, Carl E. Stafstrom b a
Departments of Paediatrics and Clinical Neurosciences, Alberta Children’s Hospital, University of Calgary, Calgary, Alberta, Canada b Department of Neurology, University of Wisconsin, Madison, WI, USA Received 3 February 2011; received in revised form 28 April 2011; accepted 1 May 2011 Available online 30 August 2011
KEYWORDS Epilepsy; Animal models; Anticonvulsant mechanisms; Ketogenic diet; Metabolism; 2-Deoxy-D-glucose
Summary Despite intense and growing interest in studying the mechanisms of ketogenic diet (KD) action, and recently published studies implicating novel molecular interactions with metabolic substrates, there nevertheless remains the pragmatic and scientific challenge of sustaining continued research in this field. This is in part a consequence of limited research funding and perhaps skepticism regarding the ultimate need to understand underlying mechanisms, particularly when clinical studies have increasingly validated the efficacy of the KD and its variants. After a decade and a half of more concerted laboratory efforts to understand KD mechanisms, it would be prudent to ask — what has all this scientific research really taught us? In this regard, it is instructive to compare and contrast laboratory research in dietary approaches for epilepsy with that traditionally used to screen for potential antiepileptic drugs (AEDs). In this review, lessons learned from AED development are applied to the more recent experimental findings and approaches attempting to link metabolic changes induced by the KD to neuronal and network excitability in the brain. © 2011 Published by Elsevier B.V.
Introduction Many reviews of the ketogenic diet (KD) in the modern era bemoan the fact that little has changed with regard to the KD formulation since the diet was originally designed almost a hundred years ago. While some diet modifications offer effectiveness comparable to the classic KD (e.g., modified Atkins diet, low-glycemic index treatment), a validated scientific basis remains lacking for epilepsy diet treatments.
∗ Corresponding author at: Division of Paediatric Neurology, Alberta Children’s Hospital, 2888 Shaganappi Trail, NW, Calgary, Alberta T3B 6A8, Canada. Tel.: +1 403 955 2635. E-mail address: [email protected] (J.M. Rho).
0920-1211/$ — see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.eplepsyres.2011.05.021
Why should we be concerned, other than to satisfy a scientific curiosity? The KD does stop seizures in many cases of refractory childhood epilepsy, so why bother spending resources and scientific talent to figure out what already works? There are several reasons. First, as mentioned above, the KD is effective but there has been little progress in making its administration more convenient and more palatable (Rho and Sankar, 2008). Second, current KD formulations are fraught with potentially serious adverse effects and can cause metabolic derangements; these might be ameliorated if more was known about how the diet works. Third, greater knowledge about the KD’s mechanisms could provide insights into the metabolic and physiological basis of normal brain function, both in the normal state, and under pathological conditions of stress and injury, such as seizure activity.
The ketogenic diet: What has science taught us? Finally, just as the KD’s clinical efficacy has recently been established through randomized, blinded trials (Neal et al., 2008; Freeman et al., 2009), an understanding of basic mechanisms would further validate the diet and provide a template for future therapeutic improvements. The better we understand how the KD suppresses seizures, retards the formation of the epileptic state (i.e., epileptogenesis), and improves the cognition and daily function of its adherents, the more the KD will be a rational choice for our patients. Eventually, various dietary formulations could be developed for patients with specific etiologies of epilepsy, in particular age ranges, or with certain pharmacogenetic — or more specifically, metabolomic — profiles. This overview is not intended to provide a comprehensive treatise on the scientific basis of the KD. The reader is referred to several recent reviews that already provide this information (Stafstrom, 2004; Bough and Rho, 2007; Hartman et al., 2007; Kim and Rho, 2008; Maalouf et al., 2009; Bough and Stafstrom, 2010). Instead, in the present article, we offer an overview of recent trends in research on KD mechanisms, to illustrate how science has contributed to and continues to provide a better understanding of the diet (Schwartzkroin et al., 1999; Stafstrom et al., 2008). We emphasize the key questions that should be asked and the approaches required to answer them. By analogy, we discuss the process by which investigational antiepileptic drugs (AEDs) are developed and describe how the KD is both similar and unique compared to the standard AEDs used in clinical practice. In a sense, the KD challenges us with a ‘‘reverse dilemma’’ compared to most drugs used in clinical medicine. In the usual situation, a disease or pathology exists and we seek to find a therapy based on the disorder’s pathophysiology or underlying molecular dysfunction. Indeed, even though the mechanism(s) of action of AEDs remain incompletely understood, early investigational studies have often revealed specific molecular targets or interactions that have been linked to seizure genesis — for example, voltage-gated sodium channels. With the KD, we have the opposite situation — the therapy is effective, but our goal is to understand its mechanism of action. On the one hand, this is a fortuitous situation since we can continue to use the KD empirically without necessarily knowing how it works. On the other hand, refractory epilepsy, especially in children, continues to represent an urgent and intractable problem, and to comprehend the KD in greater detail can only aid in our overall therapeutic efforts. Furthermore, clinical experience has taught us that the number of patients with refractory epilepsy is not declining, despite all the new AEDs now available (Arroyo et al., 2002; Kwan and Brodie, 2010) — hence the urgent need to think outside the traditional drug screening ‘‘box’’ (Bialer and White, 2010).
Animal models: a road to understanding or a blind alley? Are animal models a route to understanding mechanistic complexity in the epilepsies or merely a trifle to occupy idealistic scientists? It is perhaps a bit of both. Animal models have been considered the gold standard for testing potential AEDs. There are animal models for most of the common
211 seizure types, for some epilepsy syndromes (but not all, due to irreconcilable species differences), and for a few pathways underlying epileptogenesis — an area in dire need of further study (Gasior et al., 2006). To address the KD specifically, what models have been informative? And what are the caveats to placing too much hope in models? Finally, have experimental systems involving more reduced systems (e.g., cellular electrophysiology on brain slices, cells in culture, etc.) been helpful in unraveling the mechanisms of the KD? No single laboratory test can fully predict the clinical utility of an AED, either established or investigational. To fully evaluate the overall spectrum of clinical activity (i.e., narrow vs. broad), all investigational AEDs should ideally be screened in a variety of different seizure and epilepsy models. And despite efficacy in one more of these models, the ultimate validation must always await the results of well-designed prospective clinical trials. Nevertheless, with few exceptions, the majority of AEDs currently on the market today were advanced to clinical assessment based on their ability to block evoked seizures in one or more animal seizure or epilepsy models (White, 2003; Gareri et al., 2005). Over the years, many animal seizure models have been studied as potentially predictive seizure models (Table 1). Historically, the maximal electroshock (MES) test, the subcutaneous pentylenetetrazol (sc PTZ) test, and the electrical kindling model have been the three in vivo model systems most commonly employed in the search for new AEDs (White, 2003). Currently, animal models that more closely resemble the human epileptic condition are being utilized, including several with known underlying genetic defects (Frankel, 2009). The MES test and the kindling model are two highly predictive models that have proven useful in the characterization of a drug’s potential to block generalized tonic—clonic and partial seizures, respectively. Further, the sc PTZ test has been considered predictive of a drug’s potential utility against generalized absence seizures. This simplified approach, unfortunately, is imperfect. For example, barbiturates are effective in the sc PTZ test in animals, yet can provoke or worsen human generalized spike-wave discharges and associated absence seizures (Sazgar and Bourgeois, 2005). This dilemma has been addressed in part through the use of additional animal models that are perhaps more predictive than the sc PTZ test for generalized absence seizures. These include the spike-wave seizures induced by the chemoconvulsant ␥-butyrolactone, the genetic absence epileptic rat of Strasbourg (GAERS), and the lethargic (lh/lh) mutant mouse (Van Luijtelaar et al., 2002). Given its name, it should not be surprising that the earliest animal studies involving the KD paid keen attention to the acute effects of ketone bodies. Keith first demonstrated that acetoacetate (ACA) protected against seizures induced by thujone, a convulsant constituent of many essential oils (Keith, 1933). This intriguing observation was confirmed decades later by Likhodii and colleagues who found that both ACA and acetone blocked seizures induced by MES and PTZ (Likhodii et al., 2003). These investigators further demonstrated that intraperitoneal injection of acetone resulted in plasma and cerebrospinal fluid (CSF) concentrations consistent with doses used to suppress seizures, supporting the distinct possibility that this volatile agent might mediate the
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Table 1 Correlation between anticonvulsant efficacy and clinical utility of established and second-generation (in italics) AEDs in experimental animal models. Note that PB, TGB, and VGB block clonic seizures induced by sc PTZ but are inactive against generalized absence seizures and may exacerbate spike-wave seizures. Abbreviations: sc, subcutaneous; Hz, Hertz; mA, milliampere; MES, maximal electroshock; PTZ, pentylenetetrazol; BZD, benzodiazepines; CBZ, carbamazepine; ESM, ethosuximide; FBM, felbamate; GBP, gabapentin; LTG, lamotrigine; LVT, levetiracetam; PB, phenobarbital; PHT, phenytoin; LCM, lacosamide; PGB, pregabalin; RUF, rufinamide; TGB, tiagabine; TPM, topiramate; VPA, valproic acid; ZNS, zonisamide; VGB, vigabatrin. Experimental model
Clinical seizure type Tonic and/or clonic generalized seizures
MES (tonic extension)
Myoclonic/generalized absence seizures
Generalized Partial seizures absence seizures
CBZ, PHT, VPA, PB FBM, GBP, LCM, LTG, PGB, RUF, TPM, ZNS
sc PTZ (clonic seizures)
ESM, VPA, PB, BZD FBM, GBP, PGB, RUF, TGB, VGB
Spike-wave discharges
ESM, VPA, BZD LTG, TPM, LVT
Electrical kindling (focal seizures)
CBZ, PHT, VPA, PB, BZD FBM, GBP, LCM, LTG, TPM, TGB, ZNS, LVT, VGB LVT, GBP, TPM, FBM, LTG VPA LVT
Phenytoin-resistant kindled rat 6 Hz (44 mA)
From: White, H.S., 2010. Epilepsy and disease modification. In: Rho, J.M., Sankar, R., Stafstrom, C.E. (Eds.), Epilepsy: Mechanisms, Models, and Translational Perspectives. CRC Press/Taylor & Francis Group, Boca Raton, Florida, USA, p. 146.
clinical effects of the KD. Curiously, however, there are no studies clearly demonstrating that the major ketone body, -hydroxybutyrate (BHB), can exert similar effects when administered acutely. Most of the existing KD animal studies have involved high-fat treatments implemented prior to acute provocation with either electrical stimulation or chemoconvulsant administration in rodents. Further, most animal diets have modeled the classic long-chain triglyceride (LCT) diet and conform closely to either a 4:1 or approximately 6:1 ketogenic ratio of fats to carbohydrates plus protein (by weight). In general, irrespective of the precise dietary formulation, as long as ketosis is seen, protective effects have been seen against seizures provoked by numerous methods, including corneal electroshock, hydration electroshock, MES, PTZ, bicuculline, semicarbizide, kainate, fluorothyl, or in the 6 Hz stimulation model (Appleton and DeVivo, 1974; Hori et al., 1997; Rho et al., 1999; Bough et al., 2000; Thavendiranathan et al., 2000; Bough et al., 2002; Rho et al., 2002; Noh et al., 2003; Zhao et al., 2004; Kwon et al., 2008; Hartman et al., 2008). Published animal studies, in attempting to mirror clinical experience, suggest that dietary effects in controlling excitability in the brain extend beyond species boundaries, but they do little to enhance our knowledge of underlying mechanisms of action. Rather, they raise more issues and highlight the necessity of developing and studying a more clinically relevant model. One critical limitation is that these laboratory investigations have been conducted in normal, not epileptic, rodent brain. What is needed is a chronic model of the KD, employing an animal with early-onset, medically refractory epilepsy, that responds to a particu-
lar formulation of a high-fat diet that best recapitulates all of the essential elements of the human experience.
Cellular electrophysiology Epilepsy is defined by the occurrence of recurrent spontaneous seizures arising from hyperexcitable and hypersynchronous brain activity. The essential currency of neuronal excitability — both normal and aberrant — is the complex array of ion channels (primarily voltage-gated and ligand-gated) that determine the firing properties of neurons and mediate synaptic transmission. Against this increasingly complex backdrop, the conventional thinking has been that seizure activity arises due to perturbations in the normal balance between inhibition and excitation in a localized region, multiple brain areas (linked in a multi-nodal network), or throughout the whole brain. Recently, however, this paradigm has been shown to be too simplified, with epilepsy mechanisms and therapeutic targets addressing heretofore unsuspected physiological functions (synaptic vesicles, brain energetics, etc. — the latter of particular relevance to the KD) (Kim and Rho, 2008). At present, there appears to be at least six important mechanisms through which the currently available AEDs exert their anticonvulsant action. These are summarized in Table 2. As should be noted, and not surprisingly, the vast majority of molecular targets are ion channels and transporters that are localized to plasmalemmal membranes. While the precise manners in which AEDs exert clinical activity remain unclear, there is a wealth of in vivo and in vitro experimental data supporting this mechanistic
The ketogenic diet: What has science taught us? Table 2
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Classification of antiepileptic drug mechanisms.
• Modulation of voltage-dependent Na+ or Ca2+ channels leading to a secondary inhibition of neurotransmitter release (particularly of glutamate) or to inhibition of intrinsic bursting • Enhancement of GABA-mediated inhibition or other modulatory effects on GABA uptake and metabolism • Inhibition of synaptic excitation mediated by ionotropic glutamate receptors • Modulation of synaptic release, particularly of glutamate, through direct actions on release machinery (SV2A and ␣2␦) • Activation of voltage-dependent K+ channels and improved spike-frequency adaptation • Enhanced activation of dendritic hyperpolarization-activated cation (HCN)-channels and suppressed action potential initiation by dendritic inputs From: White, H.S., Rho, J.M., 2010. Mechanisms of Action of Antiepileptic Drugs. Professional Communications, Inc., New York.
framework. In this light, a fundamental question pertaining to KD effects at the cellular and molecular levels has been whether any of the metabolic substrates elaborated by this ‘‘non-pharmacological’’ intervention can interact with ion channels that regulate neuronal excitability. Thio and colleagues found that acute application of BHB or ACA did not affect standard measures of synaptic transmission in hippocampus (Thio et al., 2000). Specifically, these ketones did not affect GABAA receptors, ionotropic glutamate receptors, or voltage-gated sodium channels over a wide concentration range (300 M to 10 mM). The fact that ACA was ineffective in their hands was surprising, especially given its clear anticonvulsant effects when administered in vivo (Rho et al., 2002). These negative results may have been influenced in part by the facts that: (1) ketone bodies were infused acutely, not chronically; (2) experiments were conducted in normal, not epileptic, brain; and (3) both culture and perfusion media contained glucose, which might have countered a ‘‘ketotic’’ environment. ATP-sensitive potassium (KATP ) channels are logical candidates for linking metabolic changes to cellular membrane
Table 3
excitability. KATP channels are a type of inwardly rectifying potassium channel (Kir6) that is activated when intracellular ATP levels fall. Ma et al. found that ketone bodies reduced the spontaneous firing of GABAergic neurons in rat substantia nigra pars reticulata (SNr), a putative subcortical ‘‘seizure gate’’, by opening KATP channels (Ma et al., 2007). Despite the intuitive appeal and novelty of this observation, an inherent discrepancy remains to be reconciled. Earlier studies had shown that the KD can increase levels of ATP and other bioenergetic substrates through enhanced mitochondrial respiration (Cullingford et al., 2002; Sullivan et al., 2004; Bough et al., 2006; Maalouf et al., 2007; Jarrett et al., 2008). Since high ATP levels block KATP channel activity, it is unclear how opening of these channels is achieved by infusion of ketone bodies in the SNr. Ma et al. suggested that the area subjacent to KATP channels may actually exhibit lower ATP levels than other cellular compartments due to excessive membrane discharge which consumes ATP (Ma et al., 2007). In other words, while ketone bodies might raise global ATP levels, ATP levels near the plasma membrane (where KATP channels are localized) could be substantially
Ketogenic diet: clinical correlates and experimental observations.
Seizure type Age range
Calorie restriction Diet type Ketosis
Fat
Latency to KD effectiveness
Reversal of protective effect when KD discontinued
Clinical correlate
Observation in animal models
KD is effective in many seizure types and epilepsy syndromes Children extract and utilize ketones from blood more efficiently than older individuals Associated with seizure reduction Classic and MCT KDs are equally efficacious Ketosis is necessary but not sufficient for seizure control
KD is effective in models employing a wide variety of seizure paradigms Younger animals respond better to the KD
Practical concerns limit the ketogenic ratio; possible role of fat chain length and degree of saturation (e.g., PUFA) Seizures may be reduced during the pre-diet fast or after a latency of days to weeks Rapid (hours)
Increases seizure threshold Classic and MCT diets both increase seizure threshold A threshold level of ketosis is necessary but not sufficient to explain anti-seizure effects Better effectiveness with higher ketogenic ratios; uncertain if type of fat is a critical variable Several days
Rapid (hours)
Adapted from Stafstrom (2004). Abbreviations: KD, ketogenic diet; MCT, medium chain triglycerides; PUFA, polyunsaturated fatty acid.
214 decreased, and as such, KATP channels could be recruited to dampen neuronal excitability. Although this is a potentially elegant solution to the dilemma posed, there are yet no data directly supporting this hypothesis. Taken together, the role of ketone bodies as direct anticonvulsant mediators remains uncertain, especially since a clear and convincing correlation between the degree of ketonemia and seizure control has yet to be established (Bough et al., 2000; Gilbert et al., 2000). Further actions of metabolic substrates have recently been reported. Kawamura and colleagues evaluated the electrophysiological effects of reduced glucose (a consistent finding in patients successfully treated with the KD) — importantly, under conditions of adequate or enhanced ATP levels — in CA3 hippocampal pyramidal neurons using wholecell recording techniques (Kawamura et al., 2010). These authors found that glucose restriction led to ATP release through pannexin hemichannels on CA3 neurons, and that the increased extracellular ATP, upon rapid degradation by ectonucleotidases to adenosine, resulted in activation of adenosine A1 receptors which was also shown to be coupled to opening of plasmalemmal KATP channels. While this study revealed a novel mechanism of metabolic autocrine regulation, involving close cooperativity among pannexin hemichannels, adenosine receptors and KATP channels, it is yet uncertain whether adenosine is the key metabolic mediator of the KD’s anticonvulsant activity. The contribution of adenosine is plausible, however, given the well-documented role of this endogenous purine in suppressing cellular excitability (Boison, 2009). There is yet another intriguing link between metabolic substrates and neuronal excitability. Juge and colleagues recently reported that ACA inhibits vesicular glutamate transporters (VGLUTs), which are required for exocytotic release of the excitatory neurotransmitter glutamate, specifically by competing with an anion-dependent regulatory site on presynaptic vesicles (Juge et al., 2010). These authors demonstrated that ACA decreased the quantal size of excitatory neurotransmission at hippocampal synapses, and suppressed glutamate release and seizures evoked by the convulsant 4-aminopyridine in rats. So, is this a plausible explanation for the acute in vivo effects of ACA observed nearly eight decades earlier? Possibly, but ACA is reported to have other actions as well, notably effects on mitochondria (Maalouf et al., 2007; Bentourkia et al., 2009). The other caveat is that ACA is highly unstable, and undergoes spontaneous decarboxylation to acetone; further, in the presence of BHB dehydrogenase, is interconverted to the major ketone body BHB. Thus, it appears that seemingly straightforward metabolic substrates are players in a more complex arena.
Metabolic pathways relevant to KD mechanisms Key clinical observations regarding KD use provide starting points from which to investigate underlying mechanisms (Table 3). In this section, we discuss some recent trends in KD research, focusing on studies of biochemical pathways that could link metabolism with neuronal excitability. The original formulation of the KD was based on the assumption
J.M. Rho, C.E. Stafstrom that the diet mimicked the fasting state, which was known to reduce seizures. The KD restricts carbohydrate intake and provides energy from fat breakdown, leading to ketosis, but the observational leap to exactly how ketosis (or some other factor) constrains cellular excitability remains unknown. (The effects of fatty acids on neuronal excitability, another possible mechanism of KD action, are discussed elsewhere in this volume.) Another key feature is that ingestion of a small amount of carbohydrate in a patient on the KD results in rapid loss of seizure control (Huttenlocher, 1976), leading to the hypothesis that carbohydrate restriction could be protective in epilepsy (Greene et al., 2003; Yamada, 2008). By utilizing ketones as the energy source, the KD bypasses glycolysis, raising the possibility that glycolytic inhibition itself might also protect against seizures. 2-Deoxy-D-glucose (2DG) is a glucose analog differing from glucose only by substitution of a single oxygen atom in the 2 -position. 2DG cannot be metabolized and inhibits glycolysis by blocking the glycolytic enzyme, phosphoglucose isomerase, thereby preventing the conversion of glucose-6phosphate to fructose-6-phosphate. 2DG has been shown to be a potent anticonvulsant and antiepileptic agent in several animal models, including kindling, audiogenic seizures in Fring’s mice and 6-Hz corneal stimulation (Garriga-Canut et al., 2006; Stafstrom et al., 2009), as well as in vitro in CA3 neurons in hippocampal slices exposed to elevated extracellular K+ , bicuculline, or 4-aminopyridine (Stafstrom et al., 2009) or metabotropic Group 1 agonists (Pan et al., 2008). Seizure-suppressing effects of 2DG are seen both acutely and chronically. The acute anticonvulsant effects of 2DG in vitro and in vivo against both ictal and interictal activity suggest that 2DG may exert direct actions at the synaptic or membrane levels, but through mechanisms independent of altered gene expression (Stafstrom et al., 2009). Acute effects of 2DG could be related to rapid-onset metabolic or electrophysiological consequences of glycolytic inhibition leading to reduction of network synchronization. For example, there could be an effect on systemic lipid metabolism, mitochondrial function, or the phosphorylation state of GABAA receptor subunits (Pumain et al., 2008), each of which can influence neuronal excitability. In whole-cell recordings of CA3 neurons in hippocampal slices, acute application of 2DG suppressed spontaneous excitatory postsynaptic currents (EPSCs) after transient epileptic activity induced by elevated extracellular potassium or metabotropic Group 1 agonists, but not in normal slices, implicating an activity-dependence to effects of glycolytic inhibition (Pan et al., 2009). These and other potential acute mechanisms are currently under study. The chronic antiepileptic effects of 2DG have been related to the molecular regulation of genes for brainderived neurotrophic factor (BDNF) and its receptor, tyrosine kinase B (trkB). Repression of both BDNF and trkB expression are required for the progression of kindling (He et al., 2004). 2DG suppresses seizure-induced increases in BDNF and trkB, mediated by the transcriptional repressor neuron restrictive silencing factor (NRSF) and its nicotinamide adenine dinucleotide (NADH)-sensitive co-repressor carboxy-terminal binding protein (CtBP). NRSF and CtBP act at the promoter regions of BDNF and trkB genes (GarrigaCanut et al., 2006). During seizure activity, when glycolysis
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Figure 1 Glucose metabolism and points at which interventions could affect neuronal excitability and seizure control Glucose can be diverted to the pentose phosphate shunt (PPP) via fructose-1,6-diphosphate (FDP). 2-Deoxy-2-glucose (2DG) inhibits glycolysis by blocking the phosphoglucose isomerase step (see text). The ketogenic diet (KD), via ketone bodies, bypasses glycolysis by providing acetyl-CoA (ACoA) to the TCA (tricarboxylic acid cycle) after glycolysis. Anaplerotic compounds ‘‘refill’’ depleted intermediates from the TCA. Other abbreviations: -OHB, beta-hydroxybutyrate; AcAc, acetoacetate; PDH, pyruvate dehydrogenase.
and glucose production are increased, elevated levels of NADH causes dissociation of CtBP from NRSF, decreasing transcriptional repression and resulting in increased expression of BDNF and trkB. In the presence of 2DG, which reduces NADH levels as a consequence of glycolytic inhibition, the NRSF-CtBP complex maintains repression of BDNF and trkB, and kindling progression is slowed (Garriga-Canut et al., 2006; Huang and McNamara, 2006). These chronic anticonvulsant and antiepileptic effects of 2DG, coupled with its favorable safety profile (Stafstrom et al., 2009), position this compound as a viable candidate for clinical trials and raise the possibility that this agent can modify both seizure susceptibility and disease progression. Continuing the theme that a key KD mechanism might involve alteration of glucose metabolism, another component of the glycolytic pathway, fructose-1,6-diphosphate (FDP), has been shown to exert acute anticonvulsant activity in several seizure models in adult rats including kainate, pilocarpine, pentylenetetrazole, and kindling (Lian et al., 2007; Ding et al., 2010). Indeed, FDP was more effective as an anticonvulsant than 2DG, KD, and valproate in these studies. FDP increases glucose flux from glycolysis into the pentose phosphate pathway (PPP). NADPH generated in the PPP reduces glutathione, which has anticonvulsant activity. Therefore, FDP may exert an endogenous anticonvulsant (and perhaps anti-oxidant) action (Stringer and Xu, 2008), but the precise mechanism by which FDP produces an anticonvulsant effect remains unclear. Another dietary approach to epilepsy derives from the observation that seizures cause a deficiency in tricarboxylic acid cycle (TCA) intermediates (especially ␣-ketoglutarate and oxaloacetate), leading to increased excitability and possibly increased seizures. It has been hypothesized that ‘‘refilling’’ these deficient compounds, a process called ‘‘anaplerosis’’, might oppose seizure generation. One such anaplerotic compound, triheptanoin, has recently been investigated in both acute and chronic seizure models (Willis
et al., 2010). Mice fed triheptanoin exhibited delayed development of corneal kindled seizures and triheptanoin feeding increased PTZ seizure threshold in chronically epileptic mice that had undergone status epilepticus 3 weeks before PTZ testing (Willis et al., 2010). Therefore, like 2DG, anaplerotic compounds alter both acute and chronic seizure susceptibility. Anaplerosis represents a novel approach that expands the potential metabolic modifications that could be anticonvulsant or antiepileptic. Together, results from studies of the KD, 2DG, FDP, and anaplerosis suggest that modification of metabolic pathways such as glycolysis could be a possible novel mechanism for treatment of seizures (Fig. 1). A final example of the involvement of a metabolic pathway that could play a role in KD action is the endogenous peptide leptin. Leptin is part of the hormonal system that limits energy intake and expenditure and is intimately involved in appetite regulation, but it also exerts modulatory effects on neuronal excitability and suppresses seizures (Harvey, 2007; Obeid et al., 2010). Administration of leptin to rats undergoing either focal or generalized seizures resulted in shorter, less frequent seizures, possibly through alteration of AMPA receptor-mediated synaptic transmission (Xu et al., 2008). Since the KD causes a rise in leptin levels (Xu et al., 2008), it is possible that the KD’s mechanism, at least in part, may be related to leptin-associated reduction in synaptic excitability.
Conclusions It is often stated that no single mechanism is likely to explain the clinical effects of the KD. We concur with this viewpoint, and certainly, the same could be said of virtually all AEDs used in clinical practice. However, such a conclusion should not dissuade us from attempting to elucidate the multiple, likely interacting mechanisms by which the KD constrains neuronal hyperexcitability and suppresses seizures. In fact,
216 the complexity of the KD mechanism of action should be viewed as ‘‘a guide and friend’’, enabling the development of novel therapies based on the intersection between metabolism and neuronal excitability. Importantly, elucidation of critical control points in the epileptic cellular network — ones that could potentially be influenced by shifts in metabolic activity — lays the foundation of an exciting new direction in epilepsy therapeutics. Science has indeed come a long way toward unraveling KD mechanisms and we anticipate further progress, to the eventual benefit of the thousands of children suffering from intractable epilepsy.
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217 Stafstrom, C.E., 2004. Dietary approaches to epilepsy treatment: old and new options on the menu. Epilepsy Curr. 4, 215—222. Stafstrom, C.E., Ockuly, J.C., Murphee, L., Valley, M.T., Roopra, A., Sutula, T.P., 2009. Anticonvulsant and antiepileptic actions of 2-deoxy-D-glucose in epilepsy models. Ann. Neurol. 65, 435—447. Stafstrom, C.E., Zupec-Kania, B., Rho, J.M., 2008. Ketogenic diet and related dietary treatments. Epilepsia 49 (Suppl. 8), 1—133. Stringer, J.L., Xu, K., 2008. Possible mechanisms for the anticonvulsant activity of fructose-1,6-diphosphate. Epilepsia 49 (Suppl. 8), 101—103. Sullivan, P.G., Rippy, N.A., Dorenbos, K., Concepcion, R.C., Agarwal, A.K., Rho, J.M., 2004. The ketogenic diet increases mitochondrial uncoupling protein levels and activity. Ann. Neurol. 55, 576—580. Thavendiranathan, P., Mendonca, A., Dell, C., Likhodii, S.S., Musa, K., Iracleous, C., Cunnane, S.C., Burnham, W.M., 2000. The MCT ketogenic diet: effects on animal seizure models. Exp. Neurol. 161, 696—703. Thio, L., Wong, M., Yamada, K.A., 2000. Ketone bodies do not directly alter excitatory or inhibitory hippocampal transmission. Neurology 54, 325—331. Van Luijtelaar, E.L., Drinkenburg, W.H., van Rijn, C.M., Coenen, A.M., 2002. Rat models of genetic absence epilepsy: what do EEG spike-wave discharges tell us about drug effects? Meth Find. Exp. Clin. Pharmacol. 24 (Suppl. D), 65—70. White, H.S., 2003. Preclinical development of antiepileptic drugs: past, present, and future directions. Epilepsia 44 (Suppl. 7), 2—8. Willis, S., Stoll, J., Sweetman, L., Borges, K., 2010. Anticonvulsant effects of a triheptanoin diet in two mouse chronic seizure models. Neurobiol. Dis. 40 (3), 565—572. Xu, L., Rensing, N., Yang, X.F., Zhang, H.X., Thio, L.L., Rothman, S.M., Weisenfeld, A.E., Wong, M., Yamada, K.A., 2008. Leptin inhibits 4-aminopyridine- and pentylenetetrazole-induced seizures and AMPAR-mediated synaptic transmission in rodents. J. Clin. Invest. 118, 272—280. Yamada, K.A., 2008. Calorie restriction and glucose regulation. Epilepsia 49 (Suppl. 8), 94—96. Zhao, Q., Stafstrom, C.E., Fu, D.D., Hu, Y., Holmes, G.L., 2004. Detrimental effects of the ketogenic diet on cognitive function in rats. Pediatr. Res. 55, 498—506.
journal homepage: www.elsevier.com/locate/epilepsyres
The ketogenic diet: What has science taught us? Jong M. Rho a,∗, Carl E. Stafstrom b a
Departments of Paediatrics and Clinical Neurosciences, Alberta Children’s Hospital, University of Calgary, Calgary, Alberta, Canada b Department of Neurology, University of Wisconsin, Madison, WI, USA Received 3 February 2011; received in revised form 28 April 2011; accepted 1 May 2011 Available online 30 August 2011
KEYWORDS Epilepsy; Animal models; Anticonvulsant mechanisms; Ketogenic diet; Metabolism; 2-Deoxy-D-glucose
Summary Despite intense and growing interest in studying the mechanisms of ketogenic diet (KD) action, and recently published studies implicating novel molecular interactions with metabolic substrates, there nevertheless remains the pragmatic and scientific challenge of sustaining continued research in this field. This is in part a consequence of limited research funding and perhaps skepticism regarding the ultimate need to understand underlying mechanisms, particularly when clinical studies have increasingly validated the efficacy of the KD and its variants. After a decade and a half of more concerted laboratory efforts to understand KD mechanisms, it would be prudent to ask — what has all this scientific research really taught us? In this regard, it is instructive to compare and contrast laboratory research in dietary approaches for epilepsy with that traditionally used to screen for potential antiepileptic drugs (AEDs). In this review, lessons learned from AED development are applied to the more recent experimental findings and approaches attempting to link metabolic changes induced by the KD to neuronal and network excitability in the brain. © 2011 Published by Elsevier B.V.
Introduction Many reviews of the ketogenic diet (KD) in the modern era bemoan the fact that little has changed with regard to the KD formulation since the diet was originally designed almost a hundred years ago. While some diet modifications offer effectiveness comparable to the classic KD (e.g., modified Atkins diet, low-glycemic index treatment), a validated scientific basis remains lacking for epilepsy diet treatments.
∗ Corresponding author at: Division of Paediatric Neurology, Alberta Children’s Hospital, 2888 Shaganappi Trail, NW, Calgary, Alberta T3B 6A8, Canada. Tel.: +1 403 955 2635. E-mail address: [email protected] (J.M. Rho).
0920-1211/$ — see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.eplepsyres.2011.05.021
Why should we be concerned, other than to satisfy a scientific curiosity? The KD does stop seizures in many cases of refractory childhood epilepsy, so why bother spending resources and scientific talent to figure out what already works? There are several reasons. First, as mentioned above, the KD is effective but there has been little progress in making its administration more convenient and more palatable (Rho and Sankar, 2008). Second, current KD formulations are fraught with potentially serious adverse effects and can cause metabolic derangements; these might be ameliorated if more was known about how the diet works. Third, greater knowledge about the KD’s mechanisms could provide insights into the metabolic and physiological basis of normal brain function, both in the normal state, and under pathological conditions of stress and injury, such as seizure activity.
The ketogenic diet: What has science taught us? Finally, just as the KD’s clinical efficacy has recently been established through randomized, blinded trials (Neal et al., 2008; Freeman et al., 2009), an understanding of basic mechanisms would further validate the diet and provide a template for future therapeutic improvements. The better we understand how the KD suppresses seizures, retards the formation of the epileptic state (i.e., epileptogenesis), and improves the cognition and daily function of its adherents, the more the KD will be a rational choice for our patients. Eventually, various dietary formulations could be developed for patients with specific etiologies of epilepsy, in particular age ranges, or with certain pharmacogenetic — or more specifically, metabolomic — profiles. This overview is not intended to provide a comprehensive treatise on the scientific basis of the KD. The reader is referred to several recent reviews that already provide this information (Stafstrom, 2004; Bough and Rho, 2007; Hartman et al., 2007; Kim and Rho, 2008; Maalouf et al., 2009; Bough and Stafstrom, 2010). Instead, in the present article, we offer an overview of recent trends in research on KD mechanisms, to illustrate how science has contributed to and continues to provide a better understanding of the diet (Schwartzkroin et al., 1999; Stafstrom et al., 2008). We emphasize the key questions that should be asked and the approaches required to answer them. By analogy, we discuss the process by which investigational antiepileptic drugs (AEDs) are developed and describe how the KD is both similar and unique compared to the standard AEDs used in clinical practice. In a sense, the KD challenges us with a ‘‘reverse dilemma’’ compared to most drugs used in clinical medicine. In the usual situation, a disease or pathology exists and we seek to find a therapy based on the disorder’s pathophysiology or underlying molecular dysfunction. Indeed, even though the mechanism(s) of action of AEDs remain incompletely understood, early investigational studies have often revealed specific molecular targets or interactions that have been linked to seizure genesis — for example, voltage-gated sodium channels. With the KD, we have the opposite situation — the therapy is effective, but our goal is to understand its mechanism of action. On the one hand, this is a fortuitous situation since we can continue to use the KD empirically without necessarily knowing how it works. On the other hand, refractory epilepsy, especially in children, continues to represent an urgent and intractable problem, and to comprehend the KD in greater detail can only aid in our overall therapeutic efforts. Furthermore, clinical experience has taught us that the number of patients with refractory epilepsy is not declining, despite all the new AEDs now available (Arroyo et al., 2002; Kwan and Brodie, 2010) — hence the urgent need to think outside the traditional drug screening ‘‘box’’ (Bialer and White, 2010).
Animal models: a road to understanding or a blind alley? Are animal models a route to understanding mechanistic complexity in the epilepsies or merely a trifle to occupy idealistic scientists? It is perhaps a bit of both. Animal models have been considered the gold standard for testing potential AEDs. There are animal models for most of the common
211 seizure types, for some epilepsy syndromes (but not all, due to irreconcilable species differences), and for a few pathways underlying epileptogenesis — an area in dire need of further study (Gasior et al., 2006). To address the KD specifically, what models have been informative? And what are the caveats to placing too much hope in models? Finally, have experimental systems involving more reduced systems (e.g., cellular electrophysiology on brain slices, cells in culture, etc.) been helpful in unraveling the mechanisms of the KD? No single laboratory test can fully predict the clinical utility of an AED, either established or investigational. To fully evaluate the overall spectrum of clinical activity (i.e., narrow vs. broad), all investigational AEDs should ideally be screened in a variety of different seizure and epilepsy models. And despite efficacy in one more of these models, the ultimate validation must always await the results of well-designed prospective clinical trials. Nevertheless, with few exceptions, the majority of AEDs currently on the market today were advanced to clinical assessment based on their ability to block evoked seizures in one or more animal seizure or epilepsy models (White, 2003; Gareri et al., 2005). Over the years, many animal seizure models have been studied as potentially predictive seizure models (Table 1). Historically, the maximal electroshock (MES) test, the subcutaneous pentylenetetrazol (sc PTZ) test, and the electrical kindling model have been the three in vivo model systems most commonly employed in the search for new AEDs (White, 2003). Currently, animal models that more closely resemble the human epileptic condition are being utilized, including several with known underlying genetic defects (Frankel, 2009). The MES test and the kindling model are two highly predictive models that have proven useful in the characterization of a drug’s potential to block generalized tonic—clonic and partial seizures, respectively. Further, the sc PTZ test has been considered predictive of a drug’s potential utility against generalized absence seizures. This simplified approach, unfortunately, is imperfect. For example, barbiturates are effective in the sc PTZ test in animals, yet can provoke or worsen human generalized spike-wave discharges and associated absence seizures (Sazgar and Bourgeois, 2005). This dilemma has been addressed in part through the use of additional animal models that are perhaps more predictive than the sc PTZ test for generalized absence seizures. These include the spike-wave seizures induced by the chemoconvulsant ␥-butyrolactone, the genetic absence epileptic rat of Strasbourg (GAERS), and the lethargic (lh/lh) mutant mouse (Van Luijtelaar et al., 2002). Given its name, it should not be surprising that the earliest animal studies involving the KD paid keen attention to the acute effects of ketone bodies. Keith first demonstrated that acetoacetate (ACA) protected against seizures induced by thujone, a convulsant constituent of many essential oils (Keith, 1933). This intriguing observation was confirmed decades later by Likhodii and colleagues who found that both ACA and acetone blocked seizures induced by MES and PTZ (Likhodii et al., 2003). These investigators further demonstrated that intraperitoneal injection of acetone resulted in plasma and cerebrospinal fluid (CSF) concentrations consistent with doses used to suppress seizures, supporting the distinct possibility that this volatile agent might mediate the
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J.M. Rho, C.E. Stafstrom
Table 1 Correlation between anticonvulsant efficacy and clinical utility of established and second-generation (in italics) AEDs in experimental animal models. Note that PB, TGB, and VGB block clonic seizures induced by sc PTZ but are inactive against generalized absence seizures and may exacerbate spike-wave seizures. Abbreviations: sc, subcutaneous; Hz, Hertz; mA, milliampere; MES, maximal electroshock; PTZ, pentylenetetrazol; BZD, benzodiazepines; CBZ, carbamazepine; ESM, ethosuximide; FBM, felbamate; GBP, gabapentin; LTG, lamotrigine; LVT, levetiracetam; PB, phenobarbital; PHT, phenytoin; LCM, lacosamide; PGB, pregabalin; RUF, rufinamide; TGB, tiagabine; TPM, topiramate; VPA, valproic acid; ZNS, zonisamide; VGB, vigabatrin. Experimental model
Clinical seizure type Tonic and/or clonic generalized seizures
MES (tonic extension)
Myoclonic/generalized absence seizures
Generalized Partial seizures absence seizures
CBZ, PHT, VPA, PB FBM, GBP, LCM, LTG, PGB, RUF, TPM, ZNS
sc PTZ (clonic seizures)
ESM, VPA, PB, BZD FBM, GBP, PGB, RUF, TGB, VGB
Spike-wave discharges
ESM, VPA, BZD LTG, TPM, LVT
Electrical kindling (focal seizures)
CBZ, PHT, VPA, PB, BZD FBM, GBP, LCM, LTG, TPM, TGB, ZNS, LVT, VGB LVT, GBP, TPM, FBM, LTG VPA LVT
Phenytoin-resistant kindled rat 6 Hz (44 mA)
From: White, H.S., 2010. Epilepsy and disease modification. In: Rho, J.M., Sankar, R., Stafstrom, C.E. (Eds.), Epilepsy: Mechanisms, Models, and Translational Perspectives. CRC Press/Taylor & Francis Group, Boca Raton, Florida, USA, p. 146.
clinical effects of the KD. Curiously, however, there are no studies clearly demonstrating that the major ketone body, -hydroxybutyrate (BHB), can exert similar effects when administered acutely. Most of the existing KD animal studies have involved high-fat treatments implemented prior to acute provocation with either electrical stimulation or chemoconvulsant administration in rodents. Further, most animal diets have modeled the classic long-chain triglyceride (LCT) diet and conform closely to either a 4:1 or approximately 6:1 ketogenic ratio of fats to carbohydrates plus protein (by weight). In general, irrespective of the precise dietary formulation, as long as ketosis is seen, protective effects have been seen against seizures provoked by numerous methods, including corneal electroshock, hydration electroshock, MES, PTZ, bicuculline, semicarbizide, kainate, fluorothyl, or in the 6 Hz stimulation model (Appleton and DeVivo, 1974; Hori et al., 1997; Rho et al., 1999; Bough et al., 2000; Thavendiranathan et al., 2000; Bough et al., 2002; Rho et al., 2002; Noh et al., 2003; Zhao et al., 2004; Kwon et al., 2008; Hartman et al., 2008). Published animal studies, in attempting to mirror clinical experience, suggest that dietary effects in controlling excitability in the brain extend beyond species boundaries, but they do little to enhance our knowledge of underlying mechanisms of action. Rather, they raise more issues and highlight the necessity of developing and studying a more clinically relevant model. One critical limitation is that these laboratory investigations have been conducted in normal, not epileptic, rodent brain. What is needed is a chronic model of the KD, employing an animal with early-onset, medically refractory epilepsy, that responds to a particu-
lar formulation of a high-fat diet that best recapitulates all of the essential elements of the human experience.
Cellular electrophysiology Epilepsy is defined by the occurrence of recurrent spontaneous seizures arising from hyperexcitable and hypersynchronous brain activity. The essential currency of neuronal excitability — both normal and aberrant — is the complex array of ion channels (primarily voltage-gated and ligand-gated) that determine the firing properties of neurons and mediate synaptic transmission. Against this increasingly complex backdrop, the conventional thinking has been that seizure activity arises due to perturbations in the normal balance between inhibition and excitation in a localized region, multiple brain areas (linked in a multi-nodal network), or throughout the whole brain. Recently, however, this paradigm has been shown to be too simplified, with epilepsy mechanisms and therapeutic targets addressing heretofore unsuspected physiological functions (synaptic vesicles, brain energetics, etc. — the latter of particular relevance to the KD) (Kim and Rho, 2008). At present, there appears to be at least six important mechanisms through which the currently available AEDs exert their anticonvulsant action. These are summarized in Table 2. As should be noted, and not surprisingly, the vast majority of molecular targets are ion channels and transporters that are localized to plasmalemmal membranes. While the precise manners in which AEDs exert clinical activity remain unclear, there is a wealth of in vivo and in vitro experimental data supporting this mechanistic
The ketogenic diet: What has science taught us? Table 2
213
Classification of antiepileptic drug mechanisms.
• Modulation of voltage-dependent Na+ or Ca2+ channels leading to a secondary inhibition of neurotransmitter release (particularly of glutamate) or to inhibition of intrinsic bursting • Enhancement of GABA-mediated inhibition or other modulatory effects on GABA uptake and metabolism • Inhibition of synaptic excitation mediated by ionotropic glutamate receptors • Modulation of synaptic release, particularly of glutamate, through direct actions on release machinery (SV2A and ␣2␦) • Activation of voltage-dependent K+ channels and improved spike-frequency adaptation • Enhanced activation of dendritic hyperpolarization-activated cation (HCN)-channels and suppressed action potential initiation by dendritic inputs From: White, H.S., Rho, J.M., 2010. Mechanisms of Action of Antiepileptic Drugs. Professional Communications, Inc., New York.
framework. In this light, a fundamental question pertaining to KD effects at the cellular and molecular levels has been whether any of the metabolic substrates elaborated by this ‘‘non-pharmacological’’ intervention can interact with ion channels that regulate neuronal excitability. Thio and colleagues found that acute application of BHB or ACA did not affect standard measures of synaptic transmission in hippocampus (Thio et al., 2000). Specifically, these ketones did not affect GABAA receptors, ionotropic glutamate receptors, or voltage-gated sodium channels over a wide concentration range (300 M to 10 mM). The fact that ACA was ineffective in their hands was surprising, especially given its clear anticonvulsant effects when administered in vivo (Rho et al., 2002). These negative results may have been influenced in part by the facts that: (1) ketone bodies were infused acutely, not chronically; (2) experiments were conducted in normal, not epileptic, brain; and (3) both culture and perfusion media contained glucose, which might have countered a ‘‘ketotic’’ environment. ATP-sensitive potassium (KATP ) channels are logical candidates for linking metabolic changes to cellular membrane
Table 3
excitability. KATP channels are a type of inwardly rectifying potassium channel (Kir6) that is activated when intracellular ATP levels fall. Ma et al. found that ketone bodies reduced the spontaneous firing of GABAergic neurons in rat substantia nigra pars reticulata (SNr), a putative subcortical ‘‘seizure gate’’, by opening KATP channels (Ma et al., 2007). Despite the intuitive appeal and novelty of this observation, an inherent discrepancy remains to be reconciled. Earlier studies had shown that the KD can increase levels of ATP and other bioenergetic substrates through enhanced mitochondrial respiration (Cullingford et al., 2002; Sullivan et al., 2004; Bough et al., 2006; Maalouf et al., 2007; Jarrett et al., 2008). Since high ATP levels block KATP channel activity, it is unclear how opening of these channels is achieved by infusion of ketone bodies in the SNr. Ma et al. suggested that the area subjacent to KATP channels may actually exhibit lower ATP levels than other cellular compartments due to excessive membrane discharge which consumes ATP (Ma et al., 2007). In other words, while ketone bodies might raise global ATP levels, ATP levels near the plasma membrane (where KATP channels are localized) could be substantially
Ketogenic diet: clinical correlates and experimental observations.
Seizure type Age range
Calorie restriction Diet type Ketosis
Fat
Latency to KD effectiveness
Reversal of protective effect when KD discontinued
Clinical correlate
Observation in animal models
KD is effective in many seizure types and epilepsy syndromes Children extract and utilize ketones from blood more efficiently than older individuals Associated with seizure reduction Classic and MCT KDs are equally efficacious Ketosis is necessary but not sufficient for seizure control
KD is effective in models employing a wide variety of seizure paradigms Younger animals respond better to the KD
Practical concerns limit the ketogenic ratio; possible role of fat chain length and degree of saturation (e.g., PUFA) Seizures may be reduced during the pre-diet fast or after a latency of days to weeks Rapid (hours)
Increases seizure threshold Classic and MCT diets both increase seizure threshold A threshold level of ketosis is necessary but not sufficient to explain anti-seizure effects Better effectiveness with higher ketogenic ratios; uncertain if type of fat is a critical variable Several days
Rapid (hours)
Adapted from Stafstrom (2004). Abbreviations: KD, ketogenic diet; MCT, medium chain triglycerides; PUFA, polyunsaturated fatty acid.
214 decreased, and as such, KATP channels could be recruited to dampen neuronal excitability. Although this is a potentially elegant solution to the dilemma posed, there are yet no data directly supporting this hypothesis. Taken together, the role of ketone bodies as direct anticonvulsant mediators remains uncertain, especially since a clear and convincing correlation between the degree of ketonemia and seizure control has yet to be established (Bough et al., 2000; Gilbert et al., 2000). Further actions of metabolic substrates have recently been reported. Kawamura and colleagues evaluated the electrophysiological effects of reduced glucose (a consistent finding in patients successfully treated with the KD) — importantly, under conditions of adequate or enhanced ATP levels — in CA3 hippocampal pyramidal neurons using wholecell recording techniques (Kawamura et al., 2010). These authors found that glucose restriction led to ATP release through pannexin hemichannels on CA3 neurons, and that the increased extracellular ATP, upon rapid degradation by ectonucleotidases to adenosine, resulted in activation of adenosine A1 receptors which was also shown to be coupled to opening of plasmalemmal KATP channels. While this study revealed a novel mechanism of metabolic autocrine regulation, involving close cooperativity among pannexin hemichannels, adenosine receptors and KATP channels, it is yet uncertain whether adenosine is the key metabolic mediator of the KD’s anticonvulsant activity. The contribution of adenosine is plausible, however, given the well-documented role of this endogenous purine in suppressing cellular excitability (Boison, 2009). There is yet another intriguing link between metabolic substrates and neuronal excitability. Juge and colleagues recently reported that ACA inhibits vesicular glutamate transporters (VGLUTs), which are required for exocytotic release of the excitatory neurotransmitter glutamate, specifically by competing with an anion-dependent regulatory site on presynaptic vesicles (Juge et al., 2010). These authors demonstrated that ACA decreased the quantal size of excitatory neurotransmission at hippocampal synapses, and suppressed glutamate release and seizures evoked by the convulsant 4-aminopyridine in rats. So, is this a plausible explanation for the acute in vivo effects of ACA observed nearly eight decades earlier? Possibly, but ACA is reported to have other actions as well, notably effects on mitochondria (Maalouf et al., 2007; Bentourkia et al., 2009). The other caveat is that ACA is highly unstable, and undergoes spontaneous decarboxylation to acetone; further, in the presence of BHB dehydrogenase, is interconverted to the major ketone body BHB. Thus, it appears that seemingly straightforward metabolic substrates are players in a more complex arena.
Metabolic pathways relevant to KD mechanisms Key clinical observations regarding KD use provide starting points from which to investigate underlying mechanisms (Table 3). In this section, we discuss some recent trends in KD research, focusing on studies of biochemical pathways that could link metabolism with neuronal excitability. The original formulation of the KD was based on the assumption
J.M. Rho, C.E. Stafstrom that the diet mimicked the fasting state, which was known to reduce seizures. The KD restricts carbohydrate intake and provides energy from fat breakdown, leading to ketosis, but the observational leap to exactly how ketosis (or some other factor) constrains cellular excitability remains unknown. (The effects of fatty acids on neuronal excitability, another possible mechanism of KD action, are discussed elsewhere in this volume.) Another key feature is that ingestion of a small amount of carbohydrate in a patient on the KD results in rapid loss of seizure control (Huttenlocher, 1976), leading to the hypothesis that carbohydrate restriction could be protective in epilepsy (Greene et al., 2003; Yamada, 2008). By utilizing ketones as the energy source, the KD bypasses glycolysis, raising the possibility that glycolytic inhibition itself might also protect against seizures. 2-Deoxy-D-glucose (2DG) is a glucose analog differing from glucose only by substitution of a single oxygen atom in the 2 -position. 2DG cannot be metabolized and inhibits glycolysis by blocking the glycolytic enzyme, phosphoglucose isomerase, thereby preventing the conversion of glucose-6phosphate to fructose-6-phosphate. 2DG has been shown to be a potent anticonvulsant and antiepileptic agent in several animal models, including kindling, audiogenic seizures in Fring’s mice and 6-Hz corneal stimulation (Garriga-Canut et al., 2006; Stafstrom et al., 2009), as well as in vitro in CA3 neurons in hippocampal slices exposed to elevated extracellular K+ , bicuculline, or 4-aminopyridine (Stafstrom et al., 2009) or metabotropic Group 1 agonists (Pan et al., 2008). Seizure-suppressing effects of 2DG are seen both acutely and chronically. The acute anticonvulsant effects of 2DG in vitro and in vivo against both ictal and interictal activity suggest that 2DG may exert direct actions at the synaptic or membrane levels, but through mechanisms independent of altered gene expression (Stafstrom et al., 2009). Acute effects of 2DG could be related to rapid-onset metabolic or electrophysiological consequences of glycolytic inhibition leading to reduction of network synchronization. For example, there could be an effect on systemic lipid metabolism, mitochondrial function, or the phosphorylation state of GABAA receptor subunits (Pumain et al., 2008), each of which can influence neuronal excitability. In whole-cell recordings of CA3 neurons in hippocampal slices, acute application of 2DG suppressed spontaneous excitatory postsynaptic currents (EPSCs) after transient epileptic activity induced by elevated extracellular potassium or metabotropic Group 1 agonists, but not in normal slices, implicating an activity-dependence to effects of glycolytic inhibition (Pan et al., 2009). These and other potential acute mechanisms are currently under study. The chronic antiepileptic effects of 2DG have been related to the molecular regulation of genes for brainderived neurotrophic factor (BDNF) and its receptor, tyrosine kinase B (trkB). Repression of both BDNF and trkB expression are required for the progression of kindling (He et al., 2004). 2DG suppresses seizure-induced increases in BDNF and trkB, mediated by the transcriptional repressor neuron restrictive silencing factor (NRSF) and its nicotinamide adenine dinucleotide (NADH)-sensitive co-repressor carboxy-terminal binding protein (CtBP). NRSF and CtBP act at the promoter regions of BDNF and trkB genes (GarrigaCanut et al., 2006). During seizure activity, when glycolysis
The ketogenic diet: What has science taught us?
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Figure 1 Glucose metabolism and points at which interventions could affect neuronal excitability and seizure control Glucose can be diverted to the pentose phosphate shunt (PPP) via fructose-1,6-diphosphate (FDP). 2-Deoxy-2-glucose (2DG) inhibits glycolysis by blocking the phosphoglucose isomerase step (see text). The ketogenic diet (KD), via ketone bodies, bypasses glycolysis by providing acetyl-CoA (ACoA) to the TCA (tricarboxylic acid cycle) after glycolysis. Anaplerotic compounds ‘‘refill’’ depleted intermediates from the TCA. Other abbreviations: -OHB, beta-hydroxybutyrate; AcAc, acetoacetate; PDH, pyruvate dehydrogenase.
and glucose production are increased, elevated levels of NADH causes dissociation of CtBP from NRSF, decreasing transcriptional repression and resulting in increased expression of BDNF and trkB. In the presence of 2DG, which reduces NADH levels as a consequence of glycolytic inhibition, the NRSF-CtBP complex maintains repression of BDNF and trkB, and kindling progression is slowed (Garriga-Canut et al., 2006; Huang and McNamara, 2006). These chronic anticonvulsant and antiepileptic effects of 2DG, coupled with its favorable safety profile (Stafstrom et al., 2009), position this compound as a viable candidate for clinical trials and raise the possibility that this agent can modify both seizure susceptibility and disease progression. Continuing the theme that a key KD mechanism might involve alteration of glucose metabolism, another component of the glycolytic pathway, fructose-1,6-diphosphate (FDP), has been shown to exert acute anticonvulsant activity in several seizure models in adult rats including kainate, pilocarpine, pentylenetetrazole, and kindling (Lian et al., 2007; Ding et al., 2010). Indeed, FDP was more effective as an anticonvulsant than 2DG, KD, and valproate in these studies. FDP increases glucose flux from glycolysis into the pentose phosphate pathway (PPP). NADPH generated in the PPP reduces glutathione, which has anticonvulsant activity. Therefore, FDP may exert an endogenous anticonvulsant (and perhaps anti-oxidant) action (Stringer and Xu, 2008), but the precise mechanism by which FDP produces an anticonvulsant effect remains unclear. Another dietary approach to epilepsy derives from the observation that seizures cause a deficiency in tricarboxylic acid cycle (TCA) intermediates (especially ␣-ketoglutarate and oxaloacetate), leading to increased excitability and possibly increased seizures. It has been hypothesized that ‘‘refilling’’ these deficient compounds, a process called ‘‘anaplerosis’’, might oppose seizure generation. One such anaplerotic compound, triheptanoin, has recently been investigated in both acute and chronic seizure models (Willis
et al., 2010). Mice fed triheptanoin exhibited delayed development of corneal kindled seizures and triheptanoin feeding increased PTZ seizure threshold in chronically epileptic mice that had undergone status epilepticus 3 weeks before PTZ testing (Willis et al., 2010). Therefore, like 2DG, anaplerotic compounds alter both acute and chronic seizure susceptibility. Anaplerosis represents a novel approach that expands the potential metabolic modifications that could be anticonvulsant or antiepileptic. Together, results from studies of the KD, 2DG, FDP, and anaplerosis suggest that modification of metabolic pathways such as glycolysis could be a possible novel mechanism for treatment of seizures (Fig. 1). A final example of the involvement of a metabolic pathway that could play a role in KD action is the endogenous peptide leptin. Leptin is part of the hormonal system that limits energy intake and expenditure and is intimately involved in appetite regulation, but it also exerts modulatory effects on neuronal excitability and suppresses seizures (Harvey, 2007; Obeid et al., 2010). Administration of leptin to rats undergoing either focal or generalized seizures resulted in shorter, less frequent seizures, possibly through alteration of AMPA receptor-mediated synaptic transmission (Xu et al., 2008). Since the KD causes a rise in leptin levels (Xu et al., 2008), it is possible that the KD’s mechanism, at least in part, may be related to leptin-associated reduction in synaptic excitability.
Conclusions It is often stated that no single mechanism is likely to explain the clinical effects of the KD. We concur with this viewpoint, and certainly, the same could be said of virtually all AEDs used in clinical practice. However, such a conclusion should not dissuade us from attempting to elucidate the multiple, likely interacting mechanisms by which the KD constrains neuronal hyperexcitability and suppresses seizures. In fact,
216 the complexity of the KD mechanism of action should be viewed as ‘‘a guide and friend’’, enabling the development of novel therapies based on the intersection between metabolism and neuronal excitability. Importantly, elucidation of critical control points in the epileptic cellular network — ones that could potentially be influenced by shifts in metabolic activity — lays the foundation of an exciting new direction in epilepsy therapeutics. Science has indeed come a long way toward unraveling KD mechanisms and we anticipate further progress, to the eventual benefit of the thousands of children suffering from intractable epilepsy.
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