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Noebels JL, Avoli M, Rogawski MA, et al., editors. Jasper's Basic Mechanisms of the Epilepsies. 5th edition. New York: Oxford University Press; 2024. doi: 10.1093/med/9780197549469.003.0079
At the turn of the first century of ketogenic diet (KD) use for medically intractable epilepsy, investigators have uncovered a multiplicity of potential mechanisms that may underlie the clinical effects of this metabolism-based treatment. In the process, there have emerged important insights into the biochemical and metabolic bases of epilepsy, and novel approaches and targets that can be exploited for experimental and clinical therapeutics. Historically, metabolic changes thought to mediate the KD’s antiseizure effects have included—but are not limited to—ketosis, glycolytic restriction, increased purinergic and GABAergic neurotransmission, fatty acid modulation of ion channels, improved cellular and mitochondrial bioenergetics, and a reduction in oxidative stress. More recently, it has been shown that the KD and its variants, such as the medium-chain triglyceride diet, may induce anti-inflammatory, neuroprotective, epigenetic, and even antiepileptogenic effects. Finally, the gut microbiome has been linked to enhanced central inhibitory-excitatory balance through changes in the blood metabolome. As dietary treatments have been increasingly shown to evoke a wide array of metabolic, physiologic, and hormonal effects, future research will undoubtedly reveal an even more complex mechanistic framework for KD action, but one which should enable the development of improved dietary formulations, or simplified treatments based on the biology of specific biochemical substrates and enzymes, not only for epilepsy but also potentially for a broader range of neurological disorders.
The ketogenic diet (KD) is a high-fat, low-carbohydrate, adequate protein diet that has been employed as a treatment for medically refractory epilepsy for a century. This “alternative” therapy was originally designed to mimic the biochemical changes associated with fasting, a treatment reported anecdotally over millennia to control seizure activity (Freeman et al., 2006). The hallmark features of KD treatment are the production of ketone bodies (principally β-hydroxybutyrate [BHB], acetoacetate [ACA], and acetone)—products of fatty acid oxidation in the liver—and reduced blood glucose levels. Ketone bodies provide an alternative substrate to glucose for energy utilization, and, in the developing brain, also constitute essential building blocks for the biosynthesis of cell membranes and lipids (Morris, 2005; Puchalska and Crawford, 2017).
Throughout much of the past century, the popularity of the KD has waxed and waned. Initial enthusiasm was fueled by dramatic success rates, reported entirely in uncontrolled studies, but was quickly supplanted by new antiseizure medications (ASMs) such as phenytoin that became available in the 1930s (Rho and White, 2018). Clinicians found it more convenient to administer a drug than to implement a regimen requiring scrupulous attention to foodstuffs and avoidance of antiketogenic carbohydrates. Notwithstanding the prolonged stigma of being a fad therapy and one without a credible scientific basis, the KD experienced a major resurgence in the late 1990s, mostly due to serendipitous media attention and the continued failure of new ASMs to offer significantly enhanced clinical efficacy (Chen et al., 2018).
Today, the KD and its modern variants such as the medium-chain triglyceride (MCT) diet, modified Atkins diet (MAD), and low-glycemic index therapy (LGIT) all have been shown to be effective in controlling seizures in patients who have failed to respond to ASMs (Neal et al., 2008, 2009; Muzykewicz et al., 2009; Martin-McGill et al., 2020; Sondhi et al., 2020; Lyons et al., 2020; Sourbron et al., 2020; Husari and Cervenka, 2020). The growing number of clinical KD treatment centers throughout the world is a testament to the notion that irrespective of cultural and ethnic differences that define dietary and nutritional practices, a fundamental shift from carbohydrate-based consumption to fatty acid oxidation results in similar clinical effects. Despite such broad use and the heightened basic-translational research over the past decade or two, surprisingly little is understood about the KD’s underlying mechanisms of action. This may be due to the inherently complex interplay between the network dynamics of the human brain (particularly in disease states and during neurodevelopment) and the myriad biochemical and physiological changes evoked by consumption of a strikingly modified diet. The whole may be greater than the sum of its parts, and it has not always been straightforward to determine cause-and-effect relationships, that is, whether specific molecular and cellular alterations observed are relevant or simply represent epiphenomena. This knowledge gap and inherit complexity have hindered efforts to develop improved or simplified treatments (such as a “KD in a pill”) that obviate the strict adherence to protocol that the KD requires (Rho and Sankar, 2008; Kossoff et al., 2018). However, research efforts have intensified over the past decade, and recent investigations have provided new insights and molecular targets.
Herein we outline the most prominent mechanisms underlying KD action. We introduce these mechanisms chronologically as they were proposed, integrate these ideas with more recent findings, and examine the evidence for the broad neuroprotective properties of the KD—the latter, if validated, would highlight the clinical potential of the KD as a more encompassing disease-modifying intervention. Whether the cellular mechanisms responsible for the clinical utility of the KD for human epilepsies are identical to those observed in animal models, or whether they overlap with the mechanisms that afford clinical benefits for other neurological conditions, remains to be determined. An integration of older and newer ideas regarding underlying mechanisms of KDs might represent the ideal scientific strategy to eventually unlock the secrets of this metabolism-based therapy.
Initial studies into the mechanisms underlying KD action focused on concepts of acidosis, dehydration, and increased ketone body concentrations—largely because these were the readily apparent ideas stemming from clinical implementation and observations. Mild dehydration was postulated as necessary, possibly to maximize concentrations of ketone bodies, which were believed to render anticonvulsant effects. As discussed later, however, peripheral ketone body concentrations alone (whether measured in urine or blood) are not tightly correlated with seizure control, and there is no evidence that dehydration or fluid restriction is necessary for clinical efficacy. Nonetheless, because ketolytic metabolism generates protons and pH-lowering metabolic products, decreased pH (i.e., acidosis) was also considered initially to be a key aspect of the KD but with mostly evidenced in blood (Dekaban, 1966; Yancy et al., 2007). The prevailing notion has been that diet-induced ketosis does not cause cerebral acidosis (Al-Mudallal et al., 1996). To date, there is no clear evidence that a KD significantly lowers brain pH or that changes in pH are associated with its anticonvulsant activity.
Despite these negative results, however, it is possible that the KD may induce dynamic and differential pH changes in local microdomains; this possibility has yet to be assayed during KD treatment. Indeed, local compartments have been shown to exhibit differential pH regulation during neuronal activity, and recent work has highlighted pH-related anticonvulsant mechanisms. For example, acidosis in vivo reduced seizure activity via activation of a depolarizing acid-sensing ion channel 1a (ASIC1a) localized to hippocampal interneurons (Weng et al., 2010). Other studies have shown that decreased intracellular pH during periods of increased neuronal excitability releases adenosine and decreases excitatory synaptic transmission and bursting in in vitro hippocampal slices (Dulla et al., 2009). Thus, pH-related mechanisms may offer new prospects for anticonvulsant therapies, and techniques enabling higher resolution studies of pH dynamics in vivo and within neuron-glia microdomains may resolve definitively whether the KD acts in part by changing pH—which can influence proton-sensitive ion channels such as glutamatergic N-methyl-D-aspartate (NMDA) receptors and specific GABAA receptor isoforms.
From decades of clinical experience, it has been observed that almost any diet resulting in ketonemia and/or reduced blood glucose levels can produce an anticonvulsant effect. Comparable clinical efficacy has been seen using KDs comprised of either long-chain triglycerides (LCTs) or MCTs (Neal et al., 2009). Since the early 2000s, utilization and validation of both MAD and the LGIT have increased (Sondhi et al., 2020); the former allows for more liberal carbohydrate consumption and does not significantly restrict protein intake compared to the classic KD, whereas the latter was developed to mirror reduced but steady glucose levels during KD treatment; as such, it is based on a fundamental adherence to foods with low glycemic indices. The glycemic index is a value that describes the extent to which a carbohydrate is absorbed and elevates blood glucose, and lower indices correlate with slower insulin responses compared to glucose. Intriguingly, the LGIT does not induce the prominent ketosis seen with the classic KD, MCT diets, or the MAD (Muzykewicz et al., 2009). Figure 79–1 summarizes the relative composition and biochemistry of the major ketogenic diets.
Ketogenic diets, metabolism, and the production of bioactive compounds associated with seizure control. A. Several ketogenic diets have shown efficacy in seizure control, particularly in patients with drug-resistant epilepsy. Differences between the (more...)
Although there are patients with epilepsy who respond dramatically within days of initiating the KD, maximum efficacy is not generally achieved immediately and can take 1–3 months, suggesting that longer-term adaptive metabolic and/or epigenetic mechanisms may be recruited (Freeman et al., 2006; Kossoff et al., 2018; and see below). These adaptations are likely generalized throughout the epileptic brain, irrespective of underlying pathology or genetic predisposition to seizures, because the KD is an effective treatment for diverse epileptic conditions (Kossoff et al., 2018).
The bulk of the existing experimental literature pertaining to the KD involves studies in which various high-fat treatments are implemented prior to acute provocation with either electrical or chemoconvulsant stimulation in rodents. In these studies, animal diets have modeled the classic LCT (long-chain triglyceride) diet with a range from 4:1 to approximately 6:1 ketogenic ratio of fats to carbohydrates plus protein (by weight). In general, irrespective of precise dietary formulation—insofar as ketosis is seen (but not always), reflecting a shift from primarily glycolysis to intermediary metabolism–anticonvulsant effects have been observed. Indeed, whether seizures are provoked by corneal electroshock, hydration electroshock, maximal electroshock, pentylenetetrazol (PTZ), bicuculline, semicarbazide, kainate, flurothyl, or 6 Hz stimulation, chronic pretreatment with a KD appears to render anticonvulsant effects (Bough and Eagles, 1999; Likhodii et al., 2000; Su et al., 2000; Bough et al., 2002; Nylen et al., 2005; Raffo et al., 2008; Lusardi et al., 2015; Dustin and Stafstrom, 2016; Sanya et al., 2016). It should be noted that the KD is not anticonvulsant in all acute animal models, particularly in mice, and its effects may be of limited duration or can even exacerbate seizures under some circumstances (Bough et al., 2000). These mixed results may raise valid concerns about the relevance of these studies. And consistent with these laboratory observations, the KD is also not universally effective in patients with medically intractable epilepsy.
Further complicating interpretation of the animal literature, the highly variable methodologies used (e.g., use of calorie restriction, age at initiation and duration of therapy, dietary ratios and formulations, timing of treatment, mode of seizure induction, etc.) have made robust cross-comparisons nearly impossible. Regarding observations in rodent models, anticonvulsant efficacy of 4:1 or 6:1 KDs may be confounded by the fact that intake of vitamins, minerals, and antioxidants is not always controlled for. Highlighting this cautionary note, when a balanced KD was utilized in the PTZ, kainate, or flurothyl models, anticonvulsant efficacy was not observed (Samala et al., 2008). Thus, acute animal studies highlight additional complexities with experimental approaches and challenges in our ability to translate animal research to the human epileptic condition.
Chronic seizure models of medically intractable epilepsy have repeatedly demonstrated responsiveness to clinically validated formulations of the KD. That said, it is also important to recognize that species differences, particularly with respect to fatty acid metabolism and blood–brain barrier properties, may dictate incongruency of experimental results.
Muller-Schwarze and colleagues (1999) provided the first evidence that a KD can retard epileptogenesis in a chronic animal model. In this study, rats were first subjected to kainate-induced status epilepticus, and then treated with a KD. Seizure frequency and duration, recorded after the latent period, were both significantly lower in the KD-treated group compared to controls. Further, there was a significant reduction in the extent of mossy fiber sprouting in the KD-fed group. In another model of chronic epilepsy, the KD was shown to prolong lifespan in succinic semialdehyde dehydrogenase (SSADH)-deficient (Aldh5a1−/−) mice, which are characterized by GABA deficiency, recurrent seizures, and early demise. Interestingly, KD treatment restored spontaneous inhibitory postsynaptic currents (IPSCs) to control levels, effects that were later attributed to an increase in the number of mitochondria in hippocampus, as well as restoration of reduced hippocampal ATP to control levels (Nylen et al., 2008).
In line with results in induced chronic models, the KD was also effective in genetic epilepsy models. The EL mouse is a seizure-susceptible inbred strain believed to represent a model of multifactorial idiopathic focal epilepsy with secondary generalization (Meidenbauer et al., 2011). Environmental stimulation such as repetitive handling can induce seizures in EL mice and facilitate epileptogenesis beginning at postnatal day 30 (P30). Generalized seizures normally manifest by the second postnatal month and persist throughout later life. When fed a 4.75:1 KD formula over a 10-week period, seizure susceptibility scores were significantly reduced compared to controls after 3 weeks, but this difference disappeared by week 7 (Todorova et al., 2000). These transient results were comparable to what had been reported earlier in the kindling model (Hori et al., 1997). Similar effects were seen using a 6.3:1 KD in spontaneously epileptic Kcna1-null mice (lacking the gene encoding the delayed rectifier potassium channel α subunit, Kv1.1) (Fenoglio-Simeone et al., 2009; Kim et al., 2015a; Simeone et al., 2021). The frequency of spontaneous recurrent seizures was significantly reduced in KD-fed mice compared to wild-type controls.
Ultimately, to produce anticonvulsant effects, the KD must reduce neuronal hyperexcitability and hypersynchrony. The essential currency of neuronal excitability—both normal and aberrant—is the complex array of primarily voltage-gated and ligand-gated ion channels that determine the firing properties of neurons and mediate synaptic neurotransmission. At present, there appear to be at least six important mechanisms through which the currently available ASMs exert their effects (Rogawski et al., 2016), and the great majority of molecular targets are ion channels and transporters localized to plasmalemmal membranes. In this light, a fundamental question in the early renaissance era of KD research pertaining to effects at the cellular and molecular levels has been whether any of the metabolic substrates (e.g., ketone bodies) resulting from this “nonpharmacological” intervention can interact with ion channels known to regulate neuronal excitability. While this has been a long-standing consideration, more recent research demonstrates mostly otherwise. Most of the postulated mechanisms discussed below are considered in the context of multiple lines of evidence, from human data as well as in vivo and in vitro model systems.
Over the past century of research into KD mechanisms, ketone body generation has been widely considered to be a key biochemical step in providing therapeutic benefits, as a result of low glucose conditions. However, effective seizure control may depend upon both ketone bodies and glucose reduction, but in some cases just glycolytic restriction or ketone bodies alone may be sufficient to reduce or eliminate seizures (Garriga-Canut et al., 2006, Kim et al., 2015a).
Ketone bodies are produced through β-oxidation of fatty acids in the liver, yielding three distinct molecules, the most abundant being BHB and AcAc, which can be interconverted by BHB dehydrogenase (Morris, 2005; Newman and Verdin, 2017). The third ketone body, acetone, is found at low levels and is generated by spontaneous decarboxylation of AcAc (Laffel, 1999); BHB and the AcAc are the core molecules that supply energy to the body, and in the relative absence of glucose, they provide an alternative energy source during starvation, or with low-carbohydrate diets (Sherrier and Li, 2019). β-oxidation of fatty acids results in a very efficient source of energy, yielding many more molecules of ATP per fatty acid compared to glucose (Sherrier and Li, 2019). Hence, ketone bodies are thought to provide highly accessible energy for the brain, something fundamentally required for maintaining bioenergetics homeostasis, and through multiple mechanisms detailed below, to prevent seizure activity.
Initial studies into the efficacy of KDs sought to identify a direct role for ketone bodies in seizure control (Keith, 1933; Rho et al., 2002; Likhodii et al., 2003). Many of these studies focused on BHB, since it is the most abundant of the ketone bodies, reaching concentrations of 6–8 mM in the blood during prolonged starvation (Cahill, 2006) and up to 3.5 mM in therapeutic ketosis (van Delft et al., 2010). However, studies attempting to correlate serum ketone body levels with seizure control have led to discordant results. In some studies, BHB levels directly correlated with seizure control at 3, 6, and 12 months after dietary initiation (Gilbert et al., 2000; van Delft et al., 2010). However, BHB levels have not been consistently shown to correlate with seizure control in either clinical or animal studies (Bough et al., 1999; Likhodii et al., 2000; Thavendiranathan et al., 2000; Rho et al., 2002; Hartman et al., 2008; Samala et al., 2008; Buchhalter et al., 2017). By contrast, either acetoacetate or acetone has been demonstrated to control seizures in multiple animal models (Keith, 1933; Rho et al., 2002; Likhodii and Burnham, 2002; Likhodii et al., 2003).
Given the above findings, investigators have sought to determine whether ketone bodies can exert direct effects on membrane excitability much like most ASMs. In this regard, ketone bodies themselves did not alter excitatory or inhibitory hippocampal synaptic neurotransmission or induced epileptiform activity (Thio et al., 2010), at least not acutely under traditional recording conditions. BHB and AcAc also reduced the spontaneous firing of a key brain region implicated in seizure control through activation of KATP channels (Ma et al., 2007; Li et al., 2017), and BHB activated a free fatty acid receptor (FFA3), resulting in the inhibition of N-type voltage-gated calcium channels (Won et al., 2013). BHB has also been shown to induce antiseizure effects through inhibition of mitochondrial permeability transition (Kim et al., 2015b). However, no clear consensus has yet emerged for a direct role for ketone bodies in seizure control.
More recent studies have specifically investigated effects of ketone bodies on cellular functions that indirectly activate processes that can modulate seizure activity. Chronic BHB treatment demonstrated a range of neuro-protective actions in organotypic hippocampal slice cultures (Samoilova et al., 2010; Kim et al., 2015b). Acute BHB and AcAc reduced intracellular reactive oxygen species (ROS) levels (Kim et al., 2007) and enhanced catalase activity in hippocampal slices (Kim et al., 2010). Another study demonstrated that the KD raised oxygen levels in the hippocampus to reduce postictal hypoxia, and attenuated subsequent learning impairment, but not through a BHB-dependent mechanism (Gom et al., 2021). The above studies indicate that ketone bodies alone may be insufficient to provide direct seizure control but could induce a range of beneficial effects important for neuroprotection and possibly prevention of epileptogenesis (Simeone et al., 2018).
While investigators have long been focused on ketone bodies, clinicians (and more recently, researchers) have paid more attention to the role of glucose regulation, owing in part to the observation that seizure control during KD treatment can be abruptly lost when carbohydrates (which would counter ketosis) are ingested (Huttenlocher, 1976). This has led over time to the notion that carbohydrate restriction alone might protect against seizure activity (Greene et al., 2003). Certainly, inhibition of glycolysis by 2-deoxyglucose (2-DG) has been subsequently shown to retard epileptogenesis in an animal model through downstream regulation of transcription (Garriga-Canut et al., 2006). Further amplifying this are studies indicating that reducing total caloric intake can suppress seizures and provide neuroprotection (Greene et al., 2003; Yuen and Sander, 2014; Pani, 2015; Ingram and Roth, 2021). Additionally, cellular electrophysiological experiments have revealed important cooperativity between reduced glucose and regulation of cellular membrane excitability mediated by inhibitory adenosine receptors and ATP-sensitive potassium (KATP) channels (Kawamura et al., 2010). But the strongest evidence to date is the fact that the LGIT is clinically effective in controlling seizure activity in the medically intractable population (Muzykewicz et al., 2009).
To date, there are many interactions that have been documented between KD-induced changes and neurotransmitters and neuromodulators known to influence seizures. Most prominently are increased glutamate, GABA, and the purines ATP and adenosine.
Perhaps paradoxically, glutamate and ATP can each be powerful excitatory neurotransmitters. How can increasing them be beneficial? For both molecules, their KD induction appears to mobilize inhibition indirectly via their role as substrates or via activation of mechanisms outside of the synaptic cleft. For example, glutamate is the precursor to GABA, and increased GABA synthesis is observed during administration of a KD (Yudkoff et al., 2004), thereby providing an opportunity for increased synaptic inhibition. Glutamate is also a substrate for anaplerosis, a biochemical process that maintains the levels of metabolic pathway intermediates in the TCA cycle (Owen et al., 2002), and enabling normal TCA cycle and mitochondrial function. The KD overall stabilizes or increases cellular ATP levels, thereby increasing resilience to stress and bolstering cell energy reserve (DeVivo et al., 1978; Nakazawa et al., 1983; Nylen et al., 2009b; Deng-Bryant et al., 2011; Ahn et al., 2020). A net effect of increased ATP can be an increase in its breakdown products, particularly adenosine, a powerful anticonvulsant molecule with direct presynaptic and postsynaptic effects (Kawamura et al., 2010, 2014). Adenosine also mobilizes epigenetic changes, including many related to metabolism, as discussed in more detail below.
Regarding KD-induced changes in synaptic glutamate, BHB can decrease the activity of the vesicular glutamate transporter (VGLUT) (Juge et al., 2010), thereby decreasing glutamate content in synaptic vesicles and potentially increasing its availability for other roles noted above. Decreased glucose can increase adenosine levels, and KD-induced decreases in adenosine kinase (ADK) can also increase available adenosine (Masino et al., 2011). Either of these changes would increase extracellular adenosine in the synapse, thereby decreasing presynaptic glutamate release and hyperpolarizing postsynaptic membrane potentials. The anticonvulsant effects of medium-chain fatty acids (MCFAs) are also in part via activity of the adenosinergic system and are associated with reduced blood glucose in the absence of reduced dietary carbohydrate (Socała et al., 2015). As discussed below, detailed recent work on postsynaptic changes in glutamate receptor activity found direct fatty acid–induced effects on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor function (Chang et al (2016), opening new possibilities for metabolic approaches for experimental therapeutics. In addition to a glutamate-induced increase in GABA, increased central GABA levels were linked to KD-induced changes in the gut microbiome (Olson et al., 2018), findings that represent an important new line of research (see below).
In the absence of a direct role for ketone bodies or glucose in seizure control, numerous studies have examined potential effects of long-chain fatty acids (LCFAs), which are a principal constituent of the classical KD. This LCFA diet led to profound changes in brain polyunsaturated FA (PUFAs) levels (Fraser et al., 2003, Taha et al., 2005), suggesting that they may play a role in therapeutic efficacy. This is supported by the fact that PUFAs can regulate the activity of a range of ion channels leading to seizure control (Lauritzen et al., 2000; Vreugdenhil et al., 2003), and through reduced neuro-inflammation and oxidative stress (Michael-Titus and Priestley, 2014). However, subsequent clinical studies into high-PUFA diets did not clearly show a reduction in seizure activity (Schlanger et al., 2002; Yuen et al., 2005).
MCFAs, the lipase-generated products of the MCT KD (Huttenlocher et al., 1971; Neal et al., 2008; Liu and Wang, 2013), have also been investigated as a mechanism for seizure control. The MCT KD diet is just as effective in controlling seizures as the classical KD in children with medically intractable epilepsy (Neal et al., 2009), but it is often a more palatable alternative. The MCT diet was initially thought to increase ketosis, since most fats are provided as octanoic acid (60%) and decanoic acid (40%), which would result in enhanced ketone body generation compared to LCFAs (Schonfeld and Wojtczak, 2016). Interestingly, the combined use of both decanoic acid and octanoic acid may lead to preferential oxidation of octanoic acid to elevate decanoic acid levels (Khabbush et al., 2017), establishing a rationale for concomitant use in experimental models, and ultimately in humans. Early studies demonstrated that these MCFAs were found in the plasma during administration of the diet (Haidukewych et al., 1982; Sills et al., 1986a, 1986b), suggesting a potential role for these MCFAs in seizure prevention. Several studies then linked MCFAs to seizure control, where octanoic acid (Wlaź et al., 2012) and decanoic acid (Wlaź et al., 2015) exhibited brain penetrance and activity in several in vivo seizure models, with combined treatment affording enhanced activity. Subsequently, a series of investigations in a model system (Schaf et al., 2019) revealed that these two MCFAs attenuated phosphoinositide turnover (Xu et al., 2007; Chang et al., 2012), consistent with that shown for the widely employed ASM, valproic acid. In addition, these studies identified a range of related MCFAs, demonstrating this effect. These compounds exerted seizure control in several in vitro and in vivo models (Chang et al., 2012, 2013, 2016; Augustin et al., 2018). Importantly, these compounds lacked histone deacetylase activity inhibition, which has been linked to the teratogenic effects of valproic acid (Chang et al., 2013, 2014, 2015, 2016). Some of these MCFAs also function through reducing phosphoinositide recycling, through the modulation of diacylglycerol kinase (DGK) (Kelly et al., 2018), and mutations in multiple DGK isoforms have been linked to seizure activity (Rodriguez de Turco et al., 2001; Leach et al., 2007). Interestingly, this mechanism has also been widely associated with therapeutic approaches for the treatment of bipolar disorder (Wang and Friedman, 1996; Squassina et al., 2009). Thus, these studies identified a new family of compounds, including MCFAs provided by the MCT KD, which may exert direct seizure control.
Glutamate signaling has also been proposed as a mechanistic target of the KD, since this key neurotransmitter is critical for neuronal excitability, initially through effects of altered metabolism in ketosis (Hartman et al., 2007; Yudkoff et al., 2008; Lutas and Yellen, 2013; Hernandez et al., 2019). Recent studies have also identified a direct role for decanoic acid through inhibition of AMPA receptors at therapeutically relevant concentrations (Chang et al., 2016) (Fig. 79–2). These excitatory neurotransmitter receptors are necessary for seizure activity and represent a defined target for the ASM perampanel (Frampton, 2015) in seizure control (Traynelis et al., 2010; Rogawski, 2011). Decanoic acid is a noncompetitive inhibitor of AMPA receptors, with both voltage- and subunit-dependent specificity. Moreover, combination treatment with decanoic acid and perampanel provided direct synergistic inhibition of AMPA receptors, as well as seizure control in both in vitro rodent and human cellular models (Augustin et al., 2018). This synergistic effect is likely due to positive allosteric interactions since perampanel and MCFAs binds to distinct sites of on AMPA receptors (Yelshanskaya et al., 2020).
Novel molecular mechanisms proposed for the medium-chain triglyceride (MCT) ketogenic diet and related compounds. A. The direct inhibition of AMPA receptors, initially identified as a target of decanoic acid, and extended to include related medium-chain (more...)
Inhibition of the key cell signaling complex, mechanistic target of rapamycin complex 1 (mTORC1), has also been proposed as a therapeutic mechanism of KDs. The classical KD involves reduced glucose and insulin signaling (McDaniel et al., 2011; Ostendorf and Wong, 2015), and functions through the PI3K/PKB pathway to reduce mTORC1 activity. Inhibition of this target is widely recognized as a novel approach for developing drug-based approaches for epilepsy treatment (Peng et al., 2017; Brandt et al., 2018; Liu et al., 2020). Recent studies have also identified this mechanism for decanoic acid in down-regulating mTORC1, independent of both glucose and insulin signaling (Warren et al., 2020) (Fig. 79–2). This effect was also demonstrated in both a rat hippocampal slice model, and in iPSC-derived human astrocytes from healthy individuals and those with tuberous sclerosis complex mutations (Warren et al., 2020).
In support of the above preclinical work, the clinical relevance for the role of decanoic acid in seizure control has recently been demonstrated. Through a prospective open-label feasibility study (Schoeler et al., 2021), a new MCT diet that elevates blood levels of decanoic acid was demonstrated to reduce seizure activity in both adults and children, notably in the absence of systemic ketosis. This study further supports a role for decanoic acid rather than ketone bodies as a principal mechanism for the clinical effects of the MCT diet. Whether decanoic acid is an important mediator of any of the other dietary treatments for epilepsy remains unclear.
The mitochondrion is the central organelle in the regulation of energy production and is the site of β-oxidation of fatty acids and the production of ketone bodies in the liver and in astrocytes (Thevenet et al., 2016). Ketone bodies, subsequently liberated through the systemic circulation, provide an alternate energy source when the supply of glucose is limited. In the brain, ketones provide a more effective source of energy per unit of oxygen consumed than glucose (Veech et al., 2001). Long-chain fatty acids are taken up by a carrier system involving three enzymes (carnitine palmitoyl transferase I, carnitine palmitoyl transferase II, and the carnitine-acylcarnitine translocase) and undergo fatty acid oxidation to produce acetyl-CoA. Acetyl-CoA is either used to fuel the TCA cycle for energy production or under high-fat conditions to produce acetoacetyl-CoA, and the subsequent production of BHB and AcAc, with the latter leading to production of acetone through spontaneous decarboxylation (Masino and Rho, 2019). These ketone bodies, and some fats, are then transported through the blood to both neurons and glia via monocarboxylic acid transporters, whereupon in brain cells ketone bodies are converted back into acetyl-CoA, which is used by the TCA cycle to produce energy. The role of energy metabolism in seizure genesis has been clearly demonstrated in the glucose transporter 1 deficiency syndrome (GLUT1-DS), where loss-of-function mutations impair the uptake of glucose at the blood–brain barrier and produce a spectrum of neurological abnormalities— importantly, drug-resistant seizures early in postnatal life (Klepper et al., 2020). The KD is the treatment of choice for GLUT1-DS since ketone bodies can bypass the glucose transport defect through monocarboxylic acid transporters (MCTs) and serve as a critical energy source in this medical condition.
Many studies have demonstrated effects of the KD on mitochondrial function and ATP levels. The classical KD increases hippocampal mitochondrial load and expression of genes associated with the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OxPhos) and decreases expression of glycolysis-associated genes in rodent models (Bough et al., 2006; Noh et al., 2004). In addition, the KD can also reverse the phenotype due to a mutation in succinic semialdehyde dehydrogenase (SSADH) that interrupts GABA metabolism and results in epilepsy (Nylen et al., 2009a), likely through enhancing mitochondrial function (and perhaps biogenesis as well). The MCT KD has also been proposed as inducing mitochondrial load, where TCA cycle enzyme (i.e., citrate synthase) levels were elevated in conjunction with an increase in mitochondrial number following a 6-day period of decanoic acid exposure, through the activation of peroxisome proliferator-activated receptor γ (PPARγ) (Hughes et al., 2014)—an effect that is likely to contribute to the effect of KDs on seizure activity (Knowles et al., 2018). PPARγ activation can lead to reduced inflammation via inhibition of cytokine transcription (Tyagi et al., 2011; Racke and Drew, 2008). In further support of this mechanism, decanoic acid increased citrate synthase levels in primary cells derived from patients with Leigh syndrome—a severe mitochondrial disease (Fassone and Rahman, 2012). Moreover, in this study, decanoic acid increased and decreased the expression of genes involved in fatty acid metabolism and glucose metabolism, respectively (Kanabus et al., 2016). Thus, these studies highlight a key role for the KD in improving mitochondrial capacity—both structurally and functionally, effects that are hypothesized to be important for seizure control.
Another mitochondrial function potentially relevant to KDs is anaplerosis. Low carbohydrate and elevated dietary fat intake in KDs, leading to a reduction in glycolysis and increased fatty acid oxidation and elevations in acetyl-CoA levels, has been proposed for MCFAs of uneven carbon number (Borges and Sonnewald, 2012) and may also be applicable to other MCFAs (Chang et al., 2016). This anaplerotic mechanism is thought to reverse reductions in glutamate, GABA, aspartate, and glutamine (Brekke et al., 2016; Peng et al., 1993), and it may also be a consequence of glycolytic restriction (Mantis et al., 2004; Yudkoff et al., 2005; Melø et al., 2006). In support of the concept that maintaining anaplerosis can be an effective strategy against epileptic seizures, both preclinical and clinical studies have demonstrated early proof of principle (Willis et al., 2010; Thomas et al., 2012; Kim et al., 2013; Pascual et al., 2014; Calvert et al., 2018; Fogle et al., 2019).
Mitochondria also play a pivotal role in the production of reactive oxygen species (ROS), that when elevated to pathological levels can induce oxidative stress and redox imbalance (Jones, 2006). The formation of ROS is an essential part of normal cell function, but under certain conditions where there is an abnormally high mitochondrial membrane potential, ROS generation can increase (Votyakova and Reynolds, 2001) in a deleterious fashion to precipitate seizure activity (Ray et al., 2012). The KD decreases the mitochondrial production of ROS in the brain through enhanced mitochondrial uncoupling (Sullivan et al., 2004) or through the transcription factor PPARγ2 (Simeone et al., 2017a; Knowles et al., 2018), and later studies have provided further evidence that ketone bodies alone can exert similar activity (Maalouf et al., 2007; Kim et al., 2007; Haces et al., 2008; Julio-Amilpas et al., 2015). Furthermore, the MCT KD also reduces ROS production (Balietti et al., 2009), as does decanoic acid, again independent of ketosis (Mett and Muller, 2021). Finally, there is long-standing evidence that the KD increases glutathione biosynthesis (Jarrett et al., 2008), and although the KD may initially produce mild oxidative and electrophilic stress, the subsequent activation of the Nrf2 pathway via redox signaling ultimately leads to an overall improvement in the mitochondrial redox state (Milder et al., 2010, and see below). Taken together, there have emerged multiple lines of evidence indicating that the KD and some of its important constituents afford beneficial antioxidant activity.
Neuroinflammation, which includes microglial and astroglial activation, is a pathological hallmark of acquired epilepsies, and it is thought to be a driving factor for the development and progression of epilepsy (Terrone et al., 2017; Klein et al., 2018). Several mechanisms implicated in KD action result in anti-inflammatory and neuroprotective effects, which together might support the potential antiepileptogenic properties of the diet. Although PUFAs have anti-inflammatory properties, it was found that KD therapy can exert similar effects through alternative mechanisms (Dupuis et al., 2015), including the inhibition of nuclear factor kappa-B (NF-kB) and NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome activation (Youm et al., 2015; Pinto et al., 2018). Anti-inflammatory mechanisms involved in KD therapy also include the activation of transcription factor PPARs (Simeone et al., 2017a). Anti-inflammatory PPARs can be activated through several mechanisms. Specifically, PPARα can be activated by X-box binding protein 1 (XBP1), which is activated by hepatic serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1), which in turn regulates fasting-induced metabolic adaptive programs through the XBP1-PPARα signaling axis (Shao et al., 2014). In addition, as detailed above, decanoic acid was also shown to increase the number of mitochondria by activation of PPARγ (Hughes et al., 2014) as a potential anti-inflammatory and antioxidant mechanism.
Recent studies have provided evidence that the dampening of inflammation is antiepileptogenic. For example, the nonsteroidal anti-inflammatory drug etoricoxib, a selective cyclooxygenase-2 inhibitor, inhibited the development of absence seizures in WAG/Rij rats, a model of absence epilepsy and epileptogenesis (Citraro et al., 2015). In line with those findings, a drug cocktail targeting multiple inflammatory signaling pathways, including IL-1r and COX-2, retarded the development of spontaneous recurrent seizures and limited the extent of mossy fiber sprouting in the lithium-pilocarpine model of status epilepticus in rats (Kwon et al., 2013). Although these findings appear promising, it is important to determine whether the anti-inflammatory properties of the KD play a key role in the antiepileptogenic effects seen with KD treatment.
An additional mechanism through which KD therapy might affect the inflammatory cascade involved in epileptogenesis is the reduction of oxidative stress and the improvement of mitochondrial respiratory complex activity (Greco et al., 2016; Pearson-Smith and Patel, 2017; and see above). Specifically, KD treatment results in potent neuroprotective effects through the reduction of ROS production via activation of mitochondrial uncoupling proteins and the Nrf2 signaling pathway (Sullivan et al., 2004; Milder et al., 2010). These antioxidant properties of the KD are mainly mediated by BHB, which stimulates the cellular endogenous antioxidant system by activation of nuclear factor erythroid-derived 2-related factor 2 (Nrf2). Nrf2 modulates the ratio between oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD+/NADH) and increases the efficiency of the electron transport chain (Pinto et al., 2018). The ability of the KD to influence the Nrf2 pathway is of special interest due to its neuroprotective role in epilepsy (Mazzuferi et al., 2013; Shekh-Ahmad et al., 2019; Pauletti et al., 2019). Increased Nrf2 expression via gene therapy after epilepsy onset reduced spontaneous recurrent seizures evoked by pilocarpine injection in mice (Mazzuferi et al., 2013). In addition, the activation of Nrf2 by the KD also mediates glutathione biosynthesis, enhances mitochondrial antioxidant status, and protects mitochondrial DNA from oxidant-induced damage (Milder et al., 2010). However, whether activation of the Nrf2 pathway contributes to the anticonvulsant or antiepileptogenic effects of the KD remains to be determined. The KD also mediates cytochrome P450 4A-dependent ω- and ω-1-hydroxylation of reactive lipid species, a novel mechanism that might contribute to the anti-inflammatory properties of the KD (Jin et al., 2014). Through mechanisms outlined above the KD is uniquely suited to interfere with several processes of the inflammatory cascade and thus to interfere with the epileptogenic process.
Bidirectional interactions between the gastrointestinal (GI) tract and the central nervous system (CNS) form the “gut-brain-axis.” While the CNS regulates GI tract function through the autonomic nervous system and the neuroendocrine system of the hypothalamic-pituitary-adrenal axis (HPA) (Wang and Wang, 2016), the GI system affects brain function through synthesis of neurotransmitters in the CNS, augmentation of synaptogenesis, activation of the stress response, and the integrity of the blood–brain barrier (BBB). This bidirectional relationship is based on the activity of enteric microbes, a community referred to as the “gut microbiota” (Cryan et al., 2020). This complex interplay has now led firmly to the concept of the “microbiota-gut-brain (MGB) axis.”
As outlined above, inflammation is a major contributor to the development of epilepsy (Vezzani et al., 2013). Because gut microbiota in turn affect immune and endocrine systems (Wang and Wang, 2016), there is a strong scientific rationale for positing that the gut microbiota may affect inflammatory processes in the brain, which are intrinsic to epileptogenesis. Importantly, dysbiosis of the gut microbiome has been associated with a wide range of neuroinflammatory pathologies (Fung et al., 2017; Olson et al., 2018; Marques et al., 2018; Cerovic et al., 2019). Mechanistically, the gut microbiome is the source for a variety of metabolites, which can have a direct impact on the balance of cytokines and immunomodulation, the production of neurotransmitters and neuromodulators, and the function of neurons and glia (Erny et al., 2015; Fung et al., 2017; Lum et al., 2020). Notably, a healthy gut microbiome appears to relate to higher fecal and brain GABA levels (Strandwitz et al., 2019; Altaib et al., 2021). Consequently, dysbiosis of the microbiome is expected to have profound effects on the brain. Consistent with this notion, the use of antibiotics in susceptible patients has been shown to increase the propensity of developing seizures (Sutter et al., 2015). Several studies have shown associations between gut microbiota composition and epilepsy, with marked changes in gut microbiota composition between infants with refractory epilepsy compared to healthy controls (Xie et al., 2017), or in drug-resistant patients compared to drug-sensitive patients, who had a microbiome composition similar to healthy controls (Peng et al., 2018). Exciting new findings suggest that the KD exerts at least part of its antiseizure effects through modulation of the composition of the gut microbiome—specifically through an increased relative abundance of Akkermansia muciniphila and Parabacteroides, a reduction of gamma-glutamyl amino acids in the blood, and a subsequent increase in the hippocampal GABA-glutamate ratio (Olson et al., 2018), changes that correlated with anticonvulsant activity in two mouse models of seizures/epilepsy.
Epileptogenesis—commonly triggered by injuries to the brain—is a battery of spatial-temporal plastic changes that lead to the development of spontaneous recurrent seizures in a previously healthy brain (Klein et al., 2018). Although the underlying mechanisms remain elusive, neuroinflammation, neurodegeneration, and epigenetic changes are well-accepted contributors to the progression of epileptogenesis (Klein et al., 2018). Epigenetic modifications are highly plastic chemical changes that affect gene expression without alterations in the DNA sequence itself. These epigenetic changes are extremely powerful since they can preserve short-lived cellular signals and/or changes in neuronal activity as long-lasting influences on gene expression (Qureshi and Mehler, 2014; Henshall and Kobow, 2015). An increasing number of studies report that epigenetic processes such as DNA methylation, histone acetylation, and noncoding RNA expression are significantly altered in the epigenome of epileptic brains (Graff et al., 2011; Sweatt, 2013; Boison and Rho, 2020). Since epigenetic modifications play a crucial role in the regulation of gene expression, these mechanisms can affect the expression of several genes simultaneously and can represent risk factors for epilepsy. Furthermore, unlike genetic mutations, epigenetic changes are potentially reversible and may constitute a novel target for therapeutic intervention. In this section, we highlight the emerging antiepileptogenic potential of epigenetic modulators, specifically those regulated by the KD.
DNA methylation is the most prominently investigated epigenetic mechanism. The methylation of DNA is catalyzed by DNA methyltransferases (DNMTs) and typically results in transcriptional repression of genes. Changes in global DNA methylation have been observed in epileptic hippocampus in both clinical and experimental settings. In human temporal lobe epilepsy (TLE) samples from resected hippocampus, gene targets with both increased and decreased methylation were identified. Of note, 146 protein-coding genes exhibit altered DNA methylation in the hippocampus of patients with TLE when compared to controls, of which approximately 80% of these gene promoters display hypermethylation, a common and prominent biomarker of sclerotic hippocampal tissue (Miller-Delaney et al., 2015). In rodent models of TLE, similar patterns of hypermethylation in the epileptic hippocampus have been demonstrated in kainic acid–induced status epilepticus (KASE) models (Williams-Karnesky et al., 2013; Ryley Parrish et al., 2013) and in pilocarpine-induced status epilepticus models (Kobow et al., 2013; Lusardi et al., 2015). In support of this, adenosine was identified to be a key regulator of DNA methylation (Williams-Karnesky et al., 2013). Adenosine has a mass effect on biochemical enzyme reactions and is an obligatory end-product of the S-adenosylmethionine (SAM) dependent transmethylation pathway, necessary for the transfer of methyl groups onto DNA (Boison et al., 2002; Williams-Karnesky et al., 2013). As predicted by this biochemical pathway, exogenous application of either adenosine or its complementary end-product homocysteine inhibited the reaction and reduced DNA methylation, whereas addition of the methyl group donor SAM increased DNA methylation in the naive rodent brain (Williams-Karnesky et al., 2013). In further support of this, pharmacological augmentation of adenosine using 5-iodotubercidin (5-ITU), a pharmacological inhibitor of adenosine kinase (ADK), reduced hippocampal DNA methylation by 50%.
In an epileptic brain, the expression of the adenosine metabolizing enzyme ADK is upregulated particularly in astrocytes and causes adenosine deficiency in epileptogenic sclerotic tissue in a variety of rodent models of epilepsy (Gouder et al., 2004; Li et al., 2008) as well as in human specimens resected from patients with TLE and hippocampal sclerosis (Aronica et al., 2011, 2013). Hence, it is believed that lowered adenosine levels in the epileptic brain shift the equilibrium of the S-adenosylhomocysteine (SAH) hydrolase reaction away from the formation of SAH (Mandaviya et al., 2014), an inhibitor of DNA methyltransferase activity (James et al., 2002), thereby increasing the flux of DNA methylation reactions in the epileptic brain. Seizures resulting from the proconvulsant L-methionine-dl-sulfoximine, which increases the methylation flux by increasing the SAM/SAH ratio, can be blocked by adenosine and homocysteine (Schatz et al., 1983; Sellinger et al., 1984; Gill and Schatz, 1985). In the rat systemic KASE model, the direct ventricular administration of adenosine for 10 days significantly reduced epilepsy disease progression, including the progressive increase of spontaneous convulsive seizures and additional mossy fiber sprouting, and restored global DNA methylation to control levels, lasting well after the conclusion of the adenosine delivery (Williams-Karnesky et al., 2013). These findings show that global DNA methylation levels are under the direct control of adenosine, and that disruption of adenosine homeostasis (due to ADK upregulation at the epileptogenic focus) affected DNA methylation levels and altered gene expression in the epileptic brain.
The KD augments adenosine signaling and can affect epileptogenesis through adenosine receptor-independent mechanisms via interference with the transmethylation pathway (Lusardi et al., 2015; Williams-Karnesky et al., 2013), in addition to the adenosine receptor-dependent mechanisms demonstrated by pharmacological and transgenic approaches (Masino et al., 2011) (Fig. 79–3 and Table 79–1). In line with expected short- and long-term effects, a study showed that KDs, but not a conventional ASM (valproic acid), suppressed kindling-induced epileptogenesis, an effect that persisted even after a return to a standard lab diet, while valproic acid attenuated only the seizures without blocking the epileptogenic process. These data demonstrate enduring effects of the KD that are not merely due to seizure suppression (Lusardi et al., 2015). When fed to rats following status epilepticus, the KD not only inhibited the development of spontaneous seizures but also reduced DNA methylation levels both during diet administration and after a return to standard diet (Lusardi et al., 2015; Kobow et al., 2013). Though a direct link between the KD and DNA methylation levels must still be demonstrated, taken collectively, these studies indicate that the lasting effects of the KD may be conferred via adenosine regulation of the DNA methylome, supporting a key mechanism implicated in epilepsy and epileptogenesis.
Seizure suppression by a ketogenic diet (KD) depends on A1R activation. Representative EEG recordings from the CA3 of wild-type (WT) and transgenic mice reflect seizure distribution over a 1-hour time span (top traces) and individual seizures at higher (more...)
Seizure Frequency and Duration of Adenosine 1 Receptor (A1R) and Adenosine Kinase (Adk) Transgenic Mice.
Histones are important proteins that maintain the chromatin structure in eukaryotic cells and regulate gene expression. Histone modifications such as acetylation and deacetylation are essential parts of gene regulation and are mediated by the enzymes histone acetyltransferase and histone deacetylase (HDAC), respectively (Henshall and Kobow, 2015). Altered histone acetylation has been noted in epilepsy patients as well as in animal models of epilepsy and is thought to be associated with epileptogenesis (Hartman et al., 2015; Hauser et al., 2018b, Boison and Rho, 2020). Epileptic seizures triggered the deacetylation of histone H4 at the GluR2 locus (Huang et al., 2002; Tsankova et al., 2004), which is associated with increased neuronal excitability and the initiation of epileptogenesis (Tanaka et al., 2000). Experimental findings support the idea that KD, as well as ketone bodies formed from fatty acid oxidation, such as BHB, ACA, and acetone may have antiepileptogenic potential by inhibiting HDACs (Shimazu et al., 2013; Tanaka et al., 2000; Hartman et al., 2015; Simeone et al., 2017b; Hauser et al., 2018a; Boison and Rho, 2020). In support of this concept, inhibition of HDAC activity by chronic administration of butyrate retarded the development of limbic epileptogenesis and prevented epileptogenic mossy fiber axonal sprouting in a mouse hippocampal kindling model of TLE (Reddy et al., 2018). Another study used tuberous sclerosis complex genetically modified mice (TSC2+/– mice), a mouse model with characteristic developmental deficits, including cognitive abnormalities, autism, and epilepsy. This study showed that altered mTORC1 signaling led to aberrant hippocampal synaptic plasticity, which was prevented by the inhibition of HDAC using trichostatin A (Basu et al., 2019). These studies confirm that the KD reverses seizure-induced histone deacetylation primarily via the BHB-HDAC axis, thus contributing to antiepileptogenesis.
From the above, and while a detailed discussion of the expanding literature on the neuroprotective properties of the KD is beyond the scope of this chapter, it is becoming clearer that the KD may not only retard the processes of epileptogenesis but may also provide direct neuroprotective effects in epileptic brain (Noh et al., 2003, Simeone et al., 2021) through a multitude of mechanisms (Noh et al., 2003, 2005, 2006; Gano et al., 2014), findings that may extend to an expanding number of neurological disorders. The reader is referred to recent reviews for more details on this subject (Gano et al., 2014; McDougall et al., 2018; Yang et al., 2019; Włodarek, 2019; Jensen et al., 2020; Murugan and Boison, 2020).
Based on the foregoing discussion, it is becoming increasingly clear that there are likely broader clinical implications of KD-based therapies than epilepsy alone. As the mechanisms underlying the neuroprotective activity of the KD are fundamental to many disease processes, it should be no surprise that diet can profoundly influence brain function, and in a growing number of instances exert protective and potentially disease-modifying effects as detailed above. As examples, the KD (or various formulations of its key metabolic substrates and enzymes) has been found to ameliorate a range of clinical disorders and/or experimental models such as autism spectrum disorder and Rett syndrome (Mantis et al., 2009; Cheng et al., 2017); pain and other inflammation disorders (Koh et al., 2020; Ruskin et al., 2021); traumatic brain injury (McDougall et al., 2018; Yarar-Fisher et al., 2018); neurodegenerative diseases such as Alzheimer and Parkinson disease (Pinto et al., 2018; Cerovic et al., 2019; Włodarek, 2019; Choi et al., 2021); brain cancer (Thomas and Veznedaroglu, 2020; Talib et al., 2021); diabetes and obesity (Kumar et al., 2021; Tinguely et al., 2021); and headache (Di Lorenzo et al., 2021); among many others—including a growing list of psychiatric disorders—that have been reported in the literature (Stafstrom and Rho, 2012; Branco et al., 2016; Operto et al., 2020; Norwitz et al., 2020; Rawat et al., 2021; Zweers et al., 2021). More recently, it has been suggested that the KD or ketone bodies may serve as a preventative or therapeutic avenue to mitigate the adverse consequences of COVID-19 infection (Ryu et al., 2020, 2021). It should be noted that some of these disorders are comorbid with each other, especially epilepsy, thus presenting the opportunity to ameliorate multiple diseases with a single therapy.
To expand upon the above, and of the many potential uses of the KD, brain cancer is perhaps one of the most compelling targets, given the rising interest in identifying metabolic targets for intervention. The theoretical basis for using the KD to treat cancer is that tumorigenesis relies heavily on glucose, whereas normal cells retain metabolic flexibility and can use ketone bodies for fuel (Seyfried and Mukherjee, 2005; Seyfried et al., 2019). A calorie-restricted KD or other glycolysis-limiting treatment forces higher ketone body production, putting maximal stress on the tumor cells and minimal stress on the normal cells (Seyfried and Mukherjee, 2005; Seyfried et al., 2019). Paradoxically, some aspects of the current standard of care for brain cancer may paradoxically support tumor growth (D’Alterio et al., 2020).
Neurodegenerative diseases are universally associated not only with mitochondrial dysfunction (Lin and Beal, 2006; Pathak et al., 2013) but also inflammation (Stephenson et al., 2018), and inflammation is a hallmark of virtually every chronic disease process throughout the body. In addition to reducing pain and inflammation, the KD appears to enhance motor and cognitive functioning in a model of multiple sclerosis (Kim et al., 2012). Further evidence is provided by recent reports of the KD mitigating MPTP-induced neurotoxicity and microglial activation (Yang and Cheng, 2010), and encephalopathy and seizures in forms of fever-induced epilepsy (Koh et al., 2020, 2021). Ultimately, reducing inflammation could be one of the most important disease-modifying effects of a KD.
In addition to inflammation, disrupted sleep and circadian rhythms are common comorbidities with many diseases. There is emerging evidence in humans and animal models that a KD can improve sleep in children with epilepsy (Hallböök et al., 2007) and circadian rhythmicity in epileptic mice (Fenoglio-Simeone et al., 2009). It is well-known that circadian disruption is associated with epilepsy, psychiatric disorders, and the prevalent metabolic syndrome. Normalization of circadian rhythms alone could yield enormous clinical benefits (Greenhill, 2017).
In 2021, the National Institute of Neurological Disorders and Stroke (NINDS) at the U.S. National Institutes of Health held the first-ever workshop on the KD (Cervenka et al., 2021). While constrained to a virtual platform due to the COVID-19 pandemic, this meeting attracted a multitude of practitioners and researchers throughout the world. One of the primary themes was that of scientific rigor and reproducibility. While this is important for all biomedical research, the challenges are particularly vexing in metabolic paradigms with many possible protocols, variables, and mechanistic angles. New tools, checklists, and “rigor champions” were proposed to help mobilize more compatible and comparative experimental designs and approaches toward data analysis in research involving metabolism-based therapies.
The participants expressed a strong interest in and need for developing additional model systems, both in vitro and in vivo, depending on the specific scientific question, and given the limitations of the aforementioned studies, there was a clear acknowledgment of the inherent difficulties of bench-to-bedside translation in a field as complex as metabolism-based treatments for epilepsy. The special features, particularly metabolic, anatomical, and behavioral, and known similarities versus differences between species (e.g., with respect to whole organism/body physiology and metabolism), are always important considerations. Randomized clinical trials involving humans of all ages have been designed to evaluate the efficacy of KD in drug-resistant epilepsy and specific epileptic syndromes. While the clinical studies to date have been encouraging, the pace has been very slow: enrollment and compliance, among other factors, will continue to be challenging.
Emerging research strategies appear on the verge of deepening and potentially transforming our knowledge about the scientific and translational basis of the KD and related therapies—for example, detailed cell and circuit mapping, biomarker studies, multi-omics and big data approaches, and novel paradigms for clinical research. Such developments promise to reveal a clearer picture of how the varied mechanistic variables work in concert to produce anticonvulsant (and neuroprotective) effects, and are likely to uncover additional relevant mechanisms or to definitively establish whether any of the putative mechanisms revealed in earlier decades are causally related to clinical efficacy. A noninvasive biomarker that could predict efficacy and/or untoward side effects would be particularly useful and would help differentiate patients who would benefit most from metabolic therapy, or those that may need a specific type of dietary or metabolic approach—whether alone or in combination with other treatments. Wearable devices and customizable smartphone apps can also provide feedback to patients and would enable the collection of real-world data, dynamically and comprehensively, to further help identify the important variables related to seizure control.
Finally, it was acknowledged that significant advances in the field of KD therapies may come from attracting clinical, translational, and basic researchers from outside the field of epilepsy (e.g., cancer biologists, experts in artificial intelligence and machine learning, etc). Epilepsy research itself would benefit from a diverse influx of new disciplines and adoption of team science approaches dedicated to deciphering treatments that work more effectively against drug-resistant seizures. Conversely, new scientific insights from fundamental studies of the epileptic brain may shed important light on many other neurological conditions characterized either wholly or in part by metabolic derangements. Ultimately, there is hope that the future of KD research will generate innovative dietary and metabolic approaches that are less restrictive and safe, rendering improved clinical benefits for the third of patients with epilepsy who remain medically refractory, despite the advent of dozens of new ASMs over the past few decades (Chen et al., 2018).
To date, research efforts aimed at elucidating the mechanisms of KD action have not yielded simple or straightforward answers. It is becoming increasingly apparent that the relevant mechanisms are likely diverse and operate in a coordinated and potentially synergistic fashion. The ongoing quest is challenged by the inherent difficulties in understanding neuronal network activity, let alone metabolism, in the context of disease states, and importantly, in the intact patient or animal. Despite these issues, the research reviewed herein provides a clearer link between metabolism and neuronal excitability, and further validates the emerging field of neurometabolism, especially as it relates to epilepsy. It also validates the potential for neuroprotection as a broader opportunity for metabolic approaches, one that will likely be relevant for disease modification. The key proposed mechanisms of the KD and related treatments—including and beyond those described above—are listed in Table 79–2, and Figure 79–4 illustrates the separate yet converging metabolic pathways and fundamental biochemical effects—down to the level of the mitochondrion—that are implicated in KD action.
Tocome.
Major changes in important biochemical pathways reported to exert anticonvulsant and neuroprotective effects in experimental models. Glycolytic restriction or diversion of glucose metabolism, increased fatty acid oxidation leading to the production of (more...)
The evidence for a KD as a successful epilepsy treatment is clear. Multiple retrospective, multicenter and randomized prospective studies document consistent and significant clinical benefits (Neal et al., 2008; Martin-McGill et al., 2020; Sondhi et al., 2020; Lyons et al., 2020; Sourbron et al., 2020; Husari and Cervenka, 2020). The true efficacy of dietary treatments for epilepsy may be underestimated as the KD is rarely used as a first-line therapy. Certainly, by the time the KD is initiated to thwart medically refractory epilepsy, in some instances the severity of the epileptic condition may be too difficult to overcome. Remarkably though, the KD can work in patients who failed to respond to ASMs. A detailed understanding of key KD mechanisms could offer a meaningful adjuvant or ultimately the development of a “diet in a pill,” which may be comprised of multiple compounds modulating key mechanistic targets in a favorable manner (Rho and Sankar, 2008). But while clinical applications of metabolism-based therapy appear to be growing rapidly, there is a continuing need to develop modified dietary formulations with improved efficacy and tolerability (as well as palatability), and to identify new pharmacological targets for drug discovery. Furthermore, the increasing development of more accessible yet effective dietary approaches for treating patients with drug-resistant epilepsy may lead to a significant adoption of these approaches, such that a potential metabolic “first-line” treatment could be offered such as 2-DG (Bialer et al., 2020), one distinct from current pharmacological agents.
It is often stated that no single mechanism is likely to explain the clinical effects of ASMs (Rogawski et al., 2016), and certainly the same could be said for the KD and its variants. The challenge of finding key mediators of KD action is made ever more difficult by the intrinsic complexity of metabolic activity within neurons and glia, which, to be relevant to the epileptic condition, must be interpreted at network levels. In this chapter, we have reviewed many seemingly disparate variables proposed to collectively exert anticonvulsant (and potentially neuroprotective and antiepileptogenic) effects. The fact that a fundamental modification in diet can have such profound, therapeutic effects on a neurological disease underscores the importance of elucidating mechanisms of KD action. In summary, mounting interest and insight into the mechanisms of KD action have laid a promising foundation for metabolic therapy as an emerging strategy for a broad array of neurological disorders.
RSBW is funded by Vitaflo, Ltd, GW Pharma, Ltd, NC3Rs United Kingdom, the Wellcome Trust, and BBSRC United Kingdom. DB receives grant support from the National Institutes of Health (NIH R01NS103740, R01NS065957) and Citizens United for Research in Epilepsy (Epilepsy Catalyst Award). SAM is funded by NIH NS065975 and NIH AT008742. JMR is supported by the Canadian Institutes of Health Research and NIH R21 NS104513. The authors wish to thank David Ruskin for editorial assistance.
RSBW has patents related to MCFAs for therapeutic use (WO2012069790A1, WO2016038379A1, WO2018189113, WO2019002435A1). SAM has no competing interests. JMR has been a paid consultant to Aquestive Pharmaceuticals, Danone Nutricia, Mallinckrodt, Eisai Pharma, and Zogenix. JMR has also served on the Scientific Advisory Board of The Charlie Foundation for Ketogenic Therapies (Santa Monica, California, USA) and for Matthew’s Friends (United Kingdom). DB is a cofounder of PrevEp, and JMR is the Chief Medical Officer for Path Therapeutics, Inc.
This is an open access publication, available online and distributed under the terms of a Creative Commons Attribution-Non Commercial-No Derivatives 4.0 International licence (CC BY-NC-ND 4.0), a copy of which is available at https://creativecommons.org/licenses/by-nc-nd/4.0/. Subject to this license, all rights are reserved.
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