ARTICLE
Auteur(s) : Joseph Vamecq1,
Pierre Maurois2, Pierre Bac2, Bernadette
Delplanque3, Nicole Pages2,4
1INSERM Univ 045131, EA1046, Lille, France
2Neuropharmacology, Pharmacy, Châtenay-Malabry,
France
3NMPA, Univ Paris XI, Orsay, France
4Toxicology, Pharmacy, Illkirch, France
Introduction
We have previously developed evaluation of anticonvulsant and
neuroprotective drugs [1] along with interest in understanding
molecular basis of antiepileptic activity of the ketogenic diet
[2]. In a previous review, emphasis was made on relationships
between the great many changes induced by ketogenic diet and some
putative biological targets potentially capable of inducing
anticonvulsant protection such as ATP-sensitive potassium channels
and the more recently described two pore domains potassium channels
[2]. In this context, the metabolic interactions existing between
neurons and astrocytes were also evoked. Activation of
ATP-sensitive potassium channels by ketogenic diet metabolites has
been recently shown [3] although experimental support for
activation of the two pore domains potassium channels is still
awaited.
The present communication is aimed at focusing on the
astrocyte/neuron metabolic interactions and on the way in which
they might be affected by the ketogenic diet. It gives an account
successively for the historical background of anticonvulsant diets,
the part of ketogenic diet which provides anticonvulsant
protection, the neuron/astrocyte metabolic interactions in normal
feeding conditions (astrocyte/neuron lactate shuttle,
glutamate-induced astrocytic glycolysis activation,
glutamate/glutamine cycle), the neurovascular coupling, the
neuron/astrocyte metabolic interactions upon ketogenic diet
(astrocyte/neuron ketone body shuttle), the putative metabolic
anticonvulsant changes induced by the ketogenic diet (increased CNS
GABAergic tone and reduced glutamate availability for
neurotransmission), other and concluding remarks emphasizing the
convergent metabolic changes of ketogenic diet to protect brain
against epilepsy.
Brief historical account for anticonvulsant diets
Evocations of dietary measures adopted to counteract seizures have
been made in Biblical times and later in Middle Ages by submitting
epileptic patients to “water diet” [4]. In water diet, the uptake
of food is suspended and only access to water is allowed, a
condition which by definition corresponds to starvation.
In the early last century, water diet was replaced by ketogenic
diet in order to improve the compliance of patients to the
therapeutic diet [5, 6].
The prototype nutritional profile of ketogenic diet corresponds
to a diet composition of 80% fats, 15% proteins and 5% hydrates of
carbon. Introduced in the 1920’s, it was eclipsed later in the
following years and decades due to the development of the first
efficacious antiepileptic drugs, phenobarbital and phenytoin [2].
Since the last decade there has been a large regain of interest for
ketogenic diet as an anticonvulsant therapy. This diet has proven
efficacy in many animal seizure models and in human clinical trials
[2]. Interestingly, ketogenic diet may be active against
pharmaco-resistant epilepsy, and because intractable epilepsy can
be an indication for brain surgery, the diet has avoided some
patient to undergo chirurgical brain intervention.
Due to the emerging success of ketogenic diet in improving
epileptic patients, another fat diet, the Atkins’diet, has been
recently challenged as an anticonvulsant diet. This diet was
introduced by Dr Atkins in the 1950’s initially to counteract the
development of obesity. The prototype nutritional profile of the Dr
Atkins diet slightly differs in composition from ketogenic diet and
is composed by 60% fats, 30% proteins and 10% hydrates of carbon.
The Atkins’diet has now also proven efficacy in animal models and
in human epileptic patients [7, 8]. In contrast to ketogenic diet,
cooking preparations compatible with the Atkins’diet may be found
in restaurants and cafeterias, and this is a factor which may help
patient to comply the diet. Modified Atkins’diets are currently
evaluated in terms of threshold for glucose supply allowed to
maintain antiepileptic protection [8].
Another dietary measure which presents with preliminary
experimental anticonvulsant properties is caloric restriction [9].
Finally, some other diets that have been proposed to exhibit
anticonvulsant potentialities, as for instance gluten-free diet
[10, 11] remain to be investigated on a large scale.
What is part of ketogenic diet giving anticonvulsant
protection?
In contrast to several protective and preconditioning effects
induced by fats in which protection may be given by individual
fatty acids (alpha-linolenic acid and tolerance to stroke and
epilepsy) [12] there has been until now a failure to demonstrate
that the anticonvulsant protection results from the presence of
individual fatty acids in the diet. Instead, anticonvulsant
protection might be linked to the metabolic state induced by
ketogenic diet. Though metabolic changes induced by ketogenic diet
on astrocyte/neuron metabolic interactions account for only part of
the pleiotropic effects of ketogenic diet, these changes, in which
neuronal glucose or lactate oxidation are shifted towards ketone
body utilization, include convergent anticonvulsant mechanisms as
developed below in the following sections. Nevertheless,
supplementation with specific fatty acids such as ω-3 fatty acids
has been recommended to protect the cardiovascular system against
the hyperlipaemia associated to ketogenic diet [13].
Neuron/astrocyte metabolic interactions in normal feeding
In normal feeding conditions, glucose represents the main energetic
fuel for brain metabolism. Moreover, brain glucose consumption is
about 20-25% of total body glucose consumption. The glucose may be
oxidized by both neurons and astrocytes. The possibility also
exists for part of glucose being partially oxidized in astrocytes
to complete its oxidation in neurons, using lactate as a shuttle
between astrocytes and neurons (astrocyte/neuron lactate shuttle),
glutamate being capable of boosting astrocytic glycolytic
activation in the same time as taking place in the neuron/astrocyte
glutamate/glutamine cycle.
For neuronal/astrocyte metabolic interactions presented in this
section and the following section Neurovascular coupling), the
reader may be referred to valuable information given elsewhere on
metabolite shuttles and neurovascular coupling [14-17] or on
glutamate/glutamine cycle [18, 19].
The astrocyte/neuron lactate shuttle
Figure 1
represents a classic neuro-glial-vascular unit and stresses the
metabolic supply and utilization of glucose. Glucose may be
transferred from the blood stream to the astrocytes thanks to the
successive interventions of endothelial and astrocytic plasma
membrane GLUT 1 (Glucose Transporter 1). In the astrocyte, glucose
is submitted to glycolysis with as a net result the formation of
pyruvate, NADH and ATP. To proceed to completion, glycolytic
glucose oxidation needs recovery of NAD+ from NADH. This
NADH oxidation is performed during the oxidation of pyruvate to
lactate catalyzed by LDH5 (Lactico-DeHydrogenase 5). The resulting
lactate may be then transferred from the astrocyte to the neuron,
and this occurs upon intervention of astrocytic and neuronal plasma
membrane MCT (MonoCarboxylate Transporters) 1 and 2, respectively.
On the other hand glucose can enter the neuron via neuronal plasma
membrane GLUT3. Both glucose and lactate are energetic fuel for
neuronal metabolism. Lactate is converted back by LDH1 to pyruvate
which may be also formed by glycolysis from glucose. Lactate- and
glucose- derived pyruvate can enter mitochondria to be oxidized by
pyruvate dehydrogenase to acetyl-CoA units further oxidized in the
Krebs’cycle. Glycolytic (cytosolic) and total (cytosolic plus
mitochondrial) oxidations of glucose lead to the formation of 2 and
36-38 ATP, respectively.
When the neuron is supplied in both lactate and glucose, it
oxidizes preferentially lactate. This preference results from the
competition existing between lactate dehydrogenase and glycolytic
glyceraldehyde-3-phosphate for cytosolic NAD+. This
competition is in favour of lactate dehydrogenase, explaining why,
when available, lactate is preferentially used by the neuron.
The glutamate-induced astrocytic glycolysis activation
As mentionned above, astrocytic glycolysis needs NAD+
recycling from NADH, an oxidative reaction ensured by astrocytic
lactate dehydrogenase which converts pyruvate and NADH into lactate
and NAD+. To proceed to completion, astrocytic
glycolysis also needs ADP recycled from ATP. Such a recycling
reaction occurs upon synaptic glutamate neurotransmission (figure 2). After
release by the presynaptic neuron of glutamate in the synaptic
cleft, this neurotransmettor stimulates postsynaptic (and
astrocytic) ionotropic (NMDA and AMPA receptors) and metabotropic
receptors. Subsequent removal of excess glutamate from synaptic
cleft involves presynaptic and astrocytic uptake mechanisms. The
astrocytic glutamate uptake is by far more active than the
presynaptic re-uptake.
Glutamate uptake by the astrocyte is associated to the
concomitant entry of sodium ions (Na+). This entry in
cells of both sodium ions and glutamate catalysed by the astrocytic
glutamate transporters (for a review on astrocytic glutamate
transporters, the reader may be referred to the review of Anderson
and Swanson [20]) triggers a boost in cytosolic ATP hydrolysis to
ADP and therefore induces a huge increase in ADP availability for
glycolysis. The mechanisms are as follows and detailled in figure 2. The amount
of sodium having entered the astrocyte is extruded outside the cell
through the action of the Na+/K+ATPase which
catalyzes the entry of 2 K+ against the exit of 3
Na+ along with hydrolysis of 1 ATP and which then
represents a first site for regenerating ADP from ATP. Astrocytic
glutamate (plus ammonium) is converted to glutamine by glutamine
synthase in a reaction which consumes one molecule of ATP and which
represents a second site for regenerating ADP from ATP. So,
astrocytic handling and metabolism of glutamate may increase hugely
the cytosolic ADP/ATP ratio and hence the glycolytic rates (glucose
to pyruvate conversion) and subsequent formation of lactate,
addressing of which to the neurons provides an immediate important
source of metabolic fuel. This fuel supply is devoted to help
neurons to face the energetic demand required for restoration of
ionic gradients previously modified by the action potential
propagation, for synthesis of new neurotransmitter and their
vesicles and for the forecoming neurotransmitter release which all
represent energy expensive processes. A last remark in this
subsection concerns the stoechiometry between astrocytic glutamate
uptake and astrocytic glucose oxidation. Glucose entering the
glycolysis pathway was thought for a long time to be linked to
glutamate uptake in a 1/1 stoechiometric ratio because as mentioned
above astrocytic glutamate uptake generates 2 ADP from 2 ATP, and
glycolyis 2 ATP from 2ADP. However, recent evidence indicates that
glutamate may be itself a energetic substrate for the astrocyte,
therefore escaping ATP-consuming glutamine conversion and
generating on the opposite ATP production, thus lowering the
astrocytic glucose oxidation/glutamate uptake ratio.
The glutamate/glutamine shuttle
The astrocytic ATP-consuming glutamate synthase mentioned above
allows the conversion of glutamate to the neuro-inactive compound
glutamine. Glutamine may be transferred back to neurons as a
glutamate precursor. Indeed, in neurons it may be converted back to
glutamate by a phosphate-dependent glutaminase. The resulting newly
formed glutamate can then be stored in vesicles for future release
in the synaptic cleft. As mentioned above, its release in the
synaptic cleft induces post-synaptic (and astrocytic) receptor
activation, and its subsequent uptake by astrocytes generates a
pool of intracellular glutamate available for glutamine conversion.
This astrocytic glutamate to glutamine conversion followed by
glutamine export to the neuron in which glutamate is regenerated
back from glutamine, is “vesicled” and released in the synapse, and
subsequently up-taken by the astrocyte represents a metabolic cycle
referred to as the “glutamate/glutamine cycle”.
The neurovascular coupling
In the same time as synaptically released glutamate which is
up-taken by the astrocytes boosts metabolism in these cells, it
also stimulates metabolic exchanges and supply involving the blood
capillary via the induction of a local vasodilation. This
phenomenon is referred to as neurovascular coupling, it is depicted
in figure 3 and
has been reviewed and commented by Bonvento, Sibson and Pellerin
[14].
Neuron/astrocyte metabolic interactions in high fat diet
As mentioned above, in high fat diet, fatty acids represent the
vast majority of energetic substrates and, simply stated, glucose
supply is not far from being turned off. In these conditions, fatty
acids represent the main energetic fuel for the whole body. In this
respect, the main energetic fuel oxidized by neurons is derived
from fatty acids and is represented by ketone bodies
(β-hydroxybutyrate, acetoacetate, acetone). The precise mechanisms
by which these ketones are exactly supplied to neurons have been
recently revisited [21]. Until recently, neuronal supply in ketones
was considered to result from blood delivery of ketone bodies
produced by liver. Hepatic production of ketone bodies from fatty
acids involves two main successive pathways: fatty acid oxidation
to acetyl-CoA units (mitochondrial β-oxidation) and synthesis of
ketone bodies from these acetyl-CoA units (mitochondrial
ketogenesis). This classic view was based on the prerogative of
liver (plus kidney and intestine) to catalyse ketogenesis.
Nevertheless, it was recently realised that astrocytes, like
hepatocytes, also contain the mitochondrial enzymic equipment
required for catalyzing not only fatty acid oxidation but also
ketogenesis [21]. This has led to propose the existence of an
active astrocyte/neuron ketone body shuttle model [21] in which
fatty acids are supplied from blood to astrocytes to be
intra-mitochondrially oxidized in these cells in order to generate
energy covering their needs and in order to generate ketone bodies,
energetic fuel preferentially utilized by neurons although to a
lesser extend by astrocytes.
Putative anticonvulsant changes induced by ketogenic diet
Extensive consideration of many putative anticonvulsant mechanisms
were reviewed in detail elsewhere [2, 22]. Focusing on the
metabolic interactions between astrocytes and neurons, two
mechanisms were proposed by Yudkoff and coworkers: increased
cerebral GABAergic tone and decreased glutamatergic tone [23, 24].
The increased GABAergic tone [24] is explained by the fact that
in ketogenic diet-induced neuronal oxidation of ketone bodies
(3-hydroxybutyrate and subsequently acetoacetate) the entirety of
these metabolic substrates is readily converted to acetyl-CoA. In
contrast, in glucose feeding-induced neuronal oxidation of glucose
and lactate, a portion, even if limited, of the metabolic
substrates do not readily recover as acetyl-CoA, being “buffered”
in the form of lactate or as glycolytic intermediates. The higher
rates of acetyl-CoA formation from ketone bodies versus lactate
plus glucose have been proposed to “aspirate” in the Krebs’cycle
oxaloacetate for condensation with acetyl-CoA and further
metabolism. As a consequence, lesser oxaloacetate becomes available
for the reversible conversion of oxaloacetate and glutamate into
α-ketoglutarate and aspartate catalyzed by aspartate/glutamate
transaminase. A support to this view has been given by showing that
acetoacetate lowered aspartate formation rate in cultured
astrocytes incubated with glutamate [24]. Importantly, it was shown
also on synaptosomes that more glutamate was consequently available
for GABA synthesis, acetoacetate being finally shown to increase
GABA levels [24]. The putative dynamics of these events are
illustrated on figure
4.
In the same time as a higher amount of glutamate is directed
towards GABA formation (see the preceding paragraph), it has been
proposed that less glutamate was available for glutamatergic
neurotransmission [23, 24]. Two mechanisms involving the escape of
metabolites from the glutamate/glutamine cycle for notably removal
in blood have been proposed in ketotic states (figure 5). The increase of
blood leucine (starvation) has been proposed to enhance the
leucine/glutamine exchange between blood capillary and astrocyte
with as a result increased astrocytic leucine levels and blood
glutamine removal [24]. The other mechanism results from an
increase in the ratio of brain on blood levels in alanine but not
in other aminoacids such as glutamine and leucine (starvation and
ketogenic diet versus normal feeding) [23]. In these conditions, it
has been proposed that the abnormally high gradient of alanine
concentrations existing between brain and blood should favour brain
to blood transit of this aminoacid in starvation and high fat diet
[23]. This tendency for a removal of alanine out of the astrocyte
(to blood) has been suggested to displace the equilibrium of
glutamate/alanine transaminase in favour of glutamate (plus
pyruvate) conversion into alanine (plus α-ketoglutarate), inducing
a leak of glutamate from the glutamate/glutamine cycle and hence
some loss of glutamate for glutamatergic neurotransmission
[23].
Concluding remarks and perspective
Though many properties of ketogenic diet have been proposed to
account for antiepileptic activity, astrocyte/neuron metabolic
interactions (astrocyte/neuron lactate shuttle, glutamate-induced
astrocytic glycolysis activation, glutamate/glutamine cycle,
astrocyte/neuron ketone body shuttle) also appear to be affected in
dictinct degrees by ketogenic diet and in some way to contribute to
anticonvulsant protection. In normal feeding, astrocyte/neuron
metabolic interactions result in supplying neurons with lactate
and/or glucose as a metabolic fuel. In high fat diet, ketone bodies
represent the main metabolic fuel for neurons. Although ketone
bodies are available from the blood stream in ketogenic diet, local
astrocytic genesis of ketone bodies from fatty acids of blood
origin which also are available in large amounts might be the
preponderant mechanism for neuronal supply in ketones. Neuronal
ketone metabolism has been proposed to generate acetyl-CoA in a way
mobilizing oxaloacetate more intensively than when acetyl-CoA is
formed during the course of lactate and/or glucose metabolism. This
enhanced mobilization of oxaloacetate in the Krebs’cycle influences
neuronal glutamate /aspartate transamination reaction in favour of
glutamate formation, and hence increases neuronal GABA production
(by decarboxylation of glutamate). On the other hand, if more
glutamate provides more neuronal GABA in brain upon ketogenic diet,
less glutamate is however available for glutaminergic
neurotransmission as a result of a astrocytic leak from the
glutamate/glutamine cycle of either glutamine or glutamate which
are more abundantly than normally removed in blood or consumed
consequently to an increase in blood leucine or in brain to blood
ratio in alanine. Along with the antioxidant activity of ketones
[25] and modifications of NADH-related signaling pathways recently
described for glucose deprivation associated to ketogenic diet [26]
all the presently reviewed impacts of the diet on the
neuron/astrocyte metabolic interactions may be concluded to be
convergent at the point-of-view of anti-seizure activity, making by
otherwise the mimic of modifying these interactions a valuable goal
for pharmaceutical intervention.
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