A ketogenic diet suppresses seizures in mice through adenosine A₁ receptors

Abstract

A ketogenic diet (KD) is a high-fat, low-carbohydrate metabolic regimen; its effectiveness in the treatment of refractory epilepsy suggests that the mechanisms underlying its anticonvulsive effects differ from those targeted by conventional antiepileptic drugs. Recently, KD and analogous metabolic strategies have shown therapeutic promise in other neurologic disorders, such as reducing brain injury, pain, and inflammation. Here, we have shown that KD can reduce seizures in mice by increasing activation of adenosine A1 receptors (A1Rs). When transgenic mice with spontaneous seizures caused by deficiency in adenosine metabolism or signaling were fed KD, seizures were nearly abolished if mice had intact A1Rs, were reduced if mice expressed reduced A1Rs, and were unaltered if mice lacked A1Rs. Seizures were restored by injecting either glucose (metabolic reversal) or an A1R antagonist (pharmacologic reversal). Western blot analysis demonstrated that the KD reduced adenosine kinase, the major adenosine-metabolizing enzyme. Importantly, hippocampal tissue resected from patients with medically intractable epilepsy demonstrated increased adenosine kinase. We therefore conclude that adenosine deficiency may be relevant to human epilepsy and that KD can reduce seizures by increasing A1R-mediated inhibition.

Figure 1. Seizure suppression by KD depends on A₁R activation.

Representative EEG recordings from the CA3 of WT and transgenic mice reflect seizure distribution over a 1-hour time span (top traces) and individual seizures at higher resolution (1 minute; bottom traces). Asterisks in top traces denote the individual seizures chosen. Beginning and end of seizures are marked by vertical arrows. Traces from CD-fed animals showed baseline seizure activity in all mutants and lack of seizures in WT. KD almost completely abolished seizures in Adk-Tg mice; rare seizures were of reduced duration, as shown. KD reduced seizure activity in A₁R^+/– mice, had no effect in A₁R^–/– mice. Treatment with glucose or DPCPX reversed KD effects. See Table 1 for quantitation.

Figure 2. KD leads to downregulation of ADK.

(A) Representative Western blot from brain extracts of WT mice fed CD or KD for 3 and 4 weeks. Note the 2 different splice variants of ADK in the ADK-reactive bands. Anti-tubulin immunoreactivity was used to normalize for equal loading. Lanes were run on the same gel but were noncontiguous (white line). (B) Brain ADK from mice fed CD or KD for 3–4 weeks, expressed relative to CD (n = 4 per group). Data are mean ± SEM. **P < 0.01 vs. CD.

Figure 3. ADK immunoreactivity in hippocampus of control and TLE patients with medial temporal HS.

(A–D) Sections were counterstained with hematoxylin. Shown are representative CA1 (A and B) and hilus (C and D) from the same sample. (A and C) Control hippocampus showed weak ADK immunoreactivity. Histologically normal surgical hippocampus displayed an immunoreactivity pattern similar to that in control autopsy hippocampus (not shown). (B and D) The HS specimen demonstrated increased ADK expression in both residual pyramidal and hilar neurons (arrows and B, top inset) and in reactive astrocytes (arrowheads and B, bottom inset). Insets in D show expression of ADK (red) in a reactive astrocyte (GFAP, green). Scale bars: 160 μm (A and B); 80 μm (C and D); 40 μm (A, inset, and B, top inset); 15 μm (B, bottom inset, and D, insets). (E and F) Western blot analysis of ADK of total homogenates from control autopsy hippocampus and HS specimens. (E) Representative immunoblots. (F) Densitometric data, expressed relative to optical density of β-actin (n = 5 per group). Data are mean ± SEM. *P < 0.05 vs. control.