Abstracts

Rationale: The ketogenic diet (KD) is a high-fat, low carbohydrate diet that is used as a therapy for intractable epilepsy. However, to date the mechanism(s) by which the KD achieves seizure control and neuroprotection is not yet known. We hypothesize that the protective function of the ketogenic diet results from an improved mitochondrial redox status, which in part, is due to the induction of glutathione (GSH) de novo biosynthesis. Methods: To address this hypothesis, adolescent Sprague-Dawley rats (P28) were fed a KD or control diet (CD) for 3 weeks and ketosis was confirmed by measuring blood levels of β-hydroxybutyrate. To investigate the protective properties of the KD, rats were injected with single high dose of kainate and latency to seizure and seizure score was determined using a modified Racine’s scale. GSH levels were determined by high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) in freshly isolated mitochondria from KD and CD rat hippocampi. To determine whether elevated GSH during ketosis was attributable to increased de novo synthesis, protein levels of the catalytic (GCLC) and modulatory (GCLM) subunits of γ-glutamate cysteine ligase (γ-GCL) (the rate limiting enzyme in GSH biosynthesis) was analyzed by immunoblot analysis. To determine if the upregulation of GSH biosynthesis was due to ketone bodies, mixed primary cortical or astrocytes were incubated with ketone bodies (β-hydroxybutyrate, acetoacetate, and acetone) and cellular GSH was measured by HPLC-EC. Since GSH is a major mitochondrial antioxidant that protects the mitochondrial genome (mtDNA) against oxidative damage, we used quantitative polymerase chain reaction (QPCR) to compare H2O2-induced mtDNA damage in freshly isolated hippocampal mitochondria from KD and CD-fed rats. Results: KD-fed rats showed an increased latency to limbic seizures with a lower seizure severity compared to CD rats (P<0.05). Interestingly, KD-fed rats showed a 2-fold increase in mitochondrial GSH compared to CD-fed rats (P<0.05). Western blot analysis revealed an up-regulation of both GCLC and GCLM, in the KD-fed hippocampus (P<0.05), but not the CD-fed rats. Incubation of astrocytes or mixed neuronal-glial cultures with ketone bodies did not modulate GSH levels suggesting alternate mechanisms may underlie GSH regulation by the KD. H2O2 exposure to isolated mitochondria caused a greater increase in mtDNA lesions in the CD-fed rats compared to the KD-fed rats (P<0.05). Conclusions: Together, these studies demonstrate that the ketogenic diet enhances the mitochondrial redox status in part by upregulating GSH biosynthesis. Funded by grants from the Epilepsy Foundation of America Research and Fellowship Program (S.J) and NINDS RO1NS039587 (M.P.).