The ketogenic diet increases mitochondrial glutathione levels

Abstract The ketogenic diet (KD) is a high‐fat, low carbohydrate diet that is used as a therapy for intractable epilepsy. However, the mechanism(s) by which the KD achieves neuroprotection and/or seizure control are not yet known. We sought to determine whether the KD improves mitochondrial redox status. Adolescent Sprague–Dawley rats (P28) were fed a KD or control diet for 3 weeks and ketosis was confirmed by plasma levels of β‐hydroxybutyrate (BHB). KD‐fed rats showed a twofold increase in hippocampal mitochondrial GSH and GSH/GSSG ratios compared with control diet‐fed rats. To determine whether elevated mitochondrial GSH was associated with increased de novo synthesis, the enzymatic activity of glutamate cysteine ligase (GCL) (the rate‐limiting enzyme in GSH biosynthesis) and protein levels of the catalytic (GCLC) and modulatory (GCLM) subunits of GCL were analyzed. Increased GCL activity was observed in KD‐fed rats, as well as up‐regulated protein levels of GCL subunits. Reduced CoA (CoASH), an indicator of mitochondrial redox status, and lipoic acid, a thiol antioxidant, were also significantly increased in the hippocampus of KD‐fed rats compared with controls. As GSH is a major mitochondrial antioxidant that protects mitochondrial DNA (mtDNA) against oxidative damage, we measured mitochondrial H 2 O 2 production and H 2 O 2 ‐induced mtDNA damage. Isolated hippocampal mitochondria from KD‐fed rats showed functional consequences consistent with the improvement of mitochondrial redox status i.e. decreased H 2 O 2 production and mtDNA damage. Together, the results demonstrate that the KD up‐regulates GSH biosynthesis, enhances mitochondrial antioxidant status, and protects mtDNA from oxidant‐induced damage.