Abstracts

Rationale: The molecular events underlying the efficacy of the ketogenic diet (KD) remain unknown. Recently our laboratory showed increased mitochondrial glutathione (GSH) and activity of glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH biosynthesis (J. Neurochem. In press). A prominent mechanism of GCL activation is via the nuclear factor E2-related factor 2 (Nrf2) transcription factor pathway which can be activated by mild oxidative stress or electrophiles such as 4-hydroxynonenal, and in turn coordinately regulate a wide variety of protective target genes. Therefore, we hypothesized that an initial period of oxidative stress leads to the nuclear translocation of Nrf2, transcriptional activation of its target genes, and chronic neuroprotection. Methods: Adolescent (P28) male Sprague-Dawley rats were fed either a KD or control diet for periods of 1 day to 3 weeks. H₂O₂ production was assessed in isolated brain mitochondria using the Amplex UltraRed assay (Invitrogen). Free 4-hydroxynonenal (4-HNE) levels were assessed using HPLC with UV detection. Nrf2 activation was assessed by nuclear translocation with immunoblot analysis. Additionally, the activity of a Nrf2 target, NAD(P)H:quinone oxidoreductase-1 (NQO1), was assessed. Results: Mitochondrial production of H₂O₂ was significantly increased in the hippocampus of rats fed a KD for 1 and 3 days compared to controls. In contrast, by 1 week on the KD, H₂O₂ production levels decreased below control levels. Free 4-HNE levels were found to be significantly increased in hippocampus and frontal cortex of rats fed a KD for 3 weeks. Examination of nuclear translocation of Nrf2 showed an increase in hippocampus from KD-fed rats, and was maintained at a higher level than controls at 3 weeks on the diet. Finally, NQO1 activity was significantly higher in hippocampus of KD-fed rats compared to controls. Conclusions: These results demonstrate: 1) an initial increase in production of mitochondrial H₂O₂ during the first 3 days on the KD, 2) chronic elevation of 4-HNE, 3) nuclear translocation of Nrf2 and 4) increased activity of another Nrf2 target, NQO1, by the KD. The data suggest that the KD initially produces mild oxidative stress which activates the Nrf2 pathway leading to chronic cellular adaptation, induction of neuroprotective gene expression (such as GCL and NQO1) and a chronic decrease in mitochondrial oxidative stress. Thus, the protective effects of the KD may be the result of chronic adaptation to low levels of oxidative stress.