Acute oxidative stress and systemic Nrf2 activation by the ketogenic diet

Abstract

The mechanisms underlying the efficacy of the ketogenic diet (KD) remain unknown. Recently, we showed that the KD increased glutathione (GSH) biosynthesis. Since the NF E2-related factor 2 (Nrf2) transcription factor is a primary responder to cellular stress and can upregulate GSH biosynthesis, we asked whether the KD activates the Nrf2 pathway. Here we report that rats consuming a KD show acute production of H(2)O(2) from hippocampal mitochondria, which decreases below control levels by 3 weeks, suggestive of an adaptive response. 4-Hydroxy-2-nonenal (4-HNE), an electrophilic lipid peroxidation end product known to activate the Nrf2 detoxification pathway, was also acutely increased by the KD. Nrf2 nuclear accumulation was evident in both the hippocampus and liver, and the Nrf2 target, NAD(P)H:quinone oxidoreductase (NQO1), exhibited increased activity in both the hippocampus and liver after 3 weeks. We also found chronic depletion of liver tissue GSH, while liver mitochondrial antioxidant capacity was preserved. These data suggest that the KD initially produces mild oxidative and electrophilic stress, which may systemically activate the Nrf2 pathway via redox signaling, leading to chronic cellular adaptation, induction of protective proteins, and improvement of the mitochondrial redox state.

Figure 1

Mitochondrial H₂O₂ production as a function of time on KD or control diet. Hippocampal mitochondria were isolated from rats fed a control or KD for 1 day, 3 days, 1 week, or 3 weeks. Substrate-driven H₂O₂ production was assessed using a fluorometric method. Bars represent mean ± standard error of the mean. *p ≤ 0.05, **p ≤ 0.01 by one-way ANOVA.

Figure 2

Levels of 4-HNE in hippocampus of rats fed KD or control diet. 4-HNE levels were assessed in the hippocampus of rats fed a control or KD for 3 days, 1 week, or 3 weeks by HPLC with UV detection. Bars represent mean ± standard error of the mean. **p ≤ 0.01 by one-way ANOVA.

Figure 3

Nrf2 nuclear expression in the hippocampus of rats fed KD or control diet. Nuclear fractions were prepared from rats fed a control or KD for 3 days, 1 week, or 3 weeks. (A) Western blot analysis of Nrf2 protein in nuclear fractions of rat hippocampus. Actin was used as a loading control. (B) Densitometry of Nrf2 protein expression exhibiting a time-dependent increase in the hippocampus of rats fed a KD (black bars) compared to control (open bars). Bars represent mean ± standard error of the mean. **p ≤ 0.01.

Figure 4

Activity of the Nrf2 target, NQO1, in hippocampus of rats fed KD or control diet. NQO1 activity was assessed in the hippocampus of rats fed a control or KD for 3 weeks. Activity was assessed with a spectrofluorometric assay. Bars represent mean ± standard error of the mean. *p ≤ 0.05 by two-tailed t-test.

Figure 5

GSH and CoASH levels in liver of rats fed KD or control diet. (A) Liver GSH levels in rats fed KD or control diets for 3 days, 1 week, and 3 weeks. (B) Liver CoASH levels in rats fed KD or control diets for 3 days, 1 week, and 3 weeks. Bars represent mean ± standard error of the mean. *p ≤ 0.05, ***p ≤ 0.001 by one-way ANOVA.

Figure 6

Nrf2 nuclear expression in the liver of rats fed KD or control diet. Nuclear fractions were prepared from liver of rats fed a control or KD for 3 days, 1 week, or 3 weeks. (A) Western blot analysis of Nrf2 protein in nuclear fractions of rat liver. Actin was used as a loading control. (B) Densitometry of Nrf2 protein expression exhibiting a time-dependent increase in the liver of rats fed a KD (black bars) compared to control (open bars). Bars represent mean ± standard error of the mean. **p ≤ 0.01, ***p ≤ 0.001 by one-way ANOVA.

Figure 7

Nrf2 target upregulation in liver of rats fed KD or control diet for 3 weeks. (A) HO-1 protein was assessed in liver of rats fed a control or KD for 3 weeks. Western blot densitometry analysis of HO-1 protein expression in liver, normalized to actin. (B) NQO1 activity was assessed in liver of rats fed a control or KD for 3 weeks. Bars represent mean ± standard error of the mean. *p ≤ 0.05 by two-tailed t-test.