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. 2014 Feb 1;457(3):415-24.
doi: 10.1042/BJ20130863.

Nrf2 affects the efficiency of mitochondrial fatty acid oxidation

Marthe H R Ludtmann  1 , Plamena R Angelova  1 , Ying Zhang  2 , Andrey Y Abramov  1 , Albena T Dinkova-Kostova
Affiliations

Nrf2 affects the efficiency of mitochondrial fatty acid oxidation

Marthe H R Ludtmann et al. Biochem J. .

Abstract

Transcription factor Nrf2 (NF-E2 p45-related factor 2) regulates the cellular redox homoeostasis and cytoprotective responses, allowing adaptation and survival under conditions of stress. The significance of Nrf2 in intermediary metabolism is also beginning to be recognized. Thus this transcription factor negatively affects fatty acid synthesis. However, the effect of Nrf2 on fatty acid oxidation is currently unknown. In the present paper, we report that the mitochondrial oxidation of long-chain (palmitic) and short-chain (hexanoic) fatty acids is depressed in the absence of Nrf2 and accelerated when Nrf2 is constitutively active. Addition of fatty acids stimulates respiration in heart and liver mitochondria isolated from wild-type mice. This effect is significantly weaker when Nrf2 is deleted, whereas it is stronger when Nrf2 activity is constitutively high. In the absence of glucose, addition of fatty acids differentially affects the production of ATP in mouse embryonic fibroblasts from wild-type, Nrf2-knockout and Keap1 (Kelch-like ECH-associated protein 1)-knockout mice. In acute tissue slices, the rate of regeneration of FADH2 is reduced when Nrf2 is absent. This metabolic role of Nrf2 on fatty acid oxidation has implications for chronic disease conditions including cancer, metabolic syndrome and neurodegeneration.

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Figures

Figure 1
Figure 1. Nrf2 alters the efficiency of mitochondrial oxidation of palmitic acid
(A) Compared with WT cells, the levels of Nrf2 are higher in Keap1-KO, and undetectable in Nrf2-KO MEFs. (B) The enzyme activity of the Nrf2-target protein NQO1 is 10-fold lower in Nrf2-KO and 10-fold higher in Keap1-KO MEFs in comparison with WT cells. (C) Oxygen consumption was measured in aliquots of WT, Nrf2-KO and Keap1-KO MEF cell suspensions (~2×106) in glucose-free HBSS in the presence of palmitic acid (0.4 mM). Compared with WT, oxygen consumption was higher in Keap1-KO cells and lower in Nrf2-deficient cells. (D) Oxygen consumption rate in cell suspensions of WT (black bars), Keap1-KO (dark grey bars) and Nrf2-KO (light grey bars) MEFs in HBSS in the presence of palmitic acid (0.4 mM) and the absence of glucose. The contribution of oxidative phosphorylation was estimated through application of oligomycin (F1Fo-ATP synthase inhibitor; 2 μg/ml). The maximal rate of respiration was estimated after addition of FCCP (1 μM). Data are presented as means ± S.E.M. *P <0.01 compared with WT.
Figure 2
Figure 2. Nrf2 affects the production of ATP following mitochondrial oxidation of palmitic acid
Live-cell measurements of the ATP levels in WT (A), Keap1-KO (B) and Nrf2-KO (C) MEFs show differences among the genotypes in response to palmitic acid (0.4 mM) and oligomycin (2 μg/ml). (D) Quantification of the decrease in the ATP levels in response to oligomycin. Data are presented as means ± S.E.M. *P < 0.01 compared with WT.
Figure 3
Figure 3. Nrf2 affects the production of ATP following mitochondrial oxidation of palmitoylcarnitine acid in the presence of inhibitors of complexes I and II
(A) Live–cell measurements of the mitochondrial ATP levels in WT, Keap1-KO and Nrf2-KO MEFs show differences among the genotypes in response to palmitoylcarnitine (0.1 mM) in the presence of rotenone (5 μM) and malonate (20 μM). (B) Quantification of the increase in the ATP levels in response to palmitoylcarnitine in WT and Keap1-KO MEFs. (C) Live-cell measurements of the ATP levels in response to palmitoylcarnitine (0.1 mM) in the presence of rotenone (5 μM) and malonate (20 μM) in Nrf2-KO MEFs which had been pre-treated for 6 h with the antioxidant trolox. Two representative traces are shown. Data are presented as means ± S.E.M. *P <0.01.
Figure 4
Figure 4. Nrf2 alters mitochondrial respiration upon stimulation of FAO
(A) Isolated mitochondria from the brain did not show any acceleration of oxygen consumption in response to palmitic acid or its conjugate. Activation of respiration through application of glutamate demonstrated that these mitochondria were intact. Oxygen consumption in WT brain mitochondria was monitored at basal state, and after the sequential addition of palmitic acid (0.4 mM), palmitoylcarnitine (50 μM) and glutamate (5 mM). (B and C) Oxygen consumption was strongly accelerated in mitochondria from the liver (B) and heart (C) following the addition of palmitic acid (0.4 mM). (D) State 2 respiration (respiration with substrates, but before application of ADP) in isolated liver mitochondria from WT (black bars), Keap1-KD (dark grey bars) and Nrf2-KO (light grey bars) mice in the presence of palmitic acid (0.4 mM) or palmitoylcarnitine (50 μM). The value of State 2 respiration is highest in Keap1-KD and lowest in Nrf2-KO when compared with WT liver mitochondria. (E) Respiratory control ratio, State 3 respiration [ADP (substrate for ATP synthesis)-dependent] to State 4 respiration (ADP-independent, ADP consumed by mitochondria), in isolated mitochondria from the heart and liver of WT (black bars), Keap1-KD (dark grey bars) and Nrf2-KO (light grey bars) mice. (F) State 2 respiration in isolated liver mitochondria from WT (black bars), Keap1-KD (dark grey bars) and Nrf2-KO (light grey bars) mice in the presence of hexanoic acid (0.5 mM). The effect of hexanoic acid mirrored the results obtained with palmitic acid. Data are presented as means ± S.E.M. *P < 0.01 compared with WT.
Figure 5
Figure 5. Nrf2 affects the rates of FADH2 generation and utilization
FAD levels were determined in acute heart slices prepared from WT (A), Keap1-KD (B) and Nrf2-KO (C) mice. The maximal rate of complex II-dependent respiration with the highest levels of FAD was estimated through application of the uncoupler FCCP, and the lowest FAD levels were measured through application of the complex IV inhibitor sodium cyanide. Each slice was placed in glucose-free HBSS, and the FAD autofluorescence was continuously monitored at the basal state and after sequential addition of hexanoic acid (0.5 mM), followed by FCCP (1 μM) and sodium cyanide (1 mM). (D) FAD pool recovery rate after addition of sodium cyanide. Data are presented as means ± S.E.M. *P < 0.05 compared with WT. NaCN, sodium cyanide.
Figure 6
Figure 6. Effect of Nrf2 on the transcript levels of acyl-CoA dehydrogenases
The amount of mRNA for SCADs (A), MCADs (B), LCADs (C) and VLCADs (D) were analysed by real-time PCR, using β-actin mRNA as an internal control. In each group, the mRNA species was measured separately in triplicate. Data are presented as means ± S.D. and are expressed relative to WT. *P <0.05 compared with WT.
Figure 7
Figure 7. Effect of trolox on NADH and FAD levels and redox state
The mitochondrial NADH (A) and FAD (B) levels were determined by application of 1 μM FCCP (giving maximal values for FAD and minimal for NADH) and 1 mM sodium cyanide (giving minimal values for FAD and maximal for NADH). The redox index (C and D) is expressed as a percentage of the basal autofluorescence of NADH or FAD before application of 1 μM FCCP (taken as 0 for NADH and 100% for FAD). Parallel experiments were performed using MEFs that were pre-incubated with 100 μM trolox for 6 h or solvent control (0.1% DMSO). Data are presented as means ± S.E.M. *P <0.05 compared with control (no trolox).
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