Nuclear factor erythroid-derived 2-like 2 (NFE2L2, Nrf2) mediates exercise-induced mitochondrial biogenesis and the anti-oxidant response in mice

Abstract

Key points: Reactive oxygen species (ROS) and nitric oxide (NO) regulate exercise-induced nuclear factor erythroid 2-related factor 2 (NFE2L2) expression in skeletal muscle. NFE2L2 is required for acute exercise-induced increases in skeletal muscle mitochondrial biogenesis genes, such as nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A, and anti-oxidant genes, such as superoxide dismutase (SOD)1, SOD2 and catalase. Following exercise training mice with impaired NFE2L2 expression have reduced exercise performance, energy expenditure, mitochondrial volume and anti-oxidant activity. In muscle cells, ROS and NO can regulate mitochondrial biogenesis via a NFE2L2/NRF-1-dependent pathway.

Abstract: Regular exercise induces adaptations to skeletal muscle, which can include mitochondrial biogenesis and enhanced anti-oxidant reserves. These adaptations and others are at least partly responsible for the improved health of physically active individuals. Reactive oxygen species (ROS) and nitric oxide (NO) are produced during exercise and may mediate the adaptive response to exercise in skeletal muscle. However, the mechanisms through which they act are unclear. In the present study, we aimed to determine the role of the redox-sensitive transcription factor nuclear factor erythroid-derived 2-like 2 (NFE2L2) in acute exercise- and training-induced mitochondrial biogenesis and the anti-oxidant response. We report that ROS and NO regulate acute exercise-induced expression of NFE2L2 in mouse skeletal muscle and muscle cells, and that deficiency in NFE2L2 prevents normal acute treadmill exercise-induced increases in mRNA of the mitochondrial biogenesis markers, nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (mtTFA), and the anti-oxidants superoxide dismutase (SOD) 1 and 2, as well as catalase, in mouse gastrocnemius muscle. Furthermore, after 5 weeks of treadmill exercise training, mice deficient in NFE2L2 had reduced exercise capacity and whole body energy expenditure, as well as skeletal muscle mitochondrial mass and SOD activity, compared to wild-type littermates. In C2C12 myoblasts, acute treatment with exogenous H2 O2 (ROS)- and diethylenetriamine/NO adduct (NO donor) induced increases in mtTFA, which was prevented by small interfering RNA and short hairpin RNA knockdown of either NFE2L2 or NRF-1. Our results suggest that, during exercise, ROS and NO can act via NFE2L2 to functionally regulate skeletal muscle mitochondrial biogenesis and anti-oxidant defence gene expression.

Figure 1. NFE2L2 expression in skeletal muscle following exercise

Mouse skeletal muscle (gastrocnemius) NFE2L2 and NFE2L2 target (GST and GSR) mRNA was increased 30 min following an acute (1 h) bout of treadmill exercise (A and B) and following 6 weeks of treadmill exercise training (C). Representative blot showing that NFE2L2 antibodies tested detected a band at the reported size (∼95–110 kDa) of NFE2L2 in cell lysates that was not seen in NFE2L2 shRNA‐treated cells; however, these antibodies were unable to detect a band in tissue lysates at the correct molecular weight that was not seen in lysates from NFE2L2 KO mice (D). NFE2L2 KO mice did not express NFE2L2 mRNA in skeletal muscle, liver or heart (E). Acute (5 h) treatment of C2C12 myoblasts with AICAR (1 mm) and H₂O₂ (50 μm) or Deta/NO (100 μm) increased NFE2L2 protein expression (F). Three days of treatment of mice with a NO synthase inhibitor (G) or anti‐oxidant (NAC) (H) attenuates acute (1 h) exercise‐induced increases in skeletal muscle (gastrocnemius) NFE2L2, GST and GSR mRNA expression and (I) increases the blood GSH:GSSG ratio. Results are shown as the mean ± SE (n = 5–7 per group for the cell culture experiments; n for mouse experiments is shown individually). Significance was determined using a two‐tailed Student's t test or one‐way ANOVA with Fisher's least significant difference post hoc analysis. ^* P < 0.05 and ^*** P < 0.001 vs. rest/control. ^# P < 0.05 vs. untreated of the same condition. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2. NFE2L2 is required for exercise training induced mitochondrial biogenesis

mtDNA (A), citrate synthase activity (B) and mitochondrial biogenesis‐associated genes mRNA (C) were measured in the skeletal muscle (gastrocnemius) of WT and NFE2L2 KO mice following 6 weeks of treadmill exercise training (trained) or normal sedentary behaviour (untrained). Four‐week treadmill trained NRFE2L2 KO mice had reduced exercise performance (D and E) and energy expenditure (G) compared to trained WT mice. WT and NFE2L2 KO mice had a similar body weight and activity levels (F and H). Results are shown as the mean ± SE (n is shown individually). Significance was determined using one‐way ANOVA with Fisher's least significant difference post hoc analysis. ^* P < 0.05 ^** P < 0.001 vs. untrained or pre‐training of the same genotype. ^# P < 0.05 vs. WT of the same condition. EE, energy expenditure. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3. NFE2L2 is required for acute exercise, NO and H₂O₂ induced increases in mRNA of mitochondrial biogenesis‐associated genes

Mitochondrial biogenesis‐associated genes mRNA levels in the skeletal muscle (gastrocnemius) of WT and NFE2L2 KO mice 4 h following acute exercise (A). Mitochondrial biogenesis‐associated genes mRNA expression in control shRNA or NFE2L2 shRNA C2C12 myoblasts following 5 h treatment with AICAR (1 mm) (B), Deta/NO (100 μm) (D) or H₂O₂ (50 μm) (E). mtDNA was determined in control shRNA or NFE2L2 shRNA C2C12 myoblasts following 5 days (5 h day^–1) treatment with AICAR (1 mm) (C), Deta/NO (100 μm) or H₂O₂ (5 μm) (F). mtTFA mRNA expression was assessed in C2C12 myoblasts stimulated for 5 h with Deta/NO (100 μm) or H₂O₂ (50 μm) 24 h following control, NRF‐1 (100 nm) (G) or PGC1α (10 nm) (H) siRNA transfection. Results are shown as the mean ± SE (n = 6–8 per group for cell culture experiments; n for mouse experiments is shown individually). Significance was determined using one‐way ANOVA with Fisher's least significant difference post hoc analysis. ^* P < 0.05, ^** P < 0.01 ^*** P < 0.001 vs. rest or untreated of same genotype or si/shRNA. ^# P < 0.05 vs. control si/shRNA of same condition. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4. NFE2L2, ROS and NO regulate the exercise‐induced anti‐oxidant response

Mice were treated for 3 days with the NO synthase inhibitor (l‐NAME) (A) or the anti‐oxidant (NAC) (B) and skeletal muscle (gastrocnemius) anti‐oxidant gene mRNA expression was assessed 30 min following acute (1 h) treadmill exercise. Anti‐oxidant genes mRNA or SOD activity were determined in the skeletal muscle (gastrocnemius) of WT and NFE2L2 KO mice 4 h following an acute treadmill exercise (C) or following 6 weeks of treadmill exercise training (trained) or normal sedentary behaviour (untrained) (D and E). WT and NFE2L2 KO gastrocnemius muscle showed similar levels of protein carbonylation and this was not affected by exercise training (F). Results are shown as the mean ± SE (n is shown individually). Significance was determined using one‐way ANOVA with Fisher's least significant difference post hoc analysis. ^* P < 0.05 and ^*** P < 0.001 vs. rest or untrained. ^# P < 0.05 vs. untreated or WT of the same condition. CAT, catalase; Prx, peroxiredoxin; Trx, theroxiredoxin. [Colour figure can be viewed at wileyonlinelibrary.com]