Nuclear factor (erythroid derived 2)-like 2 activation increases exercise endurance capacity via redox modulation in skeletal muscles

Abstract

Sulforaphane (SFN) plays an important role in preventing oxidative stress by activating the nuclear factor (erythroid derived 2)-like 2 (Nrf2) signalling pathway. SFN may improve exercise endurance capacity by counteracting oxidative stress-induced damage during exercise. We assessed running ability based on an exhaustive treadmill test (progressive-continuous all-out) and examined the expression of markers for oxidative stress and muscle damage. Twelve- to 13-week-old Male wild-type mice (Nrf2 ^+/+) and Nrf2-null mice (Nrf2 ^-/-) on C57BL/6J background were intraperitoneally injected with SFN or vehicle prior to the test. The running distance of SFN-injected Nrf2 ^+/+ mice was significantly greater compared with that of uninjected mice. Enhanced running capacity was accompanied by upregulation of Nrf2 signalling and downstream genes. Marker of oxidative stress in SFN-injected Nrf2 ^+/+ mice were lower than those in uninjected mice following the test. SFN produced greater protection against muscle damage during exhaustive exercise conditions in Nrf2 ^+/+ mice than in Nrf2 ^-/- mice. SFN-induced Nrf2 upregulation, and its antioxidative effects, might play critical roles in attenuating muscle fatigue via reduction of oxidative stress caused by exhaustive exercise. This in turn leads to enhanced exercise endurance capacity. These results provide new insights into SFN-induced upregulation of Nrf2 and its role in improving exercise performance.

Figure 1

Experimental protocol. For 3 days, all mice performed 10 min of adaptive training at low speed for 5–10 m/min. Then, all mice were injected with SFN or vehicle four times for 3 days (72, 48, 24, and 3 h prior the exhaustive treadmill tests). On the day of the test, all mice conducted the experimental protocol. Exercise intensity was incrementally increased from running with the treadmill set at an initial speed of 5 m/min to a maximum speed of 28 m/min every 3 min. The maximal velocity was maintained until mice were exhausted. Red arrows indicate the time course of in vivo imaging after SFN or vehicle injection. Results are presented in Fig. 3A[(a)~(b)].

Figure 2

Fibre types in gastrocnemius muscle obtained by ATPase staining at pH 10.8 following SFN or vehicle injection (A). Fibre area (B) and fibre-type distribution (C) in the gastrocnemius muscle were determined by ATPase staining. Daily EE (D) and RQ (E) values assessed during each dark and light cycle using an indirect calorimeter. ATP (F), Glycogen (G) and cAMP (H) levels were measured in gastrocnemius muscle.

Figure 3

Effect of SFN injection. SFN or vehicle was injected four times for 3 days and mitochondrial biogenesis markers were measured in mice gastrocnemius muscle. (A) protein expression levels (western blot strips) of mitochondrial biogenesis markers. The amount of phosphorylated AMPKα was normalized to the amount of AMPKα protein. Protein content of mitochondrial biogenesis markers SirT1 and PGC1α were also measured. The loading volume was normalized by the expression levels of actin and lamin A/C, respectively. (B) mRNA expression of mitochondrial biogenesis markers in AA. mRNA levels of NRF1, TFAM, p53R2, COX IV, SCO1, and SCO2 were determined by real-time PCR. Values were normalized to the level of the housekeeping gene, GAPDH. (C) mtDNA copy number in the gastrocnemius muscle. This number factors into the ratio of nuclear and mtDNA (mtDNA/nDNA); brackets **P < 0.01.

Figure 4

SFN-induced Nrf2-Luc activity detected using an In Vivo Imaging System and results of genotyping of Nrf2 and OKD48 transgenic mice (A) in the prone position. Time course [(a: baseline) ~ (b: following SFN injections)] of luciferase assay is shown in Fig. 1. Nrf2-Luc activity in gastrocnemius muscle after the fourth SFN injection measured using a luminometer (B). mRNA levels of Nrf2 target genes in gastrocnemius (C) and soleus (D) muscles following four injections of SFN or vehicle. Brackets *P < 0.05, **P < 0.01.

Figure 5

Running distance of each group in exhaustive treadmill test (A) and VO₂ (circle) and RQ (triangle) per running time in mouse recorded median running distance in each group (B). Average levels of energy substrates: VO₂ (C), RQ (D), glucose (E), and FFA (F) in each group after the 50-min exhaustive treadmill test for comparison among groups at the same time course. Brackets *P < 0.05, **P < 0.01.

Figure 6

Oxidative, TBARS and GSSG/GSH (A) markers in the gastrocnemius muscle, and muscle damage, CPK and LDH (B) markers, muscle fatigue, lactate (C) marker in the blood. Brackets *P < 0.05, **P < 0.01, ^† P < 0.05 vs. after the 50-min exhaustive treadmill test, ^‡ P < 0.05 vs. Nrf2^+/+ groups.