Vitamin C and E Supplementation Inhibits Acute Exercise-induced Skeletal Muscle Signaling but does not Alter Maker of Muscle Adaptations

Aim: The aim of this study was to investigate the of vitamin C and E supplementation on acute exercise-induced changes of makers of skeletal muscle adaptation and its in mice. (PGC-1α), citrate synthase (CS) and vascular endothelial growth factor (VEGF). On the other hand, vitamin C and E supplementation prevented the phosphorylation of AMP activated kinase (AMPK) and p38 mitogen-activated protein kinase (p38 MAPK) following the treadmill running (P<0.05). Conclusion: These results suggest that reactive oxygen species (ROS) inhibits exercise-induced skeletal muscle signaling but does not alter mitochondrial biogenesis and angiogenesis in skeletal muscle.

Acute endurance exercise induces the generation of reactive oxygen species (ROS) in muscle [10]. It has been shown that exerciseinduced production of ROS also regulates skeletal muscle adaptations. First, Gomez-Cabrera et al. [11] has reported that antioxidant vitamin C supplementation prevents the mitochondrial biogenesis induced by exercise training in rat skeletal muscle. Since then, evidence supporting that antioxidant supplementation attenuates exercise traininginduced adaptations in human and rodent skeletal muscle has been published [12][13][14]. On the other hand, we and another research group have shown that antioxidant supplementation does not alter exercise training-induced adaptations [15][16][17][18][19]. Accordingly, the effects of antioxidant supplementation on endurance exercise training-induced skeletal muscle adaptations are still not definitive.
To further understand the role of exerciseinduced ROS, it is necessary to investigate the effects of antioxidants on intracellular signaling and markers of skeletal muscle adaptation during acute exercise, because exercise training adaptation reflects an accumulation of acute exercise stimulus. The effects of acute exerciseinduced ROS on skeletal muscle signals and adaptations have been evaluated using allopurinol inhibitor of xanthine oxidase (XO) or nonspecific antioxidant vitamin C. It was shown that ROS generated by XO during acute exercise regulates some intracellular signals [20,21]. However, these results only reflect the effects of ROS derived from XO on exercise-induced mitochondrial biogenesis but do not reflect the effects of other sources of ROS such as mitochondria [22] and leukocytes [23]. Wadley and McConell [24] reported that vitamin C does not alter the acute exercise-induced increases in makers of mitochondrial biogenesis. Also, they reported that vitamin C supplementation did not prevent the exercise-induced increase of oxidative stress. Thus, it is likely that their methodology was not suitable to determine the effects of exercise-induced ROS on mitochondrial biogenesis. Vitamin C is watersoluble antioxidant and scavenges radicals [25]. However, it is possible that vitamin C act as a pro-oxidant [26]. On the other hand, vitamin C can react with vitamin E radicals to regenerate vitamin E [27] and a combination of vitamin C and E has high antioxidant capacity and decreases exercise-induced oxidative stress [15,28]. In addition, to our knowledge, there are no studies investigating the effects of antioxidant supplementation and/or non-exhaustive endurance exercise on skeletal muscle gene expression of NCoR1 that coregulator of oxidative adaptation.
The aim of this study was to investigate the effects of vitamin C and E supplementation on acute exercise-induced changes of makers of skeletal muscle adaptation and its signaling pathways in mice. We hypothesized that vitamin C and E supplementation would inhibit the activation of AMPK and p38 MAPK during acute exercise and prevent increases in the markers of muscle adaptation after acute exercise.

Experimental Animals and Protocol
Male C57BL/6 mice (8 weeks old) were purchased from Takasugi experimental animals supply (Kasukabe, Japan). Five animals were housed together in 1 cage (27×17×13 cm) in a controlled environment under a light-dark cycle (lights on at 0900 and off at 2100). The experimental procedures followed the Guiding Principles for the Care and Use of Animals in the Waseda University Institutional Animal Care and Use Committee. The mice were derived into two groups (non-supplemented (NS) and vitamin C and E supplemented (VS), n=40/ groups) and then allocated to either an exercise (n=20) or a sedentary (n=20) group.
Mice in VS group were given vitamin C (750 mg/kg weight/day) and vitamin E (150 mg/kg weight/day) with a feeding needle. Mice in NS group were given vehicle (200 mg of triolein and 20 mg of Tween in 1 ml of saline). Mice were received the antioxidants or vehicle for two weeks. One hour after the last supplementation exercise group mice ran on a treadmill at 25 m/min, 8% grade for 120 min. Immediately or three hours after the treadmill runexercising and sedentary mice were sacrificed under light anesthesia with the inhalant isoflurane (Abbott, Tokyo, Japan). The gastrocnemius muscle was excised, quickly frozen in liquid nitrogen and stored at -80°C until analysis. A portion of gastrocnemius was quickly immersed in RNAlater (Applied Biosystems, Foster City, CA) and stored at -80°C.
Gastrocnemius muscle was homogenized in tissue protein extraction reagent (T-PER; Pierce, Rockford, IL) containing protease inhibitor (Complete mini protease inhibitor cocktail tablets; Roche, Mannheim, Germany) and phosphatase inhibitor (Roche) at 4°C. The homogenate was centrifuged at 10,000 × g for 15 min at 4°C and the protein content of the supernatant was determined by a bicinchoninic acid (BCA) Protein Assay Kit (Thermo, Rockford, IL). This supernatant was used for the measurement of hydro peroxide (H 2 O 2 ), thiobarbituric acid reactive substances (TBARS) and trolox equivalent antioxidant capacity (TEAC) and immunoblot analysis.  Green PCR Master Mix (Applied Biosystems). The thermal profiles consisted of 10 min at 95°C for denaturation followed by 40 cycles of 95°C for 3 s and annealing at 60°C for 15 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as the housekeeping gene, and the ΔΔCT method was used as previously described [30] to quantify target gene expression. All data are represented relative to its expression as fold change based on the values of the sedentary group. Specific PCR primer pairs for each studied gene are shown in Table 1.

Immunoblot Analysis
Homogenized samples were diluted with homogenizing buffer to 5.5 mg/mL protein.
Samples were then mixed with 4 × Laemmli sample buffer (Bio-Rad, Richmond, CA) in 10% 2-mercaptoethanol to 4 mg protein/mL and heated at 60°C for 10 min. Aliquots of samples

Statistical Analysis
Data are presented as mean±SE. A two-way analysis of variance (ANOVA) was performed (SPSS V17.0, IBM Japan, Ltd, Tokyo, Japan) to determine the main effects of exercise and/or antioxidant supplementation. If this analysis revealed a significant interaction, Bonferroni's post-hoc test was used to determine the significance among the means. Statistical significance was defined as P<0.05.

Effects of Acute Exercise and Antioxidant Supplementation on Markers of Skeletal Muscle Adaptation
Gene expression levels of citrate synthase (CS), a marker of mitochondrial biogenesis, and vascular endothelial growth factor (VEGF), a marker of angiogenesis, were significantly increased 3 hour following 120 min treadmill running (P<0.01, Figs. 2A and B). However, antioxidant supplementation did not alter expression levels of CS and VEGF ( Fig. 2A and  B).

Effects of Acute Exercise and Antioxidant Supplementation on Phosphorylation of Protein Kinase
Phosphorylation of AMPK and p38 MAPK were significantly increased immediately following endurance exercise (P<0.01, Figs. 3A

DISCUSSION
The purpose of the present study was to investigate whether antioxidant supplementation affects acute exercise-induced change in makers of skeletal muscle adaptation and its signaling pathways. In contrast to our hypothesis, the present study showed that combined supplementation with vitamins C and E did not alter the acute exercise-induced increase in markers of mitochondrial biogenesis and angiogenesis. However, interestingly, vitamin C and E supplementation prevented the phosphorylation of AMPK and p38 MAPK following the treadmill running. These results are in accordance with the rodent study of Wadley et al. [21].
It has been reported that a combination of vitamin C and vitamin E reduces oxidative stress in blood and organs [15,28]. In agreement with these studies, we have shown th supplementation with vitamin C (750 weight) and E (150 mg/kg body weight) inhibited exercise-induced oxidative stress (H TBARS) and increased the basal level of antioxidant capacity (TEAC). Thus, in this study, it seems that combination of vitamin C and E was Fig. 1. Hydrogen peroxide (H2O2; A)  appropriate for the evaluation of the effects of induced ROS production on the adaptations in skeletal muscle.

, thiobarbituric acid reactive substances (TBARS; B) and trolox equivalent antioxidant capacity (TEAC; C) in the gastrocnemius muscle of mice after 2 weeks of vehicle (NS) or vitamin C and E supplementation (VS) under sed (sedentary) or immediately after 120 min of treadmill running(exercise
Acute exercise increases markers of skeletal muscle adaptations such as mitochondrial and angiogenesis [32]. In this study, we measured CS and VEGF gene ression as markers of skeletal muscle known that CS protein content and activity reflect the and VEGF is involved 34]. The results of this study indicate that exercise increased in CS and VEGF gene expression but vitamin C and E did not affect the exercise-induced increase in CS and VEGF gene expression. These results are consistent with the studies suggesting that antioxidant supplementation did induced adaptations In addition, we evaluated the effects of antioxidant supplementation on exercise changes in intracellular signaling i.e., AMPK, p38 MAPK, PGC-1α and NCoR1. We observed that vitamin C and E supplementation attenuated the exercise-induced phosphorylation of AMPK and p38 MAPK. These proteins regulate activation and/or expression of PGC-1α [4, despite these effects of supplementation, in the present study, gene expression of PGC not affected by antioxidant supplementation PGC-1α is the key factor of skeletal muscle adaptations and modulates CS [35 [36] expression. Thus, it is likely that significantly increased CS and VEGF gene expression levels observed in both exercise groups (with or without In addition, we evaluated the effects of supplementation on exercise-induced e., AMPK, p38 NCoR1. We observed that vitamin C and E supplementation attenuated the induced phosphorylation of AMPK and p38 MAPK. These proteins regulate activation ,5]. However, despite these effects of supplementation, in the present study, gene expression of PGC-1α was not affected by antioxidant supplementation. 1α is the key factor of skeletal muscle 35] and VEGF expression. Thus, it is likely that significantly increased CS and VEGF gene expression levels observed in both exercise groups (with or without vitamin C and E) arose from the augmentation of the PGC-1α by exercise, irrespective of antioxidant supplementation. These observations agree with findings of the study of Wadley et al. [21], who reported that XO inhibition by allopurinol prevented the exerc phosphorylation of p38 MAPK and ERK but did not alter the increases in acute exercise signal gene expression levels such as PGC and the training adaptations in rat skeletal muscle. In the present study, it is unclear why PGC-1α and makers of skeletal muscle adaptation were not affected by antioxidant supplementation, even though phosphorylation of AMPK and p38 MAPK were prevented by the antioxidant supplement. It has been reported that vitamin C and E) arose from the augmentation of 1α by exercise, irrespective of idant supplementation. These observations agree with findings of the study of Wadley et al. , who reported that XO inhibition by allopurinol prevented the exercise-induced phosphorylation of p38 MAPK and ERK but did not alter the increases in acute exercise-induced signal gene expression levels such as PGC-1α and the training adaptations in rat skeletal muscle. In the present study, it is unclear why akers of skeletal muscle adaptation were not affected by antioxidant supplementation, even though phosphorylation of AMPK and p38 MAPK were prevented by the antioxidant supplement. It has been reported that PGC-1α is regulated by several intracellular signals such as AMPK [4], p38 MAPK [5], CaMK [6] and SIRT1 [7]. The limitation of the present study is that we were unable to examine the effect of vitamin supplementation on intracellular signals except for AMPK and p38 MAPK. Hence, it remains unclear what kind of signal activated PGC-1α in present study. However, it is possible that the increase in PGC-1α gene expression was induced by intracellular signals except for AMPK and p38 MAPK.
NCoR1 is transcription coregulator that negatively regulate skeletal muscle oxidative metabolism [9]. However, it is unclear whether expression level of this gene is changed by exercise and/or antioxidants. Especially, to our knowledge, there are no studies investigating the effects of antioxidant supplementation and/or non-exhaustive acute exercise on skeletal muscle NCoR1. Our results show that NCoR1 mRNA was decreased 3 hours after the nonexhaustive treadmill exercise but was not altered by antioxidant supplementation. Thus, it is likely that exercise-induced gene expression of CS in this study does not only result from the increase in PGC-1α but also the decrease in NCoR1 with exercise.

CONCLUSION
In conclusion, the combined supplementation with vitamin C and E prevented the acute exercise-induced phosphorylation of AMPK and p38 MAPK. However, despite these effects on signaling, supplementation with vitamin C and E