In the present study, glutathione supplementation resulted in higher levels of PGC-1α and mtDNA biogenesis in mouse skeletal muscle and prevented the exercise-induced reduction in intermuscular pH in mice. Moreover, in humans, glutathione supplementation suppressed fatigue-related parameters during and after exercise. While it has been well documented that glutathione plays a central role in the antioxidant network in animal cells and redox balance can act as a marker of antioxidant status in various pathological and physiological conditions including exercise [3,4,14,15], the role of exogenous glutathione on phenotypic changes relating to physical exercise has not been explored. To the best of our knowledge, the present study is the first to demonstrate that glutathione supplementation improves aerobic metabolism in skeletal muscle, leading to reduced exercise-induced muscle fatigue.

During muscle contraction, lactic acid, a major source of protons, is rapidly produced by increased glycolytic metabolism, lowering the pH and inhibiting muscle contraction [16]. The protons generated from cytosolic lactic acid are immediately buffered in the cell or exported to the interstitial fluid and further transported to the blood. The buffering capacity is relatively high in the cytosol and blood, whereas this capacity is low in interstitial fluid where the presence of buffering factors, such as proteins, is limited [17,18]. Therefore, the pH of the interstitial fluid in muscle tissues can drastically change in response to muscle contraction and can also be a marker of acid–base conditions in muscle tissue. In contrast, the majority of lactate anions are released into the circulation or immediately metabolized as an energy substrate through aerobic metabolism [19-21]; therefore, their levels are not suitable as a marker. The results of both the animal and human studies indicate that glutathione supplementation inhibited the decrease in intermuscular pH after exercise; in the human study, this was demonstrated by the differences in blood lactate concentrations following exercise between the placebo and glutathione trials. These results may also explain the differences observed between the trials in RPE and subjective fatigue during and after exercise; in particular, an improvement in muscular acidosis results in less fatigue.

Circulating NEFA concentrations are regulated by a balance between catabolic processes in adipose tissue and fatty acid substrate utilization by the skeletal muscle. Circulating catecholamines such as adrenalin and noradrenalin are increased in response to exercise and stimulate lipolysis of triglycerides in adipose tissue [22], which causes an elevation of circulating fatty acids. In contrast, muscle contraction increases uptake of fatty acids from the circulation into muscle cells [23], which leads to a decrease in circulating fatty acids. Therefore, we suggest that the reduction of NEFA observed in the glutathione-supplemented mice was due to an increase in muscle utilization rather than a release from adipose tissue. Because energy consumed in muscle during exercise is mainly supplied by carbohydrates and lipids, glutathione-induced lipid utilization can decrease energy obtained from carbohydrates, which may lead to a decrease in lactate/proton production. Collectively, this indicates that glutathione improves metabolic acidosis through the activation of lipid metabolism, which leads to suppression of exercise-induced fatigue.

PGC-1α is a central member of a family of transcriptional co-activators involved in aerobic metabolism. Activation of PGC-1α alters the metabolic phenotype through interactions with nuclear respiratory factor and peroxisome proliferator-activated receptor-α [8-10], which leads to increased mitochondrial biogenesis and activity. It has been reported that PGC-1α activation causes significant improvements in athletic performance [24,25], prevention and treatment of muscle weakness in the elderly, obesity, and other metabolic diseases such as mitochondrial myopathies and diabetes [10,11,26]. Here, we detected an increase in PGC-1α with 2 weeks of glutathione intake, along with an increase in mtDNA content, indicating the activation of mitochondrial biogenesis. Therefore, the observed elevation of PGC-1α by glutathione intake strongly suggests an acceleration in lipid metabolism. In addition, the increase of mitochondria content could also lead to a decrease of lactate generation by accelerating aerobic metabolism of glucose, which would prevent muscle acidosis during exercise even further.

The regulatory mechanism for glutathione-induced increases in PGC-1α is unclear. One explanation is the elevation of AMPK, which is an upstream factor of PGC-1α regulation [27,28]. Recently, it has been argued that oral intake of other antioxidants, including vitamin C and E, do not elevate PGC-1α in the skeletal muscle of mice and humans [29,30]; thus, this may be a specific action of glutathione as a signal factor, but not its antioxidant properties. We found that 2 weeks of glutathione supplementation did not affect plasma glutathione concentration in the basal state. However, glutathione is transported across the intestines with in its intact form [12], and its plasma concentration, along with the glutathione-derived dipeptides γ-glutamyl-cysteine and cysteinyl-glycine, is markedly elevated during the 60–120-min period after oral administration, as shown in our previous report [13]. Therefore, the transient elevation of glutathione or the derived dipeptides following supplementation over 2 weeks may indicate stimulation of specific signaling factors that lead to elevated AMPK and PGC-1α. Alternatively, glutathione content in muscle tissues may also increase with supplementation, leading to the up-regulation of these factors. Further studies are needed to determine the specific mechanism(s) by which glutathione affects muscle aerobic metabolism. In addition, we also observed that reduction of the protein-bound glutathione concentration in plasma after exercise was suppressed following glutathione supplementation, which may also be related to the regulation of energy metabolism or fatigue. Future studies should also aim to identify the bound protein in plasma and examine the mechanism of protein binding or release from the protein and its source.