Background
Stress causes activation of the hypothalamic-pituitary-adrenal (HPA) axis [
1,
2]. The pathway is initiated by the release of corticotropin-releasing hormone (CRH) from the hypothalamus, which then stimulates the anterior pituitary to release adrenocorticotropin (ACTH) into the circulation. ACTH then stimulates the adrenal cortex to release the glucocorticoid cortisol (in humans). Many studies have shown that exercise-induced stress also stimulates the HPA axis and increases cortisol secretion [
3‐
6]. It was also demonstrated that decreases in the blood glucose concentration trigger the pituitary-adrenocortical axis to enhance secretion of ACTH and cortisol during prolonged exercise in humans [
7,
8]. Moreover, there were significant correlations between the cortisol response and the sense of fatigue during stressful exercise [
9,
10]. These reports point out that cortisol is released in response to glycogen depletion or reduced blood glucose levels to maintain the blood glucose level, delaying fatigue. Luger et al. reported that physical conditioning is associated with a reduction in pituitary-adrenal activation in response to exercise (i.e., highly trained athletes have diminished responses of ACTH and cortisol to CRH) [
11]. This indicates that the increase in mitochondrial capacity by training shifts fuels to more fat burning and glycogen sparing, thus preventing reduced blood glucose levels, which can contribute to a reduction in the pituitary-adrenal response. In addition, many studies have shown that cortisol elevation induces protein breakdown [
12‐
15], which might trigger glucocorticoid-induced muscle atrophy [
16,
17]. Thus, the suppression of the level of cortisol means less stress or better physical condition (sparing glycogen, maintaining blood glucose level, etc.) during exercise and contributes to the reduction in the post-workout catabolic breakdown of protein.
There are some nutritional strategies for the suppression of cortisol levels during exercise. For instance, several studies have demonstrated that the consumption of carbohydrates during aerobic exercise contributes to reducing postexercise cortisol levels [
18‐
20]. Although there have also been several reports evaluating the effects of amino acid (AA) supplementation on the exercise-induced cortisol response, few studies have elucidated the suppressive effects of amino acids on the level of cortisol during exercise in humans. It was reported that branched-chain amino acid (BCAA) administration before exercise did not affect the response of cortisol, while BCAA administration decreased the growth hormone concentration during exercise and increased the level of testosterone during the recovery period [
21]. A powder supplement containing 5.2 g of BCAAs, 4.3 g of essential amino acids, and 1.5 g of taurine did not have a significant effect on the acute cortisol or testosterone response to resistance exercise [
22]. On the other hand, some reports indicate that some AAs may have the potential to reduce exercise-induced cortisol. It was reported that arginine administration promoted lipid metabolism in rodents and humans [
23‐
25], which might lead to the maintenance of glucose or glycogen levels during exercise and therefore suppress the cortisol increase. The acute supplementation of valine is reported to effectively decrease the level of glucocorticoid (corticosterone in rodents) by maintaining liver glycogen and blood glucose in a rodent study [
26]. Serine is known as a substrate in the production of phosphatidylserine, which is reported to blunt the increases in cortisol levels during exercise [
27]. In addition, we demonstrated that an AA mixture containing 1.8 g of arginine, 1.1 g of valine, and 0.1 g of serine supplement could reduce exercise-induced fatigue in a previous study [
28]. In that study, however, the AA mixture supplementation did not significantly suppress the cortisol concentration compared to the placebo. A certain number of subjects showed very little change in plasma cortisol concentration during exercise in the study. The exercise condition was probably not enough for them to stimulate the cortisol response. To evaluate the effect of the supplement on the cortisol response, it is important to exclude noncortisol responders. We hypothesized that the above-described AA mixture containing arginine, valine, and serine could suppress exercise-induced cortisol in subjects who have a high cortisol response to the exercise protocol. First, arginine, which promotes lipid metabolism [
23‐
25], might contribute to suppressing the cortisol response by maintaining glucose or glycogen levels during exercise. Second, valine might also act to decrease the cortisol level via the maintenance of glycogen and blood glucose [
26]. In addition, serine might blunt the increase in cortisol levels by producing phosphatidylserine, which is a different mechanism from arginine or valine [
27].
Here, we conducted a randomized, double-blinded, placebo-controlled crossover study in recreationally active healthy subjects. The aim of the present study was to examine the effect of acute supplementation of the AA mixture on the exercise-induced cortisol response in humans. We hypothesized that AA mixture supplementation would suppress the cortisol response during exercise by combining the positive effects of arginine, valine, and serine synergistically.
Discussion
The present study demonstrated that supplementation with the AA mixture containing arginine, valine, and serine suppressed the exercise-induced increase in plasma cortisol, which supported our hypothesis that AA mixture supplementation would suppress the cortisol response during exercise.
Cortisol is released by exercise-induced stress [
3‐
6] in response to glycogen depletion or reduced blood glucose levels to delay fatigue, which is also reported to increase proteolysis [
12‐
15]. In this study, prolonged exercise significantly increased the level of cortisol in the placebo condition, which demonstrated that the exercise condition was high enough to induce cortisol release in the study subjects. In contrast, the level of cortisol did not change in the AA condition, and there was a significant difference between the AA condition and the placebo condition in the cortisol concentration after exercise. These results indicated that acute supplementation with the AA mixture significantly suppressed the increase in cortisol during prolonged exercise, which suggested that the AA mixture might contribute to less stress or better physical condition during exercise and the reduction in muscle protein breakdown after exercise.
ACTH is known as one of the factors involved in the stress response of the HPA axis. ACTH secreted from the pituitary induces cortisol release from the adrenal gland [
1,
2]. In the present study, the level of ACTH was significantly elevated by exercise in the placebo condition, which indicated that the cortisol increase in the placebo condition was largely caused by the increased ACTH release. In the AA condition, while no significant differences were observed between before and after exercise on the ACTH concentration, there was a trend toward an increased ACTH level between before and after exercise. In addition, there were no significant differences in the level of ACTH between the two conditions. These results suggested that AA supplementation might not directly affect ACTH secretion during exercise in this study. There were no significant differences in the cortisol/ACTH ratio, which was used as a measure to assess adrenal responsivity [
30‐
32], between the AA condition and the placebo condition in the study. In addition, the correlations between the changes in cortisol and ACTH were evaluated in the study. In the case that the degree of suppression of cortisol and ACTH release by the AA mixture was different, it was expected that there was a difference in the regression lines between the AA condition and the placebo condition. However, in the present study, the regression lines of the relationship between the levels of cortisol and ACTH in the AA condition and the placebo condition were almost equal, which indicated that the ACTH release from the pituitary and the cortisol release from the adrenal gland were suppressed at almost the same level by the AA mixture supplement. However, because one subject’s data whose values seemed to differ greatly from the other data might have had a certain impact on the generation of the regression lines in the study, further investigations with an increased sample size should be conducted to obtain more accurate findings. Future studies are also needed to clarify the effect of the AA mixture on CRH release from the hypothalamus, the most upstream factor of the HPA axis.
Blood glucose levels are strongly related to cortisol release. Tabata et al. reported that decreases in the blood glucose concentration lead to enhanced secretion of ACTH and cortisol during prolonged exercise in humans [
7,
8]. In this study, the blood glucose levels were significantly decreased both in the AA condition and the placebo condition, and there were no significant differences between the two conditions. Thus, the blood glucose level was not related to the cortisol suppressive effect of the AA mixture in the current study. Cortisol breaks down muscle or liver glycogen to maintain the blood glucose level when blood glucose levels decrease [
33,
34]. It was reported that acute valine supplementation maintained the liver glycogen content and suppressed the cortisol level in rodents [
26]. These findings suggest that muscle or liver glycogen might be maintained by the AA mixture supplement in this study.
Arginine was reported to improve lipid metabolism [
23‐
25]. Lucoti et al. reported that long-term oral arginine treatment (8.3 g/day) decreased fat mass and waist circumference as well as improved glucose metabolism and insulin sensitivity [
35]. Hurt et al. reported that 3 g of L-arginine three times a day for 12 weeks could be effective at reducing central adiposity in obese patients [
36]. In the present study, there were no significant differences in the levels of serum total ketone body or free fatty acid between the two conditions before and after exercise, which suggested that acute supplementation with the AA mixture containing 1.8 g of arginine did not exert significant effects related to lipid metabolism in this study. The lipid metabolism-related biomarkers were not related to the effect of the AA mixture in the study.
CPK has been used as a marker of muscle cell damage because it is released into blood when a disruption occurs in the sarcomere [
37,
38]. It was reported that BCAA or phosphatidylserine supplementation could suppress the increase in the level of CPK after exercise [
39,
40]. Ammonia, which is reported to have neurotoxicity, is produced by the catabolism of muscle proteins when energy is depleted during exercise [
41,
42]. It was reported that arginine could suppress the increase in the level of ammonia during exercise [
43]. In the present study, there were no differences between the two conditions in the levels of CPK and ammonia, which indicated that these biomarkers were not related to the effect of the AA mixture on the level of cortisol.
Although the mechanism of the suppressive effect of the AA mixture could not be clarified in the study, we propose several possibilities for the mechanism of the effect of the AA mixture. First, supplementation with the AA mixture might have contributed to suppressing cortisol release via NO synthesis. Arginine is known as a substrate for nitric oxide (NO) synthesis [
44]. It was also reported that NO activity modulates the response of the neuroendocrine component of the HPA axis [
45,
46]. Second, the effect of the AA mixture might be caused by the attenuation of serotonin synthesis. It was revealed that serotonin activates the HPA axis via serotonin receptor stimulation [
47]. We have also clarified that supplementation with the AA mixture used in the study decreased the tryptophan/BCAA ratio during exercise in a previous study, suggesting that the AA mixture might attenuate the synthesis of serotonin [
28]. In addition, the AA mixture might contribute to reducing the cortisol response via phosphatidylserine production, which was reported to attenuate the release of ACTH and cortisol during moderate intensity exercise [
27]. It was also demonstrated that oral administration of serine could increase the concentration of serine in the brain and synthesize phosphatidylserine [
48,
49].
There are several limitations of the present investigation. We did not control subjects’ diets except for breakfast on the exercise trial day. Although subjects were instructed to consume their usual diets, it is possible that the differences in their energy intake limited the evaluation of the effect of the AA mixture supplement. A diet record analysis should be conducted in future studies. We also did not limit dietary supplements other than asking subjects not to consume any supplements on the trial day. Furthermore, we did not measure the levels of CRH, NO, serotonin, or phosphatidylserine, which might be involved in the mechanism of the effect of the AA mixture in this study. Further examinations measuring the effect of the AA mixture on the production of these candidate factors will support the results of this study.
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