In this study, we investigated the impact of exhaustive exercise on salivary metabolites and the relationship between fatigue and metabolites. The main findings of this study were as follows: first, exhaustive exercise led to changes in metabolic profile. Second, bioinformatic techniques including OPLS-DA analysis found that the 29 metabolites related to carbohydrate, fatty acid, protein, and TCA cycle metabolism were associated with the changes in metabolites after exercise. Furthermore, the changes in cyclohexylamine levels were significantly correlated with subjective fatigue. Therefore, these metabolites could play an important role in fatigue during exhaustive exercise.
The metabolomics approaches have revealed that acute exercise changed the metabolic profile associated with adenosine triphosphate production, including glycolysis (e.g., pyruvate and lactate), fatty acid oxidation (e.g., palmitate), protein metabolism (e.g., alanine), and ketone bodies (e.g., hydroxybutyrate).[11.12] Consistent with previous studies, the present study found that exhaustive exercise increased these energy substrates related to carbohydrate, fatty acid, and amino acid metabolism (Table 2). These metabolic responses were depicted in principal components of the PCA scores plot (Fig. 2) accounting for about 40% of the total variance. Therefore, these results suggest that exhaustive exercise acutely changes the metabolic profile related to the energy production pathway.
Prolonged exhaustive exercise was associated with an increase in long chain fatty acids, medium chain fatty acids, fatty acid oxidation products, and ketone bodies.[13] Nieman et al. (2017)[14] have showed that these fatty acid metabolites changed after exhaustive running at 70% maximum oxygen uptake for a means duration of 2.26 h. Moreover, Manaf et al. (2018)[7] have found changes in metabolites related to fatty acid, indole, neurotransmitter substances, and amino acid pathways after exhaustive cycling at an intensity of 3 mmol/L of lactate for a means of 1.2 h. The present study investigates the metabolomic profile response to a step-load cycling exercise until exhaustion (below 0.5 hours), a protocol that would be able to elicit central fatigue in a relatively short duration by high intensity exercise.[8] The results of the present study demonstrated that our exhaustive exercise changes the metabolomic profile mainly associated with glycolysis (e.g., lactate, pyruvate, dihydroxyacetone phosphate, and 2-hydroxyvaleric acid) and ketoacidosis (e.g., 2-oxousovaleric acid and 4-methyl-2oxovaleric acid). Therefore, metabolite response may be related to the nature of exercise factors such as duration and intensity.
Metabolite profile clustering was different before and after exhaustive exercise, and carbohydrate glucose metabolites showed increased VIP scores and fold changes compared to others (Table 2). In addition, TCA cycle intermediates, ketoacidosis, and some amino acid metabolites were associated with post-exercise metabolic profile changes, although most TCA cycle intermediates did not increase significantly. TCA cycle is the final pathway of carbohydrate, lipid, and some amino acid to produce energy via oxidative phosphorylation mainly during aerobic exercise. Taken together, the exhaustive exercise in the present study would reflects mostly anaerobic glycolysis metabolism, rather than the aerobic oxidation pathway, although both pathways were included in the exercise protocol. During relatively high intensity exercise, glucose metabolism is facilitated and pyruvate and lactate were increased. Under these conditions, pyruvate, lactate, and amino acid (alanine) are involved in restoration of glucose metabolism in the liver.[15] Indeed, we observed that pyruvate, lactate, and alanine were increased post-exercise in this study, suggesting that the glucose metabolism pathway was accelerated at the end of the exercise.
In this study, the changes in fatigue VAS were correlated with an increase in cyclohexylamine within the VIP metabolites (Fig. 3). Cyclohexylamine is an acyclic aliphatic amine, and known as a metabolite produced by the artificial sweetener cyclamate.[16] An early study has reported that ingestion of cyclohexylamine increased blood pressure and plasma free fatty acids, and that cyclohexylamine have a sympathomimetic capability.[17] Animal studies demonstrated that cyclohexylamine was deaminated to the corresponding ketones by microsomes in the rabbit liver.[18] Interestingly, metabolomics analysis in the present study could not detect cyclohexylamine at the baseline; however, cyclohexylamine increase was detected in several subjects after exercise. These findings may imply that exhaustive exercise-induced fatigue is in part mediated by cyclohexylamine. In this regard, further studies are necessary to elucidate a relationship between exercise and cyclohexylamine.
In conclusion, this study investigated the salivary metabolomic profile following exhaustive exercise-induced fatigue. We have identified energy-related metabolites that were significantly increased after exhaustive exercise. Our findings show an increase in metabolites related to glycolysis, lipolysis, amino acid metabolism, and ketone bodies. Furthermore, changes in cyclohexylamine levels were associated with an increase in fatigue. This metabolomic profile signature would serve as a pilot understanding of exercise induced fatigue.