Neuronal adenosine A2A receptors signal ergogenic effects of caffeine
Caffeine is one of the most used ergogenic aid for physical exercise and sports. However, its mechanism of action is still controversial. The adenosinergic hypothesis is promising due to the pharmacology of caffeine, a nonselective antagonist of adenosine A1 and A2A receptors. We now investigated A2AR as a possible ergogenic mechanism through pharmacological and genetic inactivation. Forty-two adult females (20.0 ± 0.2 g) and 40 male mice (23.9 ± 0.4 g) from a global and forebrain A2AR knockout (KO) colony ran an incremental exercise test with indirect calorimetry (V̇O2 and RER). We administered caffeine (15 mg/kg, i.p., nonselective) and SCH 58261 (1 mg/kg, i.p., selective A2AR antagonist) 15 min before the open field and exercise tests. We also evaluated the estrous cycle and infrared temperature immediately at the end of the exercise test. Caffeine and SCH 58621 were psychostimulant. Moreover, Caffeine and SCH 58621 were ergogenic, that is, they increased V̇O2max, running power, and critical power, showing that A2AR antagonism is ergogenic. Furthermore, the ergogenic effects of caffeine were abrogated in global and forebrain A2AR KO mice, showing that the antagonism of A2AR in forebrain neurons is responsible for the ergogenic action of caffeine. Furthermore, caffeine modified the exercising metabolism in an A2AR-dependent manner, and A2AR was paramount for exercise thermoregulation.
The natural plant alkaloid caffeine (1,3,7-trimethylxantine) is one of the most common ergogenic substances for physical activity practitioners and athletes1,2,3,4,5,6,7,8,9,10. Caffeine increases endurance1,8,9,10,11,12, intermittent7,13,14 and resistance4,15 exercise in humans. In rodents, its ergogenic effects are conserved because caffeine increases running time on the treadmill at constant16,17 and accelerated speeds18,19. Sports sciences promote nonselective phosphodiesterase (PDE) inhibition7,8 and increased calcium mobilization2,7,8 as mechanisms for these ergogenic effects. However, the primary pharmacological effect of caffeine is the nonselective antagonism of adenosine A1 and A2A receptors (A1R, A2AR)20,21,22,23.
Adenosine can act as an inhibitory modulator of the Central Nervous System (CNS) associated with tiredness and drowsiness24,25,26,27,28,29. During exercise, circulating ADP/AMP/adenosine levels increase due to ATP hydrolysis30,31. However, there is still no substantial evidence on the role of adenosine in exercise-induced fatigue. It is just known that the nonselective A1R and A2AR agonist 5′-(N-ethylcarboxamido)adenosine (NECA), injected into the rat brain, abolishes the ergogenic effects of caffeine16.
Since there is increasing evidence that the adenosine modulation system critically controls allostasis29 and A2AR have a crucial role in the ability of caffeine to normalize brain function30, we hypothesized that caffeine decreases fatigue during exercise through antagonism of A2AR in the CNS. We combined the use of pharmacology (SCH 58261 and caffeine) and transgenic mice with tissue-selective deletion of A2AR, to test this hypothesis in an incremental running test with indirect calorimetry (or ergospirometry). A2AR knockout (KO) mice allow assessing if the ergogenic effect of caffeine persists in the absence of A2AR; the use of SCH 58261, the current reference for A2AR antagonists32,33, allows directly assessing the ergogenic role of A2AR. SCH 58261 has excellent selectivity and affinity for A2AR32,33, and affords motor benefits in animal models of Parkinson’s disease as does caffeine, supporting the recent FDA approval of the A2AR antagonist Istradefylline for PD treatment33. Our goal is to assess the ergogenicity of A2AR using the pharmacological and genetic tools described above.
SCH 58261: pharmacological inactivation of A2AR is ergogenic
SCH 58261 was psychostimulant for males, but not for females, since SCH 58261 only increased male locomotion in the open field (F1,39 = 4.5, η2 = 0.1, β = 0.54, 95% CI 58.8–72.1, P < 0.05, Fig. 1A).
The running power of females (F7,77 = 221, P < 0.05, Fig. 1B) and males (F7,84 = 183, P < 0.05, Fig. 1B’) increased at each stage of the exercise test. Submaximal V̇O2 also increased to the maximum (V̇O2max, dotted line) of females (F8,77 = 168, P < 0.05, Fig. 1B) and males (F7,84 = 14.3, P < 0.05, Fig. 1B’). Female (F8,70 = 180, P < 0.05, Fig.S2A) and male (F8,70 = 164, P < 0.05, Fig.S2B) submaximal V̇CO2 kinetics was similar to V̇O2. SCH 58261 had no effect on these submaximal values.
We demonstrated for the first time that SCH 58261 is ergogenic since SCH 58261 increased V̇O2max (F1,36 = 27.7, η2 = 0.44, β = 0.99, 95% CI 0.16–0.2, P < 0.5, Fig. 1C) and running power (F1,35 = 55, η2 = 0.61, β = 1.0, 95% CI 1.0–1.3, P < 0.05, Fig. 1D) in both sexes.
SCH 58261 had no effect on increasing RER of females (F7,70 = 6.9, P < 0.5, η2 = 0.43, β = 0.99, Fig. 1E) and males (F7,84 = 9.4, η2 = 0.57, β = 0.99, P < 0.5, Fig. 1E’). Exercise test raised the animals’ core (F1,26 = 5.5, η2 = 0.17, β = 0.62, 95% CI 28.7–29.39, P < 0.05, Fig. 1F) and tail temperature (F1,22 = 81, η2 = 0.78, β = 0.99, 95% CI 24.2–25.6, P < 0.05, Fig. 1G), with no effect of SCH 58261. Figure 1 shows the heating of the mouse’s tail in post-exercise recovery (rec) in relation to rest. Three females at estrus (Fig.S3C) were excluded from temperature experiments due to large exercise-induced tail hyperthermia40. The previous results refer to females in diestrus (Fig.S3A), proestrus (Fig.S3B), and metestrus (Fig.S3D).
Neuronal A2AR antagonism is ergogenic
Caffeine increases exercise performance in rodents16,17,19,26,40 and humans1,4,8,9,10,11,12,13,14,15,24,28,51,52. Our results show the key role of A2AR in the ergogenic effects of caffeine using pharmacological and genetic tools. Thus, the potent and selective A2AR antagonist SCH 58261 displayed an ergogenic effect similar to that of caffeine, and the ergogenic effect of caffeine was abrogated in A2AR KO mice.
SCH 58261 and caffeine improved V̇O2max, running and critical power of wild type mice. These results are in line with the improved running time observed in caffeine-treated rats16,26,53 and mice19. Further evidence for the ergogenic effect of caffeine is based on its ability to increase muscle power and endurance output in rodents54,55,56,57,58. For the first time, we demonstrated that the selective antagonism of A2AR is ergogenic. Also, for the first time, we demonstrated that the genetic inactivation of A2AR impaired the ergogenic effects of caffeine. Tissue-specific A2AR KO selectively in forebrain neurons further allowed showing that these ergogenic effects of caffeine are due to the antagonism of A2AR in forebrain neurons. Thus, we suggest that caffeine decreases central fatigue during exercise. Moreover, caffeine decreased RER in the submaximal stages of the exercise test, an effect also abrogated in A2AR KO mice. However, exercise-induced core and tail hyperthermia were similar among animals treated with SCH 58261 or caffeine, except for A2AR KO mice, suggesting possible A1R-A2AR-mediated interactions56,57 in the temperature control51.
In summary, we have now demonstrated that A2AR antagonism is a mechanism of action for ergogenicity, as SCH 58261 was ergogenic. Furthermore, we showed that the antagonism of forebrain A2AR was the mechanism underlying the ergogenic effect of caffeine since caffeine was not ergogenic in fb-A2AR KO. The use of selective A2AR KO in forebrain neurons further reinforces the ergogenic role of caffeine in decreasing central fatigue, with possible involvement of decreased perceived exertion, pain, and mental fatigue in humans. Despite methodological limitations, our data further suggest that caffeine modified the exercising metabolism in an A2AR-dependent manner and that A2AR is essential for exercise thermoregulation.
Authors: Aderbal S.Aguiar Jr1,2*; Ana Elisa Speck1,2; Paula M. Canas1; RodrigoA. Cunha1,3
1 CNC‑Center for Neuroscience and Cell Biology, University of Coimbra, 3004‑504 Coimbra, Portugal.
2 Biology of Exercise Lab, Department of Health Sciences, UFSC-Federal University of Santa Catarina, Araranguá, SC 88905‑120, Brazil.
3 FMUC – Faculty of Medicine, University of Coimbra, 3004‑504 Coimbra, Portugal.
published in: Nature Scientific Reports 10, 13414 (2020).