**1. Introduction**

Endurance training is essential to maximally improve cardiopulmonary fitness and delay the disease process. However, this may be intolerable due to the overload of the cardiopulmonary system to elderly individuals or patients with chronic diseases, traditional concentric work at usual training intensity [1]. Eccentric endurance training has the ability to overcome these limitations because of less respiratory requirement and metabolic oxygen, as well as lower heart rate (H), cardiac index and blood lactate concentration than concentric type at equivalent workload [2]. The benefits of using eccentric cycling training (ECT) in chronic heart failure patients [3], elderly individuals [4] and chronic obstructive

**Citation:** Huang, Y.-C.; Cheng, M.-L.; Tang, H.-Y.; Huang, C.-Y.; Chen, K.-M.; Wang, J.-S. Eccentric Cycling Training Improves Erythrocyte Antioxidant and Oxygen Releasing Capacity Associated with Enhanced Anaerobic Glycolysis and Intracellular Acidosis. *Antioxidants* **2021**, *10*, 285. https://doi.org/ 10.3390/antiox10020285

Academic Editor: Gareth Davison and Conor McClean

Received: 25 January 2021 Accepted: 10 February 2021 Published: 13 February 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

pulmonary disease [5] have been confirmed. Conventionally, most studies have focused on the contribution to elicit neuromuscular adaptations of eccentric work [6]; nevertheless, a recent study further demonstrated that either acute bout of concentric or eccentric cycling at moderate intensity induced increased enzymatic antioxidant capacity and decreased oxidative stress markers [7]. Moreover, ECT induces greater fat utilization compared to concentric cycling training (CCT) at a fixed workload [8] and greater fat loss in obese adolescents [9]. Therefore, the different cardiopulmonary loading and metabolic oxygen demands in ECT and CCT may result in distinct changes in antioxidative metabolism and O2 release adaptations [10]. However, there is very limited evidence regarding these mechanisms of chronic physiological responses to eccentric cycling [11].

Erythrocytes are vital to humans because of their abundance and the irreplaceable function they have of delivering O2. However, they are susceptible to sustained free radical damage during circulation, which impairs their O2 release capacity and reduces their lifespan [12]. Previous studies have reported that blood antioxidation capacity is impaired with acute exercise [13]; in contrast, regular exercise may increase antioxidative capacity [14]. The lower oxygen and energy consumed in ECT may avoid repeated, excessive exposure to oxidative stress, which progressively impair the erythrocyte [15]. However, whether this lower metabolic stress in comparable ECT might be enough to elicit physiological adaptations as CCT or not is another concern [16]. To date, the adaptations of the antioxidation capacity and regulatory mechanism of erythrocytes under different exercise regimens remain unclear. Here, we identified the key regulatory mechanisms using metabolomics profiling technology.

When exercising, erythrocytes must accelerate O2 release into peripheral tissue according to the Bohr effect [17] and enhance the demand for glycolytically derived ATP to restore intracellular ion balances. This process is at a constant rate when ATP consumption is steady, but the activity of the process changes rapidly in response to enhanced ATP utilization [18]. Importantly, erythrocytes are also exposed to dramatically enhanced oxidative stress that must be controlled by accelerated production of reducing equivalents derived from the pentose phosphate pathway (PPP), which is the sole source of NADPH and produces GSH as an antioxidant. In the sickle cells, the impaired antioxidant capacity leaves to a loss of glycolysis and the PPP shifting mechanism control and further homeostasis rupture, contributing to a decreased lifespan of cells [19]. Moreover, altering glycolytic pathway dominance has been demonstrated to limit antioxidation capacity under hypoxia [20]. Therefore, exercise may introduce continuous substrate competition between the main glycolysis pathway and the PPP, although this needs to be further elucidated.

2,3-BPG is a strong allosteric modulator that leads to O2 unloading [21]. However, the generation of 2,3-BPG bypasses the main phosphoglycerate kinase reaction so that the overall production of ATP per mole of glucose is decreased to zero. GSH de novo synthesis is ATP dependent and is therefore impaired when the stocks of intracellular ATP are depleted. In addition, lactate is the only end product of glycolysis in erythrocytes, and it also helps create a low pH value environment to decrease Hb-O2 affinity [22] and influence GSH synthesis [23]. Therefore, one of the biggest puzzles regarding erythrocyte metabolism during exercise is how the programming of erythrocyte glucose metabolism, 2,3-BPG production, and antioxidative capacity is regulated.

To address the abovementioned questions, this study elucidated the pathways underlying the regulation of the main glycolysis and the PPP and explored the effects of oxidation and antioxidation capacity in erythrocytes after six weeks of interventions. In addition, we also investigated the capacity for O2 release under different lactate concentrations under hypoxic and normoxic conditions. The aim of this study was to provide direct evidence that both ECT and CCT induce metabolic adaptations within erythrocytes that counteract the high oxidative stress evoked by vigorous exercise.
