Monocarboxylate transporter-dependent mechanism is involved in the adaptability of the body to exercise-induced fatigue under high-altitude hypoxia environment

Under high-altitude hypoxia environment, the body is more prone to fatigue, which occurs in both peripheral muscles and the central nervous system (CNS). The key factor determining the latter is the imbalance of brain energy metabolism, which makes it difficult to maintain the central nervous system to send peripheral nerve impulse continuously. During strenuous exercise, lactate released from astrocytes is taken up by neurons stored for energy to maintain synaptic transmission, a process mediated by monocarboxylate transporters (MCTs) in CNS. The present study investigated the correlation among the adaptability to exercise-induced fatigue, brain lactate metabolism and neuronal hypoxia injury under high-altitude hypoxia environment. Rats were subjected to exhaustive incremental load treadmill exercise under either normal pressure and normoxic conditions or simulated high-altitude low pressure and hypoxic conditions, with subsequent evaluation of the average exhaustive time as well as the expression of monocarboxylate transporters 2 (MCT2), MCT4, the average neuronal density in the cerebral motor cortex, and the lactate content in rat brain. At the early stage of simulated high-altitude environment, the average exhaustive time and neuronal density of rats decreased rapidly, then gradually recovered to some extent with the extension of altitude acclimatization time. The expression of MCT2, MCT4 and the lactate content in rat brain also increased gradually with the extension of altitude acclimatization time. After the application of lactate transport inhibitor, the recovery of exercise capacity of rats after altitude acclimatization was quickly blocked, and the neuronal injury in the cerebral motor cortex of rats was also significantly aggravated. These findings demonstrate that MCT-dependent mechanism is involved in the adaptability of the body to central fatigue, and provide a potential basis for medical intervention for exercise-induced fatigue under high-altitude hypoxia environment.


Introduction
Fatigue refers to the decreased capacity or complete inability of an organism, organ, or part to function normally because of excessive stimulation or prolonged exertion, it is a feeling of tiredness. Physical fatigue is the transient inability of muscles to maintain optimal physical performance, and is made more severe by intense physical exercise (Gandevia, 1992;Hagberg, 1981;Hawley and Reilly, 1997). Physical fatigue can be caused by a lack of energy in the muscle, by a decrease in the efficiency of the neuromuscular junction or by a reduction in the drive originating from the central nervous system (Gandevia, 2001), which can be restored after proper rest time and adjustment. It is an extremely complex body comprehensive reaction process. Critical power (CP) represents the boundary between the intensity domains of heavy and severe exercise (Galán-Rioja et al., 2020). It represents an important parameter of aerobic function and is the greatest average effort that can be sustained for a period of time without fatigue (Lipková et al., 2022). Fatigue is more likely to occur at high altitudes and is usually attributed to low oxygen levels. However, millions of people live and work at high altitudes, and many of them are able to adjust successfully to the hypoxic environment of very high altitudes, which is called altitude acclimatization and is a slow physiological adaptation resulting from prolonged exposure to significantly reduced atmospheric pressure. Discovery of the mechanisms responsible for human acclimatization to hypoxia could lead to new ways to improve acclimatization and retention.
Fatigue is considered to be an interactive effect resulting from both peripheral muscles and CNS factors. The key factor determining the latter is the imbalance of brain energy metabolism, which makes it difficult to maintain sufficient peripheral nerve impulses to the muscles continuously (Girard and Millet, 2008;Secher et al., 2006). The mechanism is associated with the decrease of brain electrical activity caused by excessive oxygen consumption during exhaustive exercise and the feedback inhibition of activity in the CNS and anterior motoneurons (Amann and Dempsey, 2016;Davis et al., 2000;Davis and Bailey, 1997).
Under strenuous exercise or a hypoxic environment, the body maintains the balance of energy metabolism through anaerobic glycolysis of carbohydrates such as glycogen, and lactate is the metabolite of this process. The rapid decline in body exercise capacity after rush entry into altitude is related to the accumulation of lactate, which triggers a series of pathological responses to damage neurons by reducing the pH under the hypoxic high-altitude environment (Dhillon et al., 1999(Dhillon et al., , 1997. However, long-term altitude life leads to altitude acclimatization, and body physiological functions such as anti-fatigue ability are recovered to some extent. The best strategy to enhance the exercise capacity of the body at high altitude is to search for measures that are effective in improving the acclimation ability and shortening the acclimation time (Andrew et al., 2014). However, the molecular mechanism of altitude acclimatization in the CNS is not known. In recent years, studies have proven that lactate can be used as a source of energy metabolism under certain circumstances. During sustained exercise, the turnover and oxidation rates of lactate exceed those of glucose. The diversion of lactate carbon to oxidation during exercise and recovery represents an irreversible loss of gluconeogenic precursors because the processes of protein proteolysis and gluconeogenesis from amino acids are insufficient to achieve complete glycogen restitution after exhausting exercise (Brooks, 1986). The "astrocyte-neuron lactate shuttle hypothesis" (Pellerin and Magistretti, 1994;Chih and Roberts, 2003) indicates that under extreme exercise or hypoxic conditions, lactate released from glial cells by anaerobic metabolism is able to be taken up by neurons used as an energy substrate (Schurr, 2018;Schurr and Gozal, 2012), and that lactate is oxidized preferentially over glucose for neuronal utilization (Gao et al., 2015). The body lactate exchange carriers are monocarboxylate transporters (MCTs), which are widely distributed on tissue cell membranes and can be divided into 14 subtypes (Halestrap, 2013). The central lactate shuttle is mainly achieved by a closed loop formed by MCT2 and MCT4 specifically expressed by neurons and astrocytes, respectively (Gao et al., 2014a(Gao et al., , 2014b. Therefore, we speculated that the mechanism of altitude acclimation in the CNS is related to the metabolism and utilization of lactate in the brain. To test this hypothesis, we designed a randomized controlled trial based on an animal model of altitude exercise-induced fatigue. The variation trend in the exercise capacity of rats was recorded under simulated high-altitude hypoxic environment, with a subsequent evaluation of the expression of MCT2 and MCT4 in the cerebral motor cortex as well as neuronal hypoxia injury and the lactate content in the rat brain. To explore the correlation between exercise-induced fatigue and brain lactate metabolism, so as to provide a potential basis for medical intervention for exercise-induced fatigue under high-altitude hypoxic environment.

Materials and methods
Reagents were obtained from Sigma-Aldrich Corporation (Sigma-Aldrich, St. Louis, MO, USA) except where noted otherwise.

Experimental animals and grouping
This study was approved by the Animal Care and Use Committee of the 940th Hospital of Joint Logistics Support Force of Chinese People's Liberation Army (Lanzhou, China), and followed the National Guidelines for Animal Experimentation. Experiments were performed on 63 male Sprague-Dawley rats (8 weeks old) weighing 280 ± 10 g were obtained from the experimental animal center at the hospital. Animals were individually housed in a room maintained at 18-24 • C, and the relative humidity of the air is 40-60% under a 12:12 h light/dark cycle, with access to food and water ad libitum. Experimental rats were randomly divided into seven groups (n = 9 per group): a control group that was fed under normal pressure and normoxic conditions without incremental load exercise; a normal exercise group (NE group) that was fed under normal pressure and normoxic conditions for 2 weeks and then subjected to incremental load exercise; a rush entry into altitude group (REIA group) that was fed under normal pressure and normoxic conditions for 2 weeks and then transferred to simulated high-altitude low pressure and hypoxic conditions for incremental load exercise; a 3-day-altitude acclimatization group (3 d AA group) that was fed under normal pressure and normoxic conditions for 11 days, and then transferred to simulated high-altitude low pressure and hypoxic conditions for 3 days, and then subjected to incremental load exercise; a 1-weekaltitude acclimatization group (1 week AA group) that was successively fed under normal pressure and normoxic conditions or simulated high-altitude low pressure and hypoxic conditions for 1 week, and then subjected to incremental load exercise; a 2-weeks-altitude acclimatization group (2 weeks AA group) that was fed under simulated highaltitude low pressure and hypoxic conditions for 2 weeks, and then subjected to incremental load exercise; a monocarboxylate transporter inhibitor group (MTI group) that was fed under simulating high-altitude low pressure and hypoxic conditions for 2 weeks, and then injected with monocarboxylate transporter inhibitor before exposing to incremental load exercise.

Simulated high-altitude low pressure and hypoxic conditions
High-altitude low pressure and hypoxic conditions approximately 5000 m above sea level were simulated in the low pressure and hypoxic animal experiment module (Model: FLYDWC50-IIA; Avic Guizhou Wind Thunder Aviation Ordnance Co. Ltd. Guizhou, China). The specific parameters were as follows: pressure 0.054 Mpa, partial pressure of oxygen 11.3 Kpa, temperature 12 • C (8 o ′ clock to 20 o ′ clock every day) and 2 • C (20 o ′ clock to 8 o ′ clock the next day), and relative humidity 30%− 40%.

Exercise-induced fatigue model construction and inhibitor injection
The incremental load exercise protocol of Bedford (Bedford et al., 1979) was used to establish an exercise-induced fatigue model in rats. Except for the control group, rats in the other groups were subjected to training on the animal treadmill for 3 days of adaptation (speed: 15 m/min, time: 5 min/d, slope: 0 • ). After that, incremental load exercise was performed at a speed of 8.2 m/min × 15 min + 15 m/min × 15 min + 20 m/min, until exhaustion. The criteria for exhaustion were as follows: The running gait of rats changed from a pedal ground type with the tail raised to a crawl ground type, called "belt riding". Additionally, rats were stuck at the posterior one-third of the treadmill belt more than 3 times, failed to be driven by various stimuli, and exhibited shortness of breath, tiredness, and unresponsiveness after stopping running (Zaretsky et al., 2018).
A-cyano-4-hydroxycinnamate (4-CIN; 90 mg/kg), which is a specific covalent inhibitor of mitochondrial lactate and pyruvate transport (Schurr et al., 2001) was intraperitoneally injected 1 h before load exercise in the MTI group.
The exhaustive time of load exercise in rats was recorded and averaged in each group.

Rat brain section preparation and double-labeling immunofluorescence
3 rats randomly selected from each group were transcardially perfused with 0.1 M phosphate buffer saline (PBS, pH 7.4) and 4% paraformaldehyde in PBS after anesthetized with isoflurane. Then rats were decapitated and brains were removed and post-fixed in paraformaldehyde (PFA) for 24 h at 4 • C, and then dehydrated using graded sucrose phosphate buffer for 48 h. Coronal sections were cut on a freezing microtome at a thickness of 6 µm and serial sections were collected consecutively in separate wells of an incubation chamber containing 0.1 M PBS with 60% glycerin.

Neuropathological evaluation
Neuropathological evaluation of the cerebral motor cortex of rats in each group via Nissl's staining with thionine (Solarbio, Beijing, China), was performed to determine the delayed neuron death by histological grade (HG) and neuronal density (ND) (Gong et al., 2012). Rat brain slices were dehydrated with alcohol, cleared with xylene and stained with thionine. Then the thionine-stained brain tissues were subjected to HG and ND assessments. HG was divided into four grades and assigned as follows Gao et al., 2019): grade 0, no neuron death; grade I, scattered single neuron death; grade II, mass neuron death; grade III, almost complete neuron death. ND was determined by counting the number of surviving pyramidal neurons with intact cell membranes, full nuclei, and clear nucleoli within a 250 µm linear length of the cerebral motor cortex. The average number of pyramidal neurons in six random areas of the cerebral motor cortex was calculated as the ND value.

Determination of the lactate content in the rat brain
The remaining 3 rats in each group were decapitated after anesthetized with isoflurane. The brain tissue samples were weighed accurately and homogenized according to the weight (g): volume (ml) = 1:9 with normal saline to generate 10% brain tissue homogenate. After separation at 3500 r/min for 10 min at 4 • C, the supernatant was loaded into EP tubes, and the lactate content was detected by colorimetric method (Li et al., 2022). The operation was performed in strict accordance with the instructions of the kit.

Statistical analysis
Statistical analyses were performed using SPSS v.16.0 for Windows (SPSS Inc., Chicago, IL, USA). All values are presented as mean ± standard deviation (SD) and differences were determined using one-way ANOVA with Bonferroni or Tamhane's T2 post-hoc tests. P values ﹤0.05 were considered statistically significant. Fig. 1) The average exhaustive time in the REIA group, 3 d AA group, 1 week AA group, 2 weeks AA group and MTI group were 61.00 ± 6.55 min, 66.38 ± 4.72 min, 73.13 ± 7.02 min, 100.25 ± 9.74 min and 71.25 ± 9.59 min, respectively, which were significantly lower than that in NE group (124.75 ± 9.36 min) (P < 0.05). As the time of altitude acclimatization was prolonged, the average exhaustive time in the 2 weeks AA group significantly recovered to 100.25 ± 9.74 min, and there was a significant difference compared with that in the REIA group and MTI group (P < 0.05), but no statistical difference compared with that in control group NE group (P > 0.05). Therefore, the exhaustive time of rats in each group under simulated high-altitude low pressure and hypoxic conditions showed a sharp decline after entering altitude, and then gradually increased with the prolongation of altitude acclimatization..

Expression of MCT2 and MCT4 in the cerebral motor cortex of rats
The results of Western Blotting showed that MCT2 and MCT4 proteins in the cerebral motor cortex of rats were respectively visible as 40 and 43-kDa bands, respectively, in the immunoblot ( Fig. 2A.), and the results of relative quantitative analysis are shown as Fig. 2B. MCT2 and MCT4 were expressed minimally and normally in the cerebral motor cortex of rats in control group. Compared with the control group, there was no significant difference in the NE group and REIA group (P ﹥ 0.05). After altitude acclimatization, the expression of MCT2 in the 3d AA group, 1 week AA group, 2 weeks AA group and MTI group increased earlier than that of MCT4, and was significantly higher than that in the control group by 144.3%, 176.1%, 174.6% and 168.8%, respectively (P < 0.05). The expression of MCT4 in the 1 week AA group, 2 weeks AA group and MTI group were significantly higher than that in the control group by 81.5%, 120.6% and 164.4%, respectively (P < 0.05).
Double-labeling immunofluorescence was also used to detect the expression of MCT2 in neurons and MCT4 in astrocytes in the cerebral motor cortex of rats, respectively. As shown in Fig. 3, compared with the control group, the expression of MCT2 on neurons in the cerebral motor cortex of rats increased significantly and remained stable after 1 week of altitude acclimatization. The expression of MCT4 on astrocytes began to increase after 3 days of altitude acclimatization, and remained stable after 2 weeks of altitude acclimatization. The variation trend of immunofluorescent detection was consistent with the results of Western blotting results, which confirmed each other.

Neuropathological evaluation of delayed neuronal death in the cerebral motor cortex of rats
Delayed neuronal death was evaluated by Nissl's staining of brain sections in the cerebral motor cortex of rats. The HG grades and mean ND values of rat brain neurons in each group are shown in Table 1. The mean ND values in the REIA group, 3 d AA group, 1 week AA group and MTI group were significantly lower than those in the control group (P < 0.05), but there was no significant difference between the control group and the NE group or 2 weeks AA group (P > 0.05). Representative Nissl's staining of brain sections in the cerebral motor cortex of rats in each group is shown as Fig. 4. The cortical neurons in the control group, NE group, 1week AA group and 2 weeks AA group were arranged regularly and evenly. The boundary between the cells was clear. The cell membrane and nucleus were intact, the nucleoli were clear, and the chromatin was evenly distributed. In the REIA group, 3d AA group and MTI group, the cortical neurons were arranged loosely and disorderly, the boundary between cells was not clear, and the chromatin gathered. In particular, the cytoskeleton and organelles were obviously destroyed, and some apoptotic or necrotic cells showed pyknosis and deep staining in the REIA group and 3 d AA group.

Lactate content of rats brain (Table 2)
The lactate content of rat brains in the REIA group, 3 d AA group, 1 week AA group, 2 weeks AA group and MTI group were 0.239 ± 0.017 mmol/g, 0.247 ± 0.012 mmol/g, 0.397 ± 0.017 mmol/g, 0.412 ± 0.024 mmol/g and 0.537 ± 0.011 mmol/g, respectively, and were significantly higher than that in NE group (0.175 ± 0.021 mmol/ g) (P < 0.05), and showed a gradual upwards trend with the prolongation of altitude acclimatization. After 1 week of altitude acclimatization, the increasing trend of the lactate content in the rat brain was significantly accelerated, and the lactate content of the rat brain in the MTI group was the highest, which was significantly different from all of the other groups (P < 0.05)..

Correlation between altitude acclimatization and lactate in CNS
The most significant and lasting effect of altitude hypoxia on the body is the reduction of exercise capacity. Studies have shown that the maximum exercise capacity of the human body at an altitude of 4500 m can decline to 50% of that in plain areas. With the prolongation of the residence time in high altitude areas, the body has become acclimated to high-altitude environments, and the exercise capacity is gradually recovered (Fan and Kayser, 2016). As one of the core contents of altitude sports medicine research, altitude acclimatization has become a focus in altitude physiology. Previous studies have shown that central lactate accumulation can cause neuronal injury, and can also be used by neurons as an energy substrate in exhaustive exercise or under anaerobic conditions (Pellerin and Magistretti, 1994;Tanaka et al., 2004). Lactate transport between cells is dependent on MCTs, which are divided into 14 subtypes (Gao et al., 2014a(Gao et al., , 2014b, among which MCT2 and MCT4 are essential for lactate transport in CNS during exercise (Eydoux et al., 2000). This study indicates that the whole link between lactate excretion and uptake in rat brain shows positive changes in adaptation to exercise-induced fatigue under simulated high-altitude hypoxic environment. Therefore, it is speculated that the altitude acclimatization phenomenon is closely related to the balance between neuronal injury caused by central lactate accumulation and the oxidative energy supply.

Inhibition of lactate transport and blockade of altitude acclimatization
In 1974, British researchers first reported MCT1 and its inhibitor 4-CIN in human erythrocytes. 4-CIN is a specific mitochondrial covalent transport inhibitor of lactate and pyruvate (Órdenes et al., 2021;Gonçalves et al., 2020;Guan and Morris, 2020;Yu et al., 2021). It can effectively inhibit the lactate transport function of MCTs. On the basis of previous studies, this study explores the relationship between the changes of altitude exercise capacity, cortical neuron injury and brain lactate metabolism via 4-CIN as an interference factor. Different subtypes of MCTs have different sensitivities to 4-CIN, which is a competitive inhibitor. 4-CIN has the most obvious inhibitory effect on the transport function of MCT2, however, the sensitivity of MCT4 is lower (Garcia et al., 1995;Bröer et al., 1999). The "astrocyte-neuron lactate shuttle hypothesis (Pellerin and Magistretti, 1994;Chih and Roberts, 2003)" indicates that lactate is released from glial cells via MCT4, and then transported into neurons via MCT2 as a substrate for energy metabolism. Regardless of which link in this closed energy metabolic loop is effectively inhibited by competitive inhibition, the entire process can be effectively blocked. Therefore, the effect of regulating lactate transportation on exercise-induced fatigue at altitude was studied by intraperitoneal injection of 4-CIN in rats as an interfering factor. The results showed that the average exhaustive time of the rats was significantly shortened after rush entry into altitude, and the exercise capacity gradually recovered with the extension of altitude acclimatization. The expressions levels of MCT2 and MCT4 in the cerebral motor cortex of rats were positively correlated with the altitude acclimatization time. Western Blotting and immunofluorescent detection showed the same variation trend, thereby confirming each other. The recovery of exercise capacity after altitude acclimatization can be rapidly blocked by using 4-CIN as an interference factor. From the perspective of 4-CIN as a competitive inhibitor of lactate transport, we believe that the blockage of exercise capacity recovery is mainly due to the effective inhibition of the lactate transport function of MCT2 in the energy metabolism loop, which then affects the energy metabolism of neurons. The results also showed that 4-CIN only inhibited the lactate transport function of MCT2 and MCT4, but did not affect the expression in molecular level.

Table 1
The histological grade (HG) and pyramidal neuronal density (ND) of the cerebral motor cortex of rats in each group.

The balance between brain lactate injury and energy supply under exercise fatigue status
Hypoxia regulates the expression of a subset of astrocyte-neuron specific transporters, including glucose and glutamate transporters as well as MCTs (Halestrap and Meredith, 2004). Previous studies have shown that the expression of MCT4 is increased in the astrocyte-neuron co-cultures during hypoxia, and neurons use lactate produced by glial cells as an energy substrate to resist oxygen and glucose deprivation injury in vitro (Gao et al., 2015(Gao et al., , 2014a(Gao et al., , 2014b. Lactate is thus emerging as a potential neuroprotective agent (Pellerin and Magistretti, 2012). In vivo studies have shown that the existence of a blood-brain barrier makes it difficult for peripheral lactate to enter the brain, so intracerebral extracellular lactate is independent of blood lactate (Erlichman et al., 2008). During hypoxia and exercise, the increase in brain lactate levels is less affected by the change in blood lactate levels. In 1998, Pellerin et al. (1998) found a special distribution of lactate between neurons and glial cells, suggesting that brain lactate is derived from astrocytes, while neurons serve as sites for lactate uptake and utilization. Lactate accumulation in the brain can not only cause delayed neuronal injury (Dhillon et al., 1999), but can also act as an energy substrate for neuronal activity under certain circumstances (Gao et al., 2015). In this study, pathological evaluation of neuronal death and determination of the brain lactate content were specifically designed to further investigate the mechanism of lactate damage and energy supply under exercise-induced fatigue. The results showed that with the extension of altitude acclimatization, the exercise capacity of rats gradually recovered, the brain lactate content of rats gradually increased, and the delayed neuronal injury and death were gradually alleviated. This recovery process was significantly blocked by 4-CIN, a lactate transport inhibitor. Based on the comprehensive analysis, we believe that brain lactate accumulation caused obvious neuronal injury after rush entry into altitude. MCTs expression increases with the prolongation of altitude acclimatization. Then, brain lactate produced by glial cells can be taken up in part by neurons and used as an energy substrate in exercise-induced fatigue status, thus alleviating the neuronal injury caused by lactate accumulation, and the exercise capacity is gradually recovered. This protective effect was abolished when lactate transport was blocked by 4-CIN. We also believe that lactate accumulation is a relatively slow process. It can be seen that the increasing trend of the lactate content in rat brain in this study was gradually slowed down with the extension of altitude acclimatization, which is considered to be related to the utilization of part of the lactate by neurons for energy metabolism. We speculated that the expression of MCTs, the brain lactate content and the delayed neuronal injury would enter a stable state with the extension of altitude acclimatization. The above results fully illustrate the speculated mechanism involving the balance between brain lactate injury and energy supply in the state of exercise fatigue after altitude acclimatization.
In conclusion, we believe that MCTs can be an important target for medical interventions for exercise-induced fatigue in high-altitude hypoxic environments. In this study, 4-CIN only blocked the lactate transport function of MCTs, but did not affect its molecular expression. In futher studies, siRNA interference and gene silencing should be used to artificially regulate MCTs expression, which will provide more powerful evidence for this mechanism. In addition, in vitro studies have shown that MCTs and excitatory amino acid transporters are involved in the protective mechanism of delayed neuronal injury (Parkin et al., 2018;Liu et al., 2014), which is also worthy of further exploration in high altitude sports medicine.

Medical ethics statement
All experimental animal protocols in this study were reviewed and approved by the Experimental Animal Ethics Committee of the 940th Hospital of Joint Logistics Support Force of Chinese People's Liberation Army (Approval Letter 2020KYLL032), and all experimental protocols were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments.

Data Availability
Data will be made available on request. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.