Mitochondrial dysfunction causes neurodegenerative diseases, such as Alzheimer’s disease, and Parkinson’s disease and metabolic diseases, such as type 2 diabetes; it is caused by physiological deterioration owing to aging and lack of exercise (Short et al., 2005; Sutherland et al., 2009; Safdar et al., 2011; Zhang et al., 2012; Rodriguez-Martinez et al., 2013). Mitochondrial biogenesis, the formation of new mitochondria in cells, is vital for mitochondrial function in various tissues. Endurance exercise training induces brain and skeletal muscle mitochondrial biogenesis (Steiner et al., 2011; Halling et al., 2019).

Peroxisome proliferator-activated receptor gamma-coactivator 1-α (PGC-1α) is the master regulator of mitochondrial biogenesis in various cell types (Sutherland et al., 2009; Wareski et al., 2009; Lezi et al., 2013). Eight weeks of treadmill exercise training induces an increase in PGC-1α mRNA expression in skeletal muscle and brain regions, including the cortex and hippocampus, with a concomitant increase in mitochondrial DNA (mtDNA) copy number, enhancing exercise performance (Steiner et al., 2011). Training adaptation reflects the accumulation of the beneficial physiological functions produced from single bouts of exercise; thus, to produce a better exercise training strategy, it is essential to understand the beneficial effects of a single bout of exercise and elucidate the mechanism underlying exercise. However, it remains unclear whether a single bout of exercise would increase mitochondrial biogenesis in the brain.

Research indicates that a single bout of exercise (low, moderate, and high intensity) increases the transcriptional regulators of mitochondrial biogenesis [PGC-1α, mitochondrial transcription factor A (TFAM), and nuclear respiratory factor1 (NRF1) mRNA expression] in mice and human skeletal muscle (Safdar et al., 2009; Little et al., 2010; Tadaishi et al., 2011; Saleem and Hood, 2013; Gidlund et al., 2015), proportional to the exercise intensity used (Ikeda et al., 2008). These results suggest that even a single bout of exercise enhances mitochondrial biogenesis in skeletal muscle, and the extent of its enhancement depends on the intensity of exercise performed. On the other hand, it is unclear whether hippocampal mitochondrial biogenesis is also affected by a single bout of exercise and whether its effect, if any, depends on exercise intensity.

Skeletal muscles release lactate into the blood during exercise; blood lactate concentrations range from ~3mM at rest to ~10mM in mice following high-intensity exercises, which is above the lactate threshold (LT; Hu et al., 2021). LT is the time point when blood lactate concentrations start to rise from the resting level if the subjects continue to exercise with increasing exercise intensity. This time point is thought to express the increased usage of both muscle glycogen and fast muscle fibers (Billat et al., 2005). Lactate was thought to be only a waste product derived from glycolysis metabolism. However, recent studies revealed that circulating blood lactate serves as energy substrates for the skeletal muscle (Takahashi et al., 2020) and enhances skeletal muscle mitochondrial biogenesis (Rowe et al., 2013; Kitaoka et al., 2016). Additionally, lactate crosses the blood–brain barrier (BBB) through monocarboxylate transporters (MCT) in brain cells (Riske et al., 2017; El Hayek et al., 2019) and supplies the energy substrates to neurons (Kobayashi et al., 2019; Lev-Vachnish et al., 2019). For example, 6-week high-intensity interval training (HIIT) increased hippocampal lactate concentration and enhanced mitochondrial biogenesis (Hu et al., 2021). In addition, HIIT also increased hippocampal brain-derived neurotrophic factor (BDNF) expression and protein, which is a necessary regulator for enhancing mitochondrial quality control, and maintaining neuronal function and survival (Freitas et al., 2018; Hu et al., 2021; Okamoto et al., 2021).

Exogenous intraperitoneally (i.p.) or orally administered lactate works the same way as endogenous lactate released from skeletal muscle during exercise. Lactate administrated chronically to mice functioned as a signal molecule that promoted adult hippocampal neurogenesis (Lev-Vachnish et al., 2019). Acute and chronic peripheral lactate administration increased extracellular lactate concentrations in hippocampal tissue, which produced antidepressant-like effects in mice (Carrard et al., 2018). Lactate administered to hippocampal cells in vitro enhanced ATP levels, PGC-1α, BDNF protein level, mtDNA copy number, and potentiated mitochondrial function (Hu et al., 2021). We hypothesized that blood lactate increased by high-intensity exercise could promote PGC-1α mRNA expression in the hippocampus and trigger hippocampal mitochondrial biogenesis.

Lactate derived from brain glycogen is critical for neurons. In brief, lactate generated by glycogen degradation in astrocytes is transferred to the neurons via MCTs and acts as energy sources for neurons, helping maintain their function; this system is known as the astrocyte-neuronal lactate shuttle (ANLS; Brooks, 2002). The ANLS has been referred to play a key role in lactate transport and neuronal activity (Pellerin et al., 1998), which is involved in the hippocampal function, such as long-term potentiation and long-term memory as well as endurance exercise capacity (Newman et al., 2011; Suzuki et al., 2011; Lev-Vachnish et al., 2019). Matsui et al. found that a bout of acute moderate-intensity exercise increased hippocampal lactate and ATP levels and decreased brain glycogen concentrations, which was abolished by 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) administration, a glycogenolysis inhibitor, and diminished endurance exercise capacity (Matsui et al., 2017). We thus hypothesized that glycogenolysis inhibition during exercise might also attenuate exercise-induced PGC-1α and MCTs mRNA expression in the hippocampus. Furthermore, intracerebral DAB administration decreased neuron lactate supply from astrocytes and was deleterious to rat’s cognitive function (Suzuki et al., 2011; Matsui et al., 2017). These results suggest that lactate derived from brain glycogenolysis is vital for maintaining brain functions. However, no reports exist on whether lactate derived from brain glycogenolysis contributes to mitochondrial biogenesis after high-intensity exercise.

This study assessed the effects of a single bout of low-, moderate-, or high-intensity treadmill exercise on PGC-1α mRNA expression and mtDNA copy number in the mouse hippocampus. Next, we examined whether exogenous lactate administration increased hippocampal extracellular lactate concentration and mimicked the effects of high-intensity exercise. Then, we examined if exogenous lactate or UK5099, a lactate transporter inhibitor, administered before high-intensity exercise alters mouse hippocampus PGC-1α mRNA expression and mtDNA copy number as well as BDNF expression. Finally, we examined whether pre-exercise intra-hippocampal injection of DAB affected the mouse hippocampal PGC-1α and MCTs mRNA expression following high-intensity exercise.

Regular exercise training enhances skeletal muscle and hippocampal PGC-1α mRNA and mtDNA copy number (Zhang et al., 2012; Halling et al., 2019). Six weeks of HIIT accompanied by increased blood and hippocampal lactate concentrations has been reported to increase hippocampal expression of mitochondrial biogenesis-related genes PGC-1α, NRF2, and mtDNA copy number (Hu et al., 2021). The effects of regular exercise reflect an accumulation of physiological adaptations produced by bouts of high-intensity exercise. Thus, it is essential to understand the beneficial effects of a single bout of high-intensity exercise and elucidate the mechanisms underlying their effects to produce better high-intensity exercise training strategies. However, the effects of acute high-intensity exercise on hippocampal mitochondrial biogenesis are unclear.

In the current study, an increase in hippocampal PGC-1α mRNA expression was observed 12h after exercise. An increase in hippocampal mtDNA copy number followed 48h later, and its increment depended on exercise intensity. Running time to exhaustion (26.1±0.37min) is slightly below in the high-intensity exercise group compared to the low- and moderate-intensity exercise groups (30min); running distance in the high-intensity group did not differ from that of the moderate-intensity exercise group. However, the blood lactate concentrations immediately after exercise and post-exercise hippocampal mitochondrial biogenesis significantly differed between the three exercise groups. These results are consistent with the previous reports that 20min of a single bout of high-intensity exercise induced the activation of skeletal muscle mitochondrial transcription (Hoshino et al., 2015), and exercise intensity affects skeletal muscle PGC-1α protein levels (Terada et al., 2005; Tadaishi et al., 2011). Moreover, the present results are consistent with the results of the human study, which indicated that a high-intensity interval cycle ergometer for a total duration of 20min showed higher BDNF serum concentration levels than at rest and moderate-intensity exercise group in the human study (Saucedo Marquez et al., 2015).

A blood lactate concentration >12mM was required to increase hippocampal PGC-1α mRNA and mtDNA copy number. In contrast, low- or moderate-intensity exercise correlated with 3–4mM blood lactate concentration, which did not increase the hippocampal PGC-1α mRNA and mtDNA copy number. These results are consistent with previous findings that exercise intensity above the LT increased PGC-1α mRNA expression in human skeletal muscle but that below the LT did not (Tobina et al., 2011). Thus, these results suggested that elevated blood lactate induced by high-intensity exercise above the LT would contribute to the induction of mtDNA copy number through PGC-1α expression in the hippocampus.

To our knowledge, the current study is the first to show that an increase in mitochondrial biogenesis in the brain, especially the hippocampus, depends on exercise intensity in the same manner as seen in skeletal muscle. In contrast, a single bout of low- or moderate-intensity exercise did not affect mitochondrial biogenesis in the hippocampus. Thus, single bouts of low- or moderate-intensity exercise are not likely to induce hippocampal mitochondrial biogenesis, suggesting that these exercises would need to be regularly repeated over 4weeks to promote hippocampal mitochondrial biogenesis (Steiner et al., 2011; Zhang et al., 2012).

Brain cell MCTs transport lactate across the BBB (El Hayek et al., 2019), which is enhanced during exercise (Quistorff et al., 2008; van Hall et al., 2009). Acute (Takimoto and Hamada, 2014) and chronic exercise (El Hayek et al., 2019) above LT enhances MCT expression in the hippocampus, enhancing cognitive function by increasing BDNF expression through the induction of PGC-1α (El Hayek et al., 2019). Besides, Overgaard et al. (2012) indicated that the brain uptakes lactate from the blood and releases lactate into the blood concomitantly, and that net lactate uptake is a plus, and net brain lactate uptake is increased by the increase in circulating blood lactate level during high-intensity exercise. Based on these data, we predicted that some of the blood lactate increased by a single bout of high-intensity exercise or lactate administration would be taken up into the brain via MCTs, where it would promote PGC-1α expression, followed by mitochondrial biogenesis. To test this hypothesis, we examined hippocampal extracellular lactate concentration by using in vivo microdialysis in the presence and absence of UK5099, a potent MCT1–4 inhibitor, after lactate injection. In this experiment, to eliminate the influence of lactate production derived from astrocytes glycogenolysis, which would be expected to occur during high-intensity exercise, I.P. lactate administration and microdialysis were performed using the resting instead of the exercised mice.

We found that the hippocampal extracellular lactate concentration increased following lactate injection, consistent with previous studies (Machler et al., 2016; Carrard et al., 2018). However, this lactate concentration increase was suppressed by UK5099 administration, indicating that MCT inhibition prevents lactate transport from the blood into hippocampal extracellular space. Furthermore, UK5099 administration also suppressed lactate-induced increases in hippocampal PGC-1α and TFAM mRNA, and mtDNA copy number.

The results demonstrate that high-intensity exercise or lactate administration increases hippocampal extracellular lactate concentration, which could induce hippocampal PGC-1α mRNA expression and increase mtDNA copy number. Moreover, hippocampal BDNF mRNA expression was also increased after a single bout of high-intensity exercise, which was consistent with a previous study showing that increased PGC-1α led to the induction of BDNF expression (Wrann et al., 2013; Mikami et al., 2021). In addition, the lactate-induced hippocampal BDNF expression was suppressed by injecting MCT1/2 inhibitor, AR-C155858 (El Hayek et al., 2019). However, the present study does not elucidate how the increased hippocampal lactate upregulated PGC-1α and BDNF mRNA expression. Yang et al. showed that exposure of neurons to lactate modified the intracellular NADH/NAD ratio, promoted N-methyl-D-aspartate (NMDA) receptor activity and its downstream signaling cascade Erk1/2 (Yang et al., 2014). The activating of the NMDA receptors caused an increase in intracellular Ca²⁺ concentration, thereby inducing the gene expression of PGC-1α (Luo et al., 2009). We hypothesize that lactate taken up from blood into the brain could have altered intracellular NADH/NAD ratio and could have triggered changed mRNA expressions related to mitochondrial biogenesis. Further studies are necessary to prove our hypothesis.

Exogenous lactate administration increased blood lactate concentration, induced hippocampal PGC-1α and MCT mRNA expression, and increased mtDNA copy number, similar to high-intensity exercise. These results indicated that systemic administration of lactate mimics the effect of high-intensity exercise on hippocampal mitochondria biogenesis. However, a single lactate administration was insufficient to increase MCT2 and BDNF mRNA, and mtDNA copy number to the same extent as a single bout of high-intensity exercise.

We speculate that the differences in hippocampal mRNA expression and mtDNA copy number between exogenous lactate administration and bouts of high-intensity exercise may result from the production of myokines and other factors induced by high-intensity exercise but not by exogenous lactate administration. Irisin, a myokine released from skeletal muscle, is transferred to the brain and induces hippocampal PGC-1α and BDNF expression (Wrann et al., 2013; Azimi et al., 2018; Lourenco et al., 2019). In the future studies, we plan to investigate the effect of lactate administration on myokines in the circulating blood.

The role of lactate produced by astrocytic glycolysis in mediating memory processes (Descalzi et al., 2019) and exercise endurance capacity has recently been characterized (Machler et al., 2016; Matsui et al., 2017). The glycogen in astrocytes is broken down and metabolized to lactate, shuttled to neurons via MCT2, and acts as an energy source in nerve cells (Pellerin et al., 1998; Brooks, 2020). Inhibiting hippocampal glycogenolysis via DAB injection impaired memory and decreased endurance exercise capacity, indicating that the lactate supplied by astrocytes to neurons is critical for regulating memory processing (Suzuki et al., 2011) and maintaining endurance capacity (Matsui et al., 2017). We speculate that glycogen-derived lactate released during exercise might mediate the promotion of hippocampal mitochondrial biogenesis after high-intensity exercise.

Our results showed that compared with mice injected with saline, DAB injection before exercise inhibited hippocampal glycogenolysis after exercise; however, hippocampal lactate concentration following high-intensity exercise was elevated in the presence or absence of DAB. Moreover, hippocampal PGC-1α, MCT1, and MCT2 mRNA expression following high-intensity exercise were unaffected by DAB injection before exercise. These results indicate that the lactate derived from hippocampal glycogenolysis during high-intensity exercise does not promote hippocampal PGC-1α, MCT1, and MCT2 mRNA expression. This was inconsistent with the previous findings that lactate derived from astrocytic glycogenolysis is vital for cognitive memory and regulating exercise performance. Based on these results, although a possible effect of brain glycogen-derived lactate release cannot be ruled out, we speculate that the induction of hippocampal PGC-1α, MCT1, and MCT2 mRNA expression after exhaustive and short-term exercise is attributable to the marked increase in the lactate taken into the brain from the blood, rather than from astrocytic glycogenolysis.

The following results support our speculation; (1) hippocampal extracellular lactate, taken up from blood into the brain, was suppressed by MCT inhibitors, followed by a marked decrease in PGC-1α, MCT1, and MCT2 mRNA expression; (2) the inhibition of hippocampal glycogenolysis by DAB did not affect PGC-1α, MCT1, and MCT2 mRNA expression. The type of exercise used in the present study was short-term and exhaustive; if the exercise were different, the contribution of lactate derived from brain glycogenolysis on gene expression related to mitochondrial biogenesis would be altered. Further study is necessary to investigate the contribution of lactate, which is incorporated from circulating blood or produced from brain glycogenolysis, and the beneficial effect of different types of exercises on brain function. Our results provide a helpful strategy to enhance brain function via high-intensity exercise training, which may contribute to developing exercise programs in human subjects.

Our results show that a single bout of exercise above the LT intensity enhanced hippocampal mitochondrial biogenesis. A single injection of exogenous lactate increased hippocampal extracellular lactate concentration, which partially mimicked the effects of a single bout of high-intensity exercise. Additionally, the UK5099 administration abolished the increase in hippocampal extracellular lactate concentration. However, DAB did not affect hippocampal lactate, PGC-1α, and MCT mRNA concentration despite inhibiting hippocampal glycogenolysis. Therefore, the lactate released into the blood from skeletal muscle during high-intensity exercise may be a crucial stimulator for hippocampal mitochondria biogenesis. These data improve our understanding of the effects of high-intensity exercise and may be a basis for further clinical or athletic applications.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by the Animal Care and Use Committee of Nippon Medical School (approval no. 30-030).

JP and TM conceived and designed experiments, interpreted the results of experiments, and drafted the manuscript. JP, JK, and TM performed experiments and analyzed data. JP prepared figures. All authors have approved to submit the final version manuscript.

This study was supported by a Grant-in-Aid for Early-Career Scientists (19K20051) from the Japan Society for the Promotion of Science (JSPS).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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