Yuki Tamura

Postexercise whole body heat stress additively enhances endurance training-induced mitochondrial adaptations in mouse skeletal muscle

A recent study demonstrated that heat stress induces mitochondrial biogenesis in C2C12 myotubes, thereby implying that heat stress may be an effective treatment to enhance endurance training-induced mitochondrial adaptations in skeletal muscle. However, whether heat stress actually induces mitochondrial adaptations in skeletal muscle in vivo is unclear. In the present study, we report the novel findings that 1) whole body heat stress produced by exposure of ICR mice to a hot environment (40°C, 30 min/day, 5 days/wk, 3 wk) induced mitochondrial adaptations such as increased mitochondrial enzyme activity (citrate synthase and 3-hydroxyacyl CoA dehydrogenase) and respiratory chain protein content (complexes I-V) in skeletal muscle in vivo and 2) postexercise whole body heat stress additively enhanced endurance training-induced mitochondrial adaptations (treadmill running, 25 m/min, 30 min/day, 5 days/wk, 3 wk). Moreover, to determine the candidate mechanisms underlying mitochondrial adaptations, we investigated the acute effects of postexercise whole body heat stress on the phosphorylation status of cellular signaling cascades that subsequently induce mitochondrial gene transcription. We found that whole body heat stress boosted the endurance exercise-induced phosphorylation of p38 MAPK, increased the phosphorylation status of p70S6K, a biomarker of mammalian target of rapamycin complex 1 activity, and unexpectedly dephosphorylated AMP-activated protein kinase and its downstream target acetyl-CoA carboxylase in skeletal muscle. Our present observations suggest that heat stress can act as an effective postexercise treatment. Heat stress treatment appeared to be clinically beneficial for people who have difficulty participating in sufficient exercise training, such as the elderly, injured athletes, and patients.

Daily heat stress treatment rescues denervation-activated mitochondrial clearance and atrophy in skeletal muscle

Traumatic nerve injury or nerve disease leads to denervation and severe muscle atrophy. Recent evidence shows that mitochondrial loss could be a key mediator of skeletal muscle atrophy. Here, we show that daily heat stress treatment rescues denervation‐induced loss of mitochondria and concomitant muscle atrophy. We also found that denervation‐activated autophagy‐dependent mitochondrial clearance (mitophagy) was suppressed by daily heat stress treatment. The molecular basis of this observation is explained by our results showing that heat stress treatment attenuates the increase of key proteins that regulate the tagging step for mitochondrial clearance and the intermediate step of autophagosome formation in denervated muscle. These findings contribute to the better understanding of mitochondrial quality control in denervated muscle from a translational perspective and provide a mechanism behind the attenuation of muscle wasting by heat stress.

Effects of decreased lactate accumulation after dichloroacetate administration on exercise training–induced mitochondrial adaptations in mouse skeletal muscle

Recent studies suggested that lactate accumulation can be a signal for mitochondrial biogenesis in skeletal muscle. We investigated whether reductions in lactate concentrations in response to dichloroacetate (DCA), an activator of pyruvate dehydrogenase, attenuate mitochondrial adaptations after exercise training in mice. We first confirmed that DCA administration (200 mg/kg BW by i.p. injection) 10 min before exercise decreased muscle and blood lactate concentrations after high‐intensity interval exercise (10 bouts of 1 min treadmill running at 40 m/min with a 1 min rest). At the same time, exercise‐induced signal cascades did not change by pre‐exercise DCA administration. These results suggested that DCA administration affected only lactate concentrations after exercise. We next examined the effects of acute DCA administration on mRNA expressions involved with mitochondrial biogenesis after same high‐intensity interval exercise and the effects of chronic DCA administration on mitochondrial adaptations after high‐intensity interval training (increasing intensity from 38 to 43 m/min by the end of training period). Acute DCA administration did not change most of the exercise‐induced mRNA upregulation. These data suggest that lactate reductions by DCA administration did not affect transcriptional activation after high‐intensity interval exercise. However, chronic DCA administration attenuated, in part, mitochondrial adaptations such as training‐induced increasing rates of citrate synthase (P = 0.06), β‐hydroxyacyl CoA dehydrogenase activity (P < 0.05), cytochrome c oxidase IV (P < 0.05) and a fatty acid transporter, fatty acid translocase/CD36 (P < 0.05), proteins after exercise training. These results suggest that lactate accumulation during high‐intensity interval exercise may be associated with mitochondrial adaptations after chronic exercise training.

Lactate administration increases mRNA expression of PGC-1α and UCP3 in mouse skeletal muscle

To examine the potential role of lactate as a signalling molecule in skeletal muscle, we performed global gene expression analysis of the mouse gastrocnemius muscle, 3 h after lactate administration using the Affymetrix GeneChip system (Affymetrix, Santa Clara, Calif., USA). Among the top 15 genes with the largest fold change, increased expression of Ppargc1a, Pdk4, and Ucp3 was confirmed using real-time quantitative polymerase chain reaction. Our findings suggest that lactate serves as a signal for upregulating genes related to mitochondrial function.

Effect of electrical stimulation-induced resistance exercise on mitochondrial fission and fusion proteins in rat skeletal muscle.

It is well known that resistance exercise increases muscle protein synthesis and muscle strength. However, little is known about the effect of resistance exercise on mitochondrial dynamics, which is coupled with mitochondrial function. In skeletal muscle, mitochondria exist as dynamic networks that are continuously remodeling through fusion and fission. The purpose of this study was to investigate the effect of acute and chronic resistance exercise, which induces muscle hypertrophy, on the expression of proteins related to mitochondrial dynamics in rat skeletal muscle. Resistance exercise consisted of maximum isometric contraction, which was induced by percutaneous electrical stimulation of the gastrocnemius muscle. Our results revealed no change in levels of proteins that regulate mitochondrial fission (Fis1 and Drp1) or fusion (Opa1, Mfn1, and Mfn2) over the 24-h period following acute resistance exercise. Phosphorylation of Drp1 at Ser616 was increased immediately after exercise (P < 0.01). Four weeks of resistance training (3 times/week) increased Mfn1 (P < 0.01), Mfn2 (P < 0.05), and Opa1 (P < 0.01) protein levels without altering mitochondrial oxidative phosphorylation proteins. These observations suggest that resistance exercise has little effect on mitochondrial biogenesis but alters the expression of proteins involved in mitochondrial fusion and fission, which may contribute to mitochondrial quality control and improved mitochondrial function.

Nrf2 deficiency does not affect denervation‐induced alterations in mitochondrial fission and fusion proteins in skeletal muscle

Oxidative stress‐induced mitochondrial dysfunction is associated with age‐related and disuse‐induced skeletal muscle atrophy. However, the role of nuclear factor erythroid 2‐related factor 2 (Nrf2) during muscle fiber atrophy remains to be elucidated. In this study, we examined whether deficiency of Nrf2, a master regulator of antioxidant transcription, promotes denervation‐induced mitochondrial fragmentation and muscle atrophy. We found that the expression of Nrf2 and its target antioxidant genes was upregulated at 2 weeks after denervation in wild‐type (WT) mice. The response of these antioxidant genes was attenuated in Nrf2 knockout (KO) mice. Nrf2 KO mice exhibited elevated levels of 4‐hydroxynonenal in the skeletal muscle, whereas the protein levels of the mitochondrial oxidative phosphorylation complex IV was declined in the denervated muscle of these mice. Increased in mitochondrial fission regulatory proteins and decreased fusion proteins in response to denervation were observed in both WT and KO mice; however, no difference was observed between the two groups. These findings suggest that Nrf2 deficiency aggravates denervation‐induced oxidative stress, but does not affect the alterations in mitochondrial morphology proteins and the loss of skeletal muscle mass.

Exercise-induced expression of monocarboxylate transporter 2 in the cerebellum and its contribution to motor performance

Monocarboxylate transporter 2 (MCT2) is an important component of the lactate transport system in neurons of the adult brain. Purkinje cells in the cerebellum have been shown to have high levels of MCT2, suggesting that this protein has a key function in energy metabolism and neuronal activities in these cells. However, it is not known whether inhibition of lactate transport via MCT2 in the cerebellum affects motor performance. To address this question, we examined motor performance in mice following the inhibition of lactate transport via MCT2 in the cerebellum using α-cyano-4-hydroxycinnamate (4-CIN). 4-CIN or saline was injected into the subarachnoidal space of the cerebellum of mice and motor performance was analyzed by a rotarod test both before and after injection. 4-CIN injection reduced retention time in the rotarod test by approximately 80% at 1h post-injection compared with pre-injection. No effect was observed at 2h post-injection or in mice treated with the vehicle control. Because we observed that MCT2 plays an important role in motor performance, we next investigated the effects of acute exercise on MCT2 transcription and protein levels in mice sampled pre-exercise and at 0 and 5h after 2h of treadmill running. We found a significant increase in MCT2 mRNA levels, but not of protein levels, in the cerebellum at 5h after exercise. Our results indicate that lactate transport via MCT2 in the cerebellum may play an important role in motor performance and that exercise can increase MCT2 expression at the transcriptional level.

Effects of Heat Stress Treatment on Age-dependent Unfolded Protein Response in Different Types of Skeletal Muscle

Mitochondrial and endoplasmic reticulum (ER) stress, and subsequently activated responses (mitochondrial/ER unfolded protein responses; UPRmt/UPRER), are involved in the pathogenesis of sarcopenia. To extend both basic and translational knowledge, we examined (i) whether age-induced mitochondrial and ER stress depend on skeletal muscle type in mice and (ii) whether heat stress treatment, a suggested strategy for sarcopenia, improves age-induced mitochondrial and ER stress. Aged (21-month-old) mice showed more severe mitochondrial stress and UPRmt than young (12-week-old) mice, based on increased oxidative stress, mitochondrial proteases, and mitochondrial E3 ubiquitin ligase. The aged mice also showed ER stress and UPRER, based on decreased ER enzymes and increased ER stress-related cell death. These changes were much more evident in soleus muscle than in gastrocnemius and plantaris muscles. After daily heat stress treatment (40 °C chamber for 30 minutes per day) for 4 weeks, mice showed remarkable improvements in age-related changes in soleus muscle. Heat stress had only minor effects in gastrocnemius and plantaris muscles. Based on these findings, age-associated mitochondrial stress, ER stress, and UPRmt/ER vary qualitatively with skeletal muscle type. Our results suggest a molecular basis for the beneficial effects of heat stress on muscle atrophy with age in soleus muscle.

Comparison between pre–exercise casein peptide and intact casein supplementation on glucose tolerance in high–fat diet–fed mice

We hypothesized that along with exercise, casein peptide supplementation would have a higher impact on improving glucose tolerance than intact casein. Male 6-week-old ICR mice were provided a high-fat diet to induce obesity and glucose intolerance. The mice were randomly divided into 4 treatment groups: control (Con), endurance training (Tr), endurance training with intact casein supplementation (Cas+Tr), and endurance training with casein peptide supplementation (CP+Tr). The mice in each group were orally administrated water, intact casein, or casein peptide (1.0 mg/g body weight, every day), and then subjected to endurance training (15-25 m/min, 60 min, 5 times/week for 4 weeks) on a motor-driven treadmill 30 min after ingestion. Our results revealed that total intra-abdominal fat was significantly lower in CP+Tr than in Con (p < 0.05). Following an oral glucose tolerance test, the blood glucose area under the curve (AUC) was found to be significantly smaller for CP+Tr than for Con (p < 0.05). Moreover, in the soleus muscle, glucose transporter 4 (GLUT4) protein levels were significantly higher in CP+Tr than in Con (p < 0.01). However, intra-abdominal fat, blood glucose AUC, and GLUT4 protein content in the soleus muscle did not alter in Tr and Cas+Tr when compared with Con. These observations suggest that pre-exercise casein peptide supplementation has a higher effect on improving glucose tolerance than intact casein does in mice fed a high-fat diet.

Past injurious exercise attenuates activation of primary calcium‐dependent injury pathways in skeletal muscle during subsequent exercise

Past contraction‐induced skeletal muscle injury reduces the degree of subsequent injury; this phenomenon is called the “repeated bout effect (RBE).” This study addresses the mechanisms underlying the RBE, focusing on primary calcium‐dependent injury pathways. Wistar rats were subdivided into single injury (SI) and repeated injury (RI) groups. At age 10 weeks, the right gastrocnemius muscle in each rat in the RI group was subjected to strenuous eccentric contractions (ECs). Subsequently, mild ECs were imposed on the same muscle of each rat at 14 weeks of age in both groups. One day after the exercise, the RI group showed a lower strength deficit than did the SI group, and neither group manifested any increase in membrane permeability. The concentration of protein carbonyls and activation of total calpain increased after ECs given at the age of 14 weeks. Nonetheless, these increases were lower in the RI group than in the SI group. Furthermore, calcium‐dependent autolysis of calpain‐1 and calpain‐3 in the RI group was diminished as compared with that in the SI group. Although peak ankle joint torque and total force generation during ECs at the age of 14 weeks were similar between the two groups, phosphorylation of JNK (Thr183/Tyr185), an indicator of mechanical stress applied to a muscle, was lower in the RI group than in the SI group. These findings suggest that activation of the primary calcium‐dependent injury pathways is attenuated by past injurious exercise, and mechanical stress applied to muscle fibers during ECs may decrease in the RBE.