Effects of endurance training under calorie restriction on energy substrate metabolism in mouse skeletal muscle and liver.

We investigated whether calorie restriction (CR) enhances metabolic adaptations to endurance training (ET). Ten-week-old male Institute of Cancer Research (ICR) mice were fed ad libitum or subjected to 30% CR. The mice were subdivided into sedentary and ET groups. The ET group performed treadmill running (20-25 m/min, 30 min, 5 days/week) for 5 weeks. We found that CR decreased glycolytic enzyme activity and monocarboxylate transporter (MCT) 4 protein content, while enhancing glucose transporter 4 protein content in the plantaris and soleus muscles. Although ET and CR individually increased citrate synthase activity in the plantaris muscle, the ET-induced increase in respiratory chain complex I protein content was counteracted by CR. In the soleus muscle, mitochondrial enzyme activity and protein levels were increased by ET, but decreased by CR. It has been suggested that CR partially interferes with skeletal muscle adaptation to ET.

Better maintenance of enzymatic capacity and higher levels of substrate transporter proteins in skeletal muscle of aging female mice.

This study investigated sex-specific differences in high-energy phosphate, glycolytic, and mitochondrial enzyme activities and also metabolite transporter protein levels in the skeletal muscles of adult (5 months old), middle-aged (12 months old), and advanced-aged (24 months old) mice. While gastrocnemius glycogen content increased with age regardless of sex, gastrocnemius triglyceride levels increased only in advanced-aged female mice. Aging decreased creatine kinase and adenylate kinase activities in the plantaris muscle of both sexes and in the soleus muscle of male mice but not in female mice. Irrespective of sex, phosphofructokinase and lactate dehydrogenase (LDH) activities decreased in the plantaris and soleus muscles. Additionally, hexokinase activity in the plantaris muscle and LDH activity in the soleus muscle decreased to a greater extent in aged male mice compared with those in aged female mice. Mitochondrial enzyme activities increased in the plantaris muscle of aged female mice but did not change in male mice. The protein content of the glucose transporter 4 in the aged plantaris muscle and fatty acid translocase/cluster of differentiation 36 increased in the aged plantaris and soleus muscles of both sexes, with a significantly higher content in female mice. These findings suggest that females possess a better ability to maintain metabolic enzyme activity and higher levels of metabolite transport proteins in skeletal muscle during aging, despite alterations in lipid metabolism. Our data provide a basis for studying muscle metabolism in the context of age-dependent metabolic perturbations and diseases that affect females and males differently.

Metabolomic responses to high-intensity interval exercise in equine skeletal muscle: effects of rest interval duration.

High-intensity interval training has attracted considerable attention as a time-efficient strategy for inducing physiological adaptations, but the underlying mechanisms have yet to be elucidated. By using metabolomics techniques, we investigated changes in the metabolic network responses in Thoroughbred horses to high-intensity interval exercise performed with two distinct (15 min or 2 min) rest intervals. The peak plasma lactate level was higher during high-intensity exercise with a 2 min rest duration than that with a 15 min rest duration (24.5±6.8 versus 13.3±2.7 mmol l-1). The arterial oxygen saturation was lower at the end of all exercise sessions with a 2 min rest duration than that with a 15 min rest duration. Metabolomic analysis of skeletal muscle revealed marked changes in metabolite concentrations in the first and third bouts of the 15 min rest interval conditions. In contrast, there were no metabolite concentrations or pathways that significantly changed during the third bout of exercise performed with a 2 min rest interval. Our findings suggest that the activity of each energy production system is not necessarily reflected by apparent changes in metabolite concentrations, potentially due in part to a better match between metabolite flux into and out of the pathway and cycle, as well as between metabolite production and disposal. This study provides evidence that changes in metabolite concentrations vary greatly depending on the number of repetitions and the length of rest periods between exercises, even if the exercises themselves are identical.

Sex-specific differences in the metabolic enzyme activity and transporter levels in mouse skeletal muscle during postnatal development.

Although sex-associated differences in energy metabolism in adults are well-characterized, developmental sex-specific changes in skeletal muscle metabolism are largely unknown. This study investigated sex differences in high-energy phosphate, glycolytic, and mitochondrial enzyme activities and metabolite transporter protein levels in mouse skeletal muscles during the early postnatal period (day 10), post-weaning (day 28), sexual maturity (day 56), and adult life (day 140). No significant sex-specific differences were observed on days 10 and 28, except for glucose transporter (GLUT) 4 level. The hexokinase, phosphofructokinase, and lactate dehydrogenase activities of skeletal muscle were higher and the citrate synthase, cytochrome c oxidase, and ß-hydroxyacyl-CoA dehydrogenase activities were lower in female mice than those in male mice on days 56 and 140. The GLUT4 and FAT/CD36 protein levels were higher and the monocarboxylate transporter 4 level was lower in the skeletal muscles of female mice than those of male mice, particularly on days 56 and 140. At 140 days of age, the respiratory exchange ratio during treadmill running (15 m/min, 60 min) was lower in females than that in males, despite no sex differences at rest. In summary, sex differences were not evident in the early postnatal and post-weaning periods but became apparent after the mice reached sexual maturity. These findings indicate that sexually mature animals are a better model for investigating sex differences, particularly in the context of studying energy metabolism in mice.

Effects of Endurance Training on Metabolic Enzyme Activity and Transporter Proteins in Skeletal Muscle of Ovariectomized Mice.

PURPOSE: Estrogen deficiency or insufficiency can occur under several conditions, leading to negative health outcomes. To establish an effective countermeasure against estrogen loss, we investigated the effects of endurance training on ovariectomy (OVX)-induced metabolic disturbances. METHODS: Female Institute of Cancer Research mice underwent OVX or sham operations. On day 7 of recovery, the mice were randomized to remain either sedentary or undergo 5 wk of treadmill running (15-20 m·min -1 , 60 min, 5 d·wk -1 ). During week 5 of the training, all animals performed a treadmill running test (15 m·min -1 , 60 min). RESULTS: After the experimental period, OVX resulted in greater gains in body mass, fat mass, and triglyceride content in the gastrocnemius muscle. OVX enhanced phosphofructokinase activity in the plantaris muscle and decreased lactate dehydrogenase activity in the plantaris and soleus muscles. OVX decreased the protein content of NDUFB8, a mitochondrial respiratory chain subunit, but did not decrease other mitochondrial proteins or enzyme activities. Endurance training significantly enhanced mitochondrial enzyme activity and protein content in the skeletal muscles. Although OVX increased the respiratory exchange ratio during the treadmill running test, and postexercise blood lactate levels, endurance training normalized these parameters. CONCLUSIONS: The present findings suggest that endurance training is a viable strategy to counteract the negative metabolic consequences in hypoestrogenism.

Branched-chain amino acid supplementation suppresses the detraining-induced reduction of mitochondrial content in mouse skeletal muscle.

Exercise training enhances oxidative capacity whereas detraining reduces mitochondrial content in skeletal muscle. The strategy to suppress the detraining-induced reduction of mitochondrial content has not been fully elucidated. As previous studies reported that branched-chain amino acid (BCAA) ingestion increased mitochondrial content in skeletal muscle, we evaluated whether BCAA supplementation could suppress the detraining-induced reduction of mitochondrial content. Six-week-old male Institute of Cancer Research (ICR) mice were randomly divided into four groups as follows: control (Con), endurance training (Tr), detraining (DeTr), and detraining with BCAA supplementation (DeTr + BCAA). Mice in Tr, DeTr, and DeTr + BCAA performed treadmill running exercises [20-30 m/min, 60 min, 5 times/week, 4 weeks]. Then, mice in DeTr and DeTr + BCAA were administered with water or BCAA [0.6 mg/g of body weight, twice daily] for 2 weeks of detraining. In whole skeletal muscle, mitochondrial enzyme activities and protein content were decreased after 2 weeks of detraining, but the reduction was suppressed by BCAA supplementation. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) protein content, a master regulator of mitochondrial biogenesis, was decreased by detraining irrespective of BCAA ingestion. Regarding mitochondrial degradation, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), a mitophagy-related protein, was significantly higher in the Tr group than in the DeTr + BCAA group, but not different from in the DeTr group. With respect to mitochondrial quality, BCAA ingestion did not affect oxygen consumption rate (OCR) and reactive oxygen species (ROS) production in isolated mitochondria. Our findings suggest that BCAA ingestion suppresses the detraining-induced reduction of mitochondrial content partly through inhibiting mitophagy.

Regulation of protein and oxidative energy metabolism are down-regulated in the skeletal muscles of Asiatic black bears during hibernation.

Hibernating animals exhibit an unexplained physiological characteristic of skeletal muscles being atrophy resistance, in which case muscle mass and strength remain almost unchanged both before and after hibernation. In this study, we examined the alterations in the regulatory systems of protein and energy metabolism in the skeletal muscles of Asiatic black bears during hibernation. Skeletal muscle samples (vastus lateralis muscle) were collected from identical individuals (n = 8) during the active (July) and hibernating (February) periods, while histochemical and biochemical analyses were performed. We observed no significant alterations in body weight, muscle fiber size, and fiber type composition during the active and hibernating periods, indicating that the skeletal muscles of bears are very well preserved during hibernation. In hibernating bear skeletal muscles, both regulatory pathways of muscle protein synthesis (Akt/mechanistic target of rapamycin and mitogen-activated protein kinase systems) and proteolysis (ubiquitin-proteasome and autophagy systems) were down-regulated. Gene expression levels of factors regulating oxidative metabolism were also decreased in hibernating bear skeletal muscles. This is likely an adaptive strategy to minimize the energy wasting of amino acids and lipids during hibernation, which is accompanied by a prolonged period of disuse and starvation.

Inhibition of monocarboxylate transporters (MCT) 1 and 4 reduces exercise capacity in mice.

The concept of lactate shuttle is widely accepted in exercise physiology. Lactate transport is mediated by monocarboxylate transporters (MCT), which enable cells to take up and release lactate. However, the role of lactate during exercise has not yet been fully elucidated. In this study, we investigated the effects of lactate transport inhibition on exercise capacity and metabolism in mice. Here, we demonstrated that MCT1 inhibition by α-cyano-4-hydroxycinnamate administration (4-CIN, 200 mg/g of body weight) reduced the treadmill running duration at 20 m/min. The administration of 4-CIN increased the blood lactate concentration immediately after exercise. With matched exercise duration, the muscle lactate concentration was higher while muscle glycogen content was lower in 4-CIN-administered mice. Further, we showed that MCT4 inhibition by bindarit administration (50 mg/kg of body weight) reduced the treadmill running duration at 40 m/min. Bindarit administration increased the muscle lactate but did not alter the blood lactate and glucose concentrations, as well as muscle glycogen content, immediately after exercise. A negative correlation was observed between exercise duration at 40 m/min and muscle lactate concentration immediately after exercise. Our results suggest that lactate transport via MCT1 and MCT4 plays a pivotal role in sustaining exercise.

Effects of Lactate Administration on Mitochondrial Respiratory Function in Mouse Skeletal Muscle.

Recent evidence has shown that mitochondrial respiratory function contributes to exercise performance and metabolic health. Given that lactate is considered a potential signaling molecule that induces mitochondrial adaptations, we tested the hypothesis that lactate would change mitochondrial respiratory function in skeletal muscle. Male ICR mice (8 weeks old) received intraperitoneal injection of PBS or sodium lactate (1 g/kg BW) 5 days a week for 4 weeks. Mitochondria were isolated from freshly excised gastrocnemius muscle using differential centrifugation and were used for all analyses. Lactate administration significantly enhanced pyruvate + malate- and glutamate + malate-induced (complex I-driven) state 3 (maximal/ATP synthesis-coupled) respiration, but not state 2 (basal/proton conductance) respiration. In contrast, lactate administration significantly decreased succinate + rotenone-induced (complex II-driven) state 3 and 2 respiration. No significant differences were observed in malate + octanoyl-l-carnitine-induced state 3 or 2 respiration. The enzymatic activity of complex I was tended to increase and those of complexes I + III and IV were significantly increased after lactate administration. No differences were observed in the activities of complexes II or II + III. Moreover, lactate administration increased the protein content of NDUFS4, a subunit of complex I, but not those of the other components. The present findings suggest that lactate alters mitochondrial respiratory function in skeletal muscle.

Effects of endurance training on metabolic enzyme activity and transporter protein levels in the skeletal muscles of orchiectomized mice.

This study investigated whether endurance training attenuates orchiectomy (ORX)-induced metabolic alterations. At 7 days of recovery after sham operation or ORX surgery, the mice were randomized to remain sedentary or undergo 5 weeks of treadmill running training (15-20 m/min, 60 min, 5 days/week). ORX decreased glycogen concentration in the gastrocnemius muscle, enhanced phosphofructokinase activity in the plantaris muscle, and decreased lactate dehydrogenase activity in the plantaris and soleus muscles. Mitochondrial enzyme activities and protein content in the plantaris and soleus muscles were also decreased after ORX, but preserved, in part, by endurance training. In the treadmill running test (15 m/min, 60 min) after 4 weeks of training, orchiectomized sedentary mice showed impaired exercise performance, which was restored by endurance training. Thus, endurance training could be a potential therapeutic strategy to prevent the hypoandrogenism-induced decline in muscle mitochondrial content and physical performance.

Moderate-intensity training in hypoxia improves exercise performance and glycolytic capacity of skeletal muscle in horses.

We investigated whether moderate-intensity training of horses in moderate hypoxia for 4 weeks elicits greater adaptations in exercise performance, aerobic capacity, and glycolytic/oxidative metabolism in skeletal muscle compared to normoxic training. In a randomized crossover study design, seven untrained Thoroughbred horses (5.9 ± 1.1 years, 508 ± 9 kg) completed 4 weeks (3 sessions/week) of two training protocols consisting of 3-min cantering at 70% of maximal oxygen consumption ( V˙O2max ) in hypoxia (HYP; FI O2  = 14.7%) and normoxia (NOR; FI O2  = 21.0%) with a 4-month washout period. Normoxic incremental exercise tests (IET) were conducted before and after training. Biopsy samples were obtained from the middle gluteal muscle before IET and monocarboxylate transporter (MCT) protein expression and glycolytic/mitochondrial enzyme activities were analyzed. Data were analyzed using mixed models (p < 0.05). Running speed was 7.9 ± 0.2 m/s in both groups and arterial oxygen saturation during training in NOR and HYP were 92.9 ± 0.9% and 75.7 ± 3.9%, respectively. Run time in HYP (+9.7%) and V˙O2max in both groups (NOR, +6.4%; HYP, +4.3%) at IET increased after 4 weeks of training. However, cardiac output, arterial-mixed venous O2 difference, and hemoglobin concentration at exhaustion were unchanged in both conditions. While MCT1 protein and citrate synthase activity did not increase in both conditions after training, MCT4 protein (+13%), and phosphofructokinase activity (+42%) increased only in HYP. In conclusion, 4 weeks of moderate-intensity hypoxic training improves exercise performance and glycolytic capacity of skeletal muscle in horses.

Effects of glucose ingestion at different frequencies on glycogen recovery in mice during the early hours post exercise.

BACKGROUND: When a high-carbohydrate diet is ingested, whether as small frequent snacks or as large meals, there is no difference between the two with respect to post-exercise glycogen storage for a period of 24 h. However, the effect of carbohydrate intake frequency on glycogen recovery a few hours after exercise is not clear. Athletes need to recover glycogen quickly after physical exercise as they sometimes exercise multiple times a day. The aim of this study was to determine the effect of carbohydrate intake at different frequencies on glycogen recovery during the first few hours after exercise. METHODS: After 120 min of fasting, 6-week-old male ICR mice were subjected to treadmill running exercise (20 m/min for 60 min) to decrease the levels of muscle and liver glycogen. Mice were then given glucose as a bolus (1.2 mg/g of body weight [BW], immediately after exercise) or as a pulse (1.2 mg/g of BW, every 15 min × 4 times). Following this, the blood, tissue, and exhaled gas samples were collected. RESULTS: In the bolus group, blood glucose concentration was significantly lower and plasma insulin concentration was significantly higher than those in the pulse group (p < 0.05). The plantaris muscle glycogen concentration in the bolus group was 25.3% higher than that in the pulse group at 60 min after glucose ingestion (p < 0.05). Liver glycogen concentration in the pulse group was significantly higher than that in the bolus group at 120 min after glucose ingestion (p < 0.05). CONCLUSIONS: The present study showed that ingesting a large amount of glucose immediately after exercise increased insulin secretion and enhanced muscle glycogen recovery, whereas frequent and small amounts of glucose intake was shown to enhance liver glycogen recovery.

Effects of post-exercise glucose ingestion at different solution temperatures on glycogen repletion in mice.

Carbohydrate ingestion is essential for glycogen recovery after exercise. Although studies have investigated methods for enhancement of glycogen repletion with regard to nutrients and their amounts, no studies have examined the effect of temperature of the ingested solution on glycogen recovery. Therefore, this study aimed to investigate the effect of the temperature of glucose solution ingested after exercise on glycogen recovery. Seven-week-old male ICR mice were fasted for 16 h and subjected to treadmill running exercise (20 m/min for 60 min) to decrease glycogen storage. Then, the mice were administered glucose (1.5 mg/g body weight) at three different solution temperatures: 4°C, cold solution group (Cold); 37°C, mild solution group (Mild); and 55°C, hot solution group (Hot). Our results revealed that blood glucose, plasma insulin, and muscle glycogen concentrations did not differ among the three groups. In contrast, liver glycogen concentration in the Hot group was significantly higher than that in the post-exercise and Cold groups (p < 0.05). Furthermore, portal glucose concentration was significantly higher in the Hot group than in the Cold group (p < 0.01). These observations suggest that postexercise muscle glycogen repletion occurs regardless of glucose solution temperature, and that ingesting hot glucose solution after exercise can be an effective means for liver glycogen repletion compared with cold glucose solution ingestion.

Lactate administration does not affect denervation-induced loss of mitochondrial content and muscle mass in mice.

Lactate is considered to be a signaling molecule that induces mitochondrial adaptation and muscle hypertrophy. The purpose of this study was to examine whether lactate administration attenuates denervation-induced loss of mitochondrial content and muscle mass. Eight-week-old male Institute of Cancer Research mice underwent unilateral sciatic nerve transection surgery. The contralateral hindlimb served as a sham-operated control. From the day of surgery, mice were injected intraperitoneally with PBS or sodium lactate (equivalent to 1 g·kg-1 body weight) once daily for 9 days. After 10 days of denervation, gastrocnemius muscle weight decreased to a similar extent in both the PBS- and lactate-injected groups. Denervation significantly decreased mitochondrial enzyme activity, protein content, and MCT4 protein content in the gastrocnemius muscle. However, lactate administration did not have any significant effects. The current observations suggest that daily lactate administration for 9 days does not affect denervation-induced loss of mitochondrial content and muscle mass.

Short-term hypoxic training increases monocarboxylate transporter 4 and phosphofructokinase activity in Thoroughbreds.

The aim of this study was to investigate effects of short-term hypoxic training on lactate metabolism in the gluteus medius muscle of Thoroughbreds. Using crossover design (3 months washout), eight Thoroughbred horses were trained for 2 weeks in normoxia (FI O2  = 21%) and hypoxia (FI O2  = 18%) each. They ran at 95% maximal oxygen consumption (V̇O2max ) on a treadmill inclined at 6% for 2 min (3 days/week) measured under normoxia. Before and after each training period, all horses were subjected to an incremental exercise test (IET) under normoxia. Following the 2-week trainings, V̇O2max in IET increased significantly under both oxygen conditions. The exercise duration in IET increased significantly only after hypoxic training. The monocarboxylate transporter (MCT) 1 protein levels remained unchanged after training under both oxygen conditions, whereas MCT4 protein levels increased significantly after training in hypoxia but not after training in normoxia. Phosphofructokinase activity increased significantly only after hypoxic training, whereas cytochrome c oxidase activity increased significantly only after normoxic training. Our results suggest that hypoxic training efficiently enhances glycolytic capacity and levels of the lactate transporter protein MCT4, which facilitates lactate efflux from the skeletal muscle.

Oral Lactate Administration Additively Enhances Endurance Training-Induced Increase in Cytochrome C Oxidase Activity in Mouse Soleus Muscle.

We tested the hypothesis that oral lactate supplementation increases mitochondrial enzyme activity given the potential role of lactate for inducing mitochondrial biogenesis. In this study, mice were assigned to a saline-ingested sedentary group (S+S; n = 8), a lactate-ingested sedentary group (L+S; n = 9), a saline-ingested training group (S+T; n = 8), and a lactate-ingested training group (L+T; n = 8). Mice in the S+S and S+T groups received saline, whereas mice in the L+S and L+T groups received sodium lactate (equivalent to 5 g/kg of body weight) via oral gavage 5 days a week for 4 weeks. At 30 min after the ingestion, mice in the S+T and L+T groups performed endurance training (treadmill running, 20 m/min, 30 min, 5 days/week). At 30 min after lactate ingestion, the blood lactate level reached peak value (5.8 ± 0.4 mmol/L) in the L+S group. Immediately after the exercise, blood lactate level was significantly higher in the L+T group (9.3 ± 0.9 mmol/L) than in the S+T group (2.7 ± 0.3 mmol/L) (p < 0.01). Following a 4-week training period, a main effect of endurance training was observed in maximal citrate synthase (CS) (p < 0.01; S+T: 117 ± 3% relative to S+S, L+T: 110 ± 3%) and cytochrome c oxidase (COX) activities (p < 0.01; S+T: 126 ± 4%, L+T: 121 ± 4%) in the plantaris muscle. Similarly, there was a main effect of endurance training in maximal CS (p < 0.01; S+T: 105 ± 3%, L+T: 115 ± 2%) and COX activities (p < 0.01; S+T: 113 ± 3%, L+T: 122 ± 3%) in the soleus muscle. In addition, a main effect of oral lactate ingestion was found in maximal COX activity in the soleus (p < 0.05; L+S: 109 ± 3%, L+T: 122 ± 3%) and heart muscles (p < 0.05; L+S: 107 ± 3%, L+T: 107 ± 2.0%), but not in the plantaris muscle. Our results suggest that lactate supplementation may be beneficial for increasing mitochondrial enzyme activity in oxidative phenotype muscle.

Effect of post-exercise lactate administration on glycogen repletion and signaling activation in different types of mouse skeletal muscle.

Lactate is not merely a metabolic intermediate that serves as an oxidizable and glyconeogenic substrate, but it is also a potential signaling molecule. The objectives of this study were to investigate whether lactate administration enhances post-exercise glycogen repletion in association with cellular signaling activation in different types of skeletal muscle. Eight-week-old male ICR mice performed treadmill running (20 m/min for 60 min) following overnight fasting (16 h). Immediately after the exercise, animals received an intraperitoneal injection of phosphate-buffered saline or sodium lactate (equivalent to 1 g/kg body weight), followed by oral ingestion of water or glucose (2 g/kg body weight). At 60 min of recovery, glucose ingestion enhanced glycogen content in the soleus, plantaris, and gastrocnemius muscles. In addition, lactate injection additively increased glycogen content in the plantaris and gastrocnemius muscles, but not in the soleus muscle. Nevertheless, lactate administration did not significantly alter protein levels related to glucose uptake and oxidation in the plantaris muscle, but enhanced phosphorylation of TBC1D1, a distal protein regulating GLUT4 translocation, was observed in the soleus muscle. Muscle FBP2 protein content was significantly higher in the plantaris and gastrocnemius muscles than in the soleus muscle, whereas MCT1 protein content was significantly higher in the soleus muscle than in the plantaris and gastrocnemius muscles. The current findings suggest that an elevated blood lactate concentration and post-exercise glucose ingestion additively enhance glycogen recovery in glycolytic phenotype muscles. This appears to be associated with glyconeogenic protein content, but not with enhanced glucose uptake, attenuated glucose oxidation, or lactate transport protein.

Effects of Nutrient Intake Timing on Post-Exercise Glycogen Accumulation and its Related Signaling Pathways in Mouse Skeletal Muscle.

We investigated the effects of nutrient intake timing on glycogen accumulation and its related signals in skeletal muscle after an exercise that did not induce large glycogen depletion. Male ICR mice ran on a treadmill at 25 m/min for 60 min under a fed condition. Mice were orally administered a solution containing 1.2 mg/g carbohydrate and 0.4 mg/g protein or water either immediately (early nutrient, EN) or 180 min (late nutrient, LN) after the exercise. Tissues were harvested at 30 min after the oral administration. No significant difference in blood glucose or plasma insulin concentrations was found between the EN and LN groups. The plantaris muscle glycogen concentration was significantly (p < 0.05) higher in the EN group-but not in the LN group-compared to the respective time-matched control group. Akt Ser473 phosphorylation was significantly higher in the EN group than in the time-matched control group (p < 0.01), while LN had no effect. Positive main effects of time were found for the phosphorylations in Akt substrate of 160 kDa (AS160) Thr642 (p < 0.05), 5'-AMP-activated protein kinase (AMPK) Thr172 (p < 0.01), and acetyl-CoA carboxylase Ser79 (p < 0.01); however, no effect of nutrient intake was found for these. We showed that delayed nutrient intake could not increase muscle glycogen after endurance exercise which did not induce large glycogen depletion. The results also suggest that post-exercise muscle glycogen accumulation after nutrient intake might be partly influenced by Akt activation. Meanwhile, increased AS160 and AMPK activation by post-exercise fasting might not lead to glycogen accumulation.

Effects of lactate administration on mitochondrial enzyme activity and monocarboxylate transporters in mouse skeletal muscle.

Growing evidence shows that lactate is not merely an intermediate metabolite, but also a potential signaling molecule. However, whether daily lactate administration induces physiological adaptations in skeletal muscle remains to be elucidated. In this study, we first investigated the effects of daily lactate administration (equivalent to 1 g/kg of body weight) for 3 weeks on mitochondrial adaptations in skeletal muscle. We demonstrated that 3-week lactate administration increased mitochondrial enzyme activity (citrate synthase, 3-hydroxyacyl CoA dehydrogenase, and cytochrome c oxidase) in the plantaris muscle, but not in the soleus muscle. MCT1 and MCT4 protein contents were not different after 3-week lactate administration. Next, we examined whether lactate administration enhances training-induced adaptations in skeletal muscle. Lactate administration prior to endurance exercise training (treadmill running, 20 m/min, 60 min/day), which increased blood lactate concentration during exercise, enhanced training-induced mitochondrial enzyme activity in the skeletal muscle after 3 weeks. MCT protein content and blood lactate removal were not different after 3-week lactate administration with exercise training compared to exercise training alone. In a single bout experiment, lactate administration did not change the phosphorylation state of AMPK, ACC, p38 MAPK, and CaMKII in skeletal muscle. Our results suggest that lactate can be a key factor for exercise-induced mitochondrial adaptations, and that the efficacy of high-intensity training is, at least partly, attributed to elevated blood lactate concentration.

Effects of Nrf2 deficiency on mitochondrial oxidative stress in aged skeletal muscle.

Oxidative stress and mitochondrial dysfunction are associated with the aging process. However, the role of nuclear factor erythroid 2 -related factor 2 (Nrf2) in skeletal muscle during aging remains to be clarified. In the current study, we assessed whether the lack of Nrf2, which is known as a master regulator of redox homeostasis, promotes age-related mitochondrial dysfunction and muscle atrophy in skeletal muscle. Here, we demonstrated that mitochondrial 4-hydroxynonenal and protein carbonyls, markers of oxidative stress, were robustly elevated in aged Nrf2 knockout (KO) mice because of the decreased expression of Nrf2-target antioxidant genes. Mitochondrial respiration declined with aging; however, there was no difference between Nrf2 KO and age-matched WT mice. Similarly, cytochrome c oxidase activity was lower in aged WT and Nrf2 KO mice compared with young WT mice. The expression of Mfn1 and Mfn2 mRNA was lower in aged Nrf2 KO muscle. Mitochondrial reactive oxygen species production per oxygen consumed was elevated in aged Nrf2 KO mice. There was no effect of Nrf2 KO on muscle mass normalized to body weight. These results suggest that Nrf2 deficiency exacerbates age-related mitochondrial oxidative stress but does not affect the decline of respiratory function in skeletal muscle.