Kentaro Kawanaka

Effect of exercise intensity and AICAR on isoform-specific expressions of murine skeletal muscle PGC-1α mRNA: a role of β2-adrenergic receptor activation

There are three isoforms of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) mRNA, which promotes mitochondrial biogenesis in skeletal muscles. Compared with PGC-1α-a mRNA, PGC-1α-b or PGC-1α-c mRNA is transcribed by a different exon 1 of the PGC-1α gene. In this study, effects of exercise intensity and 5-aminoimidazole-4-carboxamide-1β-d-ribofuranoside (AICAR) on isoform-specific expressions of PGC-1α were investigated. All isoforms were increased in proportion to exercise intensity of treadmill running (10-30 m/min for 30 min). Preinjection of β₂-adrenergic receptor (AR) antagonist (ICI 118551) inhibited the increase in PGC-1α-b and PGC-1α-c mRNAs, but not the increase in PGC-1α-a mRNA, in response to high-intensity exercise. Although high-intensity exercise activated α2-AMP-activated protein kinase (α2-AMPK) in skeletal muscles, inactivation of α2-AMPK activity did not affect high-intensity exercise-induced mRNA expression of all PGC-1α isoforms, suggesting that activation of α2-AMPK is not mandatory for an increase in PGC-1α mRNA by high-intensity exercise. A single injection in mice of AICAR, an AMPK activator, increased mRNAs of all PGC-1α isoforms. AICAR increased blood catecholamine concentrations, and preinjection of β₂-AR antagonist inhibited the increase in PGC-1α-b and PGC-1α-c mRNAs but not the increase in PGC-1α-a mRNA. Direct exposure of epitrochlearis muscle to AICAR increased PGC-1α-a but not the -b isoform. These data indicate that exercise-induced PGC-1α expression was dependent on the intensity of exercise. Exercise or AICAR injection increased PGC-1α-b and PGC-1α-c mRNAs via β₂-AR activation, whereas high-intensity exercise increased PGC-1α-a expression by a multiple mechanism in which α2-AMPK is one of the signaling pathways.

Decreased insulin-stimulated GLUT-4 translocation in glycogen-supercompensated muscles of exercised rats

It was recently found that the effect of an exercise-induced increase in muscle GLUT-4 on insulin-stimulated glucose transport is masked by a decreased responsiveness to insulin in glycogen-supercompensated muscle. We evaluated the role of hexosamines in this decrease in insulin responsiveness and found that UDP- N-acetyl hexosamine concentrations were not higher in glycogen-supercompensated muscles than in control muscles with a low glycogen content. We determined whether the smaller increase in glucose transport is due to translocation of fewer GLUT-4 to the cell surface with the 2- N-4-(1-azi-2,2,2-trifluroethyl)-benzoyl-1,3-bis(d-mannose-4-yloxy)-2-propylamine (ATB-[2-3H]BMPA) photolabeling technique. The insulin-induced increase in GLUT-4 at the cell surface was no greater in glycogen-supercompensated exercised muscle than in muscles of sedentary controls and only 50% as great as in exercised muscles with a low glycogen content. We conclude that the decreased insulin responsiveness of glucose transport in glycogen-supercompensated muscle is not due to increased accumulation of hexosamine biosynthetic pathway end products and that the smaller increase in glucose transport is mediated by translocation of fewer GLUT-4 to the cell surface.It was recently found that the effect of an exercise-induced increase in muscle GLUT-4 on insulin-stimulated glucose transport is masked by a decreased responsiveness to insulin in glycogen-supercompensated muscle. We evaluated the role of hexosamines in this decrease in insulin responsiveness and found that UDP-N-acetyl hexosamine concentrations were not higher in glycogen-supercompensated muscles than in control muscles with a low glycogen content. We determined whether the smaller increase in glucose transport is due to translocation of fewer GLUT-4 to the cell surface with the 2-N-4-(1-azi-2,2,2-trifluroethyl)-benzoyl-1, 3-bis(D-mannose-4-yloxy)-2-propylamine (ATB-[2-3H]BMPA) photolabeling technique. The insulin-induced increase in GLUT-4 at the cell surface was no greater in glycogen-supercompensated exercised muscle than in muscles of sedentary controls and only 50% as great as in exercised muscles with a low glycogen content. We conclude that the decreased insulin responsiveness of glucose transport in glycogen-supercompensated muscle is not due to increased accumulation of hexosamine biosynthetic pathway end products and that the smaller increase in glucose transport is mediated by translocation of fewer GLUT-4 to the cell surface.

Brain glycogen supercompensation following exhaustive exercise

Non‐technical summary  Exercise training elicits an increase in the basal level of muscular glycogen. This happens when glycogen recovers to above its basal level (supercompensation) after it decreases with acute exercise. Although untested, it is hypothesized that, similar to that of skeletal muscle, brain glycogen supercompensation occurs after acute exhaustive exercise. We provide evidence that exhaustive exercise induces glycogen supercompensation not only in skeletal muscles, but also in the brain. Furthermore, we observed exercise training‐induced increases in basal glycogen levels in the cortex and hippocampus, which are involved in motor control and cognitive function. This suggests that, like skeletal muscles, the brain adapts metabolically, probably to meet the increased energy demands of exercise training.

Brain glycogen decreases during prolonged exercise

Non‐technical summary  Energy sources for the brain include not only blood glucose, but also astrocytic glycogen, especially when the blood‐born glucose supply is short (e.g. hypoglycaemia). Although untested, it is hypothesized that during prolonged exercise that induces hypoglycaemia, the resultant hypoglycaemia may cause a decrease in brain glycogen. Here, we tested this hypothesis and provide evidence that brain glycogen decreases during prolonged exercise with hypoglycaemia. Furthermore, in the cortex, we show that the decrease in brain glycogen levels during prolonged exercise is associated with activation of monoamine metabolism, which could be a factor inducing central fatigue. Since the discovery of muscle glycogen depletion as a candidate of peripheral fatigue during prolonged exercise, this is the first study to our knowledge to show that brain glycogen can decrease with prolonged exercise. These findings may provide a clue towards understanding the mechanisms related to central fatigue.

Insulin resistance of muscle glucose transport in male and female rats fed a high-sucrose diet.

It has been reported that, unlike high-fat diets, high-sucrose diets cause insulin resistance in the absence of an increase in visceral fat and that the insulin resistance develops only in male rats. This study was done to 1) determine if isolated muscles of rats fed a high-sucrose diet are resistant to stimulation of glucose transport when studied in vitro and 2) obtain information regarding how the effects of high-sucrose and high-fat diets on muscle insulin resistance differ. We found that, compared with rat chow, semipurified high-sucrose and high-starch diets both caused increased visceral fat accumulation and insulin resistance of skeletal muscle glucose transport. Insulin responsiveness of 2-deoxyglucose (2-DG) transport measured in epitrochlearis and soleus muscles in vitro was decreased ∼40% ( P < 0.01) in both male and female rats fed a high-sucrose compared with a chow diet. The high-sucrose diet also caused resistance of muscle glucose transport to stimulation by contractions. There was a highly significant negative correlation between stimulated muscle 2-DG transport and visceral fat mass. In view of these results, the differences in insulin action in vivo observed by others in rats fed isocaloric high-sucrose and high-starch diets must be due to additional, specific effects of sucrose that do not carry over in muscles studied in vitro. We conclude that, compared with rat chow, semipurified high-sucrose and high-cornstarch diets, like high-fat diets, cause increased visceral fat accumulation and severe resistance of skeletal muscle glucose transport to stimulation by insulin and contractions.

Muscle contractile activity regulates Sirt3 protein expression in rat skeletal muscles

Sirt3, a member of the sirtuin family, is known to control cellular mitochondrial function. Furthermore, because sirtuins require NAD for their deacetylase activity, nicotinamide phosphoribosyltransferase (Nampt), which is a rate-limiting enzyme in the intracellular NAD biosynthetic pathway, influences their activity. We examined the effects of exercise training and normal postural contractile activity on Sirt3 and Nampt protein expression in rat skeletal muscles. Male rats were trained by treadmill running at 20 m/min, 60 min/day, 7 days/wk for 4 wk. This treadmill training program increased the Sirt3 protein expression in the soleus and plantaris muscles by 49% and 41%, respectively (P < 0.05). Moreover, a 4-wk voluntary wheel-running program also induced 66% and 95% increases in Sirt3 protein in the plantaris and triceps muscles of rats, respectively (P < 0.05). Treadmill-running and voluntary running training induced no significant changes in Nampt protein expression in skeletal muscles. In resting rats, the soleus muscle, which is recruited during normal postural activity, possessed the greatest expression levels of the Sirt3 and Nampt proteins, followed by the plantaris and triceps muscles. Furthermore, the Sirt3, but not Nampt, protein level was reduced in the soleus muscles from immobilized hindlimbs compared with that shown in the contralateral control muscle. These results demonstrated that 1) Sirt3 protein expression is upregulated by exercise training in skeletal muscles and 2) local postural contractile activity plays an important role in maintaining a high level of Sirt3 protein expression in postural muscle.

Changes in insulin-stimulated glucose transport and GLUT-4 protein in rat skeletal muscle after training

After running training, which increased GLUT-4 protein content in rat skeletal muscle by <40% compared with control rats, the training effect on insulin-stimulated maximal glucose transport (insulin responsiveness) in skeletal muscle was short lived (24 h). A recent study reported that GLUT-4 protein content in rat epitrochlearis muscle increased dramatically ( approximately 2-fold) after swimming training (J.-M. Ren, C. F. Semenkovich, E. A. Gulve, J. Gao, and J. O. Holloszy. J. Biol. Chem. 269, 14396-14401, 1994). Because GLUT-4 protein content is known to be closely related to skeletal muscle insulin responsiveness, we thought it possible that the training effect on insulin responsiveness may remain for >24 h after swimming training if GLUT-4 protein content decreases gradually from the relatively high level and still remains higher than control level for >24 h after swimming training. Therefore, we examined this possibility. Male Sprague-Dawley rats swam 2 h a day for 5 days with a weight equal to 2% of body mass. Approximately 18, 42, and 90 h after cessation of training, GLUT-4 protein concentration and 2-[1,2-3H]deoxy-D-glucose transport in the presence of a maximally stimulating concentration of insulin (2 mU/ml) were examined by using incubated epitrochlearis muscle preparation. Swimming training increased GLUT-4 protein concentration and insulin responsiveness by 87 and 85%, respectively, relative to age-matched controls when examined 18 h after training. Forty-two hours after training, GLUT-4 protein concentration and insulin responsiveness were still higher by 52 and 51%, respectively, in muscle from trained rats compared with control. GLUT-4 protein concentration and insulin responsiveness in trained muscle returned to sedentary control level within 90 h after training. We conclude that 1) the change in insulin responsiveness during detraining is directly related to muscle GLUT-4 protein content, and 2) consequently, the greater the increase in GLUT-4 protein content that is induced by training, the longer an effect on insulin responsiveness persists after the training.

Development of Glucose-induced Insulin Resistance in Muscle Requires Protein Synthesis

Muscles and fat cells develop insulin resistance when exposed to high concentrations of glucose and insulin. We used an isolated muscle preparation incubated with high levels of glucose and insulin to further evaluate how glucose-induced insulin resistance (GIIR) is mediated. Incubation with 2 milliunits/ml insulin and 36 mm glucose for 5 h resulted in an ∼50% decrease in insulin-stimulated muscle glucose transport. The decrease in insulin responsiveness of glucose transport induced by glucose was not due to impaired insulin signaling, as insulin-stimulated phosphatidylinositol 3-kinase activity and protein kinase B phosphorylation were not reduced. It has been hypothesized that entry of glucose into the hexosamine biosynthetic pathway with accumulation of UDP-N-acetylhexosamines (UDP-HexNAcs) mediates GIIR. However, inhibition of the rate-limiting enzyme GFAT (glutamine:fructose-6-phosphate amidotransferase) did not protect against GIIR despite a marked reduction of UDP-HexNAcs. The mRNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide both completely protected against GIIR despite the massive increases in UDP-HexNAcs and glycogen that resulted from increased glucose entry. Activation of AMP-activated protein kinase also protected against GIIR. These results provide evidence that GIIR can occur in muscle without increased accumulation of hexosamine pathway end products, that neither high glycogen concentration nor impaired insulin signaling is responsible for GIIR, and that synthesis of a protein with a short half-life mediates GIIR. They also suggest that dephosphorylation of a transcription factor may be involved in the induction of GIIR.

Exercise Training Increases Glucose Transporter Content in Skeletal Muscles More Efficiently From Aged Obese Rats Than Young Lean Rats

Glucose uptake in rat skeletal muscles decreases with age and obesity, but increases with chronic exercise training. The purpose of our study was to determine whether the GLUT4 content in several skeletal muscles from 1-mo-old young, lean rats and 12-mo-old aged, obese rats alters with exercise training. For exercise, a treadmill run of ∼ 1 km/day was made for 4 wk by both groups of rats. The concentration of GLUT4 per protein in membrane fraction from several skeletal muscles was measured by immunoblotting. The amount of GLUT4 in the gastrocnemius and white quadriceps from aged rats slightly but significantly decreased to 73% and 78% of that from young rats, respectively. However, no significant difference in GLUT4 amount in the soleus, plantaris, and red quadriceps was observed between young and aged rats. The exercise training resulted in a larger increase in the amount of GLUT4 in each muscle from aged rats than in muscles from young rats. In aged rats, GLUT4 amount increased significantly with exercise training by 30, 33, 41, and 27% in the soleus, plantaris, gastrocnemius, and red quadriceps, respectively, compared with the sedentary controls. However, in young rats, exercise-induced increase of GLUT4 amount was significant only in the plantaris, and the increase was 17%. In exercised aged, obese rats, decreases of body weight, plasma triglyceride levels, and plasma free fatty acid were also observed. These findings suggest that the amount of GLUT4 in some skeletal muscles decreases slightly in aged, obese rats, exercise training increases the amount of GLUT4 more efficiently in aged rats than in young rats and ameliorates the GLUT4 decrease observed in aged rats, and lipid metabolism may be related to the regulation of GLUT4 protein content in muscles.

Post-exercise whey protein hydrolysate supplementation induces a greater increase in muscle protein synthesis than its constituent amino acid content.

It is well known that ingestion of a protein source is effective in stimulating muscle protein synthesis after exercise. In addition, there are numerous reports on the impact of leucine and leucine-rich whey protein on muscle protein synthesis and mammalian target of rapamycin (mTOR) signalling. However, there is only limited information on the effects of whey protein hydrolysates (WPH) on muscle protein synthesis and mTOR signalling. The aim of the present study was to compare the effects of WPH and amino acids on muscle protein synthesis and the initiation of translation in skeletal muscle during the post-exercise phase. Male Sprague–Dawley rats swam for 2 h to depress muscle protein synthesis. Immediately after exercise, the animals were administered either carbohydrate (CHO), CHO plus an amino acid mixture (AA) or CHO plus WPH. At 1 h after exercise, the supplements containing whey-based protein (AA and WPH) caused a significant increase in the fractional rate of protein synthesis (FSR) compared with CHO. WPH also caused a significant increase in FSR compared with AA. Post-exercise ingestion of WPH caused a significant increase in the phosphorylation of mTOR levels compared with AA or CHO. In addition, WPH caused greater phosphorylation of ribosomal protein S6 kinase and eukaryotic initiation factor 4E-binding protein 1 than AA and CHO. In contrast, there was no difference in plasma amino acid levels following supplementation with either AA or WPH. These results indicate that WPH may include active components that are superior to amino acids for stimulating muscle protein synthesis and initiating translation.