Endurance exercise induces increases in mitochondria and the GLUT4 isoform of the glucose transporter in muscle. Although little is known about the mechanisms underlying these adaptations, new information has accumulated regarding how mitochondrial biogenesis and GLUT4 expression are regulated. This includes the findings that the transcriptional coactivator PGC-1 promotes mitochondrial biogenesis and that NRF-1 and NRF-2 act as transcriptional activators of genes encoding mitochondrial enzymes. We tested the hypothesis that increases in PGC-1, NRF-1, and NRF-2 are involved in the initial adaptive response of muscle to exercise. Five daily bouts of swimming induced increases in mitochondrial enzymes and GLUT4 in skeletal muscle in rats. One exercise bout resulted in approximately twofold increases in full-length muscle PGC-1 mRNA and PGC-1 protein, which were evident 18 h after exercise. A smaller form of PGC-1 increased after exercise. The exercise induced increases in muscle NRF-1 and NRF-2 that were evident 12 to 18 h after one exercise bout. These findings suggest that increases in PGC-1, NRF-1, and NRF-2 represent key regulatory components of the stimulation of mitochondrial biogenesis by exercise and that PGC-1 mediates the coordinated increases in GLUT4 and mitochondria.

Exercise results in rapid increases in expression of the transcription coactivator peroxisome proliferator-activated receptor ␥ coactivator-1␣ (PGC-1␣) and in mitochondrial biogenesis in skeletal muscle. PGC-1␣ regulates and coordinates mitochondrial biogenesis, and overexpression of PGC-1␣ in muscle cells results in increases in mitochondrial content. In this context, it has been proposed that the increase in PGC-1␣ protein expression mediates the exercise-induced increase in mitochondrial biogenesis. However, we found that mitochondrial proteins with a short half-life increase as rapidly as, or more rapidly than, PGC-1␣ protein. This finding led us to hypothesize that activation, rather than increased expression, of PGC-1␣ mediates the initial phase of the exercise-induced increase in mitochondria. In this study, we found that most of the PGC-1␣ in resting skeletal muscle is in the cytosol. Exercise resulted in activation of p38 MAPK and movement of PGC-1␣ into the nucleus. In support of our hypothesis, binding of the transcription factor nuclear respiratory factor 1 (NRF-1) to the cytochrome c promoter and NRF-2 to the cytochrome oxidase subunit 4 promoter increased in response to exercise prior to an increase in PGC-1␣ protein. Furthermore, exercise-induced increases in the mRNAs of cytochrome c, ␦-aminolevulinate synthase, and citrate synthase also occurred before an increase in PGC-1 protein. Thus, it appears that activation of PGC-1␣ may mediate the initial phase of the exercise-induced adaptive increase in muscle mitochondria, whereas the subsequent increase in PGC-1␣ protein sustains and enhances the increase in mitochondrial biogenesis.

Previous studies have shown that raising cytosolic calcium in myotubes induces increases in peroxisome proliferator-activated receptor ␥ coactivator-1␣ expression and mitochondrial biogenesis. This finding suggests that the increases in cytosolic calcium in skeletal muscle during exercise may mediate the exercise-induced increase in mitochondria. The initial aim of this study was to determine whether raising calcium in skeletal muscle induces the same adaptations as in myotubes. We found that treatment of rat epitrochlearis muscles with a concentration of caffeine that raises cytosolic calcium to a concentration too low to cause contraction induces increases in peroxisome proliferator-activated receptor ␥ coactivator-1␣ expression and mitochondrial biogenesis. Our second aim was to elucidate the pathway by which calcium induces these adaptations. Raising cytosoliccalciumhasbeenshowntoactivatecalcium/calmodulindependent protein kinase in muscle. In the present study raising cytosolic calcium resulted in increases in phosphorylation of p38 mitogen-activated protein kinase and activating transcription factor-2, which were blocked by the calcium/calmodulindependent protein kinase inhibitor KN93 and by the p38 mitogen-activated protein kinase inhibitor SB202190. The increases in peroxisome proliferator-activated receptor ␥ coactivator-1␣ expression and mitochondrial biogenesis were also prevented by inhibiting p38 activation. We interpret these findings as evidence that p38 mitogen-activated protein kinase is downstream of calcium/calmodulindependent protein kinase in a signaling pathway by which increases in cytosolic calcium lead to increases in peroxisome proliferator-activated receptor ␥ coactivator-1␣ expression and mitochondrial biogenesis in muscle.

Skeletal muscle adapts to endurance exercise with an increase in mitochondria. Muscle contractions generate numerous potential signals. To determine which of these stimulates mitochondrial biogenesis, we are using L6 myotubes. Using this model we have found that raising cytosolic Ca2+ induces an increase in mitochondria. In this study, we tested the hypothesis that raising cytosolic Ca2+ in L6 myotubes induces increased expression of PGC-1, NRF-1, NRF-2, and mtTFA, factors that have been implicated in mitochondrial biogenesis and in the adaptation of muscle to exercise. Raising cytosolic Ca2+ by exposing L6 myotubes to caffeine for 5 h induced significant increases in PGC-1 and mtTFA protein expression and in NRF-1 and NRF-2 binding to DNA. These adaptations were prevented by dantrolene, which blocks Ca2+ release from the SR. Exposure of L6 myotubes to caffeine for 5 h per day for 5 days induced significant increases in mitochondrial marker enzyme proteins. Our results show that the adaptive response of L6 myotubes to an increase in cytosolic Ca2+ mimics the stimulation of mitochondrial biogenesis by exercise. They support the hypothesis that an increase in cytosolic Ca2+ is one of the signals that mediate increased mitochondrial biogenesis in muscle.

There is evidence suggesting that adaptive increases in GLUT4 and mitochondria in skeletal muscle occur in parallel. It has been reported that raising cytosolic Ca2+ in myocytes induces increases in mitochondrial enzymes. In this study, we tested the hypothesis that an increase in cytosolic Ca2+ induces an increase in GLUT4. We found that raising cytosolic Ca2+ by exposing L6 myotubes to 5 mM caffeine for 3 h/day for 5 days induced increases in GLUT4 protein and in myocyte enhancer factor (MEF)2A and MEF2D, which are transcription factors involved in regulating GLUT4 expression. The caffeine-induced increases in GLUT4 and MEF2A and MEF2D were partially blocked by dantrolene, an inhibitor of sarcoplasmic reticulum Ca2+ release, and completely blocked by KN93, an inhibitor of Ca2+-calmodulin-dependent protein kinase (CAMK). Caffeine also induced increases in MEF2A, MEF2D, and GLUT4 in rat epitrochlearis muscles incubated with caffeine in culture medium. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR), which activates AMP-activated protein kinase (AMPK), also induced approximately twofold increases in GLUT4, MEF2A, and MEF2D in L6 myocytes. Our results provide evidence that increases in cytosolic Ca2+and activation of AMPK, both of which occur in exercising muscle, increase GLUT4 protein in myocytes and skeletal muscle. The data suggest that this effect of Ca2+ is mediated by activation of CAMK and indicate that MEF2A and MEF2D are involved in this adaptive response.