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.

Contractile activity induces mitochondrial biogenesis and increases glucose transport capacity in muscle. There has been much research on the mechanisms responsible for these adaptations. The present paper reviews the evidence, which indicates that the decrease in the levels of highenergy phosphates, leading to activation of AMP kinase (AMPK), and the increase in cytosolic Ca 2+ , which activates Ca 2+ /calmodulin-dependent protein kinase (CAMK), are signals that initiate these adaptative responses. Although the events downstream of AMPK and CAMK have not been well characterized, these events lead to activation of various transcription factors, including: nuclear respiratory factors (NRF) 1 and 2, which cause increased expression of proteins of the respiratory chain; PPAR-a, which up regulates the levels of enzymes of b oxidation; mitochondrial transcription factor A, which activates expression of the mitochondrial genome; myocyte-enhancing factor 2A, the transcription factor that regulates GLUT4 expression. The well-orchestrated expression of the multitude of proteins involved in these adaptations is mediated by the rapid activation of PPARg co-activator (PGC) 1, a protein that binds to various transcription factors to maximize transcriptional activity. Activating AMPK using 5-aminoimidizole-4-carboxamide-1-b-D-riboside (AICAR) and increasing cytoplasmic Ca 2+ using caffeine, W7 or ionomycin in L6 myotubes increases the concentration of mitochondrial enzymes and GLUT4 and enhances the binding of NRF-1 and NRF-2 to DNA. AICAR and Ca-releasing agents also increase the levels of PGC-1, mitochondrial transcription factor A and myocyte-enhancing factors 2A and 2D. These results are similar to the responses seen in muscle during the adaptation to endurance exercise and show that L6 myotubes are a suitable model for studying the mechanisms by which exercise causes the adaptive responses in muscle mitochondria and glucose transport.Mitochondrial biogenesis: GLUT4: Ca 2+ /calmodulin-dependent protein kinase: AMP kinase: Transcription factors

Exercise acutely stimulates muscle glucose transport and also brings about an adaptive increase in the capacity of muscle for glucose uptake by inducing increases in GLUT-4 and hexokinase.(1) Recent studies have provided evidence that activation of AMP protein kinase (AMPK) is involved in the stimulation of glucose transport by exercise. The purpose of this study was to determine whether activation of AMPK is also involved in mediating the adaptive increases in GLUT-4 and hexokinase. To this end, we examined the effect of incubating rat epitrochlearis muscles in culture medium for 18 h in the presence or absence of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), which enters cells and is converted to the AMP analog ZMP, thus activating AMPK. Exposure of muscles to 0.5 mM AICAR in vitro for 18 h resulted in an approximately 50% increase in GLUT-4 protein and an approximately 80% increase in hexokinase. This finding provides strong evidence in support of the hypothesis that the activation of AMPK that occurs in muscle during exercise is involved in mediating the adaptive increases in GLUT-4 and hexokinase.

Muscle contractions cause numerous disturbances in intracellular homeostasis. This makes it impossible to use contracting muscle to identify which of the many signals generated by contractions are responsible for stimulating mitochondrial biogenesis. One purpose of this study was to evaluate the usefulness of L6 myotubes, which do not contract, for studying mitochondrial biogenesis. A second purpose was to evaluate further the possibility that increases in cytosolic Ca2+ can stimulate mitochondrial biogenesis. Continuous exposure to 1 μM ionomycin, a Ca2+ ionophore, for 5 days induced an increase in mitochondrial enzymes but also caused a loss of myotubes, as reflected in an ∼40% decrease in protein per dish. However, intermittent (5 h/day) exposure to ionomycin, or to caffeine or W7, which release Ca2+ from the sarcoplasmic reticulum, did not cause a decrease in protein per dish. Raising cytosolic Ca2+intermittently with these agents induced significant increases in mitochondrial enzymes. EGTA blocked most of this effect of ionomycin, whereas dantrolene, which blocks Ca2+ release from the sarcoplasmic reticulum, largely prevented the increases in mitochondrial enzymes induced by W7 and caffeine. These findings provide evidence that intermittently raising cytosolic Ca2+stimulates mitochondrial biogenesis in muscle cells.

Nuclear respiratory factor 1 (NRF-1) is a transcriptional activator of nuclear genes that encode a range of mitochondrial proteins including cytochrome c, various other respiratory chain subunits, and delta-aminolevulinate synthase. Activation of NRF-1 in fibroblasts has been shown to induce increases in cytochrome c expression and mitochondrial respiratory capacity. To further evaluate the role of NRF-1 in the regulation of mitochondrial biogenesis and respiratory capacity, we generated transgenic mice overexpressing NRF-1 in skeletal muscle. Cytochrome c expression was increased approximately twofold and delta-aminolevulinate synthase was increased approximately 50% in NRF-1 transgenic muscle. The levels of some mitochondrial proteins were increased 50-60%, while others were unchanged. Muscle respiratory capacity was not increased in the NRF-1 transgenic mice. A finding that provides new insight regarding the role of NRF-1 was that expression of MEF2A and GLUT4 was increased in NRF-1 transgenic muscle. The increase in GLUT4 was associated with a proportional increase in insulin-stimulated glucose transport. These results show that an isolated increase in NRF-1 is not sufficient to bring about a coordinated increase in expression of all of the proteins necessary for assembly of functional mitochondria. They also provide the new information that NRF-1 overexpression results in increased expression of GLUT4.

Smith JA, Kohn TA, Chetty AK, Ojuka EO. CaMK activation during exercise is required for histone hyperacetylation and MEF2A binding at the MEF2 site on the Glut4 gene. Am J Physiol Endocrinol Metab 295: E698 -E704, 2008. First published July 22, 2008 doi:10.1152/ajpendo.00747.2007.-The role of CaMK II in regulating GLUT4 expression in response to intermittent exercise was investigated. Wistar rats completed 5 ϫ 17-min bouts of swimming after receiving 5 mg/kg KN93 (a CaMK II inhibitor), KN92 (an analog of KN93 that does not inhibit CaMK II), or an equivalent volume of vehicle. Triceps muscles that were harvested at 0, 6, or 18 h postexercise were assayed for 1) CaMK II phosphorylation by Western blot, 2) acetylation of histone H3 at the Glut4 MEF2 site by chromatin immunoprecipitation (ChIP) assay, 3) bound MEF2A at the Glut4 MEF2 cis-element by ChIP, and 4) GLUT4 expression by RT-PCR and Western blot. Compared with controls, exercise caused a twofold increase in CaMK II phosphorylation. Immunohistochemical stains indicated increased CaMK II phosphorylation in nuclear and perinuclear regions of the muscle fiber. Acetylation of histone H3 in the region surrounding the MEF2 binding site on the Glut4 gene and the amount of MEF2A that bind to the site increased approximately twofold postexercise. GLUT4 mRNA and protein increased ϳ2.2-and 1.8-fold, respectively, after exercise. The exercise-induced increases in CaMK II phosphorylation, histone H3 acetylation, MEF2A binding, and GLUT4 expression were attenuated or abolished when KN93 was administered to rats prior to exercise. KN92 did not affect the increases in pCaMK II and GLUT4. These data support the hypothesis that CaMK II activation by exercise increases GLUT4 expression via increased accessibility of MEF2A to its cis-element on the gene. myocyte enhancer factor; glucose transporter 4; chromatin immunoprecipitation assay; histone H3 acetylation; KN93; Ca 2ϩ /calmodulindependent kinase II phosphorylation

In vitro binding assays have indicated that the exercise-induced increase in muscle GLUT4 is preceded by increased binding of myocyte enhancer factor 2A (MEF2A) to its cis-element on the Glut4 promoter. Because in vivo binding conditions are often not adequately recreated in vitro, we measured the amount of MEF2A that was bound to the Glut4 promoter in rat triceps after an acute swimming exercise in vivo, using chromatin immunoprecipitation (ChIP) assays. Bound MEF2A was undetectable in nonexercised controls or at 24 h postexercise but was significantly elevated approximately 6 h postexercise. Interestingly, the increase in bound MEF2A was preceded by an increase in autonomous activity of calcium/calmodulin-dependent protein kinase (CaMK) II in the same muscle. To determine if CaMK signaling mediates MEF2A/DNA associations in vivo, we performed ChIP assays on C(2)C(12) myotubes expressing constitutively active (CA) or dominant negative (DN) CaMK IV proteins. We found that approximately 75% more MEF2A was bound to the Glut4 promoter in CA compared with DN CaMK IV-expressing cells. GLUT4 protein increased approximately 70% 24 h after exercise but was unchanged by overexpression of CA CaMK IV in myotubes. These results confirm that exercise increases the binding of MEF2A to the Glut4 promoter in vivo and provides evidence that CaMK signaling is involved in this interaction.