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Item 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, 2007) Smith, James A. H.; Kohn, Tertius A.; Chetty, Ashley K.; Ojuka, Edward O.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 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 2.2- 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; Ca2 /calmodulindependent kinase II phosphorylationItem Exercise and CaMK activation both increase the binding of MEF2A to the Glut4 promoter in skeletal muscle in vivo(Am J Physiol Endocrinol Metab 292, 2006) Smith, James A. H.; Collins, Malcolm; Grobler, Liesl A.; Magee, Carrie J.; Ojuka, Edward O.Exercise and CaMK activation both increase the binding of MEF2A to the Glut4 promoter in skeletal muscle in vivo. Am J Physiol Endocrinol Metab 292: E413–E420, 2007. First published September 19, 2006; doi:10.1152/ajpendo.00142.2006.—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 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 C2C12 myotubes expressing constitutively active (CA) or dominant negative (DN) CaMK IV proteins. We found that 75% more MEF2A was bound to the Glut4 promoter in CA compared with DN CaMK IV-expressing cells. GLUT4 protein increased 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. rats; C2C12 myotubes; chromatin immunoprecipitation assay; autonomous calcium/calmodulin-dependent protein kinase activity; myocyte enhancer factor 2A; glucose transporter-4Item Increased expression of GLUT-4 and hexokinase in rat epitrochlearis muscles exposed to AICAR in vitro(J. Appl. Physiol., 2000) Ojuka, Edward O.; Nolte, Lorraine A.; Holloszy, John O.Increased expression of GLUT-4 and hexokinase in rat epitrochlearis muscles exposed to AICAR in vitro. J. Appl. Physiol. 88: 1072–1075, 2000.—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 mMAICAR in vitro for 18 h resulted in an ,50% increase in GLUT-4 protein and an ,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. AMP kinase; exercise; gene expression; skeletal muscle; tissue culture; 5-aminoimidazole-4-carboxamide ribonucleosideItem Intermittent increases in cytosolic Ca2 stimulate mitochondrial biogenesis in muscle cells(Am J Physiol Endocrinol Metab 283, 2002) Ojuka, Edward O.; Jones, Terry E.; Han, Dong-Ho; Chen, May; Wamhoff, Brain R.; Sturek, Micheal; Holloszy, John O.Intermittent increases in cytosolic Ca2 stimulate mitochondrial biogenesis in muscle cells. Am J Physiol Endocrinol Metab 283: E1040–E1045, 2002. First published July 24, 2002; 10.1152/ajpendo.00242.2002.—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 Mionomycin, 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. caffeine; exercise; gene expression; ionomycin; L6 myotubesItem Regulation of GLUT4 biogenesis in muscle: evidence for involvement of AMPK and Ca2+(Am J Physiol Endocrinol Metab 282, 2002) Ojuka, Edward O.; Jones, Terry E.; Nolte, Lorraine A.; Chen, May; Wamhoff, Brain R.; Sturek, Micheal; Holloszy, John O.Regulation of GLUT4 biogenesis in muscle: evidence for involvement of AMPK and Ca2 . Am J Physiol Endocrinol Metab 282: E1008–E1013, 2002; 10.1152/ ajpendo.00512.2001.—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. 5 -adenosine monophosphate-activated protein kinase; gene expression; skeletal muscle; tissue culture; myocyte enhancer factor 2; Ca2 -calmodulin-dependent protein kinaseItem Role of calcium and AMP kinase in the regulation of mitochondrial biogenesis and GLUT4 levels in muscle(Proceedings of the Nutrition Society, 2004) Ojuka, Edward O.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 Ca2+, which activates Ca2+/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 Ca2+ 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.Item The role of CaMKII in regulating GLUT4 expression in skeletal muscle(Am J Physiol Endocrinol Metab, 2012) Ojuka,Edward O.; Goyaram, Veeraj; Smith, James A. H.The role of CaMKII in regulating GLUT4 expression in skeletal muscle. Am J Physiol Endocrinol Metab 303: E322–E331, 2012. First published April 10, 2012; doi:10.1152/ajpendo.00091.2012.—Contractile activity during physical exercise induces an increase in GLUT4 expression in skeletal muscle, helping to improve glucose transport capacity and insulin sensitivity. An important mechanism by which exercise upregulates GLUT4 is through the activation of Ca2 /calmodulin-dependent protein kinase II (CaMKII) in response to elevated levels of cytosolic Ca2 during muscle contraction. This review discusses the mechanism by which Ca2 activates CaMKII, explains research techniques currently used to alter CaMK activity in cells, and highlights various exercise models and pharmacological agents that have been used to provide evidence that CaMKII plays an important role in regulating GLUT4 expression. With regard to transcriptional mechanisms, the key research studies that identified myocyte enhancer factor 2 (MEF2) and GLUT4 enhancer factor as the major transcription factors regulating glut4 gene expression, together with their binding domains, are underlined. Experimental evidence showing that CaMK activation induces hyperacetylation of histones in the vicinity of the MEF2 domain and increases MEF2 binding to its cis element to influence MEF2-dependent Glut4 gene expression are also given along with data suggesting that p300 might be involved in acetylating histones on the Glut4 gene. Finally, an appraisal of the roles of other calcium- and non-calcium-dependent mechanisms, including the major HDAC kinases in GLUT4 expression, is also given. calcium/calmodulin-dependent protein kinase II; glucose transporter 4; myocyte enhancer factor 2; histone hyperacetylation; histone deacetylase kinasesItem Uric acid and transforming growth factor in fructose-induced production of reactive oxygen species in skeletal muscle(Oxford University Press, 2016) Madlala, Hlengiwe P.; Maarman, Gerald J.; Ojuka, EdwardThe consumption of fructose, a major constituent of the modern diet, has raised increasing concern about the effects of fructose on health. Research suggests that excessive intake of fructose (>50 g/d) causes hyperuricemia, insulin resistance, mitochondrial dysfunction, de novo lipogenesis by the liver, and increased production of reactive oxygen species (ROS) in muscle. In a number of tissues, uric acid has been shown to stimulate the production of ROS via activation of transforming growth factor b1 and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 4. The role of uric acid in fructose-induced production of ROS in skeletal muscle, however, has not been investigated. This review examines the evidence for fructose-induced production of ROS in skeletal muscle, highlights proposed mechanisms, and identifies gaps in current knowledge.