Department of Physiology

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Browsing Department of Physiology by Author "Smith, James A. H."

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    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 phosphorylation
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    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-4
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    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 kinases