Regulation of Glut-4 Expression in Skeletal Muscle cells: The Roles of Nuclear Respiratory Factor-1 and calcium/calmodulin dependent protein Kinase

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dc.contributor.advisorOjuka, Edwarden_ZA
dc.contributor.authorMukwevho, Emmanuelen_ZA
dc.date.accessioned2014-07-28T18:17:36Z
dc.date.available2014-07-28T18:17:36Z
dc.date.issued2010en_ZA
dc.description.abstractGLUT4 protein is the major glucose transporter in skeletal muscle and is vital in the maintenance of euglycemia (17; 108). Underexpression of GLUT4 or impairement of its translocation from intracellular compartments to the cell surface, are linked to diminished glucose transport, hyperglycemia and type II diabetes (59; 61; 153). Type II diabetes can be alleviated by increasing GLUT4 expression (223). Previous reports have shown that overexpression of NRF-1 and activation of CaMKII increases GLUT4 expression but the mechanisms involved have not be characterized (10; 173). Therefore, the objective of this thesis was to investigate the molecular mechanisms by which NRF-1 and CaMK II regulate GLUT4 expression in C2C12 myocytes. We engineered C2C12 cells that overexpressed NRF-1 in response to doxycycline (Dox) using a Tet-On gene expression system and assessed the effects of NRF-1 overexpression on: a) MEF2A, GLUT4 and δALAS proteins by western blot, and b) the binding of NRF-1 to mef2a and δalas genes and MEF2A to the glut4 gene, by chromatin immunoprecipitation assay (ChIP). The importance of MEF2A in NRF-1-induced increase in GLUT4 expression was investigated by silencing MEF2A expression using small interference RNA (siRNA). CaMK II was activated in wild-type C2C12 myocytes using 10 mM caffeine and was inhibited by 25 μ M KN93. Acetylation of histones in the vicinity of NRF-1 and MEF2A binding sites on the mef2a and glut4 genes, respectively, were assessed by ChIP assay. HDAC5 nuclear export was assessed by immunocytochemistry and mRNA levels by qRT-PCR. Overexpression of NRF-1 resulted in ~3-fold increases in mef2a-bound NRF-1 and glut4 -bound MEF2A at 6 h and 8 h post Dox treatment, respectively. MEF2A and GLUT4 proteins were both increased ~1.6-fold at 6 h and 18 h post Dox treatment. Silencing of MEF2A caused a marked downregulation of GLUT4 expression in NRF-1-overexpressing cells.en_ZA
dc.identifier.apacitationMukwevho, E. (2010). <i>Regulation of Glut-4 Expression in Skeletal Muscle cells: The Roles of Nuclear Respiratory Factor-1 and calcium/calmodulin dependent protein Kinase</i>. (Thesis). University of Cape Town ,Faculty of Health Sciences ,Department of Human Biology. Retrieved from http://hdl.handle.net/11427/3272en_ZA
dc.identifier.chicagocitationMukwevho, Emmanuel. <i>"Regulation of Glut-4 Expression in Skeletal Muscle cells: The Roles of Nuclear Respiratory Factor-1 and calcium/calmodulin dependent protein Kinase."</i> Thesis., University of Cape Town ,Faculty of Health Sciences ,Department of Human Biology, 2010. http://hdl.handle.net/11427/3272en_ZA
dc.identifier.citationMukwevho, E. 2010. Regulation of Glut-4 Expression in Skeletal Muscle cells: The Roles of Nuclear Respiratory Factor-1 and calcium/calmodulin dependent protein Kinase. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Mukwevho, Emmanuel AB - GLUT4 protein is the major glucose transporter in skeletal muscle and is vital in the maintenance of euglycemia (17; 108). Underexpression of GLUT4 or impairement of its translocation from intracellular compartments to the cell surface, are linked to diminished glucose transport, hyperglycemia and type II diabetes (59; 61; 153). Type II diabetes can be alleviated by increasing GLUT4 expression (223). Previous reports have shown that overexpression of NRF-1 and activation of CaMKII increases GLUT4 expression but the mechanisms involved have not be characterized (10; 173). Therefore, the objective of this thesis was to investigate the molecular mechanisms by which NRF-1 and CaMK II regulate GLUT4 expression in C2C12 myocytes. We engineered C2C12 cells that overexpressed NRF-1 in response to doxycycline (Dox) using a Tet-On gene expression system and assessed the effects of NRF-1 overexpression on: a) MEF2A, GLUT4 and &Icirc;&acute;ALAS proteins by western blot, and b) the binding of NRF-1 to mef2a and &Icirc;&acute;alas genes and MEF2A to the glut4 gene, by chromatin immunoprecipitation assay (ChIP). The importance of MEF2A in NRF-1-induced increase in GLUT4 expression was investigated by silencing MEF2A expression using small interference RNA (siRNA). CaMK II was activated in wild-type C2C12 myocytes using 10 mM caffeine and was inhibited by 25 &Icirc;¼ M KN93. Acetylation of histones in the vicinity of NRF-1 and MEF2A binding sites on the mef2a and glut4 genes, respectively, were assessed by ChIP assay. HDAC5 nuclear export was assessed by immunocytochemistry and mRNA levels by qRT-PCR. Overexpression of NRF-1 resulted in ~3-fold increases in mef2a-bound NRF-1 and glut4 -bound MEF2A at 6 h and 8 h post Dox treatment, respectively. MEF2A and GLUT4 proteins were both increased ~1.6-fold at 6 h and 18 h post Dox treatment. Silencing of MEF2A caused a marked downregulation of GLUT4 expression in NRF-1-overexpressing cells. DA - 2010 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2010 T1 - Regulation of Glut-4 Expression in Skeletal Muscle cells: The Roles of Nuclear Respiratory Factor-1 and calcium/calmodulin dependent protein Kinase TI - Regulation of Glut-4 Expression in Skeletal Muscle cells: The Roles of Nuclear Respiratory Factor-1 and calcium/calmodulin dependent protein Kinase UR - http://hdl.handle.net/11427/3272 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/3272
dc.identifier.vancouvercitationMukwevho E. Regulation of Glut-4 Expression in Skeletal Muscle cells: The Roles of Nuclear Respiratory Factor-1 and calcium/calmodulin dependent protein Kinase. [Thesis]. University of Cape Town ,Faculty of Health Sciences ,Department of Human Biology, 2010 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/3272en_ZA
dc.language.isoeng
dc.publisher.departmentDepartment of Human Biologyen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherCell Biologyen_ZA
dc.titleRegulation of Glut-4 Expression in Skeletal Muscle cells: The Roles of Nuclear Respiratory Factor-1 and calcium/calmodulin dependent protein Kinaseen_ZA
dc.typeDoctoral Thesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnamePhDen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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