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Regulation of Glut-4 Expression in Skeletal Muscle cells: The Roles of Nuclear Respiratory Factor-1 and calcium/calmodulin dependent protein Kinase
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 t...
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| Format: | Thesis |
| Language: | English |
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Department of Human Biology
2014
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| _version_ | 1867613260659818496 |
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| access_status_str | Open Access |
| author | Mukwevho, Emmanuel |
| author2 | Ojuka, Edward |
| author_browse | Mukwevho, Emmanuel Ojuka, Edward |
| author_facet | Ojuka, Edward Mukwevho, Emmanuel |
| author_sort | Mukwevho, Emmanuel |
| collection | Thesis |
| description | 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 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. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/3272 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:33:19.547Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2014 |
| publishDateRange | 2014 |
| publishDateSort | 2014 |
| publisher | Department of Human Biology |
| publisherStr | Department of Human Biology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/3272 Regulation of Glut-4 Expression in Skeletal Muscle cells: The Roles of Nuclear Respiratory Factor-1 and calcium/calmodulin dependent protein Kinase Mukwevho, Emmanuel Ojuka, Edward Cell Biology 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 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. 2014-07-28T18:17:36Z 2014-07-28T18:17:36Z 2010 Doctoral Thesis Doctoral PhD http://hdl.handle.net/11427/3272 eng application/pdf Department of Human Biology Faculty of Health Sciences University of Cape Town |
| spellingShingle | Cell Biology Mukwevho, Emmanuel Regulation of Glut-4 Expression in Skeletal Muscle cells: The Roles of Nuclear Respiratory Factor-1 and calcium/calmodulin dependent protein Kinase |
| thesis_degree_str | Doctoral |
| title | Regulation of Glut-4 Expression in Skeletal Muscle cells: The Roles of Nuclear Respiratory Factor-1 and calcium/calmodulin dependent protein Kinase |
| title_full | Regulation of Glut-4 Expression in Skeletal Muscle cells: The Roles of Nuclear Respiratory Factor-1 and calcium/calmodulin dependent protein Kinase |
| title_fullStr | Regulation of Glut-4 Expression in Skeletal Muscle cells: The Roles of Nuclear Respiratory Factor-1 and calcium/calmodulin dependent protein Kinase |
| title_full_unstemmed | Regulation of Glut-4 Expression in Skeletal Muscle cells: The Roles of Nuclear Respiratory Factor-1 and calcium/calmodulin dependent protein Kinase |
| title_short | Regulation of Glut-4 Expression in Skeletal Muscle cells: The Roles of Nuclear Respiratory Factor-1 and calcium/calmodulin dependent protein Kinase |
| title_sort | regulation of glut 4 expression in skeletal muscle cells the roles of nuclear respiratory factor 1 and calcium calmodulin dependent protein kinase |
| topic | Cell Biology |
| url | http://hdl.handle.net/11427/3272 |
| work_keys_str_mv | AT mukwevhoemmanuel regulationofglut4expressioninskeletalmusclecellstherolesofnuclearrespiratoryfactor1andcalciumcalmodulindependentproteinkinase |