title: Caffeine induces hyperacetylation of histones at the MEF2 site on the Glut4 promoter and increases MEF2A binding to the site via a CaMK-dependent mechanism.
abstract: This study was conducted to explore the mechanism by which caffeine increases GLUT4 expression in C(2)C(12) myotubes. Myoblasts were differentiated in DMEM containing 2% horse serum for 13 days and the resultant myotubes exposed to 10 mM caffeine in the presence or absence of 25 microM KN93 or 10 mM dantrolene for 2 h. After the treatment, cells were kept in serum-free medium and harvested between 0 and 6 h later, depending on the assay. Chromatin immunoprecipitation (ChIP) assays revealed that caffeine treatment caused hyperacetylation of histone H3 at the myocyte enhancer factor 2 (MEF2) site on the Glut4 promoter (P < 0.05) and increased the amount of MEF2A that was bound to this site approximately 2.2-fold (P < 0.05) 4 h posttreatment compared with controls. These increases were accompanied by an approximately 1.8-fold rise (P < 0.05 vs. control) in GLUT4 mRNA content at 6 h post-caffeine treatment. Both immunoblot and immunocytochemical analyses showed reduced nuclear content of histone deacetylase-5 in caffeine-treated myotubes compared with controls at 0-2 h posttreatment. Inclusion of 10 mM dantrolene in the medium to prevent the increase in cytosolic Ca(2+), or 25 microM KN93 to inhibit Ca(2+)/calmodulin-dependent protein kinase (CaMK II), attenuated all the above caffeine-induced changes. These data indicate that caffeine increases GLUT4 expression by acetylating the MEF2 site to increase MEF2A binding via a mechanism that involves CaMK II.
authors.affiliation: UCT/MRC Research Unit for Exercise Science and Sports Medicine, Dept. of Human Biology, Univ. of Cape Town, Cape Town 7700, South Africa, P. O. Box 115, Newlands, 7725, South Africa.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Mukwevho
authors.forename: Tertius A
authors.initials: TA
authors.lastname: Kohn
authors.forename: Dirk
authors.initials: D
authors.lastname: Lang
authors.forename: Edward
authors.initials: E
authors.lastname: Nyatia
authors.forename: James
authors.initials: J
authors.lastname: Smith
authors.forename: Edward O
authors.initials: EO
authors.lastname: Ojuka
mesh_terms: D000107:Acetylation / Q000187:drug effects; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D002110:Caffeine / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000502:physiology*; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000235:genetics; D006655:Histone Deacetylases / Q000032:analysis / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D006801:Humans; D026161:MADS Domain Proteins / Q000378:metabolism*; D064326:MEF2 Transcription Factors; D032342:Muscle Cells / Q000648:ultrastructure; D018006:Myogenic Regulatory Factors / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis
chemical_list: D051275:Glucose Transporter Type 4; D006657:Histones; D026161:MADS Domain Proteins; D064326:MEF2 Transcription Factors; C578035:MEF2A protein, human; D018006:Myogenic Regulatory Factors; D012333:RNA, Messenger; C495692:SLC2A4 protein, human; D002110:Caffeine; D004247:DNA; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; C404500:HDAC5 protein, human; D006655:Histone Deacetylases; D002118:Calcium