title: Insulin acutely triggers transcription of Slc2a4 gene: participation of the AT-rich, E-box and NFKB-binding sites.
abstract: OBJECTIVE
  The insulin-sensitive glucose transporter protein GLUT4 (solute carrier family 2 member 4 (Slc2a4) gene) plays a key role in glycemic homeostasis. Decreased GLUT4 expression is a current feature in insulin resistant conditions such as diabetes, and the restoration of GLUT4 content improves glycemic control. This study investigated the effect of insulin upon Slc2a4/GLUT4 expression, focusing on the AT-rich element, E-box and nuclear factor NF-kappa-B (NFKB) site.
  
  
  METHODS
  Rat soleus muscles were incubated during 180 min with insulin, added or not with wortmannin (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (PI3K)-inhibitor), ML9 (serine/threonine protein kinase (AKT) inhibitor) and tumor necrosis factor (TNF, GLUT4 repressor), and processed for analysis of GLUT4 protein (Western blotting); Slc2a4, myocyte enhancer factor 2a/d (Mef2a/d), hypoxia inducible factor 1a (Hif1a), myogenic differentiation 1 (Myod1) and nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (Nfkb1) messenger ribonucleic acids (mRNAs) (polymerase chain reaction (PCR)); and AT-rich- (myocyte-specific enhancer factor 2 (MEF2)-binding site), E-box- (hypoxia inducible factor 1 alpha (HIF1A)- and myoblast determination protein 1 (MYOD1)-binding site), and NFKB-binding activity (electrophoretic mobility assay).
  
  
  RESULTS
  Insulin increased Slc2a4 mRNA expression (140%) and nuclear proteins binding to AT-rich and E-box elements (~90%), all effects were prevented by wortmannin and ML9. Insulin also increased Mef2a/d and Myod1 mRNA expression, suggesting the participation of these transcriptional factors in the Slc2a4 enhancing effect. Conversely, insulin decreased Nfkb1 mRNA expression and protein binding to the NFKB-site (~50%). Furthermore, TNF-induced inhibition of GLUT4 expression (~40%) was prevented by insulin in an NFKB-binding repressing mechanism. GLUT4 protein paralleled the Slc2a4 mRNA regulations.
  
  
  CONCLUSIONS
  Insulin enhances the Slc2a4/GLUT4 expression in the skeletal muscle by activating AT-rich and E-box elements, in a PI3K/AKT-dependent mechanism, and repressing NFKB-site activity as well. These results unravel how post-prandial increase of insulin may guarantee GLUT4 expression, and how the insulin signaling impairment can participate in insulin resistance-induced repression of GLUT4.
authors.affiliation: Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil.
authors.forename: Paulo Alexandre
authors.initials: PA
authors.lastname: Moraes
authors.affiliation: Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil.
authors.forename: Caio Yogi
authors.initials: CY
authors.lastname: Yonamine
authors.affiliation: Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil.
authors.forename: Danilo Correa
authors.initials: DC
authors.lastname: Pinto Junior
authors.affiliation: Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil.
authors.forename: João Victor DelConti
authors.initials: JV
authors.lastname: Esteves
authors.affiliation: Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil.
authors.forename: Ubiratan Fabres
authors.initials: UF
authors.lastname: Machado
authors.affiliation: Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil. Electronic address: morirc-icb@hotmail.com.
authors.forename: Rosana Cristina
authors.initials: RC
authors.lastname: Mori
mesh_terms: D020856:AT Rich Sequence / Q000235:genetics; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D024721:E-Box Elements / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D051275:Glucose Transporter Type 4 / Q000235:genetics*; D007328:Insulin / Q000494:pharmacology*; D007333:Insulin Resistance; D008297:Male; D018482:Muscle, Skeletal / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D014157:Transcription Factors / Q000235:genetics
chemical_list: D051275:Glucose Transporter Type 4; D007328:Insulin; D016328:NF-kappa B; D012333:RNA, Messenger; C495694:Slc2a4 protein, rat; D014157:Transcription Factors; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt
keywords: GLUT4; HIF1A; MEF2; MYOD1; PI3K/AKT; Skeletal muscle