title: An inositol 1,4,5-triphosphate (IP3)-IP3 receptor pathway is required for insulin-stimulated glucose transporter 4 translocation and glucose uptake in cardiomyocytes.
abstract: Intracellular calcium levels ([Ca2+]i) and glucose uptake are central to cardiomyocyte physiology, yet connections between them have not been studied. We investigated whether insulin regulates [Ca2+]i in cultured cardiomyocytes, the participating mechanisms, and their influence on glucose uptake via SLC2 family of facilitative glucose transporter 4 (GLUT4). Primary neonatal rat cardiomyocytes were preloaded with the Ca2+ fluorescent dye fluo3-acetoxymethyl ester compound (AM) and visualized by confocal microscopy. Ca2+ transport pathways were selectively targeted by chemical and molecular inhibition. Glucose uptake was assessed using [3H]2-deoxyglucose, and surface GLUT4 levels were quantified in nonpermeabilized cardiomyocytes transfected with GLUT4-myc-enhanced green fluorescent protein. Insulin elicited a fast, two-component, transient increase in [Ca2+]i. Nifedipine and ryanodine prevented only the first component. The second one was reduced by inositol-1,4,5-trisphosphate (IP3)-receptor-selective inhibitors (xestospongin C, 2 amino-ethoxydiphenylborate), by type 2 IP3 receptor knockdown via small interfering RNA or by transfected Gβγ peptidic inhibitor βARKct. Insulin-stimulated glucose uptake was prevented by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM, 2-amino-ethoxydiphenylborate, and βARK-ct but not by nifedipine or ryanodine. Similarly, insulin-dependent exofacial exposure of GLUT4-myc-enhanced green fluorescent protein was inhibited by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM and xestospongin C but not by nifedipine. Phosphatidylinositol 3-kinase and Akt were also required for the second phase of Ca2+ release and GLUT4 translocation. Transfected dominant-negative phosphatidylinositol 3-kinase γ inhibited the latter. In conclusion, in primary neonatal cardiomyocytes, insulin induces an important component of Ca2+ release via IP3 receptor. This component signals to glucose uptake via GLUT4, revealing a so-far unrealized contribution of IP3-sensitive Ca2+ stores to insulin action. This pathway may influence cardiac metabolism in conditions yet to be explored in adult myocardium.
authors.affiliation: Centro Estudios Moleculares de la Célula, Facultad de Medicina, and Departamento de Bioquímica y Biología Molecular, Universidad de Chile, Olivos 1007, Santiago 838-0492, Chile.
authors.forename: A E
authors.initials: AE
authors.lastname: Contreras-Ferrat
authors.forename: B
authors.initials: B
authors.lastname: Toro
authors.forename: R
authors.initials: R
authors.lastname: Bravo
authors.forename: V
authors.initials: V
authors.lastname: Parra
authors.forename: C
authors.initials: C
authors.lastname: Vásquez
authors.forename: C
authors.initials: C
authors.lastname: Ibarra
authors.forename: D
authors.initials: D
authors.lastname: Mears
authors.forename: M
authors.initials: M
authors.lastname: Chiong
authors.forename: E
authors.initials: E
authors.lastname: Jaimovich
authors.forename: A
authors.initials: A
authors.lastname: Klip
authors.forename: S
authors.initials: S
authors.lastname: Lavandero
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D005947:Glucose / Q000378:metabolism / Q000493:pharmacokinetics*; D051275:Glucose Transporter Type 4 / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism / Q000502:physiology*; D007328:Insulin / Q000494:pharmacology*; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D051275:Glucose Transporter Type 4; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007328:Insulin; D015544:Inositol 1,4,5-Trisphosphate; D005947:Glucose; D002118:Calcium