An inositol 1,4,5-triphosphate (IP3)-IP3 receptor pathway is required for insulin-stimulated glucose transporter 4 translocation and glucose uptake in cardiomyocytes.
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.
Contreras-Ferrat
A E
AE
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.
Toro
B
B
Bravo
R
R
Parra
V
V
Vásquez
C
C
Ibarra
C
C
Mears
D
D
Chiong
M
M
Jaimovich
E
E
Klip
A
A
Lavandero
S
S
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
D051275:Glucose Transporter Type 4; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007328:Insulin; D015544:Inositol 1,4,5-Trisphosphate; D005947:Glucose; D002118:Calcium