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.

Alteration in mitochondrial Ca 2+ uptake disrupts insulin signaling in hypertrophic cardiomyocytes

“…In this context, our group has previously demonstrated that inhibition of InsP 3 R with XeC and the Ca 2+ chelating agent BAPTA-AM also reduces Akt phosphorylation [19]. Moreover, we showed that the induction of mitochondrial fragmentation reduces insulin-induced Akt phosphorylation by reducing mitochondrial Ca 2+ uptake [35].…”

“…Ca 2+ has also been identified as an important second messenger acting downstream of the insulin receptor and shown to be essential in insulin-mediated glucose uptake in cardiac and skeletal muscle [38][39][40]. Our group showed that Ca 2+ release from the ER through the InsP 3 R is an important component of the Ca 2+ -mediated insulin response in cardiomyocytes [19]. Mitochondria have a substantial capacity for Ca 2+ storage that is facilitated by their physical and functional interaction with InsP 3 Rs in the ER membrane [41].…”

Insulin elicits a ROS-activated and an IP3-dependent Ca2+ release; both impinge on GLUT4 translocation

“…Images were collected for 10 min using laser scanning confocal microscope in the frame-by-frame mode as previously reported (Contreras-Ferrat et al, 2010). L6 myotubes were preloaded with Fluo4 am (5 mM) at 37˚for 30 min in Krebs Ringer buffer and stimulus were added to the microscopy chamber as indicated by arrows.…”

“…A recent report proposes that RyR1 channels, by controlling passive Ca 2+ efflux from the SR to the cytoplasm, represent key factors in the management of the resting muscle cytoplasmic Ca 2+ concentration (Eltit et al, 2010). Interestingly, our groups have reported that: (1) insulin elevates intracellular Ca 2+ levels via NOX2 activation in primary neonatal myotubes (Espinosa et al, 2009), (2) NOX2-dependent ROS production promotes RyR1 S-glutathionylation and stimulates RyR1-mediated Ca 2+ release from triad-enriched vesicles isolated from adult skeletal muscle , and (3) intracellular Ca 2+ contributes to the insulin-dependent increase in glucose uptake in cardiomyocytes (Contreras-Ferrat et al, 2010). The physiological mechanism whereby insulin activates NOX2, however, remains unknown.…”

Promiscuous affairs of PKB/AKT isoforms in metabolism

“…Since the identification of PKB as a serine/threonine kinase, almost 20 years ago, PKB isoforms were studied intensively and are now considered as major regulators of elementary cellular process, such as proliferation, survival, cell growth and energy metabolism (Bozulic et al, 2008, Contreras-Ferrat et al, 2010, Haga et al, 2005, Heron-Milhavet et al, 2006. Consequently, PKB isoforms play pivotal roles in physiology and their deregulation engenders diseases, such as cancer, neurodegeneration and metabolic disorders (Altomare andTesta, 2005, Zhao andTownsend, 2009).…”