Abstract 926: Oxidative Stress Induces GLUT4 Translocation by dual AMPK Kinase Activation in Cardiac Myocytes
GLUT4, the most abundant glucose transporter, resides in intracellular vesicles under basal conditions, and translocates to the plasma membrane in response to insulin, ischemia, and hypoxia. Hearts from mice with cardiac-selective GLUT4 deficiency develop profound and irreversible systolic and diastolic dysfunction after ischemia and reperfusion. Therefore, the translocation of GLUT4 may play an important role in protecting against ischemic injury. To investigate the precise mechanisms of GLUT4 translocation in cardiomyocytes, we have established a method for quantifying the relative proportion of sarcolemmal GLUT4 to total GLUT4 in these cells. Stimulation of cardiomyocytes with H2O2 led to a concentration-dependent increase in GLUT4 translocation, which peaked at 15 min after stimulation. H2O2 also activated AMP-activated protein kinase (AMPK) α2, a serine/threonine protein kinase playing a central role in hypoxia/ischemia-induced signaling. The dominant-negative form of AMPKα2 (DN-AMPKα2) inhibited the H2O2-induced translocation of GLUT4 as early as 15 min after H2O2 stimulation. We further examined the role of two known AMPK kinases, calmodulin-dependent protein kinase kinase (CaMKK)β and LKB1, in the translocation of GLUT4. DN-CaMKKβ and DN-LKB1 inhibited translocation only partially compared to the inhibition produced by DN-AMPKα2. However, DN-LKB1 and DN-CaMKKβ had an additive effect on the inhibition of GLUT4 translocation. CaMKKβ can activate AMPK in a manner that is stimulated by Ca2+ and calmodulin. Pretreatment with BAPTA-AM significantly reduced the translocation of GLUT4 as well as blocking H2O2-induced rapid increase in [Ca2+]i. We further measured the AMP: ATP ratio in H2O2-treated cardiac myocytes by an HPLC-based method. Changes in the AMP: ATP ratio were not evident until 15 min after treatment with H2O2. However, this ratio dramatically increased at 25 min after treatment. Thus, these results demonstrate that oxidative stress causes the translocation of myocardial GLUT4 to the sarcolemma through the activation of AMPK by dual action of CaMKK and LKB1, which is followed by an increase in the AMP: ATP ratio.