Oxidative stress induces GLUT4 translocation by activation of P13-K/Akt and dual AMPK kinase in cardiac myocytes

In response to metabolic stress, GLUT4, the most abundant glucose transporter, translocates from intracellular vesicles to the plasma membrane. This appears to play an important role in protecting cardiac myocytes from 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 with H(2)O(2) resulted in a concentration-dependent increase in GLUT4 translocation, which peaked at 15 min after stimulation. The dominant-negative form (DN) of AMP-activated protein kinase (AMPK) alpha 2 inhibited the H(2)O(2)-induced translocation of GLUT4. We further examined the role of two known AMPK kinases (AMPKKs), calmodulin-dependent protein kinase kinase (CaMKK)beta and LKB1. The DN of CaMKK beta or LKB1 alone inhibited H(2)O(2)-induced GLUT4 translocation only partially compared to the inhibition produced by the DN of AMPK alpha 2. However, the combination of DN-LKB1 and DN-CaMKK beta inhibited translocation to an extent similar to with DN-AMPK alpha 2. Stimulation with H(2)O(2) also activated Akt and the inhibition of P13-K/Akt prevented GLUT4 translocation to the same extent as with AMPK inhibition. When the DN of AMPKa2 was applied with DN-P13-K, there was a complete reduction in the GLUT4 membrane level similar to that seen at the 0 time-point. These results demonstrate that AMPK and P13-K/Akt have an additive effect on oxidative stress-mediated GLUT4 translocation.