Abstract 3924: Identification of Signaling Pathways that Mediate the Oxidative Stress-induced Translocation of Myocardial GLUT4 Using a Novel Lenti Viral-mediated GLUT4 Reporter Gene
GLUT4 is a major isoform of glucose transporter proteins that is expressed in the heart and is translocated from the cytoplasm to the sarcolemma in response to ischemia. Hearts from mice with cardiac-selective GLUT4 deficiency develop profound and irreversible dysfunction after ischemia. Therefore, the translocation of GLUT4 represents an important protection mechanism against ischemic injury. To identify the signaling pathways that mediate GLUT4 translocation in myocardial cells, we have established a novel method for quantifying the relative proportion of sarcolemmal GLUT4 to total GLUT4. We constructed a GLUT4 reporter containing a c-Myc epitope tag in the first exofacial loop and fused GFP to the C-terminus of this gene in the lenti virus vector. Cell-surface GLUT4 reporter was detected using an anti-Myc primary antibody and a PE-conjugated secondary antibody by FACS analysis. The ratio of cell-surface GLUT4 to total cellular GLUT4 was represented as PE/FITC (GFP fluorescence). We infected lenti virus containing this reporter into cardiac myocytes in culture and found that more than 90% of these cells expressed GFP. Stimulation with H2O2 resulted in a concentration-dependent increase in GLUT4 translocation, which peaked at 15 min after stimulation. We also confirmed the translocation of both exogenous (lenti virus-infected) and endogenous GLUT4 by Western blot and immunocytochemistry. The PI3K inhibitor LY 294002 and dominant-negative forms of PI3K and AMPK almost completely inhibited the H2O2-induced translocation of GLUT4 as well as the phosphorylation of Akt. We further examined the roles of two known AMPKKs, CaMKK and LKB1, in the translocation of GLUT4. Dominant-negative CaMKK and the CaMKK inhibitor ST-609 inhibited translocation in an earlier phase (~15 minutes after stimulation), whereas dominant-negative LKB1 inhibited translocation in a later phase (15~ minutes after stimulation). Thus, this study provides a novel approach quantifying the translocation of GLUT4 to the sarcolemma in myocardial cells. The results suggest that oxidative stress causes the translocation of GLUT4 through the serial activation of CaMKK and LKB1, which leads to activation of an AMPK-PI3K/Akt pathway.