We thank Drs. Kjøbsted and Wojtaszewski (1) for their comments on our article. In our study, we identified a cooperative AMPK-RabGTPase–activating protein (RabGAP) signaling axis in the metabolic response to exercise/contraction using a novel mouse model deficient in active skeletal muscle AMPK combined with knockout of either Tbc1d1, Tbc1d4, or both RabGAPs and pharmacological inactivation of Rac1. In their letter, the authors pointed out a limitation in our study, as we did not discriminate glucose uptake during and immediately after muscle contraction.
Cell surface levels of GLUT4 at steady state are determined by the rates of both exocytosis and endocytosis, and the latter has been notoriously difficult to determine experimentally (2). In a recent collaborative study, we proposed that AMPK/TBC1D1 signaling inhibits endocytosis of GLUT4 in skeletal muscle, which is consistent with an observed elevation of plasma membrane GLUT4 in the period postcontraction (3). In fact, studies involving surface labeling of GLUT4 in cultured L6 muscle cells and cardiomyocytes support the hypothesis that AMPK signaling lowers the rate of clearance of glucose transporters from the plasma membrane, as discussed in the letter (1), although the pertinent AMPK targets remain unidentified.
Previously, we and others demonstrated a critical role of TBC1D1 in insulin- and AICAR-stimulated glucose transport in skeletal muscle (4,5). We found that TBC1D1 interacts with the cytoplasmic domain of the insulin-regulated aminopeptidase, a resident protein of GLUT4 storage vesicles, and this binding is disrupted by phosphorylation of TBC1D1 by AKT or AMPK (6). These findings suggest that recruitment of TBC1D1 to GLUT4-containing vesicles is regulated by insulin- and contraction-mediated phosphorylation. However, as we did not specifically investigate the contribution of internalization and recycling of GLUT4 to the contraction-mediated glucose uptake in the different muscle types, genotypes, and treatment regiments, we certainly agree that further studies are required to mechanistically investigate the molecular basis for the regulation of GLUT4 endocytosis downstream of the RabGAP signaling axis. Nevertheless, our data unequivocally demonstrate that AMPK/RabGAP signaling is necessary but not sufficient for contraction-mediated GLUT4 translocation in skeletal muscle.