Predoctoral Fellowship for Melissa Smith: Mechanical Signal Transduction in Glucose Transport of Skeletal Muscle

The long term goal of this study is to understand the mechanism by which muscle derived reactive oxygen species (ROS) activate glucose uptake independent of insulin. The systematic investigation of this insulinindependent stimulation of muscular glucose uptake could lead to new therapies for people with diabetes by providing an alternative pathway for glucose regulation. This proposal complements the mission of the American Heart Associations "Heart Of Diabetes" program by raising our awareness about how exercise may regulate blood glucose levels. Exercise leads to increased glucose uptake by skeletal muscle. Exercise also causes an increase in ROS-production. We hypothesize that ROS may act as a second messenger to stimulate exercise induced glucose uptake. Studies have shown that exogenous ROS increase glucose uptake in multiple cell types. However, effects of muscle-derived ROS on glucose uptake have not been examined. In order to explore the possibility that muscle-derived ROS direct glucose uptake independent of insulin, we use mechanical loading to induce ROS production. We propose that this muscle derived ROS will stimulate AMPactivated kinase (AMPK) activity. The resulting increased AMPK activity will subsequently lead to increased glucose uptake. My first aim will evaluate muscle-derived ROS as regulators of mechanically-stimulated glucose transport. To test the hypotheses for this aim, I plan to use an animal model for in vitro muscle measurements, a dichlorofluorescin oxidation detection assay, and a radioactive glucose transport assay. My second aim will determine if ROS stimulate glucose uptake via AMPK activation. To investigate this aim I will use excised mouse limb muscles and tissue culture (C2C12 myotubes). The additional techniques to be used will include immunoblotting and a kinase assay.