AS160 Regulates Insulin- and Contraction-stimulated Glucose Uptake in Mouse Skeletal Muscle

Abstract

Insulin and contraction are potent stimulators of GLUT4 translocation and increase skeletal muscle glucose uptake. We recently identified the Rab GTPase-activating protein (GAP) AS160 as a putative point of convergence linking distinct upstream signaling cascades induced by insulin and contraction in mouse skeletal muscle. Here, we studied the functional implications of these AS160 signaling events by using an in vivo electroporation technique to overexpress wild type and three AS160 mutants in mouse tibialis anterior muscles: 1) AS160 mutated to prevent phosphorylation on four regulatory phospho-Akt-substrate sites (4P); 2) AS160 mutated to abolish Rab GTPase activity (R/K); and 3) double mutant AS160 containing both 4P and R/K mutations (2M). One week following gene injection, protein expression for all AS160 isoforms was elevated over 7-fold. To determine the effects of AS160 on insulin- and contraction-stimulated glucose uptake in transfected muscles, we measured [3H]2-deoxyglucose uptake in vivo following intravenous glucose administration and in situ muscle contraction, respectively. Insulin-stimulated glucose uptake was significantly inhibited in muscles overexpressing 4P mutant AS160. However, this inhibition was completely prevented by concomitant disruption of AS160 Rab GAP activity. Transfection with 4P mutant AS160 also significantly impaired contraction-stimulated glucose uptake, as did overexpression of wild type AS160. In contrast, overexpressing mutant AS160 lacking Rab GAP activity resulted in increases in both sham and contraction-stimulated muscles. These data suggest that AS160 regulates both insulin- and contraction-stimulated glucose metabolism in mouse skeletal muscle in vivo and that the effects of mutant AS160 on the actions of insulin and contraction are not identical. Our findings directly implicate AS160 as a critical convergence factor for independent stimulators of skeletal muscle glucose uptake.