Abstract 3781: Identification Of AS160 As An Insulin, Ischemia, And Exercise Regulated Signaling Protein In The Heart
The maintenance of glucose utilization is necessary for normal cardiac function, yet the distal signaling mechanisms mediating myocardial glucose uptake are not fully understood. Studies using adipocytes and skeletal muscle demonstrate that the Akt substrate of 160 kDa (AS160) is essential for GLUT4 translocation and glucose uptake. AS160 is a Rab GTPase-activating protein (GAP) containing 6 consensus Akt phosphorylation sites collectively referred to as “PAS” sites. Phosphorylation of PAS sites inactivates the GAP domain, resulting in GLUT4 translocation. Despite the importance of AS160 in glucose uptake, there have been no studies of AS160 in the heart. Using phosphoproteomics we identified a novel phosphorylation site, Ser711, which is an AMPK consensus sequence. We next determined if AS160 is phosphorylated on PAS and Ser711 sites in response to 3 stimulators of glucose uptake in the heart, insulin (max dose, ip, 10 min), ischemia (global, 3 min), and exercise (treadmill running, 30 min). In addition, we used Akt2 knockout (KO) and AMPKα2 inactive transgenic mice (AMPKα2i TG) to assess upstream signaling to AS160. Insulin robustly increased AS160 PAS phosphorylation (450% above basal; p<0.0001), an effect partially inhibited in Akt2KO mice (P<0.05). Surprisingly, despite being an AMPK consensus sequence, insulin increased phosphorylation of Ser711 (30%; P<0.01), an increase abolished in Akt2KO mice. Neither ischemia nor exercise increased AS160 PAS phosphorylation. However, ischemia increased Ser711 phosphorylation (70%; P<0.01), an effect partially inhibited in AMPKα2i TG mice. Exercise slightly increased Ser711 phosphorylation (20%; P<0.02), which was abolished in AMPKα2i TG mice. To determine if a model of decreased cardiac glucose uptake has altered AS160 signaling, we used the db/db mouse and found a significant impairment in insulin-stimulated PAS and Ser711 phosphorylation. In conclusion, this is the first report showing that insulin, ischemia, and exercise increase AS160 phosphorylation in the heart. These stimuli elicit distinct patterns of phosphorylation on PAS and the novel Ser711 site. Akt2 and AMPKα2 are upstream signals mediating AS160 phosphorylation. AS160 is likely to be a critical signaling mechanism in the heart.