Optogenetic activation reveals distinct roles of PI(3,4,5)P3 and Akt in adipocyte insulin action

Abstract

Glucose transporter 4 (GLUT4) resides on intracellular vesicles in muscle and adipose cells and translocates to the plasma membrane in response to insulin. The PI3K/Akt signaling pathway plays a major role in GLUT4 translocation, however a challenge has been to unravel potentially distinct contributions of PI3K and Akt to overall insulin action. Here we describe new optogenetic tools based on CRY2/CIBN (‘Opto’) modules to activate PI3K and Akt selectively in time and space in 3T3-L1 adipocytes. We validated these tools using biochemical assays and performed live cell kinetic analyses of IRAP-pHluorin translocation (where IRAP is a surrogate marker for GLUT4). Strikingly, Opto-PIP3 largely mimicked maximal insulin stimulation, whereas Opto-Akt only partially triggered translocation. Conversely, drug inhibition of Akt only partially dampened the translocation response of Opto-PIP3. In spatial optogenetic studies, focal targeting of Akt to a region of the cell marked the sites where IRAP-pHluorin vesicles fused, supporting the idea that local Akt-mediated signaling regulates exocytosis. Together these results indicate that PI3K and Akt play distinct roles and that PI3K stimulates Akt-independent pathways that are important for GLUT4 translocation.