Optogenetic activation reveals distinct roles of PIP3 and Akt in adipocyte insulin action

Abstract

Glucose transporter 4 (GLUT4; also known as SLC2A4) resides on intracellular vesicles in muscle and adipose cells, and translocates to the plasma membrane in response to insulin. The phosphoinositide 3-kinase (PI3K)-Akt signaling pathway plays a major role in GLUT4 translocation; however, a challenge has been to unravel the potentially distinct contributions of PI3K and Akt (of which there are three isoforms, Akt1-Akt3) to overall insulin action. Here, we describe new optogenetic tools based on CRY2 and the N-terminus of CIB1 (CIBN). We used these '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 (IRAP is also known as LNPEP and acts as a surrogate marker for GLUT4 here). Strikingly, Opto-PIP 3 largely mimicked the maximal effects of insulin stimulation, whereas Opto-Akt only partially triggered translocation. Conversely, drug-mediated inhibition of Akt only partially dampened the translocation response of Opto-PIP 3 . 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 Aktmediated signaling regulates exocytosis. Taken together, these results indicate that PI3K and Akt play distinct roles, and that PI3K stimulates Akt-independent pathways that are important for GLUT4 translocation.

Spatiotemporal Regulators for Insulin-Stimulated GLUT4 Vesicle Exocytosis

“…In addition, the drug inhibitor for Akt activation, Akti, does not fully abolish insulin-stimulated GLUT4 translocation in adipocytes [25, 32]. Using an optogenetic approach to control Akt activation, a study from our group has demonstrated that Akt only accounts for about two-third of a maximal insulin effect on GLUT4 translocation [25], which disagrees with the previous studies that claim Akt is sufficient for insulin-stimulated GLUT4 translocation [33–35]. Whether Akt and PI3K play equivalent roles in GLUT4 translocation needs to be further tested in other cell types or compared in a more physiological condition.…”

Induction of Signal Transduction by Using Non‐Channelrhodopsin‐Type Optogenetic Tools

“…Akt is a downstream effector of PIP3, directly binds with PIP3 through its PH domain at the plasma membrane, and is activated through phosphorylation by PDK2 . This simple activation mechanism enables us to regulate Akt activation easily by using optogenetic tools . These optogenetic tools also harness the translocation system of Akt based on the CRY2/CIB1 system by utilizing a combination of CRY2‐Akt and CIB1‐CAAX.…”

“…These optogenetic tools also harness the translocation system of Akt based on the CRY2/CIB1 system by utilizing a combination of CRY2‐Akt and CIB1‐CAAX. With these optotools, it was revealed that Akt regulates local exocytosis of GLUT4‐containing vesicles in response to insulin stimulation …”

Reciprocal regulation among TRPV1 channels and phosphoi nos itide 3-kinase in response to nerve growth factor

“…It has been previously established that PI3K activity is required for NGF-induced trafficking of TRPV1 to the PM (Bonnington andMcNaughton, 2003, Stein et al, 2006), but the role of PIP 3 , the product of PI3K activity, was unclear. We hypothesized that PIP 3 constitutes part of the required signal for TRPV1 trafficking (Fig 1), as it does for trafficking of membrane/membrane proteins in other systems (Cheatham et al, 1994, Martin et al, 1996, Xu et al, 2016. To test this hypothesis, we used two-color TIRF microscopy to examine PIP 3 production and TRPV1 trafficking simultaneously.…”