title: 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-PIP3 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-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. 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.
authors.affiliation: Department of Biomedical Engineering, MOE Key Laboratory of Biomedical Engineering, Zhejiang Provincial Key Laboratory of Cardio-Cerebral Vascular Detection Technology and Medicinal Effectiveness Appraisal, Zhejiang University, Hangzhou 310027, China Department of Cell Biology, Yale University School of Medicine, New Haven, 06510, USA yingkexu@zju.edu.cn derek.toomre@yale.edu.
authors.forename: Yingke
authors.identifier: 0000-0002-8317-0608
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Department of Biomedical Engineering, MOE Key Laboratory of Biomedical Engineering, Zhejiang Provincial Key Laboratory of Cardio-Cerebral Vascular Detection Technology and Medicinal Effectiveness Appraisal, Zhejiang University, Hangzhou 310027, China.
authors.forename: Di
authors.initials: D
authors.lastname: Nan
authors.affiliation: Department of Biomedical Engineering, MOE Key Laboratory of Biomedical Engineering, Zhejiang Provincial Key Laboratory of Cardio-Cerebral Vascular Detection Technology and Medicinal Effectiveness Appraisal, Zhejiang University, Hangzhou 310027, China.
authors.forename: Jiannan
authors.identifier: 0000-0002-6392-8493
authors.initials: J
authors.lastname: Fan
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, 06510, USA Section of Endocrinology and Metabolism, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520-8020, USA.
authors.forename: Jonathan S
authors.identifier: 0000-0001-6463-8466
authors.initials: JS
authors.lastname: Bogan
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, 06510, USA yingkexu@zju.edu.cn derek.toomre@yale.edu.
authors.forename: Derek
authors.identifier: 0000-0002-6827-8127
authors.initials: D
authors.lastname: Toomre
mesh_terms: D016475:3T3 Cells; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D005089:Exocytosis / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000235:genetics*; D006801:Humans; D007328:Insulin / Q000008:administration & dosage / Q000378:metabolism; D051379:Mice; D062308:Optogenetics; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics*; D021381:Protein Transport / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics*; D015398:Signal Transduction
chemical_list: D051275:Glucose Transporter Type 4; D007328:Insulin; C495693:Slc2a4 protein, mouse; D019869:Phosphatidylinositol 3-Kinases; C514862:Akt1 protein, mouse; C491753:Akt3 protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D005947:Glucose
keywords: Exocytosis; Glucose transporter; Insulin signaling; Optogenetics; TIRFM