biostudies-literatureDiaz-Vegas AMedical Research Council Career Development AwardDiabetes Australian Research ProgramWellcome Trust Major AwardMedical Research CouncilNational Health and Medical Research CouncilWellcome-MRC, Institute of Metabolic Science, Metabolic Research Laboratories, Imaging CoreNHMRC IdeasWellcome TrustMitochondrial FoundationWellcome Institution Strategic Support Funde202201585https://www.ebi.ac.uk/biostudies/studies/S-EPMC9595207biostudies-literatureUnknown6(1)Insulin-induced GLUT4 translocation to the plasma membrane in muscle and adipocytes is crucial for whole-body glucose homeostasis. Currently, GLUT4 trafficking assays rely on overexpression of tagged GLUT4. Here we describe a high-content imaging platform for studying endogenous GLUT4 translocation in intact adipocytes. This method enables high fidelity analysis of GLUT4 responses to specific perturbations, multiplexing of other trafficking proteins and other features including lipid droplet morphology. Using this multiplexed approach we showed that Vps45 and Rab14 are selective regulators of GLUT4, but <i>Trarg1</i>, <i>Stx6</i>, <i>Stx16</i>, <i>Tbc1d4</i> and <i>Rab10</i> knockdown affected both GLUT4 and TfR translocation. Thus, GLUT4 and TfR translocation machinery likely have some ovLife science allianceA high-content endogenous GLUT4 trafficking assay reveals new aspects of adipocyte biology.PMC9595207MR/S007091/1204845/Z/16/ZGNT1120201G057Y22G-DIAAGNT2013621GNT1061122208363/Z/17/ZFazakerley DJBurchfield JGCooke KCvan Gerwen JHumphrey SJJames DEBaird HJPotter MDiaz-Vegas AConway OJNorris DMJall-Rogg SShun-Shion ASDuan XfalseA high-content endogenous GLUT4 trafficking assay reveals new aspects of adipocyte biology.Insulin-induced GLUT4 translocation to the plasma membrane in muscle and adipocytes is crucial for whole-body glucose homeostasis. Currently, GLUT4 trafficking assays rely on overexpression of tagged GLUT4. Here we describe a high-content imaging platform for studying endogenous GLUT4 translocation in intact adipocytes. This method enables high fidelity analysis of GLUT4 responses to specific perturbations, multiplexing of other trafficking proteins and other features including lipid droplet morphology. Using this multiplexed approach we showed that Vps45 and Rab14 are selective regulators of GLUT4, but <i>Trarg1</i>, <i>Stx6</i>, <i>Stx16</i>, <i>Tbc1d4</i> and <i>Rab10</i> knockdown affected both GLUT4 and TfR translocation. Thus, GLUT4 and TfR translocation machinery likely have some ov2023-01-01T00:00:00Z2023 Jan2026-05-28T04:03:05.604Z2024-11-08T20:01:40.694ZS-EPMC95952073628370310.26508/lsa.202201585