A high-content endogenous GLUT4 trafficking assay reveals new aspects of adipocyte biology.

Autor:	Diaz-Vegas A; Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, Australia., Norris DM; Metabolic Research Laboratories, Wellcome-Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge, UK., Jall-Rogg S; Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, Australia., Cooke KC; Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, Australia., Conway OJ; Metabolic Research Laboratories, Wellcome-Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge, UK., Shun-Shion AS; Metabolic Research Laboratories, Wellcome-Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge, UK., Duan X; Metabolic Research Laboratories, Wellcome-Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge, UK., Potter M; Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, Australia., van Gerwen J; Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, Australia., Baird HJ; Metabolic Research Laboratories, Wellcome-Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge, UK., Humphrey SJ; Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, Australia., James DE; Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, Australia james.burchfield@sydney.edu.au djf72@medschl.cam.ac.uk david.james@sydney.edu.au.; School of Medical Sciences, University of Sydney, Sydney, Australia., Fazakerley DJ; Metabolic Research Laboratories, Wellcome-Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge, UK., Burchfield JG; Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, Australia james.burchfield@sydney.edu.au djf72@medschl.cam.ac.uk david.james@sydney.edu.au.
Jazyk:	angličtina
Zdroj:	Life science alliance [Life Sci Alliance] 2022 Oct 25; Vol. 6 (1). Date of Electronic Publication: 2022 Oct 25 (Print Publication: 2023).
DOI:	10.26508/lsa.202201585
Abstrakt:	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 Trarg1 , Stx6 , Stx16 , Tbc1d4 and Rab10 knockdown affected both GLUT4 and TfR translocation. Thus, GLUT4 and TfR translocation machinery likely have some overlap upon insulin-stimulation. In addition, we identified Kif13A, a Rab10 binding molecular motor, as a novel regulator of GLUT4 traffic. Finally, comparison of endogenous to overexpressed GLUT4 highlights that the endogenous GLUT4 methodology has an enhanced sensitivity to genetic perturbations and emphasises the advantage of studying endogenous protein trafficking for drug discovery and genetic analysis of insulin action in relevant cell types. (© 2022 Diaz-Vegas et al.)
Databáze:	MEDLINE
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