Abstract
Facilitative glucose transporter isoform 4 (GLUT4) in rat adipocytes is largely sequestered in intracellular sites, and insulin recruits GLUT4 from these sites to the cell surface. The process is known to involve multiple intracellular compartments and associated proteins, many of which are yet to be identified. Recently, we purified three distinct insulin-sensitive intracellular GLUT4 compartments (G4T(L), G4H, and G4L) in rat adipocytes and unraveled several new resident proteins in these compartments. Here, we describe one of them, a 62-kDa protein, purified and identified as rat adipose tissue carboxyl esterase (p62/CE) by matrix-assisted laser desorption/ionization time of flight mass spectroscopy, reverse transcription-polymerase chain reaction, gene cloning, and immunological and enzymatic activity measurements. p62/CE in rat adipocytes was 80% cytosolic and 20% microsome-associated. It was found in all of the three insulin-sensitive intracellular GLUT4 compartments, and particularly enriched in G4T(L,) a compartment thought to represent GLUT4 endocytic vesicles. Significantly, an antibody against p62/CE introduced into rat adipocytes completely abolished the insulin-induced GLUT4 recruitment to the plasma membrane in host cells without affecting the basal GLUT4 distribution. Together, these findings suggest that p62/CE plays a key role in insulin-induced GLUT4 recruitment in rat adipocytes, probably by hydrolyzing acylglycerols or acyl-CoA esters to the respective free acids that are required for GLUT4 transport vesicle budding and/or fusion.
Highlights
The uptake of glucose by muscle and adipose cells is primarily catalyzed by GLUT4,1 a facilitative glucose transporter isoform, and further regulated by hormones, by insulin [1, 2]
Clear identification of the individual intracellular GLUT4 compartments and the associated protein functions involved in these processes has been elusive
Recent success in separation and partial purification of three distinct, intracellular GLUT4 compartments from hypotonically lysed rat adipocytes [19] allowed us to detect and study novel association of certain proteins with GLUT4 compartments that hitherto escaped from identification
Summary
Vol 275, No 14, Issue of April 7, pp. 10041–10046, 2000 Printed in U.S.A. Association of Carboxyl Esterase with Facilitative Glucose Transporter Isoform 4 (GLUT4) Intracellular Compartments in Rat Adipocytes and Its Possible Role in Insulin-induced GLUT4 Recruitment*. An antibody against p62/CE introduced into rat adipocytes completely abolished the insulin-induced GLUT4 recruitment to the plasma membrane in host cells without affecting the basal GLUT4 distribution. GLUT4 is found in association with tubulovesicular structures, small vesicules, clathrin-coated lattices, and clathrin-coated vesicles [6, 7], indicating that multiple subcellular compartments are involved in GLUT4 sequestration, recycling, and recruitment Exact roles of these intracellular compartments or organelles in GLUT4 regulation remain largely undefined. The p62/CE antibody selectively abolished the insulininduced GLUT4 redistribution to the cell surface in semipermeabilized rat adipocytes, without affecting the basal GLUT4 distribution Based on these findings, we propose a working hypothesis that p62/CE modulates lipid metabolites in GLUT4 compartments and regulates membrane budding and fusion. Carboxyl Esterase in GLUT4 Compartments in Rat Adipocytes required for the insulin-induced GLUT4 recruitment in rat adipocytes
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