Sortilin is essential and sufficient for the formation of GLUT4-storage vesicles and acquisition of insulin responsiveness in 3T3-L1 cells

Impaired translocation of the glucose transporter isoform 4 (GLUT4) to the plasma membrane in fat and skeletal muscle cells may represent a primary defect leading to the development of type-2 diabetes mellitus. The insulin-responsive pool of GLUT4 is localized in specialized storage vesicles (GSVs), the biological nature and biogenesis of which are not known. In order to address this problem, a developmental approach was taken, and the intracellular localization of stably expressed myc7-GLUT4 was examined in differentiating 3T3-L1 adipocytes. In undifferentiated 3T3-L1 cells, ectopically expressed myc7-GLUT4 was rapidly degraded (t1/2 ∼ 2 hrs) via the endosomal-lysosomal system. Between days 2 and 3 of differentiation, the major pool of myc7-GLUT4 was redistributed from rapidly sedimenting endosomes/lysosomes to light vesicles that had all the characteristics of the GSVs. Formation of this vesicular compartment led to the stabilization of the transporter (t1/2 > 16 hrs) and to a dramatic increase in its insulin responsiveness. Vesicle reconstitution assay in vitro demonstrated that formation of these vesicles depended on integral membrane protein(s), which was expressed in 3T3-L1 cells on day 2 of differentiation. Experiments were then designed in order to determine whether the formation of GSVs was driven by sortilin, a putative sorting receptor that had previously been found in GSVs but whose biological functions remained unknown. Indeed, our results showed that sortilin was induced on day 2 of differentiation immediately prior to the massive re-distribution of GLUT4 from endosomes/lysosomes to GSVs. In addition, sortilin interacted with GLUT4 via its lumenal domain. Overexpression of sortilin in 3T3-L1 adipocytes increased formation of GSVs both in vivo and in vitro and stimulated insulin-regulated glucose uptake. Knock down of sortilin with shRNA had the opposite effect. Finally, formation of functional GSVs as well as insulin-stimulated glucose uptake was reconstituted in 3T3-L1, NIH 3T3 and Swiss fibroblasts by double expression of sortilin-myc/His and myc7-GLUT4. In conclusion, sortilin is essential and sufficient for biogenesis of GSVs and acquisition of insulin-regulated glucose transport in adipose cells.