Adipsin and the glucose transporter GLUT4 traffic to the cell surface via independent pathways in adipocytes

Insulin increases the exocytosis of many soluble and membrane proteins in a dipocytes. This may reflect a general effect of insulin on protein export f rom the trans Golgi network. To test this hypothesis, we have compared the trafficking of the secreted serine protease adipsin and the integral membra ne proteins GLUT4 and transferrin receptors in 3T3-L1 adipocytes. We show t hat adipsin is secreted from the trans Golgi network to the endosomal syste m, as ablation of endosomes using transferrin-HRP conjugates strongly inhib ited adipsin secretion. Phospholipase D has been implicated in export from the trans Golgi network, and we show that insulin stimulates phospholipase D activity in these cells. Inhibition of phospholipase D action with butan- 2-ol blocked adipsin secretion and resulted in accumulation of adipsin in T rans Golgi network-derived vesicles. In contrast, butan-1-ol did not affect the insulin-stimulated movement of transferrin receptors to the plasma mem brane. whereas this was abrogated following endosome ablation. GLUT4 traffi cking to the cell surface does not utilise this pathway, as insulin-stimula ted GLUT4 translocation is still observed after endosome ablation or inhibi tion of phospholipase D activity. Immunolabelling revealed that adipsin and GLUT4 are predominantly localised to distinct intracellular compartments. These data suggest that insulin stimulates the activity of the constitutive secretory pathway in adipocytes possibly by increasing the budding step at the TGN by a phospholipase D-dependent mechanism. This may have relevance for the secretion of other soluble molecules from these cells. This is not the pathway employed to deliver GLUT4 to the plasma membrane, arguing that insulin stimulates multiple pathways to the cell surface in adipocytes.