A Novel Assay for Insulin Transcytosis Reveals an Unexpected Role for Clathrin

Before insulincan reach its target tissues it must cross the micovasculature, a process which has been shown to be rate-limiting in insulinaction. Most evidence to date suggests that the major route by which insulinis transported across the endothelium is through individual cells ( transcytosis); however, little is known about how this is regulated due to technical limitations. We devised a novel method of studying insulin transcytosisacross live human adipose microvascular endothelial cells (HAMECs) using total internal reflectionfluorescence microscopy. Using this assay we study the route by which insulindestined for transcytosisis internalized. We show that insulin transcytosisis dynamin-dependent as treatment with Dyngo 4a ablated insulinuptake and transcytosis. Intriguingly, insulin transcytosiswas unaffected by cholesterol depletionby methyl-β-cyclodextrin or nystatin. Moreover, caveolin-1 depletion by siRNA had no effect and there was little colocalization of insulinwith caveolin-1 by im...