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

Before insulin can reach its target tissues it must cross the micovasculature, a process which has been shown to be rate‐limiting in insulin action. Most evidence to date suggests that the major route by which insulin is 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 transcytosis across live human adipose microvascular endothelial cells (HAMECs) using total internal reflection fluorescence microscopy. Using this assay we study the route by which insulin destined for transcytosis is internalized. We show that insulin transcytosis is dynamin‐dependent as treatment with Dyngo 4a ablated insulin uptake and transcytosis. Intriguingly, insulin transcytosis was unaffected by cholesterol depletion by methyl‐β‐cyclodextrin or nystatin. Moreover, caveolin‐1 depletion by siRNA had no effect and there was little colocalization of insulin with caveolin‐1 by immunofluorescence (IF). Instead, interfering with clathrin‐mediated endocytosis with Pitstop 2 or siRNA to clathrin heavy chain significantly impaired transcytosis. Insulin also colocalized with clathrin‐heavy chain by IF. Together, these data indicate that insulin transcytosis in HAMECs is clathrin‐dependent, in contrast to what has been reported previously for insulin uptake in macrovascular endothelial cells. Elucidating the mechanisms of insulin transcytosis may identify novel targets for insulin resistance.