Clathrin-dependent entry and vesicle-mediated exocytosis define insulin transcytosis across microvascular endothelial cells.
Bottom Line: Instead, insulin transcytosis was significantly inhibited by the clathrin-mediated endocytosis inhibitor Pitstop 2 or siRNA-mediated clathrin depletion.Accordingly, insulin internalized for 1 min in HAMECs colocalized with clathrin far more than with caveolin-1.This study constitutes the first evidence of vesicle-mediated insulin transcytosis and highlights that its initial uptake is clathrin dependent and caveolae independent.
Affiliation: Institute of Medical Sciences, University of Toronto, Toronto, ON M5S 1A8, Canada Keenan Research Centre, St. Michael's Hospital, Toronto, ON M5B 1W8, Canada Programme in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.Show MeSH
Related in: MedlinePlus
Mentions: To begin to analyze the fate of insulin in microvascular endothelial and muscle cells, we delivered a pulse of 500 nM native insulin to monolayers of HAMEC cells (illustrated in Supplemental Figure S1) or L6 myoblasts. After 5 min, the insulin was washed off, and over time, the amount of insulin within cells and that appearing in the supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Under these conditions, HAMECs took up ∼10 times more insulin than L6 myoblasts (Figure 1A). Moreover, insulin taken up by myoblasts progressively disappeared, and there was no concomitant, detectable insulin in the overlying media (Figure 1B). In contrast, insulin internalized by HAMECs decreased over time by ∼20% in parallel with a progressive recovery of insulin in the supernatant (Figure 1, A and B). To ensure that this behavior of insulin in HAMECs was not the result of saturation of the insulin-processing cellular machinery due to the high levels of insulin used, we repeated these experiments using 50-fold-lower insulin concentration in the pulse, with correspondingly similar results (Supplemental Figure S2).
Affiliation: Institute of Medical Sciences, University of Toronto, Toronto, ON M5S 1A8, Canada Keenan Research Centre, St. Michael's Hospital, Toronto, ON M5B 1W8, Canada Programme in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.