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
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Mentions: Dynamin is an essential component of both clathrin-dependent and caveolar-dependent endocytosis. Of interest, Wang et al. (2011) observed that insulin uptake into macrovascular endothelial cells from bovine aortae is mediated by caveolae. Caveolae are cholesterol-rich lipid microdomains, and, accordingly, cholesterol depletion or sequestration causes caveolar disassembly. However, depletion of cholesterol using either methyl-β-cyclodextrin (MBCD) or nystatin did not inhibit insulin uptake or transcytosis in HAMECs and instead tended to increase them (Figure 6, A–C). Caveolin-1 is the major protein constituent of caveolae and is required for caveolae generation (Williams and Lisanti, 2004). To our surprise, overexpression of dominant-negative caveolin-1 tended to promote insulin transcytosis events (Figure 6D), and knockdown of caveolin-1 by small interefering RNA (siRNA) induced a similar trend (Figure 6, E and F). Consistent with these findings, insulin-FITC internalized for 1 min exhibited little colocalization with caveolin-1 in HAMECs (Manders coefficient of 0.196 ± 0.011; Figure 6G). Taken together, these data suggest that insulin transcytosis across adipose microvascular endothelial cells is dynamin dependent but does not occur via caveolae.
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.