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Mol Biol Cell. 2015 Feb 15;26(4):740-50. doi: 10.1091/mbc.E14-08-1307. Epub 2014 Dec 24.

Clathrin-dependent entry and vesicle-mediated exocytosis define insulin transcytosis across microvascular endothelial cells.

Author information

1
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.
2
Keenan Research Centre, St. Michael's Hospital, Toronto, ON M5B 1W8, Canada.
3
Programme in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.
4
Department of Microbiology and Immunology, University of Western Ontario, London, ON N6A 5C1, Canada.
5
Institute of Medical Sciences, University of Toronto, Toronto, ON M5S 1A8, Canada Programme in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.
6
Institute of Medical Sciences, University of Toronto, Toronto, ON M5S 1A8, Canada Keenan Research Centre, St. Michael's Hospital, Toronto, ON M5B 1W8, Canada Department of Microbiology and Immunology, University of Western Ontario, London, ON N6A 5C1, Canada Interdepartmental Division of Critical Care, Department of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada leew@smh.ca amira@sickkids.ca.

Abstract

Transport of insulin across the microvasculature is necessary to reach its target organs (e.g., adipose and muscle tissues) and is rate limiting in insulin action. Morphological evidence suggests that insulin enters endothelial cells of the microvasculature, and studies with large vessel-derived endothelial cells show insulin uptake; however, little is known about the actual transcytosis of insulin and how this occurs in the relevant microvascular endothelial cells. We report an approach to study insulin transcytosis across individual, primary human adipose microvascular endothelial cells (HAMECs), involving insulin uptake followed by vesicle-mediated exocytosis visualized by total internal reflection fluorescence microscopy. In this setting, fluorophore-conjugated insulin exocytosis depended on its initial binding and uptake, which was saturable and much greater than in muscle cells. Unlike its degradation within muscle cells, insulin was stable within HAMECs and escaped lysosomal colocalization. Insulin transcytosis required dynamin but was unaffected by caveolin-1 knockdown or cholesterol depletion. 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.

PMID:
25540431
PMCID:
PMC4325843
DOI:
10.1091/mbc.E14-08-1307
[Indexed for MEDLINE]
Free PMC Article

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