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

Azizi PM, Zyla RE, Guan S, Wang C, Liu J, Bolz SS, Heit B, Klip A, Lee WL - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

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

Insulin uptake and transcytosis are dynamin dependent. (A) Cells were treated with insulin-FITC or transferrin-AF555 for 10 min after pretreatment with 30 uM dyngo 4a (right) or vehicle (left) for 30 min to impair dynamin function. White scale, 15 μm. (B) Quantification of insulin-FITC uptake in HAMECs after pretreatment with dyngo 4a. **p < 0.01 by one sample t test; data are normalized to control cells. (C) Average transcytosis events after pretreatment with dyngo 4a. **p < 0.01 by one sample t test; data are normalized to control cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4325843&req=5

Figure 5: Insulin uptake and transcytosis are dynamin dependent. (A) Cells were treated with insulin-FITC or transferrin-AF555 for 10 min after pretreatment with 30 uM dyngo 4a (right) or vehicle (left) for 30 min to impair dynamin function. White scale, 15 μm. (B) Quantification of insulin-FITC uptake in HAMECs after pretreatment with dyngo 4a. **p < 0.01 by one sample t test; data are normalized to control cells. (C) Average transcytosis events after pretreatment with dyngo 4a. **p < 0.01 by one sample t test; data are normalized to control cells.

Mentions: As shown, a saturable step of insulin internalization defines the number of insulin transcytosis events. To analyze further the molecular route of this internalization, we explored the endocytic machineries that may be mediating this mechanism. Treatment of HAMECs with dyngo 4a, a specific inhibitor of the large GTPase dynamin (McCluskey et al., 2013), before and during insulin-AF568 pulsing essentially abrogated its ensuing internalization and transcytosis (Figure 5). Use of dyngo 4a also prevented internalization of Alex Fluor 555–conjugated transferrin (transferrin-AF555, Figure 5A), consistent with the well-known dependence of this phenomenon on dynamin (van Dam and Stoorvogel, 2002).


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

Azizi PM, Zyla RE, Guan S, Wang C, Liu J, Bolz SS, Heit B, Klip A, Lee WL - Mol. Biol. Cell (2014)

Insulin uptake and transcytosis are dynamin dependent. (A) Cells were treated with insulin-FITC or transferrin-AF555 for 10 min after pretreatment with 30 uM dyngo 4a (right) or vehicle (left) for 30 min to impair dynamin function. White scale, 15 μm. (B) Quantification of insulin-FITC uptake in HAMECs after pretreatment with dyngo 4a. **p < 0.01 by one sample t test; data are normalized to control cells. (C) Average transcytosis events after pretreatment with dyngo 4a. **p < 0.01 by one sample t test; data are normalized to control cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4325843&req=5

Figure 5: Insulin uptake and transcytosis are dynamin dependent. (A) Cells were treated with insulin-FITC or transferrin-AF555 for 10 min after pretreatment with 30 uM dyngo 4a (right) or vehicle (left) for 30 min to impair dynamin function. White scale, 15 μm. (B) Quantification of insulin-FITC uptake in HAMECs after pretreatment with dyngo 4a. **p < 0.01 by one sample t test; data are normalized to control cells. (C) Average transcytosis events after pretreatment with dyngo 4a. **p < 0.01 by one sample t test; data are normalized to control cells.
Mentions: As shown, a saturable step of insulin internalization defines the number of insulin transcytosis events. To analyze further the molecular route of this internalization, we explored the endocytic machineries that may be mediating this mechanism. Treatment of HAMECs with dyngo 4a, a specific inhibitor of the large GTPase dynamin (McCluskey et al., 2013), before and during insulin-AF568 pulsing essentially abrogated its ensuing internalization and transcytosis (Figure 5). Use of dyngo 4a also prevented internalization of Alex Fluor 555–conjugated transferrin (transferrin-AF555, Figure 5A), consistent with the well-known dependence of this phenomenon on dynamin (van Dam and Stoorvogel, 2002).

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.

View Article: PubMed Central - PubMed

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