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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.

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Insulin uptake and transcytosis do not require cholesterol or caveolin-1. (A) Cells were treated with insulin-FITC or D4 membrane cholesterol probe for 10 min after pretreatment with either 1 μM methyl-β-cyclodextrin (MBCD) or 50 μg/ml nystatin to deplete cells of cholesterol. White scale, 15 μm. (B) Quantification of insulin-FITC uptake in HAMECs after pretreatment with MBCD or nystatin; data are normalized to control cells. (C) Average transcytosis events after pretreatment with MBCD or nystatin. (D) Average transcytosis events of insulin-AF568 after transfection with wild-type or dominant-negative (DN) caveolin-1 construct. (E) Average transcytosis events of insulin-AF568 after caveolin-1 was knocked down by siRNA. (F) Immunoblot of caveolin-1 protein after knockdown via siRNA. (G) Insulin-FITC colocalizes only modestly with caveolin-1 (red). Colocalization was quantified via the Manders coefficient, which is 0.196 ± 0.011. Dashed box indicates area enlarged on the right; white scale, 15 μm.
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Figure 6: Insulin uptake and transcytosis do not require cholesterol or caveolin-1. (A) Cells were treated with insulin-FITC or D4 membrane cholesterol probe for 10 min after pretreatment with either 1 μM methyl-β-cyclodextrin (MBCD) or 50 μg/ml nystatin to deplete cells of cholesterol. White scale, 15 μm. (B) Quantification of insulin-FITC uptake in HAMECs after pretreatment with MBCD or nystatin; data are normalized to control cells. (C) Average transcytosis events after pretreatment with MBCD or nystatin. (D) Average transcytosis events of insulin-AF568 after transfection with wild-type or dominant-negative (DN) caveolin-1 construct. (E) Average transcytosis events of insulin-AF568 after caveolin-1 was knocked down by siRNA. (F) Immunoblot of caveolin-1 protein after knockdown via siRNA. (G) Insulin-FITC colocalizes only modestly with caveolin-1 (red). Colocalization was quantified via the Manders coefficient, which is 0.196 ± 0.011. Dashed box indicates area enlarged on the right; white scale, 15 μm.

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.


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 do not require cholesterol or caveolin-1. (A) Cells were treated with insulin-FITC or D4 membrane cholesterol probe for 10 min after pretreatment with either 1 μM methyl-β-cyclodextrin (MBCD) or 50 μg/ml nystatin to deplete cells of cholesterol. White scale, 15 μm. (B) Quantification of insulin-FITC uptake in HAMECs after pretreatment with MBCD or nystatin; data are normalized to control cells. (C) Average transcytosis events after pretreatment with MBCD or nystatin. (D) Average transcytosis events of insulin-AF568 after transfection with wild-type or dominant-negative (DN) caveolin-1 construct. (E) Average transcytosis events of insulin-AF568 after caveolin-1 was knocked down by siRNA. (F) Immunoblot of caveolin-1 protein after knockdown via siRNA. (G) Insulin-FITC colocalizes only modestly with caveolin-1 (red). Colocalization was quantified via the Manders coefficient, which is 0.196 ± 0.011. Dashed box indicates area enlarged on the right; white scale, 15 μm.
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Related In: Results  -  Collection

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Figure 6: Insulin uptake and transcytosis do not require cholesterol or caveolin-1. (A) Cells were treated with insulin-FITC or D4 membrane cholesterol probe for 10 min after pretreatment with either 1 μM methyl-β-cyclodextrin (MBCD) or 50 μg/ml nystatin to deplete cells of cholesterol. White scale, 15 μm. (B) Quantification of insulin-FITC uptake in HAMECs after pretreatment with MBCD or nystatin; data are normalized to control cells. (C) Average transcytosis events after pretreatment with MBCD or nystatin. (D) Average transcytosis events of insulin-AF568 after transfection with wild-type or dominant-negative (DN) caveolin-1 construct. (E) Average transcytosis events of insulin-AF568 after caveolin-1 was knocked down by siRNA. (F) Immunoblot of caveolin-1 protein after knockdown via siRNA. (G) Insulin-FITC colocalizes only modestly with caveolin-1 (red). Colocalization was quantified via the Manders coefficient, which is 0.196 ± 0.011. Dashed box indicates area enlarged on the right; white scale, 15 μm.
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.

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