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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 are clathrin dependent. (A) Cells were treated with insulin-FITC or transferrin-AF555 for 10 min after pretreatment with 10 μM Pitstop 2 to impair clathrin-mediated uptake. White scale, 15 μm. (B) Quantification of insulin-FITC uptake in HAMECs after pretreatment with Pitstop 2. (C) Average insulin-AF568 transcytosis events after pretreatment with Pitstop 2. (D) Average insulin-AF568 transcytosis events after clathrin heavy chain was knocked down by siRNA. (E) Immunoblot of clathrin heavy chain protein after knockdown by siRNA. (F) Insulin-FITC (green) colocalizes with clathrin heavy chain (red). Colocalization was quantified via the Manders coefficient, which is 0.491 ± 0.020. Dashed box indicates area enlarged on the right; white scale, 15 μm.
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Figure 7: Insulin uptake and transcytosis are clathrin dependent. (A) Cells were treated with insulin-FITC or transferrin-AF555 for 10 min after pretreatment with 10 μM Pitstop 2 to impair clathrin-mediated uptake. White scale, 15 μm. (B) Quantification of insulin-FITC uptake in HAMECs after pretreatment with Pitstop 2. (C) Average insulin-AF568 transcytosis events after pretreatment with Pitstop 2. (D) Average insulin-AF568 transcytosis events after clathrin heavy chain was knocked down by siRNA. (E) Immunoblot of clathrin heavy chain protein after knockdown by siRNA. (F) Insulin-FITC (green) colocalizes with clathrin heavy chain (red). Colocalization was quantified via the Manders coefficient, which is 0.491 ± 0.020. Dashed box indicates area enlarged on the right; white scale, 15 μm.

Mentions: The dependence of insulin uptake and transcytosis on dynamin but not on caveolae suggested an unsuspected role for clathrin in insulin transcytosis. Pitstop 2 is a cell-permeant small molecule that blocks the association of amphiphysin with the terminal domain of clathrin, thereby inhibiting clathrin-mediated endocytosis (von Kleist et al., 2011). We first confirmed that Pitstop 2 effectively blocked the internalization of transferrin, a canonical clathrin-dependent process (Figure 7A). Under these conditions, uptake and transcytosis of fluorescent insulin were reduced by >50% (Figure 7, A–C). Because the specificity of Pitstop 2 has recently been questioned (Dutta et al., 2012), we confirmed our findings by knocking down clathrin heavy chain via cognate siRNA. Depletion of clathrin significantly diminished insulin-AF568 transcytosis (Figure 7, D and E). Consistent with this finding and in contrast to what we observed with caveolin-1, we observed significant colocalization between clathrin and insulin-FITC internalized for 1 min (Manders coefficient is 0.491 ± 0.020; Figure 7F). Hence these results suggest that in HAMECs, insulin internalizes via a clathrin- and dynamin-dependent mechanism, which defines its subsequent availability for quantal exocytosis. In an attempt to reconcile these findings with the caveolar dependence of insulin uptake in bovine aortic endothelial cells (Wang et al., 2011), and cognizant of the heterogeneous nature of endothelial cells, depending on the size and source of their vessels of provenance (Aird, 2007a, b), we explored the possible colocalization of internalized insulin with clathrin or caveolin in human aortic endothelial cells. As anticipated, in these large vessel–derived human endothelial cells, insulin colocalized significantly more with caveolin-1 than with clathrin (Manders coefficient 0.411 ± 0.068 for caveolin-1; 0.196 ± 0.013 for clathrin; Figure 8), paralleling the observations for bovine aortic endothelial cells. These findings underscore the selective insulin uptake processes that take place in endothelial cells of different vascular beds, irrespective of whether they are of bovine or human origin.


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 clathrin dependent. (A) Cells were treated with insulin-FITC or transferrin-AF555 for 10 min after pretreatment with 10 μM Pitstop 2 to impair clathrin-mediated uptake. White scale, 15 μm. (B) Quantification of insulin-FITC uptake in HAMECs after pretreatment with Pitstop 2. (C) Average insulin-AF568 transcytosis events after pretreatment with Pitstop 2. (D) Average insulin-AF568 transcytosis events after clathrin heavy chain was knocked down by siRNA. (E) Immunoblot of clathrin heavy chain protein after knockdown by siRNA. (F) Insulin-FITC (green) colocalizes with clathrin heavy chain (red). Colocalization was quantified via the Manders coefficient, which is 0.491 ± 0.020. 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 7: Insulin uptake and transcytosis are clathrin dependent. (A) Cells were treated with insulin-FITC or transferrin-AF555 for 10 min after pretreatment with 10 μM Pitstop 2 to impair clathrin-mediated uptake. White scale, 15 μm. (B) Quantification of insulin-FITC uptake in HAMECs after pretreatment with Pitstop 2. (C) Average insulin-AF568 transcytosis events after pretreatment with Pitstop 2. (D) Average insulin-AF568 transcytosis events after clathrin heavy chain was knocked down by siRNA. (E) Immunoblot of clathrin heavy chain protein after knockdown by siRNA. (F) Insulin-FITC (green) colocalizes with clathrin heavy chain (red). Colocalization was quantified via the Manders coefficient, which is 0.491 ± 0.020. Dashed box indicates area enlarged on the right; white scale, 15 μm.
Mentions: The dependence of insulin uptake and transcytosis on dynamin but not on caveolae suggested an unsuspected role for clathrin in insulin transcytosis. Pitstop 2 is a cell-permeant small molecule that blocks the association of amphiphysin with the terminal domain of clathrin, thereby inhibiting clathrin-mediated endocytosis (von Kleist et al., 2011). We first confirmed that Pitstop 2 effectively blocked the internalization of transferrin, a canonical clathrin-dependent process (Figure 7A). Under these conditions, uptake and transcytosis of fluorescent insulin were reduced by >50% (Figure 7, A–C). Because the specificity of Pitstop 2 has recently been questioned (Dutta et al., 2012), we confirmed our findings by knocking down clathrin heavy chain via cognate siRNA. Depletion of clathrin significantly diminished insulin-AF568 transcytosis (Figure 7, D and E). Consistent with this finding and in contrast to what we observed with caveolin-1, we observed significant colocalization between clathrin and insulin-FITC internalized for 1 min (Manders coefficient is 0.491 ± 0.020; Figure 7F). Hence these results suggest that in HAMECs, insulin internalizes via a clathrin- and dynamin-dependent mechanism, which defines its subsequent availability for quantal exocytosis. In an attempt to reconcile these findings with the caveolar dependence of insulin uptake in bovine aortic endothelial cells (Wang et al., 2011), and cognizant of the heterogeneous nature of endothelial cells, depending on the size and source of their vessels of provenance (Aird, 2007a, b), we explored the possible colocalization of internalized insulin with clathrin or caveolin in human aortic endothelial cells. As anticipated, in these large vessel–derived human endothelial cells, insulin colocalized significantly more with caveolin-1 than with clathrin (Manders coefficient 0.411 ± 0.068 for caveolin-1; 0.196 ± 0.013 for clathrin; Figure 8), paralleling the observations for bovine aortic endothelial cells. These findings underscore the selective insulin uptake processes that take place in endothelial cells of different vascular beds, irrespective of whether they are of bovine or human origin.

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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Related in: MedlinePlus