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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 in microvascular endothelium is retained in a transferrin-positive compartment. (A) Insulin-FITC (green) colocalizes moderately with transferrin (red) at early and late time points in HAMECs. Dashed box indicates area enlarged on the right; white scale, 15 μm. (B) Insulin-FITC colocalization with transferrin-AF555 decreases over time in L6 myoblasts. Dashed box indicates area enlarged on the right; white scale, 15 μm. (C) Quantification of insulin colocalizing with transferrin over time using the Manders coefficient. *p < 0.05, ***p < 0.001 compared with initial time point.
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Figure 3: Insulin in microvascular endothelium is retained in a transferrin-positive compartment. (A) Insulin-FITC (green) colocalizes moderately with transferrin (red) at early and late time points in HAMECs. Dashed box indicates area enlarged on the right; white scale, 15 μm. (B) Insulin-FITC colocalization with transferrin-AF555 decreases over time in L6 myoblasts. Dashed box indicates area enlarged on the right; white scale, 15 μm. (C) Quantification of insulin colocalizing with transferrin over time using the Manders coefficient. *p < 0.05, ***p < 0.001 compared with initial time point.

Mentions: The permanence of a large fraction of internalized insulin within HAMECs and its contrasting loss within myoblasts is in keeping with the physiological handling of the hormone in the corresponding tissues in vivo. Indeed, circulating insulin should be transported intact across the microvascular endothelium to access its target tissues (e.g., fat, muscle) in order to initiate signaling, where it is eventually degraded through the combined action of insulin-degrading enzyme and muscle/fat lysosomal hydrolysis (Hammons and Jarett, 1980; Duckworth et al., 1998). Accordingly, we examined whether internalized insulin is routed differentially inside microvascular and muscle cells. Fluorescein isothiocyanate (FITC)–conjugated insulin (insulin-FITC) internalized by myoblasts accumulated progressively in lysosomes, as shown by the colocalization of the FITC signal with that of LysoTracker, an acidotropic probe that concentrates in lysosomes (Bucci et al., 2000; Figure 2, B and C). In contrast, there was little colocalization of internalized insulin-FITC with lysosomes in HAMECs for the duration of the analysis (Figure 2, A and C). A significant fraction of the insulin-FITC internalized by HAMECs colocalized with transferrin, suggesting their joint retention in early or recycling endosomes (Figure 3). Insulin-FITC retained its bioactivity, as determined by the activation of Akt assayed in muscle cells (Supplemental Figure S3).


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 in microvascular endothelium is retained in a transferrin-positive compartment. (A) Insulin-FITC (green) colocalizes moderately with transferrin (red) at early and late time points in HAMECs. Dashed box indicates area enlarged on the right; white scale, 15 μm. (B) Insulin-FITC colocalization with transferrin-AF555 decreases over time in L6 myoblasts. Dashed box indicates area enlarged on the right; white scale, 15 μm. (C) Quantification of insulin colocalizing with transferrin over time using the Manders coefficient. *p < 0.05, ***p < 0.001 compared with initial time point.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: Insulin in microvascular endothelium is retained in a transferrin-positive compartment. (A) Insulin-FITC (green) colocalizes moderately with transferrin (red) at early and late time points in HAMECs. Dashed box indicates area enlarged on the right; white scale, 15 μm. (B) Insulin-FITC colocalization with transferrin-AF555 decreases over time in L6 myoblasts. Dashed box indicates area enlarged on the right; white scale, 15 μm. (C) Quantification of insulin colocalizing with transferrin over time using the Manders coefficient. *p < 0.05, ***p < 0.001 compared with initial time point.
Mentions: The permanence of a large fraction of internalized insulin within HAMECs and its contrasting loss within myoblasts is in keeping with the physiological handling of the hormone in the corresponding tissues in vivo. Indeed, circulating insulin should be transported intact across the microvascular endothelium to access its target tissues (e.g., fat, muscle) in order to initiate signaling, where it is eventually degraded through the combined action of insulin-degrading enzyme and muscle/fat lysosomal hydrolysis (Hammons and Jarett, 1980; Duckworth et al., 1998). Accordingly, we examined whether internalized insulin is routed differentially inside microvascular and muscle cells. Fluorescein isothiocyanate (FITC)–conjugated insulin (insulin-FITC) internalized by myoblasts accumulated progressively in lysosomes, as shown by the colocalization of the FITC signal with that of LysoTracker, an acidotropic probe that concentrates in lysosomes (Bucci et al., 2000; Figure 2, B and C). In contrast, there was little colocalization of internalized insulin-FITC with lysosomes in HAMECs for the duration of the analysis (Figure 2, A and C). A significant fraction of the insulin-FITC internalized by HAMECs colocalized with transferrin, suggesting their joint retention in early or recycling endosomes (Figure 3). Insulin-FITC retained its bioactivity, as determined by the activation of Akt assayed in muscle cells (Supplemental Figure S3).

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