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Distinct internalization pathways of human amylin monomers and its cytotoxic oligomers in pancreatic cells.

Trikha S, Jeremic AM - PLoS ONE (2013)

Bottom Line: In contrast a majority of the oligomers at both early (1 hour) and late times (24 hours) traffic with a fluid-phase marker, dextran, to the same endocytotic compartments, the uptake of which is blocked by potent macropinocytotic inhibitors.This led to a significant increase in extra-cellular PM accumulation, in turn potentiating amylin toxicity in pancreatic cells.Our studies suggest that macropinocytosis is a major but not the only clearance mechanism for both amylin's molecular forms, thereby serving a cyto-protective role in these cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, The George Washington University, Washington, District of Columbia, United States of America.

ABSTRACT
Toxic human amylin oligomers and aggregates are implicated in the pathogenesis of type 2 diabetes mellitus (TTDM). Although recent studies have shown that pancreatic cells can recycle amylin monomers and toxic oligomers, the exact uptake mechanism and trafficking routes of these molecular forms and their significance for amylin toxicity are yet to be determined. Using pancreatic rat insulinoma (RIN-m5F) beta (β)-cells and human islets as model systems we show that monomers and oligomers cross the plasma membrane (PM) through both endocytotic and non-endocytotic (translocation) mechanisms, the predominance of which is dependent on amylin concentrations and incubation times. At low (≤ 100 nM) concentrations, internalization of amylin monomers in pancreatic cells is completely blocked by the selective amylin-receptor (AM-R) antagonist, AC-187, indicating an AM-R dependent mechanism. In contrast at cytotoxic (µM) concentrations monomers initially (1 hour) enter pancreatic cells by two distinct mechanisms: translocation and macropinocytosis. However, during the late stage (24 hours) monomers internalize by a clathrin-dependent but AM-R and macropinocytotic independent pathway. Like monomers a small fraction of the oligomers initially enter cells by a non-endocytotic mechanism. In contrast a majority of the oligomers at both early (1 hour) and late times (24 hours) traffic with a fluid-phase marker, dextran, to the same endocytotic compartments, the uptake of which is blocked by potent macropinocytotic inhibitors. This led to a significant increase in extra-cellular PM accumulation, in turn potentiating amylin toxicity in pancreatic cells. Our studies suggest that macropinocytosis is a major but not the only clearance mechanism for both amylin's molecular forms, thereby serving a cyto-protective role in these cells.

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Macropinocytosis is involved in late phase of amylin oligomer internalization in RIN-m5F cells.Cells were either treated with EIPA, CytD and Wort for 1 hour or transfected with dynamin mutant construct, DN dyn1K44A for 16–18 hours followed by human amylin (10 µM) incubation for an additional 24 hours at 37°C. Dextran was finally added for 30 minutes. Confocal microscopy (A) and whole cell analysis (B–C) demonstrated a significant reduction in internalization and increase in PM accumulation of oligomers (B) and dextran (C) in the presence of macropinocytotic inhibitors. **P<0.01, hA vs. hA/inhibitors and ##P<0.01, dextran vs. dextran/inhibitors, n = 9. In contrast, expression of DN dyn1K44A in these cells failed to prevent entry of both oligomers and dextran. NS P>0.1, hA vs. hA/dyn1K44A and NS P>0.1, dextran vs. dextran/dyn1K44A, n = 9. Significance established by ANOVA followed by Dunnett-Square test. Bar 5µm.
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pone-0073080-g007: Macropinocytosis is involved in late phase of amylin oligomer internalization in RIN-m5F cells.Cells were either treated with EIPA, CytD and Wort for 1 hour or transfected with dynamin mutant construct, DN dyn1K44A for 16–18 hours followed by human amylin (10 µM) incubation for an additional 24 hours at 37°C. Dextran was finally added for 30 minutes. Confocal microscopy (A) and whole cell analysis (B–C) demonstrated a significant reduction in internalization and increase in PM accumulation of oligomers (B) and dextran (C) in the presence of macropinocytotic inhibitors. **P<0.01, hA vs. hA/inhibitors and ##P<0.01, dextran vs. dextran/inhibitors, n = 9. In contrast, expression of DN dyn1K44A in these cells failed to prevent entry of both oligomers and dextran. NS P>0.1, hA vs. hA/dyn1K44A and NS P>0.1, dextran vs. dextran/dyn1K44A, n = 9. Significance established by ANOVA followed by Dunnett-Square test. Bar 5µm.

Mentions: Following the transfections with DN AP180CFLAG or wild type construct, we observed that the amount of oligomers internalized was similar between controls, wt-AP180- and DN AP180CFLAG-transfected cells (Figure S10A–B), indicating that clathrin was dispensable for oligomer uptake in these cells. Like monomers (Figure S9A–D), oligomer internalization was significantly reduced at low temperatures (≤4°C) (Figure S10A–B), suggesting that amylin oligomers follow endocytosis. A rather minor fraction of oligomers (9±3%) underwent translocation across the PM at this late stage (Figure S10A–B). Internalization of both amylin oligomers (Figure 7A–B, Figure S11A–B) and dextran (Figure 7A, C) was also unaffected by down regulation of dynamin endocytotic function using DN Dyn1K44A. However, the same construct efficiently inhibited CTX and Trf uptakes in RIN-m5F cells (Figure S11A, C and D).


Distinct internalization pathways of human amylin monomers and its cytotoxic oligomers in pancreatic cells.

Trikha S, Jeremic AM - PLoS ONE (2013)

Macropinocytosis is involved in late phase of amylin oligomer internalization in RIN-m5F cells.Cells were either treated with EIPA, CytD and Wort for 1 hour or transfected with dynamin mutant construct, DN dyn1K44A for 16–18 hours followed by human amylin (10 µM) incubation for an additional 24 hours at 37°C. Dextran was finally added for 30 minutes. Confocal microscopy (A) and whole cell analysis (B–C) demonstrated a significant reduction in internalization and increase in PM accumulation of oligomers (B) and dextran (C) in the presence of macropinocytotic inhibitors. **P<0.01, hA vs. hA/inhibitors and ##P<0.01, dextran vs. dextran/inhibitors, n = 9. In contrast, expression of DN dyn1K44A in these cells failed to prevent entry of both oligomers and dextran. NS P>0.1, hA vs. hA/dyn1K44A and NS P>0.1, dextran vs. dextran/dyn1K44A, n = 9. Significance established by ANOVA followed by Dunnett-Square test. Bar 5µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760900&req=5

pone-0073080-g007: Macropinocytosis is involved in late phase of amylin oligomer internalization in RIN-m5F cells.Cells were either treated with EIPA, CytD and Wort for 1 hour or transfected with dynamin mutant construct, DN dyn1K44A for 16–18 hours followed by human amylin (10 µM) incubation for an additional 24 hours at 37°C. Dextran was finally added for 30 minutes. Confocal microscopy (A) and whole cell analysis (B–C) demonstrated a significant reduction in internalization and increase in PM accumulation of oligomers (B) and dextran (C) in the presence of macropinocytotic inhibitors. **P<0.01, hA vs. hA/inhibitors and ##P<0.01, dextran vs. dextran/inhibitors, n = 9. In contrast, expression of DN dyn1K44A in these cells failed to prevent entry of both oligomers and dextran. NS P>0.1, hA vs. hA/dyn1K44A and NS P>0.1, dextran vs. dextran/dyn1K44A, n = 9. Significance established by ANOVA followed by Dunnett-Square test. Bar 5µm.
Mentions: Following the transfections with DN AP180CFLAG or wild type construct, we observed that the amount of oligomers internalized was similar between controls, wt-AP180- and DN AP180CFLAG-transfected cells (Figure S10A–B), indicating that clathrin was dispensable for oligomer uptake in these cells. Like monomers (Figure S9A–D), oligomer internalization was significantly reduced at low temperatures (≤4°C) (Figure S10A–B), suggesting that amylin oligomers follow endocytosis. A rather minor fraction of oligomers (9±3%) underwent translocation across the PM at this late stage (Figure S10A–B). Internalization of both amylin oligomers (Figure 7A–B, Figure S11A–B) and dextran (Figure 7A, C) was also unaffected by down regulation of dynamin endocytotic function using DN Dyn1K44A. However, the same construct efficiently inhibited CTX and Trf uptakes in RIN-m5F cells (Figure S11A, C and D).

Bottom Line: In contrast a majority of the oligomers at both early (1 hour) and late times (24 hours) traffic with a fluid-phase marker, dextran, to the same endocytotic compartments, the uptake of which is blocked by potent macropinocytotic inhibitors.This led to a significant increase in extra-cellular PM accumulation, in turn potentiating amylin toxicity in pancreatic cells.Our studies suggest that macropinocytosis is a major but not the only clearance mechanism for both amylin's molecular forms, thereby serving a cyto-protective role in these cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, The George Washington University, Washington, District of Columbia, United States of America.

ABSTRACT
Toxic human amylin oligomers and aggregates are implicated in the pathogenesis of type 2 diabetes mellitus (TTDM). Although recent studies have shown that pancreatic cells can recycle amylin monomers and toxic oligomers, the exact uptake mechanism and trafficking routes of these molecular forms and their significance for amylin toxicity are yet to be determined. Using pancreatic rat insulinoma (RIN-m5F) beta (β)-cells and human islets as model systems we show that monomers and oligomers cross the plasma membrane (PM) through both endocytotic and non-endocytotic (translocation) mechanisms, the predominance of which is dependent on amylin concentrations and incubation times. At low (≤ 100 nM) concentrations, internalization of amylin monomers in pancreatic cells is completely blocked by the selective amylin-receptor (AM-R) antagonist, AC-187, indicating an AM-R dependent mechanism. In contrast at cytotoxic (µM) concentrations monomers initially (1 hour) enter pancreatic cells by two distinct mechanisms: translocation and macropinocytosis. However, during the late stage (24 hours) monomers internalize by a clathrin-dependent but AM-R and macropinocytotic independent pathway. Like monomers a small fraction of the oligomers initially enter cells by a non-endocytotic mechanism. In contrast a majority of the oligomers at both early (1 hour) and late times (24 hours) traffic with a fluid-phase marker, dextran, to the same endocytotic compartments, the uptake of which is blocked by potent macropinocytotic inhibitors. This led to a significant increase in extra-cellular PM accumulation, in turn potentiating amylin toxicity in pancreatic cells. Our studies suggest that macropinocytosis is a major but not the only clearance mechanism for both amylin's molecular forms, thereby serving a cyto-protective role in these cells.

Show MeSH
Related in: MedlinePlus