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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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Inhibition of macropinocytosis increases amylin oligomerization in human islets.Cells were first treated with macropinocytotic inhibitors (EIPA, CytD or Wort) for 1 hour followed by addition of human amylin (10 µM) either in the absence or presence of MB for 24 hours at 37°C. Dextran was finally added for an additional 30 minutes. (A) Immunoconfocal microscopy revealed a significant increase in the PM accumulation of both amylin oligomers (green), detected with A11 antibody and dextran (red) when treated with these inhibitors as compared to the controls (human amylin-treated cells) (left panel). However, in the presence of MB (100 µM), only dextran but not oligomer accumulation was detected on the PM (right panel). Bar 10µm. Extracellular content of amylin oligomers was analyzed by (B) western blot and (C) densitometry. Inhibition of macropinocytosis stimulated oligomer accumulation in the culturing medium, which was reversed by addition of MB. **P<0.01, hA vs. hA/inhibitors, n = 3, ANOVA followed by Dunnett-Square test and ##P<0.01, hA/inhibitors vs. hA/inhibitors/MB, n = 3, ANOVA followed by Newman-Keul post hoc test.
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pone-0073080-g011: Inhibition of macropinocytosis increases amylin oligomerization in human islets.Cells were first treated with macropinocytotic inhibitors (EIPA, CytD or Wort) for 1 hour followed by addition of human amylin (10 µM) either in the absence or presence of MB for 24 hours at 37°C. Dextran was finally added for an additional 30 minutes. (A) Immunoconfocal microscopy revealed a significant increase in the PM accumulation of both amylin oligomers (green), detected with A11 antibody and dextran (red) when treated with these inhibitors as compared to the controls (human amylin-treated cells) (left panel). However, in the presence of MB (100 µM), only dextran but not oligomer accumulation was detected on the PM (right panel). Bar 10µm. Extracellular content of amylin oligomers was analyzed by (B) western blot and (C) densitometry. Inhibition of macropinocytosis stimulated oligomer accumulation in the culturing medium, which was reversed by addition of MB. **P<0.01, hA vs. hA/inhibitors, n = 3, ANOVA followed by Dunnett-Square test and ##P<0.01, hA/inhibitors vs. hA/inhibitors/MB, n = 3, ANOVA followed by Newman-Keul post hoc test.

Mentions: Next, we sought to determine if macropinocytosis of human amylin is specific for RIN-m5F cells or it may also account for uptake of amylin monomers and oligomers in human pancreatic islet cells (Figure 10–11). Confocal microscopy revealed a significant accumulation (72±3%) of monomers on the PM of dissociated human islet cells following incubation with amylin (Figure 10A–B). Internalization of monomers in human islets remained unchanged in the presence of specific macropinocyotic inhibitors (Figure 10A–B). However, dextran internalization was significantly reduced to 9–12% by these inhibitors with a concomitant increase in its PM accumulation (Figure 10A, C). A11 positive-amylin oligomers entered islet cells by macropinocytosis as evident by a marked decrease in their internalization by EIPA, CytD or Wort (Figure 11A, left panel). The stimulatory effect of these macropinocytotic inhibitors on amylin oligomer accumulation on the PM was completely blocked by addition of MB (Figure 11A, right panel) while dextran turnover was unaffected by MB (Figure 11A, right panel). Western blot analyses followed by densitometry (Figure 11B–C) confirmed a 22–41% increase in accumulation of intermediate sized amylin oligomers in the culture medium of human islets treated with macropinocytotic inhibitors as compared to cells incubated with human amylin only. As for PM, MB completely abolished oligomer accumulation in the medium (Figure 11B–C).


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

Trikha S, Jeremic AM - PLoS ONE (2013)

Inhibition of macropinocytosis increases amylin oligomerization in human islets.Cells were first treated with macropinocytotic inhibitors (EIPA, CytD or Wort) for 1 hour followed by addition of human amylin (10 µM) either in the absence or presence of MB for 24 hours at 37°C. Dextran was finally added for an additional 30 minutes. (A) Immunoconfocal microscopy revealed a significant increase in the PM accumulation of both amylin oligomers (green), detected with A11 antibody and dextran (red) when treated with these inhibitors as compared to the controls (human amylin-treated cells) (left panel). However, in the presence of MB (100 µM), only dextran but not oligomer accumulation was detected on the PM (right panel). Bar 10µm. Extracellular content of amylin oligomers was analyzed by (B) western blot and (C) densitometry. Inhibition of macropinocytosis stimulated oligomer accumulation in the culturing medium, which was reversed by addition of MB. **P<0.01, hA vs. hA/inhibitors, n = 3, ANOVA followed by Dunnett-Square test and ##P<0.01, hA/inhibitors vs. hA/inhibitors/MB, n = 3, ANOVA followed by Newman-Keul post hoc test.
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pone-0073080-g011: Inhibition of macropinocytosis increases amylin oligomerization in human islets.Cells were first treated with macropinocytotic inhibitors (EIPA, CytD or Wort) for 1 hour followed by addition of human amylin (10 µM) either in the absence or presence of MB for 24 hours at 37°C. Dextran was finally added for an additional 30 minutes. (A) Immunoconfocal microscopy revealed a significant increase in the PM accumulation of both amylin oligomers (green), detected with A11 antibody and dextran (red) when treated with these inhibitors as compared to the controls (human amylin-treated cells) (left panel). However, in the presence of MB (100 µM), only dextran but not oligomer accumulation was detected on the PM (right panel). Bar 10µm. Extracellular content of amylin oligomers was analyzed by (B) western blot and (C) densitometry. Inhibition of macropinocytosis stimulated oligomer accumulation in the culturing medium, which was reversed by addition of MB. **P<0.01, hA vs. hA/inhibitors, n = 3, ANOVA followed by Dunnett-Square test and ##P<0.01, hA/inhibitors vs. hA/inhibitors/MB, n = 3, ANOVA followed by Newman-Keul post hoc test.
Mentions: Next, we sought to determine if macropinocytosis of human amylin is specific for RIN-m5F cells or it may also account for uptake of amylin monomers and oligomers in human pancreatic islet cells (Figure 10–11). Confocal microscopy revealed a significant accumulation (72±3%) of monomers on the PM of dissociated human islet cells following incubation with amylin (Figure 10A–B). Internalization of monomers in human islets remained unchanged in the presence of specific macropinocyotic inhibitors (Figure 10A–B). However, dextran internalization was significantly reduced to 9–12% by these inhibitors with a concomitant increase in its PM accumulation (Figure 10A, C). A11 positive-amylin oligomers entered islet cells by macropinocytosis as evident by a marked decrease in their internalization by EIPA, CytD or Wort (Figure 11A, left panel). The stimulatory effect of these macropinocytotic inhibitors on amylin oligomer accumulation on the PM was completely blocked by addition of MB (Figure 11A, right panel) while dextran turnover was unaffected by MB (Figure 11A, right panel). Western blot analyses followed by densitometry (Figure 11B–C) confirmed a 22–41% increase in accumulation of intermediate sized amylin oligomers in the culture medium of human islets treated with macropinocytotic inhibitors as compared to cells incubated with human amylin only. As for PM, MB completely abolished oligomer accumulation in the medium (Figure 11B–C).

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