Limits...
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.

Show MeSH

Related in: MedlinePlus

The late phase of amylin monomer internalization does not require macropinocytosis in human islets.Cells were treated with EIPA, CytD or Wort for 1 hour 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) revealed no change in cellular distributions of amylin monomers (green) (B), detected with human amylin antibody in the presence of EIPA, CytD or Wort when compared to controls. Dextran (red) internalization (C) was however significantly reduced with these macropinocytotic inhibitors. NS P>0.1, hA vs. hA/inhibitors, ##P<0.01, dextran vs. dextran/inhibitors, n = 9. Significance established by ANOVA followed by Dunnett-Square test. Bar 10µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3760900&req=5

pone-0073080-g010: The late phase of amylin monomer internalization does not require macropinocytosis in human islets.Cells were treated with EIPA, CytD or Wort for 1 hour 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) revealed no change in cellular distributions of amylin monomers (green) (B), detected with human amylin antibody in the presence of EIPA, CytD or Wort when compared to controls. Dextran (red) internalization (C) was however significantly reduced with these macropinocytotic inhibitors. NS P>0.1, hA vs. hA/inhibitors, ##P<0.01, dextran vs. dextran/inhibitors, n = 9. Significance established by ANOVA followed by Dunnett-Square test. Bar 10µm.

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)

The late phase of amylin monomer internalization does not require macropinocytosis in human islets.Cells were treated with EIPA, CytD or Wort for 1 hour 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) revealed no change in cellular distributions of amylin monomers (green) (B), detected with human amylin antibody in the presence of EIPA, CytD or Wort when compared to controls. Dextran (red) internalization (C) was however significantly reduced with these macropinocytotic inhibitors. NS P>0.1, hA vs. hA/inhibitors, ##P<0.01, dextran vs. dextran/inhibitors, n = 9. Significance established by ANOVA followed by Dunnett-Square test. Bar 10µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760900&req=5

pone-0073080-g010: The late phase of amylin monomer internalization does not require macropinocytosis in human islets.Cells were treated with EIPA, CytD or Wort for 1 hour 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) revealed no change in cellular distributions of amylin monomers (green) (B), detected with human amylin antibody in the presence of EIPA, CytD or Wort when compared to controls. Dextran (red) internalization (C) was however significantly reduced with these macropinocytotic inhibitors. NS P>0.1, hA vs. hA/inhibitors, ##P<0.01, dextran vs. dextran/inhibitors, n = 9. Significance established by ANOVA followed by Dunnett-Square test. Bar 10µm.
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