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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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Late phase of amylin monomer internalization in RIN-m5F cells is independent of macropinocytosis.Cells were either treated with EIPA, CytD and Wort for 1 hour or transfected with the dynamin mutant construct, DN dyn1K44A for 16–18 hours followed by human amylin incubation at either 100 nM or 10 µM for an additional 24 hours at 37°C. Dextran was sequentially added after incubating cells with 10 µM human amylin. Confocal microscopy (A) and whole cell analysis (B–D) showed no change in cellular distributions of monomers at 100 nM (B) or 10 µM (C) in the presence of EIPA, CytD, and Wort or DN dyn1K44A when compared to controls. NS P>0.1, hA vs. hA/treatments, n = 9. However, dextran (D) internalization was significantly reduced when treated with macropinocytotic inhibitors but not with DN dyn1K44A. ##P<0.01, dextran vs. dextran/inhibitors 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-g005: Late phase of amylin monomer internalization in RIN-m5F cells is independent of macropinocytosis.Cells were either treated with EIPA, CytD and Wort for 1 hour or transfected with the dynamin mutant construct, DN dyn1K44A for 16–18 hours followed by human amylin incubation at either 100 nM or 10 µM for an additional 24 hours at 37°C. Dextran was sequentially added after incubating cells with 10 µM human amylin. Confocal microscopy (A) and whole cell analysis (B–D) showed no change in cellular distributions of monomers at 100 nM (B) or 10 µM (C) in the presence of EIPA, CytD, and Wort or DN dyn1K44A when compared to controls. NS P>0.1, hA vs. hA/treatments, n = 9. However, dextran (D) internalization was significantly reduced when treated with macropinocytotic inhibitors but not with DN dyn1K44A. ##P<0.01, dextran vs. dextran/inhibitors and NS, P>0.1, dextran vs. dextran/dyn1K44A, n = 9. Significance established by ANOVA followed by Dunnett-Square test. Bar 5µm.

Mentions: We further investigated if macropinocytosis also plays a role in amylin internalization at later times (24 hours). The cells were pre-incubated with EIPA, CytD or Wort for 1 hour and then incubated with low (100 nM) or high (10 µM) amylin for additional 24 hours (Figure 5). This procedure minimizes the toxic effects of these inhibitors, which may interfere with amylin uptake. Following the treatments, dextran was added to the cells. Under control conditions, 55±4% of the cell-associated amylin monomers accumulated inside the cells when incubated with the 100 nM amylin concentration (Figure 5A, B), whereas 62±5% of monomers internalized when challenged with high (10 µM) amylin concentration (Figure 5A, C). This result indicates a saturable uptake mechanism for human amylin, not a characteristic of fluid phase endocytosis [47]. Furthermore, the macropinocytotic inhibitors had no modulatory effect on amylin monomer internalization or PM accumulation respective to controls at 24 hours (Figure 5A–C), while dextran internalization was significantly inhibited by EIPA, CytD and Wort (Figure 5A, D). A very low co-localization value (R = 0.05±0.01) was obtained between amylin monomers and dextran either in the absence or in the presence of these inhibitors (Figure S7A top panel, Figure S7B). Thus, amylin monomers and dextran follow distinct internalization pathways.


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

Trikha S, Jeremic AM - PLoS ONE (2013)

Late phase of amylin monomer internalization in RIN-m5F cells is independent of macropinocytosis.Cells were either treated with EIPA, CytD and Wort for 1 hour or transfected with the dynamin mutant construct, DN dyn1K44A for 16–18 hours followed by human amylin incubation at either 100 nM or 10 µM for an additional 24 hours at 37°C. Dextran was sequentially added after incubating cells with 10 µM human amylin. Confocal microscopy (A) and whole cell analysis (B–D) showed no change in cellular distributions of monomers at 100 nM (B) or 10 µM (C) in the presence of EIPA, CytD, and Wort or DN dyn1K44A when compared to controls. NS P>0.1, hA vs. hA/treatments, n = 9. However, dextran (D) internalization was significantly reduced when treated with macropinocytotic inhibitors but not with DN dyn1K44A. ##P<0.01, dextran vs. dextran/inhibitors 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-g005: Late phase of amylin monomer internalization in RIN-m5F cells is independent of macropinocytosis.Cells were either treated with EIPA, CytD and Wort for 1 hour or transfected with the dynamin mutant construct, DN dyn1K44A for 16–18 hours followed by human amylin incubation at either 100 nM or 10 µM for an additional 24 hours at 37°C. Dextran was sequentially added after incubating cells with 10 µM human amylin. Confocal microscopy (A) and whole cell analysis (B–D) showed no change in cellular distributions of monomers at 100 nM (B) or 10 µM (C) in the presence of EIPA, CytD, and Wort or DN dyn1K44A when compared to controls. NS P>0.1, hA vs. hA/treatments, n = 9. However, dextran (D) internalization was significantly reduced when treated with macropinocytotic inhibitors but not with DN dyn1K44A. ##P<0.01, dextran vs. dextran/inhibitors and NS, P>0.1, dextran vs. dextran/dyn1K44A, n = 9. Significance established by ANOVA followed by Dunnett-Square test. Bar 5µm.
Mentions: We further investigated if macropinocytosis also plays a role in amylin internalization at later times (24 hours). The cells were pre-incubated with EIPA, CytD or Wort for 1 hour and then incubated with low (100 nM) or high (10 µM) amylin for additional 24 hours (Figure 5). This procedure minimizes the toxic effects of these inhibitors, which may interfere with amylin uptake. Following the treatments, dextran was added to the cells. Under control conditions, 55±4% of the cell-associated amylin monomers accumulated inside the cells when incubated with the 100 nM amylin concentration (Figure 5A, B), whereas 62±5% of monomers internalized when challenged with high (10 µM) amylin concentration (Figure 5A, C). This result indicates a saturable uptake mechanism for human amylin, not a characteristic of fluid phase endocytosis [47]. Furthermore, the macropinocytotic inhibitors had no modulatory effect on amylin monomer internalization or PM accumulation respective to controls at 24 hours (Figure 5A–C), while dextran internalization was significantly inhibited by EIPA, CytD and Wort (Figure 5A, D). A very low co-localization value (R = 0.05±0.01) was obtained between amylin monomers and dextran either in the absence or in the presence of these inhibitors (Figure S7A top panel, Figure S7B). Thus, amylin monomers and dextran follow distinct internalization pathways.

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