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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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Dynamin-independent macropinocytosis regulates an early stage of amylin monomer internalization in RIN-m5F cells.Cells were treated with EIPA, CytD, Wort or Dyn for 1 hour followed by human amylin (10 µM) incubation for an additional 1 hour at 37°C. Dextran at 40µg/ml was then added for 30 minutes. Confocal microscopy (A) and whole cell analysis (B–C) revealed reduced internalization and increased PM accumulation of both monomers (B) and dextran (C) in the presence of the macropinocytotic inhibitors as compared to controls. **P<0.01, hA vs. hA/treatments and ##P<0.01, dextran vs. dextran/treatments, n = 9. On the contrary, there was no change in their cellular distributions when treated with Dyn. NS P>0.1, hA vs. hA/Dyn and NS P>0.1, dextran vs. Dextran/Dyn, n = 9. Significance established by ANOVA followed by Dunnett-Square test. Bar 5µm.
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pone-0073080-g003: Dynamin-independent macropinocytosis regulates an early stage of amylin monomer internalization in RIN-m5F cells.Cells were treated with EIPA, CytD, Wort or Dyn for 1 hour followed by human amylin (10 µM) incubation for an additional 1 hour at 37°C. Dextran at 40µg/ml was then added for 30 minutes. Confocal microscopy (A) and whole cell analysis (B–C) revealed reduced internalization and increased PM accumulation of both monomers (B) and dextran (C) in the presence of the macropinocytotic inhibitors as compared to controls. **P<0.01, hA vs. hA/treatments and ##P<0.01, dextran vs. dextran/treatments, n = 9. On the contrary, there was no change in their cellular distributions when treated with Dyn. NS P>0.1, hA vs. hA/Dyn and NS P>0.1, dextran vs. Dextran/Dyn, n = 9. Significance established by ANOVA followed by Dunnett-Square test. Bar 5µm.

Mentions: To further dissect the molecular mechanism of amylin monomer and oligomer internalization, confocal microscopy along with specific fluorescent endocytotic markers and pharmacological inhibitors were used. We first examined the mechanism that operates during an early phase (1 hour) of human amylin internalization (Figure 3–4, Figure S4–6). It was previously shown that the small and soluble oligomeric forms of brain derived β-amyloid peptide were avidly taken up by microglia cells through fluid phase-macropinocyotsis [47] which may also play a role in the initial uptake of human amylin in RIN-m5F cells. To test this hypothesis, cells were pre-treated with macropinocytotic inhibitors, EIPA, CytD or Wort, and amylin uptake examined. Under control conditions (no inhibitors), a sizable fraction (52±5%) of amylin monomers internalized in these cells (Figure 3), a portion of which trafficked to dextran-positive intracellular compartments in RIN-m5F cells as evident by their partial co-localization (R = 0.48±0.02, Figure S4A top panel, Figure S4B). This suggests a common macropinocytotic dependent internalization mechanism for dextran and amylin monomers. To further confirm that macropinocytosis is involved in the uptake of amylin monomers, macropinocytotic inhibitors were used (Figure 3). Whole cell analyses revealed that 52±5% of cell-associated amylin monomers (Figure 3A–B) and 50±4% dextran (Figure 3A, C) accumulated in these cells under control conditions, the rest being associated with PM. Inhibition of macropinocytosis by EIPA [45], [46], [48], markedly decreased the internalization of monomers to 10±3% and dextran to 10±4%, and in turn increased their PM accumulation to ≥90% (Figure 3A–C). Consequently, a significant decrease in intracellular co-localization between amylin monomers and dextran was observed (R = 0.07±0.01, Figure S4A top panel, Figure S4B). Macropinocytosis is known to be dependent on actin polymerization. The latter is required for PM ruffling and subsequent formation of macropinosomes [45]–[47].Consistent with fluid-phase uptake mechanism, an inhibitor of actin polymerization, cytD [4], [45]–[47] inhibited internalization of both monomers and dextran by ∼40% causing a comparable increase in their PM accumulation (Figure 3A–C). This treatment also prevented their intracellular co-localization as evident by a very low co-localization (R = 0.07±0.02, Figure S4A top panel, Figure S4B). Other than actin, phosphatidylinositide-3-kinase (PI 3-kinase) is required for macropinocytosis by directing the proper closure of membrane ruffles that leads to the formation of macropinosomes [48], [56], [66], [67]. Hence, cells were pre-incubated with a specific inhibitor of PI 3-kinase, Wort [48], [56], [66], [67] and its effect on amylin internalization examined. As with other macropinocytotic inhibitors, Wort significantly reduced internalization of both monomers and dextran, thereby stimulating their PM accumulation (Figure 3A–C). It has been widely debated whether dynamin is required for macropinocytosis. Both dynamin dependent and independent macropinocytosis were found to operate in cells [46]. To investigate the possible involvement of dynamin in amylin internalization, we used a general dynamin inhibitor, Dyn [46], [48]. Confocal microscopy revealed that Dyn failed to abrogate the internalization of both monomers and dextran (Figure 3A–C, Figure S5A–B) in RIN-m5F cells. However, Dyn effectively blocked internalization of Cholera Toxin (CTX) and Transferrin (Trf) (Figure S5–6), known endocytotic markers of dynamin-dependent pathways [45], [46], [48], [52], [54]. Taken together, our results (Figure 3, Figure S4–5) suggest that amylin monomers, when added at higher (10 µM) concentration, initially internalized in RIN-m5F cells by a dynamin-independent fluid phase-macropinocytotic pathway.


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

Trikha S, Jeremic AM - PLoS ONE (2013)

Dynamin-independent macropinocytosis regulates an early stage of amylin monomer internalization in RIN-m5F cells.Cells were treated with EIPA, CytD, Wort or Dyn for 1 hour followed by human amylin (10 µM) incubation for an additional 1 hour at 37°C. Dextran at 40µg/ml was then added for 30 minutes. Confocal microscopy (A) and whole cell analysis (B–C) revealed reduced internalization and increased PM accumulation of both monomers (B) and dextran (C) in the presence of the macropinocytotic inhibitors as compared to controls. **P<0.01, hA vs. hA/treatments and ##P<0.01, dextran vs. dextran/treatments, n = 9. On the contrary, there was no change in their cellular distributions when treated with Dyn. NS P>0.1, hA vs. hA/Dyn and NS P>0.1, dextran vs. Dextran/Dyn, n = 9. Significance established by ANOVA followed by Dunnett-Square test. Bar 5µm.
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pone-0073080-g003: Dynamin-independent macropinocytosis regulates an early stage of amylin monomer internalization in RIN-m5F cells.Cells were treated with EIPA, CytD, Wort or Dyn for 1 hour followed by human amylin (10 µM) incubation for an additional 1 hour at 37°C. Dextran at 40µg/ml was then added for 30 minutes. Confocal microscopy (A) and whole cell analysis (B–C) revealed reduced internalization and increased PM accumulation of both monomers (B) and dextran (C) in the presence of the macropinocytotic inhibitors as compared to controls. **P<0.01, hA vs. hA/treatments and ##P<0.01, dextran vs. dextran/treatments, n = 9. On the contrary, there was no change in their cellular distributions when treated with Dyn. NS P>0.1, hA vs. hA/Dyn and NS P>0.1, dextran vs. Dextran/Dyn, n = 9. Significance established by ANOVA followed by Dunnett-Square test. Bar 5µm.
Mentions: To further dissect the molecular mechanism of amylin monomer and oligomer internalization, confocal microscopy along with specific fluorescent endocytotic markers and pharmacological inhibitors were used. We first examined the mechanism that operates during an early phase (1 hour) of human amylin internalization (Figure 3–4, Figure S4–6). It was previously shown that the small and soluble oligomeric forms of brain derived β-amyloid peptide were avidly taken up by microglia cells through fluid phase-macropinocyotsis [47] which may also play a role in the initial uptake of human amylin in RIN-m5F cells. To test this hypothesis, cells were pre-treated with macropinocytotic inhibitors, EIPA, CytD or Wort, and amylin uptake examined. Under control conditions (no inhibitors), a sizable fraction (52±5%) of amylin monomers internalized in these cells (Figure 3), a portion of which trafficked to dextran-positive intracellular compartments in RIN-m5F cells as evident by their partial co-localization (R = 0.48±0.02, Figure S4A top panel, Figure S4B). This suggests a common macropinocytotic dependent internalization mechanism for dextran and amylin monomers. To further confirm that macropinocytosis is involved in the uptake of amylin monomers, macropinocytotic inhibitors were used (Figure 3). Whole cell analyses revealed that 52±5% of cell-associated amylin monomers (Figure 3A–B) and 50±4% dextran (Figure 3A, C) accumulated in these cells under control conditions, the rest being associated with PM. Inhibition of macropinocytosis by EIPA [45], [46], [48], markedly decreased the internalization of monomers to 10±3% and dextran to 10±4%, and in turn increased their PM accumulation to ≥90% (Figure 3A–C). Consequently, a significant decrease in intracellular co-localization between amylin monomers and dextran was observed (R = 0.07±0.01, Figure S4A top panel, Figure S4B). Macropinocytosis is known to be dependent on actin polymerization. The latter is required for PM ruffling and subsequent formation of macropinosomes [45]–[47].Consistent with fluid-phase uptake mechanism, an inhibitor of actin polymerization, cytD [4], [45]–[47] inhibited internalization of both monomers and dextran by ∼40% causing a comparable increase in their PM accumulation (Figure 3A–C). This treatment also prevented their intracellular co-localization as evident by a very low co-localization (R = 0.07±0.02, Figure S4A top panel, Figure S4B). Other than actin, phosphatidylinositide-3-kinase (PI 3-kinase) is required for macropinocytosis by directing the proper closure of membrane ruffles that leads to the formation of macropinosomes [48], [56], [66], [67]. Hence, cells were pre-incubated with a specific inhibitor of PI 3-kinase, Wort [48], [56], [66], [67] and its effect on amylin internalization examined. As with other macropinocytotic inhibitors, Wort significantly reduced internalization of both monomers and dextran, thereby stimulating their PM accumulation (Figure 3A–C). It has been widely debated whether dynamin is required for macropinocytosis. Both dynamin dependent and independent macropinocytosis were found to operate in cells [46]. To investigate the possible involvement of dynamin in amylin internalization, we used a general dynamin inhibitor, Dyn [46], [48]. Confocal microscopy revealed that Dyn failed to abrogate the internalization of both monomers and dextran (Figure 3A–C, Figure S5A–B) in RIN-m5F cells. However, Dyn effectively blocked internalization of Cholera Toxin (CTX) and Transferrin (Trf) (Figure S5–6), known endocytotic markers of dynamin-dependent pathways [45], [46], [48], [52], [54]. Taken together, our results (Figure 3, Figure S4–5) suggest that amylin monomers, when added at higher (10 µM) concentration, initially internalized in RIN-m5F cells by a dynamin-independent fluid phase-macropinocytotic pathway.

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