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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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Time lapse analyses of extracellular amylin monomers in RIN-m5F cells.(A) Human amylin (10 µM) was added to the cell culture media without (w/o) or containing cells for 24 hours. 50 µl of medium was collected at regular time intervals and amylin resolved on a Tris-Tricine gel. Amylin content was then analyzed by western blot using human amylin antibody (hA Ab). (B) Real time changes in extracellular amylin concentrations were analyzed by human amylin-specific ELISA. Upon treatments with macropinocytotic inhibitors (EIPA, CytD or Wort), western blot (C) and ELISA (D) analyses revealed no significant change in the extracellular human amylin content when compared to only human amylin-treated cells at 24 hours. NS, P>0.1, hA vs. hA/inhibitors, n = 5. Significance established by ANOVA followed by Dunnett-Square test.
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pone-0073080-g006: Time lapse analyses of extracellular amylin monomers in RIN-m5F cells.(A) Human amylin (10 µM) was added to the cell culture media without (w/o) or containing cells for 24 hours. 50 µl of medium was collected at regular time intervals and amylin resolved on a Tris-Tricine gel. Amylin content was then analyzed by western blot using human amylin antibody (hA Ab). (B) Real time changes in extracellular amylin concentrations were analyzed by human amylin-specific ELISA. Upon treatments with macropinocytotic inhibitors (EIPA, CytD or Wort), western blot (C) and ELISA (D) analyses revealed no significant change in the extracellular human amylin content when compared to only human amylin-treated cells at 24 hours. NS, P>0.1, hA vs. hA/inhibitors, n = 5. Significance established by ANOVA followed by Dunnett-Square test.

Mentions: The time lapse western blot analyses of extracellular amylin monomers revealed that the majority gradually disappeared from the cell culturing medium within the first 7 hours, with a complete loss of detectable signal at 24 hours (Figure S8). In contrast, amylin monomer levels in cell-free medium did not drop until very late (Figure 6A, left panel, graph). This delayed disappearance of amylin monomers from cell-free medium can be attributed to the peptide’s slow aggregation process described previously [69]. Addition of cells accelerated amylin monomer clearance from the medium, such that at 12 h, ∼5% of the monomers remained in the medium containing cells versus >80% in a cell-free medium (Figure 6A, left panel, graph). In the absence of cells, amylin monomer disappearance could be only due to amylin oligomerization/aggregation and would be independent of cellular uptake/degradation. The marked difference observed in the kinetics of monomer clearance from extracellular medium as compared to cell free medium strongly indicates that endocytosis and/or other cellular process but not aggregation is the major clearance mechanism for extracellular amylin, although peptide polymerization may also be responsible to lesser extent. Using ELISA, we observed that the concentration of human amylin dropped from 8±0.4 µM to 0.24±0.08 µM (Figure 6B) over a period of 24 hours. The macropinocytotic inhibitors had no effect on the clearance of amylin monomers from the culture media measured at 24 hours by western blot (Figure 6C). This finding was further confirmed with ELISA (Figure 6D) showing no significant change in the extra-cellular content of human amylin-treated cells either in the absence or presence of macropinocytotic inhibitors. Therefore, it is likely, that this late phase of amylin internalization is via clathrin and dynamin-dependent endocytotic pathways.


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

Trikha S, Jeremic AM - PLoS ONE (2013)

Time lapse analyses of extracellular amylin monomers in RIN-m5F cells.(A) Human amylin (10 µM) was added to the cell culture media without (w/o) or containing cells for 24 hours. 50 µl of medium was collected at regular time intervals and amylin resolved on a Tris-Tricine gel. Amylin content was then analyzed by western blot using human amylin antibody (hA Ab). (B) Real time changes in extracellular amylin concentrations were analyzed by human amylin-specific ELISA. Upon treatments with macropinocytotic inhibitors (EIPA, CytD or Wort), western blot (C) and ELISA (D) analyses revealed no significant change in the extracellular human amylin content when compared to only human amylin-treated cells at 24 hours. NS, P>0.1, hA vs. hA/inhibitors, n = 5. Significance established by ANOVA followed by Dunnett-Square test.
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Related In: Results  -  Collection

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pone-0073080-g006: Time lapse analyses of extracellular amylin monomers in RIN-m5F cells.(A) Human amylin (10 µM) was added to the cell culture media without (w/o) or containing cells for 24 hours. 50 µl of medium was collected at regular time intervals and amylin resolved on a Tris-Tricine gel. Amylin content was then analyzed by western blot using human amylin antibody (hA Ab). (B) Real time changes in extracellular amylin concentrations were analyzed by human amylin-specific ELISA. Upon treatments with macropinocytotic inhibitors (EIPA, CytD or Wort), western blot (C) and ELISA (D) analyses revealed no significant change in the extracellular human amylin content when compared to only human amylin-treated cells at 24 hours. NS, P>0.1, hA vs. hA/inhibitors, n = 5. Significance established by ANOVA followed by Dunnett-Square test.
Mentions: The time lapse western blot analyses of extracellular amylin monomers revealed that the majority gradually disappeared from the cell culturing medium within the first 7 hours, with a complete loss of detectable signal at 24 hours (Figure S8). In contrast, amylin monomer levels in cell-free medium did not drop until very late (Figure 6A, left panel, graph). This delayed disappearance of amylin monomers from cell-free medium can be attributed to the peptide’s slow aggregation process described previously [69]. Addition of cells accelerated amylin monomer clearance from the medium, such that at 12 h, ∼5% of the monomers remained in the medium containing cells versus >80% in a cell-free medium (Figure 6A, left panel, graph). In the absence of cells, amylin monomer disappearance could be only due to amylin oligomerization/aggregation and would be independent of cellular uptake/degradation. The marked difference observed in the kinetics of monomer clearance from extracellular medium as compared to cell free medium strongly indicates that endocytosis and/or other cellular process but not aggregation is the major clearance mechanism for extracellular amylin, although peptide polymerization may also be responsible to lesser extent. Using ELISA, we observed that the concentration of human amylin dropped from 8±0.4 µM to 0.24±0.08 µM (Figure 6B) over a period of 24 hours. The macropinocytotic inhibitors had no effect on the clearance of amylin monomers from the culture media measured at 24 hours by western blot (Figure 6C). This finding was further confirmed with ELISA (Figure 6D) showing no significant change in the extra-cellular content of human amylin-treated cells either in the absence or presence of macropinocytotic inhibitors. Therefore, it is likely, that this late phase of amylin internalization is via clathrin and dynamin-dependent endocytotic 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