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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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Methylene blue has no inhibitory effect on amylin monomers and dextran in RIN-m5F cells.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 in the presence of MB (100 µM) for 24 hours at 37°C. Dextran was added for an additional 30 minutes. Confocal microscopy revealed no significant change in the cellular distributions of amylin monomers (green) when treated with macropinocytotic inhibitors with or without MB (left and right panel). Also, in the presence of MB, dextran (red) turnover was unaffected (right panel). Bar 10µm.
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pone-0073080-g009: Methylene blue has no inhibitory effect on amylin monomers and dextran in RIN-m5F cells.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 in the presence of MB (100 µM) for 24 hours at 37°C. Dextran was added for an additional 30 minutes. Confocal microscopy revealed no significant change in the cellular distributions of amylin monomers (green) when treated with macropinocytotic inhibitors with or without MB (left and right panel). Also, in the presence of MB, dextran (red) turnover was unaffected (right panel). Bar 10µm.

Mentions: Studies show that oligomerization of human amylin is a rapid process, which can be further accelerated by its interaction with lipids in the membrane [4], [23]. Given that macropinocytosis regulates the internalization of both molecular forms of amylin during an early phase, it is unclear whether inhibition of monomer or oligomer uptake or both contributes to the extracellular amylin oligomer accumulation at later times (24 hours). It is quite possible that inhibition of amylin monomer internalization could lead to oligomerization and extracellular accumulation. Hence, the stimulatory effect of macropinocytotic inhibitors on amylin oligomer accumulation on the PM of RIN-m5F cells was tested in the absence and presence of the potent and specific amyloid oligomer inhibitor, methylene blue (MB) [4], [74]. Confocal microscopy revealed that MB specifically blocked the formation and deposition of amylin oligomers but not dextran on the PM of these cells (Figure 8A). Similar to dextran, the binding and internalization of amylin monomers were not affected by MB either in the absence or the presence of macropinocytotic inhibitors (Figure 9A). This finding indicates that inhibition of amylin oligomer (Figure 7, 8A) but not monomer (Figure 5, 9A) internalization by these macropinocytotic inhibitors accounts for an increased extracellular oligomer accumulation at later times (24 hours).


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

Trikha S, Jeremic AM - PLoS ONE (2013)

Methylene blue has no inhibitory effect on amylin monomers and dextran in RIN-m5F cells.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 in the presence of MB (100 µM) for 24 hours at 37°C. Dextran was added for an additional 30 minutes. Confocal microscopy revealed no significant change in the cellular distributions of amylin monomers (green) when treated with macropinocytotic inhibitors with or without MB (left and right panel). Also, in the presence of MB, dextran (red) turnover was unaffected (right panel). Bar 10µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073080-g009: Methylene blue has no inhibitory effect on amylin monomers and dextran in RIN-m5F cells.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 in the presence of MB (100 µM) for 24 hours at 37°C. Dextran was added for an additional 30 minutes. Confocal microscopy revealed no significant change in the cellular distributions of amylin monomers (green) when treated with macropinocytotic inhibitors with or without MB (left and right panel). Also, in the presence of MB, dextran (red) turnover was unaffected (right panel). Bar 10µm.
Mentions: Studies show that oligomerization of human amylin is a rapid process, which can be further accelerated by its interaction with lipids in the membrane [4], [23]. Given that macropinocytosis regulates the internalization of both molecular forms of amylin during an early phase, it is unclear whether inhibition of monomer or oligomer uptake or both contributes to the extracellular amylin oligomer accumulation at later times (24 hours). It is quite possible that inhibition of amylin monomer internalization could lead to oligomerization and extracellular accumulation. Hence, the stimulatory effect of macropinocytotic inhibitors on amylin oligomer accumulation on the PM of RIN-m5F cells was tested in the absence and presence of the potent and specific amyloid oligomer inhibitor, methylene blue (MB) [4], [74]. Confocal microscopy revealed that MB specifically blocked the formation and deposition of amylin oligomers but not dextran on the PM of these cells (Figure 8A). Similar to dextran, the binding and internalization of amylin monomers were not affected by MB either in the absence or the presence of macropinocytotic inhibitors (Figure 9A). This finding indicates that inhibition of amylin oligomer (Figure 7, 8A) but not monomer (Figure 5, 9A) internalization by these macropinocytotic inhibitors accounts for an increased extracellular oligomer accumulation at later times (24 hours).

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