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

Inhibition of macropinocytosis potentiates amylin toxicity in human islets.The effects on cell viability were assessed with MTT reduction (A), LDH release (B) and Caspase-3/7 cleavage (C) cytotoxic assays. Inhibition of macropinocytosis by its inhibitors (EIPA, CytD or Wort) enhanced amylin toxicity relative to the control cells (human amylin only), which in turn was reversed by adding MB to these cells. *P<0.05, **P<0.01, hA vs. hA/inhibitors, n = 9 ANOVA followed by Dunnett-Square test and #P<0.05, ##P<0.01, hA/inhibitors vs. hA/inhibitors/MB, n = 9, ANOVA followed by Newman-Keul post hoc test.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3760900&req=5

pone-0073080-g013: Inhibition of macropinocytosis potentiates amylin toxicity in human islets.The effects on cell viability were assessed with MTT reduction (A), LDH release (B) and Caspase-3/7 cleavage (C) cytotoxic assays. Inhibition of macropinocytosis by its inhibitors (EIPA, CytD or Wort) enhanced amylin toxicity relative to the control cells (human amylin only), which in turn was reversed by adding MB to these cells. *P<0.05, **P<0.01, hA vs. hA/inhibitors, n = 9 ANOVA followed by Dunnett-Square test and #P<0.05, ##P<0.01, hA/inhibitors vs. hA/inhibitors/MB, n = 9, ANOVA followed by Newman-Keul post hoc test.

Mentions: Since macropinocytosis is implicated in the clearance of cytotoxic amyloid oligomers in the pancreatic cells and other cell types [47], we investigated the inhibition of macropinocytosis with respect to amylin toxicity in RIN-m5F cells (Figure 12) and human islets (Figure 13). Amylin alone induced a significant 30% decrease in mitochondrial activity (Figure 12A), increased LDH release (leakage) by 33% from cells (Figure 12B) and stimulated apoptotic caspase-3 activity by 34% (Figure 12C) relative to the controls (set at 100%). A similar detrimental effect of human amylin on viability was demonstrated in human islets as evident by a 32±8% decrease in mitochondrial activity (Figure 13A), a 34±6% increase in LDH release (Figure 13B) and a 36±5% increase in apoptotic caspase-3 activity respective of the controls (Figure 13C). We next determined the modulatory effects of the macropinocytotic inhibitors (EIPA, CytD or Wort) in RIN-m5F cells (Figure 12A) and human islets (Figure 13A). Although, these inhibitors were not toxic to the cells on their own, they augmented the inhibitory effect of human amylin on cell’s metabolic activity (MTT reduction) by 30–34% in both cell types (Figure 12A, 13A). This increase in amylin toxicity was reversed by MB that also blocked oligomer formation and hence, their extracellular accumulation. Because the MTT assay reflects both reversible and irreversible patho-physiological events that may or may not lead to cell death [4], we verified the effect of the same inhibitors on amylin toxicity using LDH release assay (Figure 12B, 13B). EIPA, CytD or Wort stimulated 32–37% increase in human amylin-evoked LDH release in RIN-m5F cells (Figure 12B) and human islet cells (Figure 13B). Cell viability was restored by the addition of anti-oligomer inhibitor, MB [4], [74]. Macropinocytotic inhibitors increased human amylin evoked apoptosis in both cell types by 30–40% revealed by the caspase3/7 apoptotic assay, which again was reversed by MB (Figure 12C, 13C). This suggests that an increase in extracellular oligomer content is toxic to cells, which is augmented by impaired macropinocytosis.


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

Trikha S, Jeremic AM - PLoS ONE (2013)

Inhibition of macropinocytosis potentiates amylin toxicity in human islets.The effects on cell viability were assessed with MTT reduction (A), LDH release (B) and Caspase-3/7 cleavage (C) cytotoxic assays. Inhibition of macropinocytosis by its inhibitors (EIPA, CytD or Wort) enhanced amylin toxicity relative to the control cells (human amylin only), which in turn was reversed by adding MB to these cells. *P<0.05, **P<0.01, hA vs. hA/inhibitors, n = 9 ANOVA followed by Dunnett-Square test and #P<0.05, ##P<0.01, hA/inhibitors vs. hA/inhibitors/MB, n = 9, ANOVA followed by Newman-Keul post hoc test.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073080-g013: Inhibition of macropinocytosis potentiates amylin toxicity in human islets.The effects on cell viability were assessed with MTT reduction (A), LDH release (B) and Caspase-3/7 cleavage (C) cytotoxic assays. Inhibition of macropinocytosis by its inhibitors (EIPA, CytD or Wort) enhanced amylin toxicity relative to the control cells (human amylin only), which in turn was reversed by adding MB to these cells. *P<0.05, **P<0.01, hA vs. hA/inhibitors, n = 9 ANOVA followed by Dunnett-Square test and #P<0.05, ##P<0.01, hA/inhibitors vs. hA/inhibitors/MB, n = 9, ANOVA followed by Newman-Keul post hoc test.
Mentions: Since macropinocytosis is implicated in the clearance of cytotoxic amyloid oligomers in the pancreatic cells and other cell types [47], we investigated the inhibition of macropinocytosis with respect to amylin toxicity in RIN-m5F cells (Figure 12) and human islets (Figure 13). Amylin alone induced a significant 30% decrease in mitochondrial activity (Figure 12A), increased LDH release (leakage) by 33% from cells (Figure 12B) and stimulated apoptotic caspase-3 activity by 34% (Figure 12C) relative to the controls (set at 100%). A similar detrimental effect of human amylin on viability was demonstrated in human islets as evident by a 32±8% decrease in mitochondrial activity (Figure 13A), a 34±6% increase in LDH release (Figure 13B) and a 36±5% increase in apoptotic caspase-3 activity respective of the controls (Figure 13C). We next determined the modulatory effects of the macropinocytotic inhibitors (EIPA, CytD or Wort) in RIN-m5F cells (Figure 12A) and human islets (Figure 13A). Although, these inhibitors were not toxic to the cells on their own, they augmented the inhibitory effect of human amylin on cell’s metabolic activity (MTT reduction) by 30–34% in both cell types (Figure 12A, 13A). This increase in amylin toxicity was reversed by MB that also blocked oligomer formation and hence, their extracellular accumulation. Because the MTT assay reflects both reversible and irreversible patho-physiological events that may or may not lead to cell death [4], we verified the effect of the same inhibitors on amylin toxicity using LDH release assay (Figure 12B, 13B). EIPA, CytD or Wort stimulated 32–37% increase in human amylin-evoked LDH release in RIN-m5F cells (Figure 12B) and human islet cells (Figure 13B). Cell viability was restored by the addition of anti-oligomer inhibitor, MB [4], [74]. Macropinocytotic inhibitors increased human amylin evoked apoptosis in both cell types by 30–40% revealed by the caspase3/7 apoptotic assay, which again was reversed by MB (Figure 12C, 13C). This suggests that an increase in extracellular oligomer content is toxic to cells, which is augmented by impaired macropinocytosis.

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