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Time-resolved studies define the nature of toxic IAPP intermediates, providing insight for anti-amyloidosis therapeutics.

Abedini A, Plesner A, Cao P, Ridgway Z, Zhang J, Tu LH, Middleton CT, Chao B, Sartori DJ, Meng F, Wang H, Wong AG, Zanni MT, Verchere CB, Raleigh DP, Schmidt AM - Elife (2016)

Bottom Line: These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species.They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure.Aromatic interactions modulate, but are not required for toxicity.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Research Program, Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, New York University School of Medicine, New York, United States.

ABSTRACT
Islet amyloidosis by IAPP contributes to pancreatic β-cell death in diabetes, but the nature of toxic IAPP species remains elusive. Using concurrent time-resolved biophysical and biological measurements, we define the toxic species produced during IAPP amyloid formation and link their properties to induction of rat INS-1 β-cell and murine islet toxicity. These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species. They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure. Aromatic interactions modulate, but are not required for toxicity. Not all IAPP oligomers are toxic; toxicity depends on their partially structured conformational states. Some anti-amyloid agents paradoxically prolong cytotoxicity by prolonging the lifetime of the toxic species. The data highlight the distinguishing properties of toxic IAPP oligomers and the common features that they share with toxic species reported for other amyloidogenic polypeptides, providing information for rational drug design to treat IAPP induced β-cell death.

No MeSH data available.


Related in: MedlinePlus

I26P-IAPP inhibits h-IAPP amyloid formation, but prolongs cytotoxicity.(A) Time-resolved Alamar Blue reduction assays of β-cells treated with: I26P-IAPP (♦), h-IAPP (●) and 1:1 I26P-IAPP/h-IAPP (■). Light microscopy: (†) Viable β-cells treated with h-IAPP amyloid fibrils formed after 14 h- of incubation; (‡) shrunken apoptotic β-cells after treatment with lag phase intermediates of 1:1 I26P-IAPP/h-IAPP produced after 14 h of incubation. (B) Thioflavin-T monitored kinetics of amyloid formation by I26P-IAPP (♦), h-IAPP (●) and 1:1 I26P-IAPP/h-IAPP (■). The same color coding is used in panels A and B. TEM images of aliquots of: (§) h-IAPP and (φ) 1:1 I26P-IAPP/h-IAPP obtained from the same samples monitored in panel B and applied to β-cells in panel A (Scale bars: 200 nm). Concurrent Alamar Blue reduction assays, thioflavin-T binding assays, light microscopy and TEM studies used aliquots from the same peptide solutions containing 40 µM (single peptide samples) or 80 µM peptide (1:1 mixture). The final peptide concentrations after dilution into β-cell assays were 28 µM and 56 µM, respectively. Data represent mean ± SD of three to six replicate wells per condition and three replicate experiments per group. Some of the error bars in panels A and B are the same size or smaller than the symbols in the graphs.DOI:http://dx.doi.org/10.7554/eLife.12977.042
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fig10: I26P-IAPP inhibits h-IAPP amyloid formation, but prolongs cytotoxicity.(A) Time-resolved Alamar Blue reduction assays of β-cells treated with: I26P-IAPP (♦), h-IAPP (●) and 1:1 I26P-IAPP/h-IAPP (■). Light microscopy: (†) Viable β-cells treated with h-IAPP amyloid fibrils formed after 14 h- of incubation; (‡) shrunken apoptotic β-cells after treatment with lag phase intermediates of 1:1 I26P-IAPP/h-IAPP produced after 14 h of incubation. (B) Thioflavin-T monitored kinetics of amyloid formation by I26P-IAPP (♦), h-IAPP (●) and 1:1 I26P-IAPP/h-IAPP (■). The same color coding is used in panels A and B. TEM images of aliquots of: (§) h-IAPP and (φ) 1:1 I26P-IAPP/h-IAPP obtained from the same samples monitored in panel B and applied to β-cells in panel A (Scale bars: 200 nm). Concurrent Alamar Blue reduction assays, thioflavin-T binding assays, light microscopy and TEM studies used aliquots from the same peptide solutions containing 40 µM (single peptide samples) or 80 µM peptide (1:1 mixture). The final peptide concentrations after dilution into β-cell assays were 28 µM and 56 µM, respectively. Data represent mean ± SD of three to six replicate wells per condition and three replicate experiments per group. Some of the error bars in panels A and B are the same size or smaller than the symbols in the graphs.DOI:http://dx.doi.org/10.7554/eLife.12977.042

Mentions: (A) Alamar Blue reduction assays and (B) DHE fluorescence assays of β-cells treated for 1 h with h-IAPP lag phase intermediates (blue), amyloid fibrils (red) or buffer (gold) show significant toxicity and ROS production induced by the lag phase intermediates but not by the amyloid fibrils. The final peptide concentrations after dilution into β-cell assays was 28 µM. Peptide solutions were incubated on cells for a shorter amount of time (1 h) in oxidative stress experiments and concurrent control β-cell viability assays shown in panels A and B, than generally employed in our standard β-cell viability assays (5 h), since the production and detection of transient ROS occurs prior to detectable loss in cell viability. The degree of loss in β-cell viability after 1 h incubation with toxic h-IAPP lag phase intermediates shown in panel A is significantly less than that detected after 5 h of incubation at the same peptide concentration, shown in Figure 10A. Data represent mean ± SD of three to ten technical replicates per condition and a minimum of three biological replicate experiments per group (*p<0.01, **p<0.01).


Time-resolved studies define the nature of toxic IAPP intermediates, providing insight for anti-amyloidosis therapeutics.

Abedini A, Plesner A, Cao P, Ridgway Z, Zhang J, Tu LH, Middleton CT, Chao B, Sartori DJ, Meng F, Wang H, Wong AG, Zanni MT, Verchere CB, Raleigh DP, Schmidt AM - Elife (2016)

I26P-IAPP inhibits h-IAPP amyloid formation, but prolongs cytotoxicity.(A) Time-resolved Alamar Blue reduction assays of β-cells treated with: I26P-IAPP (♦), h-IAPP (●) and 1:1 I26P-IAPP/h-IAPP (■). Light microscopy: (†) Viable β-cells treated with h-IAPP amyloid fibrils formed after 14 h- of incubation; (‡) shrunken apoptotic β-cells after treatment with lag phase intermediates of 1:1 I26P-IAPP/h-IAPP produced after 14 h of incubation. (B) Thioflavin-T monitored kinetics of amyloid formation by I26P-IAPP (♦), h-IAPP (●) and 1:1 I26P-IAPP/h-IAPP (■). The same color coding is used in panels A and B. TEM images of aliquots of: (§) h-IAPP and (φ) 1:1 I26P-IAPP/h-IAPP obtained from the same samples monitored in panel B and applied to β-cells in panel A (Scale bars: 200 nm). Concurrent Alamar Blue reduction assays, thioflavin-T binding assays, light microscopy and TEM studies used aliquots from the same peptide solutions containing 40 µM (single peptide samples) or 80 µM peptide (1:1 mixture). The final peptide concentrations after dilution into β-cell assays were 28 µM and 56 µM, respectively. Data represent mean ± SD of three to six replicate wells per condition and three replicate experiments per group. Some of the error bars in panels A and B are the same size or smaller than the symbols in the graphs.DOI:http://dx.doi.org/10.7554/eLife.12977.042
© Copyright Policy
Related In: Results  -  Collection

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fig10: I26P-IAPP inhibits h-IAPP amyloid formation, but prolongs cytotoxicity.(A) Time-resolved Alamar Blue reduction assays of β-cells treated with: I26P-IAPP (♦), h-IAPP (●) and 1:1 I26P-IAPP/h-IAPP (■). Light microscopy: (†) Viable β-cells treated with h-IAPP amyloid fibrils formed after 14 h- of incubation; (‡) shrunken apoptotic β-cells after treatment with lag phase intermediates of 1:1 I26P-IAPP/h-IAPP produced after 14 h of incubation. (B) Thioflavin-T monitored kinetics of amyloid formation by I26P-IAPP (♦), h-IAPP (●) and 1:1 I26P-IAPP/h-IAPP (■). The same color coding is used in panels A and B. TEM images of aliquots of: (§) h-IAPP and (φ) 1:1 I26P-IAPP/h-IAPP obtained from the same samples monitored in panel B and applied to β-cells in panel A (Scale bars: 200 nm). Concurrent Alamar Blue reduction assays, thioflavin-T binding assays, light microscopy and TEM studies used aliquots from the same peptide solutions containing 40 µM (single peptide samples) or 80 µM peptide (1:1 mixture). The final peptide concentrations after dilution into β-cell assays were 28 µM and 56 µM, respectively. Data represent mean ± SD of three to six replicate wells per condition and three replicate experiments per group. Some of the error bars in panels A and B are the same size or smaller than the symbols in the graphs.DOI:http://dx.doi.org/10.7554/eLife.12977.042
Mentions: (A) Alamar Blue reduction assays and (B) DHE fluorescence assays of β-cells treated for 1 h with h-IAPP lag phase intermediates (blue), amyloid fibrils (red) or buffer (gold) show significant toxicity and ROS production induced by the lag phase intermediates but not by the amyloid fibrils. The final peptide concentrations after dilution into β-cell assays was 28 µM. Peptide solutions were incubated on cells for a shorter amount of time (1 h) in oxidative stress experiments and concurrent control β-cell viability assays shown in panels A and B, than generally employed in our standard β-cell viability assays (5 h), since the production and detection of transient ROS occurs prior to detectable loss in cell viability. The degree of loss in β-cell viability after 1 h incubation with toxic h-IAPP lag phase intermediates shown in panel A is significantly less than that detected after 5 h of incubation at the same peptide concentration, shown in Figure 10A. Data represent mean ± SD of three to ten technical replicates per condition and a minimum of three biological replicate experiments per group (*p<0.01, **p<0.01).

Bottom Line: These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species.They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure.Aromatic interactions modulate, but are not required for toxicity.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Research Program, Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, New York University School of Medicine, New York, United States.

ABSTRACT
Islet amyloidosis by IAPP contributes to pancreatic β-cell death in diabetes, but the nature of toxic IAPP species remains elusive. Using concurrent time-resolved biophysical and biological measurements, we define the toxic species produced during IAPP amyloid formation and link their properties to induction of rat INS-1 β-cell and murine islet toxicity. These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species. They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure. Aromatic interactions modulate, but are not required for toxicity. Not all IAPP oligomers are toxic; toxicity depends on their partially structured conformational states. Some anti-amyloid agents paradoxically prolong cytotoxicity by prolonging the lifetime of the toxic species. The data highlight the distinguishing properties of toxic IAPP oligomers and the common features that they share with toxic species reported for other amyloidogenic polypeptides, providing information for rational drug design to treat IAPP induced β-cell death.

No MeSH data available.


Related in: MedlinePlus