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Aspirin, diabetes, and amyloid: re-examination of the inhibition of amyloid formation by aspirin and ketoprofen.

Tu LH, Noor H, Cao P, Raleigh DP - ACS Chem. Biol. (2014)

Bottom Line: The loss of β-cell function and β-cell death are key features of diabetes.There are no therapeutic strategies for the treatment or prevention of islet amyloidosis.Ketoprofen is similarly ineffective at inhibiting amylin amyloid formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Stony Brook University , Stony Brook, New York 11794-3400, United States.

ABSTRACT
The loss of β-cell function and β-cell death are key features of diabetes. A range of mechanisms are thought to contribute to β-cell loss, including islet amyloid formation by the neuropancreatic hormone amylin (islet amyloid polypeptide, IAPP). Islet amyloid deposition also contributes to the failure of islet transplants. There are no therapeutic strategies for the treatment or prevention of islet amyloidosis. Aspirin and the nonsteroid anti-inflammatory drug (NSAID) ketoprofen, at clinically relevant doses, have been proposed to inhibit amyloid formation by amylin and thus may hold promise for treatment of islet amyloidosis. These compounds are potentially attractive given the importance of inflammation in islet amyloidosis and given the fact that there are no anti-islet amyloid agents in the clinic. We show that aspirin, even in 20-fold excess, has no effect on the kinetics of amyloid formation by amylin as judged by thioflavin-T binding, right angle light scattering, and transmission electron microscopy, nor does it alter the morphology of resulting amyloid fibrils. Aspirin showed no ability to disaggregate preformed amylin amyloid fibrils under the conditions of these studies, 25 °C and pH 7.4. Ketoprofen is similarly ineffective at inhibiting amylin amyloid formation. The compounds do, however, interfere with circular dichroism- and Congo Red-based assays of amylin amyloid formation. This study highlights the importance of using multiple methods to follow amyloid formation when screening inhibitors.

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Aspirindoes not disaggregate preformed human amylin amyloid fibrils.(A) Thioflavin-T fluorescence assays of the time course of amyloidformation. A 20-fold excess of aspirin was added at the time pointindicated by the red arrow. (B) TEM image of the sample just beforethe addition of a 20-fold excess aspirin (indicated by the black star).(C) TEM image of the sample just after the addition of a 20-fold excessaspirin (indicated by the green star). (D) TEM image of the sample48 h after the addition of 20-fold excess aspirin (indicated by theblue star). Scale bars represent 100 nm. Experiments were conductedat pH 7.4 and 25 °C in 20 mM Tris buffer with 0.25% DMSO (v/v)in the absence of any fluorinated alcohol cosolvent. The concentrationof amylin was 16 μM, and the concentration of aspirin was 320μM.
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fig4: Aspirindoes not disaggregate preformed human amylin amyloid fibrils.(A) Thioflavin-T fluorescence assays of the time course of amyloidformation. A 20-fold excess of aspirin was added at the time pointindicated by the red arrow. (B) TEM image of the sample just beforethe addition of a 20-fold excess aspirin (indicated by the black star).(C) TEM image of the sample just after the addition of a 20-fold excessaspirin (indicated by the green star). (D) TEM image of the sample48 h after the addition of 20-fold excess aspirin (indicated by theblue star). Scale bars represent 100 nm. Experiments were conductedat pH 7.4 and 25 °C in 20 mM Tris buffer with 0.25% DMSO (v/v)in the absence of any fluorinated alcohol cosolvent. The concentrationof amylin was 16 μM, and the concentration of aspirin was 320μM.

Mentions: We next examined the ability ofaspirin to disaggregate preformedamylin amyloid fibrils. Some, but not all, inhibitors of amyloid formationhave this property. We monitored amyloid formation using thioflavin-Tassays (Figure 4) and then added a 20-foldexcess of aspirin after amyloid formation was complete and the reactionhad reached the saturation phase. Aliquots were removed for TEM analysisjust before addition of aspirin, immediately afterward, and 48 h later.Addition of the compound did not perturb the thioflavin-T time course;in contrast, compounds that disaggregate amyloid fibrils lead to adecay in the thioflavin-T signal as a function of time.10 The TEM images recorded before and after additionof aspirin are very similar and reveal extensive deposits of amyloidfibrils, confirming that the compound does not disaggregate amylinamyloid (Figure 4B–D). In addition,CD spectra recorded before and after the addition of aspirin havean identical shape, consistent with a high degree of β-structure,although there is a modest decrease in intensity. Furthermore, nochanges in the CD spectra are observed upon further incubation ofup to 12 h (Supporting Information). TheCD studies are fully consistent with thioflavin-T, TEM, and RALS experiments.


Aspirin, diabetes, and amyloid: re-examination of the inhibition of amyloid formation by aspirin and ketoprofen.

Tu LH, Noor H, Cao P, Raleigh DP - ACS Chem. Biol. (2014)

Aspirindoes not disaggregate preformed human amylin amyloid fibrils.(A) Thioflavin-T fluorescence assays of the time course of amyloidformation. A 20-fold excess of aspirin was added at the time pointindicated by the red arrow. (B) TEM image of the sample just beforethe addition of a 20-fold excess aspirin (indicated by the black star).(C) TEM image of the sample just after the addition of a 20-fold excessaspirin (indicated by the green star). (D) TEM image of the sample48 h after the addition of 20-fold excess aspirin (indicated by theblue star). Scale bars represent 100 nm. Experiments were conductedat pH 7.4 and 25 °C in 20 mM Tris buffer with 0.25% DMSO (v/v)in the absence of any fluorinated alcohol cosolvent. The concentrationof amylin was 16 μM, and the concentration of aspirin was 320μM.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4215902&req=5

fig4: Aspirindoes not disaggregate preformed human amylin amyloid fibrils.(A) Thioflavin-T fluorescence assays of the time course of amyloidformation. A 20-fold excess of aspirin was added at the time pointindicated by the red arrow. (B) TEM image of the sample just beforethe addition of a 20-fold excess aspirin (indicated by the black star).(C) TEM image of the sample just after the addition of a 20-fold excessaspirin (indicated by the green star). (D) TEM image of the sample48 h after the addition of 20-fold excess aspirin (indicated by theblue star). Scale bars represent 100 nm. Experiments were conductedat pH 7.4 and 25 °C in 20 mM Tris buffer with 0.25% DMSO (v/v)in the absence of any fluorinated alcohol cosolvent. The concentrationof amylin was 16 μM, and the concentration of aspirin was 320μM.
Mentions: We next examined the ability ofaspirin to disaggregate preformedamylin amyloid fibrils. Some, but not all, inhibitors of amyloid formationhave this property. We monitored amyloid formation using thioflavin-Tassays (Figure 4) and then added a 20-foldexcess of aspirin after amyloid formation was complete and the reactionhad reached the saturation phase. Aliquots were removed for TEM analysisjust before addition of aspirin, immediately afterward, and 48 h later.Addition of the compound did not perturb the thioflavin-T time course;in contrast, compounds that disaggregate amyloid fibrils lead to adecay in the thioflavin-T signal as a function of time.10 The TEM images recorded before and after additionof aspirin are very similar and reveal extensive deposits of amyloidfibrils, confirming that the compound does not disaggregate amylinamyloid (Figure 4B–D). In addition,CD spectra recorded before and after the addition of aspirin havean identical shape, consistent with a high degree of β-structure,although there is a modest decrease in intensity. Furthermore, nochanges in the CD spectra are observed upon further incubation ofup to 12 h (Supporting Information). TheCD studies are fully consistent with thioflavin-T, TEM, and RALS experiments.

Bottom Line: The loss of β-cell function and β-cell death are key features of diabetes.There are no therapeutic strategies for the treatment or prevention of islet amyloidosis.Ketoprofen is similarly ineffective at inhibiting amylin amyloid formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Stony Brook University , Stony Brook, New York 11794-3400, United States.

ABSTRACT
The loss of β-cell function and β-cell death are key features of diabetes. A range of mechanisms are thought to contribute to β-cell loss, including islet amyloid formation by the neuropancreatic hormone amylin (islet amyloid polypeptide, IAPP). Islet amyloid deposition also contributes to the failure of islet transplants. There are no therapeutic strategies for the treatment or prevention of islet amyloidosis. Aspirin and the nonsteroid anti-inflammatory drug (NSAID) ketoprofen, at clinically relevant doses, have been proposed to inhibit amyloid formation by amylin and thus may hold promise for treatment of islet amyloidosis. These compounds are potentially attractive given the importance of inflammation in islet amyloidosis and given the fact that there are no anti-islet amyloid agents in the clinic. We show that aspirin, even in 20-fold excess, has no effect on the kinetics of amyloid formation by amylin as judged by thioflavin-T binding, right angle light scattering, and transmission electron microscopy, nor does it alter the morphology of resulting amyloid fibrils. Aspirin showed no ability to disaggregate preformed amylin amyloid fibrils under the conditions of these studies, 25 °C and pH 7.4. Ketoprofen is similarly ineffective at inhibiting amylin amyloid formation. The compounds do, however, interfere with circular dichroism- and Congo Red-based assays of amylin amyloid formation. This study highlights the importance of using multiple methods to follow amyloid formation when screening inhibitors.

Show MeSH
Related in: MedlinePlus