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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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Aspirin does not inhibit amyloid formation by human amylin. (A)Thioflavin-T fluorescence assays of the time course of amyloid formationin the absence (black) and presence (red) of a 20-fold excess of aspirin.(B) TEM image of the sample of amylin without aspirin recorded atthe end of the kinetic experiment. (C) TEM image of the sample ofamylin with a 20-fold excess aspirin recorded at the end of the kineticexperiment. 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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fig2: Aspirin does not inhibit amyloid formation by human amylin. (A)Thioflavin-T fluorescence assays of the time course of amyloid formationin the absence (black) and presence (red) of a 20-fold excess of aspirin.(B) TEM image of the sample of amylin without aspirin recorded atthe end of the kinetic experiment. (C) TEM image of the sample ofamylin with a 20-fold excess aspirin recorded at the end of the kineticexperiment. 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: Mature, fully processed amylin is 37 residuesin length, containsa disulfide bond between residues 2 and 7, and has an amidated C-terminus(Figure 1). We first examined the effects ofaspirin on the kinetics of amyloid formation using fluorescence-detectedthioflavin-T binding assays. This is the standard assay in this field.Thioflavin-T is a small fluorescent dye that binds to the cross β-structureof amyloid fibrils, presumably in the surface grooves formed by theparallel β-sheets. Binding constrains the conformation of thedye and relieves self-quenching, resulting in an increase in quantumyield.25 Figure 2 displays the results of kinetic experiments conducted in the presenceof aspirin. The expected sigmoidal time course is observed in theabsence of aspirin, with a T50, defined as the time requiredto reach half of the total signal change in the thioflavin-T assay,of 20 h. Addition of aspirin, up to even a 20-fold excess, had nodetectable effect on the rate of amyloid formation, as judged by thevalues of T50. The compound also had no detectable effecton the final thioflavin-T intensity. Thioflavin-T binding assays areindirect because they rely on the binding of an extrinsic probe andcan sometimes give misleading results,26 but they do have the advantage that they report on the kineticsof amylin amyloid formation in the absence of conflicting factors.We also used transmission electron microscopy (TEM) to monitor theeffects of aspirin. Aliquots were removed from each sample at theend of kinetic experiments, blotted onto TEM grids, and imaged. Extensivemats of fibrils were observed in the sample of amylin alone and inall of the samples that contained aspirin.


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)

Aspirin does not inhibit amyloid formation by human amylin. (A)Thioflavin-T fluorescence assays of the time course of amyloid formationin the absence (black) and presence (red) of a 20-fold excess of aspirin.(B) TEM image of the sample of amylin without aspirin recorded atthe end of the kinetic experiment. (C) TEM image of the sample ofamylin with a 20-fold excess aspirin recorded at the end of the kineticexperiment. 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.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Aspirin does not inhibit amyloid formation by human amylin. (A)Thioflavin-T fluorescence assays of the time course of amyloid formationin the absence (black) and presence (red) of a 20-fold excess of aspirin.(B) TEM image of the sample of amylin without aspirin recorded atthe end of the kinetic experiment. (C) TEM image of the sample ofamylin with a 20-fold excess aspirin recorded at the end of the kineticexperiment. 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: Mature, fully processed amylin is 37 residuesin length, containsa disulfide bond between residues 2 and 7, and has an amidated C-terminus(Figure 1). We first examined the effects ofaspirin on the kinetics of amyloid formation using fluorescence-detectedthioflavin-T binding assays. This is the standard assay in this field.Thioflavin-T is a small fluorescent dye that binds to the cross β-structureof amyloid fibrils, presumably in the surface grooves formed by theparallel β-sheets. Binding constrains the conformation of thedye and relieves self-quenching, resulting in an increase in quantumyield.25 Figure 2 displays the results of kinetic experiments conducted in the presenceof aspirin. The expected sigmoidal time course is observed in theabsence of aspirin, with a T50, defined as the time requiredto reach half of the total signal change in the thioflavin-T assay,of 20 h. Addition of aspirin, up to even a 20-fold excess, had nodetectable effect on the rate of amyloid formation, as judged by thevalues of T50. The compound also had no detectable effecton the final thioflavin-T intensity. Thioflavin-T binding assays areindirect because they rely on the binding of an extrinsic probe andcan sometimes give misleading results,26 but they do have the advantage that they report on the kineticsof amylin amyloid formation in the absence of conflicting factors.We also used transmission electron microscopy (TEM) to monitor theeffects of aspirin. Aliquots were removed from each sample at theend of kinetic experiments, blotted onto TEM grids, and imaged. Extensivemats of fibrils were observed in the sample of amylin alone and inall of the samples that contained aspirin.

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