Benzbromarone, Quercetin, and Folic Acid Inhibit Amylin Aggregation

Abstract

Human Amylin, or islet amyloid polypeptide (hIAPP), is a small hormone secreted by pancreatic β-cells that forms aggregates under insulin deficiency metabolic conditions, and it constitutes a pathological hallmark of type II diabetes mellitus. In type II diabetes patients, amylin is abnormally increased, self-assembled into amyloid aggregates, and ultimately contributes to the apoptotic death of β-cells by mechanisms that are not completely understood. We have screened a library of approved drugs in order to identify inhibitors of amylin aggregation that could be used as tools to investigate the role of amylin aggregation in type II diabetes or as therapeutics in order to reduce β-cell damage. Interestingly, three of the compounds analyzed-benzbromarone, quercetin, and folic acid-are able to slow down amylin fiber formation according to Thioflavin T binding, turbidimetry, and Transmission Electron Microscopy assays. In addition to the in vitro assays, we have tested the effect of these compounds in an amyloid toxicity cell culture model and we have found that one of them, quercetin, has the ability to partly protect cultured pancreatic insulinoma cells from the cytotoxic effect of amylin. Our data suggests that quercetin can contribute to reduce oxidative damage in pancreatic insulinoma β cells by modulating the aggregation propensity of amylin.

Keywords: aggregation; amylin; amyloid; benzbromarone; folic acid; quercetin; type II diabetes.

Figure 1

Inhibition of amylin (8–37) aggregation by benzbromarone, quercetin, and folic acid, followed by an increase in Thioflavin T (ThT) emission fluorescence at 482 nm. Black dots: positive aggregation control, only amylin; white dots: negative aggregation control (no amylin); Red, green, and cyan dots: aggregation of amylin in presence of benzbromarone, quercetin, and folic acid, respectively.

Figure 2

Kinetic aggregation assays of amylin (8–37) in the presence of benzbromarone, quercetin, and folic acid, followed by solution turbidity. Turbidity was measured as absorbance at 360 nm. Black dots: positive aggregation control (only amylin); white dots: negative aggregation control (no amylin); Red, green, and cyan dots: aggregation of amylin with the addition of benzbromarone, quercetin, and folic acid, respectively.

Figure 3

Amylin aggregation inhibition visualized using transmission electron microscopy (TEM). Amylin (8–37) at 50 µM was incubated in the presence or absence of the compounds (at 100 µM), fibers were negatively stained and imaged using TEM. Positive control (amylin alone): panel (a); negative control (no amylin): panel (b); amylin in presence of benzbromarone, quercetin, and folic acid: panels (c, d, or e), respectively. The length of the scale bars is 0.2 µm. Compounds strongly inhibit amylin amyloid fibril formation.

Figure 4

Quercetin reduction of amylin (8–37) cytotoxicity in pancreatic rat insulinoma Rat insulinoma pancreatic (RIN-m5F) β-cells. Cells were treated for 48 h with amylin (75 µM) in the presence or absence of benzbromarone (BBM, 10 µM), quercetin (QR, 10 µM), folic acid (FA, 10 µM), or Congo red (CR, 100 µM), and cell viability was measured using a 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide: XTT assay. The results obtained were normalized to control cells (dimethyl sulfoxide: DMSO treated) and expressed as mean values ± SD (n = 7 independent experiments). No statistical differences were obtained between the conditions where only the compounds were added and the control. Statistical analysis was carried out using an unpaired t test. * p < 0.05; ** p < 0.01; **** p < 0.0001.