Fiber-dependent and -independent toxicity of islet amyloid polypeptide

Abstract

The 37-residue peptide hormone islet amyloid polypeptide (IAPP) plays a central role in diabetes pathology. Although its amyloid fiber aggregation kinetics and cytotoxicity to β-cells are well documented, few reports have directly assessed the role of fibers in cell-based toxicity experiments. Here, we report that amyloid formation of IAPP can be strongly inhibited by the extracellular environment of live cells. For example, fiber formation is more strongly suppressed in cell culture medium than in aqueous buffer. The serum component of the medium is responsible for this inhibition. Although amyloid formation was previously shown to be catalyzed by both synthetic and chloroform-extracted phospholipid surfaces, it is instead inhibited by membrane surfaces prepared directly from the plasma membranes of an immortal β-cell line. This disparity is reconciled by direct assessment of fibers in cell-culture-based toxicity experiments. We discovered that fibers are nontoxic if they are washed free of adsorbed nonfibrillar components. Moreover, toxicity is not only rescued when monomers are added back to fibers but is greater than what is observed from the precursor alone. Our results are interpreted in light of the capacity of the fiber surface to template amyloid nucleation.

Figure 1

Cell culture medium inhibits hIAPP fiber formation. Fiber formation is initiated by dilution of hIAPP stock in water to 40 μM in reactions containing 50 mM phosphate buffer and 100 mM KCl (pH = 7.4) diluted to the indicated percentage of medium (RPMI, phenol-red free). (a) Representative normalized kinetic traces (monitored using 100 nM ThT fluorescence) are presented. (b) Statistics of reaction midpoints, t₅₀, from repeat analyses of a. Open bars are t₅₀ values (45 μM hIAPP) obtained for reactions at the indicated percentage of culture medium divided by the t₅₀ of buffer-only reactions. Dark gray bars are the relative t₅₀ obtained for reactions containing RPMI medium without the fetal bovine serum component. Light gray bars are the relative t₅₀ values of a reaction in buffer containing 0.6 μM purified bovine or human serum albumin. This corresponds to a concentration approximately equivalent to that present in 2% RPMI cell culture medium.

Figure 2

Cell-derived plasma membrane vesicles inhibit fiber formation. GPMVs were used as a reagent added to standardized 40 μM IAPP fiber formation reactions. (a) Schematic depicting the stepwise concentration of GPMVs using a 100 kDa molecular mass cutoff centrifugal concentrator for both NEM+ and NEM− samples. GPMVs are depicted as blue open circles, and residual medium components are depicted as red dots. (b) Representative kinetic traces are shown for the GPMVs added directly to the fiber formation reaction (zero spin, NEM+), and for washes that have been concentrated ∼3× (one spin, NEM+) and ∼15× (two spins, NEM+). For comparison, vesicle-free conditions were created using washes that did not contain the vesiculating reagent (two spins, NEM−). A representative trace is shown in which 11 μM 50:50 DOPC/DOPG synthetic lipid vesicles (in monomer units) were prepared in the same buffer. (c) Statistics of reaction midpoints, t₅₀, from fits to repeated analyses of b. Open bars are data from INS-1 GPMVs. Solid bars indicate data from analyses using COS-1 GPMVs. A further control is shown from NEM-containing samples that were never in contact with cells (NEM cell free). In panels b and c, green corresponds to the two-spin NEM− sample, pink corresponds to the zero-spin NEM+ sample, orange corresponds to the one-spin NEM+ sample, and purple corresponds to the two-spin NEM+ sample. To see this figure in color, go online.

Figure 3

IAPP binds GPMVs. Samples were prepared as described in Fig. 2 except for the addition of 2.5 μM-labeled IAPP. Representative white-light (right) and fluorescence (left) image pairs are shown for GPMVs derived from INS-1 cells (top) and COS-1 cells (bottom), respectively. Scale bar is 10 μm.

Figure 4

The toxicity of fresh IAPP is increased by addition of nontoxic fibers. IAPP toxicity to INS-1 cells was measured colorimetrically 72 h after exposure to the indicated total (fiber + monomer) concentration of IAPP. Cells were exposed to IAPP_fresh (open bars) or 1 μM of preformed IAPP_fib mixed with IAPP_fresh (gray bars). Horizontal lines are drawn at the toxicity levels measured for 10 μM IAPP_fresh (56% ± 6%, dotted) or 10 μM IAPP_fib (5% ± 2%, dashed).

Figure 5

The medium inhibits fiber formation seeded with preformed fibers. (a) Representative normalized kinetic traces for 40 μM IAPP_fresh, 2 μM IAPP_fib, and the indicated percentage of RPMI medium. The dotted line demarcates the time taken for 10 μM IAPP_fresh (a toxic concentration) to partition from the medium to cultured cells (18). (b) Statistics of reaction midpoints, t₅₀, from fits to repeat analyses of (a).