Concentration-dependent transitions govern the subcellular localization of islet amyloid polypeptide

Abstract

Islet amyloid polypeptide (IAPP) is a peptide hormone cosecreted with insulin by pancreatic β-cells. In type II diabetes, IAPP aggregates in a process that is associated with β-cell dysfunction and loss of β-cell mass. The relationship between IAPP's conformational landscape and its capacity to mediate cell death remains poorly understood. We have addressed these unknowns by comparing the cytotoxic effects of sequence variants with differing α-helical and amyloid propensities. IAPP was previously shown to oligomerize cooperatively on binding to lipid bilayers. Here, comparable transitions are evident in cell culture and are associated with a change in subcellular localization to the mitochondria under toxic conditions. Notably, we find that this toxic gain of function maps to IAPP's capacity to adopt aggregated membrane-bound α-helical, and not β-sheet, states. Our findings suggest that upon α-helical mediated oligomerization, IAPP acquires cell-penetrating peptide (CPP) properties, facilitating access to the mitochondrial compartment, resulting in its dysfunction.

Figure 1.

Primary sequence of IAPP. Shown are human and rat sequences of IAPP, with amino acid differences indicated in bold. Large horizontal arrows indicate areas of unambiguous secondary structure reported for both hIAPP and rIAPP on membranes (14) and for fibrillar states of hIAPP (32). Small arrows indicate additional constructs assayed in this work: hIAPP_L12N/N14L, hIAPP_H18R, and rIAPP_R18H.

Figure 2.

Complementary measures of the effects of hIAPP on cultured cells. A, B) Dose-dependent loss of mitochondrial (MTT, red) or cytosolic reductase (CTB, blue) activity. INS-1 (A) and COS-1 (B) cells were treated with the indicated concentrations of hIAPP. Change in reductase activity is expressed as a percentage of control using protein-free carrier. For comparison, inhibition of mitochondrial and cytosolic reductase activity is also presented for 5 μM melittin. C, D) Time-dependent loss of reductase activity on incubation of INS-1 (C, solid bars) and COS-1 (D, solid bars) cells with 10 μM hIAPP (C, open bars). Reductase activity was measured 48 h after culture medium was exchanged with hIAPP-free medium. Gray bars show direct measure of viability/toxicity performed by manual counting of live, adherent cells (C) or release of cytosolic LDH to the culture medium (D).

Figure 3.

Role of conformation in IAPP cytotoxicity. A) Time-dependent loss of MTT (red) or CTB (blue) response in INS-1 cells following incubation with 200 μM of the nonamyloidogenic rIAPP. B–D) Role of the helical subdomain was probed by measurement of reductase activity in INS-1 cells following incubation with 10 μM of the hIAPP mutants, L12N/N14L (B) or H18R (C), or 50 μM of the rIAPP mutant, R18H (D). Inset open bars (C) indicate loss of reductase activity at 10 μM hIAPP. Dashes (D) indicate loss of reductase activity at 50 μM rIAPP.

Figure 4.

Cellular uptake of IAPP. A, B) Loss of hIAPP (A) and rIAPP (B) from the extracellular medium of INS-1 cells. Open symbols indicate nontoxic levels (100 nM hIAPP₄₈₈ or rIAPP₄₈₈); solid symbols indicate toxic levels (100 nM labeled hIAPP₄₈₈ or rIAPP₄₈₈, with 10 or 200 μM unlabeled hIAPP or rIAPP, respectively). Comparisons are made between physiological temperature (37°C; red) and a temperature at which all endocytotic, and other energy-dependent processes, are inhibited (4°C; blue). Inset (A): effect of inhibition of endocytosis on hIAPP uptake, under toxic conditions (100 nM hIAPP₄₈₈ with 10 μM unlabeled hIAPP). Cells were pretreated with an inhibitor of clathrin-mediated endocytosis (30 μM chlorpromazine, solid black diamonds), an inhibitor of caveolae-mediated endocytosis (5 mM methyl-β-cyclodextrin, solid orange diamonds), or an inhibitor of macropinocytosis (10 μM cytochalasin D, solid green diamonds). Data are plotted on the same scale as the main figure (A). Data are calculated from loss of probe from the extracellular medium, measured by FCS. This is expressed as amount of probe per unit cell per unit time (fmol/cell/min). C) Confocal imaging of hIAPP at toxic concentrations following a 20-min incubation at 4°C. D) Effect of introducing hIAPP directly to the cytoplasm. COS-1 cells were scrape-loaded in the presence of 10 μM hIAPP. Cells were then washed and replated with IAPP-free medium. Loss of reductase activity (MTT or CTB) or release of LDH was measured after 72 h and compared to controls scraped with IAPP-free carrier. Dashes indicate effect of 72 h continuous incubation of COS-1 cells with 10 μM hIAPP. E) Measurement of internalized IAPP. Scrape indicates peptide taken up by scrape loading and assessed by FACS/FCS method, as described in Materials and Methods. Exogen indicates peptide taken up over 72 h exposure to 10 μM hIAPP and 100 nM hIAPP_A488 in culture medium, calculated by extrapolation from A (solid red ovals). Scale bar = 10 μm.

Figure 5.

Intracellular localization of IAPP. Colocalization of hIAPP_A488 (A), hIAPP_L12N/N14L (B), or rIAPP (C) with MitoTracker (top panels) or LysoTracker (bottom panels) in INS-1 cells. Conditions were 100 nM labeled IAPP with or without indicated concentration of unlabeled protein. Incubations were conducted for 72 h prior to imaging. For hIAPP and rIAPP, the addition of 10 and 200 μM unlabeled material, respectively, reflects conditions when toxicity is observed (Figs. 2 and 3A). White and cyan arrows indicate examples of extracellular aggregates and mitochondrial colocalization, respectively. Scale bars = 10 μm.