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.