Heparan Sulfate Proteoglycans Are Important for Islet Amyloid Formation and Islet Amyloid Polypeptide-induced Apoptosis

Abstract

Deposition of β cell toxic islet amyloid is a cardinal finding in type 2 diabetes. In addition to the main amyloid component islet amyloid polypeptide (IAPP), heparan sulfate proteoglycan is constantly present in the amyloid deposit. Heparan sulfate (HS) side chains bind to IAPP, inducing conformational changes of the IAPP structure and an acceleration of fibril formation. We generated a double-transgenic mouse strain (hpa-hIAPP) that overexpresses human heparanase and human IAPP but is deficient of endogenous mouse IAPP. Culture of hpa-hIAPP islets in 20 mm glucose resulted in less amyloid formation compared with the amyloid load developed in cultured islets isolated from littermates expressing human IAPP only. A similar reduction of amyloid was achieved when human islets were cultured in the presence of heparin fragments. Furthermore, we used CHO cells and the mutant CHO pgsD-677 cell line (deficient in HS synthesis) to explore the effect of cellular HS on IAPP-induced cytotoxicity. Seeding of IAPP aggregation on CHO cells resulted in caspase-3 activation and apoptosis that could be prevented by inhibition of caspase-8. No IAPP-induced apoptosis was seen in HS-deficient CHO pgsD-677 cells. These results suggest that β cell death caused by extracellular IAPP requires membrane-bound HS. The interaction between HS and IAPP or the subsequent effects represent a possible therapeutic target whose blockage can lead to a prolonged survival of β cells.

Keywords: amyloid; cell death; heparan sulfate; pancreatic islet; proteoglycan.

FIGURE 1.

Heparanase overexpression and HS fragmentation in hpa-hIAPP islets. A, extracts of isolated islets from hIAPP and hpa-hIAPP mice were analyzed by Western blot using antiserum 733 reactive against the active 50-kDa subunit of heparanase. A single band corresponding to 50 kDa was detected in hpa-hIAPP islets, whereas no reactivity was detected in hIAPP islets. Bottom blot, insulin reactivity in both islet extracts. Molecular weight is shown in kilodaltons. B, pancreas sections were double-immunostained for heparanase (733, red) and insulin (green). Bottom panels and inset, merged images demonstrating the colocalization of heparanase (733) and insulin reactivity in hpa-hIAPP islet β cells. hIAPP islets were only positive for insulin. Nuclei stained with DAPI are shown in blue. Scale bars = 30 and 3 μm (inset). C, gel chromatography analysis of ³⁵S-labeled HS isolated from the pancreata of a heparanase overexpressing mouse (hpa-tg, red) and a wild-type C57BL mouse (blue). The arrow indicates the expected elution position of heparin (14 kDa). A right shift of the elution profile from the hpa-tg mouse relative to the wild-type mouse reflects a significant reduction in HS chain length.

FIGURE 2.

Hpa-hIAPP islets develop less amyloid during culture in high glucose. Isolated islets from hIAPP and hpa-hIAPP mice were cultured in 20 mm glucose for 19 days, fixed, and stained for amyloid with thioflavin S. A and B, encircled hIAPP islet (A) and hpa-hIAPP islet (B) containing amyloid stained with thioflavin S (bright green). Scale bar = 20 μm. C, amyloid load (islet amyloid volume / total islet volume) was determined in islets from hIAPP mice (n = 6) and hpa-hIAPP mice (n = 7) cultured in 20 mm glucose (HG, black columns) or 11 mm glucose (normal glucose, NG, gray columns) (n = 2). From each mouse, an average of 19 islets were analyzed per condition. There was a significant decrease in amyloid load in islets cultured in HG from hpa-hIAPP mice compared with islets from hIAPP mice. **, p = 0.007; Student's t test; mean ± S.E. D and E, electron micrograph of an ultrathin section from an hIAPP islet (D) and an hpa-hIAPP islet (E) cultured in 20 mm glucose for 19 days. Amyloid fibrils present extracellularly are reactive with human IAPP-specific antiserum detected with 10-nm gold particles. Scale bars = 200 nm.

FIGURE 3.

Overexpression of heparanase does not affect α/β cell fractions, islet hormone secretion, or content. A, percentage of α cells (glucagon-positive, white columns) and β cells (insulin-positive, black columns) per islet as determined from immunostaining of pancreas sections from hIAPP and hpa-hIAPP mice (n = 3, ≥47 islets analyzed/genotype). B and D, glucose-stimulated release of insulin (B, n = 4) and IAPP (D, n = 2) during basal conditions (1.67 mm glucose, white columns) and stimulating conditions (16.7 mm glucose, black columns) from newly isolated islets. C and E, islet insulin content (C) and IAPP content (E) in the same isolated islet samples. No difference in α/β cell fractions, hormone release, or content could be observed between hpa-hIAPP mice and hIAPP mice (p > 0.05, Student's t test; mean ± S.E.).

FIGURE 4.

Heparin binds to the N-terminal region of hIAPP, and binding is dependent on chain size. A, ³H-labeled heparin binds hIAPP, rIAPP, and hCGRP to the same extent when analyzed with a nitrocellulose filter binding assay. ³H-labeled heparin (1400 cpm) was incubated with 6 μm peptide in PBS (pH 7.4), 0.1% BSA, and 5% DMSO for 1 h, and then the percentage of peptide-bound heparin was determined (n = 2). Bottom panel, aligned amino acid sequences of peptides bound (bold) or not bound by heparin. The gray box highlights the region important for heparin binding. B, percentage of ³H-labeled heparin fragments (10,000 cpm) bound to 6 μm hIAPP1–37 in PBS (pH 7.4), 0.1% BSA, and 2.5% DMSO increases with heparin fragment size (n = 3). Data are mean ± S.E.

FIGURE 5.

Addition of heparin fragments to cultured islets reduces the amyloid load. A, isolated islets from single transgenic hIAPP mice (hIAPP[FVB/N]) were cultured for 19 days in 11 mm glucose (normal glucose, NG, gray column) or 20 mm glucose medium (HG, black columns) alone or supplemented with heparin fragments (150 nm) of different sizes: 4-, 8-, 12-, or 18-mer heparin fragments or full-length heparin (Hep). Islets were stained with thioflavin S, and the amyloid load was determined (islet amyloid volume/total islet volume). Islets cultured in HG with 12-mers had a reduced amyloid load compared with islets cultured in HG only (*, p < 0.05 versus HG, ****, p < 0.0001 versus HG; ANOVA; Bonferroni correction). The numbers in parenthesis define the number of experiments (n = 1, performed on islets pooled from ≥6 mice). B, islets isolated from human donors (n = 3) were cultured for 19 days in 5.5 mm glucose (normal glucose, gray column) or 20 mm glucose (HG, black columns) alone or supplemented with 12-mer heparin fragments (150 or 300 nm). The amyloid load was determined by thioflavin S staining. A significant decrease in amyloid load was seen in islets cultured in HG with 150 nm 12-mer compared with islets cultured in HG only (*, p < 0.05 versus HG; ANOVA; Bonferroni correction; mean ± S.E.).

FIGURE 6.

Heparin promotes IAPP fibril formation in vitro. A, amyloid fibril formation kinetics of 1.4 μm hIAPP1–37 in PBS (pH 7.4) with 10 μm thioflavin T were analyzed by monitoring 480-nm fluorescence intensity at 440-nm excitation. IAPP was incubated alone (green) or in the presence of equimolar full-length heparin (Hep, red) or 12-mer heparin fragments (blue) (n = 2). The presence of heparin promoted IAPP fibril formation, seen by the dramatically reduced lag phase compared with IAPP alone. 12-mers also promoted fibril formation, although less potently compared with full-length heparin. B, CHO^WT cells were stably transfected with a vector encoding protein pairs for FRET (ECFP/EYFP) linked via residues DEVD. Activation of cellular caspase-3 during apoptosis cleaves the DEVD link, measured as loss of FRET signal. Incubation of CHO^WT-DEVD cells with 50 μm IAPP and sonicated seeds (corresponding to 125 nm monomeric IAPP) led to detectable caspase-3 activation at ≥6 h (green columns), reaching a maximal effect at ∼18 h. The presence of equimolar full-length heparin (red columns) or 12-mers (blue columns) significantly reduced the activation of cellular caspase-3 induced by seeded IAPP (n = 3; *, p < 0.05 versus IAPP; two-way ANOVA; Bonferroni correction). The addition of 12-mers alone was used as a negative control (gray columns). Data are presented relative to untreated cells (mean ± S.E.).

FIGURE 7.

HS is required for hIAPP-induced caspase-3 activation. CHO^WT cells and HS-deficient pgsD-677 cells were stably transfected with a vector encoding protein pairs for FRET (ECFP/EYFP) linked via residues DEVD (CHO^WT-DEVD and pgsD-677-DEVD). Activated cellular caspase-3 during apoptosis cleaves the DEVD link, measured as loss of FRET signal, i.e. reduced 540/480 nm ratio. FRET analysis was performed with monomeric IAPP with the addition of sonicated IAPP fibrils (seed) to initiate the fibril propagation process. Incubation with seed only was used as a negative control (neg. ctrl). A, incubation of CHO^WT-DEVD cells with 50 μm IAPP and sonicated IAPP seeds (corresponding to 125 nm monomeric IAPP) resulted in a progressive loss of FRET over time (green; n = 4; *, p < 0.05 versus negative control (seeds, gray); two-way ANOVA; Bonferroni correction). Stauro, staurosporine. B, in HS-deficient pgsD-677-DEVD cells, there was no difference in FRET signal between cells incubated with seeded 50 μm IAPP (green) and the negative control (gray; n = 4; p > 0.05 versus negative control (seeds, gray); two-way ANOVA; Bonferroni correction). Incubation with staurosporine was used as a positive control (blue). ns, not significant. C, cells expressing ECFP/EYFP linked via the KEAF residues, which is not recognized by caspase-3, displayed no loss of FRET during exposure to staurosporine (n = 4). D, ThT assay demonstrates aggregation kinetics of the seeded IAPP used in A and B (representative of four individual experiments), and the insets show transmission electron microscopy images of negatively stained samples removed from the ThT assay at the indicated time points. Scale bars = 100 nm. E, immunofluorescence using antiserum A133 specific for hIAPP of cells incubated with seeded IAPP for 12 h. IAPP reactivity (red) was mainly associated with cell membranes and was more abundant on CHO^WT-DEVD cells compared with pgsD-677-DEVD cells. Cell cytoplasm with ECFP/EYFP is shown in green, and nuclei were stained with DAPI (blue). Scale bars = 5 μm. F, treatment of CHO^WT-DEVD cells with caspase-8 inhibitor (purple) prevented IAPP-induced loss of FRET over time, whereas no effect was seen with caspase-9 inhibitor (black) (n = 3; *, p < 0.05 versus IAPP; two-way ANOVA; Bonferroni correction). G, summary of the effect shown in F of caspase-8 and caspase-9 inhibitors on IAPP-induced apoptosis in CHO^WT-DEVD cells at 12 h, including negative controls where IAPP was omitted (gray columns). *, p > 0.05 versus IAPP; two-way ANOVA; Bonferroni correction. Data are mean ± S.E.

FIGURE 8.

HS-deficient pgsD-677 cells have intact activation of apoptosis via the extrinsic pathway. A, no difference in Fas receptor gene expression was found between CHO^WT-DEVD and pgsD-677-DEVD cells at initiation of the FRET assay. Data are presented as the -fold change in Fas receptor gene expression normalized to an endogenous reference (GADPH) and relative to CHO^WT cells (n = 2). B and C, both CHO^WT-DEVD (B) and pgsD-677-DEVD (C) cells demonstrated a significant reduction in FRET signal after incubation with mouse Fas Ligand His₆ and polyhistidine antibody for ≥1 h. Data are representative of n = 2; *, p < 0.05 versus control; two-way ANOVA; Bonferroni correction; mean ± S.E.