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Cell Biology International (2008) 32, 447–455 (Printed in Great Britain)
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β-Amyloid peptides 1–40βA and 25–35βA suppress human amylin-mediated death of RINm5F islet β-cells with distinct actions on fibril formation
Ji‑Zhong Bai*
Department of Physiology, School of Medical and Health Sciences, University of Auckland, Private Bag 9201, Auckland, New Zealand

Abstract

Amyloid deposition is a common feature of Alzheimer’s disease and type 2 diabetes related to β-amyloid peptides (βA) and human amylin (hA), respectively. Both βA and hA form aggregates and fibrils and kill cultured cells. To investigate whether βA and hA display peptide-specific toxicity on cultured islet β-cells, we examined the effects of 1–40βA and 25–35βA peptides on hA-mediated cell death and [125I-Tyr37]hA precipitation. Synthetic hA aggregated in solution and evoked both conformation- and sequence-dependent cell death. While neither 1–40βA nor 25–35βA was toxic to islet β-cells, they suppressed hA-evoked cell death in a concentration-dependent and saturable manner. Only 1–40βA, but not 25–35βA, showed trophic effects on cultured islet β-cells and inhibited the precipitation of [125I]hA caused by hA. These results suggest that 25–35βA does not interfere with hA-mediated fibril formation. Suppression of hA-evoked death of cultured pancreatic islet β-cells by the βA peptides is likely to occur through a competing interaction at these cells.

Keywords: β-Amyloid peptide, Amylin, Islet β-cell death, Aggregation.

*Tel.: +64 9 373 7599x86205; fax: +64 9 373 7499.

1 Introduction

β-Amyloid (βA) (Glenner and Wong, 1984) and human Amylin (hA) (Cooper et al., 1987) or islet amyloid polypeptide (IAPP) (Westermark et al., 1987) are peptides of similar size, which comprise the deposits of amyloid associated with Alzheimer’s disease (AD) and type 2 diabetes mellitus, respectively. The two peptides share only 38% sequence identity but show a similar feature of spontaneously forming amyloid fibrils in solution (Lorenzo and Yankner, 1994; Westermark et al., 1990). hA evokes death in cultured islet β-cells (Bai et al., 1999; Lorenzo et al., 1994; Meier et al., 2006; Tatarek-Nossol et al., 2005; Yan et al., 2006) and neurons (Kayed et al., 2003; Jhamandas and MacTavish, 2004; Mattson and Goodman, 1995; Schubert et al., 1995; Tucker et al., 1998; Wogulis et al., 2005), and βA similarly elicits death in cultured neurons (Behl et al., 1994; Kayed et al., 2003; Jhamandas and MacTavish, 2004; Loo et al., 1993; Lorenzo and Yankner, 1994; Shearman et al., 1994; Tucker et al., 1998; Wogulis et al., 2005).

hA also evokes islet β-cell death and diabetes mellitus when expressed in the islet β-cells of both transgenic mice (Janson et al., 1996; Verchere et al., 1996) and rats (Butler et al., 2004). Likewise, the full-length precursor of βA, the amyloid precursor protein causes an AD-like syndrome when over-expressed in hippocampal neurons of transgenic mice (Games et al., 1995; LaFerla et al., 1995). A large number of similar biochemical and genetic studies suggest the implication of hA in the disease mechanisms of type 2 diabetes (for review, see Hull et al., 2004), and βA in those of AD (for review, see Selkoe, 2003). Recent reports further suggest that certain species of soluble oligomeric intermediates are substantially more cytotoxic than insoluble fibrils of amyloidogenic peptides, including βA and hA, (Meier et al., 2006; Stefani and Dobson, 2003). These findings indicate that there are substantial structural and functional similarities between hA and βA. Indeed, a recent report demonstrated that polyclonal antibodies raised against pre-fibrillar aggregates of βA peptides are able to recognize similar aggregates of hA and others, and suppress toxicity of these amyloidogenic peptides to cultured human neuroblastoma cells (Kayed et al., 2003). Therefore, a common mechanism has been proposed for cytotoxicity of βA and hA through their shared structural features of pre-fibrillar aggregates but not monomer forms.

To evoke cell death, the exogenously applied hA and βA aggregates have to first interact with the cell plasma membrane. However, it is still uncertain how exogenously applied hA and βA aggregates interact with cell membrane to initiate death in target cells (Stefani and Dobson, 2003). While hA has been consistently demonstrated to evoke both islet β-cell and neuronal cell death, the amyloidogenic βA has never been reported to kill islet β-cells. Thus, it is also not clear whether the mechanisms by which the two classes of peptides evoke cell death display aspects of their monomer specificity. Here, we examined the cytotoxicity of hA and βA peptides on cultured islet β-cells, and investigated the effects of the βA peptides, 1–40βA and 25–35βA on hA-mediated islet β-cell death and [125I-Tyr37]hA precipitation. We found that while 1–40βA and 25–35βA were not toxic to the cultured islet β-cells, they both inhibited hA-evoked islet β-cell death. However, only 1–40βA but not 25–35βA was found to promote cell growth and inhibit the precipitation of [125I]hA caused by hA. These results suggest that 25–35βA does not interfere with hA-mediated fibril formation and that the suppression of hA-evoked death by the βA peptides is possibly via a competing interaction at the cultured islet β-cells. This study provides important evidence that βA-based short peptides can modulate hA fibrillogenesis and its cytotoxicity and could therefore be promising candidates for therapeutic application in diabetes and tools for understanding hA fibrillogenesis and cytotoxicity.

2 Materials and methods

2.1 Peptides and chemicals

Synthetic human (PCPE60) and rat amylin (lot ZM275), 1–40βA (PNPE271) and 25–35βA (PNPE276) were all HPLC-purified products from Bachem California (Torrance, CA, USA). Peptides were stored under argon at −80°C until required, freshly dissolved in deionised Milli Q water at a concentration of 500μM, then diluted into tissue culture medium before addition to cultures to final concentrations. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (M2128) was from Sigma (St. Louis, MO). Fetal calf serum was purchased from Gibco-BRL-Life Technologies (Auckland, New Zealand). All other chemicals used were of analytical grade or higher.

2.2 Cell culture

The insulin-producing cell line RINm5F (Gazdar et al., 1980) were cultured at 37°C in 5% CO2/95% air (vol/vol) in RPMI 1640 medium supplemented with 10% (vol/vol) fetal calf serum, 290mg/ml l-glutamine, 100μg/ml penicillin and 100μg/ml streptomycin, as described previously (Bai et al., 1999). Cells were maintained in T75 tissue culture flasks and media changed every 3days. After reaching confluence, cells were harvested and replated by dissociation with 0.25% trypsin (v/v)/0.02% EDTA (w/v), then replated at required density. All experiments were performed with cells from passages 30 to 50.

2.3 Live/dead fluorescent assay

Islet β-cells, seeded at 4×104cells/ml, were cultured overnight on glass coverslips in 24-well plates, and treated with various concentrations of hA in culture medium for 24h. Following two gentle washes with phosphate-buffered saline (PBS), cell viability was determined by calcein-AM (1μM) and ethidium homodimer-1 (2μM) double staining (L3224, Molecular Probes, OR, USA) in PBS for 15min at 37°C. The live cells fluoresence bright green due to cleavage of calcein–AM to calcein. The dead cells take up ethidium homodimer-1 into the nucleus, resulting in a fluorescent orange-red colour. Green and red fluorescence were simultaneously visualised and the cells were thus manually counted using a fluorescent microscope equipped with a double-pass filter (FITC, excitation 490nm, emission 525nm; TMRITC, excitation 540nm, emission 570nm). Results are expressed as the percentage of dead cells relative to the total number of cells in the same culture. Values represent mean±SEM. Four 0.8-mm2 fields were scored per well, in triplicate 16-mm wells for each of 3 separate experiments. At least 400 cells were scored for each treatment.

2.4 Cellular MTT reduction assay

RINm5F cells, seeded at 8×105cells/ml, were cultured overnight in a 96-well plate, treated with peptides, and then incubated with 100μl of MTT (final concentration 0.5mg/ml) at 37°C for 4h. Following overnight addition of 100μl cell lysis buffer containing 20% SDS and 50% N,N-dimethylformamide (pH 4.7), Absorbance at 562nm was determined using an ELISA plate reader (Spectramax 340, Molecular Devices, CA, USA). Results are expressed as a percentage of MTT reduction (mean±SEM) relative to medium culture controls (defined as 100%) after removal of non-cell-derived background absorbance by subtracting relevant blank well readings.

2.5 Lactate dehydrogenase (LDH) release assay

Release of LDH was used to measure cytotoxicity in cultured islet β-cells, as previously used in assessing βA toxicity in neurons (Behl et al., 1994). Enzyme activity was determined using the LDH–glutamate/pyruvate transaminase–diaphorase method (Koh and Choi, 1987). Following peptide treatment, 100μl aliquots of culture medium were added to 900μl of reaction mixture containing 0.8mmol of sodium pyruvate and 0.1μmol of β-NADH in 0.1M K2HPO4/KH2PO4 buffer, pH 7.4. Results are expressed as percentage LDH release (mean±SEM) relative to total cellular LDH activity treated with culture medium controls determined following cell lysis with 0.1M K2HPO4/KH2PO4 containing 0.2% (vol/vol) Triton X-100, and after subtraction of the background activity in the culture medium.

2.6 DNA fragmentation assay

Total genomic DNA isolated from controls or hA-treated cells was analysed by agarose gel electrophoresis, as we previously described (Bai et al., 1999). In brief, cells were incubated in lysis buffer (10mM Tris–HCl (pH 8.0)/10mM NaCl/10mM EDTA/100μg/ml proteinase K/1%SDS) overnight at 55°C. DNA was extracted with phenol/chloroform (1:1, v/v), precipitated with cold 0.3M sodium acetate in ethanol. The DNA pellet was resuspended in 10mM Tris–HCl (pH 8.0)/1mM EDTA and incubated for 2h at 37°C with 0.5mg/ml DNase-free RNase (lot 1,119,915; Boehringer Mannheim, Mannheim, Germany). After re-extraction by the same method, DNA was analysed with 1.1% (w/v) agarose gels and detected with ethidium bromide.

2.7 In vitro [125I-Tyr37]hA precipitation assay

Tracer amounts of [125I-Tyr37]hA (4.5pM final concentration containing 4×104 counts per minute (cpm) per tube of test, Peninsula Laboratories, Belmont, CA) in aqueous solution (total volume 400μl in 1.5ml Eppendorf tubes) were incubated in the absence or presence of 10μM hA at various time points. To test the effects of βA peptides, 1–40βA and 25–35βA on [125I-Tyr37]hA precipitation by hA, the peptides were simultaneously included in the incubation mixtures at the stated concentrations for 24h. The incubation mixtures were then centrifuged (15,000×g, 20min), and the amount of [125I-Tyr37]hA in both supernatants and pellets determined (1480 WIZARD™ 3″ Automatic Gamma Counter; Wallac Oy, Turku, Finland). Results were expressed as percentage of the cpm in supernatants relative to the total cpm.

2.8 Statistical analysis

All results are expressed as mean±SEM for at least three separate experiments. Statistical analysis was performed with Statistica (StatSoft, Tulsa, OK, USA). Comparisons were made by analysis of variance (ANOVA) with post hoc analysis by the Tukey test.

3 Results

3.1 Cytotoxicity of synthetic hA to cultured islet β-cells

Cultured RINm5F islet β-cells were treated with aqueous solutions of synthetic hA. As shown in Fig. 1B, live/dead double-fluorescence assay revealed a distinct pattern of cell death in the treated β-cells from those with vehicle control (Fig. 1A). By contrast, rat amylin, which is highly homologous to hA but differs at six amino acids and does not form amyloid fibrils (Nishi et al., 1989; Westermark et al., 1990), was not toxic to islet β-cells (Fig. 1C). Similarly, there were no cytotoxic effects of cultured RINm5F cells observed for the AD-associated 1–40βA peptide and its fragment 25–35βA, as determined by the same method (Fig. 1D,E). When cell viability was examined by an alternative cellular MTT reduction assay, exposure of RINm5F cells to synthetic hA evoked both concentration- and time-dependent cell death (Fig. 2A,B). After 24h treatment, cell death was evident at 5μM hA, and reached a maximum level of about 80% at 20μM (Fig. 2A). The measurement of cell death was quantitatively reproducible when calibrated against viability assays performed with calcein-AM/ethidium homodimer-1 (Fig. 2C), and also MTT reduction against cell counts by seeding cells in 96-well plates in a dilution series (results not shown). Consistent with previous observations of primary rat and human islet β-cells (Bai et al., 1999; Lorenzo et al., 1994), the calculated EC50 value for the concentration response of hA cytotoxicity was 10μM. Time dependence studies of cell killing at 10μM hA indicated that cell death is asynchronous and detectable by 12h as expected, and reached 50% by 24h (Fig. 2B).

Fig. 1

Live/dead double-staining of cultured RINm5F cells exposed to synthetic human amylin or other peptides. RINm5F cells treated with either (A) vehicle (water) control, (B) 10μM hA, (C) 20μM rat amylin, (D) 50μM 1–40βA or (E) 50μM 25–35βA were stained with calcein-AM (1μM)/ethidium homodimer-1 (2μM) in PBS for 15min at 37°C. Green staining indicates living cells which are capable of concentrating the vital cytopasmic dye, calcein–AM, whereas red staining shows nuclei of dead cells that have lost the ability to exclude the DNA-staining fluorescent dye, ethidium homodimer. Yellow-orange cells are stained by both dyes, and are therefore in a relatively early stage of death. Micrographs represent 100× final magnification under epifluorescence microscopy (Olympus Model BH-2).

Fig. 2

Human amylin induces concentration- and time-dependent cytotoxicity in cultured RINm5F cells. Cellular 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release from damaged cells showing (A) concentration- and (B) time-dependent responses of hA-mediated cytotoxicity in cultured islet β-cells. MTT reduction and LDH release were calibrated (C) against viability assay of live/dead double fluorescent staining as stated in Fig. 1. (D) Agarose gel electrophoresis of islet β-cell DNA showing concentration-dependent internucleosomal DNA cleavage after hA treatment. Lane l, DNA marker of 100bp; lane rA, rat amylin. Cultured RINm5F cells were exposed to indicate concentrations of hA for 24h (A, C and D) or fixed concentration of 10μM hA for the indicated time periods (B). Levels of MTT reduction by cells and LDH activity in culture medium were then quantified. Results of time-dependence studies are expressed relative to corresponding controls at different time points. All results are means±SEM, n=12 from three separate experiments, *P<0.05, **P<0.001 relative to corresponding vehicle controls.

Since loss of MTT reduction is not necessarily a valid indicator of cell lysis (Behl et al., 1994; Shearman et al., 1994), hA toxicity was further examined using an LDH-release assay. However, in the presence of hA, the amount of LDH released into the culture media did not correlate with the extent of MTT reduction (Fig. 2A,B) or live/dead determinations (Fig. 2C). Following a 24h exposure to 10μM hA, there was only &007E;15% increase in LDH release relative to controls (P<0.001) (Fig. 2A). In contrast, there was &007E;50% of MTT reduction at 10μM hA concentration. Similarly, LDH release in 10μM hA-treated cultures was not significantly different from controls until 16h post-treatment; being &007E;5% compared with &007E;30% MTT reduction (Fig. 2B). Since LDH release was shown to be proportional to the number of cells lysed by 0.1% (w/v) Triton X-100, and the enzyme itself is stable in culture medium during a 24h incubation (results not shown), data from both concentration- and time-dependent LDH release in hA-treated RINm5F cell cultures strongly suggest that membrane integrity was still preserved in the early stage of hA toxicity as determined by cellular MTT reduction and live/dead assays. Late membrane lysis is one important characteristic of apoptosis (Loo et al., 1993).

To directly determine the mechanism by which hA evokes β-cell killing, we extracted genomic DNA from cells treated with hA. Agarose gel analysis of the DNA revealed internucleosomal DNA fragmentation (Fig. 2D), a further hallmark of apoptosis (Loo et al., 1993; Lorenzo et al., 1994). DNA fragmentation, with the smallest fragment in the ladder being approximate 180–200bp, was detected in hA-treated cells in a concentration-dependent manner. In contrast, treatment with rat amylin caused no detectable DNA fragmentation (Fig. 2D). This is consistent with our previous observation that dying hA-treated RINm5F islet β-cells show other ultrastructural features of apoptosis (Bai et al., 1999). Taken together, these results suggest that synthetic hA but neither rat amylin nor βA peptides evoked apoptotic cell death in continuous cultures of RINm5F pancreatic islet β-cell lines.

3.2 Effects of amyloid-associated peptides on hA-mediated cell death

To determine the effect of amyloid-associated peptides on hA-evoked islet β-cell death, quantitative concentration-response studies of the AD-associated 1–40βA peptide and its fragment 25–35βA were performed on hA-mediated cell death at a fixed concentration of 10μM hA. As shown in Fig. 3, none of the peptides tested reduced MTT reduction by the cultured islet β-cells, indicating that βA peptides are not toxic to the cultured islet β-cells as determined by the live/dead assay (Fig. 1D,E). It was also noticed that 1–40βA (Fig. 3A) but not 25–35βA (Fig. 3B) could significantly enhance MTT reduction by the islet β-cells at concentrations 100μM and below, indicating that the longer βA peptide is cytotrophic to the cultured RINm5F islet β-cells. However, both the 1–40βA (Fig. 3A), and 25–35βA (Fig. 3B) elicited concentration-dependent suppression of death evoked by hA, as determined by MTT assay. Non-fibril forming rat amylin (Fig. 3C) did not inhibit hA-mediated cell death. The calculated EC50 values for 1–40βA and 25–35βA to suppress hA-evoked cell death were both approximately 50μM, at a molar ratio of βA:hA of 5:1. At concentrations above 100μM with a molar ratio of βA:hA of 10:1, both βA-peptide completely suppressed hA-evoked cell death. These findings indicate that, whereas the amyloid-forming peptides 1–40βA and 25–35βA are not toxic to islet β-cells, they can suppress hA-evoked death in RINm5F islet β-cells.

Fig. 3

Effect of amyloid-associated peptides on hA-evoked islet β-cell killing. RINm5F cells cultured in 96-well plates (4×104 cells/well) were treated for 24h with 10μM hA (black bars) or with vehicle control (white bars) plus various concentrations of (A) 1–40βA; (B) 25–35βA; or (C) rat amylin. Following incubation, MTT reduction assays were performed as described in Fig. 2 legend and results expressed as percentages of MTT reduction (means±SEM, n=18 from four separate experiments). *P<0.05, **P<0.001 relative to hA treatment controls, and ##P<0.001 relative to vehicle controls at zero concentration of peptides.

3.3 Effects of amyloid-associated peptides on hA fibril formation

To explore the mechanisms by which these amyloid peptides suppress hA-evoked cell death, [125I-Tyr37]hA precipitation assays were carried out to determine if these peptides also affect fibril formation by hA. Tracer quantities of [125I]hA were incubated for 24h in the presence of water alone (control) or 10μM non-radiolabelled hA. The effects of amyloid peptides on hA-mediated [125I]hA precipitation were determined with the simultaneous addition of non-labelled peptides.

As shown in Fig. 4A, 90% of [125I]hA was precipitated in the presence of 10μM unlabelled hA, compared with 10% in water alone control. Further addition of 500μM hA to [125I]hA tracer resulted in almost complete precipitation of [125I]hA (Fig. 4E). These results indicate that [125I]hA was incorporated into hA-fibrils, leading to precipitation. Rat amylin (500μM, Fig. 4B), 25–35βA (500μM, Fig. 4C) and 1–40βA (200μM, Fig. 4D) caused modest significant enhancement in precipitation of [125I]hA compared with water alone control (Fig. 4A). Neither rat amylin (Fig. 4B) nor 25–35βA (Fig. 4C) significantly altered hA-mediated precipitation of [125I]hA. On the other hand, 1–40βA (Fig. 4D) completely suppressed the precipitation of [125I]hA caused by hA. These results suggest that 1–40βA and hA can form mixed fibrils, whereas 25–35βA and hA cannot. Therefore, the suppression of hA-evoked cell killing by 25–35βA is unlikely to occur through direct protein–protein interactions during fibril formation, although it is possible for the suppression of hA-evoked β-cell killing by 1–40βA.

Fig. 4

Amyloid-associated peptides on fibril formation by hA. Tracer amounts of [125I-Tyr37]hA (4×104cpm in 20μl per tube) were incubated in aqueous solutions for 24h at 37°C with the simultaneous addition of (A) vehicle (water), (B) rat amylin (500μM), (C) 25–35βA (200μM), (D) 1–40βA (200μM), or (E) human amylin (500μM) in 360μl to give the indicated final concentrations in a total volume of 400μl in the absence (white bars) or presence of 10μM hA (black bars). The amounts of [125I-Tyr37]hA in both supernatants (SN) and pellets were measured following centrifugation at 15,000×g for 20min. Results are expressed as percentages of cpm in SN relative to the total cpm at each point (mean±SEM, n=6 for each configuration from three separate experiments). Approximately 20% of [125I-Tyr37]hA was absorbed to the wall of Eppendorf test tubes (1.5ml volume) in water only control (A). ##P<0.001 relative to vehicle (water) only control, and **P<0.001 relative to hA treatment control in the water control of (A) using analysis of variance (ANOVA) with post hoc analysis by the Tukey test.

4 Discussion

This study confirms that hA, but not rat amylin, forms amyloid fibrils and evokes apoptotic cell death in cultured islet β-cells, suggesting that hA-evoked β-cell death is dependent on its ability to aggregate into amyloid fibrils. We further demonstrate for the first time that (1) hA-evoked β-cell killing was suppressed by the AD-associated amyloid peptides 1–40βA and 25–35βA, whereas they were both non-toxic to the cultured islet β-cells with 1–40βA being trophic at even lower concentrations; and (2) 1–40βA but not 25–35βA inhibited fibril formation by hA. We interpret these data as suggesting that hA-mediated β-cell death induced by a sequence and conformation-specific aggregate-cell interaction, which is inhibited by 25–35βA. Knowledge of the nature of such an interaction is of crucial importance to better understand amyloid associated-diseases and to identify the correct targets for drug design for type 2 diabetes therapy.

In agreement with our results, synthetic hA has been demonstrated to form amyloid fibrils in vitro and to evoke apoptosis in both rat and human islet β-cells through interaction of the fibrillar form of hA with the cell surface (Bai et al., 1999; Lorenzo et al., 1994). By contrast, the non-amyloidogenic rat amylin, which is 90% identical in sequence to hA but has no β-structure (Nishi et al., 1989; Westermark et al., 1990), has consistently been observed to be non-toxic (Bai et al., 1999; Lorenzo et al., 1994; Schubert et al., 1995). Conformation-dependent cytotoxicity of amyloid peptides has also been observed in neuronal apoptosis induced by the AD-associated βA (Jhamandas and MacTavish, 2004; Lorenzo and Yankner, 1994; Pike et al., 1993; Wogulis et al., 2005). In addition, studies have also shown that Congo Red can inhibit toxicity of βA (Burgevin et al., 1994; Lorenzo and Yankner, 1994) and hA (Lorenzo et al., 1994) by either binding to fibrils/aggregates or by inhibiting formation of fibrils/aggregates. There is also a report that rifampicin prevents hA fibril formation but not formation of toxic hA oligomers and β-cell apoptosis induced by either overexpression or application of hA (Meier et al., 2006). Therefore, it appears that hA is probably only cytotoxic in certain structural conformation of polymers.

In this regard, it has previously been demonstrated that the β-sheet structure is not by itself sufficient to cause cytotoxicity (Schubert et al., 1995; Yankner et al., 1990). In accord, the amyloidogenic βA has never been reported to kill islet β-cells, although hA has been consistently demonstrated to evoke both islet β-cell and neuronal cell death. We have shown here that the 1–40βA peptide at 200μM is sufficient in itself to form fibrils (Fig. 4D) but is not toxic to RINm5F cells. However, at concentrations 100μM and below it is trophic to the islet β-cells (Fig. 3A). This together suggests that the mechanism of islet β-cell toxicity of βA peptides is distinct from hA, and that hA-mediated islet β-cell toxicity is sequence-specific.

Accumulating data including our unpublished data suggest that small hA aggregates recovered in the soluble preparation are more likely to be the cell killing agents (Janson et al., 1999; Meier et al., 2006; Stefani and Dobson, 2003). This is consistent with the in vivo observations in diabetic transgenic mice or rats expressing hA in their islet β-cells (Butler et al., 2004; Janson et al., 1996; Verchere et al., 1996). These studies have also shown that antibodies specific to the toxic hA oligomers that are distinct from the hA monomers or insoluble amyloid fibrils suppress hA toxicity in cultured human neuroblastoma cells (Kayed et al., 2003; Meier et al., 2006). Although the relationship between the size or morphologies of the amyloid deposits and cytotoxicity has apparently not been defined, immunocytochemical staining combined with phase contrast microscopy did demonstrate the close association of amylin aggregates with dying β-cells following exposure to hA (Lorenzo et al., 1994).

Interestingly, insulin-like growth factors (IGFs) have been demonstrated to protect and rescue neurones against hA- and βA-induced toxicity at nanomolar concentrations (Dore et al., 1997). Substantial evidence also suggests that hA mediates islet β-cell death via activation of the c-Jun N-terminal kinase, p38 MAP kinase and caspase signalling pathways (Rumora et al., 2002; Zhang et al., 2003). Activation of these same pathways have also been observed in βA-treated hippocampal neurons (Wang et al., 2003) and βA-evoked neuronal apoptosis by binding a nerve growth factor co-receptor p75 to activate receptor signalling pathways (Costantini et al., 2005; Hashimoto et al., 2004; Yaar et al., 2002). Downstream activation of several functional transcription factors of these signalling pathways have also been reported, such as activating transcription factor 2 for hA-evoked β-cell apoptosis (Zhang et al., 2006), oxidative stress-related transcription factors NFKB (Costantini et al., 2005; Lezoualc'h and Behl, 1997) and cyclooxygenase-2 or other immediate-early genes (Tucker et al., 1998) for hA or βA-mediated death in neurons and other mammalian cells. Recent observation of Jhamandas and MacTavish (2004) that AC187, an amylin receptor antagonist, attenuates βA-mediated activation of caspases and death in basal forebrain neuronal cultures further support that βA toxicity may occur through the amylin receptor. These data strongly suggest the possible involvement of cell surface receptor pathway in amyloid-mediated cell death.

Here, we have showed that the βA peptides, 1–40βA and 25–35βA can suppress hA-evoked RINm5F islet β-cell death in a concentration-dependent and saturable manner. These results can be interpreted in light of [125I]hA precipitation studies. The concentration of 1–40βA employed in this study, 200μM, was sufficient for this peptide itself to form fibrils (Fig. 4D), but was not toxic to RINm5F cells (Fig. 3A). Thus, 1–40βA-mediated suppression of hA-evoked β-cell killing could possibly be due to protein–protein interactions between the two peptides during fibril formation. Alternatively, 1–40βA may also offset the cytotoxic effect of hA treatment by promoting the growth of the cultured islet β-cells as demonstrated by increased MTT reduction at lower concentrations (Fig. 3A). However, the suppression of hA-evoked cell killing elicited by 25–35βA was not accompanied by its trophic effect on the cultured RINm5F islet β-cells nor its inhibition of hA-mediated [125I]hA precipitation. 25–35βA was not trophic to the cultured RINm5F islet β-cells (Fig. 3B). It did not inhibit the precipitation of [125I]hA caused by hA (Fig. 4C), suggesting it does not interfere with hA-mediated fibril formation, being similar to the effect of the non-amyloidogenic and non-cytotoxic rat amylin. Nevertheless, it suppressed hA-evoked cell killing in a concentration-dependent and saturable manner. Taken together, data presented in this study strongly suggest that βA peptides could suppress hA-mediated islet β-cell death by competing with hA for a cell-surface receptor in a conformation-dependent manner. This is consistent with the fact that toxicity of hA and βA is dependent on the formation of β-pleated oligomeric aggregates although they share only 38% sequence identity (Lorenzo and Yankner, 1994; Lorenzo et al., 1994).

There are also suggestions that both βA and hA kill primary and clonal neuron cells by intercalating into the plasma membrane through the amphiphilic natures of their β structure which leads to activation of enzyme or signal pathways (Schubert et al., 1995), or by forming the oligomeric aggregate at or near the cell surface through free radical production and oxidative stress (Mattson and Goodman, 1995; Tucker et al., 1998; Wogulis et al., 2005). Although we cannot exclude the possibility of non-receptor-mediated mechanisms for the neuronal cell death caused by βA and hA, this mechanism of toxicity to islet β-cell appears unlikely from the results of the present studies that only hA, but not the similarly fibril-forming βA peptides, was toxic to cultured islet β-cells, and that both 1–40βA and 25–35βA dose-dependently inhibited hA-evoked β-cell death regardless of their distinct effects on islet β-cell growth and hA fibril formation. While it is not known why 1–40βA and 25–35βA have distinct effects on islet β-cell growth and hA fibril formation, our results imply that other amino acid sequences within the longer βA peptide may be of crucial importance. It also appears that the cytotoxic effects of the amyloid-forming peptides are both sequence- and cell type-dependent, as discussed above. Yankner et al. (1990) have also observed both neurotrophic and neurotoxic effects of the βA protein which are dependent on the age of the neuron and the concentration and sequence of the βA peptides.

Another mechanism of islet β-cell death by direct membrane disruption of hA aggregates (Harroun et al., 2001; Janson et al., 1999) or formation of ion-permeable channels by non-aggregated hA within the cell membrane (Mirzabekov et al., 1996) is not consistent with our previous observation that hA-mediated islet β-cell apoptosis was not associated with changes in intracellular free Ca2+ concentration (Bai et al., 1999). Therefore, it appears more plausible that there are structural factors that enable amylin oligomers to bind to islet β-cell membrane leading to subsequent activation of cell death. While the precise molecular mechanism(s) underlying this interaction remains to be identified, our results support the possibility that non-fibrillar hA aggregates (rather than its mature fibrils) may be the primary toxic species associated with the type 2 diabetic diseases (Janson et al., 1999; Kayed et al., 2003; Meier et al., 2006; Stefani and Dobson, 2003), and the rational for peptide-based design of potent amyloid disease therapeutics (Tatarek-Nossol et al., 2005; Yan et al., 2006). In summary, βA-based short peptides with their distinct actions on hA-mediated islet β-cell viability and fibril formation may help in better understanding of how hA aggregates interact with the cell to initiate cell death, and therefore are promising candidates for therapeutic application in diabetes and related disorders.

Acknowledgements

Professor Garth Cooper is thanked for allowing the author to pursue this project in his laboratory and providing early discussions. Help from Dr Jun Hiyama with discussions is also appreciated. This work was supported by a University of Auckland Graduate Research Fund.

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Received 12 June 2007/29 August 2007; accepted 22 December 2007

doi:10.1016/j.cellbi.2007.12.016
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