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Cell Biology International (2008) 32, 447455 (Printed in Great Britain)
β-Amyloid peptides 1–40βA and 25–35βA suppress human amylin-mediated death of RINm5F islet β-cells with distinct actions on fibril formation
Department of Physiology, School of Medical and Health Sciences, University of Auckland, Private Bag 9201, Auckland, New Zealand
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.
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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
2.2 Cell culture
The insulin-producing cell line RINm5F (Gazdar et al., 1980) were cultured at 37
2.3 Live/dead fluorescent assay
Islet β-cells, seeded at 4
2.4 Cellular MTT reduction assay
RINm5F cells, seeded at 8
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, 100
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 (10
2.7 In vitro [125I-Tyr37]hA precipitation assay
Tracer amounts of [125I-Tyr37]hA (4.5
2.8 Statistical analysis
All results are expressed as mean
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 24
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) 10
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 100
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 24
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 180–200
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 10
Effect of amyloid-associated peptides on hA-evoked islet β-cell killing. RINm5F cells cultured in 96-well plates (4
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 24
As shown in Fig. 4A, 90% of [125I]hA was precipitated in the presence of 10
Amyloid-associated peptides on fibril formation by hA. Tracer amounts of [125I-Tyr37]hA (4
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 200
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 NF
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, 200
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.
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 2007doi:10.1016/j.cellbi.2007.12.016