In this study, we sought to determine the mechanism(s) by which islet amyloid formation, under conditions of endogenous hIAPP production, results in beta cell apoptosis. We chose to examine the role of JNK signalling, a pro-apoptotic pathway that mediates beta cell apoptosis in response to several stressors including exogenous hIAPP treatment [25
]. We found that JNK signalling is activated during the process of islet amyloid formation, and this occurs in part downstream of amyloid formation. We have also shown that the JNK pathway is a critical mediator of islet amyloid-induced beta cell apoptosis and identified potential downstream mediators in both the extrinsic (Fas
) and intrinsic (Bim
) apoptotic pathways. Last, we observed that the anti-apoptotic molecule Bclxl
is upregulated in a JNK-dependent manner downstream of islet amyloid formation.
Culture of hIAPP
transgenic islets in high glucose (16.7 mmol/l) results in light microscopy-visible amyloid deposition and associated beta cell apoptosis [15
]. We observed JNK activation, manifest as increased levels of p-JNK and p-cJUN protein and cJun
mRNA in hIAPP
transgenic islets only under amyloidogenic (16.7 mmol/l glucose) conditions, indicating that the observed JNK activation was not merely secondary to the expression of the hIAPP
transgene, but rather was dependent on the process of amyloid formation. It has been previously shown that culture of human islets in increased glucose (33.3 mmol/l) for 18 h induces JNK activation and beta cell apoptosis [20
]. All human islets have the propensity to develop islet amyloid, although this was not examined in the study by Maedler et al. [20
], making it difficult to discern whether the observed JNK activation was secondary to high glucose or the presence of amyloid. To control for the potential effect of glucose independent of amyloid, we cultured non-transgenic islets in 11.1 and 16.7 mmol/l glucose and observed no difference in either p-JNK or p-cJUN protein levels or cJun
mRNA levels, demonstrating that increased glucose alone was insufficient for JNK activation. In fact, under increased glucose conditions in non-transgenic islets there was a tendency for a decrease in the activation of JNK that did not coincide with a reduction in phosphorylation of cJUN, an observation that would not be in line with the canonical mechanism of JNK. Thus, we conclude that the observed JNK activation occurs only in the presence of islet amyloid formation.
There are several potential mechanism(s) that underlie islet amyloid-induced JNK activation including oxidative stress, which we have shown to be an important mediator of islet amyloid-induced cytotoxicity [15
]. Another possible mechanism is inflammation. Culture of human islets under high glucose, a potentially amyloidogenic condition, stimulates IL-1β production, which mediates beta cell apoptosis [35
]. Our recent study demonstrated that fibrillar hIAPP elicits an inflammatory response (including IL-1β production) from bone marrow-derived dendritic cells and showed IL-1β co-localisation with islet amyloid deposits in vivo [36
]. It is therefore certainly possible that islet amyloid formation could also induce IL-1β production and JNK activation in beta cells, a scenario requiring further study.
As the process of islet amyloid formation is time dependent [33
], we also determined whether JNK activation persists with increasing amyloid formation over time. We demonstrated that JNK is activated in the early stages of amyloid formation (48 h), and remains elevated under more chronic conditions of amyloid deposition (144 h). In contrast, non-transgenic islets demonstrated no increase in JNK activation over time. At 48 h, JNK signalling manifested as increased p-cJUN without a significant increase in total cJUN, although cJun
mRNA is increased at this time point. Under the condition of prolonged amyloid formation, increases in p-cJUN, total cJUN and cJun
mRNA were observed. This pattern of activation is somewhat consistent with that reported in 16 h studies in which micromolar concentrations of hIAPP were applied to beta cell lines and increased phosphorylation of cJUN preceded increases in total cJUN [25
]. To capture the kinetics of JNK activation in relation to the process of islet amyloid formation in our model, it would have been ideal to examine islets prior to the formation of light microscopy-visible amyloid deposits (prior to 24 h in culture). However, it is well documented that the islet isolation procedure itself induces stress and JNK activation, precluding this early time point [37
]. As isolated islets recover to a low basal rate of JNK signalling following 48 h in culture [37
], we controlled for the stress of isolation by using an overnight recovery period, followed by a minimum of 48 h in culture prior to making any stress signalling measurements. The low, reproducible p-cJUN levels observed in non-transgenic islets suggest that this recovery period is sufficient to control for the confounding effects of the stress of isolation and culture.
To determine whether the JNK pathway is activated upstream or downstream of islet amyloid formation, we interrogated the culture system with the amyloid inhibitor Congo Red. This compound inhibits islet amyloid formation resulting in a rate of beta cell apoptosis in hIAPP transgenic islets similar to that in non-transgenic islets [15
]. Treatment with Congo Red resulted in a significant but incomplete reduction in p-cJUN levels. We propose two possible explanations for this observation. First, the JNK signalling pathway is activated in part downstream of islet amyloid but there may be an additional upstream role of JNK signalling. This upstream JNK signalling may (1) play a role in mediating amyloid formation itself or (2) mediate other cytotoxic effects of hIAPP that are potentially amyloid independent. We believe the former is unlikely given that hIAPP
transgenic islets treated with a JNK inhibitor demonstrated no change in amyloid severity. The latter explanation is plausible, although it would be a challenging paradigm to explore in our model in which amyloid is induced as early as 24 h in culture, and studies prior to this time point are subject to the confounding effects of the stress of isolation.
We have shown that JNK signalling is a critical mediator by which islet amyloid induces beta cell apoptosis. JNK inhibition resulted in reduced beta cell apoptosis in hIAPP
transgenic islets to levels identical to those in non-transgenic islets. Furthermore, JNK signalling under conditions of amyloid formation upregulated mRNA levels of the pro-apoptotic signalling molecules Fas
in the extrinsic pathway, Bim
in the intrinsic pathway and, as expected, the terminal effector caspase, Casp3
, through which both the extrinsic and intrinsic pathways converge to induce apoptosis. The critical role of CASP3 in mediating the toxicity of islet amyloid has been underscored by a recent in vivo study in hIAPP
transgenic mice, which demonstrated that prevention of CASP3 activation protected beta cells from amyloid-induced apoptosis and resulted in preservation of beta cell mass and improved beta cell function [38
The extrinsic pathway is typically initiated by cell surface death receptors, which includes the FAS ligand receptor, resulting in downstream JNK activation and caspase 8 activation. A recent study has shown that exogenous hIAPP treatment of beta cell lines over an acute culture period of 8 h results in upregulation of FAS and FAS-associated death domain (FADD), both at the mRNA and protein levels, with associated increases in beta cell apoptosis [28
]. Furthermore, this study demonstrated that hIAPP induces FAS through an interaction at the cell surface, which results in JNK activation. JNK signalling is then able to feedback to upregulate levels of FAS, perpetuating a cycle of pro-apoptotic signalling. These observations are consistent with those in our model of endogenous hIAPP production and islet amyloid formation in which we observe JNK-dependent upregulation of Fas
The intrinsic pathway of apoptosis involves a dynamic interplay between pro-apoptotic B-cell leukaemia/lymphoma 2 (BCL2) homology domain 3 (BH3)-only proteins and anti-apoptotic BCL2-like proteins. In response to pro-apoptotic stimuli, BH3-only proteins bind anti-apoptotic BCL2-like proteins, resulting in the release of BCL2-associated agonist of cell death (BAD) and/or BCL2 agonist killer 1 (BAK), which induce changes in mitochondrial membrane permeability and membrane potential causing cytochrome c
release and activation of effector caspases to induce apoptosis [39
]. We show for the first time that the process of islet amyloid formation and its resultant toxicity activates the pro-apoptotic BH3-only molecule Bim
, in a JNK-dependent manner. The involvement of the intrinsic pathway of apoptosis in the cytotoxicity of islet amyloid is consistent with our previous findings that islet amyloid formation is associated with induction of oxidative stress [15
], and oxidative stress is known to activate the intrinsic pathway [41
]. In islets, BCL2-interacting mediator of cell death (BIM) has also been shown to be a critical mediator of glucotoxicity and is induced with cytokine treatment, underscoring its importance in mediating apoptosis in response to other types of stress in the islet [40
]. Furthermore, exogenous hIAPP treatment of a beta cell line has been shown to (1) upregulate several BH3-only proteins including BCL2-associated protein X (BAX), BAD, p53-upregulated modulator of apoptosis (PUMA) and truncated BH3-interacting domain death agonist (t-BID), (2) induce cytochrome c
release and (3) activate caspase 7 and 9 [43
The role of the anti-apoptotic proteins and how their dynamic regulation impacts the fate of the beta cell in response to the classic stressors of diabetes is largely unrecognised. Given that we have shown that islet amyloid formation results in beta cell apoptosis, we hypothesised that we may observe downregulation in the anti-apoptotic BCL2-like family of proteins as a potential mechanism of islet amyloid-induced beta cell death. Interestingly, we demonstrated no significant change in the levels of Bcl2
, and found a JNK-dependent increase in Bclxl
in the presence of islet amyloid formation. This is in contrast to studies with exogenous hIAPP treatment, which have found no difference in BCL2-like protein 1 (BCLXL) levels and a small decrease in BCL2 protein levels [43
]. The increase in Bclxl
in our study may represent a compensatory pro-survival mechanism by which the beta cell is attempting to offset the toxicity of amyloid formation. The induction of anti-apoptotic factors in response to beta cell stress has been observed in islets treated with cytokines [44
] and islets up to 1 week post transplantation [45
]. Although the JNK signalling pathway has classically been thought to be pro-apoptotic, there is growing evidence that JNK signalling can induce pro-survival pathways in certain cell types, including neurons, T cells and B lymphocytes [46
]. The anti-apoptotic response in islets challenged with cytokines is JNK3-dependent, which is in contrast to the JNK1 and JNK2 isoforms, which are pro-apoptotic [30
]. By using a non-specific JNK inhibitor, we were unable to differentiate between the contributions of the different JNK isoforms. The significance of JNK-dependent Bclxl
upregulation in the presence of islet amyloid as a potential compensatory protective mechanism will require further exploration, and could represent a potential therapeutic target that could be modulated to protect the beta cell from amyloid-induced cytotoxicity.
We used Congo Red, an amyloid inhibitor, which we have shown reproducibly inhibits islet amyloid formation and reduces associated beta cell apoptosis [15
]. Interestingly, we observed changes in mRNA levels of some pro-apoptotic genes in our non-transgenic islets, suggesting that Congo Red may have amyloid-independent effects in islets. Whether this would have long-term deleterious effects is unclear, although a recent report in Caenorhabditis elegans
described an extension of lifespan and a slowing of ageing with another amyloid-binding dye, Thioflavin T [47
]. Other amyloid inhibitors, including WAS-406 (2-acetamido-1,3, 6-tri-O
-xylo-hexopyranose), although effective at reducing amyloid deposition, also have amyloid-independent effects and may impact islet cell viability, and as such are limited in their utility [15
]. Thus, a consideration in the future development of amyloid inhibitors would be to maximise specificity for amyloid while minimising potential off-target effects.
In summary, we have demonstrated that JNK signalling is a critical mediator of islet amyloid-induced beta cell apoptosis and that pro-apoptotic molecules in the extrinsic and intrinsic pathway are induced by JNK signalling downstream of amyloid formation. Based on these findings, we believe that future studies should evaluate JNK signalling in relation to oxidative stress and the relative contribution and importance of the downstream signalling molecules in the extrinsic and intrinsic pathways in mediating islet amyloid-induced beta cell apoptosis. Interventions targeted to prevent the activation of the JNK pathway and/or downstream mediators induced by islet amyloid may offer therapeutic benefit in minimising toxicity of islet amyloid and the preservation of beta cells in type 2 diabetes.