**1. Introduction**

212 Type 1 Diabetes – Complications, Pathogenesis, and Alternative Treatments

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Similarities Reveals Common Functional Modules Enriched for Pluripotent Drug

#### **1.1 Pathophysiology of type I diabetes mellitus: Role of pro-inflammatory cytokines**

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterised by the destruction of insulin-producing β-cells in the pancreatic islets of Langerhans (Fig.1), which is mediated by autoreactive T cells, macrophages and pro-inflammatory cytokines (Fig.2). This leads to an inability to produce sufficient insulin resulting in elevated blood glucose levels and pathological effects (Eizirik & Mandrup-Poulsen, 2001).

T1DM is believed to be initiated by physiological -cell death or islet injury triggering the homing of macrophages and dendritic cells that in turn launch an inflammatory reaction. The infiltrating macrophages secrete pro-inflammatory cytokines, namely interleukin-1β (IL-1 and tumour necrosis factor (TNF as well as various chemokines that attract immune cells such as dendritic cells, macrophages and T lymphocytes. T cells recognising cell-specific antigens become activated, infiltrate the inflamed islets and attack the -cells (Baekkeskov et al., 1990, Elias et al., 1995, Lieberman et al., 2003, Nakayama et al., 2005). In a normally functioning immune system, T cells with a high affinity for self-antigens are eliminated during their differentiation resulting in immune 'tolerance'. Autoreactive cells that have escaped these mechanisms are subject to 'peripheral immune regulation' that blocks their activation and clonal expansion, preventing development of an autoimmune disease (Mathis & Benoist, 2004). For reasons we do not fully understand, these immune regulatory mechanisms either fail to launch, or are ineffective in stopping the immune attack against the -cells in T1DM, and a positive feedback cycle is established (Mathis & Benoist, 2004). This forward-feeding process of T cell- and cytokine-mediated -cell killing can be ongoing for years progressively destroying the -cells. When over 80 % of the -cells are deleted by this continuous T lymphocyte and inflammatory cytokine-driven attack the insulin secretory capacity falls below a certain threshold and the disease manifests itself.

Activated T cells induce death of a target cell by (1) secreting perforin and granzymes, (2) releasing pro-inflammatory cytokines including interferon-γ (IFNγ) and TNFα or (3) activation of Fas receptors on the surface of target cells. All these factors have also been described to contribute to β-cell killing in T1DM (Kägi et al., 1997, D. Liu et al., 2000, Petrovsky et al., 2002, Suk et al., 2001). In particular, recent evidence suggests that the

Cytokine-Induced -Cell Stress and Death in Type 1 Diabetes Mellitus

Fig. 2. Cytokine-induced β-cell death. Initial β-cell death caused by injury, infection or physiologically during development can activate an autoimmune response that leads to activation and infiltration of cytokine-secreting macrophages, dendritic cells (DC) and T cells (TC). Pro-inflammatory cytokines IL-1β, TNFα and IFNγ secreted by macrophages and

TCs cause β-cell stress and death and secretion of chemokines that further stimulate

secretion, β-cell stress and death are detailed below (also see Fig. 3).

cause stress in -cells which eventually activates the cell's death machinery. The signal transduction pathways activated by these pro-inflammatory cytokines leading to chemokine

It is very important to note that any of the above pro-inflammatory cytokines alone has limited effects in terms of cell stress or death, on -cells. However, combinations of IL-1β/IFN or TNF/IFN have very strong, synergistic effects that trigger serious levels of

The main mediator of IL-1β signalling is the transcription factor nuclear factor kappa B (NF- B) (Flodström et al., 1996, Kwon et al., 1995). The pathway by which IL-1β activates NF-B has been delineated in a number of cell types and experimental models (Fig.3). It is thought that the same mechanisms are involved in pancreatic -cells. IL-1β, secreted by activated macrophages and T cells, binds to the IL-1 receptor 1 (IL-1R1) on the surface of target cells. IL-1R1 then recruits IL-1 receptor accessory protein (IL-1RAcP) (Dinarello, 1997). This allows binding of the adaptor protein myeloid differentiation factor 88 (MyD88) and recruitment of IL-1R1 activated kinase 1 (IRAK1) and/or IRAK2 (Burns et al., 1998, Muzio et al., 1997, Wesche et al., 1997). IRAK proteins are in complex with a protein named Tollip prior to recruitment to the receptor (Burns et al., 2000). Tollip associates with IL-1RacP when the IRAK/Tollip complex is recruited to the activated receptor. TNF-receptor-associated

autoimmune cell infiltration.

stress culminating in cell death.

**1.2.1 IL-1β signalling** 

215

Fig. 1. β-cell islets in the pancreas of (A) pre-diabetic and (B) diabetic NOD mice. The yellow arrows indicate the islets in the haematoxylin-eosin stained tissue section (original magnification 200X).

cytokines IL-1β, TNFα and IFNγ that are secreted by macrophages and T cells have a broader role in the development of T1DM than previously thought. They are the main inducers of β-cell stress responsible for significant levels of -cell death in both rodent (Iwahashi et al., 1996, Rabinovitch et al., 1994) and human (Delaney et al., 1997) experimental models of T1DM.

Underlining the importance of the cytokines, it has been shown that neutralisation of the pro-inflammatory cytokines by antibodies and/or soluble cytokine receptors against IL-1, IFN IL-6 and TNF can inhibit the development of T1DM in NOD mice or BB rats (Mandrup-Poulsen, 1996). Transgenic mice expressing IFN in -cells develop severe insulitis (pre-diabetes) and destruction of -cells. Treatment of these mice with anti-IFNantibody prevents the development of T1DM. IFN-deficient mice as well as mice injected with neutralising anti-IFN receptor antibodies were resistant to development of experimentally-induced T1DM (Cailleau et al., 1997, Seewaldt et al., 2000, B. Wang et al., 1997). Similar to IFN, genetic or pharmacological abrogation of IL-1β action also reduces disease development in animal models of T1DM (Mandrup-Poulsen et al., 2010).

Although many factors contribute to β–cell destruction during T1DM, in this book chapter we review current knowledge regarding the role of cytokines mediating β–cell stress and death in T1DM.

#### **1.2 Signal transduction of pro-inflammatory cytokines in -cells**

IL-1β, IFNγ and TNFα exert a variety of effects on -cells. They sensitise -cells to apoptosis by increasing the expression of pro-apoptotic proteins, such as the Fas receptor (Stassi et al., 1997). They drive and stabilise the autoimmune response by triggering the secretion of chemokines (e.g. CXCL9 and CXCL10) by -cells (Frigerio et al., 2002), which results in constant recruitment of autoreactive T cells. Finally, pro-inflammatory cytokines directly

Fig. 1. β-cell islets in the pancreas of (A) pre-diabetic and (B) diabetic NOD mice. The yellow

cytokines IL-1β, TNFα and IFNγ that are secreted by macrophages and T cells have a broader role in the development of T1DM than previously thought. They are the main inducers of β-cell stress responsible for significant levels of -cell death in both rodent (Iwahashi et al., 1996, Rabinovitch et al., 1994) and human (Delaney et al., 1997)

Underlining the importance of the cytokines, it has been shown that neutralisation of the pro-inflammatory cytokines by antibodies and/or soluble cytokine receptors against IL-1, IFN IL-6 and TNF can inhibit the development of T1DM in NOD mice or BB rats (Mandrup-Poulsen, 1996). Transgenic mice expressing IFN in -cells develop severe insulitis (pre-diabetes) and destruction of -cells. Treatment of these mice with anti-IFNantibody prevents the development of T1DM. IFN-deficient mice as well as mice injected with neutralising anti-IFN receptor antibodies were resistant to development of experimentally-induced T1DM (Cailleau et al., 1997, Seewaldt et al., 2000, B. Wang et al., 1997). Similar to IFN, genetic or pharmacological abrogation of IL-1β action also reduces

Although many factors contribute to β–cell destruction during T1DM, in this book chapter we review current knowledge regarding the role of cytokines mediating β–cell stress and

IL-1β, IFNγ and TNFα exert a variety of effects on -cells. They sensitise -cells to apoptosis by increasing the expression of pro-apoptotic proteins, such as the Fas receptor (Stassi et al., 1997). They drive and stabilise the autoimmune response by triggering the secretion of chemokines (e.g. CXCL9 and CXCL10) by -cells (Frigerio et al., 2002), which results in constant recruitment of autoreactive T cells. Finally, pro-inflammatory cytokines directly

arrows indicate the islets in the haematoxylin-eosin stained tissue section (original

disease development in animal models of T1DM (Mandrup-Poulsen et al., 2010).

**1.2 Signal transduction of pro-inflammatory cytokines in -cells** 

magnification 200X).

death in T1DM.

experimental models of T1DM.

Fig. 2. Cytokine-induced β-cell death. Initial β-cell death caused by injury, infection or physiologically during development can activate an autoimmune response that leads to activation and infiltration of cytokine-secreting macrophages, dendritic cells (DC) and T cells (TC). Pro-inflammatory cytokines IL-1β, TNFα and IFNγ secreted by macrophages and TCs cause β-cell stress and death and secretion of chemokines that further stimulate autoimmune cell infiltration.

cause stress in -cells which eventually activates the cell's death machinery. The signal transduction pathways activated by these pro-inflammatory cytokines leading to chemokine secretion, β-cell stress and death are detailed below (also see Fig. 3).

It is very important to note that any of the above pro-inflammatory cytokines alone has limited effects in terms of cell stress or death, on -cells. However, combinations of IL-1β/IFN or TNF/IFN have very strong, synergistic effects that trigger serious levels of stress culminating in cell death.

#### **1.2.1 IL-1β signalling**

The main mediator of IL-1β signalling is the transcription factor nuclear factor kappa B (NF- B) (Flodström et al., 1996, Kwon et al., 1995). The pathway by which IL-1β activates NF-B has been delineated in a number of cell types and experimental models (Fig.3). It is thought that the same mechanisms are involved in pancreatic -cells. IL-1β, secreted by activated macrophages and T cells, binds to the IL-1 receptor 1 (IL-1R1) on the surface of target cells. IL-1R1 then recruits IL-1 receptor accessory protein (IL-1RAcP) (Dinarello, 1997). This allows binding of the adaptor protein myeloid differentiation factor 88 (MyD88) and recruitment of IL-1R1 activated kinase 1 (IRAK1) and/or IRAK2 (Burns et al., 1998, Muzio et al., 1997, Wesche et al., 1997). IRAK proteins are in complex with a protein named Tollip prior to recruitment to the receptor (Burns et al., 2000). Tollip associates with IL-1RacP when the IRAK/Tollip complex is recruited to the activated receptor. TNF-receptor-associated

Cytokine-Induced -Cell Stress and Death in Type 1 Diabetes Mellitus

chapter (under section 2.2).

**1.2.2 TNFα signalling** 

A large number of NF-κB target genes were identified using DNA microarray technology in cytokine-treated primary rat β-cells (Cardozo et al., 2001a). Cytokines induced NF-κBdependent up-regulation of genes involved in stress responses (including CHOP, C/EBPβ and δ, Hsp27 and MnSOD), immune responses (e.g. MHC-II-associated invariant chain γ and MHC-I) and down-regulation of genes involved in β-cell function (glucose transporter-2 (Glut-2)), insulin production (Isl-1), insulin processing (PC-1), insulin release (PLD-1,

In addition to NF-κB, IL-1β signalling also activates the mitogen activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) 1/2 and induces suppressor of cytokine signalling-3 (SOCS-3) (Emanuelli et al., 2004). Signal transduction pathways induced by MAPKs and SOCS-3 are interlinked with the NF-κB-regulated pathways; MAPK activation potentiates IL-1β-dependent NF-κB activation and subsequent iNOS induction, and (ERK)1/2 activation was shown to contribute to cytokine-induced apoptosis in rat pancreatic -cells (Pavlovic et al., 2000). While MAPKs positively affect NF-κB signalling and enhance β-cell death, SOCS-3 has a negative effect. SOCS-3 belongs to a family of proteins that provide a negative feedback for cytokine-induced signalling. It was also identified as an inhibitor of insulin signalling (Emanuelli et al., 2000) as SOCS-3 can bind to the insulin receptor and block its insulin-induced autophosphorylation and activation (Emanuelli et al., 2004). SOCS-3 inhibits IL-1β signalling upstream and thus negatively regulates nearly all effects of IL-1β. SOCS-3 suppresses the expression of several IL-1βinduced pro-apoptotic genes, many of them known to be NF-κB-dependent (Karlsen et al., 2004) and protects rat β-cells from IL-1β- and TNFα-induced cell death (Bruun et al., 2009). As mentioned above, over 200 genes have been identified to be NF-κB-regulated in β-cells treated with pro-inflammatory cytokines. However, which of these genes are targets of IL-1β signalling, or to what extent their expression is regulated by IL-1β alone is currently unknown. Determining the individual targets of the cytokines would lead to a better

understanding of how the cytokines synergise to cause β-cell stress and death.

al., 2008). This effect was more pronounced in response to IL-1β than TNFα.

TNFα was also shown to lead to activation of NF-κB in pancreatic β-cells (Ortis et al., 2006). TNFα binds to and activates the TNF receptor (TNFR1), which is present on the surface of βcells (Kägi et al., 1999). TNFα binding to TNFR1 leads to the latter's trimerisation and activation (Fig. 3). Upon activation, the cytosolic death domain of TNFR1 recruits TNF receptor-associated death domain (TRADD) (Hsu et al., 1995), TRAF2 (Hsu et al., 1996b) and the death domain kinase receptor interacting protein (RIP) (Hsu et al., 1996a). TRAF2, in turn, recruits IκB kinase (IKK) and induces its activation in a RIP-dependent manner via activation of an IKK kinase (e.g. NIK) (Devin et al., 2000). Activated IKK phosphorylates IκB proteins leading to their proteasomal degradation and release of NF-κB. The activation of NF-κB by both TNFα and IL-1β has a pro-apoptotic effect in rat pancreatic β-cells (Ortis et

CCKA-receptor) and Ca2+ homeostasis (SERCA2, IP 3-kinase) (Cardozo et al., 2001a). Inducible nitric oxide synthase (iNOS) is strongly induced and is the best characterised NFκB target in both rat β-cells (Cardozo et al., 2001a, Kutlu et al., 2003) and human pancreatic islets (Flodström et al., 1996). Induction of iNOS increases nitric oxide (NO) production in βcells, resulting in the generation of reactive oxygen species (ROS) and oxidative stress. The cellular stress triggered by NO in rodent and human cells will be discussed later in this

217

Fig. 3. Cytokine-signalling in pancreatic β-cells. IL-1β, TNFα and IFNγ activate receptors on the surface of β-cells inducing a signalling cascade leading to the activation of transcription factors STAT1 and NF-κB that control numerous genes involved in β-cell function, inflammation, stress responses and apoptosis.

factor-6 (TRAF6) is recruited to IRAK1 and IRAK2 (Muzio et al., 1997, Yamin & Miller, 1997) leading to the activation of inhibitor of NF-κB (IκB) kinase (IKK) *via* NF-κB inducing kinase (NIK). IKK then phosphorylates IκB which triggers its degradation and the release of the transcription factor NF-κB from the inhibitory interaction.

In addition, phosphatidyl inositol-3 kinase (PI3K) is recruited to the activated IL1-R1 complex where it becomes activated (Reddy et al., 1997, Reddy et al., 2004). PI3K activity is required, but not sufficient for NF-κB activation (Reddy et al., 1997).

NF-κB can regulate the transcription of numerous target genes (for review see (Pahl, 1999)). The target genes include cytokines (e.g. IL-1β, TNFα, IFNγ), chemokines, immunoreceptors, proteins involved in antigen presentation, cell adhesion molecules, stress response genes, regulators of apoptosis (both pro- and anti-apoptotic), growth factors and other transcription factors. The effects of NF-κB signalling are highly cell type-specific. In most cell types the net effect of NF-κB activation is to promote cell survival. In contrast, in β-cells NF-κB activation has a pro-apoptotic effect (Eldor et al., 2006, Ortis et al., 2008). These studies demonstrate that inhibition of NF-κB protects rodent pancreatic β-cells from the damaging effects of cytokineexposure *in vitro* and prevents streptozocin-induced diabetes *in vivo*.

Fig. 3. Cytokine-signalling in pancreatic β-cells. IL-1β, TNFα and IFNγ activate receptors on the surface of β-cells inducing a signalling cascade leading to the activation of transcription

factor-6 (TRAF6) is recruited to IRAK1 and IRAK2 (Muzio et al., 1997, Yamin & Miller, 1997) leading to the activation of inhibitor of NF-κB (IκB) kinase (IKK) *via* NF-κB inducing kinase (NIK). IKK then phosphorylates IκB which triggers its degradation and the release of the

In addition, phosphatidyl inositol-3 kinase (PI3K) is recruited to the activated IL1-R1 complex where it becomes activated (Reddy et al., 1997, Reddy et al., 2004). PI3K activity is

NF-κB can regulate the transcription of numerous target genes (for review see (Pahl, 1999)). The target genes include cytokines (e.g. IL-1β, TNFα, IFNγ), chemokines, immunoreceptors, proteins involved in antigen presentation, cell adhesion molecules, stress response genes, regulators of apoptosis (both pro- and anti-apoptotic), growth factors and other transcription factors. The effects of NF-κB signalling are highly cell type-specific. In most cell types the net effect of NF-κB activation is to promote cell survival. In contrast, in β-cells NF-κB activation has a pro-apoptotic effect (Eldor et al., 2006, Ortis et al., 2008). These studies demonstrate that inhibition of NF-κB protects rodent pancreatic β-cells from the damaging effects of cytokine-

factors STAT1 and NF-κB that control numerous genes involved in β-cell function,

inflammation, stress responses and apoptosis.

transcription factor NF-κB from the inhibitory interaction.

required, but not sufficient for NF-κB activation (Reddy et al., 1997).

exposure *in vitro* and prevents streptozocin-induced diabetes *in vivo*.

A large number of NF-κB target genes were identified using DNA microarray technology in cytokine-treated primary rat β-cells (Cardozo et al., 2001a). Cytokines induced NF-κBdependent up-regulation of genes involved in stress responses (including CHOP, C/EBPβ and δ, Hsp27 and MnSOD), immune responses (e.g. MHC-II-associated invariant chain γ and MHC-I) and down-regulation of genes involved in β-cell function (glucose transporter-2 (Glut-2)), insulin production (Isl-1), insulin processing (PC-1), insulin release (PLD-1, CCKA-receptor) and Ca2+ homeostasis (SERCA2, IP 3-kinase) (Cardozo et al., 2001a).

Inducible nitric oxide synthase (iNOS) is strongly induced and is the best characterised NFκB target in both rat β-cells (Cardozo et al., 2001a, Kutlu et al., 2003) and human pancreatic islets (Flodström et al., 1996). Induction of iNOS increases nitric oxide (NO) production in βcells, resulting in the generation of reactive oxygen species (ROS) and oxidative stress. The cellular stress triggered by NO in rodent and human cells will be discussed later in this chapter (under section 2.2).

In addition to NF-κB, IL-1β signalling also activates the mitogen activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) 1/2 and induces suppressor of cytokine signalling-3 (SOCS-3) (Emanuelli et al., 2004). Signal transduction pathways induced by MAPKs and SOCS-3 are interlinked with the NF-κB-regulated pathways; MAPK activation potentiates IL-1β-dependent NF-κB activation and subsequent iNOS induction, and (ERK)1/2 activation was shown to contribute to cytokine-induced apoptosis in rat pancreatic -cells (Pavlovic et al., 2000). While MAPKs positively affect NF-κB signalling and enhance β-cell death, SOCS-3 has a negative effect. SOCS-3 belongs to a family of proteins that provide a negative feedback for cytokine-induced signalling. It was also identified as an inhibitor of insulin signalling (Emanuelli et al., 2000) as SOCS-3 can bind to the insulin receptor and block its insulin-induced autophosphorylation and activation (Emanuelli et al., 2004). SOCS-3 inhibits IL-1β signalling upstream and thus negatively regulates nearly all effects of IL-1β. SOCS-3 suppresses the expression of several IL-1βinduced pro-apoptotic genes, many of them known to be NF-κB-dependent (Karlsen et al., 2004) and protects rat β-cells from IL-1β- and TNFα-induced cell death (Bruun et al., 2009). As mentioned above, over 200 genes have been identified to be NF-κB-regulated in β-cells treated with pro-inflammatory cytokines. However, which of these genes are targets of IL-1β signalling, or to what extent their expression is regulated by IL-1β alone is currently unknown. Determining the individual targets of the cytokines would lead to a better understanding of how the cytokines synergise to cause β-cell stress and death.

#### **1.2.2 TNFα signalling**

TNFα was also shown to lead to activation of NF-κB in pancreatic β-cells (Ortis et al., 2006). TNFα binds to and activates the TNF receptor (TNFR1), which is present on the surface of βcells (Kägi et al., 1999). TNFα binding to TNFR1 leads to the latter's trimerisation and activation (Fig. 3). Upon activation, the cytosolic death domain of TNFR1 recruits TNF receptor-associated death domain (TRADD) (Hsu et al., 1995), TRAF2 (Hsu et al., 1996b) and the death domain kinase receptor interacting protein (RIP) (Hsu et al., 1996a). TRAF2, in turn, recruits IκB kinase (IKK) and induces its activation in a RIP-dependent manner via activation of an IKK kinase (e.g. NIK) (Devin et al., 2000). Activated IKK phosphorylates IκB proteins leading to their proteasomal degradation and release of NF-κB. The activation of NF-κB by both TNFα and IL-1β has a pro-apoptotic effect in rat pancreatic β-cells (Ortis et al., 2008). This effect was more pronounced in response to IL-1β than TNFα.

Cytokine-Induced -Cell Stress and Death in Type 1 Diabetes Mellitus

stimuli and intracellular stress.

**2. Cytokine-induced β-cell death** 

death can also contribute to β-cell loss.

proteolysis that dismantles the cells.

**2.1 Mechanisms of cytokine-induced β-cell death** 

Tullberg et al., 2003) and protected β-cells against infiltrating autoreactive T cells (Chong et al., 2004). In summary, the effect of IFNγ in β-cells is primarily mediated by STAT-1 through which IFNγ controls key processes culminating in loss of β-cell function, stress and finally death. IFNγ regulates a number of genes that increase the sensitivity of β-cells to apoptotic

During the development of T1DM, there are two waves of β-cell death. It is believed that βcell death is the initial trigger for the autoimmune attack. While autoimmune attack was thought to be initiated by cytolytic activity or immune-stimulation of viruses (Jun & Yoon, 2003), it is also possible that physiological -cell death might be a trigger. Instead of an exogenous impact, or environmental effect, induction of diabetes might be initiated during physiological tissue remodelling of the pancreas peaking at age 2-3 weeks in rodents. At this time, an increased level of β-cell death occurs in the islets and might be the primary trigger of the autoimmune attack (Turley et al., 2003). Programmed cell death associated with normal tissue remodelling does not induce inflammation. However, if the dead cells are not removed promptly by phagocytosis they can disintegrate and release cellular contents in a manner similar to pathological tissue damage which can trigger inflammation. In fact, accumulation of dead cells has been noticed in NOD mice and similarly, disintegrating, so called secondary necrotic cells were sufficient to induce inflammation, macrophage

The second wave of β-cell death is driven by the autoimmune reaction. This is an ongoing process gradually killing the β-cells and culminating in the disease phenotype. The mechanism of β-cell death induced by the autoreactive leukocytes has been extensively examined with consensus that the major form of β-cell death is apoptosis, however, under certain conditions and especially in rodent experimental models of T1DM, necrotic β-cell

**Apoptosis** is a physiological form of cell death involved in the elimination of cells that have served their function, are no longer needed or are damaged. It is an active, highly ordered and rapid process characterised by the detachment of the dying cell from its neighbours, cell shrinkage, condensation of chromatin, fragmentation of the nucleus and finally fragmentation of the cell into membrane bound particles, called apoptotic bodies which are engulfed by neighbouring cells or professional phagocytic cells (Samali et al., 1996). By this means, cells are eliminated without leakage of otherwise inflammatory cellular material. The morphological changes typical of apoptosis are orchestrated by the caspase family of proteases (Samali et al., 1999). Caspases are activated by two distinct mechanisms. The extrinsic pathway is triggered by an extracellular pro-apoptotic stimulus, usually a cytokine that belongs to the death ligand subfamily of the TNF superfamily. Upon engagement of the death ligand with its cognate death receptor on the cell surface of the target cell, the receptors trimerise and induce the formation of a protein complex, called the death-inducing signalling complex (DISC). The DISC is an activation platform for caspases-8 and/or -10 (Peter & Krammer, 2003). Once these initiator caspases are activated they activate downstream effector caspases, which leads to a burst of caspase activity and subsequent

The second, so called intrinsic pathway is initiated at the level of mitochondria. Upon intracellular stress these organelles release cytochrome *c* that associates with the adaptor

infiltration and pre-diabetic insulitis in NOD mice (H.S. Kim et al., 2007).

219

TNFα signalling can lead to RIP-dependent activation of three MAPKs (c-Jun N-terminal kinase JNK, p38 and ERK) in a cell type-specific manner (Devin et al., 2003). In rat pancreatic β-cells, TNFα treatment induced activation of JNK and p38 which has been suggested to contribute to an inhibitory effect of TNFα on glucose-stimulated insulin secretion (H.-E. Kim et al., 2008) and hence β-cell dysfunction in response to TNFα.
