**2. Cytokine-induced β-cell death**

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

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

IFNγ is a homodimeric cytokine. It binds to two IFNγ receptor α (IFNγRα) chains (Fig. 3). A third unit of IFNγRα and two molecules of IFNγ receptor β (IFNγRβ, also termed accessory factor 1, AF-1) bind to the IFNγRα (Thiel et al., 2000). This leads to the activation and transphosphorylation of Janus tyrosine kinase 1 and 2 (JAK1 and JAK2) which are associated with IFNγRα and IFNγRβ, respectively, and are brought together upon receptor oligomerisation (Igarashi et al., 1994, Kotenko et al., 1995). JAK1 and JAK2 phosphorylate IFNγR leading to the recruitment of two molecules of the transcription factor, signal transducer and activator of transcription-1 (STAT-1). After phosphorylation and activation by JAK2, STAT-1 homodimerises and translocates to the nucleus where it stimulates the expression of target genes (Takeda & Akira, 2000). Islet cells isolated from STAT-1-/- nonobese diabetic (NOD) mice were resistant to apoptosis induced by combined treatment with IFNγ and TNFα or IFNγ and IL-1β (S. Kim et al., 2007). In support of this, blockade of STAT-1 protected against diabetes induced by injection of multiple low doses of streptozotocin in mice (Callewaert et al., 2007, C.A. Gysemans et al., 2005). A recent gene expression analysis showed that nearly two thousand genes are regulated by STAT-1 in response to cytokine exposure (IL-1β and IFNγ) in β-cells (Moore et al., 2011). STAT-1 was found to regulate the IL-1β/IFNγ-mediated induction of chemokines, including CXCL9, CXCL10, CXCL11 and CCL20 (Moore et al., 2011) and islets from STAT-1-/- mice have decreased production of CXCL10 upon cytokine exposure both *in vitro* and *in vivo* (C.A. Gysemans et al., 2005). STAT-1 also down-regulates several genes specific to β-cell functions, such as insulin, glucokinase, Glut2, prohormone convertases, as well as many transcription factors involved in the differentiation and maintenance of β-cell phenotype (e.g. Pdx1, MafA, Nkx2.2) (Moore

Finally, STAT-1 is an important regulator of genes mediating intracellular stress and apoptotic pathways. Several apoptosis-related genes such as Puma, CHOP, Bax, Bid, caspase-3, -4, -7, DP5/Hrk and endoplasmic reticulum stress-transducing genes (XBP1, ATF4) are regulated by STAT-1 (Eizirik & Darville, 2001, Moore et al., 2011, Anastasis Stephanou et al., 2000). IFNγ has been found to profoundly accelerate IL-1β-mediated iNOS induction and thus cause oxidative stress. We have demonstrated that treatment of a rat insulinoma cell line (RIN-r) with a combination of IL-1β and IFNγ induces the mitochondrial apoptotic pathway in an iNOS-dependent manner (Holohan et al., 2008). This

The inflammatory effects of IFNγ are controlled by negative feedback regulation, exerted by interferon regulated factor-1 (IRF-1) (Moore et al., 2011) and SOCS-1 and -3 (Alexander, 2002). IRF-1 is likely to exert its STAT-1 regulatory role by up-regulation of SOCS-1 (Moore et al., 2011). IRF-1 expression reduces chemokine expression in β-cells and resulting T cell infiltration in Langerhans islets (C. Gysemans et al., 2008, Moore et al., 2011), however the effect of IRF-1 on STAT-1-mediated β-cell de-differentiation (loss of β-cell function) and cell stress is minor (Moore et al., 2011). In line with this, transgenic expression of SOCS-1 in β-cells reduced diabetes development in non-obese diabetic (NOD) mice (Flodström-

et al., 2008) and hence β-cell dysfunction in response to TNFα.

**1.2.3 IFNγ signalling** 

et al., 2011, Perez-Arana et al., 2010).

is in line with reports from other groups (Gurzov et al., 2009).

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

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

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 death can also contribute to β-cell loss.

**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 proteolysis that dismantles the cells.

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

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

IFNγ is NO-dependent in a rat insulinoma cell line (Storling et al., 2005).

inflicted by NO (Welsh et al., 1994).

of RIN-r cells (Holohan et al., 2008).

**2.3 Role of the Bcl-2 family proteins** 

cytokine-induced induction of necrosis seems to be dependent on iNOS-induced production of NO as the level of necrotic cell death was greatly reduced in purified β-cells from iNOSdeficient mice (D. Liu et al., 2000). Another study found that inhibition of iNOS in rat islets reduced both necrosis and apoptosis induction (Saldeen, 2000). In any case, the cytokineinduced production of NO seems to play a major role in mediating β-cell death in rodent experimental models of T1DM. Additionally, we recently demonstrated that a combination of IL-1β and IFNγ induces the intrinsic apoptosis pathway in a synergistic manner in a rat insulinoma cell line (RIN-r) and showed that iNOS-mediated production of NO was both required and sufficient for apoptosis induction (Holohan et al., 2008). This is in agreement with previous findings that showed that apoptosis induced by a combination of IL-1β and

Human islets have been shown to be less sensitive to NO-induced damage compared to rodent cells. As such, inhibition of iNOS could not protect human islets from cytokineinduced cell death suggesting a NO-independent cytotoxicity. (Delaney et al., 1997, Eizirik & Mandrup-Poulsen, 2001, Hoorens et al., 2001). The resistance of human islets towards NO compared to rodent islets is speculated to be due to higher levels of heat shock protein 70 (Hsp70) in human β-cells (Burkart et al., 2000) which protects cells from the oxidative stress

Cytokines can modulate the expression and/or activity of several members of the Bcl-2 family (Gowda et al., 2008, A. Stephanou et al., 2000, P. Wang et al., 2009, L. Zhang et al., 2008). The various interactions between the pro- and anti-apoptotic members of this family of proteins lie at the heart of the intrinsic pathway of apoptosis (Youle & Strasser, 2008). Bcl-2 family members are characterised by up to four conserved regions termed Bcl-2 homology (BH) domains. The pro-apoptotic multidomain family members Bax and Bak contain three BH domains and can be activated to form oligomeric structures in the outer mitochondrial membrane that trigger cytochrome *c* release, which then initiates the intrinsic pathway of caspase activation. Activation proceeds through interaction with BH3-only family members (harbouring only the third BH domain) that are induced or activated by cellular stress signals. Activation of Bax or Bak is counteracted by anti-apoptotic multidomain Bcl-2 family members (such as Bcl-2, Bcl-xL, or Mcl-1), which bind and sequester the BH3-only proteins. Viral transduction of Bcl-2, the prototype member of the family, was shown to protect human islet cells from cytokine-induced apoptosis, giving a first indication that regulation of Bcl-2 family proteins by cytokines might contribute to β-cell apoptosis (Rabinovitch et al., 1999). Likewise, adenoviral transduction of Bcl-XL prevented cytokine-mediated apoptosis

Several recent studies have addressed the involvement of Bcl-2 family proteins in cytokineinduced β-cell death in more detail. Treatment of human or rat islets with inflammatory cytokines resulted in activation of the intrinsic pathway of apoptosis and involved activation of the pro-apoptotic BH3-only protein Bad by dephosphorylation (Grunnet et al., 2009). Dephosphorylation of Bad was also found in a second study analysing cytokinetreated rat islets, and in addition up-regulation of pro-apoptotic BH3-only proteins Bim and Bid was also detected (Mehmeti et al., 2011). In a different study it was shown that in primary rat β-cells cytokines as well as ER stress lead to increased expression of the proapoptotic BH3-only protein DP5/Hrk in a JNK-dependent manner (Gurzov et al., 2009). Up-

221

protein APAF-1 to build a multimeric cytoplasmic protein complex termed the apoptosome, which functions to activate another initiator caspase, caspase-9 (Riedl & Salvesen, 2007). Mitochondrial release of cytochrome *c*, and thus activation of the intrinsic apoptosis pathway, is controlled by members of the Bcl-2 family of proteins (see section 2.3). Once cytochrome *c* is released and caspase-9 is activated, the same caspase cascade is triggered as during the extrinsic apoptotic pathway that leads to the final demise of the cell.

Interestingly, TNFα was shown to induce expression of an endogenous caspase inhibitor in -cells that prevents apoptosis, the X-linked inhibitor of apoptosis protein (XIAP). This NFkB-mediated induction of XIAP is inhibited by IFNγ signalling, providing a mechanism for synergistic cytoxicity of TNFα and IFNγ in β-cells (H.S. Kim et al., 2005).

Apoptosis is distinguished from **necrosis**, a pathological, mostly uncontrolled mode of cell death. During necrosis cells swell, their membranes disintegrate and their content is released, inducing inflammation. Recently, an active mode of necrosis, termed necroptosis, has been described that can be induced upon activation of TNFR1 when caspase-8 activation is blocked (Vandenabeele et al., 2010). A possible role of necroptosis in initiation of diabetes seems worthy of further investigation in light of the known involvement of TNFR1 signalling in diabetes and of a recent study that provided evidence of necrotic β-cell death playing a role in initiating autoimmune-type diabetes (Steer et al., 2006).

#### **2.2 The role of nitric oxide in cytokine-mediated β-cell loss**

It is thought that cytokine-induced β-cell stress and death is partly caused by intracellular production of ROS and NO. NO is a gaseous hydrophobic signalling molecule that readily diffuses through membranes and plays an essential role in various neurological, immunological and cardiovascular processes. The biosynthesis of NO is catalysed by nitric oxide synthases (NOS). In β-cells IL-1β signals up-regulation of iNOS and subsequent generation of NO. The main physiological effect of NO is mediated via the direct activation of guanylyl cyclase by NO leading to production of cyclic GMP (cGMP) and activation of cGMP-dependent signal transduction pathways. However, if present for a prolonged period or in high quantities, NO can nitrosylate specific cysteine residues of various proteins (Snitrosylation) forming nitrosothiols and thereby affect the protein's activity, stability and localisation (Hess et al., 2005). In most cases this leads to rapid degradation of the nitrosylated proteins but a small subgroup of proteins have been shown to gain stability after nitrosylation (Paige et al., 2008). NO can have anti-apoptotic and cytoprotective effects in some cell types (McCabe et al., 2006), but can become toxic if present at high levels due to formation of ROS and protein nitrosylation which, amongst other things, also causes mitochondrial damage.

It has been shown that NO can induce both necrotic and apoptotic cell death (Bonfoco et al., 1995). With respect to β-cell destruction, it has been shown that endogenous levels of NO are sufficient to induce β-cell injury in rodent models of T1DM (Thomas et al., 2002) and increased levels of NO caused by cytokine-mediated iNOS induction cause cell death by both necrosis (Hoorens et al., 2001, Welsh et al., 1994) and apoptosis (Holohan et al., 2008). The relative involvement of NO in the destruction of β-cells in human and rodent islets is not fully elucidated. Several studies have shown that a combination of IL-1β with IFNγ or TNFα induces cell death in rodent pancreatic islet cells, predominantly by induction of apoptosis but also partly by necrosis (D. Liu et al., 2000, Saldeen, 2000). In rodent β-cells the

protein APAF-1 to build a multimeric cytoplasmic protein complex termed the apoptosome, which functions to activate another initiator caspase, caspase-9 (Riedl & Salvesen, 2007). Mitochondrial release of cytochrome *c*, and thus activation of the intrinsic apoptosis pathway, is controlled by members of the Bcl-2 family of proteins (see section 2.3). Once cytochrome *c* is released and caspase-9 is activated, the same caspase cascade is triggered as

Interestingly, TNFα was shown to induce expression of an endogenous caspase inhibitor in -cells that prevents apoptosis, the X-linked inhibitor of apoptosis protein (XIAP). This NFkB-mediated induction of XIAP is inhibited by IFNγ signalling, providing a mechanism for

Apoptosis is distinguished from **necrosis**, a pathological, mostly uncontrolled mode of cell death. During necrosis cells swell, their membranes disintegrate and their content is released, inducing inflammation. Recently, an active mode of necrosis, termed necroptosis, has been described that can be induced upon activation of TNFR1 when caspase-8 activation is blocked (Vandenabeele et al., 2010). A possible role of necroptosis in initiation of diabetes seems worthy of further investigation in light of the known involvement of TNFR1 signalling in diabetes and of a recent study that provided evidence of necrotic β-cell death

It is thought that cytokine-induced β-cell stress and death is partly caused by intracellular production of ROS and NO. NO is a gaseous hydrophobic signalling molecule that readily diffuses through membranes and plays an essential role in various neurological, immunological and cardiovascular processes. The biosynthesis of NO is catalysed by nitric oxide synthases (NOS). In β-cells IL-1β signals up-regulation of iNOS and subsequent generation of NO. The main physiological effect of NO is mediated via the direct activation of guanylyl cyclase by NO leading to production of cyclic GMP (cGMP) and activation of cGMP-dependent signal transduction pathways. However, if present for a prolonged period or in high quantities, NO can nitrosylate specific cysteine residues of various proteins (Snitrosylation) forming nitrosothiols and thereby affect the protein's activity, stability and localisation (Hess et al., 2005). In most cases this leads to rapid degradation of the nitrosylated proteins but a small subgroup of proteins have been shown to gain stability after nitrosylation (Paige et al., 2008). NO can have anti-apoptotic and cytoprotective effects in some cell types (McCabe et al., 2006), but can become toxic if present at high levels due to formation of ROS and protein nitrosylation which, amongst other things, also causes

It has been shown that NO can induce both necrotic and apoptotic cell death (Bonfoco et al., 1995). With respect to β-cell destruction, it has been shown that endogenous levels of NO are sufficient to induce β-cell injury in rodent models of T1DM (Thomas et al., 2002) and increased levels of NO caused by cytokine-mediated iNOS induction cause cell death by both necrosis (Hoorens et al., 2001, Welsh et al., 1994) and apoptosis (Holohan et al., 2008). The relative involvement of NO in the destruction of β-cells in human and rodent islets is not fully elucidated. Several studies have shown that a combination of IL-1β with IFNγ or TNFα induces cell death in rodent pancreatic islet cells, predominantly by induction of apoptosis but also partly by necrosis (D. Liu et al., 2000, Saldeen, 2000). In rodent β-cells the

during the extrinsic apoptotic pathway that leads to the final demise of the cell.

synergistic cytoxicity of TNFα and IFNγ in β-cells (H.S. Kim et al., 2005).

playing a role in initiating autoimmune-type diabetes (Steer et al., 2006).

**2.2 The role of nitric oxide in cytokine-mediated β-cell loss** 

mitochondrial damage.

cytokine-induced induction of necrosis seems to be dependent on iNOS-induced production of NO as the level of necrotic cell death was greatly reduced in purified β-cells from iNOSdeficient mice (D. Liu et al., 2000). Another study found that inhibition of iNOS in rat islets reduced both necrosis and apoptosis induction (Saldeen, 2000). In any case, the cytokineinduced production of NO seems to play a major role in mediating β-cell death in rodent experimental models of T1DM. Additionally, we recently demonstrated that a combination of IL-1β and IFNγ induces the intrinsic apoptosis pathway in a synergistic manner in a rat insulinoma cell line (RIN-r) and showed that iNOS-mediated production of NO was both required and sufficient for apoptosis induction (Holohan et al., 2008). This is in agreement with previous findings that showed that apoptosis induced by a combination of IL-1β and IFNγ is NO-dependent in a rat insulinoma cell line (Storling et al., 2005).

Human islets have been shown to be less sensitive to NO-induced damage compared to rodent cells. As such, inhibition of iNOS could not protect human islets from cytokineinduced cell death suggesting a NO-independent cytotoxicity. (Delaney et al., 1997, Eizirik & Mandrup-Poulsen, 2001, Hoorens et al., 2001). The resistance of human islets towards NO compared to rodent islets is speculated to be due to higher levels of heat shock protein 70 (Hsp70) in human β-cells (Burkart et al., 2000) which protects cells from the oxidative stress inflicted by NO (Welsh et al., 1994).

#### **2.3 Role of the Bcl-2 family proteins**

Cytokines can modulate the expression and/or activity of several members of the Bcl-2 family (Gowda et al., 2008, A. Stephanou et al., 2000, P. Wang et al., 2009, L. Zhang et al., 2008). The various interactions between the pro- and anti-apoptotic members of this family of proteins lie at the heart of the intrinsic pathway of apoptosis (Youle & Strasser, 2008). Bcl-2 family members are characterised by up to four conserved regions termed Bcl-2 homology (BH) domains. The pro-apoptotic multidomain family members Bax and Bak contain three BH domains and can be activated to form oligomeric structures in the outer mitochondrial membrane that trigger cytochrome *c* release, which then initiates the intrinsic pathway of caspase activation. Activation proceeds through interaction with BH3-only family members (harbouring only the third BH domain) that are induced or activated by cellular stress signals. Activation of Bax or Bak is counteracted by anti-apoptotic multidomain Bcl-2 family members (such as Bcl-2, Bcl-xL, or Mcl-1), which bind and sequester the BH3-only proteins.

Viral transduction of Bcl-2, the prototype member of the family, was shown to protect human islet cells from cytokine-induced apoptosis, giving a first indication that regulation of Bcl-2 family proteins by cytokines might contribute to β-cell apoptosis (Rabinovitch et al., 1999). Likewise, adenoviral transduction of Bcl-XL prevented cytokine-mediated apoptosis of RIN-r cells (Holohan et al., 2008).

Several recent studies have addressed the involvement of Bcl-2 family proteins in cytokineinduced β-cell death in more detail. Treatment of human or rat islets with inflammatory cytokines resulted in activation of the intrinsic pathway of apoptosis and involved activation of the pro-apoptotic BH3-only protein Bad by dephosphorylation (Grunnet et al., 2009). Dephosphorylation of Bad was also found in a second study analysing cytokinetreated rat islets, and in addition up-regulation of pro-apoptotic BH3-only proteins Bim and Bid was also detected (Mehmeti et al., 2011). In a different study it was shown that in primary rat β-cells cytokines as well as ER stress lead to increased expression of the proapoptotic BH3-only protein DP5/Hrk in a JNK-dependent manner (Gurzov et al., 2009). Up-

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

reduced insulin production and reduced cell proliferation (Feng et al., 2009).

**3.1.2 Involvement of the UPR in cytokine-induced β-cell death** 

2001, P. Zhang et al., 2002). β-cell loss was associated with damaged rough ER and high levels of apoptosis in the pancreas (P. Zhang et al., 2002). However, a subsequent study discovered that the onset of diabetes in PERK-/- mice is due to developmental defects during β-cell proliferation and differentiation leading to a reduction in β-cell mass (W. Zhang et al., 2006). At the molecular level, down-regulation of PERK in rat β-cells was shown to induce deregulation of ER chaperones Grp78 and ERp72 and disruption of ER function leading to

There is some evidence to suggest that cytokines induce β-cell apoptosis by stimulating proapoptotic signalling of the UPR. Ca2+ levels in the ER are about four times higher than in the cytosol as high Ca2+ levels are required for ER function in aiding protein folding and posttranslational processing. Disruption of Ca2+ homeostasis causes severe ER stress resulting in accumulation of unfolded proteins in the ER and activation of the UPR. It was shown that cytokine-exposure leads to elevated basal cytosolic Ca2+ levels selectively in mouse pancreatic β-cells compared to glucagon-secreting -cells and this was associated with cytokine-induced apoptosis (L. Wang et al., 1999). In line with these results, it was shown that increased production of NO in rodent β-cells leads to depletion of ER Ca2+ levels (Oyadomari et al., 2001). Furthermore, overexpression of the ER-located Ca2+-binding protein, calreticulin increased levels of Ca2+ in the ER and made cells more resistant to NOinduced apoptosis (Oyadomari et al., 2001). This suggests that NO-induced apoptosis in rodent β-cells is at least partly caused by ER stress induced by NO-mediated Ca2+ depletion. Some evidence suggests that NO may regulate Ca2+ levels in β-cells through downregulation of the sarcoendoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) (Cardozo et al., 2005). SERCA pumps Ca2+ from the cytoplasm into the ER thus maintaining ER Ca2+ levels. Rodent and human islet cells have been reported to express the isoforms SERCA2b and SERCA3 (Varadi et al., 1996). Treatment of rodent pancreatic β-cells with a combination of IL-1β and IFNγ induced transcriptional down-regulation of SERCA2b and this was partially prevented by inhibition of iNOS. Furthermore, after inhibition of SERCA, the effect of cytokine exposure on ER Ca2+ was abolished (Cardozo et al., 2005). This suggests that NOinduced depletion of ER Ca2+ is at least in part mediated by SERCA down-regulation. The SERCA isoform SERCA2a has been shown to be specifically inactivated by peroxynitrite (ONOO-)-mediated nitration of a tyrosine residue within the channel-like domain *in vitro* (Viner et al., 1999). Peroxynitrite is produced in cells by a reaction between NO and the free radical superoxide (Pacher et al., 2007). SERCA2a differs from SERCA2b only in regions of the C-terminus (Dode et al., 1998) and it could be hypothesised that cytokine-induced NO production inhibits SERCA2b Ca2+-ATPase activity by peroxynitrite-mediated nitration in the same way. Another possible mechanism by which NO might mediate reduction of ER Ca2+ levels is via activation of the ryanodine receptor-2. Ryanodine receptor-2 is a calcium channel located in the ER membrane that releases Ca2+ from the ER into the cytosol and has been reported to be expressed in mouse pancreatic β-cells (Islam et al., 1998). NO-induced poly-S-nitrosylation enhances the activity of this calcium channel (Xu et al., 1998) but whether this mechanism is relevant to cytokine-exposed β-cells remains to be determined. Treatment of rodent β-cells with a combination of IL-1β and IFNγ induces the expression of CHOP in an NO-dependent manner (Fig. 4). This is in line with a number of other reports (Cardozo et al., 2001b, Cardozo et al., 2005). Inhibition of iNOS by N5-(1-iminoethyl)-Lornithine (L-NIO) or NG-methyl-L-arginine (LMA) blocked cytokine-induced NO

223

regulation of DP5 in β-cells is mediated by the transcription factor STAT-1 which is regulated by IFNγ (Moore et al., 2011). In addition, inflammatory cytokines led to upregulation of the pro-apoptotic BH3-only protein PUMA in primary rat β-cells as well as in human islets through a pathway involving NF-κB signalling, iNOS activation and ER stress (Gurzov et al., 2010). Furthermore, down-regulation of the anti-apoptotic multidomain Bcl-2 family member Mcl-1 turned out to be critically involved in the cytokine-induced apoptosis of the rat insulinoma cell line INS-1E (Allagnat et al., 2011). In summary, exposure to cytokines leads to alterations in expression of several Bcl-2 family members in β-cells in a manner that favours activation of the intrinsic pathway of apoptosis.
