**3. β-cell stress in type 1 diabetes**

### **3.1 Endoplasmic reticulum stress**

It has been suggested that endoplasmic reticulum (ER) stress is involved in β-cell destruction. Pancreatic -cells are specialised cells that rapidly synthesise and secrete insulin in response to fluctuations in blood glucose levels (Pirot et al., 2007). This imparts a heavy burden on the ER and, consequently, -cells are particularly susceptible to cellular conditions that impair the ER's ability to correctly fold nascent proteins. Under such conditions, the resultant accumulation of unfolded or damaged proteins within the ER lumen triggers the unfolded protein response (UPR), an adaptive signalling pathway that increases the folding capacity of the ER and restores homeostasis (Szegezdi et al., 2006). Although the initial UPR is a protective response, prolonged ER stress can lead to the initiation of apoptosis. Thus while under physiological conditions the UPR acts as a prosurvival mechanism in β-cells, chronic ER stress can lead to redirection of the UPR towards pro-apoptotic signalling.

Three ER-localized transmembrane proteins sense the accumulation of unfolded proteins in the ER lumen and initiate the UPR, PKR-like ER kinase (PERK), inositol-requiring enzyme 1 α (IRE1) and activating transcription factor 6 (ATF6). These proteins transduce information from the ER to the nucleus by activating transcription factors that control genes involved in restoring ER function (Szegezdi et al., 2006). The PERK arm of the UPR has been the main focus of studies with regard to β-cell stress in diabetes, therefore this chapter will focus on PERK signalling in more detail. Upon accumulation of unfolded proteins, PERK is activated and induces a translational block by phosphorylating eukaryotic initiation factor 2 α (eIF2). Phosphorylation of eIF2α by PERK leads to inhibition of cap-dependent protein synthesis. This reduces the protein load of the ER while allowing cap-independent translation to persist and leads to preferential translation of the transcription factor ATF4. One target gene induced by ATF4 (in conjunction with ATF6) is C/EBP homologous protein CHOP, a transcription factor that is known to promote apoptosis (Zinszner et al., 1998).

#### **3.1.1 The role of PERK in β-cell function**

The PERK signalling branch of the UPR appears to be essential for the regulation of β-cell function. Stimulation of insulin production in mouse pancreatic islets leads to dephosphorylation of eIF2α (P. Zhang et al., 2002) reversing the translational block caused by PERK signalling and allowing for increased biosynthesis of insulin. Studies with knockout mice showed that PERK is essential for β-cell function and survival (Harding et al., 2001, P. Zhang et al., 2002). Pancreatic β-cells of PERK-/- mice degenerated within the first four weeks after birth, and a diabetic phenotype could be observed (Harding et al.,

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

It has been suggested that endoplasmic reticulum (ER) stress is involved in β-cell destruction. Pancreatic -cells are specialised cells that rapidly synthesise and secrete insulin in response to fluctuations in blood glucose levels (Pirot et al., 2007). This imparts a heavy burden on the ER and, consequently, -cells are particularly susceptible to cellular conditions that impair the ER's ability to correctly fold nascent proteins. Under such conditions, the resultant accumulation of unfolded or damaged proteins within the ER lumen triggers the unfolded protein response (UPR), an adaptive signalling pathway that increases the folding capacity of the ER and restores homeostasis (Szegezdi et al., 2006). Although the initial UPR is a protective response, prolonged ER stress can lead to the initiation of apoptosis. Thus while under physiological conditions the UPR acts as a prosurvival mechanism in β-cells, chronic ER stress can lead to redirection of the UPR towards

Three ER-localized transmembrane proteins sense the accumulation of unfolded proteins in the ER lumen and initiate the UPR, PKR-like ER kinase (PERK), inositol-requiring enzyme 1 α (IRE1) and activating transcription factor 6 (ATF6). These proteins transduce information from the ER to the nucleus by activating transcription factors that control genes involved in restoring ER function (Szegezdi et al., 2006). The PERK arm of the UPR has been the main focus of studies with regard to β-cell stress in diabetes, therefore this chapter will focus on PERK signalling in more detail. Upon accumulation of unfolded proteins, PERK is activated and induces a translational block by phosphorylating eukaryotic initiation factor 2 α (eIF2). Phosphorylation of eIF2α by PERK leads to inhibition of cap-dependent protein synthesis. This reduces the protein load of the ER while allowing cap-independent translation to persist and leads to preferential translation of the transcription factor ATF4. One target gene induced by ATF4 (in conjunction with ATF6) is C/EBP homologous protein CHOP, a

The PERK signalling branch of the UPR appears to be essential for the regulation of β-cell function. Stimulation of insulin production in mouse pancreatic islets leads to dephosphorylation of eIF2α (P. Zhang et al., 2002) reversing the translational block caused by PERK signalling and allowing for increased biosynthesis of insulin. Studies with knockout mice showed that PERK is essential for β-cell function and survival (Harding et al., 2001, P. Zhang et al., 2002). Pancreatic β-cells of PERK-/- mice degenerated within the first four weeks after birth, and a diabetic phenotype could be observed (Harding et al.,

transcription factor that is known to promote apoptosis (Zinszner et al., 1998).

manner that favours activation of the intrinsic pathway of apoptosis.

**3. β-cell stress in type 1 diabetes 3.1 Endoplasmic reticulum stress** 

pro-apoptotic signalling.

**3.1.1 The role of PERK in β-cell function** 

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

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

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

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

death may be partly mediated by induction of CHOP.

conditions of cytokine-induced β-cell stress.

**3.2 Oxidative stress** 

Tiedge et al., 1998).

**4. Therapeutic strategies** 

autoimmunity (Kort et al., 2011).

that CHOP is not required for β-cell death and the development of diabetes in NOD mice (Satoh et al., 2011). However, CHOP-/- NOD mice showed delayed production of insulin autoimmune antibodies (Satoh et al., 2011) suggesting a role for CHOP in the early onset of the autoimmune reaction leading to β-cell destruction. Therefore, cytokine-induced β-cell

While a functional UPR (at least the PERK branch) appears to be essential for β-cell function and development, its downstream target CHOP has been associated with cytokine-induced β-cell destruction suggesting that PERK signalling regulates β-cell function and survival under physiological conditions but may switch towards pro-death signalling under

Cytokines induce multiple stress pathways in β-cells. Oxidative stress is induced by increased production of ROS and an imbalanced, low level of antioxidant enzymes (Sies, 1997). IL-1β, TNFα and IFNγ induce the production of ROS and NO by inducing iNOS (Rabinovitch & Suarez-Pinzon, 1998). Free oxygen and nitrogen radicals generated can react to form peroxynitrite, which is a very strong oxidant. Oxygen free radicals, nitrogen free radicals as well as the radical peroxynitrite can react with and damage a range of cellular proteins and in this way block metabolic functions and induce β-cell death (Azevedo-Martins et al., 2003). β-cells have been shown to be especially susceptible to such oxidative stress because they have particularly low levels of antioxidant enzymes (Lenzen et al., 1996,

The number of people affected by T1DM is approximately 20 million worldwide and is rapidly rising (Chabot, 2002). While exogenous administration of insulin is an effective treatment for acute hyperglycaemia in T1DM, it does not prevent secondary complications (White et al., 2008) and can in some cases lead to hypoglycaemia (Kort et al., 2011). Alternative therapeutic strategies include pancreas transplantation and islet transplantation. While whole pancreas transplantation is an invasive surgical method associated with major complications, islet transplantation is less invasive and associated with significantly lower morbidity and mortality. Successful islet transplantation would result in insulin independence, protection from hypoglycaemia, improvement of microvascular complications, improved patient survival and enhanced quality of life (Kort et al., 2011). The method is currently in clinical trials and has been used to treat around 1,000 individuals worldwide (Kort et al., 2011). Islet transplantation has many limitations, including limited availability of suitable islet graft donors, high cost and high rate of partial or total graft failure. Islet graft failure can be caused by allorejection, toxicity of immunosuppressive drugs that are required to reduce immune rejection, glucotoxicity, and recurrence of

An approach to reduce β-cell death in islet grafts is the transfer of therapeutically useful genes into islet cells prior to transplantation (McCabe et al., 2006). The development of gene therapy techniques that can protect β-cells from autoimmune destruction may not only improve outcomes after islet transplantation but may also lead to preventive therapies for

patients at high risk of developing T1DM (McCabe et al., 2006).

225

production and expression of CHOP. In addition to CHOP, the UPR marker proteins Grp78 and phosphorylated eIF2 were also found to be up-regulated after cytokine treatment, without affecting expression of spliced X-box binding protein 1 (sXBP-1) which is induced downstream of IRE1α (Fig. 4). Overexpression of iNOS alone was sufficient for CHOP expression (Fig. 4) and treatment with the NO-donor molecule S-nitroso-N-acetyl-D,Lpenicillamine (SNAP) induced expression of Grp78 and CHOP (Oyadomari et al., 2001). This suggests that NO is sufficient to activate the UPR in rodent pancreatic β-cells. Pancreatic islet cells from CHOP-/- mice were shown to be resistant to cytokine- and NOmediated apoptosis compared to cells from CHOP+/+ and CHOP+/- mice (Oyadomari et al., 2001). Together these results suggest that the apoptotic effects of cytokine-induced NO are mediated by activation of CHOP.

Fig. 4. IL-1β/IFNγ induced the expression of the pro-apoptotic transcription factor CHOP and other UPR markers. (A) A time-course cytokine treatment (IL-1β and IFNγ, 60 U/ml of each) was carried out and samples assayed for NO production. (B) The same samples were then analysed by Western blotting for iNOS and CHOP expression. This data demonstrates an increase in NO production, iNOS expression and CHOP induction occurring at 6 h post cytokine treatment, although CHOP is not strongly expressed until 9 h. (C) Samples were assayed for CHOP expression following cytokine treatment in the presence and absence of the iNOS inhibitor L-NIO. Cytokine-induced CHOP expression was decreased in the presence of L-NIO indicating that this is an NO-dependent process. (D) Alterations in the expression of ER stress-associated proteins after cytokine treatment were analysed by Western blotting. The expression of UPR markers Grp78 and phosphorylated eIF2 (peIF2were up in response to cytokine treatment. (E) Production of spliced XBP-1 mRNA after cytokine treatment was determined by RT-PCR. Thapsigargin (Tg) treatment was used as a positive control. Cytokine treatment did not show an effect on the level of spliced XBP-1 mRNA. The images presented are representative of three independent experiments.

Conversely, another study suggested that although cytokine signalling induces ER stress as demonstrated by activation of PERK and JNK, induction of CHOP is not required for -cell death in rodents (Åkerfeldt et al., 2008). In support of these findings, a recent study suggests that CHOP is not required for β-cell death and the development of diabetes in NOD mice (Satoh et al., 2011). However, CHOP-/- NOD mice showed delayed production of insulin autoimmune antibodies (Satoh et al., 2011) suggesting a role for CHOP in the early onset of the autoimmune reaction leading to β-cell destruction. Therefore, cytokine-induced β-cell death may be partly mediated by induction of CHOP.

While a functional UPR (at least the PERK branch) appears to be essential for β-cell function and development, its downstream target CHOP has been associated with cytokine-induced β-cell destruction suggesting that PERK signalling regulates β-cell function and survival under physiological conditions but may switch towards pro-death signalling under conditions of cytokine-induced β-cell stress.

### **3.2 Oxidative stress**

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

production and expression of CHOP. In addition to CHOP, the UPR marker proteins Grp78 and phosphorylated eIF2 were also found to be up-regulated after cytokine treatment, without affecting expression of spliced X-box binding protein 1 (sXBP-1) which is induced downstream of IRE1α (Fig. 4). Overexpression of iNOS alone was sufficient for CHOP expression (Fig. 4) and treatment with the NO-donor molecule S-nitroso-N-acetyl-D,Lpenicillamine (SNAP) induced expression of Grp78 and CHOP (Oyadomari et al., 2001). This suggests that NO is sufficient to activate the UPR in rodent pancreatic β-cells. Pancreatic islet cells from CHOP-/- mice were shown to be resistant to cytokine- and NOmediated apoptosis compared to cells from CHOP+/+ and CHOP+/- mice (Oyadomari et al., 2001). Together these results suggest that the apoptotic effects of cytokine-induced NO are

Fig. 4. IL-1β/IFNγ induced the expression of the pro-apoptotic transcription factor CHOP and other UPR markers. (A) A time-course cytokine treatment (IL-1β and IFNγ, 60 U/ml of each) was carried out and samples assayed for NO production. (B) The same samples were then analysed by Western blotting for iNOS and CHOP expression. This data demonstrates an increase in NO production, iNOS expression and CHOP induction occurring at 6 h post cytokine treatment, although CHOP is not strongly expressed until 9 h. (C) Samples were assayed for CHOP expression following cytokine treatment in the presence and absence of the iNOS inhibitor L-NIO. Cytokine-induced CHOP expression was decreased in the presence of L-NIO indicating that this is an NO-dependent process. (D) Alterations in the expression of ER stress-associated proteins after cytokine treatment were analysed by Western blotting. The expression of UPR markers Grp78 and phosphorylated eIF2 (peIF2were up in response to cytokine treatment. (E) Production of spliced XBP-1 mRNA after cytokine treatment was determined by RT-PCR. Thapsigargin (Tg) treatment was used as a positive control. Cytokine treatment did not show an effect on the level of spliced XBP-1

mRNA. The images presented are representative of three independent experiments.

Conversely, another study suggested that although cytokine signalling induces ER stress as demonstrated by activation of PERK and JNK, induction of CHOP is not required for -cell death in rodents (Åkerfeldt et al., 2008). In support of these findings, a recent study suggests

mediated by activation of CHOP.

Cytokines induce multiple stress pathways in β-cells. Oxidative stress is induced by increased production of ROS and an imbalanced, low level of antioxidant enzymes (Sies, 1997). IL-1β, TNFα and IFNγ induce the production of ROS and NO by inducing iNOS (Rabinovitch & Suarez-Pinzon, 1998). Free oxygen and nitrogen radicals generated can react to form peroxynitrite, which is a very strong oxidant. Oxygen free radicals, nitrogen free radicals as well as the radical peroxynitrite can react with and damage a range of cellular proteins and in this way block metabolic functions and induce β-cell death (Azevedo-Martins et al., 2003). β-cells have been shown to be especially susceptible to such oxidative stress because they have particularly low levels of antioxidant enzymes (Lenzen et al., 1996, Tiedge et al., 1998).
