**5. The role of ER stress in β cell destruction**

ER stress regulates the expression of cytokines, while cytokines in turn may also induce ER stress via pathways including inducible nitric oxide synthase (iNOS) and JNK. JNK pathway is activated by IL-1β. Suppression of JNK by its inhibitor SP600125 can protectβ cells from IL-1β-induced apoptosis (85). Inflammatory cytokines induce iNOS expression in β cells and produce copious amount of nitric oxygen (86).Nitric oxygen is an important mediator of βcell death in type 1 diabetes. Excessive nitric oxygencan induce DNA damage, which leads to β cell apoptosis through p53 pathway or necrosis through poly (ADP-ribose) polymerase pathway (87). In addition, nitric oxygencan also deplete ER Ca2+ stores by activating Ca2+ channels or inhibiting Ca2+ pumps (88-90). Depletion of Ca2+ then leads to the activation of

Given the involvement of ER stress in both innate and adaptive immune systems, pathways of ER stress play a role in the autoimmune process of type 1 diabetes. For example, mice de‐ ficient in PERK, a molecule responsible for regulating UPR, are extremely susceptible to dia‐ betes. Although the exocrine and endocrine pancreas developed normally, the *null* mice display a progressive loss of β mass and insulin insufficiency postnatally (93) (93). A severe defect of β cell proliferation and differentiation was also found in *PERK null* mice, resulting in low pancreatic β mass and proinsulin trafficking defects (94). Consistent with those obser‐ vations in mice, some infant-onset diabetic cases in humans are confirmed to be associated with the mutations in PERK. For example, loss of *EIF2AK3* (the gene encodes PERK) devel‐ ops Wolcott-Rallison syndrome, an autosomal recessive disorder featured by early infancy insulin-dependency and multiple systemic manifestations including growth retardation, hepatic/renal dysfunction, mental retardation, and cardiovascular abnormalities (86;95). Similarly, disruption of UPR by mutating eIF2α, the downstream molecule of PERK signal‐ ing, enhances the sensitivity to ER stress-induced apoptosis and results in defective gluco‐ neogenesis. Mice carrying a homozygous Ser51Ala mutation for eIF2α show multiple defects in pancreatic β cells including the smaller core of insulin-secreting β cells and attenu‐ ated insulin secretion (41). Altogether, defects in PERK/eIF2α signaling render β cells highly vulnerable to ER stress in both humans and mice (87;96). In addition to PERK/eIF2α signal‐ ing, the other two pathways of ER stress, IRE1 and ATF6, are also implicated in the func‐ tionality of β cells. The activation of IRE1 signaling is involved in the insulin biosynthesis induced by hyperglycemia. Transient exposure to high glucose enhances IRE1α phosphory‐ lation without activation of XBP-1 and BiP dissociation. IRE1α activation induced by transi‐ ent exposure to high glucose induces insulin biosynthesis by up-regulating WFS1, a component involved in UPR and maintaining ER homeostasis (10;97). However, chronic ex‐ posure of β cells to high glucose may cause activation of IRE1 but with a different down‐ stream signaling, leading to the suppression of insulin biosynthesis (10). The activation of ATF6 induced by ER stress also suppressed the expression of insulin by up-regulating or‐

CHOP and induces ER stress and apoptosis of β cells (91;92).

206 Type 1 Diabetes

phan nuclear receptor small heterodimer partner (98).

**4.4. ER stress in the autoimmune process of type 1 diabetes**

#### **5.1. The involvement of ER stress in β cell destruction**

Increasing evidence suggests an important role of ER stress in autoimmune-mediated β cell destruction (99;100). It was noted that β cell loss is the direct causing factor for insufficient insulin secretion in type 1 diabetes patients. Pancreatic β cells have a very well-developed ER to fulfill their biological function for secreting insulin and other glycoproteins, causing the high sensitivity of β cells to ER stress and the subsequent UPR. Severe or long-term ER stress would direct β cells undergoing apoptosis (99). As described earlier, all the three path‐ ways of ER stress are important in the execution of β cell function and involved in the auto‐ immune responses during the process of type 1 diabetes.

Pro-inflammatory cytokines are believed as the major mediators contributing to ER stress in β cell mediated by autoimmune response. Autoreactive immune cells infiltrated in pancreas produce pro-inflammatory cytokines, the primary causing factor for β cell death in type 1 diabetes(101). Autoreactive macrophages and T-lymphocytes present in the pancreatic islets in the early stage of type 1 diabetes and secrete massive pro-inflammatory cytokines includ‐ ing IL-1β, IFN-γ and TNF-α. Pro-inflammatory cytokines have been confirmed as strong in‐ ducers of ER stress in pancreatic β cells. Insult of β cells with IL-1β and IFN-γ was reported to induce the expression of death protein 5, a protein involved in the cytokine-induced ER stress and β cell death (102). Suppression of death protein 5 by siRNA provides protection for β cells against pro-inflammatory cytokine-induced ER stress (102). In addition, stimula‐ tion of β cells with IL-1β and IFN-γ can decrease the expression of sarcoendoplasmic reticu‐ lum pump Ca2+ ATPase 2b, leading to subsequent depletion of Ca2+ in the ER (103). It has been well demonstrated that altered ER Ca2+ concentration induces the accumulation of un‐ folded proteins in ER associated with the induction of UPR and ER stress in β cells (104). Reactive oxygen species such as nitric oxygen produced during inflammation are believed to play a critical role in ER stress-induced β cell death. Excessive nitric oxygen production during insulitis induces β cell apoptosis in a CHOP-dependent manner (91).

In addition to cytokine-induced ER stress, defective protein processing and trafficking are also a direct cause of ER stress in β cell. For instance, mis-folding of insulin in β cells directly induces chronic ER stress as evidenced by the observations in Akita mice. The mutation of *Ins2* gene in Akita mouse disrupts a disulfide bond betweenα and β chain of proinsulin, leading to the mis-folding of the mutated insulin. This mutation therefore induces chronic ER stress in β cells and finally causes diabetes in Akita mouse (105). The inefficiency of pro‐ tein trafficking from ER to Golgi apparatus also causes ER stress in β cells (106).

Hyperglycemia occurs only when β cells fail to compensate the increased demand for insu‐ lin. Therefore, β cells are usually "exhausted" in diabetic patients (87). The increased insulin demandrequires the remaining functional β cellsto increase insulin synthesis to compensate the decrease of β mass. The altered insulin synthesis causes ER stress in the β cells of pa‐ tients with type 1 diabetes. In later case, this compensation is beneficial for control of blood glucose homeostasisin a short term.However, the long term alterations of insulin synthesis in the β cells also induce ER stress which in turn exacerbates β cell dysfunction and pro‐ motes disease progression. Collectively, there is convincing evidence that ER stress plays an essential role in β cell destruction during the course of type 1 diabetes.

Furthermore, IRE1 can also activate JNK signaling by interacting with c-Jun N-terminal

Endoplasmic Reticulum (ER) Stress in the Pathogenesis of Type 1 Diabetes

http://dx.doi.org/10.5772/52644

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Although the purpose of UPR is to maintain the homeostasis of ER, apoptosis could occur when the insult of ER stress exceeds the cellular regulatory capacity. Apoptosis is initiated by the activation of several proteases including caspase-12, caspase-4, caspase-2, and cas‐ pase-9. Studies in rodents suggest that caspase-12 is activated by IRE1 and is involved in ER stress-induced apoptosis. Mice deficient for caspase-12 are resistant to ER stress-induced apoptosis, but remain susceptible to apoptosis induced by other stimuli (114). Caspase-12 can also be activated by TRAF2, a downstream molecule of IRE1 (113). In response to ER stress, caspase-7 is translocated from the cytosol to the ER surface, and then activates pro‐ caspase-12 (115). Human caspase-4, the closest paralog of rodent caspase-12, can only be ac‐ tivated by ER stress-inducing reagents not by the other apoptotic reagents. Knockdown of caspase-4 by siRNA reduces ER stress-induced apoptosis in neuroblastoma cells, suggesting the involvement of human caspase-4 in ER stress-induced cell death (116). Similarly, cas‐ pase-2 and caspase-9 are also activated in the early phase of ER stress. Inhibition of their ac‐ tivation either by inhibitors or siRNA reduces ER stress-induced apoptosis (117). Other than caspase proteins, Ask1 kinase and CHOP are also critical mediators for ER stress-induced cell death. IRE1/TRAF2 complex recruits Ask1 and activates subsequent JNK signaling. The activation of JNK then induces apoptosis by inhibiting anti-apoptotic protein BCL-2 (118) and inducing pro-apoptotic protein Bim (119;120). Deficiency of Ask1 suppresses ER stressinduced JNK activation and protects cells against ER stress-induced apoptosis (121). CHOP, a transcription factor belonging to basic leucine zipper transcription factor family, can be ac‐ tivated by many inducers of UPR including ATF4, ATF6, and XBP-1. Upon activation, CHOP induces cells undergoing apoptosis through suppressing anti-apoptotic protein

Although exogenous insulin therapy partly compensates the function of β cells, it cannot regulate blood glucose as accurately as the action of endogenous insulin. As a result, long-term improperly control of blood glucose homeostasis predisposes patients with type 1 diabetes to the development of diverse complications such as diabetic retinopathy (125-127), nephropathy (128;129), neuropathy (130-132), foot ulcers (133-135), and cardio‐ vascular diseases (136-138). Due to the long-term health consequences of diabetes, impact of insulin dependence on life quality, and increasing appearance in both young and old populations, understanding the pathophysiology of diabetes and finding a better way to treat diabetes has become a high priority. Although the underlying mechanisms leading to type 1 diabetes have yet to be fully addressed, accumulating evidence suggests that ER stress plays a critical role in autoimmune-mediated β cell destruction during the course of type 1 diabetes. ER stress in β cells can be triggered by either autoimmune responses against β-cell self-antigens or the increase of compensated insulin synthesis. During the course of type 1 diabetes, autoreactive immune cells secrete copious amount of inflamma‐

inhibitory kinase (JIK) (113).

BCL-2 (122-124).

**6. Conclusions and future directions**

#### **5.2. Mechanisms underlying ER stress-induced β cell death**

The primary purpose of ER stress response is to compensate the damage caused by the dis‐ turbances of normal ER function. However, persistence of ER dysfunction would eventually render cells undergoing apoptosis. The mechanisms underlying ER stress induced cell death are not fully elucidated, due to the fact that multiple potential participants involved but lit‐ tle clarity on the dominant death effectors in a particular cellular context. Generally, the process of cell death by ER stress can be illustrated in three phases: adaptation, alarm, and apoptosis (39).

The adaptation response phase is to protect cells from damage induced by the disturban‐ ces of ER function and restore the homeostasis of ER. As described earlier, UPR signaling involves three axes of responses: IRE1, PERK, and ATF6. These axes interact between each other and form a feedback regulatory mechanism to control the activity of UPR. The accumulation of unfolded proteins in ER results in the engagement of ER resident chaper‐ on BiP, and as a consequence, IRE1, PERK, and ATF6 are released from BiP. Therefore, over-expression of BiP can prevent cell death induced by oxidative stress, Ca2+ disturban‐ ces, and hypoxia (107). Upon ER stress, the transcription of BiP is enhanced by ATF6p50, the cleaved form of ATF6 (108). PERK is oligomerized and phosphorylated upon the re‐ lease from BiP. Activated PERK inactivates eIF2α to reduce mRNA translation and pro‐ tein load on ER. Therefore, PERK deficiency results in an abnormally elevated protein synthesis in response to ER stress, and renders cells highly sensitive to ER stress-induced apoptosis (109). Consistently, as a downstream molecule of PERK, eIF2α is required for cell survival upon the insult of ER stress. A mutation at the phosphorylation site of eIF2α (Ser51Ala) abolishes the translational suppression in response to ER stress (41). When re‐ leased from BiP, IRE1 becomes dimerized and activated. Activated IRE1 then induces XBP-1 by promoting the splicing of its mRNA (44). XBP-1 is responsible for the transcrip‐ tion of many adaptation genes implicated in UPR. Unlike PERK and IRE1, ATF6 translo‐ cates into the Golgi apparatus once released from BiP. The transmembrane domain of ATF6 is cleaved in the Golgi apparatus and is then relocated into the nucleus, by which it regulates gene expression (51).

During the alarm phase, many signal pathways are activated to alert the system. For in‐ stance, the cytoplasmic part of IRE1 can bind to TNF receptor-associated factor 2 (TRAF2), a key adaptor mediating TNF-induced innate immune response. TRAF2 then activates NFκB pathway via activating IKK and activates the signaling for c-Jun N-terminal kinas‐ es (JNK) by apoptosis signal-regulating kinase 1 (Ask1). It is reported that dominant neg‐ ative TRAF2 suppresses the activation of JNK in response to ER stress (110). In addition, TRAF2 is also a critical component for E3 ubiquitin-protein ligase complex (111). E3 ubiq‐ uitin-protein ligase complex binds to Ubc13 and mediates the noncanonical ubiquitina‐ tion of substrates, which is suggested to be required for the activation of JNK (112). Furthermore, IRE1 can also activate JNK signaling by interacting with c-Jun N-terminal inhibitory kinase (JIK) (113).

Although the purpose of UPR is to maintain the homeostasis of ER, apoptosis could occur when the insult of ER stress exceeds the cellular regulatory capacity. Apoptosis is initiated by the activation of several proteases including caspase-12, caspase-4, caspase-2, and cas‐ pase-9. Studies in rodents suggest that caspase-12 is activated by IRE1 and is involved in ER stress-induced apoptosis. Mice deficient for caspase-12 are resistant to ER stress-induced apoptosis, but remain susceptible to apoptosis induced by other stimuli (114). Caspase-12 can also be activated by TRAF2, a downstream molecule of IRE1 (113). In response to ER stress, caspase-7 is translocated from the cytosol to the ER surface, and then activates pro‐ caspase-12 (115). Human caspase-4, the closest paralog of rodent caspase-12, can only be ac‐ tivated by ER stress-inducing reagents not by the other apoptotic reagents. Knockdown of caspase-4 by siRNA reduces ER stress-induced apoptosis in neuroblastoma cells, suggesting the involvement of human caspase-4 in ER stress-induced cell death (116). Similarly, cas‐ pase-2 and caspase-9 are also activated in the early phase of ER stress. Inhibition of their ac‐ tivation either by inhibitors or siRNA reduces ER stress-induced apoptosis (117). Other than caspase proteins, Ask1 kinase and CHOP are also critical mediators for ER stress-induced cell death. IRE1/TRAF2 complex recruits Ask1 and activates subsequent JNK signaling. The activation of JNK then induces apoptosis by inhibiting anti-apoptotic protein BCL-2 (118) and inducing pro-apoptotic protein Bim (119;120). Deficiency of Ask1 suppresses ER stressinduced JNK activation and protects cells against ER stress-induced apoptosis (121). CHOP, a transcription factor belonging to basic leucine zipper transcription factor family, can be ac‐ tivated by many inducers of UPR including ATF4, ATF6, and XBP-1. Upon activation, CHOP induces cells undergoing apoptosis through suppressing anti-apoptotic protein BCL-2 (122-124).
