2. ER stress and insulin

#### 2.1. Proinsulin translation and folding in the ER

Human has INS gene as only insulin gene, whereas rodents have INS1 and INS2 genes for insulin. Human insulin gene encodes preproinsulin that has 110 amino acids containing Nterminal signal peptide following B chain, C-peptide, and A chain. Preproinsulin mRNA translation begins in the cytosol in pancreatic β-cells, and the signal peptide is recognized by signal recognition particle (SRP) to translate proinsulin across the membrane of the ER. In the ER, signal peptide is cleaved by signal peptidase to produce proinsulin that has 86 amino acids consisting of B chain, C-peptide, and A chain (Figure 3). Proinsulin is folded in the ER by chaperones including protein disulfide isomerase (PDI) family and BiP. Molecular chaperons and PDIs bind to the hydrophobic regions of proteins to promote folding and inhibit the aggregation of proteins [8]. Proinsulin has three disulfide bonds in A6–A11, A7–B7, and A20– B19 (Figure 3) [9]. N-glycosylation is often used as a marker for proper folding of newly synthesized proteins in the ER; however, proinsulin does not have N-glycosylation site.

#### 2.2. ER stress

The high demand of insulin synthesis under a high plasma glucose condition causes ER stress that could cause β-cell dysfunction. Generally, the secretory proteins and transmembrane proteins are folded and acquire a variety of modification in the ER. Environmental and genetic factors affect protein folding in the ER. If protein folding is inhibited, unfolded proteins accumulate in the ER, leading to ER stress. Cells that sense ER stress cause unfolded protein response (UPR) that includes the inhibition of general protein translation, the induction of expression of ER chaperons, and ER-associated degradation (ERAD). UPR is a cellular response to recover ER homeostasis. In mammalian cells, there are three ER stress sensors, PERK, IRE1α, and ATF6 (Figure 4).

Protein kinase RNA-like ER kinase (PERK) is type-I transmembrane protein that localizes in the ER. Under ER stress, PERK undergoes autophosphorylation to be activated and oligomerized. Oligomerized PERK phosphorylates translation initiation factor, eIF2 α-subunit to inhibit protein translation. The inhibition of protein translation attenuates the accumulation of unfolded ER proteins as well as ER stress [10]. On the other hand, this inhibition of protein translation promotes the translation of ATF4, a transcription factor that induces genes related to apoptosis, amino acid metabolism, and antioxidants. Still, when cells cannot deal with ER stress even by these measures and ER stress is continued, ATF4 induces the transcription of C/EBP homologous

proteins (CHOP/GADD153). CHOP and ATF4 form a heterodimer that induces the transcription

Figure 3. Insulin biosynthesis in the ER. The schematic structure of human insulin is shown. Insulin has signal peptide in its N-terminus followed by B chain, C-peptide, and A chain. Insulin mRNA translation is initiated in the cytosol as preproinsulin and cotranslationally inserted to the ER. Signal peptide is cleaved by endopeptidase during insertion into

ER Stress, Secretory Granule Biogenesis, and Insulin http://dx.doi.org/10.5772/intechopen.76131 41

the ER and proinsulin is generated. In the ER, proinsulin is folded by three disulfide bonds of A and B chain.

Inositol-requiring enzyme 1 (IRE1) has two isoforms: IRE1α that is expressed ubiquitously [13] and IRE1β that is expressed in goblet cells that secrete mucin in the digestive tract and lung [14, 15]. IRE1α is the type-I transmembrane protein localized in the ER. IRE1α has kinase and ribonuclease domains in its cytoplasmic region, and its lumenal domain has the binding site

of each downstream genes and promotes apoptosis [11, 12].

located in the center and the other cells are in the periphery [2]. Human islets of Langerhans do not have such clear structures. β-cells are mixed with the other cells. In avian pancreas, α-cells are in the center of islets. In zebrafish, their pancreas shares the basic structure with mammalian pancreas [3]. Recent studies show that zebrafish is a good model to study pancreatic develop-

Human has INS gene as only insulin gene, whereas rodents have INS1 and INS2 genes for insulin. Human insulin gene encodes preproinsulin that has 110 amino acids containing Nterminal signal peptide following B chain, C-peptide, and A chain. Preproinsulin mRNA translation begins in the cytosol in pancreatic β-cells, and the signal peptide is recognized by signal recognition particle (SRP) to translate proinsulin across the membrane of the ER. In the ER, signal peptide is cleaved by signal peptidase to produce proinsulin that has 86 amino acids consisting of B chain, C-peptide, and A chain (Figure 3). Proinsulin is folded in the ER by chaperones including protein disulfide isomerase (PDI) family and BiP. Molecular chaperons and PDIs bind to the hydrophobic regions of proteins to promote folding and inhibit the aggregation of proteins [8]. Proinsulin has three disulfide bonds in A6–A11, A7–B7, and A20– B19 (Figure 3) [9]. N-glycosylation is often used as a marker for proper folding of newly synthesized proteins in the ER; however, proinsulin does not have N-glycosylation site.

The high demand of insulin synthesis under a high plasma glucose condition causes ER stress that could cause β-cell dysfunction. Generally, the secretory proteins and transmembrane proteins are folded and acquire a variety of modification in the ER. Environmental and genetic factors affect protein folding in the ER. If protein folding is inhibited, unfolded proteins accumulate in the ER, leading to ER stress. Cells that sense ER stress cause unfolded protein response (UPR) that includes the inhibition of general protein translation, the induction of expression of ER chaperons, and ER-associated degradation (ERAD). UPR is a cellular response to recover ER homeostasis. In mammalian cells, there are three ER stress sensors, PERK, IRE1α, and ATF6

Protein kinase RNA-like ER kinase (PERK) is type-I transmembrane protein that localizes in the ER. Under ER stress, PERK undergoes autophosphorylation to be activated and oligomerized. Oligomerized PERK phosphorylates translation initiation factor, eIF2 α-subunit to inhibit protein translation. The inhibition of protein translation attenuates the accumulation of unfolded ER proteins as well as ER stress [10]. On the other hand, this inhibition of protein translation promotes the translation of ATF4, a transcription factor that induces genes related to apoptosis, amino acid metabolism, and antioxidants. Still, when cells cannot deal with ER stress even by these measures and ER stress is continued, ATF4 induces the transcription of C/EBP homologous

ment and diabetes mellitus [4–7].

40 Ultimate Guide to Insulin

2. ER stress and insulin

2.2. ER stress

(Figure 4).

2.1. Proinsulin translation and folding in the ER

Figure 3. Insulin biosynthesis in the ER. The schematic structure of human insulin is shown. Insulin has signal peptide in its N-terminus followed by B chain, C-peptide, and A chain. Insulin mRNA translation is initiated in the cytosol as preproinsulin and cotranslationally inserted to the ER. Signal peptide is cleaved by endopeptidase during insertion into the ER and proinsulin is generated. In the ER, proinsulin is folded by three disulfide bonds of A and B chain.

proteins (CHOP/GADD153). CHOP and ATF4 form a heterodimer that induces the transcription of each downstream genes and promotes apoptosis [11, 12].

Inositol-requiring enzyme 1 (IRE1) has two isoforms: IRE1α that is expressed ubiquitously [13] and IRE1β that is expressed in goblet cells that secrete mucin in the digestive tract and lung [14, 15]. IRE1α is the type-I transmembrane protein localized in the ER. IRE1α has kinase and ribonuclease domains in its cytoplasmic region, and its lumenal domain has the binding site

into the nucleus to function as a transcription factor that induces the transcription of genes related to ER chaperones [10, 32]. Prolonged ER stress promotes ATF6 to bind to ATF4 to

ER Stress, Secretory Granule Biogenesis, and Insulin http://dx.doi.org/10.5772/intechopen.76131 43

Pancreatic β-cells are specialized cells to synthesize and secrete a large amount of insulin. Insulin biosynthesis in pancreatic β-cells accounts for 10–50% of total protein synthesis [36, 37]. Therefore, the burden to the ER (ER stress) in pancreatic β-cells is constitutively high even in physiological condition. It is also known that pancreatic β-cells are sensitive against oxidative stress and hypoxia [38] as well as ER stress. The expression level of glutathione peroxidase, an antioxidant, is very low in pancreatic β-cells; therefore, pancreatic β-cells are sensitive to oxidative stress [39]. The islets of Langerhans are surrounded by blood vessels and supplied with nutrients and oxygen. Hypoxia affects insulin secretion of pancreatic islets and the survival rate of grafted

In type-II diabetes, it was reported that pancreatic β-cell mass is decreased [42, 43]. Huang et al. reported that the rat model of type-II diabetes expressing human islet amyloid polypeptide (hIAPP) showed the decrease of β-cell mass due to β-cell apoptosis, and the proteins related to ER stress including CHOP is highly expressed in β-cells [44]. The relationship between ER stress and diabetes has been studied by a variety of animal models and human genetic diseases. Akita mouse, another mouse model of diabetes named by Akio Koizumi in Akita University, has a single mutation in insulin 2 gene. Although there are no gross defects in the transcription of the wild-type insulin 2 allele and the two alleles of insulin 1, the phenotype of a single mutation of insulin 2 is dominant. Insulin 2 gene in Akita mouse has tyrosine instead of cysteine 96 (C96Y), and the mutated proinsulin does not form the disulfide bond between A chain and B chain (A7–B7). The mutated proinsulin cannot be transported to the Golgi apparatus and its secretion is inhibited [45]. The mutated proinsulin is accumulated in the ER that causes UPR to result in the induction of the expression of GRP78, XBP1, and CHOP. Eventually, pancreatic β-cells die by apoptosis. The necessity of ER stress for β-cell death was demonstrated by the delay of the development of diabetes in mouse produced by crossing Akita mouse with CHOP–knock-out mouse [46]. It was reported that human also has

Wolcott-Rallison syndrome (WRS) is caused by the malfunction of Eif2ak3 gene that encodes PERK [48]. WRS is an autosomal-recessive disorder that has neonatal diabetes, epiphyseal dysplasia, osteoporosis, and growth retardation. Patients with WRS have the point mutation in the kinase domain of PERK or the mutation that causes the deletion mutant of PERK. The mutation causing kinase dead of PERK develops diabetes after several months of birth, whereas the mutation that still maintains kinase activity of PERK delays the development of diabetes after 30 months. As well as WRS, PERK knock-out mice showed the secretory defects in many tissues causing diabetes and growth defects [49–51]. Furthermore, the knock-in mice having the mutation of phosphorylation site of eIF2α, the downstream molecule of PERK signaling, are unable to inhibit translation leading to over-synthesis of insulin and resulting in

induce the transcription of CHOP, resulting in apoptosis [33–35].

2.3. ER stress and insulin

islets [40, 41].

the same mutation [47].

the dysfunction of pancreatic β-cells and β-cell death [52].

Figure 4. ER stress and activation of the unfolded protein response (UPR) pathways in mammalian cells. Accumulation of unfolded protein in the ER can be recognized by ER stress sensors, IRE1α, PERK and ATF6. These proteins are activated and mediate ER stress response, including inhibition of translation and induction of transcription of ER chaperones.

for BiP. In normal condition, IRE1α is in inactive form and binds to BiP, as well as other ERstress sensors PERK and ATF6. In ER stress condition, BiP is released from IRE1α and IRE1α forms oligomer to be in an active form [16–21]. Autophosphorylation of the kinase domain in cytoplasmic region of IRE1α activates the ribonuclease (RNase) domain in the C-terminal region of cytoplasmic domain of IRE1α [22–25]. The activated RNase domain cleaves the precursor form of XBP1 (XBP1 unspliced; XBP1u mRNA) in specific two sites to produce mature XBP1 mRNA (XBP1 spliced; XBP1s mRNA) that produces functional transcription factor to induce the transcription of genes related to ER chaperones, ERAD, and lipid metabolism to recover ER homeostasis [26, 27].

Activating transcription factor 6 (ATF6) is a type-II transmembrane protein functioning as a transcription factor. ATF6 localizes in the ER but has the Golgi-localizing signal in its lumenal region that is inhibited in normal condition by binding to BiP. Under ER-stress condition, BiP is released from the lumenal region of ATF6, and its Golgi-localizing signal is exposed to transport ATF6 to the Golgi apparatus [28, 29]. In the Golgi, site 1 protease (S1P) and site 2 protease (S2P) cleave the transmembrane region of ATF6 and produce ATF6 that has only cytoplasmic region containing DNA-binding site [30, 31]. The cleaved ATF6 is translocated into the nucleus to function as a transcription factor that induces the transcription of genes related to ER chaperones [10, 32]. Prolonged ER stress promotes ATF6 to bind to ATF4 to induce the transcription of CHOP, resulting in apoptosis [33–35].

### 2.3. ER stress and insulin

for BiP. In normal condition, IRE1α is in inactive form and binds to BiP, as well as other ERstress sensors PERK and ATF6. In ER stress condition, BiP is released from IRE1α and IRE1α forms oligomer to be in an active form [16–21]. Autophosphorylation of the kinase domain in cytoplasmic region of IRE1α activates the ribonuclease (RNase) domain in the C-terminal region of cytoplasmic domain of IRE1α [22–25]. The activated RNase domain cleaves the precursor form of XBP1 (XBP1 unspliced; XBP1u mRNA) in specific two sites to produce mature XBP1 mRNA (XBP1 spliced; XBP1s mRNA) that produces functional transcription factor to induce the transcription of genes related to ER chaperones, ERAD, and lipid metab-

Figure 4. ER stress and activation of the unfolded protein response (UPR) pathways in mammalian cells. Accumulation of unfolded protein in the ER can be recognized by ER stress sensors, IRE1α, PERK and ATF6. These proteins are activated and mediate ER stress response, including inhibition of translation and induction of transcription of ER

Activating transcription factor 6 (ATF6) is a type-II transmembrane protein functioning as a transcription factor. ATF6 localizes in the ER but has the Golgi-localizing signal in its lumenal region that is inhibited in normal condition by binding to BiP. Under ER-stress condition, BiP is released from the lumenal region of ATF6, and its Golgi-localizing signal is exposed to transport ATF6 to the Golgi apparatus [28, 29]. In the Golgi, site 1 protease (S1P) and site 2 protease (S2P) cleave the transmembrane region of ATF6 and produce ATF6 that has only cytoplasmic region containing DNA-binding site [30, 31]. The cleaved ATF6 is translocated

olism to recover ER homeostasis [26, 27].

chaperones.

42 Ultimate Guide to Insulin

Pancreatic β-cells are specialized cells to synthesize and secrete a large amount of insulin. Insulin biosynthesis in pancreatic β-cells accounts for 10–50% of total protein synthesis [36, 37]. Therefore, the burden to the ER (ER stress) in pancreatic β-cells is constitutively high even in physiological condition. It is also known that pancreatic β-cells are sensitive against oxidative stress and hypoxia [38] as well as ER stress. The expression level of glutathione peroxidase, an antioxidant, is very low in pancreatic β-cells; therefore, pancreatic β-cells are sensitive to oxidative stress [39]. The islets of Langerhans are surrounded by blood vessels and supplied with nutrients and oxygen. Hypoxia affects insulin secretion of pancreatic islets and the survival rate of grafted islets [40, 41].

In type-II diabetes, it was reported that pancreatic β-cell mass is decreased [42, 43]. Huang et al. reported that the rat model of type-II diabetes expressing human islet amyloid polypeptide (hIAPP) showed the decrease of β-cell mass due to β-cell apoptosis, and the proteins related to ER stress including CHOP is highly expressed in β-cells [44]. The relationship between ER stress and diabetes has been studied by a variety of animal models and human genetic diseases. Akita mouse, another mouse model of diabetes named by Akio Koizumi in Akita University, has a single mutation in insulin 2 gene. Although there are no gross defects in the transcription of the wild-type insulin 2 allele and the two alleles of insulin 1, the phenotype of a single mutation of insulin 2 is dominant. Insulin 2 gene in Akita mouse has tyrosine instead of cysteine 96 (C96Y), and the mutated proinsulin does not form the disulfide bond between A chain and B chain (A7–B7). The mutated proinsulin cannot be transported to the Golgi apparatus and its secretion is inhibited [45]. The mutated proinsulin is accumulated in the ER that causes UPR to result in the induction of the expression of GRP78, XBP1, and CHOP. Eventually, pancreatic β-cells die by apoptosis. The necessity of ER stress for β-cell death was demonstrated by the delay of the development of diabetes in mouse produced by crossing Akita mouse with CHOP–knock-out mouse [46]. It was reported that human also has the same mutation [47].

Wolcott-Rallison syndrome (WRS) is caused by the malfunction of Eif2ak3 gene that encodes PERK [48]. WRS is an autosomal-recessive disorder that has neonatal diabetes, epiphyseal dysplasia, osteoporosis, and growth retardation. Patients with WRS have the point mutation in the kinase domain of PERK or the mutation that causes the deletion mutant of PERK. The mutation causing kinase dead of PERK develops diabetes after several months of birth, whereas the mutation that still maintains kinase activity of PERK delays the development of diabetes after 30 months. As well as WRS, PERK knock-out mice showed the secretory defects in many tissues causing diabetes and growth defects [49–51]. Furthermore, the knock-in mice having the mutation of phosphorylation site of eIF2α, the downstream molecule of PERK signaling, are unable to inhibit translation leading to over-synthesis of insulin and resulting in the dysfunction of pancreatic β-cells and β-cell death [52].

ATF6 knock-out mice do not show gross defects in normal diet, whereas high-fat diet causes the dysfunction of pancreatic β-cells [53, 54]. Furthermore, strong ER stress promotes the death of pancreatic β-cells [55, 56].

The knock-out mice of IRE1α specifically deleted in pancreatic β-cells cause diabetic phenotype [57, 58]. The mRNA levels of preproinsulin are not impaired; however, the protein level of proinsulin and mature insulin decreases, and protein and mRNA levels of five PDI protein families, PDI, PDIR, P5, ERp44, and ERp46, also decrease. These results indicate that these five PDI families are involved in proinsulin folding downstream of IRE1α, and upregulation of these PDI families could be the next approach for the treatment of diabetes.
