3. Biogenesis of insulin secretory granules

After reaching the Golgi from the ER, secretory proteins are sorted in the trans-Golgi network (TGN) (Figure 1). One of the secretory pathways is the constitutive pathway in which proteins are constitutively secreted. When there is no sorting signal, proteins are thought to follow this pathway in mammalian cells. By contrast, another secretory pathway is the regulated pathway in which secretory proteins are packaged into the immature secretory granules (ISGs) (Figure 5). ISGs mature into mature secretory granules (MSGs), then MSGs are fused with the plasma membrane (PM) upon the stimulation of secretagogues to secrete the contents of MSGs. Proinsulin follows the regulated secretory pathway after the Golgi apparatus.

Secretory proteins destined for regulated pathway are segregated from other proteins and packaged into ISGs. This is termed as sorting by entry. On the other hand, in the process of formation and maturation of ISGs, other proteins are eliminated from ISGs. It is termed as sorting by exit, or sorting by retention [59–61].

#### 3.1. Proinsulin transport to immature secretory granules

The molecular mechanisms of proinsulin sorting in the TGN are yet to be understood. It is thought that the selective aggregation of proinsulin occurs in the TGN [62, 63]. Insulin secretory granules contain a clear electron-dense core structure suggesting that insulin is crystallized in the granules. In pituitary AtT-20 cells, insulin granules can be formed by transfecting insulin gene, and hemagglutinin that flows in a constitutive pathway is segregated from the dense-core structure. Hemagglutinin is distributed evenly through the Golgi stacks as well as proinsulin; however, they are segregated after the TGN [62]. Therefore, the proinsulin sorting from constitutive pathway could occur in the TGN.

pancreatic β-cells is questionable [59]. While research to find out the sorting receptor has been going on, another possibility was proposed; cargo aggregation/oligomerization is the sorting

Figure 5. Insulin Secretory Granule (SG) formation. After folded in the ER, proinsulin is transported to the Golgi apparatus, then packaged into immature secretory granules (ISG) from the trans-Golgi network (TGN). ISGs mature into mature secretory granules (MSGs). There are several steps to make SGs; segregation from constitutive secretory proteins,

ER Stress, Secretory Granule Biogenesis, and Insulin http://dx.doi.org/10.5772/intechopen.76131 45

It is proposed that aggregated proteins directly bind to lipid micro-domain in the TGN membranes and these micro-domains could be recognized by cytosolic machineries [59, 67]. Secretory granules (SGs) contain a high amount of cholesterol, and the depletion or addition of cholesterol affects glucose-stimulated insulin secretion (GSIS) [68]. Secretogranin III is one of

signal for ISGs [60, 61, 66].

possibly homotypic fusion and removal of proteins not required for MSGs.

The sorting receptor that recognizes proinsulin and transport proinsulin into ISGs remains unidentified [59]. Carboxypeptidase E (CPE), an enzyme involved in insulin processing, was proposed to play a role as the sorting receptor [64]; however, the islets from mice that lost CPE by its mutation showed that insulin is efficiently secreted by secretagogues as well as in control islets, whereas the constitutive secretion of insulin remains as low as 1% similar to that in control islets [65]. Therefore, the possibility that CPE plays a role as a sorting receptor in

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

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

After reaching the Golgi from the ER, secretory proteins are sorted in the trans-Golgi network (TGN) (Figure 1). One of the secretory pathways is the constitutive pathway in which proteins are constitutively secreted. When there is no sorting signal, proteins are thought to follow this pathway in mammalian cells. By contrast, another secretory pathway is the regulated pathway in which secretory proteins are packaged into the immature secretory granules (ISGs) (Figure 5). ISGs mature into mature secretory granules (MSGs), then MSGs are fused with the plasma membrane (PM) upon the stimulation of secretagogues to secrete the contents of MSGs. Proin-

Secretory proteins destined for regulated pathway are segregated from other proteins and packaged into ISGs. This is termed as sorting by entry. On the other hand, in the process of formation and maturation of ISGs, other proteins are eliminated from ISGs. It is termed as

The molecular mechanisms of proinsulin sorting in the TGN are yet to be understood. It is thought that the selective aggregation of proinsulin occurs in the TGN [62, 63]. Insulin secretory granules contain a clear electron-dense core structure suggesting that insulin is crystallized in the granules. In pituitary AtT-20 cells, insulin granules can be formed by transfecting insulin gene, and hemagglutinin that flows in a constitutive pathway is segregated from the dense-core structure. Hemagglutinin is distributed evenly through the Golgi stacks as well as proinsulin; however, they are segregated after the TGN [62]. Therefore, the proinsulin sorting

The sorting receptor that recognizes proinsulin and transport proinsulin into ISGs remains unidentified [59]. Carboxypeptidase E (CPE), an enzyme involved in insulin processing, was proposed to play a role as the sorting receptor [64]; however, the islets from mice that lost CPE by its mutation showed that insulin is efficiently secreted by secretagogues as well as in control islets, whereas the constitutive secretion of insulin remains as low as 1% similar to that in control islets [65]. Therefore, the possibility that CPE plays a role as a sorting receptor in

these PDI families could be the next approach for the treatment of diabetes.

sulin follows the regulated secretory pathway after the Golgi apparatus.

3. Biogenesis of insulin secretory granules

sorting by exit, or sorting by retention [59–61].

3.1. Proinsulin transport to immature secretory granules

from constitutive pathway could occur in the TGN.

of pancreatic β-cells [55, 56].

44 Ultimate Guide to Insulin

Figure 5. Insulin Secretory Granule (SG) formation. After folded in the ER, proinsulin is transported to the Golgi apparatus, then packaged into immature secretory granules (ISG) from the trans-Golgi network (TGN). ISGs mature into mature secretory granules (MSGs). There are several steps to make SGs; segregation from constitutive secretory proteins, possibly homotypic fusion and removal of proteins not required for MSGs.

pancreatic β-cells is questionable [59]. While research to find out the sorting receptor has been going on, another possibility was proposed; cargo aggregation/oligomerization is the sorting signal for ISGs [60, 61, 66].

It is proposed that aggregated proteins directly bind to lipid micro-domain in the TGN membranes and these micro-domains could be recognized by cytosolic machineries [59, 67]. Secretory granules (SGs) contain a high amount of cholesterol, and the depletion or addition of cholesterol affects glucose-stimulated insulin secretion (GSIS) [68]. Secretogranin III is one of the components of SGs and known to bind to cholesterol-rich membranes [69]. Although the role of secretogranin III in SG biogenesis in mice is not clear [70], it could be important to investigate the role of cholesterol.

convertases (PC1/3 and PC2) and carboxypeptidase E (CPE) [88] (Figure 6). PC1 (also known as PC3) cleaves 32–33 junction between B chain and C-peptide, and then CPE removes 31, 32 arginine residues. The intermediate form of proinsulin that is cleaved in B–C junction but is yet to be cleaved in A–C junction is termed as des-31, 32 split proinsulin. PC2 cleaves 65–66 junction between A chain and C peptide, and CPE removes 64, 65 lysine and arginine residues to form another intermediate termed des-64, 65 split proinsulin [9]. The cleavage of B–C junction tends to occur first before the cleavage of A–C junction [89]. PC1/3 and PC2 are Ca2+ and pH-dependent endopeptidases. The optimal pH of both enzymes is pH 5.5 [90, 91]. The pH at the TGN is reported to be ~6.0 [92]. The pH of ISGs varies from 5.5 to 7.0 and the pH of

ER Stress, Secretory Granule Biogenesis, and Insulin http://dx.doi.org/10.5772/intechopen.76131 47

Figure 6. Proinsulin processing. Proinsulin processing is thought to be initiated in the TGN and continue to undergo in ISGs. Prohormone convertases PC1/3 and PC2 cleave C-peptide from proinsulin then Carboxypeptidase E (CPE) removes

di-basic residues (Arg-Arg or Arg-Lys) to produce mature insulin and C-peptide.

There is an interesting phenomenon using SEGFP that is the green fluorescent protein (GFP) having a signal peptide in its N-terminus. In cultured insulin-secreting cells (INS-1 cells), SEGFP is sorted to secretory granules and secreted by secretagogues similar to insulin, whereas secreted alkaline phosphatase (SEAP), a model protein of constitutive pathway, is constitutively secreted [71]. SEGFP forms oligomer by a disulfide bond, and its oligomerization could unexpectedly function as a sorting signal to ISGs. The results may support the idea that cargo oligomerization itself, rather than specific sequences on cargo, is required for sorting. The involvement of lipids or other sorting proteins in this case is unclear.

Zinc and calcium ions play important roles in insulin oligomerization. Structural studies showed that insulin forms a dimer, and in the presence of zinc and calcium ions, it forms a hexamer [72, 73]. It is thought that the concentration of zinc and calcium ions rises in the TGN [9], and these ions are enriched in SGs [74, 75]. The oligomerization regions of insulin and proinsulin are essentially the same with or without C-peptide [72]. The cleavage of C-peptide from proinsulin hexamer decreases the solubility of insulin hexamer leading to crystallization of insulin in mature secretory granules (MSGs). Insulin crystals are thought to be stable and can be stored in MSGs for a long time without being degraded [73]. ZnT8 zinc transporter, the product of SLC30A8 gene, is highly expressed in pancreatic β-cells, and the combined deletion of ZnT8 and ZnT7 inhibits GSIS [76]. However, ZnT8 mutation is protective against type 2 diabetes [77]. The precise function of ZnT8 in insulin biosynthetic pathway and its relationship with the development of diabetes remains unclarified.

Although the molecular mechanisms of sorting are yet to be understood, recent studies revealed the molecules to be involved in the fission process of SGs from the TGN. Arfaptin 1 has a lipid-binding domain termed Bin/Amphiphysin/Rvs (BAR) domain that binds to a curved membrane structure [78, 79] and implicated in a regulating membrane fission [80]. Arfaptin-1 binds to small GTP-binding proteins, Arf1- and Arf-like protein 1 (Arl1), and recruited to the Golgi membrane by a GTP-bound form of Arf1 and Arl1 [81, 82]. Arfaptin 1 is phosphorylated by Protein Kinase D (PKD) that is activated by diacylglycerol (DAG) enriched in the neck of budding vesicles [83]. Non-phosphorylated mutant of Arfaptin 1 (S132A) or PKD inhibitor blocks insulin SG fission from the TGN [84]. The expression of Arfaptin 1 (S132A) or Arfaptin 1 depletion inhibits GSIS. As Arfaptin-1 was reported to be involved in other transport pathways [82, 85, 86], the specificity of Arfaptin 1 in SG biogenesis needs to be carefully addressed. Although Arfaptin 1 is proposed to play a role in membrane fission [84, 87], it could be interesting to investigate the upstream molecules of Arfaptin 1 to look for the sorting machinery for SG biogenesis.

#### 3.2. Maturation of secretory granules

#### 3.2.1. Insulin processing

The excursion of C-peptide decreases the solubility of insulin hexamer and causes insulin crystallization within SGs [73]. Proinsulin is processed into mature insulin by prohormone convertases (PC1/3 and PC2) and carboxypeptidase E (CPE) [88] (Figure 6). PC1 (also known as PC3) cleaves 32–33 junction between B chain and C-peptide, and then CPE removes 31, 32 arginine residues. The intermediate form of proinsulin that is cleaved in B–C junction but is yet to be cleaved in A–C junction is termed as des-31, 32 split proinsulin. PC2 cleaves 65–66 junction between A chain and C peptide, and CPE removes 64, 65 lysine and arginine residues to form another intermediate termed des-64, 65 split proinsulin [9]. The cleavage of B–C junction tends to occur first before the cleavage of A–C junction [89]. PC1/3 and PC2 are Ca2+ and pH-dependent endopeptidases. The optimal pH of both enzymes is pH 5.5 [90, 91]. The pH at the TGN is reported to be ~6.0 [92]. The pH of ISGs varies from 5.5 to 7.0 and the pH of

the components of SGs and known to bind to cholesterol-rich membranes [69]. Although the role of secretogranin III in SG biogenesis in mice is not clear [70], it could be important to

There is an interesting phenomenon using SEGFP that is the green fluorescent protein (GFP) having a signal peptide in its N-terminus. In cultured insulin-secreting cells (INS-1 cells), SEGFP is sorted to secretory granules and secreted by secretagogues similar to insulin, whereas secreted alkaline phosphatase (SEAP), a model protein of constitutive pathway, is constitutively secreted [71]. SEGFP forms oligomer by a disulfide bond, and its oligomerization could unexpectedly function as a sorting signal to ISGs. The results may support the idea that cargo oligomerization itself, rather than specific sequences on cargo, is required for

Zinc and calcium ions play important roles in insulin oligomerization. Structural studies showed that insulin forms a dimer, and in the presence of zinc and calcium ions, it forms a hexamer [72, 73]. It is thought that the concentration of zinc and calcium ions rises in the TGN [9], and these ions are enriched in SGs [74, 75]. The oligomerization regions of insulin and proinsulin are essentially the same with or without C-peptide [72]. The cleavage of C-peptide from proinsulin hexamer decreases the solubility of insulin hexamer leading to crystallization of insulin in mature secretory granules (MSGs). Insulin crystals are thought to be stable and can be stored in MSGs for a long time without being degraded [73]. ZnT8 zinc transporter, the product of SLC30A8 gene, is highly expressed in pancreatic β-cells, and the combined deletion of ZnT8 and ZnT7 inhibits GSIS [76]. However, ZnT8 mutation is protective against type 2 diabetes [77]. The precise function of ZnT8 in insulin biosynthetic pathway and its relationship

Although the molecular mechanisms of sorting are yet to be understood, recent studies revealed the molecules to be involved in the fission process of SGs from the TGN. Arfaptin 1 has a lipid-binding domain termed Bin/Amphiphysin/Rvs (BAR) domain that binds to a curved membrane structure [78, 79] and implicated in a regulating membrane fission [80]. Arfaptin-1 binds to small GTP-binding proteins, Arf1- and Arf-like protein 1 (Arl1), and recruited to the Golgi membrane by a GTP-bound form of Arf1 and Arl1 [81, 82]. Arfaptin 1 is phosphorylated by Protein Kinase D (PKD) that is activated by diacylglycerol (DAG) enriched in the neck of budding vesicles [83]. Non-phosphorylated mutant of Arfaptin 1 (S132A) or PKD inhibitor blocks insulin SG fission from the TGN [84]. The expression of Arfaptin 1 (S132A) or Arfaptin 1 depletion inhibits GSIS. As Arfaptin-1 was reported to be involved in other transport pathways [82, 85, 86], the specificity of Arfaptin 1 in SG biogenesis needs to be carefully addressed. Although Arfaptin 1 is proposed to play a role in membrane fission [84, 87], it could be interesting to investigate the upstream molecules of Arfaptin 1 to

The excursion of C-peptide decreases the solubility of insulin hexamer and causes insulin crystallization within SGs [73]. Proinsulin is processed into mature insulin by prohormone

sorting. The involvement of lipids or other sorting proteins in this case is unclear.

with the development of diabetes remains unclarified.

look for the sorting machinery for SG biogenesis.

3.2. Maturation of secretory granules

3.2.1. Insulin processing

investigate the role of cholesterol.

46 Ultimate Guide to Insulin

Figure 6. Proinsulin processing. Proinsulin processing is thought to be initiated in the TGN and continue to undergo in ISGs. Prohormone convertases PC1/3 and PC2 cleave C-peptide from proinsulin then Carboxypeptidase E (CPE) removes di-basic residues (Arg-Arg or Arg-Lys) to produce mature insulin and C-peptide.

MSGs is ~5.0 [89]. PC1/3 could be active in the TGN as well as in ISGs [93], whereas PC2 is thought to be active in ISGs and MSGs [91, 94].

sensors regulate proinsulin translation and folding? Is it relevant to prevent β-cell death by preventing UPR? The inhibition of CHOP has been studied to prevent β-cell death for the treatment of diabetes [46, 107–109]; however, it should be addressed carefully that even if β-cells survive by preventing CHOP, and too much accumulation of unfolded proteins in the ER may prevent normal proinsulin folding and would not support the function of islets of Langerhans. Decreasing the continuous high demand of insulin synthesis is anyway the primary importance for diabetes; then thinking about how to support proinsulin folding, packaging proinsulin into secretory granules, and elimination of unfolded proteins from

ER Stress, Secretory Granule Biogenesis, and Insulin http://dx.doi.org/10.5772/intechopen.76131 49

β-cells would help for developing new treatments of diabetes.

\*

1 Bio-Science Research Center, Kyoto Pharmaceutical University, Kyoto, Japan

[1] Ronald Kahn C, Weir G, King G, Jacobson A, Smith R, Moses A. Joslin's Diabetes

[2] Halban PA et al. Spontaneous reassociation of dispersed adult rat pancreatic islet cells into aggregates with three-dimensional architecture typical of native islets. Diabetes.

[3] Kinkel MD, Prince VE. On the diabetic menu: Zebrafish as a model for pancreas devel-

[4] Zang L, Shimada Y, Nishimura N. Development of a novel zebrafish model for type 2

[5] Tehrani Z, Lin S. Antagonistic interactions of hedgehog, Bmp and retinoic acid signals control zebrafish endocrine pancreas development. Development. 2011;138(4):631-640 [6] Kimmel RA, Meyer D. Zebrafish pancreas as a model for development and disease.

[7] Prince VE, Anderson RM, Dalgin G. Zebrafish pancreas development and regeneration: Fishing for diabetes therapies. Current Topics in Developmental Biology. 2017;124:235-276

[8] Otero JH, Lizak B, Hendershot LM. Life and death of a BiP substrate. Seminars in Cell &

2 Faculty of Science and Engineering, Iwate University, Morioka, Japan

Mellitus. 14th ed. Lippincott Williams & Wilkins; 2004

opment and function. BioEssays. 2009;31(2):139-152

diabetes mellitus. Scientific Reports. 2017;7(1):1461

Methods in Cell Biology. 2016;134:431-461

Developmental Biology. 2010;21(5):472-478

\*Address all correspondence to: shibay@iwate-u.ac.jp

Author details

References

Michiko Saito<sup>1</sup> and Yoko Shiba<sup>2</sup>

1987;36(7):783-790

#### 3.2.2. Sorting by retention

As ISGs mature, ISGs produce vesicles to remove proteins that are not required for MSGs, whereas insulin is retained in MSGs. For example, ISGs are known to produce constitutive-like vesicles that contain excess C-peptide than insulin to be secreted [95, 96]. Also, in contrast to constitutive secretory proteins, lysosomal enzymes are thought to be segregated from ISGs in pancreatic β-cells [97, 98]. Generally, lysosomal enzymes are synthesized in the ER as well as secretory proteins and transported to the Golgi apparatus (Figure 1). In the TGN and endosomes, lysosomal enzymes are recognized by mannose 6-phosphate receptors (MPRs) and then packaged into clathrin-coated vesicle (CCVs). Clathrin is a coat protein that forms a cage-like structure to produce CCVs in the post-Golgi compartment [99]. AP-1 is a clathrin adaptor that binds to MPRs and clathrin and mediates to form clathrin/AP-1-coated vesicles [100, 101]. Proinsulin ISGs have clathrin and AP-1 on their surfaces as well as MPRs [98]. Also, MSGs lose the signal of cathepsin B, a lysosomal protease, whereas ISGs still have a strong cathepsin B signal. These results suggest that lysosomal enzymes recognized by MPRs are removed from ISGs [98].

#### 3.2.3. Homotypic fusion

Syntaxin 6, a SNARE protein that is reported to be important for the homotypic fusion of ISGs in neuroendocrine cells [102], is also removed from insulin ISGs [98]. In neuroendocrine cells, it is thought that homotypic fusion plays an important role in SG maturation, and the fusion machineries required for homotypic fusion are different than that required for MSGs fusion to the PM [67, 102]. It is proposed that membrane fusion machinery is remodeled in the end of ISG maturation. The role of Syntaxin 6 and homotypic fusion in insulin granule maturation is not clear [103]. However, recent study showed that homotypic fusion could also be important in insulin granule maturation [104]. In islets from HID-1 KO mice, Vamp-4, another SNARE protein that is proposed in ISG-derived vesicle fusion to the PM in neuroendocrine cells [67], is mislocalized. Proinsulin processing and acidification are delayed, and by 3D electron microscopy, there are less homotypic fusion events [104]. Although the role of HID-1 and Vamp-4 in homotypic fusion in pancreatic β-cells should be addressed in the future, it is possible that homotypic fusion might also be important for insulin granule formation.

The MSGs are fused with the PM, and insulin and C-peptide are secreted upon stimulation. For details about the exocytosis of insulin granules, see the review articles [59, 105, 106].
