General Aspects

The two-chain structure of insulin was the first complete protein structure to be elucidated through the pioneering work of Fred Sanger in 1955.⁹ In 1967, the discovery of the single-chain insulin precursor molecule, proinsulin,¹¹ then solved the problem of assembly of the insulin chains (Fig. 6-1). The human proinsulin polypeptide begins with the 30-amino-acid B chain domain, extends through a 35-amino-acid connecting segment, and ends with the 21-amino-acid A chain domain, comprising a single 9000-D polypeptide chain.^11–14 It contains paired basic residue cleavage sites (Arg-Arg or Lys-Arg) at each end of the connecting segment which are removed during the proteolytic conversion of proinsulin to insulin within maturing secretory granules. However, cell-free translation of the initial polypeptide products encoded in insulin messenger ribonucleic acid (mRNA) extracted from islets or islet cell tumors led in 1976 to the discovery of preproinsulin (Fig. 6-2). This extended form of the prohormone has a hydrophobic N-terminal 24-residue signal peptide.¹⁵ Such prepeptide extensions interact with the signal recognition particle (SRP), a cytosolic ribonucleoprotein particle that assists in transfer of the nascent secretory protein from the cytosolic compartment, where its synthesis is initiated, into the secretory pathway via a complex series of molecular interactions that result in the translocation of the nascent peptide across the membrane of the rough endoplasmic reticulum (RER) into its internal compartments, or cisternae.¹⁶ During or shortly after translocation, the signal sequence is cleaved by the signal peptidase, which is located on the inner surface of the RER membrane, and the signal peptide is rapidly degraded.¹⁷ The proinsulin molecule then folds and undergoes rapid formation of disulfide bonds to achieve its native structure. Evidence suggests that this process is catalyzed by protein disulfide isomerase,¹⁸ a resident protein of the RER having a C-terminal Lys-Glu-Asp-Leu (KDEL) localization sequence.¹⁹ The folded proinsulin is then transferred in small coated vesicles from the endoplasmic reticulum (ER) to the cis region of the Golgi apparatus.^20,21 It then passes from the cis to the trans Golgi, where it is sorted into immature secretory vesicles, or progranules, where it is proteolytically cleaved to release insulin and the connecting segment, minus the four basic residues, from the cleavage sites. The connecting peptide fragment released by cleavage of proinsulin is designated the C peptide. Both insulin and C peptide are stored in the secretory granules along with small amounts of residual proinsulin and partially cleaved intermediate forms²² as well as a variety of other minor β cell secretory products.²³ Stimulation of secretion results in the release of the granule contents into the hepatic portal circulation.

Morphologic Organization of the Insulin Biosynthetic Machinery

The β cells of the islets of Langerhans share many features with other endocrine and neurosecretory cells. Preproinsulin is the initial translational product of insulin mRNA, as discussed earlier, and is rapidly cleaved to proinsulin in the RER. After folding and disulfide oxidation, proinsulin is transported from the ER to the Golgi apparatus in small coated vesicles²⁰ (Fig. 6-3) in an energy-dependent process that requires about 20 minutes.^22,24,25 The biochemical basis of the energy requirement for the intracellular transport of secretory proteins is associated with the budding and/or fusion of small vesicles that transfer secretory products from the ER to and through the cis to trans Golgi cisternae.^26,27

The role of the Golgi apparatus in forming insulin-containing secretory vesicles, or β-granules, was first recognized in the 1940s and subsequently confirmed and extended by Munger,²⁸ who used electron microscopy to identify pale, homogeneous-appearing, immature “progranules” near the Golgi body. The structural organization and various functions of this complex organelle continue to engage the attention of cell biologists and biochemists.²⁹ Orci and coworkers demonstrated that these newly formed clathrin-clad vesicles are derived from the trans-Golgi network (TGN) cisternae and contain a high proportion of proinsulin, definitively confirming their proposed role as the major sites of proinsulin processing to insulin.³⁰ Conversion of proinsulin to insulin in vivo behaves kinetically as a pseudo–first-order reaction having a half-time ranging from about 20 minutes to 1 hour.^22,31,32 Peak labeling of proteins in the Golgi apparatus occurs 30 to 40 minutes after pulse labeling islets with ³H-amino acids, and relatively little radioactivity remains in this region after 1 hour.³³ A similar pattern for proinsulin is observed by means of electron microscopic immunocytochemistry. Thus, although small amounts of proinsulin conversion may be initiated in the TGN, most of it occurs in newly formed secretory vesicles, or progranules, which mature biochemically in the cytosol³⁰ (see Fig. 6-3). Numerous subsequent studies have confirmed that newly synthesized neuroendocrine precursor peptides pass via the Golgi apparatus into secretory granules of the regulated secretory pathway, where they are cleaved and processed to their mature products and then stored until secretion in response to signals such as glucose.¹⁰

Structure and Properties of Proinsulin

Mammalian proinsulins range in size from 81 (cow) to 86 (human, horse, rat) amino acid residues, owing to variations in the length of the C peptide. Various C-peptide sequences are compared in Fig. 6-4. Proinsulin is closely similar to insulin in many properties, including solubility, isoelectric point,²² self-associative properties,³⁴ and reactivity with insulin antisera.^11,35 Indeed, the conformation of the insulin moiety in proinsulin is essentially identical to that of insulin itself.³⁶ It is noteworthy that the highly flexible C peptide is much longer than necessary simply to bridge the short 8-Å gap between the ends of the B and A chains as they exist in the folded insulin molecule (see Fig. 6-2). The C-peptide segment overlays a portion of the surface of the insulin monomer, reducing its potency to about 3% to 5% of that of insulin in vitro,^22,37 and it undergoes little if any proteolysis or activation in the circulation or tissues.³⁸ The presence of the C peptide in proinsulin also does not prevent the dimerization or hexamerization of its insulin moiety³⁹ (Fig. 6-5). It lies outside the zinc-stabilized globular insulin hexamer in a disordered loop, as assessed by nuclear magnetic resonance (NMR) spectroscopy. Since low levels of proinsulin can form mixed hexamers with insulin, it can be efficiently incorporated into insulin crystals.⁴⁰ This property accounts for the presence of 1% to 2% of proinsulin in most crystalline preparations of insulin prepared from animal pancreata.⁴¹ However, x-ray analysis of crystals of proinsulin have failed to reveal any details regarding the structure of the C-peptide segment.^42,43

The major intermediate cleavage products of proinsulin consist mainly of forms cleaved at only one site and lacking the exposed basic residues (i.e., des-31,32 or des-64,65 intermediates).12,22,41,44,45 Their relative ratios in the circulation differ among species, depending on differences in the amino acid sequences surrounding the cleavage sites.⁴⁶ Proinsulin-like proteins having comparably sized C peptides have been found throughout the vertebrates and also in a variety of invertebrates. Although it seems likely that the single-chain insulin-like growth factors, IGF-1 and IGF-2, arose from a single proinsulin-like gene in protochordates,⁴⁷ some invertebrates, such as Caenorhabditis elegans,⁴⁸ have numerous insulin-like peptides that lack defined C peptides and typical paired basic residue sites for cleavage by the evolutionarily well-conserved prohormone convertases.

Enzymatic Basis of the Conversion of Proinsulin to Insulin

The major proteolytic cleavages required for converting proinsulin to insulin are summarized in Fig. 6-6. In early studies, it was shown that the joint action of pancreatic trypsin and carboxypeptidase B gives rise to the naturally occurring conversion products—C peptide and native insulin in high yields—quantitatively converting proinsulin to insulin in vitro.⁴⁹ This model also explains the major intermediate forms found in pancreatic extracts,^44,50 and it led to a search for a cellular trypsin-like convertase, assumed to be a serine protease related to trypsin,²² and for carboxypeptidases related to carboxypeptidase B, an exocrine pancreatic exopeptidase with specificity for C-terminal basic residues.⁴⁹ Early studies with isolated islet secretory granules revealed that these were major sites of proinsulin conversion, and that conversion resulted in the release of insulin along with free arginine, rather than basic dipeptides,⁵¹ confirming the likely participation of both trypsin-like and carboxypeptidase B–like proteases in maturing secretory granules.⁵² Carboxypeptidase E (or H), a homolog of carboxypeptidase B but with a more acidic pH optimum, was first identified in islets⁵² and subsequently by Fricker in brain.⁵³ Molecular cloning confirmed its structural and evolutionary relationship to the pancreatic carboxypeptidases. Additional processing carboxypeptidases have been uncovered in brain and other tissues.⁵⁴

The first authentic processing endoprotease, kexin, was identified through studies on the processing of the yeast alpha mating–factor precursor and proved to be a calcium-dependent serine protease related to bacterial subtilisin and the subtilases rather than to trypsin.55–57 This information enabled the discovery of mammalian homologs, which form a family of seven closely related endoproteases with differing points of action within the secretory pathway.^10,58,59 These have been designated as subtilisin-like proprotein convertases (SPCs), or more simply as PCs. Two members of this family, PC1/3 and PC2, have proven to be the principal effectors of neuroendocrine peptide precursor processing and are largely confined in their expression to brain and endocrine tissues in most animal species.¹⁰ For further information on the extended SPC/PC family and its multiple functions, please see the reviews in Refs. 60 to 62.

Secretory Granule Formation and Maturation

One of the unsolved puzzles of neuroendocrine and other secretory cells is the nature of the mechanism underlying the efficient sorting of proteins destined for regulated secretion into immature secretory vesicles in the TGN. In the β cell, this process is remarkably efficient, resulting in very low levels of unregulated or “constitutive” release of proinsulin (<1% to 2%). However, the newly formed secretory granules have a clathrin coat (Fig. 6-8) which appears to be involved in some reorganization of the granule contents after and/or during their formation in the TGN.⁸¹ This passive sorting presumably occurs via small clathrin-clad vesicles that transport some proteins that are excluded from the condensing granule cores into endosomal pathways that either recycle to the TGN or to the cell surface. As a consequence of this “constitutive-like” pathway, proteins such as furin, procathepsin B, and possibly others briefly pass through the immature granule compartment, where they may play an active albeit transient role (e.g., furin may participate in processing of some prohormones).^81,82 Also, small amounts of abundant soluble granule components such as proinsulin and/or C peptide may exit the granules within these vesicles.⁸³ Passive sorting may also play a role in maintaining synchrony between granule membrane area and granule volume as maturation proceeds.⁸¹

Newly formed secretory vesicles in neuroendocrine cells undergo biochemical and morphologic maturation to typical dense-core granules. In β cells, the progranules are larger, less dense, and more uniform in appearance.^29,30 The most important biochemical change taking place as these progranules mature is the proteolytic conversion of proinsulin to insulin, accompanied by reorganization of the products.³⁰ Electron microscopic studies indicate that they progressively acquire a crystalline dense central core (Fig. 6-9). High magnification reveals repeat-unit spacings in the cores that are closely similar to those observed in ordinary zinc insulin crystals.⁸⁴ Thus, as insulin is liberated from proinsulin, it tends to crystallize with zinc that is concentrated by the β cells. Recent studies have demonstrated that zinc is taken up into maturing granules via the ZnT8 zinc transporter, and they do not assume the normal dense-core appearance in its absence, even though proinsulin conversion is not impaired.⁸⁵ Biochemical fractionation of mature islet secretory granules confirms that the cores contain only insulin, whereas the C peptide liberated in the conversion process remains in solution in the clear fluid space surrounding the dense crystalline core.⁸⁴ There is no evidence for co-crystallization of the C peptide with insulin under these conditions or in vitro.

Most of the zinc in islets is present in the β granules and is liberated proportionately to insulin during secretion, in keeping with its role in crystallization of the hormone.⁸⁶ The insulins of some species, including the guinea pig, coypu, and other hystricomorph rodents⁴² and the primitive hagfish,⁸⁷ lack the histidine residue at position 10 of the B chain required for zinc binding during the association of insulin dimers into hexamers.⁸⁸ A human subject heterozygous for a mutation at B10 (His B10 Asp) had elevated levels of this proinsulin in the blood because it tended to be released constitutively at much higher levels than normal proinsulin.⁸⁹ However, in a transgenic mouse model of this mutation, there was no defect in its conversion to insulin in the granules, despite its altered self-association and sorting properties.⁹⁰

The pH of the interior of the mature secretory granule appears to be between pH 5.0 and 6.0,^51,91 an optimal pH range for insulin crystallization in vitro. The neutral or slightly alkaline pH in the cisternal spaces of the RER favors proinsulin folding and sulfhydryl oxidation. The pH remains near neutral throughout the Golgi apparatus but becomes more acidic (pH 6.1) in the TGN as the secretory products are sorted into granules and proteolytic processing begins. Vesicular proton pumps may begin to increase the uptake of protons, which then displace the cationic arginine and lysine residues liberated during conversion. As these move out of the granules and are replaced by hydrogen ions, a downward shift in intragranular pH may occur. Thus the initially mildly acidic progranules⁹¹ undergo gradual acidification as they mature in the cytosol (see Fig. 6-8), creating appropriate conditions for the conversion and crystallization of the newly formed insulin. One recently discovered role of the constitutive pathway convertase, furin, in the β cell is to activate the proton pump, which acidifies the secretory granules. As a result, proinsulin processing is impaired in β cells lacking furin.⁹²

The cellular processes related to the biosynthesis of insulin via preproinsulin and proinsulin and their intracellular transport, sorting, proteolysis, and ultimate storage in secretory granules are remarkably well integrated, both topologically and biochemically. This delicately poised integration of processes leading to the formation and storage of insulin is disturbed in islet cell tumors, which often show unregulated release of insulin together with large amounts of proinsulin; measurements of the latter can provide a useful diagnostic indicator⁹³ (see Chapter 21).

Biosynthetic Role and Biological Actions of the C Peptide

Because of its cosecretion with insulin in essentially equimolar amounts,22,94,95 the C peptide has been of great value as a marker of insulin secretion in humans under a variety of conditions. Representative vertebrate C-peptide amino acid sequences are compared in Fig. 6-4.⁹⁶ These peptides exhibit a 15-fold higher rate of mutation acceptance than do the corresponding insulins, a finding that has often been interpreted as indicating that this region in the proinsulin molecule is unlikely to have any specific hormonal function. Nonetheless, several acidic residues are consistently present at certain positions in mammalian C peptides. These offset the added cationic charges due to the pairs of basic residues at the proinsulin cleavage sites, such that the isoelectric pH of proinsulin is nearly the same as that of insulin (i.e., in the range of pH 5.1 to 5.5).²²

A large body of evidence supports a biosynthetic role for the C-peptide segment in proinsulin. Primarily, it converts the insulin A and B chain interaction from an inefficient bimolecular reaction to a highly efficient and concentration-independent unimolecular reaction.⁹⁷ Certain regions of the connecting peptide may also facilitate the folding of the proinsulin polypeptide chain and the formation of the correct disulfide bonds, or guide the enzymatic cleavage of proinsulin to insulin by helping to orient the basic residue pairs for efficient binding and cleavage by the convertases.^98,99 Recent molecular modeling studies indicate that the C peptide may assist in achieving important conformational changes in the N-terminal helical region of the A-chain segment, aiding its interaction with the convertases during processing.¹⁰⁰

The conservation of a length of 30 to 35 amino acids in almost all proinsulin C peptides, including those of insects, mollusks, and nematodes, despite the short 8- to 10-angstrom separation between A1 and B30 in the native insulin molecule⁸⁸ (see Fig. 6-2), supports the existence of an important length-related function. Since much shorter connecting segments do not impair sulfhydryl oxidation and formation of the native structure,^99,101 it is clear that this is not a critical constraint. A highly plausible reason for the evolutionary retention of a relatively long C peptide in proinsulin may be to facilitate the translocation of proinsulin across the RER membrane during its synthesis. The length of polypeptide chain required to span the large ribosomal subunit and the RER membrane has been estimated to be about 65 residues in extended configuration.¹⁰² Moreover, the arrest of translation by the signal recognition particle (SRP) occurs only after synthesis of a nascent chain of about this length and thus may play a role in translational control of insulin biosynthesis.¹⁰² Efficient intracellular transport and correct targeting to secretory granules may impose additional demands on the primary (and tertiary) structure of proinsulin and other precursor proteins. However, miniproinsulins with deleted or greatly truncated C peptides do not fold efficiently but are correctly targeted to the regulated secretory pathway.^98,101,103

Another very important function of the C-peptide is to facilitate its removal from proinsulin after it has served its biosynthetic functions, as mentioned earlier. The kinetics of insulin metabolism in vivo, as well as its biological activity, are both dependent upon its high affinity for the insulin receptor. Proinsulin’s much lower receptor-binding affinity leads to a much slower rate of clearance, which explains its relatively higher apparent biological activity in vivo. The rapid clearance of insulin is adaptively as important a characteristic as its bioactivity. To assure its interaction with the convertases, proinsulin must present more than just a pair of basic residues to the convertases. Their active sites require an extended β strand that is at least eight residues in length for recognition at each cleavage site. Molecular modeling studies have suggested how the full length of the connecting segment contributes to achieving the necessary conformations for effective proinsulin binding and cleavage by the convertases.¹⁰⁰

Since the early 1990s, a number of reports have appeared describing a variety of biological actions of the C peptide and/or smaller peptides derived from it.¹⁰⁴ These putative effects include enhancement of glucose transport and utilization; improvements in microcirculation in muscle, skin, retina, and nerve in diabetics; and stimulation of renal tubular Na⁺,K⁺-adenosine triphosphatase (ATPase) activity and other parameters of renal function. It has been suggested that tissue receptors for C peptide might exist, and that the circulating C peptide may contribute to improved glycemic control and help to slow the development of the vascular and neural complications of diabetes.¹⁰⁵ Further study is needed in this area in view of reports that some C-peptide effects do not follow the usual rules of ligand-receptor chemistry (i.e., chirality does not matter). Thus, a C peptide synthesized entirely of d amino acids is equally as active as a peptide made with l amino acids in reversed order,¹⁰⁶ whereas a random sequence of the same amino acids leads to loss of activity. These tantalizing findings suggest the need for a controlled clinical trial of combined insulin and C-peptide therapy in diabetics to fully evaluate its therapeutic potential.

The availability of biosynthetic human proinsulin and insulin, as well as of human C peptide, has opened many new possibilities for studies of the role, metabolism, and antigenicity of these peptides.107–109 In recent years, active insulin analogs with shortened linking peptides have also been developed. Such miniproinsulins and/or “single-chain insulins” appear to readily oxidize to form correct disulfide bridges and either can be used intact or after cleavage to insulin.^98,99,101 Other efforts have been directed towards synthesis of modified insulins with improved absorption, aggregation, and/or pharmacokinetic properties for therapeutic use.

Islet Amyloid Polypeptide

Another intriguing islet protein is the 37-amino-acid neuropeptide-like molecule, islet amyloid polypeptide (IAPP), or amylin, a major protein constituent of the amyloid deposits that occur in the islets of patients with type 2 diabetes and in many benign insulinomas of the pancreas, as well as in the normal pancreases of the aged23,207,208 (Fig. 6-14). Although first noted in specimens of human pancreas as early as 1901,²⁰⁹ it was not until 1986 that efforts to solubilize islet amyloid were successful, revealing a single peptide, IAPP. Its amino acid sequence was related to the 37-amino-acid calcitonin gene–related peptide types 1 and 2 (CGRP-1, CGRP-2).^207,208 Cloning of its mRNA and gene further support its evolutionary relationship to CGRP/calcitonin.^23,210,211 Its precursor has a signal peptide, followed by a short propeptide ending in Lys-Arg at the N terminus of IAPP, and on its C-terminal side is followed by Gly-Lys-Arg and a second short propeptide (Fig. 6-15). The presence of the glycine residue in the latter cleavage site indicates that IAPP is normally carboxyamidated,²¹² as is CGRP.²³ The human IAPP gene is related to the genes encoding CGRP-1 and CGRP-2 and is located on the short arm of chromosome 12.²¹⁰

Biosynthesis and Levels of IAPP in Islets

Immunocytochemical studies indicate that IAPP normally is localized to the secretory granules of the β cells and some delta (δ) cells.^23,213 In β cells, proIAPP is synthesized at levels of a few percent that of proinsulin and transferred with it into newly forming secretory vesicles in the TGN, where it is then processed into the mature 37-residue carboxyamidated peptide, stored, and subsequently cosecreted with insulin.²¹⁴ Like proinsulin, the efficient processing of proIAPP requires the actions of both β-cell convertases, PC2²¹⁵ and PC1/3.²¹⁶ However, PC2 is more critical, since it alone is able to process the N-terminal cleavage site of proIAPP, whereas both PC2 and PC1/3 can process the C-terminal site, although PC2 does so more efficiently.²¹⁷ The dominant role of PC2 explains how IAPP can be produced in the δ cells of the islet, which express only PC2.^10,67 It has been proposed that the greater dependence of IAPP upon PC2 for its processing in the β cell, unlike proinsulin where PC1/3 plays the major role, may tend to allow greater accumulation of incompletely processed proIAPP in the diabetic β cell, where granule maturation processes may be perturbed by hyperglycemia.²¹⁸

The expression of IAPP is stimulated by glucose in parallel with insulin under normal conditions, but relative expression levels may be altered in pathologic states.²¹⁹ Very low levels of IAPP mRNA have also been detected in the stomach and other regions of the gastrointestinal tract, lung, and in dorsal root ganglia of the spinal cord.²²⁰ The relative levels of IAPP to insulin in the β cell are very low. HPLC analysis of freshly isolated rat islets shows amounts of IAPP in the range of 1% to 2% of insulin.²³ Most studies^221–223 agree that the levels of IAPP are in the range of 0.2% to 3% of the level of insulin in normal adult rat islets or normal human pancreas. Studies with isolated rat islets have shown that IAPP secretion is stimulated by glucose, and that IAPP amounts to about 5% of the amount of insulin released in 1 hour at 16.7 mM glucose.²²³

Biosynthesis and Processing of Glucagon, GLP1, GLP2, Somatostatin, Pancreatic Polypeptide, and Ghrelin

The hormones of the alpha (α), delta (δ), gamma (γ), and epsilon (ε) cells of the islets are glucagon, somatostatin, pancreatic polypeptide (PP), and ghrelin, respectively. All these peptides are derived from larger protein precursors (preproproteins) that traverse the regulated secretory pathway and are processed and stored in dense-core granules analogous to the insulin/IAPP storage granules in the β cells.²⁴⁷ But unlike the β cells, the other islet endocrine cells normally express only PC2 and not PC1/3.²⁴⁸ Not surprisingly then, as illustrated in Fig. 6-15, all of these hormones with the exception of ghrelin are cleaved from their precursors by PC2 acting alone. Thus in the PC2-null mouse, there is a total absence of mature glucagon in the islets and circulation and the presence only of large amounts of unprocessed inactive precursor forms.⁶⁷ As a consequence of the lack of active glucagon, there is chronic hypoglycemia and extensive hyperplasia of both the α and δ cells, which form a greatly enlarged mantle surrounding the β cells in the islets.^67,249 Similar changes are seen in mice lacking the glucagon receptor.²⁵⁰ In the case of the PC2-null mice, this hyperplastic state can be reversed by the intraabdominal administration of glucagon via minipumps over a period of several weeks.²⁵¹

Proglucagon is a multifunctional precursor that contains two additional peptides related to glucagon¹⁰ (see Fig. 6-15). The two glucagon-like peptides, GLP-1 and GLP-2, are located within the inactive C-terminal half of the precursor molecule (major proglucagon fragment [MPGF], residues 72 to 160), which follows the 69-residue inactive N-terminal half, known as glicentin. Glicentin contains the 29-amino-acid glucagon molecule, preceded by an N-terminal propeptide of 30 amino acids and followed by another short propeptide segment which links it via a Lys-Arg doublet (residues 70 and 71) to MPGF. This “interdomain cleavage site,” as it is known, is the most rapidly cleaved site in proglucagon and can be cleaved by either PC1/3 or PC2. The other cleavage sites are then cleaved by either PC2 or PC1/3 but usually not both, as illustrated in Fig. 6-15, which allows the α cell to produce only glucagon by virtue of expressing only PC2, and the intestinal proglucagon-expressing l cells to produce only GLP-1 and GLP-2 via the exclusive expression of PC1/3.^10,247 Thus a single precursor embodies a glucose-elevating hormone (glucagon), a β cell–stimulating hormone or “incretin” (GLP-1), and a third hormone which augments intestinal function and growth (GLP2). Selectivity of secretory product(s) is then determined by the convertase available to process the precursor at various sites. However, in the embryonic pancreas, the early-developing α cells express both PC1/3 and PC2,²⁵² and this also has been reported to occur in the α cells in some poorly controlled diabetics with elevated numbers of islet α cells that express some PC1/3 and thus can secrete some active GLP-1.²⁵³ Perhaps this local production of GLP-1 may protect or augment β-cell function via its actions as an incretin. (For further discussion of the important actions of the glucagon peptide family, see Chapter 9 in this volume.)

Somatostatin, the product of the islet δ cells, is a widely distributed inhibitory peptide that influences endocrine secretion, intestinal smooth muscle contractility, and islet, brain, and gastrointestinal function generally.²⁴⁷ The islets and the brain in PC2-null mice contain only somatostatin 28 (SS-28) instead of the normally predominant somatostatin 14 (SS-14).^67,247 This quite dramatic change is not likely to have much functional impact, however, inasmuch as somatostatin receptors do not appear to discriminate greatly between these two forms.²⁵⁴ As shown in Fig. 6-15, prosomatostatin can be processed only by PC2 at the unusual but highly conserved RK↓G cleavage site within SS-28 at the N-terminus of SS-14. The presence of SS-28 in the PC2-null mice indicates that cleavage at the single arginine required for its production still occurs in the absence of any other known convertase in the δ-cell secretory granules.^247,248 This cleavage also occurs in yeast that lack kexin and is believed to be due to a different type of convertase, possibly an aspartyl protease.²⁵⁵

Pancreatic polypeptide (PP), the product of the γ cells of the islets, mainly regulates gastrointestinal functions, including gall-bladder emptying and pancreatic secretion, rather than influencing carbohydrate metabolism.²⁴⁷ PC2-null mice fail to process propancreatic polypeptide (A. Zhou and D.F. Steiner, unpublished results), which indicates that both of the cleavages required for its maturation are accomplished by PC2 (see Fig. 6-15). Cleavage of proPP at the Pro-Arg↓ site by PC2 is rather surprising, since in proghrelin, a similar Pro-Arg↓ site is cleaved only by PC1/3.²⁵⁶

The ghrelin prohormone²⁵⁶ (not shown in the figure) is similar to propancreatic polypeptide in having the ghrelin sequence at the N-terminus, but it is followed by a considerably larger C-terminal segment that contains another putative hormonal sequence called obestatin. However, in contrast to proPP, it requires PC1/3 for its processing, as demonstrated in extracts of stomach in PC1/3-null mice.²⁵⁶ An unusual feature of the mature ghrelin peptide that is required for most of its known biological functions is the modification of serine 3 by O-acylation with octanoic acid, a modification carried out by a specialized acyltransferase.²⁵⁷ Ghrelin is produced mainly in the stomach and functions as an orexigenic and metabolic hormone. Only small numbers of ghrelin-positive ε cells occur in normal adult human and rodent pancreatic islets.²⁵⁸ Although ghrelin cells are more prominent during developmental phases, ghrelin has not been shown to play a significant role in normal pancreatic islet development.²⁵⁸

1. Von Mering, J, Minkowski, O. Diabetes mellitus nach pankreas extirpation. Arch Exp Pathol Pharmacol Leipzig. 1890;26:371.

2. Bliss, M. The discovery of insulin. Chicago: University of Chicago Press; 1982.

3. Scott, EL. On the influence of intravenous injections of an extract of the pancreas on experimental pancreatic diabetes. Am J Physiol. 1912;29:306.

4. Banting, FG, Best, CH, Collip, JB. Insulin patent. Chem Abstracts. 1923;17:3571.

5. Macleod, JJR, Campbell, WR. Insulin: Its use in the treatment of diabetes. In: Medicine monographs, vol VI, parts I and II. Baltimore: Williams & Wilkins; 1925.

6. Murnaghan, JH, Talalay, P. John Jacob Abel and the crystallization of insulin. Perspect Biol Med. 1967;10:334–380.

7. Falkmer, S, El-Salhy, M, Titlbach, M. Evolution of the neuroendocrine system in vertebrates: a review with particular reference to the phylogeny and postnatal maturation of the islet parenchyma. In: Falkmer S, Håkanson R, Sundler F, eds. Evolution and tumour pathology of the neuroendocrine system. Amsterdam: Elsevier; 1984:59.

8. Humbel, RE, Bosshard, HR, Zahn, H. Chemistry of insulin. In: Steiner DF, Freinkel N, eds. Handbook of physiology, endocrinology I. Baltimore: Williams & Wilkins; 1972:111.

9. Sanger, F. Chemistry of insulin. Science. 1959;129:1340.

10. Rouillé, Y, Duguay, SJ, Lund, K, et al. Proteolytic processing mechanisms in the biosynthesis of neuroendocrine peptides: the subtilisin-like proprotein convertases. Front Neuroendocrinol. 1995;16:322–361.

11. Steiner, DF, Oyer, PE. The biosynthesis of insulin and a probable precursor of insulin by a human islet cell adenoma. Proc Natl Acad Sci U S A. 1967;57:473.

12. Chance, RE, Ellis, RM, Bromer, WW. Porcine proinsulin: characterization and amino acid sequence. Science. 1968;161:165.

13. Steiner, DF, Clark, JL, Nolan, D, et al. Proinsulin and the biosynthesis of insulin. Recent Prog Horm Res. 1969;25:207.

14. Oyer, PE, Cho, E, Peterson, JD, et al. Studies on human proinsulin: isolation and amino acid sequence of the human pancreatic C-peptide. J Biol Chem. 1971;246:1375.

15. Chan, SJ, Keim, P, Steiner, DF. Cell-free synthesis of rat preproinsulins: characterization and partial amino acid sequence determination. Proc Natl Acad Sci U S A. 1976;73:1964.

16. Sanders, SL, Schekman, R. Polypeptide translocation across the endoplasmic reticulum membrane. J Biol Chem. 1992;267:13791.

17. Patzelt, C, Labrecque, AD, Duguid, JR, et al. Detection and kinetic behavior of preproinsulin in pancreatic islets. Proc Natl Acad Sci U S A. 1978;75:1260.

18. Frand, AR, Cuozzo, JW, Kaiser, CA. Pathways for protein disulphide bond formation. Trends Cell Biol. 2000;10:203.

19. Munro, S, Pelham, HRB. A C-terminal signal prevents secretion of luminal ER proteins. Cell. 1987;48:899.

20. Orci, L, Perrelet, A, Ravazzola, et al. Coatomer-rich endoplasmic reticulum. Proc Natl Acad Sci U S A. 1994;91:11924.

21. Pagano, A, Letourneur, F, Garcia-Estefania, D, et al. Sec24 proteins and sorting at the endoplasmic reticulum. J Biol Chem. 1999;274:7833.

22. Steiner, DF, Kemmler, W, Clark, JL, et al. The biosynthesis of insulin. In: Steiner DF, Freinkel N, eds. Handbook of physiology, Endocrinology I. Baltimore: Williams & Wilkins; 1972:175.

23. Nishi, M, Sanke, T, Nagamatsu, S, et al. Islet amyloid polypeptide: a new β cell secretory product related to islet amyloid deposits. J Biol Chem. 1990;265:4173.

24. Steiner, DF, Clark, JL, Nolan, C, et al. The biosynthesis of insulin and some speculation regarding the pathogenesis of human diabetes. In: Cerasi E, Luft R, eds. The pathogenesis of diabetes mellitus. New York: John Wiley & Sons; 1970:57.

25. Howell, SL. Role of ATP in the intracellular translocation of proinsulin and insulin in the rat pancreatic B cell. Nat New Biol. 1972;235:85.

26. Wattenberg, BW, Rothman, JE. Multiple cytosolic components promote intra-Golgi protein transport: resolution of a protein acting at a late state, prior to membrane fusion. J Biol Chem. 1986;61:2208.

27. Chappell, TG, Welch, WF, Schlossman, DM, et al. Uncoating ATPase is a member of the 70 kDa family of stress proteins. Cell. 1986;45:3.

28. Munger, BL. A light and electron microscopic study of cellular differentiation in the pancreatic islets of the mouse. Am J Anat. 1958;103:275.

29. Mogelsvang, S, Howell, KE. Global approaches to study Golgi function. Curr Opin Cell Biol. 2006;18:438–443.

30. Orci, L, Ravazzola, M, Amherdt, M, et al. Direct identification of prohormone conversion site in insulin-secreting cells. Cell. 1985;42:671.

31. Steiner, DF. Evidence for a precursor in the biosynthesis of insulin. Trans N Y Acad Sci. 1967;30:60.

32. Nagamatsu, S, Bolaffi, JL, Grodsky, GM. Direct effects of glucose on proinsulin synthesis and processing during desensitization. Endocrinology. 1987;120:1225.

33. Orci, L, Lambert, AE, Kanazawa, Y, et al. Morphological and biochemical studies of B cells in fetal rat endocrine pancreas in organ culture: evidence for proinsulin biosynthesis. J Cell Biol. 1971;50:565.

34. Frank, BH, Veros, AJ. Physical studies on proinsulin: association behavior and conformation in solution. Biochem Biophys Res Commun. 1968;32:155.

35. Rubenstein, AH, Mako, M, Welbourne, WP, et al. Comparative immunology of bovine, porcine, and human proinsulin and C-peptides. Diabetes. 1970;19:546.

36. Weiss, MA, Frank, BH, Khait, I, et al. NMR and photo-CIDNP studies of human proinsulin and prohormone processing intermediates with application to endopeptidase recognition. Biochemistry. 1990;29:8389.

37. Gliemann, J, Sorenson, HH. Assay of insulin-li activity by the isolated fa cell method: IV. The biological activity of proinsulin. Diabetologia. 1970;6:499.

38. Given, BD, Cohen, RM, Shoelson, SE, et al. Biochemical and clinical implications of proinsulin conversion intermediates. J Clin Invest. 1985;76:1398.

39. Frank, BH, Veros, AJ. Interaction of zinc with proinsulin. Biochem Biophys Res Commun. 1970;38:284.

40. Steiner, DF. Cocrystallization of proinsulin and insulin. Nature. 1973;243:528.

41. Steiner, DF, Hallund, O, Rubenstein, AH, et al. Isolation and properties of proinsulin, intermediate forms and other minor components from crystalline bovine insulin. Diabetes. 1968;17:725.

42. Blundell, T, Wood, S. The conformation, flexibility, and dynamics of polypeptide hormones. Annu Rev Biochem. 1982;51:123–154.

43. Low, BW, Fullerton, WW, Rosen, LS. Insulin/proinsulin, a new crystalline complex. Nature. 1974;248:339.

44. Nolan, C, Margoliash, E, Peterson, JD, et al. The structure of bovine proinsulin. J Biol Chem. 1971;246:2780.

45. Kuzuya, J, Chance, RE, Steiner, DF, et al. On the preparation and characterization of standard materials for natural human proinsulin and C-peptide. Diabetes. 1978;27:161.

46. Furuta, M, Carroll, R, Martin, S, et al. Incomplete processing of proinsulin to insulin accompanied by elevation of des-31,32 proinsulin intermediates in islets of mice lacking active PC2. J Biol Chem. 1998;273:3431.

47. Chan, SJ, Cao, Q-P, Steiner, DF. Evolution of the insulin superfamily: cloning of a hybrid insulin/insulin-like growth factor cDNA from amphioxus. Proc Natl Acad Sci U S A. 1990;87:9319.

48. Li, C, Kim, K. Neuropeptides. WormBook. 2008;25:1–36.

49. Kemmler, W, Peterson, JD, Steiner, DF. Studies on the conversion of proinsulin to insulin: I. Conversion in vitro with trypsin and carboxypeptidase B. J Biol Chem. 1971;246:6786.

50. Steiner, DF, Cho, S, Oyer, PE, et al. Isolation and characterization of proinsulin C-peptide from bovine pancreas. J Biol Chem. 1971;246:1365.

51. Kemmler, W, Steiner, DF, Borg, J. Studies on the conversion of proinsulin to insulin: III. Studies in vitro with a crude secretion granule fraction isolated from islets of Langerhans. J Biol Chem. 1973;248:4544.

52. Zühlke, H, Steiner, DF, Lernmark, NA, et al. Carboxypeptidase B-like and trypsin-like activities in isolated rat pancreatic islets. In: CIBA Foundation, ed. Polypeptide hormones: molecular and cellular aspects. Amsterdam: Excerpta Medica North-Holland; 1976:183.

53. Fricker, LD, Evans, CJ, Esch, FS, et al. Cloning and sequence analysis of cDNA for bovine carboxypeptidase E. Nature. 1986;323:461.

54. Song, W, Fricker, LD, Day, R. Carboxypeptidase D is a potential candidate to carry out redundant processing functions of carboxypeptidase E based on comparative distribution studies in the rat central nervous system. Neuroscience. 1999;89:1301.

55. Julius, D, Brake, A, Blair, L, et al. Isolation of the putative structural gene for the lysine-arginine-cleavage endopeptidase required for processing of yeast prepro-α-factor. Cell. 1984;37:1075.

56. Fuller, RS, Sterne, RE, Thorner, J. Enzymes required for yeast prohormone processing. Annu Rev Physiol. 1988;50:345.

57. Mizuno, K, Nakamura, T, Ohshima, T, et al. Yeast KEX2 gene encodes an endopeptidase homologous to subtilisin-like serine proteases. Biochem Biophys Res Commun. 1988;156:246.

58. Smeekens, SP, Steiner, DF. Identification of a human insulinoma cDNA encoding a novel mammalian protein structurally related to the yeast diabasic processing protease Kex2. J Biol Chem. 1990;265:2997.

59. Smeekens, SP, Avruch, AS, LaMendola, J, et al. Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans. Proc Natl Acad Sci U S A. 1991;88:340.

60. Seidah, NG, Chrétien, M, Day, R. The family of subtilisin/kexin like pro-protein and pro-hormone convertases: divergent or shared functions. Biochimie. 1994;76:197.

61. Zhou, A, Webb, G, Zhu, X, et al. Proteolytic processing in the secretory pathway. J Biol Chem. 1999;274:20745.

62. Seidah, NG, Prat, A. The proprotein convertases are potential targets in the treatment of dyslipidemia. J Mol Med. 2007;85(7):685–696. [Jul. Epub Mar 10].

63. Davidson, HW, Rhodes, CJ, Hutton, JC. Intraorganellar calcium and pH control proinsulin cleavage in the pancreatic β cell via two distinct site-specific endopeptidases. Nature. 1988;333:93.

64. Guest, PC, Bailyes, EM, Hutton, JC. Endoplasmic reticulum Ca²⁺ is important for the proteolytic processing and intracellular transport of proinsulin in the pancreatic β-cell. Biochem J. 1997;323:445.

65. Smeekens, SP, Albiges-Rizo, C, Carroll, R, et al. Proinsulin processing by the subtilisin-related proprotein convertases furin, PC2 and PC3. Proc Natl Acad Sci U S A. 1992;89:8822.

66. Rhodes, CJ, Lincoln, B, Shoelson, SE. Preferential cleavage of des-31,32-proinsulin over intact proinsulin by the insulin secretory granule type II endopeptidase: implications of a favored route for prohormone processing. J Biol Chem. 1992;267:22719.

67. Furuta, M, Yano, H, Zhou, A, et al. Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2. Proc Natl Acad Sci U S A. 1997;94:6646.

68. Zhu, X, Zhou, A, Dey, A, et al. Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects. Proc Natl Acad Sci U S A. 2002;99:10293.

69. Zhu, X, Orci, L, Carroll, R, et al. Severe block in processing of proinsulin to insulin accompanied by elevation of des-64,65 proinsulin intermediates in islets of mice lacking prohormone convertase 1/3. Proc Natl Acad Sci U S A. 2002;99:10299.

70. Jackson, RS, Creemers, JWM, Ohagi, S, et al. Obesity and impaired prohormone processing associated with mutations in the human prohormone convertase 1 gene. Nat Genet. 1997;16:303.

71. Zethelius, B, Byberg, L, Hales, CN, et al. Proinsulin and acute insulin response independently predict type 2 diabetes mellitus in men—report from 27 years of follow-up study. Diabetologia. 2003;46:20.

72. Rhodes, CJ, Alarcon, C. What β-cell defect could lead to hyperproinsulinemia in NIDDM? Some clues from recent advances made in understanding the proinsulin-processing mechanism. Diabetes. 1994;43:511.

73. Sizonenko, S, Irminger, J-C, Buhler, L, et al. Kinetics of proinsulin conversion in human islets. Diabetes. 1993;42:933.

74. Skelly, R, Schuppin, G, Ishihara, H, et al. Glucose-regulated translational control of proinsulin biosynthesis with that of the proinsulin endopeptidases PC2 and PC3 in the insulin-producing MIN6 cell line. Diabetes. 1996;45:37.

75. Schuppin, GT, Rhodes, CJ. Specific co-ordinated regulation of PC3 and PC2 gene expression with that of preproinsulin in insulin-producing beta TC3 cells. Biochem J. 1996;313(Pt 1):259–268.

76. Kalidas, K, Dow, E, Saker, P, et al. Prohormone convertase 1 in obesity, gestational diabetes mellitus, and NIDDM: no evidence for a major susceptibility role. Diabetes. 1998;47:287.

77. Yoshida, H, Ohagi, S, Sanke, T, et al. Association of the prohormone convertase 2 gene (PCSK2) on chromosome 20 with NIDDM in Japanese subjects. Diabetes. 1995;44:389.

78. Utsunomiya, N, Ohagi, S, Sanke, T, et al. Organization of the human carboxypeptidase E gene and molecular scanning for mutations in Japanese subjects with NIDDM or obesity. Diabetologia. 1998;41:701.

79. Benzinou, M, Creemers, JW, Choquet, H, et al. Common nonsynonymous variants in PCSK1 confer risk of obesity. Nat. Genet. 2008;40:943–945.

80. Wen, JH, Chen, YY, Song, SJ, et al. Paired box 6 (PAX6) regulates glucose metabolism via proinsulin processing mediated by prohormone convertase 1/3 (PC1/3). Diabetologia. 2009;52:504–513.

81. Arvan, P, Castle, D. Protein sorting and secretion granule formation in regulated secretory cells. Trends Cell Biol. 1992;2:327.

82. Kuliawat, R, Klumperman, J, Ludwig, T, et al. Differential sorting of lysosomal enzymes out of the regulated secretory pathway in pancreatic beta-cells. J Cell Biol. 1997;137:595.

83. Arvan, P, Kuliawat, R, Prabakaran, D, et al. Protein discharge from immature secretory granules displays both regulated and constitutive characteristics. J Biol Chem. 1991;266:14171.

84. Michael, J, Carroll, R, Swift, H, et al. Studies on the molecular organization of rat insulin secretory granules. J Biol Chem. 1987;262:16531.

85. Nicolson, TJ, Bellomo, EA, Wijesekara, N, et al. Insulin storage and glucose homeostasis in mice null for the granule zinc transporter ZnT8 and studies of the type 2 diabetes-associated variants. Diabetes.. 2009. [[Epub ahead of print]].

86. Logothetopoulos, J, Maneko, M, Wrenshall, GA, et al, Zinc, granulation, and extractable insulin of islet cells following hyperglycemia or prolonged treatment with insulin. The structure and metabolism of the pancreatic islets Wenner-Gren Center International Symposium Series. Brolin, SE, Hellman, B, Knutson, H, eds. The structure and metabolism of the pancreatic islets Wenner-Gren Center International Symposium Series, vol 3. Oxford: Pergamon Press, 1964:333.

87. Peterson, JD, Steiner, DF, Emdin, SO, et al. The amino acid sequence of the insulin from a primitive vertebrate, the Atlantic hagfish (Myxine glutinosa). J Biol Chem. 1975;250:5183.

88. Blundell, TL, Dodson, GG, Hodgkin, DC, et al. Insulin: the structure in the crystals and its reflection in chemistry and biology. Adv Protein Chem. 1972;26:279.

89. Chan, SJ, Seino, S, Gruppuso, PA, et al. A mutation in the B chain coding region of the human insulin gene is associated with impaired proinsulin conversion in a family with hyperproinsulinemia. Proc Natl Acad Sci U S A. 1987;84:2194.

90. Carroll, RJ, Hammer, RE, Chan, SJ, et al. A mutant human proinsulin is secreted from islets of Langerhans in increased amounts via an unregulated pathway. Proc Natl Acad Sci U S A. 1988;85:8943.

91. Orci, L. The insulin factory: a tour of the plant surroundings and a visit to the assembly line. Diabetolgia. 1985;28:528.

92. Louagie, E, Taylor, NA, Flamez, D, et al. Role of furin in granular acidification in the endocrine pancreas: identification of the V-ATPase subunit Ac45 as a candidate substrate. Proc Natl Acad Sci U S A. 2008;105(34):112319–112324.

93. Cohen, RM, Given, BD, Licinio-Paixao, J, et al. Proinsulin radioimmunoassay in the evaluation of insulinomas and familial hyperproinsulinemia. Metabolism. 1986;35:1137.

94. Halban, PA. Structural domains and molecular lifestyles of insulin and its precursors in the pancreatic beta cell. Diabetologia. 1991;34:767.

95. Polonsky, K, Rubenstein, AH. Current approaches to measurement of insulin secretion. Diabetes Metab Rev. 1986;2:315.

96. Steiner, DF:, The biosynthesis of insulin. Insulin—handbook of experimental pharmacology. Cuatrecasas, P, Jacobs, S, eds. Insulin—handbook of experimental pharmacology, Vol. 92. Berlin: Springer-Verlag, 1990:67–92.

97. Steiner, DF, Clark, JL. The spontaneous reoxidation of reduced beef and rat proinsulins. Proc Natl Acad Sci U S A. 1968;60:622.

98. Guo, ZY, Qiao, ZS, Feng, YM. The in vitro oxidative folding of the insulin superfamily. Antioxid Redox Signal. 2008;10(1):127–139.

99. Kjeldsen, T, Ludvigsen, S, Diers, I, et al. Engineering-enhanced protein secretory expression in yeast with application to insulin. J Biol Chem. 2002;24:277. [(21):18245–18248, Epub Mar 28].

100. Lipkind, G, Steiner, DF. Predicted structural alterations in proinsulin during its interactions with prohormone convertases. Biochemistry. 1999;38:890.

101. Liu, M, Ramos-Cstaneda, J, Arvan, P. Role of the connecting peptide in insulin biosynthesis. J Biol Chem. 2003;278:14798.

102. Okun, MM, Shields, D. Translocation of preproinsulin across the endoplasmic reticulum membrane. J Biol Chem. 1992;267:11476.

103. Powell, S, Orci, L, Craik, CS, et al. Efficient targeting to storage granules of human proinsulins with altered propeptide domain. J Cell Biol. 1988;106:1843.

104. Wahren, J, Johansson, B-L, Wallberg-Henriksson, H. Does C-peptide have a physiological role? Diabetologia. 1994;37:99.

105. Wahren, J, Ekberg, K, Jörnvall, H. C-peptide is a bioactive peptide. Diabetologia. 2007;50:503–509.

106. Ido, Y, Vindigni, A, Chang, K, et al. Prevention of vascular and neural dysfunction in diabetic rats by C-peptide. Science. 1997;277:563.

107. Galloway, JA, Hooper, SA, Spradlin, CT, et al. Biosynthetic human proinsulin—review of chemistry, in vitro and in vivo receptor binding, animal and human pharmacology studies, and clinical experience. Diabetes Care. 1992;15:666.

108. Glauber, HS, Henry, RR, Wallace, P. The effects of biosynthetic human proinsulin on carbohydrate metabolism in non-insulin-dependent diabetes mellitus. N Engl J Med. 1987;316:443.

109. Madsen, OD, Frank, BH, Steiner, DF. Human proinsulin specific antigenic determinants identified by monoclonal antibodies. Diabetes. 1984;33:1012.

110. Rhodes, CJ, Halban, PA. Newly-synthesized proinsulin/insulin and stored insulin are released form pancreatic B-cells via a regulated, rather than a constitutive pathway. J Cell Biol. 1987;105:145–153.

111. Uchizono, Y, Alarcon, C, Wicksteed, BL, et al. The balance between proinsulin biosynthesis and insulin secretion: where can imbalance lead? Diabetes Obes Metab. 2007;9(Suppl 2):56–66.

112. Rhodes, CJ. Processing of the insulin molecule. In: LeRoith D, Taylor SI, Olefsky JM, eds. Diabetes mellitus: a fundamental and clinical text. Philadelphia: Lippincott-Raven Publishers; 2004:27–50.

113. Wicksteed, BL, Alarcón, C, Briaud, I, et al. Glucose-induced translational control of proinsulin biosynthesis is proportional to preproinsulin mRNA levels, but not regulated via a positive feedback of secreted insulin on in islet β-cells. J Biol Chem. 2003;278:42080–42090.

114. Bollheimer, LC, Skelly, RH, Chester, M, et al. Chronic exposure to free fatty acid reduces pancreatic β-cell insulin content by increasing basal insulin secretion that is not compensated for a corresponding increase in proinsulin biosynthesis translation. J Clin Invest. 1998;101:1094–1101.

115. Prentki, M, Matchinsky, FM. Ca⁺⁺, cAMP, and phospholipid-derived messengers in coupling mechanisms of insulin secretion. Physiol Rev. 1987;67:1185–1248.

116. Alarcón, C, Wicksteed, BL, Prentki, M, et al. Elevation in pancreatic β-cell cytosolic succinyl-CoA is a candidate for specific metabolic stimulus coupling of proinsulin biosynthesis translation, but not insulin secretion. Diabetes. 2002;51:2496–2504.

117. Drucker, DJ. The biology of incretin hormones. Cell Metab. 2006;3:153–165.

118. Alarcon, C, Wicksteed, B, Rhodes, CJ. Exendin 4 controls insulin production in rat islet beta cells predominantly by potentiation of glucose-stimulated proinsulin biosynthesis at the translational level. Diabetologia. 2006;49:2920–2929.

119. Herbert, TP, Alarcón, C, Skelly, RH, et al. Regulation of prohormone conversion by coordinated control of processing endopeptidase biosynthesis with that of the prohormone substrate. In: Hook VYH, ed. proteolytic and cellular mechanisms in prohormone and proprotein processing. Austin, TX.: R.G. Landes Company; 1998:105–120.

120. Guest, PC, Bailyes, EM, Rutherford, NG, et al. Insulin secretory granule biogenesis: co-ordinate regulation of the biosynthesis of the majority of constituent proteins. Biochem J. 1991;274:73–78.

121. Alarcón, C, Lincoln, B, Rhodes, CJ. The biosynthesis of the subtilisin-related proprotein convertase PC3, but not that of the PC2 convertase, is regulated by glucose in parallel to proinsulin biosynthesis in rat pancreatic islets. J Biol Chem. 1993;268:4276–4280.

122. Itoh, N, Okamoto, H. Translational control of proinsulin synthesis by glucose. Nature. 1980;283:100–102.

123. Welsh, M, Scherberg, N, Gilmore, R, et al. Translational control of insulin biosynthesis. Evidence for regulation of elongation, initiation and signal-recognition-particle-mediated translational arrest by glucose. Biochem J. 1986;235:459–467.

124. Welsh, M, Nielsen, DA, MacKrell, AJ, et al. Control of insulin gene expression in pancreatic β-cells and in an insulin producing cell line, RIN-5F cells. 2. Regulation of insulin mRNA stability. J Biol Chem. 1985;260:13590–13594.

125. German, M. Insulin gene regulation. In: LeRoith D, Taylor SI, Olefsky JM, eds. Diabetes mellitus—a fundamental and clinical text. Philadelphia: Lippincott Williams & Wilkins; 2004:15–26.

126. Van Lommel, L, Janssens, K, Quintens, R, et al. Probe-independent and direct quantification of insulin mRNA and growth hormone mRNA in enriched cell preparations. Diabetes. 2006;55:3214–3220.

127. Permutt, MA, Kipnis, DM. Insulin biosynthesis and secretion. Fed Proc. 1975;34:1549–1555.

128. Wicksteed, B, Herbert, TP, Alarcón, C, et al. Cooperation between 5′- and 3′-untranslated regions of preproinsulin mRNA control proinsulin biosynthesis. J Biol Chem. 2001;276:22553–22558.

129. Fred, RG, Tillmar, L, Welsh, N. The role of PTB in insulin mRNA stability control. Curr Diabetes Rev. 2006;2:363–366.

130. Wicksteed, BL, Uchizono, Y, Alarcon, C, et al. A cis-element in the 5′-untranslated region of the preproinsulin mRNA (ppIGE) is required for glucose regulation of proinsulin translation. Cell Metab. 2007;5:221–227.

131. German, MS, Moss, LG, Rutter, WJ. Regulation of gene expression by glucose and calcium in transfected primary islet cultures. J Biol Chem. 1990;265:22063–22066.

132. Swenne, I. The role of glucose in the in vitro regulation of cell cycle kinetics and proliferation of fetal pancreatic β-cells. Diabetes. 1982;31:754–760.

133. Lingohr, MK, Buettner, R, Rhodes, CJ. Pancreatic beta-cell growth and survival—a role in obesity-linked type 2 diabetes? Trends Mol Med. 2002;8:375–384.

134. Orci, L, Ravazzola, M, Amherdt, M, et al. Insulin, not C-peptide (proinsulin), is present in crinophagic bodies of the pancreatic B-cell. J Cell Biol. 1984;98:222–228.

135. Schnell, AH, Swenne, I, Borg, LA. Lysosomes and pancreatic islet function. A quantitative estimation of crinophagy in the mouse pancreatic B-cell. Cell Tissue Res. 1988;252:9–15.

136. Marsh, BJ, Soden, C, Alarcón, C, et al. Regulated autophagy controls hormone content in secretory-deficient pancreatic endocrine β-cells. Mol Endocrinol. 2007;21:2255–2269.

137. Halban, PA, Wollheim, CB. Intracellular degradation of insulin stores by rat pancreatic islets in vitro. An alternative pathway for homeostasis of pancreatic insulin content. J Biol Chem. 1980;255:6003–6006.

138. Skoglund, G, Ahren, B, Lundquist, I. Biochemical determination of islet lysosomal enzyme activities following crinophagy-stimulating treatment with diazoxide in mice. Diabetes Res. 1987;6:81–84.

139. Ebato, C, Uchida, T, Arakawa, M, et al. Autophagy is important in islet homeostasis and compensatory increase of beta cell mass in response to high-fat diet. Cell Metab. 2008;8:325–332.

140. Grasso, D, Sacchetti, ML, Bruno, L, et al. Autophagy and VMP1 expression are early cellular events in experimental diabetes. Pancreatology. 2008;9:81–88.

141. Ashcroft, FM, Rorsman, P. Electrophysiology of the pancreatic beta cell. Prog Biophys Mol Biol. 1989;54:87–143.

142. Gloyn, AL, Pearson, ER, Antcliff, JF, et al. Activating mutations in the gene encoding the ATP-sensitive potassium-channel subunit Kir6.2 and permanent neonatal diabetes. N Engl J Med. 2004;350:1838–1849.

143. Babenko, AP, Polak, M, Cave, H, et al. Activating mutations in the ABCC8 gene in neonatal diabetes mellitus. N Engl J Med. 2006;355:456–466.

144. Byrne, MM, Sturis, J, Clément, K, et al. Insulin secretory abnormalities in subjects with hyperglycemia due to glucokinase mutations. J Clin Invest. 1994;93:1120–1130.

145. Jacobson, DA, Kuznetsov, A, Lopez, JP, et al. Kv2.1 ablation alters glucose-induced islet electrical activity, enhancing insulin secretion. Cell Metab. 2007;6:229–235.

146. Aguilar-Bryan, L, Bryan, J. Neonatal diabetes mellitus. Endocr Rev. 2008;29:265–291.

147. Hou, JC, Min, L, Pessin, JE. Insulin granule biogenesis, trafficking and exocytosis. Vitam Horm. 2009;80:473–506.

148. Stoy, J, Edghill, EL, Flanagan, SE, et al. Insulin gene mutations as a cause of permanent neonatal diabetes. Proc Natl Acad Sci U S A. 2007;104:15040–15044.

149. Murphy, R, Ellard, S, Hattersley, AT. Clinical implications of a molecular genetic classification of monogenic beta-cell diabetes. Nat Clin Pract Endocrinol Metab. 2008;4:200–213.

150. Sayed, S, Langdon, DR, Odili, S, et al. Extremes of clinical and enzymatic phenotypes in children with hyperinsulinism caused by glucokinase activating mutations. Diabetes. 2009;58:1419–1427.

151. De Leon, DD, Stanley, CA. Mechanisms of disease: advances in diagnosis and treatment of hyperinsulinism in neonates. Nat Clin Pract Endocrinol Metab. 2007;3:57–68.

152. Miller, SP, Anand, GR, Karschnia, EJ, et al. Characterization of glucokinase mutations associated with maturity-onset diabetes of the young type 2 (MODY-2): different glucokinase defects lead to a common phenotype. Diabetes. 1999;48:1645–1651.

153. Matschinsky, FM, Magnuson, MA, Zelent, D, et al. The network of glucokinase-expressing cells in glucose homeostasis and the potential of glucokinase activators for diabetes therapy. Diabetes. 2006;55:1–12.

154. Chimienti, F, Devergnas, S, Favier, A, et al. Identification and cloning of a beta-cell-specific zinc transporter, ZnT-8, localized into insulin secretory granules. Diabetes. 2004;53:2330–2337.

155. Wenzlau, JM, Juhl, K, Yu, L, et al. The cation efflux transporter ZnT8 (Slc30A8) is a major autoantigen in human type 1 diabetes. Proc Natl Acad Sci U S A. 2007;104:17040–17045.

156. Sladek, R, Rocheleau, G, Rung, J, et al. A genome-wide association study identifies novel risk loci for type 2 diabetes. Nature. 2007;445:881–885.

157. Fu, Y, Tian, W, Pratt, EB, et al. Down-regulation of ZnT8 expression in INS-1 rat pancreatic beta cells reduces insulin content and glucose-inducible insulin secretion. PLoS One. 2009;4:e5679.

158. Conget, I, Zhang, TM, Eizirik, DL, et al. SAM prevents impairment of glucose-stimulated insulin secretion caused by hexose deprivation or starvation. Am J Physiol. 1995;268:E580–E587.

159. Maechler, P, Kennedy, ED, Pozzan, T, et al. Mitochondrial activation directly triggers the exocytosis of insulin in permeabilized pancreatic beta-cells. EMBO J. 1997;16:3833–3841.

160. Maechler, P, Wollheim, CB. Mitochondrial glutamate acts as a messenger in glucose-induced insulin exocytosis. Nature. 1999;402:685–689.

161. Wiederkehr, A, Wollheim, CB. Minireview: implication of mitochondria in insulin secretion and action. Endocrinology. 2006;147:2643–2649.

162. Ashcroft, FM, Proks, P, Smith, PA, et al. Stimulus-secretion coupling in pancreatic β-cells. J Cell Biochem. 1994;55S:54–65.

163. Misler, S, Barnett, DW, Gillis, KD, et al. Electrophysiology of stimulus-secretion coupling in human B-cells. Diabetes. 1992;41:1221–1228.

164. Fridlyand, LE, Harbeck, MC, Roe, MW, et al. Regulation of cAMP dynamics by Ca²⁺ and G protein–coupled receptors in the pancreatic beta-cell: a computational approach. Am J Physiol Cell Physiol. 2007;293:C1924–C1933.

165. Rutter, GA, Tsuboi, T, Ravier, MA. Ca²⁺ microdomains and the control of insulin secretion. Cell Calcium. 2006;40:539–551.

166. Henquin, JC. Regulation of insulin secretion: a matter of phase control and amplitude modulation. Diabetologia. 2009;52:739–751.

167. Nesher, R, Anteby, E, Yedovizky, M, et al. Beta-cell protein kinases and the dynamics of the insulin response to glucose. Diabetes. 2002;51(Suppl 1):S68–S73.

168. Drucker, DJ. The biology of incretin hormones. Cell Metab. 2006;3:153–165.

169. Wek, RC, Anthony, TG. Extending beta cell survival by upregulating ATF4 translation. Cell Metab. 2006;4:333–334.

170. Holz, GG, Kang, G, Harbeck, M, et al. Cell physiology of cAMP sensor Epac. J Physiol. 2006;577:5–15.

171. Philipson, LH. Beta-agonists and metabolism. J Allergy Clin Immunol. 2002;110:S313–S317.

172. Toyota, T, Kakizaki, M, Kimura, K, et al. Islet activating protein (IAP) derived from the culture supernatant fluid of Bordetella pertussis: effect on spontaneous diabetic rats. Diabetologia. 1978;14:319–323.

173. Draznin, B, Steinberg, JP, Goodman, M, et al. Control of secretion, vesicle margination, and lysis by glucose, IBMX, and glyburide. Am J Physiol. 1985;248:E375–E380.

174. Dov, A, Abramovitch, E, Warwar, N, et al. Diminished phosphodiesterase-8B potentiates biphasic insulin response to glucose. Endocrinology. 2008;149:741–748.

175. Landa, LR, Jr., Harbeck, M, Kaihara, K, et al. Interplay of Ca²⁺ and cAMP signaling in the insulin-secreting MIN6 beta-cell line. J Biol Chem. 2005;280:31294–31302.

176. Dyachok, O, Isakov, Y, Sagetorp, J, et al. Oscillations of cyclic AMP in hormone-stimulated insulin-secreting beta-cells. Nature. 2006;439:349–352.

177. Gilon, P, Henquin, JC. Mechanisms and physiological significance of the cholinergic control of pancreatic beta-cell function. Endocr Rev. 2001;22:565–604.

178. Covington, DK, Briscoe, CA, Brown, AJ, et al. The G-protein-coupled receptor 40 family (GPR40-GPR43) and its role in nutrient sensing. Biochem Soc Trans. 2006;34:770–773.

179. Brownlie, R, Mayers, RM, Pierce, JA, et al. The long-chain fatty acid receptor, GPR40, and glucolipotoxicity: investigations using GPR40-knockout mice. Biochem Soc Trans. 2008;36:950–954.

180. Tan, CP, Feng, Y, Zhou, YP, et al. Selective small-molecule agonists of G protein-coupled receptor 40 promote glucose-dependent insulin secretion and reduce blood glucose in mice. Diabetes. 2008;57:2211–2219.

181. Steiner, DF, Chan, SJ, Welsh, JM, et al. Structure and evolution of the insulin gene. Annu Rev Genet. 1985;19:463.

182. Pugliese, A, Zeller, M, Fernandez, A, Jr., et al. The insulin gene is transcribed in the human thymus and transcription levels correlated with allelic variation at the INS VNTR-IDDM2 susceptibility locus for type 1 diabetes. Nat Genet.. 1997;15:293–297.

183. Bell, GI, Selby, MJ, Rutter, WJ. The highly polymorphic region near the human insulin gene is composed of simple tandemly repeating sequences. Nature. 1982;295:31.

184. Steiner, DF, Tager, HS, Chan, SJ, et al. Lessons learned from molecular biology of insulin-gene mutations. Diabetes Care. 1990;13:600–609.

185. Rafiq, M, Flanagan, SE, Patch, AM, et al. Effective treatment with oral sulfonylureas in patients with diabetes due to sulfonylurea receptor 1 (SUR1) mutations. Diabetes Care. 2008;31:204–209.

186. Edghill, E, Flanagan, S, Patch, AM, et al. Insulin mutation screening in 1044 patients with diabetes mutations in the INS gene are a common cause of neonatal diabetes but a rare cause of diabetes diagnosed in childhood or adulthood. Diabetes. 2008;57:1034–1042.

187. Molven, A, Ringdal, M, Nordbø, AM, et al. Mutations in the insulin gene can cause MODY and autoantibody-negative type I diabetes. Diabetes. 2008;57:1131–1135.

188. Polak, M, Dechaume, A, Cave, H, et al. Heterozygous missense mutations in the insulin gene are linked to permanent diabetes appearing in the neonatal period or in early infancy. Diabetes. 2008;57:1115–1119.

189. Colombo, C, Porzio, O, Liu, M, et al. Seven mutations in the human insulin gene linked to permanent neonatal/infancy-onset diabetes mellitus. J Clin Invest. 2008;118:2148–2156.

190. Hua, QX, Liu, M, Hu, SQ, et al. A conserved histidine in insulin is required for the foldability of human proinsulin: structure and function of an ALAB5 analog. J Biol Chem. 2006;281:24889–24899.

190a. Rajan, S, Eames, SC, Park, SY, et al. In vitro processing and secretion of mutant insulin proteins that cause permanent neonatal diabetes. Am J Physiol Endocrinol Metab. 2009 Dec 1. [[Epub ahead of print]].

190b. Park, SY, Ye, H, Steiner, DF, et al. Mutant proinsulin proteins associated with neonatal diabetes are retained in the endoplasmic reticulum and not efficiently secreted. Biochem Biophys Res Commun. 2009 Dec 23. [[Epub ahead of print]].

191. Bonfanti, R, Colombo, C, Nocerino, V, et al. Insulin gene mutations as cause of diabetes in children negative for five type 1 diabetes autoantibodies. Diabetes Care. 2009;32:123–125.

192. Qiao, ZS, Min, CY, Hua, QX, et al. In vitro refolding of human proinsulin. Kinetic intermediates, putative disulfide-forming pathway, folding initiation site and potential role of C-peptide in folding process. J Biol Chem. 2003;278:17800–17809.

193. Nakagawa, SH, Hua, QX, Hu, SQ, et al. Chiral mutagenesis of insulin. Contribution of the B20-B23 beta-turn to activity and stability. J Biol Chem. 2006;281:22386–22396.

194. Wang, J, Takeuchi, T, Tanaka, S, et al. A mutation in the insulin 2 gene induces diabetes with severe pancreatic β-cell dysfunction in the MODY mouse. J Clin Invest. 1999;103:27–37.

195. Herbach, N, Rathkolb, B, Kemter, E, et al. Dominant-negative effects of a novel mutated Ins2 allele causes early-onset diabetes and severe β-cell loss in Munich Ins2^C95S mutant mice. Diabetes. 2007;56:1268–1276.

196. Shimizu, Y, Hendershot, LM. Oxidative folding: cellular strategies for dealing with the resultant equimolar production of reactive oxygen species. Antioxid Redox Signal. 2009. [Feb 25. [Epub ahead of print]].

197. Sevier, CS, Kaiser, CA. Ero1 and redox homeostasis in the endoplasmic reticulum. Biochim Biophys Acta. 2008;1783:549–556.

198. Back, SH, Scheuner, D, Han, J, et al. Translation attenuation through elF2α phosphorylation prevents oxidative stress and maintains the differentiated state in beta cells. Cell Metab. 2009;10:13–26.

199. Zhu, YL, Abdo, A, Gesmonde, JF, et al. Aggregation and lack of secretion of most newly synthesized proinsulin in non-β-cell lines. Endocrinology. 2004;145:3840–3849.

200. Zuber, C, Fan, JY, Guhl, B, et al. Misfolded proinsulin accumulates in expanded pre-Golgi intermediates and endoplasmic reticulum subdomains in pancreatic beta cells of Akita mice. FASEB J. 2004;18:917–999.

201. Hutton, JC. The insulin secretory granule. Diabetologia. 1989;32:271.

202. Taupenot, L, harper, KL, O’Connor, DT. The chromogranin-secretogranin family. N Engl J Med. 2003;348:1134.

203. Hutton, JC, Nielsen, E, Kastern, W. The molecular cloning of the chromogranin A-like precursor of β-granin and pancreastatin from the endocrine pancreas. FEBS Lett. 1988;236:269.

204. Schmidt, WE, Siegel, EG, Kratzin, H, et al. Isolation and primary structure of tumor-derived peptides related to human pancreastatin and chromogranin A. Proc Natl Acad Sci U S A. 1988;85:8231.

205. Tatemoto, K, Efendic, S, Mutt, V, et al. Pancreastatin, a novel pancreatic peptide that inhibits insulin secretion. Nature. 1986;324:476.

206. Hendy, GN, Li, T, Girard, M, et al. Targeted ablation of the chromogranin a (CHGA) gene: normal neuroendocrine dense-core secretory granules and increased expression of other granins. Mol Endocrinol. 2006;8:1935–1947.

207. Westermark, P, Wernstedt, C, Wilander, E, et al. A novel peptide in the calcitonin gene related peptide family as an amyloid fibril protein in the endocrine pancreas. Biochem Biophys Res Commun. 1986;140:827–831.

208. Cooper, GJS, Willis, AC, Clark, A, et al. Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients. Proc Natl Acad Sci U S A. 1987;84:8628.

209. Opie, E. The relation of diabetes mellitus to lesions of the pancreas: hyaline degeneration of the islands of Langerhans. J Exp Med. 1901;5:527.

210. Nishi, M, Sanke, T, Seino, S, et al. Human islet amyloid polypeptide gene: sequence, chromosomal localization and evolutionary history. Mol Endocrinol. 1989;3:1775.

211. Mosselman, S, Höppener, JWM, Lips, CJM, et al. The complete islet amyloid polypeptide precursor is encoded by two exons. FEBS Lett. 1989;247:154.

212. Prigge, ST, Eipper, BA, Mains, RE, et al. New insignts into copper monoxygenases and peptide amidation: structure, mechanism and function. Cell Mol Life Sci. 2000;57:1236–1259.

213. Iki, K, Pour, PM. Distribution of pancreatic endocrine cells including IAPP-expressing cells in non-diabetic and type 2 diabetic cases. J Histochem Cytochem. 2007;55:111–118.

214. Nagamatsu, S, Nishi, M, Steiner, DF. Biosynthesis of islet amyloid polypeptide. J Biol Chem. 1991;266:13737.

215. Wang, J, Xu, J, Finnerty, J, et al. The prohormone convertase enzyme 2 (PC2) is essential for processing pro-amyloid polypeptide at the NH2-terminal cleavage site. Diabetes. 2001;50:534.

216. Marzban, L, Trigo-Gonzalez, G, Zhu, X, et al. Role of beta-cell prohormone convertase (PC) 1/3 in processing of pro-islet amyloid polypeptide. Diabetes. 2004;53:141.

217. Marzban, L, Park, K, Verchere, CB. Islet amyloid polypeptide and type 2 diabetes. Exp Gerontol. 2003;38:347–351.

218. Marzban, L, Rhodes, CJ, Steiner, DF, et al. Impaired NH₂-terminal processing of human proislet amyloid polypeptide by the prohormone convertase PC2 leads to amyloid formation and cell death. Diabetes. 2006;55:2192–2201.

219. Mulder, H, Ahren, B, Stridsberg, M, et al. Non-parallelism of islet amyloid polypeptide (amylin) and insulin gene expression in rat islets following dexamethasone treatment. Diabetologia. 1995;38:395.

220. Ferrier, GJLM, Pierson, AM, Jones, PM, et al. Expression of the rat amylin (IAPP/DAP) gene. J Mol Endocrinol. 1989;3:R1.

221. Nakazato, M, Asai, J, Kangawa, K, et al. Establishment of radioimmunoassay for human islet amyloid polypeptide and its tissue content and plasma concentration. Biochem Biophys Res Commun. 1989;164:394.

222. Asai, J, Nakazato, M, Kangawa, K, et al. Regional distribution and molecular forms of rat islet amyloid polypeptide. Biochem Biophys Res Commun. 1990;169:788.

223. Kanatsuka, A, Makino, H, Ohsawa, H, et al. Secretion of islet amyloid polypeptide in response to glucose. FEBS Lett. 1989;259:199.

224. Cooper, GJS, Amylin and related proteins: physiology and pathology. Handbook of physiology, The Endocrine System. Jefferson, LS, Cunningham, AD, eds. Handbook of physiology, The Endocrine System, Vol. II. Oxford University Press, 2001:303–396. [Chapter 10].

225. Sowa, R, Sanke, T, Hirayama, J, et al. Islet amyloid polypeptide amide causes peripheral insulin resistance in vivo. Diabetologia. 1990;33:118.

226. Scherbaum, WA. The role of amylin in the physiology of glycemic control. Exp Clin Endocrinol Diabetes. 1998;106:97.

227. Gebre-Medhin, S, Mulder, H, Pekny, M, et al. Increased insulin secretion and glucose tolerance in mice lacking islet amyloid polypeptide (amylin). Biochem Biophys Res Commun. 1998;250:271.

228. Ahrén, B, Oosterwijk, C, Lips, CJM, et al. Transgenic overexpression of human islet amyloid polypeptide inhibits insulin secretion and glucose elimination after gastric glucose gavage in mice. Diabetologia. 1998;41:1374.

229. Datta, HK, Saidi, M, Wimalawansa, SJ, et al. In vivo and in vitro effects of amylin and amylin-amide on calcium metabolism in the rat and rabbit. Biochem Biophys Res Commun. 1989;162:876.

230. MacIntyre, I. Amylinamide, bone conservation, and pancreatic β-cells. Lancet. 1989;2:1026.

231. Christopoulos, G, Perry, KJ, Morfis, M, et al. Multiple amylin receptors arise from receptor activity-modifying protein interaction with the calcitonin receptor gene product. Mol Pharmacol. 1999;56:235.

232. Morfis, M, Tilakaratne, N, Furness, SG, et al. Receptor activity-modifying proteins differentially modulate the G protein-coupling efficiency of amylin receptors. Endocrinology. 2008;149:5423–5431.

233. Schmitz, O, Brock, B, Rungby, J. Amylin agonists: a novel approach in the treatment of diabetes. Diabetes. 2004;53:S233–S238.

234. O’Brien, TD, Butler, PC, Westermark, P, et al. Islet amyloid polypeptide: a review of its biology and potential roles in the pathogenesis of diabetes mellitus. Vet Pathol. 1993;30:317–332.

235. Glenner, GG, Eanes, ED, Wiley, CA. Amyloid fibrils formed from a segment of the pancreatic islet amyloid protein. Biochem Biophys Res Commun. 1988;155:608.

236. Hallman, U, Wernstedt, C, Westermark, P, et al. Amino acid sequence from degu islet amyloid-derived insulin shows unique sequence characteristics. Biochem Biophys Res Commun. 1980;169:571.

237. Nishi, M, Steiner, DF. Cloning of complementary DNAs encoding islet amyloid polypeptide, insulin, and glucagon precursors from a New World rodent, the degu, Octodon degus. Mol Endocrinol. 1990;4:1192.

238. Lukinius, A, Wilander, E, Westermark, GT, et al. Co-localization of islet amyloid polypeptide and insulin in the B cell secretory granules of the human pancreatic islets. Diabetologia. 1989;32:240.

239. Clark, A, Wells, CA, Buley, ID, et al. Islet amyloid, increased A-cells, reduced B-cells and exocrine fibrosis: quantitative changes in the pancreas. Diabetes Res. 1988;9:1519.

240. Clark, A, Edwards, CA, Ostle, LR, et al. Localisation of islet amyloid peptide in lipofuscin bodies and secretory granules of human β-cells and in islets of type-2 diabetic subjects. Cell Tissue Res. 1989;259:179.

241. Westermark, G, Westermark, P, Eizirik, DL, et al. Differences in amyloid deposition in islets of transgenic mice expressing human islet amyloid polypeptide versus human islets implanted into nude mice. Metabolism. 1999;48:448.

242. Kahn, SE, Andrikopoulos, S, Verchere, CB. Islet amyloid: a long-recognized but underappreciated pathological feature of type 2 diabetes. Diabetes. 1999;48:241.

243. Nishi, M, Bell, GI, Steiner, DF. Islet amyloid polypeptide (amylin): no evidence of an abnormal precursor sequence in 25 type 2 (non–insulin-dependent) diabetic patients. Diabetologia. 1990;33:628.

244. Anderson, A, Bohman, S, Borg, LA, et al. Amyloid deposition in transplanted human pancreatic islets: a conceivable cause of their long-term failure. Exp Diabetes Res. Epub 2009.

245. Porat, Y, Mazor, Y, Efrat, S, et al. Inhibition of islet amyloid polypeptide fibril formation: a potential role for heteroaromatic interactions. Biochemistry. 2004;43:14454–14462.

246. Tatarek-Nossol, M, Yan, L-M, Schmauder, A, et al. Inhibition of hIAPP amyloid-fibril formation and apoptotic cell death by a designed hIAPP amyloid-core-containing hexapeptide. Chem and Biol. 2005;12:797–809.

247. Steiner, DF, Bell, GI, Rubenstein, AH, et al, Chemistry and biosynthesis of the islet hormones: insulin, islet amyloid polypeptide (amylin), glucagon, somatostatin, and pancreatic polypeptide, ed 5. DeGroot L, Jameson JL, eds. Endocrinology. W.B. Saunders: Philadelphia, 2006:925–960. [Chapter 48].

248. Tanaka, S, Kurabuchi, S, Mochida, H, et al. Immunocytochemical localization of prohormone convertases PC1/PC3 and PC2 in rat pancreatic islets. Arch Histol Cytol. 1996;59:261–271.

249. Furuta, M, Zhou, A, Webb, G, et al. Severe defect in proglucagon processing in islet A-cells of prohormone convertase 2 null mice. J Biol Chem. 2001;276:27197–27202.

250. Gelling, RW, Du, XQ, Dichmann, DS. Lower blood glucose, hyperglucagonemia, and pancreatic alpha cell hyperplasia in glucagon receptor knockout mice. Proc Natl Acad Sci U S A. 2003;100:1438–1443.

251. Webb, GC, Akbar, MS, Zhao, C, et al. Glucagon replacement via micro-osmotic pump corrects hypoglycemia and alpha-cell hyperplasia in PC2 knockout mice. Diabetes. 2002;51:398–405.

252. Wilson, ME, Kalamaras, JA, German, MS. Expression pattern of IAPP and prohormone convertase 1/3 reveals a distinctive set of endocrine cells in the embryonic pancreas. Mech Dev. 2002;115:171–176.

253. Nie, Y, Nakashima, M, Brubaker, PL, et al. Regulation of pancreatic PC1 and PC2 associated with increased glucagon-like peptide 1 in diabetic rats. J Clin Invest. 2000;105:955–965.

254. Csaba, Z, Dournaud, P. Cellular biology of somatostatin receptors. Neuropeptides. 2001;35:1.

255. Egel-Mitani, M, Flygenring, HP, Hansen, MT. A novel aspartyl protease allowing KEX2-independent MFα propheromone processing in yeast. Yeast. 1990;6:127.

256. Zhu, X, Cao, Y, Voogd, K, et al. On the processing of proghrelin to ghrelin. J Biol Chem. 2006;281:38867–38870.

257. Yang, J, Brown, MS, Liang, G, et al. Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. Cell. 2008;132:387–396.

258. Andralojc, KM, Mercalli, A, Nowak, KW, et al. Ghrelin-producing epsilon cells in the developing and adult human pancreas. Diabetologia. 2009;52:486–493.

259. Webb, GB, Akbar, MS, Zhao, C, et al. Expression profiling of pancreatic beta cells: glucose regulation of secretory and metabolic pathway genes. Proc Natl Acad Sci U S A. 2000;97:5773.

260. Canaff, L, Bennett, HPJ, Hou, Y, et al. Proparathyroid hormone processing by the proprotein convertase-7: comparison with furin and assessment of modulation of parathyroid convertase messenger ribonucleic acid levels by calcium and 1,25-dihydroxyvitamin D₃. Endocrinology. 1999;140:3633–3642.