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To model islet amyloidogenesis in NIDDM and explore the glucoregulatory role of islet amyloid polypeptide (IAPP), we have created transgenic micye containing a rat insulin-I promoter-human IAPP fusion gene. Expression of human IAPP was localized to the islets of Langerhans, anterior pituitary and brain in transgenic animals; blood IAPP levels were elevated 5-fold while fasting glucose levels remained normal. Amyloid deposits have not been detected in transgenic islets suggesting that other co-existing abnormalitites in NIDDM may be required for the formation of islet amyloid. These animals provide a unique model for exploring this hypothesis and other proposed functions of IAPP.

Recent studies have shown that vertebrates, including teleostean fishes, amphibians, birds and mammals, contain two distinct insulin-like growth factor (IGF) genes. In contrast agnathans, represented by hagfish, apparently have only one IGF that has features characteristic of both IGF-I and IGF-II. Between these groups the elasmobranchs occupy a critical position in terms of the phylogeny of IGFs. We sought to determine if gene duplication and divergence of IGF-I and IGF-II occurred before or after divergence of elasmobranchs from other vertebrates by cloning IGF-like molecules from Squalus acanthias. Our analysis shows that Squalus liver produces two distinct IGF-like molecules. One has greater sequence identity to, and conserved features characteristic of, known IGF-I molecules, while the other is more IGF-II like. These results suggest that the prototypical IGF molecule duplicated and diverged in an ancestor of the extant gnathostomes.

xelerences It has recently been established in several laboratories [l-4] that a precursor larger than proinsulin is involved in the biosynthesis of insulin. This new precursor form has been less readily detected because it evidently turns over so rapidly in the beta cell of the pancreas that it is virtually invisible to the whole-cell labeling techniques that have been used to identify and study the synthesis and conversion of proinsulin into insulin [5]. Preproinsulin and structurally similar precursors of many other secreted proteins appear to provide the solution to a long-standing problem in the biosynthesis of secretory proteins, namely, the mechanism by which the sequestration of peptide chains destined for secretion occurs within cells. Thus, although it has been known for a long time that secretory proteins are synthesized in the rough endoplasmic reticulum by membranebound ribosomes, it has not been at all clear how the rough endoplasmic reticulum is formed, or how the messenger RNA (mRNA) for secretory proteins is made available exclusively to the membranebound ribosomes. originally been extracted were incubated in vitro, they synthesized only normal light chains. However, after lysis of the microsomal membranes by detergent treatment, the free polyribosomes synthesized only the larger precursor form. Milstein and coworkers suggested that the microsomes contained a protease which rapidly processed the precursors to normal light chains under normal conditions [7]. 1

To examine further the mechanism by which the synthesis of proteins translated on the rough endoplasmic reticulum is regulated in pancreatic beta-cells, the synthesis of growth hormone in islets from transgenic mice carrying the metallothionein-rat growth hormone gene fusion was studied. High glucose (17 mM) stimulated the synthesis and secretion of an apparently normally processed growth hormone. The stimulation of synthesis of growth hormone was less efficient than the stimulation of insulin synthesis in these islets, whereas the stimulation of release of labeled growth hormone paralleled that of insulin. These results are consistent with the hypothesis that signal recognition particle-mediated mechanism(s) may be involved in regulating the translational efficiency of secreted proteins in isolated islets (Welsh, M., Scherberg, N., Gilmore, R., and Steiner, D. F. (1986) Biochem. J. 235, 459-467). Furthermore, in the beta-cells, growth hormone follows the normal regulated pathway of secretory granule transport and exocytosis.

BIOSYNTHETIC studies with human islet cell tumours1,2, rat2,3 and codfish4 islets, and foetal calf pancreas5 have shown that insulin is formed from proinsulin, a single-chain polypeptide precursor. This protein has subsequently been purified from commercial preparations of crystalline insulin6, and the amino-acid sequences of porcine7 and bovine8 proinsulin have been determined. The molecule begins at the N-terminus with the B-chain sequence of insulin, terminates with the insulin A-chain and bears a connecting segment of thirty (bovine) or thirty-three (porcine) amino-acids linking the two chains. Although 30 per cent of the amino-acid sequences of these connecting segments differ, identical pairs of basic amino-acids occur at each end in both species. Limited tryptic digestion in each case liberates desalanyl–insulin, alanyl–arginine, free arginine and the remainder of the connecting segment as an intact peptide6,7.

Reductive methylation of the three amino groups of porcine insulin was accomplished by incubation with formaldehyde and sodium cyanoborohydride. The two amino termini and the epsilon amino group of B29 lysine were each dimethylated within 1 h of incubation. The fully methylated insulin bound more tightly to a reverse phase column than did native insulin, had a slightly more acid isoelectric point, and maintained approximately 50% biological activity when examined with an insulin sensitive cultured cell line. Reductive methylation with sodium cyanoboro [3H] hydride resulted in a [3H] methylated insulin with a specific activity of 6 Ci/mmol.

Dispersed rat pancreatic islet cells were mixed into a short column of Bio-Gel P-2 polyacrylamide beads and perifused with an antiserum containing islet cell surface antibodies. The release of radioactive chromium from prelabeled cells, as a measure of cell membrane permeability, was not affected by cell surface antibodies alone, but increased dramatically in the presence of complement. While there was an eightfold increase in glucose-stimulated insulin release from β-cells exposed to control serum and complement, insulin release was completely blocked from β-cells exposed to islet-cell-specific antibodies and complement. These findings suggest that islet cell surface antibodies can mediate complement-dependent cytotOXicity.

Methods have been developed for the isolation on a semi-micro scale of a plasma membrane-enriched fraction from rat islets of Langerhans. An important feature of these experiments is the use of 125I-labeled wheat germ agglutinin as a specific probe for plasma membrane-containing fractions. The partly purified plasma membrane fraction had a density in sucrose of about 1.10 and was enriched in the activities of 5'-nucleotidase, alkaline phosphatase, sodium-potassium, and magnesium-dependent ATPase and adenylate cyclase. It contained only very low levels of acid phosphatase, cytochrome c oxidase, insulin, and RNA. Further purification was hampered by the relatively small amounts of fresh plasma membrane material that could be obtained from 16-24 rats in each experiment. When islets were prelabeled with radioactive fucose, the plasma membrane-enriched fraction contained radioactivity at a four- to fivefold higher specific acivity than the whole islet homogenate. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of plasma membrane-enriched fractions pooled from several experiments revealed a distinctive pattern of protein bands as compared with other less pure fractions. With respect to rapidity, apparent specificity, and easy reversibility of the labeling of the plasma membrane fraction, 125I-wheat germ agglutinin provides a highly useful tool for the detection of microgram quantities of plasma membrane components which should be applicable to many other systems as well.

INSULIN is a well defined globular protein made up of two small peptide chains linked together through disulphide bonds1,2. It contains an unusually high proportion of hydrophobic residues and thus tends to readily self-associate3,4. In many species the hormone forms dimers in solution and, in the presence of Zn2,4ions, hexamers4,5. Recently, Hodgkin and coworkers6,7 have determined the crystal structure of the rhombohedral form of porcine Zn-insulin at 1.9 A resolution6,7. Two molecules of insulin occur in the asymmetric unit, related through an approximate twofold axis to form a nearly symmetrical dimer. Three such dimers in turn associate through coordination of histidine residues B10 with two zinc ions to form a nearly spherical hexamer. In spite of the availability of the amino acid sequences of a wide variety of vertebrate insulins2,8, however, comparative studies on the detailed structure and modes of association of vertebrate insulins have been lacking.

Many small biologically active peptides are derived from larger precursor forms which fulfil a variety of roles in the synthesis, segregation and intracellular migration of secretory products. Limited proteolysis may occur at several stages during this process, giving rise to products that are either degraded (e. g. the prepeptides) or discharged coordinately from their cells of origin during exocytosis (e. g. insulin and C-peptide). Molecular defects have recently been found to occur at cleavage sites in proinsulin as well as in other proproteins, and these point mutations may, in some instances, be responsible for familial metabolic disorders. The nature and cell specificity of the proteolytic enzymes involved in the conversion of the various precursor forms remains unresolved. Recent studies in our laboratory have led to the identification of precursors of glucagon and somatostatin in rat islets of Langerhans. Analysis of tryptic maps of these precursors has shown that a trypsin-like enzyme would be sufficient to cleave the C-terminally located somatostatin sequence from its precursor (relative molecular mass 12500), but that both trypsin-like and carboxypeptidase B-like enzymes would be necessary to cleave the internal glucagon sequence from its prohormone (relative molecular mass 18000). Molecular cloning techniques have provided valuable new approaches to analysing the structures of a variety of precursor forms, including those for insulin, gastrin, growth hormone, adrenocorticotrophic hormone and the endorphins, and in the future will undoubtedly shed more light on the structures of their chromosomal genes, the mechanisms regulating their expression, and their evolutionary origins.

Proteolytic processing of precursor proteins is a phylogenetically ancient and widely used mechanism for producing biologically active peptides. Proteolytic cleavage of proproteins begins only after transport to the Golgi apparatus has been completed and in most systems may continue for many hours within newly formed secretory vesicles as these are stored in the cytosol or transported along axons to more peripheral sites of release. Paired basic residues are required for efficient proteolysis in most precursors, suggesting that a small number of specialized tryptic proteases exist that have great site selectivity but can process many sites within the same precursor or in different precursors within the same cell, or in different cells or tissues. Cleavage-site choice may be strongly influenced by other factors, such as secondary and tertiary structure, but definitive structural information on precursor proteins is lacking. Modifications such as glycosylation, phosphorylation, and sulfation also are Golgi associated but are not known to influence proteolytic processing patterns. Golgi/granule processing also rarely occurs at sites other than pairs of basic amino acids, including single basic residues ( trypsinlike ), Leu-Ala, Leu-Ser, or Tyr-Ala bonds ( chymotrysinlike ) as well as other specialized nontryptic cleavages, suggesting that mixtures of proteases coexist in the Golgi/granule system. Cathepsin B-like thiol proteases, or their precursors, have been implicated as the major processing endopeptidases in several systems. Carboxypeptidase B-like enzymes also have been identified in secretion granules in several tissues and appear to be metalloenzymes similar in mechanism to the pancreatic carboxypeptidases, but with a lower pH optimum. The role of the Golgi apparatus in sorting newly formed secreted products from lysosomal hydrolases may have permitted the development in evolution of an intimate relationship between certain of the lysosomal degradative enzymes, such as cathepsin B or its precursors, and the Golgi/granule processing systems. The sequestration of the proteolytic products of precursors within secretion granules leads to the coordinate discharge of highly complex mixtures of peptides having related or overlapping biological activities. The cosecretion of nonfunctional peptide " leftovers ," such as the proinsulin C-peptide, can serve as useful markers of secretion or cellular localization, as well as of evolutionary relation ships. Errors in cleavage due to point mutations in precursors have been identified in several systems, leading to the accumulation of incorrectly processed materials in the circulation. These and/or defects (ABSTRACT TRUNCATED AT 400 WORDS)

Summary Synthesis of human C-peptide (human proinsulin 33–63 ) (I), [64-formyllysine]-human proinsulin 31–65 (II) and [41-glutamic acid, 43-glutamine, 64-formyllysine]-human proinsulin 31–65 (III) are described. Immunological reactivities of these synthetic polypeptides were examined using a guinea pig antiserum to the natural human C-peptide conjugated with rabbit serum albumin. I, II and the natural human C-peptide displaced 125 I-tyrosylated natural human C-peptide from the antibody almost identically, while III failed to completely displace the labeled peptide.

be further conveniently subdivided into the preproteins and proproteins. These two large groups differ significantly in their processing kinetics, in the subcellular localizations of cleavage enzymes, and in their functions. This review focuses primarily on these forms, with special emphasis on recent progress in elucidating their primary enzymic cleavage mechanisms. At this stage it seems most likely that for each subgroup a single proteolytic mecha­ nism accounts for the major processing events, with some specialized fea­ tures requiring additional enzymes for several of the prohormonal systems.

A coding mutation in the human insulin gene (His-B10----Asp) is associated with familial hyperproinsulinemia. To model this syndrome, we have produced transgenic mice that express high levels of the mutant prohormone in their islets of Langerhans. Strain 24-6 mice, containing about 100 copies of the mutant gene, were normoglycemic but had marked increases of serum human proinsulin immunoreactive components. Biosynthetic studies on isolated islets revealed that approximately 65% of the proinsulin synthesized in these mice was the human mutant form. Unlike the normal endogenous mouse proinsulin, which was almost exclusively handled via a regulated secretory pathway, up to 15% of the human [Asp10]proinsulin was rapidly secreted after synthesis via an unregulated or constitutive pathway, and approximately 20% was degraded within the islet cells. The secreted human [Asp10]proinsulin was not processed proteolytically. However, the processing of the normal mouse and human mutant proinsulins within the islets from transgenic mice was not significantly impaired. These findings suggest that the hyperproinsulinemia of the patients is the result of the continuous secretion of unprocessed mutant prohormone from the islets via this alternative unregulated pathway.

The availability of C-peptide measurement continues to provide new and useful information about the state of beta cell secretory function and the natural history of diabetes. Measurement of proinsulin is of value in the diagnosis of insulin-secreting tumors.

Normal adult rat islets usually remained intact and encapsulated, even after many days in culture. In contrast, islets cultured in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1 mM) attached more readily to the surface of plastic culture dishes and almost uniformly formed monolayers of endocrine cells. The mechanism of this effect is not known but presumably involves increases in cellular cyclic AMP content. Fibroblast growth did not appear to be stimulated by the inhibitor. These adult pancreatic endocrine monolayer cultures can be produced readily and provide useful preparations for further morphological and biochemical studies of factors affecting the differentiation, growth, and regenerative capacity of islet cells.

PROINSULIN is a single-chain polypeptide in which the A and B chains of insulin are linked together by the connecting peptide1. The terminals of the connecting peptides are occupied by the basic dipeptides, Arg–Arg and Lys–Arg, respectively, and proinsulin C-peptide is defined as connecting peptide minus the basic dipeptides. The immunological reactivity of synthetic [59-formyllysine]-bovine proinsulin 31–60, which comprises the entire sequence of the connecting peptide segment of bovine proinsulin, is indistinguishable from that of natural bovine proinsulin when compared on an equimolar basis2. The immunoassay system involved a bovine proinsulin antiserum3, which was purified by the removal of the antibodies directed towards the insulin region of the molecule. We have now evaluated the structural features of bovine connecting peptide segment which determine its reactivity with bovine proinsulin antisera. For this purpose, homogeneous synthetic peptides of defined structures serve as excellent substrates. We synthesised twelve peptides related to bovine proinsulin C-peptide which has the following amino acid sequence

Using an indirect immunofluorescence test on suspensions of viable, insulin-producing islet cells from rats, we found that 32 per cent (28/88) of insulin-treated patients with juvenile diabetes have isletcell-surface antibodies in their circulation. These antibodies also occurred in four of nine children with glucose intolerance, in one of 24 healthy children and in nondiabetic children with thyroid disorders. In the diabetic children, the immunofluorescent reaction was inhibited by preadsorption of serum to islet cells but was little affected by preadsorption to rat hepatocytes or erythrocytes or to acetone powders of various rat tissues, including pancreas. These results show that organ-specific, non-species-specific antibodies reactive with the cell surface of the islet cells can be present in serum from diabetic children, and provide an approach to investigation of immunopathological aspects of diabetes mellitus. (N Engl J Med 299:375–380, 1978)

Intracellular cleavage of protein and polypeptide precursors is now recognized as a widely occurring biosynthetk: mechanism. As this field has developed, proinsulin and its cleavage patterns and secretory products have served as useful models for investigations of other systems. A particularly relevant aspect of the proprotein concept is the simple mechanism it provides for the coordinate synthesis and discharge of related peptides from endocrine or other secretory cells. This report reviews briefly the role of the proinsulin C-peptide, first in terms of its special biosynthetk: functions, which are unique to the assembly of the two-chain insulin structure, and then with regard to its more general implications for other biosynthetk and secretory systems.

The gene-encoding human islet amyloid polypeptide (hIAPP), a recently discovered 37 amino acid hormone-like polypeptide which is expressed in the insulin-producing beta-cells of the endocrine pancreas, has been isolated and characterized. The coding region of the gene is interrupted in the 5'-untranslated region and NH2-terminal propeptide by introns of 330 and 4808 base pairs (bp), respectively. Exon 1 (104 bp) encodes most of the 5'-untranslated region of the mRNA; exon 2 (95 bp) encodes 15 nucleotides of 5'-untranslated region, the putative 22 amino acid signal peptide and five residues of the NH2-terminal propeptide; exon 3 (1246 bp) encodes the remainder of the NH2-terminal propeptide (residues 6-9), the IAPP moiety and its processing signals and the 16 amino acid COOH-terminal propeptide, as well as the 3'-untranslated region of the mRNA (1059 bp). Analysis of the nucleotide and predicted amino acid sequence of intron 2 of the hIAPP gene did not reveal any homology with the structurally related calcitonin/calcitonin-gene-related peptide genes and indicated that, in contrast to these latter genes, the hIAPP gene apparently gives rise to only a single hormonal product. The transcriptional initiation site was identified about 28 bp downstream from a TATAA sequence. The hIAPP gene was localized to the p12.3 region of chromosome 12.

Publisher Summary This chapter discusses several molecular approaches used for the study of hormonal evolution. In the case of insulin, pro-insulin messenger RNA fractions from islet tissues have been identified by their translation in cell-free protein synthesizing systems to yield material that is reactive with insulin antisera. The study of evolutionary relationships among cells that produce secretory proteins, such as insulin or glucagon, can be undertaken via the comparative analyses of the primary structures of the product molecules and, wherever possible, also via the studies of their secondary, tertiary, and quaternary structures. An interesting alternative approach to the studies of hormonal evolution would be via the comparative studies of the structures of their respective messenger RNA molecules or even of their structural genes in the chromosomal DNA. Current methods for the molecular hydridization of DNA and RNA permit direct comparisons to be made among the genomes of various species.

The radioimmunoassay for hagfish insulin described employs a specific anti-hagfish-insulin serum raised in guinea pigs, 125 I-labeled hagfish insulin, and purified hagfish insulin as standard. The apparent dissociation constant of the antiserum was 0.24 ± 0.05 n M . The cross-reactivity with ox insulin was on the order of magnitude of 0.01%. Equilibrium binding was reached within 24 hr at 4°. Insulin values, ranging from 50 pg to 5–10 ng, could be measured. The free and the bound label could be separated conveniently either with polyethylene glycol or a second precipitating antibody. Immunofluorescence microscopy showed that the antiserum reacted with hagfish islet cells similarly to what has earlier been reported using antiserum against mammalian insulins.

Both alleles of the insulin gene of a patient with mild diabetes [maturity-onset-diabetes-of-the-young (MODY)-type syndrome] associated with hyperinsulinemia have been cloned, and the sequences have been determined. One allele contained a mutation (single nucleotide transition) in the coding sequence for the B chain at position 24 (TTC leads to TCC), resulting in the loss of a restriction enzyme (Mbo II) cleavage site in the gene. This mutation results in the substitution of serine for phenylalanine in a critically important region of the insulin molecule that is intimately involved in receptor binding. Both insulin alleles were of the alpha type and, aside from a single nucleotide deletion in the 5' region of the normal allele, their sequences were identical to those previously determined.

The biosynthesis of insulin in the islets of Langerhans is strongly controlled at the translational level by glucose. We have used a variety of experimental approaches in efforts to dissect the mechanisms underlying the stimulatory effect of glucose. To assess its effects on rates of peptide-chain elongation, isolated rat islets were labelled with [3H]leucine at different glucose concentrations in the presence or absence of low concentrations of cycloheximide. Under these conditions, at glucose concentrations up to 5.6 mM, endogenous insulin mRNA did not become rate-limiting for the synthesis of insulin, whereas stimulation of non-insulin protein synthesis was abolished by cycloheximide at all glucose concentrations, indicating either that insulin synthesis is selectively regulated at the level of elongation at glucose concentrations up to 5.6 mM, or that at these concentrations inactive insulin mRNA is transferred to an actively translating pool. Glucose-induced changes in the intracellular distribution of insulin mRNA in cultured islets were assessed by subcellular fractionation and blot-hybridization using insulin cDNA probes. At glucose concentrations above 3.3 mM, cytoplasmic insulin mRNA was increasingly transferred to fractions co-sedimenting with ribosomes, and relatively more of the ribosome-associated insulin mRNA became membrane-associated, consistent with effects of glucose above 3.3 mM on both the initiation of insulin mRNA and SRP (signal recognition particle)-mediated transfer of cytosolic nascent preproinsulin to the endoplasmic reticulum. When freshly isolated islets were homogenized and incubated with 125I-Tyr-tRNA, run-off incorporation of 125I into preproinsulin was increased by prior incubation of the islets at 16.7 mM-glucose. The addition of purified SRP receptor increased the run-off incorporation of [125I]iodotyrosine into preproinsulin, especially when the islets had been preincubated at 16.7 mM-glucose. These findings taken together suggest that glucose may stimulate elongation rates of nascent preproinsulin at concentrations up to 5.6 mM, stimulates initiation of protein synthesis involving both insulin and non-insulin mRNA at concentrations above 3.3 mM, and increases the transfer of initiated insulin mRNA molecules from the cytoplasm to microsomal membranes by an SRP-mediated mechanism that involves the modification of interactions between SRP and its receptor.

Using a DNA-binding gel mobility shift assay, a protein-DNA complex was detected using the rat II insulin promoter and a nuclear extract from rat insulinoma cells. This complex was detected using extracts from other insulin-producing cell lines but was not found using nuclear extracts from non-insulin-producing pancreatic cell lines or other cell types. Binding of the tissue-specific protein to the labelled promoter fragment was effectively competed by both rat and human insulin genes but not by the SV40 TATA box. This protein-DNA interaction may be involved in tissue-specific regulation of insulin gene expression.

Polyclonal antibodies reactive with a cleavage site in human proinsulin (HPI) (C-peptide-A-chain junction) have been raised (rabbit, guinea pig) using a synthetic peptide antigen coupled with keyhole limpet hemocyanin. These antisera recognize native HPI and des-31,32-HPI equally well but react 20-50 times less well with des-64,65-HPI, the intermediate cleaved at the C-peptide-A-chain junction and lacking the Lys-Arg pair. The guinea pig antisera did not recognize insulin but reacted weakly with C peptide at high concentrations; the rabbit antisera reacted with neither insulin nor C peptide. Immunocytochemical studies with human islet tissue localized the immunoreactivity of these antisera to clathrin-coated (pro)secretory vesicles derived from the trans Golgi, indicating that cleavage of the C-peptide-A-chain junction of proinsulin occurs mainly, if not exclusively, in this compartment of the beta cell.

The concentration of thyrotropin-releasing hormone (TRH, thyroliberin) in rat islets of Langerhans is 30-fold higher than in whole rat pancreas, indicating that the islets are the main source of pancreatic TRH. The TRH extracted from islets is indistinguishable from synthetic TRH in its immunological and biological properties and in its inactivation by human serum. The physiologic function of islet TRH is unknown. However, because TRH is antagonistic to somatostatin in other systems, and somatostatin also is concentrated in islets in high concentrations, it is possible that islet TRH may serve a similar antagonistic function in the regulation of islet cell secretory activity.

The development of a simple microscale solid phase screening RIA and improved methods for cell cloning has lead to the establishment of 2 prohormone- and species-specific mouse monoclonal antibodies against human proinsulin (HPI). These antibodies react to determinant(s) only expressed on the HPI molecule. In addition, 11 rat monoclonal antibodies were generated which react with both human C-peptide (HCP) and HPI. All 11 rat antibodies recognize a very similar antigenic determinant in the C-peptide that appears to be made up of residues 40-45 and 57-63, which are probably brought into close proximity by a beta-turn near the center of the connecting segment. The identical behavior of both HPI-specific mouse antibodies in competition experiments indicates that the antigenic structure recognized in proinsulin might be the same for both antibodies. This structure could not be regenerated by mixing equimolar amounts of human insulin and C-peptide, including the chemically synthesized complete proinsulin connecting segment (Arg X Arg X HCP X Lys X Arg), which contains the entire sequence removed from proinsulin in the conversion to mature insulin. Indirect evidence is provided that a HPI molecule simultaneously can bind a C-peptide-directed rat antibody and a HPI-specific mouse antibody when the first antibody is presented to HPI in solution phase. However, when HPI is immobilized on a plastic surface, the binding of 1 type of antibody completely blocks the binding of the other. These antibodies provide useful new tools for studying the biosynthesis and 3-dimensional structure of HPI and HCP.

SUMMARY Monolayer cultures of minimal deviation hepatoma cells (H4-II-E-C3′) bound and degraded insulin specifically, the apparent Ki value for insulin inhibition of both processes being 1 × 10−8 M, indicating that cellbound 125l-insulin is the substrate for subsequent hormone degradation in these cells as in isolated hepatocytes.1 The time course of insulin binding to its receptor depended on hormone concentration and temperature. Degradation of insulin also depended highly on temperature, with little or no degradation occurring at less than 20°C, a temperature below which a membrane–lipid phase transition may block hormone translocation or uptake. The effects of various agents on the binding and degradation of 125l-insulin also were tested. Agents believed to inhibit intralysosomal degradation of various proteins also inhibited the degradation of 125l-insulin by H4 cells (chloroquine, ammonium chloride, procaine, and lidocaine); inhibitors of energy production (dinitrophenol, sodium cyanide) inhibited degradation; an agent which inhibits microtubule function (vinblastine) blocked insulin degradation; and methylamine, reported to prevent receptor aggregation,2 also interfered with insulin processing. These findings are consistent with a model for cellular insulin processing, comprising receptor binding, clustering of receptors, endocytotic uptake, intralysosomal degradation, and extracellular release of some degradation products. H4 cells were highly sensitive to insulin. The KE for a half-maximal response of hormone-stimulated incorporation of 14Cglucose into glycogen was 10−11 M insulin, corresponding to less than 1% receptor occupancy. This response was also mimicked by concanavalin A at a concentration of 10 μug/ml. Vinblastine and chloroquine both significantly inhibited insulin-stimulated glucose incorporation into glycogen without affecting basal levels. However, since these inhibitory effects were not relieved by addition of excess insulin, it seemsunlikely that their action on glycogen synthesis was exerted only at the level of the generation of an active intermediate or degradation product from hormone-receptor complexes. The hormone-sensitive H4 cells thus provide a useful system for further studies examining the role of insulin-receptor uptake in hormone action, receptor regulation, and signal termination.

Publisher Summary Since the discovery of proinsulin, post-translational modification of proteins by limited intracellular proteolysis has been recognized increasingly as a normal biosynthetic mechanism in the production of a wide variety of secretory, along with some viral, proteins. Little is known about the processing mechanisms involved, aside, in a few instances, from their tentative intracellular localization to the Golgi area, and some elements of their cleavage specificity. This chapter presents several interesting recent examples of pro-proteins and discusses the general significance of these formsMost small polypeptide hormones are derived via cleavage of larger precursor molecules. A number of examples are summarized; however, additional suggestive evidence of the existence of larger forms of somatostatin, pancreatic polypeptide, calcitonin, and vasopressin, as well as others is accumulating. These larger forms may fulfill a number of important functions in biosynthesis and secretion; however, a requirement for a minimum chain length of 65–70 residues for successful vectorial discharge and sequestration in the rough endoplasmic reticulum may provide an explanation for the existence of many of them.

We have previously identified a structurally abnormal insulin in the serum and pancreas of a middle-aged man with diabetes mellitus which arose from a leucine for phenylalanine substitution at position 24 or 25 of the insulin B chain; further analysis of the patient's leukocyte DNA showed that one of the patient's insulin alleles had undergone mutation resulting in loss of an MboII restriction site normally present in the human insulin gene. Two additional and unrelated patients with the same clinical syndrome have now been identified (ref. 4 and unpublished results). All of these patients showed hyperglycaemia typical of diabetes and with marked hyperinsulinaemia typical of insulin resistance, but all three show normal tolerance to exogenously administered insulin. As the opportunity of examining pancreatic tissue from patients suspected of secreting insulin variants is rare, we have developed a method combining HPLC and radioimmunoassay to identify insulin variants isolated from human sera. By this method we have shown that all three patients noted above secrete structurally variant and chemically distinct insulins. In correction of our original assignment, one is identified as [LeuB25]insulin.

The effects of various agents on the binding and degradation of 125I-labelled insulin by isolated rat hepatocytes and cultured H4 hepatoma cells were studied. Various lysosomotropic agents, including chloroquine, ammonium chloride, and the topical anesthetics, lidocaine and procaine, inhibited insulin degradation by H4 hepatoma cells but had little effect on the binding of the hormone. Similarly, tosyl-L-lysyl chloromethyl ketone selectively inhibited the degradation of 125I-labelled insulin by isolated hepatocytes, as did the sulfhydryl reagents, p-hydroxy- and p-chloromercuriphenyl sulfonic acid. Inhibitors of energy production, including sodium fluoride, sodium azide, and dinitrophenol, also selectively inhibited the degradation of insulin by hepatocytes, although cyanide had no effect under the conditions used. Lectins and anti-microtubular agents, which are known to affect the mobility of plasma membrane proteins or of intracytoplasmic vesicles, selectively inhibited insulin degradation by hepatocytes to varying degrees, whereas agents which inhibit the function of microfilaments had no effect. At temperatures below 20 °C, insulin degradation was negligible but rose rapidly between 20 and 37 °C, suggesting that a membrane-related step is rate limiting in the overall degradative process. These results are all consistent with a model of insulin uptake by target tissue involving pinocytosis of receptor-bound hormone followed by intralysosomal degradation.

Publisher Summary This chapter discusses the biosynthesis of the islet cell hormones insulin and glucagon. It was widely believed that insulin was formed by the combination of separately synthesized A and B chains. The discovery of pro-insulin disproved this hypothesis, and it is now known that a connecting peptide attaches the COOH terminus of the B chain to the NH 2 terminus of the A chain in the single-chain precursor. The C peptide connects the two insulin chains by pairs of dibasic amino acid residues (Lys–Arg and Arg–Arg at the A and B chains, respectively) and forms a bridge between the two portions of the insulin molecule. Studies on the translation of insulin messenger ribonucleic acid (mRNA) extracted from rat islets in cell-free systems have shown that the product is not pro-insulin but is a larger peptide that bears a 24-residue, NH 2 -terminal extension on the prohormone sequence. This molecule, called “preproinsulin,” has also been identified during the cell-free translation of insulin mRNA from cattle, anglerfish, sea raven, hagfish, and humans. An early indication of proglucagon structure arose from an examination of crystalline glucagon for higher molecular weight, hormone-related peptides. Because the COOH-terminal sequence is connected to the hormone structure by a pair of dibasic amino acid residues (Lys–Arg), both trypsin- and carboxypeptidase B-like enzymes would be necessary to convert this proglucagon fragment to glucagon. The use of trypsin and carboxypeptidase B digestions to probe the structures of glucagon-related peptides is discussed in the chapter.

Publisher Summary This chapter discusses the clinical significance of circulating pro-insulin and c-peptide. Till 1967, it was believed that the two chains of insulin were synthesized separately and then joined by means of two disulfide bonds at a post-ribosomal site. However, studies by Steiner and his colleagues using slices of a pancreatic islet cell tumor demonstrated the presence of a large molecular weight, single chain precursor molecule, which was named pro-insulin. This protein consists of the insulin A and B chains linked by an additional polypeptide segment of approximately 30 to 35 amino acids, depending on the species. The enzymes involved in the transformation of pro-insulin to insulin are probably localized in the granules or their membranes, and the conversion process takes place as the granules mature and move toward the plasma membrane of the cell. These pro-teolytic enzymes cleave pro-insulin at specific sites where two pairs of basic amino acids join the connecting peptide (C-peptide) to the insulin chains. The major products of this reaction are insulin and the C-peptide.

We have used a synthetic deoxydecanucleotide to generate an insulin-specific cDNA probe suitable for selecting transformants that contain nearly full-length cDNAs corresponding to the mRNAs coding for rat insulins I and II. Double-stranded cDNA was synthesized from x-ray-induced rat insulinoma poly(A)-RNA, inserted in pBR322 plasmid DNA by the homopolymeric tailing technique, and cloned in Escherichia coli chi 1776. Colony hybridization with oligonucleotide-primed cDNA yielded 16 positive clones of which 7 corresponded to rat insulin I mRNA and 9 to rat insulin II mRNA. Restriction endonuclease maps of representative clones of each group indicated that these contained the complete coding sequences, as was confirmed by nucleotide sequence analysis of the 5' region of the cloned DNA for rat insulin II. Nucleotide sequence analysis also established the amino acid sequence of the prepeptide of rat preproinsulin II. Comparison of the amino acid sequence of the prepeptides of rat preproinsulin I and II shows that three conservative amino acid substitutions have occurred in this region of the molecule.

SUMMARY Recent studies have established that limited intracellular proteolysis may participate at several stages in the biosynthesis of many small polypeptide hormones, as exemplified by insulin. Conversion of proinsulin to insulin by trypsin-and carboxypeptidase B-like enzymes occurs in the pancreatic B cell after the peptide has been sequestered from the cytosolic compartment and transported to the Golgi area. An earlier role for proteolysis concerns the conversion of the initial translation product, preproinsulin, to proinsulin during its compartmentalization in the rough endoplasmic reticulum. Preproinsulin, a methionine-initiated peptide which contains a hydrophobic 24-residue NH 2 -terminal extension, was originally identified as the product of the cell-free translation of insulin mRNA, but more recently it has also been detected in small amounts in intact rat islets. Its rapid formation and disappearance during pulse-chase studies are consistent with its role as a biosynthetic precursor and with the proposal that the prepeptide region serves as a leader sequence in the formation of the ribosome-membrane junction and in the vectorial discharge of the peptide. A model for segregation is presented, based on important structural features and physical properties of the prepeptides which may enable these to enter and span the microsomal membrane. Studies on insulin mRNA obtained from transplantable islet cell tumors indicate that the major component contains approximately 600 nucleotides and possesses a 5′ “cap” structure as well as a 3′ polyadenylate “tail”. A closer examination of the insulin tumor mRNA has also revealed two larger polyadenylated RNA fractions having apparent molecular weights of 280,000 and 360,000. These forms, which hybridize to the rat insulin genes and which were shown by ribonuclease T 1 digests to share nucleotide sequences with the major mRNA component, may represent precursors of the mature template. These results indicate that several sequential stages of macromolecular processing are required in the formation of the secreted hormone.

A liver metastasis (MSL) with a remarkable in vitro proliferation potential has been identified in an NEDH rat carrying a transplantable x-ray-induced islet cell tumor. Two insulin-secreting cell lines, MSL-G and MSL-H, with doubling times of 3-5 d were established by repeated limiting dilution cloning. In vivo inoculation of MSL-G cells induced severe hypoglycemia caused by a small but highly heterogeneous tumor as revealed by immunocytochemistry. Whereas most cells stained for the islet hormones, insulin, glucagon, and somatostatin, clustered cells were discovered to contain cholecystokinin (CCK). Additional in vitro-limiting dilution cloning, followed by immunocytochemical characterization, clearly demonstrated the capacity of single cell clones to simultaneously express the same four hormones. Radioimmunoassays with a panel of site-specific antisera of culture supernatants and purified cell extracts showed the MSL-G2 cells to produce, store, and secrete readily detectable amounts of processed and unprocessed CCK. Gastrin was not detected while coexpression of glucagon and CCK were demonstrated. Mutant clones selected for resistance to 6-thioguanine (frequency, 2 X 10(-7] and checked for HAT (hypoxanthine, aminopterin, thymidine) sensitivity retained the capacity for multi-hormone expression. We propose that the MSL tumor contains pluripotent endocrine stem cells. The MSL tumor and the MSL-G2 cells in particular will allow studies of not only CCK biosynthesis and processing but also of mechanisms involved in tumor and islet cell differentiation.

INTRODUCTION 463 STRUCTURE OF THE INSULIN GENE 465 REGULATION OF INSULIN GENE EXPRESSION 468 EVOLUTIONARY ASPECTS OF (PREPRO)lNSULIN GENE STRUCTURE 469 THE INSULIN SUPERFAMILY ..... .... . . .. . ... ....... . . .... . .... . .. .. . . ...... ... . 473

Whole nucleic acid fractions of isolated rat islets of Langerhans greatly stimulate incorporation of radioactive amino acids into protein in a wheat germ ribosomal system. Approximately 30% of the synthetic product is precipitated with antisera to insulin or proinsulin. Characterization of this material by gel chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates a molecular mass of 11,500 daltons. Trypsin digestion releases intact A chain as well as tryptic fragments of the C-peptides and B chains of the two rat proinsulins. Automated sequence determination of labeled cell-free product purified by immunoprecipitation discloses the presence of 23 additional amino acids NH2-terminal to the B chain sequence of proinsulin. The partial amino acid sequence of this extension is as follows: NH2-X-Leu (Lys) Met-x-Phe-Leu-Phe-Leu-Leu (Lys) Leu-Leu-x-leu-X-X-X-X-X-X-X-X-proinsulin. On the basis of the above evidence we have designated this peptide preproinsulin.

Immunoprecipitation and tryptic peptide analysis of newly synthesized proteins from rat islets have identified an 18,000 molecular weight (MW) protein as proglucagon. Conversion of this precursor was kinetically similar to the conversion of proinsulin and resulted in the formation of both pancreatic glucagon and a 10,000-MW protein lacking this hormonal sequence.

Electrophoretically homogeneous messenger RNA for rat preproinsulin has been prepared from an islet cell tumor by the use of oligo(dT(-cellulose chromatography and sucrose density gradient centrifugation. The molecular weight of the mRNA is about 210,000, as determined by polyacrylamide gel electrophoresis in formamide, and its sedimentation coefficient is 9.3 S in sucrose gradients containing 0.2 M NaCl. These results indicate that the synthesis of preproinsulin is directed by a monocistronic mRNA 600 nucleotides in length and requires approximately 55% of the molecule's coding capacity.

Publisher Summary The discovery of proinsulin, the biosynthetic precursor of insulin, has stimulated a search for the existence of precursors of other polypeptides. Growing evidence suggests that such forms exist not only for many peptide hormones including, along with insulin, parathyroid hormone, glucagon, and gastrin, but also for structural proteins including collagen, and virus capsule proteins. It is difficult to provide specific methods for the identification and analysis of all polypeptide precursors because of the divergent origin and chemical properties of the possible forms. The approach in each ease must consist of an imaginative synthesis of currently available methods for detecting and separating related proteins. A critical assessment of the precursor–product relationship of any pair of polypeptides requires both biochemical and chemical approaches. The methods used for the determination of this relationship and for the final characterization of the precursor form are largely those traditionally used in the detection and isolation of a variety of proteins and peptides. The final proof for the existence of a precursor polypeptide must rely on several carefully met criteria, each of which may only be suggestive in the absence of the others.