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Home , Cholecystokinin, Dipeptide, Gastrointestinal hormone, Lipotropin, Motilin, Pancreatic polypeptide

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Gut, 1981, 22, 1048-1069

Progress report Multiple forms of gastroenteropancreatic

The isolation and characterisation of many hormones has been facilitated by their secretion from and localisation in specific endocrine glands. Although it is now well established that the gut and produce a large number of polypeptide hormones, these organs form a diffuse system in which different types of endocrine cells are interspersed among a pre- dominance of non-endocrine tissue. This arrangement, while ideal for regulating the of a varied diet as it transverses the length of the , makes it difficult to isolate the tiny quantities of polypeptide hormones, typically present at concentrations of 100 pmol/g wet weight of tissue. In addition, whereas most endocrine glands can be extirpated and the resulting physiological changes observed, the diffuse anatomical location of endocrine cells in the gut precludes this approach. Consequently, the biological effects of some gut hormones remain uncertain. Traditionally, physiological effects have been used to monitor hormones during their purification. The classical gut hormones , , and http://gut.bmj.com/ were characterised after identification of a biological ac- tivity and isolation of the active component by fractionations in which the biological effect was used to assay each fraction. Complications in the interpretation of bioassays can arise, however, when different hormones have a common biological activity. Gastric inhibitory polypeptide, secretin,

and , for example, have -releasing activities and their immu- on September 27, 2021 by guest. Protected copyright. noreactivities can be detected in the same region of the gut. The advent of radioimmunoassay' some 20 years ago heralded an era of realisation that hormones are present in the circulation and in their tissues of origin in a variety of molecular forms. Consequently, it may be difficult to assign hormonal status to just one of these forms. Normally, this would accrue to the most abundant and most active form present in the circulation. Such a definition may be simplistic, however, as in the case with gastrin, where the 17 peptide (G-1 7), although present in lower concentration than the 34 amino acid form (G-34), accounts for most of the postprandial circulating gastrin activity in man because of its greater potency.2 In contrast, G-34 is not released into the circulation in the even though it is detectable in extracts of feline antral mucosa.3 When immunoreactive smaller than the active are detected in the circulation they are usually regarded as degradation products. These forms, however, may also be biologically active as is again illustrated by gastrin; the 14 amino acid form (G-14) is nearly as potent as G-17 but is present in much lower amounts.4 Indeed, the smallest peptide which exhibits gastrin activity is the amide that constitutes the C-terminus of both gastrin and 1048 Gut: first published as 10.1136/gut.22.12.1048 on 1 December 1981. Downloaded from

Multipleforms ofgastroenteropancreatic hormones 1049 cholecystokinin.5 The gastroenterologist is therefore confronted by a com- plex system containing a variety of hormones each probably present in more than one molecular form which may or may not be active and which may or may not be secreted into the circulation. Pro-forms and pre-forms The theme of biosynthesis of precursors of peptide hormones and sub- sequent conversion to smaller active species is common to other . The best early illustrations were provided by digestive such as , chymotrypsin, and which are synthesised as inactive proen- zymes or zymogens to prevent proteolysis occurring in tissues of synthesis. Other zymogens which are activated extracellularly include factors in the complement and blood clotting systems. Activation is normally accom- plished by cleavage of one peptide bond near the N-terminus of the proenzyme. This highly selective snipping is often referred to as limited proteolysis. In the late 1960s the concept of limited proteolysis was extended beyond zymogen activation and is now recognised as an integral event in the biosynthesis of most secreted proteins. The first evidence for the existence of prohormones came through the work of Steiner and his colleagues6 on a possible precursor of insulin. Two extractable proteins became labelled during incubation of tritiated amino acids with slices from a human islet cell adenoma. The larger was converted to material the same size as the smaller one, which, in turn, was indistinguishable from insulin, when subjected to digestion with trypsin. Material similar to the larger protein was detected in small amounts in a preparation of crystalline porcine insulin and, after isolation and determin- ation of its amino acid sequence, was found to be a proinsulin.7 In porcine http://gut.bmj.com/ proinsulin the A and B chains of insulin are joined through a segment of 33 amino acids. Significantly, pairs of basic amino acid residues form the points of attachment making these sites susceptible to proteolysis by trypsin-like enzymes. The discovery of proinsulin stimulated a search for precursors of other polypeptide hormones. The pulse-chase technique was spectacularly useful for detecting proteins which are rapidly converted to smaller forms. Slices of on September 27, 2021 by guest. Protected copyright. tissue in which the hormone is produced are incubated for a short period (the pulse) with radioactively labelled amino acids known to be present in the hormone. This is followed by incubation with unlabelled amino acids or the addition of an inhibitor of protein synthesis (the chase). A highly labelled component which is detectable after the pulse but is replaced during the chase by labelled material of lower molecular weight is indicative of a precursor. In pulse-chase experiments with slices of , a protein of molecular weight 11 500 was replaced quantitatively by para- hormone during the chase. This protein was shown to be propara- thyroid hormone based on tryptic mapping and its reactivity with antisera to .8 A milligram of the precursor was isolated from several thousand bovine glands and its sequence was determined.9 The prohormone has an extra six amino acids linked to the N-terminus of the hormone through basic amino acids. This is analogous to the relationship between proinsulin and insulin. A cell-free system from wheat germ capable of translating crude mRNA preparations in an environment devoid of proteolytic enzymes was used to Gut: first published as 10.1136/gut.22.12.1048 on 1 December 1981. Downloaded from

1050 Irvine and Murphy determine whether proparathyroid hormone was the protein specified by mRNA isolated from parathyroid gland.'0 Microsequencing of the tiny quantities of protein available was indicative of a further extension of 25 amino acids at the N-terminus of proparathyroid hormone. This initial product of translation was termed preproparathyroid hormone. Its exis- tence is in accord with the signal hypothesis proposed by Blobel and Sabatini some years earlier." They suggested that, in proteins destined for export from the cell of synthesis, a hydrophobic area is first synthesised near the N-terminus of the nascent chain. This structure is capable of binding to a receptor located on the membrane of the endoplasmic reticulum, thereby connecting the ribosome with the membrane to create the rough endo- plasmic reticulum. Binding to the receptor is thought to facilitate transport of the nascent polypeptide chain through a pore in the membrane into the cisternal space. (A modified version of the signal hypothesis, in which the polypeptide chain loops across the membrane leaving the extreme N- terminal region of the pre-peptide outside the membrane, has been proposed by Steiner and associates.' 2) During passage across the membrane and before completion of the nascent chain the pre-sequence is cleaved by 'signalase', a membrane-bound which is possibly associated with the signal/receptor-pore complex. The protein, now minus the pre-sequence, passes through the smooth endoplasmic reticulum to the Golgi apparatus where packaging into secretory granules occurs. The list of secreted proteins known to have pre-sequences is growing and now includes insulin, , lactalbumin, ovomucoid, lysozyme, and ovalbumin. The pre- region of the latter protein is presently unique in not forming the N-

terminal portion of the nascent chain and hence not being cleaved.'3 For http://gut.bmj.com/ this reason the term 'signal-sequence' rather than 'pre-sequence' may be more appropriate, as the latter term has structural implications. Pre-sequences Contrasting properties of pre- and pro-regions may be distinguished. Pre- sequences (1) form the N-terminal segment of the polypeptide, with one exception which has just been mentioned; (2) contain a hydrophobic area on September 27, 2021 by guest. Protected copyright. which can bind to a receptor on the membrane of the endoplasmic re- ticulum, thus performing a. definite function; (3) are usually linked to the main chain through an amino acid with a small side chain (Ala, Gly, Ser or Cys). These would be susceptible to cleavage by signalase which appears to be a metalloendopeptidase with a specificity similar to that of thermo- lysin14; (4) do not normally survive the completion of synthesis of the polypeptide, so that preproteins are neither detected in the circulation nor, under most conditions, in tissues where they have but a transitory existence. Pro-regions Pro-regions, on the other hand, (1) may extend the active polypeptide at either the N- or C-terminus, or both, or may be internal as in the case of proinsulin; (2) have no well-defined biological function. Possible roles will be discussed later; (3) are linked to the active protein via a pair of basic amino acid residues, except in the case of promellitin,"5 making them highly susceptible to trypsin-like enzymes; (4) are relatively long lived compared with pre-sequences. After sequestration into the cisternae of the rough endoplasmic reticulum the polypeptide passes in vesicles to the Golgi Gut: first published as 10.1136/gut.22.12.1048 on 1 December 1981. Downloaded from

Multipleforms ofgastroenteropancreatic hormones 1051 apparatus. Processing, probably by membrane bound proteinases, begins here and continues in the maturing secretory granules. Completion of the processing may require a period of hours or even days. A small proportion of molecules may escape proteolysis and be secreted with the active form into the circulation. In some endocrine tumours relatively large amounts of the precursor forms may be secreted. Our interest, then, lies with the prohormones rather than with the preprohormones. Before going on to discuss individual gut hormones we must consider the criteria for identification of a prohormone. The first clue that a prohormone may exist is often the presence of peaks of hormone-like immunoreactivity emerging before the hormone during gel filtration of tissue extracts or serum, especially from patients with tumours. Such peaks may, however, 'be due to polymerisation or binding of the hormone to carrier proteins. These possibilities can be examined by performing the gel filtration under dissociating conditions such as in 8M urea or 4M guanidine hydrochloride. Putative prohormones which survive exhaustive scrutiny may be further investigated by several experimental approaches. These include (1) pulse-chase experiments; (2) incubation of high molecular weight fractions with trypsin and carboxypeptidase B which are known to convert some prohormones to the active form; (3) isolation and chemical characteri- sation of the prohormone; (4) immunocytochemical studies using antibodies raised against the pro-region should locate the prohormone in the same endocrine cells as are responsible for the production of the hormone; (5) determination of the nucleotide sequence of the coding for the hor- mone. This provides final proof of the relationship between prohormone

and hormone. Recent developments in recombinant DNA technology have http://gut.bmj.com/ enabled the structure of the insulin in '6 and man17 to be determined. Individual hormones: molecular species and intrarelationships INSULIN Appropriately proinsulin (Table), having been the first prohormone to be discovered,6 is now the only gastroenteropancreatic hormone for which the on September 27, 2021 by guest. Protected copyright. sequence of the human gene is known.17 Two intervening sequences occur in the human gene. There are two non-allelic genes, I and II, for insulin in the rat.16 Two intervening sequences occur in the rat I gene in positions similar to the locations of those in the human gene. The rat II gene, however, has only one intervening sequence. Bell and associates17 speculate that the ancestral form of the insulin gene had, therefore, two intervening sequences. The function of prohormones and the mechanisms by which they are processed are still poorly understood. Proinsulin has been extensively studied in model systems with enzymes which are candidates for the converting role. Incubation of proinsulin with trypsin and carboxypeptidase B was shown to release insulin.18 Tryptic cleavage after Arg60 in bovine proinsulin forms the A chain but after Arg32 must be followed by removal of the basic amino acids by carboxypeptidase B to liberate the B chain. This model system is capable of converting other prohormones such as pro- glucagon to smaller species.19 Additional model systems which convert proinsulin to insulin or to insulin-like products include cathepsin B,20 Gut: first published as 10.1136/gut.22.12.1048 on 1 December 1981. Downloaded from

1052 Irvine and Murphy

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Multipleforms ofgastroenteropancreatic hormones 1053 kallikrein plus kininase,21 and plasmin.22 Virgi and colleagues23 have recently extended the plasmin model by demonstrating that secretion of plasminogen activator by rat islets is increased by and that this effect is abolished by alloxan, a diabetogenic agent. An alternative approach to using selected candidate enzymes has been to isolate proinsulin- converting activity from f, cells. Such an enzyme has been purified from a side fraction obtained during the purification of bovine insulin24 but has not yet been characterised. Likewise, proinsulin-converting activity from a secretion granular and mitochondrial fraction from islet cells was shown not to contain trypsin but to be a serine proteinase.25 Sun and colleagues26 located proinsulin-converting activity in the membrane fraction of lysed granules prepared from rat islets. Only endogenous proinsulin was con- verted in whole granule preparations, whereas the granule membrane frac- tion was capable of processing exogenous proinsulin. The activity thus appears to reside within, and be bound to, a membrane. The synthesis of insulin in tissues other than the pancreas is deemed probable by Rosenzweig et al.,27 as they could detect the hormone in tissues and in cultured lymphocytes and fibroblasts in concentrations considerably greater than the levels in plasma or culture media. This finding is repre- sentative of reports of gastroenteropancreatic hormones in many other organs, especially the . GLUCAGON The 29 amino acid sequence of glucagon, identical in most mammals, has been known for more than 20 years.28 The development of a radio- immunoassay for glucagon led to the discovery of glucagon-like immunoreactivity (GLI) in gut extracts29 and subsequently numerous http://gut.bmj.com/ species of GLI have been reported in tissue extracts and in the circulation. Antibodies raised against glucagon are of two types: one binds the C-terminal region of glucagon, while the other binds a region in the N- terminal to central portion of the hormone.30 Almost all the GLI extract- able from the gut binds only to the latter type of antibody.31 GLI, purified from porcine ileum32 and colon33 by immunoaffinity chromatography on N-terminal specific antibodies immobilised on agarose, consists mainly of on September 27, 2021 by guest. Protected copyright. material having a molecular weight of 12 000, termed large GLI. This material is electrophoretically heterogeneous and apparently biologically inert. Minor fractions of molecular weight 8000, 5000, and 3000 have also been encountered.33 The concept that extrapancreatic forms of GLI are mainly due to pro- glucagon, in cells which do not possess the enzymic mechanismh to generate biologically active species,34 is supported by the convergence of evidence obtained with gut as well as pancreatic materials. Tager and Steiner35 detected numerous immunoreactive peptides, with molecular weights rang- ing from 3700 to 9000, as contaminants of crystalline glucagon. One of these peptides, of molecular weight 4500, was purified and found to be glucagon extended at the C-terminus by an octapeptide. Flanagan et al.31 found that large gut GLI contains, in addition to the region which reacts with N-terminal specific antibodies, a structure which is homologous with the C-terminal region of glucagon. While the C-terminal structure is buried in native GLI this region was uncovered when the large GLI was subjected to limited proteolysis with serum proteinases. This result, indicative of a Gut: first published as 10.1136/gut.22.12.1048 on 1 December 1981. Downloaded from

1054 Irvine and AMurphy possible precursor role for large GLI, was later confirmed by degradative studies using solid-phase trypsin.36 Purification and partial sequence de- termination of a large gut GLI (Table) provided chemical evidence for its relationship to glucagon.37 This material was named glicentin, being a GLI containing 100 amino acids, and was found to have an octapeptide exten- sion similar to the pancreatic GLI purified by Tager and Steiner. Furthermore, peptide maps of chymotryptic digests of radiolabelled gluca- gon and radiolabelled glicentin suggest the presence of two similar peptides in both materials.38 Digestion of glicentin with trypsin and carboxypep- tidase B, enzymes capable of releasing insulin from proinsulin,18 gave rise to peptides which resemble fragments 1-17 and 19-29 of glucagon.9 Sequence studies have already shown that the C-terminal octapeptide of glicentin is connected to the glucagon sequence through a Lys-Arg sequence.38 The results obtained by enzymic digestion39 make it likely that a pair of basic residues also connect the N-terminal area of glicentin to the glucagon sequence. GLIs, reactive with N-terminal specific antisera, have also been detected in rabbit pancreatic and gut extracts.40 These peptides, of molecu- lar weight 8000 and 12 000, can be converted to material reactive towards C-terminal specific antibodies by digestion with trypsin and carboxypep- tidase B. An additional peptide of molecular weight 9000 which reacts with both types of antisera was detected only in pancreatic extracts. Tager and Markese40 conclude that any extension at the C-terminus of glucagon precludes reactivity towards C-terminal specific antisera and propose that in pancreatic tissue a number of intermediates of varying sizes and with or without the C-terminal octapeptide result from limited proteolysis at precursor. different sites within the initial http://gut.bmj.com/ Immunohistochemical studies, carried out using antisera specific for the C-terminal and N-terminal regions of glucagon and an antiserum (R64) raised against glicentin and unreactive with glucagon, have provided ad- ditional evidence of the relationship between glucagon and glicentin. Thus, R64 stains the same pancreatic A cells as does C-terminal specific anti- serum.4' The glucagon and glicentin stained regions of thea granules are, however, segregated.42 Glucagon and glicentin were stained respectively in on September 27, 2021 by guest. Protected copyright. the dense core and the peripheral mantle. L cells in the are stained by R64 and by N-terminal specific antiserum but not by C-terminal specific antiserum. After incubation of the cells with trypsin and carboxypeptidase B, however, the L cells are also stained by the latter antiserum. If GLI from the gut is a product of the same gene that codes for glucagon in the A cell then the enzymic mechanism responsible for glucagon production within the A cells must be lacking in the L cells. The concept that primitive cells, such as tumour cells, lack the converting enzymes present in more specialised cells has been discussed previously. 34 From the results obtained during recent pulse-chase experiments on rat islet tissue the initial would appear to be a protein of molecular weight 18 000.44 A post-translational modification, which is not glycosyl- ation yet results in an apparent, albeit slight, increase in molecular weight, follows during 10-30 minutes. Then, during the next three hours a protein of molecular weight 10 000, lacking the glucagon sequence, is formed possibly via an intermediate of molecular weight 13 000. During this period peptides of molecular weight 4500 and 3500 accumulate, the latter one Gut: first published as 10.1136/gut.22.12.1048 on 1 December 1981. Downloaded from

Multipleforms ofgastroenteropancreatic hormones 1055 probably being glucagon. A recent report is suggestive of the biosynthesis of anglerfish glucagon as a preprohormone containing a signal sequence of molecular weight 2000.45 GLI has recently joined the growing list of peptides detected in both gut and brain.46 Immunoreactivity is localised in nerve fibres located in the and the thalamus.47 Somatostatin is a 14 amino acid peptide originally isolated from ovine hy- pothalami.48 Several, sometimes conflicting, reports of the molecular species of somatostatin in plasma and tissue extracts have appeared in recent years. Unger and his associates, investigating the forms of somatos- tatin present in the plasma of dog49 and man,50 found that, under denatur- ing conditions, all the somatostatin-like immunoreactivity (SLI) was indistinguishable from synthetic somatostatin. Kronheim et al.51 reported similar results for one group (92%) of subjects. A second group (8%), however, in which the basal levels of SLI were higher than in the first group-1i0+00041 ng/ml as against 0.274+0.009 ng/ml-had two extra peaks of SLI of larger molecular weight than somatostatin. These were stable in 6M urea. Large molecular weight forms are more frequently encountered in extracts of tissue than in plasma. Arimura and associates52 first reported multiple forms of somatostatin in extracts of rat and pancreas. Subsequently, using techniques similar to those applied during the studies with plasma, Unger and co-workers have found that extracts of canine pancreas,53 stomach, and brain54 all contain three species of SLI. Much of the largest form (molecular weight 12 000) could be converted by dithiothreitol to material the same size as somatostatin. A second form http://gut.bmj.com/ (molecular weight 3000-5000) was reduced in size only by proteolysis with trypsin. The authors speculate that the SLI of molecular weight 12 000 may represent a precursor that is processed by cleavage of peptide bonds and a disulphide bridge. The order of cleavage could then lead to the various intermediates detected. Large forms of SLI with molecular weights ranging from less than 5000 to 25 000 have been detected in the brain of ro- dents55 56 57 and sheep.58 Dupont and Alvardo-Urbina,59 however, found on September 27, 2021 by guest. Protected copyright. that high molecular weight SLI from rat pancreas could be converted to material the same size as somatostatin by treatment with 8M urea. Using pulse-chase techniques with anglerfish islets Noe et al.60 detected accumulation of labelled material the size of somatostatin during a chase in the presence of cycloheximide. A concomitant diminution of labelled ma- terial of molecular weight 8000-15 000 occurred. This material, though stable in denaturing agents, was converted to SLI the size of somatostatin by incubation with trypsin. Using similar techniques with rat islet tissue Steiner and coworkers6l were able to detect tryptic conversion of a putative prosomatostatin (molecular weight 12 500) to somatostatin. During the isolation of somatostatin from porcine hypothalami Schally et al.62 detected SLI slightly larger and more basic than somatostatin and speculated that it might be a precursor. A probable prohormone for somatostatin has recently been isolated and its sequence determined.63 This peptide (Table) is comprised of somatostatin extended at the N-terminus by 14 amino acids. The extension is linked through a pair of basic amino acids, Arg-Lys, a feature shared by other prohormones. The sequence of a Gut: first published as 10.1136/gut.22.12.1048 on 1 December 1981. Downloaded from

1056 Irvine and Murphy somatostatin-like immunoreactive peptide from pancreatic tissue of catfish has been reported.64 This 22 amino acid peptide is homologous with somatostatin in its 14 C-terminal residues, including the cystine ring struc- ture. The extra N-terminal octapeptide is not, however, linked through basic amino acids, possibly accounting for the fact that it is not processed further. Goodman and coworkers, investigating the products of translation in a cell-free system of mRNA from islets of anglerfish, have detected prepro- somatostatins65 as well as preproglucagon.45 Two proteins (molecular weight 16 000 and 14 000) were found to be immunoprecipitable by anti- serum to somatostatin, suggestive of the existence of two separate genes.

PANCREATIC POLYPEPTIDE (PP) is a 36 amino acid peptide (Table, bovine peptide) discovered as a contaminant of partially purified insulin.66 Homologous mammalian peptides have been characterised but no phy- siological role has yet been ascribed to pancreatic polypeptide. Large increases in pancreatic polypeptide levels, however, have been detected in patients with-endocrine tumours of the pancreas. As much as 50% of this immunoreactive material may be due to several large molecular forms.67 Normal human plasma contains large PP-like immunoreactivity which is unlike pancreatic polypeptide in neither being stimulated by insulin- induced hypoglycaemia68 nor being removed by immunoabsorption.67 Consequently, it is likely to be an artefact similar to large gastrin-like immunoreactivity. PP-rich and glucagon-rich islets are found in the embryologically derived ventral and dorsal regions of the pancreas respectively.68 Slab gel elec- http://gut.bmj.com/ trophoresis in sodium dodecyl sulphate of pulse-chase labelled islets has revealed material, of molecular weight 18 000, similar to the glucagon prohormone. Intermediate-sized bands formed during processing of pro- glucagon are, however, absent. It has been suggested, therefore, that pan- creatic polypeptide and glucagon may be derived from the same precursor in different pancreatic cells.70 Precedents for a precursor common to more on September 27, 2021 by guest. Protected copyright. than one hormone exist in the -neurophysin71 and the ACTH- endorphin prohormones.72 The latter precursor is converted to ACTH, ,B- LPH, and ,B-endorphin in the but to a-MSH, CLIP, and B-endorphin in the intermediate lobe.72 CHOLECYS1TOKININ (CCK) Two forms of cholecystokinin, isolated from pig intestine, have been char- acterised as peptides containing 33 amino acids (CCK-33) and 39 amino acids (CCK-39) (Table). The latter peptide is CCK-33 extended at the N- terminus.73 Surprisingly, CCK-33 is produced by cleavage between, rather than after, the pair of basic residues Arg6-Lys7 of CCK-39. These two peptides account for most of the CCK-like immunoreactivity extractable from pig intestine by acid.74 Dockray74 found, however, that boiling water extracts of this tissue contain smaller immunoreactive peptides CCK-8 and a peptide intermediate in size between CCK-8 and CCK-33. CCK-8 has about three times and 12 times the potency of CCK-33 in stimulating gall bladder contraction and acid secretion respectively.2 The N-terminal por- tion of cholecystokinin thus decreases its activity on parietal cells more Gut: first published as 10.1136/gut.22.12.1048 on 1 December 1981. Downloaded from

Multipleforms ofgastroenteropancreatic hormones 1057 strongly than on gall bladder. CCK-8 is the smallest peptide isolated which retains the classical cholecystokinin effects of gall bladder contraction and induction of pancreatic enzyme secretion. A sulphate at position 7 from the C-terminus is essential. The C-terminal tetrapeptide alone has gastrin-like activity.5 Interest in this long-established gut hormone has been stimulated by the recent discovery of CCK-like immunoreactivity in the brain.75 Initially Vanderhaeghen et al.76 reported gastrin-like immunoreactivity in brain extracts. Using antisera of different regional specificities raised against gastrin and cholecystokinin, however, it was shown that these brain pep- tides are more closely related to cholecystokinin than to gastrin.75 The work of Rehfeld77 particularly exemplifies the correct approach for differen- tiation of molecular species by using four antisera, three containing anti- bodies specific for three different regions of cholecystokinin and the fourth reactive with gastrin-17 but not cholecystokinin. Three fractions, CCK-33 (5%), CCK-8 (80%) and CCK-4 in human and porcine brain were charac- terised. Pulse-chase experiments in the cerebral cortex78 and subcortical regions79 of rat are indicative of an immunoreactive precursor (molecular weight 10 000) which is converted to CCK-8. Conflicting results have been reported by Straus and Yalow,80 who found high levels of both CCK-33 and CCK-8 in brain and gut and attribute the failure of other workers to detect higher levels of CCK-33 in brain to the presence of two CCK- converting enzymes in brain which rapidly degrade CCK-33. These enzymes have trypsin-like specificities: one enzyme which is eluted at the void volume of Sephadex G-75 cleaves CCK-33 between Arg21-11e22 producing CCK-

12; proteolytic activity at the void volume of Sephadex G-50, however, also http://gut.bmj.com/ cleaves CCK-33 between Arg25-Asp26 to give both CCK-12 and CCK-8, so that a second enzyme is apparently present.80 The processing of CCK-39 is rather unusual in that cleavage points giving CCK-33, CCK-12, and CCK-8 all occur after a single basic amino acid rather than after a pair of basic residues so frequently encountered in other prohormones. This feature is not unique, however, as chicken proalbumin is cleaved after a single residue.82 The nature of the processing site may not be universally on September 27, 2021 by guest. Protected copyright. more important than the specificity of the converting enzyme in directing the cleavages essential for bioactivation. The CCK-converting enzymes in brain have a high specificity for cholecystokinin and do not cleave gastrin- 34 to gastrin-17.83 The CCK-12 producing enzyme cleaves the synthetic Arg-Ile, while the enzymes producing CCK-12 and CCK-8 cleave both Arg-Ile and Arg-Asp.84 Production of CCK-4 from larger forms requires another unusual processing event-namely, cleavage of a Gly-Trp bond. This must be mediated by an enzyme unlike trypsin in specificity. A parallel may be drawn with the conversion of promellitin to mellitin, found in honeybee venom. The scissile bond in this instance lies between alanine and residues.85 Recently cholecystokinin has been detected in porcine pancreas.86 The molecular species present are similar to those found in gut, except that CCK-4 is the most abundant form and the most biologically active in causing the release of insulin and other islet hormones from the pancreas. The immunoreactivity was located in nerve terminals. The existence of CCK-4 has been called in question by Dockray and Gregory,87 who were Gut: first published as 10.1136/gut.22.12.1048 on 1 December 1981. Downloaded from

1058 Irvine and Murphy

unable to detect it in extracts of pancreas or antral mucosa using an antiserum raised against CCK-4. CCK-39 may be solely an intermediate in the biosynthetic pathway or may have intrinsic activity. The stimulation of gall bladder contraction by CCK-33 released from the is clearly an endocrine function, whereas CCK-891 and CCK-4 may have paracrine or neurocrine effects in brain and pancreatic tissue respectively. GASTRIN A sulphated tyrosine residue, essential for the activity of cholecystokinin, has no effect on gastrin activity. All the molecular species of gastrin yet isolated exist in both forms. The major species found in the antral mucosa are the heptadecapeptide, G-17, sometimes referred to as little gastrin, and the N-terminally extended form G-34 or big gastrin, which is present in smaller amounts. The bigger form is the major constituent of the much lower quantities of gastrin in the duodenum88 89 but duodenal G-cells differ morphologically from antral G-cells.90 In the circulation of man and dog G-34 predominates because of its longer half life.92 It is, however, less potent than G-17, which is regarded as the active hormone. G-34, the precursor of G-17, is itself produced from a larger precursor. Dockray and coworkers used an antibody specific for the N-terminal region to identify and localise this peptide.93 Small amounts of an immunoreactive fraction are eluted between the void volume and G-34 after gel filtration of antral extracts.92 94 Even larger material has been detected in extracts of a ,94 although large material of similar size in normal plasma95 may be artefactual as it is not removed by immunoadsorption.94 Small amounts of a 'mini gastrin', G-14, have been isolated from a gastrinoma.96 http://gut.bmj.com/ G-14 is also present in normal plasma in small amounts, probably formed by degradation of G-17.92 Immunocytochemical studies on gastrointestinal tissue of monkey and pig have recently revealed a type of cell which reacts with antibodies specific for the tetrapeptide amide common to gastrin and CCK but does not react with antibodies specific for other regions ofthese hormones.97 These cells are most on September 27, 2021 by guest. Protected copyright. numerous in the distal ileum and in the Bruner gland area. Noyes et al.98 isolated mRNA for gastrin using an oligonucleotide probe deduced from a sequence in gastrin. Their elegant approach has enabled them to predict that the gastrin prohormone contains 110-140 amino acids, that it is extended at both ends of G-34, and that the extension at the N- terminal end of G-34 is linked through the sequence -His-Arg-Arg- (Table). A C-terminal extension to gastrin was not unexpected, as Bradbury et al.99 proposed that C-terminal amides are formed by transamidation with simul- taneous cleavage of a peptide bond. A precursor to mellitin has been isolated in which the C-terminal sequence is Gln-Gln-Gly rather than the Gln-Gln-NH2 sequence of mellitin.84 By characterisation of rat mRNA, Amara et al.100 have shown that the C-terminal residue, which is amidated in the active hormone, is extended through a glycine residue in the prohormone. The structure of procalcitonin and promellitin are thus consistent with a requirement for an additional amino acid residue, probably limited to glycine, at the C-terminus of the prohormone to facilitate amidation. C-terminally-extended have now been detected in serum from a patient with a gastrinoma and in some antral extracts from Gut: first published as 10.1136/gut.22.12.1048 on 1 December 1981. Downloaded from

Multipleforms ofgastroenteropancreatic hormones 1059 pig using two antisera, one specific for the N-terminal and the other for the C-terminal region of G-17.101 The C-terminally-extended gastrins do not react with antisera specific for the C-terminal region of G-17, a parallel situation to that of glucagon and C-terminally-extended GLI.

SECRETIN Immunoreactive forms larger than secretin (Table) were not detected in extracts of intestinal mucosa from pig, dog, rat, monkey, rabbit, and guinea-pig.102 In contrast, Boden et al.103 found 50-66% of secretin-like immunoreactivity to emerge between the 21 000 and 6000 molecular weight- markers after gel filtration on Sephadex G-50. A further 10% was eluted at the void volume and the remainder with radiolabelled secretin. In our laboratories, secretin-like immunoreactivity from porcine duodenum has been purified by immunoaffinity chromatography followed by gel filtration on Sephadex G-50.104 It was resolved into four fractions with molecular weight less than 2000 (5%), 3500 (20%), 6000 (70%), and 12 000 (5%). This pattern was unaltered in 2M guanidine hydrochloride or after treatment with 0.1% sodium dodecyl sulphate. Similar techniques have also been applied to the characterisation of molecular species present in the plasma of fed and fasted subjects.'05 In fed subjects and those who had fasted for 12 hours most ofthe immunoreactivity was smaller than secretin but 10-20% had a molecular weight ofabout 12 000. After a 36 hour fast, plasma levels immunoreactivity had risen sixfold. More than 90% ofthis immunoreactive material was smaller than secretin and may be due to degradation products.

A peptide with secretin-like biological activity has been isolated from http://gut.bmj.com/ porcine brain.'06 The purified peptide is probably secretin, based on its behaviour in several analytical systems.

GASTRIC INHIBITORY POLYPEPTIDE (GIP) GIP (Table), originally given as an acronym for gastric inhibitory polypep-

tide, can also stand for glucose-dependent insulinotrophic polypeptide, on September 27, 2021 by guest. Protected copyright. another biological activity of the hormone.107 GIP is released into the circulation after ingestion of glucose or . Peak levels of GIP are attained within 45 minutes after ingestion of glucose but not until two to three hours after ingestion of fat. Brown et al.'08 noted that the rapid GIP response to glucose would be consistent with one function of the peptide, regulation of insulin secretion, while the delayed response to fat would come at a time more suitable for inhibition of release. They suggested that different GIP-like immunoreactivities might be released in response to either glucose or fat. Three peaks of immunoreactivity were resolved by gel filtration of serum samples, taken at intervals after ingestion offat or glucose. One peak was the same size as GIP, while the largest peak was reduced in size by treatment with 6M urea and is probably an aggregate ofGIP and serum proteins. 107 Material in the third peak (molecular weight 7500-8000) may, however, be a precursor. The ingestion of fat or glucose resulted in similar elution profiles after gel filtration. In a recent study'09 Sarson et al. also reported two major forms of GIP in the circulation. Gut: first published as 10.1136/gut.22.12.1048 on 1 December 1981. Downloaded from

1060 Irvine and Murphy

VASOACTIVE INTESTINAL POLYPEPTIDE (VIP) VIP, a 28 amino acid member of the secretin/glucagon family (Table), is distributed widely in the body, particularly in the gut, brain, and geni- tourinary tract. Its localisation in neurons may indicate that VIP functions as a ."10 VIP-like immunoreactivity, partially purified from various tissues from a number of species, co-elutes with porcine VIP on gel filtration."10 Bryant et al.,1"' however, reported the occurrence of a larger molecular form of VIP accounting for 6% of the total immunoreactivity extractable from porcine brain. Immunoreactive peptides more acidic than VIP have been detected in human colonic mucosal"2 and in brain and gut of dog, rat, and pig."3 114 Although most of these peptides were of comparable size to VIP one"12 was smaller and may be a degradation product. Extracts of , , and brain contain enzymic activity that is highly specific for degradation.of VIP."5 Using antibodies specific for different regions of VIP, Dimaline et al.1"6 have shown that VIP and the more acidic species react equally with N-terminal-specific antisera but that the acidic forms react less well with mid- and C-terminal-specific antisera. Recently Yamaguchi and coworkers'17 reported that a macromolecular VIP with molecular weight about 15 000 comprises 5-17.5% of the total VIP immunoreactivity in extracts of tumours from patients with watery diarrhoea-hypokalaemia-achlorhydria syndrome, but immunological char- acterisation with antibodies specific for different regions of VIP was not sufficient to demonstrate the presence of the complete VIP structure. Fahrenkrugl"8 has reported that studies on the incorporation of [35S] into rat cerebral VIP are indicative of a precursor of large

molecular weight. Bodansky et al.' 19 have suggested that VIP itself may be http://gut.bmj.com/ a prohormone as it contains two pairs of basic amino acids. Glucagon, GIP, pancreatic polypeptide, and , however, each contain a pair of basic residues but these remain intact in the hormone. Prohormone status for VIP would require the detection of smaller, more active peptides derived from VIP.

MOTILIN on September 27, 2021 by guest. Protected copyright. The 22 amino acid peptide (Table) was isolated from porcine intestine as a component causing an increase in motor activity in canine stomach.120 Antisera raised against motilin are of two types reacting specifically with either the N-terminal or the C-terminal region. Although motilin is detected by both antibodies, a larger immunoreactive component present in human plasma is detected only poorly by the C-terminal-specific antisera.'2' This might be motilin extended at the C-terminus. The use of antibodies having different regional specificities is the probable cause of reports of two types of motilin-containing cells, 'argentaffin enterochromaf- fin' and 'non-argentaffin enterochromaffin' cells, stained by immunocyto- chemical techniques. C-terminal specific antisera appear to detect only the latter type of cells. 122

BOMBESIN , a 14 amino acid peptide (Table) isolated from frog skin'23 has gastrin-releasing and CCK-like activities in mammals.'24 Antibodies raised against bombesin react with material present in gut and brain of mammals. Bombesin-like immunoreactivity has been purified from ovine hypo- Gut: first published as 10.1136/gut.22.12.1048 on 1 December 1981. Downloaded from

Multipleforms ofgastroenteropancreatic hormones 1061

thalamus'25 and shown to be a peptide of about 32 amino acids. On treatment with trypsin, this peptide is converted to material equal in size to bombesin. A bombesin-like immunoreactive peptide detected in porcine non-antral gastric tissue has similar biological properties to bombesin. It has been purified by following the gastrin-releasing activity which co- purified with gall-bladder contracting activity and immunoreactivity. The peptide (Table) has 27 amino acids and nine of the residues of its C-terminal decapeptide are identical with those in the corresponding sequence of bombesin. 126 Recently, Brown and coworkers127 demonstrated that the gastrin releasing peptide had other similarities to bombesin in lowering body temperature, increasing plasma levels of noradrenaline and glucose and in producing stereotypic scratching behaviour in . The porcine peptide, however, was less potent than bombesin. Bombesin-like peptides in the brain were reported as probably not identical with the peptide from the gut. Walsh and associates128 also report that the bombesin-like immunoreactivity from hypothalamus differs from that in the fundus. The material from the latter tissue was separated into two main peaks by gel-filtration. The smaller could be resolved into two further fractions by ion exchange chromatography.

Precursor role, regulation, and relevance

ROLE Whereas a clearly defined role has been ascribed to pre-regions in the signal hypothesis, a unified and universally applicable corollary for the function of http://gut.bmj.com/ pro-regions in prohormones cannot yet be expounded. It is unlikely that the prohormones are synthesised as biologically inactive molecules in order to prevent activity being expressed in the wrong location, which is the bio- logical rationale for the existence of zymogens of proteinases. Prohormones may in fact exhibit a considerably higher relative activity than zymogens. Possible roles for prohormones may be summarised as follows:

1. Pro-regions may be extensions which are required for proteins destined on September 27, 2021 by guest. Protected copyright. for secretion. Steiner and associates'2 suggest that the minimum length of a nascent polypeptide chain which can cross the membrane of the rough endoplasmic reticulum is 65-70 amino acids. These authors adduce a comparison of insulin and insulin-like as evidence. The latter protein has a shortened C area but this is counterbalanced by an extended A chain, conservation of size being the crucial requirement. One objection to this hypothesis is the existence of pro-regions in proteins, such as albumin, which are much larger than 70 amino acids. 2. The pro-sequence may direct the assembly of the peptide into the correct conformation. The C peptide, for example, is important for align- ment of the cysteine residues in proinsulin for disulphide bond formation. Structural requirements, however, must be less critical for the synthesis of smaller peptide hormones. 3. The pro-region may be the modulator of intracellular transport, ensuring passage to the Golgi apparatus and effecting encapsulation into secretory granules. 4. Prohormones or pro-regions may themselves have an endocrine func- tion or may be activated in the circulation by proteinases. This depends on Gut: first published as 10.1136/gut.22.12.1048 on 1 December 1981. Downloaded from

1062 Irvine and Murphy the molecular species stored in cells and on the forms released. Gastrin activity is exhibited by G-34 as well as by G-17 and conversion of G-34 to the more active species occurs in plasma. Again glicentin may have a physiological effect different from that of glucagon, while CCK-33 has a definite activity distinct from CCK-4 (see glucagon and cholecystokinin). 5. Hales129 has proposed that polypeptide hormones and secreted pro- teins have evolved as a result of lysosomal proteolysis and exocytosis. He emphasises similarities between the structures, enzymic activities, and behaviour of secretory granules and lysosomes. The pro-sequence, which no longer has a specific function, is regarded as a consequence of this evo- lutionary relationship. REGULATION The most striking feature of prohormone structure is the presence of a pair of basic residues immediately adjacent to the site of cleavage. A mechanism for enzymic cleavage by a trypsin-like enzyme (followed by removal of the C-terminal basic residues by carboxypeptidase B if the pro-region extends the C-terminal end of the hormone) has been proposed.130 Each hormone might be produced by a converting enzyme specific for one prohormone. Alternatively, a few enzymes may be responsible for the conversion of all precursors due to similarities in structure around the processed sites. Current evidence favours the latter scheme. The ability of several enzymes to convert proinsulin (see insulin) suggests that the scissile bonds are uniquely vulner- able. Pairs of basic residues occur within hormones such as glucagon, VIP, and GIP yet these sites are not cleaved. Geisow13' has proposed that these

unprocessed sequences occur in a-helical regions where the rigid confor- http://gut.bmj.com/ mation of the polypeptide chain represents a poor substrate. Processed sites, on the other hand, are present in areas without secondary structure or close to ,B turns. An increase in the secondary structure off,-lipoprotein has been shown to alter the specificity of trypsin towards this substrate.132 While the Arg5 1-Trp52 and Arg60-Trp61 bonds are cleaved in aqueous solution, after addition of trifluoroethanol, a reagent which increases secondary structure, only the Arg60-Trp61 bond is split. The Arg51-Trp52 bond lies in a region on September 27, 2021 by guest. Protected copyright. of high ax-helical potential. Burbach et al.'33 have found that ,B-endorphin is processed in different ways by synaptosomal plasma membranes from brain at pH 5.9 and 6.7. Differential processing may be either a reflection of changes in the secondary structure of the protein or a consequence of altered enzyme specificity. Loh and Gainer134 have shown that glycosyl- ation may be structurally important for regulation of limited proteolysis. Inhibition of glycosylation of the ACTH-fl- precursor results in its rapid degradation to peptides atypical of normal degradation. The nature of the converting enzymes is still uncertain. Enzymes, includ- ing trypsin plus carboxypeptidase B, cathepsin B, plasmin, and kallikrein plus kininase, capable of converting proinsulin have already been reviewed. Trypsin plus carboxypeptidase B have also been shown to convert glicentin to glucagon fragments. Cathepsin B has been shown to convert proalbumin to albumin.'35 An interesting aspect of this system is its stimulation by Ca2 + ion-dependent vesicle fusion. The enzyme and prohormone thus appear to be segregated in separate vesicles and only after fusion can the contents be secreted. The role of cathepsin B as a major converting enzyme is not supported by the discovery that glucagon is a very good substrate for Gut: first published as 10.1136/gut.22.12.1048 on 1 December 1981. Downloaded from

Multipleforms ofgastroenteropancreatic hormones 1063 this enzyme.'36 Newly formed glucagon may, however, be protected from further proteolysis by formation of oligomers in the secretory granule. Three growth factor-processing enzymes have been tested for kallikrein activity and their immunological relationship with submaxillary gland kallikrein examined.'37 y subunit, -binding protein, and fl-nerve growth factor endopeptidase all have kallikrein-like activity and have structural similarities. fl-nerve growth factor -endopeptidase appears to be identical to submaxillary gland kalli- krein. Bothwell et al.'37 speculate that these three enzymes are members of a closely related family of precursor-processing enzymes. The localisation of proinsulin-converting enzymes in a membrane bound fraction of secretory granules from ,B cells has been discussed (see insulin). It is reasonable to expect that in vivo all the enzyme activities involved in prohormone acti- vation will be located in similar subcellular fractions. In the case of hormones which have an amino acid amide at the C-terminus there must exist a mechanism for converting the extended C-terminal region by pro- teolysis and transamidation (see gastrin). RELEVANCE The almost universal dependence on radioimmunoassays for detecting the multiple forms of gut hormones found in the circulation is often associated with controversy and erroneous conclusions. This is because assay anti- bodies detect only a comparatively small region, about six residues, of any polypeptide. A single antiserum, however, is commonly used for radio- immunoassay in clinical laboratories and this may not discriminate between precursors, active hormones, and fragments in which the immunogenic site is intact. Alternatively immunogenic sites on the active hormone may be http://gut.bmj.com/ buried in circulating precursors and inaccessible to the assay antibody, thus remaining undetected unless the precursors are subjected to limited pro- teolysis.138 Ideally, a series of antibodies specific for overlapping sequences in each polypeptide should be used for simultaneous assay. It would be premature, if not impossible, at this stage to review the

significance of molecular variation of GEP hormones in pathogenesis, on September 27, 2021 by guest. Protected copyright. diagnosis, and patient management because of the paucity'39 140 of relevant clinical studies. Exceptions include patients with hormone-secreting tumours where increased circulatory levels of prohormones are to be expected as in insulinoma patients14' and the Zollinger-Ellison syndrome. Diagnostic difficulties can arise because the peptide patterns vary as in patients with gastrinomas142 and also in patients with glucago- nomas. 143-146 Most initial progress can still be expected with the gastrin, cholecystokinin, and glucagon families of peptides because a wider range of antibodies is available internationally for these than for the other GEP hormones. The range is still increasing: Yanaihara and associates have recently made two further useful contributions in raising antibodies against a synthetic region (1-15) of human gastrin 34147 and against synthetic C- terminal sequences of glicentin.'48 Future progress is also likely to be accelerated by combining high pressure liquid chromatography with immunoassays to study GEP hormones in tissues and the circulation in the light of considerable recent success with endorphins149 and neurotensins.150 The full complexity of the gastroenteropancreatic hormones has yet to be revealed. Tatemoto and Mutt's' describe two more candidate hormones Gut: first published as 10.1136/gut.22.12.1048 on 1 December 1981. Downloaded from

1064 Irvine and Murphy PHI and PYY (Table) which are structurally related to the secretin/glucagon family and pancreatic polypeptide respectively. These peptides will also, no doubt, be supplemented by their prohormones. This contribution may provide some clinical workers with an interim com- pendium of the multiple forms so far discovered.

G B IRVINE AND R F MURPHY Department ofBiochemistry The Queen's University ofBelfast, Belfast BT9 7BL, N. Ireland and Department ofSurgery, University ofCincinnati Medical Center, Cincinnati, Ohio 45267 USA Received for publication 6 January 1981

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