PHYSIOLOGICAL REVIEWS
`Vol. 78, No. 4, October 1998
`Printed in U.S.A.
`
`The New Biology of Gastrointestinal Hormones
`
`JENS F. REHFELD
`
`Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
`
`I. Introduction
`II. The Classic Concept
`III. The New Concepts
`A. Many hormones and their families
`B. Multiple phenotypes of a hormone gene
`C. Widespread gene expression
`D. Cell-specic prohormone processing
`E. Cell-specic peptide release
`IV. Perspective
`
`1087
`1088
`1089
`1089
`1092
`1097
`1099
`1099
`1101
`
`Rehfeld, Jens F. The New Biology of Gastrointestinal Hormones. Physiol. Rev. 78: 1087  1108, 1998. © The classic
`concept of gastrointestinal endocrinology is that of a few peptides released to the circulation from endocrine cells,
`which are interspersed among other mucosal cells in the upper gastrointestinal tract. Today more than 30 peptide
`hormone genes are known to be expressed throughout the digestive tract, which makes the gut the largest endocrine
`organ in the body. Moreover, development in cell and molecular biology now makes it feasible to describe a new
`biology for gastrointestinal hormones based on ve characteristics. 1) The structural homology groups the hormones
`into families, each of which is assumed to originate from a common ancestral gene. 2) The individual hormone
`gene is often expressed in multiple bioactive peptides due to tandem genes encoding different hormonal peptides,
`alternative splicing of the primary transcript, or differentiated processing of the primary translation product. By
`these mechanisms, more than 100 different hormonally active peptides are produced in the gastrointestinal tract.
`3) In addition, gut hormone genes are widely expressed, also outside the gut. Some are expressed only in neuroendo-
`crine cells, whereas others are expressed in a multitude of different cells, including cancer cells. 4) The different
`cell types often express different products of the same gene, ``cell-specic expression.'' 5) Finally, gastrointestinal
`hormone-producing cells release the peptides in different ways, so the same peptide may act as an acute blood-
`borne hormone, as a local growth factor, as a neurotransmitter, and as a fertility factor. The new biology suggests
`that gastrointestinal hormones should be conceived as intercellular messengers of general physiological impact
`rather than as local regulators of the upper digestive tract.
`
`I. INTRODUCTION
`
`Gastrointestinal hormones are by all criteria ordinary
`hormones. Nevertheless, they have never been fully ac-
`cepted in endocrinology. Endocrinology textbooks and
`general endocrinology meetings witness how gastrointes-
`tinal hormones often occupy only little room in today's
`concept of endocrinology. The restrained acceptance is a
`paradox in three ways. 1) Endocrinology was conceptu-
`ally put on a rm scientic footing in the gut with the
`discovery of secretin (12, 13) and with the subsequent
`introduction of the word hormone (227). 2) The gut is
`the largest hormone-producing organ in the body, both in
`terms of number of endocrine cells and number of hor-
`mones (for comprehensive reviews, see Refs. 211 and
`251). 3) The widespread expression of gastrointestinal
`hormone genes outside the gastrointestinal tract makes
`
`the hormones multifunctional regulators of general physi-
`ological interest. Hence, gastrointestinal hormones may
`at the same time act as acute metabolic hormones, as
`neurotransmitters, as local growth factors, and as fertility
`factors. This widespread gene expression with tissue-spe-
`cic prohormone processing combined with differential
`functions constitutes essential parts of what may be con-
`ceived as a new and fascinating biology, which should
`appeal also to general endocrinology and physiology.
`The basic concept of endocrinology, blood-borne reg-
`ulation by specic messenger molecules, was discovered
`in 1902 by the British physiologists William Maddox Bay-
`liss and Ernest Henry Starling (12, 13). Following up on
`the observation of Pavlov and co-workers, that acidica-
`tion of the upper intestine resulted in secretion of pancre-
`atic juice (for review, see Ref. 178), Bayliss and Starling
`extracted from the duodenal mucosa a substance, which
`
`0031-9333/98 $15.00 Copyright ! 1998 the American Physiological Society
`
`MSN Exhibit 1011 - Page 1 of 22
`MSN v. Bausch - IPR2023-00016
`
`1087
`
`

`

`1088
`
`JENS F. REHFELD
`
`&,*/+) "#
`
`II. THE CLASSIC CONCEPT
`
`injected into blood stimulated pancreatic bicarbonate se-
`are discussed here. In other words, this review attempts
`cretion irrespective of whether the pancreas was inner-
`to present general principles governing structure and ex-
`vated or not. They called the substance secretin, a some-
`pression of gastrointestinal hormones. Readers interested
`what unspecic designation. However, in 1905, Starling in
`in details about individual hormones and their effects
`his Croonian lecture proposed the word hormone as a
`should consult comprehensive multi-author volumes com-
`general designation for blood-borne chemical messengers
`prising the entire range of gastrointestinal endocrinology
`(227). That same year, John Sidney Edkins, also from (211, 251) or the many volumes about individual gut hor-
`University College in London, discovered another hor-
`mones published in the 1990s. Before the characteristics
`and principles of the new biology of gastrointestinal hor-
`monal substance in extracts of the antral mucosa (67, 68).
`This substance stimulated gastric acid secretion. In view
`mones are presented, a summary of the old concept may
`be pertinent.
`of its origin, Edkins called it ``gastric secretin,'' but abbre-
`viated it to gastrin. Hence, the two hormones rst discov-
`ered in history were both gastrointestinal hormones. In
`the following decades, however, endocrinology as such
`blossomed by the discovery and isolation of steroid hor-
`The old concept about gastrointestinal hormones,
`mones from the adrenals, ovaries, and testes; larger pro-
`which prevailed unaffected through the century until the
`tein hormones from the anterior lobe of the pituitary and
`late 1970s, still dominates most general textbooks in phys-
`oxytocin and vasopressin from the posterior pituitary
`iology, biochemistry, and endocrinology. According to the
`lobe; and insulin from the pancreatic islets. In light of
`classic biology, a gut hormone is a substance produced
`the immediate and often life-saving implications of these
`by one type of endocrine cell dispersed in a relatively
`breakthroughs, the interest for secretin and gastrin faded well-dened region of the proximal gastrointestinal tract.
`to a degree that guratively returned them to the darkness
`From here it is released to blood by a specic stimulus
`to reach its target organ that subsequently elicits an acute
`of the bowel. Subsequently, only a small priesthood of
`physiologists maintained an interest in the hormonal con-
`response (secretion or muscle contraction).
`In the 1960s, it was shown that gastrointestinal hor-
`trol of digestion. One of them was Andrew Ivy in Chicago,
`who in 1928 assisted by Eric Oldberg found evidence of
`mones could be peptides of some 20  30 amino acid resi-
`dues. Hence, secretin, a 27-amino acid peptide, is released
`a gallbladder-emptying hormone in extracts of the small
`intestine (115). He called the substance cholecystokinin
`by gastric acid from S cells in the duodenum to stimulate
`bicarbonate secretion from exocrine pancreatic cells.
`(CCK). A stimulator of pancreatic enzyme secretion,
`termed pancreozymin, was subsequently discovered in the
`Gastrin, a 17-amino acid peptide, is by protein-rich food
`in the stomach released from antral G cells to stimulate
`1940s in small intestinal extracts by Harper and Raper in
`Newcastle (99). However, as shown in the 1960s by Erik
`acid secretion from parietal cells in the fundic mucosa.
`Jorpes and Viktor Mutt in Stockholm, CCK and pancreo- And CCK, a 33-amino acid peptide, is by fat, protein, and
`zymin are one and the same substance (124, 165), for
`acid released from small intestinal I cells to stimulate
`pancreatic enzyme secretion and gallbladder contraction.
`which now only the acronym CCK is used.
`Secretin, gastrin, and CCK are the classical gut hor-
`On the basis of simple physiological studies mainly in
`dogs, a number of additional gastrointestinal hormonal
`mones. The troika was not only discovered rst but struc-
`turally identied rst (91, 93, 165  167). It was also by
`mechanisms were proposed in the rst half of this century
`and as candidate hormones named according to function
`many believed that the endocrine regulation of digestion
`might be excreted only by these three hormones. A lead-
`and origin (incretin, enterogastrone, duocrinin, antral
`chalone, villikinin, and others). However, when the classic
`ing gastrointestinal physiologist in the 1960s and 1970s,
`Morton Grossman, even suggested that they acted via the
`troika of hormones became available as pure peptides in
`the late 1960s and early 1970s, proper experimentation
`same receptor (95). The trinity doctrine soon, however,
`turned out to be incorrect. There are many more gut hor-
`showed that several of the observed hormonal mecha-
`nisms could be explained either by interaction and/or ad-
`mones, and each has its own or even more receptors,
`although, vice versa, there are also examples showing that
`ditional effects of secretin, gastrin, and CCK or by newly
`identied gut hormones.
`different gastrointestinal peptides may act on the same
`receptor. This complexity is also part of the new biology.
`The new biology of gastrointestinal hormones main-
`However, so as not to lose sight in the avalanche of new tains in accordance with the classic conceptions that the
`information, the general and fundamental characteristics
`hormones are peptides, which are released to blood from
`cells in the gastrointestinal tract upon appropriate stimu-
`of the new biology are primarily exemplied with data for
`the classical gastrointestinal hormones. Moreover, only
`lation. However, the new biology contains a wealth of
`additional features revealed by modern molecular and cell
`the molecular and cellular biology of the gastrointestinal
`hormones as such is reviewed. Neither transport mecha-
`biology. These features have been collected under ve
`headings in this review.
`nisms, receptors, nor signal transduction in target cells
`
`/ 9j0c$$oc05
`
`P6-8
`
`09-24-98 13:48:59
`
`pra
`
`MSN Exhibit 1011 - Page 2 of 22
`MSN v. Bausch - IPR2023-00016
`APS-Phys Rev
`
`

`

`%(.,')- !$$#
`
`NEW BIOLOGY OF GASTROINTESTINAL HORMONES
`
`1089
`
`For the sake of completeness, it should be added
`here that a number of monoamines (including histamine
`and dopamine) and various eicosanoids with hormonelike
`activities also are produced in the gastrointestinal tract.
`This review is, however, restricted to proper gut hor-
`mones, which are peptides. It should also be emphasized
`that the designations gastrointestinal hormones and gut
`hormones are used synonymously in this review.
`
`III. THE NEW CONCEPTS
`
`A. Many Hormones and Their Families
`
`transcript to express CGRP is not the only example (3).
`Also, the secretin gene is expressed in different molecular
`forms in the gut because of alternative splicing (84, 131).
`Additional features contributing to the plurality are
`physiological studies which have indicated that there still
`are gut hormonal activities that are not easily explained
`by known peptides and, therefore, require identication of
`new hormones. Hence, as shown in Figure 1, 10 hormonal
`factors are still awaiting structural determination of both
`peptides and genes. Perhaps some of them can be partly
`explained by identied peptides. Hence, the incretin effect
`(135, 155) is probably exerted by a combination GIP (66)
`and GLP-I (54, 109) stimulations, whereas the entero-,
`vago-, and bulbogastrone effects may be explained by vari-
`Gastrointestinal endocrinology has since 1970, when
`ous combinations of somatostatin, GIP, EGF, and TGF-
`it was conned to only three identied peptides (91, 166, !. However, villikinin, duo- and enterocrinins, and the
`167), virtually exploded in number of regulatory gut pep-
`recently suggested gastrocalcin (181) still await a struc-
`tural identication of new substances, most probably pep-
`tides, i.e., hormones, peptide transmitters, and growth fac-
`tors (Fig. 1). Not only have new peptides with all features
`tides.
`It may be difcult to overlook the multiplicity of the
`of a hormone been found in gut extracts [gastric inhibitory
`polypeptide (GIP), Refs. 32, 34, 36; motilin, Refs. 31, 33,
`gut peptide systems. The structural identications, how-
`ever, have simplied the matter by showing striking ho-
`35, 210; peptide tyrosine tyrosine (PYY), Refs. 236, 237;
`galanin, Refs. 72, 238; glucagon-like peptides (GLPs), Refs.
`mologies between groups of peptides. Consequently, one-
`half of the hormones can be classied in families based
`16, 109, 176, 177, 234, 239, 245], but also neuropeptides
`isolated from the central nervous system and hormones
`on homology. Table 1 shows the major gastro-entero-pan-
`creatic hormone families. The expression of several pep-
`identied rst in other endocrine organs have been found
`to be present in endocrine cells and/or neurons in the
`tide genes both in the gut and the pancreas reŽects that
`the pancreas is of intestinal origin, both in onto- and phy-
`gastrointestinal tract [substance P, Refs. 43, 46, 71; the
`enkephalins, Refs. 73, 113; dynorphin, Ref. 224; neuroten-
`logenetic terms.
`The nature of the homology varies from family to
`sin, Refs. 42, 98, 130; neuropeptide Y (NPY), Refs. 157,
`232; the neurokinins, Ref. 55; pituitary adenylate cyclase-
`family. It may be an overall similarity in the primary struc-
`ture as illustrated by the PP-fold family, which comprises
`activating peptide, Refs. 158, 231; somatostatin, Refs. 8,
`29, 47; pancreatic polypeptide (PP), Refs. 45, 129; and
`PP, PYY, and NPY (Table 1). The family members display
`similarities varying between 45 and 70% (Fig. 2). The ex-
`calcitonin gene-related peptide (CGRP), Refs. 2, 3, 164,
`205, 241]. Moreover, potent regulatory peptides originally
`tensive similarity of the primary structures is coupled to
`an almost identical and stable tertiary structure because
`believed to be classical hormones but later shown to be
`widespread neurotransmitters have been isolated from
`the homology explicitly comprise residues, which are im-
`portant for stabilization of the three-dimensional PP-fold
`gut extracts [vasoactive intestinal polypeptide (VIP), Refs.
`137, 168, 207; peptide histidine isoleucine, Refs. 20, 114,
`structure (88). The PP-fold motif consists of a polyproline-
`like helix (residues 1 8) and an amphiphilic !-helix (resi-
`148, 237; and gastrin-releasing peptide (GRP), Refs. 153,
`154, 156, 160]. Finally, a number of growth factors with
`dues 15  30). The two helixes are joined by a type I "-
`turn (residues 9 12) and held in the folded conguration
`hormonal effects have now been shown to be present in
`the gut: epidermal growth factor (EGF), originally isolated
`by hydrophobic interdigitations between side chains of
`the !-helix residues and the NH2-terminal proline residues
`as a gut hormone; urogastrone, from urine independently
`of Cohen's EGF identication (49, 50), the isolation being
`(Fig. 2). Not only are the bioactive 36-amino acid peptides
`in the family highly homologous, the cDNA-deduced pre-
`monitored by its effect on gastric acid secretion by Harold
`Gregory (90); insulin-like growth factor I and II (30, 56,
`propeptides also display remarkable similarities in their
`organization (21, 147, 157).
`209, 229); transforming growth factor (TGF)-! and -" (14,
`44, 134, 250); and amphiregulin (182).
`Another type of homology is that of the gastrin family
`(Table 1 and Fig. 3). The family comprises, in addition
`The complexity is further increased by the fact that
`individual genes for gut regulatory peptides encode differ-
`to the mammalian hormones, gastrin and CCK, also the
`protochordean neuropeptide cionin (120), and the frog
`ent peptides, which in a tissue- and cell-specic manner
`release a number of different bioactive peptides. Several
`skin peptide cerulein (4). The decisive homology of this
`family is concentrated in and around the precisely dened
`principles for gene expression operate to provide such
`variety. Hence, alternative splicing of the calcitonin gene
`active site, the common COOH-terminal tetrapeptide am-
`
`/ 9j0c$$oc05
`
`P6-8
`
`09-24-98 13:48:59
`
`pra
`
`MSN Exhibit 1011 - Page 3 of 22
`MSN v. Bausch - IPR2023-00016
`APS-Phys Rev
`
`

`

`1090
`
`JENS F. REHFELD
`
`&,*/+) "#
`
`FIG. 1. Discovery and identication of regulatory peptides in gastrointestinal tract. Peptides may act as hormones,
`neurotransmitters, and growth factors. Sometimes 1 peptide acts in 2 or all of the 3 roles. Discovery is indicated by
`year of rst report. Solid circles indicate structural identication, and open circles indicate hormonal activities, which
`still require identication of responsible hormone(s). Some of structurally unidentied hormonal activities can be partly
`explained by activity of later identied hormones; for instance, incretin activity is partly due to gastric inhibitory
`polypeptide (GIP) and glucagon-like peptide I (GLP-I) activities. Commonly used acronyms are indicated in brackets
`after full name, except for PACAP, which is an acronym for pituitary adenylate cyclase-activating peptide.
`
`ide -Trp-Met-Asp-Phe-NH2 (the box in Fig. 3). Any modi-
`regulatory peptides in the gut. On the contrary, it is com-
`cation of this site grossly reduces or abolishes the recep-
`mon among all kinds of regulatory peptides, enzymes, and
`tor binding and consequently the biological effects of the
`other proteins in the organism (for reviews, see Refs. 1,
`hormones (162). Comparison of propeptide and gene
`64, 65, 118, 260). Each family is assumed to reŽect the
`structures reveals a complex pattern with an overall simi-
`phylogenetic evolution by duplication and subsequent mu-
`larity in the organization of progastrin, proCCK, and proci-
`tations of an ancestral gene. It is, however, possible that
`onin as well as their genes (22, 57, 58, 161, 203, 256, 257,
`the homology not only reŽects divergent evolution. The
`259), but with only little similarity in the amino acid resi-
`occurrence of homologous peptides in submammalian
`dues and DNA sequences outside the sequence corre-
`species may also demonstrate the existence of several
`sponding to the common active site and its COOH-termi-
`related genes, of which some do not evolve into genes of
`nal Žanking peptide. Hence, this family is in contrast to the mammalian species or of other vertebrates.
`PP-fold family and the secretin family primarily dened by
`The question of phylogenetic origin has recently
`been examined in detail for the gastrin family by John-
`the conserved active site sequence and by the neighboring
`O-sulfated tyrosyl residues (Fig. 3).
`sen (118). His study tested an immunochemically based
`hypothesis suggesting that CCK and gastrin originate
`The frequent occurrence of homology among gastro-
`intestinal hormones, peptide neurotransmitters in the gut,
`from a single common ancestral gene, which during evo-
`lution duplicated into separate CCK and gastrin genes
`and intestinal growth factors is not a feature specic for
`
`/ 9j0c$$oc05
`
`P6-8
`
`09-24-98 13:48:59
`
`pra
`
`MSN Exhibit 1011 - Page 4 of 22
`MSN v. Bausch - IPR2023-00016
`APS-Phys Rev
`
`

`

`%(.,')- !$$#
`
`NEW BIOLOGY OF GASTROINTESTINAL HORMONES
`
`1091
`
`TABLE 1. Gastroenteropancreatic peptide families
`
`*
`
`Secretin family
`Secretin
`Glucagon and
`glucagon-like peptides
`Gastric inhibitory polypeptide
`Vasoactive intestinal polypeptide and
`Peptide histidine isoleucine
`Growth hormone releasing hormone
`Pituitary adenylyl cyclase-activating
`peptide
`
`*
`
`Insulin family
`Insulin
`Insulin-like growth factor I
`Insulin-like growth factor II
`Relaxin
`
`EGF family
`Epidermal growth factor
`Transforming growth factor-!
`Amphiregulin
`
`Gastrin family
`Gastrin
`Cholecystokinin
`Cerulein
`Cionin
`
`PP-fold family
`Pancreatic polypeptide
`Peptide YY
`Neuropeptide Y
`
`Tachykinin family
`Substance P
`Neurokinin A
`Neurokinin B
`
`Somatostatin family
`Somatostatin
`Corticostatin
`
`* Peptides encoded by one gene.
`
` Not present in mammals.
`
`at the level of reptiles (140). Identication of peptides
`from brain and gut tissues of species representing the
`entire animal kingdom (including invertebrates) showed,
`however, the following: so far it has not been possible in
`invertebrates to identify true CCK-/gastrin-like peptides
`
`having an intact COOH-terminal tetrapeptide amide (Fig.
`3), although invertebrate neurons may express a family
`of less related -Asp-Phe-NH2 peptides (122). Possibly,
`however, the gastrin family may represent a subclass of
`a larger DFamide family. The earliest occurrence of true
`gastrin/CCK peptides in evolution is apparently cionin as
`expressed in protochordean neurons (120; Fig. 3). Procio-
`nin, the cionin gene, and its expression pattern resemble
`mammalian CCK rather than gastrin (161). So far, there-
`fore, the gastrin family appears to originate in protochor-
`dates with expression of the CCK-like cionin gene at the
`evolutionary level, where vertebrates branch off from in-
`vertebrates #500 million years ago or earlier. In cartilagi-
`nous sh or elasmobranchs (the earliest animals to se-
`crete gastric acid), the CCK-like gene has duplicated to
`express two very similar peptides of which one is likely
`to regulate gastric acid secretion (119). Two such CCK-
`like peptides are also expressed in the gut of bony sh,
`amphibians, reptiles, and birds (18, 121). Only mammals
`express gastrins with a structure that differs grossly from
`CCK outside the active site sequence (22, 118, 259).
`The phylogenetic story of the gastrin family shows
`that gastrointestinal hormones indeed are very old. So far,
`the data also support the idea that each gut peptide family
`has evolved from a single ancestor. An associated trait is
`that gastrointestinal hormones, at least in the gastrin fam-
`ily, to a large degree have preserved their tissue-specic
`
`FIG. 2. Structural homology of members of pancreatic polypeptide (PP)-fold family: PP, peptide tyrosine tyrosine
`(PYY), and neuropeptide tyrosine (NPY). Top: PP-fold conguration (tertiary structure) of the three 36-amino acid
`peptides. Bottom: amino acid sequences (primary structure) of the three porcine peptides.
`
`/ 9j0c$$oc05
`
`P6-8
`
`09-24-98 13:48:59
`
`MSN Exhibit 1011 - Page 5 of 22
`MSN v. Bausch - IPR2023-00016
`pra
`APS-Phys Rev
`
`

`

`1092
`
`JENS F. REHFELD
`
`&,*/+) "#
`
`FIG. 3. Structural homology of members of
`gastrin family: gastrin, cholecystokinin (CCK),
`cerulein, and cionin. The common COOH-termi-
`nal tetrapeptide amide in box constitutes mini-
`mal structure necessary for bioactivity. Note po-
`sition of characteristic O-sulfated tyrosine in the
`4 peptides. Shown poly-Glu sequence in gastrin
`decapeptide is that of human, pig, rat, and
`mouse.
`
`sites of expression during evolution, both primary and
`secondary sites (206). Accordingly, both the structural
`and cell-specic evolutionary conservation emphasize the
`general biological signicance of gut hormones.
`
`B. Multiple Phenotypes of a Hormone Gene
`
`described later for gastrin, true molecular heterogeneity
`most often reŽects an elaborate posttranslational phase of
`the expression cascade, which results in cellular synthesis
`and release of several different forms of a hormone. An-
`other and apparently less common way in which a gene
`can express different molecular forms is by alternative
`splicing of RNA transcripts.
`Alternative splicing, as a mechanism by which pep-
`A quarter of a century ago, gastrointestinal endocri-
`tide hormone complexity increases, was discovered 15
`nology was, as mentioned, believed to comprise three dif-
`years ago, when Amara and co-workers (2, 3, 205) showed
`ferent peptides (secretin, gastrin, and CCK). At that time
`that calcitonin gene transcription generates different
`it was also believed that one gene encoded one hormonal mRNA encoding either calcitonin peptides or CGRP. It
`molecule, in accordance with what was learned about the
`is now also well established that CGRP are abundantly
`master hormone, insulin. In other words, one gene equals
`expressed in intestinal neurons throughout the digestive
`one hormonal peptide. The complexity of gastrointestinal
`tract (164, 241; for review, see Ref. 110). Hence, alterna-
`endocrinology rst grew in a straightforward manner with
`tive splicing does play a signicant role in the control of
`the discovery and recognition of new hormones (Fig. 1).
`digestive functions.
`However, the synchronous rapid development in peptide
`The calcitonin gene is, however, not the only gene
`chemistry and molecular biology soon added new and
`encoding a gut peptide, which expresses itself by alterna-
`more intricate dimensions to the complexity. The ``one
`tive splicing. The alternative splicing of a tachykinin gene
`gene, one hormonal peptide'' dogma turned out to be too
`(171) and also one of the peptides encoded by the secretin
`simplistic for many gut hormones. Instead, and concur-
`gene is a result of alternative splicing of the primary tran-
`rently with the elucidation of eukaryotic gene structures
`scripts (84, 131).
`and improved understanding of the mechanisms govern-
`For many years, secretin was believed to exist only
`ing gene expression cascades, it became obvious that hor-
`as a carboxyamidated peptide of 27 amino acid residues
`mone genes often express several different bioactive pep-
`(166, 167). However, in the mid 1980s, two additional se-
`tides. Exactly which peptides appear to be regulated in a
`cretins with full bioactivity were identied in porcine gut
`cell-specic manner? Today we know three different ways
`extracts. One was the immediate precursor of amidated
`in which a gastrointestinal hormone gene can express
`secretin-27, i.e., glycine-extended secretin-28 (41), and the
`different bioactive peptides (Fig. 4).
`other was secretin-30 extended by a Lys-Arg sequence
`(83). In contrast to members of the gastrin, tachykinin,
`and PP-fold families (Table 1 and Figs. 2 and 3), COOH-
`terminal extensions of the amidated end products of se-
`cretin, VIP, and other members of the secretin family do
`not eliminate or reduce the bioactivity, because the activ-
`ity resides mainly in the NH2 terminus of the peptides.
`The existence of glycine- and glycyl-lysyl-arginine-ex-
`tended forms of secretin and VIP is not surprising. They
`are in fact to be expected from what is known about the
`biosynthesis of carboxyamidated peptides. Unexpected
`
`1. Alternative splicing of transcripts
`
`Existence of different molecular forms of a gut hor-
`mone or of an intestinal peptide neurotransmitter is quite
`common. It is important to emphasize, however, that the
`concept of molecular heterogeneity only refers to the exis-
`tence of more than one bioactive form. The coexistence
`of more or less inactive prohormones and processing in-
`termediates and/or degradation fragments with a single
`proper hormone, as well known for insulin, is trivial. As
`
`/ 9j0c$$oc05
`
`P6-8
`
`09-24-98 13:48:59
`
`pra
`
`MSN Exhibit 1011 - Page 6 of 22
`MSN v. Bausch - IPR2023-00016
`APS-Phys Rev
`
`

`

`%(.,')- !$$#
`
`NEW BIOLOGY OF GASTROINTESTINAL HORMONES
`
`1093
`
`FIG. 4. Multiple phenotypes (molecular het-
`erogeneity) of 3 gut hormone genes. The chole-
`cystokinin (CCK) gene encodes a prepropeptide,
`which through differentiated endoproteolytic
`cleavages is processed to 6 CCK peptides vary-
`ing in length from 83 to 8 amino acid residues.
`The six peptides have the same COOH-terminal
`bioactive octapeptide sequence (see also Fig. 3).
`The secretin gene encodes a prepropeptide that
`through endoproteolytic cleavages and variable
`COOH-terminal trimming is processed to 3 bio-
`active secretin peptides of almost similar size
`(secretin-27, -28, and -30). In addition, bioactive
`secretin-71 is produced by splicing out RNA en-
`coding midsequence of preprosecretin (i.e., bro-
`ken line of secretin-71). The glucagon gene en-
`codes a prepropeptide that through cell-specic
`endoproteolytic cleavages is processed to either
`genuine pancreatic glucagon (in pancreatic !-
`cells) or to glucagon-like peptides I and II (GLP-
`I, GLP-II; in intestinal L cells).
`
`was, however, the discovery of secretin-71 (84), which
`lengths with the same bioactive COOH terminus are syn-
`contains the sequence of nonamidated secretin-27 NH2
`thesized (Table 2). Although the different bioactive prod-
`terminally, followed by a Gly-Lys-Arg extension and a fur-
`ucts of the same precursor are bound to the same recep-
`ther COOH-terminal extension of 41 amino acid residues
`tor, their varying metabolic clearances from plasma affect
`(Fig. 4). With the exception of an arginine residue, which
`their hormonal signicance considerably. Hence, it mat-
`follows the Gly-Lys-Arg sequence directly, the COOH-ter-
`ters indeed whether intestinal proCCK is processed
`minal sequence of secretin-71 is identical to the COOH- mainly to CCK-58 or to CCK-8 or whether prosomatostatin
`terminal 40 amino acid residue fragment of porcine pre-
`is processed to somatostatin-28 or -14.
`So far, the processing of progastrin in antral G cells
`prosecretin (Fig. 4). Thus what corresponds to secretin
`RNA encoding a 32-amino acid sequence has been spliced
`has been examined particularly thoroughly. It will there-
`fore be used to illustrate the second way in which one
`out from the primary secretin gene transcript. For reasons
`mentioned above, secretin-71 has full secretin bioactivity.
`gastrointestinal hormone gene can encode different bioac-
`tive peptides.
`It remains to be settled how large a fraction secretin-71
`constitutes of the circulating secretins. The large molecu-
`In vertebrates, antral G cells synthesize by far most
`of the gastrin of the total organism (Table 3). Through a
`lar size suggests a slow metabolic clearance from blood.
`Secretin-71 should therefore accumulate in plasma and
`combination of knowledge about progastrin structure (22,
`80, 82, 85, 123, 149, 206, 259, see also Fig. 5), classic bio-
`constitute a signicant fraction. It also remains to be set-
`tled how common alternative splicing of the primary se-
`cretin gene and other gastrointestinal hormone gene tran-
`scripts are among vertebrates.
`
`TABLE 2. Molecular forms in primary sites/cells
`of synthesis
`
`2. Multiple products of prohormones with one
`active sequence
`
`Members of the somatostatin and gastrin families rep-
`resent peptide systems in which the gene encodes only
`one prohormone and in which only one prohormone with
`one active site is expressed, but nevertheless processed
`in a way so that a number of molecular forms of different
`
`Hormone
`
`Cell
`
`Bioactive Prohormone Products
`
`Secretin
`Gastrin
`Cholecystokinin
`
`S cells
`G cells
`I cells
`
`Secretin-71, -30, -28, and -27
`Gastrin-71, -34, -17, and -6
`CCK-83, -58, -39, -33, -22, -8, and -5
`
`Secretin-71 is synthesized by alternative splicing of secretin gene
`transcript. Gastrins are both O-sulfated and nonsulfated (except gastrin-
`6, which is fully sulfated). Cholecystokinins (CCK) are completely O-
`sulfated (except CCK-5 without a tyrosyl residue).
`
`/ 9j0c$$oc05
`
`P6-8
`
`09-24-98 13:48:59
`
`pra
`
`MSN Exhibit 1011 - Page 7 of 22
`MSN v. Bausch - IPR2023-00016
`APS-Phys Rev
`
`

`

`1094
`
`JENS F. REHFELD
`
`&,*/+) "#
`
`TABLE 3. Expression of progastrin and its products in
`mammalian tissue
`
`Tissue
`
`Gastrointestinal tract
`Antral mucosa
`Duodenal mucosa
`Jejunal mucosa
`Ileal mucosa
`Colonic mucosa
`Neuroendocrine tissue
`Cerebellum
`Vagal nerve
`Adenohypophysis
`Neurohypophysis
`Adrenal medulla
`Pancreas
`Genital tract
`Ovaries
`Testicles
`Spermatozoa
`Respiratory tract
`Bronchial mucosa
`
`Total Translation
`Product, pmol/g
`tissue
`
`Precursor
`Percentage
`
`10,000
`400
`40
`20
`0.2
`
`5
`8
`200
`30
`2
`2
`
`0.5
`6
`2
`
`0.3
`
`5
`20
`30
`85
`100
`
`20
`10
`98
`5
`100
`95
`
`100
`100
`55
`
`100
`
`Orders of magnitude are based on examination of different mam-
`malian species in the laboratory of the author (10, 26, 28, 79, 89, 117,
`150, 151, 185, 186, 189, 191, 193, 196, 197, 199, 221, 222, 244).
`
`synthesis studies (28, 62, 106, 230, 246, 247), identication
`of progastrin products in antral extracts (91, 93, 94, 192,
`194), and modern cell biology (38, 40), the biosynthetic
`pathway and the way in which G cells provide different
`gastrins are now well established (Fig. 6).
`After translation of gastrin mRNA in the rough endo-
`plasmic reticulum and cotranslational removal of the NH2-
`terminal signal peptide from preprogastrin, intact progas-
`trin is transported to the Golgi apparatus. In the trans-
`Golgi network, the rst posttranslational modications
`occur. These are O-sulfation of the tyrosyl-66 residue
`neighboring the active site and the rst of the endoproteo-
`lytic prohormone convertase cleavages. From the trans-
`Golgi network, vesicles carry the processin