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Department of Surgery University of Texas Medical Branch Galveston, Texas 77555
Address all correspondence and requests for reprints to: George H. Greeley, Jr., Ph.D., School of Medicine, Department of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555. E-mail: ggreeley{at}utmb.edu.
Many studies have demonstrated that cholecystokinin (CCK)/gastrin peptides have a long evolutionary history. The existence of CCK/gastrin-like peptides, first identified in the mammalian small intestine and stomach, was demonstrated throughout the chordates including the tunicates, which are the lowest chordate subphylum, and the arthropods (insects, arachnids, and crustaceans).
In this issue of Endocrinology, Janssen and co-workers (1), by means of a reverse pharmacology strategy, isolate and characterize two novel CCK/gastrin-like neuropeptides, called NLP-12, as endogenous ligands for CCK receptors (CKR-2a/2b) from the nematode, Caenorhabditis elegans. NLP-12 peptides show a high degree of sequence similarity with the vertebrate CCK/gastrin peptides and the arthropod sulfakinins. In addition, they demonstrate that NLP-12 peptides are recognized by a CCK-specific antibody, confirming structural similarities with the CCK/gastrin and sulfakinin sequences. NLP-12 peptides are not sulfated and have a very limited neuronal expression. NLP-12 peptides stimulate digestive enzyme secretion and fat storage, two biological activities associated with CCK/gastrin and sulfakinin peptides. Interestingly, NLP-12 peptides do not seem to exert a satiety effect. Discovery of the NLP-12 peptides indicates that the CCK/gastrin ligand-CCK1R/2R receptor signaling pathway had evolved before the divergence of protostomes and deuterostomes, and more importantly, the NLP-12-CKR-2a/2b axis represents the most ancient equivalent of the mammalian vertebrate CCK/gastrin-receptor signaling system.
The science of endocrinology was initiated 100 yr ago with the discovery of the very first hormone, a gut hormone called secretin (2). Secretin is just one of over 50 different factors described in the mammalian gastrointestinal (GI) tract that regulate processes associated with GI function (Fig. 1
). A majority of these factors are small peptides; many have been cloned and their cognate G protein-coupled receptors (GPCR) identified. Not all of these gut peptides are hormones.
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Gut hormones are produced in enteroendocrine cells found in distinct regions of the GI tract. There are at least 10 different gut hormone-producing cell types (4). Enteroendocrine cells probably evolved first at the level of protochordates from sensory gut neurons similar to those in the invertebrates. In mammals, CCK is produced in I cells of the upper small intestine, and gastrin is produced in stomach G cells. A distinct feature of gut hormone cells is that they are solitary and scattered throughout the GI tract. Many of the gut peptides without hormonal activity are found in neural structures and act as neurotransmitters or neuromodulators in the gut.
It is important to appreciate that although these peptides are called gut peptides or brain-gut peptides, they are not synthesized only in the gut and brain; many of these peptides with their cognate receptors are expressed widely in the body and exert an exceptionally diverse array of activities not linked primarily with gut physiology.
The ancient CCK/gastrin family appears to be represented in the whole chordate phylum (5, 6). Cholecystokinin was first isolated as a substance that is secreted from the upper small intestine to cause gallbladder contraction (7). An extract of the pig small intestine was shown to contain a substance that stimulates pancreatic enzyme secretion; this substance was called pancreozymin (8). Later, it was demonstrated that both activities are produced by the same substance, CCK. Gastrin was described originally as a factor contained in stomach extracts that stimulates gastric acid secretion (9, 10). Both peptides have an invariant, amidated C-terminal tetrapeptide sequence, Trp-Met-Asp-Phe-NH2, implying that CCK and gastrin evolved from a common ancestor. In addition to mammals, CCK and gastrin peptides have been identified in many nonmammalian species representing the major vertebrate classes, including fish, amphibians, reptiles, and birds. Because CCK and gastrin have been identified as separate genes in the dogfish, a duplication of the ancestral gene most likely occurred during evolution of cartilaginous fishes or earlier, giving rise to two distinct hormones, CCK and gastrin. Interestingly, the acid-stimulatory property of gastrin appeared concurrently with gastrin peptide in sharks. In amphibians, two distinct CCK and gastrin peptide systems can be identified including structurally different genes, cDNAs, peptides, expression profiles, and biological activities. CCK sequences are fairly well conserved across vertebrate species. Nonmammalian gastrins are structurally more similar to mammalian CCK than to mammalian gastrin, implying that CCK is phylogenetically older.
In chordates, the CCK-gastrin family includes, cionin, a CCK/gastrin-like peptide found in the sea squirt (Ciona intestinalis), a member of the subphylum Tunicata, which is the lowest subphylum of the phylum Chordata (6, 11). Cionin is a peptide of eight amino acids that was purified from the neural ganglion (brain) and is presumed to be the common ancestor of CCK and gastrin because the last four amino acids (Trp-Met-Asp-Phe-NH2) are identical in all three peptides. Cionin’s discovery dates the emergence of CCK-gastrin peptides to at least 500 million yr ago. In contrast to CCK and gastrin peptides, cionin has two sulfated tyrosines in positions 6 and 7 (counting from the C terminus) and is a structural hybrid of CCK and gastrin. Like the receptors for CCK and gastrin, cionin receptors are GPCR with multiple structural similarities to CCK1R/2R. In contrast to mammals, there is only a single copy of the cionin receptor that may be the common ancestor of the mammalian CCK1R and CCK2R receptors. The function of cionin in the brain is thought to be control of body wall and siphon movements. These activities resemble those of vertebrate CCK because CCK influences GI smooth muscle motility.
In arthropods (insects, arachnids, and crustaceans), there is a family of several neuropeptides structurally and functionally homologous to vertebrate CCK and gastrin called sulfakinins (12). In many cases, sulfakinins contain a single, sulfated tyrosine residue, hence the name sulfakinins. The first sulfakinins discovered were from insect extracts. Leucosulfakinin, the first member of the sulfakinin peptide family to be identified, was isolated based on its effect on the frequency of spontaneous contractions in isolated cockroach hindgut preparations. Sulfakinins are also known as myotropic peptides. They can increase the frequency and amplitude of hindgut contractions and increase the frequency of heartbeat in the cockroach. Sulfakinins contain the C-terminal hexapeptide sequence, Tyr(SO3H)-Gly-His-Met-Arg-Phe-NH2 which is the active core sequence. Like CCK and gastrin, all sulfakinins are amidated at their C terminus. Sulfakinins will activate a Drosophila GPCR; in this assay, the sulfated tyrosine is essential for biological activity because unsulfated sulfakinin is 3000 times less potent than a sulfated variant in activation of this receptor. Recent reports show that nonsulfated sulfakinins decrease the frequency of larval anterior midgut contractions, indicating that sulfation is not always required for full biological activity (13). Sulfakinin immunoreactivity is detected in the insect nervous system, suggesting a neurotransmitter role. In the cockroach, sulfakinin is found in neural structures and in endocrine cells of the midgut. Localization of sulfakinin immunoreactivity in retrocerebral nerves and in the corpora cardiaca suggests a neurohemal release (hormonal). Like CCK in mammals, sulfakinins have been shown to reduce food intake as well as stimulate digestive enzyme secretion. Sulfakinins will stimulate 
-amylase secretion in the beetle, Rhynchophorus ferrugineus. Interestingly, replacement of an Asp residue with Arg in mammalian CCK or gastrin confers activity to otherwise inactive mammalian hormones in insect assays. More interestingly, mammalian CCK-8 and insect sulfakinin can inhibit feeding behavior in the cockroach and goldfish, respectively. Together, these findings suggest that CCK/gastrin and sulfakinin peptides are homologous and that the peptide-receptor systems and mechanisms underlying regulation of ingestive behavior between arthropods and vertebrates have been conserved throughout evolution. The CCK/gastrin and sulfakinin signaling systems are excellent examples of the coevolution of peptides and their receptor systems.
CCK and gastrin constitute a family of homologous peptide hormones, characteristic of vertebrates (5, 6, 14). Both are physiological ligands for the CCK2R, whereas the CCK1R binds only sulfated CCK peptides. CCK and gastrin occur in different sizes, the dominant plasma variants are CCK-58, CCK-33, CCK-22, and CCK-8. CCK-8 is thought to be the major transmitter variant and is a potent neurotransmitter in the brain and the periphery. The major gastrin variants in tissue and plasma are gastrin-34 and gastrin-17. Although CCK and gastrin peptides are active as sulfated and nonsulfated forms, sulfation of the tyrosine residue at position 7 from the C terminus is necessary for full potency of CCK.
As indicated earlier, CCK is produced in enteroendocrine I cells of the upper small intestine and in brain neurons. CCK stimulates pancreatic enzyme secretion, gallbladder contraction, and intestinal motility and inhibits gastric emptying. CCK was one of the first gut hormones shown to exert a satiety action. The inhibitory action of CCK on gastric emptying may enhance the central satiety effect of CCK. In vertebrates, CCK inhibits food intake by targeting the vagus nerve, which relays parasympathetic activity between the gut and the brain. Gastric vagal afferents linked to CCK terminate on cell bodies in the nucleus tractus solitarius, the primary area of the brain that processes information from the gastrointestinal tract. Hormonal CCK is released from I cells into the general circulation after ingestion of dietary fats, proteins, and specific amino acids. Like mammalian CCK, fish CCK has a wide range of digestive functions. CCK stimulates pancreatic enzyme secretion, gallbladder contraction and gut peristalsis. Additionally, CCK in the mammalian brain may have roles in memory, sleep, sexual behavior, and anxiety. Gastrin does not affect food intake.
In the rat and humans, regulation of intestinal CCK secretion is exceptionally intriguing because evidence shows that its secretion is regulated by a luminal-releasing factor mechanism (15). Evidence indicates that the intestinal mucosa releases a peptide CCK-releasing factor (CCK-RF) into the intestinal lumen where it apparently triggers CCK secretion. Levels of this putative CCK-RF in the intestinal lumen are thought to be regulated by proteolytic enzymes secreted by the pancreas (i.e. trypsin).
Gastrin is produced in stomach G cells residing in the stomach antrum. Like CCK, gastrin is released into the systemic circulation in response to ingestion of specific nutrients, primarily intact and digested proteins, and certain amino acids. Gastrin regulates gastric acid secretion and mucosal epithelial cell growth.
In this issue of Endocrinology, the work of Janssen and co-workers (1) elegantly defends the hypothesis that the mammalian CCK/gastrin-CCK1R/2R signaling system is an ancient signaling system with counterparts throughout the evolutionary tree. These workers point out that the lack of CK (NLP-12 peptides, i.e. NLP-12a, NLP-12b) and CKR-2 in C. elegans did not compromise the pharyngeal pumping and elimination rates, implying that CK signaling does not affect the rate of food intake; this contradicts the satiety action of sulfakinins in insects and of CCK in vertebrates. Interestingly, a recent report indicates that cGMP, insulin, and TGF-β signaling pathways have roles in inducing quiescent behavior in C. elegans; quiescent behavior is reminiscent of satiety in mammals (16). At this point, Janssen and co-workers (1), however, do not discount hyperphagia as the underlying cause for increased fat content in NLP-12 and CKR-2 mutant worms and don’t dismiss a role for CK signaling in the regulation of satiety in nematodes. In humans, where a parallel exists, there is an ongoing debate whether hormonal CCK acts on the exocrine pancreas directly to stimulate enzyme secretion because data show that CCK can act via vagal cholinergic pathways to stimulate pancreatic enzyme secretion (17). The notion that CCK does not act directly on the pancreatic acinar cell may be considered heresy by some. CCK was discovered and characterized initially as a primary secretagogue for the exocrine pancreas, hence the name pancreozymin. In fact, ever since CCK was discovered, students of mammalian physiology have been taught that hormonal CCK is a major regulator of pancreatic secretion and that it acts directly on pancreatic exocrine cells to stimulate pancreatic enzyme secretion. However, the concept that CCK’s action on the pancreas is mediated by the vagal nerve is based upon solid data and is an excellent example of how our understanding of hormonal regulation of enteric function is still evolving. It also gives researchers like Janssen and co-workers (1) better insight as to which activities they will sleuth out when characterizing the biological functions of NLP-12 in C. elegans.
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Abbreviations: CK, NLP-12 peptides, i.e. NLP-12a, NLP-12b; CCK, cholecystokinin; CKR, CCK receptor; CCK-RF, CCK-releasing factor; GI, gastrointestinal; GPCR, G protein-coupled receptors.
Received March 18, 2008.
Accepted for publication March 26, 2008.
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