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Endocrinology 149(6):3184 –3186
Copyright © 2008 by The Endocrine Society
doi: 10.1210/en.2008-0371
Evidence for Intelligent Design in Gastrointestinal
Endocrinology: Identification of Novel Cholecystokinin/
Gastrin-Like Peptides in the Nematode Caenorhabditis
elegans
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-CKR2a/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.
That the GI tract is a rich source of regulatory peptides
should be no surprise because gut hormones are major players in regulation of metabolism. Gut peptides regulate feedAbbreviations: 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.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
ing behavior, digestion, absorption, and transport of nutrients; assimilation, partitioning, and use of energy; and the
elimination of waste (3).
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, TrpMet-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 con-
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Greeley • News & Views
Endocrinology, June 2008, 149(6):3184 –3186
Cumulative Number of Gut Peptides
Obestatin
Galanin-related Peptide
Ghrelin
Apelin
Orexins
PACAP
Secretin RF
CCK-RFs
40
Pancreastatin
Galanin
GLI
NPY
PYY
PHI
ACTH
TRH
Enkephalin
Neurotensin
Glucagon
Endorphin
Bombesin (GRP)
PP
Somatostatin
Substance P
Chymodenin
Vagogastrone
Motilin
Entero-oxyntin
VIP
GIP
Bulbogastrone
30
20
10
Insulin
Antral Chalone
Enteroglucagon
Urogastrone
Gastrone
Enterocrinin
Duocrinin
Villikinin
Enterogastrone
Incretin
Cholecystokinin
Gastrin
Secretin
0
1900
1920
1940
1960
1980
1990
2000
Year
FIG. 1. Discovery of biologically active factors in extracts of the mammalian gastrointestinal tract during the last century. The science of
endocrinology began with the discovery of the first hormone, an intestinal hormone called secretin that regulates pancreatic bicarbonate and fluid secretion. Many of the originally isolated factors were
characterized based upon biological activities; subsequently, many of
the factors that exert these biological activities have been cloned. GIP,
Glucose-dependent insulinotropic polypeptide; GLI, glicentin; GRP,
gastrin-releasing peptide; NPY, neuropeptide tyrosine; PACAP, pituitary adenylate cyclase-activating polypeptide; PHI, peptide histidine isoleucine; PP, pancreatic polypeptide; PYY, peptide tyrosine
tyrosine; VIP, vasoactive intestinal polypeptide. [Modified with permission from J. F. Rehfeld. Gut Hormones (edited by S. R. Bloom and
J. M. Polak), Churchill Livingstone, New York, p. 11 (18).]
served 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
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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
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Endocrinology, June 2008, 149(6):3184 –3186
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-␤ 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
Greeley • News & Views
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 coworkers (1) better insight as to which activities they will
sleuth out when characterizing the biological functions of
NLP-12 in C. elegans.
George H. Greeley, Jr.
Department of Surgery
University of Texas Medical Branch
Galveston, Texas 77555
Acknowledgments
Received March 18, 2008. Accepted March 26, 2008.
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: [email protected].
Disclosure Statement: The author has nothing to disclose.
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Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the
endocrine community.