Endocrine Reviews 20 (6): 876-913
Copyright © 1999 by The Endocrine Society
The Glucagon-Like Peptides
Timothy James Kieffer1 and
Joel Francis Habener2
Departments of Medicine and Physiology (T.J.K.), University of
Alberta, Edmonton, Alberta, Canada T6G 2S2; and Laboratory of Molecular
Endocrinology (J.F.H.), Massachusetts General Hospital, Howard Hughes
Medical Institute, Harvard Medical School, Boston, Massachusetts
02114
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Abstract
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- I. Introduction
- II. History of the Incretin Concept: Discovery of Gastric Inhibitory Polypeptide
- III. Discovery of GLP-1
- IV. Structures of GLPs and Family of Glucagon-Related Peptides
- V. Tissue Distribution of the Expression of GLPs
- A. Pancreatic
-cells
- B. Intestinal L cells
- C. Central nervous system
- VI. Proglucagon Biosynthesis
- A. Organization/structure of the proglucagon gene
- B. Regulation of glucagon gene expression
- C. Posttranslational processing of proglucagon
- VII. Regulation of GLP Secretion
- A. Overview
- B. Intracellular signals
- C. Carbohydrates
- D. Fats
- E. Proteins
- F. Endocrine
- G. Neural
- H. GLP-2
- VIII. Metabolism of GLPs
- A. GLP-1
- B. GLP-2
- IX. Physiological Actions of GLPs
- A. Overview
- B. Pancreatic islets
- C. Counterregulatory actions of GLP-1 and leptin on ß-cells
- D. Stomach
- E. Lung
- F. Brain
- G. Liver, skeletal muscle, and fat
- H. Pituitary, hypothalamus, and thyroid
- I. Cardiovascular system
- J. GLP-2
- X. GLP Receptors
- A. Structure
- B. Signaling
- C. Distribution
- D. Regulation
- E. GLP-2
- XI. Pathophysiology of GLP-1
- XII. GLP-1 as a Potential Treatment for Diabetes Mellitus
- XIII. Future Directions
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I. Introduction
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IT HAS been 15 yr since the initial discovery of the
glucagon-like peptides (GLPs) as potential bioactive peptides encoded
in the preproglucagon gene. The GLPs and glucagon are formed by
alternative tissue-specific cleavages in the L cells of the intestine,
the -cells of the endocrine pancreas, and neurons in the
brain. Glucagon-like peptide-1 (GLP-1) is now known to be a potent
glucose-dependent insulinotropic hormone, which has important actions
on gastric motility, on the suppression of plasma glucagon levels, and
possibly on the promotion of satiety and stimulation of glucose
disposal in peripheral tissues independent of the actions of
insulin. As a consequence of these properties,
GLP-1 is under investigation as a potential
treatment of diabetes mellitus. GLP-2 was recognized only recently to
have potent growth-promoting activities on intestinal epithelium.
The interest in the GLPs has grown exponentially. By 1988 there were
170 publications describing the properties of the GLPs. Five years
later this number grew to 426 and currently (1999) more than 1,000
publications appear in the database of the National Library of Medicine
(PubMed).
Since the last comprehensive review of GLP-1 appeared in
Endocrine Reviews in 1995 (1), many new developments have
occurred and are described in this review. The purpose of this article
is to emphasize the newer and what are perceived to be the more current
and important aspects of the biology of the GLPs. For additional
information and references, the reader is referred to several
informative earlier reviews (1-12).
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II. History of the Incretin Concept: Discovery of Gastric
Inhibitory Polypeptide
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As a result of their discovery of secretin in 1902, Bayliss and
Starling (13) speculated that signals arising from the gut after
ingestion of nutrients might elicit pancreatic endocrine responses and
affect the disposal of carbohydrates. In 1906 Moore et al.
(14) postulated that the duodenum produced a ‘chemical excitant‘ for
pancreatic secretion and attempted to treat diabetes by injecting gut
extracts. Zunz and Labarre (15, 16) pursued this factor and prepared an
intestinal extract free of secretin activity that was able to produce
hypoglycemia in dogs. Labarre (16) introduced the term ‘incretin‘ to
describe the humoral activity of the gut that might enhance the
endocrine secretion of the pancreas (16). Although other investigators
also reported the presence of hypoglycemic factors in duodenal extracts
(17-20), Loew and colleagues (21) were unable to lower blood glucose
levels in dogs with extracts of dog or hog intestinal mucosa obtained
by a number of methods. Although these later extracts were tested only
in fasting animals, after this report, interest in isolating an
intestinal hypoglycemic factor declined.
The development of a reliable RIA for insulin in the 1960s by Yalow and
Berson (22), which allowed measurements of the circulating levels of
this hormone, renewed interest in the search for incretins. It was
demonstrated by both immunoassay (23, 24) and bioassay (25, 26) that
the action of glucose on the pancreas could not account completely for
the insulin response observed in the blood. These reports demonstrated
that iv glucose administration resulted in a lower plasma insulin
response than when given by intrajejunal infusion, even though lower
blood glucose levels were achieved by the later (Fig. 1
). Perley and Kipnis (27) estimated the
alimentary component to be close to 50% by subtracting from the
insulin secretory response seen after oral glucose that insulin
response obtained with the infusion of iv glucose, which duplicated the
oral blood glucose profile.
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Figure 1. Demonstration of the incretin concept. Blood
glucose and insulin responses after either intravenous or intrajejunal
glucose infusion in normal subjects. Although plasma glucose levels
after intravenous glucose infusion were higher than those after
intrajejunal glucose infusion, the latter generated a larger insulin
response. Based on these results, McIntrye et al. (23 )
suggested that a humoral substance was released from the jejunum during
glucose absorption, acting in concert with glucose to stimulate insulin
release from pancreatic ß-cells. [Reproduced with permission from N.
McIntyre et al.: Lancet 2:20-21, 1964
(23 ) © The Lancet Ltd.].
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In 1969, Unger and Eisentraut (28) named the connection between the gut
and the pancreatic islets the ‘enteroinsular axis.‘ Creutzfeldt (29)
suggested that this axis encompasses nutrient, neural, and hormonal
signals from the gut to the islet cells secreting insulin, glucagon,
somatostatin, or pancreatic polypeptide (Fig. 2). Furthermore, Creutzfeldt (29) defined
the criteria for fulfillment of the hormonal or incretin part of the
enteroinsular axis as: 1) it must be released by nutrients,
particularly carbohydrates, and 2) at physiological levels, it must
stimulate insulin secretion in the presence of elevated blood glucose
levels.
One hormone that clearly fits the requirements to be an incretin is
glucose-dependent insulinotropic polypeptide (GIP). GIP was originally
isolated as an ‘enterogastrone,‘ or hormone secreted in response to
fat or its digestive products in the intestinal lumen that inhibits
gastric acid secretion (30). Brown and colleagues (31-33) isolated GIP
from impure preparations of cholecystokinin (CCK) that contained
acid-inhibitory activity using the canine Heidenhain pouch as a
bioassay. GIP was shown to be a potent inhibitor of gastric acid and
pepsin secretion and was thus originally named ‘gastric inhibitory
polypeptide‘ (34, 35). Earlier, Dupré and Beck (26) had
demonstrated that a crude preparation of CCK also possessed
insulinotropic activity. In 1972, Rabinovitch and Dupré (36)
found that this insulinotropic action could be removed by further
purification of the CCK. This observation resembled the loss of the
acid-inhibitory activity reported previously by Brown and Pederson (33)
during the purification of GIP from CCK and led Dupré et
al. (37) to the hypothesis that GIP may also possess
insulin-releasing capabilities. In 1973, Dupré et al.
(37) demonstrated that a purified preparation of porcine GIP infused
intravenously in humans in concert with glucose stimulated the release
of significantly greater quantities of immunoreactive insulin than when
the same dose of glucose was administered alone. The insulin response
was sustained for the duration of the GIP infusion and was not observed
during the euglycemic state (37). The glucose-dependent nature for the
insulinotropic activity of GIP was later demonstrated in
vivo in dogs (38) and humans (39) and in the perfused rat pancreas
(40). Furthermore, GIP released in response to the oral ingestion of
fat yielded no increase in plasma insulin levels unless intravenous
glucose was also administered (41-43). The glucose dependency of
GIP-stimulated insulin secretion appeared to provide an important
safeguard against inappropriate stimulation of insulin release during a
high-fat, low-carbohydrate meal. The recognition of this additional
important physiological function of GIP led to the alternate
designation glucose-dependent insulinotropic polypeptide (GIP) (44).
In accordance with the roles of GIP as an enterogastrone and an
incretin, immunoreactive GIP cells have been located in the
upper small intestine of ruminants (45), humans, pigs, dogs (46), and
rats (47). In the gastrointestinal tract of dog and man, immunoreactive
GIP is present in cells predominantly in the midzone of the duodenal
villi and, to a lesser extent, in the jejunum (48). Levels of GIP rise
several fold shortly after ingestion of a meal containing fat (41-43)
or glucose (38, 49, 50). It appears glucose may act directly at the
level of the GIP-secreting K cells to stimulate GIP release (51, 52).
Studies employing GIP antisera to immunoneutralize endogenous GIP
indicated that intestinal hormones other than GIP contribute
substantially to the incretin effect (53, 54). These findings were
supported by the observation that insulinotropic activity remained in
intestinal extracts after removal of GIP by immunoadsorption (55).
Finally, a major contribution to the incretin effect from the lower
gastrointestinal tract was shown in studies of patients after varying
degrees of resection of the small intestine (56). The incretin effect
of oral glucose correlated positively to the total length of residual
small bowel rather than to an integrated release of GIP. Patients with
preserved ileal residues had much larger incretin effects than patients
with no ileal residues, despite equal integrated increases in plasma
GIP, findings indicative of the presence of incretins other than GIP in
the ileum (56).
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III. Discovery of GLP-1
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In the interim between the discovery in the 1970s of GIP as an
important intestinal incretin hormone to the actual discovery of
GLP-1, it was suspected that there must be a second
incretin hormone in addition to GIP (54-56). The ushering in of
the era of recombinant DNA technology in the late 1970s provided the
means necessary for the identification of the ‘missing‘ incretin
hormone. In the early 1980s, the cloning of cDNAs encoding the
preproglucagons from pancreata of the anglerfish was accomplished (57, 58). The anglerfish was found to have two separate nonallelic
preproglucagon genes, I and II, both encoding a glucagon and a
glucagon-related peptide (GRP) (58). Notably, the glucagon-related
peptide encoded in the anglerfish, preproglucagon-I, located carboxy
proximal to the sequence of glucagon, bore a strong homology to the
sequence of GIP, leading Lund et al. (57) to suggest that
the anglerfish GRP-1 may be an intestinal incretin hormone. In support
of this supposition Lund et al. (59) showed that similar
preproglucagon mRNAs were expressed in the anglerfish pancreas and
intestine, a finding that strongly supported the prediction that GRP
could be an incretin hormone. Subsequently, preproglucagon mRNAs were
cloned from human (60) and rat (61) gut and shown to be identical in
sequences to the mRNAs in pancreas.
Shortly after the discovery of anglerfish GRP, the preproglucagon cDNAs
of mammals were cloned (62-64) as well as the human gene (65). It
became clear that the anglerfish GRP-I is a homolog of the
GLP-1s encoded in the mammalian preproglucagons, which
were subsequently proven to be potent insulinotropic incretins. There
was, however, some uncertainty regarding the identification of the
bioactive isoform of GLP-1 that had true insulinotropic
actions. Based on the amino acid sequence of the mammalian
preproglucagons, the sites that would be predicted for
posttranslational processing into peptide hormones were somewhat
ambiguous. At the time it was generally believed that the
yet-to-be-identified prohormone convertases (PCs) that enzymatically
split prohormones into bioactive peptides required two adjacent basic
amino acids, combinations of arginine, and lysine. The
GLP-1 sequence begins with a histidine as the
amino-terminal residue, as do most of the peptide hormones in the
glucagon-related superfamily of hormones (Fig. 3). In the preproglucagon sequence, the
first histidine is preceded by two basic amino acids, Lys-Arg, followed
by four residues, another single basic residue, arginine, and a second
histidine. The thinking at that time was that the putative bioactive
peptide that would theoretically be cleaved from the preproglucagon
during posttranslational processing would be at the Lys-Arg yielding a
peptide of 37 or 36 amino acids, depending on whether the C-terminal
glycine was present or absent and whether the penultimate
C-terminal arginine was amidated in the absence of the C-terminal
glycine. Thus, the 1-37 and 1-36 GLP-1 peptide isoforms
were the first to be synthesized and tested for biological activity.
The results of the experimental testing were disappointing. One report
questioned whether GLP-1 had any relevant activity: ‘How
glucagon-like is glucagon-like peptide?‘ (66), as it had no effect on
plasma glucose and insulin levels when administered to rabbits. Another
report showed a weak stimulation of insulin secretion in cultured rat
pancreatic islets at superpharmacological doses (25 nM) of
GLP-1(1-36)amide and suggested that an N-terminally
truncated peptide, GLP-1(7-36)amide, may be more active
(67), as was suggestedfor GLP-1(7-37), an N-terminally
truncated form of GLP-1(1-37) (67). These ideas were based
upon alignment of the sequence of GLP-1 with the other
members of the glucagon superfamily of peptide hormones (see Fig. 3),
which revealed that the best alignment was with the histidine at
position 7, and not position 1 of GLP-1 (12, 63, 67). In
1986 it was discovered that GLP-1 was indeed further
N-terminally truncated by posttranslational processing in the
intestinal L cells (68, 69). In contrast to GLP-1(1-37),
GLP-1(7-37) and (7-36)amide were found to be potent
insulinotropic hormones in the isolated perfused pancreas of rats (70)
and pigs (71), and in humans (72). Further, it was suggested that the
weak insulinotropic actions of GLP-1(1-37) at micromolar
concentrations were probably artefactual due to a 0.1% level of
nonspecific cleavage of GLP-1(1-37) to
GLP-1(7-37) by nonspecific cathepsins in the
serum-implemented tissue culture media (73). At present it is well
established that the GLP-1 isoforms
GLP-1(7-37) and GLP-1(7-36)amide are the
bioactive insulinotropic peptides derived from preproglucagon in the
intestine and the hind brain. The functions of the lesser
GLP-1 isoforms GLP-1(1-37) and
GLP-1(1-36)amide remain unknown.
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Figure 3. Amino acid sequences of the members of the
superfamily of glucagon-related peptides. Sequences include human
glucagon, human GLPs, human GIP, exendins (Heloderma
horridum), human secretin, human peptide histidine methionine
(PHM), helospectins (Heloderma horridum), helodermin
(Heloderma suspectum), human PACAP, human PACAP-related
peptide (PRP), human GRF, and human VIP. Residues identical to those of
glucagon in the same position are shaded.
Standard single letter abbreviations are used for amino
acids (IUPAC-IUB Commission on Biochemical Nomenclature): A, Ala; C,
Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M,
Met; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y,
Tyr.
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IV. Structures of GLPs and Family of Glucagon-Related Peptides
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The GLPs belong to a larger family referred to as the glucagon
superfamily of peptide hormones. These hormones are classified within
this family based on their considerable sequence homology, having
anywhere from 21% to 48% amino acid identity with glucagon. Included
in this family are: glucagon, GLP-1(7-37) and
-(7-36)amide, GIP, exendin-3 and -4, secretin, peptide
histidine-methionine amide (PHM), GLP-2, helospectin-1 and -2,
helodermin, pituitary adenyl cyclase-activating polypeptides
(PACAP)-38, and -27, PACAP-related peptide (PRP), GH-releasing factor
(GRF), and vasoactive intestinal polypeptide (VIP) (Fig. 3
). These
peptide hormones are produced in the gut, pancreas, and the central and
peripheral nervous systems and exhibit a wide variety of biological
actions in which several act as neurotransmitters. Notably, even
peptide hormones that are coencoded within the same precursor, such as
the peptide hormones derived from the cleavages of preproglucagon,
differ significantly in the physiological processes that they regulate.
For example, the major function of glucagon is to maintain blood
glucose levels during fasting, whereas GLP-1 functions
primarily during feeding to stimulate insulin release and to lower
blood glucose levels. On the other hand, GLP-2 appears to regulate the
growth of intestinal epithelial cells.
Exendin-3, exendin-4, helospectin-1, helospectin-2, and helodermin were
all isolated from lizard (Heloderma) venom. They are potent
secretagogues of the exocrine pancreas (74). Helodermin shares 53% and
42% homology with human PACAP and VIP, respectively, and has high
affinity for the VIP2 receptor (75). Exendin-4 is 53%
homologous to mammalian GLP-1 and acts as a high-affinity
agonist on the GLP-1 receptor (76, 77). Exendin-4 and
GLP-1 may interact with specific receptors yet to be
identified in guinea pig pancreatic acini tissue (78). In contrast, the
amino-terminally truncated form of exendin-3(9-39) is a potent
antagonist of GLP-1 actions (76, 77). Lizard helodermin,
exendin, VIP/PHI, PACAP, and glucagon/GLP-1 cDNAs have
been cloned, revealing that separate genes exist for these peptides
(79, 80). To date, no evidence has been uncovered for the existence of
mammalian homologues of the lizard helodermin or exendin (79, 80). It
appears that helodermin and exendin-4 are not the evolutionary
precursors to mammalian PACAP/VIP or GLP-1 but represent a
distinct family of peptides. It seems likely that the high-affinity and
biological activities of helodermin and exendin-4 on the mammalian
VIP2 and GLP-1 receptors, respectively, are a
result of convergent evolution (80).
It is proposed that the proglucagon gene arose by the duplication of an
ancestral gene approximately 800-1,000 million years ago (81). The
structural organization of the genes of the glucagon superfamily of
peptide hormones suggests that the ancestral gene consisted of four
exons, which encoded the 5'-untranslated region of the mRNA, the signal
peptide, the hormone and the 3'-untranslated region of the mRNA,
respectively (82). The glucagon superfamily of hormones may have arisen
by duplication and amplification of this basic gene, followed by a
further duplication and amplification of the exon encoding the glucagon
hormone domain to generate the multiple GLPs observed in preproglucagon
(82). Based on statistical analysis of DNA sequences of the
preproglucagon genes from bovine, human, hamster, and anglerfish, Lopez
et al. (83) postulated that the two anglerfish genes arose
from gene duplication approximately 160 million years ago (83).
Furthermore, this analysis suggested that the GLP-2 sequence originated
by duplication of the glucagon or GLP-1 sequence before
the earliest divergence of fish (83). However, until recently, it was
believed that GLP-2 was not expressed in either fish or birds (11, 84).
Irwin and Wong (85) discovered that, unlike pancreatic proglucagon of
fish and birds, the intestinal proglucagon does contain the sequence of
GLP-2. Therefore, fish and bird proglucagon mRNAs from pancreas and
intestine have different 3'-ends that are due to alternative mRNA
splicing (85). The recent cloning of the frog (Xenopus)
proglucagon cDNAs revealed the presence of three distinct
GLP-1 peptides in addition to glucagon and GLP-2 (86). It
has been postulated that the first exon duplication event resulting in
the appearance of glucagon and GLP occurred at least 405-800 million
years ago (81, 83). A duplication of the GLP-containing exon, giving
rise to GLP-1 and GLP-2, may have occurred between 365
(divergence of mammals and amphibians) and 405 (divergence of
cartilaginous fish and tetrapods) million years ago (81). The amino
acid sequences of the preproglucagon genes are highly conserved among
mammals (Fig. 4), and the products
derived from proglucagon, glucagon (Fig. 5), and GLP-1 (Fig. 6) are highly conserved throughout the
evolution of animal species. The amino acid sequence of glucagon is
highly conserved during the evolution of tetrapods (3 substitutions
between salamander and human), even more than the sequences of either
GLP-1 (7 substitutions) or GLP-2 (15 substitutions). The
high degree of conservation of the glucagon and GLP
sequences during evolution indicates the importance of the
physiological processes regulated by these hormones.
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Figure 4. Amino acid sequences of proglucagon from seven
mammalian species. GenBank accession numbers are given in
parentheses. Major proglucagon products are indicated by
bars; GRPP, glicentin-related pancreatic
peptide; IP-1 and IP-2, intervening peptides; GLP-1 and
GLP-2, GLPs. Shaded residues are completely conserved
between the seven species. Standard single letter
abbreviations are used for amino acids (IUPAC-IUB Commission on
Biochemical Nomenclature): A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G,
Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; P, Pro; Q, Gln; R, Arg; S,
Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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Figure 5. Amino acid sequences of vertebrate glucagons.
Classes are as indicated and residues identical to those of human
glucagon in the same position are shaded.
Standard single letter abbreviations are used for amino
acids (IUPAC-IUB Commission on Biochemical Nomenclature): A, Ala; C,
Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M,
Met; P, Pro; Q, Gln, R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y,
Tyr. Reference numbers indicate the source of the corresponding
sequence.
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Figure 6. Amino acid sequences of vertebrate
GLP-1s. Classes are as indicated and residues identical to
those of human GLP-1s in the same position are
shaded. Standard single letter
abbreviations are used for amino acids (IUPAC-IUB Commission on
Biochemical Nomenclature): A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G,
Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; P, Pro; Q, Gln; R, Arg; S,
Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. Reference numbers indicate the
source of the corresponding sequence.
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The conservation of GLP-1 also reflects the fact that
essentially the entire amino acid sequence of GLP-1 is
required for full biological activity. Removal of the N-terminal
histidine (= GLP-1 8-37) results in drastic loss of
receptor binding and insulinotropic activity (87-90). The positive
charge of the imidazole side chain of the histidine residue appears to
be crucial for GLP-1 actions (91). Likewise, N-terminal
truncation of this histidine from the related insulinotropic peptide
exendin-4(1-39) reduces agonist activity by approximately 10-fold (92).
Notably, N-terminal truncation of exendin-4 by two residues yields a
peptide that binds with the same affinity as full-length exendin but
antagonizes GLP-1 action (92). In contrast, an N-terminal
truncation of GLP-1 by two residues reduces binding
affinity to approximately 1% that of the intact molecule (92, 93).
Also, addition of an amino acid to the N terminus of
GLP-1(6-37) also reduces biological activity (87, 89).
Truncation at the C terminus also reduces the biological activity of
GLP-1 considerably (87, 88, 90, 93). Substitution in the
N-terminal part of the GLP-1 molecule with the
corresponding glucagon residues impaired the affinity for the
GLP-1 receptor only moderately whereas exchanges in the
C-terminal portion of GLP-1 decreased the affinity for the
GLP-1 receptor more than 100-fold (94). In
contrast, the binding affinity of GLP-1 to its receptor is
more sensitive to GIP-like changes in the N-terminal region than to
changes in the C-terminal region (95).
Another approach to understanding the structure-activity relationships
of GLP-1 has been obtained from studies of peptide analogs
in which individual amino acids are substituted. These studies revealed
that the residues in positions 1 (His), 4 (Gly), 6 (Phe), 7 (Thr), 9
(Asp) 22 (Phe), and 23 (Ile) are important for the binding affinity and
biological activity of GLP-1 (96-98). Two-dimensional
nuclear magnetic resonance of GLP-1 in a membrane-like
environment (a dodecylphosphocholine micelle) revealed that
GLP-1 consists of an N-terminal random coil segment
(residues 1-7), two helical segments (7-14 and 18-29), and a linker
region (15-17) – a structure similar to that observed for glucagon
(99). Thus far, attempts to generate smaller active fragments of
GLP-1 that retain potent insulinotropic activity have
failed (89, 98, 100).
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V. Tissue Distribution of the Expression of GLPs
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A. Pancreatic -cells
Pancreatic -cells were discovered in 1907 as
histologically distinct cells from the ß-cells of the islets of
Langerhans (101). It was not until 1962 that -cells were shown by
immunofluorescence staining studies to be the source of glucagon (102).
The -cells are one of four distinct polypeptide-secreting islet cell
types: glucagon-secreting -cells, insulin- and amylin-secreting
ß-cells, somatostatin-secreting 
-cells, and
pancreatic-polypeptide-secreting F cells. These cells are arranged in
highly organized patterns within the islets. In rodents, -cells and
-cells exist on the surface or mantle of the islet surrounding the
core of ß-cells, although the patterns of distribution of the -,
-, and ß-cells differ among animal species (103-105).
Uncertainties in the islet vascular architecture and the direction of
blood flow within the islets (106) still cloud the functional
significance of the anatomic arrangement of hormone-producing islet
cells.
mRNA encoding proglucagon can be detected by PCR early in the wall of
the embryonic foregut at the 20-somite stage and is restricted to the
area of the duodenum from which the pancreas will develop 10-12 h later
(107). The pancreas arises as two outbuddings of the gut tube shortly
after it is formed early in development at embryonic day 9 (ED 9) in
the mouse (for review see Ref. 108). By ED 10, the budding anlaga fuse
to become the dorsal and ventral pancreas with their respective ducts.
By immunofluorescence, glucagon-positive cells are identifiable at ED
10.5 in the dorsal bud and at ED 11.5 in the ventral bud (109).
Individual hormone-containing cells are located within the epithelium
of pancreatic ducts, and clusters of endocrine cells are found in the
pancreatic interstitium. Starting on ED 16.5, islets begin to form, and
by day 18.5 the islets consist of centrally located ß-cells with the
adult ‘one cell-one hormone‘ phenotype (109). The expression of
specific transcription factors is involved in the determination of cell
lineages that determine the development of islet-specific cells of the
endocrine pancreas. For example, recent findings indicate that
disruption of the gene for the transcription factor Pax6 in mice
results in a near-complete failure in the development of -cells
(110), whereas disruption of Pax4 results in the absence of mature ß-
and -cells (111) (Fig. 7). Mice
lacking the transcription factor Nkx2.2 have diabetes due to arrested
differentiation of pancreatic ß-cells (112).
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Figure 7. Proposed developmental pathway of the endocrine
pancreas in the mouse, showing interruptions of development in response
to disruptions of the transcription factor genes, IDX-1, Isl-1, Pax-4,
and Pax-6. Knockouts of IDX-1 and Isl-1 result in early failure of the
development of epithelial cells derived from the endodermal stem cell.
IDX-1 is a key factor in the very early development of all pancreatic
epithelial cells, whereas Isl-1 is required for the development of the
dorsal mesenchyme, and its failure leads to a specific arrest of
development of the epithelial cells of the dorsal pancreas; the mice
die at ED 9.5. Inactivation of Pax-4 by homologous recombination
prevents development of the ß- and -cells and shunts development
to the -cell lineage. The Pax-6 knockout does the opposite:
-cells do not develop, but some development occurs in ß- and
-cells. Recently, the knockout in mice of transcription factor
Nkx2.2 showed a phenotype of arrested differentiation of ß-cells
(112 ). GLU, Glucagon; INS, insulin; SOM, somatostatin; PP, pancreatic
polypeptide. Days of embryonic development are indicated on the
left of the figure (E10-E17) and postnatal days are
indicated by P1-P21. [Reproduced with permission from J. F.
Habener and D. A. Stoffers: Proc Assoc Am Phys
110:12-21, 1998 (585 )].
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As illustrated in Fig. 8, cell-specific
processing of proglucagon in pancreatic -cells leads primarily to
the production of glucagon. However, immunoreactive GLP-1
is detectable in rat pancreatic -cells by immunocytochemistry (113).
Fully processed GLP-1 (7-36 amide and 7-37) is also
visualized in pancreatic rat extracts by using chromatographic
techniques and RIAs (114, 115). A recent investigation detected
predominantly GLP-1 (1-36) amide in extracts of rat
pancreas (116). Using similar techniques, small amounts of N-terminally
extended GLP-1 (1-36 amide and 1-37) are also found in
extracts from porcine and human pancreas (117, 118). In addition,
immunoreactive GLP-1 is secreted from the
arginine-perfused rat pancreas and glucose-stimulated isolated rat
islets, as detected by RIA (113, 114). The relatively small quantity of
GLP-1 produced by the pancreas might have important local
actions within the islets.
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Figure 8. Expression of the preproglucagon gene. A,
Diagram of the proglucagon gene and encoded mRNA. The gene consists of
six exons (E1-E6) and five introns (IA-IE). Alternative splicing of
exons E4 and E5 occurs in salmonid fishes but not in mammals. The exons
encode functional domains of the preproglucagon: S, signal peptide; N,
amino-terminal sequence of proglucagon; Gluc, glucagon; IP, intervening
peptides. The pairs of basic residues that serve as posttranslational
sites of processing of the preproglucagon encoded by the mRNA are
shown. M, Methionine encoded by AUG codon that initiates translation;
Q, glutamine; H, histidine; K, lysine; R, arginine; UN-TX, untranslated
regions of mRNA [Adapted from S. Mojsov et al.:
J Biol Chem 261:11880-11889, 1986 (69 )]. B,
Alternative posttranslational processing of proglucagon in pancreas,
intestine, and brain. Enzymatic cleavages at specific pairs of basic
residues in proglucagon produces numerous multifunctional peptide
hormones involved in nutrient metabolism. K, Lysine; R, arginine. The
major bioactive hormones derived from proglucagon are glucagon in the
pancreatic -cells and GLP-1 (two isoforms, 7-37 and
7-36 NH2) and GLP-2 in the intestinal L cells and brain.
Numbers on proglucagon denote amino acid positions. GRPP,
Glicentin-related pancreatic peptide; Gluc, glucagon; IP-1 and IP-2,
intervening peptides; MPF, major proglucagon fragment.
GLP-1(7-36) is -amidated on the carboxyl-terminal
arginine residue.
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B. Intestinal L cells
Intestinal cells are reported to react with glucagon-specific
C-terminal antisera, although the intestinal immunodeterminant
responsible for the immune reaction appears to differ chemically from
pancreatic glucagon (152, 153). Antibodies directed against the midpart
of glucagon and antibodies against the nonglucagon part of the
glicentin molecule reveal a large population of endocrine cells in the
small and large bowel that express proglucagon and its fragments
(154-156). In contrast to the pancreas where GLPs represent minor
products, GLPs are fully processed in abundance in the intestine,
representing the major source of circulating GLPs (115-117). By virtue
of their ultrastructure as assessed by electron microscopy, these cells
are designated as L cells that clearly differ from pancreatic -cells
in the morphology of the granules (154, 156-158). The intestinal L
cells are flask shaped and open-type, the microvilli reach the
intestinal lumen, and a domain rich in endocrine granules exists near
the basal lamina (159, 160) (Fig. 9). The
shape of the L cells suggests that the cells can respond to changes in
the environment within the intestinal lumen, resulting in a basal
discharge of their granular contents.
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Figure 9. GLP-1-immunoreactive cells in the human rectal
mucosa. The cells occur in all regions of the crypts with a
predominance in the basal region (A). They reach the lumen via slender
apical processes (B and C). Bars = 25 µm.
Short arrows indicate basolateral secretory vesicles;
long arrow indicates luminal villi. [Reproduced with
permission from R. Eissele et al: Eur J Clin
Invest 22:283-291, 1992 (160 ).]
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The L cells are the second most abundant population of endocrine cells
in the human intestine, exceeded only by the population of
enterochromaffin cells. A high abundance of L cells is present in the
distal jejunum and ileum, and an increasing abundance of L cells is
demonstrable along the colon, with the highest concentration in the
rectum (160-163). L cells first appear in human fetuses at the 8th week
of gestation in the ileum, the 10th week in the oxyntic mucosa and
proximal small intestine, and at the 12th week in the colon (164). This
distribution of L cells differs greatly from that of the cells that
secrete GIP, which are located in the more proximal regions of the
jejunum (8, 45-48, 165, 166). The L cells of the small and large bowel
are thought to arise from pluripotent stem cells in the crypts that
also give rise to enterocytes, goblet cells, and Paneth cells (167).
The L cells have a lower rate of turnover than other cell types in the
crypts (168). Most of the L cells reside in the crypts of
Lieberkühn, but a few cells can also be observed in the
intestinal villi.
C. Central nervous system
Before the identification of GLP-1 as a separate
product of the posttranslational processing of proglucagon, it was
recognized that gut-type glucagon immunoreactivity existed within the
central nervous system of several mammalian species (169-173). The
subsequent development of specific antisera allowed for the analysis of
the precise anatomical distribution of GLP-1.
GLP-1-immunoreactive nerve fibers and terminals are widely
distributed throughout the brain; the highest density occurs in the
hypothalamus, thalamus, and septal regions, and the lowest occurs in
the cortex and hindbrain (174-179). Chromatographic analyses of
extracts of rat brainstem and hypothalamus revealed that proglucagon is
processed in a manner similar to that in the intestine, preferentially
giving rise to oxyntomodulin, glicentin, GLP-1, and GLP-2
(116, 177, 179-181). Posttranslational processing of proglucagon may
undergo changes during development, as the predominance of glicentin
and oxyntomodulin over glucagon in the rat hypothalamus increases
dramatically from fetus to adult (182). In rats, monkeys, and man,
GLP-1 has been detected in neuronal cell bodies within the
medulla oblongata (174-176, 179, 183). Within the caudal medulla,
immunostained cell bodies were located within the nucleus of the
solitary tract and the dorsal and ventral parts of the medullary
reticular nucleus (175). GLP-1 neurons of the solitary
tract constitute a distinct noncatecholaminergic cell group that
projects to many sites within the brain, one of which is the
hypothalamic paraventricular nucleus (179). De novo
synthesis of proglucagon occurs in these cell bodies as proglucagon
mRNA is detected by in situ hybridization experiments using
oligonucleotide probes (176, 179, 184).
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VI. Proglucagon Biosynthesis
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A. Organization/structure of the proglucagon gene
The gene encoding glucagon and the GLPs is expressed as a 2-kb
transcript that consists of a 5'-untranslated region, the
protein-coding region comprised of the N-terminal signal sequence, the
proglucagon consisting of the glicentin-specific peptide and followed
in order by the sequences that encode glucagon, GLP-1, and
GLP-2 (Fig. 10). The glucagon,
GLP-1, and GLP-2 sequences are interrupted by short spacer
sequences that encode intervening peptides (Fig. 10). The N-terminal
signal sequence is typical of preprohormones that are destined to
cross-membranes during the biosynthesis of the protein, and the details
of their function are well documented [for review see Ref. 185). It is
somewhat remarkable that the six exons that comprise the transcribed
region of the gene consist of distinct functional domains of the mRNA
and encoded preproglucagon (65, 69). Namely, these regions are the
5'-untranslated sequence, the signal and N-terminal glicentin sequence,
glucagon, GLP-1, GLP-2, and the 3'-untranslated region
(Fig. 10). This exonic arrangement of the preproglucagon gene is a
representative example of the modular arrays of exons that often encode
specific functional domains of proteins (186).
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Figure 10. DNA control elements and interactive transacting
protein factors in the 2,300-bp promoter of the rat glucagon gene.
ISEs, Intestine-specific enhancers [includes the glucagon upstream
enhancer (190 )]; CAP, CREB-associated protein; CBS, CAP-binding site;
CREB, cAMP response element-binding protein; CRE, cAMP response
element; IRBP, insulin responsive binding protein; CES, C/EBP enhancer
site; HNF3, hepatic nuclear factor-3; ETS, ubiquitous developmental
transcription factors; Beta2, Beta2/NeuroD basic helix-loop-helix
factor; Isl-1, islet lim-homeodomain protein; Brn4, brain-4; Cdx2,
caudal-related homeobox-2; Pax6, paired homeobox-6; G1, G2, G3, G4,
major -cell/islet enhancers; TATA, TATA box; TRX, transcription.
[Adapted from J. F. Habener, In: H. C. Fehmann, B. Göke
(eds) The Insulinotropic Gut Hormone Glucagon-Like
Peptide 1, vol 13:15-23, 1997 (586 )].
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B. Regulation of glucagon gene expression
There are three known sites of expression of the proglucagon gene:
the -cells of the pancreatic islets, the L cells predominantly
located in the distal ileum, colon, and rectum, and the nucleus tractus
solitarius in the hindbrain, which is the nucleus of the vagus (X)
nerve. There is also expression of the proglucagon gene in
magnacellular neurons of the hypothalamus. In many instances, nutrients
and effectors that either stimulate or suppress secretion of glucagon
or GLPs (see below) also likewise similarly control expression of the
proglucagon gene at one or more levels of gene transcription, mRNA
stability, or translation. Several factors that stimulate the secretion
of rat intestinal glucagon-like immunoreactive peptides, such as
(Bu)2cAMP, forskolin, and cholera toxin, also elevate
intestinal proglucagon mRNA levels, whereas other factors, phorbol
esters and bombesin, stimulate secretion but not mRNA levels in
intestine (61, 187-189). It is also theoretically possible that
regulation may be exerted at the level of posttranslational processing
of proglucagon to glucagon and GLPs, but that has not been demonstrated
yet. Actually, there is not a great deal known about the mechanisms
involved in the control of proglucagon gene expression.
The promoter of the proglucagon gene has been analyzed in some detail
by several groups of investigators over the past 10-12 yr. Five
important transcriptional DNA control elements have been identified in
the 2.5-kb of DNA sequence that 5' flanks the initiation of
transcription of the rat preproglucagon gene (Fig. 11). The five DNA control sequences of
approximately 20-40 bp have been designated G1, G2, G3, CRE (cAMP
response element), and ISE (intestinal specific element) or GUE
(glucagon upstream enhancer) (190). A sixth subelement within G1 has
been designated G4 (191). The G1 element confers -cell-specific
expression of the glucagon gene in the pancreas, the G2 and G3 elements
are enhancers specific to islet cells (192), the CRE lends cAMP
responsivity to transcription of the preproglucagon gene (193), and ISE
is a determinant for the transcriptional expression of the gene in
intestinal L cells (194). One caveat is that essentially all of the
information on the cis-acting control elements and the
transactivating DNA-binding proteins has been derived from studies in
cultured insulinoma cells that express the glucagon gene. These are
transformed immortalized cells that may or may not be entirely
representative of normal - or L cells in the context of the living
animal.
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Figure 11. Secretory responses of GLP-1
isopeptides GLP-1(7-37) and GLP-1(7-36)amide
to a meal in six nondiabetic subjects. RIAs are relatively specific for
detection of the differences in the COOH-termini of the two
isopeptides. Approximately 80% of the total GLP-1
consists of the GLP-1(7-36)amide. [Adapted with
permission from C. Orskov et al.:
Diabetes 43:535-539,1994 (117 )].
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The G1 element of the preproglucagon gene promoter has been the most
extensively studied of the five control elements identified so far. It
consists of 30-40 nucleotides, is located close to the TATA-box to
which the basal RNA polymerase complex of basal transcription factors
are assembled, and seems clearly to be responsible for allowing the
expression of the preproglucagon gene in -cells (192, 195). The
exclusion of, or mutations within, the G1 element precludes expression
of the gene in -cells. Deoxyribonuclease I (DNase I) footprint
analysis of the G1 element indicates that a large, complex array of
DNA-binding proteins interacts in this region of the promoter (192).
Some progress has been made in the identification of the specific
DNA-binding proteins involved in interactions with the G1 element. As
might have been anticipated, three of the five DNA-binding proteins
thus far identified to act on G1, Brn4 (196), Cdx2 (196-199), and Pax6
(200), are homeotic selector proteins, so called homeoproteins,
critically involved in the determination of the body plan and
organogenesis during development. The other two proteins, E47 and
Beta2/NeuroD, are basic helix-loop-helix proteins also known to be
important in development (201). After completing their roles in
embryonic development, homeodomain proteins typically exert a second
role in the regulation of the expression of key genes in the fully
differentiated cells, namely the -cells with respect to Brn4, Cdx2,
and Pax6. In this regard, it is noteworthy that targeted disruptions of
either the Brn4 (M. G. Rosenfeld, University of California, San
Diego, personal communication) or Pax6 (110, 202) genes in mice results
in the failure of -cells to develop in the pancreas.
The G2 and G3 DNA-control elements in the promoter are enhancers of
proglucagon gene transcription function in islet cells, but are not
restricted to islet cells. Transcription factors in the hepatocyte
nuclear factor family (HNF), HNF3ß and HNF, have been shown to
interact with G2 and G3 (203, 204). Isoforms of HNF-3 thereby either
enhance or repress transcription of the proglucagon gene promoted by
the G1 element and its cognate DNA-binding proteins described above.
The involvement of HNF transcription factors in the regulation of the
expression of the glucagon gene is interesting because the liver and
pancreas (and spleen) are derived from adjacent regions of the gut
endoderm during development [for review see Ref. 108). In conditions
of chronic injury to the pancreas, such as invoked by dietary
deficiencies of methionine or copper, pancreatic acinar tissue
undergoes metaplasia to liver tissue. Evidence has been reported that
the G3 element may serve as a negative insulin response element, and
thereby may account for the paracrine actions of insulin to suppress
glucagon gene expression (194). Transcription of the proglucagon gene
in islet -cell lines is enhanced by phorbol ester-mediated
activation of protein kinase C (205). However, in rat intestine,
phorbol esters have no effect on proglucagon mRNA levels (61). Recent
studies identify the G2 element as the target of the stimulatory
actions of phorbol esters and the interactions of the transcription
factors HNF-3ß and members of the Ets-related transcription factors
(206).
The cAMP response element (CRE) is located in the promoter of the
proglucagon gene adjacent to the G3 element. The CRE confers cAMP
responsivity to the transcription of the proglucagon gene (193, 207).
In studies in vitro in glucagon-producing insulinoma cells,
it is clear that the CRE in the promoter of the proglucagon gene is a
target for interactions with CRE-binding protein (CREB), the
CRE-binding protein involved in mediating cAMP responses of multiple
genes (193, 207-210). Proteins that bind to sites adjacent to the CRE
and inhibit the CREB-mediated cAMP stimulation of glucagon expression,
designated CAPs (CREB-associated proteins), have been described (210).
Notably, NF-Y is reported to bind to DNA sites immediately adjacent to
the CRE of the rat insulin-1 gene promoter and to inhibit
cAMP-responsive gene transcription (211). The identification of a
functional CRE in the promoter of the proglucagon gene is consistent
with the reported findings of cAMP-coupled GLP-1 and GIP
receptors on pancreatic -cells and that GLP-1 and GIP
stimulate the secretion of glucagon from -cells by cAMP-dependent
mechanisms (212-214).
The intestinal L cell permissive enhancer in the promoter of the
proglucagon gene is less well defined compared with the pancreatic
-cell-specific promoter element G1. The identification of an
intestinal-specific promoter element (ISE) was accomplished by
transient transfection-expression studies in SV40 transformed cell
lines obtained from intestinal L cell tumors that arose in mice made
transgenic with a SV40 large T antigen driven by the 2.5-kb 5'-flanking
region of the proglucagon gene (190, 208). The expression of the
proglucagon gene in L cells requires DNA control elements located
between 1,300 and 2,300 bp 5' upstream of the G1, G2, G3, and CRE
elements of the promoter.
C. Posttranslational processing of proglucagon
When the modular exonic arrangement of the proglucagon gene was
first noted (65), it seemed likely that alternative exon splicing would
generate distinct mRNAs each encoding either glucagon or the GLPs.
Indeed, in the frog, lizard, chicken, and fish, alternative RNA
splicing of two proglucagon genes generates proglucagon mRNA
transcripts that encode glucagon and GLP-1, but not GLP-2
in the pancreas, whereas mRNAs for all three are generated in the
intestine (85). However, in mammals, the diversification of the
expression of the preproglucagon gene occurs at the level of
alternative posttranslational processing of proglucagon (60, 68, 69, 215) (Fig. 8). There is a remarkably specific alternative processing of
proglucagon: the predominant bioactive peptide produced in the
pancreatic -cells is glucagon, whereas in the intestines and the
brain the bioactive products produced are predominantly GLPs. Thus, the
alternative processing reflects a dichotomy between the expression of
hormones essential for the regulation of glucose metabolism in the
fasting vs. the fed state. Glucagon is operative during
fasting in mobilizing glucose from peripheral tissues to maintain blood
glucose levels, whereas GLP-1 comes into play during
feeding to augment glucose-dependent insulin release, and possibly to
promote satiety.
Several of the enzymes that posttranslationally cleave proproteins into
peptides or hormones have been identified. These enzymes comprise a
family known as subtilisins or subtilisin-like proprotein convertases,
otherwise known as prohormone convertases (PCs) (216). Two of the five
or six identified convertases, PC2 and PC1/3, are expressed at high
levels in pancreatic islets. Several studies using cell culture models
transfected with expression vectors for recombinant PC2 and PC1/3 have
uniformly established that processing by PC1/3 results in the formation
of GLPs similar to those found in the intestine and PC2 (and possibly
another yet-to-be-identified convertase) contributes to processing
proglucagon in the pancreatic pattern to produce glucagon (124,
217-222). Further, PC2 null mice defective in the expression of PC2
manifest severe fasting hypoglycemia and a reduced rise in blood
glucose levels during an intraperitoneal glucose tolerance test,
consistent with a deficiency of circulating glucagon (223).
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VII. Regulation of GLP Secretion
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A. Overview
Determinations of circulating profiles of immunoreactive
GLP-1 levels have provided information regarding the
physiological processes that regulate GLP-1 secretion.
Before the development of specific GLP-1 RIAs in the late
1980s, L cell secretion was usually quantified as gut glucagon-like
immunoreactivity (gGLI), which includes glicentin plus oxyntomodulin.
Because gGLI is produced in quantitatively identical amounts to
GLP-1 after posttranslational processing of proglucagon
(69, 115, 117, 224-227), studies reporting secretion of gGLI reflect
that of GLP-1. More recently, assays have been
developed utilizing antisera that specifically detect
GLP-1. These procedures may detect both pancreatic and
intestinal derived GLP-1. Although small quantities of
GLP-1 (7-37 and 7-36 amide) may be produced by pancreatic
-cells and cosecreted with glucagon (113-115), the major source of
circulating GLP-1 is the intestinal L cell (116-118). As
discussed below, numerous studies have revealed that the release of
GLP-1 is under the control of nutrients, hormones, and
neural inputs. The result is a biphasic mechanism of release, with both
hormonal and neural mediation of early GLP-1 release
(15-30 min), and direct nutrient contact with L cells mediating later
GLP-1 secretion (30-60 min).
To allow for a direct assessment of the interactions among paracrine,
endocrine, neural, and luminal influences at the level of the L cell,
new in vitro techniques were required. A major factor
impeding studies at the cellular level is the diffuse nature of the
distribution of intestinal L cells. However, models consisting of
primary cultures of rat intestinal cells or canine ileal mucosal cells
have been successfully developed as in vitro strategies to
study the production of GLP-1 (188, 189, 228, 229). The
limited numbers and viability of cells obtained by these techniques in
addition to the heterogeneity of the isolated cells prevent extensive
analysis of proglucagon gene regulation. The development of
tumor-derived cell lines that express proglucagon-derived peptides has
aided in this regard. The GLUTag cell line was developed from
intestinal tumors in proglucagon-SV40 large T antigen transgenic mice
(230, 231) whereas the STC-1 cell line was derived from an intestinal
endocrine tumor that developed in mice carrying the transgenes for the
rat insulin promoter linked to SV40 large T antigen and the polyoma
virus small T antigen (232).
B. Intracellular signals
The development of in vitro methods to study
GLP-1 release at the cellular level has enabled the
analysis of intracellular signal pathways that regulate the secretion
and expression of GLP-1. Studies with intestinal cell
cultures and the L cell line, GLUTag, indicate that the activation of
protein kinase A stimulates both GLP-1 release and
synthesis (61, 188, 189, 208, 229, 233-235). In contrast, activation of
protein kinase C results in an increased secretion of
GLP-1 in intestinal cell cultures (188, 189, 233, 236) and
the GLUTag and STC-1 cell lines (208, 235, 237), but does not appear to
increase transcription of the proglucagon gene (61, 235). Treatment
with the phospholipase C activator -ketoisocaproic acid does not
enhance GLP-1 secretion by either fetal rat intestinal
cultures or GLUTag cells (235). Inhibition of GLP-1
secretion by a calcium channel blocker (CoCl2) and
stimulation of GLP-1 release by increasing intracellular
calcium concentrations indicate a primary role of calcium in basal
secretion by the L cell (233). Thus there may be multiple signals
involved in the L cell response that are perhaps important in allowing
for an integrated response to a variety of different L cell effectors.
C. Carbohydrates
GLP-1 is released into the circulation after a meal
(72, 117, 225, 238-243). Significantly more GLP-1 is
released after a liquid meal than a solid meal of identical composition
(244). The majority of GLP-1 released appears to be in the
form of GLP-1 (7-36 amide) with levels reaching
approximately 50 pM, whereas GLP-1 (7-37)
rises to 10 pM (Fig. 12).
In keeping with the role of GLP-1 as an incretin hormone,
the oral intake of glucose alone stimulates GLP-1 release
in humans (72, 241, 245-250), pigs (251, 252), dogs (253-255), and rats
(116, 256). In contrast to oral glucose administration, elevation of
plasma glucose by the administration of glucose systemically does not
stimulate GLP-1 secretion, indicating the glucose sensing
machinery is distributed on the luminal side of the intestine (236, 241, 257). Infusion of glucose into the intestinal lumen stimulates
GLP-1 release in humans (249), rats (241, 257-261), dogs
(255, 262, 263), and pigs (224). These observations are consistent with
the role of GLP-1 as an important incretin hormone acting
on the pancreatic ß-cells to stimulate appropriate insulin release
after glucose absorption.
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Figure 12. A, Effects of different concentrations of
synthetic GLP-1(7-37) and glucagon on insulin secretion
from the perfused rat pancreas. Background perfusate contains 6.6
mM glucose. B, Glucose dependency of effect of
10-9 M GLP-1(7-37) on insulin
secretion from isolated perfused rat pancreas. Insulin responses at 2.8
and 6.6 mM glucose determined by scale at
left; those at 16.7 mM determined by scale
at right. [Adapted with permission from G. C. Weir
et al.: Diabetes 38:338-342, 1989
(587 )].
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The release of GLP-1 from the isolated perfused ileum
requires sodium (241, 264), implicating the brush-border sodium/glucose
cotransporter in the glucose effect. Consistent with these findings,
other sugars that utilize this cotransporter for absorption across the
intestinal epithelium, e.g., galactose, also stimulate
GLP-1 release (241, 261, 262). Nontransportable sugars,
e g., 2-deoxyglucose, or sugars using a different mechanism
of transport, e.g., fructose and lactose, do not stimulate
the release of GLP-1 (255, 262). Furthermore,
GLP-1 release from canine or rat ileum perfused in
response to the carbohydrates methyl- D-glucoside and
3-O-methyl-D-glucose indicate that intracellular metabolism
and intracellular removal, respectively, are not essential to induce
GLP-1 secretion in rats (261, 262).
Although high concentrations of glucose (28 mM) have been
demonstrated to stimulate GLP-1 secretion from isolated
rat intestinal cell cultures (228), it is unlikely that glucose
normally acts directly on L cells. Indeed, a recent study did not
observe any effect of glucose (5-25 mM) on
GLP-1 secretion from isolated canine intestinal cells
(189). Under normal feeding conditions, the majority of glucose is
absorbed before reaching the ileum (265). In addition, the rapid
GLP-1 secretory response to oral glucose (72, 240, 241, 245, 247) suggests that glucose must activate the release of
GLP-1 by means other then a direct effect on L cells.
D. Fats
In addition to glucose, fats appear to stimulate the release of
proglucagon-derived peptides, perhaps related to the roles of both
oxyntomodulin and GLP-1 as enterogastrones, or inhibitors
of gastric function (266-271). The secretion of GLP-1 is
increased by ingestion of mixed fats or triglycerides in humans (241,
247, 249, 272-275), and dogs (274) and by placement of mixed fats
directly into the intestinal lumen of rats (257, 276) and pigs (252).
Interestingly, Roberge and Brubaker (276) discovered that placement of
fat in the duodenum of rats stimulates GLP-1 secretion
independently of the contact of nutrients with the distal L cells.
Furthermore, duodenal fat increased the secretion of GLP-1
into the circulation to the same extent as was observed after the
direct administration of fat into the ileum (257, 276). These
observations suggest the existence of a proximal-distal loop regulating
the L cell response to ingested nutrients (276). Such a mechanism could
contribute to the significant increase in circulating
GLP-1 levels observed within 5-10 min of ingesting a meal,
before contact of nutrients with the L cells (72, 240, 241, 247, 248, 250, 277). As discussed below, among the potential mediators of such a
loop are various endocrine and neuroendocrine peptides, as well as
neurotransmitters.
The observation of fatty acid-induced GLP-1 release from
isolated intestinal cell cultures suggests that fatty acids can act
directly on the L cell (187, 278, 279). Interestingly, bile acids
appear to increase the secretion of proglucagon-derived peptides in
humans (280), dogs (281), and rats (260), suggesting that the arrival
of bile into the ileum may play an important feedback message for the
release of GLP-1. Results obtained with fatty acids
indicate that both the chain length and degree of saturation of the
fatty acids affect the ability of fats to stimulate GLP-1
secretion. Monounsaturated long-chain fatty acids (=C16) are preferred
over short-chain or medium-chain, polyunsaturated or saturated fatty
acids (187, 233, 235, 251, 273, 278, 279). However, long-term exposure
of rats to short-chain fatty acids derived from a diet containing
readily fermentable fibers increases proglucagon mRNA levels and
secretion of GLP-1 in response to a glucose challenge
(282).
E. Proteins
Mixed meals that contain proteins increase GLP-1
secretion in humans (72, 117, 225, 238-243, 247) and rats (227, 283).
However, either amino acids or protein alone did not consistently
increase GLP-1 release in in vivo studies in
humans (241, 247,
Top
Abstract
I. Introduction
