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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


    Abstract
 Top
 Abstract
 I. Introduction
 II. History of the...
 III. Discovery of GLP-1
 IV. Structures of GLPs...
 V. Tissue Distribution of...
 VI. Proglucagon Biosynthesis
 VII. Regulation of GLP...
 VIII. Metabolism of GLPs
 IX. Physiological Actions of...
 X. GLP Receptors
 XI. Pathophysiology of GLP-1
 XII. GLP-1 as a...
 XIII. Future Directions
 References
 

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 {alpha}-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


    I. Introduction
 Top
 Abstract
 I. Introduction
 II. History of the...
 III. Discovery of GLP-1
 IV. Structures of GLPs...
 V. Tissue Distribution of...
 VI. Proglucagon Biosynthesis
 VII. Regulation of GLP...
 VIII. Metabolism of GLPs
 IX. Physiological Actions of...
 X. GLP Receptors
 XI. Pathophysiology of GLP-1
 XII. GLP-1 as a...
 XIII. Future Directions
 References
 
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 {alpha}-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).


    II. History of the Incretin Concept: Discovery of Gastric Inhibitory Polypeptide
 Top
 Abstract
 I. Introduction
 II. History of the...
 III. Discovery of GLP-1
 IV. Structures of GLPs...
 V. Tissue Distribution of...
 VI. Proglucagon Biosynthesis
 VII. Regulation of GLP...
 VIII. Metabolism of GLPs
 IX. Physiological Actions of...
 X. GLP Receptors
 XI. Pathophysiology of GLP-1
 XII. GLP-1 as a...
 XIII. Future Directions
 References
 
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. 1Go). 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.].

 
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. 2Go). 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.



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Figure 2. The enteroinsular axis. After ingestion of nutrients, hormone secretion from different cell types of the pancreatic islets [A ({alpha}), B (ß), D ({delta}), PP] may be modified by one or more modalities of: I, endocrine transmission; II, neuro-transmission; and III, direct substrate stimulation. [Reproduced with permission from W. Creutzfeldt: Diabetologia 16:75-85, 1979 (29 ) © Springer-Verlag].

 
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).


    III. Discovery of GLP-1
 Top
 Abstract
 I. Introduction
 II. History of the...
 III. Discovery of GLP-1
 IV. Structures of GLPs...
 V. Tissue Distribution of...
 VI. Proglucagon Biosynthesis
 VII. Regulation of GLP...
 VIII. Metabolism of GLPs
 IX. Physiological Actions of...
 X. GLP Receptors
 XI. Pathophysiology of GLP-1
 XII. GLP-1 as a...
 XIII. Future Directions
 References
 
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. 3Go). 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. 3Go), 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.

 

    IV. Structures of GLPs and Family of Glucagon-Related Peptides
 Top
 Abstract
 I. Introduction
 II. History of the...
 III. Discovery of GLP-1
 IV. Structures of GLPs...
 V. Tissue Distribution of...
 VI. Proglucagon Biosynthesis
 VII. Regulation of GLP...
 VIII. Metabolism of GLPs
 IX. Physiological Actions of...
 X. GLP Receptors
 XI. Pathophysiology of GLP-1
 XII. GLP-1 as a...
 XIII. Future Directions
 References
 
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. 3Go). 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. 4Go), and the products derived from proglucagon, glucagon (Fig. 5Go), and GLP-1 (Fig. 6Go) 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.

 
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).


    V. Tissue Distribution of the Expression of GLPs
 Top
 Abstract
 I. Introduction
 II. History of the...
 III. Discovery of GLP-1
 IV. Structures of GLPs...
 V. Tissue Distribution of...
 VI. Proglucagon Biosynthesis
 VII. Regulation of GLP...
 VIII. Metabolism of GLPs
 IX. Physiological Actions of...
 X. GLP Receptors
 XI. Pathophysiology of GLP-1
 XII. GLP-1 as a...
 XIII. Future Directions
 References
 
A. Pancreatic {alpha}-cells
Pancreatic {alpha}-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 {alpha}-cells were shown by immunofluorescence staining studies to be the source of glucagon (102). The {alpha}-cells are one of four distinct polypeptide-secreting islet cell types: glucagon-secreting {alpha}-cells, insulin- and amylin-secreting ß-cells, somatostatin-secreting {delta}-cells, and pancreatic-polypeptide-secreting F cells. These cells are arranged in highly organized patterns within the islets. In rodents, {alpha}-cells and {delta}-cells exist on the surface or mantle of the islet surrounding the core of ß-cells, although the patterns of distribution of the {alpha}-, {delta}-, 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 {alpha}-cells (110), whereas disruption of Pax4 results in the absence of mature ß- and {delta}-cells (111) (Fig. 7Go). 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 {delta}-cells and shunts development to the {alpha}-cell lineage. The Pax-6 knockout does the opposite: {alpha}-cells do not develop, but some development occurs in ß- and {delta}-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 )].

 
As illustrated in Fig. 8Go, cell-specific processing of proglucagon in pancreatic {alpha}-cells leads primarily to the production of glucagon. However, immunoreactive GLP-1 is detectable in rat pancreatic {alpha}-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 {alpha}-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 {alpha}-amidated on the carboxyl-terminal arginine residue.

 
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 {alpha}-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. 9Go). 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 ).]

 
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).


    VI. Proglucagon Biosynthesis
 Top
 Abstract
 I. Introduction
 II. History of the...
 III. Discovery of GLP-1
 IV. Structures of GLPs...
 V. Tissue Distribution of...
 VI. Proglucagon Biosynthesis
 VII. Regulation of GLP...
 VIII. Metabolism of GLPs
 IX. Physiological Actions of...
 X. GLP Receptors
 XI. Pathophysiology of GLP-1
 XII. GLP-1 as a...
 XIII. Future Directions
 References
 
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. 10Go). The glucagon, GLP-1, and GLP-2 sequences are interrupted by short spacer sequences that encode intervening peptides (Fig. 10Go). 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. 10Go). 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 {alpha}-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 )].

 
B. Regulation of glucagon gene expression
There are three known sites of expression of the proglucagon gene: the {alpha}-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. 11Go). 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 {alpha}-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 {alpha}- 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 )].

 
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 {alpha}-cells (192, 195). The exclusion of, or mutations within, the G1 element precludes expression of the gene in {alpha}-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 {alpha}-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 {alpha}-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{alpha}, 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 {alpha}-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 {alpha}-cells and that GLP-1 and GIP stimulate the secretion of glucagon from {alpha}-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 {alpha}-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. 8Go). There is a remarkably specific alternative processing of proglucagon: the predominant bioactive peptide produced in the pancreatic {alpha}-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).


    VII. Regulation of GLP Secretion
 Top
 Abstract
 I. Introduction
 II. History of the...
 III. Discovery of GLP-1
 IV. Structures of GLPs...
 V. Tissue Distribution of...
 VI. Proglucagon Biosynthesis
 VII. Regulation of GLP...
 VIII. Metabolism of GLPs
 IX. Physiological Actions of...
 X. GLP Receptors
 XI. Pathophysiology of GLP-1
 XII. GLP-1 as a...
 XIII. Future Directions
 References
 
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 {alpha}-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 {alpha}-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. 12Go). 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 )].

 
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-{alpha} 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,