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Endocrine Unit, Department of Metabolic Medicine, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom W12 0NN
Address all correspondence and requests for reprints to: Prof. S. R. Bloom, Department of Metabolic Medicine, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom W12 0NN. E-mail: s.bloom{at}ic.ac.uk
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Abstract |
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Introduction |
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GLP-1 is a potent physiological insulinotropic incretin hormone in both rat and man (4, 5, 6, 7). However, when administered into the rat cerebroventricular system (ICV), GLP-1 acts a potent inhibitor of food intake (8, 9, 10) The paraventricular nucleus (PVN) of the hypothalamus is an area involved in the control of food intake and has a high density of GLP-1 receptors (GLP-1R) (11, 12). When GLP-1 is injected into the PVN of fasted rats, a marked anorectic effect has been observed (13). Recently, GLP-2 has also been found to be an inhibitor of food intake in the rat (14). Glicentin acts within peripheral tissues, with its roles including the inhibition of gastric acid secretion in the rat (15). No effect of glicentin in the CNS has been reported to date.
OXM, originally isolated from porcine jejuno-ileal cells, is a 37-amino acid peptide, containing the entire sequence of glucagon, with a basic octapeptide C-terminal extension, known as spacer peptide-1 (SP-1) (16, 17, 18). OXM potently inhibits gastric acid secretion and pancreatic enzyme secretion when infused iv (19, 20). The C-terminal fragment of OXM, SP-1, has been described as the minimal active structure of OXM (21), as it has been found to mimic the actions of OXM in gastric mucosa, albeit less potently (19, 22, 23).
It is not known whether OXM has a specific receptor distinct from those of the other proglucagon products. Originally, Depigny et al. (24) reported that OXM mediates its actions via a specific receptor in oxyntic glands. However, this was later disputed when Gros et al. (25) reported that OXM could activate second messenger systems via a GLP-1-preferring receptor in the somatostatin-secreting cells line, RIN T3. More recently, it was demonstrated by Schepp et al. (26) that the actions of OXM are mediated via the GLP-1R in rat parietal cells. To date, no specific binding site for OXM in the CNS has been identified.
Little is known about the neuroanatomy of endogenous OXM. Furthermore, RIAs using antisera that are not specific for both the C- and N-termini of OXM cross-react with glucagon and glicentin. For this reason, specific assays for OXM, as described previously (27), might not be accurate. However, glucagon-like immunoreactivity, 90% of which is OXM and glicentin, has been detected at low levels in the rat CNS in the medulla oblongata, olfactory bulbs, cerebellum, and cortex. Higher levels of glucagon-like immunoreactivity have been detected in the hypothalamus, but none has been identified in the pituitary (27).
Exendin-4 is a 39-amino acid peptide isolated from the salivary glands of the Gila monster (Heloderma suspectum) (28). It is a potent agonist at the GLP-1R (29, 30, 31) and when injected ICV inhibits food intake in a dose-dependent manner (32). A fragment of exendin-4, exendin-(9–39), has been reported to be a potent and selective antagonist at the GLP-1R and, when coadministered, blocks the actions of GLP-1 in both the CNS and the periphery (33, 34).
Using both fasted and nonfasted rat models, we report here the relative effects of proglucagon-derived and related peptides on food intake and feeding behavior after injection ICV or iPVN compared with the effects of GLP-1. In addition, using the GLP-1R antagonist, exendin-(9–39), together with in vitro receptor binding assays, we investigated the activity of OXM at the GLP-1R.
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Materials and Methods |
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Animals
Adult male Wistar rats (Imperial College School of Medicine,
Hammersmith Hospital) were maintained in individual cages under
controlled conditions of temperature (21-23 C) and light (12 h of
light, 12 h of darkness) with ad libitum access to food
(RM1 diet, Special Diet Services UK Ltd., Witham, UK) and tap water.
Animals were handled daily after recovery from surgery until completion
of the studies. All animal procedures undertaken were approved by the
British Home Office Animals (Scientific Procedures) Act 1986 (Project
License PIL 90/1077).
ICV and iPVN cannulation and infusions of test compounds
Animals had permanent stainless steel guide cannulas (Plastics
One, Roanoke, VA) stereotactically implanted ICV or iPVN (35, 36). Substances were administered as previously described
(35, 36). All fasted animal studies were carried out in
the early light phase, between 0900–1100 h, after a 24-h fast, and
food intake was measured 1, 2, 4, 8, and 24 h postinjection.
Nonfasted animal studies were carried out at the onset of the dark
phase.
Feeding study protocols
Comparison of the effect of proglucagon-derived products and
related peptides on food intake. Rats were injected ICV with 10
µl saline, GLP-1 [3 nmol, a dose that we have
consistently found to significantly inhibit food intake (unpublished
observations)], OXM (3 nmol), glucagon (3 nmol), or glicentin (3 nmol;
n = 8/group).
Rats were injected iPVN with 1 µl saline, GLP-1 [1.0 nmol; a dose that we have found to inhibit food intake (unpublished observations)], OXM (1.0 nmol), glicentin (1.0 nmol), glucagon (1.0 nmol), or SP-1 (3.0 nmol; n = 12–15/group). Exendin-4, when injected ICV, inhibits food intake more potently than GLP-1 (32). Therefore, exendin-4 was injected iPVN at a dose of 0.03 nmol.
Investigation of the effect of increasing doses of OXM on food intake. In study 2a, rats were injected ICV with saline, GLP-1 (3 nmol), or OXM (0.3, 1, 3, or 10 nmol; n = 8/group). In study 2b, rats were injected iPVN with saline, GLP-1 (1.0 nmol), or OXM (0.1, 0.3, or 1.0 nmol; n = 12–15/group). To assess whether OXM acts via the GLP-1 receptor, a study using the GLP-1 receptor antagonist exendin-(9–39) was performed. We have shown that the ratio of exendin-(9–39) to GLP-1 required to antagonize the GLP-1R is approximately 10:1 (8).
To assess whether OXM acts via the GLP-1R: study using GLP-1R antagonist, exendin-(9–39). In study 3a, rats were injected ICV with saline, GLP-1 (3 nmol), GLP-1 (3 nmol) plus exendin-(9–39) (30 nmol), OXM (3 nmol), OXM (3 nmol) plus exendin-(9–39) (30 nmol), or exendin-(9–39) alone (30 nmol). In study 3b, rats were iPVN injected with saline, GLP-1 (1 nmol), GLP-1 (1 nmol) plus exendin-(9–39) (10 nmol), OXM (1 nmol), OXM (1 nmol) plus exendin-(9–39) (10 nmol), or exendin-(9–39) alone (10 nmol; n = 10–12/group).
Nighttime feeding and behavioral analysis. It is possible
that OXM inhibits food intake via nonspecific taste aversion, and that
it is not a true satiety factor. Therefore, ICV cannulated rats were
administered GLP-1 (3 nmol), OXM (3 nmol), or saline
(n = 6/group) at the onset of the dark phase. Food intake was
measured 1 h postinjection, and behavior was assessed. Rats were
observed for 1 h postinjection using a behavioral score sheet as
described previously (37). A summary of behaviors is shown
in Table 1
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-counter. Specific (saturable) binding was calculated as the
difference between the amount of
[125I]GLP-1 bound in the absence
(total binding) and presence of 1 µM GLP-1
or OXM (nonsaturable binding). All curves were performed with points in
triplicate. IC50 values were calculated using the
Prism 3 program (GraphPad Software, Inc., San Diego,
CA).
Statistics
For food intake analyses, data are presented as the mean ±
SEM. Statistical differences between experimental groups
were determined by ANOVA, followed by a post-hoc least
significant difference test (Systat 8.0, Evanston, IL). For behavioral
analyses, data are expressed as the median number of occurrences of
each behavior and the range. Comparisons between groups were made using
the Mann-Whitney U test (Systat 8.0). In all cases, P
< 0.05 was considered statistically significant.
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Results |
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ICV administration. OXM and GLP-1 (3 nmol)
significantly reduced refeeding. This inhibition of
food intake lasted until 4 h postinjection (Fig. 1A). Glucagon and glicentin (3 nmol)
failed to affect food intake at any time point (Fig. 1A and Table 2A).
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and
Table 2B). It is known that exendin-4 potently inhibits refeeding when
injected ICV (32). However, it was found that exendin-4
also inhibits refeeding when administered iPVN, at all time points
until 24 h postinjection. Glicentin, glucagon (1 nmol), and SP-1
(3 nmol) failed to affect food intake at any time point when injected
iPVN.
Effects of increasing doses of OXM on food intake
ICV administration. When injected ICV, OXM reduced refeeding
in a dose-dependent manner, reaching a maximal effect at a dose of 3
nmol 1, 2, and 4 h postinjection (Fig. 2A and Table 3A).
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and Table 3B). Effect of OXM in ICV-cannulated nonfasted rats at the onset of the dark phase. The dark phase is the rats’ natural feeding time. Therefore, assessing the effect of a putative satiety factor in nonfasted animals at this time would represent a more physiological effect.
Effect of OXM on food intake. When injected in the early dark
phase, both GLP-1 and OXM (3 nmol) significantly reduced
food intake compared with that of saline-treated animals 1 h
postinjection [OXM (3 nmol), 1.0 ± 0.2 g;
GLP-1 (3 nmol), 1.1 ± 0.1 g; saline, 2.3
± 0.2 g; P < 0.05; Fig. 3A].
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]. There was no change in
grooming, still, head down, burrowing, or locomotion episodes.
To assess whether OXM acts via the GLP-1R, a study using the GLP-1R
antagonist, exendin-(9–39) was performed
ICV administration. ICV coadministration of the
GLP-1 receptor antagonist exendin-(9–39) with
GLP-1 at a ratio of 10:1 (antagonist/agonist) blocked the
anorectic effects of GLP-1 [1 h:
GLP-1 (3 nmol), 1.3 ± 0.3 g (P
< 0.005); GLP-1 (3 nmol) plus exendin-(9–39) (30 nmol),
4.2 ± 1.0 g (P = NS); saline, 6.2 ±
0.4 g; Fig. 4A]. Furthermore,
coadministration of exendin-(9–39) with OXM resulted in
attenuation of the anorectic effect of OXM [1 h: OXM (3 nmol),
1.6 ± 0.7 g (P < 0.005); OXM (3 nmol) plus
exendin-(9–39) (30 nmol), 5.3 ± 0.4 g (P =
NS); saline, 5.0 ± 0.4 g; Fig. 4A].
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].
Receptor binding assays
The affinity (IC50) of GLP-1
for the GLP-1 receptor in rat hypothalamic membrane
preparations was 0.16 nM (Fig. 5), which was similar to values
previously reported (40, 41). The affinity of OXM for the
GLP-1 receptor in the same membrane preparations was 8.2
nM (Fig. 5), which is approximately 2 orders of magnitude
weaker than that of GLP-1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been suggested that there is an OXM-specific binding site in gastric mucosa (24). However, no such binding site has been identified in the CNS. Therefore, it was proposed that OXM mediated its effects via the hypothalamic GLP-1R, as GLP-1 and OXM have similar potency in feeding studies. We have shown that OXM has a nanomolar affinity for the GLP-1R (IC50 = 8.2 nM). This affinity is approximately 2 orders of magnitude weaker than that of GLP-1 (IC50 = 0.16 nM). Yet despite this reduced affinity for the GLP-1R, OXM reduces food intake to the same magnitude. One explanation for this is that OXM might act through both the GLP-1R and its own receptor in the hypothalamus. Thus, OXM could elicit a response comparable to that of GLP-1 despite its lower affinity for the GLP-1R.
Exendin-(9–39), a fragment of the GLP-1R agonist exendin-4, is reported to be a potent and selective antagonist at the GLP-1R (33, 34). When GLP-1 and exendin-(9–39) are coinjected, the anorectic actions of GLP-1 are blocked (8). We have shown that in addition to this, when OXM is coinjected with exendin-(9–39), the anorectic effects of OXM are also completely blocked. This would strengthen the argument that OXM is mediating its effects via the GLP-1R. However, the specificity of exendin-(9–39) for the GLP-1R has recently been questioned. Tang-Christensen et al. (14) reported that exendin-(9–39) blocked the anorectic effect of GLP-2. However, they thought it unlikely that the anorectic effects of GLP-2 were mediated via the GLP-1R. Rather, they suggested that GLP-2 exerts its actions through a specific GLP-2R (14, 43, 44). Therefore, exendin-(9–39) might bind to receptors other than the GLP-1R, perhaps a putative OXM-receptor.
Exendin-4 reduces food intake with greater potency than GLP-1 (32) or OXM. Here, we have shown that exendin-4 at a dose of only 30 pmol continued to inhibit feeding until 24 h postinjection when administered iPVN. The resistance of exendin-4 to dipeptidyl peptidase IV, the endopeptidase responsible for the short half-life of GLP-1 (45), could in part explain its longer action.
To further investigate the potential roles of other members of the proglucagon family, we investigated the effects of glicentin and glucagon after an acute ICV injection in fasted rats. No effect on fasting-induced food intake was seen after the administration of these peptides. In addition, there was no effect of these peptides when they were administered iPVN. When SP-1, the putative minimal active structure of OXM, was injected iPVN, no inhibition of food intake was observed.
We have shown for the first time that ICV- and iPVN-injected OXM inhibits food intake in fasted and nonfasted animals potently and in a sustained manner. Coadministration of OXM with exendin-(9–39) blocks the anorectic effect of OXM, implying that OXM might be mediating its actions via the GLP-1R. However, recent evidence questioning the specificity of exendin-(9–39) for the GLP-1R coupled with the finding that OXM has a reduced affinity for the GLP-1R suggest that OXM mediates its actions partly via the GLP-1R and partly via an OXM-specific receptor that remains to be identified.
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Acknowledgments |
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Footnotes |
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1 Current address: AstraZeneca, 12F24
Mereside, Alderley Park, Macclesfield, Cheshire, United Kingdom SK10
4TG. 
Abbreviations: CNS, Central nervous system; GLP-1, glucagon-like peptide-1; GLP-1R, glucagon-like peptide-1 receptor; ICV, intracerebroventricularly; iPVN, into the hypothalamic paraventricular nucleus; OXM, oxyntomodulin; SP-1, spacer peptide-1.
Received March 6, 2001.
Accepted for publication June 18, 2001.
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-melanocyte-stimulating hormone. Diabetes 49:177–182[Abstract]
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