Address correspondence and reprint requests to Dr. E. Blázquez at Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Complutense, 28040-Madrid, Spain.
Abstract : This study was designed to gain better insight into the relationship between glucagon-like peptide-1 (GLP-1) (7-36) amide and vasopressin (AVP) and oxytocin (OX). In situ hybridization histochemistry revealed colocalization of the mRNAs for GLP-1 receptor, AVP, and OX in neurons of the hypothalamic supraoptic and paraventricular nuclei. To determine whether GLP-1(7-36)amide alters AVP and/or OX release, both in vivo and in vitro experimental study designs were used. In vivo, intravenous administration of 1 μg of GLP-1(7-36)amide into the jugular vein significantly decreased plasma AVP and OX concentrations. In vitro incubation of the neurohypophysis with either 0.1 or 1 μg of GLP-1(7-36)amide did not modify the release of AVP. However, addition of 1 μg of GLP-1(7-36)amide to the incubation medium increased slightly the secretion of OX. The coexpression of GLP-1 receptor and AVP mRNAs in hypothalamic supraoptic and paraventricular nuclei gives further support to the already reported central effects of GLP-1(7-36)amide on AVP. Our findings also suggest a dual secretory response of AVP and OX to the effect of GLP-1(7-36)amide, which most likely is related to the amount and/or the route of peptide administration.
The identification of specific binding sites for GLP-1(7-36)amide in both the anterior (Kanse et al., 1988 ; Calvo et al., 1995a ; Alvarez et al., 1996) and posterior (Göke et al., 1995) lobe of the rat pituitary gland has been reported. Significant amounts of the GLP-1 receptor have been found in the pituitary gland, and this correlates well with our studies using in situ hybridization in which specific labeling for the GLP-1 receptor mRNA was evenly distributed throughout the anterior pituitary (Alvarez et al., 1996). It is noteworthy that by using quantitative in vitro autoradiography, a high density of specific binding sites for GLP-1(7-36)amide was characterized in sections of the posterior pituitary lobe (Göke et al., 1995). These findings may suggest a relationship between GLP-1(7-36)amide and oxytocin (OX) and vasopressin (AVP) activities. To gain further insight into these processes, the possible colocalization of the mRNAs for the GLP-1 receptor with OX and AVP in the magnocellular neurons of the supraoptic (SON) and paraventricular (PVN) nuclei of the rat hypothalamus was studied, in addition to the GLP-1(7-36)amide induced release of both peptides.
Male Wistar rats (Criffa, Barcelona, Spain) weighing 250 g were maintained in a temperature- and light-controlled environment on a 12-h light/dark cycle (lights on at 8 : 00 a.m.) with free access to food and water. All procedures were carried out according to ECC ethical regulations for animal research.
In situ hybridization histochemistry
In situ hybridization was performed as previously described (Chowen et al., 1993). Antisense or sense digoxigenin-labeled cRNA probes were generated with SP6 or T7 RNA polymerase in pGLPR-1 (Thorens, 1992) using the Dig RNA labeling kit (Boehringer Mannheim, Barcelona, Spain). The GLP-1 receptor probe was hydrolyzed in bicarbonate buffer to an average length of 150 bases. Detection of the digoxigenin-labeled probe was carried out according to the manufacturer's instructions (Boehringer Mannheim). The sense cRNA probe was used as a specificity control and under identical conditions showed no detectable labeling.
In situ hybridization double-labeling experiments were done using the antisense digoxigenin-labeled cRNA probe complementary to GLP-1 receptor mRNA and 35S-labeled cRNA probes for the localization of AVP or OX mRNA transcripts. AVP and OX riboprobes were synthesized by using T3 polymerase in the pGEM3-AVP4c plasmid containing a 241-bp fragment of exon C of AVP or in pNCO3-OTexC, containing a 105-bp NcoI6BglI fragment of exon C of OX. The plasmids containing the AVP and OX cDNAs were gifts from Dr. Thomas G. Sherman (Pittsburgh, PA, U.S.A.), and the pGLPR-1 plasmid was generously provided by Dr. B. Thorens (Lausanne, Switzerland). In vitro transcription for radioactive probes was carried out with a total UTP concentration of 25 mM in which the proportion of 35S-labeled UTP was 10%.
In vivo and in vitro secretion of AVP and OX
To study the effect of GLP-1(7-36)amide on the release of both AVP and OX, an experimental design was developed that included two approaches. For the in vivo studies, 1 day before the experiment rats were lightly anesthetized with ether, and a Silastic catheter was introduced into the jugular vein and anchored in place. In an attempt to maintain catheter permeability, it was filled with heparin. The rats recovered rapidly, and the effect of GLP-1(7-36)amide (Peninsula Laboratories, St. Helens, Merseyside, U.K.) on in vivo AVP and OX release was studied the following day in conscious animals. One milliliter blood samples were collected in prechilled tubes containing EDTA (1.2 mg/ml) at 0, 5, 10, 15, and 30 min after intravenous administration of 1 μg of GLP-1(7-36)amide dissolved in 0.9% NaCl containing 0.25% bovine serum albumin. After centrifugation, plasma was kept frozen at -20°C until analyzed.
For the in vitro studies, the posterior lobe of the pituitary gland was dissected out and preincubated for 30 min at 37°C in Krebs-Ringer-bicarbonate buffer, pH 7.4, plus glucose (1 mg/ml) and bovine serum albumin (0.25%) in an atmosphere of 95% O2/5% CO2. Thereafter, the medium was removed, 1 ml of the same buffer was added, and the tissue was incubated for 1 h under the same experimental conditions, except for the tubes in which 0.1 or 1 μg of GLP-1(7-36)amide was added. At the end of the incubation periods, the supernatants were kept frozen at -20°C until analyzed.
(Arg8)-AVP and OX were determined by radioimmunoassay by using kits purchased from Peninsula Laboratories. Before the peptides were measured, plasma was extracted and separated from potentially interfering substances using C18 SEP columns (Peninsula Laboratories). Rabbit antisera for the peptides were highly specific.
Results are expressed as means ± SEM. Statistical analysis of the results was performed by one-way ANOVA followed by the Fisher test for multiple group comparisons. Statistical analysis was conducted with the Statview 512 program for Macintosh.
In situ hybridization histochemistry
The PVN and SON were found to express the GLP-1 receptor gene at relatively abundant levels (Fig. 1). In both the PVN and SON, GLP-1 receptor mRNA was colocalized in neurons expressing either AVP (Fig. 1B and E) or OX (Fig. 1C and F). In the SON, ~80% of the neurons expressing OX or AVP also contained detectable levels of GLP-1 receptor mRNA. In the PVN, ~50% of the OX or AVP mRNA-positive neurons also expressed detectable levels of GLP-1 receptor mRNA.
In vivo effect of GLP-1(7-36)amide on the release of both AVP and OX
As shown in Fig. 2, intravenous administration of the placebo did not significantly modify AVP levels during the 30 min of the test. In contrast, intravenous administration of 1 μg of GLP-1(7-36)amide reduced plasma AVP concentrations. This reduction was statistically significant (p < 0.05) 15 min after the administration of the peptide as compared with the values at time 0 in the same experimental group, or when the values obtained at 15 min were compared between the two groups of animals.
In addition, intravenous administration of GLP-1(7-36)amide produced a significant (p < 0.05) reduction in plasma OX concentrations (Fig. 3) as determined by intragroup (time 0 versus 5, 10, and 15 min after peptide administration) or intergroup (at 10 and 30 min between experimental groups) statistical comparisons.
Effect of GLP-1(7-36)amide on the in vitro release of AVP and OX from the posterior lobe of the pituitary
In an attempt to determine whether the effect of GLP-1(7-36)amide on AVP and OX release in vivo was due to a direct action, the isolated neurohypophysis was incubated in vitro in the absence or presence of 0.1 or 1 μg of GLP-1(7-36)amide. When AVP concentrations were determined in the incubation media, no statistical difference (p > 0.05) among the three experimental groups was found (Fig. 4). Similar results were obtained when the release of OX by the neurohypophysis incubated with or without 0.1 μg of GLP-1(7-36)amide was compared (Fig. 5). However, the addition of 1 μg of GLP-1(7-36)amide to the incubation medium increased the secretion of OX.
GLP-1 receptors have been characterized in the anterior pituitary gland (Kanse et al., 1988 ; Calvo et al., 1995a ; Alvarez et al., 1996) and neurohypophysis (Göke et al., 1995), suggesting a physiological role of GLP-1(7-36)amide in these organs, which may include regulation of the secretion of the hormones synthesized or stored in the cells of the rat pituitary. Indeed, specific binding sites and mRNA of GLP-1 receptors have been found in the α-thyrotropin thyrotroph cell line, whereas this peptide increases cyclic AMP concentrations and thyrotropin release from dispersed anterior pituitary cells in a dose-dependent manner (Beak et al., 1996). Furthermore, the results presented here showing the colocalization of GLP-1 receptors with AVP or OX mRNAs in neurons of the PVN and SON of the hypothalamus suggest that GLP-1(7-36)amide may be involved in certain metabolic aspects of magnocellular neurons, such as regulating the synthesis and/or secretion of the latter two neuropeptides. Further support for this view comes from studies demonstrating that intracerebroventricular injection of GLP-1(7-36)amide induces c-fos expression in the medial parvicellular region of the PVN, as well as in the magnocellular neurons of the PVN and SON (Larsen et al., 1997).
Moreover, central and peripheral administration of GLP-1 (7-36)amide or its agonist exendin-4 significantly inhibits water intake (Navarro et al., 1996 ; Tang-Christensen et al., 1996). This effect is blocked by the GLP-1 antagonist exendin(9-39), whereas the inactive GLP-1(1-36)amide has no effect on fluid intake. Exendin-4 is an agonist and exendin(9-39) a potent antagonist of the GLP-1 receptor, which is useful in defining the role of GLP-1(7-36)amide in different physiological effects.
Central injection of GLP-1(7-36)amide significantly increases plasma concentrations of AVP (Tang-Christensen et al., 1996 ; Larsen et al., 1997), but does not modify statistically the levels of circulating OX (Larsen et al., 1997). Furthermore, administration of this peptide produces a dose-dependent increase in urine excretion, natriuresis, reduction in the K+/Na+ ratio, and no changes in plasma osmolarity (Tang-Christensen et al., 1996). It is interesting that our results presented in this article show that peripheral intravenous administration of GLP-1(7-36)amide reduces significantly the circulating concentrations of AVP and OX, in contrast to the findings reported by others in which the central administration of the peptide produces an important increase in the secretion of AVP (Tang-Christensen et al., 1996 ; Larsen et al., 1997). The different effects of GLP-1(7-36)amide when administered centrally or peripherally may be related to the localization of its brain receptors. In this sense, it is believed that peripheral GLP-1(7-36)amide abolishes angiotensin II-induced drinking via the subfornical organ, whereas intracerebroventricular injection of the peptide produces the same effect through an inhibitory effect on the neurons of Av3V (Tang-Christensen et al., 1996). However, intraperitoneal administration of up to 20 μg of GLP-1(7-36)amide failed to elicit a diuretic response, in contrast to the positive response obtained after central injection of the peptide (Tang-Christensen et al., 1996). This is experimental evidence for the existence of two distinct GLP-1 receptor populations in the brain, one of which is only accessible via the peripheral blood circulation. Another explanation for the central action of GLP-1(7-36)amide on AVP release may be related to the very high amounts of the peptide administered intracerebroventricularly (Tang-Christensen et al., 1996 ; Larsen et al., 1997) as compared with the 10-fold lower concentrations used in the peripheral studies. Indeed, using low and higher doses of GLP-1(7-36)amide intracerebroventricularly produces a biphasic response on food intake (Navarro et al., 1996), which may indicate a direct interaction with presynaptic autoreceptors regulating peptide release, as described for corticotropin-releasing factor (Koob and Bloom 1985) or dopamine receptor agonists (Wolf and Roth, 1987). Alternatively, it may point to a possible indirect mechanism mediated through the secretion of hypothalamic regulatory transmitters that can act as either releasing or inhibiting factors on the synthesis of anterior pituitary hormones or their release into the general circulation. Nevertheless, GLP-1(7-36)amide may act centrally through a paracrine effect in which greater amounts of the peptide might be presented to target cells as compared with endocrine effects.
It is also interesting that central administration of GLP-1(7-36)amide activates c-fos in corticotropin-releasing hormone-positive neurons and magnocellular oxytocinergic neurons, whereas only a few of the vasopressinergic magnocellular neurons of the PVN/SON were activated (Larsen et al., 1997). Regardless of the fact that the lack of c-fos expression does not necessarily indicate that vasopressinergic neurons are unresponsive to the central injection of GLP-1(7-36)amide, it has been proposed that the effect of this peptide on AVP secretion should be mediated at the level of neurohypophysial terminals, either via GLP-1(7-36)amide itself or via a transmitter coexisting with OX in magnocellular neurons. Despite the existence of GLP-1 receptors in the posterior pituitary, in vitro incubation of the neurohypophysis with 0.1 or 1 μg of GLP-1(7-36)amide failed to modify the basal secretion of AVP, although at higher concentrations it induced a minor increase in the release of OX. In addition, several neuropeptides have been shown to be coexpressed with OX in magnocellular neurons, and some of them, such as corticotropin-releasing hormone, cholecystokinin, and neuropeptide Y, stimulate the secretion of AVP from isolated neurointermediate lobes (Bondy and Gainer, 1989).
GLP-1(7-36)amide has several routes through which it can produce a central or peripheral effect. Because the posterior lobe of the pituitary is not shielded by the blood-brain barrier, GLP-1 receptors could be stimulated by the peptide released either from nerve fibers within the hypophysis or from the gut L-cells. Also, GLP-1(7-36)amide secreted from the intestine might enter the brain by binding to blood-brain barrier-free organs, such as the subfornical organ or the area postrema (Orskov et al., 1996), or may be transported into the brain through the choroid plexus, which has a high density of GLP-1 receptors (Alvarez et al., 1996). Hence, GLP-1(7-36)amide released peripherally may be responsible for some of its central effects, in addition to the brain peptide, which may be used for the same or other biological activities in the CNS.
In conclusion, we have shown colocalization of the GLP-1 receptor with AVP and OX mRNAs in neurons of the hypothalamic SON and PVN, which supports the central stimulating effects of GLP-1(7-36)amide on AVP release described by others and other actions, i.e., behavioral effects, induced by this peptide. However, our results also indicate that in vivo the peripheral administration of GLP-1(7-36)amide significantly decreases the circulating levels of both AVP and OX, whereas in vitro incubation of the posterior lobe of the pituitary with this peptide does not modify AVP secretion, but slightly increases OX release. These findings therefore suggest a dual secretory response of AVP and OX to the effect of GLP-1(7-36)amide, which most likely is related to the route of peptide administration or to the amount of GLP-1(7-36)amide administered.