Behavioral and neuroendocrine effects of the selective CRF2 receptor agonists urocortin II and urocortin III

doi:10.1016/j.peptides.2004.01.008

Peptides

Volume 25, Issue 4, April 2004, Pages 659-666

https://doi.org/10.1016/j.peptides.2004.01.008 Get rights and content

Abstract

We compared the in vivo efficacy of two selective CRF₂ agonists, mouse urocortin II (mUcn II) and human urocortin III (hUcn III), using food intake, anxious behavior, or ACTH release in CD-1 or Balb/c mice as indices of biological stress responses. All three peptides produced anorexia (Minimal Effective Dose (M.E.D.) for CRF and mUcn II=0.03 nmol; M.E.D. for hUcn III=0.3 nmol). Only mUcn II and CRF appeared to increase anxious behaviors in the elevated plus maze test (M.E.D.=0.3 and 0.01 nmol, respectively). CRF increased the release of plasma ACTH (M.E.D. of 0.3 nmol), while mUcn II and hUcn III had no effect on ACTH release. These data suggest that the CRF₂ receptor subtype plays a primary role in the activation of behavioral, but not neuroendocrine, stress responses.

Section snippets

Animals

Male Balb/c (18–20 g) or female CD1 mice (22–25 g) were housed in groups of five in the Neurocrine Biosciences vivarium, under a standard 12 h/12 h light/dark cycle (7.00 a.m./7.00 p.m.) where the ambient room temperature was maintained at 23–25 °C. Mice were allowed food and water ad libitum. All testing was conducted according to the standards set by the Institutional Animal Care and Use Committee at Neurocrine Biosciences.

Design

The role of the CRF₂ receptor in the central response to stress was tested by

Food intake

CRF reduced the refeeding response to overnight food deprivation at all time points (F(3,108)=21.1; P<0.0001). The M.E.D. 1 and 2 h after food exposure was 0.03 nmol (P<0.0001 versus vehicle). The potency of this effect gradually diminished with time, with the M.E.D. 4 and 6 h after food exposure one log higher, at 0.3 nmol (Ps<0.006–0.0001 versus vehicle; Fig. 1A). Ucn II also produced a robust reduction in food intake over time (F(3,105)=40.2; P<0.0001). The M.E.D. 1 h after food presentation was

Discussion

Both Ucn II and Ucn III significantly attenuated refeeding after a 16-h period of food restriction. mUcn II was 10-fold more potent than hUcn III in reducing food intake, which is consistent with the binding profile of these agonists at the CRF_2α receptor [17], and with previously published in vivo findings in rats and mice [14], [15], [27]. In addition, the anorexia induced by both of these peptides was dose-dependently reversed by the CRF₂-selective antagonist, ASV-30, which is consistent

Acknowledgements

This work was supported in part by Small Business Innovation Research Grant 1 R43 MH65106-01 from the National Institute of Mental Health.

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In addition, plasma ACTH concentrations of sheep and rat were significantly increased after intravenous (i.v.) injection of 10 μg UCN3 [71,72]. Moreover, as mentioned earlier, studies showed that either i.c.v. or i.p. injection UCN3 could both inhibit food intake of animals [50,70]. Therefore, UCN3 plays an inhibitory role in animal feeding probably by promoting the level of ACTH in circulatory system.
Urocortin3 (UCN3), the newest member of corticotrophin releasing hormone (CRH) family polypeptides, is an anorexic factor discovered in 2001, which has a strong inhibitory effect on animal appetite regulation. UCN3 is widely distributed in various tissues of animals and has many biological functions. Based on the research progress of UCN3 on mammals and non-mammals, this paper summarized the discovery, tissue distribution, appetite regulation and mechanism of UCN3 in animals, in order to provide a reference for feeding regulation and growth in mammals and fish in further research and production.
The effects of the urocortins on the hypothalamic-pituitary-adrenal axis - similarities and discordancies between rats and mice
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The urocortins (Ucn I, Ucn II and Ucn III) are structural analogues of corticotropin-releasing factor (CRF). The aim of our present experiments was to compare the effects of the urocortins on the hypothalamic-pituitary-adrenal (HPA) axis in rats and mice, including the hypothalamic adrenocorticotropic hormone (ACTH) secretagogues, such as CRF and arginine vasopressin (AVP). Therefore, male CFLP mice and male Wistar rats were injected intracerebroventricularly (icv) with 0.5, 1, 2 and 5 μg/2 μl of Ucn I, Ucn II or Ucn III. After 30 min the animals were decapitated, and then, hypothalamic CRF and AVP concentrations and plasma ACTH and corticosterone (CORT) levels were measured. All measurements were performed by enzyme-linked immunosorbent assays (ELISA), except that of the plasma CORT level, which was determined by chemofluorescent assay. Ucn I increased significantly the hypothalamic CRF and AVP concentrations in both rats and mice. Ucn II and Ucn III influenced significantly only the hypothalamic CRF concentration in rats, without affecting the hypothalamic AVP concentration. In contrast, Ucn II and Ucn III increased significantly only the hypothalamic AVP concentration in mice, without affecting the hypothalamic CRF concentration. The hypothalamic changes were reflected more or less accurately by changes of the plasma ACTH and CORT levels. The present experiments demonstrate that the urocortins regulate the HPA axis centrally via modulation of the hypothalamic ACTH secretagogues and that there are some similarities and discordancies between rats and mice regarding this regulation.
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Corticotropin-releasing factor (CRF) and the urocortins (Ucn1, Ucn2, and Ucn3) belong to the mammalian CRF family of peptides, having similar amino acidic structures but different pharmacological profiles. Their actions are mediated by two distinct receptors, CRF1 and CRF2. CRF and Ucn1 bind to both CRF receptors, whereas Ucn2 and Ucn3 bind selectively to CRF2. Several studies reported that the administration of selective CRF1 antagonists prevents the dysphoria and the reward deficit observed during nicotine withdrawal. Another study referred that the administration of nonselective CRF2 agonists, such as CRF and Ucn1, prevents the hyperphagia and the weight gain assessed during nicotine withdrawal. However, our study was the first to demonstrate that intracerebroventricular administration of Ucn2 and Ucn3 ameliorates the anxiety- and depression-like state developed during chronic nicotine treatment and consequent acute withdrawal, suggesting that the selective CRF2 agonists can be potential candidates in the therapy of nicotine withdrawal.
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The aim of the present study was to investigate the potential anxiolytic- and antidepressant-like actions of Urocortin 2 (Ucn2) and its two fragments, Ucn2 (1–21) and Ucn2 (22–38), in mice, in an attempt to identify the biologically active sequence of this 38 amino acid neuropeptide. In this purpose, male C57BL/6 mice were treated intracerebroventricularly (icv) with 0.125, 0.25, 0.5 and 1 µg/2 µl of Ucn2, Ucn2 (1–21) or Ucn2 (22–38). After 30 min, the mice were evaluated in an elevated plus-maze test and a forced swim test for anxiety- and depression-like behavior, respectively. Each test lasted 5 min. Ucn2 at dose of 0.25 µg/2 µl and Ucn2 (1–21) at dose of 0.125 µg/2 µl, but not Ucn2 (22–38), increased significantly the number of entries into and the time spent in the open-arms, without influencing the total number of entries. In parallel, the same doses of Ucn2 and Ucn2 (1–21), but not Ucn2 (22–38), increased significantly the climbing and the swimming activity, while decreasing significantly the time of immobility. In addition, Ucn2 at doses of 0.125 µg/2 µl and 0.5 µg/2 µl decreased significantly the time of immobility, but they did not change the other parameters. The present study demonstrates that Ucn2 exerts anxiolytic- and antidepressant-like effects in C57BL/6 mice, which are mediated by the N-terminal, but not the C-terminal fragment of the peptide. The establishment of the smallest active sequence by further fragmentation of Ucn2 (1–21) may allow the synthesis of new anxiolytic and antidepressant drugs.
Altered functionality of the corticotrophin-releasing hormone receptor-2 in the hypothalamic paraventricular nucleus of hyperphagic maternally separated rats
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We hypothesized that MS may promote a chronic activation of CRH and Ucns synthesis that will induce an impairment of their receptor's signaling, in such a way that their high food intake due to the chronic early-life stress, does not correspond to the known anorexic effects of these peptides. CRH and the Ucns have different affinities for the two identified CRH receptors, CRH-R1 and CRH-R2, being CRH-R2 the main mediator of the anorexic effects of these peptides (Pelleymounter et al., 2004; Stengel and Tache, 2014). The PVN is one of the brain regions where CRH-R2 is more expressed than CRH-R1 (Chalmers et al., 1995) and where its activation reduces food intake (de Gortari et al., 2009; Fekete et al., 2007).
Early-life stress induces endocrine and metabolic alterations that increase food intake and overweight in adulthood. The stress response activates the corticotropin-releasing hormone (CRH) and urocortins' (Ucns) system in the hypothalamic paraventricular nucleus (PVN). These peptides induce anorexic effects through CRH-R2 receptor activation; however, chronic stressed animals develop hyperphagia despite of high PVN CRH expression.
We analyzed this paradoxical behavior in adult rats subjected to maternal separation (MS) for 180 min/daily during post-natal days 2–14, evaluating their body weight gain, food intake, serum corticosterone and vasopressin concentrations, PVN mRNA expression of CRH-R1, CRH-R2, CRH, Ucn2, Ucn3, vasopressin and CRH-R2 protein levels.
MS adults increased their feeding, weight gain as well as circulating corticosterone and vasopressin levels, evincing chronic hyperactivity of the stress system. MS induced higher PVN CRH, Ucn2 and CRH-R2 mRNA expression and protein levels of CRH-R2 showed a tendency to decrease in the cellular membrane fraction. An intra-PVN injection of the CRH-R2 antagonist antisauvagine-30 in control adults increased receptor's mRNA expression, mimicking the observed PVN receptor's up-regulation of early-life MS adults.
An injection of Ucn-2 directly into the PVN reduced food intake and increased PVN pCREB/CREB ratio in control animals; in contrast, Ucn-2 was unable to reduce food intake and enhance phosphorylated-CREB levels in PVN of MS rats.
In conclusion, the chronic hyperactivity of the stress axis and PVN CRH-R2 resistance to Ucn2 effects, supported impaired receptor functionality in MS animals, probably due to its chronic stimulation by CRH or Ucn2, induced by early-life stress.
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The aim of our study was to determine the role of corticotropin-releasing factor (CRF), the urocortins (Ucn1, Ucn2 and Ucn3) and their receptors (CRF₁ and CRF₂) in the sociability of mice. Male CFLP mice were administered intracerebroventricularly (icv) with CRF and urocortins alone or in combination with antalarmin (specific CRF₁ antagonist) and astressin_2B (specific CRF₂ antagonist) and then investigated in a Crawley social interaction test arena, that consists of three chambers. An unknown male in a cage was put in the first chamber and an empty cage was put in the opposite chamber. The tested male was habituated with the middle chamber for 5 min and then allowed to explore the remaining chambers for 5 min, during which the number of entries and the time of interaction were measured. Intracerebroventricular administration of CRF decreased significantly the number of entries and the time of interaction with the unknown male and these effects were blocked by antalarmin, but not astressin_2B. In contrast, central administration of Ucn1 increased significantly the number of entries into the chamber of the unknown male, without changing the time of interaction and this effect was blocked by astressin_2B, but not antalarmin. Central administration of Ucn2 and Ucn3 didn’t influence remarkably the number of entries, but it reduced the time of interaction between the male mice. Our study suggests that CRF and Ucn1 may play important, but different roles in sociability, and that Ucn2 and Ucn3, playing similar roles, must be also involved in social interactions.

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¹: Present address: Department of Metabolic Research, Bristol-Myers Squibb, 311 Pennington-Rocky Hill Road, Pennington, NJ 08534, USA.

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Behavioral and neuroendocrine effects of the selective CRF2 receptor agonists urocortin II and urocortin III

Abstract

Section snippets

Animals

Design

Food intake

Discussion

Acknowledgements

Pharmacol. Biochem. Behav.

Behav. Brain Res.

Neuropharmacology

Mol. Brain Res.

J. Neurosci. Methods

Behav. Brain Res.

Peptides

Neuron

Brain Res.

Brain Res.

Brain Res.

Brain Res.

Mice deficient for corticotropin-releasing hormone receptor-2 display anxiety-like behavior and are hypersensitive to stress

Nat. Genet.

Mice deficient for both corticotropin-releasing factor receptor-1 (CRFR1) and CRFR2 have an impaired stress response and display sexually dichotomous anxiety-like behavior

J. Neurosci.

Localization of novel corticotropin releasing factor receptor (CRF2) mRNA expression to specific subcortical nuclei in rat brain: comparison with CRF1 receptor mRNA expression

J. Neurosci.

Quantitative trait loci affecting the behavior of A/J and CBA/J intercross mice in the elevated plus maze

Mamm. Genome

Dissociation of locomotor activation and suppression of food intake induced by CRF in CRFR1-deficient mice

Endocrinology

Abnormal adaptations to stress and impaired cardiovascular function in mice lacking corticotropin-releasing hormone receptor-2

Nat. Genet.

Urocortin, corticotropin releasing factor-2 receptors and energy balance

Endocrinology

J. Pharmacol. Exp. Ther.

Behavioral and neuroendocrine effects of the selective CRF₂ receptor agonists urocortin II and urocortin III

Localization of novel corticotropin releasing factor receptor (CRF2) mRNA expression to specific subcortical nuclei in rat brain: comparison with CRF₁ receptor mRNA expression