Actions of cocaine- and amphetamine-regulated transcript (CART) peptide on regulation of appetite and hypothalamo–pituitary axes in vitro and in vivo in male rats

doi:10.1016/S0006-8993(00)03312-6

Brain Research

Volume 893, Issues 1–2, 2 March 2001, Pages 186-194

https://doi.org/10.1016/S0006-8993(00)03312-6 Get rights and content

Abstract

Cocaine- and amphetamine-regulated transcript (CART) and CART peptide are abundant in hypothalamic nuclei controlling anterior pituitary function. Intracerebroventricular (ICV) injection of CART peptide results in neuronal activation in the paraventricular nucleus (PVN), rich in corticotrophin-releasing factor (CRH) and thyrotrophin-releasing factor (TRH) immunoreactive neurons. The aims of this study were three-fold. Firstly, to examine the effects of CART peptide on hypothalamic releasing factors in vitro, secondly, to examine the effect of ICV injection of CART peptide on plasma pituitary hormones and finally to examine the effect of PVN injection of CART peptide on food intake and circulating pituitary hormones. CART(55–102) (100 nM) peptide significantly stimulated the release of CRH, TRH and neuropeptide Y from hypothalamic explants but significantly reduced alpha melanocyte stimulating hormone release in vitro. Following ICV injection of 0.2 nmol CART(55–102), a dose which significantly reduces food intake, plasma prolactin (PRL), growth hormone (GH) and adrenocorticotrophin hormone (ACTH) and corticosterone increased significantly. Following PVN injection of CART(55–102), food intake was significantly reduced only at 0.2 and 0.6 nmol. However, PVN injection of 0.02 nmol CART(55–102) produced a significant increase in plasma ACTH. ICV injection of CART peptide significantly reduces food intake. Unlike many anorexigenic peptides, there is no increased sensitivity to PVN injection of CART(55–102). In contrast, both ICV and PVN injection of CART(55–102) significantly increased plasma ACTH and release of hypothalamic CRH is significantly increased by CART peptide in vitro. This suggests that CART peptide may play a role in the control of pituitary function and in particular the hypothalamo–pituitary adrenal axis.

Introduction

The hypothalamus is essential in the integration of energy homeostasis and endocrine function and hypothalamic neuropeptides play an important role in the control of both appetite and hypothalamo–pituitary axes. Cocaine- and amphetamine-regulated transcript (CART) peptide is a recently identified powerful regulator of food intake [24], [26].

Although originally identified in the rat striatum and dramatically up-regulated by psychoactive drugs [12], in situ hybridization studies have shown expression of CART mRNA to be widespread in the central nervous system [11]. Additionally, CART is one of the most abundant hypothalamic specific transcripts [16]. It is concentrated in particular hypothalamic nuclei: the paraventricular nucleus (PVN), arcuate nucleus (ARC), dorsomedial hypothalamus (DMH) and lateral hypothalamus (LHA) [10], [11].

The peptide product of CART is also abundant in the hypothalamus [21], [22]. It is present in the cell bodies of the PVN, ARC, DMH and LHA, in axons of the internal layer of the median eminence and in the fibres of the external layer of the median eminence that lie adjacent to the hypophyseal portal plexus [22].

Hypothalamic CART mRNA and peptide appear to play a role in the acute control of energy homeostasis. Arcuate nucleus CART mRNA expression is reduced by fasting and suppressed in the leptin deficient ob/ob mouse where it is restored by leptin replacement [24]. Intracerebroventricular (ICV) injection of recombinant CART fragments, CART(55–102), CART(55–76) and CART(62–76) (nomenclature after Kristensen et al.) inhibited food intake in fasted and freely feeding rats and ICV injection of CART antiserum increased night-time feeding [24], [26]. However, the reduction in DMH and LHA CART mRNA observed in ob/ob mice, which is independent of leptin [24], and the widespread hypothalamic distribution of CART mRNA and peptide suggest actions beyond the control of food intake.

Sequence homology and anatomical evidence suggests CART peptide may play a role in the control of hypothalamo–pituitary axes. CART peptide shows a high degree of species conservation, over 90% between rat, sheep and man [11] but also sequence homology with ovine somatostatin [38], known to influence endocrine function. CART mRNA and immunoreactivity (CART-IR) have been co-localised with neurotransmitters known to modulate hypothalamo–pituitary control. In the ARC, pro-opiomelanocortin (POMC) mRNA and CART mRNA [15] are found in the same cell population whilst CART mRNA and melanocyte concentrating hormone (MCH) are co-localised in the LHA [5]. CART peptide is present in the parvocellular division of the PVN, an area rich in neurons containing peptides important in the control of neuroendocrine circuits; corticotrophin-releasing hormone (CRH), thyrotrophin-releasing hormone (TRH), vasoactive intestinal peptide (VIP) and galanin [8], [17]. In addition, immunohistochemical studies have demonstrated co-existence of CART mRNA with TRH mRNA in a subpopulation of neurons in the PVN [5] and CART-IR with somatostatin in the periventricular region of the hypothalamus. However, CART does not appear to colocalise with CRH [5]. Examination of expression of the early gene, c-fos, following ICV administration of CART peptide showed cell activation occurs in the PVN where there is co-localisation of c-fos and CRH expression and in the ARC, an area known to contain growth hormone releasing hormone (GHRH) [42]. However, such studies do not indicate if the actions of CART are directly on the nuclei controlling endocrine function.

Thus, there is mounting evidence to suggest a role for CART peptide in the control of hypothalamo–pituitary function. Previous studies have examined the effect of third-ventricle injection of CART peptide to increase plasma corticosterone and oxytocin only [41]. We have, therefore, examined firstly, the actions of CART peptide on the release of hypothalamic releasing factors and peptides in vitro, secondly, the effect of ICV injection of CART peptide on circulating pituitary hormones and finally the effect of intraPVN injection of CART peptide on food intake and circulating pituitary hormones to determine if CART acts via PVN CRH neurons to increase plasma corticosterone.

Section snippets

Materials

Rat CART(55–102), the putative active C-terminal fragment [24], was purchased from Peptide Institute Inc. (Osaka, Japan). Reagents for basal hypothalamic explant experiments were supplied by BDH (Poole, Dorset, UK).

Animals

Male Wistar rats (Specific pathogen free, Imperial College School of Medicine, London, UK) weighing 250–300 g were maintained in individual cages under controlled temperature (21–23°C) and light (12 h light, 12 h dark, lights on at 07:00 h) with ad libitum access to food (RM1 diet,

Effect of CART on the release of hypothalamic releasing factors and neuropeptides from medial basal hypothalamic explants (Table 1)

Administration of CART(55–102) (100 nM) significantly increased the release of both CRH and TRH from static basal hypothalamic incubations when compared to basal release (CRH; basal 27±4 fmol/explant, CART(55–102) 39±2 fmol/explant, P<0.05 vs. basal. TRH; basal 34±10 fmol/explant, CART(55–102) 63±11 fmol/explant, P<0.05 vs. basal). The secretion of hypothalamic neuropeptides in response to CART(55–102) was also examined. The secretion of the hypothalamic NPY was significantly elevated in the

Discussion

Increasing evidence suggests a role for CART peptide as a neurotransmitter. CART peptide and mRNA are co-localised to neurons containing classical neurotransmitters; POMC, MCH, gamma amino butyric acid (GABA) and tyrosine hydroxylase [8], [15], [23], [36], [37]. CART immunoreactivity is located in dendritic dense core vesicles [37]. The peptide has an initial signal sequence [12] and its release is calcium dependant [30]. Although it plays a role in the acute control of food intake, DMH and LHA

Acknowledgements

The authors wish to express their thanks to the hypothalamic group for their assistance with the in vivo experiments and the MRC for funding this programme of research. S.A.S and L.J.S are Wellcome research fellows. K.M., D.S. and C.D. have UK MRC studentships.

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Research reportActions of cocaine- and amphetamine-regulated transcript (CART) peptide on regulation of appetite and hypothalamo–pituitary axes in vitro and in vivo in male rats

Abstract

Introduction

Section snippets

Materials

Animals

Effect of CART on the release of hypothalamic releasing factors and neuropeptides from medial basal hypothalamic explants (Table 1)

Discussion

Acknowledgements

Life Sci.

Brain Res.

J. Chem. Neuroanat.

Gene

Brain Res.

Neuron

Neuron

Peptides

Eur. J. Pharmacol.

Regul. Pep.

Peptides

Brain Res.

Brain Res.

In vivo and in vitro effects of dexamethasone on pituitary thyrotrophin-releasing hormone-like peptide concentrations in the rat

J. Endocrinol.

Glucagon-like peptide-1 stimulates luteinizing hormone-releasing hormone secretion in a rodent hypothalamic neuronal cell line

J. Clin. Invest.

Glucagon-like peptide-1 (GLP-1) releases thyrotropin (TSH): characterization of binding sites for GLP-1 on alpha-TSH cells

Endocrinology

Subtypes Y1 and Y2 of the neuropeptide Y receptor are respectively expressed in pro-opiomelanocortin- and neuropeptide-Y-containing neurons of the rat hypothalamic arcuate nucleus

Neuroendocrinology

Distribution and coexistence of corticotropin-releasing factor-, neurotensin-, enkephalin-, cholecystokinin-, galanin- and vasoactive intestinal polypeptide/peptide histidine isoleucine-like peptides in the parvocellular part of the paraventricular nucleus

Neuroendocrinology

PCR differential display identifies a rat brain mRNA that is transcriptionally regulated by cocaine and amphetamine

J. Neurosci.

Research report
Actions of cocaine- and amphetamine-regulated transcript (CART) peptide on regulation of appetite and hypothalamo–pituitary axes in vitro and in vivo in male rats