Effects of cocaine–amphetamine regulated transcript (CART) on hormone release

Regulatory Peptides 122 (2004) 55 – 59 www.elsevier.com/locate/regpep

Effects of cocaine–amphetamine regulated transcript (CART) on hormone release BoguslCawa Baranowska a,*, Ewa Wolin´ska-Witort a, Lidia Martyn´ska a, Magdalena Chmielowska a, Agnieszka Baranowska-Bik b a

Neuroendocrinology Department, Medical Centre of Postgraduate Education, Boguslawa Baranowska, Marymoncka 99, 01-813 Warsaw, Poland b Department of Internal Medicine, Endocrinology and Haemotology, Central Hospital of Ministry of Home Affairs and Administration, Woloska 137, 02-507 Warsaw, Poland Received 6 October 2003; received in revised form 26 April 2004; accepted 12 May 2004 Available online 10 August 2004

Abstract Objective: Cocaine- and amphetamine-regulated transcript (CART) is a recently described neuropeptide widely expressed in the rat brain. CART is abundant in hypothalamus nuclei controlling anterior pituitary function. In the paraventricular nucleus CART mRNA is colocalized with vasopressin and corticotrophin-releasing factor containing neurons. The data may suggest that CART plays a role in hypothalamic regulation of neuroenocrine functions. Material and methods: Male Wistar – Kyoto rats were investigated. Experiment I: CART was administered intracerebroventricularly (i.c.v.) in a dose of 0.5 Ag dissolved in 5 Al vehicle. At 60, 120 min after the infusion of CART or vehicle animals were decapitated and trunk blood was collected until hormonal estimations. Experiment II: CART in a dose of 10 Ag was injected intravenously (i.v.). At 60, 120, 240 min the rats were decapitated and the trunk blood was collected. Serum rLH, rFSH, rPRL, rTSH, rGH and plasma leptin, NPY concentrations were measured by RIA methods. Results: CART administered centrally (i.c.v.) simulated significantly GH release after 60 min ( p < 0.05) and after 120 min ( p < 0.01). CART increased also PRL after 60 min ( p < 0.05). A marked increase of corticosterone level was observed at 60 and 120 min ( p < 0.01, p < 0.01). We did not observe significant changes in LH, FSH and TSH. We found an increase of serum leptin concentrations at 60 min after CART administration ( p < 0.01). However, serum NPY levels did not change. After intravenous injection (i.v.) of CART an increase of GH was observed at 120, 240 min ( p < 0.01, p < 0.01, respectively). A rise in serum PRL was found at 240 min ( p < 0.05). Corticosterone concentrations were enhanced at 60, 120, 240 min ( p < 0.01, p < 0.01, p < 0.01, respectively). We did not observe significant changes in LH, FSH and TSH. Conclusions: CART may play a modulating role in the mechanism of pituitary hormone release. D 2004 Elsevier B.V. All rights reserved. Keywords: CART; Pituitary; Adrenal; Hormones

1. Introduction Cocaine- and amphetamine-regulated transcript (CART) is a recently described neuropeptide widely expressed in the rat brain. CART mRNA is found in hypothalamic nuclei such as the paraventricular nucleus (PVN), the supraoptic nucleus (SON), the lateral hypothalamic area (LHA), the dorsomedial nucleus (DMH), the periventricular nucleus (Pe), and the ventral premammillary nucleus (PMV) [1]. * Corresponding author. Tel./fax: +48-22-834-47-12. E-mail addresses: [email protected], [email protected] (B. Baranowska). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.05.006

The immunohistochemical investigations indicate the presence of CART in the hypothalamus, pituitary and adrenal gland [2]. CART is abundant in hypothalamic nuclei controlling anterior pituitary function. CART may play a role in the regulation of energy homeostasis in animals and in humans. It has been known that intracerebroventricular (i.c.v.) administration of CART inhibits food intake and induces the expression of c-fos in hypothalamic nuclei involved in the regulation of food intake [3]. Central injection of CART decreases food intake and CART mRNA levels in Arc are regulated by leptin [1]. It has been reported that leptin inhibits food intake and increases energy expenditure through interaction with specific leptin receptors located in the hypothalamus. Leptin receptors

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have been identified in CART, NPY, galanin-containing neurons in the arcuate nucleus and in CRH containing neurons of the paraventricular nucleus [4]. These findings suggest that CART, NPY and galanin are mediators of leptin’s action in the hypothalamus [4]. The anorexigenic effect of leptin may be mediated by stimulation of both CART, CRH, aMSH and inhibition of NPY [5]. Leptin increases the hypothalamic expression of anorexigenic peptides POMC, CART and CRH [4]. The decrease in leptin and CART may reflect a compensatory down-regulation in response to the energy deprived state [5]. The characteristic fall in leptin with fasting causes a rise in NPY mRNA as well as a fall in POMC and CART mRNA [5,6]. The hypothalamic CART mRNA expression was found to be higher in obese rats [7]. The peripheral administration of leptin to obese mice stimulates CART mRNA expression [8]. The central injection of leptin induced an increase in CART mRNA [9,10]. Leptin directly activates POMC/CART neurons that project to the lateral hypothalamic area (LHA), paraventricular nucleus and final sympathetic preganglionic neurons [11]. Leptin activates also CART/POMC neurons innervating sympathetic preganglionic neurons in the thoracic, spinal cords and these findings suggest that this pathway may contribute to the increased thermogenesis and energy expenditure observed following leptin administration [12]. Additionally CART in addition to glucagon-like peptide (GLP 1), Agouti Related Peptide (Agrp) and CRF primarily acts via independent pathway [13]. Broberger [14] demonstrated the histochemical relation of CART with neuropeptides, TRH, MCH (melanocortin concentrating hormone), orexin/hypocretin and neuropeptide Y (NPY). It has been reported that CART markedly stimulated CRH, TRH and NPY from hypothalamic explants in vitro [15]. Dhillo et al. [16] investigated interactions between CART, NPY and Agrp in vitro using a rat hypothalamic explants. They showed that injection of NPY to hypothalamic explants significantly increased release of Agrp IR. On the other hand administration of CART to hypothalamic explants significantly increases Agrp and NPY-IR (16). The relationship between leptin, NPY, CART is very important in the control of food intake, energy expenditure and neuroendocrine functions. The aim of this study was to evaluate the effects of CART on hormone release and on leptin and NPY release.

2. Materials and methods

2.2. Experiment 1: Intracerebroventricular (i.c.v.) administration of CART The animals were anesthetized i.p. with ketamine and implanted with a stainless-steel guide cannula, 23 gauge cannula was located in the third cerebroventricle (0.8 mm posterior and 7.0 mm ventral to the bergma at the midline) according to the atlas of Paxinos and Watson [17]. The inside of the cannula was closed by a removable stainless-steel plug. The placement of the intracerebroventricular cannula was verified by an injection of methylene blue dye after decapitation. The brain was inspected for complete spread of the dye in the third ventricle. Data from any subject with inadequate spread of the marker were discarded. After the surgery, the rats were transferred to individual cages with food and water freely available. During a 7-day period of recovery, rats were handled daily to minimize any stress associated with handling on the day of the experiment. On the day of the experiment, 2 h before CART administration, a stainless-steel guide cannula was opened and controlled its patency. Intracerebroventricular infusion of CART-(55 –102) (rat) (Bachem), was performed to freely moving rats. CART at a concentration of 0.5 Ag in 5 Al vehicle (artificial cerebrospinal fluid) or equal volume of the vehicle was slowly (1 Al/min) infused into the third ventricle with an automatic pump (CMA/100; Sweden) through an inner cannula inserted into the guide cannula. After the end of the infusion the rats were transferred to their home cages with free access to food and water. At 60, 120 min after the infusion of CART or vehicle, animals were decapitated and trunk blood was collected in plastic tubes containing 1000 IU aprotinine (inhibitor of protease) per each ml of blood. The time-span from removal of the animals from their cages to decapitation was approximately 2 min. 2.3. Experimental II: intravenous (i.v.) injection of CART CART (55 – 102) in a dose of 10 Ag in 300 Al of saline or 300 Al of saline alone was injected into the tail vein. After the injection the animals were transferred to individual cages with free access to food and water. At 60, 120, 240 min after the injection of CART or saline, animals were decapitated, and trunk blood was collected in plastic tubes containing 1000 IU of aprotinine (Transcolan). The blood samples were centrifuged (3000 rpm for 20 min at 4 jC). Serum samples were frozen until hormonal analyses were performed.

2.1. Animals and surgery 2.4. The hormone measurement Male Wistar-Kyoto rats (240 – 260 g) were maintained under controlled conditions (14 L:10 D, lights on at 06:00 h, temperature at 23 F 1 jC) with free access to food and water. All experimental procedures were approved by the First Warsaw Ethic Committee for Experiments on Animals (the M. Nencki Institute of Experimental Biology, the Polish Academy of Science).

Serum concentrations of rLH, rFSH and rPRL were measured by RIA in duplicates using reagents prepared by Dr. A.F. Parlow and provided by the NIDDK (Bethesda, MD). Values were expressed in terms of LH-RP3, FSH-RP2 and PRL-RP3 of reference standard, respectively. Serum concentrations of the rTSH and rGH were measured by kits

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Table 1 Effects of cocaine – ampethamine-regulated transcript (CART) administered centrally (i.c.v.) on serum pituitary hormones, leptin, NPY and corticosterone concentrations in male rats Peptides

rLH, ng/ml

rFSH, ng/ml

rPRL, ng/ml

rTSH, ng/ml

GH, ng/ml

Leptin, ng/ml

NPY, ng/ml

Corticosterone, ng/ml

After 60 min CART n = 9

0.3 F 0.1

9.4 F 1.7

5.1 F 1.5 ( p < 0.05)

2.4 F 0.1

9.4 F 2.6 ( p < 0.05)

2.5 F 0.2 ( p < 0.01)

13.0 F 1.8

320.4 F 43.0 ( p < 0.01)

After 120 min CART n = 9

0.5 F 0.1

15.3 F 1.0

2.6 F 0.3

3.3 F 0.7

2.2 F 0.2

8.2 F 0.9

0.3 F 0.2

13.0 F 1.0

2.3 F 0.2

3.1 F 0.3

21.9 F 6.3 ( p < 0.01) 4.9 F 2.0

1.3 F 0.1

10.0 F 1.0

274.8 F 19.8 ( p < 0.01) 120 F 10

CONTROL (aCSF) (Mean of all the controls after 60 min, 120 min, n = 18)

provided by Biocode, France. The limit of detection for TSH was 0.1 and 1 ng/ml for GH. All measurements were made in duplicates, in one assay. Intra-assay for all hormones were: LH 6.5%, FSH 6.7%, PRL 6.0%, GH 6.8, TSH 6.0%, corticosterone 6.6%. Leptin was evaluated in the serum samples using rat leptin RIA kit (Linco Research, St. Charles, MO). The sensitivity for this assay was 0.5 ng/ml. Serum NPY concentrations were determined using the kit from Peninsula Laboratories. The limit of detection was 10 pg/ml. Corticosterone concentrations were measured using a commercial kit from ICN Biomedicals USA. The limit of detection for corticosterone was 25 ng/ml. The serum samples for each hormone was analysed in one assay. Since we found substantial amount of variation between the controls at the various time points, the responses of hormones to CART were compared to the common control (mean of all controls). The statistical analysis was performed with unpaired ttest and one-way ANOVA as appropriate p < 0.05 was considered significant.

3. Results Serum pituitary hormones concentrations (rLH, rFSH, rPRL, rTSH, rGH), adrenal hormone-corticosterone levels as well as plasma leptin and NPY concentration after intacerebroventricular administration (i.c.v.) of CART are shown in Table 1. A marked increase of corticosterone levels was observed at 60 and 120 min ( p < 0.01, p < 0.01) after i.c.v. injection of CART. CART administered centrally (i.c.v.) stimulated significantly GH release after 60 min ( p < 0.05) and after 120 min ( p < 0.01). CART increased also PRL after 60 min ( p < 0.05). We did not observe significant changes in TSH, LH, and FSH release. We found a significant increase of serum leptin concentration ( p < 0.01) at 60 min after i.c.v. injection of CART. However, serum NPY levels did not change. Table 2 presented serum pituitary hormones, adrenal hormone-corticosterone concentrations and serum leptin and NPY levels after intravenous injection (i.v.) of CART. Corticosterone concentration were significantly increased at 60, 120, 240 min ( p < 0.01, p < 0.01, p < 0.01, respectively).

Table 2 Effects of cocaine-ampethamine-regulated transcript (CART) administered peripherally (i.v.) on serum pituitary hormones, leptin, NPY and corticosterone concentrations in male rats Peptides

rLH, ng/ml

rFSH, ng/ml

rPRL, ng/ml

rTSH, ng/ml

rGH, ng/ml

Leptin, ng/ml

After 60 min CART n = 9

0.4 F 0.03

17.8 F 3.7

2.5 F 0.2

4.2 F 0.6

15.7 F 4.7

1.9 F 0.2

7.5 F 0.7

275 F 23 ( p < 0.01)

After 120 min CART n = 9

0.3 F 0.1

1.9 F 0.4

2.7 F 0.3

42.2 F 11.1 ( p < 0.01)

1.9 F 0.2

10.9 F 1.0

295 F 26 ( p < 0.01)

After 240 min CART n = 9

0.6 F 0.1

11.4 F 1.7

3.2 F 0.5

9.5 F 1.1

11.9 F 2.1

41.4 F 10.0 ( p < 0.01) 18.9 F 5.0

1.9 F 0.2

0.5 F 0.1

4.1 F 1.3 ( p < 0.05) 2.5 F 0.4

1.9 F 0.2

8.9 F 1.0

312.8 F 35 ( p < 0.01) 143 F 10

CONTROL (Saline) (Mean of all the controls after 60 min, 120 min, 240 min n = 27)

9.6 F 0.08

2.7 F 0.8

NPY, ng/ml

Corticosterone, ng/ml

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An increase of GH was observed at 120, 240 min ( p < 0.01, p < 0.01, respectively). A rise in serum PRL was found at 240 min ( p < 0.05). Serum LH, FSH, and TSH and serum NPY and leptin levels concentrations did not change significantly.

4. Discussion The data indicating the immunohistochemical localisation of CART peptides in rat hypothalamus, pituitary and adrenal gland suggest the role of CART in hypothalamic, pituitary and adrenal functions [2]. Intracerebroventricular (i.c.v.) injection of CART leads to neuronal activation in paraventricular nucleus rich in corticotrophin-releasing factor (CRH) and thyrotrophin releasing factor (TRH) immunoreactive neurons [15]. CART immunoreactive fibres opposed in c-Fos positive CRH neurons, suggestive of a direct action of CART on PVN CRH neurons. The effect of CART on CRH neurons may lead to corticosterone secretion from adrenal gland [3]. Our findings demonstrate that both intracerebroventricular (i.c.v.) and intravenous (i.v.) injection of CART lead to a marked increase of serum corticosterone. The results confirm the investigations of other authors. Vrang et al. [3] showed that CART administered intracerebroventricular (i.c.v.) caused a strong increase in corticosterone. They suggested that CART may activate central CRH neurons as well as both neurohypophysiol and descending oxytocinergic neurons of the PVN [3]. The effect of CART on CRH neurons leads to corticosterone secretion which may additionally contribute to the inhibition effects of CART on feeding behavior. Vrang et al. [18] examined the interaction between glucocorticoids and leptin on POMC/CART mRNA expression. Leptin influences POMC and CART mRNA expression directly but this effect is not modulated by corticosterone [18]. It could be speculated that two factors may be involved in the mechanism of stimulation of corticosterone release: stimulation via CRH-ACTH and direct effect on adrenals. This suggestion may be confirmed after presentation of the results of our experiments ‘‘in vivo’’ and ‘‘in vitro’’ which we are going to do in the future. We are going to measure serum ACTH and corticosterone after i.c.v. injection of CART as well as to evaluate corticosterone release from cultured adrenal cells after administration of CART. The effects of stress response on corticosterone and GH release after CART administration could be considered but we did not observed any motor and behavioral abnormalities following the injection of CART. Our results showed that CART administered centrally (i.c.v.) and peripherally (i.v.) stimulates GH release. It is an open question whether these effects are the results of the inhibition of somatostatinergic or of stimulation of GH-RH neurons rather than the direct influence of CART on pituitary. In our pervious experiments in vitro CART did not change GH release from cultured pituitary cells [19].

Our previous data [19] demonstrate an increase of PRL in response to CART administration in cell culture. In these studies we showed that CART injected centrally (i.c.v.) and peripherally (i.v.) stimulated PRL release. It could be speculated that CART may stimulate PRL centrally through inhibition of dopaminergic neurons or through stimulation PRL releasing peptides. CART is also able to stimulate directly pituitary cells. Stanley et al. [15] observed that CART administered in a dose, which significantly reduces food intake, inhibits PRL and GH and stimulates ACTH and corticosterone. It has been reported that CART may also be contained in the fibres that innervate TRH neurons and modulate TRH gene expression and biosynthesis of TRH [20]. Double-label in situ hybridization showed that CART is co-expressed with TRH in hypothalamic paraventricular nucleus neurons [14]. On the other hand, thyroid hormones may have effect on CART activity. Hyperthyroidism induces a reduction in CART mRNA levels in PVN [21]. Our experiments in vitro showed the inhibition of TSH release from cultured pituitary cells in response to CART administration [19]. However, CART injected i.c.v. and i.v. did not change TSH release. We did not observe significant change in LH and FSH after central and peripheral injections of CART. However, CART administered to cell cultured leads to inhibition of LH release [19]. The comparison of experiments in vitro and in vivo may suggest that CART acts directly on pituitary cells and inhibits TSH and LH. The discrepancies between experiments in vivo and in vitro may be explained that in vitro CART acts directly on cultured pituitary cells. However, CART injected centrally and peripherally may act in cooperation with endogenous CART and other neuropeptides which may be involved in the mechanism of hormone release [14,16,18,20]. It has been published that CART caused a reduction of GnRH interpulse interval [22,23] and the leptin causes specific faciliatory effects on GnRH amplitude which is mediated by CART [22,23]. We found that intracerebroventricular injection of CART caused a rise in plasma leptin. Our results may indicate that CART modulates pituitary hormone release and leptin may be one of the many factors involved in the mechanism of CART’s action on hormone release [11,15,16,20,22 –24]. It has been reported that administration of CART to hypothalamic explants significantly increased the NPY-IR [16]. In our experiments CART injected centrally and peripherally did not change NPY release. The effects of CART leptin and NPY on the hypothalamic control of pituitary hormones release should be considered and the problems required further investigation.

5. In summary CART administered centrally and peripherally stimulated corticosterone, GH and PRL release.

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Intracebroventricular (i.c.v.) injection of CART leads to an increase of plasma leptin.

6. Conclusion CART may play a modulating role in the mechanism of hormones release.

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