Participation of ghrelin signalling in the reciprocal regulation of hypothalamic NPY/POMC-mediated appetite control in amphetamine-treated rats

Abstract

Hypothalamic neuropeptide Y (NPY) and proopiomelanocortin (POMC) have been documented to participate in amphetamine (AMPH)-induced appetite suppression. This study investigated whether ghrelin signalling is associated with changes in NPY/POMC-mediated appetite control. Rats were given AMPH daily for four days, and changes in food intake, body weight, plasma ghrelin, hypothalamic NPY, melanocortin 3 receptor (MC3R), ghrelin O-acyltransferase (GOAT), acyl ghrelin (AG) and ghrelin receptor (GHSR1a) were examined and compared. Food intake, body weight and NPY expression decreased, while MC3R expression increased and expressed reciprocally to NPY expression during AMPH treatment. Plasma ghrelin and hypothalamic AG/GOAT/GHSR1a expression decreased on Day 1 and Day 2, which was associated with the positive energy metabolism, and returned to normal levels on Day 3 and Day 4, which was associated with the negative energy metabolism; this expression pattern was similar to that of NPY. Infusion with a GHSR1a antagonist or an NPY antisense into the brain enhanced the decrease in NPY and AG/GOAT/GHSR1a expression and the increase in MC3R expression compared to the AMPH-treated group. Peripheral ghrelin and the central ghrelin system participated in the regulation in AMPH-induced appetite control. These results shed light on the involvement of ghrelin signalling in reciprocal regulation of NPY/POMC-mediated appetite control and may prove useful for the development of anti-obesity drugs.

Introduction

Ghrelin was first discovered in 1999 (Kojima et al., 1999), and has since been studied extensively for its role in the regulation of energy balance, feeding, stress, anxiety, and reward (Wellman & Abizaid, 2015a). Ghrelin is secreted in the stomach, and is the first peripheral hormone to be identified in the hypothalamic peptide system that exerts a dose-dependent orexigenic effect in obese and lean subjects (Druce et al., 2005, Kojima and Kangaw, 2010, Nakazato et al., 2001, Shintani et al., 2001). Moreover, ghrelin is also synthesized locally in the hypothalamus (Cowley et al., 2003), where it exerts a paracrine effect by activating the arcuate neuropeptide Y (NPY) neurons and inhibiting the proopiomelanocortin (POMC) neurons, leading to an increase in appetite (Chen et al., 2004). Ghrelin can be activated via acylation of the enzyme ghrelin O-acyltransferase (GOAT), which mediates the attachment of fatty acids to lipids and proteins (Gutierrez et al., 2008). The growth hormone secretagogue-receptor (GHS-R) is a ghrelin receptor that has two variants, GHS-R1a and GHS-R1b. GHS-R1a is predominantly expressed in several nuclei of the hypothalamus, such as arcuate nucleus (ARC) and dorsal medial hypothalamus (DMH) (Verhulst and Depoortere, 2012, Willesen et al., 1999). Three components of the ghrelin system have been targeted in the research on energy metabolism: (1) acyl ghrelin (AG), which is an active acylated form of ghrelin; (2) GOAT, the enzyme that activates ghrelin and promotes it binding with its receptor; and (3) the ghrelin receptor-GHSR1a (Wellman & Abizaid, 2015a).

Amphetamine (AMPH) is a well-known appetite suppressant (Chu, Chen, Hsieh, Yu, & Kuo, 2014). The appetite-suppressing effect of AMPH is brought about via the central release of dopamine, which decreases NPY and increases POMC expression in the hypothalamus (Chen et al., 2001, Kuo et al., 2012). Orexigenic NPY and anorexigenic POMC present in the hypothalamus play a prominent role in the regulation of appetite (Rocha et al., 2014), and these two molecules may function reciprocally in the regulation of AMPH-induced appetite control (Hsieh, Chen, Yu, & Kuo, 2014). Melanocortin 3 receptor (MC3R) and MC4R are members of the POMC system, and activation of MC3R and MC4R are associated with an anorectic effect of AMPH (Cowley et al., 1999, Hsieh et al., 2014). In addition to the central neuropeptides, peripheral hormones also participate in the central control of appetite. Peripheral hormones, such as ghrelin, leptin and insulin, primarily bind with their receptors directly in the hypothalamus or in the dorsal vagal complex in the medulla, which communicates with the hypothalamus in the control of energy metabolism (Kim, Lin, Blomain, & Waldman, 2014).

Although ghrelin receptor is expressed more abundantly in the ARC (Perello et al., 2012) and participates in regulating food intake and energy balance (Currie et al., 2005, Kirchner et al., 2012), it is unclear whether plasma ghrelin and hypothalamic AG/GOAT/GHS-R1a signalling are involved in the regulation of NPY/POMC-mediated appetite control in AMPH-treated rats. Moreover, as the result for the action of ghrelin antagonist on GHS-R1a is contrary; when peripheral administration, ghrelin antagonist may block growth hormone (GH) release in ARC, but may stimulate GH release in DMH (Halem et al., 2005, Hassouna et al., 2013). Thus, we will explore whether central administration of ghrelin antagonist can modulate ghrelin system and NPY/POMC expression, and the response of feeding behavior. In order to study this, we will examine the possible involvement of the ghrelin system-by central administration of either a GHS-R1a antagonist or an NPY antisense oligoneucleotide (i.e. NPY knockdown) in AMPH-treated rats.