Unacylated Ghrelin Suppresses Ghrelin-Induced Neuronal Activity in the Hypothalamus and Brainstem of Male Rats

et al. (2014) Unacylated Ghrelin Suppresses Ghrelin-Induced Neuronal Activity in the Hypothalamus and Brainstem of Male Rats. PLoS ONE 9(5): e98180. doi:10.1371/journal.pone.0098180 Unacylated Ghrelin Suppresses Ghrelin-Induced Neuronal Activity in the Hypothalamus and Brainstem of Male Rats Darko M. Stevanovic 0 Aldo Grefhorst 0 Axel P. N. Themmen 0 Vera Popovic 0 Joan Holstege 0 Elize Haasdijk 0 Vladimir Trajkovic 0 Aart-Jan van der Lely 0 Patric J. D. Delhanty 0 Thierry Alquier, CRCHUM-Montreal Diabetes Research Center, Canada 0 1 Department of Internal Medicine, Erasmus Medical Center , Rotterdam , The Netherlands , 2 Institute of Medical Physiology, School of Medicine, University of Belgrade, Belgrade, Serbia, 3 Institute of Endocrinology, Diabetes and Diseases of Metabolism, School of Medicine, University of Belgrade, Belgrade, Serbia, 4 Department of Neuroscience, Erasmus Medical Center , Rotterdam , The Netherlands , 5 Institute of Microbiology and Immunology, School of Medicine, University of Belgrade , Belgrade , Serbia Ghrelin, the endogenous growth hormone secretagogue, has an important role in metabolic homeostasis. It exists in two major molecular forms: acylated (AG) and unacylated (UAG). Many studies suggest different roles for these two forms of ghrelin in energy balance regulation. In the present study, we compared the effects of acute intracerebroventricular administration of AG, UAG and their combination (AG+UAG) to young adult Wistar rats on food intake and central melanocortin system modulation. Although UAG did not affect food intake it significantly increased the number of c-Fos positive neurons in the arcuate (ARC), paraventricular (PVN) and solitary tract (NTS) nuclei. In contrast, UAG suppressed AGinduced neuronal activity in PVN and NTS. Central UAG also modulated hypothalamic expression of Mc4r and Bmp8b, which were increased and Mc3r, Pomc, Agrp and Ucp2, which were decreased. Finally, UAG, AG and combination treatments caused activation of c-Fos in POMC expressing neurons in the arcuate, substantiating a physiologic effect of these peptides on the central melanocortin system. Together, these results demonstrate that UAG can act directly to increase neuronal activity in the hypothalamus and is able to counteract AG-induced neuronal activity in the PVN and NTS. UAG also modulates expression of members of the melanocortin signaling system in the hypothalamus. In the absence of an effect on energy intake, these findings indicate that UAG could affect energy homeostasis by modulation of the central melanocortin system. - Funding: This work was assisted by the ENDO/ESE International Endocrine Scholars Program Fellowship 2011 (DS) supported by the European Society of Endocrinology, and Serbian Ministry of Science and Technological Development proj. no. III 41025. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The prevalence of obesity and related diseases worldwide has catalyzed the need for a greater understanding of how physiological signals of energy intake and/or energy expenditure converge within the brain to regulate energy homeostasis. The brain melanocortin system represents a fundamental component of centrally regulated energy balance. It consists of circuits of neurons expressing either anorexigenic pro-opiomelanocortin (POMC)derived melanocortin 3 (MC3) and 4 (MC4) receptor agonists, as well as MC3R and MC4R expressing cells, which are targets of these neurons. The system also includes orexigenic neurons that express the melanocortin receptor inverse agonist agouti-related peptide (AgRP). Distinct populations of AgRP and POMC expressing neurons are found within the arcuate nucleus of the hypothalamus (ARC) and are co-expressed with neuropeptide Y (NPY) and cocaine- and amphetamine-regulated transcript (CART), respectively [1]. These first order neurons are able to receive peripheral signals about current energy balance via a wide range of circulating hormones (e.g. leptin, insulin, ghrelin, peptide YY3-36) and nutrients (e.g. glucose, fatty acids, amino acids), mediate anabolic or catabolic effects on energy balance and hence modulate food intake and energy expenditure. Melanocortin neurons in the ARC send projections to downstream secondary neuronal populations within proximal nuclei of the hypothalamus, especially to the paraventricular nucleus (PVN). The ARC and PVN, which contain neurons that express MC3R and MC4R, serve as branch points for activation of many central melanocortin-induced circuits involved in body weight regulation [2]. POMC-positive neurons and neural projections are also located within the nucleus of the solitary tract (NTS) of the caudal brainstem. This area receives and integrates both vagal afferent satiation and blood born energy status signals, and issues output commands essential to energy balance control [35]. The function of POMC neurons within the NTS may differ significantly from those in the ARC. Only a small number of studies address this issue, but they suggest divergent roles for hindbrain and forebrain POMC neurons in energy homeostasis [69]. Ghrelin is a 28-amino acid peptide hormone that can be acylated on its third serine residue (acylated ghrelin, AG) by ghrelin O-acyl transferase (GOAT), and is produced predominantly by the gastric oxyntic mucosa in mammals [1012]. Acylation is required for ghrelin to bind to its receptor, the growth hormone secretagogue receptor (GHSR) type 1a [13], located in the hypothalamo-pituitary unit, leading to stimulation of food intake and growth hormone (GH) secretion [10]. Recent studies have revealed that central and peripheral administration of AG results in increased NTS activation, suggesting a role for the NTS in mediating the feed-forward mechanisms of food intake [1416]. However, Kobelt et al. (2008) did not find any change in c-Fos positive neurons in the NTS after peripheral administration of AG [17]. Unacylated ghrelin (UAG) also occurs in the circulation [13]. Although UAG does not activate GHSR1a, it has physiological activity [1824]. A number of studies report that UAG suppresses food intake in rodents both centrally and peripherally [2527], and the effect is likely mediated via ARC and PVN neurons [26]. At the level of the NTS UAG has been shown to disrupt motor activity in the gastric antrum under fasting conditions, which could potentially modulate food intake [28]. In contrast, Toshinai et al. (2006) reported that centrally applied UAG stimulates food intake, while other reports suggest its peripheral administration has no effect on food intake in rodents and humans [18,29,30]. Because it is currently unclear if central UAG has an effect on food intake, we investigated whether central acute administration of AG, UAG or their combination affect neuronal activity in the ARC, (...truncated)