Ghrelin Causes Hyperphagia and Obesity in Rats

Alison M. Wren 0 Caroline J. Small 0 Caroline R. Abbott 0 Waljit S. Dhillo 0 Leighton J. Seal 0 Mark A. Cohen 0 Rachel L. Batterham 0 Shahrad Taheri 0 Sarah A. Stanley 0 Mohammad A. Ghatei 0 Stephen R. Bloom 0 0 From the Endocrine Unit, Imperial College School of Medicine, Hammersmith Hospital, London, U.K. Endocrine Unit, Imperial College School of Medicine, Hammersmith Hospital , Du Cane Rd., London, W12 ONN, U.K Ghrelin, a circulating growth hormone-releasing peptide derived from the stomach, stimulates food intake. The lowest systemically effective orexigenic dose of ghrelin was investigated and the resulting plasma ghrelin concentration was compared with that during fasting. The lowest dose of ghrelin that produced a significant stimulation of feeding after intraperitoneal injection was 1 nmol. The plasma ghrelin concentration after intraperitoneal injection of 1 nmol of ghrelin (2.83 0.13 pmol/ml at 60 min postinjection) was not significantly different from that occurring after a 24-h fast (2.79 0.32 pmol/ml). After microinjection into defined hypothalamic sites, ghrelin (30 pmol) stimulated food intake most markedly in the arcuate nucleus (Arc) (0 -1 h food intake, 427 43% of control; P < 0.001 vs. control, P < 0.01 vs. all other nuclei), which is potentially accessible to the circulation. After chronic systemic or intracerebroventricular (ICV) administration of ghrelin for 7 days, cumulative food intake was increased (intraperitoneal ghrelin 13.6 3.4 g greater than saline-treated, P < 0.01; ICV ghrelin 19.6 5.5 g greater than saline-treated, P < 0.05). This was associated with excess weight gain (intraperitoneal ghrelin 21.7 1.4 g vs. saline 10.6 1.9 g, P < 0.001; ICV ghrelin 15.3 4.3 g vs. saline 2.2 3.8 g, P < 0.05) and adiposity. These data provide evidence that ghrelin is important in long-term control of food intake and body weight and that circulating ghrelin at fasting concentrations may stimulate food intake. Diabetes 50: 2540 -2547, 2001 - cleus (VMN) (35). During the development of synthetic GHSs, weight gain was noted after chronic systemic administration in immature rodents (1). Ghrelin is a circulating 28-amino acid peptide that was recently purified from rat stomach, and the gene was subsequently cloned in rats and humans (6). It is the first identified endogenous ligand for the GHS-R and is highly conserved across species, differing by only two amino acids between rat and human (6). Ghrelin is synthesized primarily in X/A-like endocrine cells in the oxyntic glands of the stomach and is present in the circulation (7). Circulating ghrelin is elevated after a 48-h fast and subsequently lowered by 50% glucose administration into the stomach but not by the same volume of water (8). Ghrelin is found at lower levels in the hypothalamus, where ghrelin immunoreactivity is confined to the arcuate nucleus of colchicine-treated rats (6). The Arc is an important site in the control of food intake (9). The potent orexigenic neurotransmitters neuropeptide Y (NPY) and Agouti-related protein (AgRP) are colocalized in neurons in the medial Arc (10). After systemic administration of ghrelin or GHSs, c-foslike immunoreactivity (FLI), an indicator of neuronal activation, is evident only in the Arc (11,12). A proportion of these FLI-positive cells are NPY/ AgRP neurons (12), which have been shown to express the GHS-R (13). Thus, arcuate neurons that produce wellcharacterized orexigenic signals are potential targets for circulating ghrelin. The mechanisms that determine normal body weight regulation are not fully understood but are thought to involve hypothalamic neuronal systems responsive to peripheral signals of nutritional status. Leptin is a wellcharacterized satiety signal derived from adipose tissue, which acts on hypothalamic neurons, particularly those in the Arc (14,15). By analogy, ghrelin, released from the stomach in response to fasting, may act as a counterregulatory orexigenic signal to the hypothalamus. Ghrelin has been shown to stimulate food intake after acute systemic (intraperitoneal) or intracerebroventricular (ICV) administration (8,16). The systemic doses of ghrelin used in these studies resulted in plasma ghrelin concentrations much higher than those seen physiologically (8). It is not known how relevant this potent pharmacological stimulation of feeding is to the physiological regulation of food intake. We aimed to establish whether systemic administration of low-dose ghrelin, resulting in plasma ghrelin levels similar to those that occur during fasting, would stimulate feeding. The lowest dose of ghrelin to significantly stimulate feeding after intraperitoneal injection was investiMPO, medial preoptic area; SON, supraoptic nucleus; LHA, lateral hypothalamic area. gated, and the resulting circulating ghrelin concentration was compared with that seen during fasting. The Arc is a likely target for circulating ghrelin, but the GHS-R is also expressed in other discrete hypothalamic sites. To investigate which nuclei are involved in the feeding response, we measured food intake in response to microinjection of ghrelin into defined hypothalamic sites. Finally, to assess the possible role of ghrelin in long-term body weight control, we also examined the effect of chronic ghrelin administration on food intake and body weight and composition. RESEARCH DESIGN AND METHODS Animals. Male Wistar rats (250 300 g) were maintained in individual cages under controlled temperature (2123C) and light (12 h light, 12 h dark, lights on at 0700 h) with ad libitum access to food (RM1 diet; SDS UK) and water. All animal procedures undertaken were approved by the British Home Office Animals Scientific Procedures Act 1986 (Project license no. 90/1077). Intraperitoneal injections. Rats were accustomed to intraperitoneal injection by sham injections of 0.5 ml saline 2 days before study. For all studies, rats received an intraperitoneal injection of ghrelin or saline in 0.5-ml volume. Intranuclear and ICV cannulation and injection. Animal surgical procedures and handling were carried out as previously described (17,18). Animals were anesthetized by intraperitoneal injection of a mixture of ketamine (Ketalar HCl 60 mg/kg; Parke-Davis, Pontypool, UK) and xylazine (Rompun 12 mg/kg; Bayer UK, Bury St. Edmunds, UK) and placed in a Kopf stereotaxic frame. For intranuclear cannulation, permanent 26-gauge stainless steel guide cannulae were implanted in the animals (Plastics One, Roanoke, VA) projecting into the Arc, PVN, VMN, medial preoptic area, supraoptic nucleus, anterior hypothalamic area (AHA), dorsomedial nucleus (DMN), and lateral hypothalamic area of the hypothalamus, according to coordinates of Paxinos and Watson (19) (Table 1, Fig. 1). For ICV cannulation, permanent 22-gauge stainless steel guide cannulae were placed into the third cerebral ventricle (0.8 mm posterior to the bregma on the mid-sagittal line 6.5 mm below the outer surface of the skull, coordinates (...truncated)