Ghrelin, an Endogenous Growth Hormone Secretagogue, Is a Novel Orexigenic Peptide That Antagonizes Leptin Action Through the Activation of Hypothalamic Neuropeptide Y/Y1 Receptor Pathway

Rapid Publication Ghrelin, an Endogenous Growth Hormone Secretagogue, Is a Novel Orexigenic Peptide That Antagonizes Leptin Action Through the Activation of Hypothalamic Neuropeptide Y/Y1 Receptor Pathway Mitsuyo Shintani, Yoshihiro Ogawa, Ken Ebihara, Megumi Aizawa-Abe, Fumiko Miyanaga, Kazuhiko Takaya, Tatsuya Hayashi, Gen Inoue, Kiminori Hosoda, Masayasu Kojima, Kenji Kangawa, and Kazuwa Nakao Ghrelin, an endogenous ligand for growth hormone secretagogue (GHS) receptor originally isolated from the stomach, occurs in the hypothalamic arcuate nucleus and may play a role in energy homeostasis. Synthetic GHSs have activated the hypothalamic arcuate neurons containing neuropeptide Y (NPY), suggesting the involvement of NPY in some of ghrelin actions. This study was designed to elucidate the role of ghrelin in the regulation of food intake. A single intracerebroventricular (ICV) injection of ghrelin (5–5,000 ng/rat) caused a significant and dose-related increase in cumulative food intake in rats. Ghrelin (500 ng/rat) was also effective in growth hormone–deficient spontaneous dwarf rats. Hypothalamic NPY mRNA expression was increased in rats that received a single ICV injection of ghrelin (500 ng/rat) (~160% of that in vehicle-treated groups, P < 0.05). The ghrelin’s orexigenic effect was abolished dose-dependently by ICV co-injection of NPY Y1 receptor antagonist (10–30 µg/rat). The leptininduced inhibition of food intake was reversed by ICV co-injection of ghrelin in a dose-dependent manner (5–500 ng/rat). Leptin reduced hypothalamic NPY mRNA expression by 35% (P < 0.05), which was abolished by ICV co-injection of ghrelin (500 ng/rat). This study provides evidence that ghrelin is an orexigenic peptide that antagonizes leptin action through the activation of hypothalamic NPY/Y1 receptor pathway. Diabetes 50:227–232, 2001 From the Department of Medicine and Clinical Science (M.S., Y.O., K.E., M.A.-A., F.M., K.T., T.H., G.I., K.H., K.N.), Kyoto University Graduate School of Medicine, Kyoto; and the Department of Biochemistry (M.K., K.K.), National Cardiovascular Center Research Institute, Osaka, Japan. Address correspondence and reprint requests to Yoshihiro Ogawa, Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507 Japan. E-mail: . Received for publication 16 August 2000 and accepted 9 November 2000. Posted on the World Wide Web at www.diabetes.org/diabetes on <<insert date> 2001. AGRP, agouti-related protein; GH, growth hormone; GHRH, growth hormone–releasing hormone; GHS, growth hormone secretagogue; GHS-R, GHS receptor; ICV, intracerebroventricular; NPY, neuropeptide Y. DIABETES, VOL. 50, FEBRUARY 2001 G rowth hormone secretagogues (GHSs) are synthetic compounds that cause the release of growth hormone (GH) from the pituitary (1). They act through the GHS receptor (GHS-R) (2), a previously orphaned G-protein–coupled receptor that is expressed in the hypothalamus, pituitary, pancreas, etc. (2–4). GHSs stimulate GH release by a direct pituitary action (5,6), but several lines of evidence have suggested that it does so via a hypothalamic mechanism as well (7,8). Clinically, GHSs alone or in combination with growth hormone–releasing hormone (GHRH) have been used for diagnosis and treatment of various forms of GH deficiency (1,9,10). Using cells expressing GHS-R as assay systems, Kojima et al. (11) have recently isolated from the rat stomach a novel GHreleasing acylated peptide of 28 amino acids. Termed ghrelin, it can specifically stimulate GH release in vivo and in vitro. Previous studies have demonstrated that central as well as peripheral administration of GHS increases food intake and body weight in rats (12–16). Furthermore, GHS administration induces c-fos mRNA expression in the hypothalamic arcuate neurons containing neuropeptide Y (NPY), a potent stimulator of food intake (17). Indeed, GHS-R is colocalized with NPY in the rat hypothalamic arcuate nucleus (18). Expressed in the hypothalamic arcuate nucleus (11), it is likely that ghrelin acts also as an orexigenic peptide that regulates hypothalamic NPY production. However, the effect of ghrelin on feeding behavior has not been tested so far. Leptin is an adipocyte-derived blood-born satiety factor that acts directly on the hypothalamus, where it regulates a large number of molecules implicated in energy homeostasis (19–21). We and others have demonstrated that the satiety effect of leptin is mediated through the activation of the hypothalamic melanocortin system (22–24). Several lines of evidence have suggested that hypothalamic NPY also mediates some aspects of leptin actions (25–28). Indeed, it has been reported that the leptin receptor is expressed in the majority of the arcuate NPY neurons (29). Currently, there is evidence for the existence of at least six functional NPY receptor sub227 GHRELIN, AN OREXIGENIC PEPTIDE types (Y1–Y6) (30,31). NPY Y1 receptor antagonists suppress endogenous and exogenous NPY-induced feeding, suggesting that Y1 receptor is a major NPY receptor subtype for its orexigenic action (32–34). Furthermore, fasting-induced refeeding is severely affected in Y1-deficient mice (35), suggesting that the hypothalamic NPY/Y1 receptor pathway is activated in response to fasting when plasma leptin concentrations are reduced (36–38). It has been reported that leptin can antagonize the action of exogenously administered NPY (39). Given that leptin reduces the otherwise increased hypothalamic arcuate NPY mRNA expression in fasted rats (28,40) and that fasting-induced refeeding is inhibited by leptin treatment (28,40,41), it is conceivable that the satiety effect of leptin is mediated at least partly through the inhibition of the hypothalamic NPY/Y1 receptor pathway. The aim of this study is to elucidate the role of ghrelin in the regulation of food intake. Here we provide evidence that ghrelin is an orexigenic peptide that antagonizes leptin action through the activation of the hypothalamic NPY/Y1 receptor pathway. RESEARCH DESIGN AND METHODS Seven-week-old male Sprague-Dawley rats (200–220 g) and 9- to 11-week-old spontaneous dwarf rats (SDRs) (100–110 g) (42,43) were purchased from Japan SLC (Hamamatsu, Japan). They were housed in a temperature-, humidity-, and light-controlled room (12-h light/12-h dark cycle) and allowed free access to standard rat food (CE-2,352 kcal/100 g; Japan CLEA, Tokyo), unless otherwise indicated. All experimental procedures were approved by the Kyoto University Graduate School of Medicine Committee on Animal Research. Intracerebroventricular injection experiments. A stainless steel intracerebroventricular (ICV) cannula (outer diameter 1.09 mm) (Becton Dickinson, Sparks, MD) was implanted under anesthesia in the skull of rats 5–7 days before the injection experiments, using coordinates (6.5 mm anterior to the lambdoidal suture; ± 1.4 mm lateral to the midline; 4.5 mm from the du (...truncated)