Ghrelin, a stomach-derived orexigenic hormone, has stimulated great interest as a potential target for obesity control. Pharmacological evidence indicates that ghrelin's effects on food intake are mediated by neuropeptide Y (NPY) and agouti-related protein (AgRP) in the central nervous system. These include intracerebroventricular application of antibodies to neutralize NPY and AgRP, and the application of an NPY Y1 receptor antagonist, which blocks some of the orexigenic effects of ghrelin. Here we describe treatment of Agrp(-/-);Npy(-/-) and Mc3r(-/-);Mc4r(-/-) double knockout mice as well as Npy(-/-) and Agrp(-/-) single knockout mice with either ghrelin or an orally active nonpeptide ghrelin agonist. The data demonstrate that NPY and AgRP are required for the orexigenic effects of ghrelin, as well as the involvement of the melanocortin pathway in ghrelin signaling. Our results outline a functional interaction between the NPY and AgRP pathways. Although deletion of either NPY or AgRP caused only a modest or nondetectable effect, ablation of both ligands completely abolished the orexigenic action of ghrelin. Our results establish an in vivo orexigenic function for NPY and AgRP, mediating the effect of ghrelin.

Melanin-concentrating hormone (MCH) is a cyclic 19-aa hypothalamic neuropeptide derived from a larger prohormone precursor of MCH (Pmch), which also encodes neuropeptide EI (NEI) and neuropeptide GE (NGE). Pmch-deficient (PmchM elanin-concentrating hormone (MCH) is expressed in the central nervous system predominantly in neurons in the lateral hypothalamus and zona incerta, which project broadly throughout the brain (1, 2). MCH mRNA levels are increased in response to fasting and are elevated in leptin-deficient ob͞ob mice relative to control mice (3), suggesting that leptin negatively regulates MCH. Rodent pharmacology further supports a role for MCH in the control of energy homeostasis, as centrally administered MCH stimulates food intake in rats (3, 4).In addition to MCH, prohormone precursor of MCH (Pmch) also encodes neuropeptide EI (NEI) and neuropeptide GE (NGE) (5) and may potentially give rise to an alternative splice variant termed MCH-gene-overprinted-polypeptide (MGOP; ref. 6), as well as encode a portion of the recently identified antisense-RNA-overlapping-MCH (AROM; ref. 7). Two recently described mouse genetic models further implicate MCH in the regulation of energy homeostasis. Pmch Ϫ/Ϫ mice are lean, hypophagic, and have an increased metabolic rate (8). In contrast, transgenic mice overexpressing Pmch develop mild obesity, are hyperphagic, and become insulin-resistant (9). As both these models represent genetic manipulations of Pmch, one must consider the possibility that in addition to alterations in MCH, changes in the levels of NEI and NGE, as well as potentially MGOP and AROM, may also contribute to the phenotypes of these models.The MCH 1 receptor (MCH1R) was initially identified as an orphan G protein-coupled receptor that bound MCH with high affinity (10). Subsequently, a second high-affinity MCH receptor (MCH2R) with moderate amino acid identity to MCH1R was identified in humans (11-15). Both receptors are highly selective for MCH and are not activated by NEI, neuropeptide GE, or MCH-gene-overprinted-polypeptide (13, 16, 17); however, in vivo validation for these receptors is still lacking. We generated Mch1r Ϫ/Ϫ mice to evaluate the physiological function of MCH1R, and to determine whether it is involved in mediating the effects of MCH on energy homeostasis. Additionally, we hoped to gain insight into what aspects of the Pmch Ϫ/Ϫ and Pmch overexpressing phenotypes are likely attributed to MCH. Materials and MethodsAnimal Care and Maintenance. All animal protocols used in these studies were approved by the Merck Research Laboratories Institutional Animal Care and Use Committee in Rahway, NJ. We housed mice in microisolator cages (Lab Products, Maywood, NJ) in a barrier facility with an air shower entrance or in a specific pathogen-free facility. Mice were maintained on either regular chow [Teklad (Madison, WI) 7012: 14.8% kcal from fat; Harlan Teklad], a moderate-fat diet (D12266B: 32% kcal from fat; Research Diets, New Brunswick, NJ), or a high-fat diet (Teklad 97070: 60% kcal from fat)...

Agouti-related protein (AgRP), a neuropeptide abundantly expressed in the arcuate nucleus of the hypothalamus, potently stimulates feeding and body weight gain in rodents. AgRP is believed to exert its effects through the blockade of signaling by ␣-melanocyte-stimulating hormone at central nervous system (CNS) melanocortin-3 receptor (Mc3r) and Mc4r. We generated AgRP-deficient (Agrp

Bombesin receptor subtype 3 (BRS-3) is a G protein coupled receptor whose natural ligand is unknown. We developed potent, selective agonist (Bag-1, Bag-2) and antagonist (Bantag-1) ligands to explore BRS-3 function. BRS-3-binding sites were identified in the hypothalamus, caudal brainstem, and several midbrain nuclei that harbor monoaminergic cell bodies. Antagonist administration increased food intake and body weight, whereas agonists increased metabolic rate and reduced food intake and body weight. Prolonged high levels of receptor occupancy increased weight loss, suggesting a lack of tachyphylaxis. BRS-3 agonist effectiveness was absent in Brs3(-/Y) (BRS-3 null) mice but was maintained in Npy(-/-)Agrp(-/-), Mc4r(-/-), Cnr1(-/-), and Lepr(db/db) mice. In addition, Brs3(-/Y) mice lost weight upon treatment with either a MC4R agonist or a CB1R inverse agonist. These results demonstrate that BRS-3 has a role in energy homeostasis that complements several well-known pathways and that BRS-3 agonists represent a potential approach to the treatment of obesity.

P EROXISOME PROLIFERATOR-activated receptor (PPAR)-␥ belongs to the nuclear receptor family that serves as a ligand-regulated transcription factor. PPAR␥ forms a heterodimer with the retinoid X receptor (RXR) and regulates gene expression by either binding to specific DNA sequences termed peroxisome proliferator response elements (PPREs) or interacting with other transcription factors in a DNA binding-independent manner (1). The role of PPAR␥ in regulation of glucose and lipid metabolism has been well established, as illustrated by the applications of the thiazolidinedione (TZD) type of PPAR␥ agonists (2). TZDs such as rosiglitazone and pioglitazone improve insulin sensitivity and relieve type 2 diabetes primarily by up-regulating genes involved in glucose and lipid metabolism in adipose tissue, liver, and skeleton muscle (3). PPAR␥ agonists are also shown to reduce triglyceride (TG) content in liver and/or skeleton muscle in both animal models (4, 5) and type 2 diabetes patients (6 -8).Hormone-sensitive lipase (HSL) is an intracellular neutral lipase with a broad specificity for lipid substrates such as TG, diglycerides, cholesteryl esters, and retinyl esters. HSL is the major enzyme responsible for the hydrolysis of stored TG in adipose tissue and has a pivotal role in the mobilization of fatty acids in many other tissues, including liver and muscle (9, 10). Whereas many studies have focused on the posttranslational mechanisms in HSL regulation and demonstrated the importance of the HSL protein phosphorylation in enzyme activity (11), relatively fewer investigations have been carried out in terms of the transcriptional regulation of the HSL gene.The HSL gene is known to be involved in various metabolic disorders. For example, HSL knockout mice develop hyperglycemia and hyperinsulinemia, suggesting that lack of HSL leads to impaired insulin sensitivity (12, 13). The insulin resistance was observed in skeletal muscle and liver in those studies. Human studies also support a role for HSL in insulin sensitivity and show that maximum stimulated lipolysis is defective in patients with the insulin-resistance syndrome (14, 15), and decreased expression and function of HSL are present in fat cells from obese subjects (16). Furthermore, genetic studies suggest that the polygenic background of HSL may be involved in the pathogenesis of type 2 diabetes (17,18).Based on the importance of both PPAR␥ and HSL in metabolism and insulin sensitivity, we studied the regulatory effects of PPAR␥ and its agonists on the HSL gene expression. In this report, we show the evidence that expression of the HSL gene is up-regulated by PPAR␥ and PPAR␥ agonists, which requires the involvement of the transcription factor specificity protein-1 (Sp1).