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

Genome-wide association studies (GWAS) have demonstrated the ability to identify the strongest causal common variants in complex human diseases. However, to date, the massive data generated from GWAS have not been maximally explored to identify true associations that fail to meet the stringent level of association required to achieve genome-wide significance. Genetics of gene expression (GGE) studies have shown promise towards identifying DNA variations associated with disease and providing a path to functionally characterize findings from GWAS. Here, we present the first empiric study to systematically characterize the set of single nucleotide polymorphisms associated with expression (eSNPs) in liver, subcutaneous fat, and omental fat tissues, demonstrating these eSNPs are significantly more enriched for SNPs that associate with type 2 diabetes (T2D) in three large-scale GWAS than a matched set of randomly selected SNPs. This enrichment for T2D association increases as we restrict to eSNPs that correspond to genes comprising gene networks constructed from adipose gene expression data isolated from a mouse population segregating a T2D phenotype. Finally, by restricting to eSNPs corresponding to genes comprising an adipose subnetwork strongly predicted as causal for T2D, we dramatically increased the enrichment for SNPs associated with T2D and were able to identify a functionally related set of diabetes susceptibility genes. We identified and validated malic enzyme 1 (Me1) as a key regulator of this T2D subnetwork in mouse and provided support for the association of this gene to T2D in humans. This integration of eSNPs and networks provides a novel approach to identify disease susceptibility networks rather than the single SNPs or genes traditionally identified through GWAS, thereby extracting additional value from the wealth of data currently being generated by GWAS.

Objective: To examine the effect of a high-fat diet on gene expression in adipose tissues and to determine induction kinetics of adipose monocyte chemoattractant protein-1 and -3 (MCP-1 and MCP-3) in diet-induced obesity (DIO) and the effect of a lack of MCP-1 signaling on DIO susceptibility and macrophage recruitment into adipose tissue. Research Methods and Procedures:Obese and lean adipose tissues were profiled for expression changes. The time-course of MCP-1 and MCP-3 expression was examined by reverse transcriptase-polymerase chain reaction. Plasma MCP-1 levels were determined by enzyme-linked immunosorbent assay (ELISA). Chemokine receptor-2 (CCR2) knockout mice were placed on the high-fat diet to determine DIO susceptibility. Macrophage infiltration in adipose tissue was examined by immunohistochemistry with F4/80 antibody. Results: DIO elevated adipose expression of many inflammatory genes, including MCP-1 and MCP-3. Adipose MCP-1 and MCP-3 mRNA levels increased within 7 days of starting a high-fat diet, with elevation of plasma MCP-1 detected after 4 weeks on the diet. The induction of MCP-1 and MCP-3 expression preceded that of tumor necrosis factor-␣. The elevated plasma MCP-1 concentration in obese mice was partially reversed by treatment with AM251. No change in DIO susceptibility and macrophage accumulation in adipose tissue were observed in CCR2 knockout mice, which lack the MCP-1 receptor CCR2. Discussion: A high-fat diet elevated adipose expression of inflammatory genes, including early induction of MCP-1 and MCP-3, supporting the view that obese adipose tissues contribute to systemic inflammation. However, despite increased MCP-1 in obesity, disruption of MCP-1 signaling did not confer resistance to DIO in mice or reduce adipose tissue macrophage infiltration.