Identifying variations in DNA that increase susceptibility to disease is one of the primary aims of genetic studies using a forward genetics approach. However, identification of disease-susceptibility genes by means of such studies provides limited functional information on how genes lead to disease. In fact, in most cases there is an absence of functional information altogether, preventing a definitive identification of the susceptibility gene or genes. Here we develop an alternative to the classic forward genetics approach for dissecting complex disease traits where, instead of identifying susceptibility genes directly affected by variations in DNA, we identify gene networks that are perturbed by susceptibility loci and that in turn lead to disease. Application of this method to liver and adipose gene expression data generated from a segregating mouse population results in the identification of a macrophage-enriched network supported as having a causal relationship with disease traits associated with metabolic syndrome. Three genes in this network, lipoprotein lipase (Lpl), lactamase beta (Lactb) and protein phosphatase 1-like (Ppm1l), are validated as previously unknown obesity genes, strengthening the association between this network and metabolic disease traits. Our analysis provides direct experimental support that complex traits such as obesity are emergent properties of molecular networks that are modulated by complex genetic loci and environmental factors.

The expression pattern of mRNA encoding two orexin receptors (OX I R and OX P R) in the rat brain was examined. OX I R and OX P R exhibited marked differential distribution. Within the hypothalamus, OX I R mRNA is most abundant in the ventromedial hypothalamic nucleus whereas OX P R is predominantly expressed in the paraventricular nucleus. High levels of OX I R mRNA were also detected in tenia tecta, the hippocampal formation, dorsal raphe, and locus coeruleus. OX P R mRNA is mainly expressed in cerebral cortex, nucleus accumbens, subthalamic and paraventricular thalamic nuclei, anterior pretectal nucleus. The presence of orexin receptor mRNA in the hypothalamus is in support of its proposed role in feeding regulation. Broad central distribution of orexin receptors may indicate additional functions for orexins.z 1998 Federation of European Biochemical Societies.

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)...

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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

A major task in dissecting the genetics of complex traits is to identify causal genes for disease phenotypes. We previously developed a method to infer causal relationships among genes through the integration of DNA variation, gene transcription, and phenotypic information. Here we validated our method through the characterization of transgenic and knockout mouse models of candidate genes that were predicted to be causal for abdominal obesity. Perturbation of eight out of the nine genes, with Gas7, Me1 and Gpx3 being novel, resulted in significant changes in obesity related traits. Liver expression signatures revealed alterations in common metabolic pathways and networks contributing to abdominal obesity and overlapped with a macrophage-enriched metabolic network module that is highly associated with metabolic traits in mice and humans. Integration of gene expression in the design and analysis of traditional F2 intercross studies allows high confidence prediction of causal genes and identification of involved pathways and networks.

Motilin is a 22-amino acid peptide hormone expressed throughout the gastrointestinal (GI) tract of humans and other species. It affects gastric motility by stimulating interdigestive antrum and duodenal contractions. A heterotrimeric guanosine triphosphate-binding protein (G protein)-coupled receptor for motilin was isolated from human stomach, and its amino acid sequence was found to be 52 percent identical to the human receptor for growth hormone secretagogues. The macrolide antibiotic erythromycin also interacted with the cloned motilin receptor, providing a molecular basis for its effects on the human GI tract. The motilin receptor is expressed in enteric neurons of the human duodenum and colon. Development of motilin receptor agonists and antagonists may be useful in the treatment of multiple disorders of GI motility.