Genes, environment, neuroendocrine circuits, and energy balance

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Drug Discovery Today: Disease Models

DRUG DISCOVERY

TODAY

DISEASE

MODELS

Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Denis Noble – University of Oxford, UK

Developmental defects

Genes, environment, neuroendocrine circuits, and energy balance Virginie Tolle1, Malcolm J. Low1,2,3,* 1

Vollum Institute, L474, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA Department of Behavioral Neuroscience, Oregon Health and Science University, Portland, OR, USA 3 Center for the Study of Weight Regulation and Associated Disorders, Oregon Health and Science University, Portland, OR, USA 2

Our understanding of how the brain maintains energy homeostasis is based heavily on rodent genetic models. Increasingly research with these models has focused on gene–environment interactions including stress, diet, nutrition and maternal influences. Here, we review the evolution of transgenic mouse models to provide sophisticated spatial and temporal resolution to analyses of relevant neuroendocrine circuits. We also summarize advances in the environmental disruption of energy balance and the dissection of polygenic obe-

Section Editor: Thomas Braun – Institute of Physiological Chemistry, University of Halle-Witlenberg, Halle, Germany The massive increase of obesity not only in the western world but worldwide has become an increasingly severe medical problem that is well beyond life style attitudes. In addition, revelation of the code that enables small clusters of neurons to exert a strict control on complex behavioral actions is fascinating and might be a paradigm for other even more complex models. The authors pioneered visualization of neuronal networks and their activities that control hyperphagia and obesity. They are among the forefront of researchers who use transgenic mouse models and primary neuronal cells to decipher regulatory networks that control food consumption and energy balance.

sity in rodent models. Introduction Obesity has developed rapidly in the past 20 years, from a syndrome primarily confined to Western cultures to a worldwide epidemic with severe associated health consequences. The most parsimonious explanation for the current obesity explosion appears to be an interaction of paramount genetic factors with an abundance of calorically dense food and declines in physical activity [1]. Hypothalamic and brainstem nuclei within the central nervous system (CNS) decode information about peripheral energy stores and nutritional status conveyed by circulating hormones, and integrate these data with other interoceptive signals to maintain energy intake and energy expenditure in a balanced state [2]. Spontaneous mutations in a handful of homologous genes, ob (leptin), db (leptin receptor), POMC (proopiomelanocortin), MC4-R (melanocortin-4 receptor) *Corresponding author: (M.J. Low) [email protected] 1740-6757/$ ß 2004 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.ddmod.2004.10.007

and PC1 (protein convertase 1) produce identical obesity phenotypes in rodents and humans [1], suggesting that the homeostatic control of feeding and body weight involves similar neuroendocrine pathways among species (Box 1). In particular, the mouse Mus musculus, whose genome and environment can be manipulated easily, has been used extensively as a model organism to elucidate the multiple factors contributing to human obesity.

In vitro models Neuron-specific expression of green fluorescent proteins A major obstacle to the detailed analysis of mammalian neuroendocrine circuits at the cellular and electrophysiologic level has been the non-destructive identification or selection of specific neuronal subpopulations. Unlike principal neurons of commonly studied structures including the cerebral cortex, cerebellum and hippocampus, neuropeptidergic neurons in the hypothalamus and nucleus tractus solitarius (NTS) www.drugdiscoverytoday.com

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Glossary Diet-induced obesity: (DIO) is an experimental form of obesity that occurs in genetically susceptible rodents in response to special diets containing either high-caloric density (high-percentage fat) or highly preferred tastants (sucrose). C57BL/6J mice are commonly used for DIO studies as they are susceptible to diet-induced weight gain, hyperglycemia and insulin and leptin-resistance. Energy homeostasis: is the process that maintains a relatively constant balance between energy intake and energy expenditure in an organism. A positive energy balance, as observed in obesity, is characterized by Energy intake > Energy expenditure. Interoception: is the sensitivity to stimuli originating inside the body and conveyed by peripheral afferent nerves to the central nervous system. Leptin: is a hormone produced by the adipose tissue in proportion to fat content that signals the repletion of body energy stores to the CNS. It acts at the hypothalamic level to decrease appetite and increase metabolism. Most obese individuals develop relative leptin resistance at the CNS level, that is, their neuroendocrine circuits respond weakly despite elevated circulating leptin levels. Obesity: is defined by a body mass index >30. BMI = body weight (kg)/ height (m)2.

Box 1. Control of food intake by hypothalamic neural circuits In the central nervous system, the hypothalamus is a regulator of multiple and complex neuroendocrine functions, including the control of energy homeostasis. To maintain energy intake and energy expenditure in a balanced state the hypothalamus integrates information conveyed by circulating hormones, including leptin, about peripheral energy stores and nutritional status. These signals are processed in specific brain nuclei (arcuate nucleus, paraventricular nucleus and lateral hypothalamus) by a complex network of neurons containing neuropeptide modulators that interact with each other inside the hypothalamus and with the rest of the brain by synaptic connections. Arcuate neurons producing either POMC/CART/GABA or NPY/AgRP/ GABA contain leptin receptors and are important targets of leptin action. In conditions of positive energy balance, when leptin is produced in excess, the anorexigenic POMC neurons are activated and the orexigenic NPY/AgRP neurons are inhibited to decrease food intake and increase energy expenditure. The opposite modifications are observed when energy balance is negative and leptin levels are low. Located downstream of POMC and NPY neurons, the orexigenic MCH and orexin neurons, and the anorexigenic CRH and TRH neurons are also sensitive to leptin levels. They are second order neurons potentially regulated by POMC and NPY/AgRP projections via MC4-R and NPY receptors, but they could also be direct targets of leptin signaling.

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Box 2. Genetic tools for manipulation of the mouse genome Green fluorescent protein or GFP is a spontaneously fluorescent protein isolated from a Pacific jellyfish, Aequoria victoria, that absorbs ultraviolet (UV) energy and then emits it at a lower-energy wavelength. GFP can be expressed as a functional transgene in bacteria, yeast, plants, drosophila, zebrafish and mammalian cells and in a tissue and cell specific manner from heterologous gene promoters to yield tissue-specific expression in transgenic mice. Used as a soluble cytoplasmic or fusion protein, continuously expressed in the neurons and retaining activity, it constitutes a non-invasive marker in viable cells to allow the monitoring of gene expression and protein localization. Enhanced green fluorescent protein (EGFP) has been widely used in transgenic mice because it is more efficiently expressed in mammalian systems and exhibits brighter fluorescence than wild-type GFP. Modification of the GFP by mutagenesis has produced multiple GFP variants with different absorbance and emission spectra, allowing co-localization studies. Among the most commonly used are the blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), red fluorescent protein (mRFP1), topaz and sapphire fluorescent proteins. Additional fluorescent proteins discovered from other marine organisms are now available in the red wavelength range including DsRed or HcRed. These fluorescent proteins are available commercially from BD Biosciences Clontech (http://www.bdbiosciences.com/clontech/gfp/index.shtml), MBL International Corporation (http://www.mblintl.com/mbli/index.asp) and Evrogen (http://www.evrogen.com/). The Conditional Gene Knockout approach using the Cre–LoxP system is fully described in [12]. Basically, it requires two mouse lines: (1) The first is a Cre transgenic line in which Cre recombinase is expressed in selected tissues or at a specific stage of embryonic or post-natal development. For example both the nestin promoterenhancer or synapsin I promoter generally restrict Cre expression to neural precursor cells or mature neurons; and (2) The second line carries the gene of interest flanked by two LoxP recombination sites (a floxed gene), which are the DNA recognition sequences for Cre recombinase. After crossing the two lines, the portion of the gene of interest between the LoxP sites is excised specifically in cells expressing Cre recombinase but is left intact in all other cells, thereby creating a tissue-specific gene-disruption event.

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Box 2. (Continued) Bacterial Artificial Chromosomes (BACs) are circular double stranded DNA cloning vectors that are particularly suitable for the production of transgenic mice from large (up to 250 kb) chromosomal genomic fragments. Fragments of this size typically contain a single gene and frequently retain all the regulatory sequences necessary to assure accurate cell and developmentalspecific expression of that gene. Methods have been developed to reliably insert GFP coding sequences into the transcriptional unit of large numbers of CNSexpressed genes cloned in the BAC vectors and then produce a library of BAC transgenic mouse strains that represent a functional atlas of gene expression patterns in the CNS [32]. BAC vectors are also used to direct Cre expression to specific cell populations for use in Cre– LoxP conditional gene targeting studies [17].

exhibit a complex diversity of neurochemical phenotypes, yet are present in very small numbers (102–104) without characteristic morphologies or stratified localization. Therefore, the transgenic expression of green fluorescent protein (GFP) (Box 2) in these neurons has had a potent impact on basic research concerning the mechanisms of both hormonal and synaptic signaling and gene expression that underlie the neural regulation of food intake and metabolism (Table 1). The combination of epifluorescence and infrared differential interference contrast optics has made it possible to routinely patch-clamp GFP-expressing neuronal populations in acute brain slice preparations. Our laboratory was the first to utilize this electrophysiological approach in the analysis of a hypothalamic neural circuit involved in energy balance. Initial studies conducted on proopiomelanocortin-enhanced GFP (POMC-EGFP) neurons, using a strain of transgenic mice expressing EGFP under the transcriptional control of mouse Pomc genomic sequences, indicated that leptin increased their activity by a combination of direct postsynaptic actions and indirect effects on local presynaptic gamma-aminobutyric acid (GABA) release [3]. Subsequently numerous other factors including opioids, GABAB receptor agonists, serotonin, peptide YY (PYY3-36), extracellular glucose and the KATP channel opener diazoxide were shown to alter the firing properties of hypothalamic POMC neurons in brain slices. Most recently Hentges et al. [4] developed a primary culture preparation of dissociated hypothalamic neurons in which autocrine synapses (autapses) developed and showed that a subpopulation of POMC-EGFP neurons released the rapid inhibitory neurotransmitter GABA. The electrophysiologic properties of other hypothalamic neurons participating in the regulation of feeding have been described in additional newly developed hypocretin/orexin-GFP, neuropeptide Y

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(NPY)-tau-sapphire-GFP and POMC-tau-topaz-GFP transgenic mice [5,6]. Gene promoter-targeting of GFP has also been used to perform co-localization studies among multiple signaling proteins or neurotransmitters. Recently, neurons expressing the melanocortin-4 receptor (MC4-R) were identified by GFP labeling and contained thyrotropin-releasing hormone (TRH) and acetylcholine [7]. GFP expressing transgenic mice also constitute a powerful tool for combined in vivo/in situ experiments that detect signals of cellular activation (like cFos) in specific neuronal populations after drug administration or environmental manipulation. With this latter approach Fan et al. [8] showed that brainstem NTS POMCEGFP neurons were activated by cholecystokinin (CCK), a satiety hormone released after meals. CCK-mediated inhibition of feeding occurred via a MC4-R-dependent pathway, likely involving a brainstem-hypothalamic circuit [8]. Apart from the published studies of transgenic mice with fluorescently labeled neurons, at least two additional important uses of these models can be considered. The first is an extension of the cellular and signaling analyses of identified neuronal subpopulations in vitro to screen for novel pharmaceutical compounds with selected biological activity on neuroendocrine circuits regulating feeding. The second is a large-scale cataloging by cDNA microarray genomic or proteomic approaches of all gene products expressed in specific neuronal populations. This effort is likely to uncover potential therapeutic targets for regulating neuroendocrine circuits.

In vivo models Genetic models Manipulation of gene expression

Both spontaneous and genetically engineered mouse mutants have been heavily exploited in recent years to characterize the genes and central pathways that are physiologically relevant in the control of energy balance. Contemporary neuroendocrine obesity research crystallized around the discovery in 1994 of leptin, a hormone secreted from adipocytes that controls body weight by decreasing appetite and increasing energy utilization via the activation/inhibition of specific populations of neuronal populations (Box 1). Ob/ob mice have a null mutation in the leptin gene causing obesity and have been used extensively to decipher the hormone’s physiological actions. Similarly, a lack of leptin receptor function in db/db mice and Koletsky fa(k)/fa(k) rats produces severe hyperphagia and obesity. Although there are rare mutations in the human leptin and leptin receptor genes that cause morbid obesity, most clinical forms of obesity are associated with elevated serum leptin levels and leptin resistance in the CNS. The molecular basis of neuronal leptin resistance is not fully understood but is speculated to be because of a defect(s) in leptin receptorsignaling pathways [9]. In vitro studies have demonstrated www.drugdiscoverytoday.com

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Table 1. Rodent models to dissect the genetic and neuroendocrine pathways of energy balance In vitro genetic models Transgenic mice with fluorescent protein tagged neural circuits Pros  Non-invasive marker expressed in living cells  Direct visualization of viable neurons by fluorescence/infra-red differential contrast  Simultaneous detection of multiple cell types with different fluorescent proteins Cons

 Ectopic expression of the fluorescent marker  In vitro slice preparation isolates the neural circuit from crucial afferent signals

Best use of models

 Direct recording of synaptic activity in identified populations of neurons  Anatomic mapping of specific neuronal populations that can not otherwise be detected by immunocytochemistry; co-localization of multiple signaling proteins/neurotransmitters  Screening for novel pharmaceutical compounds affecting cellular and synaptic activity  Phenotypic characterization of specific neurons by genomic or proteomic approaches

How to get access to the models See bibliographic references for published models [3–7] In vivo genetic models Global knockout (KO) or knock-in mice Pros  Analysis of endogenous peptides/signaling pathways in an integrated system  Reproduction of human mutations and diseases  Characterization of specific genes responsible for obesity phenotypes Cons

 Possibility of embryonic lethality  Gene disruption during development might affect the phenotype (developmental compensation) in a manner unrelated to the gene’s function in the adult animal  Influence of genetic background on the phenotype requiring congenic strain production

Best use of models

 Characterization of individual molecular components in neuroendocrine circuits in vivo  Determination of the effect of orexigenic/anorexigenic drugs in absence of endogenous ligands

How to get access to the models See bibliographic references [10,11] and PubMed library (http://www.ncbi.nih.gov/entrez/query.fcgi) for published models In vivo genetic models Inducible and tissue-specific conditional KOs using Cre transgenic mice Pros  Provides disruption of gene function at a specific time or in a specific tissue  Permits the study of gene function at a specific age or developmental stage Cons

 Difficulty to engineer, time and labor intensive  Retains some disadvantages of the classic KO strategy (influence of genetic background)

Best use of models

To achieve the most specific degree of cellular and temporal gene expression or disruption and avoid embryonic lethality and developmental compensation effects

How to get access to the models

 Visit Artemis Pharmaceuticals GmbH (http://www.artemispharma.de/) and Jackson Laboratory (http://www.jaxmice.jax.org/) websites for Cre transgenic mice and Gensat (http://www.gensat.org/) for BAC gene expression strategy  See also bibliographic references for published models of tissue-specific or temporal conditional KO mice [13–18]

In vivo genetic models Advanced breeding strategies and QTL analysis Pros  Reproduces epistatic gene interactions contributing to obesity in animal population  Identification of novel candidate genes and polymorphisms associated with obesity risk  Ability to control genetic background and environment in mice Cons

 Identification of specific genes responsible for QTLs is an arduous process  Requires genotyping and phenotypic characterization of hundreds of animals

Best use of models

Genotypic mapping of QTLs associated with obesity traits including energy intake, energy expenditure, body composition, fat distribution, glucose tolerance, insulin sensitivity, sensitivity to high-fat diets and leptin levels

How to get access to the models

 10 RCS strains of polygenic obesity and type II diabetes are available on Jackson Laboratory website (http://www.jaxmice.jax.org/)  See also bibliographic references for published models [23–25]

In vivo gene/environment interaction models Dietary maternal environment and diet Pros  Testing the influence of environmental factors on obesity  Reproduces environmental, social, stress events that can influence feeding behavior

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Cons

Rodent behavior differs from human behavior, particularly cognitive and hedonic

Best use of models

Study of gene/environment interactions related to obesity traits

How to get access to the models

Mice with diet-induced obesity are available from the Jackson Laboratory and Taconic Farms (http://www.taconic.com/). Research Diets (http://www.researchdiets.com/) provides specific diet formulas. See also bibliographic reference for DIO/DR rats [26]

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that leptin receptor activation is coupled to multiple downstream effectors including Jak2/STAT3/SOCS3, SHP-2/GRB2/ ERK and IRS/PI 3-kinase. The significance of each of these biochemical pathways in vivo and the identity of leptin target neurons responsible for leptin resistance have been major objectives of recent gene targeting and transgenic mouse experiments. A landmark accomplishment is the work of Bates and Myers who used a gene knock-in approach with a mutant leptin receptor allele to substitute a serine residue for tyrosine 1138, the crucial target of Jak2 phosphorylation and subsequent recruitment of signal transducer and activator of transcription-3 (STAT3). The resulting s/s homozygous mice expressed normal levels of hypothalamic leptin receptor mRNA but leptin administration failed to activate phosphorylated STAT3. Consequently, the mice developed hyperphagia and obesity with suppressed POMC expression similar to db/db mice, but unlike the latter animals, which are deficient in all leptin receptor intracellular signaling pathways, s/s mice had increased linear growth, normal fertility and unaltered NPY gene expression indicating the retention of distinct non-STAT3-dependent signaling pathways by the mutant S1138Y leptin receptors [10]. Complementary experiments based on the loss of suppressor of cytokine signaling-3 (SOCS3) function have been reported by other laboratories. SOCS3 was previously identified as a leptin-induced negative regulator of leptin receptor signaling and hence a potential mediator of leptin resistance. However, like many signaling molecules that are diffusely expressed in multiple tissues and developmental stages, the initial generation of constitutive SOCS3 homozygous knockout mice resulted in embryonic lethality, precluding the testing of hypotheses related to a role of SOCS3 and leptin receptor signaling in adult mice. Howard and Flier postulated that haploinsufficiency of SOCS3 might be sufficient to produce a measurable phenotype, and therefore, they studied heterozygous SOCS3 deficient mice and demonstrated an elevated sensitivity to exogenous leptin manifested by enhanced weight loss and leptin receptor signaling in addition to an attenuation of high-fat diet-induced obesity [11]. In a further refinement using the technology of the Cre–loxP recombination system [12], which can disrupt gene function in specific tissues or in a time-dependent manner (Box 2), Mori et al. [13] produced a unique mouse model with a neuron-specific loss of SOCS3. Like the mice with a haploinsufficiency of SOCS3 in all tissues, deletion of SOCS3 specifically in the brain elevated sensitivity to leptin, resulting in greater weight loss and suppression of food intake. By contrast, mutant mice designed with either a pan-neuronal or hypothalamic neuronal deficiency of STAT3 expression exhibited decreased leptin responsiveness and developed obesity and glucose intolerance reminiscent of the s/s mice described above [14,15].

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Using a similar Cre–loxP conditional gene knockout strategy, Cohen et al. [16] showed that selective deletion of neuronal leptin receptors was sufficient to produce leptin resistance and obesity while a selective deletion of hepatic leptin receptors did not alter body weight or produce a fatty liver, suggesting that the effects of leptin on body weight are independent of liver activation but are mediated directly via its receptors in the brain. Most recently, a refinement on this theme utilized a bacterial artificial chromosome (BAC) transgenic mouse line (Box 2) to drive Cre expression in POMC neurons and specifically delete leptin receptors from those neurons, thus confirming the importance of leptin receptor signaling in POMC neurons for a portion of leptin’s central actions on body weight regulation [17]. The availability of certain crucial genetic tools such as transgenic mice containing a tamoxifen-inducible Cre–estrogen receptor fusion (Table 1) [18] should lead to further refinements because a successful temporal conditional gene knockout has yet to be reported in the field of energy balance. An alternative strategy to determine the physiological significance of a specific gene’s function within neuroendocrine circuits is a gain-of-function experiment with suitable transgenic constructs. Ob/ob mice exhibit decreased expression of the Pomc gene in the arcuate nucleus. Transgenic expression of POMC from a neuron-specific enolase gene promoter in the ob/ob background partially reversed the obesity and hyperphagia and effectively corrected the glucose intolerance suggesting that decreased POMC expression secondary to leptin deficiency is responsible for many of the metabolic abnormalities [19]. Conceptually similar gene rescue experiments have been performed in leptin receptordeficient rats by the stereotaxic injection of a leptin receptor expressing adenoviral vector into the hypothalamus with resultant partial correction of many of the obesity and metabolic phenotypes [20]. Mapping and isolation of naturally occurring obesity genes using advanced breeding strategies and quantitative trait locus (QTL) analysis

Although numerous rodent models of monogenic obesity have been developed and characterized to understand the regulation of energy balance, the individual effect of most genes that contribute to a complex phenotype is small and usually dependent on interactions with the genetic background, a phenomenon referred to as epistasis. It is generally accepted that multiple gene alleles contribute to common obesity susceptibility in humans and therefore a variety of different strategies have been used to identify candidate genes for further characterization. Foremost among these genetic strategies are experimental crosses utilizing various mouse and rat inbred strains to identify quantitative trait loci (QTL). Specific phenotypes that are commonly measured include energy expenditure, www.drugdiscoverytoday.com

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body composition, fat distribution, glucose tolerance, insulin sensitivity, serum leptin and lipid levels, and sensitivity of body weight gain to high-fat diets. Important mouse models used to identify QTLs for complex traits are the following: congenic strains, recombinant congenic strains (RCS), chromosome substitution strains (CSS), recombinant inbred strains (RIS) and advanced intercross lines (AIL) (See http:// jaxmice.jax.org/info for detailed definitions). These genetic models, their associated statistical strategies and relevance to gene discovery for human obesity have been comprehensively reviewed elsewhere [21–23]. Singer et al. [24] recently demonstrated the power of the CSS approach through the development of a panel of 22 mouse strains, each of which carries a single donor A/J chromosome introgressed onto a common C57BL/6J host background and identification of 150 QTLs affecting diet-induced obesity among other surveyed traits. The Jackson Laboratory (Bar Harbor, Maine) has developed a series of ten RCS models of polygenic obesity and type II diabetes to be used as tools for the genetic dissection of human obesity syndromes associated with glucose intolerance [25]. Phenotypic characteristics of the 10 strains, each based on introgression of mutiple donor chromosomal segments from the New Zealand Obese (NZO/HILt) background onto the non-obese non-diabetic (NON/Lt) host background, are available on the Jackson Laboratory website and the genome-wide scan profiles can be accessed at Dr Leiter’s website (Table 1). A major advantage in studying mice to learn about human energy balance is the ability to rigidly control both genetic background and environment (food availability, type of diet, housing, stress). Indeed, some QTLs are active only in certain environmental conditions like a high-fat diet. Many of the chromosomal regions identified in mice to date correspond to loci associated with human obesity and therefore interspecies comparisons can help to determine which specific genes are conferring obesity risk in humans.

Behavioral and dietary manipulations Genetic factors alone are not responsible for obesity, rather they interact with the environment to increase the risk of obesity and related diseases. Studies in humans suggest that the perinatal maternal environment might have a major impact on the development of obesity in offspring. Therefore, Levin and colleagues capitalized on their extensively investigated polygenic rat model of diet-induced obesity (DIO) and diet-resistance (DR) to determine the independent effects of maternal environment and genotype on the development of obesity in offspring. Rats selectively bred to develop DIO were incrementally more susceptible to develop obesity as adults if their dams were obese during gestation and lactation. Conversely, the obesity of DIO adults was attenuated if they had been cross-fostered to a DR dam. Neither DIO rats nor mice 370

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are fully responsive to the effects of leptin, in part because of a reduction of central leptin receptor-mediated STAT3 phosphorylation and nuclear translocation in the hypothalamus and a parallel activation of SOCS3 [26,27]. Neonatal rodents exhibit a reduced sensitivity to the anorexigenic effects of leptin, emphasizing the importance of developmental stage in the neuroendocrine circuits that regulate energy balance. Bouret et al. [28] recently demonstrated that in neonatal rodents crucial projections from the arcuate nucleus to other parts of the hypothalamus implicated in energy homeostasis are not completely mature and, remarkably, leptin plays an essential organizing function in the maturation of axonal projections from the hypothalamic arcuate to paraventricular nucleus during this period. Other environmental perturbations including maternal malnutrition induced by a 50% reduction in food intake or diabetes induced by streptozotocin treatment during pregnancy and lactation are associated with the development of obesity-associated diseases such as hypertension, hypercholesterolemia, impaired glucose tolerance and type II diabetes in the offspring [29,30]. Stress is another external factor that affects feeding behavior. Prenatal stress, neonatal handling and neonatal isolation have all been shown to alter feeding behavior of adult rats and induce type II diabetes [31]. It seems likely that interactions of similar environmental variables with genetic load are equally pertinent to humans and that more or less permanent modifications in the brain’s neuroendocrine circuits could underlie a subset of pathologic metabolic states during adulthood.

Conclusion Arguably the greatest impact provided by rodent genetic models in the field of energy homeostasis over the past five years has been the elucidation of central neuroendocrine circuits that control feeding and metabolic expenditure. Key experiments include: (1) the direct measurement in fluorescently tagged hypothalamic neurons of rapid cellular and synaptic responses to hormones and neurotransmitters known to influence energy balance; (2) the genetic dissection of molecular components of the leptin receptor signaling pathway in hypothalamic neurons; and (3) the demonstration that leptin exerts organizational effects on the development of hypothalamic feeding circuits in addition to its more completely understood activational effects on the same neuroendocrine circuits in the adult. QTL analyses of rodent polygenic models for moderate obesity and related metabolic traits hold great promise for the identification of many additional genes that individually exert smaller effects than those so far characterized by transgenic technology. An important issue in the analysis of any animal model of a human disorder is the extent to which the model accurately captures information relevant to the pathogenesis, diagnosis, or treatment of the disorder. The fidelity with which mono-

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genic obesity syndromes of the mouse reflect the phenotype in humans with mutations of six homologous gene loci (leptin, leptin receptor, POMC, melanocortin receptor 4, carboypeptidase E and proprotein convertase 1) all converging on a central hypothalamic energy circuit provides strong support for an evolutionarily highly conserved mechanism to regulate caloric balance in mammals [1]. There is less certainty that more peripheral effector arms of the energy homeostasis pathways are as conserved, because there are clearly large differences in the flux of metabolic building blocks and structure of end-organs like brown adipose tissue between humans and rodents. Many of these differences are related to the scaling effects of overall body mass and surface area. Other differences are behavioral and related to the more complex influence of cognitive and hedonic processes, relative to primary homeostatic drive, for control of feeding in humans. Ongoing efforts by multiple laboratories to map obesity related QTLs in both rodent models and natural human populations are likely to be strongly synergistic in the future. Comparative genomics can influence the choice of candidate genes within a captured QTL and transgenic mouse technology provides a powerful means to test functional hypotheses about gene function that can only be inferred statistically from human associational genetic comparisons. Additionally, brain slice preparations from transgenic strains with fluorescently tagged neurons can provide a platform for drug discovery efforts aimed at specific targets within the neuroendocrine circuits. Rapid advances are being made in gene delivery and mutation technology, including lentiviral vectors with the capacity to deliver genes to particular tissues, together with novel embryological manipulations that will allow a broader range of animal models including rats to further contribute to the characterization of gene–environment interactions and the development and prevention of obesity.

Competing financial interests MJL is a co-inventor on patents and has a significant financial interest in biotechnology related to the neuroendocrine control of obesity licensed to Orexigen Therapeutics. This potential conflict of interest has been reviewed and managed by the OHSU Conflict of Interest in Research Committee.

Acknowledgements Research in MJL’s laboratory is supported by NIH grants DK66604 and DK68400. VT is the recipient of an ASPET/ Merck postdoctoral fellowship in integrative pharmacology.

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