The floating blueprint of hypothalamic feeding circuits

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OPINION

The floating blueprint of hypothalamic feeding circuits Tamas L. Horvath and Sabrina Diano Early lesion studies and subsequent genetic findings identified the hypothalamus as an important regulator of appetite, food intake and energy expenditure. Over the past 10 years, increasing attention has been dedicated to delineating the hypothalamic blueprint of metabolism regulation, in the hope of developing successful strategies to combat metabolic disorders. However, recent developments indicate that the hypothalamic wiring of feeding circuits changes continuously in the face of varying metabolic parameters. These unexpected findings indicate that new therapeutic avenues might emerge from a complete understanding of synaptic plasticity in the hypothalamus.

For more than a century, increasingly sophisticated methods have been applied to the problem of how the brain contributes to the physiology of energy homeostasis and the pathogenesis of obesity. In the last decade, the combination of genetic and physiological techniques has made possible great progress in the identification of metabolic hormones and their relationships to key neuronal systems in the hypothalamus1–6. The adipose hormone, leptin, is a crucial signal that conveys metabolic information from the periphery to the hypothalamus1–6, where the melanocortin system seems to have a fundamental role in orchestrating the subsequent brain response to peripheral metabolic status1–5. This article begins with a brief description of the development of the present view of hypothalamic regulation of metabolism, followed by a discussion of how synaptic plasticity associated with metabolism affects that picture. The hypothalamus affects feeding

The roles of hypothalamic regions in the regulation of energy homeostasis were first revealed by lesion studies in which destruction of the hypothalamic ventromedial, paraventricular and dorsomedial nuclei induced hyperphagia (an abnormal increase in appetite and food intake)7–12. By contrast, lesions of the lateral hypothalamus12 induced

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hypophagia (reduced food intake). These lesion studies were strikingly precise in identifying those subregions of the hypothalamus in which circuits promote or suppress feeding. Physiological observations of obese animal strains13,14 also supported the idea that humoral signals, arising from the periphery, might inform brain sites about overall energy needs. This idea echoed Sherrington’s suggestion, made decades before the lesion studies, that feeding might be regulated in a similar way to respiration, so that peripheral signals affect the composition of the blood and thereby influence the brain15. The observation that some mouse or rat mutants, including db/db (lepr/lepr) and ob/ob (lep/lep) mice and fa/fa (lepr/lepr) rats, become strikingly obese13,14 led to the crucial discovery of the adipose hormone, leptin16, as a humoral signal that can centrally regulate metabolism16–20. The primary genetic defect in these animals is either abolished leptin production (ob/ob mice) or impaired leptin receptors (db/db mice and fa/fa rats) 16–20 . In humans, similar examples of obesity are associated with either a lack of circulating leptin21 or leptin receptor mutation22. Leptin is released by adipose tissue and acts as a humoral signal that carries information about fat stores1–6. Leptin receptors are found in the hypothalamus, particularly in the arcuate nucleus, which is thought to be the primary target for feedback signalling by leptin23,24. These original findings, and the intrigue generated by the nature of leptin signalling, raised expectations that a practical medical approach to fight obesity would be found. Time, so far, has proved otherwise. Other peripheral, primary metabolic signals that seem to be important for central metabolic regulation include glucose25,26, insulin27,28, cholecystokinin29,30, glucagon-like peptide-1 (REF. 31), ghrelin32 and pancreatic polypeptide33,34. Ghrelin is produced by the stomach32, and its discovery has revived the once discarded35 proposition from the beginning of the twentieth century that stomach-driven mechanisms can regulate food intake36,37. Nevertheless, none of these peripheral signals is as closely associated with

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the central regulation of metabolism as leptin. In addition, none, including leptin, has formed the basis for an effective drug that can be used to treat metabolic disorders. The most promising therapies for weight control emerged when no metabolism-specific neurotransmitter signalling pathways were targeted38–41. Arguably, the most successful prescription medication for weight loss, so far, has been a combination therapy that targets serotonin and noradrenaline re-uptake38,42. Most recently, the cannabinoid-1 receptor has emerged as a viable target for the development of a weight-loss drug40,41,43. Although there is evidence that both of these therapies affect components of the melanocortin system39,41, it is also logical that some of the appetite-reducing effects of these approaches lie outside the hypothalamic feeding circuits, for example in the cortex or reward circuitry. However, the leptin experience has provided an invaluable new approach to the understanding of central weight regulation by identifying the CNS, and neuronal communication in particular, as the site where the ‘Holy Grail’ of metabolism should be sought. Hypothalamic signalling pathways

The idea that the CNS, and the hypothalamus in particular, is key in metabolism regulation was reinforced by the discovery of leptin. Before that time, research focused on various neuropeptides and classical neurotransmitters, including GABA (γ-aminobutyric acid)44, glutamate45, neuropeptide Y (NPY)33,46,47, galanin48, serotonin49 and noradrenaline50, in relation to the regulation of feeding and energy expenditure. Interest in these neuromodulators stemmed, at least in part, from their presence in the hypothalamus. GABA, glutamate, NPY and galanin were found to be predominantly orexigenic (pro-feeding) when injected into the third ventricle or various hypothalamic regions, whereas serotonin and noradrenaline seemed to be anorexigenic (anti-feeding)3. However, some of these substances could trigger the opposite response, depending on the site of injection and dose3. No research on central neuromodulators in relation to feeding regulation has exceeded in quality (or in overall impact in the field) that which has been carried out on NPY. Research on NPY-associated food intake was initiated by the landmark study of Clark et al. in 1984, in which the first evidence of NPY-induced feeding was presented33. When applied intracerebroventricularly, NPY induced an acute and robust feeding response33 that has been unparalleled by

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Lateral hypothalamus

Model of a floating blueprint

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Figure 1 | Hypothalamic peptidergic systems involved in regulating energy balance and food intake. Schematic drawing showing known interactions and projection targets of various hypothalamic peptidergic systems, including lateral hypothalamic neurons producing hypocretin/orexin (Hcrt) and melanin concentrating hormone (MCH) and components of the melanocortin system, the neuropeptide Y/ agouti-related peptide (NPY/AGRP)- and α-melanocyte-stimulating hormone (α-MSH)-producing neurons. These neuronal populations are targeted from the circulation by white adipose tissue-derived leptin and stomach-derived ghrelin. They are synaptically interconnected and project, to some degree, in an overlapping manner to intra- and extra-hypothalamic sites. It is a recurring mistake to consider peptide circuits as independent entities: almost without exception, peptidergic neurons contain various other neuromodulators, including the classical neurotransmitters GABA (γ-aminobutyric acid) or glutamate (Glut). With varying artistic characteristics, most of the schemes proposed to date are similar to FIG. 1 in that they depict connectivity as a still picture implying hard wiring. While such mapping of connectivity will continue to provide important building blocks for evolving concepts, they lack both synaptic resolution as well as indication of soft wiring or plasticity. Question marks indicate putative GABA or glutamate content.

administration of any other substances, including the lateral hypothalamic orexigenic peptides, melanin-concentrating hormone51 and orexin/hypocretin52. Subsequently, the leptin story seemed to be linked to the NPY research as, for example, arcuate nucleus NPY neurons were found to be direct targets of leptin53. NPY and the arcuate neurons that produce it emerged as effectors that respond to changes in leptin levels. In conjunction with this, it was found that the obesity of leptindeficient mice is reduced by elimination of NPY54. Research into NPY continues, in part owing to the expression of NPY in a particular subset of neurons in the arcuate nucleus of the hypothalamus. These neurons also produce agouti related-peptide (AGRP)55, which has been shown to be an essential component of the melanocortin system, the putative ‘heart’ of hypothalamic feeding regulation. A more recent breakthrough was the revelation that the central melanocortin system is a key mediator of energy balance56,57 and leptin-induced satiety58. Of particular interest was the ‘yin–yang’ relationship between two sets of neurons in the arcuate nucleus — those that contain NPY/AGRP and those that contain proopiomelanocortin (POMC) — as the main regulator of appetite, satiety and energy expenditure regulation2–4,59. The

POMC cells, which produce α-melanocyte stimulating hormone (α-MSH), maintain an anorexigenic tone, whereas the NPY/ AGRP neurons maintain an orexigenic tone in which AGRP antagonizes α-MSH on the melanocortin 4 receptor 60. It is not unreasonable to suggest that melanocortin signalling represents a main ‘funnel’ that gathers information for the integration of peripheral metabolic signals to the final output of the hypothalamus in energy expenditure regulation. It is not surprising, therefore, that increasing attention has been paid to mapping the distribution of various hypothalamic peptide systems and integrating their signalling with that of the melanocortin system (FIG. 1). Such a blueprint, if assembled at the level of synapses, and correlated with peripheral hormone receptor distribution and classical neurotransmitter (GABA, glutamate, noradrenaline and serotonin) signalling61, will provide an invaluable tool for addressing conditions in which there is an imbalance in energy homeostasis regulation, such as obesity, diabetes or anorexia. However, this blueprint would represent only a snapshot of the system: for a greater understanding of how metabolism and food intake are regulated, we must consider the presence of redundancy and plasticity in the system.

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Redundancy indicates that there is a significant amount of flexibility or plasticity in the interactions of hypothalamic neuronal circuits. Recent developments, discussed below, have revealed that ongoing plasticity in adult feeding circuits might be an inherent component of energy balance control. These mechanisms might help to resolve some of the burning issues of obesity research, including how redundancy and leptin resistance arise or can be controlled. The idea that there is plasticity in the adult hypothalamus is not new. Ten years before the discovery of leptin, Theodosis, Poulain and colleagues showed that the magnocellular system in the hypothalamus exhibits morphological plasticity62 that is regulated by oxytocin63 and occurs within hours64. Around the same time, Garcia-Segura and colleagues65 found that oestradiol also causes a rapid rearrangement of synapses in the arcuate nucleus, an observation that was in line with the earlier work of Matsumoto and Arai, who showed the effect of oestradiol on arcuate nucleus synapses in adult rats with lesions66,67. Strikingly, subsequent studies showed that changes induced by oestradiol in neuronal membrane composition that are probably associated with synaptic plasticity occur within minutes of neuronal exposure to this gonadal steroid68. This oestrogen-induced synaptic remodelling of arcuate nucleus circuitry seems to be an inherent element of the central regulation of gonadotropin secretion69,70. These findings, together with previous anatomical data indicating that oestrogen and leptin have overlapping sites of action in the hypothalamus71,72, gave impetus to our investigations into whether leptin action is also associated with synaptic plasticity in central feeding circuits. In collaboration with the laboratory of Jeffrey M. Friedman at the Rockefeller Univeristy, we studied the two basic components of the melanocortin system, NPY/ AGRP neurons and POMC neurons, in the arcuate nucleus of the leptin-deficient ob/ob mouse. Increased activity of NPY/AGRP neurons suppresses melanocortin tone73 and promotes feeding; conversely, elevated POMC tone supports satiety. Classical neuroanatomical studies indicated that the perikarya of POMC neurons in ob/ob mice would have an increased number of connections, because inhibitory inputs, which should suppress POMC activity, are usually situated on the perikaryal membrane. Surprisingly, however, this was not the case: there were fewer synapses on the perikarya of POMC neurons

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PERSPECTIVES from ob/ob mice74. By contrast, there were more synapses on the perikarya of NPY/ AGRP neurons in ob/ob mice than in wildtype mice. A more detailed analysis showed that, even though there are fewer synapses on the perikarya of POMC neurons in ob/ob mice, symmetrical, putative inhibitory synapses dominate over asymmetrical, putative stimulatory ones75. This synaptic organization is consistent with the finding that ob/ob POMC neurons show higher spontaneous and mini inhibitory postsynaptic currents in slice cultures than do wild-type neurons74. In NPY/AGRP neurons from ob/ob mice, asymmetrical (putative excitatory) synapses dominated over symmetrical (putative inhibitory) connections, and this was paralleled by elevated spontaneous and mini excitatory postsynaptic currents. It is likely that the altered synaptic organization of the NPY/AGRP and POMC cells in ob/ob mice arises during development and is due to a lack of leptin. Consistent with a developmental role for leptin, a fluorescence microscopy study found that leptin can cause irreversible alterations in some melanocortin efferents in subregions of the hypothalamic paraventricular nucleus76. Bouret et al. analysed arcuate nucleus efferents using 3,3′-dioctadecylindocarbocyanine (DiI) labelling and found that a latero-dorsal region of the parvicellular paraventricular nucleus receives significantly less input from the arcuate nucleus in ob/ob animals than in wild-type littermates76. Some of these arcuate inputs probably arise from the melanocortin system, as both AGRP and α-MSH labelling are affected, to various degrees76. There is a crucial early postnatal developmental period in mice during which leptin must be present to diminish these gross anatomical differences76,77. These observations, if confirmed in non-human primates and humans, will be important for the development of a better understanding of the aetiology of childhood obesity and its influence on adult metabolic disorders. Interestingly, however, although leptin replacement in adult mice could not reverse the altered paraventricular input, it could rescue the main metabolic phenotype of ob/ob mice18,74. So, it is reasonable to suggest that at least one essential component of the feeding circuitry remains plastic in adulthood74. In accordance with this, leptin replacement in adult ob/ob animals re-wired both NPY/AGRP and POMC perikarya to support an anorexigenic tone and weight loss74. Further support for the idea that synaptic plasticity is an inherent and perhaps mandatory component in the regulation of energy homeostasis is provided by the fact

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Figure 2 | Schematic illustration of the unmasked synaptic plasticity of components of the melanocortin system. Perikaryal inputs of NPY/AGRP neurons are dominated by inhibitory (blue) connections when circulating leptin levels are high. These connections are rearranged (coloured arrows indicate changes and dotted outlines indicate new locations) when leptin levels diminish (and ghrelin levels increase). Under these circumstances, stimulatory synapses (orange) dominate over inhibitory inputs. On POMC perikarya, the changes occur in the opposite direction of that described for the NPY/AGRP inputs. Some of the inhibitory inputs on POMC cells are likely to originate from the NPY/AGRP neurons (yellow dots in a blue axon)66, and some of the stimulatory inputs on both cell types originate in the lateral hypothalamic orexin/hypocretin neurons (yellow dots in an orange axon) 82. Because rapid synaptic changes were observed in wild-type animals66, it is reasonable to propose that synaptic rearrangement of feeding circuits is a continuous phenomenon, which will also occur in a circadian fashion responding to the changing daily metabolic environment. The synaptic changes themselves might not directly trigger alterations in postsynaptic events, but could readily alter the probability of either stimulation or inhibition.

that the orexigenic, stomach-born hormone, ghrelin, also robustly alters the synaptic input of POMC neurons in wild-type animals74. The ghrelin-induced rearrangement of POMC efferents was the opposite of the leptininduced effect, resulting in an overall inhibitory tone on POMC perikarya associated with increased feeding74. These results make it clear that a static blueprint of hypothalamic signalling, like that shown in FIG. 1, cannot explain the dynamics of hypothalamic regulation of feeding and energy expenditure. Instead, we suggest that the ever-increasing information on hypothalamic circuits should be integrated into a fluid scheme (FIG. 2) and that the stagnant blueprint of hypothalamic feeding pathways be considered as only a momentary reflection of the metabolic state. To illustrate this floating blueprint, FIG. 2 depicts NPY/AGRP and POMC neurons of the arcuate nucleus. When circulating leptin concentrations are high, and circulating ghrelin concentrations are low, the perikaryal inputs of NPY/AGRP neurons are dominated by inhibitory connections (satiety or anorexigenic tone). These connections are rearranged when leptin concentrations diminish and ghrelin concentrations rise,

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so that excitatory synapses dominate over inhibitory inputs (hunger or orexigenic tone). During satiety, excitatory synapses dominate on the perikarya of POMC neurons (anorexigenic tone), but changing concetrations of leptin (diminishing) and ghrelin (elevating) result in the emergence of inhibitory connections that dominate over excitatory ones during negative energy balance (hunger or orexigenic tone). Some of the inhibitory inputs on POMC cells probably originate from the NPY/AGRP neurons74, and some of the stimulatory inputs, particularly on the NPY/AGRP cells, arise from the lateral hypothalamic orexin/hypocretin neurons78. Because these rapid synaptic changes were observed in wild-type animals as well 74, it is reasonable to propose that synaptic rearrangement of feeding circuits is a continuous phenomenon, which will also occur in a circadian fashion in response to the changing daily metabolic environment. Although the synaptic changes themselves might not directly trigger alterations in postsynaptic events, they could readily alter the probability of either stimulation or inhibition of neuronal firing. There are several issues relating to metabolism-regulated neuronal connectivity that

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PERSPECTIVES require immediate attention. For example, whether rearrangement of feeding circuits occurs in all species, including primates, and whether it happens under all types of metabolic perturbation need to be resolved. Equally important is to find out whether impaired central responses to the peripheral metabolic state can occur as a result of failures in central plasticity. Examples of such situations include all examples of leptin resistance, as develops during diet-induced obesity. Another intriguing line of inquiry would establish the temporal kinetics of synaptic rearrangements during changing metabolic states, and the origin of the axons that are involved. Although we found statistically significant changes as early as 6 h after leptin administration74, those changes must have been initiated substantially earlier. In addition, it would be helpful to determine whether synaptic inputs of other hypothalamic neuronal populations associated with feeding are also dynamically reorganized. Mechanisms of hypothalamic plasticity

Deciphering the molecular basis for plasticity in adult feeding circuits is essential for addressing the above issues. With regard to leptin’s effect, it is likely that the long-form leptin receptor is an important component in triggering synaptic remodelling, because it is the essential leptin receptor type for anorexigenic leptin signalling1–6. The action of leptin through the long-form leptin receptor requires the activation of the signal transducer and activator of transcription 3 (STAT3) signalling pathway to decrease food intake and increase energy expenditure79–81. The availability of transgenic and knockout animal models that have various impairments in leptin signalling will make it easier to investigate the role of these signalling pathways in synaptic plasticity. On the other hand, although synaptic plasticity is triggered by leptin, the rearrangement of synapses is obviously the consequence of gene products that are involved in membrane organization and maintenance, receptor trafficking and recycling, as well as in cell–cell interactions. The elucidation of these intracellular mechanisms will aid our understanding of plasticity in feeding circuits, and might lead to the identification of novel targets for pharmacological strategies to treat metabolic disorders. Some candidates for involvement in hypothalamic plasticity can already be identified. For example, one of the most abundant celladhesion molecules in the arcuate nucleus is polysialylated neuronal-cell-adhesion molecule (PSA-NCAM)82,83. PSA-NCAM is widely expressed in the embryonic brain, but remains

in only selected areas of the adult brain where synaptic plasticity is maintained in adulthood, such as the arcuate nucleus and hippocampus. It is logical, then, that like oestradiol83, leptin might mediate synaptic plasticity by altering PSA-NCAM in the arcuate nucleus. We base this prediction on the following observations: both leptin and oestradiol can cause rapid synaptic rearrangement in the arcuate nucleus involving putative inhibitory synapses74,84; leptin receptors are co-localized with oestradiol receptors72; and the removal of inhibitory synapses depends on PSA-NCAM83. We further anticipate that the ability of the adult hypothalamus to maintain plasticity must, at least in part, derive from maintained expression of certain homeodomain genes that are also associated with the immature, developing brain. For example, OTP, the product of the homeodomain gene orthopedia, is essential for pattern formation in the embryonic hypothalamus85 and is expressed in the adult hypothalamus (T.L.H and S.D., unpublished data). On the basis of the retention of these developmentally vital molecules in the adult hypothalamus, it is tempting to speculate that appropriate regulation of endocrine and metabolic functions requires a hypothalamus that retains substantial amounts of immaturity. The aforementioned are only examples of candidate molecules that might mediate the effect of leptin on synaptic plasticity in the arcuate nucleus. To acquire a more comprehensive picture, gene array analyses of the arcuate nuclei of subjects with diverse metabolic phenotypes, aided by already available information about plasticity, are needed. These studies will provide candidate genes, the roles of which can then be tested. ‘Learning’ within feeding circuits

Regardless of the cellular mechanism of metabolism-associated synaptic plasticity, the fact that it occurs must be considered and incorporated into our knowledge of the central regulation of metabolism. An intriguing consequence of synaptic rearrangement on components of the melanocortin system is that, depending on peripheral metabolic need, the activation threshold of the orexigenic and/or anorexigenic components might be altered. For example, when there is a positive energy balance (reflected by high levels of leptin), the predominance of stimulatory inputs on POMC cells will increase the likelihood of an elevated POMC tone being induced by acute anorexigenic signals, and will consequently decrease food intake and increase expenditure. Conversely, during negative energy balance when leptin is low and stimulatory synapses predominate on the

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perikarya of NPY/AGRP neurons, these neurons will be more easily activated by peripheral orexigenic signals, such as ghrelin, which will lead to an increase in food intake and a decrease in energy expenditure. Of course, synaptic plasticity alone does not explain the behavioural and endocrine responses to changing metabolic states. Even the probability of excitation or inhibition of components of the melanocortin system is probably affected by various mechanisms. An intriguing new possibility is emerging in the relationship between glial cells (astrocytes and radial glial cells, also called tanycytes) in the arcuate nucleus, and neurons of the melanocortin system. The energy level-dependent production of active thyroid hormones (T3) by type II deiodinase (D2) in glial cells86 might directly affect the metabolism of NPY/ AGRP neurons that express mitochondrial uncoupling protein 2 (UCP2)87. Because the activity and transcription of UCP2 are both induced by T3 (REF. 88), and increased UCP2 activity elevates neuronal mitochondrial number and ATP production89, it is likely that T3 formed locally by astrocytes and tanycytes will boost the metabolism of NPY/AGRP neurons during periods of negative energy balance. UCP2 expression and activation in NPY/AGRP cells might also offer a potential mechanism for the newly discovered effect of AMP kinase on induction of food intake90,91. AMP kinase enhances the transport of fatty acid to the mitochondria92, which is a ratelimiting step in UCP2 activation93. So, an AMP kinase-associated elevation in fatty-acid transport to the mitochondria could influence NPY/AGRP neuronal activity by enhancing uncoupling activity (in a similar way as described above for T3), resulting in increased mitochondrial proliferation and ATP production in these orexigenic cells. NPY/AGRP neurons with elevated intracellular fuel storage (induced by this glial–neuronal interplay) and altered synapses will then be more likely to be activated and to engage in, for example, burst firing94. Whatever the mechanism, the dynamic rearrangement of connectivity underlies changes in the threshold that must be reached for a given signal, a mechanism that fulfills a requirement for a basic learning process. The availability of animals that express green fluorescent protein in almost all hypothalamic peptidergic systems will also allow us to test whether long-term potentiation and long-term depression occur on these cells under various metabolic states. Undoubtedly, once unmasked, synaptic plasticity74 and developmental effects76 induced by metabolic cues will alter our views on hypothalamic feeding circuits. These new

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PERSPECTIVES avenues might provide innovative targets through which we can interfere with central components of energy homeostasis under pathological conditions. Equally tantalizing is the potential that these peripheral metabolic cues could affect synaptic plasticity outside the hypothalamus, including neuronal processing in higher brain regions, such as the cortex and hippocampus. The likelihood of these effects is again supported by findings that relate to oestradiol, which both directly95 and indirectly96 affects hippocampal synaptic morphology. Questions such as whether temporal or chronic alterations in metabolic state influence learning and memory are not only intriguing but might have direct medical implications. For example, there is a positive correlation between adiposity and Alzheimer’s disease97, and leptin has effects on learning and hippocampal function98. Studies need to be conducted to better understand the overall importance of rapidly changing neural connectivity in both healthy and pathological conditions. Tamas L. Horvath and Sabrina Diano are at the Department of Obstetrics, Gynecology & Reproductive Sciences, Yale University School of Medicine, 333 Cedar Street FMB 339, New Haven, Connecticut 06520, USA. Tamas L. Horvath is also at the Department of Neurobiology, Yale University School of Medicine, 333 Cedar Street FMB 339, New Haven, Connecticut 06520, USA. Correspondence to T.L.H. e-mail: [email protected] doi: 1038/nrn1479 1.

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Acknowledgements Research projects of the authors associated with mechanisms discussed in this paper have been supported by the following institutes of the National Institute of Health (NIH): NCRR, NIA, NIDDK, NIMH and NINDS.

Competing interests statement The authors declare no competing financial interests

Online links DATABASES The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene AGRP | NPY | POMC1 | STAT3 | UCP2 Access to this interactive links box is free online.

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