Obesity and its related disorders are among the most pervasive diseases in contemporary societies, and there is an urgent need for new therapies and preventive approaches. Given (i) our poor social capacity to correct unhealthy habits, and (ii) our evolutionarily genetic predisposition to store excess energy as fat, the current environment of caloric surplus makes the treatment of obesity extremely difficult. During the last few decades, an increasing number of methodological approaches have increased our knowledge of the neuroanatomical basis of the control of energy balance. Compelling evidence underlines the role of the hypothalamus as a homeostatic integrator of metabolic information and its ability to adjust energy balance. A greater understanding of the neural basis of the hypothalamic regulation of energy balance might indeed pave the way for new therapeutic targets. In this regard, it has been shown that several important peripheral signals, such as leptin, thyroid hormones, oestrogens and bone morphogenetic protein 8B, converge on common energy sensors, such as AMP-activated protein kinase to modulate sympathetic tone on brown adipose tissue. This knowledge may open new ways to counteract the chronic imbalance underlying obesity. Here, we review the current state of the art on the role of hypothalamus in the regulation of energy balance with particular focus on thermogenesis.
Obesity and the central nervous system
Throughout the last few decades, the incidence of obesity and its related disorders has increased to epidemic proportions. Recent analysis indicates that at the end of last decade, ∼36% of men and 38% of women of the world population were overweight or obese causing ∼3–4 million deaths in 2010 . Despite the global awareness, the promotion of healthy lifestyles and the development of new medical strategies remain inadequate to deal with the growing rate of obesity. This might lead to a society with a shorter life expectancy and poorer quality of life, and also a less productive one with overspending in the public health systems .
Obesity results from a sustained imbalance between energy intake and energy expenditure [3–5]. It is therefore required that mechanisms for energy storage and energy expenditure are properly balanced with those involved in energy intake. Excess caloric intake in a meal is efficiently stored as fat in white adipose tissue (WAT), leading to obesity over time. Lipid storage and WAT expansion are normal physiological processes, but excess lipid accumulation in WAT may lead to adipose tissue dysfunction and give rise to obesity-associated metabolic disorders and comorbidities [6–9]. Hence, in the context of modern lifestyles with increased accessibility of palatable hypercaloric food in addition to our genetic predisposition , it essential to understand the physiological mechanisms modulating energy balance in order to combat obesity. Currently, we know that the homeostatic mechanisms regulating energy balance involve complex and overlapping interactions between peripheral organs and the central nervous system (CNS). With this background, a greater understanding of the neural basis of energy homeostasis and how this is disturbed in metabolic disease has become a main challenge of metabolic research during the last few decades.
Hypothalamic networks and sympathetic drive to burning fat
Data spanning from early lesion studies of discrete brain regions to more recent approaches with genetically engineered murine models or monogenic human disorders have made it clear that the CNS plays a key role in energy metabolism. The brain receives metabolic information through vagal afferent or circulating metabolites (such as glucose and lipids) and hormones (such as leptin, insulin, thyroid and gonadal hormones) to integrate the energy status and modulate energy balance. Among the several brain regions of the CNS involved that control energy homeostasis, the hypothalamus is the undisputed centre integrating metabolic information. The hypothalamus is the lower structure of the diencephalon (Figure 1) and is organized in multiple clusters of neurons, called nuclei. The role of the arcuate nucleus (ARC) on feeding behaviour is well established, but cumulative data indicate that several other hypothalamic sites, such as the paraventricular (PVH), the dorsomedial (DMH) and the ventromedial (VMH) nuclei, as well as the preoptic area (POA) and the lateral hypothalamic area (LHA), also play critical roles in the control of energy homeostasis [3,11]. An integrated hypothalamic response to nutrient intake requires a complex cross-talk between hypothalamic nuclei, the brainstem, the spinal cord and peripheral tissues in response to environmental signals [3,11,12]. Also, hypothalamic nuclei act as a network, forming complex intra-hypothalamic neuronal circuits, which respond to changes in energy status by altering the expression of specific neuropeptides resulting in changes both in food intake and in energy expenditure [13,14]. In addition to the regulation of neuropeptide expression, recent evidence indicates that nutritional status is able to modulate the hypothalamic physiology, adapting the permeability of the blood–brain barrier (BBB) to circulating metabolic signals [15,16].
Neuroanatomical model of the hypothalamic control of adaptive thermogenesis.
In summary, since the early studies conducted in the middle of the last century, which essentially identified two feeding centres (a satiety and hunger centre in the ventromedial and lateral hypothalamus, respectively), we have now gained detailed knowledge about how the hypothalamus regulates energy expenditure, metabolism and nutrient partitioning, beyond the simple control of food intake [3,11,13,14]. Because hypothalamic networks responsible for the regulation of food intake have been extensively and recently reviewed in the literature [13,14], we will here mostly focus on the mechanisms through which hypothalamic nuclei control energy expenditure to modulate energy balance. Our understanding of this field has improved in recent years by the development of new methodological approaches such as mapping of brain regions using neuronal tracers such as pseudorabies viruses [17,18], comparative imaging technologies such as magnetic resonance imaging  and positron emission tomography [20,21] applied to both animal and humans, virogenetics , optogenetics  and designer receptors exclusively activated by designer drugs . The use of these experimental approaches has allowed the uncovering of the neuroanatomical basis of central control of energy homeostasis and the interaction among the different hypothalamic nuclei involved. Furthermore, it is now feasible to silence or stimulate the activity of specific subsets of hypothalamic neurons, thus determining in real-time in vivo functional responses. Using these approaches, our knowledge on how different central and peripheral signals act at the hypothalamic level to influence other aspects of energy balance, such as brown adipose tissue (BAT) activity, is gaining pace.
Sympathetic control of BAT and WAT
BAT is a special kind of fat that dissipates chemical energy as heat through adaptive thermogenesis, defined as non-shivering heat production [5,25–28]. Adaptive thermogenesis is an essential process to maintain body temperature in small and hibernating animals through uncoupled mitochondrial fatty acid oxidation [25,27,29]. Brown adipocytes contain a large number of mitochondria that express high levels of uncoupling protein 1 (UCP1), a protein that increases the permeability of the inner mitochondrial membrane, diverting protons from the oxidative cycle, ultimately leading to the dissipation of energy through the production of heat. BAT is fully innervated by sympathetic efferent fibres that ensure central control of thermogenesis [5,30].
The potential therapeutic targeting of adaptive thermogenesis resides in the extraordinary energy cost of the uncoupled fatty acid oxidation. BAT can account for up to 75% of the increased metabolic rate induced by noradrenaline (NA) in cold-adapted animals . Also, BAT can burn up to 50% of ingested triacylglycerols and 75% of ingested glucose . Accordingly, cold exposure drastically increases plasma clearance of triacylglycerols and ameliorates hyperlipidaemia . Otherwise, BAT transplantation improves insulin sensitivity and glucose homeostasis in intolerant murine recipients [34,35]. In addition, recent data estimate that intracellular triacylglycerol pools contribute to up to 84% of the thermogenic cost during acute cold exposure . All these results indicate that dissipating excess of energy as heat might be an appealing and realistic treatment strategy against obesity [5,29,30,37].
Brown adipocytes are classified as either classic brown adipocytes located in the BAT  or ‘inducible brown adipocytes’/‘beige’/‘brite’ cells that are discretely distributed in WAT depots [19,25,27,28,39]. The process of recruiting and activating beige adipocytes is referred to as ‘browning’ [5,25,27,28]. Brown adipocytes used to be considered relevant merely in new-born humans, but recent data have challenged that view [19–21,25,39–46]. In fact, recent data have reported that the gene expression profile of human supraclavicular adipose tissue is closer to beige adipocytes rather than the expected classic brown cells, suggesting that human BAT is composed mainly by inducible brown adipocytes [19,47–49]. Although the transdifferentiation of white adipocytes might contribute to browning [50,51], a specific lineage of beige adipocytes has recently been proven to exist, with low basal expression of UCP1-like white adipocytes but able to increase UCP1 expression and respiratory rates in response to sympathetic drive , potentially opening the door to new strategies. In this sense, during the last few years, several central neural modulators able to trigger adaptive thermogenesis in BAT, browning of WAT and enlarge energy expenditure have been identified (see below). Hence, understanding the molecular mechanisms involved in this ‘recruiting’ is the main challenge to boost adaptive thermogenesis and the design of future therapies.
Sympathetic signalling to BAT  and WAT  is mainly governed through β3-adrenergic receptors (β3-ARs). β-AR signalling in adipocytes is necessary to activate lipolysis [53–55] and thermogenesis  in white and brown adipocytes, respectively. Hence, administration of adrenergic agonists stimulates thermogenesis in rodents and humans [53,56,57], as well as browning of white adipocytes [58–60]. While numerous studies have shown dense sympathetic innervation of BAT [18,61–63], evidence of direct sympathetic innervation of WAT was recently addressed to also play a key role in how activation of WAT can regulate energy balance [53,55]. Initial reports suggested that innervation of white depots is scarce [64–67], exclusively perivascular, and that white adipocytes are not directly innervated . On the other hand, it was suggested that lipolysis is locally induced by catecholamines produced by infiltrated macrophages . However, recent studie finally demonstrated the existence of a sympathetic neuro-adipose junction that ‘envelops’ the adipocyte, where the catecholamines are released and mediate the lipolytic effects of leptin through β-ARs . As in BAT, recent data indicate the existence of sympathetic-sensory feedback circuits in WAT . Thus, cumulative data suggest that lipolysis is stimulated or supressed via the adenylate cyclase/PKA pathway, which is activated or inhibited through β3-AR or α2-AR, respectively . A growing body of evidence supports an essential role of several hypothalamic areas in the regulation of the sympathetic drive to BAT and WAT [71–73], and the specific effect of each hypothalamic nuclei is reviewed below.
Hypothalamic control of BAT thermogenesis
Several hypothalamic nuclei are involved in the regulation of BAT thermogenesis. In this section, we will summarize the specific role of those hypothalamic cell populations
The hypothalamic POA placed in the rostral hypothalamus (Figure 1) is known as the ‘fever centre’ or the ‘thermoregulatory centre’. Both cutaneous and central thermal signals are integrated in the POA, where heat- and cold-sensitive neurons are located to regulate adaptive thermogenesis [74,75]. Skin cold receptors signal to cold-sensitive neurons in the median preoptic nucleus, triggering adaptive thermogenesis in BAT [76–78] while inhibiting warm-sensitive neuronal activity in the medial POA [77–79].
Several studies have shown that alternative hypothalamic and extra-hypothalamic areas, innervated by cold- and warm-sensitive neurons, are implicated in POA-mediated thermoregulatory responses. One important relay point that regulates POA-induced sympathetic discharge to BAT and WAT is the DMH. Sympathoexcitatory neurons in the DMH are tonically inhibited by GABAergic inputs from warm-sensitive neurons. This counteracts the sympathetic activation of premotor neurons in the rostral raphe pallidus nucleus (rRPa) preventing adrenergic activity in BAT (adaptive thermogenesis) and WAT (browning) [63,73,80,81]. This DMH relay point is essential in both the febrile and the cold-induced thermogenic response. Fever is mediated by prostaglandin receptor-expressing neurons in the POA that suppresses GABAergic projections to the DMH [76,82]. A second key relay point in thermoregulation is the VMH, a central nucleus in the regulation of the non-shivering thermogenesis. Thus, increased VMH neuronal activity eliciting selective sympathetic outflow on BAT (see below). γ-Aminobutyric acid (GABA) agonist administration into the VMH abolishes non-shivering thermogenesis induced by prostaglandin injection into the POA [83,84], which indicates that the DMH [76,82] and VMH [83,84] are essential in the febrile response mediated by the POA. Accordingly, VMH lesions counteract cold-induced thermoregulatory responses by the POA [85,86].
In line with the high energy cost of the thermogenic response, POA neuronal activity is modulated by adiposity signals that inform of energy status [87–90]. Recent data indicate that cold-induced sympathoexcitation of BAT is mediated by leptin-responsive neurons in the POA [88–90]. Otherwise, intra-POA injection of insulin increases BAT thermogenesis and body temperature . The melanocortin system also plays a key role in POA-mediated thermoregulatory responses. POA neurons represent the second largest hypothalamic population that express the melanocortin receptor 4 (MC4R) [91,92]. As in the PVH (see below), central injection of the melanocortin receptor agonist melanotan-II (MTII) into the medial preoptic area (MPO) increases sympathetic outflow in BAT . Selective lesion studies by kainic acid demonstrate that melanocortinergic regulation of thermogenic response into the MPO is mediated by DMH sympathoexcitatory neurons .
Arcuate nucleus of the hypothalamus
The ARC has been identified as one of the most important hypothalamic sites for the integration of peripheral information and the control of homeostatic responses [3,11,15,16,93,94]. To access neuronal populations, circulating factors have to cross the BBB across the tight junctions between endothelial cells lining the brain microvasculature. However, there are discrete regions of the brain, such as the median eminence (ME), where the endothelium is fenestrated to facilitate the access of peripheral signals. The ARC is located in a privileged area for this purpose, situated at the ventral part of the hypothalamus, bilaterally surrounding the third ventricle, below the VMH and immediately adjacent to the semi-permeable BBB of the ME [3,11,15,16,93,94] (Figure 1).
Within the ARC there are two major ‘first-order’ populations of neurons, which integrate nutritional, metabolic and humoral information to subsequently modulate appetite and energy expenditure by: (i) a set of neurons that express the orexigenic factors neuropeptide Y (NPY) and agouti-related peptide (AgRP), and (ii) a second set of neurons that express the anorexigenic neuropeptide cocaine- and amphetamine-regulated transcript (CART) and α-melanocyte-stimulating hormone (α-MSH, a product from the proteolytic processing of pro-opiomelanocortin, POMC).
AgRP/NPY and downstream target neurons that express melanocortin receptor 3 (MC3R) and MC4R constitute the central melanocortin system. In general, POMC and AgRP neurons have opposite metabolic functions related to α-MSH agonism and AgRP antagonism on MCRs [95–99]. Thus, under a negative energy balance such as fasting, the expression of AgRP/NPY increases while POMC expression decreases in the ARC, whereas in a context of energy surplus the opposite happens. In fact, activated NPY/AgRP neurons directly inhibit POMC neurons through GABAergic synapses [100,101]. The antagonistic relationship between POMC and AgRP neurons is clearly reflected in the feeding response to leptin and ghrelin. Leptin inhibits AgRP and NPY expression [102,103] while increasing α-MSH production in POMC neurons [104,105]. Simultaneously, leptin reduces GABAergic inputs in POMC neurons, predominantly through direct inhibition of presynaptic GABAergic neurons  and also by decreasing GABAergic tone from AgRP neurons [101,107]. Ghrelin, on the other hand, increases the expression of NPY and AgRP [108–110], which directly hyperpolarize POMC neurons . Also, ghrelin increases AgRP GABAergic inhibitory synapses on POMC neurons . In addition to neuropeptide expression, data demonstrate that synaptic plasticity and neuronal responsiveness are also regulated by both ghrelin and leptin during homeostatic responses . Furthermore, AgRP and POMC neuronal activity is rapidly regulated after sensory detection of food .
ARC neurons heavily innervate other hypothalamic places, such as the PVH, LHA, DMH and VMH, as well as other extra-hypothalamic areas. Several surgical  and optogenetic [115,116] studies have demonstrated the importance of ARC as a relay point  in the neuronal circuits involved in feeding control [114–117]. Moreover, interactions between hypothalamic nuclei are bilateral, which strengthen the function of this network. For instance, glutamatergic inputs from the VMH  and the PVH  regulate ARC-mediated orexigenic response, which in turn are regulated by GABAergic afferences from the LHA .
In keeping with the orexigenic role of NPY and AgRP, their expression in the ARC in response to energy demand also requires that adaptive thermogenesis is suppressed to preserve energy balance . In fact, increased NPY expression in the ARC counteracts adaptive thermogenesis in BAT directly, by suppression of sympathetic outflow to brown adipocytes, and indirectly by reducing the expression of tyrosine hydroxylase (the key limiting enzyme for catecholamine synthesis) in the PVH . Accordingly, reduced expression of NPY in the ARC is associated with a marked increase in UCP1 activity in the BAT . Similarly, it has also been demonstrated that loss of half of the AgRP neurons in the ARC increases sympathetic outflow and elevates UCP1 levels in brown adipocytes , while increased AgRP neuronal activity counteracts adaptive thermogenesis . Moreover, ARC AgRP neurons are also critical in the development of beige adipocytes in WAT . In line with this evidence, genetic ablation of O-linked β-N-acetylglucosamine (O-GlcNAc) transferase in AgRP neurons prevents neuronal excitability, which finally stimulates WAT browning and protects against diet-induced obesity .
Besides the actions of AgRP/NPY neurons, cumulative data show that sympathetic outflow and therefore BAT thermogenesis  and lipid metabolism in WAT [91,126–129] are also modulated through the melanocortin system. Transneuronal tracing studies co-localizing psuedorabies virus inmunoreactivity with MC4R expression reveal that many brain areas across the sympathetic neuroaxis to BAT and WAT contain MC4R neurons [91,92], suggesting a direct function of MC4R in regulating sympathetic activity. In line with this, central administration of the MC4R agonist MTII increases NA turnover, BAT temperature  and WAT lipolysis [91,127], whereas MC4R-null mice display an obese phenotype . Coherently, decreased secretion of the endogenous agonist α-MSH increases lipid storage  and impairs lipolysis . Finally, data indicate that diet- and cold-induced adaptive thermogenesis, as well as inguinal WAT browning, requires MC4R expression in sympathetic preganglionic neurons [131,132]. MC4R-regulated autonomic activity of preganglionic neurons also controls insulin levels and the development of obesity-induced hypertension, which underlies the impact of MC4R in energy metabolism even in extra-hypothalamic areas [131,132].
The relevance of the melanocortin system in metabolic control is further highlighted by cumulative data, revealing that POMC neurons mediate leptin-induced sympathetic drive through the melanocortin system. Leptin triggers α-MSH expression in POMC neurons, leading to increased UCP1 expression both in BAT  and in WAT . In line with this, MC4R-null mice display an obese phenotype that lack leptin-induced  and diet-induced  thermogenic responses.
Data indicate that GABAergic RIP-Cre (Cre-mediated expression of rat insulin II promoter) cells in the ARC mediate the ability of leptin to stimulate BAT thermogenesis . Acute activation of RIP-Cre neurons stimulates BAT and increases energy expenditure due to direct inhibition of PVH neurons that project to the nucleus of the solitary tract in the brainstem [136,137]. The absence of these GABAergic outputs impairs the ability of leptin to stimulate adaptive thermogenesis, but not to reduce food intake . In fact, mice lacking synaptic GABA release from RIP-Cre neurons show reduced UCP1 expression in BAT and are extremely sensitive to diet-induced obesity . Finally, evidence has shown that mitofusin-mediated increase of endoplasmic reticulum stress (ERS) in POMC neurons is involved in the regulation of energy expenditure [138,139].
Paraventricular nucleus of the hypothalamus
The PVH is located in the anterior hypothalamus, just above the third ventricle (Figure 1). The PVH is considered the major autonomic output area of the hypothalamus [140,141]. Given the high number and the diversity of neuronal afferences from several hypothalamic nuclei and extra-hypothalamic areas involved in energy homeostasis, the PVH used to be considered primarily as an integrative area. However, in the last few years, it has been shown that PVH neurons are also directly targeted by metabolic factors involved in the regulation of energy homeostasis, including leptin, ghrelin and insulin [142–145]. In fact, the loss of PVH neurons by defective maturation or physical destruction leads to obesity and metabolic derangement [146–148].
Neurochemical characterization of the PVH identifies two relevant subsets of neurons controlling energy balance: thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH) neurons. Anatomical studies indicate a central role of PVH neurons in thermogenic response. First, polysynaptic retrograde tracing links BAT to the PVH [18,56,62]. In addition, lesions of the PVH counteract febrile responses [149–151], which indicate the loss of PVH-mediated sympathoexcitatory drive. Also, although electrical stimulation of PVH neurons failed to increase the BAT thermogenic activity [137,152], it has been described that CRH administration increases sympathetic drive and UCP1 expression in brown adipocytes, as well as BAT temperature . These data indicate a central role of CRH in PVH-induced thermogenic responses. In addition to leptin, PVH neurons are directly targeted by fibroblast growth factor 21 (FGF21). Central FGF21-induced sympathetic stimulation [154,155] requires CRH expression in the PVH neurons . FGF21-mediated weight loss involves a marked increase in energy expenditure independently of feeding behaviour . Accordingly, recent data indicate that central FGF21 stimulates sympathetic drive to BAT  and browning of WAT , which lead to enhanced thermogenesis. Paradoxically, studies indicate the existence of inhibitory projections from the PVH to premotor neurons in the rRPa, blocking sympathetic outflow [137,157]. Accordingly, stimulation of the PVH abolished sympathetic drive to BAT induced by cooling or glutamatergic stimulation of the rRPa [137,157].
Cumulative evidence indicates that the PVH acts as an essential relay point of ARC neurons in the regulation of energy balance. NPY receptors in the PVH neurons mediate the NPY-induced decrease in UCP1 expression and thermogenic activity in BAT [122,158,159]. In this context, taking into account NPY/AgRP co-localization in ARC neurons  and their common orexigenic role, it is tempting to consider that AgRP control of BAT thermogenesis may also be mediated by PVH neurons. Likewise, given that ARC POMC and NPY/AgRP neurons are functionally antagonistic, a hypothetical ARC–PVH axis might also contribute to POMC-mediated thermogenic activity . Accordingly, TRH neurons in the PVH are strongly innervated by ARC AgRP and POMC neurons, but also have a high density of melanocortin receptors. Different studies have shown that TRH expression is regulated by both components of the melanocortin system, with α-MSH having a stimulatory effect on TRH expression in the PVH, which can be suppressed by both AgRP and NPY [161,162], linking ARC neurons with the regulation of the hypothalamic–pituitary–thyroid axis and nutritional stress adaptation. In addition to central suppression of TRH, increased fasting levels of NPY stimulate hepatic thyroid hormone metabolism in fasting conditions .
Several pharmacological approaches support a role for the melanocortin system in PVH-mediated energy expenditure. The PVH contains a large number of neurons that express MC4R and project to BAT. The injection of the melanocortin receptor agonist MTII into the PVH increases sympathetic outflow , BAT thermogenesis  and energy expenditure in rodents . Moreover, the lack of MC4R produces massive obesity in both humans  and rodents [130,166]. In contrast, MC4R restoration in the glutamatergic neurons of the PVH in MC4R-null mice counteracts obesity through the regulation of feeding behaviour [167,168], but also involves increased energy expenditure . Therefore, selective disruption of glutamate release counteracts melanocortin-induced energy expenditure . Recent data also indicate that MC4R-mediated catabolic activity in the PVH is modulated by the endocannabinoid system [169,170].
Dorsomedial nucleus of the hypothalamus
The DMH is placed centrally in the hypothalamus, immediately above the VMH (Figure 1). The DMH is reciprocally connected with the VMH and LHA and also receives inputs from other hypothalamic areas, especially the ARC and the POA. Cumulative data recognize the DMH as a central place in the control of feeding, metabolic regulation and thermoregulatory responses  and emphasize the role of the DMH as a key intermediary in the sympathetic activity triggered in higher centres . It is remarkable that despite this strong association between DMH neuronal activity and sympathetic drive, DMH neurons do not directly project to sympathetic preganglionic neurons in the spinal cord, but relay to the rRPa  and the inferior olive (IO) neurons  in the brainstem. As noted above, DMH sympathetic neurons are tonically inhibited by GABAergic inputs from the POA [172,174–176]. Accordingly, it has been reported that pharmacological or physiological disinhibition of glutamatergic neurons in the DMH neurons is sufficient to increase sympathoexcitatory signals to the rRPa leading to BAT thermogenesis [171,172,177]. In contrast, tonic inhibition of DMH neurons counteracts glutamatergic signals descending to the rRPa  and decreases sympathetic outflow to BAT [81,171,177]. Recent data identified a novel subset of heat-activated cholinergic neurons in the DMH that signal to rRPa serotonergic neurons and decrease UCP1 expression and thermogenic activity in BAT, which suggest a central neuroprotective response against higher environmental temperatures .
Cumulative data indicate that the DMH controls sympathetic outflow, especially in response to nutrition-related metabolic signals. Data show a main role of DMH neurons in the leptin [89,179] and NPY-induced  sympathetic outflow to BAT. In conditions of energy demand, NPY is expressed by DMH neurons [181,182]. Its expression is also increased in the DMH of diet-induced obese (DIO) mice, in contrast with the reduced NPY expression in the ARC . In this sense, NPY expression in the DMH not only regulates food intake , but also plays a main role in the control of sympathetic activity to BAT and WAT . Thus, increased NPY levels in the DMH of overfed subject reduce sympathoexcitatory activity , which contributes to exacerbate the diet-induced obese phenotype. Accordingly, ablation of NPY in the DMH counteracts diet-induced obesity through boosted sympathetic drive to both fat depots [180,184]. NPY expression increases in the DMH upon cold exposure [180,184], indicating that these neurons seem to prioritize food intake to cope with increased energy demand rather than regulating cold-induced thermogenesis .
Leptin-responsive neurons are abundantly present in the DMH , with leptin receptor (LepR)-expressing neurons in the DMH playing a critical role in the increased sympathetic tone in fat depots in obese animals [24,179]. The lack of the LepR in the DMH neurons counteracts BAT thermogenic response and promotes weight gain . In this regard, recent data have designated a subset of prolactin-releasing peptide-positive neurons in the DMH as mediators of leptin-induced thermogenesis [24,186].
Ventromedial nucleus of the hypothalamus
The VMH, placed just above the ARC (Figure 1), was the first hypothalamic site identified to be involved in thermoregulation. Subsequent data indicated that other hypothalamic sites might be of greater importance for thermogenic function, but in recent years the VMH has re-emerged as a key nucleus in the hypothalamic regulation of energy balance. VMH neurons receive projections from AgRP and POMC neurons in the ARC and also project to hypothalamic nuclei and extra-hypothalamic areas strongly related with sympathetic regulation of BAT thermogenesis [5,77]. Early studies showed that administration of glutamate [83,187], tryptophan, NA and serotonin  into the VMH regulates sympathetic activity to BAT. Although some studies questioned the role of the VMH as a thermoregulatory centre  and the anatomical relationship between the VMH and fat depots is controversial , recent studies have used conditional adenoviral tracers to show a strong association between the VMH and autonomic centres in the brainstem involved in the regulation of adaptive thermogenic responses . Accordingly, selective VMH lesions strongly counteract sympathetic drive to BAT in murine models [85,86,190]. Also, sympathetic blockade by denervation or β-adrenergic antagonist treatment abolished thermogenic stimuli in BAT induced by electrical and hormonal stimulation of the VMH [26,78,152,191–198]. In summary, functional studies over the last decade underline the major position of the VMH in energy homeostasis, through feeding behaviour [199,200] but especially through its role in adaptive thermogenic responses [26,78,193–198,201–203]
In addition to anatomical evidence, genetic manipulation studies have supported the role of the VMH in the modulation of adaptive thermogenesis. First, silencing of oestrogen receptor α (ERα) in the VMH was shown to trigger increased food intake while suppressing diet-induced thermogenesis [201,204], which indicates that ERα VMH neurons mediate oestradiol-induced homeostatic responses . However, not all of the ERα-positive neurons are equally involved in energy expenditure . Data have identified steroidogenic factor 1 (SF1) neurons, the largest population in the VMH, as key in the energy expenditure triggered by ERα signalling . Thus, selective deletion of ERα in SF1 neurons reduces β3-AR and UCP1 expression and therefore thermogenic activity in BAT [201,206,207]. In fact, obesity in SF1-knockout mice mainly results from decreased energy expenditure rather than increased food intake . Accordingly, VMH-specific SF1-knockout mice showed impaired thermogenesis and an increased susceptibility to DIO . SF1 neurons are also indispensable to support an adequate response to the thermogenic and anti-obesogenic properties of leptin [206,207]. Selective LepR ablation in SF1 neurons blunts sympathetic drive to lower autonomic centres, reducing catecholamine secretion [209,210], which ultimately leads to reduced adrenergic drive, lowered UCP1 expression, impaired BAT thermogenesis and greater adiposity [206,207]. Cumulative data suggest that leptin-induced BAT thermogenesis [206–210] is mediated through the phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K)/Akt/forkhead box protein O1 (FOXO1) pathway [194,211,212] (Figure 2). PI3K is a major kinase mediating leptin and insulin actions; its activation is followed by Akt activation and inhibition of FOXO1 after a complex cascade of events. It has been demonstrated that PI3K signalling in SF1 cells is critical for leptin-induced activation of VMH neurons . In this sense, reduced PI3K activity increases sensitivity to diet-induced obesity, indicating that PI3K is essential in energy balance . Accordingly, mice lacking PI3K in SF1 neurons blunt the acute anorexigenic effect of leptin and show reduced energy expenditure . FOXO1 plays a central role in metabolic homeostasis by regulating leptin and insulin activity in many cell types, including VMH neurons. Mice lacking FOXO1 in VMH SF1 neurons showed increased energy expenditure . Likewise, recent data indicate that signalling through mechanistic (or mammalian) target of rapamycin (mTOR) complexes (mTORCs) play a main role in sympathetic activation by leptin through PI3K [213–215] (Figure 2). Ribosomal S6 kinase (S6K), a downstream target of mTOR, has been demonstrated to phosphorylate hypothalamic AMP-activated protein kinase (AMPK) to mediate leptin effects on feeding . Whether this interaction is involved in the control of thermogenesis is currently unknown.
Integrative hormonal cross-talk within the VHM to modulate thermogenesis.
An important feature of the VMH is the fact that it acts as a main integrator of hormonal actions on energy balance. Besides leptin, it is known that insulin acts as an inhibitory signal on VMH neurons controlling thermogenesis in BAT [83,187,217]. Although intracerebroventricular administration of insulin increases sympathetic drive to BAT , insulin microinjection into the VMH suppresses the thermogenic response to glutamate [83,187], as well as to cold . The VMH also plays a role in the thermogenic actions of thyroid hormones (THs): thyroxine (T4) and tri-iodothyronine (T3). Although it is clear that TH indirectly modulates sympathetic tone to BAT induced by NA , current evidence indicates that the VMH plays a key role in the metabolic effects of THs [193,220,221]. In fact, targeted inhibition of thyroid receptors in the VMH counteracts the thermogenic programme in BAT, leading to weight gain .
Data have suggested a main role of VMH neurons in the regulation of BAT activity by endocannabinoids. Cannabinoid type 1 receptor (CB1R) mRNA is highly expressed in the VMH . CB1R hypothalamic deletion increases sympathetic drive to BAT, as well as β3-AR and UCP1 mRNA levels without changes in feeding behaviour . Also, specific ablation of CB1R in SF1-expressing neurons in the VMH decreases body adiposity by increasing sympathetic activity and lipolysis, and also facilitates metabolic effects of leptin . Also, CB1R in VMH neurons supports an adaptive response to dietary changes: whereas the lack of CB1R in the VMH increases lipolysis and sympathetic activity under a standard diet, high-fat diet decreases lipolysis through decreased sympathetic activity .
In addition to anatomical and genetic evidence, functional studies demonstrated a central role of the VMH in the sympathetic-induced BAT thermogenesis and WAT browning in response to circulating factors. Electrical stimulation in the VMH increases sympathetic activity and lipolytic response in WAT . Over the last few years, accumulating evidence suggests that the energy sensor AMPK [226–231] is involved in the hypothalamic regulation of energy balance by responding to hormones and nutrient signals as a negative regulator of adaptive thermogenesis [193,195,220,232–235]. Oestradiol , THs [193,220,221], leptin  and bone morphogenetic protein 8B (BMP8B)  increase sympathetic drive to BAT through selective AMPK inhibition in the VMH and enhanced sympathetic outflow (Figure 2). Also, inhibition of AMPK activity in the VMH mediates pharmacological activation of sympathetic BAT activity by drugs such as nicotine [234,236] and the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide , suggesting a therapeutic potential. In addition to BAT thermogenesis [235,237], central injection of liraglutide  and T3  stimulates browning independently of nutrient intake. Of note, constitutive activation of AMPK in the VMH is sufficient to blunt both central GLP-1 agonist-induced thermogenesis and adipocyte browning [235,239], as well as the thermogenic responses of T3, oestradiol, BMP8B, liraglutide and nicotine [26,193,232]. At this point, it would be interesting to know whether other central thermogenic responses induced by recently described promising therapeutic targets, such as FGF21 [154,240] or amylin [241,242], are also mediated by this AMPK (VMH)–sympathetic nervous system–adipose tissues axis.
Lateral hypothalamic area
Neurochemical characterization of the LHA identifies two main orexigenic populations, namely melanin-concentrating hormone (MCH)  and orexin (OX) neurons [244–247] (Figure 1). MCH and OX are both expressed by a large number of neurons in the LHA and maintain many overlapping projections, but they do not colocalize [248–250]. MCH projections are mainly localized inside the LHA , whereas OX neurons project to other hypothalamic and extra-hypothalamic areas [246–248,252]. Viral retrograde transport from the BAT identified neurons in the caudal raphe nuclei with OX inputs , as well as OX-containing neurons in the hypothalamus .
Cumulative data show that OX and MCH neurons play a key role in energy balance through the regulation of adaptive thermogenesis in BAT and lipid metabolism in WAT depots. MCH overexpression in the LHA is enough to trigger obesity in mice fed on a chow diet . MCH-knockout mice are leaner due to reduced food intake , but especially due to a strong thermogenic phenotype resulting from increased UCP1 expression in brown adipocytes . In this sense, MCH deletion is able to reduce adiposity in leptin-deficient mice regardless of hyperphagia . In addition, central administration of MCH controls liver and WAT metabolism through the parasympathetic and sympathetic innervation, respectively, which leads to increased lipid synthesis, larger adipocyte size and elevated weight of specific fat depots .
The effect of OX is different, with ox-null mice displaying an obese phenotype despite the reduced food intake, indicating that OX neurons are also largely involved in energy expenditure [258,259]. OX neurons are critical for the development of BAT; reduced thermogenesis in ox-null mice is partly due to the lack of mature brown adipocytes . Thus, OX injections to ox-null dams allow a complete BAT development in ox-null neonates . In addition, OX neurons regulate sympathetic drive to BAT. Thus, OX neurons in the LHA are positively correlated with direct sympathetic outflow to BAT and also indirect through premotor neurons in the rRPa [253,260]. Cold exposure increases hypothalamic OX expression, but ox-null mice do not exhibit lower diurnal body temperature [247,261]. Central injection of OX elevates sympathetic outflow, physical activity, metabolic rate and body temperature [262–264]. Studies have also demonstrated that OX neurons in the LHA mediate the effects of BMP8B on BAT thermogenesis and browning of WAT [232,265]. Specifically, the thermogenic effect of BMP8B is mediated by the inhibition of AMPK in the VMH and the subsequent increase in OX signalling via OX receptor 1 (OX1R) . Accordingly, the thermogenic effect of BMP8B is totally absent from ox-null mice . The thermogenic of BMP8B effect and its impact on OX expression and thermogenesis is abolished by knockdown of glutamate vesicular transporter 2 (VGLUT2), implicating glutamatergic signalling .
However, several studies also found that UCP1 expression in BAT is not induced by central administration of OX [266,267], suggesting an alternative ‘stress fever’ mechanism. In agreement with this, handling stress [264,268] or cold exposure increased neuronal activity and OX expression . Stress-induced thermogenic response is blunted in OX neuron-ablated mice but not in ox-null mice , which indicate the existence of other neurotransmitter/modulators in the OX neurons for stress-induced hyperthermia. In addition to sympathetic activity in BAT, central administration of OX shows a biphasic dose–response-regulating sympathetic drive to WAT, with low OX levels supressing sympathetic drive to WAT and decreasing lipolysis through histamine receptor, whereas high OX levels lead to increased lipolysis in WAT and elevated plasma fatty acids 
As has been outlined in this review, the regulation of energy balance is a complex process regulated by many factors acting in concert, sometimes redundantly, at multiple levels within a neuronal network. This complex circuitry allows triggering of a homeostatic response that meets whole-body metabolic needs. It is therefore not surprising that the hypothalamic areas implicated in the control of food intake also modulate energy expenditure, and, in particular, adaptive thermogenesis. Long-term energy imbalance, for example mainly from easy availability of high-calorie foods and a sedentary lifestyle, leads to adverse consequences expressed as different disease states, such as the metabolic syndrome and Type 2 diabetes .
Changing to a healthy lifestyle has been proved inefficient to combat obesity and should be combined with therapeutic approaches. Recent findings of functional brown and beige adipocytes in adult humans [19–21,25,39–46] provide a therapeutic expectancy to fight obesity, since their inducible activity is reduced in obese subjects . Studies in animal models reveal that key hypothalamic nuclei regulate sympathetic outflows to thermogenic adipocytes in response to signals of elevated energy status, and that discrete neuronal populations project independently to both BAT and WAT depots. During the last two decades, our knowledge on this subject has increased exponentially due to the continuous incorporation of innovative techniques, such as genetically modified animals, virogenetics, neuronal tracing and, more recently, optogenetics [17,19–24]. These developments allow a more detailed mapping of the neuronal circuits, and it is critical to understand how the system works as a redundant homeostatic structure, originally evolved to exploit the scarce energy resources available during evolutionary times. The increasing insight into the intracellular mechanisms and the discrete hypothalamic areas involved in modulation of the thermogenic response may give rise to the new therapeutic targets that are urgently needed. Most recent development of tools [22–24] to precisely control neuronal activity, avoiding off-target and development/compensatory effects of pharmacological or genetic manipulations, respectively, will bring us closer to a future therapy against obesity.
AMP-activated protein kinase
arcuate nucleus of the hypothalamus
brown adipose tissue
bone morphogenetic protein 8B
cannabinoid type 1 receptor
central nervous system
dorsomedial nucleus of the hypothalamus
oestrogen receptor α
endoplasmic reticulum stress
fibroblast growth factor 21
forkhead box protein O1
lateral hypothalamic area
medial preoptic area
mechanistic target of rapamycin
phosphatidylinositol 4,5-bisphosphate 3-kinase
paraventricular nucleus of the hypothalamus
Cre-mediated expression of rat insulin II promoter
rostral raphe pallidus nucleus
steroidogenic factor 1
uncoupling protein 1
glutamate vesicular transporter 2
ventromedial nucleus of the hypothalamus
white adipose tissue
The research leading to these results has received funding from the European Community's Seventh Framework Programme [FP7/2007-2013] under grant agreement number 281854 — the ObERStress project (M.L.): Xunta de Galicia [M.L.: 2015-CP079; R.N.: 2015-CP080 and PIE13/00024], MINECO co-funded by the FEDER Program of EU [R.N.: BFU2015-70664-R; M.L.: SAF2015-71026-R]. CIBER de Fisiopatología de la Obesidad y Nutrición is an initiative of ISCIII. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
We thank Dr Cristina Contreras and Dr Noelia Martínez-Sánchez (USC) for their comments and suggestions.
The Authors declare that there are no competing interests associated with the manuscript.