- Email: [email protected]
Using brown adipose tissue to treat obesity – the central issue
Review
Using brown adipose tissue to treat obesity – the central issue Andrew J. Whittle1, Miguel Lo´pez2,3 and Antonio Vidal-Puig1 1
University of Cambridge Metabolic Research Laboratories, Level 4, Institute of Metabolic Science, Box 289, Addenbrooke’s Hospital, Cambridge, UK, CB2 0QQ 2 Department of Physiology, School of Medicine, University of Santiago de Compostela- Instituto de Investigacio´n Sanitaria, Santiago de Compostela (A Corun˜a), 15782, Spain 3 CIBER Fisiopatologı´a de la Obesidad y Nutricio´n (CIBERobn), Santiago de Compostela (A Corun˜a), 15706, Spain
Current therapeutic strategies are proving inadequate to deal with growing obesity rates because of the inherent resistance of the human body to weight loss. The activation of human brown adipose tissue (BAT) represents an opportunity to increase energy expenditure and weight loss alongside improved lipid and glucose homeostasis. Research into the regulation of BAT has made increasing the thermogenic capacity of an individual to treat metabolic disease a plausible strategy, despite thermogenesis being under tight central nervous system control. Previous therapies targeted at the sympathetic nervous system have had deleterious effects because of a lack of organ specificity, but advances in our understanding of central BAT regulatory systems might open up better strategies to specifically stimulate BAT in obese individuals to aid weight reduction. Allostatic adaptation perpetuates the obesity epidemic In 2010, obesity overtook smoking as the leading contributor to the overall disease burden in the US [1]; an event not entirely unexpected given the prediction 10 years earlier that by 2005 obesity would have become the leading cause of preventable death [2]. This pattern is not unique to North America but is echoed in most of the developed and developing world. Currently, 65% of the global population lives in countries where obesity kills more people than malnutrition (WHO - Global strategy on diet, physical activity and health, 2010). In the face of this epidemic, current health strategies to combat obesity are proving woefully inadequate at reducing its prevalence as well as the associated morbidity and mortality. Meanwhile, the economic impact of obesity continues to rise steeply. Some of the greatest barriers to reducing obesity are the allostatic regulatory mechanisms of the body that control energy balance [3]. These allostatic regulatory mechanisms respond so effectively to environmental change that even large reductions in energy intake (25%) are countered by reductions in total energy expenditure that are equal in scale. These reductions go well beyond those predicted to occur because of a loss of body mass and are facilitated by a reduced basal metabolic rate and behavioural adaptations that decrease physical activity [4], persisting for many Corresponding authors: Whittle, A.J. ([email protected]); Vidal-Puig, A. ([email protected]).
years after a dietary intervention [5]. Such adaptations probably cause the plateau in body weight encountered approximately 6 months after the initiation of caloric reduction as well as the propensity for individuals to regain lost weight. If obesity were viewed simply as an imbalance between food intake and energy expenditure, then the obvious redress would be an increase in energy expenditure alongside caloric restriction to ensure weight loss. However, the apparent simplicity of this solution should not imply it is easy in clinical practice. Although it is true that a modest exercise plan can partially offset the adaptive reduction in energy expenditure, for many individuals the extent of their obesity precludes long periods of daily aerobic exercise [4]. In these cases, the activation of brown adipose tissue (BAT), with its ability to oxidize lipids and dissipate large amounts of energy as heat, offers an attractive alternative mechanism for increasing energy expenditure. There is now little doubt that this unique tissue exists and is thermogenically active in humans [6–12], and thereby represents an additional resource that antiobesity strategies can utilize. This review will outline the potential therapeutic benefits of using BAT for the treatment of obesity and focus on recent advances in our understanding of the endogenous central mechanisms regulating its activation. Such mechanisms might provide novel targets for the more specific activation of thermogenesis in BAT. BAT and the thermogenic disconnect When stimulated, BAT is a highly metabolically active organ serving to generate heat to maintain core body temperature. This is facilitated by the presence of the mitochondrial uncoupling protein UCP1, which dissociates the electron transport chain from ATP production by Glossary Homeostasis: The ability of a biological system to sustain life. Allostasis: The maintenance of the stability of a biological system, facilitated by change. This includes homeostatic mechanisms as well as the concept that the putative ‘set points’ that they defend are continually altered in response to environmental factors. Blood pressure, for instance, is constantly maintained but can be adjusted depending on the needs of the organism, for instance during exercise or before arousal. Sympathetic tone: The rate and pattern of electrical activity travelling along the axon of sympathetic nerves to elicit an appropriate catecholamine release from nerve terminals in the target tissue.
1471-4914/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2011.04.001 Trends in Molecular Medicine, August 2011, Vol. 17, No. 8
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Table 1. Actions and limitations of molecules that activate thermogenesis in BAT Molecule 2,4-dinitrophenol Ephedrine
Sibutramine FGF21
Mechanism of action Ubiquitous chemical uncoupler. Facilitates increased mitochondrial proton leak to elevate metabolic rate in all cells. Sympathomimetic amine. Increases the action of noradrenaline on a and b receptors. Increases metabolic rate via increased SNS activity. Nonspecific SNS activation, tachycardia, hypertension, stroke. Serotonin-norepinephrine reuptake inhibitor. Suppresses appetite and maintains increased metabolic rate via increased SNS activity. Liver-derived hormone. Promotes lipolysis in white adipose tissue and activates thermogenic genes in BAT.
Facilitate increased thyroid action by inducing deiodinase 2 expression. Increase energy expenditure and BAT activity. b3 adrenergic receptor agonists Selectively stimulate BAT and protect against diet-induced obesity in rodents. Bile acids
allowing the free movement of protons back across the mitochondrial membrane. The energy dissipating capacity of BAT is significant, with the potential to increase daily energy expenditure by up to 20% in humans and its validity from a bioenergetic perspective for the treatment of obesity has been the subject of a recent review [13]. However, there are additional factors that make BAT particularly attractive from a therapeutic standpoint. When active, BAT is able to uptake and dispose of large quantities of lipid and glucose from the circulation [14,15], a trait that has enabled its detection in humans and would be of substantial benefit to obese patients suffering from insulin resistance and hyperlipidemia. This characteristic of BAT might also go some way to explain why diabetes induced in rats by streptozotocin treatment is ameliorated by housing them in the cold [16] and also the anecdotal case of an extremely insulin-resistant female who ceased to require insulin following thyroxine-induced BAT expansion [17]. A recently highlighted and fundamental quality of thermogenesis is the disconnect between the amount of energy dissipated by heat production and the allostatic mechanisms regulating compensatory increases in food intake. For this reason, housing rodents in the cold or administering b-adrenergic receptor agonists or chemical uncoupling agents can offset the effects of feeding a high fat diet on body weight [18]. An important role for BAT thermogenesis in preventing obesity might be further inferred from the inverse correlation between thermogenic capacity and susceptibility to diet-induced obesity in different mouse strains [19,20], with the possibility of the same relationship existing in humans. Increasing evidence indicates that the amounts of detectable BAT positively correlate with resting metabolic rate, and inversely correlate with BMI and fat mass [8–10,21]. It therefore follows that elucidating mechanisms for activating BAT is likely to have multiple positive implications for obese individuals. Mechanisms for activating BAT in humans Activating thermogenesis to reduce body weight is not a new idea. As early as the 1930s, compounds aimed at increasing energy expenditure in this way were being used to treat obesity. Pharmacological molecules with greater specificity have since followed, and these molecules target the major regu406
Complications Hyperthermia leading to death and the formation of cataracts.
Hypertension, ischemia, stroke. Not validated in humans. Potential for off target effects in liver, increased ketogenesis. Hepatic toxicity – not validated in humans. Extremely low efficacy in humans.
latory mechanism of BAT, the sympathetic nervous system (SNS) [22]. These drugs effectively reduce weight by acting centrally and peripherally to increase the SNS activation of BAT, exploiting the disconnect between thermogenesis and food intake. However, there is still a degree of promiscuity in their actions that causes deleterious effects on the cardiovascular system, which also expresses adrenergic receptors. These off target effects have prevented their administration to humans, particularly obese patients already at an increased risk of cardiovascular complications (Table 1). More attractive perhaps is the use of endogenous molecules that have recently begun to emerge as potential thermogenic agents, able to specifically stimulate BAT via mechanisms separate from the adrenergic system. These potential thermogenic agents include fibroblast growth factor 21 (FGF21), which is expressed in response to dietary lipid uptake and induces the expression of the thermogenic machinery (including UCP1) in BAT [23] and bile acids such as cholic acid, which enhance the actions of thyroid hormones specifically in muscle and BAT to increase energy expenditure [24,25]. However, it should be noted that such molecules might prove to have their own limitations and that the validation of such strategies in humans remains a distant prospect. There does exist, however, a highly specific regulatory mechanism within the central nervous system governing the thermogenic activity of BAT, the dissection of which has advanced greatly in recent years. Central regulation of thermogenesis and energy balance Thermoregulation in mammals is coordinated and governed by the hypothalamus, in particular the preoptic area, which is evident from the effects of its lesioning in rodents [26]. Changes in temperature are sensed by the transient receptor potential family of cation channels, such as TRPA1 and TRPM8, which sense cold, and TRPV3 and TRPV4, which sense heat. These receptors are present in neurons in the skin and the intestine, and are expressed on afferent neurons feeding back information to the preoptic area, which in turn coordinates thermoregulatory responses such as vasodilation/constriction and BAT activation/suppression via the SNS [27]. In BAT, these actions are propagated through the b-adrenergic receptors on brown adipocytes and the activation of downstream p38 mitogen-activated protein
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kinase [28]. In addition to cold exposure, other physiological conditions result in the activation of thermogenesis in BAT, such as fever [29] or, more relevant to obesity, increased caloric intake. This is evident from studying mice housed at thermoneutrality, where no cold-stimulated thermogenesis occurs. In this setting, feeding a high fat diet leads to increased thermogenic capacity, increased BAT mass and elevated UCP1 levels. Mice lacking UCP1, by contrast, have reduced thermogenic capacity and are more susceptible to diet-induced obesity under these conditions [30]. This phenomenon is termed diet-induced thermogenesis, which suggests a level of interaction between the mechanisms regulating energy balance and those controlling BAT. However, because of disagreement over the existence of dietinduced thermogenesis, the mechanisms responsible for its activation have been vastly under-researched until recently. It seems feasible that regulatory circuits already exist and that they could be exploited for the specific activation of BAT to treat obesity. Evidence that central thermoregulatory mechanisms respond to nutritional signals Elegant retrograde tracing studies have identified populations of neurons in the hypothalamus and brainstem that have efferent projections to BAT, with a notable concentration of these residing in the paraventricular nucleus of the hypothalamus (PVH). An interesting characteristic of these neurons is that many of them also express the melanocortin-4 receptor, which promotes anorexia when activated and is known to harbour common genetic variants associated with obesity [31,32]. There is evidence
[()TD$FIG]
that the PVH is a potent negative regulator of BAT, both when directly stimulated and when treated with the nutritionally regulated hormone ghrelin [33,34]. Evidence is also available that suggests the specific modulation of endogenous neuropeptides within the PVH can lead to marked increases in BAT thermogenesis, probably through the inhibition of specific subsets of neurons therein [35]. Neurons in the arcuate nucleus of the hypothalamus (ARC) play a major role in feeding control and in the modulation of BAT thermogenesis [36–38]. Several pharmacological studies have highlighted this fact in the past two decades, but this effect is exemplified by the finding that the postembryonic ablation of orexigenic neurons in the arcuate nucleus leads to a lean phenotype in mice, associated with hypophagia and the activation of the thermogenic program in BAT [39]. Despite the fact that the neurons of the arcuate nucleus are considered the ‘master centre’ for the control of energy homeostasis, ‘second-order’ nuclei also play a major role. The lateral hypothalamic area (LHA), where a large number of orexin-producing neurons are located [40,41], has attracted attention from those studying the modulation of energy expenditure, following the observation that the central administration of orexin-A induces the sympathetic and thermogenic activation of BAT [42]. Additionally, blocking the action of melanin concentrating hormone (an orexigenic neuropeptide expressed in the LHA) leads to a significant drop in body weight accompanied by increased interscapular BAT mass and UCP1 expression [43]. These mechanisms might also be viewed as potential targets for increasing energy expenditure, but some specific details
Anterior
Posterior
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PVN
DMH
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VMH
LHA ARC
TRPA1 TRPM8 Cold
TRPV3 TRPV4 Heat
Skin / intestine
RPa β3 Adrenergic receptor
Leptin, Thyroid Hormone, Calcitonin gene-related peptide (CGRP)
Ghrelin, Heat Circulation
Heat BAT
UCP1
TRENDS in Molecular Medicine
Figure 1. Thermal and nutritional regulation of the sympathetic activation of BAT. Blue and red lines represent neuronal thermogenic circuits responding to cold and warm temperatures, respectively. Green lines represent those responding to nutritional signals. Afferent neurons from skin and intestinal mucous membranes that express thermoreceptors feedback information on temperature to the preoptic area. This is integrated by the PVH and DMH before neurons innervating the inferior olive (IO) and raphe pallidus (RPa) of the medulla oblongata signal to the brainstem to elicit the appropriate sympathetic tone to BAT. A range of nutritionally regulated factors in the circulatory system can also act in nuclei in the LHA, such as the ARC, VMH, PVH and DMH, to elicit the same SNS effects on thermogenesis.
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Review still need far more characterization, such as the resistance of animals to the effects of melanocortin receptor agonism in the ‘sleeping’ phase of the light cycle and the divergent effects of orexin receptor agonism in different hypothalamic nuclei [35]. Further to this, the manipulation of the orexin system leads to profound alterations in sleep–wake patterns themselves [44]. Other peripheral molecules known to be potent regulators of energy balance and appetite can also elicit effects on thermogenesis in BAT via actions in the hypothalamus (Figure 1). As well as its potent effects on food intake, leptin also activates thermogenesis in BAT in a manner dependant on several hypothalamic nuclei, including the preoptic area, the dorsomedial (DMH) and the ventromedial (VMH) hypothalamic nuclei [45–47]. In this sense, current evidence suggests that leptin might be a drugable target to control BAT activation. The pharmacological blockade of the extracellular signal-regulated kinase in the hypothalamus, which mediates the leptin signalling pathway, reverses the anorectic effect of leptin while specifically increasing sympathetic nerve activity to BAT [48]. Whether this action displays anatomical specificity requires further investigation, but supporting this evidence, recent data have also demonstrated that leptininduced glucose uptake by BAT is increased when leptin is administrated within the VMH and the ARC but not in other hypothalamic areas, such as the DMH and the PVH [46]. Opposing the actions of leptin, ghrelin is secreted by the stomach to increase appetite in the fasted state but with equal potency its central administration results in the suppression of the sympathetic activation of BAT by reducing noradrenaline release, presumably to reduce energy expenditure [34]. In support of the antithermogenic role of ghrelin, the ablation of the ghrelin receptor results in increased BAT thermogenesis and energy expenditure [49]. Another peptide released by the gut following the ingestion of a meal is calcitonin gene-related peptide, which has been shown to bind to the calcitonin-like receptor in the brain. The efficacy of this interaction is significantly increased by receptor activity-modifying proteins, and the overexpression of RAMP1 in the brain results in a lean mouse that has significantly elevated sympathetic tone (see Glossary) to BAT and increased energy expenditure [50]. Thermogenesis in BAT is clearly intertwined with both thermoregulatory and nutrient-sensing mechanisms in the brain. Maintaining core body temperature in the cold requires a significant increase in energy intake. However, if diet-induced thermogenesis does exist as an endogenous component of energy balance then its key stimulus would itself be increased energy availability. As such, it would be unlikely to drive a further positive effect on food intake and perhaps even induce efferent sympathetic tone to BAT via different mechanisms altogether. This is supported by the analysis of hypothalamic gene induction, where of the 1176 genes measured only two (NMDA receptor 2B and GTPbinding protein G-alpha-i1) were similarly regulated following cold exposure and high fat diet feeding [51]. The importance of identifying such a mechanism stems from the fact that although cold exposure in rodents is effective 408
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at preventing obesity, no assessment has ever been made of its efficacy for reducing body weight in animals that are already obese. Artificially stimulating diet-induced thermogenesis is likely to remove any compensatory food intake increase alongside thermogenesis and thus further reduce the metabolic efficiency of an organism with more potent effects on body weight. The VMH integrates thermogenesis with energy balance Nuclei do exist that respond to specific cues to increase SNS tone to BAT and thereby thermogenesis, but these do not incur concomitant increases in appetite. In this sense, one specific nucleus of interest when examining positive thermogenic control is the VMH. With nerve projections from and to the ARC, PVH and DMH, as well as to the LHA, plus an abundance of leptin, ghrelin, oestrogen, thyroid hormone and neuropeptide receptors, the VMH should perhaps be considered a ‘reception nucleus’ for peripheral and central signals informing energy balance [52–56]. Of these signals, thyroid hormone is the beststudied endogenous metabolic stimulant. Thyroid hormone functions regulate energy utilization by acting as a global, long-term regulator of cellular metabolic status. However, it is promiscuous in its peripheral action across tissues, giving rise to the wide spectrum of conditions associated with hyperthyroidism such as hypermetabolism, hyperphagia, weight loss, heat intolerance and cardiac arrhythmia. The peripheral effects exerted by the actions of the thyroid hormone in the central nervous system seem far more specific and potent in their nature, presenting opportunities for the selective manipulation of efferent stimulatory pathways to BAT. Work by Vennstro¨m and colleagues, who removed the hypermetabolic and hyperphagic effects of a genetic form of hyperthyroidism by placing mice at thermoneutrality, gave the first suggestion that the central regulation of thermogenesis in BAT was a crucial and overriding component of this pathology [57]. In keeping with these data, it was recently demonstrated that central T3 (the activated form of thyroid hormone) has the ability to increase thermogenesis in BAT via its suppression of AMP-activated protein kinase (AMPK) activation in the VMH [58]. The central T3 treatment or genetic inhibition of AMPK in the VMH results in rapid weight loss despite no change in food intake, indicating an increased metabolic rate attributable to BAT (evident from elevated UCP1, PGC1a and PGC1b expression) following increased SNS activation. What is striking about this finding is that metabolic effects previously attributed to peripheral increases in thyroid action could be entirely replicated with far smaller doses administered to a single hypothalamic nucleus, namely the VMH. Also of note, this increase in energy expenditure seems possible without negation by increased feeding. In further support of this mechanism being VMH-specific, the genetic inhibition of the thyroid hormone receptor in the VMH reverses hyperthyroidismassociated effects on energy balance and thermogenesis, allowing hyperthyroid rats to gain weight [58]. The hypothalamic fatty acid synthesis pathway has also been shown to be essential for mediating the orexigenic effects of
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ghrelin [59]. Alterations to the activity of this pathway in the VMH have robust effects on energy expenditure in BAT and might also facilitate the integration of other circulating factors into the sympathetic control of energy balance [55]. Whether other peripheral hormones that modulate feeding and energy expenditure, such as oestrogens [60], act through the VMH–BAT axis will require further investigation.
at reducing weight, but to the detriment of the cardiovascular system or psychological health [62]. As such, further centrally focused therapies should be approached with caution, but identifying specific molecular targets, such as the hypothalamic fatty acid synthesis pathway, might enable us to produce SNS effects specific to BAT [63]. A more fundamental problem facing BAT-activating strategies might lie in the fact that the extent to which an organism can undertake diet-induced thermogenesis is proportional to the amount of cold-induced thermogenesis induced by environmental temperature [64,65]. This is largely because of the amount of BAT in an organism that is regulated by sympathetic activation. Humans have the advantage of adjusting their housing temperature to their thermal preference, usually thermoneutrality, and as such largely remove cold-induced thermogenesis from the energy balance equation. Home temperatures over the past 30 years have steadily increased thanks to modern insulation and heating systems and this correlates to a reduction in daily expenditure [66]. This drop in energy expenditure following reduced thermal stress could account for increased body weight, as in theory a reduction of just 0.6% of total energy expenditure could lead to weight gain of 1 kg per year [67]. Chronically reduced BAT activity might also directly contribute to a reduction in BAT mass in humans and, along with other factors, might potentiate the reduction that occurs with increasing age [68]. The
[()TD$FIG]
Future strategies for activating BAT in obese individuals The VMH is ideally located to integrate information pertaining to nutritional status with the traditional coldresponsive mechanisms driving sympathetic tone to BAT because it sits between the arcuate and paraventricular nuclei and close to the preoptic area (Figures 1 and 2). Apart from the gene expression study cited in the previous section, there have been virtually no studies designed to assess changes to central thermoregulatory circuits in response to acute nutritional challenges. The few that have been designed to assess these changes continue to support the existence of numerous specific mechanisms, which result in the appropriate SNS response in a diverse range of nutritional settings [61]. Further dissections at a central level will help clarify the phenomenon of diet-induced thermogenesis while exposing novel pathways for potential therapeutic manipulation. Previous strategies aimed broadly at the central nervous system have been effective
Hypothalamus LHA
PVH
PVH
DMH
LHA
Hypothalamic manipulation:
DMH
-PVH inhibition
Decreasing energy expenditure
Increased insulative capacity (obesity)
VMH
VMH
ARC
3V
acid synthesis
ARC
Pro-thermogenic Caloric restriction
SNS tone
hormonal milieu Cold environment
Ageing Increased brown adipogenesis
Increasing energy expenditure
-Increased fatty Warm environment
BAT activation (βadrenergic receptor agonism, FGF21, bile acids
BAT TRENDS in Molecular Medicine
Figure 2. Factors limiting thermogenesis versus strategies to activate BAT. Warmer living environments, increasing obesity and ageing all serve to reduce the activation of central thermogenic regulatory pathways in addition to the major therapeutic strategy currently applied to obesity – caloric restriction. To counter the range of antithermogenic factors active in obese patients several measures are available. Emerging central pathways might be targeted to specifically increase the sympathetic activation of BAT, which might itself be expanded and directly stimulated using endogenous signalling molecules whose functions are becoming increasingly understood.
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Review result is that many individuals, particularly those who are obese and more insulated, might not possess sufficient amounts of BAT to make a meaningful contribution to a weight loss strategy. We have proposed that lowering ambient temperatures to increase thermogenesis might provide therapeutic advantages and reduce the need for pharmacology. However, as any human volunteer will testify, long-term exposure to even mild cold temperatures is unpleasant at best and unlikely to be adhered to. Although intermittent short-term cold exposure might help stimulate adaptive increases in BAT mass, it is likely to be only part of a more concerted effort to increase the thermogenic capacity of obese patients. For instance, our knowledge of brown adipogenesis has itself benefited greatly from recent studies showing that human white adipose tissue contains significant numbers of precursor cells able to differentiate into functional brown adipocytes [69]. Further to this, transcription factors and secretory molecules, such as PR domain-containing 16 [70] and bone morphogenetic protein 7 [71], have been identified that activate specific pathways to drive the formation brown adipocytes from similar precursor cells in mice. Capitalizing on the adipogenic pathways these molecules function through will probably increase the size of the available pool of brown adipocytes with which to counter obesity. However, finding mechanisms to maintain the thermogenic activation of brown adipocytes will be equally fundamental to therapeutic success (Figure 2). Concluding remarks Thermogenesis in BAT is a feasible strategy by which to maintain elevated daily energy expenditure and as such improve the long-term effectiveness of weight loss strategies. However, thermogenesis is regulated centrally via a tightly controlled set of nuclei mediating changes in sympathetic tone to BAT and other tissues to defend core body temperature. It seems, however, that the arcuate and ventromedial nuclei, traditionally thought to be more important for appetite regulation, are also able to interact with the thermogenic pathway to elicit changes in energy balance. That this is dependent on nutritional status suggests that the same hypothalamic nuclei integrate both sides of the energy balance equation, namely feeding and energy expenditure. Mechanisms have recently been identified in the VMH that might be manipulated to elicit specific effects on sympathetic tone to BAT. Further studies are likely to reveal additional molecular pathways in other nuclei or perhaps even suggest subtle nutritional alterations that manipulate the hormonal milieu to maximize centrally driven thermogenesis in BAT. Ultimately, nutritional stimuli are likely to remain dependant on the more intensive induction of BAT by cold temperatures to provide the adequate thermogenic machinery. As the modern living environment provides no such stimulus and factors of increasing age and body weight conspire to reduce detectable BAT in humans even further, it is probable that multiple BAT-targeted approaches will be required in combination to successfully treat obesity. However, adaptive environmental conditioning, combined with methods exploiting endogenous brown 410
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adipogenic factors, would be one possible strategy to increase the thermogenic impact of hypothalamic manipulations at the peripheral level. Acknowledgements AW is funded by a BBSRC PhD studentship. ML is supported by the Xunta de Galicia (ML: 10PXIB208164PR), the Fondo Investigationes Sanitarias (ML: PS09/01880) and the Ministerio de Ciencia e Innovacio´n (ML: RyC2007-00211). CIBER de Fisiopatologı´a de la Obesidad y Nutricio´n is an initiative of ISCIII. AVP is supported by an MRC programme grant and FP7 - BetaBAT.
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Using brown adipose tissue to treat obesity – the central issue Andrew J. Whittle1, Miguel Lo´pez2,3 and Antonio Vidal-Puig1 1
University of Cambridge Metabolic Research Laboratories, Level 4, Institute of Metabolic Science, Box 289, Addenbrooke’s Hospital, Cambridge, UK, CB2 0QQ 2 Department of Physiology, School of Medicine, University of Santiago de Compostela- Instituto de Investigacio´n Sanitaria, Santiago de Compostela (A Corun˜a), 15782, Spain 3 CIBER Fisiopatologı´a de la Obesidad y Nutricio´n (CIBERobn), Santiago de Compostela (A Corun˜a), 15706, Spain
Current therapeutic strategies are proving inadequate to deal with growing obesity rates because of the inherent resistance of the human body to weight loss. The activation of human brown adipose tissue (BAT) represents an opportunity to increase energy expenditure and weight loss alongside improved lipid and glucose homeostasis. Research into the regulation of BAT has made increasing the thermogenic capacity of an individual to treat metabolic disease a plausible strategy, despite thermogenesis being under tight central nervous system control. Previous therapies targeted at the sympathetic nervous system have had deleterious effects because of a lack of organ specificity, but advances in our understanding of central BAT regulatory systems might open up better strategies to specifically stimulate BAT in obese individuals to aid weight reduction. Allostatic adaptation perpetuates the obesity epidemic In 2010, obesity overtook smoking as the leading contributor to the overall disease burden in the US [1]; an event not entirely unexpected given the prediction 10 years earlier that by 2005 obesity would have become the leading cause of preventable death [2]. This pattern is not unique to North America but is echoed in most of the developed and developing world. Currently, 65% of the global population lives in countries where obesity kills more people than malnutrition (WHO - Global strategy on diet, physical activity and health, 2010). In the face of this epidemic, current health strategies to combat obesity are proving woefully inadequate at reducing its prevalence as well as the associated morbidity and mortality. Meanwhile, the economic impact of obesity continues to rise steeply. Some of the greatest barriers to reducing obesity are the allostatic regulatory mechanisms of the body that control energy balance [3]. These allostatic regulatory mechanisms respond so effectively to environmental change that even large reductions in energy intake (25%) are countered by reductions in total energy expenditure that are equal in scale. These reductions go well beyond those predicted to occur because of a loss of body mass and are facilitated by a reduced basal metabolic rate and behavioural adaptations that decrease physical activity [4], persisting for many Corresponding authors: Whittle, A.J. ([email protected]); Vidal-Puig, A. ([email protected]).
years after a dietary intervention [5]. Such adaptations probably cause the plateau in body weight encountered approximately 6 months after the initiation of caloric reduction as well as the propensity for individuals to regain lost weight. If obesity were viewed simply as an imbalance between food intake and energy expenditure, then the obvious redress would be an increase in energy expenditure alongside caloric restriction to ensure weight loss. However, the apparent simplicity of this solution should not imply it is easy in clinical practice. Although it is true that a modest exercise plan can partially offset the adaptive reduction in energy expenditure, for many individuals the extent of their obesity precludes long periods of daily aerobic exercise [4]. In these cases, the activation of brown adipose tissue (BAT), with its ability to oxidize lipids and dissipate large amounts of energy as heat, offers an attractive alternative mechanism for increasing energy expenditure. There is now little doubt that this unique tissue exists and is thermogenically active in humans [6–12], and thereby represents an additional resource that antiobesity strategies can utilize. This review will outline the potential therapeutic benefits of using BAT for the treatment of obesity and focus on recent advances in our understanding of the endogenous central mechanisms regulating its activation. Such mechanisms might provide novel targets for the more specific activation of thermogenesis in BAT. BAT and the thermogenic disconnect When stimulated, BAT is a highly metabolically active organ serving to generate heat to maintain core body temperature. This is facilitated by the presence of the mitochondrial uncoupling protein UCP1, which dissociates the electron transport chain from ATP production by Glossary Homeostasis: The ability of a biological system to sustain life. Allostasis: The maintenance of the stability of a biological system, facilitated by change. This includes homeostatic mechanisms as well as the concept that the putative ‘set points’ that they defend are continually altered in response to environmental factors. Blood pressure, for instance, is constantly maintained but can be adjusted depending on the needs of the organism, for instance during exercise or before arousal. Sympathetic tone: The rate and pattern of electrical activity travelling along the axon of sympathetic nerves to elicit an appropriate catecholamine release from nerve terminals in the target tissue.
1471-4914/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2011.04.001 Trends in Molecular Medicine, August 2011, Vol. 17, No. 8
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Table 1. Actions and limitations of molecules that activate thermogenesis in BAT Molecule 2,4-dinitrophenol Ephedrine
Sibutramine FGF21
Mechanism of action Ubiquitous chemical uncoupler. Facilitates increased mitochondrial proton leak to elevate metabolic rate in all cells. Sympathomimetic amine. Increases the action of noradrenaline on a and b receptors. Increases metabolic rate via increased SNS activity. Nonspecific SNS activation, tachycardia, hypertension, stroke. Serotonin-norepinephrine reuptake inhibitor. Suppresses appetite and maintains increased metabolic rate via increased SNS activity. Liver-derived hormone. Promotes lipolysis in white adipose tissue and activates thermogenic genes in BAT.
Facilitate increased thyroid action by inducing deiodinase 2 expression. Increase energy expenditure and BAT activity. b3 adrenergic receptor agonists Selectively stimulate BAT and protect against diet-induced obesity in rodents. Bile acids
allowing the free movement of protons back across the mitochondrial membrane. The energy dissipating capacity of BAT is significant, with the potential to increase daily energy expenditure by up to 20% in humans and its validity from a bioenergetic perspective for the treatment of obesity has been the subject of a recent review [13]. However, there are additional factors that make BAT particularly attractive from a therapeutic standpoint. When active, BAT is able to uptake and dispose of large quantities of lipid and glucose from the circulation [14,15], a trait that has enabled its detection in humans and would be of substantial benefit to obese patients suffering from insulin resistance and hyperlipidemia. This characteristic of BAT might also go some way to explain why diabetes induced in rats by streptozotocin treatment is ameliorated by housing them in the cold [16] and also the anecdotal case of an extremely insulin-resistant female who ceased to require insulin following thyroxine-induced BAT expansion [17]. A recently highlighted and fundamental quality of thermogenesis is the disconnect between the amount of energy dissipated by heat production and the allostatic mechanisms regulating compensatory increases in food intake. For this reason, housing rodents in the cold or administering b-adrenergic receptor agonists or chemical uncoupling agents can offset the effects of feeding a high fat diet on body weight [18]. An important role for BAT thermogenesis in preventing obesity might be further inferred from the inverse correlation between thermogenic capacity and susceptibility to diet-induced obesity in different mouse strains [19,20], with the possibility of the same relationship existing in humans. Increasing evidence indicates that the amounts of detectable BAT positively correlate with resting metabolic rate, and inversely correlate with BMI and fat mass [8–10,21]. It therefore follows that elucidating mechanisms for activating BAT is likely to have multiple positive implications for obese individuals. Mechanisms for activating BAT in humans Activating thermogenesis to reduce body weight is not a new idea. As early as the 1930s, compounds aimed at increasing energy expenditure in this way were being used to treat obesity. Pharmacological molecules with greater specificity have since followed, and these molecules target the major regu406
Complications Hyperthermia leading to death and the formation of cataracts.
Hypertension, ischemia, stroke. Not validated in humans. Potential for off target effects in liver, increased ketogenesis. Hepatic toxicity – not validated in humans. Extremely low efficacy in humans.
latory mechanism of BAT, the sympathetic nervous system (SNS) [22]. These drugs effectively reduce weight by acting centrally and peripherally to increase the SNS activation of BAT, exploiting the disconnect between thermogenesis and food intake. However, there is still a degree of promiscuity in their actions that causes deleterious effects on the cardiovascular system, which also expresses adrenergic receptors. These off target effects have prevented their administration to humans, particularly obese patients already at an increased risk of cardiovascular complications (Table 1). More attractive perhaps is the use of endogenous molecules that have recently begun to emerge as potential thermogenic agents, able to specifically stimulate BAT via mechanisms separate from the adrenergic system. These potential thermogenic agents include fibroblast growth factor 21 (FGF21), which is expressed in response to dietary lipid uptake and induces the expression of the thermogenic machinery (including UCP1) in BAT [23] and bile acids such as cholic acid, which enhance the actions of thyroid hormones specifically in muscle and BAT to increase energy expenditure [24,25]. However, it should be noted that such molecules might prove to have their own limitations and that the validation of such strategies in humans remains a distant prospect. There does exist, however, a highly specific regulatory mechanism within the central nervous system governing the thermogenic activity of BAT, the dissection of which has advanced greatly in recent years. Central regulation of thermogenesis and energy balance Thermoregulation in mammals is coordinated and governed by the hypothalamus, in particular the preoptic area, which is evident from the effects of its lesioning in rodents [26]. Changes in temperature are sensed by the transient receptor potential family of cation channels, such as TRPA1 and TRPM8, which sense cold, and TRPV3 and TRPV4, which sense heat. These receptors are present in neurons in the skin and the intestine, and are expressed on afferent neurons feeding back information to the preoptic area, which in turn coordinates thermoregulatory responses such as vasodilation/constriction and BAT activation/suppression via the SNS [27]. In BAT, these actions are propagated through the b-adrenergic receptors on brown adipocytes and the activation of downstream p38 mitogen-activated protein
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kinase [28]. In addition to cold exposure, other physiological conditions result in the activation of thermogenesis in BAT, such as fever [29] or, more relevant to obesity, increased caloric intake. This is evident from studying mice housed at thermoneutrality, where no cold-stimulated thermogenesis occurs. In this setting, feeding a high fat diet leads to increased thermogenic capacity, increased BAT mass and elevated UCP1 levels. Mice lacking UCP1, by contrast, have reduced thermogenic capacity and are more susceptible to diet-induced obesity under these conditions [30]. This phenomenon is termed diet-induced thermogenesis, which suggests a level of interaction between the mechanisms regulating energy balance and those controlling BAT. However, because of disagreement over the existence of dietinduced thermogenesis, the mechanisms responsible for its activation have been vastly under-researched until recently. It seems feasible that regulatory circuits already exist and that they could be exploited for the specific activation of BAT to treat obesity. Evidence that central thermoregulatory mechanisms respond to nutritional signals Elegant retrograde tracing studies have identified populations of neurons in the hypothalamus and brainstem that have efferent projections to BAT, with a notable concentration of these residing in the paraventricular nucleus of the hypothalamus (PVH). An interesting characteristic of these neurons is that many of them also express the melanocortin-4 receptor, which promotes anorexia when activated and is known to harbour common genetic variants associated with obesity [31,32]. There is evidence
[()TD$FIG]
that the PVH is a potent negative regulator of BAT, both when directly stimulated and when treated with the nutritionally regulated hormone ghrelin [33,34]. Evidence is also available that suggests the specific modulation of endogenous neuropeptides within the PVH can lead to marked increases in BAT thermogenesis, probably through the inhibition of specific subsets of neurons therein [35]. Neurons in the arcuate nucleus of the hypothalamus (ARC) play a major role in feeding control and in the modulation of BAT thermogenesis [36–38]. Several pharmacological studies have highlighted this fact in the past two decades, but this effect is exemplified by the finding that the postembryonic ablation of orexigenic neurons in the arcuate nucleus leads to a lean phenotype in mice, associated with hypophagia and the activation of the thermogenic program in BAT [39]. Despite the fact that the neurons of the arcuate nucleus are considered the ‘master centre’ for the control of energy homeostasis, ‘second-order’ nuclei also play a major role. The lateral hypothalamic area (LHA), where a large number of orexin-producing neurons are located [40,41], has attracted attention from those studying the modulation of energy expenditure, following the observation that the central administration of orexin-A induces the sympathetic and thermogenic activation of BAT [42]. Additionally, blocking the action of melanin concentrating hormone (an orexigenic neuropeptide expressed in the LHA) leads to a significant drop in body weight accompanied by increased interscapular BAT mass and UCP1 expression [43]. These mechanisms might also be viewed as potential targets for increasing energy expenditure, but some specific details
Anterior
Posterior
Hypothalamus POA
PVN
DMH
Medulla oblongata IO
VMH
LHA ARC
TRPA1 TRPM8 Cold
TRPV3 TRPV4 Heat
Skin / intestine
RPa β3 Adrenergic receptor
Leptin, Thyroid Hormone, Calcitonin gene-related peptide (CGRP)
Ghrelin, Heat Circulation
Heat BAT
UCP1
TRENDS in Molecular Medicine
Figure 1. Thermal and nutritional regulation of the sympathetic activation of BAT. Blue and red lines represent neuronal thermogenic circuits responding to cold and warm temperatures, respectively. Green lines represent those responding to nutritional signals. Afferent neurons from skin and intestinal mucous membranes that express thermoreceptors feedback information on temperature to the preoptic area. This is integrated by the PVH and DMH before neurons innervating the inferior olive (IO) and raphe pallidus (RPa) of the medulla oblongata signal to the brainstem to elicit the appropriate sympathetic tone to BAT. A range of nutritionally regulated factors in the circulatory system can also act in nuclei in the LHA, such as the ARC, VMH, PVH and DMH, to elicit the same SNS effects on thermogenesis.
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Review still need far more characterization, such as the resistance of animals to the effects of melanocortin receptor agonism in the ‘sleeping’ phase of the light cycle and the divergent effects of orexin receptor agonism in different hypothalamic nuclei [35]. Further to this, the manipulation of the orexin system leads to profound alterations in sleep–wake patterns themselves [44]. Other peripheral molecules known to be potent regulators of energy balance and appetite can also elicit effects on thermogenesis in BAT via actions in the hypothalamus (Figure 1). As well as its potent effects on food intake, leptin also activates thermogenesis in BAT in a manner dependant on several hypothalamic nuclei, including the preoptic area, the dorsomedial (DMH) and the ventromedial (VMH) hypothalamic nuclei [45–47]. In this sense, current evidence suggests that leptin might be a drugable target to control BAT activation. The pharmacological blockade of the extracellular signal-regulated kinase in the hypothalamus, which mediates the leptin signalling pathway, reverses the anorectic effect of leptin while specifically increasing sympathetic nerve activity to BAT [48]. Whether this action displays anatomical specificity requires further investigation, but supporting this evidence, recent data have also demonstrated that leptininduced glucose uptake by BAT is increased when leptin is administrated within the VMH and the ARC but not in other hypothalamic areas, such as the DMH and the PVH [46]. Opposing the actions of leptin, ghrelin is secreted by the stomach to increase appetite in the fasted state but with equal potency its central administration results in the suppression of the sympathetic activation of BAT by reducing noradrenaline release, presumably to reduce energy expenditure [34]. In support of the antithermogenic role of ghrelin, the ablation of the ghrelin receptor results in increased BAT thermogenesis and energy expenditure [49]. Another peptide released by the gut following the ingestion of a meal is calcitonin gene-related peptide, which has been shown to bind to the calcitonin-like receptor in the brain. The efficacy of this interaction is significantly increased by receptor activity-modifying proteins, and the overexpression of RAMP1 in the brain results in a lean mouse that has significantly elevated sympathetic tone (see Glossary) to BAT and increased energy expenditure [50]. Thermogenesis in BAT is clearly intertwined with both thermoregulatory and nutrient-sensing mechanisms in the brain. Maintaining core body temperature in the cold requires a significant increase in energy intake. However, if diet-induced thermogenesis does exist as an endogenous component of energy balance then its key stimulus would itself be increased energy availability. As such, it would be unlikely to drive a further positive effect on food intake and perhaps even induce efferent sympathetic tone to BAT via different mechanisms altogether. This is supported by the analysis of hypothalamic gene induction, where of the 1176 genes measured only two (NMDA receptor 2B and GTPbinding protein G-alpha-i1) were similarly regulated following cold exposure and high fat diet feeding [51]. The importance of identifying such a mechanism stems from the fact that although cold exposure in rodents is effective 408
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at preventing obesity, no assessment has ever been made of its efficacy for reducing body weight in animals that are already obese. Artificially stimulating diet-induced thermogenesis is likely to remove any compensatory food intake increase alongside thermogenesis and thus further reduce the metabolic efficiency of an organism with more potent effects on body weight. The VMH integrates thermogenesis with energy balance Nuclei do exist that respond to specific cues to increase SNS tone to BAT and thereby thermogenesis, but these do not incur concomitant increases in appetite. In this sense, one specific nucleus of interest when examining positive thermogenic control is the VMH. With nerve projections from and to the ARC, PVH and DMH, as well as to the LHA, plus an abundance of leptin, ghrelin, oestrogen, thyroid hormone and neuropeptide receptors, the VMH should perhaps be considered a ‘reception nucleus’ for peripheral and central signals informing energy balance [52–56]. Of these signals, thyroid hormone is the beststudied endogenous metabolic stimulant. Thyroid hormone functions regulate energy utilization by acting as a global, long-term regulator of cellular metabolic status. However, it is promiscuous in its peripheral action across tissues, giving rise to the wide spectrum of conditions associated with hyperthyroidism such as hypermetabolism, hyperphagia, weight loss, heat intolerance and cardiac arrhythmia. The peripheral effects exerted by the actions of the thyroid hormone in the central nervous system seem far more specific and potent in their nature, presenting opportunities for the selective manipulation of efferent stimulatory pathways to BAT. Work by Vennstro¨m and colleagues, who removed the hypermetabolic and hyperphagic effects of a genetic form of hyperthyroidism by placing mice at thermoneutrality, gave the first suggestion that the central regulation of thermogenesis in BAT was a crucial and overriding component of this pathology [57]. In keeping with these data, it was recently demonstrated that central T3 (the activated form of thyroid hormone) has the ability to increase thermogenesis in BAT via its suppression of AMP-activated protein kinase (AMPK) activation in the VMH [58]. The central T3 treatment or genetic inhibition of AMPK in the VMH results in rapid weight loss despite no change in food intake, indicating an increased metabolic rate attributable to BAT (evident from elevated UCP1, PGC1a and PGC1b expression) following increased SNS activation. What is striking about this finding is that metabolic effects previously attributed to peripheral increases in thyroid action could be entirely replicated with far smaller doses administered to a single hypothalamic nucleus, namely the VMH. Also of note, this increase in energy expenditure seems possible without negation by increased feeding. In further support of this mechanism being VMH-specific, the genetic inhibition of the thyroid hormone receptor in the VMH reverses hyperthyroidismassociated effects on energy balance and thermogenesis, allowing hyperthyroid rats to gain weight [58]. The hypothalamic fatty acid synthesis pathway has also been shown to be essential for mediating the orexigenic effects of
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ghrelin [59]. Alterations to the activity of this pathway in the VMH have robust effects on energy expenditure in BAT and might also facilitate the integration of other circulating factors into the sympathetic control of energy balance [55]. Whether other peripheral hormones that modulate feeding and energy expenditure, such as oestrogens [60], act through the VMH–BAT axis will require further investigation.
at reducing weight, but to the detriment of the cardiovascular system or psychological health [62]. As such, further centrally focused therapies should be approached with caution, but identifying specific molecular targets, such as the hypothalamic fatty acid synthesis pathway, might enable us to produce SNS effects specific to BAT [63]. A more fundamental problem facing BAT-activating strategies might lie in the fact that the extent to which an organism can undertake diet-induced thermogenesis is proportional to the amount of cold-induced thermogenesis induced by environmental temperature [64,65]. This is largely because of the amount of BAT in an organism that is regulated by sympathetic activation. Humans have the advantage of adjusting their housing temperature to their thermal preference, usually thermoneutrality, and as such largely remove cold-induced thermogenesis from the energy balance equation. Home temperatures over the past 30 years have steadily increased thanks to modern insulation and heating systems and this correlates to a reduction in daily expenditure [66]. This drop in energy expenditure following reduced thermal stress could account for increased body weight, as in theory a reduction of just 0.6% of total energy expenditure could lead to weight gain of 1 kg per year [67]. Chronically reduced BAT activity might also directly contribute to a reduction in BAT mass in humans and, along with other factors, might potentiate the reduction that occurs with increasing age [68]. The
[()TD$FIG]
Future strategies for activating BAT in obese individuals The VMH is ideally located to integrate information pertaining to nutritional status with the traditional coldresponsive mechanisms driving sympathetic tone to BAT because it sits between the arcuate and paraventricular nuclei and close to the preoptic area (Figures 1 and 2). Apart from the gene expression study cited in the previous section, there have been virtually no studies designed to assess changes to central thermoregulatory circuits in response to acute nutritional challenges. The few that have been designed to assess these changes continue to support the existence of numerous specific mechanisms, which result in the appropriate SNS response in a diverse range of nutritional settings [61]. Further dissections at a central level will help clarify the phenomenon of diet-induced thermogenesis while exposing novel pathways for potential therapeutic manipulation. Previous strategies aimed broadly at the central nervous system have been effective
Hypothalamus LHA
PVH
PVH
DMH
LHA
Hypothalamic manipulation:
DMH
-PVH inhibition
Decreasing energy expenditure
Increased insulative capacity (obesity)
VMH
VMH
ARC
3V
acid synthesis
ARC
Pro-thermogenic Caloric restriction
SNS tone
hormonal milieu Cold environment
Ageing Increased brown adipogenesis
Increasing energy expenditure
-Increased fatty Warm environment
BAT activation (βadrenergic receptor agonism, FGF21, bile acids
BAT TRENDS in Molecular Medicine
Figure 2. Factors limiting thermogenesis versus strategies to activate BAT. Warmer living environments, increasing obesity and ageing all serve to reduce the activation of central thermogenic regulatory pathways in addition to the major therapeutic strategy currently applied to obesity – caloric restriction. To counter the range of antithermogenic factors active in obese patients several measures are available. Emerging central pathways might be targeted to specifically increase the sympathetic activation of BAT, which might itself be expanded and directly stimulated using endogenous signalling molecules whose functions are becoming increasingly understood.
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Review result is that many individuals, particularly those who are obese and more insulated, might not possess sufficient amounts of BAT to make a meaningful contribution to a weight loss strategy. We have proposed that lowering ambient temperatures to increase thermogenesis might provide therapeutic advantages and reduce the need for pharmacology. However, as any human volunteer will testify, long-term exposure to even mild cold temperatures is unpleasant at best and unlikely to be adhered to. Although intermittent short-term cold exposure might help stimulate adaptive increases in BAT mass, it is likely to be only part of a more concerted effort to increase the thermogenic capacity of obese patients. For instance, our knowledge of brown adipogenesis has itself benefited greatly from recent studies showing that human white adipose tissue contains significant numbers of precursor cells able to differentiate into functional brown adipocytes [69]. Further to this, transcription factors and secretory molecules, such as PR domain-containing 16 [70] and bone morphogenetic protein 7 [71], have been identified that activate specific pathways to drive the formation brown adipocytes from similar precursor cells in mice. Capitalizing on the adipogenic pathways these molecules function through will probably increase the size of the available pool of brown adipocytes with which to counter obesity. However, finding mechanisms to maintain the thermogenic activation of brown adipocytes will be equally fundamental to therapeutic success (Figure 2). Concluding remarks Thermogenesis in BAT is a feasible strategy by which to maintain elevated daily energy expenditure and as such improve the long-term effectiveness of weight loss strategies. However, thermogenesis is regulated centrally via a tightly controlled set of nuclei mediating changes in sympathetic tone to BAT and other tissues to defend core body temperature. It seems, however, that the arcuate and ventromedial nuclei, traditionally thought to be more important for appetite regulation, are also able to interact with the thermogenic pathway to elicit changes in energy balance. That this is dependent on nutritional status suggests that the same hypothalamic nuclei integrate both sides of the energy balance equation, namely feeding and energy expenditure. Mechanisms have recently been identified in the VMH that might be manipulated to elicit specific effects on sympathetic tone to BAT. Further studies are likely to reveal additional molecular pathways in other nuclei or perhaps even suggest subtle nutritional alterations that manipulate the hormonal milieu to maximize centrally driven thermogenesis in BAT. Ultimately, nutritional stimuli are likely to remain dependant on the more intensive induction of BAT by cold temperatures to provide the adequate thermogenic machinery. As the modern living environment provides no such stimulus and factors of increasing age and body weight conspire to reduce detectable BAT in humans even further, it is probable that multiple BAT-targeted approaches will be required in combination to successfully treat obesity. However, adaptive environmental conditioning, combined with methods exploiting endogenous brown 410
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adipogenic factors, would be one possible strategy to increase the thermogenic impact of hypothalamic manipulations at the peripheral level. Acknowledgements AW is funded by a BBSRC PhD studentship. ML is supported by the Xunta de Galicia (ML: 10PXIB208164PR), the Fondo Investigationes Sanitarias (ML: PS09/01880) and the Ministerio de Ciencia e Innovacio´n (ML: RyC2007-00211). CIBER de Fisiopatologı´a de la Obesidad y Nutricio´n is an initiative of ISCIII. AVP is supported by an MRC programme grant and FP7 - BetaBAT.
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