Feeding behaviour and energy storage are both crucial aspects of survival. Thus, it is of fundamental importance to understand the molecular mechanisms regulating these basic processes. The AMP-activated protein kinase (AMPK) has been revealed as one of the key molecules modulating energy homoeostasis. Indeed, AMPK appears to be essential for translating nutritional and energy requirements into generation of an adequate neuronal response, particularly in two areas of the brain, the hypothalamus and the hindbrain. Failure of this physiological response can lead to energy imbalance, ultimately with extreme consequences, such as leanness or obesity. Here, we will review the data that put brain AMPK in the spotlight as a regulator of appetite.
- energy balance
- food intake
Just as many electronic devices run on batteries, living organisms usually function on immediately accessible internal energy. However, although battery-operated machines eventually deplete their energy source, it is common for living organisms to be able to store surplus energy and access it in a controlled manner without recourse to an external energy source. Despite their structural differences, artificial machines and cells are comparable in terms of energy flux, which can be summarized as the ratio between particles depleted of energy and particles full of energy. In cells, this comprises the dynamic ratio of levels of the adenine nucleotides: adenosine mono-, di- and tri-phosphate (AMP:ATP and ADP:ATP), and reflects the relative rates of cellular catabolism (ATP production) and anabolism (ATP consumption) .
It is therefore effective that cells sense energy by detecting alterations in AMP:ATP and ADP:ATP ratios, by means of a single molecule, the AMP-activated protein kinase, AMPK . Indeed, as AMP, ADP and ATP constitute the most immediate recipients and donators of energy in eukaryotic cells, understanding how nutrients, hormones and other signals modulate their ratios will help to elucidate the basis of survival. Indeed, managing cellular energy resources is likely be the most important aspect of survival, because other vital processes such as growth, reproduction and even aging all depend on it. Given its crucial role in energy homoeostasis, it is therefore not surprising that AMPK can have a major role in human health and a clinical impact. Impaired AMPK function is now known to be involved in a range of pathologies, including diabetes , heart disease , cancer , Alzheimer's disease , viral infection  and also in the immune response .
AMPK is a highly conserved serine/threonine protein kinase with an ancient stress-response activity which requires an elevation of ADP:ATP and AMP:ATP ratios to be activated [9–12]. AMPK is an almost-ubiquitous energy sensor in eukaryotic cells [13,14], with one exception: the obligate mammalian parasite, Encephalitozoon cuniculi, which seems to have lost the AMPK gene from its genome due to its ability to extract ATP from its host .
AMPK is a heterotrimeric complex consisting of three subunits, a catalytic α subunit (α1 or α2), a regulatory β subunit (β1 or β2) and a γ subunit (γ1, γ2 or γ3) [10,11,16–21] (Figure 1). The catalytic subunit α contains a kinase domain at its N-terminus, followed by an auto-inhibitory domain (AID)  and a C-terminal serine-threonine insert (ST loop) , with its C-terminal being connected to the regulatory core of the enzyme across the β-subunit interacting domain (β-SID) [10,11]. The β subunit also anchors the α and γ subunits, shaping the regulatory core . C-terminal residues of the β subunit bind to C-terminal residues of the α subunit through extensive hydrophobic bonds and to the C-terminal of the γ subunit with short hydrogen β-strands. The γ subunit is defined by four cystathione β-synthase (CBS) domains, paired 1+2 and 3+4 and forming two Bateman modules . Although the β subunit contains a mid-molecule carbohydrate binding molecule (CMB) required to bind glycogen, subunit γ contains one nucleotide-binding site in each CBS, providing the enzyme with four potential nucleotide-binding sites (sites 1, 2, 3 and 4) [10,24–26]. Site 1 and site 3 are situated opposite each other and can bind either AMP, ADP or ATP, with site 1 having a higher affinity for all three nucleotides. Site 4, however, has a permanent molecule of AMP which is not exchanged under physiological conditions, although in the laboratory it can be . On the other hand, site 2 is unable to bind nucleotides due to its lack of a critical aspartate residue . Together, these data suggest that sites 1 and 3 are crucial for mammalian AMPK regulation , whereas site 2 might not play a functional role in regulation of activity of the enzyme in mammals [29,30].
The precise roles of the nucleotide-binding sites, however, are still unknown, as the structure of AMPK has not been fully described and the presence of different subunits makes it difficult to ascribe each site with a specific regulatory action. Intriguingly, recent reports of the crystal structure of the human AMPK α2β1γ1  and α1β1γ1  show that even though all the binding sites behave as expected from their aforementioned properties, binding of ATP at site 3 can cause rotations in the AID. This change in conformation inactivates the inhibitory role of the AID. Different studies seem to converge on the fact that site 3 prevents the auto-inhibitory effect when ATP is bound [24,31,33,34]. A more detailed description of AMPK structure and regulation is beyond the scope of the present review, but has been excellently reviewed elsewhere [35–37].
REGULATION OF AMPK ACTIVITY
Regulation of the AMPK complex can be achieved by different pathways. Although the complex has an auto-inhibitory regulation through structural elements in the α subunit, it can also be allosterically activated by AMP or ADP binding to the γ subunit. AMP-independent activation can also take place in the α subunit (see below). AMPK activity can be strengthened either by AMP binding to phosphorylated AMPK  or by phosphorylation subsequent to AMP binding to AMPK . Combination of the two means of activation leads to a 1000-fold increase in AMPK enzymatic activity , allowing AMPK to sense very small changes in energy status. Due to the fact that phosphorylation of Thr172 (P-Thr172) is the major change responsible for increasing AMPK activity, researchers have used P-Thr172 levels as an indicator of AMPK activity. Recently, however, it has been suggested that it does not provide an accurate measure . Thus, using A769662, a compound which mimics AMP allosteric activation , AMPK has been shown to have a high allosteric activation without phosphorylation of Thr172, both in wild type and mutant carrying a non-phosphorylatable residue. For maximal activation, however, phosphorylation of an additional residue (Ser108) was also required. Furthermore, this compound in the presence of AMP, reached an enzymatic activity enhancement in AMPK comparable with the maximal activity obtained in wild type with P-Thr172 .
The AID brings about inhibitory regulation by causing a change in structural conformation which interferes with substrate binding and thus prevents kinase catalytic activity [22,24,31,33,34,42]. Although the structure of the AID is not fully described in humans , it is known that on AMP binding, it undergoes a rotation which causes it to interact with the γ subunit which is itself responsible for auto-inhibition. It seems that this autoregulation is predominant in allosteric regulation , when AMPK changes from a high- to low-activity conformation on binding with AMP, ultimately removing the effect of the AID on kinase activation and on Thr172 phosphorylation . A second sequence, situated in the CBS2 domain within the γ2 isoform, has been reported to act as a pseudo-domain substrate, able to bind to the catalytic Thr172 loop and thus prevent it from being phosphorylated . That conformational change was found to occur after AMP bound to the γ subunit, preventing an inhibitory interaction between the CBS2 domain sequence and the kinase domain.
AMPK upstream kinases
Phosphorylation at the conserved Thr172 residue by upstream kinases is the major source of AMPK activation and occurs within the catalytic α subunit . Although α1 and α2 subunits have comparable specific activities, they show differences related to substrate specificity . Although many of the substrates that mediate AMPK's effect on metabolism have been well studied, those connecting AMPK to non-metabolic roles remain uncertain. In this sense, recent evidence using a chemical genetic screen has identified 57 previously unknown AMPK phosphorylation sites in human cancer cells involved in diverse aspects, such as cell motility, adhesion and invasion .
The Thr172 site is phosphorylated mainly by three upstream AMPK kinases (AMPKKs) (Figure 1), the tumour suppressor liver kinase B1 (LKB1) [48,49], the Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) [50,51] and transforming growth factor-β-activated kinase 1 (TAK1) [52,53]. The kinase suppressor of Ras 2 (KSR2) also modulates AMPK activity through an interaction with the α1 isoform . Thr172 phosphorylation by CaMKKβ depends upon an increase in intracellular Ca2+ levels triggered by the action of a third compound, such as a drug or a hormone (e.g. liraglutide , ghrelin  or adiponectin (ADPN) ), and does not require any change in AMP or ADP levels. Phosphorylation by LKB1, the main upstream kinase of AMPK, on the other hand, does require a change in the level of at least one of these nucleotides. LKB1 forms a major heterotrimeric complex with the pseudokinase STE-related adaptor protein (STRAD) and a scaffold mouse protein 25 (MO25), the complex LKB1–STRAD–MO25 [16,48,49], whose basal kinase activity is increased by the binding of adenine nucleotides to AMPK . When AMP binds to the γ subunit of AMPK it produces a conformational change that promotes Thr172 phosphorylation by LKB1 without requiring any other substrate . The same authors also suggest that binding of ADP prevents dephosphorylation of LKB1 by protein phosphatases, such as protein phosphatase 2Cα (PP2Cα) . Indeed, LKB1-AMPK activation might in fact be more complicated, with some authors suggesting a required involvement of one or more third party co-operators. A study knocking down the scaffold protein axin in mice found that AMPK activity was reduced . Using co-precipitation studies the authors found that LKB1 and axin formed a complex, whereas AMP binding to AMPK led to formation of a major complex that enhanced Thr172 phosphorylation.
Although the Ca2+-dependent and AMP-dependent activation pathways function independently, both AMP and ADP induce AMPK phosphorylation at Thr172 by means of either LKB1 or CaMKKβ [12,30]. Nonetheless, Thr172 at the α subunit can also be inactivated by dephosphorylation by members of the PPP family of proteases, specifically 2A and 2C [60–62]. Besides the PPP family, members of the protein phosphatase Mn2+-dependent family (PPM), such as Ppm1E and maybe the closely related Ppm1F interact with AMPK to dephosphorylate it . A protein phosphatase holoenzyme composed of the catalytic subunit of protein phosphatase-1 (PP1) and the regulatory subunit R6 participate in the in vivo glucose-induced dephosphorylation and inactivation of AMPK  and possibly in the control of AMPKβ2 by PP1-R6 upon glycogen depletion in muscle . It has also recently been shown that p70S6K [ribosomal S6 kinase, which is part of the mechanistic target of rapamycin (mTOR) pathway] phosphorylates and thus inhibits AMPK at Ser491 (in the α2 subunit), and that this effect is necessary to mediate leptin's anorectic action (see below) . Finally, the β subunit of AMPK has been found to be involved in cellular degradation following action of the cell death activator CIDEA .
AMPK AND ENERGY METABOLISM
Since depletion of ATP activates AMPK, it can lead to various metabolic events, with the overall function of increasing ATP production and inhibiting ATP consumption in peripheral tissues. These events encompass not only carbohydrate, lipid and protein metabolism, but also mitochondrial biogenesis, cell growth and proliferation, apoptosis and autophagy [9,68]. Regulation of the metabolic pathways includes a central role for AMPK, situated at the intersection of several signalling networks involving a substantial number of hormones and cytokines . In this context, hypothalamic AMPK has been claimed to be the major molecular switch in lipid metabolism and therefore of central importance to energy balance, as discussed later.
When ATP levels are depleted, AMPK promotes catabolic pathways to generate ATP while inhibiting anabolic pathways involved in cell growth and other processes that consume ATP. AMPK also causes the cell cycle to be arrested in G1  and modulates mitosis , partly by inhibiting p53 turnover  and preventing cell division which requires energy. AMPK also modulates biogenesis of mitochondria, critical for ATP production; AMPK activates not only the biogenesis transcription co-activator peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α), by phosphorylation , but also the lysine deacetylase activator of sirtuin 1 (SIRT1), which in turn deacetylates and activates PGC-1α . Recent evidence has also demonstrated that AMPK regulates mitochondrial fission by modulating mitochondrial fission factor (MFF) . Protein synthesis is also a target of AMPK, reducing ATP consumption in favour of immediate cell survival. This effect is mainly driven by inhibition of upstream activators of mTOR1, the tuberous sclerosis complex 2 (TSC2)  or the Raptor component of the TORC complex .
As energy is long-term stored as lipid, understanding the role of AMPK in regulating lipid biosynthesis and degradation is of great importance. AMPK inhibits de novo fatty acid synthesis. The early fatty acid synthesis pathway, also known as de novo lipogenesis, is one of the best characterized pathways in which AMPK plays a critical role. When ATP levels are low and AMPK is active, the first enzyme in this pathway, acetyl-CoA carboxylase (ACC), is inhibited by direct phosphorylation by AMPK. Malonyl-CoA decarboxylase (MCD) which catalyses the opposite reaction, moreover, is activated, thus reducing the flux of substrate for fatty acid synthase (FAS) to produce fatty acids. In addition, FAS is negatively modulated by AMPK through its transcription factor sterol regulatory element-binding protein 1 (SREBP1) [69,78,79] (Figure 2). Activated AMPK not only reduces ATP usage by inhibiting fatty acid synthesis but also increases ATP production through mitochondrial fatty acid oxidation, since carnitine palmitoyltransferase 1 (CPT1) is no longer affected by its allosteric inhibitor malonyl-CoA as a consequence of diminished ACC activity. This enables CPT1 to import long chain fatty acids into the mitochondria for fatty acid oxidation [69,78,79]. Furthermore, AMPK inhibits 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), thus reducing cholesterol synthesis . The control that AMPK exerts over the lipogenic pathway has been thoroughly investigated in recent decades, revealing its importance, not only in regulation of cellular energy usage, but also in modulation of feeding behaviour and energy expenditure at the whole body level (see below) [80–86].
CENTRAL REGULATION OF FEEDING
The hypothalamus is a major brain site involved in feeding regulation. It constitutes a key node that translates nutritional and hormonal information regarding whole body energy status into hunger or satiety, thus allowing the body to manage energy requirements [87–89]. Hypothalamic structures were first associated with appetite and body weight in the early 1900s from clinical reports on patients with tumours [90,91]. During the 1940s, lesion experiments in animals established a key role for discrete hypothalamic areas in feeding control. The original theories explaining central control of food intake were based on a ‘Dual Centre Hypothesis’ [92,93]. In this model, supported by hypothalamic lesioning experiments, feeding was controlled by two hypothalamic areas: the lateral hypothalamic ‘feeding centre’ and ventromedial hypothalamic ‘satiety centre’. Lesions of the lateral hypothalamic area (LHA) decreased food intake and eventually led to starvation and death. Conversely, lesions of several of the mediobasal hypothalamic nuclei resulted in obesity. In recent years, the availability of various techniques such as electrophysiology, chemogenetics and optogenetics, together with the development of mouse genetics, has led to much more detailed information on the roles of different neuronal populations in the regulation of energy balance. The principal concept, however, remains the same, that anatomically defined hypothalamic areas regulate food intake. Such hypothalamic nuclei form interconnected neuronal circuits which respond to changes in energy status by altering the expression of specific neuropeptides, resulting in changes in energy intake and expenditure.
The arcuate nucleus of the hypothalamus (ARC) is one of the best characterized nuclei controlling feeding regulation. This is due to two primary neuronal populations that exert direct effects on feeding, the anorexigenic proopiomelanocortin (POMC) neurons and the orexigenic agouti-related peptide/neuropeptide Y (AgRP/NPY) neurons. Activation of POMC neurons suppresses feeding [94,95], whereas their ablation induces hyperphagia and obesity [96–98]. Activation of AgRP neurons in the ARC, on the other hand, induces food intake [94,99], whereas their ablation induces hypophagia and starvation [100,101]. The key feature of the ARC is its anatomical position in the ventral part of the hypothalamus, closely apposed to the median eminence (ME), an area which lacks blood–brain barrier (BBB) and thus allows neurons to receive rapid information regarding nutritional status via the bloodstream, sensing levels of compounds ranging from nutrients (e.g. glucose, fatty acids) to hormones (e.g. leptin, insulin, ADPN) [102,103]. POMC neurons, for example, express the leptin receptor (LepR) which, when stimulated, triggers a reduction in food intake [104–107]. By contrast, ghrelin is sensed by the growth hormone secretagogue receptor (GHS-R), leading to suppression of POMC neurons and excitation of NPY/AgRP neurons by different mechanisms [83,108–110]. Endocannabinoids, moreover, induce feeding by inhibiting POMC neurons via the endocannabinoid receptor 1 (CB1R) .
BRAIN AMPK AND REGULATION OF FEEDING
As AMPK is a key modulator of energy expenditure it might also be expected to be an important mediator of neuronal regulation of feeding behaviour . Indeed, AMPK is ubiquitously expressed in the major hypothalamic nuclei . Accordingly, a normal physiological condition such as starvation induces increased levels of AMPK in multiple hypothalamic regions, such as the ARC, the ventromedial nucleus of the hypothalamus (VMH), the dorsomedial nucleus of the hypothalamus (DMH) and the paraventricular nucleus of the hypothalamus (PVH) nuclei, as well as the LHA, whereas refeeding returns AMPK levels to pre-starvation levels [83,112,113].
The role of hypothalamic AMPK in the regulation of energy balance is also evident in genetic models. Global AMPKα2 knockout (KO) mice fed a high-fat diet (HFD) show a larger increase in body weight and adiposity than their wild-type (WT) counterparts . To address the precise roles of AMPK in individual hypothalamic nuclei and discrete populations of neurons, several approaches have been used. First, inhibition of hypothalamic AMPK using dominant negative isoforms (AMPK-DN) was shown to reduce mRNA expression of the orexigenic neuropeptides AgRP and NPY in the ARC . In line with that, over-expression of a constitutively active (AMPK-CA) isoform increased levels of AgRP and NPY . These data suggest that AMPK has nuclei-specific effects on feeding control. This idea was validated elegantly by generating mice with a conditional deletion of the catalytic subunit of AMPKα2 in POMC or AgRP neurons of the ARC. Notably, both murine models displayed divergent phenotypes in terms of energy balance. Thus, whereas AMPKα2-POMC KO mice developed obesity due to hyperphagia, AMPKα2-AgRP KO mice developed an age-dependent lean phenotype . Interestingly, LKB1-POMC KO mice showed impaired glucose homoeostasis but normal body mass . Perhaps more importantly, several lines of evidence have indicated that the chronic effects of manipulating hypothalamic AMPK, specifically within the VMH, on whole body mass are also related to altered energy expenditure, specifically through the control of hormone-induced brown adipose tissue (BAT) thermogenesis [81,84–86,116] (Figure 3). Together, these findings are particularly significant as they offer the potential to modulate both sides of the energy balance equation, i.e. feeding and energy expenditure, by targeting hypothalamic AMPK . This could be translated into more successful therapies in humans for controlling obesity and its related disorders than those which target only feeding .
The importance of central AMPK's role in feeding, moreover, might also be said to lie more in its mediation of central and peripheral stimuli on feeding behaviour than its physiological regulation of feeding per se. Anorectic hormones (feeding inhibitors), e.g. leptin, insulin, glucagon-like peptide 1 (GLP-1) and oestradiol (E2), inhibit AMPK [85,86,112]; whereas in contrast, orexigenic hormones (feeding promoters) such as ghrelin, ADPN and endocannabinoids activate hypothalamic AMPK [80,82,83,119,120]. Of note, it is important to highlight that the regulation of some of these factors exert opposite effects on AMPK in the hypothalamus when compared with peripheral organs. For example, leptin inhibits hypothalamic AMPK  whereas activates it in the skeletal muscle 
AMPK has been suggested to mediate the satiating effects of several hormones and peptides . On the other hand, feeding and glucose administration promote AMPK inactivation [112,123], accompanied by increased levels of POMC and reduced levels of AgRP mRNA in the ARC . Similarly, administration of end products, such as citrate, lipoic acid or lactate, which signal an energy surplus, reduce food intake [125,126] with a correlated reduction in AMPK levels [125,126]. Nutrient enriched diets, such as HFD or with increased amino acids, which reduce the amount of food eaten, also reduce AMPK phosphorylation . Meanwhile, peripheral signals such as the gut hormone GLP-1 and the sex steroid E2 reduce food intake by inhibiting AMPK while simultaneously reducing body mass through activation of the thermogenic programme in the BAT [85,86,128]. Thyroid hormones (THs)  and bone morphogenetic protein 8B (BMP8B)  also activate BAT thermogenesis .
The anorectic effect of leptin is mediated by hypothalamic AMPK [112,113]. Inhibition of AMPK by leptin is placed downstream or in a parallel pathway to STAT3 (the canonical transduction signal pathway for leptin), since pSTAT3 levels remain elevated following a leptin injection in mice with constitutively active AMPK . In this regard, this is also supported by the fact that leptin can also override AMPK's effect, as shown by its activation of the mTOR pathway  which in turn inactivates AMPK and also directly activates ACC . It has, further, been shown that the downstream target of mTOR, S6K1, can inhibit AMPK . In animals on HFD, however, leptin is unable to suppress AMPK , suggesting that AMPK may be part of a collateral pathway by which leptin influences food intake.
Several drugs are known to modulate feeding acting on brain AMPK. For example, nicotine and liraglutide (a GLP-1 agonist) exert a catabolic action by inhibiting feeding and increasing BAT thermogenesis through hypothalamic AMPK [85,132–134]. Metformin, a synthetic biguanide, activates AMPK indirectly by inhibiting the mitochondrial respiratory chain [3,135]. Notably, when given by gastric gavage metformin exerts a profound anorectic and weight reducing effect, acting in both hypothalamic and extra-hypothalamic areas ; however the fact that metformin was given peripherally in that study may be indicative of indirect (non-AMPK mediated) effects. In any case, supporting an AMPK-dependent action, it has been reported that central administration of metformin inhibits ghrelin-induced feeding (see below) and AMPK signalling . Further work will be necessary to clarify the central action of metformin on energy balance.
Various signals promoting feeding behaviour have been found to exert their effects by activating AMPK through phosphorylation . Hunger, which can be mimicked by a physiological state of fasting, produces an increase in AMPK phosphorylation, triggering activation of the orexigenic neuropeptide AgRP and reduction of mRNA expression of the anorexigenic POMC, thus inducing feeding [83,112]. Of note, activation of hypothalamic AMPK by starvation is not observed in the spontaneously hypophagic Lou/C rats, a strain resistant to obesity . Animals under glucose deprivation produce the same effect, increasing activation of AMPK . Peripheral signals related to energy uptake and storage, such as the adipocyte derived hormone ADPN, increase hypothalamic AMPK activity and stimulate food intake, increasing AgRP expression while inhibiting POMC mRNA translation . The stomach-released peptide ghrelin has been shown to increase food intake  and to induce fat deposition . These effects of ghrelin are mediated centrally by phosphorylation of AMPK in the ARC and the VMH to induce AgRP and NPY gene expression [80,83,113]. Endocannabinoids have also been suggested to promote feeding by activating hypothalamic AMPK , thus inhibiting POMC expression in the ARC . Recently, it has been shown that CB1 mediates this feeding effect in the ARC POMC neurons .
Among the different signals modulating hypothalamic AMPK, ghrelin is the best characterized. Ghrelin increases intracellular Ca2+ levels and AMPK levels in AgRP neurons , as well as NPY gene expression , suggesting that the AMPK upstream kinase CaMKKβ is involved [142,143]. Interestingly, AMPK activation in the VMH by ghrelin also induces food intake by modulating ARC neuropeptides [83,119]. However, it has also been shown that mTOR in the ARC can drive ghrelin effects on feeding , suggesting the existence of an additional pathway for the regulation of appetite. At the cellular level, ghrelin-induced AMPK activation decreases ACC activity, reduces malonyl-CoA concentration and releases inhibition of CPT1. Thus, when CPT1a is released within the mitochondria, it leads to increased β-oxidation and elevated reactive oxygen species (ROS) levels, which are buffered mainly by uncoupling protein 2 (UCP2) . Ultimately, this leads to increased expression of npy and agrp genes in the ARC . Moreover, the decreased concentration of hypothalamic malonyl-CoA promotes disinhibition of CPT1c in the endoplasmic reticulum (ER); this in turn increases ceramide synthesis which also induces agrp and npy gene expression . AMPK also seems to be involved in a positive feedback loop mediating the effects of ghrelin and leptin on AgRP neurons to drive hunger . AMPK also induces p53 expression  which is itself negatively regulated by SIRT1 . SIRT1 activity has been found to be increased after fasting and following ghrelin administration, whereas SIRT1 blockers blunt not only ghrelin-induced feeding  but also expression of AMPK, NPY and AgRP. It is not clear, however, whether CaMKKβ or SIRT1 interact upstream of AMPK to modulate its activity.
The fact that ARC neuropeptides constitute the final effectors of ghrelin might suggest that they are the primary mediators of ghrelin's orexigenic effects. There are, however, also indications that other hypothalamic nuclei play equally, if not more, important roles. For example, the fact that AMPKα2-AgRP KO and AMPKα2-POMC KO mice remain sensitive to a reduction in feeding triggered by leptin and insulin , and AMPK in the VMH controls the orexigenic effect of ghrelin on AgRP and NPY neurons [119,151], suggests that this has a more important role in second order neurons than in those of the ARC .
Finally, a role for hindbrain AMPK in the regulation of food intake has been proposed. Food deprivation increases AMPK activity in the nucleus tractus solitarius (NTS). Pharmacological targeting of AMPK using the non-specific activator AICAR or the non-specific inhibitor compound C in the fourth ventricle ameliorated the anorectic action of leptin or induced anorexia with weight loss respectively . Knockdown of the LepR in neurons of the medial NTS (mNTS) and the area postrema (AP) (LepRKD) in rats, using an adeno-associated shRNA-interference virus (AAV-shRNAi), resulted in significant hyperphagia, increased body weight and adiposity, due to impaired leptin action and a reduction in the potentiation of CCK signalling by leptin. LepRKD rats showed increased basal AMPK activity in the mNTS and AP . Although treatment with compound C ameliorated LepRKD-induced hyperphagia, the lack of specificity of compound C limits the conclusions that can be drawn as to AMPK's mechanistic involvement . Administration of the GLP-1 agonist exendin-4 into the NTS decreased feeding and central AMPK activation. This effect was blunted with AICAR, suggesting that AMPK in the NTS mediates the anorectic action of GLP-1 receptor agonism . Finally, a role for E2 on hindbrain AMPK has been also proposed ; however, the physiological outcomes of that effect are still unclear.
Obesity causes thousands of deaths per year worldwide, directly and due to comorbidities including cancer, cardiovascular disease and type 2 diabetes, and yet it is the most preventable epidemic [118,157]. However, in spite of significant investments in education and public engagement government-led policies are relatively ineffective. This is shown in the World Health Organization (WHO)’s latest report, which states that 13% of adults globally are obese. In healthy individuals, maintaining normal weight is a matter of lifestyle. However, such apparent simplicity also necessitates an understanding of how the body manages what, how, when and why we eat, as well as how we expend calories. Each of these functions is carried out by different hormones and peptides that respond to the various physiological states occurring in arousal and sleep, with some having circadian rhythms.
Data obtained over the last decade have demonstrated an unequivocally key role for central AMPK in the regulation of almost every aspect of energy balance: feeding, energy expenditure and peripheral metabolism . Activation of AMPK in peripheral organs is one of the mechanisms of action of the widely used antidiabetic drug metformin (for an extensive review see ). However, central AMPK [83,112] is regulated differently from peripheral AMPK , and activating central AMPK, is not indicated for obesity treatment, as it would increase feeding and decrease BAT thermogenesis. On the other hand, inhibition of AMPK in peripheral tissues could also cause deleterious consequences, increasing insulin resistance and worsening diabetes. Therefore, the best strategy would be to specifically target hypothalamic AMPK, although that would appear to be highly complex. Although many known compounds can achieve this goal with high selectivity and few secondary effects, safety is also a major challenge because some of AMPK's major main physiological roles originate in the brain . Thus, therapies selectively directed at regulating feeding by inhibiting AMPK in the brain remain difficult to realize. One option might be the use of nanoparticles, but directing these to specific hypothalamic populations appears challenging. Inhibiting AMPK neurons in the VMH, for example, could promote anorexia, increased thermogenesis and weight loss. Another alternative might be the optogenetic modulation of hypothalamic AMPK, which has been already smartly applied to rodents . However, implementation of optogenetics for hypothalamic intervention in humans seems a distant goal which might not be feasible due to the very short duration of neuronal excitation that would be achieved. Perhaps, the most realistic approach would be the use of peptide conjugates, composed of putative inhibitors of AMPK and coupled to other peptides or steroid hormones that would allow targeting of specific neuronal populations . For example, a chimaera comprising GLP-1 and an oestrogen, which has already been reported , or one of GLP-1 plus TH would allow targeting of AMPK neurons in the VMH.
There are also other obstacles to finding a workable AMPK modulator. First, AMPK is not only involved in feeding behaviour, but also in a wide range of intracellular housekeeping processes, such as metabolism, proliferation and mitosis. This makes it difficult to predict the short- and long-term consequences of inhibiting AMPK. Second, AMPK is found in all cells, reflecting its universal role as an energy sensor. However, as it regulates different energy metabolic pathways depending on the tissue or cell type (e.g. glucose production in the liver and glucose uptake in skeletal muscle) different specific compounds should be able to be designed and delivered. Thus, understanding the precise role of AMPK in the cell and its effects at the whole-body level will be essential to the development of any therapeutic compound to target its activity for the treatment of obesity and other metabolic disorders.
This work was supported by the European Community's Seventh Framework Programme (FP7/2007-2013) the ObERStress project [grant number 281854 (to M.L.)]; Xunta de Galicia [grant number 2015-CP079 (to M.L.)]; the Ministerio de Economía y Competitividad (MINECO) co-funded by the FEDER Program of EU [grant numbers SAF2015-71026-R and BFU2015-70454-REDT/Adipoplast (to M.L.)]; and the CIBER de Fisiopatología de la Obesidad y Nutrición is an initiative of ISCIII.
We thank Ismael González-García for his comments and criticism. The manuscript was edited for English language by Dr Pamela V. Lear.
Abbreviations: ACC, acetyl-CoA carboxylase; ADPN, adiponectin; AgRP, agouti-related peptide; AID, auto-inhibitory domain; AMPK, AMP-activated protein kinase; AP, area postrema; ARC, arcuate nucleus of the hypothalamus; BAT, brown adipose tissue; BMP8B, bone morphogenetic protein 8B; CaMKKβ, Ca2+/calmodulin-dependent protein kinase kinase β; CBS, cystathione β-synthase; CPT1, carnitine palmitoyltransferase 1; DMH, dorsomedial nucleus of the hypothalamus; E2, oestradiol; FAS, fatty acid synthase; GLP-1, glucagon-like peptide 1; HFD, high-fat diet; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; KSR2, kinase suppresor of Ras 2; LepR, leptin receptor; LHA, lateral hypothalamic area; LKB1, liver kinase B1; MCD, Malonyl-CoA decarboxylase; MO25, mouse protein 25; mTOR, mechanistic target of rapamycin; NPY, neuropeptide Y; NTS, Nucleus tractus solitarius; PGC-1α, proliferator-activated receptor-γ coactivator-1α; POMC, proopiomelanocortin; PP1, protein phosphatase-1; PP2Cα, protein phosphatase 2Cα; PVH, paraventricular nucleus of the hypothalamus; β-SID, β-subunit interacting domain; SIRT1, sirtuin 1; SREBP1, sterol regulatory element-binding protein 1; STRAD, STE-related adaptor protein; TAK1, transforming growth factor-β-activated kinase 1; TH, thyroid hormone; TSC2, tuberous sclerosis complex 2; VMH, ventromedial nucleus of the hypothalamus
- © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society