Molecular Regulation of Energy Balance
Summary and Keywords
AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that monitors the levels of AMP and ADP relative to ATP. If increases in AMP:ATP and/or ADP:ATP ratios are detected (indicating a reduction in cellular energy status), AMPK is activated by the canonical mechanism involving both allosteric activation and enhanced net phosphorylation at Thr172 on the catalytic subunit. Once activated, AMPK phosphorylates dozens of downstream targets, thus switching on catabolic pathways that generate ATP and switching off anabolic pathways and other energy-consuming processes. AMPK can also be activated by non-canonical mechanisms, triggered either by glucose starvation by a mechanism independent of changes in adenine nucleotides, or by increases in intracellular in response to hormones, mediated by the alternate upstream kinase CaMKK2.
AMPK is expressed in almost all eukaryotic cells, including neurons, as heterotrimeric complexes comprising a catalytic α subunit and regulatory β and γ subunits. The α subunits contain the kinase domain and regulatory regions that interact with the other two subunits. The β subunits contain a domain that, with the small lobe of the kinase domain on the α subunit, forms the “ADaM” site that binds synthetic drugs that are potent allosteric activators of AMPK, while the γ subunits contain the binding sites for the classical regulatory nucleotides, AMP, ADP, and ATP.
Although much undoubtedly remains to be discovered about the roles of AMPK in the nervous system, emerging evidence has confirmed the proposal that, in addition to its universal functions in regulating energy balance at the cellular level, AMPK also has cell- and circuit-specific roles at the whole-body level, particularly in energy homeostasis. These roles are mediated by phosphorylation of neural-specific targets such as ion channels, distinct from the targets by which AMPK regulates general, cell-autonomous energy balance. Examples of these cell- and circuit-specific functions discussed in this review include roles in the hypothalamus in balancing energy intake (feeding) and energy expenditure (thermogenesis), and its role in the brainstem, where it supports the hypoxic ventilatory response (breathing), increasing the supply of oxygen to the tissues during systemic hypoxia.
Introduction: Sensing Cellular Energy
Energy-requiring processes are usually made possible in living cells by being coupled to the hydrolysis of ATP to ADP or AMP. Under ideal conditions, cells maintain a high ATP:ADP ratio (typically 10:1). Since this is many orders of magnitude away from the equilibrium position for ATP breakdown , it can act as a store of energy, analogous to a fully charged battery. Animal cells maintain a high ATP:ADP ratio primarily by means of two catabolic processes, i.e. glycolysis and mitochondrial oxidative metabolism, with the latter responsible for the bulk of ATP synthesis, especially in non-dividing, differentiated cells such as neurons. This high ATP:ADP ratio is used to drive energy-requiring processes by obligate coupling of the latter to the breakdown of ATP, thus rendering the overall reactions more energetically favorable. Examples of this include the transporters that pump ions across the plasma membrane against a concentration gradient (see reaction 1 below) and the enzymes that catalyze the modifications to glucose, amino acids, and fatty acids (reactions 2–4) that are the first steps in their incorporation into their respective macromolecular forms (glycogen, proteins, phospholipids and triglycerides):(1) (2) (3) (4)
Note that most of these reactions (e.g. 1 and 2) generate ADP with or without phosphate (Pi), although a few (e.g. 3 and 4) generate AMP and pyrophosphate (PPi) instead. If the rate of ATP-consuming reactions exceeds the rate at which ATP can be regenerated by catabolism, then increases in cellular ADP:ATP and/or AMP:ATP ratios will occur, these being key indicators that cellular energy status is compromised. An additional player here is the enzyme adenylate kinase, whose almost energy-neutral (and thus reversible) reaction (2ADP ↔ ATP + AMP) appears to be maintained close to equilibrium in almost all eukaryotic cells. The high ATP:ADP ratio in a fully energized cell means that the adenylate kinase reaction would normally run in a leftward direction (toward ADP), thus maintaining AMP at very low levels (for example, if ATP:ADP is 10:1 and the adenylate kinase reaction is at equilibrium, then ATP:AMP will be ≈100:1). However, if ADP rises relative to ATP, the adenylate kinase reaction will be displaced in a rightward direction (toward ATP and AMP). Thus, increases in cellular ADP will always be accompanied by increases in AMP, and because AMP starts at such a low level any increase will be large, much larger than the changes in ADP or ATP. In fact, if the adenylate kinase reaction is at equilibrium, the AMP:ATP ratio will vary as the square of the ADP:ATP ratio (Hardie & Hawley, 2001). It is revealing that the small number of metabolic enzymes that directly sense the energy status of cells, including glycogen phosphorylase (Cori, Colowick, & Cori, 1938), 6-phosphofructo-1-kinase (Ramaiah, Hathaway, & Atkinson, 1964), and fructose-1,6-bisphosphatase (Taketa & Pogell, 1965), all appear to primarily sense AMP and ATP rather than ADP and ATP, like AMP-activated protein kinase (AMPK).
AMPK: Multiple Subunits and Regulation
AMP-activated protein kinase (AMPK) occurs universally in eukaryotes as heterotrimeric complexes comprised of a catalytic α subunit and regulatory β ανδ γ subunits (Lin & Hardie, 2017; Ross, MacKintosh, & Hardie, 2016). The α subunit contains at its N-terminal end a serine/threonine kinase domain with a small N- terminal lobe and larger C-terminal lobe, and the catalytic site is located in the cleft between them, as is typical of such domains. As in many other protein kinases (Taylor & Kornev, 2011), the C-lobe contains a conserved residue within the “activation loop” that must be phosphorylated to render the kinase active—in AMPK this is a threonine, usually referred to as Thr172 for historical reasons (see Hawley et al., 1996)—although its exact numbering depends on the species and isoform). The principal upstream kinase that phosphorylates this site was identified in 2003 to be the tumor suppressor LKB1 (Hawley et al., 2003; Shaw et al., 2004; Woods et al., 2003).
AMP was identified as an allosteric activator of AMPK as early as 1980 (Yeh, Lee, & Kim, 1980). In the presence of a physiologically relevant concentration of ATP (5 mM), allosteric activation of AMPK complexes already phosphorylated on Thr172 can be as much as 10-fold, with a half-maximal effect at just over 100 µM AMP, which is within the physiological range (Gowans, Hawley, Ross, & Hardie, 2013; Ross, Jensen, & Hardie, 2016). AMP binding to AMPK also makes Thr172 a better substrate for the upstream kinase LKB1 (Hawley et al., 1995), and a worse substrate for protein phosphatases that dephosphorylate Thr172 (Davies, Helps, Cohen, & Hardie, 1995). There are thus three independent mechanisms by which AMP binding activates the kinase (Fig. 1), with the end result being that any small change in AMP concentration is converted into a much larger change in final output in the form of kinase activity. It has been reported that ADP mimics the effects of AMP on Thr172 phosphorylation (effect #1) (Xiao et al., 2011) and dephosphorylation (effect #2) (Oakhill et al., 2011), although not on allosteric activation (effect #3). However, in our hands this requires concentrations of ADP around 10-fold higher than those of AMP. Although ADP concentrations are indeed often higher than those of AMP, and changes in ADP might therefore contribute to activation, the latter tend to be quite small and therefore AMP may be the primary activating signal (Gowans et al., 2013).
Using the three complementary mechanisms shown in Fig. 1 (which we now refer to as the canonical pathway for activation), the activity of AMPK varies markedly as a function of the energy status of cells. However, it has become clear that there are additional, non-canonical mechanisms by which the kinase can be activated. In many cell types, including neurons, it can be activated by Thr172 phosphorylation catalyzed by the /calmodulin-dependent protein kinase, CaMKK2. By this mechanism, hormones that increase intracellular , such as thrombin (Stahmann, Woods, Carling, & Heller, 2006) or vascular endothelial growth factor (Stahmann et al., 2010) acting on endothelial cells, or ghrelin acting on hypothalamic neurons (Yang, Atasoy, Su, & Sternson, 2011) can activate AMPK in the absence of any changes in cellular AMP:ATP ratios. Another non-canonical mechanism occurs in response to glucose deprivation, which is often used as a routine method to activate AMPK in cell culture. Because the original study, which was conducted using insulinoma cells derived from pancreatic β cells, demonstrated that glucose deprivation was associated with increases in cellular AMP:ATP/ADP:ATP ratios (Salt, Johnson, Ashcroft, & Hardie, 1998), it has generally been assumed that activation by glucose deprivation is always mediated by changes in AMP and/or ADP, i.e. by the canonical pathway. However, in some cells (e.g. mouse embryo fibroblasts), as long as an alternate carbon source such as glutamine is present, the removal of glucose from the medium is not associated with any increases in AMP:ATP/ADP:ATP ratios, yet AMPK activation still occurs. Under these circumstances, activation is associated with the formation of what we term a “super-complex” at the lysosomal membrane involving the vacuolar proton pump (v-ATPase), the Ragulator complex containing Lamtor1, and the adapter protein Axin, as well as LKB1 and AMPK (C. S. Zhang et al., 2014). There is now evidence that glucose availability is sensed by the binding of the glycolytic intermediate fructose-1,6-bisphosphate (FBP) to FBP aldolase, which is also associated with the lysosomal v-ATPase. Lack of occupancy of aldolase by FBP appears to cause a change in conformation of the aldolase:v-ATPase:Ragulator complex, such that the Axin:LKB1 complex is recruited from the cytoplasm. This brings LKB1 into proximity with AMPK, which also appears to translocate to the lysosomal membrane (although some may already be there), leading to Thr172 phosphorylation and activation of AMPK (C. S. Zhang et al., 2017).
AMP: Subunit Isoforms and Structure
In mammals, each of the three subunits of AMP-activated protein kinase (AMPK) occurs as two or three isoforms (α1/α2; β1/β2; γ1/γ2/γ3) encoded by distinct genes (PRKAA1/2; PRKAB1/2; PRKAG1-3). These paralogues appear to have arisen during the two rounds of whole genome duplication that occurred during early evolution of the vertebrates (Ross, MacKintosh et al., 2016). All possible combinations of α, β, ανδ γ subunit can form complexes, so that there are up to 12 different αβγ complexes with subtle variations in their regulatory properties, expressed variably in different cell types (Ross, Jensen et al., 2016; Ross, MacKintosh et al., 2016).
Crystal structures of αβγ complexes are available for at least three combinations, that is, α2β1γ1 (Xiao et al., 2013), α1β2γ1 (Li et al., 2015), and α1β1γ1 (Calabrese et al., 2014), and a schematic representation of these structures is shown in Fig. 2. They were generated using constructs that were in active conformations with Thr172 phosphorylated and allosteric activators bound, and our understanding of regulatory mechanisms is therefore somewhat hampered by the lack of structures in inactive states. Nevertheless, plausible models for allosteric activation (mechanism #3) and protection against Thr172 dephosphorylation (mechanism #2) by AMP have been suggested. The kinase domain at the N-terminus of the α subunit (α-KD) is immediately followed by a small bundle of three α-helices termed the auto-inhibitory domain (α-AID). This domain received its name because bacterially expressed constructs containing the α-KD and α-AID were 10-fold less active than constructs containing the α-KD only, even when phosphorylated on Thr172 (Crute, Seefeld, Gamble, Kemp, & Witters, 1998; Goransson et al., 2007; Pang et al., 2007). In the structure of a construct containing just the α-KD and the α-AID (albeit from the fission yeast orthologue of AMPK), the α-AID binds to the “rear” of the α-KD (i.e. opposite side to the active site), interacting with both the N- and C-lobes and clamping the α-KD in an inactive conformation (Chen et al., 2009). However, in the three structures of mammalian heterotrimers, which are all in active conformations, the α-AID appears to have rotated away from the N-lobe such that it now interacts with the C-lobe and the γ subunit instead (Fig. 2).
Immediately following the α-AID in the α subunit sequence is the α-linker, a region of polypeptide in extended conformation (depicted schematically as a chain in Fig. 2), which connects the α-AID to the globular C-terminal domain (α-CTD). In the three heterotrimer structures, this α-linker loops down over one surface of the nucleotide-binding γ subunit, suggesting that it forms the key interface between the catalytic subunit and the regulatory γ subunit, as we shall discuss later in this section.
The β subunits (β1 and β2) contain two conserved domains, a central carbohydrate-binding module (β-CBM) and a C-terminal domain (β-CTD), with the remaining regions (the N-terminal region and the β-linker between the CBM and CTD) being either absent or unresolved in the crystal structures. The β-CBM is known to cause a proportion of AMPK to bind to glycogen particles in intact cells (Hudson et al., 2003; Polekhina et al., 2003), although the physiological function of that binding remains uncertain. The β-CBM also forms part of the binding site (the ADaM site) for potent allosteric activators of AMPK that are discussed in the next section, “Activation of AMPK by Compounds that Bind in the ADaM Site.” The β-CTD lies at the “core” of the αβγ complex and appears to play a key structural role in stabilizing it, forming multiple interactions with both the α-CTD and the γ subunit.
The γ subunits (γ1, γ2, γ3) contain the binding sites for the regulatory adenine nucleotides AMP, ADP, and ATP (Cheung, Salt, Davies, Hardie, & Carling, 2000; Xiao et al., 2007; Xiao et al., 2011). They contain four tandem repeats (designated CBS1 through CBS4) of a sequence motif termed a CBS repeat. These motifs occur in a number of other human proteins, including the enzyme cystathione-β-synthase after which they are named (Bateman, 1997). They invariably occur as tandem repeats, although the AMPK-γ subunits are unusual in having four rather than just two repeats. In general, CBS repeats appear to be involved in binding, in the cleft between each repeat pair, regulatory ligands containing adenosine such as ATP (Scott et al., 2004). In the AMPK-γ subunits the four repeats assemble in a pseudosymmetrical manner to form a flattened disk with one repeat in each quadrant, and four potential ligand-binding sites in the center (Fig. 2). These sites are now numbered by convention (Kemp, Oakhill, & Scott, 2007) according to which repeat binds the adenosine portion of the adenine nucleotide (binding of the phosphate groups can involve residues from more than one repeat). Of the four potential sites only three, i.e. CBS1, CBS3, and CBS4, appear to be utilized for nucleotide binding. The CBS2 site may be unused partly because it lacks a conserved asparagine that in the other repeats binds to the ribose ring of the bound nucleotide (Xiao et al., 2007), and partly because the entrance to it appears to be blocked by the β-loop extending from the β-CTD (Fig. 2). The CBS1 and CBS4 sites are accessible to nucleotides entering from one side of the γ subunit (the far side in Fig. 2) and the CBS3 site from the other (the near side in Fig. 2). Of the three sites, CBS1 and CBS4 appear to constitutively bind ATP and AMP respectively; it may therefore only be at CBS3 that the regulatory nucleotides actually exchange with each other (Gu et al., 2017). It has been suggested that the function of binding of ATP at CBS1 and AMP at CBS4 may be to alter the conformation of the adjacent CBS3 site such that it has a higher affinity for AMP than ADP or ATP, thus allowing AMPK to achieve the difficult task of distinguishing small changes in AMP in the presence of much higher concentrations of ADP and/or ATP (Gu et al., 2017).
Looking at their overall shape, mammalian αβγ heterotrimers consist of two largely distinct globular regions, i.e. the catalytic module comprising the α-KD plus the β-CBM, and the nucleotide-binding module comprising the α- and β-CTDs plus the γ subunit. Interestingly, in the structures of the active heterotrimers, phospho-Thr172 lies in a deep cleft between these two modules (sandwiched between the C-lobe and the β-CTD, and not visible in Fig. 2), where its accessibility to protein phosphatases may be restricted. The “hinges” that connect the two modules are the α-linker and the unresolved β-linker connecting the β-CBM and β-CTD. Although the available crystal structures are all in the active conformation, there is evidence from measurements of small angle X-ray scattering (Riek et al., 2008) and singlet oxygen-mediated luminescence energy transfer (Li et al., 2015) that binding of ATP rather than AMP at site 3 causes these two modules to move apart into a less compact conformation. This is envisaged to occur because the α-linker, which is bound to the surface of site 3 when AMP is bound (Chen et al., 2013; Xiao et al., 2011; Xin, Wang, Zhao, Wang, & Wu, 2013), dissociates from the γ subunit when ATP replaces it. This movement is thought not only to allow the α-AID to move back into its inhibitory position behind the α-KD, but also to expose phospho-Thr172 for more rapid dephosphorylation by protein phosphatases. This model therefore accounts for two of the three effects of AMP binding to AMPK, although how AMP binding promotes phosphorylation of Thr172 by LKB1 is not explained.
Activation of AMPK by Compounds that Bind in the ADaM Site
Because of its effects on metabolism, it was proposed many years ago that activators of AMP-activated protein kinase (AMPK) might be useful in treatment of metabolic disorders such as Type 2 diabetes (Winder & Hardie, 1999). The first pharmacological activator of AMPK to be developed was the nucleoside 5-aminoimidazole-4-carboxamide riboside (AICA riboside), which is taken up into cells and converted to the equivalent ribotide, ZMP. ZMP is an AMP analogue that mimics all three effects of AMP on the AMPK system (Corton, Gillespie, Hawley, & Hardie, 1995), and AICA riboside was indeed subsequently shown to have favorable metabolic effects in rodent models of obesity and Type 2 diabetes (e.g. Song et al., 2000). However, AICA riboside has poor oral availability, and ZMP is much less potent as an AMPK activator than AMP itself (Corton et al., 1995). Moreover, ZMP has known “off-target,” AMPK-independent effects, including mimicking the regulatory effects of AMP on other AMP-sensitive enzymes such as muscle glycogen phosphorylase (Longnus, Wambolt, Parsons, Brownsey, & Allard, 2003) and liver fructose-1,6-bisphosphatase (Vincent, Marangos, Gruber, & Van den Berghe, 1991). Also, AICA riboside inhibits adenosine reuptake into cells by nucleoside transporters, which can lead to accumulation of extracellular adenosine and consequent effects mediated by adenosine receptors (Gadalla et al., 2004).
Despite these shortcomings, initial results using AICA riboside appear to have encouraged pharmaceutical companies to initiate high-throughput screens that searched for novel activators of AMPK. These resulted in several compounds including A-769662 (Cool et al., 2006), MT 63-78 (Zadra et al., 2014), PF-739 and PF-249 (Cokorinos et al., 2017), PF-06409577 (Cameron et al., 2016), and MK-8722 (Myers et al., 2017). Crystal structures of some of these bound to AMPK heterotrimers (Calabrese et al., 2014; Xiao et al., 2013) showed that they bind in a cleft located between the β-CBM and the N-lobe of the α-KD (the ADaM site, Fig. 2). Interestingly, almost all of the activators known to bind this site are synthetic molecules, so it currently represents an “orphan receptor,” and a key question is whether there are any naturally occurring ligands that bind there. Most researchers in the field suspect that a natural ligand (most likely a metabolite) must exist, hence the name Allosteric Drug and Metabolite (ADaM) site (Langendorf & Kemp, 2015). However, the only naturally occurring ligand currently known to bind there is salicylate (Calabrese et al., 2014; Hawley et al., 2012), a natural product of plants that, particularly in the form of extracts of the bark of willow (genus Salix, hence the name salicylate), has been used as a medicine by humans since ancient times. It was of course the starting point for the development of perhaps the most widely used drug of the modern era, i.e. acetyl salicylic acid (ASA or aspirin; Jeffreys, 2004). The main therapeutic targets of aspirin are thought to be the cyclo-oxygenases involved in biosynthesis of prostaglandins and other prostanoids (Ferreira, Moncada, & Vane, 1971), which are irreversibly inhibited by chemical transfer of its acetyl group to their active sites (Roth, Stanford, & Majerus, 1975). However, aspirin is broken down within minutes of entering the circulation to salicylate, which is then much more stable. Interestingly, salicylate and aspirin appear to be equipotent as anti-inflammatory agents, even though salicylate has very low potency as a cyclo-oxygenase inhibitor (Higgs, Salmon, Henderson, & Vane, 1987). This raises the possibility that some of the therapeutic effects of aspirin might be mediated by AMPK activation rather than cyclo-oxygenase inhibition (Steinberg, Dandapani, & Hardie, 2013).
Roles of AMPK in the Central Nervous System
Phosphorylation by AMPK of Non-Metabolic Targets in Neurons
It is becoming evident that AMPK phosphorylates, and thus modulates, a variety of targets that fall outside of its originally proposed role in maintenance of metabolic homeostasis (Hardie, Schaffer, & Brunet, 2016). For example, AMPK has been shown not only to phosphorylate and thus inactivate the pore-forming α subunits of multiple -activated potassium channels (KCa1.1 and KCa3.1; Klein et al., 2009; Ross et al., 2011), the voltage-gated potassium channel Kv1.5 (Andersen et al., 2015; Mia et al., 2012; Moral-Sanz et al., 2016; Moral-Sanz et al., 2018) and the ATP-inhibited KATP channel (Kir6.2) (Chang et al., 2009), but also to phosphorylate and activate the α subunit of the voltage-gated potassium channel Kv2.1 (Ikematsu et al., 2011). AMPK therefore has the potential to either increase or decrease cell excitability, in a manner determined by the cell-specific expression of AMPK subunits and of specific members of ion-channel superfamilies. Evidence is also now emerging that AMPK may directly phosphorylate and regulate: (i) enzymes involved in the biosynthesis of specific transmitters (Lipton et al., 2001; Murphy, Fakira, Song, Beuve, & Routh, 2009; J. Zhang et al., 2018); (ii) receptors for neurotransmitters (Ahmadi & Roy, 2016); and (iii) pumps and transporters (Schneider et al., 2015).
In the discussion of the roles of AMPK in the nervous system that follows, we will restrict our coverage to situations in which either the downstream targets for AMPK responsible for the effects are well understood and the critical phosphorylation sites identified, or the anatomical locations or specific cell types where AMPK plays its role are well defined. There has been much interest in the role of AMPK in pathological situations in the nervous system, such as in ischemic stroke and neurodegenerative diseases. However, some of the studies conducted in these areas are difficult to interpret because they were based on use of either the AMPK activator AICA riboside, which has many off-target effects as discussed in the previous section, “Activation of AMPK by Compounds that Bind in the ADaM Site,” or the kinase inhibitor compound C (dorsomorphin), which has even poorer selectivity for AMPK (Bain et al., 2007). In the limited number of studies where these disorders were studied more specifically using AMPK-α1 or ‑α2 knockout mice, they were often performed with global rather than cell-type-specific knockouts, once again making the results difficult to interpret. Readers interested in the role of AMPK in ischemic stroke or neurodegenerative diseases should consult other specialist reviews in those areas, bearing in mind these caveats (Domise & Vingtdeux, 2016; Manwani & McCullough, 2013).
Regulation of Kv2.1 by AMPK
Of the several channels that are direct targets for AMPK, Kv2.1 has been particularly well studied at the molecular and cellular levels. Kv2.1 is phosphorylated by AMPK, both in cell-free assays and in intact cells, at two sites within the C-terminal cytoplasmic tail (Ser440 and Ser537) (Ikematsu et al., 2011). In HEK-293 cells that were engineered to stably express Kv2.1, AMPK activation using A-769662 caused hyperpolarizing shifts in the current–voltage relationship for channel activation and inactivation, which were almost abolished by single (S440A) and completely abolished by double (S440A/S537A) phosphorylation-resistant mutations. In cells expressing wild type Kv2.1, channel activation was also observed upon the intracellular administration of active AMPK, but not an inactive control, from a patch pipette in whole-cell mode. The AMPK used was a bacterially expressed α2β2γ1 complex that had been activated by thiophosphorylation of Thr172 (note that thiophosphorylated, unlike phosphorylated, serine/threonine residues are extremely resistant to dephosphorylation), while the inactive control had a mutation in the kinase domain that abolished kinase activity (Ikematsu et al., 2011).
Kv2.1 is a voltage-gated, delayed rectifier channel. Because of its relatively slow opening and closing in response to depolarization, it is not thought to be involved in repolarizing neurons after single action potentials, but instead to contribute to adjustments in their firing frequency by raising the threshold membrane potential that must be reached before activation of voltage-gated channels. Accordingly, treatment of primary rat hippocampal neurons in culture with A-769662 caused hyperpolarizing shifts in gating that were qualitatively similar to those observed in HEK-293 cells expressing Kv2.1. This effect appeared to be mediated by Kv2.1, because it was abolished by allowing an anti-Kv2.1 antibody to diffuse in from the patch pipette. Moreover, administration of active, thiophosphorylated α2β2γ1 complexes via the patch pipette reduced the firing of action potentials in the neurons as predicted, whereas inactive control complexes had no effect (Fig. 3) (Ikematsu et al., 2011). Therefore, AMPK not only regulates metabolic homeostasis of neurons, but also neuronal activity.
The firing of action potentials, together with downstream postsynaptic events, are estimated to account for up to 80% of all energy turnover in the gray matter of the rodent brain (Attwell & Laughlin, 2001). Since Kv2.1 is widely expressed, particularly in pyramidal neurons in the hippocampus and cortex (Misonou, Mohapatra, & Trimmer, 2005), its activation by AMPK could be a cell-autonomous mechanism to conserve energy by reducing membrane excitability and firing of action potentials in response to energy stress. It is interesting to speculate that this mechanism might also contribute during sleep, during which central energy reserves are replenished. Indeed, knockdown of AMPK in the central nervous system of the fruit fly Drosophila melanogaster led to disturbances in sleep patterns and to poorer recovery from sleep deprivation (Nagy et al., 2018).
It is now clear that AMPK also has cell- and circuit-specific functions in the nervous system that are involved in key physiological processes operating at the whole-body level, such as balancing energy intake (i.e. feeding) with energy expenditure, and adjustment of oxygen supply from the lungs (i.e. breathing) according to demand. These specific outputs of AMPK, which are discussed next, may be achieved via cell-specific expression of hormone receptors and AMPK subunit isoforms, and of unique sets of AMPK targets, such as specific ion channels. This can give rise to differential sensitivities to stresses such as glucose or oxygen deprivation, or to hormones and neurotransmitters that activate AMPK via the CaMKK2 pathway, according to location.
The Role of Hypothalamic AMPK in Regulating Appetite and Feeding Behavior
Among the most well established roles of AMPK in the nervous system is in the regulation of appetite and food intake. Feeding is known to be promoted by stimulation of neurons located in the arcuate nucleus of the hypothalamus that express agouti-related protein (AGRP) and neuropeptide Y (NPY), and to be inhibited by neurons in the same anatomical location that express pro-opiomelanocortin (POMC) and cocaine-and-amphetamine-regulated transcript (CART). NPY/AGRP neurons increase food intake and decrease energy expenditure by antagonizing POMC action on melanocortin receptors in neurons of the paraventricular nucleus. The first indication for a role for AMPK came from findings that it was activated in the hypothalamus of rats by treatment in vivo with the orexigenic hormone ghrelin, and inhibited by treatment with the anorexigenic hormone leptin (Andersson et al., 2004); other orexigenic mediators, such as cannabinoids, were subsequently also found to activate AMPK (Kola et al., 2005). Moreover, injection of the AMPK activator AICA riboside into the hypothalamus led to increases in food intake (Andersson et al., 2004). Although AICA riboside is now known to have many “off-target,” AMPK-independent effects (see the section “Activation of AMPK by Compounds that Bind in the ADaM Site” above), the conclusion that its effects on feeding were AMPK-mediated was strengthened by findings that ectopic expression in mouse hypothalamus of inactive mutants of AMPK-α1 and -α2 (which lack kinase activity, but exert a dominant negative effect by competing with endogenous α subunits for binding to β ανδ γ subunits) repressed food intake and body-weight gain. Conversely, expression of an activated γ1 mutant (which behaves as if AMP is bound even when it is not) had the opposite effects. Moreover, leptin was found to decrease the activity of AMPK complexes containing α2, although not α1, in the hypothalamus (Minokoshi et al., 2004). The phenotypes of knocking out AMPK-α2 in AGRP/NPY and POMC/CART neurons, respectively, were also initially consistent with a role for AMPK in appetite control, with the former being lean while the latter were obese. However, in both cases the effects were rather modest and age-dependent, and there were no detectable changes in food intake in the AGRP/NPY knockouts, while the POMC/CART knockouts still responded normally to leptin treatment in vivo in terms of food intake and body weight, and to leptin and insulin (another anorexigenic hormone) in electrophysiological studies (Claret et al., 2007). Interestingly, a proportion of both AGRP/NPY and POMC/CART neurons respond to glucose deprivation with hyperpolarization and a consequent reduction in spike frequency, although in neither case was this evident when AMPK-α2 was knocked out in these neurons. Thus, although AMPK does not appear to be required for the response to leptin and insulin in these specific (AGRP/NPY or POMC/CART) neurons, it does seem to be required for glucose-sensing (Claret et al., 2007). The latter is intriguing given the evidence that AMPK can sense glucose via a non-canonical mechanism (C. S. Zhang et al., 2017).
The apparent lack of effect of knocking out AMPK in AGRP/NPY or POMC/CART neurons on food intake (Claret et al., 2007) may be because AMPK is required not in these actual neurons, but in other neurons acting immediately upstream or downstream. In one very interesting study, AGRP/NPY neurons were identified by their fluorescence in brain slices derived from transgenic mice expressing a fluorescent protein fused to NPY, and the activity of presynaptic neurons was assessed by measuring miniature excitatory postsynaptic currents (mEPCs) in these cells, in the presence of tetrodotoxin to suppress firing of action potentials (Yang et al., 2011). Interestingly, treatment of brain slices from fed mice with the orexigenic hormone ghrelin increased mEPCs in the AGRP/NPY neurons to the level seen in fasted mice. Studies with pharmacological agents suggested that this was mediated by stimulation of the ghrelin receptor Ghsr1, which is coupled to intracellular release of via the G protein Gq/G11. This would initiate a -dependent activation of AMPK in the presynaptic neurons via the CaMKK2 pathway. Satisfyingly, there was already evidence that CaMKK2 is involved in the response to orexigenic signals, based on studies using the CaMKK2 inhibitor STO-609 and global mouse knockouts of CaMKK2 (Anderson et al., 2008). Although the fascinating study by Yang et al. (2011) relied heavily on different pharmacological agents, some of which might have off-target effects, it does provide a plausible model to explain the role of AMPK in feeding and appetite control in specific neurons of the hypothalamus. One proposal to explain the orexigenic effects of ghrelin is that the output from hypothalamic neurons, downstream of the CaMKK2-AMPK pathway, is modulated by reductions in malonyl-CoA levels due to inhibition of acetyl-CoA carboxylase by AMPK, with consequent activation of carnitine-palmitoyl transferase-1C (CPT1C) (Lopez et al., 2008) and increased levels of ceramides (Ramirez et al., 2013).
Intriguingly, recent work (Okamoto et al., 2018) shows that expression of constitutively active AMPK in the paraventricular nucleus of the hypothalamus led to a preference for carbohydrate over fat in food-choice experiments conducted on mice. This effect appeared to be mediated through activation by AMPK of CPT1C within a subset of corticotrophin-releasing hormone-positive neurons in the rostral region of the paraventricular nucleus, promoting -dependent activation of these neurons. As we will see, regulation of physiological processes by AMPK through such cell- and system-specific outputs is common to other regions of the brain.
The Role of Hypothalamic AMPK in Regulating Energy Expenditure/Thermogenesis
In another region of the hypothalamus, the ventromedial nucleus (VMH), AMPK appears to be involved in the regulation of peripheral energy expenditure rather than energy intake, by regulating the firing of sympathetic nerves that stimulate fatty acid oxidation and hence heat production (thermogenesis) in brown adipose tissue. Thus, direct administration to the VMH by intracerebroventricular injection of the female sex hormone estradiol (Martinez de Morentin et al., 2014), the thyroid hormone T3 (Lopez et al., 2010), the GLP-1 receptor agonist liraglutide (Beiroa et al., 2014), or Bone Morphogenetic Protein-8B (BMP8B) (Martins et al., 2016; Whittle et al., 2012) all reduced phosphorylation of AMPK in the VMH and increased thermogenesis. In the cases of estradiol and T3 this was associated with increases in activity of sympathetic nerves and weight loss. Moreover, injection of adenoviruses expressing an activated mutant of AMPK reduced the weight loss associated with either hormone treatment (Lopez et al., 2010; Martinez de Morentin et al., 2014). The effects of T3 in the VMH were also mediated in part by stimulation of the vagus nerve to promote lipogenesis in the liver (Martinez-Sanchez et al., 2017). The effects on thermogenesis, and hence energy expenditure, appear to be mediated by inhibition of AMPK complexes containing the α1 isoform in neurons of the VMH expressing Steroidogenic Factor-1 (SF1), because mice with a specific knockout of AMPK-α1 in these neurons recapitulated the effect of central T3 (Martinez-Sanchez et al., 2017). These mice display increased thermogenesis and are resistant to diet-induced obesity (Seoane-Collazo et al., 2018).
Interestingly, while AMPK deficiency in sympathetic (catecholaminergic) neurons might increase thermogenesis and weight loss (and block the hypoxic ventilatory response, see next subsection, “The Role of AMPK in Regulating Breathing and Oxygen Supply”), it does not appear to markedly impact systemic arterial blood pressure regulation during normoxia or hypoxia, which is determined in great part by increased sympathetic outflow (MacMillan & Evans, 2018). The only way to reconcile this is if pathways leading to sympathetic control of thermogenesis differ from those involved in control of blood pressure, another example of cell- and system-specific outputs.
The Role of AMPK in Regulating Breathing and Oxygen Supply
Recent studies have confirmed earlier suggestions (Evans, 2006) that AMPK is critical for the regulation of central respiratory networks that accelerate breathing during hypoxia and thus increase the supply of oxygen to cells throughout the body to maintain levels of ATP generation (Mahmoud et al., 2015). The hypoxic ventilatory response (HVR)—increased breathing in response to hypoxia—is driven by afferent inputs from oxygen-sensitive chemoreceptors to the central respiratory pattern generators of the brainstem, which are distributed bilaterally in the ventrolateral medulla and generate ventilatory rhythm (Evans, Mahmoud, Moral-Sanz, & Hartmann, 2016; Smith, Ellenberger, Ballanyi, Richter, & Feldman, 1991). The HVR supports, for example, increased oxygen supply during ascent to altitude and maintenance of ventilatory function as we sleep.
AMPK is intimately coupled to mitochondrial metabolism through cellular AMP/ATP and ADP/ATP ratios, and mitochondria of oxygen-sensing cells appear to be uniquely sensitive to changes in oxygen supply because they express isoforms of cytochrome C oxidase whose kinetics respond to changes in PO2 within the physiological range (Mills & Jobsis, 1970, 1972). This is due to the constitutive expression (Aras et al., 2013; Huttemann et al., 2012; Zhou, Chien, Kaleem, & Matsunami, 2016) of two nuclear-encoded, atypical subunits of the mitochondrial electron transport chain, NDUFA4L2 (Tello et al., 2011) and COX4I2 (Fukuda et al., 2007; Huttemann, Kadenbach, & Grossman, 2001). This contrasts with other cell types, where expression of NDUFA4L2 and COX4I2 expression are generally low, although they increase during prolonged hypoxia (Fukuda et al., 2007; Huttemann et al., 2001). Constitutive expression of NDUFA4L2 and COX4I2 in oxygen-sensing cells confers, in part, their capacity to monitor changes in oxygen supply (Sommer et al., 2017), because allosteric modulation of cytochrome C oxidase is mediated in a subtype-specific manner, with COX4I1 but not COX4I2 conferring inhibition by ATP (Horvat, Beyer, & Arnold, 2006; Huttemann et al., 2001). When COX4I2 is expressed, the rate of oxygen consumption (and hence mitochondrial ATP synthesis) will therefore not increase as ATP levels fall during hypoxia (Aras et al., 2013; Fukuda et al., 2007; Horvat et al., 2006; Kocha et al., 2015). The consequent fall in ATP will in turn cause increases in the AMP:ATP ratio via the adenylate kinase reaction, thus activating AMPK.
Consistent with a role for AMPK in regulating the response of oxygen-sensing cells that adjust breathing patterns during hypoxia, the HVR is markedly attenuated by conditional deletion of both AMPK-α1 and -α2 using Cre recombinase expression from the tyrosine hydroxylase promoter [in fact, exposure of these mice to hypoxia triggers hypoventilation and apnea (cessation of breathing), rather than hyperventilation as in the wild type] (Mahmoud et al., 2015). Note that catecholaminergic cells expressing tyrosine hydroxylase span the entire respiratory network, including the type I cells of carotid bodies as well as the brainstem.
The primary peripheral arterial chemoreceptors of mammals are the carotid bodies, of which the type I cells represent the archetypal oxygen-sensing cells. The general consensus has been that it is the afferent input responses of carotid bodies that deliver the entire ventilatory response to falls in arterial PO2 (Prabhakar, 2000). Challenging this, however, AMPK deletion attenuated the HVR during mild and severe hypoxia without affecting these afferent input responses. This is consistent with findings that two compounds that activate AMPK via different mechanisms, i.e. AICA riboside and A-769662, do not precisely mimic the effects of hypoxia or induce pronounced activation of carotid body type I cells (Kim, Kang, Martin, Kim, & Carroll, 2014). Thus, peripheral chemosensors are not the sole arbiters of the HVR. This has also been suggested by investigations on the evolution of ventilatory control systems. Intriguingly, oxygen-sensing (and a component of the HVR) occurs at the level of the caudal brainstem in amphibians, in which both the location and influence of the primary peripheral chemosensors changes during metamorphosis from gill-breathing tadpole to lung-assisted, air-breathing adult (Porteus, Hedrick, Hicks, Wang, & Milsom, 2011). It has been proposed (Evans et al., 2016) that evolution periodically led to the reconfiguration of peripheral chemoreceptor inputs (Porteus et al., 2011) about a common, ancestral sensor of hypoxia within the caudal brainstem, which effects signal integration and thus acts as the gatekeeper of respiratory adjustments during hypoxia. In short, the HVR may be determined by the coordinated action of the carotid body and a hypoxia-responsive circuit within the brainstem (Evans et al., 2016; Gourine & Funk, 2017; Mahmoud et al., 2015; Teppema & Dahan, 2010).
Until recently, little emphasis has been placed on the role of hypoxia-sensing by the brainstem, perhaps because the HVR is so effectively abolished by resection of the carotid sinus nerve in humans (Wade, Larson, Hickey, Ehrenfeld, & Severinghaus, 1970). However, brainstem hypoxia induces an HVR even when it receives normoxic carotid body afferent inputs (Curran et al., 2000), and directly activates subsets of catecholaminergic neurons within the nucleus tractus solitarius (NTS) and rostral ventrolateral medulla that may support partial recovery of the HVR, in a variety of animal models (Teppema & Dahan, 2010). Consistent with the effect of AMPK deletion on the HVR, dysfunction of these neurons precipitates the hypoventilation and apnea associated with Rett syndrome, which is exacerbated during hypoxia (Roux & Villard, 2010). Moreover, it is evident that COX4I2 may, as in carotid body type I cells, be constitutively expressed by certain CNS neurons (Horvat et al., 2006), rendering mitochondrial oxidative phosphorylation sensitive to falls in local PO2. AMPK activation could thus be triggered in a specialized subset of brainstem neurons during hypoxia to support the delivery of increased respiratory drive, required to protect against hypoventilation and apnea. Supporting this, examination of brainstem function in AMPK-α1/α2 knockout mice by functional magnetic resonance imaging (fMRI) identified reduced activation of discrete dorsal and ventral nuclei of the caudal brainstem, despite the fact that carotid body afferent input responses were retained (Mahmoud et al., 2015). The location of the dorsal nucleus aligns with areas of the NTS that are activated during hypoxia, and represents the primary site of receipt of carotid body afferent inputs (Teppema & Dahan, 2010), well placed for signal integration. Here AMPK deletion may attenuate activation during hypoxia of C2 (adrenergic) neurons and/or A2 (noradrenergic) neurons (both being tyrosine hydroxylase-positive) proximal to the midline and the area postrema (Mahmoud et al., 2015). Notably, A2 neurons provide afferent inputs to determine, together with the carotid body, activation by hypoxia of A1/C1 neurons within the ventrolateral medulla (Guyenet, 2014), the position of which aligns well with the ventral active region identified by fMRI analysis (Mahmoud et al., 2015). Through these projections of the NTS, AMPK could thus support the HVR (Evans et al., 2016) by either indirect or direct modulation of the respiratory central pattern generators (Smith, Abdala, Borgmann, Rybak, & Paton, 2013; Smith et al., 1991) and by coordinating functional hyperemia (Bucher et al., 2014). Because carotid body afferent discharge remains exquisitely sensitive to falls in PO2, and ventilatory responses to hypercapnia remained unaffected even during severe hypoxia, it is unlikely that AMPK deficiency compromises the capacity during hypoxia either for activation of the peripheral carotid body type I cells or brainstem catecholaminergic neurons that govern the ventilatory response to hypercapnia, for exocytosis or for effective delivery of increased respiratory drive (Evans et al., 2016). Therefore, the mechanism by which AMPK supports the HVR is most likely neurogenic and highly localized, since AMPK deficiency in smooth muscles does not affect the HVR or systemic arterial blood pressure regulation during hypoxia, while the latter (but not the former) remains unaltered following AMPK deletion in catecholaminergic neurons (MacMillan & Evans, 2018). This represents a further example of cell- and system-specific actions of AMPK.
Accepting the possibility of a role in signal integration at the NTS, the working hypothesis (Fig. 4) is that AMPK activation by the canonical, AMP/ADP-dependent pathway may provide the capacity for sensing local hypoxic stress, i.e. decreased ATP supply, in a manner that could be effectively coupled to increased ATP usage consequent to afferent inputs from peripheral chemoreceptors to the NTS, thus increasing overall ATP demand in the receiving, activated neurons (Evans et al., 2016). Intriguingly, it is AMPK-α1 but not AMPK-α2 that supports the HVR (Mahmoud et al., 2016). Relevant to this, in high-altitude populations living in the Andes the gene encoding AMPK-α1 (PRKAA1) appears to have been influenced by natural selection through single nucleotide polymorphisms (Bigham et al., 2014).
Conclusions and Perspectives
AMP-activated protein kinase (AMPK) is almost universally expressed in cells of both unicellular and multicellular eukaryotes (Lin & Hardie, 2017). In all cells, it appears to serve the cell-autonomous role of adjusting function to ensure that the supply and demand for ATP are matched, even during fluctuations in cellular energy status and/or the availability of nutrients such as glucose. However, as multicellular organisms evolved, the roles of AMPK appear to have diversified so that it also came to serve both cell- and system-specific roles in whole body physiology; examples of this (summarized in Fig. 5) include its roles in the hypothalamus in adjusting whole-body energy balance by regulating energy intake (appetite/feeding) and expenditure, and its role in regulating ventilation of the lung during hypoxia. Mammalian AMPK is able to serve both cell-autonomous and systemic roles, in part because it exists as heterotrimers formed by up to 12 combinations of α, β, ανδ γ subunit isoforms, thus generating much potential diversity in subcellular location and function. The multiple subunit isoforms that allow this diversity appear to have arisen during the two rounds of whole genome duplication that occurred during the early development of vertebrates, with signaling proteins like AMPK being highly enriched among those genes where multiple subunit isoforms have been retained (Ross, MacKintosh et al., 2016). During evolution, the AMPK system has also been able to serve new cell- and system-specific roles by acquiring the ability to phosphorylate and regulate cell-specific proteins, such as the ion channels, receptors, and transporters that are found in neural tissue.
Recent studies in the DGH laboratory have been supported by the Wellcome Trust (204766/Z/16/Z) and Cancer Research UK (C37030/A15101). Recent studies in the AME laboratory have been supported by a British Heart Foundation Programme Grant (29885) and a Wellcome Trust Programme Grant (081195).
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