<scp>AMP</scp>‐activated protein kinase: a key regulator of energy balance with many roles in human disease

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that regulates cellular and whole body energy balance. A recent crystal structure has illuminated the complex regulatory mechanisms by which AMP and ADP cause activation of AMPK, involving phosphorylation by the upstream kinase, LKB1. Once activated by falling cellular energy status, AMPK activates catabolic pathways that generate ATP, while inhibiting anabolic pathways and other cellular processes consuming ATP. AMPK is implicated in many human diseases. Mutations in the γ2 subunit cause heart disease due to excessive glycogen storage in cardiac myocytes, leading to ventricular pre-excitation. AMPK-activating drugs reverse many of the metabolic defects associated with insulin resistance, and recent studies suggest that the insulin-sensitizing effects of the widely used anti-diabetic drug, metformin, are mediated by AMPK. The upstream kinase LKB1 is a tumor suppressor, and AMPK may exert many of its anti-tumor effects. AMPK activation promotes the oxidative metabolism typical of quiescent cells, rather than the aerobic glycolysis observed in tumor cells and cells involved in inflammation, explaining in part why AMPK activators have both anti-tumor and anti-inflammatory effects. Salicylate (the major in vivo metabolite of aspirin) activates AMPK, and this could be responsible for at least some of the anti-cancer and anti-inflammatory effects of aspirin. In addition to metformin and salicylates, novel drugs that modulate AMPK are likely to enter clinical trials soon. Finally, AMPK is implicated in viral infection: down-regulation of AMPK during hepatitis C virus infection appears to be essential for efficient viral replication.

AMPK appears to exist throughout eukaryotes as heterotrimeric complexes composed of a catalytic α subunit and regulatory β and γ subunits [ 5 ] ( Fig. 1 , top). Each subunit occurs in mammals as multiple isoforms (α1, α2; β1, β2; γ1, γ2, γ3) encoded by distinct genes. The site that upstream kinases phosphorylate to cause AMPK activation was identified within the rat α2 isoform as Thr172 [ 6 ]. Phosphorylation of this site, and the critical site on the downstream target acetyl-CoA carboxylase (Ser79 in rat ACC1 [ 7 ]), both usually monitored using phosphospecific antibodies, are now widely used as biomarkers for AMPK activation. What is the physiological significance of activation of AMPK by AMP? The major source of AMP in the cell is the reaction catalyzed by adenylate kinase (2ADP ↔ ATP + AMP), which appears to be maintained close to equilibrium in most eukaryotic cells. In unstressed cells, catabolism maintains the ATP:ADP ratio at around 10:1, and this drives the adenylate kinase reaction towards ADP, so that AMP is maintained at low levels. However, if the cells experience a metabolic stress that causes the ATP:ADP ratio to fall, the adenylate kinase reaction will tend to be displaced towards AMP. Because it starts at such a low level, the changes in AMP concentration in stressed cells are always much larger than the changes in ATP or ADP [ 8 ]. Cellular AMP concentrations are thus sensitive indicators of energy stress. The principal upstream kinase phosphorylating Thr172 was identified in 2003 to be a complex containing the protein kinase LKB1 [ 9 – 11 ]. This was an exciting discovery because LKB1 had previously been identified as the product of a tumor suppressor gene mutated in an inherited susceptibility to cancer, Peutz-Jeghers syndrome [ 12 ], although its downstream targets had not been identified. These findings introduced the first clear link between AMPK and cancer, which is discussed further below. Binding of AMP to AMPK causes &gt;10-fold allosteric activation, and also promotes net phosphorylation of Thr172, both by stimulating phosphorylation by LKB1, and by inhibiting dephosphorylation by protein phosphatases [ 8 ]. The latter effect of AMP, but not the other two, is mimicked by ADP [ 13 ], albeit only at 10-fold higher concentrations [ 8 ]. All three effects are antagonized by ATP, but concentrations of AMP observed in stressed cells cause allosteric activation and promote phosphorylation even in the presence of physiological concentrations of ATP [ 8 ]. Thr172 can also be phosphorylated by the Ca 2+ /calmodulin-dependent kinase kinases, especially CaMKKβ [ 14 – 16 ], which represents an alternate upstream pathway by which AMPK can be activated by increases in intracellular Ca 2+ in the absence of changes in AMP. There are conflicting findings concerning whether phosphorylation by CaMKKβ is promoted by AMP [ 8 , 17 ], but since AMP binding inhibits Thr172 dephosphorylation, low concentrations of the two signals can act in an additive manner in any case [ 18 ]. Activation of AMPK by CaMKKβ is now known to occur in many physiological situations, including hippocampal neurons subject to depolarization [ 14 ], T cells activated by antigen [ 19 ], and cells treated with agonists that activate G protein-coupled receptors linked (via G q /G 11 ) to release of intracellular inositol trisphosphate (IP3) and hence Ca 2+ . The latter include thrombin acting via protease-activated receptor-1 (PAR-1) in endothelial cells [ 20 ], and ghrelin acting via growth hormone secretagogue receptor-1 (GHSR1) in hypothalamic neurons [ 21 ]. To conclude this section, binding of AMP to AMPK promotes its activation by three complementary mechanisms: (i) allosteric activation; (ii) promoting phosphorylation of Thr172 by LKB1; (iii) inhibiting dephosphorylation of Thr172 by protein phosphatases, with only the third of these being mimicked by ADP. Phosphorylation of Thr172 can also occur in response to an increase in intracellular Ca 2+ . In the next section, I will discuss how structural studies on AMPK have illuminated this complex regulatory mechanism.

A full discussion of the known downstream targets for AMPK is beyond the scope of this review, and readers are referred to a previous review [ 5 ]. However, a summary of some of these, and the pathways they regulate, is shown in Figure 2 . Consistent with the findings that it is switched on by ATP depletion, and has a key role in maintaining cellular energy balance, AMPK promotes catabolic pathways that generate ATP, while inhibiting anabolic pathways involved in cell growth, and other processes that consume ATP. It also causes a cell cycle arrest in G1 phase [ 28 ], in part by phosphorylating MDM4 (MDMX), part of the E3 ubiquitin ligase complex that regulates p53 turnover [ 29 ]. Cell cycle arrest by AMPK makes sense, since DNA replication (during S phase) and mitosis (M phase) are both energy-requiring processes. In general, AMPK achieves its effects both acutely via direct phosphorylation of metabolic enzymes or regulatory proteins involved in the process being regulated, and in the longer term via effects on gene expression. I will discuss here just a few examples of downstream effects of AMPK that will become relevant later in the review: 1) AMPK accelerates glucose uptake to drive catabolism of glucose during muscle contraction, which it does, at least in part, by phosphorylating the Rab-GTPase activator protein (Rab-GAP), TBC1D1 ( Fig. 3 ). TBC1D1 is a member of the same family as TBC1D4 (also known as AS160), whose phosphorylation by the insulin-activated kinase PKB/Akt plays a key role in insulin-stimulated glucose uptake [ 30 ]. While TBC1D4 appears to be the predominant form in adipocytes, TBC1D1 is the predominant form in most muscle types [ 31 ]. Both TBC1D1 and TBC1D4 bind to intracellular vesicles containing the glucose transporter GLUT4, and their Rab-GAP domains promote the GTPase activity of members of the Rab family of small G proteins, thus maintaining them in their inactive, GDP-bound state. Phosphorylation of TBC1D1 at two sites, Ser237 and Thr596, promotes its binding to 14-3-3 proteins, which appears to repress its Rab-GAP activity. Rabs are thus converted to their active GTP-bound forms, and they then promote trafficking of the GLUT4-containing intracellular vesicles to the plasma membrane and thus increased glucose uptake [ 32 – 34 ]. While AMPK can phosphorylate both Ser237 and Thr596 in cell-free assays, available evidence suggests that only Ser237 may be phosphorylated by AMPK in intact cells, with Thr596 being phosphorylated by other kinases including the insulin-stimulated kinase, Akt [ 32 ]. 2) AMPK phosphorylates and inactivates both isoforms of acetyl-CoA carboxylase, ACC1 and ACC2, lowering the concentration of their reaction product, malonyl-CoA. Since malonyl-CoA is both an intermediate in fatty acid synthesis and an inhibitor of fatty acid uptake into mitochondria via the transport system involving carnitine:palmitoyl transferase-1 (CPT1), this has the dual effect of inhibiting fatty acid synthesis and enhancing fatty acid oxidation ( Fig. 4 ) [ 35 , 36 ]. 3) AMPK enhances mitochondrial biogenesis by effects on the transcriptional co-activator PGC-1α. This may involve direct phosphorylation of PGC-1α [ 37 ], or activation of the lysine deacetylase SIRT1, which deacetylates and activates PGC-1α [ 38 ]. 4) As well as its direct effects on acetyl-CoA carboxylase, AMPK inhibits fatty acid synthesis in the longer term by phosphorylating SREBP-1c, inhibiting its ability to promote transcription of lipogenic enzymes [ 39 ]. AMPK also inhibits cholesterol and phospholipid/triacylglycerol synthesis, the former by phosphorylating and inactivating HMG-CoA reductase (HMGR) [ 35 ], and the latter by inactivating glycerol phosphate acyl transferase (GPAT; it remains unclear whether this is via direct phosphorylation) [ 40 ]. 5) AMPK inhibits two other major biosynthetic pathways required for cell growth, i.e. protein and rRNA synthesis. It inhibits the former mainly by inhibiting the mechanistic target-of-rapamycin complex-1 (mTORC1), by phosphorylating both the upstream regulator TSC2 [ 41 ] and the mTORC1 subunit, Raptor [ 42 ] ( Fig. 5 ). AMPK also inhibits rRNA synthesis by phosphorylating TIF-1A, a transcription factor for RNA polymerase-1 [ 43 ].

Type 2 diabetes occurs when increased release of insulin from the β cells of the pancreas can no longer compensate for insulin resistance, with the latter being strongly associated with increased triacylglycerol content, particularly in liver and skeletal muscle. One hypothesis is that insulin resistance occurs in obese individuals when their capacity to store triacylglycerols in adipose tissue is exceeded, leading to their accumulation in liver and muscle. This is supported by studies of humans with lipodystrophy, who lack adipose tissue and store triacylglycerols instead in liver and muscle, which then become severely insulin resistant [ 61 ]. Despite the association between insulin resistance and triacylglycerols, the real culprit may be diacylglycerols whose levels are elevated at the same time. Diacylglycerols activate novel isoforms of protein kinase C (PKCθ in muscle and PKCε in liver) that then appear to down-regulate the insulin signaling pathway [ 61 ]. This represents one explanation of the insulin resistance associated with obesity, although other factors such as increased inflammation [ 62 ] or endoplasmic reticulum stress [ 63 ] may also play a part. However, AMPK activation may be able to reverse both of these as well, since it has anti-inflammatory effects (discussed below) while inhibition of protein synthesis by AMPK has the potential to relieve endoplasmic reticulum stress. By promoting the oxidation of fatty acids and inhibiting synthesis of fatty acids and triacylglycerols, treatments that activate AMPK would be expected to reduce lipid stores in liver and skeletal muscle and hence improve insulin sensitivity. By promoting glucose uptake by skeletal muscle [ 36 , 64 ] and inhibiting gluconeogenesis in the liver [ 65 , 66 ], they might also be expected to ameliorate the hyperglycemia associated with type 2 diabetes more directly. Indeed, pharmacological agents that activate AMPK do have many of these effects in vivo. The first to be tested was AICAR, an adenosine analogue that is taken up into cells via adenosine transporters and converted to the equivalent nucleotide, ZMP, which mimics all of the activating effects of AMP on AMPK [ 35 ]. AICAR treatment was found to reverse many metabolic abnormalities in animal models of obesity and insulin resistance, such as fa/fa rats [ 67 , 68 ], ob/ob mice [ 69 ], and rats fed a high-fat diet [ 70 ]. At about the same time, it was reported that the biguanide drug metformin, currently the first choice drug for type 2 diabetes that is prescribed to &gt;100 million patients worldwide, also activated AMPK both in intact cells and in vivo [ 71 ]. Metformin activates AMPK indirectly by inhibiting the mitochondrial respiratory chain [ 50 ], but A-769662, a direct AMPK activator that binds at the same site as 991 ( Fig. 1 ), also has similar in vivo effects in animals; it increases fatty acid oxidation in rats, and decreases plasma glucose, body weight gain, plasma and liver triacylglycerols, and hepatic expression of gluconeogenic and lipogenic enzymes in ob/ob mice [ 72 ]. Similarly, the natural product berberine used in traditional Chinese medicine, which activates AMPK by inhibiting the respiratory chain in a similar manner to metformin [ 50 ], also reduces body weight and improves glucose tolerance in db/db mice, while reducing body weight and plasma triacylglycerols, and improving insulin sensitivity, in rats fed a high-fat diet [ 73 ]. Since (with the exception of A-769662) these agents activate AMPK indirectly, it has been important to establish whether their favorable metabolic effects are indeed mediated by AMPK. Arguing against this, the ability of AICAR or metformin to reduce glucose production by hepatocytes in vitro, or of metformin to improve glucose tolerance after short-term (30 minute) treatment in vivo, was unaffected by a knockout of both isoforms of the AMPK catalytic subunit (α1 and α2) in the liver [ 74 ]. The AMPK-independent effects of AICAR may be due to effects of its intracellular metabolite, ZMP, to directly inhibit the gluconeogenic enzyme fructose-1,6-bisphosphatase [ 75 ] and/or to lower cyclic AMP (which promotes transcription of gluconeogenic enzymes) due to inhibition of adenylate cyclase [ 76 ], while the AMPK-independent effects of metformin in part may a consequence of ATP depletion, which was more severe in cells lacking AMPK [ 74 ]. Despite these negative findings, there is good evidence that the longer term insulin-sensitizing effects of metformin are mediated by AMPK. This comes from recent studies of “double knock-in” (DKI mice) in which both endogenous genes encoding acetyl-CoA carboxylase (ACC1 and ACC2) were replaced by genes encoding single amino acid substitutions that eliminate the AMPK sites [ 77 ]. As expected, the ACC1/ACC2 activities, and the cellular content of the product malonyl-CoA, were elevated in the livers of these mice, and fatty acid oxidation was reduced. This was in turn associated with incr

There is increasing evidence that AMPK has anti-inflammatory effects, and that this may be mediated by its metabolic actions [ 97 ]. Unactivated cells of the immune system, including dendritic cells, neutrophils and T cells, utilize mainly oxidative metabolism (including fatty acid oxidation) to generate ATP. However, once activated, they usually switch to aerobic glycolysis, analogous to the metabolic changes that occur in tumor cells. In dendritic cells this switch is associated with reduced AMPK activation, is inhibited by pharmacological activation of AMPK, and is promoted by AMPK down-regulation [ 98 ]. Similar effects are observed in macrophages: classically activated (M1) macrophages with a pro-inflammatory role utilize aerobic glycolysis, whereas alternatively activated (M2) macrophages, more involved in the resolution of inflammation, tend to utilize oxidative metabolism instead. Studies using macrophages where AMPK has been down-regulated suggest that AMPK normally attenuates production of inflammatory cytokines [ 99 , 100 ]. The idea that the anti-inflammatory actions of AMPK are due to its metabolic actions was strengthened by studies with mouse knockouts of AMPK-β1, the predominant β subunit isoform expressed in macrophages. AMPK-β1 deficient macrophages displayed reduced ACC phosphorylation and mitochondrial content, and reduced rates of fatty acid oxidation that promoted the accumulation of pro-inflammatory diacylglycerols. This triggered M1 skewing in vivo in macrophages derived from both bone marrow and adipose tissue. Activation of AMPK with A-769662 increased fatty acid oxidation in wild type but not β1-deficient macrophages, and the effects of A-769662 to suppress inflammation were impaired if fatty acid oxidation was blocked [ 101 ]. These findings suggest that a major component of the anti-inflammatory action of AMPK is mediated via its effects on fatty acid oxidation. The role of AMPK in macrophages has also been studied using mice with global or myeloid-specific knockouts of AMPK-α1, the catalytic subunit isoform expressed in macrophages. Regeneration of muscle in response to muscle injury was defective in these mice, and this was associated with reduced skewing towards M2 macrophages [ 102 ]. Interestingly, the author’s laboratory has shown that AMPK is directly activated by salicylate, the natural product from which aspirin (acetyl salicylate) was derived [ 103 ]. Salicylate and A-769662 appear to bind to the same site on AMPK; both are selective activators of β1-containing complexes, and they increase whole body fatty acid oxidation in wild type but not β1 knockout mice, showing that increased fat oxidation is mediated by AMPK activation in vivo. Although AMPK is not directly activated by aspirin itself [ 103 ], aspirin is rapidly broken down to salicylate. Indeed, following oral administration of aspirin the plasma half-life and peak concentration of its breakdown product, salicylate, are orders of magnitude greater than those of aspirin itself. Although blockade of prostanoid biosynthesis, via inhibition of cyclo-oxygenases, has been considered to be the main mechanism of action of aspirin, there has always been a paradox in that, while aspirin and salicylate have similar anti-inflammatory potencies, salicylate is a very poor inhibitor of cyclo-oxygenase [ 104 , 105 ]. Although significant AMPK activation requires at least 1 mM salicylate, such concentrations are reached in humans taking high doses of aspirin or another salicylate derivative, salsalate, for rheumatoid arthritis. This raises the intriguing possibility that at least some of the anti-inflammatory effects of salicylate-based drugs may be mediated by AMPK. Intriguingly, the regular use of aspirin is also associated with reduced incidence of cancer [ 106 ], although whether this effect is mediated by AMPK remains uncertain.

An important consideration in the development of novel AMPK activating drugs is that they would have to be significantly more effective than existing agents such as metformin or salicylates, which are both inexpensive and have a good safety record. One potential shortcoming with metformin is that its effects appear to be largely confined to the liver. Since it is a cation that is not readily cell-permeable, the uptake of metformin into cells requires membrane transporters of the organic cation transporter family, such as OCT1 [ 110 ]. Because hepatocytes are exposed to higher concentrations of orally delivered metformin via the portal vein than most other cells [ 71 ], and also express high levels of OCT1, effects of metformin in vivo may be largely restricted to the liver [ 110 ]. In particular, the drug may not cause much activation of AMPK in skeletal muscle because of low peripheral drug concentrations and low OCT1 expression. The doses of metformin that can be administered to humans are limited by gastrointestinal side effects, which are likely to be AMPK-independent and may be caused by inhibition of the respiratory chain and consequent lactate production in the liver or gut [ 111 , 112 ]. The more hydrophobic biguanide phenformin is more cell-permeable than metformin, and therefore much less dependent on OCT1 for cellular uptake [ 50 ]. Although formerly used for treatment of Type 2 diabetes, it was withdrawn in most countries in the 1970s due to cases where persons taking the drug developed lactic acidosis (with hindsight, an unsurprising side effect since both biguanides inhibit the respiratory chain and that is how they activate AMPK [ 50 ]). However, the frequency of lactic acidosis with phenformin, while life-threatening and around 20-fold more frequent than with metformin, was still rare (≈60 cases per 100,000 patient-years) [ 112 ]. This small risk might be more acceptable if the drug was being used to treat cancer, rather than diabetes [ 88 ]. The indirect AMPK activator AICAR, which is converted into the AMP mimetic ZMP within the cell [ 35 ], has the generic drug name acadesine and has been tested in humans [ 113 ]. However, it is not orally available and has to be injected and, as discussed above, has clear “off-target” AMPK-independent effects in mice, so its potential as an AMPK-activating drug in humans seems limited. New direct AMPK activators that are readily cell-permeable, exemplified by 991 and A-769662 [ 22 ], may have additional benefits over metformin by activating AMPK and promoting glucose uptake in organs other than the liver, especially skeletal muscle. These compounds are being developed by pharmaceutical companies, and studies of their potential toxicity are not yet available within the public domain. However, one caveat with their use is that increased glucose uptake into skeletal and cardiac muscle, in the absence of a demand for increased catabolism, would be expected to increase their glycogen content, as observed with the γ2 mutations that increase basal AMPK activity (see above). Nevertheless, while it is clear that increased glycogen content is harmful if it occurs during fetal development of the heart, it is not yet certain that it would be a problem in adults. Whether AMPK-activating drugs would also cause cardiac hypertrophy, which seems to be a secondary consequence of the activating γ2 mutations that is independent of their effects on glycogen accumulation [ 56 ], also remains unclear at present. However, adverse cardiac side effects would be a potential issue with any novel AMPK activators that would have to be carefully monitored, and the development of AMPK-activating drugs targeting isoform combinations not expressed in the heart, such as γ3 [ 57 ], might be advantageous. Are there any indications for the development of inhibitors of AMPK? These might, of course, be useful for the treatment of the heart disease associated with γ2 mutations; studies using transgenic mice expressing the γ2 N488I mutation in the heart from a promoter switched off by tetracycline administration revealed that the clinical signs of the disease were at least partially reversible in mice [ 114 ]. A larger market for AMPK inhibitors might lie with treatment of those cancers where the LKB1-AMPK pathway, rather than being down-regulated as discussed above, has remained fully functional. There is evidence from mouse models that tumors with a functional LKB1-AMPK pathway are protected against cell death induced by addition of metformin or phenformin in vivo, compared with tumors lacking the pathway [ 87 , 88 ]. In these cases, the biguanides may be acting as a kind of cytotoxic drug that acts by depleting cellular ATP, which is less harmful when AMPK is available to mount a response. It seems possible that the existence of a functional LKB1-AMPK pathway within tumor cells may also protect then against the effects of other cytotoxic drugs used for cancer treatment, in which case the latt