AMP-activated protein kinase (AMPK) is a crucial cellular energy sensor. Once activated by falling energy status, it promotes ATP production by increasing the activity or expression of proteins involved in catabolism while conserving ATP by switching off biosynthetic pathways. AMPK also regulates metabolic energy balance at the whole-body level. For example, it mediates the effects of agents acting on the hypothalamus that promote feeding and entrains circadian rhythms of metabolism and feeding behaviour. Finally, recent studies reveal that AMPK conserves ATP levels through the regulation of processes other than metabolism, such as the cell cycle and neuronal membrane excitability.

SummaryA wide variety of agents activate AMPK, but in many cases the mechanisms remain unclear. We generated isogenic cell lines stably expressing AMPK complexes containing AMP-sensitive (wild-type, WT) or AMP-insensitive (R531G) γ2 variants. Mitochondrial poisons such as oligomycin and dinitrophenol only activated AMPK in WT cells, as did AICAR, 2-deoxyglucose, hydrogen peroxide, metformin, phenformin, galegine, troglitazone, phenobarbital, resveratrol, and berberine. Excluding AICAR, all of these also inhibited cellular energy metabolism, shown by increases in ADP:ATP ratio and/or by decreases in cellular oxygen uptake measured using an extracellular flux analyzer. By contrast, A769662, the Ca2+ ionophore, A23187, osmotic stress, and quercetin activated both variants to varying extents. A23187 and osmotic stress also increased cytoplasmic Ca2+, and their effects were inhibited by STO609, a CaMKK inhibitor. Our approaches distinguish at least six different mechanisms for AMPK activation and confirm that the widely used antidiabetic drug metformin activates AMPK by inhibiting mitochondrial respiration.

Salicylate, a plant product, has been in medicinal use since ancient times. More recently it has been replaced by synthetic derivatives such as aspirin and salsalate, both rapidly broken down to salicylate in vivo. At concentrations reached in plasma following administration of salsalate, or aspirin at high doses, salicylate activates adenosine monophosphate-activated protein kinase (AMPK), a central regulator of cell growth and metabolism. Salicylate binds at the same site as the synthetic activator, A-769662, to cause allosteric activation and inhibition of dephosphorylation of the activating phosphorylation site, Thr172. In AMPK knockout mice, effects of salicylate to increase fat utilization and lower plasma fatty acids in vivo were lost. Our results suggest that AMPK activation could explain some beneficial effects of salsalate and aspirin in humans.The medicinal effects of willow bark have been known since the time of Hippocrates. The active component is salicylate, a hormone produced by plants in response to pathogen infection (1). For medicinal use it was largely replaced by aspirin (acetyl salicylate), which is rapidly broken down to salicylate in vivo (2, 3). Salicylate can also be administered as salsalate, which shows promise for treatment of insulin resistance and type 2 diabetes (4, 5). Aspirin and salicylate inhibit cyclo-oxygenases and hence prostanoid biosynthesis (6), as well as the protein kinase IKKβ in the NF-κB pathway (7). However, some effects of these drugs are still observed in mice deficient in these pathways (8).Adenosine monophosphate-activated protein kinase (AMPK) is a cellular energy sensor conserved throughout eukaryotes. This heterotrimeric enzyme is composed of catalytic α Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts subunits and regulatory β and γ subunits (9, 10). Once activated in response to metabolic stress, AMPK phosphorylates targets that switch off adenosine triphosphate (ATP) consuming processes, while switching on catabolic pathways that generate ATP. AMPK is activated >100-fold by phosphorylation at Thr172 in the α subunit by the tumour suppressor protein kinase, LKB1, or the Ca 2+ -dependent kinase, CaMKKβ (9, 10). Binding of AMP or adenosine diphosphate (ADP) to the γ subunit triggers a conformational change that promotes phosphorylation and inhibits dephosphorylation (11-15), causing a switch to the active form. Binding of AMP (but not ADP) to a second site (15) causes further allosteric activation, leading to >1,000-fold activation overall (16). Most drugs or xenobiotics that activate AMPK work by inhibiting mitochondrial ATP synthesis and increasing the concentration of AMP and ADP (17). However, a synthetic activator, A-769662 (18), which also causes allosteric activation and inhibits Thr172 dephosphorylation, binds directly to AMPK at distinct site(s) (19-21).Salicylate, but not aspirin, activated AMPK when applied to HEK-293 cells, with its effects being significant at 1 mM and above ( Fig. 1A; it appears that the estera...

SummaryWhile allosteric activation of AMPK is triggered only by AMP, binding of both ADP and AMP has been reported to promote phosphorylation and inhibit dephosphorylation at Thr172. Because cellular concentrations of ADP and ATP are higher than AMP, it has been proposed that ADP is the physiological signal that promotes phosphorylation and that allosteric activation is not significant in vivo. However, we report that: AMP is 10-fold more potent than ADP in inhibiting Thr172 dephosphorylation; only AMP enhances LKB1-induced Thr172 phosphorylation; and AMP can cause >10-fold allosteric activation even at concentrations 1–2 orders of magnitude lower than ATP. We also provide evidence that allosteric activation by AMP can cause increased phosphorylation of acetyl-CoA carboxylase in intact cells under conditions in which there is no change in Thr172 phosphorylation. Thus, AMP is a true physiological regulator of AMPK, and allosteric regulation is an important component of the overall activation mechanism.

We have studied the mechanism of A-769662, a new activator of AMP-activated protein kinase (AMPK). Unlike other pharmacological activators, it directly activates native rat AMPK by mimicking both effects of AMP, i.e. allosteric activation and inhibition of dephosphorylation. We found that it has no effect on the isolated ␣ subunit kinase domain, with or without the associated autoinhibitory domain, or on interaction of glycogen with the ␤ subunit glycogen-binding domain. Although it mimics actions of AMP, it has no effect on binding of AMP to the isolated Bateman domains of the ␥ subunit. The addition of A-769662 to mouse embryonic fibroblasts or primary mouse hepatocytes stimulates phosphorylation of acetyl-CoA carboxylase (ACC), effects that are completely abolished in AMPK-␣1 ؊/؊ ␣2 ؊/؊ cells but not in TAK1 ؊/؊ mouse embryonic fibroblasts. Phosphorylation of AMPK and ACC in response to A-769662 is also abolished in isolated mouse skeletal muscle lacking LKB1, a major upstream kinase for AMPK in this tissue. However, in HeLa cells, which lack LKB1 but express the alternate upstream kinase calmodulin-dependent protein kinase kinase-␤, phosphorylation of AMPK and ACC in response to A-769662 still occurs. These results show that in intact cells, the effects of A-769662 are independent of the upstream kinase utilized. We propose that this direct and specific AMPK activator will be a valuable experimental tool to understand the physiological roles of AMPK.The AMP-activated protein kinase (AMPK) 3 is a regulator of energy balance at both the cellular and the whole body levels (1-3). Once activated, it effects a metabolic switch from an anabolic to a catabolic state, both by acutely phosphorylating metabolic enzymes and, in the longer term, by regulating gene expression. AMPK is a heterotrimer composed of a catalytic ␣ subunit and regulatory ␤ and ␥ subunits. Binding of AMP to the two "Bateman domains" formed by four tandem CBS motifs on the ␥ subunit (4) triggers increased phosphorylation at Thr-172 on the activation loop of the ␣ subunit, causing Ͼ100-fold activation (5). AMP binding was previously thought both to promote phosphorylation (6) and to inhibit dephosphorylation (7), although recent results suggest that the effect is exclusively on dephosphorylation (8). AMP binding also causes a further allosteric activation of the phosphorylated kinase by up to 10-fold (5). Phosphorylation of Thr-172 is in most cells catalyzed by the tumor suppressor kinase LKB1 (6, 9), which appears to be constitutively active (10, 11). In some cells, Thr-172 can also be phosphorylated in a Ca 2ϩ -mediated process catalyzed by calmodulin-dependent protein kinase kinases such as CaMKK␤ (12-14). The protein kinase TGF␤-activated kinase-1 (TAK1) can also activate the Saccharomyces cerevisiae homologue of AMPK (the SNF1 complex) when overexpressed in yeast, as well as phosphorylating Thr-172 on mammalian AMPK in cell-free assays (15). It remains unclear whether this has any physiological relevance in vivo.Most of the metabolic changes...

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. It is activated, by a mechanism requiring the tumor suppressor LKB1, by metabolic stresses that increase cellular ADP:ATP and/or AMP:ATP ratios. Once activated, it switches on catabolic pathways that generate ATP, while switching off biosynthetic pathways and cell cycle progress. These effects suggest that AMPK activators might be useful for treatment and/or prevention of type 2 diabetes and cancer. Indeed, AMPK is activated by the drugs metformin and salicylate, the latter being the major breakdown product of aspirin. Metformin is widely used to treat diabetes, while there is epidemiological evidence that both metformin and aspirin provide protection against cancer. We review the mechanisms of AMPK activation by these and other drugs, and by natural products derived from traditional herbal medicines.

The AMP‐activated protein kinase (AMPK) is a sensor of cellular energy status that is expressed in essentially all eukaryotic cells, suggesting that it arose during early eukaryotic evolution. It occurs universally as heterotrimeric complexes containing catalytic α subunits and regulatory β and γ subunits. Although Drosophila melanogaster contains single genes encoding each subunit, in mammals, each subunit exists as multiple isoforms encoded by distinct genes, giving rise to up to 12 heterotrimeric combinations. The multiple isoforms of each subunit are 2R‐ohnologues generated by the two rounds of whole genome duplication that occurred at the evolutionary origin of the vertebrates. Although the differential roles of these isoform combinations remain only partly understood, there are indications that they may have different subcellular locations, different inputs and outputs, and different functions. The multiple isoforms are of particular interest with respect to the roles of AMPK in cancer because the genes encoding some isoforms, such as PRKAA1 and PRKAB2 (encoding α1 and β2), are quite frequently amplified in tumour cells, whereas the genes encoding others, such as PRKAA2 (encoding α2), tend to be mutated, which, in some but not all cases, may result in a loss of function. Thus, although AMPK acts downstream of the tumour suppressor liver kinase B1, and some of its isoform combinations may act as tumour suppressors that restrain the growth and proliferation of tumour cells, other isoform combinations may paradoxically act as oncogenes, perhaps by aiding the survival of tumour cells undergoing environmental stresses such as hypoxia or nutrient deprivation.

The insulin/IGF-1 (insulin-like growth factor 1)-activated protein kinase Akt (also known as protein kinase B) phosphorylates Ser487 in the ‘ST loop’ (serine/threonine-rich loop) within the C-terminal domain of AMPK-α1 (AMP-activated protein kinase-α1), leading to inhibition of phosphorylation by upstream kinases at the activating site, Thr172. Surprisingly, the equivalent site on AMPK-α2, Ser491, is not an Akt target and is modified instead by autophosphorylation. Stimulation of HEK (human embryonic kidney)-293 cells with IGF-1 caused reduced subsequent Thr172 phosphorylation and activation of AMPK-α1 in response to the activator A769662 and the Ca2+ ionophore A23187, effects we show to be dependent on Akt activation and Ser487 phosphorylation. Consistent with this, in three PTEN (phosphatase and tensin homologue deleted on chromosome 10)-null tumour cell lines (in which the lipid phosphatase PTEN that normally restrains the Akt pathway is absent and Akt is thus hyperactivated), AMPK was resistant to activation by A769662. However, full AMPK activation could be restored by pharmacological inhibition of Akt, or by re-expression of active PTEN. We also show that inhibition of Thr172 phosphorylation is due to interaction of the phosphorylated ST loop with basic side chains within the αC-helix of the kinase domain. Our findings reveal that a previously unrecognized effect of hyperactivation of Akt in tumour cells is to restrain activation of the LKB1 (liver kinase B1)–AMPK pathway, which would otherwise inhibit cell growth and proliferation.