Fiona A. Ross

AMPK: a nutrient and energy sensor that maintains energy homeostasis

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.

Use of Cells Expressing γ Subunit Variants to Identify Diverse Mechanisms of AMPK Activation

Summary A 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.

The Ancient Drug Salicylate Directly Activates AMP-Activated Protein Kinase

An Aspirin a Day? The protein kinase AMPK (adenosine monophosphate–activated protein kinase) directly monitors cellular energy stores as reflected by changes in cellular concentrations of AMP, adenosine diphosphate (ADP), and adenosine triphosphate (ATP). Through phosphorylation of its targets, it helps to control metabolism, polarity, autophagy, and the restraint of cell proliferation. Activation of AMPK is also proposed to be beneficial for the treatment of diseases, including cancer and diabetes. Hawley et al. (p. 918, published online 19 April; see the Perspective by Shaw and Cantley) report that AMPK can be activated by high concentrations of salicylate, a compound derived from the very commonly used drug aspirin. In mice, salicylate promoted fatty acid and carbohydrate metabolism in an AMPK-dependent fashion. A possible molecular mechanism of action for a metabolite of aspirin is described. 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 of which are rapidly broken down to salicylate in vivo. At concentrations reached in plasma after administration of salsalate or of 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, threonine-172. In AMPK knockout mice, effects of salicylate to increase fat utilization and to 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.

Mechanism of action of A-769662, a valuable tool for activation of AMP-activated protein kinase

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 α subunit kinase domain, with or without the associated autoinhibitory domain, or on interaction of glycogen with the β 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 γ 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-α1–/–α2–/– 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-β, 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.

AMP-Activated Protein Kinase: A Target for Drugs both Ancient and Modern

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.

AMP Is a True Physiological Regulator of AMP-Activated Protein Kinase by Both Allosteric Activation and Enhancing Net Phosphorylation

Summary While 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.

AMP‐activated protein kinase: a cellular energy sensor that comes in 12 flavours

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.

Phosphorylation by Akt within the ST loop of AMPK-α1 down-regulates its activation in tumour cells.

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.

Phosphorylation of the voltage-gated potassium channel Kv2.1 by AMP-activated protein kinase regulates membrane excitability

Firing of action potentials in excitable cells accelerates ATP turnover. The voltage-gated potassium channel Kv2.1 regulates action potential frequency in central neurons, whereas the ubiquitous cellular energy sensor AMP-activated protein kinase (AMPK) is activated by ATP depletion and protects cells by switching off energy-consuming processes. We show that treatment of HEK293 cells expressing Kv2.1 with the AMPK activator A-769662 caused hyperpolarizing shifts in the current–voltage relationship for channel activation and inactivation. We identified two sites (S440 and S537) directly phosphorylated on Kv2.1 by AMPK and, using phosphospecific antibodies and quantitative mass spectrometry, show that phosphorylation of both sites increased in A-769662–treated cells. Effects of A-769662 were abolished in cells expressing Kv2.1 with S440A but not with S537A substitutions, suggesting that phosphorylation of S440 was responsible for these effects. Identical shifts in voltage gating were observed after introducing into cells, via the patch pipette, recombinant AMPK rendered active but phosphatase-resistant by thiophosphorylation. Ionomycin caused changes in Kv2.1 gating very similar to those caused by A-769662 but acted via a different mechanism involving Kv2.1 dephosphorylation. In cultured rat hippocampal neurons, A-769662 caused hyperpolarizing shifts in voltage gating similar to those in HEK293 cells, effects that were abolished by intracellular dialysis with Kv2.1 antibodies. When active thiophosphorylated AMPK was introduced into cultured neurons via the patch pipette, a progressive, time-dependent decrease in the frequency of evoked action potentials was observed. Our results suggest that activation of AMPK in neurons during conditions of metabolic stress exerts a protective role by reducing neuronal excitability and thus conserving energy.

Mechanism of Action of Compound-13: An α1-Selective Small Molecule Activator of AMPK

Summary AMPK is a sensor of cellular energy status and a promising target for drugs aimed at metabolic disorders. We have studied the selectivity and mechanism of a recently described activator, C2, and its cell-permeable prodrug, C13. C2 was a potent allosteric activator of α1-complexes that, like AMP, also protected against Thr172 dephosphorylation. Compared with AMP, C2 caused only partial allosteric activation of α2-complexes and failed to protect them against dephosphorylation. We show that both effects could be fully restored by exchanging part of the linker between the autoinhibitory and C-terminal domains in α2, containing the equivalent region from α1 thought to interact with AMP bound in site 3 of the γ subunit. Consistent with our results in cell-free assays, C13 potently inhibited lipid synthesis in hepatocytes from wild-type and was largely ineffective in AMPK-knockout hepatocytes; its effects were more severely affected by knockout of α1 than of α2, β1, or β2.