The results in this paper provide new information about the mechanism of action of A-769662 and show that the compound is a useful experimental tool to study the downstream consequences of AMPK activation in intact cells and in vivo
. A-769662 activated the native αβγ complex of AMPK purified from rat liver extremely potently in cell-free assays, with a half-maximal effect at 120 nM. This is even lower than the EC50
of 800 nM reported for A-769662 by Cool et al (39
), although this may be due to differences in the preparation of AMPK used and/or the assay conditions, because our estimated EC50
for the natural activator, AMP, was also lower than that reported by Cool et al (39
) (8 versus 56 μM). The ability of A-769662 to directly activate AMPK both in cell-free assays and in intact cells makes it unique among currently known cell-permeable activators. Other activators, such as AICAR, metformin and the thiazolidinediones, do not activate AMPK directly in cell-free assays, and are either pro-drugs that are converted to active components inside the cell (e.g. AICAR, which is converted to the AMP analogue ZMP (21
)) or work even more indirectly, e.g. by inhibiting the respiratory chain (metformin) or by triggering release of adiponectin from adipocytes (thiazolidinediones) (3
Our results also suggest that A-769662 does not act by binding to any of the known ligand-binding sites on the α, β or γ subunits and must utilize a novel binding site. A-769662 had no effect on the activity of the isolated kinase domain from the α1 isoform, either using a T172D mutant that does not require prior phosphorylation (not shown), or after phosphorylation of Thr-172 on the wild type kinase domain by CaMKKβ (). Neither did A-769662 relieve inhibition of the phosphorylated kinase domain by the auto-inhibitory domain (residues 311-333) previously identified by Pang et al (51
) (). An incidental finding that came out of these experiments was that the presence of the AID did not prevent the phosphorylation of Thr-172 by CaMKKβ () or LKB1 (not shown), even though it did completely prevent activation by these upstream kinases. The AID of AMPK-α1 and -α2 aligns with, and show some sequence similarity with, the ubiquitin-associated (UBA) domains in the AMPK-related kinases (56
). Indeed, Pang et al (51
) have modelled the interaction between the kinase domain and the AID of α1 based on the structure of the kinase and UBA domain of the AMPK-related kinase MARK2. However, the functions of the UBA domain in the AMPK-related kinases and the AID in the α subunits of AMPK appear to be different, because Jaleel et al (56
) found that while the UBA domains did not inhibit the AMPK-related kinases, they were required for their phosphorylation by the LKB1 complex. By contrast, we now report that while the AID in the AMPK α subunits is not required for phosphorylation by either CaMKKβ or LKB1, it does completely prevent the activation caused by these phosphorylation events.
A-769662 did not cause dissociation of the glycogen-binding domain from glycogen (), making it unlikely that the compound binds to the glycogen-binding site on this domain. We also found using a scintillation proximity assay that A-769662 did not displace [3H]AMP from the isolated Bateman domains on the γ2 subunit significantly under conditions where unlabelled AMP clearly did (). This was a little surprising, because A-769662 mimics not just one but two of the effects of AMP on the AMPK system, i.e. (i) allosteric activation (); and (ii) inhibition of dephosphorylation (). Our finding that A-769662 did not significantly displace AMP from the isolated Bateman domains suggests that the binding sites for these two ligands must be different, although they may produce a similar change in conformation.
Our studies in intact cells reinforce the idea that A-769662 is a specific and direct activator of AMPK and are also consistent with the idea that it has a dual effect, causing both allosteric activation and inhibition of dephosphorylation. Increased phosphorylation of acetyl-CoA carboxylase (ACC) by A-769662 in mouse embryo fibroblasts and primary mouse hepatocytes was completely dependent on the expression of the two catalytic subunits of AMPK () showing that, at least when measuring ACC phosphorylation, the compound is completely dependent on AMPK for its effects.
It was noticeable in all of our intact cell studies with A-769662 that, while the effects on AMPK phosphorylation were often quite small, the effects on ACC phosphorylation were generally larger. For example, while phenformin or AICAR had much larger effects than A-769662 on AMPK phosphorylation in MEF cells and primary hepatocytes (), the effects of these agents on ACC phosphorylation were similar. Related observations were made in skeletal muscle () and in HeLa cells (). The most likely explanation for these apparent differences is that phosphorylation of ACC is such a sensitive marker of AMPK activation that maximal phosphorylation of ACC occurs when only a small proportion of AMPK has been phosphorylated. The concentrations of AICAR and phenformin chosen for study were designed to give maximal phosphorylation and activation of AMPK in these cells, and are likely to be greater than those required to give maximal ACC phosphorylation. An additional explanation in the case of the effects of the Ca2+
ionophores in HeLa cells () is that calcium ions activate phosphorylation by the upstream kinase CaMKKβ (12
), but do not cause allosteric activation of AMPK. The phosphorylation of ACC in response to A-769662 would reflect a combination of allosteric activation and increased phosphorylation, but the allosteric effect on AMPK is lost during preparation of the extracts, and is not reflected in the kinase assays. By contrast, the effect of increased intracellular Ca2+
is entirely mediated by increased phosphorylation, and the effect on AMPK activity would be fully preserved in the extract. A third potential explanation for these apparent differences is that A-769662 might be able to activate dephosphorylated AMPK, but we could obtain no evidence that this was the case. While the compound did cause some activation of purified AMPK after treatment with protein phosphatase-2Cα (data not shown), the degree of activation of the treated and untreated kinase was the same (4-fold) and A-769662 did not alleviate the large inactivation caused by protein phosphatase treatment. Therefore, the small activation of the protein phosphatase-treated enzyme was most likely due to activation of the small residual amount of phosphorylated kinase left in the preparation.
The results obtained with LKB1−/−
muscle () suggest that an upstream kinase is necessary for the effect of A-769662 on ACC phosphorylation to be observed, presumably because dephosphorylated AMPK is not activated by the compound. However, the effect appears to be independent of the particular upstream kinase being utilized. Thus in HeLa cells, which do not express LKB1 but do express CaMKKβ (6
), A-769662 still promoted phosphorylation of both AMPK and ACC in a similar manner to the Ca2+
ionophore, ionomycin. Interestingly, while the CaMKK inhibitor STO-609 greatly reduced phosphorylation of ACC in response to ionomycin, a substantial phosphorylation of ACC in response to A-769662 remained (). A likely explanation of these results is that ionomycin acts by increasing Ca2+
and thus activating CaMKKβ and, while this would increase phosphorylation of Thr-172 on AMPK, there would be no concomitant allosteric activation of the kinase. By contrast, A-769662 acts both by inhibiting dephosphorylation of Thr-172 and by causing allosteric activation of AMPK, and even the very low basal activity of CaMKKβ in STO-609-treated cells without ionomycin may be sufficient to observe these effects. This would also explain why the effect of STO-609 on ACC phosphorylation in response to A-769662 was less than its effect on the response to ionomycin.
It was recently reported that the TGFβ-activated kinase-1 (TAK1) could activate the S. cerevisiae
homologue of AMPK, the SNF1 complex, when expressed in the yeast, and could also phosphorylate Thr-172 and activate mammalian AMPK in cell-free assays (15
). Our findings that phosphorylation of AMPK and ACC by A-769662 was identical in TAK1+/+
mouse embryo fibroblasts do not support an important role for TAK1 as an upstream kinase in these cells. However, they do not rule out the possibility that it could act as an upstream kinase for AMPK in other cell types, or in other circumstances.
We would suggest that A-769662 is superior to other AMPK activators, including the nucleoside AICAR and the biguanide drugs, metformin and phenformin, for studies of the downstream actions of AMPK in intact cells and in vivo
. AICAR is taken up into cells by adenosine transporters (57
) and is converted by adenosine kinase into the mono-phosphorylated nucleotide ZMP, which mimics the effects of AMP on AMPK activation, albeit 50-fold less potently than AMP itself (21
). One problem with the use of AICAR is that ZMP has been found to regulate other AMP-sensitive enzymes such as glycogen phosphorylase (58
) and fructose-1,6-bisphosphatase (59
), which is not the case with A-769662 (39
). Another problem is that, while not itself an agonist or an antagonist of adenosine receptors, it does compete with adenosine for re-uptake into cells. In some in vitro
systems this can cause effects that are due to increased accumulation of adenosine outside cells, with consequent binding to adenosine receptors (57
). Although the biguanide drugs, metformin and phenformin, activate AMPK when incubated with intact cells (29
), they have no effect on AMPK or its phosphorylation or dephosphorylation in cell-free assays (33
). The mechanism by which they activate AMPK has not been completely elucidated, but it has been reported that their entry into cells is catalyzed by organic cation transporters such as OCT1 (31
), and that the drugs then accumulate in mitochondria (driven by the membrane potential across the inner mitochondrial membrane, which would favour uptake of cations) where they inhibit complex I of the respiratory chain (34
). This may in turn produce an increase in the cellular AMP:ATP ratio that activates AMPK. An increase in the AMP:ATP ratio has indeed been demonstrated in the case of phenformin (12
), although it has been more difficult to detect in the case of the less rapidly and potently acting biguanide, metformin, possibly because this was studied in cells that do not express OCT1 (33
). This is supported by the fact that using primary hepatocytes, which do express OCT1, metformin treatment resulted in a decrease in ATP content (36
). If this proposed mechanism of action for the biguanides is correct, they may be no more specific as pharmacological activators of AMPK than are other metabolic poisons such as the mitochondrial ATP synthase inhibitor, oligomycin (33
). As a good example of the shortcomings of these widely used AMPK activators, it has recently been shown that AICAR, metformin and oligomycin all inhibit glucose phosphorylation is isolated rodent hepatocytes by inhibiting the translocation of glucokinase from the nucleus to the cytoplasm. However, all three agents were just as effective in this regard in isolated hepatocytes from double knockout (AMPK-α1−/−
) mice as in those from wild type controls, showing that these effects do not require AMPK and are probably mediated by ATP depletion (36
). Unlike AICAR, metformin or oligomycin, A-769662 is a direct activator of AMPK in cell-free assays and it is certainly the most potent and specific pharmacological activator of AMPK available at present. This drug will be a valuable experimental tool to study the physiological roles of AMPK. A more complete understanding of the mechanism by which it activates AMPK may also facilitate the design of novel AMPK activators that could be used to treat patients with metabolic disorders.