AMPK can be activated in two ways: by allosteric binding of AMP [18
], or by phosphorylation by one or more upstream AMPK kinases [19
]. Once activated, AMPK minimizes ATP consumption by suppressing ATP-consuming anabolic pathways as well as activating ATP-generating catabolic pathways.
As its name suggests, mammalian AMPK is regulated by changes in the ratio of AMP to ATP. Conditions such as hypoxia lead to the activation of AMPK due to a failure to generate sufficient ATP for cellular functions [20
]. Thus, under hypoxic conditions, AMPK initiates various adaptive responses in response to changing cellular parameters, namely the decrease in ATP levels or the increase in the AMP: ATP ratio.
However, recent findings challenge the notion that allosteric activation triggered by increased AMP:ATP ratios is the main pathway leading to AMPK activation under hypoxic condition in heart tissues [23
]. Herein, we investigated intracellular AMP and ATP concentrations in cardiomyocytes after various durations of hypoxia. Our results demonstrated that, compared with cells cultured under normoxic conditions, neither intracellular AMP levels nor AMP/ATP ratios increased significantly within 1h of hypoxia onset. In contrast, a SAMS peptide phosphorylation assay and immunoblot analysis revealed significant increases in both AMPK activity and ACC phosphorylation within 1h of hypoxic treatment. ACC is a prototypical and well-characterized AMPK target in the heart [24
]. One of the ways by which AMPK stimulates ATP synthesis in the heart is by phosphorylating ACC. This phosphorylation inhibits the activity of ACC, leading to reduced malonyl-CoA formation and relieved inhibition of carnitine palmitoyl transferase 1, ultimately resulting in accelerated β-oxidation of fatty acids and the generation of ATP. These results indicate that, at a very early stage of hypoxia onset in cardiomyocytes, AMPK activity increases independent of AMP concentrations or of the AMP:ATP ratio.
The mechanisms that regulate the increase in AMPKK activity in anoxic cardiomyocytes were elucidated through the use of α312
as a substrate to measure AMPK phosphorylation at Thr-172. Reversible phosphorylation of the Thr-172 residue, which is situated within the activation loop of the kinase domain of the AMPK α-subunit, governs the catalytic activity of AMPK [25
]. Our results showed that within 5min of exposing cardiomyocytes to hypoxic conditions, AMPK phosphorylation levels were significantly increased, by 3- to 4-fold compared with normoxic controls (P<0.01), whereas total AMPKα protein levels did not differ between aerobic and anoxic cardiomyocytes. As hypoxia persisted, relative p-AMPK levels kept increasing, but were not significantly different between any of the time points, suggesting that AMPK phosphorylation is critically increased as of the very earliest stages of hypoxia. The fusion protein [26
] was an effective substrate for the in vitro AMPKK assay; our results revealed that incubating cardiomyocyte lysates (under hypoxic conditions for 5min and 15min) with AMPKα312
significantly increased AMPKK activity (P<0.01). Measuring AMPKK activity in the absence of AMP demonstrated the intrinsic activation of AMPKK in hypoxic cardiomyocytes. Taken together, these results thus demonstrate that AMPKK activity in cardiomyocytes is markedly increased at the very early stage of hypoxia, consistent with the increase in AMPK activity observed under the same conditions. AMPK activation in the absence of measurable changes in AMP concentrations has been implicated in the response of non-cardiac tissues to leptin [27
], osmotic stress [28
] and metformin [29
], but AMPKK activity has not been assessed in these experiments and the specific mediators of presumed AMPKK activation in these settings remain unknown.
In summary, our results demonstrate that in the early stage of hypoxia in cardiomyocytes, increases in AMPK activity occur prior to, and thus independently of, increases in AMP concentration or in the AMP:ATP ratio. Therefore, in these circumstances AMPK is primarily activated by phosphorylation of the conserved Thr-172 residue in its activation loop by its upstream kinase AMPKK.