P362Potentiation effect of the AMPK activator A-769662 on cardiac myocytes metabolism and survival

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Abstract

Purpose

The AMP-activated protein kinase (AMPK) is known to be protective during metabolic stress like cardiac ischemia or in cardiac metabolic disorders like insulin resistance. A specific AMPK activator, A-769662, has been recently discovered. The aim of this work was to study the putative action of A-769662 in the heart.

Methods

Insulin and A-769662, alone or in combination, were used to stimulate adult cardiomyocytes. The effect of A-769662 on the action of two other AMPK activators, namely oligomycin and hypoxia, has been also tested. Glucose uptake was measured by the detritriation rate of [2-3H] glucose. The phosphorylation state of signaling elements was measured by immunoblot. AMPK activity was assessed by the incorporation of radioactive phosphates on a specific synthetic peptide. Cell death was evaluated using propidium iodide.

Results

Glucose uptake was increased by both insulin (via Akt) and the hypoxia-mimetic AMPK activator oligomycin (insulin: 1.57 ± 0.28, oligomycin: 1.13 ± 0.06 vs control: 0.18 ± 0.02 μmoles/mg.h; p ≤ 0.05). In the presence of insulin, oligomycin leads to on overstimulation of glucose uptake (2.77 ± 0.31 μmoles/mg.h) by inducing an Akt overstimulation. In contrast to oligomycin, A-769662 had no insulin sensitizing effects. Indeed, A-769662 promoted AMPK phosphorylation but did not stimulate glucose uptake (0.22 ± 0.03 μmoles/mg.h) and did not induce an overphosphorylation of Akt or an overstimulation of glucose uptake in the presence of insulin. On the other hand, A-769662 was able to potentiate the effects of other AMPK activators. We showed an overphosphorylation of AMPK and of its substrate ACC, when A-769662 was added to oligomycin or hypoxia. This potentiation effect was accompanied by an overstimulation of glucose uptake (3-fold increase vs. oligomycin or hypoxia alone) and by a decrease in cell mortality (oligomycin: 27 ± 1, A-769662 + oligomycin: 22 ± 1 vs control 19 ± 1 % of cell death). The apparently contradictory results on insulin action or on others AMPK activators action, can be explained by the fact that A-769662 only activated one specific AMPK catalytic isoform.

Conclusions

A-769662 is unable to stimulate glucose uptake or to increase insulin sensitivity. However, its potentiation effect on other AMPK activators makes it useful to participate in the beneficial roles of AMPK under hypoxic conditions like during myocardial ischemia.

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