AMP‐activated protein kinase phosphorylation in brain is dependent on method of killing and tissue preparation

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Abstract
AMP‐activated protein kinase (AMPK) is activated when the catalytic α subunit is phosphorylated on Thr172 and therefore, phosphorylation of the α subunit is used as a measure of activation. However, measurement of α subunit of AMPK (α‐AMPK) phosphorylation in vivo can be technically challenging. To determine the most accurate method for measuring α‐AMPK phosphorylation in the mouse brain, we compared different methods of killing and tissue preparation. We found that freeze/thawing samples after homogenization on ice dramatically increased α‐AMPK phosphorylation in mice killed by cervical dislocation. Killing of mice by focused microwave irradiation, which rapidly heats the brain and causes enzymatic inactivation, prevented the freeze/thaw‐induced increase in α‐AMPK phosphorylation and similar levels of phosphorylation were observed compared with mice killed with cervical dislocation without freeze/thawing of samples. Sonication of samples in hot 1% sodium dodecyl sulfate blocked the freeze/thaw‐induced increase in α‐AMPK phosphorylation, but phosphorylation was higher in mice killed by cervical dislocation compared with mice killed by focused microwave irradiation. These results demonstrate that α‐AMPK phosphorylation is dependent on method of killing and tissue preparation and that α‐AMPK phosphorylation can increase in a manner that does not reflect biological alterations.

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