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

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. © 2008 The Authors.