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

作者：Scharf Matthew T^*; Mackiewicz Miroslaw; Naidoo Nirinjini; O'Callaghan James P; Pack Allan I

来源：Journal of Neurochemistry, 2008, 105(3): 833-841.

DOI：10.1111/j.1471-4159.2007.05182.x

• AMP-activated protein kinase
• brain
• microwave
• phosphorylation

AMP-activated protein kinase (AMPK) is activated when the catalytic alpha subunit is phosphorylated on Thr172 and therefore, phosphorylation of the alpha subunit is used as a measure of activation. However, measurement of alpha subunit of AMPK (alpha-AMPK) phosphorylation in vivo can be technically challenging. To determine the most accurate method for measuring alpha-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 alpha-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 alpha-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 alpha-AMPK phosphorylation, but phosphorylation was higher in mice killed by cervical dislocation compared with mice killed by focused microwave irradiation. These results demonstrate that alpha-AMPK phosphorylation is dependent on method of killing and tissue preparation and that alpha-AMPK phosphorylation can increase in a manner that does not reflect biological alterations.

• 出版日期2008-5