AMP-activated protein kinase phosphorylation in brain is dependent on method of killing and tissue preparation.
Scharf-MT; Mackiewicz-M; Naidoo-N; O'Callaghan-JP; Pack-AI
J Neurochem 2008 May; 105(3):833-841
AMP-activated protein kinase (AMPK) is activated when the catalytic a subunit is phosphorylated on Thr172 and therefore, phosphorylation of the a subunit is used as a measure of activation. However, measurement of a subunit of AMPK (a-AMPK) phosphorylation in vivo can be technically challenging. To determine the most accurate method for measuring a-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 a-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 a-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 a-AMPK phosphorylation, but phosphorylation was higher in mice killed by cervical dislocation compared with mice killed by focused microwave irradiation. These results demonstrate that a-AMPK phosphorylation is dependent on method of killing and tissue preparation and that a-AMPK phosphorylation can increase in a manner that does not reflect biological alterations.
Protein-chemistry; Cellular-reactions; Laboratory-animals; Laboratory-techniques; Brain-electrical-activity; Irradiation; Cerebrovascular-system; Biological-material
Matthew T. Scharf, Center for Sleep and Respiratory Neurobiology, University of Pennsylvania School of Medicine, Translational Research Building, Suite 2100, 125 S. 31st St., Philadelphia, PA 19104-3403
Journal of Neurochemistry