AMP-activated protein kinase phosphorylation in brain is dependent on method of killing and tissue preparation.
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.
Scharf
Matthew T
MT
Center for Sleep and Respiratory Neurobiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-3403, USA.
Mackiewicz
Miroslaw
M
Naidoo
Nirinjini
N
O'Callaghan
James P
JP
Pack
Allan I
AI
D055372:AMP-Activated Protein Kinases; D000818:Animals; D001831:Body Temperature / Q000502:physiology; D001921:Brain / Q000378:metabolism* / Q000528:radiation effects; D001923:Brain Chemistry / Q000502:physiology; D015925:Cryopreservation / Q000379:methods*; D004789:Enzyme Activation / Q000502:physiology / Q000528:radiation effects; D051379:Mice; D008810:Mice, Inbred C57BL; D008872:Microwaves; D009097:Multienzyme Complexes / Q000032:analysis / Q000378:metabolism*; D009448:Neurochemistry / Q000379:methods; D010766:Phosphorylation / Q000528:radiation effects; D011180:Postmortem Changes; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000378:metabolism*; D021122:Protein Subunits / Q000032:analysis / Q000378:metabolism; D013010:Sonication; D013124:Spinal Injuries / Q000378:metabolism; D016707:Tissue Fixation / Q000379:methods*
D009097:Multienzyme Complexes; D021122:Protein Subunits; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases