AMP-Activated Protein Kinase Phosphorylation in Brain Is Dependent on Method of Killing and Tissue Preparation

Details

• Personal Author:
  Mackiewicz M ; Naidoo N ; O'Callaghan JP ; Pack AI ; Scharf MT
• Description:
  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. [Description provided by NIOSH]
• Subjects:
  Biological Products Cardiovascular System Laboratories Nervous System Occupational Health Radiation Safety
• Keywords:
  Biological-material Brain-electrical-activity Cellular-reactions Cerebrovascular-system Irradiation Laboratory-animals Laboratory-techniques Protein-chemistry
• ISSN:
  0022-3042
• Document Type:
  Text
• Genre:
  Journal Article
• Place as Subject:
  OSHA Region 3 ; Pennsylvania ; West Virginia
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Division:
  HELD - Health Effects Laboratory Division
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Volume:
  105
• Issue:
  3
• NIOSHTIC Number:
  nn:20033731
• Citation:
  J Neurochem 2008 May; 105(3):833-841
• Contact Point Address:
  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
• Email:
  mscharf@mail.med.upenn.edu
• Federal Fiscal Year:
  2008
• NORA Priority Area:
  Manufacturing
• Peer Reviewed:
  True
• Source Full Name:
  Journal of Neurochemistry
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:2c03c3170e61d310207a6ae4189b3173c39345feda8211ba272ccf60e458855e3e75b7c90c5b3529ecf90a0a1422ced68b2ae186f198c6e384b1486081e6074e
• Download URL:
  https://stacks.cdc.gov/view/cdc/189192/cdc_189192_DS1.pdf
• File Type:
  [PDF - 229.25 KB ]