title: A method to determine insulin responsiveness in synaptosomes isolated from frozen brain tissue.
abstract: BACKGROUND
  Studying the insulin signaling response at the synapse is an important approach to understand molecular mechanisms involved in disease-related neurodegenerative processes.
  
  
  METHODS
  We developed a method for studying the insulin responsiveness at the synaptic level by isolating functional synaptosomes from fresh or frozen tissue and exposing them to insulin in the presence of ATP (a critical step) to detect insulin receptor (IR) activation.
  
  
  RESULTS
  We performed an ATP dose-response curve, insulin dose-response curve, and insulin response time course to optimize this method. We also demonstrated that our protocol reflects the degree of insulin responsiveness in vivo by using an animal model of known insulin resistance, AtENPP1-Tg mice.
  
  
  CONCLUSIONS
  This method is advantageous over other methods detecting IR in total brain homogenates due to the ability to detect IR response without confounding contributions from other cell areas and cell types also expressing IR. Furthermore, ex vivo insulin stimulation can be compared to baseline synaptosomes obtained from the same animal which improves reliability and statistical power while decreasing the number of animals required to perform individual experiments.
  
  
  CONCLUSIONS
  We have developed a reliable, efficient method to measure insulin-driven ex vivo phosphorylation of the synaptosomal insulin receptor that can reliably reflect the pre-existing insulin responsiveness status in the CNS of the animal. To the best of our knowledge, this is the first evidence of stimulation of isolated synaptosomes with insulin and a promising new technique to study the synaptic CNS insulin responsiveness under physiological or disease conditions.
authors.affiliation: Mitchell Center for Neurodegenerative Diseases, Department of Neurology, University of Texas Medical Branch, Galveston, TX, USA; Department of Neuroscience & Cell Biology, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Whitney
authors.initials: W
authors.lastname: Franklin
authors.affiliation: Mitchell Center for Neurodegenerative Diseases, Department of Neurology, University of Texas Medical Branch, Galveston, TX, USA. Electronic address: gtaglial@utmb.edu.
authors.forename: Giulio
authors.initials: G
authors.lastname: Taglialatela
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D002458:Cell Fractionation / Q000379:methods*; D015925:Cryopreservation; D016591:Histocytological Preparation Techniques / Q000379:methods*; D007328:Insulin / Q000008:administration & dosage / Q000378:metabolism*; D007333:Insulin Resistance; D008822:Mice, Transgenic; D013574:Synaptosomes / Q000378:metabolism*
chemical_list: D007328:Insulin; D000255:Adenosine Triphosphate
keywords: Insulin receptor phosphorylation; Insulin responsiveness; Insulin signaling; Synaptosomes