R01 AG058571 Type 2 diabetes, characterized by insulin resistance in peripheral tissues such as skeletal muscle, increases AD risk. However, it is unclear whether there is a similar, or stronger, relationship between insulin resistance in the brain and AD. The goal of the proposed administrative supplement to Look AHEAD-MIND, while within the original scope, is to further unravel molecular pathways linking the intensive lifestyle intervention to the development of cognitive impairment by examining neural-derived blood exosome biomarkers for brain insulin resistance and Alzheimer's disease (AD) pathologies.
Methods for the Look AHEAD-MIND Supplement R01 AG058571 We will isolate neuronal-derived exosomes (NDEs) from plasma following our published method 1. Briefly, plasma will be incubated with thromboplastin-D (Fisher Scientific, Inc., Hanover Park, IL) at room temperature for 60 minutes, followed by the addition of calcium- and magnesium-free Dulbecco's phosphatase buffered saline (DPBS) with protease inhibitor and phosphatase inhibitor cocktail (Pierce Halt, Thermo Scientific, Inc., Rockford, IL). After centrifugation at 1500 x g for 20 minutes, supernates will be mixed with ExoQuick exosome precipitation solution (EXOQ; System Biosciences, Inc., Mountainview, CA), and incubated for 1 hour at 4C. Resultant exosome suspensions (total exosomes) will be centrifuged at 1500 x g for 30 minutes at 4C and each pellet will be resuspended in DPBS with inhibitor cocktails. Each sample will be then incubated for 1 hour at 4C with mouse anti-human L1 cell adhesion molecule (L1CAM) biotinylated antibody (clone 5G3, eBioscience, San Diego, CA) for NDEs and streptavidin-agarose resin (Thermo Scientific, Inc.) in 3% bovine serum albumin. After centrifugation at 200 x g for 10 minutes at 4C and removal of the supernate, each pellet will be suspended in 0.05 M glycine-HCl (pH 3.0) by vortexing for 10 seconds and then centrifuged at 200 x g for 15 minutes at 4C and supernatant collected as NDEs. The protein concentration of NDEs will be measured using a NanoDrop. In each case, exosome concentration (number per ml) and size distribution will be analyzed by nanoparticle tracking analyses (NTA).
