Separation and partial characterization of three distinct intracellular GLUT4 compartments in rat adipocytes - Subcellular fractionation without homogenization

Insulin recruits GLUT4 from an intracellular location to the plasma membran e in rat adipocytes. The process involves multiple intracellular compartmen ts and multiple protein functions, details of which are largely unknown par tly due to our inability to separate individual GLUT4 compartments. Here, b y hypotonic lysis, differential centrifugation, and glycerol density gradie nt sedimentation, we separated intracellular GLUT4 compartments in rat adip ocytes into three fractions: plasma membrane-containing fraction T and plas ma mem brane-free fractions H and L, The GLUT4 contents in fractions T, H, and L were similar to 25, 56, and 18% of total GLUT4, respectively, in basa l adipocytes and 55, 42, and 3-4% in insulin-stimulated adipocytes, The pla sma membrane GLUT4 contents estimated separately further revealed that intr acellular GLUT4 in fraction T amounts to similar to 20% in both basal and i nsulin-stimulated adipocytes, Organelle-specific marker and membrane traffi c related protein distribution data suggested that intracellular GLUT4 in f raction T represents sorting endosomes, whereas GLUT4 in fractions H and L represents storage endosomes and exocytic vesicles, respectively. The subce llular fractionation without homogenization described here should be useful in identifying the role of the individual GLUT4 compartments and the assoc iated proteins in insulin-induced GLUT4 recruitment in rat adipocytes.