14-3-3 proteins are associated with GLUT4 in rat adipocytes / 基础医学与临床

ABSTRACT

Objective To investigate the interaction between glucose transporter 4(GLUT4) and the(14-3-3) proteins in rat adipocytes.Methods Isolated primary adipocytes were obtained from the epididymal fat pads of male Sprague-Dawley rats by collagenase Ⅰ digestion.Total cell extract as well as three fractions,referred to as Fraction T,H and L,were obtained from rat adipocytes using hypotonic lysis and glycerol gradient velocity sedimentation.Immunoadsorption of GLUT4 with Trisacryl beads cross-linked with 1F8,a specific monoclonal antibody to GLUT4,was conducted in total cell extract and the three fractions and followed by elution and Western blot analysis.Co-immunoprecipitation of(14-3-3) proteins and GLUT4 was demonstrated by detecting(14-3-3) in the eluates.Results(14-3-3) proteins were co-immunoprecipitated with GLUT4 either in the total adipocyte extract, or in each of the three fractions mentioned above.The co-precipitated(14-3-3)s were identified as two bands in Western blot analysis,the molecular weights of which were approximately 29kDa and 60kDa respectively,indicating that they might be in the state of homo-or hetero-dimers while interacting with GLUT4.Conclusion There is a physiological interaction between(143-3) proteins and GLUT4 in rat adipocyte.