Differential regulation of adipocyte PDE3B in distinct membrane compartments in response to insulin and CL316243

Abstract

PDE3B is activated by insulin and CL (CL316243, a b3‐receptor agonist) in adipocytes via PKB‐ and PKA‐ signaling, respectively. Activation of PDE3B by insulin seems important in its ability to reduce cAMP, and inhibit lipolysis. Using sucrose gradient centrifugation and confocal microscopy techniques, PDE3B was found to be localized in internal membranes and in caveolin‐enriched PM in 3T3‐L1 adipocytes. Stimulation of adipocytes with insulin and CL indicated that insulin and CL preferentially phosphorylated/activated PDE3B associated with internal membranes and caveolin‐enriched PM, respectively. Superose 6 chromatography of solubilized membrane proteins from adipocytes stimulated with insulin or CL demonstrated reversible assembly of distinct macromolecular complexes (MMC) that contain 32P‐phosphorylated PDE3B and signaling proteins involved in its activation. siRNA‐mediated knock‐down of caveolin‐1 resulted in down‐regulation of membrane associated PDE3B; insulin‐ and CL‐induced activation of PDE3B was reduced. Insulin‐ and CL‐mediated MMC formation was significantly inhibited by caveolin‐1 knock‐down. These data suggest that caveolin‐1 acts as a molecular chaperone or scaffolding molecule in cholesterol‐rich lipid rafts that may be necessary for the proper stabilization and activation of PDE3B in response to CL and insulin.