Requirement of Vimentin Filament Assembly for β3-Adrenergic Receptor Activation of ERK MAP Kinase and Lipolysis

Abstract Catecholamine stimulation of β-adrenergic receptors (βAR) in adipocytes activates the cAMP-dependent protein kinase to promote liberation of fatty acids as a fuel source. The adipocyte β3AR also activates extracellular signal-regulated kinases (ERK)-1 and -2 through direct recruitment and activation of Src kinase. This pathway together with cAMP-dependent protein kinase contributes to maximal β3AR-stimulated lipolysis. In a search for other molecules that might associate with β3AR upon agonist stimulation, we identified vimentin using a proteomics approach. Immunoprecipitation of β3AR from adipocytes in the absence or presence of the β3AR agonist CL316,243, followed by Western blotting for vimentin confirmed this specific interaction. Since vimentin has also been identified on lipid droplets, the functional consequences of blocking the expression or structural integrity of vimentin intermediate filaments on β3AR regulation of ERK activation and lipolysis was assessed. Following disruption of intermediate filaments with β,β′-iminodipropionitrile, as confirmed by confocal microscopy, β3AR-stimulated ERK activation was blocked, and lipolysis was reduced by more than 40%. Independently, depletion of vimentin by small hairpin RNA (shRNA) completely inhibited β3AR-mediated ERK activation and significantly reduced lipolysis. By contrast, disruption of actin-containing microfilaments by cytochalasin D or microtubules by nocodazole had no effect on either lipolysis or ERK activation. These results indicate that vimentin plays an essential role in the signal transduction pathway from β3AR to the activation ERK and its contribution to lipolysis.