PDE4D and PDE3B orchestrate distinct cAMP microdomains in 3T3‐L1 adipocytes

Abstract Background and Purpose Lipolysis is tightly regulated by pro‐lipolytic β ‐adrenoceptor signalling, which activates the cAMP/PKA pathway, and by antilipolytic hormones like insulin and FGF1, which counter‐regulate lipolysis through cAMP‐degrading phosphodiesterases (PDEs). While the spatial compartmentalization of cAMP signalling is recognized, comparisons between distinct cAMP pools remain under‐investigated in adipocytes. Moreover, the dynamics of cAMP around lipid droplets (LD) where lipolysis occurs, are particularly intriguing. Thus, we studied whether adipose FGF1/PDE4D and insulin/PDE3B pathways regulate distinct cAMP microdomains to execute their antilipolytic actions. Experimental Approach We evaluated the role of subcellular cAMP pools in lipolysis regulation by PDEs, or antilipolytic hormones, by utilizing EPAC1‐based FRET cAMP biosensors specifically designed to localize in the cytoplasm or at the plasma membrane of living cells. Additionally, we developed the first LD‐associated cAMP biosensor by fusing the lipid droplet‐associated protein perilipin‐1 to the EPAC1‐based probe. Key Results We identified previously unrecognized cAMP pools surrounding LDs that are distinct from cytoplasmic cAMP and resistant to PDE inhibition or antilipolytic stimuli. PDE4D exhibits a stronger effect on all three cAMP pools investigated than PDE3B. FGF1 mainly inhibits the cAMP in the initiation of the signalling at the plasma membrane, whereas insulin targets mainly cytoplasmic cAMP pools. Conclusion and Implications The discovery of LD‐associated cAMP as a distinct subcellular pool suggests that cAMP signalling in adipocytes is more compartmentalized than previously recognized. The distinct pathways by which FGF1 and insulin regulate adipose cell cAMP levels highlight that antilipolytic signalling is not uniform, refining our understanding of lipolysis regulation.