Adenylyl cyclase P-site ligands accelerate differentiation in Ob1771 preadipocytes

Differentiation of Ob1771 preadipocytes to adipocytes wascharacterized by morphological changes and elevated expression of thespecific marker enzyme, glycerol-3-phosphate dehydrogenase. Adifferentiation response substantially more complete and rapid thanthat obtained with insulin and 3,5,3'-triiodothyronine was observed with established inhibitors of adenylyl cyclases:2',5'-dideoxyadenosine (2',5'-dd-Ado),9-(cyclopentyl)adenine (9-CP-Ade), and 9-(arabinofuranosyl)adenine (9-Ara-Ade), coincident with decreased cellular cAMP levels. These ligands inhibit adenylyl cyclases noncompetitively, via a domain referred to as the P-site because of its requirement for an intact purine moiety. Differentiation was not induced by inosine, a nucleoside known not to act at the P-site, or byN⁶-(2-phenylisopropyl)adenosineor 1,3-diethyl-8-phenylxanthine, agonist and antagonist, respectively,for adenosine A₁ receptors. Alsoineffective were IBMX or forskolin, agents that can raise intracellularcAMP levels. Potency of the differentiation response followed the order2',5'-dd-Ado (1-20 µM) > 9-CP-Ade (10-100µM) = 9-Ara-Ade (10-100 µM) >> inosine, consistent withtheir potencies to inhibit adenylyl cyclases. The data suggest thatinhibition of adenylyl cyclase via the P-site and the consequentreduction in cell cAMP levels facilitate the induction ofdifferentiation in Ob1771 cells. The findings raise the questionwhether the known endogenous P-site ligands participate in thedifferentiation response induced by hormones.