Previous investigations have demonstrated that both G s -and the G i -family of GTP-binding proteins are implicated in differentiation of the 3T3-L1 preadipocyte. In order to further analyze the role of G s a vs. G i2 a, which are both involved in adenylate cyclase modulation, we transfected undifferentiated 3T3-L1 cells with two sets of G-protein cDNA: the pZEM vector with either wild type, the activating (i.e., GTP-ase inhibiting) R201C-G s a or the inactivating G226A(H21a)-G s a point mutations, or the pZIPNeoSV(X) retroviral vector constructs containing the G i2 a wild type or the missense mutations R179E-G i2 a, Q205L-G i2 a, and G204A(H21a)-G i2 a.
The activating [R201C]G s a-mutant did not significantly affect the differentiation process, i.e., increase in the steady-state levels of G-protein subunits, gross appearance, or insulin-elicited deoxy-glucose uptake into 3T3-L1 adipocytes, despite a marked initial increase in hormone-elicited adenylate cyclase activity. The [H21a]G s a-mutant, on the other hand, enhanced the degree of differentiation slightly, as evidenced by an augmented production of lipid vesicles and insulin-stimulated deoxy-glucose uptake. However, an expected increase in mRNA for hormone-sensitive lipase was not seen. Secondly, it appeared that both activating [R179E]G i2 a or [Q205L]G i2 a mutants reduced cell doubling time in non-confluent 3T3-L1 cell cultures, while [H21a]G i2 a slowed proliferation rate. Furthermore, it seemed that cell proliferation, as evidenced by thymidine incorporation, ceased at a much earlier stage prior to cell confluency when cultures were transfected with the [R179E]G i2 a or [Q205L]G i2 a mutants. Upon differentiation with insulin, dexamethasone, and iBuMeXan, the following cell characteristics emerged: the [R179E]G i2 a and [Q205L]G i2 a mutants consistently enhanced adenylate cyclase activation and cAMP accumulation stimulated by isoproterenol and corticotropin over controls. Deoxy-glucose uptake was also super-activated by the [R179E]G i2 a and [Q205L]G i2 a mutants. Finally, steady-state levels of hormone sensitive lipase mRNA were dramatically increased by [R179E]G i2 a and [Q205L]G i2 a over differentiated controls. The inactivating [H21a]G i2 a-mutant obliterated all signs of preadipocyte differentiation.
It is concluded that G i2 plays a positive and much more important role than G s in 3T3-L1 preadipocyte differentiation.