The inhibitory guanine nucleotide-binding protein G(i2)alpha induces and potentiates adipocyte differentiation

The present study further elucidates the involvement of the alpha-subunit o f the GTP-binding protein G(i2) in the differentiation of murine 3T3-L1 cel ls. Control and vector-transfected cells attained a fully differentiated ad ipocyte phenotype showing ample lipid droplets. Cells expressing wild type (WT)-G(i2)alpha or the constitutively active R179E-G(i2)alpha, however, bec ame enlarged, less confluent, and produced large amounts of lipids. Differe ntiation consistently increased the triglyceride (TAG) content in control c ells. In both WT-G(i2)alpha and R179E-G(i2)alpha clones, a marked increase in TAG could be detected even prior to insulin/dexamethasone/isobutyl methy lxanthine exposure. The activity of palmitoyl-CoA synthetase (PCS) and glyc erophosphate acyltransferase (GPAT) also increased upon differentiation. WT -G(i2)alpha and R179E-G(i2)alpha overexpression also enhanced PCS and GPAT activities even before differentiation medium was added. The total amount o f phospholipids (PL) generally increased upon differentiation; however, pre - and postdifferentiation values were insignificantly different in cells ex pressing WT-G(i2)alpha and R179E-G(i2)alpha. Differentiation altered the PL profile with a relative shift from phosphatidylcholine and phosphatidyleth anolamine to phosphatidylinositol (PI) in differentiated cells. Finally, di fferentiation yielded a general increase in the activity of basal PI-phosph olipase-C activity. Again, cells expressing WT-G(i2)alpha and R179E-G(i2)al pha demonstrated elevated enzyme activity and enhanced second messenger acc umulation subsequent to differentiation. In summary, cells with the R179E-m utants of G(i2)alpha exhibited stimulated lipid turnover and accumulation i n both undifferentiated and differentiated cells.