Cytosolic and nuclear distribution of PPAR gamma 2 in differentiating 3T3-L1 preadipocytes

In light of the pivotal role that PPAR gamma 2 plays in the expression of f at specific genes (e.g., A-FABP), we have examined the hypothesis that a ri se in PPAR gamma 2 protein is required for the expression of A-FABP, and th at the acceleration of fat cell differentiation by the thiazolidinedione ag ent, pioglitazone (PIOG), reflects an increase in the abundance of PPAR gam ma 2 mRNA and protein. Western analyses surprisingly revealed that undiffer entiated 3T3-L1 fibroblasts contained significant levels of PPAR gamma 2 pr otein; that the amount of total cellular PPAR gamma 2 only increased 2-fold during differentiation; and that the levels of PPAR gamma 2 protein and mR NA were not increased by PIOG even though fat cell differentiation was acce lerated by PIOG as revealed by a 20-fold increase in A-FABP expression, Cel l fractionation studies revealed that PPAR gamma 2 was evenly distributed b etween the cytosolic and nuclear compartments in both undifferentiated and differentiating 3T3-L1 cells. Immunocytochemical studies with a PPAR gamma 2-specific antibody indicated that PPAR gamma 2 was diffusely distributed t hroughout the cytosol of undifferentiated 3T3-L1 cells, but as the differen tiation progressed, the PPAR gamma 2 became focused around the developing l ipid droplets. In contrast to PPAR gamma 2, undifferentiated 3T3-L1 cells c ontained no measurable quantities of RXR alpha, but once fat cell different iation was initiated by treatment with IBMX and dexamethasone, the cellular content of RXRa! increased several fold. The rise in RXR alpha content par alleled the induction of A-FABP, but the expression of RXR alpha was not en hanced by PIOG, Although the amount of PPAR gamma 2 and RXR alpha was unaff ected by PIOG, gel shift assays revealed that PIOG stimulated PPAR gamma 2/ RXR alpha binding to the adipose response element of A-FABP by 5-fold in le ss than 12 h. Apparently, RXRa rather than PPAR gamma 2 is the pivotal tran s-factor essential for the initiation of terminal fat cell differentiation. However, the high cytsolic content of PPAR gamma 2 and its association wit h the lipid droplet of differentiating 3T3-L1 cells suggests PPAR gamma 2 m ay possess a cytosolic function in the developing fat cell.