Cytosolic and nuclear distribution of PPARγ2 in differentiating 3T3-L1 preadipocytes

Abstract

In light of the pivotal role that PPARγ2 plays in the expression of fat specific genes (e.g., A-FABP), we have examined the hypothesis that a rise in PPARγ2 protein is required for the expression of A-FABP, and that the acceleration of fat cell differentiation by the thiazolidinedione agent, pioglitazone (PIOG), reflects an increase in the abundance of PPARγ2 mRNA and protein. Western analyses surprisingly revealed that undifferentiated 3T3-L1 fibroblasts contained significant levels of PPARγ2 protein; that the amount of total cellular PPARγ2 only increased 2-fold during differentiation; and that the levels of PPARγ2 protein and mRNA were not increased by PIOG even though fat cell differentiation was accelerated by PIOG as revealed by a 20-fold increase in A-FABP expression. Cell fractionation studies revealed that PPARγ2 was evenly distributed between the cytosolic and nuclear compartments in both undifferentiated and differentiating 3T3-L1 cells. Immunocytochemical studies with a PPARγ2-specific antibody indicated that PPARγ2 was diffusely distributed throughout the cytosol of undifferentiated 3T3-L1 cells, but as the differentiation progressed, the PPARγ2 became focused around the developing lipid droplets. In contrast to PPARγ2, undifferentiated 3T3-L1 cells contained no measurable quantities of RXRα, but once fat cell differentiation was initiated by treatment with IBMX and dexamethasone, the cellular content of RXRα increased several fold. The rise in RXRα content paralleled the induction of A-FABP, but the expression of RXRα was not enhanced by PIOG. Although the amount of PPARγ2 and RXRα was unaffected by PIOG, gel shift assays revealed that PIOG stimulated PPARγ2/RXRα binding to the adipose response element of A-FABP by 5-fold in less than 12 h. Apparently, RXRα rather than PPARγ2 is the pivotal trans-factor essential for the initiation of terminal fat cell differentiation. However, the high cytsolic content of PPARγ2 and its association with the lipid droplet of differentiating 3T3-L1 cells suggests PPARγ2 may possess a cytosolic function in the developing fat cell.—Thuillier, P., R. Baillie, X. Sha, and S. D. Clarke. Cytosolic and nuclear distribution of PPARγ2 in differentiating 3T3-L1 preadipocytes. J. Lipid Res. 1998. 39: 2329–2338.

Keywords

• insulin
• fatty acid binding protein
• gene expression
• pioglitazone
• PPARγ2
• RXRα