Original Article

Cytosolic and nuclear distribution of PPAR2 in differentiating 3T3-L1 preadipocytes

Correspondence to: Steven D. Clarke.

In light of the pivotal role that PPAR2 plays in the expression of fat specific genes (e.g., A-FABP), we have examined the hypothesis that a rise in PPAR2 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 PPAR2 mRNA and protein. Western analyses surprisingly revealed that undifferentiated 3T3-L1 fibroblasts contained significant levels of PPAR2 protein; that the amount of total cellular PPAR2 only increased 2-fold during differentiation; and that the levels of PPAR2 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 PPAR2 was evenly distributed between the cytosolic and nuclear compartments in both undifferentiated and differentiating 3T3-L1 cells. Immunocytochemical studies with a PPAR2-specific antibody indicated that PPAR2 was diffusely distributed throughout the cytosol of undifferentiated 3T3-L1 cells, but as the differentiation progressed, the PPAR2 became focused around the developing lipid droplets. In contrast to PPAR2, undifferentiated 3T3-L1 cells contained no measurable quantities of RXR, but once fat cell differentiation was initiated by treatment with IBMX and dexamethasone, the cellular content of RXR increased several fold. The rise in RXR content paralleled the induction of A-FABP, but the expression of RXR was not enhanced by PIOG. Although the amount of PPAR2 and RXR was unaffected by PIOG, gel shift assays revealed that PIOG stimulated PPAR2/RXR binding to the adipose response element of A-FABP by 5-fold in less than 12 h.

Apparently, RXR rather than PPAR2 is the pivotal trans-factor essential for the initiation of terminal fat cell differentiation. However, the high cytsolic content of PPAR2 and its association with the lipid droplet of differentiating 3T3-L1 cells suggests PPAR2 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 PPAR2 in differentiating 3T3-L1 preadipocytes. J. Lipid Res. 1998. 39: 2329–2338.

Supplementary key words: insulin, fatty acid binding protein, gene expression, pioglitazone, PPAR2, RXR

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