Adipocyte differentiation is regulated largely through the actions of the peroxisome proliferatoractivated receptor (PPAR) γ nuclear receptor and the insulin signaling pathway. 3-Phosphoinositidedependent protein kinase-1 (PDK1) serves as a critical regulatory point in insulin signaling through its ability to phosphorylate the activation loop of several protein kinase families. The present study was undertaken to determine the interrelationships between the PDK1 and PPARγ signaling pathways, and their association with adipocyte differentiation. Coexpression of PDK1 and PPARγ1 in 293T cells stimulated PPARγ response element-dependent reporter gene activity in either the presence or absence of ligand. PDK1-mediated stimulation of PPARγ1 activity was comparable in magnitude to the coactivator activated in breast cancer-1, and was blocked by either the corepressor silencing mediator of retinoid and thyroid hormone receptor or dominant-negative PAX8-PPARγ1. Heterologous Gal4-PPARγ1 assays indicated that PDK1 interacted with the ligand binding domain, and physically associated with PPARγ1; however, PDK1-mediated stimulation was not dependent on phosphorylation of PPARγ1 by PDK1. PDK1 stimulatory activity was eliminated by mutation of the α-helical hydrophobic motifs in PDK1, L 268XII, and V313XXLL, and expression of the α-helical region encompassing these motifs stimulated PPARγ response element-dependent transcription. PDK1-PPARγ interaction was confirmed by chromatin immunoprecipitation analysis of the lipoprotein lipase and adipocyte fatty acid-binding protein promoters. In cells expressing PDK1 and PPARγ, binding to PPARγ response elements occurred, which was enhanced by treatment with a PPARγ agonist. Expression of PDK1 in 3T3-L1 or COMMA-1D mammary epithelial cells promoted adipocyte differentiation in the presence of a PPARγ agonist that was comparable to the response of PPARγ1- transfected cells in the presence of agonist; expression of PDK1 and PPARγ resulted in a synergistic effect. Adipocyte differentiation in the presence of a PPARγ agonist was markedly attenuated in PDK1 null cells. These results suggest that PDK1 can function as a PPARγ1 coactivator independently of its catalytic activity and establishes an important mechanistic link between adipocyte differentiation and the insulin signaling pathway.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Endocrinology, Diabetes and Metabolism