Identification of signalling pathways involved in the transcriptional control of adipogenesis

An elucidation of the molecular mechanisms and signalling events that regulate adipogenesis is critical to the understanding of obesity-related disorders such as Type 2 diabetes and cardiovascular disease. Adipogenesis is induced by exposure of quiescent preadipocytes to insulin, which acts as a potent mitogen and adipogenic factor by signalling through the insulin-like growth factor-1 (IGF-1) receptor. Another mitogen, basic fibroblast growth factor (bFGF), inhibits differentiation. To understand the mechanisms by which these two potent mitogens have opposing effects on adipogenesis, we focused on the phosphatidylinositol (PI) 3-kinase and p42/p44 mitogen-activated protein (MAP) kinase pathways. Inhibition of the PI 3-kinase pathway with the synthetic inhibitor LY294002, completely abrogated the expression of two key regulatory transcription factors: CCAAT enhancer binding protein-α (C/EBPα) and peroxisome proliferator-activated receptor-γ (PPARγ), thereby indicating that activation of this pathway is required for differentiation. Signalling via the MAP kinase pathway is more complex. Exposure of preadipocytes to the adipogenic inducers: dexamethasone, isobutylmethylxanthine and insulin, resulted in a robust, transient activation of MAP kinase. Basic FGF prolonged MAPK activity and blocked differentiation by preventing the expression of C/EBPα and PPARγ mRNAs. This inhibition is partially rescued by the MAP kinase kinase-1 inhibitor, PD98059, suggesting that prolongation of MAPK activity is responsible for inhibiting differentiation. Differentiation of preadipocytes is also accompanied by dramatic morphological alterations. To investigate the processes responsible for these changes, we analyzed the role of matrix metalloproteinases (MMPs) during adipogenesis. Our studies demonstrate that differentiation in the presence of insulin down-regulated MMP-2 expression with a concomitant reduction of PPARγ. Exposure of preadipocytes to bFGF, on the other hand, prevented this down-regulation of MMP-2. To identify events downstream of PPARγ in regulating MMP-2 expression, we ectopically expressed PPARγ in Swiss 3T3 fibroblasts using a retroviral system (Swiss-Pγ cells). Exposure of these cells to the adipogenic inducers and the PPARγ ligand, troglitazone, resulted in their conversion into adipocytes characterized by the expression of C/EBPα and Glut4 mRNAs. Furthermore, the down-regulation of MMP-2 requires both insulin and troglitazone, suggesting that regulation of MMP-2 is dependent on PPARγ activation.