MIX-cAMP signaling in 3T3-L1 preadipocyte differentiation

GABRIELLI,M,; MARTINI,C; RONERO,D; M C VILA

Boston

Congreso; Experimental Biology Meeting; 2013

Adipogenesis is stimulated in 3T3-L1 fibroblasts by a combination of insulin, dexamethasone (DEX), and methylisobutylxanthine (MIX). Mitotic clonal expansion (MCE) precedes differentiation of 3T3-L1 fibroblasts to adipocytes. MIX increases cAMP content, which activates protein kinase A (PKA). However, PKA independent cAMP signaling through EPAC (exchange protein activated by cAMP) has also been reported. We found that H89, a PKA inhibitor, blocks MCE but not differentiation of 3T3-L1 fibroblasts. Differentiation, evaluated by Oil-Red-O staining or quantification of triglycerides, did not occur when MIX was not present in the differentiation mixture but it was restored by addition of either dibutyryl-cAMP (db-cAMP) or 8-CPT-2-MecAMP. The latter activates EPAC but not PKA signaling. Furthermore, we found that MIX, db-cAMP or 8-CPT-2-MecAMP increased PPARγ, a transcription factor required for adipocyte differentiation. PI3K-PKB signaling is known to be required for differentiation. We found that insulin but not MIX, alone or in combination with insulin+DEX, was able to increase PKB phosphorylation. These results suggest that MIX signaling through cAMP-EPAC, which is independent of PI3K-PKB signaling, is also required for 3T3-L1 fibroblasts differentiation to adipocytes.