OBJECTIVETo optimize the condition for inducing the differentiation of 3T3-L1 preadipocytes into adipocytes and study the expression of PTEN tumor suppression gene in this process, aiming to understand the regulatory role of PTEN in normal adipocyte differentiation and collect laboratory evidence for developing drugs targeting PTEN.

METHODSThe differentiation of 3T3-L1 preadipocytes cultured in high-glucose DMEM were induced according to 2 protocols with different combinations of dexamethasone, isobutylmethylxanthine (IBMX) and insulin, and the resultant adipocytes were identified by oil red O staining. The total proteins of 3T3-L1 were extracted and analyzed by Western blotting, and PTEN homology between mice and human was analyzed by bioinformatic method.

RESULTSFor optimized 3T3-L1 differentiation, 3T3-L1 cells were initially induced with the combination of 1 micromol/L dexamethasone, 0.5 mmol/L IBMX and 5 microg/ml insulin for 48 h, followed by treatment with 5 microg/ml insulin in 4.5 g/L glucose DMEM for 48 h, which resulted in high differentiation rate of 3T3-L1 cells (up to 90% on the 10th day) with unified morphology and size. PTEN expression varied quantitatively in the process of differentiation, especially low on the 12th day as compared with those measured on days 4, 6 and 9. The mice PTEN mRNA shared 96% homology and PTEN amino acid 100% homology with their human counterparts.

CONCLUSIONEndogenous PTEN expression is down-regulated during 3T3-L1 differentiation, suggesting that PTEN may enhance insulin sensitivity and promote adipogenesis under physiological conditions.