[Induced differentiation and signaling factor PTEN expression of 3T3-L1 adipocytes].
OBJECTIVE
To 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.


METHODS
The 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.


RESULTS
For 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.


CONCLUSIONS
Endogenous PTEN expression is down-regulated during 3T3-L1 differentiation, suggesting that PTEN may enhance insulin sensitivity and promote adipogenesis under physiological conditions.
Li
Yun-xiong
YX
Research Center of Medical Sciences, People's Hospital of Guangdong Province, Guangzhou 510089, China. lyx2730@126.com
Meng
Jin-xiu
JX
Cai
Xue-zhen
XZ
Li
Dong-feng
DF
Rong
Ka-bin
KB
Jiang
Wen-ling
WL
Zhang
Ren-li
RL
Yu
Xi-yong
XY
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D051379:Mice; D051059:PTEN Phosphohydrolase / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics
D012333:RNA, Messenger; D051059:PTEN Phosphohydrolase; C487757:Pten protein, mouse; D005947:Glucose