Proteome Profiling of Mitotic Clonal Expansion during 3T3-L1 Adipocyte Differentiation Using iTRAQ-2DLC-MS/MS

Mitotic clonal expansion (MCE) is
one of the important events taking
place at the early stage during 3T3-L1 adipocyte differentiation.
To investigate the mechanism underlying this process, we carried out
a temporal proteomic analysis to profile the dynamic changes in MCE.
Using 8-plex-iTRAQ-2DLC-MS/MS analysis, 3152 proteins were quantified
during the initial 28 h of 3T3-L1 adipogenesis. Functional analysis
was performed on 595 proteins with maximum or minimum quantities at
20 h of adipogenic induction that were potentially involved in MCE,
which identified PI3K/AKT/mTOR as the most relevant pathway. Among
the 595 proteins, PKM2 (Pyruvate kinase M2), a patterned protein identified
as a potential target gene of C/EBPβ in our previous work, was
selected for further investigation. Network analysis suggested positive
correlations among C/EBPβ, PIN1, and PKM2, which may be related
with the PI3K-AKT pathway. Knockdown of PKM2 with siRNA inhibited
both MCE and adipocyte differentiation of 3T3-L1 cells. Moreover,
PKM2 was down-regulated at both the mRNA level and the protein level
upon the knockdown of C/EBPβ. And overexpressed PKM2 can partially
restore MCE, although it did not restore terminal adipocyte differentiation,
which was inhibited by siC/EBPβ. Thus, PKM2, potentially regulated
by C/EBPβ, is involved in MCE during adipocyte differentiation.
The dynamic proteome changes quantified here provide a promising basis
for revealing molecular mechanism regulating adipogenesis.