Study On The Mechanism Of Mitotic Clonal Expansion During 3T3-L1 Adipocyte Differentiation

Adipose tissue functions not only in energy storage and metabolism, but also as an endocrine organ. Excess of fat accumulation is the key factor of obesity. Improved understanding of adipocyte differentiation mechanism might lead to effective strategies for treatment of obesity and its complications as the molecular biology basis.The increase of adipocyte tissue mass is due to both an increase in the number and size of adipocytes.3T3-L1 cell line is often used as vitro model in preadipocyte differentiation process studies. It is reported that stimulated with hormonal cocktail, growth-arrested 3T3-L1 preadipocyte synchronously reenter the cell cycle and undergo two rounds (about 28 hours per round) of mitotic clonal expansion (MCE), then express genes such as C/EBPa and PPARy that produce the adipocyte phenotype, and differentiated to spherical adipocytes full of triglyceride droplets. However, the critical mechanism by which the preadipocyte reenter cell cycle and undergo MCE is not fully understood. The present studies were focused on the MCE regulated mechanism within the early 28 hours of preadipocyte differentiation program after inducers initiate.Dynamic changes of protein differential expression during the early stage of 3T3-L1 preadipocytes differentiationiTRAQ (isobaric tags for relative and absolute quantitation) is a differential proteomics technology, which is especially pertinent for low abundance proteins, and can be used to synchronously quantify proteins for 4 to 8 samples. Using iTRAQ-2DLC-MS/MS as main analysis technology, we conducted a comprehensive study on the dynamic changes of protein expression throughout the early 28 hours of 3T3-L1 preadipocytes differentiation process after hormone stimulation.After comparative analysis of proteins’ recovery, repeatability and purified efficiency of different methods, we selected acetone precipitation method for sample purification. The eight samples harvested at 0,4,8,12,16,20,24 and 28 hours were purified and labeled by 8-plex iTRAQ reagents, then mixed together and analyzed by 2DLC-MS/MS. AB SCIEX Triple TOFTM 5600 was mainly used for quantification purpose, and the data were compared with those analyzed by QSTAR XL to confirm the credibility of the quantitative results. A total number of 3,152 non-redundant proteins were quantitatively acquired.Transcriptional regulatory network and key factors in MCE during adipocytes differentiationWith bioinformatics, we carried out Box-plot analysis and principal component analysis (PCA) of all quantitative proteins. Results showed that principal components at six monitored time points (0,12,16,20,24 and 28 hour) presented concentrating distribution which made it possible for comparative analysis. Subsequently, FACS cell cycle monitoring result showed 3T3-L1 began its Gl/S transition at 16 hour and peaked at 20 hour (S-phase) after hormone induction. So we presumed differential proteins at 20 hour to be maximum or minimum, and designed four modes (first rise then flat, first drop then flat, first rise then drop and first drop then rise) for differential proteins screening. There were 595 proteins (called as patterned proteins) in accordance with the modes. Comparative analysis of molecular weight (MW) and isoelectric point (PI) to all 3152 non-redundant proteins confirmed that these 595 patterned proteins were without bias, and were appropriate for further bioinformatics analysis and functional identification.During adipocyte differentiation program, C/EBPβ is an important transcription factor which can activate expression of C/EBPa and PPARy, two crucial pro-adipogenic transcription factors. It is demonstrated that the expression and activation of C/EBPβ are also required for MCE. In our previous studies, ChIP-on-chip anslysis combined with gene expression microarrays were employed to search the target genes of C/EBPβ in 3T3-L1 preadipocyte differention process, and 68 genes potentially regulated by C/EBPβ were identified. With combing iTRAQ-2DLC-MS/MS data and gene expression microarrays results, we found out 17 genes in which PKM2 and RRP1B are accorded with our designed modes.We then performed GO enrichment analysis and pathway enrichment analysis in IPA database. Enrichment functions of patterned proteins were mainly involved in embryonic development, multiple diseases (hepatic diseases, cardiovascular diseases, tumors, e. t. c), skeletal and muscular development as well as cardiovascular development. The result indicated that these patterned proteins are related to reproductive development and dysfunctional physiological process such as cell death, abnormal proliferation. Result of pathway enrichment analysis indicated that patterned proteins were significantly enriched in PI3K/AKT/mTOR pathway.According to GO enrichment analysis, pathway enrichment analysis and targeted genes screening, we selected C/EBPβ,CDKN1B, PKM2, RRP1B, RUNX2 and MCM3 as a targeted protein group. Together with the information of 595 patterned proteins in IPA database, we constructed a transcriptional regulated network. The network showed that six selected proteins can not only interrelate with each other, but also with PI3K/AKT/mTOR pathway related proteins. In addition, sub-network diagram suggested that positive correlations may exist in C/EBPβ, PIN1 and PKM2, which may be related with PI3K-AKT pathway.Role of PKM2 in MCE during 3T3-L1 preadipocytes differentiationPKM2 (Pyruvate kinase M2) is an isoenzyme of the glycolytic enzyme pyruvate kinase. Research demonstrated that PKM2 is highly expressed in cancer cells and plays essential role in Warburg Effect. In our subject, PKM2 was detected by two mass spectrometers, Triple TOFTM 5600 and QSTAR XL. And its expression level was significantly changed which presented a continuous rising pattern in 3T3-L1 preadipocyte differentiation process (the concentration of PKM2 at 20 hour was 3.4-fold compared with that at 0 hour). Western blot analysis further supported our result. We then conducted RT-qPCR to detect PKM2 expression level of mRNA. In accordance with previous results, PKM2 mRNA expression is also significantly up-regulated.To understand the role of PKM2 in MCE and adipocyte terminal differentiation, 3T3-L1 preadipocytes were transfected with PKM2 siRNA (siPKM2). Compared with control group, cell numbers at several monitored time points after induction and S-phase cell ratio at 20 hour were clearly lower in PKM2 siRNA group. Adipogenesis was also observed to be impaired. These results showed that PKM2 is crucial in 3T3-L1 preadipocyte MCE and terminal differentiation process.ChIP-on-chip and gene expression microarray results showed that PKM2 is a potential target gene of C/EBPβ. To confirm this prediction, we constructed C/EBPβ-specific siRNA to suppress C/EBPβ expression. The effect of C/EBPP knockdown on the expression of C/EBPP and PKM2 was detected by RT-qPCR and western blot, respectively. Compared with control group, both mRNA expression and protein expression of PKM2 showed significant down-regulation in C/EBPβ siRNA group, together with the loss of adipogenesis. All our data indicated that C/EBPP regulates expression of PKM2 which is closely involved in MCE during adipocyte differentiation.