The Role And Mechanism Of Brd2during The Process Of3T3-L1Preadipocyte Differentiation

Adipocytes are the major cellular component in fat tissue and the increase of adipose tissue mass associated with obesity is due in part to an increase in the number of adipocytes (hyperplasia). Adipogenesis, the process by which adipocyte precursors develop into adipocytes, is regulated by a group of specific transcription factors.Brd2is a nuclear-localized transcription factor kinase and has broader normal functions of the ubiquitous double bromodomain-containing BET class of transcriptional co-regulators. The newly described disruption of Brd2in mice causes severe obesity without type II diabetes, but the mechanism is not well understood. The goal of our research is to define the role and mechanism of Brd2during the process of3T3-L1preadipocyte differentiation.In this article, we firstly reported that Brd2knockdown promoted adipogenic differentiation, and part cells could differentiate to adipocytes even without3-isobutyl-l-methylxanthine/dexamethasone/insulin (MDI). Then conversely, Brd2overexpression strongly suppressed adipogenic differentiation. These show that Brd2plays an impotant role during adipogenesis, and we presume that Brd2may normally function as a regulator to transcription factors of adipogenesis.Therefore we stably transfected the cells with plasmids that express the long form of mouse Brd2or Brd2shRNA. Compared with controls, knockdown of Brd2increased the induction of PPARy and C/EBPa, two key factors of adipogenesis, but overexpression of Brd2inhibited the induction of PPARy and C/EBPa during adipogenic differentiation; then we also detected the expression of other relative genes by Q-PCR, the results showed that Brd2down regulation422/aP2^Glut4> Leptin and Irs-1, while have no effect to Pfkfb1. In vitro assays demonstrate that ERK is able to phosphorylate PPARy, decreases the transcriptional activity of PPARy, and Brd2co-represses PPARy transcription. These suggest Brd2may inhibit adipogenesis through ERK signal pathway. The effect of insulin to promote disposal of circulating glucose into skeletal muscle depends on the translocation of the GLUT4glucose transporter from an intracellular compartment to the cell surface. Insulin action is initiated through the binding of the hormone to the extracellular subunits of the insulin receptor. This leads to a conformational change and subsequent activation of the intracellular tyrosine kinase domain of the insulin receptor subunit. The receptor then undergoes a series of transphosphorylation reactions that lead to tyrosine phosphorylation of different substrates, including insulin receptor substrate-1(IRS-1). When tyrosine residues are phosphorylated, they serve as docking and activating sites for phosphatidylinositol3-kinase (PI3-kinase). Insulin-stimulated activation of PI3-kinase leads to GLUT4translocation and increased glucose transport. Otherwise several studies have shown that pharmacologic activation of AMPK with AICAR effectively increases glucose transport in muscle, independent of the actions of insulin. The wholebody disruption of Brd2, causes lifelong severe obesity in mice, but lowered blood glucose. Thus we speculate Brd2regulate glycometabolism by Insulin and AMPK signal pathway in3T3-L1preadipocytes.We wondered whether Brd2regulated adipogenesis through ERK signal pathway. Thus the3T3-L1preadipocytes were stably transfected with the pSiBrd2or pmBrd2plasmids to knockdown or overexpress Brd2. We found that Brd2knockdown strongly suppressed the phosphorylation of ERK1/2; conversely, Brd2overexpression increased the activity of ERK1/2. In order to understand how ERK1/2activity is regulated by Brd2in3T3-L1preadipocytes, first we examined the activation state of the upstream MEK regulators, the Raf family members, under the condition of Brd2silencing or overexpressing. In contrast to ERK1/2activation, neither A-Raf, B-Raf nor C-Raf was significantly hyperphosphorylated in an activating manner by Brd2overexpressing, as the condition of Brd2silencing. These data suggest that the phosphorylation/activation status of the Raf family is not associated with Brd2, because Brd2may be the downstream of Raf, and it has no feedback regulation to Raf in3T3-L1preadipocytes. Moreover, U0126, a chemical inhibitor of MEK1, kinase of ERK, could partial recovery the Brd2overexpression suppressed adipogenic differentiation and422/aP2expression. Otherwise JNK is not involved in the process of Brd2regulated adipogenesis.To test the hypothesis that Brd2regulate glycometabolism by Insulin and AMPK signal pathway, Brd2expression was reduced in the3T3-L1preadipocytes by stable transfection with a plasmid encoding Brd2shRNA. As expected, the activity of Akt was increased, which could be inhibited by LY294002, a chemical inhibitor of PI3K; Conversely, overexpression of Brd2inhibited the phosphorylation of Akt. On the other hand, we found Brd2had negative accommodation to AMPK:Knockdown of Brd2increased the activity of AMPK, which could be decreased by Compound C, a chemical inhibitor of AMPK, or Insulin; otherwise overexpression of Brd2inhibited AMPK activity. To determine whether Insulin and AMPK signal pathway are both involved in the regulation of Brd2to the expression of GIut4, we blocked PI3K/Akt or/and AMPK pathway by specific chemical inhibtors, We found LY294002or Compound C or both of them could inhibit the expression of Glut4in the3T3-L1preadipocytes were stably transfected with the pSiBrd2. However, phosphorylation status of GSK-3, which was identified as the kinase that phosphorylates and inactivates glycogen synthase, the final enzyme in glycogen synthesis, had no change under the condition of Brd2silencing or overexpressing.Taken together, we identified Brd2regulated adipogenesis、glycometabolism and relative gene expressions of3T3-L1preadipocytes, the signal molecules ERK、PI3K/Akt and AMPK were responsible for these processes.