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  • br Results br Discussion Two different lineages of BAs

    2018-10-20


    Results
    Discussion Two different lineages of BAs have been known. Classical BAs are derived from Myf-5 expressing common progenitors for myoblasts and BAs (Seale et al., 2008). The myoblastic precursors originate from the mesenchymal stem buy SAG supplier (MSCs). PRDM16 is crucially involved in the differentiation of myoblasts into classical BAs (Seale et al., 2007, 2008; Kajimura et al., 2009). PRDM16 interacts with C/EBP-β to form an active transcription complex that enhances expression of genes involved in brown adipogenesis, such as PGC-1α (Kajimura et al., 2009). Ahfeldt et al. (2012) induced human iPSCs to differentiate into MSCs and subsequently transduced them with the PPAR-γ2 and C/EBP-β (with/without PRDM16) genes. The resultant cells expressed UCP1 approximately 20-fold higher than WAs (Ahfeldt et al., 2012). Nishio et al. (2012) cultured human iPSCs and ESCs with a cocktail of hematopoietins and succeeded in inducing the classical BAs. The cells improved oral fat and glucose tolerance of normal mice 16 hr after transplantation (Nishio et al., 2012). Our iBAs were induced from iPSCs via a myoblast-like state, and only PRDM16 was required for the conversion of the myoblast-like cells into iBAs (Figure 1), which is consistent with previous literature on the contribution of PRDM16 in differentiation of classical BAs (Seale et al., 2007, 2008; Kajimura et al., 2009). Meanwhile, inducible BAs, also referred to as brite (brown in white), beige, or brown-like adipocytes, are directly converted from MYF-5-negative adipocytes in white lipid depots (Giralt and Villarroya, 2013; Lo and Sun, 2013; Chechi et al., 2013). The induction of the BA-like phenotype (browning) is caused by chronic cold exposure, hormonal or β-adrenergic signals, or pharmacological activation of PPAR-γ. Again, PRDM16 plays a crucial role in the browning; it binds to CTBP1 (C-terminal-binding protein 1) and CTBP2 to repress transcription of various WA-specific genes, whereas an association of PRDM16 with PGC-1α and PGC-1β results in induction of genes involved in BA differentiation (Kajimura et al., 2008). It is also been shown that the vascular endothelium of adipose tissue can transdifferentiate into WAs and BAs (Tran et al., 2012). Unlike these physiological differentiation pathways of BAs, i.e., differentiation of classical BAs from MSCs via myoblasts, and transdifferentiation of inducible BAs from WAs, we succeeded in artificial conversion of unrelated somatic cells into highly functional BAs. In a previous report, not only myoblasts but also immortalized and normal fibroblasts were co-transduced with PRDM16 plus C/EBP-β genes (Kajimura et al., 2009). After the transduction, the immortalized mouse fibroblasts expressed Ucp1 mRNA approximately 1,000-fold higher than untransduced cells, whereas normal human and mouse fibroblasts had approximately 7 and 20 times higher expression levels than untransduced ones, respectively. Mouse BAT expresses approximately 200,000 times higher Ucp1 mRNA compared with fibroblasts that express this gene very faintly (Figure 3F). Our experiments also showed that PRDM16 plus C/EBP-β (PC) very faintly induced the BA-like phenotype in human fibroblasts (Figures 3A and 3B). In contrast, the dBAs induced by C/EBP-β and C-MYC (CM) expressed UCP1 mRNA approximately 100,000-fold higher than untransduced fibroblasts (Figure 3F) and showed a much more significant BA-like phenotype than PC-transduced cells (Figures 3A and 3B). Thus, C-MYC must have played an buy SAG supplier indispensable role in the reprogramming of normal fibroblasts into fully functional dBAs. C-MYC is one of the Yamanaka’s reprogramming factors (Takahashi and Yamanaka, 2006; Takahashi et al., 2007), and although it is not a prerequisite for iPSCs generation (Nakagawa et al., 2008), C-MYC is shown to play important roles in direct reprogramming of mouse fibroblasts into neural stem cells (Han et al., 2012; Thier et al., 2012) and chondrocytes (Hiramatsu et al., 2011). In our procedure, C-MYC induced PRDM16 gene in cooperation with C/EBP-β, which may be crucial for reprogramming of fibroblasts into dBAs (Figure 3C).