br Introduction Both the excess obesity and the lack

Introduction Both the excess (obesity) and the lack/atrophy (lipodystrophy) of adipose tissue are associated with altered metabolism and lead to major diseases. In particular, the prevalence of obesity-linked diseases has increased worldwide during the past 10years (Savage et al., 2007). The chronic diseases associated with obesity include diabetes mellitus, arterial hypertension, metabolic syndrome and hyperlipidemia among others. In this context the adipose tissue as a target of research had been neglected for many years. However, this has changed and the adipose tissue has been recognized as an endocrine organ and source of signaling molecules and not merely a storage of fat. Therefore, several models have been set up for dissection of fat cell differentiation, key transcriptional regulators of the adipocyte differentiation program and identification of novel targets for pharmacological intervention (Rosen and MacDougald, 2006; Rosen et al., 2000; Choi and Tontonoz, 2013). A major limitation of essentially all of these models is that they are unable to provide information about the entire differentiation program. Hence, the earliest stages of the differentiation are unknown and therefore the sequence of events leading to preadipocyte commitment remains largely unexplored. In principle, mouse embryonic stem cells (mESCs) and induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006) could allow one to gain insight into the early phases of any differentiation including adipogenesis on different genetic backgrounds (Rosen and MacDougald, 2006; Dani et al., 1997). However, heterogeneity of ESC cultures combined with the low efficiency of the differentiation remains the major limitation of such studies of adipogenesis (Dani et al., 1997; Schaedlich et al., 2010; Chen et al., 2007). Ascorbic TAK-875 (AsA) has been used in various cell differentiation protocols previously (Shin et al., 2004; Takahashi et al., 2003; Kawada et al., 1990; Choi et al., 2008; Weiser et al., 2009). Recently, AsA has been recognized as a novel regulator of epigenetic control of genome activity (Chung et al., 2010; Esteban and Pei, 2012; Minor et al., 2013).
Materials and methods
Results and discussion
Conclusions Adipocyte differentiation from mESC requires EB formation and ATRA dependent activation of RARβ signaling (Lee et al., 2012; Wdziekonski et al., 2007; Simandi et al., 2013) as a necessary first step for successful differentiation. Rosiglitazone treatment is dispensable from days 7 to 15, as it was shown in Fig. 5. In contrast preadipocytes can undergo adipogenesis in confluent cultures with the addition of IBMX, Insulin and Dexamethasone (Hausman et al., 2008). Although to enhance adipogenesis PPAR gamma agonist is commonly used (Kim et al., 2007; Zebisch et al., 2012; Wang et al., 2014). And in some cases as in sorted PDGFRa+ preadipocytes robust adipocyte differentiation can be achieved with the only addition of Insulin (Lee et al., 2012). The mechanism on how AsA enhances the differentiation is not well understood, however it is unlikely that the antioxidant effect of AsA is involved (Shin et al., 2004; Takahashi et al., 2003; Arrigoni and De Tullio, 2002). AsA has been postulated to contribute to other pathways relevant in adipocyte differentiation, including the regulation of the activity of HIF prolyl-hydroxylases and increasing cAMP levels and also has been linked to the activity of the JmjC histone demethylases (Esteban and Pei, 2012; Team, 2011; Pisani et al., 2011; Arrigoni and De Tullio, 2002; Floyd et al., 2007; Farmer, 2006). This aspect of the compound\'s effect on cellular differentiation needs further studies.
Acknowledgment The authors would like to acknowledge the members of the Nagy laboratory, Dr. Gerardo Alvarado and Dr. Istvan Szatmari for discussions and comments on the manuscript. L.N. is supported by a grant from the Hungarian Scientific Research Fund (OTKA K100196). L.N. and I.C.-M. are recipients of the following grant TÁMOP-4.2.2.A-11/1/KONV-2012-0023VÉD-ELEM implemented through the New Hungary Development Plan co-financed by the European Social Fund and the European Regional Development Fund. Z.S. and G.N. are recipients of TÁMOP 4.2.4.A/2-11-1-2012-0001/A2-JÁDJ-13. Microarray studies and Next Generation Sequencing were carried out at the Center for Clinical Genomics and Personalized Medicine at the University of Debrecen, Medical and Health Science Center.