Alisol B 23-acetate As natural ligands of GPR several long c
As natural ligands of GPR120, several long chain polyunsaturated fatty acids (Lc-PUFAs) have been demonstrated to modulate the 3T3-L1 adipogenic differentiation. However, the effects of the Lc-PUFAs are inconclusive. DHA is shown to inhibit, promote or even not affect the differentiation of preadipocytes to adipocytes like other fatty acids (Barber et al., 2013, Casado-Díaz et al., 2013, Kim et al., 2006, Murali et al., 2014, Wang et al., 2016). In our experiment, similar to the study of Kim et al. (2006) (Kim et al., 2006), DHA could significantly inhibit the lipid accumulation and the protein level of PPARγ in a dose-dependent manner from the 2 days after MDI-induction to the termination of differentiation. On the other hand, DHA may suppress lipid droplet formation and increase lipolysis by up-regulating adipose triglyceride lipase (ATGL) (Barber et al., 2013). Unlike the results of Kim et al. (2006), 3T3-L1 Alisol B 23-acetate pretreated with DHA for 24 h before the MDI-induction showed pro-adipogenic phenotype due to the DHA treatment (Murali et al., 2014). In addition, by using the luciferase reporter system, DHA was demonstrated as a natural ligand of PPARγ in HEK293T cells in the present study. Wang et al. (2016) reported that the enhancement of adipogenesis in mouse adipose tissue-derived stromal cells (ADSC) was observed in both ALA and LA treatments (Wang et al., 2016). In our results, only ALA could promote adipogenesis by activating PPARγ. The luciferase reporter experiments showed that the ALA, not LA, can well activate PPARγ as reported by others (Lecka-Czernik et al., 2002). Furthermore, ALA cannot improve adipogenesis in the GPR120-knockdown cells, suggesting that GPR120 may be related to the improvement of adipogenesis by ALA. Based on the different effects of fatty acids, we therefore chose the synthetic agonist TUG-891 (Shimpukade et al., 2012) to study the signaling mediated by GPR120 in adipogenesis to avoid other potential GPR120-independent mechanisms which are involved in the role of natural ligands in the present study. We showed that TUG-891 promoted adipogenic progress in both 3T3-L1 and porcine SV cells. Although GPR40 may be also activated by TUG-891 in murine cells, it is not expressed in the murine preadipocytes and adipocytes from the adipose tissues (Gotoh et al., 2007). Both in 3T3-L1 preadipocytes and adipocytes, the mRNA expression level of GPR40 was low but the expression of GPR120 was shown to be significantly increased during the differentiation of predipocytes to mature adipocytes (Oh et al., 2010). Additionally, we have previously reported that porcine GPR120 shares a higher homology with human GPR120 compared with mouse GPR120, and TUG-891 is a potent agonist for porcine GPR120 (Song et al., 2015). TUG-891 increased both mRNA and protein expression of PPARγ, indicating that GPR120 might promote adipogenesis via PPARγ. However, it should be noted that PPARγ and GPR120 have similar ligand binding pockets and share common natural agonists including eicosapentaenoic acid and DHA (Gim et al., 2013, Hudson et al., 2014, Suzuki et al., 2008). It has been demonstrated that the agonist of PPARγ also increases the expression of GPR120 in 3T3-L1 cells (Gotoh et al., 2007). Although TUG-891 is a potent agonist for GPR120, no evidence is available for the relationship between TUG-891 and PPARγ. Therefore, it is interesting to clarify whether the dose of TUG-891 used in the present study can activate PPARγ directly. By using 3 × PPRE-luc reporter assay, we showed that TUG-891 increased the activation of PPARγ in HEK293T cell transfected with GPR120-pcDNA3.1 but not with empty plasmid. The expression of GPR120 in HEK293T cells was negligible and the synthetic agonists cannot activate GPR120 signaling in HEK293T cells (Briscoe et al., 2006, Song et al., 2015). Thus, these results suggested that the responses observed were indeed mediated by GPR120, which excluded the possibility of TUG-891 to activate PPARγ directly.