# A Short S-Equol Exposure Has a Long-Term Inhibitory Effect on Adipogenesis in Mouse 3T3-L1 Cells
## 4 Discussion
In an Achilles tendon injury, ER\(\beta\) deletion stimulated adipocyte accumulation through activation of the PPAR\(\gamma\) signaling pathways; moreover, the activation of ER\(\beta\) by a specific agonist inhibited the adipogenic differentiation of Tendon-derived stem cells (TDSCs) [56]. Therefore, we hypothesize that the binding of S-equol to ER\(\beta\) during the early step of adipogenesis induction promotes the activation of the receptor, which results in reductions in PPAR\(\gamma\) and C/EBP\(\alpha\), and therefore an inhibition of adipogenesis and adipocyte functions. A remarkable point of our work is that 3T3- L1 cells were treated with S-equolfor only three days, during the induction step, and the inhibitory effect was maintained until the seventh day of adipocyte differentiation. These results indicate that treatment with a low concentration of S-equol in the early step of adipogenesis produces changes in the expression of specific genes that alter the molecular program of the differentiation and maintenance steps, and therefore the adipogenesis process and adipocyte functions. They also suggest that efficient body weight control should not require a daily S-equol administration, which would reduce the cost of the treatment for people with obesity. An interesting finding of our work is the effect of S-equol on the expression of ER\(\alpha\) and ER\(\beta\) as changes in ER\(\alpha\) and ER\(\beta\) expression have been associated with the modulation of adipogenesis. ER\(\beta\) expression decreases during adipocyte differentiation in vitro [30]. ER\(\beta\) activation is related to adipogenesis inhibition, while ER\(\alpha\) activation promotes adipogenesis, proliferation, and migration [28]. Moreover, ER\(\beta\) modulates the transcriptional activity of ER\(\alpha\) by reducing its expression [57; 58; 59]. Therefore, by attenuating the downregulation of ER\(\beta\) on day 7, S-equol could help to maintain the anti-adipogenic effect due to ER\(\beta\) activation. Moreover, by reducing ER\(\alpha\) expression on days 3 and 7, S-equol could also prevent the negative side-effects associated with ER\(\alpha\) activation. Altogether, our results confirmed the potential of ER\(\beta\) ligands, particularly the soy-derived isoflavenoid S-equol, as anti-obesity molecules. Based on our results and data previously described for estradiol, we proposed a hypothetical model to explain the anti-adipogenic effects produced by a short exposure to S-equol (Figure 7). It has been reported that ER interacts with transcription factors C/EBP\(\beta\) and NF-kB and inhibits their binding on the IL-6 gene promoter [60]. During adipogenesis, C/EBP\(\beta\) induces the expression of PPAR\(\gamma\), which, in turn, promotes C/EBP\(\alpha\) expression [61; 62] that activates the transcription of Resistin, Leptin, and Adiponectin genes [63; 64; 65; 66]. On the other hand, NF-kB is associated with the expression of MCP-1 [37; 67]. The activation of membrane ER by estradiol stimulates the activation of intracellular pathways that induce ER binding to the PPAR\(\gamma\) promoter in the nucleus, reducing its expression and inhibiting adipogenesis in differentiating 3T3-L1 and ADSC cells [68]. Therefore, we hypothesize that S-equol binds to ER\(\beta\) in the cytoplasm and is translocated to the nucleus to interact with C/EBP\(\beta\) and NF-kB, which prevents the transcription of PPAR\(\gamma\) and subsequently C/EBP\(\alpha\), reducing Leptin (LEP), Resistin (RETN), Adiponectin (ADIPO), and MCP-1 production. These effects could also be due to ER\(\beta\) activation at the plasma membrane by S-equol followed by a signaling cascade in the cytoplasm that leads to transcriptional inactivation of the PPAR\(\gamma\) gene. On the other hand, it has been shown that PA1-1 promoter activity depends on the ER\(\beta\)/ER\(\alpha\) ratio, and an increase in ER\(\beta\) modulates the induction of the PAI-1 promoter [69].