# A Short S-Equol Exposure Has a Long-Term Inhibitory Effect on Adipogenesis in Mouse 3T3-L1 Cells
## 4 Discussion
Importantly, several reports have shown that adipokines can also regulate adipocyte differentiation. Notably, the overexpression of Adiponectin promotes proliferation, lipid accumulation, and adipocyte differentiation in 3T3-L1 cells through the increased expression of pro-adipogenic markers PPAR\(\gamma\), C/EBP\(\alpha\), and ADD1/SREBP1 [42]. Similar results have been reported in differentiated 3T3-L1 cells stimulated with LPS [43]. High Leptin levels were associated with an increase in fat tissue and positively correlated with weight gain, cardiovascular risk, and triglyceride and cholesterol levels [44; 45]. Treatment with 4 and 40 nM of Leptin stimulated adipocyte differentiation, increased lipid accumulation, and enhanced the expression of PPAR\(\gamma\), PLIN1, SREBP1C, as well as TNF\(\alpha\) and Adiponectin, in differentiating 3T3-L1 cells [46]. Resistin knockdown produced an anti-adipogenic effect, reducing the formation of lipid droplets and accumulation of intracellular triglycerides in differentiating 3T3-L1 cells, independently of PPAR\(\gamma\) and C/EBP\(\alpha\), through the reduction in lipogenic genes such as ChREBP, FASN, and SCD1 [47]. The overexpression of PAI-1 in 3T3-L1 inhibited adipocyte differentiation through the reduction in PPAR\(\gamma\) and CEBP\(\alpha\) expression, while PAI-1 inhibition stimulated adipogenesis [48]. Therefore, it is possible that the lower release of Adiponectin, Leptin, and Resistin, and the higher levels of PAI-1, following exposure to S-equol and ER\(\beta\) activation, could also contribute to reduce adipogenesis in 3T3-L1 cells. Interestingly, S-equol seems to produce the same anti-adipogenic effects than estradiol [49; 50; 51], confirming that S-equol is a genuine estrogen analog. However, there were several differences in adipokines synthesis. Notably, the increase in PAI-1 release on day 9 was only observed in S-equol-treated cells, indicating that distinct molecular mechanisms may be activated by both ER\(\beta\) agonists. It has been previously reported that a high concentration of (R,S)-equol (100 uM of equol) is required to significantly repress proliferation, adipogenesis, and expression of PPAR\(\gamma\), C/EBP\(\alpha\), FAS, and CD36, while a lower concentration (10 uM) tends to promote adipocyte differentiation by activating the expression of genes related to adipocyte differentiation in MC3T3-L1 [24]. Conversely, low concentrations of equol (0-20 umol/L) increased adipogenesis and PPAR\(\gamma\) transcriptional activity in mesenchymal stem cell 10T1/2 [23]. Both enantiomeric forms of equol have different binding affinities for ER, with S-equol being the best ligand for ER\(\beta\). The fact that the effective concentration of S-equol to inhibit adipogenesis was about 10-fold lower than the concentration of the racemic mixture highlights the relevance of ER\(\beta\) for adipocyte formation. Congruently, [PERSON] et al. showed that the effects of ER\(\beta\) on body weight and adipocyte size reduction are independent of ER\(\alpha\) using ovariectomized mice lacking ER\(\alpha\)[52]. In the absence of ER\(\alpha\), ER\(\beta\) also has a protective role in reducing inflammation and extracellular matrix markers, to modulate the expansion of adipose tissue [53]. These data confirm the relevance of specific ER\(\beta\) ligands, such as S-equol, for adipogenesis control. In this context, several studies have shown that ER\(\beta\) agonists reduce body weight and WAT through the reduction in PPAR\(\gamma\) activity [11; 54]. [PERSON] et al. showed that the activation of ER\(\beta\) by the DPN agonist repressed adipogenesis and downregulated PPAR\(\gamma\), PGC-1\(\alpha\), and UCP-1 expression in adipose-derived stem cells (ASCs) of brown adipose tissue extracted from male C57 BL/6 mice; in contrast, the use of PPT, an ER\(\alpha\) agonist, was associated with proliferation and migration processes [28]. Moreover, ER\(\beta\) activation by DPN negatively correlated with Leptin expression in differentiating 3T3-L1 cells [55].