# A Short S-Equol Exposure Has a Long-Term Inhibitory Effect on Adipogenesis in Mouse 3T3-L1 Cells
## 4 Discussion  
The supplementation with dietary isoflavnes has been shown to have positive effects on body weight reduction. In this context, there are contradictory reports about the effect of equal, an analog of estradiol, on adipocyte differentiation, mainly because of the use of S-equol or the racemic mixture of (R,S)-equol, which creates some uncertainty about the effectiveness of equol as an adipogenesis inhibitor. To our knowledge, this is the first work that describes the effect of S-equol on 3T3-L1 adipocyte differentiation in vitro. Low concentrations of S-equol (1 to 10 \(\upmu\)M) did not significantly affect the viability of 3T3-L1 cells, while higher concentrations led to cell death. Interestingly, S-equol (1-30 \(\upmu\)M) did not affect the viability of differentiating cells either. Considering that a racemic mixture is a 50:50 mix of two enantiomers, this observation roughly corresponds to the previous report of [PERSON] et al., 2013, which described that the cell viability of differentiating 3T3-L1 cells remained almost constant in the presence of 0.1 to 100 \(\upmu\)M of (R,S)-equol [24]. As expected for an estradiol analog and an ER\(\beta\) ligand, S-equol at 10 \(\upmu\)M inhibited adipocyte differentiation, and this anti-adipogenic effect was associated with a reduced expression of the pro-adipogenic factors C/EBP\(\upalpha\) and PPAR? ; moreover, there was a reduction in lipid accumulation and changes in adipokine release, mainly Adiponectin, Leptin, Resistin, MCP-1, and PAI-I, indicating that S-equol also affected the adipocyte role in energy storage and endocrine function. Adipokines have important roles for body weight control and related processes. Adiponectin is involved in the modulation of insulin sensitivity/resistance [31; 32]. Leptin is considered the master regulator of homeostasis by modulating food intake and expenditure energy through the hypothalamic pathway [33]. Resistin participates in insulin resistance, glucose intolerance, and the development of inflammatory processes [34; 35]. PAI-I presents a dual role in the context of obesity, modulating various metabolic and physiologic processes mainly associated with insulin resistance [36]. MCP-1 is involved in insulin resistance and macrophage infiltration in adipose tissue, contributing to the development of the cellular obesogenic microenvironment [37; 38]. The effect of soy isoflav, including equol, on the synthesis and secretion of adipokines has been previously described in 3T3-L1 cells and animal models. For example, [PERSON] et al. reported a dose-dependent reduction in Leptin secretion in differentiated 3T3-L1 adipocytes treated with 5 to 40 \(\upmu\)M of S-equol for two days [39]. Regarding animal models, [PERSON] et al. demonstrated that a supplementation with soy isoflav reduces reduced plasma Adiponectin levels and increased  
Figure 6: Effect of S-equol on the expression of estrogen receptors \(\upalpha\) (**A**) and \(\beta\) (**B**). 3T3-L1 fibroblasts treated with S-equol (10 \(\upmu\)M) for 72 h were induced to differentiation, and mRNA levels of ER\(\alpha\) (**A**) and ER\(\beta\) (**B**) were determined by real-time qRT-PCR on days 3 and 7. Untreated cells were used as control. Data were obtained from three experiments in triplicate and expressed as mean \(\pm\) SD. All values were normalized with respect to \(\beta\)-Actin, and the change in expression was calculated and compared with control cells without treatment on day 3. Statistical analysis was performed by one-way ANOVA with [PERSON]’s multiple comparison post hoc test using the GraphPad Prism software. ******* \(p<0.0001\) vs. control on day 3; &&&& \(p<0.0001\); & \(p<0.05\) vs. control on day 7; ******* \(p<0.0001\) S-equol on day 3 vs. S-equol on day 7.  
the insulin response in obese monkeys [40]. [PERSON] et al. showed that a diet with isoflav, including (R,S)-equol, inhibits MCP-1 expression and release in mice ApoE-/-mice, contributing to atherosclerotic lesions improvement [41].