**4. Discussion**

In the present study, we showed that CCT could dose-dependently exert an antiadipogenic effect by decreasing lipid accumulation, a hallmark of antiobesity action. Our results showed that 5 μM CCT, but not 1 μM CCT, added at the early stage of cell differentiation reduced lipid accumulation. CCT exhibited the same efficacy as genistein in reducing intracellular fat. However, this result did not allow discriminating whether this decrease was the result of a lower generation of lipid droplets and, hence, a lower lipid load or if it was a cytotoxic effect of CCT on adipocytes via cell death, leading to a reduction in adipocyte mass and intracellular fat. To rule this out, lipid droplet metrics and cell viability were evaluated, with no differences observed in either analysis in the presence or absence of CCT.

Thus, we also investigated the effects of CCT on the expression of essential adipogenesis-related transcription factors involved in coordinating the adipogenic process. We analyzed the effect of CCT on the expression of early (*C*/*EBP*β and *C*/*EBP*δ) and late (*C*/*EBP*α and *PPAR*γ) genes which play key roles in adipogenesis. In this context, regulating the expression of these factors can modulate the differentiation capacity of adipocytes [43].

Typically, the mRNA levels of *C*/*EBP*β and *C*/*EBP*δ increase during early differentiation, subsequently declining after the removal of adipogenic cocktail (AC), at which point the levels of *C*/*EBP*α and *PPAR*γ increase [44,45]. It is known that *C*/*EBP*β and *C*/*EBP*δ promote adipogenesis, at least in part, by inducing *C*/*EBP*α and *PPAR*γ.

Interestingly, our results, at the ID time-point, indicate that adipocyte differentiation in the presence of the highest concentration of CCT led to a decrease in *C*/*EBP*β mRNA levels, while those of C/EBPδ were not affected. However, at the FD time-point of differentiation, both tested concentrations of CCT diminished *C*/*EBP*α mRNA levels, while those of *PPAR*γ remained unchanged.

The antiadipogenic effect of 5 μM is in line with reports showing that a knockdown of *C*/*EBP*β inhibited adipocyte differentiation in 3T3-L1 preadipocytes [45]; however, in our work, CCT did not affect *C*/*EBP*δ expression. It is possible that an unaltered *C*/*EBP*δ expression can compensate for the decrease in C/EBPβ at the early stages of differentiation [46], thus playing a role in the induction of *PPAR*γ expression. Most carotenoids inhibit the adipogenic process via the repression of *PPAR*γ [47,48]; however, CCT did not alter *PPAR*γ mRNA levels at the FD time-point. In this sense, as mentioned in Section 1, CCT is unlike β-carotene and other carotenoids, potentially exerting its effects via another mechanism [49]. On this note, it is known that β-carotene added during the differentiation of NIH 3T3-L1 preadipocytes reduced the triacylglycerol content, as well as the number and size of lipid droplets, compared with control differentiated cells by diminishing *PPAR*γ expression [50]. On the other hand, Gul T et al. [51] reported that crocins at a concentration of 30 μM (treatment for 48 h) were able to diminish intracellular fat in 3T3-L1 cells, albeit producing a decrease in viability. PPARγ is not only crucial for adipogenesis but is also required for the maintenance of differentiated adipocytes [52]. It is known that knockdown of *PPAR*γ compromises adipose tissue function, accompanied by insulin resistance, inflammation, angiogenesis, and fibrosis [53]. Thus, it appears that, by not altering *PPAR*γ mRNA levels, CCT guarantees the culmination of the preadipocyte differentiation.

The decrease in *C*/*EBP*β mRNA level seems to be CCT-dose-dependent, in contrast to that of *C*/*EBP*<sup>α</sup>. This suggests that CCT is an efficient carotenoid in that it selectively downregulates *C*/*EBP*α mRNA levels with no change in *PPAR*γ expression. It has been reported that mice adipocytes in which the *C*/*EBP*α gene was disrupted showed defects in lipid accumulation [54]. Furthermore, various bioactive compounds reduce lipid accumulation in adipocytes by downregulating the expression of *C*/*EBP*α [55]. In line with these observations, the inhibition of *C*/*EBP*α reported herein could be involved in the decrease in intracellular fat caused by CCT during the induction of differentiation.

Taking our findings into consideration, further research should be conducted in this regard to determine the exact mechanism of CCT in combination with other standard therapeutic approaches applied to the obese population. This could contribute to the development of new strategies to improve the treatment of obesity.

Through our study, we have broadened the knowledge related to the health properties of CCT isolated from saffron as an antiadipogenic compound. Our research provides evidence that CCT efficiently reduces lipid accumulation in adipocytes, presumably via downregulation of *C*/*EBP*α expression. We can conclude that CCT is more potent than genistein in reducing lipid accumulation, with a similar effect produced at a lower concentration. It has been reported that genistein has a therapeutic effect on obesity [56], whereas several studies showed that its administration may be effective for adipocyte differentiation [57]. Thus, CCT's greater efficacy than genistein suggests that this natural compound is a potential candidate to be included in dietary therapies aimed at reverting adipose tissue accumulation in obesity.

**Author Contributions:** Conceptualization, S.L.; methodology, N.M.-L., A.B.-B., S.L., M.V. and E.J.-O.; validation, E.J.-O., A.B.-B. and S.L.; formal analysis, S.L.; investigation, S.L., E.J.-O., A.B.-B., E.N. and M.B.; resources, S.L., E.N., G.L.A., N.M.-L. and A.B.-B.; data curation, S.L. and E.J.-O.; writing—original draft preparation, S.L.; writing—review and editing, E.N., A.B.-B. and S.L.; visualization, S.L.; supervision, S.L.; project administration, S.L. All authors read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** We acknowledge the administrative and technical support of Maria Carmen Cifuentes. **ConflictsofInterest:**Theauthorsdeclarenoconflict of interest.
