## 4 Discussion
The results show that C3G significantly upregulated the expression of mitochondrial synthesis-related genes _Tfam_, _NRF1_ and _NRF2_, in both the BAT of both the db/db and mice with DIO, and increased the number of mitochondria and the expression levels of mitochondrial oxidative phosphorylation-related proteins ATP5A, UQCR2, and NDUFB8 [4; 18]. This strongly suggested the promotion of BAT heat production by C3G, thus warranting further exploration of the mechanism by which C3G regulates the action of _Ucp1_ and its related heat production gene. In this study, we first verified the promotional effect of C3G on _Ucp1_ gene expression through animal models. Thereafter, we found that C3G can upregulate the BAT specific gene _Ucp1_ expression during brown adipocyte differentiation, thereby effectively increasing the mitochondrial respiration uncoupling of brown adipocytes, which is consistent with the previously reported conclusion [34]. However, our study found that there is no concentration dependence, and also that the activation of _Ucp1_ by C3G is not achieved by activating the promoter, thus suggesting the existence of other regulatory factors in the process. Numerous studies have shown that the activity of Ucp1 is inhibited by nucleotides and activated by non-lipidated fatty acids [33; 35]. Norepinephrine (NE) released by sympathetic nerves or hypothermia are the main physiological signals that activate Ucp1 synthesis in BAT cells [36]. Furthermore, the synthesis of Ucp1 is strongly regulated by the transcription level; cAMP is the main activator of _Ucp1_ gene transcription [37; 38; 39; 40]; and \(\beta\)3-AR-specific agonists L-755507, CGP12177, CL316243, and BRL35135 can significantly increase the levels of _Ucp1_ mRNA in mammalian BAT cells [38; 39; 40]. In addition, several transcription factors and coregulators found in both WAT and BAT, as well as in other tissues, including PPARs, PGC1\(\alpha\), and ATF2, have been implicated in the transcriptional activation of _Ucp1_[12; 41]. Our data show that C3G can activate _Ucp1_ by upregulating the expression of the transcription factor _Prdm16_. Since Prdm16 is related to heat production [42; 43], it is even clearer that C3G does indeed play a role in weight loss by activating heat production. Prdm16 can form transcription complexes with other transcription factors, such as C/EBP\(\beta\), PGC1\(\alpha\), PPAR\(\alpha\), and PPAR\(\gamma\), to regulate the expression of thermogenic genes [14; 44]. Furthermore, it was found in our previous research that C3G can upregulate PPAR\(\alpha\)[4]. Consequently, on the basis of the above discussion, it would be reasonable to believe that PPAR\(\alpha\) is also involved in _Ucp1_ transcriptional regulation. Through double luciferase experiments, we ascertained that PPAR\(\alpha\) can indeed activate the transcriptional expression of _Ucp1_ in a similar manner to that of Prdm16. Unlike Prdm16, however, PPAR\(\alpha\) is a fatty acid oxidation-related gene that is also expressed in WAT, which suggests that the transcriptional activation of C3G to _Ucp1_ is not limited to BAT. C3G is expected to achieve weight loss and lipid reduction by increasing WAT heat production, which would be consistent with previous conclusions [18]. On the one hand, we proved that C3G could enhance the thermogenesis of BAT. On the other hand, anthocyanins were reported to have anti-cancer and anti-inflammatory effects [45], which were closely related to their antioxidant activities. Therefore, the role of antioxidant activity of C3G in thermogenesis could be further explored. However, there are a variety of metabolites of C3G, and 41 metabolites have been identified. Among them, protocatechuic acid and vanilloid are metabolites that exert the main physiological functions of C3G in the body [46]. For example, PCA and PGA, the degradation products of C3G, inhibit the proliferation of cancer cells, which is closely related to their antioxidant activity [47].