## 4 Discussion  
This study proved, for the first time, that C3G does not upregulate the expression of Ucp1 by activating the _Ucp1_ promoter. Another novel finding herein is that Prdm16 can bind to the region \(-500\) to \(-150\) bp upstream of the transcription start site of _Ucp1_ gene, thereby activating the promoter activity. It was also proposed for the first time that C3G participates in the synthesis and thermogenesis of brown adipocyte Ucp1 by regulating _Prdm16_. Therrogenesis is one of the most important functions of BAT, a tissue that has recently been shown to play a significant role in energy and glucose metabolism in adults, making it an interesting potential target for strategies aimed at augmenting the energy metabolic rate [23]. In short, increasing BAT activity may be a highly effective therapeutic approach to combating obesity and its related metabolic diseases. Uncoupling proteins (UCPs) are a class of heat-producing proteins distributed on the inner membrane of mitochondria. Their activity is closely related to heat loss, regulated by purine nucleosides (ADP, ATP), guanosine (GDP, GTP), and free fatty acids (FFA), and they, therefore, play an important role in mammalian heat production and energy metabolism regulation [24]. There are five known main types of UCPs, namely Ucp1, UCP2, UCP3, UCP4, and UCP5. Ucp1 activity exists in mammalian BAT, while UCP2 is widely distributed in whole body tissues, such as white adipose tissue (WAT), heart, liver, kidney, spleen, thyroid, and gastrointestinal tract [25]. UCP3 is mainly distributed in skeletal muscle and UCP4 is mainly distributed in brain [26; 27], while UCP5 is mainly found in brain and neural tissues [28]. Ucp1 is the only expression of uncoupling protein found in BAT. Brown fat cells are characterized by densely packed mitochondria that contain Ucp1 in their inner mitochondrial membrane [29]. BAT thermogenesis is dependent on Ucp1 as it is a proton transporter that allows protons to leak across the mitochondrial inner membrane,  
Figure 6: Conformational investigation and interaction study of PPAR\(\alpha\)-C3G **(A)**.The molecular visualization system predicted the binding conformation of ppara to C3G. **(B)** MST technique to quantitate the affinity of ppara for C3G molecular interactions. The final C3G was added at varying concentrations (ranging from 5 \(\upmu\)M to 23,000 \(\upmu\)M). thereby dissipating the electrochemical gradient normally used for ATP synthesis [29]. The regulation of levels of the _Ucp1_ gene, thus, plays a decisive role in the control of Ucp1 content and is at the center of the physiological regulation of BAT heat production [30]. Revealing the involved signal pathway and transcription regulation of Ucp1 in BAT's energy metabolic process not only helps us to better understand the important role of Ucp1 in BAT energy metabolism control but also provides a theoretical foundation for obesity treatment based on BAT. Our previous research showed that dietary supplementation with the functional ingredients of natural plants exhibited a positive effect on BAT functionality in mice. Such plant-based ingredients include mulberry polyphenol extract, such as C3G and rutin [4; 18; 31]. Moreover, some phenolic acids, such as vanillic acid and chlorogenic acid, which are components of mulberry fruit polyphones, also induce the thermogenesis of BAT and could, therefore, be applied to the prevention of dietary-induced obesity and insulin resistance [31; 32]. We previously reported that mulberry and mulberry wine extract (ME and MWE) can not only improve the expression of BAT specific gene _Ucp1_, but also improve the mitochondrial copy number [33]. As the most abundant anthocyanin in ME and MWE, C3G has also been proven to increase the quantity of mitochondria in subsequent experiments [17]. In order to further explore the heat production mechanism of BAT promoted by C3G, we studied the mitochondrial biogenesis and function.