Black Ginseng and Ginsenoside Rb1 Promote Browning by Inducing UCP1 Expression in 3T3-L1 and Primary White Adipocytes

In this study, we investigated the effects of black ginseng (BG) and ginsenoside Rb1, which induced browning effects in 3T3-L1 and primary white adipocytes (PWATs) isolated from C57BL/6 mice. BG and Rb1 suppressed the expressions of CCAAT/enhancer-binding protein alpha (C/EBPα) and sterol regulatory element-binding transcription factor-1c (SREBP-1c), whereas the expression level of peroxisome proliferator-activated receptor gamma (PPARγ) was increased. Furthermore, BG and Rb1 enhanced the protein expressions of the brown-adipocyte-specific markers PR domain containing 16 (PRDM16), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), and uncoupling protein 1 (UCP1). These results were further supported by immunofluorescence images of mitochondrial biogenesis. In addition, BG and Rb1 induced expressions of brown-adipocyte-specific marker proteins by AMP-activated protein kinase (AMPK) activation. BG and Rb1 exert antiobesity effects by inducing browning in 3T3-L1 cells and PWATs through AMPK-mediated pathway activation. We suggest that BG and Rb1 act as potential functional antiobesity food agents.


Introduction
Obesity is a metabolic disorder characterized by hyperplasia and hypertrophy of white adipose tissue (WAT) and is associated with several comorbidities, including cardiovascular disease, hypertension, stroke, diabetes, cancer, and nonalcoholic fatty liver disease [1,2]. The important features of WAT include storage of excess energy as triglycerides and release of adiponectin and leptin for energy homeostasis [3,4]. In contrast, brown adipose tissue (BAT) utilizes energy as heat through the oxidation of fatty acids. There are two types of BAT in mammals: classical BAT and brown-like adipose tissue. Brown-like adipose tissue is present within WAT and undergoes a transition to brown-in-white tissue after induction [1,5,6]. Brown-like adipose tissue is also known as brite, beige, or induced and its structural and functional aspects are similar to classical BAT [1]. The discovery of BAT has opened new options for formulating novel strategies to combat obesity and associated risk factors [7]. Therefore, besides suggesting dietary restrictions and physical activity as remedies for diet-induced obesity, browning induction has been considered an alternative strategy [3].
The leading functional factor of BAT is uncoupling protein 1 (UCP1), which is involved in adaptive thermogenesis [8,9]. UCP1 is localized in the inner mitochondrial membrane of BAT, generating heat instead of ATP by disturbing the proton gradient of the membrane [10]. Furthermore, the other hallmark proteins of BAT include a PR domain containing 16 (PRDM16) and peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 alpha (PGC-1α). To date, the thermogenic potential of bioactive agents has been demonstrated by many studies, involving the transition of WAT to beige adipose tissue types [11][12][13][14].
Ginseng (Panax ginseng C.A. Meyer) has been a popular traditional herbal supplement in the Korean Peninsula for centuries. Black ginseng (BG) is processed by drying and steaming ginseng nine times. The steaming process results in the conversion of ginsenosides of white and red ginseng into less polar types, and 19 ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg6, F4, Rk3, Rh4, 20(S)-, 20(R)-Rg3, 20(S)-, 20(R)-Rs3, Rk1, Rg5, Rs4, and Rs5) have been newly discovered in BG [15]. BG has much more substantial biological activities than white and red ginseng, including antiobesity, antidiabetes, anticancer, anti-inflammatory, antinociceptive, antioxidant, antihyperglycemic, and immune-modulating activities [16][17][18][19][20]. In addition, the health-promoting activities of ginsenoside Rb1 have also been documented [21]. For example, Rb1 has been reported to exhibit antidiabetic potential by improving glucose tolerance [22,23], as well as antiobesity [22], anti-inflammatory, immunomodulatory [24,25], hepatoprotective [26], antiatherosclerotic [21], and neuroprotective [27] bioactivities. Furthermore, Shang et al. demonstrated the protective effects of Rb1 against adipogenesis by increasing expressions of PPARγ and CCAAT/enhancer-binding protein alpha (C/EBPα) [28]. Consistent with the study by Shang et al., recent studies have described the antiobesity effects of ginseng and ginsenosides, including the induction of browning by Rg1 in 3T3-L1 cells and subcutaneous WAT [2, 3,29]. These findings suggest that ginsenosides from ginseng play an important role as bioactive components. However, little information is available on the bioactivities of Korean BG and saponins associated with browning and obesity. Thus, the present study aimed to examine the antiobesity activity of BG and ginsenoside Rb1 in 3T3-L1 preadipocytes and primary white adipocytes (PWATs) via induction of the browning mechanism. To our knowledge, this is the first investigation into the browning effects of BG and Rb1 on 3T3-L1 cells and PWATs.

Materials
The u-HPLC analysis of BG showed that the total ginsenoside content in the roots and leaves were 21.00 and 22.94 mg/g, respectively (JGR, Park et al., 2019 in press) [30].

Cell Culture and Differentiation
The 3T3-L1 preadipocytes were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% bovine calf serum (BCS) and 1% antibiotics. The cells were grown at 37 • C in a CO 2 (5%) incubator. After the cells reached confluence, differentiation of 3T3-L1 cells was initiated by incubating the cells in differentiation induction medium (0.5 mM methylisobutylxanthine (IBMX), 1 µM dexamethasone (DEX), and 5 µg/mL of insulin) in DMEM including 10% fetal bovine serum (FBS). After 3 days, the differentiation medium was replaced with postdifferentiation medium consisting of 10% FBS and 5 µg/mL of insulin. The medium was changed every 2 days, which was continued until the 3T3-L1 preadipocytes were fully differentiated into white adipocytes. On day 7, the fully differentiated white adipocytes were used for further experiments.

Primary White Adipocyte Preparation
Subcutaneous fat tissues of 5-6-week-old male C57BL/6 mice were used for the isolation of PWATs. Briefly, subcutaneous tissues were crushed with scissors and digested with collagenase type II enzyme (Sigma-Aldrich, St. Louis, MO, USA) in a water bath at 37 • C for 1 h. The mixture was filtered using 40 µm cell strainers (SPL Life Science, Gyeonggi, Korea) and centrifuged at 300× g for 7 min. The pellet consisting of stromal vascular fractions was resuspended in DMEM supplemented with 10% BCS and 1% antibiotics. After reaching confluence, differentiation was initiated by incubating the cells for 3 days in the differentiation induction medium (the composition was the same as described in Section 2.2). The animal tissue experiments followed the Guidelines for the Care and Use of Laboratory Animals of Gachon University (reference number: GIACUC-R2018016).

Cell Viability Assay
The cytotoxicity of BG and Rb1 was measured using a Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Rockville, MD, USA) according to the manufacturer's instructions. 3T3-L1 preadipocytes were seeded in a 96-well plate at a density of 5 × 10 4 cells/well and treated with varying concentrations of BG and Rb1 for 24, 48, and 72 h. Finally, the cells were treated with 10 µL of CCK-8 and incubated at 37 • C for 2 h. The absorbance was measured at 450 nm using a microplate spectrophotometer system (BioRad, Hercules, CA, USA). The results were expressed as a percentage of cell viability and each experiment was repeated three times.

Oil Red O Staining
Lipid droplets in the differentiated 3T3-L1 adipocytes were detected by Oil Red O staining. Differentiated adipocytes were washed with phosphate-buffered saline (PBS), fixed with 10% formalin (Thermo Fisher Scientific, Waltham, MA, USA), and dried working solution (60% isopropanol and 40% distilled water, w/v) was added to each well at room temperature (RT) for 30 min. The cells were washed three times with deionized water and observed under an inverted microscope. Finally, the dye was eluted using 100% isopropanol to quantify the intracellular lipid content, and the absorbance was determined at 500 nm.

Effect of BG and Rb1 on Adipogenesis and Browning Markers
To evaluate the effect of BG and Rb1 on adipogenesis, the expression of adipogenic marker proteins, including PPARγ, C/EBPα, and SREBP-1c, were examined by Western blot analysis in both 3T3-L1 cells and PWATs. Furthermore, to evaluate browning, the expression of BAT-specific marker proteins (UCP1, PRDM16, and PGC-1α) was investigated by immunoblotting in both models with and without treatment with varying concentrations of BG and Rb1. Each experiment was repeated three times.

Immunofluorescence Staining
Immunofluorescence was used to evaluate the effects of BG and Rb1 on fundamental protein, UCP1, and mitochondrial biogenesis using both 3T3-L1 cells and PWATs. For immunocytochemical analyses, differentiated adipocytes were fixed with 4% formaldehyde and permeabilized with 0.2% Triton X-100 (Sigma, St. Louis, MO, USA), followed by blocking with 1% BSA in PBST for 30 min. After that, the cells were incubated with anti-UCP1 antibody (1:250, Abcam) overnight at 4 • C. The cells were washed with PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondary antibody (1:500, Promega, Madison, WI, USA) in 1% BSA for 1 h. Mitochondria were stained using MitoTracker Red CMXRos (500 nM; Cell Signaling Technology) for 30 min according to the manufacturer's protocol. Then, the cells were fixed and rinsed three times with PBS. The nuclei of the cells were stained with 4 ,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, MA, USA). Fluorescence images were captured by using an inverted phase-contrast microscope with fluorescence (KI-2000F, Korea Lab Tech, Gyeonggi, Korea) and analyzed with image-processing software (OptiView 3.7, Korea Lab Tech, Gyeonggi, Korea).

Induction of Browning by AMPK Activation
3T3-L1 and PWAT cells were treated with an AMPK activator (5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)) and an AMPK inhibitor (dorsomorphin; Sigma-Aldrich, St. Louis, MO, USA) to examine the browning effects caused by BG and Rb1. AICAR (10 µM) or dorsomorphin (5 µM) was added to the differentiation induction and maturation media until the cells were harvested. Each experiment was repeated three times. To further evaluate the possible mechanism of browning, we measured the expression of p-AMPK and browning hallmark proteins (UCP1, PRDM16, and PGC-1α) in the presence of AICAR and dorsomorphin in PWATs.

Statistical Analyses
All data are expressed as mean ± standard deviation (SD). All experiments were performed in triplicate. One-way ANOVA and Tukey's post hoc tests were performed using GraphPad Prism 5.03 (GraphPad Software Inc., San Diego, CA, USA). p-values less than 0.05 indicted statistical significance.

Effect of BG and Rb1 on the Differentiation in 3T3-L1 Cells
The effects of BG and Rb1 on 3T3-L1 cell differentiation are shown in Figures 1 and 2, respectively. Fully differentiated 3T3-L1adipocytes not treated with BG or Rb1 showed typical lipid accumulation by Oil Red O staining. The lipid droplets were decreased by treatment with BG ( Figure 1A) and Rb1 ( Figure 2A) and lipid accumulation in the differentiated adipocytes was significantly reduced upon treatment with BG (50 and 100 µg/mL; Figure 1B) and Rb1 (20 and 40 µM; Figure 2B). The CCK-8 assay results revealed that BG and Rb1 had no significant cytotoxicity at 100 µg/mL BG or Rb1concentrations below 100 µM ( Figures 1C and 2C, respectively).  The data are presented as mean ± SD for three different experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. Con.

Effect of BG and Rb1 on Adipogenesis in 3T3-L1 and Primary White Adipocytes
We added BG and Rb1 during the differentiation of 3T3-L1 cells and PWATs. The adipogenic markers PPARγ, C/EBPα, and SREBP-1c were examined by Western blot analysis. After differentiation, BG increased the expression levels of PPARγ in 3T3-L1 cells in both types of adipocytes in a dose-dependent manner ( Figure 3). Notably, 100 μg/mL of BG significantly increased the expression of PPARγ compared with the control group. The relative Western blot expressions of C/EBPα and SREBP-1c were inhibited by BG treatment in dose-dependent manners in both 3T3-L1 cells and PWATs. As shown in Figure 4, Rb1 treatment upregulated the expression of PPARγ in both types of adipocytes. However, the increase was not observed in a dose-dependent manner in PWATs.  The data are presented as mean ± SD for three different experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. Con.

Effect of BG and Rb1 on Adipogenesis in 3T3-L1 and Primary White Adipocytes
We added BG and Rb1 during the differentiation of 3T3-L1 cells and PWATs. The adipogenic markers PPARγ, C/EBPα, and SREBP-1c were examined by Western blot analysis. After differentiation, BG increased the expression levels of PPARγ in 3T3-L1 cells in both types of adipocytes in a dose-dependent manner ( Figure 3). Notably, 100 μg/mL of BG significantly increased the expression of PPARγ compared with the control group. The relative Western blot expressions of C/EBPα and SREBP-1c were inhibited by BG treatment in dose-dependent manners in both 3T3-L1 cells and PWATs. As shown in Figure 4, Rb1 treatment upregulated the expression of PPARγ in both types of adipocytes. However, the increase was not observed in a dose-dependent manner in PWATs. (C) Relative viability of 3T3-L1 cells in the presence of Rb1, evaluated using a CCK-8 assay. The data are presented as mean ± SD for three different experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. Con.

Effect of BG and Rb1 on Adipogenesis in 3T3-L1 and Primary White Adipocytes
We added BG and Rb1 during the differentiation of 3T3-L1 cells and PWATs. The adipogenic markers PPARγ, C/EBPα, and SREBP-1c were examined by Western blot analysis. After differentiation, BG increased the expression levels of PPARγ in 3T3-L1 cells in both types of adipocytes in a dose-dependent manner ( Figure 3). Notably, 100 µg/mL of BG significantly increased the expression of PPARγ compared with the control group. The relative Western blot expressions of C/EBPα and SREBP-1c were inhibited by BG treatment in dose-dependent manners in both 3T3-L1 cells and PWATs. As shown in Figure 4, Rb1 treatment upregulated the expression of PPARγ in both types of adipocytes. However, the increase was not observed in a dose-dependent manner in PWATs.
In contrast, Rb1 substantially inhibited C/EBPα and SREBP-1c expression in a dose-dependent manner in both 3T3-L1 cells and PWATs.
Nutrients 2018, 10, x FOR PEER REVIEW 6 of 14 dose-dependent manner in PWAT. In contrast, Rb1 substantially inhibited C/EBPα and SREBP-1c expression in a dose-dependent manner in both 3T3-L1 cells and PWAT.       ) and PWATs (C,D) or absence of Rb1 (Con). The β-actin protein was used as an internal control. The data are presented as mean ± SD for three different experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Con.

Effect of BG and Rb1 on the Expression of Brown Adipocyte Markers in 3T3-L1 Cells and PWATs
To investigate the browning effects of BG and Rb1, 3T3-L1 cells and PWATs were treated with BG and Rb1. Western blot analysis revealed that 100 µg/mL BG treatment significantly increased the expression of brown adipocyte markers (UCP1, PRDM16, and PGC-1α) in both types of adipocytes  Figure 5). In addition, UCP1 and PGC-1α were upregulated in 3T3-L1 adipocytes treated with 50 µg/mL of BG. Rb1 treatments (20 and 40 µM) also significantly increased the expression of the brown adipocyte markers in both types of adipocytes in dose-dependent manners ( Figure 6).

Effect of BG and Rb1 on the expression of brown adipocyte markers in 3T3-L1 and PWAT
To investigate the browning effects of BG and Rb1, 3T3-L1 cells and PWAT were treated with BG and Rb1. Western blot analysis revealed that 100 μg/ml BG treatment significantly increased the expression of brown adipocyte markers (UCP1, PRDM16, and PGC-1α) in both types of adipocytes ( Figure. 5). In addition, UCP1 and PGC-1α were upregulated in 3T3-L1 adipocytes treated with 50 μg/ml of BG. Rb1 treatments (20 and 40 μΜ) also significantly increased the expression of the brown adipocyte markers in both types of adipocytes in dose-dependent manners ( Figure. 6).  ) and PWAT (C, D) or absence of BG (Con). The β-actin protein was used as an internal control. The data is represented as mean ± SD for three different experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Con.   ) and PWATs (C,D) or absence of BG (Con). The β-actin protein was used as an internal control. The data are presented as mean ± SD for three different experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Con.

Effect of BG and Rb1 on the expression of brown adipocyte markers in 3T3-L1 and PWAT
To investigate the browning effects of BG and Rb1, 3T3-L1 cells and PWAT were treated with BG and Rb1. Western blot analysis revealed that 100 μg/ml BG treatment significantly increased the expression of brown adipocyte markers (UCP1, PRDM16, and PGC-1α) in both types of adipocytes ( Figure. 5). In addition, UCP1 and PGC-1α were upregulated in 3T3-L1 adipocytes treated with 50 μg/ml of BG. Rb1 treatments (20 and 40 μΜ) also significantly increased the expression of the brown adipocyte markers in both types of adipocytes in dose-dependent manners ( Figure. 6).  ) and PWAT (C, D) or absence of BG (Con). The β-actin protein was used as an internal control. The data is represented as mean ± SD for three different experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Con.   L1 (A,B) and PWATs (C,D) or absence of Rb1 (Con). The β-actin protein was used as an internal control. The data are presented as mean ± SD for three different experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Con.

Immunofluorescence
As described above, BG and Rb1 treatments resulted in increased expression of BAT-specific marker proteins (UCP1, PRDM16, and PGC-1α), which suggests the possibility of browning in 3T3-L1 cells and PWATs. Immunofluorescence was used to evaluate the effects of BG and Rb1 on fundamental protein, UCP1, and mitochondrial biogenesis. Differentiated 3T3-L1 cells and PWATs were stained with MitoTracker Red (red), then incubated with UCP1 antibody (green). As shown in Figure 7A,B, mitochondrial density was significantly increased in both BG-and Rb1-treated adipocytes, which was confirmed by enhanced red fluorescent dye binding to mitochondria. Likewise, the expression of UCP1 (green) was enhanced in the 3T3-L1 cells and PWATs by BG and Rb1 treatment.

Effect of BG and Rb1 on Activation of AMPK
To determine the possible mechanism of adipocyte browning by BG and Rb1, we measured the expression levels of AMPK and p-AMPK. The ratios of p-AMPK/AMPK were significantly increased following BG and Rb1 treatment of 3T3-L1 cells (Figures 8A and 9A) and PWATs (Figures 8B and  9B).

Effect of BG and Rb1 on Activation of AMPK
To determine the possible mechanism of adipocyte browning by BG and Rb1, we measured the expression levels of AMPK and p-AMPK. The ratios of p-AMPK/AMPK were significantly increased following BG and Rb1 treatment of 3T3-L1 cells (Figures 8A and 9A) and PWATs (Figures 8B and 9B).

Effect of BG and Rb1 on Activation of AMPK
To determine the possible mechanism of adipocyte browning by BG and Rb1, we measured the expression levels of AMPK and p-AMPK. The ratios of p-AMPK/AMPK were significantly increased following BG and Rb1 treatment of 3T3-L1 cells (Figures 8A and 9A) and PWATs (Figures 8B and  9B). Expressions were measured by Western blotting. The data are presented as mean ± SD for three different experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Con. The data are presented as mean ± SD for three different experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Con.

BG and Rb1 Induces Browning Effect via AMPK-Mediated Pathway
To further examine the mechanism of the browning effects of BG and Rb1, we measured the expression of AMPK phosphorylation and UCP1, PRDM16, and PGC-1α browning hallmark proteins following treatment with an AMPK activator (AICAR; 10 μM) and an AMPK inhibitor (dorsomorphin; 5 μM) in PWATs. As shown in Figure 10, BG treatment in the presence of AICAR increased the expression of UCP1, PRDM16, and PGC-1α and increased the p-AMPK/AMPK expression ratio, while dorsomorphin (inhibitor) abolished the expressions of these proteins compared with the control. Similarly, Rb1 treatment also increased the p-AMPK/AMPK expression ratio and the browning markers compared with controls in PWAT cells (Figure 11).  The data are presented as mean ± SD for three different experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Con.

BG and Rb1 Induces Browning Effect via AMPK-Mediated Pathway
To further examine the mechanism of the browning effects of BG and Rb1, we measured the expression of AMPK phosphorylation and UCP1, PRDM16, and PGC-1α browning hallmark proteins following treatment with an AMPK activator (AICAR; 10 µM) and an AMPK inhibitor (dorsomorphin; 5 µM) in PWATs. As shown in Figure 10, BG treatment in the presence of AICAR increased the expression of UCP1, PRDM16, and PGC-1α and increased the p-AMPK/AMPK expression ratio, while dorsomorphin (inhibitor) abolished the expressions of these proteins compared with the control. Similarly, Rb1 treatment also increased the p-AMPK/AMPK expression ratio and the browning markers compared with controls in PWAT cells ( Figure 11).

Discussion
The prevalence of obesity and associated metabolic disorders continues to increase worldwide. Since browning leads to weight loss through energy expenditure, research related to increasing thermogenesis or browning has gained momentum recently. Therefore, the use of recruitable brown adipocytes represents a promising strategy to treat and prevent obesity [31]. For this reason, functional foods or bioactive ingredients are being explored for inducing browning using in vivo and in vitro models.
In the present study, the effects of BG and Rb1 in 3T3-L1 cells and PWATs were investigated to determine whether the characteristics of white adipocytes could be converted to those of brown-like adipocytes. The browning effects were evaluated by measuring the expression of PRDM16, UCP1, and PGC-1 brown-adipocyte-specific hallmark proteins in both types of adipocytes. Our results show that BG and Rb1 significantly upregulated the expression of PRDM16, UCP1, and PGC-1 in dosedependent manners in both models. Moreover, mechanistic implications suggest that the target compounds used in the present study activated the AMPK signaling cascade responsible for the increased expression of hallmark proteins, thereby promoting browning in 3T3-L1 cells and PWATs.
UCP1 is a hallmark brown adipocyte protein that plays an important role in thermogenesis by uncoupling electron transport from ATP [10]. In this study, increased expression of UCP1 showed that mitochondrial activity was essential for the browning effect. Our results were further supported by immunofluorescence assays, which showed that BG and Rb1 increased UCP1 expression and mitochondrial activity. An increase in the number of mitochondria is an important feature of beige adipocytes [1]. Consistent with our findings, Lee et al. demonstrated that ginsenoside Rg1 induced browning by increasing UCP1 expression and mitochondrial activity in 3T3-L1 cells and subcutaneous WAT. According to the authors, the antiobesity effects of Rg1 were mediated through AMPK activation [3].
PGC-1α is an important modulator of mitochondrial biogenesis and function and its expression in WAT drives the development of BAT-like characteristics. We demonstrated that BG and Rb1 increased the expression of PGC-1α in 3T3-L1 cells and PWATs in dose-dependent manners, which suggests that the target compounds accelerated energy expenditure. In addition, PGC-1α activation has been reported to induce the expression of essential energy hemostasis proteins, namely, UCP1 and PRDM16. PRDM16 is a coregulatory transcriptional marker and a key switch in the development of BAT and brown-like adipose tissue in WAT [32]. In this study, PRDM16 expression was upregulated by BG and Rb1 treatments. According to Wang and Seale, transcriptional factors, Figure 11. Effect of AICAR and dorsomorphin in the presence of Rb1 on the expression of brown adipocyte markers. AICAR (A) and dorsomorphin (B) were added to PWATs. Protein expression levels of PRDM16, UCP1, PGC-1α, and p-AMPK/AMPK were evaluated by Western blotting. The data are presented as mean ± SD for three different experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Con.

Discussion
The prevalence of obesity and associated metabolic disorders continues to increase worldwide. Since browning leads to weight loss through energy expenditure, research related to increasing thermogenesis or browning has gained momentum recently. Therefore, the use of recruitable brown adipocytes represents a promising strategy to treat and prevent obesity [31]. For this reason, functional foods or bioactive ingredients are being explored for inducing browning using in vivo and in vitro models.
In the present study, the effects of BG and Rb1 in 3T3-L1 cells and PWATs were investigated to determine whether the characteristics of white adipocytes could be converted to those of brown-like adipocytes. The browning effects were evaluated by measuring the expression of PRDM16, UCP1, and PGC-1 brown-adipocyte-specific hallmark proteins in both types of adipocytes. Our results show that BG and Rb1 significantly upregulated the expression of PRDM16, UCP1, and PGC-1 in dose-dependent manners in both models. Moreover, mechanistic implications suggest that the target compounds used in the present study activated the AMPK signaling cascade responsible for the increased expression of hallmark proteins, thereby promoting browning in 3T3-L1 cells and PWATs.
UCP1 is a hallmark brown adipocyte protein that plays an important role in thermogenesis by uncoupling electron transport from ATP [10]. In this study, increased expression of UCP1 showed that mitochondrial activity was essential for the browning effect. Our results were further supported by immunofluorescence assays, which showed that BG and Rb1 increased UCP1 expression and mitochondrial activity. An increase in the number of mitochondria is an important feature of beige adipocytes [1]. Consistent with our findings, Lee et al. demonstrated that ginsenoside Rg1 induced browning by increasing UCP1 expression and mitochondrial activity in 3T3-L1 cells and subcutaneous WAT. According to the authors, the antiobesity effects of Rg1 were mediated through AMPK activation [3].
PGC-1α is an important modulator of mitochondrial biogenesis and function and its expression in WAT drives the development of BAT-like characteristics. We demonstrated that BG and Rb1 increased the expression of PGC-1α in 3T3-L1 cells and PWATs in dose-dependent manners, which suggests that the target compounds accelerated energy expenditure. In addition, PGC-1α activation has been reported to induce the expression of essential energy hemostasis proteins, namely, UCP1 and PRDM16. PRDM16 is a coregulatory transcriptional marker and a key switch in the development of BAT and brown-like adipose tissue in WAT [32]. In this study, PRDM16 expression was upregulated by BG and Rb1 treatments. According to Wang and Seale, transcriptional factors, including PRDM16, which are expressed on brown and beige cells, cooperate with other adipogenic transcriptional factors, such as PPARγ, to drive browning and thermogenic activities [33]. PPARγ is a key regulator in the coordination of various molecular actions that confirm the normal physiological function of white, brown, and beige adipocytes. Formation of a functional transcriptional complex (PPARγ/PRDM16/PGC-1α) is believed to be involved in the regulation of brown and beige-like characteristics [34]. Our results showed that BG and Rb1 significantly increased the expression of PPARγ in both adipocyte models. Mu et al. demonstrated that Rb1 promoted browning in 3T3-L1 cells by activating PPARγ, a result that was supported by increased mRNA expressions of PRDM16, UCP1, and PGC-1α [35].
Furthermore, the process of transdifferentiation is further promoted by downregulating the protein expression of lipogenic transcriptional factors, including C/EBPα and SREBP-1c. The reduced size and accumulation of lipids in the form of small lipid droplets observed after Oil Red O staining provide further evidence. These findings imply that BG and Rb1 treatments resulted in reduced expressions of C/EBPα and SREBP1-c.
AMPK is a well-known regulator of metabolism, and activation of the AMPK-mediated pathway is associated with cellular energy homeostasis sensing [36,37]. AMPK plays an important role in the activation of UCP1, a key protein in the browning event [38]. Our data showed that the target compounds significantly upregulated the AMPK expression, along with its phosphorylated form (activated) in both models. Furthermore, a direct interaction between PGC-1α and AMPK has also been reported to be essential for thermogenic activity [39]. Besides increasing mitochondrial number and the expression of brown-specific marker proteins, BG and Rb1 significantly increased the relative expressions of p-AMPK and AMPK.
To further evaluate the roles of BG and Rb1 in AMPK activation, PWATs isolated from C57BL/6 mice were treated with AICAR and dorsomorphin in the presence of BG and Rb1. As shown in Figures 10 and 11, both target compounds enhanced the phosphorylation of AMPK and the relative expressions of PRDM16, UCP1, and PGC-1α in the presence of AICAR, while the expressions were abolished in the presence of dorsomorphin. Thus, our data demonstrated that both BG and Rb1 played a crucial role in the activation of the AMPK signaling cascade, which subsequently led to the enhanced expression of brown-specific marker proteins. Consistently, many studies have reported the browning effects of natural bioactive compounds by activation of the AMPK-mediated pathway [13,14,40]. Hence, we suggest that BG and Rb1 treatments might stimulate brown-like adipose tissue in PWATs by upregulating the expression of hallmark proteins, including PRDM16, UCP1, and PGC-1α. Overall, our data suggest that BG and Rb1 promoted browning by inducing the expression of brown-specific hallmark proteins through AMPK activation in 3T3-L1 cells and PWATs. These results are the first to report the browning effects of BG and Rb1 using in vitro and ex vivo models.
We elucidated the role of BG and Rb1 in the browning of 3T3-L1 cells and PWATs. The browning effects were mediated by increasing expressions of UCP1, PRDM16, PGC-1, and PPARγ, as well as decreased expressions of C/EBPα and SREBP1, inhibition of lipid accumulation, and activation of the AMPK signaling cascade. Therefore, the data suggest that BG and Rb1 might be potential therapeutic candidates for the prevention and treatment of obesity.