Ginsenoside Rg1 promotes browning by inducing UCP1 expression and mitochondrial activity in 3T3-L1 and subcutaneous white adipocytes

Abstract

Background: Panax ginseng Meyer is known as a conventional herbal medicine, and ginsenoside Rg1, a steroid glycoside, is one of its components. Although Rg1 has been proved to have an antiobesity effect, the mechanism of this effect and whether it involves adipose browning have not been elucidated.

Methods: 3T3-L1 and subcutaneous white adipocytes from mice were used to access the thermogenic effect of Rg1. Adipose mitochondria and uncoupling protein 1 (UCP1) expression were analyzed by immunofluorescence. Protein level and mRNA of UCP1 were also evaluated by Western blotting and real-time polymerase chain reaction, respectively.

Results: Rg1 dramatically enhanced expression of brown adipocyte-specific markers, such as UCP1 and fatty acid oxidation genes, including carnitine palmitoyltransferase 1. In addition, it modulated lipid metabolism, activated 5' adenosine monophosphate (AMP)-activated protein kinase, and promoted lipid droplet dispersion.

Conclusions: Rg1 increases UCP1 expression and mitochondrial biogenesis in 3T3-L1 and subcutaneous white adipose cells isolated from C57BL/6 mice. We suggest that Rg1 exerts its antiobesity effects by promoting adipocyte browning through activation of the AMP-activated protein kinase pathway.

Keywords: Adipocytes; Browning; Ginsenoside Rg1; Thermogenesis; Uncoupling protein 1.

Fig. 1

Effect of Rg1 on fat accumulation and the expression of key adipogenic factors in scWAT cells. oil red O staining was used to assess adipocyte differentiation after 8 days in the presence or absence of Rg1. (A) Photomicrographs of cells. (B) Intensity-densitometric analysis. (C–D) Concentration-dependent effect of Rg1 on the expression of adipogenesis markers, analyzed by Western blotting. (E) Viability of scWATs, evaluated using an MTT assay. These data are presented as the mean and standard deviation of four replicates. Data were analyzed using one-way ANOVA and Duncan's test. Values with different letters are significantly different, p < 0.05.

Fig. 2

Dose-dependent effect of Rg1 on the expression of BAT-specific markers. (A, C) Rg1 (25, 50, or 100 μM) was added to scWAT and 3T3-L1 (B, D), which was followed by the measurement of protein expression by Western blotting. Data were analyzed using one-way ANOVA and Duncan's test. There was a statistically significant difference between the control and Rg1-treated groups (p < 0.05).

Fig. 3

Known inducers of browning and Rg1 induce the expression of browning markers in scWATsand 3T3-L1. Browning was induced by the treatment of cells with 50 nM T₃ and 1 μM rosiglitazone. (A, C) scWAT were treated with 50 μM Rg1, and the protein expression levels of BAT-specific markers were measured by Western blotting. (B, D) Quantitative data, representing the mean and standard deviation of six replicates. Data were analyzed using one-way ANOVA and Duncan's test. Values with different letters are significantly different, p < 0.05.

Fig. 4

Rg1 treatment enhances mitochondrial activity and expression of UCP1. (A) Rg1-treated scWAT and (C) 3T3-L1 adipocytes were stained with MitoTracker Red and immunostained for UCP1. (B, D) Quantitative data for MitoTracker Red and UCP1 staining intensity in scWAT and 3T3-L1 adipocytes, respectively. Immunofluorescent images were captured at ×800 magnification. Data are presented as the mean and standard deviation of three replicates and were analyzed using one-way ANOVA and Duncan's test. Values with different letters are significantly different, p < 0.05.

Fig. 5

Effect of Rg1 on the expression of markers of fatty acid oxidation in adipocytes. (A, C) Western blots for p-AMPK, p-p38, PPARα, and CPT1 in scWAT and 3T3-L1s, respectively, and (B, D) their quantitation. Cells were treated with 50 nM T₃ and 1 μM rosiglitazone. (E) Relative mRNA expression of fatty acid oxidation genes was measured using real-time PCR and normalized to 18s rRNA expression. These data are presented as the mean and standard deviation of six replicates. Protein data were analyzed using one-way ANOVA and Duncan's test. Values with different letters are significantly different, p < 0.05. mRNA data were analyzed using Student t test, and values were considered significant when *p < 0.05.

Fig. 6

Effects of AICAR on lipid droplet morphology and mitochondrial biogenesis in scWATs. (A) Morphology was evaluated by optical microscopy (×400). Adipocytes were differentiated for 16 days. The scale bar represents 100 μM. (C) Representation of the mitochondrial activity, UCP1 expression, and lipid droplet size in differentiated scWATs, (B, D) Quantitative data for MitoTracker Red and UCP1 immunofluorescence staining intensity. These data are presented as the mean and standard deviation of triplicates. Data were analyzed using one-way ANOVA and Duncan's test. Values with different letters are significantly different, p < 0.05.

Fig. 7

Effects of AICAR and dorsomorphin on the expression of UCP1 in scWAT. To assess the importance of AMPK activation, adipocytes were differentiated in the presence of AICAR or dorsomorphin. 3T3-L1 cells were treated with 10 μM AICAR or 10 μM dorsomorphin, and the effects on specific protein expression levels were investigated using Western blotting. These data are presented as the mean and standard deviation of triplicates. Results were analyzed using one-way ANOVA and Duncan's test. Values with different letters are significantly different, p < 0.05.

Fig. 8

Suggested mechanisms for the induction of browning by Rg1. Rg1 induces a brown fat-like phenotype and lipid metabolism through AMPK activation. → indicates stimulation, yellow indicates the hydrolysis pathway, blue indicates the fatty acid oxidation pathway, and pink indicates the thermogenesis pathway.