Ginsenoside Rg3 Induces Browning of 3T3-L1 Adipocytes by Activating AMPK Signaling

Abstract

Ginsenoside Rg3, one of the major components in Panax ginseng, has been reported to possess several therapeutic effects including anti-obesity properties. However, its effect on the browning of mature white adipocytes as well as the underlying mechanism remains poorly understood. In this study, we suggested a novel role of Rg3 in the browning of mature 3T3-L1 adipocytes by upregulating browning-related gene expression. The browning effects of Rg3 on differentiated 3T3-L1 adipocytes were evaluated by analyzing browning-related markers using quantitative PCR, immunoblotting, and immunostaining. In addition, the size and sum area of lipid droplets in differentiated 3T3-L1 adipocytes were measured using Oil-Red-O staining. In mature 3T3-L1 adipocytes, Rg3 dose-dependently induced the expression of browning-related genes such as Ucp1, Prdm16, Pgc1α, Cidea, and Dio2. Moreover, Rg3 induced the expression of beige fat-specific genes (CD137 and TMEM26) and lipid metabolism-associated genes (FASN, SREBP1, and MCAD), which indicated the activation of lipid metabolism by Rg3. We also demonstrated that activation of 5' adenosine monophosphate-activated protein kinase (AMPK) is required for Rg3-mediated up-regulation of browning gene expression. Moreover, Rg3 inhibited the accumulation of lipid droplets and reduced the droplet size in mature 3T3-L1 adipocytes. Taken together, this study identifies a novel role of Rg3 in browning of white adipocytes, as well as suggesting a potential mechanism of an anti-obesity effect of Panax ginseng.

Keywords: AMPK; Rg3; anti-obesity; beige adipocytes; browning effect; ginsenoside.

Figure 1

Rg3 promoted the expression of brown and beige adipocyte marker genes in mature 3T3-L1 adipocytes. (A) Chemical structure of Rg3. (B) The mRNA levels of uncoupling protein 1 (Ucp1) and Prdm16 in differentiated 3T3-L1 cells treated with indicated concentration of Rg3 for 24 h. (C) Immunoblot analysis of differentiated 3T3-L1 cells treated with indicated concentration of Rg3 for 24 h (upper) and its quantitative graphs (lower). (D) Immunostaining of fully differentiated 3T3-L1 cells treated with indicated concentration of Rg3 for 24 h. (E) The mRNA levels of brown adipocyte marker genes in differentiated 3T3-L1 cells treated with indicated concentration of Rg3 for 24 h. (F) The mRNA levels of beige adipocyte marker genes in differentiated 3T3-L1 cells treated with indicated concentration of Rg3 for 24 h. Data represent means ± SEM for n = 3. Asterisks indicate significant differences from the control (one-way ANOVA; n.s.: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2

Rg3 reduced white adipocyte marker genes and increased lipid metabolism. (A) The mRNA levels of white adipocyte marker genes in differentiated 3T3-L1 cells treated with indicated concentration of Rg3 for 24 h. (B) The mRNA levels of lipogenic genes in differentiated 3T3-L1 cells treated with indicated concentration of Rg3 for 24 h. (C) The mRNA levels of MCAD in differentiated 3T3-L1 cells treated with indicated concentration of Rg3 for 24 h. (D) Oil-Red-O staining showing the accumulation of lipid droplets in differentiated 3T3-L1 cells treated with Rg3 (20, 40 μM) for 24 h (left) and its quantification graph (right) using Gen5 (Bio Tek, Winooski, VT, USA). Data represent means ± SEM for n = 3. Asterisks indicate significant differences from the control (one-way ANOVA; n.s.: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

Rg3 induced browning of differentiated 3T3-L1 cells via activation of AMPK. (A) Immunoblot analysis of differentiated 3T3-L1 cells treated with indicated concentration of Rg3 for 24 h (left) and its quantitative graphs (right). (B) The mRNA levels of Ucp1 and Prdm16 in differentiated 3T3-L1 cells treated with Rg3 (40 μM) in combination with an AMP-activated protein kinase (AMPK) inhibitor, compound C (CC), for 24 h. (C) Immunoblot analysis of differentiated 3T3-L1 cells treated with Rg3 (40 μM) in combination with compound C (CC) for 24 h (left) and its quantitative graphs (right). (D) Immunostaining of fully differentiated 3T3-L1 cells treated with Rg3 (40 μM), co-treated with or without compound C (CC) for 24 h. (E) The mRNA levels of beige adipocyte marker genes in differentiated 3T3-L1 cells treated with Rg3 (40 μM) in combination with compound C (CC) for 24 h. Data represent means ± SEM for n = 3. Asterisks indicate significant differences between marked samples (one-way ANOVA; n.s.: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4

Rg3 altered lipid metabolism without affecting white adipocyte marker gene expression, and the effects were reversed by AMPK inhibitor. (A) The mRNA levels of white adipocyte marker genes in differentiated 3T3-L1 cells treated with Rg3 (40 μM) in combination with compound C (CC) for 24 h. (B) The mRNA levels of lipogenic genes in differentiated 3T3-L1 cells treated with Rg3 (40 μM) in combination with compound C (CC) for 24 h. (C) Oil-Red-O staining showing the accumulation of lipid droplets in differentiated 3T3-L1 cells treated with Rg3 (40 μM) in combination with compound C (CC, 10 uM) for 24 h (left) and its quantitative graph (right) using Gen5 (Bio Tek, Winooski, VT, USA). Data represent means ± SEM for n = 3. Asterisks indicate significant differences between marked samples (one-way ANOVA; n.s.: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001).