Ginsenoside Rb1 Facilitates Browning by Repressing Wnt/β-Catenin Signaling in 3T3-L1 Adipocytes

Abstract

BACKGROUND The discovery of browning in white adipose tissue has provided new ideas for treating obesity. Many studies have reported that ginsenoside Rb1 (G-Rb1) has activity against diabetes, inflammation, and obesity, but further investigation is needed on the effect and mechanism of G-Rb1 on browning. MATERIAL AND METHODS We treated 3T3-L1 adipocytes with 0-200 μM G-Rb1, and 0.5 μM Compound 3f and 30 μM SKL2001 were used to activate Wnt/b-catenin signaling. Adipocyte activity was evaluated by Cell Counting Kit-8. Oil Red O staining was used to detect the lipid droplets. Quantitative real-time polymerase chain reaction was used to measure the expression of Cd-137, Cited-1, Txb-1, Prdm-16, and Ucp-1 mRNA. Western blotting was used to measure the expression of Ucp-1, pGSK-3ß (Ser 9), GSK- 3ß, and ß-catenin proteins. The expression of Ucp-1 was also detected with immunofluorescence. RESULTS Adipocyte activity was not affected by 0-100 μM G-Rb1. However, G-Rb1 dose-dependently reduced the accumulation of lipid droplets; increased the expression of Cd-137, Cited-1, Txb-1, Prdm-16, and Ucp-1 mRNA; and increased the expression of Ucp-1, pGSK-3ß (Ser 9), GSK-3ß, and ß-catenin proteins. The accumulation of lipid droplets and the expression of Ucp-1 protein decreased as b-catenin increased. CONCLUSIONS G-Rb1 at various concentrations (0-100 μM) promoted the browning of adipocytes in a dose-dependent manner. Further, we confirmed that activation of Wnt/ß-catenin signaling could inhibit browning. Therefore, the browning promoted by G-Rb1 may be associated with the inhibition of Wnt/ß-catenin signaling.

Figure 1

Effect of ginsenoside Rb1 (G-Rb1) on the adipocyte viability. (A) The structure of G-Rb1. (B) 3T3-L1 adipocytes were treated with G-Rb1 (0, 10, 50, 100, and 200 μM) for 24, 48, and 72 h. Data are expressed as mean±SD (n=10). **** P<0.0001 vs. 0 μM.

Figure 2

Effect of ginsenoside Rb1 (G-Rb1; 0, 10, 50, and 100 μM) on the browning of adipocytes. (A) Lipid droplets were measured by Oil Red O staining. Scale bar=100 μm. (B) Lipid content assessing according to the absorbance at 490 nm. (C) ImageJ measured the average diameter of lipid droplets. (D–H) Quantitative real-time polymerase chain reaction measured the mRNA expression of browning-related genes (Cd-137, Cited-1, Tbx-1, Prdm-16, and Ucp-1). (I, J) Western blotting measured the protein expression of Ucp-1. Data are expressed as mean±SD (n=3). * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 vs. 0 μM.

Figure 3

Effect of ginsenoside Rb1 (G-Rb1; 0, 10, 50, and 100 μM) on Wnt/β-catenin signaling. (A) Western blotting results of browning-related genes in 3T3-L1 adipocytes. (B–E) ImageJ quantized the gray value of each protein band. Data are expressed as mean±SD (n=3). * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 vs. 0 μM.

Figure 4

Effect of ginsenoside Rb1 (G-Rb1) on browning treated with Compound 3f. (A) Oil Red O staining detected the lipid droplets. Scale bar=100 μm. (B) Optical density values at 490 nm. (C) Lipid droplets’ diameter of ORS was measured by ImageJ. (D) Western blotting results for Ucp-1 and β-catenin, with Gapdh as the internal reference. (E, F) ImageJ quantized the gray value of each protein band. (G) Immunofluorescence was performed to detect the expression of Ucp-1. Scale bar=50 μm. Data are expressed as mean±SD (n=3). * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

Figure 5

Effect of ginsenoside Rb1 (G-Rb1) on browning treated with SLK2001. (A) Oil Red O staining (ORS) detected the lipid droplets. Scale bar=100 μm. (B) Optical density values at 490 nm. (C) Lipid droplets’ diameter of ORS was measured by ImageJ. (D) Western blotting results of Ucp-1 and β-catenin, with Gapdh as the internal reference. (E, F) ImageJ quantized the gray value of each protein band. (G) Immunofluorescence was performed to detect the expression of Ucp-1. Scale bar=50 μm. Data are expressed as mean±SD (n=3). * P<0.05, ** P<0.01, **** P<0.0001.