Amber Extract Reduces Lipid Content in Mature 3T3-L1 Adipocytes by Activating the Lipolysis Pathway

Abstract

Amber-the fossilized resin of trees-is rich in terpenoids and rosin acids. The physiological effects, such as antipyretic, sedative, and anti-inflammatory, were used in traditional medicine. This study aims to clarify the physiological effects of amber extract on lipid metabolism in mouse 3T3-L1 cells. Mature adipocytes are used to evaluate the effect of amber extract on lipolysis by measuring the triglyceride content, glucose uptake, glycerol release, and lipolysis-related gene expression. Our results show that the amount of triacylglycerol, which is stored in lipid droplets in mature adipocytes, decreases following 96 h of treatment with different concentrations of amber extract. Amber extract treatment also decreases glucose uptake and increases the release of glycerol from the cells. Moreover, amber extract increases the expression of lipolysis-related genes encoding perilipin and hormone-sensitive lipase (HSL) and promotes the activity of HSL (by increasing HSL phosphorylation). Amber extract treatment also regulates the expression of other adipocytokines in mature adipocytes, such as adiponectin and leptin. Overall, our results indicate that amber extract increases the expression of lipolysis-related genes to induce lipolysis in 3T3-L1 cells, highlighting its potential for treating various obesity-related diseases.

Keywords: adipocytes; amber; hormone-sensitive lipase; lipid metabolism; lipolysis.

Figure 1

Effects of amber extract on cell viability and lipid accumulation. (A) After treatment of 3T3-L1 cells with amber extract (0, 10, 25, and 50 µg/mL) for 96 h, cell viability was measured using the MTT assay. (B) Photographs of lipid droplets in cells stained with Oil Red O stain. (C) Triacylglycerol (TAG) levels in the lipid droplets were measured using Oil Red O staining. The results are presented as the mean ± SD; n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. 0 µg/mL.

Figure 2

Effects of amber extract on lipid metabolism. After mature adipocytes were treated with amber extract (0, 10, 25, and 50 µg/mL) for 6, 12, 18, and 24 h, the culture medium was collected. The amount of (A) glucose and (B) glycerol in the medium at 6, 12, 18, and 24 h after treatment with amber extract was measured. The decrease in glucose content in the medium compared with that at 0 h was recognized as the amount of glucose uptake. The results are indicated as the mean ± SD; n = 4; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. 0 µg/mL.

Figure 3

Effects of amber extract on lipolysis-related gene expression. Mature 3T3-L1 cells treated with amber extract (0, 10, 25, and 50 µg/mL) were collected after 24 h, in which mRNA levels were measured by RT-PCR, and protein levels were detected by western blotting. Expression of (A) adipose triglyceride lipase (ATGL), (B) perilipin, and (D) hormone-sensitive lipase (HSL) at the mRNA level was detected by RT-PCR; the data were directly obtained from the machine. Western blot images of (C) perilipin, (E) HSL, (F) pHSL^S563, (G) pHSL^S565, and (H) pHSL^S660 analyzed by ImageJ. Statistical analysis of western blot bar graphs was performed in three independent experiments. The results are expressed as the mean ± SD; n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. 0 µg/mL.

Figure 4

Effects of amber extract treatment on adipocytokine-associated gene expression in 3T3-L1 cells. Mature 3T3-L1 cells treated with amber extract (0, 10, 25, and 50 µg/mL) were collected after 96 h, in which mRNA levels were measured by RT-PCR, and protein levels were detected by western blotting. Expression of (A) adiponectin and (C) leptin at the mRNA level was detected by RT-PCR; the data were directly obtained from the machine. Western blot images of (B) adiponectin analyzed by ImageJ. Statistical analysis of western blot bar graphs was performed in three independent experiments. The results are presented as the mean ± SD; n = 3; * p < 0.05, *** p < 0.001 vs. 0 µg/mL.