Amber (Succinite) Extract Enhances Glucose Uptake through the Up-Regulation of ATP and Down-Regulation of ROS in Mouse C2C12 Cells

Abstract

Traditionally, amber (Succinite) has been used to alleviate all types of pain, skin allergies, and headaches. However, no studies have been conducted on its antidiabetic and antioxidant effects. In this study, differentiated skeletal muscle C2C12 cells were used to demonstrate the protective effects of amber (AMB) against H₂O₂-induced cell death. In addition, the effects of AMB on glucose uptake and ATP production were investigated. Our results showed that AMB at 10, 25, and 50 μg/mL suppressed the elevation of ROS production induced by H₂O₂ in a dose-dependent manner. Moreover, AMB enhanced glucose utilization in C2C12 cells through the improvement of ATP production and an increase in PGC-1α gene expression resulting in an amelioration of mitochondrial activity. On the other hand, AMB significantly increased the gene expression of glucose transporters GLUT4 and GLUT1. Our finding suggests that AMB can be used as a natural supplement for diabetes treatment and for the promotion of skeletal muscle function.

Keywords: ATP; C2C12 cells; GLUT1; GLUT4; ROS; amber; glucose uptake.

Figure 1

Protective properties of AMB against H₂O₂-induced C2C12 cell cytotoxicity. C2C12 cells were seeded at 0.5 × 10⁴ cells/well in a 96-well microplate and treated with (a) AMB (10, 25, 50, 75, and 100 µg/mL) for 24 and 48 h; (b) C2C12 cells were treated with H₂O₂ (50, 100, 150, 200, 250, and 300 µM); (c) C2C12 cells were pretreated with AMB for 24 h and then treated with 200 µM H₂O₂ for another 24 h. Cell viability was determined using an MTT assay as explained in the Materials and Methods. Each bar represents the mean of 3 independent trials ± SD. ## indicate a significant difference (p < 0.01) versus untreated control group; * p < 0.05, ** indicate a significant difference (p < 0.01) versus vehicle group (Student’s t-test).

Figure 2

Effect of AMB on H₂O₂-induced ROS elevation in C2C12 cells. After 5 days of differentiation, C2C12 cells were treated with amber (10, 25, and 50 µg/mL) for 24 h and then exposed to 200 µM of H₂O₂ for 3 h. Asterisks indicate a significant difference (p < 0.01) compared with the vehicle-only control group (cells treated with H₂O₂ only). The hash indicates a significant difference (p < 0.01) compared with the control (Ctrl) group. The results represent three independent experiments; each experiment contained triplicate samples.

Figure 3

Effects of AMB on glucose uptake in C2C12 cells. Cells were seeded at 3 × 10⁴ cells/well in a 24-well microplate. After 48 h, the DMEM medium was changed to 2% HS medium to start differentiation for 5 days. Then, a fresh medium containing AMB at 10, 25, and 50 µg/mL was added to cells for 24 h. Asterisks indicate a significant difference (p < 0.01) compared with the control (Ctrl) group at each time of sampling. The results represent three independent experiments; each experiment contained triplicate samples.

Figure 4

Effects of H₂O₂-induced ATP depletion in C2C12 cells incubated with AMB extract (10, 25, and 50 µg/mL) for 24 h and then exposed to 200 µM of H₂O₂ for 3 h. Asterisks indicate a significant difference (p < 0.01) compared with the vehicle-only control group (cells treated with H₂O₂ only). Hash indicates a significant difference (p < 0.05) compared with the control (Ctrl) group. The results represent three independent experiments; each experiment contained triplicate samples.

Figure 5

Effects of AMB on the mitochondrial activity in C2C12 cells. Differentiated C2C12 cells were cultured with AMB (10, 25, and 50 µg/mL) for 48 h. The mitochondrial number was measured by fluorescent staining. (a) The mitochondria are indicated in green; images were captured at 10 magnifications using a fluorescence microscope. (b) Fluorescence intensity. Asterisks indicate a significant difference (* p < 0.05, ** p < 0.01) compared with the control group.

Figure 6

Effects of AMB on mRNA expression of (a) Glut1, (b) Glut4, and (c) PGC-1α in C2C12 cells. C2C12 cells were seeded at 20 × 10⁴ cells/well in a 6 cm dish and were treated with amber (25 and 50 µg/mL) after 7 days of differentiation, for 24 h, and then they were treated with 200 µM H₂O₂ for 6 h. The mRNA expression of genes was normalized to GAPDH mRNA expression and was expressed as a ratio compared to the control. Each bar represents the mean of duplicate ± SD. * p < 0.05, ** p < 0.01 versus the vehicle group, and # p < 0.05 versus the control group (Student’s t-test).