Mangiferin Improved Palmitate-Induced-Insulin Resistance by Promoting Free Fatty Acid Metabolism in HepG2 and C2C12 Cells via PPAR α: Mangiferin Improved Insulin Resistance

Abstract

Elevated free fatty acid (FFA) is a key risk factor for insulin resistance (IR). Our previous studies found that mangiferin could decrease serum FFA levels in obese rats induced by a high-fat diet. Our research was to determine the effects and mechanism of mangiferin on improving IR by regulating FFA metabolism in HepG2 and C2C12 cells. The model was used to quantify PA-induced lipid accumulation in the two cell lines treated with various concentrations of mangiferin simultaneously for 24 h. We found that mangiferin significantly increased insulin-stimulated glucose uptake, via phosphorylation of protein kinase B (P-AKT), glucose transporter 2 (GLUT2), and glucose transporter 4 (GLUT4) protein expressions, and markedly decreased glucose content, respectively, in HepG2 and C2C12 cells induced by PA. Mangiferin significantly increased FFA uptake and decreased intracellular FFA and triglyceride (TG) accumulations. The activity of the peroxisome proliferator-activated receptor α (PPARα) protein and its downstream proteins involved in fatty acid translocase (CD36) and carnitine palmitoyltransferase 1 (CPT1) and the fatty acid β-oxidation rate corresponding to FFA metabolism were also markedly increased by mangiferin in HepG2 and C2C12 cells. Furthermore, the effects were reversed by siRNA-mediated knockdown of PPARα. Mangiferin ameliorated IR by increasing the consumption of glucose and promoting the FFA oxidation via the PPARα pathway in HepG2 and C2C12 cells.

Figure 1

The cytotoxic effects of mangiferin and PA on HepG2 cells and C2C12 myotubes. The two cells lines were given 0, 3.13, 6.25, 12.5, 25, 50, 100, 200, and 400 μM of mangiferin for 24 h. (a) MTT assay. (b) LDH release in culture media. (c) LDH release in culture media. The experiments were repeated 3 times. Data are presented as means ± SD (n = 3). ^∗ P < 0.05.

Figure 2

The glucose uptake, AKT, GLUT2 and GLUT4 expressions, and glucose content in HepG2 cells and C2C12 myotubes. HepG2 cells and C2C12 myotubes were treated with 0.25 mM of PA and 12.5, 25, and 50 μM of mangiferin for 24 h. The cells were incubated in transport buffer in the presence of 100 nM insulin for 30 min before the addition of 100 μL 2-NBDG for 30 min at 37°C. The glucose uptake (a–b), AKT (c–d), GLUT2 (e) and GLUT4 (f) protein expressions, and glucose content (g–h) in HepG2 cells and C2C12 myotubes. The experiments were repeated 3 times. Data are presented as means ± SD (n = 3). ^∗ P < 0.05 compared with the PA group.

Figure 3

Effects of mangiferin on AKT expressions by regulating SOCS3 and PTP1B in HepG2 cells and C2C12 myotubes. The two cell lines were transfected with pEX-RB-SOCS3 or pEX-RB-PTP1B recombinant plasmid using Lipofectamine 2000 for 48 h. Then the cells were treated with 0.25 mM of PA and 50 μM of mangiferin for 24 h. The expressions of AKT and P-AKT were determined by western blot method in HepG2 cells (a, c) and C2C12 myotubes (b, d). The experiments were repeated 3 times. Data are presented as means ± SD (n = 3). ^∗ P < 0.05 compared with the PA group and ^# P < 0.05 compared with the mangiferin group.

Figure 4

Effects of mangiferin on PA and TG in HepG2 cells and C2C12 myotubes. HepG2 cells and C2C12 myotubes were treated with 0.25 mM of PA and 12.5, 25, and 50 μM of mangiferin for 24 h. The concentrations of PA in medium (a) and intracellular (b) PA were determined by GC-MS. The TG (c) mass was quantified by using a TG test kit. The experiments were repeated 3 times. Data are presented as means ± SD (n = 3). ^∗ P < 0.05 compared with the PA stimulation group.

Figure 5

Effects of mangiferin on the ratio of AMP to ATP in HepG2 and C2C12 cells. The two cell lines were exposed to 0.25 mM of palmitate only or with 12.5, 25, and 50 μM of mangiferin for 24 h. The experiments were repeated 3 times. Data are presented as means ± SD (n = 3). ^∗ P < 0.05 compared with the PA group.

Figure 6

Effects of mangiferin on CD36, CPT1, FFA oxidation rate, and PPARα in HepG2 cells and C2C12 myotubes. HepG2 cells and C2C12 myotubes were incubated with 0.25 mM of PA and 12.5, 25, and 50 μM of mangiferin for 24 h. The CD36 and CPT1 expressions, FFA oxidation rate, and PPARα expression in HepG2 cells (a, c, e, g) and C2C12 myotubes (b, d, f, h). The experiments were repeated 3 times. Data are presented as means ± SD (n = 3). ^∗ P < 0.05 compared with the PA stimulation group.

Figure 7

Effects of mangiferin on related indicators via siPPARα in HepG2 cells and C2C12 myotubes. PPARα expression, glucose uptake, AKT, GLUT2 and GLUT4 expressions, and glucose content in HepG2 cells (a, c, e, g, i) and C2C12 myotubes (b, d, f, h, j). The experiments were repeated 3 times. Data are presented as means ± SD (n = 3). ^∗ P < 0.05 compared with the PA stimulation group.

Figure 8

Effects of mangiferin on FFA oxidation rate, CPT1, and CD36 via siPPARα in HepG2 cells and C2C12 myotubes. FFA oxidation rate, CPT1 expression, and CD36 expression in HepG2 cells (a, c, e) and C2C12 myotubes (b, d, f). The experiments were repeated 3 times. Data are presented as means ± SD (n = 3). ^∗ P < 0.05 compared with the PA stimulation group.