2-Bromo-4'-methoxychalcone and 2-Iodo-4'-methoxychalcone Prevent Progression of Hyperglycemia and Obesity via 5'-Adenosine-Monophosphate-Activated Protein Kinase in Diet-Induced Obese Mice

Abstract

Obesity and diabetes are global health-threatening issues. Interestingly, the mechanism of these pathologies is quite different among individuals. The discovery and development of new categories of medicines from diverse sources are urgently needed for preventing and treating diabetes and other metabolic disorders. Previously, we reported that chalcones are important for preventing biological disorders, such as diabetes. In this study, we demonstrate that the synthetic halogen-containing chalcone derivatives 2-bromo-4'-methoxychalcone (compound 5) and 2-iodo-4'-methoxychalcone (compound 6) can promote glucose consumption and inhibit cellular lipid accumulation via 5'-adenosine-monophosphate-activated protein kinase (AMPK) activation and acetyl-CoA carboxylase 1 (ACC) phosphorylation in 3T3-L1 adipocytes and C2C12 skeletal myotubes. In addition, the two compounds significantly prevented body weight gain and impaired glucose tolerance, hyperinsulinemia, and insulin resistance, which collectively help to delay the progression of hyperglycemia in high-fat-diet-induced obese C57BL/6 mice. These findings indicate that 2-bromo-4'-methoxychalcone and 2-iodo-4'-methoxychalcone could act as AMPK activators, and may serve as lead compounds for a new class of medicines that target obesity and diabetes.

Keywords: adipocyte; chalcone; flavonoid; halogen; skeletal muscle.

Figure 1

Chemical structures of rosiglitazone, pioglitazone, and chalcone derivatives.

Figure 2

Effects of chalcone derivatives on glucose consumption and lipid accumulation in preadipocytes and adipocytes. 3T3-L1 preadipocytes or adipocytes were incubated with vehicle (0.1% dimethyl sulfoxide; Control), insulin (0.32 µM; Ins), rosiglitazone (30 µg/mL; Rosi), pioglitazone (30 µg/mL; Pio) or 30 µg/mL of chalcone derivatives (compounds 1–6). (A) Percentage of medium glucose consumed by the preadipocytes; (B) percentage of medium glucose consumed by the adipocytes; (C) cellular lipid accumulation in the preadipocytes; (D) cellular lipid accumulation in the adipocytes. Red color shows the cellular lipid droplets stained with Oil-Red O; (E,F) cellular lipid amount was quantified by dissolving the Oil Red O in isopropanol. Data are mean ± SD from three or four independent experiments. ^a: p < 0.001 compared to Pre-Con; ^b: p < 0.001 compared to Con; ^c: p < 0.05 compared to Con; ^d: p < 0.05 compared to Pre-Con. Pre-Con: control for preadipocytes; Con: control for adipocytes. The original magnification of the cell images in panel C and D is 100× under the light microscope.

Figure 3

Effects of chalcone derivatives on 5′-adenosine-monophosphate-activated protein kinase (AMPK) and Protein kinase B (Akt) signaling in 3T3-L1 adipocytes. (A) 3T3-L1 adipocytes were treated with or without the tested compounds (30 µg/mL) for 24 h, and then phosphorylated and total AMPK and acetyl-CoA carboxylase (ACC) proteins were detected by Western blotting; (B,C) quantitative results for the blots in panel A; (D) 3T3-L1 adipocytes were pre-treated with or without Dorsomorphin (10 µM; AI) for 30 min and then administered with low dosage (15 µg/mL; L) or high dosage (30 µg/mL; H) of compound 5 or 6, or 30 µg/mL of metformin. The medium glucose consumption was detected after 12 h of incubation and the cells were harvested for protein analysis; (E) Western blotting for phosphorylated and total AMPK and ACC proteins; (F,G) quantitative results for the blots in panel E; (H) 3T3-L1 adipocytes were treated with 30 µg/mL of compounds 5 or 6 combined with or without insulin (0.32 µM) for 30 min to observe the phosphorylated and total Akt proteins; (I) quantitative results for the blots in panel H. Data are mean ± SD from three independent experiments. ^a: p < 0.001; ^b: p < 0.01; ^c: p < 0.05.

Figure 4

Effects of chalcone derivatives on the expression of CCAAT/enhancer binding protein-α and -β (C/EBPα, C/EBPβ), and peroxisome proliferator activated receptor γ (PPARγ) proteins in 3T3-L1 adipocytes. (A) 3T3-L1 adipocytes were treated with or without 0.32 µM of insulin, low dosage (15 µg/mL; L) or high dosage (30 µg/mL; H) of compound 5 or compound 6, or 30 µg/mL of pioglitazone (Pio) for 24 h. C/EBPα, C/EBPβ, and PPARγ proteins were detected by Western blotting; (B–D) the quantitative results for the blots in panel A. Data are mean ± SD from three independent experiments. ^a: p < 0.001; ^b: p < 0.01; ^c: p < 0.05.

Figure 5

Effects of chalcone derivatives on the glucose consumption, and AMPK and Akt signaling in C2C12 myotubes. (A) C2C12 myotubes were pre-treated with or without dorsomorphin (10 µM; AI) for 30 min and then administered a low dosage (15 µg/mL; L) or high dosage (30 µg/mL; H) of compounds 5 or 6, or 30 µg/mL of metformin. The medium glucose consumption was detected after 12 h of incubation and the cells were harvested for protein analysis; (B) Western blotting for phosphorylated and total AMPK and ACC proteins; (C,D) the quantitative results for the blots in panel B; (E) C2C12 myotubes were treated with 30 µg/mL of compounds 5 or 6 combined with or without insulin (0.32 µM) for 30 min, and then the cells were harvest for phosphorylated and total Akt proteins by Western blotting; (F) the quantitative results for the blots in panel E. Data are mean ± SD from three independent experiments. ^a: p < 0.001; ^b: p < 0.01; ^c: p < 0.05.

Figure 6

Anti-obesity and antidiabetic effects of compounds 5 and 6 in the HF-fed mice. (A) Illustration of the animal protocol; (B) results of OGTT in the mice; (C) area under curve (AUC) of the OGTT results; (D) body weight changes in the mice; (E) the quantitative results of the adipocyte size in panel F; (F) morphology of the visceral adipose tissues were evaluated by hematoxylin and eosin staining. The original magnification of the tissue images in panel F is 200× under the light microscope. Con: normal-diet-fed; HF: high-fat-diet-fed; HF + 5L: high-fat-diet fed with compound 5 (6.75 mg/kg/day); HF + 5H: high-fat-diet fed with compound 5 (33.75 mg/kg/day); HF + 6L: high-fat-diet fed with compound 6 (6.75 mg/kg/day); HF + 6H: high-fat-diet fed with compound 6 (33.75 mg/kg/day); HF + Pio: high-fat-diet fed with pioglitazone (6.75 mg/kg/day). Data are mean ± SD. In panel B, ^a: p < 0.001, HF group compared with all the other groups; ^b: p < 0.01, HF group compared with Con, HF + 5L, HF + 6H, and HF + Pio; ^c: p < 0.05, HF group compared with HF + 5H, and HF + 6L. ^a: p < 0.001, compared with HF group in panel C and E. In panel D, ^a: p < 0.001, HF group compared with all the other groups; ^b: p < 0.01, HF group compared with HF + 5L, HF + 6H, and HF + Pio; ^c: p < 0.05, HF group compared with HF + 6L. For detailed statistical results of (B,D) see Table S2.

Figure 7

Effects of compounds 5 and 6 on AMPK signaling pathway in the HF-fed mice. (A) Proteins from adipose tissues were extracted from the mice, and then phosphorylated and total AMPK and ACC proteins were detected by Western blotting; (B,C) the quantitative results for the blots in panel A; (D) proteins from skeletal muscles were extracted from the mice, and then phosphorylated and total AMPK and ACC proteins were detected by Western blotting; (E,F) the quantitative results for the blots in panel D. Con: normal-diet-fed; HF: high-fat-diet-fed; HF + 5L: high-fat-diet fed with compound 5 (6.75 mg/kg/day); HF + 5H: high-fat-diet fed with compound 5 (33.75 mg/kg/day); HF + 6L: high-fat-diet fed with compound 6 (6.75 mg/kg/day); HF + 6H: high-fat-diet fed with compound 6 (33.75 mg/kg/day); HF + Pio: high-fat-diet fed with pioglitazone (6.75 mg/kg/day). Data are mean ± SD. ^a: p < 0.001; ^b: p < 0.01; ^c: p < 0.05, compared with HF group.

Figure 8

Summary of the antidiabetic and anti-obesity effects of 2-bromo-4′-methoxychalcone and 2-iodo-4′-methoxychalcone.