(A) Effects of DBM on body weight in an HFD-induced obesity model.

<p>The three groups were as follows: LFD, low-fat (standard) diet; HFD, high-fat diet; and HFD supplemented with 100 mg/kg/d DBM. (B) Effects of DBM on epididymal fat in an HFD-induced obesity model. (C) Effects of DBM on peri-renal fat in an HFD-induced obesity model. (D) Effects of DBM on fatty liver in an HFD-induced obesity model. (E) Effects of DBM on insulin level of HFD-induced animals. * p < 0.05, as compared with basal condition. _* + p < 0.05, as compared with HFD condition (F) Effects of DBM on fasting blood glucose (FBG) level of HFD-induced animals. * p < 0.05, as compared with basal condition. (G) Effects of DBM on leptin level of HFD-induced animals. * p < 0.05, as compared with basal condition. (H) 3T3-L1 pre-adipocyte cells were stimulated for indicated times with DBM. The cells were then lysed with lysis buffer, and the phosphorylation of acetyl CoA carboxylase (ACC) was assessed by western blot using antibodies specific for the phosphorylate protein. The levels of ACC were also assessed. The levels of β-actin were also measured as a control for protein loading. * p < 0.05, as compared with basal condition. The results are representative of three independent experiments. (I) Total mRNA was prepared from DBM-treated 3T3-L1 cells, and RT-PCR was conducted using specific indicated primers. The PCR products were then separated on 1% agarose gels and visualized under ultraviolet light. Beta-actin was used as a loading control. * p < 0.05, as compared with basal condition. (J) ST3-L1 cells were transiently transfected with AMPKα2 siRNA for 48 h. The cells were then stimulated with 30 μM DBM for 1 h. Total mRNA was prepared from DBM-treated 3T3-L1 cells and RT-PCR was conducted using specific fatty acid synthase (FAS) primers. The PCR products were then separated on 1% agarose gels and visualized under ultraviolet light. Beta-actin was used as a loading control. * p < 0.05, as compared with basal condition.</p

(A) L6 myotubes were differentiated for 7 days and then treated with 30 μM DBM for 1 h either in the presence or absence of compound C (1 μM). 2-deoxy-D [H³] glucose (2-DG) uptake was then assayed, as described in the Methods.

<p>*p < 0.05, compared with control. **p < 0.05, compared with DBM-treated cells. (B) L6 myotubes were differentiated and then transiently transfected with 50 nM AMPKα2 siRNA for 48 h. The cells were then stimulated with 30 μM DBM for 18 hours, and 2-DG uptake was assayed. *p < 0.05, compared with control. **p < 0.05, compared with DBM-treated cells. This result is representative of four independent experiments. (C) L6 myotubes were differentiated and then transiently transfected with green fluorescent protein (GFP)-AMPKα2 K45R plasmid for 48 h. The cells were then stimulated with 30 μM DBM for 18 hours, and 2-DG uptake was measured. *p < 0.05, compared with control. **p < 0.05, compared with DBM-treated cells. This result is representative of four independent experiments. D, L6 myotubes were transiently transfected with GFP-AMPKα2 K45R plasmid for 48 h. The green fluorescent signal was detected using confocal microscopy.</p

(A) Total mRNA was prepared from DBM-treated C2C12 cells, and RT-PCR was conducted using specific GLUT4 primers.

<p>The PCR products were separated on 1% agarose gels and visualized under ultraviolet light. Beta-actin was used as a positive control. * p < 0.05, as compared with basal condition. (B) C2C12 cells were stimulated for various periods with 30 μM DBM and 100 nM insulin. The cells were lysed with lysis buffer, and the expression of GLUT4 was evaluated by western blot. The levels of β-actin were also measured as a control for protein loading. * p < 0.05, as compared with basal condition. The results are representative of three independent experiments. (C) Myoblasts stably expressing L6-GLUT4myc were differentiated for 7 days and then pre-treated with compound C (2 μM), then incubated with DBM for 60 min. Insulin was treated for 30 minutes. Cell surface expression of GLUT4myc was detected using an antibody-coupled colorimetric absorbance assay. *p < 0.05, compared with control. **p < 0.05, compared with DBM-treated cells.</p

(A) C2C12 cells were stimulated for 1 h with various concentrations of DBM.

<p>The cells were then lysed with lysis buffer, and the phosphorylation of AMPKα was assessed by western blot using antibodies specific for the phosphorylated protein. The level of total AMPKα was also assessed as a control for protein loading. (B) C2C12 cells were treated with 30 μM DBM for the times indicated. The cells were lysed with lysis buffer, and the phosphorylation of AMPKα was evaluated by western blot using antibodies specific for the phosphorylated protein. The level of total AMPKα was also assessed as a control for protein loading. (C) C2C12 cells were treated with DBM and curcumin for 1 h. The cells were lysed with lysis buffer, and the phosphorylation of AMPKα and ACC was evaluated using antibodies specific for the phosphorylated protein. The level of total AMPKα and ACC was also assessed as a control for protein loading. (D) L6 myotubes were differentiated for 7 days and then treated with 30 μM DBM and 2 mM metformin for 18 h. 2-deoxy-D [H³] glucose (2-DG) uptake was then assayed, as described in the Methods. * p < 0.05, as compared with basal condition. The results presented are representative of three individual experiments.</p

(A) C2C12 cells were stimulated with different concentrations of DBM for 1 h.

<p>The cells were lysed with lysis sample buffer, and the phosphorylation of p38 MAPK was evaluated by western blot using antibodies specific for the phosphorylated protein. The levels of total p38 MAPK were also assessed as a control for protein loading. * p < 0.05, as compared with basal condition. The results are representative of four independent experiments. (B) C2C12 cells were stimulated with 30 μM DBM for 1 h in the presence of compound C (1 μM). The cells were lysed with lysis buffer, and the phosphorylation of p38 MAPK was evaluated by western blot using phosphorylation-specific antibody. The levels of total p38 MAPK were also assessed as a control for protein loading. * p < 0.05, as compared with basal condition. Data are representative of four independent experiments. (C) Myoblast L6 cells were differentiated for 7 days and then incubated with the p38 MAPK inhibitor, SB203580, for 20 min, before cells were incubated with DBM for 18 hours. 2-DG uptake was then measured. *p < 0.05, compared with control. **p < 0.05, compared with DBM-treated cells. This result is representative of four independent experiments.</p

(A) C2C12 muscle cells pre-treated with Fluo-3, AM for 30 min were then treated with DBM (30 μM).

<p>The green fluorescent signal was detected using confocal microscopy. (B) C2C12 cells pre-treated with STO-609, a CaMKK inhibitor, for 30 min and then treated with 30 μM DBM for 1 h. The cells were then lysed with lysis buffer, and the phosphorylation of AMPKα2 was assessed by western blot using phosphorylation-specific antibodies. The level of total AMPKα2 was also assessed as a control for protein loading. * p < 0.05, as compared with basal condition. (C) Myoblast L6 cells were differentiated for 7 days and then pre-treated with STO-609 (1 μM) and then DBM (30 μM) for 1 h. Glucose uptake was then assayed for 2-DG uptake as described in the Methods. *p < 0.05, compared with control. **p < 0.05, compared with DBM-treated cells.</p