Figure 1.
BMS309403 stimulates glucose uptake and phosphorylation of AMPK and p38 in a time dependent manner.
C2C12 myotubes were starved for 2 hours in serum free high glucose DMEM containing 0.2% BSA followed by incubation with 20 µM BMS30943 for various time periods as indicated. (A) Glucose uptake by C2C12 myotubes. The results were expressed as fold over DMSO group; (B) Western blotting analysis of AMPK signaling pathway and Akt in C2C12 cells.
Figure 2.
BMS309403 stimulates glucose uptake in C2C12 myotubes and AMPK signaling pathway in a dose dependent manner.
C2C12 myotubes were starved for 2 hours in serum free high glucose DMEM containing 0.2%BSA followed by incubation for 2 h with the indicated concentrations of BMS309403. (A) 2-deoxy-D[1-3H]-glucose was added for 15 min at 37°C, followed by glucose uptake measurement. (B) Western blotting analysis of AMPK and Akt signaling molecules.
Figure 3.
Western blotting analysis of AMPK signaling pathway following treatment with BMS309403 in L6 myotubes.
L6 myotubes were starved for 2 hours in serum free high glucose DMEM containing 0.2%BSA followed by incubation for 2 h with 30 µM of BMS309403.
Figure 4.
AMPK activation is indispensable for BMS309403 induced glucose uptake.
C2C12 myotubes were pretreated with compound C (C.C) for 30 min followed by the addition of BMS309403 and/or insulin. (A) Glucose uptake in C2C12 myotubes following indicated treatments. (B) Western blotting analysis of related signaling molecules in C2C12 myotubes.
Figure 5.
AMPK activation by BMS309403 is independent of hFABP3.
Western blotting analysis of AMPK phosphorylation in C2C12 myobubes overexpressing FABP3 with or without BMS309403 treatment.
Figure 6.
BMS309403 triggered AMPK activation remains intact upon knockdown of endogenous FABP3 in C2C12 myotubes.
(A) Microscopy photography (×100) of C2C12 cell lines stably expressing lentiviral HIVU6-EGFP or HIVU6-shRNA against mFABP3 (shFABP3). (B) Quantitative real-time PCR analysis of FABP3 gene expression in differentiated C2C12 stable myotubes. (C) Western blotting analysis of AMPK signaling pathway following BMS309403 treatment in the control and FABP3 knocked-down C2C12 myotubes. (D) Densitometric analysis of phospho-ACC and phospho-AMPKα in C2C12 stable myotubes of EGFP or shFABP3 treated with DMSO or 20 µM BMS309403. C2C12 stable myotubes of EGFP treated with DMSO was used as control. There was significant difference between DMSO and BMS group, but no significant difference between EGFP + BMS and shFABP3+ BMS group.
Table 1.
AMPKα2β1γ1 Activity by AMP and BMS309403.
Figure 7.
BMS309403 is not a direct activator of AMPK.
Results of in vitro AMPK assay for AMPKα2β1γ1 incubated with BMS309403 or AMP.
Figure 8.
BMS309403 reduces mitochondrial membrane potential in a dose-dependent manner and increases cytosolic AMP/ATP ratio in C2C12 myotubes.
(A) Mitochondrial membrane potentials of C2C12 myotubes were measured on day 5 of differentiation after pretreated with BMS309403 at 37°C for 2hours. (B) Adenine nucleotides from perchloric acid extracts of C2C12 myotubes were measured by HPLC. Values were means ± SE for three independent experiments. **p<0.01 vs DMSO.