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. 2016 Jul 8;11(7):e0159191. doi: 10.1371/journal.pone.0159191

Fig 4. PPARγ activation was involved in APL-mediated FGF21 up-regulation in skeletal muscle myotubes.

Fig 4

(A) Differentiated L6 cells were untreated or pretreated with palmitate (PA,0.75 mM) for 16 h, then incubated with 10 μM APL for 24 h in the presence or absence of insulin (100 nM). PPARγ was detected by western blot. (B) Differentiated L6 cells were pretreated with palmitate (PA,0.75 mM) for 16 h, and then incubated with 1, 5 or 10 μM APL for 24 h. PPARγ was detected by western blot. (C) Differentiated L6 cells were pretreated with palmitate (PA,0.75 mM) for 16 h, and then incubated with (10 μM) APL for 6, 12 or 24 h. PPARγ was detected by western blot. (D) Differentiated L6 cells were pretreated with palmitate (PA,0.75 mM) for 16 h, then cells were treated with GW9662 (10 μM) for 1 h, or following by treated with 10 μM APL or Rosiglitazone (Ros) (10 μM) for 24 h in the presence of insulin (100 nM). Total L6 cell lysates were used for western blots. (E) Differentiated L6 cells were pretreated with palmitate (PA,0.75 mM) for 16 h, then transfected with PPARγ siRNA for 24h, following by treated with 10 μM APL or Rosiglitazone (Ros) (10 μM) for 24 h in the presence of insulin (100 nM). Total L6 cell lysates were used for western blots. (F) Molecular modeling of the interaction between APL and PPARγ. A close-up view of the consensus orientation for APL is shown. The PPARγ protein is depicted as a ribbon representation and colored by secondary structures (i.e., helix, strand, and loop) (left panel); the hydrogen bonds between APL and PPARγ(distance<3.2 Å) are depicted as red dotted lines that include the names of the residues and distances (right panel). (G) Differentiated L6 cells were pretreated with palmitate (PA,0.75 mM) for 16 h, then cells were treated with GW9662 for 1 h or PPARγ siRNA for 24 h before addition of APL (10 μM) or Ros(10 μM) in the presence or absence of insulin (100 nM). Cells were collected and 2-NBDG glucose uptake was assessed. Values are means ± SEM. n = 3, ap < 0.05 versus palmitate -treated group; bp < 0.05 versus APL and palmitate co-treated group; cp < 0.05 versus Ros and palmitate co-treated group. All results are representative western blots of three independent experiments with similar results. (H)Activation effect of APL and rosiglitazone (Ros) on hPPARγ. The activity of the vehicle control was set at 1 and the relative luciferase activities are presented as fold induction relative to the vehicle control. ap < 0.05 versus control group. n = 3. Mean±SD.