Pparα

At the end of experiments, the lenses (n = 3 per group) were washed with PBS and lysed in RIPA buffer containing protease inhibitors (Aidlab Biotechnologies, Beijing, China) for 30 min on ice. The lens lysates were centrifuged at 12,000×g for 20 min at 4 °C, and the proteins were quantified using the BCA kit. Protein samples (80 μg) were separated by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Billerica MA, USA). The membranes were blocked with 5% fat-free dry milk for 2 h and then incubated with a rabbit polyclonal antibody against SOD (1:300, R&D Systems, Minneapolis, MN, USA), CAT (1:300, Abcam, Cambridge, MA, USA), peroxisome proliferator-activated receptor alpha (PPARα) (1:500, Abcam, Cambridge, MA, USA), or GAPDH (1:1000, R&D Systems MN, USA) at 4 °C overnight. Then, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:1000, Cell Signaling Technology Inc., USA) for 2 h. Finally, the protein bands were developed using enhanced chemiluminescence reagents (Millipore). Images were obtained using a Kodak Image Station 4000R (Eastman Kodak Co., Rochester, NY, USA) and analyzed using Kodak Image Software. The optical densities of specific immunopositive bands were normalized to the GAPDH band in the same sample.

After four weeks of feeding, six groups of mice (NCD, HFD, Fenofibrate, ON50, ON100, and ON200) were sacrificed to collect liver and adipose tissues. Murine liver and fat tissues were homogenized in 1 mL of RIPA buffer (10 mM Tris pH 7.8, 1 mM EDTA, 150 mM NaCl, phosphatase inhibitor, and protease inhibitor) using a homogenizer. Tissue lysate was collected and protein analysis was performed using Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Total protein (40 µg) was then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and PVDF membrane transfer (Millipore, Bedford, MA, USA). Rabbit primary antibodies against carnitine palmitoyltransferase (CPT1α), UCP1α, adipocyte protein 2 (AP2), PPARγ, CCAAT/enhancer-binding protein α (c/EBPα) (Cell Signaling Technology, MA, USA), PPARα, peroxisomal acyl-coenzyme oxidase 1 (ACOX1), and SPHK2 (Abcam, Cambridge, MA, USA) were then used respectively to detect target proteins on PVDF membrane. These samples were normalized to mouse primary antibody against β-actin (Millipore, Cambridge, MA, USA). After incubation with secondary horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG antibody (Millipore, Bilerica, MA, USA), blots were developed with enhanced chemiluminescent substrate (Millipore, Bilerica, MA, USA). Bands were detected using a Chemiluminescence imaging equipment (Viber Lourmat, France).

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco BRL (Carlsbad, CA, USA). OEA, kojic acid, MK886, L-DOPA, dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), α-melanocyte-stimulating hormone (α-MSH), PD98059 and PTU were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Antibodies against MC1R (1:1000, Cell Signaling Technology, Boston, USA), MITF (1:1000, Cell Signaling Technology), TRP-1 (1:1000, Cell Signaling Technology), Tyrosinase (1:1000, Cell Signaling Technology), PPARα (1:1000, Abcam, Cambridge, UK), total-ERK (1:1000, Cell Signaling Technology), phospho-ERK (Thr202/Thr204) (1:1000, Cell Signaling Technology), total-Akt (1:1000, Cell Signaling Technology), phospho-Akt (Ser473) (1:1000, Cell Signaling Technology), total-CREB (1:1000, Cell Signaling Technology), phospho-CREB (Ser133) (1:1000, Cell Signaling Technology), total-p38 (1:1000, Cell Signaling Technology), phospho-p38 (1:1000, Cell Signaling Technology) and mouse monoclonal anti-β-actin (1:10000, Sigma) were obtained from the indicated manufacturers. OEA, kojic acid, MK886, PD98059 and PTU were dissolved in dimethyl sulfoxide (DMSO), and the final DMSO concentration was less than 0.05%. L-DOPA was dissolved in phosphate buffered saline (PBS).