Phospho-PPAR alpha (Ser21) Antibody - #AF8054

Figure 7The ATX–LPA axis suppresses hepatic production of FGF21 via ERK-mediated inhibition of PPARα. Six-week-old C57BL/6N male mice (n = 6–8) were fed with either STC or HFD for 8 weeks, and subsequently injected with 1 mg/kg body weight antibody (anti-ATX or nonimmune IgG [control]) every 10 days for another 7 weeks as in Figure 6. Mice were killed at the 15th week. (A) Real-time PCR analysis of mRNA expression levels of several hepatokines in liver (shown as fold change against the STC–Control group). (B) Real-time PCR analysis for mRNA expression levels of several adipokines in epididymal adipose tissue (shown as fold change against the STC–Control group). n = 6–8. Data are presented as the means ± SEM. (C) Serum levels of FGF21. (D) Hepatic mRNA expression levels of Fgf21 determined by real-time PCR analysis. (E) Serum levels of adiponectin. (F–H) Six-week-old C57BL/6N male mice were fed with STC or HFD for 15 weeks after intravenous injection with 1 × 1011 vector genome of AAV encoding shRNA specific to autotaxin (KD) or scramble control (SC) as in Figure 4. (F) Serum levels of FGF21. (G) Hepatic mRNA expression levels of FGF21 determined by real-time PCR analysis. (H) Serum levels of adiponectin. (I–J) Huh7 cells grown in serum-free DMEM were treated with or without the indicated dosage of rhATX, PF8380 (inhibitor of autotaxin), LPA, or Ki16425 (LPA-receptor antagonist) for 24 hours. (I) The mRNA expression levels of FGF21 (expressed as fold changes relative to vehicle [dimethyl sulfoxide]-treated controls) and its (J) protein concentrations in the conditioned medium were determined by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. n = 5. Data are presented as the means ± SEM. (K–L) Huh7 hepatocytes were pretreated with rhATX, LPA, PF8380 (inhibitor of autotaxin), Ki16425 (LPA-receptor antagonist) for 30 minutes, followed by treatment with or without fenofibrate for 24 hours. (K) FGF21 protein was secreted into conditioned medium and (L) hepatic FGF21 mRNA were determined by ELISA and real-time PCR, respectively. Huh7 hepatocytes were pretreated with U0126 (ERK inhibitor), wortmannin (PI3K), or LPA for 30 minutes, (M and N) followed by treatment with or without fenofibrate for 30 minutes to collect cell lysates for Western blot analysis, or (O) 24 hours to measure the relative mRNA abundance of FGF21, PPARA, and VLCAD (very-long-chain acylcoenzyme-A dehydrogenase, a downstream target of PPARα). (P) Relative luciferase activity in Huh7 hepatocytes transfected with a luciferase reporter vector carrying PPAR response element and treated with LPA, U0126, and wortmannin in the presence or absence of fenofibrate for 48 hours. Data are normalized against human β-ACTIN and expressed as the fold change relative to dimethyl sulfoxide–treated controls. (A–L and N–P) Statistical calculations were performed by 1-way analysis of variance followed by Tukey multiple comparisons using Prism 6. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. n = 5. Data are presented as the means ± SEM. HSP90, heat shock protein 90; p-ERK, phosphorylated_extracellular signal-related kinase; p-PPARα, phosphorylated peroxisome proliferator–activated receptor α; t-ERK, total extracellular signal-related kinase; t-PPARα, total peroxisome proliferator–activated receptor α.