Fig 2: XO-dependent H2O2production is required for 12/15-LOX-induced p38MAPK-CREB-Egr1-mediated DGAT2 expression: A, H2O2 production in WAT of CD-fed WT and 12 wks of HFD-fed WT and 12/15-LOX-/- mice was measured using Amplex Red. B, WAT from WT mice fed with CD or HFD in combination with and without Allopurinol (125 mg/L of drinking water) for 12 wks were analyzed for H2O2 production using Amplex Red. C-E, WAT in panel B were analyzed by western blotting (C and E) and qRT-PCR (D) for protein levels and relative mRNA levels, respectively, of the indicated molecules. F and G, Growth-arrested 3T3-L1 adipocytes were treated with and without 12(S)-HETE (0.5 µM) in the presence or absence of Allopurinol (50 µM) for 10 min (F) or 1 h (G) and cell extracts were prepared and analyzed by western blotting for the indicated proteins using their specific antibodies. H and I, WT and 12/15-LOX-/- mice were fed with CD or HFD for 12 wks, and mRNA and protein levels of XO were measured by qRT-PCR (H) and Western blot analysis (I). J and K, WAT extracts containing equal amounts of protein from each regimen were immunoprecipitated with the indicated antibodies and the immunocomplexes were analyzed by western blotting for the indicated proteins using their specific antibodies. *, p < 0.05 versus WT mice on CD; **, p < 0.05 versus WT mice on HFD.

Fig 3: p38MAPK-CREB-Egr1 signaling is required for 12(S)-HETE induced DGAT2 expression: A and B, 3T3-L1 adipocytes were growth-arrested, treated with and without 12(S)-HETE (0.5 μM) for indicated time periods and cell extracts were prepared and analyzed by western blotting for the indicated proteins using their specific antibodies. C and D, Growth-arrested 3T3-L1 adipocytes were treated with and without 12(S)-HETE (0.5 μM) in the presence or absence of SB203580 (10 μM) for 10 min (C) or 1 h (D) and cell extracts were prepared and analyzed by western blotting for the indicated proteins using their specific antibodies. E-G, 3T3-L1 adipocytes that were transfected with siControl or siCREB (100 nmoles) (E), or infected with Ad-GFP or Ad-KCREB or Ad-Egr1 (40 moi) (F and G), and growth-arrested were treated with and without 12(S)-HETE (0.5 μM) for 1 h and cell extracts were prepared and analyzed by western blotting. The efficacy of siCREB was shown by its effect on CREB levels and over-expression of the vectors for GFP, KCREB and dn-Egr1 was shown by probing for their expression levels. H and I, White adipose tissue extracts of CD-fed WT and 12 wks of HFD-fed WT and 12/15-LOX−/− mice were analyzed by western blotting for the indicated proteins using their specific antibodies. J, Relative mRNA expression levels of Egr1 in WAT of CD-fed WT and 12 wks of HFD-fed WT and 12/15-LOX−/− mice. *, p < 0.05 versus vehicle or WT mice on CD; **, p < 0.05 WT mice on HFD. WT1, Wilmer tumor protein 1.

Fig 4: 12/15-LOX deficiency prevents HFD-induced DGAT2 expression and 12/15-LOX dependent HFD-induced DGAT2 expression requires CREB and Egr1: A-C, Epididymal WAT extracts of CD-fed WT and 12 wks of HFD-fed WT and 12/15-LOX-/- mice were analyzed by western blotting for the indicated proteins using their specific antibodies. D, Relative mRNA expression levels of DGAT2 in WAT of CD-fed WT and 12 wks of HFD-fed WT and 12/15-LOX-/- mice. E, TRANSFAC analysis of mouse DGAT2 proximal promoter sequence encompassing from -312 nt to +92 nt for the identification of potential transcriptional factor binding sites. F, The effect of site-directed mutagenesis of CREB binding site at -12 nt and Egr1 binding site at -117 nt in pGL3-mDGAT2p construct on 12(S)-HETE (0.5 µM)-induced luciferase activity in 3T3-L1 adipocytes. G, 3T3-L1 adipocytes that were infected with Ad-GFP or Ad-KCREB or Ad-Egr1 were transfected with pGL3-mDGAT2p construct, treated with and without 12(S)-HETE (0.5 µM) for 1 h, and luciferase activity was measured. H and I, Time course effect of 12(S)-HETE (0.5 µM) on protein and mDGAT2 promoter DNA binding activity by EMSA using CREB binding element at -12 nt (H), and Egr1 binding element at -117 nt (I) as biotin labeled probes. J and K, Supershift EMSA analysis of nuclear extracts of control and 12(S)-HETE (0.5 µM)-treated 3T3-L1 adipocytes for CREB and Egr1 using CREB-binding element at -12 nt (J), and Egr1-binding element at -117 nt (K) as biotin labeled probes. L, 3T3-L1 adipocytes were transfected with pGL3-mDGAT2p construct, treated with and without 12(S)-HETE (0.5 µM) for 1 h in the presence or absence of SB203580 (10 µM) and luciferase activity was measured. *, p < 0.05 versus vehicle or WT mice on CD; **, p < 0.05 versus 12(S)-HETE or WT mice on HFD.