Fig. 3: p53 upregulates FMO3 expression and TMAO production in mature adipocytes.

a A diagram showing two putative p53 responsive elements (RE) on the human FMO3 gene identified by JASPAR. Upper panel: consensus p53 RE suggested by JASPAR. Lower panel: The first and second putative p53 RE sites are located on the promoter and intron 1 of FMO3 gene as indicated. The number of nucleotides is relative to the ATG start codon. b–d 3T3-L1 adipocytes treated with DMSO as vehicle or doxorubicin (10 μM) for 24 h were subjected to qPCR analysis (b) and immunoblotting analysis (c). The bar chart in (d is the densitometry analysis of FMO3, p53 and p21 normalized with HSP90. n = 3 for vehicle. n = 4 in (b) and n = 3 in (c) for doxorubicin. e, f 3T3-L1 adipocytes (e) or SVF-derived adipocytes from Adipo-FMO3-KO mice or WT controls (f) were treated with vehicle (Veh) or doxorubicin (Doxo; 10 μM) for 24 h, followed by 200 μM d9-TMA for 16 h. d9-TMAO production in cell lysate or conditioned medium (e, f) as indicated. n = 4 for vehicle, doxorubicin and d9-TMA in (e, f). n = 5 in panel e for doxorubicin ± d9-TMA. n = 4 in the cell lysate and n = 3 in the cell culture medium of panel f for doxorubicin ± d9-TMA. g–k 16-week-old male C57BL/6 mice injected with a single dose of 2 mg and 10 mg of doxorubicin per kg of body weight or 1X PBS (Veh) were used. The day before doxorubicin injection is defined as day 0. qPCR analysis of Fmo3, p53 and Cdkn1a mRNA expression are normalized with 36b4 and 18 s in sWAT (g) and eWAT (h). g: n = 5 for vehicle and 2 mg doxorubicin injection, and n = 4 for 10 mg doxorubicin injection for Cdkn1a and p53; for Fmo3, n = 5 for vehicle and 10 mg doxorubicin injection, and n = 4 for 2 mg doxorubicin injection. h: n = 4 for Cdkn1a and p53; for Fmo3, n = 4 for vehicle and 10 mg doxorubicin injection, and n = 5 for 2 mg doxorubicin injection. TMAO level in eWAT and sWAT at day 10 (i) and in serum at day 0 and day 10 (j). i: n = 4 for eWAT. n = 4 for vehicle and 10 mg doxorubicin, and n = 5 for 2 mg doxorubicin for sWAT. j: n = 5. k qPCR analysis of Fmo3 gene expression normalized with 18 s in liver. n = 5. l 3T3-L1 adipocytes were co-transfected with a vector expressing luciferase (pGL3) under the control of Fmo3 promoter or intron-1 region containing p53 RE or no promoter (Basic) for 12 h, followed by treatment with 10 μM nutlin-3a for 24 h. n = 4. m 3T3-L1 adipocytes were co-transfected with plasmids expressing GFP or GFP-tagged p53 together with indicated luciferase vectors for 48 h, followed by measurement of luciferase activity. n = 3. The data are expressed as fold change over pGL3-Basic in the cells treated with Veh (l) or transfected with GFP (m). Data expressed as mean ± SEM, analysed by two-tailed Student’s t-test or one-way ANOVA.