Extended Data Fig. 2: Improved energy metabolism in mice and Nrg4 alleviated inflammation and adhesion response in MAECs.
(A) The weight of inguinal WAT (iWAT), epididymal WAT (eWAT) and interscapular BAT (iBAT) in WT and KO mice aged 18 weeks (n = 6, *P < 0.001 vs. WT-NCD; #P < 0.001 vs. WT-WD.). (B) The weekly food intake of WT and KO mice (n = 6, *P < 0.001 vs. WT-NCD; #P < 0.001 vs. WT-WD.). (C) The energy expenditure of WT and KO mice aged 18 weeks (n = 6, *P < 0.001 vs. WT-NCD; #P < 0.001 vs. WT-WD). (D) Schematic of the transgenic construct used to generate BAT-specific Nrg4 knockout animals. (E) Nrg4 expression in EC, kidney, WAT, BAT and liver from BKO and WT mice (n = 3, *P < 0.001). (F) The mRNA levels of adhesion molecules (VCAM-1, ICAM-1, and E-selectin) and inflammation cytokines (TNF-α, IL-1β, and IL-6) (n = 6, *P < 0.001). (G) Representative images of binding of IRB-NHS-Nrg4 to endothelium in thoracic aorta in vivo. Nrg4 (red), anti-CD31 (endothelial cells, green) and 4′,6-diamidino-2-phenylindole (DAPI) (nuclei; blue). Arrowheads indicate CD31/IRB-NHS-Nrg4 colocalization. Scale bar, 20μm (n = 3 biologically independent samples). (H) Representative bioluminescence images of mice aged 14 weeks injected AAV-Zsgreen (n = 6). (I) The circulating Nrg4 concentration weekly in KO mice after AAV intervention (n = 3). (J) The experiment schedule for the effects of Nrg4 overexpression on endothelial function, endothelial inflammation and metabolic profiles in mice (6 mice in each group). KO and WT mice aged 6 weeks were divided into four groups (WT-NCD, KO-NCD, WT-WD and KO-WD) and were fed their respective diets for 12 weeks (6 mice in each group). AAV-Nrg4 or AAV-Zsgreen at a dose of 1 × 1012 viral genomes were injected into the BAT in the interscapular region of KO mice at aged 6 weeks. Statistical significance was calculated using two-sided t-tests. Data were shown as mean ± SEM.
