Extended Data Fig. 9: Adipocyte Opa1 deletion impairs preadipocytes browning by reduction in fumarate levels.
(a) Equal amounts of protein (20 µg) from SAT isolated from littermates of the indicated genotype treated where indicated with the beta-3 adrenergic agonist CL-316,243 (CL; 10 mg/kg) for 5 days were separated by SDS-PAGE and immunoblotted using the indicated antibodies. TUB: tubulin. Each lane corresponds to an individual mouse. (b) Electron microscopy (EM) images from SAT of control and Opa1ΔAT mice treated for 5 days with ip injection of 10 mg/kg of CL316,243 (CL) every day. Scale bar: 5 µm. (c) Volcano plot of a metabolomic analysis of SAT from Wt and Opa1ΔAT mice (n = 4). The red dots indicate significantly different metabolites. (d) Metabolites in (c) were analyzed using the Pathway Analysis module of MetaboAnalyst tool. Red dots show the top significantly relevant pathways. (e) Box-dot plots of Opa1 and Ucp1 expression fold change (brite vs. preadipocytes) in Wt and Opa1ΔAT SAT pre-adipocytes differentiated into brite adipocytes. Data are normalized for β-actin gene expression, calculated by ΔΔCT (n = 7). ***, p = 7.9×10−4 for Opa1 and p = 4.1×10−4 for Ucp1 in a two-tailed Mann-Whitney U test. (f) Heat map of hierarchical clustering by Pearson Correlation of metabolomics analysis of brite-differentiated white preadipocytes infected with AdCre or AdGFP as control. Each column represents one independent experiment. (g) Dot plots of Ucp1 relative expression in pre-adipocytes isolated from Opa1flxflx mice and differentiated into brite adipocytes after adenovirus-mediated infection with Cre-GFP or GFP as control. Data are expression fold change normalized for β-actin gene expression, calculated by ΔΔCT (n = 3). *, p < 0.05 Kruskal-Wallis ANOVA test (p = 0.049). In e,g dots represent biologically independent experiments, I-shaped boxes mean ± SEM, whiskers the 10th-90th percentile.
