p38s regulate respiratory capacity of brown adipocytes.

Primary adipocytes isolated from intercapsular BAT were differentiated in vitro. (A) qRT-PCR analysis of browning genes mRNA expression from primary adipocytes isolated from WT or p38δ−/− mice. mRNA expression was normalised to the amount of Gapdh mRNA (mean ± SEM; WT n = 5 wells; p38δ−/− n = 5 wells). (B) Analysis of mitochondrial DNA content with respect to nuclear DNA by RT-PCR in adipocytes isolated from BAT of Fab-cre or p38αFab-KO mice (mean ± SEM; Fab-Cre n = 3 wells; p38αFab-KO n = 5 wells) and of (C) WT or p38δ−/− mice (mean ± SEM; WT n = 3 wells; p38δ−/− n = 4 wells). (D–E) OCR to NE (1 μM) and ISO (1 μM) in differentiated brown adipocytes from Fab-Cre and p38αFab-KO mice (mean ± SEM; Fab-Cre n = 7 or p38αFab-KO n = 7 wells treated with NE; and Fab-Cre n = 8 or p38αFab-KO n = 8 wells treated with ISO) (panel D) or from WT or p38δ−/− mice (mean ± SEM; WT n = 22 or p38δ−/− n = 12 wells treated with NE; and WT n = 12 or p38δ−/− n = 12 wells treated with ISO) (panel E) analysed by Seahorse assay. Nonmitochondrial respiration was subtracted from OCR values, and all values were normalised to protein content. Upper panels show OCR over time upon different drugs injections: oligomycin (1 μM), FCCP (1 μM), and antimycin A (1 μM) with rotenone (1 μM). Lower panels show basal and NE/ISO-induced OCR. (F) OCR induced by NE and ISO in differentiated brown adipocytes from Fab-Cre and p38αFab-KO mice was abolished by pretreatment with BIRB796 (10 μM) for 1 hour (mean ± SEM; Fab-Cre n = 6 or p38αFab-KO n = 7 wells treated with NE; and Fab-Cre n = 7 or p38αFab-KO n = 8 wells treated with ISO). See also S1 Data. BAT, brown adipose tissue; FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; ISO, isoproterenol; NE, norepinephrine; OCR, oxygen consumption rate; qRT-PCR, quantitative real-time polymerase chain reaction; WT, wild-type.