Fig. 3

Lack of barr2 promotes WAT browning/beiging and enhances signaling through adipocyte β3-ARs. a Upregulation of genes promoting mouse iWAT browning/beiging in the absence of barr2 (RNA-seq analysis; HFD for 8 weeks; n = 3 or 4 per group). b, c Enhanced expression of genes involved in iWAT browning/beiging in the absence of barr2 (qRT-PCR analysis; HFD for 8 weeks; n = 3 or 4 per group). d Western blotting studies show striking increases in CKmt2, PGC-1α, and UCP1 protein levels in iWAT prepared from adipo-barr2-KO mice (HFD for 12 weeks). e Representative H&E-stained iWAT sections from iWAT (HFD for 16 weeks). f Reduction in adipocyte size in iWAT from adipo-barr2-KO mice (HFD for 16 weeks; n = 3 per group). g Mitochondrial DNA content (iWAT; HFD mice; n = 4 or 5 per group). h Tissue cAMP concentrations in iWAT (HFD mice; n = 6 per group). i–k Glycerol release studies. Mature adipocytes were stimulated with the indicated agents (n = 3 or 4 mice per group; diet: regular chow). l–n cAMP measurements. Mature adipocytes were incubated with β-AR subtype-selective agonists, followed by cAMP measurements: CL316243 (β3-selective; (l)), fenoterol (β2-selective; (m)), and xamoterol (β1-selective; (n)). Drug effects are expressed relative to cAMP responses caused by 100 μM forskolin (n = 4 or 5 mice per group; diet: regular chow). o Efficient reduction of barr2 mRNA expression after treatment of 3T3-F442A cells with barr2 siRNA (n = 4 per group). p Cell surface β3-AR expression levels after 30 min treatment of 3T3-F442A cells with CL316243 (1 μM). Prior to CL316243 incubation, cells had been treated with either barr2 siRNA or scrambled control RNA (n = 4 per group). q Time course of disappearance of cell surface β3-ARs of 3T3-F442A cells stimulated with CL316243 (1 μM) (n = 3). r Incubation of 3T3-F442A cells with filipin (1 μg/ml) and dynasore (80 μM), but not barbadin (100 μM), prevents the loss of cell surface β3-ARs triggered by CL316243 (1 μM) treatment for 30 min (n = 3). s Oxygen consumption rate (OCR) of differentiated adipocytes derived from iWAT of adipo-barr2-KO mice is significantly increased after isoproterenol (10 μM) treatment. Normalized data are shown (basal levels = 100%) (n = 3). Male mice were used for all studies. Data are given as means ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001 (b, c, f–q two-tailed Student’s t-test; r two-way ANOVA followed by Bonferroni’s post hoc test)