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. 2019 Jan 17;9:1931. doi: 10.3389/fphys.2018.01931

FIGURE 2.

FIGURE 2

ACTH activates UCP1 via the canonical cAMP-PKA-lipolysis pathway. Oxygen consumption of primary brown adipocytes was assessed using microplate-based respirometry. First, basal respiration was determined and then, oligomycin (oligo) was added, which inhibits adenosine triphosphate (ATP) synthase resulting in basal leak respiration. UCP1 mediated uncoupled respiration was determined after injection of isoproterenol (ISO, 0.5 μM) as a positive control or adrenocorticotropic hormone (ACTH, 1 μM). Next, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler that allows assessment of maximal respiratory capacity, was added. Finally, antimycin A (anti A) was added in order to determine non-mitochondrial respiration. (A) Dose–response experiment revealing most robust effects at a ACTH concentration of 1 μM assessed as stimulated respiration as fold increase of basal leak respiration. (B) ACTH- and ISO-stimulated respiration after 1 h pretreatment with different concentrations of propranolol, a non-selective blocker of adrenergic β-receptors (n = 4). (C) Effects of different concentrations of GPS1573 (100 nM and 750 nM), a MC2R antagonist, on ACTH- and ISO-stimulated UCP1-mediated uncoupled respiration. (D) Cytosolic cAMP abundance after stimulation with increasing ACTH and ISO concentrations for 30 min (n = 3). (E) Fold increase of basal leak respiration after stimulation with ISO, ACTH and vehicle (control, assay medium) with or without protein kinase A inhibitor H89 (50 μM). Inhibitor was injected together with oligo prior to addition of stimulators (n = 4). (F) Respiration stimulated by ISO, ACTH or vehicle as fold increase of basal leak after 1 h pre-treatment with lipolysis inhibitors Ai (Atglistatin, ATGL-inhibitor, 40 μM) and Hi (Hi76-0079, HSL-inhibitor, 40 μM) (n = 4). (G) Stimulated respiration of primary brown adipocytes from UCP1+/+ and UCP1-/- mice (n = 3). Data are presented as means ± SD. (B,D,G) were analyzed by two-way ANOVA (Tukey’s test). (C,E,F) were analyzed by unpaired t-test. (D) EC50 was determined by non-linear regression analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.