PMC

Changes in PKA and AKT phosphorylation in BAT of WT and β-AR KO mice treated with saline (−) or phentolamine (10 mg/kg, intraperitoneally for WT and 8 mg/kg, intraperitoneally for β-AR KO mice). Mice were sacrificed after 30 min by cervical dislocation, and BAT was isolated for protein measurements. Phosphorylated PKA Thr¹⁹⁷ (panel A), total PKA (panel B), phosphorylated AKT Ser⁴⁷³ (panel C), and total AKT (panel D) are shown. Protein levels are normalized to GAPDH (panel E). Data are expressed as the mean ± S.E.; n = 6–7 per group. *, p < 0.05 versus all groups; +, p < 0.05 versus WT; #, p < 0.05 versus WT-saline. Food was withdrawn for 2 h before the sacrifice.

Effect of denervation on CIDEA and AMPK β Ser¹⁰⁸ phosphorylation and β subunit total protein levels in brown fat of 7–9-week-old male WT and β-AR KO mice. All data are 6 days after denervation (Den). CIDEA protein (panel A and B), Ser¹⁰⁸ phosphorylation of AMPK β subunit (panel A and C), total AMPK β (panel A and D), and the ratio of phosphorylated to total AMPK β (panel E) are shown. Protein levels are normalized to GAPDH (panel F). Data are expressed as the mean ± S.E.; n = 3–4 per group. *, p < 0.05 versus respective sham. Food was withdrawn for 2 h before the sacrifice.

Phentolamine increases AMPK α Ser^485/491 phosphorylation in BAT of β-AR KO but not WT mice. Mice were injected with saline (−) or phentolamine at a dose of 10 mg/kg intraperitoneally for WT and 8 mg/kg intraperitoneally for β-AR KO mice and sacrificed after 30 min by cervical dislocation. BAT was isolated for protein measurements. Phosphorylated AMPK α Ser^485/491 (panel A), the ratio of Ser^485/491 phosphorylated to total AMPK α1 (panel B), the ratio of Ser^485/491 phosphorylated to total AMPK α2 (panel C), and the correlation of AMPK α2 activity with the ratio of Ser^485/491 phosphorylation to total AMPK α2 (panel D) are shown. Protein levels are normalized to GAPDH (panel A). Data are expressed as the mean ± S.E.; n = 6–7 per group. *, p < 0.05 versus all groups. Food was withdrawn for 2 h before the sacrifice.

Effects of unilateral denervation (Den) on UCP1 protein and AMPK and ACC activity, protein levels, and phosphorylation in brown fat of 7–9-week-old male FVB mice. Immunoblotting of UCP1 (panel A), AMPK α2 activity (panel B), AMPK α subunit phosphorylation on Thr¹⁷² and AMPK α2 protein (panel C), and ACC activity and ACC phosphorylation on Ser⁷⁹ and total ACC protein (panel D) are shown. ACC and AMPK protein levels are normalized to β-actin (panel C and panel D), which was not altered by the treatments. Data in panel C are 6 days post-denervation. Data are expressed as the mean ± S.E.; n = 10 per group. *. p < 0.05 versus sham same day; #, p < 0.05 versus denervated day 1; +, p < 0.05 versus denervated day 6. Food was withdrawn for 2 h before the sacrifice.

Effect of denervation on UCP1 and fatty acid synthase protein levels and AMPK activity and α subunit Thr¹⁷² phosphorylation in the brown fat of 7–9-week-old male WT and β-AR KO mice. All data are 6 days after denervation (Den). UCP-protein (panel A), fatty acid synthase protein (panel B), AMPK activity (panel C and D), Thr¹⁷² phosphorylation of AMPK α subunit (panel E), total AMPK α1 and α2 (panel F and G), and the ratio of phosphorylated to total AMPK α (panel H and I) are shown. Protein levels are normalized to GAPDH (panel J). Data are expressed as the mean ± S.E.; n = 12 per group. *, p < 0.05 versus respective sham; #, p < 0.05 versus all groups; **. p < 0.05 versus WT sham. Food was withdrawn for 2 h before the sacrifice.

Acute α-adrenergic blockade in β-AR KO mice decreases AMPK activity in brown fat. Mice were injected with saline (−) or phentolamine at a dose of 8 mg/kg intraperitoneally and sacrificed after 30 min by cervical dislocation. BAT was isolated for activity and protein measurements. AMPK activity (panel A and B), Thr¹⁷² phosphorylation of AMPK α (panel C and D), total AMPK α1 and α2 (panel C, E, and F), and ratio of phosphorylated to total AMPK α1 and α2 subunit (panel G and H) are shown. Protein levels are normalized to GAPDH (panel C and I). Data are expressed as the mean ± S.E.; n = 7–8 for activity assays per group, and n = 5–6 for Western blot analysis per group. #, p < 0.05 versus saline-treated animals. Food was withdrawn for 2 h before the sacrifice.

Adrenergic signaling-mediated alterations in AMPK activity (panel A) and oxygen consumption (panel B and panel C) in brown fat explants from WT mice. Brown fat explants were isolated as described under “Experimental Procedures” and incubated with either vehicle (−), phentolamine (Phen, 100 μm), propranolol (Prop, 100 μm), or isoproterenol (Iso, 60 μm) for 30 min and then frozen for activity and protein measurements. For oxygen consumption studies, explants were incubated with vehicle (−) or 100 μm phentolamine for 30 min and then transferred to an oxygen electrode chamber, and oxygen consumption was measured in the absence and presence of succinate. Finally, NaCN was added to inhibit oxygen consumption. Data are expressed as the mean ± S.E.; n = 6–8 per group. +, p < 0.05 versus all other groups. In panel B a representative tracing of the oxygen consumption in the explants is shown. Panel C shows the mean data from n = 5–6 explant preparations in the absence (−) or presence (+) of phentolamine. *, p < 0.05 versus WT-vehicle.

Effect of inhibition of AKT and PKA on AMPK Ser^485/491 phosphorylation and activity in BAT explants from WT and β-AR KO mice. BAT explants were isolated as described under “Experimental Procedures” and incubated with vehicle (−) or phentolamine (100 μm) alone or co-incubated with either DEBC-10 (3 μm) or PKA-I-14-22 (100 nm) for 30 min and then frozen for activity and protein measurements. Phosphorylated AKT Ser⁴⁷³ and total AKT (panel A), phosphorylated PKA Thr¹⁹⁷ and total PKA (panel B), phosphorylated AMPK Ser^485/491 (panel C and E), and AMPK α2 activity (panel D and F) are shown. Protein levels are normalized to GAPDH (panel A, B, C, and D). Data are expressed as the mean ± S.E.; n = 4–5 per group. **, p < 0.05 versus all other WT groups; +, p < 0.05 versus all other β-AR KO groups; #, p < 0.05 versus β-AR KO phentolamine group; *, p < 0.05 versus β-AR KO vehicle. Food was withdrawn for 2 h before the sacrifice.

Effect of pharmacological blockade of adrenergic receptors in vivo on AMPK activity and Thr¹⁷² phosphorylation and the ratio of Thr¹⁷² phosphorylated to total AMPK α in the brown fat of 7–9-week-old male WT mice. Mice were injected with either saline (Sal) or phentolamine (Phen) or propranolol (Prop) at a dose of 10 mg/kg intraperitoneally for 30 min after which animals were sacrificed by cervical dislocation, and BAT was frozen for activity and protein measurements. AMPK activity (panel A, B, E, and F), AMPK α Thr¹⁷² phosphorylation (panel C and G), and the ratio of phosphorylated to total AMPK α2 (panel D and H) are shown. Protein levels are normalized to GAPDH (panel I and J). Data are expressed as the mean ± S.E.; n = 8 per group; *, p < 0.05 versus saline. Food was withdrawn for 2 h before the sacrifice.