Figure 7.

Uncoupling function of mitochondria under NF-κB activation. (A) Uncoupling activity of mitochondria of p65 mice. Mitochondria were isolated from fresh BAT of p65 mice and were tested for uncoupling function in MAS with 10 mmol/L succinate and 2 μmol/L rotenone. OCR was monitored in mitochondria (10 μg) in response to the chemicals and calculated for the uncoupling activity. The chemicals were ADP (4 mmol/L), oligomycin (2 μmol/L), FCCP (4 μmol/L), and antimycin (4 μmol/L) (n = 3–4). (B) Enhanced uncoupling function by ANT activator. ANT activity was tested in the isolated mitochondria with the agonist Carb (20 μmol/L) and inhibitor bongkrekic acid (BA, 5 μmol/L) of ANT. OCR was used in the calculation of ATP production and maximal OCR (n = 3). (C) Ant2 overexpression in WT brown adipocytes. Ant2 was overexpressed in the primary brown adipocytes (3 × 10⁶ cells per well) of WT mice with the Ant2 expression adenoviruses and negative control (NC) virus for 48 h. The effect of Ant2 overexpression was tested in the cells after 48 h of infection by monitoring OCR upon injection of 1 μmol/L oligomycin, 2 μmol/L FCCP, 0.5 μmol/L rotenone plus 0.5 μmol/L antimycin (Rot/AA). Proton leak under the coupling and uncoupling conditions were calculated with OCR. (D) Ant2 overexpression in p65-OE brown adipocytes. The overexpression effect was tested in the primary brown adipocyte of p65-OE mice. Uncoupling activity was calculated with OCR. (E) Mitochondrial membrane potential in brown adipocytes of p65-OE mice. The potential was quantified with the fluorescent dye of JC-1 by flow cytometry. (F) Mitochondrial membrane potential in brown adipocytes of WT mice. (G) Skin temperature over iBAT in the cold environment. Each experiment was repeated three times with consistent results. In the bar figure, each bar represents mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.