Fig. 4.

TRPV1 and UCP1 double knockout decreased functional BAT generation and lipolysis. A Immunoprecipitation (IP) with TRPV1 and UCP1 antibodies on mitochondria isolated from the brown adipose tissue of WT mice. The immunoblot bands are representative of six separate experiments. B Immunofluorescence staining of primary cultured brown adipocytes with anti-TRPV1 and UCP1 antibodies. TRPV1 was stained green, and UCP1 was stained red (upper level). Bar denotes 25 μm. The images were collected using a Nikon TE2000-U inverted fluorescence microscope. The coexpression of TRPV1 by plasmid transfection and TIMM23, a mitochondrial marker, in 293 A cells. The images were collected by a confocal microscope (A1 Nikon Co., Tokyo, Japan). Images are representative of 3 separate experiments. C Patch-clamp analysis of the inner membrane of the mitochondrial response to capsaicin, a TRPV1 agonist. D Oxygen consumption at different mitochondrial stages of primary brown adipocytes in WT, TRPV1^−/−, UCP1^−/−, and TRPV1^−/−/UCP1^−/− mice was measured by Oxygraph-2 k high-resolution respirometry. Routine values respiration in the original state, CI_OXPHOS CI-dependent oxidative phosphorylation, CII_OXPHOS CII-dependent oxidative phosphorylation, CI + II_OXPHOS oxidative phosphorylation providing CI and CII substrates, CII_ETS noncoupled respiration with CII-dependent respiration is considered the maximum capacity of the ETS state, CI + II_ETS noncoupled CI and CII substrates. E–G Dihydroetorphine hydrochloride (DHE, E), MitoSox (F), and ATP (G) levels of primary cultured brown adipocytes were measured. *P < 0.05, **P < 0.01 vs. WT mice; ^##P < 0.01 vs. TRPV1^−/− mice; ^∆∆P < 0.01 vs. UCP1^−/− mice