Fig. 4
UCP1 is necessary and sufficient for the heat production in ATM mediating lipolysis. A mRNA expression of Ucp1 in sorted ATM in setting of Fig. 3C. B Immunofluorescence staining (left) of F4/80 (green) and UCP1 (red) in eWAT obtained from WT or myeloid-specific Hif-1α KO mice fed with HFD for 12 wk. Nuclei are shown in blue with DAPI counterstaining. Inserts are magnified regions where asterisks are marked. Scale bar denotes 100 μm. Quantification of F4/80+UCP1+ cells among the total F4/80+ cells is shown on the right. Data are the mean ± s.e.m. from n ≥ 5 mice per group. C ERthermAC (red) staining in the sorted ATM in mice as in (A). Yellow arrowheads indicate cells with ERthermAC staining. Quantitative analysis of ERthermAC intensity is shown in the right bar graph. D Western blot analysis for pHSL, HSL, and UCP1 of 3T3-L1 adipocytes (5 × 105 cells) directly co-cultured with WT or myeloid-specific Hif-1α KO mice BMDM (0.5 × 105 cells) for 24 h. E FFA levels in the supernatant of 3T3-L1 adipocytes (5 × 105 cells) directly co-cultured with WT, myeloid-specific Hif-1α KO mice, or Ucp1 KO mice BMDM (0.5 × 105 cells) for 24 h. F mRNA (left) and protein expression (right) of UCP1 in RAW264.7 cells after transfection of UCP1 overexpression vector. G ERthermAC (red) staining in RAW264.7 cells of (F). Nuclei of (C) and (G) are shown in blue with DAPI counterstaining. Scale bars of (C) and (G) denotes 50 μm. H, I Western blot analysis of pHSL, HSL, and UCP1 (H) or cellular FFA secretion (I) in 3T3-L1 adipocyte co-cultured with macrophages from (F). J-K Western blot of pHSL and HSL (J) or cellular FFA levels (K) in 3T3-L1 adipocytes exposed to 37 °C or 41 °C