Figure 3.

Enhanced Ca²⁺ cycling stimulates UCP1-independent thermogenesis in beige fat. (a) Intracellular Ca²⁺ levels in differentiated beige adipocytes from wild-type (WT) and Ucp1−/−mice following NE treatment (arrow). n = 6 for all groups. ***P < 0.001 by NE treatment. (b) Basal and oligomycin-resistant (uncoupled) OCR in Ucp1−/− beige adipocytes in a Ca²⁺ depleted medium containing EGTA in the presence of NE or thapsigargin (Thapsi). n = 6 for all groups. ***P < 0.001. (c) Basal OCR in WT and Ucp1−/− beige adipocytes treated with BAPTA or vehicle. WT cells, n = 5 for all the treatment; Ucp1 −/− with vehicle or NE, n = 12; Ucp1−/− with BAPTA or BAPTA+NE, n = 15. **P < 0.01 between vehicle and NE, ##P < 0.01 between vehicle and BAPTA. n.s., not significant. (d) Basal OCR in WT and Ucp1−/− brown adipocytes incubated in a medium containing BAPTA or vehicle. WT with vehicle, n = 8; WT with NE, n = 6; WT with BAPTA, n = 8; WT with BAPTA+NE, n = 6; Ucp1−/− with vehicle n = 8; Ucp1−/− with NE, n = 6; Ucp1−/− with BAPTA, n = 8; Ucp1−/− with BAPTA+NE, n = 6. **P < 0.01, ***P < 0.01 between vehicle and NE, #P < 0.05 between WT and Ucp1−/−. (e) Basal OCR in Ucp1−/− beige adipocytes treated with NE or specific agonists for α1-AR (phenylephrine), α2-AR (clonidine), β1-AR (denopamine), and β3-AR (CL316243). Vehicle and NE, n = 4; phenylephrine, n = 5; clonidine, n = 5; denopamine, n = 5; CL316243, n = 5. *P < 0.05, **P < 0.01 relative to vehicle. (f) OCR in Ucp1−/− beige adipocytes treated with specific inhibitors for α1-R (phenoxybenzamine) and β3-AR (SR59230A) in the presence of NE. NE, n = 8; phenoxybenzamine, n = 8; SR59230A, n = 9; phenoxybenzamine+SR59230A, n = 8. **P < 0.01. (g) OCR in Ucp1−/− beige adipocytes expressing RyR2 or an empty vector. Oligomycin (Oligo), FCCP, and antimycin (AA) were added at indicated time points. n = 5 for all groups. ***P < 0.001. (h) NE-induced Ca²⁺ release in Ucp1−/− beige adipocytes expressing Calbstabin2 or an empty vector in a Ca²⁺ depleted medium. n = 5 for all groups. ***P < 0.001. (i) Basal OCR in Ucp1−/− beige adipocytes expressing Calbstabin2 or an empty vector. NE or vehicle was added at the indicated time point. Vector with vehicle n = 8; with NE, n = 5; Calstabin2 with vehicle and NE, n = 10 for both. *P < 0.05, **P < 0.01, ***P < 0.001. (j) Rectal core body temperature of Ucp1−/− mice treated with the RyR2 stabilizer S107 or vehicle under 6°C at indicated time points. Vehicle, n = 9; S107, n = 8. *P < 0.05. (k) Representative EMG traces of Ucp1−/− mice treated with vehicle or S107 under 6°C. n=4 for both groups. Data are expressed as means ± s.e.m. Data analyzed by Student’s t-test (a,g,h,j,k) and ANOVA followed by Tukey’s test (b–f,i).