Figure 6.

The SERCA2-RyR2 pathway controls glucose utilization and thermogenesis in Ucp1−/− beige fat. (a) ECAR in Ucp1−/− beige adipocytes in the culture medium with low or high glucose concentrations. Glucose, oligomycin (Oligo), and 2-DG were added at indicated time points. Vehicle, n = 7; NE, n = 8. ***P < 0.001. (b) ECAR in Ucp1−/− beige adipocytes expressing a scrambled control RNA (Scr) or shRNA targeting Atp2a2 (sh-Atp2a2). Differentiated cells were treated with NE or vehicle in the culture medium with a high glucose concentration (a) and oligomycin (b). Scr with vehicle, n = 7; Scr with NE, n = 8; sh-Atp2a2with vehicle, n = 9; sh-Atp2a2 with NE, n = 9. *P < 0.05, **P < 0.01. (c) Glucose oxidation in Ucp1−/− beige adipocytes expressing a scrambled control RNA (Scr) or shRNA targeting Atp2a2 (sh-Atp2a2). n = 6 for both groups. *P < 0.05. (d) Glucose uptake in Ucp1−/− beige adipocytes expressing a scrambled control RNA (Scr) or shRNA targeting Atp2a2 (sh-Atp2a2). n = 3 for both groups. *P < 0.05. (e) Fatty acid oxidation in Ucp1−/− beige adipocytes expressing a scrambled control RNA (Scr) or shRNA targeting Atp2a2 (sh-Atp2a2). n = 6 for both groups. n.s., not significant. (f) ECAR in Ucp1−/− beige adipocytes expressing RyR2 or an empty vector (Control). n = 10 for both groups. ***P < 0.001. (g) OCR in Ucp1−/− beige adipocytes treated with 2-DG, NE, or oligomycin (Oligo) at indicated time points. Vehicle, n = 9; 2-DG, n = 10. *P < 0.05, **P < 0.01. (h) Oil-Red-O staining of differentiated pig adipocytes expressing PRDM16 (beige) and vector (white) at low magnification (top) and high magnification (bottom). Scale bars, 50 μm. (i) mRNA expression of the indicated beige fat-selective genes in differentiated pig adipocytes expressing PRDM16 (beige) and an empty vector (white). n = 3 for both groups. ***P < 0.001. (j) Mitochondrial OXPHOS proteins in differentiated pig adipocytes expressing PRDM16 (beige) and an empty vector (white). Tissue lysates from the rat heart were used as a positive control (P.C.). Immunoblot using the α-tublin antibody was shown as a loading control. Molecular weight (kDa) is shown on the right. (k) SERCA2 protein in differentiated pig beige adipocytes expressing a scrambled control RNA (Scr) or shRNA targeting Atp2a2. β-actin was shown as a loading control. (l) Basal OCR in pig beige adipocytes expressing a scrambled control RNA (Scr) or shRNA targeting Atp2a2. Scr with vehicle, n = 12; Scr with NE, n = 17; sh-Serca2 with vehicle, n = 15; sh-Serca2 with NE, n = 15. ***P < 0.001. (m) ECAR in pig beige adipocytes expressing a scrambled control RNA (Scr) or shRNA targeting Atp2a2. Scr with vehicle, n = 5; Scr with NE, n = 6; sh-Serca2 with vehicle, n = 5; sh-Serca2 with NE, n = 6. *P < 0.05, ***P < 0.001. (n) Basal OCR in pig beige adipocytes expressing RyR2 or an empty vector (Control). Control with vehicle, n = 12; Control with NE, RYR2 with vehicle, RYR2 with NE, n = 10. *P < 0.05, **P < 0.01, ***P < 0.001. (o) A proposed model of non-canonical thermogenesis in beige fat. A full explanation of the diagram is included in the Discussion section. The acronyms used in the diagram include adrenergic receptor (AR), sarco/endoplasmic reticulum Ca²⁺-ATPase2b (SERCA2b), ryanodine receptor 2 (RyR2), inositol 1,4,5-trisphosphate receptors (IP₃Rs), calstabin2 (Cal2), voltage-dependent anion channel (VDAC), mitochondrial calcium uniporter (MCU), dehydrogenase (PDH), and mitochondrial electron transport chain (ETC). Data in (a–g,i,l–n) are expressed as means ± s.e.m. Data analyzed by Student’s t-test (a,c–g,i) and ANOVA followed by Tukey’s test (b,l–n).