Cold-induced serum amyloid A3 amplifies cAMP–PKA signaling to drive adaptive thermogenesis in brown adipocytes

Fig. 1. SAA3 expression in BAT is induced by cold and correlates with thermogenesis. (A and B) Protein A and mRNA B levels of SAA3, FGF21, and UCP1 in BAT of male C57BL/6NCrl mice housed at thermoneutrality (TN, 30 °C) or cold exposure (CE, 4 °C) for 2, 6, or 11 days. C Immunoblot analysis of SAA3 and UCP1 protein expressions in differentiated WT-1 brown adipocytes treated with norepinephrine (NE, 1 μM) or CL316,243 (CL, 10 μM) for 6 h. D Time-course protein expression of thermogenic markers in WT-1 cells in response to recombinant SAA3 (SAA3rec, 1 μg/mL). Data are presented as mean ± SEM; individual biological values are plotted in all bar graphs. For all panels, n = 3–4 biological replicates per group. Immunoblots are representative of 3 independent biological experiments (molecular weight markers shown). Statistical significance for A and B was determined by two-way ANOVA with Bonferroni post-hoc test; statistical significance for C and D was determined by one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001 vs. TN A, B or control group C, D

Fig. 2. SAA3 overexpression in BAT improves thermogenic capacity and cold tolerance. A Schematic and experimental timeline of local lentivirus injection (scrambled control or full-length Saa3 cDNA) into the BAT of male C57BL/6NCrl mice and subsequent housing at TN (30 °C) or CE (4 °C) for 11 days. B Core body temperature changes measured during the 11-day cold exposure period. C Protein levels of SAA3, UCP1, FGF21, and PGC-1α in BAT. D–G mRNA expression levels of Saa3 D, Ucp1 E, Fgf21 F, and Pgc1a G in BAT. H and I Phosphorylation levels of key signaling proteins H and expression of lipolysis-related proteins I in BAT. Data are presented as mean ± SEM; individual biological values (n = 4–8 mice per group) are plotted in all bar graphs. For mRNA analysis, three technical replicates were performed per sample. Immunoblots are representative of independent biological experiments; molecular weight markers (kDa) are indicated on the right of each blot. Statistical significance for the time-course data in (B) was determined by two-way repeated-measures ANOVA with Bonferroni post-hoc test. For (C–I), statistical significance was determined by two-way ANOVA with Bonferroni post-hoc test. ∗ p <…

Fig. 3. SAA3 knockdown in BAT impairs cold tolerance and reduces thermogenic activation. A Core body temperature changes of mice locally injected with scrambled control or shSaa3 lentivirus in BAT during the 11-day experimental period at TN (30 °C) or CE (4 °C). B Protein levels of SAA3, UCP1, FGF21, and PGC-1α in BAT. C–F mRNA expression levels of Saa3 (C), Ucp1 (D), Fgf21 (E), and Pgc1a (F) in BAT. G and H Phosphorylation levels of signaling proteins G and expression of lipolysis-related proteins H in BAT. Data are presented as mean ± SEM; individual biological values (n = 3–6 mice per group) are plotted in all bar graphs. For mRNA analysis, three technical replicates were performed per sample. Immunoblots are representative of independent biological experiments; molecular weight markers (kDa) are indicated on the right of each blot. Statistical significance for the time-course data in A was determined by two-way repeated-measures ANOVA with Bonferroni post-hoc test. For B–H, statistical significance was determined by two-way ANOVA with Bonferroni post-hoc test. ∗ p < 0.05, ∗ ∗ p < 0.01 vs. indicated groups

Fig. 4. SAA3 deficiency in brown adipocytes impairs thermoregulation and energy expenditure. A Schematic overview of the brown adipocyte-specific SAA3 knockout (UCP1-SAA3−/−) mouse model generated by crossing Saa3fl/fl mice with Ucp1-Cre mice. B and C Core body temperature B and body weight C of WT littermates and UCP1-SAA3−/− mice during a 5-h acute cold challenge at 4 °C (CE) or thermoneutrality (TN, 30 °C). D mRNA levels of thermogenesis-related and mitochondrial genes in BAT assessed by qPCR after 5-h cold exposure. E and F Protein levels of key thermogenic markers E and mitochondrial oxidative phosphorylation (OXPHOS) complexes F in BAT under TN or CE conditions. (Note: Panels E and F share the same HSP70 loading control as proteins were detected on the same membrane.). G–J Metabolic parameters including oxygen consumption (VO2, G), carbon dioxide production (VCO2, H), respiratory exchange ratio (RER, I), and total energy expenditure (EE, J) measured by indirect calorimetry. Data are presented as mean ± SEM; individual biological values (individual mice) are plotted in all bar graphs. For D–F, n = 3–4 independent biological experiments with 3 technical replicates per sample. For B,…

Fig. 5. SAA3 drives thermogenesis via a GPR3-dependent cAMP–PKA signaling axis. A Western blot analysis and quantification of thermogenesis-related proteins in primary brown adipocytes from WT and UCP1-SAA3−/− mice treated with or without CL316,243 (CL, 10 μM) for 6 h. B and C Intracellular cAMP levels B and phosphorylation of PKA substrates C in primary brown adipocytes following CL treatment. D Representative ERthermAC fluorescence images and quantification of thermogenic capacity in primary brown adipocytes treated with CL or recombinant SAA3 (SAA3rec). Scale bar, 50 μm. E Oxygen consumption rate (OCR) in primary brown adipocytes with sequential additions of SAA3rec, oligomycin, FCCP, and rotenone/antimycin A (RA). F Effect of Saa3 siRNA on CL- or SAA3rec-induced protein expression in WT-1 cells. G Protein expression in WT-1 cells treated with the ꞵ-blocker Bupranolol (10 μM) with or without CL or SAA3rec. H and I Intracellular cAMP levels H and PKA substrate phosphorylation I in WT-1 cells treated with CL or SAA3rec. J Effect of PKA inhibitor H89 (5 μM) on SAA3rec-mediated signaling in WT-1 cells. K–N mRNA levels of Gpr3 (K), cAMP levels L, signaling proteins M, and thermogenic gene…