(A) Core body temperature was recorded in 2.5-, 5-, 12- and 18-month-old male GPR3 wild type (WT) and knock out (KO) mice. Values are mean ± s.e.m of n = 6–13 samples. *P < 0.05, **P < 0.01 compared to age-matched WT littermates. (B) Relative abundance of GPR3 transcript in iBAT obtained from adult male GPR3 WT at different ages. Values are mean ± s.e.m of n = 4–6 animals. #P < 0.05, compared to 5-month-old mice. (C) Relative abundance of indicated transcripts in interscapular brown adipose tissue (iBAT) of adult (5-month-old) male GPR3 WT and KO mice at room temperature. Values are mean ± s.e.m of n = 3–9 samples. *P < 0.05, **P < 0.01, compared to the age-matched WT littermates. (D) The UCP1, MTCO2 and α-tubulin (tubulin) proteins detected in iBAT from two groups of male GPR3 WT and KO mice (1–3 and 4–6). Protein sizes corresponding to UCP1, COX and α-tubulin were 33 kDa, 25 kDa and 52 kDa, respectively. Representative fluorescent immunostaining images against UCP1 in iBAT of GPR3 WT and KO mice. Pictures were taken at 10× magnification (scale bar indicates 100 μm). Western blot band and staining intensities were calculated as described in Material and Methods section and presented as bar graphs next to images. Values are mean ± s.e.m of n = 6–8 samples. *P < 0.05, **P < 0.01 compared to age-matched WT littermates. (E) Changes in core body temperature in 5-month-old male GPR3 WT and KO mice undergoing cold challenge. Body temperature was measured at ambient conditions for baseline as well as 1, 2, 3 and 4 hours after the exposure to cold (4 °C, cold challenge) and 0.5, 1 and 4 hours during the recovery period at room temperature (rt). Values are mean ± s.e.m of n = 5–6 samples. *P < 0.05 compared to age-matched WT littermates. (F) Relative abundance of GPR3 transcript in iBAT obtained from adult male GPR3 WT and GPR3 KO mice obtained at room temperature (rt) and at the 4th-hour of cold challenge (cold). Values are mean ± s.e.m of n = 3–8 samples. ***P < 0.001, compared to age-matched GPR3 WT littermates at room temperature (rt). GPR3 transcripts were not detectable (nd) in GPR3 KO mice. All target genes in panels (B,C,F) were corrected via reference Rpl19 mRNA.