GPR84-mediated signal transduction affects metabolic function by promoting brown adipocyte activity

The G protein–coupled receptor 84 (GPR84), a medium-chain fatty acid receptor, has garnered attention because of its potential involvement in a range of metabolic conditions. However, the precise mechanisms underlying this effect remain elusive. Our study has shed light on the pivotal role of GPR84, revealing its robust expression and functional significance within brown adipose tissue (BAT). Mice lacking GPR84 exhibited increased lipid accumulation in BAT, rendering them more susceptible to cold exposure and displaying reduced BAT activity compared with their WT counterparts. Our in vitro experiments with primary brown adipocytes from GPR84-KO mice revealed diminished expression of thermogenic genes and reduced O2 consumption. Furthermore, the application of the GPR84 agonist 6-n-octylaminouracil (6-OAU) counteracted these effects, effectively reinstating the brown adipocyte activity. These compelling in vivo and in vitro findings converge to highlight mitochondrial dysfunction as the primary cause of BAT anomalies in GPR84-KO mice. The activation of GPR84 induced an increase in intracellular Ca2+ levels, which intricately influenced mitochondrial respiration. By modulating mitochondrial Ca2+ levels and respiration, GPR84 acts as a potent molecule involved in BAT activity. These findings suggest that GPR84 is a potential therapeutic target for invigorating BAT and ameliorating metabolic disorders.


Figure S1 -
Figure S1-Related to Figures 1 and 2. Validation of GPR84 KO mice and GPR84 expression in different tissues.(A) Expression of GPR84 correlated with the expression of UCP1.Data from the GTEx database.TPM normalized expression values of human BAT and sWAT (bar graph).Data from GSE150119 (1).TPM normalized GPR84 expression level of obese human sWAT before and after low calorie diet (-LCD vs. + LCD; scatter plot graph).Data from GSE95640.(B) mRNA expression pattern of GPR84 shown in different tissues or organs of mouse.n = 5 -7/sample.(C) Body temperature of WT mice housed at RT and exposed to cold.n = 9/group.(D) Western blotting analysis of GPR84 protein expression in immature (D0) and mature (D8) brown adipocytes isolated from WT mice.Image is a representative image from three independent experiments.n = 5/group.(E) Representative immunofluorescence staining of GPR84 (red) and lipid droplets (BODIPY, green) in mature brown adipocytes isolated from WT and KO mice.Images are representative images from more than 10 cells of each three independent experiments.Scale bar = 10 μm (F) GPR84 mRNA expression in various tissues from WT and GPR84 KO mice.n = 6/group.(G) Western blotting analysis of GPR84 protein expression in mature brown adipocytes (BA) isolated from BAT and muscle of WT and GPR84 KO mice.****, P < 0.0001; ***, P < 0.001; *, P < 0.05 by two-tailed Student's t-test (A, F); two-way ANOVA followed by a Bonferroni's multiple comparison test (C).

Figure S2 -
Figure S2 -Related to Figure 2. Body weight, body mass and mitochondrial function in tissues from WT and KO mice at different ages.(A) Body weight changes of WT and KO mice.n = 10/group.(B) Body composition analysis including fat and lean mass for WT and GPR84 KO mice at different ages.Fat lean mass were normalized to body weight.n = 9 -10 for old mice, n = 5 for young mice.(C) Glucose tolerance test of normal chow-fed aged WT and KO mice, n = 20 -23/group.(D) Maximal OCR of fatty acid-dependent oxidation in BAT from WT and GPR84 KO mice at different ages.n = 5/group.(E -F) OCR of gWAT (E) and iWAT (F) from young and old WT and GPR84 KO mice.Data are represented as the mean ± SEM in duplicate.n = 4/group.*, P < 0.05; **, P < 0.01; ****, P<0.0001 by twotailed Student's t-test for (B); one-tailed Student's t-test (D); two-way ANOVA followed by a Bonferroni's multiple comparison test (A, E, F).Data are represented as mean ± SEM.

Figure S3 -Figure S4 -
Figure S3 -Related to Figure 3. Gene expression and OCR in tissues of young WT and GPR84 KO mice at cold exposure.(A) Tissue weight for eWAT, BAT and liver of WT and KO mice at cold exposure.(B) White adipose tissue-selective gene expression in BAT of WT and KO mice at RT and cold for 6 days.n = 8-10/group.(C -E) OCR in gWAT (C), iWAT (D), and muscle (E) of mice at 6 days after cold exposure.Data are represented as mean ± SEM in duplicate.n = 5/group.*, P < 0.05; **, P < 0.01; ****, P<0.0001 by two-way ANOVA followed by a Bonferroni's multiple comparison test.Data are represented as mean ± SEM of at least three independent experiments.

Figure S5 -
Figure S5 -Related to Figure 4. GPR84 stimulation-mediated signal transduction in brown adipocytes.(A) Adipogenic genes were measured by qPCR in fully differentiated brown adipocytes from WT and GPR84 KO mice (left-hand side) and in BAT from WT and GPR84 KO mice (right-hand side).Data are expressed as the mean !SEM of at least three independent experiments in triplicate.n = 3/group for brown adipocytes; n = 5/group for BAT.(B) Representative images of UCP1 immunofluorescence in brown adipocytes from WT and KO mice.n = 3/group.Scale bar = 50 μm.(C) CRE-luc activity in 293 cells transiently expressed GPR84.Cells were pre-treated with forskolin and then incubated with 6-OAU, Embelin, Decanoic acid (C10:0) and Lauric acid (C12:0) for 6h.n = 4 each concentration.(D) HEK293 cells transiently expressed GPR84 were pre-treated with PTX overnight and incubated with 6-OAU for 6 h.(E) SRE-Luc activity in 293 cells transiently expressed GPR84 were stimulated with 6-OAU, Embelin, Decanoic acid and Lauric acid for 6 h.(F) Calcium mobilization in mature WT brown adipocytes treated ± 6-OAU.After incubation with Fluo-4-AM for 1h RT, WT brown adipocytes ± BAPTA-AM (25 μM).Scale bar = 50 μm.Relative fluorescence units were analyzed by ROI using Image J. (G) Schematic diagram for GPR84-mediated signal transduction in brown adipocytes.Left-handed side cascade pathway indicates GPR84-Gi-mediated pathway while right-handed side pathway indicates the GPR84-Gβγ-mediated calcium pathway.*, P < 0.05; **, P < 0.01 by two-tailed Student's t-test.Data are represented as mean ± SEM from at least three independent experiment in triplicate.

Figure S6 -
Figure S6 -Related to Figure 6.GPR84 fl/fl and GPR84 BKO mice exposed to cold for 6 days.(A-C) OCR in muscle (A) and gWAT (B) and iWAT (C) from GPR84 fl/fl and GPR84 BKO mice at 6 days after cold exposure.The data are represented as the mean ± SEM in duplicate.n = 4/group.(D) Thermogenic gene expression level was measured by qPCR in BAT of vehicle-and 6-OAU-treated GPR84 fl/fl and GPR84 BKO mice at 6 days after cold exposure.n = 3 -8/group.*, P < 0.05; **, P < 0.01 by two-tailed Student's t-test (D), two-way ANOVA followed by a Bonferroni's multiple comparison test (A, B, C).Data are represented as mean ± SEM from at least three independent experiments.