Chemokine receptor CCR1 restrains adipose tissue remodelling via cAMP/PKA/CREB signalling and adipocyte‒macrophage crosstalk in mice

FIGURE 1. Deficiency of Ccr1 suppresses the weight gain and insulin resistance of mice fed a high‐fat diet (HFD). (A) Body weights of mice (n = 6‒8). (B and C) Fat and lean mass of mice (n = 6‒8). (D) Tissue weights of mice (n = 6‒8). (E) haematoxylin and eosin (H&E)‐stained epididymal white adipose tissue (eWAT) sections (scale bars = 100 µm) and adipocyte area (n = 6‒8). (F) mRNA levels of Fas, Pparα and Pgc‐1α in eWAT of mice (n = 6‒8). (G and H) Glucose and insulin tolerance tests (n = 6‒8). (I) Immunoblots of phosphorylated Ser473 AKT (p‐AKT) and total AKT in the liver and eWAT (n = 3‒4). Data are presented as mean ± SEM. (A, D, G, H) Two‐tailed Student's t‐test. (B, C, E, F, I) Two‐way analysis of variance. * p < .05, ** p < .01; # p < .05, ## p < .01.

FIGURE 2. Deficiency of Ccr1 increases energy expenditure in diet‐induced obesity (DIO) mice. (A) VO2 of the mice (n = 6). (B) VCO2 of the mice (n = 6). (C) Respiratory exchange ratio (RER) of the mice (n = 6). (D) Energy expenditure (EE) plotted against body weight and ANCOVA predicted energy expenditure at a given body weight of 31 g in mice (n = 6). The ANCOVA body weight effect is p = .008. (E) Rectal temperature of the mice (n = 6‒8). (F and G) Uncoupling protein 1 (UCP1)‐stained brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) sections in normal chow diet (NC)‐fed and high‐fat diet (HFD)‐fed mice (n = 6). Scale bars = 100 µm. (H and I) Immunoblots of UCP1 in BAT and iWAT of mice (n = 6). Data are presented as mean ± SEM. (A‒E) Two‐tailed Student's t‐test. # p < .05, ## p < .01.

FIGURE 3. Deficiency of Ccr1 enhances cAMP/protein kinase A (PKA)/cAMP‐response element‐binding protein 1 (CREB) pathway in both brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) of high‐fat diet (HFD)‐fed mice. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes (DEGs) in the iWAT of HFD‐fed mice (n = 3). (B) GSEA of cAMP pathway in the iWAT of HFD‐fed mice (n = 3). (C) cAMP levels in plasma and iWAT of mice (n = 6‒8). (D and E) Immunoblots of p‐PKA‐C and p‐CREB and their total proteins within BAT and iWAT of mice (n = 6‒8). (F) Immunoprecipitation analysis of Gαi in iWAT‐derived adipocytes (n = 3). (G) Immunoblots of uncoupling protein 1 (UCP1) in 3T3‐L1 adipocytes (n = 3). Data are presented as mean ± SEM. (C) Two‐way analysis of variance. (F and G) Two‐tailed Student's t‐test. * p < .05.

FIGURE 4. Deficiency of Ccr1 increases adaptive thermogenesis of cold‐stimulated mice. (A) Relative mRNA expression of Ccr1 in the adipose tissues of 4°C‐stimulated mice (n = 4). (B) Rectal temperature of mice (n = 4). (C) Uncoupling protein 1 (UCP1)‐stained brown adipose tissue (BAT) sections of mice (n = 4). Scale bars = 100 µm. (D) Immunoblots of UCP1, p‐PKA‐C and p‐CREB and their total proteins in BAT of 4°C‐stimulated mice (n = 4). (E) Relative mRNA levels of thermogenic genes in BAT of mice (n = 4). (F) UCP1‐stained inguinal white adipose tissue (iWAT) sections of mice (n = 4). Scale bars = 100 µm. (G) Immunoblots of UCP1, p‐PKA‐C and p‐CREB and their total proteins in iWAT of mice (n = 4). (H) Relative mRNA levels of thermogenic genes in iWAT of 4°C‐stimulated mice (n = 4). Data are presented as mean ± SEM. (B‒H) Two‐tailed Student's t‐test. * p < .05, ** p < .01.

FIGURE 5. Deficiency of Ccr1 decreases inflammation and promotes the polarisation of macrophages towards the anti‐inflammatory phenotype in inguinal white adipose tissue (iWAT) of obese mice. (A) Relative mRNA expression of Tnfα and Il‐1β in the iWAT of mice (n = 6‒8). (B) Immunoblot analysis of p‐P38 MAPK and P38 MAPK with iWAT lysates of high‐fat diet (HFD)‐fed mice (n = 6‒8). (C) F4/80 and CD11C immunostaining in iWAT sections of HFD‐fed mice (n = 6‒8). Scale bars = 100 µm. (D) Relative mRNA expression of F4/80, Cd206 and Il‐10 in the iWAT of mice (n = 6‒8). (E) Representative flow cytometry plots of total macrophages in iWAT (n = 6). (F) M1 and M2 macrophages of iWAT (n = 6). (G) Quantitation of total macrophages, M1‐ and M2‐type macrophages and M1/M2 ratio (n = 6). Data are presented as mean ± SEM. (A, D‒G) Two‐way analysis of variance. (B) Two‐tailed Student's t‐test. * p < .05, ** p < .01; # p < .05, ## p < .01.

FIGURE 6. Adipocyte‐specific Ccr1 knockout protects mice from high‐fat diet (HFD)‐induced obesity and insulin resistance. (A) Body weights of mice (n = 4‒5). (B) Food intake of mice (n = 4‒5). (C) Tissue weight ratio of mice (n = 4‒5). (D) Fed blood glucose levels of mice (n = 4‒5). (E and F) Glucose and insulin tolerance tests (n = 4‒5). (G and H) Energy expenditure of mice (n = 4). The ANCOVA body weight effect is p = .012. (I) Body temperature of 4°C‐stimulated mice (n = 4‒5). (J and K) Immunoblots of uncoupling protein 1 (UCP1), p‐PKA‐C and p‐CREB and their total proteins in brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) of mice (n = 5). (L) F4/80, CD11c and CD163 immunostaining in iWAT sections of HFD‐fed mice (n = 4‒5). Scale bars = 100 µm. Data are presented as mean ± SEM. (A‒I) Two‐tailed Student's t‐test. * p < .05, ** p < .01.

FIGURE 7. Inhibition of CCR1 with BX471 attenuates obesity development in high‐fat diet (HFD)‐fed mice. (A and B) Body and tissue weights of HFD‐fed mice (n = 6‒8). (C) Plasma triglyceride (TG) contents of mice (n = 6‒8). (D) Fed blood glucose levels of mice (n = 6‒8). (E and F) Glucose and insulin tolerance tests of HFD‐fed mice (n = 6‒8). (G) Immunoblots of uncoupling protein 1 (UCP1), p‐PKA‐C and p‐CREB and their total proteins in brown adipose tissue (BAT) of HFD‐fed mice (n = 5). (H) Relative mRNA expression of thermogenic genes in the BAT of mice (n = 6‒8). (I) Immunoblots of UCP1, p‐PKA‐C and p‐CREB and their total proteins in inguinal white adipose tissue (iWAT) of HFD‐fed mice (n = 5). (J) Relative mRNA expression of brown‐related genes in the iWAT of mice (n = 6‒8). Data are presented as mean ± SEM. (A‒F, H, J) Two‐tailed Student's t‐test. * p < .05, ** p < .01.