The P2X7 ion channel is dispensable for energy and metabolic homeostasis of white and brown adipose tissues

Figures

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  Fig. 1. Gene expression analysis in a WAT and b BAT of lean, DIO, and ob/ob mice. C57BL6/J wild-type (WT) mice were fed a normal chow (lean) or a diabetogenic high-fat diet for 16 weeks to generate lean or diet-induced obese (DIO) mice. From these mice as well as from leptin-deficient ob/ob mice, the expression of genes important for extracellular adrenergic signaling was determined by qPCR. Data are presented as mean ± SEM, *P < 0.05

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  Fig. 2. Gene expression analysis in cell fractions from adipose tissue (a–g). Male C57BL6/J WT mice were housed for 1 day at RT (22 °C) or cold (4 °C) condition. iBAT and subWAT tissues were harvested from each group, and different cell types were isolated by MACS®. Purity of cellular fractions was verified by gene expression of cell-specific markers for adipocytes (Adipoq), endothelial cells (Gpihbp1), and tissue resident macrophages (Emr1). The expression of P2rx4, P2rx5, P2rx7, and Ucp1 was determined in all isolated cell fractions. n = 4. Statistical analysis was done between cells of RT and cold group isolated from each tissue. Data are presented as mean ± SEM, *P < 0.05

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  Fig. 3. Comparative analyses of energy expenditure in WT and P2rx7 KO mice. a Ablation of P2X7 protein in adipose tissues (iBAT, interscapular BAT; iWAT, inguinal WAT; gWAT, gonadal WAT) of male WT and P2rx7 KO mice on the BALB/c and C57BL/6 backgrounds was confirmed by Western blotting. b–g Systematic energy expenditure in obese WT and P2rx7 KO mice on the BALB/c and C57BL/6 backgrounds were determined by indirect calorimetry. Male WT and P2rx7 KO mice were kept in metabolic monitoring cages for 1 week with a gradual decrease in housing temperature from 30 to 6 °C. O2 consumption, CO2 production, and respiratory exchange ratio (RER) were determined in b–d BALB/c and e–g C57BL/6 mice. For all experiments, n = 6 mice per group. Data are presented as mean ± SEM, *P < 0.05

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  Fig. 4. Diet-induced obesity studies in WT and P2rx7 KO mice on the BALB/c and C57BL/6 backgrounds. Male WT and P2rx7 KO mice were kept on a HFD for 16 weeks. a Body weight, b body weight gain, c organ weights, and d body fat composition was determined in P2rx7 KO mice on BALB/c background. e Body weight and f body weight gain was determined in P2rx7 KO mice on C57BL/6 background. n = 5–10 mice per group. g, h Representative microphotographs of gWAT, iWAT, and iBAT after hematoxylin and eosin (H&E) staining in P2rx7 KO vs. WT mice. Data are presented as mean ± SEM, *P < 0.05

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  Fig. 5. Inflammatory status in BAT and WAT of obese WT and P2rx7 KO mice on the BALB/c and C57BL/6 backgrounds. Male WT and P2rx7 KO mice were fed HFD for 16 weeks, and the gene expression of inflammatory markers was determined in a, b BAT and c, d gWAT by qPCR. e, f Representative microphotographs of anti-MAC-2 immune staining in gonadal (gWAT) and inguinal (iWAT) adipose tissues of WT and P2rx7 KO mice on HFD. Plasma g, i CCL2 and h, j IL6 levels were determined in HFD fed WT and P2rx7 KO mice on the g, h BALB/c and i, j C57BL/6 backgrounds by ELISA. n = 5–10 mice per group. Data are presented as mean ± SEM, *P < 0.05

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  Fig. 6. Glucose homeostasis in obese WT and P2rx7 KO mice. a, b Glucose tolerance test in HFD fed male WT and P2rx7 KO mice on the a BALB/c and b C57BL/6 backgrounds. Plasma c, f insulin, d, g triglyceride, and e, h cholesterol levels were measured using commercial kits. n = 5–10 mice per group. Data are presented as mean ± SEM, *P < 0.05

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  Fig. 7. Effect on liver metabolic parameters in obese WT and P2rx7 KO mice. Male WT and P2rx7 KO mice were kept on a HFD for 16 weeks. a, b Liver weight was determined in P2rx7 KO mice compared with WT controls on the a BALB/c and b C57BL/6 backgrounds. Liver c triglyceride (TG) and d cholesterol levels were determined and normalized to protein concentration. e, f Gene expression of inflammatory markers in the liver of WT and P2rx7 KO mice on the e BALB/c and f C57BL/6 background. n = 5–10 mice per group. Data are presented as mean ± SEM, *P < 0.05