The Purinergic Receptor P2X5 Modulates Glucose Metabolism and Expression of Thermogenic Genes in Brown Adipose Tissue

Figures

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  Figure 1. P2rx5 is highly expressed by mature brown adipocytes. (a) Cell type-specific gene expression of the purinergic P2X receptors (P2rx1, P2rx2, P2rx3, P2rx4, P2rx5, P2rx6, P2rx7) in mature brown adipocytes (mA), CD11b+ myeloid cells, and CD31+ endothelial cells isolated from murine brown adipose tissue (BAT) using magnetic-activated cell sorting (MACS) (n = 4); (b) gene expression of P2rx1, P2rx2, P2rx3, P2rx4, P2rx5, P2rx6, and P2rx7 during the differentiation of stromal-vascular cell (SVC)-derived brown adipocytes (n = 4); (c) P2X5 staining in primary brown adipocytes. Red, P2X5; blue, DAPI. Scale bar: 50 µm. Data are presented as mean values ± SEM. (a) A one-way ANOVA followed by Tukey’s multiple comparisons test comparing cell fractions with each other was performed. Different letters denote significant differences between cell fractions. (b) A one-way ANOVA followed by Dunnett’s multiple comparisons test comparing the different days of differentiation to day 0. In the table, significant differences are indicated by asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001).

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  Figure 2. P2rx5 deficiency resulted in lower expression of thermogenic markers in BAT. (a–g) P2rx5 knockout mice (P2rx5−/−), heterozygous (P2rx5+/−), and controls (P2rx5+/+) were housed at 22 °C. (a) Gene expression of P2rx5 in BAT and inguinal white adipose tissue (iWAT) (n = 3). (b) Whole-mount staining of P2X5 in BAT of P2rx5−/− and P2rx5+/+ mice. Green, P2X5; blue, DAPI. Scale bar: 50 µm. (c) Adipose tissue weights, (d) plasma triglycerides, (e) plasma non-esterified fatty acids (NEFAs), and (f) plasma cholesterol levels of P2rx5 knockout mice (P2rx5−/−), heterozygous (P2rx5+/−), and controls (P2rx5+/+) (n = 7). (g) Gene expression of thermogenic marker genes in BAT of P2rx5−/−, P2rx5+/−, and P2rx5+/+ mice housed at 22 °C (n = 7). Data are presented as mean values ± SEM. A one-way ANOVA followed by Tukey’s multiple comparisons test comparing wild type, heterozygous, and homozygous P2rx5 knockout mice was performed. Different letters denote significant differences.

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  Figure 3. P2rx5 deficiency improves glucose disposal by BAT. (a–b,d–g) P2rx5 knockout mice (P2rx5−/−), and controls (P2rx5+/+) were housed at 22 °C. (a) Western blot analysis of UCP1, respiratory chain (OXPHOS) complexes, and g-Tubulin in BAT as well as (b) quantification of proteins shown in (a) (n = 7). (c) Energy expenditure in P2rx5+/+ and P2rx5−/− mice housed at indicated environmental temperatures (30 °C, 24 °C, 18 °C, 12 °C, and 6 °C with a stepwise decrease in temperature every 24 h) in metabolic chambers (n = 6). (d) Plasma insulin levels of P2rx5+/+ and P2rx5−/− mice (n = 7). (e) Blood glucose levels during insulin tolerance test (n = 12) and (f) blood glucose levels during oral glucose tolerance test (n = 12–13) of P2rx5+/+ and P2rx5−/− mice. (g) Uptake of 3H-deoxyglucose (DOG) into interscapular BAT (iBAT), inguinal WAT (iWAT), gonadal WAT (gWAT), heart, muscle, kidney, spleen, and liver (n = 12–13). Data are presented as mean values ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 by Student’s t test.

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  Figure 4. Brown adipocyte-specific P2rx5 deletion resulted in lower Ucp1 expression. (a–g) Brown adipocyte-specific P2rx5-deficient mice (P2rx5fl/fl-Ucp1Cre+) and P2rx5fl/fl-Ucp1Cre− mice were housed at 22 °C. (a) Gene expression of P2rx5 in BAT, inguinal WAT (iWAT), gonadal WAT (gWAT), heart, muscle, kidney, spleen, pancreas, and liver of the P2rx5fl/fl-Ucp1Cre model (n = 4–5). (b) Whole-mount staining of P2X5 in BAT of P2rx5fl/fl-Ucp1Cre+ and P2rx5fl/fl-Ucp1Cre− mice. Green, P2X5; blue, DAPI. Scale bar: 50 µm. (c) Adipose tissue weights, (d) plasma triglycerides, (e) plasma non-esterified fatty acids (NEFAs), and (f) plasma cholesterol levels of P2rx5fl/fl-Ucp1Cre+ and P2rx5fl/fl-Ucp1Cre− mice (n = 4–7). (g) BAT gene expression of thermogenic marker genes in P2rx5fl/fl-Ucp1Cre+ and P2rx5fl/fl-Ucp1Cre− mice housed at 22 °C (n = 4–7). (h,i) siRNA-mediated knockdown of P2rx5 was induced in primary stromal-vascular cell (SVC)-derived brown adipocytes and in immortalized brown adipocytes for 48 h. (h) Gene expression of P2rx5 and Ucp1 in primary SVC-derived (n = 9, knockdown 74%) or (i) immortalized pre-adipocytes (n = 6, knockdown 60%) differentiated to mature brown adipocytes. Data are…

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  Figure 5. Brown adipocyte-specific P2rx5 deletion impaired glucose tolerance. (a,b,d,e) Brown adipocyte specific P2rx5-deficient mice (P2rx5fl/fl-Ucp1Cre+) and P2rx5fl/fl-Ucp1Cre− mice were housed at 22 °C. (a) Western blot analysis of BAT and (b) quantification of proteins shown in (a) (n = 4–5). (c) Energy expenditure in P2rx5fl/fl-Ucp1Cre+ and P2rx5fl/fl-Ucp1Cre− mice housed at indicated environmental temperatures in metabolic chambers. Here, ambient temperature was decreased every other day starting at thermoneutrality (30 °C) to mild cold exposure (22 °C) and cold exposure (6 °C) (n = 3). (d) Plasma insulin levels in P2rx5fl/fl-Ucp1Cre+ and P2rx5fl/fl-Ucp1Cre− mice. (e) Blood glucose levels during oral glucose tolerance tests in P2rx5fl/fl-Ucp1Cre+ and P2rx5fl/fl-Ucp1Cre− mice (n = 6). Data are presented as mean values ± SEM. * p < 0.05, and *** p < 0.001 by Student’s t test.

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  Figure 6. Brown adipocyte-specific P2rx5 deficiency impaired glucose tolerance and uptake into the BAT of diet-induced obese mice. (a–i) Brown adipocyte-specific P2rx5 deficient mice (P2rx5fl/fl-Ucp1Cre+) and P2rx5fl/fl-Ucp1Cre− mice were fed a high-fat diet (HFD) for 16 weeks. (a) Body weights of P2rx5fl/fl-Ucp1Cre+ and P2rx5fl/fl-Ucp1Cre− mice during the HFD feeding period (n = 9–10). (b) Adipose tissue weights, (c) plasma triglycerides, (d) plasma non-esterified fatty acids (NEFAs), and (e) plasma cholesterol levels of P2rx5fl/fl-Ucp1Cre+ and P2rx5fl/fl-Ucp1Cre− mice (n = 8–9). (f) BAT gene expression (n = 8–9). (g) Blood glucose levels during the oral glucose tolerance test (OGTT) (n = 8–9). (h) Plasma insulin levels of P2rx5fl/fl-Ucp1Cre+ and P2rx5fl/fl-Ucp1Cre− mice (n = 8–9). (i) Uptake of 3H-deoxyglucose (DOG) per total organ into interscapular BAT (iBAT), inguinal WAT (iWAT), gonadal WAT (gWAT), heart, muscle, kidney, spleen, and liver (n = 6–9). Data are presented as mean values ± SEM. * p < 0.05 by Student’s t test.