Adenosine/A2B Receptor Signaling Ameliorates the Effects of Aging and Counteracts Obesity

Abstract

The combination of aging populations with the obesity pandemic results in an alarming rise in non-communicable diseases. Here, we show that the enigmatic adenosine A2B receptor (A2B) is abundantly expressed in skeletal muscle (SKM) as well as brown adipose tissue (BAT) and might be targeted to counteract age-related muscle atrophy (sarcopenia) as well as obesity. Mice with SKM-specific deletion of A2B exhibited sarcopenia, diminished muscle strength, and reduced energy expenditure (EE), whereas pharmacological A2B activation counteracted these processes. Adipose tissue-specific ablation of A2B exacerbated age-related processes and reduced BAT EE, whereas A2B stimulation ameliorated obesity. In humans, A2B expression correlated with EE in SKM, BAT activity, and abundance of thermogenic adipocytes in white fat. Moreover, A2B agonist treatment increased EE from human adipocytes, myocytes, and muscle explants. Mechanistically, A2B forms heterodimers required for adenosine signaling. Overall, adenosine/A2B signaling links muscle and BAT and has both anti-aging and anti-obesity potential.

Keywords: GPCR; adenosine; adenosine receptor A2B; aging; brown adipose tissue; browning; energy metabolism; muscle; obesity; sarcopenia.

Figure 1.. A2B signaling in skeletal muscle. See also Figure S1.

(A) GPCR mRNA expression in murine BAT and SKM (Abbreviations see Table S10; n=3). (B) cAMP levels in C2C12 pre-treated with A2A (MSX-2; 300 μM) or A2B (PSB603; 150 nM) antagonist prior to adenosine (1 μM) stimulation (n=6). ( C,D) ANCOVA of SKM/body weight of SKMA2B-KO and Con-A2B mice (C) or mice treated with A2B agonist for 4 weeks (D) (n=6). (E, F) Forelimb grip strength of young (2 month) and old (16 month) SKMA2B-KO and Con-A2B (E) or aged mice treated with A2B agonist (F). (G, H) Specific force of SKM explants from SKMA2B-KO and Con-A2B mice (G) or mice treated with A2B agonist for 4 weeks (H) (n=6). (I) mRNA abundance of satellite marker genes in SKM of SKMA2B-KO and Con-A2B mice (n=5). (J) Satellite cells in SKM of SKMA2B-KO and Con-A2B mice (n=6). (K) 4-hydroxynonenal (4-HNE) in SKM of SKMA2B-KO and Con-A2B mice (n=8). (L) Senescence-associated Beta-galactosidase activity in SKM of SKMA2B-KO and Con-A2B mice (n=6). (M-O) Expression of senescence (M), mitochondrial (N) and oxidative metabolism marker genes (O) in SKM of SKMA2B-KO and Con-A2B mice (n=6). (P) Oxygen consumption over 24 h at 23° C of SKMA2B-KO and Con-A2B mice (n=6). (Q) Oxygen consumption of SKM explants acutely treated with A2B agonist (300 nM) or Forskolin (1 μM) (state1: endogenous; state2: ADP; state3: succinate; state4: oligomycin; uncoupled: FCCP; n=6). * P < 0.05. Data are shown as mean + SEM (A,P), scatter plot (C,D) or boxplot (with median) and whiskers (1.5x interquartile range) (B,E-O,Q) and analyzed using two-tailed student’s t-test (K-O), ANCOVA (C,D) or ANOVA with Newman-Keuls post-hoc test (B,E-J,Q).

Figure 2.. The role of A2B in BAT activation and ageing. See also Figure S2.

(A,B) Interscapular surface temperature quantified by infrared thermography (A) and relative oxygen consumption at 4°C ( B) of ATA2B-KO and Con-A2B mice (n=5). (C,D) PET/MRI [¹⁸F]FDG uptake imageing (C) and quantification (D) of mice treated with vehicle, NE (1 mg/kg), or A2B agonist (0.1 mg/kg) (n=4). Arrows indicate interscapular BAT. (E) Expression of ageing markers in BAT of ATA2B-KO and Con-A2B mice (n=6). (F,G) Abundance malondialdehyde (F) and senescence-associated Beta-galactosidase activity (G) in BAT of ATA2B-KO and Con-A2B mice (n=6). (H) Expression of oxidative metabolism and BAT whitening marker genes of ATA2B-KO and Con-A2B mice (n=6). (I) Representative HE stain of BAT of aged ATA2B-KO and Con-A2B mice (Scale bar 100 μm). ( J,K) Time-course (J) and average (K) of whole-body O₂ consumption of ATA2B-KO and Con-A2B mice at 23° C (n=5). * P < 0.05. Data are shown as mean + SEM (J) or boxplot (with median) and whiskers (1.5x interquartile range) (A,B,D-H,K) and were analyzed with two-tailed student’s t-test (A,B,E-H,K) or ANOVA with Newman-Keuls post-hoc test (D).

Figure 3.. A2B heterodimerization. See also Figure S3.

(A) Lipolysis of murine BA treated with adenosine in the presence or absence of A2B antagonist (PSB603; 150 nM) (n=6). (B) O₂ consumption of BAT explants deficient for A2B and treated with adenosine (1 μM) (n=6). ( C) O₂ consumption of ATA2B-KO and Con-A2B mice injected with vehicle or A2A agonist (CGS21680; 1mg/kg) (n=5). (D) BRET analysis of A2A-RLuc and A2B-YFP or A2A-RLuc and Ghrelin-YFP in murine brown adipocytes (n=4). (E) Representative image after A2B/A2A proximity ligation assay in murine BAT. (F) In silico model of A2B/A2A interaction. (G) Lipolysis of murine BA treated with adenosine (1 μM) in the presence of indicated TM peptides (100 μM) (n=4). (H) In silico model of amino acid residues involved in A2B/A2A heterodimerisation. (I) A2B/A2A BRET after site-directed mutagenesis of residues identified in (H) (n=4). * P < 0.05. Data are shown as boxplot (with median) and whiskers (1.5x interquartile range) (A-C, G) or mean + SD (D,I) and analyzed using two-tailed student’s t-test (A) or ANOVA with Newman-Keuls post-hoc test (B,C,G).

Figure 4.. A2B-treatment counteracts DIO. See also Figure S4.

12-week DIO-study with A2B-stimulation: (A-M) Body weight (A), glucose tolerance (B), heart rate (C), correlation of muscle mass/body weight (D), weight lift (E), ex vivo specific muscle force (F), inguinal WAT mass (G), thermogenic (H) and mitochondrial (I) marker gene expression in BAT, representative HE (top) or UCP-1 (bottom) stain of BAT (J) and inguinal WAT sections (K) (Scale bar 100 μm), UCP-1 immunoblot of inguinal WAT ( L) and O₂ consumption at 23° C ( M). See Figure 4A for body weight of the groups analyzed. n=5 per group. * P < 0.05. Data are shown as mean + SEM (A-C), scatter plot (D) or boxplot (with median) and whiskers (1.5x interquartile range) (E-I,M) and analyzed using ANCOVA (D) or ANOVA with Newman-Keuls post-hoc test (A,C,E-I,M).

Figure 5.. A2B-signaling in human BAT and SKM. See also Figure S5.

(A) Adenosine receptor expression in human BAT of lean (n=8) and overweight subjects (n=4). (B-D) BAT A2B expression correlated with age (B) total body fat mass (C)(n=12), or with UCP-1 expression (D) (n=11). (E) Representative PET/MRI image of [¹⁸F]FDG uptake of one low BAT and high BAT subject from (D). (F) A2B expression in low BAT and high BAT subjects. (G) Correlation of [¹⁸F]FDG uptake with A2B expression of high BAT subjects (n=5). (H,I) Mean adipocyte diameter (n=405) (H) and A2B/UCP-1 correlation (I) from 10 subjects with high or low A2B expression, respectively, in subcutaneous WAT. (J) Correlation of A2B expression with age in human SKM (n=9). (K) Basal and succinate-fueled O₂ consumption in human SKM correlated with A2B expression (n=6). (L,M) Correlation of A2B expression with oxidative marker Cpt1 (L) or senescence marker p16 (M) in SKM (n=9). (N) Glucose uptake of primary human myocytes treated with A2B agonist (300 nM), insulin (100 nM), or both (n=5). (O) O₂ consumption of human SKM explants treated with A2B agonist (300 nM) (n=3). * P < 0.05. Data are shown as mean + SEM (F), scatter plot (B-D,G,I-M) or Boxplot (with median) and whiskers (1.5x interquartile range) (A,H,N,O). Figure 5H also shows outliers. Data were analyzed with ANOVA with Newman-Keuls post-hoc test (A,N), Pearson correlation coefficient (B-D,G,I-M), or two-tailed student’s t-test (H,O).