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. 2014 Mar;63(3):850-66.
doi: 10.2337/db13-0573. Epub 2013 Nov 5.

A2B adenosine receptors prevent insulin resistance by inhibiting adipose tissue inflammation via maintaining alternative macrophage activation

Balázs Csóka  1 , Balázs Koscsó, Gábor Töro, Endre Kókai, László Virág, Zoltán H Németh, Pál Pacher, Péter Bai, György Haskó
Affiliations

A2B adenosine receptors prevent insulin resistance by inhibiting adipose tissue inflammation via maintaining alternative macrophage activation

Balázs Csóka et al. Diabetes. 2014 Mar.

Abstract

Obesity causes increased classical and decreased alternative macrophage activation, which in turn cause insulin resistance in target organs. Because A2B adenosine receptors (ARs) are important regulators of macrophage activation, we examined the role of A2B ARs in adipose tissue inflammation and insulin resistance. A2B AR deletion impaired glucose and lipid metabolism in mice fed chow but not a high-fat diet, which was paralleled by dysregulation of the adipokine system, and increased classical macrophage activation and inhibited alternative macrophage activation. The expression of alternative macrophage activation-specific transcriptions factors, including CCAAT/enhancer-binding protein-β, interferon regulatory factor 4, and peroxisome proliferator-activated receptor-γ, was decreased in adipose tissue of A2B AR-deficient mice. Furthermore, in in vitro studies, we found that stimulation of A2B ARs suppressed free fatty acid-induced deleterious inflammatory and metabolic activation of macrophages. Moreover, AR activation upregulated the interleukin-4-induced expression of CCAAT/enhancer-binding protein-β, interferon regulatory factor 4, and peroxisome proliferator-activated receptor-γ in macrophages. Altogether, our results indicate that therapeutic strategies targeting A2B ARs hold promise for preventing adipose tissue inflammation and insulin resistance.

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Figures

Figure 1
Figure 1
CD-fed A2B KO mice display increased body weight and show impaired glucose and insulin homeostasis compared with WT mice. A: Body weight of WT and A2B KO mice fed CD. Results are representative of three experiments; n = 7–10 mice/group. B: Representative images and quantitative analysis of H-E–stained epididymal adipose tissues from CD-fed A2B KO and WT animals (n = 3). An FM320–9M AMSCOPE EPI-Fluorescent Microscope was used with Achromatic objective lenses at room temperature. The magnification was ×40. The images were taken with AMSCOPE 9.1 MP Low-Lux True Color Digital Camera, and the acquisition software was Photoshop. C: ipGTT of CD A2B KO and WT mice. Results are representative of three experiments; n = 7–10 mice/group. D: ipITT of CD-fed A2B KO and WT mice. E: ipPTT. n = 7–10 mice/group. Results are representative of one or three experiments; n = 7–10 mice/group. Glucose infusion rate (GIR) (F) and endogenous glucose production (EGP) (G) during the steady-state of hyperinsulinemic-euglycemic clamp; n = 7–15 mice/group. Data are presented as the mean ± SEM. H: Soleus, gastrocnemius (gastrocn.), and vastus lateralus (l. vastus), and adipose tissue–specific glucose uptake/metabolic index rate (Rg) after hyperinsulinemic-euglycemic clamp of WT and A2B KO mice. n = 6–15 mice/group. Data are presented as the mean ± SEM. Protein level of phospho-Akt (Ser473) (p-Akt) and total Akt as detected by Western blot from protein extracts of skeletal muscle (I, bottom panel), liver (J, bottom panel), and epididymal fat (K, bottom panel) 5 min after insulin injection (1 unit/kg i.p.). Results are representative of two experiments. Densitometric analysis of p-Akt/total Akt Western blots of skeletal muscle (I, top panel), liver (J, top panel), and epididymal fat (K, top panel). n = 3/group. Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 vs. WT littermates. (A high-quality color representation of this figure is available in the online issue.)
Figure 2
Figure 2
CD-fed A2B KO mice display dysregulated insulin and adipokine levels and impaired liver metabolism. Plasma levels of insulin (A) and leptin (B) in A2B KO and WT mice during the course of 16 weeks of CD. Results are representative of two experiments; n = 7–10 mice/group. C: mRNA level of leptin in epididymal adipose tissue of CD-fed A2B KO and WT animals. Results are representative of three experiments; n = 7–10 mice/group. Protein level of adiponectin (D) and resistin (E) in epididymal adipose tissue of CD-fed A2B KO and WT animals. F: Triglyceride concentrations in the livers of A2B KO and WT mice after a 16-week CD. G: FFA levels in the plasma of A2B KO and WT mice after a 16-week CD. mRNA level of glucokinase (H), FASn (I), and liver-specific lipase (Lipc) (J) in the liver of CD-fed A2B KO and WT mice. K–O: Cholesterol concentrations in the plasma of A2B KO and WT mice that were fed CD for 16 weeks. Results are representative of three experiments; n = 7–10 mice/group. Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; and ***P < 0.001 vs. WT mice.
Figure 3
Figure 3
A2B AR deletion exacerbates adipose tissue inflammation. A: Representative images from immunohistochemistry for F4/80 in epididymal adipose tissue obtained from A2B KO and WT animals after a 16-week CD. The crown-like structure is indicated by an arrow. An FM320–9M AMSCOPE EPI-Fluorescent Microscope was used with achromatic objective lenses at room temperature. The magnification was ×40. The images were taken with AMSCOPE 9.1 MP Low-Lux True Color Digital Camera, and the acquisition software was Photoshop. mRNA expression of F4/80 (B) and CD11c (C) in epididymal adipose tissue of CD-fed A2B KO and WT mice. Protein concentrations of CCL2 (D), TNF-α (E), IL-6 (F), IL-10 (G), and IFN-γ (H) in the epididymal adipose tissue of A2B KO and WT animals that were kept on CD for 16 weeks. mRNA transcript levels of CCL2 (I), TNF-α (J), CCR2 (K), serum amyloid A 3 (SAA3) (L), IL-1 receptor antagonist (IL-1Ra) (M), matrix metalloproteinase 12 (MMP12) (N), and VEGF (O) in epididymal adipose tissue of CD-fed A2B KO and WT mice. Results are representative of three experiments; n = 6–10 mice/group. Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; and ***P < 0.001 vs. WT animals. (A high-quality color representation of this figure is available in the online issue.)
Figure 4
Figure 4
Lack of A2B ARs downregulates aaMϕ-specific gene expression in adipose tissue of CD-fed mice, and AR stimulation promotes IL-4–induced aaMϕ-specific transcription factor expression in macrophages. mRNA expression of Ym1 (A), RELM-α (B), CD206 (C), mgl-1 (D), and mgl-2 (E) in epididymal adipose tissue of CD-fed A2B KO and WT mice. mRNA expression of mgl-2 (F), RELM-α (G), Ym1 (H), mgl-1 (I), CD206 (J), C/EBPβ (K), IRF4 (L), and PPARγ (M) in the SVF of epididymal adipose tissue obtained from A2B KO and WT mice that were kept on CD for 16 weeks. Results are representative of three experiments; n = 5–10 mice/group. mRNA expression of C/EBPβ (N), IRF4 (O), and PPARγ (P) in RAW 264.7 macrophages after an 8-h treatment with IL-4 and/or NECA. Results are representative of three or more experiments; n = 12–20/group. Q, top: Protein level of PPARγ, as detected by Western blot from nuclear protein fraction of RAW 264.7 cells after 8 or 24 h of treatment with IL-4 and/or NECA. Results are representative of three experiments. Q, bottom: Densitometric analysis of PPARγ Western blots; n = 3–5/group. R: Arginase activity in RAW 264.7 macrophages after 8 h of treatment with IL-4 and IL-4/NECA in the presence or absence of a specific PPARγ inhibitor. Results are representative of three experiments; n = 6/group. Data are presented as the mean ± SEM. *P < 0.05 vs. WT animals. **P < 0.001 vs. control treatment. ***P < 0.001 vs. control or IL-4 treatment. #P < 0.05; ##P < 0.01; and ###P < 0.001 vs. IL-4 or IL-4/NECA treatment. con, control.
Figure 5
Figure 5
A2B AR stimulation diminishes inflammatory activation in FFA-induced macrophages. Effect of AR agonists on the production of palmitate-induced TNF-α (A), CCL2 (B), and VEGF (C) protein in RAW 264.7 cells after an 8-h incubation. Protein levels were detected in the supernatants using ELISA. mRNA transcript levels of TNF-α (D), CCL2 (E), VEGF (F), and CD11c (G) in response to an 8-h-long palmitate or combined palmitate/NECA treatment in RAW 264.7 cells. Results are representative of three experiments; n = 5–6/group. A2 AR antagonists prevent the NECA modulation of palmitate-induced release of TNF-α (H), CCL2 (I), and VEGF (J) in macrophages after an 8-h incubation. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control; ##P < 0.01 and ###P < 0.001 vs. palmitate treatment. $P < 0.05 and $$P < 0.01 vs. palmitate/NECA treatment. con, control; Pal, palmitate; Ve, vehicle.
Figure 6
Figure 6
A2B AR stimulation diminishes TNF-α production by FFA- or LPS-induced BMDMs. Protein concentration of TNF-α secreted from BMDMs obtained from WT or A2B KO mice after 8 h (A), 24 h (B), 48 h (C), and 72 h (D) of treatment with palmitate in the presence or absence of NECA. Protein concentration of TNF-α from BMDMs obtained from WT or A2B KO mice after 8 h (E), 24 h (F), 48 h (G), and 72 h (H) of treatment with LPS in the presence or absence of NECA. I: TNF-α production by palmitate-stimulated A2A KO BMDMs in the presence or absence of NECA after 8 h of stimulation. Results are representative of three or more experiments; n = 3–6/group. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. palmitate treatment. ##P < 0.01 vs. palmitate treatment. Pal, palmitate; Ve, vehicle.
Figure 7
Figure 7
Adenosine inhibits inflammation-induced metabolic switch of palmitate-elicited macrophages. Measurement of ECAR (A) and OCR (B) after a 12-h stimulation with palmitate with or without NECA. C: Effect of A2B AR–specific antagonists on palmitate- and NECA-regulated ECAR in RAW 264.7 macrophages after a 12-h incubation. Results are representative of three or more experiments; n = 6/group. Data are mean ± SEM. ***P < 0.001 vs. control (con) treatment; ###P < 0.001 vs. palmitate treatment; $$P < 0.01 and $$$P < 0.001 vs. palmitate/NECA treatment. mpH, mili pH; PSB, PSB0788; ZM, ZM241385; Pal, palmitate.
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