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The macrophage A2B adenosine receptor regulates tissue insulin sensitivity.

Johnston-Cox H, Eisenstein AS, Koupenova M, Carroll S, Ravid K - PLoS ONE (2014)

Bottom Line: High fat diet (HFD)-induced type 2 diabetes continues to be an epidemic with significant risk for various pathologies.As the A2bAR is expressed in different tissues, here, we provide the first lead to cellular mechanism by demonstrating that the receptor's influence on tissue insulin sensitivity is mediated via its expression in macrophages.The molecular mechanism for this effect involves A2bAR-mediated changes in cyclic adenosine monophosphate in macrophages, reducing the expression and release of inflammatory cytokines, which downregulate insulin receptor-2.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, United States of America; Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
High fat diet (HFD)-induced type 2 diabetes continues to be an epidemic with significant risk for various pathologies. Previously, we identified the A2b adenosine receptor (A2bAR), an established regulator of inflammation, as a regulator of HFD-induced insulin resistance. In particular, HFD was associated with vast upregulation of liver A2bAR in control mice, and while mice lacking this receptor showed augmented liver inflammation and tissue insulin resistance. As the A2bAR is expressed in different tissues, here, we provide the first lead to cellular mechanism by demonstrating that the receptor's influence on tissue insulin sensitivity is mediated via its expression in macrophages. This was shown using a newly generated transgenic mouse model expressing the A2bAR gene in the macrophage lineage on an otherwise A2bAR background. Reinstatement of macrophage A2bAR expression in A2bAR mice fed HFD restored insulin tolerance and tissue insulin signaling to the level of control mice. The molecular mechanism for this effect involves A2bAR-mediated changes in cyclic adenosine monophosphate in macrophages, reducing the expression and release of inflammatory cytokines, which downregulate insulin receptor-2. Thus, our results illustrate that macrophage A2bAR signaling is needed and sufficient for relaying the protective effect of the A2bAR against HFD-induced tissue inflammation and insulin resistance in mice.

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Effect of macrophage A2bAR on TNF-α.Kupffer cells were extracted from the liver of WT, A2bAR KO, and CD68-Tg mice following 16 weeks of HFD. A. cAMP levels (normalized to total protein) were assessed as described in the methods following treatment of Kupffer cells with the following ligands for 10 minutes: Dimethylsulfoxide (DMSO), 1 µM BAY 60–6583 (BAY), and 2 µM Forskolin (FSK). n = 5 per genotype per treatment. WT: BAY relative to DMSO p = 0.001738, FSK relative to DMSO p-value  = 0.000932; A2bAR KO: FSK relative to DMSO p-value  = 0.026068; CD68-Tg: BAY relative to DMSO p-value  = 0.000387, FSK relative to DMSO p-value  = 0.010989. B. Expression of TNF-α in Kupffer cells. CD68-Tg vs A2bAR KO p-value  = 0.0164, WT vs A2bAR KO p-value  = 0.0032. C. Systemic inflammatory profile was assessed in WT, A2bAR KO, and CD68-Tg mice after 16 weeks of HFD. Plasma TNF-α levels were lower in WT, p-value  = 0.0269, and CD68-Tg, p-value  = 0.0291, as compared to A2bAR KO after HFD. Data are averages ± SD. *Student two-tail t-test assuming equal variance was found significant only when p-value <0.05.
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pone-0098775-g002: Effect of macrophage A2bAR on TNF-α.Kupffer cells were extracted from the liver of WT, A2bAR KO, and CD68-Tg mice following 16 weeks of HFD. A. cAMP levels (normalized to total protein) were assessed as described in the methods following treatment of Kupffer cells with the following ligands for 10 minutes: Dimethylsulfoxide (DMSO), 1 µM BAY 60–6583 (BAY), and 2 µM Forskolin (FSK). n = 5 per genotype per treatment. WT: BAY relative to DMSO p = 0.001738, FSK relative to DMSO p-value  = 0.000932; A2bAR KO: FSK relative to DMSO p-value  = 0.026068; CD68-Tg: BAY relative to DMSO p-value  = 0.000387, FSK relative to DMSO p-value  = 0.010989. B. Expression of TNF-α in Kupffer cells. CD68-Tg vs A2bAR KO p-value  = 0.0164, WT vs A2bAR KO p-value  = 0.0032. C. Systemic inflammatory profile was assessed in WT, A2bAR KO, and CD68-Tg mice after 16 weeks of HFD. Plasma TNF-α levels were lower in WT, p-value  = 0.0269, and CD68-Tg, p-value  = 0.0291, as compared to A2bAR KO after HFD. Data are averages ± SD. *Student two-tail t-test assuming equal variance was found significant only when p-value <0.05.

Mentions: Signaling through the A2bAR has been shown to reduce macrophage secretion of cytokines [31], [39]. Furthermore, the downstream effecter of A2bAR signaling, cAMP, is known to reduce macrophage cytokine expression [40]–[43]. Therefore, we determined whether reinstating A2bAR in macrophages resulted in changes in cAMP levels and subsequent cytokine release. The A2bAR specific agonist, BAY 60–6583, was able to elicit an increase in cAMP levels in Kupffer cells from WT and CD68-Tg mice (Figure 2A). Moreover, Kupffer cells that express A2bAR (from WT and CD68-Tg mice) showed reduced mRNA expression of TNF-α as compared to Kupffer cells that lack A2bAR (Figure 2B). Restoration of macrophage A2bAR in CD68-Tg mice was also able to reduce systemic plasma TNF-α (Figure 2C). Hence, our results suggest that expression of A2bAR in Kupffer cells augments cAMP signaling and reduces macrophage cytokine expression and secretion.


The macrophage A2B adenosine receptor regulates tissue insulin sensitivity.

Johnston-Cox H, Eisenstein AS, Koupenova M, Carroll S, Ravid K - PLoS ONE (2014)

Effect of macrophage A2bAR on TNF-α.Kupffer cells were extracted from the liver of WT, A2bAR KO, and CD68-Tg mice following 16 weeks of HFD. A. cAMP levels (normalized to total protein) were assessed as described in the methods following treatment of Kupffer cells with the following ligands for 10 minutes: Dimethylsulfoxide (DMSO), 1 µM BAY 60–6583 (BAY), and 2 µM Forskolin (FSK). n = 5 per genotype per treatment. WT: BAY relative to DMSO p = 0.001738, FSK relative to DMSO p-value  = 0.000932; A2bAR KO: FSK relative to DMSO p-value  = 0.026068; CD68-Tg: BAY relative to DMSO p-value  = 0.000387, FSK relative to DMSO p-value  = 0.010989. B. Expression of TNF-α in Kupffer cells. CD68-Tg vs A2bAR KO p-value  = 0.0164, WT vs A2bAR KO p-value  = 0.0032. C. Systemic inflammatory profile was assessed in WT, A2bAR KO, and CD68-Tg mice after 16 weeks of HFD. Plasma TNF-α levels were lower in WT, p-value  = 0.0269, and CD68-Tg, p-value  = 0.0291, as compared to A2bAR KO after HFD. Data are averages ± SD. *Student two-tail t-test assuming equal variance was found significant only when p-value <0.05.
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Related In: Results  -  Collection

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pone-0098775-g002: Effect of macrophage A2bAR on TNF-α.Kupffer cells were extracted from the liver of WT, A2bAR KO, and CD68-Tg mice following 16 weeks of HFD. A. cAMP levels (normalized to total protein) were assessed as described in the methods following treatment of Kupffer cells with the following ligands for 10 minutes: Dimethylsulfoxide (DMSO), 1 µM BAY 60–6583 (BAY), and 2 µM Forskolin (FSK). n = 5 per genotype per treatment. WT: BAY relative to DMSO p = 0.001738, FSK relative to DMSO p-value  = 0.000932; A2bAR KO: FSK relative to DMSO p-value  = 0.026068; CD68-Tg: BAY relative to DMSO p-value  = 0.000387, FSK relative to DMSO p-value  = 0.010989. B. Expression of TNF-α in Kupffer cells. CD68-Tg vs A2bAR KO p-value  = 0.0164, WT vs A2bAR KO p-value  = 0.0032. C. Systemic inflammatory profile was assessed in WT, A2bAR KO, and CD68-Tg mice after 16 weeks of HFD. Plasma TNF-α levels were lower in WT, p-value  = 0.0269, and CD68-Tg, p-value  = 0.0291, as compared to A2bAR KO after HFD. Data are averages ± SD. *Student two-tail t-test assuming equal variance was found significant only when p-value <0.05.
Mentions: Signaling through the A2bAR has been shown to reduce macrophage secretion of cytokines [31], [39]. Furthermore, the downstream effecter of A2bAR signaling, cAMP, is known to reduce macrophage cytokine expression [40]–[43]. Therefore, we determined whether reinstating A2bAR in macrophages resulted in changes in cAMP levels and subsequent cytokine release. The A2bAR specific agonist, BAY 60–6583, was able to elicit an increase in cAMP levels in Kupffer cells from WT and CD68-Tg mice (Figure 2A). Moreover, Kupffer cells that express A2bAR (from WT and CD68-Tg mice) showed reduced mRNA expression of TNF-α as compared to Kupffer cells that lack A2bAR (Figure 2B). Restoration of macrophage A2bAR in CD68-Tg mice was also able to reduce systemic plasma TNF-α (Figure 2C). Hence, our results suggest that expression of A2bAR in Kupffer cells augments cAMP signaling and reduces macrophage cytokine expression and secretion.

Bottom Line: High fat diet (HFD)-induced type 2 diabetes continues to be an epidemic with significant risk for various pathologies.As the A2bAR is expressed in different tissues, here, we provide the first lead to cellular mechanism by demonstrating that the receptor's influence on tissue insulin sensitivity is mediated via its expression in macrophages.The molecular mechanism for this effect involves A2bAR-mediated changes in cyclic adenosine monophosphate in macrophages, reducing the expression and release of inflammatory cytokines, which downregulate insulin receptor-2.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, United States of America; Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
High fat diet (HFD)-induced type 2 diabetes continues to be an epidemic with significant risk for various pathologies. Previously, we identified the A2b adenosine receptor (A2bAR), an established regulator of inflammation, as a regulator of HFD-induced insulin resistance. In particular, HFD was associated with vast upregulation of liver A2bAR in control mice, and while mice lacking this receptor showed augmented liver inflammation and tissue insulin resistance. As the A2bAR is expressed in different tissues, here, we provide the first lead to cellular mechanism by demonstrating that the receptor's influence on tissue insulin sensitivity is mediated via its expression in macrophages. This was shown using a newly generated transgenic mouse model expressing the A2bAR gene in the macrophage lineage on an otherwise A2bAR background. Reinstatement of macrophage A2bAR expression in A2bAR mice fed HFD restored insulin tolerance and tissue insulin signaling to the level of control mice. The molecular mechanism for this effect involves A2bAR-mediated changes in cyclic adenosine monophosphate in macrophages, reducing the expression and release of inflammatory cytokines, which downregulate insulin receptor-2. Thus, our results illustrate that macrophage A2bAR signaling is needed and sufficient for relaying the protective effect of the A2bAR against HFD-induced tissue inflammation and insulin resistance in mice.

Show MeSH
Related in: MedlinePlus