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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 restoration of macrophage A2bAR on liver.Liver was collected from WT, A2bAR KO, and CD68-Tg mice after 16 weeks of HFD as described in the methods. Relative mRNA expression was determined using the ΔΔCT method with normalization to 18s rRNA. A. mRNA expression of TNF-α in liver. A2bAR KO (n = 5) vs WT (n = 7) p-value  = 0.0271; A2bAR KO vs CD68-Tg (n = 6) p-value  = 0.0210. B. mRNA expression of IL-6 in liver. A2bAR KO (n = 8) vs WT (n = 7) p-value  = 0.0159; A2bAR KO vs CD68-Tg (n = 8) p-value  = 0.0080. C. mRNA expression of IRS-2 in liver. CD68-Tg (n = 7) vs A2bAR KO (n = 8) p-value  = 0.0300. D. Western blot analysis of liver; one representative (of 3 sets) WT, CD68-Tg and A2bAR KO group shown at 15 minutes post-insulin injection, following 16 weeks of HFD. Levels of mature SREBP-1 (68 kDa), and IRS-2 (185 kDa), were probed by Western blot analysis, using β-actin (43 kDa) as loading control. Quantification of Western Blot results was performed with Image J software (http://rsb.info.nih.gov/ij/) with normalization to β-actin. WT to A2bAR KO: IRS-2 p-value  = 0.0092, SREBP-1 p-value  = 0.0154; CD68-Tg to A2bAR KO: IRS-2 p-value  = 0.0247, SREBP-1 p-value  = 0.0170. 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-g003: Effect of restoration of macrophage A2bAR on liver.Liver was collected from WT, A2bAR KO, and CD68-Tg mice after 16 weeks of HFD as described in the methods. Relative mRNA expression was determined using the ΔΔCT method with normalization to 18s rRNA. A. mRNA expression of TNF-α in liver. A2bAR KO (n = 5) vs WT (n = 7) p-value  = 0.0271; A2bAR KO vs CD68-Tg (n = 6) p-value  = 0.0210. B. mRNA expression of IL-6 in liver. A2bAR KO (n = 8) vs WT (n = 7) p-value  = 0.0159; A2bAR KO vs CD68-Tg (n = 8) p-value  = 0.0080. C. mRNA expression of IRS-2 in liver. CD68-Tg (n = 7) vs A2bAR KO (n = 8) p-value  = 0.0300. D. Western blot analysis of liver; one representative (of 3 sets) WT, CD68-Tg and A2bAR KO group shown at 15 minutes post-insulin injection, following 16 weeks of HFD. Levels of mature SREBP-1 (68 kDa), and IRS-2 (185 kDa), were probed by Western blot analysis, using β-actin (43 kDa) as loading control. Quantification of Western Blot results was performed with Image J software (http://rsb.info.nih.gov/ij/) with normalization to β-actin. WT to A2bAR KO: IRS-2 p-value  = 0.0092, SREBP-1 p-value  = 0.0154; CD68-Tg to A2bAR KO: IRS-2 p-value  = 0.0247, SREBP-1 p-value  = 0.0170. Data are averages ± SD. *Student two-tail t-test assuming equal variance was found significant only when p-value <0.05.

Mentions: Interestingly, expression of TNF-α and IL-6 in the liver of CD68-Tg mice was reduced significantly and mildly, respectively, compared to A2bAR KO mice (Figure 3A,B). In accordance, Western blot and qPCR analyses of the liver showed increased IRS-2 protein and mRNA levels in CD68-Tg mice as compared to A2bAR KO mice, mimicking the levels found in WT mice (Figure 3C,D). We also found reduced liver SREBP-1 protein expression in the CD68-Tg mice as compared to A2bAR KO mice (Figure 3D), which corresponds to the diminished cytokine expression in the CD68-Tg mice. SREBP-1 plays a crucial role in lipid metabolism. To this end, restoration of macrophage A2bAR reduced plasma levels and liver content of triglyceride and cholesterol in CD68-Tg as compared to A2bAR KO mice (Figure 4A,B). Moreover, lipid accumulation in the livers of A2bAR KO mice was ameliorated with restoration of macrophage A2bAR (Figure 4C) despite no change in weight gain on HFD between genotypes (Figure 4D). Consistent with resolution of hyperlipidemia and reduction in liver triglyceride and cholesterol content, WT and CD68-Tg mice demonstrated lower percent fat mass relative to A2bAR KO mice (Figure 4E).


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 restoration of macrophage A2bAR on liver.Liver was collected from WT, A2bAR KO, and CD68-Tg mice after 16 weeks of HFD as described in the methods. Relative mRNA expression was determined using the ΔΔCT method with normalization to 18s rRNA. A. mRNA expression of TNF-α in liver. A2bAR KO (n = 5) vs WT (n = 7) p-value  = 0.0271; A2bAR KO vs CD68-Tg (n = 6) p-value  = 0.0210. B. mRNA expression of IL-6 in liver. A2bAR KO (n = 8) vs WT (n = 7) p-value  = 0.0159; A2bAR KO vs CD68-Tg (n = 8) p-value  = 0.0080. C. mRNA expression of IRS-2 in liver. CD68-Tg (n = 7) vs A2bAR KO (n = 8) p-value  = 0.0300. D. Western blot analysis of liver; one representative (of 3 sets) WT, CD68-Tg and A2bAR KO group shown at 15 minutes post-insulin injection, following 16 weeks of HFD. Levels of mature SREBP-1 (68 kDa), and IRS-2 (185 kDa), were probed by Western blot analysis, using β-actin (43 kDa) as loading control. Quantification of Western Blot results was performed with Image J software (http://rsb.info.nih.gov/ij/) with normalization to β-actin. WT to A2bAR KO: IRS-2 p-value  = 0.0092, SREBP-1 p-value  = 0.0154; CD68-Tg to A2bAR KO: IRS-2 p-value  = 0.0247, SREBP-1 p-value  = 0.0170. 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-g003: Effect of restoration of macrophage A2bAR on liver.Liver was collected from WT, A2bAR KO, and CD68-Tg mice after 16 weeks of HFD as described in the methods. Relative mRNA expression was determined using the ΔΔCT method with normalization to 18s rRNA. A. mRNA expression of TNF-α in liver. A2bAR KO (n = 5) vs WT (n = 7) p-value  = 0.0271; A2bAR KO vs CD68-Tg (n = 6) p-value  = 0.0210. B. mRNA expression of IL-6 in liver. A2bAR KO (n = 8) vs WT (n = 7) p-value  = 0.0159; A2bAR KO vs CD68-Tg (n = 8) p-value  = 0.0080. C. mRNA expression of IRS-2 in liver. CD68-Tg (n = 7) vs A2bAR KO (n = 8) p-value  = 0.0300. D. Western blot analysis of liver; one representative (of 3 sets) WT, CD68-Tg and A2bAR KO group shown at 15 minutes post-insulin injection, following 16 weeks of HFD. Levels of mature SREBP-1 (68 kDa), and IRS-2 (185 kDa), were probed by Western blot analysis, using β-actin (43 kDa) as loading control. Quantification of Western Blot results was performed with Image J software (http://rsb.info.nih.gov/ij/) with normalization to β-actin. WT to A2bAR KO: IRS-2 p-value  = 0.0092, SREBP-1 p-value  = 0.0154; CD68-Tg to A2bAR KO: IRS-2 p-value  = 0.0247, SREBP-1 p-value  = 0.0170. Data are averages ± SD. *Student two-tail t-test assuming equal variance was found significant only when p-value <0.05.
Mentions: Interestingly, expression of TNF-α and IL-6 in the liver of CD68-Tg mice was reduced significantly and mildly, respectively, compared to A2bAR KO mice (Figure 3A,B). In accordance, Western blot and qPCR analyses of the liver showed increased IRS-2 protein and mRNA levels in CD68-Tg mice as compared to A2bAR KO mice, mimicking the levels found in WT mice (Figure 3C,D). We also found reduced liver SREBP-1 protein expression in the CD68-Tg mice as compared to A2bAR KO mice (Figure 3D), which corresponds to the diminished cytokine expression in the CD68-Tg mice. SREBP-1 plays a crucial role in lipid metabolism. To this end, restoration of macrophage A2bAR reduced plasma levels and liver content of triglyceride and cholesterol in CD68-Tg as compared to A2bAR KO mice (Figure 4A,B). Moreover, lipid accumulation in the livers of A2bAR KO mice was ameliorated with restoration of macrophage A2bAR (Figure 4C) despite no change in weight gain on HFD between genotypes (Figure 4D). Consistent with resolution of hyperlipidemia and reduction in liver triglyceride and cholesterol content, WT and CD68-Tg mice demonstrated lower percent fat mass relative to A2bAR KO mice (Figure 4E).

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