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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 CD68-driven expression of A2bAR on tissue insulin signaling.Western blot analysis of liver and visceral fat derived from matching WT, CD68-Tg and A2bAR KO mice post 16 weeks of HFD and 15 minutes following insulin injection. A. Levels of phospho-308 Akt (p308 Akt, 60 kDa), phospho-473 Akt (p473 Akt, 60 kDa), and total Akt (60 kDa) were probed by Western blot analysis, using β-actin (43 kDa) as loading control. Shown are representative out of 3 sets. B. Quantification of Western Blot results was performed with Image J software (http://rsb.info.nih.gov/ij/). Protein levels were normalized to total Akt and β-actin. Data are averages ± SD. *Student two-tail t-test assuming equal variance was found significant only when p<0.05. WT to A2bAR KO: Liver p308 Akt p-value  = 0.0007, p473 Akt p-value  = 0.0219; Visceral fat: p308 Akt p-value  = 0.0340, p473 Akt p-value  = 0.0395; CD68-Tg to A2bAR KO: Liver p308 Akt p-value  = 0.0327, p473 Akt p-value  = 0.0349; Visceral fat: p308 Akt p-value  = 0.1054; p473 Akt p-value  = 0.0221.
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pone-0098775-g007: Effect of CD68-driven expression of A2bAR on tissue insulin signaling.Western blot analysis of liver and visceral fat derived from matching WT, CD68-Tg and A2bAR KO mice post 16 weeks of HFD and 15 minutes following insulin injection. A. Levels of phospho-308 Akt (p308 Akt, 60 kDa), phospho-473 Akt (p473 Akt, 60 kDa), and total Akt (60 kDa) were probed by Western blot analysis, using β-actin (43 kDa) as loading control. Shown are representative out of 3 sets. B. Quantification of Western Blot results was performed with Image J software (http://rsb.info.nih.gov/ij/). Protein levels were normalized to total Akt and β-actin. Data are averages ± SD. *Student two-tail t-test assuming equal variance was found significant only when p<0.05. WT to A2bAR KO: Liver p308 Akt p-value  = 0.0007, p473 Akt p-value  = 0.0219; Visceral fat: p308 Akt p-value  = 0.0340, p473 Akt p-value  = 0.0395; CD68-Tg to A2bAR KO: Liver p308 Akt p-value  = 0.0327, p473 Akt p-value  = 0.0349; Visceral fat: p308 Akt p-value  = 0.1054; p473 Akt p-value  = 0.0221.

Mentions: As we had found reduced inflammation and increased IRS-2 levels in the CD68-Tg mice, we next determined whether restoration of macrophage A2bAR affected global metabolic homeostasis. Following HFD feeding, CD68-Tg mice showed improved glucose clearance and insulin sensitivity relative to A2bAR KO mice and responded to insulin and glucose no differently than WT mice (Figure 6A-C). Fasting glucose levels were lower in the CD68-Tg mice as compared to A2bAR KO mice, whereas there was no difference in fasting insulin levels between CD68-Tg and A2bAR KO mice (Figure 6D-E). In fact, CD68-Tg mice had lower fasting glucose levels than WT mice. To determine if tissues were insulin resistant, Akt phosphorylation in liver and adipose tissue, which is indicative of insulin signaling [50], was measured. Western blot analysis of liver and visceral adipose tissue after HFD and following injection with insulin demonstrated that tissue insulin signaling was restored to that of WT mice in CD68-Tg mice as the levels of phosphorylated Akt 308 and 473 were similar in CD68-Tg and WT mice (Figure 7A,B). Thus, macrophage A2bAR expression was largely responsible for the protective effect of A2bAR signaling in HFD-induced insulin resistance and glucose tolerance.


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 CD68-driven expression of A2bAR on tissue insulin signaling.Western blot analysis of liver and visceral fat derived from matching WT, CD68-Tg and A2bAR KO mice post 16 weeks of HFD and 15 minutes following insulin injection. A. Levels of phospho-308 Akt (p308 Akt, 60 kDa), phospho-473 Akt (p473 Akt, 60 kDa), and total Akt (60 kDa) were probed by Western blot analysis, using β-actin (43 kDa) as loading control. Shown are representative out of 3 sets. B. Quantification of Western Blot results was performed with Image J software (http://rsb.info.nih.gov/ij/). Protein levels were normalized to total Akt and β-actin. Data are averages ± SD. *Student two-tail t-test assuming equal variance was found significant only when p<0.05. WT to A2bAR KO: Liver p308 Akt p-value  = 0.0007, p473 Akt p-value  = 0.0219; Visceral fat: p308 Akt p-value  = 0.0340, p473 Akt p-value  = 0.0395; CD68-Tg to A2bAR KO: Liver p308 Akt p-value  = 0.0327, p473 Akt p-value  = 0.0349; Visceral fat: p308 Akt p-value  = 0.1054; p473 Akt p-value  = 0.0221.
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pone-0098775-g007: Effect of CD68-driven expression of A2bAR on tissue insulin signaling.Western blot analysis of liver and visceral fat derived from matching WT, CD68-Tg and A2bAR KO mice post 16 weeks of HFD and 15 minutes following insulin injection. A. Levels of phospho-308 Akt (p308 Akt, 60 kDa), phospho-473 Akt (p473 Akt, 60 kDa), and total Akt (60 kDa) were probed by Western blot analysis, using β-actin (43 kDa) as loading control. Shown are representative out of 3 sets. B. Quantification of Western Blot results was performed with Image J software (http://rsb.info.nih.gov/ij/). Protein levels were normalized to total Akt and β-actin. Data are averages ± SD. *Student two-tail t-test assuming equal variance was found significant only when p<0.05. WT to A2bAR KO: Liver p308 Akt p-value  = 0.0007, p473 Akt p-value  = 0.0219; Visceral fat: p308 Akt p-value  = 0.0340, p473 Akt p-value  = 0.0395; CD68-Tg to A2bAR KO: Liver p308 Akt p-value  = 0.0327, p473 Akt p-value  = 0.0349; Visceral fat: p308 Akt p-value  = 0.1054; p473 Akt p-value  = 0.0221.
Mentions: As we had found reduced inflammation and increased IRS-2 levels in the CD68-Tg mice, we next determined whether restoration of macrophage A2bAR affected global metabolic homeostasis. Following HFD feeding, CD68-Tg mice showed improved glucose clearance and insulin sensitivity relative to A2bAR KO mice and responded to insulin and glucose no differently than WT mice (Figure 6A-C). Fasting glucose levels were lower in the CD68-Tg mice as compared to A2bAR KO mice, whereas there was no difference in fasting insulin levels between CD68-Tg and A2bAR KO mice (Figure 6D-E). In fact, CD68-Tg mice had lower fasting glucose levels than WT mice. To determine if tissues were insulin resistant, Akt phosphorylation in liver and adipose tissue, which is indicative of insulin signaling [50], was measured. Western blot analysis of liver and visceral adipose tissue after HFD and following injection with insulin demonstrated that tissue insulin signaling was restored to that of WT mice in CD68-Tg mice as the levels of phosphorylated Akt 308 and 473 were similar in CD68-Tg and WT mice (Figure 7A,B). Thus, macrophage A2bAR expression was largely responsible for the protective effect of A2bAR signaling in HFD-induced insulin resistance and glucose tolerance.

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