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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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Restoration of macrophage A2bAR improves glucose clearance, insulin sensitivity and reduces glucose levels following HFD.Glucose and insulin levels were measured in WT, A2bAR KO, and CD68-Tg mice at 28 weeks of age, following 16 weeks of HFD. Data are averages ± SD. *Student two-tail t-test assuming equal variance was found significant only when p-value <0.05. A. Glucose clearance in the blood post glucose overload (n = 12/group), Data in a. were analyzed by ANOVA followed by Bonferroni comparison test with the following p-values for indicated time points, Time 0 min: p-value <0.0001; WT vs CD68-Tg p-value <0.05, WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.01. Time 60 min: p-value 0.003487; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.01, CD68-Tg vs A2bAR KO p-value <0.01. Time 90 min: p-value 0.005068; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.01, CD68-Tg vs A2bAR KO p-value <0.05. Time 90 min: p-value 0.005855; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.05. B. Insulin levels in the plasma post glucose overload during the glucose tolerance test (n = 12/group). Data in b. were analyzed by ANOVA followed by Bonferroni comparison test with the following p-values for indicated time points. Time 0 min: p-value 0.017963; WT vs CD68-Tg p-value <0.05, WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO n.s. Time 20 min: p-value 0.017304; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.05. Time 120 min: p-value 0.019056; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO n.s. C. Glucose clearance in the plasma post insulin overload (n = 12/group). Data in c. were analyzed by ANOVA followed by Bonferroni comparison test with the following p-values for indicated time points. Time 0 min: p-value 0.006653; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.01. Time 15 min: p-value 0.017975; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.05. Time 30 min: p-value 0.016828; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.05. Time 120 min: p-value 0.003444; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.01. D. Plasma glucose levels 16 hours post starvation (n = 12/group). WT vs A2bAR KO, p-value  = 0.0404; WT vs CD68-Tg, p-value  = 0.0074; CD68-Tg vs A2bAR KO, p-value  = 0.0026. E. Plasma insulin levels 16 hours post starvation (n = 12/group), WT vs A2bAR KO p-value  = 0.0175.
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pone-0098775-g006: Restoration of macrophage A2bAR improves glucose clearance, insulin sensitivity and reduces glucose levels following HFD.Glucose and insulin levels were measured in WT, A2bAR KO, and CD68-Tg mice at 28 weeks of age, following 16 weeks of HFD. Data are averages ± SD. *Student two-tail t-test assuming equal variance was found significant only when p-value <0.05. A. Glucose clearance in the blood post glucose overload (n = 12/group), Data in a. were analyzed by ANOVA followed by Bonferroni comparison test with the following p-values for indicated time points, Time 0 min: p-value <0.0001; WT vs CD68-Tg p-value <0.05, WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.01. Time 60 min: p-value 0.003487; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.01, CD68-Tg vs A2bAR KO p-value <0.01. Time 90 min: p-value 0.005068; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.01, CD68-Tg vs A2bAR KO p-value <0.05. Time 90 min: p-value 0.005855; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.05. B. Insulin levels in the plasma post glucose overload during the glucose tolerance test (n = 12/group). Data in b. were analyzed by ANOVA followed by Bonferroni comparison test with the following p-values for indicated time points. Time 0 min: p-value 0.017963; WT vs CD68-Tg p-value <0.05, WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO n.s. Time 20 min: p-value 0.017304; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.05. Time 120 min: p-value 0.019056; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO n.s. C. Glucose clearance in the plasma post insulin overload (n = 12/group). Data in c. were analyzed by ANOVA followed by Bonferroni comparison test with the following p-values for indicated time points. Time 0 min: p-value 0.006653; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.01. Time 15 min: p-value 0.017975; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.05. Time 30 min: p-value 0.016828; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.05. Time 120 min: p-value 0.003444; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.01. D. Plasma glucose levels 16 hours post starvation (n = 12/group). WT vs A2bAR KO, p-value  = 0.0404; WT vs CD68-Tg, p-value  = 0.0074; CD68-Tg vs A2bAR KO, p-value  = 0.0026. E. Plasma insulin levels 16 hours post starvation (n = 12/group), WT vs A2bAR KO p-value  = 0.0175.

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)

Restoration of macrophage A2bAR improves glucose clearance, insulin sensitivity and reduces glucose levels following HFD.Glucose and insulin levels were measured in WT, A2bAR KO, and CD68-Tg mice at 28 weeks of age, following 16 weeks of HFD. Data are averages ± SD. *Student two-tail t-test assuming equal variance was found significant only when p-value <0.05. A. Glucose clearance in the blood post glucose overload (n = 12/group), Data in a. were analyzed by ANOVA followed by Bonferroni comparison test with the following p-values for indicated time points, Time 0 min: p-value <0.0001; WT vs CD68-Tg p-value <0.05, WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.01. Time 60 min: p-value 0.003487; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.01, CD68-Tg vs A2bAR KO p-value <0.01. Time 90 min: p-value 0.005068; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.01, CD68-Tg vs A2bAR KO p-value <0.05. Time 90 min: p-value 0.005855; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.05. B. Insulin levels in the plasma post glucose overload during the glucose tolerance test (n = 12/group). Data in b. were analyzed by ANOVA followed by Bonferroni comparison test with the following p-values for indicated time points. Time 0 min: p-value 0.017963; WT vs CD68-Tg p-value <0.05, WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO n.s. Time 20 min: p-value 0.017304; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.05. Time 120 min: p-value 0.019056; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO n.s. C. Glucose clearance in the plasma post insulin overload (n = 12/group). Data in c. were analyzed by ANOVA followed by Bonferroni comparison test with the following p-values for indicated time points. Time 0 min: p-value 0.006653; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.01. Time 15 min: p-value 0.017975; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.05. Time 30 min: p-value 0.016828; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.05. Time 120 min: p-value 0.003444; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.01. D. Plasma glucose levels 16 hours post starvation (n = 12/group). WT vs A2bAR KO, p-value  = 0.0404; WT vs CD68-Tg, p-value  = 0.0074; CD68-Tg vs A2bAR KO, p-value  = 0.0026. E. Plasma insulin levels 16 hours post starvation (n = 12/group), WT vs A2bAR KO p-value  = 0.0175.
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pone-0098775-g006: Restoration of macrophage A2bAR improves glucose clearance, insulin sensitivity and reduces glucose levels following HFD.Glucose and insulin levels were measured in WT, A2bAR KO, and CD68-Tg mice at 28 weeks of age, following 16 weeks of HFD. Data are averages ± SD. *Student two-tail t-test assuming equal variance was found significant only when p-value <0.05. A. Glucose clearance in the blood post glucose overload (n = 12/group), Data in a. were analyzed by ANOVA followed by Bonferroni comparison test with the following p-values for indicated time points, Time 0 min: p-value <0.0001; WT vs CD68-Tg p-value <0.05, WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.01. Time 60 min: p-value 0.003487; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.01, CD68-Tg vs A2bAR KO p-value <0.01. Time 90 min: p-value 0.005068; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.01, CD68-Tg vs A2bAR KO p-value <0.05. Time 90 min: p-value 0.005855; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.05. B. Insulin levels in the plasma post glucose overload during the glucose tolerance test (n = 12/group). Data in b. were analyzed by ANOVA followed by Bonferroni comparison test with the following p-values for indicated time points. Time 0 min: p-value 0.017963; WT vs CD68-Tg p-value <0.05, WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO n.s. Time 20 min: p-value 0.017304; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.05. Time 120 min: p-value 0.019056; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO n.s. C. Glucose clearance in the plasma post insulin overload (n = 12/group). Data in c. were analyzed by ANOVA followed by Bonferroni comparison test with the following p-values for indicated time points. Time 0 min: p-value 0.006653; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.01. Time 15 min: p-value 0.017975; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.05. Time 30 min: p-value 0.016828; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.05. Time 120 min: p-value 0.003444; WT vs CD68-Tg n.s., WT vs A2bAR KO p-value <0.05, CD68-Tg vs A2bAR KO p-value <0.01. D. Plasma glucose levels 16 hours post starvation (n = 12/group). WT vs A2bAR KO, p-value  = 0.0404; WT vs CD68-Tg, p-value  = 0.0074; CD68-Tg vs A2bAR KO, p-value  = 0.0026. E. Plasma insulin levels 16 hours post starvation (n = 12/group), WT vs A2bAR KO p-value  = 0.0175.
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