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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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Generation of transgenic mice expressing A2bAR in macrophages only.A. Genomic analysis by PCR of CD68-hA2bAR transgene in founder lines 1, 2, and 3 (Fo # 1, 2, 3) compared to A2bAR KO mice. Line 2 was used for the remainder of the studies based on expression analysis shown in panels c,d. B. Determination of primer efficiency. The amplification efficiency of human A2bAR (hA2bAR) and mouse A2bAR (mA2bAR) TaqMan primers was tested using the CT slope method. The target template was diluted over a log scale and CT values were determined by qPCR. A plot of CT versus log cDNA concentration is shown for hA2bAR and mA2bAR primers. Amplification efficiency (Ex) is calculated using the slope of the graph in the following equation: Ex = 10 (-1/slope) – 1. The calculated efficiencies are 1.18 and 1.03 for mA2bAR and hA2bAR, respectively. C. Human A2bAR and mouse A2bAR mRNA expression was measured by qPCR in Kupffer cells isolated from mice at 12 weeks of age (n = 5 WT, 6 A2bAR KO, 6 CD68-Tg; ns = not statistically different from WT). D. Visceral (epididymal) adipose tissue macrophages were sorted via flow cytometry-based markers (see Methods) and subjected to qPCR of A2bAR mRNA. Data are averages ± SD. Relative mRNA expression was determined using the ΔΔCT method and were normalized to 18s rRNA values. 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-g001: Generation of transgenic mice expressing A2bAR in macrophages only.A. Genomic analysis by PCR of CD68-hA2bAR transgene in founder lines 1, 2, and 3 (Fo # 1, 2, 3) compared to A2bAR KO mice. Line 2 was used for the remainder of the studies based on expression analysis shown in panels c,d. B. Determination of primer efficiency. The amplification efficiency of human A2bAR (hA2bAR) and mouse A2bAR (mA2bAR) TaqMan primers was tested using the CT slope method. The target template was diluted over a log scale and CT values were determined by qPCR. A plot of CT versus log cDNA concentration is shown for hA2bAR and mA2bAR primers. Amplification efficiency (Ex) is calculated using the slope of the graph in the following equation: Ex = 10 (-1/slope) – 1. The calculated efficiencies are 1.18 and 1.03 for mA2bAR and hA2bAR, respectively. C. Human A2bAR and mouse A2bAR mRNA expression was measured by qPCR in Kupffer cells isolated from mice at 12 weeks of age (n = 5 WT, 6 A2bAR KO, 6 CD68-Tg; ns = not statistically different from WT). D. Visceral (epididymal) adipose tissue macrophages were sorted via flow cytometry-based markers (see Methods) and subjected to qPCR of A2bAR mRNA. Data are averages ± SD. Relative mRNA expression was determined using the ΔΔCT method and were normalized to 18s rRNA values. Data are averages ± SD. *Student two-tail t-test assuming equal variance was found significant only when p-value <0.05.

Mentions: Kupffer cells and adipose tissue macrophages have been shown to play a role in obesity-induced insulin resistance in the liver and fat, respectively [8], [16], [22]–[26]. As we had previously reported a protective role for A2bAR in maintaining metabolic homeostasis in mice on HFD [30], and considering the expression of this receptor in macrophages, we sought to explore the specific role of the A2bAR in macrophages in mediating this protective effect. To examine whether gain of A2bAR function in macrophages restores the protective effect of the A2bAR, we generated transgenic mice (CD68-Tg) that express A2bAR only in the monocyte lineage by using the CD68 gene promoter to drive human A2bAR expression on an A2bAR KO background. The CD68 promoter has been used in the past to direct expression of a transgene to macrophages [32], [37], [38]. The goal was also to specifically explore the role of human A2bAR, by “humanizing” a mouse model. A founder line showing macrophage A2bAR activation similar to WT was used for this study (Figure 1A). We focused on analysis of tissue macrophages, considering their contribution to tissue insulin sensitivity. Given the similar efficiencies of the human and mouse Taqman primers (Figure 1B), we compared the expression of A2bAR in Kupffer cells, resident liver macrophages. A2bAR expression was similar in Kupffer cells from WT and CD68-Tg mice (Figure 1C). Further, visceral adipose tissue macrophages sorted by flow cytometry (considering their scarcity) showed similar expression of A2bAR in the CD68-Tg and WT mice (Figure 1D).


The macrophage A2B adenosine receptor regulates tissue insulin sensitivity.

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

Generation of transgenic mice expressing A2bAR in macrophages only.A. Genomic analysis by PCR of CD68-hA2bAR transgene in founder lines 1, 2, and 3 (Fo # 1, 2, 3) compared to A2bAR KO mice. Line 2 was used for the remainder of the studies based on expression analysis shown in panels c,d. B. Determination of primer efficiency. The amplification efficiency of human A2bAR (hA2bAR) and mouse A2bAR (mA2bAR) TaqMan primers was tested using the CT slope method. The target template was diluted over a log scale and CT values were determined by qPCR. A plot of CT versus log cDNA concentration is shown for hA2bAR and mA2bAR primers. Amplification efficiency (Ex) is calculated using the slope of the graph in the following equation: Ex = 10 (-1/slope) – 1. The calculated efficiencies are 1.18 and 1.03 for mA2bAR and hA2bAR, respectively. C. Human A2bAR and mouse A2bAR mRNA expression was measured by qPCR in Kupffer cells isolated from mice at 12 weeks of age (n = 5 WT, 6 A2bAR KO, 6 CD68-Tg; ns = not statistically different from WT). D. Visceral (epididymal) adipose tissue macrophages were sorted via flow cytometry-based markers (see Methods) and subjected to qPCR of A2bAR mRNA. Data are averages ± SD. Relative mRNA expression was determined using the ΔΔCT method and were normalized to 18s rRNA values. 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-g001: Generation of transgenic mice expressing A2bAR in macrophages only.A. Genomic analysis by PCR of CD68-hA2bAR transgene in founder lines 1, 2, and 3 (Fo # 1, 2, 3) compared to A2bAR KO mice. Line 2 was used for the remainder of the studies based on expression analysis shown in panels c,d. B. Determination of primer efficiency. The amplification efficiency of human A2bAR (hA2bAR) and mouse A2bAR (mA2bAR) TaqMan primers was tested using the CT slope method. The target template was diluted over a log scale and CT values were determined by qPCR. A plot of CT versus log cDNA concentration is shown for hA2bAR and mA2bAR primers. Amplification efficiency (Ex) is calculated using the slope of the graph in the following equation: Ex = 10 (-1/slope) – 1. The calculated efficiencies are 1.18 and 1.03 for mA2bAR and hA2bAR, respectively. C. Human A2bAR and mouse A2bAR mRNA expression was measured by qPCR in Kupffer cells isolated from mice at 12 weeks of age (n = 5 WT, 6 A2bAR KO, 6 CD68-Tg; ns = not statistically different from WT). D. Visceral (epididymal) adipose tissue macrophages were sorted via flow cytometry-based markers (see Methods) and subjected to qPCR of A2bAR mRNA. Data are averages ± SD. Relative mRNA expression was determined using the ΔΔCT method and were normalized to 18s rRNA values. Data are averages ± SD. *Student two-tail t-test assuming equal variance was found significant only when p-value <0.05.
Mentions: Kupffer cells and adipose tissue macrophages have been shown to play a role in obesity-induced insulin resistance in the liver and fat, respectively [8], [16], [22]–[26]. As we had previously reported a protective role for A2bAR in maintaining metabolic homeostasis in mice on HFD [30], and considering the expression of this receptor in macrophages, we sought to explore the specific role of the A2bAR in macrophages in mediating this protective effect. To examine whether gain of A2bAR function in macrophages restores the protective effect of the A2bAR, we generated transgenic mice (CD68-Tg) that express A2bAR only in the monocyte lineage by using the CD68 gene promoter to drive human A2bAR expression on an A2bAR KO background. The CD68 promoter has been used in the past to direct expression of a transgene to macrophages [32], [37], [38]. The goal was also to specifically explore the role of human A2bAR, by “humanizing” a mouse model. A founder line showing macrophage A2bAR activation similar to WT was used for this study (Figure 1A). We focused on analysis of tissue macrophages, considering their contribution to tissue insulin sensitivity. Given the similar efficiencies of the human and mouse Taqman primers (Figure 1B), we compared the expression of A2bAR in Kupffer cells, resident liver macrophages. A2bAR expression was similar in Kupffer cells from WT and CD68-Tg mice (Figure 1C). Further, visceral adipose tissue macrophages sorted by flow cytometry (considering their scarcity) showed similar expression of A2bAR in the CD68-Tg and WT mice (Figure 1D).

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