Mice lacking adenosine 2A receptor reveal increased severity of MCD-induced NASH

in Journal of Endocrinology
Correspondence should be addressed to C Wu: cdwu@tamu.edu

*(J Zhou and H Li contributed equally to this work)

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Adenosine 2A receptor (A2AR) exerts a protective role in obesity-related non-alcoholic fatty liver disease. Here, we examined whether A2AR protects against non-alcoholic steatohepatitis (NASH). In C57BL/6J mice, feeding a methionine- and choline-deficient diet (MCD) resulted in significant weight loss, overt hepatic steatosis, and massive aggregation of macrophages in the liver compared with mice fed a chow diet. MCD feeding also significantly increased the numbers of A2AR-positive macrophages/Kupffer cells in liver sections although decreasing A2AR amount in liver lysates compared with chow diet feeding. Next, MCD-induced NASH phenotype was examined in A2AR-disrupted mice and control mice. Upon MCD feeding, A2AR-disruptd mice and control mice displayed comparable decreases in body weight and fat mass. However, MCD-fed A2AR-disrupted mice revealed greater liver weight and increased severity of hepatic steatosis compared with MCD-fed control mice. Moreover, A2AR-disupted mice displayed increased severity of MCD-induced liver inflammation, indicated by massive aggregation of macrophages and increased phosphorylation states of Jun-N terminal kinase (JNK) p46 and nuclear factor kappa B (NFκB) p65 and mRNA levels of tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6. In vitro, incubation with MCD-mimicking media increased lipopolysaccharide (LPS)-induced phosphorylation states of JNK p46 and/or NFκB p65 and cytokine mRNAs in control macrophages and RAW264.7 cells, but not primary hepatocytes. Additionally, MCD-mimicking media significantly increased lipopolysaccharide-induced phosphorylation states of p38 and NFκB p65 in A2AR-deficient macrophages, but insignificantly decreased lipopolysaccharide-induced phosphorylation states of JNK p46 and NFκB p65 in A2AR-deficient hepatocytes. Collectively, these results suggest that A2AR disruption exacerbates MCD-induced NASH, which is attributable to, in large part, increased inflammatory responses in macrophages.

 

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    MCD feeding induces severe hepatic steatosis and inflammation while causing lipoatrophy. Male C57BL/6J mice, at 9–10 weeks of age, were fed a methionine- and choline-deficient (MCD) for 5 weeks or maintained on a chow diet (CD). (A and B) Body weight was measured during the feeding period (A). Also, foods consumed by the mice were recorded during the feeding period and used to calculate food intake (B). (C) At 1 day prior to the end of feeding period, body composition of the mice was analyzed using an EchoMRI analyzer. (D) Plasma levels of alanine aminotransferase (ALT). (E) Liver weight was measured after harvest of mice. (F) Liver sections were stained with H&E or for F4/80 expression. (G) Liver lysates were examined for the phosphorylation states of JNK p46 and the amount of αSMA. Bar graphs, quantification of blots. For A, B, C, D, E and G, numeric data are means ± s.e.m. (standard error), n = 8–10. AU, arbitrary unit. *P < 0.05 and **P < 0.01 MCD vs CD (in D, E, and G) for the same time point (in A and B) or for the same type of mass (in C). A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0198.

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    MCD feeding increases A2AR expression in liver macrophages/Kupffer cells although decreasing liver A2AR abundance. Male C57BL/6J mice, at 9–10 weeks of age, were fed an MCD for 5 weeks or maintained on a CD. (A) Liver lysates were examined for A2AR amount using Western blot analysis. Bar graph, quantification of blots. Data are means ± s.e.m. n = 6–10. AU, arbitrary unit. **P < 0.01 MCD vs CD. (B) Liver sections were examined for the expression of F4/80 and/or A2AR using immunofluorescent staining. The scale bar is 75 µm for 10× images. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0198.

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    Validation of liver A2AR disruption. Male A2AR−/− mice and A2AR+/+ mice, at 9–10 weeks of age, were fed an MCD for 5 weeks or maintained on a CD. After sacrifice of mice, liver sections were prepared and subjected to immunofluorescent staining of A2AR and/or F4/80 expression. The scale bar is 75 µm for 10× images. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0198.

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    A2AR disruption does not alter MCD-induced decreases in body weight and adiposity. Male A2AR−/− mice, A2AR+/− mice, and A2AR+/+ mice, at 9–10 weeks of age, were fed an MCD for 5 weeks or maintained on a CD. (A and B) During the feeding period, body weight of the mice was measured (A). Also, foods consumed by the mice were recorded during the feeding period and used to calculate food intake (B). (C) After harvest, the mass of epididymal, mesenteric, and perinephric fats was estimated as abdominal fats. (D) Adiposity was calculated by normalizing abdominal fats with body weight. For A, B, C and D, data are means ± s.e.m. n = 8–10.

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    A2AR disruption exacerbates MCD-induced hepatic steatosis and inflammation. Male A2AR−/− mice, A2AR+/− mice, and A2AR+/+ mice, at 9–10 weeks of age, were fed an MCD for 5 weeks or maintained on a CD. (A) Plasma levels of ALT. (B) Liver weight. (C) Liver sections were stained with H&E or Oil Red O or for F4/80. (D) Liver lysates were examined for the phosphorylation states of JNK p46 and NFκB p65. Bar graphs, quantification of blots. (E) Liver mRNA levels were examined using real-time PCR. For A, B, D, and E, numeric data are means ± s.e.m. n = 8–10 (A and B) or 6–8 (D and E). AU, arbitrary unit. *P < 0.05 and **P < 0.01 A2AR−/− vs. A2AR+/+ (in A and B, and in bar graphs of D) for the same gene (in E); P < 0.05 A2AR−/− vs A2AR+/− (in D, bar graphs) for the same gene (in E); P < 0.05 and ‡‡ P < 0.01 A2AR+/− vs A2AR+/+ (in B and bar graphs of D) for the same gene (in D). A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0198.

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    A2AR deficiency enhances the effect of MCD-mimicking media on stimulating proinflammatory responses in macrophages, but not hepatocytes. (A, B and C) Proinflammatory signaling (A and B) and cytokine expression (C) in primary hepatocytes (A) and bone marrow-derived macrophages (BMDM) (B and C). For A, B and C, male A2AR−/− mice and A2AR+/+ mice, at 10–12 weeks of age, were subjected to isolation of primary hepatocytes and bone marrow cells. Primary hepatocytes and BMDM were treated with MCD-mimicking media for 24 h in the presence or absence of LPS (100 ng/mL) for the last 30 min (A and B) or LPS (20 ng/mL) for the last 6 h. (D and E) Proinflammatory signaling (D) and cytokine expression (E) in RAW264.7 cells. At 80% confluence, RAW264.7 cells were incubated with MCD-mimicking media or control media for 24 h in the presence or absence of LPS (100 ng/mL) for the last 30 min (D) or LPS (20 ng/mL) for the last 6 h. For A, B, and D, cell lysates were examined for proinflammatory signaling using Western blot analysis. Bar graphs, quantification of blots. For all bar graphs, data are means ± s.e., n = 4–6. AU, arbitrary unit. *P < 0.05 A2AR−/− vs A2AR+/+ under the same condition (Ctrl or MCD, in A and B); P < 0.05 and †† P < 0.01 MCD vs Ctrl within the same genotype (in B) or for the same gene (in C and E) or under the same condition (PBS or LPS, in D); P < 0.05 and ‡‡ P < 0.01 LPS vs PBS within the same media (in D).

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