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. 2018 Jul;68(1):48-61.
doi: 10.1002/hep.29777. Epub 2018 May 10.

Disruption of adenosine 2A receptor exacerbates NAFLD through increasing inflammatory responses and SREBP1c activity

Yuli Cai  1   2 , Honggui Li  1 , Mengyang Liu  1   3 , Ya Pei  1 , Juan Zheng  1   4 , Jing Zhou  1 , Xianjun Luo  1 , Wenya Huang  1 , Linqiang Ma  1   5   6 , Qiuhua Yang  7   8 , Shaodong Guo  1 , Xiaoqiu Xiao  5   6 , Qifu Li  5 , Tianshu Zeng  4 , Fanyin Meng  9   10 , Heather Francis  9   10 , Shannon Glaser  9   10 , Lulu Chen  4 , Yuqing Huo  7   8 , Gianfranco Alpini  9   10 , Chaodong Wu  1
Affiliations

Disruption of adenosine 2A receptor exacerbates NAFLD through increasing inflammatory responses and SREBP1c activity

Yuli Cai et al. Hepatology. 2018 Jul.

Abstract

Adenosine 2A receptor (A2A R) exerts protective roles in endotoxin- and/or ischemia-induced tissue damage. However, the role for A2A R in nonalcoholic fatty liver disease (NAFLD) remains largely unknown. We sought to examine the effects of global and/or myeloid cell-specific A2A R disruption on the aspects of obesity-associated NAFLD and to elucidate the underlying mechanisms. Global and/or myeloid cell-specific A2A R-disrupted mice and control mice were fed a high-fat diet (HFD) to induce NAFLD. In addition, bone marrow-derived macrophages and primary mouse hepatocytes were examined for inflammatory and metabolic responses. Upon feeding an HFD, both global A2A R-disrupted mice and myeloid cell-specific A2A R-defcient mice revealed increased severity of HFD-induced hepatic steatosis and inflammation compared with their respective control mice. In in vitro experiments, A2A R-deficient macrophages exhibited increased proinflammatory responses, and enhanced fat deposition of wild-type primary hepatocytes in macrophage-hepatocyte cocultures. In primary hepatocytes, A2A R deficiency increased the proinflammatory responses and enhanced the effect of palmitate on stimulating fat deposition. Moreover, A2A R deficiency significantly increased the abundance of sterol regulatory element-binding protein 1c (SREBP1c) in livers of fasted mice and in hepatocytes upon nutrient deprivation. In the absence of A2A R, SREBP1c transcription activity was significantly increased in mouse hepatocytes.

Conclusion: Taken together, our results demonstrate that disruption of A2A R in both macrophage and hepatocytes accounts for increased severity of NAFLD, likely through increasing inflammation and through elevating lipogenic events due to stimulation of SREBP1c expression and transcription activity. (Hepatology 2018;68:48-61).

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Conflict of interest statement

CONFLICT OF INTEREST

This material, in part, is the result of work supported with resources and the use of facilities at the Central Texas Veterans Health Care System, Temple, Texas. The content is the responsibility of the author(s) alone and does not necessarily reflect the views or policies of the Department of Veterans Affairs or the United States Government.

Figures

Figure 1
Figure 1. HFD feeding increases hepatic A2AR abundance and enhances liver proinflammatory responses
Wild type C57BL/6J mice were fed as described in Methods. (A) Tissue distribution of A2AR. SI, small intestine; WAT, white adipose tissue. (B) Liver A2AR mRNA levels. AU, arbitrary unit. (C) Liver amount of A2AR, CD39 (ecto-nucleoside triphosphate diphosphohydrolase 1), and CD73 (ecto-5′-nucleotidase). (D) Liver proinflammatory signaling. (E) Liver cytokine mRNA (left panel) and protein (right two panels) levels. For A, C, and D, liver lysates were used for Western blot analysis. Blots were quantified using densitometry (bar graphs in C and D). For B and E, liver mRNAs were quantified using real-time RT-PCR. For E, liver protein levels of cytokines were quantified using ELISA kits. For all bar graphs, data are means ± SE. n = 6 – 8. Statistical difference between HFD and LFD: *, P < 0.05 and **, P < 0.01 in B and E (right two panels) or in bar graphs of C, D, and E (left two panels) for the same protein or gene.
Figure 2
Figure 2. A2AR disruption exacerbates HFD-induced hepatic steatosis and inflammation
Male A2AR−/− mice, A2AR+/− mice, and A2AR+/+ mice, at 5 – 6 weeks of age, were fed an HFD for 12 weeks. Age- and gender-matched A2AR+/+ mice were fed an LFD for 12 weeks and served as normal control. (A) Liver weight. (B) Representative images of H&E, Oil-Red-O, and/or F4/80 staining for liver sections. (C) Liver proinflammatory signaling. (D) Liver mRNA levels. (E) Liver SREBP1c abundance. pSREBP1c, precursor SREBP1c; nSREBP1c, nuclear SREBP1c. (F) Liver insulin signaling. Prior to tissue harvest, HFD-fed mice were injected with or without insulin (1 U/kg) into the portal vein for 5 min. IR, insulin receptor. For C, E, and F, liver lysates (C and F), as well as liver cytosolic and nuclear fractions (E) were subjected to Western blot analysis. Bar graphs, quantification of blots. For A – F, numeric data are means ± SE. n = 10 – 12 (A) or n = 8 – 10 (C – F). Statistical difference between A2AR−/−-HFD and A2AR+/+-HFD: *, P < 0.05 and **, P < 0.01 in bar graphs of A, C, and E, in D within the same gene, or in bar graphs of F under insulin-stimulated condition; statistical difference between A2AR−/−-HFD and A2AR+/−-HFD: †, P < 0.05 and ††, P < 0.01 in bar graphs of A, C, and E, in D within the same gene, or in bar graphs of F under insulin-stimulated condition; statistical difference between A2AR+/−-HFD and A2AR+/+-HFD: ‡, P < 0.05 and ‡‡, P < 0.01 in bar graphs of A and C, in D within the same gene, or in bar graphs of F under insulin-stimulated condition.
Figure 3
Figure 3. Myeloid cell-specific A2AR disruption exacerbates HFD-induced hepatic steatosis and proinflammatory responses
Male LysMCre+-A2ARF/F mice, LysMCre+-A2ARF/+ mice, and LysMCre+-A2AR+/+ mice, at 5 – 6 weeks of age, were fed an HFD for 12 weeks. (A) Liver weight. (B) Representative images of H&E, Oil-Red-O, and/or F4/80 staining for liver sections. (C) Liver lysates were examined for the proinflammatory signaling using Western blot analysis. Bar graphs, quantification of blots. AU, arbitrary unit. TLR4, Toll-like receptor 4. (D) Liver mRNA levels were quantified using real-time RT-PCR. For A, C, and D, numeric data are means ± SE. n = 8 – 10. Statistical difference between LysMCre+-A2ARF/F and LysMCre+-A2AR+/+: *, P < 0.05 and **, P < 0.01 in A or in bar graphs of C and D within the same protein or gene; statistical difference between LysMCre+-A2ARF/F and LysMCre+-A2ARF/+: †, P < 0.05 and ††, P < 0.01 in bar graphs of C and D within the same protein or gene; statistical difference between LysMCre+-A2ARF/+ and LysMCre+-A2AR+/+: ‡, P < 0.05 and ‡‡, P < 0.01 in A or in bar graphs of C and D within the same protein or gene.
Figure 4
Figure 4. A2AR disruption aggravates macrophage proinflammatory activation
Bone marrow-derived macrophages (BMDM) were prepared as described in Methods. (A, B) Proinflammatory signaling (A) and cytokine production (B) of BMDM from A2AR−/− mice and A2AR+/+ mice. For A, BMDM were treated with or without lipopolysaccharide (LPS, 100 ng/ml) for 30 min prior to harvest. Cell lysates were subjected to Western blot analysis. Bar graphs, quantification of blots. For B, cytokine concentrations in BMDM conditioned-media. (C) Proinflammatory signaling of BMDM from LysMCre+-A2ARF/F mice and LysMCre+-A2AR+/+ mice. Cells were treated and analyzed as described in A. Bar graphs, quantification of blots. For A – C, numeric data are means ± SE. n = 4 – 6. Statistical difference between A2AR−/− and A2AR+/+: *, P < 0.05 and **, P < 0.01 in bar graphs of A (under LPS-stimulated condition) or in B; statistical difference between LysMCre+-A2ARF/F and LysMCre+-A2AR+/+: †, P < 0.05 and ††, P < 0.01 in bar graphs of C (under LPS-stimulated condition).
Figure 5
Figure 5. A2AR disruption exacerbates the effects of macrophages on increasing hepatocyte fat deposition, cytokine expression, and insulin resistance
Macrophage-hepatocyte co-cultures were performed as described in Methods. A set of primary hepatocytes were incubated without macrophages and followed by the same treatments as co-cultures. (A) Hepatocyte fat deposition. The cells were treated with palmitate (Pal, 250 μM, conjugated in bovine serum albumin (BSA)) or BSA for the last 24 hr of the 48 hr incubation period, and stained with Oil Red O for 1 hr. Bar graphs, quantification of fat content and triglyceride levels. (B, C) The mRNA levels of genes related to fat metabolism (B) and proinflammatory cytokines (C) were examined using real-time RT-PCR. Prior to harvest, cells were treated without or with LPS (20 ng/ml) for 6 hr. (D) Insulin signaling. Prior to harvest, cells were treated with insulin (100 nM) or PBS for 30 min. Cell lysates were subjected to Western blot analysis. Bar graph, quantification of blots. For A – D, numeric data are means ± SE, n = 4 – 6. Statistical difference between co-cultures with A2AR−/− BMDM and co-cultures with A2AR+/+ BMDM: *, P < 0.05 and **, P < 0.01 in A, in B and C within the same gene, or in bar graph of D under insulin-stimulated condition; statistical difference between co-cultures with A2AR−/− BMDM and hepatocytes cultured without BMDM (None): †, P < 0.05 and ††, P < 0.01 in A, in B and C within the same gene, or in bar graph of D under insulin-stimulated condition; statistical difference between co-cultures with A2AR+/+ BMDM and hepatocytes/None: ‡, P < 0.05 in bar graph of D under insulin-stimulated condition.
Figure 6
Figure 6. A2AR disruption exacerbates hepatocyte fat deposition and proinflammatory responses and impairs hepatocyte insulin sensitivity
Primary hepatocytes were isolated from chow-diet-fed male A2AR−/− mice, A2AR+/− mice, and A2AR+/+ mice, at 10 – 12 weeks of age. (A) Hepatocyte fat deposition. After attachment, hepatocytes were treated with palmitate (Pal, 250 μM) or BSA for 24 hr, and stained with Oil Red O for 1 hr. Bar graphs, quantification of fat content and triglyceride levels. (B, C) Hepatocyte proinflammatory (B) and insulin (C) signaling. Prior to harvest, cells were treated with LPS (100 ng/ml), insulin (100 nM), or PBS for 30 min. Cell lysates were subjected to Western blot analysis. For A – C, numeric data are means ± SE, n = 4 – 6. Statistical difference between A2AR−/− vs. A2AR+/+: *, P < 0.05 and **, P < 0.01 in A, in B under LPS-stimulated condition, or in C under insulin-stimulated condition; statistical difference between A2AR−/− and A2AR+/−: †, P < 0.05 in A or in B under LPS-stimulated condition; statistical difference between A2AR+/− and A2AR+/+: ‡‡, P < 0.01 in C under insulin-stimulated condition.
Figure 7
Figure 7. A2AR deficiency elevates hepatic abundance of SREBP1c under fasting/nutrient deprivation and enhances SREBP1c transcription activity
(A) Liver SREBP1c abundance. Male A2AR−/− mice and A2AR+/+ mice, at 10 – 12 weeks of age, were free-fed or fasted for 18 hr. (B) Hepatocyte SREBP1c abundance. Primary hepatocytes were incubated in M199 in the absence of fetal bovine serum for 24 hr. For A and B, liver (A) or hepatocyte (B) cytosolic and nuclear proteins were subjected to Western blot analysis. (C) Hepatocyte SREBP1c transcription activity. Left panel, primary hepatocytes were incubated in M199 in the absence of fetal bovine serum and transfected with a reporter construct in which luciferase expression is under the control of SRE sequences on fatty acid synthase (pFAS-SRE-luc) or a control (pGL3-luc) for 24 hr; right panel, primary hepatocytes were treated as described in B. Hepatocyte chromatins were immunoprecipitated with antibodies against SREBP1c. The resultant DNA were analyzed for SRE/E sequences of FAS promoter. (D) Hepatocyte mRNAs. Cells were treated as described in B. (E) Liver AMPK phosphorylation states. Male A2AR−/− mice, A2AR+/− mice, and A2AR+/+ mice were fed as described in Figure 2. (F) Hepatocyte ADP/ATP ratio. Cells were treated as described in B. For B – D and F, primary hepatocytes were isolated from chow-diet-fed male A2AR−/− mice and A2AR+/+ mice, at 10 – 12 weeks of age. For A, B, and E, blots were quantified using densitometry. For all bar graphs, data are means ± SE, n = 4 – 6 (in A – C) or n = 6 – 8 (in D – F). Statistical difference between A2AR−/− and A2AR+/+: *, P < 0.05 and **, P < 0.01 in A and B for the same fraction, in C for the same construct under the same condition, in D for the same gene, or in E and F; statistical difference between A2AR−/− and A2AR+/−: †, P < 0.05 in E and F; statistical difference between insulin-treated A2AR+/+ cells and control-treated A2AR+/+ cells (transfected with pFAS-SRE-luc): ‡, P < 0.05 in C; statistical difference between A2AR+/− and A2AR+/+: ‡‡, P < 0.01 in F.
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