Exendin-4 decreases liver inflammation and atherosclerosis development simultaneously by reducing macrophage infiltration

Abstract

Background and purpose: The aetiology of inflammation in the liver and vessel wall, leading to non-alcoholic steatohepatitis (NASH) and atherosclerosis, respectively, shares common mechanisms including macrophage infiltration. To treat both disorders simultaneously, it is highly important to tackle the inflammatory status. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist, reduces hepatic steatosis and has been suggested to reduce atherosclerosis; however, its effects on liver inflammation are underexplored. Here, we tested the hypothesis that exendin-4 reduces inflammation in both the liver and vessel wall, and investigated the common underlying mechanism.

Experimental approach: Female APOE*3-Leiden.CETP mice, a model with human-like lipoprotein metabolism, were fed a cholesterol-containing Western-type diet for 5 weeks to induce atherosclerosis and subsequently treated for 4 weeks with exendin-4.

Key results: Exendin-4 modestly improved dyslipidaemia, but markedly decreased atherosclerotic lesion severity and area (-33%), accompanied by a reduction in monocyte adhesion to the vessel wall (-42%) and macrophage content in the plaque (-44%). Furthermore, exendin-4 reduced hepatic lipid content and inflammation as well as hepatic CD68⁺ (-18%) and F4/80⁺ (-25%) macrophage content. This was accompanied by less monocyte recruitment from the circulation as the Mac-1⁺ macrophage content was decreased (-36%). Finally, exendin-4 reduced hepatic chemokine expression in vivo and suppressed oxidized low-density lipoprotein accumulation in peritoneal macrophages in vitro, effects dependent on the GLP-1 receptor.

Conclusions and implications: Exendin-4 reduces inflammation in both the liver and vessel wall by reducing macrophage recruitment and activation. These data suggest that exendin-4 could be a valuable strategy to treat NASH and atherosclerosis simultaneously.

Keywords: cholesterol; exendin-4; inflammation; macrophage content; monocyte recruitment; oxidized LDL.

Figure 1

Exendin-4 decreases (V)LDL and slightly increases HDL. After 5 weeks of feeding a Western-type diet containing 0.4% cholesterol, mice were treated with exendin-4 (50 μg·kg⁻¹·day⁻¹) or vehicle (PBS) s.c. for 4 weeks. Blood was collected by tail bleeding after 4 h of fasting before treatment (T = 0), and after 2 (T = 2) and 4 (T = 4) weeks of treatment. Plasma cholesterol (A) and TG (B) levels were determined. After 4 weeks of treatment, group-wise pooled plasma was fractionated using fast protein liquid chromatography on a Superose 6 column, and the individual fractions were assayed for cholesterol (C). Livers were isolated, mRNA was extracted and Apoa1 mRNA was determined as normalized to cyclophilin and hypoxanthine ribosyltransferase mRNA levels. Data were calculated as fold difference compared with vehicle (D). Values are means ± SEM (n = 17 mice per group). *P < 0.05 compared with vehicle.

Figure 2

Exendin-4 reduces aortic atherosclerosis development and monocyte recruitment to the endothelium wall. After 5 weeks of feeding a Western-type diet containing 0.4% cholesterol, mice were treated with exendin-4 (50 μg·kg⁻¹·day⁻¹) or vehicle (PBS) s.c. for 4 weeks. Subsequently, hearts were isolated, fixed, dehydrated and embedded in paraffin. Cross sections of the aortic root were stained with haematoxylin-phloxine-saffron (A, B) or anti-AIA serum (C, D). Total lesion area (A), lesion severity (B), the number of adhering monocytes to the endothelium wall (C) and macrophage area (D) were quantified. Values are means ± SEM (n = 17 mice per group). *P < 0.05; ***P < 0.001 compared with vehicle.

Figure 3

Exendin-4 reduces liver inflammation and macrophage content. After 5 weeks of feeding a Western-type diet containing 0.4% cholesterol, mice were treated with exendin-4 (50 μg·kg⁻¹·day⁻¹) or vehicle (PBS) s.c. for 4 weeks. mRNA was extracted from liver pieces, and mRNA expression of inflammatory markers TNF-α, IL-1β and IL-6 (A); CD68 (B); and F4/80 (C) was determined as normalized to cyclophilin and hypoxanthine ribosyltransferase mRNA levels. Data were calculated as fold difference as compared with vehicle (A–C). Liver sections were immunostained for CD68 and F4/80, and CD68⁺ (D) F4/80⁺ (E) macrophages were quantified. Values are means ± SEM (n = 17 mice per group). *P < 0.05; **P < 0.01; ***P < 0.001 compared with vehicle.

Figure 4

Exendin-4 reduces macrophage infiltration into the liver. After 5 weeks of feeding a Western-type diet containing 0.4% cholesterol, mice were treated with exendin-4 (50 μg·kg⁻¹·day⁻¹) or vehicle (PBS) s.c. for 4 weeks. In the liver, mRNA expression of Mcp-1 (A) was determined as normalized to cyclophilin and hypoxanthine ribosyltransferase mRNA levels. Data were calculated as fold difference as compared with vehicle (A). Liver sections were immunostained for Mac-1, and Mac-1⁺ macrophages were quantified (B). Values are means ± SEM (n = 17 mice per group). *P < 0.05; **P < 0.01 compared with vehicle.

Figure 5

Exendin-4 has no effect on plasma antibodies against oxLDL, but reduces oxLDL-induced foam cell formation in peritoneal macrophages. After 5 weeks of feeding a Western-type diet containing 0.4% cholesterol, mice were treated with exendin-4 (50 μg·kg⁻¹·day⁻¹) or vehicle (PBS) s.c. for 4 weeks. Plasma anti-oxLDL antibodies were determined (A). Peritoneal macrophages were incubated with exendin-4 and/or exendin-(9–39) for 48 h. The uptake of [³H]-COEth-labelled oxLDL was quantified (B). After fixation, lipid accumulation into the cells was visualized by staining with Oil red O (C). Values are means ± SEM (n = 17 mice per group). *P < 0.05; **P < 0.01 compared with vehicle.

Figure 6

Proposed mechanism underlying the beneficial effects of exendin-4 on atherosclerosis development and NASH. Exendin-4 attenuates the development of atherosclerosis and NASH by (i) reducing the expression of chemokines and adhesion molecules, which leads to less recruitment of circulating monocytes/macrophages, and (ii) by inhibiting the uptake of oxLDL by macrophages and the subsequent formation of foam cells in the vessel wall and liver respectively. For further explanations, see text. CR, chemokine receptor(s); ICAM-1, intercellular adhesion molecule-1; mΦ, macrophage; MCP-1, monocyte chemotactic protein-1; oxLDL, oxidized LDL; SR, scavenger receptor; VCAM, vascular cell adhesion molecule-1.