Nε-(carboxymethyl)lysine promotes lipid uptake of macrophage via cluster of differentiation 36 and receptor for advanced glycation end products

Abstract

Background: Advanced glycation end products (AGEs) are diabetic metabolic toxic products that cannot be ignored. N^ε-(carboxymethyl)lysine (CML), a component of AGEs, could increase macrophage lipid uptake, promote foam cell formation, and thereby accelerate atherosclerosis. The receptor for AGEs (RAGE) and cluster of differentiation 36 (CD36) were the receptors of CML. However, it is still unknown whether RAGE and CD36 play key roles in CML-promoted lipid uptake.

Aim: Our study aimed to explore the role of RAGE and CD36 in CML-induced mac-rophage lipid uptake.

Methods: In this study, we examined the effect of CML on lipid uptake by Raw264.7 macrophages. After adding 10 mmol/L CML, the lipid accumulation in macro-phages was confirmed by oil red O staining. Expression changes of CD36 and RAGE were detected with immunoblotting and quantitative real-time polymerase chain reaction. The interaction between CML with CD36 and RAGE was verified by immunoprecipitation. We synthesized a novel N-succinimidyl-4-¹⁸F-fluorobenzoate-CML radioactive probe. Radioactive receptor-ligand binding assays were performed to test the binding affinity between CML with CD36 and RAGE. The effects of blocking CD36 or RAGE on CML-promoting lipid uptake were also detected.

Results: The study revealed that CML significantly promoted lipid uptake by macro-phages. Immunoprecipitation and radioactive receptor-ligand binding assays indicated that CML could specifically bind to both CD36 and RAGE. CML had a higher affinity for CD36 than RAGE. ARG82, ASN71, and THR70 were the potential interacting amino acids that CD36 binds to CML Anti-CD36 and anti-RAGE could block the uptake of CML by macrophages. The lipid uptake promotion effect of CML was significantly attenuated after blocking CD36 or RAGE.

Conclusion: Our results suggest that the binding of CML with CD36 and RAGE promotes macrophage lipid uptake.

Keywords: Cluster of differentiation 36; Lipid uptake; Macrophage; Nε-(carboxymethyl)lysine; Receptor for advanced glycation end products.

Figure 1

N^ε-(carboxymethyl)lysine promotes lipid uptake and cluster of differentiation 36 and receptor for advanced glycation end products expression in Raw264.7 cells. A: Oil Red O staining of lipid accumulation in Raw264.7 cells, Scale 20 μm; B: Oil Red O staining quantification of lipid accumulation in Raw264.7 cells; C-E: Protein levels of cluster of differentiation 36 (CD36) and receptor for advanced glycation end products (RAGE) in Raw264.7 macrophages; F and G: mRNA abundance of Cd36 (F) and RAGE (G) in Raw264.7 macrophages. ^aP < 0.01 compared with control (Ctrl) group. CML: N^ε-(carboxymethyl)lysine.

Figure 2

N^ε-(carboxymethyl)lysine has a higher binding affinity to cluster of differentiation 36 than to receptor for advanced glycation end products. A and B: Detection of N^ε-(carboxymethyl)lysine (CML) binding to cluster of differentiation 36 (CD36) (A) and to receptor for advanced glycation end products (RAGE) (B) by immunoprecipitation; C: Synthesis of N-succinimidyl-4-¹⁸F-fluorobenzoate-CML; D and E: Detection of the specific binding between CML and CD36 (D) and between CML and RAGE (E) with radioreceptor ligand binding assays; F: Molecular docking model of CML-CD36 binding; G: Detail of the binding site of CML to CD36; H: Amino acids for the binding interaction between CML and CD36. SB: Specific binding; NB: Non-specific binding; TB: Total binding. CPM: Counts/minute.

Figure 3

Blockade of cluster of differentiation 36 or receptor for advanced glycation end products inhibits the capture of N^ε-(carboxymethyl)lysine by macrophages. ^b P < 0.05, compared with IgG group. ^cP < 0.05, compared with Anti- cluster of differentiation 36 (CD36) group. ^dP < 0.05, compared with Anti-receptor for advanced glycation end products (RAGE) group. Ctrl: Control; CML: N^ε-(carboxymethyl)lysine; malBSA: maleylated-bovine serum albumin.

Figure 4

Blockade of cluster of differentiation 36 or receptor for advanced glycation end products inhibits the capture of N^ε-(carboxymethyl)lysine by macrophages. ^e P < 0.05, compared with IgG group. ^fP < 0.05, compared with Anti- cluster of differentiation 36 (CD36) group. ^gP < 0.05, compared with Anti-receptor for advanced glycation end products (RAGE) group. Ctrl: Control; CML: N^ε-(carboxymethyl)lysine; malBSA: maleylated-bovine serum albumin.

Figure 5

N^ε-(carboxymethyl)lysine promotes lipid uptake by macrophages through receptor for advanced glycation end products and cluster of differentiation 36. CD36: Cluster of differentiation 36; RAGE: Receptor for advanced glycation end products; CML: N^ε-(carboxymethyl)lysine.