Esculetin promotes HDL-driven cholesterol excretion involved in CD36-mediated phagocytosis of adipose tissue macrophages and CEBPbeta

Postprandial dyslipidemia is a recognized risk factor for atherosclerosis. High-density lipoprotein (HDL)-mediated reverse cholesterol transport plays a crucial role in mitigating this risk by clearing postprandial lipids. This study aimed to investigate the impact of esculetin, a 4-Hydroxycoumarin, on postprandial cholesterol metabolism and excretion after a high-fat meal. Esculetin significantly elevated postprandial HDL cholesterol levels in serum and postprandial bile acid levels in bile, and altered serum metabolomics in mice fed a high-fat meal, indicating esculetin promotes HDL-driven cholesterol excretion after a high-fat meal. Furthermore, esculetin administration in mice led to an increase in the ratio and phagocytic activity of a subset of adipose tissue macrophages (ATMs) expressing high levels of CD36 and Tim4. Inhibition of CD36 by Sulfo-N-succinimidyl oleate (SSO) blocked esculetin-induced elevation of postprandial serum HDL and bile acid levels in bile. Additionally, esculetin demonstrated the ability to increase the uptake of oxidized LDL (ox-LDL) via CD36 in a macrophage cell line, which might involve alteration of the epigenetic landscape controlled by CCAAT enhancer-binding protein beta (C/EBPß). Esculetin-induced increased uptake of ox-LDL and elevation of CD36 was inhibited in C/EBPß-deficient cells. A relatively higher expression of C/EBPß was observed in CD36+ ATMs, and esculetin increased the ratio of C/EBPß+ CD36+ ATMs in mice fed a lipid-rich meal. Moreover, the direct interaction between esculetin and C/EBPß were observed by Terahertz chemical microscope. Overall, these findings suggest esculetin promotes HDL-mediated postprandial cholesterol excretion by directly binding to C/EBPß and enhancing CD36-dependent phagocytosis in ATMs. Overall design: To study the transcriptomic alterations induced by esculetin in the liver, esculetin (0 or 90mg/kg/d) was daily administered to the mice by oral gavage for 7 days. In the first 4 days, the mice were fed with a control diet (CD) containing 10% kcal from fat. Then the mice were fed with a high- fat diet (HFD) containing 60% kcal from fat for additional 3 days to understand lipid-rich meals-induced postprandial lipid circulation. The transcriptomic alterations induced by esculetin were further analyzed in ox-LDL-treated RAW264.7 cells. RAW264. 7 cells were treated with 25 ug/mL DIL-oxLDL and esculetin (0 or 40 ug/mL) for 24 hr.