Phytosterol alleviates cholesterol accumulation by influencing intestinal esterification rather than competitive solubilization under dietary MUFA.

Phytosterols (PS) are natural compounds with well-established cholesterol-lowering properties. However, their systemic bioavailability is remarkably low compared to cholesterol. While dietary Monounsaturated fatty acids (MUFA) are known to reduce cholesterol absorption and potentially positive role in PS absorption, their specific impact on the differential absorption and esterification of PS remains unclear. This study aimed to identify the key step limiting PS absorption and to reveal the mechanism by which dietary MUFA regulates PS absorption and sterol homeostasis. In vitro digestion models received PS and cholesterol, while high-MUFA diet-fed mice were administered PS. Enterocytes were treated with oleic acid (OA) and PS. PS absorption, Acyl-Coenzyme A: cholesterol acyltransferase 2 (ACAT2) activity, and blood lipid were evaluated. Dynamic molecular simulation assessed enzyme-substrate binding, and the FXR/CDX2/ACAT2 pathway governing sterol esterification was explored. MUFA reprogrammed intestinal sterol metabolism through dual regulation of ACAT2, shifting control from enzyme abundance to catalytic efficiency. While FXR/CDX2-mediated repression reduced cholesterol esterification, MUFA-derived oleoyl-CoA allosterically stabilized the ACAT2 active site, selectively enhancing PS turnover. This asymmetric compensatory mechanism redirected ACAT2 substrate preference, promoting phytosterol ester (PE) accumulation while limiting cholesterol ester (CE) production. Evidence demonstrate that dietary MUFA enhances the catalytic preference of ACAT2 for PS while transcriptionally fine-tuning the overall enzyme activity through the FXR/CDX2/ACAT2 pathway. These finding of ACAT2 as a key mediator for nutritional strategies aimed at improving sterol homeostasis and combating metabolic diseases.