Abstract
IIAEK (Ile-Ile-Ala-Glu-Lys, lactostatin) is a novel cholesterol-lowering pentapeptide derived from bovine milk β-lactoglobulin. However, the molecular mechanisms underlying the IIAEK-mediated suppression of intestinal cholesterol absorption are unknown. Therefore, we evaluated the effects of IIAEK on intestinal cholesterol metabolism in a human intestinal model using Caco-2 cells. We found that IIAEK significantly reduced the expression of intestinal cholesterol metabolism-associated genes, particularly that of the ATP-binding cassette transporter A1 (ABCA1). Subsequently, we chemically synthesized a novel molecular probe, IIXEK, which can visualize a complex of target proteins interacting with photoaffinity-labeled IIAEK by fluorescent substances. Through photoaffinity labeling and MS analysis with IIXEK for the rat small intestinal mucosa and intestinal lipid raft fractions of Caco-2 cells, we identified intestinal alkaline phosphatase (IAP) as a specific molecule interacting with IIAEK and discovered the common IIAEK-binding amino acid sequence, GFYLFVEGGR. IIAEK significantly increased IAP mRNA and protein levels while decreasing ABCA1 mRNA and protein levels in Caco-2 cells. In conclusion, we found that IIAEK targets IAP to improve cholesterol metabolism via a novel signaling pathway involving the specific activation of IAP and downregulation of intestinal ABCA1.
Highlights
IntroductionImproving cholesterol metabolism through diet modifications is an important strategy for the prevention and treatment of hypercholesterolemia and hyperlipidemia [1]
Lifestyle diseases, especially arteriosclerosis-related disorders, are closely associated with diet.Improving cholesterol metabolism through diet modifications is an important strategy for the prevention and treatment of hypercholesterolemia and hyperlipidemia [1]
Our present results suggest that IIAEK improves cholesterol metabolism through an increase of intestinal alkaline phosphatase (IAP) mRNA levels and a decrease of ATP-binding cassette transporter A1 (ABCA1) mRNA
Summary
Improving cholesterol metabolism through diet modifications is an important strategy for the prevention and treatment of hypercholesterolemia and hyperlipidemia [1]. Dietary proteins and peptides are effective in ameliorating dyslipidemia and hypercholesterolemia in animals and humans [2]. The molecular mechanisms underlying their cholesterol-lowering effects have not yet been elucidated. We previously discovered a cholesterol metabolism-improving pentapeptide: IIAEK (lactostatin). It was obtained from β-lactoglobulin trypsin hydrolysate (LTH), which is a major component of the milk whey protein [3]. We observed that the serum cholesterol-lowering effect of IIAEK (Ile-Ile-Ala-Glu-Lys, lactostatin) was comparable to that of beta-sitosterol, which is a phytosterol that was described as having pharmaceutical potential based on a study in rats [3].
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