Identification of a novel cholesterol-lowering dipeptide, phenylalanine-proline (FP), and its down-regulation of intestinal ABCA1 in hypercholesterolemic rats and Caco-2 cells

Abstract

There has been no report about in vivo active cholesterol-lowering dipeptide in any protein origin, despite their potential health benefits. Cattle heart protein hydrolysate ultra-filtrate (HPHU, molecular weight < ca. 1,000 Da peptide mixture) exhibits cholesterol-lowering activity in hypercholesterolemic rats, but the active peptide in HPHU that lowers serum cholesterol levels and its molecular mechanism are unknown. In this study, we separated and purified HPHU to identify a novel cholesterol-lowering dipeptide (phenylalanine-proline, FP) and characterized the mechanism underlying its effects in vivo and in vitro. We identified FP as an active peptide from HPHU by MALDI-TOF mass spectrometry. FP significantly decreased serum total and non-HDL cholesterol and hepatic cholesterol levels in rats. FP significantly increased serum HDL cholesterol, accompanied by a significant decrease in the atherogenic index. FP also significantly increased fecal cholesterol and acidic steroid excretion. Moreover, FP significantly decreased ATP-binding cassette transporter A1 (ABCA1) expression in the rat jejunum and reduced cholesterol absorption in Caco-2 cells. We found a novel cholesterol-lowering dipeptide FP that could improve cholesterol metabolism via the down-regulation of intestinal ABCA1. The cholesterol-lowering action induced by FP was disappeared in PepT1KO mice. FP-induced cholesterol-lowering action is mediated via PepT1 in mice.

IIAEK Targets Intestinal Alkaline Phosphatase (IAP) to Improve Cholesterol Metabolism with a Specific Activation of IAP and Downregulation of ABCA1

“…Interestingly, a recent study in mice reported that intestinal ABCA1 knockout reduced cholesterol absorption by ≈28% and total plasma cholesterol by approximately 30% [ 22 ]. Our previous study revealed that the suppression of intestinal cholesterol absorption by a novel cholesterol-lowering dipeptide, phenylalanine-proline (FP), was associated with a reduction in intestinal ABCA1 expression levels [ 12 ]. These results suggest that the inhibitory effect IIAEK exerts on cholesterol absorption is likely to be accompanied by a decrease in the expression of ABCA1 in Caco-2 cells.…”

“…Caco-2 cells were grown in 6-well Transwell ® plates (Corning, Inc., Corning, NY, USA) containing 0.5 mL DMEM supplemented with fetal bovine serum for 14 days post-confluence. The absorption of [ 14 C]-labeled micellar cholesterol in Caco-2 cells was measured as previously described [ 12 , 13 ], with some modifications. Briefly, on day 14, [ 14 C]-labeled micellar solutions containing 3.7 kBq [4- 14 C]-cholesterol (2.0 Gbq/mmol, NEN) with or without 2 mM IIAEK, 5 mM taurocholate, and 250 μM cholesterol in serum-free DMEM were prepared by sonication and incubated with shaking at 37 °C for 24 h. Following incubation, cholesterol absorption was measured for 24 h. The cellular protein concentration was determined using a commercially available kit (Bio-Rad Protein Assay; Bio-Rad, Hercules, CA, USA).…”

“…Caco-2 cell treatment, and RNA and cDNA preparations were performed as previously described [ 12 ]. Real-time PCR was performed using the StepOnePlus TM Real-time PCR system (Applied Biosystems) and SYBR ® Premix Ex Taq (TAKARA), according to the manufacturer’s protocol.…”

“…The micelle was prepared as previously described [ 13 ]. Protein preparation from Caco-2 cells and Western blot analyses were performed as previously described [ 12 ]. For Western blot analysis, we used the following specific antibodies: anti-ABCA1 (sc-58219, Santa Cruz Biotechnology, Dallas, TX, USA), anti-IAP (ab186422, Abcam, Cambridge, UK), and anti-β-actin (sc-47778, Santa Cruz).…”

“…Protein preparation from Caco-2 cells and luciferase analyses were performed as previously described [ 12 ]. Following plasmid (ABCA1-Luc plasmid containing −928 to +107 bp, −536 to +107 bp, −126 to +107 bp, or −36 to +107 bp of the ABCA1 gene promoter, and pPGK β-galactosidase plasmid) transfection, the cells were treated with 2 mM IIAEK for 12 h with micelles, which were prepared as previously described [ 13 ].…”