Trimethylamine increases intestinal fatty acid absorption: in vitro studies in a Caco-2 cell culture system

Abstract

Although elevated blood levels of trimethylamine N-oxide (TMAO) have been associated with atherosclerosis development in humans, the role of its gut microbiota-derived precursor, TMA, in this process has not been yet deciphered. Taking this into account, and the fact that increased intestinal fatty acid absorption contributes to atherosclerosis onset and progression, this study aimed to evaluate the effect of TMA on fatty acid absorption in a cell line that mimics human enterocytes. Caco-2 cells were treated with TMA 250 μM for 24 h. Fatty acid absorption was assessed by measuring the apical-to-basolateral transport and the intracellular levels of BODIPY-C₁₂, a fluorescently labelled fatty acid analogue. Gene expression of the main intestinal fatty acid transporters was evaluated by real-time quantitative reverse transcription PCR. Compared to control conditions, TMA increased, in a time-dependent manner and by 20-50 %, the apical-to-basolateral transport and intracellular levels of BODIPY-C₁₂ fatty acid in Caco-2 cells. Fatty acid transport protein 4 (FATP4) and fatty acid translocase (FAT)/CD36 gene expression were not stimulated by TMA, suggesting that TMA-induced increase in fatty acid transport may be mediated by an increase in FAT/CD36 and/or FATP4 activity and/or fatty acid passive transport. This study demonstrated that TMA increases the intestinal absorption of fatty acids. Future studies are necessary to confirm if this may constitute a novel mechanism that partially explains the existing positive association between the consumption of a diet rich in TMA sources (e.g. red meat) and the increased risk of atherosclerotic diseases.

Keywords: Caco-2 cells; EDTA, ethylenediaminetetraacetic acid; Enterocytes; F, forward; FABP, fatty acid-binding protein; FABPpm, plasma membrane fatty acid-binding protein; FAT/CD36, fatty acid translocase; FATP4, fatty acid transport protein 4; FBS, foetal bovine serum; FSA, fluorescein sulphonic acid; Fatty acid absorption; HPRT, hypoxanthine guanine phosphoribosyltransferase; MTT, 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PBS, phosphate-buffered saline; R, reverse; TG, triacylglycerol; TMA, trimethylamine; TMAO, trimethylamine N-oxide; Trimethylamine; qRT-PCR, real-time quantitative reverse transcription polymerase chain reaction; sem, standard error of the mean.