Mechanisms of provitamin A (carotenoid) and vitamin A (retinol) transport into and out of intestinal Caco-2 cells

Abstract

The purpose of this study was to compare the mechanisms of intestinal retinol (ROL) and carotenoid transport. When differentiated Caco-2 cells were incubated with ROL for varying times, cellular ROL plateaued within 2 h, whereas retinyl ester (RE) formation increased continuously. ROL and RE efflux into basolateral medium (BM) increased linearly with time, ROL in the nonlipoprotein fraction and REs in chylomicrons (CMs). In contrast to carotenoids, ROL uptake was proportional to ROL concentration (0.5-110 mM). ROL efflux into BM occurred via two processes: a) a saturable process at low concentrations (,10 mM) and b) a nonsaturable process at higher concentrations. When ROL-loaded cells were maintained on retinoid-free medium, free ROL, but not REs, was secreted into BM. Glyburide significantly reduced ROL efflux but not ROL uptake. Inhibition of ABCA1 protein expression by small interfering RNAs decreased ROL efflux but not carotenoid efflux. Scavenger receptor class B type I (SR-BI) inhibition did not affect ROL transport but decreased carotenoid uptake. The present data suggest that a) ROL enters intestinal cells by diffusion, b) ROL efflux is partly facilitated, probably by the basolateral transporter ABCA1, and c) newly synthesized REs, but not preformed esters, are incorporated into CM and secreted. In contrast to ROL transport, carotenoid uptake is mediated by the apical transporter SR-BI, and carotenoid efflux occurs exclusively via their secretion in CM.-During, A., and E. H. Harrison. Vitamin A [or retinol (ROL)] is an essential fat-soluble nutrient required by humans for vision, cellular differentiation, development and growth, reproduction, and immunity (1). The de novo synthesis of vitamin A is limited to plants and microorganisms. Thus, humans must obtain vitamin A from the diet, either as preformed ROL or as provitamin A carotenoid precursors. Both deficiency and excess of vitamin A are known to cause pathologies. Vitamin A deficiency is usually a result of malnutrition but can also be attributable to abnormalities in the intestinal absorption of ROL or provitamin A carotenoids. Vitamin A deficiency especially affects children (100 to 140 million) in .100 countries (2). Vitamin A excess, although not as common as deficiency, is a result of excessive supplementation. Therefore, better knowledge about the mechanisms of absorption of vitamin A is important in determining the appropriate amounts of vitamin A required by humans.Vitamin A is present in the diet as retinyl esters (REs) found in foods of animal origin and as provitamin A carotenoids (mainly b-carotene, a-carotene, and b-cryptoxanthin) found in plant-derived products. Intestinal absorption of vitamin A from these dietary compounds requires several enzymatic steps. The hydrolysis of dietary REs in the intestinal lumen is catalyzed by enzymes such as the pancreatic triglyceride (TG) lipase and the intestinal brush border phospholipase B to yield unesterified ROL, which is the form absorbed into the intestinal mucosal cell. Some of th...

Comparison of tetrahydrofuran, fetal calf serum, and Tween 40 for the delivery of astaxanthin and canthaxanthin to HepG2 cells

“…O'Sullivan et al (2004), on the other hand, in agreement with During and Harrison (2007), did not report cytotoxic effects of either Tween 40 or Tween 80 to Caco-2 cells. This discrepancy may be due to the use of a different cell line (derived from a colon carcinoma instead of a liver carcinoma) and the fact that lower maximum concentrations of Tween in the cell culture medium (0.1%) were employed (O'Sullivan et al 2004;During and Harrison 2007). Similarly, the viability of freshly prepared primary rat hepatocytes was not impaired by incubation for 1, 4 or 24 h with B0.03% Tween 80, but significantly reduced when 0.3% Tween 80 were used (Bravo Gonzalez et al 2004).…”

“…Lin et al (2007) reported a better uptake of lycopene into DU145 prostate cancer cells from FCS-compared to THF-supplemented media. Lipoproteins appear to play an important role in the cellular uptake of carotenoids (During and Harrison 2007;O'Sullivan et al 2007). The involvement of the scavenger receptor class B type I (SR-BI) in carotenoid uptake has been demonstrated (van Bennekum et al 2005;During and Harrison 2007).…”

“…Lipoproteins appear to play an important role in the cellular uptake of carotenoids (During and Harrison 2007;O'Sullivan et al 2007). The involvement of the scavenger receptor class B type I (SR-BI) in carotenoid uptake has been demonstrated (van Bennekum et al 2005;During and Harrison 2007). SR-BI recognizes and binds lipoproteins, both in HepG2 cells (Rhainds et al 1999) and in vivo (Van Eck et al 2008), and mediates the internalization of their lipid contents (Rhainds and Brissette 2004).…”

“…The aim of the present investigation was to compare the two recently described carotenoid delivery systems fetal calf serum (FCS) (Lin et al 2007) and Tween 40 (O'Sullivan et al 2004;During and Harrison 2007) with the commonly used THF with respect to cytotoxicity, stability of the dissolved carotenoids, and uptake and accumulation of two of the major xanthophylls, namely astaxanthin (AX) and canthaxanthin (CX), in cultured human liver cells (HepG2).…”

Intestinal lipid absorption

“…The secretion of free retinol may require facilitated transport. This notion is supported by the marked inhibition of the free retinol efflux into the basolateral medium by glyburide, a known inhibitor of the ABCA1 transporter (46). Mechanisms regulating retinol output through these pathways are not well understood.…”