Marielle Margier

Intestinal scavenger receptors are involved in vitamin K1 absorption

Background: Vitamin K intestinal absorption is poorly understood. Results: Modulating SR-BI and CD36 expression or function in cells and in mice led to control of vitamin K1 intestinal transport. Conclusion: Intestinal scavenger receptors impact vitamin K1 transport through the enterocyte. Significance: Understanding vitamin K1 intestinal absorption is crucial to maintain an optimal vitamin K status. Vitamin K1 (phylloquinone) intestinal absorption is thought to be mediated by a carrier protein that still remains to be identified. Apical transport of vitamin K1 was examined using Caco-2 TC-7 cell monolayers as a model of human intestinal epithelium and in transfected HEK cells. Phylloquinone uptake was then measured ex vivo using mouse intestinal explants. Finally, vitamin K1 absorption was compared between wild-type mice and mice overexpressing scavenger receptor class B type I (SR-BI) in the intestine and mice deficient in cluster determinant 36 (CD36). Phylloquinone uptake by Caco-2 cells was saturable and was significantly impaired by co-incubation with α-tocopherol (and vice versa). Anti-human SR-BI antibodies and BLT1 (a chemical inhibitor of lipid transport via SR-BI) blocked up to 85% of vitamin K1 uptake. BLT1 also decreased phylloquinone apical efflux by ∼80%. Transfection of HEK cells with SR-BI and CD36 significantly enhanced vitamin K1 uptake, which was subsequently decreased by the addition of BLT1 or sulfo-N-succinimidyl oleate (CD36 inhibitor), respectively. Similar results were obtained in mouse intestinal explants. In vivo, the phylloquinone postprandial response was significantly higher, and the proximal intestine mucosa phylloquinone content 4 h after gavage was increased in mice overexpressing SR-BI compared with controls. Phylloquinone postprandial response was also significantly increased in CD36-deficient mice compared with wild-type mice, but their vitamin K1 intestinal content remained unchanged. Overall, the present data demonstrate for the first time that intestinal scavenger receptors participate in the absorption of dietary phylloquinone.

Micellar lipid composition affects micelle interaction with class B scavenger receptor extracellular loops

Scavenger receptors (SRs) like cluster determinant 36 (CD36) and SR class B type I (SR-BI) play a debated role in lipid transport across the intestinal brush border membrane. We used surface plasmon resonance to analyze real-time interactions between the extracellular protein loops and various ligands ranging from single lipid molecules to mixed micelles. Micelles mimicking physiological structures were necessary for optimal binding to both the extracellular loop of CD36 (lCD36) and the extracellular loop of SR-BI (lSR-BI). Cholesterol, phospholipid, and fatty acid micellar content significantly modulated micelle binding to and dissociation from the transporters. In particular, high phospholipid micellar concentrations inhibited micelle binding to both receptors (−53.8 and −74.4% binding at 0.32 mM compared with 0.04 mM for lCD36 and lSR-BI, respectively, P < 0.05). The presence of fatty acids was crucial for micelle interactions with both proteins (94.4 and 81.3% binding with oleic acid for lCD36 and lSR-BI, respectively, P < 0.05) and fatty acid type substitution within the micelles was the component that most impacted micelle binding to the transporters. These effects were partly due to subsequent modifications in micellar size and surface electric charge, and could be correlated to micellar vitamin D uptake by Caco-2 cells. Our findings show for the first time that micellar lipid composition and micellar properties are key factors governing micelle interactions with SRs.

Pinoresinol of olive oil decreases vitamin D intestinal absorption.

Enriching oils, such as olive oil, could be one solution to tackle the worldwide epidemic of vitamin D deficiency and to better fit with omega 3 (DHA) recommendations. However, data regarding the interactions occurring at the intestinal level between vitamin D and phenols from olive oil are scarce. We first determined the effect of polyphenols from a virgin olive oil, and a virgin olive oil enriched with DHA, on vitamin D absorption in rats. We then investigated the effects of 3 main olive oil phenols (oleuropein, hydroxytyrosol and pinoresinol) on vitamin D uptake by Caco-2 cells. The presence of polyphenols in the olive oil supplemented with DHA inhibited vitamin D postprandial response in rats (-25%, p<0.05). Similar results were obtained with a mix of the 3 polyphenols delivered to Caco-2 cells. However, this inhibitory effect was due to the presence of pinoresinol only. As the pinoresinol content can highly vary between olive oils, the present results should be taken into account to formulate an appropriate oil product enriched in vitamin D.

Comparison of the Micellar Incorporation and the Intestinal Cell Uptake of Cholecalciferol, 25-Hydroxycholecalciferol and 1-α-Hydroxycholecalciferol

In the context of the global prevalence of vitamin D insufficiency, we compared two key determinants of the bioavailability of 3 vitamin D forms with significant biopotencies: cholecalciferol, 25-hydroxycholecalciferol and 1-α-hydroxycholecalciferol. To this aim, we studied their incorporation into synthetic mixed micelles and their uptake by intestinal cells in culture. Our results show that 1-α-hydroxycholecalciferol was significantly more solubilized into mixed micelles compared to the other forms (1.6-fold and 2.9-fold improvement compared to cholecalciferol and 25-hydroxycholecalciferol, respectively). In Caco-2 TC7 cells, the hydroxylated forms were taken up more efficiently than cholecalciferol (p < 0.05), and conversely to cholecalciferol, their uptake was neither SR-BI(Scavenger-Receptor class B type I)- nor NPC1L1 (NPC1 like intracellular cholesterol transporter 1)-dependent. Besides, the apical membrane sodium–bile acid transporter ASBT (Apical Sodium-dependent Bile acid Transporter) was not involved, at least in vitro, in the uptake of any of the three vitamin D forms. Further investigations are needed to identify the uptake pathways of both 1-α-hydroxycholecalciferol and 25-hydroxycholecalciferol. However, considering its high bioavailability, our results suggest the potential interest of using 1-α-hydroxycholecalciferol in the treatment of severe vitamin D deficiency.

Opposite Effects of the Spinach Food Matrix on Lutein Bioaccessibility and Intestinal Uptake Lead to Unchanged Bioavailability Compared to Pure Lutein

SCOPEnFood matrix is generally believed to alter carotenoid bioavailability, but its effect on xanthophylls is usually limited. This study thus aims to decipher the digestion-absorption process of lutein in the presence or not of a food matrix.nnnMETHODSnLutein transfer to gastric-like lipid droplets or artificial mixed micelles was assessed when lutein was added to test meals either as a pure molecule ((all-E)-lutein) or in canned spinach ((Z) + (all-E)-lutein). The obtained mixed micelles were delivered to Caco-2 cells to evaluate lutein uptake. Finally postprandial plasma lutein responses were compared in minipigs after the two test meals.nnnRESULTSnLutein transfer to gastric-like lipid droplets and to mixed micelles was higher when lutein was added in spinach than when it was added as pure lutein (+614% and +147%, respectively, pxa0<xa00.05). Conversely, lutein uptake was less effective when micellar lutein was from a meal containing spinach than from a meal containing its pure form (-55%, pxa0<xa00.05). In minipigs, postprandial lutein response was delayed with spinach but not significantly different after the two test meals.nnnCONCLUSIONnOpposite effects at the micellarization and intestinal cell uptake steps explain the lack of effect of spinach matrix on lutein bioavailability.

Phytoene and Phytofluene Isolated from a Tomato Extract are Readily Incorporated in Mixed Micelles and Absorbed by Caco‐2 cells, as Compared to Lycopene, and SR‐BI is Involved in their Cellular Uptake

SCOPEnAbsorption mechanisms of phytoene (PT) and phytofluene (PTF) are poorly known. The main objectives of the study are to measure their micellization and intestinal cell uptake efficiencies and to compare them to those of commonly consumed carotenoids. Other objectives are to assess the involvement of protein(s) in their cellular uptake and whether they compete with other carotenoids for micellization and cellular uptake.nnnMETHODS AND RESULTSnTomato-extract-purified PT and PTF, mainly present as cis-isomers, are much better incorporated in synthetic mixed micelles than pure all-trans lycopene. PT impairs lycopene micellization (-56%, Pxa0<xa00.05) while PT and PTF do not significantly affect the micellization of other carotenoids, and vice versa. At low concentration, Caco-2 PTF uptake is higher (Pxa0<xa00.05) than that of PT and lycopene (29%, 21%, and not detectable). SR-BI, but not CD36 neither NPC1L1, is involved in PT and PTF uptake. PT and PTF impair (pxa0<xa00.05) β-carotene uptake (-13 and -22%, respectively).nnnCONCLUSIONSnThe high bioaccessibility of PT and PTF can be partly explained by their high micellization efficiency, which is likely due to their natural cis isomerization and/or to their high molecular flexibility. SR-BI is involved in their cellular uptake, which can explain competitions with other carotenoids.

O46: Implication d’ABCB1 dans l’efflux de la vitamine D et de la 25-hydroxyvitamine D chez la souris

Introduction et but de l’etude La vitamine D3 (cholecalcife-rol) est une vitamine liposoluble appartenant a la famille des sterols. Elle agit principalement sur l’absorption phosphocalcique, mais on lui connait depuis peu des effets sur le systeme immunitaire, la fonction musculaire, ou dans la prevention du risque vasculaire. Face aux carences et aux subcarences tres frequentes dans de nombreux pays, ameliorer son statut et donc optimiser son absorption sont des priorites de sante publique. Les recherches de l’equipe se sont donc portees sur les mecanismes de l’absorption intestinale de la vitamine D. Des transporteurs de cholesterol ont ete impliques dans son captage par l’enterocyte. L’ ATP-binding cassette B1 (ABCB1 ou P-glycoproteine) est un transporteurs actif membranaire implique dans l’efflux de xenobiotiques. Il contribue aussi a l’expulsion du cholesterol hors de l’enterocyte vers la lumiere intestinale. Etant donne les similarites structurales existant entre cholesterol et vitamine D, nous avons emis l’hypothese qu’il pourrait egalement participer au phenomene d’efflux enterocytaire du cholecalciferol et de son metabolite de stockage, la 25-hydroxy-vitamine D (25(OH)D). Materiel et methodes Nous avons cultive sur inserts des cellules MDCK II transfectees stablement avec le gene ABCB1 humain. La capacite des cellules a effluer le cholecalciferol et la 25(OH)D a alors ete evaluee. Le statut en 25(OH)D a ensuite ete determine chez des souris deficientes en P-gp et des souris sauvages, ainsi que la participation d’ABCB1 a la reponse postpran-diale en cholecalciferol apres gavage lipidique. La capacite de l’intestin a expulser le cholecalciferol et la 25(OH)D a ete evaluee chez ces meme souris en perfusant le duodenum in situ et par la technique in vitro des chambres d’Ussing. Resultats et Analyse statistique Les donnees obtenues montrent que le transport du cholecalciferol et de la 25(OH)D est plus important chez les cellules MDCK-II qui surexpriment ABCB1 que chez les cellules parentales. In vivo , les souris deficientes en ABCB 1 possedent un statut plasmatique en 25(OH)D superieur a celui des souris sauvage. De plus, la quantite de cholecalciferol retrouvee dans le sang des souris deficientes en P-gp apres gavage est significativement plus elevee que celle retrouvee chez les souris controles. Enfin, les resultats des experiences de chambres d’Ussing montrent que l’efflux de la 25(OH)D est plus important au travers d’un fragment d’intestin appartenant aux souris sauvages par rapport aux souris deficientes en ABCB 1. Les analyses de perfusion in situ sont encore en cours. Conclusion Ces resultats, pris dans l’ensemble, montrent qu’ABCB1 pourrait controler le statut en vitamine D. ABCB1 contribue en effet a l’efflux apical de cholecalciferol neoabsorbe, mais egalement un efflux trans-epithelial de 25(OH)D. Ainsi, ABCB1 apparait comme une cible pharmacologique d’interet pour moduler le statut en vitamine D.