Aurélie Goncalves

Vitamin D intestinal absorption is not a simple passive diffusion: Evidences for involvement of cholesterol transporters

SCOPE It is assumed that vitamin D is absorbed by passive diffusion. However, since cholecalciferol (vitamin D(3) ) and cholesterol display similar structures, we hypothesized that common absorption pathways may exist. METHODS AND RESULTS Cholecalciferol apical transport was first examined in human Caco-2 and transfected Human embryonic kidney (HEK) cells. Cholecalciferol uptake was then valuated ex vivo and in vivo, using either wild-type mice, mice overexpressing Scavenger Receptor class B type I (SR-BI) at the intestinal level or mice treated or not with ezetimibe. Cholecalciferol uptake was concentration-, temperature- and direction-dependent, and was significantly impaired by a co-incubation with cholesterol or tocopherol in Caco-2 cells. Moreover Block Lipid Transport-1 (SR-BI inhibitor) and ezetimibe glucuronide (Niemann-Pick C1 Like 1 inhibitor) significantly decreased cholecalciferol transport. Transfection of HEK cells with SR-BI, Cluster Determinant 36 and Niemann-Pick C1 Like 1 significantly enhanced vitamin D uptake, which was significantly decreased by the addition of Block Lipid Transport-1, sulfo-N-succinimidyl oleate (Cluster Determinant 36 inhibitor) or ezetimibe glucuronide, respectively. Similar results were obtained in mouse intestinal explants. In vivo, cholecalciferol uptake in proximal intestinal fragments was 60% higher in mice overexpressing SR-BI than in wild-type mice (p<0.05), while ezetimibe effect remained non-significant. CONCLUSION These data show for the first time that vitamin D intestinal absorption is not passive only but involves, at least partly, some cholesterol transporters.

CD36 and SR-BI Are Involved in Cellular Uptake of Provitamin A Carotenoids by Caco-2 and HEK Cells, and Some of Their Genetic Variants Are Associated with Plasma Concentrations of These Micronutrients in Humans

Scavenger receptor class B type I (SR-BI) and cluster determinant 36 (CD36) have been involved in cellular uptake of some provitamin A carotenoids. However, data are incomplete (e.g., there are no data on α-carotene), and it is not known whether genetic variants in their encoding genes can affect provitamin A carotenoid status. The objectives were 1) to assess the involvement of these scavenger receptors in cellular uptake of the main provitamin A carotenoids (i.e., β-carotene, α-carotene, and β-cryptoxanthin) as well as that of preformed vitamin A (i.e., retinol) and 2) to investigate the contribution of genetic variations in genes encoding these proteins to interindividual variations in plasma concentrations of provitamin A carotenoids. The involvement of SR-BI and CD36 in carotenoids and retinol cellular uptake was investigated in Caco-2 and human embryonic kidney (HEK) cell lines. The involvement of scavenger receptor class B type I (SCARB1) and CD36 genetic variants on plasma concentrations of provitamin A carotenoids was assessed by association studies in 3 independent populations. Cell experiments suggested the involvement of both proteins in cellular uptake of provitamin A carotenoids but not in that of retinol. Association studies showed that several plasma provitamin A carotenoid concentrations were significantly different (P < 0.0083) between participants who bore different genotypes at single nucleotide polymorphisms and haplotypes in CD36 and SCARB1. In conclusion, SR-BI and CD36 are involved in cellular uptake of provitamin A carotenoids, and genetic variations in their encoding genes may modulate plasma concentrations of provitamin A carotenoids at a population level.

Fat-soluble vitamin intestinal absorption: Absorption sites in the intestine and interactions for absorption

The interactions occurring at the intestinal level between the fat-soluble vitamins A, D, E and K (FSVs) are poorly documented. We first determined each FSV absorption profile along the duodenal-colonic axis of mouse intestine to clarify their respective absorption sites. We then investigated the interactions between FSVs during their uptake by Caco-2 cells. Our data show that vitamin A was mostly absorbed in the mouse proximal intestine, while vitamin D was absorbed in the median intestine, and vitamin E and K in the distal intestine. Significant competitive interactions for uptake were then elucidated among vitamin D, E and K, supporting the hypothesis of common absorption pathways. Vitamin A also significantly decreased the uptake of the other FSVs but, conversely, its uptake was not impaired by vitamins D and K and even promoted by vitamin E. These results should be taken into account, especially for supplement formulation, to optimise FSV absorption.

Phytosterols can impair vitamin D intestinal absorption in vitro and in mice

SCOPE Adequate vitamin D status is necessary and beneficial for health, although deficiency and insufficiency are very common. As cholecalciferol (vitamin D(3) ) structure is close to cholesterol structure, we hypothesized that phytosterols, frequently used to decrease cholesterol, intestinal absorption and consequently to reduce hypercholesterolemia, may also interact with cholecalciferol absorption. METHODS AND RESULTS β-Sitosterol effect on cholecalciferol postprandial response was first assessed in mice. We then evaluated the effect of different sterols on (i) cholecalciferol micellar incorporation, (ii) cholecalciferol apical uptake and (iii) basolateral efflux in vitro or ex vivo. In mice, cholecalciferol bioavailability was 15-fold lower in the presence of β-sitosterol (p<0.05). This can partly be explained by the fact that phytosterols significantly impaired cholecalciferol incorporation into mixed micelles (from -16 to -36% depending on sterol micellar composition). This can also be due to the fact that in Caco-2 cells and mouse intestinal explants, phytosterols significantly lowered cholecalciferol apical uptake (from -13 to -39%). Conversely, phytosterols had no effect on cholecalciferol secretion at the basolateral side of Caco-2 cells. CONCLUSION The present data suggest for the first time that phytosterols can interact with vitamin D(3) intestinal absorption. This interaction can be explained by a competition for micellar incorporation and for apical uptake.

Fatty acids affect micellar properties and modulate vitamin D uptake and basolateral efflux in Caco-2 cells ☆ ☆☆

We have recently shown that vitamin D3 (cholecalciferol) absorption is not a simple passive diffusion but involves cholesterol transporters. As free fatty acids (FAs) modulate cholesterol intestinal absorption and metabolism, we hypothesized that FAs may also interact with vitamin D absorption. Effects of FAs were evaluated at different levels of cholecalciferol intestinal absorption. First, the physicochemical properties of micelles formed with different FAs were analyzed. The micelles were then administered to human Caco-2 cells in culture to evaluate FA effects on (i) cholecalciferol uptake and basolateral efflux and (ii) the regulation of genes coding proteins involved in lipid absorption process. Micellar electric charge was correlated with both FA chain length and degree of unsaturation. Long-chain FAs at 500 μM in mixed micelles decreased cholecalciferol uptake in Caco-2 cells. This decrease was annihilated as soon as the long-chain FAs were mixed with other FAs. Oleic acid significantly improved cholecalciferol basolateral efflux compared to other FAs. These results were partly explained by a modulation of genes coding for lipid transport proteins such as Niemann-pick C1-like 1 and scavenger receptor class B type I. The data reported here show for the first time that FAs can interact with cholecalciferol intestinal absorption at different key steps of the absorption process. Cholecalciferol intestinal absorption may thus be optimized according to oil FA composition.

Respective contributions of intestinal Niemann-Pick C1-like 1 and scavenger receptor class B type I to cholesterol and tocopherol uptake: in vivo v. in vitro studies.

The intestinal absorption of cholesterol and lipid micronutrients such as vitamin E has been shown to share some common pathways. The present study aims to further compare the uptake of cholesterol ([3H]cholesterol v. 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3-ol (NBD-cholesterol)) and tocopherol in Caco-2 TC-7 cells and in mouse intestine, with special focus on the respective roles of scavenger receptor class B type I (SR-BI) and Niemann-Pick C1-like 1 (NPC1L1). Conversely to NBD-cholesterol, the uptakes of [3H]cholesterol and tocopherol by Caco-2 cells were impaired by both block lipid transport-1 and ezetimibe, which inhibit SR-BI and NPC1L1, respectively. These inhibitions occurred only when cholesterol or tocopherol was delivered to cells included in micelles that contained biliary acid and at least oleic acid as a lipid. In vivo, after 2 h of digestion in mice, the uptake of the two cholesterol analogues and of tocopherol all showed distinct patterns along the duodenum-jejunum axis. [3H]Cholesterol uptake, which correlated closely to NPC1L1 mRNA expression in wild-type (wt) mice, was strongly inhibited by ezetimibe. Intestinal SR-BI overexpression did not change NPC1L1 expression and led to a significant increase in [3H]cholesterol uptake in the distal jejunum. Conversely, neither ezetimibe treatment nor SR-BI overexpression had an effect on NBD-cholesterol uptake. However, in contrast with SR-BI mRNA expression, tocopherol absorption increased strongly up to the distal jejunum in wt mice where it was specifically inhibited by ezetimibe, and was increased in the proximal intestine of intestinal SR-BI-overexpressing mice. Thus, cholesterol and tocopherol uptakes share common pathways in cell culture models, but display different in vivo absorption patterns associated with distinct contributions of SR-BI and NPC1L1.

The Postprandial Chylomicron Triacylglycerol Response to Dietary Fat in Healthy Male Adults Is Significantly Explained by a Combination of Single Nucleotide Polymorphisms in Genes Involved in Triacylglycerol Metabolism

CONTEXT The postprandial chylomicron (CM) triacylglycerol (TG) response to dietary fat, which is positively associated with atherosclerosis and cardiovascular disease risk, displays a high interindividual variability. This is assumed to be due, at least partly, to polymorphisms in genes involved in lipid metabolism. Existing studies have focused on single nucleotide polymorphisms (SNPs), resulting in only a low explained variability. OBJECTIVE We aimed to identify a combination of SNPs associated with the postprandial CM TG response. PARTICIPANTS AND METHODS Thirty-three healthy male volunteers were subjected to 4 standardized fat tolerance test meals (to correct for intraindividual variability) and genotyped using whole-genome microarrays. The plasma CM TG concentration was measured at regular interval times after each meal. The association of SNPs in or near candidate genes (126 genes representing 6225 SNPs) with the postprandial CM TG concentration (0-8 h areas under the curve averaged for the 4 test meals) was assessed by partial least squares regression, a multivariate statistical approach. RESULTS Data obtained allowed us to generate a validated significant model (P = 1.3 × 10(-7)) that included 42 SNPs in 23 genes (ABCA1, APOA1, APOA5, APOB, BET1, CD36, COBLL1, ELOVL5, FRMD5, GPAM, INSIG2, IRS1, LDLR, LIPC, LPL, LYPLAL1, MC4R, NAT2, PARK2, SLC27A5, SLC27A6, TCF7L2, and ZNF664) and explained 88% of the variance. In 39 of these SNPs, univariate analysis showed that subjects with different genotypes exhibited significantly different (q < .05) postprandial CM TG responses. CONCLUSIONS Using a multivariate approach, we report a combination of SNPs that explains a significant part of the variability in the postprandial CM TG response.

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.

Cluster-determinant 36 (CD36) impacts on vitamin E postprandial response.

SCOPE A single nucleotide polymorphism in the cluster determinant 36 (CD36) gene has recently been associated with plasma α-tocopherol concentration, suggesting a possible role of this protein in vitamin E intestinal absorption or tissue uptake. METHODS AND RESULTS To investigate the involvement of CD36 in vitamin E transport, we first evaluated the effect of CD36 on α- and γ-tocopherol transmembrane uptake and efflux using transfected HEK cells. γ-Tocopherol postprandial response was then assessed in CD36-deficient mice compared with wild-type mice, after the mice had been fully characterized for their α-tocopherol, vitamin A and lipid plasma, and tissue contents. Both α- and γ-tocopherol uptake was significantly increased in cells overexpressing CD36 compared with control cells. Compared with wild-type mice, CD36-deficient mice displayed a significantly decreased cholesterol hepatic concentration, and males exhibited significantly higher triacylglycerol contents in liver, brain, heart, and muscle. Although tissue α-tocopherol concentration after adjustment for lipid content was not modified, γ-tocopherol postprandial response was significantly increased in CD36-deficient mice compared with controls, likely reflecting the postprandial hypertriglyceridemia observed in these mice. CONCLUSION Our findings show for the first time that CD36 participates-directly or indirectly-in vitamin E uptake, and that CD36 effect on postprandial lipid metabolism in turn modifies vitamin E postprandial response.

β-Lactoglobulin as a Vector for β-Carotene Food Fortification

Food fortification is a strategy to overcome vitamin A deficiency in developing countries. Our aim was to investigate the involvement of the bovine milk protein β-lactoglobulin (β-Lg), a potential retinoid carrier, in vitamin A absorption. In vivo experiments were conducted by force-feeding mice with retinol or β-carotene associated with either β-Lg or oil-in-water emulsion, with subsequent determination of both vitamin A intestinal mucosa and plasma contents. Caco-2 cells were then used to investigate the mechanisms of vitamin A uptake when delivered by either β-Lg or mixed micelles. We showed that β-Lg was as efficient as emulsion to promote β-carotene, but not retinol, absorption in mice. Similar results were obtained in vitro. Interestingly, an inhibitor of the Scavenger Receptor Class B Type I significantly decreased the uptake of micellar β-carotene but not that of β-carotene bound to β-Lg. Overall, we showed that β-Lg would be a good vector for β-carotene food fortification.