Jejunal villus absorption and paracellular tight junction permeability are major routes for early intestinal uptake of food-grade TiO2 particles: an in vivo and ex vivo study in mice.

BACKGROUND: Food-grade TiO2 (E171 in the EU) is widely used as a coloring agent in foodstuffs, including sweets. Chronic dietary exposure raises concerns for human health due to proinflammatory properties and the ability to induce and promote preneoplastic lesions in the rodent gut. Characterization of intestinal TiO2 uptake is essential for assessing the health risk in humans. We studied in vivo the gut absorption kinetics of TiO2 in fasted mice orally given a single dose (40 mg/kg) to assess the ability of intestinal apical surfaces to absorb particles when available without entrapment in the bolus. The epithelial translocation pathways were also identified ex vivo using intestinal loops in anesthetized mice. RESULTS: The absorption of TiO2 particles was analyzed in gut tissues by laser-reflective confocal microscopy and ICP-MS at 4 and 8 h following oral administration. A bimodal pattern was detected in the small intestine: TiO2 absorption peaked at 4 h in jejunal and ileal villi before returning to basal levels at 8 h, while being undetectable at 4 h but significantly present at 8 h in the jejunal Peyer's patches (PP). Lower absorption occurred in the colon, while TiO2 particles were clearly detectable by confocal microscopy in the blood at 4 and 8 h after treatment. Ex vivo, jejunal loops were exposed to the food additive in the presence and absence of pharmacological inhibitors of paracellular tight junction (TJ) permeability or of transcellular (endocytic) passage. Thirty minutes after E171 addition, TiO2 absorption by the jejunal villi was decreased by 66% (p < 0.001 vs. control) in the presence of the paracellular permeability blocker triaminopyrimidine; the other inhibitors had no significant effect. Substantial absorption through a goblet cell (GC)-associated pathway, insensitive to TJ blockade, was also detected. CONCLUSIONS: After a single E171 dose in mice, early intestinal uptake of TiO2 particles mainly occurred through the villi of the small intestine, which, in contrast to the PP, represent the main absorption surface in the small intestine. A GC-associated passage and passive diffusion through paracellular TJ spaces between enterocytes appeared to be major absorption routes for transepithelial uptake of dietary TiO2.

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.

Proprotein convertase subtilisin kexin type 9 null mice are protected from postprandial triglyceridemia.

OBJECTIVES: Proprotein convertase subtilisin kexin type 9 (PCSK9) is a natural inhibitor of the low-density lipoprotein receptor, and its deficiency in humans results in low plasma LDL-cholesterol and protection against cardiovascular disease. We explored whether PCSK9 expression impacts postprandial triglyceridemia, another important cardiovascular risk factor. METHODS AND RESULTS: Real-time PCR and confocal microscopy were used to show that PCSK9 is expressed throughout the entire small intestine and in human enterocytes. On olive oil gavage, PCSK9-deficient mice showed a dramatically decreased postprandial triglyceridemia compared with their wild-type littermates. Lymph analysis revealed that intestinal TG output is not quantitatively modified by PCSK9 deletion. However, PCSK9-/- mice present with a significant reduction of lymphatic apoB secretion compared to PCSK9+/+ mice. Modulating PCSK9 expression in polarized CaCo-2 cells confirmed the relationship between PCSK9 and apoB secretion; PCSK9-/- mice consistently secrete larger TG-rich lipoprotein than wild-type littermates. Finally, kinetic studies showed that PCSK9-deficient mice have an increased ability to clear chylomicrons compared to wild-type littermates. CONCLUSION: These findings indicate that in addition to its effect on LDL-cholesterol, PCSK9 deficiency might protect against cardiovascular disease by reducing postprandial triglyceridemia.

Lycopene absorption in human intestinal cells and in mice involves scavenger receptor class B type I but not Niemann-Pick C1-like 1.

Cholesterol membrane transporters scavenger receptor class B type I (SR-BI) and (cluster determinant 36) are involved in intestinal uptake of lutein and beta-carotene, 2 of the 3 main carotenoids of the human diet. The aim of this work was therefore to determine whether SR-BI and NPC1L1 (Niemann-Pick C1-like 1), another cholesterol transporter, are implicated in absorption of lycopene, the 3rd main carotenoid of the human diet. Anti-human SR-BI antibody and block lipid transport 1 (BLT1) (a chemical inhibitor of lipid transport by SR-BI) impaired up to 60% (all-E) and (5Z)-lycopene uptake (P < 0.05) by Caco-2 cell monolayers, which were used as a model of human intestinal epithelium. The involvement of SR-BI in lycopene absorption in vivo was then verified by comparing plasma lycopene concentrations in wild-type and SR-BI transgenic mice that were fed a diet enriched with 0.25 g/kg (all-E)-lycopene for 1 mo. Plasma lycopene concentrations were approximately 10-fold higher (P < 0.001) in mice overexpressing SR-BI in the intestine than in wild-type mice, confirming the involvement of SR-BI in lycopene absorption. Further experiments showed that (all-E)-lycopene did not affect SR-BI mRNA levels in Caco-2 cells or mouse intestine. In contrast to SR-BI, neither anti-human NPC1L1 antibody nor ezetimibe, used as inhibitors of lycopene uptake via NPC1L1, significantly impaired (all-E) or (5Z)-lycopene uptake by Caco-2 monolayers. Thus, the present data show that lycopene absorption is, at least in part, mediated by SR-BI but not by NPC1L1.

Gliotoxin from Aspergillus fumigatus affects phagocytosis and the organization of the actin cytoskeleton by distinct signalling pathways in human neutrophils.

Gliotoxin is a mycotoxin having a considerable number of immuno-suppressive actions and is produced by several moulds such as Aspergillus fumigatus. In this study, we investigated its toxic effects on human neutrophils at concentrations corresponding to those found in the blood of patients with invasive aspergillosis. Incubation of the cells for 10min with 30-100ng/ml of gliotoxin inhibited phagocytosis of either zymosan or serum-opsonized zymosan without affecting superoxide production or the exocytosis of specific and azurophil granules. Gliotoxin also induced a significant re-organization of the actin cytoskeleton which collapsed around the nucleus leading to cell shrinkage and the disappearance of filopodia. This gliotoxin-induced actin phenotype was reversed by the cAMP antagonist Rp-cAMP and mimicked by pCPT-cAMP indicating that it probably resulted from the deregulation of intracellular cAMP homeostasis as previously described for gliotoxin-induced apoptosis. By contrast, gliotoxin-induced inhibition of phagocytosis was not reversed by Rp-cAMP but by arachidonic acid, another member of a known signalling pathway affected by the toxin. This suggests that gliotoxin can affect circulating neutrophils and favour the dissemination of A. fumigatus by inhibiting phagocytosis and the consequent killing of conidia.

Food-grade TiO2 impairs intestinal and systemic immune homeostasis, initiates preneoplastic lesions and promotes aberrant crypt development in the rat colon.

Food-grade titanium dioxide (TiO2) containing a nanoscale particle fraction (TiO2-NPs) is approved as a white pigment (E171 in Europe) in common foodstuffs, including confectionary. There are growing concerns that daily oral TiO2-NP intake is associated with an increased risk of chronic intestinal inflammation and carcinogenesis. In rats orally exposed for one week to E171 at human relevant levels, titanium was detected in the immune cells of Peyer's patches (PP) as observed with the TiO2-NP model NM-105. Dendritic cell frequency increased in PP regardless of the TiO2 treatment, while regulatory T cells involved in dampening inflammatory responses decreased with E171 only, an effect still observed after 100 days of treatment. In all TiO2-treated rats, stimulation of immune cells isolated from PP showed a decrease in Thelper (Th)-1 IFN-γ secretion, while splenic Th1/Th17 inflammatory responses sharply increased. E171 or NM-105 for one week did not initiate intestinal inflammation, while a 100-day E171 treatment promoted colon microinflammation and initiated preneoplastic lesions while also fostering the growth of aberrant crypt foci in a chemically induced carcinogenesis model. These data should be considered for risk assessments of the susceptibility to Th17-driven autoimmune diseases and to colorectal cancer in humans exposed to TiO2 from dietary sources.

Exposure to dietary lipid leads to rapid production of cytosolic lipid droplets near the brush border membrane.

BACKGROUND: Intestinal absorption of dietary lipids involves their hydrolysis in the lumen of proximal intestine as well as uptake, intracellular transport and re-assembly of hydrolyzed lipids in enterocytes, leading to the formation and secretion of the lipoproteins chylomicrons and HDL. In this study, we examined the potential involvement of cytosolic lipid droplets (CLD) whose function in the process of lipid absorption is poorly understood. METHODS: Intestinal lipid absorption was studied in mouse after gavage. Three populations of CLD were purified by density ultracentrifugations, as well as the brush border membranes, which were analyzed by western-blots. Immunofluorescent localization of membranes transporters or metabolic enzymes, as well as kinetics of CLD production, were also studied in intestine or Caco-2 cells. RESULTS: We isolated three populations of CLD (ranging from 15 to 1000 nm) which showed differential expression of the major lipid transporters scavenger receptor BI (SR-BI), cluster of differentiation 36 (CD-36), Niemann Pick C-like 1 (NPC1L1), and the ATP-binding cassette transporters ABCG5/G8 but also caveolin 2 and fatty acid binding proteins. The enzyme monoacylglycerol acyltransferase 2 (MGAT2) was identified in the brush border membrane (BBM) in addition to the endoplasmic reticulum, suggesting local synthesis of triglycerides and CLD at both places. CONCLUSIONS: We show a very fast production of CLD by enterocytes associated with a transfer of apical constituents as lipid transporters. Our findings suggest that following their uptake by enterocytes, lipids can be partially metabolized at the BBM and packaged into CLD for their transportation to the ER.

Annexin 1 is secreted in situ during ulcerative colitis in humans.

Although annexin l exerts extracellular anti-inflammatory properties, little is known about its release in inflammatory diseases. Here, we characterized annexin 1 secretion in ulcerative colitis (UC) patients. Annexin 1 was detected by immunoblotting, in tissue homogenates and supernatants of colonic biopsies incubated in culture media, and in luminal colonic perfusates of UC patients. Annexin 1 was released by inflamed colonic biopsies from patients having severe UC but not by biopsies from healthy colon of the same patient or by biopsies from non-UC patients or from patients with slight or moderate UC. Annexin 1 was detected in luminal colonic perfusates of patients having moderate or slight UC but not in perfusates from control patients. The level of annexin 1 expression and secretion was unrelated to long-term glucocorticoid treatment, but annexin 1 secretion in perfusates was induced, in some patients, by short-term glucocorticoid exposure. These results show that annexin 1 is secreted endogenously in the colon of patients with UC. This secretion, which occurs both in vitro and in vivo, depends on the severity of inflammation. Given the anti-inflammatory effects of annexin 1, this protein may serve to down-regulate the inflammatory response in the course of inflammatory bowel disease.

Mediation of annexin 1 secretion by a probenecid-sensitive ABC-transporter in rat inflamed mucosa.

Annexin 1 is secreted by mammalian cells but lacks a leader signal sequence necessary to lead it to the classical secretory pathway via the endoplasmic reticulum. The mechanisms involved in the secretion of leaderless proteins remain uncertain. It has been suggested to involve membrane translocation via an ABC-transporter (ATP binding cassette). Using cultured inflamed mucosa from rectocolitis induced in rats, we studied if annexin 1 secretion followed the two main characteristics of ABC-transporter substrates: dependency on ATP hydrolysis and competitive inhibition by several other ABC-transporter substrates. Annexin 1 secretion is inhibited in a dose-dependent manner by two ATPase inhibitors. The inhibition reached 63.2+/-3.2%, 66.1+/-3.73% and 88.6+/-1.4% in the presence of 2mM vanadate, 0.5 and 1mM pervanadate, respectively. The efflux of calcein, a known ABC-transporter substrate, is similarly inhibited by 69.4+/-2.8% in the presence of 1mM pervanadate. Probenecid, an inhibitor of several ABC-transporters of the subfamilly ABCC or MRP (multidrug resistant associated protein), also inhibited annexin 1 secretion in a dose-dependent manner. As compared to control, 10mM probenecid reduced annexin 1 secretion by 72+/-20% and 20mM by 95.0+/-9%. By contrast, annexin 1 secretion is not blocked by other inhibitors of MRP1 (indomethacin, MK571), MRP2 (ochratoxin A1 or MK571), MRP5 (trequinsin or sulfinpyrazone) or by verapamil, cyclosporin A or glyburide. Taken together, our results show that annexin 1 secretion appears to share the efflux properties of ABC-transporter substrates.

Lipidomic and spatio-temporal imaging of fat by mass spectrometry in mice duodenum during lipid digestion.

Intestinal absorption of dietary fat is a complex process mediated by enterocytes leading to lipid assembly and secretion of circulating lipoproteins as chylomicrons, vLDL and intestinal HDL (iHDL). Understanding lipid digestion is of importance knowing the correlation between excessive fat absorption and atherosclerosis. By using time-of-flight secondary ion mass spectrometry (TOF-SIMS), we illustrated a spatio-temporal localization of fat in mice duodenum, at different times of digestion after a lipid gavage, for the first time. Fatty acids progressively increased in enterocytes as well as taurocholic acid, secreted by bile and engaged in the entero-hepatic re-absorption cycle. Cytosolic lipid droplets (CLD) from enterocytes were originally purified separating chylomicron-like, intermediate droplets and smaller HDL-like. A lipidomic quantification revealed their contents in triglycerides, free and esterified cholesterol, phosphatidylcholine, sphingomyelin and ceramides but also in free fatty acids, mono- and di-acylglycerols. An acyl-transferase activity was identified and the enzyme monoacylglycerol acyl transferase 2 (MGAT2) was immunodetected in all CLD. The largest droplets was also shown to contain the microsomal triglyceride transfer protein (MTTP), the acyl-coenzyme A-cholesterol acyltransferases (ACAT) 1 and 2, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL). This highlights the fact that during the digestion of fats, enterocyte CLD contain some enzymes involved in the different stages of the metabolism of diet fatty acids and cholesterol, in anticipation of the crucial work of endoplasmic reticulum in the process. The data further underlines the dual role of chylomicrons and iHDL in fat digestion which should help to efficiently complement lipid-lowering therapy.

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.

Lipid rafts: dream or reality for cholesterol transporters?

As a key constituent of the cell membranes, cholesterol is an endogenous component of mammalian cells of primary importance, and is thus subjected to highly regulated homeostasis at the cellular level as well as at the level of the whole body. This regulation requires adapted mechanisms favoring the handling of cholesterol in aqueous compartments, as well as its transfer into or out of membranes, involving membrane proteins. A membrane exhibits functional properties largely depending on its lipid composition and on its structural organization, which very often involves cholesterol-rich microdomains. Then there is the appealing possibility that cholesterol may regulate its own transmembrane transport at a purely functional level, independently of any transcriptional regulation based on cholesterol-sensitive nuclear factors controling the expression level of lipid transport proteins. Indeed, the main cholesterol "transporters" presently believed to mediate for instance the intestinal absorption of cholesterol, that are SR-BI, NPC1L1, ABCA1, ABCG1, ABCG5/G8 and even P-glycoprotein, all present privileged functional relationships with membrane cholesterol-containing microdomains. In particular, they all more or less clearly induce membrane disorganization, supposed to facilitate cholesterol exchanges with the close aqueous medium. The actual lipid substrates handled by these transporters are not yet unambiguously determined, but they likely concern the components of membrane microdomains. Conversely, raft alterations may provide specific modulations of the transporter activities, as well as they can induce indirect effects via local perturbations of the membrane. Finally, these cholesterol transporters undergo regulated intracellular trafficking, with presumably some relationships to rafts which remain to be clarified.

Accelerated lipid absorption in mice overexpressing intestinal SR-BI.

Dietary cholesterol absorption contributes to a large part of the circulating cholesterol. However, the mechanism of sterol intestinal uptake is not clearly elucidated. Scavenger receptor class B type I (SR-BI), major component in the control of cholesterol homeostasis, is expressed in the intestine, but its role in this organ remains unclear. We have generated transgenic mice overexpressing SR-BI primarily in the intestine by using the mouse SR-BI gene under the control of intestinal specific "apoC-III enhancer coupled with apoA-IV promoter." We found SR-BI overexpression with respect to the natural protein along the intestine and at the top of the villosities. After a meal containing [(14)C]cholesterol and [(3)H]triolein, SR-BI transgenic mice presented a rise in intestinal absorption of both lipids that was not due to a defect in chylomicron clearance nor to a change in the bile flow or the bile acid content. Nevertheless, SR-BI transgenic mice showed a decrease of total cholesterol but an increase of triglyceride content in plasma without any change in the high density lipoprotein apoA-I level. Thus, we described for the first time a functional role in vivo for SR-BI in cholesterol but also in triglyceride intestinal absorption.