Enhancement of D-chiro-inositol transport across intestinal cells by alpha-Lactalbumin peptides.

OBJECTIVE: This study aims to characterize in vitro D-chiro-inositol intestinal absorption and identify factors able to improve its bioavailability. D-chiro-inositol, one of the natural occurring stereoisomer of myo-inositol, acts as a second messenger in insulin-regulated glucose metabolism in complementary mode with myo-inositol. Because of their insulin-mimetic activities and safety, both myo-inositol and D-chiro-inositol are often employed as supplements in insulin-resistance treatment. MATERIALS AND METHODS: Trans-epithelial passage of D-chiro-inositol was evaluated in the human intestinal Caco-2 cell line differentiated on filter, a widely established in vitro model to study intestinal absorption. D-chiro-inositol transport was assayed in a concentration range corresponding to an estimated in vivo concentration following oral supplementation. α-Lactalbumin peptides, obtained by in vitro simulated gastrointestinal digestion, were tested as possible modulators of the intestinal permeability of D-chiro-inositol. RESULTS: The absorption of this stereoisomer was relatively low and presumably due to passive diffusion, while it was greatly enhanced by the presence of α-Lactalbumin digest. α-Lactalbumin peptides induced an increase in paracellular permeability that was completely reversible, indicating lack of cytotoxicity. This effect involved temporary rearrangement of F-actin apical cytoskeleton and of the tight junction protein ZO-1. CONCLUSIONS: Although further studies are required to identify and characterize the most effective peptides, the ability of α-Lactalbumin digest to act as absorption enhancers may have very interesting and promising applications in the fields of nutritional supplements and pharmacology.

Effects of red wine on ochratoxin A toxicity in intestinal Caco-2/TC7 cells.

Ochratoxin A (OTA) is found in a variety of foods and beverages, including red wine. OTA was reported to be nephrotoxic, immunotoxic, hepatotoxic and a potential carcinogen, with yet uncharacterized mechanisms. Consumption of contaminated wines might contribute up to 13% of OTA daily human intake. Potentially chronic exposure has therefore raised public health concern. OTA toxicity in the presence of de-alcoholated red wine was investigated in human intestinal Caco-2/TC7 cells, differentiated on filter supports, by measuring tight junction (TJ) permeability, morphological alterations of TJ proteins and occurrence of apoptosis. Cells were treated with OTA, in the presence of de-alcoholated red wine, for 48h and the ability to recover from the effects of OTA was evaluated after 24h in complete medium. OTA treatment increased TJ permeability and caused intracellular redistribution of claudin-4. However, cells were able to restore permeability and correct localization of claudin-4 following 24h recovery. Conversely, in the presence of red wine, OTA produced faster and irreversible increase in TJ permeability, intracellular delocalization of claudin-4 and extensive apoptosis. Our results point at a possible synergy between OTA and some red wine components, such as polyphenols, in the induction of apoptotic cell death.

Biphasic effect of iron on human intestinal Caco-2 cells: early effect on tight junction permeability with delayed onset of oxidative cytotoxic damage.

Treatment of differentiated human intestinal Caco-2 cells with Fe(II) ascorbate altered tight junction permeability in a dose and time-dependent way for up to 3 hr of treatment Upon iron removal and transfer to complete culture medium, the effect was reversible up to 10 microM Fe(II), while at higher concentrations a late phase toxic effect was observed. Reduction of intracellular energy abolished the short term effect of iron on tight junction permeability without affecting its cellular uptake, suggesting that active processes, other than transport, were involved. The short term effect of iron the permeability of tight junctions did not appear to result from the generation of reactive oxygen species, as it was not prevented by antioxidant treatment under normal energy conditions. Conversely, the late phase effect leading to both apoptosis and necrosis during the 24 hr following iron removal could be reduced by antioxidant treatment and was exacebated by GSH depletion. Iron induced oxidative stress may therefore be responsible for membrane damage and cellular death occurring in the late phase. The reported effects of iron on intestinal tight junction permeability followed by more widespread cytotoxicity from oxidative events should be considered in light of the extensive use of iron supplementation in different phases of human life.

Iron and copper alter tight junction permeability in human intestinal Caco-2 cells by distinct mechanisms.

Human intestinal Caco-2 cells differentiated for 15-17 days on transparent filter inserts were treated for up to 3 h with 50 and 100 microM CuCl(2) or FeSO(4) in the AP compartment at pH 6.0. Trans-epithelial electrical resistance (TEER) showed a progressive decrease during the course of the experiment that was slower in cells treated with 50 microM CuCl(2) than in those treated with 100 microM CuCl(2). Both 50 and 100 microM FeSO(4) produced a similar decrease in TEER over time, tailing off after 120 min. F-actin localization by fluorescent phalloidin binding in control cells and in cells treated for 3 h with 50 microM CuCl(2) or FeSO(4) highlighted striking differences in the two treatments. Cu(II) led to an overall reduction in F-actin staining with extensive depolymerization in areas of the monolayer, in the absence of cellular loss. Conversely, Fe(II) treatment produced disorganization of F-actin and decreased staining of the perijunctional actin filaments. No changes in the localization and intensity of staining of the junctional proteins ZO1, occludin and E-cadherin were observed after treatment with 100 microM FeSO(4) in analogy with previous observations in Cu(II)-treated cells. The data presented suggest that different mechanisms are responsible for the changes to tight junction permeability produced by the two metals.

Copper uptake and intracellular distribution in the human intestinal Caco-2 cell line.

The apical uptake of 64CuCl2 was investigated in human differentiated intestinal Caco-2 cells grown on permeable supports. At pH 6.0 in the apical compartment, the uptake of copper was linear over the first 6 min and between 10 and 80 microM CuCl2 exhibited non-saturable transport kinetics. In addition, copper uptake was energy-independent, affected by the valency state of copper, preferring Cu(II) over Cu(I), and not influenced by high (10 mM) extracellular calcium. The intracellular distribution of copper was investigated by FPLC at different times of uptake ('pulse') and of 'chase'. Intracellular copper initially bound predominantly to low molecular weight components (i.e., glutathione). and subsequently shifted to higher molecular weight components such as metallothionein and Cu,Zn superoxide dismutase.

Copper treatment alters the permeability of tight junctions in cultured human intestinal Caco-2 cells.

The effects of copper on tight-junction permeability were investigated in human intestinal Caco-2 cells, monitoring transepithelial electrical resistance and transepithelial passage of mannitol. Apical treatment of Caco-2 cells with 10-100 microM CuCl(2) (up to 3 h) produced a time- and concentration-dependent increase in tight-junction permeability, reversible after 24 h in complete medium in the absence of added copper. These effects were not observed in cells treated with copper complexed to L-histidine [Cu(His)(2)]. The copper-induced increase in tight-junction permeability was affected by the pH of the apical medium, as was the apical uptake of (64)CuCl(2), both exhibiting a maximum at pH 6.0. Treatment with CuCl(2) produced a concentration-dependent reduction in the staining of F actin but not of the junctional proteins zonula occludens-1, occludin, and E-cadherin and produced ultrastructural alterations to microvilli and tight junctions that were not observed after treatment with up to 200 microM Cu(His)(2) for 3 h. Overall, these data point to an intracellular effect of copper on tight junctions, mediated by perturbations of the F actin cytoskeleton.

The efflux of lysine from the basolateral membrane of human cultured intestinal cells (Caco-2) occurs by different mechanisms depending on the extracellular availability of amino acids.

The efflux of the nutritionally essential amino acid, L-lysine from the basolateral (BL) membrane was characterized in human cultured intestinal cells (Caco-2) grown and differentiated on permeable filter supports. Cells were loaded by incubating with 3H-lysine from the apical (AP) side in the absence of sodium (substituted with choline) in the BL medium; under these conditions, cells accumulated lysine in the intracellular soluble pool to 10- to 20-fold the extracellular concentration. L-Lysine efflux in the BL medium was then followed, and initial rates of efflux were calculated under different experimental conditions. L-Lysine efflux exhibited a strong energy dependence. The presence of an inwardly directed gradient of sodium or lithium stimulated lysine efflux; ouabain reduced efflux in both sodium- and lithium-containing medium. When zwitterionic or cationic amino acids were added to the BL medium, L-lysine efflux was strongly stimulated. The most efficient trans-stimulating amino acids were L-leucine > L-methionine = L-ornithine = L-arginine. In the presence of trans-stimulating amino acids in the BL medium, L-lysine efflux exhibited energy independence and was not affected by the presence of a sodium gradient. In addition, the sensitivity, of efflux to N-ethylmaleimide was different in the absence or in the presence of amino acids in the BL medium. These results suggest that different mechanisms may operate in the BL efflux of L-lysine from human intestinal epithelial cells, depending on the extracellular availability of other amino acids, to guarantee optimal bioavailability of this essential amino acid both in the postprandial absorptive period and between meals.

The transport of lysine across monolayers of human cultured intestinal cells (Caco-2) depends on Na(+)-dependent and Na(+)-independent mechanisms on different plasma membrane domains.

To characterize the mechanisms involved in the intestinal absorption of the essential amino acid L-lysine from the diet, the transepithelial transport of L-lysine was studied in monolayers of cultured human intestinal cells (Caco-2) grown and differentiated on microporous membrane supports. L-lysine was transported mainly in the apical (AP) to basolateral (BL) direction and the BL to AP transport was approximately one order of magnitude lower at all concentrations tested. Non-linear regression analysis of the transport in the AP to BL and the BL to AP direction identified, in both cases, single saturable components with similar Km but different Vmax and a nonsaturable diffusional component. The AP to BL L-lysine transport was highly energy- and sodium-dependent and was unaffected by an unfavorable concentration gradient. Selective replacement of sodium ions in the AP or the BL compartment and determination of both AP to BL transport and the intracellular soluble lysine pool showed that uptake occurs via a sodium-independent mechanism, not significantly influenced by membrane potential, whereas efflux is a sodium-dependent process. Competition experiments showed that L-lysine uptake is highly stereospecific and is shared by cationic and large neutral amino acids. This study demonstrates the presence of a sodium-dependent mechanism of lysine efflux across the BL membrane of intestinal cells, which may be essential for lysine transport into the blood circulation. Overall, these results support the use of the Caco-2 cell model for studies of intestinal nutrient transport.