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.

Relationship between N-terminal pro-B-type natriuretic peptide (Nt-proBNP) and cardiac cycle efficiency in cardiac surgery.

N-terminal pro-B-type natriuretic peptide (Nt-proBNP) is a peptide released from myocardium in response to ventricular wall stress and dysfunction. Nt-proBNP plasma levels are elevated in a variety of cardiovascular disorders and are largely used for diagnosis and treatment of cardiac diseases. The cardiac cycle efficiency (CCE) is a haemodynamic variable that represents the left ventricle wall stress and the heart's effort to maintain an adequate blood flow to tissues. We investigated the relationship between Nt-proBNP and CCE values in patients undergoing cardiac surgery. Twenty-five patients undergoing aortic valve replacement were studied. Plasma Nt-proBNP concentrations were performed by electroluminescence immunoassay before starting surgery (t0), at the end of extracorporeal circulation (t1) and 3 hours after surgery (t2). CCE measurements were acquired at the same intervals and correlations with Nt-proBNP levels were calculated. Nt-proBNP plasma concentration was 1430 ± 341 pg/ml at t0, peaked significantly at t1 (2129 ± 561 pg/ml, p<0.001) and moderately decreased at t2 (1924 ± 477 pg/ml, p<0.05). A direct correlation between Nt-proBNP measured at t0 and t1 was found (r=0.91, p<0.001). Overall, a negative correlation between CCE and proBNP values was found (r=-0.89, p<0.01). Correlations between CCE and Nt-proBNP were -0.91, -0.83 and -0.88, at t0, t1 and t2, respectively (p<0.01). Nt-proBNP levels reflect the severity of left ventricle dysfunction in patients undergoing cardiac surgery. CCE correlated well with serum Nt-proBNP levels and seems to be a useful variable to monitor the left ventricular stress and recovery during the various phases of surgery.

Intracellular zinc stores protect the intestinal epithelium from Ochratoxin A toxicity.

Ochratoxin A (OTA) is a harmful mycotoxin frequently contaminating foods, feeds and beverages. OTA was reported to be nephrotoxic, immunotoxic, hepatotoxic and a potential carcinogen, with yet poorly characterized mechanisms. Although intestinal cells are relatively resistant to high concentrations of OTA, interaction with other dietary factors or specific nutritional conditions may increase OTA toxicity to the intestinal mucosa. The role of intracellular zinc stores in protecting the integrity of intestinal mucosa has been investigated in human Caco-2/TC7 cells challenged with OTA. Zinc depletion of cells incubated with TPEN, a specific zinc chelator, caused an increase of tight junction permeability in OTA treated cells, accompanied by increased apoptosis. These effects were fully reverted by zinc supplementation during TPEN treatment, showing a specific role for this micronutrient in enterocyte defence mechanisms from OTA toxicity. A complex perturbation of zinc homeostasis was also demonstrated by analyzing the expression of genes coding for proteins involved in cellular zinc. In particular, zinc-dependent up-regulation of the metallothionein gene MT2A upon OTA treatment may indicate that the mycotoxin acts through generation of redox imbalance and that zinc deprivation reduces the intracellular defence mechanisms against noxious insults.

Beneficial or detrimental effects of carotenoids contained in food: cell culture models.

Epidemiological studies have suggested a correlation between consumption of carotenoid-rich food and incidence of chronic diseases. In this review chemical structure, bioavailability and mechanisms of action of carotenoids most represented in human diet, mainly beta-carotene and lycopene, are reported, with focus on results obtained with cells in culture.

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.

The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics.

The human intestinal Caco-2 cell line has been extensively used over the last twenty years as a model of the intestinal barrier. The parental cell line, originally obtained from a human colon adenocarcinoma, undergoes in culture a process of spontaneous differentiation that leads to the formation of a monolayer of cells, expressing several morphological and functional characteristics of the mature enterocyte. Culture-related conditions were shown to influence the expression of these characteristics, in part due to the intrinsic heterogeneity of the parental cell line, leading to selection of sub-populations of cells becoming prominent in the culture. In addition, several clonal cell lines have been isolated from the parental line, exhibiting in general a more homogeneous expression of differentiation traits, while not always expressing all characteristics of the parental line. Culture-related conditions, as well as the different Caco-2 cell lines utilized in different laboratories, often make it extremely difficult to compare results in the literature. This review is aimed at summarizing recent, or previously unreviewed, data from the literature on the effects of culture-related factors and the influence of line sub-types (parental vs. different clonal lines) on the expression of differentiation traits important for the use of Caco-2 cells as a model of the absorptive and defensive properties of the intestinal mucosa. Since the use of Caco-2 cells has grown exponentially in recent years, it is particularly important to highlight these methodological aspects in order to promote the standardization and optimisation of this intestinal model.

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.

Transport of the antibacterial agent oxazolidin-2-one and derivatives across intestinal (Caco-2) and renal (MDCK) epithelial cell lines.

The transepithelial passage of the orally bioavailable antibacterial agent oxazolidin-2-one (OXa) and 10 derivatives has been studied with human intestinal (Caco-2) and canine renal (MDCK) cell lines grown on polycarbonate filters. The transepithelial passage was assayed in the apical-to-basolateral (AP-to-BL) direction and in the opposite direction (BL to AP) in both cell lines. The observed passage rates of OXa were similar in both directions in the two cell lines, suggesting passive diffusion. This was further confirmed by the fact that transport kinetics were linear as a function of initial concentration. The rates of AP-to-BL passage of OXa and seven of the derivatives in both cell lines were linearly related to lipophilicity, whether expressed as high-passage liquid chromatography retention time or as the logarithm of the n-octanol-water partition coefficient (log P). These data suggest that the lipophilicity of OXa is important for its observed bioavailability after oral administration. Interestingly, three of the derivatives exhibited a higher passage rate than predicted by lipophilicity. Further studies indicated that this transport was saturable, similar in the two directions, and not affected by energy depletion, suggesting the presence of an additional carrier-mediated facilitated-transport mechanism.

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.

D-cycloserine uses an active transport mechanism in the human intestinal cell line Caco 2.

In a previous study we have shown that cultured epithelial cell lines can be used to measure the transepithelial passage of antimicrobial agents across the intestine and to obtain information on the mechanisms of transport utilized and predict the bioavailability of the antimicrobial agents after oral administration. In particular, among the drugs investigated, D-cycloserine had been shown to be transported in a polarized manner only in the intestinal cells. In the present work, further characterization of the transport of D-cycloserine in the human intestinal cell line Caco 2 has shown that this occurs in the apical-to-basolateral direction by an active mechanism which is energy dependent but only partially sodium dependent. Competition studies have also indicated that the transport of D-cycloserine occurs via a carrier for imino acids, amino acids with aliphatic side chains (L-Ala, D-Ala, and beta Ala), and L-Trp, L-Tyr, L-Cys, and alpha-amino isobutyric acid. This system may correspond to a proton-dependent system for L-proline and beta-alanine recently described for Caco 2 cells. In contrast with the cephalosporins, which are taken up by the Caco 2 cells via a dipeptide carrier, D-cycloserine transport cannot be inhibited by either cephalexin (a member of the class of cephalosporins) or dipeptides.

Epithelial cells in culture as a model for the intestinal transport of antimicrobial agents.

The bioavailabilities of orally administered drugs depend to a great extent on their capability of being transported across the intestinal mucosa. In an attempt to develop an in vitro model for studying the intestinal transport of drugs, we used an intestinal epithelial cell line (Caco 2) derived from a human colon adenocarcinoma. A renal epithelial cell line (MDCK) was also used to determine the tissue specificity of drug transport. These cell lines, which were grown on filters, form a monolayer of well-polarized cells coupled by tight junctions and can be used for transcellular transport experiments. We studied the transport of nine antimicrobial agents with different physicochemical and pharmacokinetic characteristics using these epithelial cell monolayers to determine whether this model could be predictive of oral bioavailability. The transepithelial passage was assayed from the apical (AP) to the basolateral (BL) side and in the opposite direction (BL to AP) in both cell lines. Radioactively labeled mannitol was used to monitor the intactness of the cell monolayer during drug passage. The results indicated that all antimicrobial agents tested tended to behave in vitro generally according to their known in vivo absorptive characteristics. In addition, the use of epithelia from different tissues enabled us to divide the drugs into four groups according to their behaviors and suggested the existence of different transport mechanisms. In particular, two antibiotics, gentamicin and teicoplanin, showed no passage in either direction or cell line, in accordance with their very poor in vivo absorbances after oral administration. In contrast, rifapentine, rifampin, and nalidixic acid passed very efficiently at similar rates in both directions and cell lines in a concentration-dependent, nonsaturable manner, which is suggestive of passive diffusion down a concentration gradient. Of the remaining drugs, isoniazid and novobiocin sodium showed some differences in passage between the two cell lines and, given their ionized state at the pH that was used, may use the paracellular route. Finally, trimethoprim and D-cycloserine exhibited differences in passage both with respect to polarity and cell line; in particular, trimethoprim had a faster rate of passage only in Caco 2 cells and in the BL to AP direction, while D-cycloserine was exclusively transported by Caco 2 cells in the AP to BL direction. In both cases it is possible that active transport mechanisms are involved.

'Early' mammalian myoblasts are resistant to phorbol ester-induced block of differentiation.

Mesenchymal cells were isolated from somites and limbs of mouse embryos at different developmental stages. When grown in tissue culture, some of the cells underwent muscle differentiation as indicated by synthesis of sarcomeric myosin, acetylcholine receptor and, in the case of limb cells, fusion into multinucleated myotubes. When the tumour promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) was added to these cultures, it caused differential effects, depending upon the age of the embryo from which cells were isolated. In cultures of somites or limb bud from embryos up to 12 days post coitum, TPA did not interfere with the appearance of differentiated muscle cells. When TPA was added to cultures from older embryos, it inhibited muscle differentiation with an efficiency which increased with the age of the embryo, reaching about 90% inhibition at 15 days. After this period, a new population of myogenic cells appeared in the limb, which were able to differentiate in the presence of TPA and represented the great majority of myoblasts after day 18 of embryonic development. The simplest interpretation of these data can be based on the existence of three major classes of myogenic cell precursors, which appear sequentially during muscle histogenesis: 'early' myoblasts, which appear resistant to tumour promoters; 'late' myoblasts, whose differentiation is inhibited by tumour promoters and 'satellite' cells which, like early myoblasts, show no sensitivity to TPA.