Impact of micelle characteristics on cholesterol absorption and ezetimibe inhibition: Insights from Niemann-Pick C1-like 1 binding and molecular structure.

Yamanashi et al., conducted a study on the absorption of cholesterol and ß-sitosterol, as well as the inhibitory effect of ezetimibe (EZE). They used CaCo-2 cells to simulate the intestines and investigated how different mixed micelles, acting as carriers, were absorbed into these cells through the Niemann-Pick C1-like 1 (NPC1L1) protein. The study focused on the impact of micelle shape, size, and zeta potential on absorption and the inhibitory effect of EZE. I utilized small-angle X-ray scattering and a zeta potential measuring device to measure these characteristics. The findings revealed a two-step mechanism: NPC1L1 selectively bound micelles based on their shape and size, and once bound, the absorption was regulated by the molecular structure of the micelle components. EZE's inhibitory effect changed with micelle composition, influencing micelle size and shape. EZE initially acted on the micelle's shape and size, and then NPC1L1 selectively bound micelles based on their shape and size, allowing EZE to directly inhibit absorption by interacting with NPC1L1. This groundbreaking discovery challenges existing concepts and holds significant implications for researchers in drug development, as well as physicians and pharmacists.

IPEC-J2 MDR1, a Novel High-Resistance Cell Line with Functional Expression of Human P-glycoprotein (ABCB1) for Drug Screening Studies.

The P-glycoprotein (P-gp) efflux pump has been shown to affect drug distribution and absorption in various organs and to cause drug resistance in cancer therapy. The aim of this work was to develop a cell line to serve as a screening system for potential substrates of P-gp. This requires a cell line with high paracellular tightness, low expression of nonhuman ABC transporters, and high expression of functional human P-gp (ABCB1). The porcine intestinal epithelial cell line, IPEC-J2, was selected as a transfection host, due to its ability to form extremely high-resistance monolayers (>10,000 Ω·cm(2)) and its low endogenous expression of ABC-type efflux transporters. The IPEC-J2 cells were transfected with a plasmid that contained the sequence of the human MDR1 gene, which encodes P-gp, followed by a selection of successfully transfected cells with geneticin and puromycin. The resulting cell line, IPEC-J2 MDR1, retained its high transepithelilal resistance (>15,000 Ω·cm(2)), which translated into low permeability of the small hydrophilic tracer, mannitol (P < 10(-7) cm·s(-1)). The lipophilic compound, diazepam, displayed high permeability resulting in a dynamic range of 1500 (PDiazepam/Pmannitol) to separate high and low permeability compounds. Human P-gp was expressed predominantly in the apical membrane, as demonstrated by immunocytochemistry, Western blots, and a high efflux ratios (Pbasolateral-apical/Papical-basolateral) of known P-gp substrates. P-gp was demonstrated to be responsible for the efflux transport by substrate profiling, combined with application of P-gp and BCRP inhibitors. Furthermore, the compounds atenolol, citalopram, and mitoxantrone were identified as P-gp substrates. Functional P-gp expression was shown to be stable through at least 10 cell passages. In conclusion, the IPEC-J2 MDR1 cell line displays high paracellular tightness combined with high expression of human P-gp and low expression of porcine ABC transporters, and it may serve as a useful tool in drug development studies.

The Role of Food Matrices Supplemented with Milk Fat Globule Membrane in the Bioaccessibility of Lipid Components and Adaptation of Cellular Lipid Metabolism of Caco-2 Cells.

This study aimed to evaluate the digestive efficiency of food matrices supplemented with milk fat globule membrane isolated from buttermilk (BM-MFGM), using the INFOGEST in vitro digestion protocol hyphenated with the assessment of the digested material on the lipid profile of the Caco-2 cell culture model. First, we examined lipid profiles in food matrices supplemented with BM-MFGM and their subsequent digestion. The results showed distinct lipid profiles in different food matrices and micellar fractions. The presence of BM-MFGM lipids changed the cellular lipid profiles in Caco-2 cell cultures, with diverging contents in cholesteryl esters, triacylglycerides, and neutral lipids depending on the micellar food matrix factor. Hierarchical clustering analysis revealed patterns in cellular lipid responses to micellar stimuli, while volcano plots highlighted significant changes in cellular lipid profiles post-treatment. Thus, this study underscores the importance of in vitro digestion protocols in guiding food matrix selection for bioactive ingredient supplementation, elucidating intestinal epithelium responses to digested food stimuli.

Simulated Gastrointestinal Digestion of Chestnut (Castanea sativa Mill.) Shell Extract Prepared by Subcritical Water Extraction: Bioaccessibility, Bioactivity, and Intestinal Permeability by In Vitro Assays.

Chestnut shells (CSs) are an appealing source of bioactive molecules, and constitute a popular research topic. This study explores the effects of in vitro gastrointestinal digestion and intestinal permeability on the bioaccessibility and bioactivity of polyphenols from CS extract prepared by subcritical water extraction (SWE). The results unveiled higher phenolic concentrations retained after gastric and intestinal digestion. The bioaccessibility and antioxidant/antiradical properties were enhanced in the following order: oral < gastric ≤ intestinal digests, attaining 40% of the maximum bioaccessibility. Ellagic acid was the main polyphenol in the digested and undigested extract, while pyrogallol-protocatechuic acid derivative was only quantified in the digests. The CS extract revealed potential mild hypoglycemic (<25%) and neuroprotective (<75%) properties before and after in vitro digestion, along with upmodulating the antioxidant enzymes' activities and downregulating the lipid peroxidation. The intestinal permeation of ellagic acid achieved 22.89% after 240 min. This study highlighted the efficacy of the CS extract on the delivery of polyphenols, sustaining its promising use as nutraceutical ingredient.

Microplastics: What happens in the human digestive tract? First evidences in adults using in vitro gut models.

Microplastics (MPs) are ubiquitous in the environment and humans are inevitably exposed to them. However, the effects of MPs in the human digestive environment are largely unknown. The aim of our study was to investigate the impact of repeated exposure to polyethylene (PE) MPs on the human gut microbiota and intestinal barrier using, under adult conditions, the Mucosal Artificial Colon (M-ARCOL) model, coupled with a co-culture of intestinal epithelial and mucus-secreting cells. The composition of the luminal and mucosal gut microbiota was determined by 16S metabarcoding and microbial activities were characterized by gas, short chain fatty acid, volatolomic and AhR activity analyses. Gut barrier integrity was assessed via intestinal permeability, inflammation and mucin synthesis. First, exposure to PE MPs induced donor-dependent effects. Second, an increase in abundances of potentially harmful pathobionts, Desulfovibrionaceae and Enterobacteriaceae, and a decrease in beneficial bacteria such as Christensenellaceae and Akkermansiaceae were observed. These bacterial shifts were associated with changes in volatile organic compounds profiles, notably characterized by increased indole 3-methyl- production. Finally, no significant impact of PE MPs mediated by changes in gut microbial metabolites was reported on the intestinal barrier. Given these adverse effects of repeated ingestion of PE MPs on the human gut microbiota, studying at-risk populations like infants would be a valuable advance.

Development of a villi-like micropatterned porous membrane for intestinal magnesium and calcium uptake studies.

Intestinal enterocytes are key players in the absorption of magnesium (Mg2+) and calcium (Ca2+). Understanding the exact molecular mechanisms by which their absorption behavior is regulated could greatly improve treatment strategies for stimulating intestinal absorption in diseases with Mg2+ and/or Ca2+ deficiency. However, such studies are hampered by the lack of in vitro intestinal cell models mimicking the mechanical and physiological properties of the gut. In this study we develop an in vitro gut model based on porous micropatterned membranes with villi-like surface topography and mechanical properties closely mimicking that of intestinal tissue. These membranes are prepared via phase separation micromolding using poly-ε-caprolactone/poly-lactic-glycolic acid (PCL/PLGA) polymer blend and can facilitate cellular differentiation of Caco-2 cells similar to native enterocytes. In fact, cells cultured on these micropatterned membranes form a brush border of microvilli with spatial differences in morphology and tight junction formation along the villous-base axis. Moreover, cells cultured on our membranes show a 2-fold increased alkaline phosphatase activity at the end of differentiation. Finally, we demonstrate that cells cultured on our micropatterned membranes have a 4- and 1.5-fold increased uptake of 25Mg and 45Ca, respectively, compared to non-patterned membranes. These results indicate that the new membranes can mimic the intestinal environment and therefore can have a great impact on mineral uptake in vitro. STATEMENT OF SIGNIFICANCE: This study presents the development of an in vitro gut model consisting of villi-like PCL/PLGA micropatterned membranes. These membranes are prepared via phase separation micromolding (PSµM), a technique which allows tailoring of the membrane surface topography combined with membrane porosity and interconnectivity which are important parameters for membranes used for in vitro transport studies. The culture of Caco-2 cells on these micropatterned membranes shows that they facilitate cellular differentiation similar to gut enterocytes. Our data indicate that mimicking the 3D geometry of the gut is very important for improving the physiological relevance of in vitro gut models. In the future, our micropatterned membranes with segment-specific geometries, in combination with isotopic measurements, would be applied to perform detailed ion uptake and transport studies.

Characterization of Caco-2 cells stably expressing the protein-based zinc probe eCalwy-5 as a model system for investigating intestinal zinc transport.

Intestinal zinc resorption, in particular its regulation and mechanisms, are not yet fully understood. Suitable intestinal cell models are needed to investigate zinc uptake kinetics and the role of labile zinc in enterocytes in vitro. Therefore, a Caco-2 cell clone was produced, stably expressing the genetically encoded zinc biosensor eCalwy-5. The aim of the present study was to reassure the presence of characteristic enterocyte-specific properties in the Caco-2-eCalwy clone. Comparison of Caco-2-WT and Caco-2-eCalwy cells revealed only slight differences regarding subcellular localization of the tight junction protein occludin and alkaline phosphatase activity, which did not affect basic integrity of the intestinal barrier or the characteristic brush border membrane morphology. Furthermore, introduction of the additional zinc-binding protein in Caco-2 cells did not alter mRNA expression of the major intestinal zinc transporters (zip4, zip5, znt-1 and znt-5), but increased metallothionein 1a-expression and cellular resistance to higher zinc concentrations. Moreover, this study examines the effect of sensor expression level on its saturation with zinc. Fluorescence cell imaging indicated considerable intercellular heterogeneity in biosensor-expression. However, FRET-measurements confirmed that these differences in expression levels have no effect on fractional zinc-saturation of the probe.

Influence of in vitro digestion process on polyphenolic profile of skin grape (cv. Italia) and on antioxidant activity in basal or stressed conditions of human intestinal cell line (HT-29).

White table grape cv. Italia is a typical component of the Mediterranean diet and a source of phenolic compounds, particularly abundant in the skin portion. The aim of this study was to characterize the phenolic profile of the table grape skin and to assess its stability after the in vitro digestion process. The main phenolic compounds identified by the HPLC-DAD analysis were: procyanidin B1, caftaric acid, catechin, coutaric acid, quercetin 3-glucuronide and quercetin 3-glucoside. All compounds showed a good stability after in vitro digestion (from 43 to 80%). Moreover, the influence of grape skin polyphenols on the modulation of ROS and GSH levels was evaluated in basal and in stressed conditions on human intestinal cells (HT-29). In basal conditions, a higher polyphenol concentrations exerted pro-oxidant effect corresponding to high ROS level and low GSH content. This effect was probably due to the polyphenolic oxidation in cell culture condition with consequent production of hydrogen peroxide. Otherwise, in stressed conditions, grape skin polyphenols exerted antioxidant effects up to 1.3 × 10-6 µg/g and restored the stress-related GSH reduction. The in vitro digestion process attenuated the biological effect of grape skin polyphenols on intestinal cell line (HT-29). In conclusion, grape skin polyphenols showed different behavior in relation to their concentrations and to the intracellular ROS levels.

Inhibition of cholesterol transport in an intestine cell model by pine-derived phytosterols.

We have quantified the inhibition of intestinal cholesterol transport by pine-derived phytosterols using an HT29-MTX intestine cell model that forms a mucus layer similar to that in the intestine. An artificial intestinal fluid consisting of digested fat, bile salt, cholesterol, and phytosterols was formulated in order to mimic the conditions in the intestine. The apparent permeability coefficient (Papp) of the positive control, i.e., 0.1mM of cholesterol solubilized in the artificial intestine fluid, was found to be 0.33 (±0.17)×10-6cm/s. When 0.1mM ß-sitosterol was solubilized alongside, Papp was effectively zero, corresponding to a total inhibition of cholesterol transport. A similar strong inhibition was found when commercial pine-derived phytosterols, PinVita™ FSP DuPont, were co-solubilized with cholesterol in the dietary model micelles, leading to Papp=0.06 (±0.06)×10-6cm/s, i.e., 5.5 times lower than the cholesterol positive control. Additionally, the effect of potential oral administration formulations generated by the pine-derived phytosterols was also characterized. The formulations were produced as a liquid formulation of the cholesterol-containing artificial intestine fluid. Six liquid formulations were tested of which four displayed a Papp in the range of 0-0.09×10-6cm/s. The remaining two formulations did not show any inhibition effect on cholesterol transport and even enhanced cholesterol transport. It was furthermore observed that the phytosterols were found in the collected intestine cells but not transported to the basolateral region in the intestinal cell model system.