A Shared Perspective on in Vitro and in Vivo Models to Assay Intestinal Transepithelial Transport of Food Compounds.

Assessing nutrient bioavailability is complex, as the process involves multiple digestion steps, several cellular environments, and regulatory-metabolic mechanisms. Several in vitro models of different physiological relevance are used to study nutrient absorption, providing significant challenges in data evaluation. However, such in vitro models are needed for mechanistic studies as well as to screen for biological functionality of the food structures designed. This collaborative work aims to put into perspective the wide-range of models to assay the permeability of food compounds considering the particular nature of the different molecules, and, where possible, in vivo data are provided for comparison.

Effects of Glucosinolate-Enriched Red Radish (Raphanus sativus) on In Vitro Models of Intestinal Microbiota and Metabolic Syndrome-Related Functionalities.

The gut microbiota profile is determined by diet composition, and therefore this interaction is crucial for promoting specific bacterial growth and enhancing the health status. Red radish (Raphanus sativusL.) contains several secondary plant metabolites that can exert a protective effect on human health. Recent studies have shown that radish leaves have a higher content of major nutrients, minerals, and fiber than roots, and they have garnered attention as a healthy food or supplement. Therefore, the consumption of the whole plant should be considered, as its nutritional value may be of greater interest. The aim of this work is to evaluate the effects of glucosinolate (GSL)-enriched radish with elicitors on the intestinal microbiota and metabolic syndrome-related functionalities by using an in vitro dynamic gastrointestinal system and several cellular models developed to study the GSL impact on different health indicators such as blood pressure, cholesterol metabolism, insulin resistance, adipogenesis, and reactive oxygen species (ROS). The treatment with red radish had an influence on short-chain fatty acids (SCFA) production, especially on acetic and propionic acid and many butyrate-producing bacteria, suggesting that consumption of the entire red radish plant (leaves and roots) could modify the human gut microbiota profile toward a healthier one. The evaluation of the metabolic syndrome-related functionalities showed a significant decrease in the gene expression of endothelin, interleukin IL-6, and cholesterol transporter-associated biomarkers (ABCA1 and ABCG5), suggesting an improvement of three risk factors associated with metabolic syndrome. The results support the idea that the use of elicitors on red radish crops and its further consumption (the entire plant) may contribute to improving the general health status and gut microbiota profile.

Coupling in vitro food digestion with in vitro epithelial absorption; recommendations for biocompatibility.

As food transits the gastrointestinal tract, food structures are disrupted and nutrients are absorbed across the gut barrier. In the past decade, great efforts have focused on the creation of a consensus gastrointestinal digestion protocol (i.e., INFOGEST method) to mimic digestion in the upper gut. However, to better determine the fate of food components, it is also critical to mimic food absorption in vitro. This is usually performed by treating polarized epithelial cells (i.e., differentiated Caco-2 monolayers) with food digesta. This food digesta contains digestive enzymes and bile salts, and if following the INFOGEST protocol, at concentrations that although physiologically relevant are harmful to cells. The lack of a harmonized protocol on how to prepare the food digesta samples for downstream Caco-2 studies creates challenges in comparing inter laboratory results. This article aims to critically review the current detoxification practices, highlight potential routes and their limitations, and recommend common approaches to ensure food digesta is biocompatible with Caco-2 monolayers. Our ultimate aim is to agree a harmonized consensus protocol or framework for in vitro studies focused on the absorption of food components across the intestinal barrier.

Modulatory activity of a bovine hydrolyzed collagen-hydroxyapatite food complex on human primary osteoblasts after simulating its gastrointestinal digestion and absorption.

Introduction: Introduction: osteoporosis is the most prevalent bone disease and one of the main causes of chronic disability in middle and advanced ages. Conventional pharmacological treatments are still limited, and their prolonged use can cause adverse effects that motivate poor adherence to treatment. Nutritional strategies are traditionally based on supplementing the diet with calcium and vitamin D. Recent studies confirm that the results of this supplementation are significantly improved if it is accompanied by the intake of oral hydrolyzed collagen. Objective: to evaluate the possible in vitro osteogenic activity of a peptide-mineral complex formed by bovine hydrolyzed collagen and bovine hydroxyapatite (Phoscollagen®, PHC®). Methods: the digestion and absorption of PHC® were simulated using the dynamic gastrointestinal digester of AINIA and Caco-2 cell model, respectively. Primary cultures of human osteoblasts were treated with the resulting fraction of PHC® and changes were evaluated in the proliferation of preosteoblasts and in the mRNA expression of osteogenic biomarkers at different stages of osteoblast maturation: Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteocalcin (OC) and type I collagen (ColA1). Results: an increase in preosteoblastic proliferation was observed (p ≤ 0,05). No changes were detected in the biomarkers of osteoblasts with 5 days of differentiation, but were with 14 days, registering an increase in Runx2 (p = 0.0008), ColA1 (p = 0.035), OC (p = 0.027) and ALP (without significance). Conclusion: these results show that the PHC® peptide-mineral complex stimulates the activity of mature osteoblasts, being capable of promoting bone formation.

Introducción: Introducción: la osteoporosis es la enfermedad ósea más prevalente y una de las principales causas de discapacidad crónica en las edades medias y avanzadas. Los tratamientos farmacológicos convencionales aún son limitados y su uso prolongado puede provocar efectos adversos que motiven baja adherencia al tratamiento. Las estrategias nutricionales se basan tradicionalmente en suplementar la dieta con calcio y vitamina D. Estudios recientes confirman que los resultados de esta suplementación mejoran significativamente si se acompaña de la ingesta de colágeno hidrolizado oral. Objetivo: evaluar la posible actividad osteogénica in vitro de un complejo péptido-mineral formado por colágeno hidrolizado e hidroxiapatita bovinos (Phoscollagen®, PHC®). Métodos: se simuló la digestión y absorción de PHC® utilizando el digestor dinámico gastrointestinal de AINIA y el modelo celular Caco-2, respectivamente. Cultivos primarios de osteoblastos humanos se trataron con la fracción resultante de PHC® y se evaluaron los cambios en la proliferación de los preosteoblastos y en la expresión del ARNm de los biomarcadores osteogénicos en diferentes etapas de maduración de los osteoblastos: factor de transcripción 2 relacionado con Runt (Runx2), fosfatasa alcalina (ALP), osteocalcina (OC) y colágeno tipo I (ColA1). Resultados: se observó un incremento de la proliferación preosteoblástica (p ≤ 0,05). No se detectaron cambios en los biomarcadores de osteoblastos con 5 días de diferenciación, pero sí con 14 días, registrándose un aumento de Runx2 (p = 0,0008), ColA1 (p = 0,035), OC (p = 0,027) y ALP (sin significancia). Conclusión: estos resultados muestran que el complejo péptido-mineral PHC® estimula la actividad de los osteoblastos maduros, siendo susceptible de promover la formación ósea.

Improved in vitro bioavailability of a newly developed functionalized calcium carbonate salt as a food ingredient and its comparison with available commercial calcium salts.

The bioaccessibility and bioavailability of a functionalized Calcium (Ca) salt as food ingredient, based on modified Ca carbonate and hydroxyapatite (FCC), was determined and compared with frequently used Ca sources (Ca citrate tetrahydrate (CCT), tri-Ca phosphate (triCP) and Ca carbonate (CC). Results showed a similar Ca bioaccessibility for CCT (76.44 ± 9.73%), CC (73.7 ± 8.18%) and FCC (74.4 ± 1.87%) and a lower value for tri-CP (46.07 ± 8.68%). FCC showed the highest bioavailability, 5.68 ± 0.26%, compared to CC, CCT and tri-CP (3.93 ± 0.99%, 3.41 ± 0.33%, 1.85 ± 0.34%, respectively). The innovative chemical composition and structure of FCC based on amorphous hydroxyapatite combined with Ca carbonate, a greater porosity, lower agglomerates and particle size, improve the Ca solubility in the intestinal media, explaining the similar bioaccessibility but higher bioavailability of FCC compared to CCT, tri-CP and CC.

Development of New Antiproliferative Compound against Human Tumor Cells from the Marine Microalgae Nannochloropsis gaditana by Applied Proteomics.

Proteomics is a crucial tool for unravelling the molecular dynamics of essential biological processes, becoming a pivotal technique for basic and applied research. Diverse bioinformatic tools are required to manage and explore the huge amount of information obtained from a single proteomics experiment. Thus, functional annotation and protein-protein interactions are evaluated in depth leading to the biological conclusions that best fit the proteomic response in the system under study. To gain insight into potential applications of the identified proteins, a novel approach named "Applied Proteomics" has been developed by comparing the obtained protein information with the existing patents database. The development of massive sequencing technology and mass spectrometry (MS/MS) improvements has allowed the application of proteomics nonmodel microorganisms, which have been deeply described as a novel source of metabolites. Between them, Nannochloropsis gaditana has been pointed out as an alternative source of biomolecules. Recently, our research group has reported the first complete proteome analysis of this microalga, which was analysed using the applied proteomics concept with the identification of 488 proteins with potential industrial applications. To validate our approach, we selected the UCA01 protein from the prohibitin family. The recombinant version of this protein showed antiproliferative activity against two tumor cell lines, Caco2 (colon adenocarcinoma) and HepG-2 (hepatocellular carcinoma), proving that proteome data have been transformed into relevant biotechnological information. From Nannochloropsis gaditana has been developed a new tool against cancer-the protein named UCA01. This protein has selective effects inhibiting the growth of tumor cells, but does not show any effect on control cells. This approach describes the first practical approach to transform proteome information in a potential industrial application, named "applied proteomics". It is based on a novel bioalgorithm, which is able to identify proteins with potential industrial applications. From hundreds of proteins described in the proteome of N. gaditana, the bioalgorithm identified over 400 proteins with potential uses; one of them was selected as UCA01, "in vitro" and its potential was demonstrated against cancer. This approach has great potential, but the applications are potentially numerous and undefined.

GutSelf: Interindividual Variability in the Processing of Dietary Compounds by the Human Gastrointestinal Tract.

Nutritional research is currently entering the field of personalized nutrition, to a large extent driven by major technological breakthroughs in analytical sciences and biocomputing. An efficient launching of the personalized approach depends on the ability of researchers to comprehensively monitor and characterize interindividual variability in the activity of the human gastrointestinal tract. This information is currently not available in such a form. This review therefore aims at identifying and discussing published data, providing evidence on interindividual variability in the processing of the major nutrients, i.e., protein, fat, carbohydrates, vitamins, and minerals, along the gastrointestinal tract, including oral processing, intestinal digestion, and absorption. Although interindividual variability is not a primary endpoint of most studies identified, a significant number of publications provides a wealth of information on this topic for each category of nutrients. This knowledge remains fragmented, however, and understanding the clinical relevance of most of the interindividual responses to food ingestion described in this review remains unclear. In that regard, this review has identified a gap and sets the base for future research addressing the issue of the interindividual variability in the response of the human organism to the ingestion of foods.

Integrated Evaluation of the Potential Health Benefits of Einkorn-Based Breads.

Nowadays the high nutritional value of whole grains is recognized, and there is an increasing interest in the ancient varieties for producing wholegrain food products with enhanced nutritional characteristics. Among ancient crops, einkorn could represent a valid alternative. In this work, einkorn flours were analyzed for their content in carotenoids and in free and bound phenolic acids, and compared to wheat flours. The most promising flours were used to produce conventional and sourdough fermented breads. Breads were in vitro digested, and characterized before and after digestion. The four breads having the best characteristics were selected, and the product of their digestion was used to evaluate their anti-inflammatory effect using Caco-2 cells. Our results confirm the higher carotenoid levels in einkorn than in modern wheats, and the effectiveness of sourdough fermentation in maintaining these levels, despite the longer exposure to atmospheric oxygen. Moreover, in cultured cells einkorn bread evidenced an anti-inflammatory effect, although masked by the effect of digestive fluid. This study represents the first integrated evaluation of the potential health benefit of einkorn-based bakery products compared to wheat-based ones, and contributes to our knowledge of ancient grains.

Is cytotoxicity a determinant of the different in vitro and in vivo effects of bioactives?

BACKGROUND: Foodstuffs of both plant and animal origin contain a wide range of bioactive compounds. Although human intervention studies are mandatory to assess the health effects of bioactives, the in vitro approach is often used to select the most promising molecules to be studied in vivo. To avoid misleading results, concentration and chemical form, exposure time, and potential cytotoxicity of the tested bioactives should be carefully set prior to any other experiments. METHODS: In this study the possible cytotoxicity of different bioactives (docosahexaenoic acid, propionate, cyanidin-3-O-glucoside, protocatechuic acid), was investigated in HepG2 cells using different methods. Bioactives were supplemented to cells at different concentrations within the physiological range in human blood, alone or in combination, considering two different exposure times. RESULTS: Reported data clearly evidence that in vitro cytotoxicity is tightly related to the exposure time, and it varies among bioactives, which could exert a cytotoxic effect even at a concentration within the in vivo physiological blood concentration range. Furthermore, co-supplementation of different bioactives can increase the cytotoxic effect. CONCLUSIONS: Our results underline the importance of in vitro cytotoxicity screening that should be considered mandatory before performing studies aimed to evaluate the effect of bioactives on other cellular parameters. Although this study is far from the demonstration of a toxic effect of the tested bioactives when administered to humans, it represents a starting point for future research aimed at verifying the existence of a potential hazard due to the wide use of high doses of multiple bioactives.

Understanding the gastrointestinal tract of the elderly to develop dietary solutions that prevent malnutrition.

Although the prevalence of malnutrition in the old age is increasing worldwide a synthetic understanding of the impact of aging on the intake, digestion, and absorption of nutrients is still lacking. This review article aims at filling the gap in knowledge between the functional decline of the aging gastrointestinal tract (GIT) and the consequences of malnutrition on the health status of elderly. Changes in the aging GIT include the mechanical disintegration of food, gastrointestinal motor function, food transit, chemical food digestion, and functionality of the intestinal wall. These alterations progressively decrease the ability of the GIT to provide the aging organism with adequate levels of nutrients, what contributes to the development of malnutrition. Malnutrition, in turn, increases the risks for the development of a range of pathologies associated with most organ systems, in particular the nervous-, muscoskeletal-, cardiovascular-, immune-, and skin systems. In addition to psychological, economics, and societal factors, dietary solutions preventing malnutrition should thus propose dietary guidelines and food products that integrate knowledge on the functionality of the aging GIT and the nutritional status of the elderly. Achieving this goal will request the identification, validation, and correlative analysis of biomarkers of food intake, nutrient bioavailability, and malnutrition.

Glucose 6-phosphate, rather than xylulose 5-phosphate, is required for the activation of ChREBP in response to glucose in the liver.

BACKGROUND & AIMS: In liver, the glucose-responsive transcription factor ChREBP plays a critical role in converting excess carbohydrates into triglycerides through de novo lipogenesis. Although the importance of ChREBP in glucose sensing and hepatic energy utilization is strongly supported, the mechanism driving its activation in response to glucose in the liver is not fully understood. Indeed, the current model of ChREBP activation, which depends on Serine 196 and Threonine 666 dephosphorylation, phosphatase 2A (PP2A) activity, and xylulose 5-phosphate (X5P) as a signaling metabolite, has been challenged. METHODS: We inhibited PP2A activity in HepG2 cells through the overexpression of SV40 small t antigen and addressed the importance of ChREBP dephosphorylation on Ser-196 using a phospho-specific antibody. To identify the exact nature of the metabolite signal required for ChREBP activity in liver, we focused on the importance of G6P synthesis in liver cells, through the modulation of glucose 6-phosphate dehydrogenase (G6PDH) activity, the rate-limiting enzyme of the pentose phosphate pathway in hepatocytes, and in HepG2 cells using both adenoviral and siRNA approaches. RESULTS: In contrast to the current proposed model, our study reports that PP2A activity is dispensable for ChREBP activation in response to glucose and that dephosphorylation on Ser-196 is not sufficient to promote ChREBP nuclear translocation in the absence of a rise in glucose metabolism. By deciphering the respective roles of G6P and X5P as signaling metabolites, our study reveals that G6P produced by GK, but not X5P, is essential for both ChREBP nuclear translocation and transcriptional activity in response to glucose in liver cells. CONCLUSIONS: Altogether, our study, by reporting that G6P is the glucose-signaling metabolite, challenges the PP2A/X5P-dependent model currently described for ChREBP activation in response to glucose in liver.

TOR kinase pathway and 14-3-3 proteins regulate glucose-induced expression of HXT1, a yeast low-affinity glucose transporter.

Expression of HXT1, a gene encoding a Saccharomyces cerevisiae low-affinity glucose transporter, is regulated by glucose availability, being activated in the presence of glucose and inhibited when the levels of the sugar are scarce. In this study we show that 14-3-3 proteins are involved in the regulation of the expression of HXT1 by glucose. We also demonstrate that 14-3-3 proteins, in complex with Reg1, a regulatory subunit of Glc7 protein phosphatase, interact physically with Grr1 (a component of the SCF-Grr1 ubiquitination complex), a key player in the process of HXT1 induction by glucose. In addition, we show that the TOR kinase pathway participates actively in the induction of HXT1 expression by glucose. Inhibition of the TOR kinase pathway by rapamycin treatment abolishes HXT1 glucose induction. A possible involvement of PP2A protein phosphatase complex, through the Cdc55 B-subunit, in the glucose induction of HXT1 is also discussed.

Expression of the HXT1 low affinity glucose transporter requires the coordinated activities of the HOG and glucose signalling pathways.

Expression of the HXT1 gene, which encodes a low affinity glucose transporter in Saccharomyces cerevisiae, is regulated positively in response to glucose by the general glucose induction pathway, involving the Snf3/Rgt2 membrane glucose sensors, the SCF-Grr1 ubiquitination complex and the Rgt1 transcription factor. In this study we show that, in addition to the glucose signaling pathway, regulation of HXT1 expression also requires the HOG pathway. Deletion of components in the glucose signaling pathway or in the HOG pathway results in impaired HXT1 expression. Genetic analyses showed that, whereas the glucose signaling pathway regulates HXT1 through modulation of the Rgt1 transcription factor, the HOG pathway modulates HXT1 through regulation of the Sko1-Tup1-Ssn6 complex. Coordinated regulation of the two signaling pathways is required for expression of HXT1 by glucose and in response to osmostress.

Active Snf1 protein kinase inhibits expression of the Saccharomyces cerevisiae HXT1 glucose transporter gene.

Expression of HXT1, a gene encoding a Saccharomyces cerevisiae low-affinity glucose transporter, is regulated by glucose availability, being activated in the presence of glucose and inhibited when the levels of the sugar are scarce. In this study we show that Snf1 protein kinase participates actively in the inhibition of HXT1 expression. Activation of Snf1, either by physiological conditions (growth in low-glucose conditions) or by eliminating any of its negative regulators, such as Hxk2 or Reg1, leads to an inhibition of HXT1 expression. We also show that Std1, another known negative regulator of HXT1 expression, interacts physically with active Snf1 protein kinase. Std1 also interacts physically with Rgt1, a transcription factor involved in HXT1 expression, suggesting that the transcriptional properties of Rgt1 could be modulated either directly or indirectly by Std1 and Snf1 protein kinase. Finally, we show that Rgt1 interacts physically with Ssn6, a major transcriptional repressor, to regulate negatively HXT1 expression when glucose is depleted.