Trimethylamine-N-oxide postprandial response in plasma and urine is lower after fermented compared to non-fermented dairy consumption in healthy adults

Trimethylamine-N-oxide (TMAO) can be produced by the gut microbiota from dietary substrates and is associated with cardiovascular disease. While dairy products contain TMAO precursors, the effect of fermented dairy on TMAO metabolism remains unclear. We used plasma and urine samples collected for two randomised cross-over studies to evaluate the effects of fermented dairy consumption on TMAO metabolism. In Study 1, thirteen healthy young men tested a yogurt and an acidified milk during postprandial tests and a two-week daily intervention. In Study 2, ten healthy adults tested milk and cheese during postprandial tests. TMAO and five related metabolites were measured in plasma and urine by LC-MS/MS and NMR. Faecal microbiota composition was assessed in Study 1 (16S rRNA metagenomics sequencing). Fermented milk products were associated with lower postprandial TMAO responses than non-fermented milks in urine (Study 1, p = 0.01; Study 2, p = 0.02) and in plasma, comparing yogurt and acidified milk (Study 1, p = 0.04). Daily consumption of dairy products did not differentially affect fasting TMAO metabolites. Significant correlations were observed between microbiota taxa and circulating or urinary TMAO concentrations. Fermentation of dairy products appear, at least transiently, to affect associations between dairy products and circulating TMAO levels

Inter-Species Complementation of the Translocon Beta Subunit Requires Only Its Transmembrane Domain

In eukaryotes, proteins enter the secretory pathway through the translocon pore of the endoplasmic reticulum. This protein translocation channel is composed of three major subunits, called Sec61α, β and γ in mammals. Unlike the other subunits, the β subunit is dispensable for translocation and cell viability in all organisms studied. Intriguingly, the knockout of the Sec61β encoding genes results in different phenotypes in different species. Nevertheless, the β subunit shows a high level of sequence homology across species, suggesting the conservation of a biological function that remains ill-defined. To address its cellular roles, we characterized the homolog of Sec61β in the fission yeast Schizosaccharomyces pombe (Sbh1p). Here, we show that the knockout of sbh1+ results in severe cold sensitivity, increased sensitivity to cell-wall stress, and reduced protein secretion at 23°C. Sec61β homologs from Saccharomyces cerevisiae and human complement the knockout of sbh1+ in S. pombe. As in S. cerevisiae, the transmembrane domain (TMD) of S. pombe Sec61β is sufficient to complement the phenotypes resulting from the knockout of the entire encoding gene. Remarkably, the TMD of Sec61β from S. cerevisiae and human also complement the gene knockouts in both yeasts. Together, these observations indicate that the TMD of Sec61β exerts a cellular function that is conserved across species

Inter-Species Complementation of the Translocon Beta Subunit Requires Only Its Transmembrane Domain

In eukaryotes, proteins enter the secretory pathway through the translocon pore of the endoplasmic reticulum. This protein translocation channel is composed of three major subunits, called Sec61α, β and γ in mammals. Unlike the other subunits, the β subunit is dispensable for translocation and cell viability in all organisms studied. Intriguingly, the knockout of the Sec61β encoding genes results in different phenotypes in different species. Nevertheless, the β subunit shows a high level of sequence homology across species, suggesting the conservation of a biological function that remains ill-defined. To address its cellular roles, we characterized the homolog of Sec61β in the fission yeast Schizosaccharomyces pombe (Sbh1p). Here, we show that the knockout of sbh1+ results in severe cold sensitivity, increased sensitivity to cell-wall stress, and reduced protein secretion at 23°C. Sec61β homologs from Saccharomyces cerevisiae and human complement the knockout of sbh1+ in S. pombe. As in S. cerevisiae, the transmembrane domain (TMD) of S. pombe Sec61β is sufficient to complement the phenotypes resulting from the knockout of the entire encoding gene. Remarkably, the TMD of Sec61β from S. cerevisiae and human also complement the gene knockouts in both yeasts. Together, these observations indicate that the TMD of Sec61β exerts a cellular function that is conserved across species

Accommodating a Non-Conservative Internal Mutation by WaterMediated Hydrogen-Bonding Between β-Sheet Strands: A Comparison of Human and Rat Type B (Mitochondrial) Cytochrome b5

Mammalian type B (mitochondrial) cytochromes b5 exhibit greater amino acid sequence diversity than their type A (microsomal) counterparts, as exemplified by the type B proteins from human (hCYB5B) and rat (rCYB5B). The comparison of X-ray crystal structures of hCYB5B and rCYB5B reported herein reveals a striking difference in packing involving the five-stranded β-sheet, attributable to fully buried residue 21 in strand β4. The greater bulk of Leu21 in hCYB5B in comparison to Thr21 in rCYB5B results in a substantial displacement of the first two residues in β5, and consequent loss of two of the three hydrogen bonds between β5 and β4. Hydrogen-bonding between the residues is instead mediated by two well-ordered, fully buried water molecules. In a 10 ns molecular dynamics simulation, one of the buried water molecules in the hCYB5B structure exchanged readily with solvent via intermediates having three water molecules sandwiched between β4 and β5. When the buried water molecules were removed prior to a second 10 ns simulation, β4 and β5 formed persistent hydrogen bonds identical to those in rCYB5B, but the Leu21 side chain was forced to adopt a rarely observed conformation. Despite the apparently greater ease of water access to the interior of hCYB5B than of rCYB5B suggested by these observations, the two proteins exhibit virtually identical stability, dynamic and redox properties. The results provide new insight into the factors stabilizing the cytochrome b5 fold

Microfluidic chip for monitoring Ca2+ transport through a confluent layer of intestinal cells

The absorption of dietary calcium through the intestinal barrier is essential for maintaining health in general and especially of the bone system. We propose a microfluidic model that studies free calcium (Ca2+) transport through a confluent monolayer of Caco-2 cells. The latter were cultured on a porous membrane that was positioned in between a top and bottom microfluidic chamber. Fresh cell culture medium was continuously supplied into the device at a flow rate of 5 nL s (1) and the culture progress of the cell monolayer was continuously monitored using integrated Transepithelial Electrical Resistance (TEER) electrodes. The electrical measurements showed that the Caco-2 monolayer formed a dense and tight barrier in 5 days. The transported free Ca2+ from the top microfluidic chamber to the basolateral side of the cell monolayer was measured using the calcium-sensitive dye fura-2. This is a ratiometric dye which exhibits an excitation spectrum shift from 340 nm to 380 nm, when it binds to Ca2+ with an emission peak at 510 nm. Therefore, the concentration of free Ca2+ is proportional to the ratio of fluorescence emissions obtained by exciting at 340 nm and 380 nm. The barrier function of the cell monolayer was evaluated by a measured rate of Ca2+ transport through the monolayer that was 5 times lower than that through the bare porous membrane. The continuous perfusion of cell nutrients and the resultant mechanical shear on the cell surface due to the fluid flow are two key factors that would narrow the gap between the in vivo and in vitro conditions. These conditions significantly enhance the Caco-2 cell culture model for studying nutrients bioavailability

Réponses postprandiales à l’ingestion de produits laitiers chez l’homme : effet du type de produit et de l’âge

Introduction et but de l’étude : De nombreuses études montrent que la consommation de produits laitiers (PL) est associée à des bénéfices pour la santé, mais ceux-ci peuvent différer en fonction du type de PL, notamment en raison de la fermentation. Cependant, associer la consommation de chaque PL avec des bénéfices santé spécifiques reste complexe en raison de la précision insuffisante de l'évaluation des profils de consommation par les relevés alimentaires. La métabolomique incluant des biomarqueurs de consommation de PL pourrait permettre une évaluation plus objective. Ces signatures sont aussi dépendantes des différences des réponses métaboliques, notamment au cours du vieillissement. Notre objectif est d’identifier les caractéristiques métaboliques postprandiales (PP) associées à la consommation de deux PL : lait (L) et lait fermenté avec lesbactéries du yaourt (Y) chez l’homme adulte et âgé.Matériel et méthodes : Etude clinique randomisée, contrôlée et en cross-over sur des 28 hommes jeunes (14 J, 20-35 ans) et âgés (14 A, 65-80 ans) en bonne santé. Après 3 semaines d’exclusion de PL et de produits fermentés (PF), les sujets ont ingéré à jeun 600 mL de L ou de Y. Les niveaux de glucose, d’insuline, de triglycérides, de peptide insulinotrope dépendant du glucose (GIP), et Interleukine IL-6 ont été suivis durant les 6h après l’ingestion.Résultats et Analyse statistique : L’augmentation des niveaux circulants de glucose a été plus importante 30 min après la consommation de L vs Y dans les 2 groupes d’âge (J : 6,0±0,2 vs 5,3±0,2, A : 5,9±0,2 vs 5,2±0,1 mmol/L, P<0,05), ce qui est confirmé par l’aire sous la courbe PP (AUC, J : -20±18 vs -67±12 P<0,05, A : 4±19 vs -20±17 P=0.06, mmol/L/min). L’insulinémie PP a également été plus importante après la consommation de L vs Y dans les 2 groupes (J : 204±30 vs 159±21, A : 147±17 vs 119±11 pmol/L à 30 min, P<0,05). Inversement, les taux circulants de GIP ont augmenté plus fortement 60 min après la consommation de Y vs L chez les J et les A (J : 347±33 vs 229±34, A : 406±50 vs 264±28 pg/ml, P<0,05). Concernant les effets liés à l’âge, la réponsePP des niveaux de triglycérides a été plus importante chez les A vs J, indépendamment du produit consommé, et particulièrement 180 min après l’ingestion (AUC L : 72±11 vs 26±16, Y : 77±9 vs 40±15 mmol/L/min, P<0,05). Alors que nous n’avons pas observé de changement significatif chez les J, les niveaux d’IL-6 ont diminué chez les A 1h après la consommation de Y (-14% vs t0, P<0.05), avec une tendance similaire chez les J (-19% vs t0, p=0,12) après la consommation de L.Conclusion : Ces résultats confirment que le changement de la matrice laitière suite à l’application de certains procédés (la fermentation) induit des différences de réponses métaboliques. Les réponses métaboliques peuvent être également influencées par l’âge des sujets. Les analyses métabolomiques en cours devraient permettre de préciser ces différences et d’identifier des biomarqueurs signant la consommation des différents PL.Remerciements : Ce projet bénéfice d'un soutien financier de la part du CNIEL et Syndifrais

Induced expression of microsomal cytochrome b₅ determined at mRNA and protein levels in rats exposed to ellipticine, benzo[a]pyrene, and 1-phenylazo-2-naphthol (Sudan I)

ABSTRACT: The microsomal protein cytochrome b (5), which is located in the membrane of the endoplasmic reticulum, has been shown to modulate many reactions catalyzed by cytochrome P450 (CYP) enzymes. We investigated the influence of exposure to the anticancer drug ellipticine and to two environmental carcinogens, benzo[a]pyrene (BaP) and 1-phenylazo-2-naphthol (Sudan I), on the expression of cytochrome b (5) in livers of rats, both at the mRNA and protein levels. We also studied the effects of these compounds on their own metabolism and the formation of DNA adducts generated by their activation metabolite(s) in vitro. The relative amounts of cytochrome b (5) mRNA, measured by real-time polymerase chain reaction analysis, were induced by the test compounds up to 11.7-fold in rat livers. Western blotting using antibodies raised against cytochrome b (5) showed that protein expression was induced by up to sevenfold in livers of treated rats. Microsomes isolated from livers of exposed rats catalyzed the oxidation of ellipticine, BaP, and Sudan I and the formation of DNA adducts generated by their reactive metabolite(s) more effectively than hepatic microsomes isolated from control rats. All test compounds are known to induce CYP1A1. This induction is one of the reasons responsible for increased oxidation of these xenobiotics by microsomes. However, induction of cytochrome b (5) can also contribute to their enhanced metabolism. GRAPHICAL ABSTRACT: [Image: see text