Metabolic signature of extracellular vesicles depends on the cell culture conditions.

One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography-mass spectrometry (LC-MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects.

Glycosylated Benzoxazinoids Are Degraded during Fermentation of Wheat Bran.

Benzoxazinoids are plant secondary metabolites found in whole grain cereal foods including bread. They are bioavailable and metabolized in humans, and therefore their potential bioactivity is of interest. However, effects of food processing on their content and structure are not yet studied. This study reports effects of bioprocessing on wheat bran benzoxazinoid content. Benzoxazinoid glycosides were completely degraded during fermentation, whereas metabolites of benzoxazinoid aglycones were formed. Fermentation conditions did not affect the conversion process, as both yeast and yeast/lactic acid bacteria mediated fermentations had generally similar impacts. Likewise, enzymatic treatment of the bioprocess samples did not affect the conversion, suggesting that these compounds most likely are freely bioavailable from the grain matrix and not linked to the cell wall polymers. Additionally, the results show that benzoxazinoids undergo structural conversion during the fermentation process, resulting in several unknown compounds that contribute to the phytochemical intake and necessitate further analysis.

Amino acid-derived betaines dominate as urinary markers for rye bran intake in mice fed high-fat diet--A nontargeted metabolomics study.

SCOPE: Bioprocessing of whole grain cereals may affect the bioavailability of phytochemicals associated with grain fiber and ultimately lead to different health outcomes. Here, we studied the impact of long-term feeding with intact and bioprocessed rye bran on the urinary phytochemical profile of mice. METHODS AND RESULTS: Nontargeted hydrophilic interaction chromatography-ESI-qTOF-MS metabolite profiling approach was applied on urine samples collected from three groups of diet-induced obese mice fed for 8 weeks with one of the three diets: high-fat (HF) control diet, HF diet enriched with intact rye bran, or HF diet enriched with bioprocessed rye bran. The most striking finding was the increased urinary excretion of several amino-acid derived betaines after both rye diets. These included proline betaine, alanine betaine, valine betaine, phenylalanine betaine, pipecolic acid betaine, and trigonelline, but not glycine betaine. Furthermore, bioprocessing may have improved the bioavailability of rye-derived phytochemicals, as higher increase in, e.g. ferulic acid and benzoxazinoid metabolites were observed in urine of mice fed with bioprocessed than intact rye bran. CONCLUSION: Urinary excretion of various betaines was greatly increased in mice fed rye brans. Furthermore, bioprocessing of rye bran appears to serve as a beneficial way to improve the bioavailability of various phytochemicals.

Impact of wheat aleurone structure on metabolic disorders caused by a high-fat diet in mice.

The present study investigated the potential of native and structurally modified wheat aleurone, by dry-grinding or enzymatic treatments, to counteract metabolic disorders in mice with diet-induced obesity (DIO). C57BL6/J mice were first fed ad libitum with a high-fat diet for 9 weeks to induce obesity, after which the native or treated aleurone fractions were added (13% (w/w)) in the high-fat diets for an additional 8 weeks. The effects of the aleurone-enriched diets were evaluated by assessing body weight gain, adiposity, fasting blood glucose, plasma insulin and leptin, and anti-inflammatory and oxidative stress markers. Enrichment of the diet with native or finely ground aleurone did not improve any parameter analyzed; finely ground aleurone even slightly increased (p = 0.03) body weight gain. Enrichment of the diet with enzymatically treated aleurone only had a tendency toward lower body weight gain, visceral adipose tissue accumulation, fasting plasma insulin, and leptin levels.

The postprandial plasma rye fingerprint includes benzoxazinoid-derived phenylacetamide sulfates.

The bioavailability of whole-grain rye-derived phytochemicals has not yet been comprehensively characterized, and different baking and manufacturing processes can modulate the phytochemical composition of breads and other rye products. The aim of our study was to find key differences in the phytochemical profile of plasma after the consumption of 3 breads containing rye bran when compared with a plain white wheat bread control. Plasma metabolite profiles of 12 healthy middle-aged men and women were analyzed using LC quadrupole time-of-flight mass spectrometry metabolomics analysis while fasting and at 60 min, 120 min, 240 min, and 24 h after consuming a meal that contained either 100% whole-grain sourdough rye bread or white wheat bread enriched with native unprocessed rye bran or bioprocessed rye bran. White wheat bread was used as the control. The meals were served in random order after a 12-h overnight fast, with at least 3 d between each occasion. Two sulfonated phenylacetamides, hydroxy-N-(2-hydroxyphenyl) acetamide and N-(2-hydroxyphenyl) acetamide, potentially derived from the benzoxazinoid metabolites, were among the most discriminant postprandial plasma biomarkers distinguishing intake of breads containing whole-meal rye or rye bran from the control white wheat bread. Furthermore, subsequent metabolite profiling analysis of the consumed breads indicated that different bioprocessing/baking techniques involving exposure to microbial metabolism (e.g., sourdough fermentation) have a central role in modulating the phytochemical content of the whole-grain and bran-rich breads.

Disintegration of wheat aleurone structure has an impact on the bioavailability of phenolic compounds and other phytochemicals as evidenced by altered urinary metabolite profile of diet-induced obese mice.

BACKGROUND: Phenolic acids are covalently bound to the arabinoxylan fibre matrix of wheat aleurone layer. In order to be bioavailable they need to be released by endogenous or bacterial enzymes and absorbed within the intestinal lumen. The intestinal microbiota can metabolize phenolic acids and other food-born phytochemicals. However, the effect of structure of the cereal bran or aleurone layer on these processes is not comprehensively studied. METHODS: The structure of aleurone layer was modified either by dry-grinding or by enzymatic treatments with xylanase alone or in combination with feruloyl esterase. Diet induced obese C57BL6/J mice were fed with high-fat diets containing either pure ferulic acid, or one of the four differentially treated aleurone preparations for 8 weeks. The diets were designed to be isocaloric and to have similar macronutrient composition. The urinary metabolite profiles were investigated using non-targeted LC-qTOF-MS-metabolomics approach. RESULTS: The different dietary groups were clearly separated in the principal component analysis. Enzymatic processing of aleurone caused increased excretion of ferulic acid sulfate and glycine conjugates reflecting the increase in unbound form of readily soluble ferulic acid in the diet. The urinary metabolite profile of the diet groups containing native and cryo-ground aleurone was more intense with metabolites derived from microbial processing including hippuric acid, hydroxyl- and dihydroxyphenylpropionic acids. Furthermore, aleurone induced specific fingerprint on the urinary metabolite profile seen as excretion of benzoxazinoid metabolites, several small dicarboyxlic acids, and various small nitrogen containing compounds. CONCLUSIONS: The structural modifications on wheat aleurone fraction resulted in altered metabolism of aleurone derived phenolic acids and other phytochemicals excreted in urine of diet-induced obese mice.

Betaine supplementation causes increase in carnitine metabolites in the muscle and liver of mice fed a high-fat diet as studied by nontargeted LC-MS metabolomics approach.

SCOPE: Betaine (BET) reduces diet-induced liver lipid accumulation, and may relieve obesity-related metabolic disturbances. The aim of our study was to analyze metabolite alterations after supplementation of BET, polydextrose (PDX, a soluble dietary fiber), or their combination (BET PDX) via drinking water to C57BL/6J mice fed a high-fat (HF) diet. METHODS AND RESULTS: BET supplementation increased BET levels in plasma, muscle, and liver (p < 0.05), and the nontargeted LC-MS metabolite profiling revealed an increase in several metabolites in the carnitine biosynthesis pathway after BET supplementation both in liver and muscle. These included carnitine and acetylcarnitine (1.4-fold, p < 0.05), propionylcarnitine and γ-butyrobetaine (1.5-fold, p < 0.05), and several other short-chain acylcarnitines (p < 0.05) in muscle. These changes were slightly higher in the BET PDX group. Furthermore, BET reduced the HF diet induced accumulation of triglycerides in liver (p < 0.05). The supplementations did not attenuate the HF diet induced increase in body weight gain or the increase in adipose tissue mass. Instead, the combination of BET and PDX tended to increase adiposity. CONCLUSION: Our results suggest that increased availability of BET in different tissues, especially in muscle, after BET supplementation has an impact on carnitine metabolism, and this could further explain the link between BET and lipid metabolism.

Comparative nontargeted profiling of metabolic changes in tissues and biofluids in high-fat diet-fed Ossabaw pig.

Typical clinical biomarker analyses on urine and plasma samples from human dietary interventions do not provide adequate information about diet-induced metabolic changes taking place in tissues. The aim of this study was to show how a large-scale nontargeted metabolomic approach can be used to reveal metabolite groups for generating new hypotheses of obesity-related metabolic disturbances produced in an animal model. A large spectrum of metabolites in the semipolar region, including small water-soluble molecules like betaine and dihydroxyindole, and a wide range of bile acids as well as various lipid species were detected. The high-fat diet influenced metabolic homeostasis of Ossabaw pigs, especially the lipid metabolome, throughout all the analyzed sample types, including plasma, urine, bile, liver, pancreas, brain cortex, intestinal jejunum and proximal colon. However, even dramatic metabolic changes in tissues were not necessarily observed in plasma and urine. Metabolite profiling involving multiple sample types was shown to be a feasible method for the examination of a wide spectrum of metabolic species extending from small water-soluble metabolites to an array of bile acids and lipids, thus pointing to the pathways of metabolism affected by the dietary treatment.

Somatostatin receptor 5 is palmitoylated by the interacting ZDHHC5 palmitoyltransferase.

Many G-protein coupled receptors are palmitoylated in their C-terminal, intracellular regions. So far no enzymes responsible for this modification have been described. We identified an interaction of the membrane proximal helix 8 of somatostatin receptor 5 (SSTR5) with the N-terminal region of the putative palmitoyltransferase ZDHHC5 using the Ras recruitment interaction screening system. ZDHHC5 and SSTR5 are colocalized at the plasma membrane and can be efficiently coimmunoprecipitated from transfected cells. Coexpression of ZDHHC5 in HEK293 cells increased palmitoylation of SSTR5 whereas knock-down of endogenous ZDHHC5 by siRNAs decreased it. Our data identify the first palmitoyltransferase for a G-protein coupled receptor.

Impact of dietary polyphenols on carbohydrate metabolism.

Polyphenols, including flavonoids, phenolic acids, proanthocyanidins and resveratrol, are a large and heterogeneous group of phytochemicals in plant-based foods, such as tea, coffee, wine, cocoa, cereal grains, soy, fruits and berries. Growing evidence indicates that various dietary polyphenols may influence carbohydrate metabolism at many levels. In animal models and a limited number of human studies carried out so far, polyphenols and foods or beverages rich in polyphenols have attenuated postprandial glycemic responses and fasting hyperglycemia, and improved acute insulin secretion and insulin sensitivity. The possible mechanisms include inhibition of carbohydrate digestion and glucose absorption in the intestine, stimulation of insulin secretion from the pancreatic beta-cells, modulation of glucose release from the liver, activation of insulin receptors and glucose uptake in the insulin-sensitive tissues, and modulation of intracellular signalling pathways and gene expression. The positive effects of polyphenols on glucose homeostasis observed in a large number of in vitro and animal models are supported by epidemiological evidence on polyphenol-rich diets. To confirm the implications of polyphenol consumption for prevention of insulin resistance, metabolic syndrome and eventually type 2 diabetes, human trials with well-defined diets, controlled study designs and clinically relevant end-points together with holistic approaches e.g., systems biology profiling technologies are needed.