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.

Metabolomics analysis generates vast arrays of data, necessitating comprehensive workflows involving expertise in analytics, biochemistry and bioinformatics in order to provide coherent and high-quality data that enable discovery of robust and biologically significant metabolic findings. In this protocol article, we introduce notame, an analytical workflow for non-targeted metabolic profiling approaches, utilizing liquid chromatography–mass spectrometry analysis. We provide an overview of lab protocols and statistical methods that we commonly practice for the analysis of nutritional metabolomics data. The paper is divided into three main sections: the first and second sections introducing the background and the study designs available for metabolomics research and the third section describing in detail the steps of the main methods and protocols used to produce, preprocess and statistically analyze metabolomics data and, finally, to identify and interpret the compounds that have emerged as interesting.

Background Accumulating evidence is supporting the protective effect of whole grains against several chronic diseases. Simultaneously, our knowledge is increasing on the impact of gut microbiota on our health and on how diet can modify the composition of our bacterial cohabitants. Herein, we studied C57BL/6 J mice fed with diets enriched with rye bran and wheat aleurone, conventional and germ-free C57BL/6NTac mice on a basal diet, and the colonic fermentation of rye bran in an in vitro model of the human gastrointestinal system. We performed 16S rRNA gene sequencing and metabolomics on the study samples to determine the effect of bran-enriched diets on the gut microbial composition and the potential contribution of microbiota to the metabolism of a novel group of betainized compounds. Results The bran-enriched study diets elevated the levels of betainized compounds in the colon contents of C57BL/6 J mice. The composition of microbiota changed, and the bran-enriched diets induced an increase in the relative abundance of several bacterial taxa, including Akkermansia , Bifidobacterium , Coriobacteriaceae , Lactobacillus , Parasutterella , and Ruminococcus , many of which are associated with improved health status or the metabolism of plant-based molecules. The levels of betainized compounds in the gut tissues of germ-free mice were significantly lower compared to conventional mice. In the in vitro model of the human gut, the production of betainized compounds was observed throughout the incubation, while the levels of glycine betaine decreased. In cereal samples, only low levels or trace amounts of other betaines than glycine betaine were observed. Conclusions Our findings provide evidence that the bacterial taxa increased in relative abundance by the bran-based diet are also involved in the metabolism of glycine betaine into other betainized compounds, adding another potential compound group acting as a mediator of the synergistic metabolic effect of diet and colonic microbiota. Electronic supplementary material The online version of this article (10.1186/s40168-019-0718-2) contains supplementary material, which is available to authorized users.

Novel betainized compounds in humans are associated with diets rich in whole grains, and they improve insulin resistance and insulin secretion. These results suggest that these novel compounds may contribute to the beneficial effects of whole grain-rich diets. The studies were registered at clinicaltrials.gov as NCT00945854 (Naples) and NCT00573781 (Kuopio).

Heart specific PGC-1α KO induces metabolic, functional and structural abnormalities leading to dilating cardiomyopathy and HF. The metabolic changes were limited to the cardiac tissue indicating that cardiomyocyte metabolic remodeling is not sufficient to evoke the body wide metabolic alterations usually associated with HF.

Despite epidemiological evidence showing that diets rich in whole grains reduce the risk of chronic life-style related diseases, biological mechanisms for these positive effects are mostly unknown. Increased 5-aminovaleric acid betaine (5-AVAB) levels in plasma and metabolically active tissues such as heart have been associated with consumption of diets rich in whole grains. However, biological effects of 5-AVAB are poorly understood. We evaluated 5-AVAB concentrations in human and mouse heart tissue (3–22 µM and 38–78 µM, respectively) using mass spectrometry. We show that 5-AVAB, at physiological concentration range, dose-dependently inhibits oxygen consumption due to β-oxidation of fatty acids, but does not otherwise compromise mitochondrial respiration, as measured with oxygen consumption rate in cultured mouse primary cardiomyocytes. We also demonstrate that this effect is caused by 5-AVAB induced reduction of cellular L-carnitine. Reduced L-carnitine levels are at least partly mediated by the inhibition of cell membrane carnitine transporter (OCTN2) as evaluated by in silico docking, and by siRNA mediated silencing of OCTN2 in cultured cardiomyocytes. 5-AVAB caused inhibition of β-oxidation of fatty acids is a novel mechanism on how diets rich in whole grains may regulate energy metabolism in the body. Elucidating potentially beneficial effects of 5-AVAB e.g. on cardiac physiology will require further in vivo investigations.

Background The maternal microbiota affects the development of the offspring by microbial metabolites translocating to the fetus. To reveal the spectrum of these molecular mediators of the earliest host-microbe interactions, we compared placenta, fetal intestine and brain from germ-free (GF) and specific pathogen free (SPF) mouse dams by non-targeted metabolic profiling. Results One hundred one annotated metabolites and altogether 3680 molecular features were present in significantly different amounts in the placenta and/or fetal organs of GF and SPF mice. More than half of these were more abundant in the SPF organs, suggesting their microbial origin or a metabolic response of the host to the presence of microbes. The clearest separation was observed in the placenta, but most of the molecular features showed significantly different levels also in the fetal intestine and/or brain. Metabolites that were detected in lower amounts in the GF fetal organs included 5-aminovaleric acid betaine, trimethylamine N-oxide, catechol-O-sulphate, hippuric and pipecolic acid. Derivatives of the amino acid tryptophan, such as kynurenine, 3-indolepropionic acid and hydroxyindoleacetic acid, were also less abundant in the absence of microbiota. Ninety-nine molecular features were detected only in the SPF mice. We also observed several molecular features which were more abundant in the GF mice, possibly representing precursors of microbial metabolites or indicators of a metabolic response to the absence of microbiota. Conclusions The maternal microbiota has a profound impact on the fetal metabolome. Our observations suggest the existence of a multitude of yet unidentified microbially modified metabolites which pass through the placenta into the fetus and potentially influence fetal development.

Ischemic stroke is amongst the leading causes of death and disabilities. The available treatments are suitable for only a fraction of patients and thus novel therapies are urgently needed. Blockage of one of the cerebral arteries leads to massive and persisting inflammatory reaction contributing to the nearby neuronal damage. Targeting the detrimental pathways of neuroinflammation has been suggested to be beneficial in conditions of ischemic stroke. Nuclear receptor 4A-family (NR4A) member Nurr1 has been shown to be a potent modulator of harmful inflammatory reactions, yet the role of Nurr1 in cerebral stroke remains unknown. Here we show for the first time that an agonist for the dimeric transcription factor Nurr1/retinoid X receptor (RXR), HX600, reduces microglia expressed proinflammatory mediators and prevents inflammation induced neuronal death in in vitro co-culture model of neurons and microglia. Importantly, HX600 was protective in a mouse model of permanent middle cerebral artery occlusion and alleviated the stroke induced motor deficits. Along with the anti-inflammatory capacity of HX600 in vitro, treatment of ischemic mice with HX600 reduced ischemia induced Iba-1, p38 and TREM2 immunoreactivities, protected endogenous microglia from ischemia induced death and prevented leukocyte infiltration. These anti-inflammatory functions were associated with reduced levels of brain lysophosphatidylcholines (lysoPCs) and acylcarnitines, metabolites related to proinflammatory events. These data demonstrate that HX600 driven Nurr1 activation is beneficial in ischemic stroke and propose that targeting Nurr1 is a novel candidate for conditions involving neuroinflammatory component.