Bile acids (BAs) play an important role in systemic metabolic improvements following bariatric surgery. In this study, we found that orally administered norursodeoxycholic acid (norUDCA), a conjugation-resistant C23 derivative of naturally occurring UDCA, accumulated in peripheral organs including heart and brown adipose tissue (BAT). Moreover, norUDCA decreased systemic levels of endogenous conjugated BAs, while increasing unconjugated BAs. Notably, in addition to beneficial effects in a cholestatic liver disease model, norUDCA also lowered plasma glucose and fat mass in mice, suggesting that this BA derivative could be repurposed for treating obesity-associated cardiometabolic diseases. Metabolic energy expenditure studies, however, revealed that norUDCA-treated mice have impaired BAT capacity and developed intolerance to cold stress, a phenotype exacerbated in mice lacking adipose ATGL-dependent lipolysis. Transcriptomic and metabolic analyses demonstrated tissue remodeling in heart and BAT that involved pronounced changes in energy substrate utilization, including enhanced cardiac glucose uptake and higher ketone body utilization in BAT. Importantly, co-administration of a low-carb diet prevented cold stress-induced metabolic deficits. Mechanistic studies in human engineered heart tissue indicated that norUDCA compromised contractile function. In conclusion, these data suggest that conjugation- resistant BA derivatives like norUDCA impair myocardial and BAT energetics by altering glucose, lipid, and energy metabolism, particularly during catabolic cold stress conditions.

The gut microbiome is essential for neurodevelopment via bidirectional gut-brain axis signaling, yet environmental chemicals can potentially disrupt this communication by altering community structure and xenobiotic metabolism. In this study, we investigated whether the fungicide azoxystrobin, a known metabolic disruptor, modulates microbiome composition and function to influence neurobehavior. We utilized a simplified human gut microbiota model (SIHUMIx) and a vertebrate host model (larval zebrafish) to elucidate microbiome-mediated mechanisms of xenobiotic neurotoxicity. SIHUMIx was exposed to azoxystrobin for 7 days at 10% of the acceptable daily intake, followed by recovery. Integrated metaproteomic and metabolomic analyses revealed functional reprogramming of the microbiota, characterized by upregulation of vitamin and cofactor biosynthesis, nutrient acquisition, and detoxification pathways, and decreases in carbohydrate fermentation and amino acid turnover, consistent with reduced short-chain fatty acid levels. Microbiome-depleted and SIHUMIx-inoculated larvae were exposed to azoxystrobin at 4 days post fertilization, and neurobehavioral outcomes were assessed after 24 h using the Visual and Acoustic Motor Response assay. Azoxystrobin exposure disrupted non-associative habituation learning independent of microbiome status but induced dark-phase hyperactivity only in colonized larvae, indicating a microbiome-dependent phenotype. Targeted metabolomics revealed lower serotonin levels in microbiome-depleted larvae relative to colonized controls and that azoxystrobin exposure reduced serotonin in colonized larvae toward depleted levels. These results suggest that microbiota-dependent serotonergic signaling may modulate host responses to azoxystrobin. This integrated ex vivo-in vivo approach supports the concept that the microbiome is a key determinant of neurotoxic responses and underscores the importance of incorporating microbiome-mediated effects into chemical risk assessment frameworks.

Insulin-driven gene regulation is central to adipocyte function, but the roles of many of these genes in lipid metabolism remain unclear. Here, we integrate three transcriptomic datasets to identify insulin-responsive genes and define their functions in human adipocytes using a multiparametric lipid turnover screen. Our results reveal four major clusters involved in metabolic regulation, transcription, stress responses, and lipid metabolism. Among lipid-related hits, phospholipase C X domain-containing protein-1 (PLCXD1) emerges as a regulator of insulin-stimulated lipogenesis, without affecting lipolysis or adipogenesis. PLCXD1 is induced by insulin via sterol regulatory element-binding proteins, a response attenuated in insulin-resistant states. This atypical phospholipase is genetically associated with fat mass-related traits, localizes to early endosomes and catalyzes phosphatidylinositol conversion into diacylglycerol. Through structure-function analyses, we show that PLCXD1 catalytic activity is required for insulin-stimulated lipogenesis. Altogether, our results uncover PLCXD1 as an insulin-regulated enzyme linking endosomal phosphoinositide metabolism to lipid storage in adipocytes.

This study applied a suite of human-relevant, non-animal cell models to investigate early events associated with respiratory sensitization induced by 4,4'-methylene diphenyl diisocyanate (MDI), a representative low-molecular-weight respiratory sensitizer. Phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophage-like cells (THP-1M) and primary human monocyte-derived macrophages were exposed to MDI (3-300 µM, 4 h), resulting in a restricted cytokine response characterized primarily by enhanced IL-1α and IL-1β production. In contrast, lipopolysaccharide (LPS, 1 ng/mL-10 µg/mL) induced a pronounced pro-inflammatory response consistent with classical M1-like activation. Flow cytometric analysis of THP-1M indicated a partial M2a-like immunomodulatory phenotype following MDI exposure, characterized by increased CD1a and reduced CD14 expression. This response was accompanied by changes in selected central carbon metabolites, whereas LPS induced changes more consistent with M1-like activation. In human alveolar epithelial type I-like cells (hAELVi), MDI induced changes in selected central carbon metabolites, indicating epithelial metabolic responsiveness. In epithelial-macrophage coculture (hAELVi/THP-1M), metabolic responses to MDI were attenuated while chemokine production (CCL2, CCL5, CXCL8) was enhanced, suggesting macrophage-mediated modulation of epithelial responses and immune cell recruitment potential. In an air-liquid interface coculture model, these early responses were associated with dendritic cell-like activation of THP-1 cells, marked by OX40L upregulation, indicating Th2-skewing potential. Overall, the findings support interacting epithelial-immune key events relevant to respiratory sensitization and highlight the value of incorporating epithelial-immune interactions into new approach methods (NAMs) based hazard assessment. SHORT SUMMARY: 4,4'-Methylene diphenyl diisocyanate (MDI) induced epithelial metabolic changes in vitro and promoted a partial M2a-like immunomodulatory macrophage response associated with alterations in central carbon metabolites. In epithelial-macrophage co-culture, macrophages attenuated metabolic responses while enhancing chemokine production, suggesting modulation of epithelial responses and immune cell recruitment potential. In an air-liquid interface co-culture model, these early events were associated with dendritic cell-like activation of THP-1 cells, marked by OX40L upregulation and indicative of Th2-skewing potential. Together, the findings support interacting key events relevant to proposed adverse outcome pathway concepts for respiratory sensitization and highlight candidate biomarkers and mechanistically relevant cellular responses that may support human-relevant NAM development for low-molecular-weight respiratory sensitizers.

Antibiotics represent a unique and diverse group of drugs, which are known to exert deleterious effects on non-target species and contribute to the phenomenon of antimicrobial resistance. With central inclusion on the EU Surface Water Watch List, and reported known affects in multiple model organisms, the importance of the sufficient monitoring of antibiotics in the aquatic environment has been highlighted. Most studies report the impact of individual antibiotics following exposure for a single generation in animals. In this study, we assessed the impact of four antibiotics with different modes of action (amoxicillin, trimethoprim, erythromycin, and sulfamethoxazole) and their mixture on the sentinel species Daphnia magna over three generations, via biochemical markers and a targeted metabolomic analysis of central metabolic pathways. No mortality was observed at 50 mg/L of each selected antibiotic and their composite mixture. Thus, a working concentration of 1 mg/L was chosen to progress this study. Results indicated that enzyme activity was particularly sensitive to exposure to amoxicillin and the mixture, whereas trimethoprim and the mixture induced the most metabolic changes in glycolysis and the TCA cycle. Additionally, the quaternary mixture had a stronger impact on the first generation of daphnids, altering the activity of beta-galactosidase, glutathione S-transferase, and acid and alkaline phosphatase, suggesting that Daphnia can adapt to stress caused by antibiotics.

Bempedoic acid (BA) is a recently approved drug that lowers cholesterol and hepatic lipids, yet its mechanism of action remains incompletely understood. Here, we combine transcriptomic, biochemical, and structural approaches to show that BA directly binds to and activates peroxisome proliferator-activated receptor alpha (PPARα). BA treatment robustly induced PPARα signaling and fatty acid oxidation in primary hepatocytes and mouse liver. Through X-ray crystallography, we uncovered that BA binds to the ligand-binding domain of PPARα and stabilizes its active conformation. BA activated PPARα target genes independently of very-long-chain acyl-coenzyme A (CoA) synthetase (ACSVL1), the liver-enriched enzyme that converts BA to its bempedoyl-CoA form. Notably, BA-mediated induction of fatty acid oxidation required PPARα. Together, this work reveals direct PPARα activation as a key mechanism of BA action, providing a molecular basis for its lipid-lowering effects and suggesting broader therapeutic potential beyond the liver.

Human exposure to certain environmental chemicals, including phthalates, is linked to metabolic disruption and may thereby contribute to diseases like obesity. However, regulatory methods to evaluate such effects are lacking. DINCH was introduced as a substitute for banned phthalate plasticizers, but its primary metabolite, MINCH, has been shown to promote adipogenesis in human preadipocytes and alter the lipid metabolism of mature adipocytes. To investigate its potential metabolism-disrupting effects, we assessed changes in the central carbon metabolism activity of human preadipocytes and mature adipocytes by 13C metabolic tracing. In preadipocytes, MINCH increased glycolysis, pentose phosphate pathway activity, acetyl-CoA production from glucose and glutamine, and pyruvate anaplerosis, indicating a metabolic shift toward adipogenesis. In mature adipocytes, MINCH enhanced glycolysis, glyceroneogenesis, fatty acid oxidation, and oxidative TCA cycle activity, pathways associated with the browning of adipocytes. Elevated UCP1 expression confirmed MINCH-induced browning. Most pronounced effects occurred at micromolar concentrations, whereas subtle changes were already observed at nanomolar concentrations in preadipocytes, the biological relevance of which should be further investigated. Overall, our findings demonstrate the utility of 13C metabolic tracing as a New Approach Methodology for detecting chemical-induced metabolic alterations, thus providing a new perspective for the hazard and risk assessment of environmental contaminants.

The rapid advancements of the modern world led to the creation of new materials and products at an unprecedented rate. Among these, nanoparticles have found extensive applications and frequently enter freshwater ecosystems. Daphnia magna is a widely used bioindicator species for the assessment of aquatic pollution. This study explores the impact of excreted metabolites on the toxicity mechanisms of silver nanoparticle ink using D. magna metabolite conditioned media. Two different life stages of daphnids were used to condition aqueous media and form metabolite-based nanocoronas with different compositions. The primary objective was to examine how these metabolite nanocoronas affect the observed impact of exposure on several physiological markers in daphnids including mortality, key enzyme activities and core metabolism. Conditioned media was also characterised to compare similarities in composition to assess the impact of silver nanoink. The response profiles to acute exposure varied among the tested media groups, with unfunctionalized nanoparticles exerting noticeably different responses on the investigated markers of physiology. Lethality was significantly reduced by the presence of excreted metabolites compared to unconditioned exposure to the silver nanoink, while the presence of metabolites had a more significant effect on key enzyme activities and metabolism of daphnids.

Plastic materials are ubiquitous, leading to constant human exposure to plastic additives such as plasticizers. There is growing evidence that plasticizers may contribute to obesity due to their disruptive effects on metabolism. Alternatives like diisononylcyclohexane-1,2-dicarboxylate (DINCH) are replacing traditional phthalates such as di-(2-ethylhexyl) phthalate (DEHP), which are now banned due to their proven harmful health effects. While DINCH is considered a safer alternative to DEHP and no adipogenic effects have been demonstrated in in vivo studies, recent research suggests that the primary metabolite, monoisononylcyclohexane-1,2-dicarboxylic acid ester (MINCH), promotes adipocyte differentiation and dysfunction in vitro. However, metabolic and molecular effects are not fully understood in vivo. Here, we performed a comprehensive in vivo analysis using C57BL/6N mice to investigate the effects of DINCH on adipose tissue physiology and function. Mice were exposed to two doses of DINCH for 16 weeks, followed by a 10-week recovery period. Tissue analysis confirmed the presence of DINCH and MINCH in liver and adipose tissue after treatment and recovery. After the recovery period, elevated DINCH concentrations in adipose tissue depots indicated possible bioaccumulation. Although no changes were observed in body composition and energy expenditure, sex-specific metabolic effects were identified. Female mice exhibited impaired whole-body insulin sensitivity and higher triglyceride levels, while male mice showed an altered insulin/C-peptide ratio and elevated cholesterol, HDL, and LDL levels. Proteomic profiling of serum, adipose and liver tissues revealed changes in pathways related to central energy metabolism and immune response, highlighting the systemic impact of DINCH, potentially on inflammatory processes. Most effects of DINCH, such as changes in insulin response and serum lipid levels, were diminished after the recovery period. Despite many findings consistent with the existing literature suggesting DINCH as a safer DEHP substitute, the observed sex-specific effects on insulin sensitivity, lipid metabolism and inflammatory processes, as well as potential bioaccumulation and long-term metabolic effects of DINCH exposure warrant careful consideration in further risk assessment.

Per- and polyfluoroalkyl substances are a class of synthetic chemicals detected ubiquitously in the environment, humans, and wildlife. Perfluorooctanesulfonic acid (PFOS) is one prevalent chemical previously shown to cause adverse effects on nervous system function across in vivo and in vitro models, including dark-phase hyperactivity in larval zebrafish. The objective of this study was to evaluate the role of gamma-aminobutyric acid receptors (GABARs), GABAAR and GABABR, as mediators of dark-phase hyperactivity in PFOS-exposed larval zebrafish. Zebrafish were acutely exposed to 7.87 to 120 μM PFOS, 0.68 to 12.4 μM picrotoxin (GABAAR antagonist), 0.77 to 14.05 μM propofol (GABAAR-positive allosteric modulator), 4.4 to 80 μM saclofen (GABABR antagonist), 0.43 to 7.87 μM CGP13501 (GABABR-positive allosteric modulator), or the solvent control 0.4% dimethyl sulfoxide 60 min before behavior assessment at 5 days post fertilization. Co-exposures to positive allosteric modulators and PFOS were performed. Acute exposure to PFOS caused transient dark-phase hyperactivity. Concentration-dependent dark-phase hypoactivity was observed following acute propofol or CGP13501 exposure, in contrast to the concentration-dependent hyperactivity caused by acute picrotoxin exposure. Saclofen exposure provoked a modest reduction in dark-phase motor activity at the highest concentration tested. PFOS-induced hyperactivity was rescued to baseline activity by co-exposure to propofol or CGP13501. To assess relevance across species, electrophysiological measurements were performed in cultured mouse cortical neurons and BrainSpheres derived from human-induced pluripotent stem cells. PFOS exposure reduced GABAAR-mediated currents in mouse neurons. GABAAR- and GABABR-dependent units in BrainSphere-derived neural networks exhibited increased spiking activity following PFOS exposure. This study demonstrates that PFOS antagonizes GABARs in zebrafish, mouse, and human experimental systems. Taken together, this study supports the concept that early life-stage zebrafish can be used to rapidly identify causative mechanisms, conserved across taxa, by which xenobiotic agents alter neuroactivity.

Introduction: In honey bees, division of labour is a key feature, with age-related behavioural transitions being closely associated with molecular changes in the brain, gut, and microbiota. Despite evidence of both microbiome and brain changes in honey bees, most studies focus on either aspect or a single method of investigation, limiting our understanding of their interconnected roles in development and task differentiation. Objectives: In this study, we investigated the molecular changes in the gut and brain in honey bee workers of different ages using (meta-)proteomics and metabolomics to better understand their contribution to behavioural responses and modulation. Methods: Workers were sampled at seven timepoints throughout their life. Mass spectrometry for (meta-) proteomic and metabolomic of the guts and brains allowed insights into the global structural and functional dynamics of the microbiota, as well as the functional and metabolic alterations in the host gut and brain, and their interactions. Results: Our results indicate the transport of amino acids between the gut and brain, potentially influencing functional pathways and behavioural phenotypes. We observed a correlation between concentrations of tryptophan and its metabolic products between honey bee brain and gut. This provides evidence regarding gut-brain axis as a way of internal communication for different host mechanisms in honey bees. Microbiota composition changed significantly, with protein numbers increasing significantly in the establishment phase. Proteomic results from both the host and the microbiota reveal that altered metabolic and functional pathway abundances may be due to energy expenditure, task differentiation, and age of onset of foraging. Conclusion: Overall, our findings are the first to describe the global (meta-)proteomic and metabolomic changes in the honeybee gut and brain throughout a worker’s life. This provides new insights toward developing potential biomarkers for evaluation of different functional changes related to various environmental stressors.

Single and mixture exposure to plant protection products (PPPs) can affect non-target organisms at sublethal concentrations, yet the ecological relevance of behavioural effects remains underexplored. Behavioural disruptions can compromise survival and fitness, with exposure occurring across terrestrial and aquatic ecosystems. Here, we assess the behavioural impact of environmentally relevant PPP concentrations on two ecologically and toxicologically important model species: honeybees (Apis mellifera) and zebrafish (Danio rerio). These organisms represent distinct exposure pathways: pollinator-specific routes such as oral uptake of contaminated nectar and pollen or contact during overspray and foraging, and freshwater contamination via runoff. In honeybees, in-hive behaviours were monitored using a snapshot method from days three to ten after exposure, while zebrafish behavioural endpoints were quantified using a 26-endpoint visual and acoustic motor response (VAMR) assay. Sublethal PPP exposure (1.99-7.81 ng/µL in honeybee hives, 0.0253-27.5 ng/µL in zebrafish assay) caused significant, substance-specific behavioural alterations. In honeybees, flupyradifurone (SIVANTO® prime) significantly decreased foraging and nectar processing, while boscalid (Cantus®) and terbuthylazine (ClickPro®) minimised brood-tending behaviours. Insecticides and fungicides affected honeybees most, while zebrafish embryos were especially sensitive to the herbicide terbuthylazine. They exhibited concentration-dependent neurotoxic phenotypes, with behavioural profiles of the PPPs mixture (consistent with concentrations in German streams: 41.54 % boscalid, 0.013 % flupyradifurone, 58.45 % terbuthylazine) shifting along a terbuthylazine-boscalid gradient. These findings show that PPPs can elicit pronounced behavioural changes in non-target species, even at low environmental concentrations. These results support incorporating in-hive and early-life stage behavioural assays into pesticide risk assessments and warrant mechanistic studies on PPP-induced neurotoxicity.

The microbial community present in our intestines is pivotal for converting indigestible substances into vital nutrients and signaling molecules such as short-chain fatty acids (SCFAs). These compounds have considerable influence over our immune system and the development of diverse human diseases. However, ingested environmental contaminants, known as xenobiotics, can upset the delicate balance of the microbial gut community and enzymatic processes, consequently affecting the host organism. In our study, we employed an in vitro bioreactor model system based on the simplified human microbiome model (SIHUMIx) to investigate the direct effects of specific xenobiotics, such as perfluorooctanoic acid (PFOA), perfluorohexanoic acid (PFHxA) and perfluorobutanoic acid (PFBA) or bisphenol S (BPS) and bisphenol F (BPF), either individually or in combination, on the microbiota. We observed increased SCFA production, particularly acetate and butyrate, with PFAS exposure. Metaproteomics revealed pathway alterations across treatments, including changes in vitamin synthesis and fatty acid metabolism with BPX. This study underscores the necessity of assessing the combined effects of xenobiotics to better safeguard public health. It emphasizes the significance of considering adverse effects on the microbiome in the risk assessment of environmental chemicals.

Growing evidence suggests that exposure to certain metabolism-disrupting chemicals (MDCs), such as the phthalate plasticizer DEHP, might promote obesity in humans, contributing to the spread of this global health problem. Due to the restriction on the use of phthalates, there has been a shift to safer declared substitutes, including the plasticizer diisononyl-cyclohexane-1,2-dicarboxylate (DINCH). Notwithstanding, recent studies suggest that the primary metabolite monoisononyl-cyclohexane-1,2-dicarboxylic acid ester (MINCH), induces differentiation of human adipocytes and affects enzyme levels of key metabolic pathways. Given the lack of methods for assessing metabolism-disrupting effects of chemicals on adipose tissue, we used metabolomics to analyze human SGSB cells exposed to DINCH or MINCH. Concentration analysis of DINCH and MINCH revealed that uptake of MINCH in preadipocytes was associated with increased lipid accumulation during adipogenesis. Although we also observed intracellular uptake for DINCH, the solubility of DINCH in cell culture medium was limited, hampering the analysis of possible effects in the μM concentration range. Metabolomics revealed that MINCH induces lipid accumulation similar to peroxisome proliferator-activated receptor gamma (PPARG)-agonist rosiglitazone through upregulation of the pyruvate cycle, which was recently identified as a key driver of de novo lipogenesis. Analysis of the metabolome in the presence of the PPARG-inhibitor GW9662 indicated that the effect of MINCH on metabolism was mediated at least partly by a PPARG-independent mechanism. However, all effects of MINCH were only observed at high concentrations of 10 μM, which are three orders of magnitudes higher than the current concentrations of plasticizers in human serum. Overall, the assessment of the effects of DINCH and MINCH on SGBS cells by metabolomics revealed no adipogenic potential at physiologically relevant concentrations. This finding aligns with previous in vivo studies and supports the potential of our method as a New Approach Method (NAM) for the assessment of adipogenic effects of environmental chemicals.

Background & Aims: The constitutive androstane receptor (CAR) is a nuclear receptor that binds diverse xenobiotics and whose activation leads to the modulation of the expression of target genes involved in xenobiotic detoxification and energy metabolism. Although CAR hepatic activity is considered to be higher in women than in men, its sex-dependent response to an acute pharmacological activation has seldom been investigated. Methods: The hepatic transcriptome, plasma markers, and hepatic metabolome, were analysed in Car+/+ and Car-/- male and female mice treated either with the CAR-specific agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) or with vehicle. Results: Although 90% of TCPOBOP-sensitive genes were modulated in a sex-independent manner, the remaining 10% showed almost exclusive female liver specificity. These female-specific CAR-sensitive genes were mainly involved in xenobiotic metabolism, inflammation, and extracellular matrix organisation. CAR activation also induced higher hepatic oxidative stress and hepatocyte cytolysis in females than in males. Hepatic expression of flavin monooxygenase 3 (Fmo3) was almost abolished and was associated with a decrease in hepatic trimethylamine-N-oxide (TMAO) concentration in TCPOBOP-treated females. In line with a potential role in the control of TMAO homeostasis, CAR activation decreased platelet hyper-responsiveness in female mice supplemented with dietary choline. Conclusions: More than 10% of CAR-sensitive genes are sex-specific and influence hepatic and systemic responses such as platelet aggregation. CAR activation may be an important mechanism of sexually-dimorphic drug-induced liver injury. Impact and implications: CAR is activated by many drugs and pollutants. Its pharmacological activation had a stronger impact on hepatic gene expression and metabolism in females than in males, and had a specific impact on liver toxicity and trimethylamine metabolism. Sexual dimorphism should be considered when testing and/or prescribing xenobiotics known to activate CAR.

IntroductionObesity is associated with a plethora of health complications, including increased susceptibility to infections or decreased vaccine efficacy, partly due to dysregulated immune responses. Monocytes play a crucial role in innate immunity, yet their functional alterations in obesity remain poorly understood.MethodsHere, we employed proteomic and metabolomic analyses to investigate monocyte characteristics in individuals with overweight, obesity, impaired glucose tolerance (IGT), and type 2 diabetes (T2D), compared to lean donors.Results and discussionOur results revealed distinct molecular signatures in monocytes from individuals with obesity, with significant alterations in pathways related to metabolism, cellular migration, and phagocytosis. Moreover, LPS-induced activation of monocytes unveiled heightened metabolic reprogramming towards glycolysis in subjects with obesity accompanied by dysregulated cytokine responses and elevated oxidative stress. Additionally, monocytes from donors with obesity exhibited increased lipid droplet accumulation. These findings shed light on the immunometabolic dysregulation underlying obesity-associated immune dysfunction, highlighting potential targets for therapeutic intervention.

Human health and the human microbiome are inevitably intertwined, increasing their relevance in clinical research. However, the collection, transportation and storage of faecal samples may introduce bias due to methodological differences, especially since postal shipping is a common practise in large-scale clinical cohort studies. Using four different Omics layer, we determined the structural (16S rRNA sequencing, cytometric microbiota profiling) and functional integrity (SCFAs, global metabolome) of the microbiota in relation to different easy-to-handle conditions. These conditions were storage at −20 °C, −20 °C as glycerol stock, 4 °C and room temperature with and without oxygen exposure for a maximum of one week. Storage time affected the microbiota on all Omics levels. However, the magnitude was donor-dependent, highlighting the need for purpose-optimized sample collection in clinical multi-donor studies. The effects of oxygen exposure were negligible for all analyses. At ambient temperature, SCFA and compositional profiles were stable for 24 h and 48 h, respectively, while at 4 °C, SCFA profiles were maintained for 48 h. The global metabolome was highly susceptible, already changing at 24 h in non-frozen conditions. Thus, faecal microbiota was best preserved on all levels when transported as a native sample frozen within 24 h, leading to the least biased outcomes in the analysis. We conclude that the immediate freezing of native stool samples for transportation to the lab is best suited for planned multi-Omics analyses that include metabolomics to extend standard sequencing approaches.

The intestinal microbiota and its metabolites are known to influence host metabolic health. However, little is known about the role of specific microbes. In this work, we used the minimal consortium Oligo-Mouse-Microbiota (OMM12) to study the function of Coriobacteriia under defined conditions in gnotobiotic mice. OMM12 mice with or without the addition of the dominant gut bacterium Eggerthella lenta (E. lenta ) were fed with diets varying in fat content and primary bile acids. E. lenta stably colonised the mouse caecum at high relative abundances (median: 27.5%). This was accompanied by decreased occurrence of Akkermansia muciniphila and Enterococcus faecalis , but results did not reach statistical significance in all groups depending on diet and inter-individual differences. Changes in host parameters (anthropometry, blood glucose, and cholesterol) and liver proteomes were primarily due to diet. In contrast, metabolomes in colon content differed significantly between the colonisation groups. The presence of E. lenta was associated with elevated levels of latifolicinin C acid and decreased creatine, sarcosine, N,N-dimethylarginine, and N-Acetyl-DL-methionine. In conclusion, E. lenta altered specific metabolites in the colon but did not have significant effects on the mice or liver proteomes under the conditions tested due to marked inter-individual differences.