MetaboLights

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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12989/m_MTBLS12989_NMR___metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12989/a_MTBLS12989_NMR___metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12989/s_MTBLS12989.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12989/i_Investigation.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12989Urinary metabolites were annotated in 1D-1H NMR spectra by comparison of chemical shifts and multiplicities with published reference data and confirmed by spiking authentic standards.MetaboLightsPublicNuclear Magnetic Resonance (NMR) -Inhibition of IRAK4 by microbial trimethylamine blunts metabolic inflammation and ameliorates glycemic control.Standard 1H NMR spectra were measured on a spectrometer (Bruker, Rheinstetten, Germany) operating at 600.22 MHz 1H frequency.Antonis MyridakisImperial College LondonurineNMR spectroscopyMouse urine samples were prepared by using 200 uL of urine mixed with 200 uL of water and 200 l of 0.1 M phosphate buffer solution (10% 2H2OH2O volvol, with 0.05% sodium 3-trimethylsilyl-(2,2,3,3-2H4)-1-propionate for chemical shift reference at delta 0.0. C57BL/6https://www.ebi.ac.uk/metabolights/MTBLS12989Antonis Myridakis. Section of Biomolecular Medicine, Division of Systems Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College London, United Kingdom. Burlington Danes Building, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, United Kingdom. a.myridakis@imperial.ac.uk.Marc-Emmanuel Dumas. Section of Biomolecular Medicine, Division of Systems Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College London, United Kingdom. Burlington Danes Building, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, United Kingdom. m.dumas@imperial.ac.uk.The regions delta-6.0–5.5 and delta-5.0–4.5 were removed to eliminate baseline effects of imperfect water signal presaturation. Each spectrum was normalized to a constant intensity sum, and each variable was mean centered.MonthExperiment nameAnimal IDExperiment numberClass HFDUrine bair unique IDa.myridakis@imperial.ac.ukLongitudinal HFD-feeding in miceAll experiments were approved by the ethical committee of the University of Oxford. Male mice from C57BL/6J inbred strain were bred in our animal facility by using a stock originating from The Jackson Laboratory. At 5 weeks of age, groups of n=8–10 mice were transferred to a 40% w/w HFD (65% kcal) (Special Diets Services), containing 32% lard and 8% corn oil, whereas control groups remained on a 5% Low Fat Diet (CHD) (B & K Universal) for up to 6 months.Mice were housed under a 12 h–12 h light–dark cycle. For physiological profiling, several mouse groups fed CHD or HFD were tested to assess consistency of results and discard any impact of potential batch effects. Intra peritoneal glucose tolerance tests (ipGTTs) were performed in mice after 2-, 3-, 5- and 7-month-old mice after an overnight fast, (see also metabolic phenotyping below). Four days after the GTT, 24 h urinary samples (9 a.m. to 9 a.m.) were collected from mice maintained in individual metabolic cages. Urinary samples collected in a solution of 1% (wt/vol) sodium azide were centrifuged to remove solid particles and kept at −80 °C until assayed. After an overnight fast, mice were killed by exsanguination. Plasma was separated by centrifugation and stored at −80 °C until ¹H-NMR analysis. Choline supplementation on HFDAt 5 weeks of age, mice were fed either a chow diet containing 2 g of choline/kg of diet (Research Diets, D12450J), a low-choline HFD containing 2 g of choline/kg of diet (Research Diets, D12492), a high-choline HFD containing 17 g of choline/kg of diet (Research Diets, D16100401), a HC-HFD containing 1% of DMB, or a HC-HFD combined with a cocktail of antibiotics (0.5 g/L vancomycin hydrochloride, 1 g/L neomycin trisulfate, 1 g/L metronidazole, 1 g/L ampicillin sodium) in drinking bottles (n=6–10 per group) for 8 weeks (see diet formulations in Supplementary Table 6). Mice then were killed by decapitation and organs were dissected and weighed. Irak4-/- miceIrak4-/- mice on C57BL/6J background as already described[38,45] were bred with C57BL/6J mice (The Jackson Laboratory), and the F1 offspring were subsequently bred to produce Irak4-/- mice and WT littermates used for this study. Mice were bred and genotyped at the animal facility of University of Ottawa Heart Institute. The following primers were used for genotyping: Irak4 KO 5’-tga atg gaa gga ttg gag cta cgg ggg t -3’; Irak4 common 5’- gaa cac gct ccc agg tct ctt tcc aac; and Irak4 WT 5’- tct tct acc tga aat atg aaa gat tcc t -3’. The PCR reactions were run at 94 °C 60 s, 60 °C 60 s, 72 °C 60 s for 40 cycles. Ten- to twelve-week-old mice were fed with HFD for eight weeks and were killed by decapitation, and organs were dissected and weighed at the end of the study.Chronic TMA and PF06650833 treatment in LC-HFD-fed miceFive-week-old C57BL/6J mice (Charles River) were housed a week before experiment in a controlled environment. Mice were housed under a 12 h–12 h light–dark cycle. At day 0, the 10-week-old mice were anaesthetised with isoflurane (ForeneH, Abbott). Mini-osmotic pumps were implanted subcutaneously (Model 2006, Alzet) (flow rate: 0.15 mL/h, total filling volume: 200 mL, delivery duration: 42 days). The osmotic mini-pump contained either vehicle or TMA (0.1 mM in circulation) or PF06650833 (50 nM in circulation). After six weeks of metabolite treatment, mice were killed by decapitation and organs were dissected and weighed. Septic shock mouse modelSix-week-old male mice in a C57BL/6J background were purchased from Charles River (Calco, Italy) and maintained in a controlled environment for 2 weeks to acclimate them to local conditions. Animal experiment protocol was approved by local and national committees in charge (Tor Vergata University Institutional Animal Care and Use Committee and Ministry of Health, license no. 265/2019-PR) and conducted in accordance with accepted standards of humane animal care. Mice were intraperitoneally injected with 59 mg/kg TMA (Sigma Cat.No. 72761) (treatment group, n=7) or PBS alone (control group, n=6) 30 minutes before LPS injection [30 mg/kg of LPS (Sigma Cat.No. L2630) in sterile PBS by intraperitoneal injection]. The survival of the mice was monitored every 4 h for 36 h. Physiological phenotypingAfter 4 weeks of treatment an ipGTT test (2 g/kg) was performed in conscious mice following an overnight fast. Blood was collected from the tail vein before glucose injection and 30, 60, 90 and 120 min afterwards. Blood glucose levels were determined using an Accu-Check® Performa (Roche Diagnostics, Meylan, France). Additional blood samples were collected at baseline and 30 min after glucose injection in Microvette® CB 300 Lithium Heparin (Sarstedt, Marnay, France). Plasma was separated by centrifugation and stored at −80 °C until insulin radioimmunoassay. Circulating insulin levels were determined using Insulin ELISA kits (Mercodia, Uppsala, Sweden).After 5 weeks, we performed an ITT. Five-hour-fasted mice wer Longitudinal HFD-feeding in miceAll experiments were approved by the ethical committee of the University of Oxford. Male mice from C57BL/6J inbred strain were bred in our animal facility by using a stock originating from The Jackson Laboratory. At 5 weeks of age, groups of n=8–10 mice were transferred to a 40% w/w HFD (65% kcal) (Special Diets Services), containing 32% lard and 8% corn oil, whereas control groups remained on a 5% Low Fat Diet (CHD) (B & K Universal) for up to 6 months.Mice were housed under a 12 h–12 h light–dark cycle. For physiological profiling, several mouse groups fed CHD or HFD were tested to assess consistency of results and discard any impact of potential batch effects. Intra peritoneal glucose tolerance tests (ipGTTs) were performed in mice after 2-, 3-, 5- and 7-month-old mice after an overnight fast, (see also metabolic phenotyping below). Four days after the GTT, 24 h urinary samples (9 a.m. to 9 a.m.) were collected from mice maintained in individual metabolic cages. Urinary samples collected in a solution of 1% (wt/vol) sodium azide were centrifuged to remove solid particles and kept at −80 °C until assayed. After an overnight fast, mice were killed by exsanguination. Plasma was separated by centrifugation and stored at −80 °C until ¹H-NMR analysis. Choline supplementation on HFDAt 5 weeks of age, mice were fed either a chow diet containing 2 g of choline/kg of diet (Research Diets, D12450J), a low-choline HFD containing 2 g of choline/kg of diet (Research Diets, D12492), a high-choline HFD containing 17 g of choline/kg of diet (Research Diets, D16100401), a HC-HFD containing 1% of DMB, or a HC-HFD combined with a cocktail of antibiotics (0.5 g/L vancomycin hydrochloride, 1 g/L neomycin trisulfate, 1 g/L metronidazole, 1 g/L ampicillin sodium) in drinking bottles (n=6–10 per group) for 8 weeks (see diet formulations in Supplementary Table 6). Mice then were killed by decapitation and organs were dissected and weighed. Irak4-/- miceIrak4-/- mice on C57BL/6J background as already described[38,45] were bred with C57BL/6J mice (The Jackson Laboratory), and the F1 offspring were subsequently bred to produce Irak4-/- mice and WT littermates used for this study. Mice were bred and genotyped at the animal facility of University of Ottawa Heart Institute. The following primers were used for genotyping: Irak4 KO 5’-tga atg gaa gga ttg gag cta cgg ggg t -3’; Irak4 common 5’- gaa cac gct ccc agg tct ctt tcc aac; and Irak4 WT 5’- tct tct acc tga aat atg aaa gat tcc t -3’. The PCR reactions were run at 94 °C 60 s, 60 °C 60 s, 72 °C 60 s for 40 cycles. Ten- to twelve-week-old mice were fed with HFD for eight weeks and were killed by decapitation, and organs were dissected and weighed at the end of the study.Chronic TMA and PF06650833 treatment in LC-HFD-fed miceFive-week-old C57BL/6J mice (Charles River) were housed a week before experiment in a controlled environment. Mice were housed under a 12 h–12 h light–dark cycle. At day 0, the 10-week-old mice were anaesthetised with isoflurane (ForeneH, Abbott). Mini-osmotic pumps were implanted subcutaneously (Model 2006, Alzet) (flow rate: 0.15 mL/h, total filling volume: 200 mL, delivery duration: 42 days). The osmotic mini-pump contained either vehicle or TMA (0.1 mM in circulation) or PF06650833 (50 nM in circulation). After six weeks of metabolite treatment, mice were killed by decapitation and organs were dissected and weighed. Septic shock mouse modelSix-week-old male mice in a C57BL/6J background were purchased from Charles River (Calco, Italy) and maintained in a controlled environment for 2 weeks to acclimate them to local conditions. Animal experiment protocol was approved by local and national committees in charge (Tor Vergata University Institutional Animal Care and Use Committee and Ministry of Health, license no. 265/2019-PR) and conducted in accordance with accepted standards of humane animal care. Mice were intraperitoneally injected with 59 mg/kg TMA (Sigma Cat.No. 72761) (treatment group, n=7) or PBS alone (control group, n=6) 30 minutes before LPS injection [30 mg/kg of LPS (Sigma Cat.No. L2630) in sterile PBS by intraperitoneal injection]. The survival of the mice was monitored every 4 h for 36 h. Physiological phenotypingAfter 4 weeks of treatment an ipGTT test (2 g/kg) was performed in conscious mice following an overnight fast. Blood was collected from the tail vein before glucose injection and 30, 60, 90 and 120 min afterwards. Blood glucose levels were determined using an Accu-Check® Performa (Roche Diagnostics, Meylan, France). Additional blood samples were collected at baseline and 30 min after glucose injection in Microvette® CB 300 Lithium Heparin (Sarstedt, Marnay, France). Plasma was separated by centrifugation and stored at −80 °C until insulin radioimmunoassay. Circulating insulin levels were determined using Insulin ELISA kits (Mercodia, Uppsala, Sweden).After 5 weeks, we performed an ITT. Five-hour-fasted mice were injected intraperitoneally with insulin (0.75 mU/g, Actrapid, Novo Nordisk). Blood glucose levels were measured immediately before and 15, 3 e injected intraperitoneally with insulin (0.75 mU/g, Actrapid, Novo Nordisk). Blood glucose levels were measured immediately before and 15, 30, 45, 60, 90 and 120 min after insulin injection with a standard glucose meter (Accu-Check, Roche, Basel, Switzerland) on the tip of the tail vein.The 1H NMR spectra were phase and baseline corrected by using in-house software (T. Ebbels and H. Keun,personal communication) and were imported into Matlab at high resolution.MetabolomicsNMRtrimethylamineuntargeted metabolitesInsulin resistanceInflammationIRAK4Intestinal FloraNMRtrimethylamineuntargeted metabolitesInsulin resistanceInflammationIRAK4Intestinal FloraSamples then were transferred in 96-well plates for highthroughput flow-injection NMR acquisition.tmaocholinecreatinehippuric acidtrimethylaminedimethylaminefalseInhibition of IRAK4 by microbial trimethylamine blunts metabolic inflammation and ameliorates glycemic control - NMR dataThe global type 2 diabetes epidemic is a major health crisis and there is a critical need for innovative strategies to fight it. Although the microbiome plays important roles in the onset of insulin resistance (IR) and low-grade inflammation, the microbial compounds regulating these phenomena remain to be discovered. Here, we reveal that the microbiome inhibits a central kinase, eliciting immune and metabolic benefits. Through a series of in vivo experiments based on choline supplementation, blocking trimethylamine (TMA) production then administering TMA, we demonstrate that TMA decouples inflammation and IR from obesity in the context of high-fat diet (HFD) feeding. Through in vitro kinome screens, we reveal TMA specifically inhibits Interleukin-1 Receptor-associated Kinase 4 (IRAK4), a central kinase integrating signals from various toll-like receptors and cytokine receptors. TMA blunts TLR4 signalling in primary human hepatocytes and peripheral blood monocytic cells, and improves mouse survival after a lipopolysaccharide-induced septic shock. Consistent with this, genetic deletion and chemical inhibition of IRAK4 result in similar metabolic and immune improvements in HFD. In summary, TMA appears to be a key microbial compound inhibiting IRAK4 and mediating metabolic and immune effects with benefits upon HFD. Thereby we highlight the critical contribution of the microbial signalling metabolome in homeostatic regulation of host disease and the emerging role of the kinome in microbial–mammalian chemical crosstalk.2025-10-202025-09-12MTBLS12989MTBLC15354MTBLC64700MTBLC15724MTBLC17170MTBLC57947MTBLC18089CHEBI:15354CHEBI:64700CHEBI:15724CHEBI:17170CHEBI:57947CHEBI:18089