Oral treatment with Eubacterium hallii improves insulin sensitivity in db/db mice : npj Biofilms and Microbiomes. E. Cultures were grown to the end of the exponential phase, concentrated by anaerobic centrifugation, washed with phosphate- buffered saline, diluted in a solution containing maltodextrin and glucose in 1. CFU), 1. 08 CFU and 1. CFU in 1. 00 . Viability was assessed by most probable number analysis by dilution to extinction and confirmed by microscopic analysis. Samples were stored at . Male C5. 7Bl. 6/J db/db mice (1. Jackson Laboratories USA. Animals were housed at AMC SPF vivarium in groups of 5 animals/cage and fed ad libitum with regular chow diet (Research Diets, Inc.) and water. Mice were housed under constant temperature and a 1. At 1. 6 weeks of age, the animals were daily given an oral 1. As a control, an oral 1. Twenty- four- hour faeces were collected after 4 weeks of treatment (2. In the last week of treatment and after an overnight fast, mice (n=8 per group) received an intraperitoneal insulin bolus (Actrapid 0. U/kg body weight) and blood glucose was measured (Ascensia Elite glucose meter, Bayer, Leverkusen, Germany) at t=0, 6. Thereafter, animals were sacrificed using 1. Hyperinsulinemic- euglycemic clamp. Male C5. 7Bl. 6/J6 db/db mice (1. Jackson Laboratories, Bar Harbor, ME, USA. Animals were housed at University of Gothenburg SPF vivarium and fed ad libitum with regular chow diet (Research Diets, New Brunswick, NJ, USA) and water. Marine Biofilms & Microbiomes; Facilities / Research Capacities. Overview; Sequencing Capacity; Imaging Facility;. About the npj Biofilms and Microbiomes Community. Mice were housed under constant temperature and a 1. In the last week of treatment, at least 4 days before the clamp a catheter was surgically placed in the jugular vein for infusion of insulin and glucose under isoflurane anaesthesia. Prior to the clamp, mice were fasted for 4 h and placed in individual plastic containers. Basal blood glucose (Countour Next blood glucose meter, Bayer AB, Solna, Sweden) was used from tail- blood measurements. A bolus injection of . Three consecutive blood samples were taken at steady state (t=. At t=0, a priming dose of insulin (1. U/kg; Actrapid Penfill, Novo Nordisk, Bagsv. Blood glucose was measured at 1. GIR; 3. 0% glucose Fresenius Kabi, Bad Homburg, Germany) to maintain blood glucose concentration at the basal level. At steady state, defined by stable glycemia and GIR (approximately at t=1. Rd) and hepatic glucose production (Ra) under hyperinsulinemic- euglycemic- conditions. Plasma insulin was measured at t=. Animals were killed by an overdose of pentobarbital (Apoteket Farmaci AB, Stockholm, Sweden) and tissue was collected. The blood samples were deproteinised, evaporated and resuspended in deionised water for the determination of radioactivity (Beckman LS6. Multipurpose Scintillation Counter, Providence, RI, USA). Whole- body glucose appearance (Ra) and endogenous glucose production (endogenous Ra), a measure of hepatic glucose production, were calculated as published as previously described. Metabolic chamber experiments and body composition. During a parallel experiment, male db/db mice (aged 1. Thereafter, mice were individually housed in Somedic INCA metabolic cages (Somedic AB, H. Oxygen consumption (VO2) and CO2 production (VCO2) were recorded every 2 min for 2. Temperature in the metabolic chamber was kept constant at 2. Data from the first hour was discarded to account for animal acclimatisation. The average total energy expenditure per hour was determined using Weir's equation: (3. Also, magnetic resonance imaging scanning for body composition was performed as previously described. Intestinal microbiota analysis. Abundances of E. 1. A biofilm is any group of microorganisms in which cells. S r. RNA gene amplification, in vitro transcription and labelling, and hybridisation were carried out as described. The data were normalised and analysed using a set of R- based scripts in combination with a custom- designed relational database, which operates under the My. SQL database management system. For the microbial profiling, the Robust Probabilistic Averaging signal intensities of 2. MITChip were used. Diversity calculations were performed using a microbiome R- script package (https: //github. Multivariate statistics, redundancy analysis, and principal response curves were performed in Canoco 5. Npj Aging and Mechanisms. Alzheimer Prevention, Microbes Stealing, Bacteria Hold, Health Discoveries, Alzheimers Info, Health Diet. Npj Biofilms and Microbiomes npj Biofilms. Information about the open-access journal npj Biofilms and Microbiomes in DOAJ. Introducing npj Biofilms and Microbiomes Author: Staffan Normark Subject. SCFA and bile acid profiling. Twenty- four- hour faecal samples (pooled from each cage) were collected and stored for later analysis. SFCA content was analysed by gas liquid chromatography following conversion to t- butylmethylsilyl derivate as previously described. Concentrations of different bile acids were measured twice in 2. An internal standard was added before extraction with 0. Na. OH at 8. 00 . Bile salt were trimethylsilylated with pyridine, N,O- Bis(trimethylsilyl) trifluoroacetamide and trimethylchlorosilane. Faecal bile acid profile was measured using capillary gas chromatography (Hewlett–Packert gas chromatograph; HP 6. Mountain View, CA, USA) equipped with a FID and a CP Sil 1. Plasma bile acids were determined using liquid chromatography tandem mass spectrometry as described previously (1. The primary bile acids cholic acid (CA), taurocholic acid, muricholic acid (MCA), tauroalpha muricholic acid and taurobeta muricholic acid as well as the secondary bile acids taurohyodeoxycholic acid, deoxycholic acid, taurodeoxycholic acid and omega murocholic acid were analysed in plasma and 2. The total amount of primary and secondary bile acids was calculated as the sum of the individually quantified bile salts. Quantitative real- time PCRLiver and intestinal tissues were homogenised with tissue- magna. Lyzer (Roche, Basel, Switzerland). Total RNA was extracted using Tri- pure reagent (Roche). Complementary DNA was prepared by reverse transcription of 1 . Hepatic genes involved in lipogenesis (Srebp. Fasn, Acc. 1, Acc. Dgat) and gluconeogenesis (Gck. G6. Pc, Pk and Pck. Genes involved in bile acid metabolism and transport were tested in liver (Cyp. Cyp. 8b. 1, Cyp. 7b. Cyp. 27a, Ntcp, Oatp. Mrp. 3, Bsep and Mrp. Tgr. 5, Fxr, Gata. Asbt, Ilbp Ost. Gene- specific intron–exon boundary spanning primers were used and all the results were normalised with the house keeping gene 3. B4. All samples were analysed in duplicate and data were analysed according to the 2. Primer sequences are presented in Supplementary Table S2. Statistical analysis. On the basis of distribution of the clinical data, Student t- test or Mann–Whitney tests (two- sided) were used to analyse the difference between clinical groups. Microbiota analyses were done as described above.
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