Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Insights into microbial modulation of host peripheral lipid and glucose metabolism using mouse gut organoids

ABSTRACT: The gastrointestinal microbiota is involved in the development of various diseases, such as obesity and diabetes, by affecting nutrient acquisition, energy balance, and metabolic signaling of the host. However, the underlying mechanisms of these host-microbiota interactions remain unclear. The aim of this study was to characterize effects of the microbiota on the host epithelium using a novel gastrointestinal model based on mouse organoids. We have explored the transcriptional response of organoids upon exposure to short chain fatty acids (SCFA) and products generated by two conspicuous members of the gastrointestinal microbiota; Akkermansia muciniphila and Faecalibacterium prausnitzii. We observed that A. muciniphila metabolites affect a large variety of transcription factors and genes involved in cellular lipid metabolism and growth, supporting previous in vivo findings. F. prausnitzii products exerted only a weak effect on the host transcription profile. While, A. muciniphila, and its main metabolite propionate, both stimulate fasting-induced adipose factor/angiopoietin-like protein 4 (Fiaf/Angptl4) and Ppar? expression, important regulators of lipolysis and satiety. This work illustrates that specific members of the microbiota and their metabolites differentially modulate epithelial transcription in mouse organoid lines. Organoid culture: In short, murine (WT C57BL/6) small intestinal crypts were isolated and embedded in 250 µl Matrigel (BD Biosciences) and submerged in 500 ul basal culture medium supplemented with penicillin/streptomycin, HEPES, Glutamax, N2, B27, N-acetylcysteine, and the murine growth factors EGF, noggin, and R-spondin-1 (ENR), as previously described (Sato et al., 2009. Nature 459:262–5). Organoids were passaged every 7 days with a 1:4 splitting ratio. Exposure to bacterial cultures and SCFA: A. muciniphila (ATTC BAA-835) was grown anaerobically at 37C in a basal liquid medium which contained (per liter of deionized water) the following: 0.4g KH2PO4, 0.669g, Na2HPO4 + 2H2O, 0.3g NH4CL, 0.3gNaCl, 0.1g MgCl2 + 6H2O, 10g casitone, 1mM L-threonine, 1 ml trace mineral solution, 5mM L-fucose, and 5mM D-glucose. F. prausnitzii strain A2-165 (DSM 17677) was grown anaerobically at 37ºC a liquid medium which contained (per liter of deionized water) the following: 10 g casitone, 2.5 g yeast extract, 4 g NaHCO3, 1 ml resazurine (0.1%w/v), 0.45g K2HPO4, 0.45g KH2PO4, 0.9g (NH4)2SO4, 0.9g NaCL, 0.09g MgSO4, 0.09g CaCL2, 2.5 mM acetate, 9 mM propionate, 1 mM n-valerate, 1 mM isovalerate, 1 mM isobutyrate, 0.28g KOH, 2.5 ml ethanol (95%), 10 mg haemin,, 10 ug biotin, 10 ug cobalamin, 30 ug para-aminobenzoic acid, 50 ug folic acid, 150 ug pyridoxamine, 1 g L-cysteine, and 25 mM glucose (Duncan et al., 2002. Int. J. Syst. Evol. Microbiol. 52:2141–6). The concentration of the following organic acids and sugars were determined in the culture medium before and after growth using a high performance liquid chromatography (HPLC) equipped with a Shodex Sugar SH-G and Shodex SH1821 column as has been described previously (Stams et al., 1993. Appl. Environ. Microbiol. 59:1114–9): glucose, mannose, galactose, L-fucose, glucose-N-acetylglucosamine, lactate, formate acetate, propionate, 1,2-propendiol and butyrate. Overnight-grown cultures were pelleted by centrifugation at 20 000 ×g for 10 min and supernatant was collected. At t=7 days after splitting organoids were exposed to 250 μl A. muciniphila supernatant, 250 μl unconditioned A. muciniphila culture medium, 85 μl F. prausnitzii supernatant, or 85 μl F. prausnitzii culture medium for 3 hours at 37ºC, prior to RNA extraction. 250 μl A. muciniphila supernatant and culture medium Short chain fatty acids (SCFA) sodium butyrate, sodium propionate, and sodium acetate (Sigma-Aldrich, Zwijndrecht, the Netherlands) were administered at a final concentration of 5 mM. At t=7 days after splitting mature organoids were exposed to individual SCFAs for 3 hours at 37ºC, prior to RNA extraction. Data is presented from n = 4 independent biological replicates from one line of organoids. total RNA was isolated from mouse intestinal organoids exposed for 3h to SCFA and strain-specific culture medium: Am- (organoids exposed to A. muciniphila culturing medium), Am+ (A. muciniphila-conditioned medium), Fp- (F. prausnitzii-culturing medium), Fp+ (F. prausnitzii-conditioned medium), Ace (acetate), But (butyrate), Pro (propionate) and Con (standard organoid culturing medium).

ORGANISM(S): Mus musculus

SUBMITTER: Guus Roeselers

PROVIDER: E-GEOD-59644 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:Background: Acetate, propionate, and butyrate are the main short-chain fatty acids (SCFA) produced in the colon as a result of microbial fermentation of dietary fibers. An increasing amount of evidence suggests that these SCFA have major health benefits. The composition of the microbiota is altered by dietary fat, and this is believed to impact SCFA production. Currently it is unknown whether host gene expression responses to SCFA are modulated by fat content of the diet. The aim of this study was to compare the changes in colonic gene expression profiles after acetate, propionate and butyrate infusions between a low fat and high fat diet. Methods: Male C57BL/6J mice were fed semi-synthetic low fat (10 energy%) or high fat (45 E%) diets starting 2 weeks before the SCFA treatment period. During treatment, mice received a rectal infusion of either an acetate, propionate, butyrate, or a saline (control) solution for 6 consecutive days, after which colon was subjected to gene expression profiling. Unsupervised visualization of the dataset was performed using Independent Principal Component Analysis. For each SCFA, similarities of its effects on a low fat and a high fat diet were assessed using Rank-Rank Hypergeometric Overlap. In addition, differentially expressed genes were identified, and gene set enrichment analysis was performed to determine functional implications of the regulated genes. Results: Taking into account the complete dataset, we observed that more variation in gene expression profiles was explained by fat content of the diet than by SCFA treatment. Gene expression responses to acetate and butyrate were similar on the low fat versus high fat diet, but were opposite for propionate. Functionally the expression changes reflected differential modulation of several metabolic processes; genes involved in oxidative phosphorylation, lipid catabolism, lipoprotein metabolism and cholesterol transport were suppressed by acetate and butyrate treatment, whereas propionate treatment resulted in changes in fatty acid and sterol biosynthesis, and in amino acid and carbohydrate metabolism. Conclusions: We demonstrated that dietary fat content impacts the colonic gene expression response to propionate, and to a lesser extent to acetate and butyrate. The study demonstrates that knowledge on diet composition is essential when studying effects of SCFAs on metabolism.

Project description:Background: Short-chain fatty acids (SCFAs), mainly acetate, propionate and butyrate, are produced by gut microbiota through fermentation of complex carbohydrates that cannot be digested by the human host. They affect gut health and can contribute at the distal level to the pathophysiology of several diseases, including renal pathologies. Methods: SCFA levels were measured in chronic kidney disease (CKD) patients (n = 54) at different stages of the disease and associations with renal function and inflammation parameters were examined. The impact of propionate and butyrate in pathways triggered in tubular cells under inflammatory conditions was analysed using genome-wide expression assays. Finally, a pre-clinical mouse model of folic acid-induced transition from acute kidney injury to CKD was used to analyse the preventive and therapeutic potential of these microbial metabolites in the development of CKD. Results: Faecal levels of propionate and butyrate in CKD patients gradually reduce as the disease progresses, and do so in close association with established clinical parameters for serum creatinine, blood urea nitrogen and the estimated glomerular filtration rate. Propionate and butyrate jointly downregulated the expression of 103 genes related to inflammatory processes and immune system activation triggered by TNF-α in tubular cells. In vivo, the administration of propionate and butyrate, either before or soon after injury, respectively prevented and slowed the progression of damage. This was indicated by a decrease in renal injury markers, the expression of pro-inflammatory and pro-fibrotic markers, and recovery of renal function over the long term. Conclusions: Propionate and butyrate levels are associated with a progressive loss of renal function in CKD patients. Early administration of these SCFAs prevents disease advancement in a pre-clinical model of acute renal damage, demonstrating their therapeutic potential independently of the gut microbiota.

Project description:Epithelial cells of the mammalian intestine are covered with a mucus layer that prevents direct contact with intestinal microbes but also constitutes a substrate for mucus-degrading bacteria. To study the effect of mucus degradation on the host response, germ-free mice were colonized with Akkermansia muciniphila. This anaerobic bacterium belonging to the Verrucomicrobia is specialized in the degradation of mucin, the glycoprotein present in mucus, and found in high numbers in the intestinal tract of human and other mammalian species. Efficient colonization of A. muciniphila was observed with highest numbers in the cecum, where most mucin is produced. In contrast, following colonization by Lactobacillus plantarum, a facultative anaerobe belonging to the Firmicutes that ferments carbohydrates, similar cell-numbers were found at all intestinal sites. Whereas A. muciniphila was located closely associated with the intestinal cells, L. plantarum was exclusively found in the lumen. The global transcriptional host response was determined in intestinal biopsies and revealed a consistent, site-specific, and unique modulation of about 750 genes in mice colonized by A. muciniphila and over 1500 genes after colonization by L. plantarum. Pathway reconstructions showed that colonization by A. muciniphila altered mucosal gene expression profiles toward increased expression of genes involved in immune responses and cell fate determination, while colonization by L. plantarum led to up-regulation of lipid metabolism. These indicate that the colonizers induce host responses that are specific per intestinal location. In conclusion, we propose that A. muciniphila modulates pathways involved in establishing homeostasis for basal metabolism and immune tolerance toward commensal microbiota. Keywords: Analysis of target gene regulation by using microarrays Adult germ-free female NMRI-KI mice (45 – 65 days) were used for bacterial mono-association. Two bacterial strains were used in this study, A. muciniphila MucT (ATTC BAA-835) and L. plantarum WCFS1 (NCIMB 8826). A. muciniphila was grown anaerobically in a basal mucin based medium and L. plantarum was grown anaerobically at 37°C in Man-Rogosa-Sharpe broth (MRS; Le Pont de Claix, France). After 7 days of colonization, mice were killed by cervical dislocation and terminal ileum, cecum and ascending colon specimens were sampled.

Dataset Information

Insights into microbial modulation of host peripheral lipid and glucose metabolism using mouse gut organoids

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets