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A Surgical Method for Continuous Intra-Portal Infusion of Gut Microbial Metabolites in Mice

ABSTRACT: Gut-derived microbial metabolites have been shown to play key roles in human physiology and disease. However, establishing mechanistic links between gut microbial metabolites and disease pathogenesis in animal models presents many challenges. The major route of absorption for microbe-derived small molecules is venous drainage via the portal vein to the liver. In the event of extensive liver first pass- or presystemic hepatic metabolism, the route of administration of these metabolites becomes critical. Here we describe a novel portal vein cannulation technique using a subcutaneously implanted osmotic pump to achieve continuous portal vein infusion in mice. First, the microbial metabolite trimethylamine (TMA) was administered over 4 weeks and compared to a vehicle control. Using a liquid chromatography-tandem mass spectrometry (LC-MS/MS), an increase in peripheral plasma levels of TMA and its host liver-derived co-metabolite trimethylamine-N-oxide (TMAO) were observed in a sexually-dimorphic manner. Next, 4-hydroxyphenylacetic acid (4-HPAA), a structurally-distinct microbial metabolite that undergoes extensive hepatic first pass metabolism, was administered portally to examine its effects on hepatic gene expression. Peripheral plasma levels showed no difference in free or total 4-HPAA. However, while liver tissue demonstrated no difference in free 4-HPAA, total levels were increased when compared to the control group. More importantly, significant changes were observed in hepatic gene expression using an unbiased RNA sequencing approach. Collectively, this work describes a novel method for administering gut microbe-derived metabolites via the portal vein, mimicking their physiologic delivery in vivo.

ORGANISM(S): Mus musculus

PROVIDER: GSE159775 | GEO | 2021/05/10

REPOSITORIES: GEO

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The Gut Microbe-Derived Metabolite Trimethylamine is a Biomarker of and Therapeutic Target in Alcohol-Associated Liver Disease

Project description:BACKGROUND & AIMS: There is mounting evidence that microbes resident in the human intestine contribute to diverse alcohol-associated liver diseases (ALD) including the most deadly form known as alcoholic hepatitis (AH). However, mechanisms by which gut microbiota synergize with excessive alcohol intake to promote liver injury are poorly understood. Furthermore, whether drugs that selectively target gut microbial metabolism can improve ALD has never been tested. METHODS: We used liquid chromatography tandem mass spectrometry to quantify the levels of microbe and host choline co-metabolites in healthy controls and AH patients, and identified the metabolite trimethylamine (TMA) as a gut microbe-derived biomarker of AH. In subsequent studies, we treated mice with non-lethal mechanism-based bacterial choline TMA lyase inhibitors to blunt gut microbe-dependent production of TMA in the context of chronic ethanol administration. Indices of liver injury were quantified by complementary RNA sequencing, biochemical, and histological approaches. In addition, we examined the impact of ethanol consumption and TMA lyase inhibition on gut microbiome structure via 16S rRNA sequencing. RESULTS: We show the gut microbial choline metabolite trimethylamine (TMA) is elevated in AH patients, which is correlated with reduced hepatic expression of the TMA oxygenase flavin-containing monooxygenase 3 (FMO3). Provocatively, we find that small molecule inhibition of gut microbial choline TMA lyase activity protects mice from ethanol-induced liver injury. TMA lyase inhibitor-driven improvement in ethanol-induced liver injury is associated with distinct reorganization of the gut microbiome community and host liver transcriptome. CONCLUSIONS: The microbial metabolite TMA is a biomarker of AH, and blocking TMA production from gut microbes can blunt ALD in mice.

2021-03-31 | GSE157681 | GEO

Elucidation of an anaerobic pathway for metabolism of L-carnitine-derived γ-butyrobetaine to trimethylamine in human gut bacteria

Project description:Trimethylamine (TMA) is an important gut microbial metabolite strongly associated with human disease. There are prominent gaps in our understanding of how TMA is produced from the essential dietary nutrient L-carnitine, particularly in the anoxic environment of the human gut where oxygen-dependent L-carnitine-metabolizing enzymes are likely inactive. Here, we elucidate the chemical and genetic basis for anaerobic TMA generation from the L-carnitine-derived metabolite γ-butyrobetaine (γbb) by the human gut bacterium Emergencia timonensis. We identify a set of genes upregulated by γbb and demonstrate that the enzymes encoded by the induced γbb utilization (bbu) gene cluster convert γbb to TMA. The key TMA-generating step is catalyzed by a previously unknown type of TMA-lyase enzyme that utilizes a flavin cofactor to catalyze a redox neutral transformation. We identify additional cultured and uncultured host-associated bacteria that possess the bbu gene cluster, providing insights into the distribution of anaerobic γbb metabolism. Lastly, we present genetic, transcriptional, and metabolomic evidence that confirms the relevance of this metabolic pathway in the human gut microbiota. These analyses indicate that the anaerobic pathway is a more substantial contributor to TMA generation from L-carnitine in the human gut than the previously proposed aerobic pathway. The discovery and characterization of the bbu pathway provides the critical missing link in anaerobic metabolism of L-carnitine to TMA, enabling investigation into the connection between this microbial function and human disease.

2021-02-03 | GSE165976 | GEO

Intestinal microbiota composition modulates choline bioavailability from diet and accumulation of the pro-atherogenic metabolite trimethylamine-N-oxide

Project description:Choline is a water-soluble nutrient essential for human life. Gut microbial metabolism of choline results in the production of trimethylamine (TMA), which upon absorption by the host is converted in the liver to trimethylamine N-oxide (TMAO). Recent studies revealed that TMAO exacerbates atherosclerosis in mice, and positively correlates with the severity of this disease in human. However, which microbes contribute to TMA production in the human gut; the extent to which host factors, e.g., genotype and diet, affect TMA production and colonization of these microbes; as well as the effects TMA-producing microbes have on bioavailability of dietary choline remain largely unknown. We screened a collection of 78 sequenced human intestinal isolates encompassing the major phyla found in the human gut and identified eight strains capable of producing TMA from choline in vitro. Gnotobiotic mouse studies showed that TMAO accumulates in the serum of animals colonized with TMA-producing species, but not in the serum of animals colonized with intestinal isolates that do not generate TMA from choline in vitro. Remarkably, low levels of colonization of TMA-producing bacteria significantly reduced choline levels available to the host. This effect was more pronounced as the abundance of TMA-producing bacteria increased. Our findings provide a framework for designing strategies aimed at changing the representation or activity of TMA-producing bacteria in the human gut and suggest the TMA producing status of the gut microbiota should be considered when making recommendations about choline intake requirements for humans.

2015-04-02 | GSE63461 | GEO

A Single Human-Relevant Fast Food Meal Produces Rapid Reorganization of the Gut Microbiome Revealing Unique Gut Microbe-Dependent Metabolomic Signatures

Project description:Background: More than 100 million Americans are living with metabolic syndrome, increasing their propensity to develop heart disease– the leading cause of death worldwide. A major contributing factor to this epidemic is caloric excess, often a result of consuming low cost, high calorie fast food. Several recent seminal studies have demonstrated the pivotal role of gut microbes contributing to cardiovascular disease in a diet-dependent manner. Given the central contributions of diet and gut microbiota to cardiometabolic disease, we hypothesized that novel microbial metabolites originating postprandially after fast food consumption may contribute to cardiometabolic disease progression. Methods: To test this hypothesis, we gave conventionally raised or antibiotic-treated mice a single oral gavage of a fast food slurry or a control rodent chow diet slurry and sacrificed the mice four hours later. Here, we coupled untargeted metabolomics in portal and peripheral blood, 16S rRNA gene sequencing, targeted liver metabolomics, and host liver RNA sequencing to identify novel fast food-derived microbial metabolites. Results: We successfully identified several metabolites that were enriched in portal blood, increased by fast food feeding, and essentially absent in antibiotic-treated mice. Strikingly, just four hours post-gavage, we found that fast food consumption resulted in rapid reorganization of the gut microbial community structure and drastically altered hepatic gene expression. Importantly, diet-driven reshaping of the microbiome and liver transcriptome was dependent on a non-antibiotic ablated gut microbial community. Conclusions: Collectively, these data suggest that single fast food meal is sufficient to reshape the gut microbial community yielding a unique signature of food-derived microbial metabolites. Future studies are warranted to determine if these metabolites are causally linked to cardiometabolic disease.

2021-11-14 | GSE165756 | GEO

The TMAO Generating Enzyme Flavin Monooxygenase 3 is a Central Regulator of Cholesterol Balance

Project description:Circulating levels of the gut microbe-derived metabolite trimethylamine-N-oxide(TMAO) have recently been linked to cardiovascular disease (CVD) risk. Here we performed transcriptional profiling in mouse models of altered reverse cholesterol transport (RCT), and serendipitously identified the TMAO-generating enzyme flavin monooxygenase 3 (FMO3) as a powerful modifier of cholesterol metabolism and RCT. Knockdown of FMO3 in cholesterol-fed mice alters biliary lipid secretion, blunts intestinal cholesterol absorption, and limits the production of hepatic oxysterols and cholesteryl esters. Furthermore, FMO3 knockdown stimulates basal and liver X receptor (LXR)-stimulated macrophage RCT, thereby improving cholesterol balance. Conversely, FMO3 knockdown exacerbates hepatic ER stress and inflammation in part by decreasing hepatic oxysterol levels and subsequent LXR activation. FMO3 is thus identified as a central integrator of hepatic cholesterol and triacylglycerol metabolism, inflammation, and ER stress. These studies suggest that the gut microbiota-driven TMA/FMO3/TMAO pathway is a key regulator of lipid metabolism and inflammation. To identify potential regulators of macrophage reverse cholesterol transport (RCT), liver was isolated from two independent mouse models where the non-biliary RCT pathway known as transintestinal cholesterol excretion (TICE) was either chronically (NPC1L1-liver-transgenic mice) or acutely (ACAT2 antisense oligonucleotide treatment) stimulated. Total RNA was isolated and gene expression levels were profiled on the Affymetrix GeneAtlas MG-430 PM Array Strip (Affymetrix; Santa Clara, CA, USA)

2014-12-19 | E-GEOD-64326 | biostudies-arrayexpress

The TMAO Generating Enzyme Flavin Monooxygenase 3 is a Central Regulator of Cholesterol Balance

2014-12-19 | GSE64326 | GEO

A microbiome-produced metabolite drives immunostimulatory macrophages and boosts response to immune checkpoint inhibitors in pancreatic cancer [RNA-seq]

Project description:The composition of the gut microbiome controls innate and adaptive immunity and has emerged as a key regulator of tumor growth and the success of immune checkpoint blockade (ICB) therapy. However, the underlying mechanisms remain unclear. Pancreatic ductal adenocarcinoma (PDAC) tends to be refractory to therapy, including ICB. We found that the gut microbe-derived metabolite trimethylamine N-oxide (TMAO) enhances anti-tumor immunity to PDAC. Delivery of TMAO given intraperitoneally or via dietary choline supplement to PDAC-bearing mice reduces tumor growth and is associated with an immunostimulatory tumor-associated macrophage (TAM) phenotype and activated effector T cell response in the tumor microenvironment. Mechanistically, TMAO signals through potentiating type-I interferon (IFN) pathway and confers anti-tumor effects in a type-I IFN dependent manner. Notably, delivering TMAO-primed macrophages alone produced similar anti-tumor effects. Combining TMAO with ICB (anti-PD1 and/or anti-Tim3) significantly reduced tumor burden and improved survival beyond TMAO or ICB alone. Finally, the levels of trimethylamine (TMA)-producing bacteria and of CutC gene expression associate with improved survivorship and response to anti-PD1 in cancer patients. Together, our study identifies the gut microbial metabolite TMAO as an important driver of anti-tumor immunity and lays the groundwork for new therapeutic strategies.

2022-07-22 | GSE207911 | GEO

A microbiome-produced metabolite drives immunostimulatory macrophages and boosts response to immune checkpoint inhibitors in pancreatic cancer [scRNA-seq]

2022-07-22 | GSE207946 | GEO

Gut Microbe-Targeted Choline Trimethylamine Lyase Inhibitors Improves Obesity Via Rewiring of Host Circadian Rhythms.

Project description:The increasing prevalence of obesity and related metabolic disorders represents a growing public health concern. Despite advances in other areas of medicine, a safe and effective drug treatment for obesity has been elusive. Obesity has repeatedly been linked to reorganization of the gut microbiome 1-4 , yet to this point obesity therapeutics have been targeted exclusively toward the human host. Here we show that gut microbe-targeted inhibition of the metaorganismal trimethylamine N-oxide (TMAO) pathway protects mice against the metabolic disturbances associated with diet-induced obesity (DIO) or leptin deficiency (ob/ob). Selective small molecule inhibition of the gut microbial enzyme choline TMA-lyase (CutC) does not significantly reduce food intake, but instead is associated with beneficial remodeling of the gut microbiome, improvement in glucose tolerance, and enhanced energy expenditure. Leveraging untargeted metabolomics we discovered that CutC inhibition is associated with reorganization of host circadian control of both phosphatidylcholine and energy metabolism. Collectively, this study underscores the close relationship between microbe and host metabolism, and provides evidence that gut microbe-derived trimethylamine (TMA) is a key regulator of the host circadian clock. This work also demonstrates that gut microbe-targeted enzyme inhibitors can have profound effects on host energy metabolism, and have untapped potential as anti-obesity therapeutics.

2021-03-31 | GSE157925 | GEO

Proteome of gamma-butyrobetaine-grown Eubacterium limosum ATCC 8486

Project description:Eubacterium limosum ATCC 8486 makes acetate and butyrate from various substrates and is found in the human intestine. The proteome of gamma-butyrobetaine -grown Eubacterium limosum was obtained in order to identify enzymes required for growth on gamma-butyrobetaine, in particular to identify components that are unique to growth on gamma-butyrobetaine in comparison to other substrates for acetogenesis, such as lactic acid, L-carnitine, or proline betaine. Gamma-butyrobetaine is converted to trimethylamine (TMA) by certain members of the gut microbiome. Subsequent liver metabolism of TMA is now tied to progression of cardiovascular disease. Demethylation of gamma-butyrobetaine is observed during growth of Eubacterium limosum on this substrate, and does not produce TMA. Gamma-butyrobetaine demethylation by organisms like Eubacterium limosum could lessen TMA production in the gut, thereby lessening the propensity towards atherosclerosis caused by metabolism of TMA in the body. This proteome led to discovery of gamma-butyrobetaine:tetrahydrofolate methyltransferase system. The key gamma-butyrobetaine demethylating enzyme is a member of the widespread TMA methyltransferase protein superfamily.

2021-11-04 | PXD020152 | Pride

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